Membrane-type 1 Matrix Metalloproteinase Overexpression Research Articles

Membrane type 1 matrix metalloproteinase promotes LDL receptor shedding and accelerates the development of atherosclerosis

Plasma low-density lipoprotein (LDL) is primarily cleared by LDL receptor (LDLR). LDLR can be proteolytically cleaved to release its soluble ectodomain (sLDLR) into extracellular milieu. However, the proteinase responsible for LDLR cleavage is unknown. Here we report that membrane type 1-matrix metalloproteinase (MT1-MMP) co-immunoprecipitates and co-localizes with LDLR and promotes LDLR cleavage. Plasma sLDLR and cholesterol levels are reduced while hepatic LDLR is increased in mice lacking hepatic MT1-MMP. Opposite effects are observed when MT1-MMP is overexpressed. MT1-MMP overexpression significantly increases atherosclerotic lesions, while MT1-MMP knockdown significantly reduces cholesteryl ester accumulation in the aortas of apolipoprotein E (apoE) knockout mice. Furthermore, sLDLR is associated with apoB and apoE-containing lipoproteins in mouse and human plasma. Plasma levels of sLDLR are significantly increased in subjects with high plasma LDL cholesterol levels. Thus, we demonstrate that MT1-MMP promotes ectodomain shedding of hepatic LDLR, thereby regulating plasma cholesterol levels and the development of atherosclerosis.

Abstract B62: Assessing the relationship between Mucin 16 and Membrane-type I Matrix Metalloproteinase in intra-peritoneal metastatic events

Abstract The process of epithelial ovarian cancer (EOC) metastasis is unique in that epithelial cells detach from the primary tumor and shed into the peritoneal cavity as single cells and multicellular aggregates (MCA), which adhere intraperitoneally and invade the interstitial collagen-rich submesothelial matrix, where they proliferate to anchor secondary lesions. Mucin 16 (MUC16), clinically referred to as cancer antigen 125 (CA125), is a giant mucin-like glycoprotein which is undetected in the epithelium of normal ovaries but highly expressed on the ovarian tumor cell surface. Interestingly, MUC16 tends to shed from tumors, releasing soluble proteolytic fragments into the bloodstream; 90% of women with advanced EOC have elevated blood serum levels of CA125. The catalyst(s) for this shedding is unknown. Membrane-type 1 matrix metalloproteinase (MT1-MMP), an extracellular matrix degrading transmembrane protease, has been implicated in a number of pro-metastatic events. The overexpression of MT1-MMP in EOC tumors relative to the normal ovary, with enhanced expression in metastases relative to primary tumors, suggests that it may be the protease facilitating MUC16 shedding. Immunofluorescence and flow-cytometric detection of MUC16 in ovarian cancer (ovca) cells engineered to overexpress MT1-MMP indicate that MT1-MMP overexpressors have lower surface levels of MUC16. As recent studies suggest that MUC16 binds selectively to mesothelin (potentially mediating the cell-to-cell adhesion inherent in metastasis), we tested the hypothesis that cells that overexpress MT1-MMP (and thus shed MUC16) will be less adherent to mesothelial cells. Examination of the adherence of MT1-MMP overexpressing ovca cells to a live mesothelial cell monolayer indicated decreased cell-to-cell adhesion compared to controls. We have previously shown that up-regulation of MT1-MMP promotes a collagen invasive phenotype in ovarian carcinomas. As the sub-mesothelial matrix is rich in interstitial collagen, we further investigated the potential for MT1-MMP overexpressors to invade a live mesothelial cell monolayer. To assay invasion, a modified transwell “mesothelial mimetic” assay was utilized consisting of a three dimensional collagen type I matrix atop an 8μm filter and overlaid with a confluent layer of mesothelial cells. Seeding of ovca cells +/- MT1-MMP overexpression followed by quantitation of migrating/invading cells indicates a role for MT1-MMP in sub-mesothelial anchoring. These results intimate that MUC16 expression may be key for initial cell-to-mesothelial cell contact and suggest that MT1-MMP overexpression contributes to sub-mesothelial anchoring of EOC cells. Citation Format: Lana Y. Bruney, M. Sharon Stack. Assessing the relationship between Mucin 16 and Membrane-type I Matrix Metalloproteinase in intra-peritoneal metastatic events. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B62.

