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Relation of INHBA Expression to Outcomes in Gastric Cancer after Curative Surgery

TAKASHI OSHIMA1, KAZUE YOSHIHARA1, TORU AOYAMA2, SHINICH HASEGAWA2, TSUTOMU SATO3, NAOTO YAMAMOTO2, NOZAKI AKITO1, MANABU SHIOZAWA3, TAKAKI YOSHIKAWA3, KAZUSHI NUMATA1, YASUSHI RINO2, CHIKARA KUNISAKI1, KATSUAKI TANAKA1, MAKOTO AKAIKE3, TOSHIO IMADA2 and MUNETAKA MASUDA2

1Gastroenterological Center, Yokohama City University Medical Center, Yokohama, Kanagawa, Japan; 2Department of Surgery, Yokohama City University, Yokohama, Kanagawa, Japan; 3Department of Surgery, Kanagawa Cancer Center, Yokohama, Kanagawa, Japan

Abstract. Inhibin-βA (INHBA), a ligand belonging to the instrumental for axis development and organogenesis in a transforming growth factor-β superfamily, is associated with variety of species (13). In adults, activins function as cell proliferation in cancer. We studied the relations of hormone-like feedback regulators in the reproductive system INHBA to clinicopathological factors and (14). Furthermore, presence of activin has been related to outcomes in 168 patients with gastric cancer who underwent wound healing (15). curative surgery. Relative INHBA gene expression was As for the relation between activin A and cancer, measured in surgical specimens of cancer tissue and overexpression of activin A in esophageal squamous cell adjacent normal mucosa by quantitative real-time, reverse- carcinoma has been associated with advanced status, transcription polymerase chain reaction. INHBA expression clinical stage, and poorer overall outcomes (16). In addition, levels were significantly higher in cancer tissue than in chronic exposure to activin A promotes growth, adjacent normal mucosa and were related to TNM stage and tumorigenicity, tumor invasion, and resistance to apoptosis venous invasion. High INHBA gene expression was in esophageal squamous cell carcinoma (17). Inhibin α- associated with significantly poorer 5-year overall survival knockout mice express high levels of serum activin A and than was low expression. On multivariate analysis, INHBA almost uniformly develop gonadal stromal tumors, gene expression was an independent prognostic factor. supporting the role of activin A overexpression in Overexpression of the INHBA gene is considered a useful tumorigenesis (18). Furthermore, overexpression of activin independent predictor of outcomes in patients with gastric A has been reported in colon, prostate, pancreatic, ovarian, cancer after curative surgery. endometrial, and cervical cancer (19-25). The role of INHBA in cancer has not been fully- Inhibin-βA (INHBA) is a ligand belonging to the elucidated. Recent studies have shown that INHBA transforming growth factor-β (TGF-β) superfamily (1). expression correlates with cell proliferation and outcomes in INHBA forms a disulfide-linked homodimer known as lung adenocarcinoma and esophageal adenocarcinoma (26- activin A (2, 3). Activin A has been implicated in multiple 27). In gastric cancer, the INHBA gene was reported to biological processes (4-7). One of the most important belong to a set of eight biomarkers shown to predict cancer functions of activin A is regulation of cell differentiation. and normal status of gastric tissue (28). Moreover, the Activin A controls several aspects of hematopoiesis (8-10) INHBA gene was revealed as one out of seven candidate and regulates cell differentiation in the ovary, placenta, for gastric carcinogenesis and progression by a prostate, and testis (11-12). During embryogenesis, it is combined large-scale gene expression profiling and network analysis (29). However, few studies have investigated the relation between relative expression of the INHBA gene and clinicopathological factors or outcomes in patients with Correspondence to: Takashi Oshima, Gastroenterological Center, gastric cancer. Yokohama City University Medical Center, 4-57 Urafunecho, Minami- ku, Yokohama, Kanagawa 232-0024, Japan. Tel: +81 452615656, Fax: In the present study, we measured expression levels of the +81 452619492, e-mail: [email protected] INHBA gene in specimens of cancer and adjacent normal mucosa obtained from 168 patients with gastric cancer after Key Words: Inhibin beta A, gastric cancer, prognostic factor. curative surgery. Our objective was to evaluate the relative

