EFFECTS OF E-CADHERIN (CDH1) GENE PROMOTER POLYMORPHISMS ON THE RISK AND CLINICOPATHOLOGIC DEVELOPMENT OF ORAL CANCER

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1 ORIGINAL ARTICLE EFFECTS OF E-CADHERIN (CDH1) GENE PROMOTER POLYMORPHISMS ON THE RISK AND CLINICOPATHOLOGIC DEVELOPMENT OF ORAL CANCER Ming-Hsien Chien, PhD, 1,2 Lin Shih-Shen Chou, PhD, 3,4 Tsung-Te Chung, MD, 5,6 Chien-Huang Lin, PhD, 7 Ming-Yung Chou, PhD, 3,4 Meng-Shih Weng, PhD, 8 Shun-Fa Yang, PhD, 6,9 Mu-Kuan Chen, MD, PhD 6,10,11 1 Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan 2 Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan 3 School of Dentistry, Chung Shan Medical University, Taichung, Taiwan 4 Department of Dentistry, Chung Shan Medical University Hospital, Taichung, Taiwan 5 Department of Otolaryngology, Show Chwan Memorial Hospital, Changhua, Taiwan 6 Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan 7 Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan 8 Department of Nutritional Science, Fu-Jen Catholic University, Taipei, Taiwan 9 Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan 10 Department of Otorhinolaryngology Head and Neck Surgery, Changhua Christian Hospital, Changhua, Taiwan @cch.org.tw 11 School of Medicine, Chung Shan Medical University, Taichung, Taiwan Accepted 4 January 2011 Published online 5 April 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: /hed Abstract: Background. This study investigates the association between polymorphism in the E-cadherin/CDH1 promoter region and the risk and progression of oral cancer. Methods. Genetic polymorphisms of CDH1-160 and -347 were analyzed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) in 347 noncancer controls and in 251 patients with oral cancer. Results. The statistical analysis showed that subjects with at least 1 varied GA allele of CDH1-347 polymorphic genotypes or combinations of the CDH1-160 CA/-347 GGA, CDH1-160 CC/-347 GGA, or CDH1-160 CC/-347 GAGA genotypes had a significantly higher risk, whereas subjects with CDH1-160 C/A or A/A had a significantly lower risk of developing oral cancer than those with wild-type genotypes. Furthermore, elderly patients with the CDH1-347 G/GA or GA/GA genotype were associated with a higher incidence in lymph node metastasis than were those with the G/G genotype. Conclusions. These results suggest that CDH1-347 polymorphisms are associated with increased risks of oral cancer, and may be a predictive factor for tumor lymph node metastasis. VC 2011 Wiley Periodicals, Inc. Head Neck 34: , 2012 Keywords: E-cadherin; genetic polymorphism; oral cancer; tobacco consumption; betel nut chewing Oral cancer is the most common malignant disease with a poor prognosis and is the sixth leading cause of cancer death in Taiwan. 1,2 Epidemiologic studies Correspondence to: M.-K. Chen VC 2011 Wiley Periodicals, Inc. suggest that the susceptibility of an individual to oral cancer is mediated by both environmental carcinogens, such as alcohol, tobacco consumption, and betel nut chewing, 3 6 and genetic factors. 7,8 A gene and further protein expressions were affected by a single nucleotide polymorphism (SNP) located within the promoter or other regulatory regions of a gene. Several SNPs, such as hypoxia-inducible factor-1 alpha, stromal cell-derived factor-1 and its receptor, and glutathione S-transferase genes were reported to be predictive factors related to higher risks of oral cancer To elucidate the complex process of carcinogenesis and to improve the scientific basis for preventive interventions, major genes related to the susceptibility to oral cancer should be identified. E-cadherin, a 97-kDa transmembrane glycoprotein encoded by the E-cadherin gene (CDH1) located in chromosome 16q22.1, is the main adhesion protein of the epithelia and is responsible for calcium-dependent cell attachment and the development of normal epithelial polarity and tissue architecture. 12 Previous studies reported that the loss of the adhesive function of E-cadherin is a critical step in the progression of epithelial cells to a more aggressive phenotype. E-cadherin is widely recognized as an invasion-suppressor gene, because inactivation or downregulation of E- cadherin by mutations, allelic deletions, or epigenetic changes (eg, hypermethylation of the 5 0 -promoter region) is associated with tumor aggressiveness and the potential for metastasis. 13,14 The function of E- CDH1 Gene Polymorphism in Oral Cancer HEAD & NECK DOI /hed March

