Carcinogenesis, 2015, Vol. 36, No. 3, 346–354 doi:10.1093/carcin/bgu327 Advance Access publication January 7, 2015 Original Manuscript

original manuscript

Hiroki Imaoka, Yuji Toiyama*, Susumu Saigusa, Mikio Kawamura, Aya Kawamoto, Yoshinaga Okugawa, Junichiro Hiro, Koji Tanaka, Yasuhiro Inoue, Yasuhiko Mohri and Masato Kusunoki Department of Gastrointestinal and Pediatric Surgery, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan *To whom correspondence should be addressed. Tel: +81-59-231-5294; Fax: +81-59-232-6968; Email: [email protected]

Abstract Rac GTPase-activating protein (RacGAP) 1 plays a key role in controlling various cellular phenomena including cytokinesis, transformation, invasive migration and metastasis. This study investigated the function and clinical significance of RacGAP1 expression in colorectal cancer (CRC). The intrinsic functions of RacGAP1 in CRC cells were analyzed using small interfering RNA (siRNA). We analyzed RacGAP1 mRNA expression in surgical specimens from 193 CRC patients (Cohort 1) by real-time PCR. Finally, we validated RacGAP1 protein expression using formalin-fixed paraffin-embedded samples from 298 CRC patients (Cohort 2) by immunohistochemistry. Reduced RacGAP1 expression by siRNA in CRC cell lines showed significantly decreased cellular proliferation, migration and invasion. In Cohort 1, RacGAP1 expression in CRC was significantly higher than in adjacent normal mucosa and increased according to tumor node metastasis stage progression. High RacGAP1 expression in tumors was significantly associated with progression and prognosis. In Cohort 2, RacGAP1 protein was overexpressed mainly in the nuclei of CRC cells; however, its expression was scarcely observed in normal colorectal mucosa. RacGAP1 protein expression was significantly higher in CRC patients with higher T stage, vessel invasion and lymph node and distant metastasis. Increased expression of RacGAP1 protein was significantly associated with poor disease-free and overall survival. Multivariate analyses revealed that high RacGAP1 expression was an independent predictive marker for lymph node metastasis, recurrence and poor prognosis in CRC. Our data provide novel evidence for the biological and clinical significance of RacGAP1 as a potential biomarker for identifying patients with lymph node metastasis and poor prognosis in CRC.

Introduction Colorectal cancer (CRC) is one of the most common cancers in developed countries and remains a serious public health problem (1). The survival rates of CRC patients have improved in recent years because of earlier detection and improved treatment. Nevertheless, among CRC patients who have undergone potentially curative surgery, up to 30% eventually develop local recurrence or distant metastasis, which results in an overall worse prognosis (2,3). Therefore, identification of CRC patients

at a higher risk of developing metastatic disease might enable us to stratify and select the candidate patients who need standard or intensive adjuvant chemotherapy. To move closer towards individualized therapeutic strategies, novel biomarkers for accurate identification of patients at higher risk of developing metastatic disease are urgently needed. Rac GTPase-activating protein (RacGAP) 1, which is a component of the central spindlin and essential for the induction of

Received: September 4, 2014; Revised: December 5, 2014; Accepted: December 29, 2014 © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

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RacGAP1 expression, increasing tumor malignant potential, as a predictive biomarker for lymph node metastasis and poor prognosis in colorectal cancer

H.Imaoka et al. | 347

RacGAP1 RNAi

Abbreviations confidence interval colorectal cancer disease-free survival hazard ratio immunohistochemistry messenger RNA overall survival Rac GTPase-activating protein receiver-operating characteristic curve reverse transcription–PCR small interfering RNA Tris-buffered saline–Tween 20 tumor node metastasis.

