Stimulation of cell surface plasminogen activation by membrane-bound melanotransferrin: A key phenomenon for cell invasion

Experimental Cell Research 308 (2005) 479 – 490 www.elsevier.com/locate/yexcr

Research Article

Stimulation of cell surface plasminogen activation by membrane-bound melanotransferrin: A key phenomenon for cell invasion Jonathan Michaud-Levesque, Michel Demeule, Richard Be´liveau* Laboratoire de Me´decine Mole´culaire, Service d’He´mato-Oncologie, Hoˆpital Ste-Justine-Universite´ du Que´bec a` Montre´al (UQAM), C.P. 8888, Succursale Centre-ville, Montre´al, Que´bec, Canada H3C 3P8 Received 13 January 2005, revised version received 11 April 2005 Available online 3 June 2005

Abstract The activation of plasminogen at the cell surface is a crucial step in cell migration and invasion. In the present study, the effect of membrane-bound melanotransferrin (mMTf), also known as human melanoma antigen p97, on cell surface plasminogen binding and activation was investigated by using Chinese Hamster Ovary (CHO) cells transfected with full-length melanotransferrin (MTf) cDNA and SK-MeL-28 melanoma cells. The expression of mMTf in CHO increased cell surface plasminogen binding by about 2-fold. In addition, application of the monoclonal antibody L235 against MTf as well as truncated, soluble MTf (sMTf) abolished plasminogen binding to MTftransfected and SK-MeL-28 cells, indicating that mMTf is a potential cell surface plasminogen receptor. Moreover, mMTf expression in CHO cells stimulates plasminogen activation at the cell surface by about 2.5-fold. In addition to the induced binding and activation of plasminogen, cell motility, migration and invasion were about 3-fold higher in CHO cells expressing mMTf. Both monoclonal antibody L235 and truncated sMTf inhibited mMTf-stimulated CHO cell motility, migration and invasion. Overall, our results indicate a key role for mMTf in cell surface plasminogen binding and in activation processes involved during cell migration and invasion. D 2005 Elsevier Inc. All rights reserved. Keywords: Melanotransferrin; Plasminogen; Plasminogen receptor; Plasminogen activation; Cell motility; Cell migration; Cell invasion

Introduction The cell surface plasminogen (Plg) binding proteins and activation system are key modulators of the tissue remodeling processes required for tumor cell invasion and metastasis [1,2]. Native, circulating Glu-Plg binds, in a lysineand/or carbohydrate-dependent manner, to tumor and endothelial cells (EC) with low affinity but high capacity, and a heterogeneous group of Plg receptors have been identified [2– 4]. Several studies have shown that plasmin, a proangiogenic proteinase fragment released from Plg, promotes cell migration and invasion through extracellular matrices when activated at the cell surface [5– 9]. When Glu-Plg is bound to the cell surface, plasmin generation by

* Corresponding author. E-mail address: [email protected] (R. Be´liveau). 0014-4827/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2005.05.004

plasminogen activator (PA) is markedly stimulated compared to the reaction in solution [6]. This enhanced rate of Plg activation increased levels of cell surface plasmin, and cell-bound plasmin is protected from inactivation by natural inhibitors [3]. This also allows Plg to be co-localized in an activation-susceptible form with the enhanced Plg activator levels seen in malignancy, together furnishing tumor cells with elevated tissue remodeling capacity [3,10]. Since cell migration, invasion and tissue remodeling are underpinnings of many physiological and pathological responses, the modulation of Plg receptors may serve as a primary regulatory mechanism for the control of many cellular responses. Human melanoma antigen p97, identified in the early 1980s in malignant melanoma cells, is a glycosylated protein member of the transferrin family [11,12]. The transferrin family contains serum transferrin, lactoferrin and ovotransferrin and, because of p97’s homology with

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these proteins [11], it became known as melanotransferrin (MTf) [13,14]. Slightly expressed in normal tissues, MTf is present in much larger amounts in neoplastic cells and foetal tissues [12,15,16]. More recently, there have been additional reports of human MTf being detected in sweat gland ducts, salivary glands, liver EC, brain endothelium and chondrocytes [17 – 20]. Two forms of endogenous MTf have been described to date; MTf can be secreted in a soluble form (sMTf) or remain bound to the cell membrane (mMTf) by a glycosyl phosphatidylinositol anchor which can be cleaved by phospholipase C [18,21,22]. The physiological roles of both forms of MTf are still unclear. It was first thought that MTf could serve as an iron transporter; however, it was later shown that MTf played very little role in iron transport [23 –26]. We have previously shown that human recombinant sMTf, which is a truncated form of mMTf, interacts with Plg and stimulates Plg activation by the urokinase-type PA (u-PA) [27,28]. Here, we investigated whether mMTf could bind to and stimulate Plg activation at the cell surface. Moreover, the impact of mMTf expression on cell motility, migration and invasion was also assessed by using Chinese Hamster Ovary (CHO) cells transfected with the full-length MTf cDNA (MTf-transfected and control cells). The present study indicates that mMTf stimulates CHO cell migration and invasion by increasing Plg binding and activation at the cell surface. Furthermore, L235 and truncated sMTf decrease Plg binding to SK-Mel-28 melanoma cell surface. These results are also the first to suggest that the mMTf could be considered as a possible target for reducing the Plg binding and activation which is required for cancer cell invasion and malignant progression.

