Research Article
In vivo silencing of Reptin blocks the progression of human hepatocellular carcinoma in xenografts and is associated with replicative senescence

https://doi.org/10.1016/j.jhep.2009.12.029Get rights and content

Background & Aims

We previously showed that Reptin is overexpressed in hepatocellular carcinoma (HCC), and that in vitro depletion of Reptin with siRNAs led to HCC cell growth arrest and apoptosis. Here, we asked whether in vivo targeting of Reptin in established tumours had a therapeutic effect.

Methods

We used lentiviral vectors to construct HuH7 and Hep3B cell lines with doxycycline (Dox)-dependent expression of Reptin (R2) or control shRNA (GL2). Cells were injected subcutaneously into immunodeficient mice, and Dox was given when tumours reached a volume of 250 mm3.

Results

In vitro, the growth of GL2  Dox, GL2 + Dox, and R2  Dox cells was undistinguishable whereas that of R2 + Dox cells stopped 4 days after Dox treatment. The growth decrease was associated with increased apoptosis, and evidence of replicative senescence, as shown by staining for acid β-galactosidase and the presence of senescence-associated heterochromatin foci. In xenografted mice, R2 + Dox tumour growth stagnated or even regressed with prolonged treatment in contrast with the GL2  Dox, GL2 + Dox, and R2  Dox tumours that progressed steadily. The blockage of tumour progression was associated with the induction of senescence and reduced cell proliferation.

Conclusions

In vivo Reptin depletion leads to tumour growth arrest. Reptin may prove a valuable target in HCC.

Introduction

Hepatocellular carcinoma is the fifth most common cause of cancer in the world and the third most common cause of cancer mortality [1]. The identification of new therapeutic targets is essential in order to improve HCC therapy. Through a comparative study of the proteome of HCC with that of the peri-tumour liver, we identified the deregulation of a number of proteins [2], especially the overexpression of RuvBL2/Reptin [3]. Reptin is also known as TIP49b [4], TIP48 [5], Reptin52 [6], Rvb2 [7], TAP54β [8], and ECP-51 [9]. It belongs to the AAA + family of ATPases (reviewed in [10], [11]) and shows a limited homology to the bacterial RuvB ATP-dependent DNA helicase. Reptin is required for the growth and viability of yeast [12] and is essential for the normal development of Zebrafish [13], Xenopus[14], and Drosophila[15]. Reptin is found in several high-molecular-weight complexes involved in chromatin remodelling, transcriptional regulation, or DNA damage repair [7], [8], [16]. It also interacts with proteins playing key roles in oncogenesis, such as β-catenin [6], [17], c-Myc [5], and telomerase [18].

We previously showed that Reptin overexpression was found in 75% of HCC and was associated with poor prognosis [3]. We also found that in vitro depletion of Reptin with siRNAs led to tumour cell growth arrest and apoptotic cell death whereas Reptin overexpression increased tumorigenicity in xenograft experiments.

Although these findings strongly suggest that Reptin may be a target in HCC, they require in vivo validation. For instance, tumour cells in a three dimensional setting, for example within tumours, are more resistant to apoptosis and chemotherapeutic drugs than when they are cultured in vitro in two dimensions (reviewed in [19], [20]); they might also respond differently to Reptin depletion. Thus, in this study, we used a conditional shRNA expression model that allowed switching off Reptin expression in vivo within already established tumours, therefore mimicking a therapeutic setting.

Section snippets

Construction of cell lines with the conditional expression of Reptin shRNA

We previously generated HuH7 cells with conditional, doxycycline-dependent expression of Reptin shRNA [21]. Hep3B human HCC cells were similarly engineered. The Reptin shRNA sequence was the R2 sequence previously described [3]. As a control, we used an shRNA-targeting Firefly luciferase (GL2). shRNA sequences are shown in Table 1.

Transient transfection of small interfering RNA (siRNA)

The siR1 and siR2 siRNAs targeting Reptin mRNA were described [3]. They were bought from Eurogentec (Searing, Belgium) and transfected using Lipofectamine

Expression of a siRNA-resistant Reptin rescues the cell growth phenotype

We showed that transient transfection of two different siRNAs against Reptin reduced cell proliferation whereas a control siRNA had no effect [3]. Before conducting in vivo experiments, we performed an additional control in order to rule out possible off-target effects. We generated a Reptin cDNA harbouring silent mutations in the siRNA-targeting sequence that made the mRNA insensitive to this siRNA [21]. Fig. 1B shows that transduced cells express both endogenous Reptin and the Flag-tagged

Discussion

We previously showed that overexpression of Reptin was found in the vast majority of HCC and correlated with poor prognosis. In addition, experimental evidence suggested that Reptin played a role in hepatocarcinogenesis and was a potential therapeutic target [3]. It remained, however, to be shown that targeting Reptin in established tumours had a therapeutic effect.

The constitutive expression of shRNA is commonly used for the study of the loss of function of a protein, but it is a major problem

Acknowledgements

The authors who have taken part in this study declared that they do not have anything to declare regarding funding from industry or conflict of interest with respect to this manuscript.

J.R. was supported by grants from the Institut National du Cancer, Association pour la Recherche sur le Cancer, Agence Nationale pour la Recherche sur le SIDA et les Hépatites Virales, Ligue Nationale Contre le Cancer, and Conseil Régional d’Aquitaine. V.H. was supported by a fellowship from the Agence Nationale

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    L.M. and D.T. contributed equally to this work.

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