Search

Our studies, both in vivo and in vitro, strongly indicate a critical role of DPP IV in modifying NPY’s actions in the cardiovascular system. The protein can act as an NPY converting enzyme, cleaving the full length NPY1–36 to its shorter form, NPY3–36, and shifting the peptide’s activities from Y1-mediated vasoconstriction and vascular smooth muscle cell growth to Y2/Y5-mediated angiogenesis. On the other hand, the intriguing phenomenon of DPP IV being a necessary factor in Y1/Y5-mediated vascular smooth muscle cell proliferation implicates its possible role as a co-receptor facilitating signalling of and/or ligand binding to NPY receptors. Additional studies are required to determine mechanisms of DPP IV actions in certain cellular models, e.g. why does the enzyme not inactivate NPY1–36 in the vascular smooth muscle cell-Y1/Y5 system by cleavage of the peptide, or whether or not other proteases, such as aminopeptidase P, can compensate for loss of DPP IV. If DPP IV proves to be indeed a critical step required for inhibiting NPY’s contractile and pro-atherosclerotic effect and potentiating its angiogenic activities, abnormally low DPP IV expression and activity could be a risk factor for hypertension and ischemic cardiovascular diseases, in which NPY has already been implicated28. Research into this area seems particularly necessary as DPP IV inhibitors are being considered as a potential therapy for type II diabetes — a disease, which by itself increases the risk for cardiovascular consequences.

With the discovery of NPY’s growth-regulatory effects, emerges a new role for the peptide in regulating cardiovascular, neuronal and immune functions. NPY is involved in angiogenesis, vascular remodeling, proliferation of neuronal cells, and regulation of immune responses. Its actions are cell- and receptor-specific and regulated by the proteolytic activity of DPPIV. Thus, the elucidation of the mechanisms underlying NPY actions can contribute to the development of new therapeutic strategies in the treatment of various disorders, including angiogenesis-related diseases, atherosclerosis, cancer and neuronal degeneration. The growing number of selective receptor agonists and antagonists could potentially become excellent tools to target the NPY system and clinically useful drugs. However, due to the pleiotropic activities of the peptide, the therapy would have to be carefully tailored to the type of the disease, with careful consideration given to the possible side effects.

NPY is an important factor in the regulation of growth of neural crest-derived tumors. The peptide both regulates proliferation of neuroendocrine tumor cells in an autocrine manner and induces tumor vascularization via its Y2 receptor-mediated effect on ECs. The overall effect of NPY is critically dependent on the tumor type and its receptor pattern. Thus, NPY and its receptors may become targets for novel, bidirectional therapies in the treatment of neuroendocrine tumors, directed against both tumor cell proliferation and angiogenesis.

Neuropeptide Y (NPY) is a sympathetic neurotransmitter recently found to be a potent growth and angiogenic factor. The peptide and its receptors are abundant in neural crest-derived tumors, such as sympathetic neuroblastomas and pheochromocytomas, as well as parasympathetic Ewing's sarcoma family of tumors. NPY regulates their growth directly, by an autocrine activation of tumor cell proliferation or apoptosis, and indirectly, by its angiogenic activity. The overall effect of the peptide on tumor growth depends on a balance between these processes and the type of receptors expressed in the tumor cells. Thus, NPY and its receptors may become targets for the treatment of neural tumors, directed against both tumor cell proliferation and angiogenesis.

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.

Neuropeptide Y (NPY), a sympathetic co-transmitter, acts through multiple G protein-coupled receptors (Y1 to y6) to elicit its vast range of effects in the cardiovascular, immune, and central and peripheral nervous systems. Initially, the focus of the function of NPY in the cardiovascular system involved its acute actions, such as vasoconstriction via the Y1 receptor. However, recent studies have shown that NPY is a potent growth and angiogenic factor, which acts on multiple receptor subtypes. To be more specific, NPY-mediated vascular smooth muscle cell growth, leading to neointima formation, involves Y1 and Y1 receptors, while the angiogenic effects of NPY include Y2 and Y5 receptor activation. The presence of dipeptidyl peptidase IV also influences the cardiovascular responses of NPY by acting as a converting enzyme, shifting NPY activities away from Y1. Thus, agonists and antagonists aimed at the NPY system represent a new avenue for drug treatment, which may help alleviate several cardiovascular disorders in which vascular remodeling plays a major role, such as atherosclerosis, restenosis following balloon angioplasty, hypertension and peripheral vascular disease.

