Epigenetic mechanisms of gene regulation in human breast cancer

Breast cancer represents a significant health problem and improvements in our ability to prevent, diagnose, and treat the disease requires a greater understanding of the molecular basis of breast carcinogenesis. Epigenetic mechanisms play a major role in breast carcinogenesis, with DNA methylation accounting for most epigenetic gene silencing, affecting a number of different gene targets. However, mechanisms of DNA methylationdependent silencing are poorly understood. To identify epigenetically-regulated genes in breast cancer, MCF-7 breast cancer cells were exposed to demethylating treatment and gene expression patterns were examined by microarray analysis. Genes with increased expression after demethylation treatment that returned to control levels after treatment withdrawal were directly assessed for DNA methylation by bisulfite sequencing. A group of 20 putative methylation-sensitive genes were identified that could be classified into three groups based upon their promoter CpG features. The majority of these methylation-sensitive genes lacked a conventional DNA methylation target (CpG island), resulting in an expanded model for epigenetic regulation of gene expression that recognizes the importance of all promoter CpGs. The breast tumor suppressor gene CST6 (Cystatin M) is epigenetically silenced in MCF-7 breast cancer cells. CST6 is subject to methylation-dependent regulation in multiple breast cancer cell lines, primary breast tumors, and lymph node metastases, and gene expression status correlates with promoter hypermethylation. These results suggest that methylation dependent gene silencing of CST6 represents an important mechanism for loss of CST6 during breast carcinogenesis. The mechanisms that control CpG island methylation are poorly understood. CST6 was utilized as an index gene for the identification of cis elements that direct promoter CpG methylation. The methylation-sensitive CST6 promoter was assembled into luciferase reporter constructs and transfected into model breast cancer cell lines that methylate or do not methylate the CST6 promoter. Truncation of the CST6 promoter disassociated a putative instructional cis regulatory sequence located in the 5' upstream promoter region of CST6 that functions to direct CpG methylation. The observations and results described in this dissertation significantly advance our understanding of methylation-sensitive genes and mechanisms governing DNA methylation in breast carcinogenesis