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. 2016 Sep 1;22(17):4466-77.
doi: 10.1158/1078-0432.CCR-15-2901. Epub 2016 May 2.

Targeting Androgen Receptor Activation Function-1 With EPI to Overcome Resistance Mechanisms in Castration-Resistant Prostate Cancer

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Free PMC article

Targeting Androgen Receptor Activation Function-1 With EPI to Overcome Resistance Mechanisms in Castration-Resistant Prostate Cancer

Yu Chi Yang et al. Clin Cancer Res. .
Free PMC article

Abstract

Purpose: Persistent androgen receptor (AR) transcriptional activity is clinically evident in castration-resistant prostate cancer (CRPC). Therefore, AR remains as a viable therapeutic target for CRPC. All current hormonal therapies target the C-terminus ligand-binding domain (LBD) of AR. By using EPI to target AR activation function-1 (AF-1), in the N-terminal domain that is essential for AR transactivation, we evaluate the ability of EPI to overcome several clinically relevant AR-related mechanisms of resistance.

Experimental design: To study the effect of EPI on AR transcriptional activity against overexpressed coactivators, such as SRC1-3 and p300, luciferase reporter assays were performed using LNCaP cells. AR-negative COS-1 cells were employed for reporter assays to examine whether the length of polyglutamine tract affects inhibition by EPI. The effect of EPI on constitutively active AR splice variants was studied in LNCaP95 cells, which express AR-V7 variant. To evaluate the effect of EPI on the proliferation of LNCaP95 cells, we performed in vitro BrdUrd incorporation assay and in vivo studies using xenografts in mice.

Results: EPI effectively overcame several molecular alterations underlying aberrant AR activity, including overexpressed coactivators, AR gain-of-function mutations, and constitutively active AR-V7. EPI inhibited AR transcriptional activity regardless of the length of polyglutamine tract. Importantly, EPI significantly inhibited the in vitro and in vivo proliferation of LNCaP95 prostate cancer cells, which are androgen independent and enzalutamide resistant.

Conclusions: These findings support EPI as a promising therapeutic agent to treat CRPC, particularly against tumors driven by constitutively active AR splice variants that are resistant to LBD-targeting drugs. Clin Cancer Res; 22(17); 4466-77. ©2016 AACRSee related commentary by Sharp et al., p. 4280.

Conflict of interest statement

of Potential Conflicts of Interest: CAB, NRM, JW, SP, and MDS are shareholders of ESSA Pharma Inc. MDS and SP receive compensation as consultants. MDS is a Director and Officer of ESSA Pharma Inc. No potential conflicts of interest were disclosed for the other authors.

