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Laboratory of Tumor and Development Biology,
*
Laboratory of Connective Tissues Biology, University of Liège, Sart Tilman, B-4000 Liège, Belgium; and
Victorian Breast Cancer Research Consortium Invasion and Metastasis Unit, St Vincent’s Institute of Medical Research, Fitzroy, Australia
1Correspondence: Laboratory of Biology of Tumor and Development, University of Liège, 4000 Sart-Tilman, Liège, Belgium. E-mail: agnes.noel{at}ulg.ac.be
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: matrix metalloproteinases • MCF7 cells • MT1-MMP overexpression • VEGF
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. Even though the majority of MMPs are secreted as soluble enzymes into the extracellular milieu, a subset of MMPs, the membrane type MMPs (MT-MMPs) display the common structural domains of the MMP family but are characterized by a carboxyl-terminal cytoplasmic domain and an amino-terminal furin recognition site (4)
. Of the six MT-MMPs described so far, the MT1-MMP has been proposed to play critical roles in physiological and pathological processes by remodeling the ECM. MT1-MMP displays a broad spectrum of activity against ECM components such as type I and II collagens (4
5
6)
, fibronectin, vitronectin, laminin, fibrin, and proteoglycan (5
6
7
8)
. MT1-MMP also activates pro-MMP-2 (4)
and proMMP-13 (pro-collagenase 3) (9)
. This process requires the tissue inhibitor of metalloproteinases-2 (TIMP-2), which acts as an adaptor molecule mediating pro-MMP2 binding to MT1-MMP (10
, 11)
.
The expression of MT1-MMP has been reported to correlate with the malignancy of different tumor types such as lung (4
, 12
, 13)
, gastric (14
, 15)
, colon (16
, 17)
, breast (16
, 18
, 19)
, cervical carcinomas (20)
, gliomas (21)
, and melanomas (22)
. Recent studies have shown that MT1-MMP-deficient mice exhibit damage in skeletal development manifested by craniofacial dysmorphism, dwarfism, osteopenia, and fibrosis (23
, 24)
.
MT1-MMP may function as a fibrinolytic enzyme in the absence of plasmin and promote angiogenesis in fibrin matrix (8)
. The angiogenesis in collagen or fibrin matrix can be inhibited by TIMP-2 (8
, 25)
. We previously showed that TIMP-2-mediated inhibition of tumor growth and angiogenesis in a murine model was associated with a down-regulation of VEGF expression in tumor cells (26)
. These results emphasize the key role played by TIMP-2, MT1-MMP, and MMP-2 during tumoral angiogenesis.
We have also demonstrated that overexpression of MT1-MMP in human melanoma A2058 cells that produced only pro-MMP-2 was associated with enhanced in vitro invasion and increased in vivo tumor growth and vascularization (27)
.
Human adenocarcinoma MCF7 cells, which do not produce pro-MMP-2 or MT1-MMP, were used. The stable transfection of MCF7 cells with MT1-MMP cDNA alone or in combination with MMP-2 cDNA enhanced their in vitro invasion and in vivo tumor growth. On the contrary, cells overexpressing MMP-2 alone behaved in vitro and in vivo as control cells. The in vivo angiogenic potential of MT1-MMP-overexpressing MCF7 cells was associated with a enhanced production of VEGF. Altogether, our results demonstrate that MT1-MMP, but not MMP-2, promotes tumor growth and angiogenesis, and provide a new mechanism of action of MT1-MMP through the transcriptional regulation of VEGF.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Transfection of MCF7 cells with MMP-2 and/or MT1-MMP cDNA
Stable transfection of MCF7 cells with the vectors was performed in serum-free medium at 250 V and 960 µF using a gene pulser system (Bio-Rad, Richmond, CA). Parental MCF7 cells were transfected with pCHC6-hygro vector containing only the hygromycin-B resistance gene (control plasmid) or with the same plasmid carrying the full-length MMP-2 cDNA (pCHC6-MMP2) (28)
. The transfected cells were selected with hygromycin-B (125 µg/ml); stably transfected clones were isolated and maintained in normal medium containing 60 µg/ml of hygromycin-B. Clones were screened and selected for MMP-2 expression by zymography analysis.
