Presenter(s)
Z. Liu1,2, R. Zhang2, F. Liu3, D. Chen4, and J. Yu4; 1Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 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 Immunology, School of Basic Medical Sciences, Shandong University, Jinan, China, 4Shandong Provincial Key Laboratory of Precision Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong, China
Purpose/Objective(s): Radioresistance remains a major obstacle in non-small cell lung cancer (NSCLC). This study explores the role of the E3 ligase MARCHF5 in regulating this resistance, testing the mechanism that targeting MARCHF5 stabilizes VDAC1 to activate the cGAS-STING pathway, with validation in clinical specimens.
Materials/Methods: Stable MARCHF5-knockdown NSCLC cell lines were generated using lentiviral shRNA. In vitro, effects of MARCHF5 inhibition on proliferation, survival, and apoptosis after irradiation were assessed via CCK-8, colony formation, and flow cytometry. Co-immunoprecipitation and mass spectrometry identified VDAC1 as an interacting partner, validated under irradiation by Western blot. Cycloheximide chase assays assessed VDAC1 protein stability regulated by MARCHF5. Immunofluorescence confocal microscopy examined VDAC1 co-localization with LC3 and LAMP1; autophagic flux was evaluated by Western blot. Activation of the cGAS-STING pathway and type I interferon expression were analyzed by Western blot and ELISA. In vivo, subcutaneous xenografts assessed MARCHF5 knockdown effects on radiosensitivity and CD8+ T cell infiltration/activation via flow cytometry. Rescue experiments with VDAC1 knockdown confirmed axis specificity. Clinically, immunohistochemistry on tissue microarrays (90 NSCLC specimens) analyzed MARCHF5/VDAC1 expression, prognosis, and CD8+ T cell infiltration. Patient-derived organoids validated the radiosensitizing effect of targeting MARCHF5 ex vivo.
Results: MARCHF5 knockdown significantly enhanced the radiosensitivity of NSCLC cells, as evidenced by reduced colony formation and increased apoptosis post-irradiation. Mechanistically, MARCHF5 interacted with VDAC1 in an irradiation-enhanced manner. Knockdown of MARCHF5 led to the stabilization of VDAC1 protein by suppressing its selective autophagic degradation, accompanied by decreased co-localization of VDAC1 with LC3 and LAMP1. The resultant accumulation of VDAC1 activated the cGAS-STING signaling pathway, promoting the transcription and release of type I interferons. Critically, these radiosensitizing and immunostimulatory effects were partially reversed by simultaneous knockdown of VDAC1. In clinical NSCLC specimens, MARCHF5 was significantly upregulated in patients unresponsive to radiotherapy, and its high expression inversely correlated with VDAC1 protein levels. Patients with high MARCHF5 expression exhibited significantly shorter overall survival and reduced intratumoral CD8+ T cell infiltration.
Conclusion: Targeting MARCHF5 stabilizes VDAC1 by preventing its autophagic degradation, thereby activating the cGAS-STING pathway to enhance both the direct radiosensitivity of NSCLC cells and the anti-tumor immune response. Clinical evidence further supports MARCHF5 as a prognostic biomarker and a promising therapeutic target for overcoming radioresistance and improving radiotherapy efficacy in NSCLC.