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Cancer Research 67, 4311, May 1, 2007. doi: 10.1158/0008-5472.CAN-06-4761
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Stroma-Derived Matrix Metalloproteinase (MMP)-2 Promotes Membrane Type 1-MMP–Dependent Tumor Growth in Mice

Kaori Taniwaki1, Hiroshi Fukamachi2, Kiyoshi Komori1, Yohei Ohtake1, Takahiro Nonaka1, Takeharu Sakamoto1, Takayuki Shiomi3, Yasunori Okada3, Takeshi Itoh4, Shigeyoshi Itohara5, Motoharu Seiki1 and Ikuo Yana1

1 Division of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Minato-ku, 2 Department of Molecular Oncology, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, Bunkyo-ku, and 3 Department of Pathology, School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan; 4 Department Research Laboratories, Shionogi & Co., Ltd., Fukushima-ku, Osaka, Japan; and 5 Laboratories for Proteolytic Neuroscience, RIKEN Brain Science Institute, Wako-shi, Saitama, Japan

Requests for reprints: Motoharu Seiki, Division of Cancer Cell Research, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokane-dai, Minato-ku, Tokyo 108-8639, Japan. Phone: 81-3-5449-5255; Fax: 81-3-5449-5414; E-mail: mseiki{at}ims.u-tokyo.ac.jp.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Matrix metalloproteinase-2 (MMP-2) is a stroma-derived MMP belonging to the type IV collagenase family. It is believed to mediate tumor cell behavior by degrading deposits of type IV collagen, a major component of the basement membrane. The membrane type 1-MMP (MT1-MMP) is a highly potent activator of MMP-2 and is expressed in many tumor and stromal cells. However, the roles played by stromal MMP-2 in tumor progression in vivo remain poorly understood. We established a colon epithelial cell line from an Mt1-mmp–/– mouse strain and transfected these cells with an inducible expression system for MT1-MMP (MT1rev cells). Following s.c. implantation into Mmp-2+/+ mice and induction of MT1-MMP expression, MT1rev cells grew rapidly, whereas they grew very slowly in Mmp-2–/– mice, even in the presence of MT1-MMP. This MT1-MMP–dependent tumor growth of MT1rev cells was enhanced in Mmp-2–/– mice as long as MMP-2 was supplied via transfection or coimplantation of MMP-2–positive fibroblasts. MT1rev cells cultured in vitro in a three-dimensional collagen gel matrix also required the MT1-MMP/MMP-2 axis for rapid proliferation. MT1rev cells deposit type IV collagen primarily at the cell-collagen interface, and these deposits seem scarce at sites of invasion and proliferation. These data suggest that cooperation between stroma-derived MMP-2 and tumor-derived MT1-MMP may play a role in tumor invasion and proliferation via remodeling of the tumor-associated basement membrane. To our knowledge, this is the first study demonstrating that MT1-MMP–dependent tumor growth in vivo requires stromal-derived MMP-2. It also suggests that MMP-2 represents a potential target for tumor therapeutics. [Cancer Res 2007;67(9):4311–9]


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In tumor cells, phenotypic expression of genetic and epigenetic alterations largely depends on the microenvironment in the tissue (1). These microenvironments include not only tumor cells, but also stroma cells such as fibroblasts and cells in the immune system. Within these tumor environments, complex tumor-stroma interactions are mediated by the extracellular matrix (ECM), proteases, cytokines, growth factors, and various receptors for signaling molecules (2, 3). In addition to tumors, other tissues such as the mammary glands, alveolar-bronchioles and prostate require epithelial and stromal involvement to regulate their own microenvironments during development, tissue recycling, and involution (4, 5).

Malignant tumors express multiple matrix metalloproteinases (MMP), most of which are produced by stroma cells such as fibroblasts and infiltrating macrophages (6). MMPs are thought to play roles in tumor growth through ECM remodeling via the degradation of ECM components and proteolytic conversion of bioactive molecules in tumor tissue (3, 6, 7). As a result of these ideas, many MMP inhibitors (MMPI) have been developed for therapeutic use, although most clinical trials have ended in failure (3, 7). However, it should be noted that broad-spectrum MMPIs were used in these clinical studies, without any clear knowledge of the MMPs that should have been targeted. Thus, we believe that it is important to examine the functions of individual MMPs in tumor progression.

MMP-2 (also known as gelatinase A and the 72-kDa type IV collagenase) has the ability to degrade type IV collagen in the basement membrane (BM); it is one of the major stroma-derived MMPs (8–11). In spite of the many in vitro studies that have indicated that MMP-2 plays an important role in tumor invasion of the BM (6, 7, 12), little is known about the specific functions of MMP-2 in vivo. MMP-2 is produced as an inactive zymogen (proMMP-2), which is then activated in a tumor-dependent manner (12).

The membrane type 1-MMP (MT1-MMP; also known as MMP-14) is an activator of proMMP-2, which is expressed in many tumor tissues (13). In these tissues, MT1-MMP expression levels correlate well with MMP-2 activation, as well as a poor prognosis for the patient (9, 10, 14, 15). Thus, MT1-MMP is believed to be a major activator of stroma-derived MMP-2 in tumors.

MT1-MMP is an integral membrane metalloproteinase that is expressed frequently both in tumor and stroma cells (16). It cleaves a variety of ECM proteins (collagen I, II, and III; fibronectin; laminin-5), cell adhesion molecules (CD44, integrin {alpha}v, and tissue transglutaminase), cytokines, and proMMPs (proMMP-2 and proMMP-13; refs. 3, 6, 15, 16). Among these substrates, type I collagen is a particularly important physiologic target for MT1-MMP. Specifically, MT1-MMP–null mice are defective in type I collagen turnover and display severe fibrosis in their joints, delayed bone formation, multiple tissue defects, and early death (17–19). MT1-MMP is required in vitro for the invasion of type I collagen matrix by tumor and endothelial cells (20, 21). In addition, Hotary et al. (22) reported that MT1-MMP is a critical requirement for tumor formation in vivo, and that in vitro, its activity is sufficient for growth promotion of some tumor cell lines in type I collagen matrix. Thus, it seems clear that, at least in cells surrounded by type I collagen, MT1-MMP type I collagenase activity is essential for tumor growth.

