Smoking and polymorphisms in folate metabolizing genes and their effects on the histological stage and grade for bladder tumors

ARTICLE

bdc.2011.1312

Auteur(s) : Kamel Rouissi¹ rouissik2000@yahoo.fr, Najla Stambouli¹, Raja Marrakchi¹, Mohamed R. Ben Slama², Mohamed cherif², Mohamed Sfaxi², Mohamed Chebil², Amel Benammar Elgaaied¹, Slah Ouerhani³

¹ University of El Manar I, Faculty of Sciences of Tunis, Laboratory of Genetics, Immunology and Human Pathology, 2092, Tunis, Tunisia

² Charles Nicolle Hospital, Department of Urology, Tunis, Tunisia

³ University of El Manar I, Pasteur Institute of Tunis, Laboratory of Molecular and Cellular Haematology, Tunis, Tunisia

Reprint: K. Rouissi

Introduction

Bladder cancer is the fourth most common cancer in the men and the ninth most common in the women [1]. Urothelial cell carcinomas (UCC) represent more than 90% of bladder tumors and are classified into superficial (pTa and pT1) and muscle invasive (≥ pT2) stages. The management of bladder cancer is dependent on tumor stage and grade. The pTa tumors are removed by transurethral resection, whereas invasive tumors are treated by radical cystectomy with or without postoperative chemotherapy. Cigarette smoking is the most important risk factor for bladder cancer, accounting for 50% of cases in men and 35% in women [2], although the precise mechanism by which cigarette smoking causes urinary tract cancer has yet to be clarified. Cigarette smoke contains a range of xenobiotics, including oxidants and free radicals, and accordingly cigarette smoke exposure was associated with decreased levels of serum and red blood cell folate and vitamin B12 antioxidants [3, 4]. On the other hand, reports exist that plasma total homocysteine concentration is higher in smokers than in non-smokers [5, 6]. These findings suggest that the combined effects of smoking with decreased levels of folate and vitamin B12 and an increased level of homocysteine can induce increased chromosomal damage [7]. If so, DNA damage induced by smoking may be modulated by folate metabolic pathway.

Central to folate metabolism are the enzymes 5,10-methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), methionine synthase reductase (MTRR) and thymidylate synthase (TYMS), which play important and interrelated roles in folate pathway. The MTHFR enzyme occupies a pivotal position, balancing the homeostasis between DNA synthesis and methylation by catalyzing the irreversible conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. The MTHFR substrate, 5,10-methylenetetrahydrofolate, is used by thymidylate synthase in the methylation of dUMP to dTMP, which is the sole de novo source of thymidylate required for DNA synthesis and repair. The MTHFR product, 5-methyltetrahydrofolate, is the methyl group donor for the remethylation of homocysteine to methionine catalyzed by MTR in a reaction dependent on vitamin B12 as an intermediate methyl carrier. MTR may become inactive due to oxidation of its vitamin B12 cofactor, and restoration of MTR activity is dependent on reductive remethylation of vitamin B12 by MTRR. Several genetic variants in genes coding for MTHFR, MTR, MTRR and TYMS were shown to affect directly on the function of the expressed proteins. Among them are C677T and A1298C within MTHFR [8-10], A2756G within MTR [11] and A66G as well as C524T within MTRR [12]. Within the TYMS enhancer region, a polymorphic 28 bp tandem repeat located immediately upstream of the ATG codon initiation is known to influence TYMS transcription or translation, and in addition a common G-to-C transversion in the second of the TYMSER three repeats allele also affects TYMS expression [13].

Many previous case-control studies have addressed the relationship between folate metabolic genes and risk for bladder cancer, producing, however, conflicting results [14-16]. However, the relationship between these risk factors, the stage and the grade of bladder tumors was not evaluated. In view of that, we examined in this study the combined effect of smoking and polymorphisms in folate metabolic genes in different histological subgroups of bladder tumors aiming at clarifying whether smoking and genetic variations represent risk factors for the development of tumors with high stage and grade. Efficient study design utilizing these biomarkers should accelerate the development of optimum bladder cancer prevention strategies.

Patients and methods

Patients

A total of 130 patients with UCC of bladder cancer were included in the present study. Patients were recruited from the Department of Urology at the Charles Nicole Hospital in Tunisia. All were from North of Tunisia, 88.46% of them were men and the mean age at diagnosis was 67.86 ± 9.16 years. These patients were classified according to their tobacco status. The smoker category included current smokers who smoked daily. A heavy smoker was defined as a current smoker who had smoked 20 cigarettes or more a day during 20 years or more. Non-consumers of tobacco were defined as persons who had never smoked or had consumed less than 20 packs of cigarettes or 360 g of tobacco in their lifetime or less than one cigarette per day. The intensity of tobacco use (PY) was defined as the amount of tobacco consumed during the life of patients (1 PY = 7300 cigarettes smoked during 1 year). It was found that 77.69% (101/130) of patients were current smokers and 22.31% were non-tobacco consumers. It was found that 88.11% (89/101) of smokers were heavy smokers and 53.46% (54/101) have smoked more than 40 PY.

