Attenuated TRAF3 Fosters Activation of Alternative NF-Kb and Reduced Expression of Antiviral Interferon, TP53, and RB to Promote

Published OnlineFirst June 19, 2018; DOI: 10.1158/0008-5472.CAN-17-0642

Cancer Tumor Biology and Immunology Research

Attenuated TRAF3 Fosters Activation of Alternative NF-kB and Reduced Expression of Antiviral Interferon, TP53, and RB to Promote HPV-Positive Head and Neck Cancers Jialing Zhang1,2,Tony Chen1, Xinping Yang1, Hui Cheng1, Stephan S. Spath€ 3, 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- endogenous TRAF3 inhibited NF-kB2/RELB expression, demic increase in oropharyngeal head and neck squamous nuclear localization, and NF-kB reporter activity, while cell carcinomas (HNSCC), which display viral inactivation increasing the expression of IFNA1 mRNA and protein and of tumor suppressors TP53 and RB1 and rapid regional sensitizing cells to its growth inhibition. Overexpression spread. However, the role of genomic alterations in enabling of TRAF3 also enhanced TP53 and RB tumor suppressor þ the modulation of pathways that promote the aggressive proteins and decreased HPV E6 oncoprotein in HPV cells. phenotype of these cancers is unclear. Recently, a subset of Correspondingly, TRAF3 inhibited cell growth, colony for- þ HPV HNSCC has been shown to harbor novel genetic mation,migration,andresistancetoTNFa and cisplatin- defects or decreased expression of TNF receptor–associated induced cell death. Conversely, TRAF3 knockout enhanced þ factor 3 (TRAF3). TRAF3 has been implicated as a negative colonyformationandproliferationofanHPV HNSCC line regulator of alternative NF-kB pathway activation and expressing higher TRAF3 levels. Together, these findings activator of antiviral type I IFN response to other DNA support a functional role of TRAF3 as a tumor suppressor þ þ viruses. How TRAF3 alterations affect pathogenesis of HPV modulating established cancer hallmarks in HPV HNSCC. HNSCC has not been extensively investigated. Here, we þ report that TRAF3-deficient HPV tumors and cell lines Significance: These findings report the functional role of exhibit increased expression of alternative NF-kBpathway TRAF3 as a tumor suppressor that modulates the malignant þ components and transcription factors NF-kB2/RELB. Over- phenotype of HPV head and neck cancers. Cancer Res; 78(16); þ expression of TRAF3 in HPV cell lines with decreased 4613–26. 2018 AACR.

Introduction types HPV16 and HPV18 has been established as an important risk factor for HNSCC that develop predominantly in the Head and neck squamous cell carcinoma (HNSCC) is the oropharyngeal tonsils (3). Since 1981, there has been a notable sixth most common cancer, with an annual incidence of þ 225% increase in HPV HNSCC, while the incidence of smok- 650,000 new cases and 200,000 deaths worldwide (1, 2). ing-related HPV HNSCC has declined (4, 5). Clinically, the Persistent infection with high-risk human papillomavirus sub- þ HPV subset exhibits better responses to therapies and survival þ rates than similarly advanced HPV tumors. However, HPV HNSCC are distinguished by aggressive spread and growth 1 Head and Neck Surgery Branch, National Institute on Deafness and Other within regional lymph nodes, which require major surgery or 2 Communication Disorders, NIH, Bethesda, Maryland. Clinical Medicine Research toxic chemoradiotherapy regimens (2, 3). The factors that Center, The Afﬁliated Hospital, Inner Mongolia Medical University, Hohhot, 3 contribute to the molecular pathogenesis of these unique China. Pediatric Endocrinology Unit, Department of Women's and Children's þ Health, Karolinska Institute and University Hospital, Stockholm, Sweden. features of HPV HNSCC remain incomplete. 4Department of Surgery, Otolaryngology, Yale Cancer Center, New Haven, HPV16 and 18 carry early genes E6 and E7 encoding onco- Connecticut. 5Department of Pathology, Yale School of Medicine, New Haven, proteins that target key pathways, deregulating host resistance 6 Connecticut. Women's Malignancies Branch, Center for Cancer Research, NCI, to infection and cellular proliferation, to promote the viral life NIH, Bethesda, Maryland. cycle. HPV E6 expression in keratinocytes can repress type-I IFN Note: Supplementary data for this article are available at Cancer Research and promote proliferative genes, to enhance viral protein Online (http://cancerres.aacrjournals.org/). synthesis and proliferation of virally infected cells (6). Further Corresponding Authors: Carter Van Waes and Zhong Chen, National Institute studies have shown that HPV infection can induce ubiquitin on Deafness and Other Communication Disorders, NIH, Room 7N240D, 10 carboxyl-terminal hydrolase L1 (UCHL1), which can inhibit Center Drive, Bethesda, MD 20892. Phone: 301-402-4216; Fax: 301-402-1140; K63 ubiquination, important in Tank binding kinase-immune E-mail: [email protected]; [email protected] response factor 3 (TBK-IRF3)-mediated type-I IFN expression doi: 10.1158/0008-5472.CAN-17-0642 (7). HPV E6 can also commandeer and activate the so-called 2018 American Association for Cancer Research. alternative nuclear factor-kB2 (NF-kB2) transcription factors

www.aacrjournals.org 4613

Zhang et al.

