Zebrafish reporter lines reveal in vivo signaling pathway activities involved in pancreatic cancer

Abstract

Pancreatic adenocarcinoma, one of the worst malignancies of the exocrine pancreas, is a solid tumor with increasing incidence and mortality in industrialized countries. This condition is usually driven by oncogenic KRAS point mutations and evolves into a highly aggressive metastatic carcinoma due to secondary gene mutations and unbalanced expression of genes involved in the specific signaling pathways. To examine in vivo the effects of KRASG12D during pancreatic cancer progression and time correlation with cancer signaling pathway activities, we have generated a zebrafish model of pancreatic adenocarcinoma in which eGFP-KRASG12D expression was specifically driven to the pancreatic tissue by using the GAL4/UAS conditional expression system. Outcrossing the inducible oncogenic KRASG12D line with transgenic zebrafish reporters, harboring specific signaling responsive elements of transcriptional effectors, we were able to follow TGFβ, Notch, Bmp and Shh activities during tumor development. Zebrafish transgenic lines expressing eGFP-KRASG12D showed normal exocrine pancreas development until 3 weeks post fertilization (wpf). From 4 to 24 wpf we observed several degrees of acinar lesions, characterized by an increase in mesenchymal cells and mixed acinar/ductal features, followed by progressive bowel and liver infiltrations and, finally, highly aggressive carcinoma. Moreover, live imaging analysis of the exocrine pancreatic tissue revealed an increasing number of KRAS-positive cells and progressive activation of TGFβ and Notch pathways. Increase in TGFβ, following KRASG12D activation, was confirmed in a concomitant model of medulloblastoma (MDB). Notch and Shh signaling activities during tumor onset were different between MDB and pancreatic adenocarcinoma, indicating a tissue-specific regulation of cell signaling pathways. Moreover, our results show that a living model of pancreatic adenocarcinoma joined with cell signaling reporters is a suitable tool for describing in vivo the signaling cascades and molecular mechanisms involved in tumor development and a potential platform to screen for novel oncostatic drugs.

Lunatic Fringe is a potent tumor suppressor in Kras-initiated pancreatic cancer

“…Previous studies have shown increased expression of TGF-β ligands as well as progressive activation of TGF-β signaling during pancreatic cancer progression. 18,20,33 Indeed, we observed gradual increase of Tgfb1 expression in KC pancreas in the time course (Figure 6b). Interestingly, at 1 and 3 months of age, Tgfb1, Tgfb2 and Tgfbr2 expressions in LKC pancreas were higher than those in KC pancreas.…”

Pancreatic beta cells are a sensitive target of embryonic exposure to butylparaben in zebrafish (Danio rerio)

“…For example, butylparaben‐induced cell dispersion has also been shown in rodent models; Alam et al found butylparaben to cause the collapse of rat Sertoli cell vimentin filaments, allowing spermatogenic cells to separate from the Sertoli cells (Alam & Kurohmaru, ). Vimentin filaments are found in pancreatic precursor cells and in beta‐cell neogenesis, (Ko et al, ), and an increase is a marker of pancreatic cancer progression in zebrafish (Schiavone et al, ). Studies are under‐way to determine whether vimentin disruption contributes to the aberrant beta cell architecture in the developing islet.…”

The zebrafish as a model for studying neuroblastoma

“…Therefore, the generation and use of zebrafish transgenic and mutant lines may provide new opportunities to better understand the biology of NB and to test in vivo potential chemical tumor suppressors through high-throughput drug screening. In addition, zebrafish models of NB crossed with cell signaling reporter fish might be a powerful tool for the in vivo dissection of the signaling cascades and molecular mechanisms involved in tumor development [96, 97]. …”