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Background: Socioeconomically disadvantaged and immigrant cancer survivors account for a significant and growing proportion of the breast cancer population in the US. Research on symptom burden and control among Chinese American (CA) breast cancer survivors (BCS) is scarce. Among all BCS, over 55% report treatment-related symptoms (e.g., fatigue and pain) and psychological stress (e.g., fear of recurrence). In our preliminary cross-sectional study, we found similar rates (~58%) but showed that CA (especially low-acculturated) BCS were particularly likely to report fatigue, pain, and poorer physical functioning relative to non-Hispanic White (NHW) BCS. We understand very little about whether CA and NHW BCS have different ways of managing symptoms, improving quality of life and decreasing risk for functional decline. We therefore propose a study to examine how CA and NHW BCS, two culturally distinct groups with divergent social resources, adapt to breast cancer. Study design: This longitudinal, prospective study will investigate sociocultural influences on individual coping behaviors and how they in turn affect racial differences in inflammation markers, symptom severity, and functional outcomes in breast cancer. This study will enroll 260 CA and 260 NHW female BCS to examine multifactorial pathways to breast cancer survivorship outcomes. The CA cases will be age- and stage-matched to the NHW cases. Utilizing a multilevel biobehavioral framework, we will investigate the dynamics of biological, sociocultural, and behavioral (diet and exercise) influences on symptom severity, physiologic status, and functional outcomes. Participants will complete telephone survey interviews and provide blood samples at baseline and 6- and 12-month follow-up. Pro-inflammatory cytokines (e.g., IL-1β, IL-1a, IL-6, IL8, IL10, TNFα, TNFꞵ, and CRP) and cortisol will be analyzed. In-depth individual interviews with a subset of participants will be conducted to investigate causal factors in order to develop individually and culturally appropriate interventions to improve future clinical care for targeted breast cancer survivor populations. This study is supported by NIH R01CA248413. Eligibility criteria: Eligible participants are CA and NWH women (age >= 18) who are diagnosed with invasive breast cancer (stage I, II, or III), are 1-5 years post diagnosis, and have completed primary treatment (e.g., surgery, radiation, chemotherapy, and/or targeted therapy). Patients currently on adjuvant endocrine therapy are allowed. Specific aims: Aim 1: Examine whether CA BCS’ symptom, functional, and physiologic outcomes (e.g., cytokines and cortisol), and trajectory of these outcomes differ from NHW BCS at baseline, 6- and 12-month follow-up, controlling for covariates. Aim 2: Examine to what extent social resources mediate BCS’ individual behavior (e.g., medical communication, diet, and physical activity) and to what extent such pathways explain outcome differences (Aim1) among BCS. Aim 3: Examine whether race and acculturation moderate the mediational pathways. Statistical methods: Multiple general linear mixed models will be performed to examine racial differences in the trajectory of symptom and biobehavioral outcomes across time, controlling for covariates (Aim 1). To examine mediation and moderation effects (Aims 2 and 3), we will use a cross-lagged path analysis model to simultaneously describe reciprocal relationships, or directional influences, between variables over time. Present accrual and target accrual: A total of 520 participants (260 CA and 260 NHW) will be enrolled at NYU Perlmutter Cancer Center, Columbia University Irving Medical Center, Georgetown University Medical Center, and Texas A&M University community networks. Contact information: Judy Huei-yu Wang, PhD: jw235@gunet.georgetown.edu or 202-687-6306 Maryann Kwa, MD: maryann.kwa@nyulangone.org or 212-731-6364 Citation Format: Maryann Kwa, Marc Schwartz, Katherine D. Crew, Jeanine M. Genkinger, Roger L. Brown, Leena Hilakivi-Clarke, Joanna Kitlinska, Douglas W. Roblin, Michael Antoni, Sylvia Adams, Kathie-Ann Joseph, Lei-Shih Chen, Judy Huei-yu Wang. A longitudinal investigation of sociocultural and behavioral influences on symptom management, biological response, and functioning among Chinese American and White female breast cancer survivors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT3-14-01.

Impaired angiogenesis is associated with aging and several age-related pathologies, including ischemic vascular disease and delayed wound healing. However, such a reduction in angiogenic capacity could also inform therapeutic applications aiming at inhibiting tumor vascularization. Neuropeptide Y (NPY) and its Y receptors (Rs) - Y2R and Y5R - have been implicated in angiogenic responses associated with cardiovascular disease and cancer. Yet, changes in the NPY system associated with aging are not clear. Therefore, we sought to determine whether alterations in the NPY system are involved in age-associated impairment of angiogenesis. We hypothesized that NPY-mediated angiogenesis is diminished with age due to downregulation of the peptide, its receptors, and converting enzyme - cleaving full-length NPY to a Y2R agonist, as well as downstream signaling pathways. To test our hypothesis, the angiogenic profiles of human microvascular endothelial cells (HMVECs) from 24- and 90-year-old subjects were assessed via proliferation and mRNA expression. This analysis included dose-response and cross-inhibition assays with NPY, fibroblast growth factor 2 (FGF2), and vascular endothelial growth factor (VEGF) systems. Also, basal plasma levels of NPY were compared in old (24 months) and young (3 months) Balb/c mice. Further, NPY-mediated aortic sprouting was assessed in Y2R knockout (KO) mice. Angiogenic stimulation via NPY, FGF, and VEGF was observed in HMVECs from both age groups, with lower basal proliferation levels and less responsiveness to all three factors in aged HMVECs (p NIH R03 AG-20795 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Neuroblastoma (NB) is a pediatric tumor of the peripheral nervous system. Treatment of the disease represents an unsolved clinical problem, as survival of patients with aggressive form of NB remains below 50%. Despite recent identification of numerous potential therapeutic targets, clinical trials validating them are challenging due to the rarity of the disease and its high patient-to-patient heterogeneity. Hence, there is a need for the accurate preclinical models that would allow testing novel therapeutic approaches and prioritizing the clinical studies, preferentially in personalized way. Here, we propose using conditional reprogramming (CR) technology for rapid development of primary NB cell cultures that could become a new model for such tests. This newly established method allowed for indefinite propagation of normal and tumor cells of epithelial origin in an undifferentiated state by their culture in the presence of Rho-associated kinase (ROCK) inhibitor, Y-27632, and irradiated mouse feeder cells. Using a modification of this approach, we isolated cell lines from tumors arising in the TH-MYCN murine transgenic model of NB (CR-NB). The cells were positive for neuronal markers, including Phox2B and peripherin and consisted of two distinct populations: mesenchymal and adrenergic expressing corresponding markers of their specific lineage. This heterogeneity of the CR-NB cells mimicked the different tumor cell phenotypes in TH-MYCN tumor tissues. The CR-NB cells preserved anchorage-independent growth capability and were successfully passaged, frozen and biobanked. Further studies are required to determine the utility of this method for isolation of human NB cultures, which can become a novel model for basic, translational, and clinical research, including individualized drug testing.

