NIKControlsClassicalandAlternativeNF-kBActivationandIs … · patients with PTCL (Table 1). Samples...

Human Cancer Biology

NIKControlsClassical andAlternativeNF-kBActivationand IsNecessary for the Survival of Human T-cell Lymphoma Cells

Lina Odqvist1, Margarita S�anchez-Beato1,3, Santiago Montes-Moreno1,10, Esperanza Martín-S�anchez1,Raquel Pajares2, Lydia S�anchez-Verde2, Pablo L. Ortiz-Romero4, Jose Rodriguez5,Socorro M. Rodríguez-Pinilla1,6, Francisca Iniesta-Martínez9, Juan Carlos Solera-Arroyo11,Rafael Ramos-Asensio12, Teresa Flores13, Javier Menarguez Palanca7, Federico García Bragado14,Purificaci�on Domínguez Franjo8, and Miguel A. Piris1,10

AbstractPurpose: Peripheral T-cell lymphomas (PTCL) are a heterogeneous entity of neoplasms with poor

prognosis, a lack of effective therapies, and a largely unknown molecular pathology. Deregulated NF-kBactivity has been associated with several lymphoproliferative diseases, but its importance in T-cell

lymphomagenesis is poorly understood.We investigated the function of the NF-kB–inducing kinase (NIK),

in this pathway and its role as a potential molecular target in T-cell lymphomas.

Experimental Design: We used immunohistochemistry to analyze the expression of different NF-kBmembers in primary human PTCL samples and to study its clinical impact. With the aim of inhibiting the

pathway, we used genetic silencing of NIK in several T-cell lymphoma cell lines and observed its effect on

downstream targets and cell viability.

Results:We showed that the NF-kB pathway was activated in a subset of PTCLs associated with poor

overall survival. NIK was overexpressed in a number of PTCL cell lines and primary samples, and a

pivotal role for NIK in the survival of these tumor cells was unveiled. NIK depletion led to a dramatic

induction of apoptosis in NIK-overexpressing cell lines and also showed a more pronounced effect

on cell survival than inhibitor of kappa B kinase (IKK) knockdown. NIK silencing induced a blockage of

both classical and alternative NF-kB activation and reduced expression of several prosurvival and

antiapoptotic factors.

Conclusions: The results of the present study indicate that NIK could be a promising therapeutic target

in these aggressive malignancies. Clin Cancer Res; 19(9); 2319–30. �2013 AACR.

IntroductionPeripheral T-cell lymphomas (PTCL) are a heterogeneous

family of non-Hodgkin lymphomas often associated with

an aggressive clinical course and poor outcome. First-linestandard therapy is based on the combination chemother-apy regimens usually used in B-cell lymphomas or solidtumors. As these regimens generally have poor response orhigh rates of recurrence, there is a need to develop targetedtherapies based on the signaling pathways that are aber-rantly expressed in these T-cell malignancies (1, 2).

Deregulated NF-kB activity plays a key role in thedevelopment of multiple malignancies. Thus, constitutiveactivation of NF-kB signaling has been observed in var-ious tumor types, including lymphomas, leukemias, andsolid tumors (3, 4). The NF-kB signaling pathway reg-ulates the transcription of many genes involved in cancerinitiation and progression events, such as apoptosis,proliferation, angiogenesis, and metastasis, and hence,several lymphoma types rely on NF-kB activity for theirproliferation and survival (4, 5). Genetic changes leadingto constitutive activation of the pathway have beendetected in several hematologic tumors, emphasizing itsimportance in the pathogenesis of these malignancies (6–9). NF-kB can be activated either through the classical(canonical) pathway or the alternative (noncanonical)pathway. Briefly, activation of the classical pathway

Authors' Affiliations: 1Molecular Pathology Programme and 2Biotechnol-ogy Programme, Spanish National Cancer Research Centre (CNIO);3Onco-hemathology Area, Instituto de Investigacion Sanitaria, HU Puertade Hierro-Majadahonda, Madrid; 4Department of Dermatology, Instituteiþ12, Hospital 12 de Octubre Medical School University Complutense;5Department of Internal Medicine, Hospital Universitario Severo Ochoa;6DepartmentofPathology,Fundaci�onJim�enezDíaz; 7PathologyDepartment,Hospital Gregorio Mara~non; 8Pathology Department, Hospital Infanta Sofía,Madrid; 9Hospital Virgen de la Arrixaca, Murcia; 10Pathology Department,Hospital Universitario Marques de Valdecilla, Universidad de Cantabria,IFIMAV, Santander; 11Pathology Department, Hospital Virgen de la Concha,Zamora; 12PathologyDepartment, Hospital SonEspases,PalmadeMallorca;13Pathology Department, University Hospital of Salamanca, Salamanca; and14Pathology Department, Hospital Virgen del Camino, Pamplona, Spain

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Margarita S�anchez-Beato, Instituto deInvestigaci�on Sanitaria, Hospital Universitario Puerta de Hierro-Majada-honda, Calle Joaquín Rodrígo 2, E-28222 Majadahonda-Madrid, Spain.Phone: 34-911916095; Fax: 34-911917863; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-12-3151

�2013 American Association for Cancer Research.

