Int Urol Nephrol DOI 10.1007/s11255-015-0933-0

UROLOGY - ORIGINAL PAPER

GSTO1*C/GSTO2*G haplotype is associated with risk of transitional cell carcinoma of urinary bladder Tatjana Djukic · Tatjana Simic · Tanja Radic · Marija Matic · Marija Pljesa‑Ercegovac · Sonja Suvakov · Vesna Coric · Tatjana Pekmezovic · Ivana Novakovic · Dejan Dragicevic · Ana Savic‑Radojevic 

Received: 16 January 2015 / Accepted: 14 February 2015 © Springer Science+Business Media Dordrecht 2015

Abstract  Purpose  To clarify the role of genetic polymorphisms of GSTO1 (rs4925) and GSTO2 (rs156697) in individual susceptibility to urinary bladder cancer. Methods  Case–control study consisting of 187 patients with histologically confirmed transitional cell carcinoma (TCC) of urinary bladder and 140 age- and gender-matched cancer-free controls was carried out. Genotyping of GSTO1 and GSTO2 was performed by polymerase chain reaction– restriction fragment length polymorphism (PCR–RFLP). Results  We found that carriers of mutant GSTO2*G/G genotype were at increased risk of the development of TCC (OR 2.6, 95 % CI 1.2–5.8, p = 0.041), while GSTO1 rs4925 polymorphism was not significantly associated with TCC risk (p  = 0.450). According to smoking status, smokers with GSTO2*G/G genotype had significantly higher risk of TCC of urinary bladder (OR 4.3, 95 % CI 1.6–11.2, p  = 0.003) compared to wild-type carriers with no smoking history. We further analyzed the effects of T. Djukic · T. Simic · T. Radic · M. Matic · M. Pljesa‑Ercegovac · S. Suvakov · V. Coric · A. Savic‑Radojevic (*)  Faculty of Medicine, Institute of Medical and Clinical Biochemistry, University of Belgrade, Pasterova 2, 11000 Belgrade, Serbia e-mail: [email protected] T. Pekmezovic  Faculty of Medicine, Institute of Epidemiology, University of Belgrade, Visegradska 26a, 11000 Belgrade, Serbia I. Novakovic  Faculty of Medicine, Institute of Biology and Human Genetics, University of Belgrade, Visegradska 26, 11000 Belgrade, Serbia D. Dragicevic  Clinic of Urology, Faculty of Medicine, Clinical Centre of Serbia, University of Belgrade, Resavska 51, 11000 Belgrade, Serbia

GSTO1/GSTO2 haplotypes on TCC risk, based on the linkage disequilibrium found for GSTO1 (rs4925) and GSTO2 (rs156697) (D′  = 0.309, p  = 0.001). The study subjects with GSTO1*C/GSTO2*G (GSTO1 wild-type/GSTO2 mutant) haplotype were at the highest risk of the development of transitional cell carcinoma of urinary bladder (OR 2.8, 95 % CI 1.5–5.2, p = 0.002). Conclusions  Our results indicate that GSTO1*C/GSTO2*G haplotype is associated with increased risk of TCC. The modifying effect of GSTO2*G/G genotype on individual susceptibility to TCC is more pronounced, when associated with smoking. Keywords  GSTO1 · GSTO2 · Polymorphism · Risk · Bladder cancer

Introduction Bladder cancer is the fourth most common cancer in men, estimated as the eighth leading cause of cancerrelated deaths in men [1]. Several environmental risk factors, including cigarette smoking and occupational exposure, are already recognized to be risk factors for bladder cancer. Many compounds known to be carcinogenic to uroepithelial cell are detoxified by cytosolic glutathione S-transferase (GST) family of enzymes [2]. It has been well established that several GST polymorphisms (GSTM1, GSTP1, GSTT1 and GSTA1) influence risk of bladder cancer [3–5]. Considering the unique role of newly discovered class omega (GSTO) in arsenic metabolism, several studies performed in Asian population showed association of common polymorphisms of GSTO1 and GSTO2, arsenic exposure and bladder cancer risk [6–8]. Moreover, Wang et al. [7] found a significant joint effect of cigarette smoking,

