Increased pericellular proteolysis due to an imbalance between MMPs (matrix metalloproteinases) and TIMPs (tissue inhibitors of metalloproteinases) promotes early stages of tumorigenesis. We have reported that TIMP-1 down-regulation confers tumorigenicity on immortal Swiss 3T3 fibroblasts. In pursuit of the mechanism involved in this transformation, we asked whether MMP inhibitors modulate contact inhibition and cell adhesion, because the dysregulation of these events is essential for cellular transformation. Using both genetic and biochemical means, we demonstrate that MMP inhibitors regulate fibroblast cell adhesion. TIMP-1 down-regulated cells formed dense, multilayered colonies, suggesting a loss of contact inhibition. Recombinant TIMP-1 and synthetic MMP inhibitors (MMPi) restored normal cell contact and density of these cells in a dose-dependent manner. Consequently, the effect of MMPi on both cell-extracellular matrix (ECM) and cell-cell adhesion were investigated. Upon MMPi treatment, p125 FAK was redistributed, together with vinculin, to points of cell-ECM contact. Furthermore, phosphorylation of p125 FAK was restored to levels similar to that of wild type. In parallel, MMPi treatment increased cadherin levels and stabilized cadherin-mediated cell-cell contacts. Moreover, enhanced cadherin function was evident as increased calcium-dependent cell-cell aggregation and co-localization of cadherin and ␤-catenin at the cell membrane. We also obtained independent evidence of altered cadherin function using timp-1 ؊/؊ mouse embryonic fibroblasts. Our data provide provocative evidence that increased pericellular proteolysis impacts cell adhesion systems to offset normal contact inhibition, with subsequent effects on cell transformation and tumorigenesis. Matrix metalloproteinases (MMPs)1 and their tissue inhibitors (TIMPs) constitute a key system of pericellular proteolysis within the cell microenvironment. Our understanding of the role of this proteolytic system in cancer has evolved over the past decade. Initially linked to tumor invasion and metastasis, an MMP:TIMP imbalance is now thought to function in promoting early events of tumor development (1). The current emphasis is on identifying the mechanisms underlying these early effects. A better understanding of the relationship between MMP:TIMP activity and cell-extracellular matrix (ECM) and cell-cell communication is fundamental to this effort. We had reported that down-regulation of TIMP-1 expression caused an immortal fibroblast cell line to become tumorigenic (2). Extensive literature has since led to the knowledge that cancer involves a disrupted balance between MMPs and TIMPs. Both TIMPs and MMPs have been manipulated through genetic and biochemical approaches in tissue culture systems to demonstrate that, in general, TIMPs inhibit tumor cell invasion, angiogenesis, metastasis, and tumor formation (3-9), whereas MMPs promote these events (10 -13). Transgenic and knockout animals have further supported the role of this proteolytic system in early tumo...

To test if proteolysis is involved in tumor cell extravasation, we developed an in vitro model where tumor cells cross an endothelial monolayer cultured on a basement membrane. Using this model we classified the ability of the cells to transmigrate through the endothelial cell barrier onto the underlying matrix, and scored this invasion according to the stage of passage through the endothelium. Metalloproteinase inhibitors reduced tumor cell extravasation by at least 35%. Visualization of protease and cell adhesion molecules by confocal microscopy demonstrated the cell surface localization of MMP-2, MMP-9, MT1-MMP, furin, CD44 and αvβ3, during the process of transendothelial migration. By the addition of inhibitors and bio-modulators we assessed the functional requirement of the aforementioned molecules for efficient migration. Proteolytic digestion occurred at the cell-matrix interface and was most evident during the migratory stage. All of the inhibitors and biomodulators affected the transition of the tumor cells into the migratory stage, highlighting the most prevalent use of proteolysis at this particular step of tumor cell extravasation. These data suggest that a proteolytic interface operates at the tumor cell surface within the tumor-endothelial cell microenvironment.