Abstract
Backround: Gastric adenocarcinoma is one of the most common malignant tumors and the leading cause of malignancy-related death worldwide. Studies have reported overexpression of activation-induced cytidine deaminase (AID) and protein kinase c iota (PKCi) proteins showing involvement in the regulation of carcinogenesis. In the present study, we investigated the expression of AID and PKCi in patients with gastric adenocarcinoma and determined the correlation between these proteins. Materials and Methods: This study was conducted between September 2009 and September 2010 on a total of 59 patients with gastric adenocarcinoma at the Tokushima University Hospital. AID, PKCi and mutated p53 protein expressions were evaluated by immunohistochemistry in gastric adenocarcinoma. Results: High AID and PKCi expression was significantly (p<0.05) associated with poorly-differentiated gastric adenocarcinoma. In addition, PKCi expression was significantly correlated with clinicopathological findings such as a lymph node metastasis, and venous and lymphatic invasion (p<0.05). Furthermore, AID expression was significantly correlated with PKCi and mutated p53 protein expression in gastric adenocarcinoma (p<0.05). Conclusion: High AID and PKCi expressions were significantly correlated with poorly-differentiated gastric adenocarcinoma.
- AID
- PKCi
- gastric adenocarcinoma
Gastric adenocarcinoma is one of the most common malignant tumors and the leading cause of malignancy-related death worldwide (1). Its 5-year survival rates are approximately 20% in most countries of the world (2, 3), excluding Japan. Tsubono et al. reported 5-year survival rates for gastric cancer being 60% in Japan (4).
Activation-induced cytidine deaminase (AID) is a member of the apolipoprotein B mRNA editing enzyme (APOBEC), which plays a crucial role in DNA breakage through de-amination of cytosines into uracils. However, the overexpression or persistent expression of AID protein was correlated with chronic inflammation through nuclear factor-kappa B (NF-κB) in hepatitis and gastritis (5, 6). Constitutive expression of AID also caused the development of liver tumors with the morphological characteristics of hepatocellular carcinoma (7). The expression of AID was not detected in non-cancerous lung cells, it was detected in lung cancer cells (8), which shows that AID is aberrantly expressed in primary lung cancer cells. In general, the above studies show AID expression is common in several cancer types.
PKCi protein plays different roles in epithelial cell functions, such as in cell polarity (9-11), cell survival (12), and cell growth (11). Moreover, PKCi is involved in promoting tumorigenicity and metastasis of human esophageal cancer (13). Among the PKC isozymes, only atypical PKCi has been shown to function as a proper oncogene (14, 15), and as such, is an attractive target for gastric adenocarcinoma. Both AID and PKCi expressions were observed in different cancer tissues, from that we hypothesized that these proteins might be positively correlated with each other.
p53 is a tumor-suppressor gene which plays a crucial role in growth suppression of cancer, apoptosis and DNA repair (16). Mutated p53 (mp53) can accumulate at high concentration in the nuclei of tumor cells (17), and its dysfunction may affect the biological events in cancer cells, which could lead to aggressive carcinogenesis. Futhermore, mp53 was detected in primary human gastric cancer (18).
In general AID is induced by inflammation and is involved in various types of human carcinogenesis. Abnormal expression of AID, as well as PKCi, has been reported in different types of cancer. However, the correlation between AID and PKCi remains unknown. Therefore, increasing knowledge about AID and understanding its correlation with PKCi, as well as mp53, might allow for stratification of the prognosis of various cancer types and consideration of whether targeting these proteins is beneficial for patients with adenocarcinoma. In the present study we, performed immunohistochemistry in gastric tumor tissues with the aim of a better comprehension of the correlation between AID expression with PKCi, as well as mp53, expression, in gastric adenocarcinoma and also the clinicopathological correlations with the expression of these proteins.
Patients and Methods
We enrolled a total of 59 (13 male, and 46 female) patients surgically-resected for gastric adenocarcinoma at the Tokushima University Hospital between September 2009 and September 2010. The age of the study participants ranged from 40 to 87 years, with a mean age of 62 years. The clinicopathological parameters were obtained from the pathological reports, including tumor differentiation, lymph node metastasis, and TNM stage, and all data were reviewed and confirmed by experienced pathologists.
