Zebrafish model to study signaling pathways involved in pancreatic tumour.

Background and Aims: Oncogenic KRAS point mutations (G12V or G12D) are sufficient to induce pancreatic tumours in human, mice and zebrafish. Molecular pathways involved after oncogenic KRAS activation are still under investigation. To examine the effects of oncogenic KRAS during pancreatic tumour formation in vivo and correlate their progression with specific signaling pathways, we developed a zebrafish platform based on transgenic living reporters. Methods: We exploited the inducible system developed by Park et al., 2008 in which a transgenic zebrafish line is expressing GAL4 under the regulation of zebrafish ptf1a locus region. These fish were outcrossed with zebrafish reporter lines harboring specific signaling responsive elements (TGFb, Wnt, Notch, BMP) and the resulting fertilized eggs injected with a Tol2 plasmid expressing UAS:eGFP-KRASG12D. Live imaging was performed on zebrafish larvae and juveniles by using confocal microscopy. Histology and immunohistochemistry assays were performed on adult fishes. Results: Zebrafish transgenic lines expressing oncogenic mutated KRASG12D conjugated to eGFP showed normal exocrine pancreas development up to 3 weeks. From 4 to 12 weeks we observed tumour lesions, characterized by EMT and acinar features, followed by progressive bowel and liver invasion. The in vivo analysis of reporter activities revealed an increased number of KRAS positive cells also expressing TGFb, Wnt and Notch pathways reporters in the tumor lesions. Conclusions: These results extend in vivo our knowledge of the oncogenic proprieties of KRAS during pancreatic tumour development. Furthermore, this model is a promising tool to describe the signaling pathways and molecular mechanisms involved and to develop and discover new targets for drug therapies.