Prostate cancer is the most common cancer and the second leading cause of cancer-related deaths in American men. The overarching goal of this proposal is to elucidate a novel molecular mechanism that mediates metformin anti-tumor efficacy through epigenetic alterations in prostate cancer. Our strong in vitro and in vivo preliminary data demonstrated that metformin reduces expression of methyltransferase Suv39H1 and interaction with histone deacetylase Sirt1. Metformin further reduces the methylation levels of tumor suppressor microRNA (miR)-1, reduces androgen receptor (AR) expression, reduces prostate cancer cell migration, and inhibits expression of genes that favor prostate cancer skeletal metastasis in bone marrow. Based on our preliminary data we hypothesize that metformin inhibits prostate cancer growth and metastasis via epigenetic machinery mediated by Suv39H1, Sirt1 and miR1 targeting both tumor and its microenvironment. To test our hypothesis we propose three integrated specific aims: Aim 1 is to elucidate the molecular events mediated through metformin-regulated methyltransferase Suv39H1 in restricting prostate tumor growth. We will use different Suv39H1 levels or Suve39H1 lacking SET domain: Suv39H1- null, Suv39H1?SET and over-expressing cells, to determine AR expression and Sirt1-Suv39H1 binding is affected by downregulation of Suv39H1 by metformin to suppress prostate cancer cell growth and epithelial- mesenchymal transition (EMT). Aim 2 is to elucidate the molecular events mediated by metformin up- regulation of miR-1 in restricting prostate tumor growth. miR-1 methylation levels and its expression will be measured with treatment of metformin or vehicle, in the cell lines expressing different levels of Suv39H1 generated in Aim 1. Hi-Myc prostate cancer mice treated with treated with metformin or vehicle will be used to confirm the metformin up-regulation of miR-1 in vivo. Aim 3 is to elucidate the molecular mechanism by which metformin targets bone microenvironment to restrict bone metastasis. We will use miR-1- overexpressing or silenced prostate cancer cells and in vivo model to determine the levels of CXCR4/CXCL12 and miR-1 in prostate cancer tissues. Intra-tibia injections of miR-1-null or -overexpressed C4-2b cells will be used to determine the miR-1 mediates metformin suppression of prostate cancer growth in bone. Accomplishment of the experiments outlined in this proposal will determine the molecular mechanism of metformin on epigenetic regulations of prostate cancer. More recent findings suggest that metformin has anti neoplastic properties and our study will reveal important gaps in current knowledge about role of metformin in controlling prostate cancer. The outcomes of our study are expected to have exceptionally high potential to re- shape current key concept and paradigms about the role of metformin in prostate cancer treatment.