Role of GSTM3 Polymorphism in the Risk of Developing Esophageal Cancer

178 Short Communication

Meenu Jain,1 Shaleen Kumar,2 Punita Lal,2 Anu Tiwari,2 Uday C. Ghoshal,3 and Balraj Mittal1 Departments of 1Medical Genetics, 2Radiotherapy, and 3Gastroenterology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India

Abstract

GSTM3 is involved in detoxification of carcinogens and may GSTM3 AB + BB and GSTM1 null genotypes marginally be important in modulating cancer susceptibility. GSTM3 modulated risk (OR, 2.3; 95% CI, 1.1-3.7; P = 0.01). Association genotype frequencies were determined in peripheral blood with histology (adenocarcinoma: OR, 3.4; 95% CI, 1.1-10.9; DNA of 149 esophageal cancer patients and 200 nonmalignant P = 0.03) and tumor site (middle third location: OR, 2.2; 95% controls using the PCR followed by PAGE. Patients who were CI, 1.1-4.4; P = 0.01; lower third location: OR, 2.6; 95% CI, 1.2- heterozygous carriers of GSTM3 AB genotype had an 5.6; P = 0.01) was also documented. Our results suggest that enhanced risk for developing esophageal cancer [odds ratio GSTM3 polymorphism may influence esophageal cancer (OR), 2.1; 95% confidence interval (95% CI), 1.1-3.7; P = 0.01]. susceptibility, in particular modulating the risk for adeno- In males, the risk due to GSTM3 AB genotype increased carcinoma histology and tumors of the mid and lower third further (OR, 3.4; 95% CI, 1.7-6.8; P = 0.000). Interaction of region. (Cancer Epidemiol Biomarkers Prev 2007;16(1):178–81)

Introduction

Nutritional deficiency coupled with alcohol and tobacco abuse mutation, the expression of GSTM3 can be influenced. The (both smoked and smokeless forms), environmental carcino- functional consequence of this is unclear, but both negative gens, and occupational exposure have been shown to be and positive regulatory effects have been suggested (6-8). associated with the development of esophageal cancer (1, 2). However, possible influence of GSTM3 has not been studied in Glutathione S-transferases (GST) constitute a family of esophageal cancer. Altered detoxification due to GSTM3 xenobiotic detoxifying phase II enzymes that catalyze the polymorphism alone or in combination with GSTM1 null conjugation of glutathione to a variety of electrophilic genotypes might influence cancer susceptibility. Therefore, the compounds, including polyaromatic hydrocarbons, which are present study was designed to analyze the association of widely present in the human environment and are known to GSTM3 genotypes and interaction with GSTM1 null genotypes be carcinogenic. At least five mammalian GST gene families and environmental factors with riskof developing esophageal have been identified to be polymorphic, and mutations or cancer. deletions of these genes contribute to the predisposition for several diseases, including cancer. The gene cluster of GSTM1- GSTM5 has been reported to be localized on chromosome 1p13 Materials and Methods and spans a length of nearly 100 kb. Polymorphic genes of GST family, like GSTM3 and GSTP1, have been shown to modulate This study included a total of 149 untreated, histologically the cancer risk. In the GSTM3 gene, the GSTM3*A wild-type confirmed, esophageal cancer patients with squamous or allele and the GSTM3*B variant allele have been described (3). adenocarcinomas, referred to the Departments of Gastroenter- GSTM3 plays a role in the metabolism of harmful agents, like ology and Radiotherapy of a tertiary referral hospital in polyaromatic hydrocarbons benzo(a)pyrene, and has over- Northern India along with 200 nonmalignant unrelated lapping substrate specificity with GSTM1 (4). The GSTM3 controls, during a 2.5-year period (2003 to mid-2006). The polymorphism could, therefore, confer different efficiencies in ethnic background of the subjects was similar (i.e., from the metabolism of carcinogens. In a recent study, an increase in Northern India). Patient data were collected through an larynx cancer riskassociated with GSTM3 AA genotype was interview where demographic features, clinical details, and suggested (5). In the GSTM3 gene, the GSTM3*A wild-type environmental exposure were recorded using a standard allele and GSTM3*B variant allele have deletion of 3 bp in clinical proforma. Written informed consent was obtained intron 6, resulting in the generation of a recognition sequence from each patient. Environmental carcinogens included smok- for the YY1 (Ying Yang) transcription factor. As a result of this ing of cigarette, bidi (Indian cigarette made of tobacco wrapped in tendu leaf), or hukka (Indian pipe). Smokeless tobacco use consisted of gutka (sweet flavourful chewing tobacco) or zarda (aromatic chewing tobacco). Smoking was Received 7/7/06; revised 9/21/06; accepted 11/1/06. measured in pack-years (1 pack-year = 20 sticks per day for Grant support: ICMR, DST, UGC Senior Research Fellowship (M. Jain). 1 year). Smokeless tobacco use was measured as chewing-year The costs of publication of this article were defrayed in part by the payment of page charges. (1 chewing-year = taking chewable tobacco quid once in a day This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. for 1 year). Alcohol was either consumed or not. ‘‘Occupa- Requests for reprints: Balraj Mittal, Department of Medical Genetics, Sanjay Gandhi tional exposure’’ consisted of being exposed to coal, smoke, or Postgraduate Institute of Medical Sciences, Raebareilly Road, Lucknow 226014, India. petroleum products at workand included exposure of Phone: 91-522-2668973, ext. 2322; Fax: 91-522-2668017/2668074. E-mail: [email protected] or [email protected] housewives to smoke emanating from domestic cooking fuels. Copyright D 2007 American Association for Cancer Research. The study was accorded approval by the ethical committee of doi:10.1158/1055-9965.EPI-06-0542 the Institute.

