Published OnlineFirst March 29, 2019; DOI: 10.1158/1535-7163.MCT-18-0918 Small Molecule Therapeutics Molecular Cancer Therapeutics BI1071, a Novel Nur77 Modulator, Induces Apoptosis of Cancer Cells by Activating the Nur77-Bcl-2 Apoptotic Pathway Xiaohui Chen1, Xihua Cao2, Xuhuang Tu1, Gulimiran Alitongbieke1, Zebin Xia2, Xiaotong Li1, Ziwen Chen1,MeimeiYin,DanXu1, Shangjie Guo1, Zongxi Li1, Liqun Chen1,XindaoZhang1,DingyuXu1, Meichun Gao1,JieLiu1, Zhiping Zeng1, Hu Zhou1,YingSu2, and Xiao-kun Zhang1,2 Abstract Nur77 (also called TR3 or NGFI-B), an orphan member of indole-3-carbinol metabolite, as a modulator of the Nur77- the nuclear receptor superfamily, induces apoptosis by trans- Bcl-2 apoptotic pathway. BI1071 binds Nur77 with high locating to mitochondria where it interacts with Bcl-2 to affinity, promotes Nur77 mitochondrial targeting and inter- convert Bcl-2 from an antiapoptotic to a pro-apoptotic mol- action with Bcl-2, and effectively induces apoptosis of cancer ecule. Nur77 posttranslational modification such as phos- cells in a Nur77- and Bcl-2–dependent manner. Studies with phorylation has been shown to induce Nur77 translocation animal model showed that BI1071 potently inhibited the from the nucleus to mitochondria. However, small molecules growth of tumor cells in animals through its induction of that can bind directly to Nur77 to trigger its mitochondrial apoptosis. Our results identify BI1071 as a novel Nur77- localization and Bcl-2 interaction remain to be explored. Here, binding modulator of the Nur77-Bcl-2 apoptotic pathway, we report our identification and characterization of DIM-C- which may serve as a promising lead for treating cancers with þ À pPhCF3 MeSO3 (BI1071), an oxidized product derived from overexpression of Bcl-2. Introduction Bcl-2 in regulating the apoptosis of cancer cells and in the resistance of cancer cells to a variety of radio- and chemother- Nur77 (NR4A1;alsoknownasNGFI-BandTR3)isperhaps apeutic agents, understanding how the Nur77-Bcl-2 apoptotic the most potent apoptotic member of the nuclear receptor pathway is regulated and discovering its small-molecule mod- superfamily (1–8). The death effect of Nur77 was initially ulators may offer new strategies to develop effective cancer recognized during studying the apoptosis of immature thymo- therapeutics. However, small molecules that can activate the cytes, T-cell hybridomas (9, 10). Later, we found that Nur77 Nur77-Bcl-2 apoptotic pathway by binding to Nur77 to trigger mediates the death effect of the retinoid-related molecule Nur77 mitochondrial translocation and interaction with Bcl-2 AHPN (also called CD437) in cancer cells (11). Furthermore, have not been reported. we discovered a nongenomic action of Nur77, in which Nur77 As an orphan nuclear receptor, Nur77 lacks a canonical migrates from the nucleus to the cytoplasm, where it targets ligand-binding pocket (LBP; refs. 16, 17), which excludes small mitochondria to trigger cytochrome c release and apoptosis in molecules from binding to Nur77 to regulate Nur77 functions cancer cells (12–14). Further studies demonstrated in various via the canonical LBP-binding mechanism. Recent advance has cancer types that such a Nur77 mitochondrial apoptotic path- revealed the existence of alternate small-molecule binding way is characterized by its interaction with Bcl-2 and the regions on the surface of nuclear receptors, and compounds conversion of Bcl-2 from an antiapoptotic molecule to a that bind to alternate sites other than LBP have been identified pro-apoptotic molecule (6, 15). Given the pivotal role of for some nuclear receptors (18, 19), including Nur77 (20–24). These developments inspire us to discover Nur77-binding compounds that can regulate the Nur77-Bcl-2 apoptotic path- 1 School of Pharmaceutical Sciences, Fujian Provincial Key Laboratory of Inno- way.Here,wereportthatasaltformofa3,30-diindolymethane vative Drug Target Research, Xiamen University, Xiamen, China. 2Cancer Center, (DIM) derivative (di(1H-indol-3-yl)(4-(trifluoromethyl)phe- Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California. nyl)methane; named BI1071 here) can bind to Nur77 to induce Note: Supplementary data for this article are available at Molecular Cancer apoptosis of cancer cells through the Nur77-Bcl-2 apoptotic Therapeutics Online (http://mct.aacrjournals.org/). pathway. BI1071 binds to Nur77 at submicromolar concentra- X. Chen and X. Cao contributed equally to this article. tion and induces apoptosis that is dependent on the expression Corresponding Authors: Ying Su, Sanford Burnham Prebys Medical Discovery of both Nur77 and Bcl-2. BI1071 also effectively inhibits the Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037; E-maill: growth of tumor cells in animals. Moreover, BI1071 binding to [email protected] and Xiao-kun Zhang, Xiamen University, Xiamen, China; Nur77 induces not only its mitochondrial targeting but also its E-mail: [email protected] interaction with Bcl-2. Our results therefore identify BI1071 as doi: 10.1158/1535-7163.MCT-18-0918 the first Nur77-binding small molecule that promotes the Ó2019 American Association for Cancer Research. Nur77-Bcl-2 apoptotic pathway. 886 Mol Cancer Ther; 18(5) May 2019 Downloaded from mct.aacrjournals.org on September 26, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst March 29, 2019; DOI: 10.1158/1535-7163.MCT-18-0918 A New Activator of the Nur77-Bcl-2 Apoptotic Pathway Materials and Methods Generation of Nur77 and Bcl-2 knockout cells by CRISPR/Cas9 system Cell culture Knocking out Nur77 and Bcl-2 from HeLa cells used the The following cell lines are used in our study. HCT116 colon CRISPR/Cas9 system. gRNA targeting sequence of Nur77 (50- cancer, MDA-MB-231, HS578T, BT549, MCF-7, and T47D breast ACCTTCATGGACGGCTACAC-30) and Bcl-2 (50-GAGAACAGGG- cancer, breast epithelial cell line MCF-10A, HeLa ovarian cancer, TACGATAACC-30) was cloned into gRNA cloning vector Px330 mouse embryonic fibroblast (MEF) cells and HEK293T embryonic (Addgene, 71707) and confirmed by sequencing. The accession cells were cultured in DMEM, whereas ZR-75-1, HCC1937 breast numbers of Nur77 and Bcl-2 are NM 001202233 and cancer and SW480 colon cancer were cultured in RPIM-1640 - NM 000633.2 respectively. To screen for cells lacking Nur77 or medium containing 10% FBS. Human colonic epithelial cells - Bcl-2, HeLa cells were transfected with control vector and gRNA (HCoEpiC) were cultured in colonic epithelial cell medium (CoE- expression vectors, followed by G418 selection (0.5 mg/mL). piCM, Cat. #2951). Cell lines HCT116 (ATCC, CCL-247), SW480 Single colonies were subjected to Western blotting using anti- (ATCC, CCL-228), and HEK293T (ATCC, CRL-11268) were Nur77 and anti–Bcl-2 antibody to select knockout cells. obtained from the ATCC. Cell lines MCF-10A (SCSP-660), MDA-MB-231 (SCSP-5043), HeLa (TCHu187), HS578T Cell viability determination and cell death assay (TCHu127), BT549 (TCHu 93), MCF-7 (SCSP-531), ZR-75-1 Cell viability was analyzed by using colorimetric 3-(4,5- (TCHu126), T47D (TCHu 87), and HCC1937 (TCHu148) were dimethylthiazol-dimethylthiazol-2-yl)-2,5-diphenyletetrazolium obtained from Chinese Academy of Science Shanghai Cell Bank on Bromide (MTT) assay as described previously (12–14, 24). December 09, 2016. Cell line HCoEpiC was obtained from Scien- ceCell (Cat. #2950) on October 05, 2018. MEF cells were isolated Mammalian one hybrid assay from embryonic day 13 wild-type (WT) and Nur77 knock out (KO) HEK293T cells were co-transfected with pG5 Luciferase reporter mice. The cells were grown in the cell incubator with 5% CO2 at together with the plasmid encoding RXRa-LBD fused with the 37 C. Sub-confluent cells with exponential growth were used Gal4 DNA-binding domain and other expression plasmids as throughout the experiments. Cells plated onto cell culture dishes described previously (18, 25). After transfection, cells were treated and kept in 10% FBS for 24 hours were treated with compounds or with DMSO or BI1071, and assayed by using the Dual-Luciferase transfected with plasmids. Cell transfection was carried out by Reporter Assay System (Promega). Transfection efficiency was using Lipofectamin 2000 according to the manufacturer's instruc- normalized to Renilla luciferase activity. tion. The cells were tested by using Mycoplasma Hoechst Stain Assay kit (Beyotime, C0296) every 6 months. We added the Hoechest Cell fractionation solution to stain the cells with 50% density at room temperature for For cellular fractionation (12–14, 24), cells were lysed in cold 30 minutes and then used the confocal microscope to observe the buffer A (10 mmol/L HEPES-KOH (pH 7.9), 1.5 mmol/L MgCl , cells. In the cells without Mycoplasma infection, only the blue 2 10 mmol/L KCl, 0.5 mmol/L dithiothreitol) with a cocktail of fluorescence of the nucleus was observed. Filamentous blue fluo- proteinase inhibitors on ice for 10 minutes as described previ- rescence can be observed around the nucleus in Mycoplasma con- ously. Cytoplasmic fraction was collected by centrifuging at 6,000 taminated cell samples. The cells were prevented from Mycoplasma rpm for 10 minutes. Pellets containing nuclei were resuspended in infection by using plasmocin (Invivogen, ant-mpt). No positive cold high-salt buffer C (20 mmol/L HEPES-KOH (pH 7.9), 25% Mycoplasma tests was observed during the time of our experiments. glycerol, 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.5 mmol/L dithiothreitol) with a cocktail of proteinase Plasmids inhibitors on ice for 30 minutes. Plasmids pcmv-myc-Nur77, GFP-Nur77, GFP-Nur77/LBD, GST-Bcl-2, pcmv-myc-Bcl-2, Flag-cmv-Bcl-2 were constructed as GST-pull down – described (12 14, 24). Plasmids pcmv-myc-Nur77/H372D, GST or GST-Bcl-2 fusion protein (0.5 mg) was immobilized on pcmv-myc-Nur77/H372A, pcmv-myc-Nur77/Y453L, pcmv-myc- glutathione-Sepharose beads and incubated with purified His- Nur77/C566K were constructed by using the PCR and Quick- Nur77-LBD (0.2 mg) in the presence of different concentration of Chang mutagenesis kit.