Abstract

Itraconazole is a safe and widely used antifungal drug that was recently found to possess potent antiangiogenic activity. Currently, there are four active clinical trials evaluating itraconazole as a cancer therapeutic. Tumor growth is dependent on angiogenesis which is driven by the secretion of growth factors from the tumor itself. We report here that itraconazole significantly inhibited the binding of vascular endothelial growth factor (VEGF) to VEGF receptor 2 (VEGFR2) and that both VEGFR2 and an immediate downstream substrate, PLC?1 failed to become activated after VEGF stimulation. These effects were tied to a significant increase in the electrophoretic mobility of VEGFR2. We showed that this was due to the accumulation of immature N-glycans on VEGFR2 and an associated decrease in cell surface levels of the receptor. Small molecule inducers of lysosomal cholesterol accumulation and mTOR inhibition, two previously reported itraconazole activities, failed to recapitulate effects of itraconazole on VEGFR2 glycosylation and signaling. Likewise, glycosylation inhibitors did not alter cholesterol trafficking or inhibit mTOR. Repletion of cellular cholesterol levels, which was known to rescue the effects of itraconazole on mTOR and cholesterol trafficking, was also able to restore VEGFR2 glycosylation and signaling. This suggests that the new effects of itraconazole occur in parallel to those previously reported but are downstream of a common target. We also demonstrated that itraconazole globally reduced poly-LacNAc and tetra-antennary complex N-glycans in endothelial cells and induced hypoglycosylation of the epidermal growth factor receptor in a renal cell carcinoma line suggesting that effects of itraconazole extend beyond VEGFR2.