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alcohol consumption, arsenic and occupational exposures and GSTO1/GSTO2 diplotypes on risk of urothelial cancer. In Caucasians, there are no data regarding this association. It should be noted that GSTO1 also catalyses several reactions atypical for other GST, such as glutaredoxin activity, strongly influenced by common GSTO1 polymorphism. It has been shown that GSTO1 single nucleotide polymorphism (SNP) (rs4925, 419 C–A), causing alanine to aspartate substitution in amino acid 140 (*A140D) of GSTO1, exhibits altered deglutathionylation activity [9]. Since it has been shown that glutathionylation of proteins is involved in regulation of the cell cycle, apoptosis and drug response in cancer, polymorphic GSTO1 expression might have an impact on these processes [10]. The role of another GSTO class member, GSTO2, has started to emerge recently, especially with respect to cellular redox homeostatis. Key feature of GSTO2 is its ability to regenerate ascorbic acid, by exhibiting a strong dehydroascorbate reductase activity [11]. GSTO2 polymorphism (rs156697, 424 A–G), which causes an asparagine to aspartate substitution in amino acid 142 (*N142D), has been evaluated in terms of risk of various malignancies [7, 12, 13]. As we recently showed, GSTO1 and GSTO2 mutant gene variants were independent predictors of a higher risk of death among patients with muscle invasive bladder cancer, significantly influencing five-year survival. In addition, we found significant effect of GSTO2 polymorphism on the survival in the subgroup of patients who received chemotherapy [14]. Therefore, considering disturbances in redox balance of tumor uroepithelial cells and the recently described antiapoptotic role of GSTO1, analysis of the association of GSTO1 and GSTO2 polymorphisms and the risk of bladder cancer needs to be addressed [15, 16]. This has prompted us to assess the association of common GST omega polymorphisms with the risk of bladder cancer in Caucasians, as well as, their joint effect with cigarette smoking and occupational exposure.

Materials and methods Study subjects We enrolled 187 patients (142 men and 45 women, mean age 63.13 ± 10.41 years) newly diagnosed with transitional cell carcinoma (TCC) of urinary bladder from the Clinic of urology, Clinical centre of Serbia, Belgrade, after histopathological examination by board-certified pathologists. A total of 140 cancer-free controls, age and gender matched, were recruited. In the control group, there were 105 men and 35 women, mean age 62.16 ± 7.51 years. The study protocol was approved by the Ethical Committee of the Medical faculty, University of Belgrade, and the research

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was carried out in a compliance with the Declaration of Helsinki. After giving the informed consent, each participant was interviewed using a standard questionnaire. Smoking status was categorized into never and ever-smokers according to the answer to the following question: “Have you smoked at least 100 cigarettes in your life?” The amount of pack-years was calculated using the formula: pack-years = (cigarettes/ day ÷ 20) × (smoked years). The bladder cancer high-risk occupations were a priori defined and are listed elsewhere [17]. Patients were considered to be ‘exposed’ if they had occupied one or more of these jobs for ≥1 year in the past 10 years. GST genotyping Genomic DNA was isolated from the whole blood using the QIAGEN QIAmp kit (Qiagen Inc., Valencia, CA, USA). GSTO1 rs4925 and GSTO2 rs156697 polymorphisms were determined by PCR–RFLP method [18]. The primers used were GSTO1 rs4925 forward: 5′-GAA CTT GAT GCA CCC TTG GT-3′ and GSTO1 rs4925 reverse: 5′-TGA TAG CTA GGA GAA ATA ATT AC-3′. The presence of restriction site resulting in two fragments (186 and 68 bp) indicated *C/C wild-type homozygote, and if *C/A heterozygote incurred, it resulted in one more fragment of 254 bp. The primers used were GSTO2 rs156697 forward: 5′-AGG CAG AAC AGG AAC TGG AA-3′ and GSTO2 rs156697 reverse: 5′-GAG GGA CCC CTT TTT GTA CC-3′. The presence of restriction site resulting in two fragments (122 and 63 bp) indicated *G/G homozygote of polymorphic sequence, while if *A/G heterozygote incurred, it resulted in one more fragment of 185 bp. Statistical analysis Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS, version 15.0; SPSS Inc, Chicago, Illinois, USA). Selected characteristics among TCC cases and controls were compared using the χ2 test for categorical variables and the Student’s t test for continuous variables. Hardy–Weinberg equilibrium (HWE) was examined using a goodness-of-fit χ2 test to compare frequencies of observed genotype with that of expected genotype among control subjects. The extent of linkage disequilibrium (LD) between pairs of SNPs was examined using the SNPStats. The strength of LD was expressed in terms of D′ = D/Dmax. The individual haplotype and its frequencies were estimated using the implementation of the EM algorithm coded in the haplo.stats package. Effects of genetic polymorphisms of GSTO1 and GSTO2 on risk of bladder cancer were estimated by odds ratios (ORs) and 95 % confidence intervals (CIs) using the unconditional

