Background: Barrett's esophagus (BE) is a premalignant condition where columnar metaplasia replaces the stratified squamous epithelium of the distal esophagus and is regarded as the main risk factor for esophageal adenocarcinoma (EAC). BE develops as a consequence of chronic gastroesophageal reflux disease (GERD), where acid and bile salts abnormally refluxate from the stomach into the distal region of esophagus. APE1/REF-1 is a multifunctional protein that plays an essential role in DNA repair activity and in redox-dependent transcriptional regulation. STAT3 signaling has been implicated in regulating cell survival, angiogenesis, and metastasis. EGFR is well known to facilitate the tyrosine phosphorylation of STAT3 and initiates downstream signaling cascades that facilitate proliferation, tumorigenesis, metastasis, and chemo resistance. In this study, we investigated the role of APE1 in activation of STAT3 in response to acidic bile salts (ABS) in EAC.

Methodology: Non-neoplastic Barrett's (CPA, BAR10T and CPB) and esophageal adenocarcinoma (OE33, FLO-1) cells were exposed to acidic bile salts (pH4.0 and 5.5, 100 and 200uM) for 20 minutes. The mRNA and protein expression levels of APE-1, STAT3 and target genes were evaluated by real time PCR and immunoblotting. STAT3 transcription activity was studied by luciferase reporter assay. Immunofluorescence and cytoplasmic-nuclear fractionation were used to examine localization of APE-1, EGFR and STAT3 after exposure to acidic bile salts.

Results: We demonstrate that APE1 is constitutively overexpressed in EAC whereas its expression is induced in non-neoplastic BE in response to ABS. Using overexpression and shRNA knockdown of APE1, we found that APE1 is required for phosphorylation, nuclear localization, and transcription activation of STAT3. By using APE1 redox-specific mutant (C65A) and APE1 redox inhibitor (E3330), we demonstrate that APE1 activates STAT3 in a redox-dependent manner. By using pharmacologic inhibitors and genetic knockdown systems, we found that EGFR is a required link between APE1 and STAT3. Phosphorylation of EGFR (Y1068) was directly associated with APE1 levels and redox function. By using co-immunoprecipitation and proximity ligation assays, we found that APE1 coexists and interacts with the EGFR-STAT3 protein complex. Consistent with these findings, we demonstrated a significant induction in the mRNA expression levels of STAT3 target genes (IL-6, IL-17A, BCL-xL, Survivin and c-Myc) in BE and EAC cells, following ABS treatment. ChIP assays indicated that ABS-treatment enhances binding of STAT3 to the promoter of its target genes, Survivin and BCL-xL, following ABS treatment. Inhibition of APE1/REF-1 redox activity using E3330 abrogated STAT3 DNA binding and transcriptional activity. In summary, our results provide evidence that bile reflux induced APE1 is involved in the activation of EGFR-STAT3 signaling axis in BE and EAC.

Conclusion: The redox activity of APE1 mediates EGFR- STAT3 activation as a survival mechanism in response to acidic bile salts and oxidative stress. Constitutive activation of APE1 in EAC may present a therapeutic opportunity to targeting redox vulnerabilities in cancer cells.

Citation Format: Ajaz A. Bhat, Heng Lu, Mohammad Suotto, Chen Zheng, Dunfa Peng, Mohammad Haris, Abbes Belkhiri, Wael El-Rifai. Bile reflux-induced APE1 mediates activation of EGFR-STAT3 in barret's and esophageal adenocarcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-396.