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.

Biochemistry and Molecular Biology UPV EHU Leioa Spain

Cancer Cell Signaling and Metabolism Lab CIC bioGUNE Basque Research And Technology Alliance Derio Spain

Center for the Study of Liver and Gastrointestinal Diseases Carlos 3 National Institute of Health Madrid Spain

Clinical Research Vall d'Hebron Institute of Oncology Barcelona Spain

Comprehensive Liver Research Center at University of California Los Angeles California USA

Department of Anatomical Histological Forensic Medicine and Orthopaedics Sciences Sapienza University of Rome Rome Italy

Department of Biochemistry and Genetics School of Sciences University of Navarra Pamplona Spain

Department of Chemistry and Biochemistry Mendel University in Brno Brno Czech Republic

Department of Chemistry New York University New York New York USA

Department of Liver and Gastrointestinal Diseases Biogipuzkoa Health Research Institute Donostia University Hospital University of the Basque Country Donostia San Sebastian Spain

Department of Medicine David Geffen School of Medicine Los Angeles California USA

Experimental Hepatology and Drug Targeting IBSAL University of Salamanca Salamanca Spain

Hepatology Laboratory Solid Tumors Program CIMA CCUN IdiSNA Pamplona Navarra Spain

Ikerbasque Basque Foundation for Science Bilbao Spain

Immunology Ophthalmology and ENT Complutense University of Madrid Faculty of Medicine Madrid Comunidad de Madrid Spain

Institute of Pathology University of Regensburg Regensburg Germany

Liver Disease Lab CIC bioGUNE Basque Research and Technology Alliance Derio Bizkaia Spain

Medical Oncology Fundacion Jimenez Diaz University Hospital Madrid Spain Madrid Spain

Medical Surgical and Clinical Sciences University of Sassari Sassari Italy

Proteomics Platform Bizkaia Science and Technology Park Derio Spain

Proteomics Platform CIC bioGUNE ProteoRed ISCIII Bizkaia Science and Technology Park Derio Spain CIC bioGUNE Bizkaia Spain

SilenceTherapeutics GmbH Berlin Germany

START Madrid FJD Hospital Universitario Fundación Jiménez Díaz Madrid Spain

The Research Institute for Medicines Faculty of Pharmacy University of Lisbon Lisbon Portugal

Upper Gastrointestinal and Endocrine Tumor Unit VHIO Vall d'Hebron University Hospital Barcelona Spain

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