2 cadherin is frequently lost during the development of human epithelial cancers, including carcinomas of the breast, colon, prostate, stomach, prostate, and bladder E-cadherin downregulation is also associated with tumor differentiation, and invasive and lymph node metastasis in cancers of the head, neck, and oral cavity Polymorphisms within gene promoter regions can have profound effects on the transcriptional efficiency of the genes. Two such polymorphisms that affect transcription were identified in the region of the E- cadherin promoter. 24,25 The first is a C/A SNP, 160 basepairs (bp) upstream of the transcriptional start site of CDH1. Transcription from the A allele was reported to be 68% less efficient than that from the C allele. 24 The second reported promoter variant is a G/ GA SNP, 347 bp upstream of the transcriptional start site. The -347G/GA polymorphism within the promoter region may also change the transcriptional efficiency of CDH1. The GA allele has weak transcriptional factor-binding strength and transcriptional activity compared with that of the G allele, 26 suggesting that the GA allele may be associated with tumor formation or differentiation. A correlation of the E-cadherin-160 C/A and -347G/GA SNPs with cancer susceptibility was described in a range of neoplastic diseases To our knowledge, no reports concerning the roles of the E-cadherin-160 C/A or -347G/GA polymorphisms in oral cancer have yet been reported. On these grounds, we conducted a case-control study to evaluate the contribution of these polymorphisms to the risk of developing oral cancer in Taiwanese and to determine its prognostic relevance by correlating it with the clinicopathologic characteristics of tumors. MATERIALS AND METHODS Subjects and Specimen Collection. In total, 251 patients (242 male and 9 female patients, with a mean age of years) were recruited as a case group between 2007 and 2009 at Chung Shan Medical University Hospital in Taichung and Changhua Christian Hospital and Show Chwan Memorial Hospital in Changhua, Taiwan. Meanwhile, 347 raceand ethnic group matched individuals (262 males and 85 females, with a mean age of years) were randomly selected from the same geographic area to be a control group. The patient and control groups were respectively diagnosed as having and not having oral cancer in accord with the characteristic criteria of national guidelines for oral cancer. We interviewed all subjects using a structured questionnaire to obtain information on sociodemographic characteristics (age, sex, race/ethnicity), tobacco use (ever smoker vs nonsmoker), alcohol consumption (current heavy drinker, defined by Centers for Disease Control and Prevention as consuming an average of >2 drinks per day vs not current heavy drinker), and betel nut use (ever user vs nonuser). Relevant medical information, including stage, size, histologic grading of tumor at diagnosis, and evidence of lymph nodal involvement, was also collected from cases. Whole-blood specimens were placed in tubes containing ethylenediaminetetraacetic acid (EDTA), then immediately centrifuged, and stored at 80 C for further analyses. Prior to conducting this study, approval from the Institutional Review Board was obtained, and informed written consent was obtained from each individual. Genomic DNA Extraction. Genomic DNA was extracted from whole-blood samples collected from study subjects by QIAamp DNA blood minikits (Qiagen, Valencia, CA) in accord with the manufacturer s instructions. DNA was dissolved in TE buffer (10 mm Tris, ph 7.8; and 1 mm EDTA) and then quantitated by measuring the optical density 260 (OD 260 ). The final preparation was stored at 20 C and was used as a template for a polymerase