Tumor Cell Invasion Is Promoted by Activation of Protease Activated Receptor-1 in Cooperation with the αvβ5 Integrin
2001; Elsevier BV; Volume: 276; Issue: 14 Linguagem: Inglês
10.1074/jbc.m007027200
ISSN1083-351X
AutoresSharona Even-Ram, Miriam Maoz, Elisheva Pokroy, Reuven Reich, Ben‐Zion Katz, Paul Gutwein, Peter Altevogt, Rachel Bar‐Shavit,
Tópico(s)Platelet Disorders and Treatments
ResumoThe first prototype of the protease activated receptor (PAR) family, the thrombin receptor PAR1, plays a central role both in the malignant invasion process of breast carcinoma metastasis and in the physiological process of placental implantation. The molecular mechanism underlying PAR1 involvement in tumor invasion and metastasis, however, is poorly defined. Here we show that PAR1 increases the invasive properties of tumor cells primarily by increased adhesion to extracellular matrix components. This preferential adhesion is accompanied by the cytoskeletal reorganization of F-actin toward migration-favoring morphology as detected by phalloidin staining. Activation of PAR1 increased the phosphorylation of focal adhesion kinase and paxillin, and the induced formation of focal contact complexes. PAR1 activation affected integrin cell-surface distribution without altering their level of expression. The specific recruitment of αvβ5 to focal contact sites, but not of αvβ3 or α5β1, was observed by immunofluorescent microscopy. PAR1 overexpressing cells showed selective reciprocal co-precipitation with αvβ5 and paxillin but not with αvβ3 that remained evenly distributed under these conditions. This co-immunoprecipitation failed to occur in cells containing the truncated form of PAR1 that lacked the entire cytoplasmic portion of the receptor. Thus, the PAR1 cytoplasmic tail is essential for conveying the cross-talk and recruiting the αvβ5 integrin. While PAR1 overexpressing cells were invasive in vitro, as reflected by their migration through a Matrigel barrier, invasion was further enhanced by ligand activation of PAR1. Moreover, the application of anti-αvβ5 antibodies specifically attenuated this PAR1 induced invasion. We propose that the activation of PAR1 may lead to a novel cooperation with the αvβ5 integrin that supports tumor cell invasion. The first prototype of the protease activated receptor (PAR) family, the thrombin receptor PAR1, plays a central role both in the malignant invasion process of breast carcinoma metastasis and in the physiological process of placental implantation. The molecular mechanism underlying PAR1 involvement in tumor invasion and metastasis, however, is poorly defined. Here we show that PAR1 increases the invasive properties of tumor cells primarily by increased adhesion to extracellular matrix components. This preferential adhesion is accompanied by the cytoskeletal reorganization of F-actin toward migration-favoring morphology as detected by phalloidin staining. Activation of PAR1 increased the phosphorylation of focal adhesion kinase and paxillin, and the induced formation of focal contact complexes. PAR1 activation affected integrin cell-surface distribution without altering their level of expression. The specific recruitment of αvβ5 to focal contact sites, but not of αvβ3 or α5β1, was observed by immunofluorescent microscopy. PAR1 overexpressing cells showed selective reciprocal co-precipitation with αvβ5 and paxillin but not with αvβ3 that remained evenly distributed under these conditions. This co-immunoprecipitation failed to occur in cells containing the truncated form of PAR1 that lacked the entire cytoplasmic portion of the receptor. Thus, the PAR1 cytoplasmic tail is essential for conveying the cross-talk and recruiting the αvβ5 integrin. While PAR1 overexpressing cells were invasive in vitro, as reflected by their migration through a Matrigel barrier, invasion was further enhanced