Autocrine CCL2 promotes cell migration and invasion via PKC activation and tyrosine phosphorylation of paxillin in bladder cancer cells

Cytokine 59 (2012) 423–432

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Cytokine journal homepage: www.elsevier.com/locate/issn/10434666

Autocrine CCL2 promotes cell migration and invasion via PKC activation and tyrosine phosphorylation of paxillin in bladder cancer cells Hsiao-Ying Chiu a, Kuang-Hui Sun b, Shiow-Yi Chen a, Hsiao-Hsien Wang c, Ming-Yung Lee d, Yu-Chi Tsou a, Shyh-Chuan Jwo e, Guang-Huan Sun f,⇑, Shye-Jye Tang a,⇑ a

Institute of Bioscience and Biotechnology, Center of Excellence for Marine Bioenvironment and Biotechnology (CMBB), National Taiwan Ocean University, Keelung, Taiwan Department of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan Section of Urology, Cheng-Hsin Rehabilitation Medical Center, Taipei, Taiwan d Department of Pediatrics, Tri-service General Hospital, National Defense Medical Center, Keelung, Taiwan e Department of General Surgery, Chang Gung Memorial Hospital, Keelung, Taiwan f Department of Urology and Surgery, Tri-service General Hospital, National Defense Medical Center, Taipei, Taiwan b c

a r t i c l e

i n f o

Article history: Received 15 February 2012 Received in revised form 27 March 2012 Accepted 11 April 2012 Available online 20 May 2012 Keywords: Bladder cancer MCP-1/CCL2 Paxillin PKC Cell migration

a b s t r a c t The amount of monocyte chemoattractant protein-1 (MCP-1/CCL2) produced by a transitional cell carcinoma is directly correlated with high recurrence and poor prognosis in bladder cancer. However, the mechanisms underlying the effects of CCL2 on tumor progression remain unexplored. To investigate the role played by CCL2, we examined cell migration in various bladder cancer cell lines. We found that high-grade cancer cells expressing high levels of CCL2 showed more migration activity than low-grade bladder cancer cells expressing low levels of the chemokine. Although the activation of CCL2/CCR2 signals did not appreciably affect cell growth, it mediated cell migration and invasion via the activation of protein kinase C and phosphorylation of tyrosine in paxillin. Blocking CCL2 and CCR2 with small hairpin RNA (shCCL2) or a specific inhibitor reduced CCL2/CCR2-mediated cell migration. The antagonist of CCR2 promoted the survival of mice bearing MBT2 bladder cancer cells, and CCL2-depleted cells showed low tumorigenicity compared with shGFP cells. In addition to observing high-levels of CCL2 in high-grade human bladder cancer cells, we showed that the CCL2/CCR2 signaling pathway mediated migratory and invasive activity, whereas blocking the pathway decreased migration and invasion. In conclusion, high levels of CCL2 expressed in bladder cancer mediates tumor invasion and is involved with advanced tumorigenesis. Our findings suggest that this CCL2/CCR2 pathway is a potential candidate for the attenuation of bladder cancer metastases. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Chemokines are small secretory proteins that are grouped into two major subfamilies, the CC and CXC chemokines, based on the presence or absence of an amino acid between the first two

Abbreviations: CTC, chelerythrine chloride; GPCR, G protein-coupled receptor; MCP-1, monocyte chemoattractant protein-1; pPaxillin, tyrosine 118 phosphorylated paxillin; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; RS, RS102895; shRNA, short hairpin RNA; TCC, transitional cell carcinomas; WT, wild type. ⇑ Corresponding authors. Addresses: Institute of Bioscience and Biotechnology, Center of Excellence for Marine Bioenvironment and Biotechnology (CMBB), National Taiwan Ocean University, 2 Pei-Ning Road, Keelung 202-24, Taiwan. Tel.: +886 2 24622192x5510; fax: +886 2 24622320 (S.-J. Tang), Department of Urology and Surgery, National Defense Medical Center, No. 325, Sec. 2, Cheng-Kung Road, Tri-service General Hospital, Taipei 114, Taiwan. Tel.: +886 2 87927006; fax: +886 2 87927007 (G.-H. Sun). E-mail addresses: [email protected] (G.-H. Sun), [email protected] (S.-J. Tang). 1043-4666/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cyto.2012.04.017

canonical cysteines [1]. Chemokines have been implicated in a wide variety of diseases, such as rheumatoid arthritis, multiple sclerosis, obesity-induced diabetes, and inflammatory bowel disease [1–3]. Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a member of the CC chemokine family, and it is a potent agonist for monocytes, dendritic cells, memory T cells, and basophils [1]. CCL2-induced migration of monocytes is mediated through interaction with its receptor, CCR2, a G-protein-coupled receptor (GPCR) [1]. CCR2 is regarded as predominantly signal via the Gi/o-coupled heterotrimeric G protein to transduce signals; however, new evidence has revealed that CCR2 is able to couple to a wide spectrum of G proteins to regulate numerous signaling pathways [1]. In addition to its well-characterized role as a chemoattractant for monocytes in the immune response [2], much evidence suggests that CCL2 signaling is closely linked to tumor growth and progression [1,2,4]. The chemokine has been shown to promote prostate cancer proliferation in bone [5]. Expression of CCL2

