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.2016 Nov 29;7(48):79526-79543.
doi: 10.18632/oncotarget.12755.

In pursuit of synergy: An investigation of the PI3K/mTOR/MEK co-targeted inhibition strategy in NSCLC

Affiliations

In pursuit of synergy: An investigation of the PI3K/mTOR/MEK co-targeted inhibition strategy in NSCLC

Susan Heavey et al. Oncotarget..

Abstract

Clinical PI3K inhibition has been somewhat disappointing, due to both inadequate patient stratification and compensatory cell signalling through bypass mechanisms. As such, investigation of PI3K-MEK co-targeted inhibition has been recommended. With high mortality rates and a clear need for new therapeutic intervention strategies, non-small cell lung cancer (NSCLC) is an important setting to investigate the effectiveness of this approach.Here, 174 NSCLC tumours were screened for 150 mutations by Fluidigm technology, with 15 patients being profiled for phosphoprotein expression. The effects of GDC-0941 (a pan PI3K inhibitor), GDC-0980 (a dual PI3K/mTOR inhibitor) and GDC-0973 (a MEK inhibitor) alone and in combination were assessed in 3 NSCLC cell lines.PIK3CA was mutated in 5.17% of NSCLC patients. GDC-0941 and GDC-0980 treatment induced anti-proliferative and pro-apoptotic responses across all NSCLC cell lines, while GDC-0973 treatment induced only anti-proliferative responses. GDC-0980 and GDC-0973 combined treatment led to significant increases in apoptosis and synergistic reductions in proliferation across the panel of cell lines.This study found that the PI3K/MEK co-targeted inhibition strategy is synergistic in all 3 molecular subtypes of NSCLC investigated. Consequently, we would advocate clinical trials for NSCLC patients combining GDC-0980 and GDC-0973, each of which are separately under clinical investigation currently.

Keywords: MEK; NSCLC; PI3K; co-target; lung.

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Conflict of interest statement

CONFLICTS OF INTEREST

None of the authors have any potential conflicts of interest to disclose.

