Abstract 2919: p16 modulates a novel ubiquitin signaling cascade which regulates radioresponse and offers clinically relevant therapeutic targets in squamous cell carcinoma
Authors: Molkentine David , Bridges Kathleen , Sheth Aakash , Valdecanas David , Pickering Curtis , Skinner Heath D. 
Published: August 2019

Abstract
Human papilloma virus (HPV) is associated with multiple types of squamous cell carcinoma, including that of the head and neck (HNSCC). The presence of HPV leads to increased expression of p16, to the extent that p16 is used as a surrogate marker of HPV positivity. HPV/p16 expression is associated with improved response to genotoxic therapy and improved outcome; however, the mechanism of this phenomenon is unclear. Previously, we showed that HPV/p16 expression repressed the DNA damage repair protein TRIP12 and led to radiosensitivity. In the current project we identify USP7 and HUWE1 as key links in this signaling pathway. Ubiquitin-specific-processing protease 7 (USP7) is a deubiquitinating enzyme that has previously been shown to bind to TRIP12, which we confirmed in HPV (-) HNSCC cell lines. We found that forced p16 overexpression in a panel of SCC cell lines led to decreased levels of USP7 and TRIP12 protein, while inhibition of p16 in HPV (+) HNSCC cell lines led to their increase. Forced p16 expression did not affect USP7 mRNA expression, however did lead to increased K48-linked ubiquitinylation and degradation of USP7. Chemical or shRNA mediated inhibition of USP7 in p16/HPV (-) cells led to decreased 53BP1 and BRCA1 foci and increased cell death following radiation. Expression of USP7 shRNA in a HPV (-), radioresistant cell derived xenograft (HN5) lead to a tumor growth delay (TGD) of 10 days following radiation (4 Gy x 5d), compared to a TGD of 1.4 days in control tumors (p&lt;1x10-15). To determine how p16 regulates USP7 ubquitinylation and degradation, we performed immunoprecipitation/mass spectrography (IP/MS) analysis. In 3 HPV (-) and 3 HPV (+) cell lines, pulldown of USP7 identified several E3 ubquitin ligases, however only HUWE1 and TRIM21 were identified in all cell lines tested. We then examined HUWE1 expression following forced expression of p16 and found that both HUWE1 mRNA and protein were increased. Moreover, inhibition of p16 expression in HPV (+) HNSCC cell lines led to decreased HUWE1 expression. Moreover, inhibition of HUWE1 via shRNA could partially rescue the effects of p16 overexpression on expression of DNA damage repair proteins. As HUWE1 is known to be mutated in ~10% of HNSCC, we then examined the effects of HUWE1 on outcome in the TCGA HNSCC patient cohort. In HPV (+) patients, neither HUWE1 mutation nor its gene expression were associated with survival. However, in HPV (-) patients, low HUWE1 expression was associated with worse disease-free survival (DFS)(p=0.048). Additionally, truncating mutations in HUWE1 led to a median survival of 9.4 mos, compared to 67.7 mos in the remaining patients (p=0.008). In conclusion, p16-HUWE1-USP7 signaling can modulate response to radiation. These data suggest that agents under investigation to target USP7, or other members of this signaling cascade, have the potential to improve outcomes in HNSCC.
Citation Format: David Molkentine, Kathleen Bridges, Aakash Sheth, David Valdecanas, Curtis Pickering, Heath D. Skinner. p16 modulates a novel ubiquitin signaling cascade which regulates radioresponse and offers clinically relevant therapeutic targets in squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2919.