| Summary: | 碩士 === 國立中正大學 === 分子生物研究所 === 99 === The TGF-beta signaling pathway plays an important role in growth inhibition of ovarian surface epithelial cell. However, epithelial ovarian cancer became refractory to the inhibitory function of TGF-beta, and yet TGF-beta induced metastasis or epithelial-mesenchymal transition (EMT) in advanced ovarian cancer. Several studies have shown that epigenetic mechanisms are involved in the silencing of tumor suppressor and epithelium-related gene, in promoting tumor progression in ovarian cancer. Therefore, we hypothesized that disruption of TGF-beta signaling may result in epigenetic silencing of its downstream target genes in early ovarian cancer, while sustain activation of TGF-beta signaling may induce EMT phenotype.
In the first part of this study, we investigated the mechanisms leading to the epigenetic silencing of a novel TGF-beta/SMAD4 target gene RunX1T1 in ovarian cancer. In a RunX1T1 expressing IOSE cells, knockdown of SMAD4 reduced RunX1T1 expression by decreasing the ratio of dimethylation of histone H3 K4/K9 without affecting DNA methylation status. When transiently treated with DNMT inhibitor, the expression of RunX1T1 was partially restored in MCP3 cells, but gradual re-silencing through promoter re-methylation was observed after the treatment. Interestingly, SMAD4 knockdown accelerated this re-silencing process. These results suggest that TGF-beta signaling is essential for the maintenance of RunX1T1 expression.
In the second part of this study, we disrupted TGF-beta signaling pathway by over-expression SMAD7 in a mesenchymal ovarian cancer cell, CP70 in which E-cadherin is silenced by promoter methylation. Re-expression of E-cadherin can be observed after twenty passages of SMAD7 overexpression, while E-cadherin remained silence in the control cells. This re-expression was accompanied with decreased migration and invasion in SMAD7 overexpressing cells. As compared to control cells, DNA demethylation, increased H3K9 acetylation and decreased binding of TWIST can be observed at E-cadherin promoter. Combination of expression array and bioinformatic analysis identified that 14 TGF-beta targets with CpG island at the promoter regions were upregulated in SMAD7 overexpressing cells. These up-regulations may be due to epigenetic reversal as confirmed by MBD-capture PCR and demethylation treatment. Taken together, disruption of TGF-beta signaling may lead to epigenetic silencing of tumor suppressor. On the contrary, in advanced ovarian cancer, disruption of TGF-beta signaling may be able to induce demethylation of E-cadherin promoter and reverse EMT phenotype in ovarian cancer.
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