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Proteolyzed matrix as a template for the regulation of tumor progression
Biomedicine & Pharmacotherapy 57 (2003) 223–230 www.elsevier.com/locate/biopha
Dossier : Aging and age-related diseases
Proteolyzed matrix as a template for the regulation of tumor progression William Hornebeck *, François Xavier Maquart Faculté de Médecine, Centre National de la Recherche Scientifique (CNRS, FRE 2534), IFR 53 Biomolécules, Université de Reims, Champagne Ardenne, 51, rue Cognacq Jay, 51095 Reims cedex, France Received 27 March 2003; accepted 4 April 2003
Abstract Pericellular proteolysis plays a pivotal function in cell invasion, a hallmark of tumor growth and metastasis. The minidegradome constituted of two matrix metalloproteinases (MMP), i.e. MMP-2 and MT1-MMP, associated with tissue inhibitor of metalloprotease-2 (TIMP-2) and integrin (avb3) or CD44, is mainly involved in such invasive program. It catalyzes matrix degradation but, alternatively, proteolytic exposure of matricryptic sites or matrikines liberation by those enzymes regulates either positively or negatively tumor cell migration. That applies to types I and IV collagens, elastin, laminin 5, as described here, but such phenomenon might be extended to other matrix macromolecules. The development of tumors from epithelium origin is related to aging. Senescent fibroblasts are characterized by increased expression of MMPs, (particularly collagenase-1 (MMP-1) and stromelysin-1 (MMP-3)) and deposited matrix by those aged cells was shown to favor cancer cell growth. Thus, compositional variation of matrix-surrounding tumor cells, with formation of matricryptic sites and matrikines, can be considered as one main epigenetic factor contributing to tumor progression. A matrix-directed pharmacological approach in cancer is now emerging. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Matrix; Matrix metalloprotease; Tumor progression
1. Introduction The migration of cancer cells through host tissue boundaries constitutes a main event in tumor progression and formation of micrometastases [8,14,24,32]. Such invasive program requires the combined participation of matrix receptors (mainly integrins) expressed by transformed cells, and cycles of localized proteolytic activity [47,48]. Contrary to physiological invasion, tumor invasion appears to be mostly uncontrolled through continuous adaptation of the cancer cell to its microenvironment at aims to perpetually migrate [32,48]. Matrix metalloproteinases (MMPs) encompass a family of metalloendopeptidases (family M 10; clan MB), which are involved in many facets of tumor progression, influencing the growth, the differentiation, the apoptosis and the invasive property of cancer cells [8,14,36,56]. This family comprises at least 21 members that present the ability to hydrolyze virtually all matrix components, but also many other proteins as integrins or receptors, growth factors and cytokines as well as proteases from other families and their * Corresponding author. E-mail address: [email protected] (W. Hornebeck). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. DOI: 10.1016/S0753-3322(03)00049-0
inhibitors [14,22,36,53] (Table 1). MMP activity is regulated at several levels, including transcription, mRNA stability, translational efficiency, enzyme compartmentalization, proenzyme activation, inhibition and endocytosis [56]. Activation of those enzymes proceeds via several cascades, but, somewhat paradoxically, level of active enzyme(s), as determined in physiological fluids or in cell culture medium, is low; that can be attributed to the presence in the extracellular compartment of high level of specific inhibitors as tissue inhibitors of metalloproteinases (TIMPs) or a2macroglobulin (a2M) [14]. It suggested that matrix degradation, which occurs during cell migration, must be focalized in sequestered microenvironments containing high level of plasma membrane-associated MMPs [21,24,47]. Since decades, the interplay between tumor cells and host cells was described as a prominent event in cancer, promoting tumor cell survival, favoring the formation of tumor colonies and influencing cell invasiveness [32,48]. For instance, in cancer from epithelial origin, MMPs were found to be mostly expressed by stromal cells under the paracrine action of interleukins, interferons, growth factors and emmprins secreted from tumor cells [3,14]. However, tumor cells, in turn, might produce and localize at their plasma
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Table 1 Substrate specificities of gelatinase A (MMP-2) and membrane-associated matrix metalloprotease-1 (MT1-MMP) (from [14,36,52,56]) ECM substrates Gelatinase A (MMP-2) proenzyme: 72 kDa active species: 68 kDa Membrane-associated matrix metalloprotease-1 (MT1-MMP or MMP-14) proenzyme: 64 kDa active species: 60 kDa
Collagen (I, IV, V, VII, X, XI), gelatin, elastin, fibronectin, vitronectin, aggrecan Collagen (I) gelatin, fibronectin, vitronectin, fibrin, aggrecan
membrane the upstream initiators of proteolytic cascades i.e. the membrane-type matrix metalloprotease (MT-MMP) subfamily, as well as TIMP-2, to precisely regulate protease activation [21,24,47]. An additional level of complexity recently emerged, since it was observed that MMP-directed matrix hydrolysis might reveal several cryptic sites and/or liberate peptides which might influence tumor cell growth and invasive property through further positive or negative regulation of integrins as well as MMP expression, activation and activity [9,23,34,42]. This concept probably applies to any matrix macromolecule that might contain several of those sites; generated matrix fragments were designated as matrikines in keeping with their cytokine-like properties [23,34]. Besides, it needs to be emphasized that not only matrix, but also neutral endopeptidases, including MMPs themselves, could produce similar active fragments, following proteolysis and/or autocatalysis [45]. The aberrant phenotypic expression of fibroblasts surrounding tumors, i.e. myofibroblasts, has been widely documented, [2,3,41] but altered ECM proteolysis might equally create a microenvironment favoring tumor progression. In such case, the modified stroma can be considered as a main tumorigenic agent and, as a whole, is susceptible to therapeutic intervention i.e. “the stroma-directed therapy in cancer”. We do not want to cover here the vast literature on that topic dealing with numerous matrix constituents and all MMPs; instead, we will focus our presentation mainly on types I and IV collagens and elastin and will document the relationship between those matrix constituents and the MT1-MMP/MMP-2 system in order to illustrate our matter. The properties of generated matrikines will be described and the influence of aging in MMP expression and putative matrikine production and effect will be delineated.