Methodology. Genomic DNA was prepared from frozen Table 1. Clinical characteristics and environmental factors blood samples collected in EDTA. DNA was isolated as in esophageal cancer patients described elsewhere (9, 10). Genotype frequencies at the Patients (n = 154) GSTM3 and GSTM1 loci were determined by PCR. GSTM1 null polymorphism genotyping was done using method Lymph node (n = 128)* described elsewhere (9). GSTM3 reactions were carried out in Present 86 (67.2%) a total volume of 25 AL containing 50 ng DNA template, 20 pmol Absent 42 (32.8%) GSTM3 primers (10, 11), 10 mmol/L deoxynucleotide triphos- Location (n = 142)* Â Upper 22 (14.9) phates, 10 buffer containing Tris-HCl (pH 8.6), 50 mmol/L Middle 78 (52.7) KCl, 15 mmol/L MgCl2,and1.5unitsTaqpolymerase Lower 48 (32.4) (Bangalore Genei, Bangalore, India). The reaction conditions Histopathology (n = 151)* for GSTM3 were 94jC for 3 min followed by 35 cycles of Squamous cell carcinoma 142 (94.1%) denaturation at 94jC for 1 min, annealing at 62jC for 1 min, Adenocarcinoma 9 (5.9%) extension at 72jC for 1 min, and final extension for 10 min. Tobacco use (n = 146)* Smokers 32 (21.9) Intron 6 GSTM3 PCR product was separated on 20% polyacryl- Smokers + smokeless tobacco users 49 (33.6) amide gel (Fig. 1). The band sizes corresponding to 79 bp Smokeless tobacco users 37 (35.3) (GSTM3*A allele) or 76 bp (GSTM3*B allele) were recorded. Nonusers 28 (19.2) Alcohol consumption (n = 126)* Statistical Analysis. The demographic characteristics of Drinker 46 (36.5) patients and controls were described as percentages or Nondrinker 80 (63.5) summary measures. Statistical significance of frequency differ- Occupational exposure (n = 125)* ences between patients and control groups was evaluated Present 41 (32.8) using the m2 test or t test as appropriate. Deviation from the Absent 84 (67.2) Hardy-Weinberg equilibrium in controls was assessed using *Missing data. the m2 test, and P was considered significant at <0.05 level. Unconditional logistic regression analysis was used to fit statistical models to predict the association of GSTM3 patients (56.5 F 12.4 years and 76% males) and controls (55.6 F genotypes with susceptibility to esophageal cancer, histopa- 9.4 years and 71% males). The majority of patients harbored a thology, and site of tumor, lymph node presence, and squamous cell carcinoma (94.1%), located in the middle third environmental factors, like tobacco habits, alcohol consump- of the esophagus (52.7%), with nodal metastasis in 67.2%. tion, and occupational exposure. A case-only analysis was Prevalence of smoking and smokeless tobacco users was used for estimating gene-environment interaction, and inter- around 80% (Table 1). The proportion of drinkers and action terms were included in the logistic regression model. occupationally exposed patients is also summarized in Table 1. The likelihood ratio test was used to test the goodness of fit, The GSTM3 genotype distributions in the control popula- and the Wald’s test was used to assess the coefficient of tion were in Hardy-Weinberg’s genetic equilibrium model in significance. Association was expressed as odds ratios (OR) as relation to observed and expected genotypic frequencies riskestimates with 95% confidence intervals (95% CI). ORs (P = 0.40). The genotype frequencies are presented in were adjusted for confounding factors, such as sex, age, Table 2. The GSTM3 AB + BB genotype was associated with smoking, and alcohol usage. Statistical analysis was done a 2-fold riskof esophageal cancer, due to a higher frequency of using SPSS Software version 10.0 (SPSS, Chicago, IL). the GSTM3 AB genotype and a lower frequency of GSTM3 AA genotype, in cancer patients compared with controls (Table 2). The frequency of the GSTM3 B allele was also higher and Results imposed significantly riskfor esophageal cancer (OR, 2.2; 95% CI, 1.4-3.3; P = 0.00). Effect of the GSTM3 AB + BB genotype The characteristics of the study subjects are listed in Table 1. F was slightly modified by GSTM1 null genotypes (OR, 2.3; 95% Mean age SD (years) and gender distribution were similar in CI, 0.97-4.2; P = 0.01). After gender stratification according to GSTM3 genotypes, riskfor esophageal cancer increased in males (Table 2). Analysis of clinical characteristics in patients with squamous cell carcinoma histology revealed higher GSTM3 AB + BB genotype frequency (21.2%), which increased further in adenocarcinoma (44.4%), compared with controls (Table 3). Association of lymph node metastasis with GSTM3 AA + BB genotypes showed riskin patients with absence of spread to lymph nodes (Table 3). GSTM3 AB + BB genotype frequency and riskfor esophageal cancer was higher in patients with middle and lower third location (OR, 2.2; 95% CI, 1.1-4.4; P = 0.01; OR, 2.6; 95% CI, 1.2-5.6; P = 0.01). Interaction of GSTM3 AA + AB genotypes with tobacco habits (OR, 1.0; 95% CI, 0.38- 2.8; P = 0.91), smokers (OR, 1.3; 95% CI, 0.59-2.9; P = 0.48), smokeless tobacco users (OR, 0.92; 95% CI, 0.48-1.7; P = 0.80), alcohol consumption (OR, 0.55; 95% CI, 0.21-1.4; P = 0.09), and occupational exposure (OR, 1.7; 95% CI, 1.0-7.3; P = 0.21) did not modulate the riskfurther.

