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Fibroblast activation protein-α promotes ovarian cancer cell proliferation and invasion via extracellular and intracellular signaling mechanisms
Experimental and Molecular Pathology 95 (2013) 105–110
Contents lists available at SciVerse ScienceDirect
Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp
Fibroblast activation protein-α promotes ovarian cancer cell proliferation and invasion via extracellular and intracellular signaling mechanisms WeiWei Yang a,1, Wei Han b,1, ShengQian Ye a, DuanYang Liu a, Jun Wu c, HongYu Liu d, ChunHong Li a, He Chen a,⁎ a
Department of Pathology, Harbin Medical University, Harbin 150081, China Department of Pathology, The First Affiliated Hospital of Harbin Medical University, Harbin 150081, China c Department of Thoracic Surgery, Harbin Medical University Cancer Hospital, Harbin 150081, China d Department of Pathology, The First Hospital of Qiqihar, Qiqihar 161005, China b
a r t i c l e
i n f o
Article history: Received 27 May 2013 Available online 15 June 2013 Keywords: Fibroblast activation protein-α (FAPα) Urokinase-type plasminogen activator receptor (uPAR) Integrin α3β1 Ovarian cancer
a b s t r a c t Fibroblast activation protein-α (FAPα) is secreted by activated stromal fibroblasts and can promote ovarian cancer cell proliferation, migration and invasion. However, the molecular mechanism by which FAPα promotes tumor cell proliferation and invasion is unknown. The role of the non-enzymatic activities of FAPα in tumor migration and invasion and the intracellular and extracellular signaling mechanisms of FAPα were investigated. In this study, we confirm that FAPα promote ovarian cancer cell proliferation, migration and invasion by extracellular and intracellular signaling mechanisms. These results provide evidence that FAPα, together with integrin α3β1 and the uPAR signaling complex, mediate cancer cell migration in the HO-8910PM cell line via the small GTPase Rac1. FAPα-mediated upregulation of p-ERK occurred in a time-dependent manner. © 2013 Elsevier Inc. All rights reserved.
Introduction Ovarian cancer is a common malignancy and one of the leading causes of morbidity and mortality worldwide. Fibroblast activation protein-α (FAPα), also known as seprase (Goldstein, 1997), is secreted by activated stromal fibroblasts and can promote ovarian cancer cell proliferation, migration and invasion. Here, the role of the non-enzymatic activities of FAPα in tumor migration and invasion and the intracellular and extracellular signaling mechanisms of FAPα were investigated. The integrin family of transmembrane adhesion proteins exhibits multiple functions, including adhesion to the ECM and localization to invadopodia (Mueller, 1999). One hypothesis suggests that integrins are involved in the recruitment of proteases to sites of cell invasion. The integrin α3β1 associates with FAPα, and it is thought to participate in the formation of functional invadopodia through a docking interaction (Mueller, 1999). A recent study has shown that FAPα-Urokinase-type plasminogen activator receptor (uPAR) membrane complexes associate with the invadopodia of LOX cells, suggesting a co-operative role for uPAR in tumor invasion (Artym, 2002). This interaction between uPA and integrins regulates both cell adhesion and integrin signaling activities. ⁎ Corresponding author. E-mail address: [email protected] (H. Chen). 1 Contributed equally to this work. 0014-4800/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexmp.2013.06.007
GTP-RhoA also associates with the uPAR signaling complex (Kiian, 2003). Members of the Rho GTPase family are key regulators of actomyosin cytoskeleton activity, which is required for invasion and metastasis (Jaffe and Hall, 2005). The bundling and contraction of actin– myosin fibers provide the force required for cell motility and invasion (Sahai, 2009). The Rho family members, including RhoA, Rac1 and ROCK, are crucial regulators of actin cytoskeleton dynamics. Thus, these regulators control cell shape, cell adhesion and the contractions that drive cell migration (Schmitz, 2000). Among these effectors is the serine-threonine protein kinase (ROCK), which controls the activity of myosin II indirectly by phosphorylating the myosin-binding subunit of the myosin light chain (MLC) phosphatase, thus inhibiting MLC dephosphorylation (Hartshorne, 1998; Kawano, 1999; Kimura et al., 1996).
Materials and methods Cell culture The HO-8910PM cells, a highly metastatic ovarian cancer cell line derived from HO-8910 (Shenhua, 1999). Cells were grown in RPMI 1640 10% FBS, 1% penicillin and streptomycin, 5% CO2 at 37 °C. Both HO-8910 and HO-8910PM were purchased from Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Science.
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Western blotting
Invasion assay
HO-8910PM cells grown in 100-mm dishes were washed in cold PBS (pH 8.0) for 3 times before they were collected. HO-8910PM cells were extracted and centrifuged at 12,000 rpm for 30 min at 4 °C. The samples were subjected to electrophoresis and then transferred to Hybond ECL membrane. The membrane was incubated for 1 h in blocking solution with primary antibody anti-integrin α3 (1:2000 Santa Cruz Biotechnology, Inc.), anti-integrin β1 (1:2000 Santa Cruz Biotechnology, Inc.) and anti-uPAR (1:500 Santa Cruz Biotechnology, Inc.), primary antibody incubated at 4 °C overnight. Then, the membrane was incubated with a phosphate-conjugated, secondary antibody at 37 °C for 30 min. After extensive washing, the immunoblots were visualized with western blue (Promega Corporation, USA). The methods of Western-blotting of ERK, p-ERK, MLC, p-MLC protein express the same as the front. The β-actin was assessed as an internal loading control.