2. The MT1-MMP/MMP-2 system and tumor progression The suppression by gene invalidation of any single MMP family member proved to significantly inhibit tumor progression in a series of mouse model systems [14]. However, the expression of MT1-MMP and consequent MMP-2 activation
Involvement in MMP proteolytic cascades it activates: Pro MMP 1, 2, 13
Pro MMP-2, 13
Other substrates (processing activity) Pro TGF b2: pro Il-1b, MCP-3; pro TNF a, FGF-R1; IGF-BP3,5 Pro av, a5, a3 integrins; CD-44; cell surface-bound transglutaminase
has been consistently shown to correlate with several steps of tumor progression as growth, neovascularization and metastasis [15,26,48,52]. The critical importance of MT1MMP, as one member of the membrane-associated MMP subfamily, in the migration of endothelial cells and tumor/cells is now emerging [52]. To that respect, it has to be considered that contrary to most soluble MMPs knock out mice which are nearly devoid of abnormal phenotypes, MT1MMP gene invalidation in mice led to an important deficiency in bone formation, angiogenesis as well as collagen turnover [14,52]. Six MT-MMPs have been characterized, four of them being type I transmembrane proteases (MT1, MT2, MT3 and MT5) while MT4 and MT6-MMPs are associated to the cell plasma membrane via a GPI anchor [52]. Several lines of evidence indicated that MT1-MMP is the main specific activator of MMP-2, but this enzyme, similarly to soluble MMPs, also displays matrix-degrading capacity as well as processing activity (Table 1) [36,52]. Paradoxically, MT1-MMP-dependent activation of MMP-2 necessitates the participation of TIMP-2. As a first step of the activation mechanism, the N-terminal part of the inhibitor binds to the active site of membrane-anchored MT1-MMP; the free carboxylate domain of TIMP-2 can then interact with the PEX domain of pro MMP-2, forming an inactive ternary complex at the surface of the cell [7,10,11,52]. Pro MMP-2 activation requires the oligomerization of inactive MT1-MMP in the complex with active MT1-MMP [27,31]. This bringing requires the clustering of integrins (avb3 mainly) and/or CD 44, which colocalizes with enzyme complexes at ruffling edge, invadopodia or caveolae in the invasive front of a migrating cell [28,38,46]. Either PEX or cytoplasmic domains of MT1MMP are crucially important in driving MT1-MMP dimerization and also MT1-MMP-TIMP-2 internalization, both necessary events for cell invasion [58]. The contribution of several matrix constituents, acting as templates, in such activation mechanism is well documented ([17,52]; and below). During those events, active MT1-MMP might additionally cleaves avb3, and/or CD44, thus modifying cell–matrix interactions and cell phenotype [10,28]. Therefore, migration of tumor cells and endothelial cells involves the close partnership of protease–activator–inhibitor (as an adaptor protein)–receptor–substrate and possibly
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other associated molecules within cell plasma membrane. Implicitly, it suggests that disruption of this degradome edifice at any site might somewhat empede tumor growth and invasion. Thus, in addition to specific synthetic MT1-MMP or MMP-2 inhibitors, peptides from PEX domains of MMP-2 and MT1-MMP, involved in avb3 interactions and MT1-MMP dimerization, respectively, or any compound interfering with MMP-2-avb3 interaction, could be equally effective as regulator of cell invasion.
3. MT1-MMP/MMP-2-directed matrix degradation and tumor progression 3.1. Basement membranes Basement membranes separate epithelia from the underlying mesenchyme and constitute the initial important tissue boundary for cancer cells from epithelium origin and melanoma cells. They are organized as a network of several macromolecules including different collagen types (IV, XV, XVIII), laminins, nidogens and heparan-sulfate proteoglycans [51,57]. The biomechanical stability of those structures is mainly ensured by type IV collagen constituted by a network of heterotrimer from six distinct polypeptides (a1– a6), 400 nm long. Each polypeptide contains separate domains, a cysteine-rich N-terminal domain, a central triple helical domain CB3 (IV) and at the carboxy terminal of those chains, a non-collagenous NC1 domain involved in the aggregation of two heterotrimers to form an hexameric complex [13,57]. MMP-2, i.e. gelatinase A, has been initially designated as 72 kDa type IV collagenase. Indeed, MMP-2 proved to extensively degrade type IV collagen, following reduction of disulfide bounds within CB3(IV); however, this collagen with intact disulfide bonding was found to be more resistant to MMP-2 cleavage at physiological temperature [13]. Particularly, dimeric molecules, where both parts of the triple helix are involved in molecules aggregation, are totally refractory to MMP-2 hydrolysis. In the native collagen IV network, only the area located between the N-terminal domain and CB3(IV) was found susceptible to proteolysis [13]. In a recent investigation, Xu et al. [60] evidenced that a cryptic site could be exposed, both in vitro and in vivo, in type IV collagen following proteolysis by MMP-2 and MMP-9. An antagonist antibody binding was produced, using the substractive immunization procedure in conjunction with pepsin solubilized human type IV collagen, which could strongly inhibit angiogenesis and tumor growth in several animal models [60]. Interestingly, exposure of this cryptic epitope correlated with pro MMP-2 activation in vivo, concomitant with a loss of a1b1 integrin binding, counterbalanced by a gain of avb3 binding. It suggested that interaction of such hidden structure within type IV collagen to avb3 integrin might trigger the MT1-MMP/MMP-2 proteolytic system, favoring both tumor growth and invasion.
