Acinar-to-ductal Metaplasia Formation Research Articles

Cytokine CCL9 Mediates Oncogenic KRAS-Induced Pancreatic Acinar-to-Ductal Metaplasia by Promoting Reactive Oxygen Species and Metalloproteinases.

Pancreatic ductal adenocarcinoma (PDAC) can originate from acinar-to-ductal metaplasia (ADM). Pancreatic acini harboring oncogenic Kras mutations are transdifferentiated to a duct-like phenotype that further progresses to become pancreatic intraepithelial neoplasia (PanIN) lesions, giving rise to PDAC. Although ADM formation is frequently observed in KrasG12D transgenic mouse models of PDAC, the exact mechanisms of how oncogenic KrasG12D regulates this process remain an enigma. Herein, we revealed a new downstream target of oncogenic Kras, cytokine CCL9, during ADM formation. Higher levels of CCL9 and its receptors, CCR1 and CCR3, were detected in ADM regions of the pancreas in p48cre:KrasG12D mice and human PDAC patients. Knockdown of CCL9 in KrasG12D-expressed pancreatic acini reduced KrasG12D-induced ADM in a 3D organoid culture system. Moreover, exogenously added recombinant CCL9 and overexpression of CCL9 in primary pancreatic acini induced pancreatic ADM. We also showed that, functioning as a downstream target of KrasG12D, CCL9 promoted pancreatic ADM through upregulation of the intracellular levels of reactive oxygen species (ROS) and metalloproteinases (MMPs), including MMP14, MMP3 and MMP2. Blockade of MMPs via its generic inhibitor GM6001 or knockdown of specific MMP such as MMP14 and MMP3 decreased CCL9-induced pancreatic ADM. In p48cre:KrasG12D transgenic mice, blockade of CCL9 through its specific neutralizing antibody attenuated pancreatic ADM structures and PanIN lesion formation. Furthermore, it also diminished infiltrating macrophages and expression of MMP14, MMP3 and MMP2 in the ADM areas. Altogether, our results provide novel mechanistic insight into how oncogenic Kras enhances pancreatic ADM through its new downstream target molecule, CCL9, to initiate PDAC.

Abstract LB235: Prrx1 regulates acinar cell plasticity via TGFβsignaling in pancreatic acinar-to-ductal metaplasia

Abstract Introduction: Acinar cells undergo de-differentiation after injury to a progenitor-like cell type with ductal characteristics termed acinar-to-ductal metaplasia (ADM). In the absence of oncogenic mutation, the ADM lesions resolve and reform the acinar compartment (we terms this as adaptive ADM). However, in the presence of oncogenic Kras mutations (we terms this as oncogenic ADM), acinar cells undergo neoplastic transformation after ADM and evolve to pancreatic intraepithelial neoplasia (PanIN), a well-known precursor of pancreatic ductal adenocarcinoma (PDAC). Herein, we explore the role of the Paired-Related Homeobox1 transcriptional factor (Prrx1) in adaptive ADM and the relationship between Prrx1 and mutant Kras in oncogenic ADM. Methods: We generated tamoxifen-inducible Ptf1aCreERT;Rosa26YFP/YFP (abbreviated as Prrx1cWT) mice and bred in the Prrx1fl/fl allele to make (abbreviated Prrx1cKO ) mice and subjected them to caerulin (CCK analog) induced acute pancreatitis, a well-estabished model. We created a time course for both mouse genotypes subjected to acute pancreatitis for histological analysis. Quantification of ADM was performed through automated cell-counting of immunofluorescence (IF) staining. Then, we performed 3D ex vivo acinar cultures of Prrx1cWT and Prrx1cKO mice. We used an exogenous adenoviral vector to induce Cre expression and knock out Prrx1, using an empty vector as a control. Dissociated acinar cell cultures in collagen were utilized for the evaluation of TGFβ signaling by western blotting (WB). Additionally, we generated novel Pdx1-Cre;LSLKrasG12D/+;Prrx1fl/fl;Rosa26YFP/YFP (KCY Prrx1 KO) mice, in which mutant Kras is efficiently expressed and Prrx1 is deleted in a pancreas-specific manner. KCY Prrx1 WT and KO mice were sacrificed at 3 months and 5 months for histological analysis. Results: IF staining revealed that Prrx1cKO mice had fewer ADM lesions compared to Prrx1cWT mice at Day 3 post-caerulein. Ex vivo cultures demonstrated a substantial increase of cystic structures, which are representative of ADM in Prrx1cWT cultures compared to Prrx1cKO cultures. Additionally, using a novel nucleofection protocol, we demonstrated that overexpression of the Prrx1b isoform (associated with a progenitor cell state) leads to a notable increase in ADM. WB of acinar protein lysates from either Prrx1cWT or Prrx1cKO mice showed significant differences in TGFβ levels, and a >2-fold higher expression of phosphorylated SMAD2 in Prrx1cWT mice. IF staining also showed that KCY Prrx1 KO mice had fewer ADM lesions compared to Prrx1 WT mice at at the 5 months time point. Conclusions: Prrx1 can influence adaptive ADM formation. Moreover, Prrx1 can influence ADM formation in the presence of oncogenic KRAS. Our data suggest that Prrx1 facilitates PDAC progression through ADM formation. Citation Format: Noriyuki Nishiwaki, Kensuke Sugiura, Alina Li, Kensuke Suzuki, Jason R. Pitarresi, Rohit Chandwani, Anil K. Rustgi. Prrx1 regulates acinar cell plasticity via TGFβsignaling in pancreatic acinar-to-ductal metaplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB235.

