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Extracellular Matrix Metalloproteinase Inducer Up-regulates the Urokinase-Type Plasminogen Activator System Promoting Tumor Cell Invasion

¹ Institut National de la Sante et de la Recherche Medicale U716; ² Université Paris 7- IUH; ³ Laboratory of Pharmacology, AP-HP, Hôpital Saint-Louis; ⁴ Fondation A. de Rothschild, Department of Ophthalmology, Paris, France; and ⁵ Laboratoire de Recherche sur la Croissance Cellulaire la Reparation et la Régéneration Tissulaire, Centre National de la Recherche Scientifique UMR 7149, Université Paris XII, Créteil, France

Requests for reprints: Samia Mourah, Laboratoire de Pharmacologie and Institut National de la Sante et de la Recherche Medicale U716, Hôpital Saint-Louis, 27, rue Juliette Dodu, 75010 Paris, France. Phone: 33-1-42-49-48-85; Fax: 33-1-42-49-49-89; E-mail: samia.mourah{at}sls.aphp.fr.

	Abstract

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Extracellular matrix metalloproteinase inducer (EMMPRIN) is a membrane glycoprotein overexpressed in many cancer tissues and is known for its ability to stimulate MMP expression. In this work, we show that EMMPRIN is also a regulator of the urokinase-type plasminogen activation (uPA) system of serine proteases, thus participating to the increase of the overall proteolytic function of the cancer cells. Enhanced EMMPRIN expression in a tumorigenic breast epithelial cell line NS2T2A increased the levels of uPA, uPA receptor, and the uPA inhibitor plasminogen activator inhibitor-1 (PAI-1), as measured by quantitative reverse transcription-PCR, Western blot, and plasminogen-casein zymography. This response was down-regulated by either EMMPRIN small interfering RNA or a blocking antibody to EMMPRIN. EMMPRIN-containing purified membrane fraction from Chinese hamster ovary cells when added exogenously to NS2T2A cells induced a similar activation of the uPA/PAI-1 system. Additionally, overexpression of EMMPRIN in NS2T2A cells increased uPA levels in cocultured endothelial cells, showing a paracrine regulation loop involving a tumor-stroma interaction. EMMPRIN-expressing cells also exhibited enhanced invasive potential in vitro, and the use of amiloride (uPA inhibitor) and marimastat (MMP inhibitor) showed that the two proteolytic systems reduced alone and in combination the invasive potential mediated through EMMPRIN. These data show a novel regulatory pathway for uPA activity and suggest that EMMPRIN is involved in uPA dysregulation observed in cancer. [Cancer Res 2007;67(1):9–15]

	Introduction

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A major feature of cancer cells is their ability to migrate and to invade and develop in surrounding or distant tissues. During this metastatic process, tumor cells detach from their basal membrane; migrate across the extracellular matrix barrier, including the vascular wall; and colonize new sites, through multiple protease-mediated events.

Among the proteases shown to contribute to matrix degradation and invasion, the urokinase-type plasminogen activation (uPA) system to which contribute uPA, its specific receptor uPAR, and its primary inhibitor plasminogen activator inhibitor-1 (PAI-1) has been shown to be key actors (1). uPA catalyzes the conversion of the inactive zymogen plasminogen to the active broad-spectrum plasmin, which degrades a number of matrix proteins and also activates other proteases, including some matrix metalloproteinases (MMP) (2).

The implication of the uPA system in tumor invasion is supported by in vitro experiments with cultured cells and in vivo in animal tumor models (1). This has been confirmed in breast cancer patients where elevated uPA levels in primary tumor extracts correlate with poor prognosis (3). Reducing uPAR levels using antisense oligonucleotides or small interfering RNA (siRNA) provided further evidence because they inhibited tumor growth, invasion, and metastasis in several experimental cancer models (4, 5). In other studies, targeted disruption of the uPA or plasminogen genes supported the crucial role of uPA-catalyzed plasminogen activation in tumor progression towards the metastatic phenotype (6, 7). These studies also revealed the importance of the stromal response to the development of the transplanted tumor. Indeed, immunolocalization of this proteolytic system in human breast cancer tissues by in situ hybridization and immunohistochemistry showed that uPA as well as uPAR mRNA and protein are also expressed by the stromal cells surrounding the tumors (8). The fibroblasts surrounding the cancer cells were also shown to overexpress MMPs, supporting their role in tumor invasion process (9).

