Genetic Susceptibility And Mechanisms Of Bladder Cancer In Chinese Populations

Bladder cancer is one of the common cancers worldwide, and more than 90% of bladder cancers are transitional cell carcinomas. In China, the incidence and mortality of bladder cancer have been the first in the urinary cancers. Bladder cancer is a disease with a complex, multifactor etiology, including interactions between genetic makeup and environmental factors. Epidemiologic studies have shown that the development of bladder cancer is associated with tobacco smoking and occupational exposure. Although many people have been exposed to the same risk factors, only a fraction of exposed individuals develop bladder cancer in their lifetime, suggesting that there is inter-individual variation in genetic susceptibility to bladder cancer.With the completion of Human Genome Project (HGP), it is now known that genetic variations play an important role in gene structure and function, which could change the biological functions of genes. Single nucleotide polymorphisms (SNPs) are the most common types of genetic variations. In the study of molecular epidemiology and genetics, SNPs have been widely used for gene mapping of molecular markers, by comparing the normal and patients of SNPs to locate and identify specific disease-related genes. SNPs in genes can impact the gene structure and repair enzyme function, leading to increase risk of cancer. Many studies have suggested that the genes in DNA repair, apoptosis, and folate metabolism were associated with bladder cancer risk. Most functional SNPs singly or jointly contribute to the risk of bladder cancer, and possibly have gene-gene and gene-environment interactions. Recently, two genome-wide association studies (GWAS) in European populations had identified the bladder cancer susceptibility loci. However, there are no studies conducted in Chinese populations to replicate these findings.Now, with the knowledge of human genome and genetics, as well as advances in risk of bladder cancer; (2) explore the association between genetic variants of genes in DNA repair, apoptosis, and folate metabolism and bladder cancer risk; (3) validate the identified bladder cancer susceptibility loci from Caucasians in Chinese populations. Our study would reveal the biological mechanism of bladder cancer, and search for the biomarkers for genetic susceptibility to bladder cancer for the individualized treatment and prevention.Part I Genetic susceptibility and mechanisms of bladdercancer based on candidate genesChapter I Association and functional study between geneticvariants in MDM2 and bladder cancer riskThe p53 tumor suppressor mutation has been detected in more than 50% of human tumors. Genetic variants of crucial genes in p53-mediated pathway will contribute to the individual’s cancer susceptibility. MDM2 is believed to regulate the p53 level in modulating DNA repair, cell cycle control, cell growth, and apoptosis. Therefore, genetic variants in MDM2 may be associated with the development and progression of bladder cancer.To validate the hypothesis, we firstly conducted a case-control study of 234 bladder cancer cases and 253 cancer-free controls, using the haplotype-based tagging SNPs (tagSNPs) approach involving 13 common SNPs initially identified in 100 healthy control subjects. We then examined the functionality of promoter SNPs in MDM2 by using the report gene assay, electrophoretic mobility shift assay (EMSA), and quantitive real-time RT-PCR, and so on.As a result, we found that the 1797C>G polymorphism in the MDM2 promoter region was associated with risk of bladder cancer. Specifically, compared with the 1797CC genotype, the 1797GG genotype had a significantly increased risk of bladder cancer (OR = 2.45, 95%CI = 1.02-5.72). EMSA indicated that the 1797C to 1797G transition within the C/EBPαcore sequence greatly enhanced the C/EBPαbinding affinity to the promoter region, leading the increased transcriptional activity. Additional experiments with tumor tissues revealed that the individuals with the 1797G allele had significantly increased levels of the MDM2 mRNA and protein expression.These data suggested that the novel MDM2 promoter 1797C>G polymorphism may contribute to the development and progression of bladder cancer, which could be a marker for genetic susceptibility to bladder caner in Chinese populations.Chapter II Association and functional study between geneticvariants in XPF and bladder cancer riskDNA repair plays an important role in maintaining genome stability. The variation in individual DNA repair capacity (DRC) was determined by crucial genes in the DNA repair pathway. Nucleotide excision repair (NER) is a DNA repair pathway, which is most close to tobacco-induced damage repair. XPF, as the key NER gene, not only has an endonuclease activity, but also plays as a scaffolding protein participating in NER.We selected three