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NLRP3 Inflammasome activation in macrophages regulates systemic inflammation and severity during acute pancreatitis.
Abstracts / Pancreatology 17 (2017) S1eS142
form 3D structures. PANC1 formed more compact spheroids in the presence of exosomes (p<0.001). Both cell lines demonstrated increased cytotoxicity in 3D culture in the presence of exosomes. Exosome treatment is associated with increased expression of cell adhesion genes e.g. CTGF (p<0.01), uPAR (p<0.01). We are currently undertaking next generation RNA sequencing from spheroids to determine changes in mRNA expression. Conclusion: PSC exosomes contain miRNA’s implicated in a variety of cancer related cell signalling processes and impact on the phenotype of pancreatic cancer cells.
Abstract ID: 1931. In vitro comparative analysis of Circulating Tumour Cells detection in liquid biopsy for the diagnosis of pancreatic adenocarcinoma Etienne Buscail 1, Olivier Degrandi 2, Charline Caumont 2, Jean-Philippe Merlio 2, François Moreau-Gaudry 1, Aurelie Bedel 1, Christophe Laurent 1, Laurence Chiche 1, Sandrine Dabernat 1 1 2
CHU Bordeaux-Inserm U 1035, France CHU Bordeaux, France
Introduction: Pancreatic ductal adenocarcinoma incidence has been increasing with an overall survival <5%. When the cancer is suspected, neoadjuvant chemotherapies and surgery are often delayed because the mandatory histologic proof is difficult to make. Aims: Searching for tumour elements in body fluids might provide diagnosis assistance. Materials & methods: We tested 2 enrichment methods to estimate the sensitivity of CTC detection with cell spiking experiments (10 to 30 cells) in 7.5 mL blood samples from 24 healthy volunteers using the oncospecific density gradient OncoQuick® and negative selection enrichment method RosetteSep™. We used two pancreatic tumour cell lines, MiaPaCa2, CAPAN-2, expressing the reporter gene Td-Tomato or GFP. KRAS mutations were quantified in genomic DNA of purified cells by digital droplet QPCR (dd-PCR) with allele specific primers. Results: Analytical sensitivity was 100% for OncoQuick®, regardless of the cell line, and ranged between 70 and 100% for RosetteSep™. Mean recovery rate of cells was 56±23% for OncoQuick® versus 39±27% for RosetteSep™ (p<0.001). To further identify the presence of tumour cells within the tumour-enriched blood cell population, KRAS allele-specific ddPCR was performed with positivity threshold being 0.058%. Ten to 100 spiked cells could be detected after both enrichment methods but with a mutant allele frequency (MAF) 4 to 30 times higher with RosetteSep™. Conclusion: We found that OncoQuick® is more reliable in terms of recovery efficiency than RosetteSep™. However, this onco-specific density gradient is contaminated by a much larger number of red and white blood cells than after RosetteSep™ enrichment, which may dilute KRAS mutant alleles’ detection by dd-PCR.
