NR4A3 Suppresses Lymphomagenesis through Induction of ...Molecular and Cellular Pathobiology NR4A3 Suppresses Lymphomagenesis through Induction of Proapoptotic Genes Alexander J.A

Molecular and Cellular Pathobiology

NR4A3 Suppresses Lymphomagenesis throughInduction of Proapoptotic GenesAlexander J.A. Deutsch1, Beate Rinner2, Martin Pichler3, Katharina Prochazka1,Katrin Pansy1, Marco Bischof1, Karoline Fechter1, Stefan Hatzl1, Julia Feichtinger4,5,Kerstin Wenzl1, Marie-Therese Frisch2, Verena Stiegelbauer3, Andreas Prokesch6,Anne Krogsdam7, Heinz Sill1, Gerhard G. Thallinger4,5, Hildegard T. Greinix1,Chenguang Wang8, Christine Beham-Schmid9, and Peter Neumeister1

Abstract

Nuclear orphan receptor NR4A1 exerts an essential tumorsuppressor function in aggressive lymphomas. In this study, weinvestigated the hypothesized contribution of the related NR4Afamily member NR4A3 to lymphomagenesis. In aggressivelymphoma patients, low expression of NR4A3 was associatedwith poor survival. Ectopic expression or pharmacologicalactivation of NR4A3 in lymphoma cell lines led to a signifi-cantly higher proportion of apoptotic cells. In a mouse NSGxenograft model of lymphoma (stably transduced SuDHL4

cells), NR4A3 expression abrogated tumor growth, comparedwith vector control and uninduced cells that formed massivetumors. Transcript analysis of four different aggressive lympho-ma cell lines overexpressing either NR4A3 or NR4A1 revealedthat apoptosis was driven similarly by induction of BAK, Puma,BIK, BIM, BID, and Trail. Overall, our results showed thatNR4A3 possesses robust tumor suppressor functions of similarimpact to NR4A1 in aggressive lymphomas. Cancer Res; 77(9);2375–86. �2017 AACR.

IntroductionNR4A1 (Nur77),NR4A2 (Nurr1), andNR4A3 (NOR-1) belong

to the nuclear orphan receptors of the Nur77 family. NR4A1,NR4A2, and NR4A3 are widely expressed in different types oftissues, such as skeletal muscle, adipose tissue, heart, kidney, Tcells, liver, and brain. They are immediate early- or stress-responsegenes and can be induced by awide range of physiological signals,such as fatty acids, stress, prostaglandins, growth factors, calcium,inflammatory cytokines, peptide hormones, and neurotransmit-ters. Further, NR4A1 and NR4A3 play a central role in negativeselection of T lymphocytes, as well as IgM-mediated and virallyinduced B-cell apoptosis (1–4). NR4A1 and NR4A3 were identi-fied to function as tumor suppressors in acute myeloid leukemia

(AML). Deletion of both nuclear receptors led to rapid develop-ment of AML in mice. Loss of NR4A1 and NR4A3 was also foundin leukemic blasts from human AML patients, irrespective ofkaryotype (5). Additionally,NR4A1 andNR4A3 hypoallelic mice(NR4A1þ/� and NR4A3�/�; NR4A1�/� and NR4A3þ/�)—micewith a reducedNR4A1 andNR4A3 expression—develop a chronicmyeloid malignancy that recapitulates the pathologic features ofmyelodysplastic/myeloproliferative neoplasm with progressionto AML in rare cases (6).

In our previously published study (7), we found a significantreduction of both, NR4A1 and NR4A3, in B-chronic lymphaticleukemia (CLL; 71%), follicular lymphoma (FLIII; 70%), and indiffuse large B-cell lymphoma (DLBCL; 74%) compared withnormal controls. Survival analysis revealed that low NR4A1expression is associated with poor cancer-specific survival. Fur-ther, overexpression of NR4A1 caused apoptosis in several lym-phoma cell lines. SuDHL4 lymphoma cells stably transducedwithan inducible (tet-off) NR4A1 expression construct and subcuta-neously injected in NOD/SCID/IL-2rgnull (NSG) mice led tosuppressed lymphoma outgrowth after NR4A1 induction bywithdrawal of doxycycline (7).

Because it has been reported that the NR4A3 expressionlevels correlate positively with therapy success in whole-genome expression analysis of 58 DLBCL patients (8), thatNR4A3 and NR4A1 are functionally redundant in T-cell apo-ptosis (9), and that NR4A3 correlates with NR4A1 expressionin our cohort of aggressive lymphoma patients (7), we aimedto functionally characterize NR4A3 in aggressive lymphomacells. Here, we show for the first time that overexpression ofNR4A3 in aggressive lymphoma cells causes apoptosis in vitroand leads to reduced tumor growth in a xenograft model.Additionally, the induction of NR4A3 in aggressive lymphomacells by thapsigargin (TG), a NR4A3 inducing agent (10), and

1Division of Hematology, Department of Internal Medicine, Medical University ofGraz, Graz, Austria. 2Center for Medical Research (ZMF), Medical University ofGraz, Graz, Austria. 3Division of Oncology, Department of Internal Medicine,Medical University of Graz, Graz, Austria. 4Institute of Molecular BiotechnologyGraz University of Technology, Graz, Austria. 5BioTechMed Omics Center Graz,Austria. 6Institute of Cell Biology, Histology and Embryology, Medical Universityof Graz, Graz, Austria. 7Division of Bioinformatics, Biocenter Innsbruck,InnsbruckMedical University, Graz, Austria. 8Key Laboratory of Tianjin Radiationand Molecular Nuclear Medicine; Institute of Radiation Medicine, Peking UnionMedical College and Chinese Academy of Medical Sciences, Tianjin, China.9Institute for Pathology, Medical University of Graz, Graz, Austria.

