Metastasis is the final stage in tumor progression, being the main factor associated with cancer-promoted deaths [42
]. The balance between the activities of MMPs and MMP inhibitors is the essential regulator of ECM degradation and, consequently, of cellular phenotypes related to motile and invasive capacities. Similar to other cancer types, the breast cancer progression process is positively correlated with increased MMPs and MMP inhibitors expression and activity [12
], suggesting a coordinate regulation mechanism. In this report, we demonstrated, for the first time, that TGF-β1 is able to modulate MMP, TIMP and RECK expression in MDA-MB-231 human breast cancer cell line through ERK1/2 and p38MAPK. Both of these transducer pathways were essential to the TGF-β1-enhanced migration and invasion phenotypes; however, each mediated the TGF-β1 signal for MMPs and their inhibitors in a specific manner.
The important role of TGF-β during multiple stages of cancer progression has been widely reported. However, the status of several members of this pathway in human cancers remains quite complex and unclear [43
]. The TGF-β receptors and their downstream transducers are frequently lost, mutated or attenuated in human carcinomas, including pancreatic, colon and gastric tumors [43
]. Alternatively, other tumor types, such as breast tumors, present much lower mutation frequency in these TGF-β signaling effectors, but display many alterations in their expression levels [43
]. Only few reports addressed more than one TGF-β pathway member at the same time. Due to the lack of information regarding profile complexity of the TGF-β network elements and their dependence on the cell context, we first performed a general characterization of the TGF-β isoforms and their receptors by mRNA expression analysis in a panel of five human breast cancer cell lines displaying diverse invasive and metastatic capacities. We showed that, similar to MMPs, TIMPs and RECK, the mRNA levels of TGF-β receptors I and II, are expressed at a higher level in the most aggressive cell line, as compared to the less invasive ones, except for TβRI that was also highly expressed in ZR-75-1 cells. These results corroborate prior reports in the literature from tumor tissue samples, showing that, in breast cancer models, TGF-β signaling appears to be correlated with tumor-promoting functions [23
TGF-β1 acts as a growth inhibitor at the early stages of tumorigenesis while it stimulates EMT, tumor invasion and metastasis in advanced tumors [40
]. Therefore, cancer cells in different stages of aggressiveness respond differently to TGF-β treatment. The least invasive (MCF-7) and the highly invasive (MDA-MB-231) human breast cancer cell lines are examples of this dual role of TGF-β. In this case, loss of estrogen receptor expression and ras
gene amplification, two very common alterations during breast cancer progression, are some factors involved in switching the phenotypic response of TGF-β treatment, from anti-proliferative to invasive [32
]. Thus, TGF-β1 is not able to regulate proliferation of the MDA-MB-231 cells [28
]. However, we demonstrate that this cytokine is a positive modulator of migration and invasive potential of these cells.
Previous reports have suggested a crucial function of TGF-β1 in cell motility control, some of which relate this altered phenotype to its role as a modulator of MMPs [23
]. Kim and collaborators suggested that TGF-β1 also induces invasion in pre-malignant breast cancer cells (MCF10A), by upregulation of MMP-2 and MMP-9 [21
]. Subsequent reports also indicated that MMP-2 and MMP-9 are essential in the TGF-β1-incresead invasion of MCF10 cell series in a 3D model [23
]. Similarly, the high motility phenotype presented by TGF-β1-treated MDA-MB-231 cells was associated with the upregulation of MMP-9 by this cytokine [50
]. On the other hand, in the MDA-MB-435 cell line, MMP-14 was shown to be the molecule responsible for the TGF-β1-increased migration capacity [22
]. However, none of these previous reports investigated whether TGF-β1 can also modulate the expression of MMP inhibitors, and whether these inhibitors, thought to downmodulate ECM breakdown, are also implicated in the TGF-β1-induced cell spreading. Since the balance between MMPs and their inhibitors is an important factor for ECM degradation, the identification of common regulators of MMPs, TIMPs and RECK is necessary to identify the principal factors involved in the metastatic process. Here we describe, for the first time, a molecular mechanism in which TGF-β1 modulates MMP-2 and MMP-9 as well as TIMP-2 and RECK expression. The regulation of these MMPs inhibitors expression could be related to a cellular response for reestablishment of the proteases/inhibitors balance during cancer progression.
We found some discrepancy between the mRNA and protein expression levels of some MMPs and MMPs inhibitors upon treatment with TGF-β1. For instance, while RECK was increased at the transcriptional level, its protein expression levels were inhibited by this cytokine. This divergence could be due to the influence of TGF-β1 in RECK mRNA and protein stability and degradation rates and/or to other post-transcriptional and post-translational molecular mechanisms.
Although mounting evidence supports the potential role of RECK as a molecular marker for cancer prognosis and controller of cellular metastatic capacity, no reports are available unveiling its function in breast cancer [18
]. For the first time, we have demonstrated that expression of this membrane-associated MMP inhibitor is regulated by TGF-β1 in a breast cancer cell culture model, suggesting that RECK could be involved in the molecular mechanisms of breast cancer progression.
TGF-β1 is able to signal through both Smad-dependent and Smad-independent mechanisms. However, previous evidences have established that each of these pathways is related to distinct cellular responses to TGF-β1 [35
]. Therefore, the switching of TGF-β's role from a tumor suppressor to a pro-oncogenic-factor during cancer progression could be caused by changes in the way that this cytokine modulates its downstream pathways. It has been suggested that Smads are involved in the anti-tumor process, such as inhibition of cell proliferation, while the Smad-independent pathways have been implicated in induction of tumor progression [35
Here we analyzed the involvement of ERK1/2 and p38 MAPK, two well established Smad-independent pathways, in the proposed mechanism of coordinate regulation of MMPs, TIMPs and RECK by TGF-β1 in breast cancer cell lines. Our results demonstrate that both MAPKs are important for this mechanism, each being responsible for modulating specific molecules (Figure ). Unlike previously reported data of MCF10A cells [21
], p38 MAPK as well as ERK1/2 were shown to be key components mediating the TGF-β1-induced MMPs upregulation. However, our data show that p38 MAPK mediates increased levels of MMP-2 and ERK1/2 are involved in the modulation of MMP-9 levels. Although both p38 MAPK and ERK1/2 were required for TGF-β1 induction of the TIMP-2 protein expression, we demonstrated that only ERK1/2 are responsible for the RECK downregulation induced upon TGF-β1 treatment [21
Figure 10 Scheme of the molecular mechanism proposed for TGF-β1 action as a common regulator of MMPs (MMP-2 and MMP-9) and their inhibitors (TIMP-2 and RECK), through ERK1/2 and p38 MAPK pathways, in MDA-MB-231 cell line. The dashed lines indicate regulations (more ...)