{
  "abstract": "Background and objective Cholangiocarcinoma (CCA) is a heterogeneous neoplasm of the biliary epithelium that easily infiltrates, metastasises and recurs. Magnesium disbalance is a hallmark of CCA, with the magnesium transporter cyclin M4 (CNNM4) being a key driver of various hepatic diseases. This study aims to unravel the role of CNNM4 in the initiation and progression of CCA.Design CNNM4 protein and gene expression were assessed in vitro, in vivo and in patients with CCA. Silencing of CNNM4 was effectively achieved by using small interfering RNA (siRNA) or short hairpin RNA in CCA cell lines and GalNAc-conjugated siRNA in a transposon-based CCA mice model. The impact of CNNM4 on tumour cell proliferation, migration and invasion to the lungs was evaluated using the chicken chorioallantoic membrane model. Proteomic analysis was employed to elucidate the underlying molecular mechanisms.Results CNNM4 was upregulated in CCA samples from humans, mice and cell lines. Functional studies demonstrated that CNNM4 deficiency attenuates cell growth, chemoresistance, migration, invasion, cancer stem cell properties and Warburg effect in vitro and in vivo. Proteomic analysis identified nuclear protein 1 as an upstream regulator of CNNM4-induced ferroptosis in CCA, ultimately leading to cell death. The iron chelator deferiprone could reverse the decreased proliferation induced by CNNM4 silencing, while inhibition of the heme oxygenase-1 by zinc protoporphyrin IX affected only the growth of cells with no targeted CNNM4 inhibition, highlighting the specificity of ferroptosis in CNNM4-associated effects.Conclusion This study reveals that increased CNNM4 expression drives CCA progression and malignancy and that its inhibition may be an effective therapeutic strategy to limit proliferation and metastasis in patients with CCA.",
  "authors": [
    {
      "affiliations": [
        "Liver Disease Lab, CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Derio, Bizkaia, Spain"
      ],
      "name": "Maria Mercado-Gómez"
    },
    {
      "affiliations": [
        "Liver Disease Lab, CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Derio, Bizkaia, Spain",
        "Center for the Study of Liver and Gastrointestinal Diseases (CIBERehd), Carlos III National Institute of Health, Madrid, Spain"
      ],
      "name": "Naroa Goikoetxea-Usandizaga"
    },
    {
      "affiliations": [
        "Liver Disease Lab, CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Derio, Bizkaia, Spain"
      ],
      "name": "Alvaro Eguileor Giné"
    },
    {
      "affiliations": [
        "Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, Czech Republic"
      ],
      "name": "Miguel A Merlos Rodrigo"
    },
    {
      "affiliations": [
        "The Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal"
      ],
      "name": "Marta Bento Afonso"
    },
    {
      "affiliations": [
        "Proteomics Platform, Bizkaia Science and Technology Park, Derio, Spain"
      ],
      "name": "Mikel Azkargorta"
    },
    {
      "affiliations": [
        "Liver Disease Lab, CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Derio, Bizkaia, Spain"
      ],
      "name": "Leidy Estefanía Zapata-Pavas"
    },
    {
      "affiliations": [
        "Liver Disease Lab, CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Derio, Bizkaia, Spain"
      ],
      "name": "Claudia M Rejano-Gordillo"
    },
    {
      "affiliations": [
        "Center for the Study of Liver and Gastrointestinal Diseases (CIBERehd), Carlos III National Institute of Health, Madrid, Spain",
        "Experimental Hepatology and Drug Targeting (HEVEPHARM), IBSAL, University of Salamanca, Salamanca, Spain"
      ],
      "name": "Marta R Romero"
    },
    {
      "affiliations": [
        "Cancer Cell Signaling and Metabolism Lab, CIC bioGUNE, Basque Research And Technology Alliance (BRTA), Derio, Spain",
        "Ikerbasque Basque Foundation for Science, Bilbao, Spain"
      ],
      "name": "Isabel Mendizabal"
    },
    {
      "affiliations": [
        "Center for the Study of Liver and Gastrointestinal Diseases (CIBERehd), Carlos III National Institute of Health, Madrid, Spain",
        "Ikerbasque Basque Foundation for Science, Bilbao, Spain",
        "Department of Liver and Gastrointestinal Diseases, Biogipuzkoa Health Research Institute, Donostia University Hospital, University of the Basque Country (UPV/EHU), Donostia-San Sebastian, Spain"
      ],
      "name": "Pedro M Rodrigues"
    },
    {
      "affiliations": [