by ligand activation of PAR1. Moreover, the application of anti-αvβ5 antibodies specifically attenuated this PAR1 induced invasion. We propose that the activation of PAR1 may lead to a novel cooperation with the αvβ5 integrin that supports tumor cell invasion. protease activated receptor focal adhesion complex focal adhesion kinase extracellular matrix thrombin receptor-activating peptide antisense fluorescence-activated cell sorting Dulbecco's modified Eagle's medium fetal calf serum fluorescein isothiocyanate monoclonal antibodies bovine serum albumin phosphate-buffered saline tissue factor urokinase urokinase receptor The ability of tumor cells to invade beyond controlled hemostatic boundaries and re-emerge from blood vessels to establish new metastatic colonies continuous to present a major obstacle in cancer cure. It is well known that in tumor invasion and metastasis, the pericellular proteolytic systems, consisting of proteases and their specific cell surface receptors, are tightly regulated to modulate cellular functions and degrade selective matrix barriers (1Mignatti P. Rifkin D.B. Physiol. 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Extensive proteolysis in the tumor microenvironment is also responsible for the activation of several enzymatic precursors, like plasminogen, pro-matrix metalloproteinase, and prothrombin (4Carroll V.A. Binder B.R. Semin. Thromb. Hemost. 1999; 25: 183-197Crossref PubMed Scopus (66) Google Scholar, 5Kurschat P. Zigrino P. Nischt R. Breitkopf K. Steurer P. Klein C.E. Krieg T. Mauch C. J. Biol. Chem. 1999; 274: 21056-21062Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 6Al-Mondhiry H. Thromb. Diath. Haemorrh. 1975; 34: 181-193PubMed Google Scholar). In addition, the extravascular deposition of fibrin within the tumor microenvironment is well established (7Shoji M. Hancock W.W. Abe K. Micko C. Casper K. Baine R.M. Wilcox J.N. Danave I. Dillehay D.L. Matthews E. Contrino J. Morrissey J.H. Gordon S. Edington T.S. Kudryk B. Kreutzer D.L. Rickles F.R. Am. J. 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Among these is the receptor for the serine protease urokinase, which, when bound to its cell surface receptor (uPAR), converts plasminogen to plasmin; plasmin, in turn, is known to effectively degrade various matrix glycoproteins (1Mignatti P. Rifkin D.B. Physiol. Rev. 1993; 73: 161-195Crossref PubMed Scopus (1188) Google Scholar, 11Vassali J.D. Sappino A.P. Belin D. J. Clin. Invest. 1991; 88: 1067-1072Crossref PubMed Scopus (1107) Google Scholar). It has been shown also that uPAR serves as an adhesion receptor for vitronectin (12Wei Y. Lukashev M. Simon D.I. Bodary S.C. Rosenberg S. Doyle M.V. Chapman H.A. Science. 1996; 273: 1551-1555Crossref PubMed Scopus (699) Google Scholar) and that the vitronectin receptor αvβ3 not only supports the migration of tumor cells on various matrix-proteins but also binds matrix metalloproteinase-2, thus presenting an immobilized enzyme with improved matrix-collagen degradation properties at the invasive front (13Brooks P.C. Montgomery A.M. 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Invest. 1999; 103: 789-797Crossref PubMed Scopus (147) Google Scholar, 38Ojaniemi M. Vuori K. J. Biol. Chem. 1997; 272: 25993-25998Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) on the other. The combined signals involved with the activation of focal adhesion proteins indicate that the cooperation between the signaling pathway takes place most likely within these FAC structures. In the present work, we have studied the molecular mechanism of PAR1 involvement in tumor cell invasion. We show here that PAR1 modulates the invasive phenotype of melanoma cell lines, inducing the otherwise non-invasive cells to migrate effectively through Matrigel barriers. This process is accompanied by the increased adherence of the cells to various