Vitamin D inhibits osteosarcoma by reprogramming nonsense-mediated RNA decay and SNAI2-mediated epithelial-to-mesenchymal transition.

Osteosarcomas are immune-resistant and metastatic in part due to an increase in nonsense-mediated RNA decay (NMD), reactive oxygen species (ROS), and epithelial-to-mesenchymal transition (EMT) induction. In this study, we examined the impact of vitamin D 3 and its receptor (VDR) on the NMD-ROS-EMT signaling axis in osteosarcoma cells and spheroids, as well as wound-induced cell migration and osteosarcoma metastasis in vivo models. Activated VDR signaling enriched the EMT pathway in gene set enrichment analysis, whereas the active vitamin D 3 metabolite, 1,25(OH) 2 D, inhibited EMT in osteosarcoma subtypes. Because EMT processes in cancer and tissue wounds are similar, experimentally induced EMT of mouse hair follicle stem cells revealed that Vdr signaling restricts cell migration during cutaneous wound healing. In osteosarcoma cells, ligand bound VDR inhibited EMT by interacting directly with and downregulating the EMT inducer SNAI2 . Differential epigenetic regulation of SNAI2 and VDR distinguished highly metastatic from low metastatic osteosarcoma subtypes and responsiveness to 1,25(OH) 2 D. Furthermore, epigenome-wide motif and putative target gene analysis revealed the integration of the VDR with the NMD tumor immunogenic pathway. In an autoregulatory manner, 1,25(OH) 2 D inhibited NMD machinery genes, while simultaneously inducing the overexpression of known NMD target genes involved in tumor immune recognition and cell-to-cell adhesion. Dicer substrate siRNA knockdown of SNAI2 revealed superoxide dismutase 2 (SOD2)-mediated antioxidative responses and 1,25(OH) 2 D sensitization, which involves the non-canonical SOD2 nuclear-to-mitochondrial translocalization and inhibition of ROS. Calcipotriol, a clinically relevant non-calcemic vitamin D 3 derivative, inhibited osteosarcoma metastasis in a mouse xenograft model. Our research reveals novel vitamin D 3 and calcipotriol osteosarcoma-inhibiting processes.