Abstract ID: 1933. Characterization of a novel duplication allele of carboxyl-ester lipase (CEL) - a role in chronic pancreatitis? Karianne Fjeld 1, Emmanuelle Masson 2, Patrick Michl 3, Jin-Huan Lin 4, Solrun Steine 5, Bente Berg Johansson 1, Claudia Ruffert 3, Wen-Bin Zou 4, Zhao-Shen Li 6, Pål Rasmus Njølstad 1, Jian-Min Chen 7, Zhuan Liao 6, erec 9, Anders Molven 5 Stefan Johansson 8, Jonas Rosendahl 3, Claude F 1 KG Jebsen Center for Diabetes Research, Department of Clinical Science, University of Bergen, Bergen, Norway 2 et de la Recherche Me dicale (INSERM), Institut National de la Sante U1078, Brest, France
S23
3 Department of Internal Medicine I, Martin Luther University, Halle, Germany 4 Department of Gastroenterology, Changhai Hospital, The Second Military Medical University, Shanghai, China 5 The Gade Laboratory for Pathology, Department of Clinical Medicine, University of Bergen, Bergen, Norway 6 Shanghai Institute of Pancreatic Diseases, Shanghai, China 7 Etablissement Français du Sang (EFS) Bretagne, Brest, France 8 Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway 9 de Me decine et des Sciences de la Sante , Universite de Faculte Bretagne Occidentale (UBO), Brest, France
Introduction: CEL is an extremely polymorphic gene. Point mutations in exon 11 can cause a syndrome of exocrine dysfunction/diabetes. We have previously identified copy number variants of CEL: one duplication allele (CEL-DUP1) and a recombined deletion allele (CEL-HYB), the latter being a risk factor for chronic pancreatitis (Fjeld et al., Nature Genetics 2015). Aims: To characterize the structure of a novel duplicated CEL variant (CEL-DUP2) and to investigate whether it influences the risk of idiopathic chronic pancreatitis (ICP). Patients & methods: The structure of CEL-DUP2 was determined by PCR, fragment analysis and Sanger sequencing. For screening, we developed a long-range duplex PCR assay followed by gel electrophoresis. ICP patients/controls were provided by University of Bretagne (n¼507/987), University of Halle (n¼63/89) and Shanghai Institute of Pancreatic Diseases (n¼507/508). Results: CEL-DUP2 contains an extra copy of the CEL gene, located immediately downstream for the neighbouring CEL pseudogene promoter. The allele has probably arisen from non-allelic, homologous recombination between CEL and its pseudogene because of their profound sequence similarity. Carrier frequencies of CEL-DUP2 in ICP patients/controls were, respectively, 3.9% and 2.6% in the French cohort (P¼0.16), 0% and 1.1% in the German cohort (P¼0.82), and 10.7% and 8.1% in the Chinese material (P¼0.6). Conclusion: We have identified CEL-DUP2, which contains an extra copy of the entire CEL gene. No association were found with ICP. The screening of European and Asian cohorts supports previous findings that carrier frequencies of CEL alleles may vary considerably between ethnicities (Zou et al, Gastroenterology 2016).
Abstract ID: 1941. NLRP3 Inflammasome activation in macrophages regulates systemic inflammation and severity during acute pancreatitis. Matthias Sendler 1, Cindy van den Brandt 1, Weiss Frank-Ulrich 1, Janine Golchert 2, Georg Homuth 2, Bossaller Lukas 1, Lerch Markus M 1, Mayerle Julia 3 1 Department of Medicine A, University Medicine, Ernst-Moritz-ArndtUniversity Greifswald, Greifswald, Germany 2 Interfaculty Institutes for Genetics and Functional Genomics, University Medicine, Ernst-Moritz-Arndt-University Greifswald, Germany 3 Medizinische Klinik und Poliklinik II, Klinikum der LMU MünchenGrosshadern, München, Germany
Introduction: The severity and mortality of acute pancreatitis is determined by its systemic inflammatory response. Inflammasome activation by damage-associated-molecular-patterns (DAMPs) could play a crucial role for disease severity. Aims: Here we investigated the role of the NLRP3-nflammasome in macrophages during the course of pancreatitis.
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Abstracts / Pancreatology 17 (2017) S1eS142
Materials & methods: Mild pancreatitis was induced in NLRP3-/- by hourly intraperitoneal injections of caerulein (50mg/kg bodyweight). Severe necrotizing pancreatitis was induced by partial duct ligation with additional caerulein stimulation. Inhibition of NLRP3 inflammasome was achieved with MCC950. Disease severity was determined by serum amylase, lipase and histology. Systemic inflammation was measured by MPO in lung and FACS analysis of leukocyte activation in spleen. Inflammasome activation was measured in bone marrow derived macrophages co-incubated with acinar cells and on transcription level by affymetrix chip analysis. Results: Macrophages phagocytose dying acinar cells in vitro which leads to a massive release of cytokine and a shift to a pro-inflammatory phenotype of cells (M1-like). The inflammasome complex is activated in macrophages and leads to the activation of caspase 1 and the secretion of mature IL1b. Absence of inflammasome activation by genetic deletion or administration of MCC950 results in decreased disease severity in both models of pancreatitis paralleled by less local and systemic inflammation. Affymetrix chip data from macrophages and acini in co-culture suggest that IL18 is the link to SIRS. Conclusion: Inflammasome activation within macrophages induces systemic hyperinflammationin vivo as well as in vitro. Therapeutic inhibition of inflammasome activation decrease systemic and local damage and is a promising treatment option for severe acute pancreatitis.
