We assessed the functional significance of tumor cell-associated matrix metalloproteinase (MMP)-2 in extracellular matrix remodeling compared with that of the soluble enzyme by evaluating the contraction of three-dimensional collagen lattices by human glioma U251.3 and fibrosarcoma HT-1080 cell lines. In this model, the constitutive synthesis and activation of the MMP-2 proenzyme were modulated by stable transfections of tumor cells with cDNA encoding membrane type 1-MMP (MT1-MMP). The efficiency of transfected cells in contracting collagen lattices was shown to be dependent on the MT1-MMP-mediated activation of MMP-2 accompanied by cell surface association of activated MMP-2, on the cell-matrix interactions controlled by collagen-specific integrins, and on the integrity of actin and microtubule cytoskeletons. Each one of these mechanisms was essential but was not sufficient by itself in accomplishing gel contraction by MT1-MMP-transfected cells. Both MMP-2 activation and gel contraction by transfected glioma cells were inhibited by tissue inhibitor of metalloproteinase (TIMP)-2 and the recombinant COOH-terminal domain of MMP-2. However, the kinetics and mechanisms of their inhibitory effects were different, because TIMP-2 and the COOH-terminal domain of MMP-2 preferentially inhibited the MT1-MMP-dependent and autocatalytic steps of MMP-2 activation, respectively. By contrast, TIMP-1, an efficient inhibitor of soluble MMP-2 activity, failed to affect gel contraction. In addition, soluble MMP-2 activated by either organomercurials or cells was not able to induce the contraction of collagen lattices when added to transfected cells. Therefore, soluble activated MMP-2, sensitive to TIMP-1 inhibition, does not mediate collagen gel contraction by tumor cells, whereas the activity of cell surface-associated MMP-2 plays a critical role in remodeling of the extracellular matrix in vitro. These mechanisms of functional and spatial regulation of MMP-2 may also be applicable to different aspects of tissue reorganization in vivo, including cell migration and invasion, angiogenesis, and wound healing.

Stable transfection of U251.3 glioma cells with cDNA encoding MT-MMP-1 resulted in increased cell surface expression of MT-MMP-1 and TIMP-2, constitutive activation of MMP-2 proenzyme and increased collagen degradation. In tumor spheroid outgrowth assays, cell migration of MT-MMP-1 transfectants relative to control was enhanced on collagen and decreased on vitronectin and fibronectin. These effects were reversed by TIMP-2 and were not associated with any substantial changes in cell adhesion. Binding of U251.3 cells to the C-terminal domain of MMP-2 was specifically inhibited by anti-(alpha)vss3 integrin blocking antibody indicating that MMP-2 interacts with (alpha)vss3 through the enzyme's C-terminal portion at or near the integrin's matrix adhesion sites. We propose that these mechanisms could govern directed matrix degradation in the tumor cells' microenvironment by sequestration of active MMP-2 on the cell surface. Our data suggest that activation of MMP-2 and its proteolytic activity localized to the cell surface could differentially modulate tumor cell migration in response to particular matrix proteins by altering both composition of the extracellular matrix and expression of adhesion receptors on the cell surface.

The invasion-promoting MT1-MMP is a cell surface-associated collagenase with a plethora of critical cellular functions. There is a consensus that MT1-MMP is a key protease in aberrant pericellular proteolysis in migrating cancer cells and, accordingly, a promising drug target. Because of high homology in the MMP family and a limited success in the design of selective small-molecule inhibitors, it became evident that the inhibitor specificity is required for selective and successful MT1-MMP therapies. Using the human Fab antibody library (over 1.25×109 individual variants) that exhibited the extended, 23-27 residue long, VH CDR-H3 segments, we isolated a panel of the inhibitory antibody fragments, from which the 3A2 Fab outperformed others as a specific and potent, low nanomolar range, inhibitor of MT1-MMP. Here, we report the in-depth characterization of the 3A2 antibody. Our multiple in vitro and cell-based tests and assays, and extensive structural modeling of the antibody/protease interactions suggest that the antibody epitope involves the residues proximal to the protease catalytic site and that, in contrast with tissue inhibitor-2 of MMPs (TIMP-2), the 3A2 Fab inactivates the protease functionality by binding to the catalytic domain outside the active site cavity. In agreement with the studies in metastasis by others, our animal studies in acute pulmonary melanoma metastasis support a key role of MT1-MMP in metastatic process. Conversely, the selective anti-MT1-MMP monotherapy significantly alleviated melanoma metastatic burden. It is likely that further affinity maturation of the 3A2 Fab will result in the lead inhibitor and a proof-of-concept for MT1-MMP targeting in metastatic cancers.