Tumor hypoxia causes DNA hypermethylation by reducing TET activity (BeadChip). Homo sapiens

Genome wide DNA methylation profiling of normoxic and hypoxic non-small-cell lung cancer samples for 5mC and 5hmC. The Illumina Infinium 450k Human DNA methylation Beadchip v1.2 was used to obtain DNA methylation and hydroxymethylation profiles across 485,512 CpGs from DNA extracted from fresh-frozen tumor samples. Samples included 12 hypoxic and 12 normoxic tumor samples, with hypoxia determined according to the hypoxia metagene score (Buffa et al, Br J Cancer 2010). To profile hydroxymethylation, 5hmC was glycosylated and 5mC was oxidised as described by Yu and colleagues (Nat Protoc 2012), and hydroxymethylation and methylation were differentially profiled according to the Nazor and colleagues (Genomics 2014). Hypermethylation of tumor suppressor gene (TSG) promoters confers growth advantages to cancer cells, but how these changes arise is poorly understood. Here, we report that tumor hypoxia reduces the activity of oxygen-dependent TET enzymes, which catalyze DNA de-methylation through 5-methylcytosine oxidation. This occurs independently of hypoxia-associated alterations in TET gene expression, basal metabolism, HIF activity or nuclear reactive oxygen species, but directly depends on oxygen shortage. Hypoxia-induced loss of TET activity increases hypermethylation at gene promoters in vitro, while also in patients, gene promoters are markedly more methylated in hypoxic than normoxic tumors. Affected genes are frequently involved in DNA repair, cell cycle regulation, angiogenesis and metastasis, indicating cellular selection of hypermethylation events. Overall, up to 50% of the tumor-associated hypermethylation is ascribable to hypoxia across various cancer types. Accordingly, spontaneous murine breast tumors become hypermethylated when rendered hypoxic through vessel pruning, whereas vessel normalisation rescues this effect. Tumor hypoxia thus acts as a novel regulator underlying DNA methylation. Overall design: 12 normoxic and 12 hypoxic non-small-cell lung cancer samples were compared for their 5hmC and 5mC distribution

针对5-甲基胞嘧啶（5-methylcytosine, 5mC）与5-羟甲基胞嘧啶（5-hydroxymethylcytosine, 5hmC），本研究对常氧与低氧非小细胞肺癌样本开展全基因组DNA甲基化谱分析。实验采用Illumina Infinium 450k人类DNA甲基化微珠芯片v1.2（Illumina Infinium 450k Human DNA methylation Beadchip v1.2），对新鲜冰冻肿瘤样本提取的DNA进行检测，获取横跨485,512个CpG位点的DNA甲基化与羟甲基化谱。

本次研究共纳入12例低氧肿瘤样本与12例常氧肿瘤样本，样本的低氧状态根据低氧元基因评分（hypoxia metagene score）确定（Buffa等，Br J Cancer 2010）。为实现羟甲基化谱分析，按照Yu等（Nat Protoc 2012）所述方法对5hmC进行糖基化修饰、对5mC进行氧化处理，并依据Nazor等（Genomics 2014）的方案区分检测羟甲基化与甲基化水平。

肿瘤抑制基因（tumor suppressor gene, TSG）启动子区域高甲基化可赋予癌细胞生长优势，但其具体发生机制尚不明确。本研究证实，肿瘤低氧会降低氧依赖型TET酶（TET）的活性——该酶通过氧化5-甲基胞嘧啶催化DNA去甲基化。此过程不依赖于TET基因表达、基础代谢、缺氧诱导因子（hypoxia-inducible factor, HIF）活性或细胞核活性氧水平的低氧相关改变，而是直接依赖于氧缺乏。体外实验显示，低氧诱导的TET活性丧失会增加基因启动子区域的高甲基化水平；在临床患者样本中，低氧肿瘤的基因启动子甲基化水平也显著高于常氧肿瘤。受影响的基因常参与DNA修复、细胞周期调控、血管生成与转移过程，提示高甲基化事件存在细胞选择效应。总体而言，在多种癌症类型中，高达50%的肿瘤相关高甲基化可归因于低氧。相应地，通过血管修剪构建低氧模型的自发性小鼠乳腺肿瘤会出现高甲基化表型，而血管正常化可逆转这一效应。综上，肿瘤低氧是调控DNA甲基化的新型调控因子。

总体实验设计：比较12例常氧与12例低氧非小细胞肺癌样本的5hmC与5mC分布特征。