Figure 5. Androgen receptor modulates EMT pathway via upregulation of the EMT effector protein, slug.

(a) We performed differential gene expression (DGE) analysis of 8 AR-positive relative to 14 AR-negative HCC cell lines (as listed in Figure 1C) and obtained 1058 differentially expressed genes. Gene set enrichment analysis (GSEA) on this set of genes using molecular signature database (MSigDB) revealed significant enrichment of the EMT pathway among the top 10 molecular pathways in AR-positive HCC cells. P-value < 0.01 (Fisher exact test). (b) Transcript abundance from CCLE data show a positive correlation (Spearman correlation coefficient) between SNAI2 and AR expression in AR-positive (red) relative to AR-negative (black) cell lines suggesting a putative role for AR:SNAI2(Slug) mediated migration and invasion in HCC. (c) RT-PCR of SNAI2 mRNA in SNU-423 cells treated with 1 nM R1881 for 3, 8 and 24 hours (left) as well as by dose response at 24 hours (right). SNAI2 mRNA demonstrated both time- and concentration-dependent, androgen-dependent regulation. (d) SNU-423 cells were treated with vehicle, 1 nM R1881 or 10 μM enzalutamide with 1 nM R1881 for 3 and 24hours. AR and slug protein were assessed by western blot (left) revealing androgen-dependent slug regulation (densitometry, right). (e) The cellular localization of AR and slug in SNU-423 cells were determined by immunofluorescence in the presence of 1 nM R1881 alone and in combination with 10 μM enzalutamide for 24 hours. AR and slug are cytoplasmic in the absence of androgen, but both became predominantly nuclear upon stimulation with 1 nM R1881 for 24 hours. This androgen-mediated nuclear translocation of slug was inhibited in part upon co-treatment with enzalutamide. (f) Androgen treatment with 1 nM R1881 for 48 hours promoted invasion that was both AR- and SNAI2-dependent as demonstrated by the Matrigel invasion assay (performed and analyzed as described in Figure 4E, quantification bottom right). Both AR and SNAI2 were successfully knocked down using siRNA targeting AR (as in Figure 3B) or SNAI2 (western blot inset, top right). (g) 48 hours Matrigel invasion assays were performed on SNU-423 cells transfected with either 10 μg AR-v7 expressing plasmid (pAR-v7) or control (pcw107, pControl) demonstrating increased invasive capacity for AR-v7 expressing cells.(h) 48 hours post transfection, immunofluorescence analysis of AR-v7 or control transfected cells showed AR (anti-AR mAb targeting N-terminal region of AR, red) and slug (green) were cytoplasmic in the presence of control plasmid. Upon the addition of exogenous, constitutively active AR-v7, both AR and slug staining became predominantly nuclear. Cells were also harvested and analyzed for AR and slug protein content by western blot (inset bottom left) revealing an AR-v7 mediated increase in slug protein (western blot inset, bottom). (i) Immunofluorescence analysis of HCCLM3 cells shows AR and slug co-nuclearization in the absence of androgen stimulation. (j) Correlation analysis of AR and SNAI2 expression in the HCC cohort in TCGA demonstrates a relationship between AR and SNAI2 mRNA levels in liver cancer tissue (TCGA) but not normal tissue (TCGA and GTEx). Spearman correlation analyses showed statistically significant positive correlations between AR and SNAI2 in primary samples (372 patients, left) but no correlation in matched normal samples (50 patients, middle) or normal liver tissues (150 donors, right) (43). One-way ANOVA with Dunnett’s multiple comparisons test. All panels: * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001 versus vehicle (c, f), and expression plasmid controls (g)