Mammalian target of rapamycin (mTOR) is a critical regulator of basic cell function including proliferation and survival and as such is an interesting target for anti-cancer agents. The purpose of this study was to investigate the impact of the rapamycin derivative RAD001 (everolimus) on hepatocytes in a mouse model of hepatocellular carcinoma (HCC), tyrosinemia type 1 (HT1). Multiple changes in hepatic gene expression happen very rapidly in Fah-/- mice in which medical treatment with NTBC has been discontinued. These changes are correlated with severe impairment of apoptosis and cell proliferation. Treatment with RAD001 during NTBC withdrawal completely prevented the resistance against Fas-induced apoptosis in Fah-/- mice. Mice with chronic liver damage remained sensitive to Fas-induced liver failure similar to healthy mice on NTBC. Next, we were interested whether RAD001 affects hepatocyte proliferation. We have previosly shown that p21 is responsible for the cell cycle arrest occuring in Fah-/- mice following NTBC withdrawal. Loss of p21 leads to unchecked proliferation of hepatocytes and thereby to significantly accelerates tumor development in Fah/p21-/-mice. In this study, RAD001 did not affect baseline hepatocellular proliferation in healthy mice on NTBC. Following NTBC withdrawal, RAD001 prevented the accumulation of p21 mRNA and protein in Fah-/- mice. In contrast to Fah/p21-/-mice however, proliferation of hepatocytes was completely blocked indicating that the RAD001 induced cell cycle arrest occurs independently of p21. This finding was further corroborated in Fah/p21-/- treated with RAD001 during NTBC withdrawal. Baseline proliferation of hepatocytes in healthy mice on NTBC was unaffected by RAD001. Two weeks following chronic liver damage proliferation was again completely blocked. Finally, we wondered whether RAD001 prevents liver tumor development in the Fah-/- mouse model. To this aim, WT hepatocytes were transplanted into Fah/p21-/- mice, subsequently mice were taken off NTBC treatment and either treated with RAD001 or placebo. 50% of the placebo treated mice died and the remaining mice developed liver tumors within 4 months. In contrast, all RAD001 treated mice survived and none of them developed tumors. Taken together, these data show that RAD001 prevents proliferation of damaged hepatocytes, sustains their apoptosis sensitivity and thereby prevents liver tumor development. Rapamycin and its derivates are increasingly recognized as anti-cancer drugs. These data led to the beginning of subsequent clinical trials to further evaluate mTOR inhibitors in other tumor types. HCCs are difficult to treat tumors and not many treatment options are available. Our data indicate that Rapamycin and its derivates are very interesting candidates for the treatment of HCCs and/ or chemoprevention of patients who are at high risk of tumor development.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA