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.

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; refs. 3, 4) and this determines some of its cellular functions and role in multiple diseases (2). G9A forms a heterodimeric complex with GLP/Eu-HMTase to catalyze monomethylation 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 cofactor 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 15 ARMS patient samples, G9A is overexpressed compared with 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-DIM) 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.

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 ATCC. Rh41 (RMS) was a generous gift from 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 Weston Porter Texas A&M University (College Station, TX). Mouse mammary tumor 4T1 cell line was kindly provided by Mien-Chie Hung, MD Anderson Cancer Center (Houston, TX). Hec-1B cell line was a generous gift from 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. HS578T, MDA-MB-231, MDA-MB-468, A549, HUH7, HepG2, and PC3 cells were maintained in DMEM. 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% 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 Supplementary Tables S1 and S2, 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), 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 previously (19). The purities of both compounds were >98% and their nuclear magnetic resonance spectrum are included in the Supplementary Data in Supplementary Table S1. 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% fetal bovine serum (FBS) and were allowed to attach. After 24 hours, they were transfected with 100 nmol/L of desired siRNAs using 50 μmol/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 to 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 was quantified by Bradford assay. The protein content in all the lysates was then normalized and the equal amount of protein was loaded and allowed to run on SDS polyacrylamide gel, connected to an electric source. The overall protein on the gel was then transferred to a polyvinylidene difluoride 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 Tris Buffered Saline with Tween 20 (TBST) and then incubated with horseradish peroxidase (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) 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 300 to 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 previously (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 previously (21).

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 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) 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). The relative mRNA expression of the desired genes was determined by using human TATA binding protein mRNA as a control.

Chromatin immunoprecipitation assay

The ChIP-IT Express Enzymatic Kit (Active Motif) was used and the manufacturer's protocol was followed 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 the presence of protein G–conjugated magnetic beads for overnight. The beads were then washed with provided chromatin immunoprecipitation (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 nuclear extraction kit (ab113474) was then used and the manufacturer's protocol was followed 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 and were housed at Lab Animal Care Center, Texas A&M University (College Station, TX). The protocol for the animal studies was approved by Institutional Animal Care and Use Committee at Texas A&M University (College Station, TX). 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 μL 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-μL corn oil, whereas the mice in the treatment group were injected with 100 μL 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 3 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 analysis 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% charcoal-stripped serum supplied with the desired concentration of compounds for 24 hours. The XTT cell viability kit (Cell Signaling Technology) was then used and the manufacturer's protocol was followed to calculate the percentage of cell survival. The results are illustrated in Supplementary Fig. S1.

Statistical analysis

Statistical significance of differences between the treatment groups was determined by Student 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.

Previous studies showed that G9A was highly expressed in Rh30 and Rh41 ARMS cells (16) and examination of UALCAN and The Cancer Genome Atlas databases showed that in patients with sarcoma, high expression of G9A was associated with decreased survival (Fig. 1A). In a limited dataset, G9A is also expressed more in primary tumors than in nontumor tissues (Fig. 1B). The major focus of this article is to report our studies showing that the orphan nuclear receptor NR4A1 regulates G9a expression in ARMS cells. Knockdown of NR4A1 by 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 LBD (Fig. 2B) as described previously (20), and the growth inhibitory effects of these compounds are summarized in Supplementary Fig. S1; Supplementary Fig. S 2A shows that the NR4A1 antagonists also decrease NR4A1/bisANS fluorescence intensity as described previously (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 (Supplementary Fig. S 1) and decreased G9A protein and mRNA levels in Rh30 (Fig. 2E and F) 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 coexpressed 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.

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 versus 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 Materials and Methods.

Figure 1.

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 versus 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 Materials and Methods.

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Figure 2.

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 LBD of NR4A1 were determined by fluorescent quenching of the tryptophan residue in the binding pocket as outlined in Materials and Methods. Rh30 and Rh41 cells were treated with CDIM8 (C and D), 3,5-(CH3)2 and 3-Br-5-OCH3 (EH) and effects on gene products and mRNA levels were determined by Western blots and real-time PCR, respectively, as outlined in Materials and Methods. Results (D, F, and H) are expressed as means ± SD for at least three replicated determinations for each treatment group and significantly (P < 0.05) decreased responses are indicated.

Figure 2.

