Parallelisierte und individualisierte funktionelle Charakterisierung von ausgewählten Kandidatengenen im Pankreaskarzinom.

Das duktale Pankreasadenokarzinom gehört mit einer 5-Jahres-Überlebensrate von <3% und mittlerer Überlebenszeit von ca. 6 Monaten nach Diagnosestellung zu den tödlichsten Tumor-erkrankungen weltweit. Die Vielfalt an Risikofaktoren sowie genetische Heterogenität der Pankreasneoplasien als auch die...

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Bibliographic Details
Main Author: Honstein, Tatjana
Contributors: Buchholz, Malte (PD Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2014
Online Access:PDF Full Text
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Pancreatic adenocarcinoma is one of the deadliest cancers with an overall 5-year survival rate of <3% and average survival time of 6 months. The poor patient prognosis illustrates the fact that the basic mechanisms of pancreatic cancer disease are not well understood to date. Based on the data of previously performed high-throughput analyses, 79 candidate genes were identified having a high probability to be of biological relevance or to be suitable as target genes for diagnostic or therapeutic applications and were thus selected for the further characterization. We used the technique of reverse transfection microarrays as an efficient technology to identify tumor-relevant genes and characterize important functional roles with high throughput. 11 out of 79 candidate genes with strong reproducible effects have been identified as promising candidates for the single gene characterization. Among them was the G-Protein Coupled Receptor Kinase 2 (GRK2, a.k.a. ADRBK1). We verified the high overexpression of ADRBK1 in human pancreatic cancer tissue as compared to chronic pancreatitis and healthy pancreatic tissue. Detailed functional investigation uncovered a proliferative and cell cycle modulating phenotype of this candidate gene. The second part of the systematic approach for the identification of new promising candidates consisted of a kinome-wide screening using Ambion® Silencer® Select kinome-wide siRNA library. The effects of kinase down regulation in transformed Panc1 and non-transformed Hek293 cells on apoptosis activation were measured via automated apoptosis assays. This analysis identified the dual specificity protein Kinase TTK as a candidate whose repression induced apoptosis specifically in transformed cell lines. Our results show a high rate of overexpression of TTK in humane pancreatic cancer tissue in comparism to inflamed (chronic pancreatitis) and healthy pancreas controls. Further functional analyses based on the down regulation of the TTK revealed strong effects on cell viability and confirmed the apoptotic phenotype (detected via immunoblotting as well as flow cytometry). In addition, we demonstrated the involvement of the kinase in the maintenance of genomic stability. Furthermore, our soft agar clonogenic assays indicates a strong influence of TTK on the anchorage independent tumor cell growth.