Abstract
Background: The aim of this study was to investigate the significance of signal transducer and activator of transcription 4 (STAT4) expression and the correlation between STAT4 and interferon gamma (IFN-γ) in patients with gastric cancer. Patients and Methods: Sixty-two patients who underwent gastrectomy for gastric cancer were enrolled in the study. STAT4 and IFNG mRNA expression was evaluated by quantitative real-time polymerase chain reaction (PCR). Immunohistochemistry was performed to examine CD8+ T-cells, and STAT4 and IFN-γ expression. Results: STAT4 mRNA expression was significantly correlated with IFNG mRNA expression (p<0.05). Regarding disease-free survival, there was a significant difference between the groups with high and low STAT4 expression (5-year disease-free survival: 77.8% and 56.4%, p<0.05). Univariate analysis revealed that tumor differentiation and STAT4 expression were significant factors for tumor recurrence. Conclusion: High expression of STAT4 in gastric cancer predicted a better clinical outcome. STAT4 might be a useful biomarker to identify patients at high risk of recurrence after gastrectomy.
Signal transducer and activator of transcription (STAT) proteins play a pivotal role in cancer progression. In particular, STAT3 and STAT5 proteins are activated in several types of cancer (1-5). In chronic myelogenous leukemia and myeloproliferative disease, STAT3 and STAT5 promote oncogenesis (3-5). Therefore, STAT3 or STAT5 may be molecular targets for cancer therapy. STAT4 is activated by interleukin-12 (IL12). Some reports demonstrated that STAT4 has anticancer effects (6, 7). In hepatocellular carcinoma, STAT4 mRNA was down-regulated and was correlated with worse prognosis (8, 9).
Interferon gamma (IFN-γ) has an indirect anticancer effect through IL12 signaling (10-11), which induces apoptosis in tumor cells (12). In gastric cancer cells, IFN-γ slowed proliferation by affecting the cell cycle (13). Helicobacter pylori-infected patients exhibited high levels of STAT4 compared to uninfected patients (14). Importantly, deficiencies in IFN-γ (IFNG) and STAT4 genes inhibited differentiation of naïve T-cells to CD8+ T-cells and natural killer cells (6, 7). We previously demonstrated that low STAT4 expression in hepatocellular carcinoma indicated aggressive tumor behavior, predicted a worse clinical outcome, and was correlated with IFN-γ expression and CD8+ T-cell infiltration (8). However, the clinical significance of STAT4 and the correlation between STAT4, IL12 and IFN-γ in gastric cancer are unclear.
The aim of this study was to investigate the significance of STAT4 expression, and the correlation between STAT4 and IFN-γ in patients with gastric cancer.
Patients and Methods
Patients. This study enrolled a total of 62 patients who had undergone gastrectomy for gastric cancer at Tokushima University Hospital between September 2008 and September 2009. This study was authorized by the Institutional Review Board of the University of Tokushima Graduate School (no. 2901). Informed consent was obtained from each patient. Forty patients were males, and 22 patients were female. The age of the participants ranged from 40 years to 87 years, with a mean age of 62 years. Clinicopathological parameters were obtained from the pathological reports, including tumor differentiation, lymph node metastasis, and TNM stage. All of these data were reviewed and confirmed by experienced pathologists.
Quantitative real-time polymerase chain reaction (PCR). Total ribonucleic acid (RNA) was extracted from gastric cancer tissue and non-cancer tissue using an RNeasy Mini Kit (Qiagen, Hilden, Germany) and cDNA was synthesized from 2.5 μg total RNA by reverse transcription using Super Script RT kit (Promega, Madison, WI, USA) according to the manufacturer's instructions (9). Quantitative real-time RT-PCR was performed with Taqman Universal Master Mix (gene-specific TaqMan probes on a StepOne Plus; Applied Biosystems, Foster City, CA, USA) real-time core reagents on an ABI 7500 (Applied Biosystems). Primers for quantitative real time RT-PCR were as follows: TaqMan human STAT4 (Hs01028017_m1), and human IFNG (Hs00174143_m1). Expression levels of both genes were calculated as a ratio to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Immunohistochemistry. Sample processing and immunohistochemistry were performed following standard immunohistochemical staining protocols (9). In brief, tumor sections from resected specimens were cut to a thickness of 4 μm, de-paraffinized in xylene, and dehydrated in a descending ethanolic series. Endogenous peroxidase activity was blocked by 10 minutes of incubation with 0.3% hydrogen peroxidase in 50% methanol. Antigen retrieval was performed using a multi-functional microwave histoprocessor at 100°C and microwave heating in citrate buffer (pH 6.0) for 24 min. Sections were incubated with primary antibody for 60 min at ambient temperature and were washed three times with phosphate-buffered saline for 5 minutes. Immunohistochemical staining was completed by using a mouse monoclonal antibody against STAT4 (ab57822, dilution 1:100; Abcam, Cambridge, MA, USA), a rabbit polyclonal antibody to IFN-γ (ab9657, dilution 1:100; Abcam), and mouse monoclonal antibody to human CD8+ (dilution 1:50; Dako, Copenhagen, Denmark). After washing with PBS, sections were incubated for 60 minutes at ambient temperature with secondary antibody (code K5027; Dako, Glostrup, Denmark). Diaminobenzidine tetrahydrochloride was used as the chromogen. Finally, sections were counterstained with Mayer's hematoxylin.
