Attenuated TRAF3 Fosters Activation of Alternative NF-kB ...Drs. Thomas E. Carey, Mark E. Prince,...

Tumor Biology and Immunology

Attenuated TRAF3 Fosters Activation ofAlternative NF-kB and Reduced Expression ofAntiviral Interferon, TP53, and RB to PromoteHPV-Positive Head and Neck CancersJialing Zhang1,2,Tony Chen1, Xinping Yang1, Hui Cheng1, Stephan S. Sp€ath3, Paul E. Clavijo1,Jianhong Chen1, Christopher Silvin1, Natalia Issaeva4, Xiulan Su2,Wendell G.Yarbrough4,5,Christina M. Annunziata6, Zhong Chen1, and Carter Van Waes1

Abstract

Human papilloma viruses (HPV) are linked to an epi-demic increase in oropharyngeal head and neck squamouscell carcinomas (HNSCC), which display viral inactivationof tumor suppressors TP53 and RB1 and rapid regionalspread. However, the role of genomic alterations in enablingthe modulation of pathways that promote the aggressivephenotype of these cancers is unclear. Recently, a subset ofHPVþ HNSCC has been shown to harbor novel geneticdefects or decreased expression of TNF receptor–associatedfactor 3 (TRAF3). TRAF3 has been implicated as a negativeregulator of alternative NF-kB pathway activation andactivator of antiviral type I IFN response to other DNAviruses. How TRAF3 alterations affect pathogenesis of HPVþ

HNSCC has not been extensively investigated. Here, wereport that TRAF3-deficient HPVþ tumors and cell linesexhibit increased expression of alternative NF-kB pathwaycomponents and transcription factors NF-kB2/RELB. Over-expression of TRAF3 in HPVþ cell lines with decreased

endogenous TRAF3 inhibited NF-kB2/RELB expression,nuclear localization, and NF-kB reporter activity, whileincreasing the expression of IFNA1 mRNA and protein andsensitizing cells to its growth inhibition. Overexpressionof TRAF3 also enhanced TP53 and RB tumor suppressorproteins and decreased HPV E6 oncoprotein in HPVþ cells.Correspondingly, TRAF3 inhibited cell growth, colony for-mation, migration, and resistance to TNFa and cisplatin-induced cell death. Conversely, TRAF3 knockout enhancedcolony formation and proliferation of an HPVþHNSCC lineexpressing higher TRAF3 levels. Together, these findingssupport a functional role of TRAF3 as a tumor suppressormodulating established cancer hallmarks in HPVþ HNSCC.

Significance: These findings report the functional role ofTRAF3 as a tumor suppressor that modulates the malignantphenotype of HPVþ head and neck cancers. Cancer Res; 78(16);4613–26. �2018 AACR.

IntroductionHead and neck squamous cell carcinoma (HNSCC) is the

sixth most common cancer, with an annual incidence of650,000 new cases and �200,000 deaths worldwide (1, 2).Persistent infection with high-risk human papillomavirus sub-

types HPV16 and HPV18 has been established as an importantrisk factor for HNSCC that develop predominantly in theoropharyngeal tonsils (3). Since 1981, there has been a notable225% increase in HPVþ HNSCC, while the incidence of smok-ing-related HPV� HNSCC has declined (4, 5). Clinically, theHPVþ subset exhibits better responses to therapies and survivalrates than similarly advanced HPV� tumors. However, HPVþ

HNSCC are distinguished by aggressive spread and growthwithin regional lymph nodes, which require major surgery ortoxic chemoradiotherapy regimens (2, 3). The factors thatcontribute to the molecular pathogenesis of these uniquefeatures of HPVþ HNSCC remain incomplete.

HPV16 and 18 carry early genes E6 and E7 encoding onco-proteins that target key pathways, deregulating host resistanceto infection and cellular proliferation, to promote the viral lifecycle. HPV E6 expression in keratinocytes can repress type-I IFNand promote proliferative genes, to enhance viral proteinsynthesis and proliferation of virally infected cells (6). Furtherstudies have shown that HPV infection can induce ubiquitincarboxyl-terminal hydrolase L1 (UCHL1), which can inhibitK63 ubiquination, important in Tank binding kinase-immuneresponse factor 3 (TBK-IRF3)-mediated type-I IFN expression(7). HPV E6 can also commandeer and activate the so-calledalternative nuclear factor-kB2 (NF-kB2) transcription factors

1Head and Neck Surgery Branch, National Institute on Deafness and OtherCommunicationDisorders, NIH, Bethesda,Maryland. 2ClinicalMedicineResearchCenter, The Affiliated Hospital, Inner Mongolia Medical University, Hohhot,China. 3Pediatric Endocrinology Unit, Department of Women's and Children'sHealth, Karolinska Institute and University Hospital, Stockholm, Sweden.4Department of Surgery, Otolaryngology, Yale Cancer Center, New Haven,Connecticut. 5Department of Pathology, Yale School of Medicine, New Haven,Connecticut. 6Women's Malignancies Branch, Center for Cancer Research, NCI,NIH, Bethesda, Maryland.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Carter Van Waes and Zhong Chen, National Instituteon Deafness and Other Communication Disorders, NIH, Room 7N240D, 10Center Drive, Bethesda, MD 20892. Phone: 301-402-4216; Fax: 301-402-1140;E-mail: [email protected]; [email protected]

doi: 10.1158/0008-5472.CAN-17-0642

�2018 American Association for Cancer Research.

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and antiapoptotic genes, which promote resistance of kerati-nocytes to TNF, an important mediator of antiviral immunity(8). Critically, the HPV E6 and E7 oncoproteins also strategi-cally target for degradation the tumor suppressor proteins TP53and RB, which control the cell cycle (9). Interestingly, however,few individuals exposed to HPV develop chronic infection andHNSCC. These observations suggest that additional geneticalteration(s) and host factors may also affect how HPV med-iates suppression of IFNs, NF-kB activation, inhibition of TP53and RB gene expression, and the malignant phenotype.

Recently, we and The Cancer Genome Atlas (TCGA) Networkuncovered a subset of HPVþ HNSCCs that harbor deletions ofthe chromosome region 14q32.32, deleterious truncatingmutations, and/or decreased expression, affecting the gene TNFreceptor–associated factor 3 (TRAF3; ref. 10). These deletions werenot due to viral integration and disruption of the TRAF3 gene.Intriguingly, TRAF3 is a unique adaptor protein and ubiquitinligase implicated as a negative regulator of the aforementionedalternative NF-kB2/RELB pathway (11). TRAF3 promotes cIAP-mediated ubiquitination and proteasome-dependent degrada-tion of the pivotal NF-kB inducing kinase (NIK) protein, whichmediates signal activation of the alternative pathway. Lympho-toxin-b (LTb) and other ligands, which are richly expressed inthe oropharyngeal tonsils and lymph nodes where HPVþ

HNSCC arise and spread, bind receptors to activate NIK, IKKa,processing of NF-kB2 precursor p100 to p52, and nucleartranslocation of transcriptionally active NF–kB2–p52/RELBdimers. Attenuation of TRAF3 has previously been implicatedin the transcription of genes affecting cell fate, proliferation,and survival of lymphoid cells and hematopoietic malignancies(11, 12). Strikingly, TRAF3 has also been shown to serve a dualfunction in interferon responses to other DNA viruses (11, 13).However, the functional role of genetic alterations or reducedexpression of TRAF3 in modulating alternative NF-kB pathwayactivation, IFN expression, repression of tumor suppressorsTP53 and RB, and the malignant phenotype in HPVþ HNSCChas not been established.