ClinicalCancer

Research

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on January 28, 2021. © 2013 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst March 27, 2013; DOI: 10.1158/1078-0432.CCR-12-3151

http://clincancerres.aacrjournals.org/

results in nuclear translocation of mainly p50/p65 het-erodimers through the phosphorylation of IkB and p105by the inhibitor of kappa B kinase (IKK) complex. Acti-vation of the alternative pathway results in nuclear accu-mulation of p52/RelB heterodimers and depends on theactivation of the NF-kB–inducing kinase (NIK) and IKKaphosphorylation (reviewed in ref. 10). NIK (MAP3K14) isa serine/threonine kinase described as critical for theactivation of the alternative pathway by inducing phos-phorylation of IKKa and p100, leading to p100 proces-sing with subsequent p52 generation and nuclear trans-location (11, 12). Nevertheless, the involvement of NIKin the activation of the classical pathway has also beennoticed (13, 14). Signals from CD40, B-cell activatingfactor receptor (BAFF-receptor), and lymphotoxin breceptor have been shown to induce NIK-dependentNF-kB activation (15–18). In multiple myeloma, severalgenetic abnormalities, such as deletions and inactivatingmutations of the TNF receptor–associated factor 3(TRAF3), have been shown to lead to the stabilizationof NIK (19). Similarly, in B-cell lymphomas, activatingmutations in the BAFF-receptor are known to activate NF-kB in a NIK-dependent manner (20). Knockdown of NIKin several types of malignancies, such as multiple mye-loma, diffuse large B-cell lymphoma (DLBCL), adult T-cell leukemia, and melanoma, has been associated withantitumor effects (14, 16, 21, 22), suggesting that NIKcould be a therapeutic target in some cancers. However,whether NIK is involved in NF-kB activation and tumor-igenesis or not is dependent on the cell type and cellularcontext and has not been studied in PTCL. Cutaneous T-cell lymphoma cell lines and primary samples undergoapoptosis upon treatment with the proteasome inhibitorbortezomib or an IKKb inhibitor (23–25), suggesting that

NF-kB inhibition could be used as a therapeutic strategy.However, most studies have only addressed the classicalpathway, leaving the alternative pathway poorlydescribed. There are no specific NF-kB inhibitor drugs inclinical use for PTCL today, indicating a need for moredetailed studies, the identification of new targets, and thedevelopment of novel specific inhibitors in the pathway.

In the present study, we show that both the alternativeand classical NF-kB pathways are activated in a subset ofprimary PTCL samples associated with poor clinical out-come. To block the NF-kB signaling pathway in PTCL, weshowed a key role for NIK as a regulator of both pathwaysand showed that NIK-depleted PTCL cells present stronglyreduced cell viability. These results highlight NIK as anattractive molecular target in T-cell lymphomas.

Materials and MethodsPatient samples and cell lines

Use of patient samples in this study was approved by theClinical Research Ethics Committee of Hospital Universi-tario Marqu�es de Valdecilla (HUMV; Santander, Spain).Tumor biopsies before treatment were obtained from theCNIO Tumour Bank Unit. T cells from peripheral blood ofhealthy donors or patients with S�ezary syndrome wereisolated through negative selection using the RosetteSepKit (StemCell Technologies). The proportion of T cells(CD3þ) in the samples was checked by flow cytometry andensured to be more than 90%. For the gene expression datain primary samples, 37 frozen PTCL cases were used,including 19 PTCL-not otherwise specified (PTCL-NOS),15 angioimmunoblastic T-cell lymphomas (AITL), and 3anaplastic large cell lymphomas (ALCL). The human T-celllymphoma cell lines DERL-7 (hepatosplenic g-d T-cell lym-phoma) and SR-786 (ALCL) were obtained from the Ger-man Collection of Microorganism and Cell Cultures(DSMZ). HuT 78 (S�ezary syndrome), HH (cutaneous T-cell lymphoma), and MJ (PTCL, HTLV-positive) wereobtained from the American Type Culture Collection,and the cell line My-La (Mycosis fungoides) was obtainedfrom the European Collection of Cell Cultures. All celllines were previously authenticated (year 2010–2011) byDSMZ.

ImmunohistochemistryTissue microarrays of paraffin-embedded tumor biopsies

or cell lines were used for immunohistochemistry andevaluated by the pathologists participating in the project(M.A. Piris and S.Montes-Moreno). Cases presenting nucle-ar staining inmore than 20%of tumor cellswere consideredpositive, grade 1 (posþ), whereas cases with strong nuclearstaining inmore than 50%of cells were considered positive,grade 2 (pos þþ). Antibodies and conditions are summa-rized in Supplementary Table S1.

