Orphan nuclear receptor 4A (NR4A) ligands induce ferroptosis and modify immune function in colon cancer

Abstract Orphan nuclear receptor 4A1 (NR4A1) and NR4A2 exhibit pro-oncogenic activities in most solid tumors, and this study investigates the role of both receptors and the dual NR4A1/2 1,2-bis(3’-indolyl)-1-(3,5-disubstitutedphenyl)methane (DIM-3,5) ligands as inducers of ferroptosis in colon cancer cells. DIM-3,5 ligands induced reactive oxygen species (ROS) enhancement, as indicated by BODIPY fluorescence, a marker of lipoperoxidation, and increased formation of malondialdehyde (MDA), a lipoperoxide degradation product, in colon cancer cells; this was accompanied by modulated expression of key ferroptotic genes, including the transferrin receptor (TFR/CD71, induced), glutathione peroxidase 4 (GPX4, decreased), and SLC7A11 (decreased). Molecular analysis of GPX4 regulation by NR4A using knockdown, promoter analysis, and chromatin immunoprecipitation showed that GPX4 is regulated by NR4A2/Sp4, where NR4A2 acts as a ligand-dependent cofactor of the DNA-bound Sp4 transcription factor. The 3,5-dichloro analog (DIM-3,5-CI 2 ) (2.5 mg/kg/day) also inhibits colon tumor growth in a syngeneic mouse xenograft model bearing CT26 mouse colon cancer cells, and analysis of tumor-infiltrating lymphocytes shows that DIM-3,5-CI 2 increased CD8 + T cells, decreased g-MDSC, increased M1/M2 macrophage ratios, and an increased percentage of B cells to enhance immune surveillance. Thus, the dual NR4A1/2 DIM-3,5 ligands induce ferroptosis in tumors/cancer cells, and this was accompanied by modification of the immune system.