2520 - Knockdown of AKR1C1 Enhances NEDD4-Mediated Ubiquitination of GPX4 to Promote Ferroptosis and Radiosensitization in Non-Small Cell Lung Cancer
Presenter(s)
W. Yan1, R. Zhang2, D. Chen3, and J. Yu4; 1Department of Radiation Oncology and Shandong Provincial Key Laboratory of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong, China, 2Shandong University Cancer Center;Department of Radiation Oncology and Shandong Provincial Key Laboratory of Radiation Oncology, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, China, 3Department of Radiation Oncology and Shandong Provincial Key Laboratory of Precision Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, China, 4Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, China
Purpose/Objective(s): The molecular mechanism underlying radiation resistance in non-small cell lung cancer (NSCLC) is not well understood. This study aimed to investigate the potential role of AKR1C1 and its specific regulatory mechanism in NSCLC radiotherapy resistance.
Materials/Methods: Initially, FFPE tissues from 41 SBRT-treated NSCLC (T1-2N0) patients were analyzed using Digital Spatial Profiling (DSP) to spatially resolve mRNA expression of 18,000 genes across tumor (PanCK+), leukocyte (CD45+), lymphocyte (CD3+), macrophage (CD68+), and stromal (a-SMA+) regions, identifying AKR1C1 as the most upregulated gene in recurrent versus non-recurrent tumors. Validation via multi-color immunofluorescence (mIF) and Western blotting confirmed elevated AKR1C1 expression in NSCLC versus adjacent normal tissues and in SBRT-recurrent cohorts. Functionally, stable AKR1C1 knockdown or overexpression in NSCLC cell lines revealed its role in radiosensitivity through clonogenic survival, EdU/CCK-8 proliferation, ?-H2AX/Rad51 foci assays, and xenograft models. Mechanistically, AKR1C1 non-enzymatically stabilized GPX4 to suppress ferroptosis, evidenced by altered lipid ROS, Fe²? levels, mitochondrial membrane potential, ultrastructural changes, and enzyme-dead mutagenesis. Translational validation in clinical specimens and xenografts via mIF and IHC corroborated AKR1C1-driven radioresistance
Results: This study identifies AKR1C1 as a key radioresistance gene in NSCLC, showing significant upregulation in SBRT-recurrent tumors across tumor, immune, and stromal compartments, with elevated expression in NSCLC versus normal tissues correlating with poor prognosis. Functional assays demonstrate that AKR1C1 knockdown enhances radiosensitivity by inhibiting colony formation, proliferation, and DNA repair (reduced Rad51 foci, increased ?-H2AX), while promoting lipid ROS/Fe²? accumulation, mitochondrial dysfunction, and ferroptosis. Mechanistically, AKR1C1 stabilizes GPX4 via non-enzymatic interactions, whereas its depletion activates the ROS/FOXM1 axis to upregulate NEDD4, which binds GPX4 via WW domains and induces K48-linked ubiquitination at K47/K132 sites, accelerating GPX4 proteasomal degradation. Clinical and xenograft validation confirms AKR1C1-GPX4 positive correlation, inverse AKR1C1-NEDD4 association, and AKR1C1 as an independent risk factor for OS/PFS, highlighting its dual role in radioresistance and ferroptosis suppression through GPX4-NEDD4 regulatory circuitry.
Conclusion: This study utilized spatial transcriptomic data from clinical tissue samples of NSCLC patients to screen for radiation resistance-associated gene AKR1C1.It is the first to reveal that knockdown of AKR1C1 induces GPX4 ubiquitination degradation via the ROS/FOXM1/NEDD4 axis, leading to ferroptosis in NSCLC cells, and enhancing IR-induced ferroptosis. This provides a novel therapeutic target and theoretical basis for sensitizing NSCLC to radiotherapy.