Phosphoproteomics Reveals MAPK Inhibitors Enhance MET- and EGFR-Driven AKT Signaling in KRAS-Mutant Lung Cancer

Abstract

Pathway inhibition of the RAS-driven MAPK pathway using small-molecule kinase inhibitors has been a key focus for treating cancers driven by oncogenic RAS, yet significant clinical responses are lacking. Feedback reactivation of ERK driven by drug-induced RAF activity has been suggested as one of the major drug resistance mechanisms, especially in the context of oncogenic RAS. To determine whether additional adaptive resistance mechanisms may coexist, we characterized global phosphoproteomic changes after MEK inhibitor selumetinib (AZD6244) treatment in KRAS-mutant A427 and A549 lung adenocarcinoma cell lines employing mass spectrometry-based phosphoproteomics. We identified 9,075 quantifiable unique phosphosites (corresponding to 3,346 unique phosphoproteins), of which 567 phosphosites were more abundant and 512 phosphosites were less abundant after MEK inhibition. Selumetinib increased phosphorylation of KSR-1, a scaffolding protein required for assembly of MAPK signaling complex, as well as altered phosphorylation of GEF-H1, a novel regulator of KSR-1 and implicated in RAS-driven MAPK activation. Moreover, selumetinib reduced inhibitory serine phosphorylation of MET at Ser985 and potentiated HGF- and EGF-induced AKT phosphorylation. These results were recapitulated by pan-RAF (LY3009120), MEK (GDC0623), and ERK (SCH772984) inhibitors, which are currently under early-phase clinical development against RAS-mutant cancers. Our results highlight the unique adaptive changes in MAPK scaffolding proteins (KSR-1, GEF-H1) and in RTK signaling, leading to enhanced PI3K-AKT signaling when the MAPK pathway is inhibited.

Implications: This study highlights the unique adaptive changes in MAPK scaffolding proteins (KSR-1, GEF-H1) and in RTK signaling, leading to enhanced PI3K/AKT signaling when the MAPK pathway is inhibited. Mol Cancer Res; 14(10); 1019-29. ©2016 AACR.

Figure 1. Phenotypic effect of MEK inhibitors on KRAS-mutant NSCLC cell lines

A, Relative cell viability after MEK inhibitor AZD6244 and MEK162 treatment at 1 μM. Cells were incubated with MEK inhibitors for 72 hours, followed by cell viability assay (Promega). Representative triplicates ± SD are presented, which showed similar results at least 2 times. Dose-dependent effects are shown in Supplementary Figure 1. B, p27 expression, pERK, and PARP cleavage after MEK inhibitor treatment (1 μM, 48 hours).

Figure 2. Work flow of SILAC-based phosphoproteomics approach

The SILAC-labeled KRAS-mutant NSCLC cell lines (A427 and A549) were treated with selumetinib (1 μM, 24 hours) or DMSO vehicle control. Target inhibition was confirmed by Western blotting for pERK (Supplementary Figure S2). Phosphopeptides were enriched from fractionated tryptic peptides, followed by LC-MS/MS analysis. Detailed methods for mass spectrometry and statistical analyses are described in Supplementary Materials and Methods.

Figure 3. Motif analysis and kinase prediction for selumetinib-regulated phosphosites

A and B, Representative phospho-motifs enriched in up- (A) and down- (B) regulated phosphopeptides after selumetinib treatment using Motif-x. Motifs with significance of P<10⁻⁶ are shown. C and D, Pie chart for enriched kinase groups to match up- (C) and down- (D) regulated phosphopeptides after selumetinib treatment using NetworKIN.

Figure 4. Self-consistent network composed of selumetinib-regulated phosphoproteins

Red nodes indicate increased phosphoproteins, green nodes indicate decreased phosphoproteins, and dim yellow nodes indicate genes/complexes added by GeneGo Metacore to connect significantly changed nodes but were either not observed to be significantly changed or are complexes for which no data were available. Orange arrows indicate activation/phosphorylation, and blue lines indicate inhibition/dephosphorylation. Gray dashed lines indicate membership within a protein complex. Phosphorylation is indicated by thick lines, and transcriptional regulation is indicated by narrow lines. Node size corresponds to number of edges connecting to the node. Transcription factors are shown in parallelograms, kinases are shown in hexagons, and everything else is shown in ellipses.

Figure 5. Altered phosphorylations in MAPK signaling cascade by selumetinib

The log₂-transformed fold-change of phosphopeptide abundance is shown in color (green: decreased, red: increased).

Figure 6. Effect of MAPK inhibition on MET and EGFR signaling

A and B, A549 cells were incubated with selumetinib (1 μM, 24 hours) and then treated with HGF (A) or EGF (B) at 25 ng/mL for indicated hours. C and D, A549 cells were incubated with MAPK inhibitors (1 μM, 24 hours) and then treated with HGF (C) or EGF (D) at 25 ng/mL for 30 minutes. DM, DMSO; LY, LY3009120 (pan-RAFi); AZD, AZD6244 (MEKi); GDC, GDC0623 (MEKi); SCH, SCH772984 (ERKi). E and F, H23 cells were treated as described above. G and H, A549 cells were incubated with selumetinib (1 μM, 24 hours), and then endogenous MET (G) or integrin β4 (H) was immunoprecipitated. Co-immunoprecipitated integrin β4 (G) or MET (H) was detected by Western blotting.