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

Corresponding Author: Alexander Deutsch, Medical University of Graz, Auen-bruggerplatz 15, Graz, Austria. Phone: 4331638572816; Fax: 4331638573009;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-16-2320

�2017 American Association for Cancer Research.

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BF175—a pinacolyl boronate-substituted stilbene derivate (11,12)—leads to immediate lymphoma cell apoptosis. Collective-ly, these results define NR4A3 as novel gene with tumor-suppressive properties in lymphoid malignancies.

Patients and MethodsPatients

To test the implication of NR4A3 expression levels in overallsurvival of human lymphoma samples, we measured the mRNAexpression levels of NR4A3 in a cohort of 92 histologicallyconfirmed aggressive lymphoma patients. The patients receiveda rituximab-containing standard treatment regimen at the Divi-sion of Haematology, Medical University of Graz between 2000and 2010 (with last follow-up until May 2016). Patients werecategorized to a low- and high-expression group according to acutoff value generated by receiver operating curve (ROC) analysis.The low- and high-expression groups were related to the endpointoverall survival.

Cell culture and treatment with NR4A3-inducing agentsKarpas-422 and SuDHL4 as model for germinal center-(GCB-)

DLBCL and RI-1 and U2932 as model for activated B cell-(ABC-)DLBCL were used for functional characterization of NR4A3.SuDHL4, RI-1 and U2932 were maintained in Iscove's ModifiedDulbecco's Medium (IMDM, Thermo Fisher Scientific) with 10%fetal bovine serum (FBS, Thermo Fisher Scientific), Karpas-422was cultured in IMDM with 20% FBS. To the culture media,penicillin (50 U/mL) and streptomycin (50 mg/mL) were added.Cells were periodically checked for mycoplasma by PCR and werefound to be negative. All cell lines were treated in a range from1�10�1 to 1 � 10�7 mol/L with TG (Sigma-Aldrich), a NR4A3-inducing agent (10) and BF175. NR4A1- and NR4A3-overexpres-sing cell lines were additionally treated with 20 nmol/L leptomy-cin B to inhibit shuttling of the nuclear proteins into cytoplasm.The identity of theDLBCL cell lineswas confirmedby STR analysisusing Power Plex 16 System (Promega) and verified at the onlineservice of the DSMZ cell bank (http://www.dsmz.de; Supplemen-tary Table S1).

Vector construction, generation of lentiviral vectors, andlentiviral transduction

Sequence verified full-length PCR product—generated fromperipheral blood B-cell cDNA using specific PCR primers (Sup-plementary Table S2)—was subcloned into the pTight Tet-Offviral expression system (Clontech) for lentiviral transduction andinto pEZ-M61 (Genecopoeia) for transient transfection.

Details on the generation of lentiviral vectors, transduction,and functional assays of NR4A3 induced lymphoma cells areprovided in the Supplementary Methods section.

Assessment of cell growthThapsigargin- and BF175-treated lymphoma cells and their

controls were plated at a density of 10,000/mL and culturedfor 72 hours. Seven replicates of the CellTiter 96 AQueousOne Solution Cell Proliferation Assay (Promega) were doneusing 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphe-nyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS). Theabsorbance was recorded by a BioRad spectrophotometer at490 nm.

RNA extraction and real-time semiquantitative PCRTotal RNA was extracted using TRIzol (Invitrogen) according

to the manufacturer's protocol. cDNA was synthesized usingthe RevertAid H Minus First-Strand cDNA Synthesis Kit(Fermentas).

Real-time semiquantitative PCR (RQ-PCR) for NR4A1,NR4A3, Bim 1,6, Bim 9, Puma, BCL2, BCLX, MCL1, Trail, FasL,DR4, DR5, and Fas (primers are listed in Supplementary TableS2) was performed using an ABI Prism 7000 Detection system(Applied Biosystems). PCR reaction and data analysis wereperformed as previously described by our group (7, 13, 14).GAPDH, PPIA, and HPRT1—known to exhibit the lowest var-iability among lymphoid malignancies (15)—served as house-keeping genes.

Transfection for overexpression of NR4A1 and NR4A3 andsilencing of NR4A3

Cells were transfected by electroporation using AMAXA Kit V(Lonza). The programs used were O-017 for SuDHL4 and X-001for Karpas-422, RI-1 andU2932, respectively. Briefly, 5� 106 cellsof SuDHL4, Karpas-422, RI-1, and U2932 were transfected with2.5 mg pEZ-M61 carrying NR4A1 or NR4A3 or empty pEZ-M61vector in two replicates for each cell line. Cells were seeded at 5�105 cells per mL and functional assays were performed after 24,48, and 72 hours.

Cells (4 � 106) of SuDHL4 and U2932 were transfected with175 ng siRNA targeting NR4A3 (Qiagen) or scramble controls.Cells were seeded 5 � 105 per mL, after 12 hours treated with 10mmol/L TG (Sigma-Aldrich) or BF175 and functionally tested.

Xenograft experimentsNSG-mice were purchased from The Jackson Laboratory. Ten

male NSG mice received subcutaneous flank injections of 1 �107 transduced SuDHL4 cells containing the empty pLVXplasmid as vector control on the right flank and the inducibleNR4A3 pLVX construct on the left flank resuspended in 200 mLMatrigel (BD). Doxycycline (Clontech) was administered tofive animals through their drinking water in a concentration of200 mg/mL to suppress induction of NR4A3 as controls. Tumorburden was assessed weekly by tentative inspection. At day 20,tumor volume was estimated by ultrasonic testing, and alltumors were harvested for histologic analysis. Tumor volumeand histologic stains were compared between the doxycycline-administered mice and mice without doxycycline administra-tion. All animal work was done in accordance with a protocolapproved by the Institutional Animal Care and Use Committeeat the Medical University of Graz (Graz, Austria).

