Pharmacological inhibition of noncanonical EED-EZH2 signaling overcomes chemoresistance in prostate cancer

Abstract

Rationale: Chemoresistance is a major obstacle in prostate cancer (PCa) treatment. We sought to understand the underlying mechanism of PCa chemoresistance and discover new treatments to overcome docetaxel resistance. Methods: We developed a novel phenotypic screening platform for the discovery of specific inhibitors of chemoresistant PCa cells. The mechanism of action of the lead compound was investigated using computational, molecular and cellular approaches. The in vivo toxicity and efficacy of the lead compound were evaluated in clinically-relevant animal models. Results: We identified LG1980 as a lead compound that demonstrates high selectivity and potency against chemoresistant PCa cells. Mechanistically, LG1980 binds embryonic ectoderm development (EED), disrupts the interaction between EED and enhancer of zeste homolog 2 (EZH2), thereby inducing the protein degradation of EZH2 and inhibiting the phosphorylation and activity of EZH2. Consequently, LG1980 targets a survival signaling cascade consisting of signal transducer and activator of transcription 3 (Stat3), S-phase kinase-associated protein 2 (SKP2), ATP binding cassette B 1 (ABCB1) and survivin. As a lead compound, LG1980 is well tolerated in mice and effectively suppresses the in vivo growth of chemoresistant PCa and synergistically enhances the efficacy of docetaxel in xenograft models. Conclusions: These results indicate that pharmacological inhibition of EED-EZH2 interaction is a novel strategy for the treatment of chemoresistant PCa. LG1980 and its analogues have the potential to be integrated into standard of care to improve clinical outcomes in PCa patients.

Keywords: EED inhibitor; EZH2 signaling; chemoresistance; drug discovery; prostate cancer.

Figure 1

LG1980 is a selective and potent inhibitor of chemoresistant PCa cells. (A) A two-tier phenotypic screening platform for the discovery of inhibitors of chemoresistant PCa. Primary screening is performed to identify small-molecule compounds that selectively inhibit the in vitro viability of ARCaP_E-shEPLIN but not ARCaP_E-shCtrl cells. Primary hits are further screened in a second (orthogonal) assay for their potent in vitro cytotoxicity in C4-2B-TaxR cells, but not docetaxel-sensitive parental C4-2B cells. Potential leads are evaluated for their mechanism of action and in vivo efficacy against docetaxel-resistant PCa in xenograft models. (B) The “A-B-C” 3-component structure of LG1980. Note LG1980 is an “S”-stereoisomer. (C) In vitro cytotoxicity of LG1980 in the ARCaP_E and C4-2B models (72 h). (D) Left: Flow cytometry analysis of cell cycle in C4-2B-TaxR cells following LG1980 treatment at the indicated concentrations (48 h). p < 0.05 for all pairwise comparisons between the percentages of cells in each cell cycle from the control and LG1980 treatment groups, except those in G2M phase between the control and 3 μM LG1980-treated cells; Right: Flow cytometry analysis of cell cycle in C4-2B-TaxR cells following docetaxel treatment at the indicated concentrations (48 h). p > 0.05 for all pairwise comparisons between the percentages of cells in each cell cycle from the control and docetaxel treatment groups. (E) Left: Flow cytometry analysis on Annexin V staining in C4-2B-TaxR cells following LG1980 treatment at the indicated concentrations (72 h). *p < 0.05; Right: Flow cytometry analysis on Annexin V staining in C4-2B-TaxR cells following docetaxel treatment at the indicated concentrations (72 h). p > 0.05 for all pairwise comparisons between the control and different concentrations of docetaxel. (F) Western bot analysis on the expression of apoptotic markers in C4-2B and C4-2B-TaxR cells following LG1980 treatment at the indicated concentrations (72 h). β-actin was used as the loading control.

