Activation of NOTCH signaling via DLL1 is mediated by APE1-redox-dependent NF-κB activation in oesophageal adenocarcinoma.

OBJECTIVE: Oesophageal adenocarcinoma (EAC) arises in the setting of Barrett's oesophagus, an intestinal metaplastic precursor lesion that can develop in patients with chronic GERD. Here, we investigated the role of acidic bile salts, the mimicry of reflux, in activation of NOTCH signaling in EAC. DESIGN: This study used public databases, EAC cell line models, L2-IL1ß transgenic mouse model and human EAC tissue samples to identify mechanisms of NOTCH activation under reflux conditions. RESULTS: Analysis of public databases demonstrated significant upregulation of NOTCH signaling components in EAC. In vitro studies demonstrated nuclear accumulation of active NOTCH1 cleaved fragment (NOTCH intracellular domain) and upregulation of NOTCH targets in EAC cells in response to reflux conditions. Additional investigations identified DLL1 as the predominant ligand contributing to NOTCH1 activation under reflux conditions. We discovered a novel crosstalk between APE1 redox function, reflux-induced inflammation and DLL1 upregulation where NF-κB can directly bind to and induce the expression of DLL1. The APE1 redox function was crucial for activation of the APE1-NF-κB-NOTCH axis and promoting cancer cell stem-like properties in response to reflux conditions. Overexpression of APE1 and DLL1 was detected in gastro-oesophageal junctions of the L2-IL1ß transgenic mouse model and human EAC tissue microarrays. DLL1 high levels were associated with poor overall survival in patients with EAC. CONCLUSION: These findings underscore a unique mechanism that links redox balance, inflammation and embryonic development (NOTCH) into a common pro-tumorigenic pathway that is intrinsic to EAC cells.

Reflux conditions induce E-cadherin cleavage and EMT via APE1 redox function in oesophageal adenocarcinoma.

OBJECTIVE: Chronic gastro-oesophageal reflux disease, where acidic bile salts (ABS) reflux into the oesophagus, is the leading risk factor for oesophageal adenocarcinoma (EAC). We investigated the role of ABS in promoting epithelial-mesenchymal transition (EMT) in EAC. DESIGN: RNA sequencing data and public databases were analysed for the EMT pathway enrichment and patients' relapse-free survival. Cell models, pL2-IL1ß transgenic mice, deidentified EAC patients' derived xenografts (PDXs) and tissues were used to investigate EMT in EAC. RESULTS: Analysis of public databases and RNA-sequencing data demonstrated significant enrichment and activation of EMT signalling in EAC. ABS induced multiple characteristics of the EMT process, such as downregulation of E-cadherin, upregulation of vimentin and activation of ß-catenin signalling and EMT-transcription factors. These were associated with morphological changes and enhancement of cell migration and invasion capabilities. Mechanistically, ABS induced E-cadherin cleavage via an MMP14-dependent proteolytic cascade. Apurinic/apyrimidinic endonuclease (APE1), also known as redox factor 1, is an essential multifunctional protein. APE1 silencing, or its redox-specific inhibitor (E3330), downregulated MMP14 and abrogated the ABS-induced EMT. APE1 and MMP14 coexpression levels were inversely correlated with E-cadherin expression in human EAC tissues and the squamocolumnar junctions of the L2-IL1ß transgenic mouse model of EAC. EAC patients with APE1high and EMThigh signatures had worse relapse-free survival than those with low levels. In addition, treatment of PDXs with E3330 restrained EMT characteristics and suppressed tumour invasion. CONCLUSION: Reflux conditions promote EMT via APE1 redox-dependent E-cadherin cleavage. APE1-redox function inhibitors can have a therapeutic role in EAC.