Extracellular matrix metalloproteinase inducer (EMMPRIN), a glycoprotein present on the cancer cell plasma membrane, enhances fibroblast synthesis of matrix metalloproteinases (MMPs). The demonstration that peritumoral fibroblasts synthesize most of the MMPs in human tumors rather than the cancer cells themselves has ignited interest in the role of EMMPRIN in tumor dissemination. In this report we have demonstrated a role for EMMPRIN in cancer progression. Human MDA-MB-436 breast cancer cells, which are tumorigenic but slow growing in vivo, were transfected with EMMPRIN cDNA and injected orthotopically into mammary tissue of female NCr nu/nu mice. Green fluorescent protein was used to visualize metastases. In three experiments, breast cancer cell clones transfected with EMMPRIN cDNA were considerably more tumorigenic and invasive than plasmid-transfected cancer cells. Increased gelatinase A and gelatinase B expression (demonstrated by in situ hybridization and gelatin substrate zymography) was demonstrated in EMMPRIN-enhanced tumors. In contrast to de novo breast cancers in humans, human tumors transplanted into mice elicited minimal stromal or inflammatory cell reactions. Based on these experimental studies and our previous demonstration that EMMPRIN is prominently displayed in human cancer tissue, we propose that EMMPRIN plays an important role in cancer progression by increasing synthesis of MMPs.

The balance between production and activation of MMPs and their inhibition by TIMPs is a crucial aspect of cancer invasion and metastasis. On the basis of the concept that MMPs synthesized in tissues seep into the bloodstream, we have examined MMP levels in the plasma of patients with cancer. In colorectal, breast, prostate, and bladder cancer, most patients with aggressive disease have increased plasma levels of gelatinase B. In patients with advanced colorectal cancer, high levels of either gelatinase B or TIMP complex were associated with shortened survival. We propose that these assays may be clinically useful in characterizing metastatic potential in selected kinds of cancer. In rheumatoid arthritis and systemic lupus erythematosus (SLE), serum and plasma levels of stromelysin-1 were approximately 3-5-fold increased. Fluctuating serum stromelysin-1 levels in SLE did not correspond with change in disease activity. In SLE, stromelysin-1 may be a component of the chronic tissue repair process rather than being responsible for inciting tissue damage. On the basis of these observations, we conclude that measurement of plasma/serum MMP and TIMP levels may provide important data for selecting and following patients considered for treatment with drugs that interfere with MMP activity.

SUMMARY: Pericellular matrix degradation during cancer invasion and inflammation is dependent on activation of progelatinaseA by membrane type 1-matrix metalloproteinase (MT1-MMP); a stoichiometric concentration of tissue inhibitor of metalloproteinase-2 (TIMP-2) is required. Activation of progelatinase A has generally been considered to be a slow process occurring as a result of enhanced expression of MT1-MMP. We herein report that ConA treatment of HT1080 fibrosarcoma cells is followed by MT1-MMP-induced activation of progelatinase A on the cell surface within 1 hour. Cell surface biotinylation, immunohistochemistry, and 125 I-labeled TIMP-2 binding to cell surface MT1-MMP were used to characterize the appearance and function of MT1-MMP on the plasma membrane. Treatment of HT1080 cells with ConA resulted in increased specific binding of 125 I-labeled TIMP-2 to cell surface receptors within 5 minutes. TIMP-2 binds almost exclusively to activated MT1-MMP on the surface of HT1080 cells. MT1-MMP function at the cell surface was also accelerated by treatment of cells with cytochalasin D, an inhibitor of actin filaments, PMA, a stimulator of protein kinase C, and bafilomycin A 1 , an inhibitor of lysosome/endosome function. A functional pool of intracellular MT1-MMP available for trafficking to the cell surface was demonstrated by repetitive ConA stimulation. ConA-induced expression of MT1-MMP mRNA (Northern blot analysis) in HT1080 cells was a delayed event (Ͼ6 hours). These data suggest that presynthesized MT1-MMP is sorted to a transient storage compartment (trans-Golgi network/endosomes), where it is available for rapid trafficking to the plasma membrane and cell surface proteolytic activity. (Lab Invest 2002, 82:1673-1684.

Membrane-type matrix metalloproteinases (MT-MMPs) 1 are a newly described family of MMPs (1) that are distinguished by their localization to the plasma membranes of cells by a stretch of hydrophobic amino acids (transmembrane domain) (2). MTMMPs have been the subject of intense interest because of their role in activating a secreted MMP, progelatinase A, at the cell surface (1) and in directly cleaving collagen and other extracellular matrix proteins (3). Because activation of all soluble latent MMPs described to date is accompanied by cleavage of the N-terminal propeptide, thereby exposing the active site zinc by dissociation from the conserved cysteine in the prodomain, the assumption has been made that an analogous mechanism is responsible for the activation of MT1-MMP on the cell surface.Utilizing recombinant wild-type (wt) MT1-MMP cDNA and mutant MT1-MMP cDNAs transfected into cells lacking endogenous MT1-MMP, we have recently examined the function of the N-terminal propeptide domain of membrane-bound MT1-MMP. Contrary to expectation, we demonstrated that the Nterminal prodomain of wt MT1-MMP is required for function of the intact membrane-bound enzyme in COS-1 cells. Transfected COS-1 cells containing a deletion of the N-terminal propeptide domain of MT1-MMP or a chimeric construction in which the prodomain of MT1-MMP is substituted by the Nterminal propeptide domain of collagenase-3 were functionally inactive in terms of binding 125 I-labeled TIMP-2 to the cell surface and in initiating the activation of progelatinase A (4). These data led us to hypothesize that in its native plasma membrane-inserted form, the prodomain of MT1-MMP serves to facilitate the function of MT1-MMP as a receptor for TIMP-2 that subsequently immobilizes progelatinase A, thereby forming a trimolecular complex on the cell surface. A second free MT1-MMP molecule is then required to cleave the prodomain leading to activation of membrane-bound progelatinase A. The presence of excess TIMP-2 saturates all available MT1-MMP molecules, thereby interfering with progelatinase A activation. We proposed that conformational effects induced by the plasma membrane provide functional activity to latent cell surfacebound MT1-MMP without cleavage of the molecule (4, 5). It needs to be emphasized that our understanding of the importance of the propeptide domain of MT1-MMP was possible only because COS-1 cells are defective in proteolytic processing of certain newly synthesized proteins (6). Even following co-transfection of furin cDNA and MT1-MMP cDNA, COS-1 cells do not process latent MT1-MMP (63 kDa) to mature MT1-MMP (58 kDa) (7). In contrast, both the latent and mature forms of MT1-MMP are prominently displayed in several types of nontransfected cells capable of inducing progelatinase A activation (5, 8, 9); hence it was not previously possible to distinguish between the function of native versus mature enzyme.The goal of this report is to further clarify the role of the propeptide domain of MT1-MMP in maintaining the function of the plasma memb...

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