Abstract
Gastric cancer is one of the leading causes of tumor-related death worldwide, for which the prevalence and mortality rates are very high in developed countries. Caveolin-1 (Cav-1) is the main protein in the caveolin family and plays a role in tumorigenesis signaling. The contribution of CAV1 genetic variants to gastric cancer is still largely unknown. In the present study, we aimed to investigate the role of CAV1 genotypes in gastric cancer risk. We recruited 358 gastric patients and 358 cancer-free controls for CAV1 genotyping analysis. Six single-nucleotide polymorphisms (SNPs) of CAV1, C521A (rs1997623), G14713A (rs3807987), G21985A (12672038), T28608A (rs3757733), T29107A (rs7804372), and G32124A (rs3807992), were genotyped by the polymerase chain reaction-restriction fragment length polymorphism method. There was a significant difference between the gastric cancer and control groups in the genotypic frequency distribution of the CAV1 G14713A genotypes (p=1.24*10−5), with those carrying the A allele having a higher risk for gastric cancer compared to those with the GG genotype (p=0.0001). Our findings suggested that CAV1 genotype may determine the individual susceptibility to gastric cancer, and that the CAV1 G14713A genotype may serve as a novel biomarker for early detection and prediction of gastric cancer.
Gastric cancer is reported to be more common in males and in people aged 50 years or older (1-3). In literature, obesity, smoking, salt intake and Helicobacter pylori infection are important risk factors for gastric cancer (4, 5). Geographically speaking, gastric cancer is prevalent in developing countries in East Asia, Eastern Europe and South America, while the incidence is low in North America and Africa (2). Clinically, the prognosis of gastric cancer is usual poor with a 5-year survival less than 20% for those with advanced disease (3). There have been some beneficial developments in decreasing the incidence of gastric cancer in the past two decades, such as the increasing use of refrigerators, the falling dependence on salts to preserve food, the increasing availability and intake of fresh fruits and vegetables, and the effective control of chronic infection with H. pylori. However, it remains a critical cancer threat, accounting for 8% of the total cancer incidence and 10% of the total cancer deaths worldwide (2).
The major caveolin proteins, caveolin-1, -2 and -3, serve as scaffolding proteins in charge of recruiting related signaling molecules to the caveolae and regulating their activity. Results gathered from caveolin-deficient animal models have indicated that caveolins play a role in diabetes, cancer, cardiovascular diseases, atherosclerosis, pulmonary fibrosis and a variety of degenerative muscular dystrophies (6). Among the caveolin proteins, caveolin-1 (CAV1) is a protein of 178 amino acids which was identified as a tumor suppressor in 1989 (7). However, in the subsequent years, scientists found that the expression levels of CAV1 vary among different types of cancer. For instance, CAV1 is down-regulated in sarcoma, lung carcinoma and ovarian carcinoma (8-10), but on the contrary, increased expression of CAV1 has been associated with the metastasis of prostate cancer and esophageal squamous cell carcinoma (11, 12). The reports strongly indicate that the role of CAV1 may be very complex and tissue-dependent. In literature, some epidemiological studies have investigated the association between the genotype of CAV1 and the risk for several types of cancer, including hepatoma (13), nasopharyngeal carcinoma (14), non-small cell lung carcinoma (15), prostate (16-18), breast (19, 20), oral (21), colorectal (22) and bladder cancer (23). However, there seems to be no literature investigating the association of CAV1 genotype and gastric cancer risk. Thus, the specific aim of this study was to determine the genotypic frequency of six polymorphisms of the CAV1 gene at C521A (rs1997623), G14713A (rs3807987), G21985A (12672038), T28608A (rs3757733), T29107A (rs7804372), and G32124A (rs3807992) in a Taiwanese gastric cancer population and their feasibility to serve as potential gastric cancer markers.
Distributions of selected characteristics among patients with gastric cancer and controls.
The primer sequences, polymerase chain reaction and restriction fragment length polymorphism conditions for caveolin-1 gene single nucleotide polymorphisms (SNPs).
Materials and Methods
Study population. Three hundred and fifty-eight patients diagnosed with gastric cancer were recruited at the Outpatient Clinics of General Surgery between 2001-2009 at the China Medical University Hospital, Taichung, Taiwan, Republic of China. All patients voluntarily participated, completed a self-administered questionnaire and provided their peripheral blood samples. The equal number of non-cancer healthy individuals were selected by matching for age and gender after initial random sampling from the Health Examination Cohort of the hospital as controls. The mean age of the patients and the controls was 63.8 (range=38-81, SD=11.4) and 62.1 (range=39 to 79, SD=9.5) years, respectively (see Table I). Our study was approved by the Institutional Review Board of the China Medical University Hospital and written-informed consent was obtained from all participants.
Polymerase chain reaction (PCR)-restriction fragment length polymorphism genotyping conditions. Genomic DNA of each participant was prepared from peripheral blood leucocytes using a QIAamp Blood Mini Kit (Blossom, Taipei, Taiwan, ROC) and further processed according to our previous articles (24-26). The PCR cycling conditions were: one cycle at 94°C for 5 min; 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 10 min. Pairs of PCR primer sequences and restriction enzyme for each DNA product are all listed in Table II.
Distribution of caveolin-1 genotypes among patients with gastric cancer and controls.
Distribution of alleles for caveolin-1 gene among patients with gastric cancer and controls.