Previously we showed that neuropeptide Y (NPY), a sympathetic vasoconstrictor neurotransmitter, stimulates endothelial cell migration, proliferation, and differentiation in vitro. Here, we report on NPY's actions, receptors, and mediators in ischemic angiogenesis. In rats, hindlimb ischemia stimulates sympathetic NPY release (attenuated by lumbar sympathectomy) and upregulates NPY-Y2 (Y2) receptor and a peptidase forming Y2/Y5-selective agonist. Exogenous NPY at physiological concentrations also induces Y5 receptor, stimulates neovascularization, and restores ischemic muscle blood flow and performance. NPY-mediated ischemic angiogenesis is not prevented by a selective Y1 receptor antagonist but is reduced in Y2(-/-) mice. Nonischemic muscle vascularity is also lower in Y2(-/-) mice, whereas it is increased in NPY-overexpressing rats compared with their WT controls. Ex vivo, NPY-induced aortic sprouting is markedly reduced in Y2(-/-) aortas and spontaneous sprouting is severely impaired in NPY(-/-) mice. NPY-mediated aortic sprouting, but not cell migration/proliferation, is blocked by an antifetal liver kinase 1 antibody and abolished in mice null for eNOS. Thus, NPY mediates neurogenic ischemic angiogenesis at physiological concentrations by activating Y2/Y5 receptors and eNOS, in part due to release of VEGF. NPY's effectiveness in revascularization and restoring function of ischemic tissue suggests its therapeutic potential in ischemic conditions.

Neuropeptide Y (NPY), a sympathetic cotransmitter and vasoconstrictor, also stimulates vascular smooth muscle cell (VSMC) growth, but which of its Y1–Y5 receptors are involved remains unclear. In quiescent rat VSMCs, NPY receptor mRNAs were undetectable (reverse transcription – polymerase chain reaction), but Y1, Y2, and Y5 expression were upregulated or induced following NPY treatment. Concomitantly, NPY increased up to twofold [3H]thymidine incorporation and cell number bimodally, with a high-affinity peak at pM and low affinity peak at nM concentrations. The Y1 or Y5 (not Y2) antagonist alone did not change the high-affinity peak but decreased the low affinity peak by 50% and fully blocked NPY's response when combined. In VSMCs lacking NPY receptors and responsiveness, transient Y1 cDNA transfection restored their mitogenic response (blocked by the Y1 antagonist). In VSMCs with low or no NPY responsiveness, pre-exposure to β-adrenergic receptor agonist (isoproterenol), forskolin, or dibu tyryl cAMP augmented NPY's mitogenic effect, while upregulating Y1, Y2, and Y5 receptor expression (isoproterenol only). Thus, NPY is a potent vascular mitogen acting via Y1 and Y5 receptors. However, since their expression is low in nonproliferating cells, amplification of NPY's mitogenic responses requires upregulation of at least the Y1 receptor by NPY itself or β-adrenergic, cAMP-dependent activation.Key words: neuropeptide Y, NPY receptors, beta adrenergic receptor, cyclic AMP, vascular smooth muscle cells.

Age-related changes in NPY-driven angiogenesis were investigated using Matrigel and aortic sprouting assays in young (2 months.) and aged (18 months.) mice. In both assays, NPY-induced vessel growth decreased significantly with age. In parallel, aged mice showed reduced expression (RT-PCR) of Y2 receptors and the NPY converting enzyme, dipeptidyl peptidase IV (DPPIV), in spleens. Aging of human microvascular endothelial cells in vitro led to a loss of their mitogenic responses to NPY accompanied by a lack of NPY receptor mRNAs. Thus, NPY-dependent angiogenesis is impaired with age, which is associated with a decreased expression of endothelial NPY receptors (Y2) and DPPIV.