matrix components, actin stress fiber formation, and adhesion-triggered signaling, with no alteration of the cell surface integrin levels. We demonstrate now, for the first time, that PAR1 mediates these functions via selective cross-talk with the αvβ5 integrin to confer FAC formation, distinct signaling events, and cytoskeletal reorganization. Combined, these processes promote tumor cell invasion. SB-2 non-invasive human melanoma and A375-SM "super-metastatic" human melanoma cells (kindly provided by J. Fidler and M. Bar-Eli, Department of Cell Biology, University of Texas, M. D. Anderson Cancer Center, Houston, TX) were grown in 10‥ FCS-DMEM supplemented with 50 units/ml penicillin and streptomycin (Life Technologies, Inc.) and maintained in a humidified incubator with 8‥ CO2 at 37 °C. The PAR1 stable transfectants, clone 13 and clone Mix L, were grown under the same conditions; for long term maintenance, these were supplemented also with 200 μg/ml G418 antibiotics. MCF-7 cells were maintained as previously described (3Even-Ram S. Uziely B. Cohen P. Grisaru-Granovsky S. Maoz M. Ginzburg Y. Reich R. Vlodavsky I. Bar-Shavit R. Nat. Med. 1998; 4: 909-914Crossref PubMed Scopus (409) Google Scholar). Cells were grown to 30–40‥ confluence and then transfected with 0.5–2 μ g/ml plasmid DNA in Fugene 6 transfection reagent (Roche Molecular Biochemicals) according to the manufacturer's instructions. After 10 days of selection, stable, transfected clones were established in medium containing 400 μg/ml G418. Antibiotic-resistant cell colonies were transferred to separate culture dishes and were grown in 200 μg/ml G418 medium. Forty-eight hours after transfection, transiently transfected cells were collected and tested by immunoprecipitation analyses (see below). Using polymerase chain reaction, we constructed a PAR-1 mutant protein truncated in its cytoplasmic tail after the amino acid Leu-369. As a template, we used PAR-1 cDNA in the pCDNA 3 vector. For amplification, we used a T7 sense primer and the reverse primer GGTCTAGAAAACTATAGGGGGTCGATGCACGAGCT containing a STOP codon and anXbaI site. The amplified DNA fragment was subcloned using the polymerase chain reaction-blunt technique (Invitrogen) and confirmed by DNA sequencing. The insert was released from the vector byXbaI digestion and cloned into plasmid pCDNA3. To confirm the functional integrity of the DNA constructs, wild type and mutant cDNAs were transiently expressed in 293 cells that were subsequently stained with a PAR-1-specific antibody (WEDE15, Immunotech, Cedex, France). Cells were solubilized in lysis buffer containing 10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1‥ Triton X-100, and protease inhibitors (5 μg/ml aprotinin, 1 μmphenylmethylsulfonyl fluoride, and 10 μg/ml leupeptin) at 4 °C for 30 min. The cell lysates were subjected to centrifugation at 10,000 × g at 4 °C for 20 min. The supernatants were saved and their protein contents were measured; 50 μg of the lysates were loaded onto 10‥ SDS-polyacrylamide gels. After the proteins were separated, they were transferred to an Immobilon-P membrane (Millipore). Membranes were blocked and probed with 1 μg/ml amounts of the appropriate antibodies as follows: anti-PAR1 thrombin receptor mAb, clone II aR-A (Biodesign Int.); anti-paxillin monoclonal antibody (mAb), clone 349 (Transduction Laboratories, Lexington KY); anti-human focal adhesion kinase, rabbit polyclonal IgG (Upstate Biotechnology Inc., Lake Placid, NY); anti-phosphotyrosine mAb, clone 4G10 (Upstate Biotechnology Inc.); anti-vinculin mAb (Transduction Laboratories). The antibodies were suspended in 1‥ BSA in 10 mm Tris-HCl, pH 7.5, 100 mm NaCl, and 0.5‥ Tween 20. After washes with 10 mm Tris-HCl, pH 7.5, 100 mm NaCl, and 0.5‥ Tween 20, the blots were incubated with secondary antibodies conjugated to horseradish-peroxidase. Immunoreactive