Genomic Methylation--Mechanism to Alter Tumor Phenotypes

基因组甲基化——改变肿瘤表型的机制

• 批准号: 6400048
• 负责人: Bernard W FUTSCHER
• 金额: $ 25.15万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6400048
• 关键词: 5 methylcytosine; DNA methylation; acetylation; amidohydrolases; breast neoplasms; chromatin; enzyme activity; gene induction /repression; genetic promoter element; histones; neoplasm /cancer genetics; protease inhibitor; serine proteinases; tissue /cell culture; transcription factor; tumor suppressor genes

The long-term objective of this study is to determine the molecular mechanisms of maspin gene silencing in human breast cancer and to use this information to identify strategies to reactivate its expression. Maspin is a tumor suppressor whose expression is frequently lost via transcriptional down-regulation in human breast cancers. Forced re- expression of maspin in maspin-negative breast cancer cells inhibits the growth, motility, neovascularization, and metastatic potential of breast cancer cells in mouse xenografts. Our preliminary results show that the loss of maspin expression is associated with aberrant methylation.of the maspin 5' regulatory region in human breast cancer cell lines as well as clinical breast cancer specimens. In addition, we show that it is possible to pharmacologically reactivate maspin gene expression using the demethylating agent 5-aza-2' deoxycytidine, the histone deacetylase inhibitor Trichostatin A, as well as through increased levels of Mn2+ Superoxide Dismutase (MnSOD). Based on the data described above, 3 specific aims are designed to achieve the stated long-term objective and test the hypothesis that therapeutic strategies designed to reactivate maspin expression will inhibit tumor growth and have a beneficial impact on the treatment of breast cancer. Aim #1 Determine if deacetylation of core histones in the maspin promoter is associated with 5-methylcytosine-linked chromatin condensation and transcriptional repression of the maspin gene. This will be accomplished using chromatin immunoprecipitations assays. Aim #2 Determine if the condensed chromatin structure of the maspin promoter blocks the access of transcription factors to their recognition sequence. Chromatin immunoprecipitations will be used to determine transcription factor binding in cells that contain both an unmethylated active maspin promoter and a methylated inactive maspin promoter. Aim #3 Identify and optimize pharmacological strategies for reactivation of maspin gene expression in aberrantly-methylated maspin-negative breast cancer cells. Agents that can reactivate maspin expression by distinct mechanisms of action will analyzed for their ability to maximize maspin gene reactivation in combination regimens.

本研究的长期目标是确定人类乳腺癌中 maspin 基因沉默的分子机制，并利用这些信息来确定重新激活其表达的策略。 Maspin 是一种肿瘤抑制因子，其表达经常因人类乳腺癌中的转录下调而丢失。在maspin阴性乳腺癌细胞中强制重新表达maspin会抑制小鼠异种移植物中乳腺癌细胞的生长、运动、新血管形成和转移潜力。我们的初步结果表明，maspin 表达的丧失与人乳腺癌细胞系以及临床乳腺癌样本中 maspin 5' 调控区的异常甲基化有关。此外，我们还表明，使用去甲基化剂 5-aza-2' 脱氧胞苷、组蛋白脱乙酰酶抑制剂曲古抑菌素 A 以及通过增加 Mn2+ 超氧化物歧化酶 (MnSOD) 水平，可以在药理学上重新激活 maspin 基因表达。基于上述数据，设计了 3 个具体目标来实现既定的长期目标，并检验旨在重新激活 maspin 表达的治疗策略将抑制肿瘤生长并对乳腺癌治疗产生有益影响的假设。目标#1 确定 maspin 启动子中核心组蛋白的脱乙酰化是否与 5-甲基胞嘧啶连接的染色质浓缩和 maspin 基因的转录抑制有关。这将使用染色质免疫沉淀测定来完成。目标#2 确定 maspin 启动子的浓缩染色质结构是否阻止转录因子进入其识别序列。染色质免疫沉淀将用于确定含有非甲基化活性 maspin 启动子和甲基化非活性 maspin 启动子的细胞中转录因子的结合。目标#3 确定并优化在异常甲基化的 maspin 阴性乳腺癌细胞中重新激活 maspin 基因表达的药理学策略。将分析可通过不同作用机制重新激活 maspin 表达的药物在联合治疗方案中最大化 maspin 基因重新激活的能力。