DNA preparation and genotyping

After giving informed consent, peripheral blood samples were collected from all patients into tubes with EDTA at pH 8. Genomic DNA was extracted from leukocytes using a phenol-chloroform procedure [17]. MTHFR C677T, MTHFR A1298C, MTR A2756G, MTRR A66G, MTRR C524T, TYMS 2R→3R and TYMS G/C polymorphisms were detected with polymerase chain reaction/restriction fragment length polymorphism-based approaches, as described previously [18, 19].

Statistical analysis

The relative risks (RR) were estimated with 95% confidence intervals (CI) at the 0.05 significance level [20]. Relative risk was calculated using non-smoker patients with the homozygous wild-type genotypes as reference using the software Epi Info 6.0.

Results

Tumors were staged and graded according to the criteria of the tumor-node-metastasis classification (TNM) and the WHO-International Society of Urological Pathology as follows: 1 CIS, 29 pTa GI, 17 pTa GII, 2 pTa GIII, 4 pT1 GI, 26 pT1 GII, 22 pT1 GIII and 29 invasive tumors (≥ pT2).

The percents of smokers developing superficial and invasive tumors were 75.24% (76/101) and 86.20% (25/29) respectively (table 1 table 1). The comparison of these percentages does not show a significant statistic difference (P = 0.31), which suggested that tobacco does not appear to be a factor affecting the bladder tumors stage. However, we have found that patients who have smoked 20-39 PY during their lifetime have a 1.44 fold risk for developing invasive tumors compared to non-smokers patients (table 1).

Table 1 Distribution of superficial and invasive tumors according to patients smoking status and to the folate metabolizing enzyme genotypes.

1*: reference group; RR: Relative risk; CIS, pTa/pT1: superficial tumors; ≥ pt2: invasive tumors; PY: packet years.

The distribution of altered genotypes for MTHFR, MTR and MTRR genes between patients with superficial tumors and those with invasive tumors does not show a significant statistical difference (table 1). For TYMS gene, a significant statistic difference in genotypic distribution between patients with superficial tumors and those with invasive tumors was detected for the TYMS 3R*G/3R*C genotype (P = 0.02). This genotype presented a 1.74-fold increased risk of developing invasive tumors compared to reference group (RR = 1.74; 95% CI: 0.97-3.12). The stratification of superficial and invasive tumors according to smoking status, MTHFR, MTR, MTRR and TYMS genotypes does not show a significant statistical difference (table 2 table 2).

Table 2 Stratification of the genotypes of superficial and invasive tumors according to smoking status.

1*: reference group; RR: Relative risk; CIS, pTa/pT1: superficial tumors; ≥ pt2: invasive tumors; PY: packet years.

Among superficial tumors, 76.23% (77/101) were with low grade (GI or GII) and 23.76% (24/101) were with high grade (GIII). More than 81.8% (63/77) of patients with superficial low grade tumors were smokers. This percentage was statistically different to that reported for patients with high-grade tumors (table 3 table 3). The high frequency of smokers in patients with superficial low-grade tumors (used as a reference group) compared to those with high grade led to a relative risk (RR) less than 1 (RR = 0.68; 95% CI 0.47-0.97).

Table 3 Distribution of superficial bladder cancer according to tumors grade, patients smoking status and folate metabolizing enzyme genotypes.

1*: reference group; RR: Relative risk; GI/GII: low-rade superficial tumors; GIII: high grade superficial tumors; PY: packet years.

The comparison of MTHFR, MTR and MTRR altered genotypes frequencies in patients with superficial low grade to those with superficial high-grade tumors does not show a significant statistical difference (table 3). A significant statistical differences were only obtained for the TYMS 3R*G/2R genotype. This genotype presented a 1.68-fold increased risk of developing high grade tumors compared to reference group (RR = 1.68; 95% CI: 1.12-2.54). Moreover, our data reported that patients having at least one copy of 2R allele were at high risk for developing high grade tumors compared to reference group (P = 0.022, RR = 4.23; 95% CI: 1.08-16.48).

The stratification of superficial bladder tumors according to their genotypes and tobacco (table 4 table 4) have showed that the MTR 2756AA genotype was over presented in smoker patients with superficial low grade tumors compared to those with high grade tumors (P = 0.01). The presence of this wild genotype was associated with a protective role against the development of superficial high-grade bladder tumor (RR = 0.54; 95% CI: 0.29-0.98).

Table 4 Stratification of genotypes of low and high-grade superficial tumors according to smoking status.

1*: reference group; RR: Relative risk; GI/GII: low grade superficial tumors; GIII: high grade superficial tumors; PY: packet years.

