On the basis of our results, we conclude that susceptibility to lung cancer in our cohort of Caucasian women is influenced by the genotype of rs3841324, a functional promoter polymorphism in the CHRNA5 subunit, which has been clearly identified as a susceptibility gene for lung cancer. We observed that rs3841324 SS genotype is protective against lung cancer in female smokers but not in male. Further analysis showed no significant difference in CPD or smoking duration between groups stratified by rs3841324 genotypes, sex or case-control status. In addition, no joint effect between rs3841324 and rs16969968 or rs8034191 was observed.
Previously, functional characterization of CHNRA5
using luciferase assays in human cell lines has demonstrated that the −240/+53 region, which contains the rs3841324 indel, is the core promoter (24
). Using a standardized reporter gene assay system, Buckland et al. found that the rs3841324 minor allele (S allele) decreased promoter activity in HEK293 cells by 1.5-fold (26
). Recently, Zheng et al. (27
) demonstrated that the rs3841324 S allele was associated with hypoactivity of the promoter, resulting in a 2- to 6-fold decrease in CHRNA5
transcription compared with that of major allele (L allele) in A549 cells. In addition, the rs3841324 L allele showed higher affinity to nuclear extraction proteins of A549 cells than did the S allele, suggesting a difference in CHRNA5
transcription by influencing DNA-protein interactions (27
). The potential transcription factor prediction with TFSEARCH [(28
); accessed on June 9, 2011] show that rs3841324 contains a predicted binding site for the Sp1 transcription factor, which recognizes and specially binds to GC-rich regions such as the GC-box (29
). Therefore, it is very likely that deletion of rs3841324 would reduce the number of binding sites for SP1 transcription factor, thus influencing DNA-protein interactions and cause difference in CHRNA5
In the present study, we showed that the SS variant of rs3841324 was significantly associated with reduced risk of lung cancer in female smoker. There is some biological plausibility for this protective association. In the mammalian brain, nAChRs include homopentameric α7 receptors and a variety of heteropentamers, but predominantly α4β2*. CHRNA5 is most commonly found in heteromeric receptors composed α4β2α5 subunits. Inclusion of the α5 subunit in α4β2 receptors significantly increases the rate of desensitization of α4β2* nAChRs (31
). α4β2 nAChRs play important roles in regulation of anti-inflammatory processes, immune processes, and fundamental pathways involved in cell survival (34
), therefore, desensitization of α4β2 may be an important force in the development of human cancer. Hypoactivity of the promoter of CHRNA5
decreases the rate of desensitization of α4β2 nAChRs, thereby potentially reducing the risk of lung cancer. In addition, inclusion of the α5 subunit in α4β2 nAChRs significantly increases Ca2+
signals are pivotal in regulating nAChRs-mediated gene expression and cell signaling which may lead to gene activation (in addiction) (40
) or to cell proliferation (in cancer) (42
). Therefore, further studies investigating the mechanism by which CHRNA5 affects the risk of lung cancer are needed to elucidate this new protective pattern.
Some epidemiologic evidence suggests a sex difference in the association between the 15q25 variants and lung cancer(43
). Although speculative, some data have linked nAChRs signaling to sex hormones. For example, studies have shown that steroid hormones, including progesterone, are noncompetitive antagonists of nAChRs (43
). There are reports that sex hormones regulate nAChR expression or activity in the rat hippocampus (48
). In addition, a putative progesterone responsive element was found in the promoter of α5 nAChR subunit, and progesterone has been shown to have an effect on the α5 expression level both in vitro and in vivo (49
). It is biologically plausible that the interplay between sex hormones and α5-containing nAChRs may play a direct or indirect role in the mediation of sex differences in susceptibility to lung cancer. Our present results revealed a sex-specific association of rs3841324 on lung cancer risk. In order to exclude the possibility that the observed sex-specific association could potentially be caused by a difference in variant allele frequency in male and female controls, we calculated the frequency of rs3841324 variant allele for male controls (MAF=0.45) and female controls (MAF=0.44) in the study and found there was little difference (P
=0.5949), suggesting that this hospital-based study is unlikely to have yielded any significant bias in estimating the genotype-specific ORs.
Previous studies have consistently found that the genetic variants rs16969968 and rs8034191 in 15q25 are associated with lung cancer risk and nicotine dependence (5
). The diplotype analysis showed high linkage disequilibrium between rs3841324 and both rs16969968 and rs8034191. Because rs16969968 (D398->N398) is believed to alter receptor activity while rs3841324 is assumed to correspond to mRNA expression level, the influences of these variants on lung cancer risk are independent. rs3841324 SS_rs16969968 GG diplotype was associated with decreased risk for lung cancer, and the rs3841324 LL_rs16969968 AA diplotype exhibited the highest risk. These findings suggest that the risk associated with the amino acid change might counteract the protective effect of the change in gene expression to some degree. The underlying mechanism by which rs8034191 (T->C) influences lung cancer is unclear. However, diplotype analysis of rs3841324_rs8034191 showed a trend similar to that of rs3841324 SS_rs16969968; the rs3841324 SS_rs8034191 TT diplotype was associated with decreased risk and the rs3841324 LL_rs8034191 CC diplotype with the highest risk. Together, these results suggest that although variation in 15q25 influences the risk for lung cancer and nicotine dependence, different polymorphisms and different mechanisms of action are responsible for their effects. However, these observations deserve further test due to small sample size.
In summary, the current study indicates that the rs3841324 SS genotype is protective against lung cancer in Caucasian female smokers. In contrast, there is little such effect in Caucasian male, implying that the effect is sex-specific. Our results also indicate a new association between CHNRA5 promoter activity and susceptibility to lung cancer, implying that CHRNA5 plays a complex role in lung cancer. The underlying mechanism of sex differences in susceptibility to lung cancer remains unclear and will require in-depth molecular analysis.