Molecular Mechanisms Driven by MT4-MMP in Cancer Progression.

MT4-MMP (or MMP-17) belongs to the membrane-type matrix metalloproteinases (MT-MMPs), a distinct subset of the MMP family that is anchored to the cell surface, in this case by a glycosylphosphatidylinositol (GPI) motif. Its expression in a variety of cancers is well documented. However, the molecular mechanisms by which MT4-MMP contributes to tumor development need further investigation. In this review, we aim to summarize the contribution of MT4-MMP in tumorigenesis, focusing on the molecular mechanisms triggered by the enzyme in tumor cell migration, invasiveness, and proliferation, in the tumor vasculature and microenvironment, as well as during metastasis. In particular, we highlight the putative substrates processed and signaling cascades activated by MT4-MMP that may underlie these malignancy processes and compare this with what is known about its role during embryonic development. Finally, MT4-MMP is a relevant biomarker of malignancy that can be used for monitoring cancer progression in patients as well as a potential target for future therapeutic drug development.

Emerging roles of MT-MMPs in embryonic development.

Membrane-type matrix metalloproteinases (MT-MMPs) are cell membrane-tethered proteinases that belong to the family of the MMPs. Apart from their roles in degradation of the extracellular milieu, MT-MMPs are able to activate through proteolytic processing at the cell surface distinct molecules such as receptors, growth factors, cytokines, adhesion molecules, and other pericellular proteins. Although most of the information regarding these enzymes comes from cancer studies, our current knowledge about their contribution in distinct developmental processes occurring in the embryo is limited. In this review, we want to summarize the involvement of MT-MMPs in distinct processes during embryonic morphogenesis, including cell migration and proliferation, epithelial-mesenchymal transition, cell polarity and branching, axon growth and navigation, synapse formation, and angiogenesis. We also considered information about MT-MMP functions from studies assessed in pathological conditions and compared these data with those relevant for embryonic development.

E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine.

Cadherin-based intercellular adhesions are essential players in epithelial homeostasis, but their dynamic regulation during tissue morphogenesis and remodeling remain largely undefined. Here, we characterize an unexpected role for the membrane-anchored metalloproteinase MT2-MMP in regulating epithelial cell quiescence. Following co-immunoprecipitation and mass spectrometry, the MT2-MMP cytosolic tail was found to interact with the zonula occludens protein-1 (ZO-1) at the apical junctions of polarized epithelial cells. Functionally, MT2-MMP localizes in the apical domain of epithelial cells where it cleaves E-cadherin and promotes epithelial cell accumulation, a phenotype observed in 2D polarized cells as well as 3D cysts. MT2-MMP-mediated cleavage subsequently disrupts apical E-cadherin-mediated cell quiescence resulting in relaxed apical cortical tension favoring cell extrusion and re-sorting of Src kinase activity to junctional complexes, thereby promoting proliferation. Physiologically, MT2-MMP loss of function alters E-cadherin distribution, leading to impaired 3D organoid formation by mouse colonic epithelial cells ex vivo and reduction of cell proliferation within intestinal crypts in vivo Taken together, these studies identify an MT2-MMP-E-cadherin axis that functions as a novel regulator of epithelial cell homeostasis in vivo.

Deficiency of MMP17/MT4-MMP proteolytic activity predisposes to aortic aneurysm in mice.

RATIONALE: Aortic dissection or rupture resulting from aneurysm causes 1% to 2% of deaths in developed countries. These disorders are associated with mutations in genes that affect vascular smooth muscle cell differentiation and contractility or extracellular matrix composition and assembly. However, as many as 75% of patients with a family history of aortic aneurysms do not have an identified genetic syndrome. OBJECTIVE: To determine the role of the protease MMP17/MT4-MMP in the arterial wall and its possible relevance in human aortic pathology. METHODS AND RESULTS: Screening of patients with inherited thoracic aortic aneurysms and dissections identified a missense mutation (R373H) in the MMP17 gene that prevented the expression of the protease in human transfected cells. Using a loss-of-function genetic mouse model, we demonstrated that the lack of Mmp17 resulted in the presence of dysfunctional vascular smooth muscle cells and altered extracellular matrix in the vessel wall; and it led to increased susceptibility to angiotensin-II-induced thoracic aortic aneurysm. We also showed that Mmp17-mediated osteopontin cleavage regulated vascular smooth muscle cell maturation via c-Jun N-terminal kinase signaling during aorta wall development. Some features of the arterial phenotype were prevented by re-expression of catalytically active Mmp17 or the N-terminal osteopontin fragment in Mmp17-null neonates. CONCLUSIONS: Mmp17 proteolytic activity regulates vascular smooth muscle cell phenotype in the arterial vessel wall, and its absence predisposes to thoracic aortic aneurysm in mice. The rescue of part of the vessel-wall phenotype by a lentiviral strategy opens avenues for therapeutic intervention in these life-threatening disorders.

