Ductal Metaplasia Research Articles

ANGPTL4 accelerates KRASG12D-Induced acinar to ductal metaplasia and pancreatic carcinogenesis.

Oncogenic KRASG12D induces neoplastic transformation of pancreatic acinar cells through acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN), and drives pancreatic ductal adenocarcinoma (PDAC). Angiopoietin-like 4 (ANGPTL4) is known to be involved in the regulation of cancer growth and metastasis. However, whether ANGPTL4 affects KRASG12D-mediated ADM and early PDAC intervention remains unknown. In the current study, we investigated the role of ANGPTL4 in KRASG12D-induced ADM, PanIN formation, and PDAC maintenance. We found that ANGPTL4 was highly expressed in human and mouse ADM lesions and contributed to the promotion of KRASG12D-driven ADM in mice. Consistently, ANGPTL4 rapidly induced ADM in three-dimensional culture of acinar cells with KRAS mutation and formed ductal cysts that silenced acinar genes and activated ductal genes, which are characteristic of in vivo ADM/PanIN lesions. We also found that periostin works as a downstream regulator of ANGPTL4-mediated ADM/PDAC. Genetic ablation of periostin diminished the ADM/PanIN phenotype induced by ANGPTL4. A high correlation between ANGPTL4 and periostin was confirmed in human samples. These results demonstrate that ANGPTL4 is critical for ADM/PanIN initiation and PDAC progression through the regulation of periostin. Thus, the ANGPTL4/periostin axis is considered a potential target for ADM-derived PDAC.

Omega-Class Glutathione Transferases of Carcinogenic Liver Fluke, Clonorchis sinensis, Modulate Apoptosis and Differentiation of Host Cholangiocytes.

The small liver fluke Clonorchis sinensis causes hepatobiliary ductal infections in humans. Clonorchiasis is characterized histopathologically by ductal dysplasia, hyperplasia and metaplasia, which closely resembles cholangiocarcinoma (CCA). The disruption of programmed cell death is critical for malignant transformation, while molecular events underlying these phenomena have poorly been understood in clonorchiasis-related CCA tumorigenesis. We incorporated recombinant C. sinensis omega-class glutathione transferase (rCsGSTo) 1 or 2 into human intrahepatic biliary epithelial cells (HIBECs) and analyzed pathophysiological alterations of HIBECs upon the application of oxidative stress. rCsGSTos partially but significantly rescued HIBECs from cell death by inhibiting oxidative stress-induced apoptosis (p < 0.01). rCsGSTos modulated transcriptional levels of numerous genes. We analyzed 13 genes involved in programmed cell death (the upregulation of five antiapoptotic and two apoptotic genes, and the downregulation of one antiapoptotic and five apoptotic genes) and 11 genes associated with cell differentiation (the increase in seven and decrease in four genes) that showed significant modifications (p < 0.05). The induction profiles of the mRNA and proteins of these differentially regulated genes correlated well with each other, and mostly favored apoptotic suppression and/or cell differentiation. We detected increased active, phosphorylated forms of Src, PI3K/Akt, NF-κB p65, MKK3/6 and p38 MAPK, but not JNK and ERK1/2. CsGSTos were localized in the C. sinensis-infected rat cholangiocytes, where cytokeratin 19 was distributed. Our results demonstrated that CsGSTos excreted to the biliary lumen are internalized and accumulated in the host cholangiocytes. When cholangiocytes underwent oxidative stressful condition, CsGSTos appeared to be critically involved in both antiapoptotic process and the differentiation of host cholangiocytes through the regulation of target genes following the activation of responsible signal molecules.

REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway

Persistent acinar to ductal metaplasia (ADM) is a recently recognized precursor of pancreatic ductal adenocarcinoma (PDAC). Here we show that the ADM area of human pancreas tissue adjacent to PDAC expresses significantly higher levels of regenerating protein 3A (REG3A). Exogenous REG3A and its mouse homolog REG3B induce ADM in the 3D culture of primary human and murine acinar cells, respectively. Both Reg3b transgenic mice and REG3B-treated mice with caerulein-induced pancreatitis develop and sustain ADM. Two out of five Reg3b transgenic mice with caerulein-induced pancreatitis show progression from ADM to pancreatic intraepithelial neoplasia (PanIN). Both in vitro and in vivo ADM models demonstrate activation of the RAS-RAF-MEK-ERK signaling pathway. Exostosin-like glycosyltransferase 3 (EXTL3) functions as the receptor for REG3B and mediates the activation of downstream signaling proteins. Our data indicates that REG3A/REG3B promotes persistent ADM through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway. Targeting REG3A/REG3B, its receptor EXTL3, or other downstream molecules could interrupt the ADM process and prevent early PDAC carcinogenesis.

264 METAPLASIA-INDUCED EPITHELIAL HETEROGENEITY DIRECTS PANCREATIC TISSUE INJURY AND RECOVERY

Background and Aims Despite years of research, mechanisms of pancreatic injury and healing remain poorly understood. Acute pancreatitis is a painful and debilitating condition; chronic pancreatitis may be asymptomatic, but greatly enhances the risk for development of pancreatic ductal adenocarcinoma (PDAC). Acinar to ductal metaplasia (ADM), or the transdifferentiation of digestive enzyme producing acinar cells to ductal cells, is an early event in both conditions. While ADM is thought to function in healing and regeneration under conditions of injury, it also represents a possible first step in tumorigenesis, demonstrating the duplicitous nature of this inherent plasticity. The aims of this study were to identify ADM-derived populations in pancreatic injury and to characterize their role in disease progression. Methods We performed lineage tracing and single cell RNA sequencing (scRNAseq) on injury-induced pancreatic ADM using acinar cell-specific Ptf1aCreERTM/+;RosaYFP/+(CY) mice. We used the bioinformatics techniques Monocle 2, Pseudotime, and p-Creode to assess lineage progression. Results were confirmed using lineage tracing with Ptf1aCreERTM/+;RosaBrainbow2/+ (CBbow) mice. Populations identified by scRNA-seq were confirmed using multiplex immunofluorescence, immunohistochemistry (IHC), and electron microscopy. The role of ADM in pancreatic injury was tested using Aclyfl/fl;Ptf1aCre/+mice and we examined the role of ADM-derived tuft cells in pancreatic injury using Pou2f3fl/fl;Ptf1aCre/+(tuft cell knockout) mice. Weconducted IHC to evaluate the formation of ADMpopulations discovered in mice, in human disease. Results Using lineage tracing and 10x scRNA-seq we identified and characterized over 15,000 single ADM cells from chronically injured CY pancreata. Using known markers, we discovered that ADM results in the formation of significant epithelial heterogeneity, including the formation of mucin-producing cells, tuft cells, and various subtypes of enteroendocrine cells. Monocle 2, Pseudotime, and p-Creode all agree that acinar cells pass through a mucinous (gastric spasmolytic polypeptide expressing metaplasia-like) intermediate before forming either tuft or enteroendocrine cells. Lineage tracing with CBbow demonstrates that ADM cells have the ability to clonally expand. Pancreatic injury in either ADM-impaired Aclyfl/fl;Ptf1aCre/+mice, or tuft cell knockout Pou2f3fl/fl;Ptf1aCre/+mice results in significantly more injury as compared to wild type littermates. Conclusions The carefully orchestrated plasticity in ADM generates myriad epithelial cell types with the ability to mitigate injury, providing protection from the formation of pancreatitis and PDAC. By uncovering the inherent disease suppressive mechanisms of ADM-derived cell types, we can evaluate the therapeutic benefit of co-opting or targeting these pathways in patients. Download : Download high-res image (265KB) Download : Download full-size image

Abstract PO-092: Development of an organoid assay for studying racial disparity in pancreatic acinar ductal metaplasia

Abstract Blacks have higher rates of incidence and mortality of pancreatic cancer compared to Whites and non-White Hispanics. In an effort to identify a biological basis for this disparity, we propose examining the early events in the development of pancreatic cancer. The process by which pancreatic acini transdifferentiate into ductal epithelial cells (i.e. acinar ductal metaplasia or ADM) is one of the earliest events in the development of pancreatic ductal adenocarcinoma. We adapted the classic in vitro ADM assay, by which primary mouse pancreatic acini undergo ADM once plated onto the extracellular matrix matrigel, to an assay to culture and transdifferentiate human pancreatic organoids. We have received normal, human pancreatic acini from pancreatic islet transplantation centers from Black (n=8), White (n=19) and Hispanic (n=16) donors. The gender distribution was 29 males and 14 females. The acinar cells were cultured on matrigel and allowed to undergo ADM over a 5 to 6 day period. The number of ductal epithelial cells were microscopically counted and the rate of transdifferentiation was determined by kinetic modeling of the data. We have successfully cultured and transdifferentiated 40 of 43 human pancreatic acinar cells. Data from our study suggests that females undergo ADM at a faster rate than males and among the different races, the rate of acinar transdifferentiation decreases in the following order Blacks &amp;gt; Whites &amp;gt; non-White Hispanics. In summary, we demonstrate that women and Blacks have the highest rate of pancreatic transdifferentiation. Future studies include sequencing of the RNA isolated from the tissues to identify molecular drivers of acinar transdifferentiation and their impact on health disparities of pancreatic cancer. Citation Format: Alyssa Gosling, Jinmai Jiang, Corey M. Perkins, Yingwei Yao, Thomas D. Schmittgen. Development of an organoid assay for studying racial disparity in pancreatic acinar ductal metaplasia [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-092.

