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Tumor Biology |
The Extracellular Matrix Biology Program, The Burnham Institute, La Jolla, California 92037
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A series of recent studies have established that MT-MMPs, including the most widely expressed MT-MMP, MT1-MMP (11, 12, 13)
, are directly involved in tumor cell invasion, metastasis, and vascularization (7
, 14
, 15)
. In addition to the cleavage of the ECM substrates, the physiological significance of MT1-MMP may involve its discrete surface localization in migrating cells (16
, 17)
. This characteristic is critical for the ability of MT1-MMP to proteolytically control expression and functional status of cell surface receptors, including integrins, CD44, and tissue transglutaminase (18, 19, 20)
. Overall, MT1-MMP exerts pleiotropic effects on cell behavior by cleaving both the ECM and cell surface receptors. Furthermore, it has been shown that in tumor cells overexpressing MT1-MMP, integrin 
vß3 binds the MMP-2 intermediate and promotes its conversion to the mature enzyme, thereby increasing the density of active MMP-2 at discrete regions of the cell surface (21, 22, 23)
. These mechanisms appear to be instrumental in specific clustering of active MMP-2 at the invadopodia and the invasive front of tumor cells expressing MT1-MMP (16
, 24, 25, 26, 27)
.
Numerous studies have demonstrated that MT1-MMP is up-regulated in aggressive tumors of diverse cell/tissue origin (28, 29, 30, 31, 32, 33) . In human gliomas, MT1-MMP overexpression has been strongly associated with malignant progression and invasive behavior of tumor cells (34, 35, 36) . Expression of MT1-MMP is predominantly observed in glioblastomas and is enhanced with increasing tumor grade. TIMP-2, a natural inhibitor of MMPs, shows an inverse correlation, suggesting that insufficiency in TIMP-2 may also contribute to invasion and dissemination of glioma cells (37 , 38) . Treatment with TIMP-2 decreased invasiveness of tumor cells, including glioblastomas U87 and U251 (38 , 39) .
Inhibition of tumor cell motility by TIMP-2 is associated in part with inhibition of both MT1-MMP and MT1-MMP-dependent activation of proMMP-2 (11, 12, 13) . However, TIMP-2 exerts pleiotropic effects on tumor cell behavior. For example, TIMP-2 is capable of down-regulating VEGF expression in murine mammary carcinoma cells in vitro (40) . In vivo, overexpression of TIMP-2 delayed growth and angiogenesis of the induced tumors in mice and reduced the tumor volume as well as the size and density of the angiogenic blood vessels (40) .
In this study, we investigated the effects of MT1-MMP overexpression on tumorigenicity, angiogenesis, and growth characteristics of human glioma U251 cells in a xenograft model in athymic mice. This cell line, well characterized in various functional assays, has been earlier selected to overexpress MTI-MMP, since it has low intrinsic levels of the enzyme (21) . In nude mice, glioma U251 cells stably transfected with MT1-MMP developed fast-growing, extensively vascularized tumors. This suggested that VEGF expression could be up-regulated in MT1-MMP-overexpressing cells. Elevated production of VEGF by glioma U251 cells transfected with MT1-MMP was confirmed in both tumors and tissue culture. These findings suggest that MT1-MMP and VEGF can be functionally linked in glioma growth and angiogenesis. Our results indicate that overexpression of MT1-MMP in tumor cells up-regulates VEGF production, increasing the endothelial cell response and subsequent tumor vascularization and growth.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Flow Cytometry.
All staining procedures were performed on ice in DPBS (pH 7.2; Life Technologies, Inc., Gaithersburg, MD) supplemented with 1 mM CaCl2, 1 mM MgCl2, 1% BSA, and 0.02% sodium azide (all from Sigma Chemical Co., St. Louis, MO). Cells were stained for 1 h with 10 µg/ml rabbit control IgG or antibodies against the hinge domain of MT1-MMP (both from Sigma Chemical Co.). After washing, FITC-conjugated F(ab')2 fragment of goat antirabbit IgG antibodies (Sigma Chemical Co.) was added for 45 min. Cells were analyzed on a FACStar flow cytometer using CellQuest software (Becton Dickinson, Mountain View, CA). Population gates for negative control were set using cells stained with rabbit IgG.
Immunofluorescence Confocal Microscopy.
Cells were plated in 8-well LabTek II glass chambers (Nalge Nunc International, Naperville, IL) at 1.0–1.5 x 104 cells/well. After incubation for 48 h, cells were washed with DPBS and fixed for 20 min in ice-cold methanol:acetone (1:1). Nonspecific binding was blocked by incubation of slides for 30 min at room temperature in DPBS containing 10% goat serum and 5% BSA. Cells were stained for 1 h with 10 µg/ml MT1-MMP-specific antibodies and further incubated with 20 µg/ml goat antirabbit IgG antibodies conjugated with Alexa 568 (Molecular Probes, Eugene, OR). After washing with DPBS, cells were embedded into VectaShield (Vector Laboratories, Burlingame, CA) and examined on a MRC 1024 scanning confocal microscope (Bio-Rad, Hercules, CA). Acquisition and processing of images were performed with Lasersharp (Bio-Rad) and AdobePhotoshop software (San Jose, CA).
Cell Growth Assay.
Increasing concentrations of cells (from 1.0 to 8.0 x 104 cells/well) were plated in a 96-well plate in triplicate in DMEM/FCS. After an overnight incubation, serum-containing medium was replaced with serum-free DMEM, and the cells were serum-starved for 24 h. Thereafter, the serum-free medium was exchanged with DMEM/FCS (100 µl/well), and cultures were incubated for 48 h. The Cell Titer AQueous One Solution Reagent (Promega, Madison, WI) was added at 40 µl/well for the last 4 h of incubation. The amounts of soluble formazan produced due to the reduction of a tetrazolium compound by cells were determined by measuring the absorbance at 490 nm using a SpectraCount plate reader (Packard Instrument Co., Meriden, CT).
Cell Migration in Transwells.
The haptotactic cell migration assays were performed in Transwells (Costar, Cambridge, MA) under serum-free conditions essentially as described previously (19
, 23)
. The undersurface of a 6.5-mm insert membrane with an 8-µm pore size was coated at 4°C overnight with 2 µg/ml vitronectin or 20 µg/ml type I collagen (Vitrogen 100; Collagen Corp., Fremont, CA), washed with PBS, and blocked with 1% BSA. Cells were cultured overnight in DMEM/FCS, detached with enzyme-free buffer (Specialty Media, Lavalette, NJ), and plated at 7.5 x 104 cells in 0.15 ml of serum-free AIM-V medium (Life Technologies, Inc.) per insert. The outer chamber was filled with 0.6 ml of AIM-V medium. After a 24-h incubation, the cells that migrated to the membrane’s undersurface were detached with trypsin/EDTA and counted.
Three-dimensional Collagen Spheroid Assay.
Tumor spheroids were generated essentially as described previously (41)
. Briefly, cells were cultured in DMEM/FCS at 5 x 106 cells/ml in siliconized 50-ml flasks with constant rotation. After 2–3 days, spheroids of approximate diameters of 130 ± 15 µm were selected under an inverted phase-contrast microscope with an ocular scale using an Eppendorf pipette. The selected spheroids were washed with serum-free DMEM and transferred to a dish filled with AIM-V medium. Eight parts of type I collagen were neutralized with one part of 0.1 M NaOH and mixed with one part of 10x DMEM, yielding 
2.5 mg/ml collagen solution. Spheroids were mixed with the neutralized collagen to obtain 8–14 spheroids/ml and 1.5 mg/ml collagen. The spheroid-containing mixture was placed in a well of a tissue culture 24-well cluster (0.5 ml/well) and allowed to polymerize at 37°C for 30 min in a humidified air incubator. Afterward, the wells with the polymerized gels were filled with AIM-V medium (0.5 ml/well). Where indicated, the following components were added to both neutralized collagen and AIM-V medium: 3 µg/ml TIMP-2 (Calbiochem, San Diego, CA); 50 µg/ml recombinant COOH-terminal domain of MMP-2 (PEX), purified as described previously (11)
; 50 µg/ml vitronectin (a kind gift of Dr. R. DiScipio); or 0.3 µg/ml APMA-activated MMP-2 (proMMP-2 activated with APMA and thoroughly dialyzed against serum-free DMEM). Spheroids were monitored under a Diaphot Inverted Tissue Culture Microscope equipped with an eyepiece, Nomarski differential interference contrast, and a camera for automatic exposure 35-mm photomicrography (Nikon USA, Melville, NY). Cultures were fed twice a week with fresh medium. Zymography analysis of conditioned medium was performed as described below.
Mice.
Athymic female BALB/c nu/nu mice were purchased from Benton & Kingman (San Francisco, CA) and kept under pathogen-free conditions. Five to nine 4-week-old animals were used per group. At the end of the experiments, mice were sacrificed according to the NIH guidelines.
In Vivo Tumorigenicity Assay.
Cells were harvested by trypsinization of confluent cultures, washed, and resuspended at 1.0 x 108 cells/ml in ice-cold DMEM. Mice were anesthetized with Avertin (15 ml/kg body weight), and then 50 µl of U-neo or U-MT cell suspension (5.0 x 106 cells) were injected s.c. in the area of the mammary fat pad (1 or 2 sites/animal; 1 cell type/animal). In preliminary experiments, it was found that the number of inoculation sites did not affect the size or growth rate of the developing xenografts (data not shown). Tumor growth was monitored every 6–8 days by caliper measurements of two perpendicular diameters of xenografts (D1 and D2). Tumor volume was calculated by the following formula: 
/6 (D1 x D2)3/2, and it was expressed as mean tumor volume ± SE (in mm3). At 41–55 days after the cell injection, tumors were excised free of connective tissue, washed in ice-cold PBS, cut, and fixed overnight in 4% paraformaldehyde/PBS. After two washes with PBS, the resected tumor samples were placed for 10 h in 30% sucrose/PBS and then frozen in OCT compound (Sakura Finetek USA, Inc., Torrance, CA). Otherwise, the resected tumors were transferred into 70% ethanol, processed, and embedded in paraffin.
