Abstract
Inhibin-βA (INHBA), a ligand belonging to the transforming growth factor-β superfamily, is associated with cell proliferation in cancer. We studied the relations of INHBA gene expression to clinicopathological factors and outcomes in 168 patients with gastric cancer who underwent curative surgery. Relative INHBA gene expression was measured in surgical specimens of cancer tissue and adjacent normal mucosa by quantitative real-time, reverse-transcription polymerase chain reaction. INHBA expression levels were significantly higher in cancer tissue than in adjacent normal mucosa and were related to TNM stage and venous invasion. High INHBA gene expression was associated with significantly poorer 5-year overall survival than was low expression. On multivariate analysis, INHBA gene expression was an independent prognostic factor. Overexpression of the INHBA gene is considered a useful independent predictor of outcomes in patients with gastric cancer after curative surgery.
Inhibin-βA (INHBA) is a ligand belonging to the transforming growth factor-β (TGF-β) superfamily (1). INHBA forms a disulfide-linked homodimer known as activin A (2, 3). Activin A has been implicated in multiple biological processes (4-7). One of the most important functions of activin A is regulation of cell differentiation. Activin A controls several aspects of hematopoiesis (8-10) and regulates cell differentiation in the ovary, placenta, prostate, and testis (11-12). During embryogenesis, it is instrumental for axis development and organogenesis in a variety of species (13). In adults, activins function as hormone-like feedback regulators in the reproductive system (14). Furthermore, presence of activin has been related to wound healing (15).
As for the relation between activin A and cancer, overexpression of activin A in esophageal squamous cell carcinoma has been associated with advanced nodal status, clinical stage, and poorer overall outcomes (16). In addition, chronic exposure to activin A promotes growth, tumorigenicity, tumor invasion, and resistance to apoptosis in esophageal squamous cell carcinoma (17). Inhibin α-knockout mice express high levels of serum activin A and almost uniformly develop gonadal stromal tumors, supporting the role of activin A overexpression in tumorigenesis (18). Furthermore, overexpression of activin A has been reported in colon, prostate, pancreatic, ovarian, endometrial, and cervical cancer (19-25).
The role of INHBA in cancer has not been fully-elucidated. Recent studies have shown that INHBA expression correlates with cell proliferation and outcomes in lung adenocarcinoma and esophageal adenocarcinoma (26-27). In gastric cancer, the INHBA gene was reported to belong to a set of eight biomarkers shown to predict cancer and normal status of gastric tissue (28). Moreover, the INHBA gene was revealed as one out of seven candidate genes for gastric carcinogenesis and progression by a 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 gastric cancer.
In the present study, we measured expression levels of the INHBA gene in specimens of cancer and adjacent normal mucosa obtained from 168 patients with gastric cancer after curative surgery. Our objective was to evaluate the relative expression of the INHBA gene and to determine whether such expression correlates with outcomes in patients with gastric cancer who undergo curative surgery.
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.
Materials and Methods
Patients and samples. We studied surgical specimens of cancer tissue and paired adjacent normal mucosa obtained from 168 patients with previously untreated stage I-III gastric cancer who underwent curative surgery. Tumor staging was evaluated according to the seventh edition of the International Union Against Cancer (UICC)-TNM classification of malignant tumors (30). The patients underwent surgery at the Gastroenterological Center, Yokohama City University Medical Center, and at the Department of Surgery, Yokohama City University from January 2002 through July 2007. Informed consent was obtained from each patient, and the Ethics Committees of Yokohama City University Medical Center and Yokohama City University approved the protocol before initiation of the study (approval number: 18-7A-4). All tissue samples were embedded in OCT compound (Sakura Finetechnical Co., Ltd., Tokyo, Japan) and immediately stored at −80°C until use. No patient had any other malignancies. The histopathological features of specimens stained with hematoxylin and eosin were examined, and sections that consisted of more than 80% cancer cells were used to prepare total RNA.
Cell lines. Human gastric cancer cell lines (MKN1, MKN7, MKN45, MKN74, NUGC3, and NUGC4) were provided by the Japanese Cancer Research Bank, Tokyo, Japan. Cell lines were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Equitec-Bio, Ingram, TX, USA), 100 units/ml penicillin G, and streptomycin (Invitrogen). The cells were incubated in 5% CO2 at 37°C and passaged every 3-4 days, except for MKN7 which was subcultured at 7-day intervals.
RNA extraction and complementary DNA (cDNA) synthesis. Total RNA isolated from gastric cancer tissue and adjacent normal mucosa was prepared with the use of Trizol (Gibco, Life Technology, Gaithersburg, MD, USA). cDNA was synthesized from 0.4 μg of total RNA with an iScript cDNA Synthesis Kit (Bio-Rad Laboratories. Hercules, CA, USA). After synthesis, the cDNA was diluted to 0.2 μg/μl with water and stored at −20°C until use.