Delayed Diagnosis: Breast Malignancy Camouflaged by Hamartoma

Breast hamartomas are rare benign masses composed of glandular, adipose and fibrous tissue. While it is not uncommon to see ductal hyperplasia, apocrine metaplasia or adenosis in hamartomas, rare case reports of coexistent lobular or ductal intraepithelial neoplasms and in situ carcinomas have been reported in these lesions. Imaging with ultrasound and mammography was undertaken for a patient who presented with complaints of right breast pain. Right breast malignancy was detected on mammography, and guided biopsy using USG was undertaken from the same lesion. Triple-positive right breast malignancy was detected. This case highlighted the rare occurrence of a malignancy coexisting with a fairly large breast hamartoma.

Tuft Cells Inhibit Pancreatic Tumorigenesis in Mice by Producing Prostaglandin D2

Development of pancreatic ductal adenocarcinoma (PDA) involves acinar to ductal metaplasia and genesis of tuft cells. It has been a challenge to study these rare cells because of the lack of animal models. We investigated the role of tuft cells in pancreatic tumorigenesis. We performed studies with LSL-KrasG12D/+;Ptf1aCre/+ mice (KC; develop pancreatic tumors), KC mice crossed with mice with pancreatic disruption of Pou2f3 (KPouC mice; do not develop tuft cells), or mice with pancreatic disruption of the hematopoietic prostaglandin D synthase gene (Hpgds, KHC mice) and wild-type mice. Mice were allowed to age or were given caerulein to induce pancreatitis; pancreata were collected and analyzed by histology, immunohistochemistry, RNA sequencing, ultrastructural microscopy, and metabolic profiling. We performed laser-capture dissection and RNA-sequencing analysis of pancreatic tissues from 26 patients with pancreatic intraepithelial neoplasia (PanIN), 19 patients with intraductal papillary mucinous neoplasms (IPMNs), and 197 patients with PDA. Pancreata from KC mice had increased formation of tuft cells and higher levels of prostaglandin D2 than wild-type mice. Pancreas-specific deletion of POU2F3 in KC mice (KPouC mice) resulted in a loss of tuft cells and accelerated tumorigenesis. KPouC mice had increased fibrosis and activation of immune cells after administration of caerulein. Pancreata from KPouC and KHC mice had significantly lower levels of prostaglandin D2, compared with KC mice, and significantly increased numbers of PanINs and PDAs. KPouC and KHC mice had increased pancreatic injury after administration of caerulein, significantly less normal tissue, more extracellular matrix deposition, and higher PanIN grade than KC mice. Human PanIN and intraductal papillary mucinous neoplasm had gene expression signatures associated with tuft cells and increased expression of Hpgds messenger RNA compared with PDA. In mice with KRAS-induced pancreatic tumorigenesis, loss of tuft cells accelerates tumorigenesis and increases the severity of caerulein-induced pancreatic injury, via decreased production of prostaglandin D2. These data are consistent with the hypothesis that tuft cells are a metaplasia-induced tumor attenuating cell type.

\u03baB-Ras and Ral GTPases regulate acinar to ductal metaplasia during pancreatic adenocarcinoma development and pancreatitis

Pancreatic ductal adenocarcinoma (PDAC) is associated with high mortality and therapy resistance. Here, we show that low expression of κB-Ras GTPases is frequently detected in PDAC and correlates with higher histologic grade. In a model of KRasG12D-driven PDAC, loss of κB-Ras accelerates tumour development and shortens median survival. κB-Ras deficiency promotes acinar-to-ductal metaplasia (ADM) during tumour initiation as well as tumour progression through intrinsic effects on proliferation and invasion. κB-Ras proteins are also required for acinar regeneration after pancreatitis, demonstrating a general role in control of plasticity. Molecularly, upregulation of Ral GTPase activity and Sox9 expression underlies the observed phenotypes, identifying a previously unrecognized function of Ral signalling in ADM. Our results provide evidence for a tumour suppressive role of κB-Ras proteins and highlight low κB-Ras levels and consequent loss of Ral control as risk factors, thus emphasizing the necessity for therapeutic options that allow interference with Ral-driven signalling.

