.ape1/ref-1 Gene Promoter Region Polymorphism Analysis And Functional Studies Of The Genetic Susceptibility To Lung Cancer And Glioma

Lung cancer is among the most common and life-threatening malignancies worldwide, with the highest incidence and mortality rates and an overall 5-year survival rate of<15%. Although tobacco smoking has long been established as the predominant risk factor for lung cancer, genetic variation also contribute to inter-individual susceptibility to lung cancer, suggesting the pivotal role of genetic determinants including the gene-environment interaction in lung cancer etiology.Apurinic/apyrimidinic endonuclease 1/Redox effector factor-1 (APE1/Ref-1) is a ubiquitous multifunctional protein that possesses both DNA repair and redox regulatory activities. In view of its unique multiple functions, it is not surprising that APE1/Ref-1 plays a critical role in many biological processes, such as cell proliferation and growth, cell cycle control, apoptosis, and angiogenesis.Although it was originally identified as a DNA repair enzyme, accumulating evidence supports a role of APE1/Ref-1 in tumor development. Previous studies about APE1/Ref-1 polymorphisms and their associations with lung cancer susceptibility mainly focused on the nonsynonymous single nucleotide polymorphism (nsSNP), Asp148Glu, in the coding region; however, most findings have been inconsistent, and the functional relevance of this nsSNP has not been elucidated. To investigate association between APE1/Ref-1 polymorphisms and lung cancer risk in Chinese populations, we first genotyped three variants of APEl/Ref-1 among 500 patients with incident lung cancer and 517 age- and sex-matched cancer-free control participants to screen for any risk-associated SNPs.As a result, we found that the allele distribution of rs1760944, a-141 T-to-G variant in the promoter, is significantly different between case patients and control subjects (P=0.02). The significance remained after applying 100,000-time permutation tests (P=0.0489). Further logistic regression analyses revealed that the homozygous-141GG genotype was associated with decreased risk of developing lung cancer [adjusted odds ratio (OR)=0.62; P=0.043]. No altered risk was observed for the prior reported Asp148Glu polymorphism (rsl 130409). However, the joint effects of rs1130409 variant allele and rs1760944 G allele reduced lung cancer risk in the current smokers in a significant locus dose-response manner.Then we performed another independent case-control study of 572 lung cancer patients and 547 control participants to confirm this finding and similar results were obtained. Combined data from the two studies comprising a total of 1,072 lung cancer patients and 1,064 cancer-free controls generated a more significant association (adjusted OR=0.63,95%CI 0.48-0.81; P=0.002). Trend tests showed that the protective effect followed a trend of increasing magnitude by number of G alleles (P for trend=0.00035). Subjects possessing at least one copy of the G allele had～23% reduction in risk of lung cancer (adjusted OR=0.77,95%CI 0.64-0.93; P=0.006). We also observed a statistically significant interaction between rs1760944 and smoking status (P interaction=0.031), and the protective effect of the GG genotype against lung cancer was more evident in the current smokers.Gliomas are the most common primary tumors in the central nervous system, with high rates of incidence, recurrence and mortality. In view of gliomas are characterized of uncontrolled rapid cell proliferation, reduced apotosis rate, local invasion and angiogenesis, which are all relevant to APE1/Ref-1's function and since the pathogenesis of different tumor types has certain similarities, we speculate that APE1/Ref-1 rs1760944 may also confer individual susceptibility to glioma. To test this hypothesis, we investigated the association between rs1760944 and glioma risk in a case-control study of 241 glioblastoma cases,284 astrocytoma cases (glioblastoma not included),241 other glioma cases, and 824 healthy controls.We observed statistically significant differences in allele and genotype distributions of rs1760944 between glioblastoma patients and control subjects (P= 0.03 and 0.041, respectively). Consistent with the protective effect of the -141 G allele inlung cancer, individuals with the homozygous -141GG genotype exhibited 42% reduced risk of glioblastoma (adjusted OR=0.54,95%CI 0.34-0.87), compared with the TT homozygotes.The protection followed a trend of increasing magnitude by number of G alleles (P for trend=0.014). However, no significant association of rs1760944 with risk of astrocytoma and other glioma was found in our study.These results indicate that genetic variantions in APE1/Ref-1 may modify susceptibility to lung cancer and glioblastoma. Potential gene-smoking interaction may influence its susceptible role in lung cancer. Since the -141T/G SNP (rs1760944) is located in the promoter of APE1/Ref-1, only 141 bp upstream from the transcription initiation site, it appears to be functional by possibly affecting APE1/Ref-1 transcriptional activity. To test this hypothesis, we performed further functional assays.Notably, bioinformatics analysis showed that the -141T-to-G polymorphism destroyed two binding sites for octamer-binding transcription factor-1 (Oct-1), thus it may impair the binding affinity of Oct-1 to the adjacent region of the SNP in the APE1/Ref-1 promoter. We observed an interindividual variation in APE1/Ref-1 mRNA levels in human peripheral blood mononuclear cells (PBMCs) and normal lung tissues, with a higher prevalence of the protective G allele in subjects displaying lower APE1/Ref-1 expression. Our transfection experiments also found that the plasmid containing the -141 G allele displayed significantly decreased luciferase expression than that with the T allele in both human lung adenocarcinoma (H1299) cells and human embryo lung fibroblasts (HELF).Electrophoretic mobility shift assays (EMSAs) showed that the -141G-allele probe had less affinity for the nuclear proteins from H1299 cells, since the formation of the DNA-protein complex was dramatically inhibited by unlabeled T-allele probes. Differences in the binding responses of nuclear proteins to the two oligonucleotides were also measured in real time using surface plasmon resonance (SPR) analyses and similar results were obtained, indicating that the -141G allele impaired the binding affinity of some transcription factor. Supershift assays revealed that the DNA-protein complex bands formed in EMSAs were supershifted by Oct-1-specific antibodies. Chromatin immunoprecipitation (ChIP) reconfirmed in vivo binding of Oct-1 to the APE1/Ref-1 promoter at position -141. These findings demonstrate that the protein of interest was Oct-1, and that the difference in APE1/Ref-1-promoter activities between the -141 T and G alleles was presumably due to their distinct binding affinity to Oct-1.Transactivation assays, through ectopic overexpression of Oct-1, were highly suggestive for a role of Oct-1 in preferential transactivation from the -141T allele promoter, whereas the -141G allele harbored little ability to be transcriptionally activated by Oct-1, suggesting that Oct-1 may function as a transacting factor to exert allele-specific influence on APE1/Ref-1 expression. We also found that BaP can upregulate the mRNA levels of APE1/Ref-1 and Oct-1 in H1299 and HELF cells. The pGL3-T/G transfected H1299 and HELF cells exhibited increased luciferase reporter expression after treatment with BaP. Moreover, the -141T allele-carrying construct exhibited higher capacity to be induced and still maintained significantly higher expression levels, compared with the G allele construct. This result suggests that the expression of APEl/Ref-1 might be induced by carcinogen exposure, such as smoking, in an allele-specific manner.Our study explored the functional significance of the -141T/G polymorphism. We found that it may alter APE1/Ref-1 expression by affecting the binding affinity of Oct-1 to the promoter, thus playing a causal role in lung cancer and glioblastoma susceptibility.These data also provide new insights into the mechanism by which APE1/Ref-1 exert an important effect on tumorigenesis.