Figures

Figure 1
Figure 1. PI3K activation in NSCLC patients Phosphorylation of mTOR and pS6RP was profiled in 15 matched tumour and normal NSCLC patient fresh tissue samples
Intensity of expression was quantified using Image J. A Wilcoxon signed rank test was performed to compared tumour versus normal mean expression values.
Figure 2
Figure 2. Histological and mutational characterisation of NSCLC cell lines
NSCLC cell lines used in this study were chosen based on their differing histological subtypes. Mutation status was obtained from the COSMIC database. Cell images were obtained using the InCell 1000. Each cell line was stained with Höechst blue nuclear stain, mitotracker red mitochondrial stain and phalloidin green F-actin stain.
Figure 3
Figure 3. The effect of GDC-0941 treatment on viability in a panel of NSCLC cells
H460, A549, and H1975 cells were treated at noted concentrations of GDC-0941 for 48 hours in a 96 well plate and then fixed & stained with Höechst nuclear stain, mitotracker red mitochondrial stain and phalloidin green F-actin stain.A. Stained plates were imaged using the InCell 1000 (10X objective, 10 fields per well, triplicate wells, n=3).B. Images were analysed for cell count using InCell 1000 software. Cell numbers from each treatment group were compared with untreated cell numbers and are represented here as % cell count compared to untreated cells, with mean ± SEM for treated groups. */***: p<0.05/0.001 respectively, compared to untreated cells of the same cell line.
Figure 4
Figure 4. The effect of GDC-0980 treatment on viability in a panel of NSCLC cells
H460, A549, and H1975 cells were treated at noted concentrations of GDC-0980 for 48 hours in a 96 well plate and then fixed & stained with Höechst nuclear stain, mitotracker red mitochondrial stain and phalloidin green F-actin stain.A. Stained plates were imaged using the InCell 1000 (10X objective, 10 fields per well, triplicate wells, n=3).B. Images were analysed for cell count using InCell 1000 software. Cell numbers from each treatment group were compared with untreated cell numbers and are represented here as % cell count compared to untreated cells, with mean ± SEM for treated groups. ***: p<0.001 compared to untreated cells of the same cell line.
Figure 5
Figure 5. Comparison of the anti-proliferative effects of GDC-0941 and GDC-0980 in NSCLC cell lines
Cells were treated at noted concentrations of GDC-0941 or GDC-0980 for 72 hours, then proliferation rates were examined by BrdU incorporation ELISA (triplicate wells, n=3). Raw absorbance values for each treatment group were compared with untreated sample values, which were set to 100%. Data was then expressed as % proliferation compared to untreated cells, with mean ± SEM. Data from a) was analysed by linear regression and IC50 values were determined using GraphPad Prism.
Figure 6
Figure 6. The effect of GDC-0973 treatment on viability in a panel of NSCLC cells
H460, A549, and H1975 cells were treated at noted concentrations of GDC-0973 for 48 hours in a 96 well plate and then fixed & stained with Höechst nuclear stain, mitotracker red mitochondrial stain and phalloidin green F-actin stain.A. Stained plates were imaged using the InCell 1000 (10X objective, 10 fields per well, triplicate wells, n=3).B. Images were analysed for cell count using InCell 1000 software. Cell numbers from each treatment group were compared with untreated cell numbers and are represented here as % cell count compared to untreated cells, with mean ± SEM for treated groups.
Figure 7
Figure 7. Anti-proliferative effects of GDC-0973 in a panel of NSCLC cell lines
Cells were treated at noted concentrations of GDC-0973 for 72 hours, then proliferation rates were examined by BrdU incorporation ELISA (triplicate wells, n=3).A. Raw absorbance values for each treatment group were compared with untreated sample values, which were set to 100%. Data was then expressed as % proliferation compared to untreated cells, with mean ± SEM.B. Data from a) was analysed by linear regression and IC50 values were determined using GraphPad Prism.
Figure 8
Figure 8. Effects of PI3K-mTOR and MEK co-targeted inhibition on NSCLC cell viability
H460, A549 and H1975 cells were treated with GDC-0980 (1μM), GDC-0973 (1μM), both GDC-0980 (1μM) and GDC-0973 (1μM) or left untreated for 48 hours in a 96 well plate and then fixed & stained with Höechst nuclear stain, mitotracker red mitochondrial stain and phalloidin green F-actin stain.A. Stained plates were imaged using the InCell 1000 (10X objective, 10 fields per well, triplicate wells, n=3).B. Images were analysed for cell count using InCell 1000 software. Treatment groups were compared with untreated cell numbers (raw data) and are represented here as % cell count compared to untreated cells, with mean ± SEM for treated groups. */**/***: p<0.05/0.01/0.001.
Figure 9
Figure 9. PI3K-mTOR and MEK combined inhibition induces a synergistic reduction in proliferation in NSCLC cells
A. H460,B. A549 andC. H1975 cells were treated with GDC-0980, GDC-0973 or a 1:1 mix of GDC-0980 & GDC-0973 at noted concentrations for 72 hours. Proliferation rates were then assessed by BrdU incorportation ELISA (triplicate wells, n=3). Raw absorbance values for each treatment group were compared with untreated sample values, which were set to 100%. Data was then represented as absorbance (which correlates directly with proliferation) with mean ± SEM.D. Data was analysed using CalcuSyn software which tests for synergistic, additive or antagonistic drug interactions via the Chou-Talalay method. A combination index (CI) of >1 indicates an antagonistic interaction, CI = 1 indicates an additive interaction and CI < 1 indicates a synergistic interaction. */**/***:p<0.05/0.01/0.001 respectively.
Figure 10
Figure 10. The effects of co-targeting PI3K-mTOR and MEK on phosphoprotein expression in a panel of three NSCLC cell lines
Protein was isolated from H460, A549 and H1975 cell lines at baseline and after treatment with GDC-0980 (1μM, 4hrs), GDC-0973 (2μM, 6hrs) and a mix of the 2 drugs using Cell Signalling lysis buffer. Intracellular signalling protein expression was explored using PathScan arrays (duplicate spots, n=3). Array images are shown with postive (+) and negative (−) controls identified. 1: ERK1/2 (Thr202/Tyr204), 2:Stat1(Tyr701), 3: Stat3 (Tyr705), 4: AKT (Thr308), 5: AKT (Ser473), 6: AMPKα (Thr172), 7: S6RP (Ser235/236), 8: mTOR (Ser2448), 9: HSP27 (Ser78), 10: BAD (Ser15), 11: p70S6K (Thr389), 12: PRAS40 (Thr246), 13: p53 (Ser15), 14: p38 (Thr180/Tyr182), 15: SAPK/JNK (Thr183/Tyr185), 16: PARP (Asp214), 17: Caspase-3 (Asp175), 18: GSK-3β (Ser9).
Figure 11
Figure 11. Effect of GDC-0980 and/or GDC-0973 treatment on total ERK and pERK1/2 (Thr202/Tyr204, Thr185/Tyr187) expression in a panel of NSCLC cell lines
Protein was isolated from H460, A549 and H1975 cell lines at baseline and after treatment with GDC-0980 (1μM, 4hrs), GDC-0973 (2μM, 6hrs) and a mix of the 2 drugs using MSD lysis buffer. Total ERK and pERK1/2 (Thr202/Tyr204, Thr185/Tyr187) expression was explored using MSD arrays (duplicate wells, n=3). Spot intensity was quantified through MSD software and data were expressed as % Phosphoprotein = ((2 x Phospho-signal) / (Phospho-signal + Total signal)) x 100. Percentage phosphorylation was then expressed as mean ± SEM. ***: p<0.001, compared to untreated cells unless otherwise noted.
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