Fig. 8: EHD1-dependent upregulation of oncogenic attributes of EWS cells requires the IGF-1R.

a Elevated IGF-1 signaling upon mEHD1 overexpression in EHD1-low SK-ES-1 cells requires IGF-1R expression and activity. Western blot analysis of phosphorylation of IGF-1R and key signaling pathway reporters (AKT and ERK1/2) in SK-ES-1-mEHD1 cell line. Cells were pre-starved for 48 h in serum/IGF-free medium combined with treatment with DMSO or 1 µM Linsitinib and either left unstimulated or stimulated with IGF-1 (50 ng/ml) for 30 min. b–i Mouse EHD1 (mEHD1)-overexpressing SK-ES-1 cell line was used to assess the requirement of IGF-1R in EHD1-driven pro-oncogenic attributes. SK-ES-1-mEHD1 cells transiently transfected with non-targeting control (NTC) or IGF-1R-targeted siRNA or treated with IGF-1R inhibitor linsitinib (1 µM) were studied for indicated traits. b Representative western blot confirming the effective IGF-1R knockdown upon transient IGF-1R siRNA relative to NTC siRNA transfection. c Elevated cell proliferation upon mEHD1 overexpression requires IGF-1R expression and activity. The SK-ES-1-mEHD1 cells were analyzed for IGF-1 (100 ng/ml)-dependent cell proliferation by Cell-Titer Glo assay with or without the indicated treatments. Parental SK-ES-1 cells without any treatments provided a baseline of cell proliferation without mEHD1 overexpression. d Elevated cell survival upon mEHD1 overexpression requires IGF-1R expression and activity. The SK-ES-1-mEHD1 cells grown in the presence of IGF-1 (100 ng/ml) without or with the indicated treatments for 3 days were analyzed for the proportion of apoptotic cells by FACS after Annexin-V and PI staining. e, f Elevated cell migration and invasion upon mEHD1 overexpression requires IGF-1R expression and activity. The SK-ES-1-mEHD1 cells were analyzed for IGF-1 (100 ng/ml)-dependent trans-well cell migration or invasion without or with the indicated treatments. Parental SK-ES-1 cells without any treatments provided a baseline of cell migration without mEHD1 overexpression. g–i Impact of treatment with IGF-1R inhibitory monoclonal antibody 1H7 (4 µg/ml) and its corresponding IgG control on cell proliferation (g), migration (h), and invasion (i) of mouse EHD1 (mEHD1)-overexpressing SK-ES-1 cell line. Mean +/− SEM of 3 experiments, each in triplicates. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. j Rescue of signaling by exogenous IGF-1R expression in EHD1-KO EWS cells. EHD1-KO TC71 cells were transfected with Empty vector (EV)-GFP or IGF-1R-GFP constructs, and stable lines selected in G418 were pre-starved for 24 h in low serum/IGF-free medium and either left unstimulated or stimulated with IGF-1 (50 ng/ml) for 30 mins. Western blot analysis of rescue of phosphorylation of key signaling pathway reporters (AKT and ERK1/2) in IGF-1R-GFP as compared to NTC and EHD1-KO EV-GFP cell lines is shown. IGF-1 treatment also induced robust phosphorylation of introduced GFP-IGF-1R. k–p Rescue of cell proliferation (k, l), cell migration (m, o), and invasion (n, p) in TC71 and A673 EHD1-KO cells upon GFP-IGF-1R overexpression as compared to control EV-transfected cell lines. Data represent the mean +/− SEM of 3 independent experiments. ***p < 0.001.