cytokinesis (4–7), belongs to the family of Rho GTPase-activating proteins (8,9). RacGAP1 accompanied by GTP-bound Rac1 works both as a mediator of tyrosine phosphorylation of the signal transducer and activator of transcription (STAT) family of proteins and as a nuclear localization signal-containing nuclear chaperone of phosphorylated STATs, which have a variety of functions including anti-apoptosis, proliferation, differentiation and inflammation (10,11). RacGAP1 also regulates the function of Rho proteins such as Rac and cdc42 to influence cell morphology, motility and establishment of polarity (12). In malignant tumors, RacGAP1 influences the metastatic dissemination of tumor cells via pseudopod extension and invasion (12–16). Phosphorylation of RacGAP1 by PKB/Akt, as a consequence of integrin-mediated epidermal growth factor receptor 1 trafficking and signaling, promotes recruitment to the front of invading cells, which leads to suppression of Rac activity and promotes pseudopod extension and invasion by permitting activation of RhoA (13,17–19). Collectively, RacGAP1 associates with tumor cell proliferation, migration, invasion and finally metastasis. Recently, the clinical significance of RacGAP1 expression has been reported in several malignancies such as gastric cancer, meningioma, breast cancer, lung cancer and hepatocellular carcinoma (20–24), which demonstrates that high RacGAP1 expression reflects greater tumor aggressiveness and clinical features. However, to the best of our knowledge, direct association between the clinical significance of RacGAP1 and CRC has not been fully investigated. Accordingly, the aim of this study was to elucidate the clinical significance of RacGAP1 expression in primary CRC. We also analyzed the functional role of RacGAP1 in vitro by the use of RNA interference (RNAi) in CRC cell lines. By multiple approaches, we demonstrated for the first time that high expression of RacGAP1 promoted proliferation, migration and invasion of CRC cells and affected tumor aggressiveness in CRC patients. Additionally, our data show that RacGAP1 serves as an independent predictive biomarker for poor prognosis and lymph node metastasis in CRC patients.

Materials and methods Cell lines and culture conditions Human CRC cell lines Caco2, DLD1, HT29, LoVo and SW480 were obtained from the Cell Resource Center of Biomedical Research, Institute of Development, Aging and Cancer (Tohoku University, Sendai, Japan). These cell lines were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 IU/ml penicillin and 100 µg/ml streptomycin at 37°C in a 5% humidified CO2 atmosphere. The authenticity of various cell lines was routinely monitored by analyzing DNA (short tandem repeat) profiling specific for each cell line in an approved laboratory (last tested on 15 July, 2014).

RacGAP1-specific small interfering RNA (siRNA) (Silencer Predesigned siRNA; sense: CAACUAAGCGAGGAGCAAAtt, antisense: UUUGCUCCUCGCUUAGUUGaa) and negative control siRNA (Silencer Negative Control siRNA) were purchased from Ambion (Austin, TX). Cancer cell lines were seeded at 2 × 105 cells per well in a final volume of 2 ml in six-well flat-bottom microtiter plates. The cells were cultured overnight for adherence. siRNA oligonucleotides were diluted with OptiMEM I  Reduced Serum Medium (Invitrogen, Carlsbad, CA). The diluted siRNA oligonucleotides were mixed with diluted Lipofectamine RNAiMAX Reagent (Invitrogen) and incubated for 5 min at room temperature to allow formation of siRNA-Lipofectamine RNAiMAX Reagent complexes. These complexes were added to each well at a final concentration of 10 nM. The cells were incubated at 37°C in a 5% humidified CO2 atmosphere.

Western blot analysis The CRC cell lines were washed with cold phosphate-buffered saline after removal of culture medium. Cold RIPA Buffer (Nacalai Tesque, Kyoto, Japan) was then added directly to the dish. After agitating for 5 min, the cells were scraped off the dish, harvested and incubated for 15 min on ice. The whole cell lysate was centrifuged at 10 000 r.p.m. for 10 min at 4°C, and supernatants were collected and frozen at −20°C until use. The protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL). Twenty micrograms of the protein lysate was mixed with an equal volume of 2× Laemmli sample buffer containing 2-mercaptoethanol and heated at 100°C for 5 min. Samples were electrophoretically separated on 12.5% gradient polyacrylamide gels containing 0.1% sodium dodecyl sulfate, followed by semi-dry transfer to an Immun-Blot PVDF membrane (Bio-Rad Laboratories, Hercules, CA). The membrane was then blocked using 2% skimmed milk in Tris-buffered saline, pH 7.5, supplemented with 0.1% Tween 20 (TBS-T) at room temperature for 2 h. The blots were incubated with a mouse monoclonal anti-RacGAP1 antibody (A-6; Santa Cruz Biotechnology, Santa Cruz, CA) at 1:100 dilution and a mouse monoclonal anti-β-actin antibody (C4; Santa Cruz Biotechnology) at 1:200 dilution in 2% skimmed milk in TBS-T overnight at 4°C. After washing five times with TBS-T, the blots were incubated with a horseradish-peroxidaseconjugated goat anti-mouse IgG antibody (Santa Cruz Biotechnology) at 1:1000 dilution in 2% skimmed milk in TBS-T. Following treatment with an enhanced chemiluminescence detection solution, Pierce Western Blotting Substrate Plus (Thermo Fisher Scientific), chemiluminescent signals were visualized in a CS Analyzer and AE-6962 light capture (ATTO, Tokyo, Japan).

