TNFRSF19 Inhibits TGFb Signaling through Interaction with ... · TNFRSF19 is unique because it does not bind to known TNF ligands and its ICD exhibits no sequence homology to any

Molecular Cell Biology

TNFRSF19 Inhibits TGFb Signaling throughInteraction with TGFb Receptor Type I toPromote TumorigenesisChengcheng Deng, Yu-Xin Lin, Xue-Kang Qi, Gui-Ping He, Yuchen Zhang,Hao-Jiong Zhang, Miao Xu, Qi-Sheng Feng, Jin-Xin Bei, Yi-Xin Zeng, and Lin Feng

Abstract

Genetic susceptibility underlies thepathogenesis of cancer. We and othershave previously identified a novel sus-ceptibility gene TNFRSF19, whichencodes an orphan member of the TNFreceptor superfamily known to be asso-ciated with nasopharyngeal carcinoma(NPC) and lung cancer risk. Here, weshow that TNFRSF19 is highly expressedin NPC and is required for cell prolifer-ation and NPC development. However,unlike most of the TNF receptors,TNFRSF19 was not involved in NFkBactivation or associated with TRAF pro-teins. We identified TGFb receptor type I(TbRI) as a specific binding partner forTNFRSF19. TNFRSF19 bound the kinasedomain of TbRI in the cytoplasm, there-by blocking Smad2/3 association withTbRI and subsequent signal transduc-tion. Ectopic expression of TNFRSF19 innormal epithelial cells conferred resis-tance to the cell-cycle block induced byTGFb, whereas knockout of TNFRSF19in NPC cells unleashed a potent TGFbresponse characterized by upregulationof Smad2/3 phosphorylation and TGFbtarget gene transcription. Furthermore,elevated TNFRSF19 expression correlated with reduced TGFb activity and poor prognosis in patients with NPC. Our data reveal thatgain of function of TNFRSF19 in NPC represents a mechanism by which tumor cells evade the growth-inhibitory action of TGFb.

Significance: TNFRSF19, a susceptibility gene for nasopharyngeal carcinoma and other cancers, functions as a potent inhibitor ofthe TGFb signaling pathway.

Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/13/3469/F1.large.jpg. Cancer Res; 78(13); 3469–83. �2018 AACR.

IntroductionNasopharyngeal carcinoma (NPC) is a malignant tumor that

originates in the nasopharynx epithelium. Multiple factors,including genetic susceptibility, Epstein–Barr virus (EBV)infection, and environmental factors, contribute to NPC devel-opment. NPC exhibits a striking geographic and ethnic distri-bution; the incidence of NPC is unusually high in SoutheastAsia, southern China, and North Africa. In addition, familiaraggregation of NPC and the occurrence of multiple cases of thedisease in first-degree relatives have been reported in endemicregions, strongly indicating that genetic susceptibility plays a

© 2018 American Association for Cancer Research

II II

I I

TGFβ

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Growth inhibition

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TNFRSF19 is highly expressed in cancer cells and associates with TGFβ receptor type-I to blockSmad2/3 recruitment and TGFβ signal transduction.

Department of Experimental Research, Sun Yat-sen University Cancer Center,State Key Laboratory Oncology in South China, Collaborative Innovation Centerfor Cancer Medicine, Guangzhou, China.

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

C. Deng, Y.-X. Lin, and X.-K. Qi contributed equally to this article.

CorrespondingAuthors:LinFeng,SunYat-senUniversityCancerCenter,651DongFeng Road East, Guangzhou 510060, China. Phone: 8620-8734-2626; Fax: 8620-8734-3171; E-mail: [email protected]; and Yi-Xin Zeng, [email protected]

doi: 10.1158/0008-5472.CAN-17-3205

�2018 American Association for Cancer Research.

CancerResearch

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Published OnlineFirst May 7, 2018; DOI: 10.1158/0008-5472.CAN-17-3205

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key role in NPC (1, 2). However, little is known about theprecise genetic changes attributable to the pathogenesis ofNPC (3–5).

The fundamental abnormality resulting in the development ofcancer is the uncontrolled cell proliferation. Cytokine TGFb is oneof the few classes of endogenous inhibitors of cell growth. TGFbsignals through a complex ofmembrane-bound type I (TbRI) andtype II (TbRII) receptors, both of which are serine/threoninekinases. TbRII activates TbRI upon formation of the ligand–receptor complex by phosphorylating the cytoplasmicGSdomainof TbRI,which turns on the kinase activity of TbRI, followed by thephosphorylation of receptor-regulated Smads (R-Smads) includ-ing Smad2 and Smad3 at the C-terminal SSXS motif. Phosphor-ylated R-Smads then form a trimeric complex with the commonmediator Smad4 (Co-Smad), which is translocated from thecytoplasm to nucleus to cooperate with other transcriptionalmodulators to initiate the transcriptional regulation of targetgenes. Several direct target genes of the TGFb pathway includeplasminogen activator inhibitor 1 (PAI-1), the CDK inhibitorsp15INK4b and p21Cip1, and TGFb itself. In addition to canonicalSmad-dependent TGFb signaling, the TGFb receptor complex alsomediates non-Smad signaling, including activation of the MAPK,Erk, p38, and JNK kinases (6). Under physiologic conditions,TGFb arrests the cell cycle at G1 phase to inhibit cell proliferation;in contrast, tumor cells often escape the antiproliferative effects ofTGFb by acquiring loss-of-functionmutations or deregulating theexpression of various components in the TGFb pathway (7).Aberrations in TGFb pathway genes have been reported in NPC(8–10), and NPC cells often show a loss of TGFb antiproliferativeresponse (11, 12). However, it remains unclear what geneticmutations or the aberrant activities of regulatory molecules arethe cause of resistance to TGFb in NPC.

Using a genome-wide association study (GWAS), we havepreviously identified TNFRSF19 as a genetic susceptibility genein NPC (13). Subsequently, TNFRSF19 has also been reportedto be a lung cancer susceptibility gene in Han Chinese (14), indi-cating that germline mutations in TNFRSF19 confer a predispo-sition to certain cancers. TNFRSF19 (TNF Receptor SuperfamilyMember 19), also known as TROY, belongs to the TNF receptorsuperfamily, which commonly transduces cytokine signals via aspecific adaptor protein bound to the intracellular domain (ICD).TNFRSF19 is unique because it does not bind to known TNFligands and its ICD exhibits no sequence homology to any othercharacterized members of the TNF receptor superfamily (15, 16).High expression of TNFRSF19 is associated with poor prognosisin various types of cancer (17–21). However, the signal transducedby TNFRSF19 and molecular basis of TNFRSF19 in carcinogene-sis have not been explored. In this study, we characterize TNFRSF19as a potent negative regulator of the TGFb receptor–inducedsignaling response and a key determinant of NPC pathogenesis.

