Antifungal drug itraconazole targets VDAC1 to modulate the AMPK/mTOR signaling axis in endothelial cells

Significance Tumors promote angiogenesis to facilitate their growth and metastasis; thus, inhibition of angiogenesis is a promising strategy for treating cancer. During angiogenesis, endothelial cells (EC) are stimulated by proangiogenic factors to proliferate and migrate, leading to the formation of new blood vessels. Understanding the mechanisms regulating EC function therefore is essential for the development of new antiangiogenic interventions. Here, we identify a novel mechanism of EC regulation by the recently discovered angiogenesis inhibitor itraconazole, mediated by direct binding to the mitochondrial protein voltage-dependent anion channel 1 (VDAC1). VDAC1 inhibition perturbs mitochondrial ATP production, leading to activation of the AMP-activated protein kinase pathway and subsequent inhibition of mechanistic target of rapamycin, a regulator of EC proliferation. This study suggests VDAC1 may serve as a new therapeutic target for angiogenesis inhibition. Itraconazole, a clinically used antifungal drug, was found to possess potent antiangiogenic and anticancer activity that is unique among the azole antifungals. Previous mechanistic studies have shown that itraconazole inhibits the mechanistic target of rapamycin (mTOR) signaling pathway, which is known to be a critical regulator of endothelial cell function and angiogenesis. However, the molecular target of itraconazole that mediates this activity has remained unknown. Here we identify the major target of itraconazole in endothelial cells as the mitochondrial protein voltage-dependent anion channel 1 (VDAC1), which regulates mitochondrial metabolism by controlling the passage of ions and small metabolites through the outer mitochondrial membrane. VDAC1 knockdown profoundly inhibits mTOR activity and cell proliferation in human umbilical vein cells (HUVEC), uncovering a previously unknown connection between VDAC1 and mTOR. Inhibition of VDAC1 by itraconazole disrupts mitochondrial metabolism, leading to an increase in the cellular AMP:ATP ratio and activation of the AMP-activated protein kinase (AMPK), an upstream regulator of mTOR. VDAC1-knockout cells are resistant to AMPK activation and mTOR inhibition by itraconazole, demonstrating that VDAC1 is the mediator of this activity. In addition, another known VDAC-targeting compound, erastin, also activates AMPK and inhibits mTOR and proliferation in HUVEC. VDAC1 thus represents a novel upstream regulator of mTOR signaling in endothelial cells and a promising target for the development of angiogenesis inhibitors.