Figure 2 from NXP800 Activates the Unfolded Protein Response, Altering AR and E2F Function to Impact Castration-Resistant Prostate Cancer Growth

NXP800 inhibits AR transactivation and AR signaling to inhibit the growth of AR aberrant prostate cancer models. A–C, VCaP (A), LNCaP95 (B), and 22Rv1 (C) prostate cancer cells were treated with vehicle (DMSO 0.1%) or various concentrations (5, 10, 50, 100, and 250 nmol/L) of NXP800 (active, red line) or CCT365248 (inactive, blue line), and growth was determined after 5 days by CellTiter-Glo Luminescent Cell Viability Assay. Mean growth (compared with vehicle; defined as 1) with SD from a single experiment with six replicates is shown. P values were calculated for active compound compared with inactive compound for each concentration using the unpaired Student t test. P values ≤ 0.05 are shown (*). VCaP (A), LNCaP95 (B), and 22Rv1 (C) prostate cancer cells were treated with vehicle (DMSO 0.1%) or various concentrations (50, 100, and 250 nmol/L) of NXP800 (active) or CCT365248 (inactive) for 48 hours, and AR-FL, AR-V7, PSA, and GAPDH protein expression was determined from one experiment performed in triplicate. Single Western blot representative of three is shown. VCaP (A), LNCaP95 (B), and 22Rv1 (C) prostate cancer cells were treated with vehicle (DMSO 0.1%) or various concentrations (50, 100, and 250 nmol/L) of NXP800 (active, red line), or CCT365248 (inactive, blue line) for 48 hours, and KLK2, KLK3, TMPRSS2, and FKBP5 RNA expression was determined. Mean RNA expression (normalized to average of GAPDH/B2M/HRPT1/RPLP0 and vehicle; defined as 1), with SD from a single experiment with three replicates is shown. P values were calculated for active compared with inactive compound for each concentration using the unpaired Student t test. P values ≤ 0.05 are shown (*). D, PC3 cells were transfected with CONTROL-FLAG, AR-FL-FLAG, or AR-V7-FLAG and PSA-ARE3-luciferase, prior to treatment with vehicle (DMSO 0.1%), 250 nmol/L CCT365248 (inactive), 250 nmol/L NXP800 (active), or 3 μmol/L enzalutamide for 1 hour prior to stimulation with or without 10 nmol/L DHT for 16 hours. Mean luciferase activity (compared with CONTROL-FLAG/vehicle/without DHT) with SD from two experiments with five replicates is shown. P values were calculated for each plasmid (with and without DHT stimulation) with vehicle compared with other treatments using the unpaired Student t test. P values ≤ 0.05 are shown (*). E, ChIP with an AR antibody was carried out in 22Rv1 cells that were plated in starved (charcoal-striped serum/phenol red–free) media for 72 hours prior to treatment with 250 nmol/L CCT365248 (inactive) or 250 nmol/L NXP800 (active) for 1 hour prior to stimulation with or without 10 nmol/L DHT for 5 hours. AR recruitment to AR-responsive genes (KLK2, KLK3, FKBP5, TMPRSS2, CHRNA2, and ANKRD30B) was determined. Mean binding as percentage of input with SD from two experiments with three replicates is shown. P values were calculated for 250 nmol/L CCT365248 (inactive) compared with 250 nmol/L NXP800 (active) with and without DHT stimulation using the unpaired Student t test. P values ≤ 0.05 are shown (*).