Abstract 4896: Identification of the mechanisms of radiosensitization by human papillomavirus (HPV) in head and neck cancers cell lines

Abstract

Abstract It has been shown in several clinical studies that HPV-positive head and neck squamous cell carcinoma (HNSCC) have a more favorable outcome and greater response to therapy. The reason for improved prognosis of HPV-related head and neck cancers remains speculative. One hypothesis is that HPV-positive cells are intrinsically more sensitive to standard therapies and thus, respond better to treatment. Clinical studies suggest indeed that HPV-related HNSCC actually display enhanced sensitivity to concurrent chemoradiation therapy. The goal of this project is to verify this hypothesis and elucidate the underlying mechanisms. For this purpose, we determined radiosensitivity by clonogenic survival of two HPV positive HNSCC cell lines (UPCI-SCC-154 & UPCI-SCC-90) compared to two HPV negative ones (SCC-61 & SQD9). Apoptosis was measured by Annexin V/PI staining. Cell cycle distribution and G2/M checkpoint were assessed by flow cytometry. DNA damage repair was evaluated by gamma-H2Ax assay. In addition, we investigated basal DNA damage repair efficiency by reporter gene assay for homologous recombination (HR) and non-homologous end-joining (NHEJ). The surviving fraction at 2Gy (SF2) for the two HPV+ cell lines was 0,13 and 0,15 for UPCI-SCC-90 and UPCI-SCC-154, respectively while SF2 for SQD9 was 0,49 and 0,16 for SCC-61 a HNSCC cell line described as radiosensitive. At 24h after irradiation (2Gy) there was no significant difference in apoptosis (less than 5%) between HPV- and HPV+ cell lines. However further time points still need to be analysed. Cell cycle distribution 24h after irradiation, indicated a significant increase in G2/M in HPV+ cells but also in SCC61. The kinetics of H2Ax phosphorylation after a single dose of 2 Gy correlated the SF2 data with a slower clearance of H2Ax for HPV+ cells, as well as for radiosensitive SCC61. However, analysis of remaining gH2AX depending on cell cycle phase indicated a slower clearance for cells in G2/M phase in HPV+ cell lines (p<0,001). Furthermore, HPV+ cells show decreased efficiency in NHEJ but also in HR which occurs mainly in G2/M phase. These results taken together, confirm an increased radiosensitivity of HPV+ cells and an impaired DNA damage repair mainly by HR although at this point further investigation is needed to elucidate the exact mechanisms. In order to investigate whether increased radiosensitivity is directly related to the presence of viral oncoproteins E6 and/or E7 we transfected HPV- HCT116 colon carcinoma cell line (p53 wt and -/-) with the oncoproteins and we plan to assess their radioresponse in terms of cell cycle arrest and DNA damage repair. Citation Format: Vanesa Bol, Vincent Grégoire. Identification of the mechanisms of radiosensitization by human papillomavirus (HPV) in head and neck cancers cell lines. [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 4896. doi:10.1158/1538-7445.AM2014-4896