Orphan-Nuclear-Receptors belong to the Nuclear-Receptors superfamily of ligand-dependent transcription factors that act as sensors for hormones, vitamins and dietary lipids. Orphan-Nuclear-Receptors are differentially expressed in normal mammary and breast-cancer tissues. Unlike other Nuclear-Receptors, Orphan-Nuclear-Receptors lack known physiological ligands. Limited information is available on Orphan-Nuclear-Receptors function in breast-cancer. We performed a number of silencing studies based on the use of siRNAs targeting the Orphan-Nuclear-Receptors expressed in a panel of cell-lines, recapitulating breast-cancer heterogeneity. NR2F6 function was further validated by stable shRNA-mediated silencing in estrogen-receptor-positive (MCF7) and triple-negative (MDA-MB-231) cell-lines. This was followed by assays on proliferation, clonogenicity and motility in MCF7 and MDA-MB-231 cells as well as cytokine responses in MCF7 cells. Whole-genome RNA sequencing was used to define NR2F6-dependent transcriptional networks. Immunoprecipitation coupled with flow-injection high-resolution mass-spectrometry was performed to identify endogenous molecules binding NR2F6. Significant levels of ten Orphan-Nuclear-Receptors were observed in most breast-cancer cell-lines and in mammary tumor tissues. Functional siRNA studies revealed that NR2F2 and NR4A2 silencing enhanced the proliferation of breast-cancer cells, indicating a potential tumor-suppressive role. In contrast, NR2F6 knockdown consistently reduced proliferation of breast-cancer cells, supporting an oncogenic role. NR2F6 is highly expressed in breast tumors as compared with their normal counterparts. Stable NR2F6 silencing decreased the growth and clonogenicity of MCF7 and MDA-MB-231 cell lines, corroborating the siRNA findings. In the mesenchymal and high motility MDA-MB-231 cell-line, NR2F6 silencing diminished directional cell migration. In the epithelial MCF7 cell-line, this Orphan-Nuclear-Receptor induced an antiproliferative response to immune cytokines. From a mechanistic point of view, NR2F6 knock-down up-regulated gene-networks related to cell-cell and cell–matrix interactions, while it down-regulated gene-networks involved in cell-cycle progression and proliferation. A mass-spectrometry–based ligand screening approach led to the identification of palmitoylethanolamide as the only endogenous molecule capable of binding NR2F6 with high-affinity. Our study identifies NR2F6 as an oncogenic driver of breast-cancer and a promising therapeutic target for the personalized treatment of this tumor. The discovery of palmitoylethanolamide as a physiological ligand of NR2F6 provides the molecular foundation for a rational design of NR2F6 antagonists/inhibitors to be evaluated for their therapeutic potential in breast-cancer.
Caricasulo et al. (Mon,) studied this question.