bi1071, a novel nur77 modulator, induces apoptosis of cancer … · 2019. 3. 29. · the confocal...

1

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 Alitongbieke

1, Zebin Xia

2, Xiaotong Li

1,

Ziwen Chen1, Meimei Yin, Dan Xu

1, Shangjie Guo

1, Zongxi Li

1, Liqun Chen

1, Xindao Zhang

1,

Dingyu Xu1, Meichun Gao

1, Jie Liu

1, Zhiping Zeng

1, Hu Zhou

1, Ying Su

2,*, and Xiao-kun

Zhang1,2,*

1School of Pharmaceutical Sciences, Fujian Provincial Key Laboratory of Innovative Drug

Target Research, Xiamen University, Xiamen 361102, China; 2Cancer center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA;

Running title: A new activator of the Nur77-Bcl-2 apoptotic pathway

Keywords: Nur77, Bcl-2, DIM, Mitochondria, Apoptosis

# These authors equally contributed to this work

*Corresponding Authors:

Dr. Xiao-kun Zhang

Dr. Ying Su

Cancer Center

Sanford Burnham Prebys Medical Discovery Institute

10901 N. Torrey Pines Road

La Jolla, CA 92037

USA

Phone: 858-646-3141

Fax: 858-646-3195

E-mail: [email protected] or [email protected]

This study was supported in part by grants from the Natural Science Foundation of China

(U1405229, 81672749, 31271453, 31471318) to X. Zhang, Regional Demonstration of Marine

Economy Innovative Development Project (16PYY007SF17) to X. Zhang, the Fujian Provincial

on December 31, 2020. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 29, 2019; DOI: 10.1158/1535-7163.MCT-18-0918

mailto:[email protected]

http://mct.aacrjournals.org/

2

Science & Technology Department (2017YZ0002-1) to X. Zhang and the National Institutes of

Health (R01 CA198982).

Disclose of Potential Conflicts of Interest: The authors declare no potential conflicts of interest.




3

Abstract

Nur77 (also called TR3 or NGFI-B), an orphan member of the nuclear receptor superfamily,

induces apoptosis by translocating to mitochondria where it interacts with Bcl-2 to convert Bcl-2

from an anti-apoptotic to a pro-apoptotic molecule. Nur77 posttranslational modification such as

phosphorylation has been shown to induce Nur77 translocation from the nucleus to mitochondria.

However, small molecules that can bind directly to Nur77 to trigger its mitochondrial

localization and Bcl-2 interaction remain to be explored. Here we report our identification and

characterization of DIM-C-pPhCF3+MeSO3

- (BI1071), an oxidized product derived from indole-

3-carbinol metabolite, as a modulator of the Nur77-Bcl-2 apoptotic pathway. BI1071 binds

Nur77 with high affinity, promotes Nur77 mitochondrial targeting and interaction with Bcl-2,

and effectively induces apoptosis of cancer cells in a Nur77- and Bcl-2-dependent manner.

Studies with animal model showed that BI1071 potently inhibited the growth of tumor cells in

animals through its induction of apoptosis. 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 with overexpression of Bcl-2.




4

Introduction

Nur77 (NR4A1) (also known as NGFI-B and TR3) is perhaps the most potent apoptotic member

of the nuclear receptor superfamily (1-8). The death effect of Nur77 was initially recognized

during studying the apoptosis of immature thymocytes, T-cell hybridomas (9,10). Later we found

that Nur77 mediates the death effect of the retinoid-related molecule AHPN (also called CD437)

in cancer cells (11). Furthermore, we discovered a nongenomic action of Nur77, in which Nur77

migrates from the nucleus to the cytoplasm, where it targets mitochondria to trigger cytochrome

c release and apoptosis in cancer cells (12-14). Further studies demonstrated in various cancer

types that such a Nur77 mitochondrial apoptotic pathway is characterized by its interaction with

Bcl-2 and the conversion of Bcl-2 from an anti-apoptotic molecule to a pro-apoptotic molecule

(6,15). Given the pivotal role of Bcl-2 in regulating the apoptosis of cancer cells and in the

resistance of cancer cells to a variety of radio- and chemo-therapeutic agents, understanding how

the Nur77-Bcl-2 apoptotic pathway is regulated and discovering its small molecule modulators

may offer new strategies to develop effective cancer therapeutics. However, small molecules that

can activate the Nur77-Bcl-2 apoptotic pathway by binding to Nur77 to trigger Nur77

mitochondrial translocation and interaction with Bcl-2 have not been reported.

As an orphan nuclear receptor, Nur77 lacks a canonical ligand-binding pocket (LBP) (16,17),

which excludes small molecules from binding to Nur77 to regulate Nur77 functions via the

canonical LBP binding mechanism. Recent advance has revealed the existence of alternate small

molecule binding regions on the surface of nuclear receptors, and compounds that bind to

alternate sites other than LBP have been identified for some nuclear receptors (18,19), including

Nur77 (20-24). These developments inspire us to discover Nur77-binding compounds that can




5

regulate the Nur77-Bcl-2 apoptotic pathway. Here we report that a salt form of a 3,3’-

diindolymethane (DIM) derivative (di(1H-indol-3-yl)(4-(trifluoromethyl)phenyl)methane)

(named BI1071 here) can bind to Nur77 to induce apoptosis of cancer cells through the Nur77-

Bcl-2 apoptotic pathway. BI1071 binds to Nur77 at submicromolar concentration and induces

apoptosis that is dependent on the expression of both Nur77 and Bcl-2. BI1071 also effectively

inhibits the growth of tumor cells in animals. Moreover, BI1071 binding to Nur77 induces not

only its mitochondrial targeting but also its interaction with Bcl-2. Our results therefore identify

BI1071 as the first Nur77-binding small molecule that promotes the Nur77-Bcl-2 apoptotic

pathway.

Material and Method

Cell culture

The following cell lines are used in our study. HCT116 colon cancer, MDA-MB-231, HS578T,

BT549, MCF-7, and T47D breast cancer, breast epithelial cell line MCF-10A, HeLa ovarian

cancer, mouse embryonic fibroblast (MEF) cells and HEK293T embryonic cells were cultured in

Dulbecco’s Eagle’s medium (DMEM), while ZR-75-1, HCC1937 breast cancer and SW480

colon cancer were cultured in RPIM 1640 medium containing 10% fetal bovine serum (FBS).

