Estrogen receptor a dependent regulation of estrogen related receptor β and its role in cell cycle in breast cancer

Background: Breast cancer (BC) is highly heterogeneous with similar to 60-70% of estrogen receptor positive BC patient's response to anti-hormone therapy. Estrogen receptors (ERs) play an important role in breast cancer progression and treatment. Estrogen related receptors (ERRs) are a group of nuclear receptors which belong to orphan nuclear receptors, which have sequence homology with ERs and share target genes. Here, we investigated the possible role and clinicopathological importance of ERR beta in breast cancer. Methods: Estrogen related receptor beta (ERR beta) expression was examined using tissue microarray slides (TMA) of Breast Carcinoma patients with adjacent normal by immunohistochemistry and in breast cancer cell lines. In order to investigate whether ERR beta is a direct target of ER alpha, we investigated the expression of ERR beta in short hairpin ribonucleic acid knockdown of ER alpha breast cancer cells by western blot, qRT-PCR and RT-PCR. We further confirmed the binding of ERa by electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), Re-ChIP and luciferase assays. Fluorescence-activated cell sorting analysis (FACS) was performed to elucidate the role of ERR beta in cell cycle regulation. A Kaplan-Meier Survival analysis of GEO dataset was performed to correlate the expression of ERR beta with survival in breast cancer patients. Results: Tissue microarray (TMA) analysis showed that ERR beta is significantly down-regulated in breast carcinoma tissue samples compared to adjacent normal. ER + ve breast tumors and cell lines showed a significant expression of ERR beta compared to ER-ve tumors and cell lines. Estrogen treatment significantly induced the expression of ERR beta and it was ER alpha dependent. Mechanistic analyses indicate that ER alpha directly targets ERR beta through estrogen response element and ERR beta also mediates cell cycle regulation through p18, p21(cip) and cyclin D1 in breast cancer cells. Our results also showed the up-regulation of ERR beta promoter activity in ectopically co-expressed ER alpha and ERR beta breast cancer cell lines. Fluorescence activated cell sorting analysis (FACS) showed increased G0/G1 phase cell population in ERR beta overexpressed MCF7 cells. Furthermore, ERR beta expression was inversely correlated with overall survival in breast cancer. Collectively our results suggest cell cycle and tumor suppressor role of ERR beta in breast cancer cells which provide a potential avenue to target ERR beta signaling pathway in breast cancer. Conclusion: Our results indicate that ERR beta is a negative regulator of cell cycle and a possible tumor suppressor in breast cancer. ERR beta could be therapeutic target for the treatment of breast cancer.