Immunohistochemistry. The sample processing and immunohistochemistry procedures were performed following standard immunohistochemical staining protocols. In brief, sections were cut with thickness of 4 μm, de-paraffinized in xylene, and dehydrated in descending ethanol concentration. Endogenous peroxidase activity was blocked by 10 min of incubation with 0.3% hydrogen peroxidase in 50% of methanol. Antigen retrieval was performed by using a multifunctional microwave histoprocessor at 100°C and by using a microwave heating in citrate buffer (pH 6.0) for 24 min. We used a protein block (serum-free, code x0909; Dako, Glostrup, Denmark) for blocking non-specific antigen of AID for 10 min. Sections were incubated for 60 min at ambient temperature with primary antibody and were washed with phosphate buffered saline (PBS) three times for 5 min. Immunohistochemical staining was completed using a monoclonal mouse antibody against AID (ZA001, catalog no.39-250, dilution 1:100; Invitrogen, Zymed Laboratories, San Francisco, CA, USA), PKCi (Mat.Number 610176, dilution 1:100, BD Biosciences) and human p53 (clone DO-7, code no. M 7001; dilution 1:100; Dako).
After washing with PBS, sections were incubated for 60 min at ambient temperature with second antibody of the detection reagent (code K5027, Glostrup, Denmark). Diaminobenzidine tetrahydrochloride was used as the chromogen. Finally, sections were counter-stained with Mayer's hematoxylin.
Immunohistochemistry evaluation. For positive control of AID, we used tonsillar tissue and lymph nodes, and negative controls were performed by omitting the primary antibody. The mean percentage of positive tumor cells was determined using an Image System (Nikon Digital Camera, DXM1200F, Gotenba, Shizuoka, Japan).
Cytoplasmic AID expression was evaluated by summing the staining percentage and intensity scores. The staining percentage was assigned to one of the following categories: 0= no staining, 1= up to 5% of cells stained, 2= 5%-10% of cells stained, 3= 10%-25% of cells stained, 4= 25%-50% of cells stained, 5= more than 50% of cells stained. The staining intensity was assigned as: 0=no expression, 1+= weak expression, 2+= strong expression. The cytoplasmic expression levels of AID were classified as low (score ≤4) and high (score ≥5) (19).
p53 protein immunohistochemistry staining was considered positive when nuclear staining was 10% or more (20).
PKCi was scored according to a previous method as follows: 0, no staining; 1+, weak; 2+, moderate intensity; and 3+, strong intensity. Scores of 0 and 1+ were defined as negative, scores of 2+ and 3 were defined as positive (21). Figure 1 shows representative images of immunohistochemistry.
Statistical analysis. The p-value of overall and disease-free survival was denied by using Kaplan Meier analysis with JMP10 software (SAS Campus Drive, Cary, NC). Information from the laboratory analysis and patients were entered into Stat. View 5.0 software (SAS Campus Drive, Cary, NC) and statistical analyses were performed using Student's t-test, the chi-square test. A p-value of less than 0.05 was considered statistically significant.
Results
Relationship between AID expression in the cancerous lesion of gastric adenocarcinoma and clinicopathological findings. Patients' clinicopathological characteristics are summarized in Table I.
The relationship between AID expression and the clinicopathological characteristics, such as tumor differentiation, age, gender, tumor stage, and venous or lymphatic invasion were evaluated as shown in Table II. AID was significantly correlated with poorly-differentiated gastric adenocarcinoma (p=0.01). However, there was no significant correlation between AID expression and age, gender, stage, TNM classification, nor venous or lymphatic invasion.
Correlation between PKCi expression and clinicopathological findings in gastric adenocarcinoma. The correlation of PKCi expression and the clinicopathological characteristics is examined in Table III. The positive expression of the PKCi protein was significantly correlated with lymph node metastasis, venous invasion, lymphatic invasion and poorly-differentiated gastric adenocarcinoma (p<0.05).
