Abstract LB-29: Erk-mediated phosphorylation of the orphan nuclear receptor ERRγ regulates receptor stability and contributes to Tamoxifen resistance in ER+ breast cancer

Abstract

Abstract Estrogen receptor alpha-positive (ER+) breast tumors comprise ∼70% of annually diagnosed breast cancers in the United States. While pro-tumorigenic ER signaling can be blocked by endocrine therapies that either inhibit the receptor directly (Tamoxifen) or prevent synthesis of its ligand (aromatase inhibitors), de novo and acquired resistance to ER-targeted therapies is a major barrier to the successful treatment of ER+ breast cancer. One of the most common mechanisms of acquired or de novo endocrine resistance in ER+ breast cancer is increased reliance on alternative pro-proliferative and pro-survival pathways that circumvent ER. We have been studying the role of the orphan nuclear receptor estrogen-related receptor gamma (ERRγ) in Tamoxifen-resistant breast cancer, increased expression of which induces Tamoxifen-resistant proliferation in multiple ER+ breast cancer cell lines. We have also reported that i. endogenous ERRγ shows increased levels of serine phosphorylation in Tamoxifen-resistant breast cancer cells, ii. this coincides with increased activation of extracellular signal-regulated kinase 2 (ERK2), and iii. pharmacological inhibition of the MEK/ERK pathway reduces ERRγ phosphorylation and transcriptional activity. This lead us to hypothesize that ERK-mediated phosphorylation of the orphan nuclear receptor ERR contributes to Tamoxifen resistance in ER+ breast cancer. One way in which ERK-mediated phosphorylation can regulate its substrates is through changes in target protein stability. Co-transfection of wild-type ERRγ with either a constitutively active MEK (MEK-DD) or wild-type ERK2 led to an increase in ERRγ expression. Conversely, treatment of cells with the MEK inhibitor U0126 reduced transfected ERRγ expression. These data are also supported by mRNA data publicly available through ONCOMINE showing that i. relative to MCF7, BT-474 cells (which contain amplified HER2 and very high levels of active ERK) express significantly increased levels of ERRγ, and ii. treatment of BT-474 cells with the MEK inhibitor PD0325901 reduces ERRγ expression. To confirm that ERK-mediated phosphorylation of ERRγ regulates receptor stability, we performed site-directed mutagenesis to change multiple Serine residues within ERK consensus sites to Alanine. These Ser-to-Ala ERRγ mutants are expressed at lower levels when compared to the wild-type receptor, and co-transfection with MEK-DD is unable to increase their expression. Altogether, these data strongly suggest that direct ERK-mediated phosphorylation of ERR positively stabilizes receptor expression. Ongoing studies are addressing the biochemical and biological consequences of ERK-mediated ERRγ phosphorylation in the context of Tamoxifen-resistant breast cancer; putative mechanisms include receptor dimerization, DNA binding, and co-factor recruitment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-29. doi:10.1158/1538-7445.AM2011-LB-29