Abstract 5325: Cytokine CCL9, new downstream target of oncogene Kras to modulate acinar-to-ductal metaplasia during pancreatic cancer initiation

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer with a current five-year survival rate at 12% in the US, and is dominated by oncogenic Kras mutations. Accumulating evidence showed that PDAC can be originated from acinar-to-ductal metaplasia (ADM). In this process, pancreatic acini harboring oncogenic Kras mutations are transdifferentiated to a duct-like phenotype that further progresses to become pancreatic intraepithelial neoplasia (PanIN) lesions, giving rise to PDAC. Although ADM formation is commonly observed in KrasG12D transgenic mouse models of PDAC, the exact mechanisms of how oncogenic KrasG12D modulates this process remain unclear. Here, we unveil a new downstream target of oncogenic Kras, cytokine CCL9 during ADM formation. Either exogenously treating primary pancreatic acini with recombinant CCL9 or ectopically expressing CCL9 in primary pancreatic acini resulted in ADM formation in a 3D organoid culture system. Furthermore, knockdown of CCL9 in KrasG12D-expressed acini reduced KrasG12D-induced ADM. Higher levels of reactive oxygen species (ROS) were detected in the KrasG12D-expressed pancreatic acini, and knockdown of CCL9 in these acini decreased ROS levels. Overexpression of CCL9 in primary pancreatic acini elevated cellular ROS of pancreatic acinar cells. Furthermore, depletion of ROS in 3D organoid culture of CCL9-expressed acini reduced CCL9-induced ADM formation. In transgenic mice of p48cre:KrasG12D, blockade of CCL9 through its specific neutralizing antibody attenuated ADM formation as well as PanIN structures. Altogether, our results indicated oncogenic KrasG12D initiates PDAC through a new downstream target molecule, CCL9, to enhance ROS levels of pancreatic acinar cells. Citation Format: Geou-Yarh (Stancy) Liou, Justin K. Messex, Crystal J. Byrd. Cytokine CCL9, new downstream target of oncogene Kras to modulate acinar-to-ductal metaplasia during pancreatic cancer initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5325.

Abstract 637: Cytokine CCL9 mediates oncogenic Kras-induced pancreatic acinar-to-ductal metaplasia through ROS

Abstract Pancreatic ductal adenocarcinoma (PDAC) can be originated from acinar-to-ductal metaplasia (ADM). Pancreatic acini harboring oncogenic Kras mutations are transdifferentiated to a duct-like phenotype that further progresses to become pancreatic intraepithelial neoplasia (PanIN) lesions, giving rise to PDAC. Although ADM formation is frequently observed in KrasG12D transgenic mouse models of PDAC, the exact mechanisms of how oncogenic KrasG12D regulates this process remain an enigma. Herein we reveal a new downstream target of oncogenic Kras, cytokine CCL9 during ADM formation. Exogenously treating primary pancreatic acini with recombinant CCL9 in a 3D organoid culture system drives ADM formation. Furthermore, knockdown of CCL9 in KrasG12D-expressed acini reduced KrasG12D-induced ADM. Higher levels of reactive oxygen species were detected in the KrasG12D-expressed pancreatic acini, and knockdown of CCL9 in these acini dramatically decreased ROS levels. Depletion of ROS in 3D organoid culture of CCL9-expressed acini reduced CCL9-induced ADM formation. In transgenic mice of p48cre:KrasG12D, depletion of CCL9 through its specific neutralizing antibody attenuated ADM formation as well as PanIN structures. Hence, our results provided insight into how oncogenic Kras regulates pancreatic ADM through a new downstream target molecule, CCL9. Citation Format: Geou-Yarh Liou, Justin K. Messex, Kiyah L. Adams. Cytokine CCL9 mediates oncogenic Kras-induced pancreatic acinar-to-ductal metaplasia through ROS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 637.

Abstract 788: Prrx1 regulates acinar cell plasticity in Kras-driven pancreatic acinar-to-ductal metaplasia