Materials and MethodsCell lines, transfection, and lentiviral infection

The immortalized nasopharyngeal epithelial (NPE) cellsNPEC2-Bmi1 and NPEC5-TERT were provided by Dr. Mu-ShengZeng [Sun Yat-sen University Cancer Center (SYSUCC),Guangzhou, China] and were maintained in keratinocyte/serum-free medium (Invitrogen). CNE-1 and HNE-1 cells wereprovided by Dr. Chao-Nan Qian (SYSUCC) and maintained inDMEM (Invitrogen) with 10% FBS (Invitrogen) at 37�C and 5%

CO2. All the NPE and NPC cell lines used in this study wereauthenticated using short tandem repeat profiling. All cell lineswere testedMycoplasma-free as determined by PCR-basedmethod(16s rDNA-F: 50-ACTCCTACGGGAGGCAGCAGTA-30, 16s rDNA-R: 50-TGCACCATCTGTCACTCTGTTAACCTC-30). Mycoplasmatesting was carried out every 2 or 3 weeks, and the cells were notcultured for more than 2 months.

Transfection using PEI as well as lentiviral packaging andinfection were performed as described previously (22).

Constructs, reagents, and antibodiescDNA fragments encoding TNFRSF19.1 (referred as TNFRSF19),

TNFRSF19.2, mouse Tory, TNFRSF21, LMP1, TGFbRI, TGFbRII,Smad2, Smad3, and Smad4 were subcloned into pDONR201(Invitrogen) entry clones and subsequently transferred to gate-way-compatible destination vectors. Point mutants (T204D andK232R) and deletion mutants [DGS (delete 175-204 a.a.), Dkinase(delete 208-503 a.a.), and DICD (delete 151-503 a.a.)] of TGFbR1were generated using site-directed mutagenesis PCR. All constructswere verified by sequencing.

Recombinant human TGFb1 (240-B) was obtained from R&DSystems; SB-431542 was from Selleckchem.

Rabbit anti-TNFRSF19 antisera were raised by immunizingrabbits with GST-TNFRSF19 (residues 30-140) fusion proteinsexpressed in andpurified fromEscherichia coli (E. coli). The antiserawere affinity-purified using the AminoLink Plus Immobilizationand Purification Kit (Pierce). Antibodies against P21 (2947),PAI-1 (11907), p-Smad2 (3108), Smad2 (5339), p-Smad3(9520), Smad3 (9523), Smad4 (9515), p-P38 (4511), P38(8690), p-IkBa (2859), IkBa (4814), caspase-3 (9662), HA(3724), and GST (2624) were obtained from Cell SignalingTechnology. Antibodies against TGFbRI (sc-398) and TGFbRII(sc-400) were from Santa Cruz Biotechnology. The antibodyagainst GAPDH (60004-1-Ig) was from Proteintech. Mouseanti-FLAG (F3165) and rabbit anti-FLAG(F7425) antibodieswerefrom Sigma-Aldrich.

Microarray assayTotal RNA was isolated from CNE-1 and HNE-1 cells and their

TNFRSF19-knockout (KO) counterparts using TRIzol reagent(Invitrogen Corp.) according to the manufacturer's instructions.The concentration and purity of total RNA were determined byspectrophotometry. RNA integrity was confirmed by agarose gelelectrophoresis. Control and TNFRSF19 KO cells were selected formicroarray analysis. Human Genome U133 Plus 2.0 microarrays(Affymetrix Corp.) were used to monitor changes in gene expres-sion. Total RNA was labeled and processed according to themanufacturer's instructions. The microarray analysis was per-formed by CapitalBio Corporation. A gene was considered tobe differentially expressed if it was up- or downregulated by atleast 2-fold. Online CapitalBio Molecule Annotation System(MAS) version 3.0 (http://bioinfo.capitalbio.com/mas3/) andKyoto Encyclopedia of Genes and Genomes (KEGG) databaseswere used to perform pathway analyses of the differential genes.Microarray data are available publicly at http://www.ncbi.nlm.nih.gov/geo (GEO accession numbers: GSE113328).

GSEA assayMicroarray data were downloaded from the GEO database

(http://www.ncbi.nlm.nih.gov/geo/) using the accession num-bers indicated in Fig. 5C. Gene set enrichment analysis (GSEA)

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was performed using GSEA 2.2.4 (http://www.broadinstitute.org/gsea/).

Luciferase reporter assay(CAGA)12-Luc and the control vector pRL-TK (Promega)

encoding Renilla luciferase were cotransfected into HEK293T cellsor NPC cells using PEI. Luciferase activity was measured 24 hourslater using the Dual-Luciferase Reporter Assay System (Promega).The firefly luciferase activity values were normalized to those ofRenilla, and the ratios of firefly/Renilla activities were determined.The experiments were independently performed in triplicate.

Immunofluorescence analysisImmunostaining was performed as described previously (23).

Briefly, cells were incubated with primary antibodies againstSmad2 and then with Alexa Fluor Plus 488–conjugated goatantibodies against rabbit (Invitrogen). The cells were counter-stained with DAPI and imaged with a confocal laser-scanningmicroscope (Olympus FV1000). The data were processed withAdobe Photoshop 7.0 software.

Establishment of TNFRSF19 KO NPC cell linesGene knockout was performed in cells using the CRISPR/Cas9

as described previously (24). The sequences of guide RNAs(gRNA) targeting exon 3 of human TNFRSF19 gene were asfollows: gRNA#1, CAAGAATTCAGGGATCGGTC and gRNA#2,GTGTTCCCTGCAACCAGTGT. Knockout clones were verified byWestern blotting and Sanger sequencing (see the SupplementaryMaterial for detail).

Tandem affinity purification and coimmunoprecipitationTandemaffinity purification (TAP)and coimmunoprecipitation

(co-IP) were carried out as described previously (23). Briefly,HEK293T cells were transfected with plasmids encoding C-termi-nal SFB-tagged (S-tag, flag epitope tag, and streptavidin-bindingpeptide tag) TNFRSF19 to establish stable cells via puromycin(2 mg/mL) selection. The cells were lysed in NETN buffer contain-ing50mmol/Lb-glycerophosphate, 10mmol/LNaF, and1mg/mLeach of pepstatin A and aprotinin. The lysates were centrifuged at12,000 rpm to remove debris and then incubated with streptavi-din-conjugated beads (Amersham) for 1 hour at 4�C. The beadswere washed five times with NETN buffer and followed by elutionwith NETN buffer containing 2 mg/mL biotin (Sigma). The eluteswere incubated with S-protein beads (Novagen) for 4 hours. Afterfive washes, the bound proteins were analyzed by SDS-PAGE, andmass spectrometry (MS) was performed by PTM BioLabs.

