mTORC1 and mTORC2 Converge on the Arp2/3 Complex to Promote KrasG12D-Induced Acinar-to-Ductal Metaplasia and Early Pancreatic Carcinogenesis.

BACKGROUND & AIMS: Oncogenic KrasG12D induces neoplastic transformation of pancreatic acinar cells through acinar-to-ductal metaplasia (ADM), an actin-based morphogenetic process, and drives pancreatic ductal adenocarcinoma (PDAC). mTOR (mechanistic target of rapamycin kinase) complex 1 (mTORC1) and 2 (mTORC2) contain Rptor and Rictor, respectively, and are activated downstream of KrasG12D, thereby contributing to PDAC. Yet, whether and how mTORC1 and mTORC2 impact on ADM and the identity of the actin nucleator(s) mediating such actin rearrangements remain unknown. METHODS: A mouse model of inflammation-accelerated KrasG12D-driven early pancreatic carcinogenesis was used. Rptor, Rictor, and Arpc4 (actin-related protein 2/3 complex subunit 4) were conditionally ablated in acinar cells to deactivate the function of mTORC1, mTORC2 and the actin-related protein (Arp) 2/3 complex, respectively. RESULTS: We found that mTORC1 and mTORC2 are markedly activated in human and mouse ADM lesions, and cooperate to promote KrasG12D-driven ADM in mice and in vitro. They use the Arp2/3 complex as a common downstream effector to induce the remodeling the actin cytoskeleton leading to ADM. In particular, mTORC1 regulates the translation of Rac1 (Rac family small GTPase 1) and the Arp2/3-complex subunit Arp3, whereas mTORC2 activates the Arp2/3 complex by promoting Akt/Rac1 signaling. Consistently, genetic ablation of the Arp2/3 complex prevents KrasG12D-driven ADM in vivo. In acinar cells, the Arp2/3 complex and its actin-nucleation activity mediated the formation of a basolateral actin cortex, which is indispensable for ADM and pre-neoplastic transformation. CONCLUSIONS: Here, we show that mTORC1 and mTORC2 attain a dual, yet nonredundant regulatory role in ADM and early pancreatic carcinogenesis by promoting Arp2/3 complex function. The role of Arp2/3 complex as a common effector of mTORC1 and mTORC2 fills the gap between oncogenic signals and actin dynamics underlying PDAC initiation.

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.

Omega-3 Fatty Acids Prevent Early Pancreatic Carcinogenesis via Repression of the AKT Pathway.

Pancreatic cancer remains a daunting foe despite a vast number of accumulating molecular analyses regarding the mutation and expression status of a variety of genes. Indeed, most pancreatic cancer cases uniformly present with a mutation in the KRAS allele leading to enhanced RAS activation. Yet our understanding of the many epigenetic/environmental factors contributing to disease incidence and progression is waning. Epidemiologic data suggest that diet may be a key factor in pancreatic cancer development and potentially a means of chemoprevention at earlier stages. While diets high in ω3 fatty acids are typically associated with tumor suppression, diets high in ω6 fatty acids have been linked to increased tumor development. Thus, to better understand the contribution of these polyunsaturated fatty acids to pancreatic carcinogenesis, we modeled early stage disease by targeting mutant KRAS to the exocrine pancreas and administered diets rich in these fatty acids to assess tumor formation and altered cell-signaling pathways. We discovered that, consistent with previous reports, the ω3-enriched diet led to reduced lesion penetrance via repression of proliferation associated with reduced phosphorylated AKT (pAKT), whereas the ω6-enriched diet accelerated tumor formation. These data provide a plausible mechanism underlying previously observed effects of fatty acids and suggest that administration of ω3 fatty acids can reduce the pro-survival, pro-growth functions of pAKT. Indeed, counseling subjects at risk to increase their intake of foods containing higher amounts of ω3 fatty acids could aid in the prevention of pancreatic cancer.

Loss of Pten and Activation of Kras Synergistically Induce Formation of Intraductal Papillary Mucinous Neoplasia From Pancreatic Ductal Cells in Mice.

