Triple negative breast cancers (TNBC) and its most metastatic subtype, metaplastic carcinoma (mBrCA) occur with higher frequency in African American (AA) women than Whites and have 5-year overall survival of 73% and 54%, respectively. We have recently reported their proteomic landscape using clinical samples, but the molecular mechanisms driving metastasis are still unclear. We have shown that EZH2 overexpression in breast carcinomas is associated with TNBC and mBrCA subtype, and that EZH2 is an independent biomarker of poor patient survival. While the oncogenic role of EZH2 in breast and other malignancies has been established, the mechanisms of EZH2 metastatic function are far from understood. This application will elucidate a novel mechanism driving breast cancer metastasis, a critical unmet need. EZH2 is the histone H3 lysine 27 methyltransferase of the Polycomb Repressive Complex 2 (PRC2), responsible for epigenetic silencing. However, studies show that a subset of aggressive TNBC express high EZH2 and low H3K27me3, suggesting that in these tumors EZH2 may function via H3K27me3-independent mechanism(s). A major breakthrough in our lab during the previous cycle has been the discovery that phosphorylation of EZH2 at threonine 367 (T367) by p38 MAP Kinase leads to EZH2 cytoplasmic localization and is crucial for metastasis. In clinical samples, cytoplasmic pEZH2 T367 is significantly associated with TNBC and mBrCA phenotypes compared to other breast cancer subtypes, highlighting the translational significance of our proposal. Since the initial submission, we have discovered that cytoplasmic pEZH2 T367 directly methylates p38α protein, increasing p-p38 pathway activity on pro-migratory proteins such as Vinculin. However, the mechanism(s) and functional consequences need investigation. Our OVERARCHING HYPOTHESIS is that cytoplasmic pEZH2 T367 is critical for breast cancer metastasis through direct methylation of p38α and activation of p38α cytoplasmic pro-invasive and pro-metastatic targets. We further hypothesize that detection of pEZH2 T367, p-p38, p-Vinculin, and H3K27me3 proteins may serve as specific biomarkers with prognostic and diagnostic utility in clinical tissue samples. We propose three independent aims: AIM 1. To develop inducible, mammary-specific wild-type and EZH2 phospho-mutant transgenic mice and assess their proteomic landscape and metastatic potential. AIM 2. To elucidate the molecular mechanism(s) by which pEZH2 T367 promotes progression and metastasis. AIM 3. To test the potential for pEZH2-T367 and pathway proteins as diagnostic or prognostic biomarkers in human breast tissue samples of African, AA, and Whites. We have characterized cohorts of human breast cancer tissues (n>4,000, including 200 from Ghanaian, and 275 cases of mBrCAs of all races) with >15 years of follow-up. The reagents and expertise are in place in the PI and co-Investigators’ laboratories. Our innovative studies are expected to provide new diagnostic and...