Targeting the Hedgehog Pathway Using Itraconazole to Prevent Progression of Barrett's Esophagus to Invasive Esophageal Adenocarcinoma

Abstract

Objective: The aim of the study was to investigate whether inhibition of Sonic Hedgehog (SHH) pathway would prevent progression of Barrett's Esophagus (BE) to esophageal adenocarcinoma.

Background: The hedgehog signaling pathway is a leading candidate as a molecular mediator of BE and esophageal adenocarcinoma (EAC). Repurposed use of existing off-patent, safe and tolerable drugs that can inhibit hedgehog, such as itraconazole, could prevent progression of BE to EAC.

Methods: The efficacy of itraconazole was investigated using a surgical rat reflux model of Barrett's Metaplasia (BM). Weekly intraperitoneal injections of saline (control group) or itraconazole (treatment group; 200 mg/kg) were started at 24 weeks postsurgery. Esophageal tissue was harvested at 40 weeks. The role of the Hh pathway was also evaluated clinically. Esophageal tissue was harvested after 40 weeks for pathological examination and evaluation of the SHH pathway by immunohistochemistry.

Results: BM was present in control animals 29 of 31 (93%) versus itraconazole 22 of 24 (91%). EAC was significantly lower in itraconazole 2 of 24 (8%) versus control 10 of 31 (32%), respectively (P = 0.033). Esophageal SHH levels were lower in itraconazole vs control (P = 0.12). In esophageal tissue from humans with recurrent or persistent dysplastic BE within 24 months of ablative treatment, strong SHH and Indian Hedgehog expression occurred in distal BE versus proximal squamous epithelium, odds ratio = 6.1 (95% confidence interval: 1.6, 23.4) and odds ratio = 6.4 (95% confidence interval: 1.2, 32.8), respectively.

Conclusion: Itraconazole significantly decreases EAC development and SHH expression in a preclinical animal model of BM. In humans, BE tissue expresses higher SHH, Indian Hedgehog, and bone morphogenic protein levels than normal squamous esophageal epithelium.

Conflict of interest statement

The authors report no conflicts of interest.

Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.

Figures

FIGURE 1.

Schematic illustration of esophagojejunostomy and appearance of adenocarcinoma at the site of anastomosis. After a midline abdominal incision, the esophagus was ligated and divided at the esophageal gastric junction. The esophagus end was then anastomosed to a 4mm wide jejunostomy located 4cm distal to the ligament of Treitz by 4 interrupted 6–0 nylon sutures in an end-to-side fashion.

FIGURE 2.

Study design.

FIGURE 3.

Analysis of mRNA expression of SKGT4 cells following addition of multiple concentrations of itraconazole. Analysis of mRNA expression of SKGT4 cells following addition of multiple concentrations of itraconazole (0, 1, 5, 10μM). RNA was extracted at the indicated hour after itraconazole addition and analyzed by qRT-PCR for mRNAs encoding bone morphogenic protein 4 (BMP4). The results were normalized to 0μM of itraconazole at each time point. *P < 0.05 compared to 0μM, **P < 0.005 compared to 0μM, Student t test, mean ± SD, n = 6 independent cell culture for each condition at 24 hours and n = 4 independent cell culture for each condition at 48 and 72hours.

FIGURE 4.

Esophageal Adenocarcinoma at 40 weeks in the itraconazole versus control groups. A, Gross section of distal esophageal adenocarcinoma with hematoxylin and eosin staining showing a well-differentiated adenocarcinoma. B, Percentage of the adenocarcinoma positive rats in the treatment arm versus control arm at week 40 after surgery. 8.3% (2/24) versus 32.3% (10/31), P = 0.033.

FIGURE 5.

Immunohistochemistry (IHC) stain for Sonic Hedgehog (SHH) in normal squamous and adenocarcinoma cells in treatment and control groups. A, Weak SHH expression in adenocarcinoma cells in the treatment group. B, Weak SHH expression in squamous cells in the treatment group. C, Strong SHH expression in adenocarcinoma cells in the control group. D, Strong SHH expression in squamous cells in the control group.