Statistical analysisStatistical analysis was performed by using IBM SPSS Statistics

21.0 (SPSS Inc). The nonparametric Mann–Whitney U test wasused to analyze differences in the functional assay and expres-sion analysis. The Spearmen correlation test was performed toexamine any correlation of the NR4A1 and NR4A3mRNA levelsto apoptotic and antiapoptotic genes. When the P value waslower than 0.05, a significant value was reached. Overall survivalwas defined as the time in months from the date of diagnosis todeath by any cause. Patients' overall survival was calculated withthe Kaplan–Meier method, and differences were tested by thelog-rank test.

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ResultsLow NR4A3 expression is associated with poor clinicaloutcome

Because aggressive B-cell lymphomas exhibited the lowestNR4A3 expression levels in our previous study (7), we decidedto explore the influence of mRNA expression levels on survivalin a cohort of 92 patients diagnosed with DLBCL and FLIII atour institution. A marked (more than 2-fold) downregulationof NR4A3 was detected in the vast majority of DLBCL-GCB,DLBCL-NGCB, and FLIII patients in comparison the germinalcenter B cells (CD19þ, CD77þ cells) on mRNA levels, respec-tively (Fig. 1A, P < 0.05 for DLBCL-GCB and DLBCL-NGCB, P <0.1 for FLIII). To determine whether reduced NR4A3 mRNAexpression translates to reduced protein levels, Western blotanalyses for NR4A3 was performed in selected lymphoma—and control samples as previously described and a significantpositive correlation was observed by comparing densitometri-cally quantified protein levels to mRNA levels (Spearman r ¼0.565 for NR4A3, P < 0.01; ref. 7). In addition, a significantassociation between low NR4A3 expression and poor survivalwas observed (P ¼ 0.043, log-rank test, Fig. 1B). Patients withhigh NR4A3 expression reached a plateau in their survivalapproximately after 48 months.

To investigate whether promoter hypermethylation, dele-tions, or mutations in the promoter (1 kb upstream anddownstream of the transcriptional initiation site) or codingsequence (CDS) occur, direct sequencing, gene copy numberand methylation-specific PCR analyses of NR4A3 in aggressivelymphomas were performed on selected cases. However,besides some already described single-nucleotide polymorph-isms derived from publicly available databases (http://www.ncbi.nlm.nih.gov/snp), no alterations were detected (Supple-mentary Table S3).

Overexpression of NR4A3 induces apoptosis in aggressivelymphoma cells and suppresses tumor growth in xenografts

To functionally characterize NR4A3, the SuDHL4 lymphomacell line was stably transduced with an inducible lentiviral con-struct coding for NR4A3. The transduction was performed induplicates (SuDHL4 pLVX NR4A3-1 and -2). Transduction withthe viral vector without insert (SuDHL4 pLVX empty) served asvector control. The removal of doxycycline from the culturemedium led to a�28-foldNR4A3 induction (P < 0.003), whereasNR4A3 levels remained unchanged in the vector control (Fig. 2AandB). After 48hours of doxycycline removal, a higher percentageofNR4A3-induced cells stained positive for Annexin V comparedwith vector control (48.3% vs. 5.3%, Fig. 2C, P ¼ 0.035). Addi-tionally, after 48 and 72 hours of doxycycline removal, a higherproportion of SuDHL4 pLVX NR4A3 1-2 cells exhibited anincreased cleaved caspase-3/7 activity (relative activity: 3.1 vs.0.95 after 48 hours and 3.05 vs. 0.98 after 72 hours, Fig. 2D, P <0.05), and after 72 hours, a significantly higher SubG1 peakcompared with their vector controls (18.5% vs. 5.59%, Fig. 2D;P < 0.01) was detectable.

Additionally, NR4A3 induction resulted in significantlyreduced cell proliferation after 48 hours as estimated by BrdUincorporation (13.9% vs. 55.1%; Fig. 2E, P < 0.001) and cellgrowth after 72 hours as determined by the MTS assay (Fig. 2E,P ¼ 0.0008).

To clarify whether the proapoptotic property of NR4A3 ismediated via transactivation of its target genes (nuclear localiza-tion) or its mitochondrial targeting (cytoplasmic localization),inducing mitochondrial cell death (16), we treated under doxy-cycline withdrawal SuDHL4 pLVX NR4A3-1 and SuDHL4 pLVXempty cells with leptomycin B (LMB)—an inhibitor of the nuclearexport system (17) causing nuclear retention of NR4As proteins(18). Apoptosis rates estimated by Annexin V staining of SuDHL4pLVX NR4A3-1 cells were detected to a similar extent (62.3% vs.

Figure 1.

NR4A3 expression in aggressive NHLs. A, Depiction of NR4A3 mRNA expression levels in germinal center B cells (CD19þCD77þ; n ¼ 5) and aggressive NHLpatients (n¼ 92) consisting of NGCB-DLBCL (n¼ 30), GCB-DLBCL (n¼ 22), and FLIII (n¼ 33)). � , reduced expression compared with CD19þCD77þ cells (P < 0.01);� , reduced expression compared with CD19þCD77þ cells (P ¼ 0.073). B, Probability of cancer-specific survival in DLBCL patients stratified according tothe NR4A3 mRNA expression level. Low NR4A3 expression is associated with poor survival.

