Nabendu Pore

Regulation of glut1 mRNA by Hypoxia-inducible Factor-1 INTERACTION BETWEEN H-ras AND HYPOXIA

Oncogenic transformation and hypoxia both induceglut1 mRNA. We studied the interaction between theras oncogene and hypoxia in up-regulating glut1mRNA levels using Rat1 fibroblasts transformed with H-ras (Rat1-ras). Transformation with H-ras led to a substantial increase in glut1mRNA levels under normoxic conditions and additively increasedglut1 mRNA levels in concert with hypoxia. Using a luciferase reporter construct containing 6 kilobase pairs of theglut1 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-α 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 HIF1α protein levels in these cells. Collectively these results indicate that H-Ras up-regulates theglut1 promoter, at least in part, by increasing HIF-1α protein levels leading to transactivation of promoter through the HIF-1 binding site.

EGFR Tyrosine Kinase Inhibitors Decrease VEGF Expression by Both Hypoxia-Inducible Factor (HIF)-1-Independent and HIF-1-Dependent Mechanisms

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 -1 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 -88/+54 region. In a gel shift assay, gefitinib led to decreased retardation of a labeled DNA oligonucleotide probe corresponding to the -88/-66 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-1alpha expression. This occurs due to decreased protein translation without any change in the level of HIF-1alpha 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-1alpha 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.

Akt1 activation can augment hypoxia-inducible factor-1α expression by increasing protein translation through a mammalian target of rapamycin -independent pathway

The phosphoinositide 3-kinase (PI3K)/Akt pathway is commonly activated in cancer; therefore, we investigated its role in hypoxia-inducible factor-1α (HIF-1α) 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-1α 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-1α induction in response to hypoxia and decreased HIF-1α accumulation in the presence of the proteasomal inhibitor MG132. Akt small interfering RNA (siRNA) also decreased HIF-1α induction under hypoxia and its accumulation in normoxia in the presence of dimethyloxallyl glycine, a prolyl hydroxylase inhibitor that prevents HIF-1α degradation. Metabolic labeling studies showed that Akt siRNA decreased HIF-1α 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-1α induction in a variety of cell lines, a finding that was confirmed using mTOR siRNA. Furthermore, neither mTOR siRNA nor rapamycin decreased HIF-1α translation as determined by metabolic labeling studies. Therefore, our results indicate that Akt can augment HIF-1α 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-1α expression when cells were cultured in low serum, but this seems to represent a different pathway. (Mol Cancer Res 2006;4(7):471–9)

Nelfinavir Down-regulates Hypoxia-Inducible Factor 1α and VEGF Expression and Increases Tumor Oxygenation: Implications for Radiotherapy

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway can increase vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1alpha (HIF-1alpha) expression. We examined the effect of nelfinavir, an HIV protease inhibitor that inhibits Akt signaling, on VEGF and HIF-1alpha 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-1alpha, 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-1alpha/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.

Phosphatase and Tensin Homologue Deficiency in Glioblastoma Confers Resistance to Radiation and Temozolomide that Is Reversed by the Protease Inhibitor Nelfinavir

Glioblastomas are malignant brain tumors that are very difficult to cure, even with aggressive therapy consisting of surgery, chemotherapy, and radiation. Glioblastomas frequently have loss of the phosphatase and tensin homologue (PTEN), leading to the activation of the phosphoinositide-3-kinase (PI3K)/Akt pathway. We examined whether PTEN deficiency leads to radioresistance and whether this can be reversed by nelfinavir, a protease inhibitor that decreases Akt signaling. Nelfinavir decreased Akt phosphorylation and enhanced radiosensitization in U251MG and U87MG glioblastoma cells, both of which are PTEN deficient. In the derivative line U251MG-PTEN, induction of wild-type PTEN with doxycycline decreased P-Akt expression and increased radiosensitivity to a similar extent as nelfinavir. Combining these two approaches had no greater effect on radiosensitivity than either alone. This epistasis-type analysis suggests that the nelfinavir acts along the Akt pathway to radiosensitize cells. However, nelfinavir neither decreased Akt phosphorylation in immortalized human astrocytes nor radiosensitized them. Radiosensitization was also assessed in vivo using a tumor regrowth delay assay in nude mice implanted with U87MG xenografts. The mean time to reach 1,000 mm(3) in the radiation + nelfinavir group was 71 days, as compared with 41, 34, or 45 days for control, nelfinavir alone, or radiation alone groups, respectively. A significant synergistic effect on tumor regrowth was detected between radiation and nelfinavir. (P = 0.01). Nelfinavir also increased the sensitivity of U251MG cells to temozolomide. These results support the clinical investigation of nelfinavir in combination with radiation and temozolomide in future clinical trials for patients with glioblastomas.

