Membrane Type 1 Matrix Metalloproteinase Activity Research Articles

Analysis of Matrix Metalloproteinase Activity and Inhibition in Cancer Spheroids.

The utilization of tumor spheroids and organoids has greatly facilitated mechanistic understanding of tumor growth and invasion and lead to more effective high-throughput analysis of potential chemotherapeutic agents. In spheroid and organoid systems, tumor invasion occurs in three dimensions and monitoring this behavior can be data intensive. Quantitative correlation of tumor invasion with protease activity can further exacerbate data storage issues. The present method utilizes the "Hit Pick" approach to provide quantitative analysis and correlation of tumor invasion and membrane type 1 matrix metalloproteinase (MT1-MMP) activity in a rapid fashion with greatly reduced data storage requirements compared with standard image analysis approaches. Inhibition of MT1-MMP activity in spheroids can also be monitored by the present approach.

Abstract 2757: Combination of a fluorescent substrate-based MMP activity assay and hit pick reading/imaging procedure to screen for inhibitors of MT1-MMP activity and 3D glioma tumoroid invasion

Abstract Malignant and nonmalignant brain tumors are composed of a group of more than 100 different types. Malignant gliomas, including glioblastoma multiforme (GBM) and astrocytomas, are the most common primary brain tumors in the United States, and make up 78% of malignant brain tumors. Membrane type 1 matrix metalloproteinase (MT1-MMP) has been shown to be crucial for the progression, invasion, migration, and angiogenesis of tumors. Use of a FRET substrate to quantify MT1-MMP activity, where tumoroids are embedded in a collagen hydrogel, would simplify detection of potential protease inhibitors, while using an in vivo-like cell model. In the present study, multiple glioma cell lines were formed into tumor spheroids, followed by type I collagen addition. Many known MT1-MMP inhibitors were then added to the wells, to potentially inhibit invasion of the 3D cell models. The substrate and experimental setup were then combined with the joint reading and imaging process available in the Agilent BioTek Gen5 microplate reader and imager software, to create a hit pick screening method. During the procedure, all control and test wells were read, and the signal from cleaved substrate was quantified. Uninhibited invasion within positive control wells exhibited high fluorescence values. Therefore, a hit pick criterion was established such that any wells containing test molecules that were statistically lower than average in fluorescent signal from the corresponding positive control, which should exhibit inhibited invasion, were imaged. Wells that were not significantly lower were not imaged. The result was a screening method which increased efficiency by reducing image capture time and storage requirements. Citation Format: Brad Larson, Gregg Fields, Ania Knapinska. Combination of a fluorescent substrate-based MMP activity assay and hit pick reading/imaging procedure to screen for inhibitors of MT1-MMP activity and 3D glioma tumoroid invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2757.

Screening MT1-MMP Activity and Inhibition in Three-Dimensional Tumor Spheroids

Membrane type 1 matrix metalloproteinase (MT1-MMP) has been shown to be crucial for tumor angiogenesis, invasion, and metastasis, and thus MT1-MMP is a high priority target for potential cancer therapies. To properly evaluate MT1-MMP inhibitors, a screening protocol is desired by which enzyme activity can be quantified in a tumor microenvironment-like model system. In the present study, we applied a fluorogenic, collagen model triple-helical substrate to quantify MT1-MMP activity for tumor spheroids embedded in a collagen hydrogel. The substrate was designed to be MT1-MMP selective and to possess fluorescent properties compatible with cell-based assays. The proteolysis of the substrate correlated to glioma spheroid invasion. In turn, the application of either small molecule or protein-based MMP inhibitors reduced proteolytic activity and glioma spheroid invasion. The presence of MT1-MMP in glioma spheroids was confirmed by western blotting. Thus, spheroid invasion was dependent on MT1-MMP activity, and inhibitors of MT1-MMP and invasion could be conveniently screened in a high-throughput format. The combination of the fluorogenic, triple-helical substrate, the three-dimensional tumor spheroids embedded in collagen, and Hit-Pick software resulted in an easily adaptable in vivo-like tumor microenvironment for rapidly processing inhibitor potential for anti-cancer use.

