Abstract 4147: Neuropeptide Y promotes osteolytic activity during bone invasion and metastasis in Ewing sarcoma

Ewing sarcoma (ES) is a pediatric malignancy affecting bones and soft tissues. The presence of metastases is associated with poor prognosis, particularly in patients with dissemination to the bone. ES cells produce and secrete high levels of neuropeptide Y (NPY), a 36-amino acid neurotransmitter normally released from peripheral sympathetic neurons, and express its receptors. Our previous studies in an ES xenograft model demonstrated that the severity of bone destruction in primary tumors and incidence of osseous metastases positively correlate with levels of NPY release and are significantly decreased with NPY shRNA. Thus, the goal of our study was to determine the mechanism of this NPY-induced osteolytic effect. Since tumor bone invasion and metastasis is associated with changes in osteolytic and osteogenic activity within bone, we assessed osteoblast and osteoclast content in bones adjacent to the ES xenografts. No significant difference in osteoblast number was observed between bones within ES xenografts with high and low NPY levels. However, TRAP assay showed that ES tumors secreting high NPY levels had significantly increased osteoclast density on the border of the bone-tumor interface, as compared to tumor tissues with minimal NPY secretion or treated with NPY shRNA. To identify the mechanisms underlying these osteolytic actions of NPY, we tested its effect on osteoclast recruitment and differentiation. Transwell migration assay showed that osteoclast precursors, RAW 264.7 murine macrophages, were recruited at significantly higher levels to the conditioned media from ES cells with high NPY release, as compared to those cells exposed to conditioned media from NPY-low ES cells. Similarly, ES-conditioned media with high NPY content had a higher ability to stimulate differentiation of RAW 264.7 cells into osteoclasts than those obtained from NPY-low ES cells. Both of these effects were exacerbated by hypoxic conditions, known to upregulate NPY and its Y5 receptors (Y5R) in ES cells. Blocking the expression of NPY and Y5R in ES cells with shRNA inhibited macrophage recruitment and osteoclastogenesis, and decreased RANKL content in conditioned media from NPY-high ES cells. Altogether, these data suggest that the NPY/Y5R autocrine loop stimulates the release of RANKL from ES cells, promoting macrophage recruitment and consequent osteoclast differentiation. Therefore, increased NPY secretion could have a significant impact on shifting bone homeostasis to pr