Epigenetic silencing of AATK in acinar to ductal metaplasia in murine model of pancreatic cancer

BackgroundCancer subtype switching, which involves unclear cancer cell origin, cell fate decision, and transdifferentiation of cells within a confined tumor microenvironment, remains a major problem in pancreatic cancer (PDA).ResultsBy analyzing PDA subtypes in The Cancer Genome Atlas, we identified that epigenetic silencing of apoptosis-associated tyrosine kinase (AATK) inversely was correlated with mRNA expression and was enriched in the quasi-mesenchymal cancer subtype. By comparing early mouse pancreatic lesions, the non-invasive regions showed AATK co-expression in cells with acinar-to-ductal metaplasia, nuclear VAV1 localization, and cell cycle suppression; but the invasive lesions conversely revealed diminished AATK expression in those with poorly differentiated histology, cytosolic VAV1 localization, and co-expression of p63 and HNF1α. Transiently activated AATK initiates acinar differentiation into a ductal cell fate to establish apical-basal polarization in acinar-to-ductal metaplasia. Silenced AATK and ectopically expressed p63 and HNF1α allow the proliferation of ductal PanINs in mice.ConclusionEpigenetic silencing of AATK regulates the cellular transdifferentiation, proliferation, and cell cycle progression in converting PDA-subtypes.

Abstract D112: Development of an organoid assay for studying racial disparity in pancreatic acinar ductal metaplasia

Abstract Blacks have a higher rate of incidence and mortality of pancreatic cancer compared to whites. In an effort to identify a biological basis for this disparity, we propose examining the early events in the development of pancreatic cancer. The process by which pancreatic acini transdifferentiate into ductal epithelial cells (i.e. acinar ductal metaplasia or ADM) is one of the earliest events in the development of pancreatic ductal adenocarcinoma. We adapted the classic in vitro ADM assay, by which primary mouse pancreatic acini undergo ADM once plated onto the extracellular matrix matrigel, to an assay to culture and transdifferentiate human pancreatic organoids. Human pancreatic acini was received from black (n=5), white (n=3) and Hispanic (n=6) donors from pancreatic islet transplantation centers. The gender distribution was 8 males and 6 females. The acinar cells were cultured on matrigel and allowed to undergo ADM over a 5 to 6 day period. The number of ductal epithelial cells were microscopically counted and the rate of transdifferentiation was determined. Using this technique, we successfully cultured and transdifferentiated 14 of 14 human pancreatic acinar cells. Preliminary data from our study suggests that females undergo ADM at a greater rate than males. Data on race was inconclusive due to the low number of samples. In summary, we have developed an assay to culture and study pancreatic ADM using human specimens. Future studies using increased sample size will investigate if racial disparity exists for pancreatic ADM. Citation Format: Julie Bray, Sihem Ait-Oudhia, Martha Campbell-Thompson, Thomas D Schmittgen. Development of an organoid assay for studying racial disparity in pancreatic acinar ductal metaplasia [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr D112.

Abstract A22: Kras drives changes in acinar-specific gene regulatory networks in early pancreatic neoplasia in conjunction with Bmi1

