Objective: Malignant astrocytomas are deadly, treatment-resistant brain tumors. Our goal is to identify new treatment targets in malignant astrocytomas. Previous therapies, developed using normoxic conditions, have not worked well. Astrocytic potential for gluconeogenic removal of lactate may help to counteract metabolic stress during treatment. We propose that astrocytic metabolism requires brain tumor therapy to target cellular adaptations to hypoxia, as well as normoxic functions. Phosphorylations and dephosphorylations of key proteins, including phosphatidylinositol-3-kinase (PI3K)/Akt/glycogen synthase kinase-3 (GSK-3) pathway components, are proposed to trigger rapid, critical responses to hypoxia. Mutated phosphatase and tensin homolog, PTEN, commonly seen in malignant astrocytomas, allows anomalous activation of the PI3K/Akt/GSK-3 pathway, leading to activated glycogen synthase, thus providing additional removal of lactate as glycogen in poorly-perfused, invading tumor cells. Methods: Quantifying cellular adjustments to hypoxia, within 5 hrs of exposure, provided detection of the rapid adaptations that bypass the need for transcription factor-mediated alterations of protein levels. Quantifying 5 hr migration assays of U87 astrocytoma cells (mutated PTEN) under hypoxic conditions, following restoration of PTEN-wild type (WT) with transient adenoviral transfections, revealed PTEN’s contribution to control of hypoxic cell migration via its negative regulation of the pathway. Peptide competition studies of GSK-3alpha’s phosphorylation by Akt indicated whether inhibitory drugs can be developed to shut down downstream signaling leading to glycogen synthesis in PTEN-mutated astrocytomas. Results: Previously, wortmannin (3.0 –30.0 nM), a specific PI3K inhibitor, suppressed hypoxic U87 migration with a dose response. Restoration of PTEN-WT in U87 cells, followed by functional assays showed significantly decreased, 50.4% (95% CI = 35.0 to 65.8%), migration in hypoxia compared to normoxia, when control vector hypoxic migration was maintained at levels comparable to normoxic migration (p=0.3, two-tailed, paired samples, 4 assays). Previously, whole cell and pseudopodal lysates showed that GSK-3alpha was phosphorylated at peak levels in the pseudopodal leading edge. Preliminary competition studies showed that a peptide containing an 11 residue sequence of the GSK-3alpha phosphorylation site inhibited phosphorylation by cell derived Akt with a dose response at 250 to 1000-fold greater amounts than substrate. Conclusion: Anomalous PI3K/Akt/GSK-3alpha pathway activity in PTEN-mutated astrocytoma cells offers novel targets for future drug therapies to suppress hypoxic migration required for invasion. We thank The Nick Eric Wichman Foundation for encouragement and financial support.
[Proc Amer Assoc Cancer Res, Volume 45, 2004]