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.