Fig. 4: Phosphorylated ETS1 activates a proliferation gene network that promotes drug resistance.

See also Fig. S4. a Enrichment plots from GSEA conducted with normalised differential expression from RNA seq of (top left) resistant-MDA-MB231 over parental MDA-MB231 cells, (top right) resistant-SUM-159 over parental SUM-159 cells, (bottom left) resistant MDA-MB 468 over parental MDA-MB468 cells. Plots represent statistically significant enrichment (FDR < 0.25) of indicated hallmark pathways. b GSEA plots using normalized differential expression from RNA seq of S251D-ETS1 and S251A-ETS1 expressing stable T47D cells treated with 5’FU. Plots represent statistically significant enrichment (FDR < 0.25) of indicated hallmark pathways. Heatmap of selected genes from enriched pathways in RNA seq of c Resistance and parental MDA-MB231 cells, d Patient stratified by phospho-S251 ETS1 scores. e Relative transcript levels of indicated genes from Res-MDA-MB231 cell lines transfected with siRNAs were analysed using qRT-PCR. f Relative transcript levels of genes from parental MDA-MB231 and resistance MDA-MB231 cell lines post-treatment were analysed using qRT-PCR as indicated. g Relative transcript levels of genes from biopsy tissue samples collected from the treatment relapsed subjects before (Naïve) and after chemotherapy were analysed using qrt-PCR. Experiments were performed as biological triplicates (mean ± SD, n = 3). h Exon level expression and fold-change (Log2 FC) of indicated genes from ETS1 knockdown (ETS1ShRNA) cells were matched with enriched ETS1-ChIP signals over their promoters from GSE109896, GSE109884. i) Representative FACS profile for EdU staining post DNAPKcs inhibitor treatment as indicated. Alexa-488 labelled Edu-stained proliferating cells were measured in the FITC channel and plotted for three independent experiments, SD ± 3. j Model: ETS1 stabilised by DNAPKcs-mediated phosphorylation at S251 promotes resistance phenotype by activating proliferation-related genes in TNBC (Created with BioRender.com). For 4e, f, g; Two way ANOVA with Sidak’s or Dunnett’s multiple comparison test was applied for the statistical significance, For 4i, One way ANOVA with Dunnett’s multiple comparison test was applied where ****p < .0001,***p = 0.0001,**p = 0.001, *p = 0.01, n.s=not significant.