Survival and correlation analysesThe Kaplan–Meiermethod applying the log-rank test was

used to estimate the differences in overall survival (OS)betweenNF-kB–positive and -negative cases in a series of 77

Translational RelevancePeripheral T-cell lymphomas (PTCL) are aggressive

malignancies presenting poor clinical outcome. Thereare currently no effective treatments or targeted therapiesavailable for these patients. Thus, studies aimed toelucidate the mechanisms contributing to these tumorsand to identify new therapeutic targets are required toimprove their dismal prognosis. Here, we show for thefirst time that primary PTCLs expressing nuclear NF-kBare characterized by a significantly worse clinical out-come compared with NF-kB–negative tumors, support-ing a rationale for the exploration of NF-kB–interferingstrategies. We further identify NF-kB–inducing kinase(NIK) as a novel potential therapeutic target in T-celllymphomas and show that targeting NIKmight bemoreeffective than previously suggested inhibitor of kappa Bkinase (IKK) inhibition. This study opens up opportu-nities for further translational studies and will hopefullycontribute to the future development of new targeteddrugs useful in PTCL.

Odqvist et al.

Clin Cancer Res; 19(9) May 1, 2013 Clinical Cancer Research2320




patients with PTCL (Table 1). Samples presenting nuclearstaining of NF-kB (p52, p50, RelB, p65, or c-Rel) in morethan 50% (pos þþ) of tumor cells were consideredpositive for NF-kB. Multivariate survival analysis includ-ing relevant clinical parameters (see Table 1) was con-ducted using a Cox regression analysis. To determine theindependence of variables, the Pearson x2 test was used.SPSS version 15.0 (SPSS Inc.) was used to carry out thesetests. Values of P < 0.05 were considered statisticallysignificant.

Quantitative RT-PCRRNA was isolated using the RNeasy Mini Kit (Qiagen).

cDNA was synthesized from 500-mg DNaseI-treated RNAwith SuperScript II Reverse Transcriptase (Invitrogen)and random primers. Human GUSB and 18S expressionwere used as endogenous controls in samples and celllines.

ImmunoblottingProtein was extracted using radioimmunoprecipitation

assay (RIPA) lysis buffer supplemented with protease andphosphatase inhibitors, and Western blot analysis was

conducted following standard protocols. ForNIKdetection,cell lines were treated for 3 hours with 20 mmol/L protea-some inhibitor MG132 (Sigma). MG132 was exclusivelyadded to cells forNIKdetection, and separate untreated cellswere collected to detect other proteins. Western blot anal-yses were quantified using the ImageJ program (NationalInstitutes of Health, Bethesda, MD).

Gene expression microarray analysesAll gene expression experiments used 4 � 44 K Whole

Human Genome Oligo Microarrays (Agilent Technologies,Inc.). For gene expression analysis of primary PTCL samples,37 frozen biopsies were used, and the Gene Set EnrichmentAnalysis (GSEA) tool (http://www.broad.mit.edu/gsea/)was applied to classify the expression profiles into func-tional pathways. The gene sets correlated with MAP3K14(NIK) expression in primary samples were identified usingPearson correlation. Gene sets with a false discovery rate(FDR) < 0.10 were considered significant. The microarraydata are available at the Gene Expression Omnibus underaccession number GSE36172. After NIK knockdown in celllines, pellets were collected 48 hours after siRNA transfec-tion and RNA was extracted. Three independent experi-ments were carried out in My-La and SR-786 cell lines, andeach sample was hybridized onto a separate microarray.The gene sets up- or downregulated in NIK knockdowncells were identified with the GSEA tool by applying alimma t test (FDR < 0.10). Genes differentially expressedbetween control cells and NIK knockdown cells were iden-tified using a paired t test (http://pomelo2.bioinfo.cnio.es)and visualized using Gene Cluster and Treeview(http://rana.lbl.gov/EisenSoftware.htm).

RNA interferencesiRNAs against MAP3K14 (NIK), IKBKB (IKKb), and

CHUK (IKKa), or a nontemplate control (NTC; 100nmol/L stealth siRNA; Invitrogen) were used for geneticsilencing. siRNAs were introduced into the cells by micro-poration (Microporator MP-100; Digital Bio).

NF-kB–binding activityNuclear cell fractions were isolated using the BioVision

Nuclear/Cytosol Fractionation Kit (BioVision), and 5 mg ofnuclear extract was used to quantify theNF-kB transcriptionactivation using the ELISA-based TransAM NF-kB FamilyTranscription Factor Assay Kit (Active Motif), following themanufacturer’s instructions. Nuclear extracts were plated intriplicate.

Cell viability and cell-cycle analysisCell viability and cell cycle were assessed in a FACS

Canto II flow cytometer (BD Biosciences), and FlowJosoftware (FlowJo version 7.6.1; TreeStar Inc.) was used fordata quantification. Cells staining negative for AnnexinV–allophycocyanin conjugate (APC) and 40,6-diamidino-2-phenylindole (DAPI) were considered viable. To estimatethe number of cells in each stage of the cell cycle, cells werefixed with ethanol and stained with propidium iodide.

Table 1. Characteristics of patients included inthe survival analysis

Characteristics Patients, n Percentage

GenderMale 46 59.7Female 31 40.3

Age, y�60 32 41.6>60 45 58.4

Ann Arbor stageI–II 17 22.1III–IV 60 77.9

IPI0–2 34 44.23–5 41 53.2— 2 2.6

ECOG0–1 53 68.82–4 24 31.2

LDH levelNormal 34 44.2High 42 54.5— 1 1.3

PTCL subtypeAITL 30 39.0PTCL-NOS 47 61.0

Total 77 100.0

Abbreviations: ECOG, Eastern Cooperative OncologyGroup; IPI, International Prognostic Index; LDH, lactatedehydrogenase.

NIK in T-cell Lymphoma

www.aacrjournals.org Clin Cancer Res; 19(9) May 1, 2013 2321




Reagent information, primer sequences, and additionalprotocols are described in the online SupplementaryData.

ResultsNuclear NF-kB is present in primary PTCL samples andis associated with worse clinical outcome

Even though NF-kB activation has been reported inPTCL, there are few studies of patient material, and theexpression pattern of the various NF-kB members has notbeen described in detail. We conducted immunohis-tochemistry on paraffin-embedded tissues from patientswith the most common types of PTCL: AITL, ALCL, andPTCL-NOS, to examine the nuclear expression of NF-kB.Nuclear expression of components defining both theclassical (p50 and c-Rel) and alternative (p52 and RelB)pathways (Fig. 1A and B) was detected in the majority ofPTCLs. To represent different levels of NF-kB expression,2 positivity thresholds were used: 20% (pos þ) and 50%(pos þþ) positive nuclei. Surprisingly, the commonly usedmarker for NF-kB activation, p65, showed only cytoplasmicexpression in most cases, suggesting that other factors of thefamily are more frequently involved in NF-kB signaling inPTCL. A subset of cases expressed highly elevated levels ofNF-kB (pos þþ), being most frequent in the CD30-positiveALCLs. Moreover, a significant positive correlation (P <0.05) was established between nuclear p50 and p52 expres-sion (Fig. 1C), indicating frequent activation of both path-ways in the same sample.

Contradictory results have been reported for the clin-ical correlation of NF-kB in different tumors (5, 26–28).To estimate the impact of nuclear NF-kB expression onthe clinical outcome of patients with PTCL, we did aKaplan–Meier analysis to compare the OS between NF-kB–positive and –negative tumors in a series of 77 PTCLcases (Fig. 1D; see patient characteristics in Table 1).Tumors with more than 50% positive nuclei for anyNF-kB factor were considered positive. Similar thresholdshave previously been used for immunohistochemistry ofNF-kB in other studies in the lymphoma field (6, 28, 29).Only PTCL-NOS and AITL subtypes were included in thesurvival analysis, as these patients did not present anysignificant difference in basal OS due to PTCL subtype(Supplementary Fig. S1). When NF-kB was taken intoaccount, however, patients with NF-kB–positive tumorshad a significantly worse OS compared with those in theNF–kB-negative group (log-rank test; P ¼ 0.003) with a 2-year OS of 41.7% compared with 67.9%, respectively. NF-kB activation in PTCL was still significantly associatedwith inferior OS when the impact of other clinical cov-ariates was taken into account, although the InternationalPrognostic Index (IPI) was still a more accurate estimateof prognosis in our series (Supplementary Table S2). Theobservation that NF-kB signaling is activated in a subsetof T-cell lymphomas with very poor clinical outcomesuggests the merit of evaluating NF-kB inhibition strate-gies in these tumors.

NIK is overexpressed in PTCL cell lines and primarysamples, and its expression is correlated with NF-kBactivation

Because NIK is involved in the activation of NF-kB insome cells and settings, we examined whether NIK wasinvolved in NF-kB activation in T-cell lymphomas. Weobserved a remarkable overexpression of NIK mRNA inseveral PTCL cell lines and primary S�ezary syndromesamples compared with T lymphocytes from healthydonors (Fig. 2A). The cell lines expressing high levels ofNIK mRNA also expressed elevated protein levels of NIK(Fig. 2B). Although DERL-7 lacked nuclear expression ofalternative NF-kB factors (p52 and RelB), the other cellline with low NIK levels, HuT 78, expressed both nuclearp52 and RelB (Supplementary Table S3). However, HuT78 expresses a truncated form of p100 (Fig. 2B), whichhas previously been described to activate the alternativepathway in a NIK-independent manner (30). To detectNIK protein levels in primary PTCL samples, we tested ahandful of different NIK antibodies in paraffin-embeddedtissues, but none of these provided consistent results. Forthat reason, we used gene expression microarray data tocompare the expression of NIK (MAP3K14) and NF-kBtarget genes (6) in a series of 37 PTCL samples. UsingGSEA, we found a significant positive correlation betweenthe expression of NIK and NF-kB target genes (Fig. 2C),suggesting that NIK may be involved in NF-kB signalingin these tumors. Other gene sets significantly correlatedwith the expression of NIK are listed in SupplementaryTable S4.

NIK is involved in both classical and alternative NF-kBsignaling in T-cell lymphomas

Whether NIK participates in classical NF-kB pathwayactivation as well as in alternative pathway regulation iscurrently unclear. To study the role of NIK in NF-kB sig-naling in T-cell lymphoma cells, we knocked down NIKusing 2 different siRNAs in 2 PTCL cell lines (My-La and SR-786). The 2 siRNA sequences induced different levels ofknockdown efficiencies (Fig. 3A), allowing us to study theeffect of a dose-dependent decrease in NIK. As expected, thelevels of p52 were reduced and the levels of p100 wereinitially increased after NIK knockdown, indicating anattenuation of p100 processing. We also observed a similarbut somewhat delayed decrease of p50 levels and anincrease in the levels of p105, linking NIK to the regulationof classical NF-kB activation as well (Fig. 3A and C andSupplementary Fig. S2). Figure 3B confirms that the knock-down of NIK gave rise to decreased nuclear levels of bothp52 and p50. The NF-kB DNA-binding activity of all 5proteins,measured by TransAMELISA assay, was decreased,supporting a role for NIK in both classical and alternativeNF-kB regulation in PTCL (Fig. 3D).

NIK is necessary for the survival of PTCL cell lines withhigh NIK levels

According to our data, My-La and SR-786 are character-ized by NIK overexpression and an active NF-kB pathway

Odqvist et al.





(Fig. 2A and B and Supplementary Table S3). We measuredcell viability after NIK silencing in these cells by flowcytometry using Annexin V and DAPI staining. NIK knock-down led to a dramatic increase in cell death comparedwithNTC-transfected cells, suggesting that NIK is necessary forthe survival of these cells. The cell death observed after NIKknockdown increased as knockdownbecamemore efficient(Fig. 4A). To study the effect of NIK knockdown over a

longer period, we repeated the siRNA transfection 4 daysafter the first microporation. Strikingly, after 1 week of NIKdepletion, nearly all cells had undergone apoptosis, show-ing the essential role of NIK in the viability of these tumorcells (Fig. 4B). The appearance of cleaved caspase-3 afterNIK knockdown in both My-La and SR-786 indicates aninduction of a caspase-dependent apoptotic pathway (Fig.4C). Interestingly, NIK silencing in DERL-7 or HuT 78 cell

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Figure 1. Expression of NF-kB in human PTCL samples. A, immunohistochemical staining of p100/p52, p105/p50, p65, RelB, and c-Rel in primary paraffin-embedded PTCL samples shows nuclear staining of NF-kB in most cases (B). The category pos þ indicates nuclear staining of more than 20% ofthe tumor, whereas posþþ indicates nuclear staining in more than 50% of the tumor. The percentage of the total number of cases (57 PTCL-NOS, 42 AITLs,and 28 ALCLs) in each PTCL subtype is shown. Comparison of the numbers of tumors with high levels of nuclear expression (pos þþ) of p50 or p52reveals a significant positivecorrelationbetween their expression, inwhich themajority of casespositive for p52alsowerepositive for p50 (C).D,Kaplan–Meiercurve representing the OS times of patients with NF-kB–positive and –negative PTCL. The NF-kB–positive group (red line) has a significantly worse OS(P < 0.05; log-rank test) than the NF-kB–negative group (blue line).






lines, presenting low NIK levels, had no effect on cellsurvival (Fig. 4D), suggesting that NIK targeting has aselective effect only on the cells presenting elevated NIKlevels. No obvious alterations of the cell-cycle distributionwere observed afterNIK knockdown, apart from there beingmore cells in sub-G1 (Supplementary Fig. S3).

To rule out whether the toxicity of NIK knockdown wasdue to a blockade of the classical or alternative NF-kBpathway, we knocked down either IKKa, IKKb, or both, inthese cell lines (Fig. 4E and Supplementary Fig. S4).Surprisingly, IKKa and IKKb knockdown (separately orin combination) only led to a slight increase in apoptosiscompared with NIK knockdown. Moreover, only theknockdown of NIK, but not the knockdown of IKK, wasable to strongly reduce the levels of p52 and p50 (Fig. 4Eand F). These results suggest that NIK might have, at leastin part, IKK-independent roles that confer survival onthese cells, and that targeting NIK could be a moreeffective therapeutic approach than IKK inhibition inthese tumors.

NIK knockdown leads to decreased expression of NF-kB target genes and downregulation of prosurvivalgenes

To gain a better insight into the mechanisms involved inNIK-dependent survival, we analyzed the gene expressionprofile induced 48 hours after NIK knockdown in My-Laand SR-786 cells. Knockdown efficiencies are shown inSupplementary Fig. S5. GSEA applying a limma t test com-paring siNIK1 and siNIK2with control cells was conducted.The gene sets significantly lost (FDR < 0.10) in the siNIKcells included the NF-kB target genes, the JAK–STATpathway, and targets of XBP1, and are described in Supple-mentary Table S5. NF-kB target genes were significantlyunderrepresented after NIK knockdown, indicating againa pivotal role forNIK inNF-kB activation (Fig. 5A). A pairedt test revealed 395 genes in My-La and 94 genes in SR-786,which were significantly and differentially expressed (FDR< 0.05 and log2 fold change > 0.6 in either direction)between NIK knockdown and control cells (Fig. 5B; seeSupplementary Table S6 for the complete list of genes).

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Figure 2. NIK expression in PTCL cell lines and samples. A, reverse-transcription quantitative PCR analysis of NIK expression in PTCL cell lines and isolated Tcells from patients with S�ezary syndrome (SS1–SS5) showed strongly elevated levels of NIK compared with T cells from healthy donors (control 1–3).B, NIK protein levels were detected after MG-132 treatment, and a comparable expression pattern was seen between NIK mRNA and protein levelsamong the cell lines. C, GSEA of gene expression microarray data from 37 PTCL cases shows a significant positive correlation (FDR < 0.10) betweenthe expression of NIK (MAP3K14) and NF-kB target genes.

Odqvist et al.





Reverse transcription quantitative PCR (RT-qPCR) was con-ducted on selected genes to validate the gene expressiondata (Fig. 5C andD). Several NF-kB target genes involved incancer cell survival were downregulated upon NIK silenc-ing, such as the antiapoptotic BCL2L1 [Bcl-x(L)] andCFLAR(c-FLIP) as well as several interleukins. Interleukin (IL)-6and IL-21 are cytokines with known functions in cell pro-liferation and survival of cancer cells and were stronglydownregulated after NIK depletion in both cell lines. Curi-ously, despite an activated NF-kB pathway in DERL-7 andHuT 78 as well, only the NIK-expressing My-La and SR-786express high levels of these interleukins (SupplementaryFig. S6), indicating a different NF-kB transcriptional pro-

gram in NIK-expressing versus NIK-nonexpressing cells.Apart from known NF-kB target genes, NIK depletion alsomodulated the expression of other genes involved in tumor-igenesis, such as Yes-associated protein 1 (YAP1), paraox-onase 2 (PON2), and Kruppel-like factor 2 (KLF2). Insummary, NIK knockdown leads to a decrease in theexpression of NF-kB target genes andmodulates the expres-sion of many genes involved in tumor growth and survival.

DiscussionThe molecular pathology of PTCLs has been poorly

characterized, and the dismal prognosis of these neoplasmsand the lack of efficient therapies demand further studies

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Figure 3. KnockdownofNIK in T-cell lymphomacell lines. A,NIKwasknockeddownusing2 siRNAs (siNIK1 andsiNIK2) or aNTC (siNTC) inMy-La andSR-786.After NIK knockdown, the expression of NIK, p100/p52, and p105/p50 was analyzed by Western blot analysis. B, nuclear expression of p50 and p52after NIK knockdown in My-La. The quantification of the signal is represented under each blot as a ratio against the siNTC-transfected cells. C,quantification of the immunoblots confirms a decrease in the levels of both p50 andp52 after NIK knockdown. The data are represented as themean�SDof 3independent experiments. D, DNA-binding activity of p52, RelB, p50, p65, and c-Rel 48 hours after NIK knockdown was measured using the ELISA-basedTransAM assay and reveals a reduction of classical and alternative NF-kB activation.






to clarify their molecular background and to identifynew therapeutic targets. Previous data suggest that NF-kBactivation is a key step in T-cell lymphoma pathogenesis

(21, 23). NF-kB activation has previously been reported ina subset of human PTCLs using gene expression dataand immunohistochemistry for classical NF-kB subunits

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Figure 4. The effect of NIK knockdown on PTCL cell survival. A, NIK knockdown (siNIK) induces cell death in My-La and SR-786 cells compared withuntransfected (MOCK) or nontemplate-transfected (siNTC) cells, asmeasuredbyflowcytometry usingDAPI andAnnexinVstaining. Thedataare representedasthe mean � SD of 3 independent knockdown experiments. B, only 1.26% of cells remain viable 7 days after NIK knockdown in My-La (DAPI/AnnexinV–negative). C, the cleavage of caspase-3 was detected after NIK depletion in both MyLa and SR-786 cells. D, increase in cell death in cell lines with high(My-LaandSR-786) or low (HuT78andDERL-7)NIK expression, representedas the ratiobetween thevaluesof cell death inNIKknockdown (siNIK2) andcontrol(siNTC) cells. E, knockdownof IKKa, IKKb, orNIK inMy-La and evaluation of the expressionof p100/p52andp105/p50. F, percentageof nonviableSR-786cellsafter IKKa, IKKb, or NIK silencing show a more potent induction of apoptosis after NIK knockdown compared with IKK knockdown. *, P < 0.05; **, P < 0.01.

Odqvist et al.





(24, 27, 29, 31).However, only a few studies have describedthe alternative pathway status or the clinical impact of thisactivation. Here, we found markers of activation of theclassical and alternative pathways in a subset of PTCL

samples, characterized by the nuclear expression of p50,p52, RelB, and c-Rel, whereas nuclear expression of p65wasusually absent. Nuclear expression of classical or alternativecomponents was significantly associated with worse OS in

Enrichment plot of NF-κB target genes

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Figure 5. Gene expression profile after NIK knockdown. Whole genomemicroarray analysis was conducted 48 hours after NIK knockdown in My-La and SR-786. A, GSEA enrichment plot of the NF-kB target genes. The NF-kB target genes were significantly underrepresented in the NIK knockdown cells. B,heatmaps of differentially expressed genes (FDR < 0.05 and log2 fold change > �0.6) between control and NIK knockdown cells. Negative log2 foldchanges (ratio siNIK/control) are represented in green (downregulation in siNIK cells) and positive fold changes are represented in red (upregulation in siNIKcells). C and D, relative gene expression in siNIK cells compared with the NTC measured by RT-qPCR.






patients with PTCL, suggesting that aberrant NF-kB activa-tion may confer enhanced survival or treatment resistanceon these tumors, and these data support the exploration oftherapies that interfere with NF-kB activation. These find-ings mimic those already described in DLBCL, in which themore aggressive activated B cell–like (ABC) subtype isdistinguished by increased NF-kB activation (5, 26). Incontrast with our results, Martinez-Delgado and colleagues(27) associated the expression of NF-kB–related genes,using gene expression microarrays, with a favorable clinicaloutcome in PTCL. These differences might be explained bythe genes included in the NF-kB signature (not restricted toNF-kB target genes) and the signals from the tumor stromareported by Martinez-Delgado and colleagues versus thenuclear expression of NF-kB subunits as assessed by IHC inour study.

Even though there is evidence thatNF-kB inhibitionhas anantitumor effect in some circumstances (5, 32), the needremains to identify therapeutic targets in the pathway and todevelop specificNF-kB inhibitors.NF-kB inhibitors targetingmutated or aberrantly expressed molecular targets in a par-ticular tumormight be preferable to broad NF-kB inhibitionthat also abolishes normalNF-kBactivation.We showed thatNIK was highly overexpressed in a subset of PTCL cell linesand tumor samples and that its expression was significantlyassociated with NF-kB activation. NIK has previously beenfound to be overexpressed at the RNA and/or protein level inother cancers, such as melanoma, multiple myeloma,DLBCL, mucosa-associated lymphoid tissue (MALT) lym-phoma, and adult T-cell leukemia (14, 16, 21, 22, 33). Insome cases, genetic alterations such as gene amplifications,translocations, or mutations in NIK, or alterations in genesregulating the stability of NIK protein, are described, empha-sizing the role of NIK in tumorigenesis (14, 19, 34). Fromprevious reports, the role of NIK in the alternative NF-kBpathway is clear, but the involvementofNIK in the regulationof the classical pathway seems to be signal and cell typedependent (13, 35). For example, in melanoma and pancre-atic cancer, NIK only affects the alternative pathway (22, 36),whereasNIK regulatesbothpathways inDLBCLandmultiplemyeloma (14, 16). In PTCL, we showed that NIK is involvedin both classical and alternative pathway activation, as NIKknockdown led to decreased expression and DNA-bindingactivity of both classical (p65, p50, and c-Rel) and alternative(p52 and Rel-B) NF-kB transcription factors. Moreover, NIKknockdown also altered the expression of many genes com-monly associatedwith classicalNF-kBactivation, such as IL6,IL10, andNFKBIA (37, 38). However, NIK does not seem tobe strictly essential for NF-kB activation in all PTCL cells, aswe found that nuclearNF-kBwasnot exclusively expressed inNIK-overexpressing cell lines. Thus, the pathway may beactivatedbydifferentmechanisms in these cells, and aproperidentification of upstream mechanisms is important forefficient NF-kB inhibition. The fact that NIK knockdown ledto amore efficient reduction of p50 and p52, comparedwithIKKa or IKKb knockdown, suggests that NIK can, at least inpart, regulate the NF-kB pathway by IKK-independentmechanisms. Consistent with these results, direct phosphor-

ylation of p100 in an IKKa-independent manner has beenreported (11). It is not clear whether the effect on p105processing is a direct effect of NIK or an indirect effect ofreduced alternative activity.

To investigate the possible downstream effectors of NIK-regulated tumorigenesis, we conducted gene expressionanalysis ofNIK-silenced T-cell lymphoma cells. As expected,many of the genes differentially expressed between NIK-silenced and control cells wereNF-kB target genes known tobe important in regulating tumor growth and survival. Theexpression of antiapoptotic proteins is one of the mechan-isms by which tumor cells manage to survive in the envi-ronment and to resist chemotherapy (39). After NIK knock-down, we observed a downregulation of antiapoptoticgenes such as CFLAR (Bcl-xl), BIRC3 (cIAP2), and BCL2L1(c-FLIP), which might explain the strong induction ofapoptosis observed in these cells after NIK inhibition. Theinterleukins IL6 and IL21, which were highly expressed incell lines that were sensitive to NIK knockdown but not inresistant cell lines, were strongly downregulated after NIKdepletion. These cytokines are important for B- and T-celldevelopment and a proper regulation of the immuneresponse but have also been widely studied in severaltumors owing to their protumorigenic activity and theirroles as targets for cancer therapy (40, 41). Both of thesecytokines are NF-kB target genes but are also involved inJAK–STAT signaling, which also is consistent with ourobservation that NIK knockdown leads to downregulationof the JAK–STAT pathway. The expression of other genesinvolved in tumorigenesis, not described as NF-kB targets,was alsomodulated after NIK knockdown. For example, theexpression of the oncogene YAP1 (42) was reduced and anupregulation of KLF2, a tumor suppressor with antiproli-ferative effects known to be silenced in tumor cells (43), wasobserved. This can indicate either NF-kB–independentfunctions of NIK or interaction between theNF-kB pathwayand other pathways (44).

The upstream signaling events leading to NIK over-expression and/or NF-kB activation in T-cell lymphomasremain to be clarified. Several factors are known to triggerNF-kB activation in other tumors, including signals pro-vided by the tumor microenvironment or mutations thatgive rise to a constitutive activation of the pathway. InPTCL, apart from the previously reported p100 trunca-tions (7, 45), genetic lesions in NIK or other NF-kBpathway genes have still not been reported. Other pos-sible contributions to NF-kB activation in T-cell lympho-mas could be oncogenic viruses (46), signals from CD30or the T-cell receptor (47, 48), or epigenetic mechanisms,such as the previously reported polycomb-mediatedrepression of mir31 (49). Even though we observed alack of mir31 in our cell lines, this absence was indepen-dent of the levels of NIK (data not shown). It could alsobe hypothesized that the activation of NIK and NF-kB canbe an effect of deregulated signaling of other pathwayslinked to T-cell lymphomagenesis, such as the phosphoi-nositide 3-kinase/AKT, Notch, or JAK–STAT pathway,which can all result in NF-kB activation (44).

Odqvist et al.





The present study reveals a pivotal role for NIK in thesurvival of T-cell lymphoma cells. NIK knockdown stronglyreduces the cell viability of PTCL cell lines and is shown tobe more effective than IKK inhibition in these cells. Impor-tantly, the fact that cells with a low level of expression ofNIKwere not affected by NIK knockdown suggests a selectivetoxicity ofNIK inhibition inNIK-overexpressing lymphomacells.Moreover, althoughNIK-deficientmice exhibit defectsin lymphoid organogenesis, they do not present any grossphenotypic changes (50), suggesting that pharmacologicinhibition of NIK might be safer than broad NF-kB inhi-bition. To developNF-kB–based cancer therapies efficientlyand safely, it is necessary to identifymolecular targets aswellas biomarkers with which to stratify patients who are likelyto benefit from the therapy. PTCL are highly aggressivemalignancies that currently lack efficient therapies. Ourfindings indicate that NIK is a promising molecular targetin NIK-overexpressing PTCL, a conclusion that should betaken into account in further validation anddevelopment ofspecific NIK inhibitors.

Disclosure of Potential Conflicts of InterestPablo L. Ortiz-Romero has received honoraria from serving on the speak-

ers’ bureau of Bristol-Myers Squibb and Eisay and is a consultant/advisoryboard member of AOP Orphan. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: L. Odqvist, M. S�anchez-Beato, P.L. Ortiz-Romero,M.A. PirisDevelopment of methodology: L. Odqvist, E. Martı́n-S�anchez, L. S�anchez-Verde

Acquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): L. Odqvist, S. Montes-Moreno, L. S�anchez-Verde,P.L. Ortiz-Romero, J. Rodriguez, S.M. Rodrı́guez-Pinilla, F. Iniesta-Martı́nez,R. Ramos-Asensio, T. Flores, J.M. Palanca, F.G. Bragado, P.D. Franjo, M.A.PirisAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): L. Odqvist, M. S�anchez-Beato, E. Martı́n-S�anchez, J. Rodriguez, S.M. Rodrı́guez-Pinilla, T. Flores, M.A. PirisWriting, review, and/or revision of the manuscript: L. Odqvist, M.S�anchez-Beato, P.L. Ortiz-Romero, J.M. Palanca, M.A. PirisAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): L. Odqvist, E. Martı́n-S�anchez, R.Pajares, L. S�anchez-Verde, P.L. Ortiz-Romero, S.M. Rodrı́guez-Pinilla, J.C.Solera-ArroyoStudy supervision: M. S�anchez-Beato, M.A. Piris

AcknowledgmentsThe authors thank the Tumour Bank Unit of the CNIO (Marı́a-Jesus

Artiga, Laura Cereceda, andMaria Encarnaci�onCastillo) and all themembersof the laboratory at the CNIO and IFIMAV, especially Beatriz Herreros andDaniel Martin Perez, for their help and discussion, and Pierfrancesco Vargiufor technical supervision. The authors also thank Francisco X. Real forexcellent scientific discussion.

Grant SupportThis work was supported by grants from the Ministerio de Ciencia e

Innovaci�on (SAF2008-03871), Fondo de Investigaciones Sanitarias (FIS 08/0856 and RTICC RD06/0020/0107), and Asociaci�on Espa~nola Contra elCancer. L. Odqvist is supported by a grant from the Comunidad Aut�onomadeMadrid, Spain. M. S�anchez-Beato is supported by aMiguel Servet contract(CP11/00018) from the Fondo de Investigaciones Sanitarias.

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

Received October 8, 2012; revised February 12, 2013; accepted March 10,2013; published March 27, 2013.

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Odqvist et al.





2013;19:2319-2330. Published OnlineFirst March 27, 2013.Clin Cancer Res Lina Odqvist, Margarita Sánchez-Beato, Santiago Montes-Moreno, et al. Necessary for the Survival of Human T-cell Lymphoma Cells

B Activation and IsκNIK Controls Classical and Alternative NF-

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