N-WASP coordinates the delivery and F-actin–mediated capture of MT1-MMP at invasive pseudopods

Metastasizing tumor cells use matrix metalloproteases, such as the transmembrane collagenase MT1-MMP, together with actin-based protrusions, to break through extracellular matrix barriers and migrate in dense matrix. Here we show that the actin nucleation–promoting protein N-WASP (Neural Wiskott-Aldrich syndrome protein) is up-regulated in breast cancer, and has a pivotal role in mediating the assembly of elongated pseudopodia that are instrumental in matrix degradation. Although a role for N-WASP in invadopodia was known, we now show how N-WASP regulates invasive protrusion in 3D matrices. In actively invading cells, N-WASP promoted trafficking of MT1-MMP into invasive pseudopodia, primarily from late endosomes, from which it was delivered to the plasma membrane. Upon MT1-MMP’s arrival at the plasma membrane in pseudopodia, N-WASP stabilized MT1-MMP via direct tethering of its cytoplasmic tail to F-actin. Thus, N-WASP is crucial for extension of invasive pseudopods into which MT1-MMP traffics and for providing the correct cytoskeletal framework to couple matrix remodeling with protrusive invasion.     

Actin-associated protein palladin promotes tumor cell invasion by linking extracellular matrix degradation to cell cytoskeleton

Basal-like breast carcinomas, characterized by unfavorable prognosis and frequent metastases, are associated with epithelial-to-mesenchymal transition. During this process, cancer cells undergo cytoskeletal reorganization and up-regulate membrane-type 1 matrix metalloproteinase (MT1-MMP; MMP14), which functions in actin-based pseudopods to drive invasion by extracellular matrix degradation. However, the mechanisms that couple matrix proteolysis to the actin cytoskeleton in cell invasion have remained unclear. On the basis of a yeast two-hybrid screen for the MT1-MMP cytoplasmic tail-binding proteins, we identify here a novel Src-regulated protein interaction between the dynamic cytoskeletal scaffold protein palladin and MT1-MMP. These proteins were coexpressed in invasive human basal-like breast carcinomas and corresponding cell lines, where they were associated in the same matrix contacting and degrading membrane complexes. The silencing and overexpression of the 90-kDa palladin isoform revealed the functional importance of the interaction with MT1-MMP in pericellular matrix degradation and mesenchymal tumor cell invasion, whereas in MT1-MMP–negative cells, palladin overexpression was insufficient for invasion. Moreover, this invasion was inhibited in a dominant-negative manner by an immunoglobulin domain–containing palladin fragment lacking the dynamic scaffold and Src-binding domains. These results identify a novel protein interaction that links matrix degradation to cytoskeletal dynamics and migration signaling in mesenchymal cell invasion.     

Secretion of active membrane type 1 matrix metalloproteinase (MMP-14) into extracellular space in microvesicular exosomes

Membrane type 1 matrix metalloproteinase (MT1-MMP, MMP14) is an efficient extracellular matrix (ECM) degrading enzyme that plays important roles in tissue homeostasis and cell invasion. Like a number of type I membrane proteins, MT1-MMP can be internalized from the cell surface through early and recycling endosomes to late endosomes, and recycled to the plasma membrane. Late endosomes participate in the biogenesis of small (30-100 nm) vesicles, exosomes, which redirect plasma membrane proteins for extracellular secretion. We hypothesized that some of the endosomal MT1-MMP could be directed to exosomes for extracellular release. Using cultured human fibrosarcoma (HT-1080) and melanoma (G361) cells we provide evidence that both the full-length 60 kDa and the proteolytically processed 43 kDa forms of MT1-MMP are secreted in exosomes. The isolated exosomes were identified by their vesicular structure in electron microscopy and by exosomal marker proteins CD9 and tumor susceptibility gene (TSG101). Furthermore, exosomes contained beta1-integrin (CD29). The exosomes were able to activate pro-MMP-2 and degrade type 1 collagen and gelatin, suggesting that the exosomal MT1-MMP was functionally active. The targeting of MT1-MMP in exosomes represents a novel mechanism for cancer cells to secrete membrane type metalloproteolytic activity into the extracellular space.     

