Funktionelle Charakterisierung der Gene NPC2 und ADRBK1 im Pankreaskarzinom

Das duktale Adenokarzinom des Pankreas stellt heute immer noch eines der aggressivsten und am schlechtesten behandelbaren Malignome dar. Deshalb ist es umso wichtiger, Genetik und Pathomechanismen dieser Erkrankung im Detail aufzuklären und Marker zur Früh- bzw. Differenzialdiagnose sowie potentiell...

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Bibliographic Details
Main Author: Häuser, Christin
Contributors: Buchholz, Malte (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2018
Online Access:PDF Full Text
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Ductal adenocarcinoma of the pancreas today still is one of the most aggressive and most difficult to treat malignomas. Thus it is even more important to uncover genetic drivers and pathomechanisms of this disease in detail and to find markers for early or differential diagnosis, respectively, as well as potential targets for therapies. By means of enzymatically generated transcriptome-based shRNA libraries, cooperating researchers were able to identify putative oncogenes and tumor suppressor genes specific for PDAC. NPC2 as one of those potential oncogenes was selected for further functional assays concerning cell vitality, proliferation and motility. Expression analyses on mRNA level showed a distinct overexpression of the gene both in pancreatic tumor tissue and in different cell lines of the pancreatic ductal adenocarcinoma. Out of four cell lines analyzed in short-term experiments within this work, only BxPC3 cells showed a functional effect in terms of a highly significant reduction of cell vitality after siRNA-mediated knockdown. This growth-promoting effect of NPC2 cannot be transferred to PDAC in general. Cell proliferation and –motility after repression of NPC2 were not affected. The gene ADRBK1 has likewise been identified in a high-throughput screening, which was based on cDNA-microarrays specific for PDAC and the concept of reverse transfection. ADRBK1 showed a time-dependent translocation into the nucleus, which led to the question, whether the kinase plays a direct role in gene regulation. Preliminary tests in our research group also showed that ADRBK1 is not only significantly overexpressed in human PDAC, but also mediates pro-proliferative effects. Recombinant overexpression of the kinase accelerates, while its knockdown significantly inhibits, cell growth. Potential reasons for the observed growth inhibition were either induction of cell cycle arrest or apoptosis. Protein levels of CDKN1A/p21, CDKN1B/p27, Cyclin A, Cyclin D1, pRB and c-myc subsequently examined in Western blot after repression of ADRBK1 were unchanged. Cleavage of PARP and Caspase 3, both markers of apoptosis, were detectable only in PaTu-8988t cells. Thus neither induction of apoptosis nor cell cycle arrest can be regarded as the prevailing reason of growth inhibition after ADRBK1 knockdown. To elucidate the role of the gene in PDAC, nuclear translocation and its potential effects on gene function should be further investigated. Moreover cellular target proteins of ADRBK1, its relevance in interaction between cancer cells and infiltrating immune cells and its role concerning cell motility are to be identified.