Cancer and Metastasis Reviews 22: 129–143, 2003. # 2003 Kluwer Academic Publishers. Manufactured in The Netherlands.

Role of pericellular proteolysis by membrane-type 1 matrix metalloproteinase in cancer invasion and angiogenesis Motoharu Seiki*, Naohiko Koshikawa, and Ikuo Yana Division of Cancer Cell Research, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokane-dai, Minato-ku, Tokyo 108–8639, Japan

Key words: pericellular proteolysis, MT1-MMP, MMP-2, cancer, invasion and metastasis, migration Summary Membrane-type 1 matrix metalloproteinase (MT1-MMP) is an integral membrane proteinase that is frequently expressed in malignant cancer cells and has potent invasion-promoting activity. When expressed on the cell surface, MT1-MMP degrades the extracellular matrix (ECM) barrier adjacent to the cells to maintain the migration route to traverse the tissue. But MT1-MMP is not just an enzyme that degrades ECM. MT1-MMP also introduces limited cleavage into proteins at the cell-ECM interspaces and converts their functions. The target molecules are ECM components, cell adhesion molecules, and latent forms of MMPs. Through these processing events MT1-MMP modulates the migratory and invasive behavior of the cells.

Introduction Matrix metalloproteinases (MMPs) are a family of zinc-binding endopeptidases that collectively degrade most of the components of the extracellular matrix (ECM) and have been implicated in cancer invasion and metastasis [1–3]. The major contribution of MMPs to cancer invasion has been thought to be to degrade the ECM barrier in the direction of invasion. To carry out such a function, MMPs are expected to act at the leading edge of the invading cancer cells. MT1-MMP was identified as the first membrane-anchored type MMP acting as a key enzyme responsible for the degradation of the pericellular ECM [4,5]. Before the discovery of MT1-MMP, two types of type IV collagenases (MMP-2 and MMP-9) were the major focus as invasion-promoting MMPs [6]. Even though these MMPs are soluble enzymes, they were expected to * Corresponding author. E-mail: [email protected]

associate with the surface of invasive cancer cells and carry out their actions there. Indeed, type IV collagenase activity was found to associate with the cancer cell surface [7–9]. Notably, proMMP-2 was activated specifically in a cell-mediated manner [10]. Interestingly, MMP-2 was mainly immunolocalized to cancer cell nests in tissue while mRNA signals were mainly found in surrounding stromal fibroblasts [11]. Thus, it was of interest to us how proMMP-2 produced by fibroblasts binds to cancer cells and is activated there. MT1-MMP provided the answer. The expression of MT1-MMP conferred to the cell the ability to bind and to activate proMMP-2 [4,12,13]. Many groups started to elucidate the expression, function, regulation of MT1-MMP and their relevance to cancer invasion and metastasis. Now MT1-MMP is one of the best characterized MMPs, and here, we summarize recent findings on the enzyme.

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Structure of MT1-MMP MT1-MMP (MMP-14) is the first membrane type MMP identified as an activator of proMMP-2 on the cancer cell surface [4]. In addition to MT1MMP, five MT-MMPs are now known, namely MT2-MMP (MMP15) [14], MT3-MMP (MMP16) [15], MT4-MMP (MMP17) [16,17], MT5-MMP (MMP24) [18,19], and MT6-MMP (MMP-25) [20,21]. As with all MMPs, latent forms of MTMMPs (proMT-MMPs) have a common domain structure composed of a propeptide, catalytic domain, hinge, and hemopexin-like domain (PEX) (Figure 1). In addition to the common structure, MT-MMPs have a sequence that anchors the enzyme to the plasma membrane at the C-terminus. This is either a type I transmembrane domain with a short cytoplasmic tail (MT1, MT2, MT3, and MT5-MMP) [2,22] or a signal sequence for a glycosyl phosphatidyl inositol (GPI) anchor (MT4 and MT6-MMP) [23,24]. The existence of the membrane-anchoring sequence at the C-terminus clearly differentiates MT-MMPs from other soluble MMPs except MMP-23 which anchors to the plasma membrane through a type II transmembrane sequence in the propeptide [25,26]. In the propeptide of MTMMPs, there is a conserved motif of YGYL [27] and a four basic amino acid motif [4], acting as an intramolecular chaperon and a processing site for

furin or related proteinases, respectively. There is also an eight amino acid insertion in the catalytic domain of MT1, 2, and 3, as well as MT5-MMP [5] and mutations of this sequence impaired the enzymatic activity of MT1-MMP [28]. Among the six MT-MMPs, GPI-anchored MT4-MMP and MT6-MMP are clearly different from the others in their amino acid sequence conservation and biochemical properties [2].

