Membrane-type metalloproteinases in tumor invasion
Myriam Polette*, Philippe Birembaut
INSERM U.314,Unite de Biologie Cellulaire,Laboratoire Pol Bouin,CHU Muison Blanche.45,rue Cognacy-Jay.51100 Reims.
France
Received 23 February 1998: accepted 17 June 1998
Abstract
Matrix metalloproteinases (MMPs) are members of a multigene family of zinc-dependent enzymes involved in the degradation of numerous extracellular matrix (ECM) components. Among these enzymes,membrane-type MMPs (MT-MMPs) play a major role in the activation of progelatinase A (MMP-2). The molecular structure of these enzymes is characterized by a transmembrane domain and the presence of an insertion of 11 amino-acids bctwecn the pro-peptide and the catalytic domains, which may be cleaved by furin-like enzymes leading to the activated form of the enzymes. MTI-MMP appears to play a dual role in extracellular matrix remodeling through activation of progelatinase A and procollagenase 3 and direct cleavage of some ECM macromolecules such as gelatin, type I collagen and fibronectin. Tissue inhibitor of MMPs-2 (TIMP-2) serves as an intermediate in progelatinase A activation by binding to MT1-MMP and progelatinase A on the plasma membrane. In vivo. MTI-MMP is overexpressed in malignant tumor tissues in which it was mainly localized in stromal cells surrounding the neoplastic tissue.These peritumoral fibroblasts, under particular stimuli. would be induced to overexpress MT1-MMP and consequently activate gelatinase A leading to ECM degradation. The expression of MTI-MMP is however observed in vitro in the invasive tumor cells which might represent an late stage of tumor progression.All these data confirm the important role of MT-MMPs in tumor invasion and highlight a cooperation between tumor and stromal cells for the production of these enzymes.The contribution of MMPs in a metastatic process leads to the development of novel therapies using inhibitors of these enzymes. Among a multitude of synthetic inhibitors generated.Marimastat is already clinically employed in cancer treatment. 1998 Elsevier Science Ltd. All rights reserved.
Kerwords:Metalloproteinases: MT-MMPs;Tumor cells; Cancer invasion
1.Introduction
Proteolytic degradation of the extracellular matrix and especially basement membranes is a central aspect of physiologic and pathologic tissue-remodeling processes such as trophoblastic
* Corresponding author. Fax: +33-3-26065861.

implantation, wound healing, and tumor inva-sion.Several proteinases play a major role in this degradative process, including serine proteinases. cysteine proteinases, aspartic proteinases. and metalloproteinases. Matrix metalloproteinases (MMPs) are members of a multigene family of zinc-dependent enzymes.These proteases have been classified into four broad categories orig-inally based on substrate specificity [1]. These
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specific enzymes are the collagenases(MMP-1, MMP-8 and MMP-13), the gelatinases A (MMP-2) and B (MMP-9), the stromelysins (MMP-3, MMP-10 and MMP-11) including the matrilysin (MMP-7) and a metalloelastase (MMP-12),and the recent group of membrane-type MMPs (MT-MMPs).The regulation of MMP activity occurs at several levels including gene transcriptional control,proenzyme activation and inhibition of activated enzymes by endogenous inhibitors. These native MMP inhibitors comprise a family of proteins and glycoproteins referred to by the acronym TIMPs (tissue inhibitor of matrix metal-loproteinases). Four of these inhibitors have been cloned and expressed: TIMP-1, TIMP-2,TIMP-3 and TIMP-4. Gelatinase A degrades basement membrane components such as type IV collagen and laminin and is particularly implicated in the progression of tumors. In contrast to most MMPs, progelatinase A is produced constitu-tively in high concentrations by many types of cells and is not induced by cytokines that regu-late other MMPs. The final activation of this proenzyme is therefore an important step in con-trolling tissue gelatinase A activity. This process involves a cell surface activation mechanism which requires the participation of the recently described MT-MMPs.
2.Identification and structure of MT-MMPs
The first member of the MT-MMP family (MTI-MMP)has been isolated by Sato et al.[2] who cloned a cDNA encoding a MMP of 63 kDa with a transmembrane domain. Expression of the gene product of this MT-MMP induced specific activation of progelatinase A on the cell surface in vitro and cnhanced cellular invasion of the reconstituted basement membrane (Matrigel). Following the discovery of this MT1-MMP, three other MT-MMP have been identified: MT2-MMP of 72 kDa [3], MT3-MMP of 64 kDa [4] and MT4-MMP of 70 kDa [5].
While these MT-MMPs have a common MMP domain structure with pre-, pro-,catalytic and hemopexin-like domains,they have also three

unique insertions. First there is an insertion of 11 amino-acids between the pro-peptide and the cat-alytic domains. A similar insertion has been described in the stromelysin 3/MMP-11 in the same position [6]. The conserved Arg-Arg-Lys-Arg’ sequence precedes the potential processing sites of these MMPs. This sequence is recognized by furin,a proprotein convertase present in the Golgi apparatus which is able to activate recom-binant MT1-MMP [7]. Furin specifically cleaves MTI-MMP in vitro between Arg’-Tyr which results in a progelatinase A activation.However, using mutated enzymes and furin inhibitor, Cao et al. [8] have demonstrated that furin-induced activation of MTI-MMP is not a prerequisite for the progelatinase activation. In another way, Okumura et al. [9] have found that plasmin is also able to activate the pro-MTl-MMP by cleaving downstream of Arg’08 and Arg’.These authors have suggested that pro-MT1-MMP transported to the plasma membrane is activated by plasmin extracellularly. The MT-MMPs have a second insertion of eight amino-acids in the catalytic enzyme domain whose function remains undefined.The third insertion at the C-terminus contains a hydrophobic amino-acid sequence which acts as a transmembrane domain. This transmembrane region has been considered as a functional domain required for progelatinase A activation [10].However,Pei and Weiss[11]have shown that a transmembrane-deletion mutant of MTI-MMP was also able to mediate progelati-nase A activation. Moreover, a transmembrane domainless variant of MT3-MMP has been described in the rat vascular smooth muscle cells which was able to convert progelatinase A to the intermediate form but not to the mature active one [12]. By homology screening for human MT-MMP, a variant of MT3-MMP with a novel sequence of 50 amino-acids after Lys407 instead of amino-acids containing the transmembrane domain of MT3-MMP has recently been ident-ified as a soluble type of MT3-MMP formed by alternatively spliced mRNA [13]. All these mol-ecular characteristics define a complex family of MMPs whose functions are not totally well eluci-dated. 
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3.Regulaton of MT-MMP expression
There are few studies on transcriptional regu-lation of MT-MMPs. It has been shown that in vitro a continued presence of Concanavalin A stimulates the expression of mRNA encoding MTI-MMP in MDA-MB-231 human breast cancer cells [14]. Tetradecanoyl phorbol acetate is also a potent inducer of MTI-MMP in HT 1080 cells, but not in fibroblasts. Growth factors and cytokines such as interleukin-1β, EGF. bFGF,TGFβ have negligible effects on the mRNA levels and TNF-α produces a slight increase of these transcripts in HT-1080 cells [15]. Considering potential in vivo regu-lation of MT-MMPs.type I collagen treatment of invasive breast cancer cells increases the steady state MTI-MMP mRNA levels but does not induce MTI-MMP expression in non-inva-sive breast cancer cell lines. MT3-MMP and MT4-MMP are not up-regulated by type 1 collagen [16]. Moreover,there is a close parallel expression of type I collagcn and MT1-MMP mRNAs in stromal cells of breast carcinomas. These data support a role of MTI-MMP inthe previously described collagen-induced activation of progelatinase A. These results implicate MTI-MMP in collagen type I-stimulated pro-gelatinase A activation and suggest that this mechanism may be employed in vivo by tumor associated fibroblasts to facilitate cancer pro-gression in a cooperative process between cancer and stromal cells. In a recent study, we have demonstrated that an increased expression of MTI-MMP occurs in fibroblasts treated by invasive breast tumor cells conditioned medium, but not in the presence of a noninvasive breast tumor cells conditioned medium [17]. It is likely that the ability of tumor cells to influence the stromal production of MTI-MMP would correlate with their degree of metastatic con-version. All these data emphasize the import-ant role of extracellular matrix-cell interactions and intercellular cooperation between cancer and stromal cells for the production of MT-MMPs.

4.Substrates of MT-MMPs
The first substrate discovered for MT-MMPs was progelatinase A. Recombinant MTI-MMP cleaves the propeptide sequence of progelatinase A in a sequence-specific manner, recognizing the Asn’6-Leu bond and generating an intermediate form of gelatinase A [18]. The soluble catalytic domain of MTI-MMP is also able to cleave this propeptide [19]. This enzymatic activity is inhibited by TIMP2 and TIMP3, but not by TIMPI. MTI-MMP also functions as a receptor of gelatinase A. Indeed. Strongin et al. [20] have purified a fibroblast plasma membrane MTI-MMP and demonstrated that it binds TIMP2. The C-terminal domain of TIMP2 binds to the zinc catalytic domain of MTI-MMP [21].It has been proposed that the resulting MT1-MMP/ TIMP2 complex acts as a receptor for progelati-nase A. binding to the C-terminal domain of the proenzyme regulating its concentration and potentiating its cleavage by active MTI-MMP at adjacent sites [22]. The amount of TIMP2 deter-mines the balance between the level of activator (free MTI-MMP) and receptor (MTI-MMP/ TIMP2 complex),which regulates the degree of activation of progelatinase A. Thus TIMP2 may have a dichotomous mode of action. On one hand, it can act as a specific inhibitorof MMPs,on the other hand, it is implicated in the mechanism of progelatinase activation (Table 1). However,Sato et al.[23] using COS-I cells trans-fected with MTI-MMP cDNA,have shown that MTI-MMP can bind directly progelatinase A. without any TIMP2.The proenzyme cleaved by MTI-MMP is consequently processed to an in-termediate form of 69 kDa. The processing to the fully active form of 67 kDa is dependent on the gelatinase A concentration at the cell surface and is an autoproteolytic reaction. The rate of autocatalytic activation of progelatinase A initiated by MTI-MMP cleavage could bc potentiated by concentration of the pro-enzyme by binding to heparin [22]. The plasmin system is also involved in this second step of activation [24]. A recent paper by Mazzieri et al.[25] suggests that plasmin could play a dual role in the proteolytic cascade which leads to 
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progelatinase A activation on the cell membrane. Plasmin could activate pro-MTI-MMP that binds TIMP2 and progelatinase A and bound progelatinase A may be then activated by plas-min. Finally, Lee et al. [26] have described a possible intracellular activation of gelatinase A by MTI-MMP bound to the Golgi membranes of fibroblasts.
The role of MT-MMPs as activators of MMPs is not limited to progelatinase A activation. Indeed,Knäuper et al. [27] have reported that procollagenase 3 (proMMP-13) is also activated by MT1-MMP on the cell surface. This acti-vation is accelerated in the presence of gelatinase A.All these in vitro studies reflect the complexity of the proMMPs activation process which remains not fully understood in vivo.
In addition to its role in activating proMMPs, MT1-MMP may also act as an extra-cellular matrix degrading enzyme. Imai et al. [28] have demonstrated for the first time that MT1-MMP was a gelatinolytic enzyme and was secreted from cells in a complex with TIMP2, which can form a ternary complex of MTI-MMP/TIMP2/

progelatinase A. In the same way, Pei and Weiss [11] showed that transmembrane-deletion mutants of the MT1-MMP (soluble forms of MTI-MMP) were able to express proteolytic activity against extracellular matrix components such as gelatin, fibronectin, the chain B of laminin, vitronectin and dermatan sulfate proteoglycan. Such mutants and native MTI-MMP secreted from a human breast carcinoma cell line MDA-MB-231 are active without any treatment for activation and digest type I (guinea pig), II (bovine) and III (human) collagens into characteristic 3/4 and 1/4 fragments [29]. Thus,MTl-MMP appears to play a dual role in extracellular matrix remodeling through direct cleavage of extracellular matrix components and activation of progelatinase A and procolla-genase 3.
5.Participation of MT-MMPs in tumor invasion
The mechanism of progelatinase A activation has attracted the attention of many tumor biol-
Table 1
Mechanisms of activation of progelatinase A.(1)In a stochiometric reaction,TIMP-2 binds to MT1-MMP and progelatinase A at the membrane.Progelatinase A is activated with the first cleavage(Asn6) of its N-propeptide by MT1-MMP.The fully active form of the enzyme is obtained after a second cleavage by plasmin or/and autoproteolysis. (2) In excess of TIMP-2,TIMP-2 binds to all free MTI-MMP molecules blocking the potential activation of progelatinase A
________________________________________
1-Activation of pro gelatinase A

2-Excess of TIMP-2
No activation of pro gelatinase A

MT1-MMP 
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ogists,because a close association between the expression of activated gelatinase A and tumor spread has been found in numerous cancers.The essential role of MT-MMPs in this process has been emphasized by in vitro and in vivo observations.
In vitro, MTI-MMP transfection in HT-1080 and NIH3T3 cells enhances the number of in-vasive cells compared with controls when ana-lyzed using a modified Boyden chamber assay [2]. Also,high level of MT1-MMP expression has been associated with invasiveness of human cervi-cal cancer cells [30]. We have also found a close association of fibroblastoid features,expression of MTI-MMP and gelatinase A and the invasive phenotype in bronchial cancer cell lines [31].Of particular interest is the observation of Nakahara et al.[32] concerning the preferential localization of MTI-MMP in invadopodia of melanoma cells required for cell invasion. The transmembrane domain of the enzyme mediates the spatial organization of MTI-MMP into invadopodia and subsequent degradation of the extracellular matrix. In contrast, overexpression of MT1-MMP without invadopodial docking causes acti-vation of soluble progelatinase A, but does not facilitate extracellular matrix degradation and cell invasion. Using a mouse lung carcinoma cell line Madison 109 transfected with MT1-MMP cDNA, Tsunezuka et al. [33] have observed that the survival rate of these cells was increased by three-fold compared to parental cells. The num-ber of lung metastatic nodules in mice injected increased accordingly.Thesc nodulcs cxprcssing MTI-MMP were positive for gelatinase A whereas MTI-MMP negative cells were not stained for gelatinase A. These observations sup-port the results discussed above demonstrating that MTI-MMP expressing cells acquire specific ability to bind exogenous gelatinase A.All these data emphasize the key role of MTI-MMP in the acquisition of an invasive phenotype by tumor cells.
In vivo,an elevated expression of MT1-MMP has been reported in various human carcinomas of the uterine cervix [30], the stomach [34,35], the lung [2,36][37,38],the breast [16,37,39,40J. the colon [39]. the head and neck [39,41] and in

