Sabrina Campisano1, Esther Bertran2, Daniel Caballero-Díaz2, Anabela La Colla3, Isabel Fabregat4, Andrea Nancy Chisari5. 1. TGF-β and Cancer Group, Oncobell Program, Bellvitge Biomedical Research Institute (IDIBELL), Gran Via de l'Hospitalet 199, 08908 Barcelona, Spain. 2. TGF-β and Cancer Group, Oncobell Program, Bellvitge Biomedical Research Institute (IDIBELL), Gran Via de l'Hospitalet 199, 08908 Barcelona, Spain; Oncology Program, CIBEREHD, National Biomedical Research Institute on Liver and Gastrointestinal Diseases, Instituto de Salud Carlos III, 28029 Madrid, Spain. 3. Department of Chemistry, School of Sciences, National University of Mar del Plata, Dean Funes 3350, B7602AYL Buenos Aires, Argentina. 4. TGF-β and Cancer Group, Oncobell Program, Bellvitge Biomedical Research Institute (IDIBELL), Gran Via de l'Hospitalet 199, 08908 Barcelona, Spain; Oncology Program, CIBEREHD, National Biomedical Research Institute on Liver and Gastrointestinal Diseases, Instituto de Salud Carlos III, 28029 Madrid, Spain; Department of Physiological Sciences, School of Medicine, University of Barcelona, 08907, Spain. Electronic address: ifabregat@idibell.cat. 5. Department of Chemistry, School of Sciences, National University of Mar del Plata, Dean Funes 3350, B7602AYL Buenos Aires, Argentina. Electronic address: achisari@mdp.edu.ar.
Abstract
BACKGROUND: The NADPH oxidase (NOX) 4 is an important source of ROS in signal transduction that acts as a liver tumor suppressor. Transforming Growth Factor β (TGF-β) and Epidermal Growth Factor Receptor (EGFR) pathways are involved in the modulation of NOX4 expression. Data showed that recurrent protein deprivation induces changes distinctive of a preneoplastic profile. However, the mechanisms underneath these changes have not been completely understood. METHODS: Hepatocytes that survived to the lack of amino acids (Aa) (Sel line) were cultured in complete or Aa free medium. We elucidated the molecular mechanisms that support such preneoplastic alterations employing biochemical and molecular biology assays. RESULTS: Sel line showed increased phospho-AKT and phospho-ERKs levels, diminished caspase-3 activity, augmented cell proliferation and overactivation of EGFR pathway, reminiscent of a preneoplastic phenotype. NOX4 was upregulated in these cells by TGF-β canonical pathway, however ROS levels were not found increased as a result of an increment of antioxidant enzymes. Inhibition of TGF-β receptor diminished NOX4 and strikingly, after EGFR inhibition, NOX4 levels also decreased. Therefore, both TGF-β and EGFR pathways are shown to be involved in the upregulation of NOX4 in Sel line. CONCLUSIONS: This work provides novel results regarding to the regulation of NOX4 in the preneoplastic transformation of hepatocytes in the absence of Aa, in the context of TGF-β and EGFR pathways. GENERAL SIGNIFICANCE: The advances in the understanding of the molecular mechanisms whose deregulation ultimately causes Hepatocellular Carcinoma (HCC) are essential to prevent it and to design diagnostic biomarkers and therapeutic tools.
BACKGROUND: The NADPH oxidase (NOX) 4 is an important source of ROS in signal transduction that acts as a liver tumor suppressor. Transforming Growth Factor β (TGF-β) and Epidermal Growth Factor Receptor (EGFR) pathways are involved in the modulation of NOX4 expression. Data showed that recurrent protein deprivation induces changes distinctive of a preneoplastic profile. However, the mechanisms underneath these changes have not been completely understood. METHODS: Hepatocytes that survived to the lack of amino acids (Aa) (Sel line) were cultured in complete or Aa free medium. We elucidated the molecular mechanisms that support such preneoplastic alterations employing biochemical and molecular biology assays. RESULTS: Sel line showed increased phospho-AKT and phospho-ERKs levels, diminished caspase-3 activity, augmented cell proliferation and overactivation of EGFR pathway, reminiscent of a preneoplastic phenotype. NOX4 was upregulated in these cells by TGF-β canonical pathway, however ROS levels were not found increased as a result of an increment of antioxidant enzymes. Inhibition of TGF-β receptor diminished NOX4 and strikingly, after EGFR inhibition, NOX4 levels also decreased. Therefore, both TGF-β and EGFR pathways are shown to be involved in the upregulation of NOX4 in Sel line. CONCLUSIONS: This work provides novel results regarding to the regulation of NOX4 in the preneoplastic transformation of hepatocytes in the absence of Aa, in the context of TGF-β and EGFR pathways. GENERAL SIGNIFICANCE: The advances in the understanding of the molecular mechanisms whose deregulation ultimately causes Hepatocellular Carcinoma (HCC) are essential to prevent it and to design diagnostic biomarkers and therapeutic tools.