Evaluation of immunohistochemistry. For a positive control of STAT4, we used human ovary cell carcinoma tissue, and negative controls were performed by omitting the primary antibody. For a positive control of IFN-γ expression, we used tissue from a human cervical squamous cell carcinoma. The mean percentage of positively stained tumor cells was determined using an Image System (Nikon Digital Camera, DXM1200F; Gotenba, Shizuoka, Japan) (9).
Statistical analysis. Continuous variables e presented as mean±standard deviation (SD). Pearson's chi-squared test was used to compare categorical variables, whereas Student's t-test was used for continuous variables. The average was used as a cutoff value to divide the patients into high and low mRNA expression groups. Survival curves after surgery were generated using the Kaplan–Meier method and differences were compared using the log-rank test. Information from the laboratory analysis and patients were entered into JMP 10 software (SAS Institute, Cary, NC, USA) and differences with a p-value of less than 0.05 were considered statistically significant.
Results
mRNA expression of STAT4 and IFNG. STAT4 and IFNG mRNA were significantly expressed in non-tumor tissue compared with tumor tissue (p<0.05) (Figure 1A and B). CD8+ T-cell expression was evaluated in 20 patients by immunohistochemistry. The number of CD8+ T-cells expressed in non-tumor tissue was significantly higher than that in tumor tissue (Figure 1C).
Correlations between STAT4 mRNA expression in tumor tissue and clinicopathological parameters. Correlations between STAT4 mRNA expression in tumor tissue and clinicopathological parameters, including age, gender, T factor, final tumor stage, venous invasion, lymphatic invasion, and differentiation were evaluated (Table I). The patients were divided into two groups based on the average value of STAT4 expression: a high-expressing group (30.6%; n=19) and a low-expressing group (69.4%; n=43). High STAT4 expression was significantly correlated with poor tumor differentiation (p<0.05). There was no significant correlation regarding age, gender, T factor, final stage, venous invasion, or lymphatic invasion.
Correlation between STAT4 and IFNG mRNA expression and CD8+ T-cell infiltration. There was a significant positive correlation between STAT4 mRNA expression and IFNG mRNA expression (R=0.445; p<0.05) (Figure 2). STAT4 and IFN-γ protein expression were evaluated by immunohistochemistry. Immunohistochemistry revealed both STAT4 and IFN-γ were highly expressed in non-tumor tissue (Figure 3A-D). The infiltration of CD8+ T-cells in non-tumor tissue was higher than that in tumor tissue (Figure 3E and F).
Disease-free survival (DFS) rate according to STAT4 mRNA expression. DFS was evaluated in 57 patients who underwent curative resection. The high STAT4 expression group included 18 patients (31.6%), and the low STAT4 expression group included 39 patients (68.4%). Regarding DFS, there was a significant difference between the high STAT4-expressing group and the low STAT4-expressing group (5-year DFS: 77.8% and 56.4%, respectively; p<0.05) (Figure 4). Univariate analysis showed that tumor differentiation and STAT4 expression were significant factors in tumor recurrence (Table II). Multivariate analysis using the Cox's proportional hazards model showed STAT4 expression exhibited a tendency for being related to tumor recurrence (Table II).
