GlySer Polymorphism of the Receptor for Advanced Glycation End Products Is Associated with an Increased Risk of Gastric Cancer in a Chinese Population

Purpose: It has been shown that the expression of the receptor for advanced glycation end products (RAGE) is closely associated with invasion andmetastasis in gastric cancer. AGlySer polymorphism in exon 3 of RAGE gene was identified and thought to have an effect on the functions of its protein. Therefore, the goal of the present study was to investigate whether the polymorphism is involved in the development or progression of gastric cancer. Experimental Design: In the hospital-based case-control study, the RAGE genotypes were determined using PCR-RFLP in 566 individuals (283 gastric cancer patients and 283 ageand sex-matched controls). Results: The distribution of genotype was significantly different between cases and controls (P = 0.038). Compared with the wild-type 82Gly/Gly carriers, subjects with the variant genotypes (82Gly/Ser and 82Ser/Ser) had a significantly higher risk of gastric cancer (adjusted odds ratio, 1.47; 95% confidence interval, 1.05-2.06). Moreover, the elevated gastric cancer risk was especially evident in younger individuals (ages V58 years), nonsmokers, and rural subjects. Further analyses revealed that the variant genotypes were associated with adjacent organ invasion in the subanalysis of gastric cancer patients. Conclusions: Our findings indicate that the RAGE GlySer polymorphism may confer not only an increased risk of gastric cancer but also with invasion of gastric cancer in the Chinese population. Gastric cancer occurs with a high incidence in East Asia (1). Moreover, nearly 42% of the worldwide gastric cancer cases occur in China alone (1). Gastric carcinogenesis is a complex, multistep, and multifactorial process (2). Like many malignancies, gastric cancer is the result of interactions between environmental factors and genetic factors of the host (3). Moreover, our previous epidemiologic studies also provided the evidence that the risk of gastric cancer was associated with genetic polymorphisms (4–6). The receptor for advanced glycation end products (RAGE), a cell surface molecule, is a multiligand member of the immunoglobulin superfamily (7). It has been shown to participate in several important pathologic responses, including Alzheimer’s disease, diabetes, inflammation, and cancer (8–11). RAGE has previously been suggested to stimulate growth, survival, and metastatic spread of cancers (11). In addition, RAGE expression is closely associated with invasion and metastasis in gastric cancer (12). Furthermore, it was reported that blockade of the RAGE suppressed the invasive activity of gastric cancer cells (12). The gene for RAGE is found on chromosome 6p21.3 in the MHC locus class II/III junction and is composed of a 1.7-kb 5¶ flanking region and 11 exons (13). To date, several genetic variants have been identified in RAGE gene, including T-429C, T-374A, G1704T, and A2184G (14). Several studies have suggested that polymorphisms within regulation elements and/or ligand-binding regions of RAGE gene may, alone or in combination, affect the expression or function of RAGE in a given milieu (14, 15). One of the most frequently studied and relatively high prevalence variants is the GlySer (or G82S) polymorphism (15, 16). It is at codon 82 (GGC!AGC) in exon 3 of RAGE and leads to a change from glycine to serine within the putative ligand-binding domain of the protein (16). It has been proposed as a functional polymorphism and associated with enhanced RAGE signaling (15). Recent observations also indicated that the GlySer polymorphism was associated with various diseases, including skin complications in type 2 diabetes, diabetic advanced nephropathy, coronary artery disease, and rheumatoid arthritis (15–20). However, to our Cancer Prevention and Susceptibility Authors’Affiliations: Key Laboratory of Reproductive Medicine, Department of Pharmacology, Nanjing Medical University; and Department of General Surgery, First AffiliatedHospitalofNanjingMedicalUniversity,Nanjing,JiangsuProvince,China Received11/3/07; revised12/30/07; accepted1/17/08. Grant support: National Natural Science Foundation of China (No. 30672486), Natural Science Foundation of Jiangsu Province (No. BK2006525), and ‘‘333 Project’’ and ‘‘Qinglan Project’’ Funding for theYoung Academic Leader of Jiangsu Province (B.Wang). The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Note:H. Gu, L.Yang, and Q. Sun contributed equally to this work. Requests for reprints: BinWang, Department of Pharmacology, NanjingMedical University, 140 Hanzhong Road, Nanjing 210029, Jiangsu Province, China. Phone: 86-25-86862884; Fax: 86-25-86862884; E-mail: [email protected]. F2008 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-07-4808 www.aacrjournals.org Clin Cancer Res 2008;14(11) June1, 2008 3627 Research. on May 1, 2017. © 2008 American Association for Cancer clincancerres.aacrjournals.org Downloaded from knowledge, there has been no study that examined the influence of the polymorphism with risk of cancers. Given the roles of RAGE in the pathogenesis and progression of gastric cancer as well as the effect of the polymorphism in RAGE gene on the receptor function, we propose that RAGE GlySer polymorphism may be associated with an increased risk of development or progression of gastric cancer. Therefore, to test the hypothesis, we assessed the association in a hospitalbased case-control study in the Chinese population. Materials andMethods Subjects. Briefly, this was a hospital-based case-control study, which comprised 283 gastric cancer cases and 283 cancer-free controls. Cases were inpatients newly diagnosed with histologically confirmed gastric cancer, consecutively recruited at the