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     

Parp‐1 deficiency in ES cells promotes invasive and metastatic lesions accompanying induction of trophoblast giant cells during tumorigenesis in uterine environment

Embryonic stem (ES) cells deficient in poly(ADP‐ribose) polymerase‐1 (Parp‐1) develop into teratocarcinomas with the appearance of trophoblast giant cells (TGCs) when injected subcutaneously into nude mice. Because the uterus is one of the original organs in which germ cell tumors develop with induction of trophoblast lineage, here we investigated whether Parp‐1 deficiency in ES cells affects teratocarcinoma formation processes by grafting ES cells into the horns of uteri. Teratocarcinomas developed from both wild‐type (Parp‐1^+/+) and Parp‐1^?/? ES cells. The weights of the tumors derived from Parp‐1^?/? ES cells were lower than those of the tumors derived from Parp‐1^+/+ ES cells (P < 0.05). The Parp‐1^?/? tumors showed the appearance of TGCs. Notably, organ metastasis to the lung and liver was observed for the Parp‐1^?/? tumors, but not for the Parp‐1^+/+ tumors (P < 0.05). Invasions were more frequently observed with the Parp‐1^?/? tumors compared with the Parp‐1^+/+ tumors (P < 0.05). Since TGCs are known to have invasive properties, the appearance of TGCs may have supported the metastatic process. The present findings suggest that loss of Parp‐1 during teratocarcinoma formation might augment invasive and metastatic properties of the tumors in the uterine environment.     

Poly (ADP‐ribose) polymerase‐1 deficiency does not affect ethylnitrosourea mutagenicity in liver and testis of lacZ transgenic mice

Poly (ADP‐ribose) polymerase‐1 (Parp1) has been implicated in DNA base excision repair, single‐ and double‐strand break repair pathways, as well as in cell death by apoptosis or necrosis. We used Parp1^?/? lacZ plasmid‐based transgenic mice to investigate whether Parp1 deficiency influences the in vivo mutagenic and clastogenic response to the alkylating agent N‐ethyl‐N‐Nitrosourea (ENU) in somatic and germ‐cell tissues. The comparison of the lacZ mutant frequencies (MFs) between Parp1^+/+ and Parp1^?/? mice showed that the ablation of Parp1 does not affect the spontaneous or ENU‐induced MFs in liver and testis. In addition, the spectrum of the ENU‐induced mutations was not dependent on the Parp1 status, given that similar spectra, consisting mostly of point mutations and a small fraction of deletions/insertions, wereobserved in organs of both Parp1^?/? and Parp1^+/+ mice. Sequencing of point mutations revealed a consistent significant increase in A:T → T:A base substitutions, typically induced by ENU. Overall, we observed that neither the frequency nor the spectrum of ENU‐induced mutations demonstrated a specificity that could be attributed to the Parp1 impairment in mice organs. The analysis of micronucleus frequency in peripheral blood reticulocytes showed that ENU was clastogenic in both Parp1^?/? and Parp1^+/+ mice and had a strong cytotoxic effect in Parp1^?/? mice only. The present data suggest that, at a whole‐organism level, Parp1‐independent repair mechanisms may be operative in the removal of ENU‐induced DNA lesions or that highly damaged cells may be preferentially committed to death when Parp1 is inactivated. Environ. Mol. Mutagen. 2010. © 2010 Wiley‐Liss, Inc.     

The epigenetic regulators Bmi1 and Ring1B are differentially regulated in pancreatitis and pancreatic ductal adenocarcinoma

Chronic pancreatitis and pancreatic ductal adenocarcinoma (PDAC) are associated with major changes in cell differentiation. These changes may be at the basis of the increased risk for PDAC among patients with chronic pancreatitis. Polycomb proteins are epigenetic silencers expressed in adult stem cells; up‐regulation of Polycomb proteins has been reported to occur in a variety of solid tumours such as colon and breast cancer. We hypothesized that Polycomb might play a role in preneoplastic states in the pancreas and in tumour development/progression. To test these ideas, we determined the expression of PRC1 complex proteins (Bmi1 and Ring1b) during pancreatic development and in pancreatic tissue from mouse models of disease: acute and chronic pancreatic injury, duct ligation, and in K‐Ras^G12V conditional knock‐in and caerulein‐treated K‐Ras^G12V mice. The study was extended to human pancreatic tissue samples. To obtain mechanistic insights, Bmi1 expression in cells undergoing in vitro exocrine cell metaplasia and the effects of Bmi1 depletion in an acinar cancer cell line were studied. We found that Bmi1 and Ring1B are expressed in pancreatic exocrine precursor cells during early development and in ductal and islet cells—but not acinar cells—in the adult pancreas. Bmi1 expression was induced in acinar cells during acute injury, in acinar–ductal metaplastic lesions, as well as in pancreatic intraepithelial neoplasia (PanIN) and PDAC. In contrast, Ring1B expression was only significantly and persistently up‐regulated in high‐grade PanINs and in PDAC. Bmi1 knockdown in cultured acinar tumour cells led to changes in the expression of various digestive enzymes. Our results suggest that Bmi1 and Ring1B are modulated in pancreatic diseases and could contribute differently to tumour development. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

