Abstract A08: Widespread tobacco smoking-associated changes in DNA methylation and gene expression in lung tissue of smokers
Authors: Mullen Daniel J. , Stueve T. Ryan , Yan Chunli , Siegmund Kimberly D. , Laird-Offringa Ite A. 
Published: February 2019

Abstract
Significance: Epigenome-wide association studies (EWAS) have identified thousands of CpGs showing smoking-associated DNA methylation changes in blood cell DNA of smokers. Several of these CpGs are strongly associated with lung cancer risk. Until recently, the relationship of such DNA methylation changes in the blood and epigenetic alterations in the lung, as well as their molecular link to lung cancer development, remained poorly understood. Using a limited EWAS of 237 lung tissue samples, we recently identified seven smoking-associated hypomethylated CpGs in lung tissue, five of which had been previously reported in blood, and one of which had been strongly associated with lung cancer risk. We showed that the latter CpG flanked a smoking-inducible enhancer element driving expression of a xenobiotic response gene. Here we expand these observations to identify tobacco-affected regulatory elements implicated in lung cancer.
Methods: We performed an EWAS using Inifinium HumanMethylation450K BeadChip data from The Cancer Genome Atlas (TCGA) corresponding to 393 eligible lung adenocarcinoma tumor samples. We replicated our findings in 27 adjacent normal lung tissue samples, then examined epigenetic enhancer marks near the smoking-associated altered CpGs using epigenomic data from primary lung cells and lung adenocarcinoma cell lines. We next identified potential responsive target genes of these enhancers by examining smoking-associated changes in gene expression on the 1 megabase flanks of the enhancers.
Results: 37,372 CpGs were significantly differentially methylated with respect to smoking in TCGA adenocarcinoma tumor samples after Benjamini Hochberg correction (p&lt;0.05). 2,941 probes replicated at a nominal level in the adjacent normal samples (p&lt;0.05), including 145 probes previously associated with smoking in at least one blood-based EWAS. 1,150 of the 2,941 replicated probes were located within 2 kb of a likely functional region carrying a histone mark of active enhancers in primary lung cells and/or lung cancer cell lines. 949 of these were within 1 Mb of a gene showing a significant, smoking-associated expression difference. One of the top-ranked CpGs we identified was among the 10 most-replicated CpGs in blood-based smoking EWAS, and was linked to smoking-induced upregulation of the aryl hydrocarbon receptor repressor (AHRR) gene, which is involved in the cellular xenobiotic response and has been implicated in lung cancer risk.
Conclusion: Many smoking-associated methylation changes in lung tissue are located in the vicinity of epigenetic regulators in lung tissue, and could be driving expression of smoking-associated genes that may affect lung cancer risk.
Funding: R01 HL114094 to IALO, P30 CA014089, USC Provost’s fellowship and Roy E. Thomas Foundation Graduate Scholarship to DJM.
Citation Format: Daniel J. Mullen, T. Ryan Stueve, Chunli Yan, Kimberly D. Siegmund, Ite A. Laird-Offringa. Widespread tobacco smoking-associated changes in DNA methylation and gene expression in lung tissue of smokers [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A08.