malignant brain tumors [42]. MT2-MMP expression has been described in breast carcinomas [40] whereas MT3-MMP is not often observed in tumor tissue. MT4-MMP mRNAs have been detected in breast carcinomas [5].This high expression of MT1-MMP is significantly observed in malignant lesions compared to benign proliferations. It is generally associated with gelatinase A activation and coexpression of MTI-MMP and gelatinase A in tumor increases their aggressiveness [36,38]. The immunohisto-chemical detection of MTI-MMP in various car-cinomas revealed the presence of this MMP in both tumor and stromal cells. Ohtani et al. [35] have shown a dual overexpression pattern of MTI-MMP in cancer and stromal ceils in human gastrointestinal carcinomas with in situ hybridiz-ation and electron microscopy. Immunoelectron microscopy showed that MTI-MMP was loca-lized along the plasma membrane of cancer cells. whereas it was detected in rough endoplasmic reticulum in stromal cells. These authors have suggested that the expression of MT1-MMP in cancer cells may be related to the invasive growth and to tissue remodeling process in fibroblasts. However,many authors have described, using in situ hybridization,that the expression of mRNAs encoding MTI-MMP is mostly in stromal cells in some tumors (cited above). The immunohisto-chemical detection of MT1-MMP in both tumor and stromal cells in these carcinomas could be

Fig.I.In situ hybridization reveals the presence of MTI-MMP mRNAs in fibroblasts close to tumor cells (T) in invi-sive lung carcinoma.Bar=120 μm. 
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M.Polette.P. Birembaut The International Journal of Biochemistry &Cell Biology 30 (1998) 1195-1202

Fig.2. MT1-MMP mRNAs are localized in fibroblasts (F) close to tumor cells(T) in some preinvasive areas of breast cancers with in situ hybridization,whereas they are absent in fibroblasts surrounding normal breast residual tissue (N). Bar=120μm.
Table 2
Principal steps of tumor invasion.(1)In the earlier stages of tumor progression, stromal MMPs may degrade basement membrane (BM) and stroma extracellular matrix allowing tumor cell invasion.These peritumoral fibroblats are induced to express MTI-MMP and gelatinase A under the presence of membranous or diffusible factors produced by preinvasive(a) and invasive tumor cells (b). (2) At the metastatic stage, some fibroblastoid isolated tumor cells (c) which acquired an effec-tive migratory status produce their own degradative enzymes facilitating their penetration into blood vessels and their im-plantation at the secondary organ

explained by a low expression of this enzyme in tumor cells undetectable by in situ hybridization. In our own experience on breast and lung tumors, MTl-MMP mRNA expression has been essentially found in fibroblasts in close contact to malignant cells [37,38](Fig.1). This MTI-MMP stromal induction near preinvasive and invasive tumors clusters support the release of tumor-derived diffusibles factors enhancing tumor in-vasion. In the preinvasive areas, MTl-MMP transcripts had a focal stromal localiation suggesting a specific regulation by certain tumor cells (Fig.2).These foci may represent hot spots for local degradation of the extracellular matrix leading to the local tumor cell invasion. We also observed that MT1-MMP, gelatinase A and TIMP2 mRNAs are expressed by the same stro-mal cells in some preinvasive and invasive areas emphasizing the close interrelations between these molecules for the activation of progelati-nase A. All these observations underline the major rolc of fibroblasts in extracellular matrix degradation and in tumor invasion.
6.Conclusion
All these data confirm the important role of MMPs and especially MT-MMPs in tumor invasion and in tumor and stromal cells co-operation. The differential pattern of MT-MMPs expression (fibroblasts, tumor cells) may rep-resent sequential stages towards the invasive phenotype. The MTI-MMP stromal expression, influenced by the presence of diffusible factors emanating from proximal preinvasive and invasive tumor nests, could occur in the first step of tumor invasion while MT1-MMP tumor cell production may be associated to the acquisition of a fully metastatic phenotype by these cells (Table 2).These enzymes intervene specifically at various steps of this process, by activating pro-gelatinase A and degrading directly extracellular matrix components. All the substrates of MT-MMPs are not known and their function may be more extensive,facilitating angiogenesis and/or tumor growth. 
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Acknowledgement
The Lions Club of Soissons is gratefully acknowledged for their financial support of our works on MMPs.
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