chain reaction (PCR). PCR Restriction Fragment Length Polymorphism (RFLP). The forward and reverse primers used for analysis of E-cadherin-160C/A (SNP ID: rs16260) and the -347G/GA (SNP ID: rs ) gene polymorphisms were both designed as 5 0 -GCC CCG ACT TGT CTC TCT AC-3 0 and 5 0 -GGC CAC AGC CAA TCA GCA-3 0. The PCR was performed in a 10-lL volume containing a 100 ng DNA template, 1.0 ll of10 PCR buffer (Invitrogen, Carlsbad, CA), 0.25 U of Taq DNA polymerase (Invitrogen), 0.2 mm dntps (Promega, Madison, WI), and 200 nm of each primer (MDBio, Taipei, Taiwan). The PCR cycling conditions were 5 minutes at 94 C followed by 35 cycles of 1 minute at 94 C, 1 minute at 61 C, and 2 minutes at 72 C, with a final step at 72 C for 20 minutes. The PCR product was subjected to double-digestion for 4 hours with BanII and HincII at 37 C, separated on a 3% agarose gel, and then stained with ethidium bromide. Furthermore, the genotypes determined by PCR-RFLP were confirmed by a DNA sequencing analysis. Statistical Analysis. Hardy Weinberg equilibrium was assessed using a goodness-of-fit chi-square test for biallelic markers. The Mann Whitney U test and Fisher s exact test were used to compare differences in demographic characteristic distributions between healthy control and patients with oral cancer. The adjusted odds ratios (ORs) with their 95% confidence intervals (CIs) of the association of genotype frequencies with oral cancer risk and clinicopathologic characteristics were estimated by multiple logistic regression models after controlling for other covariates. A p value of <0.05 was considered significant. Data were analyzed using SAS statistical software (vers. 9.1, 2005; SAS Institute, Cary, NC). 406 CDH1 Gene Polymorphism in Oral Cancer HEAD & NECK DOI /hed March 2012

3 Table 1. Demographic characteristics in 251 patients with oral cancer. Characteristic No. (%) Sex Male 242 (96.41) Female 9 (3.59) Age, y <50 96 (38.25) (33.47) >60 71 (28.28) Smoking status No 47 (18.73) Yes 204 (81.27) Alcohol intake No 89 (35.46) Yes 162 (64.54) Betel nut chewing No 56 (22.31) Yes 195 (77.69) TNM clinical staging Stage I þ II 119 (47.41) Stage III þ IV 132 (52.59) Primary tumor size (T) T1 þ T2 163 (64.94) T3 þ T4 163 (64.94) Lymph node involvement (N) No 174 (69.32) Yes 77 (30.68) Distant metastasis No 248 (98.8) Yes 3 (1.2) Histologic grade Poor 51 (20.32 Moderately þ Well 200 (79.68) RESULTS The statistical analysis of demographic characteristics showed that 51 persons among the 347 controls (14.7%) and 195 persons among the 251 patients with oral cancer (77.7%) had the betel nut chewing habit, 123 persons among the 347 controls (35.5%) and 204 persons among the 251 patients with oral cancer (81.3%) had a smoking habit. The distributions of betel nut chewing (p <.001) and tobacco consumption (p <.001) significantly differed between healthy controls and patients with oral cancer. To diminish the possible interference of environmental carcinogens, the adjusted ORs with 95% CIs were estimated by multiple logistic regression models after controlling for age, sex, betel nut chewing, and tobacco consumption for each comparison. Of all patients, 52.59% of patients were clinical stage III or stage IV, 35.06% were at pathologic T3 T4 classification, and 30.68% had lymph node metastasis (Table 1). Genotype and allele frequencies of the cases and controls are presented in Table 2. Distribution of the E-cadherin-160 C/A genotype in healthy controls did not deviate from that expected by Hardy Weinberg equilibrium (p ¼.843), whereas distribution of the -347 G/GA genotype in healthy controls was not in Hardy Weinberg equilibrium. A significantly increased risk of oral cancer was found for GA allele carriers compared with the homozygous G allele carriers after adjusting for other confounding variables. The risk for the heterozygous and homozygous GA allele carriers was respectively increased by approximately 3.6- and 3.8-fold (G/GA: CI ; GA/GA: CI ). However, for the 160 C/A gene polymorphism of the CDH1 gene, subjects with the A allele had a significantly lower risk of having