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

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The Atpase Activity of Reptin Is Required for Its Effects on Tumor Cell Growth and Viability in Hepatocellular Carcinoma Published OnlineFirst December 10, 2012; DOI: 10.1158/1541-7786.MCR-12-0455 Molecular Cancer Cell Death and Survival Research The ATPase Activity of Reptin Is Required for Its Effects on Tumor Cell Growth and Viability in Hepatocellular Carcinoma Aude Grigoletto1,2,Veronique Neaud1,2, Nathalie Allain-Courtois1,2, Patrick Lestienne1,2, and Jean Rosenbaum1,2 Abstract Reptin is overexpressed in most human hepatocellular carcinomas. Reptin is involved in chromatin remodeling, transcription regulation, or supramolecular complexes assembly. Its silencing leads to growth arrest and apoptosis in cultured hepatocellular carcinoma cells and stops hepatocellular carcinoma progression in xenografts. Reptin has an ATPase activity linked to Walker A and B domains. It is unclear whether every Reptin function depends on its ATPase activity. Here, we expressed Walker B ATPase-dead mutants (D299N or E300G) in hepatocellular carcinoma cells in the presence of endogenous Reptin. Then, we silenced endogenous Reptin and substituted it with siRNA-resistant wild-type (WT) or Flag-Reptin mutants. There was a significant decrease in cell growth when expressing either mutant in the presence of endogenous Reptin, revealing a dominant negative effect of the ATPase dead mutants on hepatocellular carcinoma cell growth. Substitution of endogenous Reptin by WT Flag-Reptin rescued cell growth of HuH7. On the other hand, substitution by Flag-Reptin D299N or E300G led to cell growth arrest. Similar results were seen with Hep3B cells. Reptin silencing in HuH7 cells led to an increased apoptotic cell death, which was prevented by WT Flag-Reptin but not by the D299N mutant. These data show that Reptin functions relevant for cancer are dependent on its ATPase activity, and suggest that antagonists of Reptin ATPase activity may be useful as anticancer agents. Mol Cancer Res; 1–7. Ó2012 AACR. Introduction finding confirmed by others in a number of cell lines of – Reptin (RUVBL2, TIP48) is a member of the AAAþ different origins (10 12). Remarkably, Reptin silencing in (ATPases Associated with various cellular Activities) family vivo led to regression of hepatocellular carcinoma tumor (1). Reptin and its homolog and partner protein Pontin xenografts, in association with the induction of tumor cell (RUVBL1, TIP49a) are essential for many cellular func- senescence (9). These results, which are in agreement with the tions. They are involved in the remodeling of chromatin, the reported functions of Reptin, suggest that it could be an regulation of transcription, and in DNA-damage sensing and interesting target for cancer therapy. We have recently repair (2, 3). They function as chaperones and are required reported, as a proof of principle, that it is possible to inhibit for the assembly of ribonucleoprotein complexes such as the ATPase activity of the related protein Pontin with small telomerase (4), snoRNPs (5), or, as part of the R2TP molecules (13). The same applies to Reptin (P Lestienne; complex, for the assembly and/or stabilization of the RNA unpublished data) and it is tempting to speculate that drugs polymerase II complex (6) and of all members of the PIKK inhibiting Reptin ATPase activity could have antitumor effects. family including ATM, ATR, DNA-PKcs, SMG-1, þ TRRAP, and mTOR (7). The ATPase activity of AAA proteins relies on the We have previously shown an overexpression of Reptin in presence of the conserved Walker A and B domains, respon- hepatocellular carcinoma (8) and that Reptin silencing sible for ATP binding and hydrolysis, respectively. Several reduced tumor cell growth and viability in vitro (8, 9), a studies have used mutants of these domains, designed according to the homology of Reptin and Pontin with the bacterial ATPase and DNA helicase RuvB (14), to probe the Authors' Affiliations: 1INSERM, Physiopathologie du cancer du foie, role of their ATPase activity. Studies in mammalian cells are U1053, F-33000 Bordeaux, France; and 2Univ. Bordeaux, Physiopatholo- gie du cancer du foie, U1053, F-33000 Bordeaux, France scarce and have been mainly conducted for Pontin. Over- expression of the Walker B mutant Pontin D302N in the Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). presence of endogenous Pontin, thus, inhibited cell trans- formation by several oncogenes such as E1A (15), c-myc Corresponding Author: Jean Rosenbaum, INSERM U1053, Universite b Bordeaux Segalen, 146 rue Leo Saignat, 33076 Bordeaux cedex, France. (16), or -catenin (17). With regard to Reptin, the Walker B Phone: 33-5-57-57-15-94; Fax: 33-5-56-51-40-77; E-mail: mutant D299N was not able to complement the effect of [email protected] the loss of endogenous Reptin on the amount of PIKK doi: 10.1158/1541-7786.MCR-12-0455 proteins in HEK293 cells (7), suggesting that the Ó2012 American Association for Cancer Research. ATPase activity of Reptin is indeed required for the