Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2021 Sep 1.
Published in final edited form as: Mol Cancer Ther. 2020 Dec 4;20(3):612–622. doi: 10.1158/1535-7163.MCT-20-0474

THE HISTONE METHYLTRANSFERASE GENE G9A IS REGULATED BY NUCLEAR RECEPTOR 4A1 (NR4A1) IN ALVEOLAR RHABDOMYOSARCOMA CELLS

Rupesh Shrestha 1, Kumaravel Mohankumar 2, Un-ho Jin 2, Gregory G Martin 2, Stephen Safe 1,2
PMCID: PMC7933077  NIHMSID: NIHMS1653494  PMID: 33277444

Abstract

The histone methyltransferase G9A (EHMT2) gene catalyzes methylation of histone 3 lysine 9 (H3K9) and this gene silencing activity contributes to the tumor promoter-like activity of G9A in several tumor types including alveolar rhabdomyosarcoma (ARMS). Previous studies show the orphan nuclear receptor 4A1 (NR4A1, Nur77) is overexpressed in rhabdomyosarcoma and exhibits pro-oncogenic activity. In this study, we show that knockdown of NR4A1 in ARMS cells decreased expression of G9A mRNA and protein. Moreover, treatment of ARMS cells with several bis-indole – derived NR4A1 ligands (antagonists) including 1,1-bis(3΄-indolyl)-1-(4-hydroxyphenyl)methane (CDIM8), 3,5-dimethyl (3,5-(CH3)2) and 3-bromo-5-methoxy (3-Br-5-OCH3) analogs also decreased G9A expression. Furthermore, NR4A1 antagonists also decreased G9A expression in breast, lung, liver and endometrial cancer cells confirming that G9A is an NR4A1-regulated gene in ARMS and other cancer cell lines. Mechanistic studies showed that the NR4A1/Sp1 complex interacted with the GC-rich – 511 region of the G9A promoter to regulate G9A gene expression. Moreover, knockdown of NR4A1 or treatment with NR4A1 receptor antagonists decreased overall H3K9me2, H3K9me2 associated with the PTEN promoter and PTEN-regulated phospho-Akt. In vivo studies showed that the NR4A1 antagonist (3-Br-5-OCH3) inhibited tumor growth in athymic nude mice bearing Rh30 ARMS cells and confirmed that G9A was an NR4A1 regulated gene that can be targeted by NR4A1 receptor antagonists.

Keywords: Gene regulation, Nuclear receptor, Sarcomas

INTRODUCTION

Covalent modifications of histones by acetylation, ubiquitination and methylation play a pivotal role in epigenetic modifications of gene expression required for maintaining cellular homeostasis and for abnormal pathophysiology (1, 2). Histone H3 and histone H4 are common methylation sites and the methyl transferase G9A (EHMT2) gene primarily catalyzes methylation of histone 3 lysine 9 (H3K9) (3, 4) and this determines some of its cellular functions and role in multiple diseases (2). G9A forms a heterodimeric complex with GLP/Eu -HMT-ase to catalyze mono- and dimethylation of H3K9 (4) and this significantly contributes to the reported pro-oncogenic functions of G9A in multiple tumor types. For example; G9A promotes liver cancer by epigenetic silencing of RARRES3 and also inhibits expression of multiple tumor suppressor genes in aggressive ovarian cancers. In breast cancer, expression of G9A enhances hypoxia and related genes and in endometrial cancer, G9A increases invasion by repression of E-cadherin (5–9). There are several other examples of cancer cell-specific G9A-mediated gene suppression playing a key pro-oncogenic role and this includes regulation of p21, p53 and mTOR gene expression (10–14). In addition, G9A also promotes gastric cancer metastasis through its activity as a nuclear cofactor in combination with p300 and the glucocorticoid receptor to activate expression of β3-integrin (15). A recent study also reported that G9A epigenetically regulated PTEN in alveolar rhabdomyosarcoma (ARMS) cells thereby activating Akt and downstream pro-oncogenic pathways (16).

These data illustrate the importance of G9A-dependent gene repression and in some cases, gene activation in enhancing carcinogenesis in multiple tumor types and not surprisingly, G9A inhibitors have been developed as potential cancer chemotherapeutic agents (2). Compounds that competitively bind to the substrate binding site of G9A, and the S-adenosyl methionine G9A co-factor binding site, along with G9A inhibitors with nuclear mechanisms of action, have been developed (2). The pro-oncogenic activity of G9A has been associated with cell context-specific repression of diverse genes however, the functional properties of G9A after knockdown or inhibition are similar across cell lines. For example, in ARMS cells and in fifteen ARMS patient samples, G9A is overexpressed compared to normal muscle cells and knockdown of G9A inhibits ARMS cell growth, differentiation and migration (16). Studies in this laboratory have reported that the orphan nuclear receptor 4A1 (NR4A1, Nur77) is also overexpressed in ARMS cells and knockdown of NR4A1 or treatment with bis-indole derived NR4A1 antagonists (C-DIMs) inhibit ARMS cell growth and migration, and induce apoptosis (17, 18). These responses have been linked to modulation of NR4A1 regulated genes including the PAX3-FOX01 fusion oncogene that plays an important role in ARMS carcinogenesis (17). Thus, both G9A and NR4A1 regulate comparable pro-oncogenic responses in ARMS cells and therefore we hypothesized that NR4A1 may also regulate G9A expression. Our results now demonstrate that G9A is an NR4A1 regulated gene in ARMS cells and C-DIM/NR4A1 antagonists effectively downregulate G9A and represent a novel class of G9A inhibitors. Moreover, we also show similar effects on NR4A1 regulation of G9a in a panel of cancer cell lines derived from multiple tumors.

