Abstract 2318: The role of EZH2 as an epigenetic switch of the TGF-β/SMAD4 targets in regulating EMT in ovarian cancer
Authors: Lin Jora Meng-Ju , Shay Jacqueline , Chou Jian-Liang , Yan Pearlly S. , Huang Tim H.-M. , Lai Hung Cheng , Chan Michael W.y. 
Published: May 2015

Abstract
The TGF-β signaling regulates numerous cellular processes, including cell proliferation, cell differentiation, apoptosis, migration and cell fate. It was also demonstrated that TGF-β functions as a tumor suppressor in normal ovarian surface epithelium (OSE) cells but promotes tumor proliferation and epithelial-mesenchymal transition (EMT) during ovarian cancer progression. Nevertheless, the molecular mechanisms leading to this divert role of TGF-β signaling in ovarian cancer remains to be elucidated. Our previous studies demonstrated that several TGF-β/SMAD4 regulated targeted are epigenetically silenced by DNA methylation and histone modification including H3K27me3. We therefore hypothesized that the histone-lysine N-methyltransferase, EZH2 may act as an epigenetic switch to facilitate the TGF-β mediated EMT in ovarian cancer. In this study, we utilized our previously identified TGF-β responsive targets using combined ChIP-chip and expression arrays in an immortalized ovarian surface epithelial (IOSE) cell which showed TGF-β growth inhibition. Bioinformatics analysis using ENCODE ChiP-Seq data identified that several of these TGF-β targets are marked by EZH2. To investigate the effect of TGF-β signaling on the methylome of ovarian cancer, we performed MBDcap-Seq in SMAD4 knockdown CP70 cells. Our result showed that there are 99 and 73 TGF-β targets showing hyper- or hypo-methylation respectively after SMAD4 knockdown. Analysis of the gene functions on DAVID has uncovered that some of these hypermethylated genes are associated with EMT process and the TGF-β signaling pathway. On the other hand, the hypomethylated genes are associated with transcription repressor activity. We selected one of the hypermethylated target, LTBP2 which was previously found to be a tumor suppressor for further experiment. This gene was expressed in IOSE but down regulated in a panel of ovarian cancer cells showing overexpression of EZH2. Except for IOSE, promoter hypermethylation of LTBP2 was found in ovarian cancer cells as revealed by bisulphite pyrosequencing. In consistent with deep sequencing result, knock-down of SMAD4 further increased the promoter methylation in CP70 cells. Treatment of demethylation agent, 5azaDC partially restored its expression in these cancer cells. Interestingly, synergistic treatment of 5azaDC and EZH2 inhibitor, GSK343 resulted in a dramatic increase of LTBP2 expression in MCP3 and CP70 cells. Taken together, our result suggested that EZH2 may be involved in the epigenetic silencing of TGF-β/SMAD4-regulated tumor suppressors in ovarian cancer. The differential occupancy of EZH2 into these SMAD4 loci may act as an epigenetic switch to turn the function of TGF-β from a tumor suppressor into an EMT regulator. The therapeutic potential of targeting EZH2 in the inhibition of EMT in ovarian cancer deserves further investigation.
Citation Format: Jora Meng-Ju Lin, Jacqueline Shay, Jian-Liang Chou, Pearlly S. Yan, Tim H.-M. Huang, Hung Cheng Lai, Michael W.y. Chan. The role of EZH2 as an epigenetic switch of the TGF-β/SMAD4 targets in regulating EMT in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2318. doi:10.1158/1538-7445.AM2014-2318