        "Immunology, Ophthalmology and ENT, Complutense University of Madrid Faculty of Medicine, Madrid, Comunidad de Madrid, Spain"
      ],
      "name": "Hanghang Wu"
    },
    {
      "affiliations": [
        "Liver Disease Lab, CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Derio, Bizkaia, Spain"
      ],
      "name": "Rubén Rodríguez-Agudo"
    },
    {
      "affiliations": [
        "Liver Disease Lab, CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Derio, Bizkaia, Spain"
      ],
      "name": "Marina Serrano-Maciá"
    },
    {
      "affiliations": [
        "Department of Liver and Gastrointestinal Diseases, Biogipuzkoa Health Research Institute, Donostia University Hospital, University of the Basque Country (UPV/EHU), Donostia-San Sebastian, Spain"
      ],
      "name": "Paula Olaizola"
    },
    {
      "affiliations": [
        "Liver Disease Lab, CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Derio, Bizkaia, Spain"
      ],
      "name": "Jon Ander Barrenechea-Barrenechea"
    },
    {
      "affiliations": [
        "Liver Disease Lab, CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Derio, Bizkaia, Spain"
      ],
      "name": "Irene González Recio"
    },
    {
      "affiliations": [
        "Center for the Study of Liver and Gastrointestinal Diseases (CIBERehd), Carlos III National Institute of Health, Madrid, Spain",
        "Hepatology Laboratory Solid Tumors Program CIMA CCUN IdiSNA, Pamplona, Navarra, Spain"
      ],
      "name": "Maite G Fernandez-Barrena"
    },
    {
      "affiliations": [
        "Department of Anatomical, Histological, Forensic Medicine and Orthopaedics Sciences, Sapienza University of Rome, Rome, Italy"
      ],
      "name": "Diletta Overi"
    },
    {
      "affiliations": [
        "Department of Anatomical, Histological, Forensic Medicine and Orthopaedics Sciences, Sapienza University of Rome, Rome, Italy"
      ],
      "name": "Eugenio Gaudio"
    },
    {
      "affiliations": [
        "SilenceTherapeutics GmbH, Berlin, Germany"
      ],
      "name": "Ute Schaeper"
    },
    {
      "affiliations": [
        "Cancer Cell Signaling and Metabolism Lab, CIC bioGUNE, Basque Research And Technology Alliance (BRTA), Derio, Spain"
      ],
      "name": "Saioa Garcia-Longarte"
    },
    {
      "affiliations": [
        "Upper Gastrointestinal and Endocrine Tumor Unit, VHIO, Vall d’Hebron University Hospital, Barcelona, Spain",
        "Clinical Research, Vall d’Hebron Institute of Oncology, Barcelona, Spain"
      ],
      "name": "Mariana Yáñez-Bartolomé"
    },
    {
      "affiliations": [
        "Liver Disease Lab, CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Derio, Bizkaia, Spain"
      ],
      "name": "Patricia Peña-SanFelix"
    },
    {
      "affiliations": [
        "Liver Disease Lab, CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Derio, Bizkaia, Spain"
      ],
      "name": "Clàudia Gil-Pitarch"
    },
    {
      "affiliations": [
        "Department of Liver and Gastrointestinal Diseases, Biogipuzkoa Health Research Institute, Donostia University Hospital, University of the Basque Country (UPV/EHU), Donostia-San Sebastian, Spain"
      ],
      "name": "Ainhoa Lapitz"
    },
    {
      "affiliations": [
        "Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, Czech Republic"
      ],
      "name": "Hana Michalkova"
    },
    {
      "affiliations": [
        "Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, Czech Republic"
      ],
      "name": "Zbynek Heger"
    },
    {
      "affiliations": [
        "Liver Disease Lab, CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Derio, Bizkaia, Spain"
      ],
      "name": "Carolina Conter"
    },
    {
      "affiliations": [
        "Center for the Study of Liver and Gastrointestinal Diseases (CIBERehd), Carlos III National Institute of Health, Madrid, Spain",
        "Experimental Hepatology and Drug Targeting (HEVEPHARM), IBSAL, University of Salamanca, Salamanca, Spain"
      ],
      "name": "Rocio I R Macias"
    },
    {
      "affiliations": [
        "Cancer Cell Signaling and Metabolism Lab, CIC bioGUNE, Basque Research And Technology Alliance (BRTA), Derio, Spain",
        "Ikerbasque Basque Foundation for Science, Bilbao, Spain"
      ],
      "name": "Arkaitz Carracedo"
    },
    {
      "affiliations": [
        "Center for the Study of Liver and Gastrointestinal Diseases (CIBERehd), Carlos III National Institute of Health, Madrid, Spain",
        "Ikerbasque Basque Foundation for Science, Bilbao, Spain",