Human Colonic Epithelial cells (HCoEpiC) were cultured in Colonic Epithelial Cell Medium

(CoEpiCM, Cat. #2951). Cell lines HCT116 (ATCC, CCL-247), SW480 (ATCC, CCL-228), and

HEK293T (ATCC, CRL-11268) were obtained from American Type Culture Collection (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) were obtained from Chinese Academy of Science Shanghai Cell Bank




6

on Dec. 09, 2016. Cell line HCoEpiC was obtained from ScienceCell (Cat. #2950) on Oct, 05,

2018. MEF cells were isolated from embryonic day 13 wild-type (WT) and Nur77 knock out

(KO) mice. The cells were grown in the cell incubator with 5% CO2 at 37 °C. Sub-confluent

cells with exponential growth were employed throughout the experiments. Cells plated onto cell

culture dishes and kept in 10% FBS for 24 h were treated with compounds or tranfected with

plasmids. Cell transfection was carried out by using Lipofectamin 2000 according to the

manufacture's instruction. The cells were tested by using Mycoplasma Hoechst Stain Assay kit

(Beyotime, C0296) every six months. We added the Hoechest solution to stain the cells with 50%

density at room temperature for 30 minutes and then used the confocal microscope to oberserve

the cells. In the cells without mycoplasma infection, only the blue fluorescence of the nucleus

was obersed. Filamentous blue fluorescence can be obersved around the nucleus in mycoplasma

contaminated cell samples. The cells were prevented from mycoplasma infection by using

plasmocin (Invivogen, ant-mpt). No positive mycoplasma tests was observed during the time of

our experiments.

Plasmids

Plasmids pcmv-myc-Nur77, GFP-Nur77, GFP-Nur77/LBD, GST-Bcl-2, pcmv-myc-Bcl-2, Flag-

cmv-Bcl-2 were constructed as described (12-14,24). Plasmids pcmv-myc-Nur77/H372D, pcmv-

myc-Nur77/H372A, pcmv-myc-Nur77/Y453L, pcmv-myc-Nur77/C566K were constructed by

using PCR and QuickChang mutagenesis kit.

Antibodies and Reagents

Anti-Myc (9E10) (Cat. Sc-40), anti-Ki67 (Cat. ab15580) and anti-Hsp60 (Cat. ab46798)

antibodies were purchased from Abcam (UK); anti-β-actin (Cat. 4970S), anti-Cleaved caspase-3

(Cat. 9661S), and anti-Nur77 (Cat. 3960S) antibodies were purchased from Cell Signal




7

Technology (Beverly, MA, USA); anti-Nur77 (M-210) (Cat. sc-5569), anti-PCNA (Santa Cruz

sc-7907), anti-a-tublin (Santa Cruz sc-8035), anti-Bcl-2 (Santa Cruz sc-783), anti-PARP (Santa

Cruz sc-7150), and anti-GST (sc-138) antibodies were purchased from Santa Cruz

Biotechnology (Santa Cruz, CA, USA); anti-Flag (Cat.F1804) antibody was purchased from

Sigma (St. Louis, MO, USA); 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 Knock-out cells by CRISPR/Cas9 system

Knocking out Nur77 and Bcl-2 from HeLa cells employed the CRISPR/Cas9 system. gRNA

targeting sequence of Nur77 (5’-ACCTTCATGGACGGCTACAC-3’) and Bcl-2 (5’-

GAGAACAGGGTACGATAACC-3’) was cloned into gRNA cloning vector Px330 (Addgene,

71707) and confirmed by sequencing. The accession numbers of Nur77 and Bcl-2 are NM-

001202233 and NM-000633.2 respectively. To screen for cells lacking Nur77 or Bcl-2, HeLa

cells were transfected with control vector and gRNA expression vectors, followed by G418

selection (0.5mg/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 assay.

Cell viability was analyzed by using colorimetric 3-(4,5-dimethylthiazol-dimethylthiazol-2-yl)-

2,5-diphenyletetrazolium Bromide (MTT) assay as described (12-14,24).

Mammalian one hybrid assay

HEK293T cells were co-transfected with pG5 Luciferase reporter together with the plasmid

encoding RXR-LBD fused with the Gal4 DNA-binding domain and other expression plasmids

as described (18,25). After transfection, cells were treated with DMSO or BI1071, and assayed




8

by using the Dual-Luciferase Reporter Assay System (Promega). Transfection efficiency was

normalized to Renilla luciferase activity.

Cell fractionation

For cellular fractionation (12-14,24), cells were lysed in cold buffer A (10 mM HEPES-KOH

(pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol) with a cocktail of proteinase

inhibitors on ice for 10 min as described. Cytoplasmic fraction was collected by centrifuging at

6000 rpm for 10 minutes. Pellets containing nuclei were resuspended in cold high-salt buffer C

(20 mM HEPES-KOH (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA,

0.5 mM dithiothreitol) with a cocktail of proteinase inhibitors on ice for 30 minutes.

GST-pull Down

GST or GST-Bcl-2 fusion protein (0.5 mg) was immobilized on glutathione-Sepharose beads and

incubated with purified His-Nur77-LBD (0.2 mg) in the presence of different concentration of

BI1071 as described (12-14,24). Bound Nur77-LBD was analyzed by Western blotting.

Western blotting and immunoprecipitation

Western blotting and co-immunoprecipitation (co-IP) were performed as described (12-14,24).

Generation of Nur77 and Bcl-2 Knock-out cells by CRISPR/Cas9 system

Knocking out Nur77 and Bcl-2 from HeLa cells employed the CRISPR/Cas9 system. gRNA

targeting sequence of Nur77 (5’-ACCTTCATGGACGGCTACAC-3’) and Bcl-2 (5’-

GAGAACAGGGTACGATAACC-3’) was cloned into gRNA cloning vector Px330 (Addgene,

71707) and confirmed by sequencing. To screen for cells lacking Nur77 or Bcl-2, HeLa cells

were transfected with control vector and gRNA expression vectors, followed by G418 selection

(0.5mg/ml). Single colonies were subjected to Western blotting using anti-Nur77 and anti-Bcl-2

antibody to select knockout cells.




9

Apoptosis assay

Cells were plaked on six-well plates with a density of 1X106 per well. After 24 hours, the cells

treated with different concentration of BI1071 for 6 hours, and then the suspended and the

adherent cells were collected, stained with Annexin V-FITC for 10 minutes and with propidium

iodide for 5 minutes, and analyzed immediately by cytoFLEX Flow Cytometry System

(Beckman-Coulter, Miami, FL, USA) using FITC and PC5.5.

Determination of ∆Ψm and ROS

The determination of ∆Ψm and ROS was performed as previously described elsewhere (26). JC-

1 probe was used to measure mitochondrial depolarization in cells. Cells were first treated with

different concentration of BI1071 for 6 hours and then followed with the addition of JC-1

staining solution (5 μg/ml) for 20 minutes at 37°C. After washing with PBS twice, mitochondrial

membrane potentials were analyzed immediately by cytoFLEX Flow Cytometry System using

FITC and PE. Mitochondrial depolarization was measured by a change in the ratio of green/red

fluorescence intensity. ROS was monitored with the mitoSOX Red Mitochondrial Superoxide

Indicator and analyzed by cytoFLEX Flow Cytometry System using PE.

Immunostaining

Cells were fixed in 4% paraformaldehyde. For mitochondrial staining, cells were incubated with

anti-Hsp60 goat immunoglobulin G (IgG) (Santa Cruz Biotechnology, Santa Cruz, CA),

followed by anti-goat IgG conjugated with Cy3. The nuclei were visualized by DAPI staining.