Abstract Introduction: Acinar cells in the adult pancreas demonstrate cellular plasticity and undergo de-differentiation to a progenitor-like cell type with ductal characteristics after injury. This process, termed acinar-to-ductal metaplasia (ADM), is an important feature facilitating pancreas regeneration after injury. In the absence of oncogenic mutations, the ADM lesions resolve and reform the acinar compartment (adaptive ADM). However, in the presence of oncogenic Kras mutations (oncogenic ADM), acinar cells undergo neoplastic transformation after ADM and evolve to pancreatic intraepithelial neoplasia (PanIN), a well-known precursor of pancreatic ductal adenocarcinoma (PDAC). We have characterized the role of Paired-Related Homeobox1 (PRRX1) in adaptive ADM. We demonstrated through our novel conditional Prrx1 knock-out mouse model that loss of Prrx1 abrogated ADM formation. Here we explore the relationship between Prrx1 and mutant Kras on promoting ADM and a pro-ADM microenvironment. Methods: We generated novel Pdx1-Cre;LSLKrasG12D/+;Prrx1fl/fl;Rosa26YFP/YFP (KCY Prrx1 KO) mice, in which mutant Kras is efficiently expressed and Prrx1 is deleted in a pancreas-specific manner. KCY Prrx1 WT and KO mice were sacrificed at 3 months and 5 months for histological analysis. Immunofluorescence (IF) staining for CK19, characterized for the rate of ADM formation and evaluated for F4/80 and SMA. Quantification of ADM regions, F4/80 and SMA was performed through automated cell-counting of immunofluorescence staining (IF). Dissociated acinar cell culture in collagen was utilized for the evaluation of ADM under ex vivo conditions. Results: IF staining revealed that KCY Prrx1 KO mice had fewer ADM lesions compared to Prrx1 WT mice at 3 months. This difference became dramatically apparent at the 5 month timepoint. Additionally, lower areas of fibrosis were identified via H&E staining in KCY Prrx1 KO mice, which was accompanied with lower F4/80 and SMA positivity at both 3 months and 5 months. Ex vivo cultures also demonstrated significant reduction in ADM formation in the context of oncogenic Kras and loss of Prrx1. Conclusions: PRRX1 can influence ADM formation in both a cell-intrinsic and cell-extrinsic manner in the presence of oncogenic KRAS. Our preliminary data suggest Prrx1 facilitates PDAC progression through PanIN formation. We will continue to investigate the mechanisms driving Prrx1-dependent ADM formation. Citation Format: Alina L. Li, Kensuke Sugiura, Kensuke Suzuki, Jason R. Pitarresi, Anna M. Chiarella, Gizem Efe, Rohit Chandwani, Anil K. Rustgi. Prrx1 regulates acinar cell plasticity in Kras-driven pancreatic acinar-to-ductal metaplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 788.

Statins Inhibit Inflammatory Cytokine Production by Macrophages and Acinar-to-Ductal Metaplasia of Pancreatic Cells.

BACKGROUND AND AIMS:Animal data show that the presence of an oncogenic Kras mutation in pancreatic acinar cells leads to acinar-to-ductal metaplasia (ADM), pancreatic intraepithelial neoplasia (PanIN), and pancreatic ductal adenocarcinoma (PDAC). Inflammatory macrophages play an important role in the formation of ADMs and transition to PanINs. Epidemiologically, statins are associated with a reduced risk of PDAC. We investigated whether statins inhibit inflammatory cytokine production in macrophages and whether this leads to reduced ADM formation.METHODS:The efficacy of statins on inflammatory cytokine production in 2 macrophage cell lines was measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. The effect of macrophage-conditioned medium on ADM in primary pancreatic acinar cells was investigated. Mouse pancreatic tissue samples were analyzed for macrophage numbers, cytokine levels, and neoplastic/dysplastic area.RESULTS:Lipophilic statins prevented inflammatory cytokine production in Raw264.7 and J774A.1 cells stimulated by lipopolysaccharide. The inhibitory effect of statins was mediated by inhibition of mevalonate and geranylgeranyl pyrophosphate synthesis and disruption of the actin cytoskeleton but not by a reduction in intracellular cholesterol. Treatment of macrophages with lipophilic statins also blocked ADM formation of primary pancreatic acinar cells. Furthermore, oral administration of simvastatin was associated with a reduction in the number of intrapancreatic macrophages, decreased inflammatory cytokine levels in the pancreas, and attenuated ADM/PanIN formation in mice.CONCLUSION:Our data support the hypothesis that statins oppose early PDAC development by their effects on macrophages and ADM formation. The inhibitory actions of statins on macrophages may collaborate with direct inhibitory effects on transformed pancreatic epithelial cells, which cumulatively may reduce early PDAC development and progression.

Abstract PO-075: The elucidation of the role of Prrx1 for acinar to ductal metaplasia in response to acute injury of pancreas in the novel mouse models