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Figure 1. Inhibin-βA (INHBA) and β-actin mRNA expression in 6 gastric cancer cell lines (A) and clinical samples (B) by reverse-transcription polymerase chain reaction. T: Tumor, N: adjacent normal mucosa, n: negative control, p: positive control, m: ladder. The product sizes of INHBA and β-actin were 105 bp and 171 bp, respectively.

expression of the INHBA gene and to determine whether incubated in 5% CO2 at 37˚C and passaged every 3-4 days, except such expression correlates with outcomes in patients with for MKN7 which was subcultured at 7-day intervals. gastric cancer who undergo curative surgery. RNA extraction and complementary DNA (cDNA) synthesis. Total RNA isolated from gastric cancer tissue and adjacent normal Materials and Methods mucosa was prepared with the use of Trizol (Gibco, Life Technology, Gaithersburg, MD, USA). cDNA was synthesized from Patients and samples. We studied surgical specimens of cancer 0.4 μg of total RNA with an iScript cDNA Synthesis Kit (Bio-Rad tissue and paired adjacent normal mucosa obtained from 168 Laboratories. Hercules, CA, USA). After synthesis, the cDNA was patients with previously untreated stage I-III gastric cancer who diluted to 0.2 μg/μl with water and stored at −20˚C until use. underwent curative surgery. Tumor staging was evaluated according to the seventh edition of the International Union Against Cancer Oligonucleotide primers for INHBA cDNA amplification by (UICC)-TNM classification of malignant tumors (30). The patients reverse–transcription polymerase chain reaction (RT-PCR). The underwent surgery at the Gastroenterological Center, Yokohama oligonucleotide primers for INHBA were as follows: sense primer 5’- City University Medical Center, and at the Department of Surgery, GGTATGTGGAGATAGAGGATGAC-3’ and antisense primer 5’- Yokohama City University from January 2002 through July 2007. TCCTGGCTGTTCCTGACTC-3’. We used β-actin as the internal Informed consent was obtained from each patient, and the Ethics control. The oligonucleotide primers for β-actin were as follows: Committees of Yokohama City University Medical Center and sense primer 5’-AGTTGCGTTACACCCTTTCTTGAC-3’ and Yokohama City University approved the protocol before initiation antisense primer 5’-GCTCGCTCCAACCGACTGC-3’. Amplification of the study (approval number: 18-7A-4). All tissue samples were of INHBA was performed for 40 cycles of 1 minute at 95˚C, 1 minute embedded in OCT compound (Sakura Finetechnical Co., Ltd., at 56˚C, and 1 minute at 72˚C. Amplification of β-actin was Tokyo, Japan) and immediately stored at −80˚C until use. No patient performed for 40 cycles of 1 minute at 95˚C, 1 minute at 60˚C, and 1 had any other malignancies. The histopathological features of minute at 72˚C. A 10-μl aliquot of each amplified PCR product was specimens stained with hematoxylin and eosin were examined, and electrophoresed on a 3% agarose gel containing ethidium bromide. sections that consisted of more than 80% cancer cells were used to prepare total RNA. Quantitative real-time, RT-PCR. Quantitative real-time PCR was performed with iQ SYBR Green Supermix (Bio-Rad Laboratories). Cell lines. Human gastric cancer cell lines (MKN1, MKN7, PCR reactions were carried out in a total volume of 15 μl, which MKN45, MKN74, NUGC3, and NUGC4) were provided by the included 0.2 μg of cDNA, 0.4 μM of each primer, 7.5 μl of iQ Japanese Cancer Research Bank, Tokyo, Japan. Cell lines were SYBR Green Supermix containing dATP, dCTP, dGTP, and dTTP maintained in Roswell Park Memorial Institute (RPMI) 1640 at concentrations of 400 μM each, and 50 units/ml of iTag DNA medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% polymerase. The PCR consisted of 10 minutes at 95˚C, followed by fetal bovine serum (Equitec-Bio, Ingram, TX, USA), 100 units/ml 40 cycles of denaturation of the cDNA for 10 seconds at 95˚C, penicillin G, and streptomycin (Invitrogen). The cells were annealing for 10 seconds at 56˚C (60˚C for β-actin), and a primer

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Table I. Relation of Inhibin-βA (INHBA) mRNA expression to clinicopathological features.

Variably/category INHBA mRNA expression p-Value

High (n=84) Low (n=84)

Age (years) <70 45 43 0.7574 ≥70 39 41 Gender Male 59 54 0.411 Female 25 30 Tumor size (cm) <6.5 48 46 0.7559 ≥6.5 36 38 Histological type Figure 2. Comparison of Inhibin-βA (INHBA) mRNA expression levels Differentiated 43 42 0.8774 between gastric cancer tissue and adjacent normal mucosa. Box Undifferentiated 41 42 boundaries, the 25th and 75th percentiles of the observed values; capped Depth of invasion bars, the 10th and 90th percentiles; solid line, median. p-Values were pT1 5 20 0.0067 calculated by the Wilcoxon signed-rank test. Expression levels of the INHBA pT2 17 9 gene were higher in cancer than in adjacent normal mucosa (p<0.0001). pT3 28 22 pT4 34 33 Lymph node metastasis pN0 26 33 0.308 pN1 24 15 extension for 20 seconds at 72˚C, followed by 10 minutes at 72˚C. pN2 10 21 To distinguish specific from nonspecific products and primer pN3 24 15 dimmers, melting curve analyses were carried out. To evaluate TMN stage specific mRNA expression in samples, a standard curve was I 7 22 0.004 produced for each run, measuring three points of the human control II 37 23 cDNA (Clontech Laboratories, Inc., CA, USA). The concentrations III 40 39 of each sample were calculated by relating its crossing point to Lymphatic invasion standard curve. Absent 30 34 0.5251 Present 54 50 Immunohistochemical analysis. Immunohistochemical studies of Vascular invasion INHBA were performed on formalin-fixed, paraffin-embedded Absent 28 43 0.0191 surgical specimens obtained from patients with gastric cancer. The Present 56 41 tissue sections were deparaffinized and soaked in 10 mM sodium INHBA: Inhibin-βA. citrate buffer (pH 6.0) at 121˚C for 15 minutes to retrieve cell antigens. After blocking, the sections were incubated overnight at 4˚C to allow antigen−antibody reactions to occur. Peroxidase- labeled polymer (EnVision+, rabbit; DAKO, Glostrup, Denmark) was used to detect signals of the antigen−antibody reactions. All Results sections were counterstained with hematoxylin. Primary polyclonal antibodies against INHBA (Atlas Antibodies, Stockholm, Sweden) INHBA mRNA expression in human gastric cancer. were used at a dilution of 1:200. Expression of INHBA mRNA in human gastric cancer cell lines and clinical samples was analyzed by RT-PCR (Figure Statistical analysis. Gene expression levels of gastric cancer were INHBA compared with those of adjacent normal mucosa by the Wilcoxon 1). mRNA expression was observed in the human signed-rank test. Relations between gene expression and potential gastric cell lines: MKN1, MKN7, MKN45, MKN74, explanatory variables, including age, gender, tumor size, histological NUGC3, and NUGC4 (Figure 1A). We also performed RT- type, depth of invasion, lymph node metastasis, disease stage, PCR analysis of INHBA in specimens of gastric cancer and lymphatic invasion, and venous invasion, were evaluated with the χ2 paired normal adjacent mucosa obtained from seven patients. test. The postoperative survival rate was analyzed by the INHBA mRNA expression in these clinical samples was Kaplan−Meier method, and differences in survival rates were assessed significantly higher in cancer tissue than in paired normal with the log-rank test. A Cox proportional-hazards regression model INHBA was used for multivariate analyses. All statistical analyses were adjacent mucosa (Figure 1B). We confirmed mRNA performed using IBM SPSS Statistics 18.0 (SPSS, Inc., Chicago, IL, expression in clinical samples (n=168) by real-time RT-PCR. USA). Two-sided p-values were calculated, and a difference was Expression levels of the INHBA gene were higher in cancer considered significant if the p-value was less than 0.05. than in adjacent normal mucosa (p<0.0001) (Figure 2).