BACKGROUND & AIMS: Intraductal papillary mucinous neoplasias (IPMNs) are precancerous cystic lesions that can develop into pancreatic ductal adenocarcinomas (PDACs). These large macroscopic lesions are frequently detected during medical imaging, but it is unclear how they form or progress to PDAC. We aimed to identify cells that form IPMNs and mutations that promote IPMN development and progression. METHODS: We generated mice with disruption of Pten specifically in ductal cells (Sox9CreERT2;Ptenflox/flox;R26RYFP or PtenΔDuct/ΔDuct mice) and used PtenΔDuct/+ and Pten+/+ mice as controls. We also generated KrasG12D;PtenΔDuct/ΔDuct and KrasG12D;PtenΔDuct/+ mice. Pancreata were collected when mice were 28 weeks to 14.5 months old and analyzed by histology, immunohistochemistry, and electron microscopy. We performed multiplexed droplet digital polymerase chain reaction to detect spontaneous Kras mutations in PtenΔDuct/ΔDuct mice and study the effects of Ras pathway activation on initiation and progression of IPMNs. We obtained 2 pancreatic sections from a patient with an invasive pancreatobiliary IPMN and analyzed the regions with and without the invasive IPMN (control tissue) by immunohistochemistry. RESULTS: Mice with ductal cell-specific disruption of Pten but not control mice developed sporadic, macroscopic, intraductal papillary lesions with histologic and molecular features of human IPMNs. PtenΔDuct/ΔDuct mice developed IPMNs of several subtypes. In PtenΔDuct/ΔDuct mice, 31.5% of IPMNs became invasive; invasion was associated with spontaneous mutations in Kras. KrasG12D;PtenΔDuct/ΔDuct mice all developed invasive IPMNs within 1 month. In KrasG12D;PtenΔDuct/+ mice, 70% developed IPMN, predominately of the pancreatobiliary subtype, and 63.3% developed PDAC. In all models, IPMNs and PDAC expressed the duct-specific lineage tracing marker yellow fluorescent protein. In immunohistochemical analyses, we found that the invasive human pancreatobiliary IPMN tissue had lower levels of PTEN and increased levels of phosphorylated (activated) ERK compared with healthy pancreatic tissue. CONCLUSIONS: In analyses of mice with ductal cell-specific disruption of Pten, with or without activated Kras, we found evidence for a ductal cell origin of IPMNs. We also showed that PTEN loss and activated Kras have synergistic effects in promoting development of IPMN and progression to PDAC.

Glycogen synthase kinase-3ß 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-3ß) in ADM development using in vitro 3D cultures and genetically engineered mouse models. We show that GSK-3ß promotes TGF-α-induced ADM in 3D cultured primary acinar cells, whereas deletion of GSK-3ß attenuates caerulein-induced ADM formation and PanIN progression in KrasG12D transgenic mice. Furthermore, we demonstrate that GSK-3ß ablation influences ADM formation and PanIN progression by suppressing oncogenic KRas-driven cell proliferation. Mechanistically, we show that GSK-3ß regulates proliferation by increasing the activation of S6 kinase. Taken together, these results indicate that GSK-3ß 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.

Proton Pump Inhibitors Inhibit Pancreatic Secretion: Role of Gastric and Non-Gastric H+/K+-ATPases.

The mechanism by which pancreas secretes high HCO3- has not been fully resolved. This alkaline secretion, formed in pancreatic ducts, can be achieved by transporting HCO3- from serosa to mucosa or by moving H+ in the opposite direction. The aim of the present study was to determine whether H+/K+-ATPases are expressed and functional in human pancreatic ducts and whether proton pump inhibitors (PPIs) have effect on those. Here we show that the gastric HKα1 and HKß subunits (ATP4A; ATP4B) and non-gastric HKα2 subunits (ATP12A) of H+/K+-ATPases are expressed in human pancreatic cells. Pumps have similar localizations in duct cell monolayers (Capan-1) and human pancreas, and notably the gastric pumps are localized on the luminal membranes. In Capan-1 cells, PPIs inhibited recovery of intracellular pH from acidosis. Furthermore, in rats treated with PPIs, pancreatic secretion was inhibited but concentrations of major ions in secretion follow similar excretory curves in control and PPI treated animals. In addition to HCO3-, pancreas also secretes K+. In conclusion, this study calls for a revision of the basic model for HCO3- secretion. We propose that proton transport is driving secretion, and that in addition it may provide a protective pH buffer zone and K+ recirculation. Furthermore, it seems relevant to re-evaluate whether PPIs should be used in treatment therapies where pancreatic functions are already compromised.

Endosonography for Pancreatic Duct Dilatation without Definite Pathology on Ultrasonography.