Statistical analyses. To ensure that the controls used were representative of the general population and to exclude the possibility of genotyping error, the deviation of the genotype frequencies of CAV1 single-nucleotide polymorphisms (SNPs) in the controls from those expected under the Hardy–Weinberg equilibrium was assessed using the goodness-of-fit test. Pearson's Chi-square test or Fisher's exact test (when the expected number in any cell was less than five) was used to compare the distribution of the CAV1 genotypes between cases and controls. The associations between the CAV1 polymorphisms and gastric cancer risk were estimated by computing odds ratios (ORs) and their 95% confidence intervals (CIs) from unconditional logistic regression analysis with the adjustment for possible confounders. A p-value of less than 0.05 was considered statistically significant, and all statistical tests were two-sided.
Results
In Table I, the distributions of age, gender and cigarette smoking habits of the investigated subjects are summarized and analyzed. There was no difference in the distribution of these characteristics between patient and control groups (Table I).
The genotypic frequencies for CAV1 C521A, G14713A, G21985A, T28608A, T29107A and G32124A among the controls and gastric cancer patients are shown in Table III. Among the six SNPs investigated, the genotypic pattern of CAV1 G14713A was differentially distributed among the gastric cancer patients and non-cancer controls, and the p-value for trend was significant (p=1.24×10−5). In detail, the percentage of GG, AG and AA genotypes was 65.7%, 25.1% and 9.2% for the controls, and 48.0%, 38.0% and 14.0% for the patients (Table III). The AG and AA genotypes were more frequent in the patients than in the controls (Table III). As for CAV1 C521A, G21985A, T28608A, T29107A and G32124A, there was no significant difference between patient and control groups in the genotypic frequency of these SNPs (Table III). To sum up, the genotypic frequency distribution analysis indicated that individuals carrying AG or GG on CAV1 G14713A were at higher risk of gastric cancer.
The allelic distributions of CAV1 C521A, G14713A, G21985A, T28608A, T29107A and G32124A among the controls and gastric cancer patients are shown in Table IV. Among the six SNPs investigated, only the distribution of CAV1 G14713A differed significant (p=0.0001) with the percentage of the A allele being much higher in the patients (33.0%) than in the controls (21.8%) (Table IV). As for CAV1 C521A, G21985A, T28608A, T29107A and G32124A, there was no significant differences between patient and control groups in the allelic frequencies of these SNPs (Table III). Supporting the results shown in Table III, the allelic frequency distribution analysis indicated that individuals carrying an A allele for CAV1 G14713A were at higher risk of gastric cancer.
Discussion
In literature, the association of CAV1 genotypes with several types of cancer has been studied (13-23), but as far as we are aware of, never for gastric cancer. In the current study, we investigated the association of CAV1 genotypes and gastric cancer risk in Taiwan. In the study design, we selected and genotyped six SNPs (C521A, G14713A, G21985A, T28608A, T29107A, and G32124A) for 358 gastric cancer cases and 358 non-cancer controls. On the genotypic frequency distribution analysis, it was found that individuals with the AG and AA genotypes for CAV1 G14713A were at higher risk of gastric cancer compared to those with the GG genotype (Table III). As for the other SNPs, there was no differential genotype distribution among the cases and controls (Table III). Furthermore, the results of allelic frequency distribution analysis supported the idea that individuals carrying the A allele for CAV1 G14713A were at higher risk of gastric cancer compared with those carrying the G allele (Table IV). There was no gene interaction for CAV1 G14713A and smoking habit (data not shown), however, whether CAV1 G14713A genotype has interaction with other factors, such as H. pylori. infection or fruit and vegetable intake, needs further investigation.
The CAV1 G14713A maps to the intron region of the CAV1 gene and the polymorphic alteration does not directly result in a different amino acid coding. Further investigation of whether the CAV1 G14713A SNP could influence the downstream mRNA and protein levels are of great interest. In supporting this idea, Hsu and colleagues, in a study of hepatocellular carcinoma, found that the tissues from those with CAV1 G14713A AG and AA genotypes indeed had higher expression levels of CAV1 mRNA and protein than those with the GG genotype (13). The A allele of CAV1 G14713A might somehow code for a higher level of CAV1 mRNA, which leads to an increased expression of CAV1 protein and elevated cancer risk. To the best of our knowledge, this is the first study of the genotypic role of CAV1 in gastric cancer.
The functional investigation at the mRNA and protein levels for the role of CAV1 in gastric cancer is still lacking. In literature, most of the evidence came from studies in mouse models, in which the elevation of CAV1 seemed to enhance carcinogenesis, while inhibition of CAV1 suppressed the anti-apoptotic, migration and invasion capacities of cancer cells (27-29) Although the genotype–phenotype correlation for CAV1 G14713A is still not well-understood, here we report that the A allele of CAV1 G14713A may be a biomarker associated with higher risk of gastric cancer.
In conclusion, our study reports the first association of the CAV1 G14713A AG and AA variant genotypes with a higher gastric cancer risk for Taiwanese, and the presence of an A allele for CAV1 G14713A may serve a predictor for higher gastric cancer risk.
Acknowledgements
This study was supported by research grants from the Terry Fox Cancer Research Foundation and China Medical University and Hospital (DMR-103-075). The assistance of Tsai-Ping Ho in data collection, and genotyping work of Liang-Yi Lin, Hong-Xue Ji and Chieh-Lun Hsiao were highly appreciated.
Footnotes
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↵* These Authors contributed equally to this work.
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This article is freely accessible online.
- Received March 10, 2014.
- Revision received March 20, 2014.
- Accepted March 21, 2014.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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