Histology and Immunohistochemistry.
For routine histological examination, sections of paraffin-embedded tumors (8–10-µm thick) were stained with H&E. The 10–15-µm sections of frozen tumors were used for immunohistochemical analyses. The sections were incubated for 30 min at room temperature with 0.3% H2O2 to inactivate endogenous peroxidase, blocked for 1 h with 2% normal goat serum in PBS/0.1% Tween 20, and stained overnight at 4°C with 1–4 µg/ml of the following primary antibodies: rat antimouse CD31 (PECAM-1) mAb MEC 13.3 (PharMingen, San Diego, CA); rabbit antihuman VEGF antibodies (Oncogene Research Products, Cambridge, MA); or anti-MT1-MMP antibodies. The slides were incubated with biotinylated secondary donkey antirat or goat antirabbit IgG antibodies (both from Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at room temperature. The sections were then incubated for 30 min with strepavidin-horseradish peroxidase and for 5 min with diaminobenzidine (both from Vector Laboratories). The slides were counterstained with hematoxylin. The tumor sections were examined using a Nikon Eclipse TE300 microscope equipped with a RT slider Spot camera SP402-115 (Diagnostic Instruments, Inc., Sterling Heights, MI). The images were acquired using SpotCam 32 software, version 3.04 (Diagnostic Instruments, Inc.) and then processed with the AdobePhotoshop software.
Gelatin Zymography.
Tumor pieces (about 10 mm3 each) were cut from the tumor interior and periphery, weighed, and extracted with 2x SDS sample buffer (1:2, w/v). After extraction for 2 h at ambient temperature, samples were diluted 2-fold with 1x SDS sample buffer and homogenized by sequential passages through 18- and 23- gauge needles. The solubilized material was separated from the pellet by centrifugation at 14,000 x g for 30 min. Aliquots of supernatants were analyzed by gelatin zymography (20 µl/lane; Refs. 19
and 23
).
For the analysis of gelatinolytic activity in murine plasma, blood was collected from mice in heparin, incubated for 30 min at room temperature, and centrifuged at 1000 x g. The supernatants were diluted with PBS (1:50, v/v), and 5-µl aliquots were analyzed by gelatin zymography.
VEGF ELISA.
Relative amounts of VEGF in the medium conditioned by U-neo or U-MT cells were determined using a VEGF-ELISA kit (Chemicon International, Temecula, CA) according to the manufacturer’s instructions. Cells were plated into 12-well clusters at 2.0 x 105 cells/well in DMEM/FCS with or without 5 ng/ml TNF- (Sigma Chemical Co.). After an overnight incubation, the medium was exchanged for serum-free DMEM (0.5 ml/well) containing the same amount of TNF-. Conditioned medium was harvested from cultures at 24, 48, 72, and 96 h and analyzed for the VEGF content. Each sample was tested in triplicate. Because VEGF ELISAs are frequently nonlinear at high concentrations of the antigen, we expressed VEGF levels in arbitrary units. To evaluate the effect of MT1-MMP inhibition, cells were plated for 24–72 h in 12-well clusters at 1.0 x 105 cells/well in 0.5 ml of DMEM/FCS supplemented with 50 µM hydroxamate inhibitor GM6001 (AMS Scientific, Concord, CA) or 0.2% DMSO (diluent control). Cultures were then transferred to serum-free conditions and incubated for an additional 96 h. VEGF levels in medium samples were measured by ELISA and expressed as a percentage of the VEGF level observed in the U-neo cells incubated with diluent control.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Flow cytometry and immunofluorescence analyses of cells stained with MT1-MMP-specific antibodies confirmed the up-regulation of MT1-MMP expression in the U-MT cells as compared with the U-neo controls (Fig. 1, A and B)
. Immunoprecipitation of MT1-MMP from the lysates of surface biotinylated U-MT cells revealed that MT1-MMP was represented by the Mr 60,000 enzyme and the Mr 43,000–45,000 autolysed catalytically inactive form (Fig. 1C)
. The presence of these two species of MT1-MMP in cells is in agreement with our earlier findings (42)
and the results published by others (43, 44, 45, 46, 47)
. Because the levels of MT1-MMP in the control cells were low, this cell type failed to efficiently activate endogenously produced proMMP-2. The U-MT cells, which overexpressed MT1-MMP, efficiently activated proMMP-2, converting the Mr 68,000 proenzyme into the fully activated Mr 62,000 enzyme via the Mr 64,000 activation intermediate of MMP-2 (Fig. 1D)
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The cyst-like morphology of U-MT spheroids directly correlated with the MT1-MMP-mediated activation of MMP-2 (Fig. 2B)
. Thus, 1 µg/ml TIMP-2 completely blocked both the development of the spheroid lumen in U-MT cultures and the activation of proMMP-2. Similar to TIMP-2, 50 µg/ml PEX reduced the lumen size by 90% and strongly suppressed the autocatalytic maturation of MMP-2. In agreement with our previous observations (21)
, 50 µg/ml vitronectin partially blocked MMP-2 activation as well as lumen development in U-MT spheroid cultures (Fig. 2B)
. It has been shown that coexpression of integrin vß3 and MT1-MMP promotes MMP-2 activation through binding of the MMP-2 intermediate to the ß3 subunit of the integrin (19, 20, 21)
. Because vitronectin is a ligand of vß3, it could suppress lumen formation and the maturation step of MMP-2 activation in the U-MT spheroids by competitively inhibiting the interactions of MMP-2 with the integrin.
