Inhibition of EZH2 by chemo- and radiotherapy agents and small molecule inhibitors induces cell death in castration-resistant prostate cancer

Abstract

Androgen deprivation therapy is the mainstay of treatment of advanced prostate cancer (PCa). However, a significant portion of patients experience disease relapse and tumors ultimately evolve into castration resistant prostate cancer (CRPC), for which there is no cure in the clinic. The Polycomb protein enhancer of zeste homolog 2 (EZH2) is frequently overexpressed in CRPC. It is unclear whether EZH2 can be a therapeutic target in CRPC. Here, we demonstrated that chemo- and radiotherapy agents such as camptothecin (CPT) and γ irradiation decrease EZH2 expression in various PCa cell lines. We provided evidence that functional p53 and RB proteins are required for CPT- and irradiation-induced downregulation of EZH2 in CRPC cells. We demonstrated that EZH2-specific small molecule inhibitors mitigate CRPC cell growth. We further showed that the EZH2 inhibitor GSK126 inhibits both Polycomb-dependent and -independent functions of EZH2 in PCa cells. Importantly, we found that inhibition of EZH2 by genetic and pharmacological means sensitizes CRPC cells to CPT-induced apoptotic death and growth inhibition in culture and in mice. Our data suggest that concomitant administration of small molecule inhibitors of EZH2 may significantly increase the anti-tumor efficacy of conventional chemo- and radiotherapies in CRPC.

Keywords: EZH2; castration-resistant prostate cancer; chemotherapy; radiation therapy; small molecule inhibitor.

Figure 1. Treatment of PCa cells with chemo- and radiotherapy agents downregulates EZH2 expression

A–C. effect of CPT on expression of EZH2 protein in PCa cells. LNCaP (p53-, RB-positive), PC-3 (p53-negative) and DU145 (p53-, RB-negative) PCa cell lines were treated with CPT (1.25 μM) and at different time points after treatment, cells were harvested for western blot analysis with indicated antibodies. ERK2 was used as a loading control. D–F. effect of γ irradiation on expression of EZH2 protein in PCa cells. LNCaP, PC-3 and DU145 cells were treated with different doses (Gray, Gy) of γ irradiation. At 24 h after treatment cells were harvested for western blot analysis using the indicated antibodies. Irr, irradiation. G. LNCaP cells were cultured in regular medium or medium containing charcoal-stripped-serum (CSS) for 48 h. Cells were then treated with CPT (1.25 μM) for 48 h and harvested for western blot analysis. H. LNCaP cells were cultured in regular medium and C4-2 cells were cultured in CSS medium for 48 h. Cells were then treated with CPT (1.25 μM) for 48 h and harvested for western blot analysis. ERK2 was used as a loading control.

Figure 2. Role of p53 and the RB family proteins in irradiation-induced downregulation of EZH2 expression

A–B. androgen-sensitive LNCaP (A) and castration-resistant C4-2 (B) cells were transfected with a pool of non-specific control siRNAs (siC) and p53-specific siRNAs. At 48 h after transfection cells were treated with γ irradiation, and cells were harvested at different time points for western blot analysis using the indicated antibodies. C–E. LNCaP cells were transfected with a pool of non-specific control siRNAs (siC) or RB-specific siRNAs (C), p130-specific siRNAs (D), or p107-specific siRNAs (E) At 48 h after transfection cells were treated with γ irradiation, and cells were harvested at different time points for western blot analysis using the indicated antibodies. ERK2 was used as a loading control. F. a hypothetical model depicting the causal role of p53 and RB in conventional chemotherapy (CPT) and radiotherapy (both are DNA damage-based therapeutics)-induced downregulation of EZH2 in PCa cells.

Figure 3. Inhibition of CRPC cell growth by the EZH2 small molecule inhibitor GSK126

A. The structure of GSK126 adapted from the website of Selleckchem. B. C4-2 CRPC cells were treated with the EZH2 specific inhibitor GSK126 with different doses (0, 5, 10, 20 μM). Cell viability was measured by MTS assays at different time points after GSK126 treatment. Data are means ± S.D. from six replicates. *P < 0.01. C–D. C4-2 CRPC cells were treated with the EZH2 specific inhibitor GSK126 with different doses (0, 5, 10, 20 μM). At 48 h after treatment cells were harvested for western blot analysis with indicated antibodies (C) and measurement of caspase-3 activity (D) ERK2 was used as a loading control. Data are means ± S.D. from 3 replicates. *P < 0.01. E–F. C4-2, PTEN-CaP8 and PC-3 cells were treated with vehicle (DMSO) or GSK126 (20 μM) for 48 h and then used for Matrigel invasion assays. Representative images of invasion assay are shown in (E) and the quantification results are shown in (F), respectively. Scale bar, 200 μm. Data are means ± S.D. from experiments with three replicates. *P < 0.01.

Figure 4. Inhibition of CRPC cell growth by the EZH2 small molecule inhibitors GSK343 and GSK503

A. The structures of GSK343 and GSK503 adapted from the website of Selleckchem. B–E. human C4-2 (B, C) and mouse PTEN-CaP8 (D, E) CRPC cells were treated with vehicle (DMSO), GSK343 or GSK503, two small molecule inhibitors of EZH2. At different time points, cell viability was measured by MTS assays. Data are means ± S.D. from six replicates. *P < 0.01. F, C4-2 and PTEN-CaP8 cells were harvested for western blot analysis of protein expression with indicated antibodies. ERK2 was used as a loading control. Short exp.: short exposure.

Figure 5. Effect of the EZH2 small molecule inhibitor GSK126 on expression of PcD and PcI genes in CRPC cells

C4-2 CRPC cells were treated with different doses of GSK126. At 48 h after treatment, cells were harvested for RT-qPCR analysis of mRNA expression of EZH2-repressed target genes including HOXA9 and BRACHYURY A. and EZH2-activated target genes including TMEM48, CSK2 and KIAA0101 B. GAPDH was used as an internal control.

Figure 6. The EZH2 small molecule inhibitor GSK126 sensitizes CRPC cells to CPT-induced apoptotic death and enhances CPT-mediated inhibition of cell growth

A. C4-2 CRPC cells were treated with vehicle (DMSO), CPT (100 nM), GSK126 (10 μM) or both drugs. Cell viability was measured at different time points after treatment using MTS assays. B–C. C4-2 CRPC cells were treated as in (A) At 48 h after treatment cells were harvested for western blot analysis with indicated antibodies (B) and measurement of caspase-3 activity (C) ERK2 was used as a loading control. Data are means ± S.D. from 3 replicates. *P < 0.01. D. C4-2 CRPC cells were treated with CPT (250 nM), GSK126 (20 μM) or both. At 48 h after treatment, cells were collected and fixed with 70% ethanol and subject to sub-G1 analysis flow cytometry. PI, propidium iodide. Experiments were repeated two more times and similar results were obtained. E–F. C4-2 cells infected with indicated shRNAs were injected s.c. into the right flank of NSG mice (n = 5). The tumor volume of each xenograft at each time point (E) and tumors at the end of treatment (F) are shown. Error bars, SD from five tumors. *P < 0.05.