Materials and methods Materials Truncated human recombinant sMTf, which is produced by introducing a stop codon following the glycine residue at position 711 of the full-length human MTf cDNA (27 Cterminal amino acid deletion) and the L235 monoclonal antibody (mAb) were kindly provided by Biomarin Pharmaceutical (Novato, CA). Chinese Hamster Ovary (CHO) cells transfected with full-length human MTf cDNA (p97TRVb CHO cells; referred as MTf-transfected cells in the text) or with control vector (TRVb CHO cells; referred as control cells in the text) were from Dr. Malcom Kennard of the University of British Columbia (Vancouver, BC). Other biochemical reagents were from Sigma-Aldrich (Oakville, ON). Cell culture CHO cells (control and MTf-transfected) were cultured in Ham’s F12 medium from Invitrogen (Burlington, ON)

containing 10 mM HEPES and 10% calf bovine serum (CBS) under 5% CO2/95% air atmosphere, as previously described [29]. Human melanoma SK-Mel-28 cells were obtained from American Type Culture Collection (Manassas, VA). SK-Mel-28 cells were grown in modified Eagle’s medium (MEM) from Invitrogen (Burlington, ON) containing 1 mM Na-pyruvate and 10% foetal bovine serum (FBS) under 5% CO2/95% air atmosphere. Western blot analysis Near-confluent CHO cells (control and MTf-transfected) and SK-Mel-28 cells were exposed to serum-free cell culture medium. After 6 h incubation, conditioned media were removed and the cells were solubilized in lysis buffer (1% Triton-X-100, 0.5% NP-40, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 10 mM Tris, 2% N-octylglucoside, 1 mM orthovanadate, pH 7.5). Cell lysates were subjected to sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (SDS-PAGE) and separated proteins were then transferred to polyvinylidene difluoride (PVDF) membranes (PerkinElmer Life Sciences, Boston, MA). Following transfer, immunodetection analysis was performed using mAb L235 (which recognizes a conformational epitope on MTf) as previously described [28]. Fluorescence-activated cell sorting (FACS) analysis CHO (control and MTf-transfected) and SK-Mel-28 cells were dissociated by incubation with phosphate-buffered NaCl solution (PBS)-citrate solution (138 mM NaCl, 2.8 mM KCl, 1.47 mM KH2PO4, 8.1 mM Na2HPO4, 15 mM sodium citrate, pH 7.4) for 10 min. CHO cells (1  106 cells) were resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) and incubated at 4-C for 15 min with 1 Ag/ml of either mAb L235 or with non-specific, control immunoglobulin G (IgG). The cells were then washed twice with binding buffer and incubated in the dark at 4-C for 15 min with 1 Ag/ml goat anti-mouse IgG-Alexa488 (Molecular Probes, Eugene, OR). After two washes with binding buffer, the cells were analyzed by flow cytometry on a Becton Dickinson FACSCalibur with a 488nm argon laser. Cell surface expression levels of mMTf were corrected for the background fluorescence intensity measured in the presence of a non-specific IgG and were expressed as mean fluorescence intensities. [125I]-Plasminogen binding assay Glu-Plg was radioiodinated by standard procedures using Na-[125I] (Amersham Pharmacia Biotech, Baie D’Urfe´e, QC) and an Iodo-beads kit from Pierce Chemical Co. (Rockford, IL). CHO (control and MTf-transfected) and SKMeL-28 cells were plated onto 24-well culture plates. Binding experiments were performed at 4-C to limit

