Foxm1b Transcription Factor is Essential for Development of Hepatocellular Carcinomas and is Negatively Regulated by the p19ARF Tumor Suppressor

Vladimir V. Kalinichenko, Michael L. Major, Xinhe Wang, Joseph Kuechle, Helena M. Yoder, Margaret B. Dennewitz, Brian Shin, Abhishek Datta, Pradip Raychaudhuri and Robert H. Costa*

Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, College of Medicine, Chicago, IL 60607
*robcosta@uic.edu 

INTRODUCTION. Human hepatocellular carcinoma (HCC) is the fifth most common cancer, yet the third leading cause of cancer death worldwide (1).  The Forkhead Box m1b (Foxm1b, Foxm1 or HFH-11B) protein is a proliferation-specific member (2) of the Fox family of transcription factors (3). Use of the Transthyretin (TTR) promoter to prematurely express the Foxm1b (Hfh-11B) cDNA in regenerating liver of transgenic (TG) mice accelerated the onset of hepatocyte DNA replication and mitosis by stimulating earlier expression of cell cycle regulatory genes (4,5). Furthermore, preventing the decline of hepatic expression of Foxm1b in 12-month old TTR-Foxm1b TG mice is sufficient to increase regenerating hepatocyte proliferation to levels found in young regenerating liver (6).  In addition to reestablishing expression of cell cycle regulatory genes, levels of the Cdk inhibitor p27kip1 protein were diminished when Foxm1b expression was restored in old-aged regenerating liver (7).  Using the Albumin enhancer and promoter driven Cre recombinase transgene (Alb-Cre) to mediate hepatocyte-specific deletion of the Foxm1b fl/fl allele, we demonstrated that Foxm1b is essential for hepatocyte DNA replication and mitosis during liver regeneration (8). Reduced hepatocyte proliferation was associated with increased nuclear protein levels of Cdk inhibitor p21Cip1 and reduced protein expression of Cdc25B phosphatase, leading to decreased Cdk1 and Cdk2 activation required for cell cycle progression (8).

METHODS: At 14 days postnatally, the entire mouse litter of Alb-Cre Foxm1b -/- (liver-specific knock out) and Foxm1b fl/fl (control) mice received single IP injection of the tumor initiator Diethylnitrosamine (DEN). Two weeks later, mice were administered water containing 0.05% of tumor promoter Phenobarbital (PB) for either 6, 23, 33 or 50 weeks and then sacrificed for analysis for liver tumor formation (9).

RESULTS AND DISCUSSION. Patients with HCC have poor prognoses because late detection of this liver tumor renders current cancer therapy ineffective.  Here, we provide evidence that the Foxm1b transcription factor is essential for the development of HCC. Conditionally deleted Foxm1b mouse hepatocytes fail to proliferate and are highly resistant to developing HCC in response to a DEN/PB liver tumor induction protocol. The mechanism of resistance to HCC development involves nuclear accumulation of the cell cycle inhibitor p27Kip1 protein and reduced expression of the Cdk1-activator Cdc25B phosphatase. Also, we show that Foxm1b associates with the p19ARF tumor suppressor in liver following DEN/PB exposure and that Foxm1b is a novel target for p19ARF mediated transcriptional inhibition, identifying a new pathway to suppress tumor formation. Our studies demonstrate that the Foxm1b transcription factor is required for proliferative expansion during tumor progression and constitutes a new target for human HCC tumor therapy. Because Foxm1b is expressed in a variety of cancers and tumor derived cell lines, it is likely required for proliferative expansion of many different tumors.

Acknowledgment: This work was supported by National Institute of Health Grants DK 54687-05 and RO1 AG 21842-01 (to R. H. C.). 

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