TRAF3/CYLD mutations identify a distinct subset of human papillomavirus-associated head and neck squamous cell carcinoma

Abstract

Background: The incidence of human papillomavirus (HPV)-associated (HPV-positive) head and neck squamous cell carcinoma (HNSCC) of the oropharynx has dramatically increased over the last decade and continues to rise. Newly diagnosed HPV-positive HNSCCs in the United States currently outnumber any other HPV-associated cancers, including cervical cancer. Despite introduction of the HPV vaccine, the epidemic of HPV-positive HNSCC is expected to continue for approximately 60 years. Compared with patients who have tobacco-associated HNSCC, those who have HPV-positive HNSCC have better overall survival and response to treatment. Current treatment, including chemotherapy and radiation therapy, is associated with lifelong morbidity, and there are limited treatments and no curative options for patients who develop recurrent metastatic disease. Therapeutic de-escalation (decreased radiation dose) is being tested through clinical trials; however, those studies select patients based solely on tumor and patient smoking characteristics. Mechanisms of HPV-driven carcinogenesis in HNSCC are not well understood, which limits new therapeutic strategies and hinders the appropriate selection of patients for de-escalation therapy.

Methods: The authors analyzed HNSCC data from The Cancer Genome Atlas to identify molecular characteristics that correlate with outcomes and integration status of the HPV genome.

Results: The current investigations identified a subset of HPV-positive HNSCCs with mutations in the genes TRAF3 (tumor necrosis factor receptor-associated factor 3) and CYLD (cylindromatosis lysine 63 deubiquitinase). Defects in TRAF3 and CYLD correlated with the activation of transcriptional factor nuclear factor κB, episomal HPV status of tumors, and improved patient survival.

Conclusions: Defects in TRAF3/CYLD were accompanied with the activation of nuclear factor κB signaling and maintenance of episomal HPV in tumors, suggesting that these mutations may support an alternative mechanism of HPV tumorigenesis in head and neck tumors. Cancer 2017;123:1778-1790. © 2017 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society. This is an open access article under the terms of the Creative Commons Attribution NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Keywords: head and neck squamous cell carcinoma; human papillomavirus (HPV); nuclear factor κB (NF-κB); prognosis; tumor necrosis factor receptor-associated factor 3 (TRAF3).

Figure 1

Tumor necrosis factor receptor‐associated factor 3 (TRAF3) or cylindromatosis lysine 63 deubiquitinase (CYLD) is mutated in 36% of human papillomavirus‐positive (HPV+) head and neck squamous cell carcinomas (HNSCCs). (A) Genetic alterations in the TRAF3 and CYLD genes were identified in patients with HPV‐positive (n = 36) and HPV‐negative (n = 243) HNSCC. Columns represent individual tumors. The green arrow indicates that several tumors without alterations were omitted to fit the figure. (A,B) Schematic representations of missense point (green) and truncating (black) mutations in the (B) TRAF3 and (C) CYLD genes identified in HPV‐positive HNSCC are shown. NEMO indicates NF‐κB essential modulator; phos_site, phosphorylation site; UCH, ubiquitin C‐terminal hydrolase; zf, zinc finger domain; CAP, cytoskeletal‐associated protein domain. Adapted from the cBioPortal for Cancer Genomics (available at: www.cbioportal.org).26, 27

Figure 2

Tumor necrosis factor receptor‐associated factor 3 (TRAF3) or cylindromatosis lysine 63 deubiquitinase (CYLD) genetic abnormalities are not present in human papillomavirus (HPV)‐positive uterine cervical cancer. (A) Alterations in the TRAF3 and CYLD genes are illustrated in cervical cancer (n = 191). The green arrow indicates several tumors without alterations that were omitted to fit the figure. (B) Cross‐cancer alterations are summarized for the TRAF3 and CYLD genes (combined) from 126 studies; studies that included < 5% alterations in genes were omitted to fit the figure. Adeno indicates adenocarcinoma; BCCRC, breast cancer patient xenografts; ACyC, adenoid cystic carcinoma; CCLE, Cancer Cell Line Encyclopedia; chRCC, chromophobe renal cell carcinoma; CNA, copy number alteration; CS, carcinosarcoma; DLBC, diffuse large B‐cell lymphoma; LUAD, lung adenocarcinoma; MICH, University of Michigan; MSKCC, Memorial Sloan Kettering Cancer Center; NCI, National Cancer Institute; NEPC, neuroendocrine prostate cancer; TCGA, The Cancer Genome Atlas; squ, squamous; UTSW, University of Texas Southwestern Medical Center. Adapted from the cBioPortal for Cancer Genomics (available at: www.cbioportal.org).26, 27