However, it is possible that additional MMPs are required for tumor growth, depending on the surrounding ECM environment. Therefore, it is of interest to examine the in vivo functions of MMP-2 in tumor growth, relative to its potential activator MT1-MMP. The specific aim of this study was to analyze the effects of cooperation between stroma-derived MMP-2 and tumor-derived MT1-MMP on tumor formation in vivo. This study is important because there is no clear evidence to support the long-standing ideas that these MMPs cooperate and play roles in tumor cell function in vivo. To analyze MT1-MMP–dependent tumor growth in Mmp-2+/+ and Mmp-2–/– mice, we established a mouse epithelial cell line derived from a syngenic mouse strain containing the Mt1-mmp–/– genotype. We transduced cells (MT1rev cells) with an inducible MT1-MMP expression system and examined tumor-forming activity in syngenic mice with either Mmp-2+/+ or Mmp-2–/– genotypes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and antibodies. The following MMPI were obtained: the synthetic MMPI BB-94 (batimastat) was a gift from Dr. Peter D. Brown (British Biotech Pharmaceuticals Ltd., Oxford, United Kingdom); the tissue inhibitors of metalloproteinases (TIMP) TIMP-1 and TIMP-2 were provided by the Daiichi Fine Chemical Corporation. Type I collagen (Cellmatrix; type I-A) was purchased from Nitta Gelatin. The following antibodies were used: mouse monoclonal antibody recognizing both mouse and human MT1-MMP (clone 222-1D8; Daiichi Fine Chemical Corporation); horseradish peroxidase (HRP)–conjugated anti-mouse immunoglobulin G (IgG) antibody (GE Healthcare Bio-Sciences Corp.); anti-actin monoclonal antibody (MAB1501; Chemicon); alkaline-phosphatase–conjugated anti-mouse IgG polyclonal antibody (Sigma); rabbit polyclonal antibody against mouse collagen type IV (Chemicon); rabbit anti-Src[pY418] polyclonal antibody (Invitrogen); and Alexa-Fluor-488–conjugated anti-rabbit IgG (Sigma). Recombinant proMMP-2 was obtained from Madin-Darby canine kidney cells transfected with a plasmid expressing MMP-2. ProMMP-2 was purified from the supernatant using a gelatin Sepharose 4B column (Amersham Pharmacia Biotech), as described previously (23).

MMP-deficient mice and cell lines. MT1-MMP–deficient mice (Mt1-mmp–/–) lack exons 1 to 5, which encode the catalytic domain; this region has been substituted by the gene encoding ß-galactosidase (LacZ), which is fused to a nuclear localizing signal (refs. 24, 25). Initially, we used the 129J strain to establish Mt1-mmp+/– mice, then crossed them with C57BL/6J mice (CLEA) for more than eight generations. Mt1-mmp–/– mice display systemic skeletal malformations and have short life spans (3–4 weeks), consistent with characteristics reported for other MT1-MMP–deficient strains (17, 18). Mmp-2–/– and p53–/– mice generated in the C57BL/6 genetic background were obtained from the original developer (26, 27). Mt1-mmp+/– and Mmp-2–/– mice were mated to obtain Mt1-mmp–/–/Mmp-2–/– fetuses (25). Genotypes were confirmed by PCR using specific primers or by Southern blot analysis.

Cell lines. Epithelial and mesenchymal tissues were separated by treating tissue fragments in Hank's solution containing 30 mmol/L EDTA as described previously (28). To obtain a MT1-MMP–null cell line, we selected rapidly growing cells derived from a Mt1-mmp–/–/p53–/– mouse fetus (Fig. 1A ). An MT1-MMP–null cell line that grew well on collagen and maintained epithelial cell morphology (Fig. 1B) was used to establish the cell lines described below.


Figure 1
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  		Figure 1. Colonic epithelial cell lines established from MT1-MMP–deficient mice. A, schematic illustration of the procedure for the establishment of colonic epithelial cell lines from an Mt1-mmp–/–/p53–/– mouse and for the reintroduction of an inducible MT1-MMP expression system (MT1rev.1 and MT1rev.2 cells). Mt1-mmp+/– and p53+/– mice were mated to obtain Mt1-mmp–/–/p53–/– and Mt1-mmp+/+/p53–/– fetuses. Epithelial cells were individually isolated from each mouse colon mucosa of 16.5-d-old fetuses, cultured in dishes coated with rat-tail collagen I (42). Cells were seeded in Ham's F12 medium (Sigma), supplemented with 10% bovine serum (Handai Biken), 1 mg mL–1 bovine serum albumin (BSA; Sigma), 20 ng mL–1 epidermal growth factor (Upstate Biotech), 30 µg mL–1 insulin (Sigma), 200 ng mL–1 cholera toxin (List Biological Labs), and 2 µg mL–1 hydrocortisone (Sigma). Isolated cells were transfected with the v-src oncogene, followed by pRev-Tet-Off (a regulator plasmid) and transduction of pRev-TRE, which harbors mouse MT1-MMP cDNA. A clone transfected with the vector alone (namely MT1mock) was used as a negative transfection control for MT1rev cells. B, Western blot analysis of cells transfected with the v-src oncogene (Src) using antibody against the phosphorylated form of v-Src. Epithelial cells obtained from an Mt1-mmp+/+/p53–/– mouse fetus (WT) were used as a negative control. C, transmission electron micrographs of immortalized colon epithelial cells. a, white arrowhead, a dense cell adhesion complex. Bar, 10 µmol/L. b, microvilli are projecting from the luminal surface. Bar, 5 µmol/L. Cells were fixed with 2.5% glutaraldehyde (Sigma) in a 0.1 mol/L– cacodylate (pH 7.4) at 4°C for 2 h, and further with 1% OsO4 at 4°C for 2 h, then dehydrated through a graded series of ethyl alcohol and n-butyl glycidyl ether and embedded in Epon 812 resin (TAAB Laboratories Equipment). Ultrathin sections of samples were stained with uranyl acetate and lead citrate, then viewed using an electron microscope (100CX; JEOL, Tokyo). D, Western blot analysis of cell lysates prepared from MT1rev and control cells using anti–MT1-MMP monoclonal antibody. Arrows, the positions of proMT1-MMP and the active form MT1-MMP. WT, epithelial cells obtained from an Mt1-mmp+/+/p53–/– mouse fetus; rev.1, MT1rev.1 cells; rev.2, MT1rev.2 cells; mock, MT1mock cells. Cells were cultured in the presence (+) or absence (–) of doxycycline (Dox). Actin was used as a loading control.