In a second step, parental MCF7 cells and one clone overexpressing MMP-2 selected from the first transfection were transfected with pcDNA3-neo vector containing only the neomycin resistance gene (control plasmid) or with the same plasmid carrying the full-length MT1-MMP cDNA (pc3MT1800s) (11)
. Transfectants were selected with G418 (400 µg/ml) (Life Technologies) and isolated clones were maintained in normal medium containing G418 (200 µg/ml).
Preparation of conditioned media and cell extracts
Conditioned media were prepared by incubating subconfluent cells (2x105) in 24-well-plates (Falcon, Becton Dickinson) for 24 h in serum-free DMEM in the absence or presence of an exogenous source of MMP-2 (medium conditioned by CHO cells transfected with full-length MMP-2 cDNA) (11)
. Conditioned media were harvested, clarified by centrifugation, and stored frozen at -20°C. Total cell extracts were prepared from cell monolayers incubated in RIPA buffer [50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1% Nonidet P40; 1% Triton X-100; 1% sodium deoxycholate; 0.1% sodium dodecyl sulfate (SDS), 5 mM iodoacetamide; 2 mM phenylmethylsulfonyl fluoride (PMSF)]. After centrifugation at 15,000 r.p.m. for 30 min, at 4°C, the supernatant was stored frozen at -20°C. Cell extract and conditioned media from HT1080 cells treated with concanavalin A were prepared as described (11)
and used as positive control to visualize MT1-MMP and MMP-2 production. Protein concentration was determined by using the DC protein Assay Kit (Bio-Rad Laboratories, Hercules, CA) and adjusted to 2 mg/ml.
Gelatin zymography
Aliquots (5–10 µl) of conditioned media standardized for cell DNA content (29)
were mixed with equal volume of nonreducing sample buffer (62.5 mM Tris-HCl, pH 6.8; 2% SDS; 10% glycerol; 0.1% bromphenol blue) and resolved on 10% gels SDS-PAGE containing 0.1% gelatin (w/v) according to the procedure described (11)
.
Western blotting analysis
Samples of total cell extracts were resolved by SDS-PAGE under reducing conditions and proteins were transferred to nitrocellulose membranes (Hybond-ECL membrane; Amersham, Arlington Heights, IL). The membranes were exposed to primary antibodies raised against the hemopexin domain of MT1-MMP (1/200) (monoclonal 2D-7 antibody, kindly provided by M.-C. Rio, IGBMC, Illkirch, France). After extensive washings, the membranes were incubated with a secondary horseradish peroxidase-conjugated goat anti-mouse antibody at 1/1000 (DAKO, Glostrup, Denmark). VEGF Western blotting was performed using a rabbit polyclonal antibody against human VEGF165, 189, and 121 amino acids splice variants [(A-20):sc-152 Sanver TECH] at a dilution of 1/500 and, as a secondary antibody, peroxidase-conjugated mouse anti-rabbit IgG (DAKO, Glostrup, Denmark). Signals were detected using an ECL kit (NEN, Boston, MA), according to the manufacturer’s instructions.
Proliferation assay
MCF7 cells (104 cells/well) were seeded in triplicate in 24-well plates and maintained in serum containing medium. Cells were harvested at various intervals and sonicated in PBS. Fluorimetric DNA titration was performed and used as an indicator of cell density (29)
In vitro invasion assays
Cell invasiveness was assayed using Transwell cell culture inserts (8 µm pore size, 6.5 mm diameter, Costar, Cambridge, MA). Uncoated or Matrigel-coated (25 µg/filter) filters were used for chemotaxis and chemoinvasion assays, respectively. The filters were rehydrated for 2 h with 250 µl of serum-free medium at 37°C. Cells (6x104) suspended in 300 µl of serum-free medium containing 0.1% (w/v) BSA (fraction V, Miles, Elkhart, IN) were seeded in the upper compartment of the chamber. The lower compartment was filled with 600 µl of DMEM supplemented with 20% FCS and 1% BSA as chemoattractant. After incubation at 37°C in a humid atmosphere (6 or 24 h for chemotactic and chemoinvasion assays, respectively), filters were rinsed with PBS, fixed with methanol (10 min at -20°C), and stained with Giemsa (Fluka Chemie, Buchs, Switzerland) for 15 min. Cells on the upper surface of filters were wiped away with a cotton swab and those on the lower face were counted under the microscope at a magnification of x400. Each clone was tested in triplicate in at least in two independent assays. Results were expressed as mean cell number per filter.