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internalization processes during the binding assay. The binding was initiated by adding [125I]-Glu-Plg in Ringer/ HEPES solution (150 mM NaCl, 5.2 mM KCl, 2.2 mM CaCl2, 0.2 mM MgCl2, 6 mM NaHCO3, 5 mM HEPES, 2.8 mM glucose, pH 7.4) with or without 50-fold molar excess unlabeled Glu-Plg (1.25 AM), 100 mM q-amino-n-caproic acid (qACA), truncated recombinant sMTf (0.625, 1.25 and 2.5 AM) as well as L235 or a non-specific IgG (16.25, 32.5, 65 nM). After 2 h incubation, cells were washed three times with Ringer/HEPES solution containing 0.1% ovalbumin and lysed in NaOH (0.3 M). Cell associated radioactivity was quantified after trichloroacetic acid (TCA) precipitation using a gamma counter. Plasminolytic activity assay The in vitro plasminolytic activity of CHO cells (control and MTf-transfected) was measured using a colorimetric plasmin activity assay. Briefly, CHO cells were grown to 85% confluency and were dissociated by incubation in PBScitrate solution for 10 min. Cells were then washed twice with incubation buffer (50 mM Tris – HCl buffer (pH 7.5), 150 mM NaCl and 50 mM CaCl2). In the plasminolytic assay, 1 105 cells were incubated in the presence of 25 nM Glu-Plg (Calbiochem, Novato, CA) and 15 Ag of the chromogenic plasmin substrate D-Val-Leu-Arg p-Nitroanilide (VLK-pNA), with or without mAb L235 or a nonspecific IgG (100, 250, 350, 500 and 750 nM), in 200 Al of incubation buffer containing 1% BCS. In this assay, the cleavage of VLK-pNA results in a p-Nitroanilide molecule that absorbs at 405 nm. The plasmin activity was monitored for 6 h at 405 nm at 37-C using a ThermoMAX microplate reader (Molecular Devices, Sunnyvale, CA). Cell motility (wound-healing) assay CHO cells (control and MTf-transfected) were seeded onto 55 mm Petri dishes (Costar, Corning Incorporated, NY) and cultured at 37-C under 5% CO2/95% air atmosphere. The confluent cell monolayer was wounded with pipette tips. After washing with warmed PBS (138 mM NaCl, 2.8 mM KCl, 1.47 mM KH2PO4, 8.1 mM Na2HPO4, pH 7.4), the cells were incubated in fresh culture medium with or without exogenous truncated recombinant sMTf (100 nM), mAb L235 (50 nM) or a non-specific IgG (50 nM). Fresh cell culture medium containing 10 Ag/ml cycloheximide, a protein synthesis inhibitor used to prevent cell proliferation, was added during the assay. The wound areas were photographed at the beginning (0 h) and at the end (20 h) of the assay. Cell motility was monitored at 40X magnification with a digital Nikon Coolpixi 5000 camera (Nikon Canada, Mississauga, ON) attached to a Nikon TMS-F microscope (Nikon Canada). The quantification of cell motility was assessed by measuring the wounded area cell density ratio at 20 h compared to 0 h by using IPLab Gel software (Signal Analytics Corporation, Vienna, Virginia).

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Cell migration assay CHO cell migration was performed using Transwell filters (Costar; 8 Am pore size) precoated with 0.15% gelatin. Briefly, 1  105 cells were resuspended in 100 Al of serum-free medium with or without exogenous, truncated recombinant sMTf (100 nM), qACA (100 mM), mAb L235 (50 nM) or a non-specific IgG (50 nM) and added into the upper chamber of each Transwell (the lower chamber of the Transwell contained 10% serum as well as exogenous, truncated recombinant sMTf, qACA, mAb L235 or a nonspecific IgG). The plates were then placed at 37-C in 5% CO2/95% air atmosphere for 6 h. Cells that migrated to the lower surface of the filters were fixed with 3.7% formaldehyde in PBS and stained with 0.1% crystal violet/20% MeOH. Migrating cells were visualized at 100 magnification using a digital Nikon Coolpixi 5000 camera attached to a Nikon TMS-F microscope and the average number of migrated cells per field was assessed by counting at least 5 random fields per filter using Northern Eclipse software (Empix Imaging, Mississauga, ON). Cell invasion assay CHO cell invasion was assessed using Transwell filters (Costar; 8 Am pore size) precoated with 50 Ag air-dried Matrigeli matrix (BD Bioscience, Mississauga, ON). Briefly, 1  105 cells were resuspended in 100 Al of serum-free medium with or without exogenous, truncated recombinant sMTf (100 nM), qACA (100 mM), mAb L235 (50 nM) or a non-specific IgG (50 nM) and added into the upper chamber of each Transwell (the lower chamber of the Transwell contained 10% serum as well as exogenous, truncated recombinant sMTf, qACA, L235 or a non-specific IgG). The plates were then placed at 37-C in 5% CO2/95% air for 48 h. Cells that traversed the Matrigeli matrix to the lower surface of the filters were fixed with 3.7% formaldehyde in PBS and stained with 0.1% crystal violet/20% MeOH. Invading cells were visualized at 100 magnification using a digital Nikon Coolpixi 5000 camera attached to a Nikon TMS-F microscope and the average number of cells per field was assessed by counting at least 5 random fields per filter using Northern Eclipse software.

