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 8

WT ARF26–44 peptide reduces angiogenesis and survivin expression in mouse HCC.

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WT ARF26–44 peptide reduces angiogenesis and survivin expression in mous...
The CD34 protein is a marker for newly formed sinusoid-like capillaries in HCC regions (39–41), whereas survivin is critical in preventing apoptosis of tumor cells (45–48). Antibodies specific to either CD34 or survivin were used to immunostain HCC tumor sections from mice treated with mutant ARF37–44 peptide, WT ARF26–44 peptide, or PBS or from dsRNA CKO Mx-CreFoxm1–/– mice. (A–D) Mice treated with WT ARF26–44 peptide display no CD34-positive endothelial cells in HCC capillaries, whereas CD34 staining (indicated by arrows) was abundant in endothelial cells of control mouse HCCs. (E) WT ARF26–44 peptide induces apoptosis of HMEC-1 cells. HMEC-1 cells were treated for 48 hours with 100 μM of WT ARF26–44 peptide or mutant ARF37–44 peptide or with PBS and then assayed for apoptosis as described in Methods. Shown graphically is the percent apoptosis of HMEC-1 cells in response to ARF peptide treatment. (F–I) Reduced survivin expression in WT ARF26–44 peptide–treated and Foxm1–/– liver tumors. Arrows indicate nuclear staining for survivin protein. (J) Western blot analysis reveals significant decrease in survivin protein expression in WT ARF26–44 peptide–treated mouse tumors. (K) No decrease in expression of NPM protein or p53-regulated proapoptotic PUMA protein is found in WT ARF26–44 peptide–treated mouse tumors. A slight increase in hepatic tumor levels of PUMA was found in mice treated with dsRNA. Magnification, ×400.