Dual nuclear receptor 4A1 (NR4A1/NR4A2) ligands inhibit glioblastoma growth and target TWIST1.

Abstract

1,1-Bis(3'-indolyl)-1-(3,5-disubstitutedphenyl)methane (DIM-3,5) compounds are dual receptor ligands that bind both orphan nuclear receptor 4A1 (NR4A1) and NR4A2. Knockdown of NR4A1 or NR4A2 by RNA interference in glioblastoma (GBM) cells decreased growth and induced apoptosis and comparable effects were observed for DIM-3,5 analogs, which exhibit inverse agonist activity and inhibit NR4A1- and NR4A2-mediated pro-oncogenic activity. Knockdown of NR4A1 or NR4A2 or treatment with DIM-3,5 analogs also decreased expression of TWIST1 mRNA and protein in GBM cells by 40%-90%.The proximal region of the TWIST1 gene promoter contains functional GC-rich binding sites that bind Sp1 and Sp4, and knockdown of these transcription factors also decreased TWIST1 expression in GBM cells. Further analysis by chromatin immunoprecipitation, protein-protein coimmunoprecipitation, and binding assays demonstrated that NR4A1/NR4A2 coregulate TWIST1 gene expression as ligand-dependent cofactors of Sp1 and Sp4, which interact with cis proximal GC-rich sites in the TWIST1 gene promoter. In vivo studies show that DIM-3,5 dual NR4A1/2 inverse agonists also reduced intratumoral TWIST1 expression while significantly prolonging survival of mice in a syngeneic mouse model of GBM, demonstrating that these ligands are promising new agents for targeting TWIST1 and treating GBM. SIGNIFICANCE STATEMENT: The TWIST1 gene is a pro-oncogenic factor that regulates epithelial-to-mesenchymal transition in glioblastoma cells. This paper shows that the orphan nuclear receptor 4A1 (NR4A1) and NR4A2 regulate TWIST1 expression, which can be targeted by bis-indole-derived dual NR4A1/2 inverse agonists.