BACKGROUND/AIMS: Main pancreatic duct dilatation raises concerns about the possibility of pancreatobiliary malignancy. We evaluated the etiologic yield of endosonography (EUS) for main pancreatic duct dilatation without definite pathology on Ultrasonography (US). METHODOLOGY: A retrospective review was conducted in 54 consecutive patients referred for EUS. RESULTS: No pathological finding (37.0%, 20/54), followed by periampullary cancer (35.2%, 19/54), was the most common finding. Elevated alkaline phosphatase (ALK-P) and marked common bile duct (CBD) dilatation (≥ 12 mm) were the predictors of malignancy (p < 0.05). Among the 37 subjects with available ALK-P and CBD diameter, the probability of malignancy was 84.6% (11/13) for both elevated ALK-P and marked CBD dilatation, 16.7% (1/6) for isolated elevated ALK-P, 18.2% (2/11) for isolated marked CBD dilatation, and none (0/7) was for subjects with neither elevated ALK-P nor marked CBD dilatation, respectively. The overall accuracy of EUS for periampullary carcinomas was 94.7% (18/19) and for choledocholithiasis was 100% (7/7), respectively. EUS had a 100.0% (20/20) sensitivity and a 97.1% (33/34) specificity in the diagnosis of no pathological obstruction. CONCLUSIONS: EUS is accurate for main pancreatic duct dilatation without definite pathology on US, and the presence of concomitant elevated ALK-P and CBD dilatation highly suggests malignancy.

Akt kinase mediates the prosurvival effect of smoking compounds in pancreatic ductal cells.

OBJECTIVES: Cigarette smoking is a major risk factor for pancreatic cancer (PaCa). However, the mechanisms of smoking-induced PaCa remain unknown. Here we investigated the effect of smoking compounds on cell death pathways in pancreatic ductal cells, precursors of PaCa. METHODS: Human pancreatic ductal cells (HPDE6-c7) were cultured with cigarette smoke extract (CSE) or smoking compound 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Apoptosis and autophagy were assessed by DNA fragmentation and immunofluorescence, respectively. RESULTS: Exposure to CSE or NNK decreased DNA fragmentation and up-regulated BclXL. Akt kinase was activated by smoking compounds through reactive oxygen species-dependent mechanism. Specifically, Akt activation was prevented by inhibition of nicotinamide adenine dinucleotide oxidase. Molecular or pharmacologic inhibitions of Akt prevented the antiapoptotic effect of smoking compounds. Smoking compounds stimulated rapid (1 hour) and transient activation of 5'-adenosine monophosphate-activated protein kinase and formation of autophagic vacuoles, indicating stimulation of autophagy. Repeated exposure to CSE/NNK (48 hours or longer) abolished the early activation of autophagic markers. Inhibition of Akt prevented the antiautophagic effect of long exposure to smoking compounds, indicating that smoking-induced late activation of Akt prevents autophagy. CONCLUSIONS: Long exposure of pancreatic ductal cells to smoking compounds inhibited apoptosis and autophagy. The results revealed a central role for Akt kinase in mediating key procarcinogenic effects of smoking compounds.

Pancreatic bicarbonate secretion involves two proton pumps.

Pancreas secretes fluid rich in digestive enzymes and bicarbonate. The alkaline secretion is important in buffering of acid chyme entering duodenum and for activation of enzymes. This secretion is formed in pancreatic ducts, and studies to date show that plasma membranes of duct epithelium express H(+)/HCO(3)(-) transporters, which depend on gradients created by the Na(+)/K(+)-ATPase. However, the model cannot fully account for high-bicarbonate concentrations, and other active transporters, i.e. pumps, have not been explored. Here we show that pancreatic ducts express functional gastric and non-gastric H(+)-K(+)-ATPases. We measured intracellular pH and secretion in small ducts isolated from rat pancreas and showed their sensitivity to H(+)-K(+) pump inhibitors and ion substitutions. Gastric and non-gastric H(+)-K(+) pumps were demonstrated on RNA and protein levels, and pumps were localized to the plasma membranes of pancreatic ducts. Quantitative analysis of H(+)/HCO(3)(-) and fluid transport shows that the H(+)-K(+) pumps can contribute to pancreatic secretion in several species. Our results call for revision of the bicarbonate transport physiology in pancreas, and most likely other epithelia. Furthermore, because pancreatic ducts play a central role in several pancreatic diseases, it is of high relevance to understand the role of H(+)-K(+) pumps in pathophysiology.

