This is the first study to comprehensively evaluate the relationship between methylation index in a large population of lung-cancer free smokers and common variation in 40 candidate genes involved in carcinogen metabolism, regulation of methylation, and DNA damage response pathways. Our findings support a role for genetic variation in LIG1, TP53, and BIK as predictors for the acquisition of gene promoter methylation in exfoliated cells in smokers’ sputum. Specifically, molecular validation through a genotype – expression correlation analysis indicates that the three most significant SNPs in LIG1, TP53, and BIK affect the expression of these three genes in cis in NHBECs. Thus, genetic variation in genes affecting DNA replication and apoptosis impacts the propensity for gene promoter methylation in the aerodigestive tract of smokers and this effect may be driven by the change in the endogenous expression of LIG1, TP53, and BIK.
Epigenetic silencing of TSGs in lung cells is an intermediate biomarker for lung cancer. Therefore, genetic variation that is strongly associated with epigenetic silencing of TSGs should also impact the risk for lung cancer. Indeed, this premise is supported by studies that systematically assessed the association between tag SNPs in LIG1 and risk for lung cancer (33
). The criteria for selecting these studies to be summarized include large samples size (>500 pairs cases and controls), Caucasian ethnicity, pathway or genome wide association studies, and adequate SNP coverage in LIG1. These criteria were set to minimize the confounding from the publication bias and to ensure inclusion of the sufficiently powered studies. Only association with lung cancer in moderate and heavy smokers (current or former) was presented because over 90% of cohort members enrolled in the LSC are smokers with over 20 pack-year smoking history. A case-control study conducted in the Pittsburgh Lung Screening Study cohort included 722 lung cancer patients and 929 controls who were Caucasian and current or ex-cigarette smokers with > 10 pack-year smoking history and found that subjects carrying the variant homozygote for rs10500298 that is in high LD with rs3730859 (r2
=0.89) in the 1k genome project CEU population have a 96% increased risk for lung cancer (P<0.0001) (33
). A second case-control study conducted in the Beta-Carotene and Retinol Efficacy Trial cohort also identified rs156640 and rs156641 that is in high LD with rs3730859 (r2
=0.89) in CEU as a strong risk factor for lung cancer in Caucasian smokers with >20 pack-year of smoking history (ORs=1.20, Ps<0.003, 746 cases and 1477 controls) (34
). Two lung cancer GWAS studies conducted in United States populations were queried as well and subjects with homozygous rs156641 have a 22% increased risk for lung cancer in subjects with over 20 pack-year smoking history in MD Anderson GWAS study (P<0.14, 1004 cases and 926 controls) (35
). However, no association was identified between LIG1 SNPs and risk for lung cancer in two US populations used in the discovery stage in the NCI GWAS study (1652 cases and 1212 controls) (36
). The SNPs in TP53 and BIK associated with methylation index were also queried in the MD Anderson and NCI GWAS with no significant association to lung cancer identified (not shown). Together, these studies support a moderate association for rs3730859 in LIG1 with risk for the smoking-induced lung cancer and this effect may be mediated by its affect on the predisposition for epigenetic silencing of TSGs.
Among the three DNA ligase families (LIG1, LIG3, and LIG4) in vertebrates, LIG1 plays an essential role in the joining of Okazaki fragments during lagging strand synthesis and is also implicated in multiple DNA repair pathways, although the role of LIG1 in DNA repair can be substituted by other DNA ligases (37
). Deficiency in LIG1 expression results in the delayed joining of Okazaki fragments, genome instability, and increased incidence of epithelial cancers and lymphoma (37
). Expression of LIG1 in lung tumors (n=23) is reduced by 34% compared to that in paired distant normal tissues providing further support for a functional deficiency of this gene in lung carcinogenesis (P=0.028, Leng unpublished). The strong association between genetic variation in LIG1 and methylation index found in this study provides in vivo
evidence that incomplete loss of LIG1 function due to SNPs could promote the epigenetic silencing of TSGs which in turn contributes to the epithelial carcinogenesis. The mechanism for the effect of LIG1 deficiency on gene methylation is unclear. A recent study found that the replication defect in 46BR.1G1 cells which are homozygote for the Arg771Trp mutation and retain only 3 to 5% of normal LIG1 activity results in the generation of endogenous single- and double-stranded DNA breaks during S phase (40
). These DNA breaks behind the replication fork induce a persistent activation of the ATM/CHK2 pathway without triggering the S-phase-specific DNA damage checkpoint and lead to an altered chromatin structure needed for efficient processing of the DNA damage throughout the cell cycle. Thus, the replication timing could be disrupted in cells with LIG1 deficiency because of the inefficient joining of Okazaki fragments in newly synthesized DNA and the extensively altered chromatin structure. This may be a mechanism by which the guidance of the cytosine-DNA methytransferase to regions that are to be methylated is disrupted during DNA replication and the normally unmethylated regions of DNA become aberrantly methylated (41
). Although such a scenario is clearly speculative, an in vitro
malignant transformation model or in vivo
tumorigenesis model using LIG1 knock out human epithelial cells or animals could be used to directly test this hypothesis.
