BackgroundCancer stem cells are a chemotherapy-resistant population capable of self-renewal and of regenerating the bulk tumor, thereby causing relapse and patient death. Ewing's sarcoma, the second most common form of bone tumor in adolescents and young adults, follows a clinical pattern consistent with the Cancer Stem Cell model – remission is easily achieved, even for patients with metastatic disease, but relapse remains frequent and is usually fatal.Methodology/Principal FindingsWe have isolated a subpopulation of Ewing's sarcoma cells, from both human cell lines and human xenografts grown in immune deficient mice, which express high aldehyde dehydrogenase (ALDHhigh) activity and are enriched for clonogenicity, sphere-formation, and tumor initiation. The ALDHhigh cells are resistant to chemotherapy in vitro, but this can be overcome by the ATP binding cassette transport protein inhibitor, verapamil. Importantly, these cells are not resistant to YK-4-279, a small molecule inhibitor of EWS-FLI1 that is selectively toxic to Ewing's sarcoma cells both in vitro and in vivo.Conclusions/SignificanceEwing's sarcoma contains an ALDHhigh stem-like population of chemotherapy-resistant cells that retain sensitivity to EWS-FLI1 inhibition. Inhibiting the EWS-FLI1 oncoprotein may prove to be an effective means of improving patient outcomes by targeting Ewing's sarcoma stem cells that survive standard chemotherapy.

Ewing sarcoma (ES) is an aggressive malignancy driven by an oncogenic fusion protein, EWS-FLI1. Neuropeptide Y (NPY), and two of its receptors, Y1R and Y5R are up-regulated by EWS-FLI1 and abundantly expressed in ES cells. Paradoxically, NPY acting via Y1R and Y5R stimulates ES cell death. Here, we demonstrate that these growth-inhibitory actions of NPY are counteracted by hypoxia, which converts the peptide to a growth-promoting factor. In ES cells, hypoxia induces another NPY receptor, Y2R, and increases expression of dipeptidyl peptidase IV (DPPIV), an enzyme that cleaves NPY to a shorter form, NPY3-36. This truncated peptide no longer binds to Y1R and, therefore, does not stimulate ES cell death. Instead, NPY3-36 acts as a selective Y2R/Y5R agonist. The hypoxia-induced increase in DPPIV activity is most evident in a population of ES cells with high aldehyde dehydrogenase (ALDH) activity, rich in cancer stem cells (CSCs). Consequently, NPY, acting via Y2R/Y5Rs, preferentially stimulates proliferation and migration of hypoxic ALDHhigh cells. Hypoxia also enhances the angiogenic potential of ES by inducing Y2Rs in endothelial cells and increasing the release of its ligand, NPY3-36, from ES cells. In summary, hypoxia acts as a molecular switch shifting NPY activity away from Y1R/Y5R-mediated cell death and activating the Y2R/Y5R/DPPIV/NPY3-36 axis, which stimulates ES CSCs and promotes angiogenesis. Hypoxia-driven actions of the peptide such as these may contribute to ES progression. Due to the receptor-specific and multifaceted nature of NPY actions, these findings may inform novel therapeutic approaches to ES.

The insulin-like growth factor 1 receptor (IGF-1R) has been considered an important therapeutic target in Ewing sarcoma (ES), generating a need to identify the subset of patients most likely to respond to IGF-1R inhibitors. We assessed IGF-1R expression in ES cell lines and patient tumors to understand the variable clinical responses to anti-IGF-1R therapy. Using ligand-binding displacement, we measured between 13,000 and 40,000 receptors per cell in ES cell lines. We used ELISA to quantify IGF-1R in patient tumors, which expressed 4.8%  ± 3.7 to 20.0%  ± 0.2 of the levels in a positive control cell line overexpressing IGF-1R. Flow cytometry showed markedly reduced IGF-1R expression in ES cell lines compared to a standard positive control cell line. The IGF1R gene was sequenced in 47 ES tumor samples and 8 ES cell lines; only one tumor sample showed a nonsynonymous mutation, R1353H, in a region with low functional impact. Finally, we assessed IGF-1R pathway activity in the ES stem cell (ESSC) population, to characterize its potential for resistance to anti-IGF-1R therapy, using Luminex technology. We found no significant differences in IGF-1R pathway activity between ESSCs and the total cell population. Overall, our findings suggest that IGF-1R as a therapeutic target in this sarcoma may require reevaluation.

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