oral cancer compared with individuals with the C/C wild-type (WT) homozygote (C/A: adjusted odds ratio [AOR] 0.62, CI ; A/A: AOR 0.33, CI ). The haplotype analyses did not lead to much stronger associations with oral cancer, but resembled the genotype analyses (Table 2). A homozygous haplotype, GA-C/GA-C, and two heterozygous haplotypes, G-A/GA-C and G-C/GA-C, respectively, resulted in statistically significant increased risks of (CI ), (CI ), and 3.78-fold (CI ). Interactive effects between environmental risk factors and genetic polymorphisms are shown in Tables 3 and 4. Among the 326 smokers, subjects Table 2. Relationship of E-cadherin gene genotypes in controls and in patients with oral cancer. Gene Control, N ¼ 347 (%) Case, N ¼ 251 (%) OR (95% CI) AOR (95% CI) E-cadherin-347 G/G 271 (77.10) 132 (52.59) Reference Reference G/GA 65 (18.73) 106 (42.23) 3.29 ( )* 3.64 ( )* GA/GA 11 (3.17) 13 (5.18) 2.42 ( )* 3.77 ( )* E-cadherin-160 C/C 122 (35.16) 122 (48.61) Reference Reference C/A 166 (47.84) 110 (43.82) 0.65 ( )* 0.62 ( )* A/A 59 (17.00) 19 (7.57) 0.33 ( )* 0.33 ( )* Haplotype G-C/G-A 142 (40.92) 65 (25.90) Reference Reference G-A/G-A 59 (17.00) 19 (7.57) 0.70 ( ) 0.74 ( ) G-C/G-C 70 (20.17) 48 (19.12) 1.52 ( ) 1.28 ( ) G-A/GA-C 24 (6.92) 45 (17.93) 3.93 ( )* 3.46 ( )* G-C/GA-C 41 (11.82) 61 (24.30) 3.25 ( )* 3.78 ( )* GA-C/GA-C 11 (3.17) 13 (5.18) 2.58 ( )* 3.78 ( )* Abbreviations: OR, odds ratio; AOR, adjusted odds ratio; CI, confidence interval. *Statistically significant. AOR adjusted sex, age, smoking status, and betel nut chewing. CDH1 Gene Polymorphism in Oral Cancer HEAD & NECK DOI /hed March

4 Table 3. Adjusted odds ratio and 95% CI of oral cancer associated with E-cadherin genotypic frequencies and betel nut chewing among 326 smokers. Variable Controls (n ¼ 122) (%) Patients (n ¼ 204) (%) OR (95% CI) AOR (95% CI) E-cadherin-347 G/G genotype and nonbetel nut chewing* 72 (59.0) 16 (7.8) G/GA or GA/GA genotype or betel nut chewing 44 (36.1) 105 (51.5) ( ) ( ) G/GA or GA/GA genotype with betel nut chewing 6 (4.9) 83 (40.7) ( ) ( ) (n ¼ 107) (%) (n ¼ 194) (%) G/G genotype and nonbetel nut chewing* 72 (67.3) 16 (8.2) Any genotype with betel nut chewing 35 (32.7) 178 (91.8) ( ) ( ) E-cadherin-160 (n ¼ 122) (%) (n ¼ 204) (%) CC genotype and nonbetel nut chewing* 31 (25.4) 15 (7.4) CA or AA genotype or betel nut chewing 68 (55.7) 98 (48.0) 2.98 ( ) 3.75 ( ) CA or AA genotype with betel nut chewing 23 (18.9) 91 (44.6) 8.18 ( ) 9.90 ( ) (n ¼ 66) (%) (n ¼ 193) (%) CC genotype and nonbetel nut chewing* 31 (47.0) 15 (7.8) Any genotype with betel nut chewing 35 (53.0) 178 (92.2) ( ) ( ) Abbreviations: OR, odds ratio; AOR, adjusted odds ratio; CI, confidence interval. Note: The ORs with their 95% CIs were estimated by logistic regression models; the AORs with their 95% CIs were estimated by multiple logistic regression models after controlling for age and sex. *Individual with wild genotype, but without betel nut chewing. Individual with either at least 1 mutated genotype or betel nut chewing. Individual with both at least 1 mutated genotype and betel nut chewing. with at least 1 GA allele of CDH1-347 and the betel nut chewing habit had a fold (CI ) higher risk of having oral cancer; individuals with at least 1 GA allele of CDH1-347 or the betel nut chewing habit had a fold (CI ) higher risk of having oral cancer compared with individuals with CDH1-347 G/G but without the betel nut chewing habit (Table 3). Consistently, subjects with at least 1 A allele of CDH1-160 and the betel nut chewing habit had a 9.90-fold (CI ) greater risk of having oral cancer; moreover, individuals with at least 1 A allele of CDH1-160 or the betel nut chewing habit had a 3.75-fold (CI ) greater risk of developing oral cancer compared with individuals with CDH1-160 C/C but without the betel nut chewing habit (Table 3). In addition, among the 246 betel nut consumers, subjects with at least 1 GA allele of CDH1-347 and the smoking habit had a fold (CI ) greater risk of having oral cancer. Moreover, individuals with at least 1 GA allele of CDH1-347 or the smoking habit had a