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Attenuated TRAF3 Fosters Alternative Activation of NF-Κb and Reduced Expression of Anti-Viral Interferon, TP53, and RB to Promote HPV-Positive Head and Neck Cancers

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Attenuated TRAF3 Fosters Alternative Activation of NF-Κb and Reduced Expression of Anti-Viral Interferon, TP53, and RB to Promote HPV-Positive Head and Neck Cancers Author Manuscript Published OnlineFirst on June 19, 2018; DOI: 10.1158/0008-5472.CAN-17-0642 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Attenuated TRAF3 fosters alternative activation of NF-κB and reduced expression of anti- viral interferon, TP53, and RB to promote HPV-positive head and neck cancers Jialing Zhang,1,2 Tony Chen,1 Xinping Yang,1 Hui Cheng,1 Stephan S. Späth,3 Paul E. Clavijo,1 Jianhong Chen,1 Christopher Silvin,1 Natalia Issaeva,4 Xiulan Su,2 Wendell G. Yarbrough,4,5 Christina M. Annunziata,6 Zhong Chen,1* Carter Van Waes1* 1Head and Neck Surgery Branch, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland, USA; 2Clinical Medicine Research Center, The Affiliated Hospital, Inner Mongolia Medical University, Hohhot, China; 3Pediatric Endocrinology Unit, Department of Women’s and Children’s Health, Karolinska Institute and University Hospital, Stockholm, Sweden; 4Department of Surgery, Otolaryngology, Yale Cancer Center; 5Department of Pathology, Yale School of Medicine, New Haven, CT, USA; and 6Women's Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA. *Corresponding authors: Carter Van Waes, MD, PhD, NIDCD/NIH, Bldg 10/Rm 7N240, 10 Center Drive, Bethesda, MD 20892. Email: [email protected]. Phone: 301-402-4216. Fax: 301-402-1140. Zhong Chen, MD, PhD, NIDCD/NIH, Bldg 10/Rm 7N240, 10 Center Drive, Bethesda, MD 20892. Email: [email protected]. Phone: 301-435-2073. Fax: 301-402- 1140. Disclosure of Potential Conflicts of Interest: No potential conflicts of interest were disclosed by the other authors. Running Title: Decreased TRAF3 promotes HPV+ HNSCC Key words: TRAF3, alternative NF-B, interferon HPV, HNSCC 1 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 19, 2018; DOI: 10.1158/0008-5472.CAN-17-0642 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Significance Findings report the functional role of TRAF3 as a tumor suppressor that modulates the malignant phenotype of HPV+ head and neck cancers 2 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 19, 2018; DOI: 10.1158/0008-5472.CAN-17-0642 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Human papilloma viruses (HPV) are linked to an epidemic increase in oropharyngeal head and neck squamous cell carcinomas (HNSCC), which display viral inactivation of tumor suppressors TP53 and RB1 and rapid regional spread. However, the role of genomic alterations in enabling modulation of pathways that promote the aggressive phenotype of these cancers is unclear. Recently, a subset of HPV+ HNSCC has been shown to harbor novel genetic defects or decreased expression of TNF-receptor-associated-factor-3 (TRAF3). TRAF3 has been implicated as a negative regulator of alternative NF-B pathway activation, and activator of anti-viral type-I interferon (IFN) response to other DNA viruses. How TRAF3 alterations affect pathogenesis of HPV+ HNSCC has not been extensively investigated. Here, we report that TRAF3-deficient HPV+ tumors and cell lines exhibit increased expression of alternative NF-B pathway components and transcription factors NF-B2/RELB. Overexpression of TRAF3 in HPV+ cell lines with decreased endogenous TRAF3 inhibited NF- B2/RELB expression, nuclear localization, and NF-B reporter activity, while increasing the expression of IFNA1 mRNA and protein and sensitizing cells to its growth inhibition. Overexpression of TRAF3 also enhanced TP53 and RB tumor suppressor proteins and decreased HPV E6 oncoprotein in HPV+ cells. Correspondingly, TRAF3 inhibited cell growth, colony formation, migration, and resistance to TNF-α and cisplatin-induced cell death. Conversely, TRAF3 knockout enhanced colony formation and proliferation of an HPV+ HNSCC line expressing higher TRAF3 levels. Together, these findings support a functional role of TRAF3 as a tumor suppressor modulating established cancer hallmarks in HPV+ HNSCC. 