Abstract ID: 1943. Role of carboxyl ester lipase (CEL) in pancreatic disease: Functional characterization of CEL protein variants differing in C-terminal sequence Anny Gravdal 1, Xunjun Xiao 2, Monica Dalva 3, Pll Rasmus Njr lstad 4, Mark Lowe 2, Bente Berg Johansson 4, Anders Molven 5, Karianne Fjeld 1 1 KG Jebsen Center for Diabetes Research, Department of Clinical Science, University of Bergen, Norway 2 Department of Pediatrics, Division of Gastroenterology, Hepatology and Nutrition, Children's Hospital of Pittsburgh of UPMC, United States 3 Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway 4 Department of Pediatrics, Haukeland University Hospital, Bergen, Norway 5 Department of Pathology, Haukeland University Hospital, Bergen, Norway
Introduction: We have previously identified disease-associated mutations in the last exon of the CEL gene, localized in a variable number of tandem repeats (VNTR) domain. Two single base deletions (DEL1 and DEL4) predict a new truncated C-terminus of the CEL protein. Patients with these mutations suffer from exocrine pancreatic dysfunction and diabetes. In addition, a copy number variant of CEL containing only 3 VNTR repeats is a genetic risk factor for chronic pancreatitis. Aims: To understand the role of the C-terminal domain of CEL in pancreatic disease by investigating effects of various VNTRs on protein expression, secretion, post-translational modification and cell viability. Materials & methods: HEK293 cells were transiently transfected with different CEL variants and studied by Western blotting, immunostaining, confocal imaging, and deglycosylation and viability assays. Results: Expression and secretion of CEL variants varied according to VNTR length and composition. The pathogenic CEL proteins DEL1 and DEL4 showed elevated intracellular accumulation and increased apoptosis compared with variants not associated with disease. Furthermore, these two pathogenic variants and the normal variant DEL8, which all have the same theoretical molecular weight, exhibited different patterns of Oglycosylation. Comparison of CEL variants expressed with and without V5/ His epitope tags indicated a higher intracellular level of untagged proteins. Conclusion: The cellular properties of CEL variants are influenced by length, composition and post-translational modification of the C-terminal VNTR domain. In addition, epitope tags may cause increased solubility of disease-causing CEL variants, leading to underestimation of their propensity to aggregate in cellular model systems.
Abstract ID: 1946. Recapitulation of a progenitor-like transcriptional profile in pancreatic carcinogenesis is initiated by the epigenetic modifier Ring1b. Simone Benitz 1, Stefan Czemmel 2, Britta Wingerath 3, Tatjana Unruh 3, Sabrina Deubler 4, Katja Steiger 5, Bo Kong 4, Susanne Raulefs 4, Sven Nahnsen 2, Francisco X. Real 6, Güralp O. Ceyhan 4, Irene Esposito 3, Julia €rg Kleeff 8, Ivonne Regel 1 Mayerle 1, Christoph W. Michalski 7, Jo 1 Department of Internal Medicine II, Ludwig Maximilian University of Munich, Munich, Germany 2 Quantitative Biology Center, Eberhard Karls University Tuebingen, Tuebingen, Germany 3 Institute of Pathology, Heinrich Heine University Duesseldorf, Duesseldorf, Germany 4 Department of Surgery, Technical University of Munich, Munich, Germany 5 Institute of Pathology, Technical University of Munich, Munich, Germany 6 Spanish National Cancer Research Centre (CNIO), Madrid, Spain 7 Department of Surgery, University of Heidelberg, Heidelberg, Germany 8 Department of Surgery, Martin Luther University of HalleWittenberg, Halle, Germany
Introduction: Given that inflammation-triggered acinar-to-ductal metaplasia (ADM) occurs in the absence of genetic mutations, the high cellular plasticity of acinar cells and the changes in their transcriptional profile likely depend on epigenetic modifications. Even the expression of oncogenic Kras at an adult stage requires the induction of a progenitor-like ADM status for the initiation of pancreatic cancer. Previously, we have shown that the epigenetic histone remodeler Ring1b is highly reactivated in ADM and pancreatic cancer cells, contributing to the epigenetic silencing of acinar cell fate genes. Aims: We hypothesize that Ring1b establishes a progenitor-like epigenetic profile during