 
A v-src expression plasmid was transduced into the cell line described above. As described previously (29), the RevTet-Off gene expression system (Clontech Laboratories) was used to establish stable cell lines that expressed mouse MT1-MMP under control of a Tet-regulated promoter (Fig. 1A). To suppress MT1-MMP expression, MT1rev cells were maintained in medium containing doxycycline (Dox; 1 µg mL–1; Sigma), and cells were cultured in doxycycline-free conditions to induce MT1-MMP expression. The enzymatic activities of MT1-MMP and MMP-2 are unaffected by the concentration of doxycycline (1 µg mL–1) used in these experiments.

Mmp-2–/– mice were mated with Mt1-mmp+/– and Mt1-mmp+/+ [wild-type (WT)] mice to obtain Mt1-mmp+/+/Mmp-2–/–, Mt1-mmp+/–/Mmp-2–/– and Mt1-mmp–/–/Mmp-2–/– fetuses. Mouse embryonic fibroblast (MEF) cells were obtained from 15.5-day-old fetuses as described previously (30). MEF cells were cultured in MEM (Invitrogen) supplemented with 10% fetal bovine serum and incubated in a humidified atmosphere containing 5% CO2. The MEF cells were immortalized using the SV40 T-antigen (pSvtsA58ori; Dr. Masuo Obinata, Tohoku University, Sendai, Japan). Both MEF and MT1rev cells maintained their intrinsic morphology and growth rates for at least 10 passages (data not shown). Even after immortalization, MEF cells did not display tumorigenic activity in mice (data not shown).

Western blotting. Cultured cells were harvested and then lysed in 0.125 mol/L Tris-HCl (pH 6.8) containing 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, and 0.002% bromophenol blue. Lysates were cleared by centrifugation, and protein concentrations were measured using the BCR plus kit (Pierce). Equivalent amounts of protein were separated by SDS-PAGE (8.5% acrylamide gel) and then transferred to a nitrocellulose membrane (Immobilon-P; Millipore). Membranes were blocked with 5% fat-free dry milk in PBS containing 0.05% Tween 20. To detect MT1-MMP or v-src, blots were incubated overnight at 4°C with 222-1D8 or rabbit anti-Src[pY418] polyclonal antibody, respectively. The blot was then washed thrice with PBS, treated with HRP-conjugated anti-mouse IgG polyclonal antibody, then developed for 5 min using the enhanced chemiluminescence system (ECL-Plus Western, GE Healthcare Bio-Sciences Corp.). To detect actin, blots were incubated with anti-actin monoclonal antibody (MAB1501; 1:1,000 dilution), followed by the development with an alkaline phosphatase system (Sigma).

Gelatin zymograms and degradation assay. Gelatin zymography was done as described previously (31). The method is also described briefly in the legend to Fig. 2 . Gelatin degradation assays were done using the gelatin labeled with the fluorescent dye Texas Red (Invitrogen), according to the manufacturer's instruction. The assay procedure is described in the legend to Fig. 2B.


Figure 2
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  		Figure 2. Detection of MT1-MMP on the cell surface. A, Enzymatic activity of MT1-MMP on the cell surface was evaluated by the activation of exogenous proMMP-2. MT1rev and MT1mock cells were cultured in the presence or absence of doxycycline for 4 d. Cells were then cultured for 8 h in serum-free medium containing purified mouse proMMP-2. The medium was collected, and the enzymatic activity of MMP-2 was examined using gelatin zymograms. In brief, 20 µL of conditioned medium was separated by 8% PAGE containing 0.2% porcine gelatin (Sigma) under nonreducing conditions. After electrophoresis, gels were soaked in 2.5% Triton X-100 at room temperature for 30 min, followed by incubation with 50 mmol L–1 Tris-HCl (pH 7.6) containing 150 mmol L–1 NaCl, 10 mmol L–1 CaCl2, and 0.02% Brij-35 at 37°C for 16 h. Gels were stained with 0.5% Coomassie brilliant blue R-250, followed by destaining in methanol acetate (1:1). Arrows, ProMMP-2 and active MMP-2. Conditioned medium from the wild type MEF stimulated with concanavalin A was used as a control (cont.) for pro- and active MMP-2. B, localization of MT1-MMP on the cell surface was monitored by cell-mediated gelatin degradation or by an immunocytochemical signal for MT1-MMP. Chamber slides (Nalgene Nunc International) were coated with 0.01% poly-L-lysine and 50 µg mL–1 gelatin for 30 min at room temperature, followed by cross-linking with 1% glutaraldehyde. After the slides were washed with culture medium, MT1-rev. Two cells were seeded onto the gelatin-coated slides and incubated for 16 h. Cells were fixed with 4% paraformaldehyde in PBS. Degradation of gelatin was visualized under a microscope as elimination of the gelatin background fluorescence. Subsequently, the cells were fixed in 4% paraformaldehyde for 5 min, washed twice in PBS, and incubated with 5% goat serum for 10 min, then 3% BSA in PBS for 1 h at room temperature. The cells were then treated with mouse monoclonal antibody against MT1-MMP (clone 222-1D8; 1:500 dilution) overnight at 4°C, followed by Alexa-Fluor-488-conjugated goat anti-mouse IgG antibody (Invitrogen). The cells were cultured for 16 h in the presence or absence of doxycycline and the MMPI, BB-94. a to d, gelatin degradation by MT1rev.2 cells (black area). e to h, MT1-MMP immunostaining matching a to d. Working concentrations were as follows: 10 µmol L–1 BB-94 (b and f); 1 µg mL–1 TIMP-1 (c and g); and 1 µg mL–1 TIMP-2 (d and h). a and e, controls. Bar, 0.25 mm. C, negative controls for (B); see Materials and Methods for a full description. Bar, 0.25 mm.

 
Immunostaining. The immunocytostaining procedure is summarized in the legend to Fig. 3B . To immunostain cells cultured in collagen matrix, collagen gels were embedded in Tissue-Tek OCT compound (Sakura), frozen with liquid N2, and sectioned (8 µm). The sections were treated with rabbit polyclonal antibody against mouse collagen type IV (1:300 dilution). Hoechst dye 33258 (Invitrogen) was used to stain nuclei. Cell fluorescence was observed using a charge-coupled device fluorescent microscope (IX-70; Olympus).


Figure 3
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  		Figure 3. Temporal response of tumor growth to MT1-MMP expression in vivo. A, MT1rev and MT1mock cells were cultured in vitro in the presence or absence of doxycycline, and then 1 x 106 cells were implanted into Mmp-2+/+ mice (WT mice). Starting 2 d before implantation of cells, doxycycline was given to the mice via the drinking water (2 mg doxycycline mL–1; +Dox). Points, mean volume tumor sizes monitored over 21 d; bars, SE (n = 20 for mock or rev.1 ±Dox, n = 12 for rev.2 ±Dox). Mann-Whitney U tests were done for rev.1 –Dox versus rev.2 –Dox and rev.1 –Dox versus rev.1 +Dox. *, P at day 14 = 0.479 and <0.0001; **, P at day 21 =0.029 and <0.0001, respectively. B, MT1rev.2 cells were cultured in the presence or absence of doxycycline in vitro, then implanted into Mmp-2+/+ mice. The drinking water given to the mice either contained doxycycline (+Dox) or was doxycycline-free (–Dox), with matching in vivo and in vitro treatments. Some of the mice were switched to doxycycline-free water 21 d after implantation of the doxycycline-treated cells (+Dox21day; –Dox). Tumor sizes were monitored over 42 d. Mann-Whitney U tests were done for rev.2 –Dox versus rev.2 +Dox21day and –Dox versus rev.2 –Dox. *, P at day 14 =0.0079 and <0.0044; **, P at day 21 <0.001 and 0.0005, respectively.

 
In vivo tumor growth assay. Cells (1 x 106) suspended in culture medium (200 µL) were injected s.c. into the dorsal side of the mice. For coimplantations, MT1rev (1 x 106) and MEF (2 x 106) cells were injected in the same volume of medium (200 µL). Tumor size was measured twice a week using calipers. Tumor volume was estimated using the following formula: V = (LW2){pi}/6, where V, volume (mm3); L, largest diameter (mm); and W, smallest diameter (mm). For the +Dox control, cells were treated with doxycycline (1 µg mL–1) for 48 h in vitro before implantation, and doxycycline was given to the recipient mice via their drinking water (2 mg doxycycline mL–1). For doxycycline-free (i.e., –Dox) experiments, the recipient mice were provided with doxycycline-free water before and after implantation. A subgroup of mice in the +Dox group were transferred from the +Dox to doxycycline-free water regime 21 days after implantation with doxycycline-treated cells. This change was aimed at examining whether or not tumorigenesis was affected by transfer from MT1-MMP–suppressive to MT1-MMP–inducible conditions.

Statistical analysis. All variables were presented as means ± SD. These were compared using the nonparametric Mann-Whitney U test; P < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Establishment of an epithelial cell line derived from an Mt1-mmp–/– mouse strain. An immortalized cell line was obtained using epithelial cells isolated from the large intestine of an Mt1-mmp–/–/p53–/– mouse fetus and cultured in vitro (Fig. 1A). The cells were observed using transmission electron microscopy and seemed to retain intestinal epithelial characteristics such as junctional complexes at cell-cell junctions (Fig. 1B-a) and microvilli that projected from the luminal cell surface (Fig. 1B-b). Because it has been reported that c-Src activity is up-regulated in human colon cancer (32), the cells were induced further with the v-src oncoprotein (Fig. 1C) to increase their tumorigenic potential in vivo. These v-src–transfected cells continued to retain an intestinal epithelial-like morphology similar to the cells shown in Fig. 1B (data not shown).

A doxycycline-regulated system for MT1-MMP expression was introduced into the cell line, and two MT1-MMP revertants (MT1rev.1 and MT1rev.2) were obtained. Control cells were transfected with the vector alone (MT1mock). Epithelial cells isolated from the large intestine of Mt1-mmp+/+/p53–/– mouse fetuses expressed the active form of MT1-MMP; this was absent in MT1mock cells (Fig. 1D, WT and mock, respectively). In MT1rev cells (rev.1 and rev.2), MT1-MMP expression levels were low in the presence of doxycycline (+Dox), whereas expression increased 5- to 10-fold when these cells were grown in the doxycycline-free (–Dox) culture medium (Fig. 1D). The major form of MT1-MMP induced by these conditions exhibited a slightly slower band mobility than that of WT cells. This higher molecular weight protein corresponds to the latent form (proMT1-MMP), which contains a propeptide sequence. In addition to proMT1-MMP, the active form was detected in MT1rev cells cultured under doxycycline-free conditions. Note that the levels of the active form are similar between MT1rev and WT cells, despite the higher levels of proMT1-MMP observed in the former. As with most tumor cell lines of epithelial origin (8), MMP-2 was not expressed by MT1rev cells (data not shown). Furthermore, the growth rate of MT1rev cells was unaffected by doxycycline under the culture conditions used in these studies (data not shown).

