Fig. 6

VAX2 is a key master regulator for the TNBC mesenchymal development subtype. (a) Genome browser plot showing TNBC-specific SE and key master regulator VAX2 H3K27ac signals. The data were obtained from public databases, refer to Supplementary Table 1 for details. (b) Immunoblotting detection of VAX2 expression in a panel of TNBC cell lines. (c) Top: Schematic illustrating CRISPR-knockout of SE269. Bottom: DNA blot confirming successful heterozygous knockout of SE269. (d) Immunoblotting of VAX2 in CAL51 and MDA468 upon deletion of SE269 or direct knockdown of VAX2. (e) Inhibition of VAX2 expression levels in CAL51 and MDA468 cell lines by BETi, with the inhibitory effect increasing as the drug concentration increases. (f) H3K27ac and BRD4 ChIP-qPCR of the indicated cell lines using primers amplifying VAX2 SE269. Error bars represent mean ± SD, n = 3 biological independent samples. (g) IGV map showing VAX2 peaks and VAX2 ChIP-seq binding locations around SE269 in MDA468 and VAX2-knockdown MDA468 cell lines. (h) ChIP-seq analysis of VAX2 identified 11,156 potential target genes. Among them, 828 genes were downregulated in VAX2-knockdown cells. (i) ChIP-seq signal heatmap showing that VAX2 is enriched in 828 regulated genes. (j) Functional enrichment analysis of 828 genes regulated by VAX2, identified by VAX2 ChIP-seq and RNA-seq, showed relevance to mesenchymal development functions (including EMT, ECM formation, cell proliferation, cell adhesion, etc.). (k) GSEA of differentially expressed genes between VAX2-knockdown and wild-type MDA468 cell lines showed attenuated enrichment of gene sets characteristic of the TNBC mesenchymal development subtype. The p value in (f) was determined by a two-sided Student's t-test. *** p < 0.001; ns, no significance. Data in (b–f) were representative of three independent experiments.