Genetic Associations Between The Polymorphisms Of P53 Pathway Genes And Susceptibility To Hepatocellular Carcinoma And Nasopharyngeal Carcinoma

Carcinogenesis is a long-term multistage process with the involvement of genetic and environmental factors. With the completion of Human Genome Project and International HapMap Project, the identification of susceptibility genes contributing to cancers will be of great importance for the prevention and treatment of cancers. The genes of p53 pathway have a direct correlation with the risk of onset and progression of tumors. It has been found that the single nucleotide polymorphism (SNP) in the genes of p53 pathway can influence p53 activity and contribute to the development of cancers.Previous epidemiological studies have suggested that virus infection and lifetime exposure to mycotoxin aflatoxins are two major risk factors for hepatocellular carcinoma (HCC) in China. However, not all the individuals exposed to the risk factors will progress to HCC eventually. Nasopharyngeal carcinoma (NPC) is a serious health problem with an incidence rate ranging from 15 to 50/100000 in southern China, which is almost 100-fold higher than that in the Caucasian population. Now, there is increasing evidences indicating that host genetic factors may play important roles in these disease progressions. The identification of susceptibility genes contributing to these disorders may help to clarify the pathogenesis and improve the prevention and treatment of these malignancies.The genes of p53 pathway have a direct correlation with risk of onset and progression of tumors. It has been found that some SNPs in the genes of p53 pathway can influence p53 activity and contribute to the development of cancers. Previously, we have assessed the association between two functional polymorphisms of p53 pathway (MDM2 SNP309 and p53 codon72) and HCC or NPC risk. Our results indicated that these polymorphisms did not significantly increase the risk of HCC. However, the functional polymorphism of MDM2 SNP309 was associated with NPC risk. The p53 pathway consists of many genes. Thus, in addition to the SNPs above, it is expected that the other polymorphisms in this pathway are also possibly associated with the onset and progression of HCC and NPC. These SNPs include the p53 codon47, PTEN IVS4+109ins5 and -9C→G, p73 G4C14- A4T14 and five SNPs (rs3803300, rs1130214, rs3730358, rs2498799 and rs2494732)in the AKT1 gene. To identify whether these polymorphisms were associated with HCC or NPC risk, we selected 434 patients with HCC, 593 patients with NPC and 480 controls to perform the case-control association studies. Genotyping was performed by direct DNA sequencing for the p53 codon47 polymorphism, and by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for the PTEN IVS4+109ins5 and -9C→G, p73 G4C14-A4T14 and five SNPs (rs3803300, rs1130214, rs3730358, rs2498799 and rs2494732) in the AKT1 gene. The association between these polymorphisms and HCC or NPC risk was evaluated while controlling for confounding factors.The association study results showed that in HCC overall sample, the AKT1 SNP1 was not associated with susceptibility to HCC. However, further analysis stratified by HBV status showed that in non-HBV carriers, compared with the A/A genotype, the G/G+G/A genotype was associated with increased risk of HCC(OR = 1.93, 95% CI = 1.21-3.09, P = 0.006). The haplotype analysis showed that many multi-SNP haplotypes also showed significant association with HCC, and they all shared a common core haplotype that extended from SNP1 to SNP2. In further stratification analysis, the association remained significant. In NPC, only the p73 G4C14-A4T14 polymorphism was associated with susceptibility to NPC, and G4C14-A4T14 AT allele was associated with increased risk of NPC.It was reported that the AKT1 played an important role in tumorigenesis. However, the role of AKT1 polymorphisms in susceptibility to HCC and NPC has not been investigated before. So we furthered our study by carrying out the functional research on SNP1. By electrophoretic mobility shift assays(EMSA) and reporter gene assay, we demonstrated that SNP1 was not a functional regulatory polymorphism. To identify the functional polymorphism in the AKT1 gene, we screened SNPs in approximately 40kb region of AKT1 gene and constructed linkage disequilibrium (LD) block map. The screening panel included 96 unrelated individuals randomly selected from the 480 controls of Guangxi population. Meanwhile, we downloaded the SNP genotypes data of HapMap Beijing population around the 115 Kb of AKT1 gene and constructed the LD block map. We found that the LD block map of Guangxi population was similar to that of Beijng population. Furthermore, there were two genes near AKT1 gene that had a direct correlation with risk of onset and progression of tumors. Then, we selected 17 tag SNPs from the HapMap Beijing population in AKT1-SIVA-ADSSL1 gene region by Haploview software and then carried out the association study. The association study results showed that the hSNP2 was associated with susceptibility to HCC in overall sample. Further analysis stratified by HBV status showed that in non-HBV carriers, compared with the C/C genotype, the G/G+G/C genotype was associated with increased risk of HCC. By functional assays including the EMSA , real-time quantitation of AKT1 mRNA and immunohistochemical staining analysis, we demonstrated that the hSNP2 was a functional regulatory polymorphism in the AKT1 promoter. This polymorphism could regulate the expression of AKT1 gene in an allele-specific manner and the G allele was related with increased expression of AKT1 gene.In short, through the study of genetic association between p53 pathway polymorphisms and susceptibility to HCC and NPC, we have found that hSNP2 and SNP1 of AKT1 was associated susceptibility to HCC, and p73 G4C14-A4T14 polymorphism was associated with susceptibility to NPC. Our findings may help to clarify the pathogenesis and improve the prevention and treatment of HCC and NPC.