鉴定和功能验证 HPV 介导的头颈部鳞状细胞癌中甲基化。
Identification and functional validation of HPV-mediated hypermethylation in head and neck squamous cell carcinoma.
机构信息
UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6DD ; Head and Neck Centre, University College London Hospitals NHS Trust, Euston Road, London, NW1 2PG.
UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6DD.
出版信息
Genome Med. 2013 Feb 5;5(2):15. doi: 10.1186/gm419. eCollection 2013.
BACKGROUND
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.
METHODS
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.
RESULTS AND DISCUSSION
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.
CONCLUSIONS
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.
背景
与 HPV 阴性（HPV-）头颈部鳞状细胞癌（HNSCC）相比，人乳头瘤病毒阳性（HPV+）HNSCC 代表了一种独特的临床和流行病学状况。为了检验 HPV 对 HNSCC 中表观遗传调节的可能作用，我们进行了全基因组 DNA 甲基化分析。
方法
我们使用激光捕获显微切割技术对 42 例福尔马林固定石蜡包埋（FFPE）的 HNSCC 进行了研究，使用 Infinium 450 k BeadArray 技术生成了 18 例 HPV+和 14 例 HPV-样本的 DNA 甲基化图谱。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）中验证了甲基化数据。为了进行功能分析，我们使用包含两个 HPV 致癌基因（E6 和 E7）的慢病毒构建体转导了 HPV- HNSCC 细胞系，并使用 Infinium 450 k 技术检测了对甲基化的影响。
结果与讨论
对具有最大变异的甲基化变量位置（MVP）进行无监督聚类表明，样本根据 HPV 状态进行了分类，但 HPV+肿瘤也是异质的。MVP 显著富集在转录起始位点，导致在 HPV+肿瘤的亚组中鉴定出候选 CpG 岛甲基化表型。有监督分析确定 HPV+HNSCC 中 MVP 向高甲基化的优势（87%）。对我们的 HNSCC 和公开的宫颈癌和肺癌甲基化数据进行的荟萃分析证实，所观察到的 DNA 甲基化特征是 HPV 特异性和组织独立性的。将 MVP 分组为功能上更显著的差异甲基化区域，鉴定出 43 个高甲基化启动子 DMR，包括 Polycomb 靶基因家族的三个钙粘蛋白。与独立的表达数据整合显示出强烈的负相关，特别是钙粘蛋白基因家族成员。在 HPV- HNSCC 细胞系中异位表达两个 HPV 致癌基因（E6 和 E7）部分模拟了 HPV+ HNSCC 肿瘤中观察到的高甲基化特征，并确定 E6 为主要的病毒效应基因。
结论
我们的数据表明，存档的 FFPE 组织非常适合这种类型的甲基化组分析，并表明 HPV 通过高甲基化 Polycomb 抑制复合物 2 靶基因（如钙粘蛋白）来调节 HNSCC 表观基因组，这些基因与肿瘤进展和转移有关。
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