Abstract
IntroductionDrugs targeting the VEGF‐pathway have been approved to treat various cancers. However, tumors become resistant to this therapy due to the recruitment of alternative growth factors, acting via cognate endothelial cells (EC) receptor tyrosine kinases (RTK) or g‐protein coupled receptors (GPCR), to cue neo‐angiogenesis. Facio‐genital dysplasia 5 (FGD5) regulates vascular endothelial growth factor (VEGF)‐mediated angiogenesis, and VEGF receptor‐2 trafficking at the endosome. However, the role of FGD5 to regulate other pro‐angiogenic signaling pathways that co‐operate with VEGF has not been explored. Here, we identify a novel role of FGD5 to control G protein‐coupled receptor (GPCR) signaling.MethodsHuman umbilical vein endothelial cells (HUVEC) were mounted on microcarrier beads, suspended in fibrin gels, then co‐cultured with freshly harvested patient‐derived renal cell carcinoma (PD‐RCC) samples. The effect of FGD5 loss on sprouting angiogenesis was evaluated using RNAi against FGD5 in HUVEC. Stromal cell derived factor (SDF)‐1 was used as a model GPCR ligand to study the role of FGD5 in endothelial angiogenesis and PI3K signaling. Further, FDG5 domain‐deletant mutants were transiently overexpressed to rescue the effect of FGD5 knockdown on endothelial PI3K signaling. Western blot and confocal imaging were performed to readout the role of FGD5 in the GPCR/PI3K signaling pathway.ResultsLoss of FGD5 in HUVEC decreased sprouting angiogenesis stimulated by growth factors released from PD‐RCCs. The defect in angiogenesis was accompanied by downregulation of the expression of endothelial tip cell specific marker genes. Knockdown of endothelial FGD5 abolished the SDF‐1 angiogenic effect and SDF‐1 signaling to PI3K‐β and Akt. Inhibition of Rac1, a Rho GTP‐binding protein required for PI3K‐β activity, recapitulated the signaling defects of FGD5 deficiency, suggesting that FGD5 may control PI3K‐β activity through Rac1‐GTP. Overexpression of Dbl (Rho guanine nucleotide exchange factor) domain‐deleted FGD5 (FGD5ΔDbl) reduced SDF‐1‐stimulated Akt phosphorylation, and failed to rescue defective PI3K signalling in FGD5‐deficient cells, indicating that the Dbl domain of FGD5 regulates the activity of PI3K‐β. PI3K‐β and phosphorylated Akt localized to early endosomes after SDF‐1 stimulation, but loss of FGD5 decreased endosomal phosphorylated‐Akt. Failure of Akt activation at PI3K‐β‐positive endosomes suggests a defect in PI3K‐β activity after FGD5 loss.ConclusionsWe identify a novel role of FGD5 to regulate GPCR signaling to PI3K‐β. FGD5 acts at an endosomal convergence node regulating RTK and GPCR angiogenic pathways that may serve as a target for anti‐angiogenic therapy.Support or Funding InformationUniversity of Alberta Doctoral Recruitment Scholarship, Translational Medicine Research Award, Alberta Graduate Excellence Scholarship, Canadian Cancer Society, Division of Nephrology
Published Version
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