Article Figures & Data
Figures
- Figure 1.
PTEN, EGFR, and EGFRvIII interaction regulates PI3K pathway activation and tumor cell proliferation. A, schematic diagram showing the retroviral transduction strategy for generating U87 glioblastoma cells expressing EGFR, EGFRvIII, and PTEN in all relevant combinations. B, stimulation of serum-starved cells with 10 ng/mL EGF for 15 minutes induced EGFR phosphorylation and resulted in downstream phosphorylation of Akt and Erk kinases. The ligand-independent mutant EGFRvIII was constitutively phosphorylated and increased baseline Akt and Erk phosphorylation compared with parental U87MG cells, whereas PTEN consistently diminished basal Akt activation, but not Erk, in unstimulated cells. C, cell proliferation under serum-free conditions over 5 days was dramatically increased in EGFRvIII-expressing cells compared with parental U87MG cells, whereas PTEN effectively inhibited EGFRvIII-mediated proliferation, as well as proliferation of U87MG cells.
- Figure 2.
EGFRvIII and PTEN coexpression sensitizes glioblastoma cells to erlotinib. Treatment with 10 μmol/L erlotinib for 48 hours substantially increased the G1 fraction of U87-PTEN-EGFRvIII cells relative to U87-EGFRvIII cells, and of U87-PTEN cells relative to U87MG cells. Experiments were repeated thrice and representative results from one experiment are presented.
- Figure 3.
Rapamycin enhances glioblastoma sensitivity to erlotinib, particularly in PTEN-deficient cells. A, cells were treated with 10 μmol/L erlotinib, 1 nmol/L rapamycin, or a combination of both inhibitors in serum-containing whole medium for 2 days. Erlotinib monotherapy diminished phosphorylation of both EGFR and EGFRvIII, but S6 phosphorylation was reduced only when PTEN was coexpressed in U87-PTEN and U87-PTEN-EGFRvIII cells. The presence of rapamycin in combination with erlotinib completely inhibited S6 phosphorylation, even in cells lacking PTEN. B, dose response of glioblastoma cell lines to erlotinib, rapamycin, or a combination of erlotinib and 0.1 nmol/L rapamycin over 10 to 14 days was determined in 96-well drug sensitivity assays. Concurrent administration of 0.1 nmol/L rapamycin alongside a range of erlotinib doses showed an additive effect and lowered the erlotinib IC50 in all cell lines, with considerably more decrease in PTEN-deficient cell lines (U87, U87-EGFR, and U87-EGFRvIII). This indicated that rapamycin could sensitize PTEN-deficient cells to erlotinib. The additive effect was less pronounced in U87-PTEN-EGFRvIII cells, which are already highly sensitive to erlotinib. C, the fold reduction in erlotinib IC50 value resulting from the addition of rapamycin is shown for each cell line. Combining rapamycin with erlotinib had a significantly greater erlotinib IC50-reducing effect on PTEN-deficient glioblastoma cells in each of the three matched pairs (P = 0.00001).
- Figure 4.
Combined erlotinib and rapamycin treatment induces growth arrest and cell death in PTEN-deficient SF295 glioblastoma cells. A, SF295 cells stably expressing PTEN exhibit reduced baseline levels of Akt phosphorylation. B, cells were treated with 10 μmol/L erlotinib, 1 nmol/L rapamycin, or a combination of both inhibitors in serum-free medium for 2 days before being stimulated with 10 ng/mL EGF for 15 minutes. Erlotinib (10 μmol/L) strongly inhibited Akt phosphorylation and slightly reduced S6 phosphorylation in both cell lines, whereas 1 nmol/L rapamycin only diminished S6 activation. In contrast, combined erlotinib and rapamycin treatment effectively inhibited both Akt and S6 phosphorylation. C, SF295-PTEN cells were significantly more sensitive to erlotinib-mediated cell death than SF295 cells after 3 to 4 days treatment. However, the addition of rapamycin to erlotinib sensitized PTEN-deficient SF295 cells, matching the drug-induced cell death observed in erlotinib-treated SF295-PTEN cells. Combined erlotinib and rapamycin treatment also provided significant additional benefit to SF295-PTEN cells. D, the population of cells in G1 phase was dramatically higher when treated with both erlotinib and rapamycin compared with treatment with either drug alone for 48 hours.
Additional Files
Supplementary Data Wang, et al.
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Volume 66, Issue 16
