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[Cancer Research 66, 5165-5172, May 15, 2006]
© 2006 American Association for Cancer Research

Cell, Tumor, and Stem Cell Biology

Membrane-Type 4 Matrix Metalloproteinase Promotes Breast Cancer Growth and Metastases

Vincent Chabottaux1, Nor Eddine Sounni1, Caroline J. Pennington3, William R. English4, Frédéric van den Brûle1,2, Silvia Blacher1, Christine Gilles1, Carine Munaut1, Erik Maquoi1, Carlos Lopez-Otin5, Gillian Murphy4, Dylan R. Edwards3, Jean-Michel Foidart1,2 and Agnès Noël1

1 Laboratory of Tumor and Development Biology, Centre de Recherche en Cancérologie Expérimentale, Center for Biomedical Integrative Genoproteomics, University of Liège; 2 Department of Gynecology CHU, Liège, Belgium; 3 School of Biological Sciences, University of East Anglia, Norwich, Norfolk, United Kingdom; 4 Department of Oncology, Cambridge Institute for Medical Research, Cambridge University, Cambridge, United Kingdom; and 5 Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo, Spain

Requests for reprints: Agnes Noël, Laboratory of Tumor and Development Biology, University of Liège, Tour de Pathologie (B23), Sart-Tilman, B-4000 Liège. Phone: 32-4-366-24-53; Fax: 32-4-366-29-36; E-mail: agnes.noel{at}ulg.ac.be.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Membrane-type matrix metalloproteinases (MT-MMP) constitute a subfamily of six distinct membrane-associated MMPs. Although the contribution of MT1-MMP during different steps of cancer progression has been well documented, the significance of other MT-MMPs is rather unknown. We have investigated the involvement of MT4-MMP, a glycosylphosphatidylinositol–anchored protease, in breast cancer progression. Interestingly, immunohistochemical analysis shows that MT4-MMP production at protein level is strongly increased in epithelial cancer cells of human breast carcinomas compared with normal epithelial cells. Positive staining for MT4-MMP is also detected in lymph node metastases. In contrast, quantitative reverse transcription-PCR analysis reveals similar MT4-MMP mRNA levels in human breast adenocarcinomas and normal breast tissues. Stable transfection of MT4-MMP cDNA in human breast adenocarcinoma MDA-MB-231 cells does not affect in vitro cell proliferation or invasion but strongly promotes primary tumor growth and associated metastases in RAG-1 immunodeficient mice. We provide for the first time evidence that MT4-MMP overproduction accelerates in vivo tumor growth, induces enlargement of i.t. blood vessels, and is associated with increased lung metastases. These results identify MT4-MMP as a new putative target to design anticancer strategies. (Cancer Res 2006; 66(10): 5165-72)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Matrix metalloproteinases (MMP) are a family of zinc-binding endopeptidases that can degrade virtually all extracellular matrix components and a growing number of other modulators of cell functions (1–4). Although most MMPs are secreted as soluble enzymes, six of them are membrane-type MMPs (MT-MMP), which are associated with the cell membrane by either a transmembrane domain (MT1-MMP, MT2-MMP, MT3-MMP, and MT5-MMP) or a glycosylphosphatidylinositol (GPI) anchor (MT4-MMP and MT6-MMP; ref. 5).

MT1-MMP, the first discovered member of this subfamily, was initially described as an activator of pro-MMP2 (6). MT1-MMP plays critical roles in cell migration, cell invasion, and physiologic and pathologic remodeling of the extracellular matrix (5, 7). It is a multifunctional protease for which an increasing number of substrates have been identified, including pro-MMP2, pro-MMP13, extracellular matrix components, cell surface receptors, growth factors, chemokines, and adhesion and signaling molecules (2, 5). Its implication in tumor growth, invasion, and angiogenesis is now well documented (5, 8–11). A proangiogenic effect of MT1-MMP has been linked, at least in part, to an up-regulation of vascular endothelial growth factor (VEGF; refs. 12, 13).

Although much emphasis has been placed on understanding the biochemical features and functions of MT1-MMP (5), little attention has been given to the implication of other MT-MMPs in cancer progression and angiogenesis. Different MT-MMPs (MT2-MMP, MT3-MMP, and MT5-MMP) can activate pro-MMP2 but less efficiently than MT1-MMP (5, 14, 15). Despite being able to bind tissue inhibitor of metalloproteinase-2, MT4-MMP and MT6-MMP are rather inefficient at activating pro-MMP2 (16–18). Since its original isolation from human breast carcinoma (19), MT4-MMP has been detected in several human cancers, including gliomas (20), prostate carcinomas (21), and breast carcinomas (19). However, its localization and implication during human breast cancer progression is currently unknown. The inability of MT4-MMP to activate pro-MMP2 and the lack of a cytoplasmic tail in this cell surface GPI-anchored protease suggest that MT4-MMP displays different functions than MT1-MMP. Its few known substrates are fibrinogen, fibrin, and pro-tumor necrosis factor-{alpha} and a disintegrin and metalloproteinase with thrombospondin-like motif-4 (ADAMTS-4; refs. 16, 22, 23).

