Itraconazole, a commonly used antifungal that inhibits Hedgehog pathway activity and cancer growth

Abstract

In a screen of drugs previously tested in humans we identified itraconazole, a systemic antifungal, as a potent antagonist of the Hedgehog (Hh) signaling pathway that acts by a mechanism distinct from its inhibitory effect on fungal sterol biosynthesis. Systemically administered itraconazole, like other Hh pathway antagonists, can suppress Hh pathway activity and the growth of medulloblastoma in a mouse allograft model and does so at serum levels comparable to those in patients undergoing antifungal therapy. Mechanistically, itraconazole appears to act on the essential Hh pathway component Smoothened (SMO) by a mechanism distinct from that of cyclopamine and other known SMO antagonists, and prevents the ciliary accumulation of SMO normally caused by Hh stimulation.

Copyright 2010 Elsevier Inc. All rights reserved.

Figures

Figure 1. Itraconazole inhibits Hh signaling

(A) A schematic view of the Hedgehog (Hh) signaling pathway. In the absence of Hh, Patched (Ptch) suppresses Smoothened (Smo) function. Hh, when present, binds to and inhibits Ptch, permitting Smo accumulation in the primary cilium (not shown) and causing activation of the pathway via the Gli family of transcription factors. Ptch and Gli1 are themselves transcriptional targets of the pathway. Oxysterols (dashed green bracket) act between Ptch and Smo, as pathway activators, whereas statins (dashed red bracket) act downstream of Ptch and at or upstream of Smo, as pathway inhibitors. SAG and cyclopamine activate and inhibit the pathway, respectively, by binding to the transmembrane domain of Smo. Activators and inhibitors of the pathway are labeled in green and red, respectively. (B) Hh signaling assays. Luciferase reporter activity under the control of an 8×-Gli binding site in the Shh-Light2 reporter cell line was measured upon stimulation with ShhN-containing medium. Itraconazole blocked Hh pathway activity (IC50≈800 nM). (C) Schematic view of mammalian cholesterol biosynthesis from 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA). Statins inhibit HMG-CoA reductase whereas azole antifungal drugs inhibit 14α-lanosterol demethylase (14LDM), as indicated. Lathosterol and desmosterol are cholesterol precursors downstream of 14LDM. (D) Among the azole antifungals, itraconazole was the most potent inhibitor of Hh pathway activity. (E) Hydroxy-itraconazole, the major metabolite of itraconazole in mammals, also inhibited the Hh pathway (IC50 ≈1.2 µM). All signaling assays were performed with Shh-Light2 cells in 0.5% serum media and data are shown as the mean of triplicates ± s.d. See also Figure S1 and Table S1.

Figure 2. Inhibition of Hh signaling by itraconazole is not mediated by effects on cholesterol biosynthesis

(A) Increasing serum concentrations attenuated the inhibitory activity of itraconazole but not KAAD-cyclopamine. (B) The attenuating effect of serum was abolished by lipid depletion (LD). (C) Shh-Light2 cells were pretreated with methyl-β-cyclodextrin (MβCD) 8 mM in DMEM for 45 minutes to remove sterols from the cell surface (Cooper et al., 2003). Lovastatin, an HMG-CoA reductase inhibitor, inhibited Hh pathway activity induced by ShhN. Cholesterol, in ethanol solution, reversed the pathway inhibition due to lovastatin (D, E) Lathosterol, desmosterol, and cholesterol failed to rescue Hh pathway inhibition by itraconazole under low (D) or lipid depleted (E) serum conditions. All signaling assays were performed with Shh-Light2 cells and data are mean of triplicates ± s.d. See also Figure S2.

Figure 3. Low density lipoprotein modulate the Hh pathway inhibitory effects of itraconazole

(A) High density lipoprotein (HDL) 100 µg/ml and (B) very low density lipoprotein (VLDL) 6.5 µg/ml cannot reverse Hh pathway inhibition by itraconazole. (C, D) Titration of low density lipoprotein (LDL) up to 120 µg/ml rescues either induced Hh pathway activity by ShhN ligand in Shh-Light2 cells (C) or constitutively active pathway in Ptch−/− cells (Goodrich et al., 1997; Taipale et al., 2000) (D) from the inhibitory effects of itraconazole. All signaling assays were performed in lipid depleted 10% calf serum media for Shh-Light2 cells (A–C) and 0.5% fetal bovine serum media for Ptch−/− cells. Data are mean of triplicates ± s.d.

Figure 4. Mapping of itraconazole action within the Hh pathway

(A) Itraconazole inhibited constitutive Hh pathway activity in Ptch−/− cells, measured as expression of β-galactosidase activity from the Ptch-lacZ locus (Goodrich et al., 1997; Taipale et al., 2000). The levels of β-galactosidase activity from cells treated with KAAD-cyclopamine at 40 and 200 nM (2× and 10× IC50) are shown as purple and red lines, respectively. (B) Shh-Light SmoA1 cells, stably expressing a constitutively active Smo mutant, were resistant to Hh pathway inhibition by itraconazole. Cyclopamine and forskolin (orange and blue lines, respectively) were used as negative and positive controls for the inhibition of SmoA1. (C) Itraconazole and KAAD-cyclopamine both inhibited constitutive Hh pathway activation induced by transient overexpression of Smo in NIH-3T3 cells. (D) Itraconazole inhibited Hh pathway activity induced by the combined oxysterols, 20(S)-and 22(S)-hydroxycholesterol (5 µM each). All signaling assays were performed with Shh-Light2 cells in 0.5% serum media and data are shown as the mean of triplicates ± s.d. (E) Representative immunofluorescent images of NIHT-3T3 cells. Smo accumulated in primary cilia upon stimulation with ShhN (arrowheads). This accumulation was blocked by treatment with itraconazole. Insets show enlarged views of primary cilia in the dashed box, with Smo and acetylated tubulin channels offset. Arrows show primary cilia with no Smo accumulation. As seen in the histogram, the accumulation of Smo in ~90% of primary cilia of cells stimulated with ShhN, is reduced to ~5% in the additional presence of itraconazole. The number of primary cilia containing Smo over the total number of primary cilia are shown above each bar. Data are mean of 10 images ± s.d. The scale bar represents 5 µm.

