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)
Radiation therapy is a mainstay in the treatment of glioblastomas, but these tumors are often associated with radioresistance. Activation of the phosphatidylinositol-3-OH kinase (PI3K)/Akt pathway, which occurs frequently in glioblastomas due to inactivation of the tumor suppressor phosphatase and tensin homologue (PTEN), correlates with radioresistance. To directly test the link between Akt activation and radioresistance, we utilized PTEN-deficient U251 glioblastoma cells engineered to inducibly restore PTEN upon exposure to doxycycline. These cells showed high basal levels of Akt activation (i.e. high levels of phospho-Akt), but induction of PTEN led to substantially decreased phospho-Akt and was associated with radiosensitization. To investigate whether the PTEN-induced radiosensitization was attributable to impaired sensing versus repair of DNA damage, we assessed levels of ␥-H2AX after ionizing radiation in U251 cells induced for PTEN. Initial post-radiation levels of ␥-H2AX foci were not decreased in PTEN-induced cells; however, the resolution of these foci was significantly delayed. In contrast to these results, induction of phosphatase-dead PTEN showed no appreciable effect. Finally, exposure of cells to the PI3K inhibitor LY294002 did not decrease the occurrence of ␥-H2AX foci after irradiation but did markedly delay their resolution. These results together support a direct link between Akt activation, repair of DNA damage, and radioresistance in glioblastoma. Targeting the PI3K/Akt pathway may modulate DNA repair to improve the efficacy of radiation therapy.Glioblastoma multiforme, the most common primary adult brain tumor, has a dismal prognosis. Even with aggressive surgery, radiotherapy, and chemotherapy, the median survival for patients with glioblastomas is under one year (1, 2). The phosphatidylinositol-3-OH kinase (PI3K) 3 signaling pathway is commonly activated in these tumors, often by virtue of PTEN gene mutation but possibly also by epidermal growth factor receptor expression (3, 4). PTEN encodes a phosphatase that dephosphorylates phosphatidylinositol-3,4,5 triphosphate to convert it to phosphatidylinositol-4,5 bisphosphate. Therefore, inactivation of PTEN leads to increased levels of phosphatidylinositol-3,4,5 triphosphate and increased Akt activation (5). Conversely, restoration of PTEN leads to inhibition of Akt. Chakravarti et al. (3) found significantly reduced survival times in patients whose tumors showed PI3K pathway activation. These patients were treated with a combination of surgery with postoperative radiation as the only adjuvant therapy, which suggested that this pathway might play an important role in radiation resistance.One of the factors implicated in radioresistance is activation of the Ras/PI3K/Akt pathway (6, 7). Data from numerous investigators show that inhibition of this pathway leads to radiosensitization, not just of glioblastomas but also carcinoma of the colon, bladder, prostate, head and neck, and cervix (6 -15).The precise mechanism by which the PI3K/Akt pathw...
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.
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. [Cancer Res 2007;67(9):4467-73]
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