SNAP23, Syntaxin4, and vesicle-associated membrane protein 7 (VAMP7) mediate trafficking of membrane type 1–matrix metalloproteinase (MT1-MMP) during invadopodium formation and tumor cell invasion

Movement through the extracellular matrix (ECM) requires cells to degrade ECM components, primarily through the action of matrix metalloproteinases (MMPs). Membrane type 1–matrix metalloproteinase (MT1-MMP) has an essential role in matrix degradation and cell invasion and localizes to subcellular degradative structures termed invadopodia. Trafficking of MT1-MMP to invadopodia is required for the function of these structures, and here we examine the role of N-ethylmaleimide–sensitive factor–activating protein receptor (SNARE)–mediated membrane traffic in the transport of MT1-MMP to invadopodia. During invadopodium formation in MDA-MB-231 human breast cancer cells, increased association of SNAP23, Syntaxin4, and vesicle-associated membrane protein 7 (VAMP7) is detected by coimmunoprecipitation. Blocking the function of these SNAREs perturbs invadopodium-based ECM degradation and cell invasion. Increased level of SNAP23-Syntaxin4-VAMP7 interaction correlates with decreased Syntaxin4 phosphorylation. These results reveal an important role for SNARE-regulated trafficking of MT1-MMP to invadopodia during cellular invasion of ECM.     

A RAB5/RAB4 recycling circuitry induces a proteolytic invasive program and promotes tumor dissemination

The mechanisms by which tumor cells metastasize and the role of endocytic proteins in this process are not well understood. We report that overexpression of the GTPase RAB5A, a master regulator of endocytosis, is predictive of aggressive behavior and metastatic ability in human breast cancers. RAB5A is necessary and sufficient to promote local invasion and distant dissemination of various mammary and nonmammary tumor cell lines, and this prometastatic behavior is associated with increased intratumoral cell motility. Specifically, RAB5A is necessary for the formation of invadosomes, membrane protrusions specialized in extracellular matrix (ECM) degradation. RAB5A promotes RAB4- and RABENOSYN-5–dependent endo/exocytic cycles (EECs) of critical cargos (membrane-type 1 matrix metalloprotease [MT1-MMP] and β3 integrin) required for invadosome formation in response to motogenic stimuli. This trafficking circuitry is necessary for spatially localized hepatocyte growth factor (HGF)/MET signaling that drives invasive, proteolysis-dependent chemotaxis in vitro and for conversion of ductal carcinoma in situ to invasive ductal carcinoma in vivo. Thus, RAB5A/RAB4 EECs promote tumor dissemination by controlling a proteolytic, mesenchymal invasive program.     

The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA

Invadopodia are actin-based membrane protrusions formed at contact sites between invasive tumor cells and the extracellular matrix with matrix proteolytic activity. Actin regulatory proteins participate in invadopodia formation, whereas matrix degradation requires metalloproteinases (MMPs) targeted to invadopodia. In this study, we show that the vesicle-tethering exocyst complex is required for matrix proteolysis and invasion of breast carcinoma cells. We demonstrate that the exocyst subunits Sec3 and Sec8 interact with the polarity protein IQGAP1 and that this interaction is triggered by active Cdc42 and RhoA, which are essential for matrix degradation. Interaction between IQGAP1 and the exocyst is necessary for invadopodia activity because enhancement of matrix degradation induced by the expression of IQGAP1 is lost upon deletion of the exocyst-binding site. We further show that the exocyst and IQGAP1 are required for the accumulation of cell surface membrane type 1 MMP at invadopodia. Based on these results, we propose that invadopodia function in tumor cells relies on the coordination of cytoskeletal assembly and exocytosis downstream of Rho guanosine triphosphatases.     

Inhibition of β1 integrin induces its association with MT1-MMP and decreases MT1-MMP internalization and cellular invasiveness

Integrin signaling plays a fundamental role in the establishment of focal adhesions and the subsequent formation of invadopodia in malignant cancer cells. Invadopodia facilitate localized adhesion and degradation of the extracellular matrix (ECM), which promote tumour cell invasion and metastasis. Degradation of ECM components is often driven by membrane type-1 matrix metalloproteinase (MT1-MMP), and we have recently shown that regulation of enzyme internalization is dependent on signaling downstream of β1 integrin. Phosphorylation of the cytoplasmic tail of MT1-MMP is required for its internalization and delivery to Rab5-marked early endosomes, where it is then able to be recycled to new sites of invadopodia formation and promote invasion. Here we found that inhibition of β1 integrin, using the antibody AIIB2, inhibited the internalization and recycling of MT1-MMP that is necessary to support long-term cellular invasion. MT1-MMP and β1 integrin were sequestered at the cell surface when β1-integrin was inhibited, and their association under these conditions was detected using immunoprecipitation and mass spectrometry analyses. Sequestration of β1 integrin and MT1-MMP at the cell surface resulted in the formation of large invadopodia and local ECM degradation; however, the impaired internalization and recycling of MT1-MMP and β1 integrin ultimately led to a loss of invasive behaviour.     

