Mechanisms Underlying the Pharmacologic Reversal of Genetic and Epigenetic Components of Tumor Suppressor Gene Silencing in Human Breast Cancer

Abstract

In women, tumors of the breast remain the most frequently diagnosed malignancy and the second leading cause of cancer-related deaths. One of the hallmarks of carcinogenesis is the abnormal silencing of tumor supprsssor genes by both genetic and epigenetic alterations, leading to defects in cell-cycle control, DNA repair, apoptosis and cell adhesion. This dissertation focuses on the elucidation of the genetic and epigenetic mechanisms associated with tumor suppressor gene silencing in human epithelial breast tumor cells, and the development of pharmacologic strategies aimed at reversing these types of repression through gene therapy and chromatin remodeling. Desmocollin 3 (DSC3) and MASPIN are anti-metastatic tumor suppressor genes that are silenced in a large percentage of breast tumors via aberrant DNA hypermethylation and histone hypoacetylation of their promoters. DSC3 and MASPIN are also p53-target genes, requiring its transcriptional activation to promote normal expression levels, yet a significant fraction of breast tumor cell lines express mutant forms of p53. Adenoviral-mediated re-introduction of wild type (wt) p53 into mutant p53-expressing breast tumor cells resulted in significant up-regulation of DSC3 and MASPIN expression, although not to the levels seen in normal breast epithelial cells. Mechanistically, the addition of wt p53 to these tumor cells resulted in increased histone acetylation and enhanced chromatin accessibility of the DSC3 and MASPIN promoters, despite continued cytosine hypermethylation. Pre-treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) prior to wt p53 addition produced synergistic reactivation of both DSC3 and MASPIN in breast cancer cells, approaching their levels in normal mammary cells. However, 5-aza-CdR did not significantly reduce DNA methylation in many cases as originally theorized. Therefore, follow-up studies focused on the identification of alternative, novel mechanisms of 5-aza-CdR-mediated induction of epigenetically silenced genes, finding that it consistently reduced transcriptionally repressive histone H3 lysine 9 (K9) methylation levels in the promoter regions of both DSC3 and MASPIN in breast tumor cells, by mediating global decreases in the histone H3 K9 methyltransferase, G9A. In summary, these results clearly show that cancer treatments targeting both genetic and epigenetic facets of gene regulation may be a useful strategy towards the therapeutic transcriptional reprogramming of cancer cells.