Activation and inhibition MT1-MMP is expressed in a latent form (proMT1-MMP) and the propeptide sequence has to be cleaved off to generate an active enzyme [29]. Immediately upstream of the processing site, there is a basic amino acid motif (RXR/KR) that acts as a recognition site for furin or related intracellular serine proteinases acting as proprotein convertases (PCs) [4,29–31]. Similar motifs for PCs are found in eight MMPs (e.g. MT1-6 MMPs, MMP11, and MMP23) [2] and latent forms of these enzymes are likely to be activated intracellullarly during secretion. However, alternative PCindependent activation pathways may also exist [32–35]. Nevertheless, processing at the furin site is presumably the major way to activate proMT1MMP, because the active form of MT1-MMP purified from cells was found to have N-terminal

Figure 1. Domain structure of MT-MMPs. The common domains of proMT-MMPs is composed of a propeptide, a catalytic domain, linker sequence 1 (L-1), a hemopexine-like (PEX) domain composed of four repeated units, linker sequence 2 (L-2) from the N– terminus, and they have either a type I transmembrane sequence (TM) with a short cytoplasmic tail or GPI. Characteristic insertions to this group of enzymes are indicated at the upside of the illustration. They are YGYL, RXKR (furin motif), 8 amino acid insertion (8AA), transmembrane sequence TM, and cytoplasmic tail. Instead of the TM, the translation product of MT4MMP and MT6-MMP has a hydrophobic sequence at the C-terminus which is then cleaved and substituted with a GPI moiety. The propeptide sequence has to be cleaved off to generate active enzyme and this processing event is carried out intracellularly by proprotein convertases.

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sequence corresponding to the processed one at the furin site [36,37]. Conservation of the furin motif at the processing site among the related enzymes also indicates the importance of this activation mechanism. Active MT1-MMP can be inhibited by tissue inhibitors of metalloproteinases (TIMPs) by forming a 1:1 complex [36–38]. Notably however, TIMP-1 cannot inhibit MT1-MMP and other transmembrane-type MT-MMPs (MT2, MT3 and MT5-MMP). Since TIMP-2 is expressed ubiquitously in almost every tissue as a soluble form, it acts as a major inhibitor for MT1-MMP. TIMP-2 does not act merely as an inhibitor for MT1-MMP, but also acts as an adaptor to mediate the binding of proMMP-2 to MT1MMP and promotes the activation of proMMP2 as discussed later. Another interesting inhibitor for MT1-MMP is RECK which was originally isolated due to its activity to cause reversion of the transformed phenotype of K-ras-transfected cells, and later found to act as a GPI-anchored MMP inhibitor [39,40]. Mice lacking the RECK gene die at around E10.5 showing deficiency in ECM turnover and vascular development [40]. Testican 3 including its variant N-Tes [41] and claudin [42] were identified as modulators of MT1-MMP activity by expression-cloning and the former show suppressor activity, while the latter enhances the activity.

Extracellular targets To promote cancer invasion, MT1-MMP has to degrade the ECM barrier. MT1-MMP can digest fibronectin, vitronectin, laminin-1 and -5, fibrin and dermatan sulfate proteoglycans [30,43–46]. The enzyme also degrades gelatin, casein and elastin [30,37,47] and shows activity against collagens type I, II and III [46]. A deficiency of MT1-MMP in mice also emphasized the importance of the degradation of ECM by MT1-MMP during development [48,49]. The animal showed inadequate collagen turnover, resulting in dwarfism, osteopenia, arthritis, and connective tissue disease [48]. Digestion of these ECM components by MT1-MMP does not necessarily mean degradation. Limited digestion of laminin-5 at the g2

