Itraconazole Exerts Its Antitumor Effect in Esophageal Cancer By Suppressing the HER2/AKT Signaling Pathway

Abstract

Itraconazole, an FDA-approved antifungal, has antitumor activity against a variety of cancers. We sought to determine the effects of itraconazole on esophageal cancer and elucidate its mechanism of action. Itraconazole inhibited cell proliferation and induced G1-phase cell-cycle arrest in esophageal squamous cell carcinoma and adenocarcinoma cell lines. Using an unbiased kinase array, we found that itraconazole downregulated protein kinase AKT phosphorylation in OE33 esophageal adenocarcinoma cells. Itraconazole also decreased phosphorylation of downstream ribosomal protein S6, transcriptional expression of the upstream receptor tyrosine kinase HER2, and phosphorylation of upstream PI3K in esophageal cancer cells. Lapatinib, a tyrosine kinase inhibitor that targets HER2, and siRNA-mediated knockdown of HER2 similarly suppressed cancer cell growth in vitro Itraconazole significantly inhibited growth of OE33-derived flank xenografts in mice with detectable levels of itraconazole and its primary metabolite, hydroxyitraconazole, in esophagi and tumors. HER2 total protein and phosphorylation of AKT and S6 proteins were decreased in xenografts from itraconazole-treated mice compared to xenografts from placebo-treated mice. In an early phase I clinical trial (NCT02749513) in patients with esophageal cancer, itraconazole decreased HER2 total protein expression and phosphorylation of AKT and S6 proteins in tumors. These data demonstrate that itraconazole has potent antitumor properties in esophageal cancer, partially through blockade of HER2/AKT signaling.

©2021 The Authors; Published by the American Association for Cancer Research.

Figures

Figure 1.

Itraconazole inhibits proliferation of esophageal cancer cells. A, Cell proliferation assay for OE33 and KYSE510 cells. OE33 and KYSE510 cells were plated in 6-well plates and cultured in medium supplemented with 2.5 μmol/L itraconazole or DMSO for 6 days. Cell numbers were measured at the indicated timepoints. Values represent the mean ± SEM for triplicate wells. *, P < 0.05; **, P < 0.01 versus control group by Student t test. B, Representative cell-cycle analysis using flow cytometry after staining with propidium iodide. OE33 and KYSE510 were treated with 2.5 μmol/L of itraconazole or DMSO for 24 hours. Flow cytometric histograms of each phase of the cell cycle in OE33 or KYSE510 cells after itraconazole treatment as compared with control. Values represent the means ± SEM for three replicates. Black bars, DMSO; gray bars, itraconazole. *, P < 0.05; **, P < 0.01 versus control group by Student t test.

Figure 2.

Itraconazole suppresses AKT phosphorylation in esophageal cancer cells. A, Phospho-kinase analysis of OE33 cells treated with 2.5 μmol/L itraconazole or DMSO for 48 hours. The pixel density of marked phosphorylated AKT (left) relative to internal control is shown graphically (right). B, Upregulation of AKT activity in esophageal cancer. Western blot analysis of phosphorylated and total AKT expression in immortalized human esophageal squamous (NES-G2T, NES-B10T), Barrett's esophagus (BAR-T and BAR-10T), ESCC (KYSE70 and KYSE510), and EAC (FLO-1 and OE33) cell lines. GAPDH is used as a loading control.

Figure 3.

Itraconazole inhibits AKT activity in both EAC and ESCC cell lines. A, Western blot analysis of p-AKT (Thr308 and Ser473), AKT, p-S6, and S6 expression in OE33, FLO-1, KYSE70, and KYSE510 cells treated with 2.5 μmol/L itraconazole or DMSO for 48 hours. GAPDH is used as a loading control. B, RT–qPCR analysis of CDK1, CDK2, CDK4, and E2F1 genes in OE33, FLO-1, KYSE70, and KYSE510 cells treated with 2.5 μmol/L itraconazole or DMSO for 48 hours. Values represent the mean fold change ± SEM for three experiments relative to GAPDH. Black bars, DMSO; gray bars, itraconazole. *, P < 0.05; **, P < 0.01 vs. DMSO control by Student t test.