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multivariate-adjusted logistic regression adjusted for age, gender, cigarette smoking and occupational exposure. The Bonferroni post hoc test was performed for locating differences between multiple groups.

bladder cancer risk of 2.6 (95 % CI 1.6–4.3, p  = 0.001). After stratification according to the pack-years, similar results were obtained. Patients who were occupationally exposed also had a significantly higher risk of bladder cancer (OR 2.4, 95 % CI 1.5–3.9, p = 0.001).

Results

Effects of GSTO1 and GSTO2 polymorphisms on bladder carcinoma risk

Characteristics of bladder carcinoma patients and controls The characteristics of bladder cancer patients and controls regarding smoking status and occupational exposure are presented in Table 1. As expected, the smoking prevalence and occupational exposure among patients were higher (75 and 43 %, respectively) than in controls (57 and 24 %, respectively). After adjustment for age, gender and occupational exposure, we found that the history of cigarette smoking was associated with significantly increased Table 1  Smoking status and occupational exposure in bladder cancer patients and controls Patients, n (%)

Controls, n (%)

Smoking  No 46 (25) 60 (43)  Yes 141 (75) 80 (57) Pack-years  0 46 (25) 59 (42)  1–29 49 (26) 24 (17)  ≥30 92 (49) 57 (41) Occupational exposure  No 106 (57) 106 (76)  Yes

81 (43)

34 (24)

OR (95 % CI)

p

1.0a 2.6 (1.6–4.3)

0.001

1.0a 3.0 (1.6–5.8) 2.2 (1.3–3.8)

0.001 0.004

1.0b 2.4 (1.5–3.9)

0.001

a

  Adjusted for age, gender and occupational exposure

b

  Adjusted for age, gender and cigarette smoking

Table 2  Bladder cancer risk by genetic polymorphisms of GSTO1 and GSTO2

a   Adjusted for age, gender, cigarette smoking and occupational exposure b  For GSTO2, genotyping was successful in 182 of 187 patients c   Significance at p ≤ 0.025 after Bonferroni correction

Genotype

The genotype distribution of GSTO1 and GSTO2 of patients and controls is presented in Table 2. For both polymorphisms, the more common allele was considered as referent, whereas the less common allele was assessed as the variant. In the control group, genotype frequencies of GSTO1 and GSTO2 were in Hardy–Weinberg equilibrium (p  = 0.066 and  0.510, respectively). The presence of mutant GSTO1 gene variant did not influence the risk of bladder cancer (OR 1.3, 95 % CI 0.6–3.1, p  = 0.450). However, individuals who carried both mutant GSTO2 alleles (*G/G) had significantly increased bladder carcinoma risk of 2.6 (95 % CI 1.2–5.8, p  = 0.041). We also tested the combined effect of GSTO1 and GSTO2 genotypes on bladder cancer risk. The effect of mutant GSTO2 genotype was slightly enhanced only if combined with at least one copy of wild-type GSTO1 (*C/C or *C/A) (OR 3.0, 95 % CI 1.2–7.9, p = 0.025) (Table 2). In a LD analysis, we found a D′ of 0.309 for GSTO1 rs4925 and GSTO2 rs156697 (p  = 0.001), showing a LD between these SNPs. Table 3 shows the results for haplotype analyses of GSTO1 rs4925 and GSTO2 rs156697 polymorphisms. The most prevalent haplotype among patients (49 %) and controls (57 %) is H1, consisting of GSTO1 (*C) and GSTO2 (*A) wild-type alleles. The results of haplotype analysis confirmed those obtained in analysis of combined GST omega genotypes: namely, the individuals who carried H4 haplotype, represented by one copy of Patients, n (%)

Controls, n (%)

OR (95 % CI)a

p

GSTO1 rs4925  *CC (wild type)  *CA (heterozygote)  *AA (mutant)

82 (44) 87 (46) 18 (10)

