To determine the influence of the microenvironment on changes in gene expression, we did microarray analysis on three variant lines of a human pancreatic cancer (FG, L3.3, and L3.6pl) with different metastatic potentials. The variant lines were grown in tissue culture in the subcutis (ectopic) or pancreas (orthotopic) of nude mice. Compared with tissue culture, the number of genes of which the expression was affected by the microenvironment was up-regulated in tumors growing in the subcutis and pancreas. In addition, highly metastatic L3.6pl cells growing in the pancreas expressed significantly higher levels of 226 genes than did the L3.3 or FG variant cells. Growth of the variant lines in the subcutis did not yield similar results, indicating that the orthotopic microenvironment significantly influences gene expression in pancreatic cancer cells. These data suggest that investigations of the functional consequence of gene expression require accounting for experimental growth conditions.
To determine the influence of the microenvironment on changes in gene expression, we did microarray analysis on three variant lines of a human pancreatic cancer (FG, L3.3, and L3.6pl) with different metastatic potentials. The variant lines were grown in tissue culture in the subcutis (ectopic) or pancreas (orthotopic) of nude mice. Compared with tissue culture, the number of genes of which the expression was affected by the microenvironment was up-regulated in tumors growing in the subcutis and pancreas. In addition, highly metastatic L3.6pl cells growing in the pancreas expressed significantly higher levels of 226 genes than did the L3.3 or FG variant cells. Growth of the variant lines in the subcutis did not yield similar results, indicating that the orthotopic microenvironment significantly influences gene expression in pancreatic cancer cells. These data suggest that investigations of the functional consequence of gene expression require accounting for experimental growth conditions.
Pediatric adrenocortical tumors (ACT) are rare and often fatal malignancies; little is known regarding their etiology and biology. To provide additional insight into the nature of ACT, we determined the gene expression profiles of 24 pediatric tumors (five adenomas, 18 carcinomas, and one undetermined) and seven normal adrenal glands. Distinct patterns of gene expression, validated by quantitative real-time PCR and Western blot analysis, were identified that distinguish normal adrenal cortex from tumor. Differences in gene expression were also identified between adrenocortical adenomas and carcinomas. In addition, pediatric adrenocortical carcinomas were found to share similar patterns of gene expression when compared with those published for adult ACT. This study represents the first microarray analysis of childhood ACT. Our findings lay the groundwork for establishing gene expression profiles that may aid in the diagnosis and prognosis of pediatric ACT, and in the identification of signaling pathways that contribute to this disease.
Pediatric adrenocortical tumors (ACT) are rare and often fatal malignancies; little is known regarding their etiology and biology. To provide additional insight into the nature of ACT, we determined the gene expression profiles of 24 pediatric tumors (five adenomas, 18 carcinomas, and one undetermined) and seven normal adrenal glands. Distinct patterns of gene expression, validated by quantitative real-time PCR and Western blot analysis, were identified that distinguish normal adrenal cortex from tumor. Differences in gene expression were also identified between adrenocortical adenomas and carcinomas. In addition, pediatric adrenocortical carcinomas were found to share similar patterns of gene expression when compared with those published for adult ACT. This study represents the first microarray analysis of childhood ACT. Our findings lay the groundwork for establishing gene expression profiles that may aid in the diagnosis and prognosis of pediatric ACT, and in the identification of signaling pathways that contribute to this disease.
Affymetrix U133plus2 GeneChips were used to profile 59 head and neck squamous cell cancers. A hypoxia metagene was obtained by analysis of genes whose in vivo expression clustered with the expression of 10 well-known hypoxia-regulated genes (e.g., CA9, GLUT1, and VEGF). To minimize random aggregation, strongly correlated up-regulated genes appearing in >50% of clusters defined a signature comprising 99 genes, of which 27% were previously known to be hypoxia associated. The median RNA expression of the 99 genes in the signature was an independent prognostic factor for recurrence-free survival in a publicly available head and neck cancer data set, outdoing the original intrinsic classifier. In a published breast cancer series, the hypoxia signature was a significant prognostic factor for overall survival independent of clinicopathologic risk factors and a trained profile. The work highlights the validity and potential of using data from analysis of in vitro stress pathways for deriving a biological metagene/gene signature in vivo.
Affymetrix U133plus2 GeneChips were used to profile 59 head and neck squamous cell cancers. A hypoxia metagene was obtained by analysis of genes whose in vivo expression clustered with the expression of 10 well-known hypoxia-regulated genes (e.g., CA9, GLUT1, and VEGF). To minimize random aggregation, strongly correlated up-regulated genes appearing in >50% of clusters defined a signature comprising 99 genes, of which 27% were previously known to be hypoxia associated. The median RNA expression of the 99 genes in the signature was an independent prognostic factor for recurrence-free survival in a publicly available head and neck cancer data set, outdoing the original intrinsic classifier. In a published breast cancer series, the hypoxia signature was a significant prognostic factor for overall survival independent of clinicopathologic risk factors and a trained profile. The work highlights the validity and potential of using data from analysis of in vitro stress pathways for deriving a biological metagene/gene signature in vivo.
The mammalian target of rapamycin (mTOR) pathway, a major regulator of translation, is frequently activated in hepatocellular carcinomas. We investigated the effects of mTOR activation in the human HepaRG cells, which possess potent hepatocytic differentiation capability. Differentiation of HepaRG cells into functional and polarized hepatocyte-like cells correlated with a decrease in mTOR and Akt activities. Stable cell lines expressing an activated mutant of mTOR were generated. Sustained activation of mTOR impaired the hepatocytic differentiation capability of these cells as shown by impaired formation of bile canaliculi, absence of polarity, and reduced secretion of alpha1-antitrypsin. An inhibitor of mTOR, rapamycin, was able to revert this phenotype. Furthermore, increased mTOR activity in HepaRG cells resulted in their resistance to the antiproliferative effects of transforming growth factor-beta1. Profiling of polysome-bound transcripts indicated that activated mTOR specifically targeted genes posttranscriptionally regulated on hepatocytic differentiation. Three major biological networks targeted by activated mTOR were identified: (a) cell death associated with tumor necrosis factor superfamily members, IFNs and caspases; (b) lipid homeostasis associated with the transcription factors PPARalpha, PPARdelta, and retinoid X receptor beta; and (c) liver development associated with CCAAT/enhancer binding protein alpha and hepatic mitogens. In conclusion, increased mTOR activity conferred a preneoplastic phenotype to the HepaRG cells by altering the translation of genes vital for establishing normal hepatic energy homeostasis and moderating hepatocellular growth.
The mammalian target of rapamycin (mTOR) pathway, a major regulator of translation, is frequently activated in hepatocellular carcinomas. We investigated the effects of mTOR activation in the human HepaRG cells, which possess potent hepatocytic differentiation capability. Differentiation of HepaRG cells into functional and polarized hepatocyte-like cells correlated with a decrease in mTOR and Akt activities. Stable cell lines expressing an activated mutant of mTOR were generated. Sustained activation of mTOR impaired the hepatocytic differentiation capability of these cells as shown by impaired formation of bile canaliculi, absence of polarity, and reduced secretion of alpha1-antitrypsin. An inhibitor of mTOR, rapamycin, was able to revert this phenotype. Furthermore, increased mTOR activity in HepaRG cells resulted in their resistance to the antiproliferative effects of transforming growth factor-beta1. Profiling of polysome-bound transcripts indicated that activated mTOR specifically targeted genes posttranscriptionally regulated on hepatocytic differentiation. Three major biological networks targeted by activated mTOR were identified: (a) cell death associated with tumor necrosis factor superfamily members, IFNs and caspases; (b) lipid homeostasis associated with the transcription factors PPARalpha, PPARdelta, and retinoid X receptor beta; and (c) liver development associated with CCAAT/enhancer binding protein alpha and hepatic mitogens. In conclusion, increased mTOR activity conferred a preneoplastic phenotype to the HepaRG cells by altering the translation of genes vital for establishing normal hepatic energy homeostasis and moderating hepatocellular growth.
Human papillomaviruses (HPV) are associated with nearly all cervical cancers, 20% to 30% of head and neck cancers (HNC), and other cancers. Because HNCs also arise in HPV-negative patients, this type of cancer provides unique opportunities to define similarities and differences of HPV-positive versus HPV-negative cancers arising in the same tissue. Here, we describe genome-wide expression profiling of 84 HNCs, cervical cancers, and site-matched normal epithelial samples in which we used laser capture microdissection to enrich samples for tumor-derived versus normal epithelial cells. This analysis revealed that HPV(+) HNCs and cervical cancers differed in their patterns of gene expression yet shared many changes compared with HPV(-) HNCs. Some of these shared changes were predicted, but many others were not. Notably, HPV(+) HNCs and cervical cancers were found to be up-regulated in their expression of a distinct and larger subset of cell cycle genes than that observed in HPV(-) HNC. Moreover, HPV(+) cancers overexpressed testis-specific genes that are normally expressed only in meiotic cells. Many, although not all, of the hallmark differences between HPV(+) HNC and HPV(-) HNC were a direct consequence of HPV and in particular the viral E6 and E7 oncogenes. This included a novel association of HPV oncogenes with testis-specific gene expression. These findings in primary human tumors provide novel biomarkers for early detection of HPV(+) and HPV(-) cancers, and emphasize the potential value of targeting E6 and E7 function, alone or combined with radiation and/or traditional chemotherapy, in the treatment of HPV(+) cancers.
Human papillomaviruses (HPV) are associated with nearly all cervical cancers, 20% to 30% of head and neck cancers (HNC), and other cancers. Because HNCs also arise in HPV-negative patients, this type of cancer provides unique opportunities to define similarities and differences of HPV-positive versus HPV-negative cancers arising in the same tissue. Here, we describe genome-wide expression profiling of 84 HNCs, cervical cancers, and site-matched normal epithelial samples in which we used laser capture microdissection to enrich samples for tumor-derived versus normal epithelial cells. This analysis revealed that HPV(+) HNCs and cervical cancers differed in their patterns of gene expression yet shared many changes compared with HPV(-) HNCs. Some of these shared changes were predicted, but many others were not. Notably, HPV(+) HNCs and cervical cancers were found to be up-regulated in their expression of a distinct and larger subset of cell cycle genes than that observed in HPV(-) HNC. Moreover, HPV(+) cancers overexpressed testis-specific genes that are normally expressed only in meiotic cells. Many, although not all, of the hallmark differences between HPV(+) HNC and HPV(-) HNC were a direct consequence of HPV and in particular the viral E6 and E7 oncogenes. This included a novel association of HPV oncogenes with testis-specific gene expression. These findings in primary human tumors provide novel biomarkers for early detection of HPV(+) and HPV(-) cancers, and emphasize the potential value of targeting E6 and E7 function, alone or combined with radiation and/or traditional chemotherapy, in the treatment of HPV(+) cancers.
Using Affymetrix HG-U133 Plus 2.0 array and laser capture microdissection techniques, we determined whether different zones of the same pancreatic tumor exhibited differential expression of genes. Human L3.6pl pancreatic cancer cells were implanted into the pancreas of nude mice. Three weeks later when tumors were 7 to 9 mm in diameter, gene expression patterns in tumor cells within the central and peripheral zones were compared, and 1,222 genes showed statistically significant differences. Bioinformatic functional analysis revealed that 346 up-regulated genes in the peripheral zone were related to cytoskeleton organization and biogenesis, cell cycle, cell adhesion, cell motility, DNA replication, localization, integrin-mediated signaling pathway, development, morphogenesis, and IkappaB kinase/nuclear factor-kappaB cascade; 876 up-regulated genes in the central zone were related to regulation of cell proliferation, regulation of transcription, transmembrane receptor protein tyrosine kinase signaling pathways, response to stress, small GTPase-mediated signal transduction, hexose metabolism, cell death, response to external stimulus, carbohydrate metabolism, and response to wounding. The reliability of the microarray results were confirmed by in situ hybridization analysis of the expression of two genes. Collectively, the data showed zonal heterogeneity for gene expression profiles in tumors and suggest that characterization of zonal gene expression profiles is essential if microarray analyses of genetic profiles are to produce reproducible data, predict disease prognosis, and allow design of specific therapeutics.
Using Affymetrix HG-U133 Plus 2.0 array and laser capture microdissection techniques, we determined whether different zones of the same pancreatic tumor exhibited differential expression of genes. Human L3.6pl pancreatic cancer cells were implanted into the pancreas of nude mice. Three weeks later when tumors were 7 to 9 mm in diameter, gene expression patterns in tumor cells within the central and peripheral zones were compared, and 1,222 genes showed statistically significant differences. Bioinformatic functional analysis revealed that 346 up-regulated genes in the peripheral zone were related to cytoskeleton organization and biogenesis, cell cycle, cell adhesion, cell motility, DNA replication, localization, integrin-mediated signaling pathway, development, morphogenesis, and IkappaB kinase/nuclear factor-kappaB cascade; 876 up-regulated genes in the central zone were related to regulation of cell proliferation, regulation of transcription, transmembrane receptor protein tyrosine kinase signaling pathways, response to stress, small GTPase-mediated signal transduction, hexose metabolism, cell death, response to external stimulus, carbohydrate metabolism, and response to wounding. The reliability of the microarray results were confirmed by in situ hybridization analysis of the expression of two genes. Collectively, the data showed zonal heterogeneity for gene expression profiles in tumors and suggest that characterization of zonal gene expression profiles is essential if microarray analyses of genetic profiles are to produce reproducible data, predict disease prognosis, and allow design of specific therapeutics.
Angioimmunoblastic lymphoma (AILT) is the second most common subtype of peripheral T-cell lymphoma (PTCL) and is characterized by dismal prognosis. Thus far, only a few studies have dealt with its molecular pathogenesis. We performed gene expression profile (GEP) analysis of six AILT, six anaplastic large cell lymphomas (ALCL), 28 PTCL-unspecified (PTCL/U), and 20 samples of normal T lymphocytes (including CD4(+), CD8(+), and activated and resting subpopulations), aiming to (a) assess the relationship of AILT with other PTCLs, (b) establish the relationship between AILT and normal T-cell subsets, and (c) recognize the cellular programs deregulated in AILT possibly looking for novel potential therapeutic targets. First, we found that AILT and other PTCLs have rather similar GEP, possibly sharing common oncogenic pathways. Second, we found that AILTs are closer to activated CD4(+), rather than to resting or CD8(+) lymphocytes. Furthermore, we found that the molecular signature of follicular T helper cells was significantly overexpressed in AILT, reinforcing the idea that AILT may arise from such cellular counterpart. Finally, we identified several genes deregulated in AILT, including PDGFRA, REL, and VEGF. The expression of several molecules was then studied by immunohistochemistry on tissue microarrays containing 45 independent AILT cases. Notably, we found that the vascular endothelial growth factor (VEGF) was expressed not only by reactive cells, but also by neoplastic cells, and that nuclear factor-kappaB (NF-kappaB) activation is uncommon in AILT, as suggested by frequent exclusively cytoplasmic c-REL localization. Our study provides new relevant information on AILT biology and new candidates for possible therapeutic targets such as PDGFRA (platelet-derived growth factor alpha) and VEGF.
Angioimmunoblastic lymphoma (AILT) is the second most common subtype of peripheral T-cell lymphoma (PTCL) and is characterized by dismal prognosis. Thus far, only a few studies have dealt with its molecular pathogenesis. We performed gene expression profile (GEP) analysis of six AILT, six anaplastic large cell lymphomas (ALCL), 28 PTCL-unspecified (PTCL/U), and 20 samples of normal T lymphocytes (including CD4(+), CD8(+), and activated and resting subpopulations), aiming to (a) assess the relationship of AILT with other PTCLs, (b) establish the relationship between AILT and normal T-cell subsets, and (c) recognize the cellular programs deregulated in AILT possibly looking for novel potential therapeutic targets. First, we found that AILT and other PTCLs have rather similar GEP, possibly sharing common oncogenic pathways. Second, we found that AILTs are closer to activated CD4(+), rather than to resting or CD8(+) lymphocytes. Furthermore, we found that the molecular signature of follicular T helper cells was significantly overexpressed in AILT, reinforcing the idea that AILT may arise from such cellular counterpart. Finally, we identified several genes deregulated in AILT, including PDGFRA, REL, and VEGF. The expression of several molecules was then studied by immunohistochemistry on tissue microarrays containing 45 independent AILT cases. Notably, we found that the vascular endothelial growth factor (VEGF) was expressed not only by reactive cells, but also by neoplastic cells, and that nuclear factor-kappaB (NF-kappaB) activation is uncommon in AILT, as suggested by frequent exclusively cytoplasmic c-REL localization. Our study provides new relevant information on AILT biology and new candidates for possible therapeutic targets such as PDGFRA (platelet-derived growth factor alpha) and VEGF.
Therapeutic strategies based on antiangiogenic approaches are beginning to show great promise in clinical studies. However, full realization of these approaches requires identification of key differences in gene expression between endothelial cells from tumors versus their normal counterparts. Here, we examined gene expression differences in purified endothelial cells from 10 invasive epithelial ovarian cancers and 5 normal ovaries using Affymetrix U133 Plus 2.0 microarrays. More than 400 differentially expressed genes were identified in tumor-associated endothelial cells. We selected and validated 23 genes that were overexpressed by 3.6- to 168-fold using real-time reverse transcription-PCR and/or immunohistochemistry. Among these, the polycomb group protein enhancer of Zeste homologue 2 (EZH2), the Notch ligand Jagged1, and PTK2 were elevated 3- to 4.3-fold in tumor-associated endothelial cells. Silencing these genes individually with small interfering RNA blocked endothelial cell migration and tube formation in vitro. The present study shows that tumor and normal endothelium differ at the molecular level, which may have significant implications for the development of antiangiogenic therapies.
Therapeutic strategies based on antiangiogenic approaches are beginning to show great promise in clinical studies. However, full realization of these approaches requires identification of key differences in gene expression between endothelial cells from tumors versus their normal counterparts. Here, we examined gene expression differences in purified endothelial cells from 10 invasive epithelial ovarian cancers and 5 normal ovaries using Affymetrix U133 Plus 2.0 microarrays. More than 400 differentially expressed genes were identified in tumor-associated endothelial cells. We selected and validated 23 genes that were overexpressed by 3.6- to 168-fold using real-time reverse transcription-PCR and/or immunohistochemistry. Among these, the polycomb group protein enhancer of Zeste homologue 2 (EZH2), the Notch ligand Jagged1, and PTK2 were elevated 3- to 4.3-fold in tumor-associated endothelial cells. Silencing these genes individually with small interfering RNA blocked endothelial cell migration and tube formation in vitro. The present study shows that tumor and normal endothelium differ at the molecular level, which may have significant implications for the development of antiangiogenic therapies.
Adult male germ cell tumors (GCTs) are the most commonly diagnosed solid tumors in men ages 18 to 35 years. Cytogenetic analysis has identified 12p gain as the most frequent change in GCTs, occurring in ~100% of cases, regardless of histology, and thus remains a genetic hallmark of adult male GCTs. We analyzed expression profiles from a panel of 101 GCT specimens and 5 normal testis samples using Affymetrix U133A+B microarrays, which provide ~83% coverage of 12p transcripts.
Adult male germ cell tumors (GCTs) are the most commonly diagnosed solid tumors in men ages 18 to 35 years. Cytogenetic analysis has identified 12p gain as the most frequent change in GCTs, occurring in ~100% of cases, regardless of histology, and thus remains a genetic hallmark of adult male GCTs. We analyzed expression profiles from a panel of 101 GCT specimens and 5 normal testis samples using Affymetrix U133A+B microarrays, which provide ~83% coverage of 12p transcripts.
Adult male germ cell tumors (GCTs) comprise distinct groups: seminomas and nonseminomas, which include pluripotent embryonal carcinomas as well as other histologic subtypes exhibiting various stages of differentiation. Almost all GCTs show 12p gain, but the target genes have not been clearly defined. To identify 12p target genes, we examined Affymetrix (Santa Clara, CA) U133A+B microarray ( approximately 83% coverage of 12p genes) expression profiles of 17 seminomas, 84 nonseminoma GCTs, and 5 normal testis samples. Seventy-three genes on 12p were significantly overexpressed, including GLUT3 and REA (overexpressed in all GCTs) and CCND2 and FLJ22028 (overexpressed in all GCTs, except choriocarcinomas). We characterized a 200-kb gene cluster at 12p13.31 that exhibited coordinated overexpression in embryonal carcinomas and seminomas, which included the known stem cell genes NANOG, STELLA, and GDF3 and two previously uncharacterized genes. A search for other coordinately regulated genomic clusters of stem cell genes did not reveal any genomic regions similar to that at 12p13.31. Comparison of embryonal carcinoma with seminomas revealed relative overexpression of several stem cell-associated genes in embryonal carcinoma, including several core "stemness" genes (EBAF, TDGF1, and SOX2) and several downstream targets of WNT, NODAL, and FGF signaling (FGF4, NODAL, and ZFP42). Our results indicate that 12p gain is a functionally relevant change leading to activation of proliferation and reestablishment/maintenance of stem cell function through activation of key stem cell genes. Furthermore, the differential expression of core stem cell genes may explain the differences in pluripotency between embryonal carcinomas and seminomas.
Adult male germ cell tumors (GCTs) comprise distinct groups: seminomas and nonseminomas, which include pluripotent embryonal carcinomas as well as other histologic subtypes exhibiting various stages of differentiation. Almost all GCTs show 12p gain, but the target genes have not been clearly defined. To identify 12p target genes, we examined Affymetrix (Santa Clara, CA) U133A+B microarray ( approximately 83% coverage of 12p genes) expression profiles of 17 seminomas, 84 nonseminoma GCTs, and 5 normal testis samples. Seventy-three genes on 12p were significantly overexpressed, including GLUT3 and REA (overexpressed in all GCTs) and CCND2 and FLJ22028 (overexpressed in all GCTs, except choriocarcinomas). We characterized a 200-kb gene cluster at 12p13.31 that exhibited coordinated overexpression in embryonal carcinomas and seminomas, which included the known stem cell genes NANOG, STELLA, and GDF3 and two previously uncharacterized genes. A search for other coordinately regulated genomic clusters of stem cell genes did not reveal any genomic regions similar to that at 12p13.31. Comparison of embryonal carcinoma with seminomas revealed relative overexpression of several stem cell-associated genes in embryonal carcinoma, including several core "stemness" genes (EBAF, TDGF1, and SOX2) and several downstream targets of WNT, NODAL, and FGF signaling (FGF4, NODAL, and ZFP42). Our results indicate that 12p gain is a functionally relevant change leading to activation of proliferation and reestablishment/maintenance of stem cell function through activation of key stem cell genes. Furthermore, the differential expression of core stem cell genes may explain the differences in pluripotency between embryonal carcinomas and seminomas.
Adult male germ cell tumors (GCTs) comprise distinct groups: seminomas and nonseminomas, which include pluripotent embryonal carcinomas as well as other histologic subtypes exhibiting various stages of differentiation. Almost all GCTs show 12p gain, but the target genes have not been clearly defined. To identify 12p target genes, we examined Affymetrix (Santa Clara, CA) U133A+B microarray ( approximately 83% coverage of 12p genes) expression profiles of 17 seminomas, 84 nonseminoma GCTs, and 5 normal testis samples. Seventy-three genes on 12p were significantly overexpressed, including GLUT3 and REA (overexpressed in all GCTs) and CCND2 and FLJ22028 (overexpressed in all GCTs, except choriocarcinomas). We characterized a 200-kb gene cluster at 12p13.31 that exhibited coordinated overexpression in embryonal carcinomas and seminomas, which included the known stem cell genes NANOG, STELLA, and GDF3 and two previously uncharacterized genes. A search for other coordinately regulated genomic clusters of stem cell genes did not reveal any genomic regions similar to that at 12p13.31. Comparison of embryonal carcinoma with seminomas revealed relative overexpression of several stem cell-associated genes in embryonal carcinoma, including several core "stemness" genes (EBAF, TDGF1, and SOX2) and several downstream targets of WNT, NODAL, and FGF signaling (FGF4, NODAL, and ZFP42). Our results indicate that 12p gain is a functionally relevant change leading to activation of proliferation and reestablishment/maintenance of stem cell function through activation of key stem cell genes. Furthermore, the differential expression of core stem cell genes may explain the differences in pluripotency between embryonal carcinomas and seminomas.
Adult male germ cell tumors (GCTs) comprise distinct groups: seminomas and nonseminomas, which include pluripotent embryonal carcinomas as well as other histologic subtypes exhibiting various stages of differentiation. Almost all GCTs show 12p gain, but the target genes have not been clearly defined. To identify 12p target genes, we examined Affymetrix (Santa Clara, CA) U133A+B microarray ( approximately 83% coverage of 12p genes) expression profiles of 17 seminomas, 84 nonseminoma GCTs, and 5 normal testis samples. Seventy-three genes on 12p were significantly overexpressed, including GLUT3 and REA (overexpressed in all GCTs) and CCND2 and FLJ22028 (overexpressed in all GCTs, except choriocarcinomas). We characterized a 200-kb gene cluster at 12p13.31 that exhibited coordinated overexpression in embryonal carcinomas and seminomas, which included the known stem cell genes NANOG, STELLA, and GDF3 and two previously uncharacterized genes. A search for other coordinately regulated genomic clusters of stem cell genes did not reveal any genomic regions similar to that at 12p13.31. Comparison of embryonal carcinoma with seminomas revealed relative overexpression of several stem cell-associated genes in embryonal carcinoma, including several core "stemness" genes (EBAF, TDGF1, and SOX2) and several downstream targets of WNT, NODAL, and FGF signaling (FGF4, NODAL, and ZFP42). Our results indicate that 12p gain is a functionally relevant change leading to activation of proliferation and reestablishment/maintenance of stem cell function through activation of key stem cell genes. Furthermore, the differential expression of core stem cell genes may explain the differences in pluripotency between embryonal carcinomas and seminomas.
Adult male germ cell tumors (GCTs) comprise distinct groups: seminomas and nonseminomas, which include pluripotent embryonal carcinomas as well as other histologic subtypes exhibiting various stages of differentiation. Almost all GCTs show 12p gain, but the target genes have not been clearly defined. To identify 12p target genes, we examined Affymetrix (Santa Clara, CA) U133A+B microarray ( approximately 83% coverage of 12p genes) expression profiles of 17 seminomas, 84 nonseminoma GCTs, and 5 normal testis samples. Seventy-three genes on 12p were significantly overexpressed, including GLUT3 and REA (overexpressed in all GCTs) and CCND2 and FLJ22028 (overexpressed in all GCTs, except choriocarcinomas). We characterized a 200-kb gene cluster at 12p13.31 that exhibited coordinated overexpression in embryonal carcinomas and seminomas, which included the known stem cell genes NANOG, STELLA, and GDF3 and two previously uncharacterized genes. A search for other coordinately regulated genomic clusters of stem cell genes did not reveal any genomic regions similar to that at 12p13.31. Comparison of embryonal carcinoma with seminomas revealed relative overexpression of several stem cell-associated genes in embryonal carcinoma, including several core "stemness" genes (EBAF, TDGF1, and SOX2) and several downstream targets of WNT, NODAL, and FGF signaling (FGF4, NODAL, and ZFP42). Our results indicate that 12p gain is a functionally relevant change leading to activation of proliferation and reestablishment/maintenance of stem cell function through activation of key stem cell genes. Furthermore, the differential expression of core stem cell genes may explain the differences in pluripotency between embryonal carcinomas and seminomas.
Adult male germ cell tumors (GCTs) comprise distinct groups: seminomas and nonseminomas, which include pluripotent embryonal carcinomas as well as other histologic subtypes exhibiting various stages of differentiation. Almost all GCTs show 12p gain, but the target genes have not been clearly defined. To identify 12p target genes, we examined Affymetrix (Santa Clara, CA) U133A+B microarray ( approximately 83% coverage of 12p genes) expression profiles of 17 seminomas, 84 nonseminoma GCTs, and 5 normal testis samples. Seventy-three genes on 12p were significantly overexpressed, including GLUT3 and REA (overexpressed in all GCTs) and CCND2 and FLJ22028 (overexpressed in all GCTs, except choriocarcinomas). We characterized a 200-kb gene cluster at 12p13.31 that exhibited coordinated overexpression in embryonal carcinomas and seminomas, which included the known stem cell genes NANOG, STELLA, and GDF3 and two previously uncharacterized genes. A search for other coordinately regulated genomic clusters of stem cell genes did not reveal any genomic regions similar to that at 12p13.31. Comparison of embryonal carcinoma with seminomas revealed relative overexpression of several stem cell-associated genes in embryonal carcinoma, including several core "stemness" genes (EBAF, TDGF1, and SOX2) and several downstream targets of WNT, NODAL, and FGF signaling (FGF4, NODAL, and ZFP42). Our results indicate that 12p gain is a functionally relevant change leading to activation of proliferation and reestablishment/maintenance of stem cell function through activation of key stem cell genes. Furthermore, the differential expression of core stem cell genes may explain the differences in pluripotency between embryonal carcinomas and seminomas.
Adult male germ cell tumors (GCTs) comprise distinct groups: seminomas and nonseminomas, which include pluripotent embryonal carcinomas as well as other histologic subtypes exhibiting various stages of differentiation. Almost all GCTs show 12p gain, but the target genes have not been clearly defined. To identify 12p target genes, we examined Affymetrix (Santa Clara, CA) U133A+B microarray ( approximately 83% coverage of 12p genes) expression profiles of 17 seminomas, 84 nonseminoma GCTs, and 5 normal testis samples. Seventy-three genes on 12p were significantly overexpressed, including GLUT3 and REA (overexpressed in all GCTs) and CCND2 and FLJ22028 (overexpressed in all GCTs, except choriocarcinomas). We characterized a 200-kb gene cluster at 12p13.31 that exhibited coordinated overexpression in embryonal carcinomas and seminomas, which included the known stem cell genes NANOG, STELLA, and GDF3 and two previously uncharacterized genes. A search for other coordinately regulated genomic clusters of stem cell genes did not reveal any genomic regions similar to that at 12p13.31. Comparison of embryonal carcinoma with seminomas revealed relative overexpression of several stem cell-associated genes in embryonal carcinoma, including several core "stemness" genes (EBAF, TDGF1, and SOX2) and several downstream targets of WNT, NODAL, and FGF signaling (FGF4, NODAL, and ZFP42). Our results indicate that 12p gain is a functionally relevant change leading to activation of proliferation and reestablishment/maintenance of stem cell function through activation of key stem cell genes. Furthermore, the differential expression of core stem cell genes may explain the differences in pluripotency between embryonal carcinomas and seminomas.
Adult male germ cell tumors (GCTs) comprise distinct groups: seminomas and nonseminomas, which include pluripotent embryonal carcinomas as well as other histologic subtypes exhibiting various stages of differentiation. Almost all GCTs show 12p gain, but the target genes have not been clearly defined. To identify 12p target genes, we examined Affymetrix (Santa Clara, CA) U133A+B microarray ( approximately 83% coverage of 12p genes) expression profiles of 17 seminomas, 84 nonseminoma GCTs, and 5 normal testis samples. Seventy-three genes on 12p were significantly overexpressed, including GLUT3 and REA (overexpressed in all GCTs) and CCND2 and FLJ22028 (overexpressed in all GCTs, except choriocarcinomas). We characterized a 200-kb gene cluster at 12p13.31 that exhibited coordinated overexpression in embryonal carcinomas and seminomas, which included the known stem cell genes NANOG, STELLA, and GDF3 and two previously uncharacterized genes. A search for other coordinately regulated genomic clusters of stem cell genes did not reveal any genomic regions similar to that at 12p13.31. Comparison of embryonal carcinoma with seminomas revealed relative overexpression of several stem cell-associated genes in embryonal carcinoma, including several core "stemness" genes (EBAF, TDGF1, and SOX2) and several downstream targets of WNT, NODAL, and FGF signaling (FGF4, NODAL, and ZFP42). Our results indicate that 12p gain is a functionally relevant change leading to activation of proliferation and reestablishment/maintenance of stem cell function through activation of key stem cell genes. Furthermore, the differential expression of core stem cell genes may explain the differences in pluripotency between embryonal carcinomas and seminomas.
Preoperative radiotherapy has been widely used to improve local control of disease and to improve survival in the treatment of rectal cancer. However, the response to radiotherapy differs among individual tumors. Our objective here was to identify a set of discriminating genes that can be used for characterization and prediction of response to radiotherapy in rectal cancer. Fifty-two rectal cancer patients who underwent preoperative radiotherapy were studied. Biopsy specimens were obtained from rectal cancer before preoperative radiotherapy. Response to radiotherapy was determined by histopathologic examination of surgically resected specimens and classified as responders or nonresponders. By determining gene expression profiles using human U95Av2 Gene Chip, we identified 33 novel discriminating genes of which the expression differed significantly between responders and nonresponders. Using this gene set, we were able to establish a new model to predict response to radiotherapy in rectal cancer with an accuracy of 82.4%. The list of discriminating genes included growth factor, apoptosis, cell proliferation, signal transduction, or cell adhesion-related genes. Among 33 discriminating genes, apoptosis inducers (lumican, thrombospondin 2, and galectin-1) showed higher expression in responders whereas apoptosis inhibitors (cyclophilin 40 and glutathione peroxidase) showed higher expression in nonresponders. The present study suggested the possibility that gene expression profiling may be useful in predicting response to radiotherapy to establish an individualized tailored therapy for rectal cancer. Global expression profiles of responders and nonresponders may provide insights into the development of novel therapeutic targets.
Preoperative radiotherapy has been widely used to improve local control of disease and to improve survival in the treatment of rectal cancer. However, the response to radiotherapy differs among individual tumors. Our objective here was to identify a set of discriminating genes that can be used for characterization and prediction of response to radiotherapy in rectal cancer. Fifty-two rectal cancer patients who underwent preoperative radiotherapy were studied. Biopsy specimens were obtained from rectal cancer before preoperative radiotherapy. Response to radiotherapy was determined by histopathologic examination of surgically resected specimens and classified as responders or nonresponders. By determining gene expression profiles using human U95Av2 Gene Chip, we identified 33 novel discriminating genes of which the expression differed significantly between responders and nonresponders. Using this gene set, we were able to establish a new model to predict response to radiotherapy in rectal cancer with an accuracy of 82.4%. The list of discriminating genes included growth factor, apoptosis, cell proliferation, signal transduction, or cell adhesion-related genes. Among 33 discriminating genes, apoptosis inducers (lumican, thrombospondin 2, and galectin-1) showed higher expression in responders whereas apoptosis inhibitors (cyclophilin 40 and glutathione peroxidase) showed higher expression in nonresponders. The present study suggested the possibility that gene expression profiling may be useful in predicting response to radiotherapy to establish an individualized tailored therapy for rectal cancer. Global expression profiles of responders and nonresponders may provide insights into the development of novel therapeutic targets.
Stimulation of CD40 is essential for normal B lymphocyte development and function. CD40 is first expressed before the rearrangement of immunoglobulin heavy chain genes in early B cell development; its expression is maintained through all subsequent stages of B cell development and is not lost during malignant transformation. Malignant B lymphocytes and other CD40-positive tumor cells differ from normal B cells in that they undergo apoptosis following CD40 stimulation. This gene set represents genes judged as differentially expressed in CD40-sensitive versus CD40-resistant diffuse large B-cell lymphoma (DLBCL) cell lines.
Stimulation of CD40 is essential for normal B lymphocyte development and function. CD40 is first expressed before the rearrangement of immunoglobulin heavy chain genes in early B cell development; its expression is maintained through all subsequent stages of B cell development and is not lost during malignant transformation. Malignant B lymphocytes and other CD40-positive tumor cells differ from normal B cells in that they undergo apoptosis following CD40 stimulation. This gene set represents genes judged as differentially expressed in CD40-sensitive versus CD40-resistant diffuse large B-cell lymphoma (DLBCL) cell lines.
Prostate cancer is the most commonly diagnosed noncutaneous neoplasm and second most common cause of cancer-related mortality in western men. To investigate the mechanisms of prostate cancer development and progression, we did expression profiling of human prostate cancer and benign tissues. We show that the SOX4 is overexpressed in prostate tumor samples compared with benign tissues by microarray analysis, real-time PCR, and immunohistochemistry. We also show that SOX4 expression is highly correlated with Gleason score at the mRNA and protein level using tissue microarrays. Genes affected by SOX4 expression were also identified, including BCL10, CSF1, and NcoA4/ARA70. TLE-1 and BBC3/PUMA were identified as direct targets of SOX4. Silencing of SOX4 by small interfering RNA transfection induced apoptosis of prostate cancer cells, suggesting that SOX4 could be a therapeutic target for prostate cancer. Stable transfection of SOX4 into nontransformed prostate cells enabled colony formation in soft agar, suggesting that, in the proper cellular context, SOX4 can be a transforming oncogene.
Prostate cancer is the most commonly diagnosed noncutaneous neoplasm and second most common cause of cancer-related mortality in western men. To investigate the mechanisms of prostate cancer development and progression, we did expression profiling of human prostate cancer and benign tissues. We show that the SOX4 is overexpressed in prostate tumor samples compared with benign tissues by microarray analysis, real-time PCR, and immunohistochemistry. We also show that SOX4 expression is highly correlated with Gleason score at the mRNA and protein level using tissue microarrays. Genes affected by SOX4 expression were also identified, including BCL10, CSF1, and NcoA4/ARA70. TLE-1 and BBC3/PUMA were identified as direct targets of SOX4. Silencing of SOX4 by small interfering RNA transfection induced apoptosis of prostate cancer cells, suggesting that SOX4 could be a therapeutic target for prostate cancer. Stable transfection of SOX4 into nontransformed prostate cells enabled colony formation in soft agar, suggesting that, in the proper cellular context, SOX4 can be a transforming oncogene.
we did DNA microarray analysis on LNCaP cells treated with SOX4 siRNA duplexes or transfected with a SOX4 cDNA expression plasmid and examined the effect on all SOX family members. We identified 466 genes significantly altered by SOX4 levels by SAM analysis using a false discovery rate of <1% (Fig. 4C; Supplementary Table S2), but, based on these data, it is not possible to discriminate between direct and indirect targets of SOX4. These 466 genes were those that were consistently responsive both to knockdown and overexpression of SOX4.
we did DNA microarray analysis on LNCaP cells treated with SOX4 siRNA duplexes or transfected with a SOX4 cDNA expression plasmid and examined the effect on all SOX family members. We identified 466 genes significantly altered by SOX4 levels by SAM analysis using a false discovery rate of <1% (Fig. 4C; Supplementary Table S2), but, based on these data, it is not possible to discriminate between direct and indirect targets of SOX4. These 466 genes were those that were consistently responsive both to knockdown and overexpression of SOX4.
Microarray gene expression profiling is a powerful tool for generating molecular cancer classifications. However, elucidating biological insights from these large data sets has been challenging. Previously, we identified a gene expression-based classification of primary uveal melanomas that accurately predicts metastatic death. Class 1 tumors have a low risk and class 2 tumors a high risk for metastatic death. Here, we used genes that discriminate these tumor classes to identify biological correlates of the aggressive class 2 signature. A search for Gene Ontology categories enriched in our class-discriminating gene list revealed a global down-regulation of neural crest and melanocyte-specific genes and an up-regulation of epithelial genes in class 2 tumors. Correspondingly, class 2 tumors exhibited epithelial features, such as polygonal cell morphology, up-regulation of the epithelial adhesion molecule E-cadherin, colocalization of E-cadherin and beta-catenin to the plasma membrane, and formation of cell-cell adhesions and acinar structures. One of our top class-discriminating genes was the helix-loop-helix inhibitor ID2, which was strongly down-regulated in class 2 tumors. The class 2 phenotype could be recapitulated by eliminating Id2 in cultured class 1 human uveal melanoma cells and in a mouse ocular melanoma model. Id2 seemed to suppress the epithelial-like class 2 phenotype by inhibiting an activator of the E-cadherin promoter. Consequently, Id2 loss triggered up-regulation of E-cadherin, which in turn promoted anchorage-independent cell growth, a likely antecedent to metastasis. These findings reveal new roles for Id2 and E-cadherin in uveal melanoma progression, and they identify potential targets for therapeutic intervention.
Microarray gene expression profiling is a powerful tool for generating molecular cancer classifications. However, elucidating biological insights from these large data sets has been challenging. Previously, we identified a gene expression-based classification of primary uveal melanomas that accurately predicts metastatic death. Class 1 tumors have a low risk and class 2 tumors a high risk for metastatic death. Here, we used genes that discriminate these tumor classes to identify biological correlates of the aggressive class 2 signature. A search for Gene Ontology categories enriched in our class-discriminating gene list revealed a global down-regulation of neural crest and melanocyte-specific genes and an up-regulation of epithelial genes in class 2 tumors. Correspondingly, class 2 tumors exhibited epithelial features, such as polygonal cell morphology, up-regulation of the epithelial adhesion molecule E-cadherin, colocalization of E-cadherin and beta-catenin to the plasma membrane, and formation of cell-cell adhesions and acinar structures. One of our top class-discriminating genes was the helix-loop-helix inhibitor ID2, which was strongly down-regulated in class 2 tumors. The class 2 phenotype could be recapitulated by eliminating Id2 in cultured class 1 human uveal melanoma cells and in a mouse ocular melanoma model. Id2 seemed to suppress the epithelial-like class 2 phenotype by inhibiting an activator of the E-cadherin promoter. Consequently, Id2 loss triggered up-regulation of E-cadherin, which in turn promoted anchorage-independent cell growth, a likely antecedent to metastasis. These findings reveal new roles for Id2 and E-cadherin in uveal melanoma progression, and they identify potential targets for therapeutic intervention.
Global gene expression profiles of DBCs and MBCs. A total of 88 samples representing 66 SBR grade III breast cancer tissue samples (22 MBCs and 44 DBCs), 4 normal breast tissue samples, and 18 cell lines were profiled using whole-genome DNA microarrays. Identification of a gene expression signature for MBC within basal breast cancers. Supervised analysis searched for a gene expression signature (GES) that would discriminate between the 21 basal MBCs and 16 basal DBCs.
Global gene expression profiles of DBCs and MBCs. A total of 88 samples representing 66 SBR grade III breast cancer tissue samples (22 MBCs and 44 DBCs), 4 normal breast tissue samples, and 18 cell lines were profiled using whole-genome DNA microarrays. Identification of a gene expression signature for MBC within basal breast cancers. Supervised analysis searched for a gene expression signature (GES) that would discriminate between the 21 basal MBCs and 16 basal DBCs.
Becoming invasive is a crucial step in breast cancer oncogenesis. At this point, a lesion carries the potential for spreading and metastasis--a process, whose molecular characteristics still remain poorly understood. In this article, we describe a matched-pair analysis of ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) of nine breast ductal carcinomas to identify novel molecular markers characterizing the transition from DCIS to IDC. The purpose of this study was to better understand the molecular biology of this transition and to identify candidate genes whose products might serve as prognostic markers and/or as molecular targets for treatment. To obtain cellular-based gene expression profiles from epithelial tumor cells, we combined laser capture microdissection with a T7-based two-round RNA amplification and Affymetrix oligonucleotide microarray analysis. Altogether, a set of 24 tumor samples was analyzed, comprised of nine matched DCIS/IDC and replicate DCIS/IDC preparations from three of the nine tumors. Cluster analysis on expression data shows the robustness and reproducibility of the techniques we established. Using multiple statistical methods, 546 significantly differentially expressed probe sets were identified. Eighteen candidate genes were evaluated by RT-PCR. Examples of genes already known to be associated with breast cancer invasion are BPAG1, LRRC15, MMP11, and PLAU. The expression of BPAG1, DACT1, GREM1, MEF2C, SART2, and TNFAIP6 was localized to epithelial tumor cells by in situ hybridization and/or immunohistochemistry, confirming the accuracy of laser capture microdissection sampling and microarray analysis.
Becoming invasive is a crucial step in breast cancer oncogenesis. At this point, a lesion carries the potential for spreading and metastasis--a process, whose molecular characteristics still remain poorly understood. In this article, we describe a matched-pair analysis of ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) of nine breast ductal carcinomas to identify novel molecular markers characterizing the transition from DCIS to IDC. The purpose of this study was to better understand the molecular biology of this transition and to identify candidate genes whose products might serve as prognostic markers and/or as molecular targets for treatment. To obtain cellular-based gene expression profiles from epithelial tumor cells, we combined laser capture microdissection with a T7-based two-round RNA amplification and Affymetrix oligonucleotide microarray analysis. Altogether, a set of 24 tumor samples was analyzed, comprised of nine matched DCIS/IDC and replicate DCIS/IDC preparations from three of the nine tumors. Cluster analysis on expression data shows the robustness and reproducibility of the techniques we established. Using multiple statistical methods, 546 significantly differentially expressed probe sets were identified. Eighteen candidate genes were evaluated by RT-PCR. Examples of genes already known to be associated with breast cancer invasion are BPAG1, LRRC15, MMP11, and PLAU. The expression of BPAG1, DACT1, GREM1, MEF2C, SART2, and TNFAIP6 was localized to epithelial tumor cells by in situ hybridization and/or immunohistochemistry, confirming the accuracy of laser capture microdissection sampling and microarray analysis.
Alveolar rhabdomyosarcomas (ARMS) are aggressive soft-tissue sarcomas affecting children and young adults. Most ARMS tumors express the PAX3-FKHR or PAX7-FKHR (PAX-FKHR) fusion genes resulting from the t(2;13) or t(1;13) chromosomal translocations, respectively. However, up to 25% of ARMS tumors are fusion negative, making it unclear whether ARMS represent a single disease or multiple clinical and biological entities with a common phenotype. To test to what extent PAX-FKHR determine class and behavior of ARMS, we used oligonucleotide microarray expression profiling on 139 primary rhabdomyosarcoma tumors and an in vitro model. We found that ARMS tumors expressing either PAX-FKHR gene share a common expression profile distinct from fusion-negative ARMS and from the other rhabdomyosarcoma variants. We also observed that PAX-FKHR expression above a minimum level is necessary for the detection of this expression profile. Using an ectopic PAX3-FKHR and PAX7-FKHR expression model, we identified an expression signature regulated by PAX-FKHR that is specific to PAX-FKHR-positive ARMS tumors. Data mining for functional annotations of signature genes suggested a role for PAX-FKHR in regulating ARMS proliferation and differentiation. Cox regression modeling identified a subset of genes within the PAX-FKHR expression signature that segregated ARMS patients into three risk groups with 5-year overall survival estimates of 7%, 48%, and 93%. These prognostic classes were independent of conventional clinical risk factors. Our results show that PAX-FKHR dictate a specific expression signature that helps define the molecular phenotype of PAX-FKHR-positive ARMS tumors and, because it is linked with disease outcome in ARMS patients, determine tumor behavior.
Alveolar rhabdomyosarcomas (ARMS) are aggressive soft-tissue sarcomas affecting children and young adults. Most ARMS tumors express the PAX3-FKHR or PAX7-FKHR (PAX-FKHR) fusion genes resulting from the t(2;13) or t(1;13) chromosomal translocations, respectively. However, up to 25% of ARMS tumors are fusion negative, making it unclear whether ARMS represent a single disease or multiple clinical and biological entities with a common phenotype. To test to what extent PAX-FKHR determine class and behavior of ARMS, we used oligonucleotide microarray expression profiling on 139 primary rhabdomyosarcoma tumors and an in vitro model. We found that ARMS tumors expressing either PAX-FKHR gene share a common expression profile distinct from fusion-negative ARMS and from the other rhabdomyosarcoma variants. We also observed that PAX-FKHR expression above a minimum level is necessary for the detection of this expression profile. Using an ectopic PAX3-FKHR and PAX7-FKHR expression model, we identified an expression signature regulated by PAX-FKHR that is specific to PAX-FKHR-positive ARMS tumors. Data mining for functional annotations of signature genes suggested a role for PAX-FKHR in regulating ARMS proliferation and differentiation. Cox regression modeling identified a subset of genes within the PAX-FKHR expression signature that segregated ARMS patients into three risk groups with 5-year overall survival estimates of 7%, 48%, and 93%. These prognostic classes were independent of conventional clinical risk factors. Our results show that PAX-FKHR dictate a specific expression signature that helps define the molecular phenotype of PAX-FKHR-positive ARMS tumors and, because it is linked with disease outcome in ARMS patients, determine tumor behavior.
Nonmalignant human mammary epithelial cells (HMEC) seeded in laminin-rich extracellular matrix (lrECM) form polarized acini and, in doing so, transit from a disorganized proliferating state to an organized growth-arrested state. We hypothesized that the gene expression pattern of organized and growth-arrested HMECs would share similarities with breast tumors with good prognoses. Using Affymetrix HG-U133A microarrays, we analyzed the expression of 22,283 gene transcripts in 184 (finite life span) and HMT3522 S1 (immortal nonmalignant) HMECs on successive days after seeding in a lrECM assay. Both HMECs underwent growth arrest in G0-G1 and differentiated into polarized acini between days 5 and 7. We identified gene expression changes with the same temporal pattern in both lines and examined the expression of these genes in a previously published panel of microarray data for 295 breast cancer samples. We show that genes that are significantly lower in the organized, growth-arrested HMEC than in their proliferating counterparts can be used to classify breast cancer patients into poor and good prognosis groups with high accuracy. This study represents a novel unsupervised approach to identifying breast cancer markers that may be of use clinically.
Nonmalignant human mammary epithelial cells (HMEC) seeded in laminin-rich extracellular matrix (lrECM) form polarized acini and, in doing so, transit from a disorganized proliferating state to an organized growth-arrested state. We hypothesized that the gene expression pattern of organized and growth-arrested HMECs would share similarities with breast tumors with good prognoses. Using Affymetrix HG-U133A microarrays, we analyzed the expression of 22,283 gene transcripts in 184 (finite life span) and HMT3522 S1 (immortal nonmalignant) HMECs on successive days after seeding in a lrECM assay. Both HMECs underwent growth arrest in G0-G1 and differentiated into polarized acini between days 5 and 7. We identified gene expression changes with the same temporal pattern in both lines and examined the expression of these genes in a previously published panel of microarray data for 295 breast cancer samples. We show that genes that are significantly lower in the organized, growth-arrested HMEC than in their proliferating counterparts can be used to classify breast cancer patients into poor and good prognosis groups with high accuracy. This study represents a novel unsupervised approach to identifying breast cancer markers that may be of use clinically.
Nonmalignant human mammary epithelial cells (HMEC) seeded in laminin-rich extracellular matrix (lrECM) form polarized acini and, in doing so, transit from a disorganized proliferating state to an organized growth-arrested state. We hypothesized that the gene expression pattern of organized and growth-arrested HMECs would share similarities with breast tumors with good prognoses. Using Affymetrix HG-U133A microarrays, we analyzed the expression of 22,283 gene transcripts in 184 (finite life span) and HMT3522 S1 (immortal nonmalignant) HMECs on successive days after seeding in a lrECM assay. Both HMECs underwent growth arrest in G0-G1 and differentiated into polarized acini between days 5 and 7. We identified gene expression changes with the same temporal pattern in both lines and examined the expression of these genes in a previously published panel of microarray data for 295 breast cancer samples. We show that genes that are significantly lower in the organized, growth-arrested HMEC than in their proliferating counterparts can be used to classify breast cancer patients into poor and good prognosis groups with high accuracy. This study represents a novel unsupervised approach to identifying breast cancer markers that may be of use clinically.
Nonmalignant human mammary epithelial cells (HMEC) seeded in laminin-rich extracellular matrix (lrECM) form polarized acini and, in doing so, transit from a disorganized proliferating state to an organized growth-arrested state. We hypothesized that the gene expression pattern of organized and growth-arrested HMECs would share similarities with breast tumors with good prognoses. Using Affymetrix HG-U133A microarrays, we analyzed the expression of 22,283 gene transcripts in 184 (finite life span) and HMT3522 S1 (immortal nonmalignant) HMECs on successive days after seeding in a lrECM assay. Both HMECs underwent growth arrest in G0-G1 and differentiated into polarized acini between days 5 and 7. We identified gene expression changes with the same temporal pattern in both lines and examined the expression of these genes in a previously published panel of microarray data for 295 breast cancer samples. We show that genes that are significantly lower in the organized, growth-arrested HMEC than in their proliferating counterparts can be used to classify breast cancer patients into poor and good prognosis groups with high accuracy. This study represents a novel unsupervised approach to identifying breast cancer markers that may be of use clinically.
Dysadherin, a cancer-associated membrane glycoprotein, down-regulates E-cadherin and promotes cancer metastasis. This study examined the role of dysadherin in breast cancer progression. Expression of dysadherin was found to be highest in breast cancer cell lines and tumors that lacked the estrogen receptor (ER). Knockdown of dysadherin caused increased association of E-cadherin with the actin cytoskeleton in breast cancer cell lines that expressed E-cadherin. However, knockdown of dysadherin could still suppress cell invasiveness in cells that had no functional E-cadherin, suggesting the existence of a novel mechanism of action. Global gene expression analysis identified chemokine (C-C motif) ligand 2 (CCL2) as the transcript most affected by dysadherin knockdown in MDA-MB-231 cells, and dysadherin was shown to regulate CCL2 expression in part through activation of the nuclear factor-kappaB pathway. The ability of dysadherin to promote tumor cell invasion in vitro was dependent on the establishment of a CCL2 autocrine loop, and CCL2 secreted by dysadherin-positive tumor cells also promoted endothelial cell migration in a paracrine fashion. Finally, experimental suppression of CCL2 in MDA-MB-231 cells reduced their ability to metastasize in vivo. This study shows that dysadherin has prometastatic effects that are independent of E-cadherin expression and that CCL2 could play an important role in mediating the prometastatic effect of dysadherin in ER-negative breast cancer.
In the current study, we have focused on the identification and characterization of genes that are preferentially regulated by Tam but not by E2. Affymetrix human GeneChip U133A microarrays (Affymetrix, Santa Clara, CA) were used for expression profiling following infection of MCF-7 cells with adenovirus (empty virus) or AdERh and treatment with 108 mol/L E2 or 108 mol/L trans-hydroxytamoxifen (Tam) for 4 or 24 hours. MCF-7 cells expressing endogenous ERalpha were infected with adenovirus carrying either estrogen receptor beta (AdERb) or no insert (Ad) at multiplicity of infection (moi) of 10. Cells were infected with adenovirus for a period of 48hr before treatment with ligand (vehicle control, 10nM 17beta-estradiol, or 10nM hydroxytamoxifen) for a additional period of 24hr before harvest.
In the current study, we have focused on the identification and characterization of genes that are preferentially regulated by Tam but not by E2. Affymetrix human GeneChip U133A microarrays (Affymetrix, Santa Clara, CA) were used for expression profiling following infection of MCF-7 cells with adenovirus (empty virus) or AdERh and treatment with 108 mol/L E2 or 108 mol/L trans-hydroxytamoxifen (Tam) for 4 or 24 hours. MCF-7 cells expressing endogenous ERalpha were infected with adenovirus carrying either estrogen receptor beta (AdERb) or no insert (Ad) at multiplicity of infection (moi) of 10. Cells were infected with adenovirus for a period of 48hr before treatment with ligand (vehicle control, 10nM 17beta-estradiol, or 10nM hydroxytamoxifen) for a additional period of 24hr before harvest.
Malignant glioma is the most common central nervous system tumor of adults and is associated with a significant degree of morbidity and mortality. Gliomas are highly invasive and respond poorly to conventional treatments. Gliomas, like other tumor types, arise from a complex and poorly understood sequence of genetic and epigenetic alterations. Epigenetic alterations leading to gene silencing, in the form of aberrant CpG island promoter hypermethylation and histone deacetylation, have not been thoroughly investigated in brain tumors, and elucidating such changes is likely to enhance our understanding of their etiology and provide new treatment options. We used a combined approach of pharmacologic inhibition of DNA methylation and histone deacetylation, coupled with expression microarrays, to identify novel targets of epigenetic silencing in glioma cell lines. From this analysis, we identified >160 genes up-regulated by 5-aza-2'-deoxycytidine and trichostatin A treatment. Further characterization of 10 of these genes, including the putative metastasis suppressor CST6, the apoptosis-inducer BIK, and TSPYL5, whose function is unknown, revealed that they are frequent targets of epigenetic silencing in glioma cell lines and primary tumors and suppress glioma cell growth in culture. Furthermore, we show that other members of the TSPYL gene family are epigenetically silenced in gliomas and dissect the contribution of individual DNA methyltransferases to the aberrant promoter hypermethylation events. These studies, therefore, lay the foundation for a comprehensive understanding of the full extent of epigenetic changes in gliomas and how they may be exploited for therapeutic purposes.
Malignant glioma is the most common central nervous system tumor of adults and is associated with a significant degree of morbidity and mortality. Gliomas are highly invasive and respond poorly to conventional treatments. Gliomas, like other tumor types, arise from a complex and poorly understood sequence of genetic and epigenetic alterations. Epigenetic alterations leading to gene silencing, in the form of aberrant CpG island promoter hypermethylation and histone deacetylation, have not been thoroughly investigated in brain tumors, and elucidating such changes is likely to enhance our understanding of their etiology and provide new treatment options. We used a combined approach of pharmacologic inhibition of DNA methylation and histone deacetylation, coupled with expression microarrays, to identify novel targets of epigenetic silencing in glioma cell lines. From this analysis, we identified >160 genes up-regulated by 5-aza-2'-deoxycytidine and trichostatin A treatment. Further characterization of 10 of these genes, including the putative metastasis suppressor CST6, the apoptosis-inducer BIK, and TSPYL5, whose function is unknown, revealed that they are frequent targets of epigenetic silencing in glioma cell lines and primary tumors and suppress glioma cell growth in culture. Furthermore, we show that other members of the TSPYL gene family are epigenetically silenced in gliomas and dissect the contribution of individual DNA methyltransferases to the aberrant promoter hypermethylation events. These studies, therefore, lay the foundation for a comprehensive understanding of the full extent of epigenetic changes in gliomas and how they may be exploited for therapeutic purposes.
To identify the molecular mechanisms by which EBV-associated epithelial cancers are maintained, we measured the expression of essentially all human genes and all latent EBV genes in a collection of 31 laser-captured, microdissected nasopharyngeal carcinoma (NPC) tissue samples and 10 normal nasopharyngeal tissues. Global gene expression profiles clearly distinguished tumors from normal healthy epithelium. Expression levels of six viral genes (EBNA1, EBNA2, EBNA3A, EBNA3B, LMP1, and LMP2A) were correlated among themselves and strongly inversely correlated with the expression of a large subset of host genes. Among the human genes whose inhibition was most strongly correlated with increased EBV gene expression were multiple MHC class I HLA genes involved in regulating immune response via antigen presentation. The association between EBV gene expression and inhibition of MHC class I HLA expression implies that antigen display is either directly inhibited by EBV, facilitating immune evasion by tumor cells, and/or that tumor cells with inhibited presentation are selected for their ability to sustain higher levels of EBV to take maximum advantage of EBV oncogene-mediated tumor-promoting actions. Our data clearly reflect such tumor promotion, showing that deregulation of key proteins involved in apoptosis (BCL2-related protein A1 and Fas apoptotic inhibitory molecule), cell cycle checkpoints (AKIP, SCYL1, and NIN), and metastasis (matrix metalloproteinase 1) is closely correlated with the levels of EBV gene expression in NPC.
To identify the molecular mechanisms by which EBV-associated epithelial cancers are maintained, we measured the expression of essentially all human genes and all latent EBV genes in a collection of 31 laser-captured, microdissected nasopharyngeal carcinoma (NPC) tissue samples and 10 normal nasopharyngeal tissues. Global gene expression profiles clearly distinguished tumors from normal healthy epithelium. Expression levels of six viral genes (EBNA1, EBNA2, EBNA3A, EBNA3B, LMP1, and LMP2A) were correlated among themselves and strongly inversely correlated with the expression of a large subset of host genes. Among the human genes whose inhibition was most strongly correlated with increased EBV gene expression were multiple MHC class I HLA genes involved in regulating immune response via antigen presentation. The association between EBV gene expression and inhibition of MHC class I HLA expression implies that antigen display is either directly inhibited by EBV, facilitating immune evasion by tumor cells, and/or that tumor cells with inhibited presentation are selected for their ability to sustain higher levels of EBV to take maximum advantage of EBV oncogene-mediated tumor-promoting actions. Our data clearly reflect such tumor promotion, showing that deregulation of key proteins involved in apoptosis (BCL2-related protein A1 and Fas apoptotic inhibitory molecule), cell cycle checkpoints (AKIP, SCYL1, and NIN), and metastasis (matrix metalloproteinase 1) is closely correlated with the levels of EBV gene expression in NPC.
Promoter methylation of the mismatch repair gene plays a key role in sporadic microsatellite instability (MSI) colorectal cancers. However, promoter methylation often occurs in proximal colon cancers, and molecular phenotypes underlying MSI cancers in distal colon have not been fully clarified. Our goal was to clarify the difference between MSI and microsatellite stability (MSS) cancers and, furthermore, to determine distinct characteristics of proximal and distal MSI cancers. By DNA microarray analysis of 84 cancers (33 MSI and 51 MSS), we identified discriminating genes (177 probe sets), which predicted MSI status with a high accuracy rate (97.6%).
Promoter methylation of the mismatch repair gene plays a key role in sporadic microsatellite instability (MSI) colorectal cancers. However, promoter methylation often occurs in proximal colon cancers, and molecular phenotypes underlying MSI cancers in distal colon have not been fully clarified. Our goal was to clarify the difference between MSI and microsatellite stability (MSS) cancers and, furthermore, to determine distinct characteristics of proximal and distal MSI cancers. By DNA microarray analysis of 84 cancers (33 MSI and 51 MSS), we identified discriminating genes (177 probe sets), which predicted MSI status with a high accuracy rate (97.6%).
Activating mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase domain determine responsiveness to EGFR tyrosine kinase inhibitors in patients with advanced non-small cell lung cancer (NSCLC). The modulation of transcriptional pathways by mutant EGFR signaling is not fully understood. Previously, we and others identified a single base pair change leading to a threonine to methionine (T790M) amino acid alteration in the ATP-binding pocket of the EGFR as a common mechanism of acquired resistance. The gefitinib-resistant, T790M-mutant H1975 NSCLC cell line undergoes prominent growth arrest and apoptosis when treated with the irreversible EGFR inhibitor, CL-387,785. We did a transcriptional profiling study of mutant EGFR target genes that are differentially expressed in the "resistant" gefitinib-treated and the "sensitive" CL387,785-treated H1975 cells to identify the pivotal transcriptional changes in NSCLC with EGFR-activating mutations. We identified a small subset of early gene changes, including significant reduction of cyclin D1 as a result of EGFR inhibition by CL-387,785 but not by gefitinib. The reduction in cyclin D1 transcription was associated with subsequent suppression of E2F-responsive genes, consistent with proliferation arrest. Furthermore, cyclin D1 expression was higher in EGFR-mutant lung cancer cells compared with cells with wild-type EGFR. EGFR-mutant cells were routinely sensitive to the cyclin-dependent kinase inhibitor flavopiridol, confirming the functional relevance of the cyclin D axis. These studies suggest that cyclin D1 may contribute to the emergence of EGFR-driven tumorigenesis and can be an alternative target of therapy.
Activating mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase domain determine responsiveness to EGFR tyrosine kinase inhibitors in patients with advanced non-small cell lung cancer (NSCLC). The modulation of transcriptional pathways by mutant EGFR signaling is not fully understood. Previously, we and others identified a single base pair change leading to a threonine to methionine (T790M) amino acid alteration in the ATP-binding pocket of the EGFR as a common mechanism of acquired resistance. The gefitinib-resistant, T790M-mutant H1975 NSCLC cell line undergoes prominent growth arrest and apoptosis when treated with the irreversible EGFR inhibitor, CL-387,785. We did a transcriptional profiling study of mutant EGFR target genes that are differentially expressed in the "resistant" gefitinib-treated and the "sensitive" CL387,785-treated H1975 cells to identify the pivotal transcriptional changes in NSCLC with EGFR-activating mutations. We identified a small subset of early gene changes, including significant reduction of cyclin D1 as a result of EGFR inhibition by CL-387,785 but not by gefitinib. The reduction in cyclin D1 transcription was associated with subsequent suppression of E2F-responsive genes, consistent with proliferation arrest. Furthermore, cyclin D1 expression was higher in EGFR-mutant lung cancer cells compared with cells with wild-type EGFR. EGFR-mutant cells were routinely sensitive to the cyclin-dependent kinase inhibitor flavopiridol, confirming the functional relevance of the cyclin D axis. These studies suggest that cyclin D1 may contribute to the emergence of EGFR-driven tumorigenesis and can be an alternative target of therapy.
we performed global gene expression analysis of purified BM subsets from RPS19-mutated DBA patients and unaffected control individuals. Significantly changed genes in erythroid progenitors (E population) in DBA samples. To evaluate differential gene expression between diseased and control individuals for three BM cell populations (P, E, and M), we used two separate statistical methods, geometric fold change analysis [20] and significance analysis of microarrays [21]. Genes differentially expressed in E population as judged by either SAM or geometric fold change
we performed global gene expression analysis of purified BM subsets from RPS19-mutated DBA patients and unaffected control individuals. Significantly changed genes in erythroid progenitors (E population) in DBA samples. To evaluate differential gene expression between diseased and control individuals for three BM cell populations (P, E, and M), we used two separate statistical methods, geometric fold change analysis [20] and significance analysis of microarrays [21]. Genes differentially expressed in E population as judged by either SAM or geometric fold change
Diamond-Blackfan anemia (DBA) is a broad developmental disease characterized by anemia, bone marrow (BM) erythroblastopenia, and an increased incidence of malignancy. Mutations in ribosomal protein gene S19 (RPS19) are found in approximately 25% of DBA patients; however, the role of RPS19 in the pathogenesis of DBA remains unknown. Using global gene expression analysis, we compared highly purified multipotential, erythroid, and myeloid BM progenitors from RPS19 mutated and control individuals. We found several ribosomal protein genes downregulated in all DBA progenitors. Apoptosis genes, such as TNFRSF10B and FAS, transcriptional control genes, including the erythropoietic transcription factor MYB (encoding c-myb), and translational genes were greatly dysregulated, mostly in diseased erythroid cells. Cancer-related genes, including RAS family oncogenes and tumor suppressor genes, were significantly dysregulated in all diseased progenitors. In addition, our results provide evidence that RPS19 mutations lead to codownregulation of multiple ribosomal protein genes, as well as downregulation of genes involved in translation in DBA cells. In conclusion, the altered expression of cancer-related genes suggests a molecular basis for malignancy in DBA. Downregulation of c-myb expression, which causes complete failure of fetal liver erythropoiesis in knockout mice, suggests a link between RPS19 mutations and reduced erythropoiesis in DBA.
Diamond-Blackfan anemia (DBA) is a broad developmental disease characterized by anemia, bone marrow (BM) erythroblastopenia, and an increased incidence of malignancy. Mutations in ribosomal protein gene S19 (RPS19) are found in approximately 25% of DBA patients; however, the role of RPS19 in the pathogenesis of DBA remains unknown. Using global gene expression analysis, we compared highly purified multipotential, erythroid, and myeloid BM progenitors from RPS19 mutated and control individuals. We found several ribosomal protein genes downregulated in all DBA progenitors. Apoptosis genes, such as TNFRSF10B and FAS, transcriptional control genes, including the erythropoietic transcription factor MYB (encoding c-myb), and translational genes were greatly dysregulated, mostly in diseased erythroid cells. Cancer-related genes, including RAS family oncogenes and tumor suppressor genes, were significantly dysregulated in all diseased progenitors. In addition, our results provide evidence that RPS19 mutations lead to codownregulation of multiple ribosomal protein genes, as well as downregulation of genes involved in translation in DBA cells. In conclusion, the altered expression of cancer-related genes suggests a molecular basis for malignancy in DBA. Downregulation of c-myb expression, which causes complete failure of fetal liver erythropoiesis in knockout mice, suggests a link between RPS19 mutations and reduced erythropoiesis in DBA.
Diamond-Blackfan anemia (DBA) is a broad developmental disease characterized by anemia, bone marrow (BM) erythroblastopenia, and an increased incidence of malignancy. Mutations in ribosomal protein gene S19 (RPS19) are found in approximately 25% of DBA patients; however, the role of RPS19 in the pathogenesis of DBA remains unknown. Using global gene expression analysis, we compared highly purified multipotential, erythroid, and myeloid BM progenitors from RPS19 mutated and control individuals. We found several ribosomal protein genes downregulated in all DBA progenitors. Apoptosis genes, such as TNFRSF10B and FAS, transcriptional control genes, including the erythropoietic transcription factor MYB (encoding c-myb), and translational genes were greatly dysregulated, mostly in diseased erythroid cells. Cancer-related genes, including RAS family oncogenes and tumor suppressor genes, were significantly dysregulated in all diseased progenitors. In addition, our results provide evidence that RPS19 mutations lead to codownregulation of multiple ribosomal protein genes, as well as downregulation of genes involved in translation in DBA cells. In conclusion, the altered expression of cancer-related genes suggests a molecular basis for malignancy in DBA. Downregulation of c-myb expression, which causes complete failure of fetal liver erythropoiesis in knockout mice, suggests a link between RPS19 mutations and reduced erythropoiesis in DBA.
Diamond-Blackfan anemia (DBA) is a broad developmental disease characterized by anemia, bone marrow (BM) erythroblastopenia, and an increased incidence of malignancy. Mutations in ribosomal protein gene S19 (RPS19) are found in approximately 25% of DBA patients; however, the role of RPS19 in the pathogenesis of DBA remains unknown. Using global gene expression analysis, we compared highly purified multipotential, erythroid, and myeloid BM progenitors from RPS19 mutated and control individuals. We found several ribosomal protein genes downregulated in all DBA progenitors. Apoptosis genes, such as TNFRSF10B and FAS, transcriptional control genes, including the erythropoietic transcription factor MYB (encoding c-myb), and translational genes were greatly dysregulated, mostly in diseased erythroid cells. Cancer-related genes, including RAS family oncogenes and tumor suppressor genes, were significantly dysregulated in all diseased progenitors. In addition, our results provide evidence that RPS19 mutations lead to codownregulation of multiple ribosomal protein genes, as well as downregulation of genes involved in translation in DBA cells. In conclusion, the altered expression of cancer-related genes suggests a molecular basis for malignancy in DBA. Downregulation of c-myb expression, which causes complete failure of fetal liver erythropoiesis in knockout mice, suggests a link between RPS19 mutations and reduced erythropoiesis in DBA.
Many tumors, including Hodgkin's lymphoma, are associated with decreased cellular immunity and elevated levels of prostaglandin E(2) (PGE(2)), a known inhibitor of CD4+ T cell activation, suggested to be involved in immune deviation in cancer. To address the molecular mechanisms tumor-derived PGE(2) might have on primary human CD4+ T cells, we used a whole genome-based transcriptional approach and show that PGE(2) severely limited changes of gene expression induced by signaling through the T cell receptor and CD28. This data suggests an interference of PGE(2) at an early step of T cell receptor signaling: indeed, PGE(2) stimulation of T cells leads to inactivation of lck and reduced phosphorylation of ZAP70. Antiapoptotic genes escaped PGE(2)-induced inhibition resulting in partial protection from apoptosis in response to irradiation or Fas-mediated signaling. As a functional consequence, PGE(2)-treated CD4+ T cells are arrested in the cell cycle associated with up-regulation of the cyclin/cyclin-dependent kinase inhibitor p27(kip1). Most importantly, CD4+ T cells in Hodgkin's lymphoma show similar regulation of genes that were altered in vitro by PGE(2) in T cells from healthy individuals. These data strongly suggest that PGE(2) is an important factor leading to CD4+ T cell impairment observed in Hodgkin's lymphoma.
Many tumors, including Hodgkin's lymphoma, are associated with decreased cellular immunity and elevated levels of prostaglandin E(2) (PGE(2)), a known inhibitor of CD4+ T cell activation, suggested to be involved in immune deviation in cancer. To address the molecular mechanisms tumor-derived PGE(2) might have on primary human CD4+ T cells, we used a whole genome-based transcriptional approach and show that PGE(2) severely limited changes of gene expression induced by signaling through the T cell receptor and CD28. This data suggests an interference of PGE(2) at an early step of T cell receptor signaling: indeed, PGE(2) stimulation of T cells leads to inactivation of lck and reduced phosphorylation of ZAP70. Antiapoptotic genes escaped PGE(2)-induced inhibition resulting in partial protection from apoptosis in response to irradiation or Fas-mediated signaling. As a functional consequence, PGE(2)-treated CD4+ T cells are arrested in the cell cycle associated with up-regulation of the cyclin/cyclin-dependent kinase inhibitor p27(kip1). Most importantly, CD4+ T cells in Hodgkin's lymphoma show similar regulation of genes that were altered in vitro by PGE(2) in T cells from healthy individuals. These data strongly suggest that PGE(2) is an important factor leading to CD4+ T cell impairment observed in Hodgkin's lymphoma.
Numerous studies show that nonsteroidal anti-inflammatory drugs (NSAIDs) are effective in chemoprevention or treatment of cancer. Nevertheless, the mechanisms underlying these antineoplastic effects remain poorly understood. Here, we report that induction of the cancer-specific proapoptotic cytokine melanoma differentiation associated gene-7/interleukin-24 (MDA-7/IL-24) by several NSAIDs is an essential step for induction of apoptosis and G(2)-M growth arrest in cancer cells in vitro and inhibition of tumor growth in vivo. We also show that MDA-7/IL-24-dependent up-regulation of growth arrest and DNA damage inducible 45 alpha (GADD45alpha) and GADD45gamma gene expression is sufficient for cancer cell apoptosis via c-Jun NH(2)-terminal kinase (JNK) activation and growth arrest induction through inhibition of Cdc2-cyclin B checkpoint kinase. Knockdown of GADD45alpha and GADD45gamma transcription by small interfering RNA abrogates apoptosis and growth arrest induction by the NSAID treatment, blocks JNK activation, and restores Cdc2-cyclin B kinase activity. Our results establish MDA-7/IL-24 and GADD45alpha and GADD45gamma as critical mediators of apoptosis and growth arrest in response to NSAIDs in cancer cells.
Numerous studies show that nonsteroidal anti-inflammatory drugs (NSAIDs) are effective in chemoprevention or treatment of cancer. Nevertheless, the mechanisms underlying these antineoplastic effects remain poorly understood. Here, we report that induction of the cancer-specific proapoptotic cytokine melanoma differentiation associated gene-7/interleukin-24 (MDA-7/IL-24) by several NSAIDs is an essential step for induction of apoptosis and G(2)-M growth arrest in cancer cells in vitro and inhibition of tumor growth in vivo. We also show that MDA-7/IL-24-dependent up-regulation of growth arrest and DNA damage inducible 45 alpha (GADD45alpha) and GADD45gamma gene expression is sufficient for cancer cell apoptosis via c-Jun NH(2)-terminal kinase (JNK) activation and growth arrest induction through inhibition of Cdc2-cyclin B checkpoint kinase. Knockdown of GADD45alpha and GADD45gamma transcription by small interfering RNA abrogates apoptosis and growth arrest induction by the NSAID treatment, blocks JNK activation, and restores Cdc2-cyclin B kinase activity. Our results establish MDA-7/IL-24 and GADD45alpha and GADD45gamma as critical mediators of apoptosis and growth arrest in response to NSAIDs in cancer cells.
The mechanisms underlying cellular drug resistance have been extensively studied, but little is known about its regulation. We have previously reported that activating transcription factor 4 (ATF4) is upregulated in cisplatin-resistant cells and plays a role in cisplatin resistance. Here, we find out a novel relationship between the circadian transcription factor Clock and drug resistance. Clock drives the periodical expression of many genes that regulate hormone release, cell division, sleep-awake cycle and tumor growth. We demonstrate that ATF4 is a direct target of Clock, and that Clock is overexpressed in cisplatin-resistant cells. Furthermore, Clock expression significantly correlates with cisplatin sensitivity, and that the downregulation of either Clock or ATF4 confers sensitivity of A549 cells to cisplatin and etoposide. Notably, ATF4-overexpressing cells show multidrug resistance and marked elevation of intracellular glutathione. The microarray study reveals that genes for glutathione metabolism are generally downregulated by the knockdown of ATF4 expression. These results suggest that the Clock and ATF4 transcription system might play an important role in multidrug resistance through glutathione-dependent redox system, and also indicate that physiological potentials of Clock-controlled redox system might be important to better understand the oxidative stress-associated disorders including cancer and systemic chronotherapy.
The mechanisms underlying cellular drug resistance have been extensively studied, but little is known about its regulation. We have previously reported that activating transcription factor 4 (ATF4) is upregulated in cisplatin-resistant cells and plays a role in cisplatin resistance. Here, we find out a novel relationship between the circadian transcription factor Clock and drug resistance. Clock drives the periodical expression of many genes that regulate hormone release, cell division, sleep-awake cycle and tumor growth. We demonstrate that ATF4 is a direct target of Clock, and that Clock is overexpressed in cisplatin-resistant cells. Furthermore, Clock expression significantly correlates with cisplatin sensitivity, and that the downregulation of either Clock or ATF4 confers sensitivity of A549 cells to cisplatin and etoposide. Notably, ATF4-overexpressing cells show multidrug resistance and marked elevation of intracellular glutathione. The microarray study reveals that genes for glutathione metabolism are generally downregulated by the knockdown of ATF4 expression. These results suggest that the Clock and ATF4 transcription system might play an important role in multidrug resistance through glutathione-dependent redox system, and also indicate that physiological potentials of Clock-controlled redox system might be important to better understand the oxidative stress-associated disorders including cancer and systemic chronotherapy.
Lung cancer is the leading cause of cancer-related deaths in the United States due, in large part, to the lack of early detection methods. Lung cancer arises from a complex series of genetic and epigenetic changes leading to uncontrolled cell growth and metastasis. Unlike genetic changes, epigenetic changes, such as DNA methylation and histone acetylation, are reversible with currently available pharmaceuticals and are early events in lung tumorigenesis detectable by non-invasive methods. In order to better understand how epigenetic changes contribute to lung cancer, and to identify new disease biomarkers, we combined pharmacologic inhibition of DNA methylation and histone deacetylation in non-small cell lung cancer (NSCLC) cell lines, with genome-wide expression profiling. Of the more than 200 genes upregulated by these treatments, three of these, neuronatin, metallothionein 3 and cystatin E/M, were frequently hypermethylated and transcriptionally downregulated in NSCLC cell lines and tumors. Interestingly, four other genes, cylindromatosis, CD9, activating transcription factor 3 and oxytocin receptor, were dominantly regulated by histone deacetylation and were also frequently downregulated in lung tumors. The majority of these genes also suppressed NSCLC growth in culture when ectopically expressed. This study therefore reveals new putative NSCLC growth regulatory genes and epigenetic disease biomarkers that may enhance early detection strategies and serve as therapeutic targets.
Lung cancer is the leading cause of cancer-related deaths in the United States due, in large part, to the lack of early detection methods. Lung cancer arises from a complex series of genetic and epigenetic changes leading to uncontrolled cell growth and metastasis. Unlike genetic changes, epigenetic changes, such as DNA methylation and histone acetylation, are reversible with currently available pharmaceuticals and are early events in lung tumorigenesis detectable by non-invasive methods. In order to better understand how epigenetic changes contribute to lung cancer, and to identify new disease biomarkers, we combined pharmacologic inhibition of DNA methylation and histone deacetylation in non-small cell lung cancer (NSCLC) cell lines, with genome-wide expression profiling. Of the more than 200 genes upregulated by these treatments, three of these, neuronatin, metallothionein 3 and cystatin E/M, were frequently hypermethylated and transcriptionally downregulated in NSCLC cell lines and tumors. Interestingly, four other genes, cylindromatosis, CD9, activating transcription factor 3 and oxytocin receptor, were dominantly regulated by histone deacetylation and were also frequently downregulated in lung tumors. The majority of these genes also suppressed NSCLC growth in culture when ectopically expressed. This study therefore reveals new putative NSCLC growth regulatory genes and epigenetic disease biomarkers that may enhance early detection strategies and serve as therapeutic targets.
Alveolar rhabdomyosarcomas (ARMS) are aggressive soft-tissue sarcomas affecting children and young adults. Most ARMS tumors express the PAX3-FKHR or PAX7-FKHR (PAX-FKHR) fusion genes resulting from the t(2;13) or t(1;13) chromosomal translocations, respectively. However, up to 25% of ARMS tumors are fusion negative, making it unclear whether ARMS represent a single disease or multiple clinical and biological entities with a common phenotype. To test to what extent PAX-FKHR determine class and behavior of ARMS, we used oligonucleotide microarray expression profiling on 139 primary rhabdomyosarcoma tumors and an in vitro model. We found that ARMS tumors expressing either PAX-FKHR gene share a common expression profile distinct from fusion-negative ARMS and from the other rhabdomyosarcoma variants. We also observed that PAX-FKHR expression above a minimum level is necessary for the detection of this expression profile. Using an ectopic PAX3-FKHR and PAX7-FKHR expression model, we identified an expression signature regulated by PAX-FKHR that is specific to PAX-FKHR-positive ARMS tumors. Data mining for functional annotations of signature genes suggested a role for PAX-FKHR in regulating ARMS proliferation and differentiation. Cox regression modeling identified a subset of genes within the PAX-FKHR expression signature that segregated ARMS patients into three risk groups with 5-year overall survival estimates of 7%, 48%, and 93%. These prognostic classes were independent of conventional clinical risk factors. Our results show that PAX-FKHR dictate a specific expression signature that helps define the molecular phenotype of PAX-FKHR-positive ARMS tumors and, because it is linked with disease outcome in ARMS patients, determine tumor behavior.
Alveolar rhabdomyosarcomas (ARMS) are aggressive soft-tissue sarcomas affecting children and young adults. Most ARMS tumors express the PAX3-FKHR or PAX7-FKHR (PAX-FKHR) fusion genes resulting from the t(2;13) or t(1;13) chromosomal translocations, respectively. However, up to 25% of ARMS tumors are fusion negative, making it unclear whether ARMS represent a single disease or multiple clinical and biological entities with a common phenotype. To test to what extent PAX-FKHR determine class and behavior of ARMS, we used oligonucleotide microarray expression profiling on 139 primary rhabdomyosarcoma tumors and an in vitro model. We found that ARMS tumors expressing either PAX-FKHR gene share a common expression profile distinct from fusion-negative ARMS and from the other rhabdomyosarcoma variants. We also observed that PAX-FKHR expression above a minimum level is necessary for the detection of this expression profile. Using an ectopic PAX3-FKHR and PAX7-FKHR expression model, we identified an expression signature regulated by PAX-FKHR that is specific to PAX-FKHR-positive ARMS tumors. Data mining for functional annotations of signature genes suggested a role for PAX-FKHR in regulating ARMS proliferation and differentiation. Cox regression modeling identified a subset of genes within the PAX-FKHR expression signature that segregated ARMS patients into three risk groups with 5-year overall survival estimates of 7%, 48%, and 93%. These prognostic classes were independent of conventional clinical risk factors. Our results show that PAX-FKHR dictate a specific expression signature that helps define the molecular phenotype of PAX-FKHR-positive ARMS tumors and, because it is linked with disease outcome in ARMS patients, determine tumor behavior.
Alveolar rhabdomyosarcomas (ARMS) are aggressive soft-tissue sarcomas affecting children and young adults. Most ARMS tumors express the PAX3-FKHR or PAX7-FKHR (PAX-FKHR) fusion genes resulting from the t(2;13) or t(1;13) chromosomal translocations, respectively. However, up to 25% of ARMS tumors are fusion negative, making it unclear whether ARMS represent a single disease or multiple clinical and biological entities with a common phenotype. To test to what extent PAX-FKHR determine class and behavior of ARMS, we used oligonucleotide microarray expression profiling on 139 primary rhabdomyosarcoma tumors and an in vitro model. We found that ARMS tumors expressing either PAX-FKHR gene share a common expression profile distinct from fusion-negative ARMS and from the other rhabdomyosarcoma variants. We also observed that PAX-FKHR expression above a minimum level is necessary for the detection of this expression profile. Using an ectopic PAX3-FKHR and PAX7-FKHR expression model, we identified an expression signature regulated by PAX-FKHR that is specific to PAX-FKHR-positive ARMS tumors. Data mining for functional annotations of signature genes suggested a role for PAX-FKHR in regulating ARMS proliferation and differentiation. Cox regression modeling identified a subset of genes within the PAX-FKHR expression signature that segregated ARMS patients into three risk groups with 5-year overall survival estimates of 7%, 48%, and 93%. These prognostic classes were independent of conventional clinical risk factors. Our results show that PAX-FKHR dictate a specific expression signature that helps define the molecular phenotype of PAX-FKHR-positive ARMS tumors and, because it is linked with disease outcome in ARMS patients, determine tumor behavior.
Alveolar rhabdomyosarcomas (ARMS) are aggressive soft-tissue sarcomas affecting children and young adults. Most ARMS tumors express the PAX3-FKHR or PAX7-FKHR (PAX-FKHR) fusion genes resulting from the t(2;13) or t(1;13) chromosomal translocations, respectively. However, up to 25% of ARMS tumors are fusion negative, making it unclear whether ARMS represent a single disease or multiple clinical and biological entities with a common phenotype. To test to what extent PAX-FKHR determine class and behavior of ARMS, we used oligonucleotide microarray expression profiling on 139 primary rhabdomyosarcoma tumors and an in vitro model. We found that ARMS tumors expressing either PAX-FKHR gene share a common expression profile distinct from fusion-negative ARMS and from the other rhabdomyosarcoma variants. We also observed that PAX-FKHR expression above a minimum level is necessary for the detection of this expression profile. Using an ectopic PAX3-FKHR and PAX7-FKHR expression model, we identified an expression signature regulated by PAX-FKHR that is specific to PAX-FKHR-positive ARMS tumors. Data mining for functional annotations of signature genes suggested a role for PAX-FKHR in regulating ARMS proliferation and differentiation. Cox regression modeling identified a subset of genes within the PAX-FKHR expression signature that segregated ARMS patients into three risk groups with 5-year overall survival estimates of 7%, 48%, and 93%. These prognostic classes were independent of conventional clinical risk factors. Our results show that PAX-FKHR dictate a specific expression signature that helps define the molecular phenotype of PAX-FKHR-positive ARMS tumors and, because it is linked with disease outcome in ARMS patients, determine tumor behavior.
Alveolar rhabdomyosarcomas (ARMS) are aggressive soft-tissue sarcomas affecting children and young adults. Most ARMS tumors express the PAX3-FKHR or PAX7-FKHR (PAX-FKHR) fusion genes resulting from the t(2;13) or t(1;13) chromosomal translocations, respectively. However, up to 25% of ARMS tumors are fusion negative, making it unclear whether ARMS represent a single disease or multiple clinical and biological entities with a common phenotype. To test to what extent PAX-FKHR determine class and behavior of ARMS, we used oligonucleotide microarray expression profiling on 139 primary rhabdomyosarcoma tumors and an in vitro model. We found that ARMS tumors expressing either PAX-FKHR gene share a common expression profile distinct from fusion-negative ARMS and from the other rhabdomyosarcoma variants. We also observed that PAX-FKHR expression above a minimum level is necessary for the detection of this expression profile. Using an ectopic PAX3-FKHR and PAX7-FKHR expression model, we identified an expression signature regulated by PAX-FKHR that is specific to PAX-FKHR-positive ARMS tumors. Data mining for functional annotations of signature genes suggested a role for PAX-FKHR in regulating ARMS proliferation and differentiation. Cox regression modeling identified a subset of genes within the PAX-FKHR expression signature that segregated ARMS patients into three risk groups with 5-year overall survival estimates of 7%, 48%, and 93%. These prognostic classes were independent of conventional clinical risk factors. Our results show that PAX-FKHR dictate a specific expression signature that helps define the molecular phenotype of PAX-FKHR-positive ARMS tumors and, because it is linked with disease outcome in ARMS patients, determine tumor behavior.
Alveolar rhabdomyosarcomas (ARMS) are aggressive soft-tissue sarcomas affecting children and young adults. Most ARMS tumors express the PAX3-FKHR or PAX7-FKHR (PAX-FKHR) fusion genes resulting from the t(2;13) or t(1;13) chromosomal translocations, respectively. However, up to 25% of ARMS tumors are fusion negative, making it unclear whether ARMS represent a single disease or multiple clinical and biological entities with a common phenotype. To test to what extent PAX-FKHR determine class and behavior of ARMS, we used oligonucleotide microarray expression profiling on 139 primary rhabdomyosarcoma tumors and an in vitro model. We found that ARMS tumors expressing either PAX-FKHR gene share a common expression profile distinct from fusion-negative ARMS and from the other rhabdomyosarcoma variants. We also observed that PAX-FKHR expression above a minimum level is necessary for the detection of this expression profile. Using an ectopic PAX3-FKHR and PAX7-FKHR expression model, we identified an expression signature regulated by PAX-FKHR that is specific to PAX-FKHR-positive ARMS tumors. Data mining for functional annotations of signature genes suggested a role for PAX-FKHR in regulating ARMS proliferation and differentiation. Cox regression modeling identified a subset of genes within the PAX-FKHR expression signature that segregated ARMS patients into three risk groups with 5-year overall survival estimates of 7%, 48%, and 93%. These prognostic classes were independent of conventional clinical risk factors. Our results show that PAX-FKHR dictate a specific expression signature that helps define the molecular phenotype of PAX-FKHR-positive ARMS tumors and, because it is linked with disease outcome in ARMS patients, determine tumor behavior.
To identify the molecular mechanisms by which EBV-associated epithelial cancers are maintained, we measured the expression of essentially all human genes and all latent EBV genes in a collection of 31 laser-captured, microdissected nasopharyngeal carcinoma (NPC) tissue samples and 10 normal nasopharyngeal tissues. Global gene expression profiles clearly distinguished tumors from normal healthy epithelium. Expression levels of six viral genes (EBNA1, EBNA2, EBNA3A, EBNA3B, LMP1, and LMP2A) were correlated among themselves and strongly inversely correlated with the expression of a large subset of host genes. Among the human genes whose inhibition was most strongly correlated with increased EBV gene expression were multiple MHC class I HLA genes involved in regulating immune response via antigen presentation. The association between EBV gene expression and inhibition of MHC class I HLA expression implies that antigen display is either directly inhibited by EBV, facilitating immune evasion by tumor cells, and/or that tumor cells with inhibited presentation are selected for their ability to sustain higher levels of EBV to take maximum advantage of EBV oncogene-mediated tumor-promoting actions. Our data clearly reflect such tumor promotion, showing that deregulation of key proteins involved in apoptosis (BCL2-related protein A1 and Fas apoptotic inhibitory molecule), cell cycle checkpoints (AKIP, SCYL1, and NIN), and metastasis (matrix metalloproteinase 1) is closely correlated with the levels of EBV gene expression in NPC.
To identify the molecular mechanisms by which EBV-associated epithelial cancers are maintained, we measured the expression of essentially all human genes and all latent EBV genes in a collection of 31 laser-captured, microdissected nasopharyngeal carcinoma (NPC) tissue samples and 10 normal nasopharyngeal tissues. Global gene expression profiles clearly distinguished tumors from normal healthy epithelium. Expression levels of six viral genes (EBNA1, EBNA2, EBNA3A, EBNA3B, LMP1, and LMP2A) were correlated among themselves and strongly inversely correlated with the expression of a large subset of host genes. Among the human genes whose inhibition was most strongly correlated with increased EBV gene expression were multiple MHC class I HLA genes involved in regulating immune response via antigen presentation. The association between EBV gene expression and inhibition of MHC class I HLA expression implies that antigen display is either directly inhibited by EBV, facilitating immune evasion by tumor cells, and/or that tumor cells with inhibited presentation are selected for their ability to sustain higher levels of EBV to take maximum advantage of EBV oncogene-mediated tumor-promoting actions. Our data clearly reflect such tumor promotion, showing that deregulation of key proteins involved in apoptosis (BCL2-related protein A1 and Fas apoptotic inhibitory molecule), cell cycle checkpoints (AKIP, SCYL1, and NIN), and metastasis (matrix metalloproteinase 1) is closely correlated with the levels of EBV gene expression in NPC.
BACKGROUND: Prostate cancer is characterized by heterogeneity in the clinical course that often does not correlate with morphologic features of the tumor. Metastasis reflects the most adverse outcome of prostate cancer, and to date there are no reliable morphologic features or serum biomarkers that can reliably predict which patients are at higher risk of developing metastatic disease. Understanding the differences in the biology of metastatic and organ confined primary tumors is essential for developing new prognostic markers and therapeutic targets. METHODS: Using Affymetrix oligonucleotide arrays, we analyzed gene expression profiles of 24 androgen-ablation resistant metastatic samples obtained from 4 patients and a previously published dataset of 64 primary prostate tumor samples. Differential gene expression was analyzed after removing potentially uninformative stromal genes, addressing the differences in cellular content between primary and metastatic tumors. RESULTS: The metastatic samples are highly heterogenous in expression; however, differential expression analysis shows that 415 genes are upregulated and 364 genes are downregulated at least 2 fold in every patient with metastasis. The expression profile of metastatic samples reveals changes in expression of a unique set of genes representing both the androgen ablation related pathways and other metastasis related gene networks such as cell adhesion, bone remodelling and cell cycle. The differentially expressed genes include metabolic enzymes, transcription factors such as Forkhead Box M1 (FoxM1) and cell adhesion molecules such as Osteopontin (SPP1). CONCLUSION: We hypothesize that these genes have a role in the biology of metastatic disease and that they represent potential therapeutic targets for prostate cancer.
BACKGROUND: Prostate cancer is characterized by heterogeneity in the clinical course that often does not correlate with morphologic features of the tumor. Metastasis reflects the most adverse outcome of prostate cancer, and to date there are no reliable morphologic features or serum biomarkers that can reliably predict which patients are at higher risk of developing metastatic disease. Understanding the differences in the biology of metastatic and organ confined primary tumors is essential for developing new prognostic markers and therapeutic targets. METHODS: Using Affymetrix oligonucleotide arrays, we analyzed gene expression profiles of 24 androgen-ablation resistant metastatic samples obtained from 4 patients and a previously published dataset of 64 primary prostate tumor samples. Differential gene expression was analyzed after removing potentially uninformative stromal genes, addressing the differences in cellular content between primary and metastatic tumors. RESULTS: The metastatic samples are highly heterogenous in expression; however, differential expression analysis shows that 415 genes are upregulated and 364 genes are downregulated at least 2 fold in every patient with metastasis. The expression profile of metastatic samples reveals changes in expression of a unique set of genes representing both the androgen ablation related pathways and other metastasis related gene networks such as cell adhesion, bone remodelling and cell cycle. The differentially expressed genes include metabolic enzymes, transcription factors such as Forkhead Box M1 (FoxM1) and cell adhesion molecules such as Osteopontin (SPP1). CONCLUSION: We hypothesize that these genes have a role in the biology of metastatic disease and that they represent potential therapeutic targets for prostate cancer.
Dendritic cells (DCs) are potent mediators of the immune response, and can be activated by exogenous pathogen components. Galectin-1 is a member of the conserved beta-galactoside-binding lectin family that binds galactoside residues on cell surface glycoconjugates. Galectin-1 is known to play a role in immune regulation via action on multiple immune cells. However, its effects on human DCs are unknown. In this study, we show that galectin-1 induces a phenotypic and functional maturation in human monocyte-derived DCs (MDDCs) similar to but distinct from the activity of the exogenous pathogen stimuli, LPS. Immature human MDDCs exposed to galectin-1 up-regulated cell surface markers characteristic of DC maturation (CD40, CD83, CD86, and HLA-DR), secreted high levels of IL-6 and TNF-alpha, stimulated T cell proliferation, and showed reduced endocytic capacity, similar to LPS-matured MDDCs. However, unlike LPS-matured DCs, galectin-1-treated MDDCs did not produce the Th1-polarizing cytokine IL-12. Microarray analysis revealed that in addition to modulating many of the same DC maturation genes as LPS, galectin-1 also uniquely up-regulated a significant subset of genes related to cell migration through the extracellular matrix (ECM). Indeed, compared with LPS, galectin-1-treated human MDDCs exhibited significantly better chemotactic migration through Matrigel, an in vitro ECM model. Our findings show that galectin-1 is a novel endogenous activator of human MDDCs that up-regulates a significant subset of genes distinct from those regulated by a model exogenous stimulus (LPS). One unique effect of galectin-1 is to increase DC migration through the ECM, suggesting that galectin-1 may be an important component in initiating an immune response.
Dendritic cells (DCs) are potent mediators of the immune response, and can be activated by exogenous pathogen components. Galectin-1 is a member of the conserved beta-galactoside-binding lectin family that binds galactoside residues on cell surface glycoconjugates. Galectin-1 is known to play a role in immune regulation via action on multiple immune cells. However, its effects on human DCs are unknown. In this study, we show that galectin-1 induces a phenotypic and functional maturation in human monocyte-derived DCs (MDDCs) similar to but distinct from the activity of the exogenous pathogen stimuli, LPS. Immature human MDDCs exposed to galectin-1 up-regulated cell surface markers characteristic of DC maturation (CD40, CD83, CD86, and HLA-DR), secreted high levels of IL-6 and TNF-alpha, stimulated T cell proliferation, and showed reduced endocytic capacity, similar to LPS-matured MDDCs. However, unlike LPS-matured DCs, galectin-1-treated MDDCs did not produce the Th1-polarizing cytokine IL-12. Microarray analysis revealed that in addition to modulating many of the same DC maturation genes as LPS, galectin-1 also uniquely up-regulated a significant subset of genes related to cell migration through the extracellular matrix (ECM). Indeed, compared with LPS, galectin-1-treated human MDDCs exhibited significantly better chemotactic migration through Matrigel, an in vitro ECM model. Our findings show that galectin-1 is a novel endogenous activator of human MDDCs that up-regulates a significant subset of genes distinct from those regulated by a model exogenous stimulus (LPS). One unique effect of galectin-1 is to increase DC migration through the ECM, suggesting that galectin-1 may be an important component in initiating an immune response.
Chronic lymphocytic leukemia (CLL) results in the accumulation of B cells, presumably reflecting the selection of malignant cell precursors with Ag combined with complex alterations in protein activity. Repeated BCR stimulation of normal B cells leads to anergy and CD5 expression, both of which are features of CLL. Because CD5 is phosphorylated on tyrosine following BCR engagement and negatively regulates BCR signaling in normal B cells, we investigated its phosphorylation status and found it to be naturally phosphorylated on tyrosine but not on serine residues in CLL samples. To analyze the role of CD5, we established a B cell line in which CD5 is phosphorylated. Gene profiling of vector vs CD5-transfected B cells pointed out gene groups whose expression was enhanced: Apoptosis inhibitors (BCL2), NF-kappaB (RELB, BCL3), Wnt, TGFbeta, VEGF, MAPKs, Stats, cytokines, chemokines (IL-10, IL-10R, IL-2R, CCL-3, CCL-4, and CCR7), TLR-9, and the surface Ags CD52, CD54, CD70, and CD72. Most of these gene groups are strongly expressed in CLL B cells as compared with normal B cells. Unexpectedly, metabolic pathways, namely cholesterol synthesis and adipogenesis, are also enhanced by CD5. Conversely, CD5 inhibited genes involved in RNA splicing and processing, ribosome biogenesis, proteasome, and CD80 and CD86 Ags, whose expression is low in CLL. Comparison of CD5- vs tailless CD5-transfected cells further demonstrated the role of CD5 phosphorylation in the regulation of selected genes. These results support a model where CLL cells are chronically stimulated, leading to CD5 activation and cell survival. In addition to CD5 itself, we point to several CD5-induced genes as potential therapeutic targets.
Chronic lymphocytic leukemia (CLL) results in the accumulation of B cells, presumably reflecting the selection of malignant cell precursors with Ag combined with complex alterations in protein activity. Repeated BCR stimulation of normal B cells leads to anergy and CD5 expression, both of which are features of CLL. Because CD5 is phosphorylated on tyrosine following BCR engagement and negatively regulates BCR signaling in normal B cells, we investigated its phosphorylation status and found it to be naturally phosphorylated on tyrosine but not on serine residues in CLL samples. To analyze the role of CD5, we established a B cell line in which CD5 is phosphorylated. Gene profiling of vector vs CD5-transfected B cells pointed out gene groups whose expression was enhanced: Apoptosis inhibitors (BCL2), NF-kappaB (RELB, BCL3), Wnt, TGFbeta, VEGF, MAPKs, Stats, cytokines, chemokines (IL-10, IL-10R, IL-2R, CCL-3, CCL-4, and CCR7), TLR-9, and the surface Ags CD52, CD54, CD70, and CD72. Most of these gene groups are strongly expressed in CLL B cells as compared with normal B cells. Unexpectedly, metabolic pathways, namely cholesterol synthesis and adipogenesis, are also enhanced by CD5. Conversely, CD5 inhibited genes involved in RNA splicing and processing, ribosome biogenesis, proteasome, and CD80 and CD86 Ags, whose expression is low in CLL. Comparison of CD5- vs tailless CD5-transfected cells further demonstrated the role of CD5 phosphorylation in the regulation of selected genes. These results support a model where CLL cells are chronically stimulated, leading to CD5 activation and cell survival. In addition to CD5 itself, we point to several CD5-induced genes as potential therapeutic targets.
Human macrophage genes regulated by live P.gingivalis. We attempted in the present study to identify transcriptional profiles that are modulated after treatment with P. gingivalis relative to its purified LPS or its major fimbrial protein (FimA).3 We used the cDNA microarray technique to define more precisely the individual effects of LPS, FimA, and their parental bacterium, P. gingivalis, on the transcriptional reprogramming of human macrophages.Of the 54674 genes (probe sets) assayed by the genomebased cDNA microarray, P. gingivalis, its LPS, and its FimA commonly regulated 266 genes, while 501, 286, and 291 genes were uniquely regulated by P. gingivalis, its LPS, or its FimA, respectively. The complete list of changes in macrophage transcripts transcripts in response to P. gingivalis infection and to its LPS or FimA treatment is available in the Supplemental Data.
Human macrophage genes regulated by live P.gingivalis. We attempted in the present study to identify transcriptional profiles that are modulated after treatment with P. gingivalis relative to its purified LPS or its major fimbrial protein (FimA).3 We used the cDNA microarray technique to define more precisely the individual effects of LPS, FimA, and their parental bacterium, P. gingivalis, on the transcriptional reprogramming of human macrophages.Of the 54674 genes (probe sets) assayed by the genomebased cDNA microarray, P. gingivalis, its LPS, and its FimA commonly regulated 266 genes, while 501, 286, and 291 genes were uniquely regulated by P. gingivalis, its LPS, or its FimA, respectively. The complete list of changes in macrophage transcripts transcripts in response to P. gingivalis infection and to its LPS or FimA treatment is available in the Supplemental Data.
MicroRNAs (miRNAs) are 19 to 23 nucleotide-long RNAs that post-transcriptionally regulate gene expression. Human cells express several hundred miRNAs which regulate important biological pathways such as development, proliferation, and apoptosis. Recently, 12 miRNA genes have been identified within the genome of Kaposi sarcoma-associated herpesvirus; however, their functions are still unknown. To identify host cellular genes that may be targeted by these novel viral regulators, we performed gene expression profiling in cells stably expressing KSHV-encoded miRNAs. Data analysis revealed a set of 81 genes whose expression was significantly changed in the presence of miRNAs. While the majority of changes were below 2-fold, eight genes were down-regulated between 4- and 20-fold. We confirmed miRNA-dependent regulation for three of these genes and found that protein levels of thrombospondin 1 (THBS1) were decreased >10-fold. THBS1 has previously been reported to be down-regulated in Kaposi sarcoma lesions and has known activity as a strong tumor suppressor and anti-angiogenic factor, exerting its anti-angiogenic effect in part by activating the latent form of TGF-beta. We show that reduced THBS1 expression in the presence of viral miRNAs translates into decreased TGF-beta activity. These data suggest that KSHV-encoded miRNAs may contribute directly to pathogenesis by down-regulation of THBS1, a major regulator of cell adhesion, migration, and angiogenesis.
MicroRNAs (miRNAs) are 19 to 23 nucleotide-long RNAs that post-transcriptionally regulate gene expression. Human cells express several hundred miRNAs which regulate important biological pathways such as development, proliferation, and apoptosis. Recently, 12 miRNA genes have been identified within the genome of Kaposi sarcoma-associated herpesvirus; however, their functions are still unknown. To identify host cellular genes that may be targeted by these novel viral regulators, we performed gene expression profiling in cells stably expressing KSHV-encoded miRNAs. Data analysis revealed a set of 81 genes whose expression was significantly changed in the presence of miRNAs. While the majority of changes were below 2-fold, eight genes were down-regulated between 4- and 20-fold. We confirmed miRNA-dependent regulation for three of these genes and found that protein levels of thrombospondin 1 (THBS1) were decreased >10-fold. THBS1 has previously been reported to be down-regulated in Kaposi sarcoma lesions and has known activity as a strong tumor suppressor and anti-angiogenic factor, exerting its anti-angiogenic effect in part by activating the latent form of TGF-beta. We show that reduced THBS1 expression in the presence of viral miRNAs translates into decreased TGF-beta activity. These data suggest that KSHV-encoded miRNAs may contribute directly to pathogenesis by down-regulation of THBS1, a major regulator of cell adhesion, migration, and angiogenesis.
We sought to identify the target genes and pathways involved in ST7-mediated tumor suppression of PC-3 cells. This was achieved by array-based expression analysis using Affymetrix U133 plus 2.0 arrays in four independent PC-3/ST7-1a clones and the pool of five PC-3/pcDNA3.1 controls.
We sought to identify the target genes and pathways involved in ST7-mediated tumor suppression of PC-3 cells. This was achieved by array-based expression analysis using Affymetrix U133 plus 2.0 arrays in four independent PC-3/ST7-1a clones and the pool of five PC-3/pcDNA3.1 controls.
Microarray RNA gene expression profiling analysis has shown that Sox4 (Sry-related high mobility group (HMG) box 4) is one of the most upregulated genes in adenoid cystic carcinoma (ACC), relative to non-neoplastic tissue of origin. Here, we show that Sox4 protein is similarly upregulated in ACC by immunohistochemistry of 28 primary cancers and 20 normal tissues. To elucidate the functional significance of these findings, RNA interference (RNAi)-mediated RNA silencing was used to downregulate Sox4 expression in the ACC-derived cell line, ACC3. With confirmed knockdown of Sox4 protein, cell viability was reduced by 51%, with a corresponding increase of apoptosis to 85% as compared to 12% in controls. Apoptosis was confirmed by cell morphology, DNA fragmentation and flow cytometry. Cells could be rescued from the proapoptotic effects of Sox4 RNAi by co-transfection with a construct expressing functional Sox4. Microarray gene expression profiling of RNAi knockdown experiments shows that downregulation of Sox4-modulated expression of critical genes involved in apoptosis and cell cycle control. Overall, our findings suggest that Sox4 contributes to the malignant phenotype of ACC cells by promoting cell survival.
Microarray RNA gene expression profiling analysis has shown that Sox4 (Sry-related high mobility group (HMG) box 4) is one of the most upregulated genes in adenoid cystic carcinoma (ACC), relative to non-neoplastic tissue of origin. Here, we show that Sox4 protein is similarly upregulated in ACC by immunohistochemistry of 28 primary cancers and 20 normal tissues. To elucidate the functional significance of these findings, RNA interference (RNAi)-mediated RNA silencing was used to downregulate Sox4 expression in the ACC-derived cell line, ACC3. With confirmed knockdown of Sox4 protein, cell viability was reduced by 51%, with a corresponding increase of apoptosis to 85% as compared to 12% in controls. Apoptosis was confirmed by cell morphology, DNA fragmentation and flow cytometry. Cells could be rescued from the proapoptotic effects of Sox4 RNAi by co-transfection with a construct expressing functional Sox4. Microarray gene expression profiling of RNAi knockdown experiments shows that downregulation of Sox4-modulated expression of critical genes involved in apoptosis and cell cycle control. Overall, our findings suggest that Sox4 contributes to the malignant phenotype of ACC cells by promoting cell survival.
At least one third of all cases of epithelial ovarian cancer are associated with the production of ascites, although its effect on tumor cell microenvironment remains poorly understood. This study addresses the effect of the heterologous acellular fraction of ovarian cancer-derived ascites on a cell line (OV-90) derived from the chemotherapy-na??ve ovarian cancer patient. Ascites were assayed for their effect on cell invasion, growth, and spheroid formation. When compared to either no serum or 5% serum, ascites fell into one of two categories: stimulatory or inhibitory. RNA from OV-90 cells exposed to selected ascites were arrayed on an Affymetrix HG-U133A GeneChip. A supervised analysis identified a number of differentially expressed genes and quantitative polymerase chain reaction validation based on OV-90 cells exposed to 54 independent ascites demonstrated that stimulatory ascites affected the expression of ISGF3G, TRIB1, MKP1, RGS4, PLEC1, and MOSPD1 genes. In addition, TRIB1 expression was shown to independently correlate with prognosis when its expression was ascertained in an independent set of primary cultures established from ovarian ascites. The data support the validity of the strategy to uncover molecular events that are associated with tumor cell behavior and highlight the impact of ascites on the cellular and molecular parameters of ovarian cancer.
At least one third of all cases of epithelial ovarian cancer are associated with the production of ascites, although its effect on tumor cell microenvironment remains poorly understood. This study addresses the effect of the heterologous acellular fraction of ovarian cancer-derived ascites on a cell line (OV-90) derived from the chemotherapy-na??ve ovarian cancer patient. Ascites were assayed for their effect on cell invasion, growth, and spheroid formation. When compared to either no serum or 5% serum, ascites fell into one of two categories: stimulatory or inhibitory. RNA from OV-90 cells exposed to selected ascites were arrayed on an Affymetrix HG-U133A GeneChip. A supervised analysis identified a number of differentially expressed genes and quantitative polymerase chain reaction validation based on OV-90 cells exposed to 54 independent ascites demonstrated that stimulatory ascites affected the expression of ISGF3G, TRIB1, MKP1, RGS4, PLEC1, and MOSPD1 genes. In addition, TRIB1 expression was shown to independently correlate with prognosis when its expression was ascertained in an independent set of primary cultures established from ovarian ascites. The data support the validity of the strategy to uncover molecular events that are associated with tumor cell behavior and highlight the impact of ascites on the cellular and molecular parameters of ovarian cancer.