Abstract 340: MT1-MMP triggers an epithelial to mesenchymal transition program via activation of a TGF-β signaling pathway leading to Wnt5a induction

Abstract Metastases account for the vast majority of cancer-related deaths and is thought to involve a process known as epithelial-to-mesenchymal transition (EMT). EMT alters cells from a connected, stationary epithelial state to a more motile, invasive mesenchymal state. Expression of the Membrane-type 1 matrix metalloproteinase (MT1-MMP), one of the key mediators of tumor invasion and metastasis, has been reported to trigger prostate cancer cells to undergo EMT. We have found that MT1-MMP overexpression in LNCaP prostate cancer (LNCaP/MT1-MMP-GFP) and MCF-7 breast cancer (MCF-7/MT1-MMP) cells resulted in increased Wnt5a mRNA and protein. Conversely, MT1-MMP shRNA knockdown, in DU145 cells (DU145/MT1 shRNA) that express high endogenous MT1-MMP, decreased Wnt5a. Increased Wnt5a was required for MT1-MMP-mediated EMT; Wnt5a shRNA expression in MT1-MMP overexpressing cells reversed EMT, resulting in increased E-cadherin and a more epithelial morphology. Increased Wnt5a expression occured via MT1-MMP-mediated activation of a TGF-β signaling pathway. LNCaP/ MT1-MMP-GFP cells had enhanced levels of activated TGF-β, and DU145/MT1 shRNA cells displayed reduced levels of activated TGF-β relative to their respective control cells. Inhibition of TGF-β activity with a neutralizing antibody in LNCaP/MT1-MMP-GFP resulted in decreased Wnt5a expression relative to control cells. MT1-MMP-mediated TGF-β activation was inhibited with the MMP inhibitor BB3103, and LNCaP cells overexpressing the catalytically inactive MT1-MMP (LNCaP/MT1/E240-A) had almost undetectable levels of activated TGF-β. Both LNCaP/MT1-MMP-GFP treated with BB3101 and LNCaP/MT1/E-240-A failed to induce increased Wnt5a. These results support MT1-MMP catalytic activity as a requirement for activating TGF-β and for MT1-MMP mediated upregulation of Wnt5a. The CUTL1 transcription factor has been reported to be regulated by TGF-β signaling. LNCaP/MT1-MMP-GFP and MCF-7/MT1-MMP cells showed enhanced CUTL1 expression, and DU145/MT1 shRNA cells had decreased CUTL1 relative to control DU145/GFP shRNA. shRNA targeting of CUTL1, in LNCaP/MT1-MMP-GFP cells, resulted in decreased Wnt5a expression and reversal of the EMT phenotype. Upregulation of Wnt5a promoted anchorage-independent growth; Wnt5a shRNA knockdown in either LNCaP/MT1-MMP-GFP or in DU145 cells each resulted in markedly decreased growth in soft agar. Wnt5a shRNA expression in LNCaP/MT1-MMP-GFP also significantly decreased the growth of mouse tumors derived from these cells relative to tumors from LNCaP/MT1-MMP-GFP. These results demonstrate the significance of Wnt5a expression in MT1-MMP-mediated tumor progression. Collectively, these results highlight a novel link between MT1-MMP and the Wnt pathway, mediated by TGF-β signaling, that illuminate the relationship between these factors and their roles in EMT and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 340. doi:1538-7445.AM2012-340

Laminin-332-Rich Tumor Microenvironment for Tumor Invasion in the Interface Zone of Breast Cancer

Dense fibrosis, which is caused by desmoplastic reaction, is usually found in invasive ductal carcinoma and may represent the alteration of the tumor microenvironment preceding tumor invasion. Thus, the dense fibrotic zone around invasive ductal carcinoma can be considered to be the actual tissue site of tumor microenvironment, where the precedent alterations for tumor invasion occur. To characterize the dense fibrotic zone, we classified invasive ductal carcinoma tissue into a tumor zone, a normal zone, and the novel interface zone (IZ), which shows dense fibrosis. The postulated IZ is a 5-mm-wide belt that circles the tumor margin and overlaps with normal tissue. Of the extracellular matrix components, laminin-332 was specifically overexpressed in the IZ. Events that appear to be similar to the epithelial-mesenchymal transition, a novel source of myofibroblast formation from epithelial cells, were observed in the IZ, according to the following characteristics: overexpression of matrix metalloproteinase 3, membrane type 1-matrix metalloproteinase, snail, and zinc finger E-box-binding homeobox 1, and the gain of N-cadherin expression, as well as the down-regulation of miR200c. The myofibroblasts isolated from the IZ, which were designated interface zone-fibroblast, displayed laminin-332 and membrane type 1-matrix metalloproteinase overexpression, in contrast with both cancer-associated fibroblasts and normal breast fibroblasts. Taken together, our results suggest that the IZ, which shows dense fibrosis, may provide a specialized microenvironment for guiding tumor invasion: the fibrosis caused by laminin-332 overexpressing myofibroblast formation (interface zone-fibroblast) via epithelial-mesenchymal transition.