Correlation of AID, PKCi and mp53 protein expressions in gastric adenocarcinoma. In this study, 47 (80%) cases of AID-high and 12 (20%) of AID-low expression in the carcinoma of gastric adenocarcinoma were examined. There were 40 (68%) positive and 19 (32%) negative cases of PKCi protein expression, which was significantly positively-correlated with AID protein (Table II) (p=0.01). In addition, 39 (66%) mp53-positive and 20 (34%) mp53-negative cases were observed, which correlated with AID protein expression significantly in gastric adenocarcinoma (Table II) (p=0.045).
Survival rates of patients according to AID and PKCi expression in gastric adenocarcinoma. Overall survival was evaluated in 59 patients and disease-free survival was evaluated in 51 patients who underwent resection; for disease-free survival, patients with stage IV disease were excluded. Median follow-up time was 36 months, with a range of 4 to 46 months. As shown in Figure 2A there was no significant difference, but there was tendency (p=0.12) for better 4-year overall survival in the patients' group with low AID expression. The disease-free survival of AID expression was 85% in the high- and 100% low-AID groups at 4 years (p=0.23) (Figure 2B). Disease-free survival by PKCi-positive and -negative expression (80% and 100%, respectively 3-year) is shown in Figure 2C (p=0.18).
Discussion
The present study examined AID and PKCi expression in patients surgically-resected for gastric adenocarcinoma, and these results were compared to clinicopathological characteristics.
The expression of AID has been mentioned in non-B-cells, including gastric epithelial cells, human hepatocytes, biliary cells, and colonic epithelial cells (5, 7, 22, 23). These studies have suggested that AID is a genomic mutator. Its aberrant expression was detected in several types of cancer, such as hepatocellular carcinoma, cholangiocarcinoma and colon cancer (7, 22, 23). We revealed here that high AID expression was significantly correlated with poorly-differentiated gastric adenocarcinoma.
Previously, PKCi was shown to be a key signaling component involved in the regulation of normal cell proliferation, differentiation, and cell polarity (9-12, 24). This kinase localizes to the apical domain of epithelial cells, its role is establishing cell polarity (9).
PKCi is oncogenic in several types of cancer. This study investigated the relationship between PKCi and clinicopathological characteristics, and the correlation of AID and PKCi protein expression in gastric adenocarcinoma. In previous studies, overexpression of PKCi was observed in cholangiocarcinoma, and in lung, ovarian, breast, and gastric cancer (15, 25-28). Positive expression of the PKCi protein was found here to be significantly correlated with lymph node metastasis, venous and lymphatic invasion, and poorly-differentiated gastric adenocarcinoma. Takagawa et al. reported that high expression of PKCi was significantly correlated with lymph node metastasis in gastric cancer (28). The PKCi gene was identified as a human oncogene (26, 29).
In the present study, we found a significant correlation between AID and PKCi expressions. We can speculate that these proteins may influence each other and promote carcinogenesis.
In another study, Helicobacter pylori infection induced transient activation of aPKC (30, 31), which is required for NF-κB activation (32). It has been reported that AID expression was activated through the NF-κB activation in human gastric epithelial cells and hepatocellular carcinoma (5, 7).
In addition, we explored the strong correlation between AID and mp53 protein in gastric adeocarcinoma. Aberrant expression of AID protein promotes accumulation of nucleotide alterations in the p53 gene and its overexpression in human gastric cancer (5, 33). p53 mutation has been found in well-differentiated adenocarcinoma (34). An in vitro study showed the overexpression of AID induces a mutation of p53 gene in gastric cells (7). Some studies reported the frequency of p53 alteration in gastric cancer, 18%-58% had p53 mutation, and p53 overexpression occured in 26%-65% of gastric adenocarcinomas (35, 36).
We found a tendency for a high expression of AID protein to correlate with a lower rate in gastric adenocarcinoma. In our study, positive expression of PKCi protein was related to lower rate of disease-free survival in gastric adenocarcinoma, which was comparable to the findings of Takagawa et al. whereby aPKC λ/i overexpression was a strong prognostic marker for gastric cancer recurrence (28).
Conclusion
High AID and PKCi expressions were significantly correlated with poorly-differentiated gastric adenocarcinoma.
Acknowledgements
We are grateful to the members of Department of Surgery, Institute of Health Biosciences at The University of Tokushima for their important contributions to this study.
- Received December 9, 2013.
- Revision received December 22, 2013.
- Accepted December 24, 2013.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved