Role of catechol-O-methyltransferase gene in prostate cancer

 描述(由申请人提供)： 背景：前列腺癌的发病率和死亡率在美国男性中名列前茅。前列腺癌的病因尚不清楚，但根据文献，儿茶酚-O-甲基转移酶(COMT)基因能够通过代谢儿茶酚-雌激素来阻断致癌雌激素代谢途径，使其失去活性，从而起到预防癌症发生的作用。实验室的初步数据和先前的出版物支持COMT在前列腺癌中的保护作用。研究目的：本项目的主要目的是确定COMT基因在前列腺癌中的作用。我们假设：(1)COMT降低是前列腺癌发生、发展和患者预后不良的危险因素，(2)COMT基因过表达可通过影响细胞生长和进展途径抑制前列腺癌细胞，(3)COMT在前列腺癌细胞中的表达受表观遗传因素的调节。项目设计和方法：目标1。前列腺癌患者中COMT的表达。将利用现有的前列腺组织资源，包括良性前列腺增生症和来自癌症患者的匹配的正常和癌症标本。将进行以下实验：(A)从前列腺癌组织中显微解剖正常的邻近区域和癌组织，(B)通过实时荧光聚合酶链式反应(Real-time PCR)测定COMT RNA的表达水平，(C)通过免疫组织化学测定COMT蛋白的水平，以及(D)确定COMT的表达是否与癌症的发病率、分期、分级和患者预后有关。这些目标的实现将决定COMT下调是否与前列腺癌相关，并评估COMT作为前列腺癌的生物标志物。目的#2.COMT在前列腺癌细胞中的功能意义。前列腺细胞系将被用于以下实验：(A)通过实时定量PCR和Western印迹分析正常和前列腺癌(雄激素依赖和非依赖性或去势抵抗)细胞中COMT的组成性表达水平，(B)通过建立稳定的COMT表达克隆，在COMT低表达的癌细胞系中过表达COMT，(C)通过流式细胞仪和生物学分析测定表达COMT的细胞中的甲氧基雌激素水平、细胞增殖、细胞周期分布、凋亡、细胞侵袭和迁移，(D)通过显微镜观察COMT对细胞形态的影响，并通过基因芯片和Western印迹分析评估参与细胞增殖和肿瘤进展途径的其他基因的表达，以及(E)确定COMT对体内前列腺癌生长的影响。这些实验的完成将确定COMT对前列腺癌细胞生长和进展途径的功能影响，这可能意味着COMT可能成为前列腺癌治疗的潜在治疗靶点。目的#3.前列腺癌中COMT基因的表观遗传调控。临床患者标本(AIM#1)和细胞系(AIM#2)将用于下列实验：(A)用亚硫酸氢钠修饰/甲基化特异性聚合酶链式反应分析COMT DNA甲基化；(B)用染色质免疫沉淀分析组蛋白修饰状态；(C)测量细胞中组蛋白修饰酶的活性；(D)将COMT表观遗传状态和酶活性与AIMS#1和#2中确定的RNA表达水平相关联；以及(E)确定去甲基剂和组蛋白脱乙酰酶抑制剂是否可以在前列腺癌细胞中重新表达COMT。这些实验的完成将确定COMT在前列腺癌中表达的调节机制。退伍军人临床相关性：拟议的研究将对COMT在前列腺癌病因、进展和患者预后中的作用产生重要的见解。这可能会导致改进识别患有前列腺癌风险较高的退伍军人的方法。拟议的研究还可能揭示COMT是前列腺癌的重要生物标志物和治疗靶点，可能转化为临床并使退伍军人受益。

 DESCRIPTION (provided by applicant): Background: Prostate cancer incidence and death rates are among the highest in American men. The etiology of prostate cancer is not known but based on the literature the catechol-O-methyltransferase (COMT) gene is capable of blocking the carcinogenic estrogen metabolic pathway by metabolizing catechol-estrogens rendering them inactive and thus, plays a role in preventing carcinogenesis. Preliminary data and prior publications from the laboratory support a protective role for COMT in prostate cancer. Research Objectives: The main goal of this project is to determine the role of the COMT gene in prostate cancer. We hypothesize that: (1) reduced COMT is a risk factor for prostate cancer, progression and poor patient outcome, (2) over-expressing the COMT gene can inhibit prostate cancer cells by affecting cell growth and progression pathways, and (3) COMT expression in prostate cells is regulated by epigenetic factors. Project Design and Methods: Aim #1. COMT expression in prostate cancer patients. Existing resources of prostate tissues consisting of benign prostatic hyperplasia and matched normal and cancer specimens from cancer patients will be utilized. The following experiments will be performed: (a) microdissection of normal adjacent and cancer regions from prostate cancer tissues, (b) determine COMT RNA expression levels by real-time PCR, (c) determine COMT protein levels by immunohistochemistry, and (d) determine if COMT expression is associated with cancer incidence, stage, grade, and patient outcome. Accomplishment of these goals will determine whether COMT down-regulation is associated with prostate cancer and assess COMT as a biomarker for prostate cancer. Aim #2. Functional significance of COMT in prostate cancer cells. Prostate cell lines will be used for the following experiments: (a) analyze constitutive COMT expression levels in normal and prostate cancer (androgen dependent and independent or castration resistant) cells by real-time PCR and Western blot analyses, (b) over-express COMT in those cancer cell lines with low COMT expression by establishing stable COMT-expressing clones, (c) measure methoxyestrogen level, cell proliferation, cell cycle distribution, apoptosis, cell invasion, and migration in COMT-expressing cells by flow cytometry and biological assays, (d) evaluate the effects of COMT on cell morphology by microscopy and expression of other genes involved in cell proliferation and cancer progression pathways by gene arrays and Western blot analyses, and (e) determine the effect of COMT on prostate tumor growth in vivo. Accomplishment of these experiments will determine the functional effects of COMT on cell growth and progression pathways in prostate cancer which may implicate COMT as a potential therapeutic target for prostate cancer treatment. Aim #3. Epigenetic regulation of the COMT gene in prostate cancer. Clinical patient specimens (Aim #1) and cell lines (Aim #2) will be used in the following experiments: (a) analyze COMT DNA methylation using sodium bisulfite modification / methylation-specific PCR, (b) determine the histone modification status using chromatin- immunoprecipitation analysis, (c) measure the activity of histone modifying enzymes in cells, (d) correlate COMT epigenetic status and enzyme activities with RNA expression levels determined in Aims #1 and #2, and (e) Determine if demethylating agents and histone deacetylase inhibitors can re-express COMT in prostate cancer cells. Accomplishment of these experiments will determine the mechanisms of regulation of COMT expression in prostate cancer. Veterans Clinical Relevance: The proposed research will generate important insights into COMT's role in prostate cancer etiology, progression, and patient outcome. This could lead to improved methods of identifying Veterans at higher risk for prostate cancer. The proposed research may also reveal COMT to be an important biomarker and therapeutic target for prostate cancer that could translate to the clinic and benefit Veterans.