and antiapoptotic genes, which promote resistance of kerati- pipelines and accessible through TCGA data portals. Significant nocytes to TNF, an important mediator of antiviral immunity focal copy-number alterations were extracted from the GISTIC2.0 (8). Critically, the HPV E6 and E7 oncoproteins also strategi- processing pipeline, and data aggregated on significantly altered cally target for degradation the tumor suppressor proteins TP53 lesions are plotted by false discovery rate (FDR) less than 5% and P and RB, which control the cell cycle (9). Interestingly, however, values of less than 0.05. Differential gene expression analysis of few individuals exposed to HPV develop chronic infection and tumor versus normal samples was performed using DeSeq, and all HNSCC. These observations suggest that additional genetic data were log2 transformed (Bioconductor version 2.12). Molec- alteration(s) and host factors may also affect how HPV med- ular HPV signatures were identified using gene expression and iates suppression of IFNs, NF-kB activation, inhibition of TP53 somatic substitutions. Gene set enrichment analysis was per- and RB gene expression, and the malignant phenotype. formed using the online Cancer Genomics cBioportal database Recently, we and The Cancer Genome Atlas (TCGA) Network (http://www.cbioportal.org/). þ uncovered a subset of HPV HNSCCs that harbor deletions of the chromosome region 14q32.32, deleterious truncating HNSCC lines, cell culture, genomic DNA extraction, and PCR mutations, and/or decreased expression, affecting the gene TNF UMSCC [University of Michigan (Ann Arbor, MI) series of receptor–associated factor 3 (TRAF3; ref. 10). These deletions were HNSCC], UPCI (University of Pittsburgh Cancer Institute, Pitts- not due to viral integration and disruption of the TRAF3 gene. burgh, PA), and VU (Free University Amsterdam, Amsterdam, the Intriguingly, TRAF3 is a unique adaptor protein and ubiquitin Netherlands) cell lines and clinical information were provided by ligase implicated as a negative regulator of the aforementioned Drs. Thomas E. Carey, Mark E. Prince, Carol R. Bradford (Uni- alternative NF-kB2/RELB pathway (11). TRAF3 promotes cIAP- versity of Michigan), Robert Ferris, and Susanne Gollin (Univer- mediated ubiquitination and proteasome-dependent degrada- sity of Pittsburgh), or from previous publications (Supplementary tion of the pivotal NF-kB inducing kinase (NIK) protein, which Table S1; refs. 14–16). Cell authentication of UMSCC, UPCI, and mediates signal activation of the alternative pathway. Lympho- VU lines was done at the University of Michigan by DNA geno- toxin-b (LTb) and other ligands, which are richly expressed in typing of alleles for 9 loci (D3S1358, D5S818, D7S820, D8S1179, þ the oropharyngeal tonsils and lymph nodes where HPV D13S317, D18S51, D21S11, FGA, vWA) and the amelogenin HNSCC arise and spread, bind receptors to activate NIK, IKKa, locus as described previously (15). A panel of 8 HPV-positive processing of NF-kB2 precursor p100 to p52, and nuclear (UMSCC 47, 104, 2, 90, 152, 154, 93VU147T, and 105) and 8 translocation of transcriptionally active NF–kB2–p52/RELB HPV-negative (UMSCC 1, 9, 11A, 11B, 38, 46, 74A, and 74B) dimers. Attenuation of TRAF3 has previously been implicated HNSCC cell lines was selected for evaluation of HPV type and in the transcription of genes affecting cell fate, proliferation, TRAF3 expression. These cell lines were maintained in MEM or and survival of lymphoid cells and hematopoietic malignancies DMEM with 10% fetal calf serum (Life Technologies) at 37C with (11, 12). Strikingly, TRAF3 has also been shown to serve a dual 5% CO2 for a maximum of 8 weeks. Primary human oral kera- function in interferon responses to other DNA viruses (11, 13). tinocytes (HOK) were cultured, in accordance with the supplier's However, the functional role of genetic alterations or reduced protocol (Science Cell Research). Prior to experiments, all cells expression of TRAF3 in modulating alternative NF-kBpathway were confirmed to be Mycoplasma negative (by MycoAlert kit, cat. activation, IFN expression, repression of tumor suppressors no. M7006, Thermo Fisher). Genomic DNA was extracted using a þ TP53 and RB, and the malignant phenotype in HPV HNSCC DNeasy Blood and Tissue Kit (cat. no. 51104, Qiagen) from has not been established. selected frozen cell pellets. These cell lines were further evaluated In this study, we examined and revealed a novel role for for HPV status by PCR (Human Papillomavirus Detection Set, cat. decreased TRAF3 in fostering deregulation of these pathways no. 6602, Takara Bio). þ and promoting pathogenesis of a subset of HPV HNSCC. These findings provide new opportunities for basic and clinical RNA-seq analysis research that may lead to better diagnosis, prevention, and Total RNA of 3 primary human keratinocyte and individual þ treatmentofHPV HNSCC. HNSCC cell line was isolated by combination of TRIzol (cat no. 15-596-026, Life Technologies) and QIAGEN RNeasy Mini Kit procedure (cat. no. 74104, QIAGEN). Ribosomal RNA was Materials and Methods depleted using the Ribo-Zero kit (cat no. MRZH11124, Epi- HNSCC patient samples and TCGA bioinformatics analysis centre). Multiplexed whole transcriptome libraries were gener- TCGA Project Management has collected necessary human ated by SOLiD Total RNA-Seq Kit and SOLiD RNA Barcoding subjects documentation to ensure that the project complies with Kit (cat. no. 4427046, Life Technologies), and fragmented 45-CFR-46 (the "Common Rule"). The program has obtained cDNA libraries were clonally amplified by emulsion PCR. The documentation from every contributing clinical site to verify that multiplex libraries were sequenced utilizing 75 bp forward Institutional Review Board approval and informed consent has and 35 bp reverse paired-end sequencing chemistry on the been obtained to participate in TCGA. The characteristics and data ABI SOLiD system. Reads were mapped into human NCBI repositories for 279 HNSCC specimens and 16 normal mucosa Build 37 reference genome (Hg19) using LifeScope v.2.5 Geno- þ were previously reported, including 36 HPV , 243 HPV HNSCC, mic Analysis software (https://www.appliedbiosystems.com/ and 16 normal samples (10). Exome-wide sequencing was per- lifescope). The read count of the genes was normalized using formed on all samples, and normalized genomic data for 279 DESeq (estimateSizeFactors) R package (version 3.2.0) and HNSCC and 16 normal samples displaying a squamous keratin upper quartile normalization method. Expression analysis was gene signature was obtained from the TCGA Genome Data performed by comparison with normal HOK cell line expres- Analysis Center and downloaded on September 19, 2012. All sion, and gene expression was quantified using RSEM (RNA-Seq genomic data were processed through standard TCGA analytic by Expectation Maximization).

4614 Cancer Res; 78(16) August 15, 2018 Cancer Research

Decreased TRAF3 Promotes HPVþ HNSCC

þ Plasmids, siRNA, and transduction cell lines (P < 0.05, Fig. 1D), consistent with the few HPV YFP-pTo-TRAF3 fusion expression vector was kindly provided tumors and cell lines with low expression, as well as high by Dr. Christina M. Annunziata, NCI/NIH, Bethesda, MD (16). number of HPV tumors and cell lines with gain of expression His-tagged TRAF3 wild-type was generated by GeneCopoeia. For of TRAF3. We further compared TRAF3 protein levels in the 8 þ efficient retroviral transductions, certain cell lines were engineered HPV HNSCC cell lines with normal human oral keratinocytes to express these indicated vectors. SMART pool siRNAs targeting (HOK). UMSCC47 and UMSCC104 cell lines exhibited lower human TRAF3 (cat. no. E-005252) and nontargeting siRNA con- TRAF3 protein levels as assessed by Western blot (Fig. 1E), and trol (M-HUMAN-XX-0005) were purchased from Dharmacon. these lines were selected for further study of the effects of Cell transfection was carried out using X-tremeGENE HP DNA decreased TRAF3 expression and modulation. Transfection Reagent or X-TremeGENE siRNA Transfection Reagent (cat. no. 10465500, 063662, Roche), following the man- Decreased TRAF3 is associated with increased expression of þ ufacturer's instructions. Stably transfected cell lines were allowed alternative NF-kB pathway components in HPV HNSCC to recover for 48 hours before antibiotic selection, and only tumors confirmed surviving pools were utilized for subsequent analysis. As TRAF3 was previously identified as a negative regulator of the alternative NF-kB pathway in immune and hematopoietic TRAF3 CRISPR knockout cells malignant cells (11, 12), we asked whether altered TRAF3 is UPCI-SCC-90 and UMSCC47 cells were cotransfected with associated with NF-kB pathway alterations in HNSCC tissues. TRAF3 CRISPR/Cas9 KO and TRAF3 HDR plasmids (Santa Cruz Using the HNSCC TCGA datasets, we identified an apparent co- Biotechnology) at equivalent ratios using lipofectamine 2000 occurrence between TRAF3 genomic and expression alterations (cat. no. 11668019, Thermo Fisher). Forty-eight hours after trans- with the mRNA expression of alternative NF-kB pathway compo- fection, the cells were plated at low density with the addition of nents (Fig. 2A left, top), but not with those in classical NF-kB þ 1 mg/mL puromycin (cat. no. A1113802, Thermo Fisher) for pathway (Fig. 2A left, bottom), in HPV HNSCC tumors. Con- selection of stable clones. Successful knockdown was confirmed versely, no such co-occurrence was observed in HPV HNSCC with RT-PCR. Clonogenic assay was performed as described (17). tumors (Fig. 2A, right). Supporting this, we observed a significantly higher average percentage of alterations in the NF-kB þ Statistical analysis alternative pathway in HPV tumors, compared with HPV Data are shown as means SEM. Student t test or two-way tumors, whereas no significant difference was observed in the ANOVA were performed using GraphPad. Statistical analysis is classic NF-kB pathway (Fig. 2B). Finally, the co-occurrence and specifically indicated for each experiment (, P < 0.05; , P < 0.01; mutual exclusivity of paired alterations were calculated, and the , P < 0.001; , P < 0.0001). relationships with statistical significance are indicated as color Additional methods of preparation of total RNA isolation, intensities (Fig. 2C; Supplementary Table S2). We observed more cDNA synthesis, qRT-PCR analysis, protein extracts, Western blot frequent co-occurrence of alterations in the key components of the þ analysis, reporter assay, immunofluorescence staining, treat- alternative than the classic NF-kB pathway in HPV HNSCC ments, and cell viability are presented in the Supplementary tumors (Fig. 2C, left). In contrast, fewer tumor samples with Information. strong and intermediate co-occurrences were observed in HPV HNSCC (Fig. 2C, right). These analyses indicate that deficient TRAF3 is associated with increased expression of alternative þ Results NF-kB pathway components in HPV HNSCC tumor specimens. þ TRAF3 alterations are associated with HPV HNSCC status þ The recent TCGA study revealed that a subset of HPV HNSCC TRAF3 expression inhibits the alternative but not classic NF-kB þ display novel recurrent deletions, truncating mutations, and/ signaling in HPV HNSCC cell lines or decreased expression of TRAF3 (10). Here, we further To test the hypothesis that decreased TRAF3 is permissive for þ show that TRAF3 focal or broader deletions of chromosome 14 activation of the alternative NF-kB pathway in HPV HNSCC, we þ (Chr14q32.32) are mainly observed in HPV HNSCC (Fig. 1A). examined the expression of alternative pathway components and þ Among 36 HPV tumors in this dataset, 14% homozygous and the effects of transiently expressing His-tagged TRAF3 (His- þ 25% heterozygous deletions were found, whereas in 243 HPV TRAF3) or control (His-Control) vectors in the HPV UMSCC47 tumors, only 1.2% homozygous and 10% heterozygous deletions and UMSCC104 cells that displayed relatively lower endogenous were observed (Fig. 1B, Fisher exact test, P ¼ 2.1 10 6). In TRAF3 expression. TRAF3 mRNA was significantly increased in contrast, 38% of HPV tumors exhibited TRAF3 copy-number both cell lines after TRAF3 transfection (Fig. 3A, top). Next, we þ gains compared with only 6% of HPV tumors. Together, the evaluated the mRNA expression levels of key alternative pathway different distribution of these DNA copy-number variations were subunits RELB and NF-kB2, which are modulated at transcrip- þ associated with TRAF3 mRNA expression in HPV (P ¼ 3.52 tional and posttranslational levels, as well as classic (RELA/p65) 10 5) and HPV HNSCC samples (P ¼ 2.0 1010; Fig. 1C). NF-kB subunit, which is constitutively expressed and modulated Furthermore, three cases with deleterious frameshift or nonsense posttranslationally (18). Consistent with this, transient TRAF3 þ þ mutations were observed only in HPV HNSCC. overexpression in both HPV cell lines resulted in a significant þ To determine if there are HPV HNSCC cell lines with reduction of RELB and NF-kB2 mRNA expression, without affect- decreased TRAF3 expression suitable for study, we investigated ing RELA expression (Fig. 3A). We then evaluated the effect of þ TRAF3 expression in a panel of eight HPV and HPV HNSCC cell TRAF3 expression on protein levels of key alternative NF-kB lines (Supplementary Table S1), using RNA sequencing data. pathway components in the same cell lines and an additional þ Overall, we observed significantly increased TRAF3 mRNA HPV HNSCC cell line, 93VU174T, following TRAF3 transfection þ expressioninHPV , compared with normal HOK and HPV (Fig. 3B; Supplementary Fig. S1A). TRAF3 expression significantly

