Human papillomavirus mediated inhibition of DNA damage sensing and repair drives skin carcinogenesis.
BACKGROUND
The failure to mount an effective DNA damage response to repair UV induced cyclobutane pyrimidine dimers (CPDs) results in an increased propensity to develop cutaneous squamous cell carcinoma (cSCC). High-risk patient groups, such as organ transplant recipients (OTRs) frequently exhibit field cancerization at UV exposed body sites from which multiple human papillomavirus (HPV)-associated cSCCs develop rapidly, leading to profound morbidity and increased mortality. In vitro molecular evidence indicates that HPV of genus beta-papillomavirus (β-PV) play an important role in accelerating the early stages of skin tumorigenesis.


METHODS
We investigated the effects of UV induced DNA damage in murine models of β-PV E6 oncoprotein driven skin tumorigenesis by crossing K14-HPV8-E6wt mice (developing skin tumors after UV treatment) with K14-CPD-photolyase animals and by generating the K14-HPV8-E6-K136N mutant mouse strain. Thymine dimers (marker for CPDs) and γH2AX (a marker for DNA double strand breaks) levels were determined in the murine skin and organotypic skin cultures of E6 expressing primary human keratinocytes after UV-irradiation by immunohistochemistry and in cell lines by In Cell Western blotting. Phosphorylation of ATR/Chk1 and ATM were assessed in cell lines and organotypic skin cultures by Western blots and immunohistochemistry.


RESULTS
Skin tumor development after UV-irradiation in K14-HPV8-E6wt mice could completely be blocked through expression of CPD-photolyase. Through quantification of thymine dimers after UV irradiation in cells expressing E6 proteins with point mutations at conserved residues we identified a critical lysine in the C-terminal part of the protein for prevention of DNA damage repair and p300 binding. Whereas all K14-HPV8-E6wt animals develop skin tumors after UV expression of the HPV8-E6-K136N mutant significantly blocked skin tumor development after UV treatment. The persistence of CPDs in hyperproliferative epidermis K14-HPV8-E6wt skin resulted in the accumulation of γH2AX foci. DNA damage sensing was impaired in E6 positive cells grown as monolayer culture and in organotypic cultures, due to lack of phosphorylation of ATM, ATR and Chk1.


CONCLUSIONS
We showed that cells expressing E6 fail to sense and mount an effective response to repair UV-induced DNA lesions and demonstrated a physiological relevance of E6-mediated inhibition of DNA damage repair for tumor initiation. These are the first mechanistical in vivo data on the tumorigenicity of HPV8 and demonstrate that the impairment of DNA damage repair pathways by the viral E6 protein is a critical factor in HPV-driven skin carcinogenesis.
Institute of Virology, University of Cologne, Fürst-Pückler-Str. 56, Cologne, 50935, Germany.
Martin
M
Hufbauer
Centre for Cutaneous Research, The Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, E1 2AT, UK.
James
J
Cooke
MGC, Department of Genetics, Center for Biomedical Genetics, Erasmus University Medical Center, Rotterdam, 3000, CA, The Netherlands.
Gijsbertus T J
GT
van der Horst
Institute of Virology, University of Cologne, Fürst-Pückler-Str. 56, Cologne, 50935, Germany.
Herbert
H
Pfister
Department of Oncology, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, OX3 9DS, UK.
Alan
A
Storey
Institute of Virology, University of Cologne, Fürst-Pückler-Str. 56, Cologne, 50935, Germany. baki.akguel@uk-koeln.de.
Baki
B
Akgül
D000818:Animals; D004249:DNA Damage / Q000235:genetics* / Q000528:radiation effects; D004260:DNA Repair / Q000235:genetics* / Q000528:radiation effects; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D027383:Papillomaviridae / Q000235:genetics*; D012878:Skin Neoplasms / Q000209:etiology / Q000235:genetics*; D014466:Ultraviolet Rays / Q000009:adverse effects