Dual inhibition of AKT‐mTOR and AR signaling by targeting HDAC3 in PTEN‐ or SPOP‐mutated prostate cancer

A

22Rv1 cells stably infected with SPOP F133V mutant lentivirus were treated with 3 μM of RGFP966 for 24 h and harvested for Western blot with the indicated antibodies.

B, C

Representative images of 3D Matrigel cultures 22Rv1 cells stably infected control or SPOP F133V lentivirus at day 5 post‐treatment of RGF966 are shown in (B) with the quantitative data in a box plot (C). The lentivirus transfection efficiency is at least 95% of total cells. In graph (C), the description for box plot is the same as the figure legend in Fig 7H. Data are shown as means ± SD. DMSO‐Lenti‐EV (n = 188) versus RGFP966‐Lenti‐EV (n = 139): ***P = 3.05e‐11, DMSO‐SPOP‐F1333V (n = 130) versus RGFP966‐SPOP‐F133V (n = 193): ***P = 2.85e‐12 were performed by Wilcoxon rank sum test with continuity correction. Scale bar, 100 μm.

D–F

Representatives of patient‐derived SPOP‐mutant organoids at day 5 post‐treatment of DMSO or 3 μM of RGF966 are shown in (D) with the quantitative data of OD value at 490 nm in (E). ***P = 1.43e‐04 was performed using the unpaired two‐tailed Student's t‐test. Based on the observed growth rate of untreated SPOP‐mutated organoids, greater than 50% of organoids reach 10 μm in diameter at day 5. “10 μm” was set as the cutoff value. The number of organoids with the diameter > 10 μm) from at least five fields were counted and analyzed (F). Data are shown as means ± SEM. ***P = 3.36e‐05 was performed by the unpaired two‐tailed Student's t‐test. Scale bar, 100 μm.

G, H

Patient‐derived SPOP‐mutant organoids were treated with vehicle (DMSO) or 3 μM of RGF966 for 5 days and harvested for IFC for phosphorylated AKT (S473) (G) and AR (H). Scale bars, 50 μm.

Figure 8. The HDAC3 inhibitor suppresses SPOP‐mutated prostate cancer cell growth.