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. ? ? ?
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