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recruits tumor-associated macrophages, which is responsible for the prometastatic effect in the ER-negative breast cancers [6]. Moreover, CCL2 directly interacts with CCR2 on the endothelial cell surface, leading to increased vessel sprout formation and angiogenesis [7,8]. It has been shown that polymorphisms of CCL2 and CCR2-64I are associated with transitional bladder cell carcinoma [9,10]. One report indicated that the ability of CCL2 to induce Fas ligand expression and elicit apoptosis contributes to its antitumor activity [11]. Bacillus Calmette-Guérin (BCG) therapy in bladder cancer induces CCL2-dependent local immunological responses also demonstrated an antitumor role for CCL2 [12]. A number of studies have shown that CCL2 stimulates prostate cancer migration via the p70-S6 kinase and Rac-mediated actin remodeling [5,13– 15]. These reports indicate that CCL2 may involve multiple mechanisms to regulate tumor progression and anti-tumor activity. Bladder cancer is the fourth most common malignancy in the United States, and its incidence continues to rise each year [16]. The tumor is generally classified into superficial (non-muscle invasive) or muscle-invasive cancer based on histopathological behaviors [17]. The low-grade bladder tumors (Ta and T1 stages; or G0–G1 grades) are non-muscle invasive cancers, and the highgrade tumors (T2–T4 stages; G2–G3 grades) are muscle-invasive with unfavorable prognosis [17]. Most bladder cancers are transitional cell carcinomas (TCC), and on average 20–30% of patients with non-muscle invasive bladder cancer will subsequently develop muscle-invasive TCC [18]. It has been reported that the correlation of urinary CCL2 levels with tumor stage, grade and distant metastasis is highly significant [9,18,19]. Patients with stages T2–T4 bladder cancer have a three- to four-fold higher mean CCL2 concentration in their urine than those with T1 stage tumors [18]. Moreover, the highly malignant T24 bladder cancer cell line spontaneously secretes large amounts of CCL2, whereas the low-grade RT4 bladder cancer cell line produces only traces of CCL2 [12]. Because CCL2 expression was recently shown to correlate with poor survival in the bladder cancer patients, this chemokine may play a particularly important role in tumor progression [18]. However, the mechanisms by which CCL2/CCR2 activation promotes tumor progression in bladder cancer remain to be explored. In view of the important roles played by CCL2 in cancer progression, we strived to provide insights into the regulation of CCL2-induced cell migration and to investigate molecular mechanisms involving migration in response to the chemokine. In this study, we further addressed the regulation of CCL2-mediated cell migration by focusing on PKC activation and paxillin. Our findings show that CCL2 is expressed at a particularly high level in high-grade bladder cancer cells and that it promotes cell migration by a novel PKCdependent mechanism through phosphorylating tyrosine on paxillin. 2. Materials and methods 2.1. Materials Penicillin, streptomycin, fetal bovine serum, trypsin–EDTA, RPMI1640 medium, and Lipofectamine 2000 transfection reagent were purchased from Invitrogen (Carlsbad, CA). The culture media, Modified Eagle’s medium (MEM) and F-12 medium, were from Hyclone (Pittsburgh, PA). The CCL2 ELISA Kit was purchased from Millipore (Bedford, USA), and recombinant murine JE/CCL2 (rMCP-1) was from PeproTech (New Jersey, USA). The CCR2 antagonist, (10 -[2-[4-(trifluoromethyl)phenyl]ethyl]-spiro[4H-3,1benzoxazine-4,40 -peperidin]-2(1H)-one (RS-102895); the CXCR4 antagonist, 1,10 -[1,4-phenylenebis(methylene)]bis-1,4,8,11-tetraazacyclotetradecane octahydrochloride (ADM3100); the PKC inhibitor, chelerythrine chloride (CTC) and 12-(2-cyanoethyl)6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo (2,3-a) pyrrolo

(3,4-c)-carbazole (GÖ6976); the PKC activator, phorbol 12-myristate 13-acetate (PMA); the MEK1/2 inhibitor, 1,4-diamino-2,3dicyano-1,4-bis (methylthio) butadiene (U0126); puromycin, and all other chemical compounds were obtained from Sigma–Aldrich (St. Louis, USA). Reverse Transcriptase and the PepTag nonradioactive PKC assay kit were from Promega (Madison, WI). The anti-paxillin, anti-phospho-paxillin (Y118), and peroxidase-conjugated goat anti-rabbit antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). 2.2. Cell culture Four human urinary bladder cancer cell lines (SV-HUC-1, TCC grade G0; RT4, TCC G1; and T24 and J82, TCC G3) were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Human urinary TCC grade II bladder cancer cell line TSGH8301 was obtained from the Bioresource Collection and Research Center (Taiwan, ROC). The mouse highly malignant bladder cancer cell line, MBT2 (TCC G3), was obtained from the Japanese Collection of Research Bioresources (JCRB, Japan). The RT4, T24, and TSGH8301 cells were maintained in RPMI1640, the SV-HUC1 cells were maintained in modified F-12 medium, and the J82 and MBT2 cells were maintained in MEM medium. These cells were cultured in media supplemented with 10% fetal bovine serum, 1 mM glutamine, and antibiotics (10 U/ml penicillin and 10 lg/ml streptomycin) at 37 °C in a humidified chamber with 5% CO2. 2.3. Knockdown of CCL2 in MBT2 cells The shRNA constructs targeting murine CCL2 (clone TRCN0000004469, NM_011333) were purchased from the National RNAi Core Facility (NRCF) (Taiwan, ROC). The scrambled shRNA pLKO.1 vector and the shRNA against GFP were used as control. The cells were transiently transfected with shRNA vector (4 lg) using Lipofectamine 2000 according to the manufacturer’s directions (Invitrogen). Cells stably expressing shRNA were obtained following selection with 1 lg/ml puromycin for 14 days. The knockdown of CCL2 in the transfected cells was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and western blot using CCL2 antibody. 2.4. Wound-healing assay The cells (5  105cells/well) were seeded into 24-well plates and cultured until they were 90% confluence. The monolayer of cells was then scratched with a thin disposable tip to make a wound and incubated for 96 h. Cell migration was photographed with an Olympus microscope (Olympus, Tokyo, Japan). Migration activity was expressed as the percentage of the area covered by cells measured using Olympus DP72 software. Three separate visual fields were measured from each experiment. 2.5. Cell migration assay Boyden chambers (8 mm pore size; Corning Costar Corp., Cambridge, MA, USA) containing membrane filter inserted in 24well tissue culture plates were used to analyze cell migration. Cells (3  104/100 ll) were added to the upper well of the chamber and incubated for 96 h at 37 °C. The treatment mixture of 5 lM CCR2 antagonist (RS), 20 ng/ml recombinant MCP-1, 1 lM PKC inhibitor (CTC), and 10 nM PKC activator (PMA) was added to the upper well. Cells that pass through the membrane were fixed with methanol and stained with hematoxylin and eosin. The cells that migrated were counted with a light microscope and expressed as the average cell number from three independent experiments.

