The Antifungal Drug Itraconazole Inhibits Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) Glycosylation, Trafficking, and Signaling in Endothelial Cells*

Background: Itraconazole is a widely used antifungal drug that was recently found to possess potent antiangiogenic activity. Results: We report here that itraconazole caused accumulation of immature N-glycans on VEGFR2 and inhibited VEGFR2 trafficking and signaling. Conclusion: These results suggest that itraconazole interferes with VEGFR2 trafficking, glycosylation, and signaling activity. Significance: Itraconazole possesses unique antiangiogenic potential through targeting multiple pathways that are essential for angiogenesis. 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.

[1]  A. Helenius,et al.  Roles of N-linked glycans in the endoplasmic reticulum. , 2004, Annual review of biochemistry.

[2]  J Folkman,et al.  Transplacental carcinogenesis by stilbestrol. , 1971, The New England journal of medicine.

[3]  Jun O. Liu,et al.  Impact of Absolute Stereochemistry on the Antiangiogenic and Antifungal Activities of Itraconazole. , 2010, ACS medicinal chemistry letters.

[4]  J. Folkman.  Tumor angiogenesis: therapeutic implications. , 1971, The New England journal of medicine.

[5]  Jun O. Liu,et al.  Cholesterol trafficking is required for mTOR activation in endothelial cells , 2010, Proceedings of the National Academy of Sciences.

[6]  Robert Gray,et al.  Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. , 2006, The New England journal of medicine.

[7]  Alessio Ceroni,et al.  GlycoWorkbench: a tool for the computer-assisted annotation of mass spectra of glycans. , 2008, Journal of proteome research.

[8]  Jun O. Liu,et al.  A Calcineurin-Independent Mechanism of Angiogenesis Inhibition by a Nonimmunosuppressive Cyclosporin A Analog , 2011, Journal of Pharmacology and Experimental Therapeutics.

[9]  D. Hayes.  Bevacizumab treatment for solid tumors: boon or bust? , 2011, JAMA.

[10]  J. Berlin,et al.  Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. , 2004, The New England journal of medicine.

[11]  Y. Bang,et al.  Sunitinib malate for the treatment of pancreatic neuroendocrine tumors. , 2011, The New England journal of medicine.

[12]  L. Claesson‐Welsh,et al.  FGF and VEGF function in angiogenesis: signalling pathways, biological responses and therapeutic inhibition. , 2001, Trends in pharmacological sciences.

[13]  D. Swinney,et al.  Selective inhibition of mammalian lanosterol 14 alpha-demethylase by RS-21607 in vitro and in vivo. , 1994, Biochemistry.

[14]  K. Khoo,et al.  Mass spectrometry of carbohydrate-containing biopolymers. , 1994, Methods in enzymology.

[15]  A. De Maio,et al.  The Antifungal Agent Itraconazole Induces the Accumulation of High Mannose Glycoproteins in Macrophages* , 2009, The Journal of Biological Chemistry.

[16]  E. Berger,et al.  Immunocytochemical localization of galactosyltransferase in HeLa cells: codistribution with thiamine pyrophosphatase in trans-Golgi cisternae , 1982, The Journal of cell biology.

[17]  A. Prota,et al.  Structure-function analysis of VEGF receptor activation and the role of coreceptors in angiogenic signaling. , 2010, Biochimica et biophysica acta.

[18]  M. Shibuya,et al.  The 230 kDa mature form of KDR/Flk-1 (VEGF receptor-2) activates the PLC-γ pathway and partially induces mitotic signals in NIH3T3 fibroblasts , 1997, Oncogene.

[19]  H. Dvorak,et al.  VEGF-A and the induction of pathological angiogenesis. , 2007, Annual review of pathology.

[20]  J. Sato,et al.  MAP kinases, phosphatidylinositol 3‐kinase, and p70 S6 kinase mediate the mitogenic response of human endothelial cells to vascular endothelial growth factor , 1999, Journal of cellular physiology.

[21]  C. Burd,et al.  Golgi localization of glycosyltransferases requires a Vps74p oligomer. , 2008, Developmental cell.