Disruptions of LGI1 in glioblastoma (GBM) cell lines and LGI1 mutations in families with autosomal dominant epilepsy imply a role for LGI1 in glial cells as well as in neurons. Although we and others could not find LGI1 mutations in malignant gliomas, our initial studies appeared to support the idea that LGI1 is poorly expressed or absent in these tumors. Microarray data suggested that LGI1 could be involved in the control of matrix metalloproteinases, and we found that tumors derived from U87 glioblastoma cells overexpressing LGI1 were less aggressive than U87 control tumors. To our surprise, we observed that LGI1 expression after differentiation of murine neural stem cells was robust in neurons but negligible in glial cells, in agreement with immunohistochemistry studies on rodent brain. This observation could suggest that the variable levels of LGI1 expression in gliomas reflect the presence of neurons entrapped within the tumor. To test this hypothesis, we investigated LGI1 expression in parallel with expression of the neuronal marker NEF3 by real-time PCR on 30 malignant gliomas. Results showed a strong, positive correlation between the expression levels of these two genes (P < 0.0001). Thus, our data confirm that LGI1 is involved in cell-matrix interactions but suggest that its expression is not relevant in glial cells, implying that its role as a tumor suppressor in gliomas should be reconsidered.
Disruptions of LGI1 in glioblastoma (GBM) cell lines and LGI1 mutations in families with autosomal dominant epilepsy imply a role for LGI1 in glial cells as well as in neurons. Although we and others could not find LGI1 mutations in malignant gliomas, our initial studies appeared to support the idea that LGI1 is poorly expressed or absent in these tumors. Microarray data suggested that LGI1 could be involved in the control of matrix metalloproteinases, and we found that tumors derived from U87 glioblastoma cells overexpressing LGI1 were less aggressive than U87 control tumors. To our surprise, we observed that LGI1 expression after differentiation of murine neural stem cells was robust in neurons but negligible in glial cells, in agreement with immunohistochemistry studies on rodent brain. This observation could suggest that the variable levels of LGI1 expression in gliomas reflect the presence of neurons entrapped within the tumor. To test this hypothesis, we investigated LGI1 expression in parallel with expression of the neuronal marker NEF3 by real-time PCR on 30 malignant gliomas. Results showed a strong, positive correlation between the expression levels of these two genes (P < 0.0001). Thus, our data confirm that LGI1 is involved in cell-matrix interactions but suggest that its expression is not relevant in glial cells, implying that its role as a tumor suppressor in gliomas should be reconsidered.
BACKGROUND: Chronic heart failure is characterized by left ventricular remodeling and reactivation of a fetal gene program; the underlying mechanisms are only partly understood. Here we provide evidence that cardiac microRNAs, recently discovered key regulators of gene expression, contribute to the transcriptional changes observed in heart failure. METHODS AND RESULTS: Cardiac transcriptome analyses revealed striking similarities between fetal and failing human heart tissue. Using microRNA arrays, we discovered profound alterations of microRNA expression in failing hearts. These changes closely mimicked the microRNA expression pattern observed in fetal cardiac tissue. Bioinformatic analysis demonstrated a striking concordance between regulated messenger RNA expression in heart failure and the presence of microRNA binding sites in the respective 3' untranslated regions. Messenger RNAs upregulated in the failing heart contained preferentially binding sites for downregulated microRNAs and vice versa. Mechanistically, transfection of cardiomyocytes with a set of fetal microRNAs induced cellular hypertrophy as well as changes in gene expression comparable to the failing heart. CONCLUSIONS: Our data support a novel mode of regulation for the transcriptional changes in cardiac failure. Reactivation of a fetal microRNA program substantially contributes to alterations of gene expression in the failing human heart.
BACKGROUND: Chronic heart failure is characterized by left ventricular remodeling and reactivation of a fetal gene program; the underlying mechanisms are only partly understood. Here we provide evidence that cardiac microRNAs, recently discovered key regulators of gene expression, contribute to the transcriptional changes observed in heart failure. METHODS AND RESULTS: Cardiac transcriptome analyses revealed striking similarities between fetal and failing human heart tissue. Using microRNA arrays, we discovered profound alterations of microRNA expression in failing hearts. These changes closely mimicked the microRNA expression pattern observed in fetal cardiac tissue. Bioinformatic analysis demonstrated a striking concordance between regulated messenger RNA expression in heart failure and the presence of microRNA binding sites in the respective 3' untranslated regions. Messenger RNAs upregulated in the failing heart contained preferentially binding sites for downregulated microRNAs and vice versa. Mechanistically, transfection of cardiomyocytes with a set of fetal microRNAs induced cellular hypertrophy as well as changes in gene expression comparable to the failing heart. CONCLUSIONS: Our data support a novel mode of regulation for the transcriptional changes in cardiac failure. Reactivation of a fetal microRNA program substantially contributes to alterations of gene expression in the failing human heart.
Acute promyelocytic leukemia (APL) is a clonal expansion of hematopoietic precursors blocked at the promyelocytic stage. Gene expression profiles of APL cells obtained from 16 patients were compared to eight samples of CD34+-derived normal promyelocytes. Malignant promyelocytes showed widespread changes in transcription in comparison to their normal counterpart and 1020 differentially expressed genes were identified. Discriminating genes include transcriptional regulators (FOS, JUN and HOX genes) and genes involved in cell cycle and DNA repair. The strong upregulation in APL of some transcripts (FLT3, CD33, CD44 and HGF) was also confirmed at protein level. Interestingly, a trend toward a transcriptional repression of genes involved in different DNA repair pathways was found in APL and confirmed by real-time polymerase chain reactor (PCR) in a new set of nine APLs. Our results suggest that both inefficient base excision repair and recombinational repair might play a role in APLs development. To investigate the expression pathways underlying the development of APL occurring as a second malignancy (sAPL), we included in our study eight cases of sAPL. Although both secondary and de novo APL were characterized by a strong homogeneity in expression profiling, we identified a small set of differentially expressed genes that discriminate sAPL from de novo cases.
Acute promyelocytic leukemia (APL) is a clonal expansion of hematopoietic precursors blocked at the promyelocytic stage. Gene expression profiles of APL cells obtained from 16 patients were compared to eight samples of CD34+-derived normal promyelocytes. Malignant promyelocytes showed widespread changes in transcription in comparison to their normal counterpart and 1020 differentially expressed genes were identified. Discriminating genes include transcriptional regulators (FOS, JUN and HOX genes) and genes involved in cell cycle and DNA repair. The strong upregulation in APL of some transcripts (FLT3, CD33, CD44 and HGF) was also confirmed at protein level. Interestingly, a trend toward a transcriptional repression of genes involved in different DNA repair pathways was found in APL and confirmed by real-time polymerase chain reactor (PCR) in a new set of nine APLs. Our results suggest that both inefficient base excision repair and recombinational repair might play a role in APLs development. To investigate the expression pathways underlying the development of APL occurring as a second malignancy (sAPL), we included in our study eight cases of sAPL. Although both secondary and de novo APL were characterized by a strong homogeneity in expression profiling, we identified a small set of differentially expressed genes that discriminate sAPL from de novo cases.
Recent literature suggested that cells of the microenvironment of tumors could be abnormal as well. To address this hypothesis in multiple myeloma (MM), we studied bone marrow mesenchymal stem cells (BMMSCs), the only long-lived cells of the bone marrow microenvironment, by gene expression profiling and phenotypic and functional studies in three groups of individuals: patients with MM, patients with monoclonal gamopathy of undefined significance (MGUS) and healthy age-matched subjects. Gene expression profile independently classified the BMMSCs of these individuals in a normal and in an MM group. MGUS BMMSCs were interspersed between these two groups. Among the 145 distinct genes differentially expressed in MM and normal BMMSCs, 46% may account for a tumor-microenvironment cross-talk. Known soluble factors implicated in MM pathophysiologic features (i.e. IL (interleukin)-6, DKK1) were revealed and new ones were found which are involved in angiogenesis, osteogenic differentiation or tumor growth. In particular, GDF15 was found to induce dose-dependent growth of MOLP-6, a stromal cell-dependent myeloma cell line. Functionally, MM BMMSCs induced an overgrowth of MOLP-6, and their capacity to differentiate into an osteoblastic lineage was impaired. Thus, MM BMMSCs are abnormal and could create a very efficient niche to support the survival and proliferation of the myeloma cells.
Recent literature suggested that cells of the microenvironment of tumors could be abnormal as well. To address this hypothesis in multiple myeloma (MM), we studied bone marrow mesenchymal stem cells (BMMSCs), the only long-lived cells of the bone marrow microenvironment, by gene expression profiling and phenotypic and functional studies in three groups of individuals: patients with MM, patients with monoclonal gamopathy of undefined significance (MGUS) and healthy age-matched subjects. Gene expression profile independently classified the BMMSCs of these individuals in a normal and in an MM group. MGUS BMMSCs were interspersed between these two groups. Among the 145 distinct genes differentially expressed in MM and normal BMMSCs, 46% may account for a tumor-microenvironment cross-talk. Known soluble factors implicated in MM pathophysiologic features (i.e. IL (interleukin)-6, DKK1) were revealed and new ones were found which are involved in angiogenesis, osteogenic differentiation or tumor growth. In particular, GDF15 was found to induce dose-dependent growth of MOLP-6, a stromal cell-dependent myeloma cell line. Functionally, MM BMMSCs induced an overgrowth of MOLP-6, and their capacity to differentiate into an osteoblastic lineage was impaired. Thus, MM BMMSCs are abnormal and could create a very efficient niche to support the survival and proliferation of the myeloma cells.
B-cell chronic lymphocytic leukaemia (B-CLL) is a heterogenous disease with a highly variable clinical course and analysis of zeta-associated protein 70 (ZAP-70) and CD38 expression on B-CLL cells allowed for identification of patients with good (ZAP-70-CD38-) and poor (ZAP-70+CD38+) prognosis. DNA microarray technology was employed to compare eight ZAP-70+CD38+ with eight ZAP-70-CD38- B-CLL cases. The expression of 358 genes differed significantly between the two subgroups, including genes involved in B-cell receptor signaling, angiogenesis and lymphomagenesis. Three of these genes, that is, immune receptor translocation-associated protein 4 (IRTA4)/Fc receptor homologue 2 (FcRH2), angiopoietin 2 (ANGPT2) and Pim2 were selected for further validating studies in a cohort of 94 B-CLL patients. IRTA4/FcRH2 expression as detected by flow cytometry was significantly lower in the poor prognosis subgroup as compared to ZAP-70-CD38- B-CLL cells. In healthy individuals, IRTA4/FcRH2 protein expression was associated with a CD19+CD27+ memory cell phenotype. ANGPT2 plasma concentrations were twofold higher in the poor prognosis subgroup (P<0.05). Pim2 was significantly overexpressed in poor prognosis cases and Binet stage C. Disease progression may be related to proangiogenic processes and strong Pim2 expression.
B-cell chronic lymphocytic leukaemia (B-CLL) is a heterogenous disease with a highly variable clinical course and analysis of zeta-associated protein 70 (ZAP-70) and CD38 expression on B-CLL cells allowed for identification of patients with good (ZAP-70-CD38-) and poor (ZAP-70+CD38+) prognosis. DNA microarray technology was employed to compare eight ZAP-70+CD38+ with eight ZAP-70-CD38- B-CLL cases. The expression of 358 genes differed significantly between the two subgroups, including genes involved in B-cell receptor signaling, angiogenesis and lymphomagenesis. Three of these genes, that is, immune receptor translocation-associated protein 4 (IRTA4)/Fc receptor homologue 2 (FcRH2), angiopoietin 2 (ANGPT2) and Pim2 were selected for further validating studies in a cohort of 94 B-CLL patients. IRTA4/FcRH2 expression as detected by flow cytometry was significantly lower in the poor prognosis subgroup as compared to ZAP-70-CD38- B-CLL cells. In healthy individuals, IRTA4/FcRH2 protein expression was associated with a CD19+CD27+ memory cell phenotype. ANGPT2 plasma concentrations were twofold higher in the poor prognosis subgroup (P<0.05). Pim2 was significantly overexpressed in poor prognosis cases and Binet stage C. Disease progression may be related to proangiogenic processes and strong Pim2 expression.
In this study, we provide a molecular signature of highly enriched CD34+ cells from bone marrow of untreated patients with chronic myelogenous leukemia (CML) in chronic phase in comparison with normal CD34+ cells using microarrays covering 8746 genes. Expression data reflected several BCR-ABL-induced effects in primary CML progenitors, such as transcriptional activation of the classical mitogen-activated protein kinase pathway and the phosphoinositide-3 kinase/AKT pathway as well as downregulation of the proapoptotic gene IRF8. Moreover, novel transcriptional changes in comparison with normal CD34+ cells were identified. These include upregulation of genes involved in the transforming growth factorbeta pathway, fetal hemoglobin genes, leptin receptor, sorcin, tissue inhibitor of metalloproteinase 1, the neuroepithelial cell transforming gene 1 and downregulation of selenoprotein P. Additionally, genes associated with early hematopoietic stem cells (HSC) and leukemogenesis such as HoxA9 and MEIS1 were transcriptionally activated. Differential expression of differentiation-associated genes suggested an altered composition of the CD34+ cell population in CML. This was confirmed by subset analyses of chronic phase CML CD34+ cells showing an increase of the proportion of megakaryocyte-erythroid progenitors, whereas the proportion of HSC and granulocyte-macrophage progenitors was decreased in CML. In conclusion, our results give novel insights into the biology of CML and could provide the basis for identification of new therapeutic targets.
In this study, we provide a molecular signature of highly enriched CD34+ cells from bone marrow of untreated patients with chronic myelogenous leukemia (CML) in chronic phase in comparison with normal CD34+ cells using microarrays covering 8746 genes. Expression data reflected several BCR-ABL-induced effects in primary CML progenitors, such as transcriptional activation of the classical mitogen-activated protein kinase pathway and the phosphoinositide-3 kinase/AKT pathway as well as downregulation of the proapoptotic gene IRF8. Moreover, novel transcriptional changes in comparison with normal CD34+ cells were identified. These include upregulation of genes involved in the transforming growth factorbeta pathway, fetal hemoglobin genes, leptin receptor, sorcin, tissue inhibitor of metalloproteinase 1, the neuroepithelial cell transforming gene 1 and downregulation of selenoprotein P. Additionally, genes associated with early hematopoietic stem cells (HSC) and leukemogenesis such as HoxA9 and MEIS1 were transcriptionally activated. Differential expression of differentiation-associated genes suggested an altered composition of the CD34+ cell population in CML. This was confirmed by subset analyses of chronic phase CML CD34+ cells showing an increase of the proportion of megakaryocyte-erythroid progenitors, whereas the proportion of HSC and granulocyte-macrophage progenitors was decreased in CML. In conclusion, our results give novel insights into the biology of CML and could provide the basis for identification of new therapeutic targets.
Acute promyelocytic leukemia (APL) is a clonal expansion of hematopoietic precursors blocked at the promyelocytic stage. Gene expression profiles of APL cells obtained from 16 patients were compared to eight samples of CD34+-derived normal promyelocytes. Malignant promyelocytes showed widespread changes in transcription in comparison to their normal counterpart and 1020 differentially expressed genes were identified. Discriminating genes include transcriptional regulators (FOS, JUN and HOX genes) and genes involved in cell cycle and DNA repair. The strong upregulation in APL of some transcripts (FLT3, CD33, CD44 and HGF) was also confirmed at protein level. Interestingly, a trend toward a transcriptional repression of genes involved in different DNA repair pathways was found in APL and confirmed by real-time polymerase chain reactor (PCR) in a new set of nine APLs. Our results suggest that both inefficient base excision repair and recombinational repair might play a role in APLs development. To investigate the expression pathways underlying the development of APL occurring as a second malignancy (sAPL), we included in our study eight cases of sAPL. Although both secondary and de novo APL were characterized by a strong homogeneity in expression profiling, we identified a small set of differentially expressed genes that discriminate sAPL from de novo cases.
Acute promyelocytic leukemia (APL) is a clonal expansion of hematopoietic precursors blocked at the promyelocytic stage. Gene expression profiles of APL cells obtained from 16 patients were compared to eight samples of CD34+-derived normal promyelocytes. Malignant promyelocytes showed widespread changes in transcription in comparison to their normal counterpart and 1020 differentially expressed genes were identified. Discriminating genes include transcriptional regulators (FOS, JUN and HOX genes) and genes involved in cell cycle and DNA repair. The strong upregulation in APL of some transcripts (FLT3, CD33, CD44 and HGF) was also confirmed at protein level. Interestingly, a trend toward a transcriptional repression of genes involved in different DNA repair pathways was found in APL and confirmed by real-time polymerase chain reactor (PCR) in a new set of nine APLs. Our results suggest that both inefficient base excision repair and recombinational repair might play a role in APLs development. To investigate the expression pathways underlying the development of APL occurring as a second malignancy (sAPL), we included in our study eight cases of sAPL. Although both secondary and de novo APL were characterized by a strong homogeneity in expression profiling, we identified a small set of differentially expressed genes that discriminate sAPL from de novo cases.
T-cell prolymphocytic leukemia (T-PLL) is a rare aggressive lymphoma derived from mature T cells, which is, in most cases, characterized by the presence of an inv(14)(q11q32)/t(14;14)(q11;q32) and a characteristic pattern of secondary chromosomal aberrations. DNA microarray technology was employed to compare the transcriptomes of eight immunomagnetically purified CD3+ normal donor-derived peripheral blood cell samples, with five highly purified inv(14)/t(14;14)-positive T-PLL blood samples. Between the two experimental groups, 734 genes were identified as differentially expressed, including functionally important genes involved in lymphomagenesis, cell cycle regulation, apoptosis and DNA repair. Notably, the differentially expressed genes were found to be significantly enriched in genomic regions affected by recurrent chromosomal imbalances. Upregulated genes clustered on chromosome arms 6p and 8q, and downregulated genes on 6q, 8p, 10p, 11q and 18p. High-resolution copy-number determination using single nucleotide polymorphism chip technology in 12 inv(14)/t(14;14)-positive T-PLL including those analyzed for gene expression, refined chromosomal breakpoints as well as regions of imbalances. In conclusion, combined transcriptional and molecular cytogenetic profiling identified novel specific chromosomal loci and genes that are likely to be involved in disease progression and suggests a gene dosage effect as a pathogenic mechanism in T-PLL.
T-cell prolymphocytic leukemia (T-PLL) is a rare aggressive lymphoma derived from mature T cells, which is, in most cases, characterized by the presence of an inv(14)(q11q32)/t(14;14)(q11;q32) and a characteristic pattern of secondary chromosomal aberrations. DNA microarray technology was employed to compare the transcriptomes of eight immunomagnetically purified CD3+ normal donor-derived peripheral blood cell samples, with five highly purified inv(14)/t(14;14)-positive T-PLL blood samples. Between the two experimental groups, 734 genes were identified as differentially expressed, including functionally important genes involved in lymphomagenesis, cell cycle regulation, apoptosis and DNA repair. Notably, the differentially expressed genes were found to be significantly enriched in genomic regions affected by recurrent chromosomal imbalances. Upregulated genes clustered on chromosome arms 6p and 8q, and downregulated genes on 6q, 8p, 10p, 11q and 18p. High-resolution copy-number determination using single nucleotide polymorphism chip technology in 12 inv(14)/t(14;14)-positive T-PLL including those analyzed for gene expression, refined chromosomal breakpoints as well as regions of imbalances. In conclusion, combined transcriptional and molecular cytogenetic profiling identified novel specific chromosomal loci and genes that are likely to be involved in disease progression and suggests a gene dosage effect as a pathogenic mechanism in T-PLL.
The v-Myb oncoprotein encoded by Avian Myeloblastosis Virus is highly oncogenic, induces leukemias in chickens and mice and transforms immature hematopoietic cells in vitro. The v-Myb protein is a mutated and truncated version of c-Myb, a DNA-binding transcription factor expressed in many cell types that is essential for normal hematopoiesis. Previous studies suggested that two types of differences, DNA binding domain mutations and the deletion of a C-terminal negative regulatory domain were important for increasing the transforming activity of v-Myb. Here, we combined structure-function studies of the v-Myb and c-Myb proteins with unbiased microarray-based transcription assays to compare the transcriptional specificities of the two proteins. In human cells, the v-Myb and c-Myb proteins displayed strikingly different activities and regulated overlapping, but largely distinct sets of target genes. Each type of mutation that distinguished v-Myb from c-Myb, including the N- and C-terminal deletions, DNA binding domain changes and mutations in the transcriptional activation domain, affected different sets of target genes and contributed to the different activities of c-Myb and v-Myb. The results suggest that v-Myb is not just a de-repressed version of c-Myb. Instead, it is a distinct transcriptional regulator with a unique set of activities.
The v-Myb oncoprotein encoded by Avian Myeloblastosis Virus is highly oncogenic, induces leukemias in chickens and mice and transforms immature hematopoietic cells in vitro. The v-Myb protein is a mutated and truncated version of c-Myb, a DNA-binding transcription factor expressed in many cell types that is essential for normal hematopoiesis. Previous studies suggested that two types of differences, DNA binding domain mutations and the deletion of a C-terminal negative regulatory domain were important for increasing the transforming activity of v-Myb. Here, we combined structure-function studies of the v-Myb and c-Myb proteins with unbiased microarray-based transcription assays to compare the transcriptional specificities of the two proteins. In human cells, the v-Myb and c-Myb proteins displayed strikingly different activities and regulated overlapping, but largely distinct sets of target genes. Each type of mutation that distinguished v-Myb from c-Myb, including the N- and C-terminal deletions, DNA binding domain changes and mutations in the transcriptional activation domain, affected different sets of target genes and contributed to the different activities of c-Myb and v-Myb. The results suggest that v-Myb is not just a de-repressed version of c-Myb. Instead, it is a distinct transcriptional regulator with a unique set of activities.
The v-Myb oncoprotein encoded by Avian Myeloblastosis Virus is highly oncogenic, induces leukemias in chickens and mice and transforms immature hematopoietic cells in vitro. The v-Myb protein is a mutated and truncated version of c-Myb, a DNA-binding transcription factor expressed in many cell types that is essential for normal hematopoiesis. Previous studies suggested that two types of differences, DNA binding domain mutations and the deletion of a C-terminal negative regulatory domain were important for increasing the transforming activity of v-Myb. Here, we combined structure-function studies of the v-Myb and c-Myb proteins with unbiased microarray-based transcription assays to compare the transcriptional specificities of the two proteins. In human cells, the v-Myb and c-Myb proteins displayed strikingly different activities and regulated overlapping, but largely distinct sets of target genes. Each type of mutation that distinguished v-Myb from c-Myb, including the N- and C-terminal deletions, DNA binding domain changes and mutations in the transcriptional activation domain, affected different sets of target genes and contributed to the different activities of c-Myb and v-Myb. The results suggest that v-Myb is not just a de-repressed version of c-Myb. Instead, it is a distinct transcriptional regulator with a unique set of activities.
The v-Myb oncoprotein encoded by Avian Myeloblastosis Virus is highly oncogenic, induces leukemias in chickens and mice and transforms immature hematopoietic cells in vitro. The v-Myb protein is a mutated and truncated version of c-Myb, a DNA-binding transcription factor expressed in many cell types that is essential for normal hematopoiesis. Previous studies suggested that two types of differences, DNA binding domain mutations and the deletion of a C-terminal negative regulatory domain were important for increasing the transforming activity of v-Myb. Here, we combined structure-function studies of the v-Myb and c-Myb proteins with unbiased microarray-based transcription assays to compare the transcriptional specificities of the two proteins. In human cells, the v-Myb and c-Myb proteins displayed strikingly different activities and regulated overlapping, but largely distinct sets of target genes. Each type of mutation that distinguished v-Myb from c-Myb, including the N- and C-terminal deletions, DNA binding domain changes and mutations in the transcriptional activation domain, affected different sets of target genes and contributed to the different activities of c-Myb and v-Myb. The results suggest that v-Myb is not just a de-repressed version of c-Myb. Instead, it is a distinct transcriptional regulator with a unique set of activities.
The v-Myb oncoprotein encoded by Avian Myeloblastosis Virus is highly oncogenic, induces leukemias in chickens and mice and transforms immature hematopoietic cells in vitro. The v-Myb protein is a mutated and truncated version of c-Myb, a DNA-binding transcription factor expressed in many cell types that is essential for normal hematopoiesis. Previous studies suggested that two types of differences, DNA binding domain mutations and the deletion of a C-terminal negative regulatory domain were important for increasing the transforming activity of v-Myb. Here, we combined structure-function studies of the v-Myb and c-Myb proteins with unbiased microarray-based transcription assays to compare the transcriptional specificities of the two proteins. In human cells, the v-Myb and c-Myb proteins displayed strikingly different activities and regulated overlapping, but largely distinct sets of target genes. Each type of mutation that distinguished v-Myb from c-Myb, including the N- and C-terminal deletions, DNA binding domain changes and mutations in the transcriptional activation domain, affected different sets of target genes and contributed to the different activities of c-Myb and v-Myb. The results suggest that v-Myb is not just a de-repressed version of c-Myb. Instead, it is a distinct transcriptional regulator with a unique set of activities.
A better molecular characterization of breast cell lines (BCL) may help discover new markers to apply to tumour samples. We performed gene and protein expression profiling of 31 BCL using whole-genome DNA microarrays and immunohistochemistry (IHC) on 'cell microarrays' (CMA), respectively. Global hierarchical clustering discriminated two groups of BCL: group I corresponded to luminal cell lines, group II to basal and mesenchymal cell lines. Correlations with centroids calculated from a published 'intrinsic 500-gene set' assigned 15 cell lines as luminal, eight as basal and four as mesenchymal. A set of 1.233 genes was differentially expressed between basal and luminal samples. Mesenchymal and basal subtypes were rather similar and discriminated by only 227 genes. The expression of 10 proteins (CAV1, CD44, EGFR, MET, ETS1, GATA3, luminal cytokeratin CK19, basal cytokeratin CK5/6, CD10, and ERM protein moesin) encoded by luminal vs basal discriminator genes confirmed the subtype classification and the validity of the identified markers. Our BCL basal/luminal signature correctly re-classified the published series of tumour samples that originally served to identify the molecular subtypes, suggesting that the identified markers should be useful for tumour classification and might represent promising targets for disease management.
A better molecular characterization of breast cell lines (BCL) may help discover new markers to apply to tumour samples. We performed gene and protein expression profiling of 31 BCL using whole-genome DNA microarrays and immunohistochemistry (IHC) on 'cell microarrays' (CMA), respectively. Global hierarchical clustering discriminated two groups of BCL: group I corresponded to luminal cell lines, group II to basal and mesenchymal cell lines. Correlations with centroids calculated from a published 'intrinsic 500-gene set' assigned 15 cell lines as luminal, eight as basal and four as mesenchymal. A set of 1.233 genes was differentially expressed between basal and luminal samples. Mesenchymal and basal subtypes were rather similar and discriminated by only 227 genes. The expression of 10 proteins (CAV1, CD44, EGFR, MET, ETS1, GATA3, luminal cytokeratin CK19, basal cytokeratin CK5/6, CD10, and ERM protein moesin) encoded by luminal vs basal discriminator genes confirmed the subtype classification and the validity of the identified markers. Our BCL basal/luminal signature correctly re-classified the published series of tumour samples that originally served to identify the molecular subtypes, suggesting that the identified markers should be useful for tumour classification and might represent promising targets for disease management.
A better molecular characterization of breast cell lines (BCL) may help discover new markers to apply to tumour samples. We performed gene and protein expression profiling of 31 BCL using whole-genome DNA microarrays and immunohistochemistry (IHC) on 'cell microarrays' (CMA), respectively. Global hierarchical clustering discriminated two groups of BCL: group I corresponded to luminal cell lines, group II to basal and mesenchymal cell lines. Correlations with centroids calculated from a published 'intrinsic 500-gene set' assigned 15 cell lines as luminal, eight as basal and four as mesenchymal. A set of 1.233 genes was differentially expressed between basal and luminal samples. Mesenchymal and basal subtypes were rather similar and discriminated by only 227 genes. The expression of 10 proteins (CAV1, CD44, EGFR, MET, ETS1, GATA3, luminal cytokeratin CK19, basal cytokeratin CK5/6, CD10, and ERM protein moesin) encoded by luminal vs basal discriminator genes confirmed the subtype classification and the validity of the identified markers. Our BCL basal/luminal signature correctly re-classified the published series of tumour samples that originally served to identify the molecular subtypes, suggesting that the identified markers should be useful for tumour classification and might represent promising targets for disease management.
A better molecular characterization of breast cell lines (BCL) may help discover new markers to apply to tumour samples. We performed gene and protein expression profiling of 31 BCL using whole-genome DNA microarrays and immunohistochemistry (IHC) on 'cell microarrays' (CMA), respectively. Global hierarchical clustering discriminated two groups of BCL: group I corresponded to luminal cell lines, group II to basal and mesenchymal cell lines. Correlations with centroids calculated from a published 'intrinsic 500-gene set' assigned 15 cell lines as luminal, eight as basal and four as mesenchymal. A set of 1.233 genes was differentially expressed between basal and luminal samples. Mesenchymal and basal subtypes were rather similar and discriminated by only 227 genes. The expression of 10 proteins (CAV1, CD44, EGFR, MET, ETS1, GATA3, luminal cytokeratin CK19, basal cytokeratin CK5/6, CD10, and ERM protein moesin) encoded by luminal vs basal discriminator genes confirmed the subtype classification and the validity of the identified markers. Our BCL basal/luminal signature correctly re-classified the published series of tumour samples that originally served to identify the molecular subtypes, suggesting that the identified markers should be useful for tumour classification and might represent promising targets for disease management.
A better molecular characterization of breast cell lines (BCL) may help discover new markers to apply to tumour samples. We performed gene and protein expression profiling of 31 BCL using whole-genome DNA microarrays and immunohistochemistry (IHC) on 'cell microarrays' (CMA), respectively. Global hierarchical clustering discriminated two groups of BCL: group I corresponded to luminal cell lines, group II to basal and mesenchymal cell lines. Correlations with centroids calculated from a published 'intrinsic 500-gene set' assigned 15 cell lines as luminal, eight as basal and four as mesenchymal. A set of 1.233 genes was differentially expressed between basal and luminal samples. Mesenchymal and basal subtypes were rather similar and discriminated by only 227 genes. The expression of 10 proteins (CAV1, CD44, EGFR, MET, ETS1, GATA3, luminal cytokeratin CK19, basal cytokeratin CK5/6, CD10, and ERM protein moesin) encoded by luminal vs basal discriminator genes confirmed the subtype classification and the validity of the identified markers. Our BCL basal/luminal signature correctly re-classified the published series of tumour samples that originally served to identify the molecular subtypes, suggesting that the identified markers should be useful for tumour classification and might represent promising targets for disease management.
A better molecular characterization of breast cell lines (BCL) may help discover new markers to apply to tumour samples. We performed gene and protein expression profiling of 31 BCL using whole-genome DNA microarrays and immunohistochemistry (IHC) on 'cell microarrays' (CMA), respectively. Global hierarchical clustering discriminated two groups of BCL: group I corresponded to luminal cell lines, group II to basal and mesenchymal cell lines. Correlations with centroids calculated from a published 'intrinsic 500-gene set' assigned 15 cell lines as luminal, eight as basal and four as mesenchymal. A set of 1.233 genes was differentially expressed between basal and luminal samples. Mesenchymal and basal subtypes were rather similar and discriminated by only 227 genes. The expression of 10 proteins (CAV1, CD44, EGFR, MET, ETS1, GATA3, luminal cytokeratin CK19, basal cytokeratin CK5/6, CD10, and ERM protein moesin) encoded by luminal vs basal discriminator genes confirmed the subtype classification and the validity of the identified markers. Our BCL basal/luminal signature correctly re-classified the published series of tumour samples that originally served to identify the molecular subtypes, suggesting that the identified markers should be useful for tumour classification and might represent promising targets for disease management.
Little is known of the underlying biology of estrogen receptor-negative, progesterone receptor-negative (ER(-)/PR(-)) breast cancer (BC), and few targeted therapies are available. Clinical heterogeneity of ER(-)/PR(-) tumors suggests that molecular subsets exist. We performed genome-wide expression analysis of 99 primary BC samples and eight BC cell lines in an effort to reveal distinct subsets, provide insight into their biology and potentially identify new therapeutic targets. We identified a subset of ER(-)/PR(-) tumors with paradoxical expression of genes known to be either direct targets of ER, responsive to estrogen, or typically expressed in ER(+) BC. Differentially expressed genes included SPDEF, FOXA1, XBP1, CYB5, TFF3, NAT1, APOD, ALCAM and AR (P<0.001). A classification model based on the expression signature of this tumor class identified molecularly similar BCs in an independent human BC data set and among BC cell lines (MDA-MB-453). This cell line demonstrated a proliferative response to androgen in an androgen receptor-dependent and ER-independent manner. In addition, the androgen-induced transcriptional program of MDA-MB-453 significantly overlapped the molecular signature of the unique ER(-)/PR(-) subclass of human tumors. This subset of BCs, characterized by a hormonally regulated transcriptional program and response to androgen, suggests the potential for therapeutic strategies targeting the androgen signaling pathway.
Little is known of the underlying biology of estrogen receptor-negative, progesterone receptor-negative (ER(-)/PR(-)) breast cancer (BC), and few targeted therapies are available. Clinical heterogeneity of ER(-)/PR(-) tumors suggests that molecular subsets exist. We performed genome-wide expression analysis of 99 primary BC samples and eight BC cell lines in an effort to reveal distinct subsets, provide insight into their biology and potentially identify new therapeutic targets. We identified a subset of ER(-)/PR(-) tumors with paradoxical expression of genes known to be either direct targets of ER, responsive to estrogen, or typically expressed in ER(+) BC. Differentially expressed genes included SPDEF, FOXA1, XBP1, CYB5, TFF3, NAT1, APOD, ALCAM and AR (P<0.001). A classification model based on the expression signature of this tumor class identified molecularly similar BCs in an independent human BC data set and among BC cell lines (MDA-MB-453). This cell line demonstrated a proliferative response to androgen in an androgen receptor-dependent and ER-independent manner. In addition, the androgen-induced transcriptional program of MDA-MB-453 significantly overlapped the molecular signature of the unique ER(-)/PR(-) subclass of human tumors. This subset of BCs, characterized by a hormonally regulated transcriptional program and response to androgen, suggests the potential for therapeutic strategies targeting the androgen signaling pathway.
Because the ER(-) class A cell line MDA-MB-453 demonstrated a proliferative response to androgen, we set out to determine whether this was associated with the transcriptional program characteristic of ER(-) class A human breast cancers. We monitored gene expression changes after administration of androgens, androgen antagonists or vehicle control to the ER(-) class A cell line MDA-MB-453 under a variety of growth conditions (see Materials and methods). The results of the various experiments were concordant, but the most pronounced differences in gene expression were observed between those cells first incubated in steroid-deprived conditions for 48 h, and then treated with either R-1881 or vehicle for 48 h. After trimming to eliminate genes with very low-level expression (<200 in both conditions), 497 genes were differentially expressed by at least twofold between cells exposed to R-1881 or vehicle.
Because the ER(-) class A cell line MDA-MB-453 demonstrated a proliferative response to androgen, we set out to determine whether this was associated with the transcriptional program characteristic of ER(-) class A human breast cancers. We monitored gene expression changes after administration of androgens, androgen antagonists or vehicle control to the ER(-) class A cell line MDA-MB-453 under a variety of growth conditions (see Materials and methods). The results of the various experiments were concordant, but the most pronounced differences in gene expression were observed between those cells first incubated in steroid-deprived conditions for 48 h, and then treated with either R-1881 or vehicle for 48 h. After trimming to eliminate genes with very low-level expression (<200 in both conditions), 497 genes were differentially expressed by at least twofold between cells exposed to R-1881 or vehicle.
Little is known of the underlying biology of estrogen receptor-negative, progesterone receptor-negative (ER(-)/PR(-)) breast cancer (BC), and few targeted therapies are available. Clinical heterogeneity of ER(-)/PR(-) tumors suggests that molecular subsets exist. We performed genome-wide expression analysis of 99 primary BC samples and eight BC cell lines in an effort to reveal distinct subsets, provide insight into their biology and potentially identify new therapeutic targets. We identified a subset of ER(-)/PR(-) tumors with paradoxical expression of genes known to be either direct targets of ER, responsive to estrogen, or typically expressed in ER(+) BC. Differentially expressed genes included SPDEF, FOXA1, XBP1, CYB5, TFF3, NAT1, APOD, ALCAM and AR (P<0.001). A classification model based on the expression signature of this tumor class identified molecularly similar BCs in an independent human BC data set and among BC cell lines (MDA-MB-453). This cell line demonstrated a proliferative response to androgen in an androgen receptor-dependent and ER-independent manner. In addition, the androgen-induced transcriptional program of MDA-MB-453 significantly overlapped the molecular signature of the unique ER(-)/PR(-) subclass of human tumors. This subset of BCs, characterized by a hormonally regulated transcriptional program and response to androgen, suggests the potential for therapeutic strategies targeting the androgen signaling pathway.
Little is known of the underlying biology of estrogen receptor-negative, progesterone receptor-negative (ER(-)/PR(-)) breast cancer (BC), and few targeted therapies are available. Clinical heterogeneity of ER(-)/PR(-) tumors suggests that molecular subsets exist. We performed genome-wide expression analysis of 99 primary BC samples and eight BC cell lines in an effort to reveal distinct subsets, provide insight into their biology and potentially identify new therapeutic targets. We identified a subset of ER(-)/PR(-) tumors with paradoxical expression of genes known to be either direct targets of ER, responsive to estrogen, or typically expressed in ER(+) BC. Differentially expressed genes included SPDEF, FOXA1, XBP1, CYB5, TFF3, NAT1, APOD, ALCAM and AR (P<0.001). A classification model based on the expression signature of this tumor class identified molecularly similar BCs in an independent human BC data set and among BC cell lines (MDA-MB-453). This cell line demonstrated a proliferative response to androgen in an androgen receptor-dependent and ER-independent manner. In addition, the androgen-induced transcriptional program of MDA-MB-453 significantly overlapped the molecular signature of the unique ER(-)/PR(-) subclass of human tumors. This subset of BCs, characterized by a hormonally regulated transcriptional program and response to androgen, suggests the potential for therapeutic strategies targeting the androgen signaling pathway.
In the absence of androgen, PSA gene expression is induced by activation of the PKA pathway with forscolin (FSK) by a mechanism dependent on a functional AR. To identify other genes targeted by cross-talk between the androgen and PKA pathways, experiments  were undertaken to characterize the genome-wide expression profiles for androgen and FSK. LNCaP human prostate cancer cells were employed because these cells express endogenous functional androgen receptor (AR) and prostate-specific antigen (PSA). Genes differentially expressed in LNCaP cells upon exposure to R1881 (a synthetic androgen) and FSK were identiied as those cases where the level of transcript changed significantly from levels in the relevant controls. Forty-six probe sets representing 32 genes were significantly changed at least 1.3-fold in response to both androgen and FSK. Applying the Venn diagram, expression of 22 genes was increased by both androgen and FSK (Figure 3a). Eight genes were differentially expressed by R1881 and FSK in opposite directions (Table 2). Expression of Table 1 provides a list of the common genes that increased in expression by both treatment. Expression of SESN1 and NAV1 were the only genes to be decreased in response to both R1881 and FSK (Table 3).
In the absence of androgen, PSA gene expression is induced by activation of the PKA pathway with forscolin (FSK) by a mechanism dependent on a functional AR. To identify other genes targeted by cross-talk between the androgen and PKA pathways, experiments were undertaken to characterize the genome-wide expression profiles for androgen and FSK. LNCaP human prostate cancer cells were employed because these cells express endogenous functional androgen receptor (AR) and prostate-specific antigen (PSA). Genes differentially expressed in LNCaP cells upon exposure to R1881 (a synthetic androgen) and FSK were identiied as those cases where the level of transcript changed significantly from levels in the relevant controls. Forty-six probe sets representing 32 genes were significantly changed at least 1.3-fold in response to both androgen and FSK. Applying the Venn diagram, expression of 22 genes was increased by both androgen and FSK (Figure 3a). Eight genes were differentially expressed by R1881 and FSK in opposite directions (Table 2). Expression of Table 1 provides a list of the common genes that increased in expression by both treatment. Expression of SESN1 and NAV1 were the only genes to be decreased in response to both R1881 and FSK (Table 3).
In the absence of androgen, PSA gene expression is induced by activation of the PKA pathway with forscolin (FSK) by a mechanism dependent on a functional AR. To identify other genes targeted by cross-talk between the androgen and PKA pathways, experiments were undertaken to characterize the genome-wide expression profiles for androgen and FSK. LNCaP human prostate cancer cells were employed because these cells express endogenous functional androgen receptor (AR) and prostate-specific antigen (PSA). Genes differentially expressed in LNCaP cells upon exposure to R1881 (a synthetic androgen) and FSK were identiied as those cases where the level of transcript changed significantly from levels in the relevant controls. Forty-six probe sets representing 32 genes were significantly changed at least 1.3-fold in response to both androgen and FSK. Applying the Venn diagram, expression of 22 genes was increased by both androgen and FSK (Figure 3a). Eight genes were differentially expressed by R1881 and FSK in opposite directions (Table 2). Expression of Table 1 provides a list of the common genes that increased in expression by both treatment. Expression of SESN1 and NAV1 were the only genes to be decreased in response to both R1881 and FSK (Table 3).
Wilms' tumor (WT), one of the most common pediatric solid cancers, arises in the developing kidney as a result of genetic and epigenetic changes that lead to the abnormal proliferation and differentiation of the metanephric blastema. As activation of signal transducers and activators of transcription (STATs) plays an important role in the maintenance/growth and differentiation of the metanephric blastema, and constitutively activated STATs facilitate neoplastic behaviors of a variety of cancers, we hypothesized that dysregulation of STAT signaling may also contribute to WT pathogenesis. Accordingly, we evaluated STAT phosphorylation patterns in tumors and found that STAT1 was constitutively phosphorylated on serine 727 (S727) in 19 of 21 primary WT samples and two WT cell lines. An inactivating mutation of S727 to alanine reduced colony formation of WT cells in soft agar by more than 80% and induced apoptosis under conditions of growth stress. S727-phosphorylated STAT1 provided apoptotic resistance for WT cells via upregulation of expression of the heat-shock protein (HSP)27 and antiapoptotic protein myeloid cell leukemia (MCL)-1. The kinase responsible for STAT1 S727 phosphorylation in WT cells was identified based upon the use of selective inhibitors as protein kinase CK2, not p38, MAP-kinase kinase (MEK)1/2, phosphatidylinositol 3'-kinase, protein kinase C or Ca/calmodulin-dependent protein kinase II (CaMKII). The inhibition of CK2 blocked the anchorage-independent growth of WT cells and induced apoptosis under conditions of growth stress. Our findings suggest that serine-phosphorylated STAT1, as a downstream target of protein kinase CK2, plays a critical role in the pathogenesis of WT and possibly other neoplasms with similar STAT1 phosphorylation patterns.
Serrated colorectal carcinomas (CRCs) are morphologically different from conventional CRCs and have been proposed to follow a distinct pathway of CRC formation. Despite studies of single molecular events in this tumor type, the diagnosis of serrated CRC relies on morphology and the putative unique biological character of these tumors has not been established. Here we show that the gene expression profiling of 37 CRCs separated serrated and conventional CRCs into two distinct branches in unsupervised hierarchical clustering (P-value 7.8 x 10(-7)), and revealed 201 differentially expressed genes representing potential biomarkers for serrated CRC. Immunohistochemistry was utilized to verify the key findings in the 37 CRCs examined by expression profiling, and a separate validation set of 37 serrated and 86 conventional CRCs was examined to evaluate the candidate biomarkers in an extended sample material. Ephrin receptor B2, hypoxia-inducible factor 1-alpha and patched appeared as proteins important for genesis of serrated CRC. This study establishes serrated CRCs as a biologically distinct subclass of CRC and represents a step forward in the molecular classification of these cancers. The study also provides a platform to understand the molecular basis of serrated CRC and in long term may contribute to the development of specific treatment options for this tumor type.
Serrated colorectal carcinomas (CRCs) are morphologically different from conventional CRCs and have been proposed to follow a distinct pathway of CRC formation. Despite studies of single molecular events in this tumor type, the diagnosis of serrated CRC relies on morphology and the putative unique biological character of these tumors has not been established. Here we show that the gene expression profiling of 37 CRCs separated serrated and conventional CRCs into two distinct branches in unsupervised hierarchical clustering (P-value 7.8 x 10(-7)), and revealed 201 differentially expressed genes representing potential biomarkers for serrated CRC. Immunohistochemistry was utilized to verify the key findings in the 37 CRCs examined by expression profiling, and a separate validation set of 37 serrated and 86 conventional CRCs was examined to evaluate the candidate biomarkers in an extended sample material. Ephrin receptor B2, hypoxia-inducible factor 1-alpha and patched appeared as proteins important for genesis of serrated CRC. This study establishes serrated CRCs as a biologically distinct subclass of CRC and represents a step forward in the molecular classification of these cancers. The study also provides a platform to understand the molecular basis of serrated CRC and in long term may contribute to the development of specific treatment options for this tumor type.
Malignant mesothelioma is an aggressive neoplastic proliferation derived from cells lining serosal membranes. The biological and clinical characteristics of epithelial type malignant mesothelioma are distinct from those of biphasic and sarcomatous type tumors. The goal of our study was to examine the molecular basis for this distinction. Microarray analysis confirmed that the molecular signatures of epithelial and biphasic histologic subtypes were distinct. Among the differentially expressed functional gene categories was the ubiquitin-proteasome pathway, which was upregulated in biphasic tumors. Cytotoxicity experiments indicated that 211H cells derived from biphasic tumors were synergistically sensitive to sequential combination regimens containing the proteasome inhibitor bortezomib and oxaliplatin. The mechanism of this synergistic response, which was not detected in cells of epithelial tumor origin, was apoptosis. Together, our results identify the ubiquitin-proteasome pathway as a biomarker of poor prognosis biphasic peritoneal mesothelioma tumors and suggest that proteasome inhibitors could increase the effectiveness of cytotoxic chemotherapy in this subset of patients.
Malignant mesothelioma is an aggressive neoplastic proliferation derived from cells lining serosal membranes. The biological and clinical characteristics of epithelial type malignant mesothelioma are distinct from those of biphasic and sarcomatous type tumors. The goal of our study was to examine the molecular basis for this distinction. Microarray analysis confirmed that the molecular signatures of epithelial and biphasic histologic subtypes were distinct. Among the differentially expressed functional gene categories was the ubiquitin-proteasome pathway, which was upregulated in biphasic tumors. Cytotoxicity experiments indicated that 211H cells derived from biphasic tumors were synergistically sensitive to sequential combination regimens containing the proteasome inhibitor bortezomib and oxaliplatin. The mechanism of this synergistic response, which was not detected in cells of epithelial tumor origin, was apoptosis. Together, our results identify the ubiquitin-proteasome pathway as a biomarker of poor prognosis biphasic peritoneal mesothelioma tumors and suggest that proteasome inhibitors could increase the effectiveness of cytotoxic chemotherapy in this subset of patients.
Chronic wounds represent a substantial public health problem. The development of tools that would enable sophisticated scrutiny of clinical wound tissue material is highly desirable. This work presents evidence enabling rapid specific identification and laser capture of blood vessels from human tissue in a manner which lends itself to successful high-density (U133A) microarray analysis. Such screening of transcriptome followed by real-time PCR and immunohistochemical verification of candidate genes and their corresponding products were performed by using 3 mm biopsies. Of the 18,400 transcripts and variants screened, a focused set of 53 up-regulated and 24 down-regulated genes were noted in wound-derived blood vessels compared with blood vessels from intact human skin. The mean abundance of periostin in wound-site blood vessels was 96-fold higher. Periostin is known to be induced in response to vascular injury and its expression is associated with smooth muscle cell differentiation in vitro and promotes cell migration. Forty-fold higher expression of heparan sulfate 6-O-endosulfatase1 (Sulf1) was noted in wound-site vessels. Sulf1 has been recently recognized to be anti-angiogenic. During embryonic vasculogenesis, CD24 expression is down-regulated in human embryonic stem cells. Wound-site vessels had lower CD24 expression. The findings of this work provide a unique opportunity to appreciate the striking contrast in the transcriptome composition in blood vessels collected from the intact skin and from the wound-edge tissue. Sets of genes with known vascular functions but never connected to wound healing were identified to be differentially expressed in wound-derived blood vessels paving the way for innovative clinically relevant hypotheses.
Chronic wounds represent a substantial public health problem. The development of tools that would enable sophisticated scrutiny of clinical wound tissue material is highly desirable. This work presents evidence enabling rapid specific identification and laser capture of blood vessels from human tissue in a manner which lends itself to successful high-density (U133A) microarray analysis. Such screening of transcriptome followed by real-time PCR and immunohistochemical verification of candidate genes and their corresponding products were performed by using 3 mm biopsies. Of the 18,400 transcripts and variants screened, a focused set of 53 up-regulated and 24 down-regulated genes were noted in wound-derived blood vessels compared with blood vessels from intact human skin. The mean abundance of periostin in wound-site blood vessels was 96-fold higher. Periostin is known to be induced in response to vascular injury and its expression is associated with smooth muscle cell differentiation in vitro and promotes cell migration. Forty-fold higher expression of heparan sulfate 6-O-endosulfatase1 (Sulf1) was noted in wound-site vessels. Sulf1 has been recently recognized to be anti-angiogenic. During embryonic vasculogenesis, CD24 expression is down-regulated in human embryonic stem cells. Wound-site vessels had lower CD24 expression. The findings of this work provide a unique opportunity to appreciate the striking contrast in the transcriptome composition in blood vessels collected from the intact skin and from the wound-edge tissue. Sets of genes with known vascular functions but never connected to wound healing were identified to be differentially expressed in wound-derived blood vessels paving the way for innovative clinically relevant hypotheses.
The B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B gene (BCL11B) encodes a Kr??ppel-like zinc-finger protein, which plays a crucial role in thymopoiesis and has been associated with hematopoietic malignancies. It was hypothesized that BCL11B may act as a tumor-suppressor gene, but its precise function has not yet been elucidated. Here, we demonstrate that the survival of human T-cell leukemia and lymphoma cell lines is critically dependent on Bcl11b. Suppression of Bcl11b by RNA interference selectively induced apoptosis in transformed T cells whereas normal mature T cells remained unaffected. The apoptosis was effected by simultaneous activation of death receptor-mediated and intrinsic apoptotic pathways, most likely as a result of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) upregulation and suppression of the Bcl-xL antiapoptotic protein. Our data indicate an antiapoptotic function of Bcl11b. The resistance of normal mature T lymphocytes to Bcl11b suppression-induced apoptosis and restricted expression pattern make it an attractive therapeutic target in T-cell malignancies.
The B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B gene (BCL11B) encodes a Kr??ppel-like zinc-finger protein, which plays a crucial role in thymopoiesis and has been associated with hematopoietic malignancies. It was hypothesized that BCL11B may act as a tumor-suppressor gene, but its precise function has not yet been elucidated. Here, we demonstrate that the survival of human T-cell leukemia and lymphoma cell lines is critically dependent on Bcl11b. Suppression of Bcl11b by RNA interference selectively induced apoptosis in transformed T cells whereas normal mature T cells remained unaffected. The apoptosis was effected by simultaneous activation of death receptor-mediated and intrinsic apoptotic pathways, most likely as a result of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) upregulation and suppression of the Bcl-xL antiapoptotic protein. Our data indicate an antiapoptotic function of Bcl11b. The resistance of normal mature T lymphocytes to Bcl11b suppression-induced apoptosis and restricted expression pattern make it an attractive therapeutic target in T-cell malignancies.
Cockayne syndrome (CS) is an inherited neurodevelopmental disorder with progeroid features. Although the genes responsible for CS have been implicated in a variety of DNA repair- and transcription-related pathways, the nature of the molecular defect in CS remains mysterious. Using expression microarrays and a unique method for comparative expression analysis called L2L, we sought to define this defect in cells lacking a functional CS group B (CSB) protein, the SWI/SNF-like ATPase responsible for most cases of CS. Remarkably, many of the genes regulated by CSB are also affected by inhibitors of histone deacetylase and DNA methylation, as well as by defects in poly(ADP-ribose)-polymerase function and RNA polymerase II elongation. Moreover, consistent with these microarray expression data, CSB-null cells are sensitive to inhibitors of histone deacetylase or poly(ADP-ribose)-polymerase. Our data indicate a general role for CSB protein in maintenance and remodeling of chromatin structure and suggest that CS is a disease of transcriptional deregulation caused by misexpression of growth-suppressive, inflammatory, and proapoptotic pathways.
Cockayne syndrome (CS) is an inherited neurodevelopmental disorder with progeroid features. Although the genes responsible for CS have been implicated in a variety of DNA repair- and transcription-related pathways, the nature of the molecular defect in CS remains mysterious. Using expression microarrays and a unique method for comparative expression analysis called L2L, we sought to define this defect in cells lacking a functional CS group B (CSB) protein, the SWI/SNF-like ATPase responsible for most cases of CS. Remarkably, many of the genes regulated by CSB are also affected by inhibitors of histone deacetylase and DNA methylation, as well as by defects in poly(ADP-ribose)-polymerase function and RNA polymerase II elongation. Moreover, consistent with these microarray expression data, CSB-null cells are sensitive to inhibitors of histone deacetylase or poly(ADP-ribose)-polymerase. Our data indicate a general role for CSB protein in maintenance and remodeling of chromatin structure and suggest that CS is a disease of transcriptional deregulation caused by misexpression of growth-suppressive, inflammatory, and proapoptotic pathways.
Wilms' tumor 1-associating protein (WTAP) has been reported to be a ubiquitously expressed nuclear protein. Although a relation to splicing factors has been postulated, its actual physiological function still remains to be elucidated. To investigate the role of WTAP, we generated WTAP-knockout mice and performed small interfering RNA (siRNA)-mediated knockdown analyses in primary cultured cells. In DNA microarrays using human umbilical vein endothelial cells, WTAP-targeted siRNA treatment resulted in markedly reduced expression of cell-cycle-related genes. siRNA-mediated WTAP knockdown down-regulated the stability of cyclin A2 mRNA through a nine-nucleotide essential sequence in cyclin A2 mRNA 3' UTR. WTAP knockdown induced G2 accumulation, which is partially rescued by adenoviral overexpression of cyclin A2. Moreover, WTAP-null mice exhibited proliferative failure with death resulting at approximately embryonic day 6.5, an etiology almost identical to cyclin A2-null mice. Collectively, these findings establish WTAP as an essential factor for the stabilization of cyclin A2 mRNA, thereby regulating G2/M cell-cycle transition.
Wilms' tumor 1-associating protein (WTAP) has been reported to be a ubiquitously expressed nuclear protein. Although a relation to splicing factors has been postulated, its actual physiological function still remains to be elucidated. To investigate the role of WTAP, we generated WTAP-knockout mice and performed small interfering RNA (siRNA)-mediated knockdown analyses in primary cultured cells. In DNA microarrays using human umbilical vein endothelial cells, WTAP-targeted siRNA treatment resulted in markedly reduced expression of cell-cycle-related genes. siRNA-mediated WTAP knockdown down-regulated the stability of cyclin A2 mRNA through a nine-nucleotide essential sequence in cyclin A2 mRNA 3' UTR. WTAP knockdown induced G2 accumulation, which is partially rescued by adenoviral overexpression of cyclin A2. Moreover, WTAP-null mice exhibited proliferative failure with death resulting at approximately embryonic day 6.5, an etiology almost identical to cyclin A2-null mice. Collectively, these findings establish WTAP as an essential factor for the stabilization of cyclin A2 mRNA, thereby regulating G2/M cell-cycle transition.
Tumors contain a fraction of cancer stem cells that maintain the propagation of the disease. The CD34(+)CD38(-) cells, isolated from acute myeloid leukemia (AML), were shown to be enriched leukemic stem cells (LSC). We isolated the CD34(+)CD38(-) cell fraction from AML and compared their gene expression profiles to the CD34(+)CD38(+) cell fraction, using microarrays. We found 409 genes that were at least twofold over- or underexpressed between the two cell populations. These include underexpression of DNA repair, signal transduction and cell cycle genes, consistent with the relative quiescence of stem cells, and chromosomal aberrations and mutations of leukemic cells. Comparison of the LSC expression data to that of normal hematopoietic stem cells (HSC) revealed that 34% of the modulated genes are shared by both LSC and HSC, supporting the suggestion that the LSC originated within the HSC progenitors. We focused on the Notch pathway since Jagged-2, a Notch ligand was found to be overexpressed in the LSC samples. We show that DAPT, an inhibitor of gamma-secretase, a protease that is involved in Jagged and Notch signaling, inhibits LSC growth in colony formation assays. Identification of additional genes that regulate LSC self-renewal may provide new targets for therapy.
Tumors contain a fraction of cancer stem cells that maintain the propagation of the disease. The CD34(+)CD38(-) cells, isolated from acute myeloid leukemia (AML), were shown to be enriched leukemic stem cells (LSC). We isolated the CD34(+)CD38(-) cell fraction from AML and compared their gene expression profiles to the CD34(+)CD38(+) cell fraction, using microarrays. We found 409 genes that were at least twofold over- or underexpressed between the two cell populations. These include underexpression of DNA repair, signal transduction and cell cycle genes, consistent with the relative quiescence of stem cells, and chromosomal aberrations and mutations of leukemic cells. Comparison of the LSC expression data to that of normal hematopoietic stem cells (HSC) revealed that 34% of the modulated genes are shared by both LSC and HSC, supporting the suggestion that the LSC originated within the HSC progenitors. We focused on the Notch pathway since Jagged-2, a Notch ligand was found to be overexpressed in the LSC samples. We show that DAPT, an inhibitor of gamma-secretase, a protease that is involved in Jagged and Notch signaling, inhibits LSC growth in colony formation assays. Identification of additional genes that regulate LSC self-renewal may provide new targets for therapy.
Adult T-cell leukemia (ATL) is an intractable malignancy of CD4+ T cells that is etiologically associated with infection by human T-cell leukemia virus-type I. Most individuals in the chronic stage of ATL eventually undergo progression to a highly aggressive acute stage. To clarify the mechanism responsible for this stage progression, we isolated CD4+ cells from individuals in the chronic (n=19) or acute (n=22) stages of ATL and subjected them to profiling of gene expression with DNA microarrays containing >44,000 probe sets. Changes in chromosome copy number were also examined for 24 cell specimens with the use of microarrays harboring approximately 50,000 probe sets. Stage-dependent changes in gene expression profile and chromosome copy number were apparent. Furthermore, expression of the gene for MET, a receptor tyrosine kinase for hepatocyte growth factor (HGF), was shown to be specific to the acute stage of ATL, and the plasma concentration of HGF was increased in individuals in either the acute or chronic stage. HGF induced proliferation of a MET-positive ATL cell line, and this effect was blocked by antibodies to HGF. The HGF-MET signaling pathway is thus a potential therapeutic target for ATL.
Y-box-binding protein 1 (YB-1), which is a member of the DNA-binding protein family containing a cold-shock domain, has pleiotropic functions in response to various environmental stimuli. As we previously showed that YB-1 is a global marker of multidrug resistance in ovarian cancer and other tumor types. To identify YB-1-regulated genes in ovarian cancers, we investigated the expression profile of YB-1 small-interfering RNA (siRNA)-transfected ovarian cancer cells using a high-density oligonucleotide array. YB-1 knockdown by siRNA upregulated 344 genes, including MDR1, thymidylate synthetase, S100 calcium binding protein and cyclin B, and downregulated 534 genes, including CXCR4, N-myc downstream regulated gene 1, E-cadherin and phospholipase C. Exogenous serum addition stimulated YB-1 translocation from the cytoplasm to the nucleus, and treatment with Akt inhibitors as well as Akt siRNA and integrin-linked kinase (ILK) siRNA specifically blocked YB-1 nuclear localization. Inhibition of Akt activation downregulated CXCR4 and upregulated MDR1 (ABCB1) gene expression. Administration of Akt inhibitor resulted in decrease in nuclear YB-1-positive cancer cells in a xenograft animal model. Akt activation thus regulates the nuclear translocation of YB-1, affecting the expression of drug-resistance genes and other genes associated with the malignant characteristics in ovarian cancer cells. Therefore, the Akt pathway could be a novel target of disrupting the nuclear translocation of YB-1 that has important implications for further development of therapeutic strategy against ovarian cancers.
Y-box-binding protein 1 (YB-1), which is a member of the DNA-binding protein family containing a cold-shock domain, has pleiotropic functions in response to various environmental stimuli. As we previously showed that YB-1 is a global marker of multidrug resistance in ovarian cancer and other tumor types. To identify YB-1-regulated genes in ovarian cancers, we investigated the expression profile of YB-1 small-interfering RNA (siRNA)-transfected ovarian cancer cells using a high-density oligonucleotide array. YB-1 knockdown by siRNA upregulated 344 genes, including MDR1, thymidylate synthetase, S100 calcium binding protein and cyclin B, and downregulated 534 genes, including CXCR4, N-myc downstream regulated gene 1, E-cadherin and phospholipase C. Exogenous serum addition stimulated YB-1 translocation from the cytoplasm to the nucleus, and treatment with Akt inhibitors as well as Akt siRNA and integrin-linked kinase (ILK) siRNA specifically blocked YB-1 nuclear localization. Inhibition of Akt activation downregulated CXCR4 and upregulated MDR1 (ABCB1) gene expression. Administration of Akt inhibitor resulted in decrease in nuclear YB-1-positive cancer cells in a xenograft animal model. Akt activation thus regulates the nuclear translocation of YB-1, affecting the expression of drug-resistance genes and other genes associated with the malignant characteristics in ovarian cancer cells. Therefore, the Akt pathway could be a novel target of disrupting the nuclear translocation of YB-1 that has important implications for further development of therapeutic strategy against ovarian cancers.
Activation of Wnt signaling has been implicated in gastric tumorigenesis, although mutations in APC (adenomatous polyposis coli), CTNNB1 (beta-catenin) and AXIN are seen much less frequently in gastric cancer (GC) than in colorectal cancer. In the present study, we investigated the relationship between activation of Wnt signaling and changes in the expression of secreted frizzled-related protein (SFRP) family genes in GC. We frequently observed nuclear beta-catenin accumulation (13/15; 87%) and detected the active form of beta-catenin in most (12/16; 75%) GC cell lines. CpG methylation-dependent silencing of SFRP1, SFRP2 and SFRP5 was frequently seen among GC cell lines (SFRP1, 16/16, 100%; SFRP2, 16/16, 100%; SFRP5, 13/16, 81%) and primary GC specimens (SFRP1, 42/46, 91%; SFRP2, 44/46, 96%; SFRP5, 30/46, 65%), and treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine rapidly restored SFRP expression. Ectopic expression of SFRPs downregulated T-cell factor/lymphocyte enhancer factor transcriptional activity, suppressed cell growth and induced apoptosis in GC cells. Analysis of global expression revealed that overexpression of SFRP2 repressed Wnt target genes and induced changes in the expression of numerous genes related to proliferation, growth and apoptosis in GC cells. It thus appears that aberrant SFRP methylation is one of the major mechanisms by which Wnt signaling is activated in GC.
Activation of Wnt signaling has been implicated in gastric tumorigenesis, although mutations in APC (adenomatous polyposis coli), CTNNB1 (beta-catenin) and AXIN are seen much less frequently in gastric cancer (GC) than in colorectal cancer. In the present study, we investigated the relationship between activation of Wnt signaling and changes in the expression of secreted frizzled-related protein (SFRP) family genes in GC. We frequently observed nuclear beta-catenin accumulation (13/15; 87%) and detected the active form of beta-catenin in most (12/16; 75%) GC cell lines. CpG methylation-dependent silencing of SFRP1, SFRP2 and SFRP5 was frequently seen among GC cell lines (SFRP1, 16/16, 100%; SFRP2, 16/16, 100%; SFRP5, 13/16, 81%) and primary GC specimens (SFRP1, 42/46, 91%; SFRP2, 44/46, 96%; SFRP5, 30/46, 65%), and treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine rapidly restored SFRP expression. Ectopic expression of SFRPs downregulated T-cell factor/lymphocyte enhancer factor transcriptional activity, suppressed cell growth and induced apoptosis in GC cells. Analysis of global expression revealed that overexpression of SFRP2 repressed Wnt target genes and induced changes in the expression of numerous genes related to proliferation, growth and apoptosis in GC cells. It thus appears that aberrant SFRP methylation is one of the major mechanisms by which Wnt signaling is activated in GC.
Deleted in colon cancer (DCC) and UNC5 function as netrin dependence receptors by inducing apoptosis in the absence of their ligand and accordingly were recently designated as putative conditional tumor suppressors. Herein, we determined whether netrin-1 and its receptors are implicated in cancer cell invasion and tumor progression. Expression of DCC, UNC5 and adenosine A2B-receptors (A2B-Rs) was investigated by reverse transcription polymerase chain reaction in human colon cancer cells. The impact of DCC restitution and netrin-1 was evaluated on collagen type I invasion, tumor growth and metastasis in nude mice, cancer cell survival and gene expression profiling. Flow cytometry, poly(ADP-ribose)polymerase-1 and caspase-8 activation were used to evaluate the impact of DCC on cell death. Both netrin-1 and A2B-R activation induced the invasive phenotype through the Rho-Rho kinase axis in DCC-deficient human colorectal cancer cells. Restitution of wild-type DCC blocked invasion induced by netrin-1, A2B-R agonist and other agents. Ectopic expression of netrin-1 led to increased growth of human colon tumor xenografts in athymic mice. Conversely, introduction of wt-DCC in kidney MDCKts.src-ggl cells strongly inhibited metastasis in lymph nodes and lungs and increased sensitivity to apoptosis in hypoxia. DNA microarrays revealed that netrin and DCC had common and divergent impacts on gene expression linked to cell cycle, survival, surface signaling and adhesion. Our findings underscore that netrin is a potent invasion and tumor growth-promoting agent and that DCC is a metastasis suppressor gene targeting both proinvasive and survival pathways in a cumulative manner.
Deleted in colon cancer (DCC) and UNC5 function as netrin dependence receptors by inducing apoptosis in the absence of their ligand and accordingly were recently designated as putative conditional tumor suppressors. Herein, we determined whether netrin-1 and its receptors are implicated in cancer cell invasion and tumor progression. Expression of DCC, UNC5 and adenosine A2B-receptors (A2B-Rs) was investigated by reverse transcription polymerase chain reaction in human colon cancer cells. The impact of DCC restitution and netrin-1 was evaluated on collagen type I invasion, tumor growth and metastasis in nude mice, cancer cell survival and gene expression profiling. Flow cytometry, poly(ADP-ribose)polymerase-1 and caspase-8 activation were used to evaluate the impact of DCC on cell death. Both netrin-1 and A2B-R activation induced the invasive phenotype through the Rho-Rho kinase axis in DCC-deficient human colorectal cancer cells. Restitution of wild-type DCC blocked invasion induced by netrin-1, A2B-R agonist and other agents. Ectopic expression of netrin-1 led to increased growth of human colon tumor xenografts in athymic mice. Conversely, introduction of wt-DCC in kidney MDCKts.src-ggl cells strongly inhibited metastasis in lymph nodes and lungs and increased sensitivity to apoptosis in hypoxia. DNA microarrays revealed that netrin and DCC had common and divergent impacts on gene expression linked to cell cycle, survival, surface signaling and adhesion. Our findings underscore that netrin is a potent invasion and tumor growth-promoting agent and that DCC is a metastasis suppressor gene targeting both proinvasive and survival pathways in a cumulative manner.
Deleted in colon cancer (DCC) and UNC5 function as netrin dependence receptors by inducing apoptosis in the absence of their ligand and accordingly were recently designated as putative conditional tumor suppressors. Herein, we determined whether netrin-1 and its receptors are implicated in cancer cell invasion and tumor progression. Expression of DCC, UNC5 and adenosine A2B-receptors (A2B-Rs) was investigated by reverse transcription polymerase chain reaction in human colon cancer cells. The impact of DCC restitution and netrin-1 was evaluated on collagen type I invasion, tumor growth and metastasis in nude mice, cancer cell survival and gene expression profiling. Flow cytometry, poly(ADP-ribose)polymerase-1 and caspase-8 activation were used to evaluate the impact of DCC on cell death. Both netrin-1 and A2B-R activation induced the invasive phenotype through the Rho-Rho kinase axis in DCC-deficient human colorectal cancer cells. Restitution of wild-type DCC blocked invasion induced by netrin-1, A2B-R agonist and other agents. Ectopic expression of netrin-1 led to increased growth of human colon tumor xenografts in athymic mice. Conversely, introduction of wt-DCC in kidney MDCKts.src-ggl cells strongly inhibited metastasis in lymph nodes and lungs and increased sensitivity to apoptosis in hypoxia. DNA microarrays revealed that netrin and DCC had common and divergent impacts on gene expression linked to cell cycle, survival, surface signaling and adhesion. Our findings underscore that netrin is a potent invasion and tumor growth-promoting agent and that DCC is a metastasis suppressor gene targeting both proinvasive and survival pathways in a cumulative manner.
Deleted in colon cancer (DCC) and UNC5 function as netrin dependence receptors by inducing apoptosis in the absence of their ligand and accordingly were recently designated as putative conditional tumor suppressors. Herein, we determined whether netrin-1 and its receptors are implicated in cancer cell invasion and tumor progression. Expression of DCC, UNC5 and adenosine A2B-receptors (A2B-Rs) was investigated by reverse transcription polymerase chain reaction in human colon cancer cells. The impact of DCC restitution and netrin-1 was evaluated on collagen type I invasion, tumor growth and metastasis in nude mice, cancer cell survival and gene expression profiling. Flow cytometry, poly(ADP-ribose)polymerase-1 and caspase-8 activation were used to evaluate the impact of DCC on cell death. Both netrin-1 and A2B-R activation induced the invasive phenotype through the Rho-Rho kinase axis in DCC-deficient human colorectal cancer cells. Restitution of wild-type DCC blocked invasion induced by netrin-1, A2B-R agonist and other agents. Ectopic expression of netrin-1 led to increased growth of human colon tumor xenografts in athymic mice. Conversely, introduction of wt-DCC in kidney MDCKts.src-ggl cells strongly inhibited metastasis in lymph nodes and lungs and increased sensitivity to apoptosis in hypoxia. DNA microarrays revealed that netrin and DCC had common and divergent impacts on gene expression linked to cell cycle, survival, surface signaling and adhesion. Our findings underscore that netrin is a potent invasion and tumor growth-promoting agent and that DCC is a metastasis suppressor gene targeting both proinvasive and survival pathways in a cumulative manner.
The nuclear LIM-only protein 4 (LMO4) is upregulated in breast cancer, especially estrogen receptor-negative tumors, and its overexpression in mice leads to hyperplasia and tumor formation. Here, we show that deletion of LMO4 in the mammary glands of mice leads to impaired lobuloalveolar development due to decreased epithelial cell proliferation. With the goal of discovering potential LMO4-target genes, we also developed a conditional expression system in MCF-7 cells for both LMO4 and a dominant negative (DN) form of its co-regulator, cofactor of LIM domains (Clim/Ldb/Nli). We then used DNA microarrays to identify genes responsive to LMO4 and DN-Clim upregulation. One of the genes common to both data sets was bone morphogenic protein 7 (BMP7), whose expression is also significantly correlated with LMO4 transcript levels in a large dataset of human breast cancers, suggesting that BMP7 is a bona fide target gene of LMO4 in breast cancer. Inhibition of BMP7 partially blocks the effects of LMO4 on apoptosis, indicating that BMP7 mediates at least some functions of LMO4. Gene transfer studies show that LMO4 regulates the BMP7 promoter, and chromatin immunoprecipitation studies show that LMO4 and its cofactor Clim2 are recruited to the BMP7 promoter. Furthermore, we demonstrate that HDAC2 recruitment to the BMP7 promoter is inhibited by upregulation of LMO4 and that HDAC2 knockdown upregulates the promoter. These studies suggest a novel mechanism of action for LMO4: LMO4, Clim2 and HDAC2 are part of a transcriptional complex, and increased LMO4 levels can disrupt the complex, leading to decreased HDAC2 recruitment and increased promoter activity.
The nuclear LIM-only protein 4 (LMO4) is upregulated in breast cancer, especially estrogen receptor-negative tumors, and its overexpression in mice leads to hyperplasia and tumor formation. Here, we show that deletion of LMO4 in the mammary glands of mice leads to impaired lobuloalveolar development due to decreased epithelial cell proliferation. With the goal of discovering potential LMO4-target genes, we also developed a conditional expression system in MCF-7 cells for both LMO4 and a dominant negative (DN) form of its co-regulator, cofactor of LIM domains (Clim/Ldb/Nli). We then used DNA microarrays to identify genes responsive to LMO4 and DN-Clim upregulation. One of the genes common to both data sets was bone morphogenic protein 7 (BMP7), whose expression is also significantly correlated with LMO4 transcript levels in a large dataset of human breast cancers, suggesting that BMP7 is a bona fide target gene of LMO4 in breast cancer. Inhibition of BMP7 partially blocks the effects of LMO4 on apoptosis, indicating that BMP7 mediates at least some functions of LMO4. Gene transfer studies show that LMO4 regulates the BMP7 promoter, and chromatin immunoprecipitation studies show that LMO4 and its cofactor Clim2 are recruited to the BMP7 promoter. Furthermore, we demonstrate that HDAC2 recruitment to the BMP7 promoter is inhibited by upregulation of LMO4 and that HDAC2 knockdown upregulates the promoter. These studies suggest a novel mechanism of action for LMO4: LMO4, Clim2 and HDAC2 are part of a transcriptional complex, and increased LMO4 levels can disrupt the complex, leading to decreased HDAC2 recruitment and increased promoter activity.
The nuclear LIM-only protein 4 (LMO4) is upregulated in breast cancer, especially estrogen receptor-negative tumors, and its overexpression in mice leads to hyperplasia and tumor formation. Here, we show that deletion of LMO4 in the mammary glands of mice leads to impaired lobuloalveolar development due to decreased epithelial cell proliferation. With the goal of discovering potential LMO4-target genes, we also developed a conditional expression system in MCF-7 cells for both LMO4 and a dominant negative (DN) form of its co-regulator, cofactor of LIM domains (Clim/Ldb/Nli). We then used DNA microarrays to identify genes responsive to LMO4 and DN-Clim upregulation. One of the genes common to both data sets was bone morphogenic protein 7 (BMP7), whose expression is also significantly correlated with LMO4 transcript levels in a large dataset of human breast cancers, suggesting that BMP7 is a bona fide target gene of LMO4 in breast cancer. Inhibition of BMP7 partially blocks the effects of LMO4 on apoptosis, indicating that BMP7 mediates at least some functions of LMO4. Gene transfer studies show that LMO4 regulates the BMP7 promoter, and chromatin immunoprecipitation studies show that LMO4 and its cofactor Clim2 are recruited to the BMP7 promoter. Furthermore, we demonstrate that HDAC2 recruitment to the BMP7 promoter is inhibited by upregulation of LMO4 and that HDAC2 knockdown upregulates the promoter. These studies suggest a novel mechanism of action for LMO4: LMO4, Clim2 and HDAC2 are part of a transcriptional complex, and increased LMO4 levels can disrupt the complex, leading to decreased HDAC2 recruitment and increased promoter activity.
The nuclear LIM-only protein 4 (LMO4) is upregulated in breast cancer, especially estrogen receptor-negative tumors, and its overexpression in mice leads to hyperplasia and tumor formation. Here, we show that deletion of LMO4 in the mammary glands of mice leads to impaired lobuloalveolar development due to decreased epithelial cell proliferation. With the goal of discovering potential LMO4-target genes, we also developed a conditional expression system in MCF-7 cells for both LMO4 and a dominant negative (DN) form of its co-regulator, cofactor of LIM domains (Clim/Ldb/Nli). We then used DNA microarrays to identify genes responsive to LMO4 and DN-Clim upregulation. One of the genes common to both data sets was bone morphogenic protein 7 (BMP7), whose expression is also significantly correlated with LMO4 transcript levels in a large dataset of human breast cancers, suggesting that BMP7 is a bona fide target gene of LMO4 in breast cancer. Inhibition of BMP7 partially blocks the effects of LMO4 on apoptosis, indicating that BMP7 mediates at least some functions of LMO4. Gene transfer studies show that LMO4 regulates the BMP7 promoter, and chromatin immunoprecipitation studies show that LMO4 and its cofactor Clim2 are recruited to the BMP7 promoter. Furthermore, we demonstrate that HDAC2 recruitment to the BMP7 promoter is inhibited by upregulation of LMO4 and that HDAC2 knockdown upregulates the promoter. These studies suggest a novel mechanism of action for LMO4: LMO4, Clim2 and HDAC2 are part of a transcriptional complex, and increased LMO4 levels can disrupt the complex, leading to decreased HDAC2 recruitment and increased promoter activity.
Gastric carcinoma is one of the major causes of cancer mortality worldwide. Early detection results in excellent prognosis for patients with early cancer (EGC), whereas the prognosis of advanced cancer (AGC) patients remains poor. It is not clear whether EGC and AGC are molecularly distinct, and whether they represent progressive stages of the same tumor or different entities ab initio. Gene expression profiles of EGC and AGC were determined by Affymetrix technology and quantitative polymerase chain reaction. Representative regulated genes were further analysed by in situ hybridization (ISH) on tissue microarrays. Expression analysis allowed the identification of a signature that differentiates AGC from EGC. In addition, comparison with normal gastric mucosa indicated that the majority of alterations associated with EGC are retained in AGC, and that further expression changes mark the transition from EGC to AGC. Finally, ISH analysis showed that representative genes, differentially expressed in the invasive areas of EGC and AGC, are not differentially expressed in the non-invasive areas of the same tumors. Our data are more directly compatible with a progression model of gastric carcinogenesis, whereby EGC and AGC may represent different molecular stages of the same tumor. Finally, the identification of an AGC-specific signature might help devising novel therapeutic strategies for advanced gastric cancer.
Gastric carcinoma is one of the major causes of cancer mortality worldwide. Early detection results in excellent prognosis for patients with early cancer (EGC), whereas the prognosis of advanced cancer (AGC) patients remains poor. It is not clear whether EGC and AGC are molecularly distinct, and whether they represent progressive stages of the same tumor or different entities ab initio. Gene expression profiles of EGC and AGC were determined by Affymetrix technology and quantitative polymerase chain reaction. Representative regulated genes were further analysed by in situ hybridization (ISH) on tissue microarrays. Expression analysis allowed the identification of a signature that differentiates AGC from EGC. In addition, comparison with normal gastric mucosa indicated that the majority of alterations associated with EGC are retained in AGC, and that further expression changes mark the transition from EGC to AGC. Finally, ISH analysis showed that representative genes, differentially expressed in the invasive areas of EGC and AGC, are not differentially expressed in the non-invasive areas of the same tumors. Our data are more directly compatible with a progression model of gastric carcinogenesis, whereby EGC and AGC may represent different molecular stages of the same tumor. Finally, the identification of an AGC-specific signature might help devising novel therapeutic strategies for advanced gastric cancer.
Gastric carcinoma is one of the major causes of cancer mortality worldwide. Early detection results in excellent prognosis for patients with early cancer (EGC), whereas the prognosis of advanced cancer (AGC) patients remains poor. It is not clear whether EGC and AGC are molecularly distinct, and whether they represent progressive stages of the same tumor or different entities ab initio. Gene expression profiles of EGC and AGC were determined by Affymetrix technology and quantitative polymerase chain reaction. Representative regulated genes were further analysed by in situ hybridization (ISH) on tissue microarrays. Expression analysis allowed the identification of a signature that differentiates AGC from EGC. In addition, comparison with normal gastric mucosa indicated that the majority of alterations associated with EGC are retained in AGC, and that further expression changes mark the transition from EGC to AGC. Finally, ISH analysis showed that representative genes, differentially expressed in the invasive areas of EGC and AGC, are not differentially expressed in the non-invasive areas of the same tumors. Our data are more directly compatible with a progression model of gastric carcinogenesis, whereby EGC and AGC may represent different molecular stages of the same tumor. Finally, the identification of an AGC-specific signature might help devising novel therapeutic strategies for advanced gastric cancer.
Gastric carcinoma is one of the major causes of cancer mortality worldwide. Early detection results in excellent prognosis for patients with early cancer (EGC), whereas the prognosis of advanced cancer (AGC) patients remains poor. It is not clear whether EGC and AGC are molecularly distinct, and whether they represent progressive stages of the same tumor or different entities ab initio. Gene expression profiles of EGC and AGC were determined by Affymetrix technology and quantitative polymerase chain reaction. Representative regulated genes were further analysed by in situ hybridization (ISH) on tissue microarrays. Expression analysis allowed the identification of a signature that differentiates AGC from EGC. In addition, comparison with normal gastric mucosa indicated that the majority of alterations associated with EGC are retained in AGC, and that further expression changes mark the transition from EGC to AGC. Finally, ISH analysis showed that representative genes, differentially expressed in the invasive areas of EGC and AGC, are not differentially expressed in the non-invasive areas of the same tumors. Our data are more directly compatible with a progression model of gastric carcinogenesis, whereby EGC and AGC may represent different molecular stages of the same tumor. Finally, the identification of an AGC-specific signature might help devising novel therapeutic strategies for advanced gastric cancer.
We have assessed the possibility to build a prognosis predictor (PP), based on non-neoplastic mucosa microarray gene expression measures, for stage II colon cancer patients. Non-neoplastic colonic mucosa mRNA samples from 24 patients (10 with a metachronous metastasis, 14 with no recurrence) were profiled using the Affymetrix HGU133A GeneChip. Patients were repeatedly and randomly divided into 1000 training sets (TSs) of size 16 and validation sets (VS) of size 8. For each TS/VS split, a 70-gene PP, identified on the TS by selecting the 70 most differentially expressed genes and applying diagonal linear discriminant analysis, was used to predict the prognoses of VS patients. Mean prognosis prediction performances of the 70-gene PP were 81.8% for accuracy, 73.0% for sensitivity and 87.1% for specificity. Informative genes suggested branching signal-transduction pathways with possible extensive networks between individual pathways. They also included genes coding for proteins involved in immune surveillance. In conclusion, our study suggests that one can build an accurate PP for stage II colon cancer patients, based on non-neoplastic mucosa microarray gene expression measures.
We have assessed the possibility to build a prognosis predictor (PP), based on non-neoplastic mucosa microarray gene expression measures, for stage II colon cancer patients. Non-neoplastic colonic mucosa mRNA samples from 24 patients (10 with a metachronous metastasis, 14 with no recurrence) were profiled using the Affymetrix HGU133A GeneChip. Patients were repeatedly and randomly divided into 1000 training sets (TSs) of size 16 and validation sets (VS) of size 8. For each TS/VS split, a 70-gene PP, identified on the TS by selecting the 70 most differentially expressed genes and applying diagonal linear discriminant analysis, was used to predict the prognoses of VS patients. Mean prognosis prediction performances of the 70-gene PP were 81.8% for accuracy, 73.0% for sensitivity and 87.1% for specificity. Informative genes suggested branching signal-transduction pathways with possible extensive networks between individual pathways. They also included genes coding for proteins involved in immune surveillance. In conclusion, our study suggests that one can build an accurate PP for stage II colon cancer patients, based on non-neoplastic mucosa microarray gene expression measures.
Increased numbers of endothelial cells are observed in peripheral blood of cancer patients. These circulating endothelial cells (CECs) may contribute to the formation of blood vessels in the tumor or reflect vascular damage caused by treatment or tumor growth.To identify potential markers for detection and characterization of carcinoma associated CECs, gene expression profiles from CEC-enriched samples from cancer patients were compared with CEC-enriched samples from three ??healthy?? donors (containing 1, 43, and 48 detected CECs; Supplementary Table S1A). After a global scaling procedure was used to normalize the expression data between experiments, we selected ~160 genes as potential markers for detection of CECs in peripheral blood.
Increased numbers of endothelial cells are observed in peripheral blood of cancer patients. These circulating endothelial cells (CECs) may contribute to the formation of blood vessels in the tumor or reflect vascular damage caused by treatment or tumor growth.To identify potential markers for detection and characterization of carcinoma associated CECs, gene expression profiles from CEC-enriched samples from cancer patients were compared with CEC-enriched samples from three ??healthy?? donors (containing 1, 43, and 48 detected CECs; Supplementary Table S1A). After a global scaling procedure was used to normalize the expression data between experiments, we selected ~160 genes as potential markers for detection of CECs in peripheral blood.
Human papillomavirus (HPV) is associated with a subset of head and neck squamous cell carcinoma (HNSCC). Between 15% and 35% of HNSCCs harbor HPV DNA. Demographic and exposure differences between HPV-positive (HPV+) and negative (HPV-) HNSCCs suggest that HPV+ tumors may constitute a subclass with different biology, whereas clinical differences have also been observed. Gene expression profiles of HPV+ and HPV- tumors were compared with further exploration of the biological effect of HPV in HNSCC. Thirty-six HNSCC tumors were analyzed using Affymetrix Human 133U Plus 2.0 GeneChip and for HPV by PCR and real-time PCR. Eight of 36 (22%) tumors were positive for HPV subtype 16. Statistical analysis using Significance Analysis of Microarrays based on HPV status as a supervising variable resulted in a list of 91 genes that were differentially expressed with statistical significance. Results for a subset of these genes were verified by real-time PCR. Genes highly expressed in HPV+ samples included cell cycle regulators (p16(INK4A), p18, and CDC7) and transcription factors (TAF7L, RFC4, RPA2, and TFDP2). The microarray data were also investigated by mapping genes by chromosomal location (DIGMAP). A large number of genes on chromosome 3q24-qter had high levels of expression in HPV+ tumors. Further investigation of differentially expressed genes may reveal the unique pathways in HPV+ tumors that may explain the different natural history and biological properties of these tumors. These properties may be exploited as a target of novel therapeutic agents in HNSCC treatment.
PURPOSE: To better understand the molecular mechanisms of malignant melanoma progression and metastasis, gene expression profiling was done of primary melanomas and melanoma metastases. EXPERIMENTAL DESIGN: Tumor cell-specific gene expression in 19 primary melanomas and 22 melanoma metastases was analyzed using oligonucleotide microarrays after laser-capture microdissection of melanoma cells. Statistical analysis was done by random permutation analysis and support vector machines. Microarray data were further validated by immunohistochemistry and immunoblotting. RESULTS: Overall, 308 genes were identified that showed significant differential expression between primary melanomas and melanoma metastases (false discovery rate<or=0.05). Significantly overrepresented gene ontology categories in the list of 308 genes were cell cycle regulation, mitosis, cell communication, and cell adhesion. Overall, 47 genes showed up-regulation in metastases. These included Cdc6, Cdk1, septin 6, mitosin, kinesin family member 2C, osteopontin, and fibronectin. Down-regulated genes included E-cadherin, fibroblast growth factor binding protein, and desmocollin 1 and desmocollin 3, stratifin/14-3-3sigma, and the chemokine CCL27. Using support vector machine analysis of gene expression data, a performance of >85% correct classifications for primary melanomas and metastases was reached. Further analysis showed that subtypes of primary melanomas displayed characteristic gene expression patterns, as do thin tumors (<or=1.0 mm Breslow thickness) compared with intermediate and thick tumors (>2.0 mm Breslow thickness). CONCLUSIONS: Taken together, this large-scale gene expression study of malignant melanoma identified molecular signatures related to metastasis, melanoma subtypes, and tumor thickness. These findings not only provide deeper insights into the pathogenesis of melanoma progression but may also guide future research on innovative treatments.
PURPOSE: To better understand the molecular mechanisms of malignant melanoma progression and metastasis, gene expression profiling was done of primary melanomas and melanoma metastases. EXPERIMENTAL DESIGN: Tumor cell-specific gene expression in 19 primary melanomas and 22 melanoma metastases was analyzed using oligonucleotide microarrays after laser-capture microdissection of melanoma cells. Statistical analysis was done by random permutation analysis and support vector machines. Microarray data were further validated by immunohistochemistry and immunoblotting. RESULTS: Overall, 308 genes were identified that showed significant differential expression between primary melanomas and melanoma metastases (false discovery rate<or=0.05). Significantly overrepresented gene ontology categories in the list of 308 genes were cell cycle regulation, mitosis, cell communication, and cell adhesion. Overall, 47 genes showed up-regulation in metastases. These included Cdc6, Cdk1, septin 6, mitosin, kinesin family member 2C, osteopontin, and fibronectin. Down-regulated genes included E-cadherin, fibroblast growth factor binding protein, and desmocollin 1 and desmocollin 3, stratifin/14-3-3sigma, and the chemokine CCL27. Using support vector machine analysis of gene expression data, a performance of >85% correct classifications for primary melanomas and metastases was reached. Further analysis showed that subtypes of primary melanomas displayed characteristic gene expression patterns, as do thin tumors (<or=1.0 mm Breslow thickness) compared with intermediate and thick tumors (>2.0 mm Breslow thickness). CONCLUSIONS: Taken together, this large-scale gene expression study of malignant melanoma identified molecular signatures related to metastasis, melanoma subtypes, and tumor thickness. These findings not only provide deeper insights into the pathogenesis of melanoma progression but may also guide future research on innovative treatments.
PURPOSE: Recent data indicate that cDNA microarray gene expression profile of blood cells can reflect disease states and thus have diagnostic value. We tested the hypothesis that blood cell gene expression can differentiate between bladder cancer and other genitourinary cancers as well as between bladder cancer and healthy controls. EXPERIMENTAL DESIGN: We used Affymetrix U133 Plus 2.0 GeneChip (Affymetrix, Santa Clara, CA) to profile circulating blood total RNA from 35 patients diagnosed with one of three types of genitourinary cancer [bladder cancer (n = 16), testicular cancer (n = 10), and renal cell carcinoma (n = 9)] and compared their cDNA profiles with those of 10 healthy subjects. We then verified the expression levels of selected genes from the Affymetrix results in a larger number of bladder cancer patients (n = 40) and healthy controls (n = 27). RESULTS: Blood gene expression profiles distinguished bladder cancer patients from healthy controls and from testicular and renal cancer patients. Differential expression of a combined set of seven gene transcripts (insulin-like growth factor-binding protein 7, sorting nexin 16, chondroitin sulfate proteoglycan 6, and cathepsin D, chromodomain helicase DNA-binding protein 2, nell-like 2, and tumor necrosis factor receptor superfamily member 7) was able to discriminate bladder cancer from control samples with a sensitivity of 83% (95% confidence interval, 67-93%) and a specificity of 93% (95% confidence interval, 76-99%). CONCLUSION: We have shown that the gene expression profile of circulating blood cells can distinguish bladder cancer from other types of genitourinary cancer and healthy controls and can be used to identify novel blood markers for bladder cancer.
PURPOSE: Recent data indicate that cDNA microarray gene expression profile of blood cells can reflect disease states and thus have diagnostic value. We tested the hypothesis that blood cell gene expression can differentiate between bladder cancer and other genitourinary cancers as well as between bladder cancer and healthy controls. EXPERIMENTAL DESIGN: We used Affymetrix U133 Plus 2.0 GeneChip (Affymetrix, Santa Clara, CA) to profile circulating blood total RNA from 35 patients diagnosed with one of three types of genitourinary cancer [bladder cancer (n = 16), testicular cancer (n = 10), and renal cell carcinoma (n = 9)] and compared their cDNA profiles with those of 10 healthy subjects. We then verified the expression levels of selected genes from the Affymetrix results in a larger number of bladder cancer patients (n = 40) and healthy controls (n = 27). RESULTS: Blood gene expression profiles distinguished bladder cancer patients from healthy controls and from testicular and renal cancer patients. Differential expression of a combined set of seven gene transcripts (insulin-like growth factor-binding protein 7, sorting nexin 16, chondroitin sulfate proteoglycan 6, and cathepsin D, chromodomain helicase DNA-binding protein 2, nell-like 2, and tumor necrosis factor receptor superfamily member 7) was able to discriminate bladder cancer from control samples with a sensitivity of 83% (95% confidence interval, 67-93%) and a specificity of 93% (95% confidence interval, 76-99%). CONCLUSION: We have shown that the gene expression profile of circulating blood cells can distinguish bladder cancer from other types of genitourinary cancer and healthy controls and can be used to identify novel blood markers for bladder cancer.
PURPOSE: Amplification of chromosomal region 20q13 occurs in breast cancer but remains poorly characterized. EXPERIMENTAL DESIGN: To establish the frequency of 20q13 amplification and select the amplified cases to be studied, we used fluorescence in situ hybridization of bacterial artificial chromosome probes for three 20q13 loci (MYBL2, STK6, ZNF217) on sections of tissue microarrays containing 466 primary carcinoma samples. We used Affymetryx whole-genome DNA microarrays to establish the gene expression profiles of 20q13-amplified tumors and quantitative reverse transcription-PCR to validate the results. RESULTS: We found 36 (8%) 20q13-amplified samples. They were distributed in two types: type 1 tumors showed ZNF217 amplification only, whereas type 2 tumors showed amplification at two or three loci. Examination of the histoclinical features of the amplified tumors showed two strikingly opposite data. First, type 1 tumors were more frequently lymph node-negative tumors but were paradoxically associated with a poor prognosis. Second, type 2 tumors were more frequently lymph node-positive tumors but were paradoxically associated with a good prognosis. Type 1 and type 2 showed different gene expression profiles. No 20q13 gene could be associated with type 1 amplification, whereas several 20q13 genes were overexpressed in type 2 tumors. CONCLUSIONS: Our results suggest that amplified tumors of types 1 and 2 are two distinct entities resulting from two different mechanisms and associated to different prognosis.
PURPOSE: Amplification of chromosomal region 20q13 occurs in breast cancer but remains poorly characterized. EXPERIMENTAL DESIGN: To establish the frequency of 20q13 amplification and select the amplified cases to be studied, we used fluorescence in situ hybridization of bacterial artificial chromosome probes for three 20q13 loci (MYBL2, STK6, ZNF217) on sections of tissue microarrays containing 466 primary carcinoma samples. We used Affymetryx whole-genome DNA microarrays to establish the gene expression profiles of 20q13-amplified tumors and quantitative reverse transcription-PCR to validate the results. RESULTS: We found 36 (8%) 20q13-amplified samples. They were distributed in two types: type 1 tumors showed ZNF217 amplification only, whereas type 2 tumors showed amplification at two or three loci. Examination of the histoclinical features of the amplified tumors showed two strikingly opposite data. First, type 1 tumors were more frequently lymph node-negative tumors but were paradoxically associated with a poor prognosis. Second, type 2 tumors were more frequently lymph node-positive tumors but were paradoxically associated with a good prognosis. Type 1 and type 2 showed different gene expression profiles. No 20q13 gene could be associated with type 1 amplification, whereas several 20q13 genes were overexpressed in type 2 tumors. CONCLUSIONS: Our results suggest that amplified tumors of types 1 and 2 are two distinct entities resulting from two different mechanisms and associated to different prognosis.
PURPOSE: Amplification of chromosomal region 20q13 occurs in breast cancer but remains poorly characterized. EXPERIMENTAL DESIGN: To establish the frequency of 20q13 amplification and select the amplified cases to be studied, we used fluorescence in situ hybridization of bacterial artificial chromosome probes for three 20q13 loci (MYBL2, STK6, ZNF217) on sections of tissue microarrays containing 466 primary carcinoma samples. We used Affymetryx whole-genome DNA microarrays to establish the gene expression profiles of 20q13-amplified tumors and quantitative reverse transcription-PCR to validate the results. RESULTS: We found 36 (8%) 20q13-amplified samples. They were distributed in two types: type 1 tumors showed ZNF217 amplification only, whereas type 2 tumors showed amplification at two or three loci. Examination of the histoclinical features of the amplified tumors showed two strikingly opposite data. First, type 1 tumors were more frequently lymph node-negative tumors but were paradoxically associated with a poor prognosis. Second, type 2 tumors were more frequently lymph node-positive tumors but were paradoxically associated with a good prognosis. Type 1 and type 2 showed different gene expression profiles. No 20q13 gene could be associated with type 1 amplification, whereas several 20q13 genes were overexpressed in type 2 tumors. CONCLUSIONS: Our results suggest that amplified tumors of types 1 and 2 are two distinct entities resulting from two different mechanisms and associated to different prognosis.
PURPOSE: Amplification of chromosomal region 20q13 occurs in breast cancer but remains poorly characterized. EXPERIMENTAL DESIGN: To establish the frequency of 20q13 amplification and select the amplified cases to be studied, we used fluorescence in situ hybridization of bacterial artificial chromosome probes for three 20q13 loci (MYBL2, STK6, ZNF217) on sections of tissue microarrays containing 466 primary carcinoma samples. We used Affymetryx whole-genome DNA microarrays to establish the gene expression profiles of 20q13-amplified tumors and quantitative reverse transcription-PCR to validate the results. RESULTS: We found 36 (8%) 20q13-amplified samples. They were distributed in two types: type 1 tumors showed ZNF217 amplification only, whereas type 2 tumors showed amplification at two or three loci. Examination of the histoclinical features of the amplified tumors showed two strikingly opposite data. First, type 1 tumors were more frequently lymph node-negative tumors but were paradoxically associated with a poor prognosis. Second, type 2 tumors were more frequently lymph node-positive tumors but were paradoxically associated with a good prognosis. Type 1 and type 2 showed different gene expression profiles. No 20q13 gene could be associated with type 1 amplification, whereas several 20q13 genes were overexpressed in type 2 tumors. CONCLUSIONS: Our results suggest that amplified tumors of types 1 and 2 are two distinct entities resulting from two different mechanisms and associated to different prognosis.
CD34+ hematopoietic stem/progenitor cells (HSCs) reside in the bone marrow in close proximity to the endosteal bone surface, surrounded by osteoblasts, stromal cells, and various extracellular matrix molecules. We used a bioartificial matrix of fibrillar collagen I, the major matrix component of bone, as a scaffold for ex vivo expansion of HSCs. CD34+ HSCs were isolated from umbilical cord blood and cultivated within reconstituted collagen I fibrils in the presence of fms-like tyrosine kinase-3 ligand, stem cell factor, and interleukin (IL)-3. After 7 days of culture, the cell number, number of colony-forming units (CFU-C), and gene-expression profile of the cultured cells were assessed. Although the total expansion factor of CD34+ cells was slightly lower when cells were cultivated in the collagen I gel, the frequency of CFU-C was greater than in control suspension cultures. Gene-expression analysis with microarray chip technology revealed the upregulation of more than 50 genes in the presence of collagen I. Among these, genes for several growth factors, cytokines, and chemokines (e.g., IL-8 and macrophage inhibitory protein 1alpha) could be confirmed using quantitative polymerase chain reaction. Furthermore, greater expression levels of the negative cell-cycle regulator BTG2/TIS21 and an inhibitor of the mitogen-activated protein kinase pathway, DUSP2, underline the regulatory role of the extracellular matrix. Together, these data show that the expansion of CD34+ cord blood cells in a culture system containing a three-dimensional collagen I matrix induces a qualitative change in the gene-expression profile of cultivated HSCs.
CD34+ hematopoietic stem/progenitor cells (HSCs) reside in the bone marrow in close proximity to the endosteal bone surface, surrounded by osteoblasts, stromal cells, and various extracellular matrix molecules. We used a bioartificial matrix of fibrillar collagen I, the major matrix component of bone, as a scaffold for ex vivo expansion of HSCs. CD34+ HSCs were isolated from umbilical cord blood and cultivated within reconstituted collagen I fibrils in the presence of fms-like tyrosine kinase-3 ligand, stem cell factor, and interleukin (IL)-3. After 7 days of culture, the cell number, number of colony-forming units (CFU-C), and gene-expression profile of the cultured cells were assessed. Although the total expansion factor of CD34+ cells was slightly lower when cells were cultivated in the collagen I gel, the frequency of CFU-C was greater than in control suspension cultures. Gene-expression analysis with microarray chip technology revealed the upregulation of more than 50 genes in the presence of collagen I. Among these, genes for several growth factors, cytokines, and chemokines (e.g., IL-8 and macrophage inhibitory protein 1alpha) could be confirmed using quantitative polymerase chain reaction. Furthermore, greater expression levels of the negative cell-cycle regulator BTG2/TIS21 and an inhibitor of the mitogen-activated protein kinase pathway, DUSP2, underline the regulatory role of the extracellular matrix. Together, these data show that the expansion of CD34+ cord blood cells in a culture system containing a three-dimensional collagen I matrix induces a qualitative change in the gene-expression profile of cultivated HSCs.
Oxidized phospholipids are thought to promote atherogenesis by stimulating endothelial cells (ECs) to produce inflammatory cytokines, such as IL-8. In studies with mouse models, we previously demonstrated that genetic variation in inflammatory responses of endothelial cells to oxidized lipids contributes importantly to atherosclerosis susceptibility. We now show that similar variations occur in cultured aortic ECs derived from multiple heart transplant donors. These variations were stably maintained between passages and, thus, reflect either genetic or epigenetic regulatory differences. Expression array analysis of aortic EC cultures derived from 12 individuals revealed that >1,000 genes were regulated by oxidized phospholipids. We have used the observed variations in the sampled population to construct a gene coexpression network comprised of 15 modules of highly connected genes. We show that several identified modules are significantly enriched in genes for known pathways and confirm a module enriched for unfolded protein response (UPR) genes using siRNA and the UPR inducer tunicamycin. On the basis of the constructed network, we predicted that a gene of unknown function (MGC4504) present in the UPR module is a target for UPR transcriptional activator ATF4. Our data also indicate that IL-8 is present in the UPR module and is regulated, in part, by the UPR. We validate these by using siRNA. In conclusion, we show that interindividual variability can be used to group genes into pathways and predict gene-gene regulatory relationships, thus identifying targets potentially involved in susceptibility to common diseases such as atherosclerosis.
Oxidized phospholipids are thought to promote atherogenesis by stimulating endothelial cells (ECs) to produce inflammatory cytokines, such as IL-8. In studies with mouse models, we previously demonstrated that genetic variation in inflammatory responses of endothelial cells to oxidized lipids contributes importantly to atherosclerosis susceptibility. We now show that similar variations occur in cultured aortic ECs derived from multiple heart transplant donors. These variations were stably maintained between passages and, thus, reflect either genetic or epigenetic regulatory differences. Expression array analysis of aortic EC cultures derived from 12 individuals revealed that >1,000 genes were regulated by oxidized phospholipids. We have used the observed variations in the sampled population to construct a gene coexpression network comprised of 15 modules of highly connected genes. We show that several identified modules are significantly enriched in genes for known pathways and confirm a module enriched for unfolded protein response (UPR) genes using siRNA and the UPR inducer tunicamycin. On the basis of the constructed network, we predicted that a gene of unknown function (MGC4504) present in the UPR module is a target for UPR transcriptional activator ATF4. Our data also indicate that IL-8 is present in the UPR module and is regulated, in part, by the UPR. We validate these by using siRNA. In conclusion, we show that interindividual variability can be used to group genes into pathways and predict gene-gene regulatory relationships, thus identifying targets potentially involved in susceptibility to common diseases such as atherosclerosis.
Oxidized phospholipids are thought to promote atherogenesis by stimulating endothelial cells (ECs) to produce inflammatory cytokines, such as IL-8. In studies with mouse models, we previously demonstrated that genetic variation in inflammatory responses of endothelial cells to oxidized lipids contributes importantly to atherosclerosis susceptibility. We now show that similar variations occur in cultured aortic ECs derived from multiple heart transplant donors. These variations were stably maintained between passages and, thus, reflect either genetic or epigenetic regulatory differences. Expression array analysis of aortic EC cultures derived from 12 individuals revealed that >1,000 genes were regulated by oxidized phospholipids. We have used the observed variations in the sampled population to construct a gene coexpression network comprised of 15 modules of highly connected genes. We show that several identified modules are significantly enriched in genes for known pathways and confirm a module enriched for unfolded protein response (UPR) genes using siRNA and the UPR inducer tunicamycin. On the basis of the constructed network, we predicted that a gene of unknown function (MGC4504) present in the UPR module is a target for UPR transcriptional activator ATF4. Our data also indicate that IL-8 is present in the UPR module and is regulated, in part, by the UPR. We validate these by using siRNA. In conclusion, we show that interindividual variability can be used to group genes into pathways and predict gene-gene regulatory relationships, thus identifying targets potentially involved in susceptibility to common diseases such as atherosclerosis.
Oxidized phospholipids are thought to promote atherogenesis by stimulating endothelial cells (ECs) to produce inflammatory cytokines, such as IL-8. In studies with mouse models, we previously demonstrated that genetic variation in inflammatory responses of endothelial cells to oxidized lipids contributes importantly to atherosclerosis susceptibility. We now show that similar variations occur in cultured aortic ECs derived from multiple heart transplant donors. These variations were stably maintained between passages and, thus, reflect either genetic or epigenetic regulatory differences. Expression array analysis of aortic EC cultures derived from 12 individuals revealed that >1,000 genes were regulated by oxidized phospholipids. We have used the observed variations in the sampled population to construct a gene coexpression network comprised of 15 modules of highly connected genes. We show that several identified modules are significantly enriched in genes for known pathways and confirm a module enriched for unfolded protein response (UPR) genes using siRNA and the UPR inducer tunicamycin. On the basis of the constructed network, we predicted that a gene of unknown function (MGC4504) present in the UPR module is a target for UPR transcriptional activator ATF4. Our data also indicate that IL-8 is present in the UPR module and is regulated, in part, by the UPR. We validate these by using siRNA. In conclusion, we show that interindividual variability can be used to group genes into pathways and predict gene-gene regulatory relationships, thus identifying targets potentially involved in susceptibility to common diseases such as atherosclerosis.
Oxidized phospholipids are thought to promote atherogenesis by stimulating endothelial cells (ECs) to produce inflammatory cytokines, such as IL-8. In studies with mouse models, we previously demonstrated that genetic variation in inflammatory responses of endothelial cells to oxidized lipids contributes importantly to atherosclerosis susceptibility. We now show that similar variations occur in cultured aortic ECs derived from multiple heart transplant donors. These variations were stably maintained between passages and, thus, reflect either genetic or epigenetic regulatory differences. Expression array analysis of aortic EC cultures derived from 12 individuals revealed that >1,000 genes were regulated by oxidized phospholipids. We have used the observed variations in the sampled population to construct a gene coexpression network comprised of 15 modules of highly connected genes. We show that several identified modules are significantly enriched in genes for known pathways and confirm a module enriched for unfolded protein response (UPR) genes using siRNA and the UPR inducer tunicamycin. On the basis of the constructed network, we predicted that a gene of unknown function (MGC4504) present in the UPR module is a target for UPR transcriptional activator ATF4. Our data also indicate that IL-8 is present in the UPR module and is regulated, in part, by the UPR. We validate these by using siRNA. In conclusion, we show that interindividual variability can be used to group genes into pathways and predict gene-gene regulatory relationships, thus identifying targets potentially involved in susceptibility to common diseases such as atherosclerosis.
Oxidized phospholipids are thought to promote atherogenesis by stimulating endothelial cells (ECs) to produce inflammatory cytokines, such as IL-8. In studies with mouse models, we previously demonstrated that genetic variation in inflammatory responses of endothelial cells to oxidized lipids contributes importantly to atherosclerosis susceptibility. We now show that similar variations occur in cultured aortic ECs derived from multiple heart transplant donors. These variations were stably maintained between passages and, thus, reflect either genetic or epigenetic regulatory differences. Expression array analysis of aortic EC cultures derived from 12 individuals revealed that >1,000 genes were regulated by oxidized phospholipids. We have used the observed variations in the sampled population to construct a gene coexpression network comprised of 15 modules of highly connected genes. We show that several identified modules are significantly enriched in genes for known pathways and confirm a module enriched for unfolded protein response (UPR) genes using siRNA and the UPR inducer tunicamycin. On the basis of the constructed network, we predicted that a gene of unknown function (MGC4504) present in the UPR module is a target for UPR transcriptional activator ATF4. Our data also indicate that IL-8 is present in the UPR module and is regulated, in part, by the UPR. We validate these by using siRNA. In conclusion, we show that interindividual variability can be used to group genes into pathways and predict gene-gene regulatory relationships, thus identifying targets potentially involved in susceptibility to common diseases such as atherosclerosis.
Oxidized phospholipids are thought to promote atherogenesis by stimulating endothelial cells (ECs) to produce inflammatory cytokines, such as IL-8. In studies with mouse models, we previously demonstrated that genetic variation in inflammatory responses of endothelial cells to oxidized lipids contributes importantly to atherosclerosis susceptibility. We now show that similar variations occur in cultured aortic ECs derived from multiple heart transplant donors. These variations were stably maintained between passages and, thus, reflect either genetic or epigenetic regulatory differences. Expression array analysis of aortic EC cultures derived from 12 individuals revealed that >1,000 genes were regulated by oxidized phospholipids. We have used the observed variations in the sampled population to construct a gene coexpression network comprised of 15 modules of highly connected genes. We show that several identified modules are significantly enriched in genes for known pathways and confirm a module enriched for unfolded protein response (UPR) genes using siRNA and the UPR inducer tunicamycin. On the basis of the constructed network, we predicted that a gene of unknown function (MGC4504) present in the UPR module is a target for UPR transcriptional activator ATF4. Our data also indicate that IL-8 is present in the UPR module and is regulated, in part, by the UPR. We validate these by using siRNA. In conclusion, we show that interindividual variability can be used to group genes into pathways and predict gene-gene regulatory relationships, thus identifying targets potentially involved in susceptibility to common diseases such as atherosclerosis.
Oxidized phospholipids are thought to promote atherogenesis by stimulating endothelial cells (ECs) to produce inflammatory cytokines, such as IL-8. In studies with mouse models, we previously demonstrated that genetic variation in inflammatory responses of endothelial cells to oxidized lipids contributes importantly to atherosclerosis susceptibility. We now show that similar variations occur in cultured aortic ECs derived from multiple heart transplant donors. These variations were stably maintained between passages and, thus, reflect either genetic or epigenetic regulatory differences. Expression array analysis of aortic EC cultures derived from 12 individuals revealed that >1,000 genes were regulated by oxidized phospholipids. We have used the observed variations in the sampled population to construct a gene coexpression network comprised of 15 modules of highly connected genes. We show that several identified modules are significantly enriched in genes for known pathways and confirm a module enriched for unfolded protein response (UPR) genes using siRNA and the UPR inducer tunicamycin. On the basis of the constructed network, we predicted that a gene of unknown function (MGC4504) present in the UPR module is a target for UPR transcriptional activator ATF4. Our data also indicate that IL-8 is present in the UPR module and is regulated, in part, by the UPR. We validate these by using siRNA. In conclusion, we show that interindividual variability can be used to group genes into pathways and predict gene-gene regulatory relationships, thus identifying targets potentially involved in susceptibility to common diseases such as atherosclerosis.