Control of SARS-CoV-2 infection by MT1-MMP-mediated shedding of ACE2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. Angiotensin-converting enzyme 2 (ACE2) is an entry receptor for SARS-CoV-2. The full-length membrane form of ACE2 (memACE2) undergoes ectodomain shedding to generate a shed soluble form (solACE2) that mediates SARS-CoV-2 entry via receptor-mediated endocytosis. Currently, it is not known how the physiological regulation of ACE2 shedding contributes to the etiology of COVID-19 in vivo. The present study identifies Membrane-type 1 Matrix Metalloproteinase (MT1-MMP) as a critical host protease for solACE2-mediated SARS-CoV-2 infection. SARS-CoV-2 infection leads to increased activation of MT1-MMP that is colocalized with ACE2 in human lung epithelium. Mechanistically, MT1-MMP directly cleaves memACE2 at M706-S to release solACE218-706 that binds to the SARS-CoV-2 spike proteins (S), thus facilitating cell entry of SARS-CoV-2. Human solACE218-706 enables SARS-CoV-2 infection in both non-permissive cells and naturally insusceptible C57BL/6 mice. Inhibition of MT1-MMP activities suppresses solACE2-directed entry of SARS-CoV-2 in human organoids and aged mice. Both solACE2 and circulating MT1-MMP are positively correlated in plasma of aged mice and humans. Our findings provide in vivo evidence demonstrating the contribution of ACE2 shedding to the etiology of COVID-19.

SPOCK2 Affects the Biological Behavior of Endometrial Cancer Cells by Regulation of MT1-MMP and MMP2

Abnormal expression of SPARC (osteonectin), cwcv and kazal-like domains proteoglycan 2 (SPOCK2) plays a significant role in the development and progression of various human cancers, yet a relationship between SPOCK2 and endometrial cancer (EC) has not been reported. Here, we assessed the potential role and mechanism by which SPOCK2 acts in the pathogenesis and progression of EC. First, protein expression of SPOCK2 in EC tissue from patients was detected by immunohistochemistry and associated clinical data were analyzed. Then, HEC-1A and Ishikawa cells were transfected with an adenoviral vector containing an SPOCK2 recombinant fragment and the biological behavior of transfected cells was observed. Finally, the expression of membrane type 1 matrix metalloproteinase (MT1-MMP) and MMP2 in the transfected cells was detected by Western blot and zymography gel assay to analyze the effect of SPOCK2 on the regulation of the MT1-MMP/MMP2 pathway. We found that there was significantly less SPOCK2 protein expression in the EC tissue than in the normal endometrium tissue, and lack of SPOCK2 protein expression in EC tissue was associated with distant metastasis and myometrial invasion. Upregulation of SPOCK2 in HEC-1A and Ishikawa cells inhibited cell proliferation, invasion, adhesion, and apoptosis. Upregulation of SPOCK2 inhibited the expression of MT1-MMP and MMP2 and activation of MMP2 in HEC-1A and Ishikawa cells. Collectively, our data indicated that SPOCK2 contributed to the progression of EC by regulating the biological behavior of cancer cells, which is achieved partly through regulating protein expression of MT1-MMP and MMP2 and activation of MMP2.

SPOCK2 Affects the Biological Behavior of Endometrial Cancer Cells by Regulation of MT1-MMP and MMP2.