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2.6. Invasion assays

2.11. Immunostaining

Cell invasion through a 3D-extracellular matrix was observed using Matrigel-coated transwell chambers (24 well-insert, 8 mm pore size). Briefly, cells were re-suspended in 3% FBS medium and added into the upper chamber. The bottom chamber was filled with medium supplemented with 10% FBS as a chemoattractant. A mixture of RS, CTC, PMA and recombinant MCP-1 was incubated with the cells for 96 h at 37 °C. At the end of the incubation, cells not passing through the Matrigel were removed from the insert with a cotton swab, and the invasive cells were fixed with methanol and stained with hematoxylin and eosin. Cells that had completely invaded the lower surface of the filter were counted under the microscope. All assays were performed in triplicate.

Cells (5  103/200 ll) were plated on 8-well glass chamber slides and treated with 5 lM RS, 20 ng/ml recombinant MCP-1, 1 lM CTC, and 10 nM PMA for 96 h. The cells were fixed with methanol for 20 min at 4 °C, washed three times for 10 min with TBS (200 mM Tris–Cl pH 7.4 and 1 M NaCl), and blocked with blocking solution (2% BSA). The cells were incubated with primary antibodies (1:250) for 1 h. Paxillin and phospho-paxillin (Y118) were detected by incubating for 1 h with the second antibody conjugated with Alexa Fluor 488 (1:250). The slides were counterstained with DAPI. Images were analyzed with an Olympus DP72 fluorescence microscope.

2.7. Enzyme-linked immunoadsorbent assay (ELISA) Cells (3  105) were incubated for 72 h at 37 °C in 6-well culture plate, and the medium was collected. The concentration of CCL2 in the culture medium was measured by ELISA (Millipore, Bedford, USA) according to the manufacturer’s protocol. All assays were performed in triplicate. 2.8. RNA isolation and RT-PCR Total RNA was isolated with UltraspecTM-II reagent (BIOTECX Laboratories, Houston, USA). The primer sequences were as follows: mouse CCL2 (NM_011333), sense 50 -ACCAAGCTCAAGAGAGAGGT30 , antisense 50 -CTGGATTCACAGAGAGGGAA-30 ; mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (NM_008084), sense 50 -CCTTCATTGACCTTCACTACATGGTCTA-30 , antisense 50 -GCTGTAGCCAAATTCATTGTCGTTACCA-30 . The cDNA was synthesized from 1 lg of total RNA with 0.5 ng oligo-dT primers using Reverse Transcriptase (Promega). The PCRs were performed with cDNA as the template using 32 cycles and 28 cycles for CCL2 and GAPDH, respectively. The PCR products were analyzed by electrophoresis in 1.5% agarose gels and visualized by ethidium bromide staining. Signals were quantified using Multi Gauge 3.0 software (Fujifilm) and normalized to the signal of GAPDH. 2.9. PKC activity assay The analysis was performed according to the manufacturer’s manual (Promega). Briefly, 1  106 cells were homogenized with PKC extraction buffer (25 mM Tris–HCl pH 7.4, 0.5 mM EDTA, 1 lg/ml leupeptin, 0.1 mM PMSF, 10 mM b-mercaptoethanol, and 0.05% Triton X-100). The homogenates were centrifuged at 12,000g for 5 min at 4 °C. The supernatant (8 lg) was added to PepTag C1, a nonradioactive substrate, and incubated for 30 min at 25 °C. The samples were loaded onto 0.8% agarose gel for electrophoresis at 100 V for 15 min. Signals were quantified using Multi Gauge 3.0 software. 2.10. Western blot analysis The cell lysates were prepared with PBSTD lysis buffer (1% Nonidet P-40, 50 mM Tris–HCl pH 7.4, 1 mM Na3VO4, 1 mM EDTA, 1 mM PMSF, 1% protease inhibitor cocktail). The soluble lysates (30 lg) were mixed with an equal volume of SDS-sample buffer and resolved by 12% SDS–PAGE. The proteins were then transferred to nitrocellulose membranes and incubated with antibodies as indicated. After the primary antibody incubation, membranes were washed three times with TBS-T and incubated with the appropriate HRP-conjugated (1:1000) anti-rabbit antibody. The signals were detected by Fujifilm LAS-4000 BioSpectrum, and the intensity of the selected bands was analyzed using Fujifilm software.