Oxidized phospholipids are thought to promote atherogenesis by stimulating endothelial cells (ECs) to produce inflammatory cytokines, such as IL-8. In studies with mouse models, we previously demonstrated that genetic variation in inflammatory responses of endothelial cells to oxidized lipids contributes importantly to atherosclerosis susceptibility. We now show that similar variations occur in cultured aortic ECs derived from multiple heart transplant donors. These variations were stably maintained between passages and, thus, reflect either genetic or epigenetic regulatory differences. Expression array analysis of aortic EC cultures derived from 12 individuals revealed that >1,000 genes were regulated by oxidized phospholipids. We have used the observed variations in the sampled population to construct a gene coexpression network comprised of 15 modules of highly connected genes. We show that several identified modules are significantly enriched in genes for known pathways and confirm a module enriched for unfolded protein response (UPR) genes using siRNA and the UPR inducer tunicamycin. On the basis of the constructed network, we predicted that a gene of unknown function (MGC4504) present in the UPR module is a target for UPR transcriptional activator ATF4. Our data also indicate that IL-8 is present in the UPR module and is regulated, in part, by the UPR. We validate these by using siRNA. In conclusion, we show that interindividual variability can be used to group genes into pathways and predict gene-gene regulatory relationships, thus identifying targets potentially involved in susceptibility to common diseases such as atherosclerosis.
Oxidized phospholipids are thought to promote atherogenesis by stimulating endothelial cells (ECs) to produce inflammatory cytokines, such as IL-8. In studies with mouse models, we previously demonstrated that genetic variation in inflammatory responses of endothelial cells to oxidized lipids contributes importantly to atherosclerosis susceptibility. We now show that similar variations occur in cultured aortic ECs derived from multiple heart transplant donors. These variations were stably maintained between passages and, thus, reflect either genetic or epigenetic regulatory differences. Expression array analysis of aortic EC cultures derived from 12 individuals revealed that >1,000 genes were regulated by oxidized phospholipids. We have used the observed variations in the sampled population to construct a gene coexpression network comprised of 15 modules of highly connected genes. We show that several identified modules are significantly enriched in genes for known pathways and confirm a module enriched for unfolded protein response (UPR) genes using siRNA and the UPR inducer tunicamycin. On the basis of the constructed network, we predicted that a gene of unknown function (MGC4504) present in the UPR module is a target for UPR transcriptional activator ATF4. Our data also indicate that IL-8 is present in the UPR module and is regulated, in part, by the UPR. We validate these by using siRNA. In conclusion, we show that interindividual variability can be used to group genes into pathways and predict gene-gene regulatory relationships, thus identifying targets potentially involved in susceptibility to common diseases such as atherosclerosis.
Oxidized phospholipids are thought to promote atherogenesis by stimulating endothelial cells (ECs) to produce inflammatory cytokines, such as IL-8. An important mechanism contributing to the recruitment of inflammatory cells in atherosclerosis is the induction of adhesion molecules, growth factors, and cytokines in vascular ECs by oxidized phospholipids, such as oxidized 1-palmitoyl-2- arachidonyl-sn-3-glycero-phosphorylcholine (oxPAPC) derived from lipoproteins trapped in the vessel wall. we profiled global mRNA expression levels in HAEC cultures derived from 12 individual donors, where each culture was treated with either unoxidized PAPC (40 gml) as control or with oxPAPC (40 gml) for 4 h. Each condition was profiled in duplicate, so that altogether 48 arrays were examined. A gene coexpression network was constructed. The resulting gene coexpression network was composed of 1043 oxPAPC-regulated genes separated into 15 modules of highly correlated genes. Each module was assigned a unique color identifier (Fig. 2A), with the remaining, poorly connected genes colored gray. This gene set corresponds to the green module.
Oxidized phospholipids are thought to promote atherogenesis by stimulating endothelial cells (ECs) to produce inflammatory cytokines, such as IL-8. An important mechanism contributing to the recruitment of inflammatory cells in atherosclerosis is the induction of adhesion molecules, growth factors, and cytokines in vascular ECs by oxidized phospholipids, such as oxidized 1-palmitoyl-2- arachidonyl-sn-3-glycero-phosphorylcholine (oxPAPC) derived from lipoproteins trapped in the vessel wall. we profiled global mRNA expression levels in HAEC cultures derived from 12 individual donors, where each culture was treated with either unoxidized PAPC (40 gml) as control or with oxPAPC (40 gml) for 4 h. Each condition was profiled in duplicate, so that altogether 48 arrays were examined. A gene coexpression network was constructed. The resulting gene coexpression network was composed of 1043 oxPAPC-regulated genes separated into 15 modules of highly correlated genes. Each module was assigned a unique color identifier (Fig. 2A), with the remaining, poorly connected genes colored gray. This gene set corresponds to the green module.
Oxidized phospholipids are thought to promote atherogenesis by stimulating endothelial cells (ECs) to produce inflammatory cytokines, such as IL-8. An important mechanism contributing to the recruitment of inflammatory cells in atherosclerosis is the induction of adhesion molecules, growth factors, and cytokines in vascular ECs by oxidized phospholipids, such as oxidized 1-palmitoyl-2- arachidonyl-sn-3-glycero-phosphorylcholine (oxPAPC) derived from lipoproteins trapped in the vessel wall. we profiled global mRNA expression levels in HAEC cultures derived from 12 individual donors, where each culture was treated with either unoxidized PAPC (40 gml) as control or with oxPAPC (40 gml) for 4 h. Each condition was profiled in duplicate, so that altogether 48 arrays were examined. A gene coexpression network was constructed. The resulting gene coexpression network was composed of 1043 oxPAPC-regulated genes separated into 15 modules of highly correlated genes. Each module was assigned a unique color identifier (Fig. 2A), with the remaining, poorly connected genes colored gray. This gene set corresponds to the blue module.
Oxidized phospholipids are thought to promote atherogenesis by stimulating endothelial cells (ECs) to produce inflammatory cytokines, such as IL-8. An important mechanism contributing to the recruitment of inflammatory cells in atherosclerosis is the induction of adhesion molecules, growth factors, and cytokines in vascular ECs by oxidized phospholipids, such as oxidized 1-palmitoyl-2- arachidonyl-sn-3-glycero-phosphorylcholine (oxPAPC) derived from lipoproteins trapped in the vessel wall. we profiled global mRNA expression levels in HAEC cultures derived from 12 individual donors, where each culture was treated with either unoxidized PAPC (40 gml) as control or with oxPAPC (40 gml) for 4 h. Each condition was profiled in duplicate, so that altogether 48 arrays were examined. A gene coexpression network was constructed. The resulting gene coexpression network was composed of 1043 oxPAPC-regulated genes separated into 15 modules of highly correlated genes. Each module was assigned a unique color identifier (Fig. 2A), with the remaining, poorly connected genes colored gray. This gene set corresponds to the blue module.
Oxidized phospholipids are thought to promote atherogenesis by stimulating endothelial cells (ECs) to produce inflammatory cytokines, such as IL-8. In studies with mouse models, we previously demonstrated that genetic variation in inflammatory responses of endothelial cells to oxidized lipids contributes importantly to atherosclerosis susceptibility. We now show that similar variations occur in cultured aortic ECs derived from multiple heart transplant donors. These variations were stably maintained between passages and, thus, reflect either genetic or epigenetic regulatory differences. Expression array analysis of aortic EC cultures derived from 12 individuals revealed that >1,000 genes were regulated by oxidized phospholipids. We have used the observed variations in the sampled population to construct a gene coexpression network comprised of 15 modules of highly connected genes. We show that several identified modules are significantly enriched in genes for known pathways and confirm a module enriched for unfolded protein response (UPR) genes using siRNA and the UPR inducer tunicamycin. On the basis of the constructed network, we predicted that a gene of unknown function (MGC4504) present in the UPR module is a target for UPR transcriptional activator ATF4. Our data also indicate that IL-8 is present in the UPR module and is regulated, in part, by the UPR. We validate these by using siRNA. In conclusion, we show that interindividual variability can be used to group genes into pathways and predict gene-gene regulatory relationships, thus identifying targets potentially involved in susceptibility to common diseases such as atherosclerosis.
Oxidized phospholipids are thought to promote atherogenesis by stimulating endothelial cells (ECs) to produce inflammatory cytokines, such as IL-8. In studies with mouse models, we previously demonstrated that genetic variation in inflammatory responses of endothelial cells to oxidized lipids contributes importantly to atherosclerosis susceptibility. We now show that similar variations occur in cultured aortic ECs derived from multiple heart transplant donors. These variations were stably maintained between passages and, thus, reflect either genetic or epigenetic regulatory differences. Expression array analysis of aortic EC cultures derived from 12 individuals revealed that >1,000 genes were regulated by oxidized phospholipids. We have used the observed variations in the sampled population to construct a gene coexpression network comprised of 15 modules of highly connected genes. We show that several identified modules are significantly enriched in genes for known pathways and confirm a module enriched for unfolded protein response (UPR) genes using siRNA and the UPR inducer tunicamycin. On the basis of the constructed network, we predicted that a gene of unknown function (MGC4504) present in the UPR module is a target for UPR transcriptional activator ATF4. Our data also indicate that IL-8 is present in the UPR module and is regulated, in part, by the UPR. We validate these by using siRNA. In conclusion, we show that interindividual variability can be used to group genes into pathways and predict gene-gene regulatory relationships, thus identifying targets potentially involved in susceptibility to common diseases such as atherosclerosis.
Oxidized phospholipids are thought to promote atherogenesis by stimulating endothelial cells (ECs) to produce inflammatory cytokines, such as IL-8. In studies with mouse models, we previously demonstrated that genetic variation in inflammatory responses of endothelial cells to oxidized lipids contributes importantly to atherosclerosis susceptibility. We now show that similar variations occur in cultured aortic ECs derived from multiple heart transplant donors. These variations were stably maintained between passages and, thus, reflect either genetic or epigenetic regulatory differences. Expression array analysis of aortic EC cultures derived from 12 individuals revealed that >1,000 genes were regulated by oxidized phospholipids. We have used the observed variations in the sampled population to construct a gene coexpression network comprised of 15 modules of highly connected genes. We show that several identified modules are significantly enriched in genes for known pathways and confirm a module enriched for unfolded protein response (UPR) genes using siRNA and the UPR inducer tunicamycin. On the basis of the constructed network, we predicted that a gene of unknown function (MGC4504) present in the UPR module is a target for UPR transcriptional activator ATF4. Our data also indicate that IL-8 is present in the UPR module and is regulated, in part, by the UPR. We validate these by using siRNA. In conclusion, we show that interindividual variability can be used to group genes into pathways and predict gene-gene regulatory relationships, thus identifying targets potentially involved in susceptibility to common diseases such as atherosclerosis.
Oxidized phospholipids are thought to promote atherogenesis by stimulating endothelial cells (ECs) to produce inflammatory cytokines, such as IL-8. In studies with mouse models, we previously demonstrated that genetic variation in inflammatory responses of endothelial cells to oxidized lipids contributes importantly to atherosclerosis susceptibility. We now show that similar variations occur in cultured aortic ECs derived from multiple heart transplant donors. These variations were stably maintained between passages and, thus, reflect either genetic or epigenetic regulatory differences. Expression array analysis of aortic EC cultures derived from 12 individuals revealed that >1,000 genes were regulated by oxidized phospholipids. We have used the observed variations in the sampled population to construct a gene coexpression network comprised of 15 modules of highly connected genes. We show that several identified modules are significantly enriched in genes for known pathways and confirm a module enriched for unfolded protein response (UPR) genes using siRNA and the UPR inducer tunicamycin. On the basis of the constructed network, we predicted that a gene of unknown function (MGC4504) present in the UPR module is a target for UPR transcriptional activator ATF4. Our data also indicate that IL-8 is present in the UPR module and is regulated, in part, by the UPR. We validate these by using siRNA. In conclusion, we show that interindividual variability can be used to group genes into pathways and predict gene-gene regulatory relationships, thus identifying targets potentially involved in susceptibility to common diseases such as atherosclerosis.
Highly quantitative biomarkers of neurodegenerative disease remain an important need in the urgent quest for disease-modifying therapies. For Huntington's disease (HD), a genetic test is available (trait marker), but necessary state markers are still in development. In this report, we describe a large battery of transcriptomic tests explored as state biomarker candidates. In an attempt to exploit the known neuroinflammatory and transcriptional perturbations of disease, we measured relevant mRNAs in peripheral blood cells. The performance of these potential markers was weak overall, with only one mRNA, immediate early response 3 (IER3), showing a modest but significant increase of 32% in HD samples compared with controls. No statistically significant differences were found for any other mRNAs tested, including a panel of 12 RNA biomarkers identified in a previous report [Borovecki F, Lovrecic L, Zhou J, Jeong H, Then F, Rosas HD, Hersch SM, Hogarth P, Bouzou B, Jensen RV, et al. (2005) Proc Natl Acad Sci USA 102:11023-11028]. The present results may nonetheless inform the future design and testing of HD biomarker strategies.
Highly quantitative biomarkers of neurodegenerative disease remain an important need in the urgent quest for disease-modifying therapies. For Huntington's disease (HD), a genetic test is available (trait marker), but necessary state markers are still in development. In this report, we describe a large battery of transcriptomic tests explored as state biomarker candidates. In an attempt to exploit the known neuroinflammatory and transcriptional perturbations of disease, we measured relevant mRNAs in peripheral blood cells. The performance of these potential markers was weak overall, with only one mRNA, immediate early response 3 (IER3), showing a modest but significant increase of 32% in HD samples compared with controls. No statistically significant differences were found for any other mRNAs tested, including a panel of 12 RNA biomarkers identified in a previous report [Borovecki F, Lovrecic L, Zhou J, Jeong H, Then F, Rosas HD, Hersch SM, Hogarth P, Bouzou B, Jensen RV, et al. (2005) Proc Natl Acad Sci USA 102:11023-11028]. The present results may nonetheless inform the future design and testing of HD biomarker strategies.
NUP98-HOXA9, the chimeric protein resulting from the t(7;11)(p15;p15) chromosomal translocation, is a prototype of several NUP98 fusions that occur in myelodysplastic syndromes and acute myeloid leukemia. Here we identify the effects of NUP98-HOXA9 on primary human hematopoietic progenitor/stem cells and the changes in gene expression that underlie those effects. To study the role of NUP98-HOXA9 in human leukemogenesis, human CD34+ hematopoietic progenitors from mobilized peripheral blood of two patients were retrovirally transduced to express the fusion protein.
NUP98-HOXA9, the chimeric protein resulting from the t(7;11)(p15;p15) chromosomal translocation, is a prototype of several NUP98 fusions that occur in myelodysplastic syndromes and acute myeloid leukemia. Here we identify the effects of NUP98-HOXA9 on primary human hematopoietic progenitor/stem cells and the changes in gene expression that underlie those effects. To study the role of NUP98-HOXA9 in human leukemogenesis, human CD34+ hematopoietic progenitors from mobilized peripheral blood of two patients were retrovirally transduced to express the fusion protein.
NUP98-HOXA9, the chimeric protein resulting from the t(7;11)(p15;p15) chromosomal translocation, is a prototype of several NUP98 fusions that occur in myelodysplastic syndromes and acute myeloid leukemia. Here we identify the effects of NUP98-HOXA9 on primary human hematopoietic progenitor/stem cells and the changes in gene expression that underlie those effects. To study the role of NUP98-HOXA9 in human leukemogenesis, human CD34+ hematopoietic progenitors from mobilized peripheral blood of two patients were retrovirally transduced to express the fusion protein. This set contains genes differentially expressed 3 days after viral transduction.
NUP98-HOXA9, the chimeric protein resulting from the t(7;11)(p15;p15) chromosomal translocation, is a prototype of several NUP98 fusions that occur in myelodysplastic syndromes and acute myeloid leukemia. Here we identify the effects of NUP98-HOXA9 on primary human hematopoietic progenitor/stem cells and the changes in gene expression that underlie those effects. To study the role of NUP98-HOXA9 in human leukemogenesis, human CD34+ hematopoietic progenitors from mobilized peripheral blood of two patients were retrovirally transduced to express the fusion protein. This set contains genes differentially expressed 3 days after viral transduction.
NUP98-HOXA9, the chimeric protein resulting from the t(7;11)(p15;p15) chromosomal translocation, is a prototype of several NUP98 fusions that occur in myelodysplastic syndromes and acute myeloid leukemia. Here we identify the effects of NUP98-HOXA9 on primary human hematopoietic progenitor/stem cells and the changes in gene expression that underlie those effects. To study the role of NUP98-HOXA9 in human leukemogenesis, human CD34+ hematopoietic progenitors from mobilized peripheral blood of two patients were retrovirally transduced to express the fusion protein. This set contains genes differentially expressed 8 days after viral transduction.
NUP98-HOXA9, the chimeric protein resulting from the t(7;11)(p15;p15) chromosomal translocation, is a prototype of several NUP98 fusions that occur in myelodysplastic syndromes and acute myeloid leukemia. Here we identify the effects of NUP98-HOXA9 on primary human hematopoietic progenitor/stem cells and the changes in gene expression that underlie those effects. To study the role of NUP98-HOXA9 in human leukemogenesis, human CD34+ hematopoietic progenitors from mobilized peripheral blood of two patients were retrovirally transduced to express the fusion protein. This set contains genes differentially expressed 8 days after viral transduction.
NUP98-HOXA9, the chimeric protein resulting from the t(7;11)(p15;p15) chromosomal translocation, is a prototype of several NUP98 fusions that occur in myelodysplastic syndromes and acute myeloid leukemia. Here we identify the effects of NUP98-HOXA9 on primary human hematopoietic progenitor/stem cells and the changes in gene expression that underlie those effects. To study the role of NUP98-HOXA9 in human leukemogenesis, human CD34+ hematopoietic progenitors from mobilized peripheral blood of two patients were retrovirally transduced to express the fusion protein. This set contains genes differentially expressed 10 days after viral transduction.
NUP98-HOXA9, the chimeric protein resulting from the t(7;11)(p15;p15) chromosomal translocation, is a prototype of several NUP98 fusions that occur in myelodysplastic syndromes and acute myeloid leukemia. Here we identify the effects of NUP98-HOXA9 on primary human hematopoietic progenitor/stem cells and the changes in gene expression that underlie those effects. To study the role of NUP98-HOXA9 in human leukemogenesis, human CD34+ hematopoietic progenitors from mobilized peripheral blood of two patients were retrovirally transduced to express the fusion protein. This set contains genes differentially expressed 10 days after viral transduction.
NUP98-HOXA9, the chimeric protein resulting from the t(7;11)(p15;p15) chromosomal translocation, is a prototype of several NUP98 fusions that occur in myelodysplastic syndromes and acute myeloid leukemia. Here we identify the effects of NUP98-HOXA9 on primary human hematopoietic progenitor/stem cells and the changes in gene expression that underlie those effects. To study the role of NUP98-HOXA9 in human leukemogenesis, human CD34+ hematopoietic progenitors from mobilized peripheral blood of two patients were retrovirally transduced to express the fusion protein. This set contains genes differentially expressed 16 days after viral transduction.
NUP98-HOXA9, the chimeric protein resulting from the t(7;11)(p15;p15) chromosomal translocation, is a prototype of several NUP98 fusions that occur in myelodysplastic syndromes and acute myeloid leukemia. Here we identify the effects of NUP98-HOXA9 on primary human hematopoietic progenitor/stem cells and the changes in gene expression that underlie those effects. To study the role of NUP98-HOXA9 in human leukemogenesis, human CD34+ hematopoietic progenitors from mobilized peripheral blood of two patients were retrovirally transduced to express the fusion protein. This set contains genes differentially expressed 16 days after viral transduction.
Most gene expression profiling studies of mesothelioma have been based on relatively small sample numbers, limiting their statistical power. We did Affymetrix U133A microarray analysis on 99 pleural mesotheliomas, in which multivariate analysis showed advanced-stage, sarcomatous histology and P16/CDKN2A homozygous deletion to be significant independent adverse prognostic factors. Comparison of the expression profiles of epithelioid versus sarcomatous mesotheliomas identified many genes significantly overexpressed among the former, including previously unrecognized ones, such as uroplakins and kallikrein 11, both confirmed by immunohistochemistry. Examination of the gene expression correlates of survival showed that more aggressive mesotheliomas expressed higher levels of Aurora kinases A and B and functionally related genes involved in mitosis and cell cycle control. Independent confirmation of the negative effect of Aurora kinase B was obtained by immunohistochemistry in a separate patient cohort. A role for Aurora kinases in the aggressive behavior of mesotheliomas is of potential clinical interest because of the recent development of small-molecule inhibitors. We then used our data to develop microarray-based predictors of 1 year survival; these achieved a maximal accuracy of 68% in cross-validation. However, this was inferior to prognostic prediction based on standard clinicopathologic variables and P16/CDNK2A status (accuracy, 73%), and adding the microarray model to the latter did not improve overall accuracy. Finally, we evaluated three recently published microarray-based outcome prediction models, but their accuracies ranged from 63% to 67%, consistently lower than reported. Gene expression profiling of mesotheliomas is an important discovery tool, but its power in clinical prognostication has been overestimated.
Most gene expression profiling studies of mesothelioma have been based on relatively small sample numbers, limiting their statistical power. We did Affymetrix U133A microarray analysis on 99 pleural mesotheliomas, in which multivariate analysis showed advanced-stage, sarcomatous histology and P16/CDKN2A homozygous deletion to be significant independent adverse prognostic factors. Comparison of the expression profiles of epithelioid versus sarcomatous mesotheliomas identified many genes significantly overexpressed among the former, including previously unrecognized ones, such as uroplakins and kallikrein 11, both confirmed by immunohistochemistry. Examination of the gene expression correlates of survival showed that more aggressive mesotheliomas expressed higher levels of Aurora kinases A and B and functionally related genes involved in mitosis and cell cycle control. Independent confirmation of the negative effect of Aurora kinase B was obtained by immunohistochemistry in a separate patient cohort. A role for Aurora kinases in the aggressive behavior of mesotheliomas is of potential clinical interest because of the recent development of small-molecule inhibitors. We then used our data to develop microarray-based predictors of 1 year survival; these achieved a maximal accuracy of 68% in cross-validation. However, this was inferior to prognostic prediction based on standard clinicopathologic variables and P16/CDNK2A status (accuracy, 73%), and adding the microarray model to the latter did not improve overall accuracy. Finally, we evaluated three recently published microarray-based outcome prediction models, but their accuracies ranged from 63% to 67%, consistently lower than reported. Gene expression profiling of mesotheliomas is an important discovery tool, but its power in clinical prognostication has been overestimated.
Using a target gene approach, only a few host genetic risk factors for treatment-related myeloid leukemia (t-ML) have been defined. Gene expression microarrays allow for a more genome-wide approach to assess possible genetic risk factors for t-ML. We assessed gene expression profiles (n=12 625 probe sets) in diagnostic acute lymphoblastic leukemic cells from 228 children treated on protocols that included leukemogenic agents such as etoposide, 13 of whom developed t-ML. Expression of 68 probes, corresponding to 63 genes, was significantly related to risk of t-ML. Hierarchical clustering of these probe sets clustered patients into three groups with 94, 122 and 12 patients, respectively; 12 of the 13 patients who went on to develop t-ML were overrepresented in the latter group (P<0.0001). A permutation test indicated a low likelihood that these probe sets and clusters were obtained by chance (P<0.001). Distinguishing genes included transcription-related oncogenes (v-Myb, Pax-5), cyclins (CCNG1, CCNG2 and CCND1) and histone HIST1H4C. Common transcription factor recognition elements among similarly up- or downregulated genes included several involved in hematopoietic differentiation or leukemogenesis (Maz, PU.1, ARNT). This approach has identified several genes whose expression distinguishes patients at risk of t-ML, and suggests targets for assessing germline predisposition to leukemogenesis.
Using a target gene approach, only a few host genetic risk factors for treatment-related myeloid leukemia (t-ML) have been defined. Gene expression microarrays allow for a more genome-wide approach to assess possible genetic risk factors for t-ML. We assessed gene expression profiles (n=12 625 probe sets) in diagnostic acute lymphoblastic leukemic cells from 228 children treated on protocols that included leukemogenic agents such as etoposide, 13 of whom developed t-ML. Expression of 68 probes, corresponding to 63 genes, was significantly related to risk of t-ML. Hierarchical clustering of these probe sets clustered patients into three groups with 94, 122 and 12 patients, respectively; 12 of the 13 patients who went on to develop t-ML were overrepresented in the latter group (P<0.0001). A permutation test indicated a low likelihood that these probe sets and clusters were obtained by chance (P<0.001). Distinguishing genes included transcription-related oncogenes (v-Myb, Pax-5), cyclins (CCNG1, CCNG2 and CCND1) and histone HIST1H4C. Common transcription factor recognition elements among similarly up- or downregulated genes included several involved in hematopoietic differentiation or leukemogenesis (Maz, PU.1, ARNT). This approach has identified several genes whose expression distinguishes patients at risk of t-ML, and suggests targets for assessing germline predisposition to leukemogenesis.
Lipoprotein lipase (LPL) is a prognostic marker in B-cell chronic lymphocytic leukemia (B-CLL) related to immunoglobulin V(H) gene (IgV(H))mutational status. We determined gene expression profiles using Affymetrix U133A GeneChips in two groups of B-CLLs selected for either high ('LPL+', n=10) or low ('LPL-', n=10) LPL mRNA expression. Selected genes were verified by real-time PCR in an extended patient cohort (n=42). A total of 111 genes discriminated LPL+ from LPL- B-CLLs. Of these, the top three genes associated with time to first treatment were Septin10, DMD and Gravin (P</=0.01). The relationship of LPL+ and LPL- B-CLL gene expression signatures to 52 tissues was statistically analyzed. The LPL+ B-CLL expression signature, represented by 64 genes was significantly related to fat, muscle and PB dendritic cells (P<0.001). Exploration of microarray data to define functional alterations related to the biology of LPL+ CLL identified two functional modules, fatty acid degradation and MTA3 signaling, as being altered with higher statistical significance. Our data show that LPL+ B-CLL cells have not only acquired gene expression changes in fat and muscle-associated genes but also in functional pathways related to fatty acid degradation and signaling which may ultimately influence CLL biology and clinical outcome.
Lipoprotein lipase (LPL) is a prognostic marker in B-cell chronic lymphocytic leukemia (B-CLL) related to immunoglobulin V(H) gene (IgV(H))mutational status. We determined gene expression profiles using Affymetrix U133A GeneChips in two groups of B-CLLs selected for either high ('LPL+', n=10) or low ('LPL-', n=10) LPL mRNA expression. Selected genes were verified by real-time PCR in an extended patient cohort (n=42). A total of 111 genes discriminated LPL+ from LPL- B-CLLs. Of these, the top three genes associated with time to first treatment were Septin10, DMD and Gravin (P</=0.01). The relationship of LPL+ and LPL- B-CLL gene expression signatures to 52 tissues was statistically analyzed. The LPL+ B-CLL expression signature, represented by 64 genes was significantly related to fat, muscle and PB dendritic cells (P<0.001). Exploration of microarray data to define functional alterations related to the biology of LPL+ CLL identified two functional modules, fatty acid degradation and MTA3 signaling, as being altered with higher statistical significance. Our data show that LPL+ B-CLL cells have not only acquired gene expression changes in fat and muscle-associated genes but also in functional pathways related to fatty acid degradation and signaling which may ultimately influence CLL biology and clinical outcome.
Hematopoietic stem cells in the bone marrow (BM) give rise to all blood cells. According to the classic model of hematopoiesis, the differentiation paths leading to the myeloid and lymphoid lineages segregate early. A candidate 'common lymphoid progenitor' (CLP) has been isolated from CD34(+)CD38(-) human cord blood cells based on CD7 expression. Here, we confirm the B- and NK-differentiation potential of CD34(+)CD38(-)CD7(+) cells and show in addition that this population has strong capacity to differentiate into T cells. As CD34(+)CD38(-)CD7(+) cells are virtually devoid of myeloid differentiation potential, these cells represent true CLPs. To unravel the molecular mechanisms underlying lymphoid commitment, we performed genome-wide gene expression profiling on sorted CD34(+)CD38(-)CD7(+) and CD34(+)CD38(-)CD7(-) cells. Interestingly, lymphoid-affiliated genes were mainly upregulated in the CD7(+) population, while myeloid-specific genes were downregulated. This supports the hypothesis that lineage commitment is accompanied by the shutdown of inappropriate gene expression and the upregulation of lineage-specific genes. In addition, we identified several highly expressed genes that have not been described in hematopoiesis before.
Hematopoietic stem cells in the bone marrow (BM) give rise to all blood cells. According to the classic model of hematopoiesis, the differentiation paths leading to the myeloid and lymphoid lineages segregate early. A candidate 'common lymphoid progenitor' (CLP) has been isolated from CD34(+)CD38(-) human cord blood cells based on CD7 expression. Here, we confirm the B- and NK-differentiation potential of CD34(+)CD38(-)CD7(+) cells and show in addition that this population has strong capacity to differentiate into T cells. As CD34(+)CD38(-)CD7(+) cells are virtually devoid of myeloid differentiation potential, these cells represent true CLPs. To unravel the molecular mechanisms underlying lymphoid commitment, we performed genome-wide gene expression profiling on sorted CD34(+)CD38(-)CD7(+) and CD34(+)CD38(-)CD7(-) cells. Interestingly, lymphoid-affiliated genes were mainly upregulated in the CD7(+) population, while myeloid-specific genes were downregulated. This supports the hypothesis that lineage commitment is accompanied by the shutdown of inappropriate gene expression and the upregulation of lineage-specific genes. In addition, we identified several highly expressed genes that have not been described in hematopoiesis before.
CD38 expression is an important prognostic marker in chronic lymphocytic leukemia (CLL) with high levels of CD38 associated with shorter overall survival. In this study, we used gene expression profiling and protein analysis of highly purified cell-sorted CD38(+) and CD38(-) chronic lymphocytic leukemia cells to elucidate a molecular basis for the association between CD38 expression and inferior clinical outcome. Paired CD38(+) and CD38(-) CLL cells derived from the same patient were shown to be monoclonal by V(H) gene sequencing but despite this, CD38(+) CLL cells possessed a distinct gene expression profile when compared with their CD38(-) sub-clones. Importantly, CD38(+) CLL cells relatively over expressed vascular endothelial growth factor (VEGF) and appeared to preferentially utilize an internal autocrine VEGF survival loop. Elevated VEGF expression was associated with increased expression of the anti-apoptotic protein Mcl-1. Inhibition of VEGF receptor signaling also resulted in a reduction in cell viability. In contrast, exogenous VEGF caused a significant increase in CD38(-) CLL cell viability and a marked induction of Mcl-1; both effects were less obvious in CD38(+) CLL cells. Taken together, our data provide a biological rationale for the poor prognosis of CD38(+) CLL and indicate that both VEGF and Mcl-1 may prove to be useful therapeutic targets.
CD38 expression is an important prognostic marker in chronic lymphocytic leukemia (CLL) with high levels of CD38 associated with shorter overall survival. In this study, we used gene expression profiling and protein analysis of highly purified cell-sorted CD38(+) and CD38(-) chronic lymphocytic leukemia cells to elucidate a molecular basis for the association between CD38 expression and inferior clinical outcome. Paired CD38(+) and CD38(-) CLL cells derived from the same patient were shown to be monoclonal by V(H) gene sequencing but despite this, CD38(+) CLL cells possessed a distinct gene expression profile when compared with their CD38(-) sub-clones. Importantly, CD38(+) CLL cells relatively over expressed vascular endothelial growth factor (VEGF) and appeared to preferentially utilize an internal autocrine VEGF survival loop. Elevated VEGF expression was associated with increased expression of the anti-apoptotic protein Mcl-1. Inhibition of VEGF receptor signaling also resulted in a reduction in cell viability. In contrast, exogenous VEGF caused a significant increase in CD38(-) CLL cell viability and a marked induction of Mcl-1; both effects were less obvious in CD38(+) CLL cells. Taken together, our data provide a biological rationale for the poor prognosis of CD38(+) CLL and indicate that both VEGF and Mcl-1 may prove to be useful therapeutic targets.
Adult T-cell leukemia/lymphoma (ATLL) is a malignancy slowly emerging from human T-cell leukemia virus type 1 (HTLV-I)-infected mature CD4(+) T-cells. To characterize the molecular modifications induced by HTLV-I infection, we compared HTLV-I-infected WE17/10 cells with control cells, using micro-arrays. Many calcium-related genes were progressively downmodulated over a period of 2 years. Infected cells acquired a profound decrease of intracellular calcium levels in response to ionomycin, timely correlated with decreased CD7 expression. Focusing on apoptosis-related genes and their relationship with CD7, we observed an underexpression of most antiapoptotic genes. Western blotting revealed increasing Akt and Bad phosphorylation, timely correlated with CD7 loss. This was shown to be phosphatidylinositol 3-kinase (PI3K)-dependent. Activation of PI3K/Akt induced resistance to the apoptotic effect of interleukin-2 deprivation. We thus propose the following model: HTLV-I infection induces a progressive decrease in CD3 genes expression, which eventually abrogates CD3 expression; loss of CD3 is known to perturb calcium transport. This perturbation correlates with loss of CD7 expression and induction of Akt and Bad phosphorylation via activation of PI3K. The activation of the Akt/Bad pathway generates a progressive resistance to apoptosis, at a time HTLV-I genes expression is silenced, thus avoiding immune surveillance. This could be a major event in the process of the malignant transformation into ATLL.
Adult T-cell leukemia/lymphoma (ATLL) is a malignancy slowly emerging from human T-cell leukemia virus type 1 (HTLV-I)-infected mature CD4(+) T-cells. To characterize the molecular modifications induced by HTLV-I infection, we compared HTLV-I-infected WE17/10 cells with control cells, using micro-arrays. Many calcium-related genes were progressively downmodulated over a period of 2 years. Infected cells acquired a profound decrease of intracellular calcium levels in response to ionomycin, timely correlated with decreased CD7 expression. Focusing on apoptosis-related genes and their relationship with CD7, we observed an underexpression of most antiapoptotic genes. Western blotting revealed increasing Akt and Bad phosphorylation, timely correlated with CD7 loss. This was shown to be phosphatidylinositol 3-kinase (PI3K)-dependent. Activation of PI3K/Akt induced resistance to the apoptotic effect of interleukin-2 deprivation. We thus propose the following model: HTLV-I infection induces a progressive decrease in CD3 genes expression, which eventually abrogates CD3 expression; loss of CD3 is known to perturb calcium transport. This perturbation correlates with loss of CD7 expression and induction of Akt and Bad phosphorylation via activation of PI3K. The activation of the Akt/Bad pathway generates a progressive resistance to apoptosis, at a time HTLV-I genes expression is silenced, thus avoiding immune surveillance. This could be a major event in the process of the malignant transformation into ATLL.
In childhood acute lymphoblastic leukemia (ALL), persistence of leukemic blasts during therapy is of crucial prognostic significance. In the present study, we address molecular and cell biologic features of blasts persisting after 1 week of induction glucocorticoid therapy. Genome-wide gene expression analysis of leukemic samples from precursor B-cell ALL patients (n=18) identified a set of genes differentially expressed in blasts at diagnosis day 0 (d0) and persisting on day 8 (d8). Expression changes indicate a shift towards mature B cells, inhibition of cell cycling and increased expression of adhesion (CD11b/ITGAM) and cytokine (CD119/IFNGR1) receptors. A direct comparison with normal B cells, which are largely therapy resistant, confirmed the differentiation shift at the mRNA (n=10) and protein (n=109) levels. Flow cytometric analysis in independent cohorts of patients confirmed both a decreased proliferative activity (n=13) and the upregulation of CD11b and CD119 (n=29) in d8 blasts. The differentiation shift and low proliferative activity in d8 blasts may account for the persistence of blasts during therapy and affect their sensitivity to further therapeutic treatment. CD11b and CD119 are potential specific markers for d8 blast persistence and detection of minimal residual disease, which warrant further investigation.
In childhood acute lymphoblastic leukemia (ALL), persistence of leukemic blasts during therapy is of crucial prognostic significance. In the present study, we address molecular and cell biologic features of blasts persisting after 1 week of induction glucocorticoid therapy. Genome-wide gene expression analysis of leukemic samples from precursor B-cell ALL patients (n=18) identified a set of genes differentially expressed in blasts at diagnosis day 0 (d0) and persisting on day 8 (d8). Expression changes indicate a shift towards mature B cells, inhibition of cell cycling and increased expression of adhesion (CD11b/ITGAM) and cytokine (CD119/IFNGR1) receptors. A direct comparison with normal B cells, which are largely therapy resistant, confirmed the differentiation shift at the mRNA (n=10) and protein (n=109) levels. Flow cytometric analysis in independent cohorts of patients confirmed both a decreased proliferative activity (n=13) and the upregulation of CD11b and CD119 (n=29) in d8 blasts. The differentiation shift and low proliferative activity in d8 blasts may account for the persistence of blasts during therapy and affect their sensitivity to further therapeutic treatment. CD11b and CD119 are potential specific markers for d8 blast persistence and detection of minimal residual disease, which warrant further investigation.
Somatic mutations in nucleophosmin (NPM1) occur in approximately 35% of adult acute myeloid leukemia (AML). To assess the frequency of NPM1 mutations in pediatric AML, we sequenced NPM1 in the diagnostic blasts from 93 pediatric AML patients. Six cases harbored NPM1 mutations, with each case lacking common cytogenetic abnormalities. To explore the phenotype of the AMLs with NPM1 mutations, gene expression profiles were obtained using Affymetrix U133A microarrays. NPM1 mutations were associated with increased expression of multiple homeobox genes including HOXA9, A10, B2, B6 and MEIS1. As dysregulated homeobox gene expression is also a feature of MLL-rearranged leukemia, the gene expression signatures of NPM1-mutated and MLL-rearranged leukemias were compared. Significant differences were identified between these leukemia subtypes including the expression of different HOX genes, with NPM1-mutated AML showing higher levels of expression of HOXB2, B3, B6 and D4. These results confirm recent reports of perturbed HOX expression in NPM1-mutated adult AML, and provide the first evidence that the NPM1-mutated signature is distinct from MLL-rearranged AML. These findings suggest that mutated NPM1 leads to dysregulated HOX expression via a different mechanism than MLL rearrangement.
Somatic mutations in nucleophosmin (NPM1) occur in approximately 35% of adult acute myeloid leukemia (AML). To assess the frequency of NPM1 mutations in pediatric AML, we sequenced NPM1 in the diagnostic blasts from 93 pediatric AML patients. Six cases harbored NPM1 mutations, with each case lacking common cytogenetic abnormalities. To explore the phenotype of the AMLs with NPM1 mutations, gene expression profiles were obtained using Affymetrix U133A microarrays. NPM1 mutations were associated with increased expression of multiple homeobox genes including HOXA9, A10, B2, B6 and MEIS1. As dysregulated homeobox gene expression is also a feature of MLL-rearranged leukemia, the gene expression signatures of NPM1-mutated and MLL-rearranged leukemias were compared. Significant differences were identified between these leukemia subtypes including the expression of different HOX genes, with NPM1-mutated AML showing higher levels of expression of HOXB2, B3, B6 and D4. These results confirm recent reports of perturbed HOX expression in NPM1-mutated adult AML, and provide the first evidence that the NPM1-mutated signature is distinct from MLL-rearranged AML. These findings suggest that mutated NPM1 leads to dysregulated HOX expression via a different mechanism than MLL rearrangement.
Somatic mutations in nucleophosmin (NPM1) occur in approximately 35% of adult acute myeloid leukemia (AML). To assess the frequency of NPM1 mutations in pediatric AML, we sequenced NPM1 in the diagnostic blasts from 93 pediatric AML patients. Six cases harbored NPM1 mutations, with each case lacking common cytogenetic abnormalities. To explore the phenotype of the AMLs with NPM1 mutations, gene expression profiles were obtained using Affymetrix U133A microarrays. NPM1 mutations were associated with increased expression of multiple homeobox genes including HOXA9, A10, B2, B6 and MEIS1. As dysregulated homeobox gene expression is also a feature of MLL-rearranged leukemia, the gene expression signatures of NPM1-mutated and MLL-rearranged leukemias were compared. Significant differences were identified between these leukemia subtypes including the expression of different HOX genes, with NPM1-mutated AML showing higher levels of expression of HOXB2, B3, B6 and D4. These results confirm recent reports of perturbed HOX expression in NPM1-mutated adult AML, and provide the first evidence that the NPM1-mutated signature is distinct from MLL-rearranged AML. These findings suggest that mutated NPM1 leads to dysregulated HOX expression via a different mechanism than MLL rearrangement.
Somatic mutations in nucleophosmin (NPM1) occur in approximately 35% of adult acute myeloid leukemia (AML). To assess the frequency of NPM1 mutations in pediatric AML, we sequenced NPM1 in the diagnostic blasts from 93 pediatric AML patients. Six cases harbored NPM1 mutations, with each case lacking common cytogenetic abnormalities. To explore the phenotype of the AMLs with NPM1 mutations, gene expression profiles were obtained using Affymetrix U133A microarrays. NPM1 mutations were associated with increased expression of multiple homeobox genes including HOXA9, A10, B2, B6 and MEIS1. As dysregulated homeobox gene expression is also a feature of MLL-rearranged leukemia, the gene expression signatures of NPM1-mutated and MLL-rearranged leukemias were compared. Significant differences were identified between these leukemia subtypes including the expression of different HOX genes, with NPM1-mutated AML showing higher levels of expression of HOXB2, B3, B6 and D4. These results confirm recent reports of perturbed HOX expression in NPM1-mutated adult AML, and provide the first evidence that the NPM1-mutated signature is distinct from MLL-rearranged AML. These findings suggest that mutated NPM1 leads to dysregulated HOX expression via a different mechanism than MLL rearrangement.
Somatic mutations in nucleophosmin (NPM1) occur in approximately 35% of adult acute myeloid leukemia (AML). To assess the frequency of NPM1 mutations in pediatric AML, we sequenced NPM1 in the diagnostic blasts from 93 pediatric AML patients. Six cases harbored NPM1 mutations, with each case lacking common cytogenetic abnormalities. To explore the phenotype of the AMLs with NPM1 mutations, gene expression profiles were obtained using Affymetrix U133A microarrays. NPM1 mutations were associated with increased expression of multiple homeobox genes including HOXA9, A10, B2, B6 and MEIS1. As dysregulated homeobox gene expression is also a feature of MLL-rearranged leukemia, the gene expression signatures of NPM1-mutated and MLL-rearranged leukemias were compared. Significant differences were identified between these leukemia subtypes including the expression of different HOX genes, with NPM1-mutated AML showing higher levels of expression of HOXB2, B3, B6 and D4. These results confirm recent reports of perturbed HOX expression in NPM1-mutated adult AML, and provide the first evidence that the NPM1-mutated signature is distinct from MLL-rearranged AML. These findings suggest that mutated NPM1 leads to dysregulated HOX expression via a different mechanism than MLL rearrangement.
Somatic mutations in nucleophosmin (NPM1) occur in approximately 35% of adult acute myeloid leukemia (AML). To assess the frequency of NPM1 mutations in pediatric AML, we sequenced NPM1 in the diagnostic blasts from 93 pediatric AML patients. Six cases harbored NPM1 mutations, with each case lacking common cytogenetic abnormalities. To explore the phenotype of the AMLs with NPM1 mutations, gene expression profiles were obtained using Affymetrix U133A microarrays. NPM1 mutations were associated with increased expression of multiple homeobox genes including HOXA9, A10, B2, B6 and MEIS1. As dysregulated homeobox gene expression is also a feature of MLL-rearranged leukemia, the gene expression signatures of NPM1-mutated and MLL-rearranged leukemias were compared. Significant differences were identified between these leukemia subtypes including the expression of different HOX genes, with NPM1-mutated AML showing higher levels of expression of HOXB2, B3, B6 and D4. These results confirm recent reports of perturbed HOX expression in NPM1-mutated adult AML, and provide the first evidence that the NPM1-mutated signature is distinct from MLL-rearranged AML. These findings suggest that mutated NPM1 leads to dysregulated HOX expression via a different mechanism than MLL rearrangement.
Somatic mutations in nucleophosmin (NPM1) occur in approximately 35% of adult acute myeloid leukemia (AML). To assess the frequency of NPM1 mutations in pediatric AML, we sequenced NPM1 in the diagnostic blasts from 93 pediatric AML patients. Six cases harbored NPM1 mutations, with each case lacking common cytogenetic abnormalities. To explore the phenotype of the AMLs with NPM1 mutations, gene expression profiles were obtained using Affymetrix U133A microarrays. NPM1 mutations were associated with increased expression of multiple homeobox genes including HOXA9, A10, B2, B6 and MEIS1. As dysregulated homeobox gene expression is also a feature of MLL-rearranged leukemia, the gene expression signatures of NPM1-mutated and MLL-rearranged leukemias were compared. Significant differences were identified between these leukemia subtypes including the expression of different HOX genes, with NPM1-mutated AML showing higher levels of expression of HOXB2, B3, B6 and D4. These results confirm recent reports of perturbed HOX expression in NPM1-mutated adult AML, and provide the first evidence that the NPM1-mutated signature is distinct from MLL-rearranged AML. These findings suggest that mutated NPM1 leads to dysregulated HOX expression via a different mechanism than MLL rearrangement.
Somatic mutations in nucleophosmin (NPM1) occur in approximately 35% of adult acute myeloid leukemia (AML). To assess the frequency of NPM1 mutations in pediatric AML, we sequenced NPM1 in the diagnostic blasts from 93 pediatric AML patients. Six cases harbored NPM1 mutations, with each case lacking common cytogenetic abnormalities. To explore the phenotype of the AMLs with NPM1 mutations, gene expression profiles were obtained using Affymetrix U133A microarrays. NPM1 mutations were associated with increased expression of multiple homeobox genes including HOXA9, A10, B2, B6 and MEIS1. As dysregulated homeobox gene expression is also a feature of MLL-rearranged leukemia, the gene expression signatures of NPM1-mutated and MLL-rearranged leukemias were compared. Significant differences were identified between these leukemia subtypes including the expression of different HOX genes, with NPM1-mutated AML showing higher levels of expression of HOXB2, B3, B6 and D4. These results confirm recent reports of perturbed HOX expression in NPM1-mutated adult AML, and provide the first evidence that the NPM1-mutated signature is distinct from MLL-rearranged AML. These findings suggest that mutated NPM1 leads to dysregulated HOX expression via a different mechanism than MLL rearrangement.
Somatic mutations in nucleophosmin (NPM1) occur in approximately 35% of adult acute myeloid leukemia (AML). To assess the frequency of NPM1 mutations in pediatric AML, we sequenced NPM1 in the diagnostic blasts from 93 pediatric AML patients. Six cases harbored NPM1 mutations, with each case lacking common cytogenetic abnormalities. To explore the phenotype of the AMLs with NPM1 mutations, gene expression profiles were obtained using Affymetrix U133A microarrays. NPM1 mutations were associated with increased expression of multiple homeobox genes including HOXA9, A10, B2, B6 and MEIS1. As dysregulated homeobox gene expression is also a feature of MLL-rearranged leukemia, the gene expression signatures of NPM1-mutated and MLL-rearranged leukemias were compared. Significant differences were identified between these leukemia subtypes including the expression of different HOX genes, with NPM1-mutated AML showing higher levels of expression of HOXB2, B3, B6 and D4. These results confirm recent reports of perturbed HOX expression in NPM1-mutated adult AML, and provide the first evidence that the NPM1-mutated signature is distinct from MLL-rearranged AML. These findings suggest that mutated NPM1 leads to dysregulated HOX expression via a different mechanism than MLL rearrangement.
Somatic mutations in nucleophosmin (NPM1) occur in approximately 35% of adult acute myeloid leukemia (AML). To assess the frequency of NPM1 mutations in pediatric AML, we sequenced NPM1 in the diagnostic blasts from 93 pediatric AML patients. Six cases harbored NPM1 mutations, with each case lacking common cytogenetic abnormalities. To explore the phenotype of the AMLs with NPM1 mutations, gene expression profiles were obtained using Affymetrix U133A microarrays. NPM1 mutations were associated with increased expression of multiple homeobox genes including HOXA9, A10, B2, B6 and MEIS1. As dysregulated homeobox gene expression is also a feature of MLL-rearranged leukemia, the gene expression signatures of NPM1-mutated and MLL-rearranged leukemias were compared. Significant differences were identified between these leukemia subtypes including the expression of different HOX genes, with NPM1-mutated AML showing higher levels of expression of HOXB2, B3, B6 and D4. These results confirm recent reports of perturbed HOX expression in NPM1-mutated adult AML, and provide the first evidence that the NPM1-mutated signature is distinct from MLL-rearranged AML. These findings suggest that mutated NPM1 leads to dysregulated HOX expression via a different mechanism than MLL rearrangement.
The t(8;21)(q22;q22) occurs frequently in acute myelogenous leukaemia and gives rise to the transcription factor fusion protein, RUNX1-RUNX1T1 (also known as AML1-ETO). To identify the genes dysregulated by the aberrant transcriptional activity of RUNX1-RUNX1T1, we used microarrays to determine the effect of this mutation on gene expression in human progenitor cells and during subsequent development. Gene signatures of these developmental subsets were very dissimilar indicating that effects of RUNX1-RUNX1T1 are highly context dependent. We focused on gene changes associated with the granulocytic lineage and identified a clinically relevant subset of these by comparison with 235 leukaemia patient transcriptional signatures. We confirmed the overexpression of a number of significant genes (Sox4, IL-17BR, CD200 and gamma-catenin). Further, we show that overexpression of CD200 and gamma-catenin is also associated with the inv(16) abnormality which like RUNX1-RUNX1T1 disrupts core binding factor activity. We investigated the functional significance of CD200 and gamma-catenin overexpression in normal human progenitor cells. The effect of IL17 on growth was also assessed. Individually, none of these changes were sufficient to recapitulate the effects of RUNX1-RUNX1T1 on normal development. These data provide the most comprehensive and pertinent assessment of the effect of RUNX1-RUNX1T1 on gene expression and demonstrate the highly context-dependent effects of this fusion gene.
The t(8;21)(q22;q22) occurs frequently in acute myelogenous leukaemia and gives rise to the transcription factor fusion protein, RUNX1-RUNX1T1 (also known as AML1-ETO). To identify the genes dysregulated by the aberrant transcriptional activity of RUNX1-RUNX1T1, we used microarrays to determine the effect of this mutation on gene expression in human progenitor cells and during subsequent development. Gene signatures of these developmental subsets were very dissimilar indicating that effects of RUNX1-RUNX1T1 are highly context dependent. We focused on gene changes associated with the granulocytic lineage and identified a clinically relevant subset of these by comparison with 235 leukaemia patient transcriptional signatures. We confirmed the overexpression of a number of significant genes (Sox4, IL-17BR, CD200 and gamma-catenin). Further, we show that overexpression of CD200 and gamma-catenin is also associated with the inv(16) abnormality which like RUNX1-RUNX1T1 disrupts core binding factor activity. We investigated the functional significance of CD200 and gamma-catenin overexpression in normal human progenitor cells. The effect of IL17 on growth was also assessed. Individually, none of these changes were sufficient to recapitulate the effects of RUNX1-RUNX1T1 on normal development. These data provide the most comprehensive and pertinent assessment of the effect of RUNX1-RUNX1T1 on gene expression and demonstrate the highly context-dependent effects of this fusion gene.
The t(8;21)(q22;q22) occurs frequently in acute myelogenous leukaemia and gives rise to the transcription factor fusion protein, RUNX1-RUNX1T1 (also known as AML1-ETO). To identify the genes dysregulated by the aberrant transcriptional activity of RUNX1-RUNX1T1, we used microarrays to determine the effect of this mutation on gene expression in human progenitor cells and during subsequent development. Gene signatures of these developmental subsets were very dissimilar indicating that effects of RUNX1-RUNX1T1 are highly context dependent. We focused on gene changes associated with the granulocytic lineage and identified a clinically relevant subset of these by comparison with 235 leukaemia patient transcriptional signatures. We confirmed the overexpression of a number of significant genes (Sox4, IL-17BR, CD200 and gamma-catenin). Further, we show that overexpression of CD200 and gamma-catenin is also associated with the inv(16) abnormality which like RUNX1-RUNX1T1 disrupts core binding factor activity. We investigated the functional significance of CD200 and gamma-catenin overexpression in normal human progenitor cells. The effect of IL17 on growth was also assessed. Individually, none of these changes were sufficient to recapitulate the effects of RUNX1-RUNX1T1 on normal development. These data provide the most comprehensive and pertinent assessment of the effect of RUNX1-RUNX1T1 on gene expression and demonstrate the highly context-dependent effects of this fusion gene.
The t(8;21)(q22;q22) occurs frequently in acute myelogenous leukaemia and gives rise to the transcription factor fusion protein, RUNX1-RUNX1T1 (also known as AML1-ETO). To identify the genes dysregulated by the aberrant transcriptional activity of RUNX1-RUNX1T1, we used microarrays to determine the effect of this mutation on gene expression in human progenitor cells and during subsequent development. Gene signatures of these developmental subsets were very dissimilar indicating that effects of RUNX1-RUNX1T1 are highly context dependent. We focused on gene changes associated with the granulocytic lineage and identified a clinically relevant subset of these by comparison with 235 leukaemia patient transcriptional signatures. We confirmed the overexpression of a number of significant genes (Sox4, IL-17BR, CD200 and gamma-catenin). Further, we show that overexpression of CD200 and gamma-catenin is also associated with the inv(16) abnormality which like RUNX1-RUNX1T1 disrupts core binding factor activity. We investigated the functional significance of CD200 and gamma-catenin overexpression in normal human progenitor cells. The effect of IL17 on growth was also assessed. Individually, none of these changes were sufficient to recapitulate the effects of RUNX1-RUNX1T1 on normal development. These data provide the most comprehensive and pertinent assessment of the effect of RUNX1-RUNX1T1 on gene expression and demonstrate the highly context-dependent effects of this fusion gene.
The t(8;21)(q22;q22) occurs frequently in acute myelogenous leukaemia and gives rise to the transcription factor fusion protein, RUNX1-RUNX1T1 (also known as AML1-ETO). To identify the genes dysregulated by the aberrant transcriptional activity of RUNX1-RUNX1T1, we used microarrays to determine the effect of this mutation on gene expression in human progenitor cells and during subsequent development. Gene signatures of these developmental subsets were very dissimilar indicating that effects of RUNX1-RUNX1T1 are highly context dependent. We focused on gene changes associated with the granulocytic lineage and identified a clinically relevant subset of these by comparison with 235 leukaemia patient transcriptional signatures. We confirmed the overexpression of a number of significant genes (Sox4, IL-17BR, CD200 and gamma-catenin). Further, we show that overexpression of CD200 and gamma-catenin is also associated with the inv(16) abnormality which like RUNX1-RUNX1T1 disrupts core binding factor activity. We investigated the functional significance of CD200 and gamma-catenin overexpression in normal human progenitor cells. The effect of IL17 on growth was also assessed. Individually, none of these changes were sufficient to recapitulate the effects of RUNX1-RUNX1T1 on normal development. These data provide the most comprehensive and pertinent assessment of the effect of RUNX1-RUNX1T1 on gene expression and demonstrate the highly context-dependent effects of this fusion gene.
The t(8;21)(q22;q22) occurs frequently in acute myelogenous leukaemia and gives rise to the transcription factor fusion protein, RUNX1-RUNX1T1 (also known as AML1-ETO). To identify the genes dysregulated by the aberrant transcriptional activity of RUNX1-RUNX1T1, we used microarrays to determine the effect of this mutation on gene expression in human progenitor cells and during subsequent development. Gene signatures of these developmental subsets were very dissimilar indicating that effects of RUNX1-RUNX1T1 are highly context dependent. We focused on gene changes associated with the granulocytic lineage and identified a clinically relevant subset of these by comparison with 235 leukaemia patient transcriptional signatures. We confirmed the overexpression of a number of significant genes (Sox4, IL-17BR, CD200 and gamma-catenin). Further, we show that overexpression of CD200 and gamma-catenin is also associated with the inv(16) abnormality which like RUNX1-RUNX1T1 disrupts core binding factor activity. We investigated the functional significance of CD200 and gamma-catenin overexpression in normal human progenitor cells. The effect of IL17 on growth was also assessed. Individually, none of these changes were sufficient to recapitulate the effects of RUNX1-RUNX1T1 on normal development. These data provide the most comprehensive and pertinent assessment of the effect of RUNX1-RUNX1T1 on gene expression and demonstrate the highly context-dependent effects of this fusion gene.
The t(8;21)(q22;q22) occurs frequently in acute myelogenous leukaemia and gives rise to the transcription factor fusion protein, RUNX1-RUNX1T1 (also known as AML1-ETO). To identify the genes dysregulated by the aberrant transcriptional activity of RUNX1-RUNX1T1, we used microarrays to determine the effect of this mutation on gene expression in human progenitor cells and during subsequent development. Gene signatures of these developmental subsets were very dissimilar indicating that effects of RUNX1-RUNX1T1 are highly context dependent. We focused on gene changes associated with the granulocytic lineage and identified a clinically relevant subset of these by comparison with 235 leukaemia patient transcriptional signatures. We confirmed the overexpression of a number of significant genes (Sox4, IL-17BR, CD200 and gamma-catenin). Further, we show that overexpression of CD200 and gamma-catenin is also associated with the inv(16) abnormality which like RUNX1-RUNX1T1 disrupts core binding factor activity. We investigated the functional significance of CD200 and gamma-catenin overexpression in normal human progenitor cells. The effect of IL17 on growth was also assessed. Individually, none of these changes were sufficient to recapitulate the effects of RUNX1-RUNX1T1 on normal development. These data provide the most comprehensive and pertinent assessment of the effect of RUNX1-RUNX1T1 on gene expression and demonstrate the highly context-dependent effects of this fusion gene.
The t(8;21)(q22;q22) occurs frequently in acute myelogenous leukaemia and gives rise to the transcription factor fusion protein, RUNX1-RUNX1T1 (also known as AML1-ETO). To identify the genes dysregulated by the aberrant transcriptional activity of RUNX1-RUNX1T1, we used microarrays to determine the effect of this mutation on gene expression in human progenitor cells and during subsequent development. Gene signatures of these developmental subsets were very dissimilar indicating that effects of RUNX1-RUNX1T1 are highly context dependent. We focused on gene changes associated with the granulocytic lineage and identified a clinically relevant subset of these by comparison with 235 leukaemia patient transcriptional signatures. We confirmed the overexpression of a number of significant genes (Sox4, IL-17BR, CD200 and gamma-catenin). Further, we show that overexpression of CD200 and gamma-catenin is also associated with the inv(16) abnormality which like RUNX1-RUNX1T1 disrupts core binding factor activity. We investigated the functional significance of CD200 and gamma-catenin overexpression in normal human progenitor cells. The effect of IL17 on growth was also assessed. Individually, none of these changes were sufficient to recapitulate the effects of RUNX1-RUNX1T1 on normal development. These data provide the most comprehensive and pertinent assessment of the effect of RUNX1-RUNX1T1 on gene expression and demonstrate the highly context-dependent effects of this fusion gene.
The t(8;21)(q22;q22) occurs frequently in acute myelogenous leukaemia and gives rise to the transcription factor fusion protein, RUNX1-RUNX1T1 (also known as AML1-ETO). To identify the genes dysregulated by the aberrant transcriptional activity of RUNX1-RUNX1T1, we used microarrays to determine the effect of this mutation on gene expression in human progenitor cells and during subsequent development. Gene signatures of these developmental subsets were very dissimilar indicating that effects of RUNX1-RUNX1T1 are highly context dependent. We focused on gene changes associated with the granulocytic lineage and identified a clinically relevant subset of these by comparison with 235 leukaemia patient transcriptional signatures. We confirmed the overexpression of a number of significant genes (Sox4, IL-17BR, CD200 and gamma-catenin). Further, we show that overexpression of CD200 and gamma-catenin is also associated with the inv(16) abnormality which like RUNX1-RUNX1T1 disrupts core binding factor activity. We investigated the functional significance of CD200 and gamma-catenin overexpression in normal human progenitor cells. The effect of IL17 on growth was also assessed. Individually, none of these changes were sufficient to recapitulate the effects of RUNX1-RUNX1T1 on normal development. These data provide the most comprehensive and pertinent assessment of the effect of RUNX1-RUNX1T1 on gene expression and demonstrate the highly context-dependent effects of this fusion gene.
The t(8;21)(q22;q22) occurs frequently in acute myelogenous leukaemia and gives rise to the transcription factor fusion protein, RUNX1-RUNX1T1 (also known as AML1-ETO). To identify the genes dysregulated by the aberrant transcriptional activity of RUNX1-RUNX1T1, we used microarrays to determine the effect of this mutation on gene expression in human progenitor cells and during subsequent development. Gene signatures of these developmental subsets were very dissimilar indicating that effects of RUNX1-RUNX1T1 are highly context dependent. We focused on gene changes associated with the granulocytic lineage and identified a clinically relevant subset of these by comparison with 235 leukaemia patient transcriptional signatures. We confirmed the overexpression of a number of significant genes (Sox4, IL-17BR, CD200 and gamma-catenin). Further, we show that overexpression of CD200 and gamma-catenin is also associated with the inv(16) abnormality which like RUNX1-RUNX1T1 disrupts core binding factor activity. We investigated the functional significance of CD200 and gamma-catenin overexpression in normal human progenitor cells. The effect of IL17 on growth was also assessed. Individually, none of these changes were sufficient to recapitulate the effects of RUNX1-RUNX1T1 on normal development. These data provide the most comprehensive and pertinent assessment of the effect of RUNX1-RUNX1T1 on gene expression and demonstrate the highly context-dependent effects of this fusion gene.
The t(8;21)(q22;q22) occurs frequently in acute myelogenous leukaemia and gives rise to the transcription factor fusion protein, RUNX1-RUNX1T1 (also known as AML1-ETO). To identify the genes dysregulated by the aberrant transcriptional activity of RUNX1-RUNX1T1, we used microarrays to determine the effect of this mutation on gene expression in human progenitor cells and during subsequent development. Gene signatures of these developmental subsets were very dissimilar indicating that effects of RUNX1-RUNX1T1 are highly context dependent. We focused on gene changes associated with the granulocytic lineage and identified a clinically relevant subset of these by comparison with 235 leukaemia patient transcriptional signatures. We confirmed the overexpression of a number of significant genes (Sox4, IL-17BR, CD200 and gamma-catenin). Further, we show that overexpression of CD200 and gamma-catenin is also associated with the inv(16) abnormality which like RUNX1-RUNX1T1 disrupts core binding factor activity. We investigated the functional significance of CD200 and gamma-catenin overexpression in normal human progenitor cells. The effect of IL17 on growth was also assessed. Individually, none of these changes were sufficient to recapitulate the effects of RUNX1-RUNX1T1 on normal development. These data provide the most comprehensive and pertinent assessment of the effect of RUNX1-RUNX1T1 on gene expression and demonstrate the highly context-dependent effects of this fusion gene.
The t(8;21)(q22;q22) occurs frequently in acute myelogenous leukaemia and gives rise to the transcription factor fusion protein, RUNX1-RUNX1T1 (also known as AML1-ETO). To identify the genes dysregulated by the aberrant transcriptional activity of RUNX1-RUNX1T1, we used microarrays to determine the effect of this mutation on gene expression in human progenitor cells and during subsequent development. Gene signatures of these developmental subsets were very dissimilar indicating that effects of RUNX1-RUNX1T1 are highly context dependent. We focused on gene changes associated with the granulocytic lineage and identified a clinically relevant subset of these by comparison with 235 leukaemia patient transcriptional signatures. We confirmed the overexpression of a number of significant genes (Sox4, IL-17BR, CD200 and gamma-catenin). Further, we show that overexpression of CD200 and gamma-catenin is also associated with the inv(16) abnormality which like RUNX1-RUNX1T1 disrupts core binding factor activity. We investigated the functional significance of CD200 and gamma-catenin overexpression in normal human progenitor cells. The effect of IL17 on growth was also assessed. Individually, none of these changes were sufficient to recapitulate the effects of RUNX1-RUNX1T1 on normal development. These data provide the most comprehensive and pertinent assessment of the effect of RUNX1-RUNX1T1 on gene expression and demonstrate the highly context-dependent effects of this fusion gene.
The t(8;21)(q22;q22) occurs frequently in acute myelogenous leukaemia and gives rise to the transcription factor fusion protein, RUNX1-RUNX1T1 (also known as AML1-ETO). To identify the genes dysregulated by the aberrant transcriptional activity of RUNX1-RUNX1T1, we used microarrays to determine the effect of this mutation on gene expression in human progenitor cells and during subsequent development. Gene signatures of these developmental subsets were very dissimilar indicating that effects of RUNX1-RUNX1T1 are highly context dependent. We focused on gene changes associated with the granulocytic lineage and identified a clinically relevant subset of these by comparison with 235 leukaemia patient transcriptional signatures. We confirmed the overexpression of a number of significant genes (Sox4, IL-17BR, CD200 and gamma-catenin). Further, we show that overexpression of CD200 and gamma-catenin is also associated with the inv(16) abnormality which like RUNX1-RUNX1T1 disrupts core binding factor activity. We investigated the functional significance of CD200 and gamma-catenin overexpression in normal human progenitor cells. The effect of IL17 on growth was also assessed. Individually, none of these changes were sufficient to recapitulate the effects of RUNX1-RUNX1T1 on normal development. These data provide the most comprehensive and pertinent assessment of the effect of RUNX1-RUNX1T1 on gene expression and demonstrate the highly context-dependent effects of this fusion gene.
The t(8;21)(q22;q22) occurs frequently in acute myelogenous leukaemia and gives rise to the transcription factor fusion protein, RUNX1-RUNX1T1 (also known as AML1-ETO). To identify the genes dysregulated by the aberrant transcriptional activity of RUNX1-RUNX1T1, we used microarrays to determine the effect of this mutation on gene expression in human progenitor cells and during subsequent development. Gene signatures of these developmental subsets were very dissimilar indicating that effects of RUNX1-RUNX1T1 are highly context dependent. We focused on gene changes associated with the granulocytic lineage and identified a clinically relevant subset of these by comparison with 235 leukaemia patient transcriptional signatures. We confirmed the overexpression of a number of significant genes (Sox4, IL-17BR, CD200 and gamma-catenin). Further, we show that overexpression of CD200 and gamma-catenin is also associated with the inv(16) abnormality which like RUNX1-RUNX1T1 disrupts core binding factor activity. We investigated the functional significance of CD200 and gamma-catenin overexpression in normal human progenitor cells. The effect of IL17 on growth was also assessed. Individually, none of these changes were sufficient to recapitulate the effects of RUNX1-RUNX1T1 on normal development. These data provide the most comprehensive and pertinent assessment of the effect of RUNX1-RUNX1T1 on gene expression and demonstrate the highly context-dependent effects of this fusion gene.
Most gene expression profiling studies of mesothelioma have been based on relatively small sample numbers, limiting their statistical power. We did Affymetrix U133A microarray analysis on 99 pleural mesotheliomas, in which multivariate analysis showed advanced-stage, sarcomatous histology and P16/CDKN2A homozygous deletion to be significant independent adverse prognostic factors. Comparison of the expression profiles of epithelioid versus sarcomatous mesotheliomas identified many genes significantly overexpressed among the former, including previously unrecognized ones, such as uroplakins and kallikrein 11, both confirmed by immunohistochemistry. Examination of the gene expression correlates of survival showed that more aggressive mesotheliomas expressed higher levels of Aurora kinases A and B and functionally related genes involved in mitosis and cell cycle control. Independent confirmation of the negative effect of Aurora kinase B was obtained by immunohistochemistry in a separate patient cohort. A role for Aurora kinases in the aggressive behavior of mesotheliomas is of potential clinical interest because of the recent development of small-molecule inhibitors. We then used our data to develop microarray-based predictors of 1 year survival; these achieved a maximal accuracy of 68% in cross-validation. However, this was inferior to prognostic prediction based on standard clinicopathologic variables and P16/CDNK2A status (accuracy, 73%), and adding the microarray model to the latter did not improve overall accuracy. Finally, we evaluated three recently published microarray-based outcome prediction models, but their accuracies ranged from 63% to 67%, consistently lower than reported. Gene expression profiling of mesotheliomas is an important discovery tool, but its power in clinical prognostication has been overestimated.
Most gene expression profiling studies of mesothelioma have been based on relatively small sample numbers, limiting their statistical power. We did Affymetrix U133A microarray analysis on 99 pleural mesotheliomas, in which multivariate analysis showed advanced-stage, sarcomatous histology and P16/CDKN2A homozygous deletion to be significant independent adverse prognostic factors. Comparison of the expression profiles of epithelioid versus sarcomatous mesotheliomas identified many genes significantly overexpressed among the former, including previously unrecognized ones, such as uroplakins and kallikrein 11, both confirmed by immunohistochemistry. Examination of the gene expression correlates of survival showed that more aggressive mesotheliomas expressed higher levels of Aurora kinases A and B and functionally related genes involved in mitosis and cell cycle control. Independent confirmation of the negative effect of Aurora kinase B was obtained by immunohistochemistry in a separate patient cohort. A role for Aurora kinases in the aggressive behavior of mesotheliomas is of potential clinical interest because of the recent development of small-molecule inhibitors. We then used our data to develop microarray-based predictors of 1 year survival; these achieved a maximal accuracy of 68% in cross-validation. However, this was inferior to prognostic prediction based on standard clinicopathologic variables and P16/CDNK2A status (accuracy, 73%), and adding the microarray model to the latter did not improve overall accuracy. Finally, we evaluated three recently published microarray-based outcome prediction models, but their accuracies ranged from 63% to 67%, consistently lower than reported. Gene expression profiling of mesotheliomas is an important discovery tool, but its power in clinical prognostication has been overestimated.
The composition and vulnerability of the atherosclerotic plaque are considered to be important factors in the development of arterial thrombus and embolic complications.  The aim of this study was to identify differences in gene expression between stable and unstable segments of plaque obtained from the same patient. The present gene set consists of genes judged as differentially expressed by both analytical methods (individual intra- and inter-plaque analysis).
The composition and vulnerability of the atherosclerotic plaque are considered to be important factors in the development of arterial thrombus and embolic complications.  The aim of this study was to identify differences in gene expression between stable and unstable segments of plaque obtained from the same patient. The present gene set consists of genes judged as differentially expressed by both analytical methods (individual intra- and inter-plaque analysis).
The effect of endogenous parathyroid hormone-related protein (PTHrP) on gene expression in breast cancer cells was studied. We suppressed PTHrP expression in MDA-MB-231 cells by RNA interference and analyzed changes in gene expression by microarray analysis. More than 200 genes showed altered expression in response to a PTHrP-specific small interfering (si) RNA (siPTHrP). Cell cycle-regulating gene CDC2 and genes (CDC25B and Tome-1) that control CDC2 activity showed increased expression in the presence of siPTHrP. CDC2 activity was also found to be higher in siPTHrP-treated cells. Studies with PTHrP peptides 1-34 and 67-86, forskolin, and a PTH1 receptor (PTH1R)-specific siRNA showed that PTHrP regulates CDC2 and CDC25B, at least in part, via PTH1R in a cAMP-independent manner. Other siPTHrP-responsive genes included integrin alpha6 (ITGA6), KISS-1, and PAI-1. When combined, siRNAs against ITGA6, PAI-1, and KISS-1 could mimic the negative effect of siPTHrP on migration, whereas siKISS-1 and siPTHrP similarly reduced the proliferative activity of the cells. Comparative expression analyses with 50 primary breast carcinomas revealed that the RNA level of ITGA6 correlates with that of PTHrP, and higher CDC2 and CDC25B values are found at low PTHrP expression. Our data suggest that PTHrP has a profound effect on gene expression in breast cancer cells and, as a consequence, contributes to the regulation of important cellular activities, such as migration and proliferation.
The effect of endogenous parathyroid hormone-related protein (PTHrP) on gene expression in breast cancer cells was studied. We suppressed PTHrP expression in MDA-MB-231 cells by RNA interference and analyzed changes in gene expression by microarray analysis. More than 200 genes showed altered expression in response to a PTHrP-specific small interfering (si) RNA (siPTHrP). Cell cycle-regulating gene CDC2 and genes (CDC25B and Tome-1) that control CDC2 activity showed increased expression in the presence of siPTHrP. CDC2 activity was also found to be higher in siPTHrP-treated cells. Studies with PTHrP peptides 1-34 and 67-86, forskolin, and a PTH1 receptor (PTH1R)-specific siRNA showed that PTHrP regulates CDC2 and CDC25B, at least in part, via PTH1R in a cAMP-independent manner. Other siPTHrP-responsive genes included integrin alpha6 (ITGA6), KISS-1, and PAI-1. When combined, siRNAs against ITGA6, PAI-1, and KISS-1 could mimic the negative effect of siPTHrP on migration, whereas siKISS-1 and siPTHrP similarly reduced the proliferative activity of the cells. Comparative expression analyses with 50 primary breast carcinomas revealed that the RNA level of ITGA6 correlates with that of PTHrP, and higher CDC2 and CDC25B values are found at low PTHrP expression. Our data suggest that PTHrP has a profound effect on gene expression in breast cancer cells and, as a consequence, contributes to the regulation of important cellular activities, such as migration and proliferation.
The TRAP/Mediator coactivator complex serves as a functional interface between DNA-bound transactivators and the RNA polymerase II-associated basal transcription apparatus. TRAP220/MED1 is a variably associated subunit of the complex that plays a specialized role in selectively targeting TRAP/Mediator to specific genes. Ablation of the Trap220/Med1 gene in mice impairs embryonic cell growth, yet the underlying mechanism is unknown. In this report, we identified distinct cell growth regulatory genes whose expression is affected by the loss of TRAP220/MED1 by RNA interference. Among the down-regulated genes revealed by cDNA microarray analyses, we identified Aurora-A, a centrosome kinase that plays a critical role in regulating M phase events and is frequently amplified in several types of cancer. In general, we found that TRAP220/MED1 expression is required for high basal levels of Aurora-A gene expression and that ectopic overexpression of TRAP220/MED1 coactivates transcription from the Aurora-A gene promoter. Furthermore, chromatin immunoprecipitation assays show that TRAP220/MED1-containing TRAP/Mediator complexes directly bind to the Aurora-A promoter in vivo. Finally, we present evidence suggesting that TRAP/Mediator is recruited to the Aurora-A gene via direct interactions between TRAP220/MED1 and the Ets-related transcription factor GABP. Taken together, these findings suggest that TRAP220/MED1 plays a novel coregulatory role in facilitating the recruitment of TRAP/Mediator to specific target genes involved in growth and cell cycle progression.
The TRAP/Mediator coactivator complex serves as a functional interface between DNA-bound transactivators and the RNA polymerase II-associated basal transcription apparatus. TRAP220/MED1 is a variably associated subunit of the complex that plays a specialized role in selectively targeting TRAP/Mediator to specific genes. Ablation of the Trap220/Med1 gene in mice impairs embryonic cell growth, yet the underlying mechanism is unknown. In this report, we identified distinct cell growth regulatory genes whose expression is affected by the loss of TRAP220/MED1 by RNA interference. Among the down-regulated genes revealed by cDNA microarray analyses, we identified Aurora-A, a centrosome kinase that plays a critical role in regulating M phase events and is frequently amplified in several types of cancer. In general, we found that TRAP220/MED1 expression is required for high basal levels of Aurora-A gene expression and that ectopic overexpression of TRAP220/MED1 coactivates transcription from the Aurora-A gene promoter. Furthermore, chromatin immunoprecipitation assays show that TRAP220/MED1-containing TRAP/Mediator complexes directly bind to the Aurora-A promoter in vivo. Finally, we present evidence suggesting that TRAP/Mediator is recruited to the Aurora-A gene via direct interactions between TRAP220/MED1 and the Ets-related transcription factor GABP. Taken together, these findings suggest that TRAP220/MED1 plays a novel coregulatory role in facilitating the recruitment of TRAP/Mediator to specific target genes involved in growth and cell cycle progression.
Overexpression of transferrin receptor 1 (TFRC1), a major mediator of iron uptake in mammalian cells, is a common feature of human malignancies. Therapeutic strategies designed to interfere with tumor iron metabolism have targeted TFRC1. The c-Myc oncogenic transcription factor stimulates proliferation and growth by activating thousands of target genes. Here we demonstrate that TFRC1 is a critical downstream target of c-Myc. Using in vitro and in vivo models of B-cell lymphoma, we show that TFRC1 expression is activated by c-Myc. Chromatin immunoprecipitation experiments reveal that c-Myc directly binds a conserved region of TFRC1. In light of these findings, we sought to determine whether TFRC1 is required for c-Myc-mediated cellular proliferation and cell size control. TFRC1 inhibition decreases cellular proliferation and results in G1 arrest without affecting cell size. Consistent with these findings, expression profiling reveals that TFRC1 depletion alters expression of genes that regulate the cell cycle. Furthermore, enforced TFRC1 expression confers a growth advantage to cells and significantly enhances the rate of c-Myc-mediated tumor formation in vivo. These findings provide a molecular basis for increased TFRC1 expression in human tumors, illuminate the role of TFRC1 in the c-Myc target gene network, and support strategies that target TFRC1 for cancer therapy.
Overexpression of transferrin receptor 1 (TFRC1), a major mediator of iron uptake in mammalian cells, is a common feature of human malignancies. Therapeutic strategies designed to interfere with tumor iron metabolism have targeted TFRC1. The c-Myc oncogenic transcription factor stimulates proliferation and growth by activating thousands of target genes. Here we demonstrate that TFRC1 is a critical downstream target of c-Myc. Using in vitro and in vivo models of B-cell lymphoma, we show that TFRC1 expression is activated by c-Myc. Chromatin immunoprecipitation experiments reveal that c-Myc directly binds a conserved region of TFRC1. In light of these findings, we sought to determine whether TFRC1 is required for c-Myc-mediated cellular proliferation and cell size control. TFRC1 inhibition decreases cellular proliferation and results in G1 arrest without affecting cell size. Consistent with these findings, expression profiling reveals that TFRC1 depletion alters expression of genes that regulate the cell cycle. Furthermore, enforced TFRC1 expression confers a growth advantage to cells and significantly enhances the rate of c-Myc-mediated tumor formation in vivo. These findings provide a molecular basis for increased TFRC1 expression in human tumors, illuminate the role of TFRC1 in the c-Myc target gene network, and support strategies that target TFRC1 for cancer therapy.
Overexpression of transferrin receptor 1 (TFRC1), a major mediator of iron uptake in mammalian cells, is a common feature of human malignancies. Therapeutic strategies designed to interfere with tumor iron metabolism have targeted TFRC1. The c-Myc oncogenic transcription factor stimulates proliferation and growth by activating thousands of target genes. Here we demonstrate that TFRC1 is a critical downstream target of c-Myc. Using in vitro and in vivo models of B-cell lymphoma, we show that TFRC1 expression is activated by c-Myc. Chromatin immunoprecipitation experiments reveal that c-Myc directly binds a conserved region of TFRC1. In light of these findings, we sought to determine whether TFRC1 is required for c-Myc-mediated cellular proliferation and cell size control. TFRC1 inhibition decreases cellular proliferation and results in G1 arrest without affecting cell size. Consistent with these findings, expression profiling reveals that TFRC1 depletion alters expression of genes that regulate the cell cycle. Furthermore, enforced TFRC1 expression confers a growth advantage to cells and significantly enhances the rate of c-Myc-mediated tumor formation in vivo. These findings provide a molecular basis for increased TFRC1 expression in human tumors, illuminate the role of TFRC1 in the c-Myc target gene network, and support strategies that target TFRC1 for cancer therapy.
Overexpression of transferrin receptor 1 (TFRC1), a major mediator of iron uptake in mammalian cells, is a common feature of human malignancies. Therapeutic strategies designed to interfere with tumor iron metabolism have targeted TFRC1. The c-Myc oncogenic transcription factor stimulates proliferation and growth by activating thousands of target genes. Here we demonstrate that TFRC1 is a critical downstream target of c-Myc. Using in vitro and in vivo models of B-cell lymphoma, we show that TFRC1 expression is activated by c-Myc. Chromatin immunoprecipitation experiments reveal that c-Myc directly binds a conserved region of TFRC1. In light of these findings, we sought to determine whether TFRC1 is required for c-Myc-mediated cellular proliferation and cell size control. TFRC1 inhibition decreases cellular proliferation and results in G1 arrest without affecting cell size. Consistent with these findings, expression profiling reveals that TFRC1 depletion alters expression of genes that regulate the cell cycle. Furthermore, enforced TFRC1 expression confers a growth advantage to cells and significantly enhances the rate of c-Myc-mediated tumor formation in vivo. These findings provide a molecular basis for increased TFRC1 expression in human tumors, illuminate the role of TFRC1 in the c-Myc target gene network, and support strategies that target TFRC1 for cancer therapy.
Posttranslational modifications of core histones are central to the regulation of gene expression. Histone deacetylases (HDACs) repress transcription by deacetylating histones, and class I HDACs have a crucial role in mouse, Xenopus laevis, zebra fish, and Caenorhabditis elegans development. The role of individual class I HDACs in tumor cell proliferation was investigated using RNA interference-mediated protein knockdown. We show here that in the absence of HDAC1 cells can arrest either at the G(1) phase of the cell cycle or at the G(2)/M transition, resulting in the loss of mitotic cells, cell growth inhibition, and an increase in the percentage of apoptotic cells. On the contrary, HDAC2 knockdown showed no effect on cell proliferation unless we concurrently knocked down HDAC1. Using gene expression profiling analysis, we found that inactivation of HDAC1 affected the transcription of specific target genes involved in proliferation and apoptosis. Furthermore, HDAC2 downregulation did not cause significant changes compared to control cells, while inactivation of HDAC1, HDAC1 plus HDAC2, or HDAC3 resulted in more distinct clusters. Loss of these HDACs might impair cell cycle progression by affecting not only the transcription of specific target genes but also other biological processes. Our data support the idea that a drug targeting specific HDACs could be highly beneficial in the treatment of cancer.
Posttranslational modifications of core histones are central to the regulation of gene expression. Histone deacetylases (HDACs) repress transcription by deacetylating histones, and class I HDACs have a crucial role in mouse, Xenopus laevis, zebra fish, and Caenorhabditis elegans development. The role of individual class I HDACs in tumor cell proliferation was investigated using RNA interference-mediated protein knockdown. We show here that in the absence of HDAC1 cells can arrest either at the G(1) phase of the cell cycle or at the G(2)/M transition, resulting in the loss of mitotic cells, cell growth inhibition, and an increase in the percentage of apoptotic cells. On the contrary, HDAC2 knockdown showed no effect on cell proliferation unless we concurrently knocked down HDAC1. Using gene expression profiling analysis, we found that inactivation of HDAC1 affected the transcription of specific target genes involved in proliferation and apoptosis. Furthermore, HDAC2 downregulation did not cause significant changes compared to control cells, while inactivation of HDAC1, HDAC1 plus HDAC2, or HDAC3 resulted in more distinct clusters. Loss of these HDACs might impair cell cycle progression by affecting not only the transcription of specific target genes but also other biological processes. Our data support the idea that a drug targeting specific HDACs could be highly beneficial in the treatment of cancer.
Posttranslational modifications of core histones are central to the regulation of gene expression. Histone deacetylases (HDACs) repress transcription by deacetylating histones, and class I HDACs have a crucial role in mouse, Xenopus laevis, zebra fish, and Caenorhabditis elegans development. The role of individual class I HDACs in tumor cell proliferation was investigated using RNA interference-mediated protein knockdown. We show here that in the absence of HDAC1 cells can arrest either at the G(1) phase of the cell cycle or at the G(2)/M transition, resulting in the loss of mitotic cells, cell growth inhibition, and an increase in the percentage of apoptotic cells. On the contrary, HDAC2 knockdown showed no effect on cell proliferation unless we concurrently knocked down HDAC1. Using gene expression profiling analysis, we found that inactivation of HDAC1 affected the transcription of specific target genes involved in proliferation and apoptosis. Furthermore, HDAC2 downregulation did not cause significant changes compared to control cells, while inactivation of HDAC1, HDAC1 plus HDAC2, or HDAC3 resulted in more distinct clusters. Loss of these HDACs might impair cell cycle progression by affecting not only the transcription of specific target genes but also other biological processes. Our data support the idea that a drug targeting specific HDACs could be highly beneficial in the treatment of cancer.
Posttranslational modifications of core histones are central to the regulation of gene expression. Histone deacetylases (HDACs) repress transcription by deacetylating histones, and class I HDACs have a crucial role in mouse, Xenopus laevis, zebra fish, and Caenorhabditis elegans development. The role of individual class I HDACs in tumor cell proliferation was investigated using RNA interference-mediated protein knockdown. We show here that in the absence of HDAC1 cells can arrest either at the G(1) phase of the cell cycle or at the G(2)/M transition, resulting in the loss of mitotic cells, cell growth inhibition, and an increase in the percentage of apoptotic cells. On the contrary, HDAC2 knockdown showed no effect on cell proliferation unless we concurrently knocked down HDAC1. Using gene expression profiling analysis, we found that inactivation of HDAC1 affected the transcription of specific target genes involved in proliferation and apoptosis. Furthermore, HDAC2 downregulation did not cause significant changes compared to control cells, while inactivation of HDAC1, HDAC1 plus HDAC2, or HDAC3 resulted in more distinct clusters. Loss of these HDACs might impair cell cycle progression by affecting not only the transcription of specific target genes but also other biological processes. Our data support the idea that a drug targeting specific HDACs could be highly beneficial in the treatment of cancer.
Posttranslational modifications of core histones are central to the regulation of gene expression. Histone deacetylases (HDACs) repress transcription by deacetylating histones, and class I HDACs have a crucial role in mouse, Xenopus laevis, zebra fish, and Caenorhabditis elegans development. The role of individual class I HDACs in tumor cell proliferation was investigated using RNA interference-mediated protein knockdown. We show here that in the absence of HDAC1 cells can arrest either at the G(1) phase of the cell cycle or at the G(2)/M transition, resulting in the loss of mitotic cells, cell growth inhibition, and an increase in the percentage of apoptotic cells. On the contrary, HDAC2 knockdown showed no effect on cell proliferation unless we concurrently knocked down HDAC1. Using gene expression profiling analysis, we found that inactivation of HDAC1 affected the transcription of specific target genes involved in proliferation and apoptosis. Furthermore, HDAC2 downregulation did not cause significant changes compared to control cells, while inactivation of HDAC1, HDAC1 plus HDAC2, or HDAC3 resulted in more distinct clusters. Loss of these HDACs might impair cell cycle progression by affecting not only the transcription of specific target genes but also other biological processes. Our data support the idea that a drug targeting specific HDACs could be highly beneficial in the treatment of cancer.
Posttranslational modifications of core histones are central to the regulation of gene expression. Histone deacetylases (HDACs) repress transcription by deacetylating histones, and class I HDACs have a crucial role in mouse, Xenopus laevis, zebra fish, and Caenorhabditis elegans development. The role of individual class I HDACs in tumor cell proliferation was investigated using RNA interference-mediated protein knockdown. We show here that in the absence of HDAC1 cells can arrest either at the G(1) phase of the cell cycle or at the G(2)/M transition, resulting in the loss of mitotic cells, cell growth inhibition, and an increase in the percentage of apoptotic cells. On the contrary, HDAC2 knockdown showed no effect on cell proliferation unless we concurrently knocked down HDAC1. Using gene expression profiling analysis, we found that inactivation of HDAC1 affected the transcription of specific target genes involved in proliferation and apoptosis. Furthermore, HDAC2 downregulation did not cause significant changes compared to control cells, while inactivation of HDAC1, HDAC1 plus HDAC2, or HDAC3 resulted in more distinct clusters. Loss of these HDACs might impair cell cycle progression by affecting not only the transcription of specific target genes but also other biological processes. Our data support the idea that a drug targeting specific HDACs could be highly beneficial in the treatment of cancer.
Posttranslational modifications of core histones are central to the regulation of gene expression. Histone deacetylases (HDACs) repress transcription by deacetylating histones, and class I HDACs have a crucial role in mouse, Xenopus laevis, zebra fish, and Caenorhabditis elegans development. The role of individual class I HDACs in tumor cell proliferation was investigated using RNA interference-mediated protein knockdown. We show here that in the absence of HDAC1 cells can arrest either at the G(1) phase of the cell cycle or at the G(2)/M transition, resulting in the loss of mitotic cells, cell growth inhibition, and an increase in the percentage of apoptotic cells. On the contrary, HDAC2 knockdown showed no effect on cell proliferation unless we concurrently knocked down HDAC1. Using gene expression profiling analysis, we found that inactivation of HDAC1 affected the transcription of specific target genes involved in proliferation and apoptosis. Furthermore, HDAC2 downregulation did not cause significant changes compared to control cells, while inactivation of HDAC1, HDAC1 plus HDAC2, or HDAC3 resulted in more distinct clusters. Loss of these HDACs might impair cell cycle progression by affecting not only the transcription of specific target genes but also other biological processes. Our data support the idea that a drug targeting specific HDACs could be highly beneficial in the treatment of cancer.
Posttranslational modifications of core histones are central to the regulation of gene expression. Histone deacetylases (HDACs) repress transcription by deacetylating histones, and class I HDACs have a crucial role in mouse, Xenopus laevis, zebra fish, and Caenorhabditis elegans development. The role of individual class I HDACs in tumor cell proliferation was investigated using RNA interference-mediated protein knockdown. We show here that in the absence of HDAC1 cells can arrest either at the G(1) phase of the cell cycle or at the G(2)/M transition, resulting in the loss of mitotic cells, cell growth inhibition, and an increase in the percentage of apoptotic cells. On the contrary, HDAC2 knockdown showed no effect on cell proliferation unless we concurrently knocked down HDAC1. Using gene expression profiling analysis, we found that inactivation of HDAC1 affected the transcription of specific target genes involved in proliferation and apoptosis. Furthermore, HDAC2 downregulation did not cause significant changes compared to control cells, while inactivation of HDAC1, HDAC1 plus HDAC2, or HDAC3 resulted in more distinct clusters. Loss of these HDACs might impair cell cycle progression by affecting not only the transcription of specific target genes but also other biological processes. Our data support the idea that a drug targeting specific HDACs could be highly beneficial in the treatment of cancer.
Vertebrate neural crest development depends on pluripotent, migratory precursor cells. Although avian and murine neural crest stem (NCS) cells have been identified, the isolation of human NCS cells has remained elusive. Here we report the derivation of NCS cells from human embryonic stem cells at the neural rosette stage. We show that NCS cells plated at clonal density give rise to multiple neural crest lineages. The human NCS cells can be propagated in vitro and directed toward peripheral nervous system lineages (peripheral neurons, Schwann cells) and mesenchymal lineages (smooth muscle, adipogenic, osteogenic and chondrogenic cells). Transplantation of human NCS cells into the developing chick embryo and adult mouse hosts demonstrates survival, migration and differentiation compatible with neural crest identity. The availability of unlimited numbers of human NCS cells offers new opportunities for studies of neural crest development and for efforts to model and treat neural crest-related disorders.
Vertebrate neural crest development depends on pluripotent, migratory precursor cells. Although avian and murine neural crest stem (NCS) cells have been identified, the isolation of human NCS cells has remained elusive. Here we report the derivation of NCS cells from human embryonic stem cells at the neural rosette stage. We show that NCS cells plated at clonal density give rise to multiple neural crest lineages. The human NCS cells can be propagated in vitro and directed toward peripheral nervous system lineages (peripheral neurons, Schwann cells) and mesenchymal lineages (smooth muscle, adipogenic, osteogenic and chondrogenic cells). Transplantation of human NCS cells into the developing chick embryo and adult mouse hosts demonstrates survival, migration and differentiation compatible with neural crest identity. The availability of unlimited numbers of human NCS cells offers new opportunities for studies of neural crest development and for efforts to model and treat neural crest-related disorders.
BACKGROUND: The distinction between Burkitt's lymphoma and diffuse large-B-cell lymphoma is unclear. We used transcriptional and genomic profiling to define Burkitt's lymphoma more precisely and to distinguish subgroups in other types of mature aggressive B-cell lymphomas. METHODS: We performed gene-expression profiling using Affymetrix U133A GeneChips with RNA from 220 mature aggressive B-cell lymphomas, including a core group of 8 Burkitt's lymphomas that met all World Health Organization (WHO) criteria. A molecular signature for Burkitt's lymphoma was generated, and chromosomal abnormalities were detected with interphase fluorescence in situ hybridization and array-based comparative genomic hybridization. RESULTS: We used the molecular signature for Burkitt's lymphoma to identify 44 cases: 11 had the morphologic features of diffuse large-B-cell lymphomas, 4 were unclassifiable mature aggressive B-cell lymphomas, and 29 had a classic or atypical Burkitt's morphologic appearance. Also, five did not have a detectable IG-myc Burkitt's translocation, whereas the others contained an IG-myc fusion, mostly in simple karyotypes. Of the 176 lymphomas without the molecular signature for Burkitt's lymphoma, 155 were diffuse large-B-cell lymphomas. Of these 155 cases, 21 percent had a chromosomal breakpoint at the myc locus associated with complex chromosomal changes and an unfavorable clinical course. CONCLUSIONS: Our molecular definition of Burkitt's lymphoma clarifies and extends the spectrum of the WHO criteria for Burkitt's lymphoma. In mature aggressive B-cell lymphomas without a gene signature for Burkitt's lymphoma, chromosomal breakpoints at the myc locus were associated with an adverse clinical outcome.
BACKGROUND: The distinction between Burkitt's lymphoma and diffuse large-B-cell lymphoma is unclear. We used transcriptional and genomic profiling to define Burkitt's lymphoma more precisely and to distinguish subgroups in other types of mature aggressive B-cell lymphomas. METHODS: We performed gene-expression profiling using Affymetrix U133A GeneChips with RNA from 220 mature aggressive B-cell lymphomas, including a core group of 8 Burkitt's lymphomas that met all World Health Organization (WHO) criteria. A molecular signature for Burkitt's lymphoma was generated, and chromosomal abnormalities were detected with interphase fluorescence in situ hybridization and array-based comparative genomic hybridization. RESULTS: We used the molecular signature for Burkitt's lymphoma to identify 44 cases: 11 had the morphologic features of diffuse large-B-cell lymphomas, 4 were unclassifiable mature aggressive B-cell lymphomas, and 29 had a classic or atypical Burkitt's morphologic appearance. Also, five did not have a detectable IG-myc Burkitt's translocation, whereas the others contained an IG-myc fusion, mostly in simple karyotypes. Of the 176 lymphomas without the molecular signature for Burkitt's lymphoma, 155 were diffuse large-B-cell lymphomas. Of these 155 cases, 21 percent had a chromosomal breakpoint at the myc locus associated with complex chromosomal changes and an unfavorable clinical course. CONCLUSIONS: Our molecular definition of Burkitt's lymphoma clarifies and extends the spectrum of the WHO criteria for Burkitt's lymphoma. In mature aggressive B-cell lymphomas without a gene signature for Burkitt's lymphoma, chromosomal breakpoints at the myc locus were associated with an adverse clinical outcome.
Shigella invades the human intestinal mucosa, thus causing bacillary dysentery, an acute recto-colitis responsible for lethal complications, mostly in infants and toddlers. Conversely, commensal bacteria live in a mutualistic relationship with the intestinal mucosa that is characterized by homeostatic control of innate responses, thereby contributing to tolerance to the flora. Cross-talk established between commensals and the intestinal epithelium mediate this active process, the mechanisms of which remain largely uncharacterized. Probiotics such as Lactobacillus casei belong to a subclass of these commensals that modulate mucosal innate responses and possibly display anti-inflammatory properties. We analyzed whether L. casei could attenuate the pro-inflammatory signaling induced by Shigella flexneri after invasion of the epithelial lining. Cultured epithelial cells were infected with L. casei, followed by a challenge with S. flexneri. Using macroarray DNA chips, we observed that L. casei down-regulated the transcription of a number of genes encoding pro-inflammatory effectors such as cytokines and chemokines and adherence molecules induced by invasive S. flexneri. This resulted in an anti-inflammatory effect that appeared mediated by the inhibition of the NF-kappaB pathway, particularly through stabilization of I-kappaBalpha. In a time-course experiment using GeneChip hybridization analysis, the expression of many genes involved in ubiquitination and proteasome processes were modulated during L. casei treatment. Thus, L. casei has developed a sophisticated means to maintain intestinal homeostasis through a process that involves manipulation of the ubiquitin/proteasome pathway upstream of I-kappaBalpha.
Shigella invades the human intestinal mucosa, thus causing bacillary dysentery, an acute recto-colitis responsible for lethal complications, mostly in infants and toddlers. Conversely, commensal bacteria live in a mutualistic relationship with the intestinal mucosa that is characterized by homeostatic control of innate responses, thereby contributing to tolerance to the flora. Cross-talk established between commensals and the intestinal epithelium mediate this active process, the mechanisms of which remain largely uncharacterized. Probiotics such as Lactobacillus casei belong to a subclass of these commensals that modulate mucosal innate responses and possibly display anti-inflammatory properties. We analyzed whether L. casei could attenuate the pro-inflammatory signaling induced by Shigella flexneri after invasion of the epithelial lining. Cultured epithelial cells were infected with L. casei, followed by a challenge with S. flexneri. Using macroarray DNA chips, we observed that L. casei down-regulated the transcription of a number of genes encoding pro-inflammatory effectors such as cytokines and chemokines and adherence molecules induced by invasive S. flexneri. This resulted in an anti-inflammatory effect that appeared mediated by the inhibition of the NF-kappaB pathway, particularly through stabilization of I-kappaBalpha. In a time-course experiment using GeneChip hybridization analysis, the expression of many genes involved in ubiquitination and proteasome processes were modulated during L. casei treatment. Thus, L. casei has developed a sophisticated means to maintain intestinal homeostasis through a process that involves manipulation of the ubiquitin/proteasome pathway upstream of I-kappaBalpha.
Shigella invades the human intestinal mucosa, thus causing bacillary dysentery, an acute recto-colitis responsible for lethal complications, mostly in infants and toddlers. Conversely, commensal bacteria live in a mutualistic relationship with the intestinal mucosa that is characterized by homeostatic control of innate responses, thereby contributing to tolerance to the flora. Cross-talk established between commensals and the intestinal epithelium mediate this active process, the mechanisms of which remain largely uncharacterized. Probiotics such as Lactobacillus casei belong to a subclass of these commensals that modulate mucosal innate responses and possibly display anti-inflammatory properties. We analyzed whether L. casei could attenuate the pro-inflammatory signaling induced by Shigella flexneri after invasion of the epithelial lining. Cultured epithelial cells were infected with L. casei, followed by a challenge with S. flexneri. Using macroarray DNA chips, we observed that L. casei down-regulated the transcription of a number of genes encoding pro-inflammatory effectors such as cytokines and chemokines and adherence molecules induced by invasive S. flexneri. This resulted in an anti-inflammatory effect that appeared mediated by the inhibition of the NF-kappaB pathway, particularly through stabilization of I-kappaBalpha. In a time-course experiment using GeneChip hybridization analysis, the expression of many genes involved in ubiquitination and proteasome processes were modulated during L. casei treatment. Thus, L. casei has developed a sophisticated means to maintain intestinal homeostasis through a process that involves manipulation of the ubiquitin/proteasome pathway upstream of I-kappaBalpha.
Shigella invades the human intestinal mucosa, thus causing bacillary dysentery, an acute recto-colitis responsible for lethal complications, mostly in infants and toddlers. Conversely, commensal bacteria live in a mutualistic relationship with the intestinal mucosa that is characterized by homeostatic control of innate responses, thereby contributing to tolerance to the flora. Cross-talk established between commensals and the intestinal epithelium mediate this active process, the mechanisms of which remain largely uncharacterized. Probiotics such as Lactobacillus casei belong to a subclass of these commensals that modulate mucosal innate responses and possibly display anti-inflammatory properties. We analyzed whether L. casei could attenuate the pro-inflammatory signaling induced by Shigella flexneri after invasion of the epithelial lining. Cultured epithelial cells were infected with L. casei, followed by a challenge with S. flexneri. Using macroarray DNA chips, we observed that L. casei down-regulated the transcription of a number of genes encoding pro-inflammatory effectors such as cytokines and chemokines and adherence molecules induced by invasive S. flexneri. This resulted in an anti-inflammatory effect that appeared mediated by the inhibition of the NF-kappaB pathway, particularly through stabilization of I-kappaBalpha. In a time-course experiment using GeneChip hybridization analysis, the expression of many genes involved in ubiquitination and proteasome processes were modulated during L. casei treatment. Thus, L. casei has developed a sophisticated means to maintain intestinal homeostasis through a process that involves manipulation of the ubiquitin/proteasome pathway upstream of I-kappaBalpha.
Shigella invades the human intestinal mucosa, thus causing bacillary dysentery, an acute recto-colitis responsible for lethal complications, mostly in infants and toddlers. Conversely, commensal bacteria live in a mutualistic relationship with the intestinal mucosa that is characterized by homeostatic control of innate responses, thereby contributing to tolerance to the flora. Cross-talk established between commensals and the intestinal epithelium mediate this active process, the mechanisms of which remain largely uncharacterized. Probiotics such as Lactobacillus casei belong to a subclass of these commensals that modulate mucosal innate responses and possibly display anti-inflammatory properties. We analyzed whether L. casei could attenuate the pro-inflammatory signaling induced by Shigella flexneri after invasion of the epithelial lining. Cultured epithelial cells were infected with L. casei, followed by a challenge with S. flexneri. Using macroarray DNA chips, we observed that L. casei down-regulated the transcription of a number of genes encoding pro-inflammatory effectors such as cytokines and chemokines and adherence molecules induced by invasive S. flexneri. This resulted in an anti-inflammatory effect that appeared mediated by the inhibition of the NF-kappaB pathway, particularly through stabilization of I-kappaBalpha. In a time-course experiment using GeneChip hybridization analysis, the expression of many genes involved in ubiquitination and proteasome processes were modulated during L. casei treatment. Thus, L. casei has developed a sophisticated means to maintain intestinal homeostasis through a process that involves manipulation of the ubiquitin/proteasome pathway upstream of I-kappaBalpha.
Shigella invades the human intestinal mucosa, thus causing bacillary dysentery, an acute recto-colitis responsible for lethal complications, mostly in infants and toddlers. Conversely, commensal bacteria live in a mutualistic relationship with the intestinal mucosa that is characterized by homeostatic control of innate responses, thereby contributing to tolerance to the flora. Cross-talk established between commensals and the intestinal epithelium mediate this active process, the mechanisms of which remain largely uncharacterized. Probiotics such as Lactobacillus casei belong to a subclass of these commensals that modulate mucosal innate responses and possibly display anti-inflammatory properties. We analyzed whether L. casei could attenuate the pro-inflammatory signaling induced by Shigella flexneri after invasion of the epithelial lining. Cultured epithelial cells were infected with L. casei, followed by a challenge with S. flexneri. Using macroarray DNA chips, we observed that L. casei down-regulated the transcription of a number of genes encoding pro-inflammatory effectors such as cytokines and chemokines and adherence molecules induced by invasive S. flexneri. This resulted in an anti-inflammatory effect that appeared mediated by the inhibition of the NF-kappaB pathway, particularly through stabilization of I-kappaBalpha. In a time-course experiment using GeneChip hybridization analysis, the expression of many genes involved in ubiquitination and proteasome processes were modulated during L. casei treatment. Thus, L. casei has developed a sophisticated means to maintain intestinal homeostasis through a process that involves manipulation of the ubiquitin/proteasome pathway upstream of I-kappaBalpha.
Human macrophage genes regulated by live P.gingivalis. We attempted in the present study to identify transcriptional profiles that are modulated after treatment with P. gingivalis relative to its purified LPS or its major fimbrial protein (FimA).3 We used the cDNA microarray technique to define more precisely the individual effects of LPS, FimA, and their parental bacterium, P. gingivalis, on the transcriptional reprogramming of human macrophages.Of the 54674 genes (probe sets) assayed by the genomebased cDNA microarray, P. gingivalis, its LPS, and its FimA commonly regulated 266 genes, while 501, 286, and 291 genes were uniquely regulated by P. gingivalis, its LPS, or its FimA, respectively. The complete list of changes in macrophage transcripts transcripts in response to P. gingivalis infection and to its LPS or FimA treatment is available in the Supplemental Data.
Human macrophage genes regulated by live P.gingivalis. We attempted in the present study to identify transcriptional profiles that are modulated after treatment with P. gingivalis relative to its purified LPS or its major fimbrial protein (FimA).3 We used the cDNA microarray technique to define more precisely the individual effects of LPS, FimA, and their parental bacterium, P. gingivalis, on the transcriptional reprogramming of human macrophages.Of the 54674 genes (probe sets) assayed by the genomebased cDNA microarray, P. gingivalis, its LPS, and its FimA commonly regulated 266 genes, while 501, 286, and 291 genes were uniquely regulated by P. gingivalis, its LPS, or its FimA, respectively. The complete list of changes in macrophage transcripts transcripts in response to P. gingivalis infection and to its LPS or FimA treatment is available in the Supplemental Data.
Human macrophage genes regulated by live P.gingivalis. We attempted in the present study to identify transcriptional profiles that are modulated after treatment with P. gingivalis relative to its purified LPS or its major fimbrial protein (FimA).3 We used the cDNA microarray technique to define more precisely the individual effects of LPS, FimA, and their parental bacterium, P. gingivalis, on the transcriptional reprogramming of human macrophages.Of the 54674 genes (probe sets) assayed by the genomebased cDNA microarray, P. gingivalis, its LPS, and its FimA commonly regulated 266 genes, while 501, 286, and 291 genes were uniquely regulated by P. gingivalis, its LPS, or its FimA, respectively. The complete list of changes in macrophage transcripts transcripts in response to P. gingivalis infection and to its LPS or FimA treatment is available in the Supplemental Data.
Human macrophage genes regulated by live P.gingivalis. We attempted in the present study to identify transcriptional profiles that are modulated after treatment with P. gingivalis relative to its purified LPS or its major fimbrial protein (FimA).3 We used the cDNA microarray technique to define more precisely the individual effects of LPS, FimA, and their parental bacterium, P. gingivalis, on the transcriptional reprogramming of human macrophages.Of the 54674 genes (probe sets) assayed by the genomebased cDNA microarray, P. gingivalis, its LPS, and its FimA commonly regulated 266 genes, while 501, 286, and 291 genes were uniquely regulated by P. gingivalis, its LPS, or its FimA, respectively. The complete list of changes in macrophage transcripts transcripts in response to P. gingivalis infection and to its LPS or FimA treatment is available in the Supplemental Data.
A number of solid tumors, such as alveolar rhabdomyosarcoma, synovial sarcoma, and myxoid liposarcoma, are associated with recurrent translocation events that encode fusion proteins. Ewing's sarcoma is a pediatric tumor that serves as a prototype for this tumor class. Ewing's sarcomas usually harbor the (11;22)(q24;q12) translocation. The t(11;22) encodes the EWS/FLI fusion oncoprotein. EWS/FLI functions as an aberrant transcription factor, but the key target genes that are involved in oncogenesis are largely unknown. Although some target genes have been defined, many of these have been identified in heterologous model systems with uncertain relevance to the human disease. To understand the function of EWS/FLI and its targets in a more clinically relevant system, we used retroviral-mediated RNAi to "knock-down" the fusion protein in patient-derived Ewing's sarcoma cell lines. By combining transcriptional profiling data from three of these lines, we identified a conserved transcriptional response to EWS/FLI. The gene that was most reproducibly up-regulated by EWS/FLI was NR0B1. NR0B1 is a developmentally important orphan nuclear receptor with no previously defined role in oncogenesis. We validated NR0B1 as an EWS/FLI-dysregulated gene and confirmed its expression in primary human tumor samples. Functional studies revealed that ongoing NR0B1 expression is required for the transformed phenotype of Ewing's sarcoma. These studies define a new role for NR0B1 in oncogenic transformation and emphasize the utility of analyzing the function of EWS/FLI in Ewing's sarcoma cells.
A number of solid tumors, such as alveolar rhabdomyosarcoma, synovial sarcoma, and myxoid liposarcoma, are associated with recurrent translocation events that encode fusion proteins. Ewing's sarcoma is a pediatric tumor that serves as a prototype for this tumor class. Ewing's sarcomas usually harbor the (11;22)(q24;q12) translocation. The t(11;22) encodes the EWS/FLI fusion oncoprotein. EWS/FLI functions as an aberrant transcription factor, but the key target genes that are involved in oncogenesis are largely unknown. Although some target genes have been defined, many of these have been identified in heterologous model systems with uncertain relevance to the human disease. To understand the function of EWS/FLI and its targets in a more clinically relevant system, we used retroviral-mediated RNAi to "knock-down" the fusion protein in patient-derived Ewing's sarcoma cell lines. By combining transcriptional profiling data from three of these lines, we identified a conserved transcriptional response to EWS/FLI. The gene that was most reproducibly up-regulated by EWS/FLI was NR0B1. NR0B1 is a developmentally important orphan nuclear receptor with no previously defined role in oncogenesis. We validated NR0B1 as an EWS/FLI-dysregulated gene and confirmed its expression in primary human tumor samples. Functional studies revealed that ongoing NR0B1 expression is required for the transformed phenotype of Ewing's sarcoma. These studies define a new role for NR0B1 in oncogenic transformation and emphasize the utility of analyzing the function of EWS/FLI in Ewing's sarcoma cells.
Colorectal cancers are believed to arise predominantly from adenomas. Although these precancerous lesions have been subjected to extensive clinical, pathologic, and molecular analyses, little is currently known about the global gene expression changes accompanying their formation. To characterize the molecular processes underlying the transformation of normal colonic epithelium, we compared the transcriptomes of 32 prospectively collected adenomas with those of normal mucosa from the same individuals. Important differences emerged not only between the expression profiles of normal and adenomatous tissues but also between those of small and large adenomas. A key feature of the transformation process was the remodeling of the Wnt pathway reflected in patent overexpression and underexpression of 78 known components of this signaling cascade. The expression of 19 Wnt targets was closely correlated with clear up-regulation of KIAA1199, whose function is currently unknown. In normal mucosa, KIAA1199 expression was confined to cells in the lower portion of intestinal crypts, where Wnt signaling is physiologically active, but it was markedly increased in all adenomas, where it was expressed in most of the epithelial cells, and in colon cancer cell lines, it was markedly reduced by inactivation of the beta-catenin/T-cell factor(s) transcription complex, the pivotal mediator of Wnt signaling. Our transcriptomic profiles of normal colonic mucosa and colorectal adenomas shed new light on the early stages of colorectal tumorigenesis and identified KIAA1199 as a novel target of the Wnt signaling pathway and a putative marker of colorectal adenomatous transformation.
