This study provides strong evidence that the A allele of a MGMT
promoter-enhancer SNP (rs16906252) is a key determinant in the acquisition of MGMT
methylation in lung carcinogenesis. This association was found in primary adenocarcinoma and in exfoliated cells in sputum from cancer-free smokers, a finding that reflects the ongoing field cancerization within the aerodigestive tract. The magnitude of the association between rs16906252 and MGMT
methylation in premalignant lesions from lung cancer-free smokers was much smaller than observed from the lung adenocarcinoma patients consistent with the increase in prevalence of MGMT
methylation of 25% in premalignant lesions from smokers to 43% in stage I lung cancer and 61% in stage II-IV lung cancer (36
). The reduced transcription and expression seen for the A allele of MGMT
may be one predisposing factor for methylation. This hypothesis is supported by the selective methylation and accompanied silencing of the A allele in tumors from A/G heterozygotes. Thus, some of the MGMT
unmethylated lung cancer-free smokers who carry the A allele should be more prone to become methylated as their field cancerization progresses over time (32
The lack of an association between rs16906252 with risk for lung cancer based on the similar percentage of subjects carrying the A allele in lung adenocarcinoma cases and cancer-free controls in the Lovelace and Veteran Cohorts (14.2%, 15.6%, and 12.8%, respectively) is not surprising. Our previous studies demonstrate that concomitant methylation of a panel of tumor suppressor genes including MGMT
in sputum could prospectively predict risk for lung cancer in moderate and heavy smokers (18
methylation alone in sputum only contributes to a portion of the predicted increased cancer risk. Thus, a large case-control study would be needed to more precisely estimate the association of this SNP to risk for lung cancer. Moreover, approximately 20% of subjects with G/G genotype also have MGMT
methylated in sputum, suggesting that other genetic or environmental factors may also contribute to MGMT
methylation in lung carcinogenesis, albeit probably to a lesser degree than rs16906252. Thus, the prediction for lung cancer risk by MGMT
methylation in sputum could also vary by rs16906252 genotype. Testing this hypothesis will require long-term follow-up in our smoker cohorts to accumulate incident lung cancer cases with sputum samples collected before cancer diagnosis.
The sequence dependent ASM of MGMT
is not lung tumor-specific because similar associations were also reported in colorectal cancer (21
). Studies in glioblastoma did not identify an association between rs16906252 and risk for methylation (37
). However, the primers used for genotyping had several incorrect bases that likely influenced the accuracy for identifying the variant allele (37
). Recently, methylation of GSTP1
, and MSH2
were found to be allele-specific in several tumor types (11
). Low-level methylation of MGMT
was also seen in normal colon mucosa and peripheral lymphocytes from heterozygote subjects, corroborating a likely important role for this SNP in the susceptibility of the A allele of MGMT
for silencing (22
). Zhang et al.
studying methylation of gene promoters on chromosome 21 estimated that ASM may affect 10% of human genes in leukocytes (13
). Thus, genetic differences can influence inter-individual variation for DNA methylation of many genes in normal tissues and tumors.
Evidence is accumulating that reduced transcription may be one trigger for initiation and spreading of heterochromatin and methylation along a gene promoter (34
). Song et al.
and Stirzaker et al.
) showed that reduced transcription and seeds of methylation within the GSTP1
promoter served as a stimulus for the spread of methylation and chromatin remodeling. In vivo
studies found that breast cancer patients carrying the GSTP1
promoter haplotype E comprised of four SNPs and the shortest ATAAA repeat had increased methylation (38
). The polymorphisms within haplotype E disrupted binding of c-Myb leading to reduced transcription. Our studies also support reduced transcription as a mechanism for de novo
promoter methylation based on the strong association between reduced promoter activity associated with the A allele in NHBEC and the targeting of this allele for silencing in lung tumors from heterozygote patients. Other mechanisms independent of effects on gene transcription may also influence de novo
promoter methylation. For example, a 12 bp insertion polymorphism in the RIL
promoter creates an Sp1/Sp3 binding site that protects against methylation in cancer and does not change expression in lymphoblastoid cell lines (11
). The Sp1/Sp3 binding site created by this polymorphism may through protein-DNA interactions prevent spreading of DNA methylation and heterochromatin from neighboring “methylation centers”. In silico
prediction of transcription factor-binding sites using TFSEARCH and MATCH programs did not identify any transcription factors with core motifs binding rs16906252. In addition, resequencing the 1.9 Kb 5' region of the MGMT
gene did not identify any SNP in perfect LD with rs16906252. Therefore, rs16906252 may influence the binding of an unknown transcription factor or unlisted transcription factor in these two databases. Recently, Hawkins et al.
examined six gene-associated CpG islands flanking MGMT
in colorectal tumors and found that the EBF3
gene located 500 kb downstream of MGMT
had a similar methylation pattern to MGMT
). Thus, it is possible that rs16906252 is in perfect LD with other polymorphisms present in the euchromatin and heterochromatin boundaries surrounding the MGMT
and/or EBF3 which could affect gene regulation. Currently, testing this hypothesis will necessitate the completion of the 1000 Genome Project that will provide a dense map of genetic variation by sequencing the genomes of 1200 people (43
The refractory nature of unresected lung cancer to chemotherapeutic combinations tested over three decades necessitates developing targeted therapies based on underlying genetic and epigenetic tumor profiles. Responses have been extremely positive for patients with mutations in the epidermal growth factor receptor receiving the tyrosine kinase inhibitors gefitinib or erlotinib (44
). Alkylating agents such as 1,3-bis(2-chloroethyl)-1-nitrosourea were tested as chemotherapeutics for lung cancer decades ago with response rates limited by systemic toxicity (35
). TMZ has been evaluated in a phase II study for efficacy in pretreated lung cancer, melanoma, and breast cancer patients with brain metastasis. Treatment was provided without knowledge of MGMT
methylation status, and 26% of patients had a clinical response with median overall survival greatest for lung cancer patients (47
). Our in vitro
studies in lung cancer cell lines demonstrating sensitivity to TMZ that is highly correlated with levels of MGMT
methylation suggest that a subset of lung cancer patients with relatively low MGMT expression could respond to this drug, a hypothesis that requires further preclinical studies prior to initiating clinical trials.
Epigenetic silencing of MGMT by promoter methylation is a biomarker for identifying persons at high risk for lung cancer and for predicting therapeutic response to alkylating agents in glioblastoma. The A/G allele of rs16906252 in MGMT promoter-enhancer region predicts for gene methylation in sputum from cancer free smokers. This association was increased substantially in primary lung tumors supporting this variant as a key determinant for epigenetic silencing of MGMT. Importantly, the sensitivity of lung cancer cell lines to temozolamide (TMZ) was strongly correlated with levels of MGMT methylation and expression, suggesting TMZ treatment may benefit a subset of lung cancer patients methylated for MGMT, a hypothesis that warrants further preclinical studies prior to initiating clinical trials.