p21WAF1/Cip1 limits senescence and acinar‐to‐ductal metaplasia formation during pancreatitis

Trans‐differentiation of pancreatic acinar cells into ductal‐like lesions, a process defined as acinar‐to‐ductal metaplasia (ADM), is observed in the course of organ regeneration following pancreatitis. In addition, ADM is found in association with pre‐malignant PanIN lesions and correlates with an increased risk of pancreatic adenocarcinoma (PDAC). Human PDAC samples show down‐regulation of p21^WAF1/Cip1, a key regulator of cell cycle and cell differentiation. Here we investigated whether p21 down‐regulation is implicated in controlling the early events of acinar cell trans‐differentiation and ADM formation. p21‐mediated regulation of ADM formation and regression was analysed in vivo during the course of cerulein‐induced pancreatitis, using wild‐type (WT) and p21‐deficient (p21^?/?) mice. Biochemical and immunohistochemical methods were used to evaluate disease progression over 2 weeks of the disease and during a recovery phase. We found that p21 was strongly up‐regulated in WT acinar cells during pancreatitis, while it was absent in ADM areas, suggesting that p21 down‐regulation is associated with ADM formation. In support of this hypothesis, p21^?/? mice showed a significant increase in number and size of metaplasia. In addition, p21 over‐expression in acinar cells reduced ADM formation in vitro, suggesting that the protein regulates the metaplastic transition in a cell‐autonomous manner. p21^?/? mice displayed increased expression and relocalization of β‐catenin both during pancreatitis and in the subsequent recovery phase. Finally, loss of p21 was accompanied by increased DNA damage and development of senescence. Our findings are consistent with a gate‐keeper role of p21 in acinar cells to limit senescence activation and ADM formation during pancreatic regeneration. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd     

The cellular origins of cancer with particular reference to the gastrointestinal tract

Stem cells or their closely related committed progenitor cells are the likely founder cells of most neoplasms. In the continually renewing and hierarchically organized epithelia of the oesophagus, stomach and intestine, homeostatic stem cells are located at the beginning of the cell flux, in the basal layer of the oesophagus, the isthmic region of gastric oxyntic glands and at the bottom of gastric pyloric‐antral glands and colonic crypts. The introduction of mutant oncogenes such as Kras^G12D or loss of Tp53 or Apc to specific cell types expressing the likes of Lgr5 and Mist1 can be readily accomplished in genetically engineered mouse models to initiate tumorigenesis. Other origins of cancer are discussed including ‘reserve’ stem cells that may be activated by damage or through disruption of morphogen gradients along the crypt axis. In the liver and pancreas, with little cell turnover and no obvious stem cell markers, the importance of regenerative hyperplasia associated with chronic inflammation to tumour initiation is vividly apparent, though inflammatory conditions in the renewing populations are also permissive for tumour induction. In the liver, hepatocytes, biliary epithelial cells and hepatic progenitor cells are embryologically related, and all can give rise to hepatocellular carcinoma and cholangiocarcinoma. In the exocrine pancreas, both acinar and ductal cells can give rise to pancreatic ductal adenocarcinoma (PDAC), although the preceding preneoplastic states are quite different: acinar‐ductal metaplasia gives rise to pancreatic intraepithelial neoplasia culminating in PDAC, while ducts give rise to PDAC via. mucinous cell metaplasia that may have a polyclonal origin.     

Decreased expression and promoter methylation of the menin tumor suppressor in pancreatic ductal adenocarcinoma

Loss of menin, a tumor suppressor coded by the MEN1 gene, is a key factor in the pathogenesis of multiple endocrine neoplasia type I and in a percentage of sporadic endocrine tumors of the pancreas and parathyroid glands. This study investigated expression of the menin protein in the normal exocrine pancreas and in pancreatic ductal adenocarcinoma (PDAC), the most common pancreatic tumor. Immunofluorescence (IF) analyses showed that menin is expressed at high levels in normal acinar and duct cells. Examination of 24 clinical samples of PDAC revealed a pronounced decrease in menin expression in all tumors examined. To identify alterations underlying this defect, we searched for disruption and epigenetic silencing of the MEN1 gene. Analysis of nine laser‐microdissected tumors revealed loss of heterozygosity of intragenic (one tumor) or adjacent (three tumors) MEN1 microsatellite markers. Methylation of CpG sites in the MEN1 promoter was documented in five of 24 tumors. IF analyses also revealed low to undetectable menin expression in the PDAC cell lines MiaPaCa‐2 and Panc‐1. Ectopic expression of menin in these cells resulted in a marked alteration of the cell cycle, with an increase in the G1/S+G2 ratio. These findings represent the first evidence that the MEN1 gene is a target of mutation and methylation in PDAC and that menin influences the cell cycle profile of duct cells. © 2009 Wiley‐Liss,Inc.     

