The TNF-α/ROS/HIF-1-induced Upregulation of FoxMI Expression Promotes HCC Proliferation and Resistance to Apoptosis.
ABSTRACT The proliferation-specific transcription factor Forkhead box M1 (FoxM1) acts as a master regulator of cancer cell growth and survival and plays an important role in the development of hepatocellular carcinoma. However, the molecular mechanisms that regulate FoxM1 expression remain largely unknown. In the current study, we demonstrated that tumor necrosis factor (TNF)-αα induced FoxM1 expression and transactivated its promoter activity in hepatoma cells. Serial 5″ deletion and site-directed mutagenesis revealed that the induction of FoxM1 expression by TNF-α was dependent upon the hypoxia-inducible factor 1 (HIF1)-1 and HIF1-3/4 binding sites within the FoxM1 promoter. Furthermore, at the transcriptional level, the stabilization of HIF-1α via reactive oxygen species generation led to the binding of HIF-1α to the FoxM1 promoter and resulted in increased FoxM1 expression. The inhibition of both HIF-1α expression and reactive oxygen species generation significantly decreased TNF-α-induced FoxM1 overexpression. Consequently, the upregulation of FoxM1 promoted the proliferation of hepatoma cells and enhanced their resistance to TNF-α-induced apoptosis. Consistently, there was a positive correlation between HIF-1α and FoxM1 expression in 406 human hepatocellular carcinoma tissues, and the combination of these two parameters was a powerful predictor of poor prognosis in hepatocellular carcinoma patients after curative resection. Here, we report a new molecular mechanism by which FoxM1 expression is regulated by the TNF-α/reactive oxygen species/HIF-1 pathway, and this mechanism results in the proliferation of hepatoma cells and their resistance to apoptosis.
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ABSTRACT: Hypoxia-inducible factor-1 (HIF-1) regulates the transcription of many genes induced by low oxygen conditions. Recent studies have demonstrated that non-hypoxic stimuli can also activate HIF-1 in a cell-specific manner. Here, we define two key mechanisms that are implicated in increasing the active subunit of the HIF-1 complex, HIF-1alpha, following the stimulation of vascular smooth muscle cells (VSMC) with angiotensin II (Ang II). We show that, in contrast to hypoxia, the induction of HIF-1alpha by Ang II in VSMC is dependent on active transcription and ongoing translation. We demonstrate that stimulation of VSMC by Ang II strongly increases HIF-1alpha gene expression. The activation of diacylglycerol-sensitive protein kinase C (PKC) plays a major role in the increase of HIF-1alpha gene transcription. We also demonstrate that Ang II relies on ongoing translation to maintain elevated HIF-1alpha protein levels. Ang II increases HIF-1alpha translation by a reactive oxygen species (ROS)-dependent activation of the phosphatidylinositol 3-kinase pathway, which acts on the 5'-untranslated region of HIF-1alpha mRNA. These results establish that the non-hypoxic induction of the HIF-1 transcription factor via vasoactive hormones (Ang II and thrombin) is triggered by a dual mechanism, i.e. a PKC-mediated transcriptional action and a ROS-dependent increase in HIF-1alpha protein expression. Elucidation of these signaling pathways that up-regulate the vascular endothelial growth factor (VEGF) could have a strong impact on different aspects of vascular biology.Journal of Biological Chemistry 01/2003; 277(50):48403-9. · 4.65 Impact Factor
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ABSTRACT: HIF-1 alpha is a normally labile proangiogenic transcription factor that is stabilized and activated in hypoxia. Although the von Hippel Lindau (VHL) gene product, the ubiquitin ligase responsible for regulating HIF-1 alpha protein levels, efficiently targets HIF-1 alpha for rapid proteasome-dependent degradation under normoxia, HIF-1 alpha is resistant to the destabilizing effects of VHL under hypoxia. HIF-1 alpha also associates with the molecular chaperone Hsp90. To examine the role of Hsp90 in HIF-1 alpha function, we used renal carcinoma cell (RCC) lines that lack functional VHL and express stable HIF-1 alpha protein under normoxia. Geldanamycin (GA), an Hsp90 antagonist, promoted efficient ubiquitination and proteasome-mediated degradation of HIF-1 alpha in RCC in both normoxia and hypoxia. Furthermore, HIF-1 alpha point mutations that block VHL association did not protect HIF-1 alpha from GA-induced destabilization. Hsp90 antagonists also inhibited HIF-1 alpha transcriptional activity and dramatically reduced both hypoxia-induced accumulation of VEGF mRNA and hypoxia-dependent angiogenic activity. These findings demonstrate that disruption of Hsp90 function 1) promotes HIF-1 alpha degradation via a novel, oxygen-independent E3 ubiquitin ligase and 2) diminishes HIF-1 alpha transcriptional activity. Existence of an Hsp90-dependent pathway for elimination of HIF-1 alpha predicts that Hsp90 antagonists may be hypoxic cell sensitizers and possess antiangiogenic activity in vivo, thus extending the utility of these drugs as therapeutic anticancer agents.Journal of Biological Chemistry 09/2002; 277(33):29936-44. · 4.65 Impact Factor
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ABSTRACT: Previous studies indicate unrestrained cell cycle progression in liver lesions from hepatocarcinogenesis-susceptible Fisher 344 (F344) rats and a block of G(1)-S transition in corresponding lesions from resistant Brown Norway (BN) rats. Here, the role of the Forkhead box M1B (FOXM1) gene during hepatocarcinogenesis in both rat models and human hepatocellular carcinoma (HCC) was assessed. Levels of FOXM1 and its targets were determined by immunoprecipitation and real-time PCR analyses in rat and human samples. FOXM1 function was investigated by either FOXM1 silencing or overexpression in human HCC cell lines. Activation of FOXM1 and its targets (Aurora Kinose A, Cdc2, cyclin B1, Nek2) occurred earlier and was most pronounced in liver lesions from F344 than BN rats, leading to the highest number of Cdc2-cyclin B1 complexes (implying the highest G(2)-M transition) in F344 rats. In human HCC, the level of FOXM1 progressively increased from surrounding non-tumorous livers to HCC, reaching the highest levels in tumours with poorer prognosis (as defined by patients' length of survival). Furthermore, expression levels of FOXM1 directly correlated with the proliferation index, genomic instability rate and microvessel density, and inversely with apoptosis. FOXM1 upregulation was due to extracellular signal-regulated kinase (ERK) and glioblastoma-associated oncogene 1 (GLI1) combined activity, and its overexpression resulted in increased proliferation and angiogenesis and reduced apoptosis in human HCC cell lines. Conversely, FOXM1 suppression led to decreased ERK activity, reduced proliferation and angiogenesis, and massive apoptosis of human HCC cell lines. FOXM1 upregulation is associated with the acquisition of a susceptible phenotype in rats and influences human HCC development and prognosis.Gut 02/2009; 58(5):679-87. · 10.73 Impact Factor