Although neuropeptide Y (NPY) is known mainly as a sympathetic neurotransmitter, growing evidence indicates its role in tumor biology. Previous studies from our laboratory and other groups suggested associations of high expression of NPY and its Y5 receptor (Y5R) with invasive and metastatic phenotypes of various tumor types; yet the direct evidence for the metastatic activity was lacking. Our recent work began to fill this gap. Using Ewing sarcoma (ES) as a model of NPY-rich tumor, we identified the NPY/Y5R axis as a crucial pathway responsible for hypoxia-induced ES bone metastasis. This osseous dissemination was driven by hypoxia-dependent over-activation of the NPY/Y5R/RhoA pathway, which led to cytokinesis defects and formation of the aneuploid ES cell population with high propensity to bone metastasis. Here, we sought to determine the impact of the Y5R signaling on overall, hypoxia-independent metastatic capabilities of ES cells. To this end, a doxycycline-inducible CRIPSR/Cas9 system was used to knockout Y5R in ES orthotopic xenografts and test its impact on metastasis and cell migration assessed by a transwell assay. The frequency of the NPY5R gene modifications was determined by sequencing and confirmed by immunohistochemistry. FISH was used to identify NPY5R gene gains in ES xenografts. RhoA activity was measured by immunocytochemistry and pull-down assay. As expected, ES/Y5R-sgRNA primary tumors in doxycycline-treated mice consisted of a heterogeneous ES cell population with variable Y5R levels. In contrast, metastases developing from these xenografts were primarily initiated by ES clones with an intact NPY5R gene. The frequency of NPY5R gene modifications in metastases from the doxycycline-treated group was markedly reduced, as compared to the primary tumors. No metastases arising from ES clones lacking intact NPY5R gene were detected. Similarly, metastasis from wild type ES xenografts was associated with a selection of clones with the NPY5R gene gain that was present in a small percent of the parental cells. Subsequent in vitro assays implicated Y5R-dependent ES cell motility driven by the activation of a key cytoskeleton regulator, RhoA, as the mechanism underlying the metastatic effects of NPY. The above results, along with our previously published data, provide direct evidence for the crucial role of the NPY/Y5R axis in metastasis. We have shown that under basal conditions the NPY/Y5R/RhoA axis promotes tumor cell motility and overall cancer cell dissemination, while over-activation of this pathway under hypoxic conditions leads to genomic instability and bone metastasis. While our study focused on ES due to its high endogenous expression of NPY and its receptors, our findings may be relevant to other malignancies rich in NPY and Y5R, such as neuroblastoma, breast and prostate cancer. Given availability of the FDA approved Y5R antagonist, NPY/Y5R pathway may be a promising target for therapies preventing cancer progression. Yet, further research is required to establish effectiveness of such pharmacological interventions. Citation Format: Mina Adnani, Sung-Hyeok Hong, Susana Galli, Akanksha Mahajan, Nouran Abualsaud, Lindsay Caprio, Jason U. Tilan, Olga Rodriguez, Hingkun Wang, Christopher Albanese, Luciane R. Cavalli, Joanna Kitlinska. Neuropeptide Y as a metastatic factor [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR018.

Neuropeptide Y (NPY) is a multifunctional neurotransmitter acting via G protein-coupled receptors - Y1R, Y2R and Y5R. NPY activities, such as its proliferative effects, are mediated by multiple receptors, which have the ability to dimerize. However, the role of this receptor interplay in NPY functions remains unclear. The goal of the current study was to identify NPY receptor interactions, focusing on the ligand-binding fraction, and determine their impact on the mitogenic activity of the peptide. Y1R, Y2R and Y5R expressed in CHO-K1 cells formed homodimers detectable on the cell surface by cross-linking. Moreover, Y1R and Y5R heterodimerized, while no Y2R/Y5R heterodimers were detected. Nevertheless, Y5R failed to block internalization of its cognate receptor in both Y1R/Y5R and Y2R/Y5R transfectants, indicating Y5R transactivation upon stimulation of the co-expressed receptor. These receptor interactions correlated with an augmented mitogenic response to NPY. In Y1R/Y5R and Y2R/Y5R transfectants, the proliferative response started at picomolar NPY concentrations, while nanomolar concentrations were needed to trigger proliferation in cells transfected with single receptors. Thus, our data identify direct and indirect heterotypic NPY receptor interactions as the mechanism amplifying its activity. Understanding these processes is crucial for the design of treatments targeting the NPY system.

The tumor microenvironment (TME) plays an essential role in the development and progression of neoplasms. TME consists of the extracellular matrix and numerous specialized cells interacting with cancer cells by paracrine and autocrine mechanisms. Tumor axonogenesis and neoneurogenesis constitute a developing area of investigation. Prostate cancer (PC) is one of the most common malignancies in men worldwide. During the past years, more and more studies have shown that mechanisms leading to the development of PC are not confined only to the epithelial cancer cell, but also involve the tumor stroma. Different nerve types and neurotransmitters present within the TME are thought to be important factors in PC biology. Moreover, perineural invasion, which is a common way of PC spreading, in parallel creates the neural niche for malignant cells. Cancer neurobiology seems to have become a new discipline to explore the contribution of neoplastic cell interactions with the nervous system and the neural TME component, also to search for potential therapeutic targets in malignant tumors such as PC.

Neuropeptide Y (NPY) is a sympathetic neurotransmitter with pleiotropic actions, many of which are highly relevant to tumor biology. Consequently, the peptide has been implicated as a factor regulating the growth of a variety of tumors. Among them, two pediatric malignancies with high endogenous NPY synthesis and release - neuroblastoma and Ewing sarcoma - became excellent models to investigate the role of NPY in tumor growth and progression. The stimulatory effect on tumor cell proliferation, survival, and migration, as well as angiogenesis in these tumors, is mediated by two NPY receptors, Y2R and Y5R, which are expressed in either a constitutive or inducible manner. Of particular importance are interactions of the NPY system with the tumor microenvironment, as hypoxic conditions commonly occurring in solid tumors strongly activate the NPY/Y2R/Y5R axis. This activation is triggered by hypoxia-induced up-regulation of Y2R/Y5R expression and stimulation of dipeptidyl peptidase IV (DPPIV), which converts NPY to a selective Y2R/Y5R agonist, NPY(3-36). While previous studies focused mainly on the effects of NPY on tumor growth and vascularization, they also provided insight into the potential role of the peptide in tumor progression into a metastatic and chemoresistant phenotype. This review summarizes our current knowledge of the role of NPY in neuroblastoma and Ewing sarcoma and its interactions with the tumor microenvironment in the context of findings in other malignancies, as well as discusses future directions and potential clinical implications of these discoveries.

Ewing sarcoma (ES) is the second most prevalent malignant bone tumor after osteosarcoma in children and adolescents. While survival of patients with localized ES has improved, patients with metastatic ES still lack adequate therapies, which results in poor survival rates. Thus, there is an important need for new therapies targeting ES metastases and preventing its dissemnation. Previously, we have shown that ES cells constitutively express high levels of NPY and its receptors - Y1R and Y5R. However, tumor hypoxia changes expression pattern and functions of NPY system, leading to the activation of its Y2R and Y5R, which stimulate ES cell motility and invasiveness under these conditions. Hence, the goal of this study was to determine the role of NPY in hypoxia-induced ES metastases. Here, we focused on Y5R, since our data from other malignancies implicated this receptor as the major mediator of NPY's pro-metastatic effects. To this end, we used doxycycline (DOX)-inducible CRIPSR/Cas9 system to create an SK-ES1/Y5R knockout (KO) cell line. The Y5R KO significantly decreased motility of SK-ES1 cells. To determine the impact of this phenomenon on ES metastases, SK-ES1/Cas9/Y5RsgRNA cells were injected into gastrocnemius muscles of SCID/bg mice and the animals were fed with DOX+ or control diet. The Y5R KO did not affect primary tumor growth. Once the xenografts reached a volume of 0.25cm3, a femoral artery ligation (FAL) was performed in the tumor-bearing limbs to induce hypoxic conditions. Both primary tumors and their corresponding metastases were sequenced to confirm the efficiency of gene editing. Notably, majority of metastases arising in mice bearing SKES-1/Y5RsgRNA+DOX xenografts were initiated by the clones without Y5R gene modification, confirming the role of Y5R in ES dissemination. The frequency of metastases with efficient Y5R KO originating from hypoxic tumors was markedly decreased, as compared to the control group (-DOX). Moreover, Y5R KO prevented hypoxia-induced increase in osseous metastases previously reported by our group. Collectively, these data validate NPY/Y5R axis as a crucial mediator of the hypoxia-induced ES metastasis and implicate Y5R receptor as a novel target for therapies preventing ES dissemination. Importantly, the Y5R antagonist is already available and used in clinical trials. Citation Format: Mina T. Adnani, Susana Galli, Sung Hyeok Hong, Jason U. Tilan, Joanna Kitlinska. Blocking neuropeptide Y Y5 receptor prevents hypoxia-induced Ewing sarcoma metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6159.

