Epigenome-wide analysis of DNA methylation in lung tissue shows concordance with blood studies and identifies tobacco smoke-inducible enhancers

Smoking-associated DNA hypomethylation has been observed in blood cells and linked to lung cancer risk. However, its cause and mechanistic relationship to lung cancer remain unclear. We studied the association between tobacco smoking and epigenome-wide methylation in non-tumor lung (NTL) tissue from 237 lung cancer cases in the Environment And Genetics in Lung cancer Etiology study, using the Infinium HumanMethylation450 BeadChip. We identified seven smoking-associated hypomethylated CpGs (P < 1.0 × 10-7), which were replicated in NTL data from The Cancer Genome Atlas. Five of these loci were previously reported as hypomethylated in smokers' blood, suggesting that blood-based biomarkers can reflect changes in the target tissue for these loci. Four CpGs border sequences carrying aryl hydrocarbon receptor binding sites and enhancer-specific histone modifications in primary alveolar epithelium and A549 lung adenocarcinoma cells. A549 cell exposure to cigarette smoke condensate increased these enhancer marks significantly and stimulated expression of predicted target xenobiotic response-related genes AHRR (P = 1.13 × 10-62) and CYP1B1 (P < 2.49 × 10-61). Expression of both genes was linked to smoking-related transversion mutations in lung tumors. Thus, smoking-associated hypomethylation may be a consequence of enhancer activation, revealing environmentally-induced regulatory elements implicated in lung carcinogenesis. Overall design: RNAseq of DMSO or cigarette smoke condensate (CSC)-treated A549 human lung adenocarcinoma cells. Cells were treated for either 48 hours or 2 weeks, as indicated.

吸烟相关的DNA低甲基化已在血细胞中被检测到，并与肺癌风险存在关联。然而，其具体成因以及与肺癌的机制性关联仍未阐明。本研究依托「肺癌病因学环境与遗传学研究（Environment And Genetics in Lung cancer Etiology, EAGLE）」，采用Infinium人类甲基化450K芯片（Infinium HumanMethylation450 BeadChip），对237例肺癌患者的非肿瘤肺（non-tumor lung, NTL）组织进行了全表观基因组甲基化水平与吸烟暴露的关联分析。本研究鉴定出7个与吸烟相关的低甲基化CpG位点（P < 1.0 × 10^-7），上述位点在癌症基因组图谱（The Cancer Genome Atlas, TCGA）的非肿瘤肺组织数据中得到了验证。其中5个位点此前已有研究报道称在吸烟者的血液中呈现低甲基化状态，这提示针对这些位点，血液生物标志物可反映其靶组织的甲基化水平变化。4个CpG位点的侧翼序列携带芳烃受体（aryl hydrocarbon receptor, AHR）结合位点，且在原代肺泡上皮细胞与A549肺腺癌细胞中存在增强子特异性的组蛋白修饰。将A549细胞暴露于香烟烟雾冷凝液（cigarette smoke condensate, CSC）后，上述增强子标记显著上调，同时预测的靶标基因——与异生物质应答相关的AHRR（P = 1.13 × 10^-62）与CYP1B1（P < 2.49 × 10^-61）的表达也被激活。这两个基因的表达均与肺肿瘤中吸烟相关的颠换突变存在显著关联。由此可见，吸烟相关的DNA低甲基化可能是增强子激活的下游结果，这揭示了环境诱导的调控元件参与了肺癌发生的病理过程。整体实验设计：对经二甲基亚砜（dimethyl sulfoxide, DMSO）或香烟烟雾冷凝液（CSC）处理的人肺腺癌细胞A549进行RNA测序。根据实验要求，细胞分别被培养48小时或2周。