In this study, we examined and revealed a novel role fordecreased TRAF3 in fostering deregulation of these pathwaysand promoting pathogenesis of a subset of HPVþ HNSCC.These findings provide new opportunities for basic and clinicalresearch that may lead to better diagnosis, prevention, andtreatment of HPVþ HNSCC.

Materials and MethodsHNSCC patient samples and TCGA bioinformatics analysis

TCGA Project Management has collected necessary humansubjects documentation to ensure that the project complies with45-CFR-46 (the "Common Rule"). The program has obtaineddocumentation from every contributing clinical site to verify thatInstitutional Review Board approval and informed consent hasbeen obtained to participate in TCGA. The characteristics and datarepositories for 279 HNSCC specimens and 16 normal mucosawere previously reported, including 36HPVþ, 243HPV�HNSCC,and 16 normal samples (10). Exome-wide sequencing was per-formed on all samples, and normalized genomic data for 279HNSCC and 16 normal samples displaying a squamous keratingene signature was obtained from the TCGA Genome DataAnalysis Center and downloaded on September 19, 2012. Allgenomic data were processed through standard TCGA analytic

pipelines and accessible through TCGA data portals. Significantfocal copy-number alterations were extracted from the GISTIC2.0processing pipeline, and data aggregated on significantly alteredlesions are plottedby false discovery rate (FDR) less than5%andPvalues of less than 0.05. Differential gene expression analysis oftumor versus normal sampleswas performedusingDeSeq, and alldata were log2 transformed (Bioconductor version 2.12). Molec-ular HPV signatures were identified using gene expression andsomatic substitutions. Gene set enrichment analysis was per-formed using the online Cancer Genomics cBioportal database(http://www.cbioportal.org/).

HNSCC lines, cell culture, genomic DNA extraction, and PCRUMSCC [University of Michigan (Ann Arbor, MI) series of

HNSCC], UPCI (University of Pittsburgh Cancer Institute, Pitts-burgh, PA), and VU (Free University Amsterdam, Amsterdam, theNetherlands) cell lines and clinical information were provided byDrs. Thomas E. Carey, Mark E. Prince, Carol R. Bradford (Uni-versity of Michigan), Robert Ferris, and Susanne Gollin (Univer-sity of Pittsburgh), or fromprevious publications (SupplementaryTable S1; refs. 14–16). Cell authentication of UMSCC, UPCI, andVU lines was done at the University of Michigan by DNA geno-typing of alleles for 9 loci (D3S1358,D5S818,D7S820,D8S1179,D13S317, D18S51, D21S11, FGA, vWA) and the amelogeninlocus as described previously (15). A panel of 8 HPV-positive(UMSCC 47, 104, 2, 90, 152, 154, 93VU147T, and 105) and 8HPV-negative (UMSCC 1, 9, 11A, 11B, 38, 46, 74A, and 74B)HNSCC cell lines was selected for evaluation of HPV type andTRAF3 expression. These cell lines were maintained in MEM orDMEMwith 10% fetal calf serum (Life Technologies) at 37�Cwith5% CO2 for a maximum of 8 weeks. Primary human oral kera-tinocytes (HOK) were cultured, in accordance with the supplier'sprotocol (Science Cell Research). Prior to experiments, all cellswere confirmed to beMycoplasma negative (by MycoAlert kit, cat.no. M7006, Thermo Fisher). Genomic DNAwas extracted using aDNeasy Blood and Tissue Kit (cat. no. 51104, Qiagen) fromselected frozen cell pellets. These cell lines were further evaluatedforHPV status by PCR (Human Papillomavirus Detection Set, cat.no. 6602, Takara Bio).

RNA-seq analysisTotal RNA of 3 primary human keratinocyte and individual

HNSCC cell line was isolated by combination of TRIzol (cat no.15-596-026, Life Technologies) and QIAGEN RNeasy Mini Kitprocedure (cat. no. 74104, QIAGEN). Ribosomal RNA wasdepleted using the Ribo-Zero kit (cat no. MRZH11124, Epi-centre). Multiplexed whole transcriptome libraries were gener-ated by SOLiD Total RNA-Seq Kit and SOLiD RNA BarcodingKit (cat. no. 4427046, Life Technologies), and fragmentedcDNA libraries were clonally amplified by emulsion PCR. Themultiplex libraries were sequenced utilizing 75 bp forwardand 35 bp reverse paired-end sequencing chemistry on theABI SOLiD system. Reads were mapped into human NCBIBuild 37 reference genome (Hg19) using LifeScope v.2.5 Geno-mic Analysis software (https://www.appliedbiosystems.com/lifescope). The read count of the genes was normalized usingDESeq (estimateSizeFactors) R package (version 3.2.0) andupper quartile normalization method. Expression analysis wasperformed by comparison with normal HOK cell line expres-sion, and gene expression was quantified using RSEM (RNA-Seqby Expectation Maximization).

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Plasmids, siRNA, and transductionYFP-pTo-TRAF3 fusion expression vector was kindly provided

by Dr. Christina M. Annunziata, NCI/NIH, Bethesda, MD (16).His-tagged TRAF3 wild-type was generated by GeneCopoeia. Forefficient retroviral transductions, certain cell lineswere engineeredto express these indicated vectors. SMART pool siRNAs targetinghuman TRAF3 (cat. no. E-005252) and nontargeting siRNA con-trol (M-HUMAN-XX-0005) were purchased from Dharmacon.Cell transfection was carried out using X-tremeGENE HP DNATransfection Reagent or X-TremeGENE siRNA TransfectionReagent (cat. no. 10465500, 063662, Roche), following the man-ufacturer's instructions. Stably transfected cell lines were allowedto recover for 48 hours before antibiotic selection, and onlyconfirmed surviving pools were utilized for subsequent analysis.

TRAF3 CRISPR knockout cellsUPCI-SCC-90 and UMSCC47 cells were cotransfected with

TRAF3 CRISPR/Cas9 KO and TRAF3 HDR plasmids (Santa CruzBiotechnology) at equivalent ratios using lipofectamine 2000(cat. no. 11668019, Thermo Fisher). Forty-eight hours after trans-fection, the cells were plated at low density with the addition of1 mg/mL puromycin (cat. no. A1113802, Thermo Fisher) forselection of stable clones. Successful knockdown was confirmedwith RT-PCR. Clonogenic assay was performed as described (17).

Statistical analysisData are shown as means � SEM. Student t test or two-way

ANOVA were performed using GraphPad. Statistical analysis isspecifically indicated for each experiment (�, P < 0.05; ��, P < 0.01;��, P < 0.001; ��, P < 0.0001).