Neuroblastoma (NB) is a pediatric malignancy that originates from precursors of sympathetic neurons and expresses neuronal proteins, such as neuropeptide Y (NPY) and its receptors. NB cells constitutively express NPY Y2 receptor (Y2R), which maintains their proliferation and tumor vascularization. However, pro-apoptotic conditions additionally induce expression of Y5 receptor (Y5R), which acts as a survival factor for tumor cells. High Y5R expression was also observed in angioinvasive NB cells surrounding blood vessels, suggesting a role for this receptor in NB dissemination. Indeed, in NB patients, high serum levels of NPY associated with the presence of metastases. Hence, the goal of this study was to identify mechanisms that may underlie potential pro-metastatic effects of NPY. We have found that NPY, acting via Y5R, stimulates NB cell motility and acts as a chemotactic factor for these cells in a concentration-dependent manner. Hypoxia, a known condition promoting metastasis, increased expression of Y5R in SK-N-BE(2) and SK-N-AS NB cell lines. Consequently, hypoxic NB cells had increased motility and a chemotactic response to NPY. This increase in NPY-driven migration was fully blocked by Y5R antagonist. Pre-incubation of NB cells in hypoxia before orthotopic injections into adrenal glands of SCID/bg mice exacerbated metastasis in xenografts from both NB cell lines. In mice bearing non-MYCN amplified SK-N-AS tumors, the hypoxia-driven metastases were located in soft tissues, while MYCN-amplified SK-N-BE(2) xenografts derived from hypoxic cells exhibited enhanced dissemination to lungs. The latter pattern of metastases is also observed in NB patients with MYCN amplification. Further studies are required to determine if the hypoxia-driven increase in NB metastasis is Y5R-dependent. Altogether, our data support the role of tumor hypoxia and NPY/Y5R axis in NB dissemination. Since this pathway is also activated during chemotherapy and promotes NB cells resistance to treatment, NPY/Y5R may also be involved in the secondary dissemination of the recurrent tumors. If the role of Y5R in NB metastasis is validated in preclinical models, this NPY receptor may become a potential therapeutic target preventing the disease dissemination. Citation Format: Nouran Abualsaud, Lindsay Caprio, Sung Hyeok Hong, Susana Galli, Joanna Kitlinska. Hypoxia increases motility of neuroblastoma cells induced by the neuropeptide Y/Y5 receptor pathway and exacerbates their metastatic potential [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6157.

Ewing sarcoma (ES) develops in bones or soft tissues of children and adolescents. The presence of bone metastases is one of the most adverse prognostic factors, yet the mechanisms governing their formation remain unclear. As a transcriptional target of EWS-FLI1, the fusion protein driving ES transformation, neuropeptide Y (NPY) is highly expressed and released from ES tumors. Hypoxia up-regulates NPY and activates its pro-metastatic functions. To test the impact of NPY on ES metastatic pattern, ES cell lines, SK-ES1 and TC71, with high and low peptide release, respectively, were used in an orthotopic xenograft model. ES cells were injected into gastrocnemius muscles of SCID/beige mice, the primary tumors excised, and mice monitored for the presence of metastases. SK-ES1 xenografts resulted in thoracic extra-osseous metastases (67%) and dissemination to bone (50%) and brain (25%), while TC71 tumors metastasized to the lungs (70%). Bone dissemination in SK-ES1 xenografts associated with increased NPY expression in bone metastases and its accumulation in bone invasion areas. The genetic silencing of NPY in SK-ES1 cells reduced bone degradation. Our study supports the role for NPY in ES bone invasion and provides new models for identifying pathways driving ES metastases to specific niches and testing anti-metastatic therapeutics.

BACKGROUND Ewing sarcoma (ES) is driven by fusion of the Ewing sarcoma breakpoint region 1 gene (EWSR1) with an E26 transformation-specific (ETS) transcription factor (EWS-ETS), most often the Friend leukemia integration 1 transcription factor (FLI1). Neuropeptide Y (NPY) is an EWS-FLI1 transcriptional target; it is highly expressed in ES and exerts opposing effects, ranging from ES cell death to angiogenesis and cancer stem cell propagation. The functions of NPY are regulated by dipeptidyl peptidase IV (DPPIV), a hypoxia-inducible enzyme that cleaves the peptide and activates its growth-promoting actions. The objective of this study was to determine the clinically relevant functions of NPY by identifying the associations between patients' ES phenotype and their NPY concentrations and DPP activity. METHODS NPY concentrations and DPP activity were measured in serum samples from 223 patients with localized ES and 9 patients with metastatic ES provided by the Children's Oncology Group. RESULTS Serum NPY levels were elevated in ES patients compared with the levels in a healthy control group and an osteosarcoma patient population, and the elevated levels were independent of EWS-ETS translocation type. Significantly higher NPY concentrations were detected in patients with ES who had tumors of pelvic and bone origin. A similar trend was observed in patients with metastatic ES. There was no effect of NPY on survival in patients with localized ES. DPP activity in sera from patients with ES did not differ significantly from that in healthy controls and patients with osteosarcoma. However, high DPP levels were associated with improved survival. CONCLUSIONS Systemic NPY levels are elevated in patients with ES, and these high levels are associated with unfavorable disease features. DPPIV in serum samples from patients with ES is derived from nontumor sources, and its high activity is correlated with improved survival. Cancer 2015;121:697–707. © 2014 American Cancer Society.

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

Ewing Sarcoma (ES) is an aggressive malignancy that arises in children and young adults. Although the survival for patients with localized tumors is relatively high, the metastatic form carries a dismal prognosis, particularly when bone metastases are present. The frequency of metastases and osseous dissemination increases in patients with necrotic ES tumors. In line with this, hypoxia, the major cause of tumor necrosis, has been shown to increase metastatic potential of ES cells. Previous data from our laboratory demonstrated that in an ES orthotopic xenograft model, primary tumor hypoxia specifically promotes bone metastasis, which is associated with accumulation of cells with enlarged nuclei and frequent chromosome gains in bone invasion areas. We have also shown that the progeny of hypoxia-induced polyploid ES cells (ES≈4n cells) preferentially metastasized to bone. Thus, the goal of our study was to determine the mechanisms enabling osseous dissemination of ES≈4n cells. To this end, we used FUCCI Cell Cycle Sensor followed by DNA staining with Hoechst 33342 and cell sorting to isolate tetraploid cells (4n) from hypoxia-exposed SK-ES1 ES cells. Subsequently, we compared the metastatic properties of their progeny (H-SK-ES1≈4n) with a diploid cell population selected from normoxic SK-ES1 cells (N-SK-ES1-2n cells). We have found that H-SK-ES1≈4n cells have increased motility and invasiveness, as compared to the N-SK-ES1-2n cells. H-SK-ES1≈4n cells had also an increased ability to grow in hypoxia in 2D culture and in soft agar. In line with this, H-SK-ES1≈4n cells were highly sensitive to a glycolysis inhibitor, 2D-glucose, but not an inhibitor of oxidative phosphorylation, metformin. Moreover, H-SK-ES1≈4n cells were more sensitive than controls to a growth-inhibitory effect of AICAR, a blocker of anabolic metabolism. In contrary, the tetraploid cell progeny were highly resistant to doxorubicin, particularly under hypoxic conditions, which in contrast sensitized N-SK-ES1-2n and wild type SK-ES1 cells to chemotherapy. Altogether, our data implicate the following mechanisms underlying osseous dissemination of the hypoxia-induced poplyploid cell progeny: 1) increased motility and invasiveness facilitating escape from the primary tumor; 2) ability to survive under low oxygen tension characteristic for bone tissue. Moreover, we have shown that the cells initiating bone dissemination are highly resistant to conventional chemotherapy, while strategies targeting their metabolic dependencies may be more successful in treatment of patients with bone metastases. Citation Format: Shiya Zhu, Akanksha Mahajan, Sung-Hyeok Hong, Susana Galli, Congyi Lu, You-Shin Chen, Sara Misiukiewicz, Stacey Chung, Jason Tilan, Joanna B. Kitlinska. Hypoxia-induced phenotypic and metabolic changes in Ewing sarcoma cells trigger bone metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3664.