Abstract The initial stages of pancreatic ductal adenocarcinoma (PDA) involve an oncogenic Kras-driven metaplastic transition of acinar cells to a more duct-like phenotype referred to as acinar to ductal metaplasia (ADM). The exact mechanism by which Kras reprograms acinar cells to a more duct-like cell at the genomic level is not completely understood. Developmental transcription factor (TF) networks regulate and maintain the fate of cells in other contexts and rely on appropriate chromatin context for their activity. Deletion of Bmi1, a component of the Polycomb Repressor Complex 1, completely represses Kras-driven ADM and neoplasia in genetically engineered mouse models of PDA. Thus, we hypothesized that Kras reprograms the acinar fate TF network to promote PDA in a Bmi1-dependent epigenetic context. We used a genetically engineered mouse model of early pancreatic neoplasia bearing an acinar cell-specific tamoxifen inducible Cre-recombinase under the elastase promoter (Ela-CreER) combined with the Kras G12D oncogenic allele (Kras−LSL-G12D/+). We added the conditional Bmi1 knockout allele (Bmi1−fl) and the Rosa26 tdTomato reporter to generate Ela-CreER, Kras−LSL-G12D/+, Bmi1−fl/fl, R26 tdTomato mice and their litter mate controls with and without KrasG12D and Bmi1. We activated the Cre-recombinase in 6- to 8-week-old mice with 5 daily tamoxifen gavages (4mg/day) prior to induction of acute pancreatitis by intraperitoneal caerulein administration (eight doses per day for two days) one week after the first tamoxifen dose. We used FACS to sort tdTomato+ cells one week after pancreatitis induction and analyzed RNA expression levels of 26 TFs, previously reported to play a role in pancreatic development, homeostasis and neoplasia. Expression levels of three lineage markers (amylase, elastase and cytokeratin 19) were also measured by quantitative RT-PCR. Tamoxifen gavage induced high tdTomato expression in the acinar compartment of Ela-CreER+ mice. RT-qPCR of tdTomato+ cells indicated a loss of acinar specific TFs in mice expressing mutant Kras one week after induction of pancreatitis. This is consistent with cells undergoing ADM. Genetic ablation of Bmi1 in the presence of mutant Kras restored elastase and amylase expression at the mRNA level. At the morphologic level, loss of Bmi1 also led to recovery of acinar cells from ADM and correlated with an increase in expression of three key acinar-specific fate TFs, Mist1, Hnf1a and Nr5a2. The mRNA expression levels of other acinar TFs like Pdx1 and Ptf1a/p48 did not recover with loss of Bmi1 and mutant Kras expression. Together, our data suggest that oncogenic Kras-driven ADM is controlled by changes in master TF gene regulatory networks. Bmi1 deletion leads to partial reprogramming of these networks to allow acinar cells to resist Kras-driven oncogenesis. Citation Format: Joyce K. Thompson, Jack Blumberg, Osama Alkahili, Howard C. Crawford, Marina Pasca di Magliano, Filip Bednar. Kras drives changes in acinar-specific gene regulatory networks in early pancreatic neoplasia in conjunction with Bmi1 [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A22.

The Role of FAT10 in Alcoholic Hepatitis Pathogenesis

BackgroundMorphologic features of Alcoholic Hepatitis (AH) balloon cells forming Mallory‐Denk bodies and bile duct metaplasia are two morphologic features consistently being found in AH. Increased FAT10 expression may induce increasing mitotic non‐disjunction and chromosome instability leading to tumorigenesis. FAT10 expression is highly up‐regulated by pro‐inflammatory cytokines IFNγ and TNFα in all cell types and tissues.ObjectiveTo clarify the role of FAT10 in alcoholic hepatitis pathogenesis, we studied FAT10 expression in liver biopsy samples from patients with AH.MethodsWe use gene and protein array to detect the potential changes during alcoholic hepatitis pathogenesis. The potential target, FAT10, were tested at protein expression level by using western blot, immunofluorescent (IF) staining and quantitation. And FAT10 was also studied by using knockout animal models.ResultsThe results show that FAT10 is essential to maintain the function of liver cell protein quality control and Mallory‐Denk body (MDB) formation. This is consistent with the finding with fat10−/− mice which fail to form MDBs. FAT10 also signals a switch in the 26s proteasome’s proteases to cause the proteins accumulation, overexpression, and then forming MDBs. FAT10 overexpression in AH causes an extensive change in protein content in the liver leading to balloon degeneration and MDB aggregation formation, all of which is prevented in fat10−/− mice. The pathway that increases FAT10 expression includes TNFα, IFNγ, followed by NFκB and STAT3, which were all up‐regulated in AH. Using proteins expression quantitation by measuring the intensity of immunofluorescent (IF) staining, it showed the FAT10 protein expression was 4.5 fold increased in the AH livers but not in the NASH or control livers.DiscussionMilk thistle derivatives were used to treat FAT10 overexpression experimentally. Flavanone derivatives of milk thistle inhibit IFNγ/TNFα, NFκB and STAT3 then inhibits the expression of FAT10. NFκB is the key nodal hub of the IFNα/TNFα‐response genes. Silibinin derived from milk thistle is reported to be a powerful antioxidant and has anti‐carcinoma effects including against TNFα‐induced tumor growth. Studies on Silibinin or other milk thistle derivatives to treat AH confirms that overexpressed FAT10 with NFκB and TNFα are the major key nodal molecules in these networks.ConclusionOverexpressed FAT10 with over‐activated NFκB and TNFα are the major key nodal molecules in alcoholic hepatitis pathogenesis. These molecules might be potential therapeutic targets.