Following the induction of MT1-MMP expression in doxycycline-free medium, both MT1rev.1 and MT1rev.2 cells acquired the ability to activate exogenously added proMMP-2 to MMP-2 (Fig. 2A). MEF obtained from WT mice were stimulated by concanavalin A (Con A; ref. 33) and used as a positive control for MT1-MMP–mediated activation of MMP-2 (Fig. 2A, MEF). MT1mock cells were used as a negative control (Fig. 2A, mock).

To detect the matrix-degrading activity of MT1-MMP at the cell surface, MT1rev cells were seeded and cultured on glass slides that had been coated with fluorescently labeled gelatin. Areas of degraded gelatin were visualized as dark marks against a background of red fluorescence (Fig. 2B-a–d). In addition, the presence of MT1-MMP on the cell surface was visualized by immunostaining MT1-MMP under nonpermeable conditions (Fig. 2B-e–h). Degradation of gelatin and cell-surface expression of MT1-MMP were observed for MT1rev.2 cells cultured in the absence of doxycycline (Fig. 2B-a and B-e). The gelatinolytic activity of MT1rev.2 cells (–Dox) was inhibited almost completely by the synthetic MMPI BB-94 (Fig. 2B-b) and TIMP-2 (Fig. 2B-d), but not by TIMP-1 (Fig. 2B-c). The TIMP-2–sensitive and TIMP-1–insensitive gelatinolytic activity observed for MT1rev.2 cells is consistent with the enzymatic characteristics of MT1-MMP (15, 16). Expression of MT1-MMP and degradation of gelatin were not observed in either MT1rev.2 cells cultured in the presence of doxycycline or in MT1mock cells (Fig. 2C). The results obtained for MT1rev.1 cells (data not shown) were very similar to those observed for MT1rev.2 cells (Fig. 2B and C).

MT1rev cells exhibit MT1-MMP–dependent tumor formation. To evaluate tumor-forming activity, MT1rev and MT1mock cells were implanted s.c. into syngenic Mmp-2+/+ mice, and tumor size was monitored thereafter (Fig. 3A). The control cells (MT1mock) formed very small tumors that were barely detectable from the surface, regardless of the doxycycline treatment (mock; –Dox and +Dox). Although MT1rev.1 and MT1rev.2 cells formed tumors, they also remained small in size in the absence of MT1-MMP induction. However, these cells formed markedly larger tumors under MT1-MMP–inducing conditions (–Dox). During 21 days of observation, the tumor volume of MT1rev cells expressing MT1-MMP was 7- to 10-fold greater than those implanted under MT1-MMP suppressive conditions. Thus, expression of MT1-MMP promotes tumor growth of MT1rev cells in mice. This result is consistent with previous studies done using different types of tumor cells (22, 34–37). In the following experiments, we used MT1rev.2 to represent all MT1rev cells.

To evaluate the temporal effect of MT1-MMP on tumor growth, MT1rev.2 cells were implanted into mice without MT1-MMP induction (Fig. 3B, rev.2 +Dox). After 21 days, in which the slow growth rate of uninduced MT1rev.2 cells was confirmed, some of the mice were given doxycycline-free water to induce expression of MT1-MMP in vivo (Fig. 3B, rev.2 +Dox 21day; –Dox). Tumor growth was enhanced soon after the elimination of doxycycline exposure; the rate of tumor growth reached that of MT1rev.2 cells expressing MT1-MMP continuously (rev.2 –Dox). This finding indicates that in the presence of doxycycline, the tumor growth of MT1rev.2 cells is restricted by the absence of MT1-MMP. It also suggests that in mice, these cells can survive for at least 21 days under conditions that suppress MT1-MMP expression.

Effect of stroma-derived MMP-2 on MT1-MMP–dependent tumor growth. The next step was to examine whether or not MT1-MMP–dependent tumor growth requires MMP-2 from the host stroma. MT1rev.2 and MT1mock cells were implanted into Mmp-2–/– mice, and tumor growth was monitored in the presence or absence of MT1-MMP expression (Fig. 4A ). Regardless of the presence (+) or absence (–) of doxycycline, MT1mock cells (mock) formed very small tumors (Fig. 4A), similar to those formed in Mmp-2+/+ mice (Fig. 3A). In the absence of MT1-MMP expression (+Dox), MT1rev.2 cells also formed small tumors. However, MT1-MMP (–Dox) expression in MT1rev.2 cells resulted in only a 2-fold enhancement of tumor growth in Mmp-2–/– mice (Fig. 4A), suggesting that MMP-2 plays a role in tumor promotion.


Figure 4
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  		Figure 4. Tumor formation in MMP-2–deficient mice. A, tumor growth from MT1rev.2 cells was evaluated in Mmp-2–/– mice. MT1rev.2 and MT1mock cells were implanted into Mmp-2–/– mice. Tumor sizes were monitored over 21 d. Cells and mice were treated with or without doxycycline as indicated. Points, mean volume tumor sizes; bars, SE (n = 6 for each group). Mann-Whitney U tests were done for rev.2 –Dox versus rev.2 +Dox, rev.2 +Dox versus mock –Dox, and rev.2 –Dox versus mock –Dox. *, P at day 14 =0.0148, 0.035, 0.00369; **, P at day 21 =0.0892, 0.0725, and 0.0037, respectively. B, MT1rev.2 cells were transfected with a plasmid-expressing mouse MMP-2. Production of MMP-2 by MT1erv.2/MMP-2 cells was examined using gelatin zymograms. Expression of MT1-MMP was controlled by doxycycline as indicated. Arrows, latent and active forms of MMP-2. C, MT1rev.2 cells and MMP-2–transfected MT1rev.2 cells (rev.2/MMP-2) were implanted into Mmp-2–/– mice, and tumor sizes were monitored as described above. Expression of MT1-MMP was controlled by doxycycline as indicated. Points, mean volume tumor sizes; bars, SE. n = 6 for rev.2 ±Dox, and n = 16 for rev2/MMP-2 ±Dox. Mann-Whitney U tests were done for rev.2/MMP-2 –Dox versus rev.2/MMP-2 +Dox. * and **, P < 0.0001 for both days 14 and 21.