Phosphorylation of ERK in the spinal dorsal horn following pancreatic pronociceptive stimuli with proteinase-activated receptor-2 agonists and hydrogen sulfide in rats: evidence for involvement of distinct mechanisms.

Noxious stimuli cause prompt phosphorylation of extracellular signal-regulated kinase (ERK) in the spinal dorsal horn that contributes to facilitation of pain sensation and is often used as an immediate marker for excitation of spinal neurons following somatic and colonic nociception. Here we asked whether two distinct pronociceptive stimuli with proteinase-activated receptor-2 (PAR2) agonists and hydrogen sulfide (H(2)S) in the pancreas cause phosphorylation of ERK in the spinal dorsal horn and also examined involvement of their possible downstream signaling molecules, transient receptor potential vanilloid-1 (TRPV1) and T-type Ca(2+) channels, respectively. Capsaicin (a TRPV1 agonist), trypsin (an endogenous PAR2 agonist), SLIGRL-NH(2) (a PAR2-activating peptide), and NaHS (an H(2)S donor) were infused into the pancreatic duct in anesthetized rats, and phosphorylated ERK in the spinal cord was detected by immunohistochemistry. Intraductal administration of capsaicin and trypsin caused prompt phosphorylation of ERK in the superficial layers of T9, but not T5 or T12, spinal dorsal horn. SLIGRL-NH(2) and NaHS, administered in the same manner, also produced ERK phosphorylation in the corresponding spinal regions. Mibefradil, a T-type Ca(2+) channel blocker, abolished the phosphorylation of ERK caused by intraductal NaHS but not SLIGRL-NH(2). In contrast, capsazepine, an inhibitor of TRPV1, suppressed the phosphorylation of ERK caused by intraductal SLIGRL-NH(2) but not NaHS. Our data thus demonstrate that pancreatic pronociceptive stimuli with PAR2 agonists and H(2)S cause ERK phosphorylation in the spinal dorsal horn, through activation of TRPV1 and T-type Ca(2+) channels, respectively, and that those two pronociceptive pathways are independent of each other.

Mechanisms of hepatocyte growth factor-mediated signaling in differentiation of pancreatic ductal epithelial cells into insulin-producing cells.

Pancreatic ductal epithelial cells (PDECs) were induced to differentiate into insulin-producing cells by hepatocyte growth factor (HGF) in our previous study, but the mechanism through which this induction occurs is still unknown. HGF is a ligand that activates a tyrosine kinase encoded by the c-Met proto-oncogene. This activation is followed by indirect activation of multiple downstream signal transduction pathways (including MAPKs and the PI3K/AKT signaling pathways) that initiate various biological effects. Therefore, we speculated that the differentiation of PDECs is through either the MAPK signaling pathway or the PI3K/AKT signaling pathway. To test this hypothesis, isolated PDECs from adult rats were stimulated by adding HGF to their medium for 28days. Then, the expression levels of several protein kinases, including MAPKs (ERK1/2, p38, and JNK) and AKT, were determined by Western blotting to determine if specific protein kinases are activated in these pathways. Subsequently, re-isolated from adult rats and cultured PDECs were pre-treated with specific inhibitors of proteins shown to be activated in these signaling pathways; these cells were then induced to differentiate by the addition of HGF. The expression levels of protein kinases were determined by Western blotting, and the differentiation rate of insulin-positive cells was determined by flow cytometry. The change of PDEC differentiation rates were compared between the groups in which cells with or without inhibitors pretreatment to determine the specific signaling pathway(s) that may be involved in HGF-induced differentiation of PDECs. After isolating PDECs and stimulating them with HGF for 28days, the expression levels of phosphorylated ERK1/2 as well as total and phosphorylated AKT of cultured cells were significantly increased compared to the normal control group (p<0.05), suggesting that the signaling pathways involving ERK1/2 and Akt (MEK-ERK and PI3K-AKT) are activated during HGF-induced PDEC differentiation. MEK1/2 or PI3K inhibitors were separately added to the culture medium of PDECs pre-treated with HGF. These results show that compared to the HGF-treated group, the differentiation rate of insulin-positive cells was significantly decreased in the HGF/LY294002 (PI3K inhibitor) group (13.47+/-1.57% vs. 33.47+/-1.34%, p<0.05); however, the differentiation rate of insulin-positive cells was not significantly different in the HGF/PD98059 (MEK1/2 inhibitor) group. These data suggest that HGF induces PDECs to differentiate into insulin-producing cells through the PI3K/AKT signaling pathway.

c-Jun N-terminal kinase is largely involved in the regulation of tricellular tight junctions via tricellulin in human pancreatic duct epithelial cells.