[22]  M. Steinmetz,et al.  Structure of a VEGF–VEGF receptor complex determined by electron microscopy , 2007, Nature Structural &Molecular Biology.

[23]  Yonghong Xiao,et al.  GOLPH3 modulates mTOR signaling and rapamycin sensitivity in cancer , 2009, Nature.

[24]  C. D’Souza-Schorey,et al.  Neurodegeneration in Niemann-Pick Type C disease and Huntington's disease: impact of defects in membrane trafficking. , 2009, Current drug targets.

[25]  Shenhong Wu,et al.  Treatment-related mortality with bevacizumab in cancer patients: a meta-analysis. , 2011, JAMA.

[26]  M. Waxham,et al.  Interactions of FLT-1 and KDR with phospholipase C gamma: identification of the phosphotyrosine binding sites. , 1997, Biochemical and biophysical research communications.

[27]  G. Demetri,et al.  Molecular basis for sunitinib efficacy and future clinical development , 2007, Nature Reviews Drug Discovery.

[28]  L. Ellis.  The role of neuropilins in cancer , 2006, Molecular Cancer Therapeutics.

[29]  L. Tu,et al.  Signal-Mediated Dynamic Retention of Glycosyltransferases in the Golgi , 2008, Science.

[30]  C. Kielty,et al.  Vascular endothelial growth factor can signal through platelet-derived growth factor receptors , 2007, The Journal of cell biology.

[31]  D. Kelly,et al.  Characteristics of the heterologously expressed human lanosterol 14α‐demethylase (other names: P45014DM, CYP51, P45051) and inhibition of the purified human and Candida albicans CYP51 with azole antifungal agents , 1999, Yeast.

[32]  N. Rahimi,et al.  VEGFR-1 and VEGFR-2: two non-identical twins with a unique physiognomy. , 2006, Frontiers in bioscience : a journal and virtual library.

[33]  D. Gospodarowicz,et al.  The identification and partial characterization of the fibroblast growth factor receptor of baby hamster kidney cells. , 1985, The Journal of biological chemistry.

[34]  Jun O. Liu,et al.  Inhibition of angiogenesis by the antifungal drug itraconazole. , 2007, ACS chemical biology.

[35]  Mark Sutton-Smith,et al.  Glycomic profiling of cells and tissues by mass spectrometry: fingerprinting and sequencing methodologies. , 2006, Methods in enzymology.

[36]  Jun O. Liu,et al.  Itraconazole, a commonly used antifungal that inhibits Hedgehog pathway activity and cancer growth. , 2010, Cancer cell.

[37]  G. Giaccone,et al.  Epidermal growth factor receptor inhibitors in the treatment of non-small-cell lung cancer. , 2005, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[38]  Peter L Lee,et al.  Association of beta-1,3-N-acetylglucosaminyltransferase 1 and beta-1,4-galactosyltransferase 1, trans-Golgi enzymes involved in coupled poly-N-acetyllactosamine synthesis. , 2009, Glycobiology.

[39]  M C Phillips,et al.  Use of cyclodextrins for manipulating cellular cholesterol content. , 1997, Journal of lipid research.

[40]  J. Lippincott-Schwartz,et al.  Lipids and cholesterol as regulators of traffic in the endomembrane system. , 2010, Annual review of biophysics.

[41]  D. Gospodarowicz,et al.  Characterization of the receptors for vascular endothelial growth factor. , 1990, The Journal of biological chemistry.

[42]  W. Völkel,et al.  Comparison of lanosterol-14 alpha-demethylase (CYP51) of human and Candida albicans for inhibition by different antifungal azoles. , 2006, Toxicology.

[43]  M. Shibuya,et al.  A single autophosphorylation site on KDR/Flk‐1 is essential for VEGF‐A‐dependent activation of PLC‐γ and DNA synthesis in vascular endothelial cells , 2001, The EMBO journal.

[44]  Jun O. Liu,et al.  Itraconazole inhibits angiogenesis and tumor growth in non-small cell lung cancer. , 2011, Cancer research.