Abstract 2316: Itraconazole, an antifungal drug with anti-angiogenic activity, inhibits VEGFR2 trafficking, glycosylation, and signaling in endothelial cells

Abstract

Abstract Itraconazole is a widely used antifungal drug that has been recently shown to inhibit angiogenesis in vivo and to block non-small cell lung cancer growth in animal models. Based on these and other results, itraconazole is currently being evaluated in four clinical trials as a cancer therapeutic. Angiogenesis, one of the hallmarks of cancer, is required for tumor growth and is driven by tumor-secreted growth factors including vascular endothelial growth factor (VEGF). We now report that in endothelial cells, itraconazole inhibits signaling of VEGF through the VEGF receptor 2 (VEGFR2) (80% inhibition; p < 0.05) and subsequently through a downstream substrate, PLCϒ1 (60% inhibition; p < 0.05). Mechanistically, we show that this was due to a nearly complete loss of VEGF binding to VEGR2 that was secondary to a loss of cell surface expression of the receptor. After itraconazole treatment, VEGFR2 was retained in a perinuclear aggregate which partially overlapped with the cis-Golgi marker GM130. Correlated with this loss of VEGFR2 signaling and trafficking, was a dramatic change in VEGFR2 mobility on SDS-PAGE which resulted from incomplete N-glycosylation. Itraconazole globally reduced poly-LacNAc and tetra-antennary glycans in endothelial cells and induced hypoglycosylation of EGFR in a renal cell carcinoma line, suggesting that the effects of itraconazole apply to multiple cell types and receptors. Importantly, small molecule inducers of lysosomal accumulation and mTOR inhibition, two previously known itraconazole-induced effects, did not interfere with VEGFR2 glycosylation. Similarly, glycosylation inhibitors did not affect cholesterol trafficking or inhibit mTOR. However, increasing cellular cholesterol levels, which was known to rescue the effects of itraconazole on mTOR and cholesterol trafficking, restored VEGFR2 glycosylation and signaling. Thus, the newly described properties of itraconazole occur in parallel to inhibition of cholesterol trafficking and mTOR but appear downstream of a single target. Taking these findings together, we propose a model in which itraconazole inhibits intracellular trafficking. This model could explain the loss of VEGFR2 signaling, incomplete VEGFR2 glycosylation, as well as disruption of cholesterol trafficking and mTOR signaling. Importantly, the effects of itraconazole we report here may significantly contribute to the in vivo efficacy of itraconazole and are relevant to ongoing clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2316. doi:1538-7445.AM2012-2316