2.12. Tumor model C3H/HeJ female mice (National Laboratory Animal Center, Taiwan, ROC), 6–8 weeks of age, were housed in pathogen-free conditions in accordance with the National Institutes of Health guidelines. The animal protocol was approved by the Institutional Animal Care and Taiwan Committee, National Taiwan Ocean University, Taiwan, ROC. For tumorigenesis experiments, MBT2 cells, shCCL2-MBT2 or shGFP-MBT2 cells were inoculated into the right suprascapular area of C3H/HeJ mice, and tumor volume and survival were monitored. When the tumor volume reached 100 mm3 by caliper measurement, the mice harboring MBT2 cells were given RS (0.1 mg/day) or vehicle (H2O) for 7 days orally. Tumor growth was assessed twice per week following tumor implantation. Two bisecting diameters of each tumor were measured with calipers. The volume was calculated using the formula (0.4)  (ab2), with a as the larger diameter and b as the smaller diameter. Mice bearing a tumor exceeding 10% of normal body weight were considered death, and survival rates were thus measured. 2.13. Statistical analysis All experiments were conducted in triplicate and repeated in at least three independent experiments. Statistical analysis was carried out using R software for Windows. A Student’s t-test was performed to test the significance of the correlation. A p value <0.05 was considered statistically significant. 3. Results 3.1. The expression level of CCL2 correlates with cell migration in bladder cancer cells In the bladder cancer, high levels of expression of CCL2 have been observed in high-grade and distant-metastatic tumors. To examine the correlation, we analyzed CCL2 concentration in the culture media of different grade human bladder cancer cell lines, including SV-HUC-1 (TCC histopathology grade G0), RT4 (G1), TSGH8301 (G2), T24 (G3), and J82 (G3) cells. As expected, the high-grade bladder cancer cell lines demonstrated high concentrations of CCL2 (4500 and 22,700 pg/ml in T24 and J82 cells, respectively), whereas in the low-grade cancer cell lines, SV-HUC-1, RT4, and TSGH8301 cells, there were only traces of CCL2 (Fig. 1A). This result confirms previous reports and suggests that CCL2 may contribute to malignant and invasive characteristics in bladder cancer. We assumed that CCL2 could act directly on tumor cells to promote their malignant characteristics by increasing their migratory and invasive properties. To examine our hypothesis, the migration activity of these cell lines was investigated by wound healing (lateral motility) and Boyden chamber cell migration assays. Fig. 1B shows that the high-grade bladder cancer cell lines

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Fig. 1. CCL2 is overexpressed in invasive human bladder cancer cell lines and correlated with migration activity. (A) The concentration of CCL2 (pg/ml) secreted from SV-HUC-1 (TCC G0), RT4 (TCC G1), TSGH8301 (TCC G2), T24 (TCC G3), and J82 (TCC G3) cells was examined by ELISA. The cells (3  105/ml) were cultured for 72 h, and the medium was collected for analyzing the secretion of CCL2. (B) The cells were placed to the upper compartments of Boyden chambers to investigate cell migration. Columns, mean of the number of cells counted by microscope. (C) Wound-healing assay to analyzed migration. Cells grown to confluence were scratched and cultured for 48 h. In each panel, the vertical lines represent the starting line for migration, and the distance across the starting line was measured as the % area covered by cells as shown below. Scale bar: 200 lm. The results are expressed as the mean ± SEM from three independent experiments. ⁄p < 0.05, ⁄⁄ p < 0.01 and ⁄⁄⁄p < 0.001 compared with the value of SV-HUC-1 cells.

migrated obviously faster than the low-grade cell lines, demonstrating more than a 50-fold increase in T24 and J82 cells compared to SV-HUC-1 cells. As expected, high-grade bladder cells covered more area in wound-healing assays (Fig. 1C). These findings also prompted us to study whether CCL2 mediates the migratory activity to promote tumor progression in the high-grade bladder cancer cell lines. Furthermore, CCR2 may have the similar role in CCL2-mediated cell migration. To analyze whether its expression level correlates with the CCL2 production, we used western blot and FACS with CCR2 antibody. In Supplementary Fig. s1, CCR2 were expressed in these bladder cancer cell lines and unrelated with their tumor grades as well as the migration activity. 3.2. An antagonist of CCR2 reduces CCL2-mediated cell migration To address the regulation and apparently important role of CCL2 in cell migration, we chose MBT2, a mouse malignant bladder cancer cell line, to analyze the chemokine function. We used two strategies to investigate the role of CCL2 in cell migration: treating CCR2 with the antagonist RS to reduce CCL2/CCR2 effects and knockdown of CCL2 to down-regulate the expression of the gene. Treatment of MBT2 cells with RS decreased cell migration by 50% (Fig. 2A). As expected, the wound-healing ability of these cells also decreased after RS treatment (Fig. 2B). Together, our experiments show that CCL2 binding to its receptor, CCR2, leads to enhanced