ADM formation resulting in acinar gene silencing and an enhanced susceptibility towards oncogenic Kras-mediated cell transformation. During cancer progression the epigenetic landscape is remodeled further, determining tumor cell behavior. Materials & methods: Epigenetic patterns and Ring1b target genes were identified by performing gene expression microarrays and Chromatin-IP-sequencing (ChIP-Seq) from a pure-cell in vitro carcinogenesis model. The significance of Ring1b-mediated epigenetic remodeling during cancer development was determined by studying a conditional Ring1b knockout mouse model. Results: Gene expression and ChIP-Seq data revealed that ADM cells acquire a progenitor-like profile, which is further modified in cancer cells. In both, activated genes highly correlated with developmental pathways and chromatin modifications, whereas repressed genes were associated with catabolic and metabolic processes. Ring1b knockout mice lost the ability to adapt a progenitor-like profile, and acinar differentiation genes remained expressed. Consequently, inflammation-based ADM formation and KrasG12D-dependent tumor formation was significantly impaired. Conclusion: Ring1b-dependent epigenetic changes induce acinar cell dedifferentiation in acinar-to-ductal-metaplasia and pancreatic cancer development.
Abstract ID: 1950. MiR-502 regulates non-homologous end joining (NHEJ) and reduces radioresistance in human pancreatic cancer cell line Agnieszka Smolinska, Julia Swoboda, Markus M. Lerch, Patryk Moskwa University Medicine, Ernst-Moritz-Arndt-University Greifswald, Germany
form 3D structures. PANC1 formed more compact spheroids in the presence of exosomes (p<0.001). Both cell lines demonstrated increased cytotoxicity in 3D culture in the presence of exosomes. Exosome treatment is associated with increased expression of cell adhesion genes e.g. CTGF (p<0.01), uPAR (p<0.01). We are currently undertaking next generation RNA sequencing from spheroids to determine changes in mRNA expression. Conclusion: PSC exosomes contain miRNA’s implicated in a variety of cancer related cell signalling processes and impact on the phenotype of pancreatic cancer cells.
Abstract ID: 1931. In vitro comparative analysis of Circulating Tumour Cells detection in liquid biopsy for the diagnosis of pancreatic adenocarcinoma Etienne Buscail 1, Olivier Degrandi 2, Charline Caumont 2, Jean-Philippe Merlio 2, François Moreau-Gaudry 1, Aurelie Bedel 1, Christophe Laurent 1, Laurence Chiche 1, Sandrine Dabernat 1 1 2
CHU Bordeaux-Inserm U 1035, France CHU Bordeaux, France
Introduction: Pancreatic ductal adenocarcinoma incidence has been increasing with an overall survival <5%. When the cancer is suspected, neoadjuvant chemotherapies and surgery are often delayed because the mandatory histologic proof is difficult to make. Aims: Searching for tumour elements in body fluids might provide diagnosis assistance. Materials & methods: We tested 2 enrichment methods to estimate the sensitivity of CTC detection with cell spiking experiments (10 to 30 cells) in 7.5 mL blood samples from 24 healthy volunteers using the oncospecific density gradient OncoQuick® and negative selection enrichment method RosetteSep™. We used two pancreatic tumour cell lines, MiaPaCa2, CAPAN-2, expressing the reporter gene Td-Tomato or GFP. KRAS mutations were quantified in genomic DNA of purified cells by digital droplet QPCR (dd-PCR) with allele specific primers. Results: Analytical sensitivity was 100% for OncoQuick®, regardless of the cell line, and ranged between 70 and 100% for RosetteSep™. Mean recovery rate of cells was 56±23% for OncoQuick® versus 39±27% for RosetteSep™ (p<0.001). To further identify the presence of tumour cells within the tumour-enriched blood cell population, KRAS allele-specific ddPCR was performed with positivity threshold being 0.058%. Ten to 100 spiked cells could be detected after both enrichment methods but with a mutant allele frequency (MAF) 4 to 30 times higher with RosetteSep™. Conclusion: We found that OncoQuick® is more reliable in terms of recovery efficiency than RosetteSep™. However, this onco-specific density gradient is contaminated by a much larger number of red and white blood cells than after RosetteSep™ enrichment, which may dilute KRAS mutant alleles’ detection by dd-PCR.