Ewings sarcoma (ES) is an aggressive malignancy affecting children and adolescents. The presence of metastases is the single most powerful adverse prognostic factor in ES, with a 5-year event-free survival (EFS) at 72% and 3-year EFS at 27% for patients with localized and metastatic disease, respectively. While pulmonary metastases are the most common, the prognosis is worse for patients with bone involvement, particularly when both bone and lung metastases are present (8-14% EFS), and dismal for rare cases with brain metastases (2.7 months median survival). Thus, the treatment of metastatic ES and prevention of its further dissemination is a crucial problem in clinical management of this disease. Despite this, however, our understanding of mechanisms governing ES metastases formation remains poor and appropriate animal models allowing for identifying factors involved in ES metastases are missing. Thus, the goal of our study was to establish and characterize a model recapitulating metastatic processes occurring in ES patients. To this end, ES cells were injected into gastrocnemius muscles of SCIG/bg mice. Once primary tumors reached a volume of 1cm3, tumor-bearing hindlimbs were amputated and mice monitored by periodical MRI for 1-2 months. Moreover, tumor cell dissemination was assessed by histopathological analysis and real-time RT-PCR for EWS-FLI1 transcript. Using this approach, we were able to achieve high frequency of metastases formation. The pattern of metastases was dependent on the ES cell line used. Injection of SK-ES1 cells resulted in formation of multiple distant metastases in 100% of mice. These included soft tissue tumors in the thoracic region (60% of mice), brain metastases (40%), limb metastases (20%) and jaw metastases (20%). Moreover, marked bone invasion was detected in primary tumors and limb metastases. The presence of tumor cell dissemination to bone marrow and other organs (liver, lungs) was also confirmed and quantified by EWS-FLI1 real-time RT-PCR. In contrast, orthotopic injection of TC71 cells resulted primarily in formation of macroscopic lung metastases, but no brain and soft tissue thoracic tumors. Thus, the orthotopic ES xenografts followed by limb amputation provide a robust model of ES metastases suitable for identifying factors involved in ES dissemination and testing new therapeutic strategies. Moreover, the cell line-dependent specificity of the metastatic pattern may allow evaluating and therapeutically targeting particular metastatic processes, such as bone invasion or lung colonization. Citation Format: Sung-Hyeok Hong, Susana Galli, Jason Tilan, David Christian, Meredith Horton, Olga Rodriguez, Christopher Albanese, Joanna Kitlinska. In vivo model of Ewings sarcoma metastases. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A52.

Neuroblastoma (NB) is a pediatric tumor with heterogeneous phenotypes. While low stage tumors carry favorable prognosis, over 50% of high risk NB relapses after treatment with fatal outcome. Thus, developing therapies targeting this refractory form of NB remains an unsolved clinical problem. Neuropeptide Y (NPY) is a sympathetic neurotransmitter released from NB cells. High systemic levels of NPY are associated with poor clinical outcome of the disease, which is in agreement with its proliferative effect in NB cells and angiogenic properties. While all of the above functions of NPY are mediated mainly by its Y2 receptor (Y2R), predominantly expressed in NB and endothelial cells, some NB cell lines additionally express NPY Y5R. The goal of our study was to elucidate functions of Y5R/NPY pathway in NB. We have shown that, in contrast to the constitutively expressed Y2R, expression of Y5R was induced in pro-apoptotic conditions, such as serum deprivation, hypoxia or lack of attachment. Under such cellular stress, blocking Y5R by its selective antagonist or siRNA augmented NB cell death, suggesting pro-survival activity of Y5R/NPY axis. This effect was associated with a decrease in activity of p44/42 MAPK, a known mediator of NPY neuroprotective actions. This anti-apoptotic activity of Y5R contributed to chemoresistance of NB cells. Expression of Y5R and NPY was significantly increased in NB cells treated in vitro with chemotherapy. This effect was more pronounced in cells derived from relapsing tumors of patients that were previously treated with chemotherapy, suggesting pre-activation of the pathways inducing Y5R/NPY expression in these cells. Additionally, these refractory NB cell lines had elevated basal levels of Y5R and NPY expression, as compared to corresponding cell lines derived from the same patients at diagnosis. In line with this observation, 100% of surviving NB cells in tissues derived from chemotherapy-treated NB tumors was highly positive for Y5R, while in non-treated tumors only single, isolated Y5R-positive cells were observed. Blocking Y5R in chemoresistant NB cells rich in this receptor sensitized them to chemotherapy-induced apoptosis, as shown by a decrease in the number of viable cells and increase in caspase 3/7 activity. Consequently, Y5R significantly inhibited growth of NB xenografts derived from chemoresistant NB cells, which was associated with a 4-fold increase in cell death, while no significant changes in the levels of NB cell proliferation and tumor vascularization was observed. In summary, Y5R/NPY axis is an inducible pro-survival pathway activated in NB under cellular stress. This Y5R-mediated anti-apoptotic effect contributes to NB chemoresistance, implicating this receptor as a novel therapeutic target for patients with refractory NB, thus far lacking adequate treatment. Citation Format: Emily Trinh, Magdalena Czarnecka, Sung-Hyeok Hong, Congyi Lu, Samantha Martin, Susana Galli, Ewa Izycka-Swieszewska, Anna Kuan-Celarier, David Christian, Meredith Horton, Jason U. Tilan, Joanna B. Kitlinska. Neuropeptide Y Y5 receptor in neuroblastoma chemoresistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3105. doi:10.1158/1538-7445.AM2014-3105