In vivo tumorigenicity
Subconfluent cells were trypsinized and resuspended in serum-free medium (3x106 cells/ml) and mixed with an equal volume of cold Matrigel (10 mg protein/ml). Cells (2x105/injection site) were injected subcutaneously (s.c.) into 6- to 8-wk-old female nude mice (nu/nu) (Iffa Credo, L’Arbresle, France) supplemented with estradiol pellets (Innovative Research of America, Sarasota, FL). Tumor volumes were estimated every 2 days and calculated as described (30)
. Results are expressed as the mean of tumor volumes (6 tumors per experimental group). The latency period is defined as the time required to observe 100% of tumors larger than 100 mm3. Each experiment was repeated twice. After 45 to 50 days, the animals were killed and tumors were resected. Half of the tumors was fixed in 10% buffered formalin and embedded in paraffin for histological analyses by light microscopy. The other half was frozen in liquid nitrogen for immunofluorescence studies.
Immunofluorescence and morphometry
Cryostat sections (5 µm thick) fixed in acetone at -20°C and in 80% methanol at 4°C were incubated with the primary antibodies. Antibodies raised against PECAM (rat monoclonal antibody, PharMingen, San Diego, CA: diluted 1/20) or type IV collagen (guinea pig polyclonal antibody produced in our laboratory; diluted 1/100) (31)
were incubated for 1 h at room temperature. The sections were washed in PBS three times for 10 min, then appropriate secondary antibodies conjugated to Texas red (TRITC) were applied for 30 min [rabbit anti-rat (Sigma, St. Quentin Fallavier, France: diluted 1/40) or mouse anti-guinea pig (Sigma; diluted 1/40)]. The sections were stained with bis-benzamide (10 µg/ml) added with the secondary antibodies. After three washes in PBS for 10 min each and a final rinse in 10 mM Tris-HCl buffer (pH 8.8), coverslips were mounted and labeling was observed under an inverted microscope equipped with fluorescence optics. The quantification of angiogenesis was performed by measuring vessel density under fluorescence microscope on sections of whole tumors; 10 sections were made in each tumor. Image analysis and quantification of stained vessel sections were performed on a computer using the MicroImage 3.0.1.0 software from Olympus (Bio-Rad, Brussels, Belgium). Results were expressed as mean (±SD) of vessel number per mm2.
RT-PCR VEGF and MT1-MMP
Total RNA was extracted from cell monolayers or tumors by RNA Instapure treatment (Eurogentec, Liège, Belgium). RT-PCR was performed on 10 ng of total RNA using a Perkin-Elmer kit (Foster City, CA) following the manufacturer’s instructions. Reverse transcription was carried out with 5'-CTCACCGCCTCGGCTTGTCACA-3' as VEGF primer, 5'-CCATTGGGCATCCAGAAGAGAGC-3' as MT1-MMP primer, or 5'-GATTCTGACTTAGAGGCGTTCAGT-3' as 28S primer for 15 min at 70°C. PCR products were generated with the same primers as reverse primers and with 5'-CCT GGTGGACATCTTCCAGGAGTA-3' for VEGF, 5'-GGATACCCAATGCCCATTGGCCA-3' for MT1-MMP, or 5'-GTTCACCCACTA ATAGGGAACGTGA-3' for 28S as forward primers. PCR conditions were 95°C for 2 min, followed by 29 cycles for VEGF or 25 cycles for MT1-MMP consisting of 94°C for 20 s, 66°C for 20 s, 72°C for 30 s, and a final elongation step of 72°C for 2 min. Amplification products of the RNAs coding for VEGF189, VEGF165, and VEGF121, were 479, 407, and 265 bp, respectively. To control the efficiency of the RT-PCR, we designed a synthetic RNA (CTR1) that can be reverse transcribed and amplified with the same primers, giving rise to a 311 bp for VEGF (26)
and 220 bp for MT1-MMP fragments. 8000 or 30,000 copies of the internal control were added to each sample for VEGF and MT1-MMP RT-PCR, respectively. VEGF expression was normalized to that of the 28S. PCR conditions for 28S were 95°C for 2 min, 17 cycles consisting of 94°C/15 s, 68°C/20 s, 72°C/10 s, and a final elongation step of 72°C/2 min. A synthetic RNA (CTR2) was also used to control the efficiency of RT-PCR for 28S. The amplification products were electrophoresed on a polyacrylamide gel, stained with Gelstar (Sanver Tech, Antwerpen, Belgium), scanned with FluorSImager, and analyzed using multianalyst software (Bio-Rad, Belgium). Ratios between VEGF/CTR1 and 28S/CTR2 were determined. VEGF expression was expressed as the ratio of VEGF transcripts:28S transcripts.