An EMMPRIN-γ-catenin-Nm23 complex drives ATP production and actomyosin contractility at endothelial junctions.

Cell-cell adhesions are important sites through which cells experience and resist forces. In endothelial cells, these forces regulate junction dynamics and determine endothelial barrier strength. We identify the Ig superfamily member EMMPRIN (also known as basigin) as a coordinator of forces at endothelial junctions. EMMPRIN localization at junctions correlates with endothelial junction strength in different mouse vascular beds. Accordingly, EMMPRIN-deficient mice show altered junctions and increased junction permeability. Lack of EMMPRIN alters the localization and function of VE-cadherin (also known as cadherin-5) by decreasing both actomyosin contractility and tugging forces at endothelial cell junctions. EMMPRIN ensures proper actomyosin-driven maturation of competent endothelial junctions by forming a molecular complex with γ-catenin (also known as junction plakoglobin) and Nm23 (also known as NME1), a nucleoside diphosphate kinase, thereby locally providing ATP to fuel the actomyosin machinery. These results provide a novel mechanism for the regulation of actomyosin contractility at endothelial junctions and might have broader implications in biological contexts such as angiogenesis, collective migration and tissue morphogenesis by coupling compartmentalized energy production to junction assembly.

Target engagement of an anti-MT1-MMP antibody for triple-negative breast cancer PET imaging and beta therapy.

PURPOSE: Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer that lacks effective diagnostic and therapeutic options. Membrane type 1 matrix metalloproteinase (MT1-MMP) is an attractive biomarker for improving patient selection. This study aimed to develop a theranostic tool using a highly tumour-selective anti-MT1-MMP antibody (LEM2/15) radiolabelled with 89Zr for PET and 177Lu for therapy in a TNBC murine model. METHODS: The LEM2/15 antibody and IgG isotype control were radiolabelled with 89Zr. PET imaging was performed in a TNBC orthotopic mouse model at 1, 2, 4, and 7 days after administration. Tissue biodistribution and pharmacokinetic parameters were analysed and Patlak linearisation was used to calculate the influx rate of irreversible uptake. The TNBC mice were treated with [177Lu]Lu-DOTA-LEM2/15 (single- or 3-dose regimen) or saline. Efficacy of [177Lu]Lu-DOTA-LEM2/15 was evaluated as tumour growth and DNA damage (γH2AX) in MDA 231-BrM2-831 tumours. RESULTS: At 7 days post-injection, PET uptake in tumour xenografts revealed a 1.6-fold and 2.4-fold higher tumour-to-blood ratio for [89Zr]Zr-Df-LEM2/15 in the non-blocked group compared to the blocked and IgG isotype control groups, respectively. Specific uptake of LEM2/15 in TBNC tumours mediated by MT1-MMP-binding was demonstrated by the Patlak linearisation method, providing insights into the potential efficacy of LEM2/15-based treatments. A similar uptake was found for [89Zr]Zr-Df-LEM2/15 and [177Lu]Lu-DOTA-LEM2/15 in tumours 7 days post-injection (6.80 ± 1.31 vs. 5.61 ± 0.66 %ID/g). Tumour doubling time was longer in the [177Lu]Lu-DOTA-LEM2/15 3-dose regimen treated group compared to the control (50 vs. 17 days, respectively). The percentage of cells with γH2AX-foci was higher in tumours treated with [177Lu]Lu-DOTA-LEM2/15 3-dose regimen compared to tumours non-treated or treated with [177Lu]Lu-DOTA-LEM2/15 single-dose (12 % vs. 4-5 %). CONCLUSIONS: The results showed that the 89Zr/177Lu-labelled anti-MT1-MMP mAb (LEM2/15) pair facilitated immune-PET imaging and reduced tumour growth in a preclinical TNBC xenograft model.

