Most lung cancers derive from cigarette tobacco smoke, which accounts for as much as 90% of all lung cancer cases in the US [18
]. NNK is a powerful lung carcinogen associated with tobacco smoke, and total serum NNAL is a biomarker of its exposure that has been shown to be significantly associated with lung cancer risk [8
]. In the present study, we found that the CYP1B1N453S has an interaction effect on the relation between total NNAL and lung cancer risk in addition to a main effect on risk. It is notable that the SNP would not have been identified had we looked first for a main effect alone. The main effect is statistically significant only in the presence of the interaction term. Further strengthening the result is the fact that the effect increases when the analysis is limited to adenocarcinomas, the histological subtype of lung cancer caused by NNK in laboratory animals, and the most common type of lung cancer in the U.S. This would not likely have been observed if the initial observation was a chance occurrence.
The involvement of CYP1B1 has not been previously implicated in NNK or NNAL metabolism. We considered the possibility that CYP1B1 has a direct involvement in the metabolism of NNK, but found no evidence for this. However, CYP1B1 could have an influence on the NNAL pathway by affecting transcription of CYP1A1, whose role in the metabolic activation of total NNAL has been previously described, even though its catalytic efficiency is not very great [5
Transcription of both P450 family members CYP1A1 and CYP1B1 is induced upon activation of the aryl hydrocarbon receptor (AhR) pathway [21
]. AhR is a cytosolic transcription factor that is normally inactive and bound to several co-chaperones. Following exposure to endogenous and exogenous chemicals, AhR acts as a ligand-activated receptor and transcription factor, activating the transcription of xenobiotic-metabolizing enzymes such as CYP1A1 and CYP1B1 as well as other genes [22
]. There could be a signaling loop mechanism in which CYP1B1 can also act as a ligand and mediate the AhR signaling pathway, either in an activating or a suppressingfashion.
If the SNP implicated in this study, CYP1B1N453S, has a functional significance on the protein levels of CYP1B1 such that it down-regulates or abrogates them, then this would be expected to enhance AhR activation. Significantly, one study did show that inhibition of CYP1B1 is linked to enhanced AhR activation [25
]. Consequently, enhanced AhR activation leads to an enhanced transcription of CYP1A1. In fact, a recent study showed that CYP1B1N453S has a functional impact on the protein such that the protein displays lower intracellular levels and is degraded more rapidly than all other CYP1B1 variants tested in the study [26
]. It is not clear what structural alterations are responsible for the increased rate of CYP1B1 degradation caused by the codon change Asn453SSer. This residue is located in the large meander region between the K- and L-helix and probably highly accessible to proteases. This so-called meander region is situated in the COOH terminal half of CYP1B1, important in the heme-binding and proper folding of the molecule. Moreover, it is interesting to note that the regions in which the putative non-synonymous SNPs reside in CYP1B1 are not highly conserved in mammals with the exception of the SNP at codon 453 [27
]. Two different groups reported a 2-fold reduction in the cellular level of the protein containing this polymorphism, and a significantly reduced enzyme half-life [27
]. It is therefore well established that this variation has a functional consequence on the protein cellular levels, its folding and stability. Due to CYP1B1's involvement in the metabolism of carcinogens, and the SNPs residence in a conserved region of the gene, it is not surprising that this SNP is emerging as an important player in carcinogenesis. Therefore, CYP1B1N453S connection to CYP1A1 and its consequent indirect involvement in the NNK/NNAL metabolism is a possible explanation for our findings (). AhR mediated induction of CYP1 enzymes can lead to many cancer-related processes including genotoxicity, mutation and tumor initiation [29
Figure 2 Potential molecular mechanism of CYP1B1N453S impact on lung cancer susceptibility. AhR, a ligand-dependent transcription factor, becomes activated upon ligation, to endogenous ligands, inducing chemical binding. The AhR receptor is a part of a multifactor (more ...)
This indirect impact of CYP1B1 on NNK metabolism through CYP1A1 could involve other pathways that we are not aware of due to the complexity of tobacco smoke carcinogenesis. A relationship between CYP1 inducibility and cancer has been previously shown [30
]. A group of researchers demonstrated an association between CYP1 inducibility and bronchogenic carcinoma [31
]. Furthermore, in the context of hepatoma cells or in vitro studies, CYP1A1 is a primary determinant of the metabolism of benzo [a]pyrene, a PAH likely involved in tobacco-induced lung cancer [32
]. Thus, CYP1A1's link to lung cancer has been proposed in many previous studies, although the possible relationship of our observations to CYP1A1 inducibility remains speculative.
As presented in this paper, we found an even stronger effect of CYP1B1N453S in a smaller adenocarcinoma group. A study by Chang et al. [33
] found expression of AhR and CYP1B1 to be associated regardless of smoking status and AhR overexpression to up-regulate the expression of CYP1B1 in the early stage of lung adenocarcinoma. This finding may strengthen the results of our study.
Therefore, the effect of the CYP1B1N453S we observed might be predicted–lower levels of CYB1B1 protein results in increased activation of AhR, which in turn increases CYP1A1 activity (). Based on our analysis of HapMap variants we do not believe CYP1B1 to be directly involved in the metabolism of NNAL, although further functional studies on CYP1B1's involvement in NNAL and NNK metabolism are needed.
Phenanthrene and other PAHs are substrates for CYP1B1 and CYP1A1[32
]. We did not observe an association between PheT levels and lung cancer, nor was there any interaction with CYP1B1 polymorphisms. This somewhat unexpected result may be due to the relatively small size of our study, and to the fact that phenanthrene, in contrast to NNK, is not tobacco-specific. Thus, substantial amounts of serum PheT are due to phenanthrene exposure from diet or general environment.
The study is limited by its small size, which required a focus on just a few SNPs, rather than on a broad array of polymorphisms. We chose a subset of the eleven most likely candidates for study, and found evidence that one of those SNPs may segregate the population by the risk conferred by NNAL exposure as well as by the underlying risk itself.
The evidence of a strong interaction between total serum NNAL and the CYP1B1N453S SNP from this study was unexpected and, as yet, is not fully explained. If confirmed by appropriate additional molecular and epidemiologic studies, this outcome constitutes an important step in understanding how exposure to cigarette smoke leads to inter-individual variation in risk of lung cancer.