Notch1 Is Not Required for Acinar-to-Ductal Metaplasia in a Model of Kras-Induced Pancreatic Ductal Adenocarcinoma

Kissil JL (2012) Notch1 Is Not Required for Acinar-to-Ductal Metaplasia in a Model of Kras-Induced Pancreatic Ductal Adenocarcinoma. PLoS ONE 7(12): e52133. doi:10.1371/journal.pone.0052133 Notch1 Is Not Required for Acinar-to-Ductal Metaplasia in a Model of Kras-Induced Pancreatic Ductal Adenocarcinoma Jacqueline L. Avila 0 Scott Troutman 0 Amy Durham 0 Joseph L. Kissil 0 Henrik Einwaechter, Klinikum rechts der Isar der TU Mu nchen, Germany 0 1 Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania, United States of America, 2 Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine , Philadelphia, Pennsylvania , United States of America Pancreatic ductal adenocarcinoma is believed to arise from precursor lesions termed pancreatic intraepithelial neoplasia (PanIN). Mouse models have demonstrated that targeted expression of activated K-ras to mature acinar cells in the pancreas induces the spontaneous development of PanIN lesions; implying acinar-to-ductal metaplasia (ADM) is a key event in this process. Recent studies suggest Notch signaling is a key regulator of ADM. To assess if Notch1 is required for K-ras driven ADM we employed both an in vivo mouse model and in vitro explant culture system, in which an oncogenic allele of K-ras is activated and Notch1 is deleted simultaneously in acinar cells. Our results demonstrate that oncogenic K-ras is sufficient to drive ADM both in vitro and in vivo but that loss of Notch1 has a minimal effect on this process. Interestingly, while loss of Notch1 in vivo does not affect the severity of PanIN lesions observed, the overall numbers of lesions were greater in mice with deleted Notch1. This suggests Notch1 deletion renders acinar cells more susceptible to formation of K-ras-induced PanINs. - Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive forms of human cancer, with a 5-year survival rate of less than 4% [1]. PDAC is believed to arise from precursor lesions termed pancreatic intraepithelial neoplasia (PanIN), which progress through defined stages ultimately leading to the development of adenocarcinoma [2]. The most commonly mutated gene in PDAC is K-ras, with greater than 90% of human cases harboring an activating mutation in this oncogene. K-ras mutations appear to occur early during the pathogenesis of PDAC, as low-grade PanIN lesions typically contain activating mutations at codon 12 [2]. Further proof that K-ras mutations represent an initiating event in PDAC comes from mouse models, in which expression of a mutant activated K-ras allele (K-rasG12D) in pancreatic epithelium is sufficient to induce the formation of both PanIN lesions and invasive pancreatic cancer, pathologically resembling the human disease [3]. The pancreas is composed of an exocrine and endocrine compartment, with the exocrine compartment consisting of acinar, ductal, and centroacinar cells. While the cell of origin for PDAC has remained elusive, recent studies utilizing mouse models have demonstrated that targeting oncogenic K-ras to mature acinar cells results in the spontaneous development of PanIN lesions, suggesting acinar cells represent the cell of origin for PDAC [4]. A feature of this model is the appearance of acinar-to-ductal metaplasia (ADM) preceding the development of PanIN lesions. Other studies have highlighted the importance of pancreatic injury in the development of PDAC. Work by Guerra and colleagues revealed that mature acinar cells expressing K-rasG12V are refractory to PanIN development unless mice are subjected to additional stimuli such as chronic chemically-induced pancreatitis [5]. Further, endocrine cells can be made susceptible to oncogenic K-ras induced transformation in the context of pancreatic injury [6]. These findings are especially relevant to human disease, in that chronic pancreatitis is a strong risk factor for the development of PDAC [7]. The Notch signaling family of proteins is composed of 4 transmembrane receptors (Notch14), in addition to 2 Jagged ligands, and 3 Delta-like ligands. During pancreatic development, Notch signaling is required for directing cell fate decisions and progenitor cell self renewal [8]. While the role of Notch signaling in development is well characterized, the cell types expressing Notch proteins and their function in the adult pancreas remains unclear. Recent findings indicate Notch1 plays a role in pancreatic homeostasis, since loss of Notch1 in pancreatic epithelium results in impaired acinar regeneration following acute pancreatitis [9]. Moreover, Notch signaling has been implicated in ADM in that ectopic expression of transcriptionally active forms of Notch (Nic) promote transdifferentiation in explant culture models [1011]. Conversely, inhibition of Notch signaling by a c-secretase inhibitor increases the proliferation of metaplastic exocrine cells and induces p21 expression [12]. Further work demonstrates different Notch receptors have non-overlapping functions and are expressed in unique cellular compartments, with Notch1 observed primarily in acinar cells and Notch2 expressed mainly in ductal cells [13]. Although Notch1 was originally identified as an oncogene, recent evidence indicates the Notch proteins also function as tumor suppressors in a tissue-specific manner. Conclusive evidence demonstrating Notch1 acts as a tumor suppressor came from studies in the skin, where loss of both Notch1 alleles led to development of basal cell carcinoma [14]. Subsequently, Notch receptors have been identified as tumor suppressors in hepatocellular carcinoma, chronic myelomonocytic leukaemia, and squamous cell carcinomas [15,16,17,18]. Previously unknown loss of function mutations in components of the Notch pathway have been discovered in myeloid leukaemia and squamous cell carcinomas, pointing to a cell autonomous mechanism of tumor suppression for these malignancies. Alternatively, in basal cell carcinoma, Notch1 appears to function in a non-cell autonomous manner by mechanisms impacting the tumor microenvironment [19]. Previous work by our group has identified Notch1 as a tumor suppressor in a mouse model of PDAC [20]. To further investigate the mechanism of Notch1 mediated tumor suppression in pancreatic tumorigenesis we examined the effect of Notch1 deletion on acinar-to-ductal metaplasia both in vitro and in vivo. These experiments aimed to identify a cell autonomous mechanism of Notch1 mediated tumor suppression. Additionally, we investigated a potential non-cell autonomous function of Notch1 using an orthotopic transplantation tumor model. K-ras Mediated ADM does not Require Notch1 Function We have recently demonstrated that loss of Notch1 in a mouse model of K-ras-induced PDAC leads to increased PanIN incidence and progression [20]. To further investigate the mechanism of Notch1 mediated PanIN suppression, we examined the role of Notch1 in ADM using an in vitro explant culture model (...truncated)