Mutant WT1 is Associated with DNA Hypermethylation of PRC2 Targets in AML and Responds to EZH2 Inhibition
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE62929 | GEO | 2014/11/04
SECONDARY ACCESSION(S): PRJNA266224
REPOSITORIES: GEO
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Project description:Gain-of-function mutations in exon 3 of beta-catenin (CTNNB1) are specific for Wilms' tumors that have lost WT1, but 50% of WT1-mutant cases lack such "hot spot" mutations. To ask whether stabilization of beta-catenin might be essential after WT1 loss, and to identify downstream target genes, we compared expression profiles in WT1-mutant versus WT1 wild-type Wilms' tumors. Supervised and nonsupervised hierarchical clustering of the expression data separated these two classes of Wilms' tumor. The WT1-mutant tumors overexpressed genes encoding myogenic and other transcription factors (MOX2, LBX1, SIM2), signaling molecules (TGFB2, FST, BMP2A), extracellular Wnt inhibitors (WIF1, SFRP4), and known beta-catenin/TCF targets (FST, CSPG2, CMYC). Beta-Catenin/TCF target genes were overexpressed in the WT1-mutant tumors even in the absence of CTNNB1 exon 3 mutations, and complete sequencing revealed gain-of-function mutations elsewhere in the CTNNB1 gene in some of these tumors, increasing the overall mutation frequency to 75%. Lastly, we identified and validated a novel direct beta-catenin target gene, GAD1, among the WT1-mutant signature genes. These data highlight two molecular classes of Wilms' tumor, and indicate strong selection for stabilization of beta-catenin in the WT1-mutant class. Beta-Catenin stabilization can initiate tumorigenesis in other systems, and this mechanism is likely critical in tumor formation after loss of WT1. Experiment Overall Design: Identification of WNT/Beta-Catenin or WT1 target genes. 39 individual samples.
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Project description:K-ras is one of the most frequently mutated human oncogenes. However, activation of K-ras can lead to either senescence or proliferation in primary cells. The precise mechanism governing these distinct outcomes remains unclear. Here we describe a novel loss-of-function screen to assess the role of specific genes identified as potential key regulators of K-ras driven oncogenesis. Using this approach, we identify the transcription factor Wt1 as an inhibitor of senescence in primary cells expressing oncogenic K-ras. Deletion or suppression of Wt1 expression leads to senescence of primary cells expressing oncogenic K-ras under the control of the native promotor and at physiological levels, but had no effect on cells expressing wild-type K-ras. Furthermore, loss of Wt1 in lung cancer cell lines that harbor mutant K-ras leads to apoptosis. Taken together, these observations reveal a novel role for Wt1 as a key regulator of the complex genetic network required for the oncogenic effect of the small GTPase K-ras. We compare the expression profiles of K-ras mutant mouse cell line (LKR13) upon shRNA knockdown of K-ras itself or Wt1. The study provides insights into the transcriptional role of Wt1 in the context of oncogenic K-ras.
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Project description:K-ras is one of the most frequently mutated human oncogenes. Activation of K-ras can lead to either senescence or proliferation in primary cells. The precise mechanism governing these distinct outcomes remains unclear. Here we utilized a loss-of-function screen to assess the role of specific genes identified as potential key regulators of K-ras driven oncogenesis. Using this approach, we identify the transcription factor Wt1 as an inhibitor of senescence in primary cells expressing oncogenic K-ras. Deletion or suppression of Wt1 expression leads to senescence of primary cells expressing oncogenic K-ras under the control of the native promotor at physiological levels, but has no effect on cells expressing wild-type K-ras. Wt1 contributes to K-ras driven lung tumorigenesis in vivo and loss of Wt1 is specifically deleterious to human lung cancer cell lines that are dependent on oncogenic K-ras. Taken together, these observations reveal a novel role for Wt1 as a key regulator of the complex genetic network required for the oncogenic effect of the small GTPase K-ras. We compare the expression profiles of Wild type, K-ras mutant, Wt1-null, and double K-ras mutant/Wt1-null mouse embryonic fibroblasts (MEFs). The study provides insights into the transcriptional role of Wt1 in the context of oncogenic K-ras. MEFs were infected with adenoviral Cre to activate K-ras (6 samples), knockout Wt1 (5 samples), both activate K-ras and knockout Wt1 (7 samples). As a control, MEFs were infected with adenoviral GFP (5 samples). RNA was isolated using Trizol 7 days after infection. RNA was further prepared by passage over an RNeasy column. cDNA synthesis, biotinylation of cRNA and hybridization to mouse Genechip 430A v2 containing 39,000 probes was performed according to the manufacturer's instructions (Affymetrix, Santa Clara). Microarray data was normalized with Expression Console software (Affymetrix, Santa Clara) using RMA algorithms.
