Effect of GRHL2 in HMLE+Twist-ER+4-OHT cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE36081 | GEO | 2012/02/25
SECONDARY ACCESSION(S): PRJNA151731
REPOSITORIES: GEO
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Project description:Using a retrovirus-mediated cDNA expression cloning approach we identified the grainyhead-like 2 (GRHL2) transcription factor as novel protooncogene. Overexpression of GRHL2 in NIH3T3 cells induced striking morphological changes, an increase in cell proliferation, anchorage-independent growth, and tumor growth in vivo. By combining a microarray analysis and a phylogenetic footprinting analysis with various biochemical assays we identified the epidermal growth factor receptor family member Erbb3 as a novel GRHL2 target gene. In breast cancer cell lines, shRNA-mediated knockdown of GRHL2 expression or functional inactivation of GRHL2 using dominant-negative GRHL2 proteins induce downregulation of ERBB3 gene expression, a striking reduction in cell proliferation and morphological and phenotypical alterations characteristic of an epithelial-to-mesenchymal transition (EMT), thus implying dual roles of GRHL2 in breast carcinogenesis. Interestingly, we could further demonstrate that expression of GRHL2 is directly suppressed by the transcription factor zinc-finger-enhancer binding protein 1 (ZEB1) which in turn is a direct target for repression by GRHL2, suggesting that the EMT transcription factors GRHL2 and ZEB1 form a double negative regulatory feedback loop in breast cancer cells. Finally, a comprehensive immunohistochemical analysis of GRHL2 expression in primary breast cancers showed loss of GRHL2 expression at the invasive front of primary tumors. A pathophysiological relevance of GRHL2 in breast cancer metastasis is further demonstrated by our finding of a statistically significant association between loss of GRHL2 expression in primary breast cancers and lymph-node metastasis. We thus demonstrate a crucial role of GRHL2 in breast carcinogenesis. To evaluate the role of the GRHL2 transcription factor in carcinogenessis we overexpressed the human GRHL2 protein in NIH3T3 cells. Parental NIH3T3 cells do not express the orthologic protein. Alterations in genome-wide transcriptional profiles were determined by microarray analysis.
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Project description:Epithelial ovarian cancer (EOC) is clinically heterogeneous, comprising different histological and biological subtypes. Multiple studies have implicated epithelial-mesenchymal transition (EMT), a biological process by which polarized epithelial cells convert into a mesenchymal phenotype, to contribute significantly to this molecular heterogeneity of EOC. From gene expression analyses of a collection of EMT-characterized EOC cell lines, we found that the expression of the transcription factor Grainyhead-like 2 (GRHL2) correlates with E-cadherin expression and the epithelial phenotype. EOC tumors with lower levels of GRHL2 are associated with the Mes (mesenchymal) molecular subtype and show poorer overall survival in patients. Here, we demonstrate that shRNA-mediated knockdown of GRHL2 in EOC cells with an epithelial phenotype resulted in EMT changes, with increased cell migration, invasion and motility. By ChIP-sequencing and gene expression microarray, we identified a variety of target genes regulated by GRHL2, including protein-coding and non-coding genes. Our data suggest that GRHL2 maintains the epithelial phenotype of EOC cells through the regulatory networks of miR-200b/a, ZEB1 and E-cadherin. These findings support GRHL2 as a crucial player in the molecular heterogeneity of EOC.
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Project description:Using a retrovirus-mediated cDNA expression cloning approach we identified the grainyhead-like 2 (GRHL2) transcription factor as novel protooncogene. Overexpression of GRHL2 in NIH3T3 cells induced striking morphological changes, an increase in cell proliferation, anchorage-independent growth, and tumor growth in vivo. By combining a microarray analysis and a phylogenetic footprinting analysis with various biochemical assays we identified the epidermal growth factor receptor family member Erbb3 as a novel GRHL2 target gene. In breast cancer cell lines, shRNA-mediated knockdown of GRHL2 expression or functional inactivation of GRHL2 using dominant-negative GRHL2 proteins induce downregulation of ERBB3 gene expression, a striking reduction in cell proliferation and morphological and phenotypical alterations characteristic of an epithelial-to-mesenchymal transition (EMT), thus implying dual roles of GRHL2 in breast carcinogenesis. Interestingly, we could further demonstrate that expression of GRHL2 is directly suppressed by the transcription factor zinc-finger-enhancer binding protein 1 (ZEB1) which in turn is a direct target for repression by GRHL2, suggesting that the EMT transcription factors GRHL2 and ZEB1 form a double negative regulatory feedback loop in breast cancer cells. Finally, a comprehensive immunohistochemical analysis of GRHL2 expression in primary breast cancers showed loss of GRHL2 expression at the invasive front of primary tumors. A pathophysiological relevance of GRHL2 in breast cancer metastasis is further demonstrated by our finding of a statistically significant association between loss of GRHL2 expression in primary breast cancers and lymph-node metastasis. We thus demonstrate a crucial role of GRHL2 in breast carcinogenesis.
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Project description:Ovarian cancer (OC) cells exhibit varying extents of epithelial/mesenchymal phenotype, forming an EMT spectrum based on their EMT scores. Combining 450K DNA methylation array, ChIP-sequencing of five histone H3 marks (H3K4me1, H3K4me3, H3K27Ac, H3K27me3 and H3K9me3) and transcriptomic analyses, we examined the genome-wide epigenetic profiles of OC cell lines with progressive EMT phenotypes, including a knockdown model of EMT suppressor Grainyhead-like 2 (GRHL2) which showed intermediate state transition. We identified differentially methylated CpG sites (DMCs) associated with EMT genes, found mainly at the promoters of epithelial genes including CDH1, GRHL2 and genes with GRHL2 binding sites. This prompted us to further study the epigenetic role of GRHL2. GRHL2-knockdown resulted in CpG methylation gain at GRHL2 binding sites and at DMCs associated with epithelial genes. Importantly, the changes of histone modifications occurred in GRHL2-knockdown cells are also more prominent among epithelial genes and at GRHL2 binding sites—reduction in permissive marks H3K4me3 and H3K27ac; elevated repressive mark H3K27me3—similar to the transitions observed in a four-cell-line model representing the EMT spectrum. We tested the effects of GRHL2 overexpression in conjunction with epigenetic drugs 5-azacitidine, GSK126 and mocetinostat, all of which exerted MET effects to different extents, depending on the existing cell state. Overall, GRHL2 is required for the epigenetic and chromatin remodeling of epithelial genes during EMT/MET that control intermediate phenotype switching.
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