Magdalena Czarnecka

Chronic Stress, Combined with a High‐Fat/High‐Sugar Diet, Shifts Sympathetic Signaling toward Neuropeptide Y and Leads to Obesity and the Metabolic Syndrome

In response to stress, some people lose while others gain weight. This is believed to be due to either increased β‐adrenergic activation, the bodys main fat‐burning mechanism, or increased intake of sugar‐ and fat‐rich “comfort foods.” A high‐fat, high‐sugar (HFS) diet alone, however, cannot account for the epidemic of obesity, and chronic stress alone tends to lower adiposity in mice. Here we discuss how chronic stress, when combined with an HFS diet, leads to abdominal obesity by releasing a sympathetic neurotransmitter, neuropeptide Y (NPY), directly into the adipose tissue. In vitro, when “stressed” with dexamethasone, sympathetic neurons shift toward expressing more NPY, which stimulates endothelial cell (angiogenesis) and preadipocyte proliferation, differentiation, and lipid‐filling (adipogenesis) by activating the same NPY‐Y2 receptors (Y2Rs). In vivo, chronic stress, consisting of cold water or aggression in HFS‐fed mice, stimulates the release of NPY and the expression of Y2Rs in visceral fat, increasing its growth by 50% in 2 weeks. After 3 months, this results in metabolic syndrome‐like symptoms with abdominal obesity, inflammation, hyperlipidemia, hyperinsulinemia, glucose intolerance, hepatic steatosis, and hypertension. Remarkably, local intra‐fat Y2R inhibition pharmacologically or via adenoviral Y2R knock‐down reverses or prevents fat accumulation and metabolic complications. These studies demonstrated for the first time that chronic stress, via the NPY‐Y2R pathway, amplifies and accelerates diet‐induced obesity and the metabolic syndrome. Our findings also suggest the use of local administration of Y2R antagonists for treatment of obesity and NPY‐Y2 agonists for fat augmentation in other clinical applications.

Dipeptidyl Peptidases as Survival Factors in Ewing Sarcoma Family of Tumors IMPLICATIONS FOR TUMOR BIOLOGY AND THERAPY

Ewing sarcoma family of tumors (ESFT) is a group of aggressive pediatric malignancies driven by the EWS-FLI1 fusion protein, an aberrant transcription factor up-regulating specific target genes, such as neuropeptide Y (NPY) and its Y1 and Y5 receptors (Y5Rs). Previously, we have shown that both exogenous NPY and endogenous NPY stimulate ESFT cell death via its Y1 and Y5Rs. Here, we demonstrate that this effect is prevented by dipeptidyl peptidases (DPPs), which cleave NPY to its shorter form, NPY3–36, not active at Y1Rs. We have shown that NPY-induced cell death can be abolished by overexpression of DPPs and enhanced by their down-regulation. Both NPY treatment and DPP blockade activated the same cell death pathway mediated by poly(ADP-ribose) polymerase (PARP-1) and apoptosis-inducing factor (AIF). Moreover, the decrease in cell survival induced by DPP inhibition was blocked by Y1 and Y5R antagonists, confirming its dependence on endogenous NPY. Interestingly, similar levels of NPY-driven cell death were achieved by blocking membrane DPPIV and cytosolic DPP8 and DPP9. Thus, this is the first evidence of these intracellular DPPs cleaving releasable peptides, such as NPY, in live cells. In contrast, another membrane DPP, fibroblast activation protein (FAP), did not affect NPY actions. In conclusion, DPPs act as survival factors for ESFT cells and protect them from cell death induced by endogenous NPY. This is the first demonstration that intracellular DPPs are involved in regulation of ESFT growth and may become potential therapeutic targets for these tumors.

