The Forkhead box m1 (Foxm1) gene is critical for G 1 /S transition and essential for mitotic progression. However, the transcriptional mechanisms downstream of FoxM1 that control these cell cycle events remain to be determined. Here, we show that both early-passage Foxm1 ؊/؊ mouse embryonic fibroblasts (MEFs) and human osteosarcoma U2OS cells depleted of FoxM1 protein by small interfering RNA fail to grow in culture due to a mitotic block and accumulate nuclear levels of cyclin-dependent kinase inhibitor (CDKI) proteins p21Cip1 and p27 Kip1 . Using quantitative chromatin immunoprecipitation and expression assays, we show that FoxM1 is essential for transcription of the mitotic regulatory genes Cdc25B, Aurora B kinase, survivin, centromere protein A (CENPA), and CENPB. We also identify the mechanism by which FoxM1 deficiency causes elevated nuclear levels of the CDKI proteins p21Cip1 and p27 Kip1 . We provide evidence that FoxM1 is essential for transcription of Skp2 and Cks1, which are specificity subunits of the Skp1-Cullin 1-F-box (SCF) ubiquitin ligase complex that targets these CDKI proteins for degradation during the G 1 /S transition. Moreover, early-passage Foxm1 ؊/؊ MEFs display premature senescence as evidenced by high expression of the senescence-associated -galactosidase, p19 ARF , and p16 INK4A proteins. Taken together, these results demonstrate that FoxM1 regulates transcription of cell cycle genes critical for progression into S-phase and mitosis.
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 ARF 26-44 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.
Purpose: Basal-like breast tumors are typically (ER/PR/HER2) triple-negative and are associated with a high incidence of brain metastases and poor clinical outcomes. The molecular chaperone aB-crystallin is predominantly expressed in triple-negative breast cancer (TNBC) and contributes to an aggressive tumor phenotype in preclinical models. We investigated the potential role of aB-crystallin in brain metastasis in TNBCs.Experimental Design: aB-crystallin expression in primary breast carcinomas and brain metastases was analyzed by immunohistochemistry among patients with breast cancer with brain metastases. aB-crystallin was overexpressed or silenced in two different TNBC cell lines. The effects on cell adhesion to human brain microvascular endothelial cells (HBMEC) or extracellular matrix proteins, transendothelial migration, and transmigration across a HBMEC/astrocyte coculture blood-brain barrier (BBB) model were examined. In addition, the effects of overexpressing or silencing aB-crystallin on brain metastasis in vivo were investigated using orthotopic TNBC models.Results: In a cohort of women with breast cancer brain metastasis, aB-crystallin expression in primary breast carcinomas was associated with poor overall survival and poor survival after brain metastasis, even among patients with TNBC. Stable overexpression of aB-crystallin in TNBC cells enhanced adhesion to HBMECs, transendothelial migration, and BBB transmigration in vitro, whereas silencing aB-crystallin inhibited these events. aB-crystallin promoted adhesion of TNBC cells to HBMECs, at least in part, through an a3b1 integrin-dependent mechanism. aB-crystallin overexpression promoted brain metastasis, whereas silencing aB-crystallin inhibited brain metastasis in orthotopic TNBC models.Conclusion: aB-crystallin is a novel regulator of brain metastasis in TNBC and represents a potential biomarker and drug target for this aggressive disease. Clin Cancer Res; 1-12. Ó2013 AACR.
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