www.aacrjournals.org Cancer Res; 78(16) August 15, 2018 4615

Zhang et al.

reduced protein levels of components involved in the alternative co-factor TRAF2 or alternate pathway kinase IKKa that mediates NF-kB pathway, including its co-factor and ubiquitinase cIAP1 NF-kB2-p52/RELB (Supplementary Fig. S1B). Taken together, þ together with its substrate NIK, and processed NF-kB2/p52 and these results indicate that increasing expression of TRAF3 in HPV RELB subunits (Fig. 3B and C; Supplementary Fig. S1A). HNSCC cells with lower TRAF3 can suppress mRNA and/or We observed comparatively smaller decreases in TRAF3-cIAP protein 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) HNSCC tumor specimens. The blue bar at the top right corner of chromosome represents the Chr14q32.32 region where TRAF3 is located. The color gradients depict 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 and 6 HPV status was observed (Fisher exact test, P ¼ 2.12 10 ). 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 75th percentiles of TRAF3 gene expression. The whiskers mark the minimal and maximum values, excluding the outliers. The red triangle and diamond represent frameshift and nonsense mutations. D, TRAF3 mRNA expression of selected HPVþ and HPV HNSCC cell lines compared with HOK cells (control). , P < 0.05 by Student t test. E, Whole cell lysates were harvested, and TRAF3 protein levels were examined by Western blot for HPVþ HNSCC cell lines. The histogram of relative protein expression was generated from densitometry analysis of relevant protein bands after adjustment to loading control of b-actin and normalized to HOK cells (control).

4616 Cancer Res; 78(16) August 15, 2018 Cancer Research

Decreased TRAF3 Promotes HPVþ HNSCC

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 gene was 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 members in 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.

TRAF3 expression modulates NF-kB2/p52 and RELB nuclear ing nuclear p100/p52 and RELB protein levels in independent þ localization, and NF-kB activity in the HPV UMSCC47 cell line experiments (Fig. 4A; Supplementary Fig. S2A and S2B). We To further characterize how TRAF3 modulates the cellular further tested functional NF-kB activity by utilizing a NF-kB þ distribution of alternative RELB/NF-kB2 subunits in HPV luciferase reporter gene assay in the UMSCC47 cell line. Consis- HNSCC, we examined cytoplasmic and nuclear expression of tent with the reduced nuclear RELB/p52 protein levels, we these protein subunits by Western blot. Aberrant nuclear locali- observed a signiﬁcant reduction in functional NF-kB reporter zation of RELB, NF-kB2/p52, as well as p100 was detected in activity in TRAF3 cotransfected UMSCC47 cells (Fig. 4B). control nuclear as well as cytoplasmic fractions (Fig. 4A), consis- Conversely, TRAF3 knockdown using three different siRNAs, tent with a prior report demonstrating enhanced nuclear expres- signiﬁcantly reduced TRAF3 mRNA expression in UMSCC47 cells þ sion of both p100/52 in HPV SCC and E6-expressing keratino- (Fig. 4C). Consistent with reduced TRAF3 mRNA expression cytes (19). Transfection of TRAF3 increased distribution of NF- in Fig. 4C, we observed reduced TRAF3 and increased RELB kB2 p100/52 proteins in the cytoplasmic fraction while decreas- protein by Western blot (Supplementary Fig. S2C), as well

www.aacrjournals.org Cancer Res; 78(16) August 15, 2018 4617

Zhang et al.

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 lines UMSCC47 (left) and UMSCC104 (right) were transiently transfected with TRAF3 (His-TRAF3) or control (His-Control) plasmid for 24 hours, and mRNA expression of 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, HPVþ UMSCC47 and UMSCC104 cells were transiently transfected with TRAF3 expression vector and whole-cell lysates were harvested 48 hours after transfection. Protein expression of TRAF3 and alternative NF-kB pathway components were examined by Western blots. C, Histograms for expression of indicated proteins were derived from densitometry analysis of bands after adjustment to loading control of b-actin and are presented relative to the cells transfected with control plasmid at 48 hours. Data, mean SEM from independent experiments with three HPVþ cell lines (UMSCC47, 104, and 93VU174T; Fig. 3B and Supplementary Fig. S1). Signiﬁcant differences between TRAF3-transfected samples and controls refer to , P < 0.05; , P < 0.01; , P < 0.001.

as enhanced nuclear RELB by immunofluorescence staining lymphotoxin-beta receptor (LTbR), and two classic NF-kB (Fig. 4D). TRAF3 knockdown was accompanied by increased pathway TNF receptors (TNFR1A and TNFR1B). We detected þ NF-kB reporter activity in UMSCC47 cells (Fig. 4E). These results elevated mRNA expression of LTbRinHPV compared with demonstrate that TRAF3 can inversely modulate alternative HPV HNSCC cell lines (Fig. 5A, left, HOK cells as dotted NF-kB pathway RELB and NF-kB2/p52 expression, cellular local- line), while no significant difference was observed for either þ ization, and functional activity in HPV HNSCC cells. TNFRs (Fig. 5A, right). Next, we compared the functional effects of recombinant LTb or TNFa proteinonNF-kBreporter þ TRAF3 inhibits aberrant and LTb-enhanced NF-kB2/RELB activity in HPV UMSCC47 cells and HPV UMSCC1 cells, protein, increases sensitivity to TNFa and cisplatin, as well as establishing that LTb as well as TNFa could induce NF-kB þ inhibits migration of HPV HNSCC cells reporter activity in both (Fig. 5B; Supplementary Fig. S3A). We þ HPV HNSCCs display rapid growth and spread within the found that LTb treatment attenuated TRAF3 and NF-kB2/p100 tonsils and regional lymph nodes, where ligands activating the precursor protein, while enhancing RELB and processed NF- alternative NF-kB pathway are expressed (4, 11). To investigate kB2/p52 protein levels (Fig. 5C; Supplementary Fig. S3B). þ the upstream receptor-mediated activation of NF-kBinHPV Conversely,afterknockdownofTRAF3,basalandLTb-induced þ lines, we first established if HPV and HPV HNSCC cell levels of RELB and NF-kB2/p52 proteins were substantially lines express mRNAs for the alternative pathway receptor enhanced (Fig. 5C; Supplementary Fig. S3B), supporting the

