Viral load, gene expression and mapping of viral integration sites in HPV16-associated HNSCC cell lines

Nadine C. Olthof, Christian U. Huebbers*, Jutta Kolligs, Mieke Henfling, Frans C. S. Ramaekers, Iris Cornet, Josefa A. van Lent-Albrechts, Alexander P. A. Stegmann, Steffi Silling, Ulrike Wieland, Thomas E. Carey, Heather M. Walline, Susanne M. Gollin, Thomas K. Hoffmann, Johan de Winter, Bernd Kremer, Jens P. Klussmann, Ernst-Jan M. Speel

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

60 Citations (Web of Science)

Abstract

HPV-related HNSCC generally have a better prognosis than HPV-negative HNSCC. However, a subgroup of HPV-positive tumors with poor prognosis has been recognized, particularly related to smoking, EGFR overexpression and chromosomal instability. Viral integration into the host genome might contribute to carcinogenesis, as is shown for cervical carcinomas. Therefore, all HPV16-positive HNSCC cell lines currently available have been carefully analyzed for viral and host genome parameters. The viral integration status, viral load, viral gene expression and the presence of aneusomies was evaluated in the cell lines UD-SCC-2, UM-SCC-047, UM-SCC-104, UPCI:SCC090, UPCI:SCC152, UPCI:SCC154 and 93VU147T. HPV integration was examined using FISH, APOT-PCR and DIPS-PCR. Viral load and the expression of the viral genes E2, E6 and E7 were determined via quantitative PCR. All cell lines showed integration-specific staining patterns and signals indicating transcriptional activity using FISH. APOT- and DIPS-PCR identified integration-derived fusion products in six cell lines and only episomal products for UM-SCC-104. Despite the observed differences in viral load and the number of viral integration sites, this did not relate to the identified viral oncogene expression. Furthermore, cell lines exhibited EGFR expression and aneusomy (except UPCI:SCC154). In conclusion, all HPV16-positive HNSCC cell lines showed integrated and/or episomal viral DNA that is transcriptionally active, although viral oncogene expression was independent of viral copy number and the number of viral integration sites. Because these cell lines also contain EGFR expression and aneusomy, which are parameters of poor prognosis, they should be considered suitable model systems for the development of new antiviral therapies. What's new? High-risk human papillomavirus (HPV) infection is a well-established risk factor for head and neck squamous cell carcinoma (HNSCC) development. It is still unclear however whether viral DNA integration into the host genome plays an important role in HPV carcinogenesis in HNSCC. In this study, seven HPV16-positive HNSCC cell lines showed integrated and/or episomal viral DNA that is transcriptionally active. Both viral load and expression of several viral early genes were variable. Because the cell lines also feature EGFR overexpression and aneusomy, which are parameters of poor prognosis, they may represent suitable model systems for the development of new antiviral therapies.
Original languageEnglish
Pages (from-to)E207-E218
JournalInternational Journal of Cancer
Volume136
Issue number5
DOIs
Publication statusPublished - 1 Mar 2015

Keywords

  • APOT-PCR
  • DIPS-PCR
  • integration
  • viral integration
  • genetic localization

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