Abstract Introduction: Pancreatic acinar cells can de-differentiate after acute injury (acute pancreatitis) to a progenitor-like cell type with ductal characteristics in a process termed acinar-to-ductal metaplasia (ADM). In the absence of oncogenic mutation, the ADM lesions resolve and reform the acinar compartment (adaptive ADM). However, in the presence of oncogenic Kras mutations (oncogenic ADM), ADM lesions can evolve to pancreatic intraepithelial neoplasia (PanIN) and pancreatic ductal adenocarcinoma (PDAC). Our comprehensive and unbiased approach previously identified the Paired-Related homebox1 (Prrx1) as the most up-regulated transcription factor during pancreatic development, regeneration and evolution of PanIN. We showed that Prrx1 expression is upregulated in both ADM and PanIN lesion. Here, we explore the role of Prrx1 in ADM and PanIN formation using novel mouse models, ex vivo acinar culture systems, and human pancreatitis tissue microarrays (TMA). Methods: We generated novel Ptf1acre-ERT;Prrx1fl/fl;Rosa26YFP/YFP (Prrx1 KO) mice, in which Prrx1 is deleted and YFP is expressed exclusively in adult pancreatic acinar cells upon tamoxifen induction of Cre recombinase activity. Intraperitoneal caerulein injection was administered to induce acute pancreatitis. Human pancreatitis TMAs were utilized for the comparison of Prrx1 expression between pancreatitis and normal human pancreas. We also generated Pdx1Cre;LSL-KrasG12D/+; Prrx1fl/fl;Rosa26YFP/YFP (KCYPrrx1KO) mice to evaluate the function of Prrx1 in oncogenic Kras mutation induced pancreatic PanIN. Quantification of ADM regions was performed through histological examination by a pathologist (blinded to the genetic basis of the tissues) and automated cell-counting of immunofluorescence staining (IF). Dissociated acinar cell culture in collagen was utilized for the evaluation of ADM under ex vivo conditions. We also established a novel method for the induction of Prrx1 overexpression in dissociated acinar cells via nucleofection. Results: IF staining revealed that Prrx1 expression is efficiently deleted in ADM cells of Prrx1 KO mice 3 days post-caerulein treatment, a timepoint at which we see peak ADM region formation. Additionally, areas of ADM lesions and amylase loss were significantly reduced in Prrx1 KO mice compared with Prrx1 WT mice at day 3. We also found that Prrx1 overexpression promotes ADM formation in ex vivo acinar cell cultures. In human TMAs, pancreatitis tissues had higher expression of Prrx1 than in normal pancreas. In the presence of oncogenic Kras mutation, loss of Prrx1 significantly attenuates ADM formation in ex vivo culture. Moreover, 5-month-old KCYPrrx1KO mice have very limited ADM/PanIN region compared to 5-month-old KCY mice, which have a pancreas that has predominantly replaced the ADM/PanIN. Conclusions: Prrx1 promotes ADM formation in both adaptive ADM and oncogenic ADM. Our preliminary data suggest that Prrx1 facilitates PDAC progression through PanIN formation. Citation Format: Kensuke Suzuki, Alina Li, Jason R. Pitarresi, Anna M. Chiarella, Gizem Efe, Kensuke Sugiura, Rohit Chandwani, Anil K. Rustgi. The elucidation of the role of Prrx1 for acinar to ductal metaplasia in response to acute injury of pancreas in the novel mouse models [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-075.

MiR-802 Suppresses Acinar-to-Ductal Reprogramming During Early Pancreatitis and Pancreatic Carcinogenesis

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive tumor that is almost uniformly lethal in humans. Activating mutations of KRAS are found in >90% of human PDACs and are sufficient to promote acinar-to-ductal metaplasia (ADM) during tumor initiation. The roles of miRNAs in oncogenic Kras-induced ADM are incompletely understood. The Ptf1aCre/+LSL-KrasG12D/+ and Ptf1aCre/+LSL-KrasG12D/+LSL-p53R172H/+ and caerulein-induced acute pancreatitis mice models were used. mir-802 was conditionally ablated in acinar cells to study the function of miR-802 in ADM. We show that miR-802 is a highly abundant and acinar-enriched pancreatic miRNA that is silenced during early stages of injury or oncogenic KrasG12D-induced transformation. Genetic ablation of mir-802 cooperates with KrasG12D by promoting ADM formation. miR-802 deficiency results in de-repression of the miR-802 targets Arhgef12, RhoA, and Sdc4, activation of RhoA, and induction of the downstream RhoA effectors ROCK1, LIMK1, COFILIN1, and EZRIN, thereby increasing F-actin rearrangement. mir-802 ablation also activates SOX9, resulting in augmented levels ofductal and attenuated expression of acinar identity genes.Consistently with these findings, we show that this miR-802-RhoA-F-actin network is activated in biopsies of pancreatic cancer patients and correlates with poor survival. We show miR-802 suppresses pancreatic cancer initiation by repressing oncogenic Kras-induced ADM. The role of miR-802 in ADM fills the gap in our understanding of oncogenic Kras-induced F-actin reorganization, acinar reprogramming, and PDAC initiation. Modulation of the miR-802-RhoA-F-actin network may be a new strategy to interfere with pancreatic carcinogenesis.

Toll-like receptor 3 expression in myeloid cells is essential for efficient regeneration after acute pancreatitis in mice.

Stringent regulation of the inflammatory response is crucial for normal tissue regeneration. Here, we analyzed the role of Toll-like receptor 3 (TLR3) in pancreatic regeneration after acute pancreatitis (AP). AP was induced by caerulein treatment in mice with global TLR3 deficiency (TLR3OFF ) or in mice re-expressing TLR3 exclusively in the myeloid cell lineage (TLR3Mye ). Compared to WT mice, TLR3OFF mice had a markedly increased formation of acinar-to-ductal metaplasia (ADM) that persisted until day 7 after initiation of AP. Pancreatic tissue of WT mice was completely regenerated after 5 days with no detectable ADM structures. The enhancing effect of TLR3-deficiency on ADM formation was closely linked with an increased and prolonged accumulation of macrophages in pancreata of TLR3OFF mice. Importantly, the phenotype of TLR3OFF mice was rescued in TLR3Mye mice, demonstrating the causative role of myeloid cell selective TLR3 signaling. Moreover, in vitro stimulation of macrophages through TLR3 initiated cell death by a caspase-8-associated mechanism. Therefore, these findings provide evidence that TLR3 signaling in myeloid cells is sufficient to limit inflammation and ADM formation and to promote regeneration after AP. Notably, resolution of inflammation after AP was associated with macrophage sensitivity to TLR3-mediated cell death.