tagSNPs from the HapMap and dbSNPs database for the Chinese and genotyped them in a two-stage case-control study (234 cases and 254 control subjects in the first-set, and 130 cases and 150 control subjects in the second-set) to evaluate the association and further examined the functionality of the novel promoter polymorphism. Furthermore, in the second-set study, follow-up was conducted in 79 superficial bladder cancer patients with 36 months, in order to investigate the association between the functional XPF SNPs and prognosis of bladder cancer.We found that the rs744154 polymorphism in intron 1, which was in linkage disequilibrium (LD) (D’= 1.00, r~2 = 1.00)with the -357A>C (rs6498486) in the promoter region, was associated with a protective effect on bladder cancer risk. Functional study revealed that the -357C allele decreased binding ability of transcriptional factors to the XPF promoter. The vector construct containing the -357A allele had higher luciferase expression than that of the -357C allele. The transcription factor binding site containing the -357C allele also decreased expression levels of XPF mRNA and protein in bladder cancer tissues. Furthermore, patients with the -357C allele had shorter overall recurrence-free survival time than did patients with the -357A allele.In conclusion, our results suggested that the XPF promoter -357A>C polymorphism may regulate the expression of XPF and thereby contribute to the etiology of bladder cancer. Our epidemiologic and functional variant study might provide a“proof-of-principle”approach for studying candidate genes in susceptibility to bladder cancer and other cancers.Part II Association study on the genetic susceptibility ofbladder cancer based on candidate biological pathwaysChapter I Association study between functional genetic variantsIn XRCC1, APE1 and ADPRT and bladder cancer riskHuman have a variety of DNA damage repair mechanisms, including base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), and so on. BER pathway is one of the important mechanisms responsible for the removal of the oxidative DNA damage. Several crucial genes, such as XRCC1, APE1, and ADPRT, are involved in the BER pathway. Studies have reported that genetic variants in these genes may affect BER functions, leading the increased risk of cancer. We hypothesized that the functional polymorphisms in XRCC1, APE1, and ADPRT are associated with risk of bladder cancer.To test the hypothesis, we genotyped the XRCC1 -77T>C, Arg194Trp, Arg280His, Arg399Gln, APE1 -656T>G, Asp148Glu, ADPRT -442G>A and Val762Ala polymorphisms using PCR-RFLP method in a hospital-based case-control study of 234 bladder cancer cases and 253 cancer-free controls frequency-mathed to cases by age (±5 years) and sex. We investigated their associations with risk of bladder cancer.We found an increased risk of bladder cancer associated with the XRCC1 194Trp/Trp and 280Arg/His genotypes (OR = 3.90, 95%CI = 1.69-8.98 for 194Trp/Trp and OR = 2.53, 95%CI = 1.67-3.83 for 280Arg/His) compared with the 194Arg/Arg and 280Arg/Arg genotypes, respectively. In contrast, the APE1 -656GG genotype was associated with a decreased risk of bladder cancer (OR = 0.57, 95%CI = 0.33-0.98) compared with the -656TT genotype. When we evaluated these eight polymorphisms together, we found that the combined genotypes with 9-13 risk alleles (i.e., XRCC1 -77T, 194Trp, 280His, 399Arg, APE1 -656T, 148Asp, ADPRT -442A and 762Ala) were associated with an increased risk of bladder cancer (OR = 2.25, 95%CI = 1.48-3.40) compared with those with 3-8 variants. Further stratification analysis showed that this increased risk was more pronounced among subgroups of never smokers (OR = 3.07, 95% CI = 1.71-5.52), never drinkers (OR = 3.39, 95% CI = 1.91-6.02), and males (OR = 2.21, 95% CI = 1.38-3.54).These findings suggested that the XRCC1, APE1 and ADPRT polymorphisms, alone and in combination, may contribute to susceptibility to bladder cancer.Chapter II Association study between functional genetic variantsin FAS, FASL and CASP8 and bladder cancer riskT lymphocytes can be triggered into an apoptosis process known as activation-induced cell death (AICD) activated by tumor-associated or tumor-specific antigens, which are important for the living development, immunity and homeostasis maintaining. Most individuals have genetic variants in the death receptor pathway genes, which may affect the individual’s immune status and thus influence the susceptibility of cancer. We hypothesized that genetic variants in the FAS, FASL, and CASP8 genes are associated with bladder cancer risk.In our on-going case-control study, we genotyped the functional polymorphisms FAS -1377G>A, -670A>G, FASL -844T>C and CASP8 -652 6N ins/del using PCR-RFLP method. Unconditional univariate and multivariate logistic regression analyses were performed to investigate the gene-gene and gene-environment interactions.As