The protease MT1-MMP drives a combinatorial proteolytic program in activated endothelial cells.

The mechanism by which proteolytic events translate into biological responses is not well understood. To explore the link of pericellular proteolysis to events relevant to capillary sprouting within the inflammatory context, we aimed at the identification of the collection of substrates of the protease MT1-MMP in endothelial tip cells induced by inflammatory stimuli. We applied quantitative proteomics to endothelial cells (ECs) derived from wild-type and MT1-MMP-null mice to identify the substrate repertoire of this protease in TNF-α-activated ECs. Bioinformatics analysis revealed a combinatorial MT1-MMP proteolytic program, in which combined rather than single substrate processing would determine biological decisions by activated ECs, including chemotaxis, cell motility and adhesion, and vasculature development. MT1-MMP-deficient ECs inefficiently processed several of these substrates (TSP1, CYR61, NID1, and SEM3C), validating the model. This novel concept of MT1-MMP-driven combinatorial proteolysis in angiogenesis might be extendable to proteolytic actions in other cellular contexts.

Retinoid X receptor alpha controls innate inflammatory responses through the up-regulation of chemokine expression.

The retinoid X receptor alpha (RXRalpha) plays a central role in the regulation of many intracellular receptor signaling pathways and can mediate ligand-dependent transcription by forming homodimers or heterodimers with other nuclear receptors. Although several members of the nuclear hormone receptor superfamily have emerged as important regulators of macrophage gene expression, the existence in vivo of an RXR signaling pathway in macrophages has not been established. Here, we provide evidence that RXRalpha regulates the transcription of the chemokines Ccl6 and Ccl9 in macrophages independently of heterodimeric partners. Mice lacking RXRalpha in myeloid cells exhibit reduced levels of CCL6 and CCL9, impaired recruitment of leukocytes to sites of inflammation, and lower susceptibility to sepsis. These studies demonstrate that macrophage RXRalpha plays key roles in the regulation of innate immunity and represents a potential target for immunotherapy of sepsis.

Capillary pruning couples tissue perfusion and oxygenation with cardiomyocyte maturation in the postnatal mouse heart.

Introduction: Removal of poorly perfused capillaries by pruning contributes to remodeling the microvasculature to optimize oxygen and nutrient delivery. Blood flow drives this process by promoting the intravascular migration of endothelial cells in developing networks, such as in the yolk sac, zebrafish brain or postnatal mouse retina. Methods: In this study, we have implemented innovative tools to recognize capillary pruning in the complex 3D coronary microvasculature of the postnatal mouse heart. We have also experimentally tested the impact of decreasing pruning on the structure and function of this network by altering blood flow with two different vasodilators: losartan and prazosin. Results: Although both drugs reduced capillary pruning, a combination of experiments based on ex vivo imaging, proteomics, electron microscopy and in vivo functional approaches showed that losartan treatment resulted in an inefficient coronary network, reduced myocardial oxygenation and metabolic changes that delayed the arrest of cardiomyocyte proliferation, in contrast to the effects of prazosin, probably due to its concomitant promotion of capillary expansion. Discussion: Our work demonstrates that capillary pruning contributes to proper maturation and function of the heart and that manipulation of blood flow may be a novel strategy to refine the microvasculature and improve tissue perfusion after damage.

p38 MAPK priming boosts VSMC proliferation and arteriogenesis by promoting PGC1α-dependent mitochondrial dynamics.

Vascular smooth muscle cell (VSMC) proliferation is essential for arteriogenesis to restore blood flow after artery occlusion, but the mechanisms underlying this response remain unclear. Based on our previous findings showing increased VSMC proliferation in the neonatal aorta of mice lacking the protease MT4-MMP, we aimed at discovering new players in this process. We demonstrate that MT4-MMP absence boosted VSMC proliferation in vitro in response to PDGF-BB in a cell-autonomous manner through enhanced p38 MAPK activity. Increased phospho-p38 in basal MT4-MMP-null VSMCs augmented the rate of mitochondrial degradation by promoting mitochondrial morphological changes through the co-activator PGC1α as demonstrated in PGC1α-/- VSMCs. We tested the in vivo implications of this pathway in a novel conditional mouse line for selective MT4-MMP deletion in VSMCs and in mice pre-treated with the p38 MAPK activator anisomycin. Priming of p38 MAPK activity in vivo by the absence of the protease MT4-MMP or by anisomycin treatment led to enhanced arteriogenesis and improved flow recovery after femoral artery occlusion. These findings may open new therapeutic opportunities for peripheral vascular diseases.

