Epigenetic regulation of manganese superoxide dismutase expression in human breast cancer cells.

Malignant breast cancer cells often exhibit lower expression and activity of manganese superoxide dismutase (MnSOD) than their normal cell counterparts; however, the mechanism(s) responsible for this change remains unclear. We examined whether SOD2, the gene encoding MnSOD, was epigenetically repressed in breast cancer cell lines by DNA methylation and histone acetylation. RT-PCR analysis of SOD2 mRNA showed the nontumorigenic breast epithelial cell line MCF-10A to have two to three fold higher expression levels than either UACC-893 or MDA-MB-435 breast carcinoma cells. Analysis of a region in the SOD2 promoter by sodium bisulfite genomic sequencing demonstrated significantly higher levels of CpG methylation in both human breast carcinoma cell lines assessed than in MCF-10A cells. CREB binding in vitro to a cognate site derived from this region was repressed by DNA methylation, and CREB binding to the 5' regulatory region of the SOD2 gene in vivo as determined by ChIP was significantly lower in breast carcinoma cells than in MCF-10A. Increased cytosine methylation was also accompanied by a significant decrease in the level of acetylated histones in the same region of the SOD2 promoter. Finally, a causal link between cytosine methylation and transcriptional repression was established by increasing MnSOD mRNA, protein and activity in breast carcinoma cells using the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine. These findings indicate that epigenetic silencing of SOD2 constitutes one mechanism leading to the decreased expression of MnSOD observed in many breast cancers.