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Different metabolic responses to PI3K inhibition in NSCLC cells harboring wild-type and G12C mutant KRASElisa Caiola et al. Oncotarget. 2016.
. 2016 Aug 9;7(32):51462-51472. doi: 10.18632/oncotarget.9849. AffiliationsItem in Clipboard
AbstractKRAS mutations in non-small-cell lung cancer (NSCLC) patients are considered a negative predictive factor and indicate poor response to anticancer treatments. KRAS mutations lead to activation of the PI3K/akt/mTOR pathway, whose inhibition remains a challenging clinical target. Since the PI3K/akt/mTOR pathway and KRAS oncogene mutations all have roles in cancer cell metabolism, we investigated whether the activity of PI3K/akt/mTOR inhibitors (BEZ235 and BKM120) in cells harboring different KRAS status is related to their metabolic effect. Isogenic NSCLC cell clones expressing wild-type (WT) and mutated (G12C) KRAS were used to determine the response to BEZ235 and BKM120. Metabolomics analysis indicated the impairment of glutamine in KRAS-G12C and serine metabolism in KRAS-WT, after pharmacological blockade of the PI3K signaling, although the net effect on cell growth, cell cycle distribution and caspase activation was similar. PI3K inhibitors caused autophagy in KRAS-WT, but not in KRAS-G12C, where there was a striking decrease in ammonia production, probably a consequence of glutamine metabolism impairment.These findings lay the grounds for more effective therapeutic combinations possibly distinguishing wild-type and mutated KRAS cancer cells in NSCLC, exploiting their different metabolic responses to PI3K/akt/mTOR inhibitors.
Keywords: BEZ235; BKM120; KRAS; NSCLC; metabolomics.
Conflict of interest statementThe authors declare no conflicts of interests
FiguresFigure 1. KRAS-G12C and KRAS-WT clone responses…
Figure 1. KRAS-G12C and KRAS-WT clone responses to BEZ235 and BKM120 treatments and PI3K pathway…
Figure 1. KRAS-G12C and KRAS-WT clone responses to BEZ235 and BKM120 treatments and PI3K pathway modulationPanels A, B. Responses of cells to BEZ235 (A) and BKM120 (B), detected by MTS assay. The average of three independent experiments and SD are shown. Panel C. Representative Western blot analysis reporting the expression of different proteins from the MAPK and PI3K pathways in the KRAS expressing clones treated with BEZ235 (25 nM) or BKM120 (1 μM) at 48h. Tubulin was used as loading control.
Figure 2. Metabolic responses to PI3K inhibition…
Figure 2. Metabolic responses to PI3K inhibition in NSCLC harboring KRAS-G12C or KRAS-WT isoforms
Heat…
Figure 2. Metabolic responses to PI3K inhibition in NSCLC harboring KRAS-G12C or KRAS-WT isoformsHeat map and hierarchical clustering of discriminant metabolites (OPLS-DA, s-plot) in KRAS-G12C and KRAS-WT NSCLC cell clones treated with BEZ235 (25 nM) or BKM120 (1 μM) for 6, 24, 48h. Each row represents a metabolite, each column the average metabolite concentration (three biological replicates) for each experimental condition.
Figure 3. Significantly deregulated metabolites in presence…
Figure 3. Significantly deregulated metabolites in presence of KRAS isoforms after PI3K signaling impairment
Panels…
Figure 3. Significantly deregulated metabolites in presence of KRAS isoforms after PI3K signaling impairmentPanels A, B. Significant (one-way ANOVA, Tukey-Kramer HSD) metabolite concentration ratio (treated vs untreated condition) in NSCLC harboring KRAS-G12C mutation, at 6, 24, 48h after 25 nM BEZ235 (A) or 1 μM BKM120 (B). Panels C, D. Significant (one-way ANOVA, Tukey-Kramer HSD) metabolite concentration ratio (treated vs untreated condition) in NSCLC harboring KRAS-WT, at 6, 24, 48h after 25 nM BEZ235 (C) or 1 μM BKM120 (D).
Figure 4. Ammonia release and autophagy in…
Figure 4. Ammonia release and autophagy in presence of KRAS isoforms after PI3K inhibitors
Panel…
Figure 4. Ammonia release and autophagy in presence of KRAS isoforms after PI3K inhibitorsPanel A. Ammonia release in conditioned medium of NSCLC cell clones harboring KRAS-G12C or KRAS-WT isoforms 48h after BEZ235 (25 nM) or BKM120 (1 μM) treatment. Data are the mean ± SD of three independent experiments. *P < 0.05, one-way ANOVA, Tukey-Kramer HSD. Panel B. Representative Western blot analysis reporting the expression of LC3 forms in the KRAS clones at 48 h after BEZ235 (25 nM) or BKM120 (1 μM) treatment. Panel C. Band intensities of LC3-II were quantified and individually normalized to Tubulin band intensities. Data are expressed as LC3-II expression fold change of treated clones vs controls, which where arbitrarily set to 1. Histograms represent mean ± SD of three independent experiments. **P <0.01, *** P <0.001, one-way ANOVA, Tukey-Kramer HSD.
Figure 5. Apoptosis and cell cycle phase…
Figure 5. Apoptosis and cell cycle phase distribution in KRAS-G12C and KRAS-WT cells treated with…
Figure 5. Apoptosis and cell cycle phase distribution in KRAS-G12C and KRAS-WT cells treated with PI3K inhibitorsPanel A, B. Caspases 3 and 7 activities in clones treated with 25 nM BEZ235 (A) and 1 μM BKM120 (B) at the times indicated. The averages of three different biological replicates and SD are shown. No significant differences were observed (ANOVA, Tukey-Kramer HSD). Panel C. Cell cycle phase distribution in KRAS-G12C and KRAS-WT cells treated with 25 nM BEZ235 and 1 μM BKM120.
Figure 6. Schematic overview summing up the…
Figure 6. Schematic overview summing up the effects of PI3K inhibitors in NSCLC harboring G12C…
Figure 6. Schematic overview summing up the effects of PI3K inhibitors in NSCLC harboring G12C or WT KRAS isoforms observed in this studySchematic diagram depicting the most representative KRAS/PI3K/AKT/mTOR signaling pathway and its connection to cellular metabolism. Dashed lines refers to complex connections not detailed for clarity. Colored boxes refer to metabolites (circle)/proteins (rectangle) measured in this study. Arrows indicate metabolite/protein whose level is increased/decreased after PI3K inhibitor treatment compared to untreated conditions in G12C (red) and WT (blue) KRAS clones compare to untreated conditions. Equal (=) sign indicates metabolite/protein whose level does not change after PI3K inhibitor treatment in G12C (red) and WT (blue) KRAS clones compare to untreated conditions.
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