Abstract
Cancer cells exhibit phenotypic plasticity during epithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET) involving intermediate states. To study genome-wide epigenetic remodeling associated with EMT plasticity, we integrate the analyses of DNA methylation, ChIP-sequencing of five histone marks (H3K4me1, H3K4me3, H3K27Ac, H3K27me3 and H3K9me3) and transcriptome profiling performed on ovarian cancer cells with different epithelial/mesenchymal states and on a knockdown model of EMT suppressor Grainyhead-like 2 (GRHL2). We have identified differentially methylated CpG sites associated with EMT, found at promoters of epithelial genes and GRHL2 binding sites. GRHL2 knockdown results in CpG methylation gain and nucleosomal remodeling (reduction in permissive marks H3K4me3 and H3K27ac; elevated repressive mark H3K27me3), resembling the changes observed across progressive EMT states. Epigenetic-modifying agents such as 5-azacitidine, GSK126 and mocetinostat further reveal cell state-dependent plasticity upon GRHL2 overexpression. Overall, we demonstrate that epithelial genes are subject to epigenetic control during intermediate phases of EMT/MET involving GRHL2.
Highlights
Cancer cells exhibit phenotypic plasticity during epithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET) involving intermediate states
We further demonstrate that EMT induced by Grainyhead-like 2 (GRHL2) knockdown would result in genome-wide epigenetic remodeling similar to that observed in ovarian cancer cells with progressive EMT phenotypes
We validated the 450K methylation data with bisulfite pyrosequencing at selected loci of CDH1 (E-cadherin gene) and KRT19, and the results showed an overall similar trend—the tested promoter regions were hypermethylated in cell lines with high EMT score (Supplementary Fig. 2)
Summary
Cancer cells exhibit phenotypic plasticity during epithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET) involving intermediate states. With the advancement of technologies, attempts have been made to elucidate genome-wide epigenetic changes during EMT, mainly using the TGF-βinduced[14,15,16,17] or the TWIST1-induced system[18] These studies lack the population to capture epigenetic changes associated with intermediate EMT states that occur during cancer progression, which may involve pathways independent of TGF-β or TWIST1. These different stages of cell-state transition may have distinct epigenetic regulations of epithelial/mesenchymal genes. We further demonstrate that EMT induced by GRHL2 knockdown would result in genome-wide epigenetic remodeling similar to that observed in ovarian cancer cells with progressive EMT phenotypes. GRHL2 overexpression and co-treatment of epigenetic-modifying drugs 5-azacitidine—an inhibitor of DNA methyltransferases (DNMTs), GSK126—an inhibitor of enhancer of zeste homolog 2 (EZH2), and/or mocetinostat—an inhibitor of class I histone deacetylases (HDACs), could induce MET to different extents, in cells lines with an intermediate EMT or a full EMT state
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