Expression of the EWSR1-FLI1 fusion oncogene in pancreas cells drives pancreatic atrophy and lipomatosis

BackgroundPancreatic ductal adenocarcinoma (PDAC) harbors mutant KRAS as the most common driver mutation. Studies on mouse models have uncovered the tumorigenic characteristics of the Kras oncogene driving pancreatic carcinogenesis. Similarly, Ewing sarcoma predominantly depends on the occurrence of the EWSR1-FLI1 fusion oncogene. The expression of EWSR1-FLI1 affects pro-tumorigenic pathways and induces cell transformation. In this study, we investigated whether mutant Kras could be exchanged by another potent oncogene, such as EWSR1-FLI1, to initiate pancreatic cancer development. MethodsWe generated two conditional mouse models expressing mutant KrasG12D (KC) or the EWSR1-FLI1 oncogene (E/F) in pancreas cells. Pancreatic tissue was collected from the mice at 4–6 weeks and 11–13 weeks of age as well as from survival cohorts to determine the development of spontaneous acinar-to-ductal metaplasia (ADM) and neoplastic lesions. Immunohistochemistry and immunofluorescence staining were performed to characterize and quantify changes in tissue morphology. ResultsThe expression of the EWSR1-FLI1 fusion protein in pancreas cells was confirmed by positive FLI1 immunohistochemistry staining. Notably, the EWSR1-FLI1 expression in pancreas cells resulted in a strong depletion of the acinar cell mass and an extensive lipomatosis. Although the E/F mice exhibited spontaneous ADM formation and a shorter overall survival rate compared to KC mice, no development of neoplastic lesion was observed in aging E/F mice. ConclusionsThe expression of the EWSR1-FLI1 oncogene leads to a strong pancreatic atrophy and lipomatosis. ADM formation indicates that pancreatic acinar cells are susceptible for EWSR1-FLI1-mediated oncogenic transformation to a limited extent. However, the EWSR1-FLI1 oncogene is insufficient to induce pancreatic cancer development.

Akt1 signalling supports acinar proliferation and limits acinar-to-ductal metaplasia formation upon induction of acute pancreatitis.

Molecular signalling mediated by the phosphatidylinositol-3-kinase (PI3K)-Akt axis is a key regulator of cellular functions. Importantly, alteration of the PI3K-Akt signalling underlies the development of different human diseases, thus prompting the investigation of the pathway as a molecular target for pharmacologic intervention. In this regard, recent studies showed that small molecule inhibitors of PI3K, the upstream regulator of the pathway, reduced the development of inflammation during acute pancreatitis, a highly debilitating and potentially lethal disease. Here we investigated whether a specific reduction of Akt activity, by using either pharmacologic Akt inhibition, or genetic inactivation of the Akt1 isoform selectively in pancreatic acinar cells, is effective in ameliorating the onset and progression of the disease. We discovered that systemic reduction of Akt activity did not protect the pancreas from initial damage and only transiently delayed leukocyte recruitment. However, reduction of Akt activity decreased acinar proliferation and exacerbated acinar-to-ductal metaplasia (ADM) formation, two critical events in the progression of pancreatitis. These phenotypes were recapitulated upon conditional inactivation of Akt1 in acinar cells, which resulted in reduced expression of 4E-BP1, a multifunctional protein of key importance in cell proliferation and metaplasia formation. Collectively, our results highlight the critical role played by Akt1 during the development of acute pancreatitis in the control of acinar cell proliferation and ADM formation. In addition, these results harbour important translational implications as they raise the concern that inhibitors of PI3K-Akt signalling pathways may negatively affect the regeneration of the pancreas. Finally, this work provides the basis for further investigating the potential of Akt1 activators to boost pancreatic regeneration following inflammatory insults. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Acetyl-CoA Metabolism Supports Multistep Pancreatic Tumorigenesis.

Pancreatic ductal adenocarcinoma (PDA) has a poor prognosis, and new strategies for prevention and treatment are urgently needed. We previously reported that histone H4 acetylation is elevated in pancreatic acinar cells harboring Kras mutations prior to the appearance of premalignant lesions. Because acetyl-CoA abundance regulates global histone acetylation, we hypothesized that altered acetyl-CoA metabolism might contribute to metabolic or epigenetic alterations that promote tumorigenesis. We found that acetyl-CoA abundance is elevated in KRAS-mutant acinar cells and that its use in the mevalonate pathway supports acinar-to-ductal metaplasia (ADM). Pancreas-specific loss of the acetyl-CoA-producing enzyme ATP-citrate lyase (ACLY) accordingly suppresses ADM and tumor formation. In PDA cells, growth factors promote AKT-ACLY signaling and histone acetylation, and both cell proliferation and tumor growth can be suppressed by concurrent BET inhibition and statin treatment. Thus, KRAS-driven metabolic alterations promote acinar cell plasticity and tumor development, and targeting acetyl-CoA-dependent processes exerts anticancer effects. SIGNIFICANCE: Pancreatic cancer is among the deadliest of human malignancies. We identify a key role for the metabolic enzyme ACLY, which produces acetyl-CoA, in pancreatic carcinogenesis. The data suggest that acetyl-CoA use for histone acetylation and in the mevalonate pathway facilitates cell plasticity and proliferation, suggesting potential to target these pathways.See related commentary by Halbrook et al., p. 326.This article is highlighted in the In This Issue feature, p. 305.