fold (CI ) greater risk of developing oral cancer compared with individuals with the G/G allele of CDH1-347 but without the smoking habit (Table 4). Similarly, subjects with at least 1 A allele of CDH1-160 and the smoking habit had a fold (CI ) greater risk of having oral cancer, and individuals with at least 1 A allele of CDH1-160 or the smoking habit had a 9.50-fold (CI ) greater risk of developing oral cancer compared with individuals who had the C/C allele of CDH1-160 but did not smoke (Table 4). Table 4. Adjusted odds ratio and 95% CI of oral cancer associated with E-cadherin genotypic frequencies and smokers among 246 betel nut consumers. Variable Controls (n ¼ 51) (%) Patients (n ¼ 195) (%) OR (95% CI) AOR (95% CI) E-cadherin-347 G/G genotype and nonsmoker* 13 (25.5) 6 (3.1) G/GA or GA/GA genotype or smoker 32 (62.7) 106 (54.3) 7.18 ( ) ( ) G/GA or GA/GA genotype with smoking 6 (11.8) 83 (42.6) ( ) ( ) (n ¼ 48) (%) (n ¼ 184) (%) G/G genotype and nonsmoker* 13 (27.1) 6 (3.3) Any genotype with smoking 35 (72.9) 178 (96.7) ( ) ( ) E-cadherin-160 (n ¼ 51) (%) (n ¼ 195) (%) CC genotype and nonsmoker* 5 (9.8) 4 (2.0) CA or AA genotype or smoker 23 (45.1) 100 (51.3) 5.44 ( ) 9.50 ( ) CA or AA genotype with smoking 23 (45.1) 91 (46.7) 4.95 ( ) ( ) (n ¼ 40) (%) (n ¼ 180) (%) CC genotype and nonsmoker* 5 (12.5) 4 (2.2) Any genotype with smoking 35 (87.5) 178 (97.8) 6.36 ( ) ( ) Abbreviations: OR, odds ratio; AOR, adjusted odds ratio; CI, confidence interval. Note: The ORs with their 95% CIs were estimated by logistic regression models; the AORs with their 95% CIs were estimated by multiple logistic regression models after controlling for age and sex. *Individual with wild genotype, but without smoking. Individual with either at least 1 mutated genotype or smoking. Individual with both at least 1 mutated genotype and smoking. 408 CDH1 Gene Polymorphism in Oral Cancer HEAD & NECK DOI /hed March 2012

5 Table 5. Distribution frequency of clinical statuses and E-cadherin-347 genotype frequencies in elderly patients (>60 y). Genotypic frequency Variable G/G (n ¼ 32) (%) G/GA or GA/GA (n ¼ 39) (%) OR (95% CI) TNM clinical staging Stage I/II 20 (62.5) 18 (46.2) 1.00 Stage III/IV 12 (37.5) 21 (53.8) 1.94 ( ) Primary tumor size (T) T1 þ T2 22 (68.8) 24 (61.5) 1.00 T3 þ T4 10 (31.3) 15 (38.5) 1.38 ( ) Lymph node involvement (N) No 28 (87.5) 26 (66.7) 1.00 Yes 4 (12.5) 13 (33.3) 3.50 ( ) Histologic grade Well-differentiated 7 (21.9) 7 (17.9) 1.00 Moderately or poorly differentiated 25 (78.1) 32 (82.1) 1.28 ( ) Abbreviations: OR, odds ratio; CI, confidence interval. Note: The ORs with their 95% CIs were estimated by logistic regression models. Relationships of the CDH1 gene polymorphisms of 347 G/GA and 160 C/A with clinicopathologic characteristics were also analyzed to show that there was no significant association between CDH1-160 and any clinicopathologic characteristics (data not shown). However, in elderly patients (>60 years) with oral cancer, we found that individuals with oral cancer and at least 1 GA allele of CDH1-347 had a 3.5-fold higher risk (95% CI ) of progressing to lymph node metastasis, compared with individuals who had the C/C allele of CDH1-347 (Table 5). DISCUSSION It was reported that SNPs in the E-cadherin (CDH1) gene promoter region are responsible for interindividual variations in the production of E-cadherin and, in turn, lead to individual susceptibility to cancer. 28 In this study, we found a significant association between the CDH1-347 G/GA polymorphism and oral cancer. The risk for heterozygous and homozygous GA allele carriers, respectively, increased and 3.77-fold. This finding is similar to that of a study by Shin et al, 33 who reported that the GA allele was associated with a significantly increased risk of colorectal cancer in Korea. We also found that individuals with the CDH1-160 A/A genotype had a decreased risk of oral cancer. The latter finding is surprising because it was previously reported that the C allele of the CDH1 promoter region endows this gene with stronger transcription activity than does the A allele. 