THIAZOLIDINEDIONES INHIBIT HEPATIC TUMOUR FORMATION IN HBV TRANSGENIC MICE BY A PPAR gamma-INDEPENDENT REGULATION OF NUCLEOPHOSMIN

Background: Hepatocellular carcinoma (HCC) is the most frequent primary solid tumor of the liver and counts almost half million new cases each year. Antidiabetic thiazolidinediones (TZD) have in vitro antiproliferative effects in cancer cells derived from various epitheliums, including hepatocellular carcinoma. In addition, the primary biological target of TZD, the liganddependent trancriptional factor peroxisome proliferator-activated receptorγ(PPARγ) is up-regulated in HCC and seems to provide tumor-promoting responses. The effective anticancer in vivo properties of these drugs are still being investigated. Aim: The objective of our study was to evaluate whether chronic administration of TZD is affecting hepatic carcinogenesis in a HBV-related mouse model, in relation to PPARγ expression and activity. Methods: The effect of six-month oral administration of two TZDs (rosigltazone or pioglitazone) was tested on tumor formation in HBV transgenic mice (Tg[Alb-1 HBV]Bri 44), a well know model of hepatic carcinogenesis. These animals were crossed with Albumin-Cre/PPARγ-floxed (CreKOγ) transgenic mice resulting in a selective deletion of PPARγ in hepatocytes. These Tg[HBV]CreKOγ animals were used to evaluate the TZD effects not mediated by PPARγ. To identify new TZD targets we performed an integrated proteomic analysis of freshly isolated hepatocytes by differential in gel electrophoresis (DIGE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI). The regulated proteins were further tested by real time PCR, western blot analysis and transfection with specific luciferase reporters. Results: Oral administration of TZD in Tg[Alb-1 HBV]Bri 44 mice significantly reduced tumor incidence in the liver by inhibition of hepatocyte proliferation and increased apoptosis. Genetic deficiency of PPARγ in hepatocytes of Tg[HBV]CreKOγ mice did not modify hepatic carcinogenesis but enhanced the TZD antitumorigenic effect. Proteomic analysis identified nucleophosmin (NPM) as one of TZD modulated proteins in PPARγ-deficient hepatocytes. TZD inhibited mRNA expression and decreased protein levels of NPM, as well as inhibited NPM promoter activity independently of PPARγ expression. In vitro, TZD inhibition of NPM correlated with the induction of p53 phosphorylation at the Serine-15 followed by the activation of p21. Conclusion: These findings indicate that chronic administration of TZD is protective against the development of HCC through inhibition of NPM expression and activation of p53 pathway but is independent of PPARγ activation. TZD may be a new category of drugs for prevention and treatment of liver cancer.