2551 - 2-Hydroxyisobutyrylation of ALDH1A3 Promotes Stemness Maintenance and Radiotherapy Resistance in NSCLC via Activation of the STAT3 Signaling Pathway

Purpose/Objective(s): In recent years, novel acylation modifications have been widely reported to play critical regulatory roles in various biological processes of tumor cells. As a newly discovered acylation modification, 2-hydroxyisobutyrylation (Khib) and its specific functions and molecular mechanisms in tumors remain incompletely understood. This study aims to investigate the biological functions and regulatory mechanisms of Khib in NSCLC, with the goal of providing potential therapeutic targets and strategies for clinical treatment.

Materials/Methods: We employed immunohistochemical (IHC) staining to observe the expression of Khib in NSCLC tissues. Using global 2-hydroxyisobutyrylome, we systematically identified differentially expressed proteins and modification sites between normal lung epithelial cells and NSCLC cells. Transient knockdown cell lines were constructed using siRNA transfection. Stable knockout cell lines were generated using the CRISPR/Cas9 gene editing system, followed by monoclonal screening and validation. DNA damage levels were assessed using comet assays and ?-H2AX immunofluorescence (IF) staining. Stemness characteristics were evaluated through sphere formation assays and limiting dilution assays. Radiosensitivity was determined using colony formation assays and in vivo animal models. Key modification sites were validated via site-directed mutagenesis. Further, antibody microarrays, metabolomics, Western blotting (WB), and co-immunoprecipitation (Co-IP) were utilized to explore downstream signaling pathways and regulatory mechanisms affected by Khib.

Results: IHC analysis revealed that Khib expression was significantly higher in NSCLC tissues compared to normal lung tissues, suggesting its potential role in promoting cancer progression. Proteomics analysis indicated that Khib of ALDH1A3 was most significantly upregulated in NSCLC cells compared to normal lung epithelial cells. To investigate the function of ALDH1A3, we constructed ALDH1A3 knockdown cell lines. Comet assays and ?-H2AX IF staining demonstrated that ALDH1A3 knockdown cells exhibited increased DNA damage following radiotherapy. Additionally, sphere formation assays and limiting dilution assays showed that ALDH1A3 knockdown significantly inhibited stemness characteristics in NSCLC. Colony formation assays and in vivo animal models further confirmed that ALDH1A3 knockdown significantly enhanced radiosensitivity in NSCLC. Mechanistic studies revealed that ALDH1A3 knockout suppressed the activation of the STAT3 signaling pathway, thereby regulating stemness maintenance and radiotherapy resistance in NSCLC.

Conclusion: This study demonstrates that Khib of ALDH1A3 promotes stemness maintenance and radiotherapy resistance in NSCLC by activating the STAT3 signaling pathway. These findings suggest that targeting Khib of ALDH1A3 may serve as a potential therapeutic sensitization strategy for NSCLC treatment.