Maite Merino-Azpitarte1, Elisa Lozano2, María J Perugorria3, Aitor Esparza-Baquer1, Oihane Erice1, Álvaro Santos-Laso1, Colm J O'Rourke4, Jesper B Andersen4, Raúl Jiménez-Agüero1, Adelaida Lacasta1, Mauro D'Amato5, Óscar Briz6, Nidhi Jalan-Sakrikar7, Robert C Huebert7, Kristen M Thelen8, Sergio A Gradilone8, Ana M Aransay9, José L Lavín10, Maite G Fernández-Barrena11, Ander Matheu12, Marco Marzioni13, Gregory J Gores7, Luis Bujanda14, José J G Marin6, Jesús M Banales15. 1. Department of Liver and Gastrointestinal Diseases, Biodonostia Research Institute - Donostia University Hospital, University of the Basque Country (UPV/EHU), San Sebastian, Spain. 2. Department of Liver and Gastrointestinal Diseases, Biodonostia Research Institute - Donostia University Hospital, University of the Basque Country (UPV/EHU), San Sebastian, Spain; Experimental Hepatology and Drug Targeting (HEVEFARM), Biomedical Research Institute of Salamanca (IBSAL), University of Salamanca, Salamanca, Spain; National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd, Instituto de Salud Carlos III), Spain. 3. Department of Liver and Gastrointestinal Diseases, Biodonostia Research Institute - Donostia University Hospital, University of the Basque Country (UPV/EHU), San Sebastian, Spain; National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd, Instituto de Salud Carlos III), Spain; IKERBASQUE, Basque Foundation for Science, Bilbao, Spain. 4. Biotech Research and Innovation Centre, Department of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. 5. Department of Liver and Gastrointestinal Diseases, Biodonostia Research Institute - Donostia University Hospital, University of the Basque Country (UPV/EHU), San Sebastian, Spain; IKERBASQUE, Basque Foundation for Science, Bilbao, Spain. 6. Experimental Hepatology and Drug Targeting (HEVEFARM), Biomedical Research Institute of Salamanca (IBSAL), University of Salamanca, Salamanca, Spain; National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd, Instituto de Salud Carlos III), Spain. 7. Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA. 8. The Hormel Institute, University of Minnesota, Austin, MN, USA. 9. National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd, Instituto de Salud Carlos III), Spain; Genome Analysis Platform, CIC bioGUNE, Bizkaia Technology Park, Derio, Spain. 10. Genome Analysis Platform, CIC bioGUNE, Bizkaia Technology Park, Derio, Spain. 11. Hepatology Program, CIMA of the University of Navarra, Pamplona, Spain. 12. IKERBASQUE, Basque Foundation for Science, Bilbao, Spain; Neuro-Oncology Group, Biodonostia Research Institute - Donostia University Hospital, San Sebastian, Spain. 13. Department of Gastroenterology, "Università Politecnica delle Marche", Ancona, Italy. 14. Department of Liver and Gastrointestinal Diseases, Biodonostia Research Institute - Donostia University Hospital, University of the Basque Country (UPV/EHU), San Sebastian, Spain; National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd, Instituto de Salud Carlos III), Spain. 15. Department of Liver and Gastrointestinal Diseases, Biodonostia Research Institute - Donostia University Hospital, University of the Basque Country (UPV/EHU), San Sebastian, Spain; National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd, Instituto de Salud Carlos III), Spain; IKERBASQUE, Basque Foundation for Science, Bilbao, Spain. Electronic address: jesus.banales@biodonostia.org.
