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

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 ␤-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 ␤-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/␥c 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.

The ATPase Activity of Reptin Is Required for Its Effects on Tumor Cell Growth and Viability in Hepatocellular Carcinoma

“…We have previously shown an overexpression of Reptin in hepatocellular carcinoma (8) and that Reptin silencing reduced tumor cell growth and viability in vitro (8,9), a finding confirmed by others in a number of cell lines of different origins (10)(11)(12). Remarkably, Reptin silencing in vivo led to regression of hepatocellular carcinoma tumor xenografts, in association with the induction of tumor cell senescence (9).…”

“…The siR2 siRNA-targeting Reptin mRNA and the control siGL2 siRNA were described previously (8). They were bought from Eurofins MWG Operon (Ebersberg, Germany) and transfected by reverse transfection using Lipofectamine RNAiMAX (Invitrogen) at a final concentration of 16 nmol/L.…”

“…The E300G mutant was constructed with the QuikChange site-directed mutagenesis kit from Agilent Technologies (Massy, France) and verified by sequencing. Mutants and WT Flag-Reptin were inserted in the previously described lentiviral vector pRRLSIN-MND-MCS-WPRE (8). Titers were determined by the transduction of HEK 293T cells through the serial dilution of the lentiviral supernatant and were analyzed for p24 protein expression 5 days later.…”

Proteomic Analysis of Differentially Expressed Proteins in Peripheral Cholangiocarcinoma

“…A number of these proteins could be predicted to play roles in tumorigenesis or tumor growth. Some of the identified proteins have been shown to be differentially expressed in experimental models or other tumor types such as colon or liver cancer ( [15,16,25,26]). Overall the proteins found to be over-expressed or under-expressed in tumoral tissue fell into the categories shown in Fig.…”

“…Many of these proteins were involved in cellular metabolism while a number were cytoskeletal proteins. Several of these proteins have also been reported to be differentially expressed in either hepatocellular carcinoma or other tumor types, for example alcohol dehydrogenase and aldehyde dehydrogenase [12], carbamyl phosphate synthase [13], selenium binding protein 1 and DNA helicase RuvB-like protein 2 ( [14][15][16]). …”