CYP1A1, GSTM1 and NQO1 gene polymorphisms: Genetic risk factors for small cell lung cancer

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1 Turkish Journal of Cancer Volume 35, No.4, CYP1A1, GSTM1 and NQO1 gene polymorphisms: Genetic risk factors for small cell lung cancer AYÞE DEM RKAN 1, BURÇAK VURAL 1, ZEK ÜSTÜNER 2, KAZIM UYGUN 3, BETÜL ÖZTÜRK AÇIKALIN 4, DUYGU DER N 4, PINAR SA P 4, UÚUR ÖZBEK 1 1 Istanbul University, Experimental Medical Research Institute, Department of Genetics, stanbul, 2 Eskißehir University Medical School, Department of Medical Oncology, Eskißehir, 3 Trakya University Medical School, Department of Medical Oncology, Edirne, and 4 Istanbul University, Oncology Institute, stanbul-turkey ABSTRACT Smoking plays the major role in the development of lung cancer. CYP1A1, GSTM1 and NQO1 gene polymorphisms are thought to be related with some types of cancer considering their important roles in the detoxification pathway of certain carcinogens present in tobacco smoke. These polymorphisms are being widely studied in tobacco related cancer; especially in lung cancer. In this study, we investigated the frequencies of these metabolic gene polymorphisms in our smoking small cell lung cancer patient group (n=93) and ever smoking healthy control subjects (n=126). CYP1A1 ile/val, GSTM1 null/present and NQO1 pro/ser genotype analysis were performed using standard techniques. Compared with the healthy controls, NQO1 ser/ser genotype and ser allele frequency were significantly higher in SCLC patients group [p=0.045 and p=0.013 (OR = 1.82; 95% CI ), respectively]. We have found that CYP1A1 ile/ile + NQO1 pro/pro combined genotype was significantly higher in the control group [p<0.001 (OR = 3.45; 95% CI )]. Also, CYP1A1 ile/ile+gstm1 non-null+nqo1 pro/pro combined genotype was significantly higher in the control group than the SCLC patients [p<0.001 (OR = 3.51; 95% CI )]. [Turk J Cancer 2005;35(4): ]. KEY WORDS: Gene polymorphism, NQO1, GSTM1, CYP1A1, small cell lung cancer, cigarette smoking INTRODUCTION Lung cancer is the major cause of cancer related mortalities in the western world. Far now, smoking is known as the most important carcinogen source in lung cancer development. Lung cancer is divided into two main histological groups; small cell lung cancer (SCLC) and nonsmall cell lung cancer (NSCLC). Small cell lung cancer is seen about 20% among all lung cancers (1-4). Recently, some metabolic polymorphisms are widely studied in order to understand the inheritance of lung cancer and individuals tolerance to tobacco carcinogens. These include cytochrome P4501A1 (CYP1A1) ile/val, glutathione-s-transferase M1 (GSTM1) null/present and NAD(P)H: oxidoreductase 1 (NQO1) pro/ser gene polymorphisms. These three genes encode such enzymes that play major roles in detoxification pathway of several carcinogens, including PAHs that are present in tobacco smoke (5-8). Several polymorphisms have been identified at the CYP1A1 locus including a substitution of valine for isoleusine near the heme binding site at codon 462 in exon 7 (9). In meta analysis, the CYP1A1 Ile462Val has been related to lung cancer risk in Caucasians (10). GSTM1 gene is deleted in approximately 50% of the Caucasian population. Homozygous null individuals lack any functional GSTM1 protein. This deletion apparently

2 172 Genetic Risk Factors for SCLC results from homozygous unequal crossing-over between two highly homologous regions that flank the GSTM1 gene resulting a 15 kb deletion that contains the entire GSTM1 gene (11,12). In different populations GSTM1 null/cyp1a1 val/val combined genotype have been associated with an increase risk for squamous cell lung carcinoma (13-15). Also, there has been strong association between GSTM1 null genotype and Turkish lung cancer patients (16). NAD(P)H: oxidoreductase (NQO1) is also an important enzyme in xenobyotic metabolism that catalyzes the twoelectron reduction of carcinogenic quinoid compound to their reduced form such as quinones (17). The activity of NQO1 enzyme depends on polymorphisms at the NQO1 locus. The major polymorphism involves a single C to T substitution at nucleotide 609 of exon 6 of the NQO1 cdna that causes a Pro 107 Ser amino acid change leading to two mutant phenotypes. Heterozygous phenotype with approximately three-fold decreased activity and homozygous mutant (null) phenotype with a complete lack of enzyme activity (18). NQO1 pro/ser polymorphism was found to be