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Several cryptic fragments from large molecules, as angiostatin from proteolyzed plasmin(ogen), were described to exhibit antiangiogenic activity [42]. That holds true for noncollagenous C-terminal fragment of collagen XVIII (endostatin), XV (restin), but also for type IV collagen. Depending upon the polypeptide a chain forming the heterotrimer, those fragments have been named aresten (a1), constatin (a2) or tumstatin (a3) [42]. The proteases involved in the generation of those fragments, some of them being detected in serum, have not been identified. Their biological effect is not restricted to their influence on endothelial cells morphogenesis [33,44]. A 185–203 NC1a3 (IV) peptide containing a specific SNS triplet was found to inhibit the proliferation of several cell lines from breast, stomach, pancreas and prostate tumors, through a rise in cAMP levels [24,43,54]. The b3 subunit of avb3 integrin was identified as the target of this a3 (IV) fragment; its interaction at an RGD-distinct binding site of this integrin triggered a signaling cascade involving focal adhesion kinase and PI-3 kinase which ultimately led to down-regulation of MT1-MMP and avb3 and inhibition of pro MMP-2 activation in melanoma and fibrosarcoma cell lines [43,54]. Parallely, the invasive capacity of those cells through matrigel was impaired. Thus, initial type IV collagen proteolysis could expose a cryptic site within the molecule leading to a b1–avb3 switch of matrix recognition inducing cell migration; subsequently, more extensive proteolysis could liberate NC fragment as a3 NC(IV) that, on contrary, would inhibit the avb3–MT1-MMP–MMP-2 system, acting as a regulator of cell migration. Such a b1/avb3 switch, as illustrated on Fig. 1 might apply to other collagen types and matrix macromolecules and, hence, could be considered as an important mechanism governing tumor progression. However, it needs to be emphasized that the response to individual integrin is cell-type-specific and matrix-mediated induction of MMP will also depend upon the cell type. To that respect, addition of MMP-2 to human breast epithelial cells induced migration only when cells were coated onto laminin 5, but not onto other basement membrane components as type IV collagen, laminin 1 or fibronectin [18] MMP-2-mediated cleavage of the c2 subunit of laminin 5 was shown to expose a promigratory cryptic site. Later investigations by Quaranta and colleagues demonstrated that MT1-MMP was instead the primary trigger for the induced migration of several epithelial cell lines, although both MT1MMP and MMP-2 could catalyze the exposure of the c2 cryptic site in a cumulative manner [18,29]; such MMPcatalyzed proteolysis generated a laminin fragment that shared homology with EGF family of ligands, suggesting that enhanced cell motility might be driven through erb-type receptor occupancy. Interestingly, it was also reported that plasmin might instead generate an antimigratory cryptic site within a3 subunit of laminin 5 [19]. Thus, laminin 5 could control either positively or negatively the migration of cancer cells from epithelial origin depending upon the type of “decryptase” involved.
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Fig. 1. Collagen(s)-mediated b1–b3 switch and regulation of cell migration [13,20,24,37,40,42,43,54,60]. Type IV collagen: the action of MMP-2, MMP-9 (or other enzymes) reveals a cryptic site in type IV collagen that reacts with avb3 in an apparently RGD-independent manner and favors cell migration (endothelial cell). More extensive proteolysis can lead to the liberation of a3CB IV peptide that inhibits, through b3 interaction (RGD-independent), either cell growth or migration (down-regulation of MT1-MMP and avb3). Type I collagen: a2b1-mediated interaction between type I collagen and melanoma cells induces MMP-1, MT1-MMP expression and MMP-2 activation; those enzymes can cooperate in collagen degradation and expose an RGD cryptic site. Interaction of those collagen fragments with avb3 enhances cell growth and survival and stimulates their collagen invasive capacity.
3.2. Mesenchyme Type I collagen is an important and ubiquitous component of the mesenchymal boundary, known to regulate cell adhesion, migration, viability, growth and differentiation. It interacts with several cell types through b1 integrins binding. A native triple helical conformation is required for its interaction with a1b1 and a2b1 integrins via an Asp–Gly–Glu–Ala tetrapeptide sequence [20]. Either heat denaturation or cleavage of the triple helix at Gly–Leu(Ileu) by collagenases led to the exposure of a cryptic RGD site that further bound to avb3 [24,37]. Such hydrolysis can be catalyzed by MMP-1 (collagenase-1) or MMP-13 (collagenase-3), but MT1-MMP and MMP-2 can equally degrade type I collagen in synergy [1]. The lattice culture model was widely used to delineate the implication of type I collagen in melanoma progression. Anchorage-dependent survival and growth of melanoma cells was attributed to avb3 occupancy [37]. Since interaction of type I collagen with a2b1 was shown to trigger MMP-1, MT1-MMP and MMP-2 expression and activation by melanoma cells in such a 3D environment, it can be assumed that native type I collagen contains the whole information for ensuring the survival and growth of melanoma through a b1
to b3 switch (Figs. 1 and 2). Such a mechanism, not only promoted growth but also stimulated the invasion of melanoma cells through type I collagen in transwell chambers [40]. Besides fibrillar collagens, human dermis contains a network of elastic fibers which alteration was recently reported to correlate with melanoma progression [16]. Elastin, the main amorphous component of those fibers, is susceptible to proteolysis by elastase-type neutral endopeptidases, among them MMP-2 and MMP-9. The biological activities exerted by the generated elastin fragments are diverse and widely documented [6,25,50,59]. They depend upon occupancy of a non-integrin elastin binding protein, identified as a truncated form of b-galactosidase (S-Gal), by a Gly–X–X–Pro–Gly consensus sequence adopting a type VIII b turn structure [6]. Such an interaction was recently shown to trigger MT1MMP and MMP-2 expression and activation in endothelial cells and HT 1080 fibrosarcoma cells and further, to increase the invasive capacity of those cell types [25]. Extent of such MMP-enhancing effect was found to correlate with the number of Val–Gly–Val–Ala–Pro–Gly repeats and, strikingly, intact tropoelastin containing several of those motifs in its covalent structure exhibited the most pronounced influence
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Fig. 2. Cooperative influence of type I collagen and elastin on melanoma progression [6,23–25,37,39,40,50,59]. Either a2b1 or S-GAL occupancy by type I collagen and elastin, respectively, lead to up-regulation of MT1-MMP expression and MMP-2 activation in melanoma cells. Such interactions favor cell migration in a cumulative manner. Since MMP-2 was described as a collagenase and an elastase, elastin as well as collagen fragments could be locally produced, acting in an autocrine way on cancer cells or a paracrine manner on host cells. Particularly, elastin fragments could further amplify matrix proteolysis by stimulating MMP secretion from fibroblasts, or inflammatory cells.