CD147/extracellular MMP inducer (EMMPRIN), a highly glycosylated member of the immunoglobulin super family, has been identified as a cell surface inducer of MMPs both in tumor and stromal cells (10–12). High levels of EMMPRIN were reported in numerous malignant tumors, including breast carcinoma, and were related to tumor progression (13, 14). Increased EMMPRIN levels in cancer cells have not only been linked to deregulation of epidermal growth factor receptor (EGFR) signaling (15) but may also be a response to transforming growth factor-ß stimulation (16). The pathologic consequences of such elevated EMMPRIN expression in tumor growth and invasion were directly evidenced in vivo when the transfection of EMMPRIN cDNA into human MDA-MB436 breast cancer cells resulted in a marked enhancement of tumor growth and metastasis in nude mice (17).

The implication of uPA in the tumor invasive processes and its localization in the tumor-stroma invasive front led us to investigate whether this serine protease system can, like the MMPs, be regulated by tumor EMMPRIN. In this study, we used a tumorigenic human breast epithelial cell line NS2T2A to show that the serine protease uPA system was up-regulated by EMMPRIN, thus contributing to the invasive promoting activity of EMMPRIN. These data show a novel regulatory mechanism for uPA and suggest that EMMPRIN has a key role in the activation of this proteolytic system observed in cancer tissues.

	Materials and Methods

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Cell culture and membrane preparation. Transformed NS2T2A cell line was obtained following immortalization with SV40 T antigen of normal human breast epithelial cells and selection by successive passages in nude mice. The cells were cultured as previously described (15). The microvascular endothelial cell line HMEC-1 was from Dr. Thomas J. Lawley (Atlanta, GA). MDA-MB-231, Malme-3M, and Chinese hamster ovary (CHO) cell line were from the American Type Culture Collection (Rockville, MB). CHO and CHO-EMMPRIN membranes were isolated by differential centrifugation as previously described (16).

Vector construction and cell transfection. EMMPRIN full-length cDNA was cloned into pCR3.1 expression vector (Invitrogen, San Diego, CA) and confirmed by DNA sequencing. Plasmid DNA transfections into NS2T2A and CHO cells were done using FuGENE-6 (Roche, Indianapolis, IN). After 48 h, the cells were cultured in medium containing 5% serum and 250 µg/mL G418 for screening. The stably transfected cells were designated as NS2T2A-Emp and CHO-Emp cells.

siRNA transfection. Two different EMMPRIN siRNA oligos (Ambion, Austin, TX) or scrambled siRNA oligos (33 nmol/L) were transfected into the cells using the BLOCK-iT transfection kit and LipofectAMINE 2000 (Invitrogen). Cells were then incubated for 24 h before reverse transcription-PCR (RT-PCR), Western blotting, and invasion assays and for a further 24 h in serum-free medium for zymographic analysis.

Western blotting analysis. Western blot was done as described previously (16). Membranes were immunoblotted with anti-EMMPRIN/CD-147 HIM6 monoclonal antibody (mAb; BD Biosciences, San Jose, CA), anti-uPAR, mAb R2 (Dr. N. Brûnner, Copenhagen, Denmark), or anti-PAI-1 polyclonal antibody (pAb; Calbiochem, La Jolla, CA). Proteins were visualized with enhanced chemiluminescence reagent (Pierce, Rockford, IL).

Zymographic analysis. Gelatin or casein zymography was done as described previously (16, 18). Serum-free conditioned media were analyzed on 10% SDS-PAGE gels containing either 1 mg/mL gelatin for gelatinase activity or 2 mg/mL casein (Sigma, St. Louis, MO) and 10 µg/mL plasminogen (Calbiochem) for uPA activity.

