Fig 2: Ceritinib acts via the IGF1R/AKT pathway: PhKh1 cells were stimulated after starvation with IGF2 in the presence or absence of ceritinib or vehicle (DMSO). The expression of IGF1R, AKT, ERK1/ERK2, and their respective phosphorylated forms (P-IGF1R, P-AKT, P-ERK1/P-ERK2) was analyzed by western blot with specific antibodies. GAPDH was used as loading control. A representative experiment of three independent experiments is shown.

Fig 3: Insulin treatment in basal media restores IGF-1R and INSR to normal levels. (A) hTCEpi cells were cultured in basal media and supplemented individually with each component of the bullet kit (BPE, hEGF, insulin, hydrocortisone, transferrin, epinephrine) and also with 100 ng/ml of IGF-1. Expression of IGF-1R was assessed by immunoblotting. Insulin, a component of the bullet kit, was the only growth factor that decreased expression of IGF-1R. (B) hTCEpi cells were cultured in growth, basal or basal media supplemented with 10 µg/ml insulin. For both receptors, immunoblotting showed an increase in basal media that was attenuated following treatment with insulin. (C) HCECs were also cultured overnight in growth, basal or basal media supplemented with 10 µg/ml insulin. Similar to hTCEpi cells, there was a large increase in INSR and IGF-1R expression in basal media that was reduced following the addition of insulin to the culture media. (D) Real time PCR for IGF-1R and INSR in hTCEpi cells showed that changes in receptor expression levels paralleled mRNA levels with a significant increase in IGF-1R and INSR mRNA in basal media compared to growth media or basal media supplemented with insulin (P < 0.001, Two-way ANOVA, Tukey multiple comparison test). (E) Real time PCR for IGF-1R and INSR in HCECs. There was a significant increase in IGF-1R and INSR in basal media compared to growth conditions or basal media supplemented with insulin (P < 0.001, Two-way ANOVA, Tukey multiple comparison test). There was a small, but significant difference between IGF-1R and INSR mRNA in basal media supplemented with insulin compared to the growth condition (*P = 0.002 for IGF-1R and **P = 0.027 for INSR, Two-way ANOVA, Tukey multiple comparison test). Data represented as mean ± standard deviation of fold change compared to growth condition.

Fig 4: ß-arrestin2/IGF-1R interaction and receptor ubiquitination as the underlying mechanism controlling IGF-1R expression and signalling. (a) U2OS cells transfected with isoform-specific Flag-tagged-ß-arr-encoding plasmids (+ß1/+ß2) were serum starved and stimulated with IGF-1 (50 ng/ml) for 0–60 min. ß-arrs were immunoprecipitated (IP) via their Flag-tag and association with IGF-1R detected by WB, with Flag detection used as loading controls. Expression of IGF-1R, Flag and GAPDH in the total cell lysates (TCL) before IP were anaysed by WB. (b) U2OS cells co-transfected with IGF-1R and Flag-tagged-ß-arr-encoding plasmids (+ß1/+ß2), were serum starved, stimulated with IGF-1 (50 ng/ml) for 0–60 min, and immunoprecipitated via their Flag tag. WB detection analysed level of IGF-1R in the immuno-precipitates. Expression of IGF-1R, Flag and GAPDH in the TCL before IP were analysed by WB. (c) U2OS cells co-transfected with IGF-1R and Flag-tagged-ß-arr-encoding plasmids (+ß1/+ß2), were serum starved, stimulated with IGF-1 (50 ng/ml) for 0–60 min, and immunoprecipitated via IGF-1R. WB detection analysed level of ubiquitin (Ub) in the immuno-precipitates. Expression of IGF-1R, Flag and GAPDH in the TCL before IP were analysed by WB.

Fig 5: Effects of co-targeting HER-1 and HER-2 or HER-1 and IGF-1R on receptor phosphorylation in EC or TNBC. OE19 or MDA-MB-231 cells were treated for 1 h prior to ligand stimulation with 10 ng/mL EGF/HRG for 15 min. After treatment, cells were lysed 1× in RIPA lysis buffer (Santa Cruz Biotechnology, Inc; Dallas, TX, USA). Phosphorylated HER-1, HER-2, and IGF-1R were measured via western blot or by phospho-ELISA kits from R&D Systems. Percent inhibition was calculated by taking absorbance (abs) readings at 450 nm and using the following equation: (abs. untreated-abs. treated)/abs. untreated × 100). Results displayed are representative of two independent experiments performed in triplicate. Error bars represent SD of the mean. Single peptide vaccine antibody treatments with anti-HER-1-418 or with anti-HER-2-266 (A) significantly decreased phosphorylation of both receptors as compared to negative control (* p < 0.005). The combination of anti-HER-2-266 and anti-HER-1-418 in OE19 cells showed significant down-regulation of receptor phosphorylation over single treatment alone (* p < 0.005). The peptide mimic combination of HER-1-418 and HER-2-266 (B) inhibited phosphorylation significantly more than single treatment alone (* p = 0.05) with the exception of the comparison to the HER-2 receptor in the HER-1-418 single condition (p = 0.09). Western blots of total protein lysates were performed for p-Tyr1131 IGF-IR (1:200), total IGF-IR (1:250) (both from Cell Signaling), and beta-actin (1:15,000) (Sigma-Aldrich). Goat anti-mouse secondary IRDye 800 antibody (#926-32210, 1:10,000) was purchased from Li-Cor Biosciences (Lincoln, NE, USA) and goat anti-rabbit alexa-fluor 680 secondary antibody (#1027681, 1:10,000) was purchased from Invitrogen (Grand Island, NY, USA). Protein bands were detected using the Odyssey Imaging System (Li-Cor Biosciences, Lincoln, NE, USA). Results with peptide vaccine antibodies in TNBC cells (C) showed increased inhibition of receptor phosphorylation with combination treatment, as demonstrated by western blot analysis, where treatment with anti-HER-1-418 and anti-IGF-1R-56 together markedly decreased IGF-1R phosphorylation as compared to control (normal rabbit IgG) or single peptide vaccine antibody treatment alone. Single peptide mimic treatment (D) with HER-1-418 decreases phosphorylation of both HER-1 and IGF-1R receptors significantly over negative control (* p < 0.05), while single treatment with IGF-1R-56 significantly inhibits phosphorylation of the HER-1 receptor (** p < 0.01), as determined by phospho-ELISA analysis. Combination treatment with IGF-1R-56 and HER-1-418 showed significant down-regulation of both receptor phosphorylation over single treatment with IGF-1R or HER-1-418 alone (* p < 0.05, ** p < 0.01 respectively).