on MMP expression [25]. Thus, such domain is not cryptic in the tropoelastin chain; insoluble elastin arises from its precursor by crosslink formation and association with several structural glycoproteins led to the formation of the elastin fibers network [23,24]. Such supramolecular organization can well hidden the Gly–X–X–Pro–Gly motif, but recent investigations using antibody directed against (Val–Gly–Val–Ala–Pro–Gly)3 elastin sequence suggest the presence of such domain at the periphery of skin elastin fibers [39]. Thus elastin-derived peptides could be able to potentiate MT1MMP/MMP-2 expression and activation and consequently, melanoma invasion in type I collagen lattices similarly as found with HT 1080 fibrosarcoma cell lines [25]. The participation of avb3 integrin, as documented for other ECM macromolecules, in such enhancing effects is unknown but would deserve further investigations. Therefore, intact type I collagen and elastin, the main fibrillar components of human dermis, might cooperate in promoting melanoma progression. Additionally, liberated elastin fragments by pericellular active MMP-2, could favor angiogenesis and fibroblast activation [24] (Fig. 2).
4. Fibroblast senescence, MMP expression and cancer progression Neoplastic cells, mostly from epithelial origin, are more prone to develop tumors in older animals and the relationship between cancer progression and aging is documented [30,49,50]. Within dermis, senescent fibroblasts, with distinct phenotype, and decreased dividing capacity, accumulate with age. Those cells were found to exhibit altered MMP expression; notably increased collagenase-1 and stromelysin-1 secretion was considered to represent one of the main characteristics of late passage fibroblasts [5,61]. Interestingly, AP-1 expression, particularly c-Jun, is increased in aged human skin fibroblasts and recent investigations demonstrated that MMP-1 up-regulation during replicative senescence in human fibroblasts involved AktForkhead Signaling [35]. Such an enhanced host proteolytic phenotype might interfere with tumor progression at several levels. Tumor and host-derived MMPs can cooperate and generate proteolytic cascades, thus stimulating cancer cell invasion [4]. Alternatively, MMPs secreted from senescent
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matricryptic sites or to detect the presence of liberated matrikines. As a whole, matrix composition can be considered as a main epigenetic determinant in tumor progression.
fibroblasts might modify the tumor microenvironment by exposing matricryptic sites or releasing matrikines. It was recently evidenced, in elegant studies by Campisi and Coll, that matrix deposited by senescent but not presenescent fibroblasts could promote epithelial cell growth and tumorigenesis [30]. The repertoire of matrix receptors also varies with aging. Particularly, senescent human cells in culture as well as aging skin is characterized by the high expression of a novel form of b-galactosidase which may encode enzyme activity at pH 6.0. It was suggested that it might represent an alternatively spliced form of lysosomal b-Gal, although its identity with S-Gal, i.e. the elastin receptor, needs to be demonstrated [12]. Molecular, biochemical as well as morphological differences could be assigned to carcinoma-associated fibroblasts as compared to their normal counterparts. That includes the inappropriate expression of matrix constituents as tenascin, or proteolytic enzymes as stromelysin-3 [3]. Similarly, as senescent fibroblasts, those tumor-associated cells were consistently found to sustain progression of tumorigenesis [41]. Thus, senescence as well as the modified phenotype of fibroblasts surrounding tumor could act together to create a favorable environment for tumor progression.