Figure 3

Tumor necrosis factor receptor‐associated factor 3 (TRAF3) or cylindromatosis lysine 63 deubiquitinase (CYLD) mutations correlate with the absence of human papillomavirus (HPV) integration and improved survival in HPV‐associated head and neck squamous cell carcinoma (HNSCC). Contingency graphs represent (A) the distribution of smokers or nonsmokers in the TRAF3/CYLD wild‐type (WT) and mutant (mt) groups (the P value was calculated using a 2‐sided Fisher exact test); (B) the distribution of tumors with or without HPV integration in the TRAF3/CYLD WT and mt groups (the P value was calculated using a 2‐sided Fisher exact test); and (C) the distribution of tumors with episomal HPV only, with HPV integrations only, or containing both integrated and episomal HPV DNA in the TRAF3/CYLD WT and mt groups (the P value was calculated using a 2‐sided chi‐square test). (D) Kaplan‐Meier curves illustrate the overall survival of patients who had HPV‐associated HNSCC with or without alterations in TRAF3/CYLD. Statistics were calculated using the log‐rank (Mantel‐Cox) test. Adapted from the cBioPortal for Cancer Genomics (available at: www.cbioportal.org).26, 27

Figure 4

Mutations in tumor necrosis factor receptor‐associated factor 3 (TRAF3) and cylindromatosis lysine 63 deubiquitinase (CYLD) are associated with activated nuclear factor κB (NF‐κB) in human papillomavirus (HPV)‐positive head and neck squamous cell carcinoma. The results from transcription factor target genes gene set enrichment analysis are illustrated for genes that had significantly different expression in (A) the TRAF3/CYLD‐mutant (mt) group versus (B) the wild‐type (WT) group. EGR1 indicates Early Growth Response 1; ES, enrichment score; TAF, TATA box binding protein associated factor; HNF‐3B (FOXA2), hepatocyte nuclear factor 3β; hits, genes from the dataset; P, nominal P value.

Figure 5

Classification of head and neck squamous cell carcinoma (HNSCC) based on tumor necrosis factor receptor‐associated factor 3 (TRAF3) and cylindromatosis lysine 63 deubiquitinase (CYLD) mutation status is illustrated. Results are shown from gene set enrichment analysis using data sets of (A,B) oncogenic and (C) hallmark and immunologic signatures for genes had significantly different expression in the TRAF3/CYLD mutant (mt) group versus the wild‐type (WT) group. ES indicates enrichment score; HPV−, human papillomavirus‐negative; HPV+, human papillomavirus‐positive; IFNB1, interferon β1; P, nominal P value.

Figure 6

The etiology of human papillomavirus (HPV)‐positive head and neck squamous cell carcinoma (HNSCC) is illustrated. (A) This is a model for the etiology of HPV‐positive HNSCC. (Top) After initial HPV infection, mutations in tumor necrosis factor receptor‐associated factor 3 (TRAF3) and cylindromatosis lysine 63 deubiquitinase (CYLD), or in other genes, lead to constitutively active nuclear factor κB (NF‐κB), which supports persistence of the HPV episome; these tumors will be sensitive to DNA damage. (Bottom) Initial HPV infection activates type I interferon (IFN) signaling that selects for DNA damage‐resistant tumors with HPV DNA integrated into the human genome. (B) This is a schematic model of (left) canonical and (right) noncanonical NF‐κB pathways. Inactivating mutations in TRAF3/CYLD (dashed red ovals) identified in HPV‐positive HNSCC result in the activation of both NF‐κB pathways. Inactivating (solid red ovals) or activating (green ovals) mutations that activate NF‐κB identified in other types of human cancer are shown. BAFF‐R indicates B‐cell activating factor receptor; BCL3, B‐cell leukemia/lymphoma 3; CD40, cluster of differentiation 40 (costimulatory protein on antigen‐presenting cells); cIAPs, cellular inhibitor of apoptosis proteins; dsRNA, double‐stranded RNA; IKK, inhibitor of NF‐κB kinase; IL1R, interleukin 1 receptor; MyD88, myeloid differentiation primary response 88; NEMO indicates NF‐κB essential modulator; NIK, NF‐κB B‐inducing kinase; RELB, RELB proto‐oncogene, NF‐κB subunit; Toll‐like receptor; TNFR, tumor necrosis factor receptor; TRIF, Toll/interleukin‐1 receptor (TIR)‐domain–containing adapter‐inducing interferon β.