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 Kras^G12D 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.     

UHRF1 regulation of the Keap1–Nrf2 pathway in pancreatic cancer contributes to oncogenesis

The cellular defence protein Nrf2 is a mediator of oncogenesis in pancreatic ductal adenocarcinoma (PDAC) and other cancers. However, the control of Nrf2 expression and activity in cancer is not fully understood. We previously reported the absence of Keap1, a pivotal regulator of Nrf2, in ～70% of PDAC cases. Here we describe a novel mechanism whereby the epigenetic regulator UHRF1 suppresses Keap1 protein levels. UHRF1 expression was observed in 20% (5 of 25) of benign pancreatic ducts compared to 86% (114 of 132) of pancreatic tumours, and an inverse relationship between UHRF1 and Keap1 levels in PDAC tumours (n = 124) was apparent (p = 0.002). We also provide evidence that UHRF1‐mediated regulation of the Nrf2 pathway contributes to the aggressive behaviour of PDAC. Depletion of UHRF1 from PDAC cells decreased growth and enhanced apoptosis and cell cycle arrest. UHRF1 depletion also led to reduced levels of Nrf2‐regulated downstream proteins and was accompanied by heightened oxidative stress, in the form of lower glutathione levels and increased reactive oxygen species. Concomitant depletion of Keap1 and UHRF1 restored Nrf2 levels and reversed cell cycle arrest and the increase in reactive oxygen species. Mechanistically, depletion of UHRF1 reduced global and tumour suppressor promoter methylation in pancreatic cancer cell lines, and KEAP1 gene promoter methylation was reduced in one of three cell lines examined. Thus, methylation of the KEAP1 gene promoter may contribute to the suppression of Keap1 protein levels by UHRF1, although our data suggest that additional mechanisms need to be explored. Finally, we demonstrate that K‐Ras drives UHRF1 expression, establishing a novel link between this oncogene and Nrf2‐mediated cellular protection. Since UHRF1 over‐expression occurs in other cancers, its ability to regulate the Keap1–Nrf2 pathway may be critically important to the malignant behaviour of these cancers. © 2015 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Anti‐stromal treatment together with chemotherapy targets multiple signalling pathways in pancreatic adenocarcinoma

Stromal targeting for pancreatic ductal adenocarcinoma (PDAC) is rapidly becoming an attractive option, due to the lack of efficacy of standard chemotherapy and increased knowledge about PDAC stroma. We postulated that the addition of stromal therapy may enhance the anti‐tumour efficacy of chemotherapy. Gemcitabine and all‐trans retinoic acid (ATRA) were combined in a clinically applicable regimen, to target cancer cells and pancreatic stellate cells (PSCs) respectively, in 3D organotypic culture models and genetically engineered mice (LSL‐Kras^G12D/+;LSL‐Trp53^R172H/+;Pdx‐1‐Cre: KPC mice) representing the spectrum of PDAC. In two distinct sets of organotypic models as well as KPC mice, we demonstrate a reduction in cancer cell proliferation and invasion together with enhanced cancer cell apoptosis when ATRA is combined with gemcitabine, compared to vehicle or either agent alone. Simultaneously, PSC activity (as measured by deposition of extracellular matrix proteins such as collagen and fibronectin) and PSC invasive ability were both diminished in response to combination therapy. These effects were mediated through a range of signalling cascades (Wnt, hedgehog, retinoid, and FGF) in cancer as well as stellate cells, affecting epithelial cellular functions such as epithelial–mesenchymal transition, cellular polarity, and lumen formation. At the tissue level, this resulted in enhanced tumour necrosis, increased vascularity, and diminished hypoxia. Consequently, there was an overall reduction in tumour size. The enhanced effect of stromal co‐targeting (ATRA) alongside chemotherapy (gemcitabine) appears to be mediated by dampening multiple signalling cascades in the tumour–stroma cross‐talk, rather than ablating stroma or targeting a single pathway. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Expression of urocortin in pancreatic ductal adenocarcinoma and pancreatic intraepithelial neoplasia

