New Functions of Stromal Proteases and Their Inhibitors in Tumor Progression

CANCER METASTASIS: BIOLOGICAL AND CLINICAL ASPECTS

NEW FUNCTIONS OF STROMAL PROTEASES AND THEIR INHIBITORS IN TUMOR PROGRESSION Agnes Noel, PhD, Valerie Albert, MD, Khalid Bajou, MSc, Christele Bisson, MSc, Laetitia Devy, PhD, Francis Frankenne, PhD, Erik Maquoi, PhD, Veronique Masson, MD, Nor-Eddine Sounni, MSc, and Jean Michel Foidart, MD, PhD

Degradation and remodeling of the extracellular matrix (ECM)is associated with tumor cell migration, invasion, and metastasis. Such degradation occurs at several stages of the metastatic cascade including angiogenesis, local invasion, and extravasation. Because multiple ECM components exist, a number of different proteases are likely to be required to complete the metastatic sequence. These proteases can be divided into different classes based on their catalytic mechanism: serine, cysteine, aspartic, and matrix metalloproteases (MMPs).2,5267,92 They are subject to strict regulatory mechanisms including controlling their synthesis, secretion, and catalytic activity and to the action of specific natural inhibitors. Protease expression has been studied extensively in many different types of human cancers. High concentrations in components of these proteolytic systems have been associated with poor patient out~ome.~, 26, 52,87 Several proteolytic enzymes may be operational simultaneously in a tumor and act in a cascade. A functional overlap of substrate specificity may exist This work was supported by grants from the Fonds National de la Recherche Scientifique, the Federation Belge Contre le Cancer, the Centre Anticancereux pres l'Universit6 de Liege, the CGER-Assurances, the Fondation Leon Fredericq (University of Liege) and the Fonds d'Investissements de la Recherche Scientifique (CHU, Liege, Belgium).AN is a Senior Research Associate from the National Fund for ScientificResearch (FNRS, Brussels, Belgium). KB, VM, and VA are recipients of a grant from FNRS-Televie.

From the Laboratory of Tumor and Developmental Biology, University of Liege, Tour de Pathologie, Sart-Tilman, Liege, Belgium

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between the different proteolytic pathways. Accordingly, no defined phenotype has been reported in most mice deficient in proteases suggesting that they substitute each other.17,60, 96 The proteases primarily involved in ECM degradation, tumor invasion, and metastasis are thought to be the plasminogen system and MMPs. These enzymes are produced mainly by stromal cells in cancer as previ. ~ ~ article focuses on stromal proteases with a special ously d e s ~ r i b e dThis emphasis on the emerging roles of their inhibitors, tissue inhibitors of metalloproteases (TIMPs)and plasminogen activator inhibitor type 1(PAI-1). THE PLASMINOGEN SYSTEM

