MAP2K1 is a potential therapeutic target in erlotinib resistant head and neck squamous cell carcinoma

Abstract

Epidermal growth factor receptor (EGFR) targeted therapies have shown limited efficacy in head and neck squamous cell carcinoma (HNSCC) patients despite its overexpression. Identifying molecular mechanisms associated with acquired resistance to EGFR-TKIs such as erlotinib remains an unmet need and a therapeutic challenge. In this study, we employed an integrated multi-omics approach to delineate mechanisms associated with acquired resistance to erlotinib by carrying out whole exome sequencing, quantitative proteomic and phosphoproteomic profiling. We observed amplification of several genes including AXL kinase and transcription factor YAP1 resulting in protein overexpression. We also observed expression of constitutively active mutant MAP2K1 (p.K57E) in erlotinib resistant SCC-R cells. An integrated analysis of genomic, proteomic and phosphoproteomic data revealed alterations in MAPK pathway and its downstream targets in SCC-R cells. We demonstrate that erlotinib-resistant cells are sensitive to MAPK pathway inhibition. This study revealed multiple genetic, proteomic and phosphoproteomic alterations associated with erlotinib resistant SCC-R cells. Our data indicates that therapeutic targeting of MAPK pathway is an effective strategy for treating erlotinib-resistant HNSCC tumors.

Figure 1

Erlotinib-resistant SCC-R cells show epithelial-to-mesenchymal transition phenotype: (a) Erlotinib-sensitive (SCC-S) and resistant (SCC-R) cells were treated with erlotinib and metabolic activity was evaluated using MTT assay to determine percentage of viable cells. Values were set at 100% for untreated controls. Western blot analysis of (b) EGFR and pEGFR (Y1068) (c) proteins associated with EMT in SCC-S and SCC-R cells. β-actin served as loading control. (d) Migration phenotype of SCC-S and SCC-R cells as seen at 0 hr and 12 hr post-scratch wounding.

Figure 2

Genomic alterations observed in SCC-R cells: (a) Summary of SNVs observed in SCC-R cells. (b) CNAs identified using OncoCNV in SCC-R cells. Each dot corresponds to an amplicon. (Color code – green dots: outliers; grey dots: unchanged amplicons; plum color surroundings: 1-level gain; all purple dots in red circles represent copy number amplifications >1-level gain while yellow circles represent copy number loss in SCC-R cells). Single nucleotide variant in SCC-R cells resulting in (c) p.G13R in HRAS in SCC-R cells. (d) p.K57E in gene MAP2K1. (e) Copy number profile of AKT2 and AXL in SCC-R cells. Each dot corresponds to an amplicon. (Color code – red dots: gene amplicon, green dots: other amplicons; grey dots: outliers).

Figure 3

Proteomic and phosphoproteomic alterations in SCC-R cells: (a) Distribution of log2 transformed protein fold changes comparing the expression levels in SCC-R cells over SCC-S cells. (Red dots = overexpressed by ≥2 fold, Blue dots = downregulated by ≥2 fold) (b) Scatter plot of log2 transformed phosphosite ratios with total protein expression ratios (black dots depict dysregulation of total protein and phosphosite by ≥2 fold, cyan dots depict dysregulation of phosphosite by ≥2 fold at phosphopeptide level only) (c) Circos plot representing genomic and proteomic alterations in SCC-R cells compared to SCC-S cells. Chromosome ideograms are shown around the outer ring (Track A) and are oriented pter–qter in a clockwise direction with centromeres indicated in red. Other tracks contain alterations (from outside to inside): Track B- silent SNVs (cyan dots), Track C- non-synonymous SNVs (magenta dots), Track D- copy number alterations (red dots = CNA gain, grey dots = CNA neutral, and dark green dots = CNA loss), Track E- dysregulated proteins (orange dots = overexpressed, light blue dots = downregulated, fold change ≥2), Track F- altered phosphosites (purple dots = hyperphosphorylated, green dots = hypophosphorylated, fold change ≥2).

Figure 4

Genomic and proteomic alterations in SCC-R cells: (a) Copy number alterations (CNAs) of genes and their protein expression in SCC-R cells compared to SCC-S cells. Amplification and protein overexpression of (b) YAP1 and (c) AHRGEF1. MS/MS spectra of (d) wild-type peptide of MAP2K1 (e) mutant peptide representing p.K57E point mutation in MAP2K1 gene.

Figure 5

Activation of Map kinase pathway in erlotinib-resistant cells: (a) Significantly affected signalling pathways in SCC-R cells. (b) Schematic representation of canonical MAPK pathway showing genetic, proteomic and phosphoproteomic alterations observed in SCC-R cells. (c) Western blot validation of MAPK pathway intermediates (phospho-MAP2K1 (S217/S221), MAP2K1, phospho-MAPK1/3 (T202/204) and MAPK1/3, c-JUN and phospho-c-JUN (S73). β-actin served as loading control (d) Survival curve for erlotinib-sensitive (SCC-S) and resistant (SCC-R) cells treated with various concentration of MAP2K1 inhibitor (PD0325901). Metabolic activity was evaluated using MTT assay to determine percentage of viable cells. Values were set at 100% for untreated controls. (e) Western blot showing effect of different concentration of PD-0325901 on MAPK pathway intermediates (phospho-MAP2K1 (S217/S221), MAP2K1, phospho-MAPK1/3 (T202/204) and MAPK1/3 in SCC-R cells. β-actin served as loading control.