Discussion Figure 1. Gel picture showing GSTM3 intron 6 polymorphism (3 bp deletion). Lane 1: GSTM3 BB genotype, lane 2: GSTM3 AA genotype, Esophageal cancer is known to be associated with environ- lane 3: GSTM3 AB genotype, lane 4: GSTM3 AA genotype and lane 5: mental carcinogens. GSTM3 is a phase II enzyme that plays a 50 bp ladder. role in conjugation and detoxification of environmental and

Cancer Epidemiol Biomarkers Prev 2007;16(1). January 2007 Downloaded from cebp.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. 180 GSTM3 Polymorphism and Risk of Esophageal Cancer

Table 2. GSTM3 genotype association and risk of esophageal cancer

GSTM3 genotypes, n (%)

AA AB + BB

Patients (n = 154)/controls (n = 200) 118 (76.6%)/176 (88.4%) 36 (23.4%)/24 (11.6%) OR (95% CI), P 1 (reference)* 2.1 (1.1-3.7), P = 0.01 Allele frequency c c AB Patients (n = 308 )/controls (n = 400 ) 0.89/0.93 0.11/0.07 OR (95% CI), P 1 (reference)* 2.2 (1.4-3.3), P = 0.00 Gender Male patients (n = 117)/controls (n = 142) 85 (72.6%)/128 (90.1%) 32 (27.4 %)/14 (9.9%) OR (95% CI), P 1 (reference)* 3.4 (1.7-6.8), P = 0.000 Female patients (n = 37)/controls (n = 58) 33 (89.2%)/49 (84.5%) 4 (10.8%)/9 (15.5%) OR (95% CI), P 1 (reference)* 0.66 (0.18-2.3), P = 0.51 GSTM1 present Patients (n = 86)/controls (n = 104) 67 (77.9%)/91 (87.5%) 19 (22.1%)/13 (12.5%) OR (95% CI), P 1 (reference)* 2.0 (0.96-4.3), P = 0.07 GSTM1 null Patients (n = 68)/controls (n = 96) 51 (75%)/85 (88.5%) 17 (25%)/11 (11.5%) OR (95% CI), P 1 (reference)* 2.3 (0.97-4.2), P = 0.01

*Age-, gender-, smoking-, and alcohol-adjusted ORs were calculated, using GSTM3 AA as reference. cNumber of chromosomes. occupational carcinogens. Lackof glutathione conjugation differences may be due to gender-associated expression could be an important riskfactor for development of patterns of the GST family of enzymes or the influence of sex esophageal cancer. A 3-bp deletion in GSTM3 intron 6 results hormones for which GST regulation is established in rodent in two alleles: GSTM3*A and GSTM3*B. The deletion generates models (17, 18). Lifestyle and dietary factors may also a site for the binding of transcription factor YY1. The frequency influence the difference in susceptibility to disease. Dietary of GSTM3 B mutant allele has been rated as 15% to 24% (5) in influence of potentially protective vegetable diets are known to Caucasians and 68% in African Americans (11). In the present have a more pronounced effect on GST activity in females study, frequency of GSTM3 B allele was 11.5% in controls, compared with males, which may explain lower riskin which was similar to other studies from India (10, 12). females (19). Heterozygous carriers of the mutated allele GSTM3*B were The association of GSTM3 AB + BB genotypes with clinical more frequent and at riskin the group of esophageal cancer characteristics, such as histology, showed higher riskin patients. This is similar to the other studies on larynx (5), patients with adenocarcinoma compared with those with colorectal (13), bladder (6), and breast cancer (14). In contrast, squamous cell carcinoma, suggesting contribution of altered GSTM3 AA genotype has been a riskfactor in oral cancer (12). expression of GSTM3 genotypes in susceptibility for histology According to our results, the GSTM3*B allele could be specificity in esophageal cancer. The association with the considered a riskallele as it has a YY1 site. There are many absence of lymph node metastasis and GSTM3 AB + BB promoters regulated by YY1 either by initiation, activation, or genotypes may not reflect a true association as the sample repression. The repression of cytokines or stress response size in the subgroup of patients with no nodal metastasis was genes like IFN-c by YY1 transcription factor may also enhance small, with a real possibility of a type I error. However, the cancer susceptibility (15). The GSTM3*B allele has been variable riskfor tumor development due to GSTM3 AB + BB reported to lead to variable expression in cytosol (16), which genotype with middle and lower third location of tumor may lead to alteration in detoxification efficiency of carcino- seems to support a previous theory of site-specific genetic gens predisposing an individual for cancer susceptibility. alterations (20). After subgrouping patients and controls based on gender, A case-only analysis was used to explore gene-environment the GSTM3 AB + BB genotypes were at higher riskfor interaction. Case-only approaches are believed to be better esophageal cancer in males compared with females. The than case-control studies as the latter may suffer from common