The plasminogen system is composed of an inactive proenzyme plasminogen (Plg)that can be converted to plasmin by either of two plasminogen activators (PA): urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators, which are serine p r o t e i n a s e ~ . ~Their , ~ ~ ,activity ~~ is controlled by plasminogen activator inhibitors, PAI-1, and PAI-2 (plasminogen activator inhibitor-type 1 and type 2) belonging to the serine proteinase inhibitor (serpin) family. Plasmin catalyzes the degradation of a variety of matrix glycoproteins (laminin, fibronectin), proteoglycans, and fibrin and activates other proteases, such as proMMPs. Furthermore, plasmin probably also has functions unrelated to matrix degradation, such as activation or release of growth factors from the extracellular matrix including latent transforming growth factor /3 (TGFB), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF).82 While sharing a common plasminogen converting function, the two types of plasminogen activators have distinct structural and functional features. Briefly, t-PA is thought to act malnly as a fibrin-dependent and intravascular activation enzyme that is involved primarily in clot dissolution. In contrast, u-PA operates as a fibrin-independent, largely receptorbound plasminogen activator. u-PA is released from cells as an inactive proenzyme and binds a specific, high affinity, glycosylphosphatidylinosito1 (GP1)-linkedcell-surfacereceptor (the urokinase receptor or u-PAR).'l,'' Pro-u-PA can be converted to active u-PA by plasmin when it is receptorbound and receptor-bound u-PA efficiently activates plasminogen. Concomittant binding of pro-u-PA to u-PAR and of plasminogen to a cell surface binding site enhances plasminogen activation directing plasmin activity to a cell s~rface.'~ The most abundant fast-acting inhibitor of u-PA in vivo is believed to be PAI-1, a 52 kDa single chain glycoprotein secreted in an active but conformationally unstable form. Its inhibitory activity is stabilized and prolonged by binding to the matrix glycoprotein, vitronectin (for review see references 54 and 64). PAI-1 specificallybinds to free u-PA and receptor-bound u-PA. The u-PA/PAI-1 complex is internalized and degraded in lysosomes through interaction with transmembrane a2-macroglobulinreceptor/LDL-receptor related protein (LRP), an endocytic r e c e p t ~ r . ~Thereafter, ',~~ u-PAR is recycled back to the cell surface. (For a comprehensive review of this system of proteolysis, please see the article by Rabbani et a1 elsewhere in this issue.)

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MATRIX METALLOPROTEASES

The MMPs form a family of structurally and functionally related zinc endopeptidases. Collectively, they are capable of degrading all kinds of ECM proteins, such as interstitial and basement membrane collagens, proteoglycans, fibronectin, laminin, and non-ECM proteins. MMPs are produced as pro-enzymes (zymogen) and must undergo proteolytic cleavage of the NH,-terminal domain to become catalytically active (for review see reference 67 and the article by Stetler-Stevenson elsewhere in this issue).61According to their structural and functional properties, the members of the MMP family can be separated into five subgroups: (1) collagenases (MMP-1,8,13); (2) gelatinases (MMP-2 and 9); (3) stromelysins and stromelysin-like enzymes (MMP-3, -7, -10, and -12); (4) membranetype MMPs, MT1-6 MMPs (MMP-14 to -17); and (5) the other MMPs forming a more heterogeneous subgroup including MMP-18, enamalysin (MMP-20), and MMP-19.12,45,53,74,93 In recent years, the number of known MMPs has grown rapidly, mainly because of the discovery of a series of membrane-bound enzymes belonging to the MT-MMP s ~ b f a m i l y . ~ ~ , ~ ~ The recent discovery of these membrane-associated MMPs has strengthened the concept of pericellular activation cascade mechanisms for the MMPS.~ In~addition to its ability to activate proMMP-2, MT1-MMP can directly cleave fibrillar collagen, laminin 5, and fibrin.24,4z49 MMPs are regulated by physiologic inhibitors of which four types are now recognized (TIMP-14). They differ in structure, biochemical properties, and in expression in vitro and in vivo, suggesting that each TIMP may have a specific physiologic role.40TIMPs inhibit the MMP activity by forming tight 1:l stoichiometric noncovalent complexes with activated enzymes. With one exception, MT1-MMP, which is well inhibited by TIMP-2 and TIMP-3, but not by TIMP-1; all members of the TIMP family have a similar inhibitory function toward all MMPs.27*31,100 TIMPs have the ability to form complexes with some proMMPs: TIMP-1 with proMMP-9, and TIMP-2 or TIMP-4 with proMMP-2. In this way, TIMPs control the activation of these latent MMPs. In addition to their antimetalloprotease activity, TIMPs appear to be multifunctional molecules involved in the control of cell migration and apoptosis (see later). PERICELLULAR PROTEOLYSIS: COOPERATION BETWEEN MATRIX METALLOPROTEASES AND SERINE PROTEASES

Plasminogen activation, occurring at the cell surface, is a cascadelike process leading to the formation of plasmin that can in turn activate some but not all proMMPs. Plasmin can activate proMMP-3, and thereafter, in cooperation with this MMP, plasmin activates collagenases (MMP-1, MMP-13), MMP-7, and MMP-9, resulting in a cascade of activation (Fig. 1). Although this concept that plasmin is a physiologic activator of proMMPs was based initially on in vitro data, it was confirmed by in vivo observations.'' Unlike most MMPs, MMP-2 cannot be activated directlyby plasmin.