Pancreatic Acinar-to-Ductal Metaplasia and Pancreatic Cancer.

Acinar-to-ductal metaplasia (ADM) of the pancreas is a process that pancreatic acinar cells differentiate into ductal-like cells with ductal cell traits. The metaplasia of pancreatic acinar cells manifests their ability to adapt to the genetic and environmental pressure they encounter. However, with oncogenic genetic insults and/or sustained environmental stress, ADM may lead to pancreatic intraepithelial neoplasia (PanIN), which is a common precancerous lesion that precedes pancreatic cancer. Understanding the intermediate states of ADM and important molecules that regulate ADM formation may help the development of novel preventive strategies that could be translated to the clinic to benefit the people with high risk of pancreatic cancer. Mouse model is widely used in both in vivo and ex vivo studies of ADM. In this chapter, we describe detailed protocols of injury models of the adult mouse pancreas that can function as a tool to study mechanisms of ADM formation.

Serotonin uptake is required for Rac1 activation in Kras-induced acinar-to-ductal metaplasia in the pancreas.

Pancreatic ductal adenocarcinoma (PDAC), which is the primary cause of pancreatic cancer mortality, is poorly responsive to currently available interventions. Identifying new targets that drive PDAC formation and progression is critical for developing alternative therapeutic strategies to treat this lethal malignancy. Using genetic and pharmacological approaches, we investigated in vivo and in vitro whether uptake of the monoamine serotonin [5-hydroxytryptamine (5-HT)] is required for PDAC development. We demonstrated that pancreatic acinar cells have the ability to readily take up 5-HT in a transport-mediated manner. 5-HT uptake promoted activation of the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1), which is required for transdifferentiation of acinar cells into acinar-to-ductal metaplasia (ADM), a key determinant in PDAC development. Consistent with the central role played by Rac1 in ADM formation, inhibition of the 5-HT transporter Sert (Slc6a4) with fluoxetine reduced ADM formation both in vitro and in vivo in a cell-autonomous manner. In addition, fluoxetine treatment profoundly compromised the stromal reaction and affected the proliferation and lipid metabolism of malignant PDAC cells. We propose that Sert is a promising therapeutic target to counteract the early event of ADM, with the potential to stall the initiation and progression of pancreatic carcinogenesis. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

ETV5 regulates ductal morphogenesis with Sox9 and is critical for regeneration from pancreatitis.

The plasticity of pancreatic acinar cells to undergo acinar to ductal metaplasia (ADM) has been demonstrated to contribute to the regeneration of the pancreas in response to injury. Sox9 is critical for ductal cell fate and important in the formation of ADM, most likely in concert with a complex hierarchy of, as yet, not fully elucidated transcription factors. By using a mouse model of acute pancreatitis and three dimensional organoid culture of primary pancreatic ductal cells, we herein characterize the Ets-transcription factor Etv5 as a pivotal regulator of ductal cell identity and ADM that acts upstream of Sox9 and is essential for Sox9 expression in ADM. Loss of Etv5 is associated with increased severity of acute pancreatitis and impaired ADM formation leading to delayed tissue regeneration and recovery in response to injury. Our data provide new insights in the regulation of ADM with implications in our understanding of pancreatic homeostasis, pancreatitis and epithelial plasticity. Developmental Dynamics 247:854-866, 2018. © 2018 Wiley Periodicals, Inc.

Krüppel-like Factor 5, Increased in Pancreatic Ductal Adenocarcinoma, Promotes Proliferation, Acinar-to-Ductal Metaplasia, Pancreatic Intraepithelial Neoplasia, and Tumor Growth in Mice