cell migration and that this effect may be blocked by a CCR2 antagonist. Because CCR2 is a GPCR, PKCs (downstream kinases of GPCRregulated kinases) may mediate CCL2-induced cell migration [20]. To ascertain whether PKCs affect CCL2-induced cell migration, we examined the kinases linked to this response by blocking PKC activity with the inhibitor, CTC. In the presence of the inhibitor, CCL2-induced cell migration decreased about 50% in MBT2 cells (Fig. 2A), an amount similar to that caused by RS treatment. However, cell migration inhibited by RS was restored by PMA (a PKC activator) in MBT2 cells, demonstrating that PKCs are the downstream kinases in the CCL2/CCR2 signaling pathway. To examine the invasive capacity elicited by CCL2, MBT2 cells were analyzed using Matrigel-coated filters for invasive migration through a 3D extracellular matrix. After exposure to RS or CTC, the invasive activity of MBT2 cells was reduced by 50% compared with control cells (Fig. 2C). Moreover, the RS-repressed invasive ability was rescued by PMA. Furthermore, the PKC activity in the cells was assessed by analyzing its substrate phosphorylation. Treatment of MBT2 cells with RS or CTC decreased the phosphorylation of the PKC substrate, whereas the RS-suppressed activity was restored by PMA treatment (Fig. 2D). Collectively, our findings suggest that the activation of the CCL2/CCR2-mediated signal may stimulate cell migration and invasive ability via PKC activation. 3.3. CCL2 promotes tyrosine phosphorylation of paxillin Many studies have demonstrated that Y118 in paxillin is phosphorylated in response to the stimulation of cell migration [20–22]. To investigate the mechanism by which CCL2 promotes cell migration, we tested whether CCL2/CCR2 activation may promote phosphorylation of Y118 in paxillin in MBT-2 cells. By using phospho-Y118-specific antibody and western blot, we found that RS decreased the phosphorylation of paxillin Y118, and this condition was reversed by exposure to PMA (Fig 2E). Moreover, CTC treatment decreased the phosphorylation in MBT2 cells, as shown in Fig. 2F. Taken together, our results suggest that stimulation of CCL2/CCR2 may promote cell migration and thereby the downstream PKC activation and phosphorylation of paxillin Y118. 3.4. Knockdown of CCL2 reduces cell migration and the phosphorylation of tyrosine in paxillin To examine the role of CCL2 in the stimulation of cell migration, we used short hairpin RNA (shRNA) to interfere with the expression of CCL2 in MBT2 cells. After endogenous CCL2 was depleted by shRNA, the expression level of RNA and protein were examined by RT-PCR and western blot, respectively. The results showed reduced levels of endogenous CCL2 in MBT2 cells (Fig. 3A and B). Control cells harboring shGFP or shCT (the vector control) migrated faster than MBT2 cells containing the CCL2-knockdown (Fig. 3C). The diminished cell migration caused by the depletion of CCL2 by shRNA was reversed after recombinant MCP-1 (rMCP-1) treatment, indicating that the presence of CCL2 in culture medium enhances cell migration. Concomitant with the depletion of CCL2, we detected reduced PKC activity in shCCL2 cells (Fig. 3D), while recombinant MCP-1 and PMA restored PKC activity. Together, our experiments demonstrate that increased PKC activity in response to CCL2 is, at least in part, the result of augmented cell migration. Next, we studied the phosphorylation of paxillin Y118 in shCCL2 knockdowns of MBT2 cells (Fig. 3E and F). In agreement with the decreased migration and PKC activity, we observed decreased phosphorylation of paxillin Y118 in the shCCL2 cells. Moreover, in CCL2 knockdown cells treated with recombinant MCP-1, phosphorylation was restored. The decreased tyrosine phosphorylation was also restored by PMA treatment, showing

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Fig. 2. CCL2-mediated cell migration involves CCR2, PKC activation and tyrosine phosphorylation of paxillin. MBT2 cells were treated for 96 h with RS (5 lM), PMA (10 nM) or CTC (1 lM) as indicated. (A) The cells (3  104 cells/chamber), were seeded into upper chambers of Boyden chambers and the migrating cells are represented the relative cell migration (%) compared with control. (B) MBT2 cells were grown to confluence, scratched and then treated as indicated for 48 h. Cell migration was measured as % area covered by cells as shown below. (C) Matrigel-coated transwell chambers were used to examine the invasive activity of MBT2 cells. The activity is represented as the number of invasive cells. (D) MBT2 cells were treated as indicated for 96 h. PKC activity was determined in cell lysates as described in Section 2. Values are given as the ratio of phosphopeptide in treated vs. control cells. The experiment was repeated three times, and a representative image is herein shown. (E and F) MBT2 cells were treated as indicated for 96 h. Whole-cell lysates (30 lg) were used for western blot analysis using specific antibodies against phospho-paxillin (Y118). Actin was used as the loading control. The level of phospho-paxillin (Y118) was normalized to total paxillin. The blot is representative of three independent experiments. The results are expressed as the mean ± SEM. ⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001 compared with control. ##p < 0.01 and ###p < 0.001 compared with RS-treated cells.

the important role of PKC in the CCL2-induced phosphorylation of paxillin Y118 and cell migration. Taken together, these results suggest that CCL2 binding to CCR2 stimulates cell migration and the phosphorylation of paxillin Y118 via a PKC-dependent mechanism. 3.5. Inhibition and knockdown of CCL2 reduce the tumorigenicity of MBT2 cells The above experiments suggest that cell migration, PKC activity, and the phosphorylation of paxillin Y118 were abrogated by CCR2 antagonist as well as by depletion of the expression of CCL2. We therefore examined the invasive activity of shCCL2 cells. The CCL2-depleted cells showed 51% less invasive activity than shGFP cells (Fig. 4A). Treating the cells with either recombinant MCP-1 or PMA rescued the invasive activity. To investigate changes in tumorigenicity after CCL2 was downregulated or antagonized in MBT2 cells, mice were inoculated with a pooled cells transfected with shCCL2 or shGFP, and wild-type (WT) cells. MBT2 cells showed high tumorigenicity after being inoculated subcutaneously into

syngenetic mice, and no mice survived 24 days after inoculation (Fig. 4B). Remarkably, the MBT2-bearing mice treated with RS and the mice inoculated into the pool transfected with shCCL2 cells died within 35 days. Our findings show that expression of CCL2 in bladder cancer cells may promote tumorigenicity, and that downregulation of CCL2/CCR2 reduces tumorigenicity of the cells. In the inoculated tumors, we analyzed the amount of paxillin and tyrosine phosphorylation using specific antibodies and western blotting. In shCCL2-inoculated tumors (Fig. 4D) and tumors from RS-treated mice (Fig. 4C), we observed a decrease of paxillin. In contrast, an accumulation of the phosphorylation of paxillin Y118 was found in the tumors from MBT2- and shGFP-inoculated mice. 3.6. Inhibition of CCR2 reduces cell migration in human bladder cancer cells Because only traces of CCL2 and little migration activity were observed in the low-grade bladder cell lines, SV-HUC-1 and RT4 (Fig. 1), we examined the role of CCL2/CCR2 in these cells using