Additional methods of preparation of total RNA isolation,cDNA synthesis, qRT-PCR analysis, protein extracts, Western blotanalysis, reporter assay, immunofluorescence staining, treat-ments, and cell viability are presented in the SupplementaryInformation.

ResultsTRAF3 alterations are associated with HPVþ HNSCC status

The recent TCGA study revealed that a subset of HPVþ HNSCCdisplay novel recurrent deletions, truncating mutations, and/or decreased expression of TRAF3 (10). Here, we furthershow that TRAF3 focal or broader deletions of chromosome 14(Chr14q32.32) are mainly observed in HPVþ HNSCC (Fig. 1A).Among 36 HPVþ tumors in this dataset, 14% homozygous and25% heterozygous deletions were found, whereas in 243 HPV�

tumors, only 1.2%homozygous and 10%heterozygous deletionswere observed (Fig. 1B, Fisher exact test, P ¼ 2.1 � 10�6). Incontrast, 38% of HPV� tumors exhibited TRAF3 copy-numbergains compared with only 6% of HPVþ tumors. Together, thedifferent distribution of these DNA copy-number variations wereassociated with TRAF3 mRNA expression in HPVþ (P ¼ 3.52 �10�5) and HPV� HNSCC samples (P ¼ 2.0 � 1010; Fig. 1C).Furthermore, three cases with deleterious frameshift or nonsensemutations were observed only in HPVþ HNSCC.

To determine if there are HPVþ HNSCC cell lines withdecreased TRAF3 expression suitable for study, we investigatedTRAF3 expression in a panel of eightHPVþ andHPV�HNSCC celllines (Supplementary Table S1), using RNA sequencing data.Overall, we observed significantly increased TRAF3 mRNAexpression in HPV�, compared with normal HOK and HPVþ

cell lines (P < 0.05, Fig. 1D), consistent with the few HPVþ

tumors and cell lines with low expression, as well as highnumber of HPV� tumors and cell lines with gain of expressionof TRAF3. We further compared TRAF3 protein levels in the 8HPVþ HNSCC cell lines with normal human oral keratinocytes(HOK). UMSCC47 and UMSCC104 cell lines exhibited lowerTRAF3 protein levels as assessed by Western blot (Fig. 1E), andthese lines were selected for further study of the effects ofdecreased TRAF3 expression and modulation.

Decreased TRAF3 is associated with increased expression ofalternative NF-kB pathway components in HPVþ HNSCCtumors

As TRAF3 was previously identified as a negative regulator ofthe alternative NF-kB pathway in immune and hematopoieticmalignant cells (11, 12), we asked whether altered TRAF3 isassociated with NF-kB pathway alterations in HNSCC tissues.Using the HNSCC TCGA datasets, we identified an apparent co-occurrence between TRAF3 genomic and expression alterationswith the mRNA expression of alternative NF-kB pathway compo-nents (Fig. 2A left, top), but not with those in classical NF-kBpathway (Fig. 2A left, bottom), in HPVþ HNSCC tumors. Con-versely, no such co-occurrence was observed in HPV� HNSCCtumors (Fig. 2A, right). Supporting this, we observed a signifi-cantly higher average percentage of alterations in the NF-kBalternative pathway in HPVþ tumors, compared with HPV�

tumors, whereas no significant difference was observed in theclassic NF-kB pathway (Fig. 2B). Finally, the co-occurrence andmutual exclusivity of paired alterations were calculated, and therelationships with statistical significance are indicated as colorintensities (Fig. 2C; Supplementary Table S2). We observed morefrequent co-occurrence of alterations in the key components of thealternative than the classic NF-kB pathway in HPVþ HNSCCtumors (Fig. 2C, left). In contrast, fewer tumor samples withstrong and intermediate co-occurrences were observed in HPV�

HNSCC (Fig. 2C, right). These analyses indicate that deficientTRAF3 is associated with increased expression of alternativeNF-kB pathway components in HPVþ HNSCC tumor specimens.

TRAF3 expression inhibits the alternative but not classic NF-kBsignaling in HPVþ HNSCC cell lines

To test the hypothesis that decreased TRAF3 is permissive foractivation of the alternative NF-kB pathway in HPVþHNSCC, weexamined the expression of alternative pathway components andthe effects of transiently expressing His-tagged TRAF3 (His-TRAF3) or control (His-Control) vectors in the HPVþ UMSCC47and UMSCC104 cells that displayed relatively lower endogenousTRAF3 expression. TRAF3 mRNA was significantly increased inboth cell lines after TRAF3 transfection (Fig. 3A, top). Next, weevaluated the mRNA expression levels of key alternative pathwaysubunits RELB and NF-kB2, which are modulated at transcrip-tional and posttranslational levels, as well as classic (RELA/p65)NF-kB subunit, which is constitutively expressed and modulatedposttranslationally (18). Consistent with this, transient TRAF3overexpression in both HPVþ cell lines resulted in a significantreduction of RELB and NF-kB2mRNA expression, without affect-ing RELA expression (Fig. 3A). We then evaluated the effect ofTRAF3 expression on protein levels of key alternative NF-kBpathway components in the same cell lines and an additionalHPVþHNSCC cell line, 93VU174T, following TRAF3 transfection(Fig. 3B; Supplementary Fig. S1A). TRAF3 expression significantly

Decreased TRAF3 Promotes HPVþ HNSCC

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reduced protein levels of components involved in the alternativeNF-kB pathway, including its co-factor and ubiquitinase cIAP1together with its substrate NIK, and processed NF-kB2/p52 andRELB subunits (Fig. 3B and C; Supplementary Fig. S1A).We observed comparatively smaller decreases in TRAF3-cIAP

co-factor TRAF2 or alternate pathway kinase IKKa that mediatesNF-kB2-p52/RELB (Supplementary Fig. S1B). Taken together,these results indicate that increasing expression of TRAF3 inHPVþ

HNSCC cells with lower TRAF3 can suppress mRNA and/orprotein levels of components of the alternative NF-kB pathway.

Figure 1.

TRAF3 deletion is associated with HPVþ HNSCC status. A, Copy number variations (CNV) were analyzed and data were extracted from TCGA HNSCC project.TRAF3 chromosome location and copy number variation were presented on the Chr14q32.32 segmentation map for 36 HPVþ (top) and 243 HPV� (bottom) HNSCCtumor specimens. The blue bar at the top right corner of chromosome represents the Chr14q32.32 region where TRAF3 is located. The color gradientsdepict the extent of inferred copy number loss (blue) and amplifications (red), respectively. The magnified view (marked by the black box) refers to sorted HPVþ

HNSCC tumors according to the loss of TRAF3 locus (indicated by yellow lines at the bottom) and neighboring gene regions. B, Percentage of HPVþ (left) or HPV�

(right) HNSCC specimens with TRAF3 copy number variations. Copy number variation is presented on the x-axis and divided into five categories by GISTIC:homozygous deletion, �2; heterozygous deletion, �1; diploid, 0; one copy gain, 1; two copy amplification, 2. Significance of association between TRAF3 loss andHPV statuswas observed (Fisher exact test, P¼ 2.12� 106).C, The significant association between TRAF3 expression (log2 RSEM; y-axis) and copy number variation(x-axis) was observed in both HPVþ (left) and HPV� HNSCC after assessment by Pearson correlation test. The box plot indicates 25th, 50th, and 75thpercentiles of TRAF3 gene expression. The whiskers mark the minimal and maximum values, excluding the outliers. The red triangle and diamond representframeshift and nonsense mutations. D, TRAF3 mRNA expression of selected HPVþ and HPV� HNSCC cell lines compared with HOK cells (control). � , P < 0.05 byStudent t test. E, Whole cell lysates were harvested, and TRAF3 protein levels were examined by Western blot for HPVþ HNSCC cell lines. The histogram ofrelative protein expression was generated from densitometry analysis of relevant protein bands after adjustment to loading control of b-actin and normalized toHOK cells (control).

