Neuropeptide Y (NPY) is a sympathetic neurotransmitter recently found to be potently angiogenic and growth promoting for endothelial, vascular smooth muscle and neuronal cells. NPY and its cognate receptors, Y1, Y2 and Y5, are expressed in neural crest-derived tumors; however, their role in regulation of growth is unknown. The effect of NPY on the growth and vascularization of neuroendocrine tumors was tested using three types of cells: neuroblastoma, pheochromocytoma, and Ewing's sarcoma family of tumors (ESFT). The tumors varied in expression of NPY receptors, which was linked to differential functions of the peptide. NPY stimulated proliferation of neuroblastoma cells via Y2/Y5Rs and inhibited ESFT cell growth by Y1/Y5-mediated apoptosis. In both tumor types, NPY receptor antagonists altered basal growth levels, indicating a regulatory role of autocrine NPY. In addition, the peptide released from the tumor cells stimulated endothelial cell proliferation, which suggests its paracrine angiogenic effects. In nude mice xenografts, exogenous NPY stimulated growth of neuroblastoma tumors, whereas it increased apoptosis and reduced growth of ESFT. However, in both tumors, NPY treatment led to an increase in tumor vascularization. Taken together, this is the first report of NPY being a growth-regulatory factor for neuroendocrine tumors, acting both by autocrine activation of tumor cell proliferation or apoptosis and by angiogenesis. NPY and its receptors may become targets for novel approaches in the treatment of these diseases, directed against both tumor cell proliferation and angiogenesis. (Cancer Res 2005; 65(5): 1719-28)

Neuroblastomas are pediatric tumors which develop from sympathetic precursors and express neuronal proteins, such as neuropeptide Y (NPY). NPY is a sympathetic neurotransmitter acting via multiple receptors (Y1-Y5R). Both NPY and Y2Rs are commonly expressed in neuroblastoma cell lines and tissues. The peptide secreted from neuroblastomas stimulates tumor cell proliferation and angiogenesis. Since both processes are Y2R-mediated, the goal of this study was to assess Y2R as a potential therapeutic target for neuroblastoma. In vitro, Y2R antagonist (BIIE0246) prevented activation of p44/42 MAPK induced by endogenous NPY, which resulted in decreased proliferation and induction of Bim-mediated apoptosis. Similar growth-inhibitory effects were achieved with NPY siRNA and Y2R siRNA. In vivo, Y2R antagonist significantly inhibited growth of SK-N-BE(2) and SK-N-AS xenografts, which was associated with decreased activation of p44/42 MAPK, as well as reduced proliferation (Ki67) and increased apoptosis (TUNEL). The Y2R antagonist also exerted an anti-angiogenic effect. In vitro, it reduced the proliferation of endothelial cells induced by neuroblastoma-conditioned media. Consequently, the Y2R antagonist-treated xenografts had decreased vascularization and a high degree of focal fibrosis. In human neuroblastoma tissues, the expression of Y2R was observed in both tumor and endothelial cells, while NPY was predominantly expressed in neuroblastoma cells. In summary, Y2R is a promising new target for neuroblastoma therapy affecting both cancer cells and tumor vasculature.

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.

We previously reported that the sympathetic neurotransmitter neuropeptide Y (NPY) is potently angiogenic, primarily through its Y2 receptor, and that endogenous NPY is crucial for capillary angiogenesis in rodent hindlimb ischemia. Here we sought to identify the source of NPY responsible for revascularization and its mechanisms of action. At d 3, NPY(-/-) mice demonstrated delayed recovery of blood flow and limb function, consistent with impaired collateral conductance, while ischemic capillary angiogenesis was reduced (~70%) at d 14. This biphasic temporal response was confirmed by 2 peaks of NPY activation in rats: a transient early increase in neuronally derived plasma NPY and increase in platelet NPY during late-phase recovery. Compared to NPY-null platelets, collagen-activated NPY-rich platelets were more mitogenic (~2-fold vs. ~1.6-fold increase) for human microvascular endothelial cells, and Y2/Y5 receptor antagonists ablated this difference in proliferation. In NPY(+/+) mice, ischemic angiogenesis was prevented by platelet depletion and then restored by transfusion of platelets from NPY(+/+) mice, but not NPY(-/-) mice. In thrombocytopenic NPY(-/-) mice, transfusion of wild-type platelets fully restored ischemia-induced angiogenesis. These findings suggest that neuronally derived NPY accelerates the early response to femoral artery ligation by promoting collateral conductance, while platelet-derived NPY is critical for sustained capillary angiogenesis.

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

Previously, we showed that sympathetically‐derived NPY promotes ischemic revascularization after femoral artery occlusion (FAO) in rodents by activating Y2 receptors (Rs) and dipeptidyl peptidase IV (DPPIV), which forms a Y2/Y5‐preferring agonist. The role of other NPY sources (platelets), Rs and temporal relationship to ischemic revascularization was unknown and is studied here. A critical role for NPY but not Y5R was shown in NPY−/− and Y5−/− mice whose post‐FAO limb revascularization was markedly impaired and unchanged, respectively. Within 5 days, FAO in rats (expressing platelet NPY) elevated plasma NPY levels (neuronal) and Y1, Y2, DPPIV and NPY mRNA (vs. non‐ischemic contralateral limb) in adductor (arteriogenesis zone) and gastrocnemius (angiogenesis zone) muscles; while density of CD31+‐capillaries fell. Vascular density, CD31+‐ and smooth muscle α‐actin+‐vessels, was restored by 14 days, when platelet levels doubled, and accelerated to 5 days by NPY slow‐release pellet (below FAO, 1 μg/14 days). Platelet NPY and bone marrow NPY mRNA were increased after FAO in platelet NPY‐expressing Sv129 WT mice, which were then used in a platelet depletion/transfer experiment [found (KA) to restore NPY atherosclerotic effects in resistant NPY−/−] with NPY−/−mice. Thus, early activation of neuronal and later activation of megakaryocyte/platelet‐derived NPY is essential for ischemic revascularization. NIH grants HL067357 and HL055310 to Z.Z.