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chain causes functional conversion of the molecule to a form that promotes cell migration [44]. In addition to the ECM components, MT1MMP digests cell adhesion molecules as well. CD44, a major hyaluronan receptor, is cleaved by MT1-MMP and its ectodomain is released from the cell surface accompanying the stimulation of cell migration [50]. Pro-alpha v integrin subunit is also processed by MT1-MMP into a functional mature form [51]. MT1-MMP is shown to degrade a tissue transglutaminase that acts as an integrinbinding adhesion co-receptor for fibronectin [52]. Since cell adhesion molecules bind the ECM and mediate extracellular signaling to the cells, the processing of such molecules is also a way to regulate the ECM-cell communication in addition to degradation and modulation of ECM components. MT1-MMP needs cooperation of other MMPs to degrade complex array of ECM targets. Most importantly, it activates proMMP-2 on the cell surface. The activation of proMMP-2 had been reported to occur in a cell-mediated manner which is very different from that of the other proMMPs by serine proteases or active MMPs [10]. On the cell surface, MT1-MMP binds proMMP-2 using TIMP-2 as an adaptor (Figure 2). TIMP-2 is composed of two domains; the N-terminal domain has the ability to inhibit MMPs by binding at the catalytic site and the C-terminal domain specifically binds the PEX domain of MMP-2. Thus, after MT1-MMP is inhibited by TIMP-2, its Cterminal domain provides the binding site for proMMP-2 on the cell surface [36,37]. Thus, MT1MMP/TIMP-2 complex can act as a cell surface ‘receptor’ for proMMP-2. Then, the proMMP-2 in the tri-molecular complex becomes the substrate for the adjacent MT1-MMP acting as an ‘activator’ [9,53]. Thus, at least two MT1-MMP molecules that act as a ‘receptor’ and an ‘activator’ have to be close enough for the activation reaction. To remain in close proximity to each other, MT1MMP has an ability to form a homo-oligomer through the PEX domain [54,55], though the cytoplasmic portion of MT1-MMP may have an additional role in the formation [55,56]. Thus, the level of TIMP-2 on the cell surface plays a critical role in the activation of proMMP-2. Without TIMP-2, MT1-MMP cannot mediate the activation, but an excess amount of TIMP-2 inhibits all

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Figure 2. The PEX domain is the interface for protein-protein interaction. Tow MT1-MMP molecules have to be close together to activate proMMP-2. The PEX domain of MT1-MMP has an ability to form a homo-oligomer and this formation contributes efficient activation of proMMP-2 by keeping the close arrangement of the ‘receptor’ and ‘activator’ MT1-MMP molecules. The PEX domain also binds CD44. CD44 anchors actin cytoskeleton through ERM (ezrin/radixin/moesin) proteins [99] and regulated to localize at the leading edge of migrating cells [100]. The PEX of MT1-MMP binds type I collagen as well and this binding is a part of the mechanism for the specific cleavage of the substrate. After binding, the triple helical structure of the collagen near the cleavage site is unwound and each strand is cleaved successively by the activity of the catalytic domain [101].

the activity of MT1-MMP including that to activate proMMP-2 [53]. Although there appears to be some confliction between the model in which TIMP-2 plays an essential role in the activation of proMMP-2 and the role of TIMP-2 as a MMP inhibitor, there is genetic evidence that in TIMP-2deficient mice, proMMP-2 is not activated efficiently [57,58]. The activation of proMMP-2 by cancer cells is presumably important for the invasion of the basement membrane because of the type IV collagenase activity. In the type I collagen-rich environment in the stroma, MT1MMP/MMP-2 can act as a potent type I collagen degradation system employing a combination of the collagenase activity of MT1-MMP and the gelatinase activity of MMP-2. ProMMP-13 also can be activated by MT1-MMP in a cell-mediated manner and proMMP-9 indirectly [59].

