Nuclear receptor 4A1 (NR4A1) as a drug target for treating rhabdomyosarcoma (RMS)

Abstract

The orphan nuclear receptor NR4A1 is expressed in tumors from rhabdomyosarcoma (RMS) patients and Rh30 and RD RMS cell lines, and we used RNA interference (RNAi) to investigate the role of this receptor in RMS cells. Knockdown of NR4A1 in Rh30 cells decreased cell proliferation, induced Annexin V staining and induced polyADPribose polymerase (PARP) cleavage and these results were similar to those observed in other solid tumors. Previous studies show that NR4A1 regulates expression of growth promoting/pro-survival genes with GC-rich promoters, activates mTOR through suppression of p53, and maintains low oxidative stress by regulating expression of isocitrate dehydrogenase 1 (IDH1) and thioredoxin domain containing 5 (TXNDC5). Results of RNAi studies demonstrated that NR4A1 also regulates these pathways and associated genes in RMS cells and thereby exhibits pro-oncogenic activity. 1,1-Bis(3-indolyl)-1-(p-substituted phenyl)methane (C-DIM) analogs containing p-hydroxyl (DIM-C-pPhOH) and p-carboxymethyl (DIM-C-pPhCO2Me) substituents are NR4A1 ligands that decreased NR4A1-dependent transactivation in RMS cells and inhibited RMS cell and tumor growth and induced apoptosis. Moreover, the effects of NR4A1 knockdown and the C-DIM/NR4A1 antagonists were comparable as inhibitors of NR4A1-dependent genes/pathways. Both NR4A1 knockdown and treatment with DIM-C-pPhOH and DIM-C-pPhCO2Me also induced ROS which activated stress genes and induced sestrin 2 which activated AMPK and inhibited mTOR in the mutant p53 RMS cells. Since NR4A1 regulates several growth-promoting/pro-survival pathways in RMS, the C-DIM/NR4A1 antagonists represent a novel mechanism-based approach for treating this disease alone or in combination and thereby reducing the adverse effects of current cytotoxic therapies.

Keywords: C-DIMs; NR4A1; NR4A1 antagonists; inhibition; rhabdomyosarcoma.

Figure 1. NR4A1 expression and transactivation by C-DIMs

(A) Structure of C-DIMs and (B) NR4A1-regulated pro-oncogenic pathways in cancer cells. (C) Analysis of NR4A1 gene expression in patient-derived mRNA acquired from the NCBI GEO dataset GSE2851. (D) C-DIMs inhibit NR4A1-dependent transactivation. RD cells were transfected with pGAL4-NR4A1/UAS_x5-luc, NBRE_x3-luc or NuRE_s3-luc, treated with DMSO or 15 μM DIM-C-pPhOH or DIM-C-pPhCO₂Me, and luciferase activity was determined as outlined in the Materials and Methods. Results are expressed as means ± SE for at least 3 separate experiments and significantly (p < 0.05) decreased activity is indicated (*). (E) Cellular localization of NR4A1. Rh30 (A) and RD (B) cells were treated with DMSO or 20 μM DIM-C-pPhOH for 24 hr and cells were stained with DAPI and a fluorescent NR4A1 antibody. The individual and merged staining was determined as outlined in the Materials and Methods.

Figure 2. NR4A1 regulates growth of RMS cells which can be inhibited by C-DIM/NR4A1 antagonists

(A) Rh30 and Rd cells were transfected with two different oligonucleotides targeted to NR4A1 [siNR4A1(1) and siNR4A1(2)], and after 72 hr, the cells were counted and compared to the number of cells observed after transfection with a non-specific control (siCtl) oligonucleotide. Rh30 and RD cells were treated with different concentrations of DIM-C-pPhOH (B) or DIM-C-pPhCO₂Me (C) for 24 hr, and (D) RD cells were treated with 15 μM DIM-C-pPhCO₂Me for 24, 48 or 72 hr. Cells were counted and compared to the number observed after treatment with the solvent control (DMSO, set at 100%). (E) In a preliminary in vivo study, we observed that after treatment of athymic nude mice with DIM-C-pPhOH (40 mg/kg/d for 28 days), there was a small but significant inhibition of tumor growth and future studies will use a higher dose of this compound. Results (A – E) are expressed as means ± SE for at least 3 separate treatments for each group and significant (p < 0.05) growth inhibition is indicated (*).