Cardiac Restricted Overexpression of Membrane Type-1 Matrix Metalloproteinase Causes Adverse Myocardial Remodeling following Myocardial Infarction

The membrane type-1 matrix metalloproteinase (MT1-MMP) is a unique member of the MMP family, but induction patterns and consequences of MT1-MMP overexpression (MT1-MMPexp), in a left ventricular (LV) remodeling process such as myocardial infarction (MI), have not been explored. MT1-MMP promoter activity (murine luciferase reporter) increased 20-fold at 3 days and 50-fold at 14 days post-MI. MI was then induced in mice with cardiac restricted MT1-MMPexp (n = 58) and wild type (WT, n = 60). Post-MI survival was reduced (67% versus 46%, p < 0.05), and LV ejection fraction was lower in the post-MI MT1-MMPexp mice compared with WT (41 ± 2 versus 32 ± 2%,p < 0.05). In the post-MI MT1-MMPexp mice, LV myocardial MMP activity, as assessed by radiotracer uptake, and MT1-MMP-specific proteolytic activity using a specific fluorogenic assay were both increased by 2-fold. LV collagen content was increased by nearly 2-fold in the post-MI MT1-MMPexp compared with WT. Using a validated fluorogenic construct, it was discovered that MT1-MMP proteolytically processed the pro-fibrotic molecule, latency-associated transforming growth factor-1 binding protein (LTBP-1), and MT1-MMP-specific LTBP-1 proteolytic activity was increased by 4-fold in the post-MI MT1-MMPexp group. Early and persistent MT1-MMP promoter activity occurred post-MI, and increased myocardial MT1-MMP levels resulted in poor survival, worsening of LV function, and significant fibrosis. A molecular mechanism for the adverse LV matrix remodeling with MT1-MMP induction is increased processing of pro-fibrotic signaling molecules. Thus, a proteolytically diverse portfolio exists for MT1-MMP within the myocardium and likely plays a mechanistic role in adverse LV remodeling.

Membrane Type-1 Matrix Metalloproteinase Potentiates Basic Fibroblast Growth Factor-Induced Corneal Neovascularization

Corneal neovascularization is one of the leading causes of blindness. The aim of this study was to evaluate the pro-angiogenic role of corneal fibroblast-derived membrane type-1 matrix metalloproteinase (MT1-MMP) on basic fibroblast growth factor (bFGF)-induced corneal neovascularization in vivo and in vitro. Immunohistochemical studies demonstrated that MT1-MMP was expressed in keratocytes and immortalized corneal fibroblast cell lines. Vascular endothelial growth factor protein levels were increased after bFGF-stimulation of wild-type fibroblast cells compared with MT1-MMP knockout fibroblast cells. Corneal vascularization was significantly increased after a combination of bFGF pellet implantation and naked MT1-MMP DNA injection in wild-type mouse corneas compared with either bFGF pellet implantation or naked MT1-MMP DNA-injected corneas. Western blotting analysis of the phosphorylation levels of the key signaling molecules (p38, JNK, and ERK) demonstrated that phosphorylation levels of both p38 and JNK were diminished after bFGF stimulation of MT1-MMP knockout cells compared with wild-type and MT1-MMP knockin cells. These results suggest that MT1-MMP potentiates bFGF-induced corneal neovascularization, likely by modulating the bFGF signal transduction pathway.

Membrane Type-1 Matrix Metalloproteinase (MT1-MMP) Exhibits an Important Intracellular Cleavage Function and Causes Chromosome Instability

Elevated expression of membrane type-1 matrix metalloproteinase (MT1-MMP) is closely associated with malignancies. There is a consensus among scientists that cell surface-associated MT1-MMP is a key player in pericellular proteolytic events. Now we have identified an intracellular, hitherto unknown, function of MT1-MMP. We demonstrated that MT1-MMP is trafficked along the tubulin cytoskeleton. A fraction of cellular MT1-MMP accumulates in the centrosomal compartment. MT1-MMP targets an integral centrosomal protein, pericentrin. Pericentrin is known to be essential to the normal functioning of centrosomes and to mitotic spindle formation. Expression of MT1-MMP stimulates mitotic spindle aberrations and aneuploidy in non-malignant cells. Volumes of data indicate that chromosome instability is an early event of carcinogenesis. In agreement, the presence of MT1-MMP activity correlates with degraded pericentrin in tumor biopsies, whereas normal tissues exhibit intact pericentrin. We believe that our data show a novel proteolytic pathway to chromatin instability and elucidate the close association of MT1-MMP with malignant transformation.

