bi1071, a novel nur77 modulator, induces apoptosis of cancer … · nur77-bcl-2 apoptotic pathway...

Small Molecule Therapeutics

BI1071, a Novel Nur77 Modulator, InducesApoptosis of Cancer Cells by Activating theNur77-Bcl-2 Apoptotic PathwayXiaohui Chen1, Xihua Cao2, Xuhuang Tu1, Gulimiran Alitongbieke1, Zebin Xia2,Xiaotong Li1, Ziwen Chen1, Meimei Yin, Dan Xu1, Shangjie Guo1, Zongxi Li1,Liqun Chen1, Xindao Zhang1, Dingyu Xu1, Meichun Gao1, Jie Liu1, Zhiping Zeng1,Hu Zhou1, Ying Su2, and Xiao-kun Zhang1,2

Abstract

Nur77 (also called TR3 or NGFI-B), an orphan member ofthe nuclear receptor superfamily, induces apoptosis by trans-locating to mitochondria where it interacts with Bcl-2 toconvert Bcl-2 from an antiapoptotic to a pro-apoptotic mol-ecule. Nur77 posttranslational modification such as phos-phorylation has been shown to induce Nur77 translocationfrom the nucleus to mitochondria. However, small moleculesthat can bind directly to Nur77 to trigger its mitochondriallocalization and Bcl-2 interaction remain to be explored. Here,we report our identification and characterization of DIM-C-pPhCF3

þMeSO3� (BI1071), an oxidized product derived from

indole-3-carbinol metabolite, as a modulator of the Nur77-Bcl-2 apoptotic pathway. BI1071 binds Nur77 with highaffinity, promotes Nur77 mitochondrial targeting and inter-action with Bcl-2, and effectively induces apoptosis of cancercells in a Nur77- and Bcl-2–dependent manner. Studies withanimal model showed that BI1071 potently inhibited thegrowth of tumor cells in animals through its induction ofapoptosis. Our results identify BI1071 as a novel Nur77-binding modulator of the Nur77-Bcl-2 apoptotic pathway,which may serve as a promising lead for treating cancers withoverexpression of Bcl-2.

IntroductionNur77 (NR4A1; also known as NGFI-B and TR3) is perhaps

the most potent apoptotic member of the nuclear receptorsuperfamily (1–8). The death effect of Nur77 was initiallyrecognized during studying the apoptosis of immature thymo-cytes, T-cell hybridomas (9, 10). Later, we found that Nur77mediates the death effect of the retinoid-related moleculeAHPN (also called CD437) in cancer cells (11). Furthermore,we discovered a nongenomic action of Nur77, in which Nur77migrates from the nucleus to the cytoplasm, where it targetsmitochondria to trigger cytochrome c release and apoptosis incancer cells (12–14). Further studies demonstrated in variouscancer types that such a Nur77 mitochondrial apoptotic path-way is characterized by its interaction with Bcl-2 and theconversion of Bcl-2 from an antiapoptotic molecule to apro-apoptotic molecule (6, 15). Given the pivotal role of

Bcl-2 in regulating the apoptosis of cancer cells and in theresistance of cancer cells to a variety of radio- and chemother-apeutic agents, understanding how the Nur77-Bcl-2 apoptoticpathway is regulated and discovering its small-molecule mod-ulators may offer new strategies to develop effective cancertherapeutics. However, small molecules that can activate theNur77-Bcl-2 apoptotic pathway by binding to Nur77 to triggerNur77 mitochondrial translocation and interaction with Bcl-2have not been reported.

As an orphan nuclear receptor, Nur77 lacks a canonicalligand-binding pocket (LBP; refs. 16, 17), which excludes smallmolecules from binding to Nur77 to regulate Nur77 functionsvia the canonical LBP-binding mechanism. Recent advance hasrevealed the existence of alternate small-molecule bindingregions on the surface of nuclear receptors, and compoundsthat bind to alternate sites other than LBP have been identifiedfor some nuclear receptors (18, 19), including Nur77 (20–24).These developments inspire us to discover Nur77-bindingcompounds that can regulate the Nur77-Bcl-2 apoptotic path-way. Here, we report that a salt form of a 3,30-diindolymethane(DIM) derivative (di(1H-indol-3-yl)(4-(trifluoromethyl)phe-nyl)methane; named BI1071 here) can bind to Nur77 to induceapoptosis of cancer cells through the Nur77-Bcl-2 apoptoticpathway. BI1071 binds to Nur77 at submicromolar concentra-tion and induces apoptosis that is dependent on the expressionof both Nur77 and Bcl-2. BI1071 also effectively inhibits thegrowth of tumor cells in animals. Moreover, BI1071 binding toNur77 induces not only its mitochondrial targeting but also itsinteraction with Bcl-2. Our results therefore identify BI1071 asthe first Nur77-binding small molecule that promotes theNur77-Bcl-2 apoptotic pathway.

1School of Pharmaceutical Sciences, Fujian Provincial Key Laboratory of Inno-vative Drug Target Research, Xiamen University, Xiamen, China. 2Cancer Center,Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

X. Chen and X. Cao contributed equally to this article.

Corresponding Authors: Ying Su, Sanford Burnham Prebys Medical DiscoveryInstitute, 10901 North Torrey Pines Road, La Jolla, CA 92037; E-maill:[email protected] and Xiao-kun Zhang, Xiamen University, Xiamen, China;E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-18-0918

�2019 American Association for Cancer Research.

MolecularCancerTherapeutics

Mol Cancer Ther; 18(5) May 2019886

on April 21, 2021. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst March 29, 2019; DOI: 10.1158/1535-7163.MCT-18-0918

http://crossmark.crossref.org/dialog/?doi=10.1158/1535-7163.MCT-18-0918&domain=pdf&date_stamp=2019-4-17

http://mct.aacrjournals.org/

Materials and MethodsCell culture

The following cell lines are used in our study. HCT116 coloncancer, MDA-MB-231, HS578T, BT549, MCF-7, and T47D breastcancer, breast epithelial cell line MCF-10A, HeLa ovarian cancer,mouse embryonic fibroblast (MEF) cells andHEK293T embryoniccells were cultured in DMEM, whereas ZR-75-1, HCC1937 breastcancer and SW480 colon cancer were cultured in RPIM-1640medium containing 10% FBS. Human colonic epithelial cells(HCoEpiC) were cultured in colonic epithelial cell medium (CoE-piCM, Cat. #2951). Cell lines HCT116 (ATCC, CCL-247), SW480(ATCC, CCL-228), and HEK293T (ATCC, CRL-11268) wereobtained from the ATCC. Cell lines MCF-10A (SCSP-660),MDA-MB-231 (SCSP-5043), HeLa (TCHu187), HS578T(TCHu127), BT549 (TCHu 93), MCF-7 (SCSP-531), ZR-75-1(TCHu126), T47D (TCHu 87), and HCC1937 (TCHu148) wereobtained fromChinese Academyof Science Shanghai Cell Bank onDecember 09, 2016. Cell line HCoEpiC was obtained from Scien-ceCell (Cat. #2950) on October 05, 2018. MEF cells were isolatedfromembryonicday13wild-type (WT)andNur77knockout (KO)mice. The cells were grown in the cell incubator with 5% CO2 at37�C. Sub-confluent cells with exponential growth were usedthroughout the experiments. Cells plated onto cell culture dishesand kept in 10%FBS for 24 hourswere treatedwith compounds ortransfected with plasmids. Cell transfection was carried out byusing Lipofectamin 2000 according to the manufacturer's instruc-tion. The cellswere tested byusingMycoplasmaHoechst Stain Assaykit (Beyotime, C0296) every 6 months. We added the Hoechestsolution to stain the cellswith50%densityat roomtemperature for30 minutes and then used the confocal microscope to observe thecells. In the cells without Mycoplasma infection, only the bluefluorescence of the nucleus was observed. Filamentous blue fluo-rescence can be observed around the nucleus in Mycoplasma con-taminated cell samples. The cells were prevented fromMycoplasmainfection by using plasmocin (Invivogen, ant-mpt). No positiveMycoplasma tests was observed during the time of our experiments.

