Investigations into the epigenetic mechanisms regulating the prostanoid receptor EP2 in estrogen dependent breast cancer
S. To1,3, K. Takagi2, Y. Miki2, H. Sasano2, E. Simpson1,3, K. Knower1 & C. Clyne1,3
Estrogen excess is a major contributing factor to the development and progression of post-menopausal Estrogen Receptor positive (ER+) breast cancers. Increased activity of aromatase, the enzyme that converts androgens to estrogens, and upregulation of its encoding gene CYP19A1 is often observed in breast adipose fibroblasts (BAFs) surrounding ER+ tumours. Understanding the process by which CYP19A1 is regulated between normal and diseased breast stroma may help to improve outcomes for breast cancer sufferers.
Prostaglandin E2 (PGE2), secreted by breast tumours, stimulates CYP19A1 expression and estrogen production in surrounding BAFs. Whilst hormonal regulation of this process has been examined, there is increasing evidence suggesting that epigenetic mechanisms, such as DNA methylation, serve to influence the PGE2 pathway. These mechanisms are thought to lead to CYP19A1 transcription by silencing or activating upstream factors. The present study aims to determine whether an inverse correlation existed between expression of the prostanoid receptor EP2 and their promoter DNA methylation status in the context of breast cancer derived cell lines and clinical samples of cancer-associated stroma and matching normal stroma.
Sodium bisulfite sequencing of CpG methylation within the EP2 promoter revealed differential methylation showing inverse correlation with respective mRNA levels in cell lines MDA-MB-231 (ER-), MCF7 (ER+) and MCF10A (normal epithelial). Results were confirmed with inhibition of DNA methylation using 5-aza-2-deoxycytidine (5aza), which showed that expression of EP2 in MDA-MB-231 cells could be increased upon demethylation. Analysis of 9 clinical samples comprising ductal carcinoma in situ, invasive ductal carcinoma and triple negative tumours showed that differences in expression levels of EP2 between normal and cancer-associated stroma could not be attributed to differences in promoter methylation status. This suggests that upstream factors are responsible for the epigenetic regulation of CYP19A1 and EP2. Identification of these factors will further our understanding of epigenetic gene regulation in ER+ breast cancer.
Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.
Funding: This work was supported, however funding details unavailable.