Neuropeptide Y receptor Y5 as an inducible pro-survival factor in neuroblastoma: implications for tumor chemoresistance

Neuroblastoma (NB) is a pediatric tumor of neural crest origin with heterogeneous phenotypes. Although low-stage tumors carry a favorable prognosis, >50% of high-risk NB relapses after treatment with a fatal outcome. Thus developing therapies targeting refractory NB remains an unsolved clinical problem. Brain-derived neurotrophic factor (BDNF) and its TrkB receptor are known to protect NB cells from chemotherapy-induced cell death, while neuropeptide Y (NPY), acting via its Y2 receptor (Y2R), is an autocrine proliferative and angiogenic factor crucial for maintaining NB tumor growth. Here we show that in NB cells, BDNF stimulates the synthesis of NPY and induces expression of another one of its receptors, Y5R. In human NB tissues, the expression of NPY and Y5R positively correlated with the expression of BDNF and TrkB. Functionally, BDNF triggered Y5R internalization in NB cells, whereas Y5R antagonist inhibited BDNF-induced p44/42 mitogen-activated protein kinase activation and its pro-survival activity. These observations suggested TrkB-Y5R transactivation that resulted in cross-talk between their signaling pathways. Additionally, NPY and Y5R were upregulated in a BDNF-independent manner in NB cells under pro-apoptotic conditions, such as serum deprivation and chemotherapy, as well as in cell lines and tissues derived from posttreatment NB tumors. Blocking Y5R in chemoresistant NB cells rich in this receptor sensitized them to chemotherapy-induced apoptosis and inhibited their growth in vivo by augmenting cell death. In summary, the NPY/Y5R axis is an inducible survival pathway activated in NB by BDNF or cellular stress. Upon such activation, Y5R augments the pro-survival effect of BDNF via its interactions with TrkB receptor and exerts an additional BDNF-independent anti-apoptotic effect, both of which contribute to NB chemoresistance. Therefore, the NPY/Y5R pathway may become a novel therapeutic target for patients with refractory NB, thus far an incurable form of this disease.

Sympathetic Neurotransmitters in Neuroblastoma – Between Physiology and Pathology

Neuroblastomas arise from precursors of sympathetic neurons due to defects in their normal development (Edsjo et al., 2007). Consequently, the tumors exhibit various degrees of neuronal differentiation manifested by expression of markers characteristic for sympathetic neurons and release of their physiological neurotransmitters (Bourdeaut et al., 2009). Since the levels of neuroblastoma cell differentiation determines clinical phenotype of the disease and its outcome, the factors regulating this process have been extensively studied and recently introduced to the clinic (Edsjo et al., 2007; Maris, 2010). Surprisingly, however, little attention has been paid to the role the sympathetic neurotransmitters excessively released from neuroblastoma cells play in this pathological condition. Despite their known role in the regulation of proliferation and survival of other cell types, in the neuroblastoma field those factors have been treated merely as markers of neuronal differentiation. Very often, even if studies on functional effects of neurotransmitters on neuroblastoma cells have been performed, these cells have been considered purely as a neuronal model (Laifenfeld et al., 2002; Lopes et al., 2010). Therefore, the results of such studies have been interpreted in the context of other neurological disorders, but not assessed in terms of their implications for neuroblastoma biology and therapy. Research conducted in our laboratory focuses on growth-promoting functions of one of such neurotransmitters, neuropeptide Y (NPY). We were able to show that this physiological peptide acts as a crucial mitogenic and angiogenic factor for neuroblastomas and significantly contributes to their progression (Kitlinska et al., 2005; Lu et al., 2010). However, the role of other sympathetic neurotransmitters in biology of these tumors remains understudied. This chapter summarizes our current knowledge on the role these molecules play in the regulation of neuroblastoma growth, and identifies problems which thus far have not been addressed.

Abstract 187: Dipeptidyl peptidases abolish growth inhibitory effect of neuropeptide Y in Ewing's sarcoma family of tumors

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Ewings 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

Abstract 3105: Neuropeptide Y Y5 receptor in neuroblastoma chemoresistance

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

Abstract 3950: Differential functions of neuropeptide Y in Ewing's sarcoma - hypoxia as a switch

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Ewings 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, NPYs 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

Abstract LB-186: Differential effects of stress on tumor initiation and progression - role of neuropeptide Y

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

Abstract 4337: Interactions of brain-derived neurotrophic factor (BDNF) and neuropeptide Y (NPY) systems in neuroblastoma: New insight into mechanisms of BDNF's pro-survival effects

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL 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