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Figure 2.

Ectopic NR4A3 expression induces apoptosis in SuDHL4 lymphoma cells. Transduced SuDHL4 cells carrying empty vector (SuDHL4 pLVX empty) or theinducible NR4A3 construct (SuDHL4 pLVX NR4A3 1-2) were cultured in the presence of doxycycline (DOX), resulting in no NR4A3 induction, or in the absenceof doxycycline (no DOX), resulting in induction of NR4A3. The transduction experiments were performed in duplicates (SuDHL4 pLVX NR4A3-1 and SuDHL4pLVX NR4A3-2). Additionally, all functional assays were replicates once more. (Continued on the following page.)

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60.7%; Fig. 2F,P¼0.83) andNR4A3expressionwasuninfluencedby LMB treatment (Fig. 2G), suggesting that the proapoptoticproperty of NR4A3 is mediated by its nuclear function.

To confirm the specificity of the NR4A3-mediated apoptosis,we transfected SuDHL4 pLVXNR4A3-1 cells with siRNA targetingNR4A3 under doxycycline withdrawal (Fig. 2h). Silencing ofNR4A3 under concomitant induction abrogated the apoptosisrate of NR4A3 silenced lymphoma cells in comparison withscramble controls as estimated by Annexin V staining (11.3% vs.68.7%; Fig. 2I, P < 0.001).

In order to support the tumor suppressive function ofNR4A3 invivo, stably transduced SuDHL4 lymphoma cell lines were furtherinvestigated in the NSG mouse model. Control cells (SuDHL4pLVX—containing no insert) were subcutaneously injected intothe right flank and the same number of SuDHL4 pLVX NR4A3-1lymphoma cells into the leftflankofmaleNSGmice (n¼10). Fiveof 10 mice did not receive any doxycycline to induce NR4A3expression, whereas doxycycline was administered to the residualfive mice to suppressNR4A3 expression. Within 20 days, all micedeveloped visible tumors in their right flanks, whereas tumors in

(Continued.) A, NR4A3 mRNA expression analysis after induction. Relative expression levels were calculated in comparison with SuDHL4 pLVX empty culturedwith doxycycline containing media. Each bar represents the mean values of expression levels � SD. B, Western blot analysis of NR4A3 expression in SuDHL4cells carrying the empty vector (SuDHL4 pLVX empty) or the inducible NR4A3 construct (SuDHL4 pLVX NR4A3 1-2) under doxycycline withdrawal. GAPDHserved as loading control. C–D, Apoptotic assay of SuDHL4 pLVX empty cells and SuDHL4 pLVX NR4A3 1-2 cells with and without doxycycline. To determine theapoptotic effects of NR4A3 in aggressive lymphoma cells, Annexin V (C and D), caspase-3/7 activity (D) and sub-G1 peak (D) were estimated. E, Proliferationand cell growth of SuDHL4 pLVX empty cells and SuDHL4 pLVX NR4A3 1-2 cells with and without doxycycline by using BrdU incorporation and the MTS assay.F, Annexin V staining of SuDHL4 pLVX empty and SuDHL4 pLVX NR4A3-1 cells with and without LMB after 48 hours of doxycycline withdrawal. G, Westernblot analysis of NR4A3 expression of SuDHL4 pLVX empty and SuDHL4 pLVX NR4A3-1 cells with and without LMB after 24 hours of doxycycline withdrawal.H, Western blot analysis of SuDHL4 pLVX NR4A3- 1 cells being transfected with siRNA targeting NR4A3 (siNR4A32 and siNR4A3-5) and scramble controlafter 48 hours of doxycycline withdrawal. I, Annexin V staining of NR4A3 expression of SuDHL4 pLVX NR4A3-1 cells being transfected with siRNA targeting NR4A3(siNR4A3-2 and siNR4A3-5) and scramble control after 24 hours of doxycycline withdrawal. Annexin V staining and cleaved caspase-3 percentages were estimatedby using FACS analysis with specific fluorophore-labeled peptides or antibodies. Sub-G1 peaks were determined by cell-cycle analysis using FACS analysis.� , significant difference (P < 0.05).

Figure 3.

Inducible NR4A3 overexpression abrogates tumor formation in xenografts. A, Macroscopic analysis of tumors from SuDHL4 lymphoma cells carrying theinducible NR4A3 construct (left flank) and empty vector (right flank) with and without doxycycline administration after 20 days. B, Estimation of tumor volumeby using an ultrasonic device after 20 days. C, Histological analysis by using hematoxylin and eosin staining of tumors sections derived from SuDHL4lymphoma cells carrying empty vector (left) or the inducible NR4A3 construct (right) with (left) and without doxycycline (right) administrationafter 20 days (magnification, �100 ). � , significant difference (P < 0.05).






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the left flanks were detected only in the doxycycline-administeredmice. Macroscopic inspection (Fig. 3A) showed a clear size dif-ference: Mice inoculated with SuDHL4 NR4A3-1 cells withoutdoxycycline developed no lymphomas compared with doxycy-cline-administered mice or mice inoculated with isogenic emptyvector control (Fig. 3B, P < 0.01). Histologic analysis also dem-onstrated that only mice without doxycycline from SuDHL4NR4A3-1 lymphoma cells remained tumor free (Fig. 3C).

Taken together, these data imply that NR4A3 possesses tumorsuppressor functions in aggressive lymphoma cells.