The assay was performed using chambers with polycarbonate filters with 8-μm nominal pore size (Millipore) coated on the upper side with Matrigel (BD Biosciences, Bedford, MA). The chambers were placed into a 24-well plate. Twelve groups of HO-8910PM cells (1.3 × 106/ml) were harvested and placed in the upper chamber. Cells treated with FAPα (100 pM), Y27632 (100 μmol/l), Y27632 (ROCK inhibitor) and FAPα, NSC23766 (50 μmol/l), NSC23766 (Rac1 inhibitor) and FAPα, anti-integrin α3β1 (10 ng/ml), anti-integrin α3β1 and FAPα, PAI-1 (500 ng/ml), PAI-1 and FAPα, and untreated cells (blank control). The lower chamber was filled with 800 μl RPMI 1640 containing 10% FBS and then incubated for 48 h at 37 °C in a humidified atmosphere containing 5%CO2. Cells remaining in the upper chamber were completely removed by gently swabbing. invading cells which were counted in microscope fields for each membrane at 40× magnification. Proliferation assay
Co-immunoprecipitation The HO-8910PM cells were collected and extracted by the same method as Western blotting. Three independent EP tubes were added to anti-integrin α3, anti-integrin β1 and anti-uPAR at 4 °C overnight. Then, protein G was added for 4 h. They were centrifuged and precipitation was collected, and they were washed three times with RIPA buffer. The samples were subjected to electrophoresis and then transferred to Hybond ECL membrane. The follow up steps were the same as that of Western blotting. Immunofluorescence HO-8910PM were cultured in the 6-well plate, incubated at 37 °C for 8 h, and cells were fixed in cold 4% (v/v) paraformaldehyde for 20 min at room temperature. After permeabilization and blocking with 0.3% (v/v) Triton X-100, cells were respectively probed with primary antibody of integrin α3β1 (1:200), at 37 °C for 1 h, then uPAR primary antibody was added, at 4 °C overnight. Cells were washed and probed with FITC-anti-rabbit antibody (1:200) and TRITC-anti-mouse antibody respectively (1:200) (All Sigma-Aldrich) as secondary antibodies for incubation at 37 °C for 60 min in the dark. All ovarian cancer specimens were from patients (30 primary ovarian cancers) who underwent surgery in Harbin Medical University Cancer Hospital from 2007 to 2011. None of the patients received preoperative treatments such as radiotherapy or chemotherapy. The expressions of integrin α3β1 and uPAR were examined with primary antibody dilution (1:200), fluorescence labeled second antibody (1:200), in consecutive tissue sections using the LSAB + kit (Santa Cruz Biotechnology Inc., sc-271034) according to the manufacturer's instructions. Fluorescent images were captured using a fluorescence microscope (Ti; Nikon Corporation, Japan). Migration assay Cell migration was examined in Transwell cell culture chambers with gelatin-coated polycarbonate membranes. Cells were pretreated with 0.1% FBS for 5 h at 37 °C. The HO-8910PM cells were harvested (4.7 × 105/ml) and were filled with FAPα (100 PM), Y27632 (100 μmol/l), Y27632 and FAPα, NSC23766 (50 μmol/l), NSC23766 and FAPα, anti-integrin α3β1 (10 ng/ml), anti-integrin α3β1 and FAPα, Human PAI-1 (500 ng/ml), PAI-1 and FAPα. The lower chamber was filled with 800 μl RPMI 1640 containing 10% FBS and then incubated for 48 h. All non-migrated cells were removed from the upper s of the Transwell membrane with a cotton swab, and migrated cells were fixed and stained with hematoxylin. Migration was quantified by counting the number of stained cells per 40 × magnification field.
HO-8910PM proliferation was evaluated using 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay. Briefly, HO-8910PM were trypsinized and plated at a density of 4 × 104/ml cells in 150 μl of 5% FBS for 12 h, cells were washed twice with PBS and then incubated with FAPα, anti-integrin α3β1 mAb, anti-integrin α3β1 and FAPα, NSC23766, NSC23766 and FAPα, Y27632 and FAPα, in HO-8910PM medium with 0.1% FBS. Cultured medium with 0.1% FBS was used as negative control. Cell proliferation was measured by adding 20 μl of MTT (5 μg/ml) reagent which was added to each well from 0 h period till 48 h after the treatment of different reagents. The plates were incubated for 2 h at 37 °C. Formazan crystals formed were dissolved in 150 μl of dimethyl-sulfoxide and optical density was measured at 490 nm on a Bio-Rad microplate reader. Results Integrin α3β1 and uPAR expression in HO-8910PM cells and ovarian cancer tissues Integrin α3β1 and uPAR were expressed in HO-8910PM cells by Western blotting (Fig. 1A). Immunofluorescence experiments showed the expression of integrin α3β1 and uPAR in ovarian cancer cell line HO-8910PM cells (Fig. 1B) and ovarian cancer tissues, and the expression at the same location (Fig. 1C). Co-immunoprecipitation showed that both integrin α3β1 and uPAR express and form a protein complex on the surface of HO-8910PM (Fig. 1D). FAPα and integrinα3β1- uPAR dimer are required for HO-8910PM migration in vitro Transwell invasion showed that only by using uPAR inhibitor PAI-1, the ability of FAPα promotion HO-8910PM cells to invade, migrate and proliferate was not altered, thus verifying the relationship between FAPα and uPAR (*P > 0.05). However, when the activity of integrin α3β1 was inhibited, the FAPα promoted cell proliferation; invasion and migration were significantly reduced in HO-8910PM cells (**P b 0.01) (Fig. 2). FAPα intracellular signal transduction pathway related to Rac1, but not with ROCK and Rho GTP Transwell invasion showed that using Toxin B to inhibit the Rho GTP activity, and then giving FAPα, the function of FAPα is not affected at all (P > 0.05). Y27632 (ROCK inhibitor) can inhibit the cell invasion and migration, but giving FAPα after Y27632, the function of FAPα is not affected (P > 0.05). In particular, a strong activity of NSC23766 was observed in HO-8910PM cells, which were characterized
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Fig. 1. Integrin α3β1 and uPAR expression on HO-8910 PM and ovarian cancer tissue. (A) Western blot analysis of integrin α3β1 and uPAR expression on HO-8910 PM cell lines. (B–D) Immunofluorescences show integrin α3β1 and uPAR expression on ovarian cancer cell line HO-8910 PM and ovarian cancer tissue. (C) Integrin α3β1 and uPAR formed complex in HO-8910 PM cell lines, the conclusion was assessed by co-immunoprecipitation.
