Epigenetic Silencing of the Tumor Suppressor Cystatin M Occurs during Breast Cancer Progression

Abstract

Cystatin M is a secreted inhibitor of lysosomal cysteine proteases. Several lines of evidence indicate that cystatin M is a tumor suppressor important in breast malignancy; however, the mechanism(s) that leads to inactivation of cystatin M during cancer progression is unknown. Inspection of the human cystatin M locus uncovered a large and dense CpG island within the 5 ¶ region of this gene (termed CST6). Analysis of cultured human breast tumor lines indicated that cystatin M expression is either undetectable or in low abundance in several lines; however, enhanced gene expression was measured in cells cultured on the DNA demethylating agent 5-aza-2 ¶-deoxycytidine (5-aza-dC). Increased cystatin M expression does not correlate with a cytotoxic response to 5-aza-dC; rather, various molecular approaches indicated that the CST6 gene was aberrantly methylated in these tumor lines as well as in primary breast tumors. Moreover, 60% (12 of 20) of primary tumors analyzed displayed CST6 hypermethylation, indicating that this aberrant characteristic is common in breast malignancies. Finally, preinvasive and invasive breast tumor cells were microdissected from nine archival breast cancer specimens. Of the five tumors displaying CST6 gene methylation, four tumors displayed methylation in both ductal carcinoma in situ and invasive breast carcinoma lesions and reduced expression of cystatin M in these tumors was confirmed by immunohistochemistry. In summary, this study establishes that the tumor suppressor cystatin M is a novel target for epigenetic silencing during mammary tumorigenesis and that this aberrant event can occur before development of invasive breast cancer. (Cancer Res 2006; 66(16): 7899-909)

Methylation-dependent silencing of CST6 in primary human breast tumors and metastatic lesions

“…In a subsequent study, methylation analysis of CST6 in breast cancer cell lines revealed an inverse correlation between CpG island hypermethylation and CST6 gene expression, and the extent of regional methylation in the proximal promoter was strongly correlated with the lack of CST6 expression (Rivenbark et al, 2006a). Consistent with these findings, several other studies have shown that CST6 is epigenetically regulated by DNA methylation-dependent silencing in breast cancer cell lines and primary invasive ductal carcinomas (Ai et al, 2006;Schagdarsurengin et al, 2006).…”

“…Furthermore, expression of CST6 in MDA-MB-435S breast cancer cells delays tumorigenesis by transplanted cells and suppresses spontaneous formation of liver and lung metastases (Zhang et al, 2004). More recently, it has been shown that CST6 is epigenetically regulated by DNA methylation-dependent silencing in breast cancer cell lines (Ai et al, 2006;Rivenbark et al, 2006a;Schagdarsurengin et al, 2006;Shridhar et al, 2004) and primary invasive ductal carcinomas (Ai et al, 2006;Schagdarsurengin et al, 2006). Ai et al showed that 12/20 (60%) primary breast tumors exhibit CST6 promoter hypermethylation, and microdissection of individual cells from select tumors revealed that methylation occurs in both DCIS and IDC cells (Ai et al, 2006).…”

“…More recently, it has been shown that CST6 is epigenetically regulated by DNA methylation-dependent silencing in breast cancer cell lines (Ai et al, 2006;Rivenbark et al, 2006a;Schagdarsurengin et al, 2006;Shridhar et al, 2004) and primary invasive ductal carcinomas (Ai et al, 2006;Schagdarsurengin et al, 2006). Ai et al showed that 12/20 (60%) primary breast tumors exhibit CST6 promoter hypermethylation, and microdissection of individual cells from select tumors revealed that methylation occurs in both DCIS and IDC cells (Ai et al, 2006). In a similar study, Schagdarsurengin et al showed that 24/40 (60%) breast carcinomas exhibited CST6 promoter hypermethylation, and that estrogen-receptor positive tumors were more frequently methylated than estrogen-receptor negative tumors (Schagdarsurengin et al, 2006).…”

“…It is now accepted that there are two major pathways of multi-step breast cancer progression, (i) well-differentiated ductal carcinoma in situ (DCIS) progressing to grade I invasive ductal carcinoma (IDC), and (ii) poorly differentiated DCIS progressing to grade III IDC (Simpson et al, 2005). Changes in the molecular pattern of DCIS lesions may lead to the ability to collapse the myoepithelium, escape the duct structure, and invade the surrounding stroma (Ai et al, 2006;Symmans, 2005). These IDC lesions will proliferate and grow, destroying surrounding stroma, and breast architecture.…”

“…Cystatin M is involved in regulating the activity of cathepsin B and cathepsin L, and imbalances between these proteases and cystatin M may lead to the metastatic phenotype in tumor cells (Frosch et al, 1999;Kos et al, 2000;Lah and Kos, 1998). Cystatin M expression has been reported to be diminished or lost in various forms of cancer including, (i) basal and squamous cell carcinomas of the skin (Zeeuwen, 2004), (ii) squamous cell carcinomas of the head/neck and lung (Zeeuwen et al, 2002), (iii) non-small cell lung cancer (Zhong et al, 2006), (iv) metastatic oral cancer cell lines (Vigneswaran et al, 2006), (v) malignant glioma (Kim et al, 2006), and (vi) breast cancer (Ai et al, 2006;Rivenbark et al, 2006a;Schagdarsurengin et al, 2006;Shridhar et al, 2004;Song et al, 2006;Sotiropoulou et al, 1997;Zhang et al, 2004). Cystatin M contains a large CpG island that flanks the transcription start site and spans the proximal promoter and exon 1 regions.…”