The aim of this study was to explore the expression of MT4-MMP in human breast carcinomas by reverse transcription-PCR (RT-PCR) and immunohistochemical analyses and to investigate its putative role in cancer progression through its overexpression in human breast adenocarcinomas cells. We provide evidence that MT4-MMP is produced at higher levels by human mammary carcinoma cells than by normal epithelial cells. We show for the first time that its overexpression in human breast cancer cells promotes primary tumor growth leading to increased lung metastases. A link between MT4-MMP and metastatic dissemination of human breast cancers is further supported by its strong expression in human lymph node metastases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human Tissue Samples
Human breast samples were obtained from tumor banks of the Center for Experimental Cancer Research, University of Liège and of School of Biological Sciences, University of East Anglia, United Kingdom. Breast tumor samples were obtained from 63 patients with invasive breast ductal carcinomas who underwent surgery. Normal breast tissues were obtained from 21 patients who underwent reduction mammoplasty. Twelve metastatic and six normal lymph nodes from four patients were also collected. Breast tumors were reviewed regarding histopathologic type based on the WHO classification. All cancer samples were characterized by >70% cancer cell composition. Total RNA was extracted from frozen samples with RNeasy Mini kit (Qiagen GmbH, Hilden, Germany) according to manufacturer's instructions.

Cell Culture
Human breast cancer MDA-MB-231 cells obtained from the American Type Culture Collection (Manassas, VA) were grown to 80% confluence in DMEM supplemented with 10% FCS, 2 mmol/L L-glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin at 37°C in a 5% CO2 humid atmosphere. All culture reagents were purchased from Invitrogen Corp./Life Technologies (Paisley, Scotland).

Stable Transfection of MDA-MB-231 Cells with Human MT4-MMP cDNA
Parental MDA-MB-231 cells were stably transfected by electroporation (250 V, 960 µF) with pcDNA3-neo vector containing only the neomycin resistance gene (control plasmid) or with the same plasmid carrying the full-length human MT4-MMP cDNA (pcDNA3-MT4; refs. 19, 24). Selection was done under G418 pressure (1.5 mg/mL; Invitrogen/Life Technologies), and screening of clones was based on RT-PCR analysis to determine MT4-MMP expression. Stable transfectants were maintained in medium containing G418 (500 µg/mL).

Preparation of Proteins Cell Extracts, Conditioned Media, and Total RNAs
Conditioned media of cells for ELISA were prepared by seeding cells (2.5 x 105) onto six-well plates for 24 hours in 3 mL of DMEM with serum followed by incubating cells for 48 hours in 1.5 mL of serum-free DMEM. Similarly, conditioned media for rat aortic rings assay were prepared by culturing 106 cells in 100-mm diameter Petri dishes (Falcon, Becton Dickinson, Lincoln Park, NJ). Cells were grown in 8 mL of DMEM with serum for 24 hours and then in 6 mL of serum-free DMEM for 48 hours. Conditioned media were harvested, clarified by centrifugation, and stored frozen at –20°C. Total protein cell extracts were prepared from cell monolayers incubated in radioimmunoprecipitation assay buffer (25). Protein concentration was determined by using the detergent-compatible protein assay kit (Bio-Rad Laboratories, Hercules, CA). Total RNAs were extracted from cell monolayer using High Pure RNA isolation kit (Roche Diagnostic Applied Science, Mannheim, Germany).

Semiquantitative RT-PCR Analysis
RT-PCR was done on 10 ng of total RNA extracted from cells using a GeneAmp Thermostable rTth Reverse Transcriptase RNA PCR kit (Applied Biosystems, Foster City, CA) following manufacturer's instructions. Specific pairs of primers (Eurogentec, Seraing, Belgium) for human MT4-MMP were designed as follows: forward, 5'-AAGGAGACAGGTACTGGGTGTTC-3'; reverse, 5'-TCGCCATCCAGCACTTTCCAGTA-3'. Thirty-six cycles of amplification were run for 15 seconds at 94°C, 20 seconds at 68°C, and 30 seconds at 72°C.