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TRAF3 expression modulates NF-kB2/p52 and RELB nuclearlocalization, andNF-kB activity in theHPVþUMSCC47 cell line

To further characterize how TRAF3 modulates the cellulardistribution of alternative RELB/NF-kB2 subunits in HPVþ

HNSCC, we examined cytoplasmic and nuclear expression ofthese protein subunits by Western blot. Aberrant nuclear locali-zation of RELB, NF-kB2/p52, as well as p100 was detected incontrol nuclear as well as cytoplasmic fractions (Fig. 4A), consis-tent with a prior report demonstrating enhanced nuclear expres-sion of both p100/52 in HPVþ SCC and E6-expressing keratino-cytes (19). Transfection of TRAF3 increased distribution of NF-kB2 p100/52 proteins in the cytoplasmic fraction while decreas-

ing nuclear p100/p52 and RELB protein levels in independentexperiments (Fig. 4A; Supplementary Fig. S2A and S2B). Wefurther tested functional NF-kB activity by utilizing a NF-kBluciferase reporter gene assay in the UMSCC47 cell line. Consis-tent with the reduced nuclear RELB/p52 protein levels, weobserved a significant reduction in functional NF-kB reporteractivity in TRAF3 cotransfected UMSCC47 cells (Fig. 4B).Conversely, TRAF3 knockdown using three different siRNAs,significantly reduced TRAF3mRNA expression in UMSCC47 cells(Fig. 4C). Consistent with reduced TRAF3 mRNA expressionin Fig. 4C, we observed reduced TRAF3 and increased RELBprotein by Western blot (Supplementary Fig. S2C), as well

Figure 2.

TRAF3 deletion is associated with alternative NF-kB activation in HPVþ HNSCC tissues. A, Genomic alteration profiles of the alternative (top) and classic (bottom)NF-kB signaling pathway members across the TCGA dataset are presented by oncoprint, composed of HPVþ (left) and HPV� (right) HNSCC samples.Each row represents a gene and each column represents a tumor sample, respectively. The percentage of alteration for individual genewas calculated by cBioPortal.B, Histograms representing the mean percentage of alterations for alternative (top) or classic (bottom) NF-kB signaling pathway members as presented above,respectively. ��, P < 0.001; ns, not significant. C, Heat maps summarizing co-occurrence or exclusivity association of paired NF-kB pathway gene membersin HPVþ (left) and HPV� (right) HNSCC. The association was presented as log odds ratio, and the co-occurrence or exclusivity is depicted in blue or orange,respectively. P value was calculated using Fisher exact test; � , P < 0.05; �� , P < 0.01; �� , P < 0.001.






as enhanced nuclear RELB by immunofluorescence staining(Fig. 4D). TRAF3 knockdown was accompanied by increasedNF-kB reporter activity in UMSCC47 cells (Fig. 4E). These resultsdemonstrate that TRAF3 can inversely modulate alternativeNF-kB pathway RELB and NF-kB2/p52 expression, cellular local-ization, and functional activity in HPVþ HNSCC cells.

TRAF3 inhibits aberrant and LTb-enhanced NF-kB2/RELBprotein, increases sensitivity to TNFa and cisplatin, as well asinhibits migration of HPVþ HNSCC cells

HPVþ HNSCCs display rapid growth and spread within thetonsils and regional lymph nodes, where ligands activating thealternative NF-kB pathway are expressed (4, 11). To investigatethe upstream receptor-mediated activation of NF-kB in HPVþ

lines, we first established if HPVþ and HPV� HNSCC celllines express mRNAs for the alternative pathway receptor

lymphotoxin-beta receptor (LTbR), and two classic NF-kBpathway TNF receptors (TNFR1A and TNFR1B). We detectedelevated mRNA expression of LTbR in HPVþ compared withHPV� HNSCC cell lines (Fig. 5A, left, HOK cells as dottedline), while no significant difference was observed for eitherTNFRs (Fig. 5A, right). Next, we compared the functionaleffects of recombinant LTb or TNFa protein on NF-kB reporteractivity in HPVþ UMSCC47 cells and HPV� UMSCC1 cells,establishing that LTb as well as TNFa could induce NF-kBreporter activity in both (Fig. 5B; Supplementary Fig. S3A). Wefound that LTb treatment attenuated TRAF3 and NF-kB2/p100precursor protein, while enhancing RELB and processed NF-kB2/p52 protein levels (Fig. 5C; Supplementary Fig. S3B).Conversely, after knockdown of TRAF3, basal and LTb-inducedlevels of RELB and NF-kB2/p52 proteins were substantiallyenhanced (Fig. 5C; Supplementary Fig. S3B), supporting the

Figure 3.

TRAF3 downregulates mRNA and protein expression of molecules involved in the alternative NF-kB pathway in HPVþ HNSCC cells. A, HPVþ HNSCC cell linesUMSCC47 (left) and UMSCC104 (right) were transiently transfected with TRAF3 (His-TRAF3) or control (His-Control) plasmid for 24 hours, and mRNA expressionof TRAF3 and alternative RELB, NF-kB2, and classic RELA subunits was examined by qRT-PCR. mRNA expression is represented relative to control as mean � SD(from 6 replicates). � , P < 0.05; �� , P < 0.01; �� , P < 0.001, Student t test. B,HPVþUMSCC47 and UMSCC104 cells were transiently transfected with TRAF3 expressionvector and whole-cell lysates were harvested 48 hours after transfection. Protein expression of TRAF3 and alternative NF-kB pathway components wereexamined byWestern blots.C,Histograms for expression of indicated proteinswere derived fromdensitometry analysis of bands after adjustment to loading controlof b-actin and are presented relative to the cells transfectedwith control plasmid at 48 hours. Data, mean� SEM from independent experimentswith three HPVþ celllines (UMSCC47, 104, and 93VU174T; Fig. 3B andSupplementary Fig. S1). Significant differences between TRAF3-transfected samples and controls refer to � ,P

role of TRAF3 as a negative regulator of the alternative NF-kBpathway in HPVþ HNSCC cells.

