Development of a novel in vitro tumorigenesis model to characterize the cellular perturbations induced by the distinct driver mutations in pancreatic adenocarcinoma
Final Report Abstract
In vitro models of pancreatic cancer precursors are necessary in oredr to understand the molecular perturbations occurring during the early stages of pancreatic carcinogenesis. Taking recent whole exome studies we deciphered the genetic signature in a large subset of pancreatic carcinoma patients, however we still have trouble to identifying the exact molecular mechanisms responsible for initiating this process in precursor lesions. From characterization of human pancreas specimen with varying degree of dysplastic PanIN (Pancreatic intraepithelial neoplasia) lesions, a stepwise progression in line with four driver gene alterations has been described. It is unknown why the four driver gene mutations ubiquitously occur across tumor patients and how they orchestrate the malignant features of pancreatic carcinoma cells so effectively. This project aimed to establish novel pancreatic duct cell models and subsequently drive them into invasiveness by introducing driver gene alterations using modern gene editing approaches. Despite successfully deriving duct cells from primary pancreatic tissue, it proved challenging, establishing reproducible cell lines. The immortalization strategies used (hTERT, SV40T) prolonged survival, but did not create novel duct cell lines. Additional factors or alternative models are likely needed in order to sustainably cultivate healthy duct cells. Parallel to this, using the Human Pancreatic Duct Epithelial (HPDE) cells, we investigated ways to accurately introduce main driver gene alterations. We achieved knock-in of the KRAS hotspot alteration at the G12 position and were also successful in the knock-out of the tumorsupressor SMAD4 using CRISPR/Cas9. Knock-in relied on HDR events happening at diminishingly low frequencies making cell line derivation particularly challenging. Taking statistical measures into consideration, we attempted a monoclonal HPDEG12D cell line which is still in process. Monoclonal HPDESMAD4 -/- cell clones were developed which will be helpful for molecular in vitro investigation of SMAD4. Altogether, we successfully used the CRISPR/Cas9 system to introduce driver gene alterations. This could be helpful in deciphering and characterizing the precise molecular consequences impacted by these mutations in human pancreatic ductal cells. It is conceivable, that the CRISPR/Cas9 tool will help to medically reverse respective processes in foreseeable future.
Publications
- IPMNs with co-occurring invasive cancers: neighbours but not always relatives. Gut. 2018 Sep;67(9):1652-1662
Felsenstein M, Noë M, Masica DL, Hosoda W, Chianchiano P, Fischer CG, Lionheart G, Brosens LAA, Pea A, Yu J, Gemenetzis G, Groot VP, Makary MA, He J, Weiss MJ, Cameron JL, Wolfgang CL, Hruban RH, Roberts NJ, Karchin R, Goggins MG, Wood LD
(See online at https://doi.org/10.1136/gutjnl-2017-315062) - New Developments in the Molecular Mechanisms of Pancreatic Tumorigenesis. Adv Anat Pathol. 2018 Mar;25(2):131-142
Felsenstein M, Hruban RH, Wood LD
(See online at https://doi.org/10.1097/PAP.0000000000000172)