Quantitative RT-PCR Analysis
Reverse transcription and quantitative real-time PCR were conducted as described in Nuttall et al. (20). Sequences for the primers and the probe specific to MT4-MMP were designed as follows: forward, 5'-GCGGGTATCCTTCCTCTACGT-3'; reverse, 5'-CAGCGACCACAAGATCGTCTT-3'; probe, 5'-FAM-ATTGTCCTTGAACACCCAGTACCTGTCTCCTTTAA-TAMRA-3'.

Western Blotting Analysis
Samples (20 µg of total cell protein extracts) were separated under reducing conditions on 10% polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (NEN, Boston, MA). Membranes were blocked overnight at 4°C with Gloria milk powder (5%, w/v), Tween 20 (0.05%, v/v) in PBS. Antigenic bands were detected by exposing the membranes to primary antibody: anti-human MT4-MMP sheep polyclonal antibody (16) or anti-MT1-MMP mouse monoclonal antibody (12), followed by incubation with a secondary horseradish peroxidase (HRP)–conjugated rabbit anti-sheep antibody or goat anti-mouse antibody (1:1,000; DakoCytomation, Glostrup, Denmark). Signals were detected using an enhanced chemiluminescence kit (Perkin-Elmer Life Sciences, Boston, MA), according to manufacturer's instructions, and actin production level was determined as loading control.

In vitro Growth Rate
MDA-MB-231 cells (104) were seeded in triplicate in 24-well plates and maintained in standard culture conditions. Fluorimetric DNA titration was done on samples harvested every day and used as an indicator of cell density (26). The average cell density from triplicate experiments was determined as a function of time.

In vitro Invasion Assay
Boyden chamber cell invasion was assayed using a Transwell cell culture chamber inserts system (Costar, Acton, MA) with 6.5-mm polycarbonate filters (8 µm pore size) as previously described (25). Briefly, 6 x 104 cells were allowed to migrate through inserts, precoated with 25 µg of Matrigel, for 20 hours. Cells having reached the lower surface of the filters were stained with Giemsa and counted in 30 random fields per insert (400-fold magnification). Each clone was tested thrice in triplicate.

ELISA for Quantitative Determination of VEGF
VEGF (VEGF165 and VEGF121 isoforms) levels were determined in 100 µL of conditioned media (dilution 1:4) in triplicate using a human VEGF DuoSet ELISA development kit (R&D Systems, Minneapolis, MN) following manufacturer's instructions. VEGF amounts were normalized to total protein concentration of conditioned media.

In vitro Angiogenesis: Mouse Aortic Rings Assay with Conditioned Media
In vitro angiogenesis was studied by culturing rings of rat aorta in three-dimensional collagen gels as previously described (25). Images were captured on a Zeiss microscope at day 6, and morphometric analysis was done on a Sun SPARC30 workstation with the software Visilog 5.0 from Noesis. After generating binary images, the number and the maximal length of the outgrown microvessels were automatically done (27).

In vivo Tumorigenicity
Subconfluent MDA-MB-231 cells were trypsinized, resuspended in serum-free medium (5 x 106 cells/mL), and mixed with an equal volume of cold Matrigel according to Noël et al. (28). Cell suspension (106 cells in 400 µL) was injected s.c. into RAG-1 immunodeficient mice (29) at both flanks (5 or 6 mice per clone in each assay). Tumor growth was assessed by measuring the length and width of tumors every 3 to 4 days. Tumor volumes were estimated using the formula: length x width2 x 0.4 (28). Results are expressed as the mean of tumor volumes for each experimental group. Tumor incidence represents the percentage of mice bearing tumor larger than 200 mm3 at day 45.

Immunohistochemistry
To unmask antigens on paraffin sections (5-µm thick), slides were incubated for 1 hour at 80°C in citrate buffer (pH 6; DAKO, Glostrup, Denmark; S2031), autoclaved for 11 minutes at 126°C in Target Retrieval Solution (DAKO; S1699) or in citrate buffer (pH 6), and microwaved for 4 x 5 minutes at 350 W in Target Retrieval Solution for MT4-MMP, Ki-67, {alpha}-smooth muscle actin (SMA), von Willebrand factor (vWF) stainings, respectively. Endogenous peroxidase was subsequently blocked by 3% H2O2/H2O for 20 minutes, and nonspecific binding was prevented by incubation in PBS/bovine serum albumin 10% (Fraction V, Acros Organics, NJ) for 1 hour (for MT4-MMP and Ki-67) or in normal goat serum for 30 minutes (for SMA and vWF). Sections were then incubated with a rabbit polyclonal anti-MT4-MMP (Sigma, St. Louis, MO; M3684, 1:300) overnight at 4°C, a mouse monoclonal anti-Ki-67 (DAKO; clone MIB-1, M7240, 1:100), a mouse monoclonal anti-SMA (DAKO; M0801, 1:100) or a rabbit polyclonal anti-vWF (DAKO; A00602, 1:200) for 1 hour at room temperature. Slides were then incubated with a HRP-conjugated second antibody (Envision System Labeled Polymer-HRP, DAKO; K4001, ready to use; for Ki-67) or with a biotinylated second antibody (DAKO; E0433, 1:400) at room temperature for 30 minutes followed by incubation with a streptavidin/HRP complex (DAKO; P0397, 1:500) at room temperature for 30 minutes (for MT4-MMP, SMA, and vWF). Coloring was done with 3-3'diaminobenzidine hydrochloride (DAKO; K3468) for 3 minutes. Slides were finally counterstained with hematoxylin and mounted with Eukitt medium for microscope observation. Omission of the first antibody served as negative control.