Abnormal expression of SPARC (osteonectin), cwcv and kazal-like domains proteoglycan 2 (SPOCK2) plays a significant role in the development and progression of various human cancers, yet a relationship between SPOCK2 and endometrial cancer (EC) has not been reported. Here, we assessed the potential role and mechanism by which SPOCK2 acts in the pathogenesis and progression of EC. First, protein expression of SPOCK2 in EC tissue from patients was detected by immunohistochemistry and associated clinical data were analyzed. Then, HEC-1A and Ishikawa cells were transfected with an adenoviral vector containing an SPOCK2 recombinant fragment and the biological behavior of transfected cells was observed. Finally, the expression of membrane type 1 matrix metalloproteinase (MT1-MMP) and MMP2 in the transfected cells was detected by Western blot and zymography gel assay to analyze the effect of SPOCK2 on the regulation of the MT1-MMP/MMP2 pathway. We found that there was significantly less SPOCK2 protein expression in the EC tissue than in the normal endometrium tissue, and lack of SPOCK2 protein expression in EC tissue was associated with distant metastasis and myometrial invasion. Upregulation of SPOCK2 in HEC-1A and Ishikawa cells inhibited cell proliferation, invasion, adhesion, and apoptosis. Upregulation of SPOCK2 inhibited the expression of MT1-MMP and MMP2 and activation of MMP2 in HEC-1A and Ishikawa cells. Collectively, our data indicated that SPOCK2 contributed to the progression of EC by regulating the biological behavior of cancer cells, which is achieved partly through regulating protein expression of MT1-MMP and MMP2 and activation of MMP2.

RNA interference screening identifies clathrin-B and cofilin-1 as mediators of MT1-MMP in endometrial cancer

The matrix metalloproteinases (MMPs) are implicated in tumor invasion and metastasis. Given their multiple tumor promoting roles, MMPs are promising targets for the treatment of metastatic cancer. Using a siRNA library screen of 140 membrane trafficking genes, we identified 41 genes in HEC-1B and 36 in Ishikawa cancer cells that decreased metalloproteinases activity. The 16 genes common in both cancer cell lines that decreased MMPs activity are involved in cargo sorting, vesicle formation and vesicle recycling. The top two genes clathrin-B and cofilin-1 were chosen for post hoc functional studies. Higher expression of both genes was confirmed in cancer cells and knockdown with respective siRNAs inhibited their invasive potential and matrix metalloproteinases activity. Membrane Type 1- Matrix Metalloproteinase (MT1-MMP) is a master switch proteinase and regulator of invasion and metastasis. A marked decrease in MT1-MMP expression and activity was seen in clathrin-B and cofilin-1 knockdown cancer cells which was associated with a marked decreased expression of invadopodia formation proteins. Our results suggest that the decreased expression of clathrin-B and cofilin-1 decreases the expression of MT1-MMP and results in attenuation of MT1-MMP at the cell surface, thus inhibiting tumor cell invasion and metastasis.

Abstract 1953: Expression and activity of MT1-MMP in endothelial cells is regulated by Semaphorin 4D

Abstract The semaphorins and plexins, originally shown to control axon growth cone guidance, are now known to influence many aspects of cell adhesion and migration. Our group and others have demonstrated that carcinomas over express Semaphorin 4D (S4D), particularly under conditions of hypoxia, for the purpose of supporting tumor-induced angiogenesis. However, S4D is a transmembrane protein that must be proteolytically cleaved to attract endothelial cells into a malignancy, a process that in head and neck squamous cell carcinoma (HNSCC) is controlled by membrane type 1-matrix metalloproteinase (MT1-MMP). We wanted to test the possibility that S4D acts through its receptor, Plexin-B1 (PB1), to enhance activity of endothelial cell MT1-MMP, facilitating extracellular matrix degradation and further promoting angiogenesis. We observed upregulated transcription, expression and cell surface localization of MT1-MMP in endothelial cells treated with S4D, occurring in a PB1, RhoA and NF-κB-dependent manner. We also demonstrated that S4D-induced endothelial cell collagen degradation, migration and capillary tube formation are suppressed when PB1 is silenced or its downstream effectors RhoA and NF-κB are inhibited. Endothelial MT1-MMP contributes to the ability of these cells to digest their way through the basement membrane and extracellular matrix, a requirement for the creation of the vascular networks that supply a developing tumor with oxygen and nutrients and allow it access to the systemic circulation for metastasis. These results demonstrate that S4D promotes angiogenesis at multiple levels, and suggest that it would be a valid target for anti-angiogenic intervention in the treatment of solid malignancies. Citation Format: Amr Bugshan, Zaid Khoury, Asma Buhamrah, John R. Basile. Expression and activity of MT1-MMP in endothelial cells is regulated by Semaphorin 4D [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1953.

