Figure 2. Multiple MAPK pathway mutations inhibited ErbB3 activation.

(A) Kaplan–Meier overall survival curves for head and neck squamous cell carcinoma (HNSCC) patients, whose tumors overexpressed versus underexpressed phospho-ErbB3(Y1289) (median cutoff) in TCPA HNSCC cohort (N = 344). (B) Western blot results of phospho-ErbB3(Y1289) protein levels upon ectopic expression of the MAPK1, ARAF, BRAF, HRAS, and MAP2K1 as well as MAP2K2 wild-type and mutant constructs in FaDu cells by retroviral infection (pool of at least four independent repeats). (C) Western blot results of phospho-ErbB3(Y1289) levels of HNSCC Pt.25 primary tumor cultures (carrying both HRAS p.G12S and MAPK1 p.R135K mutations), HSC-6 cell line (carrying MAPK1 p.E322K mutation), and HSC-4 (MAPK pathway WT, Cancer Cell Line Encyclopedia [CCLE]) upon MAPK inhibitor GDC-0994 treatment for 30 min. 50 µg of protein lysate was used for Pt.25 and HSC-4 samples, 50 μg of protein lysate was used, whereas for HSC-6 (because of the relatively low endogenous p-RSK levels intrinsic to this cell line), 100 μg of protein lysate was loaded for presentation of signal clarity. Bar graphs showing the quantified changes of p-ErbB3(Y1289) levels upon GDC-0994 treatment (N ≥ 4 independent experiments). (D) Negative correlation between p-ErbB3(Y1289) and p-MAPK(T202/Y204) levels in MAPK-mutant HNSCC cell lines based on the published CCLE-proteomic data (Ghandi et al, 2019) and in MAPK-mutated HNSCC patient tumors (allele frequencies [AFs] >40%) based on TCPA HNSCC RPPA cohort (Li et al, 2013, 2017a). (E) Immunohistochemical staining for p-ErbB3(Y1289) in MAPK-mutated HNSCC patient tumors with high AFs close to 40%: T40 (MAPK1 p.D321N with AF = 39.1%) and T43 (HRAS p.G12S with AF = 37.3%) versus T47 and T82 (both are MAPKWT).

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