Regulation of MT1-MMP Gene expression During mouse development, MT1-MMP is mainly expressed in cells of mesenchymal origin including fibroblasts, muscular cells, and osteoblasts, and the expression decreases with maturation after birth [60,61]. Expression can be re-induced, however, when the cells require remodeling of the ECM. For example, the expression of MT1-MMP

is induced in fibroblasts when tissue is damaged and it continues throughout the wound healing process together with the expression of MMP-2 [62]. MT1-MMP is also expressed in endothelial cells forming new vessels during angiogenesis [43,63–65]. In tumors, both cancer cells and surrounding stroma cells express MT1-MMP. Since normal epithelial cells, even during the wound healing process, rarely express MT1MMP [62], the genetic events that render normal epithelial cells cancerous cause the abnormal expression of MT1-MMP in cancer. Gene expression is regulated through the specific transcription factors assembled in the promoter region. The promoter sequences of MMP-1 [66], MMP-3 [67], MMP-7 [68], and MMP-9 [69] genes have a typical TATA box and binding sites for transcription factors such as AP-1, Ets, and NFkB etc. These are characteristic to the genes of which expression is induced by growth factors and cytokines. From this point of view, the MT1MMP promoter is different from these promoters [70,71] rather resembling that of the MMP-2 gene lacking a typical TATA box and binding motifs for the above mentioned transcription factors [72]. Therefore, although the expression of MT1-MMP can be induced by growth factors or TPA in some cell lines [73], it is unlikely that the gene expression is directly downstream of the signaling. There is a GC-rich sequence immediately upstream of the cluster of multiple transcription start sites and it

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contains overlapping binding sites for Egr-1 and Sp1. Expression of MT1-MMP can be induced when the cells are cultured in collagen gel. For this induction, Egr-1 was identified to play a critical role [70]. On the other hand, share stress on endothelial cells down-regulates MT1-MMP expression. Phosphorylation of Sp1 which increases its affinity to the binding site was induced by stress and resulted in a suppression of transcription even in the presence of Egr-1 [74]. Genetic changes in cancer alter gene expression that affects the cancer cell phenotype. Mutations of the APC gene are associated with a number of human cancers. Aberrant APC causes b-catenin to accumulate in the nucleus and induces the expression of a variety of genes regulated in the downstream of Wnt signaling [75]. The target genes include those for c-myc, cyclin D1, and AF17 that affect cell growth. The expression of genes related to the malignant phenotype of cancer cells is also induced by b-catenin. These include genes for CD44, laminin-5 g2 chain, MMP-7, and uPAR. MT1-MMP expression is also regulated by b-catenin at least in cancers having defects in the Wnt signaling pathway [76]. This is based on the observation that a depletion of b-catenin in colorectal carcinoma SW480 cells expressing the wild-type APC gene down-regulated the expression of MT1-MMP. The promoter region of the gene contains an element for b-catenin/Tcf4 complex binding. Transformation of MDCK cells by v-Src induces expression of MT1-MMP at transcription level as well and this is mediated by an additional v-Src-responsive element in the promoter [71].

Localization at the migration front MT1-MMP localizes at the leading edge of migrating cells and this localization aids in the degradation of the extracellular matrix barrier to facilitate invasion. A relatively stable association of MT1-MMP with the actin cytoskeleton can be seen when the cells are treated with cytochalasin D which disrupts polymerized actin and produces actin aggregates [77]. Although MT1-MMP has a cytoplasmic tail, the domain responsible for the association was mapped to the PEX domain. Thus, the association of MT1-MMP with actin

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must be mediated by some other cell surface molecule that has a cytoplasmic domain and anchors to actin. CD44, a major hyarunonan receptor that associates with actin within cells, also localizes at the leading edge and was identified as a linker that mediates the association of MT1-MMP with actin (Figure 2) [77]. The PEX domain of MT1-MMP was responsible for the formation of a complex with CD44and a mutant MT1-MMP lacking the PEX domain failed to form a complex with CD44 and localize at the leading edge (Figure 3). Cytoplasmic deletion of CD44 abolishes the association with the actin cytoskeleton and localization of the mutant at the leading edge remaining the ability to form a complex with MT1-MMP. Overexpression of the mutant CD44 prevented MT1-MMP from localizing at the edge (Figure 3). Thus, CD44 plays a critical role in the regulation of the polarized distribution of MT1MMP during cell migration and invasion and association with the actin cytoskeleton [77].

Homo-oligomer formation and MMP-2 activation The activation of proMMP-2 requires at least two MT1-MMP molecules and this is accomplished by forming a homophilic oligomer through the PEX domain [54,55]. Substitution of the PEX domain of MT1-MMP to that of the MT4-MMP which does not form homo-oligomer abolished ability to activate proMMP-2, though the chimera has an activity to bind proMMP-2 on the cells (Figure 4). How is such a formation regulated in relation to cell function? To visualize the formation in situ, a chimeric protein composed of the extracellular portion of MT1-MMP and transmembrane and cytoplasmic portion of the nerve growth factor receptor (NGF-R) was constructed [54]. The binding of a ligand to NGF-R induces receptor dimerization and causes auto-phosphorylation at the cytoplasmic tyrosine residues. Thus, if the ectodomain of the chimera formed homo-oligomers, it would cause auto-phosphorylation of the NGF-R portion in the cytoplasm. Expression of a constitutively active Rac1 in COS-1 cells induces the formation of lamellipodia and the MT1-MMP chimera was found to localize there with intense auto-phosphorylation signals indicating that homo-oligomers formed there (Figure 5). At the

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Figure 3. CD44 is important for localization of MT1-MMP at the lamellipodia. HT-1080 cells expressing either FLAG-tagged MT1MMP (MT1-F) or its deletion of the PEX domain (MT1-F/dPEX) were cultured on glass coverslips for 12 h (77). Cells were reacted with rabbit anti-CD44 antibody and anti-FLAG antibody. After being washed with PBS, the cells were treated with TPA for 30 min. Following fixation, the bound antibodies were visualized with Alexa488-conjugated anti rabbit IgG and Cy3-conjugated anti-mouse IgG. A mutant CD44H that lacks the cytoplasmic domain was constructed (CD44H/dCP). MT1-MMP þ CD44H-F/dCP or MT1F þ CD44H/dCP were expressed in HT-1080 cells. Cells were treated with TPA before fixation. FLAG-tagged CD44 (CD44H-F/dCP) or MT1-MMP (MT1F) was visualized using anti-FLAG antibody and F-actin was stained with Alexa-594-conjugated phalloidin. Scale bar, 10 mm.

same time, Rac1 enhanced the activation of MMP2 [54,78]. Thus, the ruffled edge that forms the migration front is likely the place for the formation of oligomer of MT1-MMP and activation of proMMP-2.

Down-regulation After being displayed as an active enzyme on the surface, MT1-MMP is inactivated either by binding natural inhibitors (TIMPs, RECK, and Testi-

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Figure 4. Critical role of the MT1-MMP PEX in activation of proMMP-2. A MT1-MMP chimera that has the PEX domain of MT4MMP was constructed as illustrated (MT1-MT4PEX) [54]. COS1 cells were transfected with the expression plasmids for MT1-MMP, MT1-MT4PEX, or vector alone (Mock), and purified proMMP-2 was added in the culture medium without serum. Cells were collected and analyzed by gelatin zymography to detect MMP-2 activities associated with the cells. The same cell lysate was also analyzed by Western blotting using anti-MT1Cat (middle panel, anti-Cat). Without expression of MT1-MMP, the cells did not bind proMMP-2 at all. By expressing MT1-MMP, cells bound proMMP-2 and activated it as shown by the zymography. Interestingly, the cells expressing MT1-MT4PEX could bind proMMP-2 indicating the chimera is active and can be inhibited by TIMP-2 that mediates binding of proMMP-2. However, the chimera failed to activate proMMP-2 presumably because the PEX of MT4-MMP lacks an ability to form oligomer.

can) or by proteolytic degradation. Cells expressing MT1-MMP at high levels frequently generate degraded fragments of which the major form can be detected as bands in the range of 43–45 kDa in SDS-PAGE [79–81]. This degradation is mainly caused by autocatalytic processing, and therefore, the appearance of this degradation pattern well reflects the active state of MT1-MMP on the cell surface including its ability to activate proMMP-2. How are these molecules cleared from the surface and substituted with newly synthesized molecules? MT1-MMP has a short cytoplasmic tail of 20 amino acids and this portion was found to mediate internalization of the enzyme [82,83]. The cytoplasmic tail has a binding site for the m2 subunit of adaptor protein 2 (AP2) that mediates incorporation of the target protein into a clathrin-coated pit [82]. Although the internalization is not selective for inactivated MT1-MMP molecules, it is presumably an important part of the mechanism to turnover MT1-MMP molecules.

Regulation of cell migration Invasion requires degradation of the ECM coupled with locomotion of the cell. MT1-MMP acts not only to eliminate the ECM barrier but also to modulate the migratory behavior of cells in multiple ways. Laminin-5, a major component of the basement membrane, is known to support the migration of epithelial cells and tumor cells. MMP-2 was first found to cleave the g2 chain of laminin-5 and promote the migration of breast epithelial cells [84]. Later, cells constitutively motile on laminin 5 were found to express MT1MMP rather than MMP-2 [44]. Antisense oligonucleotides against the MT1-MMP gene inhibited processing of the g2 chain and cell migration as well. Thus, cleavage of the g2 chain by MT1-MMP seems to convert it from an inactive to active form to promote migration presumably exposing a new functional domain that is cryptic before processing. This system might support the sustained

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Figure 5. In situ visualization of the oligomer formation. (A) A schematic illustration of the mutant MT1-MMP/NGFR chimeras [54]. TK, tyrosine kinase domain; Y, tyrosine residue to be phosphorylated by the TK activity. (B) COS1 cells were transfected with expression plasmids for MT1-F/NGFR þ Rac1DA or Rac1DA alone. After fixation, the cells were stained with either by anti-PY antibody (PY20) or Alexa-594-conjugated phalloidin.

locomotion of tumor cells that express both MT1MMP and the g2 chain. CD44 is also processed by MT1-MMP accompanied by cell migration [50]. Expression of a mutant CD44 that cannot be cleaved by MT1MMP prevented promotion of cell migration by MT1-MMP (Figure 6). Although the mechanism to promote cell migration by MT1-MMP is not clear, processing of CD44 may regulate cell adhesion via CD44 to the level appropriate for migration. Another scenario is that the processing stimulates signals to promote cell migration via CD44 by itself or associated molecules. Indeed,

activation of extracellular signal-regulated kinase (ERK) is observed when cell migration is induced by MT1-MMP [85]. In addition, the cytoplasmic portion of the processed CD44 is reported to be translocated into the nucleus [86] and may act as a transcription factor to induce the expression of migration-related genes. Integrins also compose a well-known adhesion system for cell migration [87]. The integrin avb3, which binds vitronectin, is expressed in endothelial cells and invasive tumor cells. The av chain, which is translated as a single polypeptide and converted into a two-chain form by PCs, is alternatively

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Figure 6. Effect of MT1-MMP expression on cell migration. (A) Human breast carcinoma ZR-75–1 cells which do not express both CD44 and MT1-MMP were transfected with the expression plasmids indicated in the figure together with the plasmid for GFP expression. CD44HM is a mutant that has a deletion of a region containing the processing site by MT1-MMP [50]. The motility of GFP positive cells were analyzed by phagokinetic track assay on colloidal gold-coated coverslips. The migrated area of the cell was visualized under dark-field illumination and migration area was measured using NIH Image. The average of 30 cells + SEM is shown. *P < 0.05 by Student’s t test. Although either expression of CD44 or MT1-MMP alone did not stimulate cell migration, co-expression of both did. Expression of CD44HM which cannot be processed by MT1-MMP failed to promote cell migration in the presence of MT1-MMP. Expression of CD44HM showed dominant effect against the wild type CD44. (B) Expression of MT1-MMP in CHO-K1 cells which express endogenous CD44 stimulated cell migration by itself. The effect depends on the proteolytic activity of MT1-MMP because catalytically-dead mutant (E/A) failed to promote cell migration. The cells expressing the transfected genes can be detected by the fluorescence of GFP as in the upper panel.

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processed by MT1-MMP as well into a functional form [88]. The expression of avb3 and MT1-MMP in human breast carcinoma MCF-7 cells did not alter the adhesion to vitronectin but stimulated migration on the matrix accompanying phosphorylation of FAK [51]. Additionally, the cell surface transglutaminase (tTG) that associates with the integrin b1 or b3 chain is also cleaved at multiple sites by MT1-MMP. tTG binds fibronectin as a co-receptor of integrins. Cleavage of tTG by MT1-MMP suppressed cell adhesion to fibronectin and migration there. On the other hand, MT1-MMP promoted migration of the same cells on type I collagen [52].

Invasion-promoting activity Expression of MT1-MMP in cancer cells enhances their invasive and metastatic potential [4,13]. Cancer cell lines that express MT1-MMP constitutively are in general more invasive than nonproducer cells [5,89]. In collagen gel, MDCK cells form a tubular structure in response to HGF. Expression of MT1MMP is induced in the cells cultured in the collagen gel and plays a critical role in the morphogenic response that requires invasion of the collagen gel [90]. Expression of excessive amounts of MT1-MMP in MDCK cells strongly enhanced their invasive property, but disturbed tubular formation [91]. In contrast, secreted MMPs including the collagen-degrading MMP-1 and a soluble form of MT1-MMP mutant failed to promote invasion [91]. Thus, membrane-anchoring appears crucial for MT1-MMP to promote invasion. The cytoplasmic tail of MT1-MMP is also important for the invasion-promoting activity in matrigel [82,92], but not in collagen gel [91]. Interestingly, mutations in the cytoplasmic tail that abolished the invasion-promoting activity of MT1-MMP coincided with those that impaired internalization [82]. These mutant enzymes retain proteolytic activity on the cell surface at comparable levels to that of the wild type MT1-MMP. Excessive accumulation of the inactivated MT1MMP molecules at the front of invading cells caused by the defect of internalization may hamper the normal function of MT1-MMP there.

Thus, the internalization might be a mechanism to maintain the position for the newly synthesized MT1-MMP by clearing away the used molecules.

Roles in angiogenesis Like tumor cells, endothelial cells transmigrate through the ECM in response to angiogenic stimuli and this process requires degradation of the ECM and cell locomotion. MT1-MMP is expressed in endothelial cells [65] and appears important for the formation of new vessels in both physiological and pathological situations [93]. Indeed, angiogenesis in a corneal ex vivo assay was impaired in MT1-MMP-deficient mice compared to that in the wild type [49]. Vasculogenesis in the embryo is normal in MT1-MMP-deficient mice, but the formation of a secondary ossification center during bone formation is impaired severely, presumably by the defect in the formation of new blood vessels there [48,49]. In collagen gel, expression of MT1-MMP is induced in endothelial cells and the collagen-degrading activity of MT1MMP is required for a tubular network to form [65,94]. MT1-MMP also shows potent fibrinolytic activity and supports the tubular formation of endothelial cells in fibrin gel in the absence of uPA/plasminogen system [43]. MMP-2 and its association with avb3 integrin on endothelial cells are also reported to be important for angiogenesis. Disruption of the interaction by a PEX fragment of MMP-2 inhibits angiogenesis in vitro and in vivo [95]. The PEX domain of MMP-2 also competes with proMMP-2 to bind to the C-terminal domain of TIMP-2 and eventually inhibits the activation of proMMP-2 by MT1-MMP. Before the MMP-2/ avb3 integrin system works, MT1-MMP has to activate proMMP-2 in advance. In addition to its direct roles in endothelial cells, MT1-MMP may modulate tumor-induced angiogenesis in the cancer cells. Tumor cells expressing MT1-MMP grow rapidly and the tumors that form are highly vascularized. The angiogenic phenotype of MT1MMP-producing cancer cells was found to be associated with the up-regulation of VEGF expression [96]. The similar vasculature-like structure formed by tumor cells constitutes a microcirculatory system in place of the vessels formed by endothelial cells

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in human uveal melanoma and this is known as vascular mimicry [97]. Both MT1-MMP and the laminin-5 g2 chain were expressed in the tumor cells forming the tubular structure and indispensable for the mimicry as demonstrated by a study using antisense technology and a neutralizing antibody [98].

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Conclusions MT1-MMP is a potent ECM-degrading enzyme that forms a part of the invasion machinery of cancer cells. To promote invasion, MT1-MMP is delivered to the leading edge of the migratory cells and assembles other MMPs with different substrate specificities to degrade complex ECM components in the basement membrane and in the stroma. MT1-MMP also modulates the migratory properties of cells by causing the functional conversion of target molecules such as CD44, the integrin av chain, tTg, and the laminin5 g2 chain. These activities have to be regulated in coordination with cell locomotion which requires the dynamic and appropriate actions of multiple molecules in time and space. These regulatory mechanisms are also potential therapeutic targets which should be exploited in addition to the appropriate use of MMP inhibitors.

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