Bladder cancer is the fourth most common cancer in men and a significant burden for Veterans and the VHA due to the high frequency of recurrence and progression linked to smoking and exposure to deployment- related carcinogens. Nearly 80% of bladder cancers do not invade the muscle of the bladder wall (called ?non- muscle invasive bladder cancer?, NMIBC) but the most aggressive of these tumors will progress to muscle invasion with lymph node metastasis resulting in death in 30% of patients. The primary cause of death from bladder cancer is resistance to therapy as these invasive carcinomas acquire cellular plasticity and stem cell- like properties. Identification of mechanisms that regulate this change in cellular differentiation This invasive phenotype is a hallmark of cancer and a major shift in differentiation regulated by both genetic mutations and epigenetic cellular reprogramming. The long-term goal of our research is to investigate the molecular and epigenetic pathways driving invasion of bladder cancer. By understanding these mechanisms, we may develop rational and novel therapeutics for patients with bladder cancer. To investigate the epigenetic mechanisms that contribute to invasion and proliferation as a feasible target for bladder cancer, we evaluated the histone methyltransferase Enhancer of Zeste-2 (EZH2), as part of the polycomb repressor complex-2 (PRC-2) in bladder cancer. Our preliminary data demonstrate increased expression of EZH2 and its histone target, H3K27me3, in a carcinogen-induced mouse model of bladder cancer. In multiple bladder cancer cell lines, EZH2 expression is increased compared to non-transformed urothelial cells. Destabilization of the PRC-2 complex stops cellular proliferation. Consistent with our findings, bioinformatics analysis of multiple human bladder cancer databases demonstrate that EZH2 is overexpressed in invasive bladder cancers, which we have confirmed in tumor specimens from patients with all stages of bladder cancer. Given this preliminary data, our central hypothesis is that EZH2 drives invasion of bladder cancer by causing global changes in histone methylation that shifts cellular identity to an invasive and stem cell-like phenotype via an epithelial to mesenchymal transition. Thus, given our promising preliminary data, we propose to investigate our hypothesis with the following Specific Aims: 1) Determine the role of EZH2 in bladder cancer initiation and progression; 2) Investigate aberrant histone methylation of EMT, invasive and stem cell genes by EZH2 in bladder cancer; 3) Evaluate pharmacologic inhibition of EZH2 as a treatment for bladder cancer. Currently, we have no personalized genetic or epigenetic targets for bladder cancer and our best therapy for non-muscle invasive bladder cancer is > 40 years old. Through multi-disciplinary collaboration we have demonstrated feasibility with our approach. Successful completion of the studies described in this proposal will provide an innovative approach to both investigate the mechanisms involved in the invasion of bladder cancer and utilize a novel therapeutic approach to treat bladder cancer. These EZH2-targeted agents overcome the challenge of cellular resistance and have pre-clinical investigations that will allow access to Veterans with bladder cancer.