Abstract 4790: Noncanonical functions of EZH2 in triple-negative breast cancers

Abstract

Abstract Background: Triple negative (estrogen receptor, progesterone receptor, HER2-neu negative) breast cancers (TNBC) account for 15% of all breast cancers but are responsible for a disproportionate number of deaths. Overexpression of the histone methyltransferase EZH2 (Enhancer of Zeste Homolog 2) is an independent prognostic biomarker significantly associated with poorly-differentiated TNBCs and poor patient outcome. As a member of the polycomb repressive complex 2 (PRC2), the canonical function of EZH2 is to mediate transcriptional repression of target genes via trimethylation of histone H3 at lysine 27 (H3K27me3). We previously identified a novel interaction between EZH2 and the p38 mitogen-activated protein kinase, an important mediator of progression and metastasis of TNBC. We found that EZH2 can bind to p38 and that overexpression of EZH2 leads to phosphorylation and activation of p38. Based on these data and preliminary in vitro studies, we hypothesized that EZH2 may have functions independent of its H3K27me3 activity in TNBC, where it can methylate p38 and other non-histone proteins. We further hypothesized that these methylation events may be important for p38 activation or stability. Methods: In order to test this hypothesis, we performed an in vitro methyltransferase assay with recombinant PRC2 complex, using p38α as substrate. We then employed LC MS/MS to determine the lysine residues methylated on p38α by PRC2. In order to determine the functional consequences of this modification, we assessed for changes in p38 isoform protein stability in a cycloheximide pulse-chase assay with genetic or pharmacologic inhibition of EZH2 in MDA-MB-231 and SUM149 triple-negative breast cancer cell lines. As a complementary approach, we used site-directed mutagenesis to block methylation at these sites and to determine resulting changes in protein stability. Results: EZH2 methylates p38α in vitro at two lysine residues. Enzymatic inhibition of EZH2 results in reduced levels of methylated p38α, as well as decreased stability in vivo in an isoform-specific manner. Consistent with these results, lysine-to-alanine mutagenesis of these residues results in significantly reduced protein stability. We are currently investigating the possibility that EZH2 methylation may block other modifications, such as ubiquitination, at these sites. Conclusions: We provide evidence that EZH2 directly methylates oncoprotein p38α at two novel sites and that these modifications confer stability. Taken together, our data suggest that p38 methylation may represent an additional mechanism by which EZH2 contributes to TNBC progression. Citation Format: Talha Anwar, Heather Moore, Sarah Bergholtz, Celina Kleer. Noncanonical functions of EZH2 in triple-negative breast cancers. [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 4790. doi:10.1158/1538-7445.AM2015-4790