Abstract PD3-03: EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression

Abstract

Abstract Introduction: Triple negative breast cancers (TNBC) are frequently poorly differentiated with higher propensity for metastasis than all other subtypes. Enhancer of Zeste Homolog 2 (EZH2) is a lysine methyltransferase that mediates transcriptional repression of pro-differentiation genes in normal and neoplastic cells. The oncogenic role of EZH2 through H3K27me3 is well established. However, non-canonical H3K27me3-independent functions are unclear. We have reported that p38 phosphorylates T367 of EZH2, increasing TNBC metastasis. Recent reports show that EZH2 can regulate signaling pathways through direct methylation of proteins suggesting the hypothesis that EZH2 may methylate p38 in TNBC. Methods: Human tissue samples from 16 primary invasive breast carcinomas and matched distant metastasis arrayed in tissue microarrays were interrogated for pEZH2 T367 and p-p38 by immunohistochemistry. To study p38 methylation, we performed LC-MS/MS analyses GST-p38α, and GST-EZH2 were each incubated with or without recombinant PRC2 complex (EZH2/E ED/SUZ12/RbAp48/AEBP2) and S-adenosyl methionine methyl donor. MDA-MB-231, mouse and human primary TNBC cells and murine 4T1 cells were used in funcional assays in vitro and in vivo. EZH2 knockdown and rescue was carried out using lentiviral transduction of EZH2 with pBabe-myc-EZH2 (wild-type) or pBabe-myc-EZH2 (T367A). We developed p38 methylation mutants: HA-p38a K139A, HA-p38a K165A, and HA-p38a K130A/K165K. To investigate the importance of EZH2 and p38 enzymatic activities we used the EZH2 methyltransferase inhibitors GSK-343 and EPZ-6438 and the p38 inhibitor SB202190 in vitro and in vivo. For animal studies, MDA-MB-231 and 4T1 cells were orthotopically injected into the right inguinal mammary fat pad of eight-week old NOD/SCID or BALBc mice, respectively. When tumors reached 100 mm3 mice were treated 5 days/week by intraperitoneal injection with SB202190 (1mg/kg/day), EPZ-6438 (10mg/kg/day), combination of SB202190 and EPZ-6438, or control. We monitored tumor growth for 50 days, and harvested primary tumors, lungs, and sites of metastasis for histopathology, Western blot, and immunohistochemistry. Primary xenografts from the control and treatment groups were subjected to RNA sequencing. Results: EZH2 methylates p38 at lysines 139 and 165 leading to enhanced p38 stability and increased invasion, and p38 activation requires T367 phosphorylation of EZH2. Dual inhibition of EZH2 methyltransferase and p38 kinase activities downregulates pEZH2 T367, H3K27me3, and p-p38 in vivo, reduces TNBC growth and metastasis, and results in gene expression changes towards a better differentiated phenotype. pEZH2 T367 and p-p38 proteins are significantly coexpressed in human primary and metastatic breast cancer. Conclusions: We provide direct evidence that EZH2 methylates p38 with resultant p38 activation, and that EZH2 phosphorylation at T367 is important for this function. Dual targeting of EZH2 methyltransferase and p38 kinase activities with specific inhibitors reduces breast cancer growth and metastasis indicating a cooperation between EZH2 canonical and non-canonical mechanisms and suggesting therapeutic strategies. Citation Format: Maria E Gonzalez, Shilpa R. Tekula, Talha Anwar, Shoshana A. Leflein, Celina G. Kleer. EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-03.