The G-protein-coupled estrogen receptor, a gene co-expressed with ER in breast tumors, is regulated by estrogen-ER signalling in ER positive breast cancer cells

GPER is a seven transmembrane G-protein-coupled estrogen receptor that mediates rapid estrogen actions. Large volumes of data have revealed its association with clinicopathological variables in breast tumors, role in epidermal growth factor (EGF)-like effects of estrogen, potential as a therapeutic target or a prognostic marker, and involvement in endocrine resistance in the face of tamoxifen agonism. GPER cross-talks with estrogen receptor alpha (ER & alpha;) in cell culture models implicating its role in the physiology of normal or transformed mammary epithelial cells. However, discrepancies in the literature have obfuscated the nature of their relationship, its significance, and the underlying mechanism. The purpose of this study was to assess the relationship between GPER, and ER & alpha; in breast tumors, to understand the mechanistic basis, and to gauge its clinical significance. We mined The Cancer Genome Atlas (TCGA)-BRCA data to examine the relationship between GPER and ER & alpha; expression. GPER mRNA, and protein expression were analyzed in ER & alpha;-positive or-negative breast tumors from two independent cohorts using immunohistochemistry, western blotting, or RT-qPCR. The Kaplan Meier Plotter (KM) was employed for survival analysis. The influence of estrogen in vivo was studied by examining GPER expression levels in estrus or diestrus mouse mammary tissues, and the impact of 1713-estradiol (E2) administration in juvenile or adult mice. The effect of E2, or propylpyrazoletriol (PPT, an ER & alpha; agonist) stimulation on GPER expression was studied in MCF-7 and T47D cells, with or without tamoxifen or ER & alpha; knockdown. ER & alpha;-binding to the GPER locus was explored by analysing ChIP-seq data (ERP000380), in silico prediction of estrogen response elements, and chromatin immunoprecipitation (ChIP) assay. Clinical data revealed significant positive association between GPER and ER & alpha; expression in breast tumors. The median GPER expression in ER & alpha;-positive tumors was significantly higher than ER & alpha;-negative tumors. High GPER expression was significantly associated with longer overall survival (OS) of patients with ER & alpha;-positive tumors. In vivo experiments showed a positive effect of E2 on GPER expression. E2 induced GPER expression in MCF-7 and T47D cells; an effect mimicked by PPT. Tamoxifen or ER & alpha;-knockdown blocked the induction of GPER. Estrogen-mediated induction was associated with increased ER & alpha; occupancy in the upstream region of GPER. Furthermore, treatment with 17 & beta;-estradiol or PPT significantly reduced the IC50 of the GPER agonist (G1)-mediated loss of MCF-7 or T47D cell viability. In conclusion, GPER is positively associated with ER & alpha; in breast tumors, and induced by estrogen-ER & alpha; signalling axis. Estrogen-mediated induction of GPER makes the cells more responsive to GPER ligands. More in-depth studies are warranted to establish the significance of GPER-ER & alpha; co-expression, and their interplay in breast tumor development, progression, and treatment.