Expression of Neuropeptides in Neuroblastoma

Neuropeptides represent a family of substances which are primarily synthesized in the central nervous system. Most of these neuropeptides are also synthesized and act in non-neuronal tissues like the endocrine system and neuroendocrine tumors including neuroblastoma. Production and secretion of these neuropeptides in tumors causes clinical symptoms and might influence tumor growth and differentiation. For Neuropeptide Y (NPY), association with staging and prognosis of neuroblastoma tumors occurring in paediatric patients has been described. However, for Galanin, although being functionally related to NPY, no comparable data are available. For both neuropeptides, effects on tumor growth and differentiation have not been thoroughly investigated. In addition, no information on the differential expression of NPY and Galanin receptor subtypes in neuroendocrine tumors has been published. Therefore this grant application addresses the expression of Galanin- and NPY-peptides and -receptors in neuroblastoma and the effects of this expression. Our preliminary data indeed show that Galanin and a Galanin- receptor subtype are expressed in neuroblastoma cells. Therefore, it is the first specific aim to expand on these data and precisely investigate the role of Galanin, expression in neuroblastoma in correlation to tumor stage and prognosis. Since multiple NPY- and Galanin-receptor subtypes have been cloned to date, the exact definition of the expression of these receptor subtypes in different stages of neuroblastoma will constitute the second specific aim. Spontaneous differentiation is a characteristic feature of neuroblastoma. Thus. the third specific aim is the analysis of potential effects of NPY and Galanin on the extend of differentiation in neuroblastoma. Since in several experimental systems NPY and Galanin have been shown to exert potent mitogenic effects, our fourth aim is to establish possible growth regulatory effects of NPY and Galanin in neuroblastoma. In the fifth specific aim the analysis of signal transduction pathways activated by these neuropeptides in neuroblastoma will complement the preceding experiments. The results of these studies could establish Galanin as a new factor in staging and prognosis of neuroblastoma, as well as facilitate the development of new peptideanalogues for improvement of neuroblastoma specific tumor therapy.

Neuropeptides and their receptors are expressed in several types of human neoplasia and play a role in cancer development. In this study we investigated the functional expression of galanin (GAL) and GAL receptors (GALR1-3) in human neoplasia. No GAL binding sites were found in tumors of the urinary tract and the kidney. However, different amounts of binding levels were detected in human gliomas, but in this case no correlation to tumor progression could be found. In neuroblastic tumors (28 neuroblastomas, 7 ganglioneuromas ) GAL binding was found in varying order of magnitude. Furthermore, low binding levels were inversely correlated to poor clinical prognosis for children with neuroblastoma (p=0.021) and deletion of chromosome 1p (p=0.008). All three GAL receptors cloned to date were identified in neuroblastic tumors, with the GALR1 receptor subtype being expressed most prominently. The GAL peptide concentration found in most of the neuroblastic tumors is high enough to allow GAL receptor activation via an autocrine and/or paracrine pathway. To further investigate the role of GAL receptors in neuroblastoma, the cell line NB6, which showed high amounts of GAL binding, was examined to characterise its receptor subtypes. Pharmacological receptor characterization revealed a single population of high affinity binding sites. However, signalling studies showed that the receptor in NB6 cells is not functionally coupled. Therefore, the metabolic function of GAL receptors was studied in a human melanoma cell line (HBMC) which endogeneoulsy expresses the GALR1 receptor. A bi-directional response, a rapid increase in the metabolic rate followed by a decrease below the basal level, was observed after perfusion with human GAL. The phenomenon of a bi-directional response has not been described for any other neuropeptide receptor before. The potential clinical utility of our findings awaits confirmation in a larger patient number. In addition, studies on the influence of the expression of the cloned GAL receptors GALR1-3 in neuroblastoma xenografts in nude rats is expected to give further information on the role of GAL and GAL receptors in neuroblastoma.