Figure 1. Matrix metalloprotease (MMP) activation cascade. Two cell-associated proteolysis pathways initiate MMP activation. Plasmin generated by the activity of receptor bound urokinase-type plasminogen activator (uPARluPA) is a key activator of MMPs, notably MMP-1, MMP-3, and MMP-9. Active cell sulface MT-MMPs are activators of MMP-2. MT1-MMP can also activate MMP-13. MMP-2, MMP-3, and MMP-13 can activate MMP9. MMP13 also can be activated by MMP-2 and MMP-3. Dashed arrows correspond to conversion of inactive enzyme to active enzyme. The solid arrows define the activation process.

MTI-MMP Five MT-MMPs have been shown to activate can also activate proMMP-13, which is an activator for proMMP-9. Thus, regulated pericellular proteolysis is not restricted to the u-PA system because MT-MMPs are involved also in cell surface amplification of proteolytic activity (see Fig. 1). Degradation of ECM components represents a second level of cooperation between MMPs and serine proteases. Although a certain degree of overlap in substrate specificity exists between MMPs and serine proteases, these two proteolytic systems mostly display different substrate affinity and can act in synergy. The action of one enzyme can make the substrate more available for the degradation by another protease. For instance, plasmin-mediated degradation of glycoproteins that cover collagen fibers rendering the collagen accessible to the action of M M P s . ~ ~ STROMAL PROTEASES AND CANCER

Data supporting the role of proteases in cancer progression derive from in vitro and in vivo experiments demonstrating: (1) a correlationbetween protease expression and cell invasion and metastasis; (2) a modulation of the invasive properties by cell transfection with the cDNA of

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proteases or their inhibitors; (3)a reduction of tumor growth and metastatic potential by using natural or synthetic protease inhibitors, neutralizing antibodies, or antisense oligonucleotides (for review see references 17,25, 45, and 67). Many studies have shown that levels of proteases, their receptors or inhibitors in malignant tumors are related to the outcome of the disease (for review, see references 2,81,87, and 90). Additional direct evidence for the role of protease activity in tumor growth and invasion has been provided by the recent generation of mice deficient for distinct pro tease^.'^^^^,^^ The reduction of tumorigenesis was observed in MMP-11 and in MMP-7knock-out mice in response to chemical m~tagenesis~~ deficient mice cross-bred with multiple intestinal neoplasia (Min) mice that develop spontaneous intestinal car~inornas.'~' MMP-2-deficient mice and the number showed reduced angiogenesis and tumor progre~sion,4~ of metastases was markedly reduced in plasminogen -1- mice.14 An important issue that was controversial for a long time is the source of proteases expressed in tumors. It is now well accepted that in most tumors, stromal cells are the main source of proteases (Fig. 2). Increased expression of MMP-1 was observed in different cancer types, and in all these tumors, the most abundant expression of this MMP was observed

Figure 2. Cooperation between host cells and tumor cells for protease production in human carcinomas. (1) Proteases secreted by host cells (fibroblasts, inflammatory cells, or endothelial cells) may be activated by cell surface molecule (rectangle) (MT1-MMP or u-PAR) expressed by tumor cells or host cells. (2) Other MMPs secreted by host cells may be activated extracellularly or intracellularly (as for MMP-11 produced exclusively by fibroblasts). (3) Some MMPs are produced by tumor cells. For instance, MMP-7 is exclusively expressed by carcinoma cells. (4) The protease inhibitors (TIMPs and PAI-1) appeared to be produced mainly by host cells.

