Disruption of TGF-β signaling induces demethylation of E-cadherin promoter and reverses mesenchymal phenotype in ovarian cancer

Lin Yu Chen, Rui-Lan Huang, Han Lin Chou, Jian Liang Chou, Wei-Ting Deng, Hui Wen Yang, Ru Inn Lin, Chin Yang Li, Huey-Jen Lin, Tim Hui Ming Huang, Chien Chih Chiu, Hung-Cheng Lai, Michael W Y Chan

Research output: Contribution to journalArticle

Abstract

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLThe TGF-β signaling pathway plays an important role in controlling cell growth and differentiation. In advanced ovarian cancer, frequent TGF-β-induced metastasis or epithelial-mesenchymal transition (EMT) can be observed. This phenomenon is often associated with epigenetic silencing of epithelial marker, E-cadherin which can also be observed in ovarian cancer cell lines that demethylation treatment restored E-cadherin expression. We recently hypothesized that long term activation of TGF-β signaling may induce EMT phenotype by epigenetic silencing of E-cadherin and that inhibition of the signaling may restore E-cadherin and reverse EMT in ovarian cancer (Chou et al., Expert Opin Ther Targets 2010). In this study, we cloned the cDNA of the inhibitory SMAD, SMAD7 from a human immortalized ovarian surface epithelial cell, IOSE into pcDNA3.1 mammalian expression vector. The inhibitory effect of this SMAD7 expression vector on TGF-β signaling has been confirmed by reporter assay. We then stably transfected the SMAD7 expression vector into a mesenchymal ovarian cancer cell, CP70 in which E-cadherin is silenced by complete promoter methylation. Cells over-expressing SMAD7 showed up-regulation of SMAD7 and a decrease in SMAD2 phosphorylation while the control cells maintained a hyperphosphorylation of SMAD2 thus suggesting that TGF-β signaling is disrupted in SMAD7-overexpressing cells. We further examined the expression of E-cadherin from passage 5 up to 30 of the stable transfectants. Surprisingly, stable restoration of E-cadherin can only be observed from passage 20 and thereafter, of the SMAD7-overexpressing cells, while E-cadherin remained silence in the control cells. To investigate if this restoration is due to promoter demethylation of E-cadherin, we performed bisulphite pyrosequencing on the E-cadherin promoter CpG island spanning -586 to -12 of the region. Compared with control cells, consistent demethylation of E-cadherin promoter can be observed at 2 CpG sites located at -214 and -235 of the promoter such that gradual demethylation occurred from passage 5 to 30 of the SMAD7-overexpressing cells (passage 20, methylation% control vs SMAD7: 91% vs 67% at -235; 80% vs 52% at -214); while the rest of the CpG sites remained heavily methylated. This demethylation may be due to down-regulation of transcriptional repressor, TWIST after SMAD7 transfection. Additionally, one of the SMAD7 stable expression clones with highest restoration of E-cadherin showed decreased migration and invasion ability as determined by wound healing and invasion assay. Taken together, disruption of TGF-β signaling can induce demethylation of E-cadherin promoter and reverse EMT phenotype in ovarian cancer. The therapeutic potential of targeting TGF-β signaling pathway in inhibiting metastasis of ovarian cancer deserves further investigation.Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3433. doi:10.1158/1538-7445.AM2011-3433
Original languageTraditional Chinese
Pages (from-to)3433
Number of pages1
JournalCancer Research
Volume71
Issue number8 Supplement
Publication statusPublished - Nov 13 2014
Externally publishedYes

Cite this

Chen, L. Y., Huang, R-L., Chou, H. L., Chou, J. L., Deng, W-T., Yang, H. W., Lin, R. I., Li, C. Y., Lin, H-J., Huang, T. H. M., Chiu, C. C., Lai, H-C., & Chan, M. W. Y. (2014). Disruption of TGF-β signaling induces demethylation of E-cadherin promoter and reverses mesenchymal phenotype in ovarian cancer. Cancer Research, 71(8 Supplement), 3433. http://cancerres.aacrjournals.org/content/71/8_Supplement/3433.abstract