Cystatin M, a novel tumor suppressor gene, is epigenetically regulated and silenced in breast cancer cell lines and primary breast tumors

Cystatin M (CST6) is a candidate breast cancer tumor suppressor that is expressed in normal and premalignant breast epithelium, but not in metastatic breast cancer cell lines. Gene expression analysis of 12 breast cancer cell lines revealed 7/12 (58%) lack expression of CST6, while normal mammary epithelial cell lines express CST6. Analysis of the promoter and exon 1 of CST6 identified a CpG island. CST6 is not expressed in MCF7, MDA‐MB‐436, or ZR‐75‐1 cells, but treatment of these cell lines with 5aza results in a significant increase in CST6 expression, suggesting that CST6 is subject to methylation‐dependent silencing. Bisulfite sequencing demonstrated extensive CpG island methylation in CST6 nonexpressing cells lines (MCF7, MDA‐MB‐435S, MDA‐MB‐436, MDA‐MB‐453, ZR‐75‐1). In contrast, CST6 expressing cell lines (MCF12A, BT‐20, SK‐BR‐3) lack CpG island methylation. These results establish a strong correlation between CST6 promoter methylation and expression status in breast cancer cell lines. In normal breast tissue, epithelial cells (and some myoepithelial cells) show strong immunostaining for CST6, while both DCIS and IDC cells show reduced levels or lack of CST6 expression in breast cancer tissues. These results suggest that CST6 is subject to methylation‐dependent silencing in human breast cancer cells and that this may represent an important mechanism for loss of CST6 during breast carcinogensis. Support: NIH CA78343, Komen Foundation BCTR0100575, NIH CA91785.