Real-time quantitative RT-PCR. The cDNAs for EMMPRIN, uPA, PAI-1, uPAR, MMP-2, MMP-9, and tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA were cloned using TOPO II TA cloning kit (Invitrogen). Quantitative RT-PCR was done using LightCycler (Roche). Data were normalized to the TBP housekeeping gene transcripts. Primers and probes are available upon request.

Invasion assay. In vitro invasion was assessed using a modified Boyden chamber assay (19). When used, the uPA inhibitor amiloride (20 nmol/L; Sigma) or the MMP inhibitor marimastat (10 µmol/L; British Biotechnology, Oxford, United Kingdom) were added together with the cells. After 48 h of incubation, cells were fixed, stained with Diff Quik (Dade Behring, Deerfield, IL), and counted under a microscope.

Animal experiments. nu/nu mice (Janvier, Le Genest Saint-Isle, France) were purchased at 4 weeks of age. Suspensions of NS2T2A-Emp or NS2T2A-mock cells (4 x 10⁶ in 100 µL PBS) were s.c. injected into the left side of six nude mice per group. Tumor sizes were measured every 5 days, and mice were sacrificed at 5 weeks after injection. Tumors were stored in liquid nitrogen before pathologic studies, immunohistochemistry, and zymography.

Confocal immunohistochemistry. Tumor cryostat sections (10 µm) were immunostained with anti-EMMPRIN/CD-147 HIM6 mAb, anti-PAI-1 pAb, anti-uPAR mAb, and anti-EMMPRIN pAb as previously described (16). After incubation with the secondary antibody (conjugated affinity-purified donkey anti-mouse and anti-rabbit or anti-goat IgG, Alexa 488 and 594, respectively), the slides were examined with a laser-scanning confocal microscope (Leica Lasertechnik, Heidelberg, Germany). In negative controls, the primary antibody was substituted with PBS.

Statistical analysis. Data are expressed as mean ± SD. Mann-Whitney and Student's t test were used to compare differences between groups in various experiments.

	Results

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EMMPRIN up-regulates uPA, uPAR, and PAI-1 in NS2T2A breast tumor cells. To evaluate the effect of EMMPRIN on uPA proteolytic system, we stably transfected transformed human mammary epithelial cells NS2T2A with full-length EMMPRIN cDNA (Fig. 1A ). NS2T2A-Emp cells, as expected, showed an increased activity of MMP-2 and MMP-9 but not of TIMP-1 (10). Figure 1B shows that EMMPRIN increased uPA, PAI-1, and uPAR expression compared with nontransfected and mock-transfected controls. This up-regulation was observed both at the protein level and activity as measured by Western blot (1.6-fold) and zymography (2.5-fold) and RNA levels as measured by quantitative RT-PCR (for uPA, uPAR, and PAI-1: 2-, 1.5-, and 3.5-fold, respectively).

View larger version (37K): [in this window] [in a new window] [Download PPT slide]   Figure 1. EMMPRIN up-regulates uPA, uPAR, and PAI-1 in human breast epithelial tumor cells. A, a, EMMPRIN transfection was confirmed by Western blot (WB; actin was used as a loading control; representative experiment of three). b, NS2T2A, NS2T2A-mock, and NS2T2A-Emp cells were cultured in serum-free media. After 24 h, conditioned media were harvested for MMP-2 and MMP-9 gelatinase zymography. c, EMMPRIN, MMP-2, MMP-9, and TIMP-1 transcripts were quantified using quantitative RT-PCR. Columns, mean of relative expression to TBP housekeeping gene of at least three independent experiments; bars, SD. B, a, uPA activity in the serum-free conditioned media of NS2T2A, NS2T2A-mock, and NS2T2A-Emp cells was assayed by casein-plasminogen zymography. PAI-1 and uPAR were evaluated by Western blot analysis of 10 µg cell lysates from NS2T2A, NS2T2A-mock, and NS2T2A-Emp (actin was used as a loading control). Representative experiment of three. b, uPA, PAI-1, and uPAR transcript levels were quantified by quantitative RT-PCR. Columns, mean of relative expression to TBP housekeeping gene of three independent experiments; bars, SD. C, a, CHO cells were transfected with EMMPRIN as described in Materials and Methods. Membranes were isolated from CHO-Emp and CHO control cells (CHO-Emp Mb and CHO Mb, respectively) by differential centrifugation, and 10 µg of membrane extract were analyzed for EMMPRIN content by immunoblotting. b, NS2T2A cells (80% confluence) were incubated with 20 µg/mL CHO Mb or CHO-Emp Mb for 24 h, and uPA activity was analyzed using casein zymography (one representative zymography of three independent experiments). c, uPAR protein expression was analyzed by Western blot (actin was used as a loading control; representative experiment of three). d, NS2T2A cells (80% confluence) were incubated with 20 µg/mL CHO-Emp Mb for 30 min and 1 and 2 h in serum-free medium. uPA, uPAR, and PAI-1 transcripts were quantified by quantitative RT-PCR. Columns, mean of at least three different experiments; bars, SD. For Western blot and zymography, densitometric quantitation was done. Columns, average from at least three independent experiments. *, P < 0.05; **, P < 0.01, significant difference.