4618 Cancer Res; 78(16) August 15, 2018 Cancer Research

Decreased TRAF3 Promotes HPVþ HNSCC

Figure 4. TRAF3 decreases nuclear NF-kBp52 and RELB localization, and NF-kB activity in HPVþ UMSCC47 cell line. A, UMSCC47 cells were transiently transfected with TRAF3 (GFP-TRAF3 pToCMV) or control (GFP-pToCMV) plasmids for 24 hours. Cytoplasmic and nuclear protein were harvested and assessed by Western blot. b-Actin or Lamin A was used as cytoplasmic or nuclear protein loading control, respectively. B, UMSCC47 cells were transiently transfected with TRAF3 or control expression vector with NF-kB reporter plus LacZ plasmids for 24 hours. NF-kB reporter activity was adjusted to b-galactosidase. C, TRAF3 mRNA expression decreases following TRAF3-specific siRNAs (siTRAF3a-c) at 24 hours after transfection. All depicted samples were normalized to scrambled control siRNA (si-Control) knockdown. Data, mean SEM derived from three replicates. D, Nuclear RELB localization by confocal immunofluorescence microscopy following TRAF3-specific siRNA (siTRAF3b) or scrambled control siRNA (si-Control) knockdown at 24 hours. Cells were fixed and stained with antibodies against TRAF3, RELB, and Alexa fluor–conjugated secondary antibodies. Representative images show the localization of TRAF3 (green), RELB (purple), DAPI (blue), and merged images. Scale bar, 10 mm. E, NF-kB–mediated luciferase activity increases following TRAF3-specific siRNA (siTRAF3a-c). Renilla luciferase assay was performed on the same lysates and was used to adjust the differences in transfection efficiencies. All depicted samples were normalized to scrambled si-Control knockdown. Data, mean SEM derived from 6 replicates. Significant differences between samples and controls refer to , P < 0.05; , P < 0.01.

role of TRAF3 as a negative regulator of the alternative NF-kB cIAP1 above, we observed minimal modulation of BCL family þ pathway in HPV HNSCC cells. members that contribute to survival (Supplementary Fig. S4C). To As we observed above that TRAF3 inhibited alternative NF-kB further examine the functional role of alternative NF-kB pathway, activation and cIAP1, which have been previously linked to we knocked down IKKa and RELB to evaluate cell proliferation in þ proliferation, and TNFa and chemotherapy resistance (20), we HPV UMSCC47 cells. We observed a significant reduction in the evaluated if TRAF3 overexpression could inhibit tumor cell cell proliferation after knockdown of either gene alone, with RELB proliferation, or enhance the inhibitory effects of TNFa,LTb,or knockdown having a greater effect than IKKa knockdown on cell þ chemotherapy agent cisplatin used for HNSCC. Transient TRAF3 proliferation (Fig. 5E). As HPV HNSCCs show increased pro- expression resulted in a modest but significant decrease in cell pensity for malignant spread, we next performed a cell migration proliferation and showed greater inhibitory effects in combina- assay. We observed a significant delay in cell migration over a 24- tion with TNFa, cisplatin, or both at day 3 (Supplementary hour time course in UMSCC47 cells expressing TRAF3 (Fig. 5F), Fig. S4A) and day 5 (Fig. 5D). In cell lines selected for TRAF3 before antiproliferative effects 10% of control are observed in and control vector expression, TRAF3 enhanced sensitivity of cells XTT assay by day 3 (Supplementary Fig. S4A). Together, these data to the combination of TNFa and CDDP, but greater resistance and demonstrate that TRAF3 inhibits LTb enhanced alternative path- growth in response to TNFa alone (Supplementary Fig. S4B). way NF-kB2/p52 and RELB protein levels, increased sensitivity to While sensitization to TNFa and cisplatin was associated with TNFa and chemotheraputic agent as well as inhibits migration of þ TRAF3-inhibitory effects on alternative NF-kB components and HPV HNSCC cells.

www.aacrjournals.org Cancer Res; 78(16) August 15, 2018 4619

Zhang et al.

Selection for TRAF3 expression or CRISPR knockout modulates TRAF3 or vector plasmid control by G418 selection, and exam- þ þ colony formation and proliferation of HPV HNSCC ined the colony-forming capacity and proliferation of HPV As TRAF3 appeared to reduce proliferation by 5 days following UMSCC47 cells. We observed a signiﬁcant reduction in colony transient transfection, we enriched for UMSCC47 cells expressing formation of UMSCC47 cells that expressed increased TRAF3,

Figure 5. LTBR expression and LTb-induced NF-kB activation in HPVþ HNSCC cell lines and enhancing effects of TRAF3 knockdown on alternative NF-kB pathway activation. A, mRNA-seq expression in eight cell lines, each with different HPV status were normalized to HOK cells (marked by dashed line). LTBR expression is relatively higher in HPVþ cell lines, while TNFR1A and TNFR1B exhibit no significant difference across HNSCC cell lines with different HPV status. The solid black line is the mean 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 in 96-well plate, cultured for 48 hours, then treated 10 ng/mL TNFa 16 hours or 50 ng/mL LTb for 24 hours prior to GeneBLAzer Assay at 72 hours. Mean NF-kB activity normalized 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, followed by LTb induction for 2 hours. The alternative NF-kB components RELB and NF-kB2 (p100 and p52) were examined in Western blot. b-Actin (Actin) was used as 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-well plates, and the next day were treated with TNFa (25 ng/mL), LTb (100 ng/mL), cisplatin (10 mmol/L) alone, or combination of TNFa,orLTb with cisplatin. Cells transfected with control vector, blue; TRAF3 vector, red. Statistical significance (P < 0.05) was determined by the Student t test when compared with the control vector (), untreated control (#), or cisplatin alone (þ). The data are summarized from six replicates and are represented as mean SD. E, UMSCC47 cells were transiently transfected with siRNA targeting alternative pathway mediators IKKa and RELB, and cell proliferation was measured 3 days after transfection. Knockdown of IKKa or RELB decreased cell proliferation. All data are calculated relative to the samples transfected with control siRNA from 6 replicates and are represented as mean SD. , P < 0.05; , P < 0.001; , P < 0.0001; Student t test. F, HPVþ UMSCC47 cells expressing TRAF3 or control vectors were plated in three wells each, and scratches were performed next day when the cells grew to confluency. Reduced cell migration via wound-healing assay was observed in TRAF3-transfected cells for 12 and 24 hours. D, The wound area percentage is shown for the control (blue) or TRAF3-expressing cells (red) at 12 and 24 hours after scratch. The cell migration assay was quantified from triplicates; , P < 0.01 by Student t test.

4620 Cancer Res; 78(16) August 15, 2018 Cancer Research

Decreased TRAF3 Promotes HPVþ HNSCC

Figure 6. TRAF3 expression or CRISPR knockout modulates colony formation and proliferation of HPVþ HNSCC. HPVþ UMSCC47 cells were transfected with His-TRAF3 or empty His-Control vector and selected by G418 for 2 weeks. A, The images of cell colony formation were taken using a microscope image system. B, Histogram showing the percentage of colonies produced with TRAF3-expressing cell line, compared with control vector. The colony numbers are represented as mean SD, derived from triplicates. , P < 0.01 by Student t test. C, Cell proliferation of UMSCC47 cells selected for control or TRAF3 expression vector. Cells were plated in 96-well impedance plates and increasing cell densities were measured by impendence was determined at 40, 60, 80, and 100 hours. Mean cell density units þSD for 6 replicates. , P < 0.05 Student t test for TRAF3 compared with control at each time point. D, Quantitative RT-PCR showing TRAF3 mRNA depletion in UPCI-SCC-90 CRISPR (Cr)TRAF3 knockout. E and F, Increased colony formation and cell proliferation of UPCI-SCC-90 CRISPR (Cr)TRAF3 knockout. Clonogenic survival was performed after plating 5,000 cells in 6-well plates. After 1 week (UPCI-SCC-90 CrTRAF3) or 2 weeks (UPCI-SCC-90 control), the colonies were ﬁxed and stained with methylene blue in methanol. Colonies consisting of at least 30 cells were counted. Results from two independent experiments are presented. For proliferation, 1 105 UPCI-SCC-90 control or CrTRAF3 cells were plated in 12-well plates. At indicated time points, cells were collected and counted. Results from two independent experiments are presented.