Neuroblastoma (NB) is a pediatric malignancy that originates from precursors of the sympathetic neurons and expresses neuronal proteins, such as neuropeptide Y (NPY). NPY is a neurotransmitter released from sympathetic nerves, acting via its Y1-Y5 receptors (Y1R-Y5R). NB cells constitutively express Y2R, while Y5R is induced in pro-apoptotic conditions. Our previous data indicated that Y2R is involved in NB cell proliferation and angiogenesis, while Y5R acts as a pro-survival factor. In NB patients, elevated serum NPY levels are associated with poor prognosis and metastasis. Interestingly, in NB tissues, tumor cells with angioinvasive phenotype have a strong expression of Y5R on the leading edge of the penetrating cell. Thus, the goal of our study was to determine the role of NPY and its receptors in NB dissemination. To this end, we assessed the effect of NPY system perturbations on NB cell motility (IncuCyte Live Cell Analysis system), cytoskeleton organization (phalloidin staining) and activity of a cytoskeleton regulator, RhoA (ICC, pull-down assay). The role of particular NPY receptors in these processes was validated by their overexpression in CHO-K1 cells. Treating NB cells with NPY stimulated NB cell migration in a dose-dependent manner, with a peak of activity at 10-8M and lower effects at higher and lower peptide concentrations. This effect was inhibited by Y5R antagonist, while in some cell lines further reduction was observed when combining Y5R and Y2R antagonists. The stimulatory effect of NPY on cell migration was associated with cytoskeleton remodeling, which included a higher number of filopodia positive for Y5R and co-localization of Y5R with an active form of RhoA at both leading and trailing edges of a migrating cell. In line with this role for Y5R in NB motility, expression of this receptor was up-regulated by hypoxia, a known pro-metastatic factor. Consistent with the results on native NB cells, overexpression of Y5R in CHO-K1 cells significantly increased their motility, while transfection with Y1R or Y2R had no such effect. Stimulation of CHO-K1/Y5R cells with NPY increased migration in a dose-dependent manner, while blocking Y5R inhibited cell motility. Moreover, NPY exerted a chemotactic effect for CHO-K1/Y5R cells. Like in NB cells, in CHO-K1 transfectants, Y5R co-localized with active RhoA in both leading and trailing edges of migrating cells. In line with this, CHO-K1/Y5R cells had elevated basal RhoA activity, while NPY stimulation further increased RhoA-GTP levels. Altogether, our data implicate Y5R as the main NPY receptor mediating its stimulatory effect on cell motility. In NB cells, Y2R may further enhance this effect. The Y5R-induced cell motility is associated with cytoskeleton remodeling driven by RhoA activation. The concentration-dependent activity of NPY and its chemotactic effect indicates that the peptide may spatially regulate NB cell migration within tumor tissue. Citation Format: Nouran Abualsaud, Lindsay Caprio, Abrar Bakr, Lamia Alamri, Ewa Krawczyk, Susana Galli, Sung Hyeok Hong, Joanna Kitlinska. Neuropeptide Y stimulates neuroblastoma cell migration via Y5R/RhoA pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3663.

Neuroblastoma (NB) is a pediatric malignant neoplasm of sympathoadrenal origin. Challenges in its management include stratification of this heterogeneous disease and a lack of both adequate treatments for high-risk patients and noninvasive biomarkers of disease progression. Our previous studies have identified neuropeptide Y (NPY), a sympathetic neurotransmitter expressed in NB, as a potential therapeutic target for these tumors by virtue of its Y5 receptor (Y5R)–mediated chemoresistance and Y2 receptor (Y2R)–mediated proliferative and angiogenic activities. The goal of this study was to determine the clinical relevance and utility of these findings. Expression of NPY and its receptors was evaluated in corresponding samples of tumor RNA, tissues, and sera from 87 patients with neuroblastic tumors and in tumor tissues from the TH-MYCN NB mouse model. Elevated serum NPY levels correlated with an adverse clinical presentation, poor survival, metastasis, and relapse, whereas strong Y5R immunoreactivity was a marker of angioinvasive tumor cells. In NB tissues from TH-MYCN mice, high immunoreactivity of both NPY and Y5R marked angioinvasive NB cells. Y2R was uniformly expressed in undifferentiated tumor cells, which supports its previously reported role in NB cell proliferation. Our findings validate NPY as a therapeutic target for advanced NB and implicate the NPY/Y5R axis in disease dissemination. The correlation between elevated systemic NPY and NB progression identifies serum NPY as a novel NB biomarker.

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.

Tumor dissemination and relapse are the major problems in Ewing sarcoma (ES) treatment, yet the mechanisms driving these processes are unknown. To elucidate the routes of ES metastatic spread, we used an orthotopic xenograft model. ES cells were injected into the gastrocnemius muscles of SCID/beige mice. Once the primary tumors reached the desired volume, they were excised by limb amputation. Subsequently, tumor dissemination was monitored by MRI and confirmed by histopathologic analysis. Interestingly, aside from typical hematogenous metastases, such as bone and lung lesions, we have also observed frequent perineural tumor dissemination manifested by the presence of migratory ES cells along the nerves adjacent to the primary tumors. This phenomenon was associated with formation of recurrent tumors at the amputation sites, as well as pelvic tumors with spine involvement. Interestingly, the level of perineural invasion (PNI) was dependent on the expression of neuropeptide Y (NPY) in ES cells. NPY is a neuronal protein released from peripheral sympathetic neurons, but also highly expressed in ES cells along with its receptors. The xenografts derived from ES cell lines not releasing endogenous NPY (TC71, TC32) exhibited frequent PNI in tumor-bearing limbs, as well as a high number of recurrent tumors at the surgery site and spine metastases (70% and 100% of mice with evidence of PNI for TC71 and TC32 xenografts, respectively). This phenomenon was less common in ES xenografts derived from NPY-rich SK-ES1 cells (17% of mice with signs of PNI). In line with these observations, NPY knockdown in SK-ES1 xenografts drastically accelerated formation of spinal tumors (60% of mice). Notably, in 40% of mice bearing SK-ES1/NPY shRNA xenografts the spinal tumors developed before the primary tumor growth was detectable at the site of ES cell injection. Thus, our in vivo experiments suggested that a lack of endogenous NPY in ES cells expressing high levels of its receptors triggers chemotactic effects of this peptide released from neighboring peripheral nerves, facilitating PNI. Indeed, in a transwell migration assay, NPY exerted significant chemotactic activity in SK-ES1/NPY shRNA cells, but not in the original SK-ES1 cell line. An even more profound chemotactic effect specific to the SK-ES1/NPY shRNA cells was observed with NPY-rich conditioned media obtained from neuroblastoma cells, which can serve as a model of peripheral sympathetic neurons. Further studies are required to determine which NPY receptors are responsible for its chemotactic properties. If the presence of perineural tumor growth is confirmed in human tumors, factors responsible for PNI in ES, e.g., NPY receptors, may become targets for novel therapies preventing disease dissemination and recurrence. Citation Format: Susana Galli, Sung-Hyeok Hong, Jason U. Tilan, Mina Adnani, Shiya Zhu, Yassi Fallah, Yi-Chien Lee, Olga Rodriguez, Chris Albanese, Ewa Izycka-Swieszewska, Joanna Kitlinska. Perineural invasion in Ewing sarcoma—a novel mechanism and new therapeutic opportunities [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B13.

Neuroblastomas are pediatric tumors that 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. As both processes are Y2R-mediated, the aim of this study was to assess Y2R as a potential therapeutic target for neuroblastoma. In vitro, Y2R antagonist (BIIE0246) prevented activation of p44/42 mitogen-activated protein kinase (MAPK) induced by endogenous NPY, which resulted in decreased proliferation and induction of Bim-mediated apoptosis. Similar growth-inhibitory effects were achieved with NPY small interfering RNA (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 (TdT-mediated dUTP nick end labeling; TUNEL). The Y2R antagonist also exerted an antiangiogenic 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.

