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Pancreatic stellate cells increase cancer cell expression of HMGA2 in a 3D co-culture model of pancreatic cancer
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Abstracts / Pancreatology 17 (2017) S1eS142
Abstract ID: 2019.
Abstract ID: 2023.
Exocrine pancreas lipase transcript regulation
A comprehensive prognostic signature based on interaction of immune cell infiltration with stromal composition for resected pancreatic ductal adenocarcinoma
Ruth Birk 1
Department of Nutrition, Health Sciences, Ariel University, Israel
Introduction: Pancreatic lipase (triacylglycerol lipase EC 3.1.1.3, PNLIP) is an essential enzyme in hydrolysis of dietary fat. We have previously shown that different dietary fat and especially polyunsaturated fatty acids (PUFA) regulate PNLIP. However, to date the molecular mechanism underlying this regulation is mostly unknown. Aims: The aim of our research was to Identifing PNLIP transcript regulator. Materials & methods: We identify PPARg as an essential PNLIP transcript regulator. Using in silico bioinformatics tools, reporter luciferase assay, PPARg agonists and antagonists, and PPARg overexpression in exocrine pancreas in vitro AR42J and primary cells. Results: Using in silico bioinformatics tools we mapped PPARg binding sites (PPRE) to the putative promoter region of PNLIP. Reporter luciferase assay in AR42J rat exocrine pancreas acinar cells transfected with various constructs of the putative PNLIP promoter showed that PNLIP transcription is significantly enhanced by PPARg dose-dependently, reaching maximal levels with multi PPRE sites. This effect was significantly augmented in the presence of PPARg agonists and reduced by PPARg antagonists or mutagenesis abrogating PPRE sites. Over-expression of PPARg significantly elevated PNLIP transcript and protein levels in AR42J cells and in primary pancreas cells. Moreover, PNLIP expression was up-regulated by PPARg agonists (pioglitazone and 15dPGJ2) and significantly down-regulated by PPARg antagonists in non-transfected rat exocrine pancreas AR42J cell line cells. Conclusion: PPARg transcriptionally regulates PNLIP gene expression. This transcript regulation resolves part of the missing link between dietary PUFA direct regulation of PNLIP.
Abstract ID: 2021. Pancreatic stellate cells increase cancer cell expression of HMGA2 in a 3D co-culture model of pancreatic cancer 2 € Jessica Norberg 1, Carina Strell 2, Andrea Balboni 1, Arne Ostman , Rainer €hr 1 Heuchel 1, Matthias Lo 1 Karolinska Institutet, Department of Clinical Intervention and Technology, Sweden 2 Karolinska Institutet, Department of Oncology and Pathology, Sweden
Introduction: In a cohort of PDAC patients, positivity for the High Mobility Group A-2 (HMGA2) protein has been correlated to lower survival. One of the factors known to increase the expression of HMGA2 in other cancer cells, is Transforming Growth Factor-B1 (TGFB1). Aims: The aim of this study was to investigate the mechanism behind increased HMGA2 in pancreatic cancer cells, and if this is mediated through interactions with stromal cells. Materials & methods: PDAC cells and PSCs were cultured in 3D, as mono- and co-cultures, in a newly developed model. Gene expression (mRNA) for cancer- and stellate cells within the co-culture model was investigated with real time PCR, looking at cell type specific expression. Protein expression was determined by immunohistochemistry (IHC). Results: Expression of TGFB1 mRNA increased in the co-cultured vs mono-cultured PSCs. HMGA2 was found to be higher expressed in the cocultured vs mono-cultured cancer cells, both at the mRNA and the protein level. TGFB1-treatment of cancer cell mono-cultures increased the expression of HMGA2. Conclusion: Increased expression of TGFB1 in the stellate cells in the 3D cultures leads to an increased expression of HMGA2 in the cancer cells. We are currently investigating this mechanism in more detail.