        "Department of Liver and Gastrointestinal Diseases, Biogipuzkoa Health Research Institute, Donostia University Hospital, University of the Basque Country (UPV/EHU), Donostia-San Sebastian, Spain",
        "Department of Biochemistry and Genetics, School of Sciences, University of Navarra, Pamplona, Spain"
      ],
      "name": "Jesús Bañales"
    },
    {
      "affiliations": [
        "START Madrid-FJD, Hospital Universitario Fundación Jiménez Díaz, Madrid, Spain"
      ],
      "name": "Victor Moreno"
    },
    {
      "affiliations": [
        "Medical Oncology, Fundacion Jimenez Diaz University Hospital, Madrid, Spain, Madrid, Spain"
      ],
      "name": "Angela Lamarca"
    },
    {
      "affiliations": [
        "Department of Medicine, David Geffen School of Medicine, Los Angeles, California, USA",
        "Comprehensive Liver Research Center at University of California, Los Angeles, California, USA"
      ],
      "name": "Rajat Singh"
    },
    {
      "affiliations": [
        "Liver Disease Lab, CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Derio, Bizkaia, Spain"
      ],
      "name": "Teresa Cardoso Delgado"
    },
    {
      "affiliations": [
        "Liver Disease Lab, CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Derio, Bizkaia, Spain"
      ],
      "name": "Luis Alfonso Martínez-Cruz"
    },
    {
      "affiliations": [
        "Proteomics Platform, CIC bioGUNE, ProteoRed-ISCIII, Bizkaia Science and Technology Park, Derio, Spain, CIC bioGUNE, Bizkaia, Spain"
      ],
      "name": "Felix Elortza"
    },
    {
      "affiliations": [
        "Center for the Study of Liver and Gastrointestinal Diseases (CIBERehd), Carlos III National Institute of Health, Madrid, Spain",
        "Hepatology Laboratory Solid Tumors Program CIMA CCUN IdiSNA, Pamplona, Navarra, Spain"
      ],
      "name": "Matias A Avila"
    },
    {
      "affiliations": [
        "Biochemistry and Molecular Biology, UPV/EHU, Leioa, Spain"
      ],
      "name": "César Martín"
    },
    {
      "affiliations": [
        "Upper Gastrointestinal and Endocrine Tumor Unit, VHIO, Vall d’Hebron University Hospital, Barcelona, Spain",
        "Clinical Research, Vall d’Hebron Institute of Oncology, Barcelona, Spain"
      ],
      "name": "Tian V. Tian"
    },
    {
      "affiliations": [
        "Upper Gastrointestinal and Endocrine Tumor Unit, VHIO, Vall d’Hebron University Hospital, Barcelona, Spain",
        "Clinical Research, Vall d’Hebron Institute of Oncology, Barcelona, Spain"
      ],
      "name": "Teresa Macarulla"
    },
    {
      "affiliations": [
        "Department of Chemistry, New York University, New York, New York, USA"
      ],
      "name": "Daniela Buccella"
    },
    {
      "affiliations": [
        "Center for the Study of Liver and Gastrointestinal Diseases (CIBERehd), Carlos III National Institute of Health, Madrid, Spain",
        "Immunology, Ophthalmology and ENT, Complutense University of Madrid Faculty of Medicine, Madrid, Comunidad de Madrid, Spain"
      ],
      "name": "Francisco Javier Cubero"
    },
    {
      "affiliations": [
        "Institute of Pathology, University of Regensburg, Regensburg, Germany",
        "Medical, Surgical, and Clinical Sciences, University of Sassari, Sassari, Italy"
      ],
      "name": "Diego F Calvisi"
    },
    {
      "affiliations": [
        "Department of Anatomical, Histological, Forensic Medicine and Orthopaedics Sciences, Sapienza University of Rome, Rome, Italy"
      ],
      "name": "Guido Carpino"
    },
    {
      "affiliations": [
        "Center for the Study of Liver and Gastrointestinal Diseases (CIBERehd), Carlos III National Institute of Health, Madrid, Spain",
        "Experimental Hepatology and Drug Targeting (HEVEPHARM), IBSAL, University of Salamanca, Salamanca, Spain"
      ],
      "name": "Jose J G Marin"
    },
    {
      "affiliations": [
        "The Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal"
      ],
      "name": "Cecília M P Rodrigues"
    },
    {
      "affiliations": [
        "Liver Disease Lab, CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Derio, Bizkaia, Spain",
        "Center for the Study of Liver and Gastrointestinal Diseases (CIBERehd), Carlos III National Institute of Health, Madrid, Spain"
      ],
      "name": "Maria Luz Martinez-Chantar"
    }
  ],
  "title": "Role of CNNM4 in the progression of cholangiocarcinoma: implications for ferroptosis and therapeutic potential",
  "uid": "be323fd6-c2df-5d58-8aa3-8e4936cedc6d"
}