Characterization Of A Novel Small Molecule Subtype Specific Estrogen-Related Receptor Alpha Antagonist In Mcf-7 Breast Cancer Cells

Background: The orphan nuclear receptor estrogen-related receptor alpha (ERR alpha) is a member of the nuclear receptor superfamily. It was identified through a search for genes encoding proteins related to estrogen receptor alpha (ER alpha). An endogenous ligand has not been found. Novel ERR alpha antagonists that are highly specific for binding to the ligand binding domain (LBD) of ERR alpha have been recently reported. Research suggests that ERR alpha may be a novel drug target to treat breast cancer and/or metabolic disorders and this has led to an effort to characterize the mechanisms of action of N-[(2Z)-3-(4,5-dihydro-1,3-thiazol-2- yl)-1,3-thiazolidin-2-ylidene]-5H dibenzo[a,d][7]annulen-5-amine, a novel ERR alpha specific antagonist.Methodology/Principal Findings: We demonstrate this ERR alpha ligand inhibits ERR alpha transcriptional activity in MCF-7 cells by luciferase assay but does not affect mRNA levels measured by real-time RT-PCR. Also, ER alpha (ESR1) mRNA levels were not affected upon treatment with the ERR alpha antagonist, but other ERR alpha (ESRRA) target genes such as pS2 (TFF1), osteopontin (SPP1), and aromatase (CYP19A1) mRNA levels decreased. In vitro, the ERR alpha antagonist prevents the constitutive interaction between ERR alpha and nuclear receptor coactivators. Furthermore, we use Western blots to demonstrate ERR alpha protein degradation via the ubiquitin proteasome pathway is increased by the ERR alpha-subtype specific antagonist. We demonstrate by chromatin immunoprecipitation (ChIP) that the interaction between ACADM, ESRRA, and TFF1 endogenous gene promoters and ERR alpha protein is decreased when cells are treated with the ligand. Knocking-down ERR alpha (shRNA) led to similar genomic effects seen when MCF-7 cells were treated with our ERR alpha antagonist.Conclusions/Significance: We report the mechanism of action of a novel ERR alpha specific antagonist that inhibits transcriptional activity of ERR alpha, disrupts the constitutive interaction between ERR alpha and nuclear coactivators, and induces proteasome-dependent ERR alpha protein degradation. Additionally, we confirmed that knocking-down ERR alpha lead to similar genomic effects demonstrated in vitro when treated with the ERR alpha specific antagonist.