[6]
5. Concluding remarks: a stroma-directed pharmacological approach in cancer
[7]
Originally, the tumor microenvironment was considered to only play a supporting role in cancer progression but its conspicuous active function in tumor development is now firmly established. The design and use of synthetic MMP inhibitors was the first stroma-directed pharmacological approach in cancer, given the most often host origin of those enzymes in carcinoma. Countless of those compounds have been evaluated in several cancer models and some of them are still used in clinical trials [14]. Unfortunately, data did not come up to the initial expectations; disappointment might originate from a somewhat unexpected biological function of those enzymes that might influence the early stages of tumor development [3,6,14] or liberate antiangiogenic matrikines. Considering the pivotal function of the MT1-MMP/MMP-2 system in cell invasion, i.e. angiogenesis and tumor cell invasion, substances interfering selectively with the expression, activation or activity of those enzymes might be of value. Apart from synthetic inhibitors, which design needs to take into account the corresponding S and S' subsites of those MMPs, matrikines or matrikine-mimetics could be envisaged as controlling agents. Particularly, avb3-directed matrikines, as PEX domain from MMP-2 or NC domains of type IV collagen, are promising compounds [54,55]. Advantageously, those matrix fragments could be covalently linked to an MMP inhibitor in order to give rise to targeted bifunctional agents. In keeping with the distinct phenotype of tumor associated fibroblasts, extracellular matrix surrounding cancer cells exhibits distinct qualitative and quantitative features, that need to be individually characterized to identify
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Dossier : Aging and age-related diseases
Proteolyzed matrix as a template for the regulation of tumor progression William Hornebeck *, François Xavier Maquart Faculté de Médecine, Centre National de la Recherche Scientifique (CNRS, FRE 2534), IFR 53 Biomolécules, Université de Reims, Champagne Ardenne, 51, rue Cognacq Jay, 51095 Reims cedex, France Received 27 March 2003; accepted 4 April 2003
Abstract Pericellular proteolysis plays a pivotal function in cell invasion, a hallmark of tumor growth and metastasis. The minidegradome constituted of two matrix metalloproteinases (MMP), i.e. MMP-2 and MT1-MMP, associated with tissue inhibitor of metalloprotease-2 (TIMP-2) and integrin (avb3) or CD44, is mainly involved in such invasive program. It catalyzes matrix degradation but, alternatively, proteolytic exposure of matricryptic sites or matrikines liberation by those enzymes regulates either positively or negatively tumor cell migration. That applies to types I and IV collagens, elastin, laminin 5, as described here, but such phenomenon might be extended to other matrix macromolecules. The development of tumors from epithelium origin is related to aging. Senescent fibroblasts are characterized by increased expression of MMPs, (particularly collagenase-1 (MMP-1) and stromelysin-1 (MMP-3)) and deposited matrix by those aged cells was shown to favor cancer cell growth. Thus, compositional variation of matrix-surrounding tumor cells, with formation of matricryptic sites and matrikines, can be considered as one main epigenetic factor contributing to tumor progression. A matrix-directed pharmacological approach in cancer is now emerging. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Matrix; Matrix metalloprotease; Tumor progression
1. Introduction The migration of cancer cells through host tissue boundaries constitutes a main event in tumor progression and formation of micrometastases [8,14,24,32]. Such invasive program requires the combined participation of matrix receptors (mainly integrins) expressed by transformed cells, and cycles of localized proteolytic activity [47,48]. Contrary to physiological invasion, tumor invasion appears to be mostly uncontrolled through continuous adaptation of the cancer cell to its microenvironment at aims to perpetually migrate [32,48]. Matrix metalloproteinases (MMPs) encompass a family of metalloendopeptidases (family M 10; clan MB), which are involved in many facets of tumor progression, influencing the growth, the differentiation, the apoptosis and the invasive property of cancer cells [8,14,36,56]. This family comprises at least 21 members that present the ability to hydrolyze virtually all matrix components, but also many other proteins as integrins or receptors, growth factors and cytokines as well as proteases from other families and their * Corresponding author. E-mail address: [email protected] (W. Hornebeck). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. DOI: 10.1016/S0753-3322(03)00049-0
inhibitors [14,22,36,53] (Table 1). MMP activity is regulated at several levels, including transcription, mRNA stability, translational efficiency, enzyme compartmentalization, proenzyme activation, inhibition and endocytosis [56]. Activation of those enzymes proceeds via several cascades, but, somewhat paradoxically, level of active enzyme(s), as determined in physiological fluids or in cell culture medium, is low; that can be attributed to the presence in the extracellular compartment of high level of specific inhibitors as tissue inhibitors of metalloproteinases (TIMPs) or a2macroglobulin (a2M) [14]. It suggested that matrix degradation, which occurs during cell migration, must be focalized in sequestered microenvironments containing high level of plasma membrane-associated MMPs [21,24,47]. Since decades, the interplay between tumor cells and host cells was described as a prominent event in cancer, promoting tumor cell survival, favoring the formation of tumor colonies and influencing cell invasiveness [32,48]. For instance, in cancer from epithelial origin, MMPs were found to be mostly expressed by stromal cells under the paracrine action of interleukins, interferons, growth factors and emmprins secreted from tumor cells [3,14]. However, tumor cells, in turn, might produce and localize at their plasma
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Table 1 Substrate specificities of gelatinase A (MMP-2) and membrane-associated matrix metalloprotease-1 (MT1-MMP) (from [14,36,52,56]) ECM substrates Gelatinase A (MMP-2) proenzyme: 72 kDa active species: 68 kDa Membrane-associated matrix metalloprotease-1 (MT1-MMP or MMP-14) proenzyme: 64 kDa active species: 60 kDa
Collagen (I, IV, V, VII, X, XI), gelatin, elastin, fibronectin, vitronectin, aggrecan Collagen (I) gelatin, fibronectin, vitronectin, fibrin, aggrecan
membrane the upstream initiators of proteolytic cascades i.e. the membrane-type matrix metalloprotease (MT-MMP) subfamily, as well as TIMP-2, to precisely regulate protease activation [21,24,47]. An additional level of complexity recently emerged, since it was observed that MMP-directed matrix hydrolysis might reveal several cryptic sites and/or liberate peptides which might influence tumor cell growth and invasive property through further positive or negative regulation of integrins as well as MMP expression, activation and activity [9,23,34,42]. This concept probably applies to any matrix macromolecule that might contain several of those sites; generated matrix fragments were designated as matrikines in keeping with their cytokine-like properties [23,34]. Besides, it needs to be emphasized that not only matrix, but also neutral endopeptidases, including MMPs themselves, could produce similar active fragments, following proteolysis and/or autocatalysis [45]. The aberrant phenotypic expression of fibroblasts surrounding tumors, i.e. myofibroblasts, has been widely documented, [2,3,41] but altered ECM proteolysis might equally create a microenvironment favoring tumor progression. In such case, the modified stroma can be considered as a main tumorigenic agent and, as a whole, is susceptible to therapeutic intervention i.e. “the stroma-directed therapy in cancer”. We do not want to cover here the vast literature on that topic dealing with numerous matrix constituents and all MMPs; instead, we will focus our presentation mainly on types I and IV collagens and elastin and will document the relationship between those matrix constituents and the MT1-MMP/MMP-2 system in order to illustrate our matter. The properties of generated matrikines will be described and the influence of aging in MMP expression and putative matrikine production and effect will be delineated.
