Figure 1.
Schematic representation of a network of PI3K, mTOR and MEK complex signaling.
Blue arrows and black lines represent activating and inhibitory connections, respectively.
Figure 2.
Growth inhibitory concentrations for MCF-7, T47D, SKBr3 and MDA-MB-231 exposed to different drugs.
IC50 values (50% inhibition of growth) are shown for (A) trametinib, as well as (B) everolimus, NVP-BEZ235, and GSK2126458. Cells were treated with drugs for 3 days and cell proliferation was measured by the thymidine incorporation assay. Results are shown as the mean ± standard error from triplicate experiments. Phosphorylation of (A) ERK, and (B) AKT, p70S6K and S6 in MCF-7, T47D, SKBr3 and MDA-MB-231 cell lines. Immunoblots with antibodies specific for phosphorylated proteins and their respective total protein are indicated below. Tubulin is the loading control.
Figure 3.
Growth inhibitory effects of drug treatments in MCF-7, T47D, SKBr3 and MDA-MB-231 cell line.
Cells were treated with trametinib, everolimus, NVP-BEZ235 or GSK2126458 for 3 days and [3H]-thymidine added for the last 6 hours. Results are shown as the mean ± standard error from duplicate experiments.
Figure 4.
Trametinib effect on phosphorylation of ERK, AKT, p70S6K and rpS6 in breast cancer cell lines.
Phosphorylation of ERK, AKT, p70S6K and rpS6 in (A) MCF-7, (B) T47D, (C) SKBr3 and (D) MDA-MB-231 cell lines treated with indicated concentration of trametinib for 1 or 24 h. Immunoblots for phospho-proteins and their respective total proteins are indicated below. Tubulin is the loading control.
Figure 5.
Growth inhibitory effects of combined drug treatments in MCF-7, T47D, SKBr3 and MDA-MB-231 cell lines.
Cells were treated with trametinib in association with everolimus, NVP-BEZ235 or GSK2126458 for 3 days with [3H]-thymidine added for the last 6 hours. Results were shown as the mean ± standard error from duplicate experiments.
Figure 6.
The effects of drug combinations on MCF-7, T47D and SKBr3 cell lines.
Left hand panels: Growth inhibitory effects of combinations of trametinib with everolimus (TraEvl), NVP-BEZ235 (TraBEZ) and GSK2126458 (TraGSK) using the Chou-Talalay method. The combination index (CI) theorem of Chou-Talalay defines Synergism: CI<0.9; additivity: 0.90≤CI≤1.1; antagonism: 1.1≤CI≤10 in drug combinations. X axis, FA, the ratio of the fraction affected; y axis, CI, the combination index. Results were from two independent experiments. Right hand panel: Drug combination effect on phosphorylation of ERK, AKT, p70S6K and rpS6 in (A) MCF-7, (B) T47D, (C) SKBr3 cell lines after 24 h as demonstrated by immunoblotting with anti-phospho antibodies. Tubulin is the loading control.
Table 1.
Combination index for the combination of trametinib and PI3K/mTOR inhibitors in three cell lines tested.
Figure 7.
Relationship between drug sensitivity, mutation status, receptor status and pathway utilization.
IC50 values for trametinib are represented on the y-axis and individual cell lines on the x-axis. Mutations of PTEN, PIK3CA, RAS/BRAF listed. Mutation positive status from The Roche Cancer Genome Database 2.0 [42] are colored orange. Cell lines are colored orange for estrogen receptor positive (ER), HER2 positive and triple-negative (TN) in their respective rows. Relative levels of phosphorylation in p70S6K, AKT and ERK of breast cancer cell lines are shown as bar graphs. Bands are normalized to tubulin control and bars represent changes in fold compared with BT20 and expressed as the mean from two experiments.
Figure 8.
Relationship between drug sensitivity and pathway utilization in MCF-7 parental and sub-lines.
IC50 values for trametinib are represented on the y-axis and individual cell lines on the x-axis. Relative level of phosphorylation of p70S6K, AKT and ERK of breast cancer cell lines are shown as bar graphs. Bands are normalized to tubulin or actin control and bars represent changes in fold compared with MCF-7 and expressed as the mean from two experiments. Cell lines are colored orange for estrogen receptor positive (ER), and triple-negative (TN) in their respective rows.