Abstract 3847: APE1 mediates angiogenesis by stabilizing SMAD3 and blocking its ubiquitin induced degradation in esophageal adenocarcinoma

Abstract Background: The incidence of esophageal adenocarcinoma (EAC) is rapidly rising in the United States and Western countries. Unfortunately, EAC has one of the lowest survival rates due to limited treatment options. Identifying disease drivers through functional studies is crucial for improving treatment options. This study aimed to explore the role of APE1 in regulating SMAD3 and promoting EAC progression. Methods and Results: Analysis of publicly available datasets revealed that APE1 and SMAD3 are upregulated and positively correlated in EAC compared to normal samples. Immunofluorescence staining of esophageal cancer progression tissue arrays confirmed significantly higher levels of APE1 and SMAD3 in EAC than in normal tissues. Western blot analysis showed that silencing APE1 reduced SMAD3 nuclear expression, which was also confirmed by immunofluorescence staining showing a loss of nuclear SMAD3 accumulation following APE1 knockdown. Immunoprecipitation and proximity ligation assays indicated a direct interaction between APE1 and SMAD3 in the nucleus. Further analysis revealed that APE1 binds to the C-terminal MH2 domain of SMAD3, protecting it from ubiquitin-mediated proteasomal degradation by preventing its interaction with the RING finger protein, ROC1. Notably, inhibiting APE1’s redox or endonuclease activity with pharmacological inhibitors or mutants disrupted APE1-SMAD3 binding, suggesting a conformational regulation. Interestingly, knocking down APE1 reduced angiogenesis in tube formation assays and 3D co-cultures; however, this effect was reversed by overexpressing SMAD3, suggesting that APE1 regulates angiogenesis through SMAD3. Conclusion: The findings establish the role of APE1 in regulating SMAD3 in EAC and suggest a potential therapeutic approach for treating EAC patients. Citation Format: Farah Ballout, Heng Lu, Nadeem Bhat, Lei Chen, Dunfa Peng, Zheng Chen, Alexander Zaika, Oliver McDonald, Wael El Rifai. APE1 mediates angiogenesis by stabilizing SMAD3 and blocking its ubiquitin induced degradation in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3847.