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

描述(申请人提供)：前列腺癌是美国男性最常见的癌症，也是癌症相关死亡的第二大原因。这项建议的首要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来介导二甲双胍的抗肿瘤效果。我们强大的体外和体内初步数据表明，二甲双胍减少了甲基转移酶SUV39H1的表达，并减少了与组蛋白脱乙酰基酶Sirt1的相互作用。二甲双胍进一步降低肿瘤抑制因子microRNA(MiR)-1的甲基化水平，减少雄激素受体(AR)的表达，减少前列腺癌细胞的迁移，并抑制有利于前列腺癌骨髓骨转移的基因的表达。根据我们的初步数据，我们假设二甲双胍通过SUV39H1、Sirt1和miR1靶向肿瘤及其微环境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特异性目标：目的1阐明二甲双胍调节的甲基转移酶SUV39H1介导的分子事件在限制前列腺癌生长中的作用。我们将使用不同水平的SUV39H1或缺乏SET结构域的SuV39H1：SUV39H1-空、SUV39H1？SET和过度表达的细胞来确定AR的表达，以及二甲双胍下调SUV39H1抑制前列腺癌细胞生长和上皮-间充质转化(EMT)对Sirt1-SUV39H1结合的影响。目的2阐明二甲双胍上调miR-1抑制前列腺癌生长的分子机制。在AIM 1产生的表达不同水平的SUV39H1的细胞系中，将用二甲双胍或赋形剂处理来检测miR-1甲基化水平及其表达。用二甲双胍或赋形剂处理的Hi-Myc前列腺癌小鼠将在体内证实二甲双胍上调miR-1的表达。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检测前列腺癌组织中CXCR4/CXCL12和miR-1的水平。胫骨内注射miR-1缺失或过表达的C4-2b细胞将被用来确定miR-1介导二甲双胍抑制前列腺癌在骨中的生长。这项建议中概述的实验的完成将确定二甲双胍对前列腺癌表观遗传调控的分子机制。更新的发现表明，二甲双胍具有抗肿瘤特性，我们的研究将揭示目前对二甲双胍在控制前列腺癌中的作用的认识上的重要空白。我们的研究结果有望极大地重塑目前有关二甲双胍在前列腺癌治疗中作用的关键概念和范例。

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.