Immunofluorescent cell staining Cells were fixed in cold acetone–methanol for 15 min. After washed with phosphate-buffered saline three times each for 5 min, cells were blocked with 5% normal goat serum (Vector Laboratories, Burlingame, CA) diluted in TBS-T for 15 min at room temperature. Cells were then incubated with a primary monoclonal antibody against RacGAP1 (EE-16, dilution 1:100; Santa Cruz Biotechnology) at room temperature for 1 h, followed by incubation at room temperature for 1 h with a secondary fluorescent antibody (dilution 1:400; Invitrogen). Finally, cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI) to label nuclei and then observed using a confocal microscope (Olympus America, Center Valley, PA).

Proliferation assay To assess the effect of knockdown of RacGAP1 on cell proliferation, migration and invasion, RacGAP1 siRNA and negative control siRNA were used. To evaluate changes in cell viability, WST-8 [2-(2-methoxy-4-nitrophenyl)3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt] colorimetric assay was performed. Cancer cells were seeded at 5 × 103 cells per well in 96-well plates in a final volume of 100 µl culture medium per well. After overnight preincubation, siRNA transfection was performed (0 h). At 0, 24, 48 and 72 h, the spectrophotometric absorbance of the samples were measured. At each time point, 15 µl WST-8 reagent solution (Cell Counting Kit; Dojindo Laboratories, Kumamoto, Japan) was added to the culture medium, followed by incubation for 2 h at 37°C. Cell viability was determined by colorimetric comparison by reading OD450 values from a microplate reader (SoftMax Pro; Molecular Devices, Sunnyvale, CA). Each independent experiment was performed three times in triplicate.

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CI CRC DFS HR IHC mRNA OS RacGAP ROC curve RT–PCR siRNA TBS-T TNM

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Wound healing assay Cancer cells transfected with RacGAP1 siRNA or negative control siRNA were seeded onto 24-well plates and cultured until confluent. Wounds were generated using a sterile 200 µl pipette tip. Cells were then cultured for an additional 48 h. Wound closure was assessed using an Olympus IX71 microscope (Olympus America) at ×40 magnification. Cell migration distance was measured using Adobe Photoshop 9.0.2 software and compared with baseline measurements. Each independent experiment was performed three times in triplicate.

Invasion assay

Patients and sample collection Patients with primary CRC who underwent surgical resection at Mie University Hospital, Japan from 2000 to 2011 were enrolled in this study. Patients with preoperative treatment, incomplete clinical data, inadequate follow-up and inadequate tissue samples were excluded. Surgically resected CRC tissue specimens were frozen in liquid nitrogen and kept at −80°C until RNA extraction, and 193 surgical samples from 2000 to 2005 were used for RacGAP1 messenger RNA (mRNA) expression analysis (Cohort 1). The patients included 100 men and 93 women with a median age of 67 years (SD: 11.2  years). Twenty-eight patients were diagnosed with synchronous liver metastasis. All patients were postoperatively classified based on histopathological analysis by using the Union for International Cancer Control (UICC) tumor node metastasis (TNM) staging system. There were 43 patients with Stage I, 53 with Stage II, 54 with Stage III and 43 with Stage IV cancer. Nineteen patients had poorly differentiated or mucinous adenocarcinomas, and 174 patients had well or moderately differentiated adenocarcinomas. Postoperative follow-up data were obtained from all patients, and the median follow-up duration was 27 months (range: 1–117 months). A total of 298 high-quality, formalin-fixed, paraffin-embedded samples of resected primary cancer specimens were obtained from 2006 to 2011 (Cohort 2). Patients who underwent neoadjuvant therapy, treated by endoscopic mucosal resection and who had non-colonic carcinomas were excluded. The median age of the patients was 68  years (SD: 11.1  years) including 168 men and 130 women. Forty-nine patients were diagnosed with synchronous liver metastasis. Clinicopathological findings were based on the criteria of the UICC TNM classification. There were 71 patients with Stage I, 82 with Stage II, 70 with Stage III and 75 with Stage IV disease. Thirty-five patients had poorly differentiated or mucinous adenocarcinomas, whereas 263 patients had well or moderately differentiated colorectal tumors. The median follow-up period was 24  months (range: 1–118  months). Written informed consent was obtained from all the patients according to the ethical guidelines approved by the Institutional Review Board.