Progesterone and calcitriol reduce invasive potential of endometrial cancer cells by targeting ARF6, NEDD9 and MT1-MMP.

Previously, we have demonstrated that progesterone and calcitriol synergistically inhibit growth of endometrial and ovarian cancer by enhancing apoptosis and causing cell cycle arrest. Metastasis is the main reason of mortality in cancer patients. Activation of ADP-Ribosylation Factor 6 (ARF6), Neural Precursor cell expressed Developmentally Downregulated 9 (NEDD9), and Membrane-Type-1 Matrix Metalloproteinase (MT1-MMP) have been implicated in promoting tumor growth and metastasis. We examined the effects of progesterone, calcitriol and progesterone-calcitriol combination on metastasis promoting proteins in endometrial cancer. Expression of ARF6, NEDD9, and MT1-MMP was enhanced in advanced-stage endometrial tumors and in cancer cell lines compared to normal tissues and immortalized EM-E6/E7-TERT endometrial epithelial cells. Knockdown of these proteins significantly inhibited the invasiveness of the cancer cells. The expression levels of all three proteins was reduced with progesterone and progesterone-calcitriol combination treatment, whereas calcitriol alone showed no effect on their expression but moderately decreased MT1-MMP activity. Fluorescence microscopy showed membrane expression of MT1-MMP in vehicle and calcitriol-treated endometrial cancer cells. However, progesterone and calcitriol-progesterone combination treatment revealed MT1-MMP in the cytoplasm. Furthermore, progesterone and calcitriol reduced the activity of MT1-MMP, MMP-9, and MMP-2. In addition, invadopodia regulatory proteins were attenuated in both progesterone and progesterone-calcitriol combination treated cells as well as in MT1-MMP knockdown cells. Thus, targeting the aberrant MT1-MMP signaling with progesterone-calcitriol may be a novel approach to impede MT1-MMP mediated cancer dissemination and may have therapeutic benefits for endometrial cancer patients.

Discoidin domain receptor 2 mediates collagen-induced activation of membrane-type 1 matrix metalloproteinase in human fibroblasts

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a membrane-bound MMP that is highly expressed in cells with invading capacity, including fibroblasts and invasive cancer cells. However, pathways of MT1-MMP up-regulation are not clearly understood. A potential physiological stimulus for MT1-MMP expression is fibrillar collagen, and it has been shown that it up-regulates both MT1-MMP gene and functions in various cell types. However, the mechanisms of collagen-mediated MT1-MMP activation and its physiological relevance are not known. In this study, we identified discoidin domain receptor 2 (DDR2) as a crucial receptor that mediates this process in human fibroblasts. Knocking down DDR2, but not the β1 integrin subunit, a common subunit for all collagen-binding integrins, inhibited the collagen-induced MT1-MMP-dependent activation of pro-MMP-2 and up-regulation of MT1-MMP at the gene and protein levels. Interestingly, DDR2 knockdown or pharmacological inhibition of DDR2 also inhibited the MT1-MMP-dependent cellular degradation of collagen film, suggesting that cell-surface collagen degradation by MT1-MMP involves DDR2-mediated collagen signaling. This DDR2-mediated mechanism is only present in non-transformed mesenchymal cells as collagen-induced MT1-MMP activation in HT1080 fibrosarcoma cells and MT1-MMP function in MDA-MB231 breast cancer cells were not affected by DDR kinase inhibition. DDR2 activation was found to be noticeably more effective when cells were stimulated by collagen without the non-helical telopeptide region compared with intact collagen fibrils. Furthermore, DDR2-dependent MT1-MMP activation by cartilage was found to be more efficient when the tissue was partially damaged. These data suggest that DDR2 is a microenvironment sensor that regulates fibroblast migration in a collagen-rich environment.