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 LBD of NR4A1 were determined by fluorescent quenching of the tryptophan residue in the binding pocket as outlined in Materials and Methods. Rh30 and Rh41 cells were treated with CDIM8 (C and D), 3,5-(CH3)2 and 3-Br-5-OCH3 (EH) and effects on gene products and mRNA levels were determined by Western blots and real-time PCR, respectively, as outlined in Materials and Methods. Results (D, F, and H) are expressed as means ± SD for at least three replicated determinations for each treatment group and significantly (P < 0.05) decreased responses are indicated.

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Figure 3.

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 Materials and 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 analysis as outlined in Materials and Methods.

Figure 3.

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 Materials and 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 analysis as outlined in Materials and Methods.

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Previous studies in RMS and other cell lines show that NR4A1 regulates multiple genes containing GC-rich promoters by acting as a nuclear cofactor 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 with a mutant (GC) oligonucleotide in a DNA protein binding assay (Fig. 4G).

Figure 4.

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 Materials and Methods section. 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 Materials and Methods. F, Rh30 cells were treated with DMSO, CDIM8 (20 μmol/L) or mithramycin (100 nmol/L) for 24 hours and analyzed for binding to the G9a promoter in a ChIP assay as outlined in Materials and 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 Materials and Methods. Results (E and G) are expressed as means ± SD for at least three replicate determinations for each treatment group and significant (P < 0.05) changes compared with controls are indicated (*).

Figure 4.

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 Materials and Methods section. 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 Materials and Methods. F, Rh30 cells were treated with DMSO, CDIM8 (20 μmol/L) or mithramycin (100 nmol/L) for 24 hours and analyzed for binding to the G9a promoter in a ChIP assay as outlined in Materials and 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 Materials and Methods. Results (E and G) are expressed as means ± SD for at least three replicate determinations for each treatment group and significant (P < 0.05) changes compared with controls are indicated (*).

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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.

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 μmol/L; D) on G9a expression in Rh30 and Rh41 cells were determined by Western blot analysis of whole-cell lysates as outlined in Materials and Methods. Quantitation of each blot (relative to β-actin) was also determined for each blot.

Figure 5.

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 μmol/L; D) on G9a expression in Rh30 and Rh41 cells were determined by Western blot analysis of whole-cell lysates as outlined in Materials and Methods. Quantitation of each blot (relative to β-actin) was also determined for each blot.

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Previous studies in ARMS cells reported that G9A silenced PTEN thereby activating Akt (16); 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 Supplementary Fig. 2B and C. 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, which 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 with 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 with 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.

siNR4A1/NR4A1 antagonists, mithramycin, and UNC0642 inhibit phosphorylation of Akt. ARMS cells were treated with NR4A1 antagonists (A and B), mithramycin (C) and UNC0642 (5 μmol/L) (D) and whole-cell lysates were analyzed by Western blots as outlined in Materials and Methods and blots (A and B) were quantitated in Supplementary Fig. S2B and S2C. Cells were treated with 15 μmol/L 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 μmol/L), 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 Materials and Methods.

Figure 6.

siNR4A1/NR4A1 antagonists, mithramycin, and UNC0642 inhibit phosphorylation of Akt. ARMS cells were treated with NR4A1 antagonists (A and B), mithramycin (C) and UNC0642 (5 μmol/L) (D) and whole-cell lysates were analyzed by Western blots as outlined in Materials and Methods and blots (A and B) were quantitated in Supplementary Fig. S2B and S2C. Cells were treated with 15 μmol/L 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 μmol/L), 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 Materials and Methods.

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Figure 7.

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 and C) and changes in body mass (D) were determined as outlined in Materials and 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).

Figure 7.

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 and C) and changes in body mass (D) were determined as outlined in Materials and 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).

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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 neurologic 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 the following: 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 and indirectly affects 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, 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 cofactor of Sp1 or Sp4 (17, 20, 23–27, 40).

Results illustrated in Figs. 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 (Fig. 6A and B). 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 observations in ARMS cells and tumors suggest 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.

No disclosures were reported.

R. Shrestha: Conceptualization, data curation, formal analysis, methodology, writing–original draft, project administration, writing–review and editing. K. Mohankumar: Data curation, formal analysis, methodology. U.-H. Jin: Data curation, formal analysis, methodology. G.G. Martin: Data curation, formal analysis. S. Safe: Conceptualization, formal analysis, writing–original draft, project administration, writing–review and editing.

This work was supported by the NIH P30-ES023512 (to S. Safe), Kleberg Foundation (to S. Safe), Texas A&M AgriLife Research (to S. Safe), and the Sid Kyle Chair Endowment (to S. Safe).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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