An Epigenetic Strategy To Degrade The Estrogen Receptor In Breast Cancer

Epigenetic changes in DNA methylation can lead to altered gene expression and the development and progression of breast cancer. Luminal breast tumors are associated with increased DNA hypermethylation of CpG islands while basal, triple negative tumors more commonly have lower levels of DNA methylation. Increasing evidence suggests that aberrant epigenetic alterations in DNA methylation and chromatin structure may also contribute to endocrine resistance in luminal breast cancers. We thus asked whether epigenetic therapy using the demethylating agent, 5-Azacytidine (AZA) is an effective anti-tumorigenic agent in luminal breast cancers, what epigenetic mechanisms might be involved in the response and whether AZA could play a role in the sensitization of tamoxifen resistant cells. We have demonstrated using 26 breast cancer cell lines that luminal B, ER+ breast cancer is more responsive to treatment with AZA than are Her2+ or triple negative (TN) breast cancers. Cells were treated in culture with low nanomolar doses of AZA for 72 hrs, followed by transplantation into mice and weekly palpation of tumors. Tumorgraft measurements show that 67% of luminal B, ER+ cells are sensitive to AZA, whereas only 22% of TN and 25% of Her2+ cells exhibit an anti-tumorigenic response to AZA in mice. Expression data showed that AZA leads to decreases in ER mRNA and protein expression in 50% and 67% of luminal B lines respectively. Further investigation showed that a longer AZA treatment led to lower ERα protein levels and stronger tumor growth inhibition. Protease inhibitor (MG132) treatment rescued ERα protein reduction in 50% of the luminal B cell lines, suggesting that an AZA-mediated, ubiquitin-26S proteasomal pathway may be responsible for the degradation of ER. Gene expression analysis demonstrated that the E1 ubiquitin-activating enzyme, (UBA7-UBE1L) was up-regulated at the mRNA and protein levels by AZA. UBA7 has been suggested to function as a tumor suppressor in lung cancer and elevations in UBA7 have been correlated with longer overall survival in breast cancer. The interaction of UBA7 with ERα protein was validated by immunoprecipitation and over-expression of UBA7 in ZR-75-1 cells led to a reduction in ERα protein. We further analyzed the UBA7 promoter region by Illumina 450K array and bisulfite sequencing in our cell lines and identified two CpG rich sequences in the promoter that may be important for AZA induced UBA7 promoter demethylation and gene re-expression. We also demonstrated in both tamoxifen sensitive and resistant cells that combination therapy with both AZA and tamoxifen was more effective at inhibiting tumor growth than the single agents. Studies are ongoing in our lab to better understand this response. Our data suggest that AZA may be clinically useful to inhibit the growth of some luminal breast tumors via its actions to increase expression of UBA7 and to degrade ER and may have efficacy in combination with anti-estrogen treatments. Citation Format: Huili Li, Foteinos-Ioannis Dimitrakopoulos, Meredith Stone, Lauren Murphy, Stephen Baylin, Cynthia A. Zahnow. An epigenetic strategy to degrade the estrogen receptor in breast cancer [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 1002. doi:10.1158/1538-7445.AM2017-1002