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 Thr ⁴³² , Ser ⁴⁴¹ , and Ser ⁴⁴³ 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.

Background and Purpose: Although the ongoing clinical trials of ABT‐263 and ABT‐199 in chronic lymphocytic leukaemia (CLL) have indicated that BH3 mimetics hold considerable promise, understanding the mechanism of CLL resistance to BH3 mimetics remains a challenge.Experimental Approach: The LD₅₀ values of ABT‐737, ABT‐263 and ABT‐199 in a number of primary CLL cells from 40 patients, were determined. The levels of Bcl‐2 family proteins, including phosphorylated Bcl‐2 (pBcl‐2) and their interactions were measured by immunoblotting and co‐immunoprecipitation. In vitro binding assays were performed by isothermal titration calorimetry and ELISA. BH3 profiling in isolated mitochondria was analysed.Key Results: The ratio of (Mcl‐1 + pBcl‐2) to Bcl‐2 expression provided the most significant predictive marker for the cytotoxic potential of ABT‐737, ABT‐263 and ABT‐199 in the panel of CLL samples. Mechanistically, pBcl‐2 inhibited the effects of the ABT compounds on the displacement of Bax and Bim from Bcl‐2, thereby suppressing mitochondrial apoptosis. The ABT compounds exhibited 100–300‐fold lower binding affinity to the glutamic acid, phosphomimetic, mutant of Bcl‐2 (T69E, S70E and S87E; EEE‐Bcl‐2). BH3 peptides exhibited different rank orders of binding affinities to full‐length WT‐Bcl‐2 and full‐length EEE‐Bcl‐2.Conclusions and Implications: Our study suggested that a structural alteration in the BH3‐binding groove was induced by phosphorylation of Bcl‐2. Our data also provided a framework to overcome resistance of CLL cells to the ABT compounds by combining pBcl‐2 kinase inhibitors with the ABT compounds.

O-GlcNAc modification plays critical roles in regulating the stress response program and cellular homeostasis. However, systematic and multi-omics studies on the O-GlcNAc regulated mechanism have been limited. Here, comprehensive data are obtained by a chemical reporter-based method to survey O-GlcNAc function in human breast cancer cells stimulated with the genotoxic agent adriamycin. We identify 875 genotoxic stress-induced O-GlcNAc chromatin-associated proteins (OCPs), including 88 O-GlcNAc chromatin-associated transcription factors and cofactors (OCTFs), subsequently map their genomic loci, and construct a comprehensive transcriptional reprogramming network. Notably, genotoxicity-induced O-GlcNAc enhances the genome-wide interactions of OCPs with chromatin. The dynamic binding switch of hundreds of OCPs from enhancers to promoters is identified as a crucial feature in the specific transcriptional activation of genes involved in the adaptation of cancer cells to genotoxic stress. The OCTF nuclear respiratory factor 1 (NRF1) is found to be a key response regulator in O-GlcNAc-modulated cellular homeostasis. These results provide a valuable clue suggesting that OCPs act as stress sensors by regulating the expression of various genes to protect cancer cells from genotoxic stress.