The Role of APE1/Ref-1 in Reflux-Induced Epithelial-Mesenchymal Transition in Benign Barrett's Metaplasia: A Novel Target for Preventing Recurrent Barrett's Esophagus After Radiofrequency Ablation

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Project Summary Barrett’s esophagus, the condition in which the normal squamous lining of the esophagus is replaced by a metaplastic intestinal-type lining, is a risk factor for esophageal adenocarcinoma. This deadly cancer can be prevented by radiofrequency ablation (RFA), an endoscopic procedure that burns away the cancer-prone metaplastic lining. Presently, RFA is used only to eradicate Barrett’s esophagus that has precancerous changes called dysplasia. After RFA, patients require regular endoscopic cancer surveillance because Barrett’s metaplasia recurs frequently. Although RFA potentially could prevent cancer for the millions of patients with non-dysplastic Barrett’s esophagus, RFA cannot be cost-effective for them unless it permanently eradicates Barrett’s metaplasia. A condition called subsquamous intestinal metaplasia (SSIM) might underlie the frequent recurrences of Barrett’s esophagus after RFA. In SSIM, Barrett’s cells are located under a layer of normal esophageal squamous lining that shields them from destruction by RFA. Most Barrett’s patients have SSIM, which could be the nidus for recurrent metaplasia after RFA. Our published data suggest that SSIM develops when Barrett’s cells undergo a process called epithelial-mesenchymal transition (EMT), which is a wound-healing event triggered by gastroesophageal reflux disease (GERD). EMT endows Barrett’s cells with migratory abilities that enable them to move under the adjacent squamous lining. EMT also activates cell survival pathways that could enable Barrett’s cells wounded by RFA to survive. Thus, EMT appears to underlie the development of SSIM, and EMT might well underlie the high frequency of metaplasia recurrences after RFA. In Barrett’s cells, we have reported that acid and bile (the damaging factors in gastric juice that refluxes into the esophagus in GERD patients) induce oxidative stress that results in the accumulation of a molecule called HIF-1α. We also published that acidic bile salts induce signaling through a molecular pathway that causes Barrett’s cells to increase their production of ZEB1, a molecule that plays a key role in inducing the EMT that triggers the cell motility leading to the development of SSIM. Our new experiments demonstrate that acidic bile salts activate the function of a molecule called APE1/Ref-1 that is required for activation of HIF-1α. We show that activated HIF-1α mediates increased production of the ZEB1 that induces EMT. Thus, we hypothesize that GERD-induced APE1/Ref-1 function that activates HIF-1α is the pivotal event in initiating EMT that enables Barrett’s cells to form SSIM and to survive RFA, and that these events might be prevented by drugs that inhibit APE1/Ref-1. The aims of this study are to elucidate the mechanism(s) whereby APE1/Ref- 1 signaling and HIF-1α activation contribute to the induction of EMT, and to explore the role of the APE1/Ref-1- HIF-1α signaling axis in EMT induced by exposure to acidic bile salts in Barrett’s esophagus. Our ultimate goal is to determine how SSIM develops and how a targeted treatment might be used to prevent that development, findings that could provide the means to eradicate Barrett’s esophagus and its cancer risk permanently.