In vitro angiogenesis: the aortic ring assay
Rat aortic explant cultures were prepared as described (32
, 33)
. To prepare culture wells, 30 ml of 1.5% agarose solution (type VII, cell culture tested; Sigma) was poured into 100 mm-diameter Petri dishes (Corning Costar Corporation, Cambridge, MA) and allowed to gel. Agarose rings were obtained by punching two concentric circles in the agarose 10 and 17 mm in diameter and were transferred to 100 mm-diameter Petri dishes (bacteriological polystyrene; Falcon, Becton Dickinson, Lincoln Park, NJ). The bottom of each agarose well was covered with 200 µl of rat tail tendon (type I) collagen (1.5 mg/ml) (Collagen R, Serva, Heidelberg, Germany) and allowed to gel at 37°C. One aortic ring obtained by sectioning the rat aorta at 1 mm intervals (32)
was carefully positioned in each well, which was then completely filled with collagen solution. Aorta rings were maintained at 37°C in MCDB131 (Life Technologies, Paisley, Scotland) supplemented with 25 mM NaHCO3, 100 U/ml penicillin and 100 µg/ml streptomycin. Conditioned media of different clones were prepared by incubating subconfluent cells for 48 h in serum-free MCDB medium. Cultures were examined every 2 days by phase contrast microscopy. When indicated, human recombinant rVEGF165 (20 ng/ml, Sigma) was added to the medium. In some assays, an anti-VEGF antibody [Ab-3 (JH121) Neomarkers at 10 µg/ml] was added 3 days after addition of conditioned media to the aortic ring. The identity of endothelial cells was verified by Dil-Ac-LDL (Sigma-Aldrich, Belgium) incorporation in the aorta ring before embedding into collagen gel (34)
.
Statistical analysis
Differences between the experimental conditions were evaluated using the ANOVA analysis (P values < 0.05 were considered significant).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). Cells were first transfected with the control pCHC6-hygro plasmid or the pCHC6-MMP-2 vector carrying the MMP-2 cDNA (Fig. 1A
). Whereas control clones did not express MMP-2, cells transfected with MMP-2 cDNA produced the enzyme in its proform (66 kDa) (Fig. 1B
). We selected the M1 clone overexpressing MMP-2 and C1 control clone for the second transfection. Parental cells and the selected clones were transfected with the control pcDNA3-neo plasmid or the pc3MT1800s plasmid carrying the MT1-MMP cDNA. Clones obtained by selection with G418 were screened by RT-PCR (Fig. 1C
). Western blot analysis (Fig. 2
A) revealed that MT1-MMP-expressing clones produced both the 60 kDa MT1-MMP species and prominent 43 kDa/39 kDa forms identified previously as a processed form of the enzyme (35)
. However, the MMT/4 clone produced MT1-MMP only in its proform of 63 kDa. The clones co-overexpressing MT1-MMP and MMP-2 secreted pro-MMP-2 and converted it into active 59 KDa MMP-2 form (Fig. 2B
). Whereas parental cells and control clones failed to activate pro-MMP-2 exogenously added to the conditioned media (Fig. 2B
), the clones overexpressing MT1-MMP were able to activate it. The MMT/4 clone producing MT1-MMP in its proform failed to activate pro-MMP2. These results confirm that active MT1-MMP is required for pro-MMP-2 activation.
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The in vitro growth rate of clones selected after each transfection was similar to that of control clones or parental cells, with the exception of the MMT/4 clone (Fig. 3
).
