Figure 3. AR-FL and AR-SVs are Transcriptionally Active in HCC Cells.

(a) HCC (HepG2, SNU-423 and HCCLM3) and PCa (VCaP and DU145) cells were transiently transfected with an androgen responsive inducible reporter construct (MMTV-LUC) along with constitutively active renilla luciferase (RN-LUC) transfection control. Cells were maintained for 24 hours in charcoal-stripped FBS containing media (csFBS) then treated with vehicle, 1 nM R1881 or 10 μM enzalutamide (ENZ) with 1 nM R1881 for 24 hours. In SNU-423 and VCaP cells there was significant promoter and androgen-dependent induction of transcriptional activation in R1881-treated cells that was also reversible by co-treatment with ENZ. By contrast, there was no significant activation in HCCLM3, HepG2 and DU145 cells. (b) Comparing basal MMTV-LUC activity to pGL4.24 controls (in the absence of ligand) revealed a constitutive, ligand-independent transcriptional response for VCaP and HCCLM3 cells (left). This activity was significantly reduced by siRNA targeting AR-FL and AR-SV isoforms (AR exons 3 and 7). Successful AR knockdown was confirmed by WB in VCaP and HCCLM3 using N-terminal AR mAb (right). (c) AR-SV expressing HCC cells SNU-475 shows constitutive transcriptional activity similar to HCCLM3 (as determined in Figure 3B). This activity was significantly reduced by 3 different siRNA targeting AR-FL and AR-SV isoforms (left). Successful knock down of AR-v7 in SNU-475 was confirmed by WB with an N-terminal AR mAb (right). (d) Constitutive transcriptional activity in VCaP and HCCLM3 cells (as determined in Figure 3B) was insensitive or only weakly sensitive, respectively, to 24 hour 10 μM ENZ treatment. However, the AR-dependence of the transcriptional signal was demonstrated by knockdown of AR using siRNA targeting AR-FL and AR-SV isoforms (as in Figure 3B, 24 hours). (e) AR expressing SNU-423 HCC cells were transiently transfected with pGL4.24 LUC control or MMTV-LUC and an increasing amount of AR-v7 expressing plasmid (left). Successful overexpression of AR-v7 in SNU-423 was confirmed by WB with an N-terminal AR mAb (right). Exogenous AR-v7 expression in SNU-423 cells demonstrated a concentration dependent ability to increase constitutive MMTV-LUC activation. (f) SNU-423 cells were transiently cotransfected with MMTV-LUC and 10 μg pAR-v7 or empty expression vector control (pcw107) and treated as indicated for 24 hours. Relative to the control construct (pcw107), cells demonstrated increased AR-v7-dependent transcriptional activity (red bars) that was only weakly responsive to treatment with R1881 and insensitive to antagonism with ENZ. (g) C3A cells were transiently cotransfected with pGL4.24 LUC control (black bar) or MMTV-LUC (red bars) and 10 μg pAR-v7 or empty expression vector control (pcw107) for 24 hours. Relative to the control construct (pcw107), cells demonstrated a significant promotor and AR-v7-dependent transcriptional activity. All panels: Dual Luciferase Assay (Promega) with triplicate FF/RN values reported as fold versus vehicle treated control (a, d, f), basal promoter control (b, c, e), or expression vector control (g) as mean+STD. One-way ANOVA with Dunnett’s multiple comparisons test. * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001 versus vehicle treated cells (a, d, f), basal promoter transfected cells (b,e) siRNA controls (c) and empty expression vector controls (g), respectively.