Colorectal cancers are believed to arise predominantly from adenomas. Although these precancerous lesions have been subjected to extensive clinical, pathologic, and molecular analyses, little is currently known about the global gene expression changes accompanying their formation. To characterize the molecular processes underlying the transformation of normal colonic epithelium, we compared the transcriptomes of 32 prospectively collected adenomas with those of normal mucosa from the same individuals. Important differences emerged not only between the expression profiles of normal and adenomatous tissues but also between those of small and large adenomas. A key feature of the transformation process was the remodeling of the Wnt pathway reflected in patent overexpression and underexpression of 78 known components of this signaling cascade. The expression of 19 Wnt targets was closely correlated with clear up-regulation of KIAA1199, whose function is currently unknown. In normal mucosa, KIAA1199 expression was confined to cells in the lower portion of intestinal crypts, where Wnt signaling is physiologically active, but it was markedly increased in all adenomas, where it was expressed in most of the epithelial cells, and in colon cancer cell lines, it was markedly reduced by inactivation of the beta-catenin/T-cell factor(s) transcription complex, the pivotal mediator of Wnt signaling. Our transcriptomic profiles of normal colonic mucosa and colorectal adenomas shed new light on the early stages of colorectal tumorigenesis and identified KIAA1199 as a novel target of the Wnt signaling pathway and a putative marker of colorectal adenomatous transformation.
Colorectal cancers are believed to arise predominantly from adenomas. Although these precancerous lesions have been subjected to extensive clinical, pathologic, and molecular analyses, little is currently known about the global gene expression changes accompanying their formation. To characterize the molecular processes underlying the transformation of normal colonic epithelium, we compared the transcriptomes of 32 prospectively collected adenomas with those of normal mucosa from the same individuals. Important differences emerged not only between the expression profiles of normal and adenomatous tissues but also between those of small and large adenomas. A key feature of the transformation process was the remodeling of the Wnt pathway reflected in patent overexpression and underexpression of 78 known components of this signaling cascade. The expression of 19 Wnt targets was closely correlated with clear up-regulation of KIAA1199, whose function is currently unknown. In normal mucosa, KIAA1199 expression was confined to cells in the lower portion of intestinal crypts, where Wnt signaling is physiologically active, but it was markedly increased in all adenomas, where it was expressed in most of the epithelial cells, and in colon cancer cell lines, it was markedly reduced by inactivation of the beta-catenin/T-cell factor(s) transcription complex, the pivotal mediator of Wnt signaling. Our transcriptomic profiles of normal colonic mucosa and colorectal adenomas shed new light on the early stages of colorectal tumorigenesis and identified KIAA1199 as a novel target of the Wnt signaling pathway and a putative marker of colorectal adenomatous transformation.
Colorectal cancers are believed to arise predominantly from adenomas. Although these precancerous lesions have been subjected to extensive clinical, pathologic, and molecular analyses, little is currently known about the global gene expression changes accompanying their formation. To characterize the molecular processes underlying the transformation of normal colonic epithelium, we compared the transcriptomes of 32 prospectively collected adenomas with those of normal mucosa from the same individuals. Important differences emerged not only between the expression profiles of normal and adenomatous tissues but also between those of small and large adenomas. A key feature of the transformation process was the remodeling of the Wnt pathway reflected in patent overexpression and underexpression of 78 known components of this signaling cascade. The expression of 19 Wnt targets was closely correlated with clear up-regulation of KIAA1199, whose function is currently unknown. In normal mucosa, KIAA1199 expression was confined to cells in the lower portion of intestinal crypts, where Wnt signaling is physiologically active, but it was markedly increased in all adenomas, where it was expressed in most of the epithelial cells, and in colon cancer cell lines, it was markedly reduced by inactivation of the beta-catenin/T-cell factor(s) transcription complex, the pivotal mediator of Wnt signaling. Our transcriptomic profiles of normal colonic mucosa and colorectal adenomas shed new light on the early stages of colorectal tumorigenesis and identified KIAA1199 as a novel target of the Wnt signaling pathway and a putative marker of colorectal adenomatous transformation.
The PLU-1/JARID1B nuclear protein, which is upregulated in breast cancers, belongs to the ARID family of DNA binding proteins and has strong transcriptional repression activity. To identify the target genes regulated by PLU-1/JARID1B, we overexpressed or silenced the human PLU-1/JARID1B gene in human mammary epithelial cells by using adenovirus and RNA interference systems, respectively, and then applied microarray analysis to identify candidate genes. A total of 100 genes showed inversely correlated differential expression in the two systems. Most of the candidate genes were downregulated by the overexpression of PLU-1/JARID1B, including the MT genes, the tumor suppressor gene BRCA1, and genes involved in the regulation of the M phase of the mitotic cell cycle. Chromatin immunoprecipitation assays confirmed that the metallothionein 1H (MT1H), -1F, and -1X genes are direct transcriptional targets of PLU-1/JARID1B in vivo. Furthermore, the level of trimethyl H3K4 of the MT1H promoter was increased following silencing of PLU-1/JARID1B. Both the PLU-1/JARID1B protein and the ARID domain selectively bound CG-rich DNA. The GCACA/C motif, which is abundant in metallothionein promoters, was identified as a consensus binding sequence of the PLU-1/JARID1B ARID domain. As expected from the microarray data, cells overexpressing PLU-1/JARID1B have an impaired G(2)/M checkpoint. Our study provides insight into the molecular function of the breast cancer-associated transcriptional repressor PLU-1/JARID1B.
The PLU-1/JARID1B nuclear protein, which is upregulated in breast cancers, belongs to the ARID family of DNA binding proteins and has strong transcriptional repression activity. To identify the target genes regulated by PLU-1/JARID1B, we overexpressed or silenced the human PLU-1/JARID1B gene in human mammary epithelial cells by using adenovirus and RNA interference systems, respectively, and then applied microarray analysis to identify candidate genes. A total of 100 genes showed inversely correlated differential expression in the two systems. Most of the candidate genes were downregulated by the overexpression of PLU-1/JARID1B, including the MT genes, the tumor suppressor gene BRCA1, and genes involved in the regulation of the M phase of the mitotic cell cycle. Chromatin immunoprecipitation assays confirmed that the metallothionein 1H (MT1H), -1F, and -1X genes are direct transcriptional targets of PLU-1/JARID1B in vivo. Furthermore, the level of trimethyl H3K4 of the MT1H promoter was increased following silencing of PLU-1/JARID1B. Both the PLU-1/JARID1B protein and the ARID domain selectively bound CG-rich DNA. The GCACA/C motif, which is abundant in metallothionein promoters, was identified as a consensus binding sequence of the PLU-1/JARID1B ARID domain. As expected from the microarray data, cells overexpressing PLU-1/JARID1B have an impaired G(2)/M checkpoint. Our study provides insight into the molecular function of the breast cancer-associated transcriptional repressor PLU-1/JARID1B.
ErbB receptor ligands, epidermal growth factor (EGF) and heregulin (HRG), induce dose-dependent transient and sustained intracellular signaling, proliferation, and differentiation of MCF-7 breast cancer cells, respectively. In an effort to delineate the ligand-specific cell determination mechanism, we investigated time course gene expressions induced by EGF and HRG that induce distinct cellular phenotypes in MCF-7 cells. To analyze independently the effects of ligand dosage and time for gene expression, we developed a statistical method for estimating the two effects. Our results indicated that signal transduction pathways convey quantitative properties of the dose-dependent activation of ErbB receptor to early transcription. The results also implied that moderate changes in the expression levels of a number of genes, not the predominant regulation of a few specific genes, might cooperatively work at the early stage of the transcription for determining cell fate. However, the EGF- and HRG-induced distinct signal durations resulted in the ligand-oriented biphasic induction of proteins after 20 min. The selected gene list and HRG-induced prolonged signaling suggested that transcriptional feedback to the intracellular signaling results in a graded to biphasic response in the cell determination process and that each ErbB receptor is inextricably responsible for the control of amplitude and duration of cellular biochemical reactions.
ErbB receptor ligands, epidermal growth factor (EGF) and heregulin (HRG), induce dose-dependent transient and sustained intracellular signaling, proliferation, and differentiation of MCF-7 breast cancer cells, respectively. In an effort to delineate the ligand-specific cell determination mechanism, we investigated time course gene expressions induced by EGF and HRG that induce distinct cellular phenotypes in MCF-7 cells. To analyze independently the effects of ligand dosage and time for gene expression, we developed a statistical method for estimating the two effects. Our results indicated that signal transduction pathways convey quantitative properties of the dose-dependent activation of ErbB receptor to early transcription. The results also implied that moderate changes in the expression levels of a number of genes, not the predominant regulation of a few specific genes, might cooperatively work at the early stage of the transcription for determining cell fate. However, the EGF- and HRG-induced distinct signal durations resulted in the ligand-oriented biphasic induction of proteins after 20 min. The selected gene list and HRG-induced prolonged signaling suggested that transcriptional feedback to the intracellular signaling results in a graded to biphasic response in the cell determination process and that each ErbB receptor is inextricably responsible for the control of amplitude and duration of cellular biochemical reactions.
ErbB receptor ligands, epidermal growth factor (EGF) and heregulin (HRG), induce dose-dependent transient and sustained intracellular signaling, proliferation, and differentiation of MCF-7 breast cancer cells, respectively. In an effort to delineate the ligand-specific cell determination mechanism, we investigated time course gene expressions induced by EGF and HRG that induce distinct cellular phenotypes in MCF-7 cells. To analyze independently the effects of ligand dosage and time for gene expression, we developed a statistical method for estimating the two effects. Our results indicated that signal transduction pathways convey quantitative properties of the dose-dependent activation of ErbB receptor to early transcription. The results also implied that moderate changes in the expression levels of a number of genes, not the predominant regulation of a few specific genes, might cooperatively work at the early stage of the transcription for determining cell fate. However, the EGF- and HRG-induced distinct signal durations resulted in the ligand-oriented biphasic induction of proteins after 20 min. The selected gene list and HRG-induced prolonged signaling suggested that transcriptional feedback to the intracellular signaling results in a graded to biphasic response in the cell determination process and that each ErbB receptor is inextricably responsible for the control of amplitude and duration of cellular biochemical reactions.
The Wilms' tumor suppressor gene (WT1) encodes a zinc finger transcription factor that is vital during development of several organs including metanephric kidneys. Despite the critical regulatory role of WT1, the pathways and mechanisms by which WT1 orchestrates development remain elusive. To identify WT1 target genes, we performed a genome-wide expression profiling analysis in cells expressing inducible WT1. We identified a number of direct WT1 target genes, including the epidermal growth factor (EGF)-family ligands epiregulin and HB-EGF, the chemokine CX3CL1, and the transcription factors SLUG and JUNB. The target genes were validated using quantitative reverse transcriptase-polymerase chain reaction, small interfering RNA knockdowns, chromatin immunoprecipitation, and luciferase reporter analyses. Immunohistochemistry of fetal kidneys confirmed that a number of the WT1 target genes had overlapping expression patterns with the highly restricted spatiotemporal expression of WT1. Finally, using an in vitro embryonic kidney culture assay, we found that the addition of recombinant epiregulin, amphiregulin, CX3CL1, and interleukin-11 significantly enhanced ureteric bud branching morphogenesis. Our genome-wide screen implicates WT1 in the transcriptional regulation of the EGF-family of growth factors as well as the CX3CL1 chemokine during nephrogenesis.
The Wilms' tumor suppressor gene (WT1) encodes a zinc finger transcription factor that is vital during development of several organs including metanephric kidneys. Despite the critical regulatory role of WT1, the pathways and mechanisms by which WT1 orchestrates development remain elusive. To identify WT1 target genes, we performed a genome-wide expression profiling analysis in cells expressing inducible WT1. We identified a number of direct WT1 target genes, including the epidermal growth factor (EGF)-family ligands epiregulin and HB-EGF, the chemokine CX3CL1, and the transcription factors SLUG and JUNB. The target genes were validated using quantitative reverse transcriptase-polymerase chain reaction, small interfering RNA knockdowns, chromatin immunoprecipitation, and luciferase reporter analyses. Immunohistochemistry of fetal kidneys confirmed that a number of the WT1 target genes had overlapping expression patterns with the highly restricted spatiotemporal expression of WT1. Finally, using an in vitro embryonic kidney culture assay, we found that the addition of recombinant epiregulin, amphiregulin, CX3CL1, and interleukin-11 significantly enhanced ureteric bud branching morphogenesis. Our genome-wide screen implicates WT1 in the transcriptional regulation of the EGF-family of growth factors as well as the CX3CL1 chemokine during nephrogenesis.
The Wilms' tumor suppressor gene (WT1) encodes a zinc finger transcription factor that is vital during development of several organs including metanephric kidneys. Despite the critical regulatory role of WT1, the pathways and mechanisms by which WT1 orchestrates development remain elusive. To identify WT1 target genes, we performed a genome-wide expression profiling analysis in cells expressing inducible WT1. We identified a number of direct WT1 target genes, including the epidermal growth factor (EGF)-family ligands epiregulin and HB-EGF, the chemokine CX3CL1, and the transcription factors SLUG and JUNB. The target genes were validated using quantitative reverse transcriptase-polymerase chain reaction, small interfering RNA knockdowns, chromatin immunoprecipitation, and luciferase reporter analyses. Immunohistochemistry of fetal kidneys confirmed that a number of the WT1 target genes had overlapping expression patterns with the highly restricted spatiotemporal expression of WT1. Finally, using an in vitro embryonic kidney culture assay, we found that the addition of recombinant epiregulin, amphiregulin, CX3CL1, and interleukin-11 significantly enhanced ureteric bud branching morphogenesis. Our genome-wide screen implicates WT1 in the transcriptional regulation of the EGF-family of growth factors as well as the CX3CL1 chemokine during nephrogenesis.
The Wilms' tumor suppressor gene (WT1) encodes a zinc finger transcription factor that is vital during development of several organs including metanephric kidneys. Despite the critical regulatory role of WT1, the pathways and mechanisms by which WT1 orchestrates development remain elusive. To identify WT1 target genes, we performed a genome-wide expression profiling analysis in cells expressing inducible WT1. We identified a number of direct WT1 target genes, including the epidermal growth factor (EGF)-family ligands epiregulin and HB-EGF, the chemokine CX3CL1, and the transcription factors SLUG and JUNB. The target genes were validated using quantitative reverse transcriptase-polymerase chain reaction, small interfering RNA knockdowns, chromatin immunoprecipitation, and luciferase reporter analyses. Immunohistochemistry of fetal kidneys confirmed that a number of the WT1 target genes had overlapping expression patterns with the highly restricted spatiotemporal expression of WT1. Finally, using an in vitro embryonic kidney culture assay, we found that the addition of recombinant epiregulin, amphiregulin, CX3CL1, and interleukin-11 significantly enhanced ureteric bud branching morphogenesis. Our genome-wide screen implicates WT1 in the transcriptional regulation of the EGF-family of growth factors as well as the CX3CL1 chemokine during nephrogenesis.
The Wilms' tumor suppressor gene (WT1) encodes a zinc finger transcription factor that is vital during development of several organs including metanephric kidneys. Despite the critical regulatory role of WT1, the pathways and mechanisms by which WT1 orchestrates development remain elusive. To identify WT1 target genes, we performed a genome-wide expression profiling analysis in cells expressing inducible WT1. We identified a number of direct WT1 target genes, including the epidermal growth factor (EGF)-family ligands epiregulin and HB-EGF, the chemokine CX3CL1, and the transcription factors SLUG and JUNB. The target genes were validated using quantitative reverse transcriptase-polymerase chain reaction, small interfering RNA knockdowns, chromatin immunoprecipitation, and luciferase reporter analyses. Immunohistochemistry of fetal kidneys confirmed that a number of the WT1 target genes had overlapping expression patterns with the highly restricted spatiotemporal expression of WT1. Finally, using an in vitro embryonic kidney culture assay, we found that the addition of recombinant epiregulin, amphiregulin, CX3CL1, and interleukin-11 significantly enhanced ureteric bud branching morphogenesis. Our genome-wide screen implicates WT1 in the transcriptional regulation of the EGF-family of growth factors as well as the CX3CL1 chemokine during nephrogenesis.
The Wilms' tumor suppressor gene (WT1) encodes a zinc finger transcription factor that is vital during development of several organs including metanephric kidneys. Despite the critical regulatory role of WT1, the pathways and mechanisms by which WT1 orchestrates development remain elusive. To identify WT1 target genes, we performed a genome-wide expression profiling analysis in cells expressing inducible WT1. We identified a number of direct WT1 target genes, including the epidermal growth factor (EGF)-family ligands epiregulin and HB-EGF, the chemokine CX3CL1, and the transcription factors SLUG and JUNB. The target genes were validated using quantitative reverse transcriptase-polymerase chain reaction, small interfering RNA knockdowns, chromatin immunoprecipitation, and luciferase reporter analyses. Immunohistochemistry of fetal kidneys confirmed that a number of the WT1 target genes had overlapping expression patterns with the highly restricted spatiotemporal expression of WT1. Finally, using an in vitro embryonic kidney culture assay, we found that the addition of recombinant epiregulin, amphiregulin, CX3CL1, and interleukin-11 significantly enhanced ureteric bud branching morphogenesis. Our genome-wide screen implicates WT1 in the transcriptional regulation of the EGF-family of growth factors as well as the CX3CL1 chemokine during nephrogenesis.
Studies of gene regulation by oxygen have revealed novel signal pathways that regulate the hypoxia-inducible factor (HIF) transcriptional system through post-translational hydroxylation of specific prolyl and asparaginyl residues in HIF-alpha subunits. These oxygen-sensitive modifications are catalyzed by members of the 2-oxoglutarate (2-OG) dioxygenase family (PHD1, PHD2, PHD3, and FIH-1), raising an important question regarding the extent of involvement of these and other enzymes of the same family in directing the global changes in gene expression that are induced by hypoxia. To address this, we compared patterns of gene expression induced by hypoxia and by a nonspecific 2-OG-dependent dioxygenase inhibitor, dimethyloxalylglycine (DMOG), among a set of 22,000 transcripts, by microarray analysis of MCF7 cells. By using short interfering RNA-based suppression of HIF-alpha subunits, we also compared responses that were dependent on, or independent of, the HIF system. Results revealed striking concordance between patterns of gene expression induced by hypoxia and by DMOG, indicating the central involvement of 2-OG-dependent dioxygenases in oxygen-regulated gene expression. Many of these responses were suppressed by short interfering RNAs directed against HIF-1alpha and HIF-2alpha, with HIF-1alpha suppression manifesting substantially greater effects than HIF-2alpha suppression, supporting the importance of HIF pathways. Nevertheless, the definition of genes regulated by both hypoxia and DMOG, but not HIF, distinguished other pathways most likely involving the action of 2-OG-dependent dioxygenases on non-HIF substrates.
Studies of gene regulation by oxygen have revealed novel signal pathways that regulate the hypoxia-inducible factor (HIF) transcriptional system through post-translational hydroxylation of specific prolyl and asparaginyl residues in HIF-alpha subunits. These oxygen-sensitive modifications are catalyzed by members of the 2-oxoglutarate (2-OG) dioxygenase family (PHD1, PHD2, PHD3, and FIH-1), raising an important question regarding the extent of involvement of these and other enzymes of the same family in directing the global changes in gene expression that are induced by hypoxia. To address this, we compared patterns of gene expression induced by hypoxia and by a nonspecific 2-OG-dependent dioxygenase inhibitor, dimethyloxalylglycine (DMOG), among a set of 22,000 transcripts, by microarray analysis of MCF7 cells. By using short interfering RNA-based suppression of HIF-alpha subunits, we also compared responses that were dependent on, or independent of, the HIF system. Results revealed striking concordance between patterns of gene expression induced by hypoxia and by DMOG, indicating the central involvement of 2-OG-dependent dioxygenases in oxygen-regulated gene expression. Many of these responses were suppressed by short interfering RNAs directed against HIF-1alpha and HIF-2alpha, with HIF-1alpha suppression manifesting substantially greater effects than HIF-2alpha suppression, supporting the importance of HIF pathways. Nevertheless, the definition of genes regulated by both hypoxia and DMOG, but not HIF, distinguished other pathways most likely involving the action of 2-OG-dependent dioxygenases on non-HIF substrates.
Studies of gene regulation by oxygen have revealed novel signal pathways that regulate the hypoxia-inducible factor (HIF) transcriptional system through post-translational hydroxylation of specific prolyl and asparaginyl residues in HIF-alpha subunits. These oxygen-sensitive modifications are catalyzed by members of the 2-oxoglutarate (2-OG) dioxygenase family (PHD1, PHD2, PHD3, and FIH-1), raising an important question regarding the extent of involvement of these and other enzymes of the same family in directing the global changes in gene expression that are induced by hypoxia. To address this, we compared patterns of gene expression induced by hypoxia and by a nonspecific 2-OG-dependent dioxygenase inhibitor, dimethyloxalylglycine (DMOG), among a set of 22,000 transcripts, by microarray analysis of MCF7 cells. By using short interfering RNA-based suppression of HIF-alpha subunits, we also compared responses that were dependent on, or independent of, the HIF system. Results revealed striking concordance between patterns of gene expression induced by hypoxia and by DMOG, indicating the central involvement of 2-OG-dependent dioxygenases in oxygen-regulated gene expression. Many of these responses were suppressed by short interfering RNAs directed against HIF-1alpha and HIF-2alpha, with HIF-1alpha suppression manifesting substantially greater effects than HIF-2alpha suppression, supporting the importance of HIF pathways. Nevertheless, the definition of genes regulated by both hypoxia and DMOG, but not HIF, distinguished other pathways most likely involving the action of 2-OG-dependent dioxygenases on non-HIF substrates.
Studies of gene regulation by oxygen have revealed novel signal pathways that regulate the hypoxia-inducible factor (HIF) transcriptional system through post-translational hydroxylation of specific prolyl and asparaginyl residues in HIF-alpha subunits. These oxygen-sensitive modifications are catalyzed by members of the 2-oxoglutarate (2-OG) dioxygenase family (PHD1, PHD2, PHD3, and FIH-1), raising an important question regarding the extent of involvement of these and other enzymes of the same family in directing the global changes in gene expression that are induced by hypoxia. To address this, we compared patterns of gene expression induced by hypoxia and by a nonspecific 2-OG-dependent dioxygenase inhibitor, dimethyloxalylglycine (DMOG), among a set of 22,000 transcripts, by microarray analysis of MCF7 cells. By using short interfering RNA-based suppression of HIF-alpha subunits, we also compared responses that were dependent on, or independent of, the HIF system. Results revealed striking concordance between patterns of gene expression induced by hypoxia and by DMOG, indicating the central involvement of 2-OG-dependent dioxygenases in oxygen-regulated gene expression. Many of these responses were suppressed by short interfering RNAs directed against HIF-1alpha and HIF-2alpha, with HIF-1alpha suppression manifesting substantially greater effects than HIF-2alpha suppression, supporting the importance of HIF pathways. Nevertheless, the definition of genes regulated by both hypoxia and DMOG, but not HIF, distinguished other pathways most likely involving the action of 2-OG-dependent dioxygenases on non-HIF substrates.
Studies of gene regulation by oxygen have revealed novel signal pathways that regulate the hypoxia-inducible factor (HIF) transcriptional system through post-translational hydroxylation of specific prolyl and asparaginyl residues in HIF-alpha subunits. These oxygen-sensitive modifications are catalyzed by members of the 2-oxoglutarate (2-OG) dioxygenase family (PHD1, PHD2, PHD3, and FIH-1), raising an important question regarding the extent of involvement of these and other enzymes of the same family in directing the global changes in gene expression that are induced by hypoxia. To address this, we compared patterns of gene expression induced by hypoxia and by a nonspecific 2-OG-dependent dioxygenase inhibitor, dimethyloxalylglycine (DMOG), among a set of 22,000 transcripts, by microarray analysis of MCF7 cells. By using short interfering RNA-based suppression of HIF-alpha subunits, we also compared responses that were dependent on, or independent of, the HIF system. Results revealed striking concordance between patterns of gene expression induced by hypoxia and by DMOG, indicating the central involvement of 2-OG-dependent dioxygenases in oxygen-regulated gene expression. Many of these responses were suppressed by short interfering RNAs directed against HIF-1alpha and HIF-2alpha, with HIF-1alpha suppression manifesting substantially greater effects than HIF-2alpha suppression, supporting the importance of HIF pathways. Nevertheless, the definition of genes regulated by both hypoxia and DMOG, but not HIF, distinguished other pathways most likely involving the action of 2-OG-dependent dioxygenases on non-HIF substrates.
Studies of gene regulation by oxygen have revealed novel signal pathways that regulate the hypoxia-inducible factor (HIF) transcriptional system through post-translational hydroxylation of specific prolyl and asparaginyl residues in HIF-alpha subunits. These oxygen-sensitive modifications are catalyzed by members of the 2-oxoglutarate (2-OG) dioxygenase family (PHD1, PHD2, PHD3, and FIH-1), raising an important question regarding the extent of involvement of these and other enzymes of the same family in directing the global changes in gene expression that are induced by hypoxia. To address this, we compared patterns of gene expression induced by hypoxia and by a nonspecific 2-OG-dependent dioxygenase inhibitor, dimethyloxalylglycine (DMOG), among a set of 22,000 transcripts, by microarray analysis of MCF7 cells. By using short interfering RNA-based suppression of HIF-alpha subunits, we also compared responses that were dependent on, or independent of, the HIF system. Results revealed striking concordance between patterns of gene expression induced by hypoxia and by DMOG, indicating the central involvement of 2-OG-dependent dioxygenases in oxygen-regulated gene expression. Many of these responses were suppressed by short interfering RNAs directed against HIF-1alpha and HIF-2alpha, with HIF-1alpha suppression manifesting substantially greater effects than HIF-2alpha suppression, supporting the importance of HIF pathways. Nevertheless, the definition of genes regulated by both hypoxia and DMOG, but not HIF, distinguished other pathways most likely involving the action of 2-OG-dependent dioxygenases on non-HIF substrates.
Studies of gene regulation by oxygen have revealed novel signal pathways that regulate the hypoxia-inducible factor (HIF) transcriptional system through post-translational hydroxylation of specific prolyl and asparaginyl residues in HIF-alpha subunits. These oxygen-sensitive modifications are catalyzed by members of the 2-oxoglutarate (2-OG) dioxygenase family (PHD1, PHD2, PHD3, and FIH-1), raising an important question regarding the extent of involvement of these and other enzymes of the same family in directing the global changes in gene expression that are induced by hypoxia. To address this, we compared patterns of gene expression induced by hypoxia and by a nonspecific 2-OG-dependent dioxygenase inhibitor, dimethyloxalylglycine (DMOG), among a set of 22,000 transcripts, by microarray analysis of MCF7 cells. By using short interfering RNA-based suppression of HIF-alpha subunits, we also compared responses that were dependent on, or independent of, the HIF system. Results revealed striking concordance between patterns of gene expression induced by hypoxia and by DMOG, indicating the central involvement of 2-OG-dependent dioxygenases in oxygen-regulated gene expression. Many of these responses were suppressed by short interfering RNAs directed against HIF-1alpha and HIF-2alpha, with HIF-1alpha suppression manifesting substantially greater effects than HIF-2alpha suppression, supporting the importance of HIF pathways. Nevertheless, the definition of genes regulated by both hypoxia and DMOG, but not HIF, distinguished other pathways most likely involving the action of 2-OG-dependent dioxygenases on non-HIF substrates.
Studies of gene regulation by oxygen have revealed novel signal pathways that regulate the hypoxia-inducible factor (HIF) transcriptional system through post-translational hydroxylation of specific prolyl and asparaginyl residues in HIF-alpha subunits. These oxygen-sensitive modifications are catalyzed by members of the 2-oxoglutarate (2-OG) dioxygenase family (PHD1, PHD2, PHD3, and FIH-1), raising an important question regarding the extent of involvement of these and other enzymes of the same family in directing the global changes in gene expression that are induced by hypoxia. To address this, we compared patterns of gene expression induced by hypoxia and by a nonspecific 2-OG-dependent dioxygenase inhibitor, dimethyloxalylglycine (DMOG), among a set of 22,000 transcripts, by microarray analysis of MCF7 cells. By using short interfering RNA-based suppression of HIF-alpha subunits, we also compared responses that were dependent on, or independent of, the HIF system. Results revealed striking concordance between patterns of gene expression induced by hypoxia and by DMOG, indicating the central involvement of 2-OG-dependent dioxygenases in oxygen-regulated gene expression. Many of these responses were suppressed by short interfering RNAs directed against HIF-1alpha and HIF-2alpha, with HIF-1alpha suppression manifesting substantially greater effects than HIF-2alpha suppression, supporting the importance of HIF pathways. Nevertheless, the definition of genes regulated by both hypoxia and DMOG, but not HIF, distinguished other pathways most likely involving the action of 2-OG-dependent dioxygenases on non-HIF substrates.
Studies of gene regulation by oxygen have revealed novel signal pathways that regulate the hypoxia-inducible factor (HIF) transcriptional system through post-translational hydroxylation of specific prolyl and asparaginyl residues in HIF-alpha subunits. These oxygen-sensitive modifications are catalyzed by members of the 2-oxoglutarate (2-OG) dioxygenase family (PHD1, PHD2, PHD3, and FIH-1), raising an important question regarding the extent of involvement of these and other enzymes of the same family in directing the global changes in gene expression that are induced by hypoxia. To address this, we compared patterns of gene expression induced by hypoxia and by a nonspecific 2-OG-dependent dioxygenase inhibitor, dimethyloxalylglycine (DMOG), among a set of 22,000 transcripts, by microarray analysis of MCF7 cells. By using short interfering RNA-based suppression of HIF-alpha subunits, we also compared responses that were dependent on, or independent of, the HIF system. Results revealed striking concordance between patterns of gene expression induced by hypoxia and by DMOG, indicating the central involvement of 2-OG-dependent dioxygenases in oxygen-regulated gene expression. Many of these responses were suppressed by short interfering RNAs directed against HIF-1alpha and HIF-2alpha, with HIF-1alpha suppression manifesting substantially greater effects than HIF-2alpha suppression, supporting the importance of HIF pathways. Nevertheless, the definition of genes regulated by both hypoxia and DMOG, but not HIF, distinguished other pathways most likely involving the action of 2-OG-dependent dioxygenases on non-HIF substrates.
Human cord blood (CB)-derived CD133+ cells carry characteristics of primitive hematopoietic cells and proffer an alternative for CD34+ cells in hematopoietic stem cell (HSC) transplantation. To characterize the CD133+ cell population on a genetic level, a global expression analysis of CD133+ cells was performed using oligonucleotide microarrays. CD133+ cells were purified from four fresh CB units by immunomagnetic selection. All four CD133+ samples showed significant similarity in their gene expression pattern, whereas they differed clearly from the CD133- control samples. In all, 690 transcripts were differentially expressed between CD133+ and CD133- cells. Of these, 393 were increased and 297 were decreased in CD133+ cells. The highest overexpression was noted in genes associated with metabolism, cellular physiological processes, cell communication, and development. A set of 257 transcripts expressed solely in the CD133+ cell population was identified. Colony-forming unit (CFU) assay was used to detect the clonal progeny of precursors present in the studied cell populations. The results demonstrate that CD133+ cells express primitive markers and possess clonogenic progenitor capacity. This study provides a gene expression profile for human CD133+ cells. It presents a set of genes that may be used to unravel the properties of the CD133+ cell population, assumed to be highly enriched in HSCs.
Human cord blood (CB)-derived CD133+ cells carry characteristics of primitive hematopoietic cells and proffer an alternative for CD34+ cells in hematopoietic stem cell (HSC) transplantation. To characterize the CD133+ cell population on a genetic level, a global expression analysis of CD133+ cells was performed using oligonucleotide microarrays. CD133+ cells were purified from four fresh CB units by immunomagnetic selection. All four CD133+ samples showed significant similarity in their gene expression pattern, whereas they differed clearly from the CD133- control samples. In all, 690 transcripts were differentially expressed between CD133+ and CD133- cells. Of these, 393 were increased and 297 were decreased in CD133+ cells. The highest overexpression was noted in genes associated with metabolism, cellular physiological processes, cell communication, and development. A set of 257 transcripts expressed solely in the CD133+ cell population was identified. Colony-forming unit (CFU) assay was used to detect the clonal progeny of precursors present in the studied cell populations. The results demonstrate that CD133+ cells express primitive markers and possess clonogenic progenitor capacity. This study provides a gene expression profile for human CD133+ cells. It presents a set of genes that may be used to unravel the properties of the CD133+ cell population, assumed to be highly enriched in HSCs.
Facioscapulohumeral muscular dystrophy (FSHD) is the third most frequent inherited muscle disease. Because in FSHD patients the coexistence of affected and unaffected muscles is common, myoblasts expanded from unaffected FSHD muscles have been proposed as suitable tools for autologous cell transplantation. Mesoangioblasts are a new class of adult stem cells of mesodermal origin, potentially useful for the treatment of primitive myopathies of different etiology. Here, we report the isolation and characterization of mesoangioblasts from FSHD muscle biopsies and describe morphology, proliferation, and differentiation abilities of both mesoangioblasts and myoblasts derived from various affected and unaffected muscles of nine representative FSHD patients. We demonstrate that mesoangioblasts can be efficiently isolated from FSHD muscle biopsies and expanded to an amount of cells necessary to transplant into an adult patient. Proliferating mesoangioblasts from all muscles examined did not differ from controls in terms of morphology, phenotype, proliferation rate, or clonogenicity. However, their differentiation ability into skeletal muscle was variably impaired, and this defect correlated with the overall disease severity and the degree of histopathologic abnormalities of the muscle of origin. A remarkable differentiation defect was observed in mesoangioblasts from all mildly to severely affected FSHD muscles, whereas mesoangioblasts from morphologically normal muscles showed no myogenic differentiation block. Our study could open the way to cell therapy for FSHD patients to limit muscle damage in vivo through the use of autologous mesoangioblasts capable of reaching damaged muscles and engrafting into them, without requiring immune suppression or genetic correction in vitro. Disclosure of potential conflicts of interest is found at the end of this article.
Facioscapulohumeral muscular dystrophy (FSHD) is the third most frequent inherited muscle disease. Because in FSHD patients the coexistence of affected and unaffected muscles is common, myoblasts expanded from unaffected FSHD muscles have been proposed as suitable tools for autologous cell transplantation. Mesoangioblasts are a new class of adult stem cells of mesodermal origin, potentially useful for the treatment of primitive myopathies of different etiology. Here, we report the isolation and characterization of mesoangioblasts from FSHD muscle biopsies and describe morphology, proliferation, and differentiation abilities of both mesoangioblasts and myoblasts derived from various affected and unaffected muscles of nine representative FSHD patients. We demonstrate that mesoangioblasts can be efficiently isolated from FSHD muscle biopsies and expanded to an amount of cells necessary to transplant into an adult patient. Proliferating mesoangioblasts from all muscles examined did not differ from controls in terms of morphology, phenotype, proliferation rate, or clonogenicity. However, their differentiation ability into skeletal muscle was variably impaired, and this defect correlated with the overall disease severity and the degree of histopathologic abnormalities of the muscle of origin. A remarkable differentiation defect was observed in mesoangioblasts from all mildly to severely affected FSHD muscles, whereas mesoangioblasts from morphologically normal muscles showed no myogenic differentiation block. Our study could open the way to cell therapy for FSHD patients to limit muscle damage in vivo through the use of autologous mesoangioblasts capable of reaching damaged muscles and engrafting into them, without requiring immune suppression or genetic correction in vitro. Disclosure of potential conflicts of interest is found at the end of this article.
Quiescent and dividing hemopoietic stem cells (HSC) display marked differences in their ability to move between the peripheral circulation and the bone marrow. Specifically, long-term engraftment potential predominantly resides in the quiescent HSC subfraction, and G-CSF mobilization results in the preferential accumulation of quiescent HSC in the periphery. In contrast, stem cells from chronic myeloid leukemia (CML) patients display a constitutive presence in the circulation. To understand the molecular basis for this, we have used microarray technology to analyze the transcriptional differences between dividing and quiescent, normal, and CML-derived CD34+ cells. Our data show a remarkable transcriptional similarity between normal and CML dividing cells, suggesting that the effects of BCR-ABL on the CD34+ cell transcriptome are more limited than previously thought. In addition, we show that quiescent CML cells are more similar to their dividing counterparts than quiescent normal cells are to theirs. We also show these transcriptional differences to be reflected in the altered proliferative activity of normal and CML CD34+ cells. Of the most interest is that the major class of genes that is more abundant in the quiescent cells compared with the dividing cells encodes members of the chemokine family. We propose a role for chemokines expressed by quiescent HSC in the orchestration of CD34+ cell mobilization. Disclosure of potential conflicts of interest is found at the end of this article.
Quiescent and dividing hemopoietic stem cells (HSC) display marked differences in their ability to move between the peripheral circulation and the bone marrow. Specifically, long-term engraftment potential predominantly resides in the quiescent HSC subfraction, and G-CSF mobilization results in the preferential accumulation of quiescent HSC in the periphery. In contrast, stem cells from chronic myeloid leukemia (CML) patients display a constitutive presence in the circulation. To understand the molecular basis for this, we have used microarray technology to analyze the transcriptional differences between dividing and quiescent, normal, and CML-derived CD34+ cells. Our data show a remarkable transcriptional similarity between normal and CML dividing cells, suggesting that the effects of BCR-ABL on the CD34+ cell transcriptome are more limited than previously thought. In addition, we show that quiescent CML cells are more similar to their dividing counterparts than quiescent normal cells are to theirs. We also show these transcriptional differences to be reflected in the altered proliferative activity of normal and CML CD34+ cells. Of the most interest is that the major class of genes that is more abundant in the quiescent cells compared with the dividing cells encodes members of the chemokine family. We propose a role for chemokines expressed by quiescent HSC in the orchestration of CD34+ cell mobilization. Disclosure of potential conflicts of interest is found at the end of this article.
Quiescent and dividing hemopoietic stem cells (HSC) display marked differences in their ability to move between the peripheral circulation and the bone marrow. Specifically, long-term engraftment potential predominantly resides in the quiescent HSC subfraction, and G-CSF mobilization results in the preferential accumulation of quiescent HSC in the periphery. In contrast, stem cells from chronic myeloid leukemia (CML) patients display a constitutive presence in the circulation. To understand the molecular basis for this, we have used microarray technology to analyze the transcriptional differences between dividing and quiescent, normal, and CML-derived CD34+ cells. Our data show a remarkable transcriptional similarity between normal and CML dividing cells, suggesting that the effects of BCR-ABL on the CD34+ cell transcriptome are more limited than previously thought. In addition, we show that quiescent CML cells are more similar to their dividing counterparts than quiescent normal cells are to theirs. We also show these transcriptional differences to be reflected in the altered proliferative activity of normal and CML CD34+ cells. Of the most interest is that the major class of genes that is more abundant in the quiescent cells compared with the dividing cells encodes members of the chemokine family. We propose a role for chemokines expressed by quiescent HSC in the orchestration of CD34+ cell mobilization. Disclosure of potential conflicts of interest is found at the end of this article.
Quiescent and dividing hemopoietic stem cells (HSC) display marked differences in their ability to move between the peripheral circulation and the bone marrow. Specifically, long-term engraftment potential predominantly resides in the quiescent HSC subfraction, and G-CSF mobilization results in the preferential accumulation of quiescent HSC in the periphery. In contrast, stem cells from chronic myeloid leukemia (CML) patients display a constitutive presence in the circulation. To understand the molecular basis for this, we have used microarray technology to analyze the transcriptional differences between dividing and quiescent, normal, and CML-derived CD34+ cells. Our data show a remarkable transcriptional similarity between normal and CML dividing cells, suggesting that the effects of BCR-ABL on the CD34+ cell transcriptome are more limited than previously thought. In addition, we show that quiescent CML cells are more similar to their dividing counterparts than quiescent normal cells are to theirs. We also show these transcriptional differences to be reflected in the altered proliferative activity of normal and CML CD34+ cells. Of the most interest is that the major class of genes that is more abundant in the quiescent cells compared with the dividing cells encodes members of the chemokine family. We propose a role for chemokines expressed by quiescent HSC in the orchestration of CD34+ cell mobilization. Disclosure of potential conflicts of interest is found at the end of this article.
Quiescent and dividing hemopoietic stem cells (HSC) display marked differences in their ability to move between the peripheral circulation and the bone marrow. Specifically, long-term engraftment potential predominantly resides in the quiescent HSC subfraction, and G-CSF mobilization results in the preferential accumulation of quiescent HSC in the periphery. In contrast, stem cells from chronic myeloid leukemia (CML) patients display a constitutive presence in the circulation. To understand the molecular basis for this, we have used microarray technology to analyze the transcriptional differences between dividing and quiescent, normal, and CML-derived CD34+ cells. Our data show a remarkable transcriptional similarity between normal and CML dividing cells, suggesting that the effects of BCR-ABL on the CD34+ cell transcriptome are more limited than previously thought. In addition, we show that quiescent CML cells are more similar to their dividing counterparts than quiescent normal cells are to theirs. We also show these transcriptional differences to be reflected in the altered proliferative activity of normal and CML CD34+ cells. Of the most interest is that the major class of genes that is more abundant in the quiescent cells compared with the dividing cells encodes members of the chemokine family. We propose a role for chemokines expressed by quiescent HSC in the orchestration of CD34+ cell mobilization. Disclosure of potential conflicts of interest is found at the end of this article.
Quiescent and dividing hemopoietic stem cells (HSC) display marked differences in their ability to move between the peripheral circulation and the bone marrow. Specifically, long-term engraftment potential predominantly resides in the quiescent HSC subfraction, and G-CSF mobilization results in the preferential accumulation of quiescent HSC in the periphery. In contrast, stem cells from chronic myeloid leukemia (CML) patients display a constitutive presence in the circulation. To understand the molecular basis for this, we have used microarray technology to analyze the transcriptional differences between dividing and quiescent, normal, and CML-derived CD34+ cells. Our data show a remarkable transcriptional similarity between normal and CML dividing cells, suggesting that the effects of BCR-ABL on the CD34+ cell transcriptome are more limited than previously thought. In addition, we show that quiescent CML cells are more similar to their dividing counterparts than quiescent normal cells are to theirs. We also show these transcriptional differences to be reflected in the altered proliferative activity of normal and CML CD34+ cells. Of the most interest is that the major class of genes that is more abundant in the quiescent cells compared with the dividing cells encodes members of the chemokine family. We propose a role for chemokines expressed by quiescent HSC in the orchestration of CD34+ cell mobilization. Disclosure of potential conflicts of interest is found at the end of this article.
Quiescent and dividing hemopoietic stem cells (HSC) display marked differences in their ability to move between the peripheral circulation and the bone marrow. Specifically, long-term engraftment potential predominantly resides in the quiescent HSC subfraction, and G-CSF mobilization results in the preferential accumulation of quiescent HSC in the periphery. In contrast, stem cells from chronic myeloid leukemia (CML) patients display a constitutive presence in the circulation. To understand the molecular basis for this, we have used microarray technology to analyze the transcriptional differences between dividing and quiescent, normal, and CML-derived CD34+ cells. Our data show a remarkable transcriptional similarity between normal and CML dividing cells, suggesting that the effects of BCR-ABL on the CD34+ cell transcriptome are more limited than previously thought. In addition, we show that quiescent CML cells are more similar to their dividing counterparts than quiescent normal cells are to theirs. We also show these transcriptional differences to be reflected in the altered proliferative activity of normal and CML CD34+ cells. Of the most interest is that the major class of genes that is more abundant in the quiescent cells compared with the dividing cells encodes members of the chemokine family. We propose a role for chemokines expressed by quiescent HSC in the orchestration of CD34+ cell mobilization. Disclosure of potential conflicts of interest is found at the end of this article.
Quiescent and dividing hemopoietic stem cells (HSC) display marked differences in their ability to move between the peripheral circulation and the bone marrow. Specifically, long-term engraftment potential predominantly resides in the quiescent HSC subfraction, and G-CSF mobilization results in the preferential accumulation of quiescent HSC in the periphery. In contrast, stem cells from chronic myeloid leukemia (CML) patients display a constitutive presence in the circulation. To understand the molecular basis for this, we have used microarray technology to analyze the transcriptional differences between dividing and quiescent, normal, and CML-derived CD34+ cells. Our data show a remarkable transcriptional similarity between normal and CML dividing cells, suggesting that the effects of BCR-ABL on the CD34+ cell transcriptome are more limited than previously thought. In addition, we show that quiescent CML cells are more similar to their dividing counterparts than quiescent normal cells are to theirs. We also show these transcriptional differences to be reflected in the altered proliferative activity of normal and CML CD34+ cells. Of the most interest is that the major class of genes that is more abundant in the quiescent cells compared with the dividing cells encodes members of the chemokine family. We propose a role for chemokines expressed by quiescent HSC in the orchestration of CD34+ cell mobilization. Disclosure of potential conflicts of interest is found at the end of this article.
Quiescent and dividing hemopoietic stem cells (HSC) display marked differences in their ability to move between the peripheral circulation and the bone marrow. Specifically, long-term engraftment potential predominantly resides in the quiescent HSC subfraction, and G-CSF mobilization results in the preferential accumulation of quiescent HSC in the periphery. In contrast, stem cells from chronic myeloid leukemia (CML) patients display a constitutive presence in the circulation. To understand the molecular basis for this, we have used microarray technology to analyze the transcriptional differences between dividing and quiescent, normal, and CML-derived CD34+ cells. Our data show a remarkable transcriptional similarity between normal and CML dividing cells, suggesting that the effects of BCR-ABL on the CD34+ cell transcriptome are more limited than previously thought. In addition, we show that quiescent CML cells are more similar to their dividing counterparts than quiescent normal cells are to theirs. We also show these transcriptional differences to be reflected in the altered proliferative activity of normal and CML CD34+ cells. Of the most interest is that the major class of genes that is more abundant in the quiescent cells compared with the dividing cells encodes members of the chemokine family. We propose a role for chemokines expressed by quiescent HSC in the orchestration of CD34+ cell mobilization. Disclosure of potential conflicts of interest is found at the end of this article.
Quiescent and dividing hemopoietic stem cells (HSC) display marked differences in their ability to move between the peripheral circulation and the bone marrow. Specifically, long-term engraftment potential predominantly resides in the quiescent HSC subfraction, and G-CSF mobilization results in the preferential accumulation of quiescent HSC in the periphery. In contrast, stem cells from chronic myeloid leukemia (CML) patients display a constitutive presence in the circulation. To understand the molecular basis for this, we have used microarray technology to analyze the transcriptional differences between dividing and quiescent, normal, and CML-derived CD34+ cells. Our data show a remarkable transcriptional similarity between normal and CML dividing cells, suggesting that the effects of BCR-ABL on the CD34+ cell transcriptome are more limited than previously thought. In addition, we show that quiescent CML cells are more similar to their dividing counterparts than quiescent normal cells are to theirs. We also show these transcriptional differences to be reflected in the altered proliferative activity of normal and CML CD34+ cells. Of the most interest is that the major class of genes that is more abundant in the quiescent cells compared with the dividing cells encodes members of the chemokine family. We propose a role for chemokines expressed by quiescent HSC in the orchestration of CD34+ cell mobilization. Disclosure of potential conflicts of interest is found at the end of this article.
Quiescent and dividing hemopoietic stem cells (HSC) display marked differences in their ability to move between the peripheral circulation and the bone marrow. Specifically, long-term engraftment potential predominantly resides in the quiescent HSC subfraction, and G-CSF mobilization results in the preferential accumulation of quiescent HSC in the periphery. In contrast, stem cells from chronic myeloid leukemia (CML) patients display a constitutive presence in the circulation. To understand the molecular basis for this, we have used microarray technology to analyze the transcriptional differences between dividing and quiescent, normal, and CML-derived CD34+ cells. Our data show a remarkable transcriptional similarity between normal and CML dividing cells, suggesting that the effects of BCR-ABL on the CD34+ cell transcriptome are more limited than previously thought. In addition, we show that quiescent CML cells are more similar to their dividing counterparts than quiescent normal cells are to theirs. We also show these transcriptional differences to be reflected in the altered proliferative activity of normal and CML CD34+ cells. Of the most interest is that the major class of genes that is more abundant in the quiescent cells compared with the dividing cells encodes members of the chemokine family. We propose a role for chemokines expressed by quiescent HSC in the orchestration of CD34+ cell mobilization. Disclosure of potential conflicts of interest is found at the end of this article.
Quiescent and dividing hemopoietic stem cells (HSC) display marked differences in their ability to move between the peripheral circulation and the bone marrow. Specifically, long-term engraftment potential predominantly resides in the quiescent HSC subfraction, and G-CSF mobilization results in the preferential accumulation of quiescent HSC in the periphery. In contrast, stem cells from chronic myeloid leukemia (CML) patients display a constitutive presence in the circulation. To understand the molecular basis for this, we have used microarray technology to analyze the transcriptional differences between dividing and quiescent, normal, and CML-derived CD34+ cells. Our data show a remarkable transcriptional similarity between normal and CML dividing cells, suggesting that the effects of BCR-ABL on the CD34+ cell transcriptome are more limited than previously thought. In addition, we show that quiescent CML cells are more similar to their dividing counterparts than quiescent normal cells are to theirs. We also show these transcriptional differences to be reflected in the altered proliferative activity of normal and CML CD34+ cells. Of the most interest is that the major class of genes that is more abundant in the quiescent cells compared with the dividing cells encodes members of the chemokine family. We propose a role for chemokines expressed by quiescent HSC in the orchestration of CD34+ cell mobilization. Disclosure of potential conflicts of interest is found at the end of this article.
PURPOSE: To characterize the gene expression profiles of endometrioid endometrial cancers associated with lymph node metastasis in an effort to identify genes associated with metastatic spread. EXPERIMENTAL DESIGN: Tumors from 41 patients with endometrioid endometrial cancer grossly confined to the uterine cavity were evaluated. Positive lymph nodes were noted in 12 of 41 patients. RNA was analyzed for gene expression using the Affymetrix HG133A and HG133B GeneChip set, representing 45,000 array features covering >28,000 UniGene clusters. Data analysis was done using multidimensional scaling, binary comparison, and hierarchical clustering. Gene expression for several differentially expressed genes was examined using quantitative PCR. RESULTS: Gene expression data was obtained from 30,964 genes that were detected in at least 5% of the cases. Supervised analysis of node-positive versus node-negative cases indicated that 450 genes were significantly differentially expressed between the two classes at P < 0.005, 81 of which were differentially expressed by at least 2-fold at P < 0.005. Overexpressed genes included two cell cycle checkpoint genes, CDC2 and MAD2L1, which have previously been described in association with lymph node metastasis in other cancer types. The ZIC2 zinc finger gene was overexpressed in endometrial cancers with positive nodes versus those with negative nodes. CONCLUSION: Gene expression profiling of the primary tumors in patients with endometrioid endometrial cancers seems promising for identifying genes associated with lymph node metastasis. Future studies should address whether the status of nodal metastasis can be determined from the expression profiles of preoperative tissue specimens.
PURPOSE: To characterize the gene expression profiles of endometrioid endometrial cancers associated with lymph node metastasis in an effort to identify genes associated with metastatic spread. EXPERIMENTAL DESIGN: Tumors from 41 patients with endometrioid endometrial cancer grossly confined to the uterine cavity were evaluated. Positive lymph nodes were noted in 12 of 41 patients. RNA was analyzed for gene expression using the Affymetrix HG133A and HG133B GeneChip set, representing 45,000 array features covering >28,000 UniGene clusters. Data analysis was done using multidimensional scaling, binary comparison, and hierarchical clustering. Gene expression for several differentially expressed genes was examined using quantitative PCR. RESULTS: Gene expression data was obtained from 30,964 genes that were detected in at least 5% of the cases. Supervised analysis of node-positive versus node-negative cases indicated that 450 genes were significantly differentially expressed between the two classes at P < 0.005, 81 of which were differentially expressed by at least 2-fold at P < 0.005. Overexpressed genes included two cell cycle checkpoint genes, CDC2 and MAD2L1, which have previously been described in association with lymph node metastasis in other cancer types. The ZIC2 zinc finger gene was overexpressed in endometrial cancers with positive nodes versus those with negative nodes. CONCLUSION: Gene expression profiling of the primary tumors in patients with endometrioid endometrial cancers seems promising for identifying genes associated with lymph node metastasis. Future studies should address whether the status of nodal metastasis can be determined from the expression profiles of preoperative tissue specimens.
PURPOSE: To elucidate the molecular mechanisms contributing to the unique clinicopathologic characteristics of mucinous ovarian carcinoma, global gene expression profiling of mucinous ovarian tumors was carried out. EXPERIMENTAL DESIGN: Gene expression profiling was completed for 25 microdissected mucinous tumors [6 cystadenomas, 10 low malignant potential (LMP) tumors, and 9 adenocarcinomas] using Affymetrix U133 Plus 2.0 oligonucleotide microarrays. Hierarchical clustering and binary tree prediction analysis were used to determine the relationships among mucinous specimens and a series of previously profiled microdissected serous tumors and normal ovarian surface epithelium. PathwayAssist software was used to identify putative signaling pathways involved in the development of mucinous LMP tumors and adenocarcinomas. RESULTS: Comparison of the gene profiles between mucinous tumors and normal ovarian epithelial cells identified 1,599, 2,916, and 1,765 differentially expressed in genes in the cystadenomas, LMP tumors, and adenocarcinomas, respectively. Hierarchical clustering showed that mucinous and serous LMP tumors are distinct. In addition, there was a close association of mucinous LMP tumors and adenocarcinomas with serous adenocarcinomas. Binary tree prediction revealed increased heterogeneity among mucinous tumors compared with their serous counterparts. Furthermore, the cystadenomas coexpressed a subset of genes that were differentially regulated in LMP and adenocarcinoma specimens compared with normal ovarian surface epithelium. PathwayAssist highlighted pathways with expression of genes involved in drug resistance in both LMP and adenocarcinoma samples. In addition, genes involved in cytoskeletal regulation were specifically up-regulated in the mucinous adenocarcinomas. CONCLUSIONS: These data provide a useful basis for understanding the molecular events leading to the development and progression of mucinous ovarian cancer.
PURPOSE: To elucidate the molecular mechanisms contributing to the unique clinicopathologic characteristics of mucinous ovarian carcinoma, global gene expression profiling of mucinous ovarian tumors was carried out. EXPERIMENTAL DESIGN: Gene expression profiling was completed for 25 microdissected mucinous tumors [6 cystadenomas, 10 low malignant potential (LMP) tumors, and 9 adenocarcinomas] using Affymetrix U133 Plus 2.0 oligonucleotide microarrays. Hierarchical clustering and binary tree prediction analysis were used to determine the relationships among mucinous specimens and a series of previously profiled microdissected serous tumors and normal ovarian surface epithelium. PathwayAssist software was used to identify putative signaling pathways involved in the development of mucinous LMP tumors and adenocarcinomas. RESULTS: Comparison of the gene profiles between mucinous tumors and normal ovarian epithelial cells identified 1,599, 2,916, and 1,765 differentially expressed in genes in the cystadenomas, LMP tumors, and adenocarcinomas, respectively. Hierarchical clustering showed that mucinous and serous LMP tumors are distinct. In addition, there was a close association of mucinous LMP tumors and adenocarcinomas with serous adenocarcinomas. Binary tree prediction revealed increased heterogeneity among mucinous tumors compared with their serous counterparts. Furthermore, the cystadenomas coexpressed a subset of genes that were differentially regulated in LMP and adenocarcinoma specimens compared with normal ovarian surface epithelium. PathwayAssist highlighted pathways with expression of genes involved in drug resistance in both LMP and adenocarcinoma samples. In addition, genes involved in cytoskeletal regulation were specifically up-regulated in the mucinous adenocarcinomas. CONCLUSIONS: These data provide a useful basis for understanding the molecular events leading to the development and progression of mucinous ovarian cancer.
PURPOSE: To elucidate the molecular mechanisms contributing to the unique clinicopathologic characteristics of mucinous ovarian carcinoma, global gene expression profiling of mucinous ovarian tumors was carried out. EXPERIMENTAL DESIGN: Gene expression profiling was completed for 25 microdissected mucinous tumors [6 cystadenomas, 10 low malignant potential (LMP) tumors, and 9 adenocarcinomas] using Affymetrix U133 Plus 2.0 oligonucleotide microarrays. Hierarchical clustering and binary tree prediction analysis were used to determine the relationships among mucinous specimens and a series of previously profiled microdissected serous tumors and normal ovarian surface epithelium. PathwayAssist software was used to identify putative signaling pathways involved in the development of mucinous LMP tumors and adenocarcinomas. RESULTS: Comparison of the gene profiles between mucinous tumors and normal ovarian epithelial cells identified 1,599, 2,916, and 1,765 differentially expressed in genes in the cystadenomas, LMP tumors, and adenocarcinomas, respectively. Hierarchical clustering showed that mucinous and serous LMP tumors are distinct. In addition, there was a close association of mucinous LMP tumors and adenocarcinomas with serous adenocarcinomas. Binary tree prediction revealed increased heterogeneity among mucinous tumors compared with their serous counterparts. Furthermore, the cystadenomas coexpressed a subset of genes that were differentially regulated in LMP and adenocarcinoma specimens compared with normal ovarian surface epithelium. PathwayAssist highlighted pathways with expression of genes involved in drug resistance in both LMP and adenocarcinoma samples. In addition, genes involved in cytoskeletal regulation were specifically up-regulated in the mucinous adenocarcinomas. CONCLUSIONS: These data provide a useful basis for understanding the molecular events leading to the development and progression of mucinous ovarian cancer.
PURPOSE: To elucidate the molecular mechanisms contributing to the unique clinicopathologic characteristics of mucinous ovarian carcinoma, global gene expression profiling of mucinous ovarian tumors was carried out. EXPERIMENTAL DESIGN: Gene expression profiling was completed for 25 microdissected mucinous tumors [6 cystadenomas, 10 low malignant potential (LMP) tumors, and 9 adenocarcinomas] using Affymetrix U133 Plus 2.0 oligonucleotide microarrays. Hierarchical clustering and binary tree prediction analysis were used to determine the relationships among mucinous specimens and a series of previously profiled microdissected serous tumors and normal ovarian surface epithelium. PathwayAssist software was used to identify putative signaling pathways involved in the development of mucinous LMP tumors and adenocarcinomas. RESULTS: Comparison of the gene profiles between mucinous tumors and normal ovarian epithelial cells identified 1,599, 2,916, and 1,765 differentially expressed in genes in the cystadenomas, LMP tumors, and adenocarcinomas, respectively. Hierarchical clustering showed that mucinous and serous LMP tumors are distinct. In addition, there was a close association of mucinous LMP tumors and adenocarcinomas with serous adenocarcinomas. Binary tree prediction revealed increased heterogeneity among mucinous tumors compared with their serous counterparts. Furthermore, the cystadenomas coexpressed a subset of genes that were differentially regulated in LMP and adenocarcinoma specimens compared with normal ovarian surface epithelium. PathwayAssist highlighted pathways with expression of genes involved in drug resistance in both LMP and adenocarcinoma samples. In addition, genes involved in cytoskeletal regulation were specifically up-regulated in the mucinous adenocarcinomas. CONCLUSIONS: These data provide a useful basis for understanding the molecular events leading to the development and progression of mucinous ovarian cancer.
The small molecular inhibitor MK886 is known to block 5-lipoxygenase-activating protein ALOX5AP and shows antitumor activity in multiple human cell lines. The broad antitumor therapeutic window reported in vivo for MK886 in rodents supports further consideration of this structural class. Better understanding of the mode of action of the drug is important for application in humans to take place. Affymetrix microarray study was conducted to explore MK886 pharmacologic mechanism. Ingenuity Pathway Analysis software was applied to validate the results at the transcriptional level by putting them in the context of an experimental proteomic network. Genes most affected by MK886 included actin B and focal adhesion components. A subsequent National Cancer Institute-60 panel study, RT-PCR validation followed by confocal microscopy, and Western blotting also pointed to actin B down-regulation, filamentous actin loss, and disorganization of the transcription machinery. In agreement with these observations, MK886 was found to enhance the effect of UV radiation in H720 lung cancer cell line. In light of the modification of cytoskeleton and cell motility by lipid phosphoinositide 3-kinase products, MK886 interaction with actin B might be biologically important. The low toxicity of MK886 in vivo was modeled and explained by binding and transport by dietary lipids. The rate of lipid absorbance is generally higher for tumors, suggesting a promise of a targeted liposome-based delivery system for this drug. These results suggest a novel antitumor pharmacologic mechanism.
The small molecular inhibitor MK886 is known to block 5-lipoxygenase-activating protein ALOX5AP and shows antitumor activity in multiple human cell lines. The broad antitumor therapeutic window reported in vivo for MK886 in rodents supports further consideration of this structural class. Better understanding of the mode of action of the drug is important for application in humans to take place. Affymetrix microarray study was conducted to explore MK886 pharmacologic mechanism. Ingenuity Pathway Analysis software was applied to validate the results at the transcriptional level by putting them in the context of an experimental proteomic network. Genes most affected by MK886 included actin B and focal adhesion components. A subsequent National Cancer Institute-60 panel study, RT-PCR validation followed by confocal microscopy, and Western blotting also pointed to actin B down-regulation, filamentous actin loss, and disorganization of the transcription machinery. In agreement with these observations, MK886 was found to enhance the effect of UV radiation in H720 lung cancer cell line. In light of the modification of cytoskeleton and cell motility by lipid phosphoinositide 3-kinase products, MK886 interaction with actin B might be biologically important. The low toxicity of MK886 in vivo was modeled and explained by binding and transport by dietary lipids. The rate of lipid absorbance is generally higher for tumors, suggesting a promise of a targeted liposome-based delivery system for this drug. These results suggest a novel antitumor pharmacologic mechanism.
PURPOSE: Ulcerative colitis (UC) is associated with a high risk of colorectal cancer. To identify genes that could predict the development of cancer in UC, we conducted a DNA microarray analysis using nonneoplastic rectal mucosa of UC patients. EXPERIMENTAL DESIGN: Gene expression in nonneoplastic mucosa of 53 UC patients were examined. Gene expression profiles were examined using human Genome U133 Plus 2.0 gene chip array (Affymetrix). Among 53 UC patients, 10 had UC-associated cancer (UC-Ca group) whereas 43 did not (UC-NonCa group). RESULTS: By comparing gene expression profiles of nonneoplastic rectal mucosae between the UC-Ca and UC-NonCa groups, we could identify 40 genes that were differentially expressed between two groups. The list of discriminating genes included low-density lipoprotein receptor-related protein (LRP5 and LRP6). Previous studies suggested that LRP5 and LRP6 expression promotes cancer cell proliferation and tumorigenesis and are considered as candidate oncogenes. In the present study, both LRP5 and LRP6 showed significantly higher expression in the UC-Ca group, which suggests the importance of these genes in the development of UC-associated colorectal cancers. With the 40 selected discriminating genes, we did class prediction of the development of colorectal neoplasms in UC patients. Using the k-nearest neighbor method and the support vector machine, we could predict the development of UC-associated neoplasms with an accuracy of 86.8% and 98.1%, respectively. CONCLUSIONS: These findings have important implications for the early detection of malignant lesions in UC and may provide directions for future research into the molecular mechanisms of UC-associated cancer.
PURPOSE: Ulcerative colitis (UC) is associated with a high risk of colorectal cancer. To identify genes that could predict the development of cancer in UC, we conducted a DNA microarray analysis using nonneoplastic rectal mucosa of UC patients. EXPERIMENTAL DESIGN: Gene expression in nonneoplastic mucosa of 53 UC patients were examined. Gene expression profiles were examined using human Genome U133 Plus 2.0 gene chip array (Affymetrix). Among 53 UC patients, 10 had UC-associated cancer (UC-Ca group) whereas 43 did not (UC-NonCa group). RESULTS: By comparing gene expression profiles of nonneoplastic rectal mucosae between the UC-Ca and UC-NonCa groups, we could identify 40 genes that were differentially expressed between two groups. The list of discriminating genes included low-density lipoprotein receptor-related protein (LRP5 and LRP6). Previous studies suggested that LRP5 and LRP6 expression promotes cancer cell proliferation and tumorigenesis and are considered as candidate oncogenes. In the present study, both LRP5 and LRP6 showed significantly higher expression in the UC-Ca group, which suggests the importance of these genes in the development of UC-associated colorectal cancers. With the 40 selected discriminating genes, we did class prediction of the development of colorectal neoplasms in UC patients. Using the k-nearest neighbor method and the support vector machine, we could predict the development of UC-associated neoplasms with an accuracy of 86.8% and 98.1%, respectively. CONCLUSIONS: These findings have important implications for the early detection of malignant lesions in UC and may provide directions for future research into the molecular mechanisms of UC-associated cancer.
Polymorphisms in nitrosamine metabolism, DNA repair, and immune response genes have been associated with nasopharyngeal carcinoma (NPC). Studies have suggested chromosomal regions involved in NPC. To shed light on NPC etiology, we evaluated host gene expression patterns in 31 NPC and 10 normal nasopharyngeal tissue specimens using the Affymetrix Human Genome U133 Plus 2.0 Array. We focused on genes in five a priori biological pathways and chromosomal locations. Rates of differential expression within these prespecified lists and overall were tested using a bootstrap method. Differential expression was observed for 7.6% of probe sets overall. Elevations in rate of differential expression were observed within the DNA repair (13.7%; P = 0.01) and nitrosamine metabolism (17.5%; P = 0.04) pathways. Differentially expressed probe sets within the DNA repair pathway were consistently overexpressed (93%), with strong effects observed for PRKDC, PCNA, and CHEK1. Differentially expressed probe sets within the nitrosamine metabolism pathway were consistently underexpressed (100%), with strong effects observed for NQ01, CYP2B6, and CYP2E1. No significant evidence of increases in rate of differential expression was seen within the immune/inflammatory pathway. A significant elevation in rate of differential expression was noted for chromosome 4p15.1-4q12 (13.0%; P = 0.04); both overexpression and underexpression were evident (38% and 62%, respectively). An elevation in the rate of differential expression on chromosome 14q32 was observed (11.3%; P = 0.06) with a consistent pattern of gene underexpression (100%; P < 0.0001). These effects were similar when excluding late-stage tumors. Our results suggest that nitrosamine activation and DNA repair are important in NPC. The consistent down-regulation of expression on chromosome 14q32 suggests loss of heterozygosity in this region.
Polymorphisms in nitrosamine metabolism, DNA repair, and immune response genes have been associated with nasopharyngeal carcinoma (NPC). Studies have suggested chromosomal regions involved in NPC. To shed light on NPC etiology, we evaluated host gene expression patterns in 31 NPC and 10 normal nasopharyngeal tissue specimens using the Affymetrix Human Genome U133 Plus 2.0 Array. We focused on genes in five a priori biological pathways and chromosomal locations. Rates of differential expression within these prespecified lists and overall were tested using a bootstrap method. Differential expression was observed for 7.6% of probe sets overall. Elevations in rate of differential expression were observed within the DNA repair (13.7%; P = 0.01) and nitrosamine metabolism (17.5%; P = 0.04) pathways. Differentially expressed probe sets within the DNA repair pathway were consistently overexpressed (93%), with strong effects observed for PRKDC, PCNA, and CHEK1. Differentially expressed probe sets within the nitrosamine metabolism pathway were consistently underexpressed (100%), with strong effects observed for NQ01, CYP2B6, and CYP2E1. No significant evidence of increases in rate of differential expression was seen within the immune/inflammatory pathway. A significant elevation in rate of differential expression was noted for chromosome 4p15.1-4q12 (13.0%; P = 0.04); both overexpression and underexpression were evident (38% and 62%, respectively). An elevation in the rate of differential expression on chromosome 14q32 was observed (11.3%; P = 0.06) with a consistent pattern of gene underexpression (100%; P < 0.0001). These effects were similar when excluding late-stage tumors. Our results suggest that nitrosamine activation and DNA repair are important in NPC. The consistent down-regulation of expression on chromosome 14q32 suggests loss of heterozygosity in this region.
Information on gene alterations associated to poorly differentiated (PDTC) and anaplastic thyroid carcinomas (ATC) is scarce. Using human cancer cell lines as a tool for gene discovery, we performed a cytogenetic and oligo-array analysis in five new cell lines derived from two PDTC and three ATC. In PDTC we evidenced, as important, the involvement of the MAPK/ERK kinase pathway, and downregulation of a group of suppressor genes that include E-cadherin. In ATC, downregulation of a specific group of oncosuppressor genes was also observed. Our ATC cell lines presented chromosomal markers of gene amplification, and we were able to identify for the first time the nature of the involved amplicon target genes. We found that the main molecular differences between the two cell line types were related to signal transduction pathways, cell adhesion and motility process. TaqMan experiments performed for five amplicon target genes and for two genes, which allowed a clear distinction between ATC and PDTC: CDH13 and PLAU corroborated array results, not only in the cell lines, but also in an additional set of primary 14 PDTC and three ATC. We suggest that our findings may represent new tools for the development of more effective therapies to the hitherto untreatable ATC.
Information on gene alterations associated to poorly differentiated (PDTC) and anaplastic thyroid carcinomas (ATC) is scarce. Using human cancer cell lines as a tool for gene discovery, we performed a cytogenetic and oligo-array analysis in five new cell lines derived from two PDTC and three ATC. In PDTC we evidenced, as important, the involvement of the MAPK/ERK kinase pathway, and downregulation of a group of suppressor genes that include E-cadherin. In ATC, downregulation of a specific group of oncosuppressor genes was also observed. Our ATC cell lines presented chromosomal markers of gene amplification, and we were able to identify for the first time the nature of the involved amplicon target genes. We found that the main molecular differences between the two cell line types were related to signal transduction pathways, cell adhesion and motility process. TaqMan experiments performed for five amplicon target genes and for two genes, which allowed a clear distinction between ATC and PDTC: CDH13 and PLAU corroborated array results, not only in the cell lines, but also in an additional set of primary 14 PDTC and three ATC. We suggest that our findings may represent new tools for the development of more effective therapies to the hitherto untreatable ATC.
Information on gene alterations associated to poorly differentiated (PDTC) and anaplastic thyroid carcinomas (ATC) is scarce. Using human cancer cell lines as a tool for gene discovery, we performed a cytogenetic and oligo-array analysis in five new cell lines derived from two PDTC and three ATC. In PDTC we evidenced, as important, the involvement of the MAPK/ERK kinase pathway, and downregulation of a group of suppressor genes that include E-cadherin. In ATC, downregulation of a specific group of oncosuppressor genes was also observed. Our ATC cell lines presented chromosomal markers of gene amplification, and we were able to identify for the first time the nature of the involved amplicon target genes. We found that the main molecular differences between the two cell line types were related to signal transduction pathways, cell adhesion and motility process. TaqMan experiments performed for five amplicon target genes and for two genes, which allowed a clear distinction between ATC and PDTC: CDH13 and PLAU corroborated array results, not only in the cell lines, but also in an additional set of primary 14 PDTC and three ATC. We suggest that our findings may represent new tools for the development of more effective therapies to the hitherto untreatable ATC.
Information on gene alterations associated to poorly differentiated (PDTC) and anaplastic thyroid carcinomas (ATC) is scarce. Using human cancer cell lines as a tool for gene discovery, we performed a cytogenetic and oligo-array analysis in five new cell lines derived from two PDTC and three ATC. In PDTC we evidenced, as important, the involvement of the MAPK/ERK kinase pathway, and downregulation of a group of suppressor genes that include E-cadherin. In ATC, downregulation of a specific group of oncosuppressor genes was also observed. Our ATC cell lines presented chromosomal markers of gene amplification, and we were able to identify for the first time the nature of the involved amplicon target genes. We found that the main molecular differences between the two cell line types were related to signal transduction pathways, cell adhesion and motility process. TaqMan experiments performed for five amplicon target genes and for two genes, which allowed a clear distinction between ATC and PDTC: CDH13 and PLAU corroborated array results, not only in the cell lines, but also in an additional set of primary 14 PDTC and three ATC. We suggest that our findings may represent new tools for the development of more effective therapies to the hitherto untreatable ATC.
Information on gene alterations associated to poorly differentiated (PDTC) and anaplastic thyroid carcinomas (ATC) is scarce. Using human cancer cell lines as a tool for gene discovery, we performed a cytogenetic and oligo-array analysis in five new cell lines derived from two PDTC and three ATC. In PDTC we evidenced, as important, the involvement of the MAPK/ERK kinase pathway, and downregulation of a group of suppressor genes that include E-cadherin. In ATC, downregulation of a specific group of oncosuppressor genes was also observed. Our ATC cell lines presented chromosomal markers of gene amplification, and we were able to identify for the first time the nature of the involved amplicon target genes. We found that the main molecular differences between the two cell line types were related to signal transduction pathways, cell adhesion and motility process. TaqMan experiments performed for five amplicon target genes and for two genes, which allowed a clear distinction between ATC and PDTC: CDH13 and PLAU corroborated array results, not only in the cell lines, but also in an additional set of primary 14 PDTC and three ATC. We suggest that our findings may represent new tools for the development of more effective therapies to the hitherto untreatable ATC.
Information on gene alterations associated to poorly differentiated (PDTC) and anaplastic thyroid carcinomas (ATC) is scarce. Using human cancer cell lines as a tool for gene discovery, we performed a cytogenetic and oligo-array analysis in five new cell lines derived from two PDTC and three ATC. In PDTC we evidenced, as important, the involvement of the MAPK/ERK kinase pathway, and downregulation of a group of suppressor genes that include E-cadherin. In ATC, downregulation of a specific group of oncosuppressor genes was also observed. Our ATC cell lines presented chromosomal markers of gene amplification, and we were able to identify for the first time the nature of the involved amplicon target genes. We found that the main molecular differences between the two cell line types were related to signal transduction pathways, cell adhesion and motility process. TaqMan experiments performed for five amplicon target genes and for two genes, which allowed a clear distinction between ATC and PDTC: CDH13 and PLAU corroborated array results, not only in the cell lines, but also in an additional set of primary 14 PDTC and three ATC. We suggest that our findings may represent new tools for the development of more effective therapies to the hitherto untreatable ATC.
We have used a novel variant of the human oestrogen receptor (ER)-positive MCF-7 cell line, TMX2-28, as a model to study breast cancer. TMX2-28 cells show no detectable levels of mRNA or protein expression for the ER and express basal cytokeratins (CKs) 5, 14, and 17. cDNA microarray comparison between TMX2-28 and its parent cell line, MCF-7, identified 1402 differentially expressed transcripts, one of which was, phospholipase D1 (PLD1). Using real-time RT-PCR, we confirmed that PLD1 mRNA levels are 10-fold higher in TMX2-28 cells than in MCF-7 cells. We next examined PLD1 expression in human breast carcinomas. Phospholipase D1 mRNA levels were higher in breast tumours that expressed high-mRNA levels of basal CKs 5 and/or 17, but PLD1 mRNA levels were not significantly higher in ER-negative tumours. Phospholipase D1 protein was overexpressed in 10 of 42 (24%) breast tumours examined by IHC. Phospholipase D1 was overexpressed in 6 of 31 ER-positive tumours and 4 of 11 ER-negative tumours. Phospholipase D1 was overexpressed in three of the four tumours that showed high CK5/17 expression. Five PLD1-positive tumours were negative for phospho-Akt expression, but positive for phospho-mammalian target of rapamycin (mTOR) expression. The other five PLD1-positive breast tumours showed positive expression for phospho-Akt; however, only two of these cases were positive for phospho-mTOR. In this study, we report that PLD1 and phospho-mTOR are coexpressed in a subset of phospho-Akt-negative breast carcinomas.
We have used a novel variant of the human oestrogen receptor (ER)-positive MCF-7 cell line, TMX2-28, as a model to study breast cancer. TMX2-28 cells show no detectable levels of mRNA or protein expression for the ER and express basal cytokeratins (CKs) 5, 14, and 17. cDNA microarray comparison between TMX2-28 and its parent cell line, MCF-7, identified 1402 differentially expressed transcripts, one of which was, phospholipase D1 (PLD1). Using real-time RT-PCR, we confirmed that PLD1 mRNA levels are 10-fold higher in TMX2-28 cells than in MCF-7 cells. We next examined PLD1 expression in human breast carcinomas. Phospholipase D1 mRNA levels were higher in breast tumours that expressed high-mRNA levels of basal CKs 5 and/or 17, but PLD1 mRNA levels were not significantly higher in ER-negative tumours. Phospholipase D1 protein was overexpressed in 10 of 42 (24%) breast tumours examined by IHC. Phospholipase D1 was overexpressed in 6 of 31 ER-positive tumours and 4 of 11 ER-negative tumours. Phospholipase D1 was overexpressed in three of the four tumours that showed high CK5/17 expression. Five PLD1-positive tumours were negative for phospho-Akt expression, but positive for phospho-mammalian target of rapamycin (mTOR) expression. The other five PLD1-positive breast tumours showed positive expression for phospho-Akt; however, only two of these cases were positive for phospho-mTOR. In this study, we report that PLD1 and phospho-mTOR are coexpressed in a subset of phospho-Akt-negative breast carcinomas.
PURPOSE: Increased production of Th2 cytokines characterizes Sezary syndrome, the leukemic form of cutaneous T-cell lymphomas (CTCL). To identify the molecular background and to study whether shared by the most common CTCL subtype, mycosis fungoides, we analyzed the gene expression profiles in both subtypes. EXPERIMENTAL DESIGN: Freshly isolated cells from 30 samples, representing skin, blood, and enriched CD4(+) cell populations of mycosis fungoides and Sezary syndrome, were analyzed with Affymetrix (Santa Clara, CA) oligonucleotide microarrays, quantitative PCR, or immunohistochemistry. The gene expression profiles were combined with findings of comparative genomic hybridization of the same samples to identify chromosomal changes affecting the aberrant gene expression. RESULTS: We identified a set of Th1-specific genes [e.g., TBX21 (T-bet), NKG7, and SCYA5 (RANTES)] to be down-regulated in Sezary syndrome as well as in a proportion of mycosis fungoides samples. In both Sezary syndrome and mycosis fungoides blood samples, the S100P and LIR9 gene expression was up-regulated. In lesional skin, IL7R and CD52 were up-regulated. Integration of comparative genomic hybridization and transcriptomic data identified chromosome arms 1q, 3p, 3q, 4q, 12q, 16p, and 16q as likely targets for new CTCL-associated gene aberrations. CONCLUSIONS: Our findings revealed several new genes involved in CTCL pathogenesis and potential therapeutic targets. Down-regulation of a set of genes involved in Th1 polarization, including the major Th1-polarizing factor, TBX21, was for the first time associated with CTCL. In addition, a plausible explanation for the proliferative response of CTCL cells to locally produced interleukin-7 was revealed.
PURPOSE: Increased production of Th2 cytokines characterizes Sezary syndrome, the leukemic form of cutaneous T-cell lymphomas (CTCL). To identify the molecular background and to study whether shared by the most common CTCL subtype, mycosis fungoides, we analyzed the gene expression profiles in both subtypes. EXPERIMENTAL DESIGN: Freshly isolated cells from 30 samples, representing skin, blood, and enriched CD4(+) cell populations of mycosis fungoides and Sezary syndrome, were analyzed with Affymetrix (Santa Clara, CA) oligonucleotide microarrays, quantitative PCR, or immunohistochemistry. The gene expression profiles were combined with findings of comparative genomic hybridization of the same samples to identify chromosomal changes affecting the aberrant gene expression. RESULTS: We identified a set of Th1-specific genes [e.g., TBX21 (T-bet), NKG7, and SCYA5 (RANTES)] to be down-regulated in Sezary syndrome as well as in a proportion of mycosis fungoides samples. In both Sezary syndrome and mycosis fungoides blood samples, the S100P and LIR9 gene expression was up-regulated. In lesional skin, IL7R and CD52 were up-regulated. Integration of comparative genomic hybridization and transcriptomic data identified chromosome arms 1q, 3p, 3q, 4q, 12q, 16p, and 16q as likely targets for new CTCL-associated gene aberrations. CONCLUSIONS: Our findings revealed several new genes involved in CTCL pathogenesis and potential therapeutic targets. Down-regulation of a set of genes involved in Th1 polarization, including the major Th1-polarizing factor, TBX21, was for the first time associated with CTCL. In addition, a plausible explanation for the proliferative response of CTCL cells to locally produced interleukin-7 was revealed.
PURPOSE: Increased production of Th2 cytokines characterizes Sezary syndrome, the leukemic form of cutaneous T-cell lymphomas (CTCL). To identify the molecular background and to study whether shared by the most common CTCL subtype, mycosis fungoides, we analyzed the gene expression profiles in both subtypes. EXPERIMENTAL DESIGN: Freshly isolated cells from 30 samples, representing skin, blood, and enriched CD4(+) cell populations of mycosis fungoides and Sezary syndrome, were analyzed with Affymetrix (Santa Clara, CA) oligonucleotide microarrays, quantitative PCR, or immunohistochemistry. The gene expression profiles were combined with findings of comparative genomic hybridization of the same samples to identify chromosomal changes affecting the aberrant gene expression. RESULTS: We identified a set of Th1-specific genes [e.g., TBX21 (T-bet), NKG7, and SCYA5 (RANTES)] to be down-regulated in Sezary syndrome as well as in a proportion of mycosis fungoides samples. In both Sezary syndrome and mycosis fungoides blood samples, the S100P and LIR9 gene expression was up-regulated. In lesional skin, IL7R and CD52 were up-regulated. Integration of comparative genomic hybridization and transcriptomic data identified chromosome arms 1q, 3p, 3q, 4q, 12q, 16p, and 16q as likely targets for new CTCL-associated gene aberrations. CONCLUSIONS: Our findings revealed several new genes involved in CTCL pathogenesis and potential therapeutic targets. Down-regulation of a set of genes involved in Th1 polarization, including the major Th1-polarizing factor, TBX21, was for the first time associated with CTCL. In addition, a plausible explanation for the proliferative response of CTCL cells to locally produced interleukin-7 was revealed.
PURPOSE: Increased production of Th2 cytokines characterizes Sezary syndrome, the leukemic form of cutaneous T-cell lymphomas (CTCL). To identify the molecular background and to study whether shared by the most common CTCL subtype, mycosis fungoides, we analyzed the gene expression profiles in both subtypes. EXPERIMENTAL DESIGN: Freshly isolated cells from 30 samples, representing skin, blood, and enriched CD4(+) cell populations of mycosis fungoides and Sezary syndrome, were analyzed with Affymetrix (Santa Clara, CA) oligonucleotide microarrays, quantitative PCR, or immunohistochemistry. The gene expression profiles were combined with findings of comparative genomic hybridization of the same samples to identify chromosomal changes affecting the aberrant gene expression. RESULTS: We identified a set of Th1-specific genes [e.g., TBX21 (T-bet), NKG7, and SCYA5 (RANTES)] to be down-regulated in Sezary syndrome as well as in a proportion of mycosis fungoides samples. In both Sezary syndrome and mycosis fungoides blood samples, the S100P and LIR9 gene expression was up-regulated. In lesional skin, IL7R and CD52 were up-regulated. Integration of comparative genomic hybridization and transcriptomic data identified chromosome arms 1q, 3p, 3q, 4q, 12q, 16p, and 16q as likely targets for new CTCL-associated gene aberrations. CONCLUSIONS: Our findings revealed several new genes involved in CTCL pathogenesis and potential therapeutic targets. Down-regulation of a set of genes involved in Th1 polarization, including the major Th1-polarizing factor, TBX21, was for the first time associated with CTCL. In addition, a plausible explanation for the proliferative response of CTCL cells to locally produced interleukin-7 was revealed.
PURPOSE: Increased production of Th2 cytokines characterizes Sezary syndrome, the leukemic form of cutaneous T-cell lymphomas (CTCL). To identify the molecular background and to study whether shared by the most common CTCL subtype, mycosis fungoides, we analyzed the gene expression profiles in both subtypes. EXPERIMENTAL DESIGN: Freshly isolated cells from 30 samples, representing skin, blood, and enriched CD4(+) cell populations of mycosis fungoides and Sezary syndrome, were analyzed with Affymetrix (Santa Clara, CA) oligonucleotide microarrays, quantitative PCR, or immunohistochemistry. The gene expression profiles were combined with findings of comparative genomic hybridization of the same samples to identify chromosomal changes affecting the aberrant gene expression. RESULTS: We identified a set of Th1-specific genes [e.g., TBX21 (T-bet), NKG7, and SCYA5 (RANTES)] to be down-regulated in Sezary syndrome as well as in a proportion of mycosis fungoides samples. In both Sezary syndrome and mycosis fungoides blood samples, the S100P and LIR9 gene expression was up-regulated. In lesional skin, IL7R and CD52 were up-regulated. Integration of comparative genomic hybridization and transcriptomic data identified chromosome arms 1q, 3p, 3q, 4q, 12q, 16p, and 16q as likely targets for new CTCL-associated gene aberrations. CONCLUSIONS: Our findings revealed several new genes involved in CTCL pathogenesis and potential therapeutic targets. Down-regulation of a set of genes involved in Th1 polarization, including the major Th1-polarizing factor, TBX21, was for the first time associated with CTCL. In addition, a plausible explanation for the proliferative response of CTCL cells to locally produced interleukin-7 was revealed.
PURPOSE: Increased production of Th2 cytokines characterizes Sezary syndrome, the leukemic form of cutaneous T-cell lymphomas (CTCL). To identify the molecular background and to study whether shared by the most common CTCL subtype, mycosis fungoides, we analyzed the gene expression profiles in both subtypes. EXPERIMENTAL DESIGN: Freshly isolated cells from 30 samples, representing skin, blood, and enriched CD4(+) cell populations of mycosis fungoides and Sezary syndrome, were analyzed with Affymetrix (Santa Clara, CA) oligonucleotide microarrays, quantitative PCR, or immunohistochemistry. The gene expression profiles were combined with findings of comparative genomic hybridization of the same samples to identify chromosomal changes affecting the aberrant gene expression. RESULTS: We identified a set of Th1-specific genes [e.g., TBX21 (T-bet), NKG7, and SCYA5 (RANTES)] to be down-regulated in Sezary syndrome as well as in a proportion of mycosis fungoides samples. In both Sezary syndrome and mycosis fungoides blood samples, the S100P and LIR9 gene expression was up-regulated. In lesional skin, IL7R and CD52 were up-regulated. Integration of comparative genomic hybridization and transcriptomic data identified chromosome arms 1q, 3p, 3q, 4q, 12q, 16p, and 16q as likely targets for new CTCL-associated gene aberrations. CONCLUSIONS: Our findings revealed several new genes involved in CTCL pathogenesis and potential therapeutic targets. Down-regulation of a set of genes involved in Th1 polarization, including the major Th1-polarizing factor, TBX21, was for the first time associated with CTCL. In addition, a plausible explanation for the proliferative response of CTCL cells to locally produced interleukin-7 was revealed.
PURPOSE: Increased production of Th2 cytokines characterizes Sezary syndrome, the leukemic form of cutaneous T-cell lymphomas (CTCL). To identify the molecular background and to study whether shared by the most common CTCL subtype, mycosis fungoides, we analyzed the gene expression profiles in both subtypes. EXPERIMENTAL DESIGN: Freshly isolated cells from 30 samples, representing skin, blood, and enriched CD4(+) cell populations of mycosis fungoides and Sezary syndrome, were analyzed with Affymetrix (Santa Clara, CA) oligonucleotide microarrays, quantitative PCR, or immunohistochemistry. The gene expression profiles were combined with findings of comparative genomic hybridization of the same samples to identify chromosomal changes affecting the aberrant gene expression. RESULTS: We identified a set of Th1-specific genes [e.g., TBX21 (T-bet), NKG7, and SCYA5 (RANTES)] to be down-regulated in Sezary syndrome as well as in a proportion of mycosis fungoides samples. In both Sezary syndrome and mycosis fungoides blood samples, the S100P and LIR9 gene expression was up-regulated. In lesional skin, IL7R and CD52 were up-regulated. Integration of comparative genomic hybridization and transcriptomic data identified chromosome arms 1q, 3p, 3q, 4q, 12q, 16p, and 16q as likely targets for new CTCL-associated gene aberrations. CONCLUSIONS: Our findings revealed several new genes involved in CTCL pathogenesis and potential therapeutic targets. Down-regulation of a set of genes involved in Th1 polarization, including the major Th1-polarizing factor, TBX21, was for the first time associated with CTCL. In addition, a plausible explanation for the proliferative response of CTCL cells to locally produced interleukin-7 was revealed.
PURPOSE: Increased production of Th2 cytokines characterizes Sezary syndrome, the leukemic form of cutaneous T-cell lymphomas (CTCL). To identify the molecular background and to study whether shared by the most common CTCL subtype, mycosis fungoides, we analyzed the gene expression profiles in both subtypes. EXPERIMENTAL DESIGN: Freshly isolated cells from 30 samples, representing skin, blood, and enriched CD4(+) cell populations of mycosis fungoides and Sezary syndrome, were analyzed with Affymetrix (Santa Clara, CA) oligonucleotide microarrays, quantitative PCR, or immunohistochemistry. The gene expression profiles were combined with findings of comparative genomic hybridization of the same samples to identify chromosomal changes affecting the aberrant gene expression. RESULTS: We identified a set of Th1-specific genes [e.g., TBX21 (T-bet), NKG7, and SCYA5 (RANTES)] to be down-regulated in Sezary syndrome as well as in a proportion of mycosis fungoides samples. In both Sezary syndrome and mycosis fungoides blood samples, the S100P and LIR9 gene expression was up-regulated. In lesional skin, IL7R and CD52 were up-regulated. Integration of comparative genomic hybridization and transcriptomic data identified chromosome arms 1q, 3p, 3q, 4q, 12q, 16p, and 16q as likely targets for new CTCL-associated gene aberrations. CONCLUSIONS: Our findings revealed several new genes involved in CTCL pathogenesis and potential therapeutic targets. Down-regulation of a set of genes involved in Th1 polarization, including the major Th1-polarizing factor, TBX21, was for the first time associated with CTCL. In addition, a plausible explanation for the proliferative response of CTCL cells to locally produced interleukin-7 was revealed.
Tumour onset and progression are due to the accumulation of genomic lesions, which alter gene expression and ultimately proteome activities. These lesions are thought to affect primarily the transcriptional control of gene expression. In the present study, we aimed at evaluating the genome-wide occurrence of alterations in the translational control exploiting an isogenic, phenotypically validated cellular model of colorectal cancer (CRC) transition from invasive carcinoma to metastasis. In this model, microarray profiling shows that changes in the level of messenger ribonucleic acid (mRNA) association with polysomes occur more than 2-fold than changes in the level of total cellular mRNA. When common to both the total and polysomal compartments, these changes are also homodirectional, being amplified in magnitude at the polysomal level. Comparison between the transcriptional and the translational fluctuations revealed distinct signatures of statistically over-represented gene functions, involving the program of cell proliferation for both levels of analysis, while the apoptosis and the translation programs were affected mainly at translation. Looking for an upstream determinant of translational deregulation, we found an increase in the hyperphosphorylated form of the 4E-BP1 protein in the metastatic cell line, possibly resulting in an increased activation of cap-dependent translation due to increased activity of the eIF4E protein. Analysis of the distribution profiles for the 5' untranslated region (5'-UTR) length of the changed genes showed an association between longer 5'-UTRs and the probability for the relevant gene to be altered translationally, consistent with enhanced eIF4E function. This genome-wide analysis is in favour of a model of profound alteration of translational control in late CRC progression. It also suggests polysomal mRNA profiles as a new, informative dimension for the study of transcriptome imbalance in cancer.
Tumour onset and progression are due to the accumulation of genomic lesions, which alter gene expression and ultimately proteome activities. These lesions are thought to affect primarily the transcriptional control of gene expression. In the present study, we aimed at evaluating the genome-wide occurrence of alterations in the translational control exploiting an isogenic, phenotypically validated cellular model of colorectal cancer (CRC) transition from invasive carcinoma to metastasis. In this model, microarray profiling shows that changes in the level of messenger ribonucleic acid (mRNA) association with polysomes occur more than 2-fold than changes in the level of total cellular mRNA. When common to both the total and polysomal compartments, these changes are also homodirectional, being amplified in magnitude at the polysomal level. Comparison between the transcriptional and the translational fluctuations revealed distinct signatures of statistically over-represented gene functions, involving the program of cell proliferation for both levels of analysis, while the apoptosis and the translation programs were affected mainly at translation. Looking for an upstream determinant of translational deregulation, we found an increase in the hyperphosphorylated form of the 4E-BP1 protein in the metastatic cell line, possibly resulting in an increased activation of cap-dependent translation due to increased activity of the eIF4E protein. Analysis of the distribution profiles for the 5' untranslated region (5'-UTR) length of the changed genes showed an association between longer 5'-UTRs and the probability for the relevant gene to be altered translationally, consistent with enhanced eIF4E function. This genome-wide analysis is in favour of a model of profound alteration of translational control in late CRC progression. It also suggests polysomal mRNA profiles as a new, informative dimension for the study of transcriptome imbalance in cancer.
Insulin-like growth factor (IGF) signaling is a key regulator of breast development and breast cancer. We have analyzed the expression of the IGF signaling cascade in 17 human breast cancer and 4 mammary epithelial cell lines. Five cell lines expressed high levels of IGF1 receptor, insulin (INS)/IGF receptor substrate 1, IGF-binding proteins 2 and 4, as well as the estrogen receptor (ESR), indicating a co-activation of IGF and ESR signaling. Next, we stably overexpressed IGF1 and IGF2 in MCF7 breast cancer cells, which did not affect their epithelial characteristics and the expression and localization of the epithelial marker genes E-cadherin and beta-catenin. Conversely, IGF1 and IGF2 overexpression potently increased cellular proliferation rates and the efficiency of tumor formation in mouse xenograft experiments, whereas the resistance to chemotherapeutic drugs such as taxol was unaltered. Expression profiling of overexpressing cells with whole-genome oligonucleotide microarrays revealed that 21 genes were upregulated >2-fold by both IGF1 and IGF2, 9 by IGF1, and 9 by IGF2. Half of the genes found to be upregulated are involved in transport and biosynthesis of amino acids, including several amino acid transport proteins, argininosuccinate and asparagine synthetases, and methionyl-tRNA synthetase. Upregulation of these genes constitutes a novel mechanism apparently contributing to the stimulatory effects of IGF signaling on the global protein synthesis rate. We conclude that the induction of cell proliferation and tumor formation by long-term IGF stimulation may primarily be due to anabolic effects, in particular increased amino acid production and uptake.
Caudal-related homeobox 2 (Cdx2) has been suggested as an early marker of Barrett's esophagus (BE), which is the premalignant lesion of esophageal adenocarcinoma (EAC). However, the mechanism of ectopic Cdx2 expression in the esophageal epithelial cells and its role in the development of BE remained unclear. RT-PCR, pyrosequencing and methylation-specific PCR were used to determine expression and promoter methylation of Cdx2 in human esophageal epithelial cells (HET1A and SEG1) after treatment with 5-aza-2'-deoxycytidine (DAC), acid, bile acids and their combination. HET1A cells with stable transfection of Cdx2 were characterized for morphology and gene expression profiles with Affymetrix array. We found Cdx2 was expressed in most human EAC cell lines, but not in squamous epithelial cell lines. DAC-induced demethylation and expression of Cdx2 in HET1A and SEG1 cells, and treatment with a DNA methylating agent counteracted the effect of DAC. Treatment of HET1A and SEG1 cells with acid, bile acids or both also resulted in promoter demethylation and expression of Cdx2. HET1A cells with stable transfection of human Cdx2 formed crypt-like structures in vitro. Microarray analysis and quantitative real-time PCR showed that stable transfection of Cdx2 up-regulated differentiation markers of intestinal columnar epithelial cells and goblet cells in HET1A cells. This may be partially due to modulation of Notch signaling pathway, as western blotting confirmed down-regulation of Hes1 and up-regulation of Atoh1 and Muc2. Our data suggest that exposure to acid and/or bile acids may activate Cdx2 expression in human esophageal epithelial cells through promoter demethylation, and ectopic Cdx2 expression in esophageal squamous epithelial cells may contribute to intestinal metaplasia of the esophagus.
Caudal-related homeobox 2 (Cdx2) has been suggested as an early marker of Barrett's esophagus (BE), which is the premalignant lesion of esophageal adenocarcinoma (EAC). However, the mechanism of ectopic Cdx2 expression in the esophageal epithelial cells and its role in the development of BE remained unclear. RT-PCR, pyrosequencing and methylation-specific PCR were used to determine expression and promoter methylation of Cdx2 in human esophageal epithelial cells (HET1A and SEG1) after treatment with 5-aza-2'-deoxycytidine (DAC), acid, bile acids and their combination. HET1A cells with stable transfection of Cdx2 were characterized for morphology and gene expression profiles with Affymetrix array. We found Cdx2 was expressed in most human EAC cell lines, but not in squamous epithelial cell lines. DAC-induced demethylation and expression of Cdx2 in HET1A and SEG1 cells, and treatment with a DNA methylating agent counteracted the effect of DAC. Treatment of HET1A and SEG1 cells with acid, bile acids or both also resulted in promoter demethylation and expression of Cdx2. HET1A cells with stable transfection of human Cdx2 formed crypt-like structures in vitro. Microarray analysis and quantitative real-time PCR showed that stable transfection of Cdx2 up-regulated differentiation markers of intestinal columnar epithelial cells and goblet cells in HET1A cells. This may be partially due to modulation of Notch signaling pathway, as western blotting confirmed down-regulation of Hes1 and up-regulation of Atoh1 and Muc2. Our data suggest that exposure to acid and/or bile acids may activate Cdx2 expression in human esophageal epithelial cells through promoter demethylation, and ectopic Cdx2 expression in esophageal squamous epithelial cells may contribute to intestinal metaplasia of the esophagus.
Tyrosine kinase inhibitors (TKI) of the epidermal growth factor receptor (EGFR) produce objective responses in a minority of patients with advanced-stage non-small cell lung cancer (NSCLC), and about half of all treated patients progress within 6 weeks of instituting therapy. Because the target of these agents is known, it should be possible to develop biological predictors of response, but EGFR protein levels have not been proven useful as a predictor of TKI response in patients and the mechanism of primary resistance is unclear. We used microarray gene expression profiling to uncover a pattern of gene expression associated with sensitivity to EGFR-TKIs by comparing NSCLC cell lines that were either highly sensitive or highly resistant to gefitinib. This sensitivity-associated expression profile was used to predict gefitinib sensitivity in a panel of NSCLC cell lines with known gene expression profiles but unknown gefitinib sensitivity. Gefitinib sensitivity was then determined for members of this test panel, and the microarray-based sensitivity prediction was correct in eight of nine NSCLC cell lines. Gene and protein expression differences were confirmed with a combination of quantitative reverse transcription-PCR, flow cytometry, and immunohistochemistry. This gene expression pattern related to gefitinib sensitivity was independent from sensitivity associated with EGFR mutations. Several genes associated with sensitivity encode proteins involved in HER pathway signaling or pathways that interrelate to the HER signaling pathway. Some of these genes could be targets of pharmacologic interventions to overcome primary resistance.
Tyrosine kinase inhibitors (TKI) of the epidermal growth factor receptor (EGFR) produce objective responses in a minority of patients with advanced-stage non-small cell lung cancer (NSCLC), and about half of all treated patients progress within 6 weeks of instituting therapy. Because the target of these agents is known, it should be possible to develop biological predictors of response, but EGFR protein levels have not been proven useful as a predictor of TKI response in patients and the mechanism of primary resistance is unclear. We used microarray gene expression profiling to uncover a pattern of gene expression associated with sensitivity to EGFR-TKIs by comparing NSCLC cell lines that were either highly sensitive or highly resistant to gefitinib. This sensitivity-associated expression profile was used to predict gefitinib sensitivity in a panel of NSCLC cell lines with known gene expression profiles but unknown gefitinib sensitivity. Gefitinib sensitivity was then determined for members of this test panel, and the microarray-based sensitivity prediction was correct in eight of nine NSCLC cell lines. Gene and protein expression differences were confirmed with a combination of quantitative reverse transcription-PCR, flow cytometry, and immunohistochemistry. This gene expression pattern related to gefitinib sensitivity was independent from sensitivity associated with EGFR mutations. Several genes associated with sensitivity encode proteins involved in HER pathway signaling or pathways that interrelate to the HER signaling pathway. Some of these genes could be targets of pharmacologic interventions to overcome primary resistance.
RIZ1 is a histone methyltransferase whose expression and activity are reduced in many cancers. In chronic myelogenous leukemia (CML), blastic transformation is associated with loss of heterozygosity in the region where RIZ1 is located and with decreased RIZ1 expression. Forced RIZ1 expression in model CML blast crisis (BC) cell lines decreases proliferation, increases apoptosis and enhances differentiation. We characterized molecular mechanisms that may contribute to potential CML tumor suppressor properties of RIZ1. Several RIZ1-regulated genes involved in insulin-like growth factor-1 (IGF-1) signaling were identified using cDNA microarrays. RIZ1 was shown to associate with promoter regions of IGF-1 and to increase histone H3 lysine 9 methylation using chromatin immunoprecipitation assays. IGF-1-blocking antibody was used to demonstrate the importance of autocrine IGF-1 signaling in CML-BC cell line viability. Forced RIZ1 expression in CML-BC cell lines decreases IGF-1 receptor activation and activation of downstream signaling components extracellular signal-regulated kinase 1/2 and AKT. These results highlight the therapeutic potential of inhibiting IGF-1 pathway in the acute phase of CML.
RIZ1 is a histone methyltransferase whose expression and activity are reduced in many cancers. In chronic myelogenous leukemia (CML), blastic transformation is associated with loss of heterozygosity in the region where RIZ1 is located and with decreased RIZ1 expression. Forced RIZ1 expression in model CML blast crisis (BC) cell lines decreases proliferation, increases apoptosis and enhances differentiation. We characterized molecular mechanisms that may contribute to potential CML tumor suppressor properties of RIZ1. Several RIZ1-regulated genes involved in insulin-like growth factor-1 (IGF-1) signaling were identified using cDNA microarrays. RIZ1 was shown to associate with promoter regions of IGF-1 and to increase histone H3 lysine 9 methylation using chromatin immunoprecipitation assays. IGF-1-blocking antibody was used to demonstrate the importance of autocrine IGF-1 signaling in CML-BC cell line viability. Forced RIZ1 expression in CML-BC cell lines decreases IGF-1 receptor activation and activation of downstream signaling components extracellular signal-regulated kinase 1/2 and AKT. These results highlight the therapeutic potential of inhibiting IGF-1 pathway in the acute phase of CML.
RASSF1A is a tumor suppressor gene on 3p21.3 frequently inactivated by promoter hypermethylation in nasopharyngeal carcinoma (NPC). To identify RASSF1A target genes in NPC, we have investigated the expression profile of the stable RASSF1A transfectants and controls by high-density oligonucleotide array. A total of 57 genes showed differential expression in the RASSF1A-expressing cells. These RASSF1A target genes were involved in multiple cellular regulatory processes such as transcription, signal transduction, cell adhesion and RNA processing. The RASSF1A-modulated expression of eight selected genes with the highest fold changes (ATF5, TCRB, RGS1, activin betaE, HNRPH1, HNRPD, Id2 and CKS2) by RASSF1A was confirmed in both stable and transient transfectants. Compared with the RASSF1A transfectants, an inverse expression pattern of activin betaE, Id2 and ATF5 was shown in the immortalized nasopharyngeal epithelial cells treated with siRNA against RASSF1A. The findings imply that the expression of activin betaE, Id2 and ATF5 was tightly regulated by RASSF1A and may associate with its tumor suppressor function. Strikingly, overexpression of Id2 is common in NPC and RASSF1A-induced repression of Id2 was mediated by the overexpression of activin betaE. The results suggest a novel RASSF1A pathway in which both activin betaE and Id2 are involved.
RASSF1A is a tumor suppressor gene on 3p21.3 frequently inactivated by promoter hypermethylation in nasopharyngeal carcinoma (NPC). To identify RASSF1A target genes in NPC, we have investigated the expression profile of the stable RASSF1A transfectants and controls by high-density oligonucleotide array. A total of 57 genes showed differential expression in the RASSF1A-expressing cells. These RASSF1A target genes were involved in multiple cellular regulatory processes such as transcription, signal transduction, cell adhesion and RNA processing. The RASSF1A-modulated expression of eight selected genes with the highest fold changes (ATF5, TCRB, RGS1, activin betaE, HNRPH1, HNRPD, Id2 and CKS2) by RASSF1A was confirmed in both stable and transient transfectants. Compared with the RASSF1A transfectants, an inverse expression pattern of activin betaE, Id2 and ATF5 was shown in the immortalized nasopharyngeal epithelial cells treated with siRNA against RASSF1A. The findings imply that the expression of activin betaE, Id2 and ATF5 was tightly regulated by RASSF1A and may associate with its tumor suppressor function. Strikingly, overexpression of Id2 is common in NPC and RASSF1A-induced repression of Id2 was mediated by the overexpression of activin betaE. The results suggest a novel RASSF1A pathway in which both activin betaE and Id2 are involved.
In order to obtain a comprehensive picture of the molecular events regulating cutaneous photodamage of intact human epidermis, suction blister roofs obtained after a single dose of in vivo ultraviolet (UV)B exposure were used for microarray profiling. We found a changed expression of 619 genes. Half of the UVB-regulated genes had returned to pre-exposure baseline levels at 72 h, underscoring the transient character of the molecular cutaneous UVB response. Of special interest was our finding that several of the central p53 target genes remained unaffected following UVB exposure in spite of p53 protein accumulation. We next compared the in vivo expression profiles of epidermal sheets to that of cultured human epidermal keratinocytes exposed to UVB in vitro. We found 1931 genes that differed in their expression profiles between the two groups. The expression profile in intact epidemis was geared mainly towards DNA repair, whereas cultured keratinocytes responded predominantly by activating genes associated with cell-cycle arrest and apoptosis. These differences in expression profiles might reflect differences between mature differentiating keratinocytes in the suprabasal epidermal layers versus exponentially proliferating keratinocytes in cell culture. Our findings show that extreme care should be taken when extrapolating from findings based on keratinocyte cultures to changes in intact epidermis.
In order to obtain a comprehensive picture of the molecular events regulating cutaneous photodamage of intact human epidermis, suction blister roofs obtained after a single dose of in vivo ultraviolet (UV)B exposure were used for microarray profiling. We found a changed expression of 619 genes. Half of the UVB-regulated genes had returned to pre-exposure baseline levels at 72 h, underscoring the transient character of the molecular cutaneous UVB response. Of special interest was our finding that several of the central p53 target genes remained unaffected following UVB exposure in spite of p53 protein accumulation. We next compared the in vivo expression profiles of epidermal sheets to that of cultured human epidermal keratinocytes exposed to UVB in vitro. We found 1931 genes that differed in their expression profiles between the two groups. The expression profile in intact epidemis was geared mainly towards DNA repair, whereas cultured keratinocytes responded predominantly by activating genes associated with cell-cycle arrest and apoptosis. These differences in expression profiles might reflect differences between mature differentiating keratinocytes in the suprabasal epidermal layers versus exponentially proliferating keratinocytes in cell culture. Our findings show that extreme care should be taken when extrapolating from findings based on keratinocyte cultures to changes in intact epidermis.
In order to obtain a comprehensive picture of the molecular events regulating cutaneous photodamage of intact human epidermis, suction blister roofs obtained after a single dose of in vivo ultraviolet (UV)B exposure were used for microarray profiling. We found a changed expression of 619 genes. Half of the UVB-regulated genes had returned to pre-exposure baseline levels at 72 h, underscoring the transient character of the molecular cutaneous UVB response. Of special interest was our finding that several of the central p53 target genes remained unaffected following UVB exposure in spite of p53 protein accumulation. We next compared the in vivo expression profiles of epidermal sheets to that of cultured human epidermal keratinocytes exposed to UVB in vitro. We found 1931 genes that differed in their expression profiles between the two groups. The expression profile in intact epidemis was geared mainly towards DNA repair, whereas cultured keratinocytes responded predominantly by activating genes associated with cell-cycle arrest and apoptosis. These differences in expression profiles might reflect differences between mature differentiating keratinocytes in the suprabasal epidermal layers versus exponentially proliferating keratinocytes in cell culture. Our findings show that extreme care should be taken when extrapolating from findings based on keratinocyte cultures to changes in intact epidermis.
In order to obtain a comprehensive picture of the molecular events regulating cutaneous photodamage of intact human epidermis, suction blister roofs obtained after a single dose of in vivo ultraviolet (UV)B exposure were used for microarray profiling. We found a changed expression of 619 genes. Half of the UVB-regulated genes had returned to pre-exposure baseline levels at 72 h, underscoring the transient character of the molecular cutaneous UVB response. Of special interest was our finding that several of the central p53 target genes remained unaffected following UVB exposure in spite of p53 protein accumulation. We next compared the in vivo expression profiles of epidermal sheets to that of cultured human epidermal keratinocytes exposed to UVB in vitro. We found 1931 genes that differed in their expression profiles between the two groups. The expression profile in intact epidemis was geared mainly towards DNA repair, whereas cultured keratinocytes responded predominantly by activating genes associated with cell-cycle arrest and apoptosis. These differences in expression profiles might reflect differences between mature differentiating keratinocytes in the suprabasal epidermal layers versus exponentially proliferating keratinocytes in cell culture. Our findings show that extreme care should be taken when extrapolating from findings based on keratinocyte cultures to changes in intact epidermis.
Table 1 Genes regulated by mTert overexpression in MEFs Affymetrix oligonucleotide arrays were used to study the transcriptional profile of mTert-overexpressing MEFs before and after retroviral transduction with mTert. Gene expression changes (threshold X1.5-fold) are mean values of two comparisons between two independent early passage cultures derived from individual embryos, either retrovirally transduced with mTert or infected with empty virus (controls). Gene symbols, gene names, and gene ontology annotations correspond to the entries associated with each Affymetrix probe number in the public Database for Annotation, Visualization and Integrated Discovery (DAVID). MEFs, murine embryonic fibroblasts ; mTert, mouse telomerase ; MAPKK, mitogen-activated protein kinase kinase.
