Pontin52 Is A Ppargamma Target Gene and Its Knockdown Reduces Hepatic Cancer In An Orthotopic-singenic Mouse Model

Background and aim: Pontin52 is an AAA+helicase involved in many cellular processes including cell growth and cancer. Pontin has been recently shown to be overexpressed in HCC and to be an negative prognostic factor. By proteomics approach, we identified Pontin as a gene downregulated by Rosiglitazone (RGZ) in a transgenic mice prone to HBV-related HCC (Galli et al., Hepatology 2010). Aim of this study is to asses the usefulness of Pontin inhibition in reducing HCC growth in vivo and to investigate the mechanisms its inhibition by RGZ. Material and methods: Mouse (Hepa1-6) and human (HepG2) liver cancer cells were used for in vitro assays. We developed a new orthotopic mice model that allows the growth of hepatic cancer cells directly injected in the liver. Pontin expression was evaluated by western-blot and qPCR. Pontin promoter activity was evaluated by luciferase reporter assay. Gene expression knockdown was performed by RNA interference. Results: RGZ down-regulation of Ruvbl-1 expression was confirmed both by qPCR, Western Blotting and Immunofluorescence analysis on Hepa1-6 (mouse) and HepG2 (human) liver cancer cells. After 48hs of RGZ treatment (20uM) Pontin expression was effectively reduced by a 50% at mRNA and protein levels. Surprisingly, PPARg silencing significantly reduces both Pontin mRNA levels and promoter activity. Over-expression of PPARg induces both Pontin promoter activity and mRNA levels. Moreover, the non-TZD PPARg agonist GW1929 induces Pontin expression, while the PPARg inhibitor GW9662 reduces it. On the other hand, RGZ treatment has negligible effects on Pontin promoter activity. Pontin down-regulation by either RNA interference or RGZ treatment significantly inhibits hepatoma cells proliferation in vitro. Pontin-silenced hepatoma cells form significantly less tumor foci than control cells in the orthotopic mice model. Conclusions: Pontin expression is induced by PPARg, but downregulated by RGZ most likely through PPARg-independent non-genomic mechanisms. Knockdown of Pontin effectively reduces the number of cancer lesion in a new orthotopic mice model as well as hepatoma cells proliferation in vitro. We suggest that to target Pontin expression could be of potential interest for developing novel anti-cancer strategies