Ujjwal Mukund Mahajan 1, Enno Langhoff 2, Eithne Costello 3, William Greenhalf 3, Christopher Halloran 3, Georg Beyer 1, Frank Ulrich Weiss 2, John P. Neoptolemos 3, Markus W. Büchler 4, Thomas Kohlmann 5, Markus M. Lerch 2, Julia Mayerle 1 €t Medizinische Klinik und Poliklinik II, Klinikum der Universita München, Germany 2 Department of Medicine A, University Medicine Greifswald, Germany 3 NIHR Liverpool Pancreas Biomedical Research Centre, University of Liverpool, United Kingdom 4 Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Germany 5 Department of Community Medicine, University Medicine Greifswald, Germany 1
Introduction: Remodelling of the pancreatic cancer tumour (PDAC) microenvironment and extracellular matrix (ECM) has been implicated in the prognosis of PDAC. Aims: We investigated whether infiltration of immune cell subsets affect the survival of pancreatic cancer patients and affect stromal composition (fibrogenic (aSMA high/collagen-I high); fibrolytic stroma (aSMA high/collagen-I low). Materials & methods: TMA of 404 patients from the ESPAC-3 trial were immunostained for aSMA, collagen-I, CD3, CD4, CD8, CD68, CD206 and neutrophils. Median H-Scores of individual staining were calculated for all cores by multiplying the integrated density of tumor cell staining. Event free survival was plotted against low/high expression of leukocytes using Kaplan-Meier-curves, log-rank-tests, and Cox-proportional-hazardsmodels. All statistical tests were two-sided and performed in R. Results: In 1824 TMA from 382 patients (94.6%) immune cell composition was distinctly different in fibrolytic versus fibrogenic stroma. CD3 (Median EFS high-CD3-expression: 14.3 months vs. CD3-low-expreeesion 11 months, x2LR,1DF¼14.7; p¼0.0001) count were found as stroma type independent prognostic markers while CD206 and CD68 were dependent on the type of stroma. Recursive partitioning for discrete-time-survivaltree analysis showed CD3 as strongest independent predictor but macrophage subpopulations to determine fibrogenic or fibrolytic stroma with a median EFS ranging from 6.3 month (fibrolytic stroma with low-CD3 and high-CD68 count) to 20.1 month (fibrogenic stroma, CD3-high). Conclusion: The comprehensive prognostic signature based on interaction of immune cell infiltration with stromal composition provides better stratification in predicting event free survival in PDAC. Immune cells determine the predominant type of stromal composition and this may help in understanding the effect of the host immune response.
Abstract ID: 2024. Mitochondrial distribution and function in pancreatic ductal epithelial cells zga 2, La szlo th 1, Jo zsef Mal }s 1, Zsolt Ra Tretter 3, Emese To eth 1, R eka Erdo n Rakonczay, Jr. 4, P eter Hegyi 5 Zolta 1
University of Szeged, First Department of Medicine, Hungary University of Szeged, 2Department of Pathology, Hungary 3 Semmelwes University, Department of Medical Biochemistry, Hungary 4 University of Szeged, Department of Pathophysiology, Hungary 5 University of Szeged, MTA-SZTE Momentum Translational cs, Institute for Gastroenterology Research Group, University of Pe Transl, Hungary 2
Introduction: Mitochondrial dysfunction is a hallmark of several disease pathogenesis including acute pancreatitis (AP). Our previous results
Abstracts / Pancreatology 17 (2017) S1eS142
Abstract ID: 2019.
Abstract ID: 2023.
Exocrine pancreas lipase transcript regulation
A comprehensive prognostic signature based on interaction of immune cell infiltration with stromal composition for resected pancreatic ductal adenocarcinoma
Ruth Birk 1
Department of Nutrition, Health Sciences, Ariel University, Israel
Introduction: Pancreatic lipase (triacylglycerol lipase EC 3.1.1.3, PNLIP) is an essential enzyme in hydrolysis of dietary fat. We have previously shown that different dietary fat and especially polyunsaturated fatty acids (PUFA) regulate PNLIP. However, to date the molecular mechanism underlying this regulation is mostly unknown. Aims: The aim of our research was to Identifing PNLIP transcript regulator. Materials & methods: We identify PPARg as an essential PNLIP transcript regulator. Using in silico bioinformatics tools, reporter luciferase assay, PPARg agonists and antagonists, and PPARg overexpression in exocrine pancreas in vitro AR42J and primary cells. Results: Using in silico bioinformatics tools we mapped PPARg binding sites (PPRE) to the putative promoter region of PNLIP. Reporter luciferase assay in AR42J rat exocrine pancreas acinar cells transfected with various constructs of the putative PNLIP promoter showed that PNLIP transcription is significantly enhanced by PPARg dose-dependently, reaching maximal levels with multi PPRE sites. This effect was significantly augmented in the presence of PPARg agonists and reduced by PPARg antagonists or mutagenesis abrogating PPRE sites. Over-expression of PPARg significantly elevated PNLIP transcript and protein levels in AR42J cells and in primary pancreas cells. Moreover, PNLIP expression was up-regulated by PPARg agonists (pioglitazone and 15dPGJ2) and significantly down-regulated by PPARg antagonists in non-transfected rat exocrine pancreas AR42J cell line cells. Conclusion: PPARg transcriptionally regulates PNLIP gene expression. This transcript regulation resolves part of the missing link between dietary PUFA direct regulation of PNLIP.
