Abstract
The mammalian Forkhead Box (Fox) family of transcription factors consists of more than 50-mammalian proteins that share homology in the winged helix DNA binding domain. Expression of the Foxm1 transcription factor is ubiquitous in all proliferating mammalian cells and tumor derived cell lines where it regulates expression of cell cycle genes critical for DNA replication and mitosis. We previously used the Albumin Promoter/Enhancer Cre Recombinase (Alb-Cre) transgene to mediate hepatocyte-specific deletion of the mouse Foxm1 fl/fl targeted allele prior to hepatic tumor induction by Diethylnitrosamine (DEN)/Phenobarbital (PB) treatment. Foxm1 deficient (-/-) hepatocytes failed to proliferate and were resistant to development of hepatocellular carcinomas (HCC) in response to DEN/PB exposure. Furthermore, Foxm1 activity is inhibited by a cell penetrating peptide corresponding to amino acids 26 to 44 of the ARF tumor suppressor protein fused to nine D-Arginine (D-Arg) residues. In this study, we used the Interferon a/b-inducible Mx-Cre transgene to conditionally delete the Foxm1 fl/fl allele in preexisting mouse liver tumors induced by DEN/PB treatment and genetically demonstrated that Foxm1 is required for proliferation of hepatic tumor cells during tumor progression. To reduce in vivo function of Foxm1 in HCC, mice were subjected to daily intraperitoneal injections with a cell penetrating wild type (D-Arg)9 ARF 26-44 (WT ARF 26-44) peptide. After 4 weeks of WT ARF 26-44 peptide treatment, HCC regions displayed reduced proliferation and capillary development and a significant increase in HCC apoptosis without inducing damage to the adjacent normal liver tissue. This selective apoptosis correlated with a 90% reduction in levels of the anti-apoptotic survivin protein and was independent of the p53-PUMA transcriptional pro-apoptosis pathway. In contrast, no effect on proliferation, apoptosis or capillary development of HCC was found in mice treated with the cell penetrating mutant (D-Arg)9 ARF 37-44 (Mut. ARF 37-44) peptide, which was missing sequences necessary to interact with the Foxm1 protein. Furthermore, the WT ARF 26-44 peptide was able to induce apoptosis in human hepatoma HepG2 cells in vitro. This HepG2 cell apoptosis correlated with diminished expression of the mitotic regulatory proteins polo-like kinase 1 (PLK1), aurora B kinase and survivin, which are transcriptional targets of FoxM1 that are highly expressed in cancer cells and function to prevent apoptosis. These studies indicate that the WT ARF 26-44 peptide is effective in limiting HCC tumor progression by selectively inducing liver tumor apoptosis, and therefore, this peptide can be used for HCC treatment in human patients.