MATERIALS AND METHODS

Cell lines, reagents and antibodies:

Rh30 (RMS), MDA-MB-231 and MDA-MB-468 (breast cancer), A549, H1299 and H460 (lung cancer), SNU449, HUH7, and HepG2 (liver cancer), Ishikawa (endometrial cancer), HCT116 (colon cancer), and PC3 (prostate cancer) cell lines were purchased from American Type Culture Collection (Manassas, VA). Rh41 (RMS) was a generous gift from Mr. Jonas Nance, Texas Tech University Health Sciences Center- Children’s Oncology Group (Lubbock, TX). Human mammary tumor Sum159PT and HS578T cell lines were generously provided by Dr. Weston Porter, Texas A&M University (College Station, TX). Mouse mammary tumor 4T1 cell line was kindly provided by Dr. Mien-Chie Hung, MD Anderson Cancer Center (Houston, TX). Hec-1B cell line was a generous gift from Dr. Russell Broaddus, MD Anderson Cancer Center (Houston, TX). Rh30, MDA-MB-231 and A549 cells were authenticated by Biosynthesis. All tumor cells used in these studies were Mycoplasma negative. Rh30, H1299, H460, SNU449 and HCT116 cells were maintained in RPMI medium. Rh41 cell line was maintained in IMDM medium. HS578T, MDA-MB-231, MDA-MB-468, A549, HUH7, HepG2 and PC3 cells were maintained in DMEM medium. Sum159PT, 4T1, Ishikawa, Hec-1B cells were maintained in (DMEM)/Ham’s F-12 50/50 mix containing 2.5 mmol/L L-glutamine. All of these media were supplemented with 10% fetal bovine serum (FBS) and these cells were maintained at 37°C temperature in presence of 5% CO2. All the reagents/antibodies and the oligonucleotide sequences that were used are summarized in Supplemental Table 1 and 2 respectively. Analysis of the expression of G9a (EHMT2) in sarcomas was generated from the UALCAN database (http://ualcan.path.uab.edu/index.html). The new buttressed CDIM analogs 1,1-bis(3’-indolyl)-1-(3,5-dimethyl-4-hydroxyphenyl)methane [3,5-(CH3)2] and 1,1-bis(3’-indolyl)-1-(3-bromo-4-hydroxy-5-methoxyphenyl)methane (3-Br-5-OCH3) were synthesized by condensation of indole with 3,5-dimethylbenzaldehyyde and 3-bromo-5-methoxybenzaldehyde (Sigma Aldrich, St. Louis, MO) respectively and 1,1-bis(3΄-indolyl)-1-(p-hydroxyphenyl)methane (CDIM8) was synthesized by the condensation of indole and p-hydroxybenzaldehyde. The reaction conditions for synthesis of the CDIMs was performed as described (19). The purities of both compounds were > 98% and their nuclear magnetic resonance spectrum are included in the supplemental data in Table 1. LC-MS was determined using a SHIMADZU 2010 EV using methanol as solvent.

siRNA interference assay:

Cells (2.0 × 105) were seeded in a medium supplemented with 10% FBS and were allowed to attach. After 24 hours, they were transfected with 100 nM of desired siRNAs using 50 umol/L of Lipofectamine-2000 in reduced serum medium. After 6 hours, the medium was removed and replaced with fresh medium supplemented with 10% FBS. The cells were then lysed after 48–72 hours with lysis buffer and the lysates were further used for western blot analysis.

Western blot analysis:

Cell lysates were obtained either from siRNA interference assay or by lysing the cells that have been treated with the desired compounds for 24 hours. The total protein in those lysates were quantified by Bradford assay. The protein content in all the lysates were then normalized and the equal amount of protein was loaded and was allowed to run on SDS polyacrylamide gel, connected to an electric source. The overall protein on the gel was then transferred to a PVDF membrane, which was then blocked using 5% skimmed milk for an hour. After that, it was incubated overnight with primary antibody that detects and binds the specific protein of interest. The membrane was then washed with TBST and then incubated with HRP-linked secondary antibody for 2 hours. After that, the membrane was once again washed with TBST. The chemiluminescent HRP-substrate was then added to the blot and Kodak 4000 MM Pro image station (Molecular Bioimaging, Bend, OR) was used to detect the protein of interest in the membrane.

NR4A1-CDIMs binding assays:

The purified ligand binding domain (LBD) of NR4A1 protein was incubated with different concentrations of CDIM compounds and was used to obtain tryptophan fluorescence spectra with the excitation wavelength of 285 nm (slit width = 5 nm) and an emission wavelength of range 300–420 nm (slit width = 5 nm). The binding affinity (Kd) of CDIM8 analogs to NR4A1 was further determined by measuring NR4A1 tryptophan fluorescence intensity at emission wavelength of 330 nm. The binding affinity (Kd) and binding stoichiometry (Bmax) of NR4A1/bisANS was determined as described (20). The ligand binding affinity (Kd) of CDIM8 analogs to NR4A1 was determined by measuring ligand-dependent decrease of NR4A1/ bisANS fluorescence intensity at emission wavelength of 500 nm. Ligand/bisANS fluorescence intensity at each ligand concentration was used to correct the NR4A1/bisANS/ligand fluorescence intensity as described (21).

Polymerase Chain Reaction (PCR):

Cells (2.0 × 105) were seeded in a medium containing 10% FBS and were allowed to attach for 24 hours. The medium was then removed and replaced with fresh medium supplemented with 2.5% charcoal stripped FBS that also contained the desired compounds. After 24 hours, the cells were lysed and the RNA was extracted from them using Zymo Research Quick-RNA Miniprep kit (Irvine, CA) by following the manufacturer’s protocol. The total RNA content was measured and then normalized. The high capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Waltham, MA) was then used to prepare cDNA from the isolated RNA, which was then used to quantify the total mRNA of the gene of interest by quantitative real-time PCR using amfiSure qGreen Q-PCR master mix (genDEPOT, Katy, TX). The relative mRNA expression of the desired genes was determined by using human TATA binding protein mRNA as a control.

Chromatin immunoprecipitation (ChIP) assay:

The ChIP-IT express enzymatic kit (Active Motif, Carlsbad, CA) was used and the manufacturer’s protocol was followed in order to perform this assay. Rh30 cells were treated with DMSO, CDIM8 or mithramycin for 24 hours and were then fixed with formaldehyde. Glycine was then used to stop the cross-linking reaction and the cells were scraped, collected and lysed to collect the nuclei which were then sonicated and sheared to get the chromatin fragments. Immunoprecipitation was then performed with the sheared chromatin fragments with protein specific antibodies (NR4A1, Sp1, IgG, PolII, or H3K9me2) in presence of protein G-conjugated magnetic beads for overnight. The beads were then washed with provided ChIP buffers, chromatin fragments were eluted, the protein-DNA cross-links were reversed and finally the DNA was obtained by protein K digestion. PCR was then performed with the designed primers for the promoters for specific genes (G9a or PTEN). The amplified fraction of the promoter was then resolved on 2% agarose gel in presence of ethidium bromide.

