Erbin destabilization by O-GlcNAcylation promotes 5-FU resistance via homologous recombination activation in colorectal cancer.

The emergence of 5-fluorouracil (5FU) resistance critically compromises chemotherapy efficacy in colorectal cancer (CRC). While O-GlcNAcylation-a dynamic post-translational modification linked to tumor progression-has been widely investigated, its functional role in 5FU chemoresistance remains poorly defined. Through mass spectrometry and tissue microarray analysis, we identified aberrantly elevated levels of O-GlcNAcylation and its catalytic enzyme O-GlcNAc transferase (OGT) in CRC tissues. Genetic silencing of OGT significantly reduced global O-GlcNAcylation, suppressing CRC cell proliferation, migration, and invasion while enhancing 5FU sensitivity both in vitro and in vivo. Notably, 5FU-resistant CRC models (HCT8/HCT116 5FUR) exhibited upregulated OGT and O-GlcNAcylation versus parental cells. Strikingly, OGT knockdown in resistant cells reversed chemoresistance, inhibited proliferation, and induced apoptosis. O-GlcNAcylated proteomic profiling identified Erbin, a tumor suppressor, as a hyper-modified protein with diminished expression in resistant cells, validated by co-immunoprecipitation. Remarkably, Erbin overexpression restored 5FU sensitivity in resistant cells, whereas OGT depletion stabilized Erbin by blocking ubiquitination. Site-directed mutagenesis pinpointed Thr1070 as Erbin's critical O-GlcNAcylation site; its mutation reduced ubiquitination, linking O-GlcNAcylation to Erbin degradation. Mechanistically, RNA sequencing (RNA-seq) revealed that Erbin suppresses homologous recombination (HR), causing unresolved DNA damage accumulation and heightened 5FU sensitivity, confirmed in vitro and in vivo experiments. Critically, elevated OGT in 5FU-resistant cells drives hyper-O-GlcNAcylation of Erbin at Thr1070, which facilitates its ubiquitin-dependent degradation, alleviating HR suppression to sustain chemoresistance. This defines the OGT-Erbin-HR axis as a central driver of 5FU resistance, proposing therapeutic targeting of this pathway to overcome CRC chemoresistance.