Tricellulin (TRIC) is a tight junction protein at tricellular contacts where three epithelial cells meet, and it is required for the maintenance of the epithelial barrier. To investigate whether TRIC is regulated via a c-Jun N-terminal kinase (JNK) pathway, human pancreatic HPAC cells, highly expressed at tricellular contacts, were exposed to various stimuli such as the JNK activators anisomycin and 12-O-tetradecanoylphorbol 13-acetate (TPA), and the proinflammatory cytokines IL-1ß, TNFα, and IL-1α. TRIC expression and the barrier function were moderated by treatment with the JNK activator anisomycin, and suppressed not only by inhibitors of JNK and PKC but also by siRNAs of TRIC. TRIC expression was induced by treatment with the PKC activator TPA and proinflammatory cytokines IL-1ß, TNFα, and IL-1α, whereas the changes were inhibited by a JNK inhibitor. Furthermore, in normal human pancreatic duct epithelial cells using hTERT-transfected primary cultured cells, the responses of TRIC expression to the various stimuli were similar to those in HPAC cells. TRIC expression in tricellular tight junctions is strongly regulated together with the barrier function via the JNK transduction pathway. These findings suggest that JNK may be involved in the regulation of tricellular tight junctions including TRIC expression and the barrier function during normal remodeling of epithelial cells, and prevent disruption of the epithelial barrier in inflammation and other disorders in pancreatic duct epithelial cells.

Comparative analysis of clinicopathological correlations of cyclooxygenase-2 expression in resectable pancreatic cancer.

AIM: To perform a comparative analysis of clinicopathological correlations of cyclooxygenase-2 (COX-2) expression in pancreatic cancer, examined by monoclonal and polyclonal antibodies. METHODS: The COX-2 expression in 85 resection specimens of pancreatic ductal adenocarcinoma was immunohistochemically examined using both monoclonal and polyclonal antibodies. The final immunoscores were obtained by multiplying the percentage of positive cells with the numeric score reflecting the staining intensity. COX-2 expression levels were classified into three categories (0, 1+, and 2+) and the clinicopathological correlations were statistically evaluated and analyzed. RESULTS: The positive tumor expression rates of COX-2 were 80.5% using monoclonal antibody and 69.4% using polyclonal antibody. In the Kaplan-Meier analysis, no significant correlations were found between levels of COX-2 expression and overall survival (OS), but trends to longer OS were found in COX-2 negative cases using monoclonal antibody. Significantly longer disease free survival was revealed in COX-2 negative cases using monoclonal antibody (P = 0.019). No correlations between COX-2 expression levels and grade (G), tumor (T) status and nodal (N) status were demonstrated. Low histological grade showed a strong association with a longer OS (P < 0.001). Correlation of survival and T status revealed a shorter OS in T3 tumors, but the results reached only marginal statistical significance (P = 0.070). In the multivariate Cox proportional hazards regression model, histological grade, T and N status remained valuable predictors of a worse survival with borderline significance for T [hazards ratio (HR) = 4.18 for G (if G = 3, P < 0.001); HR = 1.64 for T (if T = 3, P = 0.065); HR = 2.53 for N (if N = 1, P = 0.006)]. Higher grade, T or N status was associated with a worse OS. CONCLUSION: The immunohistochemically assessed level of COX-2 expression does not seem to represent a valuable independent prognostic factor and is not superior to the conventional prognostic factors.

Caveolin-1 regulating the invasion and expression of matrix metalloproteinase (MMPs) in pancreatic carcinoma cells.

The gelatinases B (MMP9) and A (MMP2) are two members of the matrix metalloproteinase (MMPs) family that are expressed in human cancer, and play a critical role in tumor cell invasion and metastasis. Caveolin-1 (Cav1) has recently been identified as a tumor metastasis modifier gene. However, the effect and mechanism of Cav1 in pancreatic carcinoma cell invasion remain unknown. In this study, we investigated the expression of Cav1, MMP2, and MMP9 in several different pancreatic carcinoma cell lines. We transfected pcDNA3.0-Cav1 plasmid and Cav1 siRNA into SW1990 and Bxpc3 cells, respectively. Using cell invasion assay, we found that overexpression of Cav1 inhibited cell invasion, whereas the knockdown of Cav1 in Bxpc3 cells promoted cell invasion. Moreover, to explore the mechanisms underlying these observations, we further investigated the expression of MMP2, MMP9, phospho-Akt, and phospho-Erk by Western blot, and the activities of MMP2 and MMP9 by gelatin zymography. The results indicated that Cav1 gene could inhibit pancreatic carcinoma cell invasion, at least in part, probably through Erk-MMP signal pathway, suggesting that the endogenous expression or re-expression of Cav1 might help therapeutically reduce their invasive potential in pancreatic carcinoma cells.