Quantification of MT4-MMP Production in Human Breast Cancer
For quantitative measurement of MT4-MMP immunostaining, an original automatic computer-assisted image analysis was applied on 50 representative sections by using software Aptelion 3-2 from Adsis. Staining density was determined by measuring the ratio between the surface of immunostaining and the surface of total mammary epithelium. The staining intensity (1/µm2) is defined as the sum of grey level intensities of each pixel characterizing stained region divided by surface of epithelium.

Lung Metastasis Assay From s.c. Tumors
Paraffin sections of mice lung (5 mice per clones) were immunostained for human KI-67 protein (30). Metastatic foci were counted in 65 histologic fields per section at 200-fold magnification. Incidence of metastasis is expressed as the percentage of mice with detectable metastatic cells in the lungs. Severity of metastasis was scored as minimal (score 1° = metastatic nodule involving less than three stained cells), medium (score 2° = metastatic nodule composed of 3-20 stained cells), or extensive involvement (score 3° = metastatic nodule including >20 stained cells).

Statistical Analysis
We assessed statistical differences between both experimental groups using Mann-Whitney test. Fisher's exact test was used to compare metastasis incidences. In both tests, Ps < 0.05 (*) were considered as significant. Statistical analyses were carried out using the Prism 4.0 software (GraphPad, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MT4-MMP is produced by human breast epithelial cancer cells. MT4-MMP expression levels were first determined by real-time quantitative RT-PCR analysis in 63 breast adenocarcinomas and 21 normal breast tissue samples. MT4-MMP transcripts were expressed at similar levels in breast carcinomas and normal breast tissues (Fig. 1A ). MT4-MMP production in human breast tissues was further investigated by immunohistochemistry, revealing a strong homogenous staining of tumor cells in all breast tumor samples analyzed (n = 17; Fig. 1B). Staining was detected in few interstitial cells, presumably inflammatory cells (Fig. 1B). In normal breast tissues (n = 11), epithelial cells were stained at variable intensities ranging from a negative to a moderate positivity, with myoepithelial cells being unreactive. Quantitative assessment of MT4-MMP staining was done by computer-assisted image analysis. A strong enhancement of both density and intensity staining was observed in tumor samples (Fig. 1C).


Figure 1
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  		Figure 1. Expression of MT4-MMP in human breast adenocarcinomas. A, relative mRNA expression level of MT4-MMP in 63 breast cancer and 21 normal breast samples assessed by quantitative RT-PCR. Results are expressed as ratios of MT4-MMP mRNA to 18S rRNA levels. Horizontal line, median. B, immunohistochemical detection of MT4-MMP in human breast adenocarcinoma, normal breast, and metastatic and normal lymph nodes. Breast tumor sections (breast carcinoma) displayed a strong and extensive MT4-MMP production in tumor cells (t), but not in the surrounding stroma (s).White arrow, myoepithelial cells; a, adipocytes; s, stroma; t, tumor cells; l, lymphocytes; d, ducts; m, metastatic cells; ca, node capsule; gc, germinal center; co, cortex. Left, magnification, 400-fold; bar, 50 µm. Right, magnification, 100-fold; bar, 200 µm. C, quantification of MT4-MMP immunostaining. Columns, average staining density (top) and staining intensity (bottom) were determined on 25 representative sections of each group as described in Materials and Methods; bars, SE. ***, P < 0.001.

 
Immunohistochemical analysis was also done in normal and metastatic human breast cancer lymph nodes. Interestingly, in opposite to normal human breast lymph nodes, invading cancer cells and inflammatory cells of metastatic lymph nodes (11 of 12) were strongly immunostained for MT4-MMP (Fig. 1B).

The important MT4-MMP immunostaining of tumor epithelial cells in human cancer tissues prompted us to study the contribution of this membrane-associated enzyme in cancer progression and angiogenesis. Based on the mRNA expression profile of different human breast cancer cell lines (data not shown), MDA-MB-231 cells that expressed MT4-MMP at very low levels (in accordance with refs. 19, 20, 31) were selected for transfection with human MT4-MMP cDNA.