in stromal cells.99MMP-11 mRNA and protein were found specifically in fibroblastic cells immediately surrounding carcinoma ~ e l l s . ~MMP",~~ 2 mRNA was detected in stromal cells while the protein was found in the carcinoma cells.74,77, 79 This discrepancy may be explained by the binding of MMP-2 to cell surface molecules, such as the MT1-MMP, the a,b3 integ~-in,'~ or to a yet unidentified receptor.33Such cooperation between tumor cells and fibroblasts has been reported also for u-PA produced by tumor cells, which binds to u-PAR expressed at the tumor cell surface.36The recent finding that MT1-MMP is expressed by fibroblasts suggests that it binds and activates MMP-2 at the cell surface of f i b r ~ b l a s t s . ~ "62,, ~ 74,~ 78, Infiltra~~, tion of inflammatory cells or endothelial cells, a prominent feature of many malignant tumors, provides another source of MMPs (MMP-9, MMP-12)84 or u-PA.~~ It has been proposed that the tumor stroma is required for growth of most carcinoma^.^" First evidence for the interplay between tumor cells and the stroma has been provided from findings that in the early stages of epithelial malignancy, angiogenesis is observed in the stroma. Stromal fibroblasts have been shown to cause the tumorigenic conversion of epithelial ~ e l l s .The ~ , ~ability ~ of tumor cells to grow locally and to metastasize can be affected by the presence of fibroblast^.'^,^^,^^ An important cross talk between cancer cells and fibroblasts participates in the remodelling of the extracellular matrix observed in most ~ a r c i n o m a sThus, .~~ stromal cells can actively express extracellular matrix components, supply the neovascularization, secrete growth factors, and contribute to tumor growth. More recent data support the assumption that stromal cells play a key role in promoting cancer progression by secreting proteases or their inhibitors. As mentioned previously, two transplanted tumors (Lewis lung carcinoma and B16 melanoma) showed significantly reduced metastasis to the lungs of MMP-2 -1- mice.43As the cancer cells themselves have a fully functional MMP-2 gene, this observation points to an important role for host cells as supplier of MMP-2 activity. A collaboration between fibroblasts and cancer cells has been demonstrated in the growth of xenografted breast adenocarcinoma MCF7 cells into nude mice.'j8Although coinjection of fibroblasts with MCF7 cells promoted tumor growth in vivo, this effect of fibroblasts was inhibited by physiologic TIMP-2 and synthetic (Batimastat) MMP inhibitors.'j8This suggests that fibroblasts were acting as a source of MMP that could stimulate tumor growth. In addition, fibroblasts derived from MMP-11-deficient mice failed to promote MCF7 tumor These data provide evidence that a stromal MMP contributes in a paracrine manner to epithelial cell malignancy. The expression pattern of u-PA and most MMPs by host cells suggests that cancer cells recruit or induce the stromal cells to produce molecules used for tumor growth and invasion. Thus, tumors canbe considered as a cell society composed of different types of cell, each type having its specific role. What are the factors used for the recruitment or induction of host cells? In vitro studies have shown that many cytokines and growth factors can regulate the expression of pro tease^.^^,^^ Investigationsof MMP expression '

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in cocultures of fibroblasts and cancer cells in vitro have demonstrated a mutual interaction between host and cancer cells in the regulation of MMP production and a ~ t i v a t i o n . ~Cell-cell ~ , ~ ~ , contact ~~ may be implicated in the protease upregulation as demonstrated for MMP-9.41An extracellular matrix metalloprotease inducer (EMMPRIN)has been identified. This member of the immunoglobulin family, enriched on the surface of most tumor cells, stimulates stromal cells to produce different MMPs and binds MMP-1 to the tumor cell surface.39 NEW FUNCTIONS OF PROTEASES