Results MTf expression in CHO cells and in SK-MeL-28 melanoma cells MTf expression in CHO cells transfected with a vector encoding full-length MTF (MTF-transfected), or with a control vector (control), was first characterized by Western blot (Fig. 1). Under non-reducing and denaturing conditions, the truncated recombinant sMTf positive control

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Fig. 1. MTf expression in CHO cells and in SK-MeL-28 melanoma cells. (A) Immunodetection of sMTf and MTf by Western blotting. MTf was immunodetected in cell lysates (10 Ag) from control, MTf-transfected and SK-MeL-28 cells. Truncated recombinant sMTf (5 ng) was used as a positive immunodetection control. Proteins were separated by SDS-PAGE and were transferred to PVDF membranes. MTf was immunodetected under non-reducing conditions using mAb L235 and a secondary anti-mouse IgG linked to peroxidase. (B) Flow cytometry analysis of cell surface mMTf levels was performed as described in Materials and methods. CHO (control and MTf-transfected) and SK-MeL-28 cells were labeled with anti-MTf mAb L235 (bold line) or with a non-specific IgG (thin line) and detected with goat anti-mouse IgG-Alexa488. (C) Flow cytometry results were corrected for background fluorescence intensity measured with a non-specific IgG and expressed as mean fluorescence intensities (N = 3).

migrates as 60 and 73 kDa proteins (Fig. 1A), slightly less than endogenous MTf, as previously described [28]. MTf is detected at high levels in the MTf-transfected cell lysate, whereas it is undetectable in cell lysate from control cells (Fig. 1A). Moreover, as demonstrated by Western blot analysis (Fig. 1A), the level of MTf expression in MTftransfected cells is comparable to that in SK-MeL-28 melanoma cells. In view of the fact that MTf demonstrates two forms, a soluble form and a membrane-bound form, cell surface mMTf expression in CHO and SK-Mel-28 cells was determined by FACS using the mAb L235, which recognizes a conformational epitope on MTf (Fig. 1B). The intensity of the green fluorescence (Alexa488; FL1 detection) associated with the detection of cell surface mMTf by

the mAb L235 is much higher in MTf-transfected cells and SK-Mel-28 melanoma cells. In fact, the mean fluorescence intensity associated with the detection of cell surface mMTf is increased by about 200-fold in MTf-transfected compared to control cells (Fig. 1C). As previously reported [25,30], mMTf expression level in MTf-transfected cells represent about 3-fold the expression of mMTf seen in SK-Mel-28 cells. These results indicate that mMTf is expressed at the cell surface of MTf-transfected and SK-MeL-28 cells, whereas the control cells do not express detectable levels of mMTf. Since high level of mMTf is representative of the mMTf expression in SK-Mel-28 melanoma cells, these CHO cells provide a suitable model with which to study the impact of mMTf overexpression on cell behavior.

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mMTf expression in CHO cells stimulates cell surface plasminogen binding

Table 1 Plasminogen binding dissociation constants (K D) and capacities (B MAX) on CHO cell surfaces

Recent studies from our laboratory showed that truncated sMTf interacts with Plg and stimulates its activation by uPA in vitro [27,28]. Here, the influence of mMTf expression on Plg binding at the cell surface was measured (Fig. 2). Firstly, control and MTf-transfected cells were incubated in the presence of different concentrations of [125I]-Glu-Plg for 2 h at 4-C (Fig. 2A). Analysis of these data indicates that mMTf-expressing CHO cells demonstrated a similar Plg binding dissociation constant (K D), but an higher cell surface Plg binding capacity (B MAX), as the control cells (Table 1). By using the delta B MAX (MTf-transfected cells B MAX-control cells B MAX), we also determined the number of mMTf-specific plasminogen binding sites at the cellular surface of MTf-transfected cells (Fig. 2A; dashed line). The estimated number of mMTf-specific plasminogen binding sites in MTf-transfected cells is around 1.09  106 sites/cell. These results indicate that mMTf-expressing CHO cells

CHO cells

Fig. 2. mMTf expression in CHO cells stimulates cell surface plasminogen binding. (A) Saturation binding curves of [125I]-Glu-Plg to CHO cell surfaces. Binding of [125I]-Plg was performed on control (h) and MTftransfected (D) cells as described in Materials and methods. In order to evaluate the mMTf-specific Plg binding ( : dashed line), the non-specific Plg binding measured in control cells was subtracted from the Plg binding obtained in MTf-transfected cells. (B) Effect of unlabeled Plg and qACA on [125I]-Glu-Plg binding to CHO cell surfaces. Binding of [125I]-Plg (25 nM) was performed on control (g) and MTf-transfected (h) cells in the presence or absence of unlabeled Plg (1.25 AM) and qACA (100 mM) as described in Materials and methods. Statistically significant differences, as compared to control conditions (as well as between the two control conditions), are indicated by ***P < 0.001 (Student’s t test) (N = 3).

S

Control MTf-transfected

KD ( 10 1.29 1.22

6

M)