PlasmidsPlasmids pcmv-myc-Nur77, GFP-Nur77, GFP-Nur77/LBD,

GST-Bcl-2, pcmv-myc-Bcl-2, Flag-cmv-Bcl-2 were constructed asdescribed (12–14, 24). Plasmids pcmv-myc-Nur77/H372D,pcmv-myc-Nur77/H372A, pcmv-myc-Nur77/Y453L, pcmv-myc-Nur77/C566K were constructed by using the PCR and Quick-Chang mutagenesis kit.

Antibodies and reagentsAnti-Ki67 (Cat. ab15580) and anti-Hsp60 (Cat. ab46798)

antibodies were purchased from Abcam (UK); anti–b-actin (Cat.4970S), anti-Cleaved caspase-3 (Cat. 9661S), and anti-Nur77(Cat. 3960S) antibodies were purchased from Cell Signal Tech-nology; anti-Myc (9E10; Cat. Sc-40), anti-Nur77 (M-210; Cat.sc-5569), anti-PCNA (SantaCruzBiotechnology sc-7907), anti–a-tubulin (Santa Cruz Biotechnology sc-8035), anti-Bcl-2 (SantaCruz Biotechnology sc-783), anti-PARP (Santa Cruz Biotechnol-ogy sc-7150), and anti-GST (sc-138) antibodies were purchasedfrom Santa Cruz Biotechnology (Santa Cruz Biotechnology);anti-Flag (Cat.F1804) antibody was purchased from Sigma;Mito-tracker deep red (Cat. M22426), JC-1 Probe (Cat. T3168)and mitoSOX Red Mitochondrial Superoxide Indicator (Cat.M36008) were purchased from Thermo Fisher.

Generation of Nur77 and Bcl-2 knockout cells by CRISPR/Cas9system

Knocking out Nur77 and Bcl-2 from HeLa cells used theCRISPR/Cas9 system. gRNA targeting sequence of Nur77 (50-ACCTTCATGGACGGCTACAC-30) and Bcl-2 (50-GAGAACAGGG-TACGATAACC-30) was cloned into gRNA cloning vector Px330(Addgene, 71707) and confirmed by sequencing. The accessionnumbers of Nur77 and Bcl-2 are NM-001202233 andNM-000633.2 respectively. To screen for cells lacking Nur77 orBcl-2, HeLa cells were transfected with control vector and gRNAexpression vectors, followed by G418 selection (0.5 mg/mL).Single colonies were subjected to Western blotting using anti-Nur77 and anti–Bcl-2 antibody to select knockout cells.

Cell viability determination and cell death assayCell viability was analyzed by using colorimetric 3-(4,5-

dimethylthiazol-dimethylthiazol-2-yl)-2,5-diphenyletetrazoliumBromide (MTT) assay as described previously (12–14, 24).

Mammalian one hybrid assayHEK293T cellswere co-transfectedwith pG5Luciferase reporter

together with the plasmid encoding RXRa-LBD fused with theGal4 DNA-binding domain and other expression plasmids asdescribed previously (18, 25). After transfection, cells were treatedwith DMSO or BI1071, and assayed by using the Dual-LuciferaseReporter Assay System (Promega). Transfection efficiency wasnormalized to Renilla luciferase activity.

Cell fractionationFor cellular fractionation (12–14, 24), cells were lysed in cold

buffer A (10 mmol/L HEPES-KOH (pH 7.9), 1.5 mmol/L MgCl2,10 mmol/L KCl, 0.5 mmol/L dithiothreitol) with a cocktail ofproteinase inhibitors on ice for 10 minutes as described previ-ously. Cytoplasmic fractionwas collected by centrifuging at 6,000rpm for 10minutes. Pellets containing nuclei were resuspended incold high-salt buffer C (20 mmol/L HEPES-KOH (pH 7.9), 25%glycerol, 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/LEDTA, 0.5 mmol/L dithiothreitol) with a cocktail of proteinaseinhibitors on ice for 30 minutes.

GST-pull downGST or GST-Bcl-2 fusion protein (0.5mg) was immobilized on

glutathione-Sepharose beads and incubated with purified His-Nur77-LBD (0.2mg) in the presence of different concentration ofBI1071 as described previously (12–14, 24). Bound Nur77-LBDwas analyzed by Western blotting.

Western blotting and immunoprecipitationWestern blotting and co-immunoprecipitation (co-IP) were

performed as described (12–14, 24).

Generation of Nur77 and Bcl-2 knockout cells by CRISPR/Cas9system

Knocking out Nur77 and Bcl-2 from HeLa cells used theCRISPR/Cas9 system. gRNA targeting sequence of Nur77 (50-ACCTTCATGGACGGCTACAC-30) and Bcl-2 (50-GAGAACAGGG-TACGATAACC-30) was cloned into gRNA cloning vector Px330(Addgene, 71707) and confirmed by sequencing. To screen forcells lacking Nur77 or Bcl-2, HeLa cells were transfected withcontrol vector and gRNA expression vectors, followed by G418selection (0.5 mg/mL). Single colonies were subjected toWestern

A New Activator of the Nur77-Bcl-2 Apoptotic Pathway

www.aacrjournals.org Mol Cancer Ther; 18(5) May 2019 887




blotting using anti-Nur77 and anti–Bcl-2 antibody to selectknockout cells.

Apoptosis assayCells were placed on 6-well plates with a density of 1� 106 per

well. After 24 hours, the cells treated with different concentrationof BI1071 for 6 hours, and then the suspended and the adherentcells were collected, stained with Annexin V-FITC for 10 minutesand with propidium iodide for 5 minutes, and analyzed imme-diately by cytoFLEX Flow Cytometry System (Beckman-Coulter)using FITC and PC5.5.

Determination of Dcm and ROSThe determination of Dym and ROS was performed as previ-

ously described elsewhere (26). JC-1 probe was used to measuremitochondrial depolarization in cells. Cells were first treated withdifferent concentration of BI1071 for 6 hours and then followedwith the addition of JC-1 staining solution (5 mg/mL) for 20minutes at 37�C. After washing with PBS twice, mitochondrialmembrane potentials were analyzed immediately by cytoFLEXFlow Cytometry System using FITC and PE. Mitochondrial depo-larization was measured by a change in the ratio of green/redfluorescence intensity. ROSwasmonitoredwith themitoSOXRedMitochondrial Superoxide Indicator and analyzed by cytoFLEXFlow Cytometry System using PE.

ImmunostainingCells were fixed in 4% paraformaldehyde. For mitochondrial

staining, cells were incubated with anti-Hsp60 goat immuno-globulin G (IgG; Santa Cruz Biotechnology), followed by anti-goat IgG conjugatedwith Cy3. The nuclei were visualized byDAPIstaining. Fluorescent imageswere collected and analyzed by usinga fluorescence microscopy or MRC-1024 MP laser-scanning con-focal microscope (Bio-Rad).

Animal studiesThe protocols for animal studies were approved by the Animal

Care andUse Committee of XiamenUniversity, and all mice werehandled in accordance with the "Guide for the Care and Use ofLaboratory Animals" and the "Principles for the Utilization andCare of Vertebrate Animals." ForMMTV–PyMTmice breast cancermodel, femaleMMTV-PyMTmiceof 12-weeks-oldwere randomlydivided into twogroups (n¼7each), treatedwith adaily oral doseof BI1071 (5 mg/kg) for 18 days. Standard histopathologicalanalysis of tumor tissue was performed. BI1071 was dissolvedin DMSO and diluted with normal saline containing 5.0% (V/V)Tween-80 to a final concentration 0.5mg/mL. Normal saline withDMSO and 5.0% Tween-80 was used as the vehicle control. Forxenograft nude mouse study, male BALB/c nude mice (6-weeks-old) were subcutaneously injected with log growth-phase ofSW620 cells (1 � 106 cells in 0.1 mL PBS). Mice were treatedorally after 7days of transplantationwithBI1071once aday. Bodyweight and tumor size were measured every 3 days. Tumors weremeasured andweighted. Tissues isolated from the nudemicewerefixed with 4% paraformaldehyde. TdT-mediated dUTP nick endlabeling assay was performed according to the manufacturer'sinstructions (In situ Cell Death Detection Kit; Roche).

ImmunohistochemistryFour-mm-thick sections were deparaffinized and rehydrated

using xylene and a graded series of ethanol (100%, 95%, 85%,

75%, 50%), followed by washing in PBS. Antigen retrieval wasperformed in 10 mmol/L sodium citrate buffer (pH 6.0), whichwas microwaved at 100�C for 20 minutes. After rinsed twice inPBS, sections were blocked at room temperature for 1 hour byusing 10% normal goat serum, followed by incubation with anti-ki67, anti-cleaved caspase 3 overnight at 4�C. Colors were devel-oped with a DAB horseradish peroxidase color development kit.

Docking experimentsSchrodinger's (www.schrodinger.com)Glide (27), a grid-based

docking programwas used for the docking study of BI1071 to theprotein. The crystal structure of Nur77-LBD in complex with acytosporone B analog (Protein Data Bank code 3V3Q) was used.Docking was performed with the implemented standard routinein Glide. The Glide GScore was used as docking score to rank thedocking results. Poses were further visually investigated to checkfor their interactions with the protein in the docking site.Schr€odinger's Maestro was used as the primary graphical userinterfaces for the visualization of the crystal structure and dockingresults.

Surface plasmon resonanceThe binding kinetics between Nur77-LBD and compounds

were performed on a BIAcore T200 instrument (GE Healthcare)at 25�C (24). Nur77-LBD were diluted to 0.05 mg/mL in 50mmol/L NaOAc (pH 5.0) and immobilized on a CM5 sensor chip(GE Healthcare) by amine coupling at densities approximately10,000 RU according to themanufacturer's instructions. Gradientconcentrations of compounds were injected into the flow cells inrunning buffer (PBS, 0.4%DMSO) at a flow rate of 30 mL/min for150 seconds of association phase followed by a 420 secondsdissociation phase and a 30 seconds regeneration phase(10 mmol/L Glycin-HCl, pH 2.5). The data were analyzed usingBIAcore T200 Evaluation Software 2.0 and referenced for blankinjections and reference Surface. The dissociation constant (Kd)was fitted to the standard 1:1 interaction model and calculatedusing the global fitting of the kinetic data from gradientconcentrations.

Statistical analysisData were expressed as mean� SD. Each assay was repeated in

triplicate in three independent experiments. The statistical signif-icance of the differences among the means of several groups wasdetermined using the Student t test.

ResultsA salt form of DIM-C-pPhCF3 exhibits superior apoptotic effect

In our effort to identify small molecules that modulate theNur77/Bcl-2 apoptotic pathway, we evaluated an in-house com-pound library, which includes di(1H-indol-3-yl)(4-(trifluoro-methyl)phenyl)methane (DIM-C-pPhCF3, Fig. 1A; ref. 28). Wesurprisingly observed that the freshly prepared DIM-C-pPhCF3solution was not as active as the aged solution in apoptosisinduction. In addition, the freshly made DIM-C-pPhCF3 solutionis colorless, however, it turns reddishwhen it is aged or exposed toair at room temperature. Thus, we presumed that the compoundunderwent oxidization and the oxidized DIM-C-pPhCF3 wasmore active than DIM-C-pPhCF3. To test our hypothesis, DIM-C-pPhCF3 was oxidized in the presence of methanesulfonicacid, and the products were subsequently purified to obtain the

Chen et al.

Mol Cancer Ther; 18(5) May 2019 Molecular Cancer Therapeutics888



www.schrodinger.com


Figure 1.

A salt form of DIM-C-pPhCF3 exhibits superior apoptotic effect. A, Structure of DIM-C-pPhCF3 and DIM-C-pPhCF3þMeSO3

� (BI1071). B, HCT116 cells treated withthe indicated concentration of BI1071 or DIM-C-pPhCF3 (labeled as CF3) for 3 days were assessed by MTT assay. Data are shown asmean� SD (n¼ 6). C, HCT116cells treated with the indicated concentration of BI1071 for 6 hours were analyzed for PARP cleavage byWestern blotting. D, HCT116 cells treated with 0.5 mmol/LBI1071 for 6 hours were visualized by DAPI staining. Apoptotic cells were counted in 200 cells. E, Level of PARP cleavage and cleaved caspase 3 in MDA-MB-231cells treated with the indicated concentration of BI1071 for 6 hours was determined byWestern blotting. F, Annexin V/PI staining of MDA-MB-231 cells treatedwith the indicated concentration of BI1071 for 6 hours was analyzed by flow cytometry. G,MDA-MB-231 cells treated with the indicated concentration of BI1071for 6 hours were stained with JC-1. Aggregated JC-1, red fluorescence (PE), and monomeric JC-1, green fluorescence (FITC), were measured by flow cytometry.Statistical data were mean� SEM of five independent images. � , P < 0.1; �� , P < 0.001 (Student t test). H,Mitochondrial ROS production in MDA-MB-231 treatedwith the indicated concentration of BI1071 for 6 hours was analyzed by flow cytometry. ForWestern blots and flow cytometry experiments, one of three similarexperiments is shown.






oxidized DIM-C-pPhCF3: di((1H-indol-3-yl)(4-trifluoromethyl-phenyl)methylium methanesulfonate (DIM-C-pPhCF3

þMeSO3�;

BI1071, Fig. 1A; Supplementary Methods for synthesis and puri-fication). BI1071 was then tested in comparison with DIM-C-pPhCF3 for growth inhibition and apoptosis induction. Figure1B showed that BI1071 inhibited the growth of HCT116 coloncancer cells with an IC50 of 0.06 mmol/L, which is about 25-foldmore active than DIM-C-pPhCF3 (IC50 ¼ 1.5 mmol/L). Treatmentof MDA-MB-231 cells with 0.5 mmol/L BI1071 for 6 hours effec-tively induced PARP cleavage, an indication of apoptosis, whileDIM-C-pPhCF3 had no effect under the same condition (Supple-mentary Fig. S1A). Dose-dependent study demonstrated thatBI1071 could induce PARP cleavage at submicromolar concen-trations in HCT116 cells (Fig. 1C) and other cancer cell lines(Supplementary Fig. S1B).

Interestingly, BI1071 was effective in various breast cancer celllines analyzed regardless of its hormone dependency (Supple-mentary Fig. S2). Furthermore, BI1071 did not display apoptoticeffect in the non-transformed mammary and normal colon cells(Supplementary Fig. S3A and S3B), indicating that BI1071 selec-tively induces apoptosis in cancer cells. The apoptotic effect ofBI1071 was also confirmed by its induction of extensive nuclearcondensation and fragmentation revealed by DAPI staining incells treated with 0.5 mmol/L BI1071 for 6 hours (Fig. 1D; Sup-plementary Fig. S1C) and confirmed as well by PARP and caspase3 cleavage assays (Fig. 1E). The effect of BI1071 on cell death wasfurther assessed using flow cytometry-based Annexin V/ Propi-dium iodide (PI) apoptosis assay. Dose-dependent study showedthat about 31.63% of MDA-MB-231 cells were apoptotic whentreated with 1 mmol/L of BI1071 for 6 hours, whereas only 1.31%of cells were apoptotic in vehicle control cells (Fig. 1F).

Loss of mitochondrial membrane potential (Dcm) representsone of the hallmarks of apoptosis. To assess whether the BI1071-induced apoptosis was related to the intrinsic mitochondrialpathway, we used JC-1, the mitochondrial-specific dye, to mon-itor the changes of mitochondrial membrane potential (29).MDA-MB-231 breast cancer cells treatedwith BI1071were stainedwith JC-1. JC-1 dye accumulation in mitochondria is dependentof mitochondrial membrane potential, accompanied by a shift ofJC-1 fluorescence emission from green to red. In comparison withhealthy cells, apoptotic cells display an increase in the green/redfluorescence intensity ratio. Analysis of both red and green fluo-rescence emissions by flow cytometry revealed a dose-dependentBI1071 induction ofmitochondrial membrane dysfunction. Aftertreatment with 1 mmol/L of BI1071 for 6 hours, the green to redratio increased from 100% to 345% (Fig. 1G). Mitochondrialdysfunction was also revealed by marked increase in intracellularmitochondrial reactive oxygen species (mito-ROS) in MDA-MB-231 cells exposed to BI1071 in a dose-dependent manner(Fig. 1H). Collectively, these data suggested that BI1071 inducedmitochondrion-related apoptosis in cancer cells.