Figure 4.

Itraconazole inhibits HER2/PI3K signaling in esophageal cancer. A, Western blot analysis of p-PI3K, PI3K, PTEN, HER2, p-AMPK, and AMPK expression in itraconazole or DMSO-treated OE33, FLO-1, KYSE70 and KYSE510 cells. GAPDH is used as a loading control. B, RT–qPCR analysis of ERBB2 mRNA levels in OE33, FLO-1, KYSE70 and KYSE510 cells treated with 2.5 μmol/L itraconazole or DMSO for 48 hours. Values represent the mean fold change ± SEM for three experiments relative to GAPDH. *, P < 0.05 and **, P < 0.01 versus control group by Student t test. C, Western blot analysis of p-AKT and AKT expression in OE33 and KYSE510 cells treated with DMSO, 2.5 μmol/L or 5 μmol/L lapitinib for 48 hours. GAPDH is used as a loading control. D, Cell proliferation assay for OE33 and KYSE510 cells treated with 2.5 μmol/L lapatinib. Cell numbers were counted at the indicated timepoints. The data are presented as the means ± SEM of three independent experiments. E, Western blot analysis of HER2, p-AKT, and AKT expression in OE33 and KYSE510 cells transfected with HER2-specific or NTC siRNAs for three days. GAPDH is used as a loading control. F, AlamarBlue-based cell proliferation assay for OE33 and KYSE510 cells treated with HER2-specific or NTC siRNAs for three days. Values represent the mean ± SEM for 8 wells. **, P < 0.01 versus control group by Student t test.

Figure 5.

Itraconazole inhibits OE33-derived tumor xenograft growth. A, NOD-SCID mice were injected subcutaneously with 5 × 106 OE33 cells. When tumors formed, mice were randomly divided into 2 groups (control and itraconazole, n = 8). The mice were treated with placebo or itraconazole (80 mg/kg) by oral gavage twice daily for two consecutive weeks. B, Concentrations of itraconazole and its primary metabolite hydroxyitraconazole in esophagi and tumor xenografts from mice treated with placebo or itraconazole. C, Western blot analysis of p-AKT, AKT, p-S6, S6, HER2, p-AMPK, and AMPK expression in OE33-derived xenografts treated with PBS or itraconazole for two weeks. GAPDH is used as a loading control. D, Representative IHC staining of p-S6 from xenograft sections treated with placebo or itraconazole. Scale bar, 100 μm.

Figure 6.

Itraconazole inhibits HER2/AKT signaling in primary tumors from patients with esophageal cancer. A, Top, CONSORT diagram of early phase I trial. Eighteen patients with ESCC, EAC, or gastroesophageal junction cancer signed written informed consent and were enrolled in the trial. Following initial research biopsies but before itraconazole was prescribed, 11 patients screen failed or voluntarily withdrew. Seven patients were prescribed itraconazole, with one patient discontinuing the medication after only 2 doses. Bottom: clinical and pathologic characteristics of the 6 patients who completed the full course of itraconazole. B, Western blot analysis of p-AKT, AKT, p-S6, S6, and HER2 protein in primary tumors from 6 patients with esophageal cancer before and after itraconazole treatment. GAPDH is used as a loading control. C, IHC staining of p-S6 from representative tumor sections of three different patients before (top) and after (bottom) itraconazole treatment. Patients A and B responded while patient D minimally responded. Scale bar, 100 μm. D, Schematic diagram of itraconazole's action in esophageal cancer. HER2 functional activation promotes PI3K/AKT/mTOR/S6 signaling, leading to the upregulation of AKT signaling target genes CDK1, CDK2, CDK4, and E2F1. Itraconazole suppresses this signaling pathway through downregulating ERBB2 mRNA.