## 3 Discussion  
NR4A subfamily members NR4A1, NR4A2 and NR4A3 were first identified as immediate early response genes, and subsequent studies show that they regulate important functions/genes in maintaining cellular homeostasis and pathophysiology [36, 37]. The dual NR4A1 and NR4A3 knockout mice rapidly develop acute myeloid leukemia [38], and in blood-derived cancers, NR4A1 and NR4A3 exhibit tumor suppressor-like activity. In contrast, in most solid tumors, both NR4A1 and NR4A2 exhibit pro-oncogenic activity, whereas, in limited studies, NR4A3 exhibits either minimal activity [23] or acts as a tumor suppressor [39]. In several cancer- and non-cancer-related diseases, NR4A1 and NR4A2 are over-expressed and are targetable with receptor agonists or inverse agonists to ameliorate disease, and there are increasing studies on development of new NR4A ligands [23, 30]. In this laboratory, a series of bis-indole-derived compounds were developed and 1,1-bis(3\({}^{\prime}\)-indolyl)-1-(4-hydroxy phenyl)methane (DIM-4-OH) and 1,1-bis(3\({}^{\prime}\)-indolyl)-1-(4-chlorophenyl)methane (DIM-4-CI) were used as prototypical first-generation NR4A1 and NR4A2 ligands, respectively [38]. DIM-4-OH interacted directly with the ligand binding domain (LBD) of NR4A1 [24], and modeling studies showed that DIM-4-CI preferentially interacted with a cofactor site of NR4A2 [23, 38]. Subsequent structure-activity studies identified a series of DIM-3,5 analogs that were significantly more potent as inhibitors of tumor growth in athymic nude mice bearing MDA-MB-231 breast cancer cells (orthotopic) [29]. IC\({}_{50}\) values of <1 mg/kg/day were observed in this study, which included DIM-3-CI-5-CF\({}_{3}\)- and DIM-3,5-CI\({}_{2}\) (2.5 mg/kg/day)-inhibited colon tumor growth and reversed T-cell exhaustion in a syngeneic mouse model using MC38 cells as a xenograft [25]. The high in vivo potencies of the DIM-3,5 analogs coupled with the >60% similarity of the LBDs of NR4A1 and NR4A2 suggested that the DIM-3,5 compounds may bind and inactivate both receptors, and this was confirmed in a recent publication [30]. Figure 7: DE genes and pathways and their overlap. The overlap of DE genes identified when comparing the SW480 control versus transfected siRNAs individually targeting NR4A1 or NR4A2, versus treatment with DIM-3,5-CI\({}_{2}\) (**A**), and combined NR4A1 and NR4A2 siRNAs (**B**). The top 500 up- or downregulated DE genes from each pair comparison are included in the Venn diagrams. (**C**) Consensus gene ontology terms and pathways enriched in DE genes. DE analysis was conducted between control, transfected or treated groups, i.e., control versus DIM-3,5-CI\({}_{2}\), control versus siRNA1, control versus siRNA2 and control versus siRNA1 and siRNA2 (combined). DE genes identified between each of these pairs were subjected to functional enrichment analysis. Consensus gene ontology terms and pathways are those commonly identified. In this study, the functional and genomic properties of NR4A1 and NR4A2 are contrasted through the knockdown of each receptor by RNA interference, and the results are compared to that of the DIM-3,5 analogs, which are dual NR4A1/NR4A2 ligands. The results of NR4A2 knockdown in colon cancer cells confirmed the pro-oncogenic activity of this gene and was similar to that previously observed for NR4A2 other cancer cell lines [23] and consistent with the unfavorable prognosis for colon cancer patients that overexpress NR4A2. Overall, the results show that NR4A1 and NR4A2 exhibit similar pro-oncogenic activity and that the DIM-3,5 compounds are dual NR4A1/NR4A2 inverse agonists that induce responses similar to that observed for receptor silencing. Previous studies showed that \(\beta\)1-integrin and the histone methyltransferase G9a (EHMT2) gene were regulated by NR4A1 [23; 34] and our results (Figures 2 and 5) demonstrate that both \(\beta\)1-integrin and G9a are coregulated by both receptors that function as cofactors to enhance Sp1/Sp4-mediated transcription.