Normal bronchial field basal cells show persistent methylome-wide impact of tobacco smoking, including in known cancer genes

Lung carcinogenesis is causally linked to cigarette smoking, in part by epigenetic changes. We tested whether accumulated epigenetic change in smokers is apparent in bronchial basal cells as cells of origin of squamous cell carcinoma. Using an EM-seq platform covering 53.8 million CpGs (96% of the entire genome) at an average of 7.5 sequencing reads per CpG site at a single base resolution, we evaluated cytology-normal basal cells bronchoscopically brushed from the in situ tobacco smoke-exposed ‘bronchial epithelial field’ and isolated by short-term primary culture from 54 human subjects. We found that mean methylation was globally lower in ever (former and current) smokers versus never smokers (<i>p</i> = 0.0013) across promoters, CpG shores, exons, introns, 3’-UTRs, and intergenic regions, but not in CpG islands. Among 6mers with dinucleotides flanking CpG, those containing CGCG showed no effect from smoking, while those flanked with TT and AA displayed the strongest effects. At the gene level, smoking-related differences in methylation level were observed in <i>CDKL1, ARTN</i>, <i>EDC3</i>, <i>CYP1B1</i>, <i>FAM131A</i>, and <i>MAGI2</i>. Among candidate cancer genes, smoking reduced the methylation level in <i>KRAS</i>, <i>ROS1</i>, <i>CDKN1A</i>, <i>CHRNB4</i>, and <i>CADM1</i>. We conclude that smoking reduces long-term epigenome-wide methylation in bronchial stem cells, is impacted by the flanking sequence, and persists indefinitely beyond smoking cessation. In an epigenome-wide survey of normal bronchial cells brushed and primary-cultured from 54 donors, average cytosine methylation was globally lower and, equally so, in both former and current smokers versus never smokers across almost all genome compartments.At the gene level, smoking-related genome-wide significant differences in methylation level were observed in six agnostically uncovered, previously suspected carcinogenesis genes and another five genes among 40 a priori-selected candidate cancer genes.It is concluded that smoking reduces long-term epigenome-wide methylation in bronchial stem cells in cancer-related genes and persists indefinitely beyond smoking cessation, suggesting a possible decades-long epigenetic trace of risk for lung cancer and other lung disorders. In an epigenome-wide survey of normal bronchial cells brushed and primary-cultured from 54 donors, average cytosine methylation was globally lower and, equally so, in both former and current smokers versus never smokers across almost all genome compartments. At the gene level, smoking-related genome-wide significant differences in methylation level were observed in six agnostically uncovered, previously suspected carcinogenesis genes and another five genes among 40 a priori-selected candidate cancer genes. It is concluded that smoking reduces long-term epigenome-wide methylation in bronchial stem cells in cancer-related genes and persists indefinitely beyond smoking cessation, suggesting a possible decades-long epigenetic trace of risk for lung cancer and other lung disorders.