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

## TL;DR

This paper explores a new nonhormonal treatment for endometriosis by targeting two nuclear receptors, NR4A1 and NR4A2, to inhibit disease progression.

## Contribution

The study introduces DIM-3,5 compounds as dual NR4A1/NR4A2 ligands that effectively suppress endometriosis without hormonal side effects.

## Key findings

- DIM-3,5 compounds inhibit proendometriotic pathways and EMT markers in endometriotic cells.
- NR4A1 and NR4A2 regulate overlapping pathways related to cell growth, survival, and migration.
- DIM-3,5-Cl2 reduces endometriotic lesion growth in mice without causing cytotoxicity.

## Abstract

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.