Figures & data
Table 1. Summary of PI3K/AKT pathway activating oncogenic mutations in a panel of 12 bladder cancer cell lines. Highlighted mutations include EGFR, FGFR3, c-MET, PIK3CA, RAS and AKT 1/2/3
Figure 1. Sensitivity of bladder cancer cell lines to increasing concentrations of AZ7328 as measured in 48 h MTT assays. (A) The anti-proliferative effects of AZ7328 in two relatively sensitive cell lines (UM-UC-5 and UM-UC-16). (B) The anti-proliferative effects of AZ7328 in two relatively resistant cell lines (253J B-V and T24). Note the differences in scales. C, Rank ordering of sensitivity to AZ7328 at 48 h of exposure in a panel of 12 bladder cancer cell lines by the percentage of proliferative inhibition induced at both 1 and 5 μM concentrations. (aEGFR amplification, bFGFR3 mutation, cc-MET mutation, dPIK3CA mutation, eRAS mutation)
Figure 2. Sensitivity of bladder cancer cell lines to increasing concentrations of AZ7328 as measured in a 120 h MTT assay. (A) The anti-proliferative effects of AZ7328 in two relatively sensitive cell lines (UM-UC-5 and UM-UC-16). (B) The anti-proliferative effects of AZ7328 in two relatively resistant cell lines (253J B-V and T24). (C) Rank ordering of sensitivity to AZ7328 at 120 h of exposure in a panel of 12 bladder cancer cell lines by the percentage of proliferative inhibition induced at both 1 and 5 μM concentrations. (aEGFR amplification, bFGFR3 mutation, cc-MET mutation, dPIK3CA mutation, eRAS mutation)
Figure 3. Effects of AZ7328 on apoptosis. Bladder cancer cell lines were exposed to 5 μM AZ7328 alone and in combination with either 1 ng/mL or 10 ng/mL rhTRAIL or chemotherapy. Apoptotic cells were quantified by PI-FACS. (A) Effects of AZ7328 with or without TRAIL in a sensitive cell line (UM-UC-5). (B) Effects of AZ7328 with or without TRAIL in a resistant cell line (253J B-V). (C) Effects of AZ7328 with or without various combinations of both 1 μM gemcitabine and 1 μM cisplatin in a sensitive cell line (UM-UC-5). (D) Effects of AZ7328 with or without various combinations of both 1 μM gemcitabine and 1 μM cisplatin in a resistant cell line (253J B-V).
Figure 4. Pharmacodynamic response of selected cell lines to AZ7328. Each column represents a dose response assessment of the following markers within a cell line (immunoblotting with phospho-AKT, phospho-S6K1 and phospho-GSK-3β).
Figure 5. Potential predictors of response to AKT inhibition. (A) Western blot analysis of baseline PTEN status among the panel of 12 cell lines. The corresponding relative density indicates the PTEN band intensity relative to β-actin. (B) Western blot analysis of baseline AKT Ser 473 phosphorylation status among the panel of 12 cell lines. The corresponding relative densities indicate the phospho-AKT band intensity relative to total AKT.
Figure 6. Sensitivity of bladder cancer cell lines to increasing concentrations of rapamycin as measured in a 120 h MTT assay. (A) The anti-proliferative effects of rapamycin in four cell lines (UM-UC-5, UM-UC-16, 253J B-V and T24). Note the differences in scales. (B) Rank ordering of sensitivity to rapamycin at 120 h of exposure in a panel of 12 bladder cancer cell lines by the percentage of proliferative inhibition induced at both 5 and 10 νM concentrations. (aEGFR amplification, bFGFR3 mutation, cc-MET mutation, dPIK3CA mutation, eRAS mutation)
Figure 7. Sensitivity of all 12 bladder cancer cell lines to various combinations of AZ7328 and rapamycin as measured in a 120 h MTT assay.
Figure 8. Concentration-dependent effects of AZ7328 on autophagy. (A) Immunoblot displaying LC3-I and LC3-II expression in four representative cell lines (J82, UM-UC-5, UM-UC-9 and T24). The LC3 bands were quantified using Image J software and the bar graphs show the ratio of LC3-II to LC3-I as a function of autophagy. (B) Immunofluorescence analysis of LC-3 localization in J82 and T24 cells. Note that punctate LC-3 staining (green) is characteristic of autophagy.
Figure 9. Effect of AZ7328 on apoptosis, as single agent or in combination with chloroquine. Bladder cancer cells (J82, UM-UC-5, UM-UC-9 and T24) were exposed to increasing concentrations of AZ7328 alone or in combination with a fixed dose of 50 µM chloroquine. Apoptotic cells were quantified by PI-FACS.
