By mining DNA microarray data bases at GenBank TM , we identified up-regulation of membrane type 1 matrix metalloproteinase (MT1-MMP) in human primary and metastatic prostate cancer specimens as compared with nonmalignant prostate tissues. To explore the role of up-regulated MT1-MMP in early stage cancer progression, we have employed a three-dimensional cell culture model. Minimally invasive human prostate cancer cells (LNCaP) were transfected with MT1-green fluorescent protein (GFP) chimeric cDNA as compared with GFP cDNA, and morphologic and phenotypic changes were characterized. GFP-expressing LNCaP cells formed multicellular spheroids with cuboidal-like epithelial morphology, whereas MT1-GFP-expressing cells displayed a fibroblast-like morphology and a scattered growth pattern in type I collagen gels. Cell morphologic changes were accompanied by decreased epithelial markers and enhanced mesenchymal markers, consistent with epithelial-to-mesenchymal transition. MT1-MMP-induced morphologic change and cell scattering were abrogated by target inhibition of either the catalytic domain or the hemopexin domain. We further demonstrated that MT1-MMP-induced phenotypic changes were dependent upon up-regulation of Wnt5a, which has been implicated in epithelial-to-mesenchymal transition. We conclude that MT1-MMP plays an important role in early cancer dissemination by converting epithelial cells to migratory mesenchymal-like cells.Most human cancers are epithelial in origin. Carcinoma progression is often accompanied by the loss of an epithelial phenotype and the acquisition of a fibroblastic or mesenchymal phenotype (epithelial-to-mesenchymal transition (EMT) 2 ).This transition has emerged as a critical step in the conversion of early stage cancer to invasive and metastatic cancer (1, 2). Turning an epithelial cell into a mesenchymal cell requires alterations in morphology, cellular architecture, adhesion, and migration. A molecular hallmark of EMT is the decrease or loss of expression of the adherens junction protein, E-cadherin, resulting in loss of cell-cell association and change of cell morphology. Decreased levels of E-cadherin and cytokeratins and acquisition of mesenchymal proteins like fibronectin and vimentin are indicative of a switch toward a mesenchymal dedifferentiated phenotype; these phenotypic changes result in enhanced cell motility and invasiveness (3). Enhanced production and activation of matrix metalloproteinases (MMPs), especially membrane type 1 MMP (MT1-MMP), have been described in most types of carcinoma, including commonly occurring prostate and breast cancer (4). High levels of MMPs in cancer tissues have been correlated with poor prognosis. MMPs have been linked with EMT through both autocrine and/or paracrine pathways (5). Secreted MMPs (e.g. MMP-2, -3, -9, and -28) have been associated with cancer cell EMT through various mechanisms (6 -8). Although MT1-MMP is capable of cleaving E-cadherin in transfected breast cancer cells (9), the effect of this cleavage on EMT has not been characte...

Substrate degradation and cell migration are key steps in cancer metastasis. Membrane-type 1-matrix metalloproteinase (MT1-MMP) has been linked with these processes. Using the fluorescein isothiocyanate (FITC)-labeled fibronectin degradation assay combined with the phagokinetic cell migration assay, structurefunction relationships of MT1-MMP were studied. Our data indicate that MT1-MMP initiates substrate degradation and enhances cell migration; cell migration occurs as a concurrent but independent event. Using recombinant DNA approaches, we demonstrated that the hemopexin-like domain and a nonenzymatic component of the catalytic domain of MT1-MMP are essential for MT1-MMP-mediated cell migration. Because the cytoplasmic domain of MT1-MMP was not required for MT1-MMP-mediated fibronectin degradation and cell migration, it is proposed that cross-talk between the hemopexin domain of MT1-MMP and adjacent cell surface molecules is responsible for outside-in signaling. Employing cDNAs encoding dominant negative mutations, we demonstrated that Rac1 participates in the MT1-MMP signal transduction pathway. These data demonstrated that each domain of MT1-MMP plays a distinct role in substrate degradation and cell migration.Cell migration and invasion are critical coordinated events in the cancer dissemination process (1, 2). Cell migration involves the locomotion of a cell over an extracellular matrix (ECM) 1 substratum (3). Extension of the leading edge is associated with adhesion, i.e. binding of integrins to their ECM ligands leading to subsequent migration and further invasion (4). Cancer cell invasion requires degradation of surrounding ECM and basement membrane by proteinases located at the leading edge of migrating cells. Extracellular proteolytic enzymes, i.e. matrix metalloproteinases (MMPs), serine and cysteine proteinases have long been implicated in cancer metastasis (1, 5).MMPs have been linked to the metastatic phenotype of tumor cells through both correlative and functional studies. Production and activation of MMPs in tumors are required for degradation of the ECM and dissemination of cancer cells to distant organs (2). MMPs also play an important role in tumor angiogenesis (6). The mechanism of activation of latent MMP-2 (pMMP-2) in tumors has been the focus of considerable recent interest based on the identification of a new category of intrinsic membrane-type MMPs (MT1, 2, 3, 4, 5, and 6-MMPs) (7).MT1-MMP is able to activate pMMP-2 on the surface of tumor cells by assembling a unique triplex with tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) and pMMP-2; a second MT1-MMP molecule then cleaves the propeptide of pMMP-2, thereby activating the enzyme at the cell surface (8 -10). Integrin receptors, ␣ 3 ␤ 1 and ␣ v ␤ 3 , participate in this response (11). Recombinant MT1-MMP hydrolyze collagen types I, II, and III and digests cartilage proteoglycan, fibronectin, fibrinogen, vitronectin, and laminin (12).It is now recognized that the actions of MMPs are not restricted to the simple breakdown of ECM...