As we observed above that TRAF3 inhibited alternative NF-kBactivation and cIAP1, which have been previously linked toproliferation, and TNFa and chemotherapy resistance (20), weevaluated if TRAF3 overexpression could inhibit tumor cellproliferation, or enhance the inhibitory effects of TNFa, LTb, orchemotherapy agent cisplatin used for HNSCC. Transient TRAF3expression resulted in a modest but significant decrease in cellproliferation and showed greater inhibitory effects in combina-tion with TNFa, cisplatin, or both at day 3 (SupplementaryFig. S4A) and day 5 (Fig. 5D). In cell lines selected for TRAF3and control vector expression, TRAF3 enhanced sensitivity of cellsto the combination of TNFa andCDDP, but greater resistance andgrowth in response to TNFa alone (Supplementary Fig. S4B).While sensitization to TNFa and cisplatin was associated withTRAF3-inhibitory effects on alternative NF-kB components and

cIAP1 above, we observed minimal modulation of BCL familymembers that contribute to survival (Supplementary Fig. S4C). Tofurther examine the functional role of alternative NF-kB pathway,we knocked down IKKa and RELB to evaluate cell proliferation inHPVþUMSCC47 cells. We observed a significant reduction in thecell proliferation after knockdown of either gene alone, with RELBknockdown having a greater effect than IKKa knockdown on cellproliferation (Fig. 5E). As HPVþ HNSCCs show increased pro-pensity for malignant spread, we next performed a cell migrationassay. We observed a significant delay in cell migration over a 24-hour time course in UMSCC47 cells expressing TRAF3 (Fig. 5F),before antiproliferative effects �10% of control are observed inXTT assay by day 3 (Supplementary Fig. S4A). Together, these datademonstrate that TRAF3 inhibits LTb enhanced alternative path-way NF-kB2/p52 and RELB protein levels, increased sensitivity toTNFa and chemotheraputic agent as well as inhibits migration ofHPVþ HNSCC cells.

Figure 4.

TRAF3 decreases nuclear NF-kB p52and RELB localization, and NF-kBactivity in HPVþ UMSCC47 cell line.A, UMSCC47 cells were transientlytransfected with TRAF3 (GFP-TRAF3pToCMV) or control (GFP-pToCMV)plasmids for 24 hours. Cytoplasmicand nuclear protein were harvestedand assessed byWestern blot. b-Actinor Lamin Awas used as cytoplasmic ornuclear protein loading control,respectively. B, UMSCC47 cells weretransiently transfected with TRAF3 orcontrol expression vector with NF-kBreporter plus LacZ plasmids for 24hours. NF-kB reporter activity wasadjusted to b-galactosidase. C, TRAF3mRNA expression decreases followingTRAF3-specific siRNAs (siTRAF3a-c)at 24 hours after transfection. Alldepicted samples were normalized toscrambled control siRNA (si-Control)knockdown. Data, mean � SEMderived from three replicates. D,Nuclear RELB localization by confocalimmunofluorescence microscopyfollowing TRAF3-specific siRNA(siTRAF3b) or scrambled controlsiRNA (si-Control) knockdown at 24hours. Cells were fixed and stainedwith antibodies against TRAF3, RELB,andAlexafluor–conjugated secondaryantibodies. Representative imagesshow the localization of TRAF3(green), RELB (purple), DAPI (blue),and merged images. Scale bar, 10 mm.E, NF-kB–mediated luciferase activityincreases following TRAF3-specificsiRNA (siTRAF3a-c). Renilla luciferaseassay was performed on the samelysates and was used to adjust thedifferences in transfection efficiencies.All depicted samples were normalizedto scrambled si-Control knockdown.Data, mean � SEM derived from6 replicates. Significant differencesbetween samples and controls refer to� , P < 0.05; �� , P < 0.01.






Selection for TRAF3 expression or CRISPR knockoutmodulatescolony formation and proliferation of HPVþ HNSCC

As TRAF3 appeared to reduce proliferation by 5 days followingtransient transfection, we enriched for UMSCC47 cells expressing

TRAF3 or vector plasmid control by G418 selection, and exam-ined the colony-forming capacity and proliferation of HPVþ

UMSCC47 cells. We observed a significant reduction in colonyformation of UMSCC47 cells that expressed increased TRAF3,

Figure 5.

LTBR expression and LTb-induced NF-kB activation in HPVþHNSCC cell lines and enhancing effects of TRAF3 knockdown on alternative NF-kB pathway activation.A,mRNA-seq expression in eight cell lines, eachwith different HPV statuswere normalized toHOK cells (marked by dashed line). LTBR expression is relatively higherin HPVþ cell lines, while TNFR1A and TNFR1B exhibit no significant difference across HNSCC cell lines with different HPV status. The solid black line is themean value. �� , P < 0.0001; ns, not significant, Student t test. B,NF-kB-RE reporter stably transfected UMSCC47 cells were plated at a cell density of 3,000/well in96-well plate, cultured for 48hours, then treated� 10 ng/mLTNFa 16 hours or 50ng/mLLTb for 24 hours prior toGeneBLAzerAssay at 72hours.MeanNF-kBactivitynormalized to no treatment �SD for 6 replicates; P < 0.05 Student t test. C, UMSCC47 cells were transiently transfected with TRAF3 or control siRNA for 24 hours,followedby LTb induction for 2 hours. The alternativeNF-kBcomponents RELB andNF-kB2 (p100 andp52)were examined inWestern blot. b-Actin (Actin)was usedas protein loading control. D, HPVþ UMSCC47 cells expressing TRAF3 were sensitized to TNFa and cisplatin at day 5 in the XTT assay. Cells were plated in 96-wellplates, and the next day were treated with TNFa (25 ng/mL), LTb (100 ng/mL), cisplatin (10 mmol/L) alone, or combination of TNFa, or LTb with cisplatin.Cells transfectedwith control vector, blue; TRAF3 vector, red. Statistical significance (P

compared with control (Fig. 6A and B). UMSCC47 cells selectedfor expression also demonstrated significantly reduced cell pro-liferation in TRAF3 versus vector control cells (Fig. 6C). Interest-ingly, we observed that TRAF3 expression declined with passagedespite selection, consistent with the antiproliferative activity of atumor suppressor (Supplementary Fig. S5). As the cell line UPCI-SCC-90 expressed higher endogenous levels of TRAF3 (Fig. 1E),we next examined effects of CRISPR knockout of TRAF3on colonyformation and cell proliferation. CRISPR depleted TRAF3markedly enhanced colony formation and proliferation ofUPCI-SCC-90 (Fig. 6D–F). Together, our data demonstrate thatTRAF3 expression suppresses the proliferation of HPVþ HNSCCcells.