Figures

Figure 1
Figure 1. EPI overcomes aberrant AR transcriptional activity caused by overexpressed coactivators
(A) SRC1 and SRC3 show robust binding, while SRC2 shows relatively weak interactions with AR AF1. EPI-001 does not impair the binding of SRC1, 2 or 3. The inactive analogue compound 185-9-1 (B2H) was a control. The results are for two independent experiments measured in triplicate: SRC1 and SRC2 data was pooled (n=6), while SRC3 is a representative experiment (n=3). (B) EPI-001 does not block physical interaction between endogenous AR and SRC1 in LNCaP cells exposed to R1881 or vehicle (Ethanol). IP: immunoprecipitation; WB: western blotting. (C) LNCaP cells were co-transfected with PSA-luciferase reporter (PSA6.1-LUC) and coactivators. Cells were pretreated with vehicle, 10μM enzalutamide (ENZ), or 25μM EPI (EPI-002) for 1 hr before the treatment of 1nM R1881 for 48 hr. Luciferase activities were normalized to vehicle control treatment for SRC1, SRC2, SRC3, and (D) p300. Percentage of inhibition by treatments was plotted using R1881-treatment for normalization. Coactivator overexpression in LNCaP cells was confirmed by western blot analyses using specific antibodies with β-actin as a loading control. For the reporter assays, bar graphs are mean ± SEM with n ≥ 3 independent experiments.
Figure 2
Figure 2. EPI inhibits polymorphic AR NTD with variable lengths of polyglutamine tract
COS-1 cells were co-transfected with PB-luciferase reporter and expression vectors containing AR with different lengths of polyglutamine tract. Cells were pretreated with vehicle, 10μM enzalutamide (ENZ), or 25μM EPI (EPI-002) for 1 hr prior to treatment of 1 nM R1881 for 24 hr. (A) Luciferase activities for ARs were plotted in percentage of activation using vehicle-R1881 treatment as normalization. (B) Percentage of inhibition by treatments was calculated based on vehicle-R1881 treatment and plotted. (C) R1881-induced PB-LUC activity under vehicle treatment was plotted for each AR. (D) The expression levels of ARs transfected in COS-1 cells were compared to LNCaP endogenous levels of AR by western blot using AR-N20. β-actin was a loading control. Bar graphs are mean ± SEM with n ≥ 3 independent experiments. One-way ANOVA compared the treatment groups to vehicle-R1881 control in (A), and ARs with variable length of polyglutamine tract in (C); *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 3
Figure 3. EPI blocks the transcriptional activity of ARs with clinically relevant gain-of-function mutations
COS-1 cells were co-transfected with PB-luciferase reporter and expression vectors containing wild-type (WT) AR and mutant AR. Cells were pretreated with vehicle, 10μM enzalutamide (ENZ), 10μM bicalutamide (BIC), or 10μM hydroxyflutamide (FLU), or 25μM EPI (EPI-002) for 1 hr prior to treatment of 1 nM R1881 or ethanol for 24 hr. (A) Luciferase activities were measured, and values were plotted after normalized to vehicle-ethanol treatment. (B) Percentage of inhibition by treatments was calculated based on vehicle-R1881 treatment and plotted. Bar graphs are mean ± SEM with n ≥ 3 independent experiments. One-way ANOVA compared treatment groups with vehicle controls in (A), and unpaired Student’s t test was used to compare EPI with ENZA in (B); *p < 0.05; **p < 0.01; ***p < 0.001. (C) Protein levels of ectopic expression of WT AR and AR mutants in COS-1 cells compared to endogenous levels of AR in LNCaP cells were shown in a representative western blot using antiAR N-20 antibody. β-actin was used as a loading control.
Figure 4
Figure 4. EPI inhibits transcriptional activity of full-length AR and AR-V7 in LNCaP95 cells
(A) LNCaP95 cells were transfected with AR-driven luciferase reporter PSA6.1-LUC and pretreated with vehicle, 10μM enzalutamide (ENZ) or bicalutamide (BIC), or 25μM EPI (EPI-002) for 1 hr prior to addition of 1 nM R1881 for 48 hr. Luciferase activities were measured, and values were plotted after normalized to vehicle-ethanol treatment. (B–E) LNCaP95 cells were treated the same as above. Levels of mRNA were measured and quantified for (B) canonical AR-regulated genes PSA and FKBP5, (C) AR variant-regulated genes UBE2C and CDC20, and (D) full-length (FL) AR and AR-V7. Levels of expression for each gene were normalized to mRNA levels of RPL13A. (E) AR protein levels measured by western blot using AR-N20 antibody and β-actin as a loading control. Bar graphs are mean ± SEM with n ≥ 3 independent experiments. One-way ANOVA comparing treatment groups to vehicle controls; *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 5
Figure 5. EPI inhibits the growth of CRPC driven by AR variants
(A) LNCaP95 cells were pretreated with vehicle, 10μM bicalutamide (BIC), 10μM enzalutamide (ENZ), or 25μM EPI (EPI-002) for 1 hr prior to addition of 0.1 nM R1881 for 2 days. Proliferation was measured by bromodeoxyuridine (BrdU) incorporation. Bar graphs are mean ± SEM with n ≥ 3 independent experiments. Two-way ANOVA comparing treatment groups to vehicle controls; ****p < 0.0001. (B) LNCaP95 tumor growth in castrated mice orally administered EPI (EPI-002) (n=4; 100mg/kg body weight, b.i.d.), enzalutamide (n=3; 50 mg/kg body weight, q.d.) or vehicle control (n=3; CMC/DMSO, q.d.) for a total of 26 doses. (C) Final tumor volume on day 27 prior to harvesting. (D) Photographs of representative xenografts harvested on day 27. The white scale bar represents 1 cm. (E) Transcript levels of full-length (FL)-AR, AR-V7, PSA, UBE2C, FKBP5, and UGT2B17 normalized to RPL13A using total RNA isolated from the above LNCaP95 xenografts harvested on day 27. (F) Intra-tumoral steroid levels as determined by mass spectroscopy, values are mean +/− SD in three tumors for each treatment. A significant decrease of testosterone and dihydrotestosterone following castration, p < 0.001, with no effects of EPI or enzalutamide on castrate steroid levels. There were no changes in dehydroepiandosterone (DHEA), androsterone, or 5-androstenedione (androst-5-ene-3,17-dione, or AED) pre- or post-castration.
Figure 6
Figure 6. EPI reduces proliferation and increases apoptosis in LNCaP95 xenografts
(A) Representative xenograft tumors stained for hematoxylin and eosin (HE), AR, Ki67 and TUNEL. Scale bars (red) indicate 20 μm. (B) % of Ki67 and % of TUNEL positive cells were counted in xenograft sections of each treatment. Total number of cells counted: 2361 (Control, Ki67), 2658 (EPI-002, Ki67), 2409 (ENZA, Ki67), 1209 (Control, TUNEL), 1210 (EPI-002, TUNEL) and 1137 (ENZA, TUNEL). Bar graphs are mean ± SEM with n = 3 different xenograft sections. One-way ANOVA comparing treatment groups to control; *p < 0.05; **p < 0.01.

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