In vitro remodeling of collagen matrix by tumor cells such as glioma U251 (48)
and Madin-Darby canine kidney cells (14
, 49) is critically dependent on the activity of MT-MMPs, including MT1-MMP and MT3-MMP. Indeed, the addition of exogenously activated MMP-2 at 0.3 ng/ml did not induce lumen formation in U-neo spheroids or affect the lumen size of U-MT spheroids (Fig. 2B)
. Thus, soluble MMP-2 does not appear to be sufficient for the cyst-like morphogenesis of spheroids. These findings suggest that MT1-MMP functions are likely to be important in tissue reorganization in vivo.
Increased Tumorigenicity of Glioma Cells Overexpressing MT1-MMP.
To investigate the functional role of MT1-MMP on glioma growth in vivo, the U-MT and U-neo cells were s.c. xenografted into immunodeficient mice. The size of the developing tumors was measured at 6–8-day intervals for 41–55 days. The incidence of xenografts was high in each cell type (Table 1)
, but the percentage of tumors originating from the U-MT cells was 16% higher compared with the control. After a relatively long lag period (about 20 days for both cell types), U-MT xenografts acquired a growth rate that far exceeded that of the U-neo tumors. At day 55 postimplantation, the volume of the U-MT tumors was 6-fold larger than that of the control tumors (Fig. 3)
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. In contrast, the U-neo cells did not induce any development of large-size blood vessels at the sites of the cell inoculation. A well-organized network of blood vessels at the periphery of the U-MT tumors was evident after resection (Fig. 4, B and C)
. We did not find any metastatic nodules in the lungs, spleen, or mesenterium of the recipient animals. This was not surprising because transfectants of highly invasive but poorly metastatic glioma U251 cell line were xenografted s.c. In agreement, no metastases of U251 and U87 gliomas have been observed in tissues from recipient mice by histological examination or PCR analysis (50)
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. Connective tissue capsule was well developed in U-MT xenografts and less prominent in the control tumors. By the end of the experiments, the U-MT xenografts regularly displayed necrosis and hemorrhage, mainly in the central parts, whereas no signs of necrosis or hemorrhage were present in the U-neo tumors (data not shown).
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. Staining of the tumor sections with control murine and rabbit IgG was clearly negative (data not shown).
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Next, we evaluated the expression of MT1-MMP in U-MT and U-neo xenografts by immunohistochemistry using anti-MT1-MMP antibodies. MT1-MMP staining was the most prominent at the tumor periphery and considerably more intense in the U-MT tumors as compared with the control (Fig. 6B)
. Because MT1-MMP activity is crucial for the activation of proMMP-2, zymography was performed to identify the status of MMP-2 in tumors. Both the latent and activated forms of MMP-2 were detected in the extracts of control tumors (Fig. 7A
, Lanes 1 and 2). Previously, partial activation of proMMP-2 was observed in MT1-MMP-deficient cell lines such as fibrosarcoma HT-1080 cultured at high cell density (51)
. Therefore, the presence of activated MMP-2 in the U-neo xenografts was not surprising. In contrast, only the active enzyme was identified the U-MT xenografts (Fig. 7A
, Lanes 3 and 4). In addition, higher levels of the MMP-2 enzyme were observed at the periphery as compared with the central part of the U-MT tumors (Fig. 7A)
. These results are consistent with our in vitro data (21
, 48
, 51)
, thereby confirming high efficiency of the U-MT cells in MMP-2 activation.
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Effects of MT1-MMP on Proliferation and Migration of Glioma Cells.
To analyze the mechanisms underlying the increased growth and angiogenesis in the MT1-MMP-expressing tumors, we next evaluated the proliferation and migration of the U-neo and U-MT cells. To compare the growth rates, cells were plated at increasing concentrations, serum-starved, and stimulated by changing serum-free medium to DMEM/FCS. After incubation for 48 h, the relative cell numbers/well were determined by a colorimetric assay based on the ability of cells to reduce a tetrazolium compound to formazan. Fig. 8A
shows that the U-MT cell numbers were up to 60% higher as compared with the control. Apparently, this difference is not sufficient to account for a 6.5-fold difference observed in the corresponding xenografts (Fig. 3)
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. Therefore, it is likely that the increased cell motility and proliferation rate of U-MT cells might have contributed considerably to the larger size of U-MT xenografts. However, to sustain intrinsically high invasive and proliferative potentials, the U-MT cells should be able to induce a stronger angiogenic response relative to the U-neo cells.