For co-IP experiments, cells were washed with ice-cold PBS andthen lysed in NETN buffer at 4�C for 30minutes. The crude lysateswere cleared by centrifugation at 12,000 rpm and 4�C for 30minutes, and the supernatantswere incubatedwith S-proteinbeadsor anti-HA agarose (Sigma) at 4�C for 4 hours to precipitate SFB-tagged or HA-tagged proteins, respectively. For endogenous IP, thecell lysates were incubated with control IgG or a protein-specificantibody overnight at 4�C, followed by incubation with proteinA/G PLUS-Agarose (Santa Cruz Biotechnology) at 4�C for 1 hour.The immunocomplexes were washed four times with NETN bufferand then subjected to SDS-PAGE and Western blotting.

Pull-down assayGST, GST-fused TNFRSF19 extracellular domain (ECD; 30-

170 a.a.) or ICD (192-423 a.a.), GST-fused full-length TNFRSF19without transmembrane domain (30-170 plus 192-423 a.a.), andMBP-fused TGFbRI ECD (34-134 a.a.) or ICD (148-504 a.a.) were

expressed in E. Coli BL21 cells. The GST fusion proteins werepurified with Glutathione Sepharose 4B (GEHealthcare), and theMBP fusion proteins were purified with amylose beads (NewEngland Biolabs) according to the manufacturers' instructions.For the pull-down assay, bait fusion proteins were incubated withcell lysates or target proteins in NETN buffer for 2 hours at 4�C.The beads were washed five times with NETN buffer, and thebound proteins were separated by SDS-PAGE and analyzed byWestern blotting or MS.

Patient enrollment and IHCA cohort of 140 patients with NPC (median age, 44.8 years;

range, 15–74 years) who hadundergone definitive treatmentwithcurative intent at our institute from 2003 to 2011 was evaluated.The cases were selected based on the following criteria: patho-logically confirmed NPC with available biopsy specimens fortissue microarray construction; no previous malignant disease orsecond primary tumors; and no prior history of radiotherapy,chemotherapy, or surgery. All the selectedNPC samples containedat least 70% carcinoma tissue as determined by the examinationof frozen sections. All patients were treated with standard curativeradiotherapy with or without chemotherapy. Protocols of thestudy were approved by Ethic Committees of SYSUCC(YB2013-04). This study was conducted under the provisions ofthe Declaration of Helsinki, and informed written consents wereobtained from all patients before inclusion. The clinical NPCsamples were fixed in 10% formalin and embedded in paraffin,and then sections of the embedded specimens were deparaffi-nized and rehydrated. The slides were subjected to appropriateantigen retrieval protocols, and endogenous peroxidase activitywas blocked with 10%H2O2 for 10minutes. The slides were thenexposed to anti-TNFRSF19 antibodies at 4�C overnight. Immu-nostaining was performed using the Envision System (Dako). Asemiquantitative scoring criterion was used for the IHC results,whereby both the staining intensity and positive areas wererecorded. The staining index (values 0–12) was obtained bymultiplying the intensity of TNFRSF19-positive stain (negative,0; weak, 1; moderate, 2; or strong, 3) by the proportion ofimmunopositive cells of interest (<25%, 1; 25%–50%, 2;50%–75%, 3; or �75%, 4). All scores were subdivided into twocategories according to a cut-off value of the ROC curve in thestudy cohort: low expression (�7) and high expression (>7).

Statistical analysisSPSS software version 16.0 was used to perform all statistical

analyses. Cumulative survival was calculated using Kaplan–Meieranalysis, and comparison between groups was performed usingthe log-rank test. Bivariate correlations between study variableswere determined using Pearson correlation coefficients. Eachexperiment was performed at least three times. The significanceof variances between groups was determined by the t test. Allstatistical tests were two-sided, and P < 0.05 was consideredstatistically significant. The authenticity of this article has beenvalidated by uploading the key raw data onto the Research DataDeposit public platform (www.researchdata.org.cn), with theapproval RDD number as RDDB2018000310.

Soft agar colony formation, tumor spheroid formation, andxenograft studies

All procedures were performed as described previously(24, 25). All the animal experiments were performed with theapproval of Institutional Animal Care and Use Committee of

Characterization of TNFRSF19 as a Repressor of TGFb Pathway

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Figure 1.

TNFRSF19 is highly expressed in NPC. A, Left, IHC analysis of TNFRSF19 expression in 8 normal nasopharyngeal and 140 NPC tissues (scale bar, 50 mm),together with an enlarged view of each in the corresponding inset. Right, scatterplots representing the IHC scores are shown on the left. B,Western blot assay ofTNFRSF19 expression in three normal NPE cells and 6 NPC cell lines. C, Oncomine box plots of TNFRSF19 expression levels in multiple advanced human cancers.D–F,Kaplan–Meier analysis of TNFRSF19 expression and overall survival (D), distantmetastasis-free survival (E), and recurrence-free survival (F) in 140 patientswithNPC. G, Kaplan–Meier analysis of overall survival in lung (1,145 patients) and gastric cancer (631 patients) stratified by TNFRSF19 expression. The P valueswere calculated using the log-rank test.

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TNFRSF19 is required for NPC tumorigenesis. A, Western blot analysis of TNFRSF19 in WT and knockout (KO) cells. TNFRSF19 KO cells were generated using theCRISPR-Cas9 systemwith two single-guideRNAs targetingexon3, and clones 1# and 2#were fromsgRNAs 1# and 2#, respectively.B,Proliferation curve ofHNE-1WTorTNFRSF19 KO cells as measured by CCK-8 assay. Error bars, SDs (n ¼ 3). C and D, Knockout of TNFRSF19 reduced the ability to form colonies on conventionalplates (C) and soft agar (D).E andF,Primary and secondary tumor sphere formation inultralowattachmentplatesofCNE-1 andHNE-1 cellswith orwithout TNFRSF19KO.Secondary spheroids (F) were obtained from the dissociation of primary spheroids (E) into single cells and reseeded. Error bars, SD (n ¼ 3). Scale bars, 50 mm.G, Wild-type or TNFRSF19 KO cells were subcutaneously injected into the right and left flanks of athymic BALB/c mice, respectively. Images were captured 7 weekspostimplantation. H, Growth curves of the tumors formed by wild-type or TNFRSF19 KO cells. Mean tumor volumes are plotted. Error bars, SD (n ¼ 6).






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Sun Yat-sen University (reference no. GZR2016-105), and theanimals were handled in accordance with institutional guide-lines. For xenograft studies, female BALB/c nude mice (5–6weeks old) were purchased from Shanghai Laboratory AnimalCenter (Shanghai, China).

