1102 - CDCA3 Promotes Radiotherapy Resistance in Lung Adenocarcinoma by Facilitating Non-Homologous End Joining

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:

The potential prognostic significance of CDCA3 in LUAD was assessed by analyzing the overlap between differentially expressed genes, key survival-associated genes from the TCGA LUAD database, and E3 ubiquitin ligase complex genes. Immunohistochemical (IHC) staining and Western blot (WB) were performed to investigate the expression of CDCA3 in LUAD patients. Colony formation assays and subcutaneous transplanted tumor models were utilized to evaluate cell radiosensitivity both in vitro and in vivo. We then assessed the DNA damage induced by radiation combined with CDCA3 depletion through ?-H2AX immunofluorescence (IF) staining and comet assays. Next, to determine how DNA damage is repaired, the EJ5-GFP reporter system were used to detect non-homologous end joining (NHEJ) repair efficiency. Furthermore, to identify the downstream targets of CDCA3 in regulating NHEJ, immunoprecipitation and subsequent LC–MS/MS were performed in CDCA3 overexpressed A549 cells to screen potential CDCA3 interactors. These interactors were subsequently intersected with the core genes regulating NHEJ. Finally, candidate target proteins were validated through WB.

Results:

We found that through intersection-analysis of the gene sets , CDCA3 is uniquely overexpressed in LUAD patients compared to non-tumor tissues, adversely affecting patient survival. IHC and WB findings confirmed elevated CDCA3 expression in tumor tissues compared to normal tissues, indicating a poor prognosis. Colony formation and mouse model experiments showed that knockdown of CDCA3 significantly enhanced the radiosensitivity of LUAD cells. The results of ?-H2AX IF staining and comet assay showed that CDCA3 promoted radiotherapy resistance of LUAD cells by inhibiting DNA damage. DR-GFP and EJ5-GFP reporting systems showed that overexpression of CDCA3 can enhance the efficiency of DNA repair for NHEJ. According to the LC–MS/MS identification and WB results, CDCA3 interacts with and inhibits the degradation of Ku70/Ku80 and promotes its recruitment to DNA double strand breaks (DSB), thereby promoting DNA repair and reducing DNA damage by facilitating NHEJ.

Conclusion:

Collectively, our findings propose that CDCA3 may enhance radiotherapy resistance in LUAD cells by promoting DNA repair through NHEJ. This implies that CDCA3 could serve as a promising target for radiosensitization therapy in LUAD.