3 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 19, 2018; DOI: 10.1158/0008-5472.CAN-17-0642 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common cancer, with an annual incidence of 650,000 new cases and ~200,000 deaths worldwide(1,2). Persistent infection with high-risk human papillomavirus subtypes HPV16 and HPV18 has been established as an important risk factor for HNSCC that develop predominantly in the oropharyngeal tonsils (3). Since 1981, there has been a notable 225% increase in HPV+ HNSCC, while the incidence of smoking-related HPV- HNSCC has declined (4,5). Clinically, the HPV+ subset exhibits better responses to therapies and survival rates than similarly advanced HPV- tumors. However, HPV+ HNSCC are distinguished by aggressive spread and growth within regional lymph nodes, which require major surgery or toxic chemoradiotherapy regimens (2,3). The factors that contribute to the molecular pathogenesis of these unique features of HPV+ HNSCC remain incomplete. HPV16 and 18 carry early genes E6 and E7 encoding oncoproteins that target key pathways, deregulating host resistance to infection and cellular proliferation, to promote the viral life cycle. HPV E6 expression in keratinocytes can repress type I interferon (IFN) and promote proliferative genes, to enhance viral protein synthesis and proliferation of virally infected cells (6). Further studies have shown that HPV infection can induce ubiquitin carboxyl-terminal hydrolase LI (UCHL1), which can inhibit K63 ubiquination, important in Tank Binding Kinase-Immune Response Factor 3 (TBK-IRF3)-mediated type I IFN expression (7). HPV E6 can also commandeer and activate the so-named alternative Nuclear Factor-B2 (NF-B2) transcription factors and anti-apoptotic genes, that promote resistance of keratinocytes to Tumor Necrosis Factor (TNF), an important mediator of antiviral immunity (8). Critically, the HPV E6 and E7 oncoproteins also strategically target for degradation the 4 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 19, 2018; DOI: 10.1158/0008-5472.CAN-17-0642 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. tumor suppressor proteins TP53 and RB, that control the cell cycle (9). Interestingly however, few individuals exposed to HPV develop chronic infection and HNSCC. These observations suggest that additional genetic alteration(s) and host factors may also affect how HPV mediates suppression of IFNs, NF-B activation, inhibition of TP53 and RB gene expression, and the malignant phenotype. Recently, we and The Cancer Genome Atlas Network uncovered a subset of HPV+ HNSCC that harbor deletions of Chromosome region 14q32.32, deleterious truncating mutations, and/or decreased expression, affecting the gene Tumor Necrosis Factor (TNF) receptor-associated Factor 3 (TRAF3) (10). These deletions were not due to viral integration and disruption of the TRAF3 gene. Intriguingly, TRAF3 is a unique adaptor protein and ubiquitin ligase implicated as a negative regulator of the aforementioned alternative NF- B2/RELB pathway (11). TRAF3 promotes cIAP-mediated ubiquitination and proteasome- dependent degradation of the pivotal NF-B inducing kinase (NIK) protein, that mediates signal activation of the alternative pathway. Lymphotoxin- (LT) and other ligands, that are richly expressed in the oropharyngeal tonsils and lymph nodes where HPV+ HNSCC arise and spread, bind receptors to activate NIK, Inhibitor-B kinase- (IKK), processing of NF- κB2 p100 to p52, and nuclear translocation of transcriptionally active NF-κB2p52/RELB dimers. Attenuation of TRAF3 has previously been implicated in the transcription of genes affecting cell fate, proliferation, and survival of lymphoid cells and hematopoietic malignancies (11). Strikingly, TRAF3 has also been shown to serve a dual function in interferon responses to other DNA viruses (11,12). However, the functional role of genetic alterations or reduced expression of TRAF3 in modulating alternative NF-B pathway 5 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 19, 2018; DOI: 10.1158/0008-5472.CAN-17-0642 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. activation, IFN expression, repression of tumor suppressors TP53 and RB, and the malignant phenotype in HPV+ HNSCC has not been established. In this study, we examined and reveal a novel role for decreased TRAF3 in fostering deregulation of these pathways and promoting pathogenesis of a subset of HPV+ HNSCC. These findings identify new opportunities for basic and clinical research that may lead to better diagnosis, prevention and treatment of HPV+ HNSCC. METHODS HNSCC Patient Samples and TCGA Bioinformatics Analysis TCGA Project
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