Proprotein convertases play an important role in tumorigenesis and invasiveness. Here, we report that a dibasic amino acid convertase, furin, directly cleaves proMMP-2 within the trans-Golgi network leading to an inactive form of matrix metalloproteinase-2 (MMP-2). Co-transfection of COS-1 cells with both proMMP-2 and furin cDNAs resulted in the cleavage of the N-terminal propeptide of proMMP-2. The molecular mass of cleaved MMP-2 (63 kDa), detected in both cell lysates and conditioned medium, is between the intermediate and fully activated forms of MMP-2 induced by membrane type 1-MMP. Furin-cleaved MMP-2 does not possess proteolytic activity as examined in a cell-free assay. Treatment of transfected cells with a furin inhibitor resulted in a dose-dependent inhibition of proMMP-2 cleavage; recombinant tissue inhibitor of metalloproteinase-2, which binds to the active site of membrane type 1-MMP, had no inhibitory effect. Site-directed mutagenesis of amino acids in the furin consensus recognition motif of proMMP-2(R 69 KPR 72 2) prevented propeptide cleavage, thereby identifying the scissile bond and characterizing the basic amino acids required for cleavage. Other experimental observations were consistent with intracellular furin cleavage of proMMP-2 in the trans-Golgi network. The furin cleavage site in other proMMPs was examined. MMP-3, which contains the RXXR furin consensus sequence, was cleaved in furin co-transfected cells, whereas MMP-1, which lacks an RXXR consensus sequence, was not cleaved. In conclusion, we report the novel observation that furin can directly cleave the RXXR amino acid sequence in the propeptide domain of proMMP-2 leading to inactivation of the enzyme. Matrix metalloproteinases (MMPs)1 are an important family of zinc-and calcium-dependent proteinases that degrade extracellular matrix components and numerous other proteins. MMPs are implicated in physiological and pathological processes related to extracellular matrix turnover, including wound healing, angiogenesis, tumor invasion, and metastasis (1). MMP-2 (gelatinase A, 72-kDa type IV collagenase) appears to be especially important in tumor invasion and metastasis because of its ability to degrade basement membrane type IV collagen (2).All MMPs are synthesized as preproenzymes, and most of them are secreted from cells as proenzymes consisting of a propeptide, a catalytic domain, a hinge region, and a hemopexin-like domain; MMP-7, -23, -26, and membrane-type matrix metalloproteinases (MT-MMPs) are exceptions (1, 3). The zymogens of most MMPs are activated through a two-step activation mechanism. Activator proteinases, such as trypsin or plasmin or organomercurial chemical treatment, first attack the proteinase-susceptible "bait" region located in the middle of the propeptide domain (4, 5). This cleavage induces conformational changes in the propeptide and renders the final activation site to be readily cleaved by a second proteolysis. The latter reaction is usually an intermolecular autocatalytic event (6).In contrast to other secret...

MT1-MMP (membrane type 1 matrix metalloproteinase, or MMP-14) is a key enzyme in molecular carcinogenesis, tumour-cell growth, invasion and angiogenesis. Novel and potent MMP inhibitors with a mercaptosulphide zinc-binding functionality have been designed and synthesized, and tested against human MT1-MMP and other MMPs. Binding to the MT1-MMP active site was verified by the competitive-inhibition mechanism and stereochemical requirements. MT1-MMP preferred deep P1' substituents, such as homophenylalanine instead of phenylalanine. Novel inhibitors with a non-prime phthalimido substituent had K(i) values in the low-nanomolar range; the most potent of these inhibitors was tested and found to be stable against air-oxidation in calf serum for at least 2 days. To illustrate the molecular interactions of the inhibitor-enzyme complex, theoretical docking of the inhibitors into the active site of MT1-MMP and molecular minimization of the complex were performed. In addition to maintaining the substrate-specificity pocket (S1' site) van der Waals interactions, the P1' position side chain may be critical for the peptide-backbone hydrogen-bonding network. To test the inhibition of cell-mediated substrate cleavage, two human cancer-cell culture models were used. Two of the most potent inhibitors tested reached the target enzyme and effectively inhibited activation of proMMP-2 by endogenous MT1-MMP produced by HT1080 human fibrosarcoma cells, and blocked fibronectin degradation by prostate cancer LNCaP cells stably transfected with MT1-MMP. These results provide a model for mercaptosulphide inhibitor binding to MT1-MMP that may aid in the design of more potent and selective inhibitors for MT1-MMP.