Ebselen's role in overcoming cisplatin resistance in colorectal Cancer via SQSTM1 ubiquitination modulation.

Cisplatin resistance severely limits chemotherapy efficacy in colorectal cancer (CRC). Although Ebselen has antitumor potential, its role in reversing cisplatin resistance remains unclear. Here, we combined in vitro/in vivo assays with scRNA-seq and spatial transcriptomics to evaluate Ebselen in cisplatin-resistant CRC. In HCT116/DDP cells, Ebselen inhibited proliferation, migration and invasion, promoted apoptosis, and enhanced cisplatin-induced DNA damage signaling. In a matched PBMC-humanized subcutaneous CDX model (NOD/SCID), Ebselen plus cisplatin achieved stronger tumor suppression than cisplatin alone, with reduced proliferation, increased γH2AX and elevated apoptosis. Single-cell analyses indicated that combination therapy reshaped the tumor microenvironment by shifting cellular composition and strengthening immune-cell communication, which was supported by CellChat/NicheNet and further validated by spatial transcriptomics showing altered spatial cytokine programs. Mechanistically, integrative screening identified SQSTM1 as a key regulator. Co-IP and linkage-specific IP-WB demonstrated SQSTM1 interacts with RAD51 and promotes RAD51 K48-linked polyubiquitination. Ebselen increased RAD51 protein stability in CHX-chase assays, an effect reversed by SQSTM1 reconstitution; MG132, but not chloroquine, restored RAD51 levels, indicating proteasome-dependent degradation. Functionally, SQSTM1 reconstitution attenuated Ebselen effects, while RAD51 reconstitution rescued related phenotypes. Overall, Ebselen reverses cisplatin resistance by inhibiting SQSTM1-mediated RAD51 proteasomal turnover, thereby amplifying DNA damage and remodeling antitumor immunity.