SUMMARY Initiation of pancreatic ductal adenocarcinoma (PDA) is definitively linked to activating mutations in the KRAS oncogene. However, PDA mouse models show that mutant Kras expression early in development gives rise to a normal pancreas, with tumors forming only after a long latency or pancreatitis induction. Here we show that oncogenic KRAS upregulates endogenous EGFR expression and activation, the latter being dependent upon the EGFR ligand sheddase, ADAM17. Genetic ablation or pharmacological inhibition of EGFR or ADAM17 effectively eliminates KRAS-driven tumorigenesis in vivo. Without EGFR activity, active RAS levels are not sufficient to induce robust MEK/ERK activity, a requirement for epithelial transformation.

Background & Aims Metaplasias often have characteristics of developmentally related tissues. Pancreatic metaplastic ducts are usually associated with pancreatitis and pancreatic ductal adenocarcinoma. The tuft cell is a chemosensory cell that responds to signals in the extracellular environment via effector molecules. Commonly found in the biliary tract, tuft cells are absent from normal murine pancreas. Using the aberrant appearance of tuft cells as an indicator, we tested if pancreatic metaplasia represents transdifferentiation to a biliary phenotype and what effect this has on pancreatic tumorigenesis. Methods We analyzed pancreatic tissue and tumors that developed in mice that express an activated form of Kras (KrasLSL−G12D/+;Ptf1aCre/+ mice). Normal bile duct, pancreatic duct, and tumor-associated metaplasias from the mice were analyzed for tuft cell and biliary progenitor markers, including SOX17, a transcription factor that regulates biliary development. We also analyzed pancreatic tissues from mice expressing transgenic SOX17 alone (ROSAtTa/+;Ptf1 CreERTM/+;tetO-SOX17) or along with activated Kras (ROSAtT a/+;Ptf1a CreERTM/+;tetO-SOX17;KrasLSL−G12D;+). Results Tuft cells were frequently found in areas of pancreatic metaplasia, decreased throughout tumor progression, and were absent from invasive tumors. Analysis of the pancreatobiliary ductal systems of mice revealed tuft cells in the biliary tract, but not the normal pancreatic duct. Analysis for biliary markers revealed expression of SOX17 in pancreatic metaplasia and tumors. Pancreas-specific overexpression of SOX17 led to ductal metaplasia along with inflammation and collagen deposition. Mice that overexpressed SOX17 along with KrasG12D had a greater degree of transformed tissue compared with mice expressing only KrasG12D. Immunofluorescence analysis of human pancreatic tissue arrays revealed the presence of tuft cells in metaplasia and early-stage tumors, along with SOX17 expression, consistent with a biliary phenotype. Conclusions Expression of KrasG12D and SOX17 in mice induces development of metaplasias with a biliary phenotype, containing tuft cells. Tuft cells express a number of tumorigenic factors that can alter the microenvironment. Expression of SOX17 induces pancreatitis and promotes KrasG12D-induced tumorigenesis in mice.

Aberrant activation of embryonic signaling pathways is frequent in pancreatic ductal adenocarcinoma (PDA), making developmental regulators therapeutically attractive. Here we demonstrate diverse functions for pancreatic and duodenal homeobox 1 (PDX1), a transcription factor indispensable for pancreas development, in the progression from normal exocrine cells to metastatic PDA. We identify a critical role for PDX1 in maintaining acinar cell identity, thus resisting the formation of pancreatic intraepithelial neoplasia (PanIN)-derived PDA. Upon neoplastic transformation, the role of PDX1 changes from tumor-suppressive to oncogenic. Interestingly, subsets of malignant cells lose PDX1 expression while undergoing epithelial-to-mesenchymal transition (EMT), and PDX1 loss is associated with poor outcome. This stage-specific functionality arises from profound shifts in PDX1 chromatin occupancy from acinar cells to PDA. In summary, we report distinct roles of PDX1 at different stages of PDA, suggesting that therapeutic approaches against this potential target need to account for its changing functions at different stages of carcinogenesis. These findings provide insight into the complexity of PDA pathogenesis and advocate a rigorous investigation of therapeutically tractable targets at distinct phases of PDA development and progression.

Background & Aims New drug targets are urgently needed for the treatment of pancreatic ductal adenocarcinoma (PDA). Nearly all PDAs contain oncogenic mutations in the KRAS gene. Pharmacologic inhibition of KRAS has been unsuccessful, leading to a focus on downstream effectors that are more easily targeted with small molecule inhibitors. We investigated the contributions of phosphoinositide 3 kinase (PI3K) to KRAS-initiated tumorigenesis. Methods Tumorigenesis was measured in the KrasG12D/+;Ptf1aCre/+ mouse model of PDA; these mice were crossed with mice with pancreas-specific disruption of genes encoding PI3K p110α (Pik3ca), p110β (Pik3cb), or RAC1 (Rac1).Pancreatitis was induced by 5 daily intraperitoneal injections of cerulein. Pancreata and primary acinar cells were isolated; acinar cells were incubated with an inhibitor of p110α (PIK75) followed by a broad spectrum PI3K inhibitor (GDC0941). PDA cell lines (NB490 and MiaPaCa2) were incubated PIK75 followed by GDC0941. Tissues and cells were analyzed by histology, immunohistochemistry, quantitative reverse transcription PCR, and immunofluorescence analyses for factors involved in the PI3K signaling pathway. We also examined human pancreas tissue microarrays for levels of p110α and other PI3K pathway components. Results Pancreas-specific disruption of Pik3ca or Rac1, but not Pik3cb, prevented development of pancreatic tumors in KrasG12D/+ ;Ptf1aCre/+ mice. Loss of transformation was independent of AKT regulation. Preneoplastic ductal metaplasia developed in the mice lacking pancreatic p110α, but regressed. Levels of activated and total RAC1 were higher in pancreatic tissues from Kras G12D/+ ;Ptf1aCre/+ mice, compared with controls. Loss of p110α reduced RAC1 activity and expression in these tissues. p110α was required for the upregulation and activity of the RAC guanine exchange factor (encoded by Rasgrf1) during tumorigenesis. Levels of p110α and RAC1 were increased in human pancreatic intraepithelial neoplasias and PDAs, compared with healthy pancreata. Conclusions KRAS signaling, via p110α to activate RAC1, is required for transformation in Kras G12D/+;Ptf1a Cre/+ mice.