P16(Ink4a), A Surrogate Marker Of Hpv Infection And Prognosis For Head And Neck Cancer, Delays Dna Damage Repair And Enhances Radiation Response

Human papillomavirus (HPV) type 16 is a major cause of oropharyngeal carcinoma (OPC). P16 INK4A has been suggested to be a reliable surrogate marker of HPV-associated OPC with 100% sensitivity and about 80% specificity. Increasing data showed tumor HPV positivity or p16 expression was strongly associated with significantly better prognosis in patients with OPC. As a tumor suppressor gene, p16 has biological functions including regulation of cell cycle progression at the G1/S boundary, angiogenesis, cell senescence, tumor invasion, cell spreading, apoptosis and anoikis. The present study was undertaken to assess the role of p16 in regulating tumor radioresponse and the underlying mechanisms in OPC cell lines. OPC cell lines HN-5 (HPV and p16 negative) and UMSCC-47 (HPV and p16 positive) were used. P16 overexpressing HN-5 cells and shRNA p16 knockdown UMSCC-47 cells were generated using lentivirus vectors. Treatment endpoint was clonogenic cell survival (CSA) determined 10-12 days (for HN5) or 17-20 days (for UMSCC-47) after exposing the cells to 2-10 Gy single doses of γ-radiation (IR). Compared with the control (scramble) cells, p16-overexpressing HN5 cells had significantly higher radiosensitivity (by a factor of 1.56 at 0.1 cell survival fraction); whereas, p16-knockingdown UMSCC-47 cells had less radiosensitivity (by a factor of 1.23 at 0.1 cell survival fraction). Overexpressing P16 in HN-5 cells significantly prolonged the presence of radiation-induced double-strand breaks detected on the basis of 53BP1 foci at 24h after 4 Gy IR. To directly gauge damaged DNA, an alkaline comet assay to detect both single- and double-strand DNA breaks was performed. P16 overexpressing HN-5 cells exhibited a 2.09-fold increase in the comet ‘tail moment’ 48 hours after IR. This finding was supported by increased expression of 53BP1 analyzed by Western blot. In conclusion, other than being a robust surrogate marker for tumor control and survival outcome, our findings demonstrated that p16 also functions as a potent radiation sensitizer. The major underlying mechanism of p16 regulating radiosensitivity is by inhibition of DNA damage repair. Citation Format: Li Wang, Peijing Zhang, David P Molkentine, Hailong Piao, Chunyan Chen, Jessica M Molkentine, Jinsong Zhang, David R Valdecanas, Heath Skinner, Thomas A Buchholz, Junjie Chen, Li Ma, Kathy A Mason, Kie-kian Ang, Raymond E Meyn. P16 INK4A , a surrogate marker of HPV infection and prognosis for head and neck cancer, delays DNA damage repair and enhances radiation response. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4895. doi:10.1158/1538-7445.AM2014-4895