The Role of the NPY/Y5R Axis in Ewing Sarcoma-Induced Osteolysis

Ewing sarcoma (ES) is an aggressive pediatric tumor that frequently arises in bone, causing bone degradation at the tumor-bone interface. High release of neuropeptide Y (NPY), acting via its Y receptors (Y1R, Y2R, Y5R), from ES tumors is associated with a more osteolytic ES phenotype. However, the mechanisms and osteolytic factors involved in NPY-dependent bone degradation remain unclear. This study investigates the role of the NPY/Y5R autocrine axis in ES, hypothesizing that it drives osteolysis by promoting osteoclast differentiation and macrophage migration toward the tumor-bone border. Using a mouse xenograft model of a high-NPY ES tumor (SK-ES-1) treated with Y2R or Y5R antagonists, bone degradation and osteoclast density at the tumor-bone interface were measured by hematoxylin and eosin (H&E) and tartrate-resistant acid phosphatase (TRAP) staining, respectively. Y5R antagonist treatment significantly reduced both bone degradation and osteoclast density at the tumor-bone border (p < 0.05), demonstrating the key role of the NPY/Y5R axis in osteolysis. In vitro, conditioned media from SK-ES-1 cells was shown to induce the macrophage cell (RAW 264.7) differentiation into osteoclasts. In media from SK-ES-1 cells transfected with shRNA targeting NPY or Y5R, the stimulated osteoclast differentiation was reduced (p < 0.001). An antibody array revealed that conditioned media from NPY- or Y5R-deficient cells had decreased matrix metalloproteinase-9 (MMP-9) secretion (p < 0.05). Supplementing conditioned media with MMP-9 restored osteoclastogenesis to control levels, implicating the NPY/Y5R/MMP-9 axis in ES-induced osteoclastogenesis. A transwell migration assay further demonstrated that NPY, but not Y5R, knockdown reduced macrophage cell migration (p < 0.05), suggesting that NPY itself is chemotactic or it stimulates chemotactic factor release via an alternate receptor. Collectively, these findings highlight the role of the NPY/Y5R/MMP-9 axis in osteoclastogenesis at the ES tumor-bone interface and identify NPY as a driver of macrophage recruitment. Further investigation into NPY signaling in ES may inform therapeutic strategies to mitigate bone invasion and metastasis. Funding provided by NIH grant #R01CA197964 awarded to Joanna Kitlinska and the Georgetown University School of Health Discovery Center Grant. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.