Dual Targeting of Orphan Nuclear Receptors NR4A1 and NR4A2 for Nonhormonal Endometriosis Therapy

Previous studies show that orphan nuclear receptor 4A1 (NR4A1) regulates endometriotic cell growth, survival, estrogen receptor β (ERβ), mechanistic target of rapamycin signaling and fibrosis. NR4A2 is also expressed in epithelial and stromal derived endometriotic cells, and in this study the effects of 1,1-bis(3′-indolyl)-(3,5-disubstitutedphenyl)methane (DIM-3,5) dual NR4A1/nuclear receptor 4A2 (NR4A2) ligands and knockdown of NR4A1 and NR4A2 were investigated. The dual NR4A1/2 DIM-3,5 analogs inhibited previously identified proendometriotic pathways and gene products, and they also inhibited TWIST1 and multiple markers associated with epithelial-to-mesenchymal transition (EMT). The results show that both NR4A1 and NR4A2 regulate the same pathways, including endometriotic cell growth, survival, and migration and also some of the same genes in endometriotic epithelial and stromal cells. For example, DIM-3,5 compounds downregulate ERβ in stromal but not epithelial endometriotic cells, and this response is NR4A1- and not NR4A2-dependent. Among the EMT-related markers, claudin-1 is induced by DIM-3,5 ligands and after knockdown of NR4A1 or NR4A2 in both epithelial and stromal cells. Most of the EMT markers are downregulated by DIM-3,5 ligands and are coregulated by NR4A1 and NR4A2. In vivo studies showed that DIM-3,5-Cl₂ significantly reduced the growth of endometriotic lesions in a mouse model without inducing cytotoxicity during treatment. Thus, DIM-3,5 derivatives simultaneously suppress NR4A1- and NR4A2-dependent endometriosis progression effectively and represent a promising nonhormonal therapeutic strategy to replace current hormone-based treatments that can be associated with adverse effects.