Glucocorticoids are extensively used in combination chemotherapy of advanced prostate cancer (PC). Little is known, however, about the status of the glucocorticoid receptor (GR) in PC. We evaluated over 200 prostate samples and determined that GR expression was strongly decreased or absent in 70-85% of PC. Similar to PC tumors, some PC cell lines, including LNCaP, also lack GR. To understand the role of GR, we reconstituted its expression in LNCaP cells using lentiviral approach. Treatment of LNCaP-GR cells with the glucocorticoids strongly inhibited proliferation in the monolayer cultures and blocked anchorage-independent growth. This was accompanied by upregulation of p21 and p27, down-regulation of cyclin D1 expression and c-Myc phosphorylation. Importantly, the activation of GR resulted in normalized expression of PC markers hepsin, AMACR, and maspin. On the signaling level, GR decreased expression and inhibited activity of the MAP-kinases (MAPKs) including p38, JNK/SAPK, Mek1/2 and Erk1/2. We also found that activation of GR inhibited activity of numerous transcription factors (TF) including AP-1, SRF, NF-kappaB, p53, ATF-2, CEBPalpha, Ets-1, Elk-1, STAT1 and others, many of which are regulated via MAPK cascade. The structural analysis of hepsin and AMACR promoters provided the mechanistic rationale for PC marker downregulation by glucocorticoids via inhibition of specific TFs. Our data suggest that GR functions as a tumor suppressor in prostate, and inhibits multiple signaling pathways and transcriptional factors involved in proliferation and transformation.
p16(INK4A) (p16) has been suggested to be an early biomarker for the detection of cervical cancer. However, its functional role in cervical cancer is not well characterized. In this study, we reported the consistent and significant upregulation of p16 in cervical cancer tissues when compared to both matched non-tumourous tissues of the same patient and normal cervical tissues from non-cancer patients. We have employed p16 small interfering RNA (siRNA) to dissect the role of p16 in cervical carcinogenesis. Although the silencing of p16 was accompanied by the upregulation of p53, p21 and RB in the p16 siRNA-transfected cells, no significant effect on cell cycle progression was observed. When the p16 siRNA-silenced cells were subjected to DNA damage stress including ultraviolet-irradiation and cisplatin treatments, a significantly higher percentage of apoptotic cells could be observed in the p16-siRNA silenced cells compared to control siRNA-treated cells. Moreover, induction of apoptosis was associated with the activation of p53 through phosphorylation, and this process, when studied by gene profiling experiments, involved both the intrinsic and extrinsic apoptotic pathways. The observation that silencing of p16 expression augments DNA damage-induced apoptosis in cervical cancer cells offers alternative strategies for anti-cancer therapies for human cervical cancer.
p16(INK4A) (p16) has been suggested to be an early biomarker for the detection of cervical cancer. However, its functional role in cervical cancer is not well characterized. In this study, we reported the consistent and significant upregulation of p16 in cervical cancer tissues when compared to both matched non-tumourous tissues of the same patient and normal cervical tissues from non-cancer patients. We have employed p16 small interfering RNA (siRNA) to dissect the role of p16 in cervical carcinogenesis. Although the silencing of p16 was accompanied by the upregulation of p53, p21 and RB in the p16 siRNA-transfected cells, no significant effect on cell cycle progression was observed. When the p16 siRNA-silenced cells were subjected to DNA damage stress including ultraviolet-irradiation and cisplatin treatments, a significantly higher percentage of apoptotic cells could be observed in the p16-siRNA silenced cells compared to control siRNA-treated cells. Moreover, induction of apoptosis was associated with the activation of p53 through phosphorylation, and this process, when studied by gene profiling experiments, involved both the intrinsic and extrinsic apoptotic pathways. The observation that silencing of p16 expression augments DNA damage-induced apoptosis in cervical cancer cells offers alternative strategies for anti-cancer therapies for human cervical cancer.
The purpose of this paper is to correlate the molecular phenotype of papillary thyroid carcinoma (PTC) to their biological pathology. We hybridized 26 PTC on microarrays and showed that nearly 44% of the transcriptome was regulated in these tumors. We then combined our data set with two published PTC microarray studies to produce a platform- and study-independent list of PTC-associated genes. We further confirmed the mRNA regulation of 15 genes from this list by quantitative reverse transcription-PCR. Analysis of this list with statistical tools led to several conclusions: (1) there is a change in cell population with an increased expression of genes involved in the immune response, reflecting lymphocyte infiltration in the tumor compared to the normal tissue. (2) The c-jun N-terminal kinase pathway is activated by overexpression of its components. (3) The activation of ERKK1/2 by genetic alterations is supplemented by activation of the epidermal growth factor but not of the insulin-like growth factor signaling pathway. (4) There is a downregulation of immediate early genes. (5) We observed an overexpression of many proteases in accordance with tumor remodeling, and suggested a probable role of S100 proteins and annexin A2 in this process. (6) Numerous overexpressed genes favor the hypothesis of a collective migration mode of tumor cells.
The purpose of this paper is to correlate the molecular phenotype of papillary thyroid carcinoma (PTC) to their biological pathology. We hybridized 26 PTC on microarrays and showed that nearly 44% of the transcriptome was regulated in these tumors. We then combined our data set with two published PTC microarray studies to produce a platform- and study-independent list of PTC-associated genes. We further confirmed the mRNA regulation of 15 genes from this list by quantitative reverse transcription-PCR. Analysis of this list with statistical tools led to several conclusions: (1) there is a change in cell population with an increased expression of genes involved in the immune response, reflecting lymphocyte infiltration in the tumor compared to the normal tissue. (2) The c-jun N-terminal kinase pathway is activated by overexpression of its components. (3) The activation of ERKK1/2 by genetic alterations is supplemented by activation of the epidermal growth factor but not of the insulin-like growth factor signaling pathway. (4) There is a downregulation of immediate early genes. (5) We observed an overexpression of many proteases in accordance with tumor remodeling, and suggested a probable role of S100 proteins and annexin A2 in this process. (6) Numerous overexpressed genes favor the hypothesis of a collective migration mode of tumor cells.
Mutational activation of ras genes is required for the onset and maintenance of different malignancies. Here we show, using a combination of molecular physiology, nutritional perturbations and transcriptional profiling, that full penetrance of phenotypes related to oncogenic Ras activation, including the shift of carbon metabolism towards fermentation and upregulation of key cell cycle regulators, is dependent upon glucose availability. These responses are induced by Ras activation, being specifically reverted by downregulation of the Ras pathway obtained through the expression of a dominant-negative Ras-specific guanine nucleotide exchange protein. Our data allow to link directly to ras activation the alteration in energy metabolism of cancer cells, their fragility towards glucose shortage and ensuing apoptotic death.
Mutational activation of ras genes is required for the onset and maintenance of different malignancies. Here we show, using a combination of molecular physiology, nutritional perturbations and transcriptional profiling, that full penetrance of phenotypes related to oncogenic Ras activation, including the shift of carbon metabolism towards fermentation and upregulation of key cell cycle regulators, is dependent upon glucose availability. These responses are induced by Ras activation, being specifically reverted by downregulation of the Ras pathway obtained through the expression of a dominant-negative Ras-specific guanine nucleotide exchange protein. Our data allow to link directly to ras activation the alteration in energy metabolism of cancer cells, their fragility towards glucose shortage and ensuing apoptotic death.
Mutational activation of ras genes is required for the onset and maintenance of different malignancies. Here we show, using a combination of molecular physiology, nutritional perturbations and transcriptional profiling, that full penetrance of phenotypes related to oncogenic Ras activation, including the shift of carbon metabolism towards fermentation and upregulation of key cell cycle regulators, is dependent upon glucose availability. These responses are induced by Ras activation, being specifically reverted by downregulation of the Ras pathway obtained through the expression of a dominant-negative Ras-specific guanine nucleotide exchange protein. Our data allow to link directly to ras activation the alteration in energy metabolism of cancer cells, their fragility towards glucose shortage and ensuing apoptotic death.
Mutational activation of ras genes is required for the onset and maintenance of different malignancies. Here we show, using a combination of molecular physiology, nutritional perturbations and transcriptional profiling, that full penetrance of phenotypes related to oncogenic Ras activation, including the shift of carbon metabolism towards fermentation and upregulation of key cell cycle regulators, is dependent upon glucose availability. These responses are induced by Ras activation, being specifically reverted by downregulation of the Ras pathway obtained through the expression of a dominant-negative Ras-specific guanine nucleotide exchange protein. Our data allow to link directly to ras activation the alteration in energy metabolism of cancer cells, their fragility towards glucose shortage and ensuing apoptotic death.
Mutational activation of ras genes is required for the onset and maintenance of different malignancies. Here we show, using a combination of molecular physiology, nutritional perturbations and transcriptional profiling, that full penetrance of phenotypes related to oncogenic Ras activation, including the shift of carbon metabolism towards fermentation and upregulation of key cell cycle regulators, is dependent upon glucose availability. These responses are induced by Ras activation, being specifically reverted by downregulation of the Ras pathway obtained through the expression of a dominant-negative Ras-specific guanine nucleotide exchange protein. Our data allow to link directly to ras activation the alteration in energy metabolism of cancer cells, their fragility towards glucose shortage and ensuing apoptotic death.
Mutational activation of ras genes is required for the onset and maintenance of different malignancies. Here we show, using a combination of molecular physiology, nutritional perturbations and transcriptional profiling, that full penetrance of phenotypes related to oncogenic Ras activation, including the shift of carbon metabolism towards fermentation and upregulation of key cell cycle regulators, is dependent upon glucose availability. These responses are induced by Ras activation, being specifically reverted by downregulation of the Ras pathway obtained through the expression of a dominant-negative Ras-specific guanine nucleotide exchange protein. Our data allow to link directly to ras activation the alteration in energy metabolism of cancer cells, their fragility towards glucose shortage and ensuing apoptotic death.
We characterized differential gene expression profiles of fibroblast cell lines harboring single or double-homozygous null mutations in H-ras and N-ras. Whereas the expression level of the individual H-, N- and K-ras genes appeared unaffected by the presence or absence of the other ras loci, significant differences were observed between the expression profiles of cells missing N-ras and/or H-ras. Absence of N-ras produced much stronger effects than absence of H-ras over the profile of the cellular transcriptome. N-ras(-/-) and H-ras(-/-) fibroblasts displayed rather antagonistic expression profiles and the transcriptome of H-ras(-/-) cells was significantly closer to that of wild-type fibroblasts than to that of N-ras(-/-) cells. Classifying all differentially expressed genes into functional categories suggested specific roles for H-Ras and N-Ras. It was particularly striking in N-ras(-/-) cells the upregulation of a remarkable number of immunity-related genes, as well as of several loci involved in apoptosis. Reverse-phase protein array assays demonstrated in the same N-ras(-/-) cells the overexpression and nuclear migration of tyrosine phosphorylated signal transducer and activator of transcription 1 (Stat1) which was concomitant with transcriptional activation mediated by interferon-stimulated response elements. Significantly enhanced numbers of apoptotic cells were also detected in cultures of N-ras(-/-) cells. Our data support the notion that different Ras isoforms play functionally distinct cellular roles and indicate that N-Ras is significantly involved in immune modulation/host defense and apoptotic responses.
We characterized differential gene expression profiles of fibroblast cell lines harboring single or double-homozygous null mutations in H-ras and N-ras. Whereas the expression level of the individual H-, N- and K-ras genes appeared unaffected by the presence or absence of the other ras loci, significant differences were observed between the expression profiles of cells missing N-ras and/or H-ras. Absence of N-ras produced much stronger effects than absence of H-ras over the profile of the cellular transcriptome. N-ras(-/-) and H-ras(-/-) fibroblasts displayed rather antagonistic expression profiles and the transcriptome of H-ras(-/-) cells was significantly closer to that of wild-type fibroblasts than to that of N-ras(-/-) cells. Classifying all differentially expressed genes into functional categories suggested specific roles for H-Ras and N-Ras. It was particularly striking in N-ras(-/-) cells the upregulation of a remarkable number of immunity-related genes, as well as of several loci involved in apoptosis. Reverse-phase protein array assays demonstrated in the same N-ras(-/-) cells the overexpression and nuclear migration of tyrosine phosphorylated signal transducer and activator of transcription 1 (Stat1) which was concomitant with transcriptional activation mediated by interferon-stimulated response elements. Significantly enhanced numbers of apoptotic cells were also detected in cultures of N-ras(-/-) cells. Our data support the notion that different Ras isoforms play functionally distinct cellular roles and indicate that N-Ras is significantly involved in immune modulation/host defense and apoptotic responses.
We characterized differential gene expression profiles of fibroblast cell lines harboring single or double-homozygous null mutations in H-ras and N-ras. Whereas the expression level of the individual H-, N- and K-ras genes appeared unaffected by the presence or absence of the other ras loci, significant differences were observed between the expression profiles of cells missing N-ras and/or H-ras. Absence of N-ras produced much stronger effects than absence of H-ras over the profile of the cellular transcriptome. N-ras(-/-) and H-ras(-/-) fibroblasts displayed rather antagonistic expression profiles and the transcriptome of H-ras(-/-) cells was significantly closer to that of wild-type fibroblasts than to that of N-ras(-/-) cells. Classifying all differentially expressed genes into functional categories suggested specific roles for H-Ras and N-Ras. It was particularly striking in N-ras(-/-) cells the upregulation of a remarkable number of immunity-related genes, as well as of several loci involved in apoptosis. Reverse-phase protein array assays demonstrated in the same N-ras(-/-) cells the overexpression and nuclear migration of tyrosine phosphorylated signal transducer and activator of transcription 1 (Stat1) which was concomitant with transcriptional activation mediated by interferon-stimulated response elements. Significantly enhanced numbers of apoptotic cells were also detected in cultures of N-ras(-/-) cells. Our data support the notion that different Ras isoforms play functionally distinct cellular roles and indicate that N-Ras is significantly involved in immune modulation/host defense and apoptotic responses.
We characterized differential gene expression profiles of fibroblast cell lines harboring single or double-homozygous null mutations in H-ras and N-ras. Whereas the expression level of the individual H-, N- and K-ras genes appeared unaffected by the presence or absence of the other ras loci, significant differences were observed between the expression profiles of cells missing N-ras and/or H-ras. Absence of N-ras produced much stronger effects than absence of H-ras over the profile of the cellular transcriptome. N-ras(-/-) and H-ras(-/-) fibroblasts displayed rather antagonistic expression profiles and the transcriptome of H-ras(-/-) cells was significantly closer to that of wild-type fibroblasts than to that of N-ras(-/-) cells. Classifying all differentially expressed genes into functional categories suggested specific roles for H-Ras and N-Ras. It was particularly striking in N-ras(-/-) cells the upregulation of a remarkable number of immunity-related genes, as well as of several loci involved in apoptosis. Reverse-phase protein array assays demonstrated in the same N-ras(-/-) cells the overexpression and nuclear migration of tyrosine phosphorylated signal transducer and activator of transcription 1 (Stat1) which was concomitant with transcriptional activation mediated by interferon-stimulated response elements. Significantly enhanced numbers of apoptotic cells were also detected in cultures of N-ras(-/-) cells. Our data support the notion that different Ras isoforms play functionally distinct cellular roles and indicate that N-Ras is significantly involved in immune modulation/host defense and apoptotic responses.
We characterized differential gene expression profiles of fibroblast cell lines harboring single or double-homozygous null mutations in H-ras and N-ras. Whereas the expression level of the individual H-, N- and K-ras genes appeared unaffected by the presence or absence of the other ras loci, significant differences were observed between the expression profiles of cells missing N-ras and/or H-ras. Absence of N-ras produced much stronger effects than absence of H-ras over the profile of the cellular transcriptome. N-ras(-/-) and H-ras(-/-) fibroblasts displayed rather antagonistic expression profiles and the transcriptome of H-ras(-/-) cells was significantly closer to that of wild-type fibroblasts than to that of N-ras(-/-) cells. Classifying all differentially expressed genes into functional categories suggested specific roles for H-Ras and N-Ras. It was particularly striking in N-ras(-/-) cells the upregulation of a remarkable number of immunity-related genes, as well as of several loci involved in apoptosis. Reverse-phase protein array assays demonstrated in the same N-ras(-/-) cells the overexpression and nuclear migration of tyrosine phosphorylated signal transducer and activator of transcription 1 (Stat1) which was concomitant with transcriptional activation mediated by interferon-stimulated response elements. Significantly enhanced numbers of apoptotic cells were also detected in cultures of N-ras(-/-) cells. Our data support the notion that different Ras isoforms play functionally distinct cellular roles and indicate that N-Ras is significantly involved in immune modulation/host defense and apoptotic responses.
We characterized differential gene expression profiles of fibroblast cell lines harboring single or double-homozygous null mutations in H-ras and N-ras. Whereas the expression level of the individual H-, N- and K-ras genes appeared unaffected by the presence or absence of the other ras loci, significant differences were observed between the expression profiles of cells missing N-ras and/or H-ras. Absence of N-ras produced much stronger effects than absence of H-ras over the profile of the cellular transcriptome. N-ras(-/-) and H-ras(-/-) fibroblasts displayed rather antagonistic expression profiles and the transcriptome of H-ras(-/-) cells was significantly closer to that of wild-type fibroblasts than to that of N-ras(-/-) cells. Classifying all differentially expressed genes into functional categories suggested specific roles for H-Ras and N-Ras. It was particularly striking in N-ras(-/-) cells the upregulation of a remarkable number of immunity-related genes, as well as of several loci involved in apoptosis. Reverse-phase protein array assays demonstrated in the same N-ras(-/-) cells the overexpression and nuclear migration of tyrosine phosphorylated signal transducer and activator of transcription 1 (Stat1) which was concomitant with transcriptional activation mediated by interferon-stimulated response elements. Significantly enhanced numbers of apoptotic cells were also detected in cultures of N-ras(-/-) cells. Our data support the notion that different Ras isoforms play functionally distinct cellular roles and indicate that N-Ras is significantly involved in immune modulation/host defense and apoptotic responses.
Glucocorticoids are potent inhibitors of mouse skin tumorigenesis. The glucocorticoid control of cellular functions is mediated via the glucocorticoid receptor (GR), a well-known transcription factor. Recently, we generated transgenic mice overexpressing GR under control of the keratin5 (K5) promoter, and showed that K5.GR animals are resistant to skin carcinogenesis. Follicular epithelial stem cells (SCs), located in the bulge region of the hair follicle, are believed to be one of the target cells for skin carcinogenesis. We found that the number of putative hair follicle SC detected as label-retaining cells was significantly less in the K5.GR transgenics compared to wild type (w.t.) littermates. We also showed that GR overexpression led to a reduction in the clonogenicity of the follicular epithelial SCs. We evaluated the global effect of GR on gene expression in a population of follicular SC-enriched bulge keratinocytes isolated by fluorescence activated cell sorting. We found that GR affected the expression of numerous bulge SC 'signature' genes, genes involved in the maintenance of SC and progenitor cells of non-epidermal origin and proapoptotic genes. Our findings underscore the important role of GR signaling in the homeostasis of follicular epithelial SCs, and suggest that the reduction in their number may underlie the tumor suppressor effect of GR in the skin.
Glucocorticoids are potent inhibitors of mouse skin tumorigenesis. The glucocorticoid control of cellular functions is mediated via the glucocorticoid receptor (GR), a well-known transcription factor. Recently, we generated transgenic mice overexpressing GR under control of the keratin5 (K5) promoter, and showed that K5.GR animals are resistant to skin carcinogenesis. Follicular epithelial stem cells (SCs), located in the bulge region of the hair follicle, are believed to be one of the target cells for skin carcinogenesis. We found that the number of putative hair follicle SC detected as label-retaining cells was significantly less in the K5.GR transgenics compared to wild type (w.t.) littermates. We also showed that GR overexpression led to a reduction in the clonogenicity of the follicular epithelial SCs. We evaluated the global effect of GR on gene expression in a population of follicular SC-enriched bulge keratinocytes isolated by fluorescence activated cell sorting. We found that GR affected the expression of numerous bulge SC 'signature' genes, genes involved in the maintenance of SC and progenitor cells of non-epidermal origin and proapoptotic genes. Our findings underscore the important role of GR signaling in the homeostasis of follicular epithelial SCs, and suggest that the reduction in their number may underlie the tumor suppressor effect of GR in the skin.
We have used microarray technology to identify the transcriptional targets of Rho subfamily guanosine 5'-triphosphate (GTP)ases in NIH3T3 cells. This analysis indicated that murine fibroblasts transformed by these proteins show similar transcriptomal profiles. Functional annotation of the regulated genes indicate that Rho subfamily GTPases target a wide spectrum of functions, although loci encoding proteins linked to proliferation and DNA synthesis/transcription are upregulated preferentially. Rho proteins promote four main networks of interacting proteins nucleated around E2F, c-Jun, c-Myc and p53. Of those, E2F, c-Jun and c-Myc are essential for the maintenance of cell transformation. Inhibition of Rock, one of the main Rho GTPase targets, leads to small changes in the transcriptome of Rho-transformed cells. Rock inhibition decreases c-myc gene expression without affecting the E2F and c-Jun pathways. Loss-of-function studies demonstrate that c-Myc is important for the blockage of cell-contact inhibition rather than for promoting the proliferation of Rho-transformed cells. However, c-Myc overexpression does not bypass the inhibition of cell transformation induced by Rock blockage, indicating that c-Myc is essential, but not sufficient, for Rock-dependent transformation. These results reveal the complexity of the genetic program orchestrated by the Rho subfamily and pinpoint protein networks that mediate different aspects of the malignant phenotype of Rho-transformed cells.
We have used microarray technology to identify the transcriptional targets of Rho subfamily guanosine 5'-triphosphate (GTP)ases in NIH3T3 cells. This analysis indicated that murine fibroblasts transformed by these proteins show similar transcriptomal profiles. Functional annotation of the regulated genes indicate that Rho subfamily GTPases target a wide spectrum of functions, although loci encoding proteins linked to proliferation and DNA synthesis/transcription are upregulated preferentially. Rho proteins promote four main networks of interacting proteins nucleated around E2F, c-Jun, c-Myc and p53. Of those, E2F, c-Jun and c-Myc are essential for the maintenance of cell transformation. Inhibition of Rock, one of the main Rho GTPase targets, leads to small changes in the transcriptome of Rho-transformed cells. Rock inhibition decreases c-myc gene expression without affecting the E2F and c-Jun pathways. Loss-of-function studies demonstrate that c-Myc is important for the blockage of cell-contact inhibition rather than for promoting the proliferation of Rho-transformed cells. However, c-Myc overexpression does not bypass the inhibition of cell transformation induced by Rock blockage, indicating that c-Myc is essential, but not sufficient, for Rock-dependent transformation. These results reveal the complexity of the genetic program orchestrated by the Rho subfamily and pinpoint protein networks that mediate different aspects of the malignant phenotype of Rho-transformed cells.
We have used microarray technology to identify the transcriptional targets of Rho subfamily guanosine 5'-triphosphate (GTP)ases in NIH3T3 cells. This analysis indicated that murine fibroblasts transformed by these proteins show similar transcriptomal profiles. Functional annotation of the regulated genes indicate that Rho subfamily GTPases target a wide spectrum of functions, although loci encoding proteins linked to proliferation and DNA synthesis/transcription are upregulated preferentially. Rho proteins promote four main networks of interacting proteins nucleated around E2F, c-Jun, c-Myc and p53. Of those, E2F, c-Jun and c-Myc are essential for the maintenance of cell transformation. Inhibition of Rock, one of the main Rho GTPase targets, leads to small changes in the transcriptome of Rho-transformed cells. Rock inhibition decreases c-myc gene expression without affecting the E2F and c-Jun pathways. Loss-of-function studies demonstrate that c-Myc is important for the blockage of cell-contact inhibition rather than for promoting the proliferation of Rho-transformed cells. However, c-Myc overexpression does not bypass the inhibition of cell transformation induced by Rock blockage, indicating that c-Myc is essential, but not sufficient, for Rock-dependent transformation. These results reveal the complexity of the genetic program orchestrated by the Rho subfamily and pinpoint protein networks that mediate different aspects of the malignant phenotype of Rho-transformed cells.
We have used microarray technology to identify the transcriptional targets of Rho subfamily guanosine 5'-triphosphate (GTP)ases in NIH3T3 cells. This analysis indicated that murine fibroblasts transformed by these proteins show similar transcriptomal profiles. Functional annotation of the regulated genes indicate that Rho subfamily GTPases target a wide spectrum of functions, although loci encoding proteins linked to proliferation and DNA synthesis/transcription are upregulated preferentially. Rho proteins promote four main networks of interacting proteins nucleated around E2F, c-Jun, c-Myc and p53. Of those, E2F, c-Jun and c-Myc are essential for the maintenance of cell transformation. Inhibition of Rock, one of the main Rho GTPase targets, leads to small changes in the transcriptome of Rho-transformed cells. Rock inhibition decreases c-myc gene expression without affecting the E2F and c-Jun pathways. Loss-of-function studies demonstrate that c-Myc is important for the blockage of cell-contact inhibition rather than for promoting the proliferation of Rho-transformed cells. However, c-Myc overexpression does not bypass the inhibition of cell transformation induced by Rock blockage, indicating that c-Myc is essential, but not sufficient, for Rock-dependent transformation. These results reveal the complexity of the genetic program orchestrated by the Rho subfamily and pinpoint protein networks that mediate different aspects of the malignant phenotype of Rho-transformed cells.
We used gene expression profiling, mutation analyses of FGFR3 and TP53, and LOH analyses of chromosome 9 and the TP53 region on chromosome arm 17p, to molecularly characterize 75 Ta and T1 bladder carcinomas. We identified four major cellular processes related to cell cycle, protein synthesis, immune response, and extra cellular components that contribute to the expressional heterogeneity of early-stage urothelial cell carcinoma (UCC). Activating FGFR3 mutations were found at the highest frequency in G1 tumors (80%), and showed a strong correlation with FGFR3 expression. In contrast, G3 tumors displayed mutations in less than 10% of the cases and a low level of FGFR3 expression. Even though LOH on chromosome 9 was not associated with any specific expression pattern, our data indicate that loss of chromosome 9 is associated with tumor development rather than initiation. The combined analyses suggest the existence of two types of UCC tumors, one which is characterized by FGFR3 mutation or expression, high expression of protein synthesis genes, and low expression of cell cycle genes. Furthermore, the presented data underscore FGFR3 receptor involvement in urothelial cell transformation as the presence of FGFR3 mutations has a major impact on the global gene expression profile of bladder carcinomas.
We used gene expression profiling, mutation analyses of FGFR3 and TP53, and LOH analyses of chromosome 9 and the TP53 region on chromosome arm 17p, to molecularly characterize 75 Ta and T1 bladder carcinomas. We identified four major cellular processes related to cell cycle, protein synthesis, immune response, and extra cellular components that contribute to the expressional heterogeneity of early-stage urothelial cell carcinoma (UCC). Activating FGFR3 mutations were found at the highest frequency in G1 tumors (80%), and showed a strong correlation with FGFR3 expression. In contrast, G3 tumors displayed mutations in less than 10% of the cases and a low level of FGFR3 expression. Even though LOH on chromosome 9 was not associated with any specific expression pattern, our data indicate that loss of chromosome 9 is associated with tumor development rather than initiation. The combined analyses suggest the existence of two types of UCC tumors, one which is characterized by FGFR3 mutation or expression, high expression of protein synthesis genes, and low expression of cell cycle genes. Furthermore, the presented data underscore FGFR3 receptor involvement in urothelial cell transformation as the presence of FGFR3 mutations has a major impact on the global gene expression profile of bladder carcinomas.
We used gene expression profiling, mutation analyses of FGFR3 and TP53, and LOH analyses of chromosome 9 and the TP53 region on chromosome arm 17p, to molecularly characterize 75 Ta and T1 bladder carcinomas. We identified four major cellular processes related to cell cycle, protein synthesis, immune response, and extra cellular components that contribute to the expressional heterogeneity of early-stage urothelial cell carcinoma (UCC). Activating FGFR3 mutations were found at the highest frequency in G1 tumors (80%), and showed a strong correlation with FGFR3 expression. In contrast, G3 tumors displayed mutations in less than 10% of the cases and a low level of FGFR3 expression. Even though LOH on chromosome 9 was not associated with any specific expression pattern, our data indicate that loss of chromosome 9 is associated with tumor development rather than initiation. The combined analyses suggest the existence of two types of UCC tumors, one which is characterized by FGFR3 mutation or expression, high expression of protein synthesis genes, and low expression of cell cycle genes. Furthermore, the presented data underscore FGFR3 receptor involvement in urothelial cell transformation as the presence of FGFR3 mutations has a major impact on the global gene expression profile of bladder carcinomas.
We used gene expression profiling, mutation analyses of FGFR3 and TP53, and LOH analyses of chromosome 9 and the TP53 region on chromosome arm 17p, to molecularly characterize 75 Ta and T1 bladder carcinomas. We identified four major cellular processes related to cell cycle, protein synthesis, immune response, and extra cellular components that contribute to the expressional heterogeneity of early-stage urothelial cell carcinoma (UCC). Activating FGFR3 mutations were found at the highest frequency in G1 tumors (80%), and showed a strong correlation with FGFR3 expression. In contrast, G3 tumors displayed mutations in less than 10% of the cases and a low level of FGFR3 expression. Even though LOH on chromosome 9 was not associated with any specific expression pattern, our data indicate that loss of chromosome 9 is associated with tumor development rather than initiation. The combined analyses suggest the existence of two types of UCC tumors, one which is characterized by FGFR3 mutation or expression, high expression of protein synthesis genes, and low expression of cell cycle genes. Furthermore, the presented data underscore FGFR3 receptor involvement in urothelial cell transformation as the presence of FGFR3 mutations has a major impact on the global gene expression profile of bladder carcinomas.
We used gene expression profiling, mutation analyses of FGFR3 and TP53, and LOH analyses of chromosome 9 and the TP53 region on chromosome arm 17p, to molecularly characterize 75 Ta and T1 bladder carcinomas. We identified four major cellular processes related to cell cycle, protein synthesis, immune response, and extra cellular components that contribute to the expressional heterogeneity of early-stage urothelial cell carcinoma (UCC). Activating FGFR3 mutations were found at the highest frequency in G1 tumors (80%), and showed a strong correlation with FGFR3 expression. In contrast, G3 tumors displayed mutations in less than 10% of the cases and a low level of FGFR3 expression. Even though LOH on chromosome 9 was not associated with any specific expression pattern, our data indicate that loss of chromosome 9 is associated with tumor development rather than initiation. The combined analyses suggest the existence of two types of UCC tumors, one which is characterized by FGFR3 mutation or expression, high expression of protein synthesis genes, and low expression of cell cycle genes. Furthermore, the presented data underscore FGFR3 receptor involvement in urothelial cell transformation as the presence of FGFR3 mutations has a major impact on the global gene expression profile of bladder carcinomas.
We used gene expression profiling, mutation analyses of FGFR3 and TP53, and LOH analyses of chromosome 9 and the TP53 region on chromosome arm 17p, to molecularly characterize 75 Ta and T1 bladder carcinomas. We identified four major cellular processes related to cell cycle, protein synthesis, immune response, and extra cellular components that contribute to the expressional heterogeneity of early-stage urothelial cell carcinoma (UCC). Activating FGFR3 mutations were found at the highest frequency in G1 tumors (80%), and showed a strong correlation with FGFR3 expression. In contrast, G3 tumors displayed mutations in less than 10% of the cases and a low level of FGFR3 expression. Even though LOH on chromosome 9 was not associated with any specific expression pattern, our data indicate that loss of chromosome 9 is associated with tumor development rather than initiation. The combined analyses suggest the existence of two types of UCC tumors, one which is characterized by FGFR3 mutation or expression, high expression of protein synthesis genes, and low expression of cell cycle genes. Furthermore, the presented data underscore FGFR3 receptor involvement in urothelial cell transformation as the presence of FGFR3 mutations has a major impact on the global gene expression profile of bladder carcinomas.
Mutation or epigenetic silencing of mismatch repair genes, such as MLH1 and MSH2, results in microsatellite instability (MSI) in the genome of a subset of colorectal carcinomas (CRCs). However, little is yet known of genes that directly contribute to tumor formation in such cancers. To characterize MSI-dependent changes in gene expression, we have now compared transcriptomes between fresh CRC specimens positive or negative for MSI (n=10 for each) with the use of high-density oligonucleotide microarrays harboring >44,000 probe sets. Correspondence analysis of the expression patterns of isolated MSI-associated genes revealed that the transcriptome of MSI+ CRCs is clearly distinct from that of MSI- CRCs. Such MSI-associated genes included that for AXIN2, an important component of the WNT signaling pathway. AXIN2 was silenced, apparently as a result of extensive methylation of its promoter region, specifically in MSI+ CRC specimens. Forced expression of AXIN2, either by treatment with 5'-azacytidine or by transfection with AXIN2 cDNA, resulted in rapid cell death in an MSI+ CRC cell line. These data indicate that epigenetic silencing of AXIN2 is specifically associated with carcinogenesis in MSI+ CRCs.
The Akt pathway is commonly deregulated in many cancers. Clinical trials are currently underway to test the effectiveness of breast cancer treatment by inhibition of various Akt pathway intermediates. A set of genes induced by Akt in a transgenic mouse model, a subset of which were sensitive to mammalian target of rapamycin (mTOR) inhibitor RAD001, was examined in five public gene expression profile data sets of clinical breast tumor specimens (representing >1000 different samples in all). In each of the clinical data sets, the Akt mouse model genes as a group were significantly overexpressed in human tumors having high levels of AKT1 mRNA. The subset of genes both upregulated by Akt and dependent on mTOR activity were associated with estrogen receptor-negative status, higher grade, increasing tumor size and poor prognosis in multiple patient cohorts; these associations were either not present or not as strong for the Akt-induced, mTOR-independent genes or for AKT1 expression alone. The genes shown here to be relevant to Akt-mTOR both experimentally and pathologically have the potential for use in a molecular diagnostic to determine which patients should receive mTOR antagonist treatment.
The Akt pathway is commonly deregulated in many cancers. Clinical trials are currently underway to test the effectiveness of breast cancer treatment by inhibition of various Akt pathway intermediates. A set of genes induced by Akt in a transgenic mouse model, a subset of which were sensitive to mammalian target of rapamycin (mTOR) inhibitor RAD001, was examined in five public gene expression profile data sets of clinical breast tumor specimens (representing >1000 different samples in all). In each of the clinical data sets, the Akt mouse model genes as a group were significantly overexpressed in human tumors having high levels of AKT1 mRNA. The subset of genes both upregulated by Akt and dependent on mTOR activity were associated with estrogen receptor-negative status, higher grade, increasing tumor size and poor prognosis in multiple patient cohorts; these associations were either not present or not as strong for the Akt-induced, mTOR-independent genes or for AKT1 expression alone. The genes shown here to be relevant to Akt-mTOR both experimentally and pathologically have the potential for use in a molecular diagnostic to determine which patients should receive mTOR antagonist treatment.
Breast cancer prognostic signature of 70 genes that significantly correlated with survival. Table 1 Cox-ranked signature. Cox-ranked signature genes, UG-cluster, Cytoband, Log2 expression differences between poor and good outcome and Log-ranked P- and q-values  for each gene are demonstrated.
Breast cancer prognostic signature of 70 genes that significantly correlated with survival. Table 1 Cox-ranked signature. Cox-ranked signature genes, UG-cluster, Cytoband, Log2 expression differences between poor and good outcome and Log-ranked P- and q-values  for each gene are demonstrated.
We analysed 148 primary breast cancers using BAC-arrays containing 287 clones representing cancer-related gene/loci to obtain genomic molecular portraits. Gains were detected in 136 tumors (91.9%) and losses in 123 tumors (83.1%). Eight tumors (5.4%) did not have any genomic aberrations in the 281 clones analysed. Common (more than 15% of the samples) gains were observed at 8q11-qtel, 1q21-qtel, 17q11-q12 and 11q13, whereas common losses were observed at 16q12-qtel, 11ptel-p15.5, 1p36-ptel, 17p11.2-p12 and 8ptel-p22. Patients with tumors registering either less than 5% (median value) or less than 11% (third quartile) total copy number changes had a better overall survival (log-rank test: P=0.0417 and P=0.0375, respectively). Unsupervised hierarchical clustering based on copy number changes identified four clusters. Women with tumors from the cluster with amplification of three regions containing known breast oncogenes (11q13, 17q12 and 20q13) had a worse prognosis. The good prognosis group (Nottingham Prognostic Index (NPI) <or=3.4) tumors had frequent loss of 16q24-qtel. Genes significantly associated with estrogen receptor (ER), Grade and NPI were used to build k-nearest neighbor (KNN) classifiers that predicted ER, Grade and NPI status in the test set with an average misclassification rate of 24.7, 25.7 and 35.7%, respectively. These data raise the prospect of generating a molecular taxonomy of breast cancer based on copy number profiling using tumor DNA, which may be more generally applicable than expression microarray analysis.
We analysed 148 primary breast cancers using BAC-arrays containing 287 clones representing cancer-related gene/loci to obtain genomic molecular portraits. Gains were detected in 136 tumors (91.9%) and losses in 123 tumors (83.1%). Eight tumors (5.4%) did not have any genomic aberrations in the 281 clones analysed. Common (more than 15% of the samples) gains were observed at 8q11-qtel, 1q21-qtel, 17q11-q12 and 11q13, whereas common losses were observed at 16q12-qtel, 11ptel-p15.5, 1p36-ptel, 17p11.2-p12 and 8ptel-p22. Patients with tumors registering either less than 5% (median value) or less than 11% (third quartile) total copy number changes had a better overall survival (log-rank test: P=0.0417 and P=0.0375, respectively). Unsupervised hierarchical clustering based on copy number changes identified four clusters. Women with tumors from the cluster with amplification of three regions containing known breast oncogenes (11q13, 17q12 and 20q13) had a worse prognosis. The good prognosis group (Nottingham Prognostic Index (NPI) <or=3.4) tumors had frequent loss of 16q24-qtel. Genes significantly associated with estrogen receptor (ER), Grade and NPI were used to build k-nearest neighbor (KNN) classifiers that predicted ER, Grade and NPI status in the test set with an average misclassification rate of 24.7, 25.7 and 35.7%, respectively. These data raise the prospect of generating a molecular taxonomy of breast cancer based on copy number profiling using tumor DNA, which may be more generally applicable than expression microarray analysis.
Uterine fibroids are some of the most common tumours of females, but relatively little is known about their molecular basis. Several studies have suggested that deletions on chromosome 7q could have a role in fibroid formation. We analysed 165 sporadic uterine fibroids to define a small 3.2 megabase (Mb) commonly deleted region on 7q22.3-q31.1, flanked by clones AC005070 and AC007567. We also used oligonucleotide microarrays to compare the expression profiles of 10 samples of normal myometrium and 15 fibroids, nine of which displayed 7q-deletions. Activating transcription factor 3, patched homolog (Drosophila), homeo box A5, death-associated protein kinase 1, and retinoic acid receptor responder 3 were downregulated, and excision repair crosscomplementing 3, transcription factor AP-2 gamma and protein kinase C beta 1 were upregulated in fibroids. New pathways were discovered related to fibroid formation. The presence or absence of 7q-deletions did not dramatically affect the global expression pattern of the tumours; changes, however, were observed in genes related to vesicular transport and nucleic acid binding.
Uterine fibroids are some of the most common tumours of females, but relatively little is known about their molecular basis. Several studies have suggested that deletions on chromosome 7q could have a role in fibroid formation. We analysed 165 sporadic uterine fibroids to define a small 3.2 megabase (Mb) commonly deleted region on 7q22.3-q31.1, flanked by clones AC005070 and AC007567. We also used oligonucleotide microarrays to compare the expression profiles of 10 samples of normal myometrium and 15 fibroids, nine of which displayed 7q-deletions. Activating transcription factor 3, patched homolog (Drosophila), homeo box A5, death-associated protein kinase 1, and retinoic acid receptor responder 3 were downregulated, and excision repair crosscomplementing 3, transcription factor AP-2 gamma and protein kinase C beta 1 were upregulated in fibroids. New pathways were discovered related to fibroid formation. The presence or absence of 7q-deletions did not dramatically affect the global expression pattern of the tumours; changes, however, were observed in genes related to vesicular transport and nucleic acid binding.
Uterine fibroids are some of the most common tumours of females, but relatively little is known about their molecular basis. Several studies have suggested that deletions on chromosome 7q could have a role in fibroid formation. We analysed 165 sporadic uterine fibroids to define a small 3.2 megabase (Mb) commonly deleted region on 7q22.3-q31.1, flanked by clones AC005070 and AC007567. We also used oligonucleotide microarrays to compare the expression profiles of 10 samples of normal myometrium and 15 fibroids, nine of which displayed 7q-deletions. Activating transcription factor 3, patched homolog (Drosophila), homeo box A5, death-associated protein kinase 1, and retinoic acid receptor responder 3 were downregulated, and excision repair crosscomplementing 3, transcription factor AP-2 gamma and protein kinase C beta 1 were upregulated in fibroids. New pathways were discovered related to fibroid formation. The presence or absence of 7q-deletions did not dramatically affect the global expression pattern of the tumours; changes, however, were observed in genes related to vesicular transport and nucleic acid binding.
Uterine fibroids are some of the most common tumours of females, but relatively little is known about their molecular basis. Several studies have suggested that deletions on chromosome 7q could have a role in fibroid formation. We analysed 165 sporadic uterine fibroids to define a small 3.2 megabase (Mb) commonly deleted region on 7q22.3-q31.1, flanked by clones AC005070 and AC007567. We also used oligonucleotide microarrays to compare the expression profiles of 10 samples of normal myometrium and 15 fibroids, nine of which displayed 7q-deletions. Activating transcription factor 3, patched homolog (Drosophila), homeo box A5, death-associated protein kinase 1, and retinoic acid receptor responder 3 were downregulated, and excision repair crosscomplementing 3, transcription factor AP-2 gamma and protein kinase C beta 1 were upregulated in fibroids. New pathways were discovered related to fibroid formation. The presence or absence of 7q-deletions did not dramatically affect the global expression pattern of the tumours; changes, however, were observed in genes related to vesicular transport and nucleic acid binding.
Upregulation of HER2/ErbB2/Neu occurs in 15-30% of human breast cancers and correlates with poor prognosis. Identification of ErbB2/Neu transcriptional targets should facilitate development of novel therapeutic approaches. Development of breast cancer is a multistep process; thus, to identify the transcriptomes associated with different stages of progression of tumorigenesis, we compared expression profiles of mammary tumors and preneoplastic mammary tissue from MMTV-Neu transgenic mice to expression profiles of wild-type mammary glands using Affymetrix microarrays. We identified 324 candidate genes that were unique to ErbB2/Neu-induced tumors relative to normal mammary gland tissue from wild-type controls. Expression of a subset of these genes (82) was also changed in the preneoplastic mammary glands compared to wild-type controls, indicating that they may play a pivotal role during early events of ErbB2/Neu-initiated mammary tumorigenesis. Further analysis of the microarray data revealed that expression of several known transforming growth factor (TGF)-beta target genes was altered, suggesting that the TGF-beta signaling cascade is downregulated in ErbB2/Neu-induced tumors. Western blot analysis for TGF-beta-Receptor-I/ALK5 and immunohistochemistry for TGF-beta-Receptor-I/ALK5 and phosphorylated/activated Smad2 confirmed that the Smad-dependent TGF-beta signaling cascade was inactive in these tumors. Although absent in most of the tumor, phosphorylated Smad2 was present in the periphery of tumors. Interestingly, presence of phosphorylated/activated Smad2 correlated with expression of Activin-Receptor-IB/ALK4, suggesting that although Smad-dependent TGF-beta signaling is absent in ErbB2/Neu-induced tumors, Activin signaling may be active at the leading edge of these tumors. Cumulatively, these data indicate that the TGF-beta pathway is intrinsically suppressed in ErbB2/Neu tumors via a mechanism involving loss of TGF-beta-Receptor-I/ALK5.
Upregulation of HER2/ErbB2/Neu occurs in 15-30% of human breast cancers and correlates with poor prognosis. Identification of ErbB2/Neu transcriptional targets should facilitate development of novel therapeutic approaches. Development of breast cancer is a multistep process; thus, to identify the transcriptomes associated with different stages of progression of tumorigenesis, we compared expression profiles of mammary tumors and preneoplastic mammary tissue from MMTV-Neu transgenic mice to expression profiles of wild-type mammary glands using Affymetrix microarrays. We identified 324 candidate genes that were unique to ErbB2/Neu-induced tumors relative to normal mammary gland tissue from wild-type controls. Expression of a subset of these genes (82) was also changed in the preneoplastic mammary glands compared to wild-type controls, indicating that they may play a pivotal role during early events of ErbB2/Neu-initiated mammary tumorigenesis. Further analysis of the microarray data revealed that expression of several known transforming growth factor (TGF)-beta target genes was altered, suggesting that the TGF-beta signaling cascade is downregulated in ErbB2/Neu-induced tumors. Western blot analysis for TGF-beta-Receptor-I/ALK5 and immunohistochemistry for TGF-beta-Receptor-I/ALK5 and phosphorylated/activated Smad2 confirmed that the Smad-dependent TGF-beta signaling cascade was inactive in these tumors. Although absent in most of the tumor, phosphorylated Smad2 was present in the periphery of tumors. Interestingly, presence of phosphorylated/activated Smad2 correlated with expression of Activin-Receptor-IB/ALK4, suggesting that although Smad-dependent TGF-beta signaling is absent in ErbB2/Neu-induced tumors, Activin signaling may be active at the leading edge of these tumors. Cumulatively, these data indicate that the TGF-beta pathway is intrinsically suppressed in ErbB2/Neu tumors via a mechanism involving loss of TGF-beta-Receptor-I/ALK5.
Upregulation of HER2/ErbB2/Neu occurs in 15-30% of human breast cancers and correlates with poor prognosis. Identification of ErbB2/Neu transcriptional targets should facilitate development of novel therapeutic approaches. Development of breast cancer is a multistep process; thus, to identify the transcriptomes associated with different stages of progression of tumorigenesis, we compared expression profiles of mammary tumors and preneoplastic mammary tissue from MMTV-Neu transgenic mice to expression profiles of wild-type mammary glands using Affymetrix microarrays. We identified 324 candidate genes that were unique to ErbB2/Neu-induced tumors relative to normal mammary gland tissue from wild-type controls. Expression of a subset of these genes (82) was also changed in the preneoplastic mammary glands compared to wild-type controls, indicating that they may play a pivotal role during early events of ErbB2/Neu-initiated mammary tumorigenesis. Further analysis of the microarray data revealed that expression of several known transforming growth factor (TGF)-beta target genes was altered, suggesting that the TGF-beta signaling cascade is downregulated in ErbB2/Neu-induced tumors. Western blot analysis for TGF-beta-Receptor-I/ALK5 and immunohistochemistry for TGF-beta-Receptor-I/ALK5 and phosphorylated/activated Smad2 confirmed that the Smad-dependent TGF-beta signaling cascade was inactive in these tumors. Although absent in most of the tumor, phosphorylated Smad2 was present in the periphery of tumors. Interestingly, presence of phosphorylated/activated Smad2 correlated with expression of Activin-Receptor-IB/ALK4, suggesting that although Smad-dependent TGF-beta signaling is absent in ErbB2/Neu-induced tumors, Activin signaling may be active at the leading edge of these tumors. Cumulatively, these data indicate that the TGF-beta pathway is intrinsically suppressed in ErbB2/Neu tumors via a mechanism involving loss of TGF-beta-Receptor-I/ALK5.
Upregulation of HER2/ErbB2/Neu occurs in 15-30% of human breast cancers and correlates with poor prognosis. Identification of ErbB2/Neu transcriptional targets should facilitate development of novel therapeutic approaches. Development of breast cancer is a multistep process; thus, to identify the transcriptomes associated with different stages of progression of tumorigenesis, we compared expression profiles of mammary tumors and preneoplastic mammary tissue from MMTV-Neu transgenic mice to expression profiles of wild-type mammary glands using Affymetrix microarrays. We identified 324 candidate genes that were unique to ErbB2/Neu-induced tumors relative to normal mammary gland tissue from wild-type controls. Expression of a subset of these genes (82) was also changed in the preneoplastic mammary glands compared to wild-type controls, indicating that they may play a pivotal role during early events of ErbB2/Neu-initiated mammary tumorigenesis. Further analysis of the microarray data revealed that expression of several known transforming growth factor (TGF)-beta target genes was altered, suggesting that the TGF-beta signaling cascade is downregulated in ErbB2/Neu-induced tumors. Western blot analysis for TGF-beta-Receptor-I/ALK5 and immunohistochemistry for TGF-beta-Receptor-I/ALK5 and phosphorylated/activated Smad2 confirmed that the Smad-dependent TGF-beta signaling cascade was inactive in these tumors. Although absent in most of the tumor, phosphorylated Smad2 was present in the periphery of tumors. Interestingly, presence of phosphorylated/activated Smad2 correlated with expression of Activin-Receptor-IB/ALK4, suggesting that although Smad-dependent TGF-beta signaling is absent in ErbB2/Neu-induced tumors, Activin signaling may be active at the leading edge of these tumors. Cumulatively, these data indicate that the TGF-beta pathway is intrinsically suppressed in ErbB2/Neu tumors via a mechanism involving loss of TGF-beta-Receptor-I/ALK5.
The proteasome has emerged as a novel target for antineoplastic treatment of hematological malignancies and solid tumors, including those of the central nervous system. To identify cell death pathways activated in response to inhibition of the proteasome system in cancer cells, we treated human SH-SY5Y neuroblastoma cells with the selective proteasome inhibitor (PI) epoxomicin (Epoxo). Prolonged exposure to Epoxo was associated with increased levels of poly-ubiquitinylated proteins and p53, release of cytochrome c from the mitochondria, and activation of caspases. Analysis of global gene expression using high-density oligonucleotide microarrays revealed that Epoxo triggered transcriptional activation of the two Bcl-2-homology domain-3-only (BH3-only) genes p53 upregulated modulator of apoptosis (PUMA) and Bim. Subsequent studies in PUMA- and Bim-deficient cells indicated that Epoxo-induced caspase activation and apoptosis was predominantly PUMA-dependent. Further characterization of the transcriptional response to Epoxo in HCT116 human colon cancer cells demonstrated that PUMA induction was p53-dependent; with deficiency in either p53 or PUMA significantly protected HCT116 cells against Epoxo-induced apoptosis. Our data suggest that p53 activation and the transcriptional induction of its target gene PUMA play an important role in the sensitivity of cancer cells to apoptosis induced by proteasome inhibition, and imply that antineoplastic therapies with PIs might be especially useful in cancers with functional p53.
The proteasome has emerged as a novel target for antineoplastic treatment of hematological malignancies and solid tumors, including those of the central nervous system. To identify cell death pathways activated in response to inhibition of the proteasome system in cancer cells, we treated human SH-SY5Y neuroblastoma cells with the selective proteasome inhibitor (PI) epoxomicin (Epoxo). Prolonged exposure to Epoxo was associated with increased levels of poly-ubiquitinylated proteins and p53, release of cytochrome c from the mitochondria, and activation of caspases. Analysis of global gene expression using high-density oligonucleotide microarrays revealed that Epoxo triggered transcriptional activation of the two Bcl-2-homology domain-3-only (BH3-only) genes p53 upregulated modulator of apoptosis (PUMA) and Bim. Subsequent studies in PUMA- and Bim-deficient cells indicated that Epoxo-induced caspase activation and apoptosis was predominantly PUMA-dependent. Further characterization of the transcriptional response to Epoxo in HCT116 human colon cancer cells demonstrated that PUMA induction was p53-dependent; with deficiency in either p53 or PUMA significantly protected HCT116 cells against Epoxo-induced apoptosis. Our data suggest that p53 activation and the transcriptional induction of its target gene PUMA play an important role in the sensitivity of cancer cells to apoptosis induced by proteasome inhibition, and imply that antineoplastic therapies with PIs might be especially useful in cancers with functional p53.
Metastasis via the lymphatics is a major risk factor in squamous cell carcinoma of the oral cavity (OSCC). We sought to determine whether the presence of metastasis in the regional lymph node could be predicted by a gene expression signature of the primary tumor. A total of 18 OSCCs were characterized for gene expression by hybridizing RNA to Affymetrix U133A gene chips. Genes with differential expression were identified using a permutation technique and verified by quantitative RT-PCR and immunohistochemistry. A predictive rule was built using a support vector machine, and the accuracy of the rule was evaluated using crossvalidation on the original data set and prediction of an independent set of four patients. Metastatic primary tumors could be differentiated from nonmetastatic primary tumors by a signature gene set of 116 genes. This signature gene set correctly predicted the four independent patients as well as associating five lymph node metastases from the original patient set with the metastatic primary tumor group. We concluded that lymph node metastasis could be predicted by gene expression profiles of primary oral cavity squamous cell carcinomas. The presence of a gene expression signature for lymph node metastasis indicates that clinical testing to assess risk for lymph node metastasis should be possible.
Metastasis via the lymphatics is a major risk factor in squamous cell carcinoma of the oral cavity (OSCC). We sought to determine whether the presence of metastasis in the regional lymph node could be predicted by a gene expression signature of the primary tumor. A total of 18 OSCCs were characterized for gene expression by hybridizing RNA to Affymetrix U133A gene chips. Genes with differential expression were identified using a permutation technique and verified by quantitative RT-PCR and immunohistochemistry. A predictive rule was built using a support vector machine, and the accuracy of the rule was evaluated using crossvalidation on the original data set and prediction of an independent set of four patients. Metastatic primary tumors could be differentiated from nonmetastatic primary tumors by a signature gene set of 116 genes. This signature gene set correctly predicted the four independent patients as well as associating five lymph node metastases from the original patient set with the metastatic primary tumor group. We concluded that lymph node metastasis could be predicted by gene expression profiles of primary oral cavity squamous cell carcinomas. The presence of a gene expression signature for lymph node metastasis indicates that clinical testing to assess risk for lymph node metastasis should be possible.
The reciprocal chromosomal translocation t(4;11) is correlated with infant, childhood, adult and therapy-related high-risk acute leukemia. Here, we investigated the biological effects of MLL.AF4, AF4.MLL or the combination of both reciprocal fusion proteins in a conditional in vitro cell culture model system. Several parameters like cell growth, cell cycling capacity, apoptotic behavior and growth transformation were investigated under physiological and stress conditions. Co-transfected cells displayed the highest resistance against apoptotic triggers, cell cycling capacity and loss-of-contact inhibition. These analyses were complemented by gene expression profiling experiments and specific gene signatures were established for each of the three cell lines. Interestingly, co-transfected cells strongly upregulate the homeobox gene Nanog. In combination with Oct4, the Nanog homeoprotein is steering maintenance of pluripotency and self-renewal in embryonic stem cells. Transcription of Nanog and other stem cell factors, like Oct4 and Bmi1, was verified in biopsy material of t(4;11) patient cells which express both reciprocal t(4;11) fusion genes. In conclusion, the presence of both reciprocal MLL fusion proteins confers biological properties known from t(4;11) leukemia, suggesting that each of the two fusion proteins contribute specific properties and, in combination, also synergistic effects to the leukemic phenotype.
The reciprocal chromosomal translocation t(4;11) is correlated with infant, childhood, adult and therapy-related high-risk acute leukemia. Here, we investigated the biological effects of MLL.AF4, AF4.MLL or the combination of both reciprocal fusion proteins in a conditional in vitro cell culture model system. Several parameters like cell growth, cell cycling capacity, apoptotic behavior and growth transformation were investigated under physiological and stress conditions. Co-transfected cells displayed the highest resistance against apoptotic triggers, cell cycling capacity and loss-of-contact inhibition. These analyses were complemented by gene expression profiling experiments and specific gene signatures were established for each of the three cell lines. Interestingly, co-transfected cells strongly upregulate the homeobox gene Nanog. In combination with Oct4, the Nanog homeoprotein is steering maintenance of pluripotency and self-renewal in embryonic stem cells. Transcription of Nanog and other stem cell factors, like Oct4 and Bmi1, was verified in biopsy material of t(4;11) patient cells which express both reciprocal t(4;11) fusion genes. In conclusion, the presence of both reciprocal MLL fusion proteins confers biological properties known from t(4;11) leukemia, suggesting that each of the two fusion proteins contribute specific properties and, in combination, also synergistic effects to the leukemic phenotype.
The reciprocal chromosomal translocation t(4;11) is correlated with infant, childhood, adult and therapy-related high-risk acute leukemia. Here, we investigated the biological effects of MLL.AF4, AF4.MLL or the combination of both reciprocal fusion proteins in a conditional in vitro cell culture model system. Several parameters like cell growth, cell cycling capacity, apoptotic behavior and growth transformation were investigated under physiological and stress conditions. Co-transfected cells displayed the highest resistance against apoptotic triggers, cell cycling capacity and loss-of-contact inhibition. These analyses were complemented by gene expression profiling experiments and specific gene signatures were established for each of the three cell lines. Interestingly, co-transfected cells strongly upregulate the homeobox gene Nanog. In combination with Oct4, the Nanog homeoprotein is steering maintenance of pluripotency and self-renewal in embryonic stem cells. Transcription of Nanog and other stem cell factors, like Oct4 and Bmi1, was verified in biopsy material of t(4;11) patient cells which express both reciprocal t(4;11) fusion genes. In conclusion, the presence of both reciprocal MLL fusion proteins confers biological properties known from t(4;11) leukemia, suggesting that each of the two fusion proteins contribute specific properties and, in combination, also synergistic effects to the leukemic phenotype.
The reciprocal chromosomal translocation t(4;11) is correlated with infant, childhood, adult and therapy-related high-risk acute leukemia. Here, we investigated the biological effects of MLL.AF4, AF4.MLL or the combination of both reciprocal fusion proteins in a conditional in vitro cell culture model system. Several parameters like cell growth, cell cycling capacity, apoptotic behavior and growth transformation were investigated under physiological and stress conditions. Co-transfected cells displayed the highest resistance against apoptotic triggers, cell cycling capacity and loss-of-contact inhibition. These analyses were complemented by gene expression profiling experiments and specific gene signatures were established for each of the three cell lines. Interestingly, co-transfected cells strongly upregulate the homeobox gene Nanog. In combination with Oct4, the Nanog homeoprotein is steering maintenance of pluripotency and self-renewal in embryonic stem cells. Transcription of Nanog and other stem cell factors, like Oct4 and Bmi1, was verified in biopsy material of t(4;11) patient cells which express both reciprocal t(4;11) fusion genes. In conclusion, the presence of both reciprocal MLL fusion proteins confers biological properties known from t(4;11) leukemia, suggesting that each of the two fusion proteins contribute specific properties and, in combination, also synergistic effects to the leukemic phenotype.
The reciprocal chromosomal translocation t(4;11) is correlated with infant, childhood, adult and therapy-related high-risk acute leukemia. Here, we investigated the biological effects of MLL.AF4, AF4.MLL or the combination of both reciprocal fusion proteins in a conditional in vitro cell culture model system. Several parameters like cell growth, cell cycling capacity, apoptotic behavior and growth transformation were investigated under physiological and stress conditions. Co-transfected cells displayed the highest resistance against apoptotic triggers, cell cycling capacity and loss-of-contact inhibition. These analyses were complemented by gene expression profiling experiments and specific gene signatures were established for each of the three cell lines. Interestingly, co-transfected cells strongly upregulate the homeobox gene Nanog. In combination with Oct4, the Nanog homeoprotein is steering maintenance of pluripotency and self-renewal in embryonic stem cells. Transcription of Nanog and other stem cell factors, like Oct4 and Bmi1, was verified in biopsy material of t(4;11) patient cells which express both reciprocal t(4;11) fusion genes. In conclusion, the presence of both reciprocal MLL fusion proteins confers biological properties known from t(4;11) leukemia, suggesting that each of the two fusion proteins contribute specific properties and, in combination, also synergistic effects to the leukemic phenotype.
The reciprocal chromosomal translocation t(4;11) is correlated with infant, childhood, adult and therapy-related high-risk acute leukemia. Here, we investigated the biological effects of MLL.AF4, AF4.MLL or the combination of both reciprocal fusion proteins in a conditional in vitro cell culture model system. Several parameters like cell growth, cell cycling capacity, apoptotic behavior and growth transformation were investigated under physiological and stress conditions. Co-transfected cells displayed the highest resistance against apoptotic triggers, cell cycling capacity and loss-of-contact inhibition. These analyses were complemented by gene expression profiling experiments and specific gene signatures were established for each of the three cell lines. Interestingly, co-transfected cells strongly upregulate the homeobox gene Nanog. In combination with Oct4, the Nanog homeoprotein is steering maintenance of pluripotency and self-renewal in embryonic stem cells. Transcription of Nanog and other stem cell factors, like Oct4 and Bmi1, was verified in biopsy material of t(4;11) patient cells which express both reciprocal t(4;11) fusion genes. In conclusion, the presence of both reciprocal MLL fusion proteins confers biological properties known from t(4;11) leukemia, suggesting that each of the two fusion proteins contribute specific properties and, in combination, also synergistic effects to the leukemic phenotype.
The reciprocal chromosomal translocation t(4;11) is correlated with infant, childhood, adult and therapy-related high-risk acute leukemia. Here, we investigated the biological effects of MLL.AF4, AF4.MLL or the combination of both reciprocal fusion proteins in a conditional in vitro cell culture model system. Several parameters like cell growth, cell cycling capacity, apoptotic behavior and growth transformation were investigated under physiological and stress conditions. Co-transfected cells displayed the highest resistance against apoptotic triggers, cell cycling capacity and loss-of-contact inhibition. These analyses were complemented by gene expression profiling experiments and specific gene signatures were established for each of the three cell lines. Interestingly, co-transfected cells strongly upregulate the homeobox gene Nanog. In combination with Oct4, the Nanog homeoprotein is steering maintenance of pluripotency and self-renewal in embryonic stem cells. Transcription of Nanog and other stem cell factors, like Oct4 and Bmi1, was verified in biopsy material of t(4;11) patient cells which express both reciprocal t(4;11) fusion genes. In conclusion, the presence of both reciprocal MLL fusion proteins confers biological properties known from t(4;11) leukemia, suggesting that each of the two fusion proteins contribute specific properties and, in combination, also synergistic effects to the leukemic phenotype.
The reciprocal chromosomal translocation t(4;11) is correlated with infant, childhood, adult and therapy-related high-risk acute leukemia. Here, we investigated the biological effects of MLL.AF4, AF4.MLL or the combination of both reciprocal fusion proteins in a conditional in vitro cell culture model system. Several parameters like cell growth, cell cycling capacity, apoptotic behavior and growth transformation were investigated under physiological and stress conditions. Co-transfected cells displayed the highest resistance against apoptotic triggers, cell cycling capacity and loss-of-contact inhibition. These analyses were complemented by gene expression profiling experiments and specific gene signatures were established for each of the three cell lines. Interestingly, co-transfected cells strongly upregulate the homeobox gene Nanog. In combination with Oct4, the Nanog homeoprotein is steering maintenance of pluripotency and self-renewal in embryonic stem cells. Transcription of Nanog and other stem cell factors, like Oct4 and Bmi1, was verified in biopsy material of t(4;11) patient cells which express both reciprocal t(4;11) fusion genes. In conclusion, the presence of both reciprocal MLL fusion proteins confers biological properties known from t(4;11) leukemia, suggesting that each of the two fusion proteins contribute specific properties and, in combination, also synergistic effects to the leukemic phenotype.
The reciprocal chromosomal translocation t(4;11) is correlated with infant, childhood, adult and therapy-related high-risk acute leukemia. Here, we investigated the biological effects of MLL.AF4, AF4.MLL or the combination of both reciprocal fusion proteins in a conditional in vitro cell culture model system. Several parameters like cell growth, cell cycling capacity, apoptotic behavior and growth transformation were investigated under physiological and stress conditions. Co-transfected cells displayed the highest resistance against apoptotic triggers, cell cycling capacity and loss-of-contact inhibition. These analyses were complemented by gene expression profiling experiments and specific gene signatures were established for each of the three cell lines. Interestingly, co-transfected cells strongly upregulate the homeobox gene Nanog. In combination with Oct4, the Nanog homeoprotein is steering maintenance of pluripotency and self-renewal in embryonic stem cells. Transcription of Nanog and other stem cell factors, like Oct4 and Bmi1, was verified in biopsy material of t(4;11) patient cells which express both reciprocal t(4;11) fusion genes. In conclusion, the presence of both reciprocal MLL fusion proteins confers biological properties known from t(4;11) leukemia, suggesting that each of the two fusion proteins contribute specific properties and, in combination, also synergistic effects to the leukemic phenotype.
The reciprocal chromosomal translocation t(4;11) is correlated with infant, childhood, adult and therapy-related high-risk acute leukemia. Here, we investigated the biological effects of MLL.AF4, AF4.MLL or the combination of both reciprocal fusion proteins in a conditional in vitro cell culture model system. Several parameters like cell growth, cell cycling capacity, apoptotic behavior and growth transformation were investigated under physiological and stress conditions. Co-transfected cells displayed the highest resistance against apoptotic triggers, cell cycling capacity and loss-of-contact inhibition. These analyses were complemented by gene expression profiling experiments and specific gene signatures were established for each of the three cell lines. Interestingly, co-transfected cells strongly upregulate the homeobox gene Nanog. In combination with Oct4, the Nanog homeoprotein is steering maintenance of pluripotency and self-renewal in embryonic stem cells. Transcription of Nanog and other stem cell factors, like Oct4 and Bmi1, was verified in biopsy material of t(4;11) patient cells which express both reciprocal t(4;11) fusion genes. In conclusion, the presence of both reciprocal MLL fusion proteins confers biological properties known from t(4;11) leukemia, suggesting that each of the two fusion proteins contribute specific properties and, in combination, also synergistic effects to the leukemic phenotype.
We have used microarray technology to identify the transcriptional targets of Rho subfamily guanosine 5'-triphosphate (GTP)ases in NIH3T3 cells. This analysis indicated that murine fibroblasts transformed by these proteins show similar transcriptomal profiles. Functional annotation of the regulated genes indicate that Rho subfamily GTPases target a wide spectrum of functions, although loci encoding proteins linked to proliferation and DNA synthesis/transcription are upregulated preferentially. Rho proteins promote four main networks of interacting proteins nucleated around E2F, c-Jun, c-Myc and p53. Of those, E2F, c-Jun and c-Myc are essential for the maintenance of cell transformation. Inhibition of Rock, one of the main Rho GTPase targets, leads to small changes in the transcriptome of Rho-transformed cells. Rock inhibition decreases c-myc gene expression without affecting the E2F and c-Jun pathways. Loss-of-function studies demonstrate that c-Myc is important for the blockage of cell-contact inhibition rather than for promoting the proliferation of Rho-transformed cells. However, c-Myc overexpression does not bypass the inhibition of cell transformation induced by Rock blockage, indicating that c-Myc is essential, but not sufficient, for Rock-dependent transformation. These results reveal the complexity of the genetic program orchestrated by the Rho subfamily and pinpoint protein networks that mediate different aspects of the malignant phenotype of Rho-transformed cells.
We have used microarray technology to identify the transcriptional targets of Rho subfamily guanosine 5'-triphosphate (GTP)ases in NIH3T3 cells. This analysis indicated that murine fibroblasts transformed by these proteins show similar transcriptomal profiles. Functional annotation of the regulated genes indicate that Rho subfamily GTPases target a wide spectrum of functions, although loci encoding proteins linked to proliferation and DNA synthesis/transcription are upregulated preferentially. Rho proteins promote four main networks of interacting proteins nucleated around E2F, c-Jun, c-Myc and p53. Of those, E2F, c-Jun and c-Myc are essential for the maintenance of cell transformation. Inhibition of Rock, one of the main Rho GTPase targets, leads to small changes in the transcriptome of Rho-transformed cells. Rock inhibition decreases c-myc gene expression without affecting the E2F and c-Jun pathways. Loss-of-function studies demonstrate that c-Myc is important for the blockage of cell-contact inhibition rather than for promoting the proliferation of Rho-transformed cells. However, c-Myc overexpression does not bypass the inhibition of cell transformation induced by Rock blockage, indicating that c-Myc is essential, but not sufficient, for Rock-dependent transformation. These results reveal the complexity of the genetic program orchestrated by the Rho subfamily and pinpoint protein networks that mediate different aspects of the malignant phenotype of Rho-transformed cells.
Tumors of low malignant potential (LMP) represent 20% of epithelial ovarian cancers (EOCs) and are associated with a better prognosis than the invasive tumors (TOV). Defining the relationship between LMPs and TOVs remains an important goal towards understanding the molecular pathways that contribute to prognosis, as well as providing molecular markers, for these EOCs. To this end, DNA microarray analyses were performed either in a primary culture or a tumor tissue model system and selected candidate genes showing a distinctive expression profile between LMPs and TOVs were identified using a class prediction approach based on three statistical methods of analysis. Both model systems appear relevant as candidate genes identified by either model allowed the proper reclassification of samples as either LMPs or TOVs. Selected candidate genes (CAS, CCNE1, LGALS8, ITGbeta3, ATP1B1, FLIP, KRT7 and KRT19) were validated by real-time quantitative PCR analysis and show differential expression between LMPs and TOVs. Immunohistochemistry analyses showed that the two tumor classes were distinguishable by their expression of CAS, TNFR1A, FLIP, CKS1 and CCNE1. These results define signature patterns for gene expression of LMPs and TOVs and identify gene candidates that warrant further study to deepen our understanding of the biology of EOC.
Tumors of low malignant potential (LMP) represent 20% of epithelial ovarian cancers (EOCs) and are associated with a better prognosis than the invasive tumors (TOV). Defining the relationship between LMPs and TOVs remains an important goal towards understanding the molecular pathways that contribute to prognosis, as well as providing molecular markers, for these EOCs. To this end, DNA microarray analyses were performed either in a primary culture or a tumor tissue model system and selected candidate genes showing a distinctive expression profile between LMPs and TOVs were identified using a class prediction approach based on three statistical methods of analysis. Both model systems appear relevant as candidate genes identified by either model allowed the proper reclassification of samples as either LMPs or TOVs. Selected candidate genes (CAS, CCNE1, LGALS8, ITGbeta3, ATP1B1, FLIP, KRT7 and KRT19) were validated by real-time quantitative PCR analysis and show differential expression between LMPs and TOVs. Immunohistochemistry analyses showed that the two tumor classes were distinguishable by their expression of CAS, TNFR1A, FLIP, CKS1 and CCNE1. These results define signature patterns for gene expression of LMPs and TOVs and identify gene candidates that warrant further study to deepen our understanding of the biology of EOC.
Upregulation of HER2/ErbB2/Neu occurs in 15-30% of human breast cancers and correlates with poor prognosis. Identification of ErbB2/Neu transcriptional targets should facilitate development of novel therapeutic approaches. Development of breast cancer is a multistep process; thus, to identify the transcriptomes associated with different stages of progression of tumorigenesis, we compared expression profiles of mammary tumors and preneoplastic mammary tissue from MMTV-Neu transgenic mice to expression profiles of wild-type mammary glands using Affymetrix microarrays. We identified 324 candidate genes that were unique to ErbB2/Neu-induced tumors relative to normal mammary gland tissue from wild-type controls. Expression of a subset of these genes (82) was also changed in the preneoplastic mammary glands compared to wild-type controls, indicating that they may play a pivotal role during early events of ErbB2/Neu-initiated mammary tumorigenesis. Further analysis of the microarray data revealed that expression of several known transforming growth factor (TGF)-beta target genes was altered, suggesting that the TGF-beta signaling cascade is downregulated in ErbB2/Neu-induced tumors. Western blot analysis for TGF-beta-Receptor-I/ALK5 and immunohistochemistry for TGF-beta-Receptor-I/ALK5 and phosphorylated/activated Smad2 confirmed that the Smad-dependent TGF-beta signaling cascade was inactive in these tumors. Although absent in most of the tumor, phosphorylated Smad2 was present in the periphery of tumors. Interestingly, presence of phosphorylated/activated Smad2 correlated with expression of Activin-Receptor-IB/ALK4, suggesting that although Smad-dependent TGF-beta signaling is absent in ErbB2/Neu-induced tumors, Activin signaling may be active at the leading edge of these tumors. Cumulatively, these data indicate that the TGF-beta pathway is intrinsically suppressed in ErbB2/Neu tumors via a mechanism involving loss of TGF-beta-Receptor-I/ALK5.
Upregulation of HER2/ErbB2/Neu occurs in 15-30% of human breast cancers and correlates with poor prognosis. Identification of ErbB2/Neu transcriptional targets should facilitate development of novel therapeutic approaches. Development of breast cancer is a multistep process; thus, to identify the transcriptomes associated with different stages of progression of tumorigenesis, we compared expression profiles of mammary tumors and preneoplastic mammary tissue from MMTV-Neu transgenic mice to expression profiles of wild-type mammary glands using Affymetrix microarrays. We identified 324 candidate genes that were unique to ErbB2/Neu-induced tumors relative to normal mammary gland tissue from wild-type controls. Expression of a subset of these genes (82) was also changed in the preneoplastic mammary glands compared to wild-type controls, indicating that they may play a pivotal role during early events of ErbB2/Neu-initiated mammary tumorigenesis. Further analysis of the microarray data revealed that expression of several known transforming growth factor (TGF)-beta target genes was altered, suggesting that the TGF-beta signaling cascade is downregulated in ErbB2/Neu-induced tumors. Western blot analysis for TGF-beta-Receptor-I/ALK5 and immunohistochemistry for TGF-beta-Receptor-I/ALK5 and phosphorylated/activated Smad2 confirmed that the Smad-dependent TGF-beta signaling cascade was inactive in these tumors. Although absent in most of the tumor, phosphorylated Smad2 was present in the periphery of tumors. Interestingly, presence of phosphorylated/activated Smad2 correlated with expression of Activin-Receptor-IB/ALK4, suggesting that although Smad-dependent TGF-beta signaling is absent in ErbB2/Neu-induced tumors, Activin signaling may be active at the leading edge of these tumors. Cumulatively, these data indicate that the TGF-beta pathway is intrinsically suppressed in ErbB2/Neu tumors via a mechanism involving loss of TGF-beta-Receptor-I/ALK5.
This study assessed the possibility to build a prognosis predictor, based on microarray gene expression measures, in stage II and III colon cancer patients. Tumour (T) and non-neoplastic mucosa (NM) mRNA samples from 18 patients (nine with a recurrence, nine with no recurrence) were profiled using the Affymetrix HGU133A GeneChip. The k-nearest neighbour method was used for prognosis prediction using T and NM gene expression measures. Six-fold cross-validation was applied to select the number of neighbours and the number of informative genes to include in the predictors. Based on this information, one T-based and one NM-based predictor were proposed and their accuracies were estimated by double cross-validation. In six-fold cross-validation, the lowest numbers of informative genes giving the lowest numbers of false predictions (two out of 18) were 30 and 70 with the T and NM gene expression measures, respectively. A 30-gene T-based predictor and a 70-gene NM-based predictor were then built, with estimated accuracies of 78 and 83%, respectively. This study suggests that one can build an accurate prognosis predictor for stage II and III colon cancer patients, based on gene expression measures, and one can use either tumour or non-neoplastic mucosa for this purpose.
This study assessed the possibility to build a prognosis predictor, based on microarray gene expression measures, in stage II and III colon cancer patients. Tumour (T) and non-neoplastic mucosa (NM) mRNA samples from 18 patients (nine with a recurrence, nine with no recurrence) were profiled using the Affymetrix HGU133A GeneChip. The k-nearest neighbour method was used for prognosis prediction using T and NM gene expression measures. Six-fold cross-validation was applied to select the number of neighbours and the number of informative genes to include in the predictors. Based on this information, one T-based and one NM-based predictor were proposed and their accuracies were estimated by double cross-validation. In six-fold cross-validation, the lowest numbers of informative genes giving the lowest numbers of false predictions (two out of 18) were 30 and 70 with the T and NM gene expression measures, respectively. A 30-gene T-based predictor and a 70-gene NM-based predictor were then built, with estimated accuracies of 78 and 83%, respectively. This study suggests that one can build an accurate prognosis predictor for stage II and III colon cancer patients, based on gene expression measures, and one can use either tumour or non-neoplastic mucosa for this purpose.
This study assessed the possibility to build a prognosis predictor, based on microarray gene expression measures, in stage II and III colon cancer patients. Tumour (T) and non-neoplastic mucosa (NM) mRNA samples from 18 patients (nine with a recurrence, nine with no recurrence) were profiled using the Affymetrix HGU133A GeneChip. The k-nearest neighbour method was used for prognosis prediction using T and NM gene expression measures. Six-fold cross-validation was applied to select the number of neighbours and the number of informative genes to include in the predictors. Based on this information, one T-based and one NM-based predictor were proposed and their accuracies were estimated by double cross-validation. In six-fold cross-validation, the lowest numbers of informative genes giving the lowest numbers of false predictions (two out of 18) were 30 and 70 with the T and NM gene expression measures, respectively. A 30-gene T-based predictor and a 70-gene NM-based predictor were then built, with estimated accuracies of 78 and 83%, respectively. This study suggests that one can build an accurate prognosis predictor for stage II and III colon cancer patients, based on gene expression measures, and one can use either tumour or non-neoplastic mucosa for this purpose.
This study assessed the possibility to build a prognosis predictor, based on microarray gene expression measures, in stage II and III colon cancer patients. Tumour (T) and non-neoplastic mucosa (NM) mRNA samples from 18 patients (nine with a recurrence, nine with no recurrence) were profiled using the Affymetrix HGU133A GeneChip. The k-nearest neighbour method was used for prognosis prediction using T and NM gene expression measures. Six-fold cross-validation was applied to select the number of neighbours and the number of informative genes to include in the predictors. Based on this information, one T-based and one NM-based predictor were proposed and their accuracies were estimated by double cross-validation. In six-fold cross-validation, the lowest numbers of informative genes giving the lowest numbers of false predictions (two out of 18) were 30 and 70 with the T and NM gene expression measures, respectively. A 30-gene T-based predictor and a 70-gene NM-based predictor were then built, with estimated accuracies of 78 and 83%, respectively. This study suggests that one can build an accurate prognosis predictor for stage II and III colon cancer patients, based on gene expression measures, and one can use either tumour or non-neoplastic mucosa for this purpose.
The oncogenic pathways in mitochondrial-rich thyroid carcinomas are not clearly understood. To investigate the possible implication of mitochondrial abundance in the genesis of thyroid tumors, we have explored the gene expression profile of six oncocytic carcinomas and six mitochondrial-rich papillary carcinomas using cDNA-microarray technology. A supervised approach allowed us to identify 83 genes differentially expressed in the two types of carcinoma. These genes were classified according to their ontologic profiles. Three genes, NOS3, alpha-actinin-2 and alpha-catenin, suspected of playing a role in tumor genesis, were explored by quantitative RT-PCR analysis and immunohistochemistry. Of the 59 genes overexpressed in papillary carcinomas, 51% were involved in cell communication. Of the 24 genes overexpressed in oncocytic carcinomas, 84% were involved in mitochondrial and cellular metabolism. Our results suggest that mitochondrial respiratory chain complexes III and IV play a significant role in the regulation of reactive oxygen species production by oncocytic tumors.
The oncogenic pathways in mitochondrial-rich thyroid carcinomas are not clearly understood. To investigate the possible implication of mitochondrial abundance in the genesis of thyroid tumors, we have explored the gene expression profile of six oncocytic carcinomas and six mitochondrial-rich papillary carcinomas using cDNA-microarray technology. A supervised approach allowed us to identify 83 genes differentially expressed in the two types of carcinoma. These genes were classified according to their ontologic profiles. Three genes, NOS3, alpha-actinin-2 and alpha-catenin, suspected of playing a role in tumor genesis, were explored by quantitative RT-PCR analysis and immunohistochemistry. Of the 59 genes overexpressed in papillary carcinomas, 51% were involved in cell communication. Of the 24 genes overexpressed in oncocytic carcinomas, 84% were involved in mitochondrial and cellular metabolism. Our results suggest that mitochondrial respiratory chain complexes III and IV play a significant role in the regulation of reactive oxygen species production by oncocytic tumors.
We have used microarray technology to identify the transcriptional targets of Rho subfamily guanosine 5'-triphosphate (GTP)ases in NIH3T3 cells. This analysis indicated that murine fibroblasts transformed by these proteins show similar transcriptomal profiles. Functional annotation of the regulated genes indicate that Rho subfamily GTPases target a wide spectrum of functions, although loci encoding proteins linked to proliferation and DNA synthesis/transcription are upregulated preferentially. Rho proteins promote four main networks of interacting proteins nucleated around E2F, c-Jun, c-Myc and p53. Of those, E2F, c-Jun and c-Myc are essential for the maintenance of cell transformation. Inhibition of Rock, one of the main Rho GTPase targets, leads to small changes in the transcriptome of Rho-transformed cells. Rock inhibition decreases c-myc gene expression without affecting the E2F and c-Jun pathways. Loss-of-function studies demonstrate that c-Myc is important for the blockage of cell-contact inhibition rather than for promoting the proliferation of Rho-transformed cells. However, c-Myc overexpression does not bypass the inhibition of cell transformation induced by Rock blockage, indicating that c-Myc is essential, but not sufficient, for Rock-dependent transformation. These results reveal the complexity of the genetic program orchestrated by the Rho subfamily and pinpoint protein networks that mediate different aspects of the malignant phenotype of Rho-transformed cells.
We have used microarray technology to identify the transcriptional targets of Rho subfamily guanosine 5'-triphosphate (GTP)ases in NIH3T3 cells. This analysis indicated that murine fibroblasts transformed by these proteins show similar transcriptomal profiles. Functional annotation of the regulated genes indicate that Rho subfamily GTPases target a wide spectrum of functions, although loci encoding proteins linked to proliferation and DNA synthesis/transcription are upregulated preferentially. Rho proteins promote four main networks of interacting proteins nucleated around E2F, c-Jun, c-Myc and p53. Of those, E2F, c-Jun and c-Myc are essential for the maintenance of cell transformation. Inhibition of Rock, one of the main Rho GTPase targets, leads to small changes in the transcriptome of Rho-transformed cells. Rock inhibition decreases c-myc gene expression without affecting the E2F and c-Jun pathways. Loss-of-function studies demonstrate that c-Myc is important for the blockage of cell-contact inhibition rather than for promoting the proliferation of Rho-transformed cells. However, c-Myc overexpression does not bypass the inhibition of cell transformation induced by Rock blockage, indicating that c-Myc is essential, but not sufficient, for Rock-dependent transformation. These results reveal the complexity of the genetic program orchestrated by the Rho subfamily and pinpoint protein networks that mediate different aspects of the malignant phenotype of Rho-transformed cells.
Array-based comparative genomic hybridization (CGH-array) has a powerful potential for high-throughput identification of genetic aberrations in cell genomes. We identified a homozygous loss of ADAM23 (2q33.3) in the course of a program to screen a panel of gastric cancer (GC) cell lines (1/32, 3.1%) for genomic copy-number aberrations using our custom-made CGH-array. Infrequent homozygous deletion of ADAM23 was also seen in primary gastric tumors (1/39, 2.6%). ADAM23 mRNA was expressed in normal stomach tissue, but not in the majority of GC cell lines without homozygous deletion of this gene. Expression of ADAM23 mRNA was restored to gene-silenced GC cells after treatment with 5-aza 2'-deoxycytidine. The methylation status of the ADAM23 CpG island, which showed promoter activity, correlated inversely with its expression. Methylation of this CpG island was observed both in GC cell lines and in primary GC tissues; in primary tumors with a hypermethylated CpG island, expression of ADAM23 was lower than in adjacent noncancerous tissues. Moreover, restoration of ADAM23 in GC cells reduced their numbers in colony-formation assays. These results suggest that genetic or epigenetic silencing by hypermethylation of the ADAM23 CpG-rich promoter region leads to loss of ADAM23 function, which may be a factor in gastric carcinogenesis.
Array-based comparative genomic hybridization (CGH-array) has a powerful potential for high-throughput identification of genetic aberrations in cell genomes. We identified a homozygous loss of ADAM23 (2q33.3) in the course of a program to screen a panel of gastric cancer (GC) cell lines (1/32, 3.1%) for genomic copy-number aberrations using our custom-made CGH-array. Infrequent homozygous deletion of ADAM23 was also seen in primary gastric tumors (1/39, 2.6%). ADAM23 mRNA was expressed in normal stomach tissue, but not in the majority of GC cell lines without homozygous deletion of this gene. Expression of ADAM23 mRNA was restored to gene-silenced GC cells after treatment with 5-aza 2'-deoxycytidine. The methylation status of the ADAM23 CpG island, which showed promoter activity, correlated inversely with its expression. Methylation of this CpG island was observed both in GC cell lines and in primary GC tissues; in primary tumors with a hypermethylated CpG island, expression of ADAM23 was lower than in adjacent noncancerous tissues. Moreover, restoration of ADAM23 in GC cells reduced their numbers in colony-formation assays. These results suggest that genetic or epigenetic silencing by hypermethylation of the ADAM23 CpG-rich promoter region leads to loss of ADAM23 function, which may be a factor in gastric carcinogenesis.
To investigate the relationship between Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC), we determined gene expression profiles of discrete pathological stages of esophageal neoplasia using a sequence-verified human cDNA microarray. Fifty one RNAs, comprising 24 normal esophagi (NE), 18 BEs, and nine EACs were hybridized to cDNA microarrays. Five statistical analyses were used for the data analysis. Genes showing significantly different expression levels among the three sample groups were identified. Genes were grouped into functional categories based on the Gene Ontology Consortium. Surprisingly, the expression pattern of BE was significantly more similar to EAC than to NE, notwithstanding the known histopathologic differences between BE and EAC. The pattern of NE was clearly distinct from that of EAC. Thirty-six genes were the most differentially modulated, according to these microarray data, in BE-associated neoplastic progression. Twelve genes were significantly differentially expressed in cancer-associated BE's plus EAC (as a single combined tissue group) vs noncancer-associated BE's. These genes represent potential biomarkers to diagnose EAC at its early stages. Our results demonstrate that molecular events at the transcriptional level in BE are remarkably similar to BE's-associated adenocarcinoma of the esophagus. This finding alarmingly implies that BE is biologically closer to cancer than to normal esophagus, and that the cancer risk of BE is perhaps higher than we had imagined. These findings suggest that changes modulated at the molecular biologic level supervene earlier than histologic changes, and that BE is an early intermediate stage in the process of EAC.
To investigate the relationship between Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC), we determined gene expression profiles of discrete pathological stages of esophageal neoplasia using a sequence-verified human cDNA microarray. Fifty one RNAs, comprising 24 normal esophagi (NE), 18 BEs, and nine EACs were hybridized to cDNA microarrays. Five statistical analyses were used for the data analysis. Genes showing significantly different expression levels among the three sample groups were identified. Genes were grouped into functional categories based on the Gene Ontology Consortium. Surprisingly, the expression pattern of BE was significantly more similar to EAC than to NE, notwithstanding the known histopathologic differences between BE and EAC. The pattern of NE was clearly distinct from that of EAC. Thirty-six genes were the most differentially modulated, according to these microarray data, in BE-associated neoplastic progression. Twelve genes were significantly differentially expressed in cancer-associated BE's plus EAC (as a single combined tissue group) vs noncancer-associated BE's. These genes represent potential biomarkers to diagnose EAC at its early stages. Our results demonstrate that molecular events at the transcriptional level in BE are remarkably similar to BE's-associated adenocarcinoma of the esophagus. This finding alarmingly implies that BE is biologically closer to cancer than to normal esophagus, and that the cancer risk of BE is perhaps higher than we had imagined. These findings suggest that changes modulated at the molecular biologic level supervene earlier than histologic changes, and that BE is an early intermediate stage in the process of EAC.
Testicular germ cell cancers remain one of the few solid tumors routinely cured in advanced stages with conventional cisplatin-based chemotherapy. The mechanisms remain largely unknown. Through use of gene-expression array profiling we define immediate transcriptional targets in response to cisplatin in testicular germ cell-derived human embryonal carcinoma cells. We report 46 genes upregulated and five genes repressed by cisplatin. Several of these gene products, including FAS, TRAILR3, PHLDA3, LRDD, and IER3 are previously implicated in the apoptotic death receptor pathway, while others including SESN1, FDXR, PLK3, and DDIT4 are known mediators of reactive oxygen species generation. Approximately 54% of the upregulated genes are established or suspected downstream targets of p53. Specific siRNA to p53 prevents cisplatin-mediated activation of p53 and p53 pathway genes and renders embryonal carcinoma cells relatively resistant to cisplatin cytotoxicity. Interestingly, in p53 knockdown cells nearly the entire set of identified cisplatin targets fail to respond or have a diminished response to cisplatin, suggesting that many are new direct or indirect targets of p53 including GPR87, STK17A, INPP5D, FLJ11259, and EPS8L2. The data indicate that robust transcriptional activation of p53 is linked to the known hypersensitivity of testicular germ cell tumors to chemotherapy. Many of the gene products may participate in the unique curability of this disease.
Testicular germ cell cancers remain one of the few solid tumors routinely cured in advanced stages with conventional cisplatin-based chemotherapy. The mechanisms remain largely unknown. Through use of gene-expression array profiling we define immediate transcriptional targets in response to cisplatin in testicular germ cell-derived human embryonal carcinoma cells. We report 46 genes upregulated and five genes repressed by cisplatin. Several of these gene products, including FAS, TRAILR3, PHLDA3, LRDD, and IER3 are previously implicated in the apoptotic death receptor pathway, while others including SESN1, FDXR, PLK3, and DDIT4 are known mediators of reactive oxygen species generation. Approximately 54% of the upregulated genes are established or suspected downstream targets of p53. Specific siRNA to p53 prevents cisplatin-mediated activation of p53 and p53 pathway genes and renders embryonal carcinoma cells relatively resistant to cisplatin cytotoxicity. Interestingly, in p53 knockdown cells nearly the entire set of identified cisplatin targets fail to respond or have a diminished response to cisplatin, suggesting that many are new direct or indirect targets of p53 including GPR87, STK17A, INPP5D, FLJ11259, and EPS8L2. The data indicate that robust transcriptional activation of p53 is linked to the known hypersensitivity of testicular germ cell tumors to chemotherapy. Many of the gene products may participate in the unique curability of this disease.
Alteration of methylation status has been recognized as a possible epigenetic mechanism of selection during tumorigenesis in pancreatic cancer. This type of cancer is characterized by poor prognosis partly due to resistance to conventional drug treatments. We have used microarray technology to investigate the changes in global gene expression observed after treatment of different pancreatic cancer cell lines with the methylase inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR). We have observed that this agent is able to inhibit to various degrees the growth of three pancreatic cancer cell lines. In particular, this inhibition was associated with induction of interferon (IFN)-related genes, as observed in other tumour types. Thus, expression of STAT1 seems to play a key role in the cellular response to treatment with the cytosine analogue. Moreover, we found increased p21(WAF1) and gadd45A expression to be associated with the efficacy of the treatment; this induction may correlate with activation of the IFN signalling pathway. Expression of the p16(INK) protein was also linked to the ability of cells to respond to 5-aza-CdR. Finally, genome-wide demethylation induced sensitization that significantly increased response to further treatment with various chemotherapy agents.
Alteration of methylation status has been recognized as a possible epigenetic mechanism of selection during tumorigenesis in pancreatic cancer. This type of cancer is characterized by poor prognosis partly due to resistance to conventional drug treatments. We have used microarray technology to investigate the changes in global gene expression observed after treatment of different pancreatic cancer cell lines with the methylase inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR). We have observed that this agent is able to inhibit to various degrees the growth of three pancreatic cancer cell lines. In particular, this inhibition was associated with induction of interferon (IFN)-related genes, as observed in other tumour types. Thus, expression of STAT1 seems to play a key role in the cellular response to treatment with the cytosine analogue. Moreover, we found increased p21(WAF1) and gadd45A expression to be associated with the efficacy of the treatment; this induction may correlate with activation of the IFN signalling pathway. Expression of the p16(INK) protein was also linked to the ability of cells to respond to 5-aza-CdR. Finally, genome-wide demethylation induced sensitization that significantly increased response to further treatment with various chemotherapy agents.
The tumor suppressor p53 is a key modulator of the cellular stress response, inducing cell-cycle arrest, apoptosis, senescence and cell differentiation. To evaluate further the molecular mechanism underlying p53 function, the transcriptional profiles of proliferating and senescent WI-38 cells, both wild-type p53 expressers and counterparts with an inactivated p53, were compared by DNA microarray analysis. In particular, the amyloid-beta precursor-like protein 1 (APLP1) is induced in senescent cells in a p53-dependent manner. APLP1 was confirmed to be a novel transcriptional target of p53 by in vivo and in vitro characterization of a p53 responsive element found in the first intron of the APLP1 gene locus. APLP1 knockdown experiments demonstrate that APLP1 is required for the proliferation of fibroblastic and epithelial cells. Moreover, depletion of APLP1 expression diminishes stress-induced apoptosis of neural cells, whereas ectopic APLP1 expression augments apoptosis. Based on these data, a mechanism is proposed whereby p53-dependent induction of APLP1 is involved in neural cell death, and which may exacerbate neuronal cell loss in some acute or chronic neurodegenerative disorders.
The tumor suppressor p53 is a key modulator of the cellular stress response, inducing cell-cycle arrest, apoptosis, senescence and cell differentiation. To evaluate further the molecular mechanism underlying p53 function, the transcriptional profiles of proliferating and senescent WI-38 cells, both wild-type p53 expressers and counterparts with an inactivated p53, were compared by DNA microarray analysis. In particular, the amyloid-beta precursor-like protein 1 (APLP1) is induced in senescent cells in a p53-dependent manner. APLP1 was confirmed to be a novel transcriptional target of p53 by in vivo and in vitro characterization of a p53 responsive element found in the first intron of the APLP1 gene locus. APLP1 knockdown experiments demonstrate that APLP1 is required for the proliferation of fibroblastic and epithelial cells. Moreover, depletion of APLP1 expression diminishes stress-induced apoptosis of neural cells, whereas ectopic APLP1 expression augments apoptosis. Based on these data, a mechanism is proposed whereby p53-dependent induction of APLP1 is involved in neural cell death, and which may exacerbate neuronal cell loss in some acute or chronic neurodegenerative disorders.
To investigate the relationship between Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC), we determined gene expression profiles of discrete pathological stages of esophageal neoplasia using a sequence-verified human cDNA microarray. Fifty one RNAs, comprising 24 normal esophagi (NE), 18 BEs, and nine EACs were hybridized to cDNA microarrays. Five statistical analyses were used for the data analysis. Genes showing significantly different expression levels among the three sample groups were identified. Genes were grouped into functional categories based on the Gene Ontology Consortium. Surprisingly, the expression pattern of BE was significantly more similar to EAC than to NE, notwithstanding the known histopathologic differences between BE and EAC. The pattern of NE was clearly distinct from that of EAC. Thirty-six genes were the most differentially modulated, according to these microarray data, in BE-associated neoplastic progression. Twelve genes were significantly differentially expressed in cancer-associated BE's plus EAC (as a single combined tissue group) vs noncancer-associated BE's. These genes represent potential biomarkers to diagnose EAC at its early stages. Our results demonstrate that molecular events at the transcriptional level in BE are remarkably similar to BE's-associated adenocarcinoma of the esophagus. This finding alarmingly implies that BE is biologically closer to cancer than to normal esophagus, and that the cancer risk of BE is perhaps higher than we had imagined. These findings suggest that changes modulated at the molecular biologic level supervene earlier than histologic changes, and that BE is an early intermediate stage in the process of EAC.
To investigate the relationship between Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC), we determined gene expression profiles of discrete pathological stages of esophageal neoplasia using a sequence-verified human cDNA microarray. Fifty one RNAs, comprising 24 normal esophagi (NE), 18 BEs, and nine EACs were hybridized to cDNA microarrays. Five statistical analyses were used for the data analysis. Genes showing significantly different expression levels among the three sample groups were identified. Genes were grouped into functional categories based on the Gene Ontology Consortium. Surprisingly, the expression pattern of BE was significantly more similar to EAC than to NE, notwithstanding the known histopathologic differences between BE and EAC. The pattern of NE was clearly distinct from that of EAC. Thirty-six genes were the most differentially modulated, according to these microarray data, in BE-associated neoplastic progression. Twelve genes were significantly differentially expressed in cancer-associated BE's plus EAC (as a single combined tissue group) vs noncancer-associated BE's. These genes represent potential biomarkers to diagnose EAC at its early stages. Our results demonstrate that molecular events at the transcriptional level in BE are remarkably similar to BE's-associated adenocarcinoma of the esophagus. This finding alarmingly implies that BE is biologically closer to cancer than to normal esophagus, and that the cancer risk of BE is perhaps higher than we had imagined. These findings suggest that changes modulated at the molecular biologic level supervene earlier than histologic changes, and that BE is an early intermediate stage in the process of EAC.
To investigate the relationship between Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC), we determined gene expression profiles of discrete pathological stages of esophageal neoplasia using a sequence-verified human cDNA microarray. Fifty one RNAs, comprising 24 normal esophagi (NE), 18 BEs, and nine EACs were hybridized to cDNA microarrays. Five statistical analyses were used for the data analysis. Genes showing significantly different expression levels among the three sample groups were identified. Genes were grouped into functional categories based on the Gene Ontology Consortium. Surprisingly, the expression pattern of BE was significantly more similar to EAC than to NE, notwithstanding the known histopathologic differences between BE and EAC. The pattern of NE was clearly distinct from that of EAC. Thirty-six genes were the most differentially modulated, according to these microarray data, in BE-associated neoplastic progression. Twelve genes were significantly differentially expressed in cancer-associated BE's plus EAC (as a single combined tissue group) vs noncancer-associated BE's. These genes represent potential biomarkers to diagnose EAC at its early stages. Our results demonstrate that molecular events at the transcriptional level in BE are remarkably similar to BE's-associated adenocarcinoma of the esophagus. This finding alarmingly implies that BE is biologically closer to cancer than to normal esophagus, and that the cancer risk of BE is perhaps higher than we had imagined. These findings suggest that changes modulated at the molecular biologic level supervene earlier than histologic changes, and that BE is an early intermediate stage in the process of EAC.
To investigate the relationship between Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC), we determined gene expression profiles of discrete pathological stages of esophageal neoplasia using a sequence-verified human cDNA microarray. Fifty one RNAs, comprising 24 normal esophagi (NE), 18 BEs, and nine EACs were hybridized to cDNA microarrays. Five statistical analyses were used for the data analysis. Genes showing significantly different expression levels among the three sample groups were identified. Genes were grouped into functional categories based on the Gene Ontology Consortium. Surprisingly, the expression pattern of BE was significantly more similar to EAC than to NE, notwithstanding the known histopathologic differences between BE and EAC. The pattern of NE was clearly distinct from that of EAC. Thirty-six genes were the most differentially modulated, according to these microarray data, in BE-associated neoplastic progression. Twelve genes were significantly differentially expressed in cancer-associated BE's plus EAC (as a single combined tissue group) vs noncancer-associated BE's. These genes represent potential biomarkers to diagnose EAC at its early stages. Our results demonstrate that molecular events at the transcriptional level in BE are remarkably similar to BE's-associated adenocarcinoma of the esophagus. This finding alarmingly implies that BE is biologically closer to cancer than to normal esophagus, and that the cancer risk of BE is perhaps higher than we had imagined. These findings suggest that changes modulated at the molecular biologic level supervene earlier than histologic changes, and that BE is an early intermediate stage in the process of EAC.
To investigate the relationship between Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC), we determined gene expression profiles of discrete pathological stages of esophageal neoplasia using a sequence-verified human cDNA microarray. Fifty one RNAs, comprising 24 normal esophagi (NE), 18 BEs, and nine EACs were hybridized to cDNA microarrays. Five statistical analyses were used for the data analysis. Genes showing significantly different expression levels among the three sample groups were identified. Genes were grouped into functional categories based on the Gene Ontology Consortium. Surprisingly, the expression pattern of BE was significantly more similar to EAC than to NE, notwithstanding the known histopathologic differences between BE and EAC. The pattern of NE was clearly distinct from that of EAC. Thirty-six genes were the most differentially modulated, according to these microarray data, in BE-associated neoplastic progression. Twelve genes were significantly differentially expressed in cancer-associated BE's plus EAC (as a single combined tissue group) vs noncancer-associated BE's. These genes represent potential biomarkers to diagnose EAC at its early stages. Our results demonstrate that molecular events at the transcriptional level in BE are remarkably similar to BE's-associated adenocarcinoma of the esophagus. This finding alarmingly implies that BE is biologically closer to cancer than to normal esophagus, and that the cancer risk of BE is perhaps higher than we had imagined. These findings suggest that changes modulated at the molecular biologic level supervene earlier than histologic changes, and that BE is an early intermediate stage in the process of EAC.
In all, 85% of Ewing's sarcoma family tumors (ESFT), a neoplasm of unknown histogenesis, express EWS-FLI1 transcription factor gene fusions. To characterize direct target genes avoiding artificial model systems, we cloned genomic DNA from ESFT chromatin precipitating with EWS-FLI1. We now present a comprehensive list of 99 putative transcription factor targets identified, for the first time, by a hypothesis-free approach based on physical interaction. Gene-derived chromatin fragments co-precipitating with EWS-FLI1 were nonrandomly distributed over the human genome and localized predominantly to the upstream region and the first two introns of the genes. At least 20% of putative direct EWS-FLI1 targets were neural genes. One-third of genes recovered showed a significant ESFT-specific expression pattern and were found to be altered upon RNAi-mediated knockdown of EWS-FLI1. Among them, MK-STYX, encoding a MAP kinase phosphatase-like protein, was consistently expressed in ESFT. EWS-FLI1 was found to drive MK-STYX expression by binding to a single ETS binding motif within the first gene intron. MK-STYX serves as precedence for successful recovery of direct EWS-FLI1 targets from the authentic ESFT cellular context, the most relevant system to study oncogenic mechanisms for the discovery of new therapeutic targets in this disease.
In all, 85% of Ewing's sarcoma family tumors (ESFT), a neoplasm of unknown histogenesis, express EWS-FLI1 transcription factor gene fusions. To characterize direct target genes avoiding artificial model systems, we cloned genomic DNA from ESFT chromatin precipitating with EWS-FLI1. We now present a comprehensive list of 99 putative transcription factor targets identified, for the first time, by a hypothesis-free approach based on physical interaction. Gene-derived chromatin fragments co-precipitating with EWS-FLI1 were nonrandomly distributed over the human genome and localized predominantly to the upstream region and the first two introns of the genes. At least 20% of putative direct EWS-FLI1 targets were neural genes. One-third of genes recovered showed a significant ESFT-specific expression pattern and were found to be altered upon RNAi-mediated knockdown of EWS-FLI1. Among them, MK-STYX, encoding a MAP kinase phosphatase-like protein, was consistently expressed in ESFT. EWS-FLI1 was found to drive MK-STYX expression by binding to a single ETS binding motif within the first gene intron. MK-STYX serves as precedence for successful recovery of direct EWS-FLI1 targets from the authentic ESFT cellular context, the most relevant system to study oncogenic mechanisms for the discovery of new therapeutic targets in this disease.
We analysed chromosome 16q in 106 breast cancers using tiling-path array-comparative genomic hybridization (aCGH). About 80% of ductal cancers (IDCs) and all lobular cancers (ILCs) lost at least part of 16q. Grade I (GI) IDCs and ILCs often lost the whole chromosome arm. Grade II (GII) and grade III (GIII) IDCs showed less frequent whole-arm loss, but often had complex changes, typically small regions of gain together with larger regions of loss. The boundaries of gains/losses tended to cluster, common sites being 54.5-55.5 Mb and 57.4-58.8 Mb. Overall, the peak frequency of loss (83% cancers) occurred at 61.9-62.9 Mb. We also found several 'minimal' regions of loss/gain. However, no mutations in candidate genes (TRADD, CDH5, CDH8 and CDH11) were detected. Cluster analysis based on copy number changes identified a large group of cancers that had lost most of 16q, and two smaller groups (one with few changes, one with a tendency to show copy number gain). Although all morphological types occurred in each cluster group, IDCs (especially GII/GIII) were relatively overrepresented in the smaller groups. Cluster groups were not independently associated with survival. Use of tiling-path aCGH prompted re-evaluation of the hypothetical pathways of breast carcinogenesis. ILCs have the simplest changes on 16q and probably diverge from the IDC lineage close to the stage of 16q loss. Higher-grade IDCs probably develop from low-grade lesions in most cases, but there remains evidence that some GII/GIII IDCs arise without a GI precursor.
We analysed chromosome 16q in 106 breast cancers using tiling-path array-comparative genomic hybridization (aCGH). About 80% of ductal cancers (IDCs) and all lobular cancers (ILCs) lost at least part of 16q. Grade I (GI) IDCs and ILCs often lost the whole chromosome arm. Grade II (GII) and grade III (GIII) IDCs showed less frequent whole-arm loss, but often had complex changes, typically small regions of gain together with larger regions of loss. The boundaries of gains/losses tended to cluster, common sites being 54.5-55.5 Mb and 57.4-58.8 Mb. Overall, the peak frequency of loss (83% cancers) occurred at 61.9-62.9 Mb. We also found several 'minimal' regions of loss/gain. However, no mutations in candidate genes (TRADD, CDH5, CDH8 and CDH11) were detected. Cluster analysis based on copy number changes identified a large group of cancers that had lost most of 16q, and two smaller groups (one with few changes, one with a tendency to show copy number gain). Although all morphological types occurred in each cluster group, IDCs (especially GII/GIII) were relatively overrepresented in the smaller groups. Cluster groups were not independently associated with survival. Use of tiling-path aCGH prompted re-evaluation of the hypothetical pathways of breast carcinogenesis. ILCs have the simplest changes on 16q and probably diverge from the IDC lineage close to the stage of 16q loss. Higher-grade IDCs probably develop from low-grade lesions in most cases, but there remains evidence that some GII/GIII IDCs arise without a GI precursor.
Expression microarray analysis identified over 930 genes regulated during puberty in the mouse mammary gland. Most prominent were genes whose expression increased in parallel with pubertal development and remained high thereafter. Members of the Wnt, transforming growth factor-beta and oestrogen-signalling pathways were significantly overrepresented. Comparison to expression data from CITED1 knockout mice identified a subset of oestrogen-responsive genes displaying altered expression in the absence of CITED1. Included in this subset are stanniocalcin2 (Stc2) and amphiregulin (Areg). Chromatin immunoprecipitation revealed that ERalpha binds to oestrogen response elements in both the Stc2 and Areg genes in the mammary gland during puberty. Additionally, CITED1 and ERalpha localize to the same epithelial cells of the pubertal mammary gland, supporting a role for interaction of these two proteins during normal development. In a human breast cancer data set, expression of Stc2, Areg and CITED1 parallel that of ERalpha. Similar to ERalpha, CITED1 expression correlates with good outcome in breast cancer, implying that potential maintenance of the ERalpha-CITED1 co-regulated signalling pathway in breast tumours can indicate good prognosis.
Expression microarray analysis identified over 930 genes regulated during puberty in the mouse mammary gland. Most prominent were genes whose expression increased in parallel with pubertal development and remained high thereafter. Members of the Wnt, transforming growth factor-beta and oestrogen-signalling pathways were significantly overrepresented. Comparison to expression data from CITED1 knockout mice identified a subset of oestrogen-responsive genes displaying altered expression in the absence of CITED1. Included in this subset are stanniocalcin2 (Stc2) and amphiregulin (Areg). Chromatin immunoprecipitation revealed that ERalpha binds to oestrogen response elements in both the Stc2 and Areg genes in the mammary gland during puberty. Additionally, CITED1 and ERalpha localize to the same epithelial cells of the pubertal mammary gland, supporting a role for interaction of these two proteins during normal development. In a human breast cancer data set, expression of Stc2, Areg and CITED1 parallel that of ERalpha. Similar to ERalpha, CITED1 expression correlates with good outcome in breast cancer, implying that potential maintenance of the ERalpha-CITED1 co-regulated signalling pathway in breast tumours can indicate good prognosis.
Expression microarray analysis identified over 930 genes regulated during puberty in the mouse mammary gland. Most prominent were genes whose expression increased in parallel with pubertal development and remained high thereafter. Members of the Wnt, transforming growth factor-beta and oestrogen-signalling pathways were significantly overrepresented. Comparison to expression data from CITED1 knockout mice identified a subset of oestrogen-responsive genes displaying altered expression in the absence of CITED1. Included in this subset are stanniocalcin2 (Stc2) and amphiregulin (Areg). Chromatin immunoprecipitation revealed that ERalpha binds to oestrogen response elements in both the Stc2 and Areg genes in the mammary gland during puberty. Additionally, CITED1 and ERalpha localize to the same epithelial cells of the pubertal mammary gland, supporting a role for interaction of these two proteins during normal development. In a human breast cancer data set, expression of Stc2, Areg and CITED1 parallel that of ERalpha. Similar to ERalpha, CITED1 expression correlates with good outcome in breast cancer, implying that potential maintenance of the ERalpha-CITED1 co-regulated signalling pathway in breast tumours can indicate good prognosis.
Expression microarray analysis identified over 930 genes regulated during puberty in the mouse mammary gland. Most prominent were genes whose expression increased in parallel with pubertal development and remained high thereafter. Members of the Wnt, transforming growth factor-beta and oestrogen-signalling pathways were significantly overrepresented. Comparison to expression data from CITED1 knockout mice identified a subset of oestrogen-responsive genes displaying altered expression in the absence of CITED1. Included in this subset are stanniocalcin2 (Stc2) and amphiregulin (Areg). Chromatin immunoprecipitation revealed that ERalpha binds to oestrogen response elements in both the Stc2 and Areg genes in the mammary gland during puberty. Additionally, CITED1 and ERalpha localize to the same epithelial cells of the pubertal mammary gland, supporting a role for interaction of these two proteins during normal development. In a human breast cancer data set, expression of Stc2, Areg and CITED1 parallel that of ERalpha. Similar to ERalpha, CITED1 expression correlates with good outcome in breast cancer, implying that potential maintenance of the ERalpha-CITED1 co-regulated signalling pathway in breast tumours can indicate good prognosis.