Fluorescent images were collected and analyzed by using a fluorescence microscopy or MRC-

1024 MP laser-scanning confocal microscope (Bio-Rad, Hercules, CA).

Animal studies

The protocols for animal studies were approved by the Animal Care and Use Committee of




10

Xiamen University, and all mice were handled in accordance with the “Guide for the Care and

Use of Laboratory Animals” and the “Principles for the Utilization and Care of Vertebrate

Animals”. For MMTV–PyMT mice breast cancer model, female MMTV-PyMT mice of 12

weeks old were randomly divided into two groups (n=7 each), treated with a daily oral dose of

BI1071 (5 mg/kg) for 18 days. Standard histopathological analysis of tumor tissue was

performed. BI1071 was dissolved in DMSO and diluted with normal saline containing 5.0%

(V/V) Tween-80 to a final concentration 0.5 mg/ml. Normal saline with DMSO and 5.0%

Tween-80 was employed as the vehicle control. For xenograft nude mouse study, male BALB/c

nude mice (6 weeks old) were subcutaneously injected with log growth-phase of SW620 cells (1

X 106 cells in 0.1 ml PBS). Mice were treated orally after 7 days of transplantation with BI1071

once a day. Body weight and tumor size were measured every 3 days. Tumors were measured

and weighted. Tissues isolated from the nude mice were fixed with 4% paraformaldehyde. TdT-

mediated dUTP nick end labeling assay was performed according to the manufacturer’s

instructions (In situ Cell Death Detection Kit; Roche).

Immunohistochemistry

4 µm 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 was performed in

10 mM sodium citrate buffer (pH 6.0), which was microwaved at 100°C for 20 minutes. After

rinsed twice in PBS, sections were blocked at room temperature for 1 hour by using 10% normal

goat serum, followed by incubation with anti-ki67, anti-cleaved caspase 3 overnight at 4°C.

Colors were developed with a DAB horseradish peroxidase color development kit.

Docking experiments




11

Schrodinger’s (www.schrodinger.com) Glide (27), a grid-based docking program was used for

the docking study of BI1071 to the protein. The crystal structure of Nur77-LBD in complex with

a cytosporone B analog (Protein Data Bank code 3V3Q) was used. Docking was performed with

the implemented standard routine in Glide. The Glide GScore was used as docking score to rank

the docking results. Poses were further visually investigated to check for their interactions with

the protein in the docking site. Schrödinger's Maestro was used as the primary graphical user

interfaces for the visualization of the crystal structure and docking results.

Surface plasmon resonance (SPR)

The binding kinetics between Nur77-LBD and compounds were performed on a BIAcore T200

instrument (GE Healthcare) at 25℃ (24). Nur77-LBD were diluted to 0.05 mg/mL in 50 mM

NaOAc (pH 5.0) and immobilized on a CM5 sensor chip (GE Healthcare) by amine coupling at

densities ~10000 RU according to the manufacturer’s instructions. Gradient concentrations of

compounds were injected into the flow cells in running buffer (PBS, 0.4% DMSO) at a flow rate

of 30 μL/min for 150 s of association phase followed by a 420 s dissociation phase and a 30 s

regeneration phase (10 mM Glycin-HCl, pH 2.5). The data were analyzed using BIAcore T200

Evaluation Software 2.0 and referenced for blank injections and reference Surface. The

dissociation constant (Kd) was fitted to the standard 1:1 interaction model and calculated using

the global fitting of the kinetic data from gradient concentrations.

Statistical analysis

Data were expressed as mean ± SD. Each assay was repeated in triplicate in three independent

experiments. The statistical significance of the differences among the means of several groups

was determined using Student’s t-test.

Results




12

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

In our effort to identify small molecules that modulate the Nur77/Bcl-2 apoptotic pathway, we

evaluated an in-house compound library, which includes di(1H-indol-3-yl)(4-

(trifluoromethyl)phenyl)methane (DIM-C-pPhCF3, Fig. 1A) (28). We surprisingly observed that

the freshly prepared DIM-C-pPhCF3 solution was not as active as the aged solution in apoptosis

induction. In addition, the freshly made DIM-C-pPhCF3 solution is colorless, however it turns

reddish when it is aged or exposed to air at room temperature. Thus, we presumed that the

compound underwent oxidization and the oxidized DIM-C-pPhCF3 was more active than DIM-

C-pPhCF3. To test our hypothesis, DIM-C-pPhCF3 was oxidized in the presence of

methanesulfonic acid, and the products were subsequently purified to obtain the oxidized DIM-

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

pPhCF3+MeSO3

-) (BI1071, Fig. 1A and Supplementary Methods for synthesis and purification).

BI1071 was then tested in comparison with DIM-C-pPhCF3 for growth inhibition and apoptosis

induction. Fig. 1B showed that BI1071 inhibited the growth of HCT116 colon cancer cells with

an IC50 of 0.06 M, which is about 25-fold more active than DIM-C-pPhCF3 (IC50=1.5 M).

Treatment of MDA-MB-231 cells with 0.5 M BI1071 for 6 hours effectively induced PARP

cleavage, an indication of apoptosis, while DIM-C-pPhCF3 had no effect under the same

condition (Supplementary Fig. S1A). Dose-dependent study demonstrated that BI1071 could

induce PARP cleavage at submicromolar concentrations in HCT116 cells (Fig. 1C) and other

cancer cell lines (Supplementary Fig. S1B).

Interestingly, BI1071 was effective in various breast cancer cell lines analyzed regardless of its

hormone dependency (Supplementary Fig. S2). Furthermore, BI1071 did not display apoptotic




13

effect in the non-transformed mammary and normal colon cells (Supplementary Fig. S3A-B),

indicating that BI1071 selectively induces apoptosis in cancer cells. The apoptotic effect of

BI1071 was also confirmed by its induction of extensive nuclear condensation and fragmentation

revealed by DAPI staining in cells treated with 0.5 M BI1071 for 6 hours (Fig. 1D and

Supplementary Fig. S1C) and confirmed as well by PARP and caspase 3 cleavage assays (Fig.

1E). The effect of BI1071 on cell death was further assessed using flow cytometry-based

Annexin V/ Propidium iodide (PI) apoptosis assay. Dose-dependent study showed that about

31.63% of MDA-MB-231 cells were apoptotic when treated with 1 M of BI1071 for 6 hours,

while only 1.31% of cells were apoptotic in vehicle control cells (Fig. 1F).

Loss of mitochondrial membrane potential (Δψm) represents one of the hallmarks of apoptosis.

To assess whether the BI1071-induced apoptosis was related to the intrinsic mitochondrial

pathway, we used JC-1, the mitochondrial-specific dye, to monitor the changes of

mitochondrial membrane potential (29). MDA-MB-231 breast cancer cells treated with BI1071

were stained with JC-1. JC-1 dye accumulation in mitochondria is dependent of mitochondrial

membrane potential, accompanied by a shift of JC-1 fluorescence emission from green to red. In

comparison to healthy cells, apoptotic cells display an increase in the green/red fluorescence

intensity ratio. Analysis of both red and green fluorescence emissions by flow cytometry

revealed a dose-dependent BI1071 induction of mitochondrial membrane dysfunction. After

treatment with 1 M of BI1071 for 6 hours, the green to red ratio increased from 100% to 345%

(Fig. 1G). Mitochondrial dysfunction was also revealed by marked increase in intracellular

mitochondrial 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 induced

mitochondrion-related apoptosis in cancer cells.




14

BI1071 inhibits the growth of tumor cells in vivo

To assess the apoptotic effect of BI1071 in animals, SW620 colon cancer cells were inoculated

subcutaneously in the right and left hind-side flank of nude mice. Administration of tumor-

bearing nude mice with BI1071 inhibited the growth of SW620 xenograft tumor in a dose and

time-dependent manner (Figs. 2A-C). TUNEL assay revealed extensive apoptosis in BI1071-

treated tumor specimens as compared to control tumor (Fig. 2D). MMTV-PyMT-transgenic

mouse model of breast cancer was also used to evaluate the anti-cancer effect of BI1071.

Administration of the MMTV-PyMT mice with BI1071 (5 mg/kg) potently inhibited the growth

of PyMT mammary tumor (Fig. 2E and 2F). Western blotting of tumor tissues prepared from

treated and non-treated mice revealed that the expression levels of two proliferation markers,

PCNA and Ki67, were markedly reduced by BI1071 (Fig. 2G). Immunostaining also showed a

reduced expression of Ki67 and enhanced expression of cleaved caspase 3 in tumor tissue

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 the BI1071-

treated mice in both animal models (Supplementary Fig. S4A-B). These data demonstrated that

BI1071 potently inhibited the growth of tumor cells in animals through its induction of apoptosis.

BI1071 induces Nur77 dependent apoptosis and Nur77 mitochondrial targeting

We next determined whether BI1071-induced apoptosis was Nur77-dependent by examining its

apoptotic effect in mouse embryonic fibroblast (MEF) and MEF lacking Nur77 (Nur77-/-

MEF).

BI1071 dose-dependently inhibited the growth of MEFs, but such an inhibitory effect was

significantly diminished in Nur77-/-

MEFs (Fig. 3A). Induction of PARP cleavage in MEFs by




15

BI1071 was also attenuated in Nur77-/-

MEFs (Fig. 3B). The death effect of BI1071 was also

evaluated in Nur77 genome knockout HeLa cells generated by CRISPR/Cas9 technology.

Induction of PARP cleavage and caspase 3 activation by BI1071 were strongly suppressed in

Nur77-/-

HeLa cells (Fig. 3C). Annexin V/PI staining revealed a reduced apoptotic effect of

BI1071 in Nur77-/-

HeLa cells than in the parental HeLa cells (from 35.55% to 3.25%) (Fig. 3D).

Furthermore, unlike the parental HeLa cells, Nur77-/-

HeLa cells did not display BI1071-induced

mitochondrial membrane potential loss measured by JC-1 staining (Fig. 3E) or BI1071-induced

release of mitochondrial ROS (Fig. 3F). To further address the role of Nur77, we transfected the

ligand-binding domain (LBD) of Nur77, Nur77-LBD, into HEK293T cells and asked whether

the overexpression of Nur77-LBD could influence the effect of BI1071. Indeed, transfection of

Nur77-LBD enhanced the killing effect of BI1071, with 36% of the transfected HEK293T cells

undergoing apoptosis, while 4.5% of the non-transfected cells were apoptotic (Supplementary

Fig. S5). Together, these results demonstrated that BI1071 targets Nur77 to induce cancer cell

apoptosis.

Our observation that BI1071 induced mitochondria-dependent apoptosis prompted us to

determine whether BI1071 exerted its Nur77-dependent apoptosis by promoting Nur77

mitochondrial targeting. Immunostaining showed that Nur77 was mainly localized in the nucleus

of HCT116 cells. However, it was predominantly cytoplasmic when cells were treated with 0.5

M of BI1071 for 2 hours (Fig. 3G). To confirm the effect of BI1071 on Nur77 cytoplasmic

localization, HEK293T cells were transfected with GFP-Nur77 and subsequently treated with 0.5

M BI1071. Transfected GFP-Nur77 resided in the nucleus, however it was diffusely distributed

in both the cytoplasm and nucleus upon BI1071 treatment (Fig. 3H). Cells transfected with GFP-




16

Nur77-LBD also responded well to BI1071. Only for cells treated with BI1071, GFP-Nur77-

LBD colocalized extensively with the mitochondria-specific Hsp60 protein revealed by confocal

microscopy (Fig. 3I) and co-accumulated with the Hsp60 protein in the heavy membrane fraction

shown by cellular fractionation (Fig. 3J). The effect of BI1071 on inducing Nur77 mitochondrial

targeting was further illustrated by cellular fractionation experiments showing that a significant

amount of transfected Myc-Nur77 accumulated in the mitochondria-enriched heavy membrane

(HM) fraction when cells were treated with BI1071 (Fig. 3K). Taken together, these data

demonstrated that BI1071 exerted its Nur77-dependent apoptotic effect by promoting Nur77

mitochondrial targeting.

BI1071 binds Nur77 to induce its mitochondrial targeting and apoptosis

Although Nur77 lacks a LBP and no endogenous ligands have yet been identified (16,17), recent

crystallographic studies have identified several regions on the surface of the Nur77 protein as

small molecule binding regions (20,22,30). We therefore determined whether BI1071 binds

directly to Nur77 to induce its mitochondrial targeting and apoptosis. Surface plasmon resonance

(SPR) analyses revealed that DIM-C-pPhCF3 bound to Nur77-LBD with a Kd of 3.0 M (Fig.

4A) and that BI1071 bound to Nur77-LBD protein with a Kd of 0.17 M (Fig. 4B), which

demonstrated that BI1071 binding to Nur77-LBD was 18-fold stronger than DIM-C-pPhCF3. We

also evaluated the effect of BI1071 on the transcriptional activity of Nur77/RXR-LBD

heterodimer. Co-transfection of pBind-RXR-LBD and Nur77 strongly activate the reporter

transcriptional activity when cells were treated with 9-cis-RA, a RXR ligand (Fig. 4C). BI1071

further dose-dependently induced the reporter activity (Fig. 4C), likely due to its binding to

Nur77. To exclude the possibility that BI1071 acted on RXR, Glu453 and Glu456 in the




17

activation function 2 (AF2) region of RXR (31) were substituted with Ala and the resulting

mutant, RXR-LBD/E453,6A, was used to repeat the reporter assay. As expected, 9-cis-RA

failed to induce the reporter activity in cells transfected with Nur77 and pBind-RXR-LBD-

E453,6A. However, BI1071 could still activate the reporter gene transcription (Fig. 4C),

demonstrating that BI1071 induced reporter gene transcription through Nur77 binding but not

RXR. We also excluded the possibility of BI1071 binding to other nuclear receptors using

reporter assays (Supplementary Fig. S6A). We next employed molecular docking approach to

study how BI1071 bound to Nur77-LBD. Our docking studies showed that BI1071 docked better

to a binding region formed by helices H1, H5, H7 and H8, and loops H1-H2, H5-B1 and H7-H8.

The docked mode also suggested that the indole ring of BI1071 made key interaction with the

side chains of H372 and Y453 located in H1 and H5, respectively (Fig. 4D). For comparison, we

also docked DIM-C-pPhCF3 to the same region. As shown in Fig. 4D, BI1071 fit better to the

binding groove with the bis-indolyl rings embedded deeper in the groove. The bis-indolyl rings

of BI1071 also was positioned to form π-π interaction with Y453 and to make stronger

interaction with H372. To test this binding mode, H372 was mutated into either Ala or Asp.

When tested in the Gal4 reporter assay for its response to BI1071, Nur77/H372D (Fig. 4E) or

Nur77/H372A (Supplementary Fig. S6B) could not induce the reporter 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 the heavy membrane fraction in the cellular

fractionation assay (Fig. 4F). To further characterize the binding of BI1071 and its apoptotic

effect, we made 2 more mutants: mutant Nur77/Y453L, another key residue suggested by the

docking studies, and mutant Nur77/C566K, a residue located in another reported ligand-binding

region (20-24). BI1071 failed to induce PARP cleavage in cells transfected with Nur77/H372D




18

or Nur77/Y453L, while it strongly induced PARP cleavage in cells transfected with Nur77 or

Nur77/C566K (Fig. 4G). Similarly, in cells transfected with Nur77/H372D or Nur77/Y453L, the

effect of BI1071 on inducing Mito-ROS generation (Fig. 4H), on apoptosis analyzed using dual

staining with fluorescent Annexin V and PI (Fig. 4I), or on loss of mitochondrial membrane

potential (Fig. 4J) was much attenuated when compared to cells transfected with Nur77 or

Nur77/C566K. Taken together, these data demonstrated that BI1071 exerted its Nur77-dependent

apoptotic effect by a direct Nur77 binding mechanism.

BI1071-induced Nur77 mitochondrial targeting and apoptosis is Bcl-2 dependent

We previously showed that the Nur77 mitochondria-dependent apoptotic pathway involved

Nur77 interaction with Bcl-2 (14). We therefore asked if Bcl-2 plays a role in BI1071-induced

apoptosis. Thus, the apoptotic effect of BI1071 was evaluated in MEFs and MEFs lacking Bcl-2

(Bcl-2-/-

MEF). BI1071 at 0.5 M effectively induced PARP cleavage in MEFs, while it had no

apparent effect on PARP cleavage in Bcl-2-/-

MEFs (Fig. 5A). This was confirmed by DAPI

staining showing that Bcl-2-/-

MEF cells were much more resistant than MEFs to the apoptotic

effect of BI1071 (Fig. 5B). In addition, the impaired effect of BI1071 in Bcl-2-/-

MEFs could be

rescued by re-expression of Bcl-2 (Supplementary Fig. S7). In response to 0.5 M of BI1071,

40% of MEFs displayed chromatin condensation and nuclear fragmentation, whereas only 14%

of Bcl-2-/-

MEF cells exhibited similar apoptotic features. We also used the CRISPR/Cas9

technology to generate Bcl-2 knockout HeLa cells and showed that the effect of BI1071 on

inducing PARP cleavage was almost completely suppressed in Bcl-2-/-

HeLa cells (Fig. 5C). The

role of Bcl-2 in mediating the death effect of BI1071 was also illustrated by Annexin V/PI

staining showing a reduced apoptotic effect of BI1071 in Bcl-2-/-

HeLa cells compared to its




19

effect in the parental HeLa cells (from 24.59% to 7.95%), and in Bcl-2-/-

MEF cells compared to

the parental MEF cells (from 76.65% to 10.27%) (Fig. 5D). Induction of the mitochondrial

membrane potential loss by BI1071 was also suppressed in Bcl-2-/-

MEFs and Bcl-2-/-

HeLa cells

(Fig. 5E). Furthermore, BI1071-induced release of mitochondrial ROS was compromised by loss

of Bcl-2 (Fig. 5F). These results revealed a crucial role of Bcl-2 in mediating the Nur77-

dependent apoptotic effect of B1071, demonstrating that the compound acts through the Nur77-

Bcl-2 apoptotic pathway.

BI1071 promotes Nur77 interaction with Bcl-2

In this study we have showed that BI1071 can bind to Nur77 to induce its migration from the

nucleus to mitochondria, where it interacts with Bcl-2 and triggers Bcl-2-dependent apoptosis.

However, if the binding of BI1071 to Nur77 promotes the interaction between Nur77 and Bcl-2

is not clear. Therefore, we investigated whether BI1071 binding to Nur77 enhanced the Nur77

interaction with Bcl-2. In vitro GST-pull down assays showed that Nur77-LBD was pulled down

by GST-Bcl-2 in a BI1071 concentration-dependent manner (Fig. 6A). Cell-based Co-IP showed

that Nur77 (Fig. 6B) or Nur77-LBD (Fig. 6C) transfected in HEK293T cells interacted with Bcl-

2 when cells were treated with BI1071. Endogenous Nur77 could be specifically

immunoprecipitated together with endogenous Bcl-2 by anti-Bcl-2 antibody only when cells

were treated with BI1071 (Fig. 6D). Moreover, confocal microscopy analysis revealed that

BI1071 promoted extensive mitochondrial colocalization of transfected GFP-Nur77 (Fig. 6E and

Supplementary Fig. S8A) or GFP-Nur77-LBD (Fig. 6F and Supplementary Fig. S8B) with Bcl-2

in cells. To further address the role of BI1071 on inducing Nur77 interaction with Bcl-2, the

aforementioned Nur77 mutants were analyzed. Fig. 6G showed that Nur77/C566K like the wild-

type Nur77 interacted strongly with Bcl-2 in a BI1071 dependent manner. In contrast,




20

Nur77/H372D and Nur77/Y453L failed to interact with Bcl-2 in the presence of BI1071. The

importance of the binding of BI1071 on inducing Nur77 interaction with Bcl-2 was also

illustrated by immunostaining showing extensive colocalization of transfected Flag Bcl-2 with

the wild-type Nur77 LBD, but not with Nur77 LBD H372A (Supplementary Fig. S8C). Thus,

binding of BI1071 to Nur77 promotes Nur77 interaction with Bcl-2 and mitochondrial

localization.

Discussion

BI1071 is a salt form of DIM-C-pPhCF3 (Fig. 1A) previously reported to induce Nur77-

dependent apoptosis (30,32). However, relative high concentrations (around 10 M) of DIM-C-

pPhCF3 are required for its induction of apoptosis and activation of Nur77. To our surprise, we

observed that aged DIM-C-pPhCF3. was generally more active than the freshly prepared one in

apoptosis induction. This led to our synthesis of the oxidized product of DIM-C-pPhCF3,

methanesulfonate salt of DIM-C-pPhCF3 (BI1071). Our evaluation of BI1071 revealed its

superior death effect in cancer cells. BI1071 was also very effective in other cancer cell lines and

in animal tumor models. The apoptotic effect of several DIM derivatives has been shown to be

Nur77-dependent and various pathways were proposed to account for their apoptotic effect (2).

However, the mechanism by which Nur77 mediates their death effect remains elusive, which is

conceivably due to the activation of multiple pathways by the high compound concentration used

in the studies. For instance, both transcriptional agonist such as DIM-C-pPhOCH3 and

transcriptional antagonist such as DIM-C-pPhOH were shown to induce Nur77-dependent

apoptosis (28). Our finding that oxidization of DIM-C-pPhCF3 could augment its death effect

offered an opportunity to delineate the mechanism by which Nur77 mediates the apoptotic effect




21

of DIM-related small molecules. To this end, our studies showed that the potent apoptotic effect

of BI1071 was Nur-77 dependent (Fig. 3) and was a result of its induction of Nur77

mitochondrial targeting via a direct Nur77 binding mechanism. Furthermore, our results revealed

that the death effect of BI1071 was also dependent of Bcl-2 expression and that BI1071 could

induce Nur77 interaction with Bcl-2 leading to Nur77 colocalization with Bcl-2 at mitochondria

and apoptosis.

Binding studies showed that BI1071 could bind to Nur77 better than DIM-C-pPhCF3. This is

likely due to the difference in the structural conformations between the oxidized and the

unoxidized forms of DIM-C-pPhCF3, perhaps resulted from different atomic orbital

hybridization of the central C atom. In the oxidized form the central C is sp2-hybridized,

positively charged and bonded to 3 atoms with a co-planner arrangement, while in the

unoxidized form, C is sp3-hybridized and bonded to 4 atoms with a tetrahedral arrangement.

Differences in structural conformation and charge distributions can affect how molecules bind to

proteins. Our docking results suggested that BI1071 could interact more strongly with Nur77-

LBD than DIM-C-pPhCF3 due to the different conformations adopted by the compounds.

Mutagenesis studies confirmed that H372 and Y453 were 2 key residues involving in the binding

of BI1071 as suggested by the docking studies. Previously, we located the critical region in

Nur77 responsible for its interaction with Bcl-2 and identified a peptide NuBCP-9 as Bcl-2-

converting peptide, capable of inducing apoptosis of cancer cells in vitro and in animals (12).

NuBCP-9 is located at the C-terminal portion of H7. Interestingly our docking studies suggested

that H7 was part of the BI1071 binding region and BI1071 could potentially interact directly

with amino acids D499 and A450, structurally flanking the residues from which NuBCP-9 is

derived. Therefore, it is conceivable that the BI1071-bound Nur77 offers a more suitable Bcl-2




22

interacting interface that promotes the formation of Nur77/Bcl-2 complex and thus augments the

biological effect of BI1071.

A critical step in the Nur77 mitochondrial apoptotic pathway is the interaction of Nur77 with

Bcl-2, which induces a conformation change in Bcl-2 and converts Bcl-2 from a pro-survival to a

killer (12,14). Members of the Bcl-2 family are critical regulators of apoptosis. As the funding

member of the Bcl-2 family, Bcl-2 acts as a survival molecule to protect cells from programmed

cell death. Bcl-2 overexpression is often observed in cancer cells and is associated with cancer

treatment resistance and poor prognosis (33-36). Thus, Bcl-2 has been an important drug target

(37,38). Two strategies are commonly used to develop therapeutic agents targeting Bcl-2: The

first relies on making use of antisense oligonucleotides to block Bcl-2 expression and the second

relies on designing and optimizing BH3 small-molecule or peptide mimetics that bind the Bcl-2

BH3-binding cleft, antagonizing its antiapoptotic activity (37,38). Like Bcl-2, Nur77 is

overexpressed in a variety of cancer cells and plays a dual role in mediating apoptosis and

survival of cancer cells (39,40). While the growth promoting effect of Nur77 appears to be

dependent on its nuclear action, the death effect of Nur77 involves its translocation from nucleus

to cytoplasm (41-43). Our results showed that cancer cells are sensitive to the treatment of

BI1071 as compared to normal cells, consisting with the fact that the level of Nur77 is elevated

in cancer cells. Thus, targeting Nur77 by BI1071 will have less effect on normal cells, and

therefore likely offer a high therapeutic index. The ability of Nur77 to interact with Bcl-2 to not

only suppress its anti-apoptotic function but also convert Bcl-2 into a pro-apoptotic molecule

(12,14) provides a promising strategy to target both Nur77 and Bcl-2 for cancer therapy. Agents

that can bind directly to Nur77 to promote Nur77 translocation and interaction with Bcl-2 are

unique in that they can simultaneously target both Nur77 and Bcl-2. The reported Nur77-derived




23

peptide with 9 amino acids (NuBCP-9) and its enantiomer as Bcl-2-converting peptides has

demonstrated such a potential (12,44,45). In this regard, our identification of small molecules

that can directly bind Nur77 to activate the Nur77-Bcl-2 apoptotic pathway is significant and

BI1071 represents the first lead of this class of small molecules, which warrants further

evaluation.

Acknowledgements

The authors thank Dr. Marcia Dawson for her contributions in chemistry to this work and to her

memory this paper is dedicated. We also thank Dr. Lin Li for her critical reading of this

manuscript.

This study was supported in part by grants from the Natural Science Foundation of China

(U1405229, 81672749, 31271453, 31471318) to X. Zhang, Regional Demonstration of Marine

Economy Innovative Development Project (16PYY007SF17) to X. Zhang, the Fujian Provincial

Science & Technology Department (2017YZ0002-1) to X. Zhang and the National Institutes of

Health (R01 CA198982).

References

1. Kurakula K, Koenis DS, van Tiel CM, de Vries CJ. NR4A nuclear receptors are orphans

but not lonesome. Biochimica et biophysica acta 2014;1843:2543-55

2. Lee SO, Li X, Khan S, Safe S. Targeting NR4A1 (TR3) in cancer cells and tumors.

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. Clinical cancer research :

an official journal of the American Association for Cancer Research 2012;18:3223-8




24

4. Pearen MA, Muscat GE. Minireview: Nuclear hormone receptor 4A signaling:

implications for metabolic disease. Molecular endocrinology 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 orphan receptors NR4A as

therapeutic target in cancer therapy. Anti-cancer agents in medicinal chemistry

2012;12:1001-14

8. McMorrow JP, Murphy EP. Inflammation: a role for NR4A orphan nuclear receptors?

Biochemical Society transactions 2011;39:688-93

9. Liu ZG, Smith SW, Mclaughlin KA, Schwartz LM, Osborne BA. Apoptotic Signals

Delivered through the T-Cell Receptor of a T-Cell Hybrid Require the Immediate-Early

Gene Nur77. Nature 1994;367:281-4

10. Woronicz JD, Calnan B, Ngo V, Winoto A. Requirement for the Orphan Steroid-

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. Molecular determinants of

AHPN (CD437)-induced growth arrest and apoptosis in human lung cancer cell lines.

Molecular and Cellular Biology 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 Cell 2008;14:285-98

13. Li H, Kolluri SK, Gu J, Dawson MI, Cao XH, Hobbs PD, et al. Cytochrome c release and

apoptosis induced by mitochondrial targeting of nuclear orphan receptor TR3. Science

2000;289:1159-64

14. Lin BZ, Kolluri SK, Lin F, Liu W, Han YH, Cao XH, et al. Conversion of Bcl-2 from

protector to killer by interaction with nuclear orphan receptor Nur77/TR3. Cell

2004;116:527-40

15. Moll UM, Marchenko N, Zhang XK. p53 and Nur77/TR3 - transcription factors that

directly target mitochondria for cell death induction. Oncogene 2006;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 and function

of Nurr1 identifies a class of ligand-independent nuclear receptors. 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 a Novel Site.

Chemistry & Biology 2014;21:596-607

19. Su Y, Zeng Z, Chen Z, Xu D, Zhang W, Zhang XK. Recent Progress in the Design and

Discovery of RXR Modulators Targeting Alternate Binding Sites of the Receptor. Curr

Top Med Chem 2017;17:663-75

20. Zhan YP, Du XP, Chen HZ, Liu JJ, Zhao BX, Huang DH, et al. Cytosporone B is 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 Unique

Pharmacophore for Activation of the Nuclear Orphan Receptor Nur77 In vivo and In

vitro. Cancer Research 2010;70:3628-37




25

22. Zhan YY, Chen Y, Zhang Q, Zhuang JJ, Tian M, Chen HZ, et al. The orphan nuclear

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. Orphan nuclear receptor

TR3 acts in autophagic cell death via mitochondrial signaling pathway. Nat Chem Biol

2014;10:133-40

24. Hu M, Luo Q, Alitongbieke G, Chong S, Xu C, Xie L, et al. Celastrol-Induced Nur77

Interaction with TRAF2 Alleviates Inflammation by Promoting Mitochondrial

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. Nitrostyrene Derivatives Act as

RXRalpha Ligands to Inhibit TNFalpha Activation of NF-kappaB. Cancer Res

2015;75:2049-60

26. Wei W, Chen ZJ, Zhang KS, Yang XL, Wu YM, Chen XH, et al. The activation of G

protein-coupled receptor 30 (GPR30) inhibits proliferation of estrogen receptor-negative

breast cancer cells in vitro and in vivo. Cell death & disease 2014;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 of Schrodinger'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 phenyl) methanes induces

apoptosis through nuclear pathways. J Biol Chem 2005;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. Methods in enzymology 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 in prostate cancer cells.

Oncogene 2001;20:2927-36

31. Thompson PD, Remus LS, Hsieh JC, Jurutka PW, Whitfield GK, Galligan MA, et al.

Distinct retinoid X receptor activation function-2 residues mediate transactivation in

homodimeric and vitamin D receptor heterodimeric contexts. J Mol Endocrinol

2001;27:211-27

32. Hedrick E, Lee SO, Kim G, Abdelrahim M, Jin UH, Safe S, et al. Nuclear Receptor 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. Science

1998;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 and future

prospects. Blood 2008;111:3322-30

36. Maji S, Panda S, Samal SK, Shriwas O, Rath R, Pellecchia M, et al. Bcl-2 Antiapoptotic

Family Proteins and Chemoresistance in Cancer. Advances in cancer research

2018;137:37-75

37. Ashkenazi A, Fairbrother WJ, Leverson JD, Souers AJ. From basic apoptosis discoveries

to advanced selective BCL-2 family inhibitors. Nat Rev Drug Discov 2017;16:273-84

38. Croce CM, Reed JC. Finally, An Apoptosis-Targeting Therapeutic for Cancer. Cancer

Res 2016;76:5914-20




26

39. Xie L, Jiang F, Zhang X, Alitongbieke G, Shi X, Meng M, et al. Honokiol sensitizes

breast cancer cells to TNF-alpha induction of apoptosis by inhibiting Nur77 expression.

British journal of pharmacology 2016;173:344-56

40. Wu H, Lin Y, Li W, Sun Z, Gao W, Zhang H, et al. Regulation of Nur77 expression by

beta-catenin and its mitogenic effect in colon cancer cells. FASEB journal : official

publication of the Federation of American Societies for Experimental Biology

2011;25:192-205

41. Kolluri SK, Bruey-Sedano N, Cao X, Lin B, Lin F, Han YH, et al. Mitogenic effect of

orphan receptor TR3 and its regulation by MEKK1 in lung cancer cells. Mol Cell Biol

2003;23:8651-67

42. Lee SO, Abdelrahim M, Yoon K, Chintharlapalli S, Papineni S, Kim K, et al.

Inactivation of the Orphan Nuclear Receptor TR3/Nur77 Inhibits Pancreatic Cancer Cell

and Tumor Growth. Cancer Research 2010;70:6824-36

43. Lee SO, Andey T, Jin UH, Kim K, Sachdeva M, Safe S. The nuclear receptor TR3

regulates mTORC1 signaling in lung cancer cells expressing wild-type p53. 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 based biodegradable nanoparticles:

Studies on antitumor efficacy of BCL-2 converting peptide, NuBCP-9. International

journal of pharmaceutics 2016;511:876-89

45. Kumar M, Gupta D, Singh G, Sharma S, Bhat M, Prashant CK, et al. Novel polymeric

nanoparticles for intracellular delivery of peptide Cargos: antitumor efficacy of the BCL-

2 conversion peptide NuBCP-9. Cancer Res 2014;74:3271-81




27

Figure legends

Figure 1. A salt form of DIM-C-pPhCF3 exhibits superior apoptotic effect.

A. Structure of DIM-C-pPhCF3 and DIM-C-pPhCF3+MeSO3

- (BI1071). B. HCT116 cells treated

with the indicated concentration of BI1071 or DIM-C-pPhCF3 (labeled as CF3) for 3 days were

assessed by MTT assay. Data are shown as mean ± SD (n=6). C. HCT116 cells treated with the

indicated concentration of BI1071 for 6 hours were analyzed for PARP cleavage by Western

blotting. D. HCT116 cells treated with 0.5 M BI1071 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-231 cells treated with the indicated concentration of BI1071 for 6 hours

was determined by Western blotting. F. Annexin V/PI staining of MDA-MB-231 cells treated

with the indicated concentration of BI1071 for 6 hours was analyzed by flow cytometry. G.

MDA-MB-231 cells treated with the indicated concentration of BI1071 for 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 5 independent

images. *P< 0.1, ***P< 0.001 (Student’s t test). H. Mitochondrial ROS production in MDA-

MB-231 treated with the indicated concentration of BI1071 for 6 hours was analyzed by flow

cytometry. For Western blots and flow cytometry experiments, one of three similar experiments

are shown.

Figure 2. Effect of BI1071 on tumor growth and apoptosis in animals.

A. Nude mice (n=6) injected with SW620 (2X106 cells) were administrated with the indicated

dose of BI1071 once a day and tumors were measured every three days. B-C. 12 days after

administration of BI1071, nude mice bearing SW620 tumor were sacrificed and tumors were

removed, weighted and showed (***P< 0.001, Student’s t test). D. Representative TUNEL

staining images illustrating the apoptotic effect of BI1071. The apoptotic cells were detected by

TUNEL assay in specimens of xenograft tumors. E. Representative images of MMTV–PyMT

mammary tumor model mice and tumors from mice administered with or without BI1071. For

MMTV–PyMT mammary tumor model, female wild-type MMTV-PyMT mice of 12 weeks old

were randomly divided into two groups (n=7), treated with daily oral doses of BI1071 (5 mg/kg)

for 18 days. F. Inhibition of PyMT tumor growth by BI1071. Mice treated with BI1071 as in E,

and tumors were weighted (n=7). G. Western blot analysis of the expression of PARP, Ki67 and




28

PCNA in tumor tissues prepared from 3 MMTV-PyMT mice treated with or without BI1071 (5

mg/kg) for 18 days. H. Representative immunocytochemistry staining showing the expression of

Ki67 and cleaved caspase 3 in tumor tissues prepared from MMTV-PyMT mice 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 BI1071 for 6 hours were

assessed by MTT assay. (**P< 0.01 and ***P< 0.001, Student’s t test). B. PARP cleavage in

MEFs or Nur77-/-

MEFs treated with 0.5 M BI1071 for 6 hours was determined by Western

blotting. C-F. HeLa or Nur77-/-

HeLa cells were treated with 0.5 M BI1071 for 6 hours. Cells

were then subjected to Western blotting for PARP cleavage and cleaved caspase 3 detecting (C),

Annexin V/PI staining for apoptosis measurement (D), JC-1 staining for measuring

mitochondrial membrane potential (E) or mito-SOX staining for determining the production of

mitochondrial ROS (F). One of three similar experiments is shown. NS, not significant; ***P<

0.001 (Student’s t test).G. Subcellular localization of endogenous Nur77 in HCT116 cells

treated with 0.5 M BI1071 for 2 hours was analyzed by confocal microscopy after

immunostained with anti-Nur77 antibody. Nucleus were visualized by DAPI staining. H. HeLa

cells transfected with GFP-Nur77 were treated with 0.5 M BI1071 for 2 hours and visualized

by confocal microscopy. I. HeLa cells transfected with GFP-Nur77-LBD were treated with

BI1071 (0.5 M) for 2 hours were immunostained with anti-Hsp60 antibody and visualized by

confocal microscopy. J. HEK293T cells were transfected with GFP-Nur77-LBD and treated with

0.5 M BI1071 for 2 hours. Cytosolic (Cyt) and HM fraction were then prepared and analyzed

by Western blotting. Expression of cytoplasmic IB and mitochondrial Hsp60 was shown to

indicate the purity of cytosolic and mitochondrial fraction, respectively. K. HEK293T cells were

transfected with Myc-Nur77. Whole cell lysate (WCL) and mitochondria-enriched heavy

membrane (HM) fractions were prepared from HEK293T cells treated with 0.5 M BI1071 for 2

hours, and analyzed by Western blotting. Expression of nuclear PARP and mitochondrial Hsp60

was shown to ensure the purity of HM fraction. For cellular fractionation experiments, one of

three similar experiments are shown.

Figure 4. BI1071 binds directly to Nur77.




29

A, B. Binding of DIM-C-pPhCF3 (A) or BI1071(B) to purified Nur77-LBD by SPR. C.

HEK293T cells were transfected with Gal-4 reporter plasmid and Gal-4-RXR-LBD or Gal-4-

RXR-LBD/E453,6A together with Myc-Nur77, and treated with the indicated concentration of

BI1071 or 9-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) to Nur77-LBD. E.

HEK293T cells transfected with Gal-4 reporter plasmid and Gal-4-RXR-LBD together with

Myc-Nur77 or Myc-Nur77/H372D were treated with the indicated concentration of BI1071, and

reporter activities were measured. **P< 0.01, ***P< 0.001 (Student’s t test). F. HEK293T cells

were transfected with Myc-Nur77, Myc-Nur77/H372A and treated with BI1071 (0.5 M) for 2

hours. WCL and HM fractions were then prepared and analyzed by Western blotting. G−J.

Nur77-/-

HeLa cells were transfected with the indicated Nur77 and mutant plasmids and treated

with 0.5 M BI1071 for 6 hours. Cells were then subjected to Western blotting for PARP

cleavage (G), mito-SOX staining for determining the production of mitochondrial ROS (H),

Annexin V/PI staining for apoptosis (I), or JC-1 staining for measuring mitochondrial membrane

potential (J). One of three similar experiments are shown. NS, not significant; ***P< 0.001

(Student’s t test).

Figure 5. Bcl-2 dependent induction of apoptosis by BI1071.

A, B. MEFs or Bcl-2-/-

MEFs were treated with 0.5 M BI1071 for 6 hours. Cells were then

subjected to Western blot analysis for PARP cleavage (A) or DAPI staining for apoptosis (B).

Apoptotic cells were counted in 200 cells. ***P< 0.001 (Student’s t test). C. PARP cleavage in

HeLa or Bcl-2-/-

HeLa cells treated with 0.5 M BI1071 for 6 hours was analyzed by Western

blotting. D-F. MEFs or Bcl-2-/-

MEFs, HeLa cells or Bcl-2-/-

HeLa cells were treated with 0.5 M

BI1071 for 6 hours. Cells were subjected to Annexin V/PI staining for apoptosis measurement

(D), JC-1 staining for measuring mitochondrial membrane potential (E) or mito-SOX staining for

determining the production of mitochondrial ROS (F). One of three similar experiments are

shown. *P< 0.1, ***P< 0.001 (Student’s t test).

Figure 6. BI1071 promotes Nur77 interaction with Bcl-2.




30

A. GST pull-down. Purified Nur77-LBD incubated with or without the indicated concentration

of BI1071 was pulled down by GST or GST-Bcl-2 protein and analyzed by Western blotting. B,

C. Co-immunoprecipitation assay. HEK293T transfected with Myc-Bcl-2 together with GFP-

Nur77 (B) or GFP-Nur77-LBD (C) were treated with or without 0.5 M BI1071 and analyzed by

co-immunoprecipitation assays using anti-Myc antibody. D. Interaction of endogenous Nur77

and Bcl-2 in MDA-MB-231 cells treated with or without 0.5 M BI1071 for 2 hours was

analyzed by co-immunoprecipitation assay using anti-Bcl-2 antibody. E, F. Colocalization of

Nur77 with Bcl-2. HEK293T cells were transfected with Myc-Bcl-2 together with GFP-Nur77

(E) or GFP-Nur77-LBD, treated with or without 0.5 M BI1071 for 2 hours, stained with anti-

Myc antibody, and visualized using confocal microscopy. G. HEK293T cells transfected with

the indicated expression plasmids were treated with 0.5 M BI1071 for 2 hours and analyzed by

co-immunoprecipitation assays using anti-Flag antibody. For Co-IP experiments, one of three

similar experiments are shown.




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:

Manuscript

Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been

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

Subscriptions

Reprints and

[email protected] at

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

Permissions

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

.http://mct.aacrjournals.org/content/early/2019/03/29/1535-7163.MCT-18-0918To 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/cgi/alerts

mailto:[email protected]

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


bi1071, a novel nur77 modulator, induces apoptosis of cancer … · 2019. 3. 29. · the confocal...

Documents