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Project description:Gain-of-function mutations in exon 3 of beta-catenin (CTNNB1) are specific for Wilms' tumors that have lost WT1, but 50% of WT1-mutant cases lack such "hot spot" mutations. To ask whether stabilization of beta-catenin might be essential after WT1 loss, and to identify downstream target genes, we compared expression profiles in WT1-mutant versus WT1 wild-type Wilms' tumors. Supervised and nonsupervised hierarchical clustering of the expression data separated these two classes of Wilms' tumor. The WT1-mutant tumors overexpressed genes encoding myogenic and other transcription factors (MOX2, LBX1, SIM2), signaling molecules (TGFB2, FST, BMP2A), extracellular Wnt inhibitors (WIF1, SFRP4), and known beta-catenin/TCF targets (FST, CSPG2, CMYC). Beta-Catenin/TCF target genes were overexpressed in the WT1-mutant tumors even in the absence of CTNNB1 exon 3 mutations, and complete sequencing revealed gain-of-function mutations elsewhere in the CTNNB1 gene in some of these tumors, increasing the overall mutation frequency to 75%. Lastly, we identified and validated a novel direct beta-catenin target gene, GAD1, among the WT1-mutant signature genes. These data highlight two molecular classes of Wilms' tumor, and indicate strong selection for stabilization of beta-catenin in the WT1-mutant class. Beta-Catenin stabilization can initiate tumorigenesis in other systems, and this mechanism is likely critical in tumor formation after loss of WT1. Keywords: single channel
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Project description:K-ras is one of the most frequently mutated human oncogenes. However, activation of K-ras can lead to either senescence or proliferation in primary cells. The precise mechanism governing these distinct outcomes remains unclear. Here we describe a novel loss-of-function screen to assess the role of specific genes identified as potential key regulators of K-ras driven oncogenesis. Using this approach, we identify the transcription factor Wt1 as an inhibitor of senescence in primary cells expressing oncogenic K-ras. Deletion or suppression of Wt1 expression leads to senescence of primary cells expressing oncogenic K-ras under the control of the native promotor and at physiological levels, but had no effect on cells expressing wild-type K-ras. Furthermore, loss of Wt1 in lung cancer cell lines that harbor mutant K-ras leads to apoptosis. Taken together, these observations reveal a novel role for Wt1 as a key regulator of the complex genetic network required for the oncogenic effect of the small GTPase K-ras. We compare the expression profiles of K-ras mutant mouse cell line (LKR13) upon shRNA knockdown of K-ras itself or Wt1. The study provides insights into the transcriptional role of Wt1 in the context of oncogenic K-ras. LKR13 cells were infected with plko.1s vectors carrying one of two shRNAs against K-ras or Wt1. Control cells were infected with an shRNA against GFP. 48 hours after infection, cells were selected with puromycin for 3 days. RNA was isolated using Trizol 7 days after infection. RNA was further prepared by passage over an RNeasy column. cDNA synthesis, biotinylation of cRNA and hybridization to mouse Genechip 430A v2 containing 39,000 probes was performed according to the manufacturerâ??s instructions (Affymetrix, Santa Clara). Microarray data was normalized with Expression Console software (Affymetrix, Santa Clara), using RMA algorithms.
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2014-06-01 | E-GEOD-58073 | biostudies-arrayexpress