Fig. 2. Transwell invasion test. FAP and integrin α3β1, uPAR interaction in the extracellular signaling transduction. Ovarian cancer cell line HO-8910 PM treated with integrin α3β1 antibody, the invasion ability of FAPα promoted tumor cells decreased significantly (P b 0.01). Treated with the uPAR inhibitor (PAI-1), the invasion ability of FAP α promotion tumor cells did not change significantly (P > 0.05).
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by inhibiting the invasion with Matrigel (Fig. 3). Giving FAPα following NSC23766, the function of FAPα was inhibited significantly (**P b 0.01). We conclude that, in the HO-8910PM cancer cell line, the level of active Rac1, but not of ROCK, is inversely correlated with the ability of the ovarian cancer cells to invade.
promoted the expression of phosphorylated ERK. While the cells stimulated with FAPα for 0.5 h, 1.5 h, 3 h, 6 h, 12 h, 24 h, the most significant expression of phosphorylated MLC was 0.5 h, the first time point, and its expression was decreased as the time increases (Fig. 5).
FAPα stimulates the proliferation of HO-8910PM, which can be suppressed by integrin α3β1 and Rac1
Discussion
To confirm these results, proliferation assay was performed at different time points. After 12 h, FAPα promoted the proliferation of HO-8910PM cells, while anti-integrin α3β1 antibody inhibited the proliferation of HO-8910PM cells and the effect of FAPα. After 24 h, NSC23766 reduced HO-8910PM cell proliferation significantly (**P b0.01). At 48 h, Y27632 had no any effect on the proliferation of HO-8910PM cells; on the contrary, it promoted their proliferation (Fig. 4). FAPα increases phosphorylation of MLC and ERK in HO-8910PM This study examined whether FAPα could increase the expression of phosphorylated ERK and phosphorylated MLC in HO-8910PM cells. As shown in Fig. 5, HO-8910PM cells were treated for 3 h, 6 h, 12 h with FAPα (100 pM), increased the expression of phosphorylated ERK in HO-8910PM cells, and the time of 3 h most significantly
Previous reports have shown that FAPα increases ovarian cancer cell proliferation, invasion and migration (He, 2009); however, the mechanisms by which FAPα promotes HO-8910PM tumor cell proliferation, invasion and migration are unknown. In this study, we investigated the possible relationship between FAPα, integrin α3β1, uPAR and Rho GTPase family members in ovarian cancer cell proliferation, invasion, and migration. We show that ovarian cancer cell invasion is correlated with a marked decrease in integrin α3β1 and Rac1 activity, but not with ROCK activity. The uPA/uPAR system regulates migration of various cell types. These effects extend far beyond localizing proteolytic activity to the cell surface. Recent data demonstrates that the level of uPAR protein expression affects the migratory properties of cells in a uPA-independent manner. The uPAR protein contains three domains (DI, DII and DIII) (Gardsvoll, 1999). The DI domain serves as the ligand-binding site for uPA (Gardsvoll, 1999), while the DII and DIII domains contain the binding sites for proteins
Fig. 3. Effect on invasion ability of HO8910PM cells. (A) HO8910PM cells were treated with, FAPα (100 PM), Y27632 (100 umol/l) Y27632 + FAPα, NSC23766 (50 umol/l), NSC23766 + FAPα interaction in the intracellular signaling transduction. Toxin B didn't play any role for tumor cell invasion though a plate chamber filter to the vehicle-treated controls. T-test of two independent samples with the control (*P b 0.05), t-test of two independent samples of with FAP alone (**P b 0.01).
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Fig. 4. Colorimetric MTT assay of HO-8910 PM proliferation. HO8910PM cells were treated with FAPα (100 PM), anti-integrin α3β1 (10 ng/ml), anti-integrin α3β1 + FAPα, NSC23766 (50 umol/l), NSC23766 + FAPα, Y27632 (100 umol/l) and Y27632 + FAPα or vehicle for different time points as indicated, and then tested for proliferation using colorimetric MTT assay. T-test of two independent samples with the control for the same point. T-test of two independent samples with FAP alone for the same point.
such as integrins and vitronectin (Vn) (Chaurasia, 2006; Gardsvoll and Ploug, 2007). Co-immunoprecipitation experiments confirmed that integrin α3β1 and uPAR formed a dimer that was bound to the surface of HO-8910PM cells and that FAPα mediated the signal through integrin α3β1. Upon uPAR inhibition by PAI-1, the ability of FAPα to promote HO-8910PM cells to invade, migrate and proliferate was not altered, indicating (P > 0.05). However, when the activity of integrin α3β1 was inhibited, the FAPα significantly lost its ability to promote cell proliferation, invasion and migration in HO-8910PM cells (P b 0.01). Integrin β1 expression is associated with non-small cell lung cancer lymph node metastasis, and integrin β1-positive cancer cells contain strong invasion and migration capabilities (Aguirre Ghiso, 1999). In oral squamous carcinoma cells, the uPAR-integrin α3β1 interaction potentiates cellular signal transduction, leads to the activation of uPA expression, and enhanced uPA-dependent invasive behavior (Ghosh, 2006). uPAR regulates cell migration through intracellular signaling, which is induced upon uPA binding. Various signaling pathways are reported to be induced by uPA in various cell types. The response to uPA stimulation and the subsequent increase in cell migration seem to be cell specific. Other reports indicate that (Vassalli et al., 1985) the interaction between the integrin β helix and uPAR plays a role in extracellular signal transduction. To examine the intracellular signaling pathway mechanisms, we focused on the relationship between FAPα and the Rho-GTPase family. We found that GTP-RhoA was associated with proteins of the uPAR signaling complex. In this experiment, we tested FAPα intracellular signaling through GTP-RhoA. We found that Toxin B can specifically inhibit
Fig. 5. Western blot analysis of ERK, phosphorylated ERK, MLC, phosphorylated MLC expression in HO-8910 PM cell lines. Treated with the FAPα (100 pM) 0.6 h, 12 h, 24 h, 48 h, 72 h test the protein of ERK, pERK, MLC, pMLC expression. Protein ERK without any changes, pERK began to decline significantly in 12 h. MLC and pMLC didn't have any changes.