Expression microarray analysis identified over 930 genes regulated during puberty in the mouse mammary gland. Most prominent were genes whose expression increased in parallel with pubertal development and remained high thereafter. Members of the Wnt, transforming growth factor-beta and oestrogen-signalling pathways were significantly overrepresented. Comparison to expression data from CITED1 knockout mice identified a subset of oestrogen-responsive genes displaying altered expression in the absence of CITED1. Included in this subset are stanniocalcin2 (Stc2) and amphiregulin (Areg). Chromatin immunoprecipitation revealed that ERalpha binds to oestrogen response elements in both the Stc2 and Areg genes in the mammary gland during puberty. Additionally, CITED1 and ERalpha localize to the same epithelial cells of the pubertal mammary gland, supporting a role for interaction of these two proteins during normal development. In a human breast cancer data set, expression of Stc2, Areg and CITED1 parallel that of ERalpha. Similar to ERalpha, CITED1 expression correlates with good outcome in breast cancer, implying that potential maintenance of the ERalpha-CITED1 co-regulated signalling pathway in breast tumours can indicate good prognosis.
Expression microarray analysis identified over 930 genes regulated during puberty in the mouse mammary gland. Most prominent were genes whose expression increased in parallel with pubertal development and remained high thereafter. Members of the Wnt, transforming growth factor-beta and oestrogen-signalling pathways were significantly overrepresented. Comparison to expression data from CITED1 knockout mice identified a subset of oestrogen-responsive genes displaying altered expression in the absence of CITED1. Included in this subset are stanniocalcin2 (Stc2) and amphiregulin (Areg). Chromatin immunoprecipitation revealed that ERalpha binds to oestrogen response elements in both the Stc2 and Areg genes in the mammary gland during puberty. Additionally, CITED1 and ERalpha localize to the same epithelial cells of the pubertal mammary gland, supporting a role for interaction of these two proteins during normal development. In a human breast cancer data set, expression of Stc2, Areg and CITED1 parallel that of ERalpha. Similar to ERalpha, CITED1 expression correlates with good outcome in breast cancer, implying that potential maintenance of the ERalpha-CITED1 co-regulated signalling pathway in breast tumours can indicate good prognosis.
Expression microarray analysis identified over 930 genes regulated during puberty in the mouse mammary gland. Most prominent were genes whose expression increased in parallel with pubertal development and remained high thereafter. Members of the Wnt, transforming growth factor-beta and oestrogen-signalling pathways were significantly overrepresented. Comparison to expression data from CITED1 knockout mice identified a subset of oestrogen-responsive genes displaying altered expression in the absence of CITED1. Included in this subset are stanniocalcin2 (Stc2) and amphiregulin (Areg). Chromatin immunoprecipitation revealed that ERalpha binds to oestrogen response elements in both the Stc2 and Areg genes in the mammary gland during puberty. Additionally, CITED1 and ERalpha localize to the same epithelial cells of the pubertal mammary gland, supporting a role for interaction of these two proteins during normal development. In a human breast cancer data set, expression of Stc2, Areg and CITED1 parallel that of ERalpha. Similar to ERalpha, CITED1 expression correlates with good outcome in breast cancer, implying that potential maintenance of the ERalpha-CITED1 co-regulated signalling pathway in breast tumours can indicate good prognosis.
Expression microarray analysis identified over 930 genes regulated during puberty in the mouse mammary gland. Most prominent were genes whose expression increased in parallel with pubertal development and remained high thereafter. Members of the Wnt, transforming growth factor-beta and oestrogen-signalling pathways were significantly overrepresented. Comparison to expression data from CITED1 knockout mice identified a subset of oestrogen-responsive genes displaying altered expression in the absence of CITED1. Included in this subset are stanniocalcin2 (Stc2) and amphiregulin (Areg). Chromatin immunoprecipitation revealed that ERalpha binds to oestrogen response elements in both the Stc2 and Areg genes in the mammary gland during puberty. Additionally, CITED1 and ERalpha localize to the same epithelial cells of the pubertal mammary gland, supporting a role for interaction of these two proteins during normal development. In a human breast cancer data set, expression of Stc2, Areg and CITED1 parallel that of ERalpha. Similar to ERalpha, CITED1 expression correlates with good outcome in breast cancer, implying that potential maintenance of the ERalpha-CITED1 co-regulated signalling pathway in breast tumours can indicate good prognosis.
PKCalpha and Ets1 are both associated with breast cancer progression. Our previous studies suggested that these proteins are likely to functionally interact with one another. Here, we show that attenuation of endogenous PKCalpha expression (siPalpha) by RNA interference leads to reduced Ets1 protein expression in a variety of cancer cells. Pulse-chase experiments and treatment with proteasome inhibitor MG-132 revealed that siPalpha interferes with both Ets1 protein synthesis and stability. The effect of siPalpha on Ets1 expression could be partially prevented by KN-93, suggesting that calcium/calmodulin-dependent kinase II (CaMKII), a modulator of Ets1 activity, may play a role in PKCalpha-dependent Ets1 regulation. In contrast, Ets1-regulating kinases ERK1/2 were not found to be involved in this process. To assess the importance of the PKCalpha/Ets1 interaction, we compared the biological responses of MDA-MB-231 cells to PKCalpha- and Ets1-specific siRNAs (siE1). While only siPalpha induced changes in cellular morphology and anchorage-independent growth, both siRNAs similarly affected cellular responses to the antitumor drug mithramycin A and to UV light. Microarray analyses further showed that the expression of a certain set of genes was equally affected by siPalpha and siE1. The data suggest that Ets1 serves as an effector for PKCalpha to fulfil certain functions in cancer cells.
PKCalpha and Ets1 are both associated with breast cancer progression. Our previous studies suggested that these proteins are likely to functionally interact with one another. Here, we show that attenuation of endogenous PKCalpha expression (siPalpha) by RNA interference leads to reduced Ets1 protein expression in a variety of cancer cells. Pulse-chase experiments and treatment with proteasome inhibitor MG-132 revealed that siPalpha interferes with both Ets1 protein synthesis and stability. The effect of siPalpha on Ets1 expression could be partially prevented by KN-93, suggesting that calcium/calmodulin-dependent kinase II (CaMKII), a modulator of Ets1 activity, may play a role in PKCalpha-dependent Ets1 regulation. In contrast, Ets1-regulating kinases ERK1/2 were not found to be involved in this process. To assess the importance of the PKCalpha/Ets1 interaction, we compared the biological responses of MDA-MB-231 cells to PKCalpha- and Ets1-specific siRNAs (siE1). While only siPalpha induced changes in cellular morphology and anchorage-independent growth, both siRNAs similarly affected cellular responses to the antitumor drug mithramycin A and to UV light. Microarray analyses further showed that the expression of a certain set of genes was equally affected by siPalpha and siE1. The data suggest that Ets1 serves as an effector for PKCalpha to fulfil certain functions in cancer cells.
The chromosomal translocation t(8;21) is associated with 10-15% of all cases of acute myeloid leukaemia (AML). The resultant fusion protein AML1/MTG8 interferes with haematopoietic gene expression and is an important regulator of leukaemogenesis. We studied the effects of small interfering RNA (siRNA)-mediated AML1/MTG8 depletion on global gene expression in t(8;21)-positive leukaemic cell lines and in primary AML blasts using cDNA arrays, oligonucleotide arrays and real-time reverse transcription-polymerase chain reaction (RT-PCR). Suppression of AML1/MTG8 results in the increased expression of genes associated with myeloid differentiation, such as AZU1, BPI, CTSG, LYZ and RNASE2 as well as of antiproliferative genes such as IGFBP7, MS4A3 and SLA both in blasts and in cell lines. Furthermore, expression levels of several genes affiliated with drug resistance or indicative of poor prognosis AML (BAALC, CD34, PRG2, TSPAN7) are affected by AML1/MTG8 depletion. In conclusion, siRNA-mediated suppression of AML1/MTG8 cause very similar changes in gene expression pattern in t(8;21)-positive cell lines and in primary AML blasts. Furthermore, the results suggest that the specific targeting of AML1/MTG8 function may be a promising approach for complementing existing treatment strategies.
The chromosomal translocation t(8;21) is associated with 10-15% of all cases of acute myeloid leukaemia (AML). The resultant fusion protein AML1/MTG8 interferes with haematopoietic gene expression and is an important regulator of leukaemogenesis. We studied the effects of small interfering RNA (siRNA)-mediated AML1/MTG8 depletion on global gene expression in t(8;21)-positive leukaemic cell lines and in primary AML blasts using cDNA arrays, oligonucleotide arrays and real-time reverse transcription-polymerase chain reaction (RT-PCR). Suppression of AML1/MTG8 results in the increased expression of genes associated with myeloid differentiation, such as AZU1, BPI, CTSG, LYZ and RNASE2 as well as of antiproliferative genes such as IGFBP7, MS4A3 and SLA both in blasts and in cell lines. Furthermore, expression levels of several genes affiliated with drug resistance or indicative of poor prognosis AML (BAALC, CD34, PRG2, TSPAN7) are affected by AML1/MTG8 depletion. In conclusion, siRNA-mediated suppression of AML1/MTG8 cause very similar changes in gene expression pattern in t(8;21)-positive cell lines and in primary AML blasts. Furthermore, the results suggest that the specific targeting of AML1/MTG8 function may be a promising approach for complementing existing treatment strategies.
The demonstration of the PAX8-PPAR(gamma) fusion oncogene in a subset of follicular thyroid tumors provides a new and promising starting point to dissect the molecular genetic events involved in the development of this tumor form. In the present study, we compared the gene expression profiles of follicular thyroid carcinomas (FTCs) bearing a PAX8-PPAR(gamma) fusion against FTCs that lack this fusion. Using unsupervised clustering and multidimensional scaling analyses, we show that FTCs possessing a PAX8-PPAR(gamma) fusion have a highly uniform and distinct gene expression signature that clearly distinguishes them from FTCs without the fusion. The PAX8-PPAR(gamma)(+) FTCs grouped in a defined cluster, where highly ranked genes were mostly associated with signal transduction, cell growth and translation control. Notably, a large number of ribosomal protein and translation-associated genes were concurrently underexpressed in the FTCs with the fusion. Taken together, our findings further support that follicular carcinomas with a PAX8-PPAR(gamma) rearrangement constitute a distinct biological entity. The current data represent one step to elucidate the molecular pathways in the development of FTCs with the specific PAX8-PPAR(gamma) fusion.
The demonstration of the PAX8-PPAR(gamma) fusion oncogene in a subset of follicular thyroid tumors provides a new and promising starting point to dissect the molecular genetic events involved in the development of this tumor form. In the present study, we compared the gene expression profiles of follicular thyroid carcinomas (FTCs) bearing a PAX8-PPAR(gamma) fusion against FTCs that lack this fusion. Using unsupervised clustering and multidimensional scaling analyses, we show that FTCs possessing a PAX8-PPAR(gamma) fusion have a highly uniform and distinct gene expression signature that clearly distinguishes them from FTCs without the fusion. The PAX8-PPAR(gamma)(+) FTCs grouped in a defined cluster, where highly ranked genes were mostly associated with signal transduction, cell growth and translation control. Notably, a large number of ribosomal protein and translation-associated genes were concurrently underexpressed in the FTCs with the fusion. Taken together, our findings further support that follicular carcinomas with a PAX8-PPAR(gamma) rearrangement constitute a distinct biological entity. The current data represent one step to elucidate the molecular pathways in the development of FTCs with the specific PAX8-PPAR(gamma) fusion.
Tumors of low malignant potential (LMP) represent 20% of epithelial ovarian cancers (EOCs) and are associated with a better prognosis than the invasive tumors (TOV). Defining the relationship between LMPs and TOVs remains an important goal towards understanding the molecular pathways that contribute to prognosis, as well as providing molecular markers, for these EOCs. To this end, DNA microarray analyses were performed either in a primary culture or a tumor tissue model system and selected candidate genes showing a distinctive expression profile between LMPs and TOVs were identified using a class prediction approach based on three statistical methods of analysis. Both model systems appear relevant as candidate genes identified by either model allowed the proper reclassification of samples as either LMPs or TOVs. Selected candidate genes (CAS, CCNE1, LGALS8, ITGbeta3, ATP1B1, FLIP, KRT7 and KRT19) were validated by real-time quantitative PCR analysis and show differential expression between LMPs and TOVs. Immunohistochemistry analyses showed that the two tumor classes were distinguishable by their expression of CAS, TNFR1A, FLIP, CKS1 and CCNE1. These results define signature patterns for gene expression of LMPs and TOVs and identify gene candidates that warrant further study to deepen our understanding of the biology of EOC.
Tumors of low malignant potential (LMP) represent 20% of epithelial ovarian cancers (EOCs) and are associated with a better prognosis than the invasive tumors (TOV). Defining the relationship between LMPs and TOVs remains an important goal towards understanding the molecular pathways that contribute to prognosis, as well as providing molecular markers, for these EOCs. To this end, DNA microarray analyses were performed either in a primary culture or a tumor tissue model system and selected candidate genes showing a distinctive expression profile between LMPs and TOVs were identified using a class prediction approach based on three statistical methods of analysis. Both model systems appear relevant as candidate genes identified by either model allowed the proper reclassification of samples as either LMPs or TOVs. Selected candidate genes (CAS, CCNE1, LGALS8, ITGbeta3, ATP1B1, FLIP, KRT7 and KRT19) were validated by real-time quantitative PCR analysis and show differential expression between LMPs and TOVs. Immunohistochemistry analyses showed that the two tumor classes were distinguishable by their expression of CAS, TNFR1A, FLIP, CKS1 and CCNE1. These results define signature patterns for gene expression of LMPs and TOVs and identify gene candidates that warrant further study to deepen our understanding of the biology of EOC.
Hepatocyte nuclear factor 4alpha (HNF4alpha) is a tissue-specific transcription factor known to regulate a large number of genes in hepatocytes and pancreatic beta cells. Although HNF4alpha is highly expressed in some sections of the kidney, little is known about its role in this organ and about HNF4alpha-regulated genes in the kidney cells. The abundance and activity of HNF4alpha are frequently reduced in renal cell carcinoma (RCC) indicating some tumor suppressing function of HNF4alpha in renal cells. To determine the potential role of HNF4alpha in RCC, we used Flp recombinase-mediated gene integration to generate human embryonic kidney cells (HEK293) that conditionally express wild-type or mutated HNF4alpha. Expression of wild-type HNF4alpha but not of the mutants led to reduction of proliferation and alterations of cell morphology. These effects were reversible and induced at physiological concentrations of HNF4alpha. Using gene expression profiling by microarrays, we determined genes regulated by HNF4alpha. Interestingly, many of the genes regulated by HNF4alpha have been shown to be deregulated in RCC microarray studies. These genes (ACY1, WT1, SELENBP1, COBL, EFHD1, AGXT2L1, ALDH5A1, THEM2, ABCB1, FLJ14146, CSPG2, TRIM9 and HEY1) are good candidates for genes whose activity is changed upon the decrease of HNF4alpha in RCC.
Hepatocyte nuclear factor 4alpha (HNF4alpha) is a tissue-specific transcription factor known to regulate a large number of genes in hepatocytes and pancreatic beta cells. Although HNF4alpha is highly expressed in some sections of the kidney, little is known about its role in this organ and about HNF4alpha-regulated genes in the kidney cells. The abundance and activity of HNF4alpha are frequently reduced in renal cell carcinoma (RCC) indicating some tumor suppressing function of HNF4alpha in renal cells. To determine the potential role of HNF4alpha in RCC, we used Flp recombinase-mediated gene integration to generate human embryonic kidney cells (HEK293) that conditionally express wild-type or mutated HNF4alpha. Expression of wild-type HNF4alpha but not of the mutants led to reduction of proliferation and alterations of cell morphology. These effects were reversible and induced at physiological concentrations of HNF4alpha. Using gene expression profiling by microarrays, we determined genes regulated by HNF4alpha. Interestingly, many of the genes regulated by HNF4alpha have been shown to be deregulated in RCC microarray studies. These genes (ACY1, WT1, SELENBP1, COBL, EFHD1, AGXT2L1, ALDH5A1, THEM2, ABCB1, FLJ14146, CSPG2, TRIM9 and HEY1) are good candidates for genes whose activity is changed upon the decrease of HNF4alpha in RCC.
Wilms tumor is one of the most frequent neoplasias in children. Our previous microarray screening in a large series of Wilms tumors revealed several candidate genes that are deregulated in advanced tumors and are part of the retinoic acid signaling pathway. To investigate whether retinoic acid could be employed as a novel therapeutic agent in these tumors, we treated cultured Wilms tumor cells with different concentrations of all-trans retinoic acid (ATRA) and assessed gene expression changes by real-time RT-PCR as well as microarray analysis. Several genes like RARRES1, RARRES3, CTGF, CKS2, CCNA2, IGFBP3, UBE2C, CCL2 or ITM2B that were previously found to be deregulated in advanced tumors exhibited opposite expression changes after ATRA treatment. In addition to enhanced retinoid signaling, the transforming growth factor-beta (TGFbeta) pathway was strongly activated by ATRA treatment of Wilms tumor cells. Both the retinoic acid and the TGFbeta pathway mediate inhibition of cell growth. These findings represent the first molecular evidence of a potential benefit from ATRA treatment in Wilms tumors.
Wilms tumor is one of the most frequent neoplasias in children. Our previous microarray screening in a large series of Wilms tumors revealed several candidate genes that are deregulated in advanced tumors and are part of the retinoic acid signaling pathway. To investigate whether retinoic acid could be employed as a novel therapeutic agent in these tumors, we treated cultured Wilms tumor cells with different concentrations of all-trans retinoic acid (ATRA) and assessed gene expression changes by real-time RT-PCR as well as microarray analysis. Several genes like RARRES1, RARRES3, CTGF, CKS2, CCNA2, IGFBP3, UBE2C, CCL2 or ITM2B that were previously found to be deregulated in advanced tumors exhibited opposite expression changes after ATRA treatment. In addition to enhanced retinoid signaling, the transforming growth factor-beta (TGFbeta) pathway was strongly activated by ATRA treatment of Wilms tumor cells. Both the retinoic acid and the TGFbeta pathway mediate inhibition of cell growth. These findings represent the first molecular evidence of a potential benefit from ATRA treatment in Wilms tumors.
Wilms tumor is one of the most frequent neoplasias in children. Our previous microarray screening in a large series of Wilms tumors revealed several candidate genes that are deregulated in advanced tumors and are part of the retinoic acid signaling pathway. To investigate whether retinoic acid could be employed as a novel therapeutic agent in these tumors, we treated cultured Wilms tumor cells with different concentrations of all-trans retinoic acid (ATRA) and assessed gene expression changes by real-time RT-PCR as well as microarray analysis. Several genes like RARRES1, RARRES3, CTGF, CKS2, CCNA2, IGFBP3, UBE2C, CCL2 or ITM2B that were previously found to be deregulated in advanced tumors exhibited opposite expression changes after ATRA treatment. In addition to enhanced retinoid signaling, the transforming growth factor-beta (TGFbeta) pathway was strongly activated by ATRA treatment of Wilms tumor cells. Both the retinoic acid and the TGFbeta pathway mediate inhibition of cell growth. These findings represent the first molecular evidence of a potential benefit from ATRA treatment in Wilms tumors.
Wilms tumor is one of the most frequent neoplasias in children. Our previous microarray screening in a large series of Wilms tumors revealed several candidate genes that are deregulated in advanced tumors and are part of the retinoic acid signaling pathway. To investigate whether retinoic acid could be employed as a novel therapeutic agent in these tumors, we treated cultured Wilms tumor cells with different concentrations of all-trans retinoic acid (ATRA) and assessed gene expression changes by real-time RT-PCR as well as microarray analysis. Several genes like RARRES1, RARRES3, CTGF, CKS2, CCNA2, IGFBP3, UBE2C, CCL2 or ITM2B that were previously found to be deregulated in advanced tumors exhibited opposite expression changes after ATRA treatment. In addition to enhanced retinoid signaling, the transforming growth factor-beta (TGFbeta) pathway was strongly activated by ATRA treatment of Wilms tumor cells. Both the retinoic acid and the TGFbeta pathway mediate inhibition of cell growth. These findings represent the first molecular evidence of a potential benefit from ATRA treatment in Wilms tumors.
Amplification of the 8p11-12 region occurs in 15-20% of breast cancers, but the driving oncogene at this locus has yet to be definitively identified. We mapped the 8p11-12 amplicon in breast cancer cell lines and primary human breast cancers and identified the candidate oncogene human Sm-like protein (hLsm1, LSM1) based on increases in copy number and expression level relative to human mammary epithelial cells. To examine the oncogenic role of LSM1, we overexpressed this gene in MCF10A mammary epithelial cells and inhibited its production in the SUM44 breast cancer cell line, which has a natural amplification and overexpression of LSM1. Our data confirmed that LSM1 is an oncogene from the 8p11-12 amplicon by showing that hLsm1 overexpression induced growth factor-independent proliferation and soft agar colony formation in MCF10A cells, and hLsm1 inhibition in SUM44 cells dramatically reduced soft agar growth. Little is known about hLsm1 function other than its involvement in mRNA degradation; therefore, we used expression microarray analysis to investigate how hLsm1 affects cell transformation in MCF10A and SUM44 cells. We identified numerous genes altered following hLsm1 overexpression common to SUM44 breast cancer cells that play important roles in cell cycle regulation, cell proliferation and other cancer-promoting processes. Future work will continue to characterize these important changes to achieve a more complete understanding of the mechanism of hLsm1's effect on cancer progression.
Amplification of the 8p11-12 region occurs in 15-20% of breast cancers, but the driving oncogene at this locus has yet to be definitively identified. We mapped the 8p11-12 amplicon in breast cancer cell lines and primary human breast cancers and identified the candidate oncogene human Sm-like protein (hLsm1, LSM1) based on increases in copy number and expression level relative to human mammary epithelial cells. To examine the oncogenic role of LSM1, we overexpressed this gene in MCF10A mammary epithelial cells and inhibited its production in the SUM44 breast cancer cell line, which has a natural amplification and overexpression of LSM1. Our data confirmed that LSM1 is an oncogene from the 8p11-12 amplicon by showing that hLsm1 overexpression induced growth factor-independent proliferation and soft agar colony formation in MCF10A cells, and hLsm1 inhibition in SUM44 cells dramatically reduced soft agar growth. Little is known about hLsm1 function other than its involvement in mRNA degradation; therefore, we used expression microarray analysis to investigate how hLsm1 affects cell transformation in MCF10A and SUM44 cells. We identified numerous genes altered following hLsm1 overexpression common to SUM44 breast cancer cells that play important roles in cell cycle regulation, cell proliferation and other cancer-promoting processes. Future work will continue to characterize these important changes to achieve a more complete understanding of the mechanism of hLsm1's effect on cancer progression.
The tyrosine kinase Src is frequently activated in advanced human prostate carcinomas and its activation correlates with tyrosine phosphorylation of the RNA-binding protein Sam68. Herein, we have investigated the expression and function of Sam68 in human prostate cancer cells. Analysis of specimens obtained from 20 patients revealed that Sam68 is upregulated at the protein level in 35% of the samples. Real-time polymerase chain reaction confirmed the results at the mRNA level in most patients. Downregulation of Sam68 by RNAi in LNCaP prostate cancer cells delayed cell cycle progression and reduced the proliferation rate. Moreover, depletion of Sam68 sensitized cells to apoptosis induced by DNA-damaging agents. Similarly, stable cell lines expressing a truncated GFP-Sam68(GSG) protein displayed reduced growth rates and higher sensitivity to cisplatin-induced apoptosis. Microarray analyses revealed that a subset of genes involved in proliferation and apoptosis were altered when Sam68 was knocked down in LNCaP cells. Our results indicate that Sam68 expression supports prostate cancer cells proliferation and survival to cytotoxic agents.
The tyrosine kinase Src is frequently activated in advanced human prostate carcinomas and its activation correlates with tyrosine phosphorylation of the RNA-binding protein Sam68. Herein, we have investigated the expression and function of Sam68 in human prostate cancer cells. Analysis of specimens obtained from 20 patients revealed that Sam68 is upregulated at the protein level in 35% of the samples. Real-time polymerase chain reaction confirmed the results at the mRNA level in most patients. Downregulation of Sam68 by RNAi in LNCaP prostate cancer cells delayed cell cycle progression and reduced the proliferation rate. Moreover, depletion of Sam68 sensitized cells to apoptosis induced by DNA-damaging agents. Similarly, stable cell lines expressing a truncated GFP-Sam68(GSG) protein displayed reduced growth rates and higher sensitivity to cisplatin-induced apoptosis. Microarray analyses revealed that a subset of genes involved in proliferation and apoptosis were altered when Sam68 was knocked down in LNCaP cells. Our results indicate that Sam68 expression supports prostate cancer cells proliferation and survival to cytotoxic agents.
Arsenic trioxide (As(2)O(3)) has shown considerable efficacy in treating hematological malignancies with induction of programmed cell death (PCD) type I, apoptosis. However, the mechanisms underlying the antitumor effect of As(2)O(3) on solid tumors are poorly defined. Previously, we reported that As(2)O(3) induced autophagic cell death (PCD type II) but not apoptosis in human malignant glioma cell lines. The purpose of this study was to elucidate the molecular pathway leading to autophagic cell death. In this study, we demonstrated that the cell death was accompanied by involvement of autophagy-specific marker, microtubule-associated protein light chain 3 (LC3), and damage of mitochondrial membrane integrity, but not by caspase activation. Analysis by cDNA microarray, RT-PCR, and Western blot showed that cell death members of Bcl-2 family, Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) and its homologue BNIP3-like (BNIP3L), were upregulated in As(2)O(3)-induced autophagic cell death. Exogenous expression of BNIP3, but not BNIP3L, induced autophagic cell death in malignant glioma cells without As(2)O(3) treatment. When upregulation of BNIP3 induced by As(2)O(3) was suppressed by a dominant-negative effect of the transmembrane-deleted BNIP3 (BNIP3 Delta TM), autophagic cell death was inhibited. In contrast, BNIP3 transfection augmented As(2)O(3)-induced autophagic cell death. These results suggest that BNIP3 plays a central role in As(2)O(3)-induced autophagic cell death in malignant glioma cells. This study adds a new concept to characterize the pathways by which As(2)O(3) acts to induce autophagic cell death in malignant glioma cells.
Dasatinib is an ATP-competitive, multi-targeted SRC and ABL kinase inhibitor that can bind BCR-ABL in both the active and inactive conformations. From a clinical standpoint, dasatinib is particularly attractive because it has been shown to induce hematologic and cytogenetic responses in imatinib-resistant chronic myeloid leukemia patients. The fact because the combination of imatinib and dasatinib shows the additive/synergistic growth inhibition on wild-type p210 BCR-ABL-expressing cells, we reasoned that these ABL kinase inhibitors might induce the different molecular pathways. To address this question, we used DNA microarrays to identify genes whose transcription was altered by imatinib and dasatinib. K562 cells were cultured with imatinib or dasatinib for 16 h, and gene expression data were obtained from three independent microarray hybridizations. Almost all of the imatinib- and dasatinib-responsive genes appeared to be similarly increased or decreased in K562 cells; however, small subsets of genes were identified as selectively altered expression by either imatinib or dasatinib. The distinct genes that are selectively modulated by dasatinib are cyclin-dependent kinase 2 (CDK2) and CDK8, which had a maximal reduction of <5-fold in microarray screen. To assess the functional importance of dasatinib regulated genes, we used RNA interference to determine whether reduction of CDK2 and CDK8 affected the growth inhibition. K562 and TF-1BCR-ABL cells, pretreated with CDK2 or CDK8 small interfering RNA, showed additive growth inhibition with imatinib, but not with dasatinib. These findings demonstrate that the additive/synergistic growth inhibition by imatinib and dasatinib may be mediated in part by CDK2 and CDK8.
Dasatinib is an ATP-competitive, multi-targeted SRC and ABL kinase inhibitor that can bind BCR-ABL in both the active and inactive conformations. From a clinical standpoint, dasatinib is particularly attractive because it has been shown to induce hematologic and cytogenetic responses in imatinib-resistant chronic myeloid leukemia patients. The fact because the combination of imatinib and dasatinib shows the additive/synergistic growth inhibition on wild-type p210 BCR-ABL-expressing cells, we reasoned that these ABL kinase inhibitors might induce the different molecular pathways. To address this question, we used DNA microarrays to identify genes whose transcription was altered by imatinib and dasatinib. K562 cells were cultured with imatinib or dasatinib for 16 h, and gene expression data were obtained from three independent microarray hybridizations. Almost all of the imatinib- and dasatinib-responsive genes appeared to be similarly increased or decreased in K562 cells; however, small subsets of genes were identified as selectively altered expression by either imatinib or dasatinib. The distinct genes that are selectively modulated by dasatinib are cyclin-dependent kinase 2 (CDK2) and CDK8, which had a maximal reduction of <5-fold in microarray screen. To assess the functional importance of dasatinib regulated genes, we used RNA interference to determine whether reduction of CDK2 and CDK8 affected the growth inhibition. K562 and TF-1BCR-ABL cells, pretreated with CDK2 or CDK8 small interfering RNA, showed additive growth inhibition with imatinib, but not with dasatinib. These findings demonstrate that the additive/synergistic growth inhibition by imatinib and dasatinib may be mediated in part by CDK2 and CDK8.
KIT or alpha-platelet-derived growth factor receptor (alpha-PDGFR) activating mutations are the pathogenic mechanisms that characterize gastrointestinal stromal tumors (GIST). Despite excellent responses to imatinib mesylate (IM), patients are relapsing. We developed an IM-resistant GIST cell line (GIST-R) from the IM-sensitive GIST882 cell line (GIST-S) by growing these cells in IM. Gene expression profiling (GEP) of GIST-S, GIST-R cells and two IM resistant GIST patients demonstrated that KIT is downregulated implying a major role in IM resistance. Instead, GIST-R cells have acquired IM resistance by overexpressing the oncogenic receptor tyrosine kinase - AXL - in a 'kinase switch'. Further, the two IM resistant GIST patients express AXL and not c-Kit, seen by immunohistochemistry (IHC). Real time reverse transcriptase-polymerase chain reaction and Western blotting of the GIST-S and GIST-R cells confirmed the switch from Kit to AXL. In GIST-R, AXL is tyrosine phosphorylated and its ligand growth-arrest-specific gene 6 is overexpressed implying autocrine activation. The kinase switch is associated with a morphological change from spindle to epithelioid. Molecular modeling of the kinase domain of mutant c-Kit (V654A) and AXL showed no binding to IM but efficient binding to MP470, a novel c-Kit/AXL kinase inhibitor. MP470 synergizes with docetaxel (taxotere) and is cytotoxic to GIST cells.
KIT or alpha-platelet-derived growth factor receptor (alpha-PDGFR) activating mutations are the pathogenic mechanisms that characterize gastrointestinal stromal tumors (GIST). Despite excellent responses to imatinib mesylate (IM), patients are relapsing. We developed an IM-resistant GIST cell line (GIST-R) from the IM-sensitive GIST882 cell line (GIST-S) by growing these cells in IM. Gene expression profiling (GEP) of GIST-S, GIST-R cells and two IM resistant GIST patients demonstrated that KIT is downregulated implying a major role in IM resistance. Instead, GIST-R cells have acquired IM resistance by overexpressing the oncogenic receptor tyrosine kinase - AXL - in a 'kinase switch'. Further, the two IM resistant GIST patients express AXL and not c-Kit, seen by immunohistochemistry (IHC). Real time reverse transcriptase-polymerase chain reaction and Western blotting of the GIST-S and GIST-R cells confirmed the switch from Kit to AXL. In GIST-R, AXL is tyrosine phosphorylated and its ligand growth-arrest-specific gene 6 is overexpressed implying autocrine activation. The kinase switch is associated with a morphological change from spindle to epithelioid. Molecular modeling of the kinase domain of mutant c-Kit (V654A) and AXL showed no binding to IM but efficient binding to MP470, a novel c-Kit/AXL kinase inhibitor. MP470 synergizes with docetaxel (taxotere) and is cytotoxic to GIST cells.
Although widespread metastasis is the major cause of human lung cancer-related deaths, its underlying mechanism remains largely unclear. Our genome-wide comparison of the expression profiles of a highly metastatic lung cancer cell line, NCI-H460-LNM35 (LNM35), and its parental clone, NCI-H460-N15 (N15), resulted in the identification of a cancer metastasis signature composed of 45 genes. Through gene ontology analysis, our study also provided insights into how this 45-gene metastasis signature may contribute to the acquisition of metastatic potential. By applying the signature to datasets of human cancer cases, we could demonstrate significant associations with a subset of cases with poor prognosis not only for the two datasets of cancers of the lung but also for cancers of the breast. Furthermore, we were able to show that enforced expression of the DLX4 homeobox gene, which was identified as a gene with significant downregulation in LNM35 as well as with significant association with favorable prognosis for lung cancer patients, markedly inhibited in vitro motility and invasion as well as in vivo metastasis via both hematogenous and lymphogenous routes. Taken together, these findings indicate that our combined transcriptome analysis is an efficient approach in the search for genes possessing both clinical usefulness in terms of prognostic prediction in human cancer cases and clear functional relevance for studying cancer biology in relation to metastasis.
Although widespread metastasis is the major cause of human lung cancer-related deaths, its underlying mechanism remains largely unclear. Our genome-wide comparison of the expression profiles of a highly metastatic lung cancer cell line, NCI-H460-LNM35 (LNM35), and its parental clone, NCI-H460-N15 (N15), resulted in the identification of a cancer metastasis signature composed of 45 genes. Through gene ontology analysis, our study also provided insights into how this 45-gene metastasis signature may contribute to the acquisition of metastatic potential. By applying the signature to datasets of human cancer cases, we could demonstrate significant associations with a subset of cases with poor prognosis not only for the two datasets of cancers of the lung but also for cancers of the breast. Furthermore, we were able to show that enforced expression of the DLX4 homeobox gene, which was identified as a gene with significant downregulation in LNM35 as well as with significant association with favorable prognosis for lung cancer patients, markedly inhibited in vitro motility and invasion as well as in vivo metastasis via both hematogenous and lymphogenous routes. Taken together, these findings indicate that our combined transcriptome analysis is an efficient approach in the search for genes possessing both clinical usefulness in terms of prognostic prediction in human cancer cases and clear functional relevance for studying cancer biology in relation to metastasis.
Metastasis, the aggressive spread of a malignant tumor to distant organs, is a major cause of death in cancer patients. Despite this critical role in cancer outcomes, the molecular mechanisms that control this process are just beginning to be understood. Metastasis is largely dependent upon the ability of tumor cells to invade the barrier formed by the basement membrane and to migrate through neighboring tissues. This review will summarize the evidence that tumor cell invasion is the result of oncogene-mediated signal transduction pathways that control the expression of a specific set of genes that together mediate tumor cell invasion. We focus on the role of the transcription factor AP-1 to both induce the expression of genes that function as invasion effectors and repress other genes that function as invasion suppressors. This identifies AP-1 as a critical regulator of a complex program of gene expression that defines the invasive phenotype.
Successful treatment of multiple cancer types requires early detection and identification of reliable biomarkers present in specific cancer tissues. To test the feasibility of identifying proteins from archival cancer tissues, we have developed a methodology, termed direct tissue proteomics (DTP), which can be used to identify proteins directly from formalin-fixed paraffin-embedded prostate cancer tissue samples. Using minute prostate biopsy sections, we demonstrate the identification of 428 prostate-expressed proteins using the shotgun method. Because the DTP method is not quantitative, we employed the absolute quantification method and demonstrate picogram level quantification of prostate-specific antigen. In depth bioinformatics analysis of these expressed proteins affords the categorization of metabolic pathways that may be important for distinct stages of prostate carcinogenesis. Furthermore, we validate Wnt-3 as an upregulated protein in cancerous prostate cells by immunohistochemistry. We propose that this general strategy provides a roadmap for successful identification of critical molecular targets of multiple cancer types.
Angiogenesis is regarded as essential for tumour growth. However, we have demonstrated that some other aggressive non-small-cell lung carcinomas (n-SCLC) do not have angiogenesis. In this study, using cDNA microarray analysis, we demonstrate that angiogenic and nonangiogenic tumour types can be distinguished by their gene expression profiles. Tissue samples from 42 n-SCLC patients were obtained with consent. In all, 12 tumours were nonangiogenic and 30 angiogenic. The two groups were matched by age, sex, smoking and tumour stage. Total RNAs were extracted followed by microarray hybridization and image scan procedure. Data were analysed using GeneSpring 5.1 software. A total of 62 genes were found to be able to separate angiogenic from nonangiogenic tumours. Nonangiogenic tumours have higher levels of genes concerned with mitochondrial metabolism, mRNA transcription, protein synthesis and the cell cycle. Angiogenic tumours have higher levels of genes coding for membrane vesicles, integrins, remodelling, angiogenesis and apoptosis. These results further support our first finding that nonangiogenic lung tumours are fast-growing tumours filling the alveoli in the absence of vascular remodelling. We raise the hypothesis that in nonangiogenic tumours, hypoxia leads to a higher activation of the mitochondrial respiratory chain, which allows tumour growth without triggering angiogenesis.
Angiogenesis is regarded as essential for tumour growth. However, we have demonstrated that some other aggressive non-small-cell lung carcinomas (n-SCLC) do not have angiogenesis. In this study, using cDNA microarray analysis, we demonstrate that angiogenic and nonangiogenic tumour types can be distinguished by their gene expression profiles. Tissue samples from 42 n-SCLC patients were obtained with consent. In all, 12 tumours were nonangiogenic and 30 angiogenic. The two groups were matched by age, sex, smoking and tumour stage. Total RNAs were extracted followed by microarray hybridization and image scan procedure. Data were analysed using GeneSpring 5.1 software. A total of 62 genes were found to be able to separate angiogenic from nonangiogenic tumours. Nonangiogenic tumours have higher levels of genes concerned with mitochondrial metabolism, mRNA transcription, protein synthesis and the cell cycle. Angiogenic tumours have higher levels of genes coding for membrane vesicles, integrins, remodelling, angiogenesis and apoptosis. These results further support our first finding that nonangiogenic lung tumours are fast-growing tumours filling the alveoli in the absence of vascular remodelling. We raise the hypothesis that in nonangiogenic tumours, hypoxia leads to a higher activation of the mitochondrial respiratory chain, which allows tumour growth without triggering angiogenesis.
The oncogenic fusion protein, Pax3/FKHR, is a more potent transcription factor relative to its normal counterpart, Pax3. Since Pax3 induced a mesenchymal to epithelial transition (MET) in human SaOS-2 osteosarcomas, we hypothesized that Pax3/FKHR would also induce a morphological change in SaOS-2 cells. We demonstrate here that Pax3/FKHR more potently induces a MET in SaOS-2 cells than Pax3. This greater potency was further evident where Pax3/FKHR, but not Pax3, induced a morphological alteration in U2-OS osteosarcoma cells. By microarray analysis, we determined that Pax3/FKHR altered the expression of gene targets in a manner quantitatively and qualitatively distinct from Pax3. Three classes of genes were identified: (i) genes induced or repressed by Pax3 and Pax3/FKHR, (ii) genes induced or repressed by Pax3/FKHR but not Pax3 and (iii) genes induced by Pax3/FKHR but repressed by Pax3. Chromatin immunoprecipitations confirmed the direct binding of Pax3/FKHR to the promoter region of several factors including cannabinoid receptor-1, EPHA2 and EPHA4. Verification of the microarray data also revealed coordinate alteration in the expression of factors involved in BMP4 signalling. Regulation of gene expression by Pax3 and Pax3/FKHR is, however, cell-type specific. BMP4 expression, for example, was repressed by both Pax3 and Pax3/FKHR in SaOS-2 cells, while in the rhabdomyosarcoma, RD, Pax3/FKHR, but not Pax3, induced BMP4 expression. Thus, our data reveal that Pax3/FKHR regulates a distinct but overlapping set of genes relative to Pax3 and that the global set of Pax3 and Pax3/FKHR gene targets is cell-type specific.
The oncogenic fusion protein, Pax3/FKHR, is a more potent transcription factor relative to its normal counterpart, Pax3. Since Pax3 induced a mesenchymal to epithelial transition (MET) in human SaOS-2 osteosarcomas, we hypothesized that Pax3/FKHR would also induce a morphological change in SaOS-2 cells. We demonstrate here that Pax3/FKHR more potently induces a MET in SaOS-2 cells than Pax3. This greater potency was further evident where Pax3/FKHR, but not Pax3, induced a morphological alteration in U2-OS osteosarcoma cells. By microarray analysis, we determined that Pax3/FKHR altered the expression of gene targets in a manner quantitatively and qualitatively distinct from Pax3. Three classes of genes were identified: (i) genes induced or repressed by Pax3 and Pax3/FKHR, (ii) genes induced or repressed by Pax3/FKHR but not Pax3 and (iii) genes induced by Pax3/FKHR but repressed by Pax3. Chromatin immunoprecipitations confirmed the direct binding of Pax3/FKHR to the promoter region of several factors including cannabinoid receptor-1, EPHA2 and EPHA4. Verification of the microarray data also revealed coordinate alteration in the expression of factors involved in BMP4 signalling. Regulation of gene expression by Pax3 and Pax3/FKHR is, however, cell-type specific. BMP4 expression, for example, was repressed by both Pax3 and Pax3/FKHR in SaOS-2 cells, while in the rhabdomyosarcoma, RD, Pax3/FKHR, but not Pax3, induced BMP4 expression. Thus, our data reveal that Pax3/FKHR regulates a distinct but overlapping set of genes relative to Pax3 and that the global set of Pax3 and Pax3/FKHR gene targets is cell-type specific.
Promyelocytic leukemia zinc-finger (PLZF) is a transcriptional repressor and tumor suppressor. PLZF is expressed in melanocytes but not in melanoma cells, and recovery of PLZF expression markedly suppresses melanoma cell growth. Several target genes regulated by PLZF have been identified, but the precise function of PLZF remains uncertain. Here, we searched for candidate target genes of PLZF by DNA microarray analysis. Pre-B-cell leukemia transcription factor 1 (Pbx1) was one of the prominently suppressed genes. Pbx1 was highly expressed in melanoma cells, and its expression was reduced by transduction with the PLZF gene. Moreover, the growth suppression mediated by PLZF was reversed by enforced expression of Pbx1. Knockdown of Pbx1 by specific small interfering RNAs suppressed melanoma cell growth. We also found that Pbx1 binds HoxB7. Reverse transcription-polymerase chain reaction analysis demonstrated that repression of Pbx1 by PLZF reduces the expression of HoxB7 target genes, including tumor-associated neoangiogenesis factors such as basic fibroblast growth factor, angiopoietin-2 and matrix metalloprotease 9. These findings suggest that deregulation of Pbx1 expression owing to loss of PLZF expression contributes to the progression and/or pathogenesis of melanoma.
Promyelocytic leukemia zinc-finger (PLZF) is a transcriptional repressor and tumor suppressor. PLZF is expressed in melanocytes but not in melanoma cells, and recovery of PLZF expression markedly suppresses melanoma cell growth. Several target genes regulated by PLZF have been identified, but the precise function of PLZF remains uncertain. Here, we searched for candidate target genes of PLZF by DNA microarray analysis. Pre-B-cell leukemia transcription factor 1 (Pbx1) was one of the prominently suppressed genes. Pbx1 was highly expressed in melanoma cells, and its expression was reduced by transduction with the PLZF gene. Moreover, the growth suppression mediated by PLZF was reversed by enforced expression of Pbx1. Knockdown of Pbx1 by specific small interfering RNAs suppressed melanoma cell growth. We also found that Pbx1 binds HoxB7. Reverse transcription-polymerase chain reaction analysis demonstrated that repression of Pbx1 by PLZF reduces the expression of HoxB7 target genes, including tumor-associated neoangiogenesis factors such as basic fibroblast growth factor, angiopoietin-2 and matrix metalloprotease 9. These findings suggest that deregulation of Pbx1 expression owing to loss of PLZF expression contributes to the progression and/or pathogenesis of melanoma.
Amplification and overexpression of the E2F3 gene at 6p22 in human bladder cancer is associated with increased tumour stage, grade and proliferation index, and in prostate cancer E2F3 overexpression is linked to tumour aggressiveness. We first used small interfering RNA technology to confirm the potential importance of E2F3 overexpression in bladder cancer development. Knockdown of E2F3 expression in bladder cells containing the 6p22 amplicon strongly reduced the extent of bromodeoxyuridine (BrdU) incorporation and the rate of cellular proliferation. In contrast, knockdown of CDKAL1/FLJ20342, another proposed oncogene, from this amplicon had no effect. Expression cDNA microarray analysis on bladder cancer cells following E2F3 knockdown was then used to identify genes regulated by E2F3, leading to the identification of known E2F3 targets such as Cyclin A and CDC2 and novel targets including pituitary tumour transforming gene 1, Polo-like kinase 1 (PLK1) and Caveolin-2. For both bladder and prostate cancer, we have proposed that E2F3 protein overexpression may cooperate with removal of the E2F inhibitor retinoblastoma tumor suppressor protein (pRB) to drive cellular proliferation. In support of this model, we found that ectopic expression of E2F3a enhanced the BrdU incorporation, a marker of cellular proliferation rate, of prostate cancer DU145 cells, which lack pRB, but had no effect on the proliferation rate of PC3 prostate cancer cells that express wild-type pRB. BrdU incorporation in PC3 cells could, however, be increased by overexpressing E2F3a in cells depleted of pRB. When taken together, these observations indicate that E2F3 levels have a critical role in modifying cellular proliferation rate in human bladder and prostate cancer.
Amplification and overexpression of the E2F3 gene at 6p22 in human bladder cancer is associated with increased tumour stage, grade and proliferation index, and in prostate cancer E2F3 overexpression is linked to tumour aggressiveness. We first used small interfering RNA technology to confirm the potential importance of E2F3 overexpression in bladder cancer development. Knockdown of E2F3 expression in bladder cells containing the 6p22 amplicon strongly reduced the extent of bromodeoxyuridine (BrdU) incorporation and the rate of cellular proliferation. In contrast, knockdown of CDKAL1/FLJ20342, another proposed oncogene, from this amplicon had no effect. Expression cDNA microarray analysis on bladder cancer cells following E2F3 knockdown was then used to identify genes regulated by E2F3, leading to the identification of known E2F3 targets such as Cyclin A and CDC2 and novel targets including pituitary tumour transforming gene 1, Polo-like kinase 1 (PLK1) and Caveolin-2. For both bladder and prostate cancer, we have proposed that E2F3 protein overexpression may cooperate with removal of the E2F inhibitor retinoblastoma tumor suppressor protein (pRB) to drive cellular proliferation. In support of this model, we found that ectopic expression of E2F3a enhanced the BrdU incorporation, a marker of cellular proliferation rate, of prostate cancer DU145 cells, which lack pRB, but had no effect on the proliferation rate of PC3 prostate cancer cells that express wild-type pRB. BrdU incorporation in PC3 cells could, however, be increased by overexpressing E2F3a in cells depleted of pRB. When taken together, these observations indicate that E2F3 levels have a critical role in modifying cellular proliferation rate in human bladder and prostate cancer.
The path to the discovery of suberoylanilide hydroxamic acid (SAHA, vorinostat) began over three decades ago with our studies designed to understand why dimethylsulfoxide causes terminal differentiation of the virus-transformed cells, murine erythroleukemia cells. SAHA can cause growth arrest and death of a broad variety of transformed cells both in vitro and in vivo at concentrations that have little or no toxic effects on normal cells. It was discovered that SAHA inhibits the activity of histone deacetylases (HDACs), including all 11 known human class I and class II HDACs. HDACs have many protein targets whose structure and function are altered by acetylation including histones and non-histone proteins component of transcription factors controlling gene expression and proteins that regulate cell proliferation, migration and death. SAHA is in clinical trials and has significant anticancer activity against both hematologic and solid tumors at doses well tolerated by patients. A new drug application has been approved for SAHA (vorinostat) treatment of cutaneous T-cell lymphoma.
The path to the discovery of suberoylanilide hydroxamic acid (SAHA, vorinostat) began over three decades ago with our studies designed to understand why dimethylsulfoxide causes terminal differentiation of the virus-transformed cells, murine erythroleukemia cells. SAHA can cause growth arrest and death of a broad variety of transformed cells both in vitro and in vivo at concentrations that have little or no toxic effects on normal cells. It was discovered that SAHA inhibits the activity of histone deacetylases (HDACs), including all 11 known human class I and class II HDACs. HDACs have many protein targets whose structure and function are altered by acetylation including histones and non-histone proteins component of transcription factors controlling gene expression and proteins that regulate cell proliferation, migration and death. SAHA is in clinical trials and has significant anticancer activity against both hematologic and solid tumors at doses well tolerated by patients. A new drug application has been approved for SAHA (vorinostat) treatment of cutaneous T-cell lymphoma.
Adult T-cell leukemia (ATL) is an intractable malignancy of CD4+ T cells that is etiologically associated with infection by human T-cell leukemia virus-type I. Most individuals in the chronic stage of ATL eventually undergo progression to a highly aggressive acute stage. To clarify the mechanism responsible for this stage progression, we isolated CD4+ cells from individuals in the chronic (n=19) or acute (n=22) stages of ATL and subjected them to profiling of gene expression with DNA microarrays containing >44,000 probe sets. Changes in chromosome copy number were also examined for 24 cell specimens with the use of microarrays harboring approximately 50,000 probe sets. Stage-dependent changes in gene expression profile and chromosome copy number were apparent. Furthermore, expression of the gene for MET, a receptor tyrosine kinase for hepatocyte growth factor (HGF), was shown to be specific to the acute stage of ATL, and the plasma concentration of HGF was increased in individuals in either the acute or chronic stage. HGF induced proliferation of a MET-positive ATL cell line, and this effect was blocked by antibodies to HGF. The HGF-MET signaling pathway is thus a potential therapeutic target for ATL.
Adult T-cell leukemia (ATL) is an intractable malignancy of CD4+ T cells that is etiologically associated with infection by human T-cell leukemia virus-type I. Most individuals in the chronic stage of ATL eventually undergo progression to a highly aggressive acute stage. To clarify the mechanism responsible for this stage progression, we isolated CD4+ cells from individuals in the chronic (n=19) or acute (n=22) stages of ATL and subjected them to profiling of gene expression with DNA microarrays containing >44,000 probe sets. Changes in chromosome copy number were also examined for 24 cell specimens with the use of microarrays harboring approximately 50,000 probe sets. Stage-dependent changes in gene expression profile and chromosome copy number were apparent. Furthermore, expression of the gene for MET, a receptor tyrosine kinase for hepatocyte growth factor (HGF), was shown to be specific to the acute stage of ATL, and the plasma concentration of HGF was increased in individuals in either the acute or chronic stage. HGF induced proliferation of a MET-positive ATL cell line, and this effect was blocked by antibodies to HGF. The HGF-MET signaling pathway is thus a potential therapeutic target for ATL.
von Hippel-Lindau (VHL) disease is a dominantly inherited family cancer syndrome characterized by the development of retinal and central nervous system haemangioblastomas, renal cell carcinoma (RCC) and phaeochromocytoma. Specific germline VHL mutations may predispose to haemangioblastomas, RCC and phaeochromocytoma to a varying extent. Although dysregulation of the hypoxia-inducible transcription factor-2 and JunB have been linked to the development of RCC and phaeochromocytoma, respectively, the precise basis for genotype-phenotype correlations in VHL disease have not been defined. To gain insights into the pathogenesis of RCC in VHL disease we compared gene expression microarray profiles in a RCC cell line expressing a Type 1 or Type 2B mutant pVHL (RCC-associated) to those of a Type 2A or 2C mutant (not associated with RCC). We identified 19 differentially expressed novel VHL target genes linked to RCC development. Eight targets were studied in detail by quantitative real-time polymerase chain reaction (three downregulated and five upregulated by wild-type VHL) and for six genes the effect of VHL inactivation was mimicked by hypoxia (but hypoxic-induction of smooth muscle alpha-actin 2 was specific for a RCC cell line). The potential role of four RCC-associated VHL target genes was assessed in vitro. NB thymosin beta (TMSNB) and proteinase-activated receptor 2 (PAR2) (both downregulated by wt pVHL) increased cell growth and motility in a RCC cell line, but aldehyde dehydrogenase (ALDH)1 and ALDH7 had no effect. These findings implicate TMSNB and PAR2 candidate oncogenes in the pathogenesis of VHL-associated RCC.
Epigenetic alterations and the resulting inactivation of tumor suppressor genes often contribute to the development of various cancers. To identify novel candidates that may be silenced by aberrant methylation in esophageal squamous-cell carcinoma (ESCC), we analysed ESCC cell lines by a recently developed method known as bacterial artificial chromosome array-based methylated CpG island amplification (BAMCA), and selected candidates through BAMCA-assisted strategy. In the course of this program, we identified frequent CpG methylation-dependent silencing of the gene encoding cellular retinoic acid binding protein 1 (CRABP1) in our panel of ESCC cell lines. Expression of CRABP1 mRNA was restored in gene-silenced ESCC cells after treatment with 5-aza 2'-deoxycytidine. The DNA methylation status of the CRABP1 CpG island with clear promoter activity correlated inversely with expression of this gene. CpG methylation of CRABP1 was frequently observed in primary ESCC tissues as well. Restoration of CRABP1 expression in ESCC cells lacking the protein reduced cell growth by inducing arrest at G(0)-G(1), whereas knockdown of the gene in cells expressing CRABP1 promoted cell growth. Among 113 primary ESCC tumors, the absence of immunoreactive CRABP1 was significantly associated with de-differentiation of cancer cells and with distant lymph-node metastases in the patients. These results indicate that CRABP1 appears to have a tumor-suppressor function in esophageal epithelium, and its epigenetic silencing may play a pivotal role during esophageal carcinogenesis. Its expression status in biopsies or resected tumors might serve as an index for identifying ESCC patients for whom combined therapeutic modalities would be recommended.
As alterations in retinoblastoma (RB)/E2F pathway are commonly found in human cancers, the molecular mechanism underlying cell cycle deregulation caused by the mutations in the RB/E2F pathway needs to be investigated extensively. Compared with good understanding of RB/E2F functions in G1-S cell cycle progression, it is not fully understood how an abrogated RB pathway affects the G2-M phase of the cell cycle. Here, we report that disruption of RB accelerated G2-M progression in the presence of DNA damage by elevating the expression of a set of mitotic regulatory genes. We generated RB(+)- and (-)-matched cells using short hairpin RNA. In the RB(-) cells, the G2/M checkpoint mediated by a DNA-damaging agent was over-ridden. With microarray analysis, we found that the expression of key G2-M regulatory genes was upregulated in RB(-) cells. In particular, we demonstrated that the proto-oncogene ECT2 was directly regulated by E2Fs. Furthermore, suppression of ECT2 expression by small interfering RNA in RB(-) cells resulted in cytokinesis arrest, suggesting that RB(-) cells lack the regulation of E2F-mediated cytokinesis. These results indicate that aberrant ECT2 expression, observed in various human tumors, could be the direct result of RB/E2F pathway deficiency, thereby contributing to cell division in cancers.
Inhibition of the nonsense-mediated decay (NMD) mechanism in cells results in stabilization of transcripts carrying premature translation termination codons. A strategy referred to as gene identification by NMD inhibition (GINI) has been proposed to identify genes carrying nonsense mutations. Genes containing frameshift mutations in colon cancer cell line have been identified using a modified version of GINI. To increase the efficiency of identifying mutant genes using GINI, we have now further improved the strategy. In this approach, inhibition of NMD with emetine is complemented with inhibiting NMD by blocking the phosphorylation of the hUpf1 protein with caffeine. In addition, to enhance the GINI strategy, comparing mRNA level alterations produced by inhibiting transcription alone or inhibiting transcription together with NMD following caffeine pretreatment were used for the efficient identification of false positives produced as a result of stress response to NMD inhibition. To demonstrate the improved efficiency of this approach, we analysed colon cancer cell lines showing microsatellite instability. Bi-allelic inactivating mutations were found in the FXR1, SEC31L1, NCOR1, BAT3, PHF14, ZNF294, C19ORF5 genes as well as genes coding for proteins with yet unknown functions.
The androgen receptor (AR) plays a key role as a transcriptional factor in prostate development and carcinogenesis. Identification of androgen-regulated genes is essential to elucidate the AR pathophysiology in prostate cancer. Here, we identified androgen target genes that are directly regulated by AR in LNCaP cells, by combining chromatin immunoprecipitation (ChIP) with tiling microarrays (ChIP-chip). ChIP-enriched or control DNAs from the cells treated with R1881 were hybridized with the ENCODE array, in which a set of regions representing approximately 1% of the whole genome. We chose 10 bona fide AR-binding sites (ARBSs) (P<1e-5) and validated their significant AR recruitment ligand dependently. Eight upregulated genes by R1881 were identified in the vicinity of the ARBSs. Among the upregulated genes, we focused on UGT1A and CDH2 as AR target genes, because the ARBSs close to these genes (in UGT1A distal promoter and CDH2 intron 1) were most significantly associated with acetylated histone H3/H4, RNA polymerase II and p160 family co-activators. Luciferase reporter constructs including those two ARBSs exhibited ligand-dependent transcriptional regulator/enhancer activities. The present study would be powerful to extend our knowledge of the diversity of androgen genetic network and steroid action in prostate cancer cells.
Aberrant expression of the human homeobox-containing proto-oncogene TLX1/HOX11 inhibits hematopoietic differentiation programs in a number of murine model systems. Here, we report the establishment of a murine erythroid progenitor cell line, iEBHX1S-4, developmentally arrested by regulatable TLX1 expression. Extinction of TLX1 expression released the iEBHX1S-4 differentiation block, allowing erythropoietin-dependent acquisition of erythroid markers and hemoglobin synthesis. Coordinated activation of erythroid transcriptional networks integrated by the acetyltransferase co-activator CREB-binding protein (CBP) was suggested by bioinformatic analysis of the upstream regulatory regions of several conditionally induced iEBHX1S-4 gene sets. In accord with this notion, CBP-associated acetylation of GATA-1, an essential regulator of erythroid differentiation, increased concomitantly with TLX1 downregulation. Coimmunoprecipitation experiments and glutathione-S-transferase pull-down assays revealed that TLX1 directly binds to CBP, and confocal laser microscopy demonstrated that the two proteins partially colocalize at intranuclear sites in iEBHX1S-4 cells. Notably, the distribution of CBP in conditionally blocked iEBHX1S-4 cells partially overlapped with chromatin marked by a repressive histone methylation pattern, and downregulation of TLX1 coincided with exit of CBP from these heterochromatic regions. Thus, we propose that TLX1-mediated differentiation arrest may be achieved in part through a mechanism that involves redirection of CBP and/or its sequestration in repressive chromatin domains.
Aberrant expression of the human homeobox-containing proto-oncogene TLX1/HOX11 inhibits hematopoietic differentiation programs in a number of murine model systems. Here, we report the establishment of a murine erythroid progenitor cell line, iEBHX1S-4, developmentally arrested by regulatable TLX1 expression. Extinction of TLX1 expression released the iEBHX1S-4 differentiation block, allowing erythropoietin-dependent acquisition of erythroid markers and hemoglobin synthesis. Coordinated activation of erythroid transcriptional networks integrated by the acetyltransferase co-activator CREB-binding protein (CBP) was suggested by bioinformatic analysis of the upstream regulatory regions of several conditionally induced iEBHX1S-4 gene sets. In accord with this notion, CBP-associated acetylation of GATA-1, an essential regulator of erythroid differentiation, increased concomitantly with TLX1 downregulation. Coimmunoprecipitation experiments and glutathione-S-transferase pull-down assays revealed that TLX1 directly binds to CBP, and confocal laser microscopy demonstrated that the two proteins partially colocalize at intranuclear sites in iEBHX1S-4 cells. Notably, the distribution of CBP in conditionally blocked iEBHX1S-4 cells partially overlapped with chromatin marked by a repressive histone methylation pattern, and downregulation of TLX1 coincided with exit of CBP from these heterochromatic regions. Thus, we propose that TLX1-mediated differentiation arrest may be achieved in part through a mechanism that involves redirection of CBP and/or its sequestration in repressive chromatin domains.
Aberrant expression of the human homeobox-containing proto-oncogene TLX1/HOX11 inhibits hematopoietic differentiation programs in a number of murine model systems. Here, we report the establishment of a murine erythroid progenitor cell line, iEBHX1S-4, developmentally arrested by regulatable TLX1 expression. Extinction of TLX1 expression released the iEBHX1S-4 differentiation block, allowing erythropoietin-dependent acquisition of erythroid markers and hemoglobin synthesis. Coordinated activation of erythroid transcriptional networks integrated by the acetyltransferase co-activator CREB-binding protein (CBP) was suggested by bioinformatic analysis of the upstream regulatory regions of several conditionally induced iEBHX1S-4 gene sets. In accord with this notion, CBP-associated acetylation of GATA-1, an essential regulator of erythroid differentiation, increased concomitantly with TLX1 downregulation. Coimmunoprecipitation experiments and glutathione-S-transferase pull-down assays revealed that TLX1 directly binds to CBP, and confocal laser microscopy demonstrated that the two proteins partially colocalize at intranuclear sites in iEBHX1S-4 cells. Notably, the distribution of CBP in conditionally blocked iEBHX1S-4 cells partially overlapped with chromatin marked by a repressive histone methylation pattern, and downregulation of TLX1 coincided with exit of CBP from these heterochromatic regions. Thus, we propose that TLX1-mediated differentiation arrest may be achieved in part through a mechanism that involves redirection of CBP and/or its sequestration in repressive chromatin domains.
P27kip is a key inhibitory protein of the cell-cycle progression, which is rapidly downregulated in early G1 phase by a post-translational mechanism involving the proteosomal degradation. In this study, using a wounding model that induces cell-cycle entry of human dermal fibroblasts, we demonstrate that p27mRNA is downregulated when cells progress into the G1 phase, and then it returns to its basal level when cells approach the S phase. By using a quantitative polymerase chain reaction screening we identified inhibitors of differentiation (Id3), a bHLH transcriptional repressor, as a candidate mediator accounting for p27 mRNA decrease. Id3 silencing, using an small interfering RNA approach, reversed the injury mediated p27 downregulation demonstrating that Id3 is involved in the transcriptional repression of p27. Reporter gene experiments and a chromatin immunoprecipitation assay showed that Id3 likely exerts its repressive action through ELK1 inhibition. By inhibiting early p27 downregulation, Id3 depletion blocked (i) the G1-phase progression as assessed by the inhibition of pRb phosphorylation and p130 degradation and (ii) the G1/S transition as observed by the inhibition of cyclin A induction, demonstrating that p27 mRNA decrease is required for cell proliferation. Apart from its effect on the early p27 diminution, Id3 appears also involved in the control of the steady-state level of p27 at the G1/S boundary. In conclusion, this study identifies a novel mechanism of p27 regulation which besides p27 protein degradation also implicates a transcriptional mechanism mediated by Id3.
Glioblastoma, the most aggressive and least treatable form of malignant glioma, is the most common human brain tumor. Although many regions of allelic loss occur in glioblastomas, relatively few tumor suppressor genes have been found mutated at such loci. To address the possibility that epigenetic alterations are an alternative means of glioblastoma gene inactivation, we coupled pharmacological manipulation of methylation with gene profiling to identify potential methylation-regulated, tumor-related genes. Duplicates of three short-term cultured glioblastomas were exposed to 5 microM 5-aza-dC for 96 h followed by cRNA hybridization to an oligonucleotide microarray (Affymetrix U133A). We based candidate gene selection on bioinformatics, reverse transcription-polymerase chain reaction (RT-PCR), bisulfite sequencing, methylation-specific PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Two genes identified in this manner, RUNX3 and Testin (TES), were subsequently shown to harbor frequent tumor-specific epigenetic alterations in primary glioblastomas. This overall approach therefore provides a powerful means to identify candidate tumor-suppressor genes for subsequent evaluation and may lead to the identification of genes whose epigenetic dysregulation is integral to glioblastoma tumorigenesis.
Aberrant expression of the human homeobox-containing proto-oncogene TLX1/HOX11 inhibits hematopoietic differentiation programs in a number of murine model systems. Here, we report the establishment of a murine erythroid progenitor cell line, iEBHX1S-4, developmentally arrested by regulatable TLX1 expression. Extinction of TLX1 expression released the iEBHX1S-4 differentiation block, allowing erythropoietin-dependent acquisition of erythroid markers and hemoglobin synthesis. Coordinated activation of erythroid transcriptional networks integrated by the acetyltransferase co-activator CREB-binding protein (CBP) was suggested by bioinformatic analysis of the upstream regulatory regions of several conditionally induced iEBHX1S-4 gene sets. In accord with this notion, CBP-associated acetylation of GATA-1, an essential regulator of erythroid differentiation, increased concomitantly with TLX1 downregulation. Coimmunoprecipitation experiments and glutathione-S-transferase pull-down assays revealed that TLX1 directly binds to CBP, and confocal laser microscopy demonstrated that the two proteins partially colocalize at intranuclear sites in iEBHX1S-4 cells. Notably, the distribution of CBP in conditionally blocked iEBHX1S-4 cells partially overlapped with chromatin marked by a repressive histone methylation pattern, and downregulation of TLX1 coincided with exit of CBP from these heterochromatic regions. Thus, we propose that TLX1-mediated differentiation arrest may be achieved in part through a mechanism that involves redirection of CBP and/or its sequestration in repressive chromatin domains.
Aberrant expression of the human homeobox-containing proto-oncogene TLX1/HOX11 inhibits hematopoietic differentiation programs in a number of murine model systems. Here, we report the establishment of a murine erythroid progenitor cell line, iEBHX1S-4, developmentally arrested by regulatable TLX1 expression. Extinction of TLX1 expression released the iEBHX1S-4 differentiation block, allowing erythropoietin-dependent acquisition of erythroid markers and hemoglobin synthesis. Coordinated activation of erythroid transcriptional networks integrated by the acetyltransferase co-activator CREB-binding protein (CBP) was suggested by bioinformatic analysis of the upstream regulatory regions of several conditionally induced iEBHX1S-4 gene sets. In accord with this notion, CBP-associated acetylation of GATA-1, an essential regulator of erythroid differentiation, increased concomitantly with TLX1 downregulation. Coimmunoprecipitation experiments and glutathione-S-transferase pull-down assays revealed that TLX1 directly binds to CBP, and confocal laser microscopy demonstrated that the two proteins partially colocalize at intranuclear sites in iEBHX1S-4 cells. Notably, the distribution of CBP in conditionally blocked iEBHX1S-4 cells partially overlapped with chromatin marked by a repressive histone methylation pattern, and downregulation of TLX1 coincided with exit of CBP from these heterochromatic regions. Thus, we propose that TLX1-mediated differentiation arrest may be achieved in part through a mechanism that involves redirection of CBP and/or its sequestration in repressive chromatin domains.
Aberrant expression of the human homeobox-containing proto-oncogene TLX1/HOX11 inhibits hematopoietic differentiation programs in a number of murine model systems. Here, we report the establishment of a murine erythroid progenitor cell line, iEBHX1S-4, developmentally arrested by regulatable TLX1 expression. Extinction of TLX1 expression released the iEBHX1S-4 differentiation block, allowing erythropoietin-dependent acquisition of erythroid markers and hemoglobin synthesis. Coordinated activation of erythroid transcriptional networks integrated by the acetyltransferase co-activator CREB-binding protein (CBP) was suggested by bioinformatic analysis of the upstream regulatory regions of several conditionally induced iEBHX1S-4 gene sets. In accord with this notion, CBP-associated acetylation of GATA-1, an essential regulator of erythroid differentiation, increased concomitantly with TLX1 downregulation. Coimmunoprecipitation experiments and glutathione-S-transferase pull-down assays revealed that TLX1 directly binds to CBP, and confocal laser microscopy demonstrated that the two proteins partially colocalize at intranuclear sites in iEBHX1S-4 cells. Notably, the distribution of CBP in conditionally blocked iEBHX1S-4 cells partially overlapped with chromatin marked by a repressive histone methylation pattern, and downregulation of TLX1 coincided with exit of CBP from these heterochromatic regions. Thus, we propose that TLX1-mediated differentiation arrest may be achieved in part through a mechanism that involves redirection of CBP and/or its sequestration in repressive chromatin domains.
Aberrant expression of the human homeobox-containing proto-oncogene TLX1/HOX11 inhibits hematopoietic differentiation programs in a number of murine model systems. Here, we report the establishment of a murine erythroid progenitor cell line, iEBHX1S-4, developmentally arrested by regulatable TLX1 expression. Extinction of TLX1 expression released the iEBHX1S-4 differentiation block, allowing erythropoietin-dependent acquisition of erythroid markers and hemoglobin synthesis. Coordinated activation of erythroid transcriptional networks integrated by the acetyltransferase co-activator CREB-binding protein (CBP) was suggested by bioinformatic analysis of the upstream regulatory regions of several conditionally induced iEBHX1S-4 gene sets. In accord with this notion, CBP-associated acetylation of GATA-1, an essential regulator of erythroid differentiation, increased concomitantly with TLX1 downregulation. Coimmunoprecipitation experiments and glutathione-S-transferase pull-down assays revealed that TLX1 directly binds to CBP, and confocal laser microscopy demonstrated that the two proteins partially colocalize at intranuclear sites in iEBHX1S-4 cells. Notably, the distribution of CBP in conditionally blocked iEBHX1S-4 cells partially overlapped with chromatin marked by a repressive histone methylation pattern, and downregulation of TLX1 coincided with exit of CBP from these heterochromatic regions. Thus, we propose that TLX1-mediated differentiation arrest may be achieved in part through a mechanism that involves redirection of CBP and/or its sequestration in repressive chromatin domains.
The chromosomal translocation t(2;13), characteristic for the aggressive childhood cancer alveolar rhabdomyosarcoma (aRMS), generates the chimeric transcription factor PAX3/FKHR with a well known oncogenic role. However, the molecular mechanisms mediating essential pathophysiological functions remain poorly defined. Here, we used comparative expression profiling of PAX3/FKHR silencing in vitro and PAX3/FKHR-specific gene signatures in vivo to identify physiologically important target genes. Hereby, 51 activated genes, both novel and known, were identified. We also found repression of skeletal muscle-specific genes suggesting that PAX3/FKHR blocks further differentiation of aRMS cells. Importantly, TFAP2B was validated as direct target gene mediating the anti-apoptotic function of PAX3/FKHR. Hence, we developed a pathophysiologically relevant transcriptional profile of PAX3/FKHR and identified a critical target gene for aRMS development.
p53 and p63 belong to a family of sequence-specific transcription factors regulating key cellular processes. Differential composition of the p53 and p63 DNA-binding sites may contribute to distinct functions of these protein homologues. We used SELEX (systematic evolution of ligands by exponential enrichment) methodology to identify nucleic acid ligands for p63. We found that p63 bound preferentially to DNA fragments conforming to the 20 bp sequence 5'-RRRC(A/G)(A/T)GYYYRRRC(A/T)(C/T)GYYY-3'. Relative to the p53 consensus, the p63 consensus DNA-binding site (DBS) was more degenerate, particularly at positions 10 and 11, and was enriched for A/G at position 5 and C/T at position 16 of the consensus. The differences in DNA-binding site preferences between p63 and p53 influenced their ability to activate transcription from select response elements (REs) in cells. A computer algorithm, p63MH, was developed to find candidate p63-binding motifs on input sequences. We identified genes responsive to p63 regulation that contain functional p63 REs. Our results suggest that the sequence composition of REs could be one contributing factor to target gene discrimination between p63 and p53.
p53 and p63 belong to a family of sequence-specific transcription factors regulating key cellular processes. Differential composition of the p53 and p63 DNA-binding sites may contribute to distinct functions of these protein homologues. We used SELEX (systematic evolution of ligands by exponential enrichment) methodology to identify nucleic acid ligands for p63. We found that p63 bound preferentially to DNA fragments conforming to the 20 bp sequence 5'-RRRC(A/G)(A/T)GYYYRRRC(A/T)(C/T)GYYY-3'. Relative to the p53 consensus, the p63 consensus DNA-binding site (DBS) was more degenerate, particularly at positions 10 and 11, and was enriched for A/G at position 5 and C/T at position 16 of the consensus. The differences in DNA-binding site preferences between p63 and p53 influenced their ability to activate transcription from select response elements (REs) in cells. A computer algorithm, p63MH, was developed to find candidate p63-binding motifs on input sequences. We identified genes responsive to p63 regulation that contain functional p63 REs. Our results suggest that the sequence composition of REs could be one contributing factor to target gene discrimination between p63 and p53.
p53 and p63 belong to a family of sequence-specific transcription factors regulating key cellular processes. Differential composition of the p53 and p63 DNA-binding sites may contribute to distinct functions of these protein homologues. We used SELEX (systematic evolution of ligands by exponential enrichment) methodology to identify nucleic acid ligands for p63. We found that p63 bound preferentially to DNA fragments conforming to the 20 bp sequence 5'-RRRC(A/G)(A/T)GYYYRRRC(A/T)(C/T)GYYY-3'. Relative to the p53 consensus, the p63 consensus DNA-binding site (DBS) was more degenerate, particularly at positions 10 and 11, and was enriched for A/G at position 5 and C/T at position 16 of the consensus. The differences in DNA-binding site preferences between p63 and p53 influenced their ability to activate transcription from select response elements (REs) in cells. A computer algorithm, p63MH, was developed to find candidate p63-binding motifs on input sequences. We identified genes responsive to p63 regulation that contain functional p63 REs. Our results suggest that the sequence composition of REs could be one contributing factor to target gene discrimination between p63 and p53.
Zinc-finger protein 143 (ZNF143) is a human homolog of Xenopus transcriptional activator staf that is involved in selenocystyl tRNA transcription. We previously showed that ZNF143 expression is induced by treatment with DNA-damaging agents and that it preferentially binds to cisplatin-modified DNA. In this study, the potential function of ZNF143 was investigated. ZNF143 was overexpressed in cisplatin-resistant cells. ZNF143 knockdown in prostate cancer caused increased sensitivity for cisplatin, but not for oxaliplatin, etoposide and vincristine. We also showed that ZNF143 is associated with tumor suppressor gene product p73 but not with p53. p73 could stimulate the binding of ZNF143 to both ZNF143 binding site and cisplatin-modified DNA, and modulate the function of ZNF143. We provide a direct evidence that both Rad51 and flap endonuclease-1 are target genes of ZNF143 and overexpressed in cisplatin-resistant cells. Taken together, these experiments demonstrate that an interplay of ZNF143, p73 and ZNF143 target genes is involved in DNA repair gene expression and cisplatin resistance.
Deleted in colon cancer (DCC) and UNC5 function as netrin dependence receptors by inducing apoptosis in the absence of their ligand and accordingly were recently designated as putative conditional tumor suppressors. Herein, we determined whether netrin-1 and its receptors are implicated in cancer cell invasion and tumor progression. Expression of DCC, UNC5 and adenosine A2B-receptors (A2B-Rs) was investigated by reverse transcription polymerase chain reaction in human colon cancer cells. The impact of DCC restitution and netrin-1 was evaluated on collagen type I invasion, tumor growth and metastasis in nude mice, cancer cell survival and gene expression profiling. Flow cytometry, poly(ADP-ribose)polymerase-1 and caspase-8 activation were used to evaluate the impact of DCC on cell death. Both netrin-1 and A2B-R activation induced the invasive phenotype through the Rho-Rho kinase axis in DCC-deficient human colorectal cancer cells. Restitution of wild-type DCC blocked invasion induced by netrin-1, A2B-R agonist and other agents. Ectopic expression of netrin-1 led to increased growth of human colon tumor xenografts in athymic mice. Conversely, introduction of wt-DCC in kidney MDCKts.src-ggl cells strongly inhibited metastasis in lymph nodes and lungs and increased sensitivity to apoptosis in hypoxia. DNA microarrays revealed that netrin and DCC had common and divergent impacts on gene expression linked to cell cycle, survival, surface signaling and adhesion. Our findings underscore that netrin is a potent invasion and tumor growth-promoting agent and that DCC is a metastasis suppressor gene targeting both proinvasive and survival pathways in a cumulative manner.
Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) syndromes are characterized by deficiency in nucleotide excision repair pathway, but with distinguished clinical manifestations. While XP patients exhibit a high frequency of skin cancer, TTD patients are not cancer prone. The relation between lack of DNA repair and their clinical manifestations was investigated through analysis of the transcriptional profile of 12,600 transcripts in two isogenic cell lines with different capabilities of DNA repair. These cell lines result from a stable transfection of the XPB-TTD allele into XP complementation group B fibroblasts, from an XP patient who also have clinical abnormalities corresponding to Cockayne's syndrome (CS). The microarray assays performed under normal growth conditions showed the expression of distinct groups of genes in each cell line. The UVC-transcription modulation of these cells revealed the changes in 869 transcripts. Some of these transcripts had similar modulation pattern in both cells, although with eventually different time patterns for induction or repression. However, some different 'UVC signature' for each cell line was also found, that is, transcripts that were specifically UV regulated depending on the DNA repair status of the cell. These results provide a detailed portrait of expression profiles that may potentially unravel the causes of the different phenotypes of XP/CS and TTD patients.
Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) syndromes are characterized by deficiency in nucleotide excision repair pathway, but with distinguished clinical manifestations. While XP patients exhibit a high frequency of skin cancer, TTD patients are not cancer prone. The relation between lack of DNA repair and their clinical manifestations was investigated through analysis of the transcriptional profile of 12,600 transcripts in two isogenic cell lines with different capabilities of DNA repair. These cell lines result from a stable transfection of the XPB-TTD allele into XP complementation group B fibroblasts, from an XP patient who also have clinical abnormalities corresponding to Cockayne's syndrome (CS). The microarray assays performed under normal growth conditions showed the expression of distinct groups of genes in each cell line. The UVC-transcription modulation of these cells revealed the changes in 869 transcripts. Some of these transcripts had similar modulation pattern in both cells, although with eventually different time patterns for induction or repression. However, some different 'UVC signature' for each cell line was also found, that is, transcripts that were specifically UV regulated depending on the DNA repair status of the cell. These results provide a detailed portrait of expression profiles that may potentially unravel the causes of the different phenotypes of XP/CS and TTD patients.
Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) syndromes are characterized by deficiency in nucleotide excision repair pathway, but with distinguished clinical manifestations. While XP patients exhibit a high frequency of skin cancer, TTD patients are not cancer prone. The relation between lack of DNA repair and their clinical manifestations was investigated through analysis of the transcriptional profile of 12,600 transcripts in two isogenic cell lines with different capabilities of DNA repair. These cell lines result from a stable transfection of the XPB-TTD allele into XP complementation group B fibroblasts, from an XP patient who also have clinical abnormalities corresponding to Cockayne's syndrome (CS). The microarray assays performed under normal growth conditions showed the expression of distinct groups of genes in each cell line. The UVC-transcription modulation of these cells revealed the changes in 869 transcripts. Some of these transcripts had similar modulation pattern in both cells, although with eventually different time patterns for induction or repression. However, some different 'UVC signature' for each cell line was also found, that is, transcripts that were specifically UV regulated depending on the DNA repair status of the cell. These results provide a detailed portrait of expression profiles that may potentially unravel the causes of the different phenotypes of XP/CS and TTD patients.
Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) syndromes are characterized by deficiency in nucleotide excision repair pathway, but with distinguished clinical manifestations. While XP patients exhibit a high frequency of skin cancer, TTD patients are not cancer prone. The relation between lack of DNA repair and their clinical manifestations was investigated through analysis of the transcriptional profile of 12,600 transcripts in two isogenic cell lines with different capabilities of DNA repair. These cell lines result from a stable transfection of the XPB-TTD allele into XP complementation group B fibroblasts, from an XP patient who also have clinical abnormalities corresponding to Cockayne's syndrome (CS). The microarray assays performed under normal growth conditions showed the expression of distinct groups of genes in each cell line. The UVC-transcription modulation of these cells revealed the changes in 869 transcripts. Some of these transcripts had similar modulation pattern in both cells, although with eventually different time patterns for induction or repression. However, some different 'UVC signature' for each cell line was also found, that is, transcripts that were specifically UV regulated depending on the DNA repair status of the cell. These results provide a detailed portrait of expression profiles that may potentially unravel the causes of the different phenotypes of XP/CS and TTD patients.
The mitogen-activated protein kinases (MAPKs) are a family of serine/threonine kinases that play an essential role in signal transduction by modulating gene transcription in the nucleus in response to changes in the cellular environment. They include the extracellular signal-regulated protein kinases (ERK1 and ERK2); c-Jun N-terminal kinases (JNK1, JNK2, JNK3); p38s (p38alpha, p38beta, p38gamma, p38delta) and ERK5. The molecular events in which MAPKs function can be separated in discrete and yet interrelated steps: activation of the MAPK by their upstream kinases, changes in the subcellular localization of MAPKs, and recognition, binding and phosphorylation of MAPK downstream targets. The resulting pattern of gene expression will ultimately depend on the integration of the combinatorial signals provided by the temporal activation of each group of MAPKs. This review will focus on how the specificity of signal transmission by MAPKs is achieved by scaffolding molecules and by the presence of structural motifs in MAPKs that are dynamically regulated by phosphorylation and protein-protein interactions. We discuss also how MAPKs recognize and phosphorylate their target nuclear proteins, including transcription factors, co-activators and repressors and chromatin-remodeling molecules, thereby affecting an intricate balance of nuclear regulatory molecules that ultimately control gene expression in response to environmental cues.
The mitogen-activated protein kinases (MAPKs) are a family of serine/threonine kinases that play an essential role in signal transduction by modulating gene transcription in the nucleus in response to changes in the cellular environment. They include the extracellular signal-regulated protein kinases (ERK1 and ERK2); c-Jun N-terminal kinases (JNK1, JNK2, JNK3); p38s (p38alpha, p38beta, p38gamma, p38delta) and ERK5. The molecular events in which MAPKs function can be separated in discrete and yet interrelated steps: activation of the MAPK by their upstream kinases, changes in the subcellular localization of MAPKs, and recognition, binding and phosphorylation of MAPK downstream targets. The resulting pattern of gene expression will ultimately depend on the integration of the combinatorial signals provided by the temporal activation of each group of MAPKs. This review will focus on how the specificity of signal transmission by MAPKs is achieved by scaffolding molecules and by the presence of structural motifs in MAPKs that are dynamically regulated by phosphorylation and protein-protein interactions. We discuss also how MAPKs recognize and phosphorylate their target nuclear proteins, including transcription factors, co-activators and repressors and chromatin-remodeling molecules, thereby affecting an intricate balance of nuclear regulatory molecules that ultimately control gene expression in response to environmental cues.
The mitogen-activated protein kinases (MAPKs) are a family of serine/threonine kinases that play an essential role in signal transduction by modulating gene transcription in the nucleus in response to changes in the cellular environment. They include the extracellular signal-regulated protein kinases (ERK1 and ERK2); c-Jun N-terminal kinases (JNK1, JNK2, JNK3); p38s (p38alpha, p38beta, p38gamma, p38delta) and ERK5. The molecular events in which MAPKs function can be separated in discrete and yet interrelated steps: activation of the MAPK by their upstream kinases, changes in the subcellular localization of MAPKs, and recognition, binding and phosphorylation of MAPK downstream targets. The resulting pattern of gene expression will ultimately depend on the integration of the combinatorial signals provided by the temporal activation of each group of MAPKs. This review will focus on how the specificity of signal transmission by MAPKs is achieved by scaffolding molecules and by the presence of structural motifs in MAPKs that are dynamically regulated by phosphorylation and protein-protein interactions. We discuss also how MAPKs recognize and phosphorylate their target nuclear proteins, including transcription factors, co-activators and repressors and chromatin-remodeling molecules, thereby affecting an intricate balance of nuclear regulatory molecules that ultimately control gene expression in response to environmental cues.
The mitogen-activated protein kinases (MAPKs) are a family of serine/threonine kinases that play an essential role in signal transduction by modulating gene transcription in the nucleus in response to changes in the cellular environment. They include the extracellular signal-regulated protein kinases (ERK1 and ERK2); c-Jun N-terminal kinases (JNK1, JNK2, JNK3); p38s (p38alpha, p38beta, p38gamma, p38delta) and ERK5. The molecular events in which MAPKs function can be separated in discrete and yet interrelated steps: activation of the MAPK by their upstream kinases, changes in the subcellular localization of MAPKs, and recognition, binding and phosphorylation of MAPK downstream targets. The resulting pattern of gene expression will ultimately depend on the integration of the combinatorial signals provided by the temporal activation of each group of MAPKs. This review will focus on how the specificity of signal transmission by MAPKs is achieved by scaffolding molecules and by the presence of structural motifs in MAPKs that are dynamically regulated by phosphorylation and protein-protein interactions. We discuss also how MAPKs recognize and phosphorylate their target nuclear proteins, including transcription factors, co-activators and repressors and chromatin-remodeling molecules, thereby affecting an intricate balance of nuclear regulatory molecules that ultimately control gene expression in response to environmental cues.
The mitogen-activated protein kinases (MAPKs) are a family of serine/threonine kinases that play an essential role in signal transduction by modulating gene transcription in the nucleus in response to changes in the cellular environment. They include the extracellular signal-regulated protein kinases (ERK1 and ERK2); c-Jun N-terminal kinases (JNK1, JNK2, JNK3); p38s (p38alpha, p38beta, p38gamma, p38delta) and ERK5. The molecular events in which MAPKs function can be separated in discrete and yet interrelated steps: activation of the MAPK by their upstream kinases, changes in the subcellular localization of MAPKs, and recognition, binding and phosphorylation of MAPK downstream targets. The resulting pattern of gene expression will ultimately depend on the integration of the combinatorial signals provided by the temporal activation of each group of MAPKs. This review will focus on how the specificity of signal transmission by MAPKs is achieved by scaffolding molecules and by the presence of structural motifs in MAPKs that are dynamically regulated by phosphorylation and protein-protein interactions. We discuss also how MAPKs recognize and phosphorylate their target nuclear proteins, including transcription factors, co-activators and repressors and chromatin-remodeling molecules, thereby affecting an intricate balance of nuclear regulatory molecules that ultimately control gene expression in response to environmental cues.
KIT or alpha-platelet-derived growth factor receptor (alpha-PDGFR) activating mutations are the pathogenic mechanisms that characterize gastrointestinal stromal tumors (GIST). Despite excellent responses to imatinib mesylate (IM), patients are relapsing. We developed an IM-resistant GIST cell line (GIST-R) from the IM-sensitive GIST882 cell line (GIST-S) by growing these cells in IM. Gene expression profiling (GEP) of GIST-S, GIST-R cells and two IM resistant GIST patients demonstrated that KIT is downregulated implying a major role in IM resistance. Instead, GIST-R cells have acquired IM resistance by overexpressing the oncogenic receptor tyrosine kinase - AXL - in a 'kinase switch'. Further, the two IM resistant GIST patients express AXL and not c-Kit, seen by immunohistochemistry (IHC). Real time reverse transcriptase-polymerase chain reaction and Western blotting of the GIST-S and GIST-R cells confirmed the switch from Kit to AXL. In GIST-R, AXL is tyrosine phosphorylated and its ligand growth-arrest-specific gene 6 is overexpressed implying autocrine activation. The kinase switch is associated with a morphological change from spindle to epithelioid. Molecular modeling of the kinase domain of mutant c-Kit (V654A) and AXL showed no binding to IM but efficient binding to MP470, a novel c-Kit/AXL kinase inhibitor. MP470 synergizes with docetaxel (taxotere) and is cytotoxic to GIST cells.
KIT or alpha-platelet-derived growth factor receptor (alpha-PDGFR) activating mutations are the pathogenic mechanisms that characterize gastrointestinal stromal tumors (GIST). Despite excellent responses to imatinib mesylate (IM), patients are relapsing. We developed an IM-resistant GIST cell line (GIST-R) from the IM-sensitive GIST882 cell line (GIST-S) by growing these cells in IM. Gene expression profiling (GEP) of GIST-S, GIST-R cells and two IM resistant GIST patients demonstrated that KIT is downregulated implying a major role in IM resistance. Instead, GIST-R cells have acquired IM resistance by overexpressing the oncogenic receptor tyrosine kinase - AXL - in a 'kinase switch'. Further, the two IM resistant GIST patients express AXL and not c-Kit, seen by immunohistochemistry (IHC). Real time reverse transcriptase-polymerase chain reaction and Western blotting of the GIST-S and GIST-R cells confirmed the switch from Kit to AXL. In GIST-R, AXL is tyrosine phosphorylated and its ligand growth-arrest-specific gene 6 is overexpressed implying autocrine activation. The kinase switch is associated with a morphological change from spindle to epithelioid. Molecular modeling of the kinase domain of mutant c-Kit (V654A) and AXL showed no binding to IM but efficient binding to MP470, a novel c-Kit/AXL kinase inhibitor. MP470 synergizes with docetaxel (taxotere) and is cytotoxic to GIST cells.
KIT or alpha-platelet-derived growth factor receptor (alpha-PDGFR) activating mutations are the pathogenic mechanisms that characterize gastrointestinal stromal tumors (GIST). Despite excellent responses to imatinib mesylate (IM), patients are relapsing. We developed an IM-resistant GIST cell line (GIST-R) from the IM-sensitive GIST882 cell line (GIST-S) by growing these cells in IM. Gene expression profiling (GEP) of GIST-S, GIST-R cells and two IM resistant GIST patients demonstrated that KIT is downregulated implying a major role in IM resistance. Instead, GIST-R cells have acquired IM resistance by overexpressing the oncogenic receptor tyrosine kinase - AXL - in a 'kinase switch'. Further, the two IM resistant GIST patients express AXL and not c-Kit, seen by immunohistochemistry (IHC). Real time reverse transcriptase-polymerase chain reaction and Western blotting of the GIST-S and GIST-R cells confirmed the switch from Kit to AXL. In GIST-R, AXL is tyrosine phosphorylated and its ligand growth-arrest-specific gene 6 is overexpressed implying autocrine activation. The kinase switch is associated with a morphological change from spindle to epithelioid. Molecular modeling of the kinase domain of mutant c-Kit (V654A) and AXL showed no binding to IM but efficient binding to MP470, a novel c-Kit/AXL kinase inhibitor. MP470 synergizes with docetaxel (taxotere) and is cytotoxic to GIST cells.
Fenretinide (4-HPR) is a synthetic retinoid with antitumor activity, which induces apoptosis in cancer cell lines of different histotypes. To identify genes contributing to its apoptotic activity in ovarian cancer cells, we monitored, by cDNA arrays, gene expression changes after 4-HPR exposure in A2780, a human ovarian carcinoma cell line sensitive to the retinoid. Among the differentially expressed transcripts, PLAcental Bone morphogenetic protein (PLAB), a proapoptotic gene, was the most highly induced. In a panel of ovarian carcinoma cell lines with different 4-HPR sensitivities, PLAB upregulation was associated with cellular response to 4-HPR, its overexpression increased basal apoptosis and its silencing by small interfering RNA decreased the ability of 4-HPR to induce apoptosis. PLAB induction by 4-HPR was p53- and EGR-1 independent and was regulated, at least in part, by increased stability of PLAB mRNA. PLAB up-modulation by 4-HPR also occurred in vivo: in ascitic cells collected from patients with ovarian cancer before and after 4-HPR treatment, PLAB was upmodulated in 2/4 patients. Our results in certain ovarian cancer cell lines indicate a role for PLAB as a mediator of 4-HPR-induced apoptosis. The correlation of increased PLAB in vivo with antitumor activity remains to be established.
Fenretinide (4-HPR) is a synthetic retinoid with antitumor activity, which induces apoptosis in cancer cell lines of different histotypes. To identify genes contributing to its apoptotic activity in ovarian cancer cells, we monitored, by cDNA arrays, gene expression changes after 4-HPR exposure in A2780, a human ovarian carcinoma cell line sensitive to the retinoid. Among the differentially expressed transcripts, PLAcental Bone morphogenetic protein (PLAB), a proapoptotic gene, was the most highly induced. In a panel of ovarian carcinoma cell lines with different 4-HPR sensitivities, PLAB upregulation was associated with cellular response to 4-HPR, its overexpression increased basal apoptosis and its silencing by small interfering RNA decreased the ability of 4-HPR to induce apoptosis. PLAB induction by 4-HPR was p53- and EGR-1 independent and was regulated, at least in part, by increased stability of PLAB mRNA. PLAB up-modulation by 4-HPR also occurred in vivo: in ascitic cells collected from patients with ovarian cancer before and after 4-HPR treatment, PLAB was upmodulated in 2/4 patients. Our results in certain ovarian cancer cell lines indicate a role for PLAB as a mediator of 4-HPR-induced apoptosis. The correlation of increased PLAB in vivo with antitumor activity remains to be established.
Transcription in eukaryotes is a tightly regulated, multistep process. Gene-specific transcriptional activators, several different co-activators and general transcription factors are necessary to access specific loci to allow precise initiation of RNA polymerase II transcription. As the dense chromatin folding of the genome does not allow the access of these sites by the huge multiprotein transcription machinery, remodelling is required to loosen up the chromatin structure for successful transcription initiation. In the present review, we summarize the recent evolution of our understanding of the function of two histone acetyl transferases (ATs) from metazoan organisms: GCN5 and PCAF. Their overall structure and the multiprotein complexes in which they are carrying out their activities are discussed. Metazoan GCN5 and PCAF are subunits of at least two types of multiprotein complexes, one having a molecular weight of 2 MDa (SPT3-TAF9-GCN5 acetyl transferase/TATA binding protein (TBP)-free-TAF complex/PCAF complexes) and a second type with about a size of 700 kDa (ATAC complex). These complexes possess global histone acetylation activity and locus-specific co-activator functions together with AT activity on non-histone substrates. Thus, their biological functions cover a wide range of tasks and render them indispensable for the normal function of cells. That deregulation of the global and/or specific AT activities of these complexes leads to the cancerous transformation of the cells highlights their importance in cellular processes. The possible effects of GCN5 and PCAF in tumorigenesis are also discussed.
Transcription in eukaryotes is a tightly regulated, multistep process. Gene-specific transcriptional activators, several different co-activators and general transcription factors are necessary to access specific loci to allow precise initiation of RNA polymerase II transcription. As the dense chromatin folding of the genome does not allow the access of these sites by the huge multiprotein transcription machinery, remodelling is required to loosen up the chromatin structure for successful transcription initiation. In the present review, we summarize the recent evolution of our understanding of the function of two histone acetyl transferases (ATs) from metazoan organisms: GCN5 and PCAF. Their overall structure and the multiprotein complexes in which they are carrying out their activities are discussed. Metazoan GCN5 and PCAF are subunits of at least two types of multiprotein complexes, one having a molecular weight of 2 MDa (SPT3-TAF9-GCN5 acetyl transferase/TATA binding protein (TBP)-free-TAF complex/PCAF complexes) and a second type with about a size of 700 kDa (ATAC complex). These complexes possess global histone acetylation activity and locus-specific co-activator functions together with AT activity on non-histone substrates. Thus, their biological functions cover a wide range of tasks and render them indispensable for the normal function of cells. That deregulation of the global and/or specific AT activities of these complexes leads to the cancerous transformation of the cells highlights their importance in cellular processes. The possible effects of GCN5 and PCAF in tumorigenesis are also discussed.
Transcription in eukaryotes is a tightly regulated, multistep process. Gene-specific transcriptional activators, several different co-activators and general transcription factors are necessary to access specific loci to allow precise initiation of RNA polymerase II transcription. As the dense chromatin folding of the genome does not allow the access of these sites by the huge multiprotein transcription machinery, remodelling is required to loosen up the chromatin structure for successful transcription initiation. In the present review, we summarize the recent evolution of our understanding of the function of two histone acetyl transferases (ATs) from metazoan organisms: GCN5 and PCAF. Their overall structure and the multiprotein complexes in which they are carrying out their activities are discussed. Metazoan GCN5 and PCAF are subunits of at least two types of multiprotein complexes, one having a molecular weight of 2 MDa (SPT3-TAF9-GCN5 acetyl transferase/TATA binding protein (TBP)-free-TAF complex/PCAF complexes) and a second type with about a size of 700 kDa (ATAC complex). These complexes possess global histone acetylation activity and locus-specific co-activator functions together with AT activity on non-histone substrates. Thus, their biological functions cover a wide range of tasks and render them indispensable for the normal function of cells. That deregulation of the global and/or specific AT activities of these complexes leads to the cancerous transformation of the cells highlights their importance in cellular processes. The possible effects of GCN5 and PCAF in tumorigenesis are also discussed.
Both Ki-ras mutation and hepatocyte growth factor (HGF) receptor Met overexpression occur at high frequency in colon cancer. This study investigates the transcriptional changes induced by Ki-ras oncogene and HGF/Met signaling activation in colon cancer cell lines in vitro and in vivo. The model system used in these studies included the DLD-1 colon cancer cell line with a mutated Ki-ras allele, and the DKO-4 cell line generated from DLD-1, with its mutant Ki-ras allele inactivated by targeted disruption. These cell lines were transduced with cDNAs of full-length Met receptor. Microarray transcriptional profiling was conducted on cell lines stimulated with HGF, as well as on tumor xenograft tissues. Overlapping genes between in vitro and in vivo microarray data sets were selected as a subset of HGF/Met and Ki-ras oncogene-regulated targets. Using the Online Predicted Human Interaction Database, novel HGF/Met and Ki-ras regulated proteins with putative functional linkage were identified. Novel proteins identified included histone acetyltransferase 1, phosphoribosyl pyrophosphate synthetase 2, chaperonin containing TCP1, subunit 8, CSE1 chromosome segregation 1-like (yeast)/cellular apoptosis susceptibility (mammals), CCR4-NOT transcription complex, subunit 8, and cyclin H. Transcript levels for these Met-signaling targets were correlated with Met expression levels, and were significantly elevated in both primary and metastatic human colorectal cancer samples compared to normal colorectal mucosa. These genes represent novel Met and/or Ki-ras transcriptionally coregulated genes with a high degree of validation in human colorectal cancers.
Gastric cancer (GC) is one of the most common malignancies worldwide. Genes expressed only in cancer tissue will be useful molecular markers for diagnosis and may also be good therapeutic targets. However, little is known about cancer-specific genes, at least in GC. In this study, we searched for GC-specific genes by serial analysis of gene expression (SAGE) data analysis and quantitative reverse transcription (RT)-PCR. Comparing GC SAGE libraries with those of various normal tissues in the SAGEmap database, we identified 54 candidate GC-specific genes. Quantitative RT-PCR analysis of these candidates revealed that APin protein (APIN), taxol resistance-associated gene 3 (TRAG3), cytochrome P450, family 2, subfamily W, polypeptide 1 (CYP2W1), melanoma inhibitory activity (MIA), matrix metalloproteinase-10 (MMP-10), dickkopf homolog 4 (DKK4), GW112, regenerating islet-derived family, member 4 (REGIV), and HORMA domain-containing 1 (HORMAD1) were expressed much more highly in GC than in 14 kinds of normal tissues. Immunohistochemical staining for MIA, MMP-10, and DKK4 was found in 47 (31.1%), 68 (45.0%), and two (1.3%) of 151 GCs, respectively, and staining for both MIA and MMP-10 was correlated with poor prognosis in advanced GC (P=0.0001 and 0.0141, respectively). Moreover, enzyme-linked immunosorbent assay showed high levels of MMP-10 (65/69, 94.2%) in serum samples from patients with GC. Levels of MIA were raised in a small proportion of serum samples from patients with GC (4/69, 5.8%). In Boyden chamber invasion assays, MIA-transfected GC cells were up to three times more invasive than cells transfected with empty vector. Taken together, these results suggest that MMP-10 is a good marker for the detection of GC and that MIA and MMP-10 are prognostic factors for GC. As expression of MIA and MMP-10 is narrowly restricted in cancer, these two molecules may be good therapeutic targets for GC.
The HDM2 oncoprotein is a cellular inhibitor of p53 and is frequently deregulated in human cancer. However, the HDM2 gene encodes alternatively spliced variants whose functional significance is poorly understood. We had previously reported the detection of alternative HDM2 forms in Hodgkin's lymphoma (HL)-derived cell lines. Here, we have cloned several of these transcripts, including the previously described HDM2-A, -B and -C (which encode the COOH terminus of HDM2), and two novel variants (HDM2-HL1 and -HL2) containing a complete p53 interaction domain. Real-time PCR assays demonstrated that HDM2-A and -B were selectively expressed by HL cell lines and primary tumors, compared with their non-neoplastic counterparts. In transient transfection experiments, alternatively spliced HDM2 isoforms were partially or totally localized within the cytoplasm. HDM2-HL2 was able to inhibit transactivation of a p53-inducible reporter construct and induced a partial relocalization of p53 to the cytoplasm. Expression of HDM2-A and -B caused the activation of p53/p21 and induced growth arrest in primary cells, but also increased the expression levels of cyclins D1 and E. Other possible genes regulated by HDM2-A and -B were identified using cDNA microarray technology. These results imply that HDM2 isoforms may have multiple effects on cell cycle control, and provide insight into the mechanisms through which these molecules contribute to tumorigenesis.
Dysregulation of pleiotropic growth factors, receptors and their downstream signaling pathway components represent a central protumorigenic principle in human hepatocarcinogenesis. Especially the Insulin-like Growth Factor/IGF-1 receptor (IGF/IGF-1R), Hepatocyte Growth Factor (HGF/MET), Wingless (Wnt/beta-catenin/FZD), Transforming Growth Factor alpha/Epidermal Growth Factor receptor (TGFalpha/EGFR) and Transforming Growth Factor beta (TGFbeta/TbetaR) pathways contribute to proliferation, antiapoptosis and invasive behavior of tumor cells. This review focuses on the relevant alterations in these pathways identified in human human hepatocellular carcinomas (HCCs). Resultant functional effects are modulated by multiple cross-talks between the different signaling pathways and additional tumor-relevant factors, such as cyclooxygenase-2 and p53. Several specific strategies are currently under development such as receptor kinase inhibitors, neutralizing antibodies and antagonistic proteins, which may improve the systemic treatment of human HCCs.
To identify tumor markers and differentiation markers for lung adenocarcinoma (AdC), we analysed expression profiles of 14,500 genes against three cases of type II alveolar epithelial cells, bronchiolar epithelial cells, and bronchial epithelial cells, respectively, and 10 cases of AdC cells isolated by laser capture microdissection. Hierarchical clustering analysis indicated that AdC cells and noncancerous lung epithelial cells are significantly different in their expression profiles, and that different sets of differentiation markers are expressed among alveolar, bronchiolar and bronchial epithelial cells. Nine genes were identified as being highly expressed in AdC cells, but not expressed in noncancerous lung epithelial cells. Sixteen genes were identified as differentiation markers for lung epithelial cells. Real-time RT-PCR analysis of 45 lung AdC cases further revealed that expression of four tumor markers in AdC cells was significantly higher than that in noncancerous lung cells and that expression of ten differentiation markers was retained in a considerable fraction of lung AdC cases. Five tumor markers and seven differentiation markers were not expressed in peripheral blood cells. Similarities and differences in expression profiles between normal epithelial cells from different lung respiratory compartments and AdC cells demonstrated in this study will be informative for the molecular diagnosis of lung AdC.
Mitochondria are vital for cellular bioenergetics and play a central role in determining the point-of-no-return of the apoptotic process. As a consequence, mitochondria exert a dual function in carcinogenesis. Cancer-associated changes in cellular metabolism (the Warburg effect) influence mitochondrial function, and the invalidation of apoptosis is linked to an inhibition of mitochondrial outer membrane permeabilization (MOMP). On theoretical grounds, it is tempting to develop specific therapeutic interventions that target the mitochondrial Achilles' heel, rendering cancer cells metabolically unviable or subverting endogenous MOMP inhibitors. A variety of experimental therapeutic agents can directly target mitochondria, causing apoptosis induction. This applies to a heterogeneous collection of chemically unrelated compounds including positively charged alpha-helical peptides, agents designed to mimic the Bcl-2 homology domain 3 of Bcl-2-like proteins, ampholytic cations, metals and steroid-like compounds. Such MOMP inducers or facilitators can induce apoptosis by themselves (monotherapy) or facilitate apoptosis induction in combination therapies, bypassing chemoresistance against DNA-damaging agents. In addition, it is possible to design molecules that neutralize inhibitor of apoptosis proteins (IAPs) or heat shock protein 70 (HSP70). Such IAP or HSP70 inhibitors can mimic the action of mitochondrion-derived mediators (Smac/DIABLO, that is, second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with a low isoelectric point, in the case of IAPs; AIF, that is apoptosis-inducing factor, in the case of HSP70) and exert potent chemosensitizing effects.
High-grade gliomas, including glioblastomas, are malignant brain tumors for which improved treatment is urgently needed. Genetic studies have demonstrated the existence of biologically distinct subsets. Preliminary studies have indicated that platelet-derived growth factor (PDGF) receptor signaling contributes to the growth of some of these tumors. In this study, human high-grade glioma primary cultures were analysed for sensitivity to treatment with the PDGF receptor inhibitor imatinib/Glivec/Gleevec/STI571. Six out of 15 cultures displayed more than 40% growth inhibition after imatinib treatment, whereas seven cultures showed less than 20% growth inhibition. In the sensitive cultures, apoptosis contributed to growth inhibition. Platelet-derived growth factor receptor status correlated with imatinib sensitivity. Supervised analyses of gene expression profiles and real-time PCR analyses identified expression of the chemokine CXCL12/SDF-1 (stromal cell-derived factor 1) as a predictor of imatinib sensitivity. Exogenous addition of CXCL12 to imatinib-insensitive cultures conferred some imatinib sensitivity. Finally, coregulation of CXCL12 and PDGF alpha-receptor was observed in glioblastoma biopsies. We have thus defined the characteristics of a novel imatinib-sensitive subset of glioma cultures, and provided evidence for a functional relationship between imatinib sensitivity and chemokine signaling. These findings will assist in the design and evaluation of clinical trials exploring therapeutic effects of imatinib on malignant brain tumors.
Fanconi anaemia (FA) is a rare recessive disorder associated with chromosomal fragility, aplastic anaemia, congenital abnormalities and a high risk of cancer, including acute myeloid leukaemia and squamous cell carcinomas. The identification of 11 different FA genes has revealed a complex web of interacting proteins that are involved in the recognition or repair of DNA interstrand crosslinks and perhaps other forms of DNA damage. Bi-allelic mutations in BRCA2 are associated with a rare and highly cancer-prone form of FA, and the DNA helicase BRIP1 (formerly BACH1) is mutated in FA group J. There is little convincing evidence that FA heterozygotes are at increased risk of cancer, but larger studies are needed to address the possibility of modest risk effects. Somatic inactivation of the FA pathway by mutation or epigenetic silencing has been observed in several different types of sporadic cancer, and this may have important implications for targeted chemotherapy. Inhibition of this pathway represents a possible route to sensitization of tumours to DNA crosslinking drugs such as cisplatin.
Genomic amplification of oncogenes and inactivation of suppressor genes are critical in the pathogenesis of human cancer. To identify chromosomal alterations associated with hepatocarcinogenesis, we performed allelic gene dosage analysis on 36 hepatocellular carcinomas (HCCs). Data from high-density single-nucleotide polymorphism arrays were analysed using the Genome Imbalance Map (GIM) algorithm, which simultaneously detects DNA copy number alterations and loss of heterozygosity (LOH) events. Genome Imbalance Map analysis identified allelic imbalance regions, including uniparental disomy, and predicted the coexistence of a heterozygous population of cancer cells. We observed that gains of 1q, 5p, 5q, 6p, 7q, 8q, 17q and 20q, and LOH of 1p, 4q, 6q, 8p, 10q, 13q, 16p, 16q and 17p were significantly associated with HCC. On 6q24-25, which contains imprinting gene clusters, we observed reduced levels of PLAGL1 expression owing to loss of the unmethylated allele. Finally, we integrated the copy number data with gene expression intensity, and found that genome dosage is correlated with alteration in gene expression. These observations indicated that high-resolution GIM analysis can accurately determine the localizations of genomic regions with allelic imbalance, and when integrated with epigenetic information, a mechanistic basis for inactivation of a tumor suppressor gene in HCC was elucidated.
This article serves as an introduction to the collection of reviews on nuclear factor-kappaB (NF-kappaB). It provides an overview of the discovery and current status of NF-kappaB as a research topic. Described are the structures, activities and regulation of the proteins in the NF-kappaB family of transcription factors. NF-kappaB signaling is primarily regulated by inhibitor kappaB (IkappaB) proteins and the IkappaB kinase complex through two major pathways: the canonical and non-canonical NF-kappaB pathways. The organization and focus of articles included in the following reviews are described, as well as likely future areas of research interest on NF-kappaB.
Transcription factors of the NF-kappaB family regulate hundreds of genes in the context of multiple important physiological and pathological processes. NF-kappaB activation depends on phosphorylation-induced proteolysis of inhibitory IkappaB molecules and NF-kappaB precursors by the ubiquitin-proteasome system. Most of the diverse signaling pathways that activate NF-kappaB converge on IkappaB kinases (IKK), which are essential for signal transmission. Many important details of the composition, regulation and biological function of IKK have been revealed in the last years. This review summarizes current aspects of structure and function of the regular stoichiometric components, the regulatory transient protein interactions of IKK and the mechanisms that contribute to its activation, deactivation and homeostasis. Both phosphorylation and ubiquitinatin (destructive as well as non-destructive) are crucial post-translational events in these processes. In addition to controlling induced IkappaB degradation in the cytoplasm and processing of the NF-kappaB precursor p100, nuclear IKK components have been found to act directly at the chromatin level of induced genes and to mediate responses to DNA damage. Finally, IKK is engaged in cross talk with other pathways and confers functions independently of NF-kappaB.
The nuclear factor-kappaB (NF-kappaB) transcription factors have emerged as major regulators of programmed cell death (PCD) whether via apoptosis or necrosis. In this context, NF-kappaB's activity has important ramifications for normal tissue development, homoeostasis and the physiological functions of various cell systems including the immune, hepatic, epidermal and nervous systems. However, improper regulation of PCD by NF-kappaB can have severe pathologic consequences, ranging from neurodegeneration to cancer, where its activity often precludes effective therapy. Although NF-kappaB generally protects cells by inducing the expression genes encoding antiapoptotic and antioxidizing proteins, its role in apoptosis and necrosis can vary markedly in different cell contexts, and NF-kappaB can sensitize cells to death-inducing stimuli in some instances. This article describes our current knowledge of the role of NF-kappaB in apoptosis and necrosis, and focuses on the many advances since we last reviewed this rapidly evolving topic in Oncogene 3 years ago. There has been substantial progress in understanding NF-kappaB's mode of action in apoptosis and necrosis and the mechanisms that regulate its anti- vs proapoptotic activities. These recent developments shed new light on the role of NF-kappaB in many disease conditions including tumor development, tumor progression and anticancer treatment.
