Abstract 3530: Mechanisms underlying Ewing sarcoma-induced osteolysis

Abstract

Abstract Ewing Sarcoma (ES) is an aggressive pediatric tumor, which often arises in bones and forms osseous metastases, both of which present as osteolytic lesions. Our previous studies indicated that neuropeptide Y (NPY), which is secreted from ES tumors, stimulates tumor-induced bone degradation via its effect on macrophage recruitment and their subsequent osteoclastic differentiation. This effect was dependent on the autocrine activation of the NPY Y5 receptor (Y5R) expressed by ES cells. However, the exact mechanisms of the osteolytic activity induced by the NPY/Y5R axis remained unknown. To identify the osteolytic factors released from ES cells due to the NPY/Y5R autocrine loop activity, we used shRNA to silence NPY or Y5R in SK-ES-1 ES cells and compared concentrations of known factors involved in the regulation of bone homeostasis in their conditioned media by an antibody array. Four secreted factors were found to be significantly decreased after silencing of NPY signaling: interleukin 8 (IL-8; p<0.005), matrix metalloproteinase 9 (MMP-9; p<0.05), intercellular adhesion molecule 1 (ICAM-1; p<0.005), and monocyte chemoattractant protein 1 (MCP-1; p<0.05). Among these proteins, IL-8 and MMP-9 were selected as the main candidates for mediators of NPY-induced osteolysis. To test their role in ES-mediated stimulation of osteolysis, we co-cultured mouse RAW 264.7 cells, that represent monocyte/macrophage lineage, with SK-ES-1 cells in the presence or absence of the MMP-9 inhibitor (JNJ-0966; 10-7M-10-5M) or anti-IL-8 neutralizing antibody (0.1-0.4µg/ml) and tested their effect on osteoclast differentiation detected by TRAP staining. Inhibiting MMP-9 activity significantly decreased osteoclastogenesis of RAW264.7 cells in dose-dependent manner, while blocking IL-8 had no significant effect on the number of formed osteoclasts. These findings implicate MMP-9 as the mediator of NPY osteolytic activity. Further studies are required to understand the exact mechanisms underlying these effects, as well as potential role of NPY-induced release of MCP-1 in macrophage recruitment to ES tumors. Citation Format: James J. Williams, Joanna B. Kitlinska. Mechanisms underlying Ewing sarcoma-induced osteolysis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3530.