the total activity of Rho-GTP family members. Thus, by blocking the Rho-GTP pathway with Toxin B and subsequently treating the cells with FAPα, we show that the role of FAPα in HO-8910PM cell signaling was significantly reduced (P b 0.05). These results suggest that FAPα plays a role in completing intracellular signal transduction through some Rho-GTP family member, and promotes tumor cell proliferation, invasion and migration. The Y27632, an inhibitor for blocking activation of the RhoA/ROCK pathway, failed to inhibit cell proliferation, invasion and metastasis in HO-8910PM cells upon FAPα treatment, Thus, FAPα's role is independent of the RhoA/ROCK pathway. NSC23766, an inhibitor of Rac1-GTP, inhibits the cell proliferation, invasion and metastasis of HO-8910PM cells, particularly during migration. When NSC23766 was combined with FAPα treatment in HO-8910PM cells, the role of FAPα was downplayed. These data suggest that FAPα play a role in intracellular signal transduction through the Rac1-GTP pathway. Overexpression of uPAR and its binding to vitronectin lead to Rac1 activation and cytoskeleton rearrangements (Kjøller and Hall, 2001). Furthermore, similar effects were observed even at a basal level of uPAR expression. Lipoprotein receptor-related protein (LRP-1) regulates the basal level of Rac1 activation via endocytosis and the substantial down-regulation of cell-surface uPAR (Ma, 2002). This RhoA/ROCK-mediated increase in MLC phosphorylation levels and contraction occurs in the absence of calcium (Kimura et al., 1996), whereas myosin light chain kinase (MLCK) phosphorylates MLC in a Ca2+-dependent manner (Katoh, 2001). These two kinases may play distinct roles in the spatial regulation of myosin II activity. The RhoA/ROCK signaling pathway plays an important role in generating myosin II-based contractility in the center of the cell (Kimura et al., 1996; Kawano, 1999; Chrzanowska-Wodnicka and Burridge, 1996; Totsukawa, 2000), which is necessary for cell adhesion and tail retraction during migration (Worthylake, 2001). To investigate the functional role of FAPα in ovarian cancer, we assessed the roles of p-ERK, ERK, MLC and p-MLC following FAPα stimulation of ovarian cancer cells. In this study, the FAPα intracellular signaling has no relationship with ROCK; therefore, expression of MLC and phospho-MLC was not increased in HO-8910PM cells when compared to control cells, suggesting that MLC and phospho-MLC are not significantly involved in FAPα signaling in ovarian cancer cells. Phospho-ERK expression increased in a time-dependent manner, although total ERK levels did not change. This result suggests that FAPα promotes cell proliferation by upregulating p-ERK expression.
Conflict of interest The authors declare that there are no conflicts of interest.
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Acknowledgments This work was supported by Heilongjiang Province Education Committee of China (no. 12511182).
References Aguirre Ghiso, J.A., 1999. Tumor dormancy induced by downregulation of urokinase receptor in human carcinoma involves integrin and MAPK signaling. Journal of Cell Biology 147, 89–104. Artym, V.V., 2002. Molecular receptor on malignant melanoma cell membranes: dependence on beta1 integrins and the cytoskeleton. Carcinogenesis 23, 1593–1601. Chaurasia, P., 2006. A region in urokinase plasminogen receptor domain III controlling a functional association with α5β1 integrin and tumor growth. Journal of Biological Chemistry 281, 14852–14863. Chrzanowska-Wodnicka, M., Burridge, K., 1996. Rho-stimulated contractility drives the formation of stress fibers and focal adhesions. Journal of Cell Biology 133, 1403–1415. Gardsvoll, H., 1999. Mapping part of the functional epitope for ligand binding on the receptor for urokinase-type plasminogen activator by site-directed mutagenesis. Journal of Biological Chemistry 274, 37995–38003. Gardsvoll, H., Ploug, M., 2007. Mapping of the vitronectin binding site on the urokinase receptor involvement of a coherent receptor: interface consisting of residues from both domain I and the flanking interdomain linker region. Journal of Biological Chemistry 282, 13561–13572. Ghosh, S., 2006. Functional relevance of urinary-type plasminogen activator receptorα3β1 integrin association in proteinase regulatory pathways. Journal of Biological Chemistry 281, 13021–13029. Goldstein, L.A., 1997. Molecular cloning of seprase: a serine integral membrane protease from human melanoma. Biochimica et Biophysica Acta 1361, 11–19. Hartshorne, D.J., 1998. Myosin phosphatase: subunits and interactions. Acta Physiologica Scandinavica 164, 483–493.