Treating metastatic disease remains a major obstacle in oncology. Neuropeptide Y (NPY) and its Y5 receptor (Y5R) are expressed in a number of malignancies, including pediatric tumors Ewing sarcoma (ES) and neuroblastoma (NB). Our previous clinical and experimental data implicated NPY/Y5R axis in ES and NB metastasis. We have shown that Y5R is highly expressed in angioinvasive NB cells, while elevated release of NPY from NB tumors associates with metastatic disease and poor clinical outcome. Additionally, hypoxia-induced over-activation of NPY/Y5R pathway in ES tumors, which express particularly high levels of Y5R, led to formation of polyploid cells that give rise to chromosomally unstable, metastatic progeny. This phenomenon was reproduced in CHO-K1 cells over-expressing Y5R, which became polyploid due to a cytokinesis defect. Altogether, our data suggested a role of the NPY/Y5R pathway in the regulation of cell migration and cytokinesis, processes essential in tumor growth and dissemination. Thus, the goal of our study was to determine mechanisms of NPY/Y5R actions. We have found that in both CHO-K1/Y5R transfectants and ES cells, Y5R stimulation activated an essential cytoskeleton regulator, RhoA, activity of which is tightly controlled in a spatial and temporal manner during both cell migration and cytokinesis. This data was confirmed by strong co-localization of Y5R and active RhoA in all cell types. In non-dividing cells, expression of Y5R and active RhoA was limited, while their levels were higher in mitotic cells. Notably, in CHO-K1 cells over-expressing Y5R, both Y5R and RhoA accumulated in the narrow zone of the cleavage furrow at the abscission phase of cytokinesis. Such RhoA activation at this stage was previously shown to trigger cytokinesis defects. The concurrent changes in subcellular localization of Y5R and active RhoA were also observed in migratory cells. In single cell migration, both Y5R and active RhoA accumulated on the trailing and leading edges of the cells, while in the cells migrating collectively their expression was elevated in those present at the front of the moving colony. Strong Y5R/active Rho-A co-localization was also observed in membrane blebs of cells in amoebal migration mode. The role of Y5R in stimulation of cell migration was confirmed by transwell assay, in which Y5R antagonist significantly inhibited NPY-induced migration of NB and ES cells. Altogether, our data support the role of NPY/Y5R/RhoA pathway in regulation of cytoskeleton functions. The stimulation of this pathway promotes cell migration, while its over-activation may lead to cytokinesis defects and chromosomal instability, both of which are essential processes in tumor progression. Further studies are required to determine if these functions of the NPY/Y5R/RhoA pathway contribute to metastasis and validate Y5R as a potential therapeutic target. Citation Format: Nouran Abualsaud, Congyi Lu, Akanksha Mahajan, Abrar Bakr, Shiya Zhu, Lindsay Caprio, Sung Hyeok Hong, Joanna Kitlinska. The role of neuropeptide Y and its Y5 receptor in RhoA-mediated regulation of cytokinesis and cell motility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4143.

Ewing sarcoma (ES) is a pediatric malignancy affecting bones and soft tissues. The presence of metastases is associated with poor prognosis, particularly in patients with dissemination to the bone. ES cells produce and secrete high levels of neuropeptide Y (NPY), a 36-amino acid neurotransmitter normally released from peripheral sympathetic neurons, and express its receptors. Our previous studies in an ES xenograft model demonstrated that the severity of bone destruction in primary tumors and incidence of osseous metastases positively correlate with levels of NPY release and are significantly decreased with NPY shRNA. Thus, the goal of our study was to determine the mechanism of this NPY-induced osteolytic effect. Since tumor bone invasion and metastasis is associated with changes in osteolytic and osteogenic activity within bone, we assessed osteoblast and osteoclast content in bones adjacent to the ES xenografts. No significant difference in osteoblast number was observed between bones within ES xenografts with high and low NPY levels. However, TRAP assay showed that ES tumors secreting high NPY levels had significantly increased osteoclast density on the border of the bone-tumor interface, as compared to tumor tissues with minimal NPY secretion or treated with NPY shRNA. To identify the mechanisms underlying these osteolytic actions of NPY, we tested its effect on osteoclast recruitment and differentiation. Transwell migration assay showed that osteoclast precursors, RAW 264.7 murine macrophages, were recruited at significantly higher levels to the conditioned media from ES cells with high NPY release, as compared to those cells exposed to conditioned media from NPY-low ES cells. Similarly, ES-conditioned media with high NPY content had a higher ability to stimulate differentiation of RAW 264.7 cells into osteoclasts than those obtained from NPY-low ES cells. Both of these effects were exacerbated by hypoxic conditions, known to upregulate NPY and its Y5 receptors (Y5R) in ES cells. Blocking the expression of NPY and Y5R in ES cells with shRNA inhibited macrophage recruitment and osteoclastogenesis, and decreased RANKL content in conditioned media from NPY-high ES cells. Altogether, these data suggest that the NPY/Y5R autocrine loop stimulates the release of RANKL from ES cells, promoting macrophage recruitment and consequent osteoclast differentiation. Therefore, increased NPY secretion could have a significant impact on shifting bone homeostasis to promote osteolysis, bone invasion, and osseous metastasis. Further studies are needed to identify other factors released by tumor cells secreting high NPY levels and to test Y5R antagonists as a potential therapeutic option to inhibit these processes. Importantly, aside from ES, the above findings may be relevant to other NPY-rich tumors metastasizing to the bone, such as neuroblastoma. Citation Format: Richard Garner, Emily Hong, Shiya Zhu, Mina Adnani, Sung-Hyeok Hong, Susana Galli, Joanna Kitlinska. Neuropeptide Y promotes osteolytic activity during bone invasion and metastasis in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4147.

Ewing sarcoma (ES) is a pediatric tumor driven by EWS-ETS fusion proteins that carries dismal prognosis in its metastatic form. Nevertheless, the factors triggering metastatic processes in ES remain unknown. Even though ES is not considered genomically unstable, the presence of tumor cells with complex karyotypes and high chromosome number is one of a few known adverse prognostic factors, suggesting an acquired chromosomal instability (CIN) as a driver of ES progression. Moreover, worse patient survival correlates with tumor hypoxia, which associates with multiple metastases and dissemination to the bone. Interestingly, hypoxia is also a factor up-regulating neuropeptide Y (NPY) and its Y5 receptor (Y5R) in ES cells. Such an up-regulation has been shown to activate growth-promoting and prometastatic functions of the NPY in these tumors. Previously we have shown that: 1) metastatic progression of ES associates with increase in frequency of polyploid cells and CIN; 2) this effect is triggered by tumor hypoxia; 3) tumor hypoxia promotes formation of bone metastases and the large polyploid cells accumulate in the areas of bone invasion; 4) overactivation of NPY/Y5R pathway leads to cytokinesis defects and polyploidy; and 5) expression of NPY and Y5R increases in bone, but not soft tissue metastases. Based on these observations we hypothesize that polyploid tumor cells arising in hypoxic tumors are responsible for ES bone metastases and thereby poor clinical outcome observed in ES patients with hypoxic tumors. We also propose NPY/Y5R as a trigger of these processes. Thus, the goal of our study was to test the metastatic potential of ES polyploid cells and determine if blocking NPY/Y5R pathway will prevent their formation. To this end, we performed in vitro purification of the 4N-G1 phase cell population from hypoxic SK-ES-1 cell line using FUCCI technology. The progeny of these cells, SK-ES1-4N cells, was characterized for its tumorigenic and metastatic potential in vitro and in vivo. Using soft agar and proliferation assays in standard and low attachment plates, we have shown that under hypoxic conditions SK-ES1-4N cells have enhanced anchorage-independent growth and capability to form colonies. In an orthotopic xenograft model, tissues and cells derived from primary tumors and metastases exhibited broader range of cell and nuclear sizes and increased ploidy, as compared to the tumors from original SK-ES-1 cells, confirming their high CIN. Strikingly, in this model the SK-ES1-4N xenografts metastasized almost exclusively to bones. Bone lesions constituted 83% of total metastases in mice bearing SK-ES1-4N tumors, while only 25% of metastases from the original SK-ES1 xenografts were localized to the bones. To determine if the increase in CIN accompanying ES progression can be prevented by blocking NPY/Y5R pathway, the SK-ES1 primary tumors were treated with Y5R antagonists, CGP 71683. The Y5R blockade decreased frequency of polyploid cells in the large, hypoxic tumors to the levels observed in nonhypoxic ES. Altogether, our findings support the role for polyploidy in ES bone metastasis and implicate the hypoxia-induced activation of the NPY/Y5R axis as its potential trigger. This abstract is also being presented as Poster B34. Citation Format: Akanksha Mahajan, Sung-Hyeok Hong, Susana Galli, Congyi Lu, Shiya Zhu, Jason Tilan, Luciane Cavalli, Joanna Kitlinska. Hypoxia, polyploidy, neuropeptide Y, and Ewing sarcoma bone metastases: Is there a link? [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr PR15.

Neuroblastomas are pediatric tumors of sympathetic origin, expressing neuronal markers, such as NPY and its receptors. Due to this, neuroblastomas are often associated with elevated plasma levels of NPY, which correlates with poor clinical outcome of the disease. This clinical data corroborates the recent discovery of growth-promoting actions of NPY in neuroblastomas. The peptide has been shown to stimulate proliferation of neuroblastoma cells in an autocrine manner and induce tumor vascularization. Since both processes are mediated by the same Y2 and Y5 receptors, targeting this pathway may be a potential bidirectional therapy for these children's tumors.