Figure 2

LG1980 is a novel EED inhibitor. (A) Left: key amino acid residues and bonds mediating the interaction between LG1980 and EED. Pink lines refer to hydrogen bonds and the green lines refer to pi-pi bonds; Right: docked structure of LG1980 (green) and EED protein (gray). (B) Docked structure of LG1980 (green) and the EED (gray)-EZH2 (purple) complex. The binding of H3K27Me3 (blue) is also shown. (C) Binding of LG1980 to randomly biotinylated EED protein on ForteBio Octet Red384 System. X-axis, time in seconds; Y-axis, binding in nm. R² was calculated as 0.9202. (D) Left: CETSA analysis of EED expression in C4-2B-TaxR cells treated with DMSO or LG1980 (50 µM, 1 h). MAK683 (10 µM, 1 h) was used as a positive control. Right: melting temperature curves of EED protein in the presence of DMSO, LG1980 or MAK683 in C4-2B-TaxR cells.

Figure 3

LG1980 disassembles the PRC2 complex and destabilizes EZH2 protein in chemoresistant PCa cells. (A) Protein expression of core PRC2 components in the ARCaP_E and C4-2B models. (B) Western bot analysis on the expression of EZH2 and SUZ12 in IgG- and EED antibody-immunoprecipitates in C4-2B-TaxR cells treated with LG1980 or vehicle control (7 µM, 16 h). (C) Western bot analysis on the expression of p-EZH2, EZH2, SUZ12 and EED in C4-2B and C4-2B-TaxR cells treated with LG1980 (7 µM) at the indicated time points. Both β-actin and histone H3 were used as loading controls. (D) Left: Expression of EZH2 in C4-2B-TaxR cells treated with DMSO or LG1980 (7 µM) in the presence of CHX; Right: Calculated half-life of EZH2 protein in C4-2B-TaxR cells treated with DMSO or LG1980 (7 µM). (E) Western bot analysis on the expression of polyubiquitination in IgG- and EZH2 antibody-immunoprecipitates in C4-2B-TaxR cells treated with LG1980 or vehicle control (7 µM, 16 h). Arrow indicates the approximate size of EZH2 protein.

Figure 4

EZH2 regulates noncanonical Stat3-SKP2-ABCB1/survivin survival signaling in chemoresistant PCa cells. (A) Western blot analysis on the expression of H3K27 methylation in C4-2B-TaxR cells treated with LG1980 (7 µM) at the indicated time points. (B) Protein expression of p-Stat3, Stat3, SKP2, ABCB1 and survivin in the ARCaP_E and C4-2B models. (C) Left: Western blot analysis on the expression of EZH2 in C4-2B-TaxR cells transfected with control or EZH2 siRNA (60 nM, 72 h); Right: In vitro viability of C4-2B-TaxR cells transfected with control siRNA or EZH2 siRNA (60 nM, 72 h) and in the presence of varying concentrations of docetaxel. (D) Protein expression of EZH2, p-Stat3, SKP2, ABCB1 and survivin in C4-2B-TaxR cells transfected with control or EZH2 siRNA (60 nM, 72 h). (E) Western blot analysis on the expression of Stat3, SKP2, ABCB1 and survivin in C4-2B-TaxR cells transfected with control or Stat3 siRNA (72 h). (F) Western blot analysis on the expression of p-Stat3, Stat3, SKP2, ABCB1, survivin and p27 in C4-2B-TaxR cells transfected with control or SKP2 siRNA (60 nM, 72 h). (G) Western blot analysis on the expression of SKP2, ABCB1 and survivin in C4-2B cells transfected with SKP2 expression vector or control plasmid (72 h).