Although cancer cells require a well-regulated pericellular proteolysis to migrate and invade the surrounding tissue, it has recently become increasingly apparent that ECM degradation by proteases does more than simply remove a physical barrier to invasion. Evidence supporting this concept has been provided by experiments demonstrating that an increase in MMP-7 or MMP-11 expression in tumor cells enhances primary tumor growth70,lo2rather than invasion.Different hypotheses can be formulated to explain the mechanism of protease action (Fig. 3): (1)the release of growth factors sequestrated in the ECM9'; (2) the activationof growth factors or the regulation of their bioavailability by cleaving their carrier molecules such as IGF-binding protein^^^,^^; (3) the control of cell apoptosis by destroying basement membrane integrity or inducing the release of Fas ligand9,46; (4) the shedding of cell-surface-bound growth factors or receptor^.^,^^,^' All these effects may generate an appropriate microenvironment that favors initial and sustained growth of primary tumors and metastatic foci. Furthermore, MMPs may directly regulate cell-matrix and cell-cell attachment by interacting with or cleaving matrix receptors such as i n t e g r i n ~ . ~ ~ For instance, the D4 integrin was shown to be fragmented in response to MMP-7 a~tivity.9~ More recently, it appeared that MT1-MMP expression is associated with functional activation of a,D3 integrin and increased cell motilityz9 In addition to these mechanisms of protease action, u-PA, u-PAR, and PAI-1 have been shown to influence cell migration independently of the regulation of cell surface proteolysis. These molecules also control cell adhesion, migration, and chemotaxis through different mechanisms.ll Chemotaxis is induced through a u-PA-dependent conformational change in u-PAR that uncovers a potent chemotactic epitope. Cell adhesion involves a u-PA-dependent exposure of u-PAR sites that interact with vitronectin, integrins, and ca~eolin.ll,~~ u-PA, therefore, can convert u-PAR from a simple receptor for plasminogen activator into a pleiotropic ligand for other molecules. These processes are controlled by PAI-1, which is located in the ECM in a vitronectin-bound form. When PAI-1 binds vitronectin, integrins or the u-PA/u-PAR complex can no longer interact with vitronectin because the same region of this matrix protein is required for interaction with PAI-1, u-PA/u-PAR, and integrins. PAI-1 has been considered as a molecular switch that governs u-PAR and integrin-mediated

apoptosis proliferation

d l migration

'

1 inactive

receptor integrin

inactive protease

-'u

A

= Binding Protein

ECM

= Extracellular Matri

Figure 3. Potential functions of proteases: (1) degradation of extracellular matrix components facilitating cell migration; (2) release of growth factors from the matrix; (3) enhancement of growth factor bioavailability by cleaving matrix components or binding proteins; (4) activation of other proteases; (5) cleavage of cell surface molecules involved in cell-cell adhesion; (6) activation of integrins; (7) cleavage of cell surface receptors resulting in their shedding or inactivation.

cell adhesion and relea~e.'~ It is beyond the scope of this article to review the role of proteases in cell migration, adhesion, and chemotaxis. Interested readers are referred to several recent reviews on this topicl1,54 and to several relevant articles in this issue. PROTEASE INHIBITORS IN CANCER

In healthy individuals, PAI-1 expression is low and confined mainly to vascular smooth muscle cells, adipocytes, and megakaryocytes. Its expression, however, can be stimulated strongly in other cell types, including endothelial cells, by different stimuli such as cytokines, growth factors, and angiogenic factors (bFGF and VEGF)." 17,64, 78 In colon cancer or neuroblastoma, PAI-1 mRNA could not be found in cancer cells but in endothelial cells located in the tumor ~ t r o r n a . ' ~In, ~vitro ~ assays demonstrated that expression of PAI-1 in endothelial cells was stimulated by soluble factors secreted by neuroblastoma cells.89In a rat aorta explant culture, PAI-1 expression was detected mostly in u-PA expressing cells, juxtaposed next to elongating sprout^.^ It was proposed that transient interactions between endothelial cells and cells such as fibroblasts that do not contact the endothelium under nonangiogenic circumstances induced PAI-1 expre~sion.~

