FOXA1是一个在表观遗传重编程中起作用的转录因子，对乳腺癌的进展至关重要。然而，FOXA1实现其致癌功能的机制尚不清楚。在这里，我们证明了FOXA1的O-链接β-N-乙酰葡萄糖胺修饰（O-GlcNAcylation）通过协调转录许多转移调节因子的方式来促进乳腺癌的转移。位于Thr432、Ser441和Ser443的O-GlcNAcylation调节了FOXA1的稳定性，并促使其与染色质的组合。O-GlcNAcylation塑造了FOXA1的互作组织，尤其触发了转录抑制子甲基化CpG结合蛋白2的招募，并因此促进了FOXA1染色质结合位点转移到粘附相关基因的染色质位点，包括EPB41L3和COL9A2。FOXA1上O-GlcNAcylation的位点特异性耗竭影响了各种下游基因的表达，从而在体外和体内抑制了乳腺癌的增殖和转移。我们的数据确立了异常的FOXA1 O-GlcNAcylation在乳腺癌进展中的重要性，并表明靶向O-GlcNAcylation是治疗转移性乳腺癌的一种策略。

FOXA1, a transcription factor involved in epigenetic reprogramming, is crucial for breast cancer progression. However, the mechanisms by which FOXA1 achieves its oncogenic functions remain elusive. Here, we demonstrate that the O-linked β-N-acetylglucosamine modification (O-GlcNAcylation) of FOXA1 promotes breast cancer metastasis by orchestrating the transcription of numerous metastasis regulators. O-GlcNAcylation at Thr432, Ser441, and Ser443 regulates the stability of FOXA1 and promotes its assembly with chromatin. O-GlcNAcylation shapes the FOXA1 interactome, especially triggering the recruitment of the transcriptional repressor methyl-CpG binding protein 2 and consequently stimulating FOXA1 chromatin-binding sites to switch to chromatin loci of adhesion-related genes, including EPB41L3 and COL9A2. Site-specific depletion of O-GlcNAcylation on FOXA1 affects the expression of various downstream genes and thus inhibits breast cancer proliferation and metastasis both in vitro and in vivo. Our data establish the importance of aberrant FOXA1 O-GlcNAcylation in breast cancer progression and indicate that targeting O-GlcNAcylation is a therapeutic strategy for metastatic breast cancer.