He, Chen, 2009. TGF-beta-induced fibroblast activation protein expression, fibroblast activation protein expression increases the proliferation, adhesion, and migration of HO-8910PM. Experimental and Molecular Pathology 87, 189–194. Jaffe, A.B., Hall, A., 2005. Rho GTPases: biochemistry and biology. Annual Review of Cell and Developmental Biology 21, 247–269. Katoh, K., 2001. Rho-kinase-mediated contraction of isolated stress fibers. Journal of Cell Biology 153, 569–584. Kawano, Y., 1999. Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by Rho-kinase in vivo. European Journal of Cell Biology 147, 1023–1038. Kiian, I., 2003. Urokinase-induced migration of human vascular smooth muscle cells requires coupling of the small GTPases RhoA and Rac1 to the Tyk2/PI3-K signalling pathway. Thrombosis and Haemostasis 89, 904–914. Kimura, K., et al., 1996. Regulation of myosin phosphatase by Rho and Rho associated kinase (Rho-kinase). Science 273, 245–248. Kjøller, L., Hall, A., 2001. Rac mediates cytoskeletal rearrangements and increased cell motility induced by urokinase-type plasminogen activator receptor binding to vitronectin. Journal of Cell Biology 152, 1145–1157. Ma, Z., 2002. Regulation of Rac1 activation by the low density lipoprotein receptorrelated protein. Journal of Cell Biology 159, 1061–1070. Mueller, S.C., 1999. A novel protease docking function of integrin at invadopodia. Journal of Biological Chemistry 274, 24947–24952. Olson, M.F., Sahai, E., 2009. The actin cytoskeleton in cancer cell motility. Clinical & Experimental Metastasis 26, 273–287. Schmitz, A.A., 2000. Rho GTPases:signaling, migration, and invasion. Experimental Cell Research 261, 1–12. Shenhua, X., 1999. Establishment of a highly metastatic human ovarian cancer cell line (HO-8910 PM) and its characterization. Journal of Experimental and Clinical Cancer Research 18, 233–239. Totsukawa, G., 2000. Distinct roles of ROCK (Rho-kinase) and MLCK in spatial regulation of MLC phosphorylation for assembly of stress fibers and focal adhesions in 3T3 fibroblasts. Journal of Cell Biology 150, 797–806. Vassalli, J.D., et al., 1985. A cellular binding site for the Mr 55,000 form of the human plasminogen activator, urokinase. The Journal of Cell Biology 100, 86–92. Worthylake, R.A., 2001. RhoA is required for monocyte tail retraction during transendothelial migration. Journal of Cell Biology 154, 147–160.
Contents lists available at SciVerse ScienceDirect
Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp
Fibroblast activation protein-α promotes ovarian cancer cell proliferation and invasion via extracellular and intracellular signaling mechanisms WeiWei Yang a,1, Wei Han b,1, ShengQian Ye a, DuanYang Liu a, Jun Wu c, HongYu Liu d, ChunHong Li a, He Chen a,⁎ a
Department of Pathology, Harbin Medical University, Harbin 150081, China Department of Pathology, The First Affiliated Hospital of Harbin Medical University, Harbin 150081, China c Department of Thoracic Surgery, Harbin Medical University Cancer Hospital, Harbin 150081, China d Department of Pathology, The First Hospital of Qiqihar, Qiqihar 161005, China b
a r t i c l e
i n f o
Article history: Received 27 May 2013 Available online 15 June 2013 Keywords: Fibroblast activation protein-α (FAPα) Urokinase-type plasminogen activator receptor (uPAR) Integrin α3β1 Ovarian cancer
a b s t r a c t Fibroblast activation protein-α (FAPα) is secreted by activated stromal fibroblasts and can promote ovarian cancer cell proliferation, migration and invasion. However, the molecular mechanism by which FAPα promotes tumor cell proliferation and invasion is unknown. The role of the non-enzymatic activities of FAPα in tumor migration and invasion and the intracellular and extracellular signaling mechanisms of FAPα were investigated. In this study, we confirm that FAPα promote ovarian cancer cell proliferation, migration and invasion by extracellular and intracellular signaling mechanisms. These results provide evidence that FAPα, together with integrin α3β1 and the uPAR signaling complex, mediate cancer cell migration in the HO-8910PM cell line via the small GTPase Rac1. FAPα-mediated upregulation of p-ERK occurred in a time-dependent manner. © 2013 Elsevier Inc. All rights reserved.
Introduction Ovarian cancer is a common malignancy and one of the leading causes of morbidity and mortality worldwide. Fibroblast activation protein-α (FAPα), also known as seprase (Goldstein, 1997), is secreted by activated stromal fibroblasts and can promote ovarian cancer cell proliferation, migration and invasion. Here, the role of the non-enzymatic activities of FAPα in tumor migration and invasion and the intracellular and extracellular signaling mechanisms of FAPα were investigated. The integrin family of transmembrane adhesion proteins exhibits multiple functions, including adhesion to the ECM and localization to invadopodia (Mueller, 1999). One hypothesis suggests that integrins are involved in the recruitment of proteases to sites of cell invasion. The integrin α3β1 associates with FAPα, and it is thought to participate in the formation of functional invadopodia through a docking interaction (Mueller, 1999). A recent study has shown that FAPα-Urokinase-type plasminogen activator receptor (uPAR) membrane complexes associate with the invadopodia of LOX cells, suggesting a co-operative role for uPAR in tumor invasion (Artym, 2002). This interaction between uPA and integrins regulates both cell adhesion and integrin signaling activities. ⁎ Corresponding author. E-mail address: [email protected] (H. Chen). 1 Contributed equally to this work. 0014-4800/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexmp.2013.06.007
GTP-RhoA also associates with the uPAR signaling complex (Kiian, 2003). Members of the Rho GTPase family are key regulators of actomyosin cytoskeleton activity, which is required for invasion and metastasis (Jaffe and Hall, 2005). The bundling and contraction of actin– myosin fibers provide the force required for cell motility and invasion (Sahai, 2009). The Rho family members, including RhoA, Rac1 and ROCK, are crucial regulators of actin cytoskeleton dynamics. Thus, these regulators control cell shape, cell adhesion and the contractions that drive cell migration (Schmitz, 2000). Among these effectors is the serine-threonine protein kinase (ROCK), which controls the activity of myosin II indirectly by phosphorylating the myosin-binding subunit of the myosin light chain (MLC) phosphatase, thus inhibiting MLC dephosphorylation (Hartshorne, 1998; Kawano, 1999; Kimura et al., 1996).
