Die PAC1-Agonisten und PACAP-Rezeptoren in murinen und humanen kleinzelligen Bronchialkarzinom Zelllinien

Summary The pleiotropic neuropeptides PACAP (pituitary adenylate cyclase activating peptide) and VIP (vasoactive intestinal polypeptide) are members of the VIP/PACAP/glucagon/secretin superfamily and function via G-protein coupled receptors. PACAP acts on the PAC1 receptor and like VIP on VPAC1 and VPAC2 receptors. Pharmacological effects of agonists and antagonists of the VIP-/PACAP-family on lung cancer cell lines shown by Terry Moody provided first evidence that lung cancer cell lines express functional VIP -/PACAP receptors. However the differential PAC1, VPAC1 and VPAC2 receptor profiles of lung cancer cells have not yet been deciphered. Therefore, it was the aim of this thesis to differentiate the mRNA expression profiles of PAC1 and its splice variants, as well as that of VPAC1 and VPAC2 in the murine small cell Lewis-lung carcinoma-1-(LLC1) cell line and its subcutaneous and pulmonary transplants as well as in different human small cell lung cancer cell lines and carcinomas. In addition, the functionality of the PAC1-receptor in the LLC1 cell line and the effect of PAC1 agonists on proliferation and apoptosis in the human small cell lung cancer cell line NCI-H82 should be tested. Another objective was to determine the expression of the tumor relevant PAC1 target genes stanniocalcin 1 und stathmin 1 in the murine and human cell lines and in human small cell lung cancer and to detect the influence of PAC1-specific ligands on their expression. The following major new findings were obtained. The expression profile of PAC1, VPAC1 a nd VPAC2 and all known PAC1 splice variants in the murine LLC1-tumor cell line and its subcutaneous and pulmonary transplants and human small cell lung cancer cell lines was successfully differentiated. Two groups of human cell lines of small cell lung cancer featuring reciprocal abundance of PAC1 and PACAP mRNA could be differentiated. In group 1, PAC1 showed high and PACAP not detectable mRNA expression levels while in group 2 PAC1 expression was low and that of PACAP high. Using a FRET (Förster resonance energy transfer) experimental setup a functionally relevant activation of the PAC1 receptor in LLC1 cells by subnanomolar concentrations of PACAP38 was detected, which induced the synthesis of cAMP and the release of Ca2+. While the PAC1 receptor activation in the murine LLC1 cell line resulted in a modest reduction of the proliferation rate, the PAC1 receptor activation in the human NCI-H82 cell line resulted only in a marginal change of the rate of proliferation without detectable change of the CisPt-induced apoptosis rate. The selective PAC1 ligand maxadilan, PACAP38 and PACAP27 seemed to cause a feedback inhibition of the expression of PAC1 receptor at the mRNA level in LLC1 cells. The murine LLC1-cell line and the murine pulmonary LLC1 graft exhibited mRNA coexpression of PAC1 receptor and its putative target genes stanniocalcin 1 und stathmin 1. In contrast, mRNA coexpression patterns of PAC1, stanniocalcin 1 und stathmin 1 were heterogeneous in human small cell lung cancer cell lines and small cell lu ng cancer biopsies. Neither in the murine nor in the human small cell lung cancer cell line(s) evidence for transcriptional regulation of these two PAC1 target genes could be obtained. The results imply the following conclusions. Because PAC1 and its putative target genes stanniocalcin 1 und stathmin 1 are generally highly expressed in murine and human small cell lung cancer cell lines are, they are suitable biomarkers and tumor markes in both preclinical and clinical cancer research. Thus, the development of maxadilan PET ligands for prognostic and prognostic tumor imaging is a new perspective. PAC1 receptors on small cell bronchial carcinoma could serve for "drug targeting". In the light of the heterogeneous coexpression patterns of PAC1 -receptors, stanniocalcin 1 und stathmin 1 in human small cell lung cancer cell lines, combinatorial expression profiling of PAC1, PACAP, stanniocalcin 1 and stathmin 1 in tumor biopsies, metastases and blood samples is a new biomarking approach for diagnostic and prognostic differentiation of malignancy of lung cancers and their metastases in the clinics and preclinical research. Overall, the results obtained here offer a contribution to new translational approaches in personalized cancer medicine. Keywords : small cell lung cancer , small-cell lung carcinoma cell lines, LLC1 cell line , PACAP, PAC1, VPAC1, VPAC2, PAC1 splice variants, stanniocalcin 1, stathmin 1, gene expression, maxadilan