Generation of MDA-MB-231 cells stably expressing MT4-MMP. Parental MDA-MB-231 cells were stably transfected with control pcDNA3-neo vector (clones C) or with pcDNA3-neo containing human MT4-MMP cDNA (clones M). Four clones overexpressing MT4-MMP (M2, M3, M10, and M14) and four control clones (C4, C12, C14, and C15) were selected according to MT4-MMP expression at mRNA (Fig. 2A ) and protein (Fig. 2B) levels. MT4-MMP overexpressing clones were found to stably express MT4-MMP. Western blot analysis revealed two bands at the expected molecular weight (~64 kDa), with the upper one being the pro form and the lower one being the active form (16). Clone M2, which was characterized by the highest MT4-MMP production (Fig. 2), was confirmed to express both active and pro forms by loading a lower amount of protein in Western blot analysis. In all selected clones, MT1-MMP protein expression was not affected by transfection with MT4-MMP cDNA (data not shown).


Figure 2
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  		Figure 2. Analysis of MT4-MMP expression in MDA-MB-231 clones stably transfected with MT4-MMP cDNA (M2, M3, M10, and M14) or with pcDNA3-neo control vector (C4, C12, C14, and C15). A, RT-PCR analysis of MT4-MMP mRNA level. Expression of 28S rRNA is shown as loading control. B, Western blot analysis of MT4-MMP protein. Total protein extracts of clones were analyzed using an anti-human MT4-MMP sheep polyclonal antibody raised against MT4-MMP catalytic domain. Actin production level in transfectants is shown as loading control.

 
MT4-MMP overexpression does not affect in vitro cell proliferation or invasion. Clones were then compared for their in vitro proliferation and invasive capacities through different matrices. MT4-MMP overexpression did not significantly affect in vitro growth rate as illustrated in Fig. 3A (P > 0.05). The in vitro invasion potential of all clones was then evaluated in Boyden chambers by using inserts coated with Matrigel. The numbers of invading cells was independent of MT4-MMP production (P > 0.05; data not shown). Similarly, the capacity of MT4-MMP transfectants to migrate through gelatin-coated inserts or to invade type I collagen gel-coated inserts did not differ to that of control clones (data not shown).


Figure 3
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  		Figure 3. Characterization of in vitro and in vivo properties of four MDA-MB-231 clones stably transfected with MT4-MMP cDNA (black symbols, continued lines, clones M) or with pcDNA3-neo control vector (white symbols, dotted lines, clones C). A, in vitro growth curves of transfectants. Clones were seeded in triplicate in 24-well plates and harvested each day for DNA content evaluation. Points, mean of DNA concentration of triplicates of one representative experiment out of three; bars, SE. B and C, in vivo tumor growth curves of the transfectants. Clones were s.c. injected into RAG-1–/– mice in three independent assays, and tumor volumes were assessed as described in Materials and Methods. Two representative independent experiments, in which MT4-MMP clones: M2, M3, M14 (B) and M2, M3, M10 (C) and control clones: C4, C12, and C15 (B) and C4 and C14 (C) were tested. Points, mean of tumor volumes for each experimental group; bars, SE. ***, P < 0.001; **, P < 0.01; *, P < 0.05.

 
Gelatin zymography (data not shown) revealed that MT4-MMP production by MDA-MB-231 clones, either treated or not with concanavalin-A, did not lead to a higher activation of exogenous pro-MMP2 (consistent with ref. 16).

MT4-MMP overexpression does not affect in vitro angiogenesis or VEGF expression. Because MT1-MMP has been shown to affect in vitro and in vivo angiogenesis through a modulation of VEGF-A expression (12), we measured VEGF production of all clones by RT-PCR analysis and ELISA. VEGF mRNA levels in clones expressing MT4-MMP were undistinguishable from those of control clones (data not shown). Furthermore, similar amounts of proteins were detected by ELISA in medium conditioned by MT4-MMP overexpressing clones (1,365 ± 100 pg/mL) and control clones (1,369 ± 344 pg/mL). Similarly, RT-PCR analysis of other angiogenic molecules (VEGF-B, VEGF-C, VEGF-D, and PlGF) did not reveal any difference between clones expressing or not MT4-MMP (data not shown).

In addition, we investigated the ability of selected clones to modulate in vitro angiogenesis in the rat aortic ring assay. Rat aortic rings were embedded in a collagen gel and maintained in MCDB supplemented with media conditioned by transfectants (1:6, v/v). After 6 days of incubation, computer-assisted image analysis was applied to determine geometric and morphologic variables (number of vessels = Nv and maximal length of vessels = Lmax; ref. 27). These variables did not differ in aortic ring cultures supplemented with medium conditioned by cells expressing or not MT4-MMP (Nv = 26.5 ± 5 for MT4-MMP clones versus 29 ± 2 for control clones; Lmax = 614 ± 46 µm for MT4 clones versus 543 ± 40 µm for control clones).