Activating mutations in KRAS are detected in most pancreatic ductal adenocarcinomas (PDACs). Expression of an activated form of KRAS (KrasG12D) in pancreata of mice is sufficient to induce formation of pancreatic intraepithelial neoplasia (PanINs)-a precursor of PDAC. Pancreatitis increases formation of PanINs in mice that express KrasG12D by promoting acinar-to-ductal metaplasia (ADM). We investigated the role of the transcription factor Krüppel-like factor 5 (KLF5) in ADM and KRAS-mediated formation of PanINs. We performed studies in adult mice with conditional disruption of Klf5 (Klf5fl/fl) and/or expression of KrasG12D (LSL-KrasG12D) via CreERTM recombinase regulated byan acinar cell-specific promoter (Ptf1a). Activation of KrasG12D and loss of KLF5 was achieved by administration of tamoxifen. Pancreatitis was induced in mice by administration of cerulein; pancreatic tissues were collected, analyzed by histology and immunohistochemistry, and transcriptomes were compared between mice that did or did not express KLF5. We performed immunohistochemical analyses of human tissue microarrays, comparing levels of KLF5 among 96 human samples of PDAC. UN-KC-6141 cells (pancreatic cancer cells derived from Pdx1-Cre;LSL-KrasG12D mice) were incubated with inhibitors of different kinases and analyzed in proliferation assays and by immunoblots. Expression of KLF5 was knocked down with small hairpin RNAs or CRISPR/Cas9 strategies; cells were analyzed in proliferation and gene expression assays, and compared with cells expressing control vectors. Cells were subcutaneously injected into flanks of syngeneic mice and tumor growth was assessed. Of the 96 PDAC samples analyzed, 73% were positive for KLF5 (defined as nuclear staining in more than 5% of tumor cells). Pancreata from Ptf1a-CreERTM;LSL-KrasG12D mice contained ADM and PanIN lesions, which contained high levels of nuclear KLF5 within these structures. In contrast, Ptf1a-CreERTM;LSL-KrasG12D;Klf5fl/fl mice formed fewer PanINs. After cerulein administration, Ptf1a-CreERTM;LSL-KrasG12D mice formed more extensive ADM than Ptf1a-CreERTM;LSL-KrasG12D;Klf5fl/fl mice. Pancreata from Ptf1a-CreERTM;LSL-KrasG12D;Klf5fl/fl mice had increased expression of the tumor suppressor NDRG2 and reduced phosphorylation (activation) of STAT3, compared with Ptf1a-CreERTM;LSL-KrasG12D mice. In UN-KC-6141 cells, PI3K and MEK signaling increased expression of KLF5; a high level of KLF5 increased proliferation. Cells with knockdown of Klf5 had reduced proliferation, compared with control cells, had reduced expression of ductal markers, and formed smaller tumors (71.61 ± 30.79 mm3 vs 121.44 ± 34.90 mm3 from control cells) in flanks of mice. Levels of KLF5 are increased in human PDAC samples and in PanINs of Ptf1a-CreERTM;LSL-KrasG12D mice, compared with controls. KLF5 disruption increases expression of NDRG2 and reduces activation of STAT3 and reduces ADM and PanINs formation in mice. Strategies to reduce KLF5 activity might reduce progression of acinar cells from ADM to PanIN and pancreatic tumorigenesis.

Glycogen synthase kinase-3β ablation limits pancreatitis-induced acinar-to-ductal metaplasia.

Acinar-to-ductal metaplasia (ADM) is a reversible epithelial transdifferentiation process that occurs in the pancreas in response to acute inflammation. ADM can rapidly progress towards pre-malignant pancreatic intraepithelial neoplasia (PanIN) lesions in the presence of mutant KRas and ultimately pancreatic adenocarcinoma (PDAC). In the present work, we elucidate the role and related mechanism of glycogen synthase kinase-3beta (GSK-3β) in ADM development using in vitro 3D cultures and genetically engineered mouse models. We show that GSK-3β promotes TGF-α-induced ADM in 3D cultured primary acinar cells, whereas deletion of GSK-3β attenuates caerulein-induced ADM formation and PanIN progression in KrasG12D transgenic mice. Furthermore, we demonstrate that GSK-3β ablation influences ADM formation and PanIN progression by suppressing oncogenic KRas-driven cell proliferation. Mechanistically, we show that GSK-3β regulates proliferation by increasing the activation of S6 kinase. Taken together, these results indicate that GSK-3β participates in early pancreatitis-induced ADM and thus could be a target for the treatment of chronic pancreatitis and the prevention of PDAC progression. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Metformin suppresses cancer initiation and progression in genetic mouse models of pancreatic cancer

BackgroundPancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-associated mortality worldwide with an overall five-year survival rate less than 7%. Accumulating evidence has revealed the cancer preventive and therapeutic effects of metformin, one of the most widely prescribed medications for type 2 diabetes mellitus. However, its role in pancreatic cancer is not fully elucidated. Herein, we aimed to further study the preventive and therapeutic effects of metformin in genetically engineered mouse models of pancreatic cancer.MethodsLSL-KrasG12D/+; Pdx1-Cre (KC) mouse model was established to investigate the effect of metformin in pancreatic tumorigenesis suppression; LSL-KrasG12D/+; Trp53fl/+; Pdx1-Cre (KPC) mouse model was used to evaluate the therapeutic efficiency of metformin in PDAC. Chronic pancreatitis was induced in KC mice by peritoneal injection of cerulein.ResultsFollowing metformin treatment, pancreatic acinar-to-ductal metaplasia (ADM) and mouse pancreatic intraepithelial neoplasia (mPanIN) were decreased in KC mice. Chronic pancreatitis induced a stroma-rich and duct-like structure and increased the formation of ADM and mPanIN lesions, in line with an increased cytokeratin 19 (CK19)-stained area. Metformin treatment diminished chronic pancreatitis-mediated ADM and mPanIN formation. In addition, it alleviated the percent area of Masson’s trichrome staining, and decreased the number of Ki67-positive cells. In KPC mice, metformin inhibited tumor growth and the incidence of abdominal invasion. More importantly, it prolonged the overall survival.ConclusionsMetformin inhibited pancreatic cancer initiation, suppressed chronic pancreatitis-induced tumorigenesis, and showed promising therapeutic effect in PDAC.