Table 3. Association of GSTM3 genotypes with histology, lymph nodes, and location of tumor in esophageal cancer

GSTM3 AA*, n (%) GSTM3 AB+BB,n (%)

Histology Squamous cell carcinoma patients (n = 142)/controls (n = 200) 112 (78.8%)/176 (88%) 30 (21.2%)/24 (12%) OR (95% CI), P 1 (reference)* 1.9 (1.0-3.4), P = 0.03 Adenocarcinoma patients (n = 9)/controls (n = 200) 5 (55.6%)/176 (88.5%) 4 (44.4%)/ 24 (11.5%) OR (95% CI), P 1 (reference)* 3.4 (1.1-10.9), P = 0.03 Lymph nodes Presence (n = 86)/controls (n = 200) 72 (83.7%)/176 (88.5%) 14 (16.3%)/24 (11.5%) OR (95% CI), P 1 (reference)* 1.6 (0.85-3.1), P = 0.13 Absence (n = 41)/controls (n = 200) 28 (68.3%)/176 (88.4%) 13 (31.7%)/24 (11.5%) OR (95% CI), P 1 3.1 (1.5-6.3), P = 0.001 Tumor location Upper (n = 21)/controls (n = 200) 18 (85.7%)/176 (88.5%) 3 (14.2%)/24 (11.5%) OR (95% CI), P 1 (reference)* 2.1 (0.80-5.9), P = 0.12 Middle (n = 73)/controls (n = 200) 58 (79.4%)/176 (88.5%) 15 (20.5%)/24 (11.5%) OR (95% CI), P 1 (reference)* 2.2 (1.1-4.4), P = 0.01 Lower (n = 48)/controls (n = 200) 37 (77.0%)/176 (88.5%) 11 (22.9%)/24 (11.5%) OR (95% CI), P 1 (reference)* 2.6 (1.2-5.6), P = 0.01

*Age-, gender-, smoking-, and alcohol-adjusted ORs were calculated, using GSTM3 AA as reference.

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In an article in the January 2007 issue (1), several errors were noted by the authors. On page 178, ‘‘P value = 0.000>>>’’ should read ‘‘P value = 0.0003.’’ In Table 2, p. 180, ‘‘P value = 0.00>>>’’ should read ‘‘P value = 0.00001’’ and ‘‘P value = 0.000>>>’’ should read ‘‘P value = 0.0003.’’ Also, on page 178, in the Abstract, ‘‘DNA of 149 esophageal cancer patients’’ should read as ‘‘DNA of 154 esophageal cancer patients.’’ In ‘‘Materials and Methods’’ on page 178..., ‘‘This study included a total of 149’’ should read as ‘‘This study included a total of 154....’’

Reference 1. Jain M, et al. Role of GSTM3 polymorphism in the risk of developing esophageal cancer. Cancer Epidemiol Biomarkers Prev 2007;16:178 – 81.

Cancer Epidemiol Biomarkers Prev 2007;16(5). May 2007 Role of GSTM3 Polymorphism in the Risk of Developing Esophageal Cancer

Meenu Jain, Shaleen Kumar, Punita Lal, et al.

Cancer Epidemiol Biomarkers Prev 2007;16:178-181.

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