54 (38) 74 (53) 12 (9)

1.0 0.8 (0.5–1.3) 1.3 (0.6–3.1)

0.450

GSTO2 rs156697b  *AA (wild type)  *AG (heterozygote)  *GG (mutant)

85 (47) 69 (38) 28 (15)

79 (56) 50 (36) 11 (8)

1.0 1.4 (0.8–2.4) 2.6 (1.2–5.8)

0.041

142 (78) 22 (12) 12 (7)

122 (87) 6 (4) 7 (5)

1.0 3.0 (1.2–7.9) 1.9 (0.7–5.5)

0.025 0.208

6 (3)

5 (4)

1.4 (0.4–5.0)

0.609

Combined GSTO1/GSTO2c  *CC + *CA/*AA + *AG  *CC + *CA/*GG  *AA/*AA + *AG  *AA/*GG

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Table 3  Bladder cancer risk by haplotype of GSTO1 and GSTO2 Haplotype

GSTO1

GSTO2

Patients (%)

Controls (%)

OR (95 % CI)a

p

H1 H2 H3

*C *A *A

*A *A *G

49 17 16

57 18 17

1.00 1.3 (0.8–2.1) 1.2 (0.7–1.9)

0.360 0.580

H4

*C

*G

18

8

2.8 (1.5–5.2)

0.002

Global haplotype association p value is 0.01 a

  Adjusted for age, gender, cigarette smoking and occupational exposure

Table 4  Combined effect of smoking/occupational exposure and GSTO1/GSTO2 genotype on bladder cancer risk

a

  Significance at p ≤ 0.025 after Bonferroni correction b

  Adjusted for age, gender and occupational exposure c

 For GSTO2, genotyping was successful in 182 of 187 patients d

  Adjusted for age, gender and cigarette smoking

Patients, n (%)

Controls, n (%)

OR (95 % CI)

pa

GSTO1/smoking status *CC + *CA/non-smokers *CC + *CA/smokers *AA/non-smokers *AA/smokers

39 (21) 129 (69) 6 (3) 13 (7)

52 (37) 76 (54) 4 (3) 8 (6)

1.0b 2.6 (1.5–4.7) 2.6 (0.8–8.4) 2.3 (0.5–10.2)

0.001 0.098 0.282

GSTO2/smoking statusc *AA + *AG/non-smokers *AA + *AG/smokers *GG/non-smokers *GG/smokers

36 (20) 118 (65) 8 (4) 20 (11)

57 (41) 72 (51) 3 (2) 8 (6)

1.0b 2.8 (1.6–4.9) 3.4 (0.8–14.9) 4.3 (1.6–11.2)

0.001 0.103 0.003

GSTO1/occupational exposure *CC + *CA/non-exposed *CC + *CA/exposed *AA/non-exposed *AA/exposed

92 (49) 77 (41) 14 (8) 4 (2)

97 (69) 31 (22) 9 (7) 3 (2)

1.0d 2.7 (1.6–4.5) 1.9 (0.8–4.7) 1.7 (0.4–8.3)

0.001 0.172 0.519

GSTO2/occupational exposurec *AA + *AG/non-exposed *AA + *AG/exposed *GG/non-exposed

85 (47) 69 (38) 17 (9)

97 (69) 32 (23) 9 (6)

1.0d 2.5 (1.5–4.3) 1.9 (0.8–4.8)

0.001 0.127

*GG/exposed

11 (6)

2 (2)

6.0 (1.3–29.2)

0.025

wild-type GSTO1 (*C) and mutant GSTO2 (*G), had significantly increased risk of 2.8 for bladder cancer (95 % CI 1.5–5.2, p = 0.002) (Table 3).

increased risk of developing bladder cancer (95 % CI 1.6– 11.2, p  = 0.003) compared to wild-type carriers with no smoking history (Table 4).