Tuft Cell Formation Reflects Epithelial Plasticity in Pancreatic Injury: Implications for Modeling Human Pancreatitis.

Chronic pancreatitis, a known risk factor for the development of pancreatic ductal adenocarcinoma (PDA), is a serious, widespread medical condition characterized by inflammation, fibrosis, and acinar to ductal metaplasia (ADM). ADM is a cell type transdifferentiation event where pancreatic acinar cells become ductal-like under conditions of injury or oncogenic mutation. Here, we show that chronic pancreatitis and ADM in genetically wild type mice results in the formation of a significant population of chemosensory tuft cells. Transcriptomic analyses of pancreatitis tuft cells identify expression of inflammatory mediators, consistent with a role for tuft cells in injury progression and/or resolution. Though similar to tuft cell populations in other organs and disease systems, we identified a number of key differences that suggest context-specific tuft cell functions. We evaluated seven different mouse strains for tuft cell formation in response to chronic injury and identified significant heterogeneity reflecting varying proclivity for epithelial plasticity between strains. These results have interesting implications in the role of epithelial plasticity and heterogeneity in pancreatitis and highlight the importance of mouse strain selection when modeling human disease.

Irreversible and sustained upregulation of endothelin axis during oncogene-associated pancreatic inflammation and cancer

Endothelin-1 (ET-1) and its two receptors, endothelin receptor A (ETAR) and endothelin receptor B (ETBR) exhibit deregulated overexprerssion in pancreatic ductal adenocarcinoma (PDAC) and pancreatitis. We examined the expression pattern of endothelin (ET) axis components in the murine models of chronic and acute inflammation in the presence or absence of oncogenic K-ras. While the expression of endothelin converting enzyme-1 (ECE-1), ET-1, ETAR and ETBR in the normal pancreas is restricted predominantly to the islet cells, progressive increase of ET receptors in ductal cells and stromal compartment is observed in the KC model (Pdx-1 Cre; K-rasG12D) of PDAC. In the murine pancreas harboring K-rasG12D mutation (KC mice), following acute inflammation induced by cerulein, increased ETAR and ETBR expression is observed in the amylase and CK19 double positive cells that represent cells undergoing pancreatic acinar to ductal metaplasia (ADM). As compared to the wild type (WT) mice, cerulein treatment in KC mice resulted in significantly higher levels of ECE-1, ET-1, ETAR and ETBR, transcripts in the pancreas. Similarly, in response to cigarette smoke-induced chronic inflammation, the expression of ET axis components is significantly upregulated in the pancreas of KC mice as compared to the WT mice. In addition to the expression in the precursor pancreatic intraepithelial neoplasm (PanIN lesions) in cigarette smoke-exposure model and metaplastic ducts in cerulein-treatment model, ETAR and ETBR expression is also observed in infiltrating F4/80 positive macrophages and α-SMA positive fibroblasts and high co-localization was seen in the presence of oncogenic K-ras. In conclusion, both chronic and acute pancreatic inflammation in the presence of oncogenic K-ras contribute to sustained upregulation of ET axis components in the ductal and stromal cells suggesting a potential role of ET axis in the initiation and progression of PDAC.

Interleukin 22 Signaling Regulates Acinar Cell Plasticity to Promote Pancreatic Tumor Development in Mice