BI1071 inhibits the growth of tumor cells in vivoTo assess the apoptotic effect of BI1071 in animals, SW620

colon cancer cells were inoculated subcutaneously in the rightand left hind-side flank of nude mice. Administration of tumor-bearing nude mice with BI1071 inhibited the growth of SW620xenograft tumor in a dose- and time-dependent manner (Fig. 2A–C). TUNEL assay revealed extensive apoptosis in BI1071-treatedtumor specimens as compared with control tumor (Fig. 2D).MMTV-PyMT-transgenic mouse model of breast cancer was also

used to evaluate the anticancer effect of BI1071. Administration ofthe MMTV-PyMT mice with BI1071 (5 mg/kg) potently inhibitedthe growth of PyMT mammary tumor (Fig. 2E and F). Westernblotting of tumor tissues prepared from treated and non-treatedmice revealed that the expression levels of two proliferationmarkers, PCNA and Ki67, were markedly reduced by BI1071(Fig. 2G). Immunostaining also showed a reduced expression ofKi67 and enhanced expression of cleaved caspase 3 in tumortissue specimens prepared from mice treated with BI1071(Fig. 2H). There was not significant difference in the body weight(without tumor weight) between the control mice and theBI1071-treated mice in both animal models (Supplementary Fig.S4A and S4B). These data demonstrated that BI1071 potentlyinhibited the growth of tumor cells in animals through its induc-tion of apoptosis.

BI1071 induces Nur77-dependent apoptosis and Nur77mitochondrial targeting

We next determined whether BI1071-induced apoptosiswas Nur77-dependent by examining its apoptotic effect inmouse embryonic fibroblast (MEF) and MEF lacking Nur77(Nur77�/�MEF). BI1071 dose-dependently inhibited the growthof MEFs, but such an inhibitory effect was significantly dimin-ished in Nur77�/�MEFs (Fig. 3A). Induction of PARP cleavage inMEFs by BI1071 was also attenuated in Nur77�/�MEFs (Fig. 3B).The death effect of BI1071 was also evaluated in Nur77 genomeknockout HeLa cells generated by CRISPR/Cas9 technology.Induction of PARP cleavage and caspase 3 activation by BI1071were strongly suppressed in Nur77�/�HeLa cells (Fig. 3C).Annexin V/PI staining revealed a reduced apoptotic effect ofBI1071 in Nur77�/�HeLa cells than in the parental HeLa cells(from 35.55% to 3.25%; Fig. 3D). Furthermore, unlike the paren-tal HeLa cells, Nur77�/�HeLa cells did not display BI1071-induced mitochondrial membrane potential loss measured byJC-1 staining (Fig. 3E) or BI1071-induced release of mitochon-drial ROS (Fig. 3F). To further address the role of Nur77, wetransfected the ligand-binding domain (LBD) of Nur77, Nur77-LBD, intoHEK293T cells and askedwhether the overexpression ofNur77-LBD could influence the effect of BI1071. Indeed, trans-fection of Nur77-LBD enhanced the killing effect of BI1071, with36% of the transfected HEK293T cells undergoing apoptosis,while 4.5% of the non-transfected cells were apoptotic (Supple-mentary Fig. S5). Together, these results demonstrated thatBI1071 targets Nur77 to induce cancer cell apoptosis.

Our observation that BI1071 induced mitochondria-depen-dent apoptosis prompted us to determinewhether BI1071 exertedits Nur77-dependent apoptosis by promoting Nur77 mitochon-drial targeting. Immunostaining showed that Nur77 was mainlylocalized in the nucleus of HCT116 cells. However, it was pre-dominantly cytoplasmic when cells were treated with 0.5 mmol/Lof BI1071 for 2 hours (Fig. 3G). To confirm the effect of BI1071 onNur77 cytoplasmic localization, HEK293T cells were transfectedwith GFP-Nur77 and subsequently treated with 0.5 mmol/LBI1071. Transfected GFP-Nur77 resided in the nucleus, howeverit was diffusely distributed in both the cytoplasm and nucleusupon BI1071 treatment (Fig. 3H). Cells transfected with GFP-Nur77-LBD also responded well to BI1071. Only for cells treatedwith BI1071, GFP-Nur77-LBD colocalized extensively with themitochondria-specific Hsp60 protein revealed by confocalmicroscopy (Fig. 3I) and co-accumulated with the Hsp60 proteinin the heavy membrane fraction shown by cellular fractionation

Chen et al.





Figure 2.

Effect of BI1071 on tumor growth and apoptosis in animals.A, Nudemice (n¼ 6) injected with SW620 (2� 106 cells) were administered with the indicated doseof BI1071 once a day, and tumors were measured every 3 days. B and C, 12 days after administration of BI1071, nudemice bearing SW620 tumor were sacrificedand tumors were removed, weighted, and showed (�� , P < 0.001, Student t test).D, Representative TUNEL staining images illustrating the apoptotic effect ofBI1071. The apoptotic cells were detected by TUNEL assay in specimens of xenograft tumors. E, Representative images of MMTV–PyMTmammary tumor modelmice and tumors frommice administered with or without BI1071. For MMTV–PyMTmammary tumor model, female wild-type MMTV-PyMT mice that were12 weeks old were randomly divided into two groups (n¼ 7), treated with daily oral doses of BI1071 (5 mg/kg) for 18 days. F, Inhibition of PyMT tumor growth byBI1071. Mice treated with BI1071 as in E, and tumors were weighted (n¼ 7). G,Western blot analysis of the expression of PARP, Ki67, and PCNA in tumor tissuesprepared from 3 MMTV-PyMTmice treated with or without BI1071 (5 mg/kg) for 18 days.H, Representative immunocytochemistry staining showing theexpression of Ki67 and cleaved caspase 3 in tumor tissues prepared fromMMTV-PyMTmice treated with or without BI1071 (5 mg/kg) for 18 days.






Figure 3.

BI1071 induces Nur77-dependent apoptosis and Nur77 mitochondrial targeting. A,MEFs and Nur77�/�MEFs treated with the indicated concentration of BI1071for 6 hours were assessed by MTT assay. (�� , P < 0.01 and ��, P < 0.001, Student t test). B, PARP cleavage in MEFs or Nur77�/�MEFs treated with 0.5 mmol/LBI1071 for 6 hours was determined byWestern blotting. C–F, HeLa or Nur77�/�HeLa cells were treated with 0.5 mmol/L BI1071 for 6 hours. Cells were thensubjected toWestern blotting for PARP cleavage and cleaved caspase 3 detecting (C), Annexin V/PI staining for apoptosis measurement (D), JC-1 staining formeasuring mitochondrial membrane potential (E) or mito-SOX staining for determining the production of mitochondrial ROS (F). One of three similarexperiments is shown. NS, not significant; �� , P < 0.001 (Student t test).G, Subcellular localization of endogenous Nur77 in HCT116 cells treated with 0.5 mmol/LBI1071 for 2 hours was analyzed by confocal microscopy after immunostained with anti-Nur77 antibody. Nuclei were visualized by DAPI staining. H, HeLa cellstransfected with GFP-Nur77 were treated with 0.5 mmol/L BI1071 for 2 hours and visualized by confocal microscopy. I, HeLa cells transfected with GFP-Nur77-LBDwere treated with BI1071 (0.5 mmol/L) for 2 hours were immunostained with anti-Hsp60 antibody and visualized by confocal microscopy. J, HEK293T cellswere transfected with GFP-Nur77-LBD and treated with 0.5 mmol/L BI1071 for 2 hours. Cytosolic (Cyt) and HM fractions were then prepared and analyzed byWestern blotting. Expression of cytoplasmic IkBa andmitochondrial Hsp60 was shown to indicate the purity of cytosolic and mitochondrial fractions,respectively. K, HEK293T cells were transfected with Myc-Nur77. Whole cell lysate (WCL) andmitochondria-enriched heavymembrane (HM) fractions wereprepared from HEK293T cells treated with 0.5 mmol/L BI1071 for 2 hours and analyzed byWestern blotting. Expression of nuclear PARP andmitochondrialHsp60 was shown to ensure the purity of HM fraction. For cellular fractionation experiments, one of three similar experiments is shown.