Regulation of membrane type-1 matrix metalloproteinase activity and intracellular localization in clinical thoracic aortic aneurysms

ObjectiveMembrane type-1 matrix metalloproteinase (MT1-MMP) is elevated during thoracic aortic aneurysm (TAA) development in mouse models, and plays an important role in the activation of matrix metalloproteinase (MMP)-2 and the release of matrix- bound transforming growth factor-β. In this study, we tested the hypothesis that MT1-MMP is subject to protein kinase C (PKC)–mediated regulation, which alters intracellular trafficking and activity with TAAs. MethodsLevels of MMP-2, native and phosphorylated MT1-MMP, and PKC-δ were measured in aortic tissue from patients with small TAAs (<5 cm; n = 8) and large TAAs (>6.5 cm; n = 8), and compared with values measured in normal controls (n = 8). Cellular localization of green fluorescent protein (GFP)-tagged MT1-MMP was assessed in aortic fibroblasts isolated from control and 4-week TAA mice. The effects of PKC-mediated phosphorylation on MT1-MMP cellular localization and function (active MMP-2 vs phospo-Smad2 abundance) were assessed after treatment with a PKC activator (phorbol-12-myristate-13-acetate [PMA], 100 nM) with and without a PKC-δ–specific inhibitor (röttlerin, 3 μM). ResultsCompared with controls, MT1-MMP abundance was increased in aortas from both TAA groups. Active MMP-2 was increased only in the large TAA group. The abundances of phosphorylated MT1-MMP and activated PKC-δ were enhanced in the small TAA group compared with the large TAA group. MT1-MMP was localized on the plasma membrane in aortic fibroblasts from control mice and in endosomes from TAA mice. Treatment with PMA induced MT1-MMP-GFP internalization, enhanced phospho-Smad2, and reduced MMP-2 activation, whereas röttlerin pretreatment inhibited these effects. ConclusionsPhosphorylation of MT1-MMP mediates its activity through directing cellular localization, shifting its role from MMP-2 activation to intracellular signaling. Thus, targeted inhibition of MT1-MMP may have therapeutic relevance as an approach to attenuating TAA development.

Abstract B36: NF-kappaB-dependent and -independent roles for NIK in regulating GBM cell invasion

Abstract The aggressive invasion of GBM cells into healthy brain tissue is a major factor contributing to the therapy resistance and poor prognosis of this malignancy. Our recent results demonstrated that the alternative, or noncanonical, NF-kappaB signaling pathway, predominates in the aggressive mesenchymal subtype of GBM. Additionally, NF-kappaB-inducing kinase (NIK), a key upstream activator of noncanonical NF-kappaB signaling, robustly promotes GBM cell invasion and pathogenesis. We demonstrate that expression of NIK is increased in response to exposure to collagen, a major extracellular matrix (ECM) protein present in GBM tumors, with NIK protein levels continuing to increase during invasion in 3D collagen matrices. Interestingly, NIK expression levels correlates with induction of membrane type-1 matrix metalloproteinase (MT1-MMP), a critical enzyme in many invasive tumors, functioning to degrade the basement membrane and ECM during cell migration and invasion. We provide evidence that NIK promotes MT1-MMP activation through increased phosphorylation at membrane protrusions and elevated enzymatic activity. Notably, the activation of MT1-MMP and the pro-invasive effects of NIK persist in the absence of the downstream NF-kappaB transcription factors RelA/p65 and RelB. Together, these data demonstrate a novel ability of NIK to promote GBM cell migration and invasion that is independent of NF-kappaB transcriptional activation functions. The results underscore the therapeutic potential of targeting noncanonical NF-kappaB signaling in these highly invasive tumors. Citation Format: Camille L. Duran, Linda Herrera de Lechuga, Dong W. Lee, Kayla J. Bayless, Raquel Sitcheran. NF-kappaB-dependent and -independent roles for NIK in regulating GBM cell invasion. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B36.