Development of anti-membrane type 1-matrix metalloproteinase nanobodies as immunoPET probes for triple negative breast cancer imaging.

Triple-negative breast cancer (TNBC) is characterized by aggressiveness and high rates of metastasis. The identification of relevant biomarkers is crucial to improve outcomes for TNBC patients. Membrane type 1-matrix metalloproteinase (MT1-MMP) could be a good candidate because its expression has been reported to correlate with tumor malignancy, progression and metastasis. Moreover, single-domain variable regions (VHHs or Nanobodies) derived from camelid heavy-chain-only antibodies have demonstrated improvements in tissue penetration and blood clearance, important characteristics for cancer imaging. Here, we have developed a nanobody-based PET imaging strategy for TNBC detection that targets MT1-MMP. A llama-derived library was screened against the catalytic domain of MT1-MMP and a panel of specific nanobodies were identified. After a deep characterization, two nanobodies were selected to be labeled with gallium-68 (68Ga). ImmunoPET imaging with both ([68Ga]Ga-NOTA-3TPA14 and [68Ga]Ga-NOTA-3CMP75) in a TNBC mouse model showed precise tumor-targeting capacity in vivo with high signal-to-background ratios. (68Ga)Ga-NOTA-3CMP75 exhibited higher tumor uptake compared to (68Ga)Ga-NOTA-3TPA14. Furthermore, imaging data correlated perfectly with the immunohistochemistry staining results. In conclusion, we found a promising candidate for nanobody-based PET imaging to be further investigated as a diagnostic tool in TNBC.

Intravascular Crawling of Patrolling Monocytes: A Lèvy-Like Motility for Unique Search Functions?

Patrolling monocytes (PMo) are the organism's preeminent intravascular guardians by their continuous search of damaged endothelial cells and harmful microparticles for their removal and to restore homeostasis. This surveillance is accomplished by PMo crawling on the apical side of the endothelium through regulated interactions of integrins and chemokine receptors with their endothelial ligands. We propose that the search mode governs the intravascular motility of PMo in vivo in a similar way to T cells looking for antigen in tissues. Signs of damage to the luminal side of the endothelium (local death, oxidized LDL, amyloid deposits, tumor cells, pathogens, abnormal red cells, etc.) will change the diffusive random towards a Lèvy-like crawling enhancing their recognition and clearance by PMo damage receptors as the integrin αMß2 and CD36. This new perspective can help identify new actors to promote unique PMo intravascular actions aimed at maintaining endothelial fitness and combating harmful microparticles involved in diseases as lung metastasis, Alzheimer's angiopathy, vaso-occlusive disorders, and sepsis.

Dynamic Expression of Membrane Type 1-Matrix Metalloproteinase (Mt1-mmp/Mmp14) in the Mouse Embryo.

MT1-MMP/MMP14 belongs to a subgroup of the matrix metalloproteinases family that presents a transmembrane domain, with a cytosolic tail and the catalytic site exposed to the extracellular space. Deficient mice for this enzyme result in early postnatal death and display severe defects in skeletal, muscle and lung development. By using a transgenic line expressing the LacZ reporter under the control of the endogenous Mt1-mmp promoter, we reported a dynamic spatiotemporal expression pattern for Mt1-mmp from early embryonic to perinatal stages during cardiovascular development and brain formation. Thus, Mt1-mmp shows expression in the endocardium of the heart and the truncus arteriosus by E8.5, and is also strongly detected during vascular system development as well as in endothelial cells. In the brain, LacZ reporter expression was detected in the olfactory bulb, the rostral cerebral cortex and the caudal mesencephalic tectum. LacZ-positive cells were observed in neural progenitors of the spinal cord, neural crest cells and the intersomitic region. In the limb, Mt1-mmp expression was restricted to blood vessels, cartilage primordium and muscles. Detection of the enzyme was confirmed by Western blot and immunohistochemical analysis. We suggest novel functions for this metalloproteinase in angiogenesis, endocardial formation and vascularization during organogenesis. Moreover, Mt1-mmp expression revealed that the enzyme may contribute to heart, muscle and brain throughout development.