B MAX (ng/Ag total protein) 1.25 2.56

demonstrate a similar affinity for Plg as control cells but a higher Plg binding capacity at their cell surface. Secondly, to confirm that the Plg binding observed with both control and MTf-transfected cells was a saturable process dependent on lysine residues, the binding of 25 nM [125I]-Glu-Plg was measured in the presence of unlabeled Glu-Plg and qACA, a specific lysine-analogue plasmin(ogen) binding activity inhibitor (Fig. 2B). A 50-fold molar excess of unlabeled Glu-Plg (1.25 AM) as well as 100 mM of qACA inhibited radiolabeled Plg binding on cell surfaces by approximately 90% for both control and MTf-transfected cells. Thirdly, to verify that the increased Plg binding is specific to mMTf expression, the Plg binding assay was performed in the presence of either mAb L235 or non-specific IgG (Fig. 3A). The addition of mAb L235 (65 nM) completely inhibited the increased Plg binding of MTf-transfected cells. In addition, the inhibitory effect of mAb L235 on plasminogen binding to MTf-transfected cells was dependent of the mAb concentration (Fig. 3B). Fourthly, to investigate the possibility that truncated sMTf could antagonize the binding of Plg to the cell surface, truncated sMTf was added during the binding assay (Fig. 3C). The addition of this form of sMTf decreased in a dose-dependent manner the binding of Plg to the cell surfaces of both CHO cells. The Plg binding inhibition measured in the presence of truncated sMTf (2.5 AM) for control and MTf-transfected cells reached a maximum inhibition of 50% and 70%, respectively. Altogether, these results indicate that mMTf can bind Plg directly by a saturable process in a lysine-dependent manner. We then investigated the inhibitory effect of the mAb L235 and truncated sMTf on Plg binding to SK-Mel28 melanoma cells (Fig. 4), another cell type expressing high level of mMTf. mAb L235 (65 nM) and truncated sMTf (2.5 AM) inhibit by about 40% the SK-Mel-28 cell surface Plg binding. Altogether, these results indicate that both mAb L235 and truncated sMTf decreased cell surface Plg binding in mMTf-expressing cells. mMTf expression in CHO cells stimulates cell surface plasminogen activation Because mMTf binds Plg, the impact of mMTf expression on CHO cell surface plasminolytic activity was investigated (Fig. 5). The plasmin activity at the surface of MTf-transfected cells and control cells was measured (Fig. 5A). The Plg activation initial velocity (v) was 0.028 mAU/min for control cells and reached 0.084 mAU/min for mMTf-expressing CHO cells. To establish whether mMTf is

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Fig. 4. mAb L235 and truncated recombinant sMTf inhibit SK-Mel-28 melanoma cell surface plasminogen binding. Effect of mAb L235 as well as truncated sMTf on [125I]-Glu-Plg binding to SK-Mel-28 cell surface. Binding of [125I]-Plg (25 nM) was performed on SK-Mel-28 cell in the presence or absence of either mAb L235 (65 nM) or non-specific IgG (65 nM) as well as truncated recombinant sMTf (2.5 AM) as described in Materials and methods. Statistically significant differences as compared to control conditions are indicated by **P < 0.01; ***P < 0.001 (Student’s t test) (N = 3).

responsible for the increased Plg activation by MTfexpressing CHO cells, the plasminolytic activity at the cell surface of CHO cells was assessed in the presence of either 350 nM mAb L235 or non-specific IgG (Fig. 5B). When MTf-transfected cells were pre-incubated with the mAb L235, the plasminolytic activity at the cell surface was reduced to that observed with control cells. Moreover, the addition of the mAb L235 decreased in a dose-dependent manner the cell surface plasminolytic activity of MTftransfected cells (Fig. 5C). These results indicate that inhibition of the increased Plg activation in mMTf-expressing cells by mAb L235 could be related to reduced Plg binding at their cell surface. mMTf expression in CHO cells stimulates cell motility, migration and invasion

Fig. 3. mAb L235 and truncated recombinant sMTf inhibit mMTfstimulated cell surface plasminogen binding. (A) Effect of mAb L235 on [125I]-Glu-Plg binding to CHO cell surfaces. Binding of [125I]-Plg (25 nM) was performed on control (g) and MTf-transfected (h) cells in the presence or absence of mAb L235 (65 nM) or non-specific IgG (65 nM) as described in Materials and methods. (B) Effect of mAb L235 on mMTf-specific [125I]-Glu-Plg binding to CHO cell surfaces. Binding of [125I]-Plg (25 nM) was performed on MTf-transfected cells in the presence or absence of various concentrations of mAb L235 as described in Materials and methods. Results were expressed as mMTf-specific Plg binding by subtracting the Plg binding background measured in control cells. (C) Effect of truncated recombinant sMTf on [125I]-Glu-Plg binding to CHO cell surfaces. Binding of [125I]-Plg (25 nM) was performed on control (g) and MTf-transfected (h) cells in the presence or absence of various concentrations of truncated recombinant sMTf as described in Materials and methods. Statistically significant differences, as compared to control conditions (as well as between the two control conditions), are indicated by *P < 0.05; **P < 0.01; ***P < 0.001 (Student’s t test) (N = 3).