Evading apoptosis is a hallmark of cancer and occurs during the progression from premalignant lesions to carcinoma in the lung (42
). Apoptosis occurs through two mechanisms: the death receptor FAS/FAS ligand (also called extrinsic) pathway and the mitochondrial (DNA damage–induced and p53-mediated, also called intrinsic) pathway (43
). The intrinsic apoptosis pathway plays a key role in eliminating lung epithelial cells with extensive DNA damage upon carcinogen exposure (43
). Thus, the association between functional SNPs in TP53 and BIK and methylation index support the concept that the TP53 mediated apoptosis pathway could prevent the acquisition of heritable genetic and epigenetic changes through eliminating lung cells with extensive DNA damage. The loss of TP53 function by gene mutation is a common genetic alteration found in human cancers and over 50% of lung cancer cases carry somatic mutation of TP53 (44
). TP53 mutation or reduced level of wild-type TP53 protein was associated with apoptosis suppression during progression from premalignant lesions to carcinoma in the lung (42
). Thus, rs1641511 that was associated with a 40% reduction of TP53 transcription could render lung cells more resistant to apoptosis upon extensive DNA damage in smokers, which in turn could result in an increased methylation index. Although rs1641511 is geographically closer to ATP1B2, the gene downstream of TP53, LD analysis using the Phase 2 HapMap CEU data and the 1000 genome project identify one SNP (rs72829457) around 3kb downstream of TP53 in moderate LD with rs1641511 (D'=1, r2
=0.5). Moreover, the deletion/insertion polymorphisms that usually account for 15–20% of the entire genetic variation, are not available from the above two databases. Thus, it is possible that the missing deletion/insertion polymorphisms in high LD with rs1641511 are causal for the reduced expression of TP53.
BIK (Bcl2-interacting killer) is the founding member of the pro-apoptotic BH3-only proteins (45
). BIK functions as a death sensor to mediate activation of the mitochondrial apoptosis pathway as shown in response to oncogenic stress signals or DNA damage in epithelial cancer cell lines (45
). BIK’s role in cancer development may vary by tumor type. Inactivation of BIK in some primary tissues occurs frequently by chromosomal deletion in renal cell carcinomas (40%), colorectal cancers (22%), and gliomas (42%), but only occasionally in head and neck cancers (5%; 45
). Somatic mutations that lead to amino acid changes were also detected in 8% of patients with B cell lymphomas (45
). Epigenetic silencing was also proposed as a potential mechanism to silence BIK expression in specific tumor types, although this mechanism was only indirectly tested based on BIK re-expression in tumor cell lines treated with DNA demethylating agent and/or histone deacetylase inhibitor (45
). Rs1883264 associated with reduced BIK expression in NHBECs is actually protective for gene methylation in smokers. This seems to be in conflict with the pro-apoptotic role of BIK identified in epithelial cancer cell lines. However, BIK is over expressed in lung tumors (n=23) versus paired distant normal tissues (P=0.015, Leng unpublished). Furthermore, CpG sites in the BIK promoter and exon1 are not methylated in lung adenocarcinomas interrogated on the Illumina HumanMethylation 27K BeadChip (Leng unpublished). Consistent with these findings in lung cancer, BIK over expression was also reported in breast tumors (46
). In addition, poor prognosis of non-small cell lung cancers correlated with high expression of BIK (47
). These results support different mechanisms for BIK regulation in cancer biology by tumor site and carcinogen exposure pattern. Thus, the protective effect of rs1883264 on gene methylation in smokers identified in our studies is consistent with the increased expression of BIK in lung tumors. Additional mechanistic studies are required to understand the regulation of BIK biology in cigarette smoke-induced lung cancer.
These studies identified the DNA replication and apoptosis pathways as new determinants for acquiring gene methylation in lung cells from smokers. Lung cancer GWAS studies successfully identified several loci in the human genome that are associated with risk for lung cancer at 10−8
significance level (35
). However, these validated genetic variants only explain a small fraction of lung cancer heritability. Some of the missing heritability likely exists in the genetic variants with less significant P values. The detection of promoter methylation of TSGs in exfoliated cells in sputum as an assessment of the extent of field cancerization in the lung can be used as a functional readout to help identify the true association from the candidate SNPs with GWAS P values > 10−8
. Thus, the genetic variants in LIG1 provide a proof-of-concept that SNPs strongly associated with risk for gene methylation in lung cells from smokers can be moderately associated with risk for lung cancer and should be incorporated into risk assessment models for lung cancer.