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

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 250mm³.

Results

In vitro, the growth of GL2−Dox, GL2+Dox, and R2−Dox cells was undistinguishable whereas that of R2+Dox cells stopped 4days 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.

Abbreviations: HCC, hepatocellular carcinoma, SAHF, senescence-associated heterochromatin foci, Dox, doxycycline

Keywords: Liver cancer, Apoptosis, Senescence, shRNA, Doxycycline, Reptin