Prenatal stress, psychologically and metabolically, increases the risk of obesity and diabetes in the progeny. However, the mechanisms of the pathogenesis remain unknown. In adult mice, stress activates NPY and its Y2R in a glucocorticoid-dependent manner in the abdominal fat. This increased adipogenesis and angiogenesis, leading to abdominal obesity and metabolic syndrome which were inhibited by intra-fat Y2R inactivation. To determine whether stress elevates NPY system and accelerates adipogenic potential of embryo, here we "stressed" murine embryonic stem cells (mESCs) in vitro with epinephrine (EPI) during their adipogenic differentiation. EPI was added during the commitment stage together with insulin, and followed by dexamethasone in the standard adipogenic differentiation medium. Undifferentiated embryonic bodies (EBs) showed no detectable expression of NPY. EPI markedly up-regulated the expression NPY and the Y1R at the commitment stage, followed by increased Y2R mRNA at the late of the commitment stage and the differentiation stage. EPI significantly increased EB cells proliferation and expression of the preadipocyte marker Pref-1 at the commitment stage. EPI also accelerated and amplified adipogenic differentiation detected by increasing the adipocyte markers FABP4 and PPARγ mRNAs and Oil-red O-staining at the end of the differentiation stage. EPI-induced adipogenesis was completely prevented by antagonists of the NPY receptors (Y1R+Y2R+Y5R), indicating that it was mediated by the NPY system in mESC's. Taken together, these data suggest that stress may play an important role in programming ESCs for accelerated adipogenesis by altering the stress induced hormonal regulation of the NPY system.

