There is accumulating evidence for the importance of oncogenic signaling by STAT3 in the pathogenesis of various human cancers. Thus, we focused on the contribution of STAT3 to the onset and development of colorectal cancer. Our previous work shows that STAT3 may contribute to oncogenesis in the colon epithelium by exerting a promoting effect on cell proliferation [5
], whereas this paper demonstrates its critical involvement in the enhancement of invasive cell behavior.
One of the prerequisites for the invasiveness of tumor cells is increased motility, and, of metastasis, a common feature of late-stage CRC. The pleiotropic protein STAT3 is known to exert control on cell motility in various settings. STAT3 is crucial, e.g., for cell motility during gastrulation [48
], wound healing, and blood vessel formation [49
]. We recently observed that the invasiveness of trophoblast cells in pregnancy is directly influenced through STAT3 activation [5,50
More importantly, inadequate STAT3 activity was found correlated with cancer-associated motility of breast [27,51
] and ovarian cancer [12
] as well as of choriocarcinoma cells [44
]. In mesenchymal-like and colon carcinoma cells, STAT3 plays a critical role in the transmission of invasiveness-promoting signaling by the c-Met receptor [52,53
Altered invasiveness in transformed cells is a consequence of changes in the status of cell adhesion, the cytoskeleton, and gene expression. STAT3 has been shown to act on all these aspects: In addition to its well-established role as a transcription factor, STAT3 may also function as a signaling adaptor at focal adhesions and, presumably, as a persistent scaffold factor in cytokine/growth factor receptor assemblies [12
]. As recently reported, STAT3 can signal in an as yet unknown way through Rho GTPases, thus regulating multiple cellular functions including actin cytoskeleton reorganization and cell migration [54
Transcriptional regulation of target genes provides an obvious route through which STAT3 enhances cellular malignancy and, in particular, invasiveness. A central aspect of invasiveness is the expression of proteases that render the tumor cells capable of digesting constituents of the ECM. Various reports point to a role of STAT3 in promoting invasive cell behavior by exerting influence on the transcription of protease genes. In metastatic melanoma cells, a link was found between the expression of MMP-2, invasiveness, and constitutive STAT3 activity. More importantly, inhibition of STAT3 by a dominant-negative mutant reduced MMP-2 expression and invasiveness and blocked metastasis in nude mice [55
]. In skin epithelial cells, STAT3 was shown to mediate the IL-6-provoked induction of MMP-1 and MMP-3 [56
]. However, STAT3 is involved in controlling the expression of MMP-7 in prostate carcinoma cells [26
] and of MMP-9 in cervix carcinoma cells [57
We showed that STAT3 directly drives transcription from the MMP-1 promoter in colon carcinoma cells and also defined a binding site for STAT3 in the MMP-1 as well as in the MMP-3 promoter. However, the precise role of STAT factors in MMP gene regulation in colon cancer cells requires further elucidation. Despite the presence of various additional consensus sequences, we identified only one sequence element in both the MMP-1 and the MMP-3 promoter that specifically binds to activated STAT3. This finding leaves open the possibility that the other elements interact with STAT1 or STAT5, both of which are also frequently active in CRC biopsies [5
]. Furthermore, those sequences showing no affinity for STAT3 in our assay may require additional binding sites for the interaction with STAT3 tetramers, as recently observed for other promoters [58,59
Transcription of the MMP-1 gene is regulated in a complex manner. The process appears to be influenced by the orchestrated action of various transcription factors and seems also to depend on the type of cells involved. Apart from STAT cognate elements, other regulatory elements within the MMP-1 promoter region include various transcription factor binding sites for AP-1, GATA binding, and ETS proteins [60,61
]. The cooperative contribution of AP-1 and ETS transcription factors toward control of MMP-1 expression was demonstrated by the observation that a single nucleotide polymorphism creating an additional ETS binding site close to the AP-1 recognition element at -1602 bp results in greater transcriptional activity [39
]. However, we found that, starting from different “basal” levels, STAT3 activation profoundly enhanced transcription from both variants of the MMP-1 promoter in colon carcinoma cells (data not shown). The relative importance of AP-1- and STAT-related signaling pathways in the control of MMP-1 expression apparently depends on the cell type. For instance, a specific inhibitor of JAK3, a kinase operative in the activation of STATs, almost completely inhibited MMP-1 mRNA induction and protein abundance in human chondrocytes. In contrast, inhibition of ERK1/2 kinases signaling via AP-1 had little effect on the expression of MMP-1 in these cells [62
]. We conclude that, along with its importance in CRC proliferation and transformation, the STAT3 pathway is one driving force responsible for overexpression and activity of MMP-1 in invasive colonic cancer cells. The prognostic significance of MMP-1 expression in colorectal cancer has already been reported. High levels of MMP-1 expression have been correlated with metastatic spread of tumors and poor prognosis of colorectal cancer [63–66
]. Interestingly, MMP-1 inhibitors such as batimastat blocked peritoneal carcinomatosis and liver metastasis development in experimental colon carcinoma [67
]. Of all the proteases addressed in study, MMP-1 is one whose expression level is particularly linked with STAT3 activation in colorectal cancer. There are, however, other proteases whose expression level was also found to follow the degree of STAT3 phosphorylation during the course of this study. Induction of MMP-7 (matrilysin) expression in prostate carcinoma cells by fibroblast growth factor was shown to involve STAT3 through a probable interaction with STAT binding sites within the matrilysin promoter [26
]. A recent study reported a correlation between tyrosinephosphorylated STAT3 and expression of MMP-9 in breast carcinoma cells and demonstrated a direct influence of STAT3 on MMP-9 promoter activity [27
The enzymatic activity of MMPs is specifically inhibited by TIMPs, and high levels of TIMP-1 and TIMP-2 are associated with aggressive cancers [62
]. STAT3 has been described as contributing to the downregulation of TIMP-1 expression in synovial lining cells [22
]. We recently found that STAT3 activation through leukemia inhibitory factor enhances invasiveness and coincides with a decrease of TIMP-1 expression in choriocarcinoma cells [44
]. Interestingly, in the course of this study we observed that TIMP-1 mRNA was significantly less abundant in colorectal cancer biopsies with high STAT3 activity (data not shown).
In conclusion, STAT3 activation can contribute to the malignancy of many different types of cancer. One such contribution involves the promotion of cell invasiveness through (cell type specific) alteration in the pattern of protease activity. Our data suggest that STAT3-controlled proteolysis via MMPs is a major determinant of local tumor progression and metastasis of colorectal cancer and, thus, is an attractive potential target for therapeutic intervention. Moreover, p-Y-STAT3 and MMP-1 are likely to be of prognostic value or be of diagnostic importance with regard to CRC progression.
Recent reports point to an important role of aberrant STAT3 activity in metastasis of various malignant tumor entities such as cutaneous squamous cell carcinoma [68
], melanoma [69
], or hepatocellular carcinoma [70
]. To investigate the underlying molecular mechanisms, our future work will include the analysis of patient CRC biopsy samples and will take advantage of mouse models that closely mimic stages of human CRC.