MT1-MMP Up-Regulates VEGF in Glioma Cells.
Because VEGF is one of the major regulators of angiogenesis, we further investigated the effects of MT1-MMP overexpression on VEGF production in glioma U251 cells. Therefore, we compared the levels of VEGF produced by the U-MT and U-neo cells in both tumors and cell cultures.
Immunohistochemistry was used to assess VEGF expression in tumor sections (Fig. 9)
. VEGF was detected in both U-neo and U-MT xenografts. However, immunostaining for VEGF was significantly stronger in the U-MT tumors compared with the control. Cytoplasmic staining of VEGF was demonstrated in the clusters of glioma cells (brown staining) as well as in the adjacent endothelium of blood microvessels (arrows). The U-MT cells apparently secrete VEGF in amounts sufficient to induce and support proliferation and growth of the host endothelial cells. Because murine endothelial cells are sensitive to human VEGF (54)
, these findings explain the high angiogenic response observed in the U-MT xenografts in mice.
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. The higher proliferation rate of U-MT cells relative to that of the U-neo control could partially contribute to the higher level of VEGF production observed in the U-MT cultures. However, a 30–40% difference in the number of cells (Fig. 8)
is unlikely to account for a 2–3 fold difference in the VEGF levels (Fig. 10A)
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, which has been shown to stimulate VEGF expression in gliomas, including the U251 cell line (55)
, up-regulated VEGF in the U-neo cells to levels similar to those observed in the U-MT cultures. In contrast, TNF- did not affect the amounts of VEGF secreted by the U-MT cells (Fig. 10A)
, suggesting that the production of this growth factor was at its maximum.
To confirm that MT1-MMP activity and expression of VEGF are functionally associated, we inhibited MT1-MP by using GM6001, a potent hydroxamate MMP inhibitor. For these purposes, U-neo and U-MT cells were incubated for 24–72 h in serum-containing medium supplemented with 50 µM GM6001 or 0.2% DMSO (diluent control) and then transferred the cells to serum-free DMEM. This concentration of GM6001 has been shown to fully inhibit both MT1-MMP activity and proMMP-2 activation in a variety of tumor cell lines, including glioma U-MT (20
, 42)
. At 96 h, VEGF in media was assessed by ELISA. Remarkably, GM6001 down-regulated VEGF production in the U-MT cells in a time-dependent manner to the levels found in the control U-neo cells (Fig. 10B)
. These findings suggest that MT1-MMP activity and VEGF expression are physiologically linked in tumor cells.
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DISCUSSION |
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Here we present additional evidence that expression of MT1-MMP enables the MT1-MMP-overexpressing glioma U-MT cells to reorganize collagen matrix. The development of large cell-free lumens in U-MT spheroids, the alignment of tumor cells at the edge of the lumen, and the migration of cells into the surrounding collagen suggest the involvement of MT1-MMP in matrix reorganization. The lack of lumen formation in control spheroids even in the presence of exogenously activated MMP-2 and strong inhibition of the cyst-like development of the U-MT spheroids by agents interfering with the proteolytic functions of MT1-MMP confirm the role of this enzyme in ECM remodeling.
The lumen formation is unlikely to be caused by inadequate nutrition and necrosis of tumor cells in the center of spheroids. First, the U-neo spheroids demonstrated similar size relative to the U-MT spheroids; however the U-neo spheroids never developed the central lumen. Second, TIMP-2 abrogated the cyst formation in the U-MT spheroids, making their morphology highly similar to that of the U-neo spheroids. Third, the U-MT cells appeared well aligned at the lumen edge. These observations suggest that lumen formation found in the U-MT spheroids is likely to result from the MT1-MMP-dependent directional degradation and remodeling of the collagen matrix. Because MT1-MMP-expressing cells efficiently activate proMMP-2, the enzymatic activity of MMP-2 in the vicinity of the cell surface could also facilitate tumor cell invasion.
In this study, we demonstrate that enzymatic activities of MT1-MMP and MMP-2 control the integrity of the ECM in vitro. Similar observations were obtained previously with HT-1080 fibrosarcoma cells in studies using the collagen gel contraction model (48) . Thus, nonrandom, spatially organized matrix remodeling is likely to direct cell migration and invasion. In tumors, hydrolysis of the ECM components may allow MT1-MMP-overexpressing cells to penetrate the basement membrane and migrate through blood vessel walls. Therefore, therapeutic strategies to target and suppress MT1-MMP functions may be promising for treatment of gliomas.