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An analogous stromal expression pattern has been described for TIMPs in several cancers.75In breast and bladder cancers, high levels of TIMP-2 have been reported at the interface between malignant cells and stromal Cells.25,80,94 It is conceivable that overexpression of TIMP or PAI-1 reflects a host reaction against the most invasive malignant cells. Paradoxically, increasing number of clinical studies have demonstrated that high PAI-1 or TIMP levels indicate a poor prognosis for the survival of patients suffering from a variety of cancers.35, 80, 89 It remains undetermined whether the increased inhibitor levels causally contribute to, or rather are the consequence of the malignancy. One potential explanation of this paradox could be a simultaneous enhancement in expression of proteases and inhibitors resulting in a net excess of proteolytic activity. On the other hand, the initially unexpected findings that PAI-1 and TIMP-1 and 2 are strong negative prognostic markers in cancer may be related to a potential direct role of these inhibitors in cancer progression. It is conceivable that at low doses, protease inhibitors expressed in tumors may promote tumor cell growth and angiogenesis, while, at a higher concentration, they may predominantly act as anticancer agents. 723

ROLE OF PAI-1 IN CANCER PROGRESSION AND ANGIOGENESIS Based on its ability to block u-PA proteolysis, PAI-1 would be anticipated to reduce tumor growth and metastasis formation. Pulmonary metastases from HT1080 cells were increased by exogenous administration of PAI-1 and reduced by injection of antiPAI-1 antibody.91These data may reflect novel functions for PAI-1. The impact of PAI-1 absence was evaluated in different experimental tumor model using PAI-1 deficient mice. Although host PAI-1 deficiency did not influence the primary tumor growth or metastatic potential of B16 melanoma cells,3' it impaired tumor angiogenesis and invasion of malignant keratin~cytes.~ Such reduced tumor invasion and vascularization in PAI-1-deficient hosts have been observed similarly when malignant keratinocytes plated on a collagen gel were implanted onto the dorsal muscle fascia of mice67or when cells were directly, subcutaneously injected (unpublished data). In these systems, host PAI-1 appears essential for tumor growth and vascularization and PAI-1 produced by the cancer cells was not sufficient to overcome the host cell deficiency. Although the exact mechanism of PAI-1 action remains to be elucidated, different hypotheses have been formulated and discussed previously, according to the multifunctional nature of PAI-l.64The current interpretation of the proangiogenic effect of PAI-1 is that PAI-1 may act as a natural balancer of plasmin-mediated pericellular proteolysis that protects the stroma from excessive proteolysis during endothelial cell invasion. PAI-1 may be implicated in the stabilization of the extracellular matrix surrounding sprouting neovessels. Although endothelial cell migration requires active proteolysis, it has been shown in vitro

that excessive proteolysis prevents the coordinated assembly of endothelial cells into capillary shoots." A precise balance between proteolytic enzyme and their inhibitors may be essential for endothelial cell migration and differentiation into functional vessels. If this is the case, it is likely that PAI-1 exerts proangiogenic effect at low concentration, and antiangiogenic effect at high concentration. Altogether, these observationsexplain, at least in part, the clinical apparent paradoxic data that high PAI-1 levels are predictive of a poor prognosis for survival of patients with cancer. Furthermore, they identify PAI-1 as a new putative target for antiangiogenic cancer therapies. ROLE OF TlMPs IN CANCER PROGRESSION AND ANGIOGENESIS

