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Foxo3a pathway in human intrahepatic cholangiocarcinoma (CCA)
Abstracts / Digestive and Liver Disease 49S (2017) e1–e18
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Identification of Twinfilin-1 as a key regulator of cholangiocyte biological response to injury: Evidence for a possible role of ageing in the progression of cholangiopathies
Metformin reduces cell migration and down-regulates epithelial to mesenchymal transition (EMT) by AMPK/Foxo3a pathway in human intrahepatic cholangiocarcinoma (CCA)
L. Maroni 1 , C. Pinto 1 , D.M. Giordano 1 , S. Saccomanno 1 , C. Rychlicki 1 , L. Trozzi 1 , M.C. Albertini 2 , F. Orlando 3 , E. Melum 4 , J. Banales 5 , A. Benedetti 1 , G. Svegliati Baroni 1 , M. Marzioni 1
S. Di Matteo 1 , D. Costantini 1 , L. Nevi 1 , A. Lustri 1 , C. Napoletano 3 , J. Faccioli 1 , F. Giulitti 1 , E. Manzi 2 , A.M. DeRose 4 , M.C. Bragazzi 1 , G. Grazi 2 , P.B. Berloco 5 , F. Giuliante 4 , V. Cardinale 1 , G. Carpino 6 , D. Alvaro 1
1 Dipartimento di Gastroenterologia e Epatologia, Università Politecnica delle Marche, Ancona, Italy 2 Dipartimento Scienze Biomolecolari, Università degli Studi di Urbino “Carlo Bo”, Urbino, Italy 3 Servizio di Allevamento Sperimentazione Animale, Polo Scientifico Tecnologico, Falconara, Italy 4 Norwegian PSC Research Center, Division of Surgery, Inflammatory Medicine and Transplantation, Oslo University Hospital, Rikshospitalet, Oslo, Norway 5 Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute – Donostia University Hospital, Ikerbasque, CIBERehd, University of the Basque Country (UPV/EHU), San Sebastian, Spain
1 Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Rome, Italy 2 Gastroenterology Unit, Regina Elena National Cancer Institute, Rome, Italy 3 Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy 4 Hepatobiliary Unit, Catholic University of the Sacred Heart School of Medicine, Rome, Italy 5 Department of General Surgery and Organ Transplantation, Sapienza University of Rome, Rome, Italy 6 Department of Health Science, University of Rome “Foro Italico”, Rome, Italy
Introduction: Disorders of the biliary tree such as PBC, PSC or cholangiocarcinoma develop and progress differently according to the patient age. Similarly, biliary injury after liver transplantation is affected by donor age. It is not known yet whether the ageing process plays any role in the pathophysiology of biliary disorders. The aim of the study was to identify molecular pathways associated to the ageing process and to verify their role in cholangiocyte response to injury. Materials and methods: Expression of a number of age-related microRNAs (miRs) were evaluated in cholangiocytes of 2-month old (young) and 22-month old (old) mice, either subjected or not to 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-treatment, a model of sclerosing cholangitis. Common intracellular pathways and molecular targets of elevated miRs were identified by in silico analysis. Proliferation was assessed by sulforhodamine B (SRB) assay; senescence and senescence-associated secretory phenotype (SASP) markers (p16INK4a , p21WAF1/Cip1 , Il-1, Il-6, Igf-1, Pai-1) were evaluated in vitro by qPCR. Results: Ageing-related miR-1a, miR-30e, miR-93, miR-34a, miR-146b and miR-20a were up-regulated in cholangiocytes from DDC-treated mice compared to controls. In silico analysis identified Twinfilin-1 (Twf1) as a common target of the up-regulated miR-1a, miR-30e and miR-20a. Young mice subjected to DDC and old-untreated mice showed similar expression of Twf1 and related miRs in cholangiocytes, which was markedly increased as compared to young untreated ones. Twf1 and miRs expression was further increased in DDC-treated old mice. By immunofluorescence, intrahepatic cholangiocytes of PSC patients were positive for Twf1 expression. Knock-down of Twf1 by siRNAs in cultured cholangiocytes significantly reduced cell proliferation. In parallel, senescence and SASP markers were increased in Twf1-deficient cholangiocytes. Conclusions: Twf1 is as an important mediator of both cholangiocyte adaptation to ageing processes and response to injury. Our data suggest that disease and ageing might share common pathways, which may unveil novel markers of disease progression. http://dx.doi.org/10.1016/j.dld.2017.01.028
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CCA is an aggressive cancer resistance to chemotherapeutics. We demonstrated that CCA is enriched of cancer stem cells expressing EMT traits associated with aggressiveness and drug resistance. We established primary cell cultures from human IHCCA subtypes (mucin and mixed). Treatment with the anti-diabetic drug metformin has been associated with reduced cancer incidence. In immortalized cancer cell lines, metformin showed EMT inhibitory effects by up-regulating Foxo3a signaling. We aimed evaluating the effects of metformin on proliferation, apoptosis, cell migration and expression of EMT traits in primary cultures of CCA subtypes. CCA were treated with acute increasing metformin concentrations (5–1000 M, 1–4 days) and chronically at 10 M Metformin. We evaluated proliferation by MTS assay; apoptosis by Flow cytometry analysis of Annexin V-FITC/Propidium Iodide; and cell migration by wound-healing assay. The expression of Vimentin, E-Cadherin, SNAIL1/2, TWIST1, Cytokeratin19, FOXO3a and AMPK genes were analyzed by RT-qPCR, whereas FOXO3a, Cytokeratin19 and Vimentin were analyzed by Immunofluorescence Assay. Metformin significantly inhibited cell proliferation and induced apoptosis in primary cultures ofmucin- and mixed-IHCCA; the effects were dose- and time-dependent. The migration of IHCCA cells, from both mucin and mixed CCA subtypes, was also significantly reduced by acute treatment. The effects of metformin were associated with enhanced gene expression of the epithelial marker E-Cadherin and decreased expression of Vimentin and EMT specific genes, SNAIL1/2 and TWIST1. Metformin also increased the AMPK and FOXO3a mRNA levels. FOXO3a gene expression was negatively correlated with the expression of SNAIL1 and Vimentin genes. The FOXO3a protein migrates from cytoplasm to nucleus in metformin treated cells. After chronic treatments the Mucin-IHCCA showed a high expression of Cytocheratin19 and a very low expression of Vimentin. In conclusion, we demonstrated that metformin inhibits cell proliferation, enhances apoptosis and impairs EMT traits by activating Foxo3a in primary cultures of human CCA. Therefore, metformin could play anticancer effects against human CCAs with relevant therapeutic implications. http://dx.doi.org/10.1016/j.dld.2017.01.029
OC-24
OC-25
Identification of Twinfilin-1 as a key regulator of cholangiocyte biological response to injury: Evidence for a possible role of ageing in the progression of cholangiopathies
Metformin reduces cell migration and down-regulates epithelial to mesenchymal transition (EMT) by AMPK/Foxo3a pathway in human intrahepatic cholangiocarcinoma (CCA)
L. Maroni 1 , C. Pinto 1 , D.M. Giordano 1 , S. Saccomanno 1 , C. Rychlicki 1 , L. Trozzi 1 , M.C. Albertini 2 , F. Orlando 3 , E. Melum 4 , J. Banales 5 , A. Benedetti 1 , G. Svegliati Baroni 1 , M. Marzioni 1
S. Di Matteo 1 , D. Costantini 1 , L. Nevi 1 , A. Lustri 1 , C. Napoletano 3 , J. Faccioli 1 , F. Giulitti 1 , E. Manzi 2 , A.M. DeRose 4 , M.C. Bragazzi 1 , G. Grazi 2 , P.B. Berloco 5 , F. Giuliante 4 , V. Cardinale 1 , G. Carpino 6 , D. Alvaro 1
1 Dipartimento di Gastroenterologia e Epatologia, Università Politecnica delle Marche, Ancona, Italy 2 Dipartimento Scienze Biomolecolari, Università degli Studi di Urbino “Carlo Bo”, Urbino, Italy 3 Servizio di Allevamento Sperimentazione Animale, Polo Scientifico Tecnologico, Falconara, Italy 4 Norwegian PSC Research Center, Division of Surgery, Inflammatory Medicine and Transplantation, Oslo University Hospital, Rikshospitalet, Oslo, Norway 5 Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute – Donostia University Hospital, Ikerbasque, CIBERehd, University of the Basque Country (UPV/EHU), San Sebastian, Spain
1 Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Rome, Italy 2 Gastroenterology Unit, Regina Elena National Cancer Institute, Rome, Italy 3 Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy 4 Hepatobiliary Unit, Catholic University of the Sacred Heart School of Medicine, Rome, Italy 5 Department of General Surgery and Organ Transplantation, Sapienza University of Rome, Rome, Italy 6 Department of Health Science, University of Rome “Foro Italico”, Rome, Italy
