Role of galanin receptor expression in neuroblastic tumors

Neuroblastic tumors including neuroblastoma and ganglioneuroma are the most common paediatric neuroendocrine tumors, which are characterised by a variable clinical course. The expression of several neuropeptides and their respective receptors in neuroblastoma tissue has been previously shown to be related to patient outcome. Recently, we investigated the expression of galanin and galanin receptors in tumor tissue from childhood neuroblastic tumors. Ligand binding experiments showed low to high amounts of galanin binding in all 28 primary neuroblastomas and 7 ganglioneuromas investigated. Low galanin binding (=6 fmol bound galanin/mg protein) was significantly correlated to allelic loss of chromosome lp and survival. These findings indicate a potential role for galanin in the pathophysiology of this disease and the possible use of galanin receptor binding as a new prognostic marker in neuroblastic tumors. Therefore, the analysis of additional neuroblastic tumors regarding galanin binding and prognosis is warranted. All three galanin receptor subtypes cloned to date were detected in the neuroblastic tumor tissues. However, we were not able to identify by RT-PCR the receptor subtype mainly responsible for galanin binding. In situ hybridisation for the three subtypes should allow correlation of galanin receptor autoradiography with the expression of a certain galanin receptor mRNA at the cellular level. The determination of galanin receptor mRNA correlating with galanin binding sites in the tissues will be the basis for the development of a quantitative analysis of the respective galanin receptor mRNA for possible prognostic purposes. To further investigate the role of galanin receptors in neuroblastic cells we will stably transfect the neuroblastoma cell line SH-SY5Y with the human galanin receptor subtypes GALR1, GALR2 and GALR3, respectively. The expression of the galanin receptors will be under the control of a tetracycline regulated expression system which allows repression and derepression of the receptors. The influence of galanin receptor expression and activation by galanin on SH-SY5Y proliferation and differentiation will be investigated in cell culture. In addition, the transfected cell lines will be used to induce neuroblastomas in rats to determine if the expression of a certain galanin receptor subtype influences the morphology and progression of neuroblastoma xenografts in nude rats.

Neuropeptides have a widespread distribution and diverse regulatory functions in the human body. Neuropeptides exert their cellular function via specific binding to peptide receptors. There is increasing interest for neuropeptides and the corresponding peptide receptors in cancer based on the possibility of receptor targeting, because peptide receptors are often expressed in different human tumors. We investigated the two structurally and functionally related neuropeptides galanin and galanin-like peptide and their respective receptors in a variety of neuroendocrine tumors. Especially, pheochromocytomas, brain tumors and the neuroblastic cancers ganglioneuroma and neuroblastoma, the most common solid paediatric cancers occurring in the periphery, show production of galanin and its corresponding receptors. In neuroblastic cancers a correlation to the degree of differentiation, a feature of tumour aggressiveness, and presence of galanin receptors was observed. We were able to show that activation of the galanin receptor GalR2 either by galanin or the galanin-like peptide in human neuroblastoma cells induces cell death. This fact could be an explanation for lack of galanin receptors we observed on highly aggressive types of neuroblastic tumors. By screening human neuroendocrine tumors for the presence of galanin and galanin-like peptide a shorter variant of the galanin-like peptide messenger-RNA was detected, which leads to the loss of homology to galanin and a completely novel peptide. The putative novel 25 amino acid long neuropeptide was named "alarin". We were able to show that the novel peptide alarin is located in distinct neuronal structures of human tissues and therefore might represent a novel member of the growing family of peptides of the galanin system.