The most notable functionof TIMP-2 is its capacity to form tight 1:l stoichiometric complexes with active MMPs, resulting in the inhibition of the catalytic activity of these proteinases. In this context, exogenously added TIMP-1 through TIMP-4 have been shown to suppress tumor invasion, metastasis, and tumor growth and neovascularization in several tumor models.',48,65, 97 Interestingly, intravital microscopy studies demonstrate that TIMP-1 alters the growth of injected tumor cells rather than the ability of cells to extravasate.19In transgenic mice, TIMP-1 overexpression inhibited primary growth of T-cell lymphoma but did not hinder metastatic col~nization.~' These data change the view of protease inhibitor functions from an initial focus on inhibition of cell invasion to a control of tumor cell growth. Although the exact mechanisms of TIMP-mediated antitumor effects are not yet well understood, it is likely that TIMPs modulate tumor growth by different mechanisms. First, through their antiproteolytic action, they may reduce the bioavailability of ECM-bound growth factors and maintain contact between cell and intact extracellular matrix. This latter effect may result in the maintenance of antiproliferative signals coming from in~ ~ , ~ ~TIMPs and tact matrix components such as fibrillar ~ o l l a g e n .Second, synthetic MMP inhibitors are known to inhibit angiogenesis by blocking endothelial cell proliferation and invasion.40Third, TIMPs may regulate cell apoptosis. This, at least, is the case for TIMP-3, which induces apoptotic cell death of various cancer cell lines.7This effect could be related to a stabilization of tumor necrosis factor alpha receptorss6 or to an action independent of MMP inhibitory a~tivity.~ Paradoxically, as mentioned previously, an association between a poor prognosis and high levels of expression of TIMP-1 or TIMP-2 has been reported for several cancer t y p e ~These . ~ ~ reports suggest that TIMPs have a dual effect in tumor growth. In addition to suppressing proteolysis and neovascularization, they may promote tumor cell proliferation at some TIMP-1 is known to display an erythroidstages of tumor progre~sion.~~ potentiating activity (EPA) that results in growth-stimulating effect on erythroid precursors and erythroleukemia cells.37TIMP-2 enhances the

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proliferation rate of fibroblasts or lymphoma cells3' and has no effect on breast adenocarcinoma MCF7 cells.68Therefore, it appears that the growthpromoting effect of TIMP is cell type specific. TIMP-1 may also influence the tumor mass by its antiapoptotic effect on some cells.38 Beside its inhibitory activity, TIMP-2 plays a pivotal role in the activation of proMMP-2. This process requires the participation of the 63 kDa integral plasma membrane MT1-MMP that binds TIMP-2 through its catalytic domain, thus docking soluble TIMP-2 to the cell surface. This biomolecular complex subsequently functions as a receptor for 66 kDa proMMP-2 through the interaction between the C-terminal domain of the enzyme and the C-terminal domain of TIMP-2?8,'5,103 In the presence of an adjacent free active MT1-MMP, the propeptide of MMP-2 is first cleaved between Asn37 and generating an activated 62 kDa intermediate form. When present at a sufficiently high concentration on the cell surface, this intermediate form is further processed to a fully activated 59 kDa MMP-2 by an intermolecular autocatalytic ~leavage.~ Alternatively, this second cleavage can involve the action of plasmin8Recently, it has been shown that MT1-MMPbound TIMP-2 is internalized and intracellularly degraded by different tumor cells.55This degradation process could result from the processing of MT1-MMP/TIMP-2/MMP-2 trimolecular complexes, as described for u-PAR/u-PA/PAI-1 complexes.22Alternatively, this process could represent an upstream mechanism that regulates the concentration of cell surface-associated TIMP-2, thus indirectly controlling the binding and hence the activation of proMMP-2. Taken together, these data demonstrate that the activation of proMMP2 at the cell surface is regulated by the balance between MT1-MMP complexed by TIMP-2 (which functions as a receptor for MMP-2) and TIMP2 free MT1-MMP (which functions as an activator of MT1-MMP-bound MMP-2). At low concentration, it promotes the binding of proMMP-2 to MT1-MMP, whereas at higher concentrations, it prevents the activation process because there is no free MT1-MMP available to initiate proMMP-2 activation. SUMMARY