þ þ compared with control (Fig. 6A and B). UMSCC47 cells selected gene in HPV and HPV cell lines, as well as tumors. The HPV for expression also demonstrated significantly reduced cell pro- HNSCC cell lines displayed lower IFNA1 expression than normal liferation in TRAF3 versus vector control cells (Fig. 6C). Interest- HOK or HPV HNSCC cell lines (Fig. 7A, left). IFNA1 mRNA in þ ingly, we observed that TRAF3 expression declined with passage HPV HNSCC tissues were also expressed at significantly lower despite selection, consistent with the antiproliferative activity of a levels when compared with HPV HNSCC tissues from TCGA tumor suppressor (Supplementary Fig. S5). As the cell line UPCI- data (Fig. 7A, right). To test the hypothesis that decreased TRAF3 SCC-90 expressed higher endogenous levels of TRAF3 (Fig. 1E), may contribute to reduction in IFNA and be reversed by TRAF3, we þ we next examined effects of CRISPR knockout of TRAF3 on colony compared IFNA1 expression in the two HPV HNSCC cell lines formation and cell proliferation. CRISPR depleted TRAF3 with decreased TRAF3 expression, UMSCC47 and UMSCC104, markedly enhanced colony formation and proliferation of following transient transfection with control and TRAF3 vectors. UPCI-SCC-90 (Fig. 6D–F). Together, our data demonstrate that TRAF3 induced a small but significant increase in the expression of þ TRAF3 expression suppresses the proliferation of HPV HNSCC IFNA1 in both cell lines (Fig. 7B). IFNA1 protein was also found to cells. be modestly significantly increased in cell culture supernatants þ isolated from HPV UMSCC47 cells stably transfected with þ TRAF3 expression enhances expression of and sensitizes HPV TRAF3 compared with control vector (Fig. 7C). UMSCC47 stably HNSCC to antiviral interferons expressing increased TRAF3 and IFNA1 as above exhibited TRAF3 contributes to expression of type I IFN in response to reduced proliferation and displayed enhanced sensitivity to exog- other DNA virus infections in other tissues (13), but the expres- enous IFNA1 or 2a (Fig. 7D). We explored if TRAF3 modulated sion of type-I IFN and relation to lower TRAF3 observed in mRNA expression of interferon response factors (IRF), which are þ HPV HNSCC has not been examined. We analyzed RNA-seq transcription factors that mediate the transcription of type-I IFN data from our HNSCC cell lines and TCGA HNSCC tumors to (7, 11). However, we did not see consistent changes in IRF3, 5, or 7 þ compare the mRNA expression profiles for type-I interferon IFNA mRNA expression in the HPV line selected for expression of the

www.aacrjournals.org Cancer Res; 78(16) August 15, 2018 4621

Zhang et al.

Figure 7. Expression of TRAF3 induces an antiviral interferon response, sensitizes to IFN, increases TP53 and RB protein expression, and modulates nuclear HPVE6 oncoprotein 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 HNSCC tissues 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 in supernatants by ELISA (C). Transient transfection of TRAF3 for 24 hours increased IFNA1 mRNA expression in HPVþ UMSCC47 and 104 cells and protein in supernatant of UMSCC47 cells. Data are derived from 6 replicates and represented as mean SD. , P < 0.05; ns, not significant. D, UMSCC47 cells stably transfected 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 by impedance. 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) when comparing IFNA1- or IFNA2a-treated cells with untreated cells. E, UMSCC47 selected to express TRAF3 show increased expression of tumor suppressor TP53 and RB. 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 stably transfected with TRAF3 or control vector were plated overnight and transfected with siRNA targeting CDKN1A or control siRNA. Cell proliferation was measured by impedance. 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) 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, E6 and empty pcDNA vector were transiently transfected into UMSCC47 cells and effects in E6 protein expression were compared. A slight increase in E6 protein was detected. Bottom right, A slight decrease in E6 expression was detected 72 hours after TRAF3 expression. Actin was used as the loading control.

4622 Cancer Res; 78(16) August 15, 2018 Cancer Research

Decreased TRAF3 Promotes HPVþ HNSCC

TRAF3 gene (Supplementary Fig. S6A). Further, while TRAF3- transfection is likely dependent on mechanisms other than mod- þ regulated IRF3 nuclear protein was also decreased in HPV ulation of E6 protein expression alone. UMSCC47 cells, the lack of nuclear IRF3 did not appear to be due to lower TRAF3 protein level alone, as expressing TRAF3 Discussion vector induced a minimal increase in expression of nuclear IRF3 in replicate experiments (Supplementary Fig. S6B). Moreover, TRAF3 copy loss, mutation, and/or decreased expression was þ þ even in HPV UMSCC47 with lower TRAF3 expression and low significantly enriched in a subset of HPV HNSCC tumors (10), nuclear IRF3, nuclear IRF3 and type-I IFN remained inducible suggesting that TRAF3 could be a novel suppressor of HPV viral with a non-HPV RNA virus mimic and TLR3 ligand poly-I:C, and tumor pathogenesis. Previously, TRAF3 deficiency was impli- unless TRAF3 was completely knocked out by CRISPR (Supple- cated in impaired Toll-like receptor 3 responses and increased mentary Fig. S7A–S7D). Conversely, UMSCC47 CRISPR/CAS9 susceptibility to herpes simplex virus (HSV)–induced encephalitis TRAF3-9 KO rescued with TRAF3 vector showed no increase (23), hematologic malignancies, and Epstein–Barr virus (EBV)– in baseline IRF3 or IFN, but did so following stimulation with related nasopharyngeal cancers, but not HPV cancers. We poly-I:C (20 mg/mL; Supplementary Fig. S8A–S8D). Together, observed that oropharyngeal HNSCC linked to HPV exhibit these findings are consistent with either a lack of strong activation aberrant nuclear activation of the alternative NF-kB pathway of TRAF3-IRF3 signaling by integrated HPV in UMSCC47, or that (24), but the significance and function and role of deficient TRAF3 IRF3 activation and type-I IFN expression may be further in alternative NF-kB activation have not been investigated previ- þ disrupted downstream of TRAF3 expression, as found in HPV ously in HPV HNSCC cell lines and tissues. Here, we demon- E6/7-expressing keratinocytes in prior studies (6, 7). strate for the first time that decreased TRAF3 expression is linked to aberrant expression and activation of alternative NF-kB2/RELB TRAF3 expression enhances TP53 and RB tumor suppressor and transcription factors, contributes to reduced expression of anti- inhibits E6 protein expression viral type-I IFN and tumor suppressor TP53 and RB proteins, þ As repression of tumor suppressor proteins TP53 and RB by which are deregulated in HPV HNSCC (Supplementary þ þ HPV contributes to the proliferation of HPV cancers (21, 22), we Fig. S11). Supporting this role, ectopic TRAF3 expression in HPV examined if TRAF3 may modulate expression of TP53 and RB. HNSCC with decreased endogenous TRAF3 inversely modulat- Compared with the HPV mtTP53 line UMSCC46, UMSCC47 ed these key targets and suppressed several hallmarks of cancer, expressed low basal levels of TP53 and RB protein expression including cell colony formation, proliferation, and migration, þ (Fig. 7E). Unexpectedly, TP53 and RB expression was reproduc- while sensitizing HPV HNSCC to immune IFNs, TNFa,and ibly enhanced in UMSCC47 cells selected for expression of TRAF3 chemotherapy agent cisplatin. Complementing our findings, or after transient transfection (Fig. 7E; Supplementary Fig. S9A). one of our laboratories currently obtained evidence that dele- þ UMSCC104 cells with the lowest TRAF3 expression also showed tions or mutations in TRAF3 in HPV HNSCC tumors are enhanced TP53 and RB expression after transient transfection with associated with increased expression of an NF-kBtranscription- TRAF3 (Supplementary Fig. S9B). In UMSCC47 TRAF3-expressing al program, and episomal HPV infection (25). Interestingly, þ cells, the increase in TP53 was not attributable to increased TRAF3-deficient HPV HNSCCalsoappeartobelinkedtoa þ mRNA, while RB mRNA was increased 2-fold as measured by better prognosis (25), than HPV or HPV tumors with qRT-PCR (Supplementary Fig. S9C). TP53 siRNA knockdown PIK3CA, DNp63, FADD,cIAP1/2, and other genomic alterations attenuated the antiproliferative effects of TRAF3 (Supplementary (i.e., TP53; ref. 10), implicated in modulation of NF-kBorother Fig. S7D). The increase in TP53 was associated with enhanced pathways important in pathogenicity (26–29). The findings in expression of TP53-regulated cyclin-dependent kinase CDKN1A the present study support the addition of TRAF3 as a tumor þ (p21), and proliferation was enhanced by siRNA knockdown of suppressor in HPV HNSCC. CDKN1A(p21) in UMSCC47 expressing TRAF3 (Fig. 7F and The recent discovery of deep deletions on chromosome Supplementary Fig. S9E), supporting a contribution of this cell- 14q32.32 containing the TRAF3 coding region and 3 deleterious cycle regulator to the antiproliferative effects of TRAF3 and TP53. loss-of-function mutations by TCGA project was previously þ As HPV E6 oncoprotein mediates suppression of TP53 (22), we unrecognized in HPV HNSCC (10). Here, we further defined explored whether TRAF3 may affect HPV E6 levels protein in an increased frequency of hetero- and homozygous deletions of þ UMSCC47 cells selected to express TRAF3 or control vector. HPV TRAF3 in HPV HNSCC, in contrast to HPV tumors, which often E6 protein localized predominantly in the nucleus in the control display copy gain. Furthermore, we observed that TRAF3 genetic cell line and was partially decreased in the TRAF3-expressing alterations and expression significantly correlated with expression UMSCC47 cell line (Fig. 7G). We observed minimal change in of alternative NF-kB pathway components in HNSCC tissues. transcription of E6 mRNA (Supplementary Fig. S10A). However, TRAF3 gene copy loss, inactivating mutations, and decreased we observed only a minimal increase in E6 protein after trans- expression of TRAF3 have been previously observed only in fection with an E6 expression vector that increased mRNA 400- non-HPV cancers. These include multiple myeloma (MM; ref. 16), fold (Fig. 7H; Supplementary Fig. S10B), indicating that the very B-cell lymphomas (30), and EBV–infected human nasopharyn- low level of E6 detected is not primarily determined by transcrip- geal carcinomas (NPC; ref. 31). Annunziata and colleagues found tional regulation (Fig. 7H). Conversely, we also observed only a that 4.4% of 451 patients with MM had TRAF3 genetic defects, minimal decrease in E6 protein at 72 hours after transient trans- such as silencing, homozygous deletion, or somatic mutation, fection with TRAF3 (Fig. 7H) and did not observe higher or lower and 17 MM cases exhibited TRAF3 mRNA expression 13-fold MW bands, or enhancement by proteasome inhibitors, that below the median level of the cohort, predominantly in would support a ubiquitination or proteasome-dependent mech- association with these genetic alterations (16). Chung and anism (Supplementary Fig. S10C). Together, these observations colleagues detected deletion or missense mutations of TRAF3, suggest that the increase in TP53 with TRAF3 within 24 hours after TRAF2, and A20 in 3 of 33 (9.1%) of primary NPC tumor

