Abstract 4481: Phosphorylation regulates EZH2 neoplastic functions in triple-negative breast cancer

Abstract

Abstract Background: Triple negative (estrogen receptor, progesterone receptor, HER2-neu negative) breast cancers (TNBC) comprise 15% of all breast cancers but are responsible for a disproportionately high 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. We previously identified a novel link between EZH2 and the p38 mitogen-activated protein kinase, an important mediator of progression and metastasis of TNBC. We found that EZH2 binds to p38, and that EZH2 and activated p38 are concordantly expressed in the metastases of breast cancer patients. Based on these data and previous in vitro studies, we hypothesized that p38 MAPK may also regulate EZH2 through phosphorylation in breast cancer. We further hypothesized that this phosphorylation event may be important for EZH2 contribution to malignancy. Methods: In order to test this hypothesis, we performed knockdown rescue experiments in triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-436. Stable knockdown of EZH2 was achieved using shRNA targeting the 3’UTR. Knockdown of EZH2 was rescued by reintroduction of myc-EZH2 (WT) or a T367A-EZH2 phosphorylation-deficient mutant. Cell lines were then used in functional assays of proliferation, migration, and invasion. In order to further interrogate the importance of this phosphorylation event, we developed a phospho-specific EZH2 T367 antibody. Results: p38-mediated phosphorylation of EZH2 at T367 contributes to the migratory and invasive properties of TNBC. Mechanistically, phosphorylation by p38 does not affect binding to other PRC2 members but may affect EZH2 activity. We are currently investigating the relevance of pEZH2 as a biomarker of breast cancer survival with our new antibody. Conclusions: We provide evidence that p38 phosphorylation of EZH2 at T367 contributes to malignancy of triple-negative breast cancers. Our data suggest a new mechanism by which EZH2 is regulated and may offer an additional mechanism by which EZH2 contributes to TNBC progression. Citation Format: Talha Anwar, Caroline Arellano-Garcia, Boris Burman, Celina G. Kleer. Phosphorylation regulates EZH2 neoplastic functions in triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4481.