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy that invades surrounding structures and metastasizes rapidly. Although inflammation is associated with tumor formation and progression, little is known about the mechanisms of this connection. We investigate the effects of interleukin (IL) 22 in the development of pancreatic tumors in mice. We performed studies with Pdx1-Cre;LSL-KrasG12D;Trp53+/-;Rosa26EYFP/+ (PKCY) mice, which develop pancreatic tumors, and PKCY mice with disruption of IL22 (PKCY Il22-/-mice). Pancreata were collectedat different stages of tumor development and analyzed by immunohistochemistry, immunoblotting, real-time polymerase chain reaction, and flow cytometry. Some mice were given cerulean to induce pancreatitis. Pancreatic cancer cell lines (PD2560) were orthotopically injected into C57BL/6 mice or Il22-/-mice, and tumor development was monitored. Pancreatic cells were injected into the tail veins of mice, and lung metastases were quantified. Acini were collected from C57BL/6 mice and resected human pancreata and were cultured. Cell lines and acini cultures were incubated with IL22 and pharmacologic inhibitors, and protein levels were knocked down with small hairpin RNAs. We performed immunohistochemical analyses of 26 PDACs and 5 nonneoplastic pancreas specimens. We observed increased expression of IL22 and the IL22 receptor (IL22R) in the pancreas compared with other tissues in mice; IL22 increased with pancreatitis and tumorigenesis. Flow cytometry indicated that the IL22 was produced primarily by T-helper 22 cells. PKCY Il22-/-mice did not develop precancerous lesions or pancreatic tumors. The addition of IL22 to cultured acinar cells increased their expression of markers of ductal metaplasia; these effects of IL22 were prevented with inhibitors of Janus kinase signaling to signal transducer and activator of transcription (STAT) (ruxolitinib) or mitogen-activated protein kinase kinase (MEK) (trametinib) and with STAT3 knockdown. Pancreatic cells injected into Il22-/- mice formed smaller tumors than those injected into C57BL/6. Incubation of IL22R-expressing PDAC cells with IL22 promoted spheroid formation and invasive activity, resulting in increased expression of stem-associated transcription factors (GATA4, SOX2, SOX17, and NANOG), and increased markers of the epithelial-mesenchymal transition (CDH1, SNAI2, TWIST1, and beta catenin); ruxolitinib blocked these effects. Human PDAC tissues had higher levels of IL22, phosphorylated STAT3, and markers of the epithelial-mesenchymal transition than nonneoplastic tissues. An increased level of STAT3 in IL22R-positive cells was associated with shorter survival times of patients. We found levels of IL22 to be increased during pancreatitis and pancreatic tumor development and to be required for tumor development and progression in mice. IL22 promotes acinar to ductal metaplasia, stem cell features, and increased expression of markers of the epithelial-mesenchymal transition; inhibitors of STAT3 block these effects. Increased expression of IL22 by PDACs is associated with reduced survival times.

NECROTIZING SIALOMETAPLASIA OF MINOR SALIVARY GLAND: REPORT OF 2 NEW CASES

Necrotizing sialometaplasia is a benign self-limiting inflammatory reaction that mainly occurs in the minor salivary glands of the hard palate, mimicking malignancy. Case 1 involved a 32-year-old man with a painful ulcerated swelling in the hard palate lasting 3 weeks. Case 2 involved a 63-years-old man presenting a painless ulcer of 2-week duration located in the hard palate. Microscopically, both cases showed salivary glands with preserved lobular architecture containing acini with variable degrees of degeneration and foci of ductal squamous metaplasia. Both lesions regressed completely after incisional biopsy. Oral clinicians and pathologists should consider necrotizing sialometaplasia when evaluating oral ulcerated lesions located in the hard palate.

Cholecystokinin Receptor Antagonist Therapy Decreases Inflammation and Fibrosis in Chronic Pancreatitis.

Chronic pancreatitis is associated with recurrent inflammation, pain, fibrosis, and loss of exocrine and endocrine pancreatic function and risk of cancer. We hypothesized that activation of the CCK receptor contributes to pancreatitis and blockade of this pathway would improve chronic pancreatitis. Two murine models were used to determine whether CCK receptor blockade with proglumide could prevent and reverse histologic and biochemical features of chronic pancreatitis: the 6-week repetitive chronic cerulein injection model and the modified 75% choline-deficient ethionine (CDE) diet. In the CDE-fed model, half the mice received water supplemented with proglumide, for 18weeks. After chronic pancreatitis was established in the cerulein model, half the mice were treated with proglumide and half with water. Histology was scored in a blinded fashion for inflammation, fibrosis and acinar ductal metaplasia (ADM) and serum lipase levels were measured. RNA was extracted and examined for differentially expressed fibrosis genes. Proglumide therapy decreased pancreatic weight in the CDE diet study and the cerulein-induced chronic pancreatitis model. Fibrosis, inflammation, and ADM scores were significantly reduced in both models. Lipase values improved with proglumide but not in controls in both models. Proglumide decreased pancreas mRNA expression of amylase, collagen-4, and TGFβR2 gene expression by 44, 38, and 25%, respectively, compared to control mice. New strategies are needed to decreased inflammation and reduce fibrosis in chronic pancreatitis. CCK receptor antagonist therapy may improve chronic pancreatitis by reversing fibrosis and inflammation. The decrease in ADM may reduce the risk of the development of pancreatic cancer.