Discussion

Urothelial cell carcinoma (UCC) is a heterogeneous neoplasm that presents as either superficial or muscle invasive at diagnosis. Superficial low-grade tumors are characterized by frequent recurrences. In contrast, high-grade tumors (pTa GIII and pT1 GIII) represent a significant risk of future tumors progression and death for the disease. Tobacco smoke is the most important exogenous risk factor for bladder cancer [2]. A previously findings suggest that the combined effects of smoking with decreased levels of folate and vitamin B12 and an increased level of homocysteine can induce increased chromosomal damage [7]. In view of that, we examined in this study the combined effect of smoking and polymorphisms in folate metabolic genes in different histological subgroups of bladder tumors aiming at clarifying whether smoking and genetic variations represent risk factors for the development of tumors with high stage and grade.

Our data have reported that 77.69% (101/130) of patients were current smokers and 22.31% were non-tobacco consumers. It was found that 88.11% (89/101) of smokers were heavy smokers and 53.46% (54/101) have smoked more than 40 PY. These results suggested the important role of tobacco in bladder cancer development in the Tunisian population. The distribution of patients according to their smoking status and to the histological tumors stage, has suggested that patients who have smoked 20-39 PY during their lifetime have a 1.44 fold risk for developing invasive tumors compared to non-smokers patients. However, patients who have smoked 40 PY ore more during their lifetime develop low grade but not high-grade superficial tumors group. This finding was in contradiction with others many studies, which have suggested that bladder tumors in patients who smoke tend to be large, multi-focal and demonstrate high histological grade and stage [21].

The distribution of altered genotypes for MTHFR and MTRR genes between patients with superficial tumors and those with invasive tumors does not show a significant statistical difference. This results is waited because we have previously reported for that the isolated MTHFR 677*T, MTRR 66*G and MTRR 524*T variants did not appear to influence bladder cancer susceptibility [22]. For the MTR gene, although we have previously found that MTR 2756*G variant increases the risk of bladder cancer development [22], we have not reported any association with the tumors stage and grade. As so, this gene is considered as markers for the bladder cancer development but not for the tumors presentation. With considering the TYMS gene, our data have reported that patients having at least one copy of 2R allele were at 4.23–fold increased risk for developing high-grade tumors compared to reference group. This result appears in contradiction with our previously reported data, which considered that patients having the 2R variant were protected against bladder cancer development. The mechanism by which TYMS polymorphisms influence bladder cancer susceptibility was by their effect on gene expression. Indeed Mandola et al. [13], have reported that the ATG codon initiation site containing two (2R) or three (3R) 28-base pair (bp) repeats that can influence TYMS transcription or translation. The 3R/3R genotype was shown to be associated with increased expression of the TYMS gene and/or TS protein. In addition, a common G-to-C transversion in the second of the three 28-bp repeats of TYMSER*3 has been identified. This single nucleotide polymorphism (SNP) changes a critical residue in the upstream stimulatory factor E-box consensus element, which leads to a decrease in TYMS transcription such that TYMSER*3C has lower activity than TYMSER*3G, but similar activity to TYMSER*2 3R/3R genotype. The asked question was how the 2R variant was associated in the same time with a protective role against bladder cancer development and with advanced tumors grade? This result is explained by equilibrium between methylation and thymidine synthesis. Indeed; when the activity of TYMS enzyme decrease, due to the presence of 2R variant, the methylation process is accelerated and the oncogene were deactivated which protect against bladder cancer development. After development of bladder tumors the decrease of thymidine synthesis increases the risk of DNA instability and chromosome aberration, which characterize the advanced bladder tumors grade.

The distribution of patients developing superficial bladder tumors according to genotypic frequencies of MTHFR, MTRR and TYMS, smoking status and histological tumors grade does not show a significant statistical difference. This result suggests that genetic polymorphisms in these genes don’t interact with tobacco to influence the histological tumors grade. However, we have found that smoking patients harbouring the wild genotype for MTR (MTR 2756AA) were protected against the development of superficial high-grade tumors. This protective role is explained by maintain of a normal DNA and protein methylation. Indeed it was shown that variations in MTR gene, which is crucial in the provision of methyl groups for DNA, RNA and protein methylation, as well as in purine and pyrimidine synthesis [23, 24] leads to decreased DNA methylation and such insufficiency may promote carcinogenesis by inducing genomic instability or by the derepression of proto-oncogenes [14].

Although some of the results presented in this study are novel, the study has some limitations. Firstly, the sample size is small, limiting the precision of the statistic analyses. Secondly, we have not information regarding somatic altered genes such as FGFR3 and p53, which was respectively associated to superficial low and high-grade bladder tumors. Besides that in the future, enlargement of sample sizes in the Tunisian population and analysis of somatic altered genes will be essential to assess the role that environmental factors together with the genetic factors play as predictors of differential susceptibility to the malignancy presentation.

Conflicts of interest: none.

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