Colorectal carcinoma (CRC) is a major cause of morbidity and mortality in Western countries. It has so far been molecularly defined mainly by alterations of the Wnt pathway. We show here for the first time that aberrant activities of the signal transducer and activator of transcription STAT3 actively contribute to this malignancy and, thus, are a potential therapeutic target for CRC. Constitutive STAT3 activity was found to be abundant in dedifferentiated cancer cells and infiltrating lymphocytes of CRC samples, but not in non-neoplastic colon epithelium. Cell lines derived from malignant colorectal tumors lost persistent STAT3 activity in culture. However, implantation of colon carcinoma cells into nude mice resulted in restoration of STAT3 activity, suggesting a role of an extracellular stimulus within the tumor microenvironment as a trigger for STAT activation. STAT3 activity in CRC cells triggered through interleukin-6 or through a constitutively active STAT3 mutant promoted cancer cell multiplication, whereas STAT3 inhibition through a dominant-negative variant impaired IL-6-driven proliferation. Blockade of STAT3 activation in CRC-derived xenograft tumors slowed down their development, arguing for a contribution of STAT3 to colorectal tumor growth.
Signal transducer and activator of transcription 3 (STAT3) is aberrantly activated in colorectal carcinomas (CRCs). Here, we define the relationship between STAT3 function and the malignant properties of colon carcinoma cells. Elevated activation of STAT3 enhances invasive growth of the CRC cell lines. To address mechanisms through which STAT3 influences invasiveness, the protease mRNA expression pattern of CRC biopsies was analyzed and correlated with the STAT3 activity status. These studies revealed a striking coincidence of STAT3 activation and strong expression of matrix metalloproteinases MMP-1, -3, -7, and -9. Immunohistological examination of CRC tumor specimens showed a clear colocalization of MMP-1 and activated STAT3. Experimentally induced STAT3 activity in CRC cell lines enhanced both the level of MMP-1 mRNA and secreted MMP-1 enzymatic activity. A direct connection of STAT3 activity and transcription from the MMP-1 promoter was shown by reporter gene experiments. Moreover, high-affinity binding of STAT3 to STAT recognition elements in both the MMP-1 and MMP-3 promoter was demonstrated. Xenograft tumors arising from implantation of CRC cells into nude mice showed simultaneous appearance and colocalization of p-Y-STAT3 and MMP-1 expression. Our results link aberrant activity of STAT3 in CRC to malignant tumor progression through upregulated expression of MMPs.
Aberrant activation of STAT3 in colorectal carcinoma (CRC) tissue is correlated with elevated expression of matrix metalloproteinase-1 (MMP-1). We analyzed transcriptional regulation of the human MMP-1 promoter in CRC cells by tyrosine phosphorylated (pY-) STAT3. One of six putative STAT binding elements within a 4.3 kb MMP-1 trancriptional promoter fragment showed a particular high affinity for STAT3 in vitro. However, the most profound regulatory influence on MMP-1 promoter activity resides in a proximal region relative to the transcriptional start, bearing a pair of putative binding sites for STAT3 and AP-1. Mutational analysis of the combined STAT3/AP-1 recognition element revealed that the integrity of the STAT3 binding site is necessary, but not sufficient for both DNA interaction and transcriptional regulation by activated STAT3. Instead, the adjacent AP-1 site was essential for pY-STAT3-mediated transcription on the MMP-1 promoter. DNA-protein binding assays provided strong evidence for complex formation of STAT3 and c-Jun governed by protein-protein contacts. We observed striking coincidence for concerted aberrant activation of both STAT3 and AP-1 in human colon cancer specimens. This finding supports the notion that the combination of inappropriate STAT3 and AP-1 activities drives elevated MMP-1 expression and tissue invasion in colorectal cancer and is of clinical relevance.
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