Co-recycling of MT1-MMP and MT3-MMP through the trans-Golgi network. Identification of DKV582 as a recycling signal

Members of the membrane-type matrix metalloproteinases (MT-MMPs) have been implicated in a wide range of physiological and pathological processes from normal development to tumor growth. Tethered on plasma membrane, these enzymes are potentially regulated by the trafficking machinery of the cells. Here we demonstrate that both MT1-MMP and MT3-MMP are internalized, transported to the trans-Golgi network through early endosomes, and recycled back to cell surface in 60 min in a manner distinct from the one employed by transferrin receptor. Interestingly, co-expressed MT1-MMP and MT3-MMP are localized and routed in the same vesicles throughout the trafficking process. We further demonstrated that the carboxyl-terminal sequence DKV(582) of MT1-MMP is required for its recycling, thus defining a novel recycling motif. These results suggest that MT-MMPs may coordinate their proteolytic activities through the cellular trafficking machinery.     

Proactive for invasion: Reuse of matrix metalloproteinase for structural memory

Migratory cells translocate membrane type-1 matrix metalloproteinase (MT1-MMP) to podosomes or invadosomes to break extracellular matrix barriers. In this issue, El Azzouzi et al. (2016. J. Cell. Biol. http://dx.doi.org/10.1083/jcb.201510043) describe an unexpected function for the MT1-MMP cytoplasmic domain in imprinting spatial memory for podosome reformation via assembly in membrane islets.Invasion of most normal and cancer cells across basement membranes and collagen-rich interstitial tissues involves degradation of the ECM by membrane type-1 matrix metalloproteinase (MT1-MMP/MMP14; Willis et al., 2013). To fulfill this activity, MT1-MMP is transported to podosomes, the specialized ECM-degrading membrane protrusions found in highly migratory cells such as activated macrophages, osteoclasts, endothelial cells, and smooth muscle cells (Murphy and Courtneidge, 2011). In cancer cells, MT1-MMP is transported to ECM-degrading invasive structures called invadopodia (Poincloux et al., 2009). Both these membrane protrusions, collectively called invadosomes, are composed of an actin-rich core surrounded by scaffold and adhesion proteins, and numerous mechanisms of invadosome assembly, maturation, and dynamics have been identified (Poincloux et al., 2009; Murphy and Courtneidge, 2011). MT1-MMP activity is regulated at multiple levels to achieve targeted ECM degradation, cell surface protein processing, and protease activation (Sato et al., 1994; Osenkowski et al., 2004; Sugiyama et al., 2013; Willis et al., 2013; Itoh, 2015). Potential regulatory functions of MT1-MMP toward the cytoskeleton have, however, remained unclear. In this issue, El Azzouzi et al. describe an unexpected and novel function for MT1-MMP that goes beyond its traditional proteolytic activity: they show that MT1-MMP accumulates in membrane islets that provide macrophages with spatial information, or memory, in sites of podosome dissolution so as to enable efficient podosome reassembly.El Azzouzi et al. (2016) first used total internal reflection fluorescence microscopy and a pH-sensitive version of MT1-MMP devised to fluoresce only when the MT1-MMP ectodomain is exposed to the extracellular environment’s pH. With this approach, they show that, on the ventral surface of cultured human macrophages, MT1-MMP is localized at two different membrane compartments: underneath the podosome core, as previously suggested based on matrix degradation and colocalization with podosome markers, and in distinct islets devoid of other podosome components, CD44, or integrin-mediated adhesion to the ECM (Fig. 1; Osiak et al., 2005). MT1-MMP islets were dependent on intact cortical actin, but became more apparent and persisted after podosome disruption by pharmacological perturbation of key components of podosome assembly and maturation, such as integrin adhesion, Src kinase activity, and the Arp2/3 complex essential for actin nucleation and branched actin cytoskeleton. Podosomes often reemerge at sites of previous podosome localization, and El Azzouzi et al. (2016) hypothesized that MT1-MMP islets might mark sites of podosome formation. They treated cells with an Arp2/3 inhibitor to disrupt podosomes and induce the appearance of MT1-MMP membrane clusters, and used time-lapse imaging to track what happens upon washout and podosome reformation. Interestingly, they show that these novel MT1-MMP structures serve as remarkably immobile cell membrane anchors capable of rerecruiting the podosomal actin cores/scaffolds to the same islets.Open in a separate windowFigure 1.MT1-MMP islets