MT1-MMP dependent repression of the tumor suppressor SPRY4 contributes to MT1-MMP driven melanoma cell motility

Metastatic melanoma is the deadliest of all skin cancers. Despite progress in diagnostics and treatment of melanoma, the prognosis for metastatic patients remains poor. We previously showed that Membrane-type 1 Matrix Metalloproteinase (MT1-MMP) is one of the drivers of melanoma metastasis. Classically, MT1-MMP regulates a verity of cellular functions including cell-to-cell interaction and cell-to-matrix communication. Recently, MT1-MMP has been found to also modulate gene expression. To specifically assess MT1-MMP dependent gene regulation in melanoma, microarray gene expression analysis was performed in a melanoma cell line whose metastatic properties depend on the activity of MT1-MMP. We identified the tumor suppressor gene SPRY4 as a new transcriptional target of MT1-MMP that is negatively regulated by the protease. Knockdown of MT1-MMP enhances SPRY4 expression at the mRNA and protein level. SPRY4 expression inversely correlates with that of MT1-MMP in melanoma samples and importantly, correlates with melanoma patient survival. SPRY4 modulates MT1-MMP dependent cell migration such that inhibition of SPRY4 rescues cell migration that has been impaired by MT1-MMP knock down. MT1-MMP decreases SPRY4 in part through an MMP2/RAC1 axis we previously show promotes cell motility downstream of MT1-MMP. These results identify the tumor suppressor SPRY4 as a novel molecular effector of MT1-MMP affecting melanoma cell motility.

TIMP-2 Interaction with MT1-MMP Activates the AKT Pathway and Protects Tumor Cells from Apoptosis.

Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with an extracellular catalytic domain and a short cytoplasmic tail, degrades a variety of extracellular matrix (ECM) components. In addition, MT1-MMP activates intracellular signaling through proteolysis-dependent and independent mechanisms. We have previously shown that binding of tissue inhibitor of metalloproteinases-2 (TIMP-2) to MT1-MMP controls cell proliferation and migration, as well as tumor growth in vivo by activating the Ras—extracellular signal regulated kinase-1 and -2 (ERK1/2) pathway through a mechanism that requires the cytoplasmic but not the proteolytic domain of MT1-MMP. Here we show that in MT1-MMP expressing cells TIMP-2 also induces rapid and sustained activation of AKT in a dose- and time-dependent manner and by a mechanism independent of the proteolytic activity of MT1-MMP. Fibroblast growth factor receptor-1 mediates TIMP-2 induction of ERK1/2 but not of AKT activation; however, Ras activation is necessary to transduce the TIMP-2-activated signal to both the ERK1/2 and AKT pathways. ERK1/2 and AKT activation by TIMP-2 binding to MT1-MMP protects tumor cells from apoptosis induced by serum starvation. Conversely, TIMP-2 upregulates apoptosis induced by three-dimensional type I collagen in epithelial cancer cells. Thus, TIMP-2 interaction with MT1-MMP provides tumor cells with either pro- or anti-apoptotic signaling depending on the extracellular environment and apoptotic stimulus.

The Oncogenic Response to MiR-335 Is Associated with Cell Surface Expression of Membrane-Type 1 Matrix Metalloproteinase (MT1-MMP) Activity.

MicroRNA miR-335 has been reported to have both tumor suppressor and oncogenic activities. In order to determine possible tissue and cell type differences in response to miR-335, we examined the effect of miR-335 on cell expression of MT1-MMP, a proteinase commonly expressed in tumors and associated with cell proliferation and migration. miR-335 increased cell surface expression of MT1-MMP in fibrosarcoma HT-1080 and benign prostate BPH-1 cells, but not in prostate LNCaP or breast MCF-7 tumor cells. miR-335 stimulated proliferation and cell migration in a wound healing in vitro assay in HT-1080, BPH-1, and U87 glioblastoma cells, cells which demonstrated significant cell surface expression of MT1-MMP. In contrast, miR-335 did not affect proliferation or migration in cells without a prominent plasma membrane associated MT1-MMP activity. Our data suggest that differences in response to miR-335 by tumor cells may lie in part in the mechanism of regulation of MT1-MMP production.