Total RNA extraction, cDNA synthesis and quantitative reverse transcription–PCR The surgical specimens or CRC cell lines were homogenized using a Mixer Mill MM 300 homogenizer (Qiagen, Chatsworth, CA). Total RNA was isolated using an RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. cDNA was synthesized with a random hexamers and Superscript III reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. Target gene expression was determined by quantitative real-time reverse transcription–PCR (RT–PCR) using Power SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA) performed on the StepOnePlus Real Time PCR System (Applied Biosystems). Primer

Immunohistochemistry About 3  µm thick sections were cut from the formalin-fixed, paraffinembedded specimens from the 298 CRC patients to analyze RacGAP1 protein expression. After deparaffinization and dehydration, the specimens were heated in 10 mM sodium citrate buffer (pH 6.0) at 121°C for 10 min to unmask the antigens. The sections were incubated in 3% hydrogen peroxide for 10 min, blocked with normal goat serum (Vector Laboratories) for 1 h and incubated with primary antibody overnight at 4°C. Antibody binding was detected using Envision reagents (Dako REAL EnVision Detection System, Peroxidase/DAB+; DakoCytomation, Denmark). A primary mouse monoclonal antibody against RacGAP1 (EE-16, dilution 1:100; Santa Cruz Biotechnology) was used. All sections were counterstained with hematoxylin before dehydration and mounting. Negative controls were also run simultaneously by exclusion of the primary antibody.

Evaluation of immunohistochemical staining The immunoreactivity of RacGAP1 in primary CRC was evaluated at the core of the tumor (center of the tumor in general, excluding the necrotic areas) by scanning the entire tissue specimen under low-power magnification (×40) and then confirmed under high-power magnification (×100 and ×200). We calculated immunohistochemistry (IHC) scores, as described previously (25), by multiplying the percentage score (1–25%, 1; 26–50%, 2; 51–75%, 3 and 76–100%, 4) (Supplementary Figure 1A–D, available at Carcinogenesis Online) of RacGAP1-positive cancer cells by the intensity score (no staining, 0; weak staining, 1; moderate staining, 2; and strong staining, 3) (Supplementary Figure 2A–D, available at Carcinogenesis Online). RacGAP1 IHC scores were in the range of 0–12. The slides were evaluated three times by two independent investigators who were blinded to the nature of the specimens and any clinicopathological information and antibody used. Specimens were re-scored if the difference between the scores by two investigators was >3.

Statistical analysis All statistical analyses were performed using JMP version 10 (SAS Institute, Cary, NC). The results are expressed as median values or as the mean ± SD. Comparisons were performed using the non-parametric Mann–Whitney U-test for continuous variables. Differences between groups were estimated using the Pearson’s χ2 test or Fisher’s exact probability test. Receiver-operating characteristic (ROC) curves were established for determining cutoff values for analyzing prediction of lymph node metastasis and prognosis by Youden’s index. The survival curves were obtained using the Kaplan–Meier product limit method, and comparisons were made using the log-rank test. Prognostic factors were examined by univariate and multivariate analysis (Cox proportional hazards regression model). Logistic regression analysis was used to evaluate the factors influencing lymph node metastasis. Two-sided P values