Since mMTf affects the activation of Plg in vitro and since Plg activation is required for tumor cell invasion and metastasis [1,2], the impact of mMTf overexpression on in vitro CHO cell motility, migration and invasion was examined (Fig. 6). A wound-healing assay showed that MTf-transfected cells possessed much higher cell motility (Fig. 6A; left panel). Quantification of the wound area cell density ratio at 20 h compared to 0 h revealed that MTftransfected cells motility was about 2.5-fold higher than control cells (Fig. 6A; right panel). To confirm this observation, cell movement was independently assessed by in vitro cell migration and invasion assays. When cell movement was examined using Transwell filters coated with gelatin, ¨3.5-fold more mMTf-expressing cells (MTf-transfected) migrated to the bottom chamber than did control cells (Fig. 6B). The invasive capacities of the cells were next assessed using Transwell filters coated with Matrigeli

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activates certain matrix metalloproteinases (MMPs) [10]. Thus, we also examined, by substrate gel electrophoresis (zymography), whether the increase in plasmin activity measured in MTf-transfected cells affects their gelatinase levels. The secreted gelatinase pattern in CHO cells is unaffected by mMTf expression (data not shown), suggesting that the effects of mMTf expression are independent of gelatinase expression and/or activation. mAb L235 and truncated recombinant sMTf inhibit mMTf-stimulated CHO cell motility, migration and invasion

Fig. 5. mAb L235 inhibits mMTf-stimulated cell surface plasminogen activation. (A) Effect of mMTf expression on CHO cell surfaces plasminolytic activity. The plasminolytic activity was measured on control (h) and MTf-transfected (D) cells in the presence of Plg (25 nM) as described in Materials and methods. (B) Effect of mAb L235 on CHO cell surfaces plasminolytic activity. The plasminolytic activity was measured on control and MTf-transfected cells in the presence of Plg (25 nM) with or without mAb L235 (350 nM) or non-specific IgG (350 nM) as described in Materials and methods. (C) Effect of mAb L235 on MTf-transfected cell surface plasminolytic activity. The plasminolytic activity was measured in the presence of various concentrations of mAb L235 (h) or non-specific IgG (D) as described in Materials and methods. Statistically significant differences as compared to control conditions are indicated by ***P < 0.001 (Student’s t test) (N = 3).

matrix. The invasive capacity was found to be enhanced by ¨2.5-fold in cells expressing mMTf (Fig. 6C). Thus, in addition to stimulation of the Plg activation by mMTf, cell movement assays showed that MTf-transfected cells have greatly enhanced in vitro cell motility, migration and invasive ability as compared to control cells. Plasmin is a broad-spectrum protease of tryptic specificity that directly

Since mAb L235 and the truncated sMTf affect the activation of Plg in vitro, their effect on mMTf-induced cell movement was evaluated. CHO cell motility was first assessed in the presence of either mAb L235, non-specific IgG or truncated sMTf using a wound-healing assay (Fig. 7). Quantification of the CHO cells motility showed that mAb L235 strongly inhibited the mMTf-stimulated cell motility by about 80% (Fig. 7A). Moreover, the addition of truncated sMTf to this assay also antagonized the mMTfstimulated cell motility by more than 65% (Fig. 7B). To further investigate the impact of the mAb L235 and truncated sMTf on mMTf-stimulated cell movement, in vitro cell migration (Fig. 8) and invasion (Fig. 9) were then measured. First, to determine whether CHO cell migration and invasion was dependent of plasminogen binding and/or activation, we measured CHO cells migration and invasion in the presence of qACA (Figs. 8A and 9A). qACA effectively blocked both control and MTf-transfected CHO cells migration as well as invasion, indicating that plasminogen binding and activation is involved in CHO cells movement. Second, using Transwell filters coated with gelatin, mAb L235 inhibited about 60% of the mMTfinduced migration of CHO cells to the bottom chamber without affecting control cells (Fig. 8B). Also, the addition of truncated sMTf during the assay inhibited mMTf-induced migration of CHO cells by about 81% (Fig. 8C). The inhibition by mAb L235 and the truncated sMTf of the invasive capacity of CHO cells was also assessed using Transwell filters coated with Matrigeli matrix (Figs. 9B – C). The invasive properties of CHO cells overexpressing mMTf into Matrigeli matrix in the presence of L235 or truncated sMTf were inhibited by 78% and 40%, respectively (Figs. 9B –C). Together, the different cell movement assays showed that mMTf-stimulated CHO cell movement, i.e., cell motility, migration and invasion, was inhibited by both the mAb L235 and by truncated sMTf.

Discussion Earlier observations showed that Plg binding to the cell surface imparts a kinetic advantage to activation and protects the newly generated plasmin from inhibition [31 – 34]. However, the identities of cell surface binding sites for

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Fig. 6. mMTf expression in CHO cells stimulates cell movement. (A) Monolayers of confluent control and MTf-transfected cells were wounded with pipette tips. After washing with warm PBS, the cells were incubated with fresh culture medium containing 10 Ag/ml cycloheximide. The wounded areas were photographed at the beginning (0 h) and at the end (20 h) of the assay. Photos (original magnification, 40) obtained from a representative experiment are shown. Results are expressed as the wounded area cell density ratio at 20 h compared to 0 h. (B) CHO cell migration was performed using modified Boyden chambers coated with gelatin. Cells that had migrated to the lower surface of the filters were fixed, stained with crystal violet and counted as described in Materials and methods. Photos (original magnification, 100) obtained from a representative experiment are shown. Results are expressed as a percentage of cell migration seen in control cells. (C) CHO cell invasion was performed using modified Boyden chambers coated with Matrigeli Matrix. Cells that had invaded to the lower surface of the filters were fixed, stained with crystal violet and counted as described in Materials and methods. Photos (original magnification, 100) obtained from a representative experiments are shown. Results are expressed as a percentage of cell migration seen in control cells. Statistically significant differences compared to control cells are indicated by **P < 0.01; ***P < 0.001 (Student’s t test) (N = 3).

