Dissecting the role of the NADPH oxidase NOX4 in TGF-beta signaling in hepatocellular carcinoma
Rut Espinosa-Sotelo,
Noel P. Fusté,
Irene Peñuelas-Haro,
Ania Alay,
Gabriel Pons,
Xènia Almodóvar,
Júlia Albaladejo,
Ismael Sánchez-Vera,
Ricard Bonilla-Amadeo,
Francesco Dituri,
Grazia Serino,
Emilio Ramos,
Teresa Serrano,
Mariona Calvo,
María Luz Martínez-Chantar,
Gianluigi Giannelli,
Esther Bertran,
Isabel Fabregat
Affiliations
Rut Espinosa-Sotelo
TGF-β and Cancer Group, Oncobell Program, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain; CIBEREHD, ISCIII, Spain
Noel P. Fusté
TGF-β and Cancer Group, Oncobell Program, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain
Irene Peñuelas-Haro
TGF-β and Cancer Group, Oncobell Program, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain; CIBEREHD, ISCIII, Spain
Ania Alay
Unit of Bioinformatics for Precision Oncology, Catalan Institute of Oncology (ICO), L'Hospitalet de Llobregat, Barcelona, Spain; Preclinical and Experimental Research in Thoracic Tumors (PReTT), Oncobell Program, IDIBELL, L'Hospitalet de Llobregat, Spain
Gabriel Pons
Physiological Sciences Department, University of Barcelona, Oncobell-IDIBELL, Barcelona, Spain
Xènia Almodóvar
TGF-β and Cancer Group, Oncobell Program, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain
Júlia Albaladejo
TGF-β and Cancer Group, Oncobell Program, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain
Ismael Sánchez-Vera
Physiological Sciences Department, University of Barcelona, Oncobell-IDIBELL, Barcelona, Spain
Ricard Bonilla-Amadeo
TGF-β and Cancer Group, Oncobell Program, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain
Francesco Dituri
National Institute of Gastroenterology, IRCCS Saverio De Bellis Research Hospital, Castellana Wrotte, Bari, Italy
Grazia Serino
National Institute of Gastroenterology, IRCCS Saverio De Bellis Research Hospital, Castellana Wrotte, Bari, Italy
Emilio Ramos
CIBEREHD, ISCIII, Spain; Department of Surgery, Liver Transplant Unit, University Hospital of Bellvitge and Faculty of Medicine and Health Sciences, University of Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain
Teresa Serrano
CIBEREHD, ISCIII, Spain; Pathological Anatomy Service, University Hospital of Bellvitge and Faculty of Medicine and Health Sciences, University of Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain
CIBEREHD, ISCIII, Spain; Center for Cooperative Research in Biosciences (CIC BioGUNE), Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Derio, Bizkaia, Spain
Gianluigi Giannelli
National Institute of Gastroenterology, IRCCS Saverio De Bellis Research Hospital, Castellana Wrotte, Bari, Italy
Esther Bertran
TGF-β and Cancer Group, Oncobell Program, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain; CIBEREHD, ISCIII, Spain
Isabel Fabregat
TGF-β and Cancer Group, Oncobell Program, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain; CIBEREHD, ISCIII, Spain; Corresponding author. IDIBELL, Gran Vía de l’Hospitalet, 199, 08908, L'Hospitalet de Llobregat, Barcelona, Spain.
The NADPH oxidase NOX4 has been proposed as necessary for the apoptosis induced by the Transforming Growth Factor-beta (TGF-β) in hepatocytes and hepatocellular carcinoma (HCC) cells. However, whether NOX4 is required for TGF-β-induced canonical (SMADs) or non-canonical signals is not fully understood yet, neither its potential involvement in other parallel actions induced by TGF-β. In this work we have used CRISPR Cas9 technology to stable attenuate NOX4 expression in HCC cells. Results have indicated that NOX4 is required for an efficient SMAD2/3 phosphorylation in response to TGF-β, whereas non-canonical signals, such as the phosphorylation of the Epidermal Growth Receptor or AKT, are higher in NOX4 silenced cells. TGF-β-mediated inhibition of cell proliferation and viability is attenuated in NOX4 silenced cells, correlating with decreased response in terms of apoptosis, and maintenance of high expression of MYC and CYCLIN D1. These results would indicate that NOX4 is required for all the tumor suppressor actions of TGF-β in HCC. However, analysis in human HCC tumors has revealed a worse prognosis for patients showing high expression of TGF-β1-related genes concomitant with high expression of NOX4. Deepening into other tumorigenic actions of TGF-β that may contribute to tumor progression, we found that NOX4 is also required for TGF-β-induced migratory effects. The Epithelial-Mesenchymal transition (EMT) program does not appear to be affected by attenuation of NOX4 levels. However, TGF-β-mediated regulation of cytoskeleton dynamics and focal adhesions require NOX4, which is necessary for TGF-β-induced increase in the chaperone Hsp27 and correct subcellular localization of Hic-5 within focal adhesions, as well for upregulation of the metalloprotease MMP9. All these results together point to NOX4 as a key element in the whole TGF-β signaling in HCC cells, revealing an unknown role for NOX4 as tumor promoter in HCC patients presenting activation of the TGF-β pathway.