To determine if preeclampsia (PE) is associated with dysregulation of the neuropeptide Y (NPY) system.The study enrolled 114 subjects either with normal pregnancy (NP) or with PE. Systolic blood pressure (SBP) was collected from patients using a standard sphygmomanometer. The PE patients were divided into two groups based on the gestational age (GA) at delivery - placental PE (PLPE, GA34 weeks) or maternal PE (MTPE, GA ≥34 weeks). NPY was measured in platelet rich plasma (PRP), platelet poor plasma (PPP) and in the serum of NP and PE patients utilizing radioimmunoassay. Serum levels of soluble fms-like tyrosine kinase-1 (sFlt-1) and placental growth factor (PlGF) were measured in NP and PE subjects by ELISA.SBP was higher in PE compared to NP. Circulating NPY in serum and PRP, as well as NPY content per 100,000 platelets, but not its concentrations in PPP, were elevated in PE, as compared to NP. The highest NPY concentrations were observed in sera and PRP of patients with MTPE. PE patients had also elevated levels of sFlt-1, as compared to NP, although no difference between PLPE and MTPL groups were observed. There was no increase in P1GF in PE patients.Systemic NPY is elevated in PE patients, as compared to NP. This increase is observed in blood fractions containing platelets, suggesting accumulation of the peptide in these cells. NPY concentrations are particularly high in patients with MTPE, underlying differences in etiology between PLPE and MTPE. Our study implicates NPY as a potential target in antihypertensive therapies for PE patients.

Neuroblastoma (NB) is a pediatric malignancy arising due to defects in sympathetic neuron differentiation. NB is a heterogeneous disease, with phenotypes ranging from spontaneously regressing to highly aggressive, incurable tumors. This clinical variability cannot be explained solely by genetic aberrations. Even in families with hereditary NB the penetrance of the disease is incomplete and the same genetic mutation often results in tumors with phenotypes varying from differentiating ganglioneuromas to undifferentiated, highly aggressive NBs. Thus, other, perhaps non-genetic factors can contribute to the disease development and modify its phenotype. Strikingly, the two factors promoting de-differentiation of NB cells and their malignant phenotype, hypoxia and glucocorticoids, are elevated in the fetus during maternal stress, suggesting a role for prenatal stress in NB tumorigenesis. Previously, using TH-MYCN mice as a model of aggressive NB, we have shown that an increase in maternal corticosterone levels during pregnancy attained by inserting slow release pellets resulted in increased tumor frequency in TH-MYCN offspring. The goal of the current study was to determine the effect of prenatal stress on NB metastasis. To this end, pregnant mice carrying TH-MYCN hemizygous offspring were subjected to chronic stress at embryonic days 10-17, the time of sympathetic neuroblast proliferation and differentiation. Two established stress paradigms were used - chronic unpredictable stress, in which mice were subjected daily to various stressors, and chronic cold stress comprising of daily 30 min exposure to cold. The phenotypes of the disease and its dissemination were compared between offspring of control and stressed mothers. The offspring from both prenatally stressed groups presented with more malignant disease, as manifested by the presence of advanced lung metastases disseminating from small primary tumors (1,000 mm3). Although not common, lung metastases occur preferentially in NB patients with MYCN amplification and are associated with significantly worse prognosis, as compared to patients with metastatic disease, but no pulmonary involvement (14 vs 43% 3-year event-free survival, respectively). Thus, the profound pulmonary dissemination observed in prenatally-stressed TH-MYCN mice mimics one of the most malignant NB phenotypes observed in human disease. Altogether, our data implicate maternal stress during pregnancy as a potential environmental factor modifying the effects of genetic aberrations and promoting malignant phenotype of NB. Citation Format: Sung Hyeok Hong, Larissa Wietlisbach, Susana Galli, Akanksha Mahajan, Shiya Zhu, Jason Tilan, Yichien Lee, Olga Rodriguez, Chris Albanese, Joanna Kitlinska. Prenatal stress increases malignancy of neuroblastoma tumors in TH-MYCN animal model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1940. doi:10.1158/1538-7445.AM2017-1940

Neuroblastoma (NB) is a tumor derived from neural crest cells, primitive progenitors of sympathetic ganglia. It accounts for 6-10% of all pediatric cancers with approximately 700 new cases per year throughout the US. Although there are highly effective therapies for patients with low-risk and intermediate-risk disease who have local relapses, recurrent disease in patients with high-risk NB is mostly refractory to therapy. Neuropeptide Y (NPY) is a sympathetic neurotransmitter highly expressed in NB. Its elevated release from tumor tissue is associated with unfavorable clinical outcome. NPY, acting via its Y5 receptor (Y5R), stimulates NB cell survival and chemoresistance, however the mechanisms underlying NPY/Y5R axis activation in these tumors remain unclear. The aim of this work was to investigate the correlation between the expression of NPY and NPY5R proteins and the copy number status of the NPY and NPY5R genes in pre- and post-chemotherapy NB. Eighty-five tissue samples, including specimens from the primary tumors, distant metastases and local relapses, pre- and post-chemotherapy, were collected from the Hospital Pequeno Principe, Parana, Brazil. Protein expression was investigated by immunohistochemistry. Copy number alterations (CNAs) for NPY and NPY5R genes were determined using TaqMan copy number assay. Additionally, FISH analysis was performed to assess MYCN amplification status, a genetic marker of high risk NB. Our results show that elevated extracellular NPY staining, which reflects peptide release, correlates with patients’ age above 18 months, relapse and poor clinical outcome. Moreover, the intensity of NPY staining was increased in chemotherapy-treated NBs, as compared to tumors at diagnosis. However, the differences in NPY CNAs between these samples were not statistically significant. Thus, chemotherapy-induced increase in NPY levels observed in post-treatment NB is driven by its elevated expression rather than genomic changes. For NPY5R, significantly higher level of CNAs was observed in the post-chemotherapy samples (P Citation Format: Selene Elifio-Esposito, Akanksha Mahajan, Aline S. Fonseca, Susana Galli, Lucia Noronha, Lisiane C. Poncio, Bonald C. Figueiredo, Joanna B. Kitlinska, Luciane R. Cavalli. Increase in protein expression and copy number drives the activation of NPY/Y5R pro-survival loop in chemotherapy-treated neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5822. doi:10.1158/1538-7445.AM2017-5822

Neuroblastoma (NB) is a pediatric malignancy with phenotypes varying from spontaneously regressing to metastatic tumors. Thus, disease stratification and the subsequent treatment decision is of utmost importance for NB patients. The methods, which are currently available for prognosis, require complex genetic analyses and access to the tumor tissue. Thus, there is a need for new prognostic and predictive markers that reflect the NB biology and rely on simple tests and easily accessible material. Serum metabolite profiles reflect a combination of factors released by the tumors and the general metabolic state of the patients. Thus, identifying the differences in metabolites between patients with various tumor phenotypes and responses to treatment may lead to discovery of biologically relevant prognostic biomarkers. The goal of our study was to determine blood-based metabolic signatures characteristic for patients with low and high-risk NB and identify potential biomarkers of high-risk disease. Plasma samples from NB patients at different stages of the disease were obtained from the Children’s Oncology Group. Initially, 50 samples were analyzed by Ultra Performance Liquid Chromatography (UPLC) in conjunction with electrospray-quadrupole-time of flight mass spectrometry (ESI-Q-TOF). A set of 26 independent plasma samples was then used for validation study. Statistically significant differences in metabolic profiles were observed between serum from patients with low and high risk disease, various stages of the disease (stage 1-2 vs stage 3 and/or stage 4), as well as undifferentiated vs. differentiating tumors. For all comparisons, the metabolic signatures consisting of metabolites significantly different between the groups exhibited high test sensitivity and specificity (the area under the ROC curves 0.92-1). Metabolites that were dysregulated in high risk NBs included those involved in energy and choline metabolism, as well as amino acid and lipid biosynthesis. Of particular interest was a significant increase in serum levels of oxoglutaric acid observed in patients with high-risk disease (p Citation Format: Richa Jain, Kirandeep Gill, Leon Qingliang Li, Amrita K. Cheema, Joanna B. Kitlinska. Metabolic serum signatures as potential prognostic biomarkers for neuroblastoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1954. doi:10.1158/1538-7445.AM2017-1954

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.

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.—Tilan, J. U., Everhart, L. M., Abe, K., Kuo-Bonde, L., Chalothorn, D., Kitlinska, J., Burnett, M. S., Epstein, S. E., Faber, J. E., Zukowska, Z. Platelet neuropeptide Y is critical for ischemic revascularization in mice.