Smooth muscle-specific MMP17 (MT4-MMP) regulates the intestinal stem cell niche and regeneration after damage.

Smooth muscle is an essential component of the intestine, both to maintain its structure and produce peristaltic and segmentation movements. However, very little is known about other putative roles that smooth muscle cells may have. Here, we show that smooth muscle cells may be the dominant suppliers of BMP antagonists, which are niche factors essential for intestinal stem cell maintenance. Furthermore, muscle-derived factors render epithelium reparative and fetal-like, which includes heightened YAP activity. Mechanistically, we find that the membrane-bound matrix metalloproteinase MMP17, which is exclusively expressed by smooth muscle cells, is required for intestinal epithelial repair after inflammation- or irradiation-induced injury. Furthermore, we propose that MMP17 affects intestinal epithelial reprogramming after damage indirectly by cleaving diffusible factor(s) such as the matricellular protein PERIOSTIN. Together, we identify an important signaling axis that establishes a role for smooth muscle cells as modulators of intestinal epithelial regeneration and the intestinal stem cell niche.

PGE2 induces angiogenesis via MT1-MMP-mediated activation of the TGFbeta/Alk5 signaling pathway.

The development of a new vascular network is essential for the onset and progression of many pathophysiologic processes. Cyclooxygenase-2 displays a proangiogenic activity in in vitro and in vivo models, mediated principally through its metabolite prostaglandin E(2) (PGE(2)). Here, we provide evidence for a novel signaling route through which PGE(2) activates the Alk5-Smad3 pathway in endothelial cells. PGE(2) induces Alk5-dependent Smad3 nuclear translocation and DNA binding, and the activation of this pathway involves the release of active TGFbeta from its latent form through a process mediated by the metalloproteinase MT1-MMP, whose membrane clustering is promoted by PGE(2). MT1-MMP-dependent transforming growth factor beta (TGFbeta) signaling through Alk5 is also required for PGE(2)-induced endothelial cord formation in vitro, and Alk5 kinase activity is required for PGE(2)-induced neovascularization in vivo. These findings identify a novel signaling pathway linking PGE(2) and TGFbeta, 2 effectors involved in tumor growth and angiogenesis, and reveal potential targets for the treatment of angiogenesis-related disorders.

MT1-MMP collagenolytic activity is regulated through association with tetraspanin CD151 in primary endothelial cells.

MT1-MMP plays a key role in endothelial function, as underscored by the angiogenic defects found in MT1-MMP deficient mice. We have studied the molecular interactions that underlie the functional regulation of MT1-MMP. At lateral endothelial cell junctions, MT1-MMP colocalizes with tetraspanin CD151 (Tspan 24) and its associated partner alpha3beta1 integrin. Biochemical and FRET analyses show that MT1-MMP, through its hemopexin domain, associates tightly with CD151, thus forming alpha3beta1 integrin/CD151/MT1-MMP ternary complexes. siRNA knockdown of HUVEC CD151 expression enhanced MT1-MMP-mediated activation of MMP2, and the same activation was seen in ex vivo lung endothelial cells isolated from CD151-deficient mice. However, analysis of collagen degradation in these experimental models revealed a diminished MT1-MMP enzymatic activity in confined areas around the cell periphery. CD151 knockdown affected both MT1-MMP subcellular localization and its inclusion into detergent-resistant membrane domains, and prevented biochemical association of the metalloproteinase with the integrin alpha3beta1. These data provide evidence for a novel regulatory role of tetraspanin microdomains on the collagenolytic activity of MT1-MMP and indicate that CD151 is a key regulator of MT1-MMP in endothelial homeostasis.

Remodeling of the Microvasculature: May the Blood Flow Be With You.