Urocortin (UCN) is a 40‐aminoacid neuropeptide that regulates angiogenesis and inhibits cell proliferation. Our aim was to examine the relationship of UCN expression to the clinicopathological parameters of pancreatic ductal adenocarcinoma (PDAC) and histological grade of pancreatic intraepithelial neoplasia (PanIN). Tissue microarray was used to analyze UCN protein expression in 89 surgical specimens including 21 PanIN, 3 PDAC arising from PanIN, and 65 PDAC without PanIN. UCN immunoscores ranging from 0 to 12 were obtained by multiplying intensity (scored on a 3‐point scale) by the percentage of stained cells (scored on a 4‐point scale). Strong expression of UCN was detected in 5 specimens of non‐neoplastic pancreatic ductal epithelia. UCN immunoscore was significantly higher in PanIN‐1 than in PanIN‐2 and PanIN‐3 (p = 0.038) and significantly higher in well‐differentiated PDAC or early American Joint Committee on Cancer (AJCC) stage PDAC than in poorly differentiated or advanced stage PDAC (p = 0.025, p = 0.018). Higher expression of UCN correlates with PDAC tumor grade and AJCC pathologic stage as well as PanIN grade. Immunohistochemical assessment of UCN may help clinicians predict tumor recurrence rate and help pathologists make a proper diagnosis.     

Serotonin promotes acinar dedifferentiation following pancreatitis‐induced regeneration in the adult pancreas

The exocrine pancreas exhibits a distinctive capacity for tissue regeneration and renewal following injury. This regenerative ability has important implications for a variety of disorders, including pancreatitis and pancreatic cancer, diseases associated with high morbidity and mortality. Thus, understanding its underlying mechanisms may help in developing therapeutic interventions. Serotonin has been recognized as a potent mitogen for a variety of cells and tissues. Here we investigated whether serotonin exerts a mitogenic effect in pancreatic acinar cells in three regenerative models, inflammatory tissue injury following pancreatitis, tissue loss following partial pancreatectomy, and thyroid hormone‐stimulated acinar proliferation. Genetic and pharmacological techniques were used to modulate serotonin levels in vivo. Acinar dedifferentiation and cell cycle progression during the regenerative phase were investigated over the course of 2 weeks. By comparing acinar proliferation in the different murine models of regeneration, we found that serotonin did not affect the clonal regeneration of mature acinar cells. Serotonin was, however, required for acinar dedifferentiation following inflammation‐mediated tissue injury. Specifically, lack of serotonin resulted in delayed up‐regulation of progenitor genes and delayed the formation of acinar‐to‐ductal metaplasia and defective acinar cell proliferation. We identified serotonin‐dependent acinar secretion as a key step in progenitor‐based regeneration, as it promoted acinar cell dedifferentiation and the recruitment of type 2 macrophages. Finally, we identified a regulatory Hes1–Ptfa axis in the uninjured adult pancreas, activated by zymogen secretion. Our findings indicated that serotonin plays a critical role in the regeneration of the adult pancreas following pancreatitis by promoting the dedifferentiation of acinar cells. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Pancreatic cell plasticity and cancer initiation induced by oncogenic Kras is completely dependent on wild-type PI 3-kinase p110α

Increased PI 3-kinase (PI3K) signaling in pancreatic ductal adenocarcinoma (PDAC) correlates with poor prognosis, but the role of class I PI3K isoforms during its induction remains unclear. Using genetically engineered mice and pharmacological isoform-selective inhibitors, we found that the p110α PI3K isoform is a major signaling enzyme for PDAC development induced by a combination of genetic and nongenetic factors. Inactivation of this single isoform blocked the irreversible transition of exocrine acinar cells into pancreatic preneoplastic ductal lesions by oncogenic Kras and/or pancreatic injury. Hitting the other ubiquitous isoform, p110β, did not prevent preneoplastic lesion initiation. p110α signaling through small GTPase Rho and actin cytoskeleton controls the reprogramming of acinar cells and regulates cell morphology in vivo and in vitro. Finally, p110α was necessary for pancreatic ductal cancers to arise from Kras-induced preneoplastic lesions by increasing epithelial cell proliferation in the context of mutated p53. Here we identify an in vivo context in which p110α cellular output differs depending on the epithelial transformation stage and demonstrate that the PI3K p110α is required for PDAC induced by oncogenic Kras, the key driver mutation of PDAC. These data are critical for a better understanding of the development of this lethal disease that is currently without efficient treatment.     

Expression patterns of epiplakin1 in pancreas, pancreatic cancer and regenerating pancreas