ResultsHigh expression of TNFRSF19 in NPC

As our previous work suggested that TNFRSF19 is associatedwith NPC risk (13), we first evaluated TNFRSF19 expression inNPC patient samples. A specific polyclonal antibody recognizingTNFRSF19 was raised and used for IHC in NPC biopsies (Sup-plementary Fig. S1A) and Western blotting (Supplementary Fig.S1B). We found that TNFRSF19 was highly expressed in patient-derivedNPC tissues, but it couldbebarely detected innormalNPEtissues (Fig. 1A). Similarly, TNFRSF19 was expressed in NPC celllines but not in the normal NPE cell lines NPEC2-Bmi1, NPEC5-Tert, and NP69 (Fig. 1B). In addition, Oncomine expressionanalysis revealed high expression of TNFRSF19 in other humancancers (Fig. 1C), suggesting that the high expression ofTNFRSF19 is characteristic of multiple human cancer types. Toinvestigate whether TNFRSF19 expression serves as a novel prog-nostic marker, the correlation of TNFRSF19 expression with NPCprognosis was evaluated. Kaplan–Meier survival curves showedthat patients with high TNFRSF19 expression had a significantlypoorer overall survival and distant metastasis-free survival whencompared with patients with low TNFRSF19 expression, as dem-onstrated by the log-rank test (P < 0.001, Fig. 1D; P ¼ 0.032; Fig.1E). There was no significant correlation between TNFRSF19expression and recurrence-free survival (P ¼ 0.191; Fig. 1F). Inaddition, the online database Kaplan–Meier Plotter also revealeda statistically significant inverse correlation between highTNFRSF19 expression and overall survival in lung and gastriccancers (Fig. 1G). These data indicate that TNFRSF19may play anoncogenic role.

Loss of TNFRSF19 decreases tumorigenicity of NPCTo gain insights into the role of TNFRSF19 in cancer devel-

opment, the TNFRSF19 gene was knocked out in two NPC celllines, CNE-1 and HNE-1, using the CRISPR-Cas9 genomeediting system. Two independent sgRNAs with efficient cleav-age activity were selected (Supplementary Fig. S2A), and thegenetic ablation of TNFRSF19 was confirmed by Western blot-

ting (Fig. 2A) and Sanger sequencing (Supplementary Fig. S2B).Knockout of TNFRSF19 resulted in a substantially reducedgrowth rate (Fig. 2B), lower plating efficiency (Fig. 2C) andcolony-forming capacity on soft agar (Fig. 2D), and reducedprimary and secondary tumor spheroid formation (Fig. 2E andF), indicating that the transformation ability was greatlyimpaired by TNFRSF19 loss in vitro. To determine the role ofTNFRSF19 in tumorigenesis, we established xenograft tumorsvia subcutaneous inoculation of wild-type (WT) and TNFRSF19KO NPC cells into the right and left flanks of nude mice,respectively. TNFRSF19 KO cells displayed a significant inhi-bition of tumor growth compared with control cells (Fig. 2Gand H). Furthermore, knockout of TNFRSF19 resulted in areduction of tumor-initiating ability, as determined by limitingdilution transplantation analysis of NPC xenografts (Supple-mentary Table S1). The above data indicate that TNFRSF19 isrequired for cell growth and tumorigenesis of NPC.

TNFRSF19 interacts with TGFb type-I receptorThemajor signal transducers for the TNFR superfamily are TNF

receptor–associated factors (TRAF), which are linked to NF-kBactivation (16). As amember of TNFRs, some studies have shownthat TNFRSF19 interacts with TRAF proteins and activates the NF-kBpathway (19, 21, 26). ThehumanTNFRSF19 gene encodes twotranscripts: TNFRSF19.1 and TNFRSF19.2, which share most ofexons except the last one (Supplementary Fig. S3A) and are bothexpressed in human cells (Supplementary Fig. S3B and S3C).TNFRSF19.1 has 421 a.a. and lacks the TRAF-binding motif;TNFRSF19.2 is shorter (417 a.a.) and has a distinct C-terminuswith a potential TRAF-binding consensus sequence (P/S/A/T)X(Q/E)E, 413SLQE416. Mouse Tnfrsf19, commonly named as Troy,only has one transcript and the encoded protein containing adifferent TRAF-binding motif 276TLQE279 (Supplementary Fig.S3A andS3D).However, transient transfectionof these TNFRSF19constructs into HEK293T cells did not lead to IkBa phosphory-lation or caspase-3 cleavage, while a positive control LMP1, anEBV oncoprotein acting as a constitutively active mimic of TNFRCD40 and binding TRAFs (27), potently stimulated IkBa phos-phorylation (Fig. 3A). Consistently, co-IP revealed that TNFRSF19transcript 1 and 2 or the mouse ortholog did not associate withTRAF2 or TRAF6, whereas binding of TRAF2 to LMP1 was readilydetectable (Fig. 3B). Therefore, it seems that TNFRSF19 does notshare the same set of signal transducers with other TNFRs andmayhave different signaling outputs.

Figure 3.TNFRSF19 interacts with type I TGFb receptor. A, Lysates from HEK293T cells transfected with human TNFRSF19.1, 19.2, mouse Troy, and LMP1 constructswere immunoblotted with the indicated antibodies. LMP1 served as a positive control for NFkB activation. B, Top, SFB-tagged TNFRSF19.1, 19.2, and mouse Troy orLMP1 were transfected into HEK293T cells along with HA-TRAF2. The cell lysates were precipitated with S-protein beads and immunoblotted with the indicatedantibodies. LMP1 served as a positive control for TRAF2 binding. Bottom, the interaction of SFB-tagged TNFRSF19.1, 19.2, and mouse Troy with HA-TRAF6. C,HEK293T cells stably expressing SFB-tagged TNFRSF19were used for TAP. Silver staining of TAP and the number of peptides identified byMS are shown.D, Co-IP ofexogenous TGFbRI (TbRI) and TNFRSF19 (T19). HEK293T cells were transfected with plasmids encoding C-terminal SFB-tagged TGFbRI and C-terminalHA-tagged TNFRSF19. Cell lysates were precipitated with S-protein beads and immunoblotted with the indicated antibodies. E, Interactions between endogenousTNFRSF19 and TbRI in HNE-1 and CNE-1 cells were assessed by immunoprecipitation (IP) with anti-TbRI antibody or control IgG and immunoblotting usinganti-TNFRSF19 and TbRI antibodies. Arrow, IgG heavy chain. F, Lysates from HEK293T cells expressing HA-tagged Smad2, Smad4, and TbRI along with SFB-taggedTNFRSF19 were immunoprecipitated with anti-HA agarose and immunoblotted with antibodies as indicated. G, Co-IP of SFB-tagged TNFRSF19 or TNFRSF21with HA-TbRI or TbRII in transfected HEK293T cells. H, The ICD of TNFRSF19 binds to TbRI. Bacterially expressed GST-ECD and ICD fragments of TNFRSF19 or GSTalone were incubated with HNE-1 cell lysates. Proteins bound to glutathione sepharose beads were analyzed by immunoblotting. GST fusion proteins areshown by Coomassie Brilliant Blue (CBB) staining. I, TNFRSF19 directly binds to ICD of TbRI. MBP-tagged ECD or ICD of TbRI fragments were coincubated withGST-tagged TNFRSF19; proteins bound to amylose beads were immunoblotted with anti-GST antibody. J, Left, schematic representation of the TGFbRIdomain structure. SP, signal peptide; TM, transmembrane domain. Right, lysates from HEK293T cells expressing SFB-tagged full-length (FL) or deletion mutants ofTbRIwith orwithoutHA-taggedTNFRSF19were subjected to immunoprecipitationwith anti-HAagarose and immunoblottingwith anti-FLAGandanti-HAantibodies.