Treatment of lymphoma cell lines with TG and BF175 inducesNR4A3-mediated apoptosis

To investigate the effects of pharmacologic activation ofNR4A3 with the NR4A3-inducing agent TG in lymphoma cells,we used Karpas-422 and SuDHL4 (as GCB-DLBCL model) andRI-1 and U2932 (as ABC-DLBCL model) cell lines. After 72hours of TG treatment, a concentration-dependent growthinhibition in all investigated cell lines was detected by theMTS assay (Fig. 4A). NR4A3 expression was induced by treat-ment with 1.0� 10�5 mol/L TG in all lymphoma cell lines (Fig.4B, P < 0.01) after 4, 12, 24, 48, and 72 hours of TG treatmenton mRNA levels. Furthermore, Western blot analysis confirmedTG-mediated NR4A3 induction on the protein level (Fig. 4C).In order to investigate whether TG-induced growth inhibitionis mediated by apoptosis, we determined the cleavage ofcaspase-3, the sub-G1 peak and the positivity for Annexin V.After 24 hours of treatment, TG led to a significantly highernumber of Annexin V–positive cells compared with theiruntreated controls (Fig. 4D; P < 0.005). Additionally, thepercentage of cells exhibiting cleaved caspase-3 after 24 hoursand a sub-G1 peak was also higher in TG-treated lymphomacells compared with their untreated controls (Fig. 4E, P <0.008). Finally, to investigate whether the observed apoptoticeffects are specific to TG action via induction of NR4A3, siRNA-mediated silencing was performed in SuDHL4 and in U2932followed by TG treatment. Silencing of NR4A3 entirely abro-gated the apoptotic effects of TG (Fig. 4F–H, P < 0.01).Reduction of apoptosis was also observed after 24, 48, and72 hours TG treatment inNR4A3-silenced lymphoma cells (Fig.4I, P < 0.01). Additionally, the number of viable cells signif-icantly increased in NR4A3-silenced lymphoma cells in com-parison with the respective scramble controls, in which the

number of viable cells decreased under TG treatment (Fig. 4J, P< 0.001). After 72-hour treatment, the number of viable cells ofscramble controls were just 4.2% for U2932 and 1.8% forSuDHL4 of the NR4A3-silenced lymphoma cells comparedwith scramble controls.

Furthermore, we identified an agent that induced NR4A3 inaggressive lymphoma cell lines (Fig. 5A and 5B). Similar to TGtreatment, BF175—a pinacolyl boronate-substituted stilbenederivate (11, 12)—treatment resulted in concentration-depen-dent growth inhibition of Karpas-422, RI-1, and U2932 cells(Fig. 5C). Additionally, treatment with 1.0 � 10�5 mol/L ofBF175 induced NR4A3 expression (Fig. 5A and 5B, P < 0.01)accompanied by higher Annexin V positivity (Fig. 5D, P <0.008) and by an increased cleavage of caspase-3 and signifi-cantly higher sub-G1 peak (Fig. 5E, P < 0.01) in BF175-treatedcells compared with untreated controls. Furthermore, silencingof NR4A3 in U2932 inhibited the apoptotic effects of BF175(Fig. 5F–5H). Diminished apoptosis was also observed after 24,48, and 72 hours of BF175 treatment in NR4A3-silencedlymphoma cells (Fig. 5I, P < 0.009). Likewise, the number ofviable cells significantly increased in NR4A3-silenced lympho-ma cells compared with controls, which showed a decreasednumber of viable cells under BF175 treatment (Fig. 5LJ, P <0.001). After 72-hour treatment, the number of viable cells ofscramble controls was just 1.8% of the NR4A3-silenced lym-phoma cells.

Importantly, NR4A1 mRNA expression levels were alsoinduced by TG (Supplementary Fig. S1) and BF175 (Supplemen-tary Fig. S2) treatment. However, the extent of NR4A1 inductionwas remarkably lower compared with NR4A3.

Together, these data indicate that the apoptotic effects of TGand BF175 in aggressive lymphoma cells are NR4A3 mediated.

NR4A1 and NR4A3 possess proapoptotic properties andregulate proapoptotic genes to a similar extent in aggressivelymphoma cells

NR4A1 andNR4A3were separately overexpressed in aggressivelymphoma cell lines (Karpas-422, SuDHL4, RI-1, and U2932)followed by expression analysis and various apoptotic assays. Asshown in Fig. 6A and B, NR4A1 and NR4A3 were significantlyoverexpressed at similar levels in the respective cell lines (P <0.003). A marked increased Annexin V positivity was detectedafter 48 hours in all cell lines caused by NR4A1 overexpression

Figure 4.NR4A3 is necessary for TG-mediated apoptosis in several lymphoma cell lines. A, Estimation of cell growth by the MTS assay of Karpas-422 and SuDHL4as GCB-DLBCL lymphoma cell lines, RI-1 and U2392 as ABC-DLBCL lymphoma cell lines 72 hours after TG treatment in a range from 1� 10�1 to 1� 10�7 mol/L. Cellgrowth was estimated by using the MTS assay in comparison with each cell line treated with DMSO as a vehicle control. B, NR4A3 mRNA expression analysis inKarpas-422, SuDHL4, RI-1, and U2392 after treatment with 10 mmol/L TG or DMSO. Relative expression levels were calculated in comparison to each cell linetreated with DMSO as vehicle control. Each bar represents the mean values of expression levels� SD. C,Western blot analysis of NR4A3 expression of Karpas-422,SuDHL4, RI-1, andU2392 after treatment for 24hourswith 10mmol/L TGorDMSO.D–E,Apoptosis assays inKarpas-422, SuDHL4, RI-1, andU2392 after treatmentwith10 mmol/L TG to assess the apoptotic effects of TG, Annexin V staining (D), and determination of the percentage of cleaved caspase-3 and sub-G1 peak (E) wereperformed by using FACS analysis with specific fluorophore labeled peptides or antibodies and cell-cycle distribution, respectively. Each bar represents the meanvalues of expression levels � SD for analyses for cleaved caspase-3 and sub-G1 peak. F, NR4A3mRNA expression analysis of NR4A3-silenced SuDHL4 and U2932lymphoma cells, and scramble control 24 hours after 10 mmol/L TG treatment. Relative expression levels were calculated in comparison with scramble transfectedcells treated with DMSO as vehicle control. G, Western blot analysis of NR4A3 expression of NR4A3-silenced SuDHL4 and U2932 lymphoma cells, and scramblecontrol 24 hours after 10 mmol/L TG treatment. SuDHL4 and U2932 cells were silenced with two different siRNA targetingNR4A3 termed siNR4A3-2 and siNR4A3-5,or scramble control—termed SCR. Each bar represents the mean values of expression levels � SD. H, Annexin V staining of SuDHL4 and U2932 lymphoma cellssilencedwithNR4A3-specific siRNAand vector control 24 hours after TG treatment. I,Percentage of Annexin V–positive SuDHL4 andU2932 lymphoma cells silencedwithNR4A3-specific siRNA and vector control under TG treatment after 24, 48, and 72 hours. Annexin V stainingwas performed by using FACS analysis with specificfluorophore-labeled peptides. J, Number of viable SuDHL4 and U2932 lymphoma cells silenced with NR4A3-specific siRNA and vector control under TG treatmentafter 24, 48, and 72 hours. � , significant difference (P < 0.05).






Figure 5.

A novel NR4A3-inducing substance, BF175, induces apoptosis in an NR4A3-dependent manner. A, NR4A3 mRNA expression analysis in Karpas-422, RI-1, andU2392 after treatment with 10 mmol/L BF175 or DMSO. Relative expression levels were calculated in comparison with each cell line treated with DMSO asa vehicle control. Each bar represents the mean values of expression levels � SD. (Continued on the following page.)


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(Fig. 6C; P < 0.01) and by NR4A3 overexpression (Fig. 6C, P <0.01). A similar increase in caspase-3/7 activity (Fig. 6D, P < 0.01)and sub-G1 peak positive cells (Fig. 6E, P < 0.01) was detected forNR4A1 and NR4A3 overexpression in all tested cell lines.

LMB treatment of NR4A1 overexpressing SuDHL4 (pEZ-M46NR4A1) and its controls (pEZ-M46 empty) did not influenceapoptosis estimated by Annexin V staining (59% vs. 60.3% forpEZ-M46NR4A1with andwithout LMB, Fig. 6F andG, P¼ 0.73),suggesting that the proapoptotic property of NR4A1 is mediatedby its nuclear function.

To confirm the specificity of NR4A1-mediated apoptosis, wetransfected SN1 III cells, which carry an inducible NR4A1 con-struct (Tet off -pLVX) and were previously described by our group(7), with siRNA targetingNR4A1. The apoptosis rate estimated byAnnexin V staining of NR4A1 silenced lymphoma cells wassignificantly diminished compared with scramble controls(6.6% vs. 52.8%; Fig. 6H and I, P < 0.001).

Expression analysis of potential NR4A apoptotic and anti-apoptotic target genes demonstrated that NR4A1 and NR4A3overexpression caused a similar strong induction of proapop-totic Puma, TRAIL, BID, BIK, isoform 1 and 6 of Bim and BAK(Fig. 7, P < 0.001 for all six genes), whereas expression levels oftheir inhibitors (BCL2, BCLX, and MCL1), and their receptors(Fas, DR4, and DR5) remained unchanged after 48 hours (datanot shown).

NR4A1 and NR4A3 positively correlate with proapoptoticgenes in primary aggressive lymphoma and in othermalignancies

For 82 patients diagnosed with aggressive lymphomas (31non–GCB-DLBCL, 18 GCB-DLBCL and 33 follicular lymphomagrade 3)—exhibiting reduced NR4A1 and NR4A3 expressionlevels as previously described by us (7)—expression levels of pro-and antiapoptotic genes were determined. A significant positivecorrelation of Puma (Spearman r¼ 0.460 forNR4A1 and Pearsonr ¼ 0.485 for NR4A3, P < 0.001; Supplementary Figs. S3A andS3B), isoform1 and 6 ofBim (Spearman r¼ 0.415 forNR4A1 andSpearman r ¼ 0.636 for NR4A3, P < 0.001, Supplementary Figs.S3C and S3D) and Trail (Spearman r ¼ 0.492 for NR4A1 andSpearman r ¼ 0.502 for NR4A3, P < 0.001, Supplementary Figs.S3E and S3F) were detected. These data suggest that NR4A3 andNR4A1 possess a redundant function by regulating proapoptoticgenes to a similar extent.

To investigate whether NR4A1 and NR4A3 correlate with BAK,Puma, BIM, BID, and Trail, we performed database retrieval viaThe Cancer Genome Atlas (TCGA) and analyzed 12 differenttumor entities, which containedmore than 90 patients per group.