Abstract 3518: Brg1 plays a critical role in PanIN formation through regulating Sox9 expression

Abstract Background: Mutations of SWI/SNF chromatin remodeling complexes are one of the hallmarks of human pancreatic ductal adenocarcinoma (PDA). Brahma related gene 1 (Brg1) is a catalytic ATPase subunit of SWI/SNF complexes and Brg1 is silenced in about 10% of human PDA. Recent study revealed that Brg1 inhibits formation of intraductal pancreatic mucinous neoplasm (IPMN) and IPMN-derived PDA in the context of oncogenic Kras and that IPMNs originate from adult pancreatic ductal cells, demonstrating a tumor suppressive role of Brg1. However, the role of Brg1 in the formation of pancreatic intraepithelial neoplasia (PanIN) from acinar cells and PanIN-derived PDA is not fully understood. Aim: To investigate the functional role of Brg1 in the formation of PanIN Methods and Results: We generated Ptf1a-CreER; KrasG12D; Brg1f/f (KBC) mice, in which Brg1 is deleted exclusively in adult pancreatic acinar cells along with expression of oncogenic Kras upon tamoxifen induction of Cre activity and assessed spontaneous acinar-to-ductal metaplasia (ADM) and PanIN formation. We found that ADM and PanIN formation was significantly decreased in KBC mice compared with littermate controls. Next, we induced caerulein-acute pancreatitis to KBC mice and assessed for pancreatitis-induced tumorigenesis. We found that PanINs were decreased in KBC mice compared with littermate controls, although pancreatitis-induced ADMs were widely observed in KBC mice. Since Brg1 is known to critical regulator of p53, mutant p53 allele was crossed with KBC mice. Unexpectedly, even in the context of mutant p53, loss of Brg1 significantly deceased PanIN formation. These results suggest that Brg1 plays a tumor-supportive role in acinar cell-derived PanIN formation in various contexts. Primary acinar cell culture experiments revealed that ADM formation was markedly reduced in the absence of Brg1 and that Sox9 expression was downregulated in Brg1-depleted acinar cells. Furthermore, we confirmed that Sox9 expression is downregulated in Brg1-depleted ADMs/PanINs compared with Brg1-retained ADMs/PanINs by immunohistochemistry. To determine the functional relationship between Brg1 and Sox9, we generated Ptf1a-CreER; KrasG12D; Brg1f/f; Sox9OE mice, in which Sox9 is constitutively overexpressed. Remarkably, Sox9 overexpression canceled the phenotype of decrease in PanIN formation by Brg1 deletion. Furthermore, to investigate whether Brg1/Sox9 axis is observed in human PanIN-derived PDAs, we performed immunohistochemistry of Brg1 and Sox9 in human PanIN-derived PDAs. In 27 human PDAs, all of 5 PDAs with low Brg1 expression had low Sox9 expression. Conclusions: Our results suggest that Brg1 plays a critical role in acinar cell-derived PanIN formation in part through regulating Sox9 expression positively. Our data underscore a cell-type specific, context-dependent role of Brg1 in the initiation of PDA formation. Citation Format: Motoyuki Tsuda, Akihisa Fukuda, Satoshi Ogawa, Kenji Masuo, Norihiro Goto, Yukiko Hiramatsu, Yu Muta, Kozo Ikuta, Yoshito Kimura, Yoshihide Matsumoto, Yutaka Takada, Takuto Yoshioka, Takahisa Maruno, Haruhiko Akiyama, Kyoichi Takaori, Shinji Uemoto, Tsutomu Chiba, Hiroshi Seno. Brg1 plays a critical role in PanIN formation through regulating Sox9 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3518. doi:10.1158/1538-7445.AM2017-3518

Oncogenic KRas-induced Increase in Fluid-phase Endocytosis is Dependent on N-WASP and is Required for the Formation of Pancreatic Preneoplastic Lesions

Fluid-phase endocytosis is a homeostatic process with an unknown role in tumor initiation. The driver mutation in pancreatic ductal adenocarcinoma (PDAC) is constitutively active KRasG12D, which induces neoplastic transformation of acinar cells through acinar-to-ductal metaplasia (ADM). We have previously shown that KRasG12D-induced ADM is dependent on RAC1 and EGF receptor (EGFR) by a not fully clarified mechanism. Using three-dimensional mouse and human acinar tissue cultures and genetically engineered mouse models, we provide evidence that (i) KRasG12D leads to EGFR-dependent sustained fluid-phase endocytosis (FPE) during acinar metaplasia; (ii) variations in plasma membrane tension increase FPE and lead to ADM in vitro independently of EGFR; and (iii) that RAC1 regulates ADM formation partially through actin-dependent regulation of FPE. In addition, mice with a pancreas-specific deletion of the Neural-Wiskott–Aldrich syndrome protein (N-WASP), a regulator of F-actin, have reduced FPE and impaired ADM emphasizing the in vivo relevance of our findings. This work defines a new role of FPE as a tumor initiating mechanism.