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In vitro invasion is enhanced in clones overexpressing MT1-MMP
Noninvasive adenocarcinoma MCF7 cells transfected to overexpress MT1-MMP and/or MMP-2 were tested for their ability to migrate through uncoated filters (chemotaxis) or Matrigel-coated (25 µg/filter) filters for 24 h (chemoinvasion). The expression of MT1-MMP associated or not with MMP-2 did not affect chemotaxis (data no shown), but led to a significant enhancement of chemoinvasion (Fig. 4
). In contrast, overexpression of MMP-2 alone in MCF-7 cells did not significantly affect their invasion. Invasion of the MMT/4 clone that expresses MT1-MMP and MMP-2 in proforms was similar to that of control cells. These results demonstrate the importance of active MT1-MMP in cell invasion.
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In vivo tumorigenicity of MCF7 cells overproducing MT1-MMP and/or MMP-2
Athymic nude mice were next inoculated s.c. with different clones, in the presence of Matrigel. The latency period was 47 days after injection of two control clones (C1/2 and C12); the incidence of tumor never reached 100% with the other control clones tested (Table 1
). In contrast, injection of MT1-MMP-expressing clones (‘MT1’ and ‘MT1/MMP-2’ clones) always led to 100% tumor formation with a latency period of <40 days (Table 1)
. For all clones tested, MT1-MMP overexpression in the presence or not of MMP-2 expression was associated with accelerated tumor growth. The tumor volume reached at the end of the assay was always higher than that of control tumors (Fig. 5
) (P<0.05). In contrast, the injection of MMP-2-overexpressing clones led to tumor development similar to that observed after injection of control cells (Fig. 5
and Table 1
).
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Histological analysis and quantification of tumor vascularization
The histology of tumors obtained 45 days after cell injection was observed. At low magnification, the tumors formed by MMP-2-expressing or control cells presented a large zone of necrosis at their center. Such necrotic areas were not observed in tumors issued from MT1-MMP or MT1-MMP and MMP-2-expressing cells.
The tumor vascularization was analyzed after immunostaining with anti-type IV collagen or anti-PECAM (anti-CD31) antibodies, both revealing similar vessel distribution (Fig. 6
A). Whereas control tumors and MMP-2-expressing tumors were poorly vascularized, numerous vessels were observed and homogenously distributed inside MT1-MMP-expressing tumors. Again, results obtained with MT1-MMP clones were independent of the MMP-2 expression. Angiogenesis quantification performed by image analysis revealed that MT1-MMP or MT1-MMP/MMP-2 overexpression was associated with enhanced vessel density (P<0,05) (Fig. 6B
). No significant difference was observed between control and MMP-2-expressing tumors (Fig. 6B
).
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VEGF production in overexpressing MT1-MMP and/or MMP2 MCF7 clones
Since MT1-MMP expression was associated with increased in vivo tumor vascularization, we evaluated VEGF mRNA expression in the different MCF7 clones. RT-PCR analysis of total RNA from subconfluent culture cells, showed a sixfold increase in VEGF189, VEGF165, and VEGF121 mRNA isoforms in MT1-MMP-expressing or MT1-MMP- and MMP-2-coexpressing clones vs. weakly angiogenic pro-MMP-2 producing clones or MCF7 control cells (Fig. 7
). This modulation of VEGF expression was confirmed by measuring the VEGF protein by Western blot analysis (Fig. 8
A). In accordance with these in vitro data, an enhancement of VEGF production was evidenced by Western blot analysis performed on MT1-MMP-expressing tumors (Fig. 8B
).