To gain insight into the biological pathway transduced byTNFRSF19, we studied the protein interaction network ofTNFRSF19. TAP and MS (TAP-MS) were carried out in HEK293Tcells stably expressing SFB (S-FLAG-SBP) triple-tagged TNFRSF19(transcript variant 1). Interestingly,MSdata revealed peptides thatcorresponded toTbRI (Fig. 3C). TbRIwas also identifiedvia TAP intheNPC cell lineHK1 (Supplementary Fig. S3E); besides, the pull-down assay using TNFRSF19 ICD as bait also captured TbRIpeptides in HEK293T cell lysates (Supplementary Fig. S3F), butno TRAF proteins or death domain-containing proteins werefound in these MS lists. To verify the interaction of TNFRSF19with TbRI, reciprocal co-IPswere performed inHEK293T cells andshowed that TbRI bound to TNFRSF19 in cooverexpressionexperiment (Fig. 3D). Furthermore, endogenous TNFRSF19 wascoimmunoprecipitated with TbRI in HNE-1 and CNE-1 cells(Fig. 3E). In addition, TNFRSF19 did not bind Smad2 or Smad4(Fig. 3F). To further ensure the binding specificity betweenTNFRSF19 and TbRI, another member of the TNF receptor super-family, TNFRSF21, which contains the death domain (28), wasused as a control, and the co-IP result suggested that onlyTNFRSF19 interacted with TbRI but not TbRII in transientlytransfected HEK293T cells (Fig. 3G). Collectively, these resultssuggest that TNFRSF19 specifically binds TbRI in vivo.

Next, we sought to determine the regions responsible for theTNFRSF19–TbRI interaction. To achieve this goal, bacteriallyexpressed ECDs and ICDs of TNFRSF19 and TbRI were used inpull-down assays. The GST-fused ICD, but not the ECD ofTNFRSF19, was able to pull down TbRI (Fig. 3H). Conversely,GST-fused TNFRSF19 bound to MBP-fused ICD but not to theECD of TbRI (Fig. 3I). These results suggest that TNFRSF19 bindsdirectly to TbRI through the respective cytoplasmic domains. AsTNFRSF19 ICD does not harbor any known functional motif, wefurther dissected the domains of TbRI ICD mediating the associ-ation with TNFRSF19. The cytoplasmic region of TbRI is com-posed of a glycine- and serine-rich sequence, termed the GSdomain, followed by a kinase domain. A series of deletionmutants lacking the GS domain, kinase domain, or the entireICD of TbRI was generated (Fig. 3J, right), and the co-IP assaydemonstrated that the GS domain was dispensable for TbRI–TNFRSF19 interaction, but deletion of the kinase domain or theentire ICD of TbRI abolished the TNFRSF19 association (Fig. 3J,left). Taken together, these data suggest that TNFRSF19 binds tothe kinase domain of TbRI in the cytoplasm.

TNFRSF19 blocks formation of the TGFbRI–Smad2/3complex

Having established the interaction between TNFRSF19 andTbRI, we next asked how the TbRI complex is regulated byTNFRSF19. Upon ligand stimulation, TbRI forms a heteromericcomplex with TbRII and is phosphorylated in the GS domain.Phosphorylation induces a conformational change of TbRI and itssubsequent association and phosphorylation of the R-Smads,Smad2, and Smad3 via the kinase domain (29–32) (Fig. 4A).We first examined whether the TNFRSF19 and TbRI association isTGFb-induced. As most TNFRSF19-positive NPC cells lost theirresponse to TGFb (see below), we chose HaCaT, an immortalizedhuman keratinocyte cell line that is highly responsive to TGFb andTNFRSF19-positive, to immunoprecipitate endogenous TbRIbefore and after TGFb treatment. The TbRI–TbRII complex wasinduced after 1 hour of TGFb treatment and declined after 6 hoursof treatment, which correlated with the phosphorylation of

Smad2; in contrast, a constitutive interaction between TNFRSF19and TbRI was observed regardless of the presence of TGFb (Fig.4B). In addition, we compared the interaction of TNFRSF19 witheither active or inactive forms of TbRI. The T204Dmutation in theGS domain causes ligand- and TbRII-independent activation ofTbRI, whereas the K232R mutation leads to kinase inactivation(29). Co-IP experiment suggested that TNFRSF19 bound to allforms of TbRI, and treatment with SB-431542, a small moleculethat inhibits the catalytic activity of TbRI, did not affect theirinteractions (Fig. 4C). Thus, unlike TbRII and Smad2/3, thebinding of TNFRSF19 to TbRI is TGFb independent.

Next, we questioned whether TNFRSF19 affects the interactionof TbRI with the upstream TbRII and the downstream R-Smads.Overexpression of TNFRSF19 did not affect TbRI/II heterotetra-meric receptor complex formation (Fig. 4D); however, the inter-actions of Smad2/3 with TbRI were severely impaired byTNFRSF19 overexpression (Fig. 4E), and both wild-type and theconstitutively active mutant T204D of TbRI lost their abilities toassociate with Smad2 in the presence of exogenous TNFRSF19(Fig. 4F). In agreement with the overexpression data, knockout ofTNFRSF19 in HNE-1 cells greatly induced endogenous TbRI–Smad2 complex formation (Fig. 4G). And as expected, the down-stream event of TbRI/R–Smads interaction, oligomerization ofSmad2 with the Co-Smad, Smad4 was inhibited by TNFRSF19overexpression (Fig. 4H), and depletion of TNFRSF19 in HNE-1cells substantially increased Smad2–Smad4 complex (Fig. 4I). Insummary, TNFRSF19 competes with R-Smads for binding to thekinase domain of TbRI and thereby blocks the recognition andactivation of R-Smads and the subsequent formation of an activeR-Smad/Co-Smad complex.

TNFRSF19 inhibits TGFb signalingTo investigate the biological significance of TNFRSF19 in the

TGFb pathway, we compared the global gene expression profilesof control and TNFRSF19 KO cells using microarrays. A total of143 differential geneswere found betweenWT and TNFRSF19 KOHNE-1 cells (Fig. 5A). Strikingly, functional profiling of thesedifferentially expressed genes suggested that the TGFb signalingpathway was one of the most significantly affected pathways bythe loss of TNFRSF19 (Fig. 5B). Furthermore, after analysis ofTNFRSF19 expression and TGFb-regulated gene signatures viaGSEA in GEO public NPC patient expression datasets (33), wefound that TNFRSF19 levels were inversely correlated with thegene signatures activated by TGFb (Fig. 5C).