24 A decreased transcription efficiency of the A allele can be expected to result in less translation of the E-cadherin protein. It is hard to envision how the A/A genotype operates in reducing the risk of oral cancer because recent studies demonstrated that CDH1 is a tumor-suppressor gene. However, caution should be noted because some researchers showed that there is not necessarily any direct correlation between messenger (m)rna expression and protein levels. 34 Moreover, results similar to ours were reported in Taiwanese patients with gastric cancer and the CDH1-160 A/A genotype having a decreased risk of gastric cancer 35 ; in that report, however, the authors indicated that no significant relation between CDH1 protein expression and genotypes from immunohistochemical staining data in different genotypes was seen. However, in our study, the correlation between E-cadherin protein expression and genotypes must be further investigated. Moreover, from our analysis of the haplotype data (Table 2), we found haplotypes GA-C/GA-C, G-A/GA-C, and G-C/GA-C but not G-C/G- A, G-C/G-C, or G-A/G-A resulted in a statistically significantly increased risk of oral cancer. These results indicated that the promotion effect of the 347 GA allele on oral cancer generation might overcome the suppressive effect of the 160 A allele. Alcohol, 3,8 tobacco consumption, 3,6,8 and betel nut chewing 8,36,37 are the main etiologic factors of oral cancer. In this study, we found that tobacco consumption and betel nut chewing significantly increased the risk of developing oral cancer among our 598 recruited subjects. The gene environment interaction was studied in 326 smokers and 246 betel nut consumers to determine if any synergistic effects between the gene polymorphism of CDH1-347 and tobacco or betel nut consumption significantly increased the risk of getting oral cancer. We found that these 2 etiologic factors could also overcome the protective effect of the CDH1-160 A allele against oral cancer generation (Tables 3 and 4). Previous studies showed that betel nut increased protein levels of c-fos and c-jun proto-oncogenes, induced cell transformation, and contributed to colony formation on soft agar, higher cell proliferation, and the production of a multilayer on culture dishes. 38 In addition, it was demonstrated that the cell cell contact of gingival epithelial cells was markedly impaired by a betel nut extract. This was accompanied by disruption of the distribution of E-cadherin. 39 Moreover, the major addictive component of tobacco smoke, nicotine, was CDH1 Gene Polymorphism in Oral Cancer HEAD & NECK DOI /hed March

6 proven to induce invasion and the epithelial mesenchymal transition by downregulating E-cadherin in lung, breast, pancreatic, and colon cancers. 40,41 Taken together, we believe that in individuals with variant genotypes of CDH1, there are interactive effects with tobacco and betel nut consumption to increase the risk of oral cancer, and we verified that the gene polymorphism of CDH1-347 was very important in these interactive effects. Clinical studies of E-cadherin in oral cancer patients demonstrated increased numbers of regional and distant metastases in patients with tumors expressing reduced levels of E-cadherin. 42 Recently, Pyo et al 43 also indicated that the reduced expression of E-cadherin was closely associated with lymph node metastasis in oral squamous cell carcinoma, and this suggests that the loss of E-cadherin probably supports the malignant behavior of oral cancer cells. In this study, we found that the 347 but not the 160 gene polymorphism of CDH1 was significantly associated with the onset of lymph node metastasis in elderly patients (>60 years) with oral cancer (Table 5). Other clinicopathologic aspects, including the clinical stage, tumor size, and cell differentiation of oral cancer, were not significantly associated with either the 347 or 160 gene polymorphisms of CDH1 (Table 5). We suggest that the 347 gene polymorphism of CDH1 might affect lymph node metastasis in oral cancer, especially in elderly populations. Information on alcohol, betel