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In vitro angiogenesis induced by MT1-MMP-overexpressing MCF7 clones
To assess the ability of the selected clones to modulate in vitro angiogenesis, rat aorta rings were embedded in collagen gel maintained in conditioned media (MCDB) of MT1-MMP-overexpressing clones (MT/2 and MT/5), MT1/MMP-2-overexpressing clones (MMT/1), MMP-2-overexpressing clone (M1), or control clones (C10, C1, and C2). When aorta rings were maintained in the conditioned media of MT1-MMP-overexpressing clones or an MT1-MMP- and MMP-2-overexpressing clone, a rapid microvessel outgrowth was observed within 6 days (Fig. 9
Ia, b). The extent of angiogenesis was comparable to that observed in the presence of VEGF (20 ng/ml) used as positive control (Fig. 9IIa
). In sharp contrast, the generation of microvessels was nearly undetectable from rat aorta cultured in the presence of medium conditioned by MMP-2-expressing clones or control clones (Fig. 9Ic, d
). The in vitro angiogenic effect induced by conditioned media of clones overexpressing MT1-MMP was completely inhibited after addition of an antibody against VEGF (Fig. 9IIa-d
). The data demonstrate clearly the implication of VEGF produced by MCF7 clones overexpressing MT1-MMP.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, its role during tumor angiogenesis has not clearly been established. In the present study, we show that MT1-MMP overexpression promotes tumor angiogenesis and propose that one of its functions is to up-regulate the expression of the angiogenesis inducer VEGF. Several lines of evidence support these conclusions. First, MT1-MMP overexpression in MCF7 cells resulted in enhanced tumor incidence, tumor growth, and increased vessel density as evaluated by immunostaining. Second, conditioned medium from MT1-MMP-expressing cells induced in vitro angiogenesis in the aortic ring assay. Third, enhancement of VEGF mRNA and protein levels were concomitant with MT1-MMP overexpression in cultured cells, as well as in tumor extracts. An increase in vitro invasiveness through Matrigel-coated filters was also observed. These effects could not be ascribed to a modulation of cell proliferation by MT1-MMP expression as verified by in vitro proliferation assays (Fig. 3)
. These data are in accordance with our recent demonstration that the expression of MT1-MMP by human melanoma A2058 cells promotes tumor growth and vascularization (27)
. In the present work, the use of MCF7 cells that do not produce either MT1-MMP or MMP2 allowed us to determine by separate transfection or cotransfection the specific role of MT1-MMP or MMP2 produced by tumor cells. From our data, we can conclude that the effect of MT1-MMP on in vitro invasion and in vivo tumor growth and vascularization did not require the coexpression of MMP2 by tumor cells. Indeed, MMP2 expression alone failed to affect MCF7 cells angiogenic, invasive, and tumorigenic properties and MMP2 coexpressed with MT1-MMP did not potentiate the effects of MT1-MMP observed in vitro and in vivo. However, this does not preclude that MMP2 could be involved in the in vivo processes, since we cannot exclude the possibility that reactive stromal cells may supply the tumor microenvironment with MMP2 (36
, 37)
.
The MMT/4 clone overexpressing MT1-MMP in its 63 kDa zymogen form failed to activate pro-MMP2 and induced tumor development similar to that observed with control or parental MCF7 cells. This may suggest that the tumor-promoting effect of MT1-MMP requires the activation of this transmembrane protein. However, this MMT/4 clone displayed in vitro a lower proliferation rate, which could account for a low tumorigenicity. This observation may suggest a clonal variation in parental MCF7 cells or that a recombination event leading to decreased proliferation rate has occurred during cell transfection. Our observations suggest that VEGF up-regulation can occur in the absence of MT1-MMP activation. We are testing the hypothesis that in those clones, MT1-MMP functions as a membrane-associated transducing molecule, as suggested by recent work (38)
.
One can speculate that MT1-MMP contributes to tumor angiogenesis by its capacity to activate MMP2 and/or to degrade extracellular matrix components, thereby promoting cell migration or influencing the bioavailability of growth factors. In accordance, MT1-MMP may function as a fibrinolytic enzyme and mediate pericellular proteolysis in angiogenesis (8)
. MT1-MMP expression has recently been correlated with activation of the 
vß3 integrin, which plays a major role during angiogenesis (39)
. However, the implication of vß3 in our system is unlikely since MCF7 cells used in the present study do not express the ß3 integrin subunit (40)
.
Involvement of some MMPs during the tumoral angiogenic process has been reported by different groups. MMP2 has been implicated in the angiogenic switching in subcutaneous transplant models of tumor progression (41
, 42)
. In a model of squamous carcinogenesis of the epidermis, MMP9 enhanced keratinocyte proliferation and angiogenesis (43)
. The contribution of MMP9 during tumor angiogenesis has been associated with its capacity to increase the availability of VEGF (44)
. Our study demonstrates for the first time that overexpression of an MMP, the MT1-MMP, is concomitant to a transcriptional up-regulation of VEGF and provides a new mechanism of MT1-MMP action during tumor progression.