A variety of studies have shown that some activated nuclear receptors (NRs), especially the glucorticoid receptor, the estrogen receptor and peroxisome proliferator-activated receptor, can inhibit the activity of the transcription factor nuclear factor kappaB (NF-kappaB), which plays a key role in the control of genes involved in inflammation, cell proliferation and apoptosis. This review describes the molecular mechanisms of cross-talk between NRs and NF-kappaB and the biological relevance of this cross-talk. The importance and mechanistic aspects of selective NR modulation are discussed. Also included are future research prospects, which will lead to a new era in the field of NR research with the aim of specifically inhibiting NF-kappaB-driven gene expression for anti-inflammatory, anti-tumor and immune-modulatory purposes.
Both Ki-ras mutation and hepatocyte growth factor (HGF) receptor Met overexpression occur at high frequency in colon cancer. This study investigates the transcriptional changes induced by Ki-ras oncogene and HGF/Met signaling activation in colon cancer cell lines in vitro and in vivo. The model system used in these studies included the DLD-1 colon cancer cell line with a mutated Ki-ras allele, and the DKO-4 cell line generated from DLD-1, with its mutant Ki-ras allele inactivated by targeted disruption. These cell lines were transduced with cDNAs of full-length Met receptor. Microarray transcriptional profiling was conducted on cell lines stimulated with HGF, as well as on tumor xenograft tissues. Overlapping genes between in vitro and in vivo microarray data sets were selected as a subset of HGF/Met and Ki-ras oncogene-regulated targets. Using the Online Predicted Human Interaction Database, novel HGF/Met and Ki-ras regulated proteins with putative functional linkage were identified. Novel proteins identified included histone acetyltransferase 1, phosphoribosyl pyrophosphate synthetase 2, chaperonin containing TCP1, subunit 8, CSE1 chromosome segregation 1-like (yeast)/cellular apoptosis susceptibility (mammals), CCR4-NOT transcription complex, subunit 8, and cyclin H. Transcript levels for these Met-signaling targets were correlated with Met expression levels, and were significantly elevated in both primary and metastatic human colorectal cancer samples compared to normal colorectal mucosa. These genes represent novel Met and/or Ki-ras transcriptionally coregulated genes with a high degree of validation in human colorectal cancers.
The Wnt/beta-catenin signaling pathway is activated in many human hepatocellular carcinomas (HCC). We tried to identify the genes involved in carcinogenesis and progression of HCC with beta-catenin mutations. We used PCR-based subtractive hybridization to compare gene expression between malignant and benign components of a human HCC occurring in pre-existing adenoma activated for beta-catenin. Two of the genes identified belong to the Regenerating gene (REG) family. They encode the Regenerating islet-derived 3 alpha (REG3A/HIP/PAP/REG-III) and 1 alpha (REG1A) proteins, both involved in liver and pancreatic regeneration and proliferation. Using siRNA directed against beta-catenin, we demonstrated that REG3A is a target of beta-catenin signaling in Huh7 hepatoma cells. The upregulation of REG3A and REG1A expression is significantly correlated to the beta-catenin status in 42 HCC and 28 hepatoblastomas characterized for their beta-catenin status. Thus, we report strong evidence that both genes are downstream targets of the Wnt pathway during liver tumorigenesis.
Pre-B lymphoblastic leukemia cells carrying a BCR-ABL1 gene rearrangement exhibit an undifferentiated phenotype. Comparing the genome-wide gene expression profiles of normal B-cell subsets and BCR-ABL1+ pre-B lymphoblastic leukemia cells by SAGE, the leukemia cells show loss of B lymphoid identity and aberrant expression of myeloid lineage-specific molecules. Consistent with this, BCR-ABL1+ pre-B lymphoblastic leukemia cells exhibit defective expression of IKAROS, a transcription factor needed for early lymphoid lineage commitment. As shown by inducible expression of BCR-ABL1 in human and murine B-cell precursor cell lines, BCR-ABL1 induces the expression of a dominant-negative IKAROS splice variant, termed IK6. Comparing matched leukemia sample pairs from patients before and during therapy with the BCR-ABL1 kinase inhibitor STI571 (Imatinib), inhibition of BCR-ABL1 partially corrected aberrant expression of IK6 and lineage infidelity of the leukemia cells. To elucidate the contribution of IK6 to lineage infidelity in BCR-ABL1+ cell lines, IK6 expression was silenced by RNA interference. Upon inhibition of IK6, BCR-ABL1+ leukemia cells partially restored B lymphoid lineage commitment. Therefore, we propose that BCR-ABL1 induces aberrant splicing of IKAROS, which interferes with lineage identity and differentiation of pre-B lymphoblastic leukemia cells.
The ATM protein kinase, functionally missing in patients with the human genetic disorder ataxia-telangiectasia, is a master regulator of the cellular network induced by DNA double-strand breaks. The ATM gene is also frequently mutated in sporadic cancers of lymphoid origin. Here, we applied a functional genomics approach that combined gene expression profiling and computational promoter analysis to obtain global dissection of the transcriptional response to ionizing radiation in murine lymphoid tissue. Cluster analysis revealed a prominent pattern characterizing dozens of genes whose response to irradiation was Atm-dependent. Computational analysis identified significant enrichment of the binding site signatures of NF-kappaB and p53 among promoters of these genes, pointing to the major role of these two transcription factors in mediating the Atm-dependent transcriptional response in the irradiated lymphoid tissue. Examination of the response showed that pro- and antiapoptotic signals were simultaneously induced, with the proapoptotic pathway mediated by p53 targets, and the prosurvival pathway by NF-kappaB targets. These findings further elucidate the molecular network induced by IR, point to novel putative NF-kappaB targets, and suggest a mechanistic model for cellular balancing between pro- and antiapoptotic signals induced by IR in lymphoid tissues, which has implications for cancer management. The emerging model suggests that restoring the p53-mediated apoptotic arm while blocking the NF-kappaB-mediated prosurvival arm could effectively increase the radiosensitivity of lymphoid tumors.
The ATM protein kinase, functionally missing in patients with the human genetic disorder ataxia-telangiectasia, is a master regulator of the cellular network induced by DNA double-strand breaks. The ATM gene is also frequently mutated in sporadic cancers of lymphoid origin. Here, we applied a functional genomics approach that combined gene expression profiling and computational promoter analysis to obtain global dissection of the transcriptional response to ionizing radiation in murine lymphoid tissue. Cluster analysis revealed a prominent pattern characterizing dozens of genes whose response to irradiation was Atm-dependent. Computational analysis identified significant enrichment of the binding site signatures of NF-kappaB and p53 among promoters of these genes, pointing to the major role of these two transcription factors in mediating the Atm-dependent transcriptional response in the irradiated lymphoid tissue. Examination of the response showed that pro- and antiapoptotic signals were simultaneously induced, with the proapoptotic pathway mediated by p53 targets, and the prosurvival pathway by NF-kappaB targets. These findings further elucidate the molecular network induced by IR, point to novel putative NF-kappaB targets, and suggest a mechanistic model for cellular balancing between pro- and antiapoptotic signals induced by IR in lymphoid tissues, which has implications for cancer management. The emerging model suggests that restoring the p53-mediated apoptotic arm while blocking the NF-kappaB-mediated prosurvival arm could effectively increase the radiosensitivity of lymphoid tumors.
The ATM protein kinase, functionally missing in patients with the human genetic disorder ataxia-telangiectasia, is a master regulator of the cellular network induced by DNA double-strand breaks. The ATM gene is also frequently mutated in sporadic cancers of lymphoid origin. Here, we applied a functional genomics approach that combined gene expression profiling and computational promoter analysis to obtain global dissection of the transcriptional response to ionizing radiation in murine lymphoid tissue. Cluster analysis revealed a prominent pattern characterizing dozens of genes whose response to irradiation was Atm-dependent. Computational analysis identified significant enrichment of the binding site signatures of NF-kappaB and p53 among promoters of these genes, pointing to the major role of these two transcription factors in mediating the Atm-dependent transcriptional response in the irradiated lymphoid tissue. Examination of the response showed that pro- and antiapoptotic signals were simultaneously induced, with the proapoptotic pathway mediated by p53 targets, and the prosurvival pathway by NF-kappaB targets. These findings further elucidate the molecular network induced by IR, point to novel putative NF-kappaB targets, and suggest a mechanistic model for cellular balancing between pro- and antiapoptotic signals induced by IR in lymphoid tissues, which has implications for cancer management. The emerging model suggests that restoring the p53-mediated apoptotic arm while blocking the NF-kappaB-mediated prosurvival arm could effectively increase the radiosensitivity of lymphoid tumors.
The ATM protein kinase, functionally missing in patients with the human genetic disorder ataxia-telangiectasia, is a master regulator of the cellular network induced by DNA double-strand breaks. The ATM gene is also frequently mutated in sporadic cancers of lymphoid origin. Here, we applied a functional genomics approach that combined gene expression profiling and computational promoter analysis to obtain global dissection of the transcriptional response to ionizing radiation in murine lymphoid tissue. Cluster analysis revealed a prominent pattern characterizing dozens of genes whose response to irradiation was Atm-dependent. Computational analysis identified significant enrichment of the binding site signatures of NF-kappaB and p53 among promoters of these genes, pointing to the major role of these two transcription factors in mediating the Atm-dependent transcriptional response in the irradiated lymphoid tissue. Examination of the response showed that pro- and antiapoptotic signals were simultaneously induced, with the proapoptotic pathway mediated by p53 targets, and the prosurvival pathway by NF-kappaB targets. These findings further elucidate the molecular network induced by IR, point to novel putative NF-kappaB targets, and suggest a mechanistic model for cellular balancing between pro- and antiapoptotic signals induced by IR in lymphoid tissues, which has implications for cancer management. The emerging model suggests that restoring the p53-mediated apoptotic arm while blocking the NF-kappaB-mediated prosurvival arm could effectively increase the radiosensitivity of lymphoid tumors.
The ATM protein kinase, functionally missing in patients with the human genetic disorder ataxia-telangiectasia, is a master regulator of the cellular network induced by DNA double-strand breaks. The ATM gene is also frequently mutated in sporadic cancers of lymphoid origin. Here, we applied a functional genomics approach that combined gene expression profiling and computational promoter analysis to obtain global dissection of the transcriptional response to ionizing radiation in murine lymphoid tissue. Cluster analysis revealed a prominent pattern characterizing dozens of genes whose response to irradiation was Atm-dependent. Computational analysis identified significant enrichment of the binding site signatures of NF-kappaB and p53 among promoters of these genes, pointing to the major role of these two transcription factors in mediating the Atm-dependent transcriptional response in the irradiated lymphoid tissue. Examination of the response showed that pro- and antiapoptotic signals were simultaneously induced, with the proapoptotic pathway mediated by p53 targets, and the prosurvival pathway by NF-kappaB targets. These findings further elucidate the molecular network induced by IR, point to novel putative NF-kappaB targets, and suggest a mechanistic model for cellular balancing between pro- and antiapoptotic signals induced by IR in lymphoid tissues, which has implications for cancer management. The emerging model suggests that restoring the p53-mediated apoptotic arm while blocking the NF-kappaB-mediated prosurvival arm could effectively increase the radiosensitivity of lymphoid tumors.
The ATM protein kinase, functionally missing in patients with the human genetic disorder ataxia-telangiectasia, is a master regulator of the cellular network induced by DNA double-strand breaks. The ATM gene is also frequently mutated in sporadic cancers of lymphoid origin. Here, we applied a functional genomics approach that combined gene expression profiling and computational promoter analysis to obtain global dissection of the transcriptional response to ionizing radiation in murine lymphoid tissue. Cluster analysis revealed a prominent pattern characterizing dozens of genes whose response to irradiation was Atm-dependent. Computational analysis identified significant enrichment of the binding site signatures of NF-kappaB and p53 among promoters of these genes, pointing to the major role of these two transcription factors in mediating the Atm-dependent transcriptional response in the irradiated lymphoid tissue. Examination of the response showed that pro- and antiapoptotic signals were simultaneously induced, with the proapoptotic pathway mediated by p53 targets, and the prosurvival pathway by NF-kappaB targets. These findings further elucidate the molecular network induced by IR, point to novel putative NF-kappaB targets, and suggest a mechanistic model for cellular balancing between pro- and antiapoptotic signals induced by IR in lymphoid tissues, which has implications for cancer management. The emerging model suggests that restoring the p53-mediated apoptotic arm while blocking the NF-kappaB-mediated prosurvival arm could effectively increase the radiosensitivity of lymphoid tumors.
Epithelial to mesenchymal transition (EMT) is implicated in the progression of primary tumours towards metastasis and is likely caused by a pathological activation of transcription factors regulating EMT in embryonic development. To analyse EMT-causing pathways in tumourigenesis, we identified transcriptional targets of the E-cadherin repressor ZEB1 in invasive human cancer cells. We show that ZEB1 repressed multiple key determinants of epithelial differentiation and cell-cell adhesion, including the cell polarity genes Crumbs3, HUGL2 and Pals1-associated tight junction protein. ZEB1 associated with their endogenous promoters in vivo, and strongly repressed promotor activities in reporter assays. ZEB1 downregulation in undifferentiated cancer cells by RNA interference was sufficient to upregulate expression of these cell polarity genes on the RNA and protein level, to re-establish epithelial features and to impair cell motility in vitro. In human colorectal cancer, ZEB1 expression was limited to the tumour-host interface and was accompanied by loss of intercellular adhesion and tumour cell invasion. In invasive ductal and lobular breast cancer, upregulation of ZEB1 was stringently coupled to cancer cell dedifferentiation. Our data show that ZEB1 represents a key player in pathologic EMTs associated with tumour progression.
Array-based comparative genomic hybridization (array-CGH) has good potential for the high-throughput identification of genetic aberrations in cell genomes. In the course of a program to screen a panel of oral squamous-cell carcinoma (OSCC), cell lines for genomic copy-number aberrations by array-CGH using our in-house arrays, we identified a 3-Mb homozygous deletion at 10p12 in 1 of 18 cell lines (5.6%). Among seven genes located within this region, expression of PRTFDC1 mRNA was not detected in 50% (9/18) or decreased in 5.6% (1/18) of OSCC cell lines, but detected in normal oral epithelia and restored in gene-silenced OSCC cells without its homozygous loss after treatment with 5-aza-2'-deoxycytidine. Among 17 cell lines without a homozygous deletion, the hypermethylation of the PRTFDC1 CpG island, which showed promoter activity, was observed in all nine cell lines with no or reduced PRTFDC1 expression (52.9%). Methylation of this CpG island was also observed in primary OSCC tissues (8/47, 17.0%). In addition, restoration of PRTFDC1 in OSCC cells lacking its expression inhibited cell growth in colony-formation assays, whereas knockdown of PRTFDC1 expression in OSCC cells expressing the gene promoted cell growth. These results suggest that epigenetic silencing of PRTFDC1 by hypermethylation of the CpG island leads to a loss of PRTFDC1 function, which might be involved in squamous cell oral carcinogenesis.
In the last decade, the identification of enzymes that regulate acetylation of histones and nonhistone proteins has revealed the key role of dynamic acetylation and deacetylation in various cellular processes. Mammalian histone deacetylases (HDACs), which catalyse the removal of acetyl groups from lysine residues, are grouped into three classes, on the basis of similarity to yeast counterparts. An abundance of experimental evidence has established class IIa HDACs as crucial transcriptional regulators of various developmental and differentiation processes. In the past 5 years, a tremendous effort has been dedicated to characterizing the regulation of these enzymes. In this review, we summarize the latest discoveries in the field and discuss the molecular and structural determinants of class IIa HDACs regulation. Finally, we emphasize that comprehension of the mechanisms underlying class IIa HDAC functions is essential for potential therapeutic applications.
Amplification and overexpression of putative oncogenes confer growth advantages for tumor development. We used a functional genomic approach that integrated simultaneous genomic and transcript microarray, proteomics, and tissue microarray analyses to directly identify putative oncogenes in lung adenocarcinoma. We first identified 183 genes with increases in both genomic copy number and transcript in six lung adenocarcinoma cell lines. Next, we used two-dimensional polyacrylamide gel electrophoresis and mass spectrometry to identify 42 proteins that were overexpressed in the cancer cells relative to normal cells. Comparing the 183 genes with the 42 proteins, we identified four genes - PRDX1, EEF1A2, CALR, and KCIP-1 - in which elevated protein expression correlated with both increased DNA copy number and increased transcript levels (all r > 0.84, two-sided P < 0.05). These findings were validated by Southern, Northern, and Western blotting. Specific inhibition of EEF1A2 and KCIP-1 expression with siRNA in the four cell lines tested suppressed proliferation and induced apoptosis. Parallel fluorescence in situ hybridization and immunohistochemical analyses of EEF1A2 and KCIP-1 in tissue microarrays from patients with lung adenocarcinoma showed that gene amplification was associated with high protein expression for both genes and that protein overexpression was related to tumor grade, disease stage, Ki-67 expression, and a shorter survival of patients. The amplification of EEF1A2 and KCIP-1 and the presence of overexpressed protein in tumor samples strongly suggest that these genes could be oncogenes and hence potential targets for diagnosis and therapy in lung adenocarcinoma.
Amplification and overexpression of putative oncogenes confer growth advantages for tumor development. We used a functional genomic approach that integrated simultaneous genomic and transcript microarray, proteomics, and tissue microarray analyses to directly identify putative oncogenes in lung adenocarcinoma. We first identified 183 genes with increases in both genomic copy number and transcript in six lung adenocarcinoma cell lines. Next, we used two-dimensional polyacrylamide gel electrophoresis and mass spectrometry to identify 42 proteins that were overexpressed in the cancer cells relative to normal cells. Comparing the 183 genes with the 42 proteins, we identified four genes - PRDX1, EEF1A2, CALR, and KCIP-1 - in which elevated protein expression correlated with both increased DNA copy number and increased transcript levels (all r > 0.84, two-sided P < 0.05). These findings were validated by Southern, Northern, and Western blotting. Specific inhibition of EEF1A2 and KCIP-1 expression with siRNA in the four cell lines tested suppressed proliferation and induced apoptosis. Parallel fluorescence in situ hybridization and immunohistochemical analyses of EEF1A2 and KCIP-1 in tissue microarrays from patients with lung adenocarcinoma showed that gene amplification was associated with high protein expression for both genes and that protein overexpression was related to tumor grade, disease stage, Ki-67 expression, and a shorter survival of patients. The amplification of EEF1A2 and KCIP-1 and the presence of overexpressed protein in tumor samples strongly suggest that these genes could be oncogenes and hence potential targets for diagnosis and therapy in lung adenocarcinoma.
We used gene expression profiling, mutation analyses of FGFR3 and TP53, and LOH analyses of chromosome 9 and the TP53 region on chromosome arm 17p, to molecularly characterize 75 Ta and T1 bladder carcinomas. We identified four major cellular processes related to cell cycle, protein synthesis, immune response, and extra cellular components that contribute to the expressional heterogeneity of early-stage urothelial cell carcinoma (UCC). Activating FGFR3 mutations were found at the highest frequency in G1 tumors (80%), and showed a strong correlation with FGFR3 expression. In contrast, G3 tumors displayed mutations in less than 10% of the cases and a low level of FGFR3 expression. Even though LOH on chromosome 9 was not associated with any specific expression pattern, our data indicate that loss of chromosome 9 is associated with tumor development rather than initiation. The combined analyses suggest the existence of two types of UCC tumors, one which is characterized by FGFR3 mutation or expression, high expression of protein synthesis genes, and low expression of cell cycle genes. Furthermore, the presented data underscore FGFR3 receptor involvement in urothelial cell transformation as the presence of FGFR3 mutations has a major impact on the global gene expression profile of bladder carcinomas.
We used gene expression profiling, mutation analyses of FGFR3 and TP53, and LOH analyses of chromosome 9 and the TP53 region on chromosome arm 17p, to molecularly characterize 75 Ta and T1 bladder carcinomas. We identified four major cellular processes related to cell cycle, protein synthesis, immune response, and extra cellular components that contribute to the expressional heterogeneity of early-stage urothelial cell carcinoma (UCC). Activating FGFR3 mutations were found at the highest frequency in G1 tumors (80%), and showed a strong correlation with FGFR3 expression. In contrast, G3 tumors displayed mutations in less than 10% of the cases and a low level of FGFR3 expression. Even though LOH on chromosome 9 was not associated with any specific expression pattern, our data indicate that loss of chromosome 9 is associated with tumor development rather than initiation. The combined analyses suggest the existence of two types of UCC tumors, one which is characterized by FGFR3 mutation or expression, high expression of protein synthesis genes, and low expression of cell cycle genes. Furthermore, the presented data underscore FGFR3 receptor involvement in urothelial cell transformation as the presence of FGFR3 mutations has a major impact on the global gene expression profile of bladder carcinomas.
We used gene expression profiling, mutation analyses of FGFR3 and TP53, and LOH analyses of chromosome 9 and the TP53 region on chromosome arm 17p, to molecularly characterize 75 Ta and T1 bladder carcinomas. We identified four major cellular processes related to cell cycle, protein synthesis, immune response, and extra cellular components that contribute to the expressional heterogeneity of early-stage urothelial cell carcinoma (UCC). Activating FGFR3 mutations were found at the highest frequency in G1 tumors (80%), and showed a strong correlation with FGFR3 expression. In contrast, G3 tumors displayed mutations in less than 10% of the cases and a low level of FGFR3 expression. Even though LOH on chromosome 9 was not associated with any specific expression pattern, our data indicate that loss of chromosome 9 is associated with tumor development rather than initiation. The combined analyses suggest the existence of two types of UCC tumors, one which is characterized by FGFR3 mutation or expression, high expression of protein synthesis genes, and low expression of cell cycle genes. Furthermore, the presented data underscore FGFR3 receptor involvement in urothelial cell transformation as the presence of FGFR3 mutations has a major impact on the global gene expression profile of bladder carcinomas.
To identify tumor markers and differentiation markers for lung adenocarcinoma (AdC), we analysed expression profiles of 14,500 genes against three cases of type II alveolar epithelial cells, bronchiolar epithelial cells, and bronchial epithelial cells, respectively, and 10 cases of AdC cells isolated by laser capture microdissection. Hierarchical clustering analysis indicated that AdC cells and noncancerous lung epithelial cells are significantly different in their expression profiles, and that different sets of differentiation markers are expressed among alveolar, bronchiolar and bronchial epithelial cells. Nine genes were identified as being highly expressed in AdC cells, but not expressed in noncancerous lung epithelial cells. Sixteen genes were identified as differentiation markers for lung epithelial cells. Real-time RT-PCR analysis of 45 lung AdC cases further revealed that expression of four tumor markers in AdC cells was significantly higher than that in noncancerous lung cells and that expression of ten differentiation markers was retained in a considerable fraction of lung AdC cases. Five tumor markers and seven differentiation markers were not expressed in peripheral blood cells. Similarities and differences in expression profiles between normal epithelial cells from different lung respiratory compartments and AdC cells demonstrated in this study will be informative for the molecular diagnosis of lung AdC.
To identify tumor markers and differentiation markers for lung adenocarcinoma (AdC), we analysed expression profiles of 14,500 genes against three cases of type II alveolar epithelial cells, bronchiolar epithelial cells, and bronchial epithelial cells, respectively, and 10 cases of AdC cells isolated by laser capture microdissection. Hierarchical clustering analysis indicated that AdC cells and noncancerous lung epithelial cells are significantly different in their expression profiles, and that different sets of differentiation markers are expressed among alveolar, bronchiolar and bronchial epithelial cells. Nine genes were identified as being highly expressed in AdC cells, but not expressed in noncancerous lung epithelial cells. Sixteen genes were identified as differentiation markers for lung epithelial cells. Real-time RT-PCR analysis of 45 lung AdC cases further revealed that expression of four tumor markers in AdC cells was significantly higher than that in noncancerous lung cells and that expression of ten differentiation markers was retained in a considerable fraction of lung AdC cases. Five tumor markers and seven differentiation markers were not expressed in peripheral blood cells. Similarities and differences in expression profiles between normal epithelial cells from different lung respiratory compartments and AdC cells demonstrated in this study will be informative for the molecular diagnosis of lung AdC.
To identify tumor markers and differentiation markers for lung adenocarcinoma (AdC), we analysed expression profiles of 14,500 genes against three cases of type II alveolar epithelial cells, bronchiolar epithelial cells, and bronchial epithelial cells, respectively, and 10 cases of AdC cells isolated by laser capture microdissection. Hierarchical clustering analysis indicated that AdC cells and noncancerous lung epithelial cells are significantly different in their expression profiles, and that different sets of differentiation markers are expressed among alveolar, bronchiolar and bronchial epithelial cells. Nine genes were identified as being highly expressed in AdC cells, but not expressed in noncancerous lung epithelial cells. Sixteen genes were identified as differentiation markers for lung epithelial cells. Real-time RT-PCR analysis of 45 lung AdC cases further revealed that expression of four tumor markers in AdC cells was significantly higher than that in noncancerous lung cells and that expression of ten differentiation markers was retained in a considerable fraction of lung AdC cases. Five tumor markers and seven differentiation markers were not expressed in peripheral blood cells. Similarities and differences in expression profiles between normal epithelial cells from different lung respiratory compartments and AdC cells demonstrated in this study will be informative for the molecular diagnosis of lung AdC.
To identify tumor markers and differentiation markers for lung adenocarcinoma (AdC), we analysed expression profiles of 14,500 genes against three cases of type II alveolar epithelial cells, bronchiolar epithelial cells, and bronchial epithelial cells, respectively, and 10 cases of AdC cells isolated by laser capture microdissection. Hierarchical clustering analysis indicated that AdC cells and noncancerous lung epithelial cells are significantly different in their expression profiles, and that different sets of differentiation markers are expressed among alveolar, bronchiolar and bronchial epithelial cells. Nine genes were identified as being highly expressed in AdC cells, but not expressed in noncancerous lung epithelial cells. Sixteen genes were identified as differentiation markers for lung epithelial cells. Real-time RT-PCR analysis of 45 lung AdC cases further revealed that expression of four tumor markers in AdC cells was significantly higher than that in noncancerous lung cells and that expression of ten differentiation markers was retained in a considerable fraction of lung AdC cases. Five tumor markers and seven differentiation markers were not expressed in peripheral blood cells. Similarities and differences in expression profiles between normal epithelial cells from different lung respiratory compartments and AdC cells demonstrated in this study will be informative for the molecular diagnosis of lung AdC.
Mitochondria are vital for cellular bioenergetics and play a central role in determining the point-of-no-return of the apoptotic process. As a consequence, mitochondria exert a dual function in carcinogenesis. Cancer-associated changes in cellular metabolism (the Warburg effect) influence mitochondrial function, and the invalidation of apoptosis is linked to an inhibition of mitochondrial outer membrane permeabilization (MOMP). On theoretical grounds, it is tempting to develop specific therapeutic interventions that target the mitochondrial Achilles' heel, rendering cancer cells metabolically unviable or subverting endogenous MOMP inhibitors. A variety of experimental therapeutic agents can directly target mitochondria, causing apoptosis induction. This applies to a heterogeneous collection of chemically unrelated compounds including positively charged alpha-helical peptides, agents designed to mimic the Bcl-2 homology domain 3 of Bcl-2-like proteins, ampholytic cations, metals and steroid-like compounds. Such MOMP inducers or facilitators can induce apoptosis by themselves (monotherapy) or facilitate apoptosis induction in combination therapies, bypassing chemoresistance against DNA-damaging agents. In addition, it is possible to design molecules that neutralize inhibitor of apoptosis proteins (IAPs) or heat shock protein 70 (HSP70). Such IAP or HSP70 inhibitors can mimic the action of mitochondrion-derived mediators (Smac/DIABLO, that is, second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with a low isoelectric point, in the case of IAPs; AIF, that is apoptosis-inducing factor, in the case of HSP70) and exert potent chemosensitizing effects.
DNA copy number amplifications activate oncogenes and are hallmarks of nearly all advanced tumors. Amplified genes represent attractive targets for therapy, diagnostics and prognostics. To investigate DNA amplifications in different neoplasms, we performed a bibliomics survey using 838 published chromosomal comparative genomic hybridization studies and collected amplification data at chromosome band resolution from more than 4500 cases. Amplification profiles were determined for 73 distinct neoplasms. Neoplasms were clustered according to the amplification profiles, and frequently amplified chromosomal loci (amplification hot spots) were identified using computational modeling. To investigate the site specificity and mechanisms of gene amplifications, colocalization of amplification hot spots, cancer genes, fragile sites, virus integration sites and gene size cohorts were tested in a statistical framework. Amplification-based clustering demonstrated that cancers with similar etiology, cell-of-origin or topographical location have a tendency to obtain convergent amplification profiles. The identified amplification hot spots were colocalized with the known fragile sites, cancer genes and virus integration sites, but global statistical significance could not be ascertained. Large genes were significantly overrepresented on the fragile sites and the reported amplification hot spots. These findings indicate that amplifications are selected in the cancer tissue environment according to the qualitative traits and localization of cancer genes.
DNA copy number amplifications activate oncogenes and are hallmarks of nearly all advanced tumors. Amplified genes represent attractive targets for therapy, diagnostics and prognostics. To investigate DNA amplifications in different neoplasms, we performed a bibliomics survey using 838 published chromosomal comparative genomic hybridization studies and collected amplification data at chromosome band resolution from more than 4500 cases. Amplification profiles were determined for 73 distinct neoplasms. Neoplasms were clustered according to the amplification profiles, and frequently amplified chromosomal loci (amplification hot spots) were identified using computational modeling. To investigate the site specificity and mechanisms of gene amplifications, colocalization of amplification hot spots, cancer genes, fragile sites, virus integration sites and gene size cohorts were tested in a statistical framework. Amplification-based clustering demonstrated that cancers with similar etiology, cell-of-origin or topographical location have a tendency to obtain convergent amplification profiles. The identified amplification hot spots were colocalized with the known fragile sites, cancer genes and virus integration sites, but global statistical significance could not be ascertained. Large genes were significantly overrepresented on the fragile sites and the reported amplification hot spots. These findings indicate that amplifications are selected in the cancer tissue environment according to the qualitative traits and localization of cancer genes.
DNA copy number amplifications activate oncogenes and are hallmarks of nearly all advanced tumors. Amplified genes represent attractive targets for therapy, diagnostics and prognostics. To investigate DNA amplifications in different neoplasms, we performed a bibliomics survey using 838 published chromosomal comparative genomic hybridization studies and collected amplification data at chromosome band resolution from more than 4500 cases. Amplification profiles were determined for 73 distinct neoplasms. Neoplasms were clustered according to the amplification profiles, and frequently amplified chromosomal loci (amplification hot spots) were identified using computational modeling. To investigate the site specificity and mechanisms of gene amplifications, colocalization of amplification hot spots, cancer genes, fragile sites, virus integration sites and gene size cohorts were tested in a statistical framework. Amplification-based clustering demonstrated that cancers with similar etiology, cell-of-origin or topographical location have a tendency to obtain convergent amplification profiles. The identified amplification hot spots were colocalized with the known fragile sites, cancer genes and virus integration sites, but global statistical significance could not be ascertained. Large genes were significantly overrepresented on the fragile sites and the reported amplification hot spots. These findings indicate that amplifications are selected in the cancer tissue environment according to the qualitative traits and localization of cancer genes.
DNA copy number amplifications activate oncogenes and are hallmarks of nearly all advanced tumors. Amplified genes represent attractive targets for therapy, diagnostics and prognostics. To investigate DNA amplifications in different neoplasms, we performed a bibliomics survey using 838 published chromosomal comparative genomic hybridization studies and collected amplification data at chromosome band resolution from more than 4500 cases. Amplification profiles were determined for 73 distinct neoplasms. Neoplasms were clustered according to the amplification profiles, and frequently amplified chromosomal loci (amplification hot spots) were identified using computational modeling. To investigate the site specificity and mechanisms of gene amplifications, colocalization of amplification hot spots, cancer genes, fragile sites, virus integration sites and gene size cohorts were tested in a statistical framework. Amplification-based clustering demonstrated that cancers with similar etiology, cell-of-origin or topographical location have a tendency to obtain convergent amplification profiles. The identified amplification hot spots were colocalized with the known fragile sites, cancer genes and virus integration sites, but global statistical significance could not be ascertained. Large genes were significantly overrepresented on the fragile sites and the reported amplification hot spots. These findings indicate that amplifications are selected in the cancer tissue environment according to the qualitative traits and localization of cancer genes.
DNA copy number amplifications activate oncogenes and are hallmarks of nearly all advanced tumors. Amplified genes represent attractive targets for therapy, diagnostics and prognostics. To investigate DNA amplifications in different neoplasms, we performed a bibliomics survey using 838 published chromosomal comparative genomic hybridization studies and collected amplification data at chromosome band resolution from more than 4500 cases. Amplification profiles were determined for 73 distinct neoplasms. Neoplasms were clustered according to the amplification profiles, and frequently amplified chromosomal loci (amplification hot spots) were identified using computational modeling. To investigate the site specificity and mechanisms of gene amplifications, colocalization of amplification hot spots, cancer genes, fragile sites, virus integration sites and gene size cohorts were tested in a statistical framework. Amplification-based clustering demonstrated that cancers with similar etiology, cell-of-origin or topographical location have a tendency to obtain convergent amplification profiles. The identified amplification hot spots were colocalized with the known fragile sites, cancer genes and virus integration sites, but global statistical significance could not be ascertained. Large genes were significantly overrepresented on the fragile sites and the reported amplification hot spots. These findings indicate that amplifications are selected in the cancer tissue environment according to the qualitative traits and localization of cancer genes.
DNA copy number amplifications activate oncogenes and are hallmarks of nearly all advanced tumors. Amplified genes represent attractive targets for therapy, diagnostics and prognostics. To investigate DNA amplifications in different neoplasms, we performed a bibliomics survey using 838 published chromosomal comparative genomic hybridization studies and collected amplification data at chromosome band resolution from more than 4500 cases. Amplification profiles were determined for 73 distinct neoplasms. Neoplasms were clustered according to the amplification profiles, and frequently amplified chromosomal loci (amplification hot spots) were identified using computational modeling. To investigate the site specificity and mechanisms of gene amplifications, colocalization of amplification hot spots, cancer genes, fragile sites, virus integration sites and gene size cohorts were tested in a statistical framework. Amplification-based clustering demonstrated that cancers with similar etiology, cell-of-origin or topographical location have a tendency to obtain convergent amplification profiles. The identified amplification hot spots were colocalized with the known fragile sites, cancer genes and virus integration sites, but global statistical significance could not be ascertained. Large genes were significantly overrepresented on the fragile sites and the reported amplification hot spots. These findings indicate that amplifications are selected in the cancer tissue environment according to the qualitative traits and localization of cancer genes.
DNA copy number amplifications activate oncogenes and are hallmarks of nearly all advanced tumors. Amplified genes represent attractive targets for therapy, diagnostics and prognostics. To investigate DNA amplifications in different neoplasms, we performed a bibliomics survey using 838 published chromosomal comparative genomic hybridization studies and collected amplification data at chromosome band resolution from more than 4500 cases. Amplification profiles were determined for 73 distinct neoplasms. Neoplasms were clustered according to the amplification profiles, and frequently amplified chromosomal loci (amplification hot spots) were identified using computational modeling. To investigate the site specificity and mechanisms of gene amplifications, colocalization of amplification hot spots, cancer genes, fragile sites, virus integration sites and gene size cohorts were tested in a statistical framework. Amplification-based clustering demonstrated that cancers with similar etiology, cell-of-origin or topographical location have a tendency to obtain convergent amplification profiles. The identified amplification hot spots were colocalized with the known fragile sites, cancer genes and virus integration sites, but global statistical significance could not be ascertained. Large genes were significantly overrepresented on the fragile sites and the reported amplification hot spots. These findings indicate that amplifications are selected in the cancer tissue environment according to the qualitative traits and localization of cancer genes.
DNA copy number amplifications activate oncogenes and are hallmarks of nearly all advanced tumors. Amplified genes represent attractive targets for therapy, diagnostics and prognostics. To investigate DNA amplifications in different neoplasms, we performed a bibliomics survey using 838 published chromosomal comparative genomic hybridization studies and collected amplification data at chromosome band resolution from more than 4500 cases. Amplification profiles were determined for 73 distinct neoplasms. Neoplasms were clustered according to the amplification profiles, and frequently amplified chromosomal loci (amplification hot spots) were identified using computational modeling. To investigate the site specificity and mechanisms of gene amplifications, colocalization of amplification hot spots, cancer genes, fragile sites, virus integration sites and gene size cohorts were tested in a statistical framework. Amplification-based clustering demonstrated that cancers with similar etiology, cell-of-origin or topographical location have a tendency to obtain convergent amplification profiles. The identified amplification hot spots were colocalized with the known fragile sites, cancer genes and virus integration sites, but global statistical significance could not be ascertained. Large genes were significantly overrepresented on the fragile sites and the reported amplification hot spots. These findings indicate that amplifications are selected in the cancer tissue environment according to the qualitative traits and localization of cancer genes.
DNA copy number amplifications activate oncogenes and are hallmarks of nearly all advanced tumors. Amplified genes represent attractive targets for therapy, diagnostics and prognostics. To investigate DNA amplifications in different neoplasms, we performed a bibliomics survey using 838 published chromosomal comparative genomic hybridization studies and collected amplification data at chromosome band resolution from more than 4500 cases. Amplification profiles were determined for 73 distinct neoplasms. Neoplasms were clustered according to the amplification profiles, and frequently amplified chromosomal loci (amplification hot spots) were identified using computational modeling. To investigate the site specificity and mechanisms of gene amplifications, colocalization of amplification hot spots, cancer genes, fragile sites, virus integration sites and gene size cohorts were tested in a statistical framework. Amplification-based clustering demonstrated that cancers with similar etiology, cell-of-origin or topographical location have a tendency to obtain convergent amplification profiles. The identified amplification hot spots were colocalized with the known fragile sites, cancer genes and virus integration sites, but global statistical significance could not be ascertained. Large genes were significantly overrepresented on the fragile sites and the reported amplification hot spots. These findings indicate that amplifications are selected in the cancer tissue environment according to the qualitative traits and localization of cancer genes.
DNA copy number amplifications activate oncogenes and are hallmarks of nearly all advanced tumors. Amplified genes represent attractive targets for therapy, diagnostics and prognostics. To investigate DNA amplifications in different neoplasms, we performed a bibliomics survey using 838 published chromosomal comparative genomic hybridization studies and collected amplification data at chromosome band resolution from more than 4500 cases. Amplification profiles were determined for 73 distinct neoplasms. Neoplasms were clustered according to the amplification profiles, and frequently amplified chromosomal loci (amplification hot spots) were identified using computational modeling. To investigate the site specificity and mechanisms of gene amplifications, colocalization of amplification hot spots, cancer genes, fragile sites, virus integration sites and gene size cohorts were tested in a statistical framework. Amplification-based clustering demonstrated that cancers with similar etiology, cell-of-origin or topographical location have a tendency to obtain convergent amplification profiles. The identified amplification hot spots were colocalized with the known fragile sites, cancer genes and virus integration sites, but global statistical significance could not be ascertained. Large genes were significantly overrepresented on the fragile sites and the reported amplification hot spots. These findings indicate that amplifications are selected in the cancer tissue environment according to the qualitative traits and localization of cancer genes.
The chromosomal translocation t(2;13), characteristic for the aggressive childhood cancer alveolar rhabdomyosarcoma (aRMS), generates the chimeric transcription factor PAX3/FKHR with a well known oncogenic role. However, the molecular mechanisms mediating essential pathophysiological functions remain poorly defined. Here, we used comparative expression profiling of PAX3/FKHR silencing in vitro and PAX3/FKHR-specific gene signatures in vivo to identify physiologically important target genes. Hereby, 51 activated genes, both novel and known, were identified. We also found repression of skeletal muscle-specific genes suggesting that PAX3/FKHR blocks further differentiation of aRMS cells. Importantly, TFAP2B was validated as direct target gene mediating the anti-apoptotic function of PAX3/FKHR. Hence, we developed a pathophysiologically relevant transcriptional profile of PAX3/FKHR and identified a critical target gene for aRMS development.
The chromosomal translocation t(2;13), characteristic for the aggressive childhood cancer alveolar rhabdomyosarcoma (aRMS), generates the chimeric transcription factor PAX3/FKHR with a well known oncogenic role. However, the molecular mechanisms mediating essential pathophysiological functions remain poorly defined. Here, we used comparative expression profiling of PAX3/FKHR silencing in vitro and PAX3/FKHR-specific gene signatures in vivo to identify physiologically important target genes. Hereby, 51 activated genes, both novel and known, were identified. We also found repression of skeletal muscle-specific genes suggesting that PAX3/FKHR blocks further differentiation of aRMS cells. Importantly, TFAP2B was validated as direct target gene mediating the anti-apoptotic function of PAX3/FKHR. Hence, we developed a pathophysiologically relevant transcriptional profile of PAX3/FKHR and identified a critical target gene for aRMS development.
The mechanism by which the p53 family of proteins coordinately regulates select target genes after various types of cell stress is not well understood. To further define factors that dictate regulation of target genes, we examined the binding of p53, DeltaNp63alpha and RNA polymerase II (pol II) to the regulatory regions of select target genes in primary human epidermal keratinocytes (HEKs) using chromatin immunoprecipitation. In rapidly proliferating cells, we observed constitutive binding of DeltaNp63alpha and varying levels of p53 binding, to consensus sites in target genes involved in cell cycle arrest, DNA repair and apoptosis. Following genotoxic stress, p53 occupancy increased whereas DeltaNp63alpha occupancy decreased at the majority of binding sites examined. Microarray analysis of transcripts isolated from HEKs ectopically expressing p53 and DeltaNp63alpha revealed an inverse regulation of select target genes by the two family members. Collectively, our results suggest that DeltaNp63alpha can function as a repressor of select p53 target genes involved in growth arrest, DNA repair and apoptosis, and that the location of the p53 consensus binding site(s) in a target gene may dictate whether pol II is constitutively bound in proliferating cells.
DNA copy number amplifications activate oncogenes and are hallmarks of nearly all advanced tumors. Amplified genes represent attractive targets for therapy, diagnostics and prognostics. To investigate DNA amplifications in different neoplasms, we performed a bibliomics survey using 838 published chromosomal comparative genomic hybridization studies and collected amplification data at chromosome band resolution from more than 4500 cases. Amplification profiles were determined for 73 distinct neoplasms. Neoplasms were clustered according to the amplification profiles, and frequently amplified chromosomal loci (amplification hot spots) were identified using computational modeling. To investigate the site specificity and mechanisms of gene amplifications, colocalization of amplification hot spots, cancer genes, fragile sites, virus integration sites and gene size cohorts were tested in a statistical framework. Amplification-based clustering demonstrated that cancers with similar etiology, cell-of-origin or topographical location have a tendency to obtain convergent amplification profiles. The identified amplification hot spots were colocalized with the known fragile sites, cancer genes and virus integration sites, but global statistical significance could not be ascertained. Large genes were significantly overrepresented on the fragile sites and the reported amplification hot spots. These findings indicate that amplifications are selected in the cancer tissue environment according to the qualitative traits and localization of cancer genes.
DNA copy number amplifications activate oncogenes and are hallmarks of nearly all advanced tumors. Amplified genes represent attractive targets for therapy, diagnostics and prognostics. To investigate DNA amplifications in different neoplasms, we performed a bibliomics survey using 838 published chromosomal comparative genomic hybridization studies and collected amplification data at chromosome band resolution from more than 4500 cases. Amplification profiles were determined for 73 distinct neoplasms. Neoplasms were clustered according to the amplification profiles, and frequently amplified chromosomal loci (amplification hot spots) were identified using computational modeling. To investigate the site specificity and mechanisms of gene amplifications, colocalization of amplification hot spots, cancer genes, fragile sites, virus integration sites and gene size cohorts were tested in a statistical framework. Amplification-based clustering demonstrated that cancers with similar etiology, cell-of-origin or topographical location have a tendency to obtain convergent amplification profiles. The identified amplification hot spots were colocalized with the known fragile sites, cancer genes and virus integration sites, but global statistical significance could not be ascertained. Large genes were significantly overrepresented on the fragile sites and the reported amplification hot spots. These findings indicate that amplifications are selected in the cancer tissue environment according to the qualitative traits and localization of cancer genes.
DNA copy number amplifications activate oncogenes and are hallmarks of nearly all advanced tumors. Amplified genes represent attractive targets for therapy, diagnostics and prognostics. To investigate DNA amplifications in different neoplasms, we performed a bibliomics survey using 838 published chromosomal comparative genomic hybridization studies and collected amplification data at chromosome band resolution from more than 4500 cases. Amplification profiles were determined for 73 distinct neoplasms. Neoplasms were clustered according to the amplification profiles, and frequently amplified chromosomal loci (amplification hot spots) were identified using computational modeling. To investigate the site specificity and mechanisms of gene amplifications, colocalization of amplification hot spots, cancer genes, fragile sites, virus integration sites and gene size cohorts were tested in a statistical framework. Amplification-based clustering demonstrated that cancers with similar etiology, cell-of-origin or topographical location have a tendency to obtain convergent amplification profiles. The identified amplification hot spots were colocalized with the known fragile sites, cancer genes and virus integration sites, but global statistical significance could not be ascertained. Large genes were significantly overrepresented on the fragile sites and the reported amplification hot spots. These findings indicate that amplifications are selected in the cancer tissue environment according to the qualitative traits and localization of cancer genes.
DNA copy number amplifications activate oncogenes and are hallmarks of nearly all advanced tumors. Amplified genes represent attractive targets for therapy, diagnostics and prognostics. To investigate DNA amplifications in different neoplasms, we performed a bibliomics survey using 838 published chromosomal comparative genomic hybridization studies and collected amplification data at chromosome band resolution from more than 4500 cases. Amplification profiles were determined for 73 distinct neoplasms. Neoplasms were clustered according to the amplification profiles, and frequently amplified chromosomal loci (amplification hot spots) were identified using computational modeling. To investigate the site specificity and mechanisms of gene amplifications, colocalization of amplification hot spots, cancer genes, fragile sites, virus integration sites and gene size cohorts were tested in a statistical framework. Amplification-based clustering demonstrated that cancers with similar etiology, cell-of-origin or topographical location have a tendency to obtain convergent amplification profiles. The identified amplification hot spots were colocalized with the known fragile sites, cancer genes and virus integration sites, but global statistical significance could not be ascertained. Large genes were significantly overrepresented on the fragile sites and the reported amplification hot spots. These findings indicate that amplifications are selected in the cancer tissue environment according to the qualitative traits and localization of cancer genes.
DNA copy number amplifications activate oncogenes and are hallmarks of nearly all advanced tumors. Amplified genes represent attractive targets for therapy, diagnostics and prognostics. To investigate DNA amplifications in different neoplasms, we performed a bibliomics survey using 838 published chromosomal comparative genomic hybridization studies and collected amplification data at chromosome band resolution from more than 4500 cases. Amplification profiles were determined for 73 distinct neoplasms. Neoplasms were clustered according to the amplification profiles, and frequently amplified chromosomal loci (amplification hot spots) were identified using computational modeling. To investigate the site specificity and mechanisms of gene amplifications, colocalization of amplification hot spots, cancer genes, fragile sites, virus integration sites and gene size cohorts were tested in a statistical framework. Amplification-based clustering demonstrated that cancers with similar etiology, cell-of-origin or topographical location have a tendency to obtain convergent amplification profiles. The identified amplification hot spots were colocalized with the known fragile sites, cancer genes and virus integration sites, but global statistical significance could not be ascertained. Large genes were significantly overrepresented on the fragile sites and the reported amplification hot spots. These findings indicate that amplifications are selected in the cancer tissue environment according to the qualitative traits and localization of cancer genes.
DNA copy number amplifications activate oncogenes and are hallmarks of nearly all advanced tumors. Amplified genes represent attractive targets for therapy, diagnostics and prognostics. To investigate DNA amplifications in different neoplasms, we performed a bibliomics survey using 838 published chromosomal comparative genomic hybridization studies and collected amplification data at chromosome band resolution from more than 4500 cases. Amplification profiles were determined for 73 distinct neoplasms. Neoplasms were clustered according to the amplification profiles, and frequently amplified chromosomal loci (amplification hot spots) were identified using computational modeling. To investigate the site specificity and mechanisms of gene amplifications, colocalization of amplification hot spots, cancer genes, fragile sites, virus integration sites and gene size cohorts were tested in a statistical framework. Amplification-based clustering demonstrated that cancers with similar etiology, cell-of-origin or topographical location have a tendency to obtain convergent amplification profiles. The identified amplification hot spots were colocalized with the known fragile sites, cancer genes and virus integration sites, but global statistical significance could not be ascertained. Large genes were significantly overrepresented on the fragile sites and the reported amplification hot spots. These findings indicate that amplifications are selected in the cancer tissue environment according to the qualitative traits and localization of cancer genes.
DNA copy number amplifications activate oncogenes and are hallmarks of nearly all advanced tumors. Amplified genes represent attractive targets for therapy, diagnostics and prognostics. To investigate DNA amplifications in different neoplasms, we performed a bibliomics survey using 838 published chromosomal comparative genomic hybridization studies and collected amplification data at chromosome band resolution from more than 4500 cases. Amplification profiles were determined for 73 distinct neoplasms. Neoplasms were clustered according to the amplification profiles, and frequently amplified chromosomal loci (amplification hot spots) were identified using computational modeling. To investigate the site specificity and mechanisms of gene amplifications, colocalization of amplification hot spots, cancer genes, fragile sites, virus integration sites and gene size cohorts were tested in a statistical framework. Amplification-based clustering demonstrated that cancers with similar etiology, cell-of-origin or topographical location have a tendency to obtain convergent amplification profiles. The identified amplification hot spots were colocalized with the known fragile sites, cancer genes and virus integration sites, but global statistical significance could not be ascertained. Large genes were significantly overrepresented on the fragile sites and the reported amplification hot spots. These findings indicate that amplifications are selected in the cancer tissue environment according to the qualitative traits and localization of cancer genes.
Multiple myeloma (MM) is the most common form of plasma cell dyscrasia, characterized by a marked heterogeneity of genetic lesions and clinical course. It may develop from a premalignant condition (monoclonal gammopathy of undetermined significance, MGUS) or progress from intramedullary to extramedullary forms (plasma cell leukemia, PCL). To provide insights into the molecular characterization of plasma cell dyscrasias and to investigate the contribution of specific genetic lesions to the biological and clinical heterogeneity of MM, we analysed the gene expression profiles of plasma cells isolated from seven MGUS, 39 MM and six PCL patients by means of DNA microarrays. MMs resulted highly heterogeneous at transcriptional level, whereas the differential expression of genes mainly involved in DNA metabolism and proliferation distinguished MGUS from PCLs and the majority of MM cases. The clustering of MM patients was mainly driven by the presence of the most recurrent translocations involving the immunoglobulin heavy-chain locus. Distinct gene expression patterns have been found to be associated with different lesions: the overexpression of CCND2 and genes involved in cell adhesion pathways was observed in cases with deregulated MAF and MAFB, whereas genes upregulated in cases with the t(4;14) showed apoptosis-related functions. The peculiar finding in patients with the t(11;14) was the downregulation of the alpha-subunit of the IL-6 receptor. In addition, we identified a set of cancer germline antigens specifically expressed in a subgroup of MM patients characterized by an aggressive clinical evolution, a finding that could have implications for patient classification and immunotherapy.
Multiple myeloma (MM) is the most common form of plasma cell dyscrasia, characterized by a marked heterogeneity of genetic lesions and clinical course. It may develop from a premalignant condition (monoclonal gammopathy of undetermined significance, MGUS) or progress from intramedullary to extramedullary forms (plasma cell leukemia, PCL). To provide insights into the molecular characterization of plasma cell dyscrasias and to investigate the contribution of specific genetic lesions to the biological and clinical heterogeneity of MM, we analysed the gene expression profiles of plasma cells isolated from seven MGUS, 39 MM and six PCL patients by means of DNA microarrays. MMs resulted highly heterogeneous at transcriptional level, whereas the differential expression of genes mainly involved in DNA metabolism and proliferation distinguished MGUS from PCLs and the majority of MM cases. The clustering of MM patients was mainly driven by the presence of the most recurrent translocations involving the immunoglobulin heavy-chain locus. Distinct gene expression patterns have been found to be associated with different lesions: the overexpression of CCND2 and genes involved in cell adhesion pathways was observed in cases with deregulated MAF and MAFB, whereas genes upregulated in cases with the t(4;14) showed apoptosis-related functions. The peculiar finding in patients with the t(11;14) was the downregulation of the alpha-subunit of the IL-6 receptor. In addition, we identified a set of cancer germline antigens specifically expressed in a subgroup of MM patients characterized by an aggressive clinical evolution, a finding that could have implications for patient classification and immunotherapy.
Previous microarray studies on breast cancer identified multiple tumour classes, of which the most prominent, named luminal and basal, differ in expression of the oestrogen receptor alpha gene (ER). We report here the identification of a group of breast tumours with increased androgen signalling and a 'molecular apocrine' gene expression profile. Tumour samples from 49 patients with large operable or locally advanced breast cancers were tested on Affymetrix U133A gene expression microarrays. Principal components analysis and hierarchical clustering split the tumours into three groups: basal, luminal and a group we call molecular apocrine. All of the molecular apocrine tumours have strong apocrine features on histological examination (P=0.0002). The molecular apocrine group is androgen receptor (AR) positive and contains all of the ER-negative tumours outside the basal group. Kolmogorov-Smirnov testing indicates that oestrogen signalling is most active in the luminal group, and androgen signalling is most active in the molecular apocrine group. ERBB2 amplification is commoner in the molecular apocrine than the other groups. Genes that best split the three groups were identified by Wilcoxon test. Correlation of the average expression profile of these genes in our data with the expression profile of individual tumours in four published breast cancer studies suggest that molecular apocrine tumours represent 8-14% of tumours in these studies. Our data show that it is possible with microarray data to divide mammary tumour cells into three groups based on steroid receptor activity: luminal (ER+ AR+), basal (ER- AR-) and molecular apocrine (ER- AR+).
Previous microarray studies on breast cancer identified multiple tumour classes, of which the most prominent, named luminal and basal, differ in expression of the oestrogen receptor alpha gene (ER). We report here the identification of a group of breast tumours with increased androgen signalling and a 'molecular apocrine' gene expression profile. Tumour samples from 49 patients with large operable or locally advanced breast cancers were tested on Affymetrix U133A gene expression microarrays. Principal components analysis and hierarchical clustering split the tumours into three groups: basal, luminal and a group we call molecular apocrine. All of the molecular apocrine tumours have strong apocrine features on histological examination (P=0.0002). The molecular apocrine group is androgen receptor (AR) positive and contains all of the ER-negative tumours outside the basal group. Kolmogorov-Smirnov testing indicates that oestrogen signalling is most active in the luminal group, and androgen signalling is most active in the molecular apocrine group. ERBB2 amplification is commoner in the molecular apocrine than the other groups. Genes that best split the three groups were identified by Wilcoxon test. Correlation of the average expression profile of these genes in our data with the expression profile of individual tumours in four published breast cancer studies suggest that molecular apocrine tumours represent 8-14% of tumours in these studies. Our data show that it is possible with microarray data to divide mammary tumour cells into three groups based on steroid receptor activity: luminal (ER+ AR+), basal (ER- AR-) and molecular apocrine (ER- AR+).
Previous microarray studies on breast cancer identified multiple tumour classes, of which the most prominent, named luminal and basal, differ in expression of the oestrogen receptor alpha gene (ER). We report here the identification of a group of breast tumours with increased androgen signalling and a 'molecular apocrine' gene expression profile. Tumour samples from 49 patients with large operable or locally advanced breast cancers were tested on Affymetrix U133A gene expression microarrays. Principal components analysis and hierarchical clustering split the tumours into three groups: basal, luminal and a group we call molecular apocrine. All of the molecular apocrine tumours have strong apocrine features on histological examination (P=0.0002). The molecular apocrine group is androgen receptor (AR) positive and contains all of the ER-negative tumours outside the basal group. Kolmogorov-Smirnov testing indicates that oestrogen signalling is most active in the luminal group, and androgen signalling is most active in the molecular apocrine group. ERBB2 amplification is commoner in the molecular apocrine than the other groups. Genes that best split the three groups were identified by Wilcoxon test. Correlation of the average expression profile of these genes in our data with the expression profile of individual tumours in four published breast cancer studies suggest that molecular apocrine tumours represent 8-14% of tumours in these studies. Our data show that it is possible with microarray data to divide mammary tumour cells into three groups based on steroid receptor activity: luminal (ER+ AR+), basal (ER- AR-) and molecular apocrine (ER- AR+).
DNA copy number amplifications activate oncogenes and are hallmarks of nearly all advanced tumors. Amplified genes represent attractive targets for therapy, diagnostics and prognostics. To investigate DNA amplifications in different neoplasms, we performed a bibliomics survey using 838 published chromosomal comparative genomic hybridization studies and collected amplification data at chromosome band resolution from more than 4500 cases. Amplification profiles were determined for 73 distinct neoplasms. Neoplasms were clustered according to the amplification profiles, and frequently amplified chromosomal loci (amplification hot spots) were identified using computational modeling. To investigate the site specificity and mechanisms of gene amplifications, colocalization of amplification hot spots, cancer genes, fragile sites, virus integration sites and gene size cohorts were tested in a statistical framework. Amplification-based clustering demonstrated that cancers with similar etiology, cell-of-origin or topographical location have a tendency to obtain convergent amplification profiles. The identified amplification hot spots were colocalized with the known fragile sites, cancer genes and virus integration sites, but global statistical significance could not be ascertained. Large genes were significantly overrepresented on the fragile sites and the reported amplification hot spots. These findings indicate that amplifications are selected in the cancer tissue environment according to the qualitative traits and localization of cancer genes.
DNA copy number amplifications activate oncogenes and are hallmarks of nearly all advanced tumors. Amplified genes represent attractive targets for therapy, diagnostics and prognostics. To investigate DNA amplifications in different neoplasms, we performed a bibliomics survey using 838 published chromosomal comparative genomic hybridization studies and collected amplification data at chromosome band resolution from more than 4500 cases. Amplification profiles were determined for 73 distinct neoplasms. Neoplasms were clustered according to the amplification profiles, and frequently amplified chromosomal loci (amplification hot spots) were identified using computational modeling. To investigate the site specificity and mechanisms of gene amplifications, colocalization of amplification hot spots, cancer genes, fragile sites, virus integration sites and gene size cohorts were tested in a statistical framework. Amplification-based clustering demonstrated that cancers with similar etiology, cell-of-origin or topographical location have a tendency to obtain convergent amplification profiles. The identified amplification hot spots were colocalized with the known fragile sites, cancer genes and virus integration sites, but global statistical significance could not be ascertained. Large genes were significantly overrepresented on the fragile sites and the reported amplification hot spots. These findings indicate that amplifications are selected in the cancer tissue environment according to the qualitative traits and localization of cancer genes.
The demonstration of the PAX8-PPAR(gamma) fusion oncogene in a subset of follicular thyroid tumors provides a new and promising starting point to dissect the molecular genetic events involved in the development of this tumor form. In the present study, we compared the gene expression profiles of follicular thyroid carcinomas (FTCs) bearing a PAX8-PPAR(gamma) fusion against FTCs that lack this fusion. Using unsupervised clustering and multidimensional scaling analyses, we show that FTCs possessing a PAX8-PPAR(gamma) fusion have a highly uniform and distinct gene expression signature that clearly distinguishes them from FTCs without the fusion. The PAX8-PPAR(gamma)(+) FTCs grouped in a defined cluster, where highly ranked genes were mostly associated with signal transduction, cell growth and translation control. Notably, a large number of ribosomal protein and translation-associated genes were concurrently underexpressed in the FTCs with the fusion. Taken together, our findings further support that follicular carcinomas with a PAX8-PPAR(gamma) rearrangement constitute a distinct biological entity. The current data represent one step to elucidate the molecular pathways in the development of FTCs with the specific PAX8-PPAR(gamma) fusion.
The demonstration of the PAX8-PPAR(gamma) fusion oncogene in a subset of follicular thyroid tumors provides a new and promising starting point to dissect the molecular genetic events involved in the development of this tumor form. In the present study, we compared the gene expression profiles of follicular thyroid carcinomas (FTCs) bearing a PAX8-PPAR(gamma) fusion against FTCs that lack this fusion. Using unsupervised clustering and multidimensional scaling analyses, we show that FTCs possessing a PAX8-PPAR(gamma) fusion have a highly uniform and distinct gene expression signature that clearly distinguishes them from FTCs without the fusion. The PAX8-PPAR(gamma)(+) FTCs grouped in a defined cluster, where highly ranked genes were mostly associated with signal transduction, cell growth and translation control. Notably, a large number of ribosomal protein and translation-associated genes were concurrently underexpressed in the FTCs with the fusion. Taken together, our findings further support that follicular carcinomas with a PAX8-PPAR(gamma) rearrangement constitute a distinct biological entity. The current data represent one step to elucidate the molecular pathways in the development of FTCs with the specific PAX8-PPAR(gamma) fusion.
The demonstration of the PAX8-PPAR(gamma) fusion oncogene in a subset of follicular thyroid tumors provides a new and promising starting point to dissect the molecular genetic events involved in the development of this tumor form. In the present study, we compared the gene expression profiles of follicular thyroid carcinomas (FTCs) bearing a PAX8-PPAR(gamma) fusion against FTCs that lack this fusion. Using unsupervised clustering and multidimensional scaling analyses, we show that FTCs possessing a PAX8-PPAR(gamma) fusion have a highly uniform and distinct gene expression signature that clearly distinguishes them from FTCs without the fusion. The PAX8-PPAR(gamma)(+) FTCs grouped in a defined cluster, where highly ranked genes were mostly associated with signal transduction, cell growth and translation control. Notably, a large number of ribosomal protein and translation-associated genes were concurrently underexpressed in the FTCs with the fusion. Taken together, our findings further support that follicular carcinomas with a PAX8-PPAR(gamma) rearrangement constitute a distinct biological entity. The current data represent one step to elucidate the molecular pathways in the development of FTCs with the specific PAX8-PPAR(gamma) fusion.
The demonstration of the PAX8-PPAR(gamma) fusion oncogene in a subset of follicular thyroid tumors provides a new and promising starting point to dissect the molecular genetic events involved in the development of this tumor form. In the present study, we compared the gene expression profiles of follicular thyroid carcinomas (FTCs) bearing a PAX8-PPAR(gamma) fusion against FTCs that lack this fusion. Using unsupervised clustering and multidimensional scaling analyses, we show that FTCs possessing a PAX8-PPAR(gamma) fusion have a highly uniform and distinct gene expression signature that clearly distinguishes them from FTCs without the fusion. The PAX8-PPAR(gamma)(+) FTCs grouped in a defined cluster, where highly ranked genes were mostly associated with signal transduction, cell growth and translation control. Notably, a large number of ribosomal protein and translation-associated genes were concurrently underexpressed in the FTCs with the fusion. Taken together, our findings further support that follicular carcinomas with a PAX8-PPAR(gamma) rearrangement constitute a distinct biological entity. The current data represent one step to elucidate the molecular pathways in the development of FTCs with the specific PAX8-PPAR(gamma) fusion.
The demonstration of the PAX8-PPAR(gamma) fusion oncogene in a subset of follicular thyroid tumors provides a new and promising starting point to dissect the molecular genetic events involved in the development of this tumor form. In the present study, we compared the gene expression profiles of follicular thyroid carcinomas (FTCs) bearing a PAX8-PPAR(gamma) fusion against FTCs that lack this fusion. Using unsupervised clustering and multidimensional scaling analyses, we show that FTCs possessing a PAX8-PPAR(gamma) fusion have a highly uniform and distinct gene expression signature that clearly distinguishes them from FTCs without the fusion. The PAX8-PPAR(gamma)(+) FTCs grouped in a defined cluster, where highly ranked genes were mostly associated with signal transduction, cell growth and translation control. Notably, a large number of ribosomal protein and translation-associated genes were concurrently underexpressed in the FTCs with the fusion. Taken together, our findings further support that follicular carcinomas with a PAX8-PPAR(gamma) rearrangement constitute a distinct biological entity. The current data represent one step to elucidate the molecular pathways in the development of FTCs with the specific PAX8-PPAR(gamma) fusion.
DNA copy number amplifications activate oncogenes and are hallmarks of nearly all advanced tumors. Amplified genes represent attractive targets for therapy, diagnostics and prognostics. To investigate DNA amplifications in different neoplasms, we performed a bibliomics survey using 838 published chromosomal comparative genomic hybridization studies and collected amplification data at chromosome band resolution from more than 4500 cases. Amplification profiles were determined for 73 distinct neoplasms. Neoplasms were clustered according to the amplification profiles, and frequently amplified chromosomal loci (amplification hot spots) were identified using computational modeling. To investigate the site specificity and mechanisms of gene amplifications, colocalization of amplification hot spots, cancer genes, fragile sites, virus integration sites and gene size cohorts were tested in a statistical framework. Amplification-based clustering demonstrated that cancers with similar etiology, cell-of-origin or topographical location have a tendency to obtain convergent amplification profiles. The identified amplification hot spots were colocalized with the known fragile sites, cancer genes and virus integration sites, but global statistical significance could not be ascertained. Large genes were significantly overrepresented on the fragile sites and the reported amplification hot spots. These findings indicate that amplifications are selected in the cancer tissue environment according to the qualitative traits and localization of cancer genes.
DNA copy number amplifications activate oncogenes and are hallmarks of nearly all advanced tumors. Amplified genes represent attractive targets for therapy, diagnostics and prognostics. To investigate DNA amplifications in different neoplasms, we performed a bibliomics survey using 838 published chromosomal comparative genomic hybridization studies and collected amplification data at chromosome band resolution from more than 4500 cases. Amplification profiles were determined for 73 distinct neoplasms. Neoplasms were clustered according to the amplification profiles, and frequently amplified chromosomal loci (amplification hot spots) were identified using computational modeling. To investigate the site specificity and mechanisms of gene amplifications, colocalization of amplification hot spots, cancer genes, fragile sites, virus integration sites and gene size cohorts were tested in a statistical framework. Amplification-based clustering demonstrated that cancers with similar etiology, cell-of-origin or topographical location have a tendency to obtain convergent amplification profiles. The identified amplification hot spots were colocalized with the known fragile sites, cancer genes and virus integration sites, but global statistical significance could not be ascertained. Large genes were significantly overrepresented on the fragile sites and the reported amplification hot spots. These findings indicate that amplifications are selected in the cancer tissue environment according to the qualitative traits and localization of cancer genes.
DNA copy number amplifications activate oncogenes and are hallmarks of nearly all advanced tumors. Amplified genes represent attractive targets for therapy, diagnostics and prognostics. To investigate DNA amplifications in different neoplasms, we performed a bibliomics survey using 838 published chromosomal comparative genomic hybridization studies and collected amplification data at chromosome band resolution from more than 4500 cases. Amplification profiles were determined for 73 distinct neoplasms. Neoplasms were clustered according to the amplification profiles, and frequently amplified chromosomal loci (amplification hot spots) were identified using computational modeling. To investigate the site specificity and mechanisms of gene amplifications, colocalization of amplification hot spots, cancer genes, fragile sites, virus integration sites and gene size cohorts were tested in a statistical framework. Amplification-based clustering demonstrated that cancers with similar etiology, cell-of-origin or topographical location have a tendency to obtain convergent amplification profiles. The identified amplification hot spots were colocalized with the known fragile sites, cancer genes and virus integration sites, but global statistical significance could not be ascertained. Large genes were significantly overrepresented on the fragile sites and the reported amplification hot spots. These findings indicate that amplifications are selected in the cancer tissue environment according to the qualitative traits and localization of cancer genes.
The paediatric eye tumour retinoblastoma is initiated by inactivation of RB1, a tumour suppressor on chromosome 13q. In addition to RB1 loss, many retinoblastomas show other genetic alterations including gains on chromosomes 6p21-pter and 1q31-q32. Recently, the minimal region of gains on chromosome 6 was narrowed to band p22. We examined genomic gains and expression changes in primary retinoblastomas to identify potential target genes in 6p22. Quantitative multiplex PCR detected copy numbers > or = 3 in 25 (33%) tumours and no gains in 31 of 76 (40%) tumours. The remaining 20 (26%) samples showed gains only at some loci, most often including E2F3 and DEK in 6p22.3. Analysis of RNA from 21 primary retinoblastomas showed that expression levels of these and some other genes in 6p22 correspond to DNA gains. However, KIF 13A, a reported candidate oncogene on 6p, was expressed at low levels or absent. Clinical manifestation of tumours with gains at all 6p22 loci was distinct in that distribution of age at diagnosis was markedly shifted to older age compared to tumours with no or partial gains. In summary, our results suggest that DEK and E2F3 are potential targets of 6p gains in retinoblastoma.
The demonstration of the PAX8-PPAR(gamma) fusion oncogene in a subset of follicular thyroid tumors provides a new and promising starting point to dissect the molecular genetic events involved in the development of this tumor form. In the present study, we compared the gene expression profiles of follicular thyroid carcinomas (FTCs) bearing a PAX8-PPAR(gamma) fusion against FTCs that lack this fusion. Using unsupervised clustering and multidimensional scaling analyses, we show that FTCs possessing a PAX8-PPAR(gamma) fusion have a highly uniform and distinct gene expression signature that clearly distinguishes them from FTCs without the fusion. The PAX8-PPAR(gamma)(+) FTCs grouped in a defined cluster, where highly ranked genes were mostly associated with signal transduction, cell growth and translation control. Notably, a large number of ribosomal protein and translation-associated genes were concurrently underexpressed in the FTCs with the fusion. Taken together, our findings further support that follicular carcinomas with a PAX8-PPAR(gamma) rearrangement constitute a distinct biological entity. The current data represent one step to elucidate the molecular pathways in the development of FTCs with the specific PAX8-PPAR(gamma) fusion.
Proliferation of higher eukaryotic cells is triggered by the proto-oncogene c-myc (myc), which is induced downstream of a large number of growth factor receptors. Myc, a basic helix-loop-helix leucine zipper transcription factor, transmits growth signals by up- and downregulation of target genes. The importance of Myc in growth control is well established. However, the number of growth control genes requiring Myc as an essential factor for regulation after mitogenic stimulation of cells is not yet clear. Here, we have studied the transcriptional programme of a human B-cell line, P493-6, in response to Myc and serum. P493-6 cells do not express the endogenous myc, nor is it induced by serum stimulation. Proliferation of the cells is dependent upon both the expression of a tetracycline-regulated myc gene and serum stimulation. Using DNA microarrays, expression profiling was performed following stimulation of cells with serum, with Myc, or with both. We observed serum regulation of >1000 genes. A number of these genes were synergistically or antagonistically regulated by Myc. Moreover, we identified >300 Myc-regulated genes that were almost unresponsive to serum. Gene ontology analysis revealed that a high proportion of Myc target genes are involved in ribosome biogenesis and tRNA metabolism. The data support our current notion that Myc is essential for the regulation of a large number of growth-related genes in B cells, and cannot be replaced by other serum-induced factors.
Proliferation of higher eukaryotic cells is triggered by the proto-oncogene c-myc (myc), which is induced downstream of a large number of growth factor receptors. Myc, a basic helix-loop-helix leucine zipper transcription factor, transmits growth signals by up- and downregulation of target genes. The importance of Myc in growth control is well established. However, the number of growth control genes requiring Myc as an essential factor for regulation after mitogenic stimulation of cells is not yet clear. Here, we have studied the transcriptional programme of a human B-cell line, P493-6, in response to Myc and serum. P493-6 cells do not express the endogenous myc, nor is it induced by serum stimulation. Proliferation of the cells is dependent upon both the expression of a tetracycline-regulated myc gene and serum stimulation. Using DNA microarrays, expression profiling was performed following stimulation of cells with serum, with Myc, or with both. We observed serum regulation of >1000 genes. A number of these genes were synergistically or antagonistically regulated by Myc. Moreover, we identified >300 Myc-regulated genes that were almost unresponsive to serum. Gene ontology analysis revealed that a high proportion of Myc target genes are involved in ribosome biogenesis and tRNA metabolism. The data support our current notion that Myc is essential for the regulation of a large number of growth-related genes in B cells, and cannot be replaced by other serum-induced factors.
Proliferation of higher eukaryotic cells is triggered by the proto-oncogene c-myc (myc), which is induced downstream of a large number of growth factor receptors. Myc, a basic helix-loop-helix leucine zipper transcription factor, transmits growth signals by up- and downregulation of target genes. The importance of Myc in growth control is well established. However, the number of growth control genes requiring Myc as an essential factor for regulation after mitogenic stimulation of cells is not yet clear. Here, we have studied the transcriptional programme of a human B-cell line, P493-6, in response to Myc and serum. P493-6 cells do not express the endogenous myc, nor is it induced by serum stimulation. Proliferation of the cells is dependent upon both the expression of a tetracycline-regulated myc gene and serum stimulation. Using DNA microarrays, expression profiling was performed following stimulation of cells with serum, with Myc, or with both. We observed serum regulation of >1000 genes. A number of these genes were synergistically or antagonistically regulated by Myc. Moreover, we identified >300 Myc-regulated genes that were almost unresponsive to serum. Gene ontology analysis revealed that a high proportion of Myc target genes are involved in ribosome biogenesis and tRNA metabolism. The data support our current notion that Myc is essential for the regulation of a large number of growth-related genes in B cells, and cannot be replaced by other serum-induced factors.
Proliferation of higher eukaryotic cells is triggered by the proto-oncogene c-myc (myc), which is induced downstream of a large number of growth factor receptors. Myc, a basic helix-loop-helix leucine zipper transcription factor, transmits growth signals by up- and downregulation of target genes. The importance of Myc in growth control is well established. However, the number of growth control genes requiring Myc as an essential factor for regulation after mitogenic stimulation of cells is not yet clear. Here, we have studied the transcriptional programme of a human B-cell line, P493-6, in response to Myc and serum. P493-6 cells do not express the endogenous myc, nor is it induced by serum stimulation. Proliferation of the cells is dependent upon both the expression of a tetracycline-regulated myc gene and serum stimulation. Using DNA microarrays, expression profiling was performed following stimulation of cells with serum, with Myc, or with both. We observed serum regulation of >1000 genes. A number of these genes were synergistically or antagonistically regulated by Myc. Moreover, we identified >300 Myc-regulated genes that were almost unresponsive to serum. Gene ontology analysis revealed that a high proportion of Myc target genes are involved in ribosome biogenesis and tRNA metabolism. The data support our current notion that Myc is essential for the regulation of a large number of growth-related genes in B cells, and cannot be replaced by other serum-induced factors.
Proliferation of higher eukaryotic cells is triggered by the proto-oncogene c-myc (myc), which is induced downstream of a large number of growth factor receptors. Myc, a basic helix-loop-helix leucine zipper transcription factor, transmits growth signals by up- and downregulation of target genes. The importance of Myc in growth control is well established. However, the number of growth control genes requiring Myc as an essential factor for regulation after mitogenic stimulation of cells is not yet clear. Here, we have studied the transcriptional programme of a human B-cell line, P493-6, in response to Myc and serum. P493-6 cells do not express the endogenous myc, nor is it induced by serum stimulation. Proliferation of the cells is dependent upon both the expression of a tetracycline-regulated myc gene and serum stimulation. Using DNA microarrays, expression profiling was performed following stimulation of cells with serum, with Myc, or with both. We observed serum regulation of >1000 genes. A number of these genes were synergistically or antagonistically regulated by Myc. Moreover, we identified >300 Myc-regulated genes that were almost unresponsive to serum. Gene ontology analysis revealed that a high proportion of Myc target genes are involved in ribosome biogenesis and tRNA metabolism. The data support our current notion that Myc is essential for the regulation of a large number of growth-related genes in B cells, and cannot be replaced by other serum-induced factors.
Proliferation of higher eukaryotic cells is triggered by the proto-oncogene c-myc (myc), which is induced downstream of a large number of growth factor receptors. Myc, a basic helix-loop-helix leucine zipper transcription factor, transmits growth signals by up- and downregulation of target genes. The importance of Myc in growth control is well established. However, the number of growth control genes requiring Myc as an essential factor for regulation after mitogenic stimulation of cells is not yet clear. Here, we have studied the transcriptional programme of a human B-cell line, P493-6, in response to Myc and serum. P493-6 cells do not express the endogenous myc, nor is it induced by serum stimulation. Proliferation of the cells is dependent upon both the expression of a tetracycline-regulated myc gene and serum stimulation. Using DNA microarrays, expression profiling was performed following stimulation of cells with serum, with Myc, or with both. We observed serum regulation of >1000 genes. A number of these genes were synergistically or antagonistically regulated by Myc. Moreover, we identified >300 Myc-regulated genes that were almost unresponsive to serum. Gene ontology analysis revealed that a high proportion of Myc target genes are involved in ribosome biogenesis and tRNA metabolism. The data support our current notion that Myc is essential for the regulation of a large number of growth-related genes in B cells, and cannot be replaced by other serum-induced factors.
Proliferation of higher eukaryotic cells is triggered by the proto-oncogene c-myc (myc), which is induced downstream of a large number of growth factor receptors. Myc, a basic helix-loop-helix leucine zipper transcription factor, transmits growth signals by up- and downregulation of target genes. The importance of Myc in growth control is well established. However, the number of growth control genes requiring Myc as an essential factor for regulation after mitogenic stimulation of cells is not yet clear. Here, we have studied the transcriptional programme of a human B-cell line, P493-6, in response to Myc and serum. P493-6 cells do not express the endogenous myc, nor is it induced by serum stimulation. Proliferation of the cells is dependent upon both the expression of a tetracycline-regulated myc gene and serum stimulation. Using DNA microarrays, expression profiling was performed following stimulation of cells with serum, with Myc, or with both. We observed serum regulation of >1000 genes. A number of these genes were synergistically or antagonistically regulated by Myc. Moreover, we identified >300 Myc-regulated genes that were almost unresponsive to serum. Gene ontology analysis revealed that a high proportion of Myc target genes are involved in ribosome biogenesis and tRNA metabolism. The data support our current notion that Myc is essential for the regulation of a large number of growth-related genes in B cells, and cannot be replaced by other serum-induced factors.
Multiple myeloma (MM) is the most common form of plasma cell dyscrasia, characterized by a marked heterogeneity of genetic lesions and clinical course. It may develop from a premalignant condition (monoclonal gammopathy of undetermined significance, MGUS) or progress from intramedullary to extramedullary forms (plasma cell leukemia, PCL). To provide insights into the molecular characterization of plasma cell dyscrasias and to investigate the contribution of specific genetic lesions to the biological and clinical heterogeneity of MM, we analysed the gene expression profiles of plasma cells isolated from seven MGUS, 39 MM and six PCL patients by means of DNA microarrays. MMs resulted highly heterogeneous at transcriptional level, whereas the differential expression of genes mainly involved in DNA metabolism and proliferation distinguished MGUS from PCLs and the majority of MM cases. The clustering of MM patients was mainly driven by the presence of the most recurrent translocations involving the immunoglobulin heavy-chain locus. Distinct gene expression patterns have been found to be associated with different lesions: the overexpression of CCND2 and genes involved in cell adhesion pathways was observed in cases with deregulated MAF and MAFB, whereas genes upregulated in cases with the t(4;14) showed apoptosis-related functions. The peculiar finding in patients with the t(11;14) was the downregulation of the alpha-subunit of the IL-6 receptor. In addition, we identified a set of cancer germline antigens specifically expressed in a subgroup of MM patients characterized by an aggressive clinical evolution, a finding that could have implications for patient classification and immunotherapy.
Multiple myeloma (MM) is the most common form of plasma cell dyscrasia, characterized by a marked heterogeneity of genetic lesions and clinical course. It may develop from a premalignant condition (monoclonal gammopathy of undetermined significance, MGUS) or progress from intramedullary to extramedullary forms (plasma cell leukemia, PCL). To provide insights into the molecular characterization of plasma cell dyscrasias and to investigate the contribution of specific genetic lesions to the biological and clinical heterogeneity of MM, we analysed the gene expression profiles of plasma cells isolated from seven MGUS, 39 MM and six PCL patients by means of DNA microarrays. MMs resulted highly heterogeneous at transcriptional level, whereas the differential expression of genes mainly involved in DNA metabolism and proliferation distinguished MGUS from PCLs and the majority of MM cases. The clustering of MM patients was mainly driven by the presence of the most recurrent translocations involving the immunoglobulin heavy-chain locus. Distinct gene expression patterns have been found to be associated with different lesions: the overexpression of CCND2 and genes involved in cell adhesion pathways was observed in cases with deregulated MAF and MAFB, whereas genes upregulated in cases with the t(4;14) showed apoptosis-related functions. The peculiar finding in patients with the t(11;14) was the downregulation of the alpha-subunit of the IL-6 receptor. In addition, we identified a set of cancer germline antigens specifically expressed in a subgroup of MM patients characterized by an aggressive clinical evolution, a finding that could have implications for patient classification and immunotherapy.
Previous microarray studies on breast cancer identified multiple tumour classes, of which the most prominent, named luminal and basal, differ in expression of the oestrogen receptor alpha gene (ER). We report here the identification of a group of breast tumours with increased androgen signalling and a 'molecular apocrine' gene expression profile. Tumour samples from 49 patients with large operable or locally advanced breast cancers were tested on Affymetrix U133A gene expression microarrays. Principal components analysis and hierarchical clustering split the tumours into three groups: basal, luminal and a group we call molecular apocrine. All of the molecular apocrine tumours have strong apocrine features on histological examination (P=0.0002). The molecular apocrine group is androgen receptor (AR) positive and contains all of the ER-negative tumours outside the basal group. Kolmogorov-Smirnov testing indicates that oestrogen signalling is most active in the luminal group, and androgen signalling is most active in the molecular apocrine group. ERBB2 amplification is commoner in the molecular apocrine than the other groups. Genes that best split the three groups were identified by Wilcoxon test. Correlation of the average expression profile of these genes in our data with the expression profile of individual tumours in four published breast cancer studies suggest that molecular apocrine tumours represent 8-14% of tumours in these studies. Our data show that it is possible with microarray data to divide mammary tumour cells into three groups based on steroid receptor activity: luminal (ER+ AR+), basal (ER- AR-) and molecular apocrine (ER- AR+).
Previous microarray studies on breast cancer identified multiple tumour classes, of which the most prominent, named luminal and basal, differ in expression of the oestrogen receptor alpha gene (ER). We report here the identification of a group of breast tumours with increased androgen signalling and a 'molecular apocrine' gene expression profile. Tumour samples from 49 patients with large operable or locally advanced breast cancers were tested on Affymetrix U133A gene expression microarrays. Principal components analysis and hierarchical clustering split the tumours into three groups: basal, luminal and a group we call molecular apocrine. All of the molecular apocrine tumours have strong apocrine features on histological examination (P=0.0002). The molecular apocrine group is androgen receptor (AR) positive and contains all of the ER-negative tumours outside the basal group. Kolmogorov-Smirnov testing indicates that oestrogen signalling is most active in the luminal group, and androgen signalling is most active in the molecular apocrine group. ERBB2 amplification is commoner in the molecular apocrine than the other groups. Genes that best split the three groups were identified by Wilcoxon test. Correlation of the average expression profile of these genes in our data with the expression profile of individual tumours in four published breast cancer studies suggest that molecular apocrine tumours represent 8-14% of tumours in these studies. Our data show that it is possible with microarray data to divide mammary tumour cells into three groups based on steroid receptor activity: luminal (ER+ AR+), basal (ER- AR-) and molecular apocrine (ER- AR+).
Previous microarray studies on breast cancer identified multiple tumour classes, of which the most prominent, named luminal and basal, differ in expression of the oestrogen receptor alpha gene (ER). We report here the identification of a group of breast tumours with increased androgen signalling and a 'molecular apocrine' gene expression profile. Tumour samples from 49 patients with large operable or locally advanced breast cancers were tested on Affymetrix U133A gene expression microarrays. Principal components analysis and hierarchical clustering split the tumours into three groups: basal, luminal and a group we call molecular apocrine. All of the molecular apocrine tumours have strong apocrine features on histological examination (P=0.0002). The molecular apocrine group is androgen receptor (AR) positive and contains all of the ER-negative tumours outside the basal group. Kolmogorov-Smirnov testing indicates that oestrogen signalling is most active in the luminal group, and androgen signalling is most active in the molecular apocrine group. ERBB2 amplification is commoner in the molecular apocrine than the other groups. Genes that best split the three groups were identified by Wilcoxon test. Correlation of the average expression profile of these genes in our data with the expression profile of individual tumours in four published breast cancer studies suggest that molecular apocrine tumours represent 8-14% of tumours in these studies. Our data show that it is possible with microarray data to divide mammary tumour cells into three groups based on steroid receptor activity: luminal (ER+ AR+), basal (ER- AR-) and molecular apocrine (ER- AR+).
Previous microarray studies on breast cancer identified multiple tumour classes, of which the most prominent, named luminal and basal, differ in expression of the oestrogen receptor alpha gene (ER). We report here the identification of a group of breast tumours with increased androgen signalling and a 'molecular apocrine' gene expression profile. Tumour samples from 49 patients with large operable or locally advanced breast cancers were tested on Affymetrix U133A gene expression microarrays. Principal components analysis and hierarchical clustering split the tumours into three groups: basal, luminal and a group we call molecular apocrine. All of the molecular apocrine tumours have strong apocrine features on histological examination (P=0.0002). The molecular apocrine group is androgen receptor (AR) positive and contains all of the ER-negative tumours outside the basal group. Kolmogorov-Smirnov testing indicates that oestrogen signalling is most active in the luminal group, and androgen signalling is most active in the molecular apocrine group. ERBB2 amplification is commoner in the molecular apocrine than the other groups. Genes that best split the three groups were identified by Wilcoxon test. Correlation of the average expression profile of these genes in our data with the expression profile of individual tumours in four published breast cancer studies suggest that molecular apocrine tumours represent 8-14% of tumours in these studies. Our data show that it is possible with microarray data to divide mammary tumour cells into three groups based on steroid receptor activity: luminal (ER+ AR+), basal (ER- AR-) and molecular apocrine (ER- AR+).
Previous microarray studies on breast cancer identified multiple tumour classes, of which the most prominent, named luminal and basal, differ in expression of the oestrogen receptor alpha gene (ER). We report here the identification of a group of breast tumours with increased androgen signalling and a 'molecular apocrine' gene expression profile. Tumour samples from 49 patients with large operable or locally advanced breast cancers were tested on Affymetrix U133A gene expression microarrays. Principal components analysis and hierarchical clustering split the tumours into three groups: basal, luminal and a group we call molecular apocrine. All of the molecular apocrine tumours have strong apocrine features on histological examination (P=0.0002). The molecular apocrine group is androgen receptor (AR) positive and contains all of the ER-negative tumours outside the basal group. Kolmogorov-Smirnov testing indicates that oestrogen signalling is most active in the luminal group, and androgen signalling is most active in the molecular apocrine group. ERBB2 amplification is commoner in the molecular apocrine than the other groups. Genes that best split the three groups were identified by Wilcoxon test. Correlation of the average expression profile of these genes in our data with the expression profile of individual tumours in four published breast cancer studies suggest that molecular apocrine tumours represent 8-14% of tumours in these studies. Our data show that it is possible with microarray data to divide mammary tumour cells into three groups based on steroid receptor activity: luminal (ER+ AR+), basal (ER- AR-) and molecular apocrine (ER- AR+).
Previous microarray studies on breast cancer identified multiple tumour classes, of which the most prominent, named luminal and basal, differ in expression of the oestrogen receptor alpha gene (ER). We report here the identification of a group of breast tumours with increased androgen signalling and a 'molecular apocrine' gene expression profile. Tumour samples from 49 patients with large operable or locally advanced breast cancers were tested on Affymetrix U133A gene expression microarrays. Principal components analysis and hierarchical clustering split the tumours into three groups: basal, luminal and a group we call molecular apocrine. All of the molecular apocrine tumours have strong apocrine features on histological examination (P=0.0002). The molecular apocrine group is androgen receptor (AR) positive and contains all of the ER-negative tumours outside the basal group. Kolmogorov-Smirnov testing indicates that oestrogen signalling is most active in the luminal group, and androgen signalling is most active in the molecular apocrine group. ERBB2 amplification is commoner in the molecular apocrine than the other groups. Genes that best split the three groups were identified by Wilcoxon test. Correlation of the average expression profile of these genes in our data with the expression profile of individual tumours in four published breast cancer studies suggest that molecular apocrine tumours represent 8-14% of tumours in these studies. Our data show that it is possible with microarray data to divide mammary tumour cells into three groups based on steroid receptor activity: luminal (ER+ AR+), basal (ER- AR-) and molecular apocrine (ER- AR+).
Previous microarray studies on breast cancer identified multiple tumour classes, of which the most prominent, named luminal and basal, differ in expression of the oestrogen receptor alpha gene (ER). We report here the identification of a group of breast tumours with increased androgen signalling and a 'molecular apocrine' gene expression profile. Tumour samples from 49 patients with large operable or locally advanced breast cancers were tested on Affymetrix U133A gene expression microarrays. Principal components analysis and hierarchical clustering split the tumours into three groups: basal, luminal and a group we call molecular apocrine. All of the molecular apocrine tumours have strong apocrine features on histological examination (P=0.0002). The molecular apocrine group is androgen receptor (AR) positive and contains all of the ER-negative tumours outside the basal group. Kolmogorov-Smirnov testing indicates that oestrogen signalling is most active in the luminal group, and androgen signalling is most active in the molecular apocrine group. ERBB2 amplification is commoner in the molecular apocrine than the other groups. Genes that best split the three groups were identified by Wilcoxon test. Correlation of the average expression profile of these genes in our data with the expression profile of individual tumours in four published breast cancer studies suggest that molecular apocrine tumours represent 8-14% of tumours in these studies. Our data show that it is possible with microarray data to divide mammary tumour cells into three groups based on steroid receptor activity: luminal (ER+ AR+), basal (ER- AR-) and molecular apocrine (ER- AR+).
Specific HPV DNA sequences are associated with more than 90% of invasive carcinomas of the uterine cervix. Viral E6 and E7 oncogenes are key mediators in cell transformation by disrupting TP53 and RB pathways. To investigate molecular mechanisms involved in the progression of invasive cervical carcinoma, we performed a gene expression study on cases selected according to viral and clinical parameters. Using Coupled Two-Way Clustering and Sorting Points Into Neighbourhoods methods, we identified a 'cervical cancer proliferation cluster' composed of 163 highly correlated transcripts. Most of these transcripts corresponded to E2F pathway genes controlling cell division or proliferation, whereas none was known as TP53 primary target. The average expression level of the genes of this cluster was higher in tumours with an early relapse than in tumours with a favourable course (P = 0.026). Moreover, we found that E6/E7 mRNA expression level was positively correlated with the expression level of the cluster genes and with viral DNA load. These findings suggest that HPV E6/E7 expression level plays a key role in the progression of invasive carcinoma of the uterine cervix via the deregulation of cellular genes controlling tumour cell proliferation. HPV expression level may thus provide a biological marker useful for prognosis assessment and specific therapy of the disease.
Transcription factors of the NF-kappaB family regulate hundreds of genes in the context of multiple important physiological and pathological processes. NF-kappaB activation depends on phosphorylation-induced proteolysis of inhibitory IkappaB molecules and NF-kappaB precursors by the ubiquitin-proteasome system. Most of the diverse signaling pathways that activate NF-kappaB converge on IkappaB kinases (IKK), which are essential for signal transmission. Many important details of the composition, regulation and biological function of IKK have been revealed in the last years. This review summarizes current aspects of structure and function of the regular stoichiometric components, the regulatory transient protein interactions of IKK and the mechanisms that contribute to its activation, deactivation and homeostasis. Both phosphorylation and ubiquitinatin (destructive as well as non-destructive) are crucial post-translational events in these processes. In addition to controlling induced IkappaB degradation in the cytoplasm and processing of the NF-kappaB precursor p100, nuclear IKK components have been found to act directly at the chromatin level of induced genes and to mediate responses to DNA damage. Finally, IKK is engaged in cross talk with other pathways and confers functions independently of NF-kappaB.
Molecular mechanisms contributing to initiation and progression of head and neck squamous cell carcinoma are still poorly known. Numerous genetic alterations have been described, but molecular consequences of such alterations in most cases remain unclear. Here, we performed an integrated high-resolution microarray analysis of gene copy number and expression in 20 laryngeal cancer cell lines and primary tumors. Our aim was to identify genetic alterations that play a key role in disease pathogenesis and pinpoint genes whose expression is directly impacted by these events. Integration of DNA level data from array-based comparative genomic hybridization with RNA level information from oligonucleotide microarrays was achieved with custom-developed bioinformatic methods. High-level amplifications had a clear impact on gene expression. Across the genome, overexpression of 739 genes could be attributed to gene amplification events in cell lines, with 325 genes showing the same phenomenon in primary tumors including FADD and PPFIA1 at 11q13. The analysis of gene ontology and pathway distributions further pinpointed genes that may identify potential targets of therapeutic intervention. Our data highlight genes that may be critically important to laryngeal cancer progression and offer potential therapeutic targets.
Ring chromosomes and/or giant marker chromosomes have been observed in a variety of human tumor types, but they are particularly common in a subgroup of mesenchymal tumors of low-grade or borderline malignancy. These rings and markers have been shown to contain amplified material predominantly from 12q13-15, but also sequences from other chromosomes. Such amplified sequences were mapped in detail by genome-wide array comparative genomic hybridization in ring-containing tumor samples from soft tissue (n = 15) and bone (n = 6), using tiling resolution microarrays, encompassing 32 433 bacterial artificial chromosome clones. The DNA copy number profiles revealed multiple amplification targets, in many cases highly discontinuous, leading to delineation of large numbers of very small amplicons. A total number of 356 (median size: 0.64 Mb) amplicons were seen in the soft tissue tumors and 90 (median size: 1.19 Mb) in the bone tumors. Notably, more than 40% of all amplicons in both soft tissue and bone tumors were mapped to chromosome 12, and at least one of the previously reported recurrent amplifications in 12q13.3-14.1 and 12q15.1, including SAS and CDK4, and MDM2, respectively, were present in 85% of the soft tissue tumors and in all of the bone tumors. Although chromosome 12 was the only chromosome displaying recurrent amplification in the bone tumors, the soft tissue tumors frequently showed recurrent amplicons mapping to other chromosomes, that is, 1p32, 1q23-24, 3p11-12, 6q24-25 and 20q11-12. Of particular interest, amplicons containing genes involved in the c-jun NH2-terminal kinase/mitogen-activated protein kinase pathway, that is, JUN in 1p32 and MAP3K7IP2 (TAB2) in 6q24-25, were found to be independently amplified in eight of 11 cases with 12q amplification, providing strong support for the notion that aberrant expression of this pathway is an important step in the dedifferentiation of liposarcomas.
Ring chromosomes and/or giant marker chromosomes have been observed in a variety of human tumor types, but they are particularly common in a subgroup of mesenchymal tumors of low-grade or borderline malignancy. These rings and markers have been shown to contain amplified material predominantly from 12q13-15, but also sequences from other chromosomes. Such amplified sequences were mapped in detail by genome-wide array comparative genomic hybridization in ring-containing tumor samples from soft tissue (n = 15) and bone (n = 6), using tiling resolution microarrays, encompassing 32 433 bacterial artificial chromosome clones. The DNA copy number profiles revealed multiple amplification targets, in many cases highly discontinuous, leading to delineation of large numbers of very small amplicons. A total number of 356 (median size: 0.64 Mb) amplicons were seen in the soft tissue tumors and 90 (median size: 1.19 Mb) in the bone tumors. Notably, more than 40% of all amplicons in both soft tissue and bone tumors were mapped to chromosome 12, and at least one of the previously reported recurrent amplifications in 12q13.3-14.1 and 12q15.1, including SAS and CDK4, and MDM2, respectively, were present in 85% of the soft tissue tumors and in all of the bone tumors. Although chromosome 12 was the only chromosome displaying recurrent amplification in the bone tumors, the soft tissue tumors frequently showed recurrent amplicons mapping to other chromosomes, that is, 1p32, 1q23-24, 3p11-12, 6q24-25 and 20q11-12. Of particular interest, amplicons containing genes involved in the c-jun NH2-terminal kinase/mitogen-activated protein kinase pathway, that is, JUN in 1p32 and MAP3K7IP2 (TAB2) in 6q24-25, were found to be independently amplified in eight of 11 cases with 12q amplification, providing strong support for the notion that aberrant expression of this pathway is an important step in the dedifferentiation of liposarcomas.
Mammalian Aurora-A is related to a serine/threonine protein kinase that was originally identified by its close homology with Saccharomyces cerevisiae Ipl1p and Drosophila melanogaster aurora that are key regulators in the orchestration of mitotic events. The protein level of Aurora-A, its peak kinase activity during mitosis, and its activation have been attributed to phosphorylation. Here we show that this enzyme is an arginine-directed kinase and define its substrate specificity. We also found that Thr288 within the activation loop is a critical residue for activating phosphorylation events in vitro and that it is spatiotemporally restricted to a brief window at mitosis on duplicated centrosomes and on spindle microtubules proximal to the poles in vivo. Immunodepletion assays indicated that an upstream kinase(s) of Aurora-A might exist in mammalian cells in addition to autophosphorylation. Furthermore, human activated Aurora-A forms complexes with the negative regulator protein serine/threonine phosphatase type 1 (PP1) that was negatively phosphorylated on Thr320. Interestingly, phospho-specific Aurora-A monoclonal antibodies restrain Aurora-A kinase activity in vitro, providing further therapeutic avenues to explore.
Histone deacetylase (HDAC) inhibitors are expected to be effective for refractory cancer because their mechanism of action differs from that of conventional antineoplastic agents. In this study, we examined the effect of the HDAC inhibitor FK228 on malignant melanoma, as well as its molecular mechanisms. FK228 was highly effective against melanoma compared with other commonly used drugs. By comparing the gene expression profiles of melanoma cells and normal melanocytes, we defined a subset of genes specifically upregulated in melanoma cells by FK228, which included Rap1, a small GTP-binding protein of the Ras family. The expression of Rap1 mRNA and protein increased in FK228-treated melanoma cells in both a dose- and a time-dependent manner. A decrease in the phosphorylation of c-Raf, MEK1/2, and ERK1/2 was accompanied by an increase in Rap1 expression in both FK228-treated and Rap1-overexpressing cells. Inhibition of Rap1 upregulation by small interfering RNA (siRNA) abrogated the induction of apoptosis and suppression of ERK1/2 phosphorylation in FK228-treated melanoma cells. These results indicate that the cytotoxic effects of FK228 are mediated via the upregulation of Rap1. Furthermore, we found that Rap1 was overexpressed and formed a complex with B-Raf in melanoma cell lines with a V599E mutation of B-Raf. The siRNA-mediated abrogation of Rap1 overexpression increased the viability of these cells, suggesting that Rap1 is also an endogenous regulator of Ras-MAP kinase signaling in melanomas.
p63, a p53 homolog has been shown to play a role in development and cancer. p63 is essential for both commitment of ectoderm to stratified epithelia and for the proliferative potential of epithelial stem cells. p63 knockout mice are born with severe development defects and lack organs of epithelial origin. In addition, p63 has also been shown to play a role in cancer development through the differential regulation of genes with tumor suppressor function and genes involved in metastasis. In order to understand the role of p63 in cancer and development, genes that are specifically regulated by p63 but not p53 were identified. In this study, we provide evidence that p63gamma specifically upregulates vitamin D Receptor (VDR). In contrast, p53 does not appear to be involved in upregulation of VDR expression. Additionally, we demonstrate that a naturally occurring p63 missense mutant, p63gamma (R279H) and p14(ARF), both act in a dominant negative manner to inhibit p63gamma-mediated upregulation of VDR. Furthermore, using chromatin immunoprecipitation assays, we demonstrated that p63 directly binds to the VDR promoter in vivo. Our findings clearly demonstrate that VDR is a direct target of p63 and suggests that p63 may play a role in cancer and differentiation through modulation of the VDR pathway.
Silencing of gene expression by methylation of CpG islands in regulatory elements is frequently observed in cancer. However, an influence of the most common oncogenic signalling pathways onto DNA methylation has not yet been investigated thoroughly. To address this issue, we identified genes suppressed in HRAS-transformed rat fibroblasts but upregulated after treatment with the demethylating agent 5-Aza-2-deoxycytidine and with the MEK1,2 inhibitor U0126. Analysis of gene expression by microarray and Northern blot analysis revealed the MEK/ERK target genes clusterin, matrix metalloproteinase 2 (Mmp2), peptidylpropyl isomerase C-associated protein, syndecan 4, Timp2 and Thbs1 to be repressed in the HRAS-transformed FE-8 cells in a MEK/ERK- and methylation-dependent manner. Hypermethylation of putative regulatory elements in HRAS-transformed cells as compared to immortalized fibroblasts was detected within a CpG island 14.5 kb upstream of clusterin, within the clusterin promoter and within a CpG island of the Mmp2 promoter by bisulphite sequencing. Furthermore, hypermethylation of the clusterin promoter was observed 10 days after induction of HRAS in immortalized rat fibroblasts and a clear correlation between reduced clusterin expression and hypermethlyation could also be observed in distinct rat tissues. These results suggest that silencing of individual genes by DNA methylation is controlled by oncogenic signalling pathways, yet the mechanisms responsible for initial target gene suppression are variable.
Tumors arising in BRCA1 and BRCA2 mutation carriers appear to have specific pathological and gene expression profiles, which show a high level of concordance. BRCA1 tumors are high-grade, negative for hormone receptors, have a high proliferation rate, and are positive for some cell cycle promoter genes. BRCA2 tumors present a phenotype opposite to BRCA1 tumors but very similar to sporadic tumors, except that BRCA2 overexpress some DNA repair markers such as CHEK2, show high cytoplasmic expression of RAD51, and are negative for HER-2 amplification and expression. Some of these characteristics have also been found in cDNA expression studies, although more analysis are necessary in order to obtain new markers that can be associated with a germ line mutation in BRCA1 or BRCA2. In this way, some studies in normal tissues of BRCA1/2 carriers suggest that differences exist in the level of expression of some genes when compared with noncarriers. Finally, IHC studies in tumors carrying a mutation in CHEK2 are rare and show contradictory results, probably due to the low number of these cases. However, they represent an example showing how different mutations of the same gene may be associated with specific histological subtypes of cancer.
A major obstacle for clinicians in the treatment of advanced prostate cancer is the inevitable progression to chemoresistance, especially to docetaxel. It is essential to understand the molecular events that lead to docetaxel resistance in order to identify means to prevent or interfere with chemoresistance. In initial attempts to detect these events, we analysed genomic differences between non-resistant and docetaxel-resistant prostate tumor cells and, of the genes modulated by docetaxel treatment, we observed Stat1 and clusterin gene expression heightened in the resistant phenotype. In this study, we provide biochemical and biological evidence that these two gene products are related. Stat1 and clusterin protein expression was induced upon docetaxel treatment of DU145 cells and highly overexpressed in the docetaxel-resistant DU145 cells (DU145-DR). The increase in total Stat1 corresponded to an increase in phosphorylated Stat1. Interestingly, there was no detectable difference between DU145 and DU145-DR cells expression of total Stat3 and phosphorylated Stat3. Treatment of DU145-DR cells with small interfering RNA targeted for Stat1 not only resulted in the knockdown of Stat1 expression, but it also caused the inhibition of clusterin expression. Thus, Stat1 appears to play a key role in the regulation of clusterin. Remarkably, inhibition of Stat1 or clusterin expression resulted in the re-sensitization of DU145-DR cells to docetaxel. These results offer the first evidence that Stat1, and its subsequent regulation of clusterin, are essential for docetaxel resistance in prostate cancer. Targeting this pathway could be a potential therapeutic means for intervention of docetaxel resistance.
Interactions between extracellular matrix (ECM) and mammary epithelial cells are critical for mammary gland homeostasis and apoptotic signaling. Interferon regulatory factor-1 (IRF-1) is a transcriptional regulator that promotes apoptosis during mammary gland involution and p53-independent apoptosis. We have recently shown that rapid cell surface tamoxifen (Tam) signaling promotes apoptosis in normal human mammary epithelial cells that were acutely damaged by expression of human papillomavirus type-16 E6 protein (*HMEC-E6). Apoptosis was mediated by recruitment of CREB-binding protein (CBP) to the gamma-activating sequence (GAS) element of the IRF-1 promoter, induction of IRF-1 and caspase-1/-3 activation. Here, we show that growth factor-depleted, reconstituted ECM (rECM), similar to Tam, promotes apoptosis in *HMEC-E6 cells through induction of IRF-1. Apoptosis was temporally associated with recruitment of CBP to the GAS element of the IRF-1 promoter, induction of IRF-1 expression and caspase-1/-3 activation. Small interfering RNA-mediated suppression of IRF-1 protein expression in *HMEC-E6 cells blocked (1) induction of IRF-1, (2) caspase-1/-3 activation and (3) apoptosis. These observations demonstrate that IRF-1 promotes rECM-mediated apoptosis and provide evidence that both rECM and rapid Tam signaling transcriptionally activate IRF-1 through recruitment of CBP to the IRF-1 GAS promoter complex.
Interactions between extracellular matrix (ECM) and mammary epithelial cells are critical for mammary gland homeostasis and apoptotic signaling. Interferon regulatory factor-1 (IRF-1) is a transcriptional regulator that promotes apoptosis during mammary gland involution and p53-independent apoptosis. We have recently shown that rapid cell surface tamoxifen (Tam) signaling promotes apoptosis in normal human mammary epithelial cells that were acutely damaged by expression of human papillomavirus type-16 E6 protein (*HMEC-E6). Apoptosis was mediated by recruitment of CREB-binding protein (CBP) to the gamma-activating sequence (GAS) element of the IRF-1 promoter, induction of IRF-1 and caspase-1/-3 activation. Here, we show that growth factor-depleted, reconstituted ECM (rECM), similar to Tam, promotes apoptosis in *HMEC-E6 cells through induction of IRF-1. Apoptosis was temporally associated with recruitment of CBP to the GAS element of the IRF-1 promoter, induction of IRF-1 expression and caspase-1/-3 activation. Small interfering RNA-mediated suppression of IRF-1 protein expression in *HMEC-E6 cells blocked (1) induction of IRF-1, (2) caspase-1/-3 activation and (3) apoptosis. These observations demonstrate that IRF-1 promotes rECM-mediated apoptosis and provide evidence that both rECM and rapid Tam signaling transcriptionally activate IRF-1 through recruitment of CBP to the IRF-1 GAS promoter complex.
Eucaryotic genes that are coordinately expressed tend to be clustered. Furthermore, gene clusters across chromosomal regions are often upregulated in various tumors. However, relatively little is known about how gene clusters are coordinately expressed in physiological or pathological conditions. Cofactor of BRCA1 (COBRA1), a subunit of the human negative elongation factor, has been shown to repress estrogen-stimulated transcription of trefoil factor 1 (TFF1 or pS2) by stalling RNA polymerase II. Here, we carried out a genome-wide study to identify additional physiological target genes of COBRA1 in breast cancer cells. The study identified a total of 134 genes that were either activated or repressed upon small hairpin RNA-mediated reduction of COBRA1. Interestingly, many COBRA1-regulated genes reside as clusters on the chromosomes and have been previously implicated in cancer development. Detailed examination of two such clusters on chromosome 21 (21q22) and chromosome X (Xp11) reveals that COBRA1 is physically associated with a subset of its regulated genes in each cluster. In addition, COBRA1 was shown to regulate both estrogen-dependent and -independent transcription of the gene cluster at 21q22, which encompasses the previously identified COBRA1-regulated TFF1 (pS2) locus. Thus, COBRA1 plays a critical role in the regulation of clustered gene expression at preferred chromosomal domains in breast cancer cells.
Eucaryotic genes that are coordinately expressed tend to be clustered. Furthermore, gene clusters across chromosomal regions are often upregulated in various tumors. However, relatively little is known about how gene clusters are coordinately expressed in physiological or pathological conditions. Cofactor of BRCA1 (COBRA1), a subunit of the human negative elongation factor, has been shown to repress estrogen-stimulated transcription of trefoil factor 1 (TFF1 or pS2) by stalling RNA polymerase II. Here, we carried out a genome-wide study to identify additional physiological target genes of COBRA1 in breast cancer cells. The study identified a total of 134 genes that were either activated or repressed upon small hairpin RNA-mediated reduction of COBRA1. Interestingly, many COBRA1-regulated genes reside as clusters on the chromosomes and have been previously implicated in cancer development. Detailed examination of two such clusters on chromosome 21 (21q22) and chromosome X (Xp11) reveals that COBRA1 is physically associated with a subset of its regulated genes in each cluster. In addition, COBRA1 was shown to regulate both estrogen-dependent and -independent transcription of the gene cluster at 21q22, which encompasses the previously identified COBRA1-regulated TFF1 (pS2) locus. Thus, COBRA1 plays a critical role in the regulation of clustered gene expression at preferred chromosomal domains in breast cancer cells.
The molecular pathways activated in response to acute cisplatin exposure, as well as the mechanisms involved in the long-term development of cisplatin-resistant cancer cells remain unclear. Using whole genome oligonucleotide microarrays, we have examined the kinetics of gene expression changes in a cisplatin-sensitive cell line, A2780, and its cisplatin-resistant derivative, ACRP. Both sensitive and resistant cell lines exhibited a very similar response of p53-inducible genes as early as 16 h after treatment. This p53 response was further increased at the 24-h time point. These experiments identify p53 as the main pathway producing a large-scale transcriptional response after cisplatin treatment in these cells containing wild-type p53. Consistent with a role for the p53 response in cisplatin sensitivity, knockdown of the p53 protein with small interfering RNA led to a twofold decrease in cell survival in the resistant cells. In addition, our analysis also allowed the identification of several genes that are differentially expressed between sensitive and resistant cells. These genes include GJA1 (encoding connexin 43 (Cx43)) and TWIST1, which are highly upregulated in cisplatin-resistant cells. The importance of Cx43 in drug resistance was demonstrated through functional analyses, although paradoxically, inhibition of Cx43 function in high expressing cells led to an increase in drug resistance. The pathways important in cisplatin response, as well as the genes found differentially expressed between cisplatin-resistant and -sensitive cells, may represent targets for therapy aimed at reversing drug resistance.