Fluid shear stress promotes proprotein convertase-dependent activation of MT1-MMP

During angiogenesis, endothelial cells (ECs1) initiate new blood vessel growth and invade into the extracellular matrix (ECM). Membrane type-1 matrix metalloproteinase (MT1-MMP) facilitates this process and translocates to the plasma membrane following activation to promote ECM cleavage. The N-terminal pro-domain within MT1-MMP must be processed for complete activity of the proteinase. This study investigated whether MT1-MMP activation was altered by sphingosine 1-phosphate (S1P) and wall shear stress (WSS), which combine to stimulate EC invasion in three dimensional (3D) collagen matrices. MT1-MMP was activated rapidly and completely by WSS but not S1P. Proprotein convertases (PCs) promoted MT1-MMP processing, prompting us to test whether WSS or S1P treatments increased PC activity. Like MT1-MMP, PC activity increased with WSS, while S1P had no effect. A pharmacological PC inhibitor completely blocked S1P- and WSS-induced EC invasion and MT1-MMP translocation to the plasma membrane. Further, a recombinant PC inhibitor reduced MT1-MMP activation and decreased lumen formation in invading ECs, a process known to be controlled by MT1-MMP. Thus, we conclude that PC and MT1-MMP activation are mechanosensitive events that are required for EC invasion into 3D collagen matrices.

Noncanonical Activation of Notch1 Protein by Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Controls Melanoma Cell Proliferation

Notch1 is an evolutionarily conserved signaling molecule required for stem cell maintenance that is inappropriately reactivated in several cancers. We have previously shown that melanomas reactivate Notch1 and require its function for growth and survival. However, no Notch1-activating mutations have been observed in melanoma, suggesting the involvement of other activating mechanisms. Notch1 activation requires two cleavage steps: first by a protease and then by γ-secretase, which releases the active intracellular domain (Notch1(NIC)). Interestingly, although ADAM10 and -17 are generally accepted as the proteases responsible of Notch1 cleavage, here we show that MT1-MMP, a membrane-tethered matrix metalloproteinase involved in the pathogenesis of a number of tumors, is a novel protease required for the cleavage of Notch1 in melanoma cells. We find that active Notch1 and MT1-MMP expression correlate significantly in over 70% of melanoma tumors and 80% of melanoma cell lines, whereas such correlation does not exist between Notch1(NIC) and ADAM10 or -17. Modulation of MT1-MMP expression in melanoma cells affects Notch1 cleavage, whereas MT1-MMP expression in ADAM10/17 double knock-out fibroblasts restores the processing of Notch1, indicating that MT1-MMP is sufficient to promote Notch1 activation independently of the canonical proteases. Importantly, we find that MT1-MMP interacts with Notch1 at the cell membrane, supporting a potential direct cleavage mechanism of MT1-MMP on Notch1, and that MT1-MMP-dependent activation of Notch1 sustains melanoma cell growth. Together, the data highlight a novel mechanism of activation of Notch1 in melanoma cells and identify Notch1 as a new MT1-MMP substrate that plays important biological roles in melanoma.

Angiopoietin-like protein 2 regulates endothelial colony forming cell vasculogenesis

Angiopoietin-like 2 (ANGPTL2) has been reported to induce sprouting angiogenesis; however, its role in vasculogenesis, the de novo lumenization of endothelial cells (EC), remains unexplored. We sought to investigate the potential role of ANGPTL2 in regulating human cord blood derived endothelial colony forming cell (ECFC) vasculogenesis through siRNA mediated inhibition of ANGPTL2 gene expression. We found that ECFCs in which ANGPTL2 was diminished displayed a threefold decrease in in vitro lumenal area whereas addition of exogenous ANGPTL2 protein domains to ECFCs lead to increased lumen formation within a 3 dimensional (3D) collagen assay of vasculogenesis. ECFC migration was attenuated by 36 % via ANGPTL2 knockdown (KD) although proliferation and apoptosis were not affected. We subsequently found that c-Jun NH2-terminal kinase (JNK), but not ERK1/2, phosphorylation was decreased upon ANGPTL2 KD, and expression of membrane type 1 matrix metalloproteinase (MT1-MMP), known to be regulated by JNK and a critical regulator of EC migration and 3D lumen formation, was decreased in lumenized structures in vitro derived from ANGPTL2 silenced ECFCs. Treatment of ECFCs in 3D collagen matrices with either a JNK inhibitor or exogenous rhTIMP-3 (an inhibitor of MT1-MMP activity) resulted in a similar phenotype of decreased vascular lumen formation as observed with ANGPTL2 KD, whereas stimulation of JNK activity increased vasculogenesis. Based on gene silencing, pharmacologic, cellular, and biochemical approaches, we conclude that ANGPTL2 positively regulates ECFC vascular lumen formation likely through its effects on migration and in part by activating JNK and increasing MT1-MMP expression.