associated with an increased risk for overall lung cancer (19-21). Considering the different studies about these polymorphisms, we focused on their possibility to cause small cell lung cancer. We have investigated the frequencies of CYP1A1 ile/val GSTM1 null/present and NQO1pro/ser polymorphisms among smoking SCLC patients and smoking controls and questioned the possible relation between these genotypes and clinical parameters. obtained from the entire case and control group and the study was approved by the local Ethical Committee of Istanbul Medical Faculty. Genomic DNA was extracted from whole blood using standard techniques (22). By using specific primers, we obtained PCR fragments including the polymorphic sites of CYP1A1 and NQO1 genes. To detect the CYP1A1 ile/val polymorphism, we used Msp1 enzyme digestion and Hinf 1 restriction enzyme for NQO1 pro/ser polymorphism, as previously described (23,24). GSTM1 gene was amplified with PCR using specific primers, ß-globin gene was used as an internal positive control. Fragments were electrophoresed using 3% agarose gel and visualized by ethidium bromide staining. Statistical method The distribution of variables between cases and controls were compared using the Pearson s χ 2 test. Odds ratios (ORs) were used as the estimates of the relative risk and 95% confidence interval calculated. All analyses were performed using SPSS 10.0 for Windows. A PATIENTS AND METHODS SCLC patients (n=93) were included in this study by Istanbul University Oncology Institute, between years 2001 and Before treatment age, family history of any malignancy, performance status (ECOG), serum LDH level, disease stage and smoking status (pack-years) of the patients were recorded. Also possibility of other sources of benzene exposure was noted. We constructed a healthy control group consisted of individuals with no neoplastic history. Since these polymorphisms affect through cigarette consumption, these healthy controls were current smokers with minimum one pack-year smoking history and were chosen from the DNA bank of Istanbul University Experimental Research Institute, Department of Genetics. A written informed consent was B C Fig 1 (A&B&C). (A): GSTM1 null/present polymorphism, 3% agarose gel electrophoresis; (B): NQOI pro/ser polymorphism, 3% agarose gel electrophoresis; (C): CYP1A1 ile/val polymorphism, 3% agarose gel electrophoresis M: DNA ladder; wt: present allele; null: null allele; het: heterozygous allele; mut: mutant allele; bp: base-pairs

3 Demirkan et al. 173 Table 1 Study population Age Median age >55 <55 Unknown Females Males Pack-years Median pack-years >40 <40 Females Males Serum LDH Median LDH >450 <450 Females Males Survival Median survival (months) >12 <12 Females Males Performance status (ECOG) Females Males Stage Limited Extended Females Males Metastasis ( + ) ( - ) Females Males Family history ( + ) ( - ) Females Males RESULTS We performed genotyping of 93 cases and 126 controls (Figure 1A&B&C). Control subjects were healthy volunteers aged between 19 and 56, median age was 37 in men (n=75) and 35 in women (n=51). Patients aged between 33 and 78 years. The median age was 59 in men (n=79) and 60 in women (n=13). Clinical properties of the study population are shown in table 1. Median pack-years were 53 in females (range: 33-80) and 54 (range: ) in males. Over all median survival was 13 (range: 1-39) months after diagnosis (17 months in females and 12 months in males). Median LDH level was 510 (range: ); 480 in females and 516 in males. Distribution of CYP1A1, GSTM1 and NQO1 allele frequencies between SCLC patients and healthy controls are shown in table 2. CYP1A1 ile/val polymorphism did not differ between patients and controls considering both genotype and val allele frequency [p=0.076, p=0.84 (OR = 1.06; 95% CI )]. GSTM1 null/present polymorphism was not different between the cases or controls [p=0.55 (OR = 1.18; 95% CI )]. Compared with the controls, NQO1 ser/ser genotype and ser allele frequency were found to be higher in SCLC patients in this study [p=0.045 and p=0.013 (OR = 1.82; 95% CI ), respectively]. Combined genotype analysis is shown in table 3. We found that CYP1A1-ile/ile + NQO1 pro/pro combined genotype was significantly higher in the control group [p<0.001 (OR= 3.45; 95% CI )]. Also, CYP1A1 ile/ile+gstm1 non-null+nqo1 pro/pro combined genotype was significantly higher in the control group than the SCLC patients [p<0.001 (OR = 3.51; 95% CI )].