All together, we were able to analyze 4,788 patient specimens. Astatistical significant correlation between NR4A1 and NR4A3expression could be shown in 10 of 12 tumor entities (Supple-mentary Table S4). Almost all analyzed proapoptotic genes sig-nificantly correlated with NR4A1 and NR4A3 expression in AMLand colorectal adenocarcinoma (Supplementary Tables S4 andS5). Additionally, in low-grade glioma, all apoptotic genes cor-related with NR4A3 expression (Supplementary Table S5). In theother cancer entities, a marked correlation of single proapoptoticgenes to NR4A1 and/or NR4A3 was observed (SupplementaryTables S4 and S5). However, the correlation coefficients were byfar lower. These data show that NR4A3 is functionally redundantto NR4A1 in many different cancer entities and that both NR4Aspossess a proapoptotic function at least in AML, colorectal ade-nocarcinoma, and low-grade glioma.

DiscussionThis study was designed to investigate the function of NR4A3

in aggressive lymphomas and to study whether NR4A3 hastumor suppressive properties. It was reported that NR4A3expression levels correlated positively with therapeutic successin whole-genome expression analysis of 58 DLBCL patients (8)and that NR4A3 plays an important role in tumorigenesis (1, 5,6, 19–21). Although we described a downregulation of NR4A3together with NR4A1 in aggressive lymphomas (7), the func-tion of NR4A3 in aggressive lymphomas is unknown. In ourstudy, a significant association of low NR4A3 expression andpoor survival indicated a tumor suppressive role of this proteinin human lymphoma samples. To test this hypothesis, our invitro experiments showed that overexpression of NR4A3 led toan induction of apoptosis in several aggressive lymphoma cellmodels. Likewise, xenograft experiments demonstrated thatNR4A3 induction completely suppressed lymphoma forma-tion. Until now, proapoptotic effects of NR4A3 have beenexclusively described in T cells (9, 16), AML cells (22, 23),and breast cancer cell lines (24); however, data on the under-lying mechanisms are lacking. Based on the functional redun-dancy of NR4A3 to NR4A1 (9) shown in other cell types, it ispossible that either NR4A3 transactivates proapoptotic genessimilar to NR4A1 (16) or it translocates to the mitochondria,leading to BCL2 rearrangement and ultimately to induction ofapoptosis (25–27). As Puma, Trail, Bid, Bik, Bim, and Bak1 wereupregulated by NR4A3 overexpression in aggressive lymphomacell lines and correlated to NR4A3 expression in primarylymphomas, and LMB treatment did not influence the apopto-sis rate in NR4A3-induced SuDHL4 cells, we speculate that the

(Continued.) B, Western blot analysis of NR4A3 expression in Karpas-422, RI-1, and U2392 after 24-hour treatment with 10 mmol/L BF175 or DMSO. C,Estimation of cell growth by the MTS assay of Karpas-422, RI-1, and U2392 72 hours after BF175 treatment in a range from 1� 10�1 to 1� 10�7 mol/L. Cell growthwasestimated by using the MTS assay in comparison with each cell line treated with DMSO as a vehicle control. D–E, Apoptosis assays in Karpas-422, RI-1, andU2392 after 10 mmol/L BF175 treatment. To determine apoptotic effects of BF175, Annexin V staining (D), estimation of the percentage of cleaved caspase-3 by usingFACS analysis with specific fluorophore-labeled peptides or antibodies, and sub-G1 peak (E) determination by using FACS analysis for cell-cycle distribution wereperformed. Eachbar represents themeanvalues of expression levels�SD for analyses for cleaved caspase-3 and sub-G1 peak.F,NR4A3mRNAexpression analysis ofNR4A3-silenced U2932 lymphoma cells and vector control 24 hours after 10 mmol/L BF175 treatment. Relative expression levels were calculated in comparison withscramble-transfected cells treated with DMSO as a vehicle control. G, Western blot analysis of NR4A3 expression of NR4A3-silenced U2932 lymphoma cells andscramble control 24 hours after 10 mmol/L BF175 treatment. U2932 cells were silenced with two different siRNA targetingNR4A3-termed siNR4A3-2 and siNR4A3-5,or scramble control—termed SCR. Each bar represents themean values of expression levels� SD.H,Annexin V staining of U2932 lymphoma cells silenced by siRNAspecific forNR4A3 andvector control 24 hours after BF175 treatment. I,PercentageofAnnexin V–positiveU2932 lymphomacells silencedwithNR4A3-specific siRNAand vector control under BF175 treatment after 24, 48, and 72 hours. Annexin V staining was performed by using FACS analysis with specific fluorophore-labeledpeptides. J, Number of viable U2932 lymphoma cells silenced with NR4A3-specific siRNA and vector control under BF175 treatment after 24, 48, and 72 hours.� , significant difference (P < 0.05).






Figure 6.

NR4A3 and NR4A1 possess a similar function in aggressive lymphoma cells. U2932, Karpas-422, RI-1, and SuDHL4 were transfected with a construct eithercarrying NR4A1 (pEZ-M46 NR4A1), NR4A3 (pEZ-M46 NR4A3), or empty vector (pEZ-M46 empty) and phenotypically analyzed. A, NR4A1 and NR4A3 mRNAexpression analysis 24, 48, or 72 hours after transfection. Relative expression levelswere calculated in comparisonwith vector controls. Each bar represents themeanvalues of expression levels� SD. B,Western blot analysis of NR4A1 and NR4A3 expression 24 hours after transfectionwith the indicated constructs. C–E, Apoptoticassay of transfected lymphoma cells. To determine the apoptotic effects ofNR4A1 andNR4A3 in aggressive lymphoma cells, Annexin V (C), caspase-3/7 activity (D),and sub-G1 peak (E) were estimated. Annexin V staining was estimated by using FACS analysis with specific fluorophore-labeled peptides. The caspase-3/7 activitywas calculated in comparison with vector controls. Sub-G1 peaks were determined by cell-cycle analysis using FACS analysis. F, Annexin V staining of SuDHL4 cellstransfectedwith pEZ-M46 empty or pEZ-M46NR4A1with andwithout LMB after 24 hours.G,Western blot analysis ofNR4A1 expression of SuDHL4 cells transfectedwith pEZ-M46 empty or pEZ-M46 NR4A1 with and without LMB after 24 hours. H,Western blot analysis of NR4A1 expression of SN1 III cells being transfected withsiRNA targeting NR4A1 (siNR4A1-1 and siNR4A1-9) and scramble control under doxycycline withdrawal. I, Annexin V staining of SN1 III cells—carrying an inducibleNR4A1 construct (tet off) as previously reported (7)—being transfected with siRNA targeting NR4A1 (siNR4A1-1 and siNR4A1-9) and scramble control underdoxycycline withdrawal. � , significant difference (P < 0.05).

