Abstract 3950: Differential functions of neuropeptide Y in Ewing's sarcoma - hypoxia as a switch
Authors: Tilan Jason U. , Lu Congyi , Earnest Joshua Patrick , Everhart Lindsay M. , Shabir Asim , O'Neill Alison , Wang Shuo , Czarnecka Magdalena , Toretsky Jeffrey A. , Kitlinska Joanna 
Published: August 2012

Ewing's sarcoma (ES) is a group of aggressive pediatric malignancies triggered by a fusion protein, EWS-FLI1, acting as an aberrant transcription factor. A sympathetic neurotransmitter neuropeptide Y (NPY) and two of its receptors (Rs), Y1 and Y5, have been identified as EWS-FLI1 target genes up-regulated in ES. Paradoxically, we have shown that this EWS-FLI1-driven Y1R/Y5R/NPY autocrine loop stimulates ES cell death. On the other hand, however, microarray data associated high expression of Y2Rs, which are not detectable in ES cells in vitro, with a metastatic phenotype of the disease. These seemingly contradictory observations raised the question as to the localization and functions of Y2Rs in ES tumors. Previously, we have shown that aside from being an apoptotic factor for ES cells, NPY also stimulates ES tumor vascularization via Y2Rs present on endothelial cells (ECs). This effect is further enhanced by dipeptidyl peptidase IV (DPPIV), an enzyme that converts NPY to the Y2/Y5R-selective agonist, NPY3-36. Here, we have shown that this Y2R/NPY3-36/DPPIV growth promoting system is up-regulated by hypoxia and may contribute to ES progression. In Y2R-negative ES cells, exposure to 0.1% oxygen induced expression of Y2Rs and significantly up-regulated Y5Rs, while levels of Y1Rs remained unchanged. This shift in R pattern was accompanied by an increase in the expression of DPPIV and NPY itself, leading to the elevated release of the peptide, most likely as a Y2/Y5R-agonist, NPY3-36. These changes were observed at both mRNA and protein levels and confirmed by accumulation of Y2R- and DPPIV-positive ES cells in hypoxic areas of ES xenografts. Importantly, the induction of Y2R expression was particularly apparent in ES cancer stem cells (CSCs), which were identified based on the high activity of aldehyde dehydrogenase (ALDH). This shift in NPY R expression pattern was accompanied by changes in functions of NPY, which under hypoxic conditions stimulated ES CSC proliferation and migration in a Y2/Y5R-dependent manner. In addition to its effect on ES cells, NPY's angiogenic actions are augmented by hypoxia via up-regulation of Y2 and Y5 Rs in ECs, thereby sensitizing these cells to NPY. Consequently, the proliferative effect of ES conditioned media on hypoxic ECs was increased and this effect was NPY-dependent. Moreover, simultaneous increases in NPY release and DPPIV activity in hypoxic ES cells enhanced the angiogenic potential of conditioned media derived from these cells. The clinical relevance of our findings was confirmed by Y2R expression in ES and ECs cells in human tumors and by elevated NPY in sera of ES patients. In summary, hypoxia shifts the activity of NPY in ES from Y1/Y5-mediated tumor cell death to Y2/Y5R-driven stimulatory effects on ES CSCs and ECs. These hypoxia-driven activities of NPY may contribute to ES progression and explain the clinical association between high Y2R expression and metastatic phenotype of the disease.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3950. doi:1538-7445.AM2012-3950