The concept that most human carcinomas are stroma-dependent tumors has been proposed for a long time. It is only recently, however, that this view has been supported at the molecular level. A striking finding has been that proteases and their inhibitors are produced by stromal cells rather than by cancer cells themselves. Recent studies have suggested a continuous cross talk between cancer cells, fibroblasts, and inflammatory cells during cancer progression, leading at least to an upregulation of protease expression, secretion, and activation. These proteases are involved in different stages of tumor evolution including tumor growth, angiogenesis, invasion, and dissemination. Their tumor-promoting effects have been related to their capacity to breakdown extracellular matrix components and nonmatrix proteins that regulate cellular functions. Based on their

inhibitory function against proteases, inhibitors such as PAI-1 and TIMPs were viewed initially as anti-cancer agents. However, our concept of the role of these inhibitors must expand to include important effects on cell functions such as proliferation, apoptosis, and migration. It appears now that PAI-1 and TIMPs are multifunctional molecules that can promote or block tumor progression. These opposite effects are likely to depend on tumor cell types and the precise balance between proteases and inhibitors. Altogether, these data have modified the concept of protease inhibitors and have refreshed interest in stromal-epithelial interactions during tumor progression. A better understanding of the molecular role of the different proteolyhc systems and of cancer cell-host cell interactions is required to develop appropriate anticancer strategies. References 1. Ahonen M, Baker AH, Kahari VM: Adenovirus-mediated gene delivery of tissue inhibitor of metalloproteinases-3 inhibits invasion and induces apoptosis in melanoma cells. Cancer Res 58:2310-2315,1998 2. Andreasen PA, Kjoller L, Christensen L, et al: The urokinase-type plasminogen activator system in cancer metastasis: A review. Int J Cancer 72:l-22,1997 3. Atkinson SJ, Crabbe T, Cowell S, et al: Intermolecular autolytic cleavage can contribute to the activation of progelatinase A by cell membranes. J Biol Chem 270:30479-30485,1995 4. Atula S, Grenman R, Syrjanen S: Fibroblasts can modulate the phenotype of malignant epithelial cells in vitro. Exp Cell Res 235:180-187,1997 5. Bacharach E, Itin A, Keshet E: Apposition-dependent induction of plasminogen activator inhibitor type 1 expression: A mechanism for balancing pericellular proteolysis during angiogenesis. Blood 92:939-945,1998 6. Bajou K, Noel A, Gerard RD, et al: Absence of host plasminogen activator inhibitor 1 prevents cancer invasion and vascularization. Nat Med 4923-928,1998 7. Baker AH, George SJ, Zaltsman AB, et al: Inhibition of invasion and induction of apoptotic cell death of cancer cell lines by overexpression of TIMP-3. Br J Cancer 79:1347-1355,1999 8. Baramova EN, Bajou K, Remacle A, et al: Involvement of PA/plasmin system in the ~ - in 9 the second step of p r o - ~ ~activation. ~ - j FEBS Lett processing of p r o l ~ ~ and 405:157-162,1997 9. Basbaurn CB, Werb Z: Focalized proteolysis: Spatial and temporal regulation of extracellular matrix degradation at the cell surface. Curr Opin Cell Biol8:731-738,1996 10. Basset P, Bellocq JP, Wolf C, et al: A novel metalloproteinase gene specifically expressed in stromal cells of breast carcinomas. Nature 348:699-704,1990 11. Blasi F: The urokinase receptor. A cell surface, regulated chemokine. APMIS 107:96-101, 1999 12. Bode W, Femandez-Catalan C, Tschesche H, et al: Structural properties of matrix metalloproteinases. Cell Mol Life Sci 55:639-652,1999 13. Brooks PC, Stromblad S, Sanders LC et al: Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin alpha v beta 3. Cell 85:683-693,1996 14. Bugge TH, Lund LR, Kombrinck KK, et al: Reduced metastasis of Polyoma virus middle T antigen-induced mammary cancer in plasminogen-deficient mice. Oncogene 163097-3104,1998 15. Butler GS, Butler MJ, Atkinson SJ, et al: The TIMP2 membrane type 1metalloproteinase "receptor" regulates the concentration and efficient activation of progelatinase A. A kinetic study. J Biol Chem 273:871-880,1998 16. Camps JL, Chang SM, Hsu TC, et al: Fibroblast-mediated acceleration of human epithelial tumor growth in vivo. Proc Natl Acad Sci USA 87:75-79,1990

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