Abstract ID: 1933. Characterization of a novel duplication allele of carboxyl-ester lipase (CEL) - a role in chronic pancreatitis? Karianne Fjeld 1, Emmanuelle Masson 2, Patrick Michl 3, Jin-Huan Lin 4, Solrun Steine 5, Bente Berg Johansson 1, Claudia Ruffert 3, Wen-Bin Zou 4, Zhao-Shen Li 6, Pål Rasmus Njølstad 1, Jian-Min Chen 7, Zhuan Liao 6, erec 9, Anders Molven 5 Stefan Johansson 8, Jonas Rosendahl 3, Claude F 1 KG Jebsen Center for Diabetes Research, Department of Clinical Science, University of Bergen, Bergen, Norway 2 et de la Recherche Me dicale (INSERM), Institut National de la Sante U1078, Brest, France
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3 Department of Internal Medicine I, Martin Luther University, Halle, Germany 4 Department of Gastroenterology, Changhai Hospital, The Second Military Medical University, Shanghai, China 5 The Gade Laboratory for Pathology, Department of Clinical Medicine, University of Bergen, Bergen, Norway 6 Shanghai Institute of Pancreatic Diseases, Shanghai, China 7 Etablissement Français du Sang (EFS) Bretagne, Brest, France 8 Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway 9 de Me decine et des Sciences de la Sante , Universite de Faculte Bretagne Occidentale (UBO), Brest, France
Introduction: CEL is an extremely polymorphic gene. Point mutations in exon 11 can cause a syndrome of exocrine dysfunction/diabetes. We have previously identified copy number variants of CEL: one duplication allele (CEL-DUP1) and a recombined deletion allele (CEL-HYB), the latter being a risk factor for chronic pancreatitis (Fjeld et al., Nature Genetics 2015). Aims: To characterize the structure of a novel duplicated CEL variant (CEL-DUP2) and to investigate whether it influences the risk of idiopathic chronic pancreatitis (ICP). Patients & methods: The structure of CEL-DUP2 was determined by PCR, fragment analysis and Sanger sequencing. For screening, we developed a long-range duplex PCR assay followed by gel electrophoresis. ICP patients/controls were provided by University of Bretagne (n¼507/987), University of Halle (n¼63/89) and Shanghai Institute of Pancreatic Diseases (n¼507/508). Results: CEL-DUP2 contains an extra copy of the CEL gene, located immediately downstream for the neighbouring CEL pseudogene promoter. The allele has probably arisen from non-allelic, homologous recombination between CEL and its pseudogene because of their profound sequence similarity. Carrier frequencies of CEL-DUP2 in ICP patients/controls were, respectively, 3.9% and 2.6% in the French cohort (P¼0.16), 0% and 1.1% in the German cohort (P¼0.82), and 10.7% and 8.1% in the Chinese material (P¼0.6). Conclusion: We have identified CEL-DUP2, which contains an extra copy of the entire CEL gene. No association were found with ICP. The screening of European and Asian cohorts supports previous findings that carrier frequencies of CEL alleles may vary considerably between ethnicities (Zou et al, Gastroenterology 2016).