nut, and tobacco use is dichromized into ever user versus non-user. As the result, we were not able to perform more detailed analysis by stratifying individuals based on amount, length, and past history of betel nut, alcohol, and tobacco consumption. Data collection relied on selfreports, for which some individuals may have been reluctant to report their habitual use of such substances. Thus, there may be a residual confounding effect as a result of betel nut, alcohol, and tobacco use misclassification. In conclusion, this study indicated that 347 G/ GA gene polymorphism of the CDH1 gene is an important factor in the susceptibility and lymph node metastasis of oral cancer. The combined effects of gene-to-gene and gene-to-environmental risks significantly increase the risk of developing oral cancer. REFERENCES 1. Yang SF, Yang WE, Chang HR, Chu SC, Hsieh YS. Luteolin induces apoptosis in oral squamous cancer cells. J Dent Res 2008;87: Lo WY, Tsai MH, Tsai Y, et al. Identification of over-expressed proteins in oral squamous cell carcinoma (OSCC) patients by clinical proteomic analysis. Clin Chim Acta 2007;376: Biolchini F, Pollastri G, Figurelli S, Chiarini L. Carcinogen metabolism, DNA damage repair and oral head and neck squamocellular carcinoma (HNSCC). A review. Minerva Stomatol 2005;54: Chen YJ, Chang JT, Liao CT, et al. Head and neck cancer in the betel quid chewing area: recent advances in molecular carcinogenesis. Cancer Sci 2008;99: Ko YC, Huang YL, Lee CH, Chen MJ, Lin LM, Tsai CC. Betel quid chewing, cigarette smoking and alcohol consumption related to oral cancer in Taiwan. J Oral Pathol Med 1995;24: Nagaraj NS, Beckers S, Mensah JK, Waigel S, Vigneswaran N, Zacharias W. Cigarette smoke condensate induces cytochromes P450 and aldo-keto reductases in oral cancer cells. Toxicol Lett 2006;165: Vairaktaris E, Yapijakis C, Serefoglou Z, et al. The interleukin- 10 (-1082A/G) polymorphism is strongly associated with increased risk for oral squamous cell carcinoma. Anticancer Res 2008;28: Yen CY, Liu SY, Chen CH, et al. Combinational polymorphisms of four DNA repair genes XRCC1, XRCC2, XRCC3, and XRCC4 and their association with oral cancer in Taiwan. J Oral Pathol Med 2008;37: Chen MK, Chiou HL, Su SC, et al. The association between hypoxia inducible factor-1alpha gene polymorphisms and increased susceptibility to oral cancer. Oral Oncol 2009;45:e222 e Chen MK, Tsai HT, Chung TT, et al. Glutathione S-transferase P1 and alpha gene variants; role in susceptibility and tumor size development of oral cancer. Head Neck 2010;32: Teng YH, Liu TH, Tseng HC, et al. Contribution of genetic polymorphisms of stromal cell-derived factor-1 and its receptor, CXCR4, to the susceptibility and clinicopathologic development of oral cancer. Head Neck 2009;31: Takeichi M. Cadherin cell adhesion receptors as a morphogenetic regulator. Science 1991;251: Christofori G, Semb H. The role of the cell-adhesion molecule E- cadherin as a tumour-suppressor gene. Trends Biochem Sci 1999;24: Okegawa T, Li Y, Pong RC, Hsieh JT. Cell adhesion proteins as tumor suppressors. J Urol 2002;167: Gamallo C, Palacios J, Suarez A, et al. Correlation of E-cadherin expression with differentiation grade and histological type in breast carcinoma. Am J Pathol 1993;142: Kinsella AR, Lepts GC, Hill CL, Jones M. Reduced E-cadherin expression correlates with increased invasiveness in colorectal carcinoma cell lines. Clin Exp Metastasis 1994;12: Mayer B, Johnson JP, Leitl F, et al. E-cadherin expression in primary and metastatic gastric cancer: down-regulation correlates with cellular dedifferentiation and glandular disintegration. Cancer Res 1993;53: Pignatelli M, Ansari TW, Gunter P, et al. Loss of membranous E-cadherin expression in pancreatic cancer: correlation with lymph node metastasis, high grade, and advanced stage. J Pathol 1994;174: Umbas R, Schalken JA, Aalders TW, et al. Expression of the cellular adhesion molecule E-cadherin is reduced or absent in highgrade prostate cancer. Cancer Res 1992;52: Kudo Y, Kitajima S, Ogawa I, et al. Invasion and metastasis of oral cancer cells require