Abstract PR15: Hypoxia, polyploidy, neuropeptide Y, and Ewing sarcoma bone metastases: Is there a link?

Ewing sarcoma (ES) is a pediatric tumor driven by EWS-ETS fusion proteins that carries dismal prognosis in its metastatic form. Nevertheless, the factors triggering metastatic processes in ES remain unknown. Even though ES is not considered genomically unstable, the presence of tumor cells with complex karyotypes and high chromosome number is one of a few known adverse prognostic factors, suggesting an acquired chromosomal instability (CIN) as a driver of ES progression. Moreover, worse patient survival correlates with tumor hypoxia, which associates with multiple metastases and dissemination to the bone. Interestingly, hypoxia is also a factor up-regulating neuropeptide Y (NPY) and its Y5 receptor (Y5R) in ES cells. Such an up-regulation has been shown to activate growth-promoting and prometastatic functions of the NPY in these tumors. Previously we have shown that: 1) metastatic progression of ES associates with increase in frequency of polyploid cells and CIN; 2) this effect is triggered by tumor hypoxia; 3) tumor hypoxia promotes formation of bone metastases and the large polyploid cells accumulate in the areas of bone invasion; 4) overactivation of NPY/Y5R pathway leads to cytokinesis defects and polyploidy; and 5) expression of NPY and Y5R increases in bone, but not soft tissue metastases. Based on these observations we hypothesize that polyploid tumor cells arising in hypoxic tumors are responsible for ES bone metastases and thereby poor clinical outcome observed in ES patients with hypoxic tumors. We also propose NPY/Y5R as a trigger of these processes. Thus, the goal of our study was to test the metastatic potential of ES polyploid cells and determine if blocking NPY/Y5R pathway will prevent their formation. To this end, we performed in vitro purification of the 4N-G1 phase cell population from hypoxic SK-ES-1 cell line using FUCCI technology. The progeny of these cells, SK-ES1-4N cells, was characterized for its tumorigenic and metastatic potential in vitro and in vivo. Using soft agar and proliferation assays in standard and low attachment plates, we have shown that under hypoxic conditions SK-ES1-4N cells have enhanced anchorage-independent growth and capability to form colonies. In an orthotopic xenograft model, tissues and cells derived from primary tumors and metastases exhibited broader range of cell and nuclear sizes and increased ploidy, as compared to the tumors from original SK-ES-1 cells, confirming their high CIN. Strikingly, in this model the SK-ES1-4N xenografts metastasized almost exclusively to bones. Bone lesions constituted 83% of total metastases in mice bearing SK-ES1-4N tumors, while only 25% of metastases from the original SK-ES1 xenografts were localized to the bones. To determine if the increase in CIN accompanying ES progression can be prevented by blocking NPY/Y5R pathway, the SK-ES1 primary tumors were treated with Y5R antagonists, CGP 71683. The Y5R blockade decreased frequency of polyploid cells in the large, hypoxic tumors to the levels observed in nonhypoxic ES. Altogether, our findings support the role for polyploidy in ES bone metastasis and implicate the hypoxia-induced activation of the NPY/Y5R axis as its potential trigger. This abstract is also being presented as Poster B34. Citation Format: Akanksha Mahajan, Sung-Hyeok Hong, Susana Galli, Congyi Lu, Shiya Zhu, Jason Tilan, Luciane Cavalli, Joanna Kitlinska. Hypoxia, polyploidy, neuropeptide Y, and Ewing sarcoma bone metastases: Is there a link? [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr PR15.