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.

BACKGROUND & AIMSMitogen-activated protein kinase (MAPK) signaling in the exocrine pancreas has been extensively studied in the context of pancreatic cancer, where its potential as a therapeutic target is limited by acquired drug resistance. However, its role in pancreatitis is less understood. We investigated the role of mitogen-activated protein kinase kinase (MEK)-initiated MAPK signaling in pancreatitis to determine the potential for MEK inhibition in treating pancreatitis patients.METHODSTo examine the role of MEK signaling in pancreatitis, we used both genetic and pharmacologic approaches to inhibit the MAPK signaling pathway in a murine model of cerulein-induced pancreatitis. We generated mice harboring inducible short hairpins targeting the MEK isoforms Map2k1 and/or Map2k2 specifically in the pancreatic epithelium. We also used the MEK inhibitor trametinib to determine the efficacy of systemic inhibition in mice with pancreatitis.RESULTSWe demonstrated an essential role for MEK signaling in the initiation of pancreatitis. We showed that both systemic and parenchyma-specific MEK inhibition in established pancreatitis induces epithelial differentiation and stromal remodeling. However, systemic MEK inhibition also leads to a loss of the proliferative capacity of the pancreas, preventing the restoration of organ mass.CONCLUSIONSMEK activity is required for the initiation and maintenance of pancreatitis. MEK inhibition may be useful in the treatment of chronic pancreatitis to interrupt the vicious cycle of destruction and repair but at the expense of organ regeneration.

Objective To evaluate Plectin-1 expression as a biomarker of malignant risk for intraductal papillary mucinous neoplasms (IPMNs). Methods Plectin-1 immunohistochemistry (IHC) was performed retrospectively on surgical (N=71) and cytology (N=33) specimens from Mayo Clinic Jacksonville and UCLA Medical Center, including IPMNs with low grade dysplasia (LGD), high grade dysplasia (HGD), or an associated invasive adenocarcinoma. Results Plectin-1 expression was increased in invasive adenocarcinoma compared to adjacent in situ IPMN (p=0.005), as well as the in situ HGD component of IPMNs with invasive cancer compared to HGD of IPMNs without invasive cancer (p=0.02). Plectin IHC discriminated IPMNs with invasive adenocarcinoma from non-invasive IPMN (area under the curve [AUC] of 0.79, 75% sensitivity, 85% specificity), but was insufficient for discriminating HGD IPMN from LGD IPMNs in surgical resections (AUC 0.67, 56% sensitivity, 64% specificity) or fine needle aspiration specimens (AUC 0.45). Conclusions While Plectin-1 immunohistochemistry has insufficient accuracy to be used as a definitive biomarker for malignant risk in the evaluation of IPMN biopsy or cytology specimens, increased Plectin-1 expression observed in both invasive cancer and in situ HGD of malignant IPMNs suggests it might be successfully leveraged as a cyst fluid biomarker or molecular imaging target.

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MYCN RA response is not mediated solely though the region controlling basal activity. RA may be exerting its effects via multiple non-adjacent regulatory regions, potentially including basal motifs, either within the MYCN promoter or distally, on the same or even different chromosomes. Such cooperative trans-type DNA-protein interactions could explain the inaccessibility of this mechanism to the locus-specific approaches employed up to this point.