www.aacrjournals.org Cancer Res; 78(16) August 15, 2018 4623

Zhang et al.

specimens (31). Together, these observations in different shown that HPV infection can also induce ubiquitin carboxyl- cancer types support the potential relevance of genetic terminal hydrolase L1 (UCHL1), which could inhibit TRAF3 K63 þ alterations in HPV HNSCC and suggest that decreased TRAF3 ubiquination, important in downstream TBK-IRF3–mediated facilitates the pathogenesis of tumors within the hematopoietic type-I IFN expression (7). In addition, we have shown a significant or lymphatic microenvironment. increase in both type-I IFN mRNA and protein expression by þ We previously reported that alternative pathway NF-kB2 and overexpressing TRAF3 in HPV UMSCC cell lines with low RELB subunits are often aberrantly expressed, and localized to the endogenous levels or TRAF3 rescue in TRAF3-KO cell lines. TRAF3 þ nucleus in oropharyngeal HNSCC tumors linked to HPV (24), but also sensitized HPV cells to antiproliferative effects of exogenous the possible relationship to genomic alterations, other factors and IFNA1 that may be produced by cells in the tumor microenvi- to the malignant phenotype was unknown. Here, our analysis of ronment, or IFN2A, which is used clinically. TCGA HNSCC data indicated that TRAF3 deletion is associated A novel finding of this study supporting the importance of with increased mRNA expression of molecules involved in the decreased TRAF3 in viral pathogenesis is that overexpression of alternative NF-kB pathway, such as NIK, cIAPs, RELB, and NF- TRAF3 enhanced the expression of TP53 and RB, which are critical kB2. Co-occurrence and mutual exclusivity analyses for key com- tumor suppressor proteins inactivated by HPV E6 and E7 onco- ponents of the NF-kB pathways revealed a significant association proteins (21, 22, 38, 39). Proliferation was enhanced by knock- of altered TRAF3 with molecules involved in an alternative NF-kB down of TP53 and TP53-regulated CDKN1A(p21) in cells selected þ pathway in HPV , but not in HPV HNSCC tissues. We further to express TRAF3, but not in control cells. However, the exact þ demonstrated that HPV HNSCC cell lines with decreased endog- mechanism by which TRAF3 modulates these tumor suppressors enous TRAF3 also display aberrant expression and nuclear acti- and the relationship of these effects to E6 and E7 is complicated by vation of the alternative NF-kB pathway. Furthermore, this poten- the difficulty in detecting these proteins. The minor reduction in tial of decreased TRAF3 to foster expression and activation of E6 observed with TRAF3 was not accompanied by appearance of þ alternative NF-kB in HPV HNSCC tumors could potentially be increased or decreased MW bands, and/or modulated by protea- enhanced within the local tonsillar and lymphatic microenviron- some inhibitors, that would support a ubiquitin or proteasome- ment, where alternative NF-kB signal inducers such as LTb are dependent mechanism (38). Further, the effects of TRAF3 on abundant (32). decreased E6 were detected at later time points, compared with LTb is a key signaling ligand that binds to surface receptor LTbR effects on TP53 and RB expression or NF-kB activation. The and activates the alternative NF-kB pathway (33). We originally different kinetics suggest that TRAF3 modulates E6, TP53, and detected aberrant activation of the alternative pathway in HPV RB expression by different transcriptional or posttranslational HNSCC and demonstrated that activation could be enhanced by mechanisms. LTb stimulation (34). Here, we observed increased LTbR expres- Of potential therapeutic relevance, TRAF3 re-expression in þ sion compared with HPV cell lines and showed that LTb could HPV HNSCC cells with low endogenous expression sensitized þ further enhance NF-kB activation in HPV HNSCC cells expres- them to inhibition by cisplatin chemotherapy and TNFa,an sing lower endogenous TRAF3. TRAF3 knockdown further important cell death ligand induced by radiation and immune enhanced aberrant and LTb-stimulated expression of the alterna- therapy (40, 41). Cisplatin is the most active chemotherapeutic tive NF-kB2 subunit p52 and RELB. Interestingly, in a subset of agent in HNSCC (42), leading to DNA damage and subsequently EBV-infected nasopharyngeal HNSCC that harbor TRAF3 altera- promoting cell death (43). TNFa is known to bind to TNF tions, Or and colleagues demonstrated 12p13.3 copy-number receptors and induce apoptotic cell death in normal cells, but gains and increased expression of LTbR (35). Overexpression of HNSCC are relatively resistant to TNFa (28, 44). NF-kB and cIAPs LTbR in nasopharyngeal epithelial cells resulted in increased NF- have been implicated in resistance to TNFa and chemotherapy kB activity and cell proliferation (36). Together, these data impli- (20, 45). Our data suggest that TRAF3 expression can inhibit cate TRAF3 and/or LTbR genomic alterations, along with HPV E6 cIAP1 expression and activation of the alternative NF-kB pathway, þ and EBV LMP1 viral oncogenes that promote alternative NF-kB and sensitize HPV HNSCC to TNFa and cisplatin-induced cell pathway activation, in viral pathogenesis of oro- and nasopha- death. We have recently reported that IAP1 inhibitors can sensitize ryngeal HNSCC. LMP-1 has been shown to bind TRAF3 and HPV HNSCC with gene copy gain and expression of IAP1 to TNF, enhance NIK-dependent alternative pathway activation (36). chemotherapy, and radiotherapy (28, 46, 47), supporting future þ HPV E6 is reported to promote alternative NF-kB activation via investigation of these agents in HPV HNSCCs expressing defi- a mechanism requiring its PDZ domain implicated in binding cient TRAF3 and/or cIAP1. þ phosphatases (8). Our data support a role for deficient TRAF3 Although both HPV HNSCCs and NPCs develop within the expression in enhancing NIK, cIAP1, IKKa, NF-kB2-p52, and adenotonsillar tissue and exhibit high rates of spread to regional RELB proteins, but we have found it exceedingly difficult to find lymph nodes, the mechanisms for this predilection are not well conditions to detect HPV16 E6 protein and an association with understood. Intriguingly, TRAF3 re-expression inhibited cell TRAF3 in HNSCC. migration in vitro, suggesting that deficient TRAF3 could contrib- TRAF3 has been reported to have a unique dual role in pos- ute to enhanced migration, underlying the high rates of regional þ itively regulating type-I IFNs in response to different DNA viruses spread of both HPV HNSCC and NPC in vivo. The alternative of in other tissues (11). IFNA1 potentially has antiviral effects NF-kB pathway could have a broader role in migration in HNSCC, through inhibition of viral protein expression, proliferation, as as we found that IKKa and RELB knockdown decreased cell well as immunostimulatory properties, that link innate and migration and proliferation in HPV HNSCC (34, 48). Together, adaptive immunity (37). In this study, we observed significantly the unique genomic alterations in TRAF3 and aberrantly activated þ lower expression of IFNA1 in HPV HNSCC lines, consistent with alternative NF-kB pathway components such as cIAPs or NIK, þ the lower expression of IFNA1 detected in HPV compared with could serve as potential markers and targets for novel therapies of þ HPV tumor tissues from TCGA data. Previous studies have HPV HNSCC.