Oligonucleotide primers for INHBA cDNA amplification by reverse–transcription polymerase chain reaction (RT-PCR). The oligonucleotide primers for INHBA were as follows: sense primer 5’-GGTATGTGGAGATAGAGGATGAC-3’ and antisense primer 5’-TCCTGGCTGTTCCTGACTC-3’. We used β-actin as the internal control. The oligonucleotide primers for β-actin were as follows: sense primer 5’-AGTTGCGTTACACCCTTTCTTGAC-3’ and antisense primer 5’-GCTCGCTCCAACCGACTGC-3’. Amplification of INHBA was performed for 40 cycles of 1 minute at 95°C, 1 minute at 56°C, and 1 minute at 72°C. Amplification of β-actin was performed for 40 cycles of 1 minute at 95°C, 1 minute at 60°C, and 1 minute at 72°C. A 10-μl aliquot of each amplified PCR product was electrophoresed on a 3% agarose gel containing ethidium bromide.
Quantitative real-time, RT-PCR. Quantitative real-time PCR was performed with iQ SYBR Green Supermix (Bio-Rad Laboratories). PCR reactions were carried out in a total volume of 15 μl, which included 0.2 μg of cDNA, 0.4 μM of each primer, 7.5 μl of iQ SYBR Green Supermix containing dATP, dCTP, dGTP, and dTTP at concentrations of 400 μM each, and 50 units/ml of iTag DNA polymerase. The PCR consisted of 10 minutes at 95°C, followed by 40 cycles of denaturation of the cDNA for 10 seconds at 95°C, annealing for 10 seconds at 56°C (60°C for β-actin), and a primer extension for 20 seconds at 72°C, followed by 10 minutes at 72°C. To distinguish specific from nonspecific products and primer dimmers, melting curve analyses were carried out. To evaluate specific mRNA expression in samples, a standard curve was produced for each run, measuring three points of the human control cDNA (Clontech Laboratories, Inc., CA, USA). The concentrations of each sample were calculated by relating its crossing point to standard curve.
Comparison of Inhibin-βA (INHBA) mRNA expression levels between gastric cancer tissue and adjacent normal mucosa. Box boundaries, the 25th and 75th percentiles of the observed values; capped bars, the 10th and 90th percentiles; solid line, median. p-Values were calculated by the Wilcoxon signed-rank test. Expression levels of the INHBA gene were higher in cancer than in adjacent normal mucosa (p<0.0001).
Immunohistochemical analysis. Immunohistochemical studies of INHBA were performed on formalin-fixed, paraffin-embedded surgical specimens obtained from patients with gastric cancer. The tissue sections were deparaffinized and soaked in 10 mM sodium 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 sections were counterstained with hematoxylin. Primary polyclonal antibodies against INHBA (Atlas Antibodies, Stockholm, Sweden) were used at a dilution of 1:200.
Statistical analysis. Gene expression levels of gastric cancer were compared with those of adjacent normal mucosa by the Wilcoxon signed-rank test. Relations between gene expression and potential explanatory variables, including age, gender, tumor size, histological type, depth of invasion, lymph node metastasis, disease stage, lymphatic invasion, and venous invasion, were evaluated with the χ2 test. The postoperative survival rate was analyzed by the Kaplan–Meier method, and differences in survival rates were assessed with the log-rank test. A Cox proportional-hazards regression model was used for multivariate analyses. All statistical analyses were performed using IBM SPSS Statistics 18.0 (SPSS, Inc., Chicago, IL, USA). Two-sided p-values were calculated, and a difference was considered significant if the p-value was less than 0.05.
Relation of Inhibin-βA (INHBA) mRNA expression to clinicopathological features.
Results
INHBA mRNA expression in human gastric cancer. Expression of INHBA mRNA in human gastric cancer cell lines and clinical samples was analyzed by RT-PCR (Figure 1). INHBA mRNA expression was observed in the human gastric cell lines: MKN1, MKN7, MKN45, MKN74, NUGC3, and NUGC4 (Figure 1A). We also performed RT-PCR analysis of INHBA in specimens of gastric cancer and paired normal adjacent mucosa obtained from seven patients. INHBA mRNA expression in these clinical samples was significantly higher in cancer tissue than in paired normal adjacent mucosa (Figure 1B). We confirmed INHBA mRNA expression in clinical samples (n=168) by real-time RT-PCR. Expression levels of the INHBA gene were higher in cancer than in adjacent normal mucosa (p<0.0001) (Figure 2).
