Table of Contents:
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