RANKL regulates differentially breast cancer stem cell properties through its RANK and LGR4 receptors

Breast cancer stem cells (BCSC) are a subpopulation responsible for cancer resistance and relapse. The receptor activator of nuclear factor kappa-Β ligand (RANKL) is a cytokine capable of activating RANK and LGR4 receptors. RANKL/RANK signaling maintains the self-renewal of BCSCs, however, the effect of RANKL via LGR4 remains unclear. Evidence from osteoclasts suggests that RANKL/LGR4 axis disrupts RANK signaling, leading to opposing cellular responses. Anti-RANKL inhibitors are potential agents for eradicating CSCs, but their effect on RANKL/LGR4 signal has not been demonstrated.

This project aimed to elucidate the role of RANKL in regulating stemness depending on the expression of its receptors.

We use in vitro and in vivo approaches to evaluate the effects of RANKL inhibition in stemness in low or high-LGR4 expressing cells. Furthermore, we analyze the effects of RANKL stimulation on the stemness of LGR4 or RANK overexpressing cells. Additionally, we evaluated the impact of RANKL/LGR4 signaling in the activity of Wnt/β-catenin and NF-κB signaling pathways.

Our findings indicated that elevated RANKL expression is related to a favorable prognosis in patients with high LGR4 levels. Furthermore, RANKL inhibition decreased BCSC properties in LGR4-low cell lines, while it promoted migration in LGR4-high cells. Additionally, the RANKL/RANK axis activated NF-κB signaling and enhanced BCSCs in RANK-overexpressing cells. In contrast, in LGR4-overexpressing cells, RANKL failed to activate NF-κB but instead inhibited the Wnt/β-catenin pathway, leading to a reduction in BCSCs.

Our findings suggest that RANKL exerts different responses according to the expression of its receptors.