as memory sites for podosome reformation. Migratory cells translocate MT1-MMP (red) to podosomes or invadosomes to degrade the ECM (green fibers). These membrane structures are composed of an actin-rich core (brown) surrounded by adhesion and scaffold proteins (beige) such as integrins (blue). El Azzouzi et al. (2016) show a function for MT1-MMP accumulation in membrane ”islets” (1), where they imprint spatial memory for podosome reemergence after podosome disassembly (2). Unlike dynamic mature podosomes (3), MT1-MMP assembles in stable islets via anchorage to cortical actin. Future work in the fields of inflammation, cancer, and angiogenesis will need to address the nature of the cytoskeletal dynamics mediating islet formation, the involvement of microtubules in islet formation, the exact islet protein composition, and the relevance of these memory sites to 2D or 3D environments and to other cell types beyond macrophages, including endothelial cells and invasive cancer cells.Further, by expressing mutant MT1-MMP proteins in cells silenced for the endogenous proteinase and using a podosome reformation assay (based on pharmacological dissolution of podosomes via Src inhibition, followed by podosome reformation after washout), El Azzouzi et al. (2016) pinpointed the region of MT1-MMP critical for islet formation, the LLY-sequence in its cytoplasmic domain. Moreover, when attached to the membrane by the MT1-MMP transmembrane domain, the 20–amino acid cytoplasmic tail appeared necessary and sufficient to form the islets. Considering the LLY sequence–dependent actin-binding ability of MT1-MMP (Yu et al., 2012) coupled with the observed necessity of cortical actin for islet appearance and podosome reformation, the direct interaction with unbranched cortical actin was suggested by the authors as a likely decisive mechanism for the remarkable MT1-MMP islet stabilization in podosome-free areas, although a possible indirect interaction was not ruled out. Actin binding through the MT1-MMP cytosolic tail was likewise suggested as a potential means for podosome rerecruitment by MT1-MMP memory islets.Although cortical actin is instrumental for the emergence of the spatially and temporally stable MT1-MMP islets upon podosome dissolution in macrophages and direct actin–MT1-MMP interaction has been proven in vitro and suggested as a means for retaining MT1-MMP in invadopodia, a Src-regulated interaction between MT1-MMP’s cytoplasmic domain and the actin-binding scaffold protein palladin has also been shown to regulate MT1-MMP targeting into invadopodia (Yu et al., 2012; von Nandelstadh et al., 2014). Moreover, the LLY sequence in MT1-MMP’s cytoplasmic tail harbors a Src substrate sequence and mediates an interaction between MT1-MMP and AP-2 that is important for MT1-MMP internalization and dynamics in cell migration and invasion (Uekita et al., 2001; Nyalendo et al., 2007). Intriguingly, El Azzouzi et al. (2016) did not find evidence of involvement of dynamin-dependent membrane trafficking events in the ability of MT1-MMP islets to function as memory sites. However, their results after treatment with the microtubule inhibitor nocodazole indicated that although the islets themselves remained intact, podosome reappearance was mislocalized, suggesting that microtubules contribute by as yet undefined mechanisms to the ability of MT1-MMP islets to provide spatial memory and to facilitate podosome reassembly. Therefore, further identification of drivers and specific regulatory mechanisms of the actin–MT1-MMP interaction dynamics in podosomes, of the stable actin–MT1-MMP interaction and structures in podosome-free areas, and of microtubule-dependent podosome reassembly will be of interest.A striking observation of this study is that MT1-MMP islets do not display degradative activity in matrix degradation assays. In addition, inhibition of the proteolytic activity of MT1-MMP through pharmacological agents or via an inactivating mutation did not impact islet appearance or podosome reemergence at sites of MT1-MMP clustering. Overall, on the extracellular side of the plasma membrane, the apparent lack of contact and degradation of the ECM as well as the relatively minor impact of the N-terminal MT1-MMP ectodomain on islet formation and podosome reemergence are peculiar features of the MT1-MMP islets. However, El Azzouzi et al. (2016) show evidence for somewhat impaired islet formation in cells expressing an MT1-MMP mutant lacking the entire ectodomain, and they demonstrate that endogenous MT1-MMP must be silenced for the LLY MT1-MMP mutant to disrupt islet localization. Based on these results, the authors suggest the possible influence of MT1-MMP oligomerization and of