MT4-MMP overexpression promotes in vivo primary tumor growth. RAG-1 immunodeficient mice were s.c. injected with the different MT4-MMP expressing or control clones in three separate sets of experiments. Tumor incidence and tumor growth were monitored from the 3rd to the 6th week after injection. MT4-MMP overexpression resulted in increased tumor incidence and tumor growth (Fig. 3B and C). At day 45, tumor incidence was 96% (46 of 48 tumors) in MT4-MMP expressing tumors and 50% (19 of 38 tumors) in control tumors. Thirty-two days after cell injection, all tumors expressing MT4-MMP reached higher volumes compared with control tumors (P < 0.05; Fig. 3B and C). When considering all independent experiments together, the mean tumor volumes reached at the end of the assays ranged from 639 to 1,340 mm3 for MT4-MMP–transfected cells and from 54 to 486 mm3 for control clones (P < 0.05).

Considering the previously described involvement of MT1-MMP in tumor angiogenesis (25), we further investigated whether MT4-MMP could affect tumor vascularization (Fig. 4B ). No obvious difference in vessel density was detected between experimental groups. However, interestingly, MT4-MMP production affected vessel shape and size, leading to an important enlargement of i.t. blood vessels. Immunostaining for {alpha}-SMA revealed the presence of pericytes surrounding enlarged vessels of MT4-MMP expressing tumors as well as collapsed and small vessels presenting a lumen in control tumors (Fig. 4C).


Figure 4
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  		Figure 4. Immunohistochemical analysis of primary tumors. Tumors were induced by s.c. injection of MT4-MMP–transfected MDA-MB-231 clones (MT4 clones, left) or control clones (CTR clones, right) into RAG-1–/– mice. Tumor sections were immunostained by using antibodies raised against (A) MT4-MMP, (B) vWF, (C) {alpha}-SMA. Magnification, 200-fold. Bar, 100 µm. B and C, serial sections for both experimental groups.

 
MT4-MMP overexpression is associated with an induction of lung metastases. Metastatic ability of all transfectants injected in RAG-1–/– mice was assessed by histochemical and immunohistochemical analyses of lungs harvested 45 days after tumor cell inoculation. Numerous metastatic nodules were easily distinguishable in lungs of mice inoculated with MT4-MMP-expressing cells but not in those of mice injected with control clones (Fig. 5A ). To detect small metastatic foci, lung sections were immunostained with an antibody raised against human Ki-67 protein and detecting proliferative cancer cells (Fig. 5A and B). Incidence and severity of metastases were determined by considering the size of metastatic foci (number of cells per foci; Table 1 ). All clones overexpressing MT4-MMP induced lung metastasis in 100% of injected animals. In sharp contrast, when considering control clones, metastasis incidence varied from 0% to 100% (Table 1A). The severity of metastases was also strongly increased in mice injected with MT4-MMP expressing cells. Score 3° metastatic nodules (composed of >20 cells) were only detected after injection of MT4-MMP transfectants (Table 1; Fig. 5A), whereas metastatic foci induced by inoculation of control clones were scored 1° (dispersed cells) or 2° (composed of <20 cells). In addition, MT4-MMP transfectants showed a significant increase in the number of metastatic foci (P < 0.05; Fig. 5D). When considering all clones together, it clearly appeared that both incidence and severity of metastasis were significantly higher in mice injected with MT4-MMP expressing clones compared with control clones (P < 0.001). Immunohistochemistry further confirmed MT4-MMP expression in lung metastases produced by MT4-MMP–transfected clones (Fig. 5C) and in the corresponding primary tumors (Fig. 4A).


Figure 5
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  		Figure 5. Immunodetection and quantification of lung metastases. MDA-MB-231 clones expressing MT4-MMP (MT4 clones, left) or not (CTR clones, right) were s.c. injected into RAG-1–/– mice (5-6 mice per clones) in two independent assays. At day 45, lungs were collected, formalin fixed, and paraffin embedded. A and B, metastatic nodules were immunodetected in lung tissue sections with an antibody raised against human Ki-67 protein and scored 1° (white arrow), 2° (gray arrow), or 3° (black arrow) as described in Materials and Methods. C, immunostaining analysis confirmed MT4-MMP production in metastases induced by MT4-MMP–transfected cells (left). A, magnification, 100-fold. Bar, 200 µm. B and C, magnification, 200-fold. Bar, 100 µm. B and C, serial sections for both experimental groups. bv, blood vessel. D, for each clone, metastatic foci were counted in 65 histologic fields at 200-fold magnification. Severity of metastasis was scored as illustrated in (A). Columns, average number of metastatic nodules detected per mice for both experimental groups; bars, SE. Cells transfected with MT4-MMP show a significant increase in number and severity of metastasis. Control clones never produced score 3° metastasis. ***, P < 0.001; *, P < 0.05.

