Expression analysis of cystatin C and M in laser-capture microdissectioned human breast cancer cells—a preliminary study

Summary

Cathepsins B and L, implicated in the progression of malignant tumors, are regulated by a family of endogenous inhibitors referred to as the cystatins. Cystatin M was identified by differential display as down-regulated gene in metastatic breast cancer cells. However, this finding has yet to be confirmed in clinical breast cancer specimens. Our objective is to examine the expression levels of cystatins C, M, and cathepsins B and L mRNA in breast cancer cells isolated by laser capture microdissection. The mRNA and protein levels of cathepsin B, L, and cystatin C and M in breast cancer specimens were determined utilizing laser capture microdissection/RT-PCR, Western blotting, and immunohistochemical methods. Expression levels of either cystatin M or C were not significantly different between lymph node-positive and -negative breast carcinomas. Increased expression levels of both cystatin M and C correlated significantly with larger tumor size. Cystatin M mRNA was detected by in situ hybridization in both primary and metastatic breast cancer cells. Our findings are at variance with a previous report proposing a metastasis suppressive function for cystatin M. Therefore, additional studies in a larger series with adequate follow-up are necessary to elucidate the biologic significance of cystatin M expression in breast cancer metastasis.

Introduction

Cystatins are specific endogenous inhibitors of lysosomal cysteine proteinases, namely cathepsins B, C, F, H, L, O, and X [1]. One of the primary functions of cystatins in tumor cells is regulating the activities of cathepsins B and L, which are implicated in tumor cell invasion and metastasis [7], [8], [35]. Cathepsins B and L have broad substrate specificity and are capable of degrading constituents of the extracellular matrix and basement membrane such as collagen IV, fibronectin, and laminin [2], [10], [14], [20]. Increased expression of cathepsin B mRNA and protein has been noted in breast carcinoma cells compared to adjacent noncancerous breast tissue [36]. Overexpression of cathepsin B in breast carcinomas was associated with a statistically significant higher risk for recurrence and shorter overall survival rate [4], [18].

Cystatins belong to a single superfamily of evolutionary related proteins that are further classified into three distinct types [1]. Type 1 cystatins A and B (also called stefins) with an approximate molecular weight of 11 kD lack signal peptides and, thus, function intracellularly [1]. Type 2 cystatins, namely C, D, S, SA, SN, M and F synthesized with signal peptides (13 –14 kD), are found secreted and intracellularly [1]. Type 3 cystatins are composed of L- and H-kininogen, which are complex glycosylated cytoplasmic proteins with type 2-like cystatin domain and the bradykinin moiety. Cystatin C, which is the strongest inhibitor of cathepsin B, is expressed at high levels in various tissues and does not show any tissue-specific restriction in its expression pattern. Relative ratios of cathepsin B to cystatins are reported to be increased in breast and prostate cancers, compared to their respective normal tissue [28], [36]. A higher ratio of cathepsin B to cystatin A predicted aggressive phenotype in prostate cancers [28]. Reduced expression of cystatin B was reported to be associated with lymph node metastasis in esophageal carcinomas [27]. Cystatin M (also called cystatin E) was originally cloned independently by two investigators by expressed sequence tag sequencing and by differential display, comparing the cDNA libraries of primary and metastatic breast carcinomas of the same patient [21], [30]. Cystatin M expression by primary breast carcinoma cells, but not by metastatic cells, was observed, leading to the hypothesis that loss of cystatin M expression is associated with the progression of primary breast cancer cells to metastatic phenotype [30]. However, this finding has yet to be substantiated by examining clinical breast cancer specimens.

The above-mentioned reports seem to support the theory that an increase in the ratios of cathepsins to cystatins contribute to tumor invasiveness and metastasis [29]. However, there are conflicting data regarding the role of cystatins in tumor invasion and metastasis. High levels of cystatins B and C predicted shorter survival of patients with colorectal cancer [15]. Cystatin F was identified as a novel metastasis-associated protein that is differentially expressed between murine carcinoma cells of low and high metastatic potential [19]. Increased expression of cystatin F in colorectal carcinomas showed a strong correlation with liver metastasis [31]. Increased expression of cystatin A in breast carcinomas has been found to correlate positively with tumor size, increased mitotic activity, and negative staining for the anti-apoptotic protein Bcl-2 [17].

These observations do not fit well with the expected anti-proteolytic activity of cystatins. Recently, however, it has become evident that cystatins may have additional and, as yet, poorly characterized biologic activities such as inhibition of tumor cell apoptosis [6]. Furthermore, previous studies have examined the expression levels of cathepsins and cystatins in whole tumor extracts, which consist of cathepsins and cystatins derived from both tumor and stromal cells [15], [16], [23], [27], [31], [36]. Therefore, the current study is set out to analyze tumor cell-specific transcripts of each of these genes using laser capture microdissection (LCM) combined with RT-PCR (LCM/RT-PCR). LCM can procure “pure” breast cancer cells from mixed cell population of breast tissue for gene expression analysis.

In this study, we examine the expression levels of cathepsins B and L, cystatins C and M, and their clinicopathologic significance in breast cancers. We also sought to clarify the correlation between transcript and protein expressions using immunohistochemical and immunoblotting analyses of the same tumor samples. To our knowledge, this is the first study that examines the concomitant expressions of cathepsins B and L and cystatins C and M using RT-PCR in pure breast cancer cells isolated by LCM.