Abstract 2915: Identification of the mechanisms of radiosensitization by human papillomavirus (HPV) in head and neck cancer cell lines

Abstract

Abstract Background: Several clinical studies have shown that HPV+ head and neck squamous cell carcinomas (HNSCC) present a more favorable outcome and greater response to radiotherapy. Although there are some data supporting the hypothesis that HPV-related HNSCC exhibit higher sensitivity to radiotherapy, it is difficult to conclude that the improved outcome of HPV-related HNSCC is only attributable to intrinsic radiosensitivity of the HPV+ cells. We postulate that a complex interaction occurs between intrinsic mechanisms of radio-response and the tumor micro-environment including cells of the immune system stimulated by the presence of HPV. The objectives of this work are to study mechanisms of radio-response in HPV+ cells and to investigate whether irradiated HPV+ cells exhibit increased immunogenic cell death signals including membrane exposure of endoplasmatic reticulum-resident chaperone calreticulin, secretion of High-Mobility-Group-Box 1 protein (HMGB1) and ATP release. Material and Methods: We determined radiosensitivity by clonogenic survival of two HPV+ HNSCC cell lines (UPCI-SCC-154 & UPCI-SCC90) compared to two HPV- cells (SCC61 & SQD9). Cell cycle distribution and G2/M checkpoint were assessed by flow cytometry. DNA damage repair was evaluated by gamma-H2Ax assay, HR and NHEJ reporter gene assay. In addition, cell death (apoptosis, necrosis and mitotic catastrophe) and senscence were measured in the four cell lines together with immunogenic cell death signals, through cytokines, HMGB1, and ATP-release as well as, membrane exposure of calreticulin. Results: 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 cell line already described as radiosensitive. Cell cycle distribution indicated an important block in G2/M 24h after irradiation for the HPV+ cell lines together with a slower clearance of gamma-H2Ax for HPV+ cells, compared to HPV- cells. Furthermore, HPV+ cells showed decreased DNA repair capacity by NHEJ and HR. Apoptosis rate after irradiation (2Gy) was variable depending on the cell line, but remained overall under 5%. Senescence was not increased after irradiation (2Gy) whereas mitotic catastrophe increased in HPV-negative cell lines after 24h. Cytokine secretion was cell line dependent while HMGB1 and ATP release was increased in HPV+ cell lines compared to HPV- cell lines. Finally, calreticulin membrane exposure after irradiation detected by immunofluorescence was more frequent in HPV+ compared with HPV- cells. Conclusions: Our results confirm an increased radiosenstitivity of HPV+ cells after irradiation together with increased signals recognised as “immunogenic cell death” (calreticulin exposure, HMGB1 and ATP release) providing a rationale to investigate immune responses in HPV+ tumors after radiotherapy. Note: This abstract was not presented at the meeting. Citation Format: Vanesa Bol. Identification of the mechanisms of radiosensitization by human papillomavirus (HPV) in head and neck cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2915. doi:10.1158/1538-7445.AM2015-2915