 

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  		Table 1. Effect of MT4-MMP on incidence and severity of lung metastasis in RAG-1–/– mice bearing s.c. tumors of MDA-MB-231 clones

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We investigated the potential implication of the GPI-anchored MT4-MMP in breast cancer progression. This study provides the first direct evidence for a pivotal role of MT4-MMP in primary breast cancer growth, leading to metastatic dissemination. Several lines of evidence support these conclusions. First, MT4-MMP is produced at higher levels in human breast cancer tissues than in normal breasts. Second, MT4-MMP overexpression in MDA-MB-231 cells resulted in an enhanced tumor incidence, in an acceleration of primary tumor growth with subsequent apparition of lung metastases, and in an enlargement of i.t. blood vessels. Third, the involvement of MT4-MMP in metastatic dissemination is suggested by its expression in human metastatic cells originating from human breast cancers.

Quantitative RT-PCR analyses were first done on human normal breast and mammary adenocarcinoma samples. MT4-MMP mRNA was found expressed in all human breast adenocarcinoma samples but at similar levels as in normal breast samples. However, interestingly, immunohistochemical analyses revealed a very high and homogenous production of MT4-MMP protein in human breast adenocarcinomas and metastatic lymph nodes, which is consistent with its original isolation from a human breast adenocarcinoma source (19). Although MT1-MMP was described as a stromal MMP (32–34), MT4-MMP expression seems restricted to cancerous epithelial cells with some staining in inflammatory cells. Quantitative analysis revealed a 3- to 4-fold increase of MT4-MMP staining density and intensity in tumor samples compared with normal breast tissues in which a faint and irregular immunostaining was observed. The absence of a correlation between MT4-MMP mRNA levels and tumor development might seem disappointing. However, this observation is consistent with previous studies done on human prostate cancers (21) and mammary carcinomas of PyMT mice (transgenic for polyoma virus middle T antigen under the control of the mouse mammary tumor virus promoter; ref. 35). The discrepancy between RT-PCR and immunohistochemical results could be related to mRNA heterogeneities of samples used for total RNA extractions, whereas tissue sections allow a better localization of the molecule of interest. In addition, MT4-MMP production could be controlled at posttranscriptional and/or posttranslational levels. Increased production of MT4-MMP in tumors could be related to modification of mRNA translation rate or reduction of MT4-MMP degradation, cell surface shedding, and/or internalization. Evidences exist indicating that members of MT-MMP subfamily can be internalized (36, 37) or shed from the cell membrane (5, 38). Dissection of molecular mechanisms leading to a regulation of MT4-MMP protein requires further investigations. Therefore, the increased MT4-MMP protein production in human breast adenocarcinomas might suggest a specific function of MT4-MMP in human breast cancer progression and prompted us to evaluate the effect of MT4-MMP overexpression in human breast cancer cell lines.

We next transfected human breast cancer MDA-MB-231 cells with MT4-MMP cDNA. Four clones stably expressing this MT-MMP and four control clones were characterized in vitro and in vivo. Although MT4-MMP overexpression in MDA-MB-231 cells does not significantly affect their in vitro proliferation or in vitro invasiveness in the different matrices tested, it strongly increases their in vivo tumorigenicity with subsequent apparition of lung metastases when s.c. injected into RAG-1 immunodeficient mice. Using human Ki-67 protein as an indicator of metastasis (30), we indeed observed that MT4-MMP overexpression was associated with an increase of both the number and severity of lung metastases. MT1-MMP (39) and pro-MMP-2 (40) overexpressed in cancer cells were already reported to increase experimental lung metastases by i.v. and i.c. injections, respectively. Here, we show that MT4-MMP overexpressed in breast cancer cells can lead directly or indirectly to in vivo lung metastases after s.c. injection. In addition to establishing a new association between MT-MMPs and cancer, our MT4-MMP–transfected cells provide a relevant model of lung metastases from primary tumor and involving all steps implicated in metastatic dissemination, such as intravastion, extravasation, and migration (41). Further studies are required to determine which steps of cancer progression are affected by MT4-MMP.