Induction of an angiogenic response is among the earliest and most important stages of tumorigenesis. The development of the tumor capillary network requires VEGF, a highly specific and potent mitogen for vascular endothelial cells (58 , 59) . Several VEGF isoforms produced by alternative splicing of a single gene mRNA form functionally active disulfide-linked homodimers. Numerous studies demonstrate that the expression of VEGF mRNA and protein by tumor cells directly correlates with angiogenesis and development of tumor metastases (4 , 7 , 60 , 61) . Stimulation of endothelial cell proliferation and migration by VEGF correlates with the induction of MMPs, including MT1-MMP (62, 63, 64) . Although MT1-MMP has been directly linked to angiogenesis (65 , 66) , functional association between MT1-MMP expression and VEGF production has not been demonstrated.
The present study describes an association between the functional activity of MT1-MMP and the production of VEGF in glioma cell growth, tumorigenicity, and neovascularization. Our results suggest that MT1-MMP can play an important role in these processes through the induction of angiogenic response in vessels surrounding tumor cells overexpressing VEGF. We have demonstrated for the first time that MT1-MMP expression in glioma can be associated with an up-regulation of VEGF production. In breast carcinoma MCF7 cells, similar to the U251 glioma cell line, overexpression of MT1-MMP correlated with the increase in VEGF levels.4 The higher rate of proliferation and particularly the increased production of VEGF by U-MT cells might have contributed to the accelerated tumor development of the MT1-MMP-overexpressing glioma cells in immunodeficient mice.
The increased levels of VEGF secreted by MT1-MMP-positive tumor cells apparently promoted an angiogenic response at the sites of implantation, followed by the invasion of endothelial cells and the formation of angiogenic blood vessels necessary to sustain the growth of the induced tumor foci. The presence of high levels of well-developed blood vessels in U-MT xenografts supports this suggestion. Accordingly, enhanced angiogenesis sustained accelerated growth and development of MT1-MMP-overexpressing U251 glioma. The response to hypoxic conditions within the central part of U-MT tumors may also partially contribute to the up-regulation of VEGF. An inadvertent selection of tumor clones bearing spontaneous mutations could also affect the survival and proliferation of U-MT cells in vivo.
Inhibition of MT1-MMP activity by hydroxamate down-regulated the production of VEGF, which supports our hypothesis that MT1-MMP activity and VEGF are physiologically linked in gliomas. Consistent with our results, recent studies have shown that a natural MMP inhibitor, TIMP-2, can exert its antiangiogenic effect through inhibition of MMP-2 and down-regulation of VEGF production. Thus, overexpression of TIMP-2 in murine mammary carcinoma cells delayed in vivo tumor growth and vascularization by strongly reducing VEGF expression (40) . In conjunction with these findings, our results provide evidence of a new mechanism responsible for the opposing effects of MT1-MMP and TIMP-2 on tumor development via reciprocal modulation of VEGF production.
The functional interplay between MT1-MMP and VEGF may also involve other proteins. There is emerging evidence that in addition to the direct breakdown of the ECM, MT1-MMP is able to specifically cleave cell surface receptors such as tissue transglutaminase, integrins, and CD44 (18, 19, 20) . Proteolytic modification of these essential receptors followed by changes in the downstream signal transduction may allow MT1-MMP to exert pleiotropic effects on cell physiology. It is conceivable that complex signaling mechanisms associated with MT1-MMP overexpression could also influence VEGF production.
In conclusion, our results suggest that an up-regulation of cell proliferation and VEGF production could be the mechanism underlying the enhanced growth and vascularization of MT1-MMP-overexpressing tumors. Thus, MT1-MMP may constitute a novel therapeutic target for antiangiogenic cancer therapy.
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FOOTNOTES |
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1 Supported by NIH Grants CA83017 and CA77470, California Breast Cancer Research Program Grant 5JB0094, and Susan G. Komen Breast Cancer Foundation Grant 9849. 
2 To whom requests for reprints should be addressed, at The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037. Phone: (858) 646-3100; Fax: (858) 646-3192; E-mail: strongin{at}burnham.org
3 The abbreviations used are: MMP, matrix metalloproteinase; MT, membrane-type; APMA, p-aminophenylmercuric acetate; proMMP-2, proenzyme of MMP-2; TIMP, tissue inhibitor of metalloproteinase; VEGF, vascular endothelial growth factor; ECM, extracellular matrix; DPBS, Dulbecco’s PBS; TNF-, tumor necrosis factor ; mAb, monoclonal antibody.
4 E. I. Deryugina and A. Y. Strongin, unpublished results.
Received 8/20/01. Accepted 11/14/01.