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Fig. 3. Knockdown of CCL2 reduces cell migration. MBT2 cells were transfected with CCL2 shRNA to reduce the expression of CCL2. After selection with puromycin, the stable clones were examined for the expression of RNA (A) and protein (B). Transfections with non-specific shRNA (shCT) or shGFP were used as a control. (C) Migration activity of CCL2 knockdown clones (#2 of MBT2 shCCL2 clone) was examined by treating with or without recombinant MCP-1 (rMCP-1, 20 ng/ml). After 24 h, the cells on the lower surface of the chamber were counted. Migrating cells are represented the relative cell migration (%) compared with shCT control cells. (D) The PKC activity of CCL2knockdown cells treated with recombinant MCP-1 (20 ng/ml) or PMA (10 nM) for 24 h. PKC activity was determined in cell lysates. Values are given as the ratio of phosphopeptide in treated vs. shGFP cells. The experiment was repeated three times, and a representative image is shown. (E and F) The phosphorylation of paxillin in CCL2knockdown clone cells were examined after treatment with recombinant MCP-1 (rMCP-1, 20 ng/ml) or PMA (10 nM) for 24 h. A representative blot detecting the indicated protein is shown. The level of phospho-paxillin (Y118) was normalized to total paxillin and adjusted to shGFP, which was set to 100%. The results were obtained from three independent experiments. The results are expressed as the mean ± SEM. ⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001 compared with shGFP cells. #p < 0.05 and ###p < 0.001 compared with shCCL2 control cells.

RS to block and recombinant MCP-1 or PMA to stimulate the activation of CCL2/CCR2. However, SV-HUC-1 and RT4 cells did not respond to recombinant MCP-1, PMA, or RS treatments (Fig. 5A). Furthermore, TSGH8301 cells were found to only have a marginal difference in cell migration, but treating the cells with recombinant MCP-1 or PMA increased cell migration (Fig. 5A). Our findings show that the low migration activity of TSGH8301 cells is due to the low level of expression of CCL2. Concomitant with the enhanced migration of TSGH8301 cells in response to recombinant MCP-1 or PMA, we detected higher PKC activity (Fig. 5B). We also found that the phosphorylation of paxillin Y118 was induced by recombinant MCP-1 in TSGH8301 cells (Fig. 5C), but not in SV-HUC-1 and RT4 cells. We hypothesized that in the high-grade bladder cell lines T24 (Fig. 6) and J82 (Supplementary Fig. s2) cells, CCL2/CCR2 mediates cell migration via PKC activation and the phosphorylation of paxillin. To address the hypothesis, we treated T24 cells with RS, which remarkably reduced cell migration, invasive activity (Fig. 6A and B) and the phosphorylation of paxillin Y118 (Fig. 6C). We also examined the subcellular localization of paxillin in J82 cells using specific antibodies and immunostaining. There was not exhibited obvious difference in the pattern of paxillin analyzed by anti-paxillin antibody. The phospho-paxillin (Y118) was most intense at the periphery in J82 and PMA-treated cells (Supplementary Fig. s3), whereas the intense spots disappeared in the cells treated with RS or CTC. As expected, the decreased intense spots were also restored by PMA treatment in the RS-treated cells (Supplementary Fig. s3). Moreover, CTC treatment led to reduced invasive activity

in T24 cells, whereas PMA increased migration and invasion despite treatment with RS (Fig. 6A and B). As expected, decreased PKC activity was observed in T24 cells after exposure to RS or CTC, and PMA was able to restore the RS-suppressed PKC activity (Fig. 6D). Collectively, these results provide evidence that the invasive activity of T24 bladder cancer cells correlates with the expression of CCL2 and the activation of the CCL2/CCR2-mediated signaling pathway.

4. Discussion The correlation of urinary levels of CCL2 with tumor stages and grades in patients with bladder cancer has been reported previously [18]. Moreover, high-stage and high-grade bladder cancers possessing invasive and metastatic phenotypes frequently recur regardless of treatment with surgery, chemotherapy, or BCG immunotherapy [16]. However, the roles of CCL2 in bladder cancer are obscure. In this study, we demonstrate a novel role played by CCL2 in cell migration and invasion by bladder cancer cells. In addition, T24, J82 and MBT2, the highly malignant bladder cancer cell lines expressing high levels of CCL2, have more migration activity than the low-grade cell lines, SV-HUC-1, RT4 and TSGH8301 (Fig. 1). Although TSGH8301 cell is characterized as a low-grade bladder cancer cell line, stimulation with recombinant MCP-1 could lead to enhanced cell migration and increased the phosphorylation of paxillin (Y118). However, neither SV-HUC-1 nor RT4 cells responded to recombinant MCP-1, suggesting that

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A

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Co nt ro l rM CP PM 1 A Co nt ro l rM CP PM -1 A Co nt r rM ol CP PM -1 A

WT-Vehicle shCCL2

shCCL2 1 2

Fig. 4. Knockdown of CCL2 reduces invasive activity and tumorigenicity in MBT2 cells. (A) The invasive activity of CCL2 knockdown MBT2 cells treated with recombinant MCP-1 (rMCP-1, 20 ng/ml) or PMA (10 nM) was analyzed in Matrigelcoated transwell chambers. After 96 h, the cells on the lower surface of the chamber were counted. ⁄⁄⁄p < 0.001 compared with the shGFP clone. ###p < 0.001 compared with shCCL2 control cells. (B) C3H/HeJ mice were inoculated subcutaneously with MBT2 cells (WT, n = 8) or the pooled cells transfected with shCCL2 (n = 10) or shGFP (1  107 cells/mouse, n = 10). The tumors were measured with calipers in two perpendicular diameters every 2 or 3 days. Tumor volumes and the survival of mice were calculated as described in Section 2. The tumor-bearing mice (WT) were treated orally with RS (0.1 mg/day) or vehicle as indicated. The survival (%) of inoculated mice represents the proportion of surviving at specific days after tumor implants. Inhibition of CCR2 (C) and knockdown of CCL2 (D) decreased the phosphorylation of tyrosine in paxillin in mice inoculated with MBT2 cells. Tumor tissues from two mice treated as described in (B), were dissected and collected at 19 days after inoculation. Tissue lysates (30 lg) were used for western blot analysis using specific antibodies against paxillin, paxillin (Y118), and CCL2. Actin was used as the loading control.