Immunohistochemistry of Inhibin-βA (INHBA) expression. Expression of INHBA protein 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 INHBA protein was evaluated by immunohistochemical analysis of resected specimens of gastric cancer. Positive staining for INHBA was observed in the cytoplasm and was markedly more intense in human gastric cancer cells than in stromal cells in both differentiated and undifferentiated types of gastric cancer (Figure 3).
Relations of INHBA mRNA expression levels to clinicopathological features. Expression levels of the INHBA gene were categorized as low or high according to the median value. The relations between gene expression and clinicopathological features were then examined. Expression levels of the INHBA gene were related to TNM stage and venous invasion (Table I).
Relations between INHBA mRNA expression levels and outcomes. Expression levels of the INHBA gene were categorized as low or high according to the median expression value. Overall survival was significantly poorer in the patients with high expression levels of the INHBA gene than in those with low expression levels (p=0.003) (Figure 4).
Univariate and multivariate analyses of the relations of clinicopathological factors and INHBA mRNA expression levels to outcomes. Univariate analysis revealed that gender, serosal invasion, lymph node metastasis, lymphatic invasion, vascular invasion, and INHBA gene expression were related to outcomes. On multivariate Cox proportional hazards regression analysis, INHBA gene expression was independently inversely related to outcome (p=0.0087) (Table II).
Discussion
In the present study, we examined expression levels of INHBA mRNA in gastric cancer and adjacent normal mucosa. We also studied the relations of relative expression of the INHBA gene to clinicopathological factors and outcomes in patients with gastric cancer after curative surgery.
Several previous studies have compared relative mRNA expression levels of the INHBA gene between various types of cancer tissues and adjacent normal mucosa. Reis et al. reported that INHBA mRNA expression was higher in breast cancer tissues than in normal breast tissues (31). Seder et al. found that expression levels of INHBA mRNA were 3.1-times higher in lung adenocarcinomas than in normal lung tissue (26). Another study showed that expression levels of INHBA mRNA were higher in esophageal adenocarcinoma than in Barrett's metaplasia or esophageal dysplasia (27). Wang et al. reported that INHBA mRNA levels in gastric cancer were significantly higher than those in the adjacent non-cancerous tissue (32). Our results are consistent with these findings: expression levels of INHBA mRNA were significantly higher in 168 specimens of gastric cancer tissue than in paired adjacent normal mucosa.
We next examined the relation between INHBA mRNA expression levels and clinicopathological features in gastric cancer. Shimizu et al. reported that overexpression of INHBA correlated with lymph node metastasis in head and neck squamous cell carcinoma (33). Wang et al. found that increased INHBA mRNA expression significantly correlated with tumor diameter and depth of invasion in gastric cancer (32). In our study, expression levels of INHBA mRNA were significantly related to TNM stage and vascular invasion.
Univariate and multivariate Cox proportional hazards analysis of clinicopathological factors.
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 years was significantly poorer in patients with high expression levels of the INHBA gene than in those with low expression levels of the INHBA gene. On univariate and multivariate Cox proportional-hazards regression analysis, a higher level of INHBA gene expression was a significant independent predictor of 5-year survival in patients with gastric cancer.
Relation between Inhibin-βA (INHBA) mRNA expression and postoperative survival. Expression levels of the INHBA gene were categorized as low or high according to the median value. The postoperative survival rate was analyzed by the Kaplan–Meier method, and differences in survival rates were assessed by the log-rank test. Overall survival was significantly poorer in patients with high expression levels of the INHBA gene than in those with low expression levels (p=0.003).
The molecular mechanisms and functional impact of INHBA expression in cancer remain to be fully elucidated. Seder et al. treated esophageal cancer cells with 5-aza-2’ deoxycytidine and trichodstatin A to investigate the role of epigenetic regulation in INHBA expression (27). Such treatment was found to up-regulate INHBA mRNA and protein production. They suggested that overexpression of INHBA may be affected by promoter methylation and histone acetylation and may promote proliferation of esophageal cancer cells. Yoshinaga et al. found that the expression level of neuronal cadherin (N-cadherin) was higher in esophageal cell lines transfected with INHBA than in the control cells, and a clinicopathological analysis suggested that patients with high expression of N-cadherin have significantly poorer outcomes than those with low expression of N-cadherin (17). They suggested that activin A, a homodimer of INHBA, might mediate the expression of N-cadherin, potentially leading to poor outcomes. Further molecular investigations are necessary to clarify the role of INHBA as a prognostic factor and to explain the pleiotropic functions of this protein.
In conclusion, INHBA gene expression levels were higher in cancer tissue than in normal mucosa in patients with gastric cancer, and high expression of this gene was significantly related to poor outcomes. Our findings suggest that overexpression of the INHBA gene is a useful independent predictor of outcome after curative surgery in patients with gastric cancer.
- Received January 10, 2014.
- Revision received February 24, 2014.
- Accepted February 25, 2014.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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