ORGINAL ARTICLEExpression analysis of cystatin C and M in laser-capture microdissectioned human breast cancer cells—a preliminary study
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.
Section snippets
Subjects
Our study utilized 17 fresh-frozen archival breast cancer specimens, which were immediately frozen in liquid nitrogen and stored at −70 °C. The corresponding formalin-fixed paraffin blocks were used for immunohistochemical and in situ hybridization studies. Paraffin blocks of lymph node metastasis corresponding to these cases were included to analyze the cystatin M expression by immunohistochemical and in situ hybridization studies. Case identification and accrual of clinical information were
Results
Using laser capture microdissection combined with RT-PCR, we examined the cathepsins B and L and cystatins C and M transcripts in 17 cases of infiltrative ductal carcinomas of the breast. Clinicopathologic findings of these cases are summarized in Table 1. Fig. 1 shows the results of LCM/RT-PCR analysis of cathepsins B and L and cystatins M and C expression in these breast cancer specimens. Relative expression levels of cathepsins B and L and cystatins M and C mRNA in each tumor sample were
Discussion
Cystatin M was initially characterized by differential display, as a down-regulated mRNA in metastatic breast cancer cells, but present in parent primary tumor cells. Hence, it was hypothesized that lack of cystatin M expression might be associated with the progression of primary tumor to metastatic phenotype. However, the relationship between tumor cell levels of cystatin M and lymph node status has not been investigated in clinical breast cancer specimens. Therefore, the primary goal of this
Acknowledgement
This work was funded by a research award from the Department of Defense Breast Cancer Research Program DAMD17-01-1-0643 (N.V.). We thank Dr. M. Abrahamson for providing the polyclonal cystatin M antisera.
References (37)
Cystatins
Methods Enzymol.
(1994)- et al.
Cathepsin B mediates tumor necrosis factor-induced arachidonic acid release in tumor cells
J. Biol. Chem.
(2002) - et al.
Cathepsin B efficiently activates the soluble and the tumor cell receptor-bound form of the proenzyme urokinase-type plasminogen activator (Pro-uPA)
J. Biol. Chem.
(1991) - et al.
Cystatin E is a novel human cysteine proteinase inhibitor with structural resemblance to family 2 cystatins
J. Biol. Chem.
(1997) - et al.
Identification, cloning, and characterization of cystatin M, a novel cysteine proteinase inhibitor, down-regulated in breast cancer
J. Biol. Chem.
(1997) - et al.
Upregulation of cystatin M during the progression of oropharyngeal squamous cell carcinoma from primary tumor to metastasis
Oral Oncol.
(2003) - et al.
Variable expression of cathepsin B and D correlates with highly invasive and metastatic phenotype of oral cancer
Hum. Pathol.
(2000) - et al.
Cystatin M/E expression is restricted to differentiated epidermal keratinocytes and sweat glandsa new skin-specific proteinase inhibitor that is a target for cross-linking by transglutaminase
J. Invest. Dermatol.
(2001) - et al.
Degradation of extracellular-matrix proteins by human cathepsin B from normal and tumour tissues
Biochem. J.
(1992) - et al.
Secretory leukocyte protease inhibitor promotes the tumorigenic and metastatic potential of cancer cells
Proc. Natl. Acad. Sci. USA
(2003)
Prognostic significance of cathepsins B and L in primary human breast cancer
J. Clin. Oncol.
Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor
J. Cell Biol.
Activity, expression, and transcription rate of the cathepsins B, D, H, and L in cutaneous malignant melanoma
Cancer
Molecular regulation, membrane association and secretion of tumor cathepsin B
Apmis
High levels of urokinase-type plasminogen activator and its inhibitor PAI-1 in cytosolic extracts of breast carcinomas are associated with poor prognosis
Cancer Res.
“In vitro” study of basement membrane degradation by the cysteine proteinases, cathepsins B, B-like and L. Digestion of collagen IV, laminin, fibronectin, and release of gelatinase activities from basement membrane fibronectin
Biol. Chem. Hoppe Seyler
Prognostic impact of proteolytic factors (urokinase-type plasminogen activator, plasminogen activator inhibitor 1, and cathepsins B, D, and L) in primary breast cancer reflects effects of adjuvant systemic therapy
Clin. Cancer Res.
High preoperative plasma tissue inhibitor of metalloproteinase-1 levels are associated with short survival of patients with colorectal cancer
Clin. Cancer Res.
Cited by (30)
On the role of cystatin C in cancer progression
2018, Life SciencesCitation Excerpt :It follows that, by thwarting these effects Cyst C may indirectly facilitate the growth and spread of cancer cells [94,131–139] (Fig. 1B). This phenomenon might also account for the positive association between elevated Cyst C serum levels and a larger tumor size as revealed by some clinical investigations [79–81]. Moreover, Wilder et al. [139] have recently shown that the exposure of human MDA-MB-231 breast cancer cells to physiological concentrations of Cyst C, while inhibiting cathepsin L, induced compensatory mechanisms resulting in an up-regulation of active proteinases, such as cathepsin S which appear to facilitate tumor invasion, angiogenesis and metastasis [120,140,141].
Cystatin M expression is reduced in gastric carcinoma and is associated with promoter hypermethylation
2010, Biochemical and Biophysical Research CommunicationsCitation Excerpt :Loss of cystatin M expression might contribute to increased proteolysis of tissue architecture, facilitating the spread of cancer cells [4]. Cystatin M expression has been reported to be diminished or lost in various forms of cancer including basal and squamous cell carcinomas of the skin, squamous cell carcinomas of the head/neck and lung, non-small cell lung cancer, metastatic oral cancer cell lines, malignant glioma, prostate cancer, and breast cancer [3,8,10,14–17]. Similarly, we found that cystatin M expression was downregulated in gastric cancer tissues compared with normal tissue, which was kept in line with its role of tumor suppressor gene.
The biology of cystatin ME and its cognate target proteases
2009, Journal of Investigative DermatologyCitation Excerpt :Preliminary studies using laser-capture dissection of human breast cancer cells varied in outcome. Zhang et al. (2004) reported a downregulation of cystatin M/E mRNA and protein expression in invasive ductal carcinomas compared with normal breast tissue, which is in contrast with another study that showed cystatin M/E expression in most of the breast carcinomas, including primary and metastatic tumors (Vigneswaran et al., 2005). Recently, epigenetic studies reported DNA methylation-dependent silencing of the cystatin M/E gene in breast cancer cell lines, primary breast tumors, and metastases.
Identification of gene signatures for invasive colorectal tumor cells
2007, Cancer Detection and PreventionTowards novel anti-cancer strategies based on cystatin function
2006, Cancer LettersThe candidate tumor suppressor CST6 alters the gene expression profile of human breast carcinoma cells: Down-regulation of the potent mitogenic, motogenic, and angiogenic factor autotaxin
2006, Biochemical and Biophysical Research CommunicationsCitation Excerpt :Our microarray and TaqMan data show that CST6 down-regulates ATX but up-regulates IL-8. This suggests that CST6 might behave like a tumor suppressor for some types of cancers [6,7] while acting perhaps like a tumor promoter for other types of cancers [43,44]. We have shown previously [6] that most breast cancer and melanoma cells have lost expression of CST6 while two of three prostate cancer cell lines were found to express high levels of transcript.