4 174 Genetic Risk Factors for SCLC Table 2 Genotype and allele frequencies Genotype Cases N (%) Controls N (%) p value OR (95% CI) CYP1A1 Msp1 polymorphism ile/ile 59/86 (67.6%) 63/84 (75%) ile/val 26/86 (30.2%) 16/84 (19.1%) val/val 1/86 (1.2%) 5/84 (5.6%) p=0.076 val allele frequency 28/172 (16.3) 26/168 (15.5) p= ( ) GSTM1 polymorphism Non-null 37/89 (41.6%) 53/116 (45.7%) Null 52/89 (58.4%) 63/116 (54.3%) p= ( ) NQO1 polymorphism pro/pro 47/88 (53.4%) 80/117 (68,4%) pro/ser 34/88 (38.6%) 34/117 (29.0%) ser/ser 7/88 (8%) 3/117 (2.6%) p=0.045 ser allele frequency 48/176 (27.3%) 40/234 (17.1%) p= ( ) Table 3 Combined genotype frequencies Combined genotype Cases / Controls, N (%) OR 95% CI P value N=83 N=79 CYP1A1-ile/ile + NQO1 pro/pro 23 (27.7) / 45 (56.9) <0.001 CYP1A1 ile/ile +NQO1 pro/pro + GSTM1 non-null 23 (27.4) / 45 (56.9) <0.001 We did not find a correlation between any of these genotypes or combined genotypes and clinical parameters. Also, no correlation was found between pack-years of smoke and survival among the patient group. DISCUSSION In the past few years molecular studies have revealed that there are multiple genetic lesions in the respiratory epithelium of current and former smokers and these appear directly associated with cigarette smoking (2-4). Polycyclic aromatic hydrocarbons (PAHs) in cigarette smoke, including benzo[a]pyrene (BaP) are thought to be the most responsible carcinogen group in cigarette smoke and they first require a metabolic activation by phase I enzymes e.g. cytochrome P 4501A1 (CYP1A1), to their ultimate forms and then bind to DNA, forming aromatic DNA adducts that are thought to be an early step in tumorigenesis (25). On the other hand, phase II enzymes detoxify these activated forms, especially glutathione S-transferases (GSTs) thus genetically determined susceptibility to lung cancer may depend on the metabolic balance between phase 1 and phase II enzymes (26-28). Inherited GST variants that alter GST mediated detoxification of carcinogenic chemical have been extensively investigated as candidate genes for lung cancer susceptibility. (12, 29-31). These polymorphisms also have been reported to affect survival among lung cancer patients. Among all phase I and phase II enzymes, CYP1A1 and GSTM1 and their metabolic balance is thought to be important in lung

5 Demirkan et al. 175 cancer and has been widely investigated in different populations (32). CYP1A1 Val/Val polymorphism results in an increase in the enzyme activity, whereas an inherited homozygous deletion of the GSTM1 gene (GSTM1 null genotype) results in the lack of enzyme activity. The CYP1A1 Val/Val genotype, the GSTM1 null genotype and their combined genotype have been reproducibly associated with an increased risk for squamous cell lung carcinoma (33,34). In our study, we did not find a correlation between CYP1A1 ile/val polymorphism and small cell lung cancer (OR =1.06; 95% CI ) It is known that BaP activates the aryl hydrocarbon receptors (AhR) which subsequently induces CYP1A1 expression which is involved in the conversion of BaP into 7,8-dihydroxy-9, 10 epoxy-7,8,9,10- tetrahydrobenzoapyrene (BPDE) that forms DNA adducts. (32,33,35,36). Recently, BaP and BPDE were also reported to activate several signaling pathways that triggered epigenetic effects (37). NQO1 is a flavoenzyme that converts the