IN BARRETT'S ESOPHAGUS, REFLUX INDUCES FEATURES OF EPITHELIAL-MESENCHYMAL PLASTICITY MEDIATED BY APE/REF-1 REDOX FUNCTION: EVIDENCE FROM STUDIES IN BARRETT'S PATIENTS AND ORGANOIDS

Introduction: Wounding triggers epithelial-mesenchymal plasticity (EMP), a process in which epithelial cells acquire mesenchymal cell features. Some cells undergoing EMP exhibit both epithelial and mesenchymal features, and these epi-mesenchymal (E-M) cells are susceptible to cancer initiation. In earlier studies, we found that acidic bile salt solutions (ABS) triggered EMP in immature Barrett’s esophagus (BE) cells that formed BE spheroids, and that ABS increased ZEB1 (a key EMP transcription factor) via HIF-1α-VEGF axis signaling in BE cell lines. HIF-1α is maintained in its active state through the redox function of APE1 protein, and we found that APE1 is required for ABS to trigger the HIF-1α-VEGF axis in BE cells. Now, we assessed EMP features developing in BE patients who had acute reflux esophagitis induced by stopping PPIs, and explored if we could block ABS-induced EMP by inhibiting APE1 redox function in BE cells.
Methods: We took biopsies across the squamo-BE junction in 15 BE patients at baseline on PPIs, and at 1 and 2 weeks after stopping PPIs; we evaluated ZEB1 and CDH1 (an epithelial marker) by IHC. In BE cell lines, we used siRNA to knockdown endogenous APE1; scrambled siRNA served as control. Constructs overexpressing either a redox-dead APE1 mutant or empty vector were treated with ABS for 15 minutes. Primary BE cells were suspended in 25 µl Matrigel for 5 days to develop spheroids, which we then dissociated, re-suspended in Collagen I, and treated with ABS 15 minutes/day for 2 days. Cells were treated with the APE1 redox inhibitor APX2009 or its analog RN7-58, which does not inhibit APE1 redox activity. EMP features were evaluated by morphology; ZEB1 mRNA was evaluated by qPCR.
Results: BE biopsies with reflux esophagitis exhibited regenerating crypt epithelium with cells that showed E-M features penetrating into the stroma (Fig1A). ZEB1 nuclear staining was seen in cells with E-M features in the stroma abutting regenerating crypts (Fig1B). ZEB1 nuclear staining significantly increased from baseline to week 2 after stopping PPIs, with no loss of membranous CDH1 (Fig1C,D). ABS significantly increased ZEB1 mRNA in control BE cells with empty vector, but not in cells overexpressing the APE1 redox-dead mutant (Fig1E). ABS significantly increased 1) ZEB1 mRNA expression in BE cell lines and spheroids (Fig2A,B) and 2) cell protrusions in BE spheroids treated with RN7-58, effects that were significantly reduced by APX2009 (Fig2C).
Conclusion: Biopsies of BE in patients with acute reflux esophagitis induced by stopping PPIs exhibit histologic features and markers of E-M cells. In BE cells and spheroids, ABS exposure increases ZEB1 mRNA expression and cell protrusions through the redox function of APE1. This suggests that APE1 redox function could be a target for preventing reflux-induced EMP and, thus, cancer in BE patients.