We then used RNA silencing to evaluate the specificity of these effects on the uPA system. EMMPRIN siRNA transfection of the NS2T2A-Emp cells down-regulated EMMPRIN expression both at the protein and mRNA level by 70%. This was associated with a clear decrease in the mRNA levels of uPA (55%), uPAR (65%), and PAI-1 (41%), but not of TIMP-1, compared with their levels in the scrambled control siRNA-transfected cells (Fig. 2A ). A decrease in the secreted uPA activity was also observed (50%). These inhibitions were greater than those obtained in the nontransfected NS2T2A cells when treated with siRNA (65% EMMPRIN inhibition leading to 40%, 40%, and 30% reduction in uPA, uPAR, and PAI-1, respectively; Fig. 2A). An alternative inhibition strategy using blocking anti-EMMPRIN antibodies confirmed these EMMPRIN-mediated effects (only uPA activity is shown).

View larger version (46K): [in this window] [in a new window] [Download PPT slide]   Figure 2. EMMPRIN siRNA inhibits the autocrine and paracrine regulation of the uPA system. A, a, NS2T2A and NS2T2A-Emp cells were transfected for 12 or 48 h with two different EMMPRIN siRNA (Ambion siRNA 1 ID 1608981 and 2 ID 160897) or their corresponding scrambled control siRNA at 33 nmol/L concentration. EMMPRIN protein expression was evaluated by Western blot analysis of 10 µg cell lysates (actin was used as a loading control; representative blot of three independent experiments), and conditioned medium was harvested for uPA casein zymography. b, quantitative RT-PCR analyses of EMMPRIN, uPA, PAI-1, uPAR, and TIMP-1. Columns, mean of relative expression to TBP housekeeping gene of at least three independent experiments; bars, SD. *, P < 0.05; **, P < 0.01, significant difference between the expression values of siRNA or scrambled control siRNA. c, NS2T2A-Emp cells were incubated for 24 h with 20 µg/mL anti-EMMPRIN blocking antibody (Ancell, Bayport, MN) or with an IgG control antibody in serum-free medium, after which conditioned medium was harvested for uPA casein zymography. B, a, NS2T2A-Emp (1 x 105 per well) was allowed to adhere, after which cells were transfected or not with the EMMPRIN siRNA or its corresponding scrambled control siRNA. Human microvascular endothelial cells (HMEC-1) were then added and allowed to attach in the same medium for an additional 6 h. The cells were then incubated for 48 h in serum-free DMEM, and the conditioned media were harvested for uPA casein zymography. b, HMEC-1 cells were incubated with CHO membranes containing or not EMMPRIN. Conditioned media were analyzed by casein zymography for uPA activity. CHO-Emp membranes alone do not contain endogenous uPA activity. For Western blot and zymography, densitometric quantitation was done. Columns, average from three separate experiments; bars, SD. C, EMMPRIN induces uPA in other tumor cell lines: MDA-MB-231 and Malme-3M cells (breast cancer cell line and melanoma cell line, respectively). a, cells were transfected with the two EMMPRIN siRNA (see above) or scrambled control siRNA. EMMPRIN protein expression was evaluated by Western blot analysis (actin was used as a loading control). b, quantitative RT-PCR analyses of EMMPRIN, uPA, and TIMP-1. Columns, mean of relative expression to TBP housekeeping gene of at least three independent experiments; bars, SD. *, P < 0.05; **, P < 0.01, significant difference between the expression values of siRNA or scrambled control siRNA. c, MDA-MB-231 and Malme-3M cells incubated with 20 µg/mL CHO or CHO-Emp Mb or transfected by either EMMPRIN siRNA or scrambled control. Conditioned media were harvested for uPA casein zymography.