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.     

Robust immunoglobulin class switch recombination and end joining in Parp9‐deficient mice

To mount highly specific and adapted immune responses, B lymphocytes assemble and diversify their antibody repertoire through mechanisms involving the formation of programmed DNA damage. Immunoglobulin class switch recombination (CSR) is triggered by DNA lesions induced by activation‐induced cytidine deaminase, which are processed to double‐stranded DNA break (DSB) intermediates. These DSBs activate the cellular DNA damage response and enroll numerous DNA repair factors, involving poly(ADP‐ribose) polymerases Parp1, Parp2, and Parp3 to promote appropriate DNA repair and efficient long‐range recombination. The macroParp Parp9, which is overexpressed in certain lymphomas, has been recently implicated in DSB repair, acting together with Parp1. Here, we examine the contribution of Parp9 to the resolution of physiological DSBs incurred during V(D)J recombination and CSR by generating Parp9^?/? mice. We find that Parp9‐ deficient mice are viable, fertile, and do not show any overt phenotype. Moreover, we find that Parp9 is dispensable for B‐cell development. Finally, we show that CSR and DNA end‐joining are robust in the absence of Parp9, indicating that Parp9 is not essential in vivo to achieve physiological DSB repair, or that strong compensatory mechanisms exist.     

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.     

NF‐κB1, NF‐κB2 and c‐Rel differentially regulate susceptibility to colitis‐associated adenoma development in C57BL/6 mice

NF‐κB signalling is an important factor in the development of inflammation‐associated cancers. Mouse models of Helicobacter‐induced gastric cancer and colitis‐associated colorectal cancer have demonstrated that classical NF‐κB signalling is an important regulator of these processes. In the stomach, it has also been demonstrated that signalling involving specific NF‐κB proteins, including NF‐κB1/p50, NF‐κB2/p52, and c‐Rel, differentially regulate the development of gastric pre‐neoplasia. To investigate the effect of NF‐κB subunit loss on colitis‐associated carcinogenesis, we administered azoxymethane followed by pulsed dextran sodium sulphate to C57BL/6, Nfkb1^?/?, Nfkb2^?/?, and c‐Rel^?/?mice. Animals lacking the c‐Rel subunit were more susceptible to colitis‐associated cancer than wild‐type mice, developing 3.5 times more colonic polyps per animal than wild‐type mice. Nfkb2^?/? mice were resistant to colitis‐associated cancer, developing fewer polyps per colon than wild‐type mice (median 1 compared to 4). To investigate the mechanisms underlying these trends, azoxymethane and dextran sodium sulphate were administered separately to mice of each genotype. Nfkb2^?/? mice developed fewer clinical signs of colitis and exhibited less severe colitis and an attenuated cytokine response compared with all other groups following DSS administration. Azoxymethane administration did not fully suppress colonic epithelial mitosis in c‐Rel^?/? mice and less colonic epithelial apoptosis was also observed in this genotype compared to wild‐type counterparts. These observations demonstrate different functions of specific NF‐κB subunits in this model of colitis‐associated carcinogenesis. NF‐κB2/p52 is necessary for the development of colitis, whilst c‐Rel‐mediated signalling regulates colonic epithelial cell turnover following DNA damage. © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Tissue plasminogen activator in murine exocrine pancreas cancer: selective expression in ductal tumors and contribution to cancer progression

