O-GlcNAcylation of DDB1 at Ser-764 by OGT promotes cancer cell stemness in colorectal cancer through increased polyubiquitination of p53.

Several ubiquitin E3 ligases, including MDM2, COP1, HUWE1, and CUL4A-DDB1, regulate the stability of the p53 protein, which is crucial for maintaining cell survival, promoting apoptosis, facilitating DNA repair, and preventing the stemness of cancer cells. The CUL4A-DDB1 complex is recognized as one of the non-canonical pathways that control p53 stability. However, the role of DDB1 O-GlcNAcylation has not been studied until now. Here, we present new evidence that DDB1 glycosylation mainly decreases p53 protein levels by promoting the formation of the CUL4A-DDB1-p53 complex. DDB1 is highly expressed in colorectal cancer (CRC) and plays a crucial role in supporting the functions of cancer stem cells (CSCs). Our data show that DDB1 is O-GlcNAcylated by OGT, with Ser-764 as the primary glycosylation site. Notably, the glycosylation-defective DDB1 mutant (Ser764A) reduces its interaction with CUL4A and p53, resulting in decreased p53 ubiquitination. These results suggest an inverse relationship between DDB1 glycosylation levels and p53 stability. The CSC-defective traits in DDB1-deficient CRC cells were rescued by p53 deficiency but not by its overexpression, indicating that the CUL4A-DDB1-mediated regulation of p53 stability may control cancer stem-like properties in CRC. In summary, our findings emphasize DDB1 as a key regulator of colorectal cancer stemness and p53 stability through its O-GlcNAcylation at Ser-764, which enhances the E3 ligase activity of the CUL4-DDB1 complex.