Abstract
BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a biliary malignancy linked to genetic and epigenetic abnormalities, such as hypermethylation of SOX17 promoter. Here, the role of SOX17 in cholangiocyte differentiation and cholangiocarcinogenesis was studied. METHODS: SOX17 expression/function was evaluated along the differentiation of human induced pluripotent stem cells (iPSC) into cholangiocytes, in the dedifferentiation process of normal human cholangiocytes (NHC) in culture and in cholangiocarcinogenesis. Lentiviruses for SOX17 overexpression or knockdown were used. Gene expression and DNA methylation profiling were performed. RESULTS: SOX17 expression is induced in the last stage of cholangiocyte differentiation from iPSC and regulates the acquisition of biliary markers. SOX17 becomes downregulated in NHC undergoing dedifferentiation; experimental SOX17 knockdown in differentiated NHC downregulated biliary markers and promoted baseline and Wnt-dependent proliferation. SOX17 expression is lower in human CCA than in healthy tissue, which correlates with worse survival after tumor resection. In CCA cells, SOX17 overexpression decreased their tumorigenic capacity in murine xenograft models, which was related to increased oxidative stress and apoptosis. In contrast, SOX17 overexpression in NHC did not affect their survival but inhibited their baseline proliferation. In CCA cells, SOX17 inhibited migration, anchorage-independent growth and Wnt/β-catenin-dependent proliferation, and restored the expression of biliary markers and primary cilium length. In human CCA, SOX17 promoter was found hypermethylated and its expression inversely correlates with the methylation grade. In NHC, Wnt3a decreased SOX17 expression in a DNMT-dependent manner, whereas in CCA, DNMT1 inhibition or silencing upregulated SOX17. CONCLUSIONS: SOX17 regulates the differentiation and maintenance of the biliary phenotype and functions as a tumor suppressor for CCA, being a potential prognostic marker and a promising therapeutic target. LAY SUMMARY: Understanding the molecular mechanisms involved in the pathogenesis of CCA is key in finding new valuable diagnostic and prognostic biomarkers, as well as therapeutic targets. This study provides evidence that SOX17 regulates the differentiation and maintenance of the biliary phenotype, and its downregulation promotes their tumorigenic transformation. SOX17 acts as a tumor suppressor in CCA and its genetic, molecular and/or pharmacological restoration may represent a new promising therapeutic strategy. Moreover, SOX17 expression correlates with the outcome of patients after tumor resection, being a potential prognostic biomarker.
BACKGROUND & AIMS:Cholangiocarcinoma (CCA) is a biliary malignancy linked to genetic and epigenetic abnormalities, such as hypermethylation of SOX17 promoter. Here, the role of SOX17 in cholangiocyte differentiation and cholangiocarcinogenesis was studied. METHODS:SOX17 expression/function was evaluated along the differentiation of human induced pluripotent stem cells (iPSC) into cholangiocytes, in the dedifferentiation process of normal human cholangiocytes (NHC) in culture and in cholangiocarcinogenesis. Lentiviruses for SOX17 overexpression or knockdown were used. Gene expression and DNA methylation profiling were performed. RESULTS:SOX17 expression is induced in the last stage of cholangiocyte differentiation from iPSC and regulates the acquisition of biliary markers. SOX17 becomes downregulated in NHC undergoing dedifferentiation; experimental SOX17 knockdown in differentiated NHC downregulated biliary markers and promoted baseline and Wnt-dependent proliferation. SOX17 expression is lower in human CCA than in healthy tissue, which correlates with worse survival after tumor resection. In CCA cells, SOX17 overexpression decreased their tumorigenic capacity in murine xenograft models, which was related to increased oxidative stress and apoptosis. In contrast, SOX17 overexpression in NHC did not affect their survival but inhibited their baseline proliferation. In CCA cells, SOX17 inhibited migration, anchorage-independent growth and Wnt/β-catenin-dependent proliferation, and restored the expression of biliary markers and primary cilium length. In human CCA, SOX17 promoter was found hypermethylated and its expression inversely correlates with the methylation grade. In NHC, Wnt3a decreased SOX17 expression in a DNMT-dependent manner, whereas in CCA, DNMT1 inhibition or silencing upregulated SOX17. CONCLUSIONS:SOX17 regulates the differentiation and maintenance of the biliary phenotype and functions as a tumor suppressor for CCA, being a potential prognostic marker and a promising therapeutic target. LAY SUMMARY: Understanding the molecular mechanisms involved in the pathogenesis of CCA is key in finding new valuable diagnostic and prognostic biomarkers, as well as therapeutic targets. This study provides evidence that SOX17 regulates the differentiation and maintenance of the biliary phenotype, and its downregulation promotes their tumorigenic transformation. SOX17 acts as a tumor suppressor in CCA and its genetic, molecular and/or pharmacological restoration may represent a new promising therapeutic strategy. Moreover, SOX17 expression correlates with the outcome of patients after tumor resection, being a potential prognostic biomarker.
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