Upregulation Of Membrane-type 1 Matrix Metalloproteinase Research Articles

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.

High threshold of β1 integrin inhibition required to block collagen I-induced membrane type-1 matrix metalloproteinase (MT1-MMP) activation of matrix metalloproteinase 2 (MMP-2).

BackgroundMatrix metalloproteinase-2 (MMP-2) is an endopeptidase that facilitates extracellular matrix remodeling and molecular regulation, and is implicated in tumor metastasis. Type I collagen (Col I) regulates the activation of MMP-2 through both transcriptional and post-transcriptional means; however gaps remain in our understanding of the involvement of collagen-binding β1 integrins in collagen-stimulated MMP-2 activation.MethodsThree β1 integrin siRNAs were used to elucidate the involvement of β1 integrins in the Col I-induced MMP-2 activation mechanism. β1 integrin knockdown was analyzed by quantitative RT-PCR, Western Blot and FACS analysis. Adhesion assay and collagen gel contraction were used to test the biological effects of β1 integrin abrogation. MMP-2 activation levels were monitored by gelatin zymography.ResultsAll three β1 integrin siRNAs were efficient at β1 integrin knockdown and FACS analysis revealed commensurate reductions of integrins α2 and α3, which are heterodimeric partners of β1, but not αV, which is not. All three β1 integrin siRNAs inhibited adhesion and collagen gel contraction, however only the siRNA showing the greatest magnitude of β1 knockdown inhibited Col I-induced MMP-2 activation and reduced the accompanying upregulation of MT1-MMP, suggesting a dose response threshold effect. Re-transfection with codon-swapped β1 integrin overcame the reduction in MMP-2 activation induced by Col-1, confirming the β1 integrin target specificity. MMP-2 activation induced by TPA or Concanavalin A (Con A) was not inhibited by β1 integrin siRNA knockdown.ConclusionTogether, the data reveals that strong abrogation of β1 integrin is required to block MMP-2 activation induced by Col I, which may have implications for the therapeutic targeting of β1 integrin.

Abstract LB-30: Membrane-anchored MT1-MMP downstream of p63 is essential for the transition of in situ to invasive breast carcinoma

Abstract Understanding breast tumor progression requires insights both at the molecular and cellular levels. In particular, the transition of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) is a key, yet poorly understood event. Membrane-Type 1 matrix metalloproteinase (MT1-MMP) is critical for pericellular remodelling of the basement membrane and interstitial collagen during invasion by carcinoma cells. The role of MT1-MMP during the transition from DCIS to IDC was investigated using an intraductal human-in-mouse xenograft model. Breast tumor-derived MCF10.DCIS.com cells, which express MT1-MMP were injected into the primary mammary ducts of female SCID mice. Histological analysis revealed a progression from DCIS, to microinvasive and invasive lesions by 5, 7-8 and 10-12 weeks post-injection, respectively. Immunohistochemistry (IHC) staining showed homogeneous expression of MT1-MMP over the tumor section at DCIS stage, and up-regulation at the edge of the tumours at microinvasive and invasive states. In microinvasives lesion, up-regulation of MT1-MMP in tumour cells coincided with disruption of the basement membrane. At later stages, one characteristic of invasive lesions is the increase in collagen fibers deposition. Interestingly, MT1-MMP expression was also increased at the front of infiltrating tumours in contact with the stroma. After 10 weeks, intraductally injected cells knocked down for MT1-MMP developed invasive tumours only in 20% of injected mammary glands, while 100% of glands injected with control cells developed infiltrating lesions. All together, these results demonstrated that MT1-MMP is instrumental for the transition from in situ to invasive breast tumours. IHC staining revealed a strong correlation of the expression of the basal marker p63 and MT1-MMP in tumour cells both at the microinvasive and invasive stages. One hypothesis was that contact of tumor cells with the stroma triggered induction of p63 that in turn up-regulated MT1-MMP. Along this line, we found that MCF10.DCIS.com cells plated on type I collagen up-regulated p63 and MT1-MMP, both at the mRNA and protein levels, while silencing of p63 abolished collagen-dependent MT1-MMP increase. At the functional level, silencing of MT1-MMP or p63 inhibited invasion of multicellular spheroids into 3D type collagen as well as the cells capacity to degrade collagen I fibers. Finally, MT1-MMP expression was analysed by IHC staining on human tissue microarray comprising 432 IDCs, 40 microinfiltrated lesions and 68 DCIS. MT1-MMP was upregulated in IDC as compared to DCIS. Invasive triple-negative breast (TNBC) and grade III tumours expressed highest MT1-MMP levels. To our knowledge, this is the first demonstration that up-regulation of MT1-MMP is associated with invasiveness, histopathologic grade and molecular subtypes in human breast cancer. We also observed 10% of TNBC expressing p63 and found a positive correlation with MT1-MMP expression suggesting an interplay between p63 and MT1-MMP during progression. Citation Format: Catalina Lodillinsky, Elvira Infante, Laetitia Fuhrmann, Alan Guichard, Joanna Cyrta, Emilie Lagoutte, Marie Irondelle, Sophie Vacher, Ivan Bieche, Marina Glukhova, Anne Vincent-Salomon, Philippe Chavrier. Membrane-anchored MT1-MMP downstream of p63 is essential for the transition of in situ to invasive breast carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-30. doi:10.1158/1538-7445.AM2014-LB-30

