Abstract 4353: Polyphenol-mediated epigenetic reactivation of tumor suppressor gene SEMA3A in breast cancer cells

Abstract

Abstract Alterations in DNA methylation occur in cancer, and may underlie silencing of genes with tumor suppressor functions. Reversing DNA methylation, thus potentially reactivating genes that combat cancer, constitutes a promising anti-cancer strategy. Interestingly, studies have indicated that certain dietary polyphenols, such as resveratrol present in grapes, exert anti-cancer effects through epigenetic regulation of gene expression. However, studies have been limited to candidate genes, and comprehensive and mechanistic insights are missing. In the present study, following genome-wide DNA methylation analysis with Illumina 450K BeadChip array, we identified CpG sites within regulatory regions of tumor suppressor genes that are hypomethylated upon treatment of breast cancer cells with resveratrol. Non-invasive MCF10CA1h and invasive MCF10CA1a human breast cancer cell lines were used as an experimental model. Pyrosequencing and QPCR were performed to assess respectively methylation and expression of selected genes. Chromatin immunoprecipitation (ChIP) was applied to assess binding events. We identified 990 hypomethylated CpG sites in MCF10CA1h and 1,146 hypomethylated CpG sites in MCF10CA1a cells upon 9-day treatment with 15µM resveratrol as compared with control untreated cells (differential methylation ≤-0.05, nominal p<0.05, limma t-test). Those CpG sites corresponded to approximately 650 genes that were predominantly associated with tumor suppressor function in cancer. We selected tumor suppressor gene SEMA3A as resveratrol target for further investigation. As array data indicated, resveratrol led to reduction in methylation of SEMA3A promoter region. The 18% decrease in methylation of SEMA3A was confirmed by pyrosequencing in MCF10CA1a breast cancer cells. This coincided with 23% up-regulation of SEMA3A expression. In addition, analysis of the effects on DNA methyltransferases (DNMTs) demonstrated that resveratrol decreases expression of DNMT3A. Furthermore, ChIP indicated decreased occupancy of DNMT3A at the SEMA3A promoter, suggesting that down-regulation and lower affinity of this de novo DNA methylating enzyme to DNA may mediate decrease in methylation within tumor suppressor genes upon resveratrol exposure. In support of reactivation of SEMA3A, active histone mark, H3K9ac, was significantly increased and repressive histone mark, H3K27me3, was significantly decreased after resveratrol treatment, indicating an open, more transcriptionally active chromatin state. These results demonstrate a role for polyphenol-mediated epigenetic modifications in reactivation of tumor suppressor genes in breast cancer and pave the way for further studies on the mechanism driving these changes. This study was supported by the PCCR, Indiana CTSI (UL1TR001108), Women’s Global Health Institute, and USDA National Institute of Food and Agriculture (Hatch project 1005656) granted to BS. Citation Format: Megan Beetch, Katarzyna Lubecka, Lucinda Kurzava, Kirsty Flower, James M. Flanagan, Barbara Stefanska. Polyphenol-mediated epigenetic reactivation of tumor suppressor gene SEMA3A in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4353. doi:10.1158/1538-7445.AM2017-4353