Fig 2: HDAC5 is indispensable for deacetylation of SOX9 in MCF-7 TAMR cells. a Western blot of HDAC5 protein from shNC and shHDAC5 TAMR MCF-7 cells. b Western blot of SOX9 protein in cytoplasmic and nuclear fractions from shNC and shHDAC5 MCF-7 cells. c Representative confocal images of immunofluorescence for SOX9 locations in shNC and shHDAC5 TAMR MCF-7 cells. d Immunoprecipitation in shNC and shHDAC5 MCF-7 cells with anti-SOX9 followed by immunoblotting with an antibody against acetylation proteins. e MTT assay of growth rates in shNC or shHDAC5 TAMR MCF-7 cells. f MTT assay of growth rates of TAMR MCF-7 cells treated with DMSO or HDAC4/HDAC5 inhibitor LMK-235 (12 nM). g Flow cytometry of ALDH+ cells following HDAC5 knocking down in TAMR MCF-7 cells. h Western blot analysis for CD44, ALDH1A1 and SOX2 proteins in TAMR MCF-7 cells with shNC and shHDAC5. i MTT assay of growth rates of MCF-7 transfected with HDAC5 or vector control and treated with tamoxifen (1 µM). j Bioinformatics analysis of HDAC5 expression in the public dataset (GEO: GSE9574) of healthy control and patients with ER+ breast cancer (control n = 14, breast cancer n = 14). k Cox regression to quantify hazard ratio (HR) and the corresponding confidence interval (CI) between overall survival (OS) and SOX9, HDAC5 expression in human breast cancer tissues (N = 1178, HR = 0.91, 95% CI: 0.82–1.0, P = 0.0409). Data are representative of means ± SEM of three independent experiments (unpaired t-test, *p < 0.05, **p < 0.01)

Fig 3: Associations of TB with EMT and tumor stemness. (A) Representative IHC images of E-cadherin and its associations with TB grade. (B) Representative IHC images of vimentin and its associations with TB grade. (C) Heatmap showed CSC marker expression in 229 cases of BC patients. (D) Representative IHC images of CD44 and its associations with TB grade. (E) Representative IHC images of ALDH1A1 and its associations with TB grade. (F) Representative IHC images of CD24 and its associations with TB grade. (G) Association between TB grade and CSC score. (H) The Kaplan-Meier survival curve shows disease-free survival of BC after stratification by CSC score. ** p < 0.01.

Fig 4: MiR-328-3p depletion enhances cancer cells stemness.A The relative ALDH1 expression in MDA-MB-468 cells with overexpressed miR-328-3p by RT-qPCR. Mean ± SD, ***p < 0.001 by one-way ANOVA with Dunnett t-test (n = 3). B The proportions of ALDH1+ cancer cells were monitored by flow cytometry, cells were treated with or without mimic-miR-328-3p in MDA-MB-468 CSCs spheroids. Mean ± SD, **p < 0.01 by One-way ANOVA with Dunnett t-test (n = 3). C The co-expression of cytokeratin (CK) and ALDH1 in clinical BC tissue with low or high miR-328-3p expression. Scale bar: 50 µm. D, E The spheroid forming ability of MDA-MB-468 (D) or MCF-7 (E) in the indicated treatment. Scale bar: 30 µm. F Scheme of the miR-328-3p-mediated cell stemness network in BC: The low expression of miR-328-3p in small partition BC patients who suffered tumor metastasis, which promotes CPT1A expression that enhances fatty acid ß-oxidation and stemness of cells, then promotes cancer cell metastasis.

Fig 5: SOX9 localised in the nucleus is required for tamoxifen resistance. a Kaplan–Meier plot analysis of the correlation between the expression of SOX9 and survival rates in human breast cancer with public bioinformatics dataset (SOX9 low n = 1977, high n = 1974). b Western blot analysis of SOX9 protein of cytoplasmic and nuclear fractions from MDA-MB-231 and MCF-7 cells. c Comparison of SOX9 mRNA level in human breast cancer according to its sensitivities to tamoxifen by using GSE9195 cohort (ER+ breast cancer samples from women treated with adjuvant tamoxifen monotherapy, tamoxifen-sensitive n = 64 and tamoxifen-resistance n = 13). d Kaplan–Meier plot analysis of disease-free survival based on SOX9 mRNA levels by using GSE9195 cohort (ER+ breast cancer samples from women treated with adjuvant tamoxifen monotherapy, lower n = 39, higher n = 38). e Representative confocal images of immunofluorescence for SOX9 expression in MCF-7 and TAMR MCF-7 cells. Scale bar, 20 µm. f Western blot analysis for SOX9 protein in parental and TAMR MCF-7 cells. g Western blot for SOX9 protein in control and KO-SOX9 TAMR MCF-7 cells. h MTT assay of the growth rate in control or KO-SOX9 TAMR MCF-7 cells (lower). i Flow cytometric analysis of ALDH+ cells with control and SOX9-KO TAMR MCF-7 cells. j qRT-PCR of CD44, ALDH1A1 and SOX2 mRNA in TAMR MCF-7 cells with control or SOX9-KO. k Western blot analysis for CD44, ALDH1A1 and SOX2 proteins in TAMR MCF-7 cells with control or KO-SOX9. l MTT assay of growth rate after tamoxifen (1 µM) treatment in MCF-7 cells with SOX9 overexpression or vector control. m Western blot analysis for ERa C-MYC ABCG2 p21 and Bcl2 proteins in TAMR MCF-7 cells with control or KO-SOX9. n Western blot analysis of tamoxifen-resistance related signalling pathways in MCF-7 cells with control and SOX9-KO. Data are representative of means ± SEM of three independent experiments (unpaired t-test, *p < 0.05, **p < 0.01)