M-CSF Signals through the MAPK/ERK Pathway via Sp1 to Induce VEGF Production and Induces Angiogenesis In Vivo

Background M-CSF recruits mononuclear phagocytes which regulate processes such as angiogenesis and metastases in tumors. VEGF is a potent activator of angiogenesis as it promotes endothelial cell proliferation and new blood vessel formation. Previously, we reported that in vitro M-CSF induces the expression of biologically-active VEGF from human monocytes. Methodology and Results In this study, we demonstrate the molecular mechanism of M-CSF-induced VEGF production. Using a construct containing the VEGF promoter linked to a luciferase reporter, we found that a mutation reducing HIF binding to the VEGF promoter had no significant effect on luciferase production induced by M-CSF stimulation. Further analysis revealed that M-CSF induced VEGF through the MAPK/ERK signaling pathway via the transcription factor, Sp1. Thus, inhibition of either ERK or Sp1 suppressed M-CSF-induced VEGF at the mRNA and protein level. M-CSF also induced the nuclear localization of Sp1, which was blocked by ERK inhibition. Finally, mutating the Sp1 binding sites within the VEGF promoter or inhibiting ERK decreased VEGF promoter activity in M-CSF-treated human monocytes. To evaluate the biological significance of M-CSF induced VEGF production, we used an in vivo angiogenesis model to illustrate the ability of M-CSF to recruit mononuclear phagocytes, increase VEGF levels, and enhance angiogenesis. Importantly, the addition of a neutralizing VEGF antibody abolished M-CSF-induced blood vessel formation. Conclusion These data delineate an ERK- and Sp1-dependent mechanism of M-CSF induced VEGF production and demonstrate for the first time the ability of M-CSF to induce angiogenesis via VEGF in vivo.

The chemokine receptor CXCR4: a homing device for hypoxic cancer cells?

Commentary to: Regulation of the Chemokine Receptor CXCR4 and Metastasis by Hypoxia-Inducible Factor in Non Small Cell Lung Cancer Cell Lines Yong-Lei Liu, Jin-Ming Yu, Xian-Rang Song, Xing-Wu Wang, Li-Gang Xing and Bin-Bin Gao CB&T Volume 5, Issue 10

PTEN mutation cooperates with EGFR activation in human glioblastoma cells to increase VEGF mRNA levels by transactivating an element in the proximal promoter

Abstract Purpose: Glioblastomas often express high levels of vascular endothelial growth factor (VEGF), even under normoxic conditions. Genetic alterations that commonly occur in these tumors, including PTEN mutations and epidermal growth factor receptor (EGFR) overexpression, may contribute to this increased VEGF expression. Our previous work showed that, compared to parental U87MG human glioblastoma cells, VEGF mRNA levels are decreased in U87/T691, a derivative line in which EGFR signaling is inhibited by introduction of a truncated p185Neu protein (Cancer Research 60: 5879-5886, 2000). In that study, we also showed that the effect of EGFR activation on VEGF was mediated at the level of transcription via a PI3K dependent pathway. The purpose of the current study was to assess the role of PTEN, a negative regulator of PI3K, and its interaction with EGFR activation in regulating VEGF expression. Materials and Methods: Protein lysates were obtained from U87MG and U87/T691 cells for use in Western blotting for phosphorylated Akt. U87MG and U87/T691 cells were infected with adenovirus expressing either wildtype PTEN or phosphatase dead PTEN, then RNA was harvested for Northern blotting for VEGF. Nuclear extracts were also obtained from these cells for use in gel shift assays. Luciferase assays were performed with lysates of U87MG cells transfected with luciferase reporter constructs containing fragments of the VEGF promoter. Results: The level of phosphorylated Akt, a marker for PI3K activation, was lower in U87/T691 cells compared to U87MG cells, an expected result because of inhibition of the EGFR in the former. Treatment of U87/T691 cells with the PI3K inhibitor LY294002 further decreased the level of phosphorylated Akt and VEGF mRNA. Therefore, in spite of EGFR inhibition, U87/T691 cells contain residual PI3K activity that regulates VEGF expression. To determine whether the PTEN status of these cells influences VEGF mRNA levels, wildtype PTEN was introduced into U87MG and U87/T691 cells using adenovirus, This resulted in a decrease in both phosphorylated Akt and VEGF mRNA levels. Treatment of U87MG cells with LY294002, a PI3K inhibitor, or cotransfection with a vector expressing either wildtype PTEN or dominant negative PI3K decreased activity of a luciferase reporter containing −88/+54 bp of the VEGF promoter (+1 is the transcription start site). LY294002 also blocked induction of activity of this reporter by EGF stimulation. Gel shift assays were also performed using nuclear lysates of U87MG cells infected with either wildtype or phosphatase dead PTEN adenovirus. In these assays, the radioactively labeled probe used was an oligonucleotide spanning −88 to −65. These assays showed that introduction of wildtype PTEN decreased binding of a protein complex to this region in the promoter. Conclusion: In human glioblastoma cells, PTEN mutation can cooperate with EGFR activation to increase VEGF mRNA levels by transcriptionally upregulating the proximal VEGF promoter (−88/+54) via a PI3K-dependent pathway. Furthermore, PI3K activation leads to increased binding of a protein complex to a segment of the promoter located between −88 and −65.