title: Methylation-specific digital karyotyping of HPV16E6E7-expressing human keratinocytes identifies novel methylation events in cervical carcinogenesis.
abstract: Transformation of epithelial cells by high-risk human papillomavirus (hrHPV) types can lead to anogenital carcinomas, particularly cervical cancer, and oropharyngeal cancers. This process is associated with DNA methylation alterations, often affecting tumour suppressor gene expression. This study aimed to comprehensively unravel genome-wide DNA methylation events linked to a transforming hrHPV-infection, which is driven by deregulated expression of the viral oncogenes E6 and E7 in dividing cells. Primary human keratinocytes transduced with HPV16E6E7 and their untransduced counterparts were subjected to methylation-specific digital karyotyping (MSDK) to screen for genome-wide DNA-methylation changes at different stages of HPV-induced transformation. Integration of the obtained methylation profiles with genome-wide gene expression patterns of cervical carcinomas identified 34 genes with increased methylation in HPV-transformed cells and reduced expression in cervical carcinomas. For 12 genes (CLIC3, CREB3L1, FAM19A4, LFNG, LHX1, MRC2, NKX2-8, NPTX-1, PHACTR3, PRDM14, SOST and TNFSF13) specific methylation in HPV-containing cell lines was confirmed by semi-quantitative methylation-specific PCR. Subsequent analysis of FAM19A4, LHX1, NKX2-8, NPTX-1, PHACTR3 and PRDM14 in cervical tissue specimens showed increasing methylation levels for all genes with disease progression. All six genes were frequently methylated in cervical carcinomas, with highest frequencies (up to 100%) seen for FAM19A4, PHACTR3 and PRDM14. Analysis of hrHPV-positive cervical scrapes revealed significantly increased methylation levels of the latter three genes in women with high-grade cervical disease compared to controls. In conclusion, MSDK analysis of HPV16-transduced keratinocytes at different stages of HPV-induced transformation resulted in the identification of novel DNA methylation events, involving FAM19A4, LHX1, NKX2-8, PHACTR3 and PRDM14 genes in cervical carcinogenesis. These genes may provide promising triage markers to assess the presence of (pre)cancerous cervical lesions in hrHPV-positive women.
authors.affiliation: Department of Pathology, VU University Medical Centre, Amsterdam, The Netherlands. r.steenbergen@vumc.nl
authors.forename: Renske D M
authors.initials: RD
authors.lastname: Steenbergen
authors.forename: Maté
authors.initials: M
authors.lastname: Ongenaert
authors.forename: Suzanne
authors.initials: S
authors.lastname: Snellenberg
authors.forename: Geert
authors.initials: G
authors.lastname: Trooskens
authors.forename: Wendy F
authors.initials: WF
authors.lastname: van der Meide
authors.forename: Deeksha
authors.initials: D
authors.lastname: Pandey
authors.forename: Noga
authors.initials: N
authors.lastname: Bloushtain-Qimron
authors.forename: Kornelia
authors.initials: K
authors.lastname: Polyak
authors.forename: Chris J L M
authors.initials: CJ
authors.lastname: Meijer
authors.forename: Peter J F
authors.initials: PJ
authors.lastname: Snijders
authors.forename: Wim
authors.initials: W
authors.lastname: Van Criekinge
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D002472:Cell Transformation, Viral; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005260:Female; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D052162:Human papillomavirus 16 / Q000502:physiology; D006801:Humans; D007621:Karyotyping / Q000379:methods; D015603:Keratinocytes / Q000378:metabolism* / Q000821:virology; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D050725:Papillomavirus E7 Proteins / Q000235:genetics*; D030361:Papillomavirus Infections / Q000235:genetics* / Q000821:virology; D011230:Precancerous Conditions; D060888:Real-Time Polymerase Chain Reaction; D012097:Repressor Proteins / Q000235:genetics*; D014161:Transduction, Genetic; D014162:Transfection; D002583:Uterine Cervical Neoplasms / Q000235:genetics* / Q000821:virology
chemical_list: D014408:Biomarkers, Tumor; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012097:Repressor Proteins; C059731:oncogene protein E7, Human papillomavirus type 16
keywords: DNA methylation; biomarkers; cervical cancer; epigenetic reprogramming; human papillomavirus