Cystatin M suppresses the malignant phenotype of human MDA-MB-435S cells

Abstract

Proteases are involved in many aspects of tumor progression, including cell survival and proliferation, escape from immune surveillance, cell adhesion and migration, remodeling and invasion of the extracellular matrix. Several lysosomal cysteine proteases have been cloned and shown to be overexpressed in cancer; yet, despite the great potential for development of novel therapeutics, we still know little about the regulation of their proteolytic activity. Cystatins such as cystatin M are potent endogenous protein inhibitors of lysosomal cysteine proteases. Cystatin M is expressed in normal and premalignant human epithelial cells, but not in many cancer cell lines. Here, we examined the effects of cystatin M expression on malignant properties of human breast carcinoma MDA-MB-435S cells. Cystatin M was found to significantly reduce in vitro: cell proliferation, migration, Matrigel invasion, and adhesion to endothelial cells. Reduction of cell proliferation and adhesion to an endothelial cell monolayer were both independent of the inhibition of lysosomal cysteine proteases. In contrast, cell migration and matrix invasion seemed to rely on lysosomal cysteine proteases, as both recombinant cystatin M and E64 were able to block these processes. This study provides the first evidence that cystatin M may play important roles in safeguarding against human breast cancer.

In Silico Analysis Validates Proteomic Findings of Formalin-fixed Paraffin Embedded Cutaneous Squamous Cell Carcinoma Tissue

“…The role of CST6 as a tumor suppressor is well established in many cancer types including breast (63)(64)(65)(66)(67) and prostate cancer (68). CST6 is a potent endogenous protein inhibitor of lysosomal proteases and its down-regulation, mainly through epigenetic inactivation, promotes tumor cell invasion, cell proliferation and matrix remodeling (64,67). Increased expression of KRT9, KRT10, FLG and FLG2, which are involved in terminal epidermal differentiation, was reported in cSCC using gene-expression analysis (69).…”

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

“…When exogenously expressed in human MDA-MB-435S breast cancer cells, CST6 significantly alters the neoplastic phenotype in vitro, resulting in diminished cell proliferation, loss of cell migration, inhibition of Matrigel invasion, and reduced endothelial cell adhesion (Shridhar et al, 2004). 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).…”

“…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).…”

“…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.…”

Frequent epigenetic inactivation of cystatin M in breast carcinoma

“…In wound healing, cystatin M expression was not found in the edge of migrating keratinocytes and in epidermal neoplasia cystatin M was only detected in differentiated cells and keratinized cell nests (Zeeuwen et al, 2002). Cystatin M downregulation was also observed in different cancer cells, including prostate cancer, colon cancer, glioblastoma, lung cancer and melanoma Shridhar et al, 2004). This indicates that cystatin M may be epigenetically downregulated in other cancers.…”