Neuropeptide Y in neuroblastoma: growth, angiogenesis and future therapeutics.

描述(申请人提供)：神经母细胞瘤(NBS)是一种儿童肿瘤，尽管接受了治疗，但往往会导致患者死亡。NBS起源于交感神经，表达神经肽Y(NPY)，这是一种交感神经递质，但其在这些肿瘤中的作用尚不清楚。随着我们实验室发现NPY对各种细胞具有强大的促生长和血管生成活性，我们最近发现，NPY还通过相同的Y2和Y5受体(Rs)刺激NB细胞的增殖和肿瘤血管的形成。因此，本方案的总体目标是确定NPY是否是NBS中的一种自分泌/旁分泌生长因子，确定其作用机制及其与肿瘤环境的相互作用，验证NPY及其受体是否为潜在的预后标志物，并测试阻断NPY/Y2/Y5通路是否可能是治疗NB的有效的双向治疗方法。特定的靶点将决定：1)NPY及其受体在NB组织中的表达；2)NPY与其他NB生长因子的相互作用；3)R相互作用及其细胞内信号转导；4)在NB中阻断NPY作用的有效途径。通过实时定量RT-PCR和免疫组织化学染色研究NPY及其Y2和Y5Rs在人NBS组织中的表达，以评估NPY及其受体在NBS中的临床意义和预后及治疗价值，并与患者生存、疾病表型和其他已知预后指标相关联。为了确定NPY的作用机制及其与其他已知的NB生长因子的相互作用，我们将确定：1)神经营养因子及其受体是否上调NPY及其Y2/Y5受体；2)NPY是否介导神经营养因子的促生长作用。NBS中NPY促进生长和血管生成作用的下游信号介质将通过蛋白质组学鉴定，并通过NB和内皮细胞(EC)增殖、EC迁移和主动脉萌发试验进行测试。由于初步数据表明Y2和Y5Rs的异构性是放大NPY诱导的NB和EC增殖的机制-在这里，我们将使用Y2/Y5Rs转基因的CHO-K1细胞来证明它们的二聚性，鉴定结合结构域，并设计阻止二聚体形成的多肽。阻断肽对NPY诱导的增殖的影响将在体外在NB和ECs中进行测试，并与Y2/Y5R拮抗剂一起在体内在NB异种移植中进行测试。这项拟议的研究将首次确定NPY在NBS中的作用和机制，并确定该肽及其受体的预后和治疗价值。

DESCRIPTION (provided by applicant): Neuroblastomas (NBs) are childhood tumors, which, despite therapy, often lead to the death of the patients. Being of sympathetic origin, NBs express neuropeptide Y (NPY), a sympathetic neurotransmitter, whose role, however, in these tumors remains unknown. Following our lab's discovery of NPY's potent growth-promoting and angiogenic activities for various cells, we have recently found that the peptide also stimulates NB cell proliferation and tumor vascularization, apparently via the same Y2 and Y5 receptors (Rs). Thus, the overall goal of this proposal is to establish if NPY is an autocrine/paracrine growth factor in NBs, determine mechanisms of its actions and interactions with tumor environment, validate NPY and its Rs as potential prognostic markers and test if blocking the NPY/Y2/Y5 pathway may be an effective, bi-directional therapy for NB. Specific aims will determine: 1) expression of NPY and its Rs in human NB tissues; 2) interactions of NPY with other NB growth factors; 3) R interactions and their intracellular signaling, and 4) an efficient way of blocking NPY actions in NB. Clinical relevance and the prognostic and therapeutic value of NPY and its Rs for NBs will be assessed by studying expression of NPY and its Y2 and Y5Rs in human NB tissues by Real-time RT-PCR and immunostaining, correlated with patient survival, phenotype of the disease and other known prognostic markers. To establish the mechanisms of NPY actions and its interactions with other known NB growth factors we will determine i) if neurotrophins and their Rs upregulate NPY and its Y2/Y5 Rs, and ii) if NPY mediates nuerotrophins' growth-promoting effects. The downstream signaling mediators of NPY growth-promoting and angiogenic actions in NBs will be identified by proteomics and tested using NB and endothelial cell (EC) proliferation, EC migration and aortic sprouting assays. Since the preliminary data implicated heterodimerization of Y2 and Y5Rs as a mechanism amplifying NPY-induced NB and EC proliferation - here, we will use CHO-K1 cells transfected with the Y2/Y5Rs to prove their dimerization, identify binding domains and design peptides blocking dimer formation. The effect of blocking peptide on NPY-induced proliferation will be tested in vitro in NB and ECs and, along with Y2/Y5R antagonists, in vivo in NB xenografts. The proposed research will be the first to establish the role and mechanisms of NPY's actions in NBs, and determine prognostic and therapeutic values of the peptide and its Rs.