Fibronectin induces colon cancer MMP-2 activation through a Src- and NfκB-dependent mechanism

Abstract

Background. MMP-2 is a matrix metalloproteinase implicated in colon cancer invasion. MMP-2 is secreted in latent form (72 kDa) then cleaved into the active form (69 kDa) by membrane-bound type MMP (MT1-MMP). The signals regulating MMP-2 activation are poorly understood. Fibronectin is a major extracellular matrix component of cancer-associated stroma. Because integrin-mediated interactions with fibronectin are implicated to modulate cancer cell invasion, here we tested the hypothesis that fibronectin regulates MMP-2 activation through a Src- and NFκB-dependent signal. Methods. We tested three human colon cancer cell lines, SW480, HCT116, and HT29. Cells were seeded onto noncoated and fibronectin- or laminin-coated plates. MMP-2 activation was assessed by zymography and Western blotting. MT1-MMP expression, Src activation, and NFκB activation were assayed using Western blotting, immunoprecipitation, and EMSA, respectively. We treated cells with anti-integrin blocking antibody, PP2, or PDTC to inhibit cell attachment to fibronectin, Src, or NFκB, respectively. Results. Minimal NFκB activation was detected under baseline conditions in each of the three cell lines. Fibronectin, but not laminin, induced MT1-MMP, Src phosphorylation, and NFκB activation at 6 h in all three cell lines, with concomitant induction of active form of MMP-2. Anti-α4β1, but not α5β1 integrin blocking antibody (2.5 μg/ml) suppressed MT-MMP1 expression (by 84, 80, and 82% for SW480, HCT116, and HT29 respectively, P < 0.05). PP2 (20 μM), and PDTC (100 μM) suppressed Src activation (by 93, 91, and 88% for each cell line, P < 0.05) and NFκB activation (by 95, 92, and 88% for each cell line, P < 0.05), respectively, and they each abolished fibronectin-induced MT1-MMP expression as well as MMP-2 activation. Conclusion. These findings suggested that fibronectin induces MMP-2 activation by MT1-MMP through a α4β1 integrin, -Src-, and NFκB-dependent signal in colon cancer cells. This signaling pathway may represent a therapeutic target for strategies designed to inhibit colon cancer progression.