Background
Among diverse cancer types, breast carcinoma stands out for its increasing incidence rates and high mortality worldwide [
1]. Like most solid tumors, metastatic disease rather than the primary tumor itself is responsible for death [
2‐
4]. The metastatic process involves a complex cascade of events, including the organized breakdown of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) [
5,
6]. Together, the MMPs are able to process or degrade all ECM components. Each ECM element is cleaved by a specific MMP or MMP group [
7]. The activity of these proteases is tightly regulated by specific inhibitors, known as tissue inhibitors of MMPs (TIMPs) [
7,
8]. Consistent with their role in tumor progression, high levels of a number of MMPs have been shown to correlate with poor prognosis in human cancers [
9‐
11]. Surprisingly, high levels of TIMP-1 and TIMP-2 have also been shown to predict adverse prognosis and correlate with tumor aggressiveness in several different human cancers, including breast cancer [
12‐
14]. TIMPs expression profile could be the result of its action as a multifunctional molecule [
8].
The RECK metastasis suppressor gene was isolated by screening a fibroblast expression library for cDNAs that induced flat revertants in ν-Ki-ras-transformed NIH3T3 cells [
15]. RECK encodes a membrane-associated MMP regulator protein that is able to suppress tumor invasion and metastasis by negatively regulating MMPs involved in carcinogenesis, namely: MMP-2, MMP-9 and MMP-14 (MT1-MMP) [
16,
17]. Due to these functions, RECK has been described as a good prognosis marker in several tumor types, including breast carcinomas [
18,
19].
The ECM degradation and, consequently, the invasive and metastatic potential of tumor cells is the result of the umbalance between the activities of these multiple factors that compose the proteases/inhibitors equilibrium [
20]. Furthermore, each one of these molecules is involved in different stages and processes during tumor progression [
20,
21]. Although the expression and activity profile of MMPs, TIMPs and RECK have already been described in several cell line models [
22‐
24], few reports analyze more than one MMP or MMP inhibitor in different cell lines at the same time, using the same approach [
23]. Thus, there are no studies which address the complexity of MMPs/inhibitors system in a multi-factorial context. Moreover, there are no reports comparing expression profiles of these important modulators of the metastatic process, in both a cell line model system and in breast tumor tissue samples.
Here, we analyzed the expression levels of MMPs and their inhibitors, by qRT-PCR, in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential and in 72 primary breast cancer and 30 adjacent non-tumor tissue specimens. The results demonstrate a significant and positive correlation between the levels of MMPs and their inhibitors both in the cell line models and in tumor tissue samples, suggesting that the expression of these molecules, at least at the transcriptional level, might be regulated by common factors and signaling pathways. Therefore, deregulation of only one of these components could be followed by alterations of several other members of the proteases/inhibitors families. Therefore, we suggest that a multi-factorial analysis is crucial to properly evaluate metastasis.
Discussion
Development of metastasis is the most critical parameter for determination of survival in breast cancer patients. Patients diagnosed and treated before further extension of the tumor present higher cure rate [
2‐
4]. The role of MMPs and their specific inhibitors in the metastatic process is well established. Nowadays, there are more than 20 MMPs identified in humans, and several MMP inhibitors, each one of these molecules being involved in different steps of tumor progression [
20,
21]. In spite of the complexity of MMPs/MMP inhibitors system, all previous studies which analyzed the status of these molecules during mammary tumor progression only focused on a small number of MMPs/MMP inhibitors in a limited number of models. Here, we proposed a more extensive approach by analyzing the mRNA expression levels of different MMPs (MMP-2, MMP-9 and MMP-14) and MMP inhibitors by qRT-PCR (TIMP-1, TIMP-2 and RECK) in several models (five human breast cancer cell lines, 72 primary breast tumors and 30 adjacent normal tissues).
The verified mRNA expression levels, obtained by qRT-PCR, suggested that breast cancer progression from pre-malignant stages to highly invasive and metastatic carcinomas could be accompanied by increased expression of MMPs and their inhibitors. The total RNA from a panel of five human breast cancer cell lines, displaying increased degrees of invasiveness and metastatic potential, were used to perform this quantitative RT-PCR assay. In the cell model used, the levels of all analyzed MMPs (MMP-2, MMP-9 and MMP-14) were, in almost all cases, higher in cell lines with elevated invasive and metastatic capacities (MDA-MB-435, MDA-MB-231 and Hs578T) when compared to less aggressive ones (MCF-7 and ZR-75-1). These results corroborate the data previously obtained by other groups, in which MMPs expression in breast cancer cell models positively correlates with a highly aggressive phenotype [
26,
27]. We found similar mRNA expression profiles for all analyzed MMP inhibitors (TIMP-1, TIMP-2 and RECK). At first sight, these results seem contradictory, since these molecules were described as MMP inhibitors. However, elevated mRNA expression levels of TIMP-1 and TIMP-2 have been previously detected in breast cancer cell lines with high invasive and metastatic potential [
28]. Furthermore, several reports describe TIMPs as multifunctional molecules [
8]. Thus, these molecules were both able to suppress and to promote tumor progression [
8].