DNA-protein binding assay:

Rh30 cells (2.0×106) were seeded in a medium supplemented with 10% FBS and were allowed to attach for 24 hours. The Abcam (Cambridge, UK) nuclear extraction kit (ab113474) was then used and the manufacturer’s protocol was followed in order to extract the nuclear protein from the cells. This nuclear protein was used with the Abcam DNA-protein binding assay kit (ab117139) and the manufacturer’s protocol was followed to quantify the interaction of Sp1 protein with G9a promoter. The G9a oligonucleotide probes used were: WT, 5’-CCGGGGCGGC-3’; Mutant, 5’-CCGTGTCGGC-3’.

Animal studies:

Female athymic nude mice (3–4 weeks old) were purchased from Envigo Rms, LLC (Indianapolis, IN) and were housed at Lab Animal Care Center, Texas A&M University. The protocol for the animal studies was approved by Institutional Animal Care and Use Committee (IACUC) at Texas A&M University. The mice were allowed to acclimate for a week and were fed standard chow diet. Rh30 cells (4.0 × 106) cultured in RPMI medium supplemented with 10% FBS were detached by trypsinization, washed with sterile PBS, and then resuspended in 100 ul of PBS and matrigel in 1:1 ratio. These cells were then injected into the mice subcutaneously. After the tumor size were palpable (~50 to 100 mm3 in size), the mice were randomly divided into two groups – control and treatment groups. The mice in the control group were injected with 100 ul corn oil whereas the mice in the treatment group were injected with 100 ul of 12.5 mg/kg 3-Br-5-OCH3 prepared in corn oil every other day intraperitoneally. The mice were weighed every week and the tumor volume in each mouse was calculated using a Vernier Calliper (V = L*W*W/2 mm3). After three weeks, all the mice were sacrificed. The tumor from each mouse was then removed and weighed. A small piece of fresh tumor was homogenized in lysis buffer and was further used for western blot and PCR studies.

Cell survival (XTT) assay:

Cells (1.0×104) were seeded using 10% FBS containing medium and were allowed to attach for 24 hours. The medium was then replaced with a fresh medium containing 2.5% stripped charcoal serum supplied with the desired concentration of compounds for 24 hours. The XTT cell viability kit (Cell Signaling Technology, Danvers, MA) was then used and the manufacturer’s protocol was followed to calculate the percentage of cell survival. Results for this are now illustrated in Supplemental Figure 1.

Statistical Analysis:

Statistical significance of differences between the treatment groups was determined by Student’s t-test. Each experiment was performed three times and the results were presented as means with error bars representing 95% confidence intervals. Data with a p value of less than 0.05 were considered statistically significant.

RESULTS

Previous studies showed that G9A was highly expressed in Rh30 and Rh41 ARMS cells (16) and examination of UALCAN and TCGA databases showed that in sarcoma patients, high expression of G9A was associated with decreased survival (Fig. 1A). In a limited data set, G9A is also expressed more in primary tumors than in non-tumor tissues (Fig. 1B). The major focus of this paper is to report our studies showing that the orphan nuclear receptor NR4A1 regulates G9a expression in ARMS cells. Knockdown of NR4A1 by RNA interference (RNAi) using multiple oligonucleotides in Rh30 and Rh41 cells decreased expression of NR4A1 and G9A (Fig. 1C) whereas knockdown of G9A by RNAi decreased expression of G9A but not NR4A1 proteins (Fig. 1D). These results indicate that NR4A1 regulates expression of G9A in ARMS cells whereas knockdown of G9A has minimal effects on NR4A1. 1,1-Bis(3’-indolyl)-1-(p-hydroxyphenyl)methane (CDIM8) is a prototypical NR4A1 antagonist in cancer cells including ARMS cells (18) and the 3,5-(CH3)2 and 3-Br-5-OCH3 buttressed analogs of CDIM8 (Fig. 2A) bind NR4A1 and quench fluorescence of tryptophan in the ligand binding domain (Fig. 2B) as described (20)and the growth inhibitory effects of these compounds are summarized in Supplemental Figure 1. Supplemental Figure 2A shows that the NR4A1 antagonists also decrease NR4A1/ bisANS fluorescence intensity as described (20). Treatment of Rh30 and Rh41 cells with CDIM8 decreased levels of G9A protein and this was also accompanied by decreased NR4A1 protein (Fig. 2C). CDIM8 also decreased expression of G9A mRNA levels in Rh30 and Rh41 cells (Fig. 2D). We also used buttressed CDIM8 analogs 3,5-(CH3)2 and 3-Br-5-OCH3 (21–23) and investigated their effects on G9A expression in ARMS cells. Like C-DIM8, both compounds inhibited growth of Rh30 and Rh41 cells (Supplemental Figure 1) and decreased G9A protein and mRNA levels in Rh30 (Figs 2E and 2F) and Rh41 (Figs. 2G and 2H) cells. These results suggest that NR4A1 regulates G9A expression in ARMS cells and this was further investigated in a panel of NR4A1-expressing cancer cell lines (Fig. 3). NR4A1 and G9A are also co-expressed in a panel of breast (Fig. 3A), lung and liver (Fig. 3B) and endometrial, colon and prostate (Fig. 3C) cancer cells. Moreover, in a subset of these cell lines (MDA-MB-231, H1299, SNU449, Ishikawa and Hec1B), treatment with C-DIM8 (Fig. 3D) or knockdown of NR4A1 by RNAi (Fig. 3E) decreased levels of G9A protein and these results were consistent with those observed in ARMS cells suggesting that NR4A1 regulates G9a in multiple cancer cell lines.

Figure 1.

Figure 1.

Open in a new tab

G9a (EHMT2) is an NR4A1 regulated gene in ARMS. High expression of EHMT2 is a negative prognostic factor for sarcoma patient survival (A) and is more highly expressed in tumors vs. normal (B). Rh30 and Rh41 ARMS were transfected with oligonucleotides targeting NR4A1 (siNR4A1) (C) and G9a (siG9a) (D) and whole cell lysates were analyzed by western blots as outlined in the Methods.

Figure 2.

Figure 2.

Open in a new tab

NR4A1 ligands act as antagonists and decrease G9a expression in ARMS cells. A. Structures of CDIM8 and buttressed analogs. B. The Kd values for 3,5-(CH3)2 and 3-Br-5-OCH3 interactions with the ligand binding domain of NR4A1 were determined by fluorescent quenching of the tryptophan residue in the binding pocket as outlined in the Methods. Rh30 and Rh41 cells were treated with CDIM8 (C, D), 3,5-(CH3)2 and 3-Br-5-OCH3 (E, F, G, H) and effects on gene products and mRNA levels were determined by western blots and real time PCR respectively as outlined in the Methods. Results (D, F and H) are expressed as means ± SD for at least 3 replicated determinations for each treatment group and significantly (p<0.05) decreased responses are indicated.

Figure 3.

Figure 3.

Open in a new tab

G9a is an NR4A1 regulated gene in multiple cancer cell lines. Lysates from several breast (A), lung and liver (B), endometrial, colon and prostate (C) cancer cell lines were analyzed by western blots as outlined in the Methods. Selected breast (MDA-MB-231), lung (H1299), liver (SNU449) and endometrial (Ishikawa and Hec1B) cancer cell lines were treated with the NR4A1 antagonist CDIM8 (D) or transfected with siNR4A1 (2 oligonucleotides) (E) and whole cell lysates were analyzed by western blot as outlined in the Methods.

Previous studies in RMS and other cell lines show that NR4A1 regulates multiple genes containing GC-rich promoters by acting as a nuclear co-factor for DNA bound Sp1 or Sp4, (17, 24–28) and this was previously observed for NR4A1 regulation of PAX3-FOX01 and β1-integrin in ARMS cells (17). Figure 4A illustrates that G9A contains a consensus GC-rich promoter site and knockdown of Sp1 (Fig. 4B) but not Sp4 (Fig. 4C) decreased expression of G9A in Rh30 and Rh41 cells. The role of Sp1 in regulating G9A expression was further confirmed by showing that mithramycin, a drug that binds GC-rich sites to inhibit Sp-dependent gene expression (17) also decreased expression of G9A protein (Fig. 4D) and mRNA (Fig. 4E) in Rh30 and Rh41 cells. ChIP analysis shows that Sp1, pol II and NR4A1 bind to the GC-rich region of the G9A gene promoter and after treatment of Rh30 cells with CDIM8 or mithramycin for 24 hours, we observed decreased binding of Sp1, NR4A1 and pol II to the G9A promoter (Fig. 4F). These results are consistent with previous ChIP analysis of other NR4A1/Sp-regulated genes in RMS and other cell lines (17, 24–29). In addition, we show that Sp1 protein from nuclear extracts of Rh30 cells binds to a GC-rich oligonucleotide derived from wild type G9a promoter significantly higher in comparison to a mutant (GC) oligonucleotide in a DNA protein binding assay (Fig. 4G).

Figure 4.

Figure 4.

Open in a new tab

G9a is an NR4A1/Sp1 regulated gene in ARMS cells. A. G9a promoter and GC-rich Sp1 binding site. Rh30 and Rh41 cells were transfected with oligonucleotides targeting Sp1 (B) and Sp4 (C), and whole cell lysates were analyzed by western blots as outlined in the Methods. Rh30 and Rh41 cells were treated with mithramycin and effects on G9a protein (D) and mRNA levels (E) were determined by western blots and real time PCR respectively as outlined in the Methods. F. Rh30 cells were treated with DMSO, CDIM8 (20 μM) or mithramycin (100 nM) for 24 hours and analyzed for binding to the G9a promoter in a chromatin immunoprecipitation (ChIP) assay as outlined in the Methods and the band intensities were quantitated. G. Binding of nuclear extracts from Rh30 cells to a GC-rich oligonucleotide (identical to the GC-rich/-511 G9a promoter) was determined as outlined in the Methods. Results (E and G) are expressed as means ± SD for at least 3 replicate determinations for each treatment group and significant (p<0.05) changes compared to controls are indicated (*).

The histone methyltransferase activity of G9A primarily enhances dimethylation of H3K9 and knockdown of G9A in Rh30 and Rh41 cells decreases overall H3K9me2 expression in Rh30 and Rh41 cells (Fig. 5A). Similar results were observed after knockdown of NR4A1 (Fig. 5B) or treatment of CDIM8 (Fig. 5C) with Rh30 and Rh41 cells. UNC0642 has previously been characterized as a substrate competitive inhibitor of G9A (30) and we observed that this compound also decreased levels of H3K9me2 in Rh30 and Rh41 cells (Fig. 5D). Thus, like UNC0642, knockdown of NR4A1 or treatment with CDIM8 not only decreases G9A, but also decreases G9A-dependent levels of H3K9me2(30).

Figure 5.

Figure 5.

Open in a new tab

NR4A1/G9a regulate H3K9me2 levels in ARMS cells. The effects of knockdown of G9a (A) and NR4A1 (B), treatment with CDIM8 (C) or the G9a inhibitor UNC0642 (5 μM) (D) on G9a expression in Rh30 and Rh41 cells were determined by western blot analysis of whole cell lysates as outlined in the Methods. Quantitation of each blot (relative to β-actin) was also determined for each blot.

Previous studies in ARMS cells reported that G9A silenced PTEN thereby activating Akt (16) and therefore we further investigated effects of NR4A1 antagonists on G9A, PTEN and phosphorylated Akt in ARMS cells. Treatment of Rh30 (Fig. 6A) and Rh41 (Fig. 6B) cells with C-DIM8, 3,5-(CH3)2 and 3-Br-5-OCH3 decreased expression of phospho-Akt and these results are quantified in Supplemental Fig. 2B and 2C. Similar results were observed after treatment with mithramycin (Fig. 6C) and UNC0642 (Fig. 6D) in Rh30 and Rh41 cells demonstrating that inactivation of NR4A1 inhibits G9A-dependent phosphorylation of Akt. Mithramycin also decreases levels of Akt protein (Fig. 6C) and previous studies show that other drugs that downregulate Sp1 also decrease Akt levels in some cancer cell lines (31–33). Rh30/Rh41 cells were also treated with 3-Br-5-OCH3 and 3,5-(CH3)2 for 12 hours and this results in significant induction of PTEN mRNA levels in both cell lines (Fig. 6E). Using a similar treatment protocol, we also observed increased levels of PTEN protein in Rh30 and Rh41 cells after treatment for 9 and 12 hours; the induction response was not observed after longer treatment times (≥ 24 hours) (Fig. 6F). ChIP analysis of the PTEN promoter in Rh30 cells (Fig. 6G) showed that both CDIM8 and mithramycin decrease H3K9me2 associated with the PTEN promoter and this is consistent with their effects on decreasing G9A expression in these cells. We also investigated the effects of the NR4A1 antagonist 3-Br-5-OCH3 as an inhibitor of tumor growth in athymic nude mice bearing Rh30 cells as xenografts. Tumor volumes in control (corn oil) mice were significantly increased compared to the 3-Br-5-OCH3 treated mice (12.5 mg/kg every other day) over the 21-day duration of study (Fig. 7A). After sacrifice, the volumes (Fig. 7B) and the weights (Fig. 7C) of the excised tumors in control mice were also significantly larger/higher in comparison to the 3-Br-5-OCH3 treated mice, however their body weights remained unchanged over the treatment period (Fig. 7D). Quantitative PCR and western blot analysis of the tumor extracts showed that the treatment with the NR4A1 antagonist also decreased expression of G9a mRNA (Fig. 7E) and protein (Fig 7F). Results of both in vitro and in vivo studies were complementary and demonstrate for the first time that the histone methyltransferase G9a gene is regulated by NR4A1 in ARMS and the bis-indole derived NR4A1 antagonists target G9a and represent a novel class of G9a inhibitors.

Figure 6.

Figure 6.

Open in a new tab

siNR4A1/NR4A1 antagonists, mithramycin and UNC0642 inhibit phosphorylation of Akt. ARMS cells were treated with NR4A1 antagonists (A, B), mithramycin (C) and UNC0642 (5 μM) (D) and whole cell lysates were analyzed by western blots as outlined in the Methods and blots (6A and 6B) were quantitated in Supplemental Figures 2B and 2C. Cells were treated with 15 μM 3-Br-5-OCH3 and 3,5-(CH3)2 and PTEN mRNA levels (E) and protein (F) were determined by real time PCR and western blots respectively. G. Cells were treated with DMSO, CDIM8 (20 μM) or mithramycin (100 nm) for 24 hours and association of H3K9me2 with the PTEN promoter was determined (and quantitated) in a ChIP assay as outlined in the Methods.

Figure 7.

Figure 7.

Open in a new tab

NR4A1 antagonists inhibit tumor growth and decrease G9a expression. Athymic nude mice bearing Rh30 cells as xenografts were treated with 3-Br-5-OCH3 (12.5 mg/kg/every other day) by intraperitoneal injection over a period of 3 weeks and tumor volumes (A) and weights (B, C) and changes in body mass (D) were determined as outlined in the Methods. Real time PCR and western blot analysis were performed with the tumor extracts to obtain the expression of G9a mRNA (E) and protein (F).

DISCUSSION

The orphan nuclear receptors NR4A1, NR4A2 and NR4A3 are immediate early genes induced by multiple stressors, and the NR4A receptors play an important role in maintaining cellular homeostasis and disease. There is increasing evidence for the role of these receptors in metabolic, cardiovascular and neurological functions as well as in inflammation and inflammatory diseases and in immune functions and cancer (30, 34). NR4A1 is overexpressed in multiple solid tumors and in breast, colon and lung tumor patient’s high expression of NR4A1 is a negative prognostic factor and predicts decreased survival (34–37). The functional activity of NR4A1 in cancer has been extensively investigated in cancer cell lines by either knockdown or overexpression. In blood-derived cancers, the combined loss of NR4A1 and NR4A3 in mice results in rapid development of acute myeloid leukemia symptoms and both receptors exhibit tumor suppressor-like activity (38, 39). In contrast, NR4A1 is a pro-oncogenic factor in solid tumors and regulates one or more of cancer cell proliferation, survival, cell cycle progression, migration, and invasion in lung, melanoma, lymphoma, pancreatic, colon, cervical, ovarian, rhabdomyosarcoma and gastric cancer cell lines (17, 18, 20, 22–26, 36, 37, 40, 41). NR4A1 regulates many of the same pathways in RMS and most solid tumor-derived cancer cells, and this includes regulation of thioredoxin domain containing 5 (TXNDC5) and isocitrate dehydrogenase 1 (IDH1) which maintains high reductant levels which indirectly affect mTOR signaling. Knockdown of NR4A1 or treatment with bis-indole derived NR4A1 antagonists decreases expression of TXNDC5 and IDH1 resulting in induction of ROS and ROS-dependent sestrin2 which in turn activates AMPK and inhibits mTOR signaling (18, 21, 22, 40, 42–45). NR4A1 also acts as a cofactor for several pro-oncogenic Sp-regulated genes including bcl2/survivin, epidermal growth factor receptor (EGFR), several integrins and PAX3-FOX01 in ARMS cells and knockdown of NR4A1 or NR4A1 antagonists decrease expression of these genes (17, 20, 23–27, 40).

Recent studies showed that NR4A1 regulates β1-integrin expression in breast cancer cells, and NR4A1 antagonists inhibit β1-integrin gene expression and β1-integrin – dependent cell migration/invasion (25). NR4A1 also plays an important role in TGFβ-induced breast and lung cancer invasion and CDIM8 inhibits this response (37, 44, 45). The mechanism of regulation of several genes, including survivin, TXNDC5, and several integrins by NR4A1 involves interactions of the receptor with Sp1 or Sp4 transcription factors bound to GC-rich promoter regions of these genes. ChIP analysis shows that NR4A1, Sp1 and p300 bind to the GC-rich β1-integrin gene promoter and treatment with CDIM8 or its p-carbomethoxy analog decreases these interactions with the β1-integrin promoter and decreases expression of β1-integrin in MDA-MB-231 and SKBR3 breast cancer cells (23).

NR4A1 also plays an important pro-oncogenic role in RMS cells and regulates expression of genes associated with cell proliferation, survival and migration/invasion and this includes NR4A1/Sp4-dependent regulation of the PAX3-FOX01 fusion oncogene and β1-integrin expressed in ARMS cells (17). A recent study also reported high expression of G9A in ARMS cells and like NR4A1, G9A also regulates ARMS cell growth and migration (16). This raised the possibility that pro-oncogenic functions of NR4A1 in ARMs cells and the potent antitumorigenic activity of bis-indole derived NR4A1 antagonists (17, 18) may also be linked to the regulation of G9A. Moreover, the G9A gene promoter contains a GC-rich sequence that potentially binds Sp transcription factors (Fig. 4A) and one mechanism of NR4A1 regulation of genes is due to the receptor acting as a co-factor of Sp1 or Sp4 (17, 20, 23–27, 40).

Results illustrated in Figures 1 and 2 demonstrate that knockdown of NR4A1 by RNAi decreased expression of G9A protein and NR4A1 antagonists decreased expression of G9A protein and mRNA suggesting that G9A is an NR4A1-regulated gene that can be targeted by NR4A1 antagonists. Moreover, this is supported by comparable results in multiple cancer cell lines suggesting that NR4A1 is an upstream regulator of G9A expression (Fig. 3). We also show by protein, gene and ChIP analysis that NR4A1 and Sp1 (but not Sp4) are important for regulation of G9A and both NR4A1 and Sp1 interact with the GC-rich region of the G9A gene in a ChIP assay (Fig. 4). Interestingly our previous studies showed that NR4A1/Sp4 regulates PAX3-FOX01 gene expression in ARMS cells demonstrating that the transactivation functions of NR4A1/Sp1 and NR4A1/Sp4 are gene specific and this has previously been observed for regulation of integrins by NR4A1 (17, 24–26).

Previous studies in ARMS cells showed that G9A suppressed PTEN expression and the resulting activation of Akt was a critical factor in cell and tumor growth (16). We observed that there was a decrease in activated phospho-Akt after knockdown of G9A or treatment with UNC0642, a G9A substrate binding site inhibitor (Figs. 6A and 6B). Moreover, this was also observed after treatment with NR4A1 antagonists demonstrating that G9A is regulated by NR4A1 and can be targeted by NR4A1 antagonists. Results of in vivo studies (Fig. 7) complemented data obtained in cell culture demonstrating that NR4A1 antagonists inhibit tumor growth and this is accompanied by downregulation of G9A. These observation in ARMS cells and tumors suggests that NR4A1 through regulation of G9A also enhances tumorigenesis via epigenetic pathways in ARMS and possibly other cancer cell lines. Moreover, our studies also show that bis-indole derived NR4A1 antagonists represent a new class of G9A inhibitors that inhibit transcription of this gene and thereby act as modulators of G9a-dependent epigenetic pathways in cancer cells.

Supplementary Material

1

Acknowledgements:

This work was supported by the National Institutes of Health P30-ES023512 (SS), Kleberg Foundation (SS), Texas A&M AgriLife Research (SS), and the Sid Kyle Chair Endowment (SS).

Footnotes

DISCLOSURES:

Conflicts of Interest: The authors declare that they have no conflicts of interest.

REFERENCES

  • 1.Lachner M, O’Sullivan RJ, Jenuwein T. An epigenetic road map for histone lysine methylation. J Cell Sci. 2003;116(Pt 11):2117–24. Epub 2003/05/06. doi: 10.1242/jcs.00493. [DOI] [PubMed] [Google Scholar]
  • 2.Cao H, Li L, Yang D, Zeng L, Yewei X, Yu B, Liao G, Chen J. Recent progress in histone methyltransferase (G9a) inhibitors as anticancer agents. Eur J Med Chem. 2019;179:537–46. Epub 2019/07/06. doi: 10.1016/j.ejmech.2019.06.072. [DOI] [PubMed] [Google Scholar]
  • 3.Tachibana M, Sugimoto K, Fukushima T, Shinkai Y. Set domain-containing protein, G9a, is a novel lysine-preferring mammalian histone methyltransferase with hyperactivity and specific selectivity to lysines 9 and 27 of histone H3. J Biol Chem. 2001;276(27):25309–17. Epub 2001/04/24. doi: 10.1074/jbc.M101914200. [DOI] [PubMed] [Google Scholar]
  • 4.Tachibana M, Matsumura Y, Fukuda M, Kimura H, Shinkai Y. G9a/GLP complexes independently mediate H3K9 and DNA methylation to silence transcription. EMBO J. 2008;27(20):2681–90. Epub 2008/09/27. doi: 10.1038/emboj.2008.192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Wei L, Chiu DK, Tsang FH, Law CT, Cheng CL, Au SL, Lee JM, Wong CC, Ng IO, Wong CM. Histone methyltransferase G9a promotes liver cancer development by epigenetic silencing of tumor suppressor gene RARRES3. J Hepatol. 2017;67(4):758–69. Epub 2017/05/24. doi: 10.1016/j.jhep.2017.05.015. [DOI] [PubMed] [Google Scholar]
  • 6.Hua KT, Wang MY, Chen MW, Wei LH, Chen CK, Ko CH, Jeng YM, Sung PL, Jan YH, Hsiao M, Kuo ML, Yen ML. The H3K9 methyltransferase G9a is a marker of aggressive ovarian cancer that promotes peritoneal metastasis. Mol Cancer. 2014;13:189. Epub 2014/08/15. doi: 10.1186/1476-4598-13-189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Casciello F, Al-Ejeh F, Kelly G, Brennan DJ, Ngiow SF, Young A, Stoll T, Windloch K, Hill MM, Smyth MJ, Gannon F, Lee JS. G9a drives hypoxia-mediated gene repression for breast cancer cell survival and tumorigenesis. Proc Natl Acad Sci U S A. 2017;114(27):7077–82. Epub 2017/06/21. doi: 10.1073/pnas.1618706114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Ho JC, Abdullah LN, Pang QY, Jha S, Chow EK, Yang H, Kato H, Poellinger L, Ueda J, Lee KL. Inhibition of the H3K9 methyltransferase G9A attenuates oncogenicity and activates the hypoxia signaling pathway. PLoS One. 2017;12(11):e0188051. Epub 2017/11/18. doi: 10.1371/journal.pone.0188051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Hsiao SM, Chen MW, Chen CA, Chien MH, Hua KT, Hsiao M, Kuo ML, Wei LH. The H3K9 Methyltransferase G9a Represses E-cadherin and is Associated with Myometrial Invasion in Endometrial Cancer. Ann Surg Oncol. 2015;22 Suppl 3:S1556–65. Epub 2015/01/24. doi: 10.1245/s10434-015-4379-5. [DOI] [PubMed] [Google Scholar]
  • 10.Huang T, Zhang P, Li W, Zhao T, Zhang Z, Chen S, Yang Y, Feng Y, Li F, Shirley Liu X, Zhang L, Jiang G, Zhang F. G9A promotes tumor cell growth and invasion by silencing CASP1 in non-small-cell lung cancer cells. Cell Death Dis. 2017;8(4):e2726. Epub 2017/04/07. doi: 10.1038/cddis.2017.65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Li KC, Hua KT, Lin YS, Su CY, Ko JY, Hsiao M, Kuo ML, Tan CT. Inhibition of G9a induces DUSP4-dependent autophagic cell death in head and neck squamous cell carcinoma. Mol Cancer. 2014;13:172. Epub 2014/07/17. doi: 10.1186/1476-4598-13-172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Rada M, Vasileva E, Lezina L, Marouco D, Antonov AV, Macip S, Melino G, Barlev NA. Human EHMT2/G9a activates p53 through methylation-independent mechanism. Oncogene. 2017;36(7):922–32. Epub 2016/07/28. doi: 10.1038/onc.2016.258. [DOI] [PubMed] [Google Scholar]
  • 13.Yin C, Ke X, Zhang R, Hou J, Dong Z, Wang F, Zhang K, Zhong X, Yang L, Cui H. G9a promotes cell proliferation and suppresses autophagy in gastric cancer by directly activating mTOR. FASEB J. 2019;33(12):14036–50. Epub 2019/10/28. doi: 10.1096/fj.201900233RR. [DOI] [PubMed] [Google Scholar]
  • 14.Oh ST, Kim KB, Chae YC, Kang JY, Hahn Y, Seo SB. H3K9 histone methyltransferase G9a-mediated transcriptional activation of p21. FEBS Lett. 2014;588(5):685–91. Epub 2014/02/05. doi: 10.1016/j.febslet.2014.01.039. [DOI] [PubMed] [Google Scholar]
  • 15.Hu L, Zang MD, Wang HX, Zhang BG, Wang ZQ, Fan ZY, Wu H, Li JF, Su LP, Yan M, Zhu ZQ, Yang QM, Huang Q, Liu BY, Zhu ZG. G9A promotes gastric cancer metastasis by upregulating ITGB3 in a SET domain-independent manner. Cell Death Dis. 2018;9(3):278. Epub 2018/02/17. doi: 10.1038/s41419-018-0322-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Bhat AV, Palanichamy Kala M, Rao VK, Pignata L, Lim HJ, Suriyamurthy S, Chang KT, Lee VK, Guccione E, Taneja R. Epigenetic Regulation of the PTEN-AKT-RAC1 Axis by G9a Is Critical for Tumor Growth in Alveolar Rhabdomyosarcoma. Cancer Res. 2019;79(9):2232–43. Epub 2019/03/06. doi: 10.1158/0008-5472.CAN-18-2676. [DOI] [PubMed] [Google Scholar]
  • 17.Lacey A, Rodrigues-Hoffman A, Safe S. PAX3-FOXO1A Expression in Rhabdomyosarcoma Is Driven by the Targetable Nuclear Receptor NR4A1. Cancer Res. 2017;77(3):732–41. Epub 2016/11/20. doi: 10.1158/0008-5472.CAN-16-1546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Lacey A, Hedrick E, Cheng Y, Mohankumar K, Warren M, Safe S. Interleukin-24 (IL24) Is Suppressed by PAX3-FOXO1 and Is a Novel Therapy for Rhabdomyosarcoma. Mol Cancer Ther. 2018;17(12):2756–66. Epub 2018/09/08. doi: 10.1158/1535-7163.MCT-18-0118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Li X, Tjalkens RB, Shrestha R, Safe S. Structure-dependent Activation of Gene Expression by Bis-indole and Quinoline-derived Activators of Nuclear Receptor 4A2. Chem Biol Drug Des. 2019. Epub 2019/05/19. doi: 10.1111/cbdd.13564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Lee SO, Li X, Hedrick E, Jin UH, Tjalkens RB, Backos DS, Li L, Zhang Y, Wu Q, Safe S. Diindolylmethane analogs bind NR4A1 and are NR4A1 antagonists in colon cancer cells. Molecular endocrinology. 2014;28(10):1729–39. doi: 10.1210/me.2014-1102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Mohankumar K, Lee J, Wu CS, Sun Y, Safe S. Bis-Indole-Derived NR4A1 Ligands and Metformin Exhibit NR4A1-Dependent Glucose Metabolism and Uptake in C2C12 Cells. Endocrinology. 2018;159(5):1950–63. Epub 2018/04/11. doi: 10.1210/en.2017-03049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Mohankumar K, Li X, Sridharan S, Karki K, Safe S. Nuclear receptor 4A1 (NR4A1) antagonists induce ROS-dependent inhibition of mTOR signaling in endometrial cancer. Gynecol Oncol. 2019;154(1):218–27. Epub 2019/05/06. doi: 10.1016/j.ygyno.2019.04.678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Hedrick E, Li X, Cheng Y, Lacey A, Mohankumar K, Zarei M, Safe S. Potent inhibition of breast cancer by bis-indole-derived nuclear receptor 4A1 (NR4A1) antagonists. Breast Cancer Res Treat. 2019;177(1):29–40. Epub 2019/05/24. doi: 10.1007/s10549-019-05279-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Lee SO, Abdelrahim M, Yoon K, Chintharlapalli S, Papineni S, Kim K, Wang H, Safe S. Inactivation of the orphan nuclear receptor TR3/Nur77 inhibits pancreatic cancer cell and tumor growth. Cancer research. 2010;70(17):6824–36. doi: 10.1158/0008-5472.CAN-10-1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Hedrick E, Lee SO, Doddapaneni R, Singh M, Safe S. NR4A1 Antagonists Inhibit beta1-Integrin-Dependent Breast Cancer Cell Migration. Mol Cell Biol. 2016;36(9):1383–94. Epub 2016/03/02. doi: 10.1128/MCB.00912-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Hedrick E, Li X, Safe S. Penfluridol Represses Integrin Expression in Breast Cancer through Induction of Reactive Oxygen Species and Downregulation of Sp Transcription Factors. Mol Cancer Ther. 2017;16(1):205–16. Epub 2016/11/05. doi: 10.1158/1535-7163.MCT-16-0451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Karki K, Wright GA, Mohankumar K, Jin UH, Zhang XH, Safe S. A Bis-Indole-Derived NR4A1 Antagonist Induces PD-L1 Degradation and Enhances Anti-Tumor Immunity. Cancer Res. 2020. Epub 2020/01/09. doi: 10.1158/0008-5472.CAN-19-2314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Rao M, Atay SM, Shukla V, Hong Y, Upham T, Ripley RT, Hong JA, Zhang M, Reardon E, Fetsch P, Miettinen M, Li X, Peer CJ, Sissung T, Figg WD, De Rienzo A, Bueno R, Schrump DS. Mithramycin Depletes Specificity Protein 1 and Activates p53 to Mediate Senescence and Apoptosis of Malignant Pleural Mesothelioma Cells. Clin Cancer Res. 2016;22(5):1197–210. Epub 2015/10/16. doi: 10.1158/1078-0432.CCR-14-3379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Liu F, Barsyte-Lovejoy D, Li F, Xiong Y, Korboukh V, Huang XP, Allali-Hassani A, Janzen WP, Roth BL, Frye SV, Arrowsmith CH, Brown PJ, Vedadi M, Jin J. Discovery of an in vivo chemical probe of the lysine methyltransferases G9a and GLP. J Med Chem. 2013;56(21):8931–42. Epub 2013/10/10. doi: 10.1021/jm401480r. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Pearen MA, Muscat GE. Minireview: Nuclear hormone receptor 4A signaling: implications for metabolic disease. Mol Endocrinol. 2010;24(10):1891–903. Epub 2010/04/16. doi: 10.1210/me.2010-0015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Liu X, Jutooru I, Lei P, Kim K, Lee SO, Brents LK, Prather PL, Safe S. Betulinic acid targets YY1 and ErbB2 through cannabinoid receptor-dependent disruption of microRNA-27a:ZBTB10 in breast cancer. Molecular cancer therapeutics. 2012;11(7):1421–31. doi: 10.1158/1535-7163.MCT-12-0026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Chadalapaka G, Jutooru I, Burghardt R, Safe S. Drugs that target specificity proteins downregulate epidermal growth factor receptor in bladder cancer cells. Molecular cancer research : MCR. 2010;8(5):739–50. doi: 10.1158/1541-7786.MCR-09-0493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Nair V, Sreevalsan S, Basha R, Abdelrahim M, Abudayyeh A, Rodrigues Hoffman A, Safe S. Mechanism of metformin-dependent inhibition of mammalian target of rapamycin (mTOR) and Ras activity in pancreatic cancer: role of specificity protein (Sp) transcription factors. The Journal of biological chemistry. 2014;289(40):27692–701. doi: 10.1074/jbc.M114.592576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Safe S, Jin UH, Hedrick E, Reeder A, Lee SO. Minireview: role of orphan nuclear receptors in cancer and potential as drug targets. Molecular endocrinology. 2014;28(2):157–72. doi: 10.1210/me.2013-1291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Wu H, Lin Y, Li W, Sun Z, Gao W, Zhang H, Xie L, Jiang F, Qin B, Yan T, Chen L, Zhao Y, Cao X, Wu Y, Lin B, Zhou H, Wong AS, Zhang XK, Zeng JZ. Regulation of Nur77 expression by beta-catenin and its mitogenic effect in colon cancer cells. FASEB J. 2011;25(1):192–205. Epub 2010/09/18. doi: 10.1096/fj.10-166462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Wang JR, Gan WJ, Li XM, Zhao YY, Li Y, Lu XX, Li JM, Wu H. Orphan nuclear receptor Nur77 promotes colorectal cancer invasion and metastasis by regulating MMP-9 and E-cadherin. Carcinogenesis. 2014;35(11):2474–84. Epub 2014/07/30. doi: 10.1093/carcin/bgu157. [DOI] [PubMed] [Google Scholar]
  • 37.Zhou F, Drabsch Y, Dekker TJ, de Vinuesa AG, Li Y, Hawinkels LJ, Sheppard KA, Goumans MJ, Luwor RB, de Vries CJ, Mesker WE, Tollenaar RA, Devilee P, Lu CX, Zhu H, Zhang L, Dijke PT. Nuclear receptor NR4A1 promotes breast cancer invasion and metastasis by activating TGF-beta signalling. Nat Commun. 2014;5:3388. Epub 2014/03/04. doi: 10.1038/ncomms4388. [DOI] [PubMed] [Google Scholar]
  • 38.Mullican SE, Zhang S, Konopleva M, Ruvolo V, Andreeff M, Milbrandt J, Conneely OM. Abrogation of nuclear receptors Nr4a3 and Nr4a1 leads to development of acute myeloid leukemia. Nature medicine. 2007;13(6):730–5. Epub 2007/05/23. doi: 10.1038/nm1579. [DOI] [PubMed] [Google Scholar]
  • 39.Ramirez-Herrick AM, Mullican SE, Sheehan AM, Conneely OM. Reduced NR4A gene dosage leads to mixed myelodysplastic/myeloproliferative neoplasms in mice. Blood. 2011;117(9):2681–90. Epub 2011/01/06. doi: 10.1182/blood-2010-02-267906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Lee SO, Andey T, Jin UH, Kim K, Singh M, Safe S. The nuclear receptor TR3 regulates mTORC1 signaling in lung cancer cells expressing wild-type p53. Oncogene. 2012;31(27):3265–76. doi: 10.1038/onc.2011.504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Hedrick E, Lee SO, Doddapaneni R, Singh M, Safe S. Nuclear receptor 4A1 as a drug target for breast cancer chemotherapy. Endocr Relat Cancer. 2015;22(5):831–40. Epub 2015/08/01. doi: 10.1530/ERC-15-0063. [DOI] [PubMed] [Google Scholar]
  • 42.Pastukhov AV, Ropson IJ. Fluorescent dyes as probes to study lipid-binding proteins. Proteins. 2003;53(3):607–15. Epub 2003/10/28. doi: 10.1002/prot.10401. [DOI] [PubMed] [Google Scholar]
  • 43.Hedrick E, Lee SO, Doddapaneni R, Singh M, Safe S. Nuclear receptor 4A1 (NR4A1) as a drug target for breast cancer chemotherapy. Endocrine-related cancer. 2015. doi: 10.1530/ERC-15-0063. [DOI] [PubMed] [Google Scholar]
  • 44.Hedrick E, Safe S. Transforming Growth Factor beta/NR4A1-Inducible Breast Cancer Cell Migration and Epithelial-to-Mesenchymal Transition Is p38alpha (Mitogen-Activated Protein Kinase 14) Dependent. Mol Cell Biol. 2017;37(18). Epub 2017/07/05. doi: 10.1128/MCB.00306-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Hedrick E, Mohankumar K, Safe S. TGFbeta-Induced Lung Cancer Cell Migration Is NR4A1-Dependent. Mol Cancer Res. 2018;16(12):1991–2002. Epub 2018/08/04. doi: 10.1158/1541-7786.MCR-18-0366. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1653494-supplement-1.docx (209.7KB, docx)

RESOURCES