Materials and methods Cell culture The HO-8910PM cells, a highly metastatic ovarian cancer cell line derived from HO-8910 (Shenhua, 1999). Cells were grown in RPMI 1640 10% FBS, 1% penicillin and streptomycin, 5% CO2 at 37 °C. Both HO-8910 and HO-8910PM were purchased from Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Science.
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Western blotting
Invasion assay
HO-8910PM cells grown in 100-mm dishes were washed in cold PBS (pH 8.0) for 3 times before they were collected. HO-8910PM cells were extracted and centrifuged at 12,000 rpm for 30 min at 4 °C. The samples were subjected to electrophoresis and then transferred to Hybond ECL membrane. The membrane was incubated for 1 h in blocking solution with primary antibody anti-integrin α3 (1:2000 Santa Cruz Biotechnology, Inc.), anti-integrin β1 (1:2000 Santa Cruz Biotechnology, Inc.) and anti-uPAR (1:500 Santa Cruz Biotechnology, Inc.), primary antibody incubated at 4 °C overnight. Then, the membrane was incubated with a phosphate-conjugated, secondary antibody at 37 °C for 30 min. After extensive washing, the immunoblots were visualized with western blue (Promega Corporation, USA). The methods of Western-blotting of ERK, p-ERK, MLC, p-MLC protein express the same as the front. The β-actin was assessed as an internal loading control.
The assay was performed using chambers with polycarbonate filters with 8-μm nominal pore size (Millipore) coated on the upper side with Matrigel (BD Biosciences, Bedford, MA). The chambers were placed into a 24-well plate. Twelve groups of HO-8910PM cells (1.3 × 106/ml) were harvested and placed in the upper chamber. Cells treated with FAPα (100 pM), Y27632 (100 μmol/l), Y27632 (ROCK inhibitor) and FAPα, NSC23766 (50 μmol/l), NSC23766 (Rac1 inhibitor) and FAPα, anti-integrin α3β1 (10 ng/ml), anti-integrin α3β1 and FAPα, PAI-1 (500 ng/ml), PAI-1 and FAPα, and untreated cells (blank control). The lower chamber was filled with 800 μl RPMI 1640 containing 10% FBS and then incubated for 48 h at 37 °C in a humidified atmosphere containing 5%CO2. Cells remaining in the upper chamber were completely removed by gently swabbing. invading cells which were counted in microscope fields for each membrane at 40× magnification. Proliferation assay
Co-immunoprecipitation The HO-8910PM cells were collected and extracted by the same method as Western blotting. Three independent EP tubes were added to anti-integrin α3, anti-integrin β1 and anti-uPAR at 4 °C overnight. Then, protein G was added for 4 h. They were centrifuged and precipitation was collected, and they were washed three times with RIPA buffer. The samples were subjected to electrophoresis and then transferred to Hybond ECL membrane. The follow up steps were the same as that of Western blotting. Immunofluorescence HO-8910PM were cultured in the 6-well plate, incubated at 37 °C for 8 h, and cells were fixed in cold 4% (v/v) paraformaldehyde for 20 min at room temperature. After permeabilization and blocking with 0.3% (v/v) Triton X-100, cells were respectively probed with primary antibody of integrin α3β1 (1:200), at 37 °C for 1 h, then uPAR primary antibody was added, at 4 °C overnight. Cells were washed and probed with FITC-anti-rabbit antibody (1:200) and TRITC-anti-mouse antibody respectively (1:200) (All Sigma-Aldrich) as secondary antibodies for incubation at 37 °C for 60 min in the dark. All ovarian cancer specimens were from patients (30 primary ovarian cancers) who underwent surgery in Harbin Medical University Cancer Hospital from 2007 to 2011. None of the patients received preoperative treatments such as radiotherapy or chemotherapy. The expressions of integrin α3β1 and uPAR were examined with primary antibody dilution (1:200), fluorescence labeled second antibody (1:200), in consecutive tissue sections using the LSAB + kit (Santa Cruz Biotechnology Inc., sc-271034) according to the manufacturer's instructions. Fluorescent images were captured using a fluorescence microscope (Ti; Nikon Corporation, Japan). Migration assay Cell migration was examined in Transwell cell culture chambers with gelatin-coated polycarbonate membranes. Cells were pretreated with 0.1% FBS for 5 h at 37 °C. The HO-8910PM cells were harvested (4.7 × 105/ml) and were filled with FAPα (100 PM), Y27632 (100 μmol/l), Y27632 and FAPα, NSC23766 (50 μmol/l), NSC23766 and FAPα, anti-integrin α3β1 (10 ng/ml), anti-integrin α3β1 and FAPα, Human PAI-1 (500 ng/ml), PAI-1 and FAPα. The lower chamber was filled with 800 μl RPMI 1640 containing 10% FBS and then incubated for 48 h. All non-migrated cells were removed from the upper s of the Transwell membrane with a cotton swab, and migrated cells were fixed and stained with hematoxylin. Migration was quantified by counting the number of stained cells per 40 × magnification field.