bands were detected by the enhanced chemiluminescence (ECL) reagent using Luminol and p-cumaric acid (Sigma). Cells were treated for 30–60 min with thrombin at a concentration of 1 NIH unit/ml of serum-free DMEM medium (0.5‥ BSA), and then lysed as described above. We used 400 μg of total protein for immunoprecipitation of αvβ3, αvβ5, paxillin, FAK, or both paxillin and FAK. All the antibodies were used at a concentration of 10 μg/ml. After overnight incubation, Protein A-Sepharose beads (Amersham Pharmacia Biotech) were added to the suspension (50 μl) that was subsequently rotated at 4 °C for 1 h. Elution of the reactive proteins was made by re-suspending the beads in protein 2× sample buffer (63 mm Tris-HCl, pH 6.8, 20‥ glycerol, 20‥ SDS, 0.01‥ bromphenol blue, 5‥ β-mercaptoethanol, 0.02 m dithiothreitol) and boiling for 5 min. The supernatant was loaded on a 10‥ SDS-polyacrylamide gel followed by the same procedure as in Western blotting. We used blind-well chemotaxis chambers with 13-mm diameter filters. Polyvinylpyrrolidone-free polycarbonate filters with 8-μm pores (Costar Scientific Co., Cambridge, MA) were coated with basement membrane Matrigel (25 μg/filter) as described previously (39Albini A. Pathol. Oncol. Res. 1998; 4: 230-241Crossref PubMed Scopus (156) Google Scholar). Briefly, the Matrigel was diluted to the desired final concentration with cold distilled water, applied to the filters, dried under a hood, and reconstituted with serum-free medium. In the upper compartment of the Boyden chamber, we placed 2–3 × 105 cells suspended in DMEM containing 0.1‥ bovine serum albumin. As a chemo-attractant, into the lower compartment of the Boyden chamber, we put 3T3 fibroblast conditioned medium. Assays were carried out in 5‥ CO2 at 37 °C. After 2 h of incubation, we observed that more than 90‥ of the cells were attached to the filter. At this time, the cells on the upper surface of the filter were removed by wiping with a cotton swab. The filters were fixed in DifQuick system (American Scientific Products) and stained with hematoxylin and eosin. Cells from various areas of the lower surface were counted. Each assay was performed in triplicate. For chemotaxis studies (a control of Matrigel invasion), the filters were coated with collagen type IV alone (5 mg/filter) to promote cell adhesion. Cells were added to the upper chamber and conditioned medium to the lower compartment. The medium of cells grown in 10‥ FCS was replaced by DMEM with 0.5‥ BSA, and the cells were detached from the plate by treating with 0.05‥ trypsin in a solution of 0.02‥ EDTA in 0.01 m sodium phosphate, pH 7.4 (Biological Industries, Beit Ha'emek, Israel). After washing, 0.5 × 106cells/ml cells were re-suspended in a serum-free DMEM medium (as above) and laid on 13-mm culture dishes pre-coated with either 100 μg/ml fibronectin or Th-1, a thrombin-derived RGD (arginine-glycine-aspartic acid) peptide. After a 45-min incubation period to allow cell adhesion, the excess cells were washed away. The adhered cells were fixed to the plates with 4‥ formaldehyde in PBS, pH 7.4, for at least 2 h. After fixation, the plates were washed in 1‥ boric acid solution and the cells were stained with 1‥ methylene blue reagent (Sigma) in 1‥ boric acid for 30 min. After extensive washing with tap water, the methylene stain was eluted by the addition of 500 μl of 1m HCl. The intensity of the color staining was measured by color spectrometry at a wavelength of 620 nm. Cells were plated on glass coverslips in 16-mm culture dishes; after the cells had grown to subconfluence, they were washed with PBS, permeabilized in 0.5‥ Triton X-100-containing 3.5‥ paraformaldehyde/PBS solution on ice for 2 min, and finally fixed with 3.5‥ paraformaldehyde/PBS for 20 min. Reactions with the appropriate antibodies were performed in room temperature for 60 