We have also shown that EMMPRIN (contained in CHO membranes) up-regulated uPA, and that EMMPRIN siRNA reduced uPA expression and activity in two other cell lines, the breast cancer MDA-MB231 and the melanoma Malme-3M cell lines (Fig. 2C), showing that EMMPRIN regulation of uPA is not limited to a specific cell line but represents a more common mechanism.

EMMPRIN increases in vitro tumor cell invasion through both uPA and MMP activities. EMMPRIN has already been implicated in tumor cell invasion (10). This was confirmed in our in vitro experiments using a Matrigel invasion assay, showing a mean 35% increased invasion in the NS2T2A-Emp cells compared with mock-transfected cells (Fig. 3A ). EMMPRIN siRNA reduced cell invasion in NS2T2A by 37% and in EMMPRIN-overexpressing cells by 59% (Fig. 3B). We then sought to evaluate in this model the relative contribution of the uPA and MMP systems, both induced by EMMPRIN, to the invasion capacity of the tumor cells. We conducted a series of experiments using amiloride at 20 nmol/L for uPA inhibition and marimastat at 10 µmol/L for a global MMP inhibition. When used separately, amiloride or marimastat caused a similar inhibition in the cell invasion of NS2T2A-Emp (mean reduction of 42% and 35%, respectively). These two inhibitors in combination caused a further invasion reduction reaching 74%, showing that both systems contribute to EMMPRIN-mediated invasion (Fig. 3B).

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Figure 3. EMMPRIN siRNA reduces tumor cell invasion: implication of uPA and MMP activities. A, the in vitro invasive property of the transfected cells NS2T2A-mock or NS2T2A-Emp was compared using tissue culture Transwell inserts (8-mm pore size; BD Biosciences) placed in a 24-well culture plate. Cells (1 x 105) were suspended in serum-free DMEM/F12 and seeded into the upper well of each insert onto membranes coated with growth factor–reduced Matrigel (BD Biosciences). After 48 h of incubation, cells that remained in the top compartment were removed by cotton swabs, and cells on the underside of insert filters were fixed, stained, and counted under a microscope. B, effect of EMMPRIN siRNA on the invasion of NS2T2A (left) and NS2T2A-Emp cells (right). NS2T2A-Emp cells were also treated with uPA inhibitor amiloride and MMP inhibitor marimastat. Amiloride (20 nmol/L) and marimastat (10 µmol/L) were added to the upper compartment together with the cells. Representative photographs of invaded cells on the downside face of the membrane. *, P < 0.05; **, P < 0.01, significant difference.

View larger version (101K): [in this window] [in a new window] [Download PPT slide]   Figure 4. EMMPRIN, uPA, uPAR, and PAI-1 in NS2T2A-Emp–derived tumors. A, nu/nu mice were s.c. injected with a suspension of NS2T2A-Emp or NS2T2A-mock cells. After 5 weeks, mice were sacrificed, and tumor volumes were determined according to the formula: V = 0.4 x A x B2, where A is the largest dimension of the tumor, and B is the smallest dimension. Columns, mean observed in six animals in each group injected with 4 x 106 transfected cancer cells; bars, SD. *, P < 0.05, significant difference. B, zymographic analysis of uPA and MMP expression in extracts of cryostat sections of tumors derived from NS2T2A-Emp or NS2T2A-mock cell. Total proteins (10 µg) were loaded on casein and gelatin zymographies (two tumor extracts). C, tissue sections from tumors were subjected to double-labeled confocal immunohistochemistry using mouse anti-human CD147 mAb (a and c; green), rabbit anti human PAI-1 pAb (b and c; red), goat anti-human EMMPRIN pAb antibodies (d and f; green), and mouse anti-human uPAR mAb (e and f; red) and counterstained with DAPI (c and f; blue). Level of staining seemed heterogeneous for each of the proteins, showing regions of particularly high staining, more noticeable at the tumor edge. Merged staining of EMMPRIN and PAI-1 (c; yellow) revealed areas of colocalization, particularly at the cell surface, whereas an intracellular staining of PAI-1 was frequently noted in cells with surface expression of EMMPRIN. The cell surface staining of uPAR and EMMPRIN, when merged, displayed frequent colocalization (f; yellow), in particular at the tumor edge. Bar, 50 µm.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The up-regulation of uPA and uPAR by EMMPRIN was associated with an increase in tumor cell invasion. The exact biological function of PAI-1 in tumor invasion remains elusive as it seems to be a multifunctional protein involved in multiple molecular interactions. Its up-regulation by EMMPRIN in addition to uPA and uPAR is however in accordance with its high levels in tumor cells and tissues.

Our results show that EMMPRIN is able to induce, in the same cellular model, the two proteinase systems, the serine protease urokinase and the MMPs. Their concerted action in the breakdown of the extracellular matrix, largely reported during various physiopathologic situations (1), was clearly shown in our in vitro invasion studies, where the addition of inhibitors for both classes of proteases (marimastat and amiloride) was more efficient than that obtained by each inhibitor alone. This suggests that when acting together, they can amplify the proteolytic and invasive potential of the cell. The siRNA inhibition of EMMPRIN, upstream of this proteinases induction, reduced cell invasiveness comparable with the combination of marimastat and amiloride, supporting the role of EMMPRIN as a major regulator of both systems. It also suggests that EMMPRIN inhibition should be an interesting target to reduce tumor cell invasion.

uPA and the MMPs are also regulated by a variety of growth factors, including the EGF/EGFR system, which was particularly investigated. We previously reported that amphiregulin, another EGFR ligand, increased tumor cell invasion by stimulating uPA production (20). Because the activation of the EGFR signaling pathways, commonly deregulated in a variety of cancers, was also shown to increase EMMPRIN levels in breast cancer cells (15), it is tempting to suggest that EGFR increases the level of uPA and MMPs and promotes tumor invasion and progression through the up-regulation of EMMPRIN. Indeed, the inhibition of EMMPRIN by siRNA blocked the EGF induction of both MMPs (MMP-2 and MMP-9) and uPA.⁶ Whether the signaling events downstream of EMMPRIN are common for MMP and uPA stimulation is yet to be established. Mitogen-activated protein kinase p38 has been implicated in EMMPRIN-mediated induction of MMP-1 production and activation of 5-lipoxygenase and phospholipase A2 in MMP-2 production (10). It is of interest in this respect that p38 also regulates uPA expression and the stability of uPA and uPAR mRNA (19), and it may therefore represent a common pathway for both protease systems in response to EMMPRIN.

Activation of both the MMP and the plasminogen activation systems has been related to the metastatic phenotype and poor prognosis in breast cancer. The abnormally high levels of EMMPRIN in cancer cells and its capacity to induce both these proteinase systems suggest that targeting EMMPRIN should provide an alternative strategy to the inhibition of plasmin or MMP systems, thus far unsuccessful, as it would specifically inhibit those proteases induced by the overexpressed EMMPRIN in the tumor. In addition, our results showing the induction of the uPA system provide important insight for further understanding the mechanism by which EMMPRIN functions in tumor cell invasion and metastasis.

	Acknowledgments

 
Grant support: Société Française de Pharmacologie (C. Quemener), La Ligue contre le cancer (F. Calvo), La Fondation de L'Avenir, and PPF funding from Ministère de la Recherche and Conseil Régional Ile-De-France (confocal microscopy imaging).

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 Niclas Satterblad for confocal microscopy imaging.

	Footnotes

 
⁶ Unpublished results.

Received 7/ 6/06. Revised 10/18/06. Accepted 10/26/06.

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