highly genotoxic benzoquinone to the less toxic hydroxy metabolites (38). NQO1 reduces the formation of DNA adducts induced by benzo[a]pyrene3,6 quinone, one of the important carcinogenic PAH identified in cigarette smoke (39). By this way, NQO1 specifically prevents the formation of Benzo[a]pyrene quinone- DNA adducts generated by cytochrome CYP1A1 (39). In addition, there is evidence to suggest that this enzyme is induced in the lung in response to cigarette smoke (40). NQO1 has been detected at high levels in normal respiratory epithelium and in tissue from adenocarcinoma and squamous cell carcinoma tumors. However, this enzyme has not been detected in small cell lung tumors suggesting that the lack of NQO1 may play a specific role in the pathology of SCLC. The NQO1 ser/ser genotype results a decrease in enzyme activity, which is caused by enhanced polyubiquitination and proteosomal degradation of the mutant protein (18). According to our genotyping results, NQO1 ser/ser genotype and serine allele frequency may be a risk factor for SCLC, but despite some other studies, CYP1A1 and GSTM1 genotypes solely do not seem to be associated with a risk for SCLC, also any of these three polymorphisms are not correlated with any of the clinical parameters known for our SCLC patient group. Also, our results indicate that combined wild type genotypes of these three genes may be a protective factor for SCLC probably because of their enhancing effect on BaP detoxification pathway that prevents the formation of DNA adducts. This is the first case-control study about the synergistic and additive effects of these gene polymorphisms to small cell lung cancer susceptibility in Turkish population. References 1. Hecht SS. Tobacco smoke carcinogens and lung cancer. J Natl Cancer Inst 1999;91: Wistuba, II, Lam S, Behrens C. Molecular damage in the bronchial epithelium of current and former smokers. J Natl Cancer Inst 1997;89: Mao L, Lee JS, Kurie JM. Clonal genetic alterations in the lungs of current and former smokers. J Natl Cancer Inst 1997;89: Denissenko MF, Pao A, Tang M: Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in P53. Science 1996;274: Spivack SD, Fasco MJ, Walker VE. The molecular epidemiology of lung cancer. Crit Rev Toxicol 1997;27: Hayashi S, Watanabe J, Nakachi K, et al. Genetic linkage of lung cancer-associated Msp1 polymorphisms with amino acid replacement in the heme binding region of the human cytochrome P4501A1 gene. J Biochem 1991;110: Rebbeck TR. Molecular epidemiology of the human glutathione S-transferase genotypes GSTM1 and GSTT1 in cancer susceptibility. Cancer Epidemiol Biomark Prev 1997;6: Taningher M, Malacarne D, Izzotti A, et al. Drug metabolism polymorphisms as modulators of cancer susceptibility. Mutat Res 1999;436: Hayashi S, Watanabe J, Nakachi K, et al. Genetic linkage f lung cancer-associated Msp1 polymorphisms with amino acid replacement in the heme binding region of the human cytochrome P4501A1 gene. J Biochem 1991;110: Vineis P, Veglia F, Benhamou S, et al. CYP1A1 T33801C polymorphism and lung cancer: a pooled analysis of 2451 cases and 3358 controls. Int J Cancer 2003;104: McWilliams JE, Sanderson BJ, Harris EL, et al. Glutathione S-transferase M1 (GSTM1) deficiency and lung cancer risk. Cancer Epidemiol Biomark Prev 1995;4:

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