MT1-MMP–ECM binding on islet recruitment and stabilization. Nevertheless, these observations altogether indicate that the adhesive and degradative activities of MT1-MMP memory islets toward the ECM are minor and, intriguingly, do not influence the structure or function of these islets as currently characterized in 2D cultures.Furthermore, the aforementioned results raise questions about the possible contribution of the different molecular forms of MT1-MMP (e.g., cleaved or uncleaved and inhibitor bound or not) to the stabilization and podosome reassembly function of MT1-MMP islets. In cells and conditions in which MT1-MMP activity is high, MT1-MMP turnover is typically fast via autocatalytic cleavage or shedding of the N-terminal catalytic domain (Lehti et al., 1998; Yana and Weiss, 2000; Itoh et al., 2001; Osenkowski et al., 2004). After interaction with inhibitors such as tissue inhibitors of metalloproteinases, active endocytosed MT1-MMP may dissociate from the bound inhibitor to be recycled to the plasma membrane (Jiang et al., 2001; Remacle et al., 2003). However, in the absence of interaction with a protease inhibitor or collagen/matrix substrate, MT1-MMP oligomerization facilitates MT1-MMP turnover via autocatalytic inactivating cleavage (Itoh et al., 2001; Lehti et al., 2002; Osenkowski et al., 2004). In the current study, El Azzouzi et al. (2016) used MT1-MMP proteins with a pH sensor inserted N-terminally to the transmembrane domain, so that the probe is located extracellularly on the surface-exposed protease. The fluorescence signal from these constructs is not expected to be affected by proteolytic processing or shedding of the catalytic domain, so it is unclear whether the MT1-MMP proteins clustered in islets are cleaved or not. However, FRAP results showed that the turnover of MT1-MMP molecules within the islets is relatively slow. It thus remains to be clarified if and how the proteolytically active or possibly processed or protease inhibitor–bound inactive forms of MT1-MMP are stabilized in these MT1-MMP islets.As posodomes are highly dynamic protrusions, their rapid turnover implicates a constant disassembly at the rear and formation at the front of migrating macrophages. Assembly and dissassembly are known to depend on Arp2/3-mediated actin nucleation and fission of preexisting podosomes, respectively (Linder et al., 2000). Both of these mechanisms may contribute to podosome reassembly at MT1-MMP memory sites. Considering that these islets are laterally immobile and overall stable in at least unpolarized cells, it is unclear how migrating cells coordinate their actin and microtubule cytoskeletons for podosome reassembly at the front using islets formed upon podosome dissolution at the rear of the cell (Fig. 1). Moreover, the structural and functional features of MT1-MMP islets in the scenario of 3D cell–ECM microenvironments is intriguing and will need to be investigated at high resolution, as cytoskeletal dynamics, cell polarity, and matrix stiffness greatly differ in 3D tissues and matrices from the simple 2D setting of cultured cells, and all are known to influence cell behavior. Although the transient nature of these MT1-MMP islets in bridging podosome disassembly and reassembly exemplifies and reflects the efficiency of podosome reusage, probing the protein composition of these islets as well as the dynamics of podosome reassembly will likely be challenging. Future studies comparing MT1-MMP state, dynamics, reuse, and turnover in different types of invadosomes, islets, and other subcellular compartments will be instrumental to better understand how cells integrate the different types of MT1-MMP membrane structures and cell–ECM communication with other cellular signals and with drivers of cytoskeletal dynamics.The identification of the molecular mechanisms of structural and functional podosome memory are not only relevant to the fields of macrophage biology and inflammation but also more broadly to those of tissue invasion and matrix remodeling. For instance, endothelial cells, smooth muscle cells, and cancer cells are also known to target MT1-MMP to podosomes or related invadosomes. Examining MT1-MMP memory in such specialized subcellular compartments will be interesting beyond the podosome field, as the podosome counterparts in cancer cells may display and use MT1-MMP or other metalloproteinases in a similar manner. By shedding light on the mechanisms of dynamic protrusion formation and function, this paper not only opens new avenues of investigation into the cellular structures marking protrusion sites as “memory devices” but also brings about a new concept to the fields of cell invasion, angiogenesis, and cancer.     