min, after which the cells were washed extensively in PBS. The antibodies included the following: anti-αvβ3 mAb clone LM609, anti-αvβ5 clone P1F6, and anti-α5β1 clone JBS5, (all from Chemicon Int.). After the 60-min incubation with the primary antibodies, followed by extensive washes in PBS, an additional 60-min incubation was carried out in the dark with secondary antibodies, goat-anti-rabbit or goat-anti-mouse IgG each conjugated with Cy-3 (Jackson Immunoresearch Laboratories) diluted 1:700. Labeling of filamentous actin by 1 μ g/ml FITC-conjugated phalloidin (Sigma) was performed similarly. The labeled cells were visualized and photographed by fluorescent confocal microscopy (MRC-1024 confocal imaging system, Bio-Rad). The medium of cells grown in 10‥ FCS-DMEM was replaced by serum-free DMEM containing 0.5‥ BSA. Thrombin at a concentration of 1 IU/ml was added to the plates that were activated by incubation for 60 min. The plates were washed with PBS, and the cells were detached from the plates by treatment with 0.05‥ trypsin in a solution of 0.02‥ EDTA in 0.1 m sodium phosphate at pH 7.4 (Biological Industries). After being washed twice in PBS, the cells were re-suspended in 200 μl of PBS and the appropriate antibodies were added to a concentration of 10 μg/ml. These reactions, performed at room temperature for 60 min, were followed by extensive washing in PBS. A 1-h incubation with a secondary antibody goat-anti-mouse IgG (Jackson Immunoresearch Laboratories) conjugated with FITC and diluted 1:500 was carried out in the dark. The treated cells were washed extensively, re-suspended in 100 μl of PBS, and analyzed by FACS. In previous work (3Even-Ram S. Uziely B. Cohen P. Grisaru-Granovsky S. Maoz M. Ginzburg Y. Reich R. Vlodavsky I. Bar-Shavit R. Nat. Med. 1998; 4: 909-914Crossref PubMed Scopus (409) Google Scholar), we showed that there is a direct correlation between PAR1 expression and the metastatic potential of primary tumor biopsies and tumor cell lines, as reflected by theirin vitro potential to invade through a Matrigel barrier. 2E. Pokroy, B. Uziely, S. Even-Ram Cohen, M. Maoz, I. Cohen, S. Ochayon, R. Reich, J. Pe'er, O. Drize, M. Lotem, and R. Bar-Shavit, submitted for publication. In a physiological invading model system of placenta trophoblast implantation, we have also shown that PAR1 is part of the invasive program of trophoblast, as evaluated by their villi extension and matrix metalloproteinase synthesis. 3S. Even-Ram Cohen, S. Grisaru-Granovsky, M. Maoz, S. Zaidoun, Y.-J. Yin, and R. Bar-Shavit, submitted for publication. Here, to clarify how high levels of PAR1 may confer invasiveness, we transfected a non-invasive melanoma cell line (SB-2 cells) with PAR1 cDNA and compared the properties of the transfected cells to those of the highly invasive melanoma cell line A375SM. We used PAR1 cDNA under the control of the cytomegalovirus viral promoter in the pCDNA3 expression vector. We selected several stable clones that expressed high levels of PAR1, as evaluated by Western blot analysis (Fig.1 a) and Northern blot analysis (data not shown). The selected clones were then tested for their ability to invade through Matrigel-coated filters. Indeed, clones expressing high levels of PAR1 had an increased ability to invade the Matrigel layer, as compared with control clones transfected with empty vectors or SB-2 cells that had not been transfected at all (Fig.1 b). In addition, we observed that, whereas highly invasive A375SM cells invaded Matrigel coated membranes more efficiently than did non-metastatic cells (Fig. 1 b, SB-2), activating the A375SM cells with PAR1 increased their ability to invade Matrigel to an even higher level (Fig. 1 b, activ. A375SM). In addition, the invasiveness of PAR1-transfected cells was further increased when they were either activated by thrombin, as shown in two separate