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J. R. Basile, K. Holmbeck, T. H. Bugge, and J. S. Gutkind MT1-MMP Controls Tumor-induced Angiogenesis through the Release of Semaphorin 4D J. Biol. Chem., March 2, 2007; 282(9): 6899 - 6905. [Abstract] [Full Text] [PDF]
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 L. Ramont, S. Brassart-Pasco, J. Thevenard, A. Deshorgue, L. Venteo, J. Y. Laronze, M. Pluot, J.-C. Monboisse, and F.-X. Maquart The NC1 domain of type XIX collagen inhibits in vivo melanoma growth Mol. Cancer Ther., February 1, 2007; 6(2): 506 - 514. [Abstract] [Full Text] [PDF]
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A. G. Remacle, A. V. Chekanov, V. S. Golubkov, A. Y. Savinov, D. V. Rozanov, and A. Y. Strongin O-Glycosylation Regulates Autolysis of Cellular Membrane Type-1 Matrix Metalloproteinase (MT1-MMP) J. Biol. Chem., June 23, 2006; 281(25): 16897 - 16905. [Abstract] [Full Text] [PDF]
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V. Chabottaux, N. E. Sounni, C. J. Pennington, W. R. English, F. van den Brule, S. Blacher, C. Gilles, C. Munaut, E. Maquoi, C. Lopez-Otin, et al. Membrane-type 4 matrix metalloproteinase promotes breast cancer growth and metastases. Cancer Res., May 15, 2006; 66(10): 5165 - 5172. [Abstract] [Full Text] [PDF]
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 V. W.M. van Hinsbergh, M. A. Engelse, and P. H.A. Quax Pericellular Proteases in Angiogenesis and Vasculogenesis Arterioscler Thromb Vasc Biol, April 1, 2006; 26(4): 716 - 728. [Abstract] [Full Text] [PDF]
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V. S. Golubkov, A. V. Chekanov, S. J. Doxsey, and A. Y. Strongin Centrosomal Pericentrin Is a Direct Cleavage Target of Membrane Type-1 Matrix Metalloproteinase in Humans but Not in Mice: POTENTIAL IMPLICATIONS FOR TUMORIGENESIS J. Biol. Chem., December 23, 2005; 280(51): 42237 - 42241. [Abstract] [Full Text] [PDF]
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E. I. Deryugina, A. Zijlstra, J. J. Partridge, T. A. Kupriyanova, M. A. Madsen, T. Papagiannakopoulos, and J. P. Quigley Unexpected Effect of Matrix Metalloproteinase Down-Regulation on Vascular Intravasation and Metastasis of Human Fibrosarcoma Cells Selected In vivo for High Rates of Dissemination Cancer Res., December 1, 2005; 65(23): 10959 - 10969. [Abstract] [Full Text] [PDF]
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 A. G. Remacle, D. V. Rozanov, P. C. Baciu, A. V. Chekanov, V. S. Golubkov, and A. Y. Strongin The transmembrane domain is essential for the microtubular trafficking of membrane type-1 matrix metalloproteinase (MT1-MMP) J. Cell Sci., November 1, 2005; 118(21): 4975 - 4984. [Abstract] [Full Text] [PDF]
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T. Nonaka, K. Nishibashi, Y. Itoh, I. Yana, and M. Seiki Competitive disruption of the tumor-promoting function of membrane type 1 matrix metalloproteinase/matrix metalloproteinase-14 in vivo Mol. Cancer Ther., August 1, 2005; 4(8): 1157 - 1166. [Abstract] [Full Text] [PDF]
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 E. W. Newcomb, M. A. Ali, T. Schnee, L. Lan, Y. Lukyanov, M. Fowkes, D. C. Miller, and D. Zagzag Flavopiridol downregulates hypoxia-mediated hypoxia-inducible factor-1{alpha} expression in human glioma cells by a proteasome-independent pathway: Implications for in vivo therapy Neuro-oncol, July 1, 2005; 7(3): 225 - 235. [Abstract] [PDF]
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V. S. Golubkov, S. Boyd, A. Y. Savinov, A. V. Chekanov, A. L. Osterman, A. Remacle, D. V. Rozanov, S. J. Doxsey, and A. Y. Strongin Membrane Type-1 Matrix Metalloproteinase (MT1-MMP) Exhibits an Important Intracellular Cleavage Function and Causes Chromosome Instability J. Biol. Chem., July 1, 2005; 280(26): 25079 - 25086. [Abstract] [Full Text] [PDF]
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 D. Voskas, N. Jones, P. Van Slyke, C. Sturk, W. Chang, A. Haninec, Y. O. Babichev, J. Tran, Z. Master, S. Chen, et al. A Cyclosporine-Sensitive Psoriasis-Like Disease Produced in Tie2 Transgenic Mice Am. J. Pathol., March 1, 2005; 166(3): 843 - 855. [Abstract] [Full Text] [PDF]
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L. Labrecque, C. Nyalendo, S. Langlois, Y. Durocher, C. Roghi, G. Murphy, D. Gingras, and R. Beliveau Src-mediated Tyrosine Phosphorylation of Caveolin-1 Induces Its Association with Membrane Type 1 Matrix Metalloproteinase J. Biol. Chem., December 10, 2004; 279(50): 52132 - 52140. [Abstract] [Full Text] [PDF]