Neuropeptide Y (NPY) is a neurotransmitter, which is released from normal sympathetic neurons, as well as tumors of sympathetic origin, such as neuroblastoma and pheochromocytoma. In neuroblastoma patients, the elevated systemic levels of NPY are associated with poor clinical outcome. We have found that, despite their different origin, Ewing's sarcoma cells (ES) also express and release NPY, which can stimulate angiogenesis via its endothelial Y2 receptor (Y2R) and induce apoptosis in ES cells via Y1 and Y5R. These seemingly opposing functions beg the question: Which of these NPY-mediated actions is clinically relevant? Thus, we sought to determine whether release of NPY from ES tumors will result in elevated systemic levels of the peptide and establish the potential correlation of NPY levels with clinical features of this devastating disease. Analysis of ES cell lines revealed NPY expression at the mRNA level in all investigated cell lines. NPY release (measured by ELISA) into culture media of ES cell lines bearing EWS/FLI type I translocations was not detectable. Interestingly, those bearing type II and III were found at levels comparable to that of sympathetic neuroblastoma. Analysis of serum NPY levels of 248 ES patients with localized disease (collected by Children's Oncology Group under the protocol # AEWS0031) revealed a similar trend in NPY release from tumor tissue. In particular, overall NPY levels were elevated in ES patients versus healthy controls and osteosarcoma patients (p=0.0069), with two distinct groups – ES patients that release NPY similarly to healthy controls and those at significantly higher levels. Furthermore, increased levels of NPY were associated with pelvic tumors (p<0.001), which carry a worse prognosis. The angiogenic properties of NPY are favored with a processing enzyme found to be expressed in ESFT, dipeptidyl peptidase IV (DPPIV), which converts NPY to a Y2R agonist. Thus, we also looked at the DPPIV activity in sera of ES patients and found it to be significantly lower than that of osteosarcoma patients (p=0.0254), but only slightly decreased relative to healthy controls. This suggests that the activity measured in the sera was not derived from tumor-associated DPPIV, but rather the enzyme was shed from other sources, such as immune or endothelial cells. Surprisingly, increased levels of DPPIV activity were found to have a strong predictive value of event free survival. Since DPPIV is known to be involved in immune function, this result may reflect an impaired immune response in ES patients with worse survival. In summary, this is the first report of elevated NPY levels and the first study on DPPIV activity in ES patients. We have found that NPY release was elevated in pelvic ES tumors, while higher DPPIV activity was associated with better prognosis. However, further study and analysis, such as levels of NPY and DPPIV in ES patients with metastatic disease, is needed to fully clarify the role of NPY in ES patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4352. doi:10.1158/1538-7445.AM2011-4352

Ewing's sarcoma family of tumors (ESFT) is a group of aggressive pediatric malignancies which exhibit a variable degree of neuronal differentiation. Previously, we have shown that ESFT cells express a sympathetic neurotransmitter, neuropeptide Y (NPY), and its Y1 and Y5 receptors. We have shown that both endogenous and exogenous NPY can stimulate Y1/Y5R-mediated apoptosis in ESFT cells, which can lead to inhibition of ESFT tumor growth in vivo. However, our further studies suggested that the growth-inhibitory effect of NPY in ESFT cells can be abolished by dipeptidyl peptidase IV (DPPIV) – a membrane protease which cleaves the peptide to its shorter form, NPY3-36, that is inactive at Y1Rs. DPPIV, as well as its homologs with similar activities – membrane-bound fibroblast activation protein (FAP) and cytoplasmic DPP8 and DPP9, have been identified as therapeutic targets in a variety of other tumors. Moreover, numerous broad-spectrum and selective DPP inhibitors have been developed and tested in clinical trials for diabetes and cancer. Thus, the goal of this study was to elucidate the role of particular DPPs in the regulation of ESFT growth and their potential use as therapeutic targets. We have found that aside from expressing NPY and its receptors, all ESFT cells expressed variable levels of DPPIV and its homologs. Both exogenous and endogenous NPY significantly inhibited growth of ESFT cells with low DPP activities. This effect was blocked by Y1/Y5R antagonists and abolished by transfection with DPPIV mRNA. In contrast, cells with high DPP activities did not respond to exogenous NPY and the endogenous peptide had no effect on their growth, as shown by experiments with NPY siRNA. In these DPP-rich cells, the response to both exogenous and endogenous peptide was restored by DPP siRNAs or their selective inhibitors. Surprisingly, similar levels of growth reduction were achieved by blocking DPPIV, as with inhibiting cytoplasmic DPP8 and DPP9. Both effects were mediated by AIF, suggesting caspase-independent cell death, and blocked by Y1/Y5R antagonists, confirming that this was indeed NPY-mediated. In contrast, FAP did not affect the growth-inhibitory actions of NPY in ESFT cells, which is consistent with its low activity, while cleaving substrates with Tyrosine in the first position, such as NPY. In summary, DPPs are important regulators of the growth-inhibitory effect of NPY in ESFT cells, indicating their potential value as therapeutic targets in the treatment of these tumors. However, given the multiple functions of both DPPs and NPY itself, more studies are required to determine their effects on other functions of ESFT cells, such as migration and invasiveness. Moreover, the role of particular DPPs differ in vivo, since FAP and DPPIV are known to be highly expressed in tumor-associated fibroblasts and endothelial cells, and have been shown to modify interactions between tumor and stromal cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 187. doi:10.1158/1538-7445.AM2011-187