FOXA1 O-GlcNAcylation–mediated transcriptional switch governs metastasis capacity in breast cancer

Abstract

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.

Comprehensive Glycomic and Glycoproteomic Analyses of Human Programmed Cell Death Protein 1 Extracellular Domain

“…Both of the samples were each divided into three aliquots, respectively; one aliquot of both samples was lyophilized and kept at −80 °C as a control; the other two aliquots of both samples were treated by PNGase F to remove N-glycans and then lyophilized, one of the two aliquots of each sample was kept at −80 °C, and the other aliquot was further processed via alkaline elimination to release O-glycans. All above-mentioned aliquots were used for SDS-PAGE (12% gel), followed by Coomassie blue staining or being transferred to polyvinylidene difluoride (PVDF) membrane (Millipore) for Western blot as described elsewhere . The membranes were blocked with a 5% nonfat milk solution and hybridized with primary antibodies at 4 °C overnight.…”

“…All above-mentioned aliquots were used for SDS-PAGE (12% gel), followed by Coomassie blue staining or being transferred to polyvinylidene difluoride (PVDF) membrane (Millipore) for Western blot as described elsewhere. 38 The membranes were blocked with a 5% nonfat milk solution and hybridized with primary antibodies at 4 °C overnight. After 3 tris-buffered saline with Tween 20 (TBST) washings, horseradish peroxidase (HRP)-linked secondary antibody was used at room temperature.…”

“…Its 1 "z" ion (z6) and 3 "c" ions (c5, c8, and c9) support peptide sequence identification. In addition, the z6 ion clearly indicates the absence of HexNAc on peptide fragment FS 38 Supplementary Table S9 and Supplementary Table S10 summarize all newly identified O-glycosylation sites on hPD-1 ECD expressed by HEK 293 and CHO cells as well as the Oglycan components on each site.…”