 
To confirm that MMP-2 is responsible for these differences, MT1rev.2 cells were transfected with an expression plasmid that affected the stable expression of MMP-2. These transfected cells (MT1rev.2/MMP-2) produced proMMP-2 during growth in the presence (+) and absence (–) of doxycycline, which generated active MMP-2 following the induction of MT1-MMP expression (Fig. 4B). Although proMMP-2 was expressed in the presence (+) of doxycycline, it did little to promote MT1rev.2 cell-derived tumor development in vivo (Fig. 4C, rev.2/MMP-2 +Dox). In the absence (–) of doxycycline, MT1rev.2/MMP-2 cells expressed MT1-MMP, and tumor growth was enhanced 4- to 5-fold (Fig. 4C). Expression of MT1-MMP did not enhance tumor growth in MT1rev.2/mock cells. Thus, MMP-2 is required for the MT1-MMP–dependent tumor growth of MT1rev.2 cells in mice.

Reconstitution of MMP-2–mediated tumor-stroma interaction in MMP-2–deficient mice. MMP-2 is produced primarily by fibroblasts in the stroma of tumors (8, 11). Thus, the epithelial expression of MMP-2 by cells such as MT1rev.2/MMP-2 might represent completely artificial conditions. Therefore, we investigated whether or not stroma-derived MMP-2 is sufficient for MT1-MMP–dependent tumor growth. An MMP-2–positive stromal environment was reconstructed in Mmp-2–/– mice, after which MEFs obtained from Mt1-mmp+/+/Mmp-2+/+ (WT), Mt1-mmp–/–/Mmp-2+/+ (MT1KO), or Mt1-mmp–/–/Mmp-2–/– (DKO) mice were introduced. WT MEFs produced proMMP-2; following treatment with concanavalin A (+Con A), activated MMP-2 was detected (Fig. 5A, WT ). Similar to WT MEFs, MT1KO-MEFs produced proMMP-2. However, no active MMP-2 was observed following treatment with Con A (Fig. 5A, MT1KO). Neither proMMP-2 nor MMP-2 were detected in DKO MEFs (Fig. 5A, DKO).


Figure 5
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  		Figure 5. Reconstitution of an MMP-2–positive stromal environment in MMP-2–deficient mice. A, MMP-2 production by MEF cells isolated from WT and mutant (knock-out) mouse fetuses was analyzed using gelatin zymograms. MEFs were isolated from Mt1-mmp+/+/Mmp-2+/+ (WT), Mt1-mmp–/–/Mmp-2+/+ (MT1KO), and Mt1-mmp–/–/Mmp-2–/– (DKO) mouse fetuses as described in Materials and Methods. Cells were cultured in the presence or absence of 50 µg mL–1 concanavalin A (Con. A), after which, serum-free culture supernatants were collected and analyzed using gelatin zymograms. Arrows, ProMMP-2 and active MMP-2. B, MT1rev.2 cells were coimplanted into Mmp-2–/– mice with the MEFs indicated. Expression of MT1-MMP was controlled by doxycycline as described above, and tumor sizes were monitored. Points, mean volume tumor sizes; bars, SE. n = 4 for each group. A Mann-Whitney U test was done for rev.2 –Dox/MT1KO-MEF versus rev.2 +Dox/MT1KO-MEF. *, P = 0.098 for day 14; **, P = 0.012 for day 21.

 
We coimplanted Mmp-2–/– mice with MT1rev2 cells and each of the MEFs and then monitored tumor size (Fig. 5B). Implantation of MEF cells alone did not result in tumor formation in mice (data not shown). DKO MEF cells were unable to support growth of MT1rev.2 tumors, regardless of MT1-MMP expression (Fig. 5B, rev.2/DKO). In contrast, the proMMP-2–producing MT1KO MEF cells were able to enhance MT1-MMP–dependent (–Dox) tumor growth by 3- to 4-fold (Fig. 5B, rev.2/MT1KO). Thus, the expression of MMP-2 by fibroblasts promoted MT1rev.2 tumor formation in mice in a MT1-MMP–dependent manner.

Proliferation of MT1rev.2 cells in type I collagen matrix in vitro. In vivo, type I collagen is reported to form the major ECM environment for tumors (20). MT1-MMP is a cell-associated form of collagenase; it is required and sufficient for the in vitro growth of some tumor cell lines in collagen matrix (22). Because in vivo tumor formation requires MMP-2 in addition to MT1-MMP, we investigated whether or not MMP-2 was necessary for the in vitro proliferation of MT1rev.2 cells in a type I collagen matrix. In the absence of MT1-MMP expression, the number of MT1rev.2 cells in the collagen matrix increased 4-fold within 10 days (Fig. 6A , +Dox). However, when these cells expressed MT1-MMP, their numbers increased 10-fold (Fig. 6A, –Dox). These findings suggest that MT1-MMP is required for the rapid growth of MT1rev.2 cells in a collagen matrix in vitro (Fig. 6A), similar to tumor formation in vivo (Fig. 3A). Although MT1-MMP expression (–Dox) enhanced cell growth, this promotion was suppressed by both TIMP-1 and TIMP-2, which are noninhibitory and inhibitory to MT1-MMP enzymatic activity, respectively (Fig. 6A). Thus, it would seem that a TIMP-1–sensitive MMP-2 is contained in the mouse serum that was added to the culture. Furthermore, this MMP-2 seems to be involved in MT1-MMP–dependent cell growth in a collagen matrix. MT1rev.2 cells were cultured with MMP-2–free serum obtained from Mmp-2–/– mice, and no significant enhancement of growth was observed, even when MT1-MMP was expressed (Fig. 6B, –Dox). In contrast, MT1-MMP–dependent growth recovered following the addition of recombinant proMMP-2 to the MMP-2–free culture medium (Fig. 6B). Thus, even in an in vitro type I collagen matrix, MMP-2 is critical for MT1-MMP–dependent proliferation of MT1rev.2 cells.