Expression and localization of membrane-type-1 matrix metalloproteinase, CD 44, and laminin-5?2 chain during colorectal carcinoma tumor progression

Membrane-type-1 matrix metalloproteinase (MT1-MMP) is overexpressed in many malignant tumor tissues and would be involved in tumor-cell migration. Using dual immunofluorescence of frozen sections, this study examined the expression and localization of MT1-MMP and its interacting molecules, CD44 and laminin-5gamma2 chain (LN-5gamma2) monomer, in 48 cases of colorectal tumors. Recent studies have shown that MT1-MMP, CD44 and LN-5gamma2 are direct downstream targets in the adenomatosis polyposis coli (APC)/beta-catenin (Wnt)-signaling pathway, which is upregulated in most colorectal epithelial tumors. MT1-MMP overexpression was observed in adenocarcinoma cases with moderate and/or less differentiation coinciding with CD44 downmodulation. Recent observations indicate that MT1-MMP overexpression disrupts tubulogenesis of MDCK cells in type-I collagen-rich tissues. Therefore, MT1-MMP overexpression might involve disturbances of neoplastic glandular structures during colorectal adenocarcinoma tumor progression. Intensity distribution analyses of images with dual immunofluorescence indicated that overexpressed MT1-MMP is closely associated with the enhanced expression of the LN-5gamma2 monomers at the invasive front of dedifferentiated tumor cells. Additionally, the graded expression of nuclear active beta-catenin was found in moderately differentiated and dedifferentiated areas of adenocarcinomas, where MT1-MMP overexpression was observed. Therefore, this study reveals that MT1-MMP might be a major effector of Wnt signaling in the late stage of colorectal carcinoma tumor progression.

Up-regulation of Vascular Endothelial Growth Factor-A by Active Membrane-type 1 Matrix Metalloproteinase through Activation of Src-Tyrosine Kinases

Membrane-type 1 matrix metalloproteinase (MT1-MMP) and vascular endothelial growth factor (VEGF) are two key molecules involved in pericellular proteolysis and cell proliferation during tumor growth and angiogenesis. Our previous data showed that MT1-MMP overexpression in human breast carcinoma MCF7 cells induced an up-regulation of VEGF expression. This effect was associated in vivo with accelerated tumor growth and angiogenesis. We now provide evidence that MT1-MMP overexpression specifically affected VEGF-A production and failed to influence that of other VEGF family members (VEGF, B, C, D, or PlGF) or their receptors. The up-regulation of VEGF-A by MT1-MMP was related to an increased transcriptional activation rather than to a modification of mRNA stability. It was blocked by synthetic MMP inhibitors, TIMP2, but not TIMP-1 and abolished by a partial deletion of the catalytic domain or the cytoplasmic tail of MT1-MMP. Analysis of the signal transduction mechanisms demonstrated that MT1-MMP acts through a signaling pathway involving Src tyrosine kinases. Thus, our results provide new insight into the mechanisms of action of MT1-MMP during angiogenesis and suggest that the full enzymatic activity of MT1-MMP is required for a specific up-regulation of VEGF-A through an activation of Src tyrosine kinase pathways.

Expression of membrane type 1 matrix metalloproteinase (MT1-MMP) in A2058 melanoma cells is associated with MMP-2 activation and increased tumor growth and vascularization.

Membrane-type metalloproteinase-1 (MT1-MMP) is a transmembrane metalloproteinase overexpressed in tumors, which plays a major role in the first step of pro-MMP-2 activation, leading to the generation of an intermediate 62 kDa species. The second step of MMP-2 activation that yields to the mature form is less understood and could involve an autocatalytic process and/or the activity of the plasminogen/plasmin system. Human melanoma A2058 cells, which express MMP-2 only in its pro-form, were used to determine the role of MT1-MMP during pericellular proteolysis and tumor progression. The induction of MT1-MMP overexpression by MT1-MMP cDNA transfection initiated the first step of MMP-2 activation. We provide evidence that a cooperation between the plasminogen/plasmin system and MT1-MMP endowed the cells with the ability to fully activate MMP-2 and with enhanced invasive properties in vitro. When injected subcutaneously in nude mice, MT1-MMP expressing clones induced rapid tumor growth and high tumor vascularization, while the control clones were poorly or not tumorigenic. Our data provide the first demonstration, in an experimental model, that MT1-MMP expression by tumor cells promotes tumor vascularization.