Joint effect of cigarette smoking, occupational exposure and GSTO1 and GSTO2 risk genotypes on bladder carcinoma risk

Discussion

The 2.6-fold risk conferred by association of GSTO1 genotype and smoking (95 % CI 1.5–4.7, p = 0.001) or occupational exposure (OR 2.7, 95 % CI 1.6–4.5, p  = 0.001) in carriers of either *C/C or *C/A GSTO1 genotype did not differ from that obtained for smoking or occupational exposure alone (OR 2.6, 95 % CI 1.6–4.3, p = 0.001; OR 2.4, 95 % CI 1.5–3.9, p  = 0.001; respectively). However, the combined effect of smoking and the risk GSTO2*G/G genotype was much stronger than that of smoking alone. Smoker carriers of GSTO2*G/G genotype had 4.3-fold

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Our results showed that homozygous carriers of mutant GSTO2 gene variant are at higher risk of urinary bladder cancer in comparison with carriers of at least one wild-type GSTO2 allele. Furthermore, haplotype analysis pointed out that carriers of H4 haplotype (presence of GSTO1 wildtype *C and mutant GSTO2*G) are running the highest risk of developing bladder cancer. This risk conferred by GSTO2 polymorphism was even higher if mutant homozygous genotype was associated with cigarette smoking. Our data on increased risk in carriers of mutant GSTO2 genotype are consistent with previous results, but the

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question arises which functions of this enzyme support this [7, 8]. Since GSTO2 is an enzyme with the most prominent dehydroascorbate reductase (DHAR) activity in humans, our findings are biologically compatible with its role in regeneration of dehydroascorbate [6]. Moreover, it was speculated that DHAR activity of GSTO2 might be influenced by rs156697 polymorphism [19]. Therefore, it may be speculated that low enzyme activity in subjects with both mutant GSTO2 alleles would presumably result in deficient DHAR activity and lower ascorbic acid level in the bladder. In the view of the role of oxidative stress in the biology of urothelial tumors, altered activity of GSTO2 protein might result in inter-individual differences in capacity to scavenge and detoxify reactive species produced by smoking or occupational exposure in TCC patients [15]. Indeed, we found that smoker carriers of GSTO2*G/G genotype were at approximately fourfold increased TCC risk. It is important to note that in addition to its antioxidant role, ascorbic acid (vitamin C) is also involved in regulation of hypoxia-inducible factor (HIF)1, a transcription factor that regulates many genes responsible for tumor growth, energy metabolism and apoptosis [20]. In that context, recent data speculates that vitamin C-dependent inhibition of the HIF pathway may provide additional approach for controlling tumor progression and inflammation [21]. On the other hand, the results of increased risk in occupationally exposed patients, carriers of GSTO2*G/G genotype must be taken with caution, considering the small number of controls in the subgroup of exposed GSTO2*G/G carriers. Although GSTO1 rs4925 was not significantly associated with increased risk of bladder cancer, we analyzed the effects of GSTO1 and GSTO2 haplotypes on bladder cancer risk, based on the LD found for GSTO1 (rs4925) and GSTO2 (rs156697): namely, carriers of H4 haplotype had a higher TCC risk, meaning that carriers of GSTO1 wildtype *C allelic variant and GSTO2 mutant *G allelic variant are at the highest risk of TCC. In the course of bladder cancer development, complex changes occur in terms of cellular redox regulation, shifting toward more reduced state of molecules during tumor progression [15]. In this process, post-translational modification of proteins in which glutathione is reversibly added (glutathionylation) and removed (deglutathionylation) to proteins functions as molecular switch, which tunes the activity of many proteins involved in tumor growth [10]. Recent work of Board et al. [9] pointed out several polymorphic variants of GSTO1 that could potentially impact regulation of glutathionylation, indicating that GSTO1 wild-type *C variant codes for protein with the highest deglutathionylation activity. Since glutathionylation can influence both protein structure and function, the difference in activity and the potential difference in protein specificity between the allelic variants of

GSTO1 could provide a plausible mechanism to explain the associations between this genetic polymorphism and a range of disorders [22, 23]. It might be speculated that the presence of GSTO1*C allelic variant might also favor deglutathionylation of protooncogenic proteins involved in the promotion of bladder tumors and thus contribute to overall risk in carriers of H4 haplotype. Among oncogenic proteins that are subject to regulation by this molecular mechanism is antiapoptotic GSTP1 enzyme, with welldefined role in TCC promotion and progression [15, 24]. Taken together, the presence of H4 haplotype, comprising high deglutathionylation and low DHAR activity, might underlie the increased TCC risk among our patients. Acknowledgments  This work was supported by the Grant 175052 from the Serbian Ministry of Education, Science and Technological Developmental. Conflict of interest  The authors declare that they have no conflict of interest. Ethical standard  All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.

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