Abstract 1368: 4NQO shows formation of yH2AX foci, oxidative base damage and activates DNA repair pathway preferentially in HPV positive HNSCC cells

Abstract

Abstract Background: There is an epidemic of HPV associated head and neck squamous cell carcinoma (HNSCC) There is controversy as to whether HPV positive SCC patients who have a >10 pack year smoking history have a worse prognosis than HPV associated non-smokers. Tobacco exposure is associated with alteration of DNA damage repair (DDR) pathway which is often activated in several cancers including HNSCC. A mutational profile of HPV associated smokers determined there was no significant difference in the mutational profile between HPV associated non-smokers and smokers. Hence we wanted to test this observation by analyzing the effects of 4NQO, a mutagen and carcinogen which serves as a surrogate for tobacco exposure, in HPV positive and HPV negative HNSCC cells. Methods: Protein and mRNA expression were analyzed by western blotting and real time PCR respectively. yH2AX staining and oxidative DNA damage analysis were performed using immunofluorescence and FPG-modified alkaline comet assay respectively. Results: Our preliminary data shows that 4NQO activates DDR pathway preferably in NOK E6/E7 and HPV positive UMSCC47 and SCC104 HNSCC cells as seen by increased phosphorylation of BRCA1, Rad50 and RAD51, chk1 and increased p21 levels in a time- and concentration-dependent fashion. Real time PCR data showed higher mRNA transcript levels of RAD51 and BRCA1 in NOK E6/E7 and HPV positive UMSCC47 and SCC104 HNSCC cells as compared to NOK and HPV negative SCC40 HNSCC cells upon 4NQO treatment. 4NQO causes yH2AX foci formation suggesting double strand breaks (DSBs) formation and an increase in DNA damage in HNSCC cells. 4NQO increased oxidative base damage in NOK E6/E7, UMSCC47 and SCC104 HPV positive HNSCC cells, and to a lesser degree in NOK and SCC40 HPV negative HNSCC cells. Conclusion: Our preliminary study shows for the first time that HPV E6/E7 oncogenes promote DNA repair in NOK and HPV positive HNSCC cells in response to 4NQO treatment. This could explain why HPV associated HNSCC have a better prognosis irrespective of smoking history. Citation Format: Gauri Shishodia, Rhodee Ric G. Toledo, Xiahuao Rong, Adam Y. Xiao, Lynn Harrison, Cherie Ann Nathan. 4NQO shows formation of yH2AX foci, oxidative base damage and activates DNA repair pathway preferentially in HPV positive HNSCC cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1368.