Summary of molecular mechanism of prostate cancer progression Prostate cancer is the most common cancer of men in the world. Age, ethnicity, diet, gene mutations and a number of other factors are associated with increased risk for prostate cancer. However, the cellular progression of a prostate tumor cell are not well known. Diepoxybutane (DEB) is a carcinogenic metabolite of 1,3-butadiene (BD), a hazardous chemical used in rubber production and present in automobile exhaust and tobacco smoke. Efforts to understand mechanisms of BD toxicity have recently demonstrated that DEB causes the cell cycle to stop and further induces apoptosis upon binding to DNA. However, we have found that at low concentrations (0.5 µM), DEB resulted in DU145 cell migration and activates PI3K, EMT marker E-Cadherin and the Cancer Stem Cell (CSC) markers SSEA-4 and Oct3/4 in 2D cultures of DU145 prostate cancer cells but its role is unclear under 3D cell culture conditions. We hypothesize that DEB may be effective in inducing Epithelial-to-Mesenchymal Transition (EMT) and stimulates drug resistant mechanisms through the activation of specific cell signaling pathways. We will test our hypothesis by two Aims. We will first to identify the functional role of the PI3K/AKT/ mTOR cell signaling pathway in prostate cancer progression by the use of commercially available signal transduction inhibitors. We then will investigate the new molecular mechanism by which DEB promotes prostate cancer progression due to network of signaling pathways. This Aim will be conducted single-cell transcriptome (scRNAseq) and proteomic analyses (Cell signal and Apoptotic antibody arrays) on control versus DEB-treated spheroids to identify the genes/ proteins that show altered expression in prostate cancer cells exposed to DEB. Functional studies will be conducted by performing CRISPR/Cas9 knockdown or RNAi of new key gene or genes with known association with cell migration and/or acquisition of drug resistance. The results obtained from this project will help us to understand the role of DEB on prostate cancer progression and will provide an insight on anticancer drug metabolism that leads to chemotherapy resistance. Furthermore, the results obtained from this project will provide insights into new important approaches to combat prostate cancer which may also be applicable to other types of cancer. More importantly, the knowledge acquired at the conclusion of this project may enhance our ability to fight cancer progression.