Research Papers:
Novel HBsAg mutations correlate with hepatocellular carcinoma, hamper HBsAg secretion and promote cell proliferation in vitro
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Abstract
Romina Salpini1,*, Matteo Surdo1,*, Nadia Warner2, Maria Francesca Cortese1, Danny Colledge2, Sally Soppe2, Maria Concetta Bellocchi1, Daniele Armenia1, Luca Carioti1, Fabio Continenza3, Domenico Di Carlo1, Patrizia Saccomandi1, Carmen Mirabelli1,4, Michela Pollicita1, Roberta Longo5, Sara Romano5, Giuseppina Cappiello5, Alberto Spanò5, Pascale Trimoulet6, Herve Fleury6, Jacopo Vecchiet7, Nerio Iapadre8, Angelo Barlattani9, Ada Bertoli1, Terenzio Mari10, Caterina Pasquazzi11, Gabriele Missale12, Cesare Sarrecchia13, Elisa Orecchini14, Alessandro Michienzi14, Massimo Andreoni13, Simona Francioso15, Mario Angelico15, Jens Verheyen16, Francesca Ceccherini-Silberstein1, Stephen Locarnini2, Carlo Federico Perno1, Valentina Svicher1
1Department of Experimental Medicine and Surgery, University of Rome “Tor Vergata” Rome, Italy
2Research and Molecular Development, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia
3Laboratory of Monitoring Antiviral Drugs, National Institute for Infectious Diseases (INMI) “Lazzaro Spallanzani” Rome, Italy
4Institut Pasteur, Unité de Biologie des Virus Entériques, Paris, France
5Unit of Microbiology, “S. Pertini Hospital”, Rome, Italy
6Laboratoire de Microbiologie Fondamentale et Pathogénicité, Hôpital Pellegrin Tripode, Bordeaux, France
7Department of Medicine and Aging Sciences, “SS Annunziata” Hospital, Chieti, Italy
8Infectious Diseases Unit, “S Salvatore” Hospital, L'Aquila, Italy
9Hepatology Unit, “S Giacomo” Hospital, Rome, Italy
10Hepatology Unit, “Regina Margherita” Hospital, Rome, Italy
11Hepato-Infectivology Unit, “S Andrea” Hospital, Rome, Italy
12Hospital of Parma, Parma, Italy
13Tor Vergata University Hospital, Infectious Diseases Unit, Rome, Italy
14Department of Biomedicine and Prevention, University of Rome “Tor Vergata” Rome, Italy
15Tor Vergata University Hospital, Hepatology Unit, Rome, Italy
16Institute of Virology, University Hospital, University of Duisburg-Essen, Essen, Germany
*These authors have contributed equally to this work
Correspondence to:
Carlo Federico Perno, email: [email protected]
Valentina Svicher, email: [email protected]
Keywords: hepatitis B, hepatocellular carcinoma, hepatitis B surface antigen, HBsAg mutations, cell proliferation
Received: November 25, 2016 Accepted: December 27, 2016 Published: February 01, 2017
ABSTRACT
Background: An impaired HBsAg-secretion can increase HBV oncogenic-properties. Here, we investigate genetic-determinants in HBsAg correlated with HBV-induced hepatocellular carcinoma (HCC), and their impact on HBsAg-secretion and cell-proliferation.
Methods: This study included 128 chronically HBV-infected patients: 23 with HCC (73.9% D; 26.1% A HBV-genotype), and 105 without cirrhosis/HCC (72.4% D, 27.6% A) as reference-group. The impact of mutations on HBsAg-secretion was assessed by measuring the ratio [secreted/intracellular HBsAg] until day 5 post-transfection. The impact of mutations on cell-cycle advancement was assessed by flow-cytometry.
Results: Two HBsAg mutations significantly correlated with HCC: P203Q (17.4% [4/23] in HCC vs 1.0% [1/105] in non-HCC, P=0.004); S210R (34.8% [8/23] in HCC vs 3.8% [4/105] in non-HCC, P <0.001); P203Q+S210R (17.4% [4/23] in HCC vs 0% [0/110] in non-HCC, P=0.001). Both mutations reside in trans-membrane C-terminal domain critical for HBsAg-secretion. In in-vitro experiments, P203Q, S210R and P203Q+S210R significantly reduced the ratio [secreted/intracellular HBsAg] compared to wt at each time-point analysed (P <0.05), supporting an impaired HBsAg-secretion. Furthermore, P203Q and P203Q+S210R increased the percentage of cells in S-phase compared to wt, indicating cell-cycle progression (P203Q:26±13%; P203Q+S210R:29±14%; wt:18%±9, P <0.01. Additionally, S210R increased the percentage of cells in G2/M-phase (26±8% for wt versus 33±6% for S210R, P <0.001).
Conclusions: Specific mutations in HBsAg C-terminus significantly correlate with HBV-induced HCC. They hamper HBsAg-secretion and are associated with increased cellular proliferation, supporting their involvement in HCC-development. The identification of viral genetic markers associated with HCC is critical to identify patients at higher HCC-risk that may deserve intensive liver monitoring, and/or early anti-HBV therapy.
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PII: 14944