one or more additional factors produced from high-grade bladder cancer cells cooperate with CCL2 to elicit tumor progression. CCL2 is the major CC family chemokine expressed and secreted by MBT2 cells. The expression level of CCL2 is 2- to 20-fold higher than the levels of other members of the chemokines analyzed by protein array (Supplementary Fig. s4). Since MBT2 cells are able to secrete a large amount of CCL2 by autocrine, shGFP MBT2 cells did not respond to the addition of recombinant MCP-1 (Fig. 3), but the shCCL2 MBT2 cells responded to recombinant MCP-1. Moreover, inhibiting the CCL2/CCR2 signal by shRNA or RS decreased significantly cell migration and invasion. Studies of CXCR4 expression have shown that it reflects tumor progression and regulates the motility of bladder cancer cells [23]. However, SDF-1/ CXCL12, a ligand of CXCR4, is secreted by MBT2 cells only in trace amounts, and cell migration did not respond to treatment with the CXCR4 antagonist (Supplementary Fig. s5), indicating that

rMCP-1 P-Paxillin

SV-HUC-1 +

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+

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Paxillin β-actin P-Paxiilin (Y118) pPaxillin protein (Fold of control)

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3.0 2.5 2.0 1.5 1.0 0.5 0

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Fig. 5. Low-grade bladder cancer cell lines show various responses to CCL2 treatment. (A) Cells (3  104/chamber) were seeded into the upper chambers of Boyden chambers and treated with recombinant MCP-1 (rMCP-1, 20 ng/ml) or PMA (10 nM) for 24 h. Migrating cells represented the relative cell migration (%) compared with control. (B) PKC activity of low-grade bladder cancer cells treated as indicated. PKC activity was determined in cell lysates. The values are given as the ratio of phosphopeptide in treated versus control cells. The experiment was repeated three times, and a representative image is shown. (C) The cells were treated with recombinant MCP-1 for 24 h. Whole-cell lysatse (30 lg) were used for western blot analysis using specific antibodies against phospho-paxillin (Y118). Actin was used as loading control. The level of phospho-paxillin (Y118) was normalized to total paxillin. The blot is representative of three independent experiments. The results are expressed as the mean ± SEM. ⁄⁄p < 0.01 and ⁄⁄⁄ p < 0.001 compared with control.

CXCR4-mediated motility of bladder cancer cells is unrelated to the autocrine CXCL12. In invasive malignant T24 cells, the addition of CXCL1, IGF-1, or CXCL12 may elicit cell migration, showing that these exogenous chemokines or growth factors play important roles in bladder cancer [24–26]. In our study, inhibition of the autocrine CCL2 and the CCL2/CCR2 downstream kinase resulted in reduced cell migration and invasive activity, demonstrating a direct role in tumorigenesis. In breast cancer, the expression of CCL2 is involved in promotion and progression associated with low-levels of differentiation and a poor prognosis [6,27,28]. The chemokine also mediates

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Co ntr RS ol

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1.0

**

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**

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150 100 50

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Fig. 6. High-grade bladder cancer cell lines demonstrated that invasive activity is correlated with PKC activation and tyrosine phosphorylation (Y118) of paxillin. The T24 cells (3  104 cells/well) were added to chambers and treated with RS (5 lM), PMA (10 nM), or CTC (1 lm) for 96 h. (A) Boyden chambers were used to analyze migration activity. (B) Matrigel-coated transwell chambers were used to examine the invasive activity of the cells. The activity is represented as the number of invasive cells. (C) The cells were treated as indicated for 96 h. Whole-cell lysates (30 lg) were used for western blot analysis using specific antibodies against phospho-paxillin (Y118). Actin was used as the loading control. The level of phospho-paxillin (Y118) was normalized to total paxillin. The blot is representative of three independent experiments. (D) The cells were treated as indicated for 96 h. PKC activity was determined in cell lysates. Values are given as the ratio of phosphopeptide in treated versus control cells. The experiment was repeated three times, and a representative image is herein shown. The results are expressed as the mean ± SEM of three independent experiments. ⁄⁄p < 0.01 and ⁄⁄⁄ p < 0.001 compared with control. ###p < 0.001 compared with RS-treated cells.

tumor-promoting interactions between breast tumor cells and cells of the tumor microenvironment thereby recruiting tumorassociated macrophages (TAM) that have the ability to release soluble mediators to promote tumor growth, angiogenesis and matrix degradation [28]. Our findings show that the invasive activity of bladder cancer correlates with the autocrine CCL2 and the activation of the CCL2/CCR2 signaling pathway. The cellular effect of CCL2 is mediated through interaction with CCR2, activating GPCR and inducing the intracellular signal [20]. We have examined the signaling pathways activated by CCL2 in TCC cells, and our results show that PKC activation is required for CCL2-induced motility and invasion in bladder cancer cells. The autocrine CCL2-mediated cell migration was inhibited by RS, but the inhibition was reversed by PMA, demonstrating that CCL2 binding to CCR2 activates PKC to maintain cell migration in urothelial carcinoma-derived cell lines. The PKC inhibitor CTC also reduced cell migration stimulated by autocrine CCL2 in TCC cells (Fig. 2). These results suggest that the GPCR-PLCb-PKC pathway may play a crucial role in CCL2-induced migration and invasion. Furthermore, we have shown that RS can decrease the phosphorylation of tyrosine in paxillin, whereas PMA can prevent the decrease. This finding also suggests that PKC is the downstream signal molecule involved in the regulation of CCL2-mediated tyrosine phosphorylation of paxillin. Consistently, knockdown of CCL2 by shRNA decreased the phosphorylation of tyrosine in paxillin, and the decrease could be reversed by recombinant MCP-1 and PMA (Fig. 3), suggesting that CCL2/CCR2 signaling is required to stimulate the phosphorylation of tyrosine in paxillin. To dissect the type of PKC activated by CCL2/CCR2, we used various concentrations of GÖ6976, a PKC-specific inhibitor, to analyze cell migra-

