Abstract 4674: Localization of estrogen receptor α(ERα) at the centrosome and its regulation by protein kinases in breast cancer cells

Abstract

Abstract The centrosome plays a key role in the regulation of cell cycle progression. Centrosome amplification or defects are directly linked to aneuploidy and chromosomal instability, which are frequently observed in numerous human cancers. In breast tumors, cancer cells which undergo unrestricted divisions have both structurally and functionally abnormal centrosomes. Estrogen receptor-α (ERα) is known as a nuclear transcriptional factor, which is expressed in two thirds of primary breast cancers. ERα modulates cell proliferation in breast cancer cells by influencing the expression of target genes. Measuring ERα status is of clinical importance in breast cancer diagnostics and determination of treatment response to hormone therapy. Despite extensive studies on its function and mode of regulation on transcription, the exact roles of ERα in cell cycle regulation and centrosome function in breast cancer cells are largely unknown. In this study, we use immunofluorescence assay (IF), biochemical analysis of isolated centrosomes and proximity ligation assay (PLA) to show, for the first time, that ERα is a novel centrosomal protein in the breast tumor cell line MCF7. To search for kinases possibly involved in the regulation of ERα in the centrosome, we focus on three mitotic serine/threonine kinases: Aurora A kinase, Aurora B kinase and Polo-like kinase 1 (PLK1). Aurora A is recruited to centrosomes at G2/M phase and it is required for centrosome maturation and separation, as well as G2/M transition. It was previously shown that Aurora A can phosphorylate ERα at S167 and S305. Aurora B is a chromosome passenger protein which is localized at the midbody and involved in cytokinesis. PLK1 can regulate ERα transcriptional activity during interphase, but it's more widely known functions include driving G2/M transition, and it localizes to centrosomes during metaphase and to the midbody during cytokinesis. By using co-immunoprecipitation (Co-IP) assays, we show that ERα interacts with Aurora A, Aurora B and PLK1 in ER+ MCF7 and T47D cells, and the interactions are increased for Aurora A and PLK1 in the presence of nocodazole which arrested cells at prometaphase. Moreover, PLA showed that ERα interacts with Aurora A mainly in the centrosome region in mitotic MCF7 cells. IF data confirmed that phosphorylated ERα pS118 colocalizes with Aurora A at centrosomes, Aurora B at midzone/midbody, and with PLK1 at centrosome and midbody in mitotic MCF7 and T47D cells. These data suggested that ERα might be directly involved in centrosome function in breast cancer cells, and its dysregulation might play a role in tumor initiation and progression. As a whole, this study reveals a novel and previously unrecognized function for ERα, a finding that could have further implications for finding more accurate biomarkers of prognosis, and providing an alternative treatment strategy against certain types of ER+ breast cancers. Citation Format: Yingfeng Zheng, Christine Bruce, Shihua He, Kirk McManus, Jim Davie, Leigh Murphy. Localization of estrogen receptor α(ERα) at the centrosome and its regulation by protein kinases in breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4674. doi:10.1158/1538-7445.AM2015-4674