Use of IHC and newly designed matriptase inhibitors to elucidate the role of matriptase in pancreatic ductal adenocarcinoma.

Matriptase, also known as MT-SP1, is a type II transmembrane serine protease strongly implicated in both the development and progression of a variety of epithelial cancers. Evidence comes from studies of its expression in human cancers and from mouse models of spontaneous cancer. Matriptase is considered to be a major activator of two key stimulators of invasive growth, namely hepatocyte growth factor/scatter factor and urokinase-type plasminogen activator. The aim of this study was to examine the role of matriptase in pancreatic ductal adenocarcinoma by expression analysis and functional assays in vitro. Immunohistochemical analysis of matriptase performed on microtissue arrays and large samples of 55 pancreatic ductal adenocarcinomas and on 31 samples of normal pancreatic ducts revealed that although matriptase expression differed greatly in both malignant and normal ductal pancreatic tissue, matriptase scores were significantly (p=0.02) elevated in pancreatic ductal adenocarcinoma compared to normal pancreatic ducts. To evaluate the role of matriptase during development of pancreatic cancer, we studied the effects of newly designed matriptase inhibitors on the processing of the zymogen of urokinase-type plasminogen activator in the human adenocarcinoma cell lines AsPC-1 and BxPC-3. In both cell lines, at 1 microM, all matriptase inhibitors completely prevented zymogen activation. At lower inhibitor concentrations, the degree of inhibition of zymogen processing correlated with the affinities of the inhibitors towards matriptase indicating that this is a specific result of matriptase inhibition. Furthermore, matriptase inhibitors reduced the phosphorylation of the HGF receptor/cMet and the overall cellular invasiveness of the human pancreatic adenocarcinoma cell line AsPC-1. Our findings demonstrate for the first time that matriptase may be involved in the progression of pancreatic ductal adenocarcinoma and that matriptase inhibition may contribute to preventing the progression of this devastating disease.

Carbonic anhydrase II-positive pancreatic cells are progenitors for both endocrine and exocrine pancreas after birth.

The regenerative process in the pancreas is of particular interest because diabetes results from an inadequate number of insulin-producing beta cells and pancreatic cancer may arise from the uncontrolled growth of progenitor/stem cells. Continued and substantial growth of islet tissue occurs after birth in rodents and humans, with additional compensatory growth in response to increased demand. In rodents there is clear evidence of pancreatic regeneration after some types of injury, with proliferation of preexisting differentiated cell types accounting for some replacement. Additionally, neogenesis or the budding of new islet cells from pancreatic ducts has been reported, but the existence and identity of a progenitor cell have been debated. We hypothesized that the progenitor cells are duct epithelial cells that after replication undergo a regression to a less differentiated state and then can form new endocrine and exocrine pancreas. To directly test whether ductal cells serve as pancreatic progenitors after birth and give rise to new islets, we generated transgenic mice expressing human carbonic anhydrase II (CAII) promoter: Cre recombinase (Cre) or inducible CreER(TM) to cross with ROSA26 loxP-Stop-loxP LacZ reporter mice. We show that CAII-expressing cells within the pancreas act as progenitors that give rise to both new islets and acini normally after birth and after injury (ductal ligation). This identification of a differentiated pancreatic cell type as an in vivo progenitor of all differentiated pancreatic cell types has implications for a potential expandable source for new islets for replenishment therapy for diabetes.

Pdx-1-driven overexpression of aurora a kinase induces mild ductal dysplasia of pancreatic ducts near islets in transgenic mice.