2. The MT1-MMP/MMP-2 system and tumor progression The suppression by gene invalidation of any single MMP family member proved to significantly inhibit tumor progression in a series of mouse model systems [14]. However, the expression of MT1-MMP and consequent MMP-2 activation
Involvement in MMP proteolytic cascades it activates: Pro MMP 1, 2, 13
Pro MMP-2, 13
Other substrates (processing activity) Pro TGF b2: pro Il-1b, MCP-3; pro TNF a, FGF-R1; IGF-BP3,5 Pro av, a5, a3 integrins; CD-44; cell surface-bound transglutaminase
has been consistently shown to correlate with several steps of tumor progression as growth, neovascularization and metastasis [15,26,48,52]. The critical importance of MT1MMP, as one member of the membrane-associated MMP subfamily, in the migration of endothelial cells and tumor/cells is now emerging [52]. To that respect, it has to be considered that contrary to most soluble MMPs knock out mice which are nearly devoid of abnormal phenotypes, MT1MMP gene invalidation in mice led to an important deficiency in bone formation, angiogenesis as well as collagen turnover [14,52]. Six MT-MMPs have been characterized, four of them being type I transmembrane proteases (MT1, MT2, MT3 and MT5) while MT4 and MT6-MMPs are associated to the cell plasma membrane via a GPI anchor [52]. Several lines of evidence indicated that MT1-MMP is the main specific activator of MMP-2, but this enzyme, similarly to soluble MMPs, also displays matrix-degrading capacity as well as processing activity (Table 1) [36,52]. Paradoxically, MT1-MMP-dependent activation of MMP-2 necessitates the participation of TIMP-2. As a first step of the activation mechanism, the N-terminal part of the inhibitor binds to the active site of membrane-anchored MT1-MMP; the free carboxylate domain of TIMP-2 can then interact with the PEX domain of pro MMP-2, forming an inactive ternary complex at the surface of the cell [7,10,11,52]. Pro MMP-2 activation requires the oligomerization of inactive MT1-MMP in the complex with active MT1-MMP [27,31]. This bringing requires the clustering of integrins (avb3 mainly) and/or CD 44, which colocalizes with enzyme complexes at ruffling edge, invadopodia or caveolae in the invasive front of a migrating cell [28,38,46]. Either PEX or cytoplasmic domains of MT1MMP are crucially important in driving MT1-MMP dimerization and also MT1-MMP-TIMP-2 internalization, both necessary events for cell invasion [58]. The contribution of several matrix constituents, acting as templates, in such activation mechanism is well documented ([17,52]; and below). During those events, active MT1-MMP might additionally cleaves avb3, and/or CD44, thus modifying cell–matrix interactions and cell phenotype [10,28]. Therefore, migration of tumor cells and endothelial cells involves the close partnership of protease–activator–inhibitor (as an adaptor protein)–receptor–substrate and possibly
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other associated molecules within cell plasma membrane. Implicitly, it suggests that disruption of this degradome edifice at any site might somewhat empede tumor growth and invasion. Thus, in addition to specific synthetic MT1-MMP or MMP-2 inhibitors, peptides from PEX domains of MMP-2 and MT1-MMP, involved in avb3 interactions and MT1-MMP dimerization, respectively, or any compound interfering with MMP-2-avb3 interaction, could be equally effective as regulator of cell invasion.
3. MT1-MMP/MMP-2-directed matrix degradation and tumor progression 3.1. Basement membranes Basement membranes separate epithelia from the underlying mesenchyme and constitute the initial important tissue boundary for cancer cells from epithelium origin and melanoma cells. They are organized as a network of several macromolecules including different collagen types (IV, XV, XVIII), laminins, nidogens and heparan-sulfate proteoglycans [51,57]. The biomechanical stability of those structures is mainly ensured by type IV collagen constituted by a network of heterotrimer from six distinct polypeptides (a1– a6), 400 nm long. Each polypeptide contains separate domains, a cysteine-rich N-terminal domain, a central triple helical domain CB3 (IV) and at the carboxy terminal of those chains, a non-collagenous NC1 domain involved in the aggregation of two heterotrimers to form an hexameric complex [13,57]. MMP-2, i.e. gelatinase A, has been initially designated as 72 kDa type IV collagenase. Indeed, MMP-2 proved to extensively degrade type IV collagen, following reduction of disulfide bounds within CB3(IV); however, this collagen with intact disulfide bonding was found to be more resistant to MMP-2 cleavage at physiological temperature [13]. Particularly, dimeric molecules, where both parts of the triple helix are involved in molecules aggregation, are totally refractory to MMP-2 hydrolysis. In the native collagen IV network, only the area located between the N-terminal domain and CB3(IV) was found susceptible to proteolysis [13]. In a recent investigation, Xu et al. [60] evidenced that a cryptic site could be exposed, both in vitro and in vivo, in type IV collagen following proteolysis by MMP-2 and MMP-9. An antagonist antibody binding was produced, using the substractive immunization procedure in conjunction with pepsin solubilized human type IV collagen, which could strongly inhibit angiogenesis and tumor growth in several animal models [60]. Interestingly, exposure of this cryptic epitope correlated with pro MMP-2 activation in vivo, concomitant with a loss of a1b1 integrin binding, counterbalanced by a gain of avb3 binding. It suggested that interaction of such hidden structure within type IV collagen to avb3 integrin might trigger the MT1-MMP/MMP-2 proteolytic system, favoring both tumor growth and invasion.
