Fig. 5

CCHE1 modulated LDHA phosphorylation via FGFR1. A CCHE1 was overexpressed in melanoma cells and the level of phos-LDHA (Y10) was detected by western blot. Enhanced LDHA-Y10 was observed with CCHE1 overexpression. B The phos-LDHA (Y10) was decreased upon CCHE1 depletion in both A375 and G-361 cells. (C) Melanoma cells were treated with 2.5 μM FGFR1 inhibitor PD166866 for 24 h, and the p-LDHA (Y10) was determined via western blot. Blocking FGFR1 attenuated CCHE1-promoted LDHA-Y10 phosphorylation. D The specific binding between CCHE1 and FGFR1 was determined by the RIP assay, which showed the significant enrichment of CCHE1 in the immunoprecipitates with anti-FGFR1 antibody. E Pull-down assay was performed with his tagged recombinant FGFR1 protein and biotin-labeled CCHE1. FGFR1 interacted with CCHE1 but not the antisense CCHE1. F Cells were transfected with control vector or CCHE1, and the interaction between LDHA and FGFR1 was detected by co-IP assay using anti-LDHA antibody. Enhanced abundance of FGFR1 was found in the precipitates of LDHA with CCHE1 overexpression. G, H Blockade of FGFR1 significantly inhibited CCHE1-promoted glucose uptake and lactate generation. I, J FGFR1 inhibition weaken the positive role of CCHE1 in melanoma cell proliferation. K, L Both the glucose consumption and lactate production were significantly reduced with the addition of inhibitors of FGFR1 (PD166866, 2.5 μM) and LDHA (GSK2837808A, 10 μM) in CCHE1-depleted melanoma cells. M, N Depletion of CCHE1 combined with inhibition of FGFR1 and LDHA obviously suppressed the proliferation of melanoma cells. *p < 0.05, **p < 0.01, ***p < 0.001