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P. Wang, J. Nie, and D. Pei The Hemopexin Domain of Membrane-type Matrix Metalloproteinase-1 (MT1-MMP) Is Not Required for Its Activation of proMMP2 on Cell Surface but Is Essential for MT1-MMP-mediated Invasion in Three-dimensional Type I Collagen J. Biol. Chem., December 3, 2004; 279(49): 51148 - 51155. [Abstract] [Full Text] [PDF]
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D. V. Rozanov, S. Sikora, A. Godzik, T. I. Postnova, V. Golubkov, A. Savinov, S. Tomlinson, and A. Y. Strongin Non-proteolytic, Receptor/Ligand Interactions Associate Cellular Membrane Type-1 Matrix Metalloproteinase with the Complement Component C1q J. Biol. Chem., November 26, 2004; 279(48): 50321 - 50328. [Abstract] [Full Text] [PDF]
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 I. S. Aljada, N. Ramnath, K. Donohue, S. Harvey, J. J. Brooks, S. M. Wiseman, T. Khoury, G. Loewen, H. K. Slocum, T. M. Anderson, et al. Upregulation of the Tissue Inhibitor of Metalloproteinase-1 Protein Is Associated With Progression of Human Non-Small-Cell Lung Cancer J. Clin. Oncol., August 15, 2004; 22(16): 3218 - 3229. [Abstract] [Full Text] [PDF]
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T. E. Van Meter, W. C. Broaddus, H. K. Rooprai, G. J. Pilkington, and H. L. Fillmore Induction of membrane-type-1 matrix metalloproteinase by epidermal growth factor-mediated signaling in gliomas Neuro-oncol, July 1, 2004; 6(3): 188 - 199. [Abstract] [PDF]
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A. Zijlstra, R. T. Aimes, D. Zhu, K. Regazzoni, T. Kupriyanova, M. Seandel, E. I. Deryugina, and J. P. Quigley Collagenolysis-dependent Angiogenesis Mediated by Matrix Metalloproteinase-13 (Collagenase-3) J. Biol. Chem., June 25, 2004; 279(26): 27633 - 27645. [Abstract] [Full Text] [PDF]
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 A Noel, C Maillard, N Rocks, M Jost, V Chabottaux, N E Sounni, E Maquoi, D Cataldo, and J M Foidart Membrane associated proteases and their inhibitors in tumour angiogenesis J. Clin. Pathol., June 1, 2004; 57(6): 577 - 584. [Abstract] [Full Text] [PDF]
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N. E. Sounni, C. Roghi, V. Chabottaux, M. Janssen, C. Munaut, E. Maquoi, B. G. Galvez, C. Gilles, F. Frankenne, G. Murphy, et al. Up-regulation of Vascular Endothelial Growth Factor-A by Active Membrane-type 1 Matrix Metalloproteinase through Activation of Src-Tyrosine Kinases J. Biol. Chem., April 2, 2004; 279(14): 13564 - 13574. [Abstract] [Full Text] [PDF]
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 J. Schoenberger, D. Grimm, P. Kossmehl, M. Infanger, E. Kurth, and C. Eilles Effects of PTK787/ZK222584, a Tyrosine Kinase Inhibitor, on the Growth of a Poorly Differentiated Thyroid Carcinoma: An Animal Study Endocrinology, March 1, 2004; 145(3): 1031 - 1038. [Abstract] [Full Text] [PDF]
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 C. Johnson, H.-J. Sung, S. M. Lessner, M. E. Fini, and Z. S. Galis Matrix Metalloproteinase-9 Is Required for Adequate Angiogenic Revascularization of Ischemic Tissues: Potential Role in Capillary Branching Circ. Res., February 6, 2004; 94(2): 262 - 268. [Abstract] [Full Text] [PDF]
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 Y. N. Ye, E. S. L. Liu, V. Y. Shin, W. K. K. Wu, J. C. Luo, and C. H. Cho Nicotine Promoted Colon Cancer Growth via Epidermal Growth Factor Receptor, c-Src, and 5-Lipoxygenase-Mediated Signal Pathway J. Pharmacol. Exp. Ther., January 1, 2004; 308(1): 66 - 72. [Abstract] [Full Text] [PDF]
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K. Ohno-Matsui, T. Uetama, T. Yoshida, M. Hayano, T. Itoh, I. Morita, and M. Mochizuki Reduced Retinal Angiogenesis in MMP-2-Deficient Mice Invest. Ophthalmol. Vis. Sci., December 1, 2003; 44(12): 5370 - 5375. [Abstract] [Full Text] [PDF]
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A. Jiang and D. Pei Distinct Roles of Catalytic and Pexin-like Domains in Membrane-type Matrix Metalloproteinase (MMP)-mediated Pro-MMP-2 Activation and Collagenolysis J. Biol. Chem., October 3, 2003; 278(40): 38765 - 38771. [Abstract] [Full Text] [PDF]
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A. Remacle, G. Murphy, and C. Roghi Membrane type I-matrix metalloproteinase (MT1-MMP) is internalised by two different pathways and is recycled to the cell surface J. Cell Sci., October 1, 2003; 116(19): 3905 - 3916. [Abstract] [Full Text] [PDF]
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