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.

Membrane Type 1 Matrix Metalloproteinase induces an epithelial to mesenchymal transition and cancer stem cell-like properties in SCC9 cells

BackgroundTissue invasion and metastasis are acquired abilities of cancer and related to the death in oral squamous cell carcinoma (OSCC). Emerging observations indicate that the epithelial-to-mesenchymal transition (EMT) is associated with tumor progression and the generation of cells with cancer stem cells (CSCs) properties. Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) is a cell surface proteinase, which is involved in degrading extracellular matrix components that can promote tumor invasion and cell migration.MethodsIn the current study, we utilized SCC9 cells stably transfected with an empty vector (SCC9-N) or a vector encoding human MT1-MMP (SCC9-M) to study the role of MT1-MMP in EMT development.ResultsUpon up-regulation of MT1-MMP, SCC9-M cells underwent EMT, in which they presented a fibroblast-like phenotype and had a decreased expression of epithelial markers (E-cadherin, cytokeratin18 and β-catenin) and an increased expression of mesenchymal markers (vimentin and fibronectin). We further demonstrated that MT1-MMP-induced morphologic changes increased the level of Twist and ZEB, and were dependent on repressing the transcription of E-cadherin. These activities resulted in low adhesive, high invasive abilities of the SCC9-M cells. Furthermore, MT1-MMP-induced transformed cells exhibited cancer stem cell (CSC)-like characteristics, such as low proliferation, self-renewal ability, resistance to chemotherapeutic drugs and apoptosis, and expression of CSCs surface markers.ConclusionsIn conclusion, our study indicates that overexpression of MT1-MMP induces EMT and results in the acquisition of CSC-like properties in SCC9 cells. Our growing understanding of the mechanism regulating EMT may provide new targets against invasion and metastasis in OSCC.

Three-Dimensional Collagen I Promotes Gemcitabine Resistance in Pancreatic Cancer through MT1-MMP–Mediated Expression of HMGA2

One of the hallmarks of human pancreatic ductal adenocarcinoma (PDAC) is its pronounced type I collagen-rich fibrotic reaction. Although recent reports have shown that the fibrotic reaction can limit the efficacy of gemcitabine chemotherapy, the underlying mechanisms remain poorly understood. In this article, we show that the type I collagen allows PDAC cells to override checkpoint arrest induced by gemcitabine. Relative to cells grown on tissue culture plastic, PDAC cells grown in 3-dimensional collagen microenvironment have minimal Chk1 phosphorylation and continue to proliferate in the presence of gemcitabine. Collagen increases membrane type 1 matrix metalloproteinase (MT1-MMP)-dependent ERK1/2 phosphorylation to limit the effect of gemcitabine. Collagen also increases MT1-MMP-dependent high mobility group A2 (HMGA2) expression, a nonhistone DNA-binding nuclear protein involved in chromatin remodeling and gene transcription, to attenuate the effect of gemcitabine. Overexpression of MT1-MMP in the collagen microenvironment increases ERK1/2 phosphorylation and HMGA2 expression, and thereby further attenuates gemcitabine-induced checkpoint arrest. MT1-MMP also allows PDAC cells to continue to proliferate in the presence of gemcitabine in a xenograft mouse model. Clinically, human tumors with increased MT1-MMP show increased HMGA2 expression. Overall, our data show that collagen upregulation of MT1-MMP contributes to gemcitabine resistance in vitro and in a xenograft mouse model, and suggest that targeting MT1-MMP could be a novel approach to sensitize pancreatic tumors to gemcitabine.