To validate the participation of TNFRSF19 in the TGFb path-way, we used (CAGA)12-Luc, a TGFb-responsive luciferase report-er containing a Smad3/4–binding box on the PAI-1 gene pro-moter (34), to determine whether TNFRSF19 affects TGFb-medi-ated transcriptional responses. Luciferase assays showed that lossof TNFRSF19 dramatically increased the activity of the TGFb/Smad–responsive reporter in TGFb-treated HNE-1 cells (Fig. 5D).Consistent with the reporter data, the protein levels of P21 andPAI-1, two well-characterized direct transcriptional targets ofTGFb pathway, were robustly upregulated in TNFRSF19 KO cellsthan in control cells, the latter respondedpoorly to TGFb (Fig. 5E).Next, we examined the early mediators of TGFb signaling, forexample, TGFb-induced phosphorylation of R-Smads. Smad2/3phosphorylation was almost undetectable in HNE-1 WT cellseven after TGFb stimulation; in contrast, much higher levels ofbasal and TGFb-induced Smad2/3 phosphorylation wereobserved in TNFRSF19 KO cells, while p38 phosphorylation was

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slightly increased (Fig. 5F). Thesedatawere in linewith thefindingthat TNFRSF19 prevented R-Smads from being recruited andactivated by TbRI (Fig. 4).

To further examine the role of TNFRSF19 in the TGFb pathway invivo, we analyzed the levels of P21 and phospho-Smad2 by IHC in

NPC tumor xenografts, as shown in Fig. 2H. The staining resultsrevealed that tumors derived from TNFRSF19 KO cells had higherlevels of p21 and phosphorylated Smad2 compared with tumorsderived from control cells (Fig. 5G). Consistently, IHC in clinicalspecimens demonstrated anegative correlationbetweenTNFRSF19

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TNFRSF19 blocks TGFbRI from binding to R-Smads. A, Schematic representation of TbRI activation and signal transduction via receptors. B, Cell lysates fromHaCaT cell line treated with TGFb (5 ng/mL) for the indicated times were immunoprecipitated (IP) using anti-TbRI antibody or control IgG, followed byimmunoblotting with the indicated antibodies. C, HEK293T cells were cotransfected with plasmids encoding HA-tagged WT, T204D, and K232R mutants of TbRI,along with TNFRSF19-SFB. Following 1 hour of SB-431542 (10 mmol/L) treatment or no treatment, the cell lysates were precipitated with S-protein beads andimmunoblotted with the indicated antibodies. Phospho-Smad2 and PAI-1 represent early and late response to TGFb, respectively.D, Co-IP of SFB-tagged TbRII andHA-tagged TbRI in the presence or absence of exogenous TNFRSF19 in transfected HEK293T cells. E, Co-IP of SFB-tagged Smad2 or Smad3 with HA-TbRIin the presence or absence of TNFRSF19 overexpression in transfected HEK293T cells. F, Co-IP of SFB-Smad2 with HA-taggedWT, T204D, or K232Rmutant of TbRIwith or without cotransfection of TNFRSF19 in transfected HEK293T cells. G, Interaction of endogenous TbRI and Smad2 in WT and TNFRSF19 KO HNE-1 cellswith orwithout TGFb (5 ng/mL) treatmentwas analyzedby immunoprecipitationwith anti-TbRI antibody, followedby immunoblottingwith the indicated antibodies.Arrow, IgG heavy chain. H, Co-IP of SFB-Smad2 and Myc-Smad4 with or without TNFRSF19 overexpression in transfected HEK293T cells. I, EndogenousSmad2–Smad4 interaction in WT and TNFRSF19 KO HNE-1 cells with or without TGFb (5 ng/mL) treatment as analyzed by immunoprecipitation with anti-Smad2antibody, followed by immunoblotting (IB) with anti-Smad4 antibody.






PPAR signaling pathway

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TNFRSF19 suppresses TGFb signaling in NPC. A, Double-log scatter plot comparing the differential expression of mRNAs in control and TNFRSF19 KO HNE-1 cells.Green, downregulatedgenes; red, upregulatedgenes.B,KEGGpathwayenrichment analysis ofdifferentially expressedgenesbetweenWTandTNFRSF19KOcells.C,GSEAplot showing that TNFRSF19 expression is inversely correlated with TGFb-activated gene signatures in published NPC patient gene expression datasets (GSE12452,n ¼ 31). ES, enrichment score; NES, normalized enrichment score. D, Luciferase assay of the TGFb reporter (CAGA)12-Luc in WT or TNFRSF19 KO cells treated or notwith TGFb (5 ng/mL) for 24 hours. Error bars, SD (n¼ 3). E, Effects of TNFRSF19 KO on TGFb-induced P21 and PAI-1 expression. WT, TNFRSF19 KO CNE-1, or HNE-1 cellswere starved overnight and then treated with TGFb for 6 hours; the induction of target genes was examined by Western blot analysis. F, Effects of TNFRSF19 KOon TGFb-induced phosphorylation of Smad2/3 and P38. Cells with or without TNFRSF19 deletion were starved overnight and then stimulated with TGFb for 1 hour,and the phosphorylated and total proteins were immunoblotted with the indicated antibodies. G, IHC analysis of TNFRSF19 and phosphorylated Smad2 and P21 inxenografts generated from WT and TNFRSF19-KO cells as shown in Fig. 2H. Scale bars, 100 mm. H, Expression levels of p-Smad2 and P21 were inversely associatedwith TNFRSF19 levels in 40 primary human NPC specimens. Two representative cases are shown. Scale bar, 100 mm. The inset shows a magnified view.


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and P21 and phospho-Smad2 levels (Fig. 5H). Taken together,these results indicate that TNFRSF19 inhibits TGFb-inducedR-Smads phosphorylation and transcriptional responses in NPC.

