Presenter(s)
T. Lai1, L. Du2, Y. Tan3, W. Zhou4, and Y. Wu2; 1Radiation Oncology Center, Chongqing University Cancer Hospital, Chongqing, China, 2Department of Radiation Oncology, Chongqing University Cancer Hospital, Chongqing, China, 3Chongqing University School of Medicine, Chongqing, China, 4Department of radiotherapy oncology, Chongqing University Cancer Hospital & Chongqing Cancer Institute & Chongqing Cancer Hospital, Chongqing, China
Purpose/Objective(s): Lung adenocarcinoma (LUAD), a prevalent subtype of non-small cell lung cancer, is commonly treated with radiotherapy as a primary clinical approach. Cell division cycle-associated protein 3 (CDCA3), an F-box-like protein that participates in the Skp1-cullin-F-box ubiquitin ligase complex, plays vital roles in various tumor types, and its elevated expression correlates with tumor progression and unfavorable prognoses. Nonetheless, the biological functions involving CDCA3 in LUAD in the context of radiotherapy remain inadequately elucidated.
Materials/Methods: CDCA3 expression and its prognostic value in LUAD were analyzed using TCGA database and validated in 50 paired LUAD clinical samples by immunohistochemistry and Western blot. Stable CDCA3 knockdown and overexpression LUAD cell lines (A549, H1299) were established using lentiviral shRNA and overexpression vectors. Radiosensitivity was assessed by colony formation assays after gradient irradiation (0-8 Gy). DNA repair efficiency was evaluated by ?-H2AX foci clearance, comet assay, and NHEJ/HR reporter systems. The interaction between CDCA3 and NHEJ core proteins was identified by co-immunoprecipitation coupled with mass spectrometry (Co-IP/MS) and validated by endogenous Co-IP. Ubiquitination levels and protein stability of the identified target were examined upon CDCA3 knockdown. Functional rescue experiments were performed by reconstituting the downstream effector candidates in CDCA3-knockdown cells, followed by assessment of radiosensitivity, target ubiquitination, and protein half-life.
Results: CDCA3 was significantly upregulated in LUAD tissues and correlated with poor prognosis. CDCA3 knockdown sensitized LUAD cells to irradiation, impaired DNA repair capacity, and specifically reduced NHEJ repair efficiency. Mechanistically, CDCA3 directly bound to Ku80, a core component of the NHEJ pathway. Notably, contrary to the canonical role of F-box proteins, CDCA3 knockdown unexpectedly increased Ku80 ubiquitination and accelerated its degradation, suggesting a non-canonical stabilizing function. Further screening identified a CDCA3-interacting partner that mediates Ku80 deubiquitination. Reconstitution of this downstream effector reversed CDCA3 knockdown-induced radiosensitization and restored Ku80 protein stability. Targeting this regulatory axis with a small molecule inhibitor phenocopied CDCA3 depletion and enhanced radiosensitivity.
Conclusion: Our study unveils a novel mechanism by which CDCA3 functions as a scaffold protein to stabilize Ku80, thereby enhancing NHEJ repair and promoting radioresistance in lung adenocarcinoma. Targeting the CDCA3-mediated regulatory axis represents a promising therapeutic strategy to overcome radiotherapy resistance.