Ewing sarcoma (ES) is a tumor driven by EWS-ETS fusion proteins. Yet, the same fusions are present in localized and metastatic tumors that carry strikingly different prognoses. Despite low levels of genomic instability in primary ES tumors, the presence of complex karyotypes is one of a few adverse prognostic factors, implicating an acquired chromosomal instability (CIN) in ES progression. As transcriptional targets of EWS-ETS, neuropeptide Y (NPY) and its Y5 receptor (Y5R) are highly expressed in ES and further activated by hypoxia. We have found that overexpression of Y5R leads to defects in cytokinesis, followed by formation of polyploid cells, chromosome loss and CIN. Thus, the goal of our study was to determine whether CIN that is driven by hypoxia-induced activation of NPY/Y5R axis promotes ES metastases. ES cells were injected into gastrocnemius muscles of SCID/beige mice. Hypoxia in the resulting primary tumors was created by 72h ligation of the femoral artery. Then, the tumors were excised and mice were monitored for metastases. Tissues and cells derived from primary tumors and metastases were subjected to cytogenetic analyses. ES metastasis was associated with progressive genomic changes in tumor cells. Cells derived from primary tumors exhibited increases in nuclear sizes and ploidy, as compared to the original cells. Tumor hypoxia exacerbated this effect. This initial increase in ploidy was followed by a decrease in nuclear size, increase in mitotic errors and reduced chromosome numbers in cells from metastatic tissues, suggesting that ES progression associates with increased CIN and is triggered by cell polyploidization. This notion was confirmed by increased DNA copy number alterations in tissues from ES metastases observed in xenografts derived from 2 different cell lines and a clinical case of matched primary tumor and metastasis tissue (array-CGH). In SK-ES1 xenografts, these alterations involved gains in the locus of Y5R. Consequently, FISH identified an SK-ES1 clone with 3 copies of the Y5R gene. The percent of cells with Y5R gene amplification increased with the degree of SK-ES1 progression, with 16-24% cells in the original SK-ES1 cell line, 40-60% in primary tumors and 86-100% in metastases. This was associated with an increase in Y5R expression in metastatic tissues. Thus, the metastasis in SK-ES1 xenografts associated with a selection of the clone with amplified Y5R. SK-ES1 cells subjected to hypoxia in vitro presented with similar increases in nuclear sizes and enrichment in the clone with amplified Y5R (48%), as was observed in primary tumors. Y5R activation in normoxic SK-ES1 cells mimicked this effect. Our findings support the role for acquired CIN in ES progression and metastasis and implicate the hypoxia-induced activation of the NPY/Y5R axis as its potential trigger. Thus, Y5R antagonist may serve as an adjuvant treatment to prevent ES CIN and progression. Citation Format: Akanksha Mahajan, Sung-Hyeok Hong, Jason U. Tilan, Susana Galli, Congyi Lu, Jasmine Rodgers, Anju Duttargi, Rachel Acree, Luciane R. Cavalli, Joanna B. Kitlinska. Ewing sarcoma progression associates with increasing chromosomal instability: A role for neuropeptide Y and its Y5 receptor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2443.

Ewing sarcoma (ES) is a pediatric tumor induced by EWS-ETS fusion proteins, most often EWS-FLI1. While the presence of metastases is the single most powerful adverse prognostic factor for ES patients, the mechanisms underlying their development remain unclear. Tumor hypoxia is one of the few factors implicated in ES progression. In ES patients, the presence of nonperfused areas within tumor tissue was associated with poor prognosis. In vitro, hypoxia increases invasiveness of ES cells and triggers expression of pro-metastatic genes via changes in transcriptional activity of the EWS-FLI1 gene. However, despite this line of evidence, no direct proof for this hypoxia-induced ES progression and spread has been provided. Moreover, the mechanisms by which hypoxia could exert such effects are unknown. To fill this gap, we created an in vivo model of hypoxia in ES and tested its effect on tumor metastasis. SK-ES1 ES cells were injected into gastrocnemius muscles of SCID/beige mice. Once the tumors reached a volume of 250mm3, they were either excised (control) or subjected to femoral artery ligation (FAL) for 72h prior to excision, inducing ischemia of the lower hindlimb, thus creating tumor hypoxia. Then, the mice were monitored for metastases. The extent of the metastatic disease was assessed and compared between experimental groups based on periodic MRI, necropsy and histopathology findings. FAL resulted in profound tumor hypoxia, as evidenced by inhibition of primary tumor growth, severe tissue necrosis and positive staining for a hypoxyprobe, pimonidazole. However, despite the impaired growth of primary tumors, xenografts subjected to FAL were more metastatic. The involvement of hypoxic cells in metastases was evidenced by the accumulation of pimonidazole-positive cells (hypoxic at the time of FAL) in areas of tissue invasion and intravasation. Consequently, mice bearing FAL-treated tumors exhibited a decreased latency of metastases formation and an increase in their number from an average of 0.9 to 2.3 metastases per mouse in control and FAL groups, respectively. We also observed a change in the pattern of metastases, as FAL-treated tumors metastasized more often to distant organs (average of 0.3 organ metastases per mouse in control and 1.3 in FAL group). The hypoxia-induced metastases were most often observed in adrenal gland and spine (50% and 42% of mice in FAL group, respectively), while no such metastases were observed in the control group. Moreover, 100% of FAL-treated mice had signs of bone marrow invasion, while no tumor cells were detectable in bone marrow of control mice. This data provides the first-ever direct evidence for tumor hypoxia as a driver of ES metastases. Moreover, our model of tumor hypoxia in vivo provides an excellent opportunity to identify hypoxia-induced pathways involved in ES metastatic progression that subsequently may become novel therapeutic targets for this disease. Citation Format: Jason U. Tilan, Sung-Hyeok Hong, Susana Galli, Rachel Acree, Katherine Connors, Meredith Horton, Akanksha Mahajan, Larissa Wietlisbach, Yi-Chien Lee, Olga Rodriguez, Christopher Albanese, Joanna Kitlinska. Tumor hypoxia promotes Ewing sarcoma metastases in a mouse xenograft model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2478.

Neuropeptide Y (NPY) mediates stress-induced obesity in adult male mice by activating its Y2 receptor (Y2R) in visceral adipose tissue (VAT). Here, we studied whether the NPY-Y2R system is also activated by maternal low-protein diet (LPD) and linked to obesity in offspring. Prenatal LPD offspring had lower birth weights compared to normal-protein diet (NPD) offspring. Female prenatal and lactation stress (PLS) offspring from mothers fed an LPD developed abdominal adiposity and glucose intolerance associated with a 5-fold up-regulation of NPY mRNA and a 6-fold up-regulation of Y2R mRNA specifically in VAT, in addition to elevated platelet-rich-plasma (PRP) NPY, compared to control females fed a high-fat diet (HFD). Conversely, PLS male offspring showed lower NPY in PRP, a 10-fold decrease of Y2R mRNA in VAT, lower adiposity, and improved glucose tolerance compared to control males. Interestingly, prenatal LPD offspring cross-fostered to control lactating mothers had completely inverse metabolic and NPY phenotypes. Taken together, these findings suggested that maternal LPD activates the VAT NPY-Y2R system and increases abdominal adiposity and glucose intolerance in a sex- and time-specific fashion, suggesting that the peripheral NPY system is a potential mediator of programming for the offspring's vulnerability to obesity and metabolic syndrome.—Han, R., Li, A., Li, L., Kitlinska, J. B., Zukowska, Z. Maternal low-protein diet up-regulates the neuropeptide Y system in visceral fat and leads to abdominal obesity and glucose intolerance in a sex- and time-specific manner.

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.