Chen et al.





(Fig. 3J). The effect of BI1071 on inducing Nur77 mitochondrialtargeting was further illustrated by cellular fractionation expe-riments showing that a significant amount of transfectedMyc-Nur77 accumulated in the mitochondria-enriched heavymembrane (HM) fraction when cells were treated with BI1071(Fig. 3K). Taken together, these data demonstrated that BI1071exerted its Nur77-dependent apoptotic effect by promotingNur77 mitochondrial targeting.

BI1071 binds Nur77 to induce its mitochondrial targeting andapoptosis

Although Nur77 lacks a LBP and no endogenous ligands haveyet been identified (16, 17), recent crystallographic studies haveidentified several regions on the surface of the Nur77 protein assmall-molecule binding regions (20, 22, 30). We therefore deter-mined whether BI1071 binds directly to Nur77 to induce itsmitochondrial targeting and apoptosis. Surface plasmon reso-nance (SPR) analyses revealed that DIM-C-pPhCF3 bound toNur77-LBD with a Kd of 3.0 mmol/L (Fig. 4A) and that BI1071bound to Nur77-LBD protein with a Kd of 0.17 mmol/L (Fig. 4B),which demonstrated that BI1071 binding to Nur77-LBD was 18-fold stronger than DIM-C-pPhCF3. We also evaluated the effect ofBI1071 on the transcriptional activity of Nur77/RXRa-LBD het-erodimer. Co-transfection of pBind-RXRa-LBD and Nur77strongly activate the reporter transcriptional activity when cellswere treatedwith 9-cis-RA, a RXRa ligand (Fig. 4C). BI1071 furtherdose-dependently induced the reporter activity (Fig. 4C), likelydue to its binding toNur77. To exclude the possibility that BI1071acted on RXRa, Glu453 and Glu456 in the activation function 2(AF2) region of RXRa (31) were substituted with Ala and theresulting mutant, RXRa-LBD/E453,6A, was used to repeat thereporter assay. As expected, 9-cis-RA failed to induce the reporteractivity in cells transfected with Nur77 and pBind-RXRa-LBD-E453,6A. However, BI1071 could still activate the reporter genetranscription (Fig. 4C), demonstrating that BI1071 inducedreporter gene transcription throughNur77 binding but not RXRa.We also excluded the possibility of BI1071 binding to othernuclear receptors using reporter assays (Supplementary Fig.S6A). We next employed molecular docking approach to studyhow BI1071 bound to Nur77-LBD. Our docking studies showedthat BI1071 docked better to a binding region formed by helicesH1, H5, H7 and H8, and loops H1-H2, H5-B1 and H7-H8. Thedocked mode also suggested that the indole ring of BI1071 madekey interaction with the side chains of H372 and Y453 locatedin H1 and H5, respectively (Fig. 4D). For comparison, we alsodocked DIM-C-pPhCF3 to the same region. As shown in Fig. 4D,BI1071 fit better to the binding groove with the bis-indolyl ringsembedded deeper in the groove. The bis-indolyl rings of BI1071also was positioned to form p-p interaction with Y453 and tomake stronger interaction with H372. To test this binding mode,H372wasmutated into either Ala or Asp.When tested in the Gal4reporter assay for its response to BI1071, Nur77/H372D (Fig. 4E)or Nur77/H372A (Supplementary Fig. S6B) could not induce thereporter gene transcription in response to BI1071 treatment,revealing a critical role of H372 in BI1071 binding. Nur77/H372A also failed to respond to BI1071 to accumulate in theheavy membrane fraction in the cellular fractionation assay(Fig. 4F). To further characterize the binding of BI1071 and itsapoptotic effect, wemade 2moremutants:mutant Nur77/Y453L,another key residue suggested by the docking studies, andmutantNur77/C566K, a residue located in another reported ligand-

binding region (20–24). BI1071 failed to induce PARP cleavagein cells transfected with Nur77/H372D or Nur77/Y453L, whereasit strongly induced PARP cleavage in cells transfected with Nur77or Nur77/C566K (Fig. 4G). Similarly, in cells transfected withNur77/H372D or Nur77/Y453L, the effect of BI1071 on inducingMito-ROS generation (Fig. 4H), on apoptosis analyzed using dualstaining with fluorescent Annexin V and PI (Fig. 4I), or on loss ofmitochondrial membrane potential (Fig. 4J) was much attenu-ated when compared with cells transfected with Nur77 orNur77/C566K. Taken together, these data demonstrated thatBI1071 exerted its Nur77-dependent apoptotic effect by a directNur77-binding mechanism.

BI1071-induced Nur77 mitochondrial targeting and apoptosisis Bcl-2 dependent

We previously showed that the Nur77 mitochondria-depen-dent apoptotic pathway involved Nur77 interaction with Bcl-2 (14). We therefore asked whether Bcl-2 plays a role inBI1071-induced apoptosis. Thus, the apoptotic effect of BI1071was evaluated in MEFs and MEFs lacking Bcl-2 (Bcl-2�/�MEF).BI1071 at 0.5 mmol/L effectively induced PARP cleavage inMEFs, whereas it had no apparent effect on PARP cleavage inBcl-2�/�MEFs (Fig. 5A). This was confirmed by DAPI stainingshowing that Bcl-2�/�MEF cells were much more resistant thanMEFs to the apoptotic effect of BI1071 (Fig. 5B). In addition, theimpaired effect of BI1071 in Bcl-2�/�MEFs could be rescued by re-expression of Bcl-2 (Supplementary Fig. S7). In response to0.5 mmol/L of BI1071, 40% of MEFs displayed chromatin con-densation and nuclear fragmentation, whereas only 14% ofBcl-2�/�MEF cells exhibited similar apoptotic features. We alsoused the CRISPR/Cas9 technology to generate Bcl-2 knockoutHeLa cells and showed that the effect of BI1071 on inducing PARPcleavage was almost completely suppressed in Bcl-2�/�HeLa cells(Fig. 5C). The role of Bcl-2 inmediating the death effect of BI1071was also illustrated by Annexin V/PI staining showing a reducedapoptotic effect of BI1071 inBcl-2�/�HeLa cells comparedwith itseffect in the parental HeLa cells (from 24.59% to 7.95%), and inBcl-2�/�MEF cells compared with the parental MEF cells (from76.65% to 10.27%; Fig. 5D). Induction of the mitochondrialmembrane potential loss by BI1071 was also suppressed inBcl-2�/�MEFs and Bcl-2�/�HeLa cells (Fig. 5E). Furthermore,BI1071-induced release of mitochondrial ROS was compromisedby loss of Bcl-2 (Fig. 5F). These results revealed a crucial role ofBcl-2 in mediating the Nur77-dependent apoptotic effect ofB1071, demonstrating that the compound acts through theNur77-Bcl-2 apoptotic pathway.

BI1071 promotes Nur77 interaction with Bcl-2In this study, we have showed that BI1071 can bind toNur77 to

induce its migration from the nucleus to mitochondria, where itinteracts with Bcl-2 and triggers Bcl-2–dependent apoptosis.However, if the binding of BI1071 to Nur77 promotes theinteraction between Nur77 and Bcl-2 is not clear. Therefore, weinvestigated whether BI1071 binding to Nur77 enhanced theNur77 interaction with Bcl-2. In vitro GST-pull down assaysshowed that Nur77-LBD was pulled down by GST-Bcl-2 in aBI1071 concentration-dependent manner (Fig. 6A). Cell-basedCo-IP showed that Nur77 (Fig. 6B) or Nur77-LBD (Fig. 6C)transfected in HEK293T cells interacted with Bcl-2 when cellswere treated with BI1071. Endogenous Nur77 could be specifi-cally immunoprecipitated together with endogenous Bcl-2 by






Figure 4.

