Challenges in detecting pre-malignant pancreatic lesions during acute pancreatitis using a serum microRNA assay: a study based on KrasG12D transgenic mice.
ABSTRACT: Caerulein-induced acute pancreatitis accelerates the progression of pancreatic intraepithelial neoplasia (PanIN) lesions in a pancreas-specific KrasG12D mouse model. The purpose of this study was to explore whether serum microRNAs (miRNAs) can serve as sensitive biomarkers to detect occult PanIN in the setting of acute pancreatitis. Serum miRNA profiles were quantified by an array-based method and normalized by both Variance Stabilization Normalization (VSN) and invariant methods. Individual miRNAs were validated by TaqMan real-time PCR with synthetic spike-in C. elegans miRNAs as external controls. Serum miRNA profiles distinguished KrasG12D mice with pancreatitis from wild-type mice without pancreatitis, but failed to differentiate KrasG12D mice with pancreatitis from wild-type mice with pancreatitis. Most individual miRNAs that increased in KrasG12D mice with pancreatitis were not significantly different between KrasG12D mice without pancreatitis and wild-type mice without pancreatitis. Mechanistically, Gene Set Enrichment Analysis (GSEA) of the mRNA array data and immunohistochemical assays showed that caerulein-induced acute pancreatitis involved acinar cell loss and immune cell infiltration, which might contribute to serum miRNA profile changes. This study highlighted the challenges in using sensitive serum miRNA biomarker screening for the early detection of pancreatic malignancies during acute pancreatitis.
Project description:Transgenic KrasG12D mice can recapitulate pancreas intra-epithelial neoplasia (PanIN). Caerulein is a cholecystokinin analogue and induces acute pancreatitis when injected intra-abdominally. Caerulein-induced acute pancreatitis will accelerate PanIN progression in KrasG12D mice. We compared mRNA profile changes between KrasG12D mice with acute caerulein-induced pancreatitis and wild-type mice without acute pancreatitis. The experiment had two groups. Experiment group: KrasG12D mice with acute caerulein-induced pancreatitis (N=6). Three mice in experiment group received 1-week caerulein injection, and the other three mice received 2-week caerulein injection. All experiment group mice started to receive caerulein injection at 1-month of age, and were sacrificed at the last day of caerulein injection. Control group: wild-type mice without acute pancreatitis (N=6). The mice were sacrificed at 1.5-month of age. Whole pancreas tissue lysate samples were subjected to mRNA array assay.
Project description:Consecutive caerulein injections induce an acute pancreatitis in mice. Here, we recorded gene expression levels at different stages of pancreatic regeneration in wild-type mice as well as KrasG12D-mutated mice. Tissue was collected from mice pancreata, cell sorting was not performed. t=0h refers to the time where caerulein was injected. control referes to NaCl-treated samples (no caerulein). 13 time points were used for wild-type mice, 9 time points were used for KrasG12D-mutated mice. Multiple replicates were generated for each time point. We used Affymetrix GeneChip Mouse Gene 1.0 ST arrays.
Project description:Activating mutations in the KRAS oncogene are prevalent in pancreatic ductal adenocarcinoma (PDAC). We previously demonstrated that pancreatic intraepithelial neoplasia (PanIN) formation, which precedes malignant transformation, associates with the expression of immediate early response 3 (Ier3) as part of a prooncogenic transcriptional pathway. Here, we evaluated the role of IER3 in PanIN formation and PDAC development. In human pancreatic cancer cells, IER3 expression efficiently sustained ERK1/2 phosphorylation by inhibiting phosphatase PP2A activity. Moreover, IER3 enhanced KrasG12D-dependent oncogenesis in the pancreas, as both PanIN and PDAC development were delayed in IER3-deficient KrasG12D mice. IER3 expression was discrete in healthy acinar cells, becoming highly prominent in peritumoral acini, and particularly high in acinar ductal metaplasia (ADM) and PanIN lesions, where IER3 colocalized with phosphorylated ERK1/2. However, IER3 was absent in undifferentiated PDAC, which suggests that the IER3-dependent pathway is an early event in pancreatic tumorigenesis. IER3 expression was induced by both mild and severe pancreatitis, which promoted PanIN formation and progression to PDAC in KrasG12D mice. In IER3-deficient mice, pancreatitis abolished KrasG12D-induced proliferation, which suggests that pancreatitis enhances the oncogenic effect of KRAS through induction of IER3 expression. Together, our data indicate that IER3 supports KRASG12D-associated oncogenesis in the pancreas by sustaining ERK1/2 phosphorylation via phosphatase PP2A inhibition.
Project description:The cystic fibrosis (CF) mouse pancreas has constitutively elevated expression of the Reg/PAP cell stress genes (60-fold greater Reg3alpha, and 10-fold greater PAP/Reg3beta and Reg3gamma). These genes are suggested to be involved in protection or recovery from pancreatic injury.To test this idea the supramaximal caerulein model was used to induce acute pancreatitis in wild type and CF mice. Serum amylase, pancreatic water content (as a measure of edema), pancreatic myeloperoxidase activity, and Reg/PAP expression were quantified.In both wild type and CF mice caerulein induced similar elevations in serum amylase (maximal at 12 h), pancreatic edema (maximal at 7 h), and pancreatic myeloperoxidase activity (MPO, a marker of neutrophil infiltration; maximal at 7 h). By immunohistochemistry, Reg3alpha was strongly expressed in the untreated CF pancreas but not in wild type. During pancreatitis, Reg3alpha was intensely expressed in foci of inflamed tissue in both wild type and CF.These data demonstrate that the severity of caerulein-induced pancreatitis is not ameliorated in the CF mouse even though the Reg/PAP stress genes are already highly upregulated. While Reg/PAP may be protective they may also have a negative effect during pancreatitis due to their anti-apoptotic activity, which has been shown to increase the severity of pancreatitis.
Project description:Mice lacking Sirt2 spontaneously develop tumors in multiple organs, as well as when expressed in combination with oncogenic KrasG12D, leading to pancreatic tumors. Here, we report that after caerulein-induced pancreatitis, Sirt2-deficient mice exhibited an increased inflammatory phenotype and delayed pancreatic tissue recovery. Seven days post injury, the pancreas of Sirt2-/- mice display active inflammation, whereas wild-type mice had mostly recovered. In addition, the pancreas from the Sirt2-/- mice exhibited extensive tissue fibrosis, which was still present at six weeks after exposure. The mice lacking Sirt2 also demonstrated an enhanced whole body pro-inflammatory phenotype that was most obvious with increasing age. Importantly, an accumulation of a cell population with spontaneous cancerous KrasG12D mutations was observed in the Sirt2-/- mice that is enhanced in the recovering pancreas after exposure to caerulein. Finally, transcriptome analysis of the pancreas of the Sirt2-/- mice exhibited a pro-inflammatory genomic signature. These results suggest that loss of Sirt2, as well as increased age, enhanced the immune response to pancreatic injury and induced an inflammatory phenotype permissive for the accumulation of cells carrying oncogenic Kras mutations.
Project description:The pancreatic acinar-enriched miR-216a, miR-216b and miR-217 are encoded within the miR217HG. These miRNAs have been purported to play a tumor suppressive role as their expression is reduced in both human and mouse pancreatic ductal adenocarcinoma (PDAC). To examine this possibility, we generated individual, germline knockout (KO) mice of miR-216a, miR-216b or miR-217. Unlike our previous study showing germline deletion of the miR217HG was embryonic lethal, CRISPR-Cas9 deleted portions of the 5' seed region of the miRNAs produced live births. To investigate possible phenotypes during pancreatic acinar ductal metaplasia (ADM), pancreatic acini from wild type and KO mice were plated on collagen and allowed to transdifferentiate over 4 days. Acini from each of the three miRNA KO mice produced greater numbers of ducts compared to controls. Evaluation of the gene expression during in vitro ADM demonstrated an increase in Krt19 and a reduction in acinar genes (Carboxypeptidase A1, Amylase2a) on day 4 of the transdifferentiation. Recovery was delayed for the miR-216a and miR-216b KOs following caerulein-induced acute pancreatitis. Also predominate in the caerulein treated miR-216a and miR-216b KO mice was the presence of pancreatic duct glands (PDGs). To further establish a phenotype, miRNA KO mice were crossed with EL-KRAS<sup>G12D</sup> (EK) mice and followed up to 13 months of age. While all mice developed severe dysplasia and cystic papillary neoplasms, there existed no apparent phenotypic difference in the miRNA KO/EK mice compared to EK mice. Our data does not support a tumor suppressor role for miR-216a, miR-216b or miR-217 in PDAC and emphasizes the need for phenotypic evaluation of miRNAs in complex in vivo models beyond that performed using cell culture.