Heat shock suppresses membrane type 1-matrix metalloproteinase production and progelatinase A activation in human fibrosarcoma HT-1080 cells and thereby inhibits cellular invasion.

Expression of membrane type-1 matrix metalloproteinase (MT1-MMP) is closely correlated with tumor invasiveness. We investigated the effect of hyperthermia on the production of MT1-MMP in human fibrosarcoma HT-1080 cells. Heat shock at 42 degrees C suppressed the production and gene expression of MT1-MMP in HT-1080 cells. Heat shock-induced suppression of MT1-MMP production resulted in the inhibition of progelatinase A (proMMP-2) activation and the increased release of tissue inhibitor of metalloproteinases 2 from cell surface. In addition, in vitro tumor invasion assay in a Matrigel model indicated that heat shock inhibited the invasive activity of HT-1080 cells. These results suggest that heat shock preferentially suppresses the production of MT1-MMP and thereby inhibits proMMP-2 activation, events which subsequently inhibit tumor invasion. Therefore, heat shock shows an anti-invasive effect along with the known mechanism of inhibiting tumor growth.     

TGF-β1 facilitates MT1-MMP-mediated proMMP-9 activation and invasion in oral squamous cell carcinoma cells

Matrix metalloproteinase (MMP)-2 and MMP-9, also known as gelatinases or type IV collagenases, are recognized as major contributors to the proteolytic degradation of extracellular matrix during tumor invasion. Latent MMP-2 (proMMP-2) is activated by membrane type 1 MMP (MT1-MMP) on the cell surface of tumor cells. We previously reported that cell-bound proMMP-9 is activated by the MT1-MMP/MMP-2 axis in HT1080 cells treated with concanavalin A in the presence of exogenous proMMP-2. However, the regulatory mechanism of proMMP-9 activation remains largely unknown. Transforming growth factor (TGF)-β1 is frequently overexpressed in tumor tissues and is associated with tumor aggressiveness and poor prognosis. In this study, we examined the role of TGF-β1 on MT1-MMP-mediated proMMP-9 activation using human oral squamous cell carcinoma cells. TGF-β1 significantly increased the expression of MMP-9. By adding exogenous proMMP-2, TGF-β1-induced proMMP-9 was activated during collagen gel culture, which was suppressed by the inhibition of TGF-β1 signaling or MT1-MMP activity. This MT1-MMP-mediated proMMP-9 activation was needed to facilitate TGF-β1-induced cell invasion into collagen gel. Thus, TGF-β1 may facilitate MT1-MMP-mediated MMP-9 activation and thereby stimulate invasion of tumor cells in collaboration with MT1-MMP and MMP-2.     

Involvement of syntaxin 4 in the transport of membrane-type 1 matrix metalloproteinase to the plasma membrane in human gastric epithelial cells

Membrane-type 1 matrix metalloproteinase (MT1-MMP) localized on the plasma membrane plays a central role in various normal biological responses including tissue remodeling, wound heeling, and angiogenesis and in cancer cell invasion and metastasis, by functioning as a collagenase and activating other matrix metalloproteinases. In order to elucidate the molecular mechanism of the MT1-MMP targeted localization on the plasma membrane, we examined the participation of syntaxin proteins in MT1-MMP intracellular transport to the plasma membrane in human gastric epithelial AGS cells. Western blotting showed that syntaxin 3 and 4 proteins, which are known to function in intracellular transport towards the plasma membrane, were expressed in AGS cells. Immunocytochemistry revealed that transient transfection of AGS cells with dominant-negative mutant syntaxin 4 decreased plasma membrane MT1-MMP expression. In contrast, transient transfection with either dominant-negative mutant syntaxin 3 or 7 did not affect MT1-MMP localization on the plasma membrane. Cell surface biotinylation assay and Matrigel chamber assay demonstrated that stable transfection with dominant-negative mutant syntaxin 4 decreased the amount of MT1-MMP on the plasma membranes and inhibited the cell invasiveness. We suggest that syntaxin 4 is involved in the intracellular transport of MT1-MMP toward the plasma membrane.     

Metalloproteinase MT1-MMP islets act as memory devices for podosome reemergence

Podosomes are dynamic cell adhesions that are also sites of extracellular matrix degradation, through recruitment of matrix-lytic enzymes, particularly of matrix metalloproteinases. Using total internal reflection fluorescence microscopy, we show that the membrane-bound metalloproteinase MT1-MMP is enriched not only at podosomes but also at distinct “islets” embedded in the plasma membrane of primary human macrophages. MT1-MMP islets become apparent upon podosome dissolution and persist beyond podosome lifetime. Importantly, the majority of MT1-MMP islets are reused as sites of podosome reemergence. siRNA-mediated knockdown and recomplementation analyses show that islet formation is based on the cytoplasmic tail of MT1-MMP and its ability to bind the subcortical actin cytoskeleton. Collectively, our data reveal a previously unrecognized phase in the podosome life cycle and identify a structural function of MT1-MMP that is independent of its proteolytic activity. MT1-MMP islets thus act as cellular memory devices that enable efficient and localized reformation of podosomes, ensuring coordinated matrix degradation and invasion.     