Abstract ID: 1941. NLRP3 Inflammasome activation in macrophages regulates systemic inflammation and severity during acute pancreatitis. Matthias Sendler 1, Cindy van den Brandt 1, Weiss Frank-Ulrich 1, Janine Golchert 2, Georg Homuth 2, Bossaller Lukas 1, Lerch Markus M 1, Mayerle Julia 3 1 Department of Medicine A, University Medicine, Ernst-Moritz-ArndtUniversity Greifswald, Greifswald, Germany 2 Interfaculty Institutes for Genetics and Functional Genomics, University Medicine, Ernst-Moritz-Arndt-University Greifswald, Germany 3 Medizinische Klinik und Poliklinik II, Klinikum der LMU MünchenGrosshadern, München, Germany
Introduction: The severity and mortality of acute pancreatitis is determined by its systemic inflammatory response. Inflammasome activation by damage-associated-molecular-patterns (DAMPs) could play a crucial role for disease severity. Aims: Here we investigated the role of the NLRP3-nflammasome in macrophages during the course of pancreatitis.
S24
Abstracts / Pancreatology 17 (2017) S1eS142
Materials & methods: Mild pancreatitis was induced in NLRP3-/- by hourly intraperitoneal injections of caerulein (50mg/kg bodyweight). Severe necrotizing pancreatitis was induced by partial duct ligation with additional caerulein stimulation. Inhibition of NLRP3 inflammasome was achieved with MCC950. Disease severity was determined by serum amylase, lipase and histology. Systemic inflammation was measured by MPO in lung and FACS analysis of leukocyte activation in spleen. Inflammasome activation was measured in bone marrow derived macrophages co-incubated with acinar cells and on transcription level by affymetrix chip analysis. Results: Macrophages phagocytose dying acinar cells in vitro which leads to a massive release of cytokine and a shift to a pro-inflammatory phenotype of cells (M1-like). The inflammasome complex is activated in macrophages and leads to the activation of caspase 1 and the secretion of mature IL1b. Absence of inflammasome activation by genetic deletion or administration of MCC950 results in decreased disease severity in both models of pancreatitis paralleled by less local and systemic inflammation. Affymetrix chip data from macrophages and acini in co-culture suggest that IL18 is the link to SIRS. Conclusion: Inflammasome activation within macrophages induces systemic hyperinflammationin vivo as well as in vitro. Therapeutic inhibition of inflammasome activation decrease systemic and local damage and is a promising treatment option for severe acute pancreatitis.
Abstract ID: 1943. Role of carboxyl ester lipase (CEL) in pancreatic disease: Functional characterization of CEL protein variants differing in C-terminal sequence Anny Gravdal 1, Xunjun Xiao 2, Monica Dalva 3, Pll Rasmus Njr lstad 4, Mark Lowe 2, Bente Berg Johansson 4, Anders Molven 5, Karianne Fjeld 1 1 KG Jebsen Center for Diabetes Research, Department of Clinical Science, University of Bergen, Norway 2 Department of Pediatrics, Division of Gastroenterology, Hepatology and Nutrition, Children's Hospital of Pittsburgh of UPMC, United States 3 Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway 4 Department of Pediatrics, Haukeland University Hospital, Bergen, Norway 5 Department of Pathology, Haukeland University Hospital, Bergen, Norway
Introduction: We have previously identified disease-associated mutations in the last exon of the CEL gene, localized in a variable number of tandem repeats (VNTR) domain. Two single base deletions (DEL1 and DEL4) predict a new truncated C-terminus of the CEL protein. Patients with these mutations suffer from exocrine pancreatic dysfunction and diabetes. In addition, a copy number variant of CEL containing only 3 VNTR repeats is a genetic risk factor for chronic pancreatitis. Aims: To understand the role of the C-terminal domain of CEL in pancreatic disease by investigating effects of various VNTRs on protein expression, secretion, post-translational modification and cell viability. Materials & methods: HEK293 cells were transiently transfected with different CEL variants and studied by Western blotting, immunostaining, confocal imaging, and deglycosylation and viability assays. Results: Expression and secretion of CEL variants varied according to VNTR length and composition. The pathogenic CEL proteins DEL1 and DEL4 showed elevated intracellular accumulation and increased apoptosis compared with variants not associated with disease. Furthermore, these two pathogenic variants and the normal variant DEL8, which all have the same theoretical molecular weight, exhibited different patterns of Oglycosylation. Comparison of CEL variants expressed with and without V5/ His epitope tags indicated a higher intracellular level of untagged proteins. Conclusion: The cellular properties of CEL variants are influenced by length, composition and post-translational modification of the C-terminal VNTR domain. In addition, epitope tags may cause increased solubility of disease-causing CEL variants, leading to underestimation of their propensity to aggregate in cellular model systems.