Tissue plasminogen activator (tPA) is absent from normal human pancreas and is expressed in 95% of human pancreatic adenocarcinomas. We have analyzed the expression of components of the tPA system in murine pancreatic tumors and the role of tPA in neoplastic progression. Transgenic mice expressing T antigen and c-myc under the control of the elastase promoter (Ela1-TAg and Ela1-myc, respectively) were used. tPA was undetectable in normal pancreas, acinar dysplasia, ductal complexes, and in all acinar tumors. By contrast, it was consistently detected in Ela1-myc tumors showing ductal differentiation. Crossing transgenic Ela1-myc with tPA-/- mice had no effect on the proportion of ductal tumors, indicating that tPA is not involved in the acinar-to-ductal transition. Ela1-myc:tPA-/- mice showed an increased survival in comparison to control mice. All ductal tumors, and none of the acinar tumors, overexpressed the tPA receptor annexin A2, suggesting its participation in the effects mediated by tPA. Our findings indicate that murine and human pancreatic ductal tumors share molecular alterations in the tPA system that may play a role in tumor progression.     

Card9‐dependent IL‐1β regulates IL‐22 production from group 3 innate lymphoid cells and promotes colitis‐associated cancer

Inflammatory bowel diseases (IBD) are key risk factors for the development of colorectal cancer, but the mechanisms that link intestinal inflammation with carcinogenesis are insufficiently understood. Card9 is a myeloid cell‐specific signaling protein that regulates inflammatory responses downstream of various pattern recognition receptors and which cooperates with the inflammasomes for IL‐1β production. Because polymorphisms in Card9 were recurrently associated with human IBD, we investigated the function of Card9 in a colitis‐associated cancer (CAC) model. Card9^?/? mice develop smaller, less proliferative and less dysplastic tumors compared to their littermates and in the regenerating mucosa we detected dramatically impaired IL‐1β generation and defective IL‐1β controlled IL‐22 production from group 3 innate lymphoid cells. Consistent with the key role of immune‐derived IL‐22 in activating STAT3 signaling during normal and pathological intestinal epithelial cell (IEC) proliferation, Card9^?/? mice also exhibit impaired tumor cell intrinsic STAT3 activation. Our results imply a Card9‐controlled, ILC3‐mediated mechanism regulating healthy and malignant IEC proliferation and demonstrates a role of Card9‐mediated innate immunity in inflammation‐associated carcinogenesis.     

Effect of All‐Trans Retinoic Acid on the Pancreas of Streptozotocin‐Induced Diabetic Rat

All‐trans Retinoic acid (atRA) is instructive for the development of endocrine pancreas and is an integral component of β‐cell induction protocols. We showed that atRA induces glucose‐responsive endocrine transdifferentiation of pleomorphic pancreatic ductal adenocarcinoma cells in vitro. This study aimed to detect the role of atRA in improving the histological changes of the pancreas in diabetic rats. Forty young male Wistar rats were used and divided into three groups. Group I: normal vehicle control (N = 5). Group II: streptozotocin‐induced diabetic rats (N = 20) were followed up at 0.0, 1, 2, and 4 weeks. Group III: streptozotocin‐induced diabetic rats (N = 15) treated with atRA (2.5 mg/kg/day), were followed up at 1, 2, and 4 weeks. Specimens from the pancreas were processed for light, electron microscopy and pancreatic insulin mRNA expression. Blood samples were assayed for the levels of glucose, insulin, and total peroxides. In the atRA‐treated group, the number of the islets and the islet area significantly increased. Strong insulin‐immunoreactive endocrine‐like cells were observed nearby the pancreatic acini and the interlobular ducts. Interestingly, insulin‐positive cells seemed to arise from pancreatic acinar and ductal epithelium. Ultrastructurally, ß‐cells, acinar, and ductal cells restored their normal appearance. Pancreatic insulin mRNA and blood indices were almost normalized. AtRA improved the histological changes of the pancreas and the blood indices in diabetic rats. Anat Rec, 299:334–351, 2016. © 2015 Wiley Periodicals, Inc.     

Galectin‐1 is essential for the induction of MOG35–55‐based intravenous tolerance in experimental autoimmune encephalomyelitis

