Abstract A65: Snail, a potent inducer of global DNA methylation in ovarian cancer

Abstract Snail plays a critical role in the epithelial to mesenchymal transition (EMT). To investigate the role of Snail in this event, we established inducible Snail expression in ovarian cancer cell lines. Reverse Phase Protein Assay (RPPA) revealed that Dnmt1 was upregulated after snail expression in SKOv3-Snail inducible cells. Dnmt1, a major enzyme responsible for transferring a methyl group to the cytosine in a CpG dinucleotide that commonly occurs in the promoter region of genes, is required for heterochromatin maintenance and transcriptional silencing in somatic cells. To evaluate the function of Snail in mediating DNA methylation in ovarian cancer, we first validated the correlated expression of Snail and Dnmt1 by Western blotting in SKOv3-Snail and HO8910PM-Snail inducible cells. Under non-inducible condition, Dnmt1 was mainly localized in the nucleus. However, Dnmt1 was increased in the cytoplasm and had little change in the nucleus when Snail was induced. In a parallel experiment, two other DNA methyltransferases Dnmt3a and Dnmt3b, attribute to de novo DNA methylation during embryonic development, remained localized in the nucleus and had no altered expression when Snail was induced. We also examined 5-Methylcytosine (5-MC) expression in SKOv3-Snail and HO8910PM-Snail inducible cells using immunofluorescent staining. Nuclear 5-MC, which infers global DNA methylation, became apparent when Snail was induced. To further extend this observation in ovarian cancer, we analyzed the expression of 5-MC in an ovarian cancer progression tissue array. We found that 5-MC was expressed in nucleus in all ovarian cancer and only a small portion of positive stain in normal ovarian epithelial and benign neoplasm. We further discovered that expression of 5-MC not only correlated with the stage of ovarian cancer but also significantly elevated in metastatic lesions. In our previous study, we demonstrated that Snail expression correlated with stages of ovarian cancer and its expression was elevated in metastatic leisions, which was also in accordance with the pattern of 5-MC pattern in ovarian cancer cells. To identify the underlying molecular mechanism, we performed NimbleGen Multiplex CpG Island Plus RefSeq Promoter Array using Snail inducible ovarian cancer cell lines. After normalization, a 1.25 fold of increase in total DNA methylation was found in Snail inducible cells in comparison with the un-inducible cells. The increased DNA methylation is consistent with the Snail-5-MC pattern in ovarian cancer cells. In summary, our data show that Snail can alter the global DNA methylation in ovarian cancer, through the regulation of Dnmt1. Further study will shed a new light into this intricate epigenetic regulation and may provide novel therapeutic intervention for targeting ovarian cancer. Citation Format: Jin Hongyan, Zhou Jiayi, Zhao Xiaoyan, Jia Luoqi, Lu Yilin, Yu Yinhua. Snail, a potent inducer of global DNA methylation in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A65.