Hypoxia up-regulates SERPINB3 through HIF-2α in human liver cancer cells

Abstract

// Stefania Cannito 1,* , Cristian Turato 2,* , Claudia Paternostro 1 , Alessandra Biasiolo 2 , Sebastiano Colombatto 3 , Irene Cambieri 4 , Santina Quarta 2 , Erica Novo 1 , Elisabetta Morello 1 , Gianmarco Villano 2 , Silvano Fasolato 2 , Tiziana Musso 5 , Ezio David 6 , Ignazia Tusa 7 , Elisabetta Rovida 7 , Riccardo Autelli 1 , Antonina Smedile 8 , Umberto Cillo 9 , Patrizia Pontisso 2 and Maurizio Parola 1 1 Department of Clinical and Biological Sciences, Unit of Experimental Medicine and Interuniversity Center for Liver Pathophysiology, University of Torino, Italy 2 Department of Medicine, University of Padova, Italy 3 Department of Oncology, University of Torino, Italy 4 Department of Plastic Surgery and Burn Unit Skin Bank, CTO Hospital, Torino, Italy 5 Department of Public Health and Pediatric Sciences, University of Torino, Italy 6 Pathology Unit, San Giovanni Battista Hospital, Torino, Italy 7 Department of Biomedical, Experimental and Clinical Sciences “Mario Serio”, University of Firenze, Italy 8 Gastroenterology and Hepatology Division, San Giovanni Battista Hospital, Torino, Italy 9 Unit of Hepatobiliary Surgery and Liver Transplantation, University of Padova, Italy * These authors contributed equally to this work Correspondence: Maurizio Parola, email: // Keywords : SERPINB3, hypoxia, hepatocellular carcinoma, HIF-2α, reactive oxygen species Received : September 01, 2014 Accepted : December 09, 2014 Published : December 10, 2014 Abstract SERPINB3 is a cysteine-proteases inhibitor up-regulated in a significant number of cirrhotic patients carrying hepatocellular carcinoma (HCC) and recently proposed as a prognostic marker for HCC early recurrence. SERPINB3 has been reported to stimulate proliferation, inhibit apoptosis and, similar to what reported for hypoxia, to trigger epithelial-to-mesenchymal transition (EMT) and increased invasiveness in liver cancer cells. This study has investigated whether SERPINB3 expression is regulated by hypoxia-related mechanisms in liver cancer cells. Exposure of HepG2 and Huh7 cells to hypoxia up-regulated SERPINB3 transcription, protein synthesis and release in the extracellular medium. Hypoxia-dependent SERPINB3 up-regulation was selective (no change detected for SERPINB4) and operated through hypoxia inducible factor (HIF)-2α (not HIF-1α) binding to SERPINB3 promoter, as confirmed by chromatin immuno-precipitation assay and silencing experiments employing specific siRNAs. HIF-2α-mediated SERPINB3 up-regulation under hypoxic conditions required intracellular generation of ROS. Immuno-histochemistry (IHC) and transcript analysis, performed in human HCC specimens, revealed co-localization of the two proteins in liver cancer cells and the existence of a positive correlation between HIF-2α and SERPINB3 transcript levels, respectively. Hypoxia, through HIF-2α-dependent and redox-sensitive mechanisms, up-regulates the transcription, synthesis and release of SERPINB3, a molecule with a high oncogenic potential.