Figure 5. Itraconazole acts at a site distinct from the cyclopamine-binding site of Smo

(A) BODIPY-cyclopamine (B–C) bound to tetracycline-induced Smo in 293S cells as monitored by FACS. As negative controls, the grey tracing shows fluorescence of cells not treated with B–C and the blue tracing (blue arrow) shows uninduced cells treated with B–C; the black tracing (black arrow) represents maximal fluorescence of cells induced for Smo expression and treated with B–C. Itraconazole treatment (no arrows) at concentrations up to 12.5× IC50 (10 µM) did not effectively compete for B–C binding, whereas KAAD-cyclopamine at 2× IC50 and 10× IC50 (40 nM and 200 nM; tan and red arrowheads, respectively) caused significant decreases in fluorescence. (B, C) Synergistic action of itraconazole and KAAD-cyclopamine. In signaling assays performed with Shh-Light2 cells, the presence of itraconazole caused a downward shift in the IC50 of KAAD-cyclopamine (from 21 nM to 1.8 nM in the presence of 900 nM itraconazole; panel B and Table S2); KAAD-cyclopamine caused a downward shift in the IC50 of itraconazole (from 805 to 135 nM in the presence of 30 nM KAAD-cyclopamine; panel C and Table S3). (D) KAAD-cyclopamine is a competitive inhibitor of SAG, a known Hh pathway agonist, as shown by shifts of the SAG EC50 by ~5 and ~13 fold at 40 nM and 100 nM concentrations of KAAD-cyclopamine. In contrast, itraconazole did not alter the SAG EC50 but decreased the maximal activity of SAG, thus acting as a noncompetitive inhibitor (see Table S4). All signaling assays were performed with Shh-Light2 cells in 0.5% serum media and data are mean of triplicates ± s.d. See also Tables S2, S3, and S4.

Figure 6. Itraconazole inhibits the growth of Hh-dependent medulloblastoma in vivo

(A) Itraconazole, either alone or in combination with cyclopamine, suppressed the growth of allografted medulloblastomas from a Ptch+/−p53−/− mouse, as compared to vehicle-treated tumors. Treatments were given twice per day by oral gavage. The number of tumors used in this experiment were: Cyclodextrin control n = 8, Itraconazole 100 mg/kg n = 12, Itraconazole 75 mg/kg n = 10, Cyclopamine 37.5 mg/kg n = 10, Itraconazole/cyclopamine n = 10. Data are expressed as mean percentage change in volume ± s.e.m. (B, C) Mean serum and tumor itraconazole levels obtained at the end of the allograft study in (A) are shown in panels (B) and (C), respectively. The dashed lines represent the minimum detectable levels of the assay; vehicle controls were at or below these levels. (D) Pharmacokinetics of itraconazole levels in serum of athymic nude mice after one oral dose of itraconazole 100 mg/kg or 75 mg/kg. All itraconazole levels were obtained using the bioassay method (Clemons et al., 2002; Harvey et al., 1980; Hostetler et al., 1992; Rex et al., 1991). Data for itraconazole levels in panels (BD) are the mean of three samples ± s.d. (E) Hh pathway is decreased in allografted medulloblastoma tumors when treated with either itraconazole or cyclopamine as compared to cyclodextrin control treatment. Hh pathway was monitored by Gli1 mRNA levels. Gli1 mRNA levels of drug treatments were normalized to the mean of Gli1 mRNA levels of cyclodextrin control. Treatments were given twice per day for four days by oral gavage. ** p = 0.0197; *** p = 0.0386

Figure 7. Itraconazole inhibits the growth of Hh-dependent basal cell carcinoma in vivo

(A) Compared to cyclodextrin vehicle treated tumors, itraconazole suppressed the growth of endogenous basal cell carcinomas from a K-14CreER;Ptch+/−;p53 fl/fl mouse. Treatments were given twice per day by oral gavage. Gray arrow indicates the day that itraconazole treatment was stopped. The number of tumors used in this experiment were: Cyclodextrin control n = 10, Itraconazole 100 mg/kg n = 20 until day 19 when itraconazole treatment was discontinued then n = 6 after some mice were euthanized and tumors dissected for further examination (see “Experimental Procedures”). For statistical analysis, pairwise comparisons of treatment arms were made for corresponding day of treatment with the exception of cyclodextrin control day 17 being compared with itraconazole day 18. Data are expressed as mean percentage change in tumor length ± s.e.m. (B) Cyclodextrin control treated tumors showed nests of well organized tumor cells (arrowhead), whereas itraconazole-treated tumors showed greater evidence of necrosis as seen by destruction of organized nests of basophilic tumor cells and pyknosis (arrow) and destruction of eosinophilic stroma lining the BCC nests (black arrowhead). Images are H&E sections. The scale bar represents 100 µm. (C) From the tumor growth inhibition study, BCC tumors from itraconazole treated mice (including those that had itraconazole discontinued) show more evidence of necrosis than cyclodextrin control treated tumors as measured by necrosis score (see Experimental Procedures) with higher numbers indicating worsening necrosis.