The vasculature ensures optimal delivery of nutrients and oxygen throughout the body, and to achieve this function it must continually adapt to varying tissue demands. Newly formed vascular plexuses during development are immature and require dynamic remodeling to generate well-patterned functional networks. This is achieved by remodeling of the capillaries preserving those which are functional and eliminating other ones. A balanced and dynamically regulated capillary remodeling will therefore ensure optimal distribution of blood and nutrients to the tissues. This is particularly important in pathological contexts in which deficient or excessive vascular remodeling may worsen tissue perfusion and hamper tissue repair. Blood flow is a major determinant of microvascular reshaping since capillaries are pruned when relatively less perfused and they split when exposed to high flow in order to shape the microvascular network for optimal tissue perfusion and oxygenation. The molecular machinery underlying blood flow sensing by endothelial cells is being deciphered, but much less is known about how this translates into endothelial cell responses as alignment, polarization and directed migration to drive capillary remodeling, particularly in vivo. Part of this knowledge is theoretical from computational models since blood flow hemodynamics are not easily recapitulated by in vitro or ex vivo approaches. Moreover, these events are difficult to visualize in vivo due to their infrequency and briefness. Studies had been limited to postnatal mouse retina and vascular beds in zebrafish but new tools as advanced microscopy and image analysis are strengthening our understanding of capillary remodeling. In this review we introduce the concept of remodeling of the microvasculature and its relevance in physiology and pathology. We summarize the current knowledge on the mechanisms contributing to capillary regression and to capillary splitting highlighting the key role of blood flow to orchestrate these processes. Finally, we comment the potential and possibilities that microfluidics offers to this field. Since capillary remodeling mechanisms are often reactivated in prevalent pathologies as cancer and cardiovascular disease, all this knowledge could be eventually used to improve the functionality of capillary networks in diseased tissues and promote their repair.

Macrophages promote endothelial-to-mesenchymal transition via MT1-MMP/TGFß1 after myocardial infarction.

Macrophages (Mφs) produce factors that participate in cardiac repair and remodeling after myocardial infarction (MI); however, how these factors crosstalk with other cell types mediating repair is not fully understood. Here we demonstrated that cardiac Mφs increased the expression of Mmp14 (MT1-MMP) 7 days post-MI. We selectively inactivated the Mmp14 gene in Mφs using a genetic strategy (Mmp14f/f:Lyz2-Cre). This conditional KO (MAC-Mmp14 KO) resulted in attenuated post-MI cardiac dysfunction, reduced fibrosis, and preserved cardiac capillary network. Mechanistically, we showed that MT1-MMP activates latent TGFß1 in Mφs, leading to paracrine SMAD2-mediated signaling in endothelial cells (ECs) and endothelial-to-mesenchymal transition (EndMT). Post-MI MAC-Mmp14 KO hearts contained fewer cells undergoing EndMT than their wild-type counterparts, and Mmp14-deficient Mφs showed a reduced ability to induce EndMT in co-cultures with ECs. Our results indicate the contribution of EndMT to cardiac fibrosis and adverse remodeling post-MI and identify Mφ MT1-MMP as a key regulator of this process.

Endothelial MT1-MMP targeting limits intussusceptive angiogenesis and colitis via TSP1/nitric oxide axis.

Pathological angiogenesis contributes to cancer progression and chronic inflammatory diseases. In inflammatory bowel disease, the microvasculature expands by intussusceptive angiogenesis (IA), a poorly characterized mechanism involving increased blood flow and splitting of pre-existing capillaries. In this report, mice lacking the protease MT1-MMP in endothelial cells (MT1iΔEC ) presented limited IA in the capillary plexus of the colon mucosa assessed by 3D imaging during 1% DSS-induced colitis. This resulted in better tissue perfusion, preserved intestinal morphology, and milder disease activity index. Combined in vivo intravital microscopy and lentiviral rescue experiments with in vitro cell culture demonstrated that MT1-MMP activity in endothelial cells is required for vasodilation and IA, as well as for nitric oxide production via binding of the C-terminal fragment of MT1-MMP substrate thrombospondin-1 (TSP1) to CD47/αvß3 integrin. Moreover, TSP1 levels were significantly higher in serum from IBD patients and in vivo administration of an anti-MT1-MMP inhibitory antibody or a nonamer peptide spanning the αvß3 integrin binding site in TSP1 reduced IA during mouse colitis. Our results identify MT1-MMP as a new actor in inflammatory IA and a promising therapeutic target for inflammatory bowel disease.