Role of catechol-O-methyltransferase gene in prostate cancer

儿茶酚-O-甲基转移酶基因在前列腺癌中的作用

• 批准号: 9211217
• 负责人: Yuichiro Tanaka
• 金额: --
• 依托单位: VETERANS AFFAIRS MED CTR SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9211217
• 关键词: Affect; Aging; American; Animals; Apoptosis; Apoptotic; Aromatase; Benign; Benign Prostatic Hypertrophy; Biological Assay; Biological Markers; Cancer Etiology; Cancer Patient; Cancer cell line; Cancerous; Castration; Catechol Estrogens; Catechol O-Methyltransferase; Cell Cycle; Cell Line; Cell Proliferation; Cells; Cellular Morphology; Clinic; Clinical; Cytochrome P450; DNA Methylation; DNA Sequence Alteration; Data; Death Rate; Detection; Disease; Down-Regulation; Enzymes; Epigenetic Process; Estradiol; Estrogens; Etiology; Extrahepatic; Flow Cytometry; Gene Expression; Genes; Genetic Polymorphism; Genetic Transcription; Goals; Health; Histone Deacetylase Inhibitor; Histones; Hyperplasia; Immunohistochemistry; In Vitro; Incidence; Individual; Laboratories; Lead; Literature; Malignant Neoplasms; Malignant neoplasm of prostate; Measures; Metabolic Pathway; Methods; Methylation; Methyltransferase Gene; Microdissection; Microscopy; Mission; Modification; Neoplasm Metastasis; Normal Cell; Oncogenic; Pathway interactions; Patient-Focused Outcomes; Patients; Play; Predisposition; Production; Prostate; Prostatic; Prostatic Neoplasms; Protein O-Methyltransferase; Proteins; Publications; Publishing; Regulation; Reporting; Research; Resistance; Resources; Risk; Risk Factors; Role; Specimen; Testosterone; The Cancer Genome Atlas; Therapeutic; Time; Tissues; Translating; Tumor Suppressor Genes; Urine; Veterans; Western Blotting; androgen sensitive; base; cancer cell; cancer therapy; carcinogenesis; carcinogenicity; cell growth; chromatin immunoprecipitation; chromatin modification; clinically relevant; design; enzyme activity; epigenetic regulation; experimental study; high risk; histone modification; improved; in vivo; in vivo Model; insight; men; migration; overexpression; prevent; prostate cancer cell; prostate cancer cell line; prostate carcinogenesis; public health relevance; sodium bisulfite; therapeutic target; tumor growth; tumor progression

 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.

 描述（由申请人提供）： 背景：前列腺癌的发病率和死亡率是美国男性中最高的。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-雌激素使其失活来阻断致癌雌激素代谢途径，从而起到预防癌变的作用。实验室的初步数据和先前发表的文章支持 COMT 在前列腺癌中的保护作用。研究目标：该项目的主要目标是确定 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、进展和患者预后不良的危险因素，（2）COMT 基因过度表达可以通过影响细胞生长和进展途径来抑制前列腺癌细胞，（3）前列腺细胞中 COMT 的表达受到表观遗传因素的调节。项目设计和方法：目标#1。 COMT 在前列腺癌患者中的表达。将利用现有的前列腺组织资源，包括良性前列腺增生以及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) 对前列腺癌组织中的正常邻近区域和癌症区域进行显微切割，(b) 通过实时 PCR 确定 COMT RNA 表达水平，(c) 通过免疫组织化学确定 COMT 蛋白水平，以及 (d) 确定 COMT 表达是否与癌症发病率、分期、分级和患者结果相关。这些目标的实现将确定 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。目标#2。 COMT 在前列腺癌细胞中的功能意义。前列腺细胞系将用于以下实验：（a）通过实时PCR和蛋白质印迹分析分析正常和前列腺癌（雄激素依赖性和非依赖性或去势抵抗性）细胞中的组成型COMT表达水平，（b）通过建立稳定的COMT表达克隆在低COMT表达的癌细胞系中过度表达COMT，（c）测量甲氧基雌激素水平，细胞增殖，细胞周期分布， 通过流式细胞术和生物测定法评估 COMT 表达细胞的凋亡、细胞侵袭和迁移，(d) 通过显微镜评估 COMT 对细胞形态的影响，并通过基因阵列和蛋白质印迹分析评估参与细胞增殖和癌症进展途径的其他基因的表达，以及 (e) 确定 COMT 对体内前列腺肿瘤生长的影响。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，这可能表明 COMT 作为前列腺癌治疗的潜在治疗靶点。目标#3。前列腺癌中 COMT 基因的表观遗传调控。临床患者标本（目标 #1）和细胞系（目标 #2）将用于以下实验：(a) 使用亚硫酸氢钠修饰/甲基化特异性 PCR 分析 COMT DNA 甲基化，(b) 使用染色质免疫沉淀分析确定组蛋白修饰状态，(c) 测量细胞中组蛋白修饰酶的活性，(d) 将 COMT 表观遗传状态与 目标 #1 和 #2 中确定的 RNA 表达水平的酶活性，以及 (e) 确定去甲基化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前列腺癌中 COMT 表达的调节机制。退伍军人临床相关性：拟议的研究将为 COMT 在前列腺癌病因、进展和患者结果中的作用提供重要见解。这可能会导致识别前列腺癌高风险退伍军人的方法得到改进。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，可以转化为临床并造福退伍军人。