Whether MT1-MMP controls the VEGF transcription directly or indirectly remains to be elucidated. It is possible that MT1-MMP transduces an intracellular signal through its cytoplasmic carboxyl-terminal domain. The use of MT1-MMP mutants may shed light on this. We earlier reported that overexpression of TIMP2 in murine mammary cells reduced tumor growth and angiogenesis by down-regulating VEGF (26)
. It is therefore plausible that VEGF transcription is controlled by a balance between MT1-MMP and its inhibitor TIMP2. Additional experiments are required to demonstrate whether TIMP2 may have dual functions depending on its concentration. Such a dual effect has been demonstrated for pro-MMP2 activation, which requires low concentration of TIMP2 but is inhibited by a high concentration of the MMP inhibitor.
In conclusion, our work demonstrates the key role played by MT1-MMP during tumor angiogenesis and provides a new function for this membrane type MMP in regulating VEGF expression. Although it raises some questions on the exact mechanism of MT1-MMP action, it opens opportunities for future investigations and development of new anti-angiogenic strategies combining inhibitors of MMPs and VEGF.
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ACKNOWLEDGMENTS |
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Received for publication October 1, 2001.
Revision received January 8, 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 Y. Takeda, A. R. Kazarov, C. E. Butterfield, B. D. Hopkins, L. E. Benjamin, A. Kaipainen, and M. E. Hemler Deletion of tetraspanin Cd151 results in decreased pathologic angiogenesis in vivo and in vitro Blood, February 15, 2007; 109(4): 1524 - 1532. [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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 M.-S. Lee, S.-A. Yoo, C.-S. Cho, P.-G. Suh, W.-U. Kim, and S. H. Ryu Serum Amyloid A Binding to Formyl Peptide Receptor-Like 1 Induces Synovial Hyperplasia and Angiogenesis J. Immunol., October 15, 2006; 177(8): 5585 - 5594. [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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G. Chakraborty, H. Rangaswami, S. Jain, and G. C. Kundu Hypoxia Regulates Cross-talk between Syk and Lck Leading to Breast Cancer Progression and Angiogenesis J. Biol. Chem., April 21, 2006; 281(16): 11322 - 11331. [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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M. Plaisier, P. Koolwijk, R. Hanemaaijer, R. A. Verwey, R. M.F. van der Weiden, E. K.J. Risse, C. Jungerius, F. M. Helmerhorst, and V. W.M. van Hinsbergh Membrane-type matrix metalloproteinases and vascularization in human endometrium during the menstrual cycle Mol. Hum. Reprod., January 1, 2006; 12(1): 11 - 18. [Abstract] [Full Text] [PDF]
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M. Polette, C. Gilles, B. Nawrocki-Raby, J. Lohi, W. Hunziker, J.-M. Foidart, and P. Birembaut Membrane-Type 1 Matrix Metalloproteinase Expression Is Regulated by Zonula Occludens-1 in Human Breast Cancer Cells Cancer Res., September 1, 2005; 65(17): 7691 - 7698. [Abstract] [Full Text] [PDF]
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 S. Smola-Hess, J. Pahne, C. Mauch, P. Zigrino, H. Smola, and H. J. Pfister Expression of membrane type 1 matrix metalloproteinase in papillomavirus-positive cells: role of the human papillomavirus (HPV) 16 and HPV8 E7 gene products J. Gen. Virol., May 1, 2005; 86(5): 1291 - 1296. [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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S. McMahon, F. Grondin, P. P. McDonald, D. E. Richard, and C. M. Dubois Hypoxia-enhanced Expression of the Proprotein Convertase Furin Is Mediated by Hypoxia-inducible Factor-1: IMPACT ON THE BIOACTIVATION OF PROPROTEINS J. Biol. Chem., February 25, 2005; 280(8): 6561 - 6569. [Abstract] [Full Text] [PDF]