Neuropeptide Y (NPY), a sympathetic and platelet-derived vascular mitogen and angiogenic factor, has been implicated in atherosclerosis in animal and human genetic studies. Here we evaluate its association with human and murine atherosclerosis, and assess the role of platelet-derived NPY in lesion vulnerability. NPY immunoreactivity (NPY-ir) was measured in the platelet-poor and platelet-rich (PRP) plasmas, and NPY receptors (mitogenic Y1R and angiogenic Y2 and Y5Rs), CD26/DPPIV (a protease forming Y2/Y5-selective agonist), CD31-positive vascularity, and lesion morphology assessed by histo- and immunocyto-chemistry—in patients with peripheral artery disease (PAD) and healthy volunteers, and in lard-fed ApoE−/− mice. NPY and NPY-R immunostaining was greater in lesions from PAD patients compared to normal vessels of healthy volunteers (p < 0.001), and localized to smooth muscle cells, macrophages, and adventitial/neovascular endothelial cells. CD26/DPPIV staining co-localized with CD31-positive endothelial cells only in atherosclerotic lesions. NPY-ir in PRP (but not plasma) and vascular immunostaining was higher (p < 0.05 and 0.001, respectively) in men (not women) with PAD compared to healthy subjects. A similar gender specificity was observed in mice. PRP NPY-ir levels correlated with lesion area (p = 0.03), necrotic core area, and the necrotic core-to-lesion area ratio (p < 0.01) in male, but not female, mice. Also males with neovascularized lesions had higher PRP NPY-ir levels than those lacking lesion microvessels (p < 0.05). NPY and its Rs are up-regulated in human and murine atherosclerotic lesions suggesting pathogenic role. DPPIV expression by microvascular endothelium in atherosclerotic tissue may shift NPY’s affinity toward angiogenic Y2/Y5Rs, and thus enhance angiogenesis and lesion vulnerability. Remarkably, plaque neovascularization was associated with increased NPY-ir in PRP in males but not females, suggesting that platelet NPY may be a novel mediator/marker of lesion vulnerability particularly in males, for reasons that remain to be determined. Both animal and human data suggest that NPY is an important contributor to, and platelet NPY-ir a marker of, atherosclerotic lesion burden and vulnerability but only in males, perhaps due to androgen-dependent up-regulation of NPY, previously shown in rats.

Recent evidence supports a longstanding hypothesis that chronic stress can influence tumor growth and progression. It has been shown that sympathetic neurotransmitters, such as catecholamines and neuropeptides, can affect both cancer cell growth and tumor vascularization. Depending on neurotransmitter and type of tumor, these effects can be both stimulatory and inhibitory. Norepinephrine (NE) and epinephrine (E) are potent stimulators of vascularization, acting both by inducing the release of angiogenic factors from tumor cells and directly on endothelial cell (EC) functions. As a result, activation of the adrenergic system increases growth of various types of tumors and has been shown to mediate stress-induced augmentation of tumor progression. Dopamine (DA), on the other hand, interferes with VEGF signaling in endothelial cells, blocks its angiogenic functions and inhibits tumor growth. Another sympathetic neurotransmitter coreleased with NE, neuropeptide Y (NPY), directly stimulates angiogenesis. However, proangiogenic actions of NPY can be altered by its direct effect on tumor cell proliferation and survival. In consequence, NPY can either stimulate or inhibit tumor growth, depending on tumor type. Hence, sympathetic neurotransmitters are powerful modulators of tumor growth and can become new targets in cancer therapy.

In response to stress, some people lose while others gain weight. This is believed to be due to either increased beta-adrenergic activation, the body's 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.

The relationship between stress and obesity remains elusive. In response to stress, some people lose weight, whereas others gain. Here we report that stress exaggerates diet-induced obesity through a peripheral mechanism in the abdominal white adipose tissue that is mediated by neuropeptide Y (NPY). Stressors such as exposure to cold or aggression lead to the release of NPY from sympathetic nerves, which in turn upregulates NPY and its Y2 receptors (NPY2R) in a glucocorticoid-dependent manner in the abdominal fat. This positive feedback response by NPY leads to the growth of abdominal fat. Release of NPY and activation of NPY2R stimulates fat angiogenesis, macrophage infiltration, and the proliferation and differentiation of new adipocytes, resulting in abdominal obesity and a metabolic syndrome-like condition. NPY, like stress, stimulates mouse and human fat growth, whereas pharmacological inhibition or fat-targeted knockdown of NPY2R is anti-angiogenic and anti-adipogenic, while reducing abdominal obesity and metabolic abnormalities. Thus, manipulations of NPY2R activity within fat tissue offer new ways to remodel fat and treat obesity and metabolic syndrome.

Neuroblastomas (NB) are pediatric malignancies with heterogenous phenotypes, ranging from spontaneously regressing to highly aggressive, incurable tumors. Although NB is considered a genetic disease, its etiology and heterogeneity cannot be explained solely by genetic aberrations. NB arises due to defects in sympathetic neuron (SN) differentiation occurring during fetal development. Strikingly, the two factors promoting de-differentiation of NB cells, hypoxia and glucocorticoids, are elevated in the fetus during maternal stress, suggesting a role for prenatal stress in NB tumorigenesis. To test this hypothesis we used mice expressing MYCN oncogene under tyrosine hydroxylase promoter (TH-MYCN mice), which spontaneously develop NBs. To mimic stress, pregnant mothers carrying hemizygous TH-MYCN offspring were implanted with pellets containing either the main rodent glucocorticoid, corticosterone, or placebo at the time of neuroblast proliferation (embryonic days 10-20). Tumor frequency was compared between these two experimental groups and TH-MYCN offspring from intact pregnancies. Surprisingly, in pregnant mothers from the placebo group, physiological stress associated with experimental procedures alone (animal handling, blood collections, pellet insertion and anesthesia) was sufficient to elevate their corticosterone levels and increase tumorigenicity in their hemizygous TH-MYCN offspring from 32 to 64% (p = 0.03). A similar effect was observed in offspring of corticosterone-treated mothers with its levels comparable to mice eliciting a physiological stress response in the placebo group (1200ng/ml), indicating that only physiologically relevant levels of stress mediators can accurately recapitulate the stress response in animal models. Taken together, these findings support the role for prenatal stress in NB development, as well as implicate other pathologies associated with elevated levels of glucocorticoids in its etiology. Citation Format: Sung-Hyeok Hong, David Christian, Emily Trinh, Susana Galli, Meredith Horton, Yichien Lee, Christopher Albanese, Olga Rodriguez, Jason Ulip Tilan, Joanna Kitlinska. Prenatal stress increases neuroblastoma tumorigenesis in TH-MYCN mice model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3291. doi:10.1158/1538-7445.AM2015-3291

Neuropeptide Y (NPY) is a sympathetic neurotransmitter, abundantly expressed in neuroblastoma (NB). Previously, we have shown that NPY, acting via its Y2 receptor (Y2R), stimulates proliferation of NB cells and tumor vascularization, while inducible Y5 receptor (Y5R) promotes tumor cell survival and chemoresistance. The aim of the current study was to assess the prognostic value of NPY and its receptor expression in NB. We have tested corresponding samples of RNA, tissue sections and serum from 87 NB patients at various stages of the disease, obtained from the Children's Oncology Group. Samples were analyzed in terms of NPY system expression (NPY, Y2R and Y5R). Protein levels and mRNA in tumor tissue were quantified by immunohistochemistry (IHC) and real time-PCR, respectively, while NPY concentration in serum was measured by ELISA. The expression of other factors implicated in NB development and progression (MYCN, ALK, TrkA III, TrkB and BDNF) were detected on mRNA levels by real time-PCR. For each of the categorical prognostic variables, a Wilcoxon rank-sum or Fisher's exact test were administered to compare NPY and receptor values between groups, and a log-rank test was performed to compare the event-free survival and overall survival between groups. 1) NPY mRNA was detectable in 100% of analyzed tumors. IHC staining revealed that NPY accumulated intracellularly in differentiating and maturing cells, while in undifferentiated NBs a significant amount of the peptide was observed in extracellular spaces, suggesting its free release. In line with this observation, serum concentrations of NPY were higher in the subset of NB patients with undifferentiated and poorly differentiated tumors, as compared to those with differentiating NBs (p-value = 0.03). High serum concentrations of NPY strongly correlated with several adverse prognostic factors (Stage 4, high risk, diploidy, and unfavorable histology) (p-value This study validated NPY and its receptors as targets for NB therapy. Serum NPY was the most highly prognostic variable, found to be associated with five NB prognostic factors. Higher NPY levels were correlated with a more detrimental disease phenotype. Citation Format: Susana Galli, Jason Tilan, Arlene Naranjo, Collin Van Ryn, Chao Yang, Jessica Tsuei, Emily Trinh, Joanna B. Kitlinska. Neuropeptide Y (NPY) and its receptor expression in neuroblastoma patients - associations with disease prognosis and patients’ survival. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1628. doi:10.1158/1538-7445.AM2015-1628