TRAF3 expression enhances expression of and sensitizes HPVþ

HNSCC to antiviral interferonsTRAF3 contributes to expression of type I IFN in response to

other DNA virus infections in other tissues (13), but the expres-sion of type-I IFN and relation to lower TRAF3 observed inHPVþHNSCC has not been examined. We analyzed RNA-seqdata from our HNSCC cell lines and TCGA HNSCC tumors tocompare the mRNA expression profiles for type-I interferon IFNA

gene in HPVþ and HPV� cell lines, as well as tumors. The HPVþ

HNSCC cell lines displayed lower IFNA1 expression than normalHOK or HPV� HNSCC cell lines (Fig. 7A, left). IFNA1 mRNA inHPVþ HNSCC tissues were also expressed at significantly lowerlevels when compared with HPV� HNSCC tissues from TCGAdata (Fig. 7A, right). To test the hypothesis that decreased TRAF3may contribute to reduction in IFNAandbe reversedbyTRAF3,wecompared IFNA1 expression in the two HPVþ HNSCC cell lineswith decreased TRAF3 expression, UMSCC47 and UMSCC104,following transient transfection with control and TRAF3 vectors.TRAF3 induced a small but significant increase in the expressionofIFNA1 inboth cell lines (Fig. 7B). IFNA1proteinwas also found tobe modestly significantly increased in cell culture supernatantsisolated from HPVþ UMSCC47 cells stably transfected withTRAF3 compared with control vector (Fig. 7C). UMSCC47 stablyexpressing increased TRAF3 and IFNA1 as above exhibitedreduced proliferation and displayed enhanced sensitivity to exog-enous IFNA1 or 2a (Fig. 7D). We explored if TRAF3 modulatedmRNA expression of interferon response factors (IRF), which aretranscription factors that mediate the transcription of type-I IFN(7, 11).However, wedidnot see consistent changes in IRF3, 5, or 7mRNA expression in the HPVþ line selected for expression of the

Figure 6.

TRAF3 expression or CRISPR knockout modulatescolony formation and proliferation of HPVþ HNSCC.HPVþ UMSCC47 cells were transfected with His-TRAF3or empty His-Control vector and selected by G418 for 2weeks. A, The images of cell colony formation weretaken using a microscope image system. B, Histogramshowing the percentage of colonies produced withTRAF3-expressing cell line, compared with controlvector. The colony numbers are represented as mean�SD, derived from triplicates. � , P < 0.01 by Studentt test. C, Cell proliferation of UMSCC47 cells selected forcontrol or TRAF3 expression vector. Cells were plated in96-well impedance plates and increasing cell densitieswere measured by impendence was determined at 40,60, 80, and 100 hours. Mean cell density unitsþSD for 6replicates. � ,P

Figure 7.

Expression of TRAF3 induces an antiviral interferon response, sensitizes to IFN, increases TP53 and RB protein expression, and modulates nuclear HPV E6oncoprotein in HPVþ HNSCC cells. A, Antiviral cytokine IFNA1 mRNA expression in a panel of HPVþ vs. HPV� HNSCC cell lines in this study (left) and HNSCCtissues from TCGA dataset (right). The mRNA expression for all samples is normalized to HOK cells (left) or normal tissues (right). P < 0.05 by Student t test:� , compared with normal tissues; #, comparison between HPVþ and normal controls. B and C, IFNA1 mRNA expression by qRT-PCR (B) and protein expression insupernatants by ELISA (C). Transient transfection of TRAF3 for 24 hours increased IFNA1 mRNA expression in HPVþ UMSCC47 and 104 cells and protein insupernatant of UMSCC47 cells. Data are derived from 6 replicates and represented as mean � SD. � , P < 0.05; ns, not significant. D, UMSCC47 cells stablytransfected with TRAF3 or control vector were plated overnight and treated with IFNA1 (5,000 U/ml) or IFNA2a (10,000 U/ml). Cell proliferation was measured byimpedance. Data were calculated and are presented in bar graph as a mean of 6 replicates � SD with Student t test. � , statistical significance (P < 0.05) whencomparing IFNA1- or IFNA2a-treated cellswith untreated cells. E,UMSCC47 selected to express TRAF3 show increased expression of tumor suppressor TP53 andRB.Expression of TRAF3, TP53, and RB is shown in HPV� UMSCC46 cells as a positive control. b-Actin served as a loading control. F, UMSCC47 cells stablytransfected with TRAF3 or control vector were plated overnight and transfected with siRNA targeting CDKN1A or control siRNA. Cell proliferation wasmeasured by impedance. Data were calculated and are presented in bar graph as amean of 6 replicates� SDwith Student t test. � , statistical significance (P < 0.05)when comparing cells transfected with CDKN1A siRNA with control siRNA. G, Cytoplasmic fractions (CF), nuclear fractions (NF), and whole cell lysates (WC)were isolated and HPV E6 protein was examined by Western blots. UMSCC47 cells selected to express TRAF3 show decreased E6 protein levels in nuclear lysates.H, Effects of E6 or TRAF3 overexpression on E6 protein in UMSCC47 cells. Left, E6 peptide-KLH protein–positive control for anti-E6 antibody. Top right, E6and empty pcDNA vector were transiently transfected into UMSCC47 cells and effects in E6 protein expression were compared. A slight increase in E6 proteinwas detected. Bottom right, A slight decrease in E6 expression was detected 72 hours after TRAF3 expression. Actin was used as the loading control.

Zhang et al.





TRAF3 gene (Supplementary Fig. S6A). Further, while TRAF3-regulated IRF3 nuclear protein was also decreased in HPVþ

UMSCC47 cells, the lack of nuclear IRF3 did not appear to bedue to lower TRAF3 protein level alone, as expressing TRAF3vector induced aminimal increase in expression of nuclear IRF3 inreplicate experiments (Supplementary Fig. S6B). Moreover,even in HPVþ UMSCC47 with lower TRAF3 expression and lownuclear IRF3, nuclear IRF3 and type-I IFN remained induciblewith a non-HPV RNA virus mimic and TLR3 ligand poly-I:C,unless TRAF3 was completely knocked out by CRISPR (Supple-mentary Fig. S7A–S7D). Conversely, UMSCC47 CRISPR/CAS9TRAF3-9 KO rescued with TRAF3 vector showed no increasein baseline IRF3 or IFN, but did so following stimulation withpoly-I:C (20 mg/mL; Supplementary Fig. S8A–S8D). Together,these findings are consistent with either a lack of strong activationof TRAF3-IRF3 signaling by integrated HPV in UMSCC47, or thatIRF3 activation and type-I IFN expression may be furtherdisrupted downstream of TRAF3 expression, as found in HPVE6/7-expressing keratinocytes in prior studies (6, 7).

TRAF3 expression enhances TP53 andRB tumor suppressor andinhibits E6 protein expression

As repression of tumor suppressor proteins TP53 and RB byHPV contributes to the proliferation ofHPVþ cancers (21, 22), weexamined if TRAF3 may modulate expression of TP53 and RB.Compared with the HPV� mtTP53 line UMSCC46, UMSCC47expressed low basal levels of TP53 and RB protein expression(Fig. 7E). Unexpectedly, TP53 and RB expression was reproduc-ibly enhanced inUMSCC47 cells selected for expression of TRAF3or after transient transfection (Fig. 7E; Supplementary Fig. S9A).UMSCC104 cells with the lowest TRAF3 expression also showedenhancedTP53 andRBexpression after transient transfectionwithTRAF3 (Supplementary Fig. S9B). InUMSCC47TRAF3-expressingcells, the increase in TP53 was not attributable to increasedmRNA, while RB mRNA was increased �2-fold as measured byqRT-PCR (Supplementary Fig. S9C). TP53 siRNA knockdownattenuated the antiproliferative effects of TRAF3 (SupplementaryFig. S7D). The increase in TP53 was associated with enhancedexpression of TP53-regulated cyclin-dependent kinase CDKN1A(p21), and proliferation was enhanced by siRNA knockdown ofCDKN1A(p21) in UMSCC47 expressing TRAF3 (Fig. 7F andSupplementary Fig. S9E), supporting a contribution of this cell-cycle regulator to the antiproliferative effects of TRAF3 and TP53.