Discussion
This report showed that STAT4 and IFNG mRNA in non-tumor tissue were highly expressed compared with that in tumor tissue. High STAT4 mRNA expression in tumor tissue was significantly correlated with poor differentiation. STAT4 mRNA expression was significantly correlated with IFNG mRNA expression in gastric cancer. High STAT4 mRNA expression indicated better DFS in gastric cancer.
Recently, several studies reported that STAT genes play important and differing roles in carcinogenesis. STAT3 has an oncogenic role in breast cancer, melanoma and lung cancer cell lines (15-17), and directly activates vascular endothelial growth factor (18). In contrast, STAT4 regulates transcription of IFNG via the Janus kinase (JAK)/STAT pathway (19). IFN-γ has roles in tumor growth and metastasis (20, 21), and is also correlated with IL12. IL12 promotes tumor regression and has antiproliferative effects (22). STAT4 expression was found to be activated in immune cells, and down-regulation of STAT4 correlated with decreased antiviral and antitumor activity and increased mortality in in vivo experiments (23-26). High STAT4 expression was identified in non-tumor tissue but not in tumor tissue. These findings may be explained by the effect of STAT4 on the immune system including natural killer cells or CD8+ T-cells (27). Moreover, a previous report demonstrated that natural killer cells were decreased in gastric cancer (28). To our knowledge, this is the first report regarding the role of STAT4 expression in gastric cancer, especially regarding the clinicopathological findings of gastric cancer. Interestingly, our results showed that the group with high STAT4 mRNA expression had a lower disease-free survival rate than those with low STAT4 mRNA expression. As shown in our previous report, down-regulation of STAT4 indicated aggressive tumor characteristics and worse clinical outcome in patients with hepatocellular carcinoma (9).
mRNA expression levels of signal transducer and activator of transcription 4 (STAT4) (A) and interferon gamma (IFN-γ) (B) in tumorous and non-tumorous tissue from gastric cancer. C: Average number of CD8+ cells, as stained by immunohistochemistry. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.
Correlation between signal transducer and activator of transcription 4 (STAT4) mRNA expression and clinicopathological findings.
Correlation between mRNA expression of signal transducer and activator of transcription 4 (STAT4) and interferon gamma (IFN-γ) in gastric cancer. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.
Representative histological features in non-tumorous and tumorous tissue a from gastric cancer patient by signal transducer and activator of transcription 4 (STAT4) and interferon gamma (IFN-γ) immunostaining. STAT4 expression in non-tumor (A) and in tumor (B) tissue. IFN-γ expression in non-tumor (C) and tumor (D) tissue. CD8+-positive cells in non-tumor (E) and tumor (F) tissue.
Risk factor analysis of gastric cancer recurrence in tumor tissue.
IFN-γ plays an indirect anticancer role through the immune system, and inhibited peritoneal metastasis in an animal gastric cancer model (29). The mechanisms of the anticancer effect of IFN-γ have been investigated. The regulation of T-cell migration to tumor cells, antagonizing products of immunosuppressive cytokines such as transforming growth factor-β and IL10, induce an effective antitumor immune response (20, 21, 30, 31). In this study, IFNG mRNA expression in non-tumor tissue was higher than in tumor tissue, and had a significant positive correlation with STAT4 mRNA expression. In hepatocellular carcinoma, STAT4 mRNA expression was also positively correlated with IFNG mRNA expression in hepatocellular carcinoma (9).
CD8+ T-cells play a role in the local immune response to suppress carcinogenesis, and generate IFN-γ by binding tumor-related antigens (32), and increase cancer suppressor activity by inducing apoptosis or via the tumor-killing effect of macrophages (33, 34). In our experiments, there was no significant correlation between the presence of CD8+ T-cells and IFNG or STAT4 mRNA expression, but immunohistochemistry showed significantly high proportions of CD8+ T-cells in non-tumor tissue compared with tumor tissue; this finding may indicate a relationship between these genes.
Disease-free survival (DFS) curve according to signal transducer and activator of transcription 4 (STAT4) mRNA expression in gastric cancer.
In conclusion, high expression of STAT4 in gastric cancer predicted a better clinical outcome. STAT4 might be a useful biomarker to identify patients at high risk of recurrence after gastrectomy.
Footnotes
Ethical Statement
All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964 and later versions. Informed consent or a substitute was obtained from all patients included in the study.
Conflicts of Interest
The Authors declare that they have no conflict of interest in regard to this study.
- Received August 21, 2017.
- Revision received October 5, 2017.
- Accepted October 9, 2017.
- Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved