Genetic inactivation ofNupr1acts as a dominant suppressor event in a two-hit model of pancreatic carcinogenesis
Carla E. CanoTewfik HamidiMaría Noé GarciaDaniel GrassoCéline LoncleStéphane GarciaÉzéquiel CalvoGwen LomberkNelson DusettiLaurent BartholinRaúl UrrutiaJuan Iovanna
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Nuclear protein 1 (Nupr1) is a major factor in the cell stress response required for Kras(G12D)-driven formation of pancreatic intraepithelial neoplastic lesions (PanINs). We evaluated the relevance of Nupr1 in the development of pancreatic cancer. We investigated the role of Nupr1 in pancreatic ductal adenocarcinoma (PDAC) progression beyond PanINs in Pdx1-cre;LSL-Kras(G12D);Ink4a/Arf(fl/fl)(KIC) mice. Even in the context of the second tumorigenic hit of Ink4a/Arf deletion, Nupr1 deficiency led to suppression of malignant transformation involving caspase 3 activation in premalignant cells of KIC pancreas. Only half of Nupr1-deficient;KIC mice achieved PDAC development, and incident cases survived longer than Nupr1(wt);KIC mice. This was associated with the development of well-differentiated PDACs in Nupr1-deficient;KIC mice, which displayed enrichment of genes characteristic of the recently identified human classical PDAC subtype. Nupr1-deficient;KIC PDACs also shared with human classical PDACs the overexpression of the Kras-activation gene signature. In contrast, Nupr1(wt);KIC mice developed invasive PDACs with enriched gene signature of human quasi-mesenchymal (QM) PDACs. Cells derived from Nupr1-deficient;KIC PDACs growth in an anchorage-independent manner in vitro had higher aldehyde dehydrogenase activity and overexpressed nanog, Oct-4 and Sox2 transcripts compared with Nupr1(wt);KIC cells. Moreover, Nupr1-deficient and Nurpr1(wt);KIC cells differed in their sensitivity to the nucleoside analogues Ly101-4b and WJQ63. Together, these findings show the pivotal role of Nupr1 in both the initiation and late stages of PDAC in vivo, with a potential impact on PDAC cell stemness. According to Nupr1 status, KIC mice develop tumours that phenocopy human classical or QM-PDAC, respectively, and present differential drug sensitivity, thus becoming attractive models for preclinical drug trials.Keywords:
Pancreatic Intraepithelial Neoplasia
Activating mutations in the KRAS proto-oncogene occur almost ubiquitously in pancreatic ductal adenocarcinoma (PDAC) and in its putative precursor lesions, pancreatic intraepithelial neoplasia (PanIN). Conditional expression of an activated Kras allele in the mouse pancreas produces a model that faithfully recapitulates PanIN formation and progression to PDAC. Importantly, although nearly every cell in the pancreata of these mice express activated Kras, only a very small minority of cells give rise to PanINs. How the transforming activity of Kras is constrained in the pancreas remains unknown, and the cell types from which PanINs and PDAC arise are similarly unknown. Here, we describe our recent results demonstrating that acinar cells are competent to form Kras-induced PanINs, and that active Notch signaling can synergize with Kras in PanIN initiation and progression. Further efforts to understand how Notch and Kras synergize, as well as experiments to determine how other pancreatic cell types contribute to PDAC development, should aid in the development of new therapies and early detection techniques that are desperately needed for this cancer.
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Abstract Pancreatic cancer is an aggressive disease characterized by rapid growth and early metastasis. The recepteur d'origine nantais (RON) receptor tyrosine kinase is overexpressed and/or constitutively active in several epithelial cancers, but its role in pancreatic cancer is unknown. In this study, we have characterized RON expression in both murine and human pancreatic cancer. Immunoblotting indicates that RON is expressed in pancreatic intraepithelial neoplasia (PanIN), primary, and metastatic cell lines both in the human and mouse. Immunostaining revealed that 93% of high-grade PanIN, 79% of primary, and 83% of metastatic lesions from human pancreatic tissue samples expressed RON, with minimal expression in normal ducts and low-grade PanIN (6% and 18%, respectively). Moreover, we show a dose-dependent effect of hepatocyte growth factor-like protein (HGFL), the RON-specific ligand, on pancreatic cancer cell migration and invasion, which was reversed by RON inhibition. Although stimulation with HGFL had no effect on proliferation, concurrent RON receptor blockade and gemcitabine treatment increased apoptosis of RON-expressing pancreatic cancer cells versus gemcitabine treatment alone. Finally, HGFL stimulation of pancreatic cancer cells resulted in increased expression of phospho-mitogen-activated protein kinase and phospho-Akt. Taken together, these findings suggest that RON receptor signaling may contribute to pancreatic carcinogenesis, and that further investigation is warranted to assess the potential of RON-directed therapies in this deadly disease. [Cancer Res 2007;67(13):6075–82]
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Abstract Pancreatic cancer is characterised by the prevalence of oncogenic mutations in KRAS . Previous studies have reported that altered Kras gene dosage drives progression and metastatic incidence in pancreatic cancer. While the role of oncogenic KRAS mutation is well characterised, the relevance of the partnering wild-type KRAS allele in pancreatic cancer is less well understood and controversial. Using in vivo mouse modelling of pancreatic cancer, we demonstrate that wild-type Kras restrains the oncogenic impact of mutant Kras , and drastically impacts both Kras -mediated tumourigenesis and therapeutic response. Mechanistically, deletion of wild-type Kras increases oncogenic Kras signalling through the downstream MAPK effector pathway, driving pancreatic intraepithelial neoplasia (PanIN) initiation. In addition, in the KPC mouse model, a more aggressive model of pancreatic cancer, loss of wild-type KRAS leads to accelerated initiation but delayed tumour progression. These tumours had altered stroma, downregulated Myc levels and an enrichment for immunogenic gene signatures. Importantly, loss of wild-type Kras sensitises Kras mutant tumours to MEK1/2 inhibition though tumours eventually become resistant and then rapidly progress. This study demonstrates the repressive role of wild-type Kras during pancreatic tumourigenesis and highlights the critical impact of the presence of wild-type KRAS on tumourigenesis and therapeutic response in pancreatic cancer.
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Pancreatic cancer is a human malignancy with one of the highest mortality rates and little progress has been achieved in its treatment in recent decades. Further improvement to the understanding of the biological and molecular mechanisms underlying the initiation and development of pancreatic ductal adenocarcinoma (PDAC) is required. Previous studies using genetically engineered mouse models have demonstrated that oncogenic GTPase KRas (KRAS) mutation is involved in the formation of pancreatic intraepithelial neoplasia and promotes the progression of PDAC. However, attempts to target KRAS directly by pharmacological inhibition have been unsuccessful. This has resulted in increased efforts to identify pharmacological targets and nodes associated with the mutated KRAS. The present review discusses the recent progress and prospects of KRAS signaling in pancreatic cancer.
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Pancreatic cancer is one of the deadliest human malignancies and little progress has been achieved in its treatment over the past decades. Advances in our understanding of the biology of this disease provide new potential opportunities for treatment. Pancreatic cancer is preceded by precursor lesions, the most common of which are known as Pancreatic Intraepithelial Neoplasia (PanIN). PanIN lesions, which are the focus of this review, have a high incidence of Kras mutations, and Kras mutations are a hallmark of the late-stage disease. We now know from genetically engineered mouse models that oncogenic Kras is not only driving the formation of pancreatic cancer precursor lesions, but it is also required for their progression, and for the maintenance of invasive and metastatic disease. Thus, an enormous effort is being placed in generating Kras inhibitors for clinical use. Additionally, alternative approaches, including understanding the role of Kras effector pathways at different stages of the disease progression, are being devised to target Kras effector pathways therapeutically. In particular, efforts have focused on the MAPK pathway and the PI3K pathway, for which inhibitors are widely available. Finally, recent studies have highlighted the need for oncogenic Kras to establish feedback mechanisms that maintain its levels of activity; the latter might constitute alternative ways to target Kras in pancreatic cancer. Here, we will review recent basic research and discuss potential therapeutic applications.
