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The gene CST6 was cloned by differential display based on its highly downregulated expression in 21MT-1 metastatic breast cancer cell line when compared to 21PT cell line isolated from the primary tumor of the same patient, while it was found inactivated in the majority of metastatic breast cancers (Sotiropoulou, Anisowicz, & Sager, 1997). The corresponding gene (CST6) was mapped to chromosomal locus 11q13 (Stenman et al., 1997), which is believed to harbor tumor suppressor genes because loss of heterozygosity (LOH) has been frequently observed in several types of cancer. Later the same gene was identified by expressed sequence tag (EST) sequencing in amniotic cells and fetal skin epithelial cells and was named cystatin E (Ni et al., 1997). Cystatin M, re-named cystatin E/M, is an endogenous inhibitor of asparaginyl endopeptidase/legumain and of the lysosomal cysteine proteases cathepsins B and L (Shridhar et al., 2004; J. Zhang et al., 2004). Increased proteolytic activities of lysosomal proteases are known to promote cancer progression, invasion and metastasis via degradation of the basement membrane and extracellular matrix (ECM) (Mohamed & Sloane, 2006). In this respect, it was shown that cystatin E/M is a tumor suppressor for breast cancer (Shridhar et al., 2004; J. Zhang et al., 2004). Further, cystatin E/M was shown to inhibit adhesion of breast cancer cells to endothelial cells, an important step for cancer metastasis (Shridhar et al., 2004), as well as invasion of human melanoma by suppressing legumain activity (Briggs et al., 2010). In a recent study, CST6 was also shown to suppress the proliferation, colony formation, migration and invasion of breast cancer cells (Jin et al., 2012).
Several studies have shown that aberrant hypermethylation of a CpG island located in the proximal promoter and first exon is associated with epigenetic silencing of CST6 expression in breast cancer cell lines and primary breast carcinomas, as well as in other types of cancer (Ai et al., 2006; Kioulafa et al., 2009; Qiu et al., 2008; Veena et al., 2008). In cancer cells, promoter CpG islands are prone to de novo methylation that prevents binding of regulatory transcription factors (TFs) to their cognate DNA sequences or TF-binding sites (TFBS) (Jones & Baylin, 2002). On the other hand, microRNAs (miRNAs) are small non-coding RNAs which negatively regulate gene expression at the post-transcriptional level acting by sequence-specific base-pairing at the 3’-UTR of the target mRNA that induces either mRNA degradation or translation inhibition by masking the ribosome-binding sequence. While it is now well established that miRNAs represent a distinct level of epigenetic gene regulation, an expanding body of evidence suggests a strong link between miRNAs and human diseases, including most types of cancer (Sotiropoulou, Pampalakis, Lianidou, & Mourelatos, 2009). Certain miRNAs are aberrantly expressed in cancer cells and were shown to contribute to tumor growth and/or progression functioning either as oncogenes (oncomirs) or tumor suppressors (Sotiropoulou et al., 2009).
Therefore, to this end, we searched for a potential association between miRNAs and CST6 silencing in cancer cells. In order to identify cancer-associated transcriptional and posttranscriptional regulators of CST6, we employed bioinformatics tools to search both the CST6 promoter and 3’-UTR for TFBS and miRNA targets, respectively. Moreover, the association between hypermethylation of the CpG island located in the CST6 promoter and to epigenetic silencing of cystatin E/M tumor suppressor in breast cancer cells was examined. Finally, we identified novel SNPs, as well as novel transcripts and cystatin E/M orthologous proteins by comprehensive phylogenetic analysis.