Figure 2.

ARVib disrupts the AR/AR-V7 gene expression program in enzalutamide resistant prostate cancer cells. A. Venn diagram of RNA-seq results depicting the overlap of differentially expressed genes (DEGs) in C4-2B MDVR cells treated with 1.5 ¼M ARVib-7 (vs. DMSO) or 1.5 ¼M ARVib-31 (vs. DMSO) for 24 hours. B. Hierarchical clustering and heatmap visualization of the differentially expressed genes (DEGs) in ARVib-treated C4-2B MDVR cells with fold change (FC) > 1.2, as compared to vehicle (DMSO). The genes are displayed in rows and the normalized expression counts per sample are displayed in columns. Red and blue coloring indicates up-regulated and down-regulated relative expression levels, respectively. Middle and right, AR-FL and AR-V7 activity-signature genes that were altered in expression are displayed [5]. C. GSEA of C4-2B MDVR cells treated with ARVibs (relative to DMSO control) demonstrates enrichment for suppressed expression of genes comprising PID-AR pathway (left) and the AR-V7 (right) gene signatures. The latter signature was defined by genes that are preferentially upregulated by AR-V7 [7]. D. qRT-PCR analysis of the indicated genes in C4-2B MDVR cells treated with DMSO or 1 ¼M ARVibs for 48 hours. E. C4-2B MDVR cells were treated with DMSO or 1 ¼M ARVibs in FBS conditions for 48 hours, PSA level was determined by PSA ELISA. F. C4-2B cells were transiently transfected with pcDNA with or without 1nM DHT, AR-V1, AR-V3, AR-V7, AR-V9 and AR-V12 with PSA E/P-luciferase plasmids in CS-FBS conditions, and then treated with DMSO, 5¼M abiraterone, 20 ¼M enzalutamide, 20 ¼M apalutamide or 1 ¼M ARVibs for 16 hours. PSA luciferase activity was examined. G. 293 cells were transiently transfected with vector, AR-V7, mutant AR plus PSA promoter luciferase plasmids in FBS condition, and then treated with antiandrogens or ARVibs for 16 hours. PSA luciferase activity was determined. * p<0.05. Results are the mean of three independent experiments (±S.D.). Apal: apalutamide, Bica: bicalutamide.