Fig 1: FOXA1 O-GlcNAcylation elevates the metastatic potential of breast cancer cells.(A to C) The motility (A), migration and invasion ability (B), and adhesion ability (C) of breast cancer cells were analyzed. Cells were treated with OSMI-4 (50 μM) or TMG (50 μM) for 24 hours. Mitomycin C was used to inhibit the impact of cell proliferation. Representative and quantified results of the wound-healing (A; scale bar, 200 μm), Transwell (B, scale bar, 100 μm) and adhesion (C; scale bar, 100 μm) assays in FOXA1WT- or FOXA13A-expressing breast cancer cells are shown. (A and B) n = 3 biologically independent experiments. (C) n = 6 biologically independent experiments. (D and E) The migration and invasion abilities were analyzed and quantified by Transwell assays in FOXA1WT- or FOXA13A-expressing breast cancer cells (MCF-7 FOXA1 KO and MDA-MB-231; scale bar, 100 μm). Cells were transfected with MECP2 (D) or EPB41L3 (E) shRNA (shMECP2 and shEPB41L3) or scrambled shRNA (control) for 48 hours before the assays. Mitomycin C was used to inhibit the impact of cell proliferation. n = 3 biologically independent experiments. (F) FOXA1 immunoprecipitation was performed in five breast cancer patient tumor samples, and immunoprecipitated fractions were analyzed by WB. The pathological grade and the patient no. are indicated.
Fig 2: Schematic working model of FOXA1 O-GlcNAcylation–mediated breast cancer metastasis.O-GlcNAcylation at T432/S441/S443 shapes the FOXA1 interactome, especially triggers the recruitment of transcriptional repressor MECP2, consequently stimulates FOXA1 chromatin-binding sites switch to chromatin loci of adhesion-related genes, including EPB41L3 and COL9A2, and thus promotes breast cancer proliferation and metastasis both in vitro and in vivo.
Fig 3: The chromatin-binding switch of O-GlcNAcylated FOXA1 is stimulated by MECP2.(A and B) O-GlcNAcylated FOXA1 chromatin loci shows transcriptional suppression characteristics. Average enrichment profiles of published MCF-7 cells (A) ATAC-seq (GSE179666), RNA-Pol II ChIP-seq (GSE54693), (B) H3K27me3 (GSE96363), H3K4me1 (GSE86714), H3K4me3 (GSE97481), and H3K27ac (GSE97481) ChIP-seq reads at differential quantitative FOXA1WT and FOXA13A ChIP-seq peaks are shown. (C) Heat maps of the FOXA1WT and FOXA13A ChIP-seq signal density at hg19 genome-wide CpG islands. (D) Average published MCF-7 cells genome-wide DNA methylation (GSE54693) profiles at CpG islands (left) and typical/super enhancer elements (right) of differential quantitative FOXA1WT and FOXA13A ChIP-seq peaks. (E) Heat map representation of endogenic MECP2 ChIP-seq (in FOXA1WT and FOXA13A expressed MCF-7 FOXA1 KO cells, respectively) signal enrichment at differential quantitative FOXA1WT and FOXA13A sites. (F) Cells were transfected with MECP2 shRNA (shMECP2). Average enrichment profiles of FOXA1 ChIP-seq reads at FOXA1WT and FOXA13A-biased sites are shown. (G) The box plots showing the mRNA expression changes of FOXA1-targeting genes associated with FOXA1WT and FOXA13A-biased sites. n = 2 biologically independent RNA-seq replicates. (H) Predicted regulatory network between FOXA1 and the corresponding downstream genes in different O-GlcNAcylation state. Functional enrichment is indicated. (I) Integrative Genomics Viewer tracks showing FOXA1, MECP2, RNA-Pol II ChIP-seq and DNA methylation, ATAC-seq signal at the promoter regions of the representative genes EPB41L3 and COL9A2. (J) Certain FOXA1 targeting genes were validated by qPCR and ChIP-qPCR. (K) Methylation status of EPB41L3 and COL9A2 gene promoter regions in the indicated cells were measured by methylation-specific qPCR. (L) Cells were transfected with MECP2 shRNA (shMECP2). Certain FOXA1 targeting genes were validated by qPCR and ChIP-qPCR. For (J) to (L), cells were pretreated with OSMI-4 (50 μM) or TMG (50 μM) for 24 hours. n = 4 biologically independent experiments.
Fig 4: FOXA1 O-GlcNAcylation promotes oncogenesis and metastasis of breast cancer in vivo.(A and B) Elimination of FOXA1 O-GlcNAcylation diminished xenograft tumor formation in vivo. WT MCF-7, MCF-7 FOXA1 KO, FOXA1WT, or FOXA13A MCF-7 FOXA1 KO cells were injected subcutaneously into the axillae of nude mice (n = 5 for each group). Mice were euthanized after 24 days, and their tumor masses were excised (A), measured and weighed (B). (C) HA-FOXA1 immunoprecipitation was performed with anti-HA-tag magnetic beads using xenograft tumor samples in each group, and the immunoprecipitated fractions were analyzed by WB. (D) Xenograft tumor samples were subjected to H&E, EPB41L3, and Ki67 staining. One representative experiment of n = 3 independent experiments is shown. Scale bar, 100 μm. (E) Schematics for tail vein injection of breast cancer cells in nude mice to generate experimental pulmonary metastasis. (F) MCF-7, MCF-7 FOXA1 KO, FOXA1WT, or FOXA13A MCF-7 FOXA1 KO cells were intravenously injected into the tail vein of nude mice (n = 5 for each group). Three weeks later, the animals were anesthetized, injected with luciferin, and imaged for luciferase activity every week. One representative image is shown. The fluorescence intensity represents the sizes of the breast cancer metastases. P values are indicated. (G) Gross appearances of lungs with tumors and numbers of tumor nodules. The blue dotted-line circles represent tumors (n = 5 for each group). (H) H&E staining of sections of metastasized lungs. Scale bar, 500 μm.
Supplier Page from Abcam for Anti-EPB41L3/DAL1 antibody