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D. V. Rozanov, A. Y. Savinov, V. S. Golubkov, T. I. Postnova, A. Remacle, S. Tomlinson, and A. Y. Strongin Cellular Membrane Type-1 Matrix Metalloproteinase (MT1-MMP) Cleaves C3b, an Essential Component of the Complement System J. Biol. Chem., November 5, 2004; 279(45): 46551 - 46557. [Abstract] [Full Text] [PDF]
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 M. Plaisier, K. Kapiteijn, P. Koolwijk, C. Fijten, R. Hanemaaijer, J. M. Grimbergen, A. Mulder-Stapel, P. H. A. Quax, F. M. Helmerhorst, and V. W. M. van Hinsbergh Involvement of Membrane-Type Matrix Metalloproteinases (MT-MMPs) in Capillary Tube Formation by Human Endometrial Microvascular Endothelial Cells: Role of MT3-MMP J. Clin. Endocrinol. Metab., November 1, 2004; 89(11): 5828 - 5836. [Abstract] [Full Text] [PDF]
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 E. Maquoi, N. E. Sounni, L. Devy, F. Olivier, F. Frankenne, H.-W. Krell, F. Grams, J.-M. Foidart, and A. Noel Anti-Invasive, Antitumoral, and Antiangiogenic Efficacy of a Pyrimidine-2,4,6-trione Derivative, an Orally Active and Selective Matrix Metalloproteinases Inhibitor Clin. Cancer Res., June 15, 2004; 10(12): 4038 - 4047. [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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M.-H. Lee, M. Rapti, V. Knauper, and G. Murphy Threonine 98, the Pivotal Residue of Tissue Inhibitor of Metalloproteinases (TIMP)-1 in Metalloproteinase Recognition J. Biol. Chem., April 23, 2004; 279(17): 17562 - 17569. [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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A. M. Tester, M. Waltham, S.-J. Oh, S.-N. Bae, M. M. Bills, E. C. Walker, F. G. Kern, W. G. Stetler-Stevenson, M. E. Lippman, and E. W. Thompson Pro-Matrix Metalloproteinase-2 Transfection Increases Orthotopic Primary Growth and Experimental Metastasis of MDA-MB-231 Human Breast Cancer Cells in Nude Mice Cancer Res., January 15, 2004; 64(2): 652 - 658. [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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S. Garvin and C. Dabrosin Tamoxifen Inhibits Secretion of Vascular Endothelial Growth Factor in Breast Cancer in Vivo Cancer Res., December 15, 2003; 63(24): 8742 - 8748. [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. 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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 B.-W. Wang, H. Chang, S. Lin, P. Kuan, and K.-G. Shyu Induction of matrix metalloproteinases-14 and -2 by cyclical mechanical stretch is mediated by tumor necrosis factor-{alpha} in cultured human umbilical vein endothelial cells Cardiovasc Res, August 1, 2003; 59(2): 460 - 469. [Abstract] [Full Text] [PDF]
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 B. Annabi, Y.-T. Lee, S. Turcotte, E. Naud, R. R. Desrosiers, M. Champagne, N. Eliopoulos, J. Galipeau, and R. Beliveau Hypoxia Promotes Murine Bone-Marrow-Derived Stromal Cell Migration and Tube Formation Stem Cells, May 1, 2003; 21(3): 337 - 347. [Abstract] [Full Text] [PDF]
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 A. V. SOMLYO, C. PHELPS, C. DIPIERRO, M. ETO, P. READ, M. BARRETT, J. J. GIBSON, M. C. BURNITZ, C. MYERS, and A. P. SOMLYO Rho kinase and matrix metalloproteinase inhibitors cooperate to inhibit angiogenesis and growth of human prostate cancer xenotransplants FASEB J, February 1, 2003; 17(2): 223 - 234. [Abstract] [Full Text] [PDF]
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M. Arlt, C. Kopitz, C. Pennington, K. L. M. Watson, H.-W. Krell, W. Bode, B. Gansbacher, R. Khokha, D. R. Edwards, and A. Kruger Increase in Gelatinase-specificity of Matrix Metalloproteinase Inhibitors Correlates with Antimetastatic Efficacy in a T-Cell Lymphoma Model Cancer Res., October 1, 2002; 62(19): 5543 - 5550. [Abstract] [Full Text] [PDF]
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