Activation of the extracellular signal-regulated protein kinase (ERK) cascade by membrane-type-1 matrix metalloproteinase (MT1-MMP)

The mechanisms underlying membrane-type-1 matrix metalloproteinase (MT1-MMP)-dependent induction of cell migration were investigated. Overexpression of MT1-MMP induced a marked increase in cell migration, this increase being dependent on the presence of the cytoplasmic domain of the protein. MT1-MMP-dependent migration was inhibited by a mitogen-activated protein kinase kinase 1 inhibitor, suggesting the involvement of the extracellular signal-regulated protein kinase (ERK) cascade in the induction of migration. Accordingly, MT1-MMP overexpression induced the activation of ERK, this process being also dependent on the presence of its cytoplasmic domain. MT1-MMP-induced activation of both migration and ERK required the catalytic activity of the enzyme as well as attachment of the cells to matrix proteins. The MT1-MMP-dependent activation of ERK was correlated with the activation of transcription through the serum response element, whereas other promoters were unaffected. Taken together, these results indicate that MT1-MMP trigger important changes in cellular signal transduction events, leading to cell migration and to gene transcription, and that these signals possibly originate from the cytoplasmic domain of the protein.

Neutrophil-derived serine proteinases enhance membrane type-1 matrix metalloproteinase–dependent tumor cell invasion

Background: Matrix metalloproteinase-2 degrades a variety of basement membrane components and is essential for tumor invasion. We have previously reported that membrane type-1 matrix metalloproteinase (MT1-MMP) cooperates with neutrophil-derived serine proteinases (NDPs; elastase, cathepsin G, protease-3) to activate matrix metalloproteinase-2. We therefore hypothesized that NDPs enhance tumor-cell invasion. Methods: Clones of human HT1080 fibrosarcoma cells transfected with MT1-MMP sense (HT-SE) or antisense CDNA (HT-AS) were used. These cells express either high (HT-SE) or extremely low levels (HT-AS) of MT1-MMP relative to nontransfected HT1080 cells (HT-WT). The cells were incubated in the presence or absence of purified NDP, with or without α1-antitrypsin or the MMP inhibitor batimastat. Cell invasion was measured with the use of Boyden chambers with polycarbonate membranes coated with a reconstituted extracellular matrix. Results: Under control conditions HT-WT and HT-SE cells were 4-fold more invasive than HT-AS cells. The addition of NDP increased HT-WT and HT-SE cell invasion 60% to 100% but had no effect on HT-AS cells. α1-antitrypsin or batimastat did not decrease the baseline invasiveness of HT-WT and HT-SE cells; however, they abrogated the stimulatory effect of NDP. Conclusions: HT1080 cell invasion depends on MT1-MMP expression. MT1-MMP overexpression does not increase invasiveness by itself. NDPs increase invasion by MT1-MMP expressing cells by activating matrix metalloproteinase-2. (Surgery 2000;126:142-7.)

Overexpression of MT1-MMP is insufficient to increase experimental liver metastasis of human colon cancer cells

The expression and activation of matrix metalloproteinases (MMPs) by tumor cells is correlated with invasive and metastatic potential. The purpose of this study was to examine the impact of increased membrane type 1 matrix metalloproteinase (MT1-MMP) expression on liver metastatic potential utilizing human colorectal cancer (CRC) cell lines. Three human CRC cell lines, DLD1, HCT116 and HT29, were stably transfected with the MT1-MMP cDNA, and experimental liver metastasis was established by injecting the cells into the spleens of nude mice. Reverse transcription-polymerase chain reaction (RT-PCR) confirmed increased expression of MT1-MMP mRNA in the stable tranfectants. In vitro analysis by gelatin zymography and morphological survey demonstrated that MT1-MMP transfectants displayed a matured gelatinolytic activity and invasive properties when cultured in 3D collagen gel, indicating that transduced MT1-MMP cDNA was functional. Although there was no difference in cell proliferation rate between MT1-MMP overexpressing cells and the Mock control cells, in vivo experiments indicated that the liver metastatic ability was not affected by MT1-MMP overexpression. Our findings indicated that conditional MT1-MMP overexpression was insufficient to increase experimental liver metastasis, suggesting a more complicated mechanism may be involved in the activation and regulation of MMPs cascades in vivo.