Introduction: Disorders of the biliary tree such as PBC, PSC or cholangiocarcinoma develop and progress differently according to the patient age. Similarly, biliary injury after liver transplantation is affected by donor age. It is not known yet whether the ageing process plays any role in the pathophysiology of biliary disorders. The aim of the study was to identify molecular pathways associated to the ageing process and to verify their role in cholangiocyte response to injury. Materials and methods: Expression of a number of age-related microRNAs (miRs) were evaluated in cholangiocytes of 2-month old (young) and 22-month old (old) mice, either subjected or not to 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-treatment, a model of sclerosing cholangitis. Common intracellular pathways and molecular targets of elevated miRs were identified by in silico analysis. Proliferation was assessed by sulforhodamine B (SRB) assay; senescence and senescence-associated secretory phenotype (SASP) markers (p16INK4a , p21WAF1/Cip1 , Il-1, Il-6, Igf-1, Pai-1) were evaluated in vitro by qPCR. Results: Ageing-related miR-1a, miR-30e, miR-93, miR-34a, miR-146b and miR-20a were up-regulated in cholangiocytes from DDC-treated mice compared to controls. In silico analysis identified Twinfilin-1 (Twf1) as a common target of the up-regulated miR-1a, miR-30e and miR-20a. Young mice subjected to DDC and old-untreated mice showed similar expression of Twf1 and related miRs in cholangiocytes, which was markedly increased as compared to young untreated ones. Twf1 and miRs expression was further increased in DDC-treated old mice. By immunofluorescence, intrahepatic cholangiocytes of PSC patients were positive for Twf1 expression. Knock-down of Twf1 by siRNAs in cultured cholangiocytes significantly reduced cell proliferation. In parallel, senescence and SASP markers were increased in Twf1-deficient cholangiocytes. Conclusions: Twf1 is as an important mediator of both cholangiocyte adaptation to ageing processes and response to injury. Our data suggest that disease and ageing might share common pathways, which may unveil novel markers of disease progression. http://dx.doi.org/10.1016/j.dld.2017.01.028
e13
CCA is an aggressive cancer resistance to chemotherapeutics. We demonstrated that CCA is enriched of cancer stem cells expressing EMT traits associated with aggressiveness and drug resistance. We established primary cell cultures from human IHCCA subtypes (mucin and mixed). Treatment with the anti-diabetic drug metformin has been associated with reduced cancer incidence. In immortalized cancer cell lines, metformin showed EMT inhibitory effects by up-regulating Foxo3a signaling. We aimed evaluating the effects of metformin on proliferation, apoptosis, cell migration and expression of EMT traits in primary cultures of CCA subtypes. CCA were treated with acute increasing metformin concentrations (5–1000 M, 1–4 days) and chronically at 10 M Metformin. We evaluated proliferation by MTS assay; apoptosis by Flow cytometry analysis of Annexin V-FITC/Propidium Iodide; and cell migration by wound-healing assay. The expression of Vimentin, E-Cadherin, SNAIL1/2, TWIST1, Cytokeratin19, FOXO3a and AMPK genes were analyzed by RT-qPCR, whereas FOXO3a, Cytokeratin19 and Vimentin were analyzed by Immunofluorescence Assay. Metformin significantly inhibited cell proliferation and induced apoptosis in primary cultures ofmucin- and mixed-IHCCA; the effects were dose- and time-dependent. The migration of IHCCA cells, from both mucin and mixed CCA subtypes, was also significantly reduced by acute treatment. The effects of metformin were associated with enhanced gene expression of the epithelial marker E-Cadherin and decreased expression of Vimentin and EMT specific genes, SNAIL1/2 and TWIST1. Metformin also increased the AMPK and FOXO3a mRNA levels. FOXO3a gene expression was negatively correlated with the expression of SNAIL1 and Vimentin genes. The FOXO3a protein migrates from cytoplasm to nucleus in metformin treated cells. After chronic treatments the Mucin-IHCCA showed a high expression of Cytocheratin19 and a very low expression of Vimentin. In conclusion, we demonstrated that metformin inhibits cell proliferation, enhances apoptosis and impairs EMT traits by activating Foxo3a in primary cultures of human CCA. Therefore, metformin could play anticancer effects against human CCAs with relevant therapeutic implications. http://dx.doi.org/10.1016/j.dld.2017.01.029