Overexpression and role of the ATPase and putative DNA helicase RuvB-like 2 in human hepatocellular carcinoma.
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 beta-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 beta-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/gammac mice (P = 0.016).


CONCLUSIONS
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.
Rousseau
Benoît
B
INSERM, U889, Bordeaux, F-33076 France.
Ménard
Ludovic
L
Haurie
Valérie
V
Taras
Danièle
D
Blanc
Jean-Frédéric
JF
Moreau-Gaudry
François
F
Metzler
Philippe
P
Hugues
Michel
M
Boyault
Sandrine
S
Lemière
Sylvie
S
Canron
Xavier
X
Costet
Pierre
P
Cole
Michael
M
Balabaud
Charles
C
Bioulac-Sage
Paulette
P
Zucman-Rossi
Jessica
J
Rosenbaum
Jean
J
D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D017209:Apoptosis / Q000235:genetics / Q000502:physiology; D006528:Carcinoma, Hepatocellular / Q000209:etiology / Q000378:metabolism* / Q000473:pathology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D053938:DNA Fragmentation; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008113:Liver Neoplasms / Q000209:etiology / Q000378:metabolism* / Q000473:pathology; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D008875:Middle Aged; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014183:Transplantation, Heterologous
D002352:Carrier Proteins; D012333:RNA, Messenger; D053148:Caspase 3; D000251:Adenosine Triphosphatases; D000074183:ATPases Associated with Diverse Cellular Activities; D004265:DNA Helicases; C122079:RUVBL2 protein, human