Genomic Methylation--Mechanism to Alter Tumor Phenotypes

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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.