tion. The results showed that treatment with 100 nM GÖ6976 inhibited CCL2/CCR2-mediated cell migration, indicating that PKC d, e and f but not PKC a and b may be involved in CCL2/CCR2 signaling (Supplementary Fig. s5). However, the specific PKC-mediated signaling pathway remains to be identified. In a previous report, ERK1/2 activation was required for IGF1-induced motility and invasion in T24 cells [26]. In our study, an ERK1/2 inhibitor did not reduce CCL2-mediated cell migration (Supplementary Fig. s5), suggesting that PKC activation may play a more critical role in cell migration. Malignant cells may undergo an epithelial-mesenchymal transition (EMT) to sustain the metastatic phenotype [29,30]. As for the mechanisms by which CCL2 regulates the invasive ability, we used RT-PCR to analyze genes related to EMT and matrix metalloproteinases (MMPs) in high-grade bladder cancer cells treated with RS; however, we did not find any significantly different expression of these genes. Therefore, it is probable that the autocrine CCL2 in bladder cancer affects invasion capacity by promoting cell migration via the CCL2/CCR2-PKC pathway. Tumor suppressor gene p53 has been observed frequently point mutation and deletion in advanced stage bladder tumors [31]. In this study, SV-HUC-1 [32], RT4 [33,34], and TSGH8301 cells [35] contain wild-type p53, whereas T24 [33,34], J82 [36] and MBT2 cells [37] harbor mutant-form p53, implying that mutant-form p53 may result in the autocrine CCL2 in bladder cancer. However, Hacke et al. found that non-functional p53 leads to diminished CCL2 transcription upon TNF-a treatment [38]. In bladder cancer, whether mutant-form p53 may induce the expression of CCL2 remains to be uncovered. p53 also play a role in opposing EMT and cell migration by reducing the expression of Slug or Twist [39],

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and mutant p53 proteins promote cell migration by gain of function [40]. T24 and J82 cells showed autocrine CCL2 and potent CCL2-mediated migration activity (Fig. 6 and Supplementary Fig. s2), but TSGH8031 cells not secreting CCL2 respond to the chemokine to increased cell migration, indicating that wild-type p53 in the TSGH8031 cells does not suppress CCL2-mediated cell migration. Moreover, TSGH8301 human bladder cancer cells were enhanced cisplastin-induced apoptosis by exogenous mutant p53 DNA [34]. It is interested to further study whether mutant p53 may increase the CCL2-mediated cell migration in bladder cancer. Phosphorylation of tyrosine in paxillin has been found to be involved cancer migration and spreading [21,41,42]. In myogenesis, PKC is associated with the increased phosphorylation of tyrosine in paxillin [43]. PDGF, EGF, and VEGF were able to activate PKC resulting in enhanced cell migration [44–47]. The spreading of gastric carcinoma cells is regulated via TGF-b through PKC activation, which increases the phosphorylation of paxillin [48]. In malignant pleural mesothelioma, the phosphorylation of tyrosine in paxillin, and cell migration were reduced by the inhibition of PKC activity [49]. Stromal cell-derived factor-1a stimulates the phosphorylation of tyrosine in paxillin and induces migration which requires the activation of PKC in hematopoietic cells [50]. Although an inhibitory effect of PKC on paxillin phosphorylation was also observed in other cell types [51,52], our findings confirmed the role of PKC in CCL2-stimulated cell migration via the phosphorylation of tyrosine in paxillin. It is possible that CCL2 stimulation may activate the paxillin signaling pathway by PKC, and the activation may determine the migratory activity of bladder cancer cells. Consistently, mice treated with RS or mice inoculated with shCCL2 cells were demonstrated decreased tumorigenicity elicited by MBT2 bladder cancer cell and showed decreased an accumulation of the phosphorylation of paxillin Y118 (Fig. 4), suggesting that blockage of autocrine CCL2/CCR2 signals may reduce tumor progression. In conclusion, we have characterized the critical role of CCL2, whose expression level in bladder cancer cells correlates with the tumor stage. The chemokine CCL2 stimulates cell migration and invasion via activation of CCL2/CCR2, PKC, and paxillin. Based on our results, the CCL2/CCR2 pathway appears to be a crucial step in bladder cancer progression and is a potential therapeutic target. Acknowledgments We thank professor C.-J. Wu (Department of Food Science, National Taiwan Ocean University) for assistance with animal care. This research work was supported by CHRMC/YM research Grant 98F117CY11 and NSC97-2311-B-019-004-MY3. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cyto.2012.04.017. References [1] Deshmane SL, Kremlev S, Amini S, Sawaya BE. Monocyte chemoattractant protein-1 (MCP-1): an overview. J Interferon Cytokine Res 2009;29:313–26. [2] Charo IF, Ransohoff RM. The many roles of chemokines and chemokine receptors in inflammation. N Engl J Med 2006;354:610–21. [3] Insel PA, Tang CM, Hahntow I, Michel MC. Impact of GPCRs in clinical medicine: monogenic diseases, genetic variants and drug targets. Biochim Biophys Acta 2007;1768:994–1005. [4] Raman D, Baugher PJ, Thu YM, Richmond A. Role of chemokines in tumor growth. Cancer Lett 2007;256:137–65. [5] Loberg RD, Day LL, Harwood J, Ying C, St John LN, Giles R, et al. CCL2 is a potent regulator of prostate cancer cell migration and proliferation. Neoplasia 2006;8:578–86. [6] Nam JS, Kang MJ, Suchar AM, Shimamura T, Kohn EA, Michalowska AM, et al. Chemokine (C-C motif) ligand 2 mediates the prometastatic effect of dysadherin in human breast cancer cells. Cancer Res 2006;66:7176–84.

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