Introduction

Transitional cell carcinoma of the urinary tract (UT) is one of the most common malignancies seen worldwide.1 Within the UT, urothelial carcinoma (UC) of the urinary bladder accounts for the majority of them (90–95%), while only approximately 5 to 10% of the cases occur in the upper UT.2 Comparative genomic hybridization studies have shown that certain chromosomal areas are frequently altered during the progression of bladder cancer (BC).345 This makes it necessary to fully understand the molecular mechanisms of the pathogenesis of BC.

The human gene encoding the type B urea transporter protein, namely, the SLC14A1, has been mapped to chromosome 18q12.3, adjacent to another urea transporter, namely, SLC14A2, locus (Fig. 1).6 This protein is known to facilitate the passive transport of urea, which is responsible for establishing an osmotic gradient within the inner medulla and thereby preventing intracellular toxicity within the extrarenal cells.7 It is interesting to note that SLC14A1 gene polymorphisms have been known to be related to carcinogenesis of the bladder in humans.8910 Genetic variations in SLC14A1 could provide new etiological insights into carcinogenesis of the bladder.

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Fig. 1: Mechanism of upregulation and downregulation of the SLC14A1 gene.

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Use of tobacco products, chewing and smoking, is an established risk factor for BC in both men and women.1112 Previous studies have estimated the risk in smokers to be three times.1314 The present study aimed to assess the relationship between BC and SLC14A1 gene polymorphisms (rs17674580) in patients with image and histologically confirmed BC. Our secondary objective was to assess if the use of tobacco products in these patients further increased the risk of BC.

Patients and Methods

This prospective study was conducted with the approval from the university's ethical committee (KAHER/EC/21–22/003). All patients aged ≥18 years with an image (ultrasonography/computed tomography) and histologically confirmed BC formed the study group. Age- and gender-matched controls aged ≥18 years, genetically unrelated, formed the controls. Patients with a previous history of other cancers, cancer metastasized to the bladder from another origin, and a previous history of radiotherapy were excluded from the study. The participating patients signed a written informed consent to be part of the study.

Demographic data were obtained by interviewing each one of the participants. Individuals who smoked once a day for more than 5 years were defined as smokers. Similarly, patients who chewed tobacco for more than 5 years were defined as tobacco users. The individuals who had never smoked in their lifetime were regarded as nonsmokers. After the interview, a 2-mL blood sample was collected into coded ethylenediaminetetraacetic acid (EDTA) vials and stored at –80°C.

Results

During the study period, from August 2020 to August 2023, a total of 107 patients (84 males and 23 females) with image and histologically confirmed BC formed the study group. The mean age of the patients with BC was 58.47 ± 4.5 years and that of the controls was 60.01 ± 12.5 years.

In patients with BC, 28 (26.2%) showed no polymorphisms (rs17674580) of the SLC14A1 gene, whereas 79 (73.8%) patients showed polymorphisms in the gene and the types of mutations. In comparison, 54 (51.5%) of the controls showed no polymorphisms in the gene. The different types of polymorphisms (rs17674580) are seen within the SLC14A1 gene. Heterozygous polymorphisms (CT) were noted in 46 (42.9%) patients, whereas homozygous polymorphisms were noted in 33 (30.9%) patients. The ORs were 2.772 (1.459–3.247) and 3.349 (1.610–6.922), respectively. The subgroup analysis of the association between polymorphisms of SLC14A1 in relation to tumor grade and stage is shown in Table 1. The relation between polymorphisms of SLC14A1 and the use of tobacco is shown in the graph in Fig. 2.

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Fig. 2: Association between SLC14A1 and exposure to smoking and tobacco use.

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Discussion

Single nucleotide polymorphism (SNP) is a variation that occurs in a genetic sequence and affects only one of the basic building blocks—adenine (A), guanine (G), thymine (T), or cytosine (C)—in a segment of a DNA molecule. These variations occur in more than 1% of a population.14 Roughly 90% of the genetic variation between humans is the result of SNPs. Studies have shown that both the nuclear and membranous SLC14A1 proteins play the role of tumor suppression through several signaling pathways in both in vitro and in vivo clinical specimens of UC.10 Another study on meta-analysis of genome-wide association studies of urinary bladder UCs recently reported that SLC14A1-rs10775480, a variant at intron 6, is highly associated with susceptibility in BC.15

To determine whether SLC14A1 gene polymorphisms increase the risk of invasive BC, we compared genotype distribution between tumor grade and stage and discovered significant heterogeneity in the effect of the combined CT/TT genotype for stage-wise comparison was (OR for CT/TT (Cytosine, Thymine/Thymine, Thymine) = 1.115, 95% CI = 0.460–2.705) and for grade-wise distribution (OR for CT/TT = 1.056, 95% CI = 0.436–2.558). When low- and high-risk non–muscle-invasive bladder cancer (NMIBC) patients were compared, the heterozygous genotype (CT) of SLC14A1C/T was shown to be linked with BC susceptibility. Significant risks of BC susceptibility were found among North Indians, with a threefold risk of BC susceptibility in the case of variant TT genotype and a 1.5-fold risk at the variant T allele level, which were nearly consistent with our findings. In a Genome wide association study (GWAS) conducted by Rafnar et al,8 the highest signal was provided by an SNP in intron 3, rs1764580, which demonstrated considerable risk in both the discovery and follow-up groups. Moreover, a study conducted by Chan et al10 concluded that clinical studies, animal models, and in vitro indicators provided solid evidence that the SLC14A1 gene was a novel tumor suppressor in UCs.

In India, the use of tobacco is done in several ways. Apart from cigarette smoking, people in rural areas smoke rolled dried tobacco leaves called bidis. Tobacco is also used as a component along with beetle leaves or mouth freshener (locally called as gutka) and chewed. Some also use raw tobacco or mixed with lime paste and kept in the buccal crevices over a long time. The risk of these tobacco products has not been studied in relation to BC.8 Furthermore, our data showed that the combined CT/TT genotype had a larger impact on tobacco exposure in smokers (OR for CT/TT = 1.45, 95% CI = 0.567–3.707) than in nonsmokers (OR for CT/TT = 0.236, 95% CI = 0.101–0.551; Fig. 2). Previous studies have shown more than threefold increased risk of BC in current tobacco smokers and twofold increased risk in former smokers compared with nonsmokers.1617 A 2016 meta-analysis18 of 89 observational studies from the past 50 years reported that smoking was associated with the highest risk of BC compared with any other environmental or occupational risk factor. Studies19 of patients with NMIBC, which represents approximately 75% of cases, have reported mixed results on the impact of lifetime smoking behavior on cancer outcomes. Some studies have found that current, heavy, and long-term prediagnosis smoking was associated with worse recurrence-free survival, while other studies have found no such association.20

Finally, our findings establish the role of SLC14A1C/T as major contributing genetic factors in the process of bladder carcinogenesis in North Karnataka, which is supported by significant findings with clinical parameters such as smoking and tumor grade and stage of the patients, which may have important clinical implications. The limitation of this study was a smaller sample size, which lowered the statistical power for discovering the interaction effects. More research with larger sample sizes and various ethnicities is needed to corroborate our findings.

Conclusion

Urothelial BC is a common UT tumor. It is associated with several risk factors including the use of tobacco. SLC14A1 is a human gene encoding the type B urea transporter protein. Gene polymorphisms associated with this gene are associated with patients with BC. The use of tobacco products further increases the risks in these patients.