Figure 5

LG1980 inhibits the noncanonical EZH2-Stat3-SKP2-ABCB1/survivin signaling in chemoresistant PCa cells. (A) Western blot analysis of lysine methylation in the IgG- or Stat3 antibody-immunoprecipitates in C4-2B-TaxR cells treated with DMSO or LG1980 (7 µM, 24 h). (B) Protein expression of p-Stat3, Stat3, SKP2, ABCB1, survivin and p27 in C4-2B and C4-2B-TaxR cells following LG1980 treatment (7 µM) at the indicated time points. (C) Left: qPCR analysis of RNA expression of ABCB1 and survivin in C4-2B-TaxR cells treated with LG1980 (7 µM, 24 h). * p < 0.01. Middle and right: C4-2B-TaxR cells were pre-incubated with CHX (50 µg/mL, 2h) prior to the treatment with DMSO or LG1980 (7 µM) for the indicated times. Protein expression of ABCB1 and survivin was analyzed by Western blotting and quantitated using the ImageJ program. (D) A proposed mechanism of action of LG1980. In chemoresistant PCa cells, EZH2 phosphorylation initiates noncanonical signaling via the methylation and phosphorylation of non-histone substrate Stat3, thereby activating the expression of SKP2, ABCB1 and survivin and inhibiting p27. LG1980 binds EED, disrupts EED-EZH2 interaction and causes ubiquitin-mediated degradation of EZH2, thereby reducing p-EZH2 and suppressing Stat3-dependent survival signals. These events eventually induce apoptosis in chemoresistant PCa cells and sensitize them to chemotherapeutics. (E) Fluorescence microscopy images of cellular uptake of Oregon Green 488-paclitaxel in C4-2B-TaxR cells. Cells were first treated with LG1980 (7 µM) for 72 h prior to paclitaxel incubation for the indicated times. Scale bar: 50 µm.

Figure 6

In vitro and in vivo safety profile of LG1980. (A) In vitro effect of LG1980 on the activities of CYP450 3A4 and 2D6. (B) CCK-8 assay of the in vitro cytotoxicity of LG1980 in BPH-1 and RWPE-1 cells (72 h). (C) Left: Average body weights of healthy CD-1 mice treated with vehicle (n = 3), LG1980 (50 mg/kg, n = 3) or LG1980 (100 mg/kg, n = 4) via subcutaneous route, three times per week; Right: Percentage of mouse body weight change in different treatment groups.

Figure 7

As a monotherapy, LG1980 inhibits the in vivo growth of chemoresistant PCa and enhances docetaxel efficacy in athymic nude mice. (A) Left: serum PSA values of C4-2B-TaxR xenograft-bearing mice treated with vehicle (n = 4), docetaxel (5 mg/kg, i.p, once per week; n = 3), or LG1980 (20 mg/kg, i.p, three times per week; n = 5); Right: two-way ANOVA analysis of the PSA values between different treatment groups. ** p < 0.01, *** p < 0.001. (B) Representative x-ray radiography of tumor-bearing mouse tibias in different treatment groups. Red arrow: osteoblastic lesions; green arrow: osteolytic lesions. (C) IHC expression of p-EZH2(S21) in C4-2B-TaxR bone tumor tissues. Scale bar: 50 µm.

Figure 8

As an adjunct therapy, LG1980 synergistically enhances the in vivo efficacy of docetaxel against the skeletal growth of C4-2 tumors and the subcutaneous growth of LuCaP 23.1 tumors in athymic nude mice. (A) Left: In vitro cytotoxicity of LG1980 in C4-2 cells (72 h); Right: In vitro cytotoxicity of docetaxel in C4-2 cells in the presence of varying concentrations of LG1980 (72 h). (B) Western blot analysis of protein expression of SKP2 and survivin in C4-2 cells treated with LG1980 and docetaxel. (C) Left: serum PSA values of C4-2-Luc tumor-bearing mice treated with vehicle control (n = 5), docetaxel (5 mg/kg, i.p, once per week; n = 5), LG1980 (10 mg/kg, i.p, three times per week; n = 5), or the combination of docetaxel and LG1980 (n = 5). Right: two-way ANOVA analysis of the PSA values between different treatment groups. * p < 0.05, ** p < 0.01, *** p < 0.001. (D) Representative x-ray radiography of tumor-bearing mouse tibias in different treatment groups. Red arrow: osteoblastic lesions; green arrow: osteolytic lesions. (E) Left: serum PSA values of LuCaP 23.1 tumor-bearing mice treated with vehicle (n = 3), docetaxel (5 mg/kg, once per week; n = 5), LG1980 (20 mg/kg, three times per week; n = 5), or the combination of docetaxel and LG1980 (n = 6). Right: two-way ANOVA analysis of the PSA values between different groups. ** p < 0.01, *** p < 0.001.