Plg have not been completely elucidated. The interaction between truncated sMTf and plasminogen, and its involvement in plasminogen activation stimulation by scu-PA was previously reported [27,28]. Thus, we now investigate whether mMTf could be a potential cell surface Plg binding and activation protein and determine its involvement in cell migration and invasion, by using control and MTf-transfected CHO cells as well as SK-Mel-28 melanoma cells. Western blot and FACS analysis revealed that mMTf is highly expressed and present on the cellular surface of MTftransfected cells, at a level comparable to mMTf expression in SK-MeL-28 melanoma cells. Here, we report that mMTf expression in CHO cells increased cell capacity to bind Plg at the cell surface without affecting their affinity to Plg. The K D (1.29 AM for control cells; 1.22 AM for MTf-transfected cells) measured for the binding of Plg to both CHO cell surfaces is in agreement with data already reported for other cell surface receptors. In fact, the binding of Plg by heterogeneous receptors is generally characterized by a low affinity for Plg, with K D values around 10 6 M [4]. Furthermore, the number of

plasminogen binding sites associated to mMTf in MTftransfected cells is 1.09  106 sites/cell. Since the number of mMTf sites in MTf-transfected CHO cells is 1.2  106 sites/ cell [30,35], the number of mMTf-specific plasminogen binding sites correspond to about 90% of the mMTf molecules at the cell surface. Under the conditions used here, the cell surface Plg binding of both CHO cell lines (control and MTf-transfected) is dependent on lysine residues as evidenced by the loss of cell surface Plg binding induced by qACA. Since Plg presumably binds to pericellular proteins with C-terminal lysines [10,36], lysine derivatives with free a-carboxyl groups, such as qACA, or peptides with carboxy-terminal lysine residues are effective inhibitors of Plg binding to the cells [37,38]. mMTf effectively possess a carboxyl-terminal lysine but the truncated sMTf does not [29]. Since both MTf (truncated sMTf and mMTf) interact with Plg, we hypothesized that the action of the lysine analogue qACA on Plg binding to cell surface occurs via its binding to plasminogen, which possesses high-affinity lysine-binding sites [39,40]. These lysine-binding sites have unique binding

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show the enhanced effectiveness of mMTf-expressing cells in binding and activating Plg into plasmin. Since truncated sMTf stimulates Plg activation by u-PA [27,28], mMTf can

Fig. 7. mAb L235 and truncated recombinant sMTf inhibit mMTfstimulated CHO cell motility. Monolayers of confluent control (g) and MTf-transfected (h) cells were wounded with pipette tips. After washing with warm PBS, the cell were incubated with fresh culture medium containing either 10 Ag/ml cycloheximide with mAb L235 (50 nM) or with non-specific IgG (50 nM) (A), or with and without exogenous truncated recombinant sMTf (100 nM) (B). The wounded areas were photographed at the beginning (0 h) and at the end (20 h) of the assay. Results are expressed as the wounded area cell density ratio at 20 h compared to 0 h. Statistically significant differences, as compared to control conditions (as well as between the two control conditions), are indicated by *P < 0.05; **P < 0.01 (Student’s t test) (N = 2).

affinity for N-amino acid, such as qACA. Thus, as with many Plg receptors [38], the inhibition of Plg binding as well as the inhibition of cell migration and invasion by qACA would be related to a competition between mMTf and qACA for the lysine-binding sites situated on Plg. The addition of mAb L235 inhibited Plg binding to CHO cell surface expressing mMTf as well as to SK-Mel-28 cell surface, indicating that mMTf is directly involved in the binding of Plg. Moreover, truncated sMTf antagonized the Plg binding to CHO (control and MTf-transfected) and SKMel-28 cell surfaces. In light of this, truncated sMTf could inhibit cellular Plg binding by interacting with Plg in the surrounding media, reducing the quantity of Plg able to bind to a cell surface receptor. Furthermore, mMTf expression also stimulated the conversion of Plg into plasmin at the cell surface, as demonstrated by plasminolytic assay. These data

Fig. 8. mAb L235 and truncated recombinant sMTf inhibit mMTfstimulated CHO cell migration. Control (g) and MTf-transfected (h) cell migration was performed with or without qACA (100 mM) (A), with either mAb L235 (50 nM) or non-specific IgG (50 nM) (B) as well as with exogenous truncated recombinant sMTf (100 nM) (C) using modified Boyden chambers with filters coated with gelatin. Cells that had migrated to the lower surface of the filters were fixed, stained with crystal violet and counted as described in Materials and methods. Results are expressed as a percentage of cell migration seen in control cells. Statistically significant differences, as compared to control conditions (as well as between the two control conditions), are indicated by ***P < 0.001 (Student’s t test) (N = 3).

