Identification and functional validation of HPV-mediated hypermethylation in head and neck squamous cell carcinomaReport as inadecuate




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Genome Medicine

, 5:15

6. Ethical, legal and social issues

Abstract

BackgroundHuman 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.

MethodsUsing 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 discussionUnsupervised 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.

ConclusionsOur 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.

AbbreviationsCIMPCpG island methylator phenotype

DMEMDulbecco-s modified Eagle-s medium

DMRDifferentially methylated region

DNMTDNA methyltransferase

EMTEpithelial to mesenchymal transition

FBSFetal bovine serum

FDRFalse-discovery rate

FFfresh-frozen

FFPEFormalin-fixed, paraffin wax-embedded

GADPHGlyceraldehyde-3-phosphate dehydrogenase

HEKHhuman embryonic kidney

HNSCCHead and neck squamous cell cancer

HPVHuman papillomavirus

HPV+HPV-positive

HPV-HPV-negative

MDSMulti-dimensonal scaling

MVPMethylation variable position

PcGPolycomb group proteins

PRC2Polycomb repressive complex 2

qPCRQuantitative polymerase chain reaction

RMTRandom matrix theory

TSStranscription start site

UTRuntranslated region.

Electronic supplementary materialThe online version of this article doi:10.1186-gm419 contains supplementary material, which is available to authorized users.

Tim Fenton, James West contributed equally to this work.

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Author: Matthias Lechner - Tim Fenton - James West - Gareth Wilson - Andrew Feber - Stephen Henderson - Christina Thirlwell - Harpr

Source: https://link.springer.com/







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