Figure 3. NR4A1 regulates RMS survival that can be inhibited by C-DIM/NR4A1 antagonists

Rh30 and RD cells were transfected with siNR4A1 or siNR4A2, and induction of Annexin V staining (A) or enhanced PARP cleavage (B) were determined as outlined in the Materials and Methods. Rh30 and RD cells were treated with DIM-C-pPhOH or DIM-C-pPhCO₂Me and effects on Annexin V staining (C) or enhanced PARP cleavage (D) were determined as outlined in the Materials and Methods. Results (A and C) were expressed as means ± SE for at least 3 replicate determinations per treatment group and significant (p < 0.05) induction is indicated (*).

Figure 4. NR4A1 regulation of pro-survival/growth promoting genes and their inhibition by C-DIM/NR4A1 antagonists

(A) Rh30 and RD cells were transfected with siNR4A1 or siCtl, and whole cell lysates were analyzed by Western blots as outlined in the Materials and Methods. Rh30 and RD cells were treated with DMSO (solvent control), DIM-C-pPhOH (B) or DIM-C-pPhCO₂Me (C), and whole cell lysates were analyzed by Western blot as outlined in the Materials and Methods. (D) Rh30 and RD cells were treated with DIM-C-pPhCO₂Me, and binding of NR4A1, Sp1, p300 and pol II to the survivin promoter was determined in a ChiP assay. (E) Cells were treated with DIM-C-pPhCO₂Me, and survivin, cyclin D1 and EGFR mRNA levels were determine by real time PCR. Results are expressed as means ± SE (3 replicates) and significant (p < 0.05) changes in gene expression are indicated (*).

Figure 5. Role of NR4A1 in regulating oxidative stress

Rh30 and RD cells were either transfected with siNR4A1(1)/siNR4A1(s) (A) or treated with DIM-C-pPhOH or DIM-C-pPhCO₂Me (B), and ROS was determined using the cell permeable fluorescent probe CM-H2DCFDA as outlined in the Materials and Methods. Rh30 and RD cells were either transfected with siNR4A1 (C) or treated with DIM-C-pPhOH or DIM-C-pPhCO₂Me (D), and whole cell lysates were analyzed for TXNDC5, IDH1 and various ER stress genes by Western blot analysis as outlined in the Materials and Methods. (E) Rh30 cells were treated with DMSO or 20 μM DIM-C-pPhCO₂Me, and binding of NR4A1, p300, Sp1 and pol II to the GC-rich regions of the TXNDC5 and IDH1 gene promoters were determined in a ChIP assay as outlined in the 24 hr and (F) mRNA levels were determined by real time PCR. Results are expressed as means ± SE (triplicate determinations) and significant (p < 0.05) changes in gene expression are indicated.

Figure 6. NR4A1 regulates sestrin 2 and mTOR in RMS cells

(A) Rh30 cells were either transfected with siNR4A1 or treated with DIM-C-pPhOH, and whole cell lysates were analyzed as outlined in the Materials and Methods. Rh30 and RD cells were either treated with DIM-C-pPhOH or DIM-C-pPhCO₂Me alone or in combination with GSH (B) or transfected with siCtl/siNR4A1 alone or in combination with GSH treatment (C). Whole cell lysates were analyzed for sestrin 2 (SESN2) by Western blots as outlined in the Materials and Methods. Rh30 and RD cells were treated with DMSO and DIM-C-pPhCO₂Me alone (D) or in combination with GSH (E), and expression of sestrin 2 mRNA levels were determined by real time PCR as outlined in the Materials and Methods. Results (D and E) are expressed as means ± SE (3 replicates) and significant (p < 0.05) induction (*) or inhibition of induction (**) are indicated.