Proteomics signature of early stages in pancreatic cancer

Abstract

s / Pancreatology 12 (2012) 502–597 514 Dept. of Medicine, Div. of Gastroenterology, Endocrinology and Metabolism, Philipps-University Marburg, Marburg, Germany 2 Institute for General Pathology, University Hospital, Tuebingen, Germany Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive pancreatic cancer with an overall 5-year survival rate of <5%. Despite significant advances in treatment of the disease during the past decade, the median survival rate (w6 months) has hardly improved, warranting the need to identify novel targets for therapeutic approaches. Our previous data from microarray analyses of microdissected pancreatic tumor tissues as well as parallelized cell-based assays indicated a previously unknown role for Plac8 inpancreatic cancer initiation and progression. Plac8 isa rather smallproteinwhosephysiological functions remain largelyunclear. Aim:Main aim of this project is to characterize gene Plac8 with respect to its role in PDAC. The study also intends to dissect out the pathway(s) involved in the functional role. Methods: qPCR, tissue microarrays, RNAi, cell proliferation and viability assays, FACS analysis, Western blot, shRNA inducible clones. Results: qRT-PCR as well as tissue microarray data confirmed that Plac8 has a strong ectopic expression in pancreatic cancer tissues and further demonstrated that this high Plac8 expression is also retained in the majority of pancreatic tumor cell lines in vitro, with negligible expression in non-transformed cell lines. Functional effects of Plac8 were investigated after transient knockdown in a variety of different transformed and non-transformed cell lines. Proliferation and viability were strongly impaired by down-regulation of Plac8. Western blot and flow cytometry did not show any involvement of classical apoptosis pathway (Caspase-3 and PARP cleavage). Flow cytometry analyses and time course experiments with cell cycle inhibitors demonstrated strong attenuation of cell cycle progression after Plac8 knockdown, although intriguingly classical checkpoint mechanisms (p21, p53, Chk1, Cdc25A) were not activated, thereby suggesting involvement of hitherto unknown mechanisms. Generation of stable inducible clones as well as “multiple” transgenic mice (pertinent to human PDAC condition) for further in vivo experiments is in progress. Conclusion: Our experimental data shows that ectopic expression of Plac8 is indispensible for proliferation, viability and cell cycle progression in pancreatic cancer cells. A clear insight into pathophysiological role(s) of Plac8 in the onset and progression of pancreatic cancer is expected to emerge with the generation of transgenic mice.