Abstract ID: 2021. Pancreatic stellate cells increase cancer cell expression of HMGA2 in a 3D co-culture model of pancreatic cancer 2 € Jessica Norberg 1, Carina Strell 2, Andrea Balboni 1, Arne Ostman , Rainer €hr 1 Heuchel 1, Matthias Lo 1 Karolinska Institutet, Department of Clinical Intervention and Technology, Sweden 2 Karolinska Institutet, Department of Oncology and Pathology, Sweden
Introduction: In a cohort of PDAC patients, positivity for the High Mobility Group A-2 (HMGA2) protein has been correlated to lower survival. One of the factors known to increase the expression of HMGA2 in other cancer cells, is Transforming Growth Factor-B1 (TGFB1). Aims: The aim of this study was to investigate the mechanism behind increased HMGA2 in pancreatic cancer cells, and if this is mediated through interactions with stromal cells. Materials & methods: PDAC cells and PSCs were cultured in 3D, as mono- and co-cultures, in a newly developed model. Gene expression (mRNA) for cancer- and stellate cells within the co-culture model was investigated with real time PCR, looking at cell type specific expression. Protein expression was determined by immunohistochemistry (IHC). Results: Expression of TGFB1 mRNA increased in the co-cultured vs mono-cultured PSCs. HMGA2 was found to be higher expressed in the cocultured vs mono-cultured cancer cells, both at the mRNA and the protein level. TGFB1-treatment of cancer cell mono-cultures increased the expression of HMGA2. Conclusion: Increased expression of TGFB1 in the stellate cells in the 3D cultures leads to an increased expression of HMGA2 in the cancer cells. We are currently investigating this mechanism in more detail.
Ujjwal Mukund Mahajan 1, Enno Langhoff 2, Eithne Costello 3, William Greenhalf 3, Christopher Halloran 3, Georg Beyer 1, Frank Ulrich Weiss 2, John P. Neoptolemos 3, Markus W. Büchler 4, Thomas Kohlmann 5, Markus M. Lerch 2, Julia Mayerle 1 €t Medizinische Klinik und Poliklinik II, Klinikum der Universita München, Germany 2 Department of Medicine A, University Medicine Greifswald, Germany 3 NIHR Liverpool Pancreas Biomedical Research Centre, University of Liverpool, United Kingdom 4 Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Germany 5 Department of Community Medicine, University Medicine Greifswald, Germany 1
Introduction: Remodelling of the pancreatic cancer tumour (PDAC) microenvironment and extracellular matrix (ECM) has been implicated in the prognosis of PDAC. Aims: We investigated whether infiltration of immune cell subsets affect the survival of pancreatic cancer patients and affect stromal composition (fibrogenic (aSMA high/collagen-I high); fibrolytic stroma (aSMA high/collagen-I low). Materials & methods: TMA of 404 patients from the ESPAC-3 trial were immunostained for aSMA, collagen-I, CD3, CD4, CD8, CD68, CD206 and neutrophils. Median H-Scores of individual staining were calculated for all cores by multiplying the integrated density of tumor cell staining. Event free survival was plotted against low/high expression of leukocytes using Kaplan-Meier-curves, log-rank-tests, and Cox-proportional-hazardsmodels. All statistical tests were two-sided and performed in R. Results: In 1824 TMA from 382 patients (94.6%) immune cell composition was distinctly different in fibrolytic versus fibrogenic stroma. CD3 (Median EFS high-CD3-expression: 14.3 months vs. CD3-low-expreeesion 11 months, x2LR,1DF¼14.7; p¼0.0001) count were found as stroma type independent prognostic markers while CD206 and CD68 were dependent on the type of stroma. Recursive partitioning for discrete-time-survivaltree analysis showed CD3 as strongest independent predictor but macrophage subpopulations to determine fibrogenic or fibrolytic stroma with a median EFS ranging from 6.3 month (fibrolytic stroma with low-CD3 and high-CD68 count) to 20.1 month (fibrogenic stroma, CD3-high). Conclusion: The comprehensive prognostic signature based on interaction of immune cell infiltration with stromal composition provides better stratification in predicting event free survival in PDAC. Immune cells determine the predominant type of stromal composition and this may help in understanding the effect of the host immune response.
Abstract ID: 2024. Mitochondrial distribution and function in pancreatic ductal epithelial cells zga 2, La szlo th 1, Jo zsef Mal }s 1, Zsolt Ra Tretter 3, Emese To eth 1, R eka Erdo n Rakonczay, Jr. 4, P eter Hegyi 5 Zolta 1
University of Szeged, First Department of Medicine, Hungary University of Szeged, 2Department of Pathology, Hungary 3 Semmelwes University, Department of Medical Biochemistry, Hungary 4 University of Szeged, Department of Pathophysiology, Hungary 5 University of Szeged, MTA-SZTE Momentum Translational cs, Institute for Gastroenterology Research Group, University of Pe Transl, Hungary 2
Introduction: Mitochondrial dysfunction is a hallmark of several disease pathogenesis including acute pancreatitis (AP). Our previous results