Improved bioengineered cartilage tissue formation following cyclic compression is dependent on upregulation of MT1‐MMP

The generation of bioengineered cartilage tissue suitable for transplantation is a potential therapy to treat damaged cartilage. We have shown previously that the physical and biomechanical properties of bioengineered cartilage can be improved by the application of 30 min of cyclic compression by a mechanism involving sequential upregulation of gene and protein levels of membrane type-1 matrix metalloproteinase (MT1-MMP) and MMP-13. In the current study, we demonstrated that MT1-MMP is critical to this response, as blocking the upregulation of MT1-MMP prevented the improvement in tissue formation. MT1-MMP seems to act by inducing tissue remodeling as evidenced by the presence of aggrecan degradation products by Western blot analysis and increased release of matrix molecules into the media. Release of these molecules was diminished when MT1-MMP upregulation was prevented. This matrix degradation was likely due to MT1-MMP, as under conditions where MMP-13 expression is maintained (stimulation in the presence of MT1-MMP siRNA) the release of these matrix molecules into the media was still prevented. It also appears that MT1-MMP does not regulate MMP-13 gene expression, as MT1-MMP-siRNA pretreatment had no effect on MMP-13 expression following mechanical stimulation. Further analysis of the anabolic genes and proteins involved in mechanically stimulated cartilage will lead to better understanding of the mechanism(s) underlying tissue formation yielding improved bioengineered cartilage.

Wnt signalling in mouse mesenchymal stem cells: impact on proliferation, invasion and MMP expression

Based on the capacity of mesenchymal stem cells (MSC) to differentiate into multiple cell types in vitro and in vivo, MCS may be a suitable source for cell therapy and regeneration strategies. A prerequisite for effective clinical applications of human MSC (hMSC) is a profound knowledge of signal transduction cascades that mediate processes like proliferation, targeted migration and differentiation. Recently, we identified the canonical Wnt signal transduction pathway as a key player in hMSC proliferation and invasion. To evaluate whether those findings are transferable to the equivalent counterparts in mice, we studied important steps in the wingless/int-1 (Wnt) signal transduction pathway in mouse MSC (mMSC) and mMSC carrying a T cell specific transcription factor (TCF)/lymphoid enhancer binding factor (LEF)-reporter transgene. We found that the induction of the canonical Wnt pathway resulted in the up-regulation of the known Wnt target gene cyclin D1, closely associated with an enhanced proliferation capacity of mMSC. Interestingly, the expression of the Wnt target gene membrane type 1-matrix metalloproteinase (MT1-MMP) was diminished in mMSC upon Wnt3a stimulation, which came along with an impaired invasion. In line with these findings, MMP-2 and MMP-9 expression levels in mMSC were also decreased after Wnt3a treatment. In contrast, inhibition of Wnt signalling by the knockdown of the transcriptional activator beta-catenin resulted in an up-regulation of MT1-MMP and mMSC invasion. By comparing these findings with the settings in hMSC, major differences in Wnt-regulated MMP expression were observed in mMSC. Thus, our data advice caution when mouse model systems represent the pre-clinical validation of MSC-mediated therapeutical approaches.

Identification of membrane type-1 matrix metalloproteinase as a target of hypoxia-inducible factor-2α in von Hippel–Lindau renal cell carcinoma

Metastatic renal cell carcinoma (RCC) resulting from the hereditary loss of the von Hippel-Lindau (VHL) tumor suppressor gene is the leading cause of death in VHL patients due to the deleterious effects of the metastatic tumor(s). VHL functions in the destruction of the alpha subunits of the heterodimeric transcription factor, hypoxia-inducible factor (HIF-1 alpha and HIF-2 alpha), in normoxic conditions. When VHL function is lost, HIF-alpha protein is stabilized, and target hypoxia-inducible genes are transcribed. The process of tumor invasion and metastasis involves the destruction of the extracellular matrix, which is accomplished primarily by the matrix metalloproteinase (MMP) family of enzymes. Here, we describe a connection between the loss of VHL tumor suppressor function and the upregulation of membrane type-1 MMP (MT1-MMP) gene expression and protein. Specifically, MT1-MMP is upregulated in VHL-/- RCC cells through an increase in gene transcription, which is mediated by the cooperative effects of the transcription factors, HIF-2 and Sp1. Further, we identify a functional HIF-binding site in the proximal promoter of MT1-MMP. To our knowledge, this is the first report to show direct regulation of MT1-MMP by HIF-2 and to provide a direct link between the loss of VHL tumor suppressor function and an increase in MMP gene and protein expression.

