(PQA1) Novel epigenetic mechanism mediate metformin inhibition of prostate cancer

描述（由申请人提供）：前列腺癌是美国男性中最常见的癌症，也是癌症相关死亡的第二大原因。该提案的总体目标是阐明一种新的分子机制，该机制通过前列腺癌的表观遗传学改变介导二甲双胍抗肿瘤疗效。我们强有力的体外和体内初步数据表明，二甲双胍降低甲基转移酶Suv 39 H1的表达和与组蛋白脱乙酰酶Sirt 1的相互作用。二甲双胍进一步降低肿瘤抑制微RNA（miR）-1的甲基化水平，降低雄激素受体（AR）表达，减少前列腺癌细胞迁移，并抑制有利于骨髓中前列腺癌骨转移的基因表达。基于我们的初步数据，我们假设二甲双胍通过靶向肿瘤及其微环境的Suv 39 H1、Sirt 1和miR 1介导的表观遗传机制抑制前列腺癌的生长和转移。为了验证我们的假设，我们提出了三个综合的具体目标：目的1是阐明通过二甲双胍调节的甲基转移酶Suv 39 H1介导的限制前列腺肿瘤生长的分子事件。我们将使用不同的Suv 39 H1水平或缺乏SET结构域的Suv 39 H1：Suv 39 H1- null，Suv 39 H1？SET和过表达细胞，以确定AR表达和Sirt 1-Suv 39 H1结合受二甲双胍下调Suv 39 H1以抑制前列腺癌细胞生长和上皮-间质转化（EMT）的影响。目的二是阐明二甲双胍上调miR-1抑制前列腺肿瘤生长的分子机制。在Aim 1中产生的表达不同水平Suv 39 H1的细胞系中，将用二甲双胍或溶媒处理测量miR-1甲基化水平及其表达。使用二甲双胍或媒介物治疗的Hi-Myc前列腺癌小鼠将用于确认二甲双胍在体内上调miR-1。目的3阐明二甲双胍靶向骨微环境抑制骨转移的分子机制。我们将使用miR-1过表达或沉默的前列腺癌细胞和体内模型来确定前列腺癌组织中CXCR 4/CXCL 12和miR-1的水平。胫骨内注射miR-1缺失或过表达的C4-2b细胞将用于确定miR-1介导的二甲双胍对骨中前列腺癌生长的抑制。完成本提案中概述的实验将确定二甲双胍对前列腺癌表观遗传调控的分子机制。最近的研究结果表明，二甲双胍具有抗肿瘤特性，我们的研究将揭示二甲双胍在控制前列腺癌中作用的现有知识中的重要空白。我们的研究结果有望极大地重塑二甲双胍在前列腺癌治疗中作用的当前关键概念和范式。

DESCRIPTION (provided by applicant): 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.