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Several cryptic fragments from large molecules, as angiostatin from proteolyzed plasmin(ogen), were described to exhibit antiangiogenic activity [42]. That holds true for noncollagenous C-terminal fragment of collagen XVIII (endostatin), XV (restin), but also for type IV collagen. Depending upon the polypeptide a chain forming the heterotrimer, those fragments have been named aresten (a1), constatin (a2) or tumstatin (a3) [42]. The proteases involved in the generation of those fragments, some of them being detected in serum, have not been identified. Their biological effect is not restricted to their influence on endothelial cells morphogenesis [33,44]. A 185–203 NC1a3 (IV) peptide containing a specific SNS triplet was found to inhibit the proliferation of several cell lines from breast, stomach, pancreas and prostate tumors, through a rise in cAMP levels [24,43,54]. The b3 subunit of avb3 integrin was identified as the target of this a3 (IV) fragment; its interaction at an RGD-distinct binding site of this integrin triggered a signaling cascade involving focal adhesion kinase and PI-3 kinase which ultimately led to down-regulation of MT1-MMP and avb3 and inhibition of pro MMP-2 activation in melanoma and fibrosarcoma cell lines [43,54]. Parallely, the invasive capacity of those cells through matrigel was impaired. Thus, initial type IV collagen proteolysis could expose a cryptic site within the molecule leading to a b1–avb3 switch of matrix recognition inducing cell migration; subsequently, more extensive proteolysis could liberate NC fragment as a3 NC(IV) that, on contrary, would inhibit the avb3–MT1-MMP–MMP-2 system, acting as a regulator of cell migration. Such a b1/avb3 switch, as illustrated on Fig. 1 might apply to other collagen types and matrix macromolecules and, hence, could be considered as an important mechanism governing tumor progression. However, it needs to be emphasized that the response to individual integrin is cell-type-specific and matrix-mediated induction of MMP will also depend upon the cell type. To that respect, addition of MMP-2 to human breast epithelial cells induced migration only when cells were coated onto laminin 5, but not onto other basement membrane components as type IV collagen, laminin 1 or fibronectin [18] MMP-2-mediated cleavage of the c2 subunit of laminin 5 was shown to expose a promigratory cryptic site. Later investigations by Quaranta and colleagues demonstrated that MT1-MMP was instead the primary trigger for the induced migration of several epithelial cell lines, although both MT1MMP and MMP-2 could catalyze the exposure of the c2 cryptic site in a cumulative manner [18,29]; such MMPcatalyzed proteolysis generated a laminin fragment that shared homology with EGF family of ligands, suggesting that enhanced cell motility might be driven through erb-type receptor occupancy. Interestingly, it was also reported that plasmin might instead generate an antimigratory cryptic site within a3 subunit of laminin 5 [19]. Thus, laminin 5 could control either positively or negatively the migration of cancer cells from epithelial origin depending upon the type of “decryptase” involved.
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Fig. 1. Collagen(s)-mediated b1–b3 switch and regulation of cell migration [13,20,24,37,40,42,43,54,60]. Type IV collagen: the action of MMP-2, MMP-9 (or other enzymes) reveals a cryptic site in type IV collagen that reacts with avb3 in an apparently RGD-independent manner and favors cell migration (endothelial cell). More extensive proteolysis can lead to the liberation of a3CB IV peptide that inhibits, through b3 interaction (RGD-independent), either cell growth or migration (down-regulation of MT1-MMP and avb3). Type I collagen: a2b1-mediated interaction between type I collagen and melanoma cells induces MMP-1, MT1-MMP expression and MMP-2 activation; those enzymes can cooperate in collagen degradation and expose an RGD cryptic site. Interaction of those collagen fragments with avb3 enhances cell growth and survival and stimulates their collagen invasive capacity.
3.2. Mesenchyme Type I collagen is an important and ubiquitous component of the mesenchymal boundary, known to regulate cell adhesion, migration, viability, growth and differentiation. It interacts with several cell types through b1 integrins binding. A native triple helical conformation is required for its interaction with a1b1 and a2b1 integrins via an Asp–Gly–Glu–Ala tetrapeptide sequence [20]. Either heat denaturation or cleavage of the triple helix at Gly–Leu(Ileu) by collagenases led to the exposure of a cryptic RGD site that further bound to avb3 [24,37]. Such hydrolysis can be catalyzed by MMP-1 (collagenase-1) or MMP-13 (collagenase-3), but MT1-MMP and MMP-2 can equally degrade type I collagen in synergy [1]. The lattice culture model was widely used to delineate the implication of type I collagen in melanoma progression. Anchorage-dependent survival and growth of melanoma cells was attributed to avb3 occupancy [37]. Since interaction of type I collagen with a2b1 was shown to trigger MMP-1, MT1-MMP and MMP-2 expression and activation by melanoma cells in such a 3D environment, it can be assumed that native type I collagen contains the whole information for ensuring the survival and growth of melanoma through a b1
to b3 switch (Figs. 1 and 2). Such a mechanism, not only promoted growth but also stimulated the invasion of melanoma cells through type I collagen in transwell chambers [40]. Besides fibrillar collagens, human dermis contains a network of elastic fibers which alteration was recently reported to correlate with melanoma progression [16]. Elastin, the main amorphous component of those fibers, is susceptible to proteolysis by elastase-type neutral endopeptidases, among them MMP-2 and MMP-9. The biological activities exerted by the generated elastin fragments are diverse and widely documented [6,25,50,59]. They depend upon occupancy of a non-integrin elastin binding protein, identified as a truncated form of b-galactosidase (S-Gal), by a Gly–X–X–Pro–Gly consensus sequence adopting a type VIII b turn structure [6]. Such an interaction was recently shown to trigger MT1MMP and MMP-2 expression and activation in endothelial cells and HT 1080 fibrosarcoma cells and further, to increase the invasive capacity of those cell types [25]. Extent of such MMP-enhancing effect was found to correlate with the number of Val–Gly–Val–Ala–Pro–Gly repeats and, strikingly, intact tropoelastin containing several of those motifs in its covalent structure exhibited the most pronounced influence
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Fig. 2. Cooperative influence of type I collagen and elastin on melanoma progression [6,23–25,37,39,40,50,59]. Either a2b1 or S-GAL occupancy by type I collagen and elastin, respectively, lead to up-regulation of MT1-MMP expression and MMP-2 activation in melanoma cells. Such interactions favor cell migration in a cumulative manner. Since MMP-2 was described as a collagenase and an elastase, elastin as well as collagen fragments could be locally produced, acting in an autocrine way on cancer cells or a paracrine manner on host cells. Particularly, elastin fragments could further amplify matrix proteolysis by stimulating MMP secretion from fibroblasts, or inflammatory cells.