MT1-MMP Initiates Activation of pro-MMP-2 and Integrin αvβ3 Promotes Maturation of MMP-2 in Breast Carcinoma Cells

We evaluated cellular mechanisms involved in the activation pathway of matrix prometalloproteinase-2 (pro-MMP-2), an enzyme implicated in the malignant progression of many tumor types. Membrane type-1 matrix metalloproteinase (MT1-MMP) cleaves the N-terminal prodomain of pro-MMP-2 thus generating the activation intermediate that then matures into the fully active enzyme of MMP-2. Our results provide evidence on how a collaboration between MT1-MMP and integrin αvβ3 promotes more efficient activation and specific, transient docking of the activation intermediate and, further, the mature, active enzyme of MMP-2 at discrete regions of cells. We show that coexpression of MT1-MMP and integrin αvβ3 in MCF7 breast carcinoma cells specifically enhances in trans autocatalytic maturation of MMP-2. The association of MMP-2′s C-terminal hemopexin-like domain with those molecules of integrin αvβ3 which are proximal to MT1-MMP facilitates MMP-2 maturation. Vitronectin, a specific ligand of integrin αvβ3, competitively blocked the integrin-dependent maturation of MMP-2. Immunofluorescence and immunoprecipitation studies supported clustering of MT1-MMP and integrin αvβ3 at discrete regions of the cell surface. Evidently, the identified mechanisms appear to be instrumental to clustering active MMP-2 directly at the invadopodia and invasive front of αvβ3-expressing cells or in their close vicinity, thereby accelerating tumor cell locomotion.     

Tissue inhibitor of metalloproteinases-2 binding to membrane-type 1 matrix metalloproteinase induces MAPK activation and cell growth by a non-proteolytic mechanism

Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with a short cytoplasmic domain and an extracellular catalytic domain, controls a variety of physiological and pathological processes through the proteolytic degradation of extracellular or transmembrane proteins. MT1-MMP forms a complex on the cell membrane with its physiological protein inhibitor, tissue inhibitor of metalloproteinases-2 (TIMP-2). Here we show that, in addition to extracellular proteolysis, MT1-MMP and TIMP-2 control cell proliferation and migration through a non-proteolytic mechanism. TIMP-2 binding to MT1-MMP induces activation of ERK1/2 by a mechanism that does not require the proteolytic activity and is mediated by the cytoplasmic tail of MT1-MMP. MT1-MMP-mediated activation of ERK1/2 up-regulates cell migration and proliferation in vitro independently of extracellular matrix proteolysis. Proteolytically inactive MT1-MMP promotes tumor growth in vivo, whereas proteolytically active MT1-MMP devoid of cytoplasmic tail does not have this effect. These findings illustrate a novel role for MT1-MMP-TIMP-2 interaction, which controls cell functions by a mechanism independent of extracellular matrix degradation.     

MT1-MMP proinvasive activity is regulated by a novel Rab8-dependent exocytic pathway

MT1-matrix metalloproteinase (MT1-MMP) is one of the most critical factors in the invasion machinery of tumor cells. Subcellular localization to invasive structures is key for MT1-MMP proinvasive activity. However, the mechanism driving this polarized distribution remains obscure. We now report that polarized exocytosis of MT1-MMP occurs during MDA-MB-231 adenocarcinoma cell migration into collagen type I three-dimensional matrices. Polarized trafficking of MT1-MMP is triggered by beta1 integrin-mediated adhesion to collagen, and is required for protease localization at invasive structures. Localization of MT1-MMP within VSV-G/Rab8-positive vesicles, but not in Rab11/Tf/TfRc-positive compartment in invasive cells, suggests the involvement of the exocytic traffic pathway. Furthermore, constitutively active Rab8 mutants induce MT1-MMP exocytic traffic, collagen degradation and invasion, whereas Rab8- but not Rab11-knockdown inhibited these processes. Altogether, these data reveal a novel pathway of MT1-MMP redistribution to invasive structures, exocytic vesicle trafficking, which is crucial for its role in tumor cell invasiveness. Mechanistically, MT1-MMP delivery to invasive structures, and therefore its proinvasive activity, is regulated by Rab8 GTPase.     