On the other hand, high expression levels of RECK, a membrane-associated MMP regulator protein, which is able to suppress tumor invasion and metastasis, were associated with better prognosis in several types of tumor, including breast carcinomas [
18,
19]. However, our results demonstrate that RECK mRNA expression level is increased during breast cancer progression. Thus, at first sight, these data seem to be at variance with the previous studies, suggesting that increased RECK levels could be associated not with better prognosis, but with late clinical stages. Nevertheless, other reports demonstrated that elevated levels of RECK were associated with better prognosis only for patients in late clinical stages [
29,
30]. Therefore, tumors that present overexpression of RECK could display a better clinical course, depending on the enzyme/inhibitor context. Thus, induction of RECK gene expression, observed during breast cancer progression, could be a cellular response to enhanced expression and activity of MMPs. In this context, the ECM proteolysis equilibrium could be maintained by re-establishment of the MMPs/inhibitors balance.
The role of cell-ECM elements interaction in regulation of expression and activity of these molecules was evaluated. The gelatin degradation ability of MMP-2 and MMP-9, evaluated by zymography assays, was significantly increased in cultures seeded onto Matrigel, when compared to those seeded onto uncoated plastic. Moreover, in highly aggressive cell lines, enhanced MMP activity was observed in less invasive and metastatic breast cancer cells, corroborating the data obtained in mRNA expression analysis. However, among the analyzed proteases, only MMP-2 expression was significantly modulated by cell-ECM elements interaction. These results suggest that increased proteolytic activity is independent of transcriptional activation, corroborating previously published data [
31]. Our results demonstrate that the transcript levels of all studied MMP inhibitors are significantly modulated by the reconstituted basal lamina. This regulation of TIMP-1, TIMP-2 and RECK mRNA levels by cell-Matrigel contact was significant only in highly invasive and metastatic cell lines. Therefore, the intense ECM degradation is correlated with increased MMP inhibitor expression. The ECM supplies mechanical support to cells and structural integrity to tissues [
6,
32]. Furthermore, ECM performs an instructive role, since it is a reservoir of cytokines, and of growth and differentiation factors, which can be discharged and/or activated during proteolysis of ECM elements [
33] by MMPs, contributing to the crosstalk between tumor cells and the microenvironment [
34]. Therefore, the high mRNA expression levels of MMP inhibitors, found in highly aggressive cell lines, probably reflects a cellular reaction mechanism to intense ECM remodeling, as previously discussed. Thus, during ECM degradation, an important factor involved in induction of TIMPs and RECK can be discharged and/or activated. Our results corroborate this hypothesis, since we demonstrated that ECM is important only in modulation of gene expression of MMP inhibitors and, moreover, only in highly invasive and metastatic cell lines. Therefore, we suggest that this negative feedback mechanism of induction of protease inhibitors, during breast cancer progression, occurs for the re-establishment of the MMP/inhibitors balance and, consequently, for homeostasis of ECM proteolysis.
Cellular models are commonly used in Cell and Molecular Biology studies due to the limited source of tissue samples. However, enrichment for malignant cells and absence of stroma may compromise comparisons between cell lines and tumors [
35]. Therefore, to discard possible cell culture artifacts, we also analyzed the expression of MMPs and their inhibitors in primary breast cancer tumor samples by qRT-PCR. The results obtained demonstrate, in a more general scenario, that the mRNA expression levels of MMPs are significantly higher in tumor samples than in adjacent non-tumor tissues. On the other hand, TIMP-1, TIMP-2 and RECK gene expression levels were significantly lower in tumors when compared with adjacent normal tissues. Furthermore, our results demonstrate a significant positive correlation between the gene expression levels of these proteases and their inhibitors in primary breast tumors from patients. Therefore, we suggest that increased expression of genes responsible for ECM degradation is coordinately accompanied by increased expression of their specific inhibitors and that the levels of MMPs, TIMPs and RECK transcripts may be regulated by common factors and signaling pathways.
Corroborating other studies in breast cancer [
10,
36], we found no significant association between the mRNA expression levels of the MMPs analyzed and clinical-pathological data [
10,
36]. These results suggest that the MMPs may supply independent prognostic information. However, there are conflicting results on the prognostic value of these proteases in this model. These differences can, at least in part, be due to the different methodologies used and to possible modulation of MMPs by adjuvant systemic treatment [
37,
38]. Furthermore, the TIMP-1, TIMP-2 and RECK transcript levels did not correlate with the clinical parameters from patients. The status of PR was an exception, which showed a significant correlation with TIMP-1 gene expression only. Likewise, we suggest that expression of the analyzed MMPs inhibitors may supply independent prognostic information.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
RCSF was responsible for most of the experimental work and interpretation of some of the results. LRG was responsible for data discussion and interpretation, statistical analysis and manuscript preparation. JSN, FCS and IDCGS were responsible for collecting the tumor samples used in this study. MCS participated in the study design, data discussion and supervision of the study. All authors read and approved the final manuscript.