A cell-penetrating ARF peptide inhibitor of FoxM1 in mouse hepatocellular carcinoma treatment
Galina A. Gusarova, … , Vladimir Petrovic, Robert H. Costa
Galina A. Gusarova, … , Vladimir Petrovic, Robert H. Costa
Published January 2, 2007
Citation Information: J Clin Invest. 2007;117(1):99-111. https://doi.org/10.1172/JCI27527.
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Research Article

A cell-penetrating ARF peptide inhibitor of FoxM1 in mouse hepatocellular carcinoma treatment

Abstract

The forkhead box m1 (Foxm1) transcription factor is essential for initiation of carcinogen-induced liver tumors; however, whether FoxM1 constitutes a therapeutic target for liver cancer treatment remains unknown. In this study, we used diethylnitrosamine/phenobarbital treatment to induce hepatocellular carcinomas (HCCs) in either WT mice or Arf–/–Rosa26-FoxM1b Tg mice, in which forkhead box M1b (FoxM1b) is overexpressed and alternative reading frame (ARF) inhibition of FoxM1 transcriptional activity is eliminated. To pharmacologically reduce FoxM1 activity in HCCs, we subjected these HCC-bearing mice to daily injections of a cell-penetrating ARF26–44 peptide inhibitor of FoxM1 function. After 4 weeks of this treatment, HCC regions displayed reduced tumor cell proliferation and angiogenesis and a significant increase in apoptosis within the HCC region but not in the adjacent normal liver tissue. ARF peptide treatment also induced apoptosis of several distinct human hepatoma cell lines, which correlated with reduced protein levels of the mitotic regulatory genes encoding polo-like kinase 1, aurora B kinase, and survivin, all of which are transcriptional targets of FoxM1 that are highly expressed in cancer cells and function to prevent apoptosis. These studies indicate that ARF peptide treatment is an effective therapeutic approach to limit proliferation and induce apoptosis of liver cancer cells in vivo.

Authors

Galina A. Gusarova, I-Ching Wang, Michael L. Major, Vladimir V. Kalinichenko, Timothy Ackerson, Vladimir Petrovic, Robert H. Costa

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Figure 2

The WT ARF26–44 peptide targets the liver tumor FoxM1 protein to the nucleolus.

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The WT ARF26–44 peptide targets the liver tumor FoxM1 protein to the nuc...
(A) Experimental design diagram of ARF peptide treatment of liver tumor–bearing mice. Liver tumors were induced in mice with DEN/PB exposure, and then they were subjected to daily i.p. injections of the WT ARF26–44 peptide or mutant ARF37–44 peptide (Mut. ARF37–44 peptide) as described in Methods. (B–F) GFP-FoxM1b protein is targeted to the nucleolus by the WT ARF26–44 peptide. U2OS cells were transfected with GFP-FoxM1b expression vector and were either left untreated or incubated for 48 hours with TMR fluorescently tagged WT ARF26–44 peptide (C and D) or mutant ARF37–44 peptide (E and F) and then analyzed for GFP (green) or peptide (red) fluorescence. (G) TMR fluorescence labeling revealed that the WT ARF26–44 peptide was localized to the hepatocyte cytoplasm and nucleolus (white arrow) and in the hepatic mesenchymal cells (yellow arrow). (H and I) Both mutant ARF37–44 peptide and WT ARF26–44 peptide are targeted to the hepatocyte cytoplasm and nucleolus (white arrows) as determined by laser confocal microscopy. Immunostaining of tumor sections with antibody specific to either NPM protein (black arrows) (J) or FoxM1 protein (K–M). WT ARF26–44 peptide targets tumor FoxM1 to the nucleolus (black arrows), whereas FoxM1 remains nuclear after treatment with mutant ARF37–44 peptide or PBS (red arrows). Magnification: ×400 (B–F and J–M); ×200 (G); and ×600 (H and I).