OBJECTIVES: To further explore the oncogenic activity of Aurora A kinase while attempting to develop a useful mouse model for pancreatic cancer, Aurora A kinase was targeted to pancreatic duodenal homeobox gene-1 (Pdx-1)-positive cells. METHODS: Aurora A kinase overexpression was targeted to mouse pancreas tissues using the Pdx-1 promoter in a transgenic model. The pancreas tissues of 7- to 11-month-old transgenic animals were evaluated for metastatic adenocarcinomas, preinvasive ductal neoplasia, or other histological anomalies. RESULTS: Examination of pancreatic tissue from Pdx-1-Aurora A transgenic mice revealed abnormalities, such as mild islet cell hyperplasia, lymphocytic infiltration, and general dysplasia between ductal/islet cell interfaces. However, most tissues from these transgenic mice were normal. CONCLUSIONS: The overexpression of Aurora A can potentially initiate the development of mild abnormalities in pancreatic tissue; however, neither preinvasive ductal neoplasia nor fully metastatic adenocarcinomas were observed. Combining the Pdx-1-Aurora A transgenic model with other genetic alterations may provide additional insight.

Histone deacetylase inhibitors modify pancreatic cell fate determination and amplify endocrine progenitors.

During pancreas development, transcription factors play critical roles in exocrine and endocrine differentiation. Transcriptional regulation in eukaryotes occurs within chromatin and is influenced by posttranslational histone modifications (e.g., acetylation) involving histone deacetylases (HDACs). Here, we show that HDAC expression and activity are developmentally regulated in the embryonic rat pancreas. We discovered that pancreatic treatment with different HDAC inhibitors (HDACi) modified the timing and determination of pancreatic cell fate. HDACi modified the exocrine lineage via abolition and enhancement of acinar and ductal differentiation, respectively. Importantly, HDACi treatment promoted the NGN3 proendocrine lineage, leading to an increased pool of endocrine progenitors and modified endocrine subtype lineage choices. Interestingly, treatments with trichostatin A and sodium butyrate, two inhibitors of both class I and class II HDACs, enhanced the pool of beta cells. These results highlight the roles of HDACs at key points in exocrine and endocrine differentiation. They show the powerful use of HDACi to switch pancreatic cell determination and amplify specific cellular subtypes, with potential applications in cell replacement therapies in diabetes.

NBT-PABA test to assess efficiency and kinetics of substituted proteolytic enzyme action in pancreatic duct ligated minipigs.

The NBT-PABA test is an established method for diagnosis of pancreatic exocrine insufficiency. In the present study the NBT-PABA test was used to test and compare the efficacy of two multienzyme preparations (product A and B) differing in galenic preparation in minipigs in which pancreatic exocrine insufficiency (PEI) was induced by pancreatic duct ligation. Without enzyme substitution no distinct increase in PABA was found in blood after oral administration of NBT-PABA. Administration of both enzyme preparations led to a clear dose dependent rise in PABA-concentrations in blood. Interestingly, the two preparations showed different time curves of serum PABA concentration, indicating differences in the kinetic of proteolytic enzyme action. It is concluded that the NBT-PABA test can be a very useful test for indirectly evaluating proteolytic enzyme efficacy in vivo, and also gives information about the kinetics of enzyme action, not only the end-result of enzyme action (like digestibility trials which were used traditionally). A single test is performed in a few hours and there is no need for fistulated animals.

Activation of phosphatidylinositol-3 kinase regulates pancreatic duodenal homeobox-1 in duct cells during pancreatic regeneration.

OBJECTIVE: The purpose of our study was to determine whether the phosphatidylinositol 3-kinase (PI3K)/Akt pathway contributes to expression of pancreatic duodenal homeobox-1 (PDX-1) in duct cells and the cell differentiation during pancreatic regeneration. METHODS: The role of PI3K in PDX-1 expression and duct cell differentiation with pancreatic regeneration in mice after partial pancreatectomy (Px) was examined using either wortmannin, a pharmacological PI3K inhibitor, or small-interfering RNA directed to the p85alpha regulatory subunit of PI3K. Akt phosphorylation, a marker of PI3K activation, and PDX-1 expression were assessed by Western blot analysis and immunohistochemistry. RESULTS: Both PDX-1 levels and Akt phosphorylation were concomitantly increased in pancreatic ducts after partial Px and, conversely, blocked by treatment with wortmannin or p85alpha small-interfering RNA. Pancreatic duct cell differentiation, as assessed by appearance of insulin-positive cells 3 days after partial Px, was effectively reduced by wortmannin. CONCLUSIONS: The PI3K/Akt activation plays a critical role for both PDX-1 expression and pancreatic duct cell differentiation into insulin-producing cells during pancreatic regeneration.