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

Epidemiological and experimental studies suggest that psychosocial stress contributes to cancer development and progression. Yet, the mechanism of this phenomenon is not clear. The goal of our study was to test whether the sympathetic neurotransmitter neuropeptide Y (NPY), which is up-regulated in chronic stress and acts as an angiogenic and growth factor, mediates this process. To this end, wild type (WT) and NPY knockout (NPY KO) mice were treated with a chemical carcinogen, 7,12 dimethylbenz[a]anthracene (DMBA), in the presence of medroxyprogesterone acetate (MPA). To isolate NPY-dependent effects of stress, DMBA/MPA-treated WT and NPY KO mice were subjected to chronic stress (daily cold exposure) for two weeks in the following groups: 1) Control; 2) Early stress during DMBA administration to establish its effect on tumor initiation; 3) Late stress applied when tumors were detectable to determine its effect on their progression. Tumorigenic processes were assessed by monitoring mammary tumor growth, periodical MRI imaging and histopathology of tumor tissues. DMBA/MPA treatment resulted in various neoplastic changes: lymphoma/leukemia (Leu), uterine hemangiomas and angiosarcomas, folliculomas and mammary tumors. The following differences were observed: 1) Under basal conditions, lack of NPY resulted in reduced incidence of Leu and a protective effect on mammary tumor formation (reduced frequency and increased latency) in NPY KO mice suggesting its role in development of these malignancies; 2) early stress increased incidence of uterine angiosarcomas in an NPY-dependent manner. In contrast, a stress-induced increase in frequency of Leu and ovarian folliculoma was observed only in NPY KO mice suggesting that this effect may be mediated by catecholamines (CA), which are excessively released in the absence of an inhibitor of their secretion such as NPY. Surprisingly, early stress had a protective effect on mammary tumor formation that was observed in both WT and NPY KO mice suggesting an NPY-independent mechanism; 3) Late stress accelerated the progression of Leu in an NPY-independent manner. A similar phenomenon was observed in uterine angiosarcomas induced in WT mice, while late stress in NPY KO mice caused their complete regression. These results may be explained by a growth inhibitory effect of CA excessively released in stressed animals devoid of NPY. Lastly, in mammary tumors, stress increased tumor vascularization and proliferation in an NPY-dependent manner. In summary, our data demonstrate potent and differential effects of NPY and other stress mediators on tumor development and progression. These results lay a foundation for further, more comprehensive and mechanistic studies on the effects of stress on particular tumor types. This, in turn, may open new therapeutic and perhaps also preventative avenues for cancer patients, as well as identify populations at risk. 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 LB-186. doi:1538-7445.AM2012-LB-186

BDNF is a known survival factor for neuroblastomas (NB) and is implicated in the development of their chemoresistance. NPY, on the other hand, is a sympathetic neurotransmitter, which is highly released from NB tumors. The elevated NPY levels in NB patients are associated with poor clinical outcome, which is in agreement with its Y2 receptor (Y2R)-mediated proliferative effects on NB cells and pro-angiogenic activities. In normal sympathetic neurons, BDNF has been shown to up-regulate NPY expression, while NPY itself has been implicated in promoting their survival. Therefore, we sought to determine if similar interactions between BDNF and NPY systems occur in NB cells. We have found that BDNF up-regulates expression of both NPY and its Y2Rs in native NB cells, as well as SH-SY5Y cells transfected with BDNF receptor – TrkB (SH-SY5Y/TrkB). However, even more dramatically, BDNF induces expression of another NPY receptor – Y5R, which is not detectable in most NB cell lines under basal conditions. In agreement with this, expression of Y5Rs correlated with TrkB and BDNF expression in human NB samples. In addition, Y5R seems to be directly involved in BDNF signaling. In SH-SY5Y/TrkB cells, BDNF-induced activation of p44/42 MAPK is significantly reduced by selective Y5R antagonist, CGP 71683, but not by Y2R antagonist, BIIE 0246. Interestingly, the Y5R antagonist had no effect on Akt activation stimulated by BDNF. Thus, these data suggest specific cross-talk between TrkB and Y5R signaling leading to activation of p44/42 MAPK pathway. Since BDNF is a known survival factor for NB cells, we sought to determine if pro-survival activity of BDNF is mediated by NPY. Chemotherapy was found to up-regulate the expression of both BDNF and NPY systems. This was further supported by elevated expression of NPY and its Y5Rs in cells derived from a chemotherapy-treated patient (CHLA-20, SMS-KANR) as compared to cells derived from primary tumors of the same patient (CHLA-15, SMS-KAN). In the functional studies, NPY mimicked the anti-apoptotic effect of BDNF in chemotherapy-treated NB cells. Moreover, as observed with MAPK activation, blocking Y5Rs, but not Y2Rs, reduced the pro-survival activity of BDNF in these cells. In summary, while Y2R is the main NPY R constitutively expressed in NBs and responsible for its proliferative effect, expression of Y5R is induced by BDNF and chemotherapy, subsequently enhancing the pro-survival functions of NPY. These anti-apoptotic actions of NPY can additionally augment the known direct survival effects of BDNF. 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 4337. doi:10.1158/1538-7445.AM2011-4337

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 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

Nat. Med. 13, 803–811 (2007); published online 1 July; corrected after print 24 July 2007 The version of this article initially published contained several typographical errors affecting figure citations, units of measure and figure legends, none of which change the scientific conclusions of the manuscript in any way.