Knockout of Acinar Enriched microRNAs in Mice Promote Duct Formation But Not Pancreatic Cancer

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-KRASG12D (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.

Loss of Setd2 promotes Kras-induced acinar-to-ductal metaplasia and epithelia–mesenchymal transition during pancreatic carcinogenesis

ObjectiveSETD2, the sole histone H3K36 trimethyltransferase, is frequently mutated or deleted in human cancer, including pancreatic ductal adenocarcinoma (PDAC). However, whether SETD2/H3K36me3 alteration results in PDAC remains largely unknown.DesignTCGA(PAAD) public...

Abstract 951: Setdb1 deletion protects against formation of pancreatic ductal adenocarcinoma through inducing apoptosis and upregulating p53 expression

Abstract One group of epigenetic regulators known to be altered in cancer development is histone modification. Setdb1, a histone methyltransferase that trimethylates histone H3 on lysine 9 (H3K9), has been implicated in tumorigenesis by altering the expression of oncogenes/ tumor suppressor genes. In this study, we aimed to elucidate the functional role of Setdb1 in pancreatic acinar regeneration and formation of pancreatic ductal adenocarcinoma (PDAC). Setdb1 was expressed in pancreatic ductal cells and a small subset of pancreatic acinar cells in adult wild type mice. Upon caerulein-induced pancreatitis, Setdb1 was expressed in acinar cells and acinar to ductal metaplasia (ADM). To determine the impact of pancreatic Setdb1 deletion on acinar regeneration, we generated Ptf1a-Cre; Setdb1f/f (CS) mice and induced caerulein-pancreatitis. Pancreatic deletion of Setdb1 resulted in pancreatic atrophy concomitant with increased apoptosis of acinar cells and increased expression of p53 in CS mice after caerulein-induced pancreatitis. In addition, pancreatic acinar regeneration was delayed with persistent inflammation inCS mice. To investigate the impact of pancreatic Setdb1 deletion on PDAC formation, we next generated Ptf1a-Cre; KrasG12D; Setdb1f/f (KCS) mice and compared to control Ptf1a-Cre; KrasG12D (KC) mice. Pancreatic deletion of Setdb1 dramatically accelerated spontaneous development of ADM and pancreatic intraepithelial neoplasia (PanIN), the most common precursor lesion of PDAC, in KCS mice compared to KC mice at the age of 4 weeks. Acinar cell culture experiments revealed that Setdb1 null acinar cells converted to ADM, at least in part, in a cell autonomous manner. Long term observation revealed massive pancreatic atrophy in KCS mice. Immunostaining revealed a dramatic increase in apoptotic cells and upregulated expression of p53 in KCS mice compared to KC mice at the age of 20 weeks. Consistently, microarray analysis revealed upregulated expression of p53 and apoptotic pathway genes in KCS mice compared to KC mice. We next examined the impact of pancreatic Setdb1 deletion on PDAC formation in the context of p53 deletion. Surprisingly, PDAC formation was suppressed in KCS mice in the context of heterozygous p53 deletion (KPhereroCS mice). Again, apoptosis was increased and p53 expression was upregulated in KPhereroCS mice. In contrast, in the context of homozygous p53 deletion, PDAC was formed and increased apoptosis was canceled in KPhomoCS mice. These findings suggested that Setdb1 deletion protects against PDAC formation through inducing apoptosis, which is mediated by p53. In conclusion, Setdb1 is required for normal pancreatic acinar regeneration and protects against PDAC formation though inducing apoptosis, which is mediated by p53. Thus, Setdb1 could be a therapeutic target for PDAC which retains wild p53 expression. Citation Format: Satoshi Ogawa, Akihisa Fukuda, Motoyuki Tsuda, Tomonori Masuda, Makoto Sono, Yuichi Fukunaga, Takaaki Yoshikawa, Osamu Araki, Munemasa Nagao, Norihiro Goto, Yukiko Hiramatsu, Takahisa Maruno, Yoichi Shinkai, Hiroshi Seno. Setdb1 deletion protects against formation of pancreatic ductal adenocarcinoma through inducing apoptosis and upregulating p53 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 951.