HER2 Overexpression And Its Mediated Signaling Pathway Upregulate The Expression Of Matrix Metalloproteinase-7

Human epidermal growth factor receptor 2 (HER2), also known as ErbB2, belongs to the epidermal growth factor receptor (EGFR) family of highly regulated receptor tyrosine kinases (RTKs). This family is composed of human epidermal growth factor receptors 1, 2, 3, and 4 (EGFR, HER2, HER3, and HER4). HER2 overexpression is commonly found in various types of human cancers including breast, ovary, prostate, gastric, lung, bladder and kidney carcinomas as well as osteosarcomas and neuroblastomas. Clinical evidence suggests that HER2 overexpression is associated with drug resistance, metastases and poor prognosis. Heregulin (HRG) is a family of growth factors. It binds directly to membrane receptors HER3 and HER4. Messenger RNA transcripts of HRG were detected in about 25% of human mammary carcinomas, a percentage similar to HER2 overexpression. HRG expression also correlates with poor histological grade of human breast cancer biopsies. HRG can activate the Her2 receptor via Her2/Her3 or Her2/Her4 heterodimerization and the HER2 receptor has been demonstrated to play an essential role in the activation of HER signaling by HRG. However, the mechanism of HER2 and HRG overexpression in tumorigenesis, invasion and metastasis is not yet clear. It should be noted that two distinct breast cancer populations—one overexpressing HER2 with low levels of HRG, and the other overexpressing HRG but not HER2—appear to employ the same signaling pathways controlling breast cancer chemosensitivity. This suggests that HER2 and HRG cause tumorigenesis and poor prognosis of breast cancer by the same mechanism.Matrix metalloproteinase-7 (MMP-7) is overexpressed in a variety of epithelial tumors and mesenchymal tumors. MMP-7 plays roles not only in tumor progression, but also in the early stages of cancer development. It has been reported that MMP-7 sheds the ectodomain of membrane-bound FasL (mFasL) from tumor cells membranes and protects tumor cells from chemotherapeutic drug cytotoxicity. Previous studies have shown that HER2 levels were strongly correlated with the expression of MMP-7, and HER signaling may involve in the regulation of MMP protein secretion.In this study, breast cancer cell line MCF-7 stably overexpressing HER2 (MCF-7/HER2) was established. Increased promoter activity, mRNA and protein expressions of MMP-7 were observed in MCF-7/HER2. The expression of MMP-7 gene was further significantly enhanced by HRGβinduction, indicating that HER2 overexpression and HRGβstimulation co-operate to regulate MMP-7 expression. Both STAT3 and AP-1 function as the effectors in response to a variety of signals and play roles in tumor transformation, invasion and metastases. In examining the promoter sequence of MMP-7 gene, three putative STAT3 binding sites and an AP-1 site were found within the first 296 bp of the transcription start site in human MMP-7 promoter. Constitutively activated STAT3 and AP-1 increased promoter activity, mRNA and protein expressions of MMP-7 in MCF-7/STAT3C and MCF-7/c-Jun cells. ChIP-PCR and mutagenesis of MMP-7 promoter further confirmed that Stat3 and AP-1 bind to the elements of MMP-7 promoter in vivo. The STAT3 sites at -168/-159 and -137/-122 region of MMP-7 promoter were demonstrated to be functional STAT3 binding sites. AP-1 binding site was also required for the regulation of MMP-7 expression. It is indicated that MMP-7 is a potential target gene of STAT3 and AP-1.To illustrate the signal pathway by which MMP-7 expression is regulated, we investigated the phosphorylation of STAT3 and AP-1 by HRGβinduction in MCF-7/HER2 cells. The data demonstrated that HER2 overexpression enhanced phosphorylation of HER2 and STAT3, HRGβinduction further increased level of phosphorylated HER2 and STAT3 and the activation of c-Jun and the expression of c-Jun and c-Fos were associated with HER2/HRGβtreatment. It indicates that HER2/HRGβinduced STAT3 and AP-1 activation. By cotransfection with STAT3 and AP-1 dominant negative mutants, STAT3-YF and TAM67, and mutagenesis of STAT3 and AP-1 binding sites in MMP-7 promoter, we demonstrated that HER2/HRG promoted matrix metalloproteinase-7 expression via STAT3 and AP-1 activation. We also found that constitutively activated STAT3 did not induce AP-1 activation, and that TAM67 had no inhibitory effect on MMP-7 promoter activity, suggesting that HER2/HRGβupregulate MMP-7 expression through directly inducing STAT3 and AP-1 activition.Taken together, our findings demonstrate that HER2 overexpression and HRG synergistically upregulate MMP-7 expression; STAT3 and AP-1 regulate MMP-7 expression by directly binding to MMP-7 promoter; HER2/HRGβupregulate MMP-7 expression through inducing STAT3 and AP-1 activition. Our data suggest that HER2 overexpression and HRG stimulation activate STAT3 and AP-1, which in turn upregulate MMP-7 expression.