The molecular mechanisms of MT4-MMP action in tumor progression are unknown. Its localization at the cell surface raised the possibility that it controls pericellular proteolysis and participates directly or indirectly to intracellular signaling events. Alternatively, MT4-MMP could be involved in tissue remodeling associated to tumor growth and metastatic spread of cancer cells. In this context, it is worth noting that a role for MT4-MMP in cartilage aggrecanolysis through ADAMTS-4 activation has been described (22). In addition, interleukin-1 (IL-1) has been shown to induce MT4-MMP with subsequent processing of ADAMTS-4 in IL-1-mediated aggrecanolysis of bovine cartilage (23). As assessed by quantitative RT-PCR, no ADAMTS-4 mRNAs were detected in our cultured transfectants or their corresponding in vivo xenografts (data not shown). Nevertheless, the lack of ADAMTS-4 detection in established primary tumors does not negate the possibility that the ADAMTS-4/MT4-MMP pathway contributes to earlier steps of cancer development. In addition, we can not exclude a possible implication of this pathway in host-tumor interface in tumor and metastasis microenvironments.

Shedding of MT4-MMP offers an alternative way to contribute to biological processes at a distance from the producing cells. Indeed, in accordance with the data of Itoh et al. (38), we detected large amounts of MT4-MMP protein shed in the supernatant of cells expressing MT4-MMP (data not shown). In contrast to our and other data previously reported for MT1-MMP (10, 25), the tumor-promoting effect of MT4-MMP could not be associated to higher in vitro invasive capacities, as assessed in Boyden chamber assays using Matrigel and collagen matrices. In addition, consistent with the previous report of English et al. (16), MT4-MMP production does not affect the in vitro capacity to activate exogenous pro-MMP-2, known to participate in cell invasion. These observations suggest that MT4-MMP does not contribute to cell invasion through direct extracellular matrix degradation or that it could degrade some substrates via other molecules that are absent in vitro. Alternatively, in vivo MT4-MMP activities could require other factors, such as for instances, inflammatory mediators, or growth factors (16, 19). Our original findings of MT4-MMP protein overproduction by epithelial cancerous cells of breast adenocarcinomas and metastastic lymph nodes, in addition to its detection in inflammatory cells (the present study and refs. 16, 19, 42), extend the putative functions of MT4-MMP and suggest a dual role in both inflammatory responses and cancer processes for MT4-MMP.

The higher in vivo tumor growth induced by MT4-MMP expression could not be explained by a direct angiogenic effect as described for MT1-MMP (13, 25). We previously reported that MT1-MMP tumor promoting effect could be linked, at least in part, to an up-regulation of VEGF at a transcriptional levels (12). However, in contrast to MT1-MMP, MT4-MMP overexpressed in breast cancer cells fails to increase in vitro VEGF expression. In addition, medium conditioned by transfected cells is not able to enhance in vitro angiogenesis in the aortic ring assay. Nevertheless, MT4-MMP production modulates vasculature architecture. In vivo, MT4-MMP induces enlargement of i.t. blood vessels, which were covered by pericytes reflecting their maturity (43). Altogether, our findings suggest that MT4-MMP contribution to cancer progression involves other pathways that those activated by MT1-MMP.

In conclusion, our data clearly show a pivotal role of MT4-MMP in human breast cancer growth ending up to metastatic dissemination. Both MT1-MMP and MT4-MMP contribute to cancer progression, but their mechanisms of action are divergent. These findings have direct implication for development of new anticancer strategies by highlighting the importance to target different MT-MMPs.


   Acknowledgments
 
Grant support: Communauté française de Belgique, Actions de Recherches Concertées; Commission of European Communities grants FP5 and FP6; Fonds de la Recherche Scientifique Médicale; Fonds National de la Recherche Scientifique, Belgium; Fédération Belge Contre le Cancer; Fonds spéciaux de la Recherche, University of Liège; Centre Anticancéreux près l'Université de Liège; Fortis Banque Assurances; Fondation Léon Fredericq, University of Liège; D.G.T.R.E. from the Région Wallonne; Fonds d'Investissements de la Recherche Scientifique, CHU, Liège, Belgium; Interuniversity Attraction Poles Programme/Belgian Science Policy, Brussels, Belgium; Cancer Research UK (G. Murphy); Medical Research Council, United Kingdom (G. Murphy); and Fonds National de la Recherche Scientifique-Télévie (V. Chabottaux).

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.

We thank F. Olivier, N. Lefin, M-L. Alvarez, G. Roland, I. Dasoul, P. Gavitelli, and E. Feyereisen for their technical help.


   Footnotes
 
Note: E. Maquoi, C. Gilles, and C. Munaut are Research Associates, and F. van den Brûle is Senior Research Associate, all from Fonds National de la Recherche Scientifique, Belgium. W.R. English is an Intermediate Fellow of the British Heart Foundation.

Received 8/23/05. Revised 2/24/06. Accepted 3/14/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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