Affiliated Hospital of Nanjing Medical University. Those with secondary, recurrent malignancies were excluded. Controls were selected from the inpatients admitted to the hospital during the same period, having no history or diagnosis of any cancer and genetic disease. They were matched with the cases on age (within 5 y) and sex. All of the subjects were unrelated Han nationality and from Jiangsu Province or its surrounding regions. Information on age, gender, smoking status, residence, body weight, and personal medical history was collected by questionnaire. Individuals who formerly or currently smoked z10 cigarettes per day on average were defined as smokers. Clinicopathologic variables were obtained from the medical records of the gastric cancer patients. All gastric carcinomas were classified according to the tumor-node-metastasis classification criteria of International Union Against Cancer (21). Differentiation grade was classified according to WHO classification. The study was approved by the Nanjing Medical University Affiliated Hospital Ethics Committee and informed consent was obtained from each participant. RAGE genotyping. The protocol for genomic DNA extraction was described in our previous study (4). A PCR-RFLP assay was used to determine the RAGE GlySer polymorphism. PCR was done in 20 AL reaction mixtures containing 1.625 mmol/L MgCl2, 0.14 mmol/L deoxynucleotide triphosphates, 1 unit of Taq polymerase (MBI Fermentas), 2 AL of 10 PCR buffer (MBI Fermentas), 200 ng genomic DNA, and 0.25 Amol/L of each primer (forward, 5¶-GTAAGCGGGGCTCCTGTTGCA-3¶; reverse, 5 ¶-GGCCAAGGCTGGGGTTGAAGG-3¶; ref. 15). After an initial denaturation at 95jC for 5 min, the DNA was amplified by 35 cycles of 94jC for 30s, 62jC for 40 s, and 72jC for 45s, with a final elongation at 72jC for 10 min. The 397-bp PCR products were digested by the restriction enzyme AluI (New England BioLabs), 5 units for 16 h at 37jC, followed by electrophoresis on a 2% agarose gel (15). The digestion revealed fragments of length 249, 123, and 26 bp for the wild-type Gly allele and 181, 123, 67, and 26 bp for the variant Ser allele. About 10% of the samples were randomly selected to do the repeated assays, and the results were 100% concordant. Two researchers, blinded to the clinical data, scored the genotypes independently. Statistical analysis. All statistical analyses were done by using the Stata version 8.0 (STATA Corp.). All of the tests were two sided, and statistical significance was defined as P < 0.05. Quantitative variables departing from the normal distribution were summarized as median and analyzed by Mann-Whitney rank sum test. Pearson’s m test was used to test the difference in the distribution of categorical variables. The Hardy-Weinberg equilibrium of the RAGE genotypes was evaluated by a goodness-of-fit m test. The association between the polymorphism and risk of gastric cancer was estimated by odds ratio (OR) and 95% confidence interval (95% CI). Carriers of the wild genotype 82Gly/Gly comprised reference group. The crude OR was computed using the Woolf approximation method and the adjusted OR was assessed by unconditional logistic regression method, with adjustment for age, sex, smoking status, residence, hypertension, and diabetes. Results Demographic characteristics of the study participants are summarized in Table 1. Cases and controls were well matched in terms of sex and age (within 5 years). Moreover, the two groups were similar with respect to smoking status, residence, history of hypertension, and diabetes. Patients with cancer of the gastric cardia and noncardia were 83 and 200, respectively. Most of the cases were adenocarcinoma (97.88%). Among those 275 gastric cancers with available clinicopathologic data, 48, 31, 133, and 63 were T1, T2, T3, and T4, respectively; 38, 129, and 108 were reported well, moderate, and poor differentiation, respectively. Positive lymph nodes were identified in 177 cases. Data for genotype frequency and the association between RAGE GlySer polymorphism and the risk of gastric cancer are shown in Table 2. The genotype distribution was in agreement with that predicted under Hardy-Weinberg equilibrium for both cases (P = 0.053) and controls (P = 0.081). The frequencies of the genotypes between the cases and controls were significantly different (Pearson m = 6.552; P = 0.038). Furthermore, the Ser allele frequency was significantly higher (Pearson m = 5.855; P = 0.016) in the case group (27.56%) than in the control group (21.38%). With the 82Gly/Gly genotype as reference, the OR for the variant genotypes (82Gly/ Ser and 82Ser/Ser) was 1.47 (95% CI, 1.05-2.06; P = 0.024) after adjustment for age, sex, smoking status, residence, hypertension, and diabetes. Moreover, the 82Gly/Ser heterozygotes had a 41% increased risk of gastric cancer (adjusted OR, 1.41; 95% CI, 1.00-1.99), and the 82Ser/Ser homozygotes had a 150% increased risk (adjusted OR, 2.50; 95% CI, 1.01-6.17). Table 3 presents the results of stratified analyses by the median age of controls (58 years), sex, smoking status, and residence with the RAGE variant genotypes. A significantly elevated risk associated with the variant genotypes was found for younger subjects (ages V58 years; adjusted OR, 2.49; 95% CI, 1.53-4.05) but not in older subjects (ages >58 years). Stratification by smoking status revealed a significant association of the polymorphism with an increased gastric cancer risk for nonsmokers (adjusted OR, 1.70; 95% CI, 1.16-2.49) but not in smokers. In rural subjects, possession of the variant genotypes was associated with a 75% increased risk of gastric cancer (adjusted OR, 1.75; 95% CI, 1.06-2.88), whereas the association was not statistically significant in urban subjects. Table 1. Demographic information Characteristics Cases