Deutsch et al.





proapoptotic effects of NR4A3 are achieved by transactivationof proapoptotic genes.

Treatment of aggressive lymphoma cell lines with TG—aNR4A3 inducing agent (10)—effectively induced apoptosis. TGis an endoplasmic reticulum stress inducer with apoptotic effectsin various cells (28–30) and the molecular mechanisms of TG-mediated apoptotic effects are unknown. Importantly, our obser-vation, that silencing of NR4A3 by siRNA reversed the inductionof apoptosis, suggests that TG works, at least in part, throughtranscriptional induction of NR4A3.

Our in vitro experiments, in which aggressive lymphoma celllines were treated with BF175, resulted in induction of NR4A3accompanied by apoptosis. Silencing of NR4A3 abrogated theapoptotic effects of BF175. It was reported that BF175 affects thelipid metabolism, but it did not induce apoptosis in any previ-ously investigated cell line—mainly liver cell lines—even athigher concentrations (11, 12). Thus, it seems that the apoptoticeffect of BF175 is mediated by NR4A3 and is apparently exclu-sively found in aggressive lymphoma cell lines.

Overexpressing either NR4A3 or NR4A1 in aggressive lympho-ma cell lines demonstrated that bothNR4As possess proapoptoticeffects in aggressive lymphomas. NR4A3 has been shown to befunctionally redundant with NR4A1 in T-cell apoptosis (9) andoverexpression of eitherNR4A1 orNR4A3 suppressed growth andsurvival of AML cells (22). Additionally, it was demonstrated thatin AML cells NR4A1 and NR4A3 overexpression caused almost

identical gene expression profiles (22). However, NR4A3 knock-out mice are either embryonically lethal or show an inner eardefect with partial bidirectional circling behavior. In contrast,NR4A1 knockout mice show no obvious phenotype (31–33).Together with the fact thatNR4A3 expression profiles significantlycorrelated with aggressiveness of lymphomas (7, 34), it might behypothesized that NR4A1 and NR4A3 have a functional redun-dancy at least with regard to regulation of apoptosis in aggressivelymphoma cells. Additionally, based on our TCGA analysis, itmight be hypothesized that NR4A3 also shows a tumor suppres-sive function redundant to NR4A1 in other tumor entities likeAML, colorectal adenocarcinoma, and low-grade glioma.

In conclusion, we have demonstrated that NR4A3 has proa-poptotic properties, which are functionally redundant to NR4A1and defineNR4A3 as novel tumor suppressor involved in aggres-sive lymphoma development. Hence, NR4A3 and NR4A3 induc-ing agents are novel promising future targets for drug develop-ment in lymphoma therapy.

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

Authors' ContributionsConception and design: A.J.A. Deutsch, A.M. Krogsdam, P. NeumeisterDevelopment of methodology: A.J.A. Deutsch, B. Rinner, M. Pichler, M.-T.Frisch, A. Prokesch, A.M. Krogsdam, C.Wang, C. Beham-Schmid, P. Neumeister

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Figure 7.

Overexpression of NR4A1 and NR4A3 induces a similar pattern of proapoptotic genes. mRNA expression levels of potential target genes of lymphoma cellstransfectedwith an overexpression construct either carryingNR4A1, NR4A3, or empty vector after 48hours. Relative expression levelswere calculated in comparisonwith vector controls. Each bar represents the mean values of expression levels � SD. � , significant difference (P < 0.05).






Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.J.A. Deutsch, B. Rinner, M. Pichler, M. Bischof,K. Wenzl, C. Beham-Schmid, P. NeumeisterAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.J.A. Deutsch, S. Hatzl, J. Feichtinger, K. Wenzl,H. Sill, G.G. Thallinger, H.T. Greinix, C. Beham-Schmid, P. NeumeisterWriting, review, and/or revision of the manuscript: A.J.A. Deutsch, B. Rinner,M. Pichler, K. Troppan, K. Fechter, J. Feichtinger, K. Wenzl, A. Prokesch,G.G. Thallinger, H.T. Greinix, C. Wang, P. NeumeisterAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases):A.J.A. Deutsch, B. Rinner, K. Troppan, K. Fechter,V. Stiegelbauer, A. Prokesch, A.M. Krogsdam, P. NeumeisterStudy supervision: A.J.A. Deutsch, P. Neumeister

Grant SupportP. Neumeister was supported by grants of Fellinger Krebsforschung, Land

Steiermark, Jubil€aumsfond der €ONB (N11181) and the OMICS Center Grazgrant of the Austrian Ministry for Science, Research and Economy (G.G.Thallinger). A.J.A. Deutsch was supported by the START-Funding-Program ofthe Medical University of Graz and by a research grant of MEFOgraz.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 25, 2016; revised October 12, 2016; accepted February 22,2017; published OnlineFirst March 1, 2017.

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