BI1071 binds directly to Nur77. A and B, Binding of DIM-C-pPhCF3 (A) or BI1071 (B) to purified Nur77-LBD by SPR. C, HEK293T cells were transfected with Gal-4reporter plasmid and Gal-4-RXRa-LBD or Gal-4-RXRa-LBD/E453,6A together with Myc-Nur77, and treated with the indicated concentration of BI1071 or9-cis-RA for 12 hours. Reporter activities were measured. D,Molecular modeling of the binding of BI1071 (in yellow sticks) and DIM-C-pPhCF3 (in green sticks) toNur77-LBD. E. HEK293T cells transfected with Gal-4 reporter plasmid and Gal-4-RXRa-LBD together with Myc-Nur77 or Myc-Nur77/H372D were treated withthe indicated concentration of BI1071, and reporter activities were measured. �� , P < 0.01; �� , P < 0.001 (Student t test). F, HEK293T cells were transfected withMyc-Nur77, Myc-Nur77/H372A, and treated with BI1071 (0.5 mmol/L) for 2 hours. WCL and HM fractions were then prepared and analyzed byWestern blotting.G and J,Nur77�/�HeLa cells were transfected with the indicated Nur77 andmutant plasmids and treated with 0.5 mmol/L BI1071 for 6 hours. Cells were thensubjected toWestern blotting for PARP cleavage (G), mito-SOX staining for determining the production of mitochondrial ROS (H), Annexin V/PI staining forapoptosis (I), or JC-1 staining for measuring mitochondrial membrane potential (J). One of three similar experiments is shown. NS, not significant; �� , P < 0.001(Student t test).

Chen et al.





anti-Bcl-2 antibody only when cells were treated with BI1071(Fig. 6D). Moreover, confocal microscopy analysis revealed thatBI1071 promoted extensive mitochondrial colocalization oftransfected GFP-Nur77 (Fig. 6E; Supplementary Fig. S8A) or

GFP-Nur77-LBD (Fig. 6F; Supplementary Fig. S8B) with Bcl-2 incells. To further address the role of BI1071 on inducing Nur77interaction with Bcl-2, the aforementioned Nur77 mutants wereanalyzed. Fig. 6G showed that Nur77/C566K like the wild-type

Figure 5.

Bcl-2–dependent induction of apoptosis by BI1071. A and B,MEFs or Bcl-2�/�MEFs were treated with 0.5 mmol/L BI1071 for 6 hours. Cells were then subjected toWestern blot analysis for PARP cleavage (A) or DAPI staining for apoptosis (B). Apoptotic cells were counted in 200 cells. �� , P < 0.001 (Student t test). C, PARPcleavage in HeLa or Bcl-2�/� HeLa cells treated with 0.5 mmol/L BI1071 for 6 hours was analyzed byWestern blotting. D–F,MEFs or Bcl-2�/�MEFs, HeLa cells orBcl-2�/� HeLa cells were treated with 0.5 mmol/L BI1071 for 6 hours. Cells were subjected to Annexin V/PI staining for apoptosis measurement (D), JC-1 stainingfor measuring mitochondrial membrane potential (E), or mito-SOX staining for determining the production of mitochondrial ROS (F). One of three similarexperiments is shown; � , P < 0.1; �� , P < 0.001 (Student t test).






Figure 6.

BI1071 promotes Nur77 interaction with Bcl-2. A, GST-pull down. Purified Nur77-LBD incubated with or without the indicated concentration ofBI1071 was pulled down by GST or GST-Bcl-2 protein and analyzed by Western blotting. B and C, Co-immunoprecipitation assay. HEK293Ttransfected with Myc-Bcl-2 together with GFP-Nur77 (B) or GFP-Nur77-LBD (C) was treated with or without 0.5 mmol/L BI1071 and analyzed byco-immunoprecipitation assays using anti-Myc antibody. D, Interaction of endogenous Nur77 and Bcl-2 in MDA-MB-231 cells treated with or without0.5 mmol/L BI1071 for 2 hours was analyzed by co-immunoprecipitation assay using anti-Bcl-2 antibody. E and F, Colocalization of Nur77 with Bcl-2.HEK293T cells were transfected with Myc-Bcl-2 together with GFP-Nur77 (E) or GFP-Nur77-LBD (F), treated with or without 0.5 mmol/L BI1071 for2 hours, stained with anti-Myc antibody, and visualized using confocal microscopy. G, HEK293T cells transfected with the indicated expressionplasmids were treated with 0.5 mmol/L BI1071 for 2 hours and analyzed by co-immunoprecipitation assays using anti-Flag antibody. For Co-IPexperiments, one of three similar experiments is shown.

Chen et al.





Nur77 interacted strongly with Bcl-2 in a BI1071-dependentmanner. In contrast, Nur77/H372D and Nur77/Y453L failedto interact with Bcl-2 in the presence of BI1071. The importanceof the binding of BI1071 on inducing Nur77 interaction withBcl-2 was also illustrated by immunostaining showing extensivecolocalization of transfected Flag Bcl-2 with the wild-type Nur77LBD, but not with Nur77 LBD H372A (Supplementary Fig. S8C).Thus, binding of BI1071 to Nur77 promotes Nur77 interactionwith Bcl-2 and mitochondrial localization.

DiscussionBI1071 is a salt form of DIM-C-pPhCF3 (Fig. 1A) previously

reported to induce Nur77-dependent apoptosis (30, 32). How-ever, relative high concentrations (around 10 mmol/L) ofDIM-C-pPhCF3 are required for its induction of apoptosisand activation of Nur77. To our surprise, we observed that agedDIM-C-pPhCF3. was generally more active than the freshly pre-pared one in apoptosis induction. This led to our synthesis of theoxidized product of DIM-C-pPhCF3, methanesulfonate salt ofDIM-C-pPhCF3 (BI1071). Our evaluation of BI1071 revealed itssuperior death effect in cancer cells. BI1071 was also very effectivein other cancer cell lines and in animal tumor models. Theapoptotic effect of several DIM derivatives has been shown tobe Nur77-dependent and various pathways were proposed toaccount for their apoptotic effect (2). However, the mechanismby which Nur77 mediates their death effect remains elusive,which is conceivably due to the activation of multiple pathwaysby the high compound concentration used in the studies. Forinstance, both transcriptional agonist such as DIM-C-pPhOCH3

and transcriptional antagonist such as DIM-C-pPhOH wereshown to induce Nur77-dependent apoptosis (28). Our findingthat oxidization of DIM-C-pPhCF3 could augment its deatheffect offered an opportunity to delineate the mechanism bywhich Nur77 mediates the apoptotic effect of DIM-relatedsmall molecules. To this end, our studies showed that thepotent apoptotic effect of BI1071 was Nur-77 dependent(Fig. 3) and was a result of its induction of Nur77 mitochon-drial targeting via a direct Nur77-binding mechanism. Further-more, our results revealed that the death effect of BI1071 wasalso dependent of Bcl-2 expression and that BI1071 couldinduce Nur77 interaction with Bcl-2 leading to Nur77 coloca-lization with Bcl-2 at mitochondria and apoptosis.

Binding studies showed that BI1071 could bind toNur77betterthan DIM-C-pPhCF3. This is likely due to the difference in thestructural conformations between the oxidized and the unoxi-dized forms of DIM-C-pPhCF3, perhaps resulted from differentatomic orbital hybridization of the central C atom. In the oxidizedform the central C is sp2-hybridized, positively charged andbonded to 3 atoms with a co-planner arrangement, whereas inthe unoxidized form, C is sp3-hybridized and bonded to 4 atomswith a tetrahedral arrangement. Differences in structural confor-mation and charge distributions can affect howmolecules bind toproteins.Our docking results suggested that BI1071 could interactmore strongly with Nur77-LBD than DIM-C-pPhCF3 due to thedifferent conformations adoptedby the compounds.Mutagenesisstudies confirmed that H372 and Y453 were 2 key residuesinvolving in the binding of BI1071 as suggested by the dockingstudies. Previously, we located the critical region inNur77 respon-sible for its interactionwith Bcl-2 and identified a peptideNuBCP-9 as Bcl-2-converting peptide, capable of inducing apoptosis of

cancer cells in vitro and in animals (12). NuBCP-9 is located at theC-terminal portion of H7. Interestingly, our docking studiessuggested that H7 was part of the BI1071-binding region andBI1071 could potentially interact directly with amino acids D499and A450, structurally flanking the residues fromwhichNuBCP-9is derived. Therefore, it is conceivable that the BI1071-boundNur77 offers a more suitable Bcl-2–interacting interface thatpromotes the formation of Nur77/Bcl-2 complex, and thus aug-ments the biological effect of BI1071.