Figure 6
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  		Figure 6. Cell proliferation assay in a type I collagen matrix and detection of type IV collagen. Cell culture in type I collagen matrix was performed as follows: type I collagen matrix (2.4 mg mL–1) was prepared by mixing Cellmatrix (Nitta Gelatine), 10x MEM and 0.34 N NaOH in an 8:1:1 ratio, respectively. The gel solution (200 µL) was transferred into 48-well plates and incubated at 37°C. Just before the complete polymerization, 5 µL of suspended cells (1 x 104) were seeded into the gels. Samples were cultured separately in medium supplemented with either 10% serum from WT or MMP-2–deficient mice. To analyze the effects of MMP-2 on cell growth, recombinant pro–MMP-2 (4.3 pg) was added to the culture medium. After 10 d in culture, each collagen sample was digested with bacterial collagenase, and the cells were harvested and counted. A, MT1rev.2 or MT1mock cells (1 x 104) were inoculated into a collagen matrix and cultured for 10 d under the doxycycline and inhibitor conditions indicated, after which the cells were counted. MMP-2 was supplied by serum prepared from MMP-2–positive mice (Mmp-2+/+). Expression of MT1-MMP was regulated by doxycycline; proteolytic activity was inhibited by TIMP-1 (1 µmol/L) or TIMP-2 (1 µmol/L). Columns, mean cell numbers; bars, SE (n = 3). B, MT1rev.2 cells were cultured in a collagen matrix with medium supplemented with serum obtained from MMP-2–deficient mice (Mmp-2–/–). Recombinant pro–MMP-2 (5 pg) was added to the culture medium as indicated. Columns, mean cell numbers; bars, SE (n = 3). C, expression of ECM-related genes in MT1rev.2 cells was analyzed using an oligonucleotide microarray assay (Oligo GEArray; SuperArray Bioscience Corp.) according to the supplier's protocols. Results of the hybridization are presented. Intensity of the signal was measured by densitometry; intensity relative to type IV collagen {alpha}1 is indicated. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Col1{alpha}1, type I collagen {alpha}1; Col3{alpha}1, type III collagen {alpha}1; Col4{alpha}1, type IV collagen {alpha}1; Col5{alpha}1, type V collagen {alpha}1; Col6{alpha}1, type VI collagen {alpha}1; FN, fibronectin; and Lamß1, laminin ß1. D, MT1mock (a) and MT1rev.2 (b and c) cells were cultured in a collagen matrix. After 3 d in culture, bright-field photos were taken of the cells using a phase-contrast microscope. Red arrows, cell protrusions invading the collagen matrix in the absence of doxycycline (–Dox; c). Bars (a-c), 0.25 mm. Following 5 d in the absence of doxycycline, cells were sliced into 8-µm sections and immunostained for collagen IV. d, a bright-field image showing cells extending into the collagen matrix. Cells at the invasive end exhibit filopodia (arrows). e, immunostaining of the same section using an anti-type IV collagen antibody. The signal was developed using Alexa-Fluor-488-conjugated secondary antibody. f, merge of images in (d) and (e). Type IV collagen was deposited throughout the cell layer, yet there is little deposition on invasive filopodia (arrows). Bar, 0.25 mm.

 
Production and deposition of type IV collagen by MT1rev.2 cells. MT1-MMP degrades type I collagen (38) and is sufficient to support growth of some tumor cell lines in a collagen matrix (20). Because MT1rev.2 cells require MMP-2 for growth in a type I collagen matrix, it is possible that they produce additional ECM components that require MMP-2 for degradation. Therefore, we used a membrane-based ECM gene microarray assay to identify whether or not these cells expressed other ECM components. Indeed, we detected expression of mRNAs for type IV collagen ({alpha}1), type V collagen ({alpha}1), fibronectin, and laminin (ß1), whereas no mRNAs were detected for interstitial collagens such as type I collagen ({alpha}1) or type III collagen ({alpha}1; Fig. 6C). Because type IV collagen is a well-characterized substrate of MMP-2, immunostaining was used to investigate its deposition around MT1rev.2 cells in the collagen matrix. MT1mock (Fig. 6D-a) and MT1rev.2 cells were cultured in a three-dimensional collagen matrix in the presence of WT serum (i.e., MMP-2–containing) and under MT1-MMP–suppressing conditions (Fig. 6D-b, +Dox). Under phase contrast microscopy, these cells were observed to form ball-like structures in the collagen matrix (Fig. 6D-a and D-b, respectively). In contrast, under MT1-MMP–inducing conditions, MT1rev.2 cells elongated and extended protrusions into the collagen matrix (Fig. 6D-c). Frozen sections of MT1-MMP–expressing MT1rev.2 cells (–Dox) were prepared and subjected to type IV collagen immunostaining (Fig. 6D-d–f). The cells seemed to form a sheetlike structure (Fig. 6D-d), and type IV collagen was detected along the sheet (Fig. 6D-e). Interestingly, there seemed to be little type IV collagen at the protruding tip area (Fig. 6D-f). Thus, it would seem that MT1rev.2 cells express additional ECM components, which include type IV collagen, and that these are deposited at the cell-collagen interface. MT1-MMP and MMP-2 cooperate to eliminate these deposits, thereby promoting invasion and proliferation of the cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Consistent with previous studies (22, 34–37), we observed that MT1-MMP is an essential component of the mechanisms that support growth of MT1rev cells under three-dimensional conditions such as in mouse tissue or an in vitro matrix of type I collagen. MT1rev.2 cells started to proliferate 21 days after implantation, following in vivo induction of MT1-MMP expression. In the presence of doxycycline, tumor growth of MT1rev.2 cells seems to be limited by MT1-MMP. Interestingly, similar growth rates were observed between tumors following in vivo induction of MT1-MMP and cells in which MT1-MMP had been induced previously in vitro (Fig. 3B). This finding indicates that in the absence of MT1-MMP expression, MT1rev.2 cell numbers did not decrease significantly during the 21 days following implantation. Indeed, the percentage of apoptotic cells in the tumors seemed to be unaffected by the absence of MT1-MMP expression (data not shown).