In experimental autoimmune encephalomyelitis (EAE), intravenous (i.v.) injection of the antigen, myelin oligodendrocyte glycoprotein‐derived peptide, MOG_35–55, suppresses disease development, a phenomenon called i.v. tolerance. Galectin‐1, an endogenous glycan‐binding protein, is upregulated during autoimmune neuroinflammation and plays immunoregulatory roles by inducing tolerogenic dendritic cells (DCs) and IL‐10 producing regulatory type 1 T (Tr1) cells. To examine the role of galectin‐1 in i.v. tolerance, we administered MOG_35–55‐i.v. to wild‐type (WT) and galectin‐1 deficient (Lgals1^?/?) mice with ongoing EAE. MOG_35–55 suppressed disease in the WT, but not in the Lgals1^?/? mice. The numbers of Tr1 cells and Treg cells were increased in the CNS and periphery of tolerized WT mice. In contrast, Lgals1^?/? MOG‐i.v. mice had reduced numbers of Tr1 cells and Treg cells in the CNS and periphery, and reduced IL‐27, IL‐10, and TGF‐β1 expression in DCs in the periphery. DCs derived from i.v.‐tolerized WT mice suppressed disease when adoptively transferred into mice with ongoing EAE, whereas DCs from Lgals1^?/? MOG‐i.v. mice were not suppressive. These findings demonstrate that galectin‐1 is required for i.v. tolerance induction, likely via induction of tolerogenic DCs leading to enhanced development of Tr1 cells, Treg cells, and downregulation of proinflammatory responses.     

Expression of key hypoxia sensing prolyl‐hydroxylases PHD1, ‐2 and ‐3 in pancreaticobiliary cancer

Gossage L, Zaitoun A, Fareed K R, Turley H, Aloysius M, Lobo D N, Harris A L & Madhusudan S
(2010) Histopathology 56, 908–920
Expression of key hypoxia sensing prolyl‐hydroxylases PHD1, ‐2 and ‐3 in pancreaticobiliary cancer Aims: Tumour hypoxia is associated with an aggressive phenotype and resistance to chemotherapy and radiotherapy. The aim was to investigate whether key hypoxia sensing prolyl hydroxylases PHD1, PHD2 and PHD3 are dysregulated in pancreaticobiliary cancers, and to evaluate their potential clinical significance. Methods and results: Formalin‐fixed human pancreatic tissue from 120 consecutive patients undergoing pancreatic resections between June 2001 and June 2006 was constructed into tissue microarrays. Expression of PHD1, PHD2 and PHD3 was analysed using immunohistochemistry and correlated with clinicopathological variables and disease‐specific overall survival. PHD1, PHD2 and PHD3 were significantly overexpressed in pancreaticobiliary tumours compared with normal pancreatic ductal tissues (P = 0.03, P < 0.0001 and P < 0.0001, respectively). PHD3 expression in tumour tissue was associated with a trend towards worse overall disease‐specific survival in ampullary adenocarcinomas (P = 0.035) and pancreatic adenocarcinomas (P = 0.084). Absence of PHD1 expression was significantly associated with perineural invasion in pancreatic adenocarcinomas (P = 0.02) and a trend towards significance was also seen for absence of PHD2 expression in pancreatic adenocarcinomas (P = 0.04). Conclusions: Our results provide the first clinical evidence that PHD1, PHD2 and PHD3 may be involved in pancreaticobiliary tumorigenesis.     

Immunisation route‐dependent expression of IL‐4/IL‐13 can modulate HIV‐specific CD8+ CTL avidity

All HIV‐1 ‘systemic vaccine trials’ in humans have yielded poor outcomes. Thus, it is important to understand whether the route of delivery influences the quality of protective CTL immunity. Using heterologous poxvirus immunisation we have shown that systemically (i.m./i.m.) immunised CD8⁺ T cells generated higher levels of IL‐4/IL‐13 compared to mucosal delivery and expression also correlated with i.m./i.m. immunised mice eliciting CTL of lower avidity. Studies using IL‐4^?/? and IL‐13^?/? KO mice have shown that the capacity to express IFN‐γ, IL‐4 and/or IL‐13 by K^dGag_197–205‐specific CTL differed between these groups and was inversely correlated with CTL avidity (IL‐13^?/?>IL‐4^?/?>BALB/c), although no significant differences in the magnitude of CTL responses were observed between IL‐13^?/? and wild type mice. When IL‐13 was reconstituted in IL‐13^?/? splenocytes in vitro, their ability to bind tetramers also decreased significantly. Our data reveal that total absence of IL‐13 can greatly enhance CTL avidity. In contrast, extracellular IL‐4 appears to be important in maintaining long‐term Th1/Th2 balance in CTL, even though expression of IL‐4 by CTL markedly reduced avidity. STAT6^?/? mice also showed memory CTL of higher avidity. Furthermore, CCL5 expression in K^dGag_197–205‐specific CTL was also regulated by IL‐4/IL‐13.