4624 Cancer Res; 78(16) August 15, 2018 Cancer Research

Decreased TRAF3 Promotes HPVþ HNSCC

Disclosure of Potential Conflicts of Interest Acknowledgments C. Van Waes reports receiving a commercial research grant from Astex We thank Dr. Cheng-Ming Chiang and S.-Y. Wu (UT Southwestern Pharamaceuticals. No potential conflicts of interest were disclosed by the other Medical Center) for providing HPV16E6 expression plasmid and their authors. helpful suggestions. We thank Drs. Liu Yang (University of Maryland School of Medicine) and WanJun Chen (NIDCR/NIH) for reading the manuscript Authors' Contributions and comments. This work was supported by the Intramural Research Program of Conception and design: J. Zhang, C.M. Annunziata, Z. Chen, C. Van Waes The National Institute on Deafness and Other Communication Development of methodology: J. Zhang, X. Yang, P.E. Clavijo, Z. Chen Disorders. J. Zhang, T. Chen, H. Cheng, X. Yang, P. Clavijo, J. Coupar, Acquisition of data (provided animals, acquired and managed patients, C. Silvin, Z. Chen, and C. Van Waes are supported by NIDCD intra- provided facilities, etc.): J. Zhang, T. Chen, X. Yang, S.S. Sp€ath, C. Silvin, mural projects ZIA-DC-000016, 73, 74. C.M. Annunziata is supported by N. Issaeva, Z. Chen the NCI. Analysis 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 Waes The costs of publication of this article were defrayed in part by the Writing, review, and/or revision of the manuscript: J. Zhang, T. Chen, payment of page charges. This article must therefore be hereby marked advertisement S.S. Sp€ath, P.E. Clavijo, J. Chen, W.G. Yarbrough, C.M. Annunziata, in accordance with 18 U.S.C. Section 1734 solely to indicate Z. Chen, C. Van Waes this fact. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): X. Yang, Z. Chen Received March 2, 2017; revised January 11, 2018; accepted June 7, 2018; Study supervision: Z. Chen, C. Van Waes published first June 19, 2018.

References 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin cell-containing head and neck squamous cell carcinoma cell line. Head 2016;66:7–30. Neck 2012;34:1480–91. 2. Leemans CR, Braakhuis BJ, Brakenhoff RH. The molecular biology of head 16. Brenner JC, Graham MP, Kumar B, Saunders LM, Kupfer R, Lyons RH, et al. and neck cancer. Nat Rev Cancer 2011;11:9–22. Genotyping of 73 UM-SCC head and neck squamous cell carcinoma cell 3. Ang KK, Harris J, Wheeler R, Weber R, Rosenthal DI, Nguyen-Tan PF, et al. lines. Head Neck 2010;32:417–26. Human papillomavirus and survival of patients with oropharyngeal can- 17. Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C. Clonogenic cer. N Engl J Med 2010;363:24–35. assay of cells in vitro. Nat Protoc 2006;1:2315–9. 4. Gillison ML, Koch WM, Capone RB, Spafford M, Westra WH, Wu L, 18. Oeckinghaus A, Ghosh S. The NF-kappaB family of transcription et al. Evidence for a causal association between human papilloma- factors and its regulation. Cold Spring Harb Perspect Biol 2009;1: virus and a subset of head and neck cancers. J Natl Cancer I 2000; a000034. 92:709–20. 19. Havard L, Rahmouni S, Boniver J, Delvenne P. High levels of p105 (NFKB1) 5. Chaturvedi AK, Engels EA, Pfeiffer RM, Hernandez BY, Xiao WH, Kim E, and p100 (NFKB2) proteins in HPV16-transformed keratinocytes: role of et al. Human papillomavirus and rising oropharyngeal cancer incidence in E6 and E7 oncoproteins. Virology 2005;331:357–66. the United States. J Clin Oncol 2011;29:4294–301. 20. Wang CY, Mayo MW, Korneluk RG, Goeddel DV, Baldwin AS Jr. 6. Nees M, Geoghegan JM, Hyman T, Frank S, Miller L, Woodworth CD. NF-kappaB antiapoptosis: induction of TRAF1 and TRAF2 and c- Papillomavirus type 16 oncogenes downregulate expression of inter- IAP1 and c-IAP2 to suppress caspase-8 activation. Science 1998;281: feron-responsive genes and upregulate proliferation-associated and NF- 1680–3. kappaB-responsive genes in cervical keratinocytes. J Virol 2001;75: 21. Yim EK, Park JS. The role of HPV E6 and E7 oncoproteins in HPV-associated 4283–96. cervical carcinogenesis. Cancer Res Treat 2005;37:319–24. 7. Karim R, Tummers B, Meyers C, Biryukov JL, Alam S, Backendorf C, et al. 22. Thomas MC, Chiang CM. E6 oncoprotein represses p53-dependent gene Human papillomavirus (HPV) upregulates the cellular deubiquitinase activation via inhibition of protein acetylation independently of inducing UCHL1 to suppress the keratinocyte's innate immune response. PLoS p53 degradation. Mol Cell 2005;17:251–64. Pathog 2013;9:e1003384. 23. Perez de Diego R, Sancho-Shimizu V, Lorenzo L, Puel A, Plancoulaine S, 8. James MA, Lee JH, Klingelhutz AJ. Human papillomavirus type 16 E6 Picard C, et al. Human TRAF3 adaptor molecule deﬁciency leads to activates NF-kappaB, induces cIAP-2 expression, and protects against impaired Toll-like receptor 3 response and susceptibility to herpes simplex apoptosis in a PDZ binding motif-dependent manner. J Virol 2006;80: encephalitis. Immunity 2010;33:400–11. 5301–7. 24. Allen C, Saigal K, Nottingham L, Arun P, Chen Z, Van Waes C. 9. Schiffman M, Doorbar J, Wentzensen N, de Sanjose S, Fakhry C, Monk BJ, Bortezomib-induced apoptosis with limited clinical response is accom- et al. Carcinogenic human papillomavirus infection. Nat Rev Dis Primers panied by inhibition of canonical but not alternative nuclear factor- 2016;2:16086. kappa B subunits in head and neck cancer. Clinical Cancer Research 10. Comprehensive genomic characterization of head and neck squamous cell 2008;14:4175–85. carcinomas. Nature 2015;517:576–82. 25. Hajek M, Sewell A, Kaech S, Burtness B, Yarbrough WG, Issaeva N. TRAF3/ 11. H€acker H1 TP, Karin M. Expanding TRAF function: TRAF3 as a tri-faced CYLD mutations identify a distinct subset of human papillomavirus- immune regulator. Nat Rev Immunol 2011;11:457–68. associated head and neck squamous cell carcinoma. Cancer-Am Cancer 12. Annunziata CM, Davis RE, Demchenko Y, Bellamy W, Gabrea A, Zhan F, Soc 2017;123:1778–90. et al. Frequent engagement of the classical and alternative NF-kappaB 26. Mohan S, Vander Broek R, Shah S, Eytan DF, Pierce ML, Carlson SG, pathways by diverse genetic abnormalities in multiple myeloma. Cancer et al. MEK inhibitor PD-0325901 overcomes resistance to PI3K/mTOR Cell 2007;12:115–30. inhibitor PF-5212384 and potentiates antitumor effects in human 13. Oganesyan G, Saha SK, Guo B, He JQ, Shahangian A, Zarnegar B, et al. head and neck squamous cell carcinoma. Clin Cancer Res 2015;21: Critical role of TRAF3 in the Toll-like receptor-dependent and -indepen- 3946–56. dent antiviral response. Nature 2006;439:208–11. 27. Yang X, Lu H, Yan B, Romano RA, Bian Y, Friedman J, et al. DNp63 14. Tang AL, Owen JH, Hauff SJ, Park JJ, Papagerakis S, Bradford CR, et al. Head versatilely regulates a broad NF-kB gene program and promotes squamous and neck cancer stem cells: the effect of HPV—an in vitro and mouse study. epithelial proliferation, migration, and inﬂammation. Cancer Res 2011;71: Otolaryngol Head Neck Surg 2013;149:252–60. 3688–700. 15. Tang AL, Hauff SJ, Owen JH, Graham MP, Czerwinski MJ, Park JJ, et al. 28. Eytan DF, Snow GE, Carlson S, Derakhshan A, Saleh A, Schiltz S, et al. UM-SCC-104: a new human papillomavirus-16-positive cancer stem SMAC mimetic birinapant plus radiation eradicates human head and neck

