Overexpression and role of the ATPase and putative DNA helicase RuvB-like 2 in human hepatocellular carcinoma

Authors

  • Benoît Rousseau,

    1. INSERM, U889, Bordeaux, F-33076 France
    2. Université Victor Segalen Bordeaux 2, Institut Fédératif de Recherches 66, Bordeaux, F-33076 France
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    • These authors contributed equally to this study.

  • Ludovic Ménard,

    1. INSERM, U889, Bordeaux, F-33076 France
    2. Université Victor Segalen Bordeaux 2, Institut Fédératif de Recherches 66, Bordeaux, F-33076 France
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    • These authors contributed equally to this study.

  • Valérie Haurie,

    1. INSERM, U889, Bordeaux, F-33076 France
    2. Université Victor Segalen Bordeaux 2, Institut Fédératif de Recherches 66, Bordeaux, F-33076 France
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  • Danièle Taras,

    1. INSERM, U889, Bordeaux, F-33076 France
    2. Université Victor Segalen Bordeaux 2, Institut Fédératif de Recherches 66, Bordeaux, F-33076 France
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  • Jean-Frédéric Blanc,

    1. INSERM, U889, Bordeaux, F-33076 France
    2. Université Victor Segalen Bordeaux 2, Institut Fédératif de Recherches 66, Bordeaux, F-33076 France
    3. CHU, Groupement des Spécialités Digestives, Bordeaux, F-33076 France
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  • François Moreau-Gaudry,

    1. Université Victor Segalen Bordeaux 2, Institut Fédératif de Recherches 66, Bordeaux, F-33076 France
    2. INSERM, E217, Bordeaux, F-33076 France
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  • Philippe Metzler,

    1. INSERM, U889, Bordeaux, F-33076 France
    2. Université Victor Segalen Bordeaux 2, Institut Fédératif de Recherches 66, Bordeaux, F-33076 France
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  • Michel Hugues,

    1. INSERM, U889, Bordeaux, F-33076 France
    2. Université Victor Segalen Bordeaux 2, Institut Fédératif de Recherches 66, Bordeaux, F-33076 France
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  • Sandrine Boyault,

    1. INSERM, U674, CEPH, IUH, Paris, F-75010 France
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  • Sylvie Lemière,

    1. INSERM, E113, Talence, F-33405 France
    2. Université Bordeaux 1, Institut Fédératif de Recherches 66, Talence, F-33405 France
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  • Xavier Canron,

    1. INSERM, E113, Talence, F-33405 France
    2. Université Bordeaux 1, Institut Fédératif de Recherches 66, Talence, F-33405 France
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  • Pierre Costet,

    1. Université Victor Segalen Bordeaux 2, Animalerie spécialisée, Bordeaux, F-33076 France
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  • Michael Cole,

    1. Dartmouth Medical School, Department of Pharmacology and of Genetics, Lebanon, NH 03756 USA
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  • Charles Balabaud,

    1. INSERM, U889, Bordeaux, F-33076 France
    2. Université Victor Segalen Bordeaux 2, Institut Fédératif de Recherches 66, Bordeaux, F-33076 France
    3. CHU, Groupement des Spécialités Digestives, Bordeaux, F-33076 France
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  • Paulette Bioulac-Sage,

    1. INSERM, U889, Bordeaux, F-33076 France
    2. Université Victor Segalen Bordeaux 2, Institut Fédératif de Recherches 66, Bordeaux, F-33076 France
    3. CHU, Groupement des Spécialités Digestives, Bordeaux, F-33076 France
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  • Jessica Zucman-Rossi,

    1. INSERM, U674, CEPH, IUH, Paris, F-75010 France
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  • Jean Rosenbaum

    Corresponding author
    1. INSERM, U889, Bordeaux, F-33076 France
    2. Université Victor Segalen Bordeaux 2, Institut Fédératif de Recherches 66, Bordeaux, F-33076 France
    • Unit 889, Institut National de la Santé et de la Recherche Médicale, Université Victor Segalen Bordeaux 2, 146 Rue Léo Saignat, 33076 Bordeaux, France
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    • fax: (+33) 5 56 51 40 77


  • Potential conflict of interest: Nothing to report.

Abstract

Using a proteomic analysis of human hepatocellular carcinoma (HCC), we identified the overexpression in 4 tumors of RuvB-like 2 (RUVBL2), an ATPase and putative DNA helicase known to interact with β-catenin and cellular v-myc myelocytomatosis viral oncogene homolog (c-myc). RUVBL2 expression was further analyzed in tumors with quantitative reverse-transcription polymerase chain reaction analysis and immunohistochemistry; in addition, RUVBL2 expression in a HuH7 cell line was silenced by small interfering RNA or increased with a lentiviral vector. RUVBL2 messenger RNA overexpression was confirmed in 72 of 96 HCC cases, and it was associated with poorly differentiated tumors (P = 0.02) and a poor prognosis (P = 0.02) but not with β-catenin mutations or c-myc levels. Although RUVBL2 was strictly nuclear in normal hepatocytes, tumoral hepatocytes exhibited additional cytoplasmic staining. There was no mutation in the coding sequence of RUVBL2 in 10 sequenced cases. Silencing RUVBL2 in HuH7 HCC cells reduced cell growth (P < 0.001) and increased apoptosis, as shown by DNA fragmentation (P < 0.001) and caspase 3 activity (P < 0.005). This was associated with an increased expression of several proapoptotic genes and with an increased conformational activation of Bak-1 and Bax. On the other hand, HuH7 cells with an overexpression of RUVBL2 grew better in soft agar (P < 0.03), had increased resistance to C2 ceramide–induced apoptosis (P < 0.001), and gave rise to significantly larger tumors when injected into immunodeficient Rag2/γc mice (P = 0.016). Conclusion: RUVBL2 is overexpressed in a large majority of HCCs. RUVBL2 overexpression enhances tumorigenicity, and RUVBL2 is required for tumor cell viability. These results argue for a major role of RUVBL2 in liver carcinogenesis. (HEPATOLOGY 2007.)

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