Epiplakin1 (Eppk1) is a plakin family gene with its function remains largely unknown, although the plakin genes are known to function in interconnecting cytoskeletal filaments and anchoring them at plasma membrane-associated adhesive junction. Here we analyzed the expression patterns of Eppk1 in the developing and adult pancreas in the mice. In the embryonic pancreas, Eppk1+/Pdx1+ and Eppk1+/Sox9+ pancreatic progenitor cells were observed in early pancreatic epithelium. Since Pdx1 expression overlapped with that of Sox9 at this stage, these multipotent progenitor cells are Eppk1+/Pdx1+/Sox9+ cells. Then Eppk1 expression becomes confined to Ngn3+ or Sox9+ endocrine progenitor cells, and p48+ exocrine progenitor cells, and then restricted to the duct cells and a cells at birth. In the adult pancreas, Eppk1 is expressed in centroacinar cells (CACs) and in duct cells. Eppk1 is observed in pancreatic intraepithelial neoplasia (PanIN), previously identified as pancreatic ductal adenocarcinoma (PDAC) precursor lesions. In addition, the expansion of Eppk1-positive cells occurs in a caerulein-induced acute pancreatitis, an acinar cell regeneration model. Furthermore, in the partial pancreatectomy (Px) regeneration model using mice, Eppk1 is expressed in "ducts in foci", a tubular structure transiently induced. These results suggest that Eppk1 serves as a useful marker for detecting pancreatic progenitor cells in developing and regenerating pancreas.     

Meflin is a marker of pancreatic stellate cells involved in fibrosis and epithelial regeneration in the pancreas

Pancreatic stellate cells (PSCs) are stromal cells in the pancreas that play an important role in pancreatic pathology. In chronic pancreatitis (CP) and pancreatic ductal adenocarcinoma (PDAC), PSCs are known to get activated to form myofibroblasts or cancer-associated fibroblasts (CAFs) that promote stromal fibroinflammatory reactions. However, previous studies on PSCs were mainly based on the findings obtained using ex vivo expanded PSCs, with few studies that addressed the significance of in situ tissue-resident PSCs using animal models. Their contributions to fibrotic reactions in CP and PDAC are also lesser-known. These limitations in our understanding of PSC biology have been attributed to the lack of specific molecular markers of PSCs. Herein, we established Meflin (Islr), a glycosylphosphatidylinositol-anchored membrane protein, as a PSC-specific marker in both mouse and human by using human pancreatic tissue samples and Meflin reporter mice. Meflin-positive (Meflin⁺) cells contain lipid droplets and express the conventional PSC marker Desmin in normal mouse pancreas, with some cells also positive for Gli1, the marker of pancreatic tissue-resident fibroblasts. Three-dimensional analysis of the cleared pancreas of Meflin reporter mice showed that Meflin⁺ PSCs have long and thin cytoplasmic protrusions, and are localised on the abluminal side of vessels in the normal pancreas. Lineage tracing experiments revealed that Meflin⁺ PSCs constitute one of the origins of fibroblasts and CAFs in CP and PDAC, respectively. In these diseases, Meflin⁺ PSC-derived fibroblasts showed a distinctive morphology and distribution from Meflin⁺ PSCs in the normal pancreas. Furthermore, we showed that the genetic depletion of Meflin⁺ PSCs accelerated fibrosis and attenuated epithelial regeneration and stromal R-spondin 3 expression, thereby implying that Meflin⁺ PSCs and their lineage cells may support tissue recovery and Wnt/R-spondin signalling after pancreatic injury and PDAC development. Together, these data indicate that Meflin may be a marker specific to tissue-resident PSCs and useful for studying their biology in both health and disease. © 2023 The Pathological Society of Great Britain and Ireland.     

Mesenchymal–epithelial transition of pancreatic cancer cells at perineural invasion sites is induced by Schwann cells

Epithelial–mesenchymal transition (EMT) promotes invasion and metastasis of pancreatic ductal adenocarcinoma (PDAC). However, the importance of its reverse process, mesenchymal–epithelial transition (MET), for PDAC remains unclear. We aimed to characterize the histological finding “focal differentiation” in PDAC at perineural invasion sites in the context of MET and to investigate the role of Schwann cells in inducing tumor MET. Tumor differentiation and immunohistochemical expressions of E‐cadherin, SMAD3, and vimentin at perineural invasion sites were examined in 168 PDAC tissues. Four PDAC cell lines were co‐cultured with Schwann cells to investigate cell morphology, motility, or EMT‐related markers using immunocytochemistry and quantitative PCR. Of 168 tumors, 124 (74%) showed focal differentiation with enhanced E‐cadherin membrane expression (P < 0.001) and decreased nuclear accumulation of SMAD3 (P < 0.001). Among 115 PDACs harboring grade 1/2 tumor, tumors with focal differentiation showed worse survival compared to those without focal differentiation (P = 0.019). PDAC cells co‐cultured with Schwann cells demonstrated a sheet‐like appearance, increased E‐cadherin expression, decreased expressions of SMAD3 and vimentin, and reduced cell motility. In conclusion, MET‐like change is induced by Schwann cells, suggesting that Schwann cells contribute to PDAC colonization in pancreatic nerves through activating the MET machinery inside tumor cells in the pancreatic tumor microenvironment.