Expression of MT1-MMP–enhanced MT1rev cell growth in Mmp-2+/+ mice (Fig. 3A), but this effect was diminished greatly when the cells were implanted into Mmp-2–/– mice (Fig. 4A). In the absence of MT1-MMP, expression of MMP-2 alone (presumably as proMMP-2) did not promote tumor growth (Fig. 4C). However, following transfection of MT1rev.2 cells with an MMP-2 expression vector (MT1rev.2/MMP-2 cells), tumor growth in Mmp-2–/– mice was enhanced greatly in response to MT1-MMP expression (Fig. 4C). Moreover, similar effects were observed following coimplantation of MEF cells expressing MMP-2 and MT1rev.2 cells (Fig. 5B). Thus, stroma-derived MMP-2 is important and sufficient to support the MT1-MMP–dependent tumor growth of MT1rev.2 cells. To our knowledge, this is the first demonstration that stroma-derived MMP-2 can be used for tumor growth in a MT1-MMP–dependent manner.

Presumably, the fact that MMPs are required for tumor growth in vivo indicates the involvement of the ECM environment around the tumors. Therefore, it is interesting that MT1rev.2 cells required MMP-2 for cell growth in the type I collagen matrix in vitro (Fig. 6). Because MT1-MMP is sufficient to degrade type I collagen (22, 38), the requirement of MMP-2 for the growth of these cells suggests that additional ECM components exist at the cell-collagen interface. Following the analysis of the ECM surrounding the MT1rev.2 cells in the collagen matrix, we found the deposition of type IV collagen (Fig. 6D), a major BM component and a well-known substrate of MMP-2 (6, 12, 39). Thus, MT1rev.2 cells may have the ability to form BM because they expressed additional BM components such as fibronectin and laminin (Fig. 6C). Without MT1-MMP expression, MT1rev.2 cells formed a compact ball-like structure in the collagen matrix. However, cells expressing MT1-MMP seemed to grow numerous protrusions that extended into the collagen matrix. This type of growth occurs in the presence of MMP-2, and type IV collagen seemed to be very limited at the edge of the protrusions into the matrix (Fig. 6D-f, white arrows). Thus, we speculate that MMP-2 cooperates with MT1-MMP to loosen the BM-like structure around cells before extension into the type I collagen matrix (40, 41).

MT1-MMP is expressed in both tumor and stroma cells (6), whereas expression of MMP-2 is mostly in the latter (11). Because fibroblasts express both MT1-MMP and MMP-2, these cells can activate proMMP-2 using their own MT1-MMP, as shown in Fig. 5A. However, the MMP-2 activated by fibroblasts may be less effective at supporting tumor growth because tumor formation of MT1rev cells clearly required expression of MT1-MMP, even when the cells were implanted into Mt1-mmp+/+/Mmp-2+/+ mice (Fig. 3A and B). This result implies that MMP-2 must be activated on the tumor cell surface to contribute to growth.

What clinical implications does the present study hold? Our findings suggest that MMP-2 plays a particularly important role in the early stages of tumors, when the BM remains adjacent to the tumor cells. At the same time, tumor cells in the early stages may retain the ability to express components of BM, including type IV collagen and laminin, as shown here with MT1rev cells. On the other hand, the MMP-2 requirement may not be as critical for more advanced tumors, such as those that have completed the epithelial-mesenchymal transition. Even for advanced tumors, MT1-MMP may still be important because tumor cells are surrounded by type I collagen (22). Thus, MMP-2–targeted therapy may work effectively only in the early stages of tumors. However, it is noteworthy that the phenotypic characteristics of Mmp-2–/– mice are not severe (26). Thus, specific inhibition of MMP-2 might provide the effective prevention of the early stages of tumor progression, without causing serious side effects. However, as described earlier, the phenotypic characteristics of Mt1-mmp–/– mice are severe. Thus, only MT1-MMP expressed by tumor cells or in tumor tissue should be targeted if these severe side effects are to be avoided.

Over the last few years, the therapeutic value of many MMPIs has been tested, but in clinical trials, most have proved less effective than expected. Because these MMPIs can inhibit multiple MMPs concurrently, the effect of inhibition of individual MMPs needs to be reevaluated. Thus, we believe that an improved understanding of MMP functions in in vivo tumor development will contribute to the effective and safe application of anti-MMP therapy. In particular, our findings suggest that specific inhibition of MMP-2, rather than general inhibition of MMPs, may provide a promising therapy for some epithelial tumor progressions.


   Acknowledgments
 
Grant support: Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (17014019).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The authors thank Naohiko Koshikawa (University of Tokyo, Japan) for helpful and crucial discussions, Chieko Konishi (University of Tokyo, Japan) for excellent technical assistance, and Akiko Okada (Mie University, Tsu City, Japan) for her initial work on establishing the Mt1-mmp–/– mice.

Received 12/27/06. Revised 2/21/07. Accepted 2/27/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
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Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online