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 1

The mouse Foxm1 transcription factor is required for hepatic tumor progression.

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The mouse Foxm1 transcription factor is required for hepatic tumor progr...
(A) Diagram depicting experimental design of conditional deletion of Foxm1fl/fl in preexisting liver tumors. See Methods for details of DEN/PB treatment of mice to induce HCCs as described previously (8). To create the Foxm1 CKO, mice were injected with synthetic dsRNA to induce expression of the Mx-Cre recombinase transgene (Mx-Cre) to delete the mouse Foxm1fl/fl targeted allele at 30 weeks after DEN/PB treatment, and the mice were then subjected to 10 additional weeks of PB tumor promotion and were labeled with BrdU as described in Methods. Controls included dsRNA-treated Foxm1fl/fl mice and PBS-treated Mx-CreFoxm1fl/fl mice. PN, postnatally. (B–D) dsRNA CKO Mx-CreFoxm1–/– liver tumors display no detectable nuclear staining of Foxm1 protein as determined by immunostaining with Foxm1 antibody. (E–G) H&E staining of the indicated HCC liver sections after 40 weeks of DEN/PB exposure (tumor margins indicated by arrowheads). (H–J) BrdU incorporation was detected by immunostaining of liver tumor sections with monoclonal BrdU antibody from the indicated mice at 40 weeks following DEN/PB exposure. Arrows indicate nuclear staining for either Foxm1 protein or BrdU. (K) Graph of mean number of BrdU-positive cells per square millimeter liver tumor (±SD) as described in Methods. The asterisks indicate statistically significant changes: **P ≤ 0.01 and ***P ≤ 0.001. Ad., hepatic adenomas. Magnification: ×200 (B–G); ×400 (H–J).