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Fig. 9. mAb L235 and truncated recombinant sMTf inhibit mMTfstimulated CHO cell invasion. Control (g) and MTf-transfected (h) cell invasion was performed with or without qACA (100 mM) (A), with either mAb L235 (50 nM) or non-specific IgG (50 nM) (B) as well as with exogenous truncated recombinant sMTf (100 nM) (C) using modified Boyden chambers with filters coated with Matrigel\ Matrix. Cells that had invaded to the lower surface of the filters were fixed, stained with crystal violet and counted as described in Materials and methods. Results are expressed as a percentage of cell invasion seen in control cells. Statistically significant differences, as compared to control conditions (as well as between the two control conditions), are indicated by *P < 0.05; ***P < .001 (Student’s t test) (N = 3).

also participate in the activation of Plg into plasmin at the cell surface by recruiting Plg for its activation by cell associated PA. The enhance cell surface catalytic efficiency

of plasmin production in mMTf-expressing cells could be explained by the higher plasminogen binding at their cell surface, thereby promoting cell migration and invasion. In view of the fact that mMTf is a tumor-associated antigen, upregulated in most skin melanomas [12,16], mMTf could participate in the migration and invasion of melanoma and neoplastic cells by increasing the Plg binding and its activation into plasmin by PA. We also observed that cellular motility, migration and invasion is stimulated by mMTf overexpression in CHO cells. In addition to the increase in Plg binding, MTftransfected cells possess a much higher capacity to generate plasmin from Plg. Tumor-stroma interactions play a pivotal role in regulating tumor progression and malignancy [41]. Malignant progression is characterized by inappropriately high cell surface levels of receptor-bound Plg which, in turn, stimulate plasmin generation by PA required for high cellular proteolytic potential. This increased proteolytic potential facilitates cell detachment from the primary tumor as well as cell migration and invasion through connective tissue and basement membrane [41,42]. Plasmin, a broadspecific serine proteinase capable of degrading almost all components of the extracellular matrix [42], is increased at the cell surface of MTf-transfected cells. In particular, it has been reported that plasmin induced the migration of CHO cells [43]. This latter study and the present findings suggest that the higher generation of plasmin by PA at the cell surface of CHO cells expressing mMTf contributes to their migration and invasion. Thus, the mMTf-stimulated Plg binding would lead to an increase Plg activation by PA at the cell surface. This could be a potential mechanism by which mMTf exerts its stimulatory effects on cell migration and invasion. As for other Plg binding molecules [44], the binding of Plg to mMTf could favor its conversion into plasmin by PA, such as u-PA, located in the vicinity of the cell surface, thereby promoting cell migration and invasion. In this matter, we recently proposed that MTf could be involved in EC and melanoma cell migration [27] and that truncated recombinant sMTf could inhibit EC movement and morphogenic differentiation into capillary-like structures (tubulogenesis) [28]. Thus, given the important role of plasmin in cell movement [45], a protein like mMTf that targets the formation of plasmin to the cell surface and acts on cell movement and EC tubulogenesis might be expected to affect cancer cell invasion and metastasis. The original idea of Plg activation as a ratelimiting factor in tumor invasion and metastasis has been supported by many in vitro and in vivo studies [10]. In light of these results, we can postulate that factors or stimuli that will induce the synthesis and/or release of mMTf from the cell surface or the secretion of endogenous sMTf could lead to a plasmin-mediated cascade of events. Such a mechanism could represent an important regulatory step in the invasive capacities of neoplastic cells expressing mMTf.

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Altogether, our results show that mMTf-stimulated plasmin generation as well as cell migration and invasion are, in part, dependent on the ability of mMTf to increase cell surface Plg availability for its activation by PA located at the cell surface. Moreover, these are the first results showing that mMTf, by stimulating the activation of Plg, could play an important role in cell migration and invasion. The effect of mMTf expression on cell movement is antagonized by the mAb L235 and truncated sMTf, suggesting that endogenous mMTf can be considered as a potential therapeutic target, which can be inhibited by this mAb or by a truncated form of sMTf. Overall, our findings may be clinically relevant for future therapeutic strategies based on specific molecules that will reduce the Plg activation at the cell surface, required for cancer cell invasion and malignant progression, by inhibiting the interaction of mMTf with Plg.

Acknowledgments This study was supported by grants from the Canadian Institutes of Health Research (CIHR) to R.B.. J.M.L. is a recipient of a Ph.D. scholarship from the Fonds de la Recherche en Sante´ du Que´bec (FRSQ). We also thank Julie Poirier and Constance Gagnon for their technical support.

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