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Abstract Chronic or repeated episodes of acute pancreatic inflammation, or pancreatitis, are risk factors for the development of pancreatic cancer. Pancreatic cancer is characterized by a strong fibro‐inflammatory tumor microenvironment. In pancreatitis, the same fibro‐inflammatory reaction is observed concurrently with a loss of normal pancreatic cells. Mouse models are commonly employed to study the progression of pancreatitis and pancreatic cancer, with genetic and pharmacological tools used to elucidate cellular and acellular interactions within pancreatic tumors. Described in this article is a protocol for using Kras G12D ; Pdx1‐Cre (KC) mice stimulated with caerulein, a small oligopeptide that increases secretion of digestive enzymes, as a model for pancreatitis. KRAS is mutated in 90‐95% of the tumors in patients with pancreatic cancer. The combination of this mutation with an inflammatory stimulus accelerates the development of pancreatic cancer. The protocol detailed in this report follows the progression of disease in KC mice from pancreatic intraepithelial neoplasias to invasive pancreatic adenocarcinoma. © 2018 by John Wiley & Sons, Inc.
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Abstract Pancreatic cancer is one of the most aggressive cancers and has an extremely poor prognosis. Despite recent progress in both basic and clinical research, most pancreatic cancers are detected at an incurable stage owing to the absence of disease-specific symptoms. Thus, developing novel approaches for detecting pancreatic cancer at an early stage is imperative. Our in silico and immunohistochemical analyses showed that KIAA1199 is specifically expressed in human pancreatic cancer cells and pancreatic intraepithelial neoplasia, the early lesion of pancreatic cancer, in a genetically engineered mouse model and in human patient samples. We also detected secreted KIAA1199 protein in blood samples obtained from pancreatic cancer mouse models, but not in normal mice. Furthermore, we found that assessing KIAA1199 autoantibody increased the sensitivity of detecting pancreatic cancer. These results indicate the potential benefits of using KIAA1199 as a biomarker for early-stage pancreatic cancer.
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Abstract Although the universal presence of KRAS mutations and their critical role in human pancreatic ductal adenocarcinoma (PDAC) designates it as an ideal therapeutic target, oncogenic KRAS (KRAS*) is still regarded as ‘undruggable’ to date. Therefore, to identify therapeutic points of intervention, it is critical to understand the impact of KRAS*-mediated pathways on in vivo tumor pathogenesis. We have recently generated an inducible KrasG12D-driven (iKRAS*) mouse PDAC model and established a critical role for sustained KRAS* activity in tumor maintenance, providing a model to characterize pathways required for KRAS*-dependent tumorigenicity. Cell surface proteins are relatively accessible and can not only provide candidates for biomarker discovery, but also be utilized as therapeutic targets. To characterize the KRAS*-specific organization of cell surface proteins for the development of possible diagnostic or therapeutic tools, we conducted an unbiased surfaceome analyses of tumor cells in the presence and absence of KRAS* signaling using the iKRAS* mouse PDAC model. Syndecans (SDC), a family of heparin sulfate proteoglycans, were identified as candidates whose membrane expression is correlated with KRAS* activity. SDC1 expression is increased in premalignant and malignant pancreatic lesions of primary human PDAC and various KRAS*-driven pancreatic cancer mouse models. In addition, SDC1 expression is specifically upregulated in response to KRAS* induction in acinar-ductal metaplasia (ADM) and early pancreatic intraepithelial neoplasia (PanIN), but not in cerulin-induced chromic pancretitis. Moreover, SDC1 membrane expression was abolished upon KRAS* extinction in PDAC cells and our data indicated that MAPK pathway, but not PI3K pathway, is important for KRAS*-mediated SDC1 surface localization. To further study the role of SDC1 in KRAS*-driven pancreatic cancers, SDC1 knockout mouse model was generated and crossed with LSL-KrasG12D-PDAC models. Our preliminary data indicated that genetic ablation of SDC1 expression effectively suppressed KRAS*-driven PDAC initiation and progression. The mechanisms of KRAS*-mediated SDC1 membrane localization and its impact on PDAC initiation and progression is being further explored. SDC1 might potentially be the drug target and diagnostic marker for PDAC. Citation Format: Wantong Yao, Haoqiang Ying, Giulio Draetta. Syndecan1 is required for oncogenic Kras-driven PDAC tumorigenesis and maintenance. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C157.
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