Identification and functional validation of HPV-mediated hypermethylation in head and neck squamous cell carcinoma.
Identification and functional validation of HPV-mediated hypermethylation in head and neck squamous cell carcinoma.
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作者：
Lechner M;Fenton T;West J;Wilson G;Feber A;Henderson S;Thirlwell C;Dibra HK;Jay A;Butcher L;Chakravarthy AR;Gratrix F;Patel N;Vaz F;O'Flynn P;Kalavrezos N;Teschendorff AE;Boshoff C;Beck S
人乳头瘤病毒阳性（HPV+）头颈部鳞状细胞癌（HNSCC）与HPV阴性（HPV-）HNSCC相比，代表了一种独特的临床和流行病学状况。为了测试HPV在HNSCC中的表观遗传调节的可能参与，我们进行了全基因组DNA甲基化分析。使用激光捕获显微切割42福尔马林固定石蜡包埋（FFPE）的HNSCC，我们产生了18 HPV+和14 HPV-样本的DNA甲基化谱，使用Infinium 450 k BeadArray技术。使用两种独立的方法（Infinium 450 k和全基因组甲基化DNA免疫沉淀测序（MeDIP-seq））在两组独立的HPV+/HPV- HNSCC样本（新鲜冷冻样本和细胞系）中验证甲基化数据。对于功能分析，用含有两种HPV致癌基因（E6和E7）的慢病毒构建体转导HPV-HNSCC细胞系，并使用Infinium 450 k技术测定对甲基化的影响。在具有最大变化的甲基化可变位置（MVP）上的无监督聚类显示样品根据HPV状态分离，但HPV+肿瘤也是异质的。MVP在转录起始位点显著富集，导致在HPV+肿瘤的亚组中鉴定出候选CpG岛甲基化表型。监督分析确定了HPV+ HNSCC中MVP对高甲基化的强烈优势（87%）。我们的HNSCC和宫颈癌和肺癌中公开可用的甲基化数据的荟萃分析证实了观察到的DNA甲基化特征是HPV特异性的和组织无关的。将MVP定位到功能上更显著的差异甲基化区域，鉴定了43个高甲基化的启动子DMR，包括Polycomb组靶基因的三个钙粘蛋白。与独立表达数据的整合显示出很强的负相关性，特别是对于钙粘蛋白基因家族成员。在HPV-HNSCC细胞系中两种HPV癌基因（E6和E7）的组合异位表达部分表型模仿了在HPV+ HNSCC肿瘤中观察到的超甲基化特征，并确立了E6作为主要的病毒效应基因。我们的数据证实档案FFPE组织非常适合这种类型的甲基化组分析，并表明HPV通过Polycomb抑制复合物2靶基因（如钙粘蛋白）的超甲基化来调节HNSCC表观基因组，该靶基因与肿瘤进展和转移有关。
Human papillomavirus-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) represents a distinct clinical and epidemiological condition compared with HPV-negative (HPV-) HNSCC. To test the possible involvement of epigenetic modulation by HPV in HNSCC, we conducted a genome-wide DNA-methylation analysis. Using laser-capture microdissection of 42 formalin-fixed paraffin wax-embedded (FFPE) HNSCCs, we generated DNA-methylation profiles of 18 HPV+ and 14 HPV- samples, using Infinium 450 k BeadArray technology. Methylation data were validated in two sets of independent HPV+/HPV- HNSCC samples (fresh-frozen samples and cell lines) using two independent methods (Infinium 450 k and whole-genome methylated DNA immunoprecipitation sequencing (MeDIP-seq)). For the functional analysis, an HPV- HNSCC cell line was transduced with lentiviral constructs containing the two HPV oncogenes (E6 and E7), and effects on methylation were assayed using the Infinium 450 k technology. Unsupervised clustering over the methylation variable positions (MVPs) with greatest variation showed that samples segregated in accordance with HPV status, but also that HPV+ tumors are heterogeneous. MVPs were significantly enriched at transcriptional start sites, leading to the identification of a candidate CpG island methylator phenotype in a sub-group of the HPV+ tumors. Supervised analysis identified a strong preponderance (87%) of MVPs towards hypermethylation in HPV+ HNSCC. Meta-analysis of our HNSCC and publicly available methylation data in cervical and lung cancers confirmed the observed DNA-methylation signature to be HPV-specific and tissue-independent. Grouping of MVPs into functionally more significant differentially methylated regions identified 43 hypermethylated promoter DMRs, including for three cadherins of the Polycomb group target genes. Integration with independent expression data showed strong negative correlation, especially for the cadherin gene-family members. Combinatorial ectopic expression of the two HPV oncogenes (E6 and E7) in an HPV- HNSCC cell line partially phenocopied the hypermethylation signature seen in HPV+ HNSCC tumors, and established E6 as the main viral effector gene. Our data establish that archival FFPE tissue is very suitable for this type of methylome analysis, and suggest that HPV modulates the HNSCC epigenome through hypermethylation of Polycomb repressive complex 2 target genes such as cadherins, which are implicated in tumor progression and metastasis.
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