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Figure 3. Immunohistochemistry of Inhibin-βA (INHBA) expression. Expression of INHBA was evaluated by immunohistochemical analysis of resected specimens of gastric cancer. Positive staining for INHBA was observed in cytoplasm and was markedly more intense in human gastric cancer cells than in stromal cells, in both differentiated (A) and undifferentiated (B) types of gastric cancer.

Immunohistochemistry of INHBA expression. Expression of Discussion INHBA protein was evaluated by immunohistochemical analysis of resected specimens of gastric cancer. Positive In the present study, we examined expression levels of staining for INHBA was observed in the cytoplasm and was INHBA mRNA in gastric cancer and adjacent normal markedly more intense in human gastric cancer cells than in mucosa. We also studied the relations of relative expression stromal cells in both differentiated and undifferentiated types of the INHBA gene to clinicopathological factors and of gastric cancer (Figure 3). outcomes in patients with gastric cancer after curative surgery. Relations of INHBA mRNA expression levels to clinico- Several previous studies have compared relative mRNA pathological features. Expression levels of the INHBA gene expression levels of the INHBA gene between various types were categorized as low or high according to the median of cancer tissues and adjacent normal mucosa. Reis et al. value. The relations between gene expression and reported that INHBA mRNA expression was higher in breast clinicopathological features were then examined. Expression cancer tissues than in normal breast tissues (31). Seder et al. levels of the INHBA gene were related to TNM stage and found that expression levels of INHBA mRNA were 3.1- venous invasion (Table I). times higher in lung adenocarcinomas than in normal lung tissue (26). Another study showed that expression levels of Relations between INHBA mRNA expression levels and INHBA mRNA were higher in esophageal adenocarcinoma outcomes. Expression levels of the INHBA gene were than in Barrett’s metaplasia or esophageal dysplasia (27). categorized as low or high according to the median expression Wang et al. reported that INHBA mRNA levels in gastric value. Overall survival was significantly poorer in the patients cancer were significantly higher than those in the adjacent with high expression levels of the INHBA gene than in those non-cancerous tissue (32). Our results are consistent with with low expression levels (p=0.003) (Figure 4). these findings: expression levels of INHBA mRNA were significantly higher in 168 specimens of gastric cancer tissue Univariate and multivariate analyses of the relations of than in paired adjacent normal mucosa. clinicopathological factors and INHBA mRNA expression levels We next examined the relation between INHBA mRNA to outcomes. Univariate analysis revealed that gender, serosal expression levels and clinicopathological features in gastric invasion, lymph node metastasis, lymphatic invasion, vascular cancer. Shimizu et al. reported that overexpression of INHBA invasion, and INHBA gene expression were related to correlated with lymph node metastasis in head and neck outcomes. On multivariate Cox proportional hazards regression squamous cell carcinoma (33). Wang et al. found that analysis, INHBA gene expression was independently inversely increased INHBA mRNA expression significantly correlated related to outcome (p=0.0087) (Table II). with tumor diameter and depth of invasion in gastric cancer

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Table II. Univariate and multivariate Cox proportional hazards analysis of clinicopathological factors.

Univariate Multivariate

Variable/category n Hazard ratio 95% CI p-Value Hazard ratio 95% CI p-Value

Age (years) <70 88 1 ≥70 80 1.292 0.644-2.591 0.4699 Gender Female 55 1 1 Male 113 3.077 1.183-8.001 0.0212 2.481 0.941-6.536 0.0664 Histological type Differentiated 85 1 Undifferentiated 83 1.742 0.851-3.571 0.1286 Tumor size (cm) <6.5 94 1 ≥6.5 74 2.026 0.937-4.381 0.0728 Depth of invasion pT1, pT2, pT3 101 1 1 pT4 67 2.841 1.387-5.814 0.0043 1.912 0.855-4.274 0.1143 Lymph node metastasis pN0-pN2 129 1 1 pN3 39 2.833 1.406-5.714 0.0035 1.227 0.591-2.907 0.6126 Lympatic invasion Negative 64 1 1 Positive 104 4.032 1.543-10.526 0.0044 3.125 1.134-8.621 0.0276 Vascular invasion Negative 71 1 1 Positive 97 2.967 1.282-6.849 0.0111 1.366 0.561-3.322 0.4932 INHBA mRNA expression Low 84 1 1 High 84 3.048 1.404-6.623 0.0048 2.994 1.316-6.803 0.0087

INHBA: Inhibin-βA.

(32). In our study, expression levels of INHBA mRNA were significantly related to TNM stage and vascular invasion. We finally assessed the relation between INHBA gene expression levels and outcomes in gastric cancer. Shimizu et al. reported that disease-free survival was significantly shorter in patients with squamous cell carcinomas of the head and neck with high expression levels of INHBA compared to those with low expression levels (33). Seder et al. found that patients with stage I lung adenocarcinoma who had high levels of INHBA transcripts had significantly poorer outcomes than those who had low levels (26). Wang et al. demonstrated that patients with gastric cancer who had high INHBA expression had shorter overall survival and disease-free survival than those who had low INHBA expression (32). Our results confirm and extend the findings of these previous studies. Overall survival at five Figure 4. Relation between Inhibin-βA (INHBA) mRNA expression and years was significantly poorer in patients with high expression postoperative survival. Expression levels of the INHBA gene were levels of the INHBA gene than in those with low expression categorized as low or high according to the median value. The levels of the INHBA gene. On univariate and multivariate Cox postoperative survival rate was analyzed by the Kaplan−Meier method, and differences in survival rates were assessed by the log-rank test. proportional-hazards regression analysis, a higher level of Overall survival was significantly poorer in patients with high INHBA gene expression was a significant independent predictor expression levels of the INHBA gene than in those with low expression of 5-year survival in patients with gastric cancer. levels (p=0.003).

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The molecular mechanisms and functional impact of 8 Eto Y, Tsuji T, Takezawa M, Takano S, Yokogawa Y and Shibai INHBA expression in cancer remain to be fully H: Purification and characterization of erythroid differentiation elucidated. Seder et al. treated esophageal cancer cells factor (EDF) isolated from human leukemia cell line THP- 1.Biochem Biophys Res Commun 142: 1095-1103, 1987. with 5-aza-2’ deoxycytidine and trichodstatin A to 9 Murata M, Onomichi K, Eto Y, Shibai H and Muramatsu M: investigate the role of epigenetic regulation in INHBA Expression of erythroid differentiation factor (EDF) in Chinese expression (27). Such treatment was found to up-regulate hamster ovary cells. Biochem Biophys Res Commun 151: 230- INHBA mRNA and protein production. They suggested 235, 1988. that overexpression of INHBA may be affected by 10 Shiozaki M, Kosaka M and Eto Y: Activin A: A commitment promoter methylation and histone acetylation and may factor in erythroid differentiation. Biochem Biophys Res promote proliferation of esophageal cancer cells. Commun 242: 631-635, 1998. Yoshinaga et al. found that the expression level of 11 Mather JP, Moore A and Li RH: Activins, inhibins, and follistatins: Further thoughts on a growing family of regulators. neuronal cadherin (N-cadherin) was higher in esophageal Proc Soc Exp Biol Med 215: 209-222, 1997. cell lines transfected with INHBA than in the control 12 Mather JP, Woodruff TK and Krummen LA: Paracrine regulation cells, and a clinicopathological analysis suggested that of reproductive function by inhibin and activin. Proc Soc Exp patients with high expression of N-cadherin have Biol Med 201: 1-15, 1992. significantly poorer outcomes than those with low 13 Gurdon JB, Harger P, Mitchell A and Lemaire P: Activin expression of N-cadherin (17). 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