Overexpression of TNFRSF19 confers resistance to growth-inhibitory effect of TGFb

TGFb acts as a tumor suppressor by inhibiting the growth ofnormal and premalignant cells. We next determined the effects ofTNFRSF19gainof functionon the antiproliferative role of TGFb in

normal cells. Overexpression of TNFRSF19 abolished TGFb-induced (CAGA)12-Luc reporter activation in HEK293T cells(Fig. 6A). When TNFRSF19 was transduced into a normal NPEcell line, NPEC2-Bmi1, which does not express TNFRSF19 (Fig.1B), cell proliferation was accelerated (Fig. 6B), and inhibition ofTGFb-induced growth was significantly alleviated compared withthe control cells without TNFRSF19 transduction (Fig. 6C). Inepithelial cells, TGFb stimulates the transcription of p15INK4b

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Overexpression of TNFRSF19 confers resistance to TGFb-mediated growth inhibition in normal NPE cells. A, Effect of TNFRSF19 on the (CAGA)12-Luctranscriptional response induced by TGFb (5 ng/mL) for 24 hours in 293T cells. B, NPEC2-Bmi1 cells were infected with control lentivirus or lentivirusexpressing TNFRSF19. The cell proliferation was determined by CCK-8 assay. C, NPEC2-Bmi1 cells with or without TNFRSF19 overexpression were cultured inthe absence or presence of TGFb. After 48-hour incubation, the cell viability was determined by CCK-8 assay. D, Control or TNFRSF19-overexpressing NPEC2-Bmi1cells were treated or not with various concentrations of TGFb for 6 hours; the inductions of P21 and PAI-1 are shown. E, TNFRSF19 overexpression inhibits thephosphorylation of Smad2 induced by 30 minutes of TGFb treatment in NPE cells. F, TNFRSF19 inhibits the nuclear translocation of Smad2 induced by TGFb. Left,immunofluorescence images representing the subcellular localization of Smad2 before and after 30 minutes of stimulation with TGFb in control or TNFRSF19-overexpressing NPE cells. Scale bars, 50 mm. Right, histogram representing the percentage of cells displaying Smad2 distributed in the nuclear, cytoplasmic,or both compartments.






cell-cycle progression. Consequently, overexpression of TNFRSF19substantially reduced the induction of P21 and PAI-1 by TGFb (Fig.6D), while P15 was undetectable due to homozygous deletion ofthe p15INK4b locus. The data suggest that TNFRSF19 reduces tran-scriptional activation of TGFb to bypass TGFb-induced growthinhibition.Consistently, TGFb-inducedphosphorylationofSmad2was substantially inhibitedbyectopic expressionofTNFRSF19(Fig.6E). Furthermore, the TGFb-induced cytoplasmic to nuclear trans-location of Smad2 was suppressed by TNFRSF19 in NPE cells (Fig.6F), Thus, the overexpression data were consistent with the resultsobtained from NPC knockout cells.

In addition to its tumor-suppressing activity, TGFb alsoexhibits tumor-promoting effects by assisting cell migrationand cancer metastasis via epithelial-to-mesenchymal transition(7). However, loss of TNFRSF19 did not change the level ofE-cadherin, although N-cadherin levels increased. Besides,the expression of E- and N-cadherin was insensitive to TGFbin HNE-1 cells (Supplementary Fig. S4A). Furthermore,TNFRSF19-deficient cells exhibited similar migration abilitycompared with control cells (Supplementary Fig. S4B), suggest-ing that at least in the context of NPC, TNFRSF19 mainlyregulates the growth-inhibitory function of TGFb.

In summary, our results support a model in which TNFRSF19functions as a key repressor of TGFb receptor–induced signalingresponses and of TGFb-dependent antiproliferative effects inNPC (Fig. 7).

DiscussionNPC is a special type of head and neck cancer. Strong ethnic

clustering and familiar aggregation of NPC indicates that geneticsusceptibility plays a significant role in this disease (1, 35).However, even thoughnumerousmutations/variations havebeendiscovered inNPCbymultiple whole-genome or exome sequenc-ing studies over the years, the exact genetic perturbations resultingin NPC are far from being fully understood (3).

We have previously identified TNFRSF19 as a susceptibilitygene for NPC (13), and another GWAS has reported thatTNFRSF19 is a susceptibility factor in lung cancer (14). TNFRSF19belongs to the TNFR superfamily. The family members are char-acterized by four conserved cysteine-rich domains in their ECDand a distinct ICD that is responsible for TNFR signaling. Inhuman, a total of 29 TNFRs have been described to date. Ingeneral, members of the TNFR superfamily can be divided intotwo groups: survival receptors and death receptors. Survivalreceptors activate the NF-kB pathway by binding to TRAF pro-teins. Death receptors mediate signal-induced cell death throughtheir death domains (16). TNFRSF19 is far less characterized inthis family. TNFRSF19 does not bind to TNF-related ligands andremains an orphan receptor to date (15). Its cytoplasmic domainlacks death domain; in addition, despite the presence of a putativeTRAF-binding motif in the cytoplasmic region of humanTNFRSF19.2 and mouse Troy (Supplementary Fig. S3), they wereunable to bind TRAF2 and TRAF6; accordingly, overexpression or

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Working model of TNFRSF19-mediated inhibition of TGFb signaling in cancer.

Deng et al.





knockout of TNFRSF19 did not affect NF-kB activity (Figs. 3A andB and 5E). Our results are in accordance with previous studiesconducted in other cell lines and mouse models (15, 36). More-over, unlike most TNFRs, which are expressed in the immunesystem and play roles in innate and adaptive immunity,TNFRSF19 is not present in lymphoid tissues but is highlyexpressed in skin and hair follicles (15, 37). Collectively, thesefindings indicate that TNFRSF19 is functionally distinct fromcanonical TNFRs.

TNFRs utilize adaptor proteins to transduce and amplify recep-tor information to different cell fates. To identify the adaptors forTNFRSF19, we performed affinity purification of TNFRSF19.Unexpectedly, TGFb type I receptor was captured as a specificbinding partner for the ICD of TNFRSF19. Domain mappingrevealed that TNFRSF19 bound to the kinase domain of TbRI,which overlapped with the binding region of R-Smads on TbRI(Fig. 3J; refs. 30–32). However, unlike TGFbRI/R-Smad complexformation, which is induced by TGFb, TNFRSF19 constitutivelyassociates with TbRI, thereby blocking the formation of the activeTbRI/R-Smad complex and the downstream R-Smad/Co-Smadcomplex (Fig. 4). The constitutive interaction of TNFRSF19 withTbRI and the competition of R-Smads for binding to the kinasedomain inhibit leaky activation of the receptor in the absence ofTGFb, thereby eliminating spurious signaling caused by receptoroligomerization-induced R-Smads recruitment in the absence ofligand. Indeed, depletion of TNFRSF19 results in active TbRI/R-Smad complex formation and TGFb pathway activation, even inthe absence of ligand (Figs. 4G and I and 5). To our knowledge,this is one of the very few examples of a repressor of the TGFbpathway via direct binding to TGFb receptors, and this findingmay advance our understanding of the functional divergence ofthe TNFR superfamily.

Genetic alterations in the TGFb pathway, such as the biallelicinactivation of TGFBRII, Smad2 and Smad3mutations, and Smad4deletion, are often found in human cancers (7, 38). Notably,targeted deletion of Smad4 in the head and neck epithelium ofmice is sufficient to drive spontaneous head and neck squamouscell carcinomas (39). Loss of sensitivity to TGFb-induced growthsuppression has been found in NPC (11, 12). We identifiedTNFRSF19 as a key repressor of the TGFb pathway that is oftenoverexpressed in NPC, and high levels of TNFRSF19 are inverselycorrelated with TGFb pathway activity in vivo; these evidencesindicate that the inactivation of TGFb signaling in NPC couldresult from the gain of function of TNFRSF19 rather than muta-tions in canonical TGFb components. In addition to TNFRSF19,other NPC susceptibility loci we have identified in 2010 includeMDS1-EVI1 and the CDKN2A-CDKN2B gene cluster (13). Inter-estingly, CDKN2B (p15INK4b) is a TGFb target gene that partici-pates in themediation of TGFb-induced cell-cycle arrest (40), andMDS1-EVI1 is an oncoprotein that suppresses TGFb signaling bybinding to Smad3 (41). The three NPC susceptibility genes seemto be involved in the regulation of TGFb signal transduction atdifferent levels. In addition, TGFbRII has been shown to bedownregulated in more than 50% of NPC (10). Various geneticperturbations leading to the dysfunction of the same biologicalpathway are common in cancers. Further study of the TGFbpathway status in a large cohort of patients with NPC andevaluation of its association with prognosis are warranted.

The discovery that TNFRSF19 antagonizes TGFb signaling byinteracting with the type I receptor and preventing its activation ina ligand-independent manner explains how TNFRSF19 controls

the sensitivity of cells to TGFb signals. It is conceivable thatreactivation of TNFRSF19 in normal or premalignant epithelialcells can protect them against small amounts of autocrine andparacrine TGFb and eventually cause uncontrolled cell growth.Although expression of TNFRSF19 in human normal NPE cellsis not associated with transformation, such as soft agar growthand the formation of tumors in xenograft mouse models,TNFRSF19 does exert growth-promoting effects in NPE cells(Fig. 6B). Given that the etiology of NPC is complex andinvolves predisposed genetic and environmental factors, it isconceivable that multiple factors orchestrate the initiation ofNPC. For example, EBV has been considered as another keyplayer in NPC pathogenesis, and its latent to lytic switch isinduced by TGFb (42, 43). Thus, TNFRSF19 may also play rolesin the establishment of persistent EBV infection in the earlystage of NPC development. Future studies are needed to acquirea better understanding of gene–gene and gene–environmentinteractions in the development of NPC.

In contrast to human TNFRSF19, its mouse ortholog Troy hasbeen well studied in mouse models. Using lineage tracing tech-nology, Troy has been proposed as a stem cell marker for stomach(44), kidney (45), intestine (20), and brain (46) inmice. Troy alsointeractswith TGFbRI as its humanorthologs (Supplementary Fig.S5A), and therefore, we generated tnfrsf19/troy KO mice usingCRISPR-Cas9 technology (Supplementary Fig. S5B and S5C). Theisolated mouse embryonic fibroblasts (MEF) with a homozygousdeletion of the tnfrsf19 gene were more sensitive to the antipro-liferative effects of TGFb than the WT and heterozygous MEFs(Supplementary Fig. S5D and S5E) and exhibited enhanced TGFbsignaling activity (Supplementary Fig. S5F and S5G). However,the tnfrsf19�/� mice were alive at birth and grew into adulthoodwithout gross abnormalities compared with their littermates,which is consistent with previous reports of tnfrsf19 KO mousemodels generated by different gene-editing strategies and indifferent mice strains (36, 47). It could be a result of the redun-dancy of other TNFR family members such as Edar (36). None-theless, we showed that human TNFRSF19 is essential for theproliferation and tumorigenicity of NPC cells. Future assessmentof chemically induced tumor formation, such as 4NQO(4-nitroquinoline 1-oxide)-induced head and neck squamouscell carcinoma is needed in the Troy KO mouse model. One theother side, given the gain-of-function property of TNFRSF19 inNPC, a transgenicmousemodelwouldbemore valuable to definethe role of TNFRSF19 in NPC development.

The cause of aberrant expression of TNFRSF19 in cancers is stillelusive. Tumor-specific expression of TNFRSF19 has beenobserved in NPC and other cancers (18, 19, 21). However,whether the genetic variations cause reactivation of TNFRSF19remains uncertain. GWAS identified three SNPs within theTNFRSF19 region, rs1572072, rs9510787, and rs753955, butnone of these SNPs are located in the coding region or nearthe promoter of TNFRSF19 (13, 14).Moreover, using quantitativeRT-PCR in various cell lines with or without TNFRSF19 expres-sion,wedidnotfind concordance between themRNAandproteinexpression levels (Supplementary Fig. S3C). The reasons for thepoor correlation of mRNA and protein levels include posttran-scriptional and posttranslational modifications, necessitatingfurther investigations.

In conclusion, our results suggest that gain of function ofTNFRSF19 in NPC inhibits the tumor-suppressive role of theTGFb pathway and promotes tumorigenesis. These findings help






pave theway toward a better understanding of themolecular basisand therapeutic potential of NPC.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J.-X. Bei, Y.-X. Zeng, L. FengDevelopment of methodology: C. Deng, H.-J. Zhang, L. FengAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C. Deng, G.-P. He, H.-J. Zhang, Q.-S. Feng, J.-X. BeiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C. Deng, Y.-X. Lin, X.-K. Qi, L. FengWriting, review, and/or revision of the manuscript: C. Deng, Y.-X. Lin, X.-K.Qi, J.-X. Bei, L. FengAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): G.-P. He, Y. Zhang, M. Xu, Q.-S. FengStudy supervision: J.-X. Bei, L. Feng

Acknowledgments

We thankDrs. JunjieChen andKwokWai Lo for helpful discussions. Thisworkwas supported by the National Key R&D Program of China (nos.2016YFC0902000 to Y.-X. Zeng and 2017YFA0505600 to L. Feng), Major Projectof Chinese National Programs for Fundamental Research and Development (no.2013CB910301 toY.-X. Zeng), theNationalNatural Science Foundationof China(nos. 81672980 to L. Feng and 81372882 to J.-X. Bei), the Key Program of theNational Natural Science Foundation of China (no. 81430059 to Y.-X. Zeng), theHealth & Medical Collaborative Innovation Project of Guangzhou City, China(no. 201803040003 to Y.-X. Zeng), and the Foundation of theMinistry of Scienceand Technology of Guangdong Province (no. 2015B050501005 to Y.-X. Zeng).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 17, 2017; revised March 27, 2018; accepted April 26, 2018;published first May 7, 2018.

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2018;78:3469-3483. Published OnlineFirst May 7, 2018.Cancer Res Chengcheng Deng, Yu-Xin Lin, Xue-Kang Qi, et al. Receptor Type I to Promote Tumorigenesis

β Signaling through Interaction with TGFβTNFRSF19 Inhibits TGF

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TNFRSF19 Inhibits TGFb Signaling through Interaction with ... · TNFRSF19 is unique because it does not bind to known TNF ligands and its ICD exhibits no sequence homology to any