Abstract The molecular mechanisms that control transformation and tumorigenicity of human papillomavirus-associated (HPV+) head and neck squamous cell carcinomas (HNSCCs) are currently being investigated. The number of HPV+ HNSCC cases has increased sharply in recent years, especially in tonsil and oropharyngeal cancers. These HPV+ HNSCCs often respond differently to treatments. Recently, The Cancer Genome Atlas (TCGA, 2015) identified novel loss-of-function genomic alterations of TNF receptor-associated factor 3 (TRAF3) in HPV+ HNSCCs. TRAF3 is a ring-finger E3 Ubiquitin Ligase which inhibits downstream alternative NF-κB signaling and promotes anti-viral immunity by promoting degradation of ubiquitinated proteins. To assess TRAF3’s role in HPV+ HNSCCs, we identified cell lines with lower level of TRAF3 protein, consistent with deficient TRAF3 expression identified in TCGA data. Functional studies showed that TRAF3 expression led to decreased steady-state protein levels of the alternative NF-κB pathway components RELB and NF-κB2/p52, as assessed by Western blot, reporter assays and immunofluorescence. Additionally, TRAF3 expression led to decreased cell proliferation, tumorigenic activity and migration, and increased sensitivity to chemotherapy agent cisplatin. Interestingly, TRAF3 increases the steady state protein level of the classical tumor suppressors RB and p53 in HPV+ HNSCC cell lines as assessed by Western blot. Further in vitro characterization of TRAF3’s function in HPV+ HNSCCs was assessed using clinically identified TRAF3 loss-of-function mutagenic isoforms, mimicking both TCGA data and HNSCC cell line data for TRAF3 defects. In contrast to wtTRAF3, these mutant forms of TRAF3 do not as strongly restore p53 or inhibit RELB and NF-κB2/p52 protein levels. One specific mutation which causes a frameshift at residue 210 greatly inhibits TRAF3 function as assessed by Western blot. To further assess effects of wt or TRAF3 loss-of-function mutants in vitro and in vivo, an inducible HPV+ HNSCC line expressing TRAF3 using the TET-ON system, and TRAF3 knockout in HPV+ HNSCC line and Human Oral Keratinocyte (HOK) line using CRISPR-Cas9 genomic editing are generated. In conclusion, the ring-finger E3 ubiquitin ligase TRAF3 inhibits the pro-survival alternative NF-κB signaling pathway and restores TP53 and RB, thereby serving as a tumor suppressor in HPV+ HNSCCs. (Supported by NIDCD intramural project ZIA-DC-000016, 73 and 74) Citation Format: Tony W. Chen, Jialing Zhang, Xinping Yang, Zhong Chen, Carter Van Waes. TRAF3 serves as a tumor suppressor in HPV-associated head and neck squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 537. doi:10.1158/1538-7445.AM2017-537
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