Project description:Targeting of oncogenic Kras to the pancreatic Nestin-expressing embryonic progenitor cells and subsequently to the adult acinar compartment and Nestin-expressing cells is sufficient for the development of low grade pancreatic intraepithelial neoplasia (PanIN) between 2 and 4 months. The mice die around 6 month-old of unrelated causes, and it is therefore not possible to assess whether the lesions will progress to carcinoma. We now report that two brief episodes of caerulein-induced acute pancreatitis in 2 month-old mice causes rapid PanIN progression and pancreatic ductal adenocarcinoma (PDAC) development by 4 months of age. These events occur with similar frequency as observed in animals where the oncogene is targeted during embryogenesis to all pancreatic cell types. Thus, these data show that oncogenic Kras-driven PanIN originating in a non-ductal compartment can rapidly progress to PDAC when subjected to a brief inflammatory insult.
Project description:<h4>Background and aims</h4>Heat shock protein (Hsp) 72 is a molecular chaperone which is upregulated in response to a variety of stress situations and has a general cytoprotective function. Increased Hsp72 levels were implicated in protection from acute pancreatitis; a hypothesis which was not tested in a transgenic mouse model yet.<h4>Methods</h4>To analyze the role of Hsp72 during acute pancreatitis, well-characterized transgenic animals overexpressing rat Hsp72 (Hsp72 mice) under the control of the ß-actin promoter were subjected to caerulein- and L-arginine-induced acute pancreatitis. The severity of experimental pancreatitis was determined via serum lipase levels, morphometric evaluation and quantification of pancreatic edema/inflammation.<h4>Results</h4>Hsp72 mice displayed ?100-times Hsp72 overexpression, but no changes in the remaining chaperones. Robust Hsp72 signal was observed in pancreatic acini, but not in islets or ductal cells. In both models, elevated Hsp72 did not protect from development of acute pancreatitis and the pancreatitis-associated lung injury, but accelerated recovery from caerulein-induced tissue injury (lower lipase levels, edema, inflammation and necrosis 36 h after caerulein administration). The observed protective function of Hsp72 in caerulein-induced pancreatitis is likely due to an attenuated NF-?B signalling.<h4>Conclusions</h4>Hsp72 overexpression accelerates the recovery from acute pancreatitis and may represent a potential treatment strategy.
Project description:To determine the molecular basis of gene regulation in pancreatic ductal epithelial cells, we developed methods for the isolation of this cell population during mouse development and normal adult homeostasis, as well as in conditions with ductal features (acinar-to-ductal metaplasia (ADM), pancreatic intraepithelial neoplasia (PanIN) and pancreatic ductal adenocarcinoma (PDAC)). Our technique utilizes the specificity of Dolichos biflorus Agglutinin (DBA) lectin marking the entire normal ductal tree, including terminal intercalated ducts (putative sites of stem or progenitor cells) and ductal structures in ADM and PanIN. We used ferromagnetic-labeled DBA lectin to isolate ductal structures. Ductal cells were isolated under the following conditions: (1) Embryonic Development in wild type mice: E14.5, E15.5, E16.5, and postnatal day 1 (P1); (2) Injury and regeneration (pancreatitis) 0, 1, 3, 5 days following cerulein-induced acute pancreatitis. Cerulein is a cholecystokinin analog which produces a self-limited pancreatitis with injury and subsequent regeneration and repair, completed five days after insult; and (3) Pdx1-Cre;LSL-KrasG12D/+ mice aged 10 and 20 weeks that harbor PanIN lesions and a subset develop PDAC. Ductal/PanIN cells were isolated from these mice and appropriate control mice (Pdx1-Cre;Kras+/+). Overall design: Expression profiling of ductal cells at embryonic days 14.5, 15.5, 16.5 and post-natal day 1 as well as days 0, 1, 3 and 5 after acute ceruelin-induced pancreatitis and 10/20 weeks aged Pdx1-Cre;LSL-KrasG12D/+ mice.