Abstract 3192A: Senescence may play an important role in explaining the mechanisms of increased radiosensitivity of HPV- and EBV-associated HNSCC
Authors: Anandharaj Arunkumar , Ekshyyan Oleksandr , Rong Xiaohua , Hartel Dylan , Bauerle Brandon , Nathan Sean , Harrison Lynn , Scott Rona , Shi Runhua , Nathan Cherie-Ann O. 
Published: May 2015

Background: Viral associated HPV and EBV squamous cell carcinomas are common in the oropharynx and nasopharynx respectively. In fact co-infection of these viruses is reported in 15-46% of nasopharyngeal carcinomas, 13% of oral cancers and 11% of other head and neck tumors. Viral associated HNSCC tumors are inherently radiosensitive when compared to tobacco associated malignancies. We had determined using stable clones generated in a HNSCC cell line that HPV, EBV and the combination was significantly more radiosensitive than the negative viral clone. However mechanisms of increased sensitivity had not been defined. The aim of the study is to elucidate the molecular mechanisms of increased radiosensitivity in viral associated HNSCC.

Methods: To test our hypothesis four stable clones of FaDu cells (HPV-/EBV-, HPV+/EBV-, HPV-/EBV+, HPV+/EBV+) generated by transfection with HPV E6/7 ORF constructs or/and infection with recombinant EBV strain were irradiated with 4Gy. Cell cycle was analyzed using FACS and protein expression was evaluated by western blotting. Senescence associated beta-galactosidase (SABG) staining was used to determine the level of senescence.

Results: Cell cycle analysis at various time points (0-72h) after 4Gy γ-radiation revealed the viral positive clones showed increased G1/S arrest compared to the HPV-/EBV- clone and resumed cycling earlier at 24h compared to recovery at 48h noted in the viral negative clone. Deregulated phosphorylation of DNA repair components such as ATM and ATR was observed in the viral clones while steady state phosphorylation was seen in the negative clone. Cell cycle and protein analysis did not show increased apoptosis in the viral clones. However p21 protein expression was significantly increased in the viral positive clones after radiation treatment (P&lt;0.05) compared to negative clone. Given the increase in p21, a marker of cellular senescence, SABG staining was performed. There was an accumulation of senescent cells after radiation (at 72h: 80% increase for HPV-/EBV-; 217% increase for HPV+/EBV-; 215% increase for HPV-/EBV+ and 106% increase for HPV+/EBV+ over the respective baselines). There was a significant increase in the percentage of senescent cells in the irradiated HPV+/EBV- (P&lt;0.01) and HPV-/EBV+ (P&lt;0.05) clones compared to the irradiated HPV-/EBV- clone.

Conclusion: We show that both HPV and EBV disrupt cell cycle control and DNA repair signaling. Radiation-induced upregulation of p21 expression in viral clones was associated with reduced clonogenicity, altered cell cycle progression and an increase in the percentage of cells with markers of premature cellular senescence. The faster recovery of cell cycle in the viral clones could result in incomplete DNA repair resulting in genomic instability and an increase in cellular senescence.

Citation Format: Arunkumar Anandharaj, Oleksandr Ekshyyan, Xiaohua Rong, Dylan Hartel, Brandon Bauerle, Sean Nathan, Lynn Harrison, Rona Scott, Runhua Shi, Cherie-Ann O. Nathan. Senescence may play an important role in explaining the mechanisms of increased radiosensitivity of HPV- and EBV-associated HNSCC. [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 3192A. doi:10.1158/1538-7445.AM2014-3192A