a result, we found a statistically significantly increased risk of bladder cancer associated with the FASL -844CC genotype (OR = 1.51, 95%CI = 1.03-2.23). Consistently, the FAS haplotypes with 2-4 risk alleles (i.e., -1377A and -670A) were associated with an increased risk of bladder cancer compared with those with 0-1 variants (OR = 2.14, 95%CI = 1.10-4.16). Furthermore, when we evaluated these three polymorphisms together, we found that the combined genotypes with 4-6 risk alleles (i.e., FAS -1377A, -670A and FASL -844C) were associated with an increased risk of bladder cancer (OR = 1.58, 95%CI = 1.07-2.34) compared with those with 1-3 variants. Meanwhile, A significantly decreased risk of bladder cancer was found for the CASP8 -652 6N ins/del (OR = 0.72, 95%CI = 0.53-0.99) and del/del (OR = 0.37, 95%CI = 0.18-0.77) genotypes. Furthermore, a significant additive interaction between the CASP8 polymorphism and tobacco smoking on bladder cancer risk was observed (P < 0.05).In this study, we selected the crucial genes in the death pathway to investigate the association between the functional polymorphisms and bladder cancer risk, further providing the evidence that genetic variants in apoptosis-related genes are involved in the etiology of bladder cancer and function of gene polymorphisms in the apoptosis pathway. Chapter III Association study between functional geneticvariants in MTHFR and MS and bladder cancer riskFolate deficiency and the enzyme activation changes due to the genetic variants in MTHFR and MS can affect cancer susceptibility by affecting DNA methylation and synthesis. We hypothesized that the MTHFR 677C>T, 1298A>C, and MS 2756A>G polymorphisms are associated with risk of bladder cancer.In a case-control study of 239 bladder cancer cases and 250 cancer-free controls, we found that the MTHFR 677TT genotype was associated with a statistically significantly increased risk of bladder cancer compared with the 677CC genotype (OR = 2.06, 95%CI = 1.16-3.64). Furthermore, the TA haplotype was associated with a significantly increased bladder cancer risk (OR = 1.38, 95% CI = 1.05-1.81) compared with the most common haplotype CA. We also found that the combined genotypes with 4-6 risk alleles (i.e., MTFHR 667T, 1298A and MS 2756G) were associated with an increased risk of bladder cancer (OR = 1.62, 95%CI = 1.03-2.53), and this increased risk was more pronounced among subgroup of older subjects (OR = 1.71, 95%CI = 1.03-2.83). A meta-analysis of MTHFR polymorphisms did not show a significant association between the MTHFR polymorphisms and bladder cancer risk in Caucasians.In the study, we selected the key genes in the folate metabolism to investigate the functional polymorphisms and bladder cancer risk. Our findings suggested that the MTHFR 677C>T polymorphism appears to contribute to bladder cancer risk. However, the meta-analysis showed contrast results, which may be due to the difference in ethnic background and environmental exposure. Thus, further larger and more diverse ethnic groups should be warranted to confirm these findings.Part III Replication study on the genetic susceptibility ofbladder cancer based on GWASRecently, Kiemeney et al. reported the first bladder cancer GWAS in European populations, they identified two common sequence variants, rs9642880 on chromosome 8q24 and rs710521 on chromosome 3q28, that were associated with bladder cancer risk. Subsequently, Wu et al. identified additional susceptibility loci in the second GWAS on bladder cancer, and found a significant association of rs2294008 in exon 1 of PSCA with bladder cancer risk. We hypothesized that these variants are also associated with risk of bladder cancer in Chinese populations.To validate the rs9642880 and rs710521 in the first GWAS, we genotyped them in a two-stage case-control study using PCR-RFLP method. We further examined the expression of MYC using QuantiGene and immunohistochemistry in bladder cancer tissues with different genotypes. To test the results from the second GWAS, we conducted a case-control study of 581 bladder cacner cases and 580 cancer-free controls, genotyped the rs2294008 using Taqman method.We found that the rs9642880 G>T, but not the rs710521 A>G polymorphism, was associated with an increased risk of bladder cancer. Compared with the rs9642880 GG genotype, the GT/TT genotypes were associated with an OR of 1.65 (95%CI = 1.25-2.17), and this risk was more pronounced in young men and for low-risk tumors. Additional experiments revealed that the rs9642880 GT/TT genotypes were associated with enhanced levels of both MYC mRNA and protein in bladder cancer tissues. However, further studies between rs710521 on 3q28 and bladder cancer susceptibility were warranted to validate. As for rs2294008, a significant increased risk of bladder cancer was found for rs2294008 CT/TT genotypes (OR = 1.38, 95%CI = 1.09?1.75) compared with the CC genotype.