www.aacrjournals.org OF1 Downloaded from mcr.aacrjournals.org on September 28, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst December 10, 2012; DOI: 10.1158/1541-7786.MCR-12-0455 Grigoletto et al. regulation of PIKK levels. However, the same mutant Caspase 3 activity assay wasasefficient as wild-type (WT) Reptin for the repres- Cells were transduced as described earlier. After 6 days, sion of the influenza A virus polymerase (18) or of the 2 Â 106 cells were solubilized in 100 mL of buffer and assayed transcriptional activity of ATF2 (19). In the latter case, the with the caspase-3 colorimetric activity assay from Chemi- C-terminus part of Reptin lacking both Walker domains con international (Millipore) according to the manufac- wasalmostasefficient as the complete protein. Taken turer's instructions. together, these data suggest that some functions of Reptin are independent of its ATPase activity, and may rather Western blot and immunoprecipitation depend on protein–protein interactions. Particularly, it Western blot was done as described previously (20). All remains to be shown that the effects of Reptin underlying blots were analyzed with the Odyssey system (Li-Cor Bios- its role in tumor cell growth and viability are dependent on ciences). We used anti-Reptin mouse monoclonal (BD its ATPase activity. Biosciences, Pharmingen), anti-Pontin rabbit polyclonal Thus, in this study, we have used Reptin Walker B (ProteinTech), anti-FLAG-M2, and anti-b-actin mouse mutants to systematically probe the role of the ATPase monoclonal (Sigma-Aldrich) antibodies. activity on phenotypic properties of hepatocellular carcino- For immunoprecipitation, cells were washed in PBS. Lysis ma cells relevant for carcinogenesis. buffer (20 mmol/L Tris-HCl pH 7.5, 1% Triton X-100, 1 mmol/L NaVO3, and 100 mmol/L NaF) supplemented Materials and Methods with protease inhibitor cocktail (Roche) was added to the Cell culture and siRNA transfection pellet. After 20 minutes of incubation in ice, the pellet was The human hepatocellular carcinoma cell lines HuH7 and vortexed and centrifuged at 15,000g for 5 minutes. Cell Hep3B were grown in Dulbecco's modified Eagle's medium extracts were incubated overnight at 4C with monoclonal (DMEM) supplemented with 10% fetal calf serum in a 5% anti-Flag M2 beads (Sigma-Aldrich). The beads were washed CO2 atmosphere at 37 C. Cells were authenticated using 5 times with lysis buffer and eluted with Laemmli sample short tandem repeat analysis and tested for mycoplasma buffer. Eluates were separated on a 10% SDS-PAGE for contamination on a regular basis. Western blotting. The siR2 siRNA-targeting Reptin mRNA and the control siGL2 siRNA were described previously (8). They were Senescence assay bought from Eurofins MWG Operon (Ebersberg, Ger- Senescence-Associated Heterochromatin Foci (SAHF; many) and transfected by reverse transfection using Lipo- ref. 21) were visualized with DAPI staining (0.5 mg/mL; fectamine RNAiMAX (Invitrogen) at a final concentration Sigma) and quantified by counting using a Zeiss Axioplan of 16 nmol/L. microscope. Representative pictures were obtained with a Leica SP5 scanning laser microscope (Leica Microsystems Constructs and lentiviral transduction GmbH). The D299N mutant Flag-Reptin cDNA was a gift from M Cole (Lebanon, USA). The E300G mutant was constructed Results with the QuikChange site-directed mutagenesis kit from Expression of a Reptin ATPase-dead mutant impairs Agilent Technologies (Massy, France) and verified by hepatocellular carcinoma cell growth sequencing. Mutants and WT Flag-Reptin were inserted in Following the observation that the D113N mutation in the previously described lentiviral vector pRRLSIN-MND- bacterial RuvB impairs ATP hydrolysis, it was shown that the MCS-WPRE (8). Titers were determined by the transduc- homologous D299N point mutation in the Walker B tion of HEK 293T cells through the serial dilution of the domain of human Reptin led to the loss of the ATPase lentiviral supernatant and were analyzed for p24 protein activity of Reptin–Pontin complexes (22). We, thus, used expression 5 days later. this point mutant as a tool to investigate the role of Reptin ATPase activity in hepatocellular carcinoma cells. We first Cell proliferation made sure that this mutation did not affect key properties of Cells were seeded at a density of 50,000 per well in 12-well Reptin, such as its subcellular localization and its ability to plates. Adherent cells were counted at various times with a interact with Pontin. Nuclear and cytoplasmic repartition of Coulter counter (Beckman Coulter) in triplicate wells. WT Flag-Reptin or Flag-tagged mutants was examined by Western blotting after cell fractionation. As observed for WT Propidium iodide staining and flow cytometry Flag-Reptin, the D299N and the E300G mutants were both For cell-cycle analysis, cells were collected by trypsini- expressed in the nucleus as well as in the cytoplasm (Sup- zation and fixed in 70% ethanol at 4C for a minimum of plementary Fig. S1). In addition, we examined the capacity 30 minutes.
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