HO-8910PM proliferation was evaluated using 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay. Briefly, HO-8910PM were trypsinized and plated at a density of 4 × 104/ml cells in 150 μl of 5% FBS for 12 h, cells were washed twice with PBS and then incubated with FAPα, anti-integrin α3β1 mAb, anti-integrin α3β1 and FAPα, NSC23766, NSC23766 and FAPα, Y27632 and FAPα, in HO-8910PM medium with 0.1% FBS. Cultured medium with 0.1% FBS was used as negative control. Cell proliferation was measured by adding 20 μl of MTT (5 μg/ml) reagent which was added to each well from 0 h period till 48 h after the treatment of different reagents. The plates were incubated for 2 h at 37 °C. Formazan crystals formed were dissolved in 150 μl of dimethyl-sulfoxide and optical density was measured at 490 nm on a Bio-Rad microplate reader. Results Integrin α3β1 and uPAR expression in HO-8910PM cells and ovarian cancer tissues Integrin α3β1 and uPAR were expressed in HO-8910PM cells by Western blotting (Fig. 1A). Immunofluorescence experiments showed the expression of integrin α3β1 and uPAR in ovarian cancer cell line HO-8910PM cells (Fig. 1B) and ovarian cancer tissues, and the expression at the same location (Fig. 1C). Co-immunoprecipitation showed that both integrin α3β1 and uPAR express and form a protein complex on the surface of HO-8910PM (Fig. 1D). FAPα and integrinα3β1- uPAR dimer are required for HO-8910PM migration in vitro Transwell invasion showed that only by using uPAR inhibitor PAI-1, the ability of FAPα promotion HO-8910PM cells to invade, migrate and proliferate was not altered, thus verifying the relationship between FAPα and uPAR (*P > 0.05). However, when the activity of integrin α3β1 was inhibited, the FAPα promoted cell proliferation; invasion and migration were significantly reduced in HO-8910PM cells (**P b 0.01) (Fig. 2). FAPα intracellular signal transduction pathway related to Rac1, but not with ROCK and Rho GTP Transwell invasion showed that using Toxin B to inhibit the Rho GTP activity, and then giving FAPα, the function of FAPα is not affected at all (P > 0.05). Y27632 (ROCK inhibitor) can inhibit the cell invasion and migration, but giving FAPα after Y27632, the function of FAPα is not affected (P > 0.05). In particular, a strong activity of NSC23766 was observed in HO-8910PM cells, which were characterized
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Fig. 1. Integrin α3β1 and uPAR expression on HO-8910 PM and ovarian cancer tissue. (A) Western blot analysis of integrin α3β1 and uPAR expression on HO-8910 PM cell lines. (B–D) Immunofluorescences show integrin α3β1 and uPAR expression on ovarian cancer cell line HO-8910 PM and ovarian cancer tissue. (C) Integrin α3β1 and uPAR formed complex in HO-8910 PM cell lines, the conclusion was assessed by co-immunoprecipitation.
Fig. 2. Transwell invasion test. FAP and integrin α3β1, uPAR interaction in the extracellular signaling transduction. Ovarian cancer cell line HO-8910 PM treated with integrin α3β1 antibody, the invasion ability of FAPα promoted tumor cells decreased significantly (P b 0.01). Treated with the uPAR inhibitor (PAI-1), the invasion ability of FAP α promotion tumor cells did not change significantly (P > 0.05).
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by inhibiting the invasion with Matrigel (Fig. 3). Giving FAPα following NSC23766, the function of FAPα was inhibited significantly (**P b 0.01). We conclude that, in the HO-8910PM cancer cell line, the level of active Rac1, but not of ROCK, is inversely correlated with the ability of the ovarian cancer cells to invade.
promoted the expression of phosphorylated ERK. While the cells stimulated with FAPα for 0.5 h, 1.5 h, 3 h, 6 h, 12 h, 24 h, the most significant expression of phosphorylated MLC was 0.5 h, the first time point, and its expression was decreased as the time increases (Fig. 5).
FAPα stimulates the proliferation of HO-8910PM, which can be suppressed by integrin α3β1 and Rac1
Discussion
To confirm these results, proliferation assay was performed at different time points. After 12 h, FAPα promoted the proliferation of HO-8910PM cells, while anti-integrin α3β1 antibody inhibited the proliferation of HO-8910PM cells and the effect of FAPα. After 24 h, NSC23766 reduced HO-8910PM cell proliferation significantly (**P b0.01). At 48 h, Y27632 had no any effect on the proliferation of HO-8910PM cells; on the contrary, it promoted their proliferation (Fig. 4). FAPα increases phosphorylation of MLC and ERK in HO-8910PM This study examined whether FAPα could increase the expression of phosphorylated ERK and phosphorylated MLC in HO-8910PM cells. As shown in Fig. 5, HO-8910PM cells were treated for 3 h, 6 h, 12 h with FAPα (100 pM), increased the expression of phosphorylated ERK in HO-8910PM cells, and the time of 3 h most significantly
Previous reports have shown that FAPα increases ovarian cancer cell proliferation, invasion and migration (He, 2009); however, the mechanisms by which FAPα promotes HO-8910PM tumor cell proliferation, invasion and migration are unknown. In this study, we investigated the possible relationship between FAPα, integrin α3β1, uPAR and Rho GTPase family members in ovarian cancer cell proliferation, invasion, and migration. We show that ovarian cancer cell invasion is correlated with a marked decrease in integrin α3β1 and Rac1 activity, but not with ROCK activity. The uPA/uPAR system regulates migration of various cell types. These effects extend far beyond localizing proteolytic activity to the cell surface. Recent data demonstrates that the level of uPAR protein expression affects the migratory properties of cells in a uPA-independent manner. The uPAR protein contains three domains (DI, DII and DIII) (Gardsvoll, 1999). The DI domain serves as the ligand-binding site for uPA (Gardsvoll, 1999), while the DII and DIII domains contain the binding sites for proteins
Fig. 3. Effect on invasion ability of HO8910PM cells. (A) HO8910PM cells were treated with, FAPα (100 PM), Y27632 (100 umol/l) Y27632 + FAPα, NSC23766 (50 umol/l), NSC23766 + FAPα interaction in the intracellular signaling transduction. Toxin B didn't play any role for tumor cell invasion though a plate chamber filter to the vehicle-treated controls. T-test of two independent samples with the control (*P b 0.05), t-test of two independent samples of with FAP alone (**P b 0.01).
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Fig. 4. Colorimetric MTT assay of HO-8910 PM proliferation. HO8910PM cells were treated with FAPα (100 PM), anti-integrin α3β1 (10 ng/ml), anti-integrin α3β1 + FAPα, NSC23766 (50 umol/l), NSC23766 + FAPα, Y27632 (100 umol/l) and Y27632 + FAPα or vehicle for different time points as indicated, and then tested for proliferation using colorimetric MTT assay. T-test of two independent samples with the control for the same point. T-test of two independent samples with FAP alone for the same point.
such as integrins and vitronectin (Vn) (Chaurasia, 2006; Gardsvoll and Ploug, 2007). Co-immunoprecipitation experiments confirmed that integrin α3β1 and uPAR formed a dimer that was bound to the surface of HO-8910PM cells and that FAPα mediated the signal through integrin α3β1. Upon uPAR inhibition by PAI-1, the ability of FAPα to promote HO-8910PM cells to invade, migrate and proliferate was not altered, indicating (P > 0.05). However, when the activity of integrin α3β1 was inhibited, the FAPα significantly lost its ability to promote cell proliferation, invasion and migration in HO-8910PM cells (P b 0.01). Integrin β1 expression is associated with non-small cell lung cancer lymph node metastasis, and integrin β1-positive cancer cells contain strong invasion and migration capabilities (Aguirre Ghiso, 1999). In oral squamous carcinoma cells, the uPAR-integrin α3β1 interaction potentiates cellular signal transduction, leads to the activation of uPA expression, and enhanced uPA-dependent invasive behavior (Ghosh, 2006). uPAR regulates cell migration through intracellular signaling, which is induced upon uPA binding. Various signaling pathways are reported to be induced by uPA in various cell types. The response to uPA stimulation and the subsequent increase in cell migration seem to be cell specific. Other reports indicate that (Vassalli et al., 1985) the interaction between the integrin β helix and uPAR plays a role in extracellular signal transduction. To examine the intracellular signaling pathway mechanisms, we focused on the relationship between FAPα and the Rho-GTPase family. We found that GTP-RhoA was associated with proteins of the uPAR signaling complex. In this experiment, we tested FAPα intracellular signaling through GTP-RhoA. We found that Toxin B can specifically inhibit
Fig. 5. Western blot analysis of ERK, phosphorylated ERK, MLC, phosphorylated MLC expression in HO-8910 PM cell lines. Treated with the FAPα (100 pM) 0.6 h, 12 h, 24 h, 48 h, 72 h test the protein of ERK, pERK, MLC, pMLC expression. Protein ERK without any changes, pERK began to decline significantly in 12 h. MLC and pMLC didn't have any changes.
the total activity of Rho-GTP family members. Thus, by blocking the Rho-GTP pathway with Toxin B and subsequently treating the cells with FAPα, we show that the role of FAPα in HO-8910PM cell signaling was significantly reduced (P b 0.05). These results suggest that FAPα plays a role in completing intracellular signal transduction through some Rho-GTP family member, and promotes tumor cell proliferation, invasion and migration. The Y27632, an inhibitor for blocking activation of the RhoA/ROCK pathway, failed to inhibit cell proliferation, invasion and metastasis in HO-8910PM cells upon FAPα treatment, Thus, FAPα's role is independent of the RhoA/ROCK pathway. NSC23766, an inhibitor of Rac1-GTP, inhibits the cell proliferation, invasion and metastasis of HO-8910PM cells, particularly during migration. When NSC23766 was combined with FAPα treatment in HO-8910PM cells, the role of FAPα was downplayed. These data suggest that FAPα play a role in intracellular signal transduction through the Rac1-GTP pathway. Overexpression of uPAR and its binding to vitronectin lead to Rac1 activation and cytoskeleton rearrangements (Kjøller and Hall, 2001). Furthermore, similar effects were observed even at a basal level of uPAR expression. Lipoprotein receptor-related protein (LRP-1) regulates the basal level of Rac1 activation via endocytosis and the substantial down-regulation of cell-surface uPAR (Ma, 2002). This RhoA/ROCK-mediated increase in MLC phosphorylation levels and contraction occurs in the absence of calcium (Kimura et al., 1996), whereas myosin light chain kinase (MLCK) phosphorylates MLC in a Ca2+-dependent manner (Katoh, 2001). These two kinases may play distinct roles in the spatial regulation of myosin II activity. The RhoA/ROCK signaling pathway plays an important role in generating myosin II-based contractility in the center of the cell (Kimura et al., 1996; Kawano, 1999; Chrzanowska-Wodnicka and Burridge, 1996; Totsukawa, 2000), which is necessary for cell adhesion and tail retraction during migration (Worthylake, 2001). To investigate the functional role of FAPα in ovarian cancer, we assessed the roles of p-ERK, ERK, MLC and p-MLC following FAPα stimulation of ovarian cancer cells. In this study, the FAPα intracellular signaling has no relationship with ROCK; therefore, expression of MLC and phospho-MLC was not increased in HO-8910PM cells when compared to control cells, suggesting that MLC and phospho-MLC are not significantly involved in FAPα signaling in ovarian cancer cells. Phospho-ERK expression increased in a time-dependent manner, although total ERK levels did not change. This result suggests that FAPα promotes cell proliferation by upregulating p-ERK expression.
Conflict of interest The authors declare that there are no conflicts of interest.
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Acknowledgments This work was supported by Heilongjiang Province Education Committee of China (no. 12511182).
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