Abstract 3964: APE1 protects SMAD3 against ROC1 ubiquitin mediated degradation in esophageal adenocarcinoma cells

Abstract Background: The incidence of esophageal adenocarcinoma (EAC) has increased more than six-fold over the past three decades and continues to rise in the Western world. The 5-year survival rate for EAC patients is less than 20% which underscores the need to better understand the underlying mechanisms to identify new therapeutic approaches. This study aimed at investigating the potential role of APE1 in regulating SMAD3 and promoting EAC progression. Methods and Results: Western blot data showed that APE1 and SMAD3 were highly expressed in EAC cell lines. APE1 silencing reduced SMAD3 nuclear expression and downregulated its downstream targets SERPINE1 and c-myc. These results were confirmed by immunofluorescence staining showing loss of nuclear accumulation of SMAD3 after APE1 knockdown. Mechanistically, immunoprecipitation and proximity ligation assays revealed a direct binding between APE1 and SMAD3 in the nucleus. Further investigation showed that APE1 binds to the C-terminal MH2 domain of SMAD3, and this binding protects SMAD3 from ubiquitin mediated proteasomal degradation by blocking its interaction with the RING finger protein, ROC1. Interestingly, APE1-redox-specific inhibition (APX2009) downregulated SMAD3 expression and the APE1 redox-deficient mutant (C65A) disrupted APE1-SMAD3 binding indicating that this regulation depends on APE1 redox activity. Conclusion: Our findings establish a role of APE1 in regulating SMAD3 in EAC. These findings provide a potential therapeutic approach for the treatment of EAC by the pharmacological inhibition of APE1. Citation Format: Farah Ballout, Heng Lu, Dunfa Peng, Wael El-Rifai. APE1 protects SMAD3 against ROC1 ubiquitin mediated degradation in esophageal adenocarcinoma cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3964.