MET activation confers resistance to cetuximab, and prevents HER2 and HER3 upregulation in head and neck cancer

Figure S1: Detailed summary of the history of the disease and treatment in the case report

Figure S2: RNAseq gene expression analysis (A) and Locus analysis (B) of genes expressed differentially between the Cetux^Res and the Cetux^Sen tumors.

Figure S3: (A) MET expression in the upper aerodigestive cell lines compared to other tumor cell lines extracted from the Cancer Cell Line Encyclopedia (CCLE), and MET expression level (RMA, log2) in the HNSCC cell lines used in this study (B) 5-day proliferation assay testing cetuximab (12.5ug/ml) efficacy in CAL33 and HSC2 and the FaDu HNSCC cell lines, with and without rHGF (50 ng/ml) and the MET inhibitors PHA-665752 (1uM) or MGCD265 (1uM). Statistical significance was calculated using one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001). (C) 5-day proliferation assay testing cetuximab (12.5ug/ml) efficacy in FaDu shCont and FaDu shMET with and without rHGF (50 ng/ml), low panel - Western blot of MET (D) Western blot for the indicated protein levels following 24 h treatment of cetuximab (12.5ug/ml), with different duration of rHGF (50 ng/ml) stimulation (E) Protein expression (Protein Array -Pathscan) of the SNU1076 HNSCC cell line treated with cetuximab (12.5ug/ml) for 24 h with and without rHGF (50 ng/ml) and the MET inhibitor PHA-665752 (1uM). Results are presented according to the cetuximab inhibition level. (F) Western blot for the indicated protein levels following 24 h treatment of cetuximab (12.5ug/ml), with and without rHGF (50 ng/ml) and the MET inhibitor PHA-665752 (1uM), in the CAL33 HNSCC cell lines and densitometry of phosphorylated ERK normalized to total ERK. Statistical significance was calculated using an unpaired t-test with Welch's correction (*p < 0.05, **p < 0.01). (G) Densitometry of phosphorylated ERK normalized to total ERK Statistical significance was calculated using one way ANOVA test (*p < 0.05, **p < 0.01)

Figure S4: (A) Excised tumors and the mice after 25-day treatments. (B) Tumor weight (mg) at the end point (day 25). Statistical significance was calculated using one-way ANOVA (*p < 0.05, **p < 0.01).

Figure S5: (A) Quantification of the Proteome Profiler™ Antibody Arrays (R&D systems) pixel density for the indicated phospho-RTK in the SNU1076 HNSCC cell line following 24 h treatment of cetuximab (12.5ug/ml), with and without rHGF (50 ng/ml). (B) Dotplot analysis of mRNA expression levels of MET and ERBB2/ERBB3obtained from the TCGA dataset of HNSCC. (C) 5-day proliferation assay testing cetuximab (12.5ug/ml) efficacy in SNU1076 and HSC4 HNSCC cell lines, HER2/3 blockers afatininb (500 nM) and lapatinib (500 nM) with and without rHGF (50 ng/ml) and the MET inhibitors PHA-665752 (1uM) . Representative experiment of two, each experiment was performed in biological triplicate. (D) Western blot for the indicated protein levels following 24 h treatment of cetuximab (12.5ug/ml), with and without rHGF (50 ng/ml) and the MEK1/2 inhibitor PD-0325901 (25 nM), in the HSC4 HNSCC cell line. (E) Western blot for the indicated protein levels following 24 h treatment of cetuximab (12.5ug/ml), with and without rHGF (50 ng/ml) and the MEK1/2 inhibitor PD-0325901 (25 nM), AKT inhibitor MK22016 (1 uM), and STAT3 inhibitor CAS 1041438–68-9 (25 nM) in the SNU1076 HNSCC cell line

Figure S6: (A) Correlation between mRNA expression level (log2) of HER2 or HER3 with EHF (top panels) or Foxo3 (bottom panels), in the HNSCC datasets of TCGA. Blue dots indicate that neither of the genes is mutated. (B) mRNA levels of HER2 and HER3 following 24 h treatment of cetuximab (12.5ug/ml), with either siCT or siFOXO3 in SNU1076 HNSCC. (C) Western blot for the indicated protein levels following 24 h treatment of cetuximab (12.5ug/ml), with either siCT, siEHF, or siFoxo3 in SNU1076 HNSCC. (D) Densitometry of ERBBs normalized to total actin. (E) mRNA levels of HER2 and HER3 following 24 h treatment of cetuximab (12.5ug/ml), with either siCont or siEHF in H1975 lung cancer cell line.