Genomic Methylation--Mechanism to Alter Tumor Phenotypes

本研究的长期目标是确定maspin基因沉默在人类乳腺癌中的分子机制，并利用这些信息确定重新激活其表达的策略。Maspin是一种肿瘤抑制因子，其表达在人类乳腺癌中经常通过转录下调而丢失。maspin阴性乳腺癌细胞中maspin的强制再表达抑制小鼠异种移植乳腺癌细胞的生长、运动、新生血管和转移潜能。我们的初步结果表明，maspin表达的缺失与异常甲基化有关。人类乳腺癌细胞系和临床乳腺癌标本中maspin 5'调控区。此外，我们还发现，通过使用去甲基化剂5-aza-2'脱氧胞苷、组蛋白去乙酰化酶抑制剂Trichostatin A，以及通过增加Mn2+超氧化物歧化酶（MnSOD）水平，可以从药理学上重新激活maspin基因表达。基于上述数据，我们设计了3个具体目标，以实现既定的长期目标，并验证旨在重新激活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。目的1：确定maspin启动子核心组蛋白的去乙酰化是否与5-甲基胞嘧啶连接的染色质凝聚和maspin基因的转录抑制有关。这将通过染色质免疫沉淀法来完成。确定masmasin启动子的浓缩染色质结构是否会阻碍转录因子进入其识别序列。染色质免疫沉淀法将用于确定含有未甲基化的活性maspin启动子和甲基化的非活性maspin启动子的细胞中的转录因子结合。确定并优化在异常甲基化的maspin阴性乳腺癌细胞中maspin基因表达再激活的药理学策略。可以通过不同的作用机制重新激活maspin表达的药物将分析其在联合方案中最大化maspin基因重新激活的能力。

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.