on MMP expression [25]. Thus, such domain is not cryptic in the tropoelastin chain; insoluble elastin arises from its precursor by crosslink formation and association with several structural glycoproteins led to the formation of the elastin fibers network [23,24]. Such supramolecular organization can well hidden the Gly–X–X–Pro–Gly motif, but recent investigations using antibody directed against (Val–Gly–Val–Ala–Pro–Gly)3 elastin sequence suggest the presence of such domain at the periphery of skin elastin fibers [39]. Thus elastin-derived peptides could be able to potentiate MT1MMP/MMP-2 expression and activation and consequently, melanoma invasion in type I collagen lattices similarly as found with HT 1080 fibrosarcoma cell lines [25]. The participation of avb3 integrin, as documented for other ECM macromolecules, in such enhancing effects is unknown but would deserve further investigations. Therefore, intact type I collagen and elastin, the main fibrillar components of human dermis, might cooperate in promoting melanoma progression. Additionally, liberated elastin fragments by pericellular active MMP-2, could favor angiogenesis and fibroblast activation [24] (Fig. 2).
4. Fibroblast senescence, MMP expression and cancer progression Neoplastic cells, mostly from epithelial origin, are more prone to develop tumors in older animals and the relationship between cancer progression and aging is documented [30,49,50]. Within dermis, senescent fibroblasts, with distinct phenotype, and decreased dividing capacity, accumulate with age. Those cells were found to exhibit altered MMP expression; notably increased collagenase-1 and stromelysin-1 secretion was considered to represent one of the main characteristics of late passage fibroblasts [5,61]. Interestingly, AP-1 expression, particularly c-Jun, is increased in aged human skin fibroblasts and recent investigations demonstrated that MMP-1 up-regulation during replicative senescence in human fibroblasts involved AktForkhead Signaling [35]. Such an enhanced host proteolytic phenotype might interfere with tumor progression at several levels. Tumor and host-derived MMPs can cooperate and generate proteolytic cascades, thus stimulating cancer cell invasion [4]. Alternatively, MMPs secreted from senescent
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matricryptic sites or to detect the presence of liberated matrikines. As a whole, matrix composition can be considered as a main epigenetic determinant in tumor progression.
fibroblasts might modify the tumor microenvironment by exposing matricryptic sites or releasing matrikines. It was recently evidenced, in elegant studies by Campisi and Coll, that matrix deposited by senescent but not presenescent fibroblasts could promote epithelial cell growth and tumorigenesis [30]. The repertoire of matrix receptors also varies with aging. Particularly, senescent human cells in culture as well as aging skin is characterized by the high expression of a novel form of b-galactosidase which may encode enzyme activity at pH 6.0. It was suggested that it might represent an alternatively spliced form of lysosomal b-Gal, although its identity with S-Gal, i.e. the elastin receptor, needs to be demonstrated [12]. Molecular, biochemical as well as morphological differences could be assigned to carcinoma-associated fibroblasts as compared to their normal counterparts. That includes the inappropriate expression of matrix constituents as tenascin, or proteolytic enzymes as stromelysin-3 [3]. Similarly, as senescent fibroblasts, those tumor-associated cells were consistently found to sustain progression of tumorigenesis [41]. Thus, senescence as well as the modified phenotype of fibroblasts surrounding tumor could act together to create a favorable environment for tumor progression.
[6]
5. Concluding remarks: a stroma-directed pharmacological approach in cancer
[7]
Originally, the tumor microenvironment was considered to only play a supporting role in cancer progression but its conspicuous active function in tumor development is now firmly established. The design and use of synthetic MMP inhibitors was the first stroma-directed pharmacological approach in cancer, given the most often host origin of those enzymes in carcinoma. Countless of those compounds have been evaluated in several cancer models and some of them are still used in clinical trials [14]. Unfortunately, data did not come up to the initial expectations; disappointment might originate from a somewhat unexpected biological function of those enzymes that might influence the early stages of tumor development [3,6,14] or liberate antiangiogenic matrikines. Considering the pivotal function of the MT1-MMP/MMP-2 system in cell invasion, i.e. angiogenesis and tumor cell invasion, substances interfering selectively with the expression, activation or activity of those enzymes might be of value. Apart from synthetic inhibitors, which design needs to take into account the corresponding S and S' subsites of those MMPs, matrikines or matrikine-mimetics could be envisaged as controlling agents. Particularly, avb3-directed matrikines, as PEX domain from MMP-2 or NC domains of type IV collagen, are promising compounds [54,55]. Advantageously, those matrix fragments could be covalently linked to an MMP inhibitor in order to give rise to targeted bifunctional agents. In keeping with the distinct phenotype of tumor associated fibroblasts, extracellular matrix surrounding cancer cells exhibits distinct qualitative and quantitative features, that need to be individually characterized to identify
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