The molecular pathways activated in response to acute cisplatin exposure, as well as the mechanisms involved in the long-term development of cisplatin-resistant cancer cells remain unclear. Using whole genome oligonucleotide microarrays, we have examined the kinetics of gene expression changes in a cisplatin-sensitive cell line, A2780, and its cisplatin-resistant derivative, ACRP. Both sensitive and resistant cell lines exhibited a very similar response of p53-inducible genes as early as 16 h after treatment. This p53 response was further increased at the 24-h time point. These experiments identify p53 as the main pathway producing a large-scale transcriptional response after cisplatin treatment in these cells containing wild-type p53. Consistent with a role for the p53 response in cisplatin sensitivity, knockdown of the p53 protein with small interfering RNA led to a twofold decrease in cell survival in the resistant cells. In addition, our analysis also allowed the identification of several genes that are differentially expressed between sensitive and resistant cells. These genes include GJA1 (encoding connexin 43 (Cx43)) and TWIST1, which are highly upregulated in cisplatin-resistant cells. The importance of Cx43 in drug resistance was demonstrated through functional analyses, although paradoxically, inhibition of Cx43 function in high expressing cells led to an increase in drug resistance. The pathways important in cisplatin response, as well as the genes found differentially expressed between cisplatin-resistant and -sensitive cells, may represent targets for therapy aimed at reversing drug resistance.
The cytokine scatter factor (SF) (hepatocyte growth factor) transduces various biologic actions, including cell motility, invasion, angiogenesis and apoptosis inhibition. The latter is relevant to understanding the role of SF in promoting tumor cell survival in different contexts, for example, detachment from basement membrane, growth in metastatic sites and responses to chemo- and radiotherapy. Previously, we showed that SF protects cells against apoptosis owing to DNA damage, by a mechanism involving phosphoinositol-3-kinase/c-Akt signaling. Here, we used DNA microarray assays to identify c-Akt-regulated genes that might contribute to cell protection. DU-145 human prostate cancer cells were transfected+/-a dominant-negative mutant Akt, treated+/-SF and analysed for gene expression using Affymetrix arrays. These studies identified SF-regulated genes for which induction was c-Akt-dependent vs -independent. Selected microarray findings were confirmed by semiquantitative and quantitative reverse transcription-polymerase chain reaction. We tested the contribution of four SF-inducible/c-Akt-dependent genes (AMPD3, EPHB2, MX1 and WNT4) to protection against adriamycin (a DNA topoisomerase IIalpha inhibitor) using RNA interference. Knockdown of each gene except EPHB2 caused a small but significant reduction in the SF cell protection. The lack of effect of EPHB2 knockdown may be due to the fact that DU-145 cells contain a single-mutant EPHB2 allele. A combination of three small interfering RNAs blocked most of the protection by SF in both DU-145 and T47D cells. These findings identify novel c-Akt-regulated genes, some of which contribute to SF-mediated cytoprotection.
The cytokine scatter factor (SF) (hepatocyte growth factor) transduces various biologic actions, including cell motility, invasion, angiogenesis and apoptosis inhibition. The latter is relevant to understanding the role of SF in promoting tumor cell survival in different contexts, for example, detachment from basement membrane, growth in metastatic sites and responses to chemo- and radiotherapy. Previously, we showed that SF protects cells against apoptosis owing to DNA damage, by a mechanism involving phosphoinositol-3-kinase/c-Akt signaling. Here, we used DNA microarray assays to identify c-Akt-regulated genes that might contribute to cell protection. DU-145 human prostate cancer cells were transfected+/-a dominant-negative mutant Akt, treated+/-SF and analysed for gene expression using Affymetrix arrays. These studies identified SF-regulated genes for which induction was c-Akt-dependent vs -independent. Selected microarray findings were confirmed by semiquantitative and quantitative reverse transcription-polymerase chain reaction. We tested the contribution of four SF-inducible/c-Akt-dependent genes (AMPD3, EPHB2, MX1 and WNT4) to protection against adriamycin (a DNA topoisomerase IIalpha inhibitor) using RNA interference. Knockdown of each gene except EPHB2 caused a small but significant reduction in the SF cell protection. The lack of effect of EPHB2 knockdown may be due to the fact that DU-145 cells contain a single-mutant EPHB2 allele. A combination of three small interfering RNAs blocked most of the protection by SF in both DU-145 and T47D cells. These findings identify novel c-Akt-regulated genes, some of which contribute to SF-mediated cytoprotection.
The cytokine scatter factor (SF) (hepatocyte growth factor) transduces various biologic actions, including cell motility, invasion, angiogenesis and apoptosis inhibition. The latter is relevant to understanding the role of SF in promoting tumor cell survival in different contexts, for example, detachment from basement membrane, growth in metastatic sites and responses to chemo- and radiotherapy. Previously, we showed that SF protects cells against apoptosis owing to DNA damage, by a mechanism involving phosphoinositol-3-kinase/c-Akt signaling. Here, we used DNA microarray assays to identify c-Akt-regulated genes that might contribute to cell protection. DU-145 human prostate cancer cells were transfected+/-a dominant-negative mutant Akt, treated+/-SF and analysed for gene expression using Affymetrix arrays. These studies identified SF-regulated genes for which induction was c-Akt-dependent vs -independent. Selected microarray findings were confirmed by semiquantitative and quantitative reverse transcription-polymerase chain reaction. We tested the contribution of four SF-inducible/c-Akt-dependent genes (AMPD3, EPHB2, MX1 and WNT4) to protection against adriamycin (a DNA topoisomerase IIalpha inhibitor) using RNA interference. Knockdown of each gene except EPHB2 caused a small but significant reduction in the SF cell protection. The lack of effect of EPHB2 knockdown may be due to the fact that DU-145 cells contain a single-mutant EPHB2 allele. A combination of three small interfering RNAs blocked most of the protection by SF in both DU-145 and T47D cells. These findings identify novel c-Akt-regulated genes, some of which contribute to SF-mediated cytoprotection.
The cytokine scatter factor (SF) (hepatocyte growth factor) transduces various biologic actions, including cell motility, invasion, angiogenesis and apoptosis inhibition. The latter is relevant to understanding the role of SF in promoting tumor cell survival in different contexts, for example, detachment from basement membrane, growth in metastatic sites and responses to chemo- and radiotherapy. Previously, we showed that SF protects cells against apoptosis owing to DNA damage, by a mechanism involving phosphoinositol-3-kinase/c-Akt signaling. Here, we used DNA microarray assays to identify c-Akt-regulated genes that might contribute to cell protection. DU-145 human prostate cancer cells were transfected+/-a dominant-negative mutant Akt, treated+/-SF and analysed for gene expression using Affymetrix arrays. These studies identified SF-regulated genes for which induction was c-Akt-dependent vs -independent. Selected microarray findings were confirmed by semiquantitative and quantitative reverse transcription-polymerase chain reaction. We tested the contribution of four SF-inducible/c-Akt-dependent genes (AMPD3, EPHB2, MX1 and WNT4) to protection against adriamycin (a DNA topoisomerase IIalpha inhibitor) using RNA interference. Knockdown of each gene except EPHB2 caused a small but significant reduction in the SF cell protection. The lack of effect of EPHB2 knockdown may be due to the fact that DU-145 cells contain a single-mutant EPHB2 allele. A combination of three small interfering RNAs blocked most of the protection by SF in both DU-145 and T47D cells. These findings identify novel c-Akt-regulated genes, some of which contribute to SF-mediated cytoprotection.
The cytokine scatter factor (SF) (hepatocyte growth factor) transduces various biologic actions, including cell motility, invasion, angiogenesis and apoptosis inhibition. The latter is relevant to understanding the role of SF in promoting tumor cell survival in different contexts, for example, detachment from basement membrane, growth in metastatic sites and responses to chemo- and radiotherapy. Previously, we showed that SF protects cells against apoptosis owing to DNA damage, by a mechanism involving phosphoinositol-3-kinase/c-Akt signaling. Here, we used DNA microarray assays to identify c-Akt-regulated genes that might contribute to cell protection. DU-145 human prostate cancer cells were transfected+/-a dominant-negative mutant Akt, treated+/-SF and analysed for gene expression using Affymetrix arrays. These studies identified SF-regulated genes for which induction was c-Akt-dependent vs -independent. Selected microarray findings were confirmed by semiquantitative and quantitative reverse transcription-polymerase chain reaction. We tested the contribution of four SF-inducible/c-Akt-dependent genes (AMPD3, EPHB2, MX1 and WNT4) to protection against adriamycin (a DNA topoisomerase IIalpha inhibitor) using RNA interference. Knockdown of each gene except EPHB2 caused a small but significant reduction in the SF cell protection. The lack of effect of EPHB2 knockdown may be due to the fact that DU-145 cells contain a single-mutant EPHB2 allele. A combination of three small interfering RNAs blocked most of the protection by SF in both DU-145 and T47D cells. These findings identify novel c-Akt-regulated genes, some of which contribute to SF-mediated cytoprotection.
The cytokine scatter factor (SF) (hepatocyte growth factor) transduces various biologic actions, including cell motility, invasion, angiogenesis and apoptosis inhibition. The latter is relevant to understanding the role of SF in promoting tumor cell survival in different contexts, for example, detachment from basement membrane, growth in metastatic sites and responses to chemo- and radiotherapy. Previously, we showed that SF protects cells against apoptosis owing to DNA damage, by a mechanism involving phosphoinositol-3-kinase/c-Akt signaling. Here, we used DNA microarray assays to identify c-Akt-regulated genes that might contribute to cell protection. DU-145 human prostate cancer cells were transfected+/-a dominant-negative mutant Akt, treated+/-SF and analysed for gene expression using Affymetrix arrays. These studies identified SF-regulated genes for which induction was c-Akt-dependent vs -independent. Selected microarray findings were confirmed by semiquantitative and quantitative reverse transcription-polymerase chain reaction. We tested the contribution of four SF-inducible/c-Akt-dependent genes (AMPD3, EPHB2, MX1 and WNT4) to protection against adriamycin (a DNA topoisomerase IIalpha inhibitor) using RNA interference. Knockdown of each gene except EPHB2 caused a small but significant reduction in the SF cell protection. The lack of effect of EPHB2 knockdown may be due to the fact that DU-145 cells contain a single-mutant EPHB2 allele. A combination of three small interfering RNAs blocked most of the protection by SF in both DU-145 and T47D cells. These findings identify novel c-Akt-regulated genes, some of which contribute to SF-mediated cytoprotection.
The cytokine scatter factor (SF) (hepatocyte growth factor) transduces various biologic actions, including cell motility, invasion, angiogenesis and apoptosis inhibition. The latter is relevant to understanding the role of SF in promoting tumor cell survival in different contexts, for example, detachment from basement membrane, growth in metastatic sites and responses to chemo- and radiotherapy. Previously, we showed that SF protects cells against apoptosis owing to DNA damage, by a mechanism involving phosphoinositol-3-kinase/c-Akt signaling. Here, we used DNA microarray assays to identify c-Akt-regulated genes that might contribute to cell protection. DU-145 human prostate cancer cells were transfected+/-a dominant-negative mutant Akt, treated+/-SF and analysed for gene expression using Affymetrix arrays. These studies identified SF-regulated genes for which induction was c-Akt-dependent vs -independent. Selected microarray findings were confirmed by semiquantitative and quantitative reverse transcription-polymerase chain reaction. We tested the contribution of four SF-inducible/c-Akt-dependent genes (AMPD3, EPHB2, MX1 and WNT4) to protection against adriamycin (a DNA topoisomerase IIalpha inhibitor) using RNA interference. Knockdown of each gene except EPHB2 caused a small but significant reduction in the SF cell protection. The lack of effect of EPHB2 knockdown may be due to the fact that DU-145 cells contain a single-mutant EPHB2 allele. A combination of three small interfering RNAs blocked most of the protection by SF in both DU-145 and T47D cells. These findings identify novel c-Akt-regulated genes, some of which contribute to SF-mediated cytoprotection.
The cytokine scatter factor (SF) (hepatocyte growth factor) transduces various biologic actions, including cell motility, invasion, angiogenesis and apoptosis inhibition. The latter is relevant to understanding the role of SF in promoting tumor cell survival in different contexts, for example, detachment from basement membrane, growth in metastatic sites and responses to chemo- and radiotherapy. Previously, we showed that SF protects cells against apoptosis owing to DNA damage, by a mechanism involving phosphoinositol-3-kinase/c-Akt signaling. Here, we used DNA microarray assays to identify c-Akt-regulated genes that might contribute to cell protection. DU-145 human prostate cancer cells were transfected+/-a dominant-negative mutant Akt, treated+/-SF and analysed for gene expression using Affymetrix arrays. These studies identified SF-regulated genes for which induction was c-Akt-dependent vs -independent. Selected microarray findings were confirmed by semiquantitative and quantitative reverse transcription-polymerase chain reaction. We tested the contribution of four SF-inducible/c-Akt-dependent genes (AMPD3, EPHB2, MX1 and WNT4) to protection against adriamycin (a DNA topoisomerase IIalpha inhibitor) using RNA interference. Knockdown of each gene except EPHB2 caused a small but significant reduction in the SF cell protection. The lack of effect of EPHB2 knockdown may be due to the fact that DU-145 cells contain a single-mutant EPHB2 allele. A combination of three small interfering RNAs blocked most of the protection by SF in both DU-145 and T47D cells. These findings identify novel c-Akt-regulated genes, some of which contribute to SF-mediated cytoprotection.
The cytokine scatter factor (SF) (hepatocyte growth factor) transduces various biologic actions, including cell motility, invasion, angiogenesis and apoptosis inhibition. The latter is relevant to understanding the role of SF in promoting tumor cell survival in different contexts, for example, detachment from basement membrane, growth in metastatic sites and responses to chemo- and radiotherapy. Previously, we showed that SF protects cells against apoptosis owing to DNA damage, by a mechanism involving phosphoinositol-3-kinase/c-Akt signaling. Here, we used DNA microarray assays to identify c-Akt-regulated genes that might contribute to cell protection. DU-145 human prostate cancer cells were transfected+/-a dominant-negative mutant Akt, treated+/-SF and analysed for gene expression using Affymetrix arrays. These studies identified SF-regulated genes for which induction was c-Akt-dependent vs -independent. Selected microarray findings were confirmed by semiquantitative and quantitative reverse transcription-polymerase chain reaction. We tested the contribution of four SF-inducible/c-Akt-dependent genes (AMPD3, EPHB2, MX1 and WNT4) to protection against adriamycin (a DNA topoisomerase IIalpha inhibitor) using RNA interference. Knockdown of each gene except EPHB2 caused a small but significant reduction in the SF cell protection. The lack of effect of EPHB2 knockdown may be due to the fact that DU-145 cells contain a single-mutant EPHB2 allele. A combination of three small interfering RNAs blocked most of the protection by SF in both DU-145 and T47D cells. These findings identify novel c-Akt-regulated genes, some of which contribute to SF-mediated cytoprotection.
The cytokine scatter factor (SF) (hepatocyte growth factor) transduces various biologic actions, including cell motility, invasion, angiogenesis and apoptosis inhibition. The latter is relevant to understanding the role of SF in promoting tumor cell survival in different contexts, for example, detachment from basement membrane, growth in metastatic sites and responses to chemo- and radiotherapy. Previously, we showed that SF protects cells against apoptosis owing to DNA damage, by a mechanism involving phosphoinositol-3-kinase/c-Akt signaling. Here, we used DNA microarray assays to identify c-Akt-regulated genes that might contribute to cell protection. DU-145 human prostate cancer cells were transfected+/-a dominant-negative mutant Akt, treated+/-SF and analysed for gene expression using Affymetrix arrays. These studies identified SF-regulated genes for which induction was c-Akt-dependent vs -independent. Selected microarray findings were confirmed by semiquantitative and quantitative reverse transcription-polymerase chain reaction. We tested the contribution of four SF-inducible/c-Akt-dependent genes (AMPD3, EPHB2, MX1 and WNT4) to protection against adriamycin (a DNA topoisomerase IIalpha inhibitor) using RNA interference. Knockdown of each gene except EPHB2 caused a small but significant reduction in the SF cell protection. The lack of effect of EPHB2 knockdown may be due to the fact that DU-145 cells contain a single-mutant EPHB2 allele. A combination of three small interfering RNAs blocked most of the protection by SF in both DU-145 and T47D cells. These findings identify novel c-Akt-regulated genes, some of which contribute to SF-mediated cytoprotection.
Secreted protein acidic and rich in cysteine (SPARC) is an extracellular glycoprotein expressed in several solid cancers, including malignant gliomas, upon adoption of metastatic or invasive behaviors. SPARC expression in glioma cells promotes invasion and survival under stress, the latter process dependent on SPARC activation of AKT. Here we demonstrate that downregulation of SPARC expression with short interfering RNA (siRNA) in glioma cells decreased tumor cell survival and invasion. SPARC siRNA reduced the activating phosphorylation of AKT and two cytoplasmic kinases, focal adhesion kinase (FAK) and integrin-linked kinase (ILK). We determined the contributions of FAK and ILK to SPARC effects using SPARC protein and cell lines engineered to overexpress SPARC. SPARC activated FAK and ILK in glioma cells previously characterized as responsive to SPARC. Downregulation of either FAK or ILK expression inhibited SPARC-mediated AKT phosphorylation, and targeting both FAK and ILK attenuated AKT activation more potently than targeting either FAK or ILK alone. Decreased SPARC-mediated AKT activation correlated with a reduction in SPARC-dependent invasion and survival upon the downregulation of FAK and/or ILK expression. These data further demonstrate the role of SPARC in glioma tumor progression through the activation of intracellular kinases that may provide novel therapeutic targets for advanced cancers.
Secreted protein acidic and rich in cysteine (SPARC) is an extracellular glycoprotein expressed in several solid cancers, including malignant gliomas, upon adoption of metastatic or invasive behaviors. SPARC expression in glioma cells promotes invasion and survival under stress, the latter process dependent on SPARC activation of AKT. Here we demonstrate that downregulation of SPARC expression with short interfering RNA (siRNA) in glioma cells decreased tumor cell survival and invasion. SPARC siRNA reduced the activating phosphorylation of AKT and two cytoplasmic kinases, focal adhesion kinase (FAK) and integrin-linked kinase (ILK). We determined the contributions of FAK and ILK to SPARC effects using SPARC protein and cell lines engineered to overexpress SPARC. SPARC activated FAK and ILK in glioma cells previously characterized as responsive to SPARC. Downregulation of either FAK or ILK expression inhibited SPARC-mediated AKT phosphorylation, and targeting both FAK and ILK attenuated AKT activation more potently than targeting either FAK or ILK alone. Decreased SPARC-mediated AKT activation correlated with a reduction in SPARC-dependent invasion and survival upon the downregulation of FAK and/or ILK expression. These data further demonstrate the role of SPARC in glioma tumor progression through the activation of intracellular kinases that may provide novel therapeutic targets for advanced cancers.
Genomic instability can be found in most cancer cells. Cell proliferation is under tight control to ensure accurate DNA replication and chromosome segregation. Cyclin-dependent kinases (Cdks) and their activating subunits, the cyclins, are the driving forces of the cell division cycle. Regulation of cyclin oscillation by ubiquitin-dependent proteolysis thereby has a central role in cell cycle regulation. The anaphase-promoting complex (APC) is a specific ubiquitin ligase and is essential for chromosome segregation, exit from mitosis and a stable subsequent G1 phase allowing cell differentiation or accurate DNA replication in the following S phase. The APC is activated by the regulatory subunits Cdc20 (APC(Cdc20)) and Cdh1 (APC(Cdh1)) to target securin, mitotic cyclins and other cell cycle regulatory proteins for proteasomal degradation. This review is focused on the role of APC-dependent proteolysis in cell cycle regulation and how its deregulation may lead to genomic instability of cancer cells.
Our laboratory has delineated that the phosphatidylinositol 3' kinase (PI3K)/AKT/I kappa B kinase (IKK) pathway positively regulates NF kappa B and beta-catenin, both important transcriptional regulators in colorectal cancer (CRC). Therefore, we investigated the effect of inhibiting the PI3K/AKT/IKK alpha pathway in regulating the inappropriate constitutive activation of NF kappa B and beta-catenin in CRC cell lines. SW480 and RKO CRC cell lines demonstrate constitutive activation of AKT as well as both NF kappa B- and beta-catenin-dependent transcription. The constitutive activation of NF kappa B- and beta-catenin-dependent transcription is inhibited by transiently transfecting either kinase dead (KD) IKK alpha, which blocks IKK alpha kinase activity, KD AKT, which blocks AKT activity, or wildtype (WT) PTEN, which inhibits PI3K and AKT activity. The ability of KD IKK alpha, KD AKT or WT PTEN to decrease beta-catenin-dependent transcription is independent of their effects on NF kappa B. Inducible expression of either KD IKK alpha or WT PTEN strongly inhibits both the constitutive NF kappa B- and beta-catenin-dependent promoter and endogenous gene activation. Targeted array-based gene expression analysis of this inducible system reveals that many of the genes downregulated upon inhibition of this pathway are involved in tumor angiogenesis and metastasis. The activation of this pathway and the expression of the three most repressed genes was further analysed in samples of CRC. These results indicate a role of this pathway in controlling gene expression important in tumor progression and metastasis.
In acute myeloid leukemia (AML), constitutive activation of the FLT3 receptor tyrosine kinase, either by internal tandem duplications (FLT3-ITD) of the juxtamembrane region or by point mutations in the second tyrosine kinase domain (FLT3-TKD), as well as point mutations of the NRAS gene (NRAS-PM) are among the most frequent somatic gene mutations. To elucidate whether these mutations cause aberrant signal transduction in AML, we used gene expression profiling in a series of 110 newly diagnosed AML patients with normal karyotype. The different algorithms used for data analysis revealed highly concordant sets of genes, indicating that the identified gene signatures are specific for each analysed subgroup. Whereas samples with FLT3-ITD and FLT3-TKD could be separated with up to 100% accuracy, this did not apply for NRAS-PM and wild-type samples, suggesting that only FLT3-ITD and FLT3-TKD are associated with an apparent signature in AML. The set of discriminating genes included several known genes, which are involved in cell cycle control (CDC14A, WEE1), gene transcription (HOXB5, FOXA1), and signal transduction (SMG1). In conclusion, we showed that unique gene expression patterns can be correlated with FLT3-ITD and FLT3-TKD. This might lead to the identification of further pathogenetic relevant candidate genes particularly in AML with normal karyotype.
In acute myeloid leukemia (AML), constitutive activation of the FLT3 receptor tyrosine kinase, either by internal tandem duplications (FLT3-ITD) of the juxtamembrane region or by point mutations in the second tyrosine kinase domain (FLT3-TKD), as well as point mutations of the NRAS gene (NRAS-PM) are among the most frequent somatic gene mutations. To elucidate whether these mutations cause aberrant signal transduction in AML, we used gene expression profiling in a series of 110 newly diagnosed AML patients with normal karyotype. The different algorithms used for data analysis revealed highly concordant sets of genes, indicating that the identified gene signatures are specific for each analysed subgroup. Whereas samples with FLT3-ITD and FLT3-TKD could be separated with up to 100% accuracy, this did not apply for NRAS-PM and wild-type samples, suggesting that only FLT3-ITD and FLT3-TKD are associated with an apparent signature in AML. The set of discriminating genes included several known genes, which are involved in cell cycle control (CDC14A, WEE1), gene transcription (HOXB5, FOXA1), and signal transduction (SMG1). In conclusion, we showed that unique gene expression patterns can be correlated with FLT3-ITD and FLT3-TKD. This might lead to the identification of further pathogenetic relevant candidate genes particularly in AML with normal karyotype.
Latent membrane protein 1 (LMP1), an oncoprotein encoded by Epstein-Barr virus (EBV), is an integral membrane protein, which acts like a constitutively active receptor. LMP1 is critical for some facet of EBV's induction and maintenance of proliferation of infected B cells. It, in part, mimics signaling by the CD40 receptor and has been implicated in regulating proliferation, survival, or both properties of EBV-infected cells. We established a conditional LMP1 allele in the context of the intact EBV genome to define the immediate-early cellular target genes regulated by LMP1 in order to assess its contributions to infected human B cells. The functional analysis of this conditional system indicated that LMP1 specifically induces mitogenic B-cell activation through c-myc and Jun/AP1 family members and confirms its direct role in upregulating expression of multiple genes with opposing activities involved in cell survival. LMP1's signals were found to be essential for the G1/S transition in human B cells; cells lacking LMP1's signals are cell cycle arrested and survive quiescently. LMP1's activities are therefore not required to maintain survival in nonproliferating cells. LMP1 does induce both pro- and antiapoptotic genes whose balance seems to permit survival during LMP1's induction and maintenance of proliferation.
On the basis of epidemiological studies, infection was suggested to play a role in the etiology of human cancer. While for some cancers such a role was indeed demonstrated, there is no direct biological support for the role of viral pathogens in the pathogenesis of childhood leukemia. Using a novel bioinformatic tool that alternates between clustering and standard statistical methods of analysis, we performed a 'double-blind' search of published gene expression data of subjects with different childhood acute lymphoblastic leukemia (ALL) subtypes, looking for unanticipated partitions of patients, induced by unexpected groups of genes with correlated expression. We discovered a group of about 30 genes, related to the interferon response pathway, whose expression levels divide the ALL samples into two subgroups; high in 50, low in 285 patients. Leukemic subclasses prevalent in early childhood (the age most susceptible to infection) are over-represented in the high-expression subgroup. Similar partitions, induced by the same genes, were found also in breast and ovarian cancer but not in lung cancer, prostate cancer and lymphoma. About 40% of breast cancer samples expressed the 'interferon-related' signature. It is of interest that several studies demonstrated mouse mammary tumor virus-like sequences in about 40% of breast cancer samples. Our discovery of an unanticipated strong signature of an interferon-induced pathway provides molecular support for a role for either inflammation or viral infection in the pathogenesis of childhood leukemia as well as breast and ovarian cancer.
Gene expression responses of human cell lines exposed to a diverse set of stress agents were compared by cDNA microarray hybridization. The B-lymphoblastoid cell line TK6 (p53 wild-type) and its p53-null derivative, NH32, were treated in parallel to facilitate investigation of p53-dependent responses. RNA was extracted 4 h after the beginning of treatment when no notable decrease in cell viability was evident in the cultures. Gene expression signatures were defined that discriminated between four broad general mechanisms of stress agents: Non-DNA-damaging stresses (heat shock, osmotic shock, and 12-O-tetradecanoylphorbol 13-acetate), agents causing mainly oxidative stress (arsenite and hydrogen peroxide), ionizing radiations (neutron and gamma-ray exposures), and other DNA-damaging agents (ultraviolet radiation, methyl methanesulfonate, adriamycin, camptothecin, and cis-Platinum(II)diammine dichloride (cisplatin)). Within this data set, non-DNA-damaging stresses could be discriminated from all DNA-damaging stresses, and profiles for individual agents were also defined. While DNA-damaging stresses showed a strong p53-dependent element in their responses, no discernible p53-dependent responses were triggered by the non-DNA-damaging stresses. A set of 16 genes did exhibit a robust p53-dependent pattern of induction in response to all nine DNA-damaging agents, however.
Genomes of solid tumors are characterized by gains and losses of regions, which may contribute to tumorigenesis by altering gene expression. Often the aberrations are extensive, encompassing whole chromosome arms, which makes identification of candidate genes in these regions difficult. Here, we focused on narrow regions of gene amplification to facilitate identification of genetic pathways important in oral squamous cell carcinoma (SCC) development. We used array comparative genomic hybridization (array CGH) to define minimum common amplified regions and then used expression analysis to identify candidate driver genes in amplicons that spanned <3 Mb. We found genes involved in integrin signaling (TLN1), survival (YAP1, BIRC2), and adhesion and migration (TLN1, LAMA3, MMP7), as well as members of the hedgehog (GLI2) and notch (JAG1, RBPSUH, FJX1) pathways to be amplified and overexpressed. Deregulation of these and other members of the hedgehog and notch pathways (HHIP, SMO, DLL1, NOTCH4) implicates deregulation of developmental and differentiation pathways, cell fate misspecification, in oral SCC development.
Substantial genomic and functional evidence from primary tumors and cell lines indicates that a consistent region of distal chromosome 1p is deleted in a sizable proportion of human neuroblastomas, suggesting that this region contains one or more tumor suppressor genes. To determine systematically and precisely the location and extent of 1p deletion in neuroblastomas, we performed allelic loss studies of 737 primary neuroblastomas and genotype analysis of 46 neuroblastoma cell lines. Together, the results defined a single region within 1p36.3 that was consistently deleted in 25% of tumors and 87% of cell lines. Two neuroblastoma patients had constitutional deletions of distal 1p36 that overlapped the tumor-defined region. The tumor- and constitutionally-derived deletions together defined a smallest region of consistent deletion (SRD) between D1S2795 and D1S253. The 1p36.3 SRD was deleted in all but one of the 184 tumors with 1p deletion. Physical mapping and DNA sequencing determined that the SRD minimally spans an estimated 729 kb. Genomic content and sequence analysis of the SRD identified 15 characterized, nine uncharacterized, and six predicted genes in the region. The RNA expression profiles of 21 of the genes were investigated in a variety of normal tissues. The SHREW1 and KCNAB2 genes both had tissue-restricted expression patterns, including expression in the nervous system. In addition, a novel gene (CHD5) with strong homology to proteins involved in chromatin remodeling was expressed mainly in neural tissues. Together, these results suggest that one or more genes involved in neuroblastoma tumorigenesis or tumor progression are likely contained within this region.
The oncogenic fusion protein, Pax3/FKHR, is a more potent transcription factor relative to its normal counterpart, Pax3. Since Pax3 induced a mesenchymal to epithelial transition (MET) in human SaOS-2 osteosarcomas, we hypothesized that Pax3/FKHR would also induce a morphological change in SaOS-2 cells. We demonstrate here that Pax3/FKHR more potently induces a MET in SaOS-2 cells than Pax3. This greater potency was further evident where Pax3/FKHR, but not Pax3, induced a morphological alteration in U2-OS osteosarcoma cells. By microarray analysis, we determined that Pax3/FKHR altered the expression of gene targets in a manner quantitatively and qualitatively distinct from Pax3. Three classes of genes were identified: (i) genes induced or repressed by Pax3 and Pax3/FKHR, (ii) genes induced or repressed by Pax3/FKHR but not Pax3 and (iii) genes induced by Pax3/FKHR but repressed by Pax3. Chromatin immunoprecipitations confirmed the direct binding of Pax3/FKHR to the promoter region of several factors including cannabinoid receptor-1, EPHA2 and EPHA4. Verification of the microarray data also revealed coordinate alteration in the expression of factors involved in BMP4 signalling. Regulation of gene expression by Pax3 and Pax3/FKHR is, however, cell-type specific. BMP4 expression, for example, was repressed by both Pax3 and Pax3/FKHR in SaOS-2 cells, while in the rhabdomyosarcoma, RD, Pax3/FKHR, but not Pax3, induced BMP4 expression. Thus, our data reveal that Pax3/FKHR regulates a distinct but overlapping set of genes relative to Pax3 and that the global set of Pax3 and Pax3/FKHR gene targets is cell-type specific.
In all, 85% of Ewing's sarcoma family tumors (ESFT), a neoplasm of unknown histogenesis, express EWS-FLI1 transcription factor gene fusions. To characterize direct target genes avoiding artificial model systems, we cloned genomic DNA from ESFT chromatin precipitating with EWS-FLI1. We now present a comprehensive list of 99 putative transcription factor targets identified, for the first time, by a hypothesis-free approach based on physical interaction. Gene-derived chromatin fragments co-precipitating with EWS-FLI1 were nonrandomly distributed over the human genome and localized predominantly to the upstream region and the first two introns of the genes. At least 20% of putative direct EWS-FLI1 targets were neural genes. One-third of genes recovered showed a significant ESFT-specific expression pattern and were found to be altered upon RNAi-mediated knockdown of EWS-FLI1. Among them, MK-STYX, encoding a MAP kinase phosphatase-like protein, was consistently expressed in ESFT. EWS-FLI1 was found to drive MK-STYX expression by binding to a single ETS binding motif within the first gene intron. MK-STYX serves as precedence for successful recovery of direct EWS-FLI1 targets from the authentic ESFT cellular context, the most relevant system to study oncogenic mechanisms for the discovery of new therapeutic targets in this disease.
Wound healing and cancer are both characterized by cell proliferation, remodeling of extracellular matrix, cell invasion and migration, new blood vessel formation, and modulation of blood coagulation. The mechanisms that link wound healing and cancer are poorly understood. We report here that Stat3, a common signaling mechanism involved in oncogenesis and tissue injury, regulates a common set of genes involved in wound healing and cancer. Using oligonucleotide gene arrays and quantitative real-time PCR, we evaluated changes in global gene expression resulting from expression of Stat3 in lung epithelial cells. We report here previously uncharacterized genes induced by Stat3 implicated in signaling pathways common to both wound healing and cancer including cell invasion and migration, angiogenesis, modulation of coagulation, and repression of interferon-inducible genes. Consistent with these results, we found increased Stat3 activity associated with wound healing in chronically inflamed mouse lungs and increased Stat3 activity was identified at the leading edge of lung tumors invading adjacent nontumor stroma. These findings provide a molecular basis for understanding cancer as a deregulation of normal wound healing processes.
Wound healing and cancer are both characterized by cell proliferation, remodeling of extracellular matrix, cell invasion and migration, new blood vessel formation, and modulation of blood coagulation. The mechanisms that link wound healing and cancer are poorly understood. We report here that Stat3, a common signaling mechanism involved in oncogenesis and tissue injury, regulates a common set of genes involved in wound healing and cancer. Using oligonucleotide gene arrays and quantitative real-time PCR, we evaluated changes in global gene expression resulting from expression of Stat3 in lung epithelial cells. We report here previously uncharacterized genes induced by Stat3 implicated in signaling pathways common to both wound healing and cancer including cell invasion and migration, angiogenesis, modulation of coagulation, and repression of interferon-inducible genes. Consistent with these results, we found increased Stat3 activity associated with wound healing in chronically inflamed mouse lungs and increased Stat3 activity was identified at the leading edge of lung tumors invading adjacent nontumor stroma. These findings provide a molecular basis for understanding cancer as a deregulation of normal wound healing processes.
Male adult germ cell tumors (GCTs) comprise two major histologic groups: seminomas and nonseminomas. Nonseminomatous GCTs (NSGCTs) can be further divided into embryonal carcinoma (EC), teratoma (T), yolk sac tumor (YS), and choriocarcinoma (CC) on the basis of the lineage differentiation that they exhibit. NSGCTs frequently present as mixed tumors consisting of two or more histological subtypes, often limiting correlative studies of clinical and molecular features to histology. We sought to develop a molecular classifier that could predict the predominant histologic subtype within mixed NSGCT tumor samples. The expression profiles of 84 NSGCTs (42 pure and 42 mixed) and normal age-matched testes were obtained using Affymetrix microarrays. Using prediction analysis for microarrays, we identified 146 transcripts that classified the histology of pure NSGCTs samples with 93% accuracy. When applied to mixed NSGCTs, the classifier predicted a histology that was consistent with one of the reported components in 93% of cases. Among the predictive transcripts were CGB (high in CC), LCN2 (high in T), BMP2 (high in YS), and POU5F1 (high in EC). Thus, the expression-based classifier accurately assigned a single predominant histology to mixed NSGCTs, and identified transcripts differentially expressed between histologic components with relevance to NSGCT differentiation.
Male adult germ cell tumors (GCTs) comprise two major histologic groups: seminomas and nonseminomas. Nonseminomatous GCTs (NSGCTs) can be further divided into embryonal carcinoma (EC), teratoma (T), yolk sac tumor (YS), and choriocarcinoma (CC) on the basis of the lineage differentiation that they exhibit. NSGCTs frequently present as mixed tumors consisting of two or more histological subtypes, often limiting correlative studies of clinical and molecular features to histology. We sought to develop a molecular classifier that could predict the predominant histologic subtype within mixed NSGCT tumor samples. The expression profiles of 84 NSGCTs (42 pure and 42 mixed) and normal age-matched testes were obtained using Affymetrix microarrays. Using prediction analysis for microarrays, we identified 146 transcripts that classified the histology of pure NSGCTs samples with 93% accuracy. When applied to mixed NSGCTs, the classifier predicted a histology that was consistent with one of the reported components in 93% of cases. Among the predictive transcripts were CGB (high in CC), LCN2 (high in T), BMP2 (high in YS), and POU5F1 (high in EC). Thus, the expression-based classifier accurately assigned a single predominant histology to mixed NSGCTs, and identified transcripts differentially expressed between histologic components with relevance to NSGCT differentiation.
Male adult germ cell tumors (GCTs) comprise two major histologic groups: seminomas and nonseminomas. Nonseminomatous GCTs (NSGCTs) can be further divided into embryonal carcinoma (EC), teratoma (T), yolk sac tumor (YS), and choriocarcinoma (CC) on the basis of the lineage differentiation that they exhibit. NSGCTs frequently present as mixed tumors consisting of two or more histological subtypes, often limiting correlative studies of clinical and molecular features to histology. We sought to develop a molecular classifier that could predict the predominant histologic subtype within mixed NSGCT tumor samples. The expression profiles of 84 NSGCTs (42 pure and 42 mixed) and normal age-matched testes were obtained using Affymetrix microarrays. Using prediction analysis for microarrays, we identified 146 transcripts that classified the histology of pure NSGCTs samples with 93% accuracy. When applied to mixed NSGCTs, the classifier predicted a histology that was consistent with one of the reported components in 93% of cases. Among the predictive transcripts were CGB (high in CC), LCN2 (high in T), BMP2 (high in YS), and POU5F1 (high in EC). Thus, the expression-based classifier accurately assigned a single predominant histology to mixed NSGCTs, and identified transcripts differentially expressed between histologic components with relevance to NSGCT differentiation.
Male adult germ cell tumors (GCTs) comprise two major histologic groups: seminomas and nonseminomas. Nonseminomatous GCTs (NSGCTs) can be further divided into embryonal carcinoma (EC), teratoma (T), yolk sac tumor (YS), and choriocarcinoma (CC) on the basis of the lineage differentiation that they exhibit. NSGCTs frequently present as mixed tumors consisting of two or more histological subtypes, often limiting correlative studies of clinical and molecular features to histology. We sought to develop a molecular classifier that could predict the predominant histologic subtype within mixed NSGCT tumor samples. The expression profiles of 84 NSGCTs (42 pure and 42 mixed) and normal age-matched testes were obtained using Affymetrix microarrays. Using prediction analysis for microarrays, we identified 146 transcripts that classified the histology of pure NSGCTs samples with 93% accuracy. When applied to mixed NSGCTs, the classifier predicted a histology that was consistent with one of the reported components in 93% of cases. Among the predictive transcripts were CGB (high in CC), LCN2 (high in T), BMP2 (high in YS), and POU5F1 (high in EC). Thus, the expression-based classifier accurately assigned a single predominant histology to mixed NSGCTs, and identified transcripts differentially expressed between histologic components with relevance to NSGCT differentiation.
Treatment of the breast cancer cell line, MDAMB468 with the DNA methylation inhibitor, 5-azacytidine (5-AzaC) results in growth arrest, whereas the growth of the normal breast epithelial line DU99 (telomerase immortalized) is relatively unaffected. Comparing gene expression profiles of these two lines after 5-AzaC treatment, we identified 36 genes that had relatively low basal levels in MDAMB468 cells compared to the DU99 line and were induced in the cancer cell line but not in the normal breast epithelial line. Of these genes, 33 have associated CpG islands greater than 300 bp in length but only three have been previously described as targets for aberrant methylation in human cancer. Northern blotting for five of these genes (alpha-Catenin, DTR, FYN, GADD45a, and Zyxin) verified the array results. Further analysis of one of these genes, GADD45a, showed that 5-AzaC induced expression in five additional breast cancer cell lines with little or no induction in three additional lines derived from normal breast epithelial cells. The CpG island associated with GADD45a was analysed by bisulfite sequencing, sampling over 100 CpG dinucleotides. We found that four CpG's, located approximately 700 bp upstream of the transcriptional start site are methylated in the majority of breast cancer cell lines and primary tumors but not in DNA from normal breast epithelia or matched lymphocytes from cancer patients. Therefore, this simple method of dynamic transcriptional profiling yielded a series of novel methylation-sensitive genes in breast cancer including the BRCA1 and p53 responsive gene, GADD45a.
DNA copy number alterations are believed to play a major role in the development and progression of human neoplasms. Although most of these genomic imbalances have been associated with dysregulation of individual genes, their large-scale transcriptional consequences remain unclear. Pancreatic carcinomas frequently display gene copy number variation of entire chromosomes as well as of chromosomal subregions. These changes range from homozygous deletions to high-level amplifications and are believed to constitute key genetic alterations in the cellular transformation of this tumor type. To investigate the transcriptional consequences of the most drastic genomic changes, that is, genomic amplifications, and to analyse the genome-wide transcriptional effects of DNA copy number changes, we performed expression profiling of 29 pancreatic carcinoma cell lines and compared the results with matching genomic profiling data. We show that a strong association between DNA copy numbers and mRNA expression levels is present in pancreatic cancer, and demonstrate that as much as 60% of the genes within highly amplified genomic regions display associated overexpression. Consequently, we identified 67 recurrently overexpressed genes located in seven precisely mapped commonly amplified regions. The presented findings indicate that more than one putative target gene may be of importance in most pancreatic cancer amplicons.
Amplification of the MYCN oncogene contributes to the malignant progression of human neuroblastomas, but the mechanisms have remained unclear. We have previously demonstrated that N-Myc facilitates angiogenesis by downregulating an angiogenesis inhibitor identified as the inhibin betaA homodimer activin A. Here, we have sought to define the molecular, biological and clinical consequences of activin A expression in human neuroblastoma. We report that enhanced activin A expression suppresses proliferation and colony formation of human neuroblastoma cells with amplified MYCN in vitro; that it inhibits neuroblastoma growth and angiogenesis in vivo; that it is highly expressed in differentiated, but not undifferentiated human neuroblastomas; and that it correlates with favourable outcome of neuroblastoma patients. Our results indicate that high activin A expression plays an important beneficial role in human neuroblastoma.
Several investigations have supposed that tumor suppressor genes might be located on human chromosome 8. We used microcell-mediated transfer of chromosome 8 into MDA-MB-231 breast cancer cells and generated independent hybrids with strongly reduced tumorigenic potential. Loss of the transferred chromosome results in reappearance of the malignant phenotype. Expression analysis identified a set of 109 genes (CT8-ps) differentially expressed in microcell hybrids as compared to the tumorigenic MDA-MB-231 and rerevertant cells. Of these, 44.9% are differentially expressed in human breast tumors. The expression pattern of CT8-ps was associated with prognostic factors such as tumor size and grading as well as loss of heterozygosity at the short arm of chromosome 8. We identified CT8-ps networks suggesting that these genes act cooperatively to cause reversion of tumorigenicity in MDA-MB-231 cells. Our findings provide a conceptual basis and experimental system to identify and evaluate genes and gene networks involved in the development and/or progression of breast cancer.
Several investigations have supposed that tumor suppressor genes might be located on human chromosome 8. We used microcell-mediated transfer of chromosome 8 into MDA-MB-231 breast cancer cells and generated independent hybrids with strongly reduced tumorigenic potential. Loss of the transferred chromosome results in reappearance of the malignant phenotype. Expression analysis identified a set of 109 genes (CT8-ps) differentially expressed in microcell hybrids as compared to the tumorigenic MDA-MB-231 and rerevertant cells. Of these, 44.9% are differentially expressed in human breast tumors. The expression pattern of CT8-ps was associated with prognostic factors such as tumor size and grading as well as loss of heterozygosity at the short arm of chromosome 8. We identified CT8-ps networks suggesting that these genes act cooperatively to cause reversion of tumorigenicity in MDA-MB-231 cells. Our findings provide a conceptual basis and experimental system to identify and evaluate genes and gene networks involved in the development and/or progression of breast cancer.
Activating mutations of KIT and platelet-derived growth factor receptor alpha (PDGFRA) are known to be alternative and mutually exclusive genetic events in the development of gastrointestinal stromal tumors (GISTs). We examined the effect of the mutations of these two genes on the gene expression profile of 22 GISTs using the oligonucleotide microarray. Mutations of KIT and PDGFRA were found in 17 cases and three cases, respectively. The remaining two cases had no detectable mutations in either gene. The mutation status of KIT and PDGFRA was directly related to the expression levels of activated KIT and PDGFRA, and was also related to the different expression levels of activated proteins that play key roles in the downstream of the receptor tyrosine kinase III family. To evaluate the impact of mutation status and the importance of the type of mutation in gene expression and clinical features, microarray-derived data from 22 GISTs were interpreted using a principal component analysis (PCA). Three relevant principal component representing mutation of KIT, PDGFRA and chromosome 14q deletion were identified from the interpretation of the oligonucleotide microarray data with PCA. After supervised analysis, there was at least a two fold difference in expression between GISTs with KIT and PDGFRA mutation in 70 genes. Our findings demonstrate that mutations of KIT and PDGFRA affect differential activation and expression of some genes, and can be used for the molecular classification of GISTs.
Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) syndromes are characterized by deficiency in nucleotide excision repair pathway, but with distinguished clinical manifestations. While XP patients exhibit a high frequency of skin cancer, TTD patients are not cancer prone. The relation between lack of DNA repair and their clinical manifestations was investigated through analysis of the transcriptional profile of 12,600 transcripts in two isogenic cell lines with different capabilities of DNA repair. These cell lines result from a stable transfection of the XPB-TTD allele into XP complementation group B fibroblasts, from an XP patient who also have clinical abnormalities corresponding to Cockayne's syndrome (CS). The microarray assays performed under normal growth conditions showed the expression of distinct groups of genes in each cell line. The UVC-transcription modulation of these cells revealed the changes in 869 transcripts. Some of these transcripts had similar modulation pattern in both cells, although with eventually different time patterns for induction or repression. However, some different 'UVC signature' for each cell line was also found, that is, transcripts that were specifically UV regulated depending on the DNA repair status of the cell. These results provide a detailed portrait of expression profiles that may potentially unravel the causes of the different phenotypes of XP/CS and TTD patients.
Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) syndromes are characterized by deficiency in nucleotide excision repair pathway, but with distinguished clinical manifestations. While XP patients exhibit a high frequency of skin cancer, TTD patients are not cancer prone. The relation between lack of DNA repair and their clinical manifestations was investigated through analysis of the transcriptional profile of 12,600 transcripts in two isogenic cell lines with different capabilities of DNA repair. These cell lines result from a stable transfection of the XPB-TTD allele into XP complementation group B fibroblasts, from an XP patient who also have clinical abnormalities corresponding to Cockayne's syndrome (CS). The microarray assays performed under normal growth conditions showed the expression of distinct groups of genes in each cell line. The UVC-transcription modulation of these cells revealed the changes in 869 transcripts. Some of these transcripts had similar modulation pattern in both cells, although with eventually different time patterns for induction or repression. However, some different 'UVC signature' for each cell line was also found, that is, transcripts that were specifically UV regulated depending on the DNA repair status of the cell. These results provide a detailed portrait of expression profiles that may potentially unravel the causes of the different phenotypes of XP/CS and TTD patients.
Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) syndromes are characterized by deficiency in nucleotide excision repair pathway, but with distinguished clinical manifestations. While XP patients exhibit a high frequency of skin cancer, TTD patients are not cancer prone. The relation between lack of DNA repair and their clinical manifestations was investigated through analysis of the transcriptional profile of 12,600 transcripts in two isogenic cell lines with different capabilities of DNA repair. These cell lines result from a stable transfection of the XPB-TTD allele into XP complementation group B fibroblasts, from an XP patient who also have clinical abnormalities corresponding to Cockayne's syndrome (CS). The microarray assays performed under normal growth conditions showed the expression of distinct groups of genes in each cell line. The UVC-transcription modulation of these cells revealed the changes in 869 transcripts. Some of these transcripts had similar modulation pattern in both cells, although with eventually different time patterns for induction or repression. However, some different 'UVC signature' for each cell line was also found, that is, transcripts that were specifically UV regulated depending on the DNA repair status of the cell. These results provide a detailed portrait of expression profiles that may potentially unravel the causes of the different phenotypes of XP/CS and TTD patients.
Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) syndromes are characterized by deficiency in nucleotide excision repair pathway, but with distinguished clinical manifestations. While XP patients exhibit a high frequency of skin cancer, TTD patients are not cancer prone. The relation between lack of DNA repair and their clinical manifestations was investigated through analysis of the transcriptional profile of 12,600 transcripts in two isogenic cell lines with different capabilities of DNA repair. These cell lines result from a stable transfection of the XPB-TTD allele into XP complementation group B fibroblasts, from an XP patient who also have clinical abnormalities corresponding to Cockayne's syndrome (CS). The microarray assays performed under normal growth conditions showed the expression of distinct groups of genes in each cell line. The UVC-transcription modulation of these cells revealed the changes in 869 transcripts. Some of these transcripts had similar modulation pattern in both cells, although with eventually different time patterns for induction or repression. However, some different 'UVC signature' for each cell line was also found, that is, transcripts that were specifically UV regulated depending on the DNA repair status of the cell. These results provide a detailed portrait of expression profiles that may potentially unravel the causes of the different phenotypes of XP/CS and TTD patients.
Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) syndromes are characterized by deficiency in nucleotide excision repair pathway, but with distinguished clinical manifestations. While XP patients exhibit a high frequency of skin cancer, TTD patients are not cancer prone. The relation between lack of DNA repair and their clinical manifestations was investigated through analysis of the transcriptional profile of 12,600 transcripts in two isogenic cell lines with different capabilities of DNA repair. These cell lines result from a stable transfection of the XPB-TTD allele into XP complementation group B fibroblasts, from an XP patient who also have clinical abnormalities corresponding to Cockayne's syndrome (CS). The microarray assays performed under normal growth conditions showed the expression of distinct groups of genes in each cell line. The UVC-transcription modulation of these cells revealed the changes in 869 transcripts. Some of these transcripts had similar modulation pattern in both cells, although with eventually different time patterns for induction or repression. However, some different 'UVC signature' for each cell line was also found, that is, transcripts that were specifically UV regulated depending on the DNA repair status of the cell. These results provide a detailed portrait of expression profiles that may potentially unravel the causes of the different phenotypes of XP/CS and TTD patients.
Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) syndromes are characterized by deficiency in nucleotide excision repair pathway, but with distinguished clinical manifestations. While XP patients exhibit a high frequency of skin cancer, TTD patients are not cancer prone. The relation between lack of DNA repair and their clinical manifestations was investigated through analysis of the transcriptional profile of 12,600 transcripts in two isogenic cell lines with different capabilities of DNA repair. These cell lines result from a stable transfection of the XPB-TTD allele into XP complementation group B fibroblasts, from an XP patient who also have clinical abnormalities corresponding to Cockayne's syndrome (CS). The microarray assays performed under normal growth conditions showed the expression of distinct groups of genes in each cell line. The UVC-transcription modulation of these cells revealed the changes in 869 transcripts. Some of these transcripts had similar modulation pattern in both cells, although with eventually different time patterns for induction or repression. However, some different 'UVC signature' for each cell line was also found, that is, transcripts that were specifically UV regulated depending on the DNA repair status of the cell. These results provide a detailed portrait of expression profiles that may potentially unravel the causes of the different phenotypes of XP/CS and TTD patients.
Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) syndromes are characterized by deficiency in nucleotide excision repair pathway, but with distinguished clinical manifestations. While XP patients exhibit a high frequency of skin cancer, TTD patients are not cancer prone. The relation between lack of DNA repair and their clinical manifestations was investigated through analysis of the transcriptional profile of 12,600 transcripts in two isogenic cell lines with different capabilities of DNA repair. These cell lines result from a stable transfection of the XPB-TTD allele into XP complementation group B fibroblasts, from an XP patient who also have clinical abnormalities corresponding to Cockayne's syndrome (CS). The microarray assays performed under normal growth conditions showed the expression of distinct groups of genes in each cell line. The UVC-transcription modulation of these cells revealed the changes in 869 transcripts. Some of these transcripts had similar modulation pattern in both cells, although with eventually different time patterns for induction or repression. However, some different 'UVC signature' for each cell line was also found, that is, transcripts that were specifically UV regulated depending on the DNA repair status of the cell. These results provide a detailed portrait of expression profiles that may potentially unravel the causes of the different phenotypes of XP/CS and TTD patients.
We wanted to identify genes regulated by LMP1's signaling to determine its role in directing the proliferation and survival of its host cell. For this, we developed a derivative of EBV in which LMP1 is an oncoprotein encoded by Epstein-Barr virus (EBV). it is an integral membrane protein and acts like a constitutively active receptor. We have developed a derivative of EBV in which LMP1 could be expressed conditionally dependent on tetracycline. B2264-19/3 cells are primary human B-lymphocytes infected with the recombinant EBV virus. Cluster 3 includes genes upregulated late after the activation of LMP1???s signaling, which are regulators of transcription, differentially expressed signaling mediators, and nearly all of the identi???ed LMP1-induced metabolic and structural proteins. identi???ed LMP1-induced metabolic and structural
We wanted to identify genes regulated by LMP1's signaling to determine its role in directing the proliferation and survival of its host cell. For this, we developed a derivative of EBV in which LMP1 is an oncoprotein encoded by Epstein-Barr virus (EBV). it is an integral membrane protein and acts like a constitutively active receptor. We have developed a derivative of EBV in which LMP1 could be expressed conditionally dependent on tetracycline. B2264-19/3 cells are primary human B-lymphocytes infected with the recombinant EBV virus. Cluster 3 includes genes upregulated late after the activation of LMP1???s signaling, which are regulators of transcription, differentially expressed signaling mediators, and nearly all of the identi???ed LMP1-induced metabolic and structural proteins. identi???ed LMP1-induced metabolic and structural
DNA copy number alterations are believed to play a major role in the development and progression of human neoplasms. Although most of these genomic imbalances have been associated with dysregulation of individual genes, their large-scale transcriptional consequences remain unclear. Pancreatic carcinomas frequently display gene copy number variation of entire chromosomes as well as of chromosomal subregions. These changes range from homozygous deletions to high-level amplifications and are believed to constitute key genetic alterations in the cellular transformation of this tumor type. To investigate the transcriptional consequences of the most drastic genomic changes, that is, genomic amplifications, and to analyse the genome-wide transcriptional effects of DNA copy number changes, we performed expression profiling of 29 pancreatic carcinoma cell lines and compared the results with matching genomic profiling data. We show that a strong association between DNA copy numbers and mRNA expression levels is present in pancreatic cancer, and demonstrate that as much as 60% of the genes within highly amplified genomic regions display associated overexpression. Consequently, we identified 67 recurrently overexpressed genes located in seven precisely mapped commonly amplified regions. The presented findings indicate that more than one putative target gene may be of importance in most pancreatic cancer amplicons.
DNA copy number alterations are believed to play a major role in the development and progression of human neoplasms. Although most of these genomic imbalances have been associated with dysregulation of individual genes, their large-scale transcriptional consequences remain unclear. Pancreatic carcinomas frequently display gene copy number variation of entire chromosomes as well as of chromosomal subregions. These changes range from homozygous deletions to high-level amplifications and are believed to constitute key genetic alterations in the cellular transformation of this tumor type. To investigate the transcriptional consequences of the most drastic genomic changes, that is, genomic amplifications, and to analyse the genome-wide transcriptional effects of DNA copy number changes, we performed expression profiling of 29 pancreatic carcinoma cell lines and compared the results with matching genomic profiling data. We show that a strong association between DNA copy numbers and mRNA expression levels is present in pancreatic cancer, and demonstrate that as much as 60% of the genes within highly amplified genomic regions display associated overexpression. Consequently, we identified 67 recurrently overexpressed genes located in seven precisely mapped commonly amplified regions. The presented findings indicate that more than one putative target gene may be of importance in most pancreatic cancer amplicons.
DNA copy number alterations are believed to play a major role in the development and progression of human neoplasms. Although most of these genomic imbalances have been associated with dysregulation of individual genes, their large-scale transcriptional consequences remain unclear. Pancreatic carcinomas frequently display gene copy number variation of entire chromosomes as well as of chromosomal subregions. These changes range from homozygous deletions to high-level amplifications and are believed to constitute key genetic alterations in the cellular transformation of this tumor type. To investigate the transcriptional consequences of the most drastic genomic changes, that is, genomic amplifications, and to analyse the genome-wide transcriptional effects of DNA copy number changes, we performed expression profiling of 29 pancreatic carcinoma cell lines and compared the results with matching genomic profiling data. We show that a strong association between DNA copy numbers and mRNA expression levels is present in pancreatic cancer, and demonstrate that as much as 60% of the genes within highly amplified genomic regions display associated overexpression. Consequently, we identified 67 recurrently overexpressed genes located in seven precisely mapped commonly amplified regions. The presented findings indicate that more than one putative target gene may be of importance in most pancreatic cancer amplicons.
DNA copy number alterations are believed to play a major role in the development and progression of human neoplasms. Although most of these genomic imbalances have been associated with dysregulation of individual genes, their large-scale transcriptional consequences remain unclear. Pancreatic carcinomas frequently display gene copy number variation of entire chromosomes as well as of chromosomal subregions. These changes range from homozygous deletions to high-level amplifications and are believed to constitute key genetic alterations in the cellular transformation of this tumor type. To investigate the transcriptional consequences of the most drastic genomic changes, that is, genomic amplifications, and to analyse the genome-wide transcriptional effects of DNA copy number changes, we performed expression profiling of 29 pancreatic carcinoma cell lines and compared the results with matching genomic profiling data. We show that a strong association between DNA copy numbers and mRNA expression levels is present in pancreatic cancer, and demonstrate that as much as 60% of the genes within highly amplified genomic regions display associated overexpression. Consequently, we identified 67 recurrently overexpressed genes located in seven precisely mapped commonly amplified regions. The presented findings indicate that more than one putative target gene may be of importance in most pancreatic cancer amplicons.
Gene expression responses of human cell lines exposed to a diverse set of stress agents were compared by cDNA microarray hybridization. The B-lymphoblastoid cell line TK6 (p53 wild-type) and its p53-null derivative, NH32, were treated in parallel to facilitate investigation of p53-dependent responses. RNA was extracted 4 h after the beginning of treatment when no notable decrease in cell viability was evident in the cultures. Gene expression signatures were defined that discriminated between four broad general mechanisms of stress agents: Non-DNA-damaging stresses (heat shock, osmotic shock, and 12-O-tetradecanoylphorbol 13-acetate), agents causing mainly oxidative stress (arsenite and hydrogen peroxide), ionizing radiations (neutron and gamma-ray exposures), and other DNA-damaging agents (ultraviolet radiation, methyl methanesulfonate, adriamycin, camptothecin, and cis-Platinum(II)diammine dichloride (cisplatin)). Within this data set, non-DNA-damaging stresses could be discriminated from all DNA-damaging stresses, and profiles for individual agents were also defined. While DNA-damaging stresses showed a strong p53-dependent element in their responses, no discernible p53-dependent responses were triggered by the non-DNA-damaging stresses. A set of 16 genes did exhibit a robust p53-dependent pattern of induction in response to all nine DNA-damaging agents, however.
Gene expression responses of human cell lines exposed to a diverse set of stress agents were compared by cDNA microarray hybridization. The B-lymphoblastoid cell line TK6 (p53 wild-type) and its p53-null derivative, NH32, were treated in parallel to facilitate investigation of p53-dependent responses. RNA was extracted 4 h after the beginning of treatment when no notable decrease in cell viability was evident in the cultures. Gene expression signatures were defined that discriminated between four broad general mechanisms of stress agents: Non-DNA-damaging stresses (heat shock, osmotic shock, and 12-O-tetradecanoylphorbol 13-acetate), agents causing mainly oxidative stress (arsenite and hydrogen peroxide), ionizing radiations (neutron and gamma-ray exposures), and other DNA-damaging agents (ultraviolet radiation, methyl methanesulfonate, adriamycin, camptothecin, and cis-Platinum(II)diammine dichloride (cisplatin)). Within this data set, non-DNA-damaging stresses could be discriminated from all DNA-damaging stresses, and profiles for individual agents were also defined. While DNA-damaging stresses showed a strong p53-dependent element in their responses, no discernible p53-dependent responses were triggered by the non-DNA-damaging stresses. A set of 16 genes did exhibit a robust p53-dependent pattern of induction in response to all nine DNA-damaging agents, however.
Interleukin-2 (IL-2) mediates cell cycle progression and antiapoptosis in human T cells via several signal transduction pathways. The Tax protein of the human T-cell leukemia virus type I (HTLV-1) deregulates cell growth and alters the role of IL-2 in infected cells. However, Tax-immortalized cells stay dependent on IL-2, suggesting that events besides HTLV-1 gene expression are required for leukemia to develop. Here, IL-2-dependent and -independent events were analysed in a human T cell line immortalized by Tax. These studies show that, of the signaling pathways evaluated, only STAT5 remains dependent. Microarray analyses revealed several genes, including il-5, il-9 and il-13, are uniquely upregulated by IL-2 in the presence of Tax. Bioinformatics and supporting molecular biology show that some of these genes are STAT5 targets, explaining their IL-2 upregulation. These results suggest that IL-2 and viral proteins work together to induce gene expression, promoting the hypothesis that deregulation via the constitutive activation of STAT5 may lead to the IL-2-independent phenotype of HTLV-1-transformed cells.
Interleukin-2 (IL-2) mediates cell cycle progression and antiapoptosis in human T cells via several signal transduction pathways. The Tax protein of the human T-cell leukemia virus type I (HTLV-1) deregulates cell growth and alters the role of IL-2 in infected cells. However, Tax-immortalized cells stay dependent on IL-2, suggesting that events besides HTLV-1 gene expression are required for leukemia to develop. Here, IL-2-dependent and -independent events were analysed in a human T cell line immortalized by Tax. These studies show that, of the signaling pathways evaluated, only STAT5 remains dependent. Microarray analyses revealed several genes, including il-5, il-9 and il-13, are uniquely upregulated by IL-2 in the presence of Tax. Bioinformatics and supporting molecular biology show that some of these genes are STAT5 targets, explaining their IL-2 upregulation. These results suggest that IL-2 and viral proteins work together to induce gene expression, promoting the hypothesis that deregulation via the constitutive activation of STAT5 may lead to the IL-2-independent phenotype of HTLV-1-transformed cells.
Interleukin-2 (IL-2) mediates cell cycle progression and antiapoptosis in human T cells via several signal transduction pathways. The Tax protein of the human T-cell leukemia virus type I (HTLV-1) deregulates cell growth and alters the role of IL-2 in infected cells. However, Tax-immortalized cells stay dependent on IL-2, suggesting that events besides HTLV-1 gene expression are required for leukemia to develop. Here, IL-2-dependent and -independent events were analysed in a human T cell line immortalized by Tax. These studies show that, of the signaling pathways evaluated, only STAT5 remains dependent. Microarray analyses revealed several genes, including il-5, il-9 and il-13, are uniquely upregulated by IL-2 in the presence of Tax. Bioinformatics and supporting molecular biology show that some of these genes are STAT5 targets, explaining their IL-2 upregulation. These results suggest that IL-2 and viral proteins work together to induce gene expression, promoting the hypothesis that deregulation via the constitutive activation of STAT5 may lead to the IL-2-independent phenotype of HTLV-1-transformed cells.
Interleukin-2 (IL-2) mediates cell cycle progression and antiapoptosis in human T cells via several signal transduction pathways. The Tax protein of the human T-cell leukemia virus type I (HTLV-1) deregulates cell growth and alters the role of IL-2 in infected cells. However, Tax-immortalized cells stay dependent on IL-2, suggesting that events besides HTLV-1 gene expression are required for leukemia to develop. Here, IL-2-dependent and -independent events were analysed in a human T cell line immortalized by Tax. These studies show that, of the signaling pathways evaluated, only STAT5 remains dependent. Microarray analyses revealed several genes, including il-5, il-9 and il-13, are uniquely upregulated by IL-2 in the presence of Tax. Bioinformatics and supporting molecular biology show that some of these genes are STAT5 targets, explaining their IL-2 upregulation. These results suggest that IL-2 and viral proteins work together to induce gene expression, promoting the hypothesis that deregulation via the constitutive activation of STAT5 may lead to the IL-2-independent phenotype of HTLV-1-transformed cells.
Homologous recombination can induce tumorigenic sequence rearrangements. Here, we show that persistent hyper-recombination can be induced following exposure to a bifunctional alkylating agent, mitomycin C (MMC), and that the progeny of exposed cells induce a hyper-recombination phenotype in unexposed neighboring cells. Residual damage cannot be the cause of delayed recombination events, since recombination is observed after drug and template damage are diluted over a million-fold. Furthermore, not only do progeny of MMC-exposed cells induce recombination in unexposed cells (bystanders), but these bystanders can in turn induce recombination in their unexposed neighbors. Thus, a signal to induce homologous recombination can be passed from cell to cell. Although the underlying molecular mechanism is not yet known, these studies reveal that cells suffer consequences of damage long after exposure, and that can signal unexposed neighboring cells to respond similarly. Thus, a single acute exposure to a chemotherapeutic agent can cause long-term changes in genomic stability. If the results of these studies of mouse embryonic stem (ES) cells are generally applicable to many cell types, these results suggest that a relatively small number of cells could potentially induce a tissue-wide increase in the risk of de novo homologous recombination events.
The authors characterized the expression of 6912 genes in human primary fibroblasts after exposure to three different kinds of cellular stress that introduces DNA damage: the carcinogen 4-nitroquinoline-1-oxide (4NQO), gamma-irradiation, or UV-irradiation. Analysis of the transcriptional response to individual DNA-damaging agents revealed that a common set of genes was regulated after all of the stress types tested (Fig 2 of the paper). For consistency with the terminology established in yeast, the authors referred to these genes as the human environmental stress response (H-ESR). In addition, each DNA-damaging agent was able toEach DNA-damaging agent was able to exclusively alter the expression of specific subsets of genes. This gene set lists genes altered in response to 4NQO only.
Homologous recombination can induce tumorigenic sequence rearrangements. Here, we show that persistent hyper-recombination can be induced following exposure to a bifunctional alkylating agent, mitomycin C (MMC), and that the progeny of exposed cells induce a hyper-recombination phenotype in unexposed neighboring cells. Residual damage cannot be the cause of delayed recombination events, since recombination is observed after drug and template damage are diluted over a million-fold. Furthermore, not only do progeny of MMC-exposed cells induce recombination in unexposed cells (bystanders), but these bystanders can in turn induce recombination in their unexposed neighbors. Thus, a signal to induce homologous recombination can be passed from cell to cell. Although the underlying molecular mechanism is not yet known, these studies reveal that cells suffer consequences of damage long after exposure, and that can signal unexposed neighboring cells to respond similarly. Thus, a single acute exposure to a chemotherapeutic agent can cause long-term changes in genomic stability. If the results of these studies of mouse embryonic stem (ES) cells are generally applicable to many cell types, these results suggest that a relatively small number of cells could potentially induce a tissue-wide increase in the risk of de novo homologous recombination events.
Homologous recombination can induce tumorigenic sequence rearrangements. Here, we show that persistent hyper-recombination can be induced following exposure to a bifunctional alkylating agent, mitomycin C (MMC), and that the progeny of exposed cells induce a hyper-recombination phenotype in unexposed neighboring cells. Residual damage cannot be the cause of delayed recombination events, since recombination is observed after drug and template damage are diluted over a million-fold. Furthermore, not only do progeny of MMC-exposed cells induce recombination in unexposed cells (bystanders), but these bystanders can in turn induce recombination in their unexposed neighbors. Thus, a signal to induce homologous recombination can be passed from cell to cell. Although the underlying molecular mechanism is not yet known, these studies reveal that cells suffer consequences of damage long after exposure, and that can signal unexposed neighboring cells to respond similarly. Thus, a single acute exposure to a chemotherapeutic agent can cause long-term changes in genomic stability. If the results of these studies of mouse embryonic stem (ES) cells are generally applicable to many cell types, these results suggest that a relatively small number of cells could potentially induce a tissue-wide increase in the risk of de novo homologous recombination events.
Homologous recombination can induce tumorigenic sequence rearrangements. Here, we show that persistent hyper-recombination can be induced following exposure to a bifunctional alkylating agent, mitomycin C (MMC), and that the progeny of exposed cells induce a hyper-recombination phenotype in unexposed neighboring cells. Residual damage cannot be the cause of delayed recombination events, since recombination is observed after drug and template damage are diluted over a million-fold. Furthermore, not only do progeny of MMC-exposed cells induce recombination in unexposed cells (bystanders), but these bystanders can in turn induce recombination in their unexposed neighbors. Thus, a signal to induce homologous recombination can be passed from cell to cell. Although the underlying molecular mechanism is not yet known, these studies reveal that cells suffer consequences of damage long after exposure, and that can signal unexposed neighboring cells to respond similarly. Thus, a single acute exposure to a chemotherapeutic agent can cause long-term changes in genomic stability. If the results of these studies of mouse embryonic stem (ES) cells are generally applicable to many cell types, these results suggest that a relatively small number of cells could potentially induce a tissue-wide increase in the risk of de novo homologous recombination events.
The authors characterized the expression of 6912 genes in human primary fibroblasts after exposure to three different kinds of cellular stress that introduces DNA damage: the carcinogen 4-nitroquinoline-1-oxide (4NQO), gamma-irradiation, or UV-irradiation. Analysis of the transcriptional response to individual DNA-damaging agents revealed that a common set of genes was regulated after all of the stress types tested (Fig 2 of the paper). For consistency with the terminology established in yeast, the authors referred to these genes as the human environmental stress response (H-ESR). In addition, each DNA-damaging agent was able toEach DNA-damaging agent was able to exclusively alter the expression of specific subsets of genes. This gene set lists genes altered in response to gamma or UV irradiation but not to 4NQO.
The authors characterized the expression of 6912 genes in human primary fibroblasts after exposure to three different kinds of cellular stress that introduces DNA damage: the carcinogen 4-nitroquinoline-1-oxide (4NQO), gamma-irradiation, or UV-irradiation. Analysis of the transcriptional response to individual DNA-damaging agents revealed that a common set of genes was regulated after all of the stress types tested (Fig 2 of the paper). For consistency with the terminology established in yeast, the authors referred to these genes as the human environmental stress response (H-ESR). In addition, each DNA-damaging agent was able toEach DNA-damaging agent was able to exclusively alter the expression of specific subsets of genes. This gene set lists genes altered in response to UV and 4NQO but not to gamma irradiation.
p63 and p73 show a high degree of structural homology to p53 and are members of a family of transcriptional factors that can activate transcription of p53-responsive genes. p53 is mutated in more than 50% of human cancers, whereas p63 and p73 are rarely mutated. Studies of knockout mice also revealed an unexpected functional diversity among the p53 family. To determine how p63 and p73 are involved in tumorigenesis and normal development, we used cDNA microarray to examine 9216 genes in human colorectal cancer cells. We discovered that the expression of pigment epithelium-derived factor (PEDF) was specifically induced by either p63 or p73, but not by p53. We also report here that the PEDF gene contains a response element specific for p63 and p73 in its promoter region and is a direct target of p63 and p73. Collectively, p63 and p73 may be involved in cell fate by inducing PEDF expression.
Gain-of-function mutation of the K-ras gene is one of the most common genetic changes in human tumors. In tumors carrying K-ras mutation, the presence of oncogenic K-Ras is necessary for maintenance of the transformed phenotype. ESXR1 is a human paired-like homeodomain-containing protein expressed primarily in the testis. In cells, the 65-kDa full-length ESXR1 protein is proteolytically processed into an N-terminal 45-kDa fragment containing the homeodomain, which localizes exclusively within the nucleus, and a C-terminal 20-kDa fragment consisting of a proline-rich repeat region, which is located in the cytoplasm. In this work, we demonstrated that the N-terminal ESXR1 fragment specifically recognizes the TAATNNNATTA P3 consensus sequence for the paired-like homeodomain and functions as a sequence-specific transcriptional repressor. We also showed that the N-terminal ESXR1 fragment binds to the TAATGTTATTA sequence present within the first intron of the human K-ras gene and inhibits its expression at both mRNA and protein levels. Ectopic expression of the N-terminal ESXR1 fragment in human carcinoma cells that carry mutated K-ras reduces the level of K-Ras and thereby inhibits the tumor cell proliferation. Identification of ESXR1 as a transcriptional repressor of K-ras has an important implication for the development of cancer therapy that inhibits oncogenic K-Ras expression.
Tumor hypoxia is an adverse prognostic factor. In a recent study, we could demonstrate that cyclic hypoxia selects for hypoxia-tolerant tumor cells, which are cross-resistant to other stimuli of mitochondrial death pathways. In contrast, sensitivity of the cells to death-receptor ligands was mainly not affected. The aim of the present study was to further elucidate cellular changes induced by cyclic hypoxia and to identify alterations in gene expression pattern upon hypoxic selection by means of DNA-microarray analysis. Our data reveal that cyclic hypoxia resulted in the selection of cells with resistance to doxorubicine and radiation. Furthermore, hypoxic selection was accompanied by constitutive changes of the gene expression pattern with downregulation of 156 and upregulation of 82 genes. Most of the differentially regulated genes were involved in cellular responses to hypoxia and reoxygenation. While many of the genes that were downregulated upon hypoxic selection represent genes that are usually upregulated by acute hypoxia, the genes that were upregulated represent genes that are involved in stress resistance and anti-apoptotic signalling. Most importantly, hypoxic selection was not associated with changes of single apoptosis relevant genes, but with alterations in gene expression levels of a wide variety of genes indicating a more complex adaptation process.
Tumor hypoxia is an adverse prognostic factor. In a recent study, we could demonstrate that cyclic hypoxia selects for hypoxia-tolerant tumor cells, which are cross-resistant to other stimuli of mitochondrial death pathways. In contrast, sensitivity of the cells to death-receptor ligands was mainly not affected. The aim of the present study was to further elucidate cellular changes induced by cyclic hypoxia and to identify alterations in gene expression pattern upon hypoxic selection by means of DNA-microarray analysis. Our data reveal that cyclic hypoxia resulted in the selection of cells with resistance to doxorubicine and radiation. Furthermore, hypoxic selection was accompanied by constitutive changes of the gene expression pattern with downregulation of 156 and upregulation of 82 genes. Most of the differentially regulated genes were involved in cellular responses to hypoxia and reoxygenation. While many of the genes that were downregulated upon hypoxic selection represent genes that are usually upregulated by acute hypoxia, the genes that were upregulated represent genes that are involved in stress resistance and anti-apoptotic signalling. Most importantly, hypoxic selection was not associated with changes of single apoptosis relevant genes, but with alterations in gene expression levels of a wide variety of genes indicating a more complex adaptation process.
To investigate the molecules that regulate the acquisition of cis-diamminedichloroplatinum (II) (cisplatin) resistance, we performed cDNA microarrays using two pairs of parental and cisplatin-resistant bladder cancer cell lines. We found a markedly reduced expression of inositol 1,4,5-trisphosphate (IP3) receptor type1 (IP3R1), endoplasmic reticulum membrane protein, in cisplatin-resistant cells. The suppression of IP3R1 expression using small interfering RNA in parental cells prevented apoptosis and resulted in decreased sensitivity to cisplatin. Contrarily, overexpression of IP3R1 in resistant cells induced apoptosis and increased sensitivity to cisplatin. These results suggest that cisplatin-induced downregulation of IP3R1 expression was closely associated with the acquisition of cisplatin resistance in bladder cancer cells.
To investigate the molecules that regulate the acquisition of cis-diamminedichloroplatinum (II) (cisplatin) resistance, we performed cDNA microarrays using two pairs of parental and cisplatin-resistant bladder cancer cell lines. We found a markedly reduced expression of inositol 1,4,5-trisphosphate (IP3) receptor type1 (IP3R1), endoplasmic reticulum membrane protein, in cisplatin-resistant cells. The suppression of IP3R1 expression using small interfering RNA in parental cells prevented apoptosis and resulted in decreased sensitivity to cisplatin. Contrarily, overexpression of IP3R1 in resistant cells induced apoptosis and increased sensitivity to cisplatin. These results suggest that cisplatin-induced downregulation of IP3R1 expression was closely associated with the acquisition of cisplatin resistance in bladder cancer cells.
Upregulation of hypoxia-inducible factors HIF-1 and HIF-2 is frequent in human cancers and may result from tissue hypoxia or genetic mechanisms, in particular the inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene (TSG). Tumours with VHL inactivation are highly vascular, but it is unclear to what extent HIF-dependent and HIF-independent mechanisms account for pVHL tumour suppressor activity. As the identification of novel pVHL targets might provide insights into pVHL tumour suppressor activity, we performed gene expression microarray analysis in VHL-wild-type and VHL-null renal cell carcinoma (RCC) cell lines. We identified 30 differentially regulated pVHL targets (26 of which were 'novel') and the results of microarray analysis were confirmed in all 11 novel targets further analysed by real-time RT-PCR or Western blotting. Furthermore, nine of 11 targets were dysregulated in the majority of a series of primary clear cell RCC with VHL inactivation. Three of the nine targets had been identified previously as candidate TSGs (DOC-2/DAB2, CDKN1C and SPARC) and all were upregulated by wild-type pVHL. The significance for pVHL function of two further genes upregulated by wild-type pVHL was initially unclear, but re-expression of GNG4 (G protein gamma-4 subunit/guanine nucleotide-binding protein-4) and MLC2 (myosin light chain) in a RCC cell line suppressed tumour cell growth. pVHL regulation of CDKN1C, SPARC and GNG4 was not mimicked by hypoxia, whereas for six of 11 novel targets analysed (including DOC-2/DAB2 and MLC2) the effects of pVHL inactivation and hypoxia were similar. For GPR56 there was evidence of a tissue-specific hypoxia response. Such a phenomenon might, in part, explain organ-specific tumorigenesis in VHL disease. These provide insights into mechanisms of pVHL tumour suppressor function and identify novel hypoxia-responsive targets that might be implicated in tumorigenesis in both VHL disease and in other cancers with HIF upregulation.
p53 prevents cancer development by inhibiting the proliferation of stressed or abnormal cells. The interaction between MDM2 and p53 is critical for cell viability; loss of Mdm2 causes cell death in vitro and in vivo in a p53-dependent manner. MDM4 has some of the same properties as MDM2, but unlike MDM2, it does not cause nuclear export or degradation of p53. MDM4 knock out mouse embryos die at midgestational stages exhibiting p53-dependent cell cycle arrest and apoptosis. As the interaction between Mdm4 and p53 is critical for cell  cycle progression and survival, the authors used this mouse model to search for new key downstream effectors of p53-dependent cell cycle arrest and apoptosis.  With the aim of identifying changes in gene expression in MDM4 knockout mice in apoptotic (neuroepithelium) tissue, they used Affymetrix DNA microarrays (MG_U74Av2) to compare expression ofof the euroepithelial tissue (Neur) derived from wild type (WT) and Mdm4 knockout (KO) embryos at day E10.5.
p53 prevents cancer development by inhibiting the proliferation of stressed or abnormal cells. The interaction between MDM2 and p53 is critical for cell viability; loss of Mdm2 causes cell death in vitro and in vivo in a p53-dependent manner. MDM4 has some of the same properties as MDM2, but unlike MDM2, it does not cause nuclear export or degradation of p53. MDM4 knock out mouse embryos die at midgestational stages exhibiting p53-dependent cell cycle arrest and apoptosis. As the interaction between Mdm4 and p53 is critical for cell  cycle progression and survival, the authors used this mouse model to search for new key downstream effectors of p53-dependent cell cycle arrest and apoptosis.  With the aim of identifying changes in gene expression in MDM4 knockout mice in apoptotic (neuroepithelium) tissue, they used Affymetrix DNA microarrays (MG_U74Av2) to compare expression ofof the euroepithelial tissue (Neur) derived from wild type (WT) and Mdm4 knockout (KO) embryos at day E10.5.
The p53 tumour suppressor functions as a transcriptional activator, and several p53-inducible genes that play a critical proapoptotic role have been described. Moreover, p53 regulates the expression of various proteins participating in autoregulatory feedback loops, including proteins that negatively control p53 stability (Mdm2 and Pirh2) or modulate stress-induced phosphorylation of p53 on Ser-46 (p53DINP1 or Wip1), a key event for p53-induced apoptosis. Here, we describe a new systematic analysis of p53 targets using oligonucleotide chips, and report the identification of dapk1 as a novel p53 target. We demonstrate that dapk1 mRNA levels increase in a p53-dependent manner in various cellular settings. Both human and mouse dapk1 genomic loci contain DNA sequences that bind p53 in vitro and in vivo. Since dapk1 encodes a serine/threonine kinase previously shown to suppress oncogene-induced transformation by activating a p19ARF/p53-dependent apoptotic checkpoint, our results suggest that Dapk1 participates in a new positive feedback loop controlling p53 activation and apoptosis.
The p53 tumour suppressor functions as a transcriptional activator, and several p53-inducible genes that play a critical proapoptotic role have been described. Moreover, p53 regulates the expression of various proteins participating in autoregulatory feedback loops, including proteins that negatively control p53 stability (Mdm2 and Pirh2) or modulate stress-induced phosphorylation of p53 on Ser-46 (p53DINP1 or Wip1), a key event for p53-induced apoptosis. Here, we describe a new systematic analysis of p53 targets using oligonucleotide chips, and report the identification of dapk1 as a novel p53 target. We demonstrate that dapk1 mRNA levels increase in a p53-dependent manner in various cellular settings. Both human and mouse dapk1 genomic loci contain DNA sequences that bind p53 in vitro and in vivo. Since dapk1 encodes a serine/threonine kinase previously shown to suppress oncogene-induced transformation by activating a p19ARF/p53-dependent apoptotic checkpoint, our results suggest that Dapk1 participates in a new positive feedback loop controlling p53 activation and apoptosis.
The aminothiol WR1065 exerts selective cytoprotective effects in normal cells compared to cancer cells and has clinical applications for the protection of normal cells in cancer patients undergoing radio- or chemotherapy. There is evidence that p53 is activated in response to WR1065. To examine the effects of WR1065 on the signalling pathways controlled by p53, isogeneic human colon carcinoma cell lines (HCT116) differing only in the presence or absence of wild-type p53 were used. Treatment with WR1065 resulted in G1 cell cycle arrest in the p53-positive cell line but not in the p53-negative cell line. Long-term exposure resulted in minimal apoptosis of either cell line. Changes in gene expression in p53-positive or -negative cells treated with WR1065 were examined using commercial human stress and cancer gene arrays (Clontech Atlas arrays). Genes found to be specifically upregulated in a p53-dependent manner included coproporphyrinogen oxidase, ICErel-II cysteine protease, macrophage inhibitory cytokine-1 (also known as placental transforming growth factor beta), S100A4, and Waf1/p21. However, most proapoptotic genes typically upregulated by p53 in response to DNA damage were not activated. These studies show that WR1065 specifically modulates a subset of p53 target genes in a colon carcinoma cell line, consistent with the observation that this agent elicits essentially p53-dependent, cell cycle arrest responses.
The aminothiol WR1065 exerts selective cytoprotective effects in normal cells compared to cancer cells and has clinical applications for the protection of normal cells in cancer patients undergoing radio- or chemotherapy. There is evidence that p53 is activated in response to WR1065. To examine the effects of WR1065 on the signalling pathways controlled by p53, isogeneic human colon carcinoma cell lines (HCT116) differing only in the presence or absence of wild-type p53 were used. Treatment with WR1065 resulted in G1 cell cycle arrest in the p53-positive cell line but not in the p53-negative cell line. Long-term exposure resulted in minimal apoptosis of either cell line. Changes in gene expression in p53-positive or -negative cells treated with WR1065 were examined using commercial human stress and cancer gene arrays (Clontech Atlas arrays). Genes found to be specifically upregulated in a p53-dependent manner included coproporphyrinogen oxidase, ICErel-II cysteine protease, macrophage inhibitory cytokine-1 (also known as placental transforming growth factor beta), S100A4, and Waf1/p21. However, most proapoptotic genes typically upregulated by p53 in response to DNA damage were not activated. These studies show that WR1065 specifically modulates a subset of p53 target genes in a colon carcinoma cell line, consistent with the observation that this agent elicits essentially p53-dependent, cell cycle arrest responses.
Although the monomeric GTPases RalA and RalB have been shown to regulate a variety of transcription factors, little is known regarding the differences or similarities in transcriptional programs regulated by RalA compared to RalB. Further, the association of these transcriptional pathways to human carcinogenesis and progression remains unclear. Here, we studied the role of RalA and/or RalB in transcriptional regulation by combining short interfering RNA depletion of Ral with gene expression profiling via microarray in the human bladder cancer cell line, UMUC-3. A large number of genes were found to be similarly modulated in cells with RalA and RalB depletion, suggesting that RalA and RalB impinge on overlapping transcriptional signaling pathways. However, smaller sets of genes were modulated by depletion of RalA or RalB, indicating that these closely related proteins also regulate nonoverlapping transcriptional pathways. Computational analysis of upstream sequences of genes modulated by Ral depletion identified Ras-responsive element-binding protein (RREB)-1, as a putative Ral transcriptional target, which we verified experimentally. Importantly, as a group, Ral-regulated probe sets identified here were disproportionally represented among those differentially expressed as a function of human bladder transformation. Taken together, these data strongly suggest that Ral family members mediate both common and specific transcriptional programs that are associated with human cancer and identify RREB-1 as a novel transcriptional effector of Ral.
Metaplastic carcinoma of the breast (MCB) is a poorly understood subtype of breast cancer. It is generally characterized by the coexistence of ductal carcinomatous and transdifferentiated sarcomatous components, but the underlying molecular alterations, possibly related to epithelial-mesenchymal transition (EMT), remain elusive. We performed transcriptional profiling using half-a-genome oligonucleotide microarrays to elucidate genetic profiles of MCBs and their differences to those of ductal carcinoma of breasts (DCBs) using discarded specimens of four MCBs and 34 DCBs. Unsupervised clustering disclosed distinctive expression profiles between MCBs and DCBs. Supervised analysis identified gene signatures discriminating MCBs from DCBs and between MCB subclasses. Notably, many of the discriminator genes were associated with downregulation of epithelial phenotypes and with synthesis, remodeling and adhesion of extracellular matrix, with some of them have known or inferred roles related to EMT. Importantly, several of the discriminator genes were upregulated in a mutant Snail-transfected MCF7 cell known to exhibit features of EMT, thereby indicating a crucial role for EMT in the pathogenesis of MCBs. Finally, the identification of SPARC and vimentin as poor prognostic factors reinforced the role of EMT in cancer progression. These data advance our understanding of MCB and offer clues to the molecular alterations underlying EMT.
Metaplastic carcinoma of the breast (MCB) is a poorly understood subtype of breast cancer. It is generally characterized by the coexistence of ductal carcinomatous and transdifferentiated sarcomatous components, but the underlying molecular alterations, possibly related to epithelial-mesenchymal transition (EMT), remain elusive. We performed transcriptional profiling using half-a-genome oligonucleotide microarrays to elucidate genetic profiles of MCBs and their differences to those of ductal carcinoma of breasts (DCBs) using discarded specimens of four MCBs and 34 DCBs. Unsupervised clustering disclosed distinctive expression profiles between MCBs and DCBs. Supervised analysis identified gene signatures discriminating MCBs from DCBs and between MCB subclasses. Notably, many of the discriminator genes were associated with downregulation of epithelial phenotypes and with synthesis, remodeling and adhesion of extracellular matrix, with some of them have known or inferred roles related to EMT. Importantly, several of the discriminator genes were upregulated in a mutant Snail-transfected MCF7 cell known to exhibit features of EMT, thereby indicating a crucial role for EMT in the pathogenesis of MCBs. Finally, the identification of SPARC and vimentin as poor prognostic factors reinforced the role of EMT in cancer progression. These data advance our understanding of MCB and offer clues to the molecular alterations underlying EMT.
The Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all virus-associated tumors where it plays an essential role in the maintenance, replication and transcription of the EBV genome. Transcriptional profiling of EBNA1-expressing carcinoma cells demonstrated that EBNA1 also influences the expression of a range of cellular genes including those involved in translation, transcription and cell signaling. Of particular interest was the ability of EBNA1 to enhance expression of STAT1 and sensitize cells to interferon-induced STAT1 activation with resultant enhancement of major histocompatibility complex expression. A negative effect of EBNA1 on the expression of TGFbeta1-responsive betaig-h3 and PAI-1 genes was confirmed at the protein level in EBV-infected carcinoma cells. This effect resulted from the ability of EBNA1 to repress TGFbeta1-induced transcription via a reduction in the interaction of SMAD2 with SMAD4. More detailed analysis revealed that EBNA1 induces a lower steady-state level of SMAD2 protein as a consequence of increased protein turnover. These data show that EBNA1 can influence cellular gene transcription resulting in effects that may contribute to the development of EBV-associated tumors.
The Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all virus-associated tumors where it plays an essential role in the maintenance, replication and transcription of the EBV genome. Transcriptional profiling of EBNA1-expressing carcinoma cells demonstrated that EBNA1 also influences the expression of a range of cellular genes including those involved in translation, transcription and cell signaling. Of particular interest was the ability of EBNA1 to enhance expression of STAT1 and sensitize cells to interferon-induced STAT1 activation with resultant enhancement of major histocompatibility complex expression. A negative effect of EBNA1 on the expression of TGFbeta1-responsive betaig-h3 and PAI-1 genes was confirmed at the protein level in EBV-infected carcinoma cells. This effect resulted from the ability of EBNA1 to repress TGFbeta1-induced transcription via a reduction in the interaction of SMAD2 with SMAD4. More detailed analysis revealed that EBNA1 induces a lower steady-state level of SMAD2 protein as a consequence of increased protein turnover. These data show that EBNA1 can influence cellular gene transcription resulting in effects that may contribute to the development of EBV-associated tumors.
Expression of homeobox A1 (HOXA1) results in oncogenic transformation of immortalized human mammary epithelial cells with aggressive tumor formation in vivo. However, the mechanisms by which HOXA1 mediates oncogenic transformation is not well defined. To identify molecules that could potentially be involved in HOXA1-mediated oncogenic transformation, microarray analysis was utilized to characterize and compare the gene expression pattern in response to forced expression or depletion of HOXA1 in human mammary carcinoma cells. Gene expression profiling identified that genes involved in the p44/42 mitogen-activated protein (MAP) kinase activation pathway (GRB2, MAP kinase kinase (MEK1) and SDFR1) or p44/42 MAP kinase-regulated genes (IER3, EPAS1, PCNA and catalase) are downstream expression targets of HOXA1. Forced expression of HOXA1 increased GRB2 and MEK1 mRNA and protein expression and increased p44/42 MAP kinase phosphorylation, activity and Elk-1-mediated transcription. Use of a MEK1 inhibitor demonstrated that increased p44/42 MAP kinase activity is required for the HOXA1-mediated increase in cell proliferation, survival, oncogenicity and oncogenic transformation. Thus, modulation of the p44/42 MAP kinase pathway is one mechanism by which HOXA1 mediates oncogenic transformation of the human mammary epithelial cell.
Expression of homeobox A1 (HOXA1) results in oncogenic transformation of immortalized human mammary epithelial cells with aggressive tumor formation in vivo. However, the mechanisms by which HOXA1 mediates oncogenic transformation is not well defined. To identify molecules that could potentially be involved in HOXA1-mediated oncogenic transformation, microarray analysis was utilized to characterize and compare the gene expression pattern in response to forced expression or depletion of HOXA1 in human mammary carcinoma cells. Gene expression profiling identified that genes involved in the p44/42 mitogen-activated protein (MAP) kinase activation pathway (GRB2, MAP kinase kinase (MEK1) and SDFR1) or p44/42 MAP kinase-regulated genes (IER3, EPAS1, PCNA and catalase) are downstream expression targets of HOXA1. Forced expression of HOXA1 increased GRB2 and MEK1 mRNA and protein expression and increased p44/42 MAP kinase phosphorylation, activity and Elk-1-mediated transcription. Use of a MEK1 inhibitor demonstrated that increased p44/42 MAP kinase activity is required for the HOXA1-mediated increase in cell proliferation, survival, oncogenicity and oncogenic transformation. Thus, modulation of the p44/42 MAP kinase pathway is one mechanism by which HOXA1 mediates oncogenic transformation of the human mammary epithelial cell.
An improved understanding of cell immortalization and its manifestation in clinical tumors could facilitate novel therapeutic approaches. However, only rare tumor cells, which maintain telomerase expression in vitro, immortalize spontaneously. By expression-profiling analyses of limited-life primary breast tumor cultures pre- and post-hTERT transduction, and spontaneously immortalized breast cancer cell lines, we identified a common signature characteristic of tumor cell immortalization. A predominant feature of this immortalization signature (ImmSig) was the significant overexpression of oxidoreductase genes. In contrast to epithelial cells derived from low histologic grade primary tumors, which required hTERT transduction for the acquisition of ImmSig, spontaneously immortalizing high-grade tumor cultures displayed similar molecular changes independent of exogenous hTERT. Silencing the hTERT gene reversed ImmSig expression, increased cellular reactive oxygen species levels, altered mitochondrial membrane potential and induced apoptotic and proliferation changes in immortalized cells. In clinical breast cancer samples, cell-proliferation-pathway genes were significantly associated with ImmSig. In these cases, ImmSig expression itself was inversely correlated with patient survival (P=0), and was particularly relevant to the outcome of estrogen receptor-positive tumors. Our data support the notion that ImmSig assists in surmounting normal barriers related to oxidative and replicative stress response. Targeting a subset of aggressive breast cancers by reversing ImmSig components could be a practical therapeutic strategy.
An improved understanding of cell immortalization and its manifestation in clinical tumors could facilitate novel therapeutic approaches. However, only rare tumor cells, which maintain telomerase expression in vitro, immortalize spontaneously. By expression-profiling analyses of limited-life primary breast tumor cultures pre- and post-hTERT transduction, and spontaneously immortalized breast cancer cell lines, we identified a common signature characteristic of tumor cell immortalization. A predominant feature of this immortalization signature (ImmSig) was the significant overexpression of oxidoreductase genes. In contrast to epithelial cells derived from low histologic grade primary tumors, which required hTERT transduction for the acquisition of ImmSig, spontaneously immortalizing high-grade tumor cultures displayed similar molecular changes independent of exogenous hTERT. Silencing the hTERT gene reversed ImmSig expression, increased cellular reactive oxygen species levels, altered mitochondrial membrane potential and induced apoptotic and proliferation changes in immortalized cells. In clinical breast cancer samples, cell-proliferation-pathway genes were significantly associated with ImmSig. In these cases, ImmSig expression itself was inversely correlated with patient survival (P=0), and was particularly relevant to the outcome of estrogen receptor-positive tumors. Our data support the notion that ImmSig assists in surmounting normal barriers related to oxidative and replicative stress response. Targeting a subset of aggressive breast cancers by reversing ImmSig components could be a practical therapeutic strategy.
Infection with Helicobacter pylori cagA-positive strains is associated with gastric adenocarcinoma. Intestinal metaplasia is a precancerous lesion of the stomach characterized by transdifferentiation of the gastric mucosa to an intestinal phenotype. The H. pylori cagA gene product, CagA, is delivered into gastric epithelial cells, where it undergoes tyrosine phosphorylation by Src family kinases. Tyrosine-phosphorylated CagA specifically binds to and activates SHP-2 phosphatase, thereby inducing cell-morphological transformation. We report here that CagA physically interacts with E-cadherin independently of CagA tyrosine phosphorylation. The CagA/E-cadherin interaction impairs the complex formation between E-cadherin and beta-catenin, causing cytoplasmic and nuclear accumulation of beta-catenin. CagA-deregulated beta-catenin then transactivates beta-catenin-dependent genes such as cdx1, which encodes intestinal specific CDX1 transcription factor. In addition to beta-catenin signal, CagA also transactivates p21(WAF1/Cip1), again, in a phosphorylation-independent manner. Consequently, CagA induces aberrant expression of an intestinal-differentiation marker, goblet-cell mucin MUC2, in gastric epithelial cells that have been arrested in G1 by p21(WAF1/Cip1). These results indicate that perturbation of the E-cadherin/beta-catenin complex by H. pylori CagA plays an important role in the development of intestinal metaplasia, a premalignant transdifferentiation of gastric epithelial cells from which intestinal-type gastric adenocarcinoma arises.
Oligoarray analysis of a matched pair of prostate cancer and normal cell lines derived from the same radical prostatectomy specimen identified 113 candidate hypomethylated genes that were overexpressed in the cancer cells and contained CpG islands. Hypomethylation of wingless-related MMTV integration site 5A (WNT5A), S100 calcium-binding protein P (S100P) and cysteine-rich protein 1(CRIP1) was confirmed in the cancer cells by bisulfite sequencing. Treatment of the corresponding normal prostate epithelial cells 1542-NPTX with the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-aza-CdR) induced higher levels of mRNA expression and partial loss of methylation on these genes. Primary prostate cancers were tested using methylation-specific polymerase chain reaction. WNT5A was hypomethylated in 11/17 (65%) tumors, S100P in 8/16 (50%) and CRIP1 in 13/20 (65%). Bisulfite sequencing of a section of the 5' untranslated region (UTR) of WNT5A revealed that three CpG sites (15, 24 and 35) were consistently methylated (93%) in the normal cell line and normal tissues, but not in the prostate cancer cell line and eight primary prostate cancers. Multiple putative binding sites for the transcription factors SP1 and AP-2 were found adjacent to CpG sites 15 and 24. A putative c-Myb binding site was located within the CpG site 35. Anti-c-Myb antibody co-precipitation with WNT5A was methylation-sensitive in 1542-NPTX cells. It is likely that an epigenetic mechanism regulates WNT5A expression in prostate cancer.
Upregulation of hypoxia-inducible factors HIF-1 and HIF-2 is frequent in human cancers and may result from tissue hypoxia or genetic mechanisms, in particular the inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene (TSG). Tumours with VHL inactivation are highly vascular, but it is unclear to what extent HIF-dependent and HIF-independent mechanisms account for pVHL tumour suppressor activity. As the identification of novel pVHL targets might provide insights into pVHL tumour suppressor activity, we performed gene expression microarray analysis in VHL-wild-type and VHL-null renal cell carcinoma (RCC) cell lines. We identified 30 differentially regulated pVHL targets (26 of which were 'novel') and the results of microarray analysis were confirmed in all 11 novel targets further analysed by real-time RT-PCR or Western blotting. Furthermore, nine of 11 targets were dysregulated in the majority of a series of primary clear cell RCC with VHL inactivation. Three of the nine targets had been identified previously as candidate TSGs (DOC-2/DAB2, CDKN1C and SPARC) and all were upregulated by wild-type pVHL. The significance for pVHL function of two further genes upregulated by wild-type pVHL was initially unclear, but re-expression of GNG4 (G protein gamma-4 subunit/guanine nucleotide-binding protein-4) and MLC2 (myosin light chain) in a RCC cell line suppressed tumour cell growth. pVHL regulation of CDKN1C, SPARC and GNG4 was not mimicked by hypoxia, whereas for six of 11 novel targets analysed (including DOC-2/DAB2 and MLC2) the effects of pVHL inactivation and hypoxia were similar. For GPR56 there was evidence of a tissue-specific hypoxia response. Such a phenomenon might, in part, explain organ-specific tumorigenesis in VHL disease. These provide insights into mechanisms of pVHL tumour suppressor function and identify novel hypoxia-responsive targets that might be implicated in tumorigenesis in both VHL disease and in other cancers with HIF upregulation.
Upregulation of hypoxia-inducible factors HIF-1 and HIF-2 is frequent in human cancers and may result from tissue hypoxia or genetic mechanisms, in particular the inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene (TSG). Tumours with VHL inactivation are highly vascular, but it is unclear to what extent HIF-dependent and HIF-independent mechanisms account for pVHL tumour suppressor activity. As the identification of novel pVHL targets might provide insights into pVHL tumour suppressor activity, we performed gene expression microarray analysis in VHL-wild-type and VHL-null renal cell carcinoma (RCC) cell lines. We identified 30 differentially regulated pVHL targets (26 of which were 'novel') and the results of microarray analysis were confirmed in all 11 novel targets further analysed by real-time RT-PCR or Western blotting. Furthermore, nine of 11 targets were dysregulated in the majority of a series of primary clear cell RCC with VHL inactivation. Three of the nine targets had been identified previously as candidate TSGs (DOC-2/DAB2, CDKN1C and SPARC) and all were upregulated by wild-type pVHL. The significance for pVHL function of two further genes upregulated by wild-type pVHL was initially unclear, but re-expression of GNG4 (G protein gamma-4 subunit/guanine nucleotide-binding protein-4) and MLC2 (myosin light chain) in a RCC cell line suppressed tumour cell growth. pVHL regulation of CDKN1C, SPARC and GNG4 was not mimicked by hypoxia, whereas for six of 11 novel targets analysed (including DOC-2/DAB2 and MLC2) the effects of pVHL inactivation and hypoxia were similar. For GPR56 there was evidence of a tissue-specific hypoxia response. Such a phenomenon might, in part, explain organ-specific tumorigenesis in VHL disease. These provide insights into mechanisms of pVHL tumour suppressor function and identify novel hypoxia-responsive targets that might be implicated in tumorigenesis in both VHL disease and in other cancers with HIF upregulation.
Pancreatic ductal adenocarcinoma is the eighth most common cancer with the lowest overall 5-year relative survival rate of any tumor type today. Expression profiling using microarrays has been widely used to identify genes associated with pancreatic cancer development. To extract maximum value from the available gene expression data, we applied a meta-analysis to search for commonly differentially expressed genes in pancreatic ductal adenocarcinoma. We obtained data sets from four different gene expression studies on pancreatic cancer. We selected a consensus set of 2984 genes measured in all four studies and applied a meta-analysis approach to evaluate the combined data. Of the genes identified as differentially expressed, several were validated using RT-PCR and immunohistochemistry. Additionally, we used a class discovery algorithm to identify a gene expression signature. Our meta-analysis revealed that the pancreatic cancer gene expression data sets shared a significant number of up- and downregulated genes, independent of the technology used. This interstudy crossvalidation approach generated a set of 568 genes that were consistently and significantly dysregulated in pancreatic cancer. Of these, 364 (64.1%) were upregulated and 204 (35.9%) were downregulated in pancreatic cancer. Only 127 (22%) were described in the published individual analyses. Functional annotation of the genes revealed that genes presumably associated with the cell adhesion-mediated drug resistance pathway are frequently overexpressed in pancreatic cancer. Meta-analysis is an important tool for the identification and validation of differentially expressed genes. These could represent good candidates for novel diagnostic and therapeutic approaches to pancreatic cancer.
Pancreatic ductal adenocarcinoma is the eighth most common cancer with the lowest overall 5-year relative survival rate of any tumor type today. Expression profiling using microarrays has been widely used to identify genes associated with pancreatic cancer development. To extract maximum value from the available gene expression data, we applied a meta-analysis to search for commonly differentially expressed genes in pancreatic ductal adenocarcinoma. We obtained data sets from four different gene expression studies on pancreatic cancer. We selected a consensus set of 2984 genes measured in all four studies and applied a meta-analysis approach to evaluate the combined data. Of the genes identified as differentially expressed, several were validated using RT-PCR and immunohistochemistry. Additionally, we used a class discovery algorithm to identify a gene expression signature. Our meta-analysis revealed that the pancreatic cancer gene expression data sets shared a significant number of up- and downregulated genes, independent of the technology used. This interstudy crossvalidation approach generated a set of 568 genes that were consistently and significantly dysregulated in pancreatic cancer. Of these, 364 (64.1%) were upregulated and 204 (35.9%) were downregulated in pancreatic cancer. Only 127 (22%) were described in the published individual analyses. Functional annotation of the genes revealed that genes presumably associated with the cell adhesion-mediated drug resistance pathway are frequently overexpressed in pancreatic cancer. Meta-analysis is an important tool for the identification and validation of differentially expressed genes. These could represent good candidates for novel diagnostic and therapeutic approaches to pancreatic cancer.
The transforming growth factor-beta (TGF-beta)-Smad signaling pathway inhibits the growth of human epithelial cells and plays a role in tumor suppression. The Smad4 gene is mutated or deleted in 50% of pancreatic cancers. In this study, we succeeded in establishing Smad4 knockdown (S4KD) pancreatic cancer cell lines using the stable RNA interference (RNAi) method. Smad4 protein expression was reduced dramatically and TGF-beta-Smad signaling was markedly inhibited in the S4KD cell lines. The S4KD and control cells were stimulated with TGF-beta and analysed using a cDNA microarray that contained 3756 genes, in order to screen for target molecules downstream of TGF-beta. The microarray analysis revealed that 187 S4KD genes and 155 genes in the control cells were regulated immediately upon TGF-beta stimulation. Quantitative RT-PCR analysis on several of these genes produced results that corroborated the outcome of the microarray analysis. Most of the genes in the S4KD and control cells identified by the array differed, which suggests signaling pathways that differ according to Smad4 status. Of the identified genes, 246 have not been reported previously as genes that lie downstream of TGF-beta. Genes that are involved in cell proliferation, adhesion, and motility were found to be regulated differentially with respect to S4KD and control cells. Cell migration induced by TGF-beta was inhibited in the S4KD cells, which might be associated with a different regulation of integrin beta7. The knock down of a specific gene using stable RNAi appears to be a promising tool for analysing endogenous gene function.
The transforming growth factor-beta (TGF-beta)-Smad signaling pathway inhibits the growth of human epithelial cells and plays a role in tumor suppression. The Smad4 gene is mutated or deleted in 50% of pancreatic cancers. In this study, we succeeded in establishing Smad4 knockdown (S4KD) pancreatic cancer cell lines using the stable RNA interference (RNAi) method. Smad4 protein expression was reduced dramatically and TGF-beta-Smad signaling was markedly inhibited in the S4KD cell lines. The S4KD and control cells were stimulated with TGF-beta and analysed using a cDNA microarray that contained 3756 genes, in order to screen for target molecules downstream of TGF-beta. The microarray analysis revealed that 187 S4KD genes and 155 genes in the control cells were regulated immediately upon TGF-beta stimulation. Quantitative RT-PCR analysis on several of these genes produced results that corroborated the outcome of the microarray analysis. Most of the genes in the S4KD and control cells identified by the array differed, which suggests signaling pathways that differ according to Smad4 status. Of the identified genes, 246 have not been reported previously as genes that lie downstream of TGF-beta. Genes that are involved in cell proliferation, adhesion, and motility were found to be regulated differentially with respect to S4KD and control cells. Cell migration induced by TGF-beta was inhibited in the S4KD cells, which might be associated with a different regulation of integrin beta7. The knock down of a specific gene using stable RNAi appears to be a promising tool for analysing endogenous gene function.
The transforming growth factor-beta (TGF-beta)-Smad signaling pathway inhibits the growth of human epithelial cells and plays a role in tumor suppression. The Smad4 gene is mutated or deleted in 50% of pancreatic cancers. In this study, we succeeded in establishing Smad4 knockdown (S4KD) pancreatic cancer cell lines using the stable RNA interference (RNAi) method. Smad4 protein expression was reduced dramatically and TGF-beta-Smad signaling was markedly inhibited in the S4KD cell lines. The S4KD and control cells were stimulated with TGF-beta and analysed using a cDNA microarray that contained 3756 genes, in order to screen for target molecules downstream of TGF-beta. The microarray analysis revealed that 187 S4KD genes and 155 genes in the control cells were regulated immediately upon TGF-beta stimulation. Quantitative RT-PCR analysis on several of these genes produced results that corroborated the outcome of the microarray analysis. Most of the genes in the S4KD and control cells identified by the array differed, which suggests signaling pathways that differ according to Smad4 status. Of the identified genes, 246 have not been reported previously as genes that lie downstream of TGF-beta. Genes that are involved in cell proliferation, adhesion, and motility were found to be regulated differentially with respect to S4KD and control cells. Cell migration induced by TGF-beta was inhibited in the S4KD cells, which might be associated with a different regulation of integrin beta7. The knock down of a specific gene using stable RNAi appears to be a promising tool for analysing endogenous gene function.
The transforming growth factor-beta (TGF-beta)-Smad signaling pathway inhibits the growth of human epithelial cells and plays a role in tumor suppression. The Smad4 gene is mutated or deleted in 50% of pancreatic cancers. In this study, we succeeded in establishing Smad4 knockdown (S4KD) pancreatic cancer cell lines using the stable RNA interference (RNAi) method. Smad4 protein expression was reduced dramatically and TGF-beta-Smad signaling was markedly inhibited in the S4KD cell lines. The S4KD and control cells were stimulated with TGF-beta and analysed using a cDNA microarray that contained 3756 genes, in order to screen for target molecules downstream of TGF-beta. The microarray analysis revealed that 187 S4KD genes and 155 genes in the control cells were regulated immediately upon TGF-beta stimulation. Quantitative RT-PCR analysis on several of these genes produced results that corroborated the outcome of the microarray analysis. Most of the genes in the S4KD and control cells identified by the array differed, which suggests signaling pathways that differ according to Smad4 status. Of the identified genes, 246 have not been reported previously as genes that lie downstream of TGF-beta. Genes that are involved in cell proliferation, adhesion, and motility were found to be regulated differentially with respect to S4KD and control cells. Cell migration induced by TGF-beta was inhibited in the S4KD cells, which might be associated with a different regulation of integrin beta7. The knock down of a specific gene using stable RNAi appears to be a promising tool for analysing endogenous gene function.
Previous microarray studies on breast cancer identified multiple tumour classes, of which the most prominent, named luminal and basal, differ in expression of the oestrogen receptor alpha gene (ER). We report here the identification of a group of breast tumours with increased androgen signalling and a 'molecular apocrine' gene expression profile. Tumour samples from 49 patients with large operable or locally advanced breast cancers were tested on Affymetrix U133A gene expression microarrays. Principal components analysis and hierarchical clustering split the tumours into three groups: basal, luminal and a group we call molecular apocrine. All of the molecular apocrine tumours have strong apocrine features on histological examination (P=0.0002). The molecular apocrine group is androgen receptor (AR) positive and contains all of the ER-negative tumours outside the basal group. Kolmogorov-Smirnov testing indicates that oestrogen signalling is most active in the luminal group, and androgen signalling is most active in the molecular apocrine group. ERBB2 amplification is commoner in the molecular apocrine than the other groups. Genes that best split the three groups were identified by Wilcoxon test. Correlation of the average expression profile of these genes in our data with the expression profile of individual tumours in four published breast cancer studies suggest that molecular apocrine tumours represent 8-14% of tumours in these studies. Our data show that it is possible with microarray data to divide mammary tumour cells into three groups based on steroid receptor activity: luminal (ER+ AR+), basal (ER- AR-) and molecular apocrine (ER- AR+).