PAR1-transfected clones (Fig. 1 b,clones 13 and Mix L), or when they were treated with the thrombin-receptor-activating peptide (TRAP) that corresponds to PAR1 internal ligand SFFLRN (data not shown). Circulating tumor cells can invade into a new metatastic site only if they can adhere to the basement membrane. We analyzed the adhesion properties of cells suspended in a serum-free medium and then incubated for 60 min on plates coated with either fibronectin, a major component of the ECM, or with Th-1, an 11-amino acid peptide, corresponding to the thrombin RGD motif (40Bar-Shavit R. Sabbah V. Lampugnani M.G. Marchisio P.C. Fenton I.I.J.W. Vlodavsky I. Dejana E. J. Cell Biol. 1991; 112: 335-344Crossref PubMed Scopus (87) Google Scholar). Highly invasive A375 SM melanoma cells adhered strongly to both Th-1 and fibronectin; however, under the same conditions, the non-invasive SB-2 cells failed to adhere. We observed a marked increase in the adherence to both of these matrices of PAR1-transfected SB-2 cells (Fig. 2,a and b). The level of adherence of these PAR1 transfectants was directly correlated both with their level of PAR1 expression and with their ability to invade the Matrigel barrier. To assure that this increase in their adherence was actually caused by the presence of PAR1, we asked if reducing the expression of PAR1 in malignant cells would reduce the adhesion properties of these cells. To do this, we evaluated the effect of transfection by PAR1 antisense DNA on the adhesion properties of the invasive A375SM cells. We used a 462-base pair oligonucleotide fragment corresponding to the 5′ region of PAR1 that included part of the near promoter sequence and the coding region for the internal ligand. We cloned this DNA segment into pCDNA3 mammalian expression vector in an antisense orientation, selecting for stable clones expressing the plasmid bearing the PAR1 antisense DNA as compared with cells transfected by empty vectors or non-transfected control cells. Northern blot analysis (Fig.2 d) indicated that, whereas empty vector transfection (Fig.2 d, B) had no significant effect on PAR1 expression (Fig. 2 d, A), clones AS -3 (Fig.2 d, C) and AS-4 (Fig. 2 d,D), which were transfected by the PAR1 antisense DNA, did exhibit reduced PAR1 expression. When we analyzed clones AS-3 and AS-4 for their adhesion properties, we found that the cell adherence properties to fibronectin (Fig. 2 c) and to Th-1 (data not shown) of both of these clones were significantly lower than those of the A375 SM parental cells. The organization of the cytoskeleton is critically influenced by adhesion interactions. To explore the effect of PAR1 activation on cytoskeletal reorganization, we plated PAR1-transfected cells (clone 13) and control non-transfected cells (SB-2 cells) on glass coverslips and then treated them with TRAP for various periods of time (Fig.2 e). After activation by TRAP, the cells were permeabilized, fixed, and stained with FITC-labeled phalloidin to detect filamentous actin (F-actin). Cytoskeletal reorganization was observed as early as 15 min after activation by TRAP (Fig. 2 e). Thirty to 60 min after PAR1 activation, we observed a transition in the PAR1 transfectants from elongated spindle-like shapes to spreading, jellyfish-like structures. Ninety minutes to 2 h after activation, the cells became rounder and we observed the appearance of a ringlike bundle of actin filaments at the base of the cells (typical of migrating cells). These changes occurred more rapidly and were more dramatic in PAR1-overexpressing cells than they did in the non-transfected control cells. Altogether, these data show that the adhesive properties of tumor cells were affected by changes in PAR1 expression. Integrin activation typically leads to the assembly of focal adhesion contacts
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