Oncogenic transformation and hypoxia both induce glut1 mRNA. We studied the interaction between the ras oncogene and hypoxia in up-regulating glut1 mRNA levels using Rat1 fibroblasts transformed with H-ras (Rat1-ras). Transformation with H-ras led to a substantial increase in glut1 mRNA levels under normoxic conditions and additively increased glut1 mRNA levels in concert with hypoxia. Using a luciferase reporter construct containing 6 kilobase pairs of the glut1 promoter, we showed that this effect was mediated at the transcriptional level. Promoter activity was much higher in Rat1-ras cells than in Rat1 cells and could be down-regulated by cotransfection with a dominant negative Ras construct (RasN17). A 480-base pair (bp) cobalt/hypoxia-responsive fragment of the promoter containing a HIF-1 binding site showed significantly higher activity in Rat1-ras cells than in Rat1 cells, suggesting that Ras might mediate its effect through HIF-1 even under normoxic conditions. Consistent with this, Rat1-ras cells displayed higher levels of HIF1-␣ protein under normoxic conditions. In addition, a promoter construct containing a 4-bp mutation in the HIF1 binding site showed lower activity in Rat1-ras cells than a construct with an intact HIF1 binding site. The activity of the latter construct but not the former could be down-regulated by RasN17, supporting the importance of the HIF1 binding site in regulation by Ras. The phosphatidylinositol 3-kinase inhibitor LY29004 down-regulated glut1 promoter activity and mRNA levels under normoxia and also decreased HIF1␣ protein levels in these cells. Collectively these results indicate that H-Ras up-regulates the glut1 promoter, at least in part, by increasing HIF-1␣ protein levels leading to transactivation of promoter through the HIF-1 binding site.Oncogenic transformation of mammalian cells is associated with many alterations in metabolism (see Ref. 1 for review). An increased rate of glucose transport is among the most characteristic biochemical markers of the transformed phenotype. The Glut1 glucose transporter is one of the proteins responsible (reviewed in Ref. 2). A number of oncogenes, including fps, src, and ras have been shown to increase glucose transport and to up-regulate glut1 mRNA and protein levels (3-5). glut1 gene expression and glucose transport are also stimulated in a variety of cells under hypoxic conditions, a response that is mediated by the transcription factor HIF-1.1 HIF-1 binds to a cis-acting binding sites located within the 5Ј-flanking region of the glut1 gene (8, 9).Because hypoxia and oncogenic mutations are both commonly present in tumors, we set out to examine the interaction between the two in up-regulating glut1 mRNA levels. Mutations in Ras are seen in a third of human cancers (6); therefore, as our model system we used Rat1 fibroblasts transformed with H-ras. Transformation with H-ras led to a substantial increase in glut1 mRNA levels under normoxic conditions and additively increased glut1 mRNA levels in concert with hypoxia. Our results ind...
Increased expression of vascular endothelial growth factor (VEGF) contributes to the growth of many tumors by increasing angiogenesis. Although hypoxia is a potent inducer of VEGF, we previously showed that epidermal growth factor receptor amplification and loss of PTEN, both of which can increase phosphatidylinositol-3-kinase (PI3K) activity, increase VEGF expression. Using both adenoviral vectors and a cell line permanently expressing constitutively active myristoylated Akt (myrAkt), we show that activation of Akt, which is downstream of PI3K, increases VEGF expression in vitro and increases angiogenesis in a Matrigel plug assay. Transient transfection experiments using reporter constructs containing the VEGF promoter showed that up-regulation of VEGF by Akt is mediated through Sp1 binding sites located in the proximal promoter. Small interfering RNA directed against Sp1 prevented the induction of VEGF mRNA in response to myrAkt but not to hypoxia. Expression of myrAkt is associated with increased phosphorylation of Sp1 and its increased binding to a probe corresponding to the ؊88/؊66 promoter region. In conclusion, our results indicate that Sp1 is required for transactivation of the VEGF by Akt. Others have proposed that the PI3K/Akt pathway can increase VEGF expression via the hypoxia-inducible factor 1 (HIF-1); however, our results suggest an alternative mechanism can also operate. INTRODUCTIONVascular endothelial growth factor (VEGF), which is an important mediator of angiogenesis in a variety of settings (Ferrara, 2002), is often overexpressed in cancers and may be important for their continued growth beyond a certain size. This is underscored by the fact that strategies to inhibit VEGF expression and function, including antibodies, kinase inhibitors, and soluble VEGF receptors, efficiently inhibit tumor growth in animal models of gliomas and other tumors (Manley et al., 2002;Bogler and Mikkelsen, 2003). Therefore, defining the mechanisms that regulate VEGF expression in cancer cells may have important implications for understanding tumor biology.VEGF is strongly induced by hypoxia, and undoubtedly this is an important mechanism of induction in tumors (Shweiki et al., 1992). However, when grown in standard tissue culture conditions under ambient oxygen conditions, many cell lines express high levels of VEGF expression, indicating that other factors can also play a role in regulating VEGF (Feldkamp et al., 1999). Our previous work suggested that genetic changes found in many cancers, specifically activation of the epidermal growth factor receptor (EGFR) and mutation of the tumor suppressor gene PTEN, may lead to up-regulation of VEGF (Maity et al., 2000;Pore et al., 2003).PTEN (phosphatase and tensin homolog deleted on chromosome ten), also known as MMAC-1 or TEP-1, functions primarily as a lipid phosphatase to dephosphorylate the D-3 position of phosphoinositide phosphates such as PI(3,4,5)P 3 to convert them to PI(4,5)P 2 (Vivanco and Sawyers, 2002). The PTEN tumor suppressor gene product therefore ac...
Epidermal growth factor receptor (EGFR) inhibitors can decrease vascular endothelial growth factor (VEGF) expression and tumor angiogenesis. In the current study, we investigate the molecular pathways by which this occurs using two drugs that have been used in the clinic, gefitinib (Iressa) and erlotinib (Tarceva). The decrease in VEGF expression by gefitinib in SQ20B squamous cell carcinoma cells was opposed by adenoviral expression of Akt in these cells. The hypoxia-inducible factor-1 (HIF-1) binding site located at approximately À1 kbp in the VEGF promoter was not required for down-regulation of promoter activity by gefitinib under normoxia. Furthermore, the drug decreased activity of a reporter containing the À88/ +54 region. In a gel shift assay, gefitinib led to decreased retardation of a labeled DNA oligonucleotide probe corresponding to the À88/À66 region of the VEGF promoter, which contains Sp1 binding sites. These effects of gefitinib on VEGF promoter activity and DNA binding were both reversed by Akt expression. Phosphorylation of Sp1 was decreased in the presence of gefitinib. Gefitinib also decreases VEGF expression by decreasing HIF-1A expression. This occurs due to decreased protein translation without any change in the level of HIF-1A mRNA. Together, these results suggest that gefitinib decreases VEGF expression both by decreasing Sp1 binding to the proximal core VEGF promoter and by down-regulating HIF-1A expression. Similar results were obtained with erlotinib in SQ20B and gefitinib in HSC3 squamous carcinoma cells. These results indicate that there are at least two separate mechanisms by which EGFR inhibitors decrease VEGF expression. (Cancer Res 2006; 66(6): 3197-204)
The phosphoinositide 3-kinase (PI3K)/Akt pathway is commonly activated in cancer; therefore, we investigated its role in hypoxia-inducible factor-1A (HIF-1A) regulation. Inhibition of PI3K in U87MG glioblastoma cells, which have activated PI3K/Akt activity secondary to phosphatase and tensin homologue deleted on chromosome 10 (PTEN) mutation, with LY294002 blunted the induction of HIF-1A protein and its targets vascular endothelial growth factor and glut1 mRNA in response to hypoxia. Introduction of wild-type PTEN into these cells also blunted HIF-1A induction in response to hypoxia and decreased HIF-1A accumulation in the presence of the proteasomal inhibitor MG132. Akt small interfering RNA (siRNA) also decreased HIF-1A induction under hypoxia and its accumulation in normoxia in the presence of dimethyloxallyl glycine, a prolyl hydroxylase inhibitor that prevents HIF-1A degradation. Metabolic labeling studies showed that Akt siRNA decreased HIF-1A translation in normoxia in the presence of dimethyloxallyl glycine and in hypoxia. Inhibition of mammalian target of rapamycin (mTOR) with rapamycin (10-100 nmol/L) had no significant effect on HIF-1A induction in a variety of cell lines, a finding that was confirmed using mTOR siRNA. Furthermore, neither mTOR siRNA nor rapamycin decreased HIF-1A translation as determined by metabolic labeling studies. Therefore, our results indicate that Akt can augment HIF-1A expression by increasing its translation under both normoxic and hypoxic conditions; however, the pathway we are investigating seems to be rapamycin insensitive and mTOR independent. These observations, which were made on cells grown in standard tissue culture medium (10% serum), were confirmed in PC3 prostate carcinoma cells. We did find that rapamycin could decrease HIF-1A expression when cells were cultured in low serum, but this seems to represent a different pathway. (Mol Cancer Res 2006;4(7):471 -9)
The phosphatidylinositol 3-kinase (PI3K)/Akt pathway can increase vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1A (HIF-1A) expression. We examined the effect of nelfinavir, an HIV protease inhibitor that inhibits Akt signaling, on VEGF and HIF-1A expression and on angiogenesis, tumor oxygenation, and radiosensitization. Nelfinavir decreases VEGF expression under normoxia via the transcription factor Sp1, which regulates the proximal core VEGF promoter. Nelfinavir decreased Sp1 phosphorylation and decreased Sp1 binding to a probe corresponding to the proximal VEGF promoter in a gel shift assay. Nelfinavir also decreased the hypoxic induction of HIF-1A, which also regulates the VEGF promoter, most likely by decreasing its translation. The effect of nelfinavir on VEGF expression had the functional consequence of decreasing angiogenesis in an in vivo Matrigel plug assay. To determine the effect this might have on tumor radiosensitization, we did tumor regrowth assays with xenografts in nude mice. The combination of nelfinavir and radiation increased time to regrowth compared with radiation alone whereas nelfinavir alone had little effect on tumor regrowth. This radiosensitizing effect was greater than suggested by in vitro clonogenic survival assays. One possible explanation for the discordance is that nelfinavir has an effect on tumor oxygenation. Therefore, we examined this with the hypoxia marker EF5 and found that nelfinavir leads to increased oxygenation within tumor xenografts. Our results suggest that nelfinavir decreases HIF-1A/VEGF expression and tumor hypoxia, which could play a role in its in vivo radiosensitizing effect. These data support the use of nelfinavir in combination with radiation in future clinical trials.
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