Abstract B17: Epigenetic regulation of NOTCH oncogenic signaling in breast cancer

Abstract

Abstract Alterations in DNA methylation occur at different stages of cancer, including initiation, and may underlie up-regulation of genes with oncogenic functions. The NOTCH pathway is often overactive in breast cancer and plays roles in cancer development and progression. It is therefore a possible target for anti-cancer strategies. However, the mechanisms of NOTCH regulation in mammary carcinogenesis remain unclear which hinders the development of effective approaches to target this oncogenic pathway. Interestingly, certain dietary compounds such as polyphenols with a stilbenoid structure suppress the NOTCH signals in cancer and were shown in our study to modify epigenetic marks in genes positively regulating the pathway, including MAML2. In the present study, using two polyphenols, resveratrol from grapes and pterostilbene from blueberries, we investigate the implication of DNA methylation in regulation of NOTCH in breast cancer. Non-invasive MCF10CA1h and invasive MCF10CA1a human breast cancer cell lines were used as an experimental model. Following genome-wide DNA methylation analysis with Illumina 450K BeadChip array, pyrosequencing and QPCR were performed to assess methylation and expression of MAML2 and NOTCH target genes. Chromatin immunoprecipitation (ChIP) was applied to determine binding of epigenetic enzymes to MAML2 regulatory region. Depletion with siRNA was applied to establish functional link between MAML2 silencing and NOTCH activity. We found 4,293 CpG sites differentially methylated upon 9-day treatment of MCF10CA1h breast cancer cells with 15µM resveratrol as compared to untreated cells (0.05<differential methylation<-0.05, p<0.03, Wilcoxon rank-sum test). Among these changes, 3,508 CpGs corresponding to 1,707 genes were hypermethylated and functionally associated with oncogenic signaling pathways, including NOTCH. As the array data indicated, resveratrol led to re-methylation of enhancer region of MAML2 that is a coactivator of NOTCH target genes. The 15-20% increase in methylation of MAML2 was confirmed by pyrosequencing in breast cancer cells with both high and low invasive potential. Pterostilbene, an analog of resveratrol, exerted similar effects on MAML2 methylation state at 10µM concentration. Along with methylation of the MAML2 enhancer region, the compounds decreased MAML2 expression by 30-60%. Stronger MAML2 down-regulation was achieved in invasive cancer cells as compared to non-invasive cells after exposure to either resveratrol or pterostilbene. Epigenetic silencing of MAML2 was associated with downregulation of NOTCH target genes, including HES1 (30-50%), HEY1 (30-60%), and NOTCH1 (40-65%). Depletion of MAML2 with siRNA mimicked polyphenols' effects leading to suppression of NOTCH pathway. It supports a functional link between MAML2 and NOTCH signaling. The most profound effects were observed in invasive MCF10CA1a cells where further analyses revealed 2-fold increase in trimethylation of H3K27 (repressive histone mark) and 30% decrease in acetylation of H3K9 (activating mark) at MAML2 enhancer region upon exposure to resveratrol. The condensed chromatin structure was associated with binding of DNMT3B to the tested fragment of MAML2 upon polyphenol treatment whereas no binding was detected in untreated cells. It suggests the role of DNMT3B in increased methylation of MAML2, which is consistent with increased DNMT3B expression upon treatment with polyphenols. Our results establish a role for epigenetic modifications in regulation of NOTCH oncogenic signaling in breast cancer. It constitutes a novel insight into regulation of oncogenic signals in cancer and provides support for epigenetic-targeting strategies as an effective anti-cancer approach. This study was supported by the Purdue University Center for Cancer Research, Indiana CTSI (UL1TR001108), and Women's Global Health Institute granted to BS. Citation Format: Katarzyna Lubecka, Lucinda Kurzava, Kirsty Flower, Hannah Buvala, Dorothy Teegarden, Ignacio Camarillo, James M. Flanagan, Barbara Stefanska. Epigenetic regulation of NOTCH oncogenic signaling in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B17.