As HPV E6 oncoproteinmediates suppression of TP53 (22), weexplored whether TRAF3 may affect HPV E6 levels protein inUMSCC47 cells selected to express TRAF3 or control vector. HPVE6 protein localized predominantly in the nucleus in the controlcell line and was partially decreased in the TRAF3-expressingUMSCC47 cell line (Fig. 7G). We observed minimal change intranscription of E6 mRNA (Supplementary Fig. S10A). However,we observed only a minimal increase in E6 protein after trans-fection with an E6 expression vector that increased mRNA �400-fold (Fig. 7H; Supplementary Fig. S10B), indicating that the verylow level of E6 detected is not primarily determined by transcrip-tional regulation (Fig. 7H). Conversely, we also observed only aminimal decrease in E6 protein at 72 hours after transient trans-fection with TRAF3 (Fig. 7H) and did not observe higher or lowerMW bands, or enhancement by proteasome inhibitors, thatwould support a ubiquitination or proteasome-dependent mech-anism (Supplementary Fig. S10C). Together, these observationssuggest that the increase in TP53with TRAF3within 24 hours after

transfection is likely dependent on mechanisms other than mod-ulation of E6 protein expression alone.

DiscussionTRAF3 copy loss, mutation, and/or decreased expression was

significantly enriched in a subset of HPVþ HNSCC tumors (10),suggesting that TRAF3 could be a novel suppressor of HPV viraland tumor pathogenesis. Previously, TRAF3 deficiency was impli-cated in impaired Toll-like receptor 3 responses and increasedsusceptibility toherpes simplex virus (HSV)–induced encephalitis(23), hematologic malignancies, and Epstein–Barr virus (EBV)–related nasopharyngeal cancers, but not HPV cancers. Weobserved that oropharyngeal HNSCC linked to HPV exhibitaberrant nuclear activation of the alternative NF-kB pathway(24), but the significance and function and role of deficient TRAF3in alternative NF-kB activation have not been investigated previ-ously in HPVþ HNSCC cell lines and tissues. Here, we demon-strate for thefirst time that decreased TRAF3expression is linked toaberrant expression and activation of alternative NF-kB2/RELBtranscription factors, contributes to reduced expression of anti-viral type-I IFN and tumor suppressor TP53 and RB proteins,which are deregulated in HPVþ HNSCC (SupplementaryFig. S11). Supporting this role, ectopic TRAF3 expression inHPVþ

HNSCC with decreased endogenous TRAF3 inversely modulat-ed these key targets and suppressed several hallmarks of cancer,including cell colony formation, proliferation, and migration,while sensitizing HPVþ HNSCC to immune IFNs, TNFa, andchemotherapy agent cisplatin. Complementing our findings,one of our laboratories currently obtained evidence that dele-tions or mutations in TRAF3 in HPVþ HNSCC tumors areassociated with increased expression of an NF-kB transcription-al program, and episomal HPV infection (25). Interestingly,TRAF3-deficient HPVþ HNSCC also appear to be linked to abetter prognosis (25), than HPVþ or HPV� tumors withPIK3CA, DNp63, FADD, cIAP1/2, and other genomic alterations(i.e., TP53; ref. 10), implicated in modulation of NF-kB or otherpathways important in pathogenicity (26–29). The findings inthe present study support the addition of TRAF3 as a tumorsuppressor in HPVþ HNSCC.

The recent discovery of deep deletions on chromosome14q32.32 containing the TRAF3 coding region and 3 deleteriousloss-of-function mutations by TCGA project was previouslyunrecognized in HPVþ HNSCC (10). Here, we further definedan increased frequency of hetero- and homozygous deletions ofTRAF3 inHPVþHNSCC, in contrast toHPV� tumors, which oftendisplay copy gain. Furthermore, we observed that TRAF3 geneticalterations and expression significantly correlatedwith expressionof alternative NF-kB pathway components in HNSCC tissues.TRAF3 gene copy loss, inactivating mutations, and decreasedexpression of TRAF3 have been previously observed only innon-HPV cancers. These includemultiplemyeloma (MM; ref. 16),B-cell lymphomas (30), and EBV–infected human nasopharyn-geal carcinomas (NPC; ref. 31). Annunziata and colleagues foundthat 4.4% of 451 patients with MM had TRAF3 genetic defects,such as silencing, homozygous deletion, or somatic mutation,and 17 MM cases exhibited TRAF3 mRNA expression 13-foldbelow the median level of the cohort, predominantly inassociation with these genetic alterations (16). Chung andcolleagues detected deletion or missense mutations of TRAF3,TRAF2, and A20 in 3 of 33 (9.1%) of primary NPC tumor






specimens (31). Together, these observations in differentcancer types support the potential relevance of geneticalterations in HPVþ HNSCC and suggest that decreased TRAF3facilitates the pathogenesis of tumors within the hematopoieticor lymphatic microenvironment.

We previously reported that alternative pathway NF-kB2 andRELB subunits are often aberrantly expressed, and localized to thenucleus in oropharyngealHNSCC tumors linked toHPV (24), butthe possible relationship to genomic alterations, other factors andto the malignant phenotype was unknown. Here, our analysis ofTCGA HNSCC data indicated that TRAF3 deletion is associatedwith increased mRNA expression of molecules involved in thealternative NF-kB pathway, such as NIK, cIAPs, RELB, and NF-kB2. Co-occurrence and mutual exclusivity analyses for key com-ponents of the NF-kB pathways revealed a significant associationof altered TRAF3 withmolecules involved in an alternative NF-kBpathway in HPVþ, but not in HPV� HNSCC tissues. We furtherdemonstrated that HPVþHNSCC cell lines with decreased endog-enous TRAF3 also display aberrant expression and nuclear acti-vation of the alternative NF-kB pathway. Furthermore, this poten-tial of decreased TRAF3 to foster expression and activation ofalternative NF-kB in HPVþ HNSCC tumors could potentially beenhanced within the local tonsillar and lymphatic microenviron-ment, where alternative NF-kB signal inducers such as LTb areabundant (32).

LTb is a key signaling ligand that binds to surface receptor LTbRand activates the alternative NF-kB pathway (33). We originallydetected aberrant activation of the alternative pathway in HPV�

HNSCC and demonstrated that activation could be enhanced byLTb stimulation (34). Here, we observed increased LTbR expres-sion compared with HPV� cell lines and showed that LTb couldfurther enhance NF-kB activation in HPVþ HNSCC cells expres-sing lower endogenous TRAF3. TRAF3 knockdown furtherenhanced aberrant and LTb-stimulated expression of the alterna-tive NF-kB2 subunit p52 and RELB. Interestingly, in a subset ofEBV-infected nasopharyngeal HNSCC that harbor TRAF3 altera-tions, Or and colleagues demonstrated 12p13.3 copy-numbergains and increased expression of LTbR (35). Overexpression ofLTbR in nasopharyngeal epithelial cells resulted in increased NF-kB activity and cell proliferation (36). Together, these data impli-cate TRAF3 and/or LTbR genomic alterations, along with HPV E6and EBV LMP1 viral oncogenes that promote alternative NF-kBpathway activation, in viral pathogenesis of oro- and nasopha-ryngeal HNSCC. LMP-1 has been shown to bind TRAF3 andenhance NIK-dependent alternative pathway activation (36).HPV E6 is reported to promote alternative NF-kB activation viaa mechanism requiring its PDZ domain implicated in bindingphosphatases (8). Our data support a role for deficient TRAF3expression in enhancing NIK, cIAP1, IKKa, NF-kB2-p52, andRELB proteins, but we have found it exceedingly difficult to findconditions to detect HPV16 E6 protein and an association withTRAF3 in HNSCC.