www.aacrjournals.org Cancer Res; 78(16) August 15, 2018 4625

Zhang et al.

cancers with genomic ampliﬁcations of cell death genes FADD and BIRC2. 39. Boyer SN, Wazer DE, Band V. E7 protein of human papilloma virus-16 Cancer Res 2016;76:5442–54. induces degradation of retinoblastoma protein through the ubiquitin- 29. Lee TL, Yeh J, Friedman J, Yan B, Yang X, Yeh NT, et al. A signal proteasome pathway. Cancer Res 1996;56:4620–24. network involving coactivated NF-kappaB and STAT3 and altered 40. Hallahan DE, Spriggs DR, Beckett MA, Kufe DW, Weichselbaum p53 modulates BAX/BCL-XL expression and promotes cell survival RR. Increased tumor necrosis factor alpha mRNA after cellular of head and neck squamous cell carcinomas. Int J Cancer 2008;122: exposure to ionizing radiation. Proc Natl Acad Sci U S A 1989; 1987–98. 86:10104–7. 30. Mambetsariev N, Lin WW, Stunz LL, Hanson BM, Hildebrand JM, Bishop 41. Zhang M, Kong F, Qian J, Chen M, Lawrence TS. Radiation-induced lung GA. Nuclear TRAF3 is a negative regulator of CREB in B cells. Proc Natl Acad apoptosis is mediated by TNF-alpha action. Int J Radiat Oncol 2005;63: Sci U S A 2016;113:1032–37. S83–S84. 31. Chung GT, Lou WP, Chow C, To KF, Choy KW, Leung AW, et al. Consti- 42. Seiwert TY, Salama JK, Vokes EE. The chemoradiation paradigm in head tutive activation of distinct NF-kappaB signals in EBV-associated nasopha- and neck cancer. Nat Clin Pract Oncol 2007;4:156–71. ryngeal carcinoma. J Pathol 2013;231:311–22. 43. Ummat A, Rechkoblit O, Jain R, Roy Choudhury J, Johnson RE, Silverstein 32. Ganeff C, Remouchamps C, Boutaffala L, Benezech C, Galopin G, Vande- TD, et al. Structural basis for cisplatin DNA damage tolerance by human paer S, et al. Induction of the alternative NF-kappaB pathway by lympho- polymerase eta during cancer chemotherapy. Nat Struct Mol Biol 2012; toxin alphabeta (LTalphabeta) relies on internalization of LTbeta receptor. 19:628–32. Mol Cell Biol 2011;31:4319–34. 44. Duffey DC1 C-BC, Chen Z, Ondrey FG, Nejad-Sattari M, Dong G, Van Waes 33. Sun SC. Non-canonical NF-kappaB signaling pathway. Cell Res 2011; C. Inhibition of transcription factor nuclear factor-kappaB by a mutant 21:71–85. inhibitor-kappaBalpha attenuates resistance of human head and neck 34. Nottingham LK, Yan CH, Yang X, Si H, Coupar J, Bian Y, et al. Aberrant squamous cell carcinoma to TNF-alpha caspase-mediated cell death. Br IKKalpha and IKKbeta cooperatively activate NF-kappaB and induce EGFR/ J Cancer 2000;83:1367–74. AP1 signaling to promote survival and migration of head and neck cancer. 45. Tse BW, Scott KF, Russell PJ. Paradoxical roles of tumour necrosis Oncogene 2014;33:1135–47. factor-alpha in prostate cancer biology. Prostate Cancer 2012;2012: 35. Or YY, Chung GT, To KF, Chow C, Choy KW, Tong CY, et al. Identiﬁcation 128965. of a novel 12p13.3 amplicon in nasopharyngeal carcinoma. J Pathol 46. Eytan DF, Snow GE, Carlson SG, Schiltz S, Chen Z, Van Waes C. 2010;220:97–107. Combination effects of SMAC mimetic birinapant with TNFalpha, 36. Xie P, Bishop GA. Roles of TNF receptor-associated factor 3 in signaling TRAIL, and docetaxel in preclinical models of HNSCC. Laryngoscope to B lymphocytes by carboxyl-terminal activating regions 1 and 2 of the 2015;125:E118–24. EBV-encoded oncoprotein latent membrane protein. J Immunol 47. Xiao R, An Y, Derakhshan A, Chen Z, Schmitt N, Van Waes C. Novel dual 2004;173:5546–55. cIAP1/XIAP antagonist ASTX660 activity in preclinical models of human 37. Ivashkiv LB, Donlin LT. Regulation of type I interferon responses. Nat Rev papillomavirus(þ) and (-) head and neck squamous cell carcinoma. Amer Immunol 2014;14:36–49. Assn Cancer Res 2017;abstr 1086. 38. Huibregtse JM, Scheffner M, Howley PM. Localization of the E6-Ap 48. Das R CJ, Saleh A, Clavijo PE, Chen Z, Van Waes C. LTb/LTb receptor regions that direct human papillomavirus E6 binding, association with mediates alternative NF-kB activation through NIK and RELB/NF-kB2 to P53, and ubiquitination of associated proteins. Mol Cell Biol 1993; promote cell migration of HNSCC. Proc Amer Assn Cancer Res 2017; 13:4918–27. abstr 358.

4626 Cancer Res; 78(16) August 15, 2018 Cancer Research

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

Jialing Zhang, Tony Chen, Xinping Yang, et al.

Cancer Res 2018;78:4613-4626. Published OnlineFirst June 19, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-17-0642

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2018/06/29/0008-5472.CAN-17-0642.DC2 http://cancerres.aacrjournals.org/content/suppl/2018/06/19/0008-5472.CAN-17-0642.DC1

Cited articles This article cites 46 articles, 15 of which you can access for free at: http://cancerres.aacrjournals.org/content/78/16/4613.full#ref-list-1

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/78/16/4613. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.