A critical step in theNur77mitochondrial apoptotic pathway isthe interaction of Nur77 with Bcl-2, which induces a conforma-tion change in Bcl-2 and converts Bcl-2 from a pro-survival to akiller (12, 14). Members of the Bcl-2 family are critical regulatorsof apoptosis. As the fundingmember of the Bcl-2 family, Bcl-2 actsas a survival molecule to protect cells from programmed celldeath. Bcl-2 overexpression is often observed in cancer cells and isassociated with cancer treatment resistance and poor progno-sis (33–36). Thus, Bcl-2 has been an important drug target(37, 38). Two strategies are commonly used to develop therapeu-tic agents targeting Bcl-2: The first relies on making use of anti-sense oligonucleotides to block Bcl-2 expression and the secondrelies on designing and optimizing BH3 small-molecule or pep-tidemimetics that bind the Bcl-2 BH3-binding cleft, antagonizingits antiapoptotic activity (37, 38). Like Bcl-2, Nur77 is overex-pressed in a variety of cancer cells and plays a dual role inmediating apoptosis and survival of cancer cells (39, 40).Although the growth promoting effect of Nur77 appears to bedependent on its nuclear action, thedeath effect ofNur77 involvesits translocation from nucleus to cytoplasm (41–43). Our resultsshowed that cancer cells are sensitive to the treatment of BI1071 ascompared with normal cells, consisting with the fact that the levelof Nur77 is elevated in cancer cells. Thus, targeting Nur77 byBI1071 will have less effect on normal cells, and therefore likelyoffer a high therapeutic index. The ability ofNur77 to interactwithBcl-2 to not only suppress its antiapoptotic function but alsoconvert Bcl-2 into a pro-apoptotic molecule (12, 14) provides apromising strategy to target both Nur77 and Bcl-2 for cancertherapy. Agents that can bind directly toNur77 to promoteNur77translocation and interaction with Bcl-2 are unique in that theycan simultaneously target both Nur77 and Bcl-2. The reportedNur77-derived peptide with 9 amino acids (NuBCP-9) and itsenantiomer as Bcl-2–converting peptides has demonstrated sucha potential (12, 44, 45). In this regard, our identification ofsmall molecules that can directly bind Nur77 to activate theNur77-Bcl-2 apoptotic pathway is significant and BI1071 repre-sents the first lead of this class of small molecules, which warrantsfurther evaluation.

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

Authors' ContributionsConception and design: X. Chen, X. Cao, G. Alitongbieke, Z. Xia, Y. Su,X.-K. ZhangDevelopment of methodology: X. Chen, X. Cao, X. Tu, G. Alitongbieke, M. Yin,D. Xu, S. Guo, Y. SuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): X. Chen, X. Cao, X. Tu, G. Alitongbieke, X. Li,D. Xu, S. Guo, Z. Li, L. Chen, X. ZhangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): X. Chen, X. Cao, X. Tu, G. Alitongbieke, Z. Xia, X. Li,Z. Chen, D. Xu, S. Guo, Z. Li, H. Zhou, Y. Su, X.-K. Zhang






Writing, review, and/or revision of the manuscript: X. Chen, X. Cao, M. Gao,Y. Su, X.-K. ZhangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): X. Chen, X. Cao, X. Tu, G. Alitongbieke, Z. Xia,X. Li, M. Yin, D. Xu, S. Guo, Z. Li, L. Chen, D. Xu, J. Liu, Z. Zeng, H. Zhou, Y. SuStudy supervision: X. Cao, X. Li, H. Zhou, Y. Su, X.-K. Zhang

AcknowledgmentsThe authors thank Dr. Marcia Dawson for her contributions in chemistry

(including conception anddesign) to thiswork, and to hermemory this article isdedicated. We also thank Dr. Lin Li for her critical reading of this article. Thisstudy was supported in part by grants from the Natural Science Foundation ofChina (U1405229, 81672749, 31271453, 31471318; to X.-k. Zhang), Regional

Demonstration of Marine Economy Innovative Development Project(16PYY007SF1; to X.-k. Zhang), the Fujian Provincial Science and TechnologyDepartment (2017YZ0002-1; to X.-k. Zhang), and the National Institutes ofHealth (R01 CA198982; to X.-k. Zhang).

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 August 13, 2018; revised December 28, 2018; accepted March 14,2019; published first March 29, 2019.

References1. Kurakula K, Koenis DS, van Tiel CM, de Vries CJ. NR4A nuclear receptors

are orphans but not lonesome. Biochim Biophys Acta 2014;1843:2543–55.

2. Lee SO, Li X, Khan S, Safe S. Targeting NR4A1 (TR3) in cancer cells andtumors. Expert Opin Ther Targets 2011;15:195–206.

3. Mohan HM, Aherne CM, Rogers AC, Baird AW, Winter DC, Murphy EP.Molecular pathways: the role of NR4A orphan nuclear receptors in cancer.Clin Cancer Res 2012;18:3223–8.

4. Pearen MA, Muscat GE. Minireview: nuclear hormone receptor 4Asignaling: implications for metabolic disease. Mol Endocrinol 2010;24:1891–903.

5. To SK, Zeng JZ, Wong AS. Nur77: a potential therapeutic target in cancer.Expert Opin Ther Targets 2012;16:573–85.

6. Zhang XK. Targeting Nur77 translocation. Expert Opin Ther Targets 2007;11:69–79.

7. Deutsch AJ, Angerer H, Fuchs TE, Neumeister P. The nuclear orphanreceptors NR4A as therapeutic target in cancer therapy. Anticancer AgentsMed Chem 2012;12:1001–14.

8. McMorrow JP, Murphy EP. Inflammation: a role for NR4A orphan nuclearreceptors? Biochem Soc Trans 2011;39:688–93.

9. Liu ZG, Smith SW, Mclaughlin KA, Schwartz LM, Osborne BA. Apoptoticsignals delivered through the T-cell receptor of a T-cell hybrid require theimmediate-early gene Nur77. Nature 1994;367:281–4.

10. Woronicz JD, Calnan B, Ngo V, Winoto A. Requirement for the orphansteroid-receptor Nur77 in apoptosis of T-cell hybridomas. Nature 1994;367:277–81.

11. Li Y, Lin BZ, Agadir A, Liu R, Dawson MI, Reed JC, et al. Moleculardeterminants of AHPN (CD437)-induced growth arrest and apoptosis inhuman lung cancer cell lines. Mol Cell Biol 1998;18:4719–31.

12. Kolluri SK, Zhu XW, Zhou X, Lin BZ, Chen Y, Sun K, et al. A short Nur77-derived peptide converts Bcl-2 from a protector to a killer. Cancer Cell2008;14:285–98.

13. Li H, Kolluri SK, Gu J, DawsonMI, Cao XH, Hobbs PD, et al. Cytochrome crelease and apoptosis induced by mitochondrial targeting of nuclearorphan receptor TR3. Science 2000;289:1159–64.

14. Lin BZ, Kolluri SK, Lin F, LiuW,Han YH, Cao XH, et al. Conversion of Bcl-2fromprotector to killer by interactionwith nuclear orphan receptorNur77/TR3. Cell 2004;116:527–40.

15. Moll UM, Marchenko N, Zhang XK. p53 and Nur77/TR3 - transcriptionfactors that directly targetmitochondria for cell death induction.Oncogene2006;25:4725–43.

16. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K,et al. The nuclear receptor superfamily: the second decade. Cell 1995;83:835–9.