The hepatitis B virus X protein promotes tumor cell invasion by inducing membrane-type matrix metalloproteinase-1 and cyclooxygenase-2 expression.

Hepatocellular carcinoma is strongly associated with chronic infection by the hepatitis B virus (HBV) and has poor prognosis due to intrahepatic metastasis. HBx is often the only HBV protein detected in hepatic tumor cells; however, its contribution to tumor invasion and metastasis has not been established so far. In this work, we show that HBx enhances tumor cell invasion, both in vivo and in vitro. The increased invasive capacity induced by HBx is mediated by an upregulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) expression, which in turn activates matrix metalloproteinase-2. Induction of both MT1-MMP expression and cell invasion by HBx is dependent on cyclooxygenase-2 (COX-2) activity. In addition, HBx upregulates the expression of COX-2, which is mediated by the transcriptional activation of the COX-2 gene promoter in a nuclear factor of activated T cell-dependent (NF-AT-dependent) manner. These results demonstrate the ability of HBx to promote tumor cell invasion by a mechanism involving the upregulation of MT1-MMP and COX-2 and provide new insights into the mechanism of action of this viral protein and its involvement in tumor metastasis and recurrence of hepatocellular carcinoma.

The hepatitis B virus X protein promotes tumor cell invasion by inducing membrane-type matrix metalloproteinase-1 and cyclooxygenase-2 expression

Hepatocellular carcinoma is strongly associated with chronic infection by the hepatitis B virus (HBV) and has poor prognosis due to intrahepatic metastasis. HBx is often the only HBV protein detected in hepatic tumor cells; however, its contribution to tumor invasion and metastasis has not been established so far. In this work, we show that HBx enhances tumor cell invasion, both in vivo and in vitro. The increased invasive capacity induced by HBx is mediated by an upregulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) expression, which in turn activates matrix metalloproteinase-2. Induction of both MT1-MMP expression and cell invasion by HBx is dependent on cyclooxygenase-2 (COX-2) activity. In addition, HBx upregulates the expression of COX-2, which is mediated by the transcriptional activation of the COX-2 gene promoter in a nuclear factor of activated T cell–dependent (NF-AT–dependent) manner. These results demonstrate the ability of HBx to promote tumor cell invasion by a mechanism involving the upregulation of MT1-MMP and COX-2 and provide new insights into the mechanism of action of this viral protein and its involvement in tumor metastasis and recurrence of hepatocellular carcinoma.

Ligation of α4β1 Integrin on Human Intestinal Mucosal Mesenchymal Cells Selectively Up-Regulates Membrane Type-1 Matrix Metalloproteinase and Confers a Migratory Phenotype

Human intestinal lamina propria mesenchymal cells show high surface expression of the alpha4ss1 integrin. Ligation of alpha4ss1 on mesenchymal cell lines with an activating monoclonal anti-alpha4 antibody or vascular cell adhesion molecule-immunoglobulin (VCAM-IgG) leads to the appearance of activated forms of gelatinase A in culture supernatants, and the de novo expression of activated membrane type-1-matrix metalloproteinase (MT1-MMP). In functional assays, signaling through alpha4ss1 results in an increased capacity of mesenchymal cells to migrate through an artificial extracellular matrix, an effect inhibitable by excess tissue inhibitor of metalloproteinase-2. In organ cultures of human intestine, VCAM-IgG also up-regulates MT1-MMP, and in mucosal ulcers of inflammatory bowel disease patients, MT1-MMP transcripts are abundant, coincident with expression of VCAM-1 on cells at the ulcer margin. Collectively these results suggest that alpha4ss1-induced up-regulation of MT1-MMP may be a crucial factor in the migration of mesenchymal cells into ulcer beds during restitution of diseased gut mucosa.