Abstract 4975: Identification of a novel interaction between EZH2 and p38 in triple negative breast cancer

Abstract

Abstract Background: Breast cancer accounts for more than 40,000 deaths yearly in the United States, the majority of which are due to metastatic disease. The histone methyltransferase EZH2 (Enhancer of Zeste Homolog 2) is a member of the polycomb repressive complex 2 (PRC2) responsible for transcriptional repression by trimethylation of histone H3 at lysine 27. EZH2 is an independent predictor of recurrence and mortality that is overexpressed in 55% of invasive breast carcinomas. We previously identified a novel link between EZH2 and the p38 mitogen-activated protein kinase, an important mediator of breast cancer progression and metastasis. EZH2 binds to p38, and overexpression of EZH2 leads to phosphorylation and activation of p38. Based on in vitro evidence and recent studies implicating EZH2 in the methylation of non-histone targets, we hypothesized that EZH2 may methylate p38 in breast cancer cells. We further hypothesized that this methylation event may be important for p38 activation or stability. Methods: In order to test this hypothesis, we used stable shRNA knockdown of EZH2 in MDA-MB-231 and SUM149 breast cancer cell lines. We performed co-immunoprecipitation using p38 and pan-methyl lysine antibodies to assess changes in methylated p38 by immunoblot. We are currently performing LC MS/MS analysis to identify the specific residues that are methylated by PRC2 both in vitro and in vivo. Additionally, in order to localize this interaction, we performed co-immunoprecipitation experiments on fractionated MDA-MB-231 and SUM149 lysates. Results: Stable knockdown of EZH2 resulted in reduced methylated p38 compared to vector controls, suggesting that EZH2 methylates p38 in vivo. We found that EZH2 and p38 interact in nuclear and, surprisingly, cytoplasmic fractions. Conclusions. We provide evidence that in addition to being a nuclear protein, EZH2 can be found in the cytoplasm of TNBCs, where it is able to complex with p38. Taken together, our data suggest that p38 methylation may represent an additional mechanism by which EZH2 mediates breast cancer progression in TNBC. Citation Format: Talha Anwar, Heather Moore, Maria E. Gonzalez, Celina Kleer. Identification of a novel interaction between EZH2 and p38 in triple negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4975. doi:10.1158/1538-7445.AM2014-4975