17. Wang ZL, Benoit G, Liu JS, Prasad S, Aarnisalo P, Liu XH, et al. Structure andfunction of Nurr1 identifies a class of ligand-independent nuclear recep-tors. Nature 2003;423:555–60.

18. Chen LQ,Wang ZG, Aleshin AE, Chen F, Chen JB, Jiang FQ, et al. Sulindac-derived RXR alpha modulators inhibit cancer cell growth by binding to anovel site. Chem Biol 2014;21:596–607.

19. Su Y, Zeng Z, Chen Z, Xu D, Zhang W, Zhang XK. Recent progress in thedesign and discovery of RXRmodulators targeting alternate binding sites ofthe receptor. Curr Top Med Chem 2017;17:663–75.

20. Zhan YP,DuXP, ChenHZ, Liu JJ, ZhaoBX,HuangDH, et al. Cytosporone Bis an agonist for nuclear orphan receptor Nur77. Nat Chem Biol 2008;4:548–56.

21. Liu JJ, Zeng HN, Zhang LR, Zhan YY, Chen Y, Wang YA, et al. A uniquepharmacophore for activation of the nuclear orphan receptor nur77 in vivoand in vitro. Cancer Res 2010;70:3628–37.

22. Zhan YY, Chen Y, Zhang Q, Zhuang JJ, Tian M, Chen HZ, et al. The orphannuclear receptor Nur77 regulates LKB1 localization and activates AMPK.Nat Chem Biol 2012;8:897–904.

23. Wang WJ, Wang Y, Chen HZ, Xing YZ, Li FW, Zhang Q, et al. Orphannuclear receptor TR3 acts in autophagic cell death via mitochondrialsignaling pathway. Nat Chem Biol 2014;10:133–40.

24. HuM, LuoQ, AlitongbiekeG, Chong S, XuC, Xie L, et al. Celastrol-inducedNur77 interaction with TRAF2 alleviates inflammation by promotingmitochondrial ubiquitination and autophagy. Mol Cell 2017;66:141–53.e6.

25. Zeng Z, Sun Z, Huang M, Zhang W, Liu J, Chen L, et al. Nitrostyrenederivatives act as RXRalpha ligands to inhibit TNFalpha activation of NF-kappaB. Cancer Res 2015;75:2049–60.

26. WeiW, Chen ZJ, Zhang KS, Yang XL,Wu YM, Chen XH, et al. The activationof G protein-coupled receptor 30 (GPR30) inhibits proliferation of estro-gen receptor-negative breast cancer cells in vitro and in vivo. Cell DeathDis2014;5:e1428.

27. Murphy R, Repasky M, Zhou ZY, Abel R, Krilov G, Tubert-Brohman I, et al.Evaluation of docking and scoring accuracy using a new version ofSchrodinger's Glide XP and Induced Fit Docking (IFD) methodologies.Abstr Pap Am Chem S 2011;241.

28. Chintharlapalli S, Burghardt R, Papineni S, Ramaiah S, Yoon K, Safe S.Activation of Nur77 by selected 1,1-bis(3'-indolyl)-1-(p-substituted phe-nyl) methanes induces apoptosis through nuclear pathways. J Biol Chem2005;280:24903–14.

29. Reers M, Smiley ST, Mottola-Hartshorn C, Chen A, Lin M, Chen LB.Mitochondrial membrane potential monitored by JC-1 dye. MethodsEnzymol 1995;260:406–17.

30. Chinni SR, Li YW, Upadhyay S, Koppolu PK, Sarkar FH. Indole3-carbinol(I3C) induced cell growth inhibition, G1 cell cycle arrest and apoptosis inprostate cancer cells. Oncogene 2001;20:2927–36.

31. Thompson PD, Remus LS, Hsieh JC, Jurutka PW, Whitfield GK, GalliganMA, et al. Distinct retinoid X receptor activation function-2 residuesmediate transactivation in homodimeric and vitamin D receptor hetero-dimeric contexts. J Mol Endocrinol 2001;27:211–27.

32. Hedrick E, Lee SO, Kim G, Abdelrahim M, Jin UH, Safe S, et al. Nuclearreceptor 4A1 (NR4A1) as a drug target for renal cell adenocarcinoma.PLoS ONE 2015;10.

33. Adams JM, Cory S.The Bcl-2 protein family: arbiters of cell survival. Science1998;281:1322–6.

34. Green DR, Reed JC. Mitochondria and apoptosis. Science 1998;281:1309–12.

35. Reed JC. Bcl-2-family proteins and hematologic malignancies: history andfuture prospects. Blood 2008;111:3322–30.

36. Maji S, Panda S, Samal SK, Shriwas O, Rath R, Pellecchia M, et al. Bcl-2antiapoptotic family proteins and chemoresistance in cancer. Adv CancerRes 2018;137:37–75.

Chen et al.





37. Ashkenazi A, Fairbrother WJ, Leverson JD, Souers AJ. From basic apoptosisdiscoveries to advanced selective BCL-2 family inhibitors. Nat Rev DrugDiscov 2017;16:273–84.

38. Croce CM, Reed JC. Finally, an apoptosis-targeting therapeutic for cancer.Cancer Res 2016;76:5914–20.

39. Xie L, Jiang F, Zhang X, Alitongbieke G, Shi X, Meng M, et al. Honokiolsensitizes breast cancer cells to TNF-alpha induction of apoptosis byinhibiting Nur77 expression. Br J Pharmacol 2016;173:344–56.

40. Wu H, Lin Y, Li W, Sun Z, Gao W, Zhang H, et al. Regulation of Nur77expression by beta-catenin and its mitogenic effect in colon cancer cells.FASEB J 2011;25:192–205.

41. Kolluri SK, Bruey-Sedano N, Cao X, Lin B, Lin F, Han YH, et al. Mitogeniceffect of orphan receptor TR3 and its regulation by MEKK1 in lung cancercells. Mol Cell Biol 2003;23:8651–67.

42. Lee SO, AbdelrahimM, Yoon K, Chintharlapalli S, Papineni S, Kim K, et al.Inactivation of the orphan nuclear receptor TR3/Nur77 inhibits pancreaticcancer cell and tumor growth. Cancer Res 2010;70:6824–36.

43. Lee SO, Andey T, Jin UH, Kim K, Sachdeva M, Safe S. The nuclear receptorTR3 regulates mTORC1 signaling in lung cancer cells expressing wild-typep53. Oncogene 2012;31:3265–76.

44. Kapoor S, Gupta D, Kumar M, Sharma S, Gupta AK, Misro MM, et al.Intracellular delivery of peptide cargos using polyhydroxybutyrate basedbiodegradable nanoparticles: studies on antitumor efficacy of BCL-2 con-verting peptide, NuBCP-9. Int J Pharm 2016;511:876–89.

45. Kumar M, Gupta D, Singh G, Sharma S, Bhat M, Prashant CK, et al. Novelpolymeric nanoparticles for intracellular delivery of peptide Cargos: anti-tumor efficacyof theBCL-2 conversionpeptideNuBCP-9. Cancer Res 2014;74:3271–81.






2019;18:886-899. Published OnlineFirst March 29, 2019.Mol Cancer Ther Xiaohui Chen, Xihua Cao, Xuhuang Tu, et al. Cells by Activating the Nur77-Bcl-2 Apoptotic PathwayBI1071, a Novel Nur77 Modulator, Induces Apoptosis of Cancer

Updated version

10.1158/1535-7163.MCT-18-0918doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2019/03/29/1535-7163.MCT-18-0918.DC1

Access the most recent supplemental material at:

Cited articles

http://mct.aacrjournals.org/content/18/5/886.full#ref-list-1

This article cites 43 articles, 15 of which you can access for free at:

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

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/18/5/886To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-18-0918

http://mct.aacrjournals.org/content/suppl/2019/03/29/1535-7163.MCT-18-0918.DC1

http://mct.aacrjournals.org/content/18/5/886.full#ref-list-1

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/18/5/886


bi1071, a novel nur77 modulator, induces apoptosis of cancer … · nur77-bcl-2 apoptotic pathway...

Documents