Abstract P5-12-05: Novel pEZH2 T367-PRC2 interaction and methyltransferase activity in the nuclear and cytoplasmic fractions of breast cancer cells

Abstract

Abstract Introduction: EZH2 is the main enzymatic subunit of the Polycomb Repressive Complex 2 (PRC2) that promotes transcriptional repression by trimethylating histone H3 at lysine 27 (H3K27me3). Several reports evidenced EZH2 overexpression in many types of cancers, including breast cancer. In invasive breast carcinoma high levels of EZH2 are associated with triple-negative breast cancer (TNBC) status, presence of metastasis and poor clinical outcome. Despite an oncogenic role of EZH2 as a transcriptional silencer is well known, recent studies showed the association between high levels of EZH2 and low H3K27me3 in TNBC, suggesting that EZH2 may act via non-canonical mechanisms in breast cancer initiation, development, and progression. Interestingly, emerging data highlight that EZH2 can methylate non-histone substrates, regulating numerous cellular functions in a PRC2-dependent or -independent manner. We have established a critical role of p38-mediated EZH2 T367 phosphorylation in the cytoplasmic compartment in promoting cell migration, invasion, and metastasis in TNBC. Here, we tested the hypothesis that EZH2 phosphorylation at T367 induces EZH2 binding to PRC2 in the cytoplasmic compartment of triple-negative breast cancer cells to methylate non-histone proteins. Methods: In vitro and in vivo studies were carried out by using MDA-MB-231 cells. Stable knockdown followed by rescue of EZH2 was attained using lentiviral transduction of EZH2 with pBabe-myc-EZH2 (wild-type), phospho-deficient pBabe-myc-EZH2 (T367A) or HMTase-deficient p-Babe-myc-EZH2 (H694A) in MDA-MB-231 cells. To better evaluate the relevance of cytoplasmic EZH2 in breast cancer progression, we transiently overexpressed EZH2 full length (WT-EZH2) and EZH2 mutant lacking the nuclear localization signaling (ΔNLS-EZH2) adenoviral constructs in MDA-MB-231 shEZH2 cells. The EZH2-catalytic activity inhibition, in vitro and in vivo, was performed through two different drugs, GSK126 and EPZ6438, which exhibit high selectivity for EZH2 lysine N-methyltransferase (KMTase) activity maintaining PRC2 complex integrity. To investigate the interaction between p-EZH2 T367 and PRC2 complex components we carried out Co-immunoprecipitation assay (Co-IP) in the nuclear and cytoplasmic compartments of MDA-MB-231 cells. The interaction was validated by Proximity ligation assays (PLA). We evaluated EZH2 KMTase in the cytoplasmic and nuclear compartments using MTase-Glo Methyltransferase assay in vitro and in vivo. Xenografts obtained from NOD/SCID mice orthotopically injected with MDA-MB-231 cells and treated 5 days/weeks by intraperitoneal injection with 4% DMSO-30% PEG 300-5% Tween 80 (control), or EPZ-6438 (10mg/kg/day) were used to test EZH2 KMTase in vivo. Results: Co-IP and PLA revealed the binding of p-EZH2 T367 to PRC2-complex members in the cytoplasmic compartment of MDA-MB-231 cells expressing WT-EZH2, H694A-EZH2 and ΔNLS-EZH2. In contrast, T367A-EZH2 showed reduced binding to PRC2 members in the cytoplasm. To test the biological relevance of this interaction we measured the KMTase of WT-EZH2 and mutants-EZH2 in the cytoplasmic fraction of MDA-MB-231 cells. Methyltransferase assays using p38 and histone H3 as substrates, showed that WT-EZH2, H694A-EZH2 and ΔNLS-EZH2 are able to methylate p38 protein in the cytoplasmic fraction of MDA-MB-231 cells, and that this requires T367 phosphorylation of EZH2. Similar results were obtained in xenografts of MDA-MB-231 cells in vivo. Conclusion: Our results provide evidence that EZH2 phosphorylation at T376 induces EZH2 binding to PRC2-complex members in the cytoplasm of triple-negative breast cancer cells to methylate non-histone proteins in vitro and in vivo. These data deepen our understanding of the mechanism(s) by which pEZH2 T367 functions in TNBC progression Citation Format: Giuseppina Daniela Naimo, Maria Elena Gonzalez, Shilpa Reddy Tekula, Jessica Camille Gauss, Loredana Mauro, Sebastiano Andò, Celina Graciela Kleer. Novel pEZH2 T367-PRC2 interaction and methyltransferase activity in the nuclear and cytoplasmic fractions of breast cancer cells [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 P5-12-05.