Non-destructive and Selective Imaging of the Functionally Active, Pro-invasive Membrane Type-1 Matrix Metalloproteinase (MT1-MMP) Enzyme in Cancer Cells

Proteolytic activity of cell surface-associated MT1-matrix metalloproteinase (MMP) (MMP-14) is directly related to cell migration, invasion, and metastasis. MT1-MMP is regulated as a proteinase by activation and conversion of the latent proenzyme into the active enzyme, and also via inhibition by tissue inhibitors of MMPs (TIMPs) and self-proteolysis. MT1-MMP is also regulated as a membrane protein through its internalization and recycling. Routine immunohistochemistry, flow cytometry, reverse transcription-PCR, and immunoblotting methodologies do not allow quantitative imaging and assessment of the cell-surface levels of the active, TIMP-free MT1-MMP enzyme. Here, we developed a fluorescent reporter prototype that targets the cellular active MT1-MMP enzyme alone. The reporter (MP-3653) represents a liposome tagged with a fluorochrome and functionalized with a PEG chain spacer linked to an inhibitory hydroxamate warhead. Our studies using the MP-3653 reporter and its inactive derivative demonstrated that MP-3653 can be efficiently used not only to visualize the trafficking of MT1-MMP through the cell compartment, but also to quantify the femtomolar range amounts of the cell surface-associated active MT1-MMP enzyme in multiple cancer cell types, including breast carcinoma, fibrosarcoma, and melanoma. Thus, the levels of the naturally expressed, fully functional, active cellular MT1-MMP enzyme are roughly equal to 1 × 10(5) molecules/cell, whereas these levels are in a 1 × 10(6) range in the cells with the enforced MT1-MMP expression. We suggest that the reporter we developed will contribute to the laboratory studies of MT1-MMP and then, ultimately, to the design of novel, more efficient prognostic approaches and personalized cancer therapies.

Insights into Ectodomain Shedding and Processing of Protein-tyrosine Pseudokinase 7 (PTK7)

The membrane PTK7 pseudokinase, a component of both the canonical and noncanonical/planar cell polarity Wnt pathways, modulates cell polarity and motility in biological processes as diverse as embryo development and cancer cell invasion. To determine the individual proteolytic events and biological significance of the ectodomain shedding in the PTK7 function, we used highly invasive fibrosarcoma HT1080 cells as a model system. Current evidence suggested a likely link between PTK7 shedding and cell invasion in our HT1080 cell model system. We also demonstrated that in HT1080 cells the cleavage of the PTK7 ectodomain by an ADAM proteinase was coupled with the membrane type-1 matrix metalloproteinase (MT1-MMP) cleavage of the PKP(621)↓LI site in the seventh Ig-like domain of PTK7. Proteolytic cleavages led to the generation of two soluble, N-terminal and two matching C-terminal, cell-associated fragments of PTK7. This proteolysis was a prerequisite for the intramembrane cleavage of the C-terminal fragments of PTK7 by γ-secretase. γ-Secretase cleavage was predominantly followed by the efficient decay of the resulting C-terminal PTK7 fragment via the proteasome. In contrast, in HT1080 cells, which overexpressed the C-terminal PTK7 fragment, the latter readily entered the nucleus. Our data imply that therapeutic inhibition of PTK7 shedding may be used to slow cancer progression.