methylation of E-cadherin and/or degradation of membranous beta-catenin. Clin Cancer Res 2004;10: Mattijssen V, Peters HM, Schalkwijk L, et al. E-cadherin expression in head and neck squamous-cell carcinoma is associated with clinical outcome. Int J Cancer 1993;55: Shinohara M, Hiraki A, Ikebe T, et al. Immunohistochemical study of desmosomes in oral squamous cell carcinoma: correlation with cytokeratin and E-cadherin staining, and with tumour behaviour. J Pathol 1998;184: Takes RP, Baatenburg De Jong RJ, Alles MJ, et al. Markers for nodal metastasis in head and neck squamous cell cancer. Arch Otolaryngol Head Neck Surg 2002;128: Li LC, Chui RM, Sasaki M, et al. A single nucleotide polymorphism in the E-cadherin gene promoter alters transcriptional activities. Cancer Res 2000;60: Nakamura A, Shimazaki T, Kaneko K, et al. Characterization of DNA polymorphisms in the E-cadherin gene (CDH1) promoter region. Mutat Res 2002;502: Shin Y, Kim IJ, Kang HC, et al. The E-cadherin 347G!GA promoter polymorphism and its effect on transcriptional regulation. Carcinogenesis 2004;25: Fei Y, Hu J, Liu S, Liu X, Wang F, Gong J. E-cadherin-160 C/A promoter polymorphism and risk of pancreatic carcinoma in Chinese population. Cancer Genet Cytogenet 2010;197: Kiemeney LA, van Houwelingen KP, Bogaerts M, et al. Polymorphisms in the E-cadherin (CDH1) gene promoter and the risk of bladder cancer. Eur J Cancer 2006;42: CDH1 Gene Polymorphism in Oral Cancer HEAD & NECK DOI /hed March 2012

7 29. Pittman AM, Twiss P, Broderick P, et al. The CDH1-160C!A polymorphism is a risk factor for colorectal cancer. Int J Cancer 2009;125: Zou XP, Dai WJ, Cao J. CDH1 promoter polymorphism (-347G!GA) is a possible prognostic factor in sporadic colorectal cancer. World J Gastroenterol 2009;15: Zhang XF, Wang YM, Wang R, et al. Correlation of E-cadherin polymorphisms to esophageal squamous cell carcinoma and gastric cardiac adenocarcinoma [in Chinese]. Ai Zheng 2005;24: Zhang B, Pan K, Liu Z, et al. Genetic polymorphisms of the E- cadherin promoter and risk of sporadic gastric carcinoma in Chinese populations. Cancer Epidemiol Biomark Prev 2008;17: Shin Y, Kim IJ, Kang HC, et al. A functional polymorphism (-347 G!GA) in the E-cadherin gene is associated with colorectal cancer. Carcinogenesis 2004;25: Anderson L, Seilhamer J. A comparison of selected mrna and protein abundances in human liver. Electrophoresis 1997;18: Wu MS, Huang SP, Chang YT, et al. Association of the 160 C!A promoter polymorphism of E-cadherin gene with gastric carcinoma risk. Cancer 2002;94: Lai KC, Lee TC. Genetic damage in cultured human keratinocytes stressed by long-term exposure to areca nut extracts. Mutat Res 2006;599: Ramachandran S, Ramadas K, Hariharan R, Rejnish Kumar R, Radhakrishna Pillai M. Single nucleotide polymorphisms of DNA repair genes XRCC1 and XPD and its molecular mapping in Indian oral cancer. Oral Oncol 2006;42: Lin MH, Wang CJ, Huang HP, Chou MY, Chou FP. The tumorigenic characteristics of lime-piper betel quid-transformed JB6 cells. Arch Toxicol 2004;78: Tseng YH, Chang KW, Liu CJ, Lin CY, Yang SC, Lin SC. Areca nut extract represses migration and differentiation while activating matrix metalloproteinase-9 of normal gingival epithelial cells. J Periodontal Res 2008;43: Dasgupta P, Rizwani W, Pillai S, et al. Nicotine induces cell proliferation, invasion and epithelial-mesenchymal transition in a variety of human cancer cell lines. Int J Cancer 2009;124: Wei PL, Chang YJ, Ho YS, et al. Tobacco-specific carcinogen enhances colon cancer cell migration through alpha7-nicotinic acetylcholine receptor. Ann Surg 2009;249: Tanaka N, Odajima T, Ogi K, Ikeda T, Satoh M. Expression of E-cadherin, alpha-catenin, and beta-catenin in the process of lymph node metastasis in oral squamous cell carcinoma. Br J Cancer 2003;89: Pyo SW, Hashimoto M, Kim YS, et al. Expression of E-cadherin, P-cadherin and N-cadherin in oral squamous cell carcinoma: correlation with the clinicopathologic features and patient outcome. J Craniomaxillofac Surg 2007;35:1 9. CDH1 Gene Polymorphism in Oral Cancer HEAD & NECK DOI /hed March