Differential regulation of membrane type 1-matrix metalloproteinase activity by ERK 1/2- and p38 MAPK-modulated tissue inhibitor of metalloproteinases 2 expression controls transforming growth factor-beta1-induced pericellular collagenolysis

Acquisition of matrix metalloproteinase-2 (MMP-2) activity is temporally associated with increased migration and invasiveness of cancer cells. ProMMP-2 activation requires multimolecular complex assembly involving proMMP-2, membrane type 1-MMP (MT1-MMP, MMP-14), and tissue inhibitor of metalloproteinases-2 (TIMP-2). Because transforming growth factor-beta1 (TGF-beta1) promotes tumor invasion in advanced squamous cell carcinomas, the role of TGF-beta1 in the regulation of MMP activity in a cellular model of invasive oral squamous cell carcinoma was examined. Treatment of oral squamous cell carcinoma cells with TGF-beta1 promoted MMP-dependent cell scattering and collagen invasion, increased expression of MMP-2 and MT1-MMP, and enhanced MMP-2 activation. TGF-beta1 induced concomitant activation of ERK1/2 and p38 MAPK, and kinase inhibition studies revealed a negative regulatory role for ERK1/2 in modulating acquisition of MMP-2 activity. Thus, a reciprocal effect on proMMP-2 activation was observed whereupon blocking ERK1/2 phosphorylation promoted proMMP-2 activation and MT1-MMP activity, whereas inhibiting p38 MAPK activity decreased proteolytic potential. The cellular mechanism for the control of MT1-MMP catalytic activity involved concurrent reciprocal modulation of TIMP-2 expression by ERK1/2 and p38 MAPKs, such that inhibition of ERK1/2 phosphorylation decreased TIMP-2 production, and down-regulation of p38 MAPK activity enhanced TIMP-2 synthesis. Further, p38 MAPK inhibition promoted ERK1/2 phosphorylation, providing additional evidence for cross-talk between MAPK pathways. These observations demonstrate the complex reciprocal effects of ERK1/2 and p38 MAPK in the regulation of MMP activity, which could complicate the use of MAPK-specific inhibitors as therapeutic agents to down-regulate the biologic effects of TGF-beta1 on pericellular collagen degradation and tumor invasion.     

MT-LOOP-dependent Localization of Membrane Type I Matrix Metalloproteinase (MT1-MMP) to the Cell Adhesion Complexes Promotes Cancer Cell Invasion

Localization of membrane type I matrix metalloproteinase (MT1-MMP) to the leading edge is thought to be a crucial step during cancer cell invasion. However, its mechanisms and functional impact on cellular invasion have not been clearly defined. In this report, we have identified the MT-LOOP, a loop region in the catalytic domain of MT1-MMP (¹⁶³PYAYIREG¹⁷⁰), as an essential region for MT1-MMP to promote cellular invasion. Deletion of the MT-LOOP effectively inhibited functions of MT1-MMP on the cell surface, including proMMP-2 activation, degradation of gelatin and collagen films, and cellular invasion into a collagen matrix. This is not due to loss of the catalytic function of MT1-MMP but due to inefficient localization of the enzyme to β1-integrin-rich cell adhesion complexes at the plasma membrane. We also found that an antibody that specifically recognizes the MT-LOOP region of MT1-MMP (LOOP_Ab) inhibited MT1-MMP functions, fully mimicking the phenotype of the MT-LOOP deletion mutant. We therefore propose that the MT-LOOP region is an interface for molecular interactions that mediate enzyme localization to cell adhesion complexes and regulate MT1-MMP functions. Our findings have revealed a novel mechanism regulating MT1-MMP during cellular invasion and have identified the MT-LOOP as a potential exosite target region to develop selective MT1-MMP inhibitors.     

Prointegrin maturation follows rapid trafficking and processing of MT1-MMP in Furin-Negative Colon Carcinoma LoVo Cells

Understanding the function of invasion-promoting membrane type-1 matrix metalloproteinase (MT1-MMP) is of paramount importance for understanding cancer biology. MT1-MMP is synthesized in cells as a latent zymogen that requires the cleavage of its prodomain to exert the proteolytic activity. The mature alphav integrin subunit is also generated by endoproteolytic cleavage of the alphav subunit precursor (pro-alphav). Cleavage by furin is considered to be a principal event in the activation of both MT1-MMP and pro-alphav. To elucidate the alternative activation pathway of MT1-MMP and pro-alphav, we employed furin-negative LoVo cells, which co-express MT1-MMP with integrin alphavbeta3. In these cells the MT1-MMP proenzyme was rapidly trafficked to the plasma membrane via an unconventional Brefeldin A-resistant pathway and, then, autocatalytically processed on the cell surface. Next, the MT1-MMP activity converted the cell surface-associated pro-alphav into the mature alphav integrin, represented by the disulfide-bonded heavy and light chains, and promoted the formation of the functional integrin alphavbeta3 heterodimer. These events stimulated cell motility in vitro, and malignant invasion and tumor growth in vivo. Our data suggest that in furin-negative colon carcinoma cells MT1-MMP is autocatalytically processed and the active protease then operates as a prointegrin convertase. Our findings argue strongly that the processing by furin is not a prerequisite for the activation of MT1-MMP.