The antifungal drug itraconazole inhibits vascular endothelial growth factor receptor 2 (VEGFR2) glycosylation, trafficking, and signaling in endothelial cells

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, phospholipase C γ1, failed to become activated after VEGF stimulation. These effects were due to a defect in VEGFR2 trafficking, leading to a decrease in cell surface expression, and were associated with the accumulation of immature N-glycans on VEGFR2. Small molecule inducers of lysosomal cholesterol accumulation and mammalian target of rapamycin (mTOR) inhibition, two previously reported itraconazole activities, failed to recapitulate itraconazole's effects 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-N-acetyllactosamine 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 itraconazole's effects extend beyond VEGFR2.

Figures

FIGURE 1.

Itraconazole induces a change in the migration pattern of VEGFR2. A, HUVEC were treated for 24 h with the indicated doses of itraconazole or vehicle (DMSO), and VEGFR2 was analyzed by Western blot. B, HUVEC were treated as in A with three inhibitors of fungal 14DM (terconazole, fluconazole, and ketoconazole) and an inhibitor of human 14DM (azalanstat).

FIGURE 2.

Itraconazole blocks VEGF165 binding to VEGFR2 and inhibits VEGFR2 signaling. A, HUVEC were grown in low serum medium for 24 h in the presence of vehicle (DMSO), itraconazole (Ita), or the VEGFR2 inhibitor sunitinib. The cells were then stimulated for the indicated times with VEGF165, lysed, and analyzed by Western blot for an activating phosphorylation on VEGFR2 (Tyr-1175), its immediate downstream binding partner PLCγ1 (Tyr-783), and a downstream effector ERK1/2 (Thr-202/Tyr-204). Total protein levels are shown as controls. B, for the 400 nm itraconazole samples, the phosphorylated signals were quantitated, normalized to the levels of total protein and then to vehicle control. n = 3 independent experiments; error bars, S.E.; *, p < 0.05. C, HUVEC were treated with 800 nm itraconazole or vehicle under the same conditions as in A and then were incubated with 125I-VEGF165 with or without competition with cold VEGF165 (20-fold excess). The ligand was cross-linked to VEGFR2, which was then immunoprecipitated and subject to both autoradiography and Western blotting (IB) for VEGFR2.

FIGURE 3.

Itraconazole alters N-linked glycosylation in HUVEC. A, Western blot of VEGFR2 from HUVEC treated for 24 h with itraconazole (Ita) or N-linked glycosylation inhibitors Sws, dMM, or castanospermine (Cast). HUVEC were treated for the indicated times with 800 nm itraconazole (B), tunicamycin, or dMM (C) prior to analysis of VEGFR2 by Western blot. D, lysates from itraconazole- or vehicle-treated HUVEC were digested with sialidase, endo H, or PNGase F, and the migration pattern of VEGFR2 was analyzed by Western blot. E, partial MALDI-TOF mass spectra of the high molecular mass region of the global population of N-glycans from HUVEC treated with 800 nm itraconazole or vehicle alone. The inset shows a Western blot for VEGFR2 from samples of the cell pellets used for the global profiling. See supplemental Fig. 1 for complete spectra and a more detailed description of the annotations. F, cell surface proteins from itraconazole-treated HUVEC or vehicle controls were biotinylated, captured on streptavidin beads, and subjected to Western blotting. Tubulin, a control intracellular protein, was present only in the input fraction. Yellow circles, galactose; green circles, mannose; blue squares, N-acetylglucosamine; yellow squares, N-acetylgalactosamine; red triangles, fucose; purple diamonds, N-acetylneuraminic acid.

FIGURE 4.

Subcellular localization of VEGFR2 after treatment with itraconazole or other glycosylation inhibitors. HUVEC were treated for 24 h with itraconazole (Ita; 800 nm) or vehicle (DMSO), fixed, and stained with anti-VEGFR2 and anti-GM130 (A), a cis-Golgi marker, or anti-PDI (B), an ER marker. C, cells were treated with 500 μm castanospermine (Cast), 500 μm dMM, 50 μm Sws, 800 nm itraconazole, or vehicle (DMSO) and treated as in A. Representative confocal micrographs are shown. Bars, 20 μm. Experiments were repeated in triplicate for GM130 staining experiments and in duplicate for PDI staining.

FIGURE 5.

Itraconazole's effects on glycosylation extend beyond VEGFR2 and HUVEC. A, HUVEC were treated with the indicated doses of itraconazole (Ita) or DMSO vehicle (0 μm itraconazole dose), and the migration pattern of VEGFR1 was analyzed by Western blot. B, ACHN cells were treated with the indicated concentrations of itraconazole in 2% serum, and EGFR was analyzed by Western blot. C, the effects of itraconazole on the proliferation of the renal cell carcinoma line, ACHN, in the presence of 2 or 10% serum. D, lysates from itraconazole-treated ACHN cells were digested with either endo H or PNGase F and analyzed by Western blot for EGFR. Error bars, S.E.

FIGURE 6.

Itraconazole's effects on VEGFR2 glycosylation occur in parallel to other itraconazole-induced effects. A, cholesterol localization was visualized by filipin staining of HUVEC treated with 800 nm itraconazole (Ita), 500 μm castanospermine (Cast), 500 μm dMM, or 50 μm Sws. Bar, 20 μm. B, mTOR inhibition was determined based on the phosphorylation status of its substrate S6 kinase (S6K) after treatment with itraconazole, castanospermine, dMM, or Sws, as determined by Western blot. C, VEGFR2 migration patterns were analyzed by Western blot in lysates from HUVEC treated for 24 h with itraconazole, the mTOR inhibitor rapamycin, or two inhibitors of cholesterol trafficking, U18666A and imipramine. D, activation of VEGFR2 phosphorylation by VEGF165 after treatment with the indicated compounds was assessed by Western blotting.

FIGURE 7.

Itraconazole-induced VEGFR2 hypoglycosylation and signaling inhibition is rescued by supplementation of cellular cholesterol. A, HUVEC were treated with itraconazole or vehicle alone in the presence of free cholesterol or free β-estradiol, free methyl-β-cyclodextrin, methyl-β-cyclodextrin·cholesterol, or methyl-β-cyclodextrin·β-estradiol complexes. VEGFR2 migration patterns in lysates were analyzed by Western blot. B, the ability of methyl-β-cyclodextrin·cholesterol to rescue itraconazole (Itra)-induced VEGFR2 signaling inhibition in HUVEC after VEGF165 stimulation was determined by Western blot. C, the capacity of methyl-β-cyclodextrin·cholesterol to rescue dMM-induced migration changes was determined as in A.