Project description:DNA methylation profiling of four DLD1 colorectal cancer cell derivatives that recapitulate EMT/MET transitions during metastasis. The Illumina Infinium 450k Human DNA methylation Beadchip was used to obtain DNA methylation profiles across approximately 450,000 CpGs.
Project description:H3K4me3, H3K27me3, and H3K79me3 profiling of a cellular model of colorectal cancer progression, which is based on four defined derivates of a CRC cell line that resulted from EMT/MET transitions and phenotypically recapitulate the metastatic cascade: epithelial-like primary tumor, mesenchymal-like tumor cells growing in soft agar or as colonospheres, and mouse xenografts of the latter resulting in lung-metastasis comprised of epithelial-state cells.
Project description:microRNA profiling of a cellular model of colorectal cancer progression, which is based on four defined derivates of a CRC cell line that resulted from EMT/MET transitions and phenotypically recapitulate the metastatic cascade: epithelial-like primary tumor, mesenchymal-like tumor cells growing in soft agar or as colonospheres, and mouse xenografts of the latter resulting in lung-metastasis comprised of epithelial-state cells.
Project description:RNA profiling of a cellular model of colorectal cancer progression, which is based on four defined derivates of a CRC cell line that resulted from EMT/MET transitions and phenotypically recapitulate the metastatic cascade: epithelial-like primary tumor, mesenchymal-like tumor cells growing in soft agar or as colonospheres, and mouse xenografts of the latter resulting in lung-metastasis comprised of epithelial-state cells.
Project description:Our goal was to assess gene expression changes that occur when Lymphoid Enhancer Factor-1 (LEF-1) promotes epithelial-mesenchymal transition (EMT), the primary mechanism of tumor metastasis. To observe this phenomenon without interference from other signaling pathways, we selected DLD1 colon carcinoma cells (ATCC) which contain a mutation in APC. APC is a necessary component of a ubiquitin protein complex (including GSK-3beta, Axin, etc.) that is responsible for degrading cytoplasmic beta-catenin. Therefore, sufficient levels of LEF-1 can be easily activated by forming complexes with the abundant beta-catenin located in the cytoplasm of DLD1 cells. These complexes can then promote transcription of genes that stimulate EMT. We treated DLD1 cells with an adenoviral LEF-1 expression construct, which induced EMT within 48 hours. RNA was then extracted from these cells along with untreated DLD1 cells, then subjected to microarray analysis. From this analysis, we acquired several gene expression profiles by which epithelial colon carcinoma cells transform to an invasive, mesenchymal phenotype to initiate metastasis. Keywords: epithelial-mesenchymal transition, tumor metastasis, cancer progression, epithelial cell plasticity
Project description:Our goal was to assess gene expression changes that occur when Lymphoid Enhancer Factor-1 (LEF-1) promotes epithelial-mesenchymal transition (EMT), the primary mechanism of tumor metastasis. To observe this phenomenon without interference from other signaling pathways, we selected DLD1 colon carcinoma cells (ATCC) which contain a mutation in APC. APC is a necessary component of a ubiquitin protein complex (including GSK-3beta, Axin, etc.) that is responsible for degrading cytoplasmic beta-catenin. Therefore, sufficient levels of LEF-1 can be easily activated by forming complexes with the abundant beta-catenin located in the cytoplasm of DLD1 cells. These complexes can then promote transcription of genes that stimulate EMT. We treated DLD1 cells with an adenoviral LEF-1 expression construct, which induced EMT within 48 hours. RNA was then extracted from these cells along with untreated DLD1 cells, then subjected to microarray analysis. From this analysis, we acquired several gene expression profiles by which epithelial colon carcinoma cells transform to an invasive, mesenchymal phenotype to initiate metastasis. Experiment Overall Design: DLD1 cells were treated with an adenoviral LEF-1 expression construct as described by Kim et al. (2002). Total RNA was extracted from both untreated and treated DLD1 cells using the RNeasy mini extraction kit (Qaigen). RNA amplification, biotin labeling, microarray hybridization, and fluidics were performed following the eukaryotic sample and array processing protocol (Affymetrix). Chips were scanned using an Affymetrix Gene Array Scanner (Hewlett-Packard). Raw data was compiled using Microarray Suite 5.0 software (Affymetrix).
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.
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.