TRAF3 has been reported to have a unique dual role in pos-itively regulating type-I IFNs in response to different DNA virusesin other tissues (11). IFNA1 potentially has antiviral effectsthrough inhibition of viral protein expression, proliferation, aswell as immunostimulatory properties, that link innate andadaptive immunity (37). In this study, we observed significantlylower expression of IFNA1 inHPVþHNSCC lines, consistent withthe lower expression of IFNA1 detected in HPVþ compared withHPV� tumor tissues from TCGA data. Previous studies have

shown that HPV infection can also induce ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), which could inhibit TRAF3 K63ubiquination, important in downstream TBK-IRF3–mediatedtype-I IFNexpression (7). In addition,wehave showna significantincrease in both type-I IFN mRNA and protein expression byoverexpressing TRAF3 in HPVþ UMSCC cell lines with lowendogenous levels or TRAF3 rescue in TRAF3-KO cell lines. TRAF3also sensitizedHPVþ cells to antiproliferative effects of exogenousIFNA1 that may be produced by cells in the tumor microenvi-ronment, or IFN2A, which is used clinically.

A novel finding of this study supporting the importance ofdecreased TRAF3 in viral pathogenesis is that overexpression ofTRAF3 enhanced the expression of TP53 and RB, which are criticaltumor suppressor proteins inactivated by HPV E6 and E7 onco-proteins (21, 22, 38, 39). Proliferation was enhanced by knock-downof TP53 andTP53-regulatedCDKN1A(p21) in cells selectedto express TRAF3, but not in control cells. However, the exactmechanism by which TRAF3 modulates these tumor suppressorsand the relationship of these effects to E6 andE7 is complicated bythe difficulty in detecting these proteins. The minor reduction inE6 observed with TRAF3 was not accompanied by appearance ofincreased or decreased MW bands, and/or modulated by protea-some inhibitors, that would support a ubiquitin or proteasome-dependent mechanism (38). Further, the effects of TRAF3 ondecreased E6 were detected at later time points, compared witheffects on TP53 and RB expression or NF-kB activation. Thedifferent kinetics suggest that TRAF3 modulates E6, TP53, andRB expression by different transcriptional or posttranslationalmechanisms.

Of potential therapeutic relevance, TRAF3 re-expression inHPVþ HNSCC cells with low endogenous expression sensitizedthem to inhibition by cisplatin chemotherapy and TNFa, animportant cell death ligand induced by radiation and immunetherapy (40, 41). Cisplatin is the most active chemotherapeuticagent in HNSCC (42), leading to DNA damage and subsequentlypromoting cell death (43). TNFa is known to bind to TNFreceptors and induce apoptotic cell death in normal cells, butHNSCC are relatively resistant to TNFa (28, 44). NF-kB and cIAPshave been implicated in resistance to TNFa and chemotherapy(20, 45). Our data suggest that TRAF3 expression can inhibitcIAP1 expression and activation of the alternativeNF-kBpathway,and sensitize HPVþ HNSCC to TNFa and cisplatin-induced celldeath.Wehave recently reported that IAP1 inhibitors can sensitizeHPV�HNSCCwith gene copy gain and expression of IAP1 to TNF,chemotherapy, and radiotherapy (28, 46, 47), supporting futureinvestigation of these agents in HPVþ HNSCCs expressing defi-cient TRAF3 and/or cIAP1.

Although both HPVþ HNSCCs and NPCs develop within theadenotonsillar tissue and exhibit high rates of spread to regionallymph nodes, the mechanisms for this predilection are not wellunderstood. Intriguingly, TRAF3 re-expression inhibited cellmigration in vitro, suggesting that deficient TRAF3 could contrib-ute to enhanced migration, underlying the high rates of regionalspread of both HPVþ HNSCC and NPC in vivo. The alternative ofNF-kBpathway couldhave abroader role inmigration inHNSCC,as we found that IKKa and RELB knockdown decreased cellmigration and proliferation in HPV� HNSCC (34, 48). Together,the unique genomic alterations in TRAF3 and aberrantly activatedalternative NF-kB pathway components such as cIAPs or NIK,could serve as potential markers and targets for novel therapies ofHPVþ HNSCC.

Zhang et al.





Disclosure of Potential Conflicts of InterestC. Van Waes reports receiving a commercial research grant from Astex

Pharamaceuticals. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: J. Zhang, C.M. Annunziata, Z. Chen, C. Van WaesDevelopment of methodology: J. Zhang, X. Yang, P.E. Clavijo, Z. ChenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Zhang, T. Chen, X. Yang, S.S. Sp€ath, C. Silvin,N. Issaeva, Z. ChenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Zhang, T. Chen, X. Yang, H. Cheng, S.S. Sp€ath,P.E. Clavijo, J. Chen, N. Issaeva, W.G. Yarbrough, Z. Chen, C. Van WaesWriting, review, and/or revision of the manuscript: J. Zhang, T. Chen,S.S. Sp€ath, P.E. Clavijo, J. Chen, W.G. Yarbrough, C.M. Annunziata,Z. Chen, C. Van WaesAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): X. Yang, Z. ChenStudy supervision: Z. Chen, C. Van Waes

AcknowledgmentsWe thank Dr. Cheng-Ming Chiang and S.-Y. Wu (UT Southwestern

Medical Center) for providing HPV16E6 expression plasmid and theirhelpful suggestions. We thank Drs. Liu Yang (University of Maryland Schoolof Medicine) and WanJun Chen (NIDCR/NIH) for reading the manuscriptand comments.

This work was supported by the Intramural Research Program ofThe National Institute on Deafness and Other CommunicationDisorders. J. Zhang, T. Chen, H. Cheng, X. Yang, P. Clavijo, J. Coupar,C. Silvin, Z. Chen, and C. Van Waes are supported by NIDCD intra-mural projects ZIA-DC-000016, 73, 74. C.M. Annunziata is supported bythe NCI.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 2, 2017; revised January 11, 2018; accepted June 7, 2018;published first June 19, 2018.

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Zhang et al.




2018;78:4613-4626. Published OnlineFirst June 19, 2018.Cancer Res Jialing Zhang, Tony Chen, Xinping Yang, et al. Promote HPV-Positive Head and Neck CancersReduced Expression of Antiviral Interferon, TP53, and RB to

B andκAttenuated TRAF3 Fosters Activation of Alternative NF-

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