Fig. 7: FOXA1 associates with the differentiation plasticity of SNAI1 knockout cells via regulation of AR expression.

A RT-qPCR analysis of ER, PGR, HER2 and AR in ZR-75-1, MCF-7, MDA-MB-231-WT and SNAI1-KO cells. B Boxplots showing the expression of AR in various human BRCA subtypes in TCGA datasets (Basal n = 171, HER2 n = 78, Luminal A n = 499, Luminal B n = 197). The statistical significance is derived using the Wilcoxon rank-sum test (U test). C Scatter plot demonstrating the negative correlation between SNAI1 and AR expression in BRCA samples obtained from TCGA datasets. D Representative phase contrast images from three biological repeats showing the morphology of MDA-MB-231-WT, SNAI1-KOs and ZR-75-1 cells at day 5 of treatment with 0.05 μM of the androgen antagonist enzalutamide. Scale bars = 100 μM. E Representative phase contrast images from three biological repeats showing the morphology of mammospheres derived from MDA-MB-231-WT, SNAI1-KOs and ZR-75-1 cells at day 5 of treatment with either DMSO or 0.05 μM enzalutamide. Scale bars = 200 μM. F Quantification of the cross-section area of the mammospheres. G Scatter plot demonstrating the positive correlation between FOXA1 and AR expression in BRCA samples obtained from TCGA datasets. H RT-qPCR analysis of FOXA1 and AR expression in CS16 SNAI1-KO after knocking down FOXA1 expression using two independent siRNAs. Values in A and H represent fold-change of mRNA expression normalized to GAPDH. Data in A, F and H are presented as mean values of three biological replicates ± SEM, each in technical triplicates and p-values are shown based on two-tailed unpaired Student’s t-test. I ChIP-qPCR analysis of FOXA1 binding in three different regions (900, 600 and 300 bp) upstream of the AR transcription starting site. Values represent fold-enrichment over the IgG and FOXA1 binding to GAPDH is used as a negative control. Data are presented as mean values of three biological replicates ± SEM, each in technical triplicates and p-values are shown based on one-tailed unpaired Student’s t-test. J Schematic illustration of the proposed model of cellular plasticity in breast cancer based on the SNAI1/FOXA/AR regulatory module. Dark letters: high expression; light letters: low expression. p-values *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.