Abstract ID: 1946. Recapitulation of a progenitor-like transcriptional profile in pancreatic carcinogenesis is initiated by the epigenetic modifier Ring1b. Simone Benitz 1, Stefan Czemmel 2, Britta Wingerath 3, Tatjana Unruh 3, Sabrina Deubler 4, Katja Steiger 5, Bo Kong 4, Susanne Raulefs 4, Sven Nahnsen 2, Francisco X. Real 6, Güralp O. Ceyhan 4, Irene Esposito 3, Julia €rg Kleeff 8, Ivonne Regel 1 Mayerle 1, Christoph W. Michalski 7, Jo 1 Department of Internal Medicine II, Ludwig Maximilian University of Munich, Munich, Germany 2 Quantitative Biology Center, Eberhard Karls University Tuebingen, Tuebingen, Germany 3 Institute of Pathology, Heinrich Heine University Duesseldorf, Duesseldorf, Germany 4 Department of Surgery, Technical University of Munich, Munich, Germany 5 Institute of Pathology, Technical University of Munich, Munich, Germany 6 Spanish National Cancer Research Centre (CNIO), Madrid, Spain 7 Department of Surgery, University of Heidelberg, Heidelberg, Germany 8 Department of Surgery, Martin Luther University of HalleWittenberg, Halle, Germany
Introduction: Given that inflammation-triggered acinar-to-ductal metaplasia (ADM) occurs in the absence of genetic mutations, the high cellular plasticity of acinar cells and the changes in their transcriptional profile likely depend on epigenetic modifications. Even the expression of oncogenic Kras at an adult stage requires the induction of a progenitor-like ADM status for the initiation of pancreatic cancer. Previously, we have shown that the epigenetic histone remodeler Ring1b is highly reactivated in ADM and pancreatic cancer cells, contributing to the epigenetic silencing of acinar cell fate genes. Aims: We hypothesize that Ring1b establishes a progenitor-like epigenetic profile during ADM formation resulting in acinar gene silencing and an enhanced susceptibility towards oncogenic Kras-mediated cell transformation. During cancer progression the epigenetic landscape is remodeled further, determining tumor cell behavior. Materials & methods: Epigenetic patterns and Ring1b target genes were identified by performing gene expression microarrays and Chromatin-IP-sequencing (ChIP-Seq) from a pure-cell in vitro carcinogenesis model. The significance of Ring1b-mediated epigenetic remodeling during cancer development was determined by studying a conditional Ring1b knockout mouse model. Results: Gene expression and ChIP-Seq data revealed that ADM cells acquire a progenitor-like profile, which is further modified in cancer cells. In both, activated genes highly correlated with developmental pathways and chromatin modifications, whereas repressed genes were associated with catabolic and metabolic processes. Ring1b knockout mice lost the ability to adapt a progenitor-like profile, and acinar differentiation genes remained expressed. Consequently, inflammation-based ADM formation and KrasG12D-dependent tumor formation was significantly impaired. Conclusion: Ring1b-dependent epigenetic changes induce acinar cell dedifferentiation in acinar-to-ductal-metaplasia and pancreatic cancer development.
Abstract ID: 1950. MiR-502 regulates non-homologous end joining (NHEJ) and reduces radioresistance in human pancreatic cancer cell line Agnieszka Smolinska, Julia Swoboda, Markus M. Lerch, Patryk Moskwa University Medicine, Ernst-Moritz-Arndt-University Greifswald, Germany