miR-124 and Androgen Receptor Signaling Inhibitors Repress Prostate Cancer Growth by Downregulating Androgen Receptor Splice Variants, EZH2, and Src

Abstract

miR-124 targets the androgen receptor (AR) transcript, acting as a tumor suppressor to broadly limit the growth of prostate cancer. In this study, we unraveled the mechanisms through which miR-124 acts in this setting. miR-124 inhibited proliferation of prostate cancer cells in vitro and sensitized them to inhibitors of androgen receptor signaling. Notably, miR-124 could restore the apoptotic response of cells resistant to enzalutamide, a drug approved for the treatment of castration-resistant prostate cancer. We used xenograft models to examine the effects of miR-124 in vivo when complexed with polyethylenimine-derived nanoparticles. Intravenous delivery of miR-124 was sufficient to inhibit tumor growth and to increase tumor cell apoptosis in combination with enzalutamide. Mechanistic investigations revealed that miR-124 directly downregulated AR splice variants AR-V4 and V7 along with EZH2 and Src, oncogenic targets that have been reported to contribute to prostate cancer progression and treatment resistance. Taken together, our results offer a preclinical rationale to evaluate miR-124 for cancer treatment.

©2015 American Association for Cancer Research.

Figures

Figure 1. WST-1 analyses of proliferation of CaP cells

22Rv1 cells (A) and C4-2B cells (B) grown in androgen-deprived medium, and LNCaP cells (C) grown in FBS medium, were treated with miR-124 and enzalutamide (Enz.), alone or in combination. (D) C4-2B cells grown in androgen-deprived medium were treated with miR-124 and bicalutamide (Bic), alone or in combination. These experiments were repeated three times with similar results obtained each time. The representative results are shown as Mean ± SD (n = 3). The bars represent SDs. untreat., untreated; miR-NC, miRNA-negative control; both, combination of miR-124 and enzalutamide.

Figure 2

The inhibitory effect of miR-124 on colony formation in enzalutamide-resistant 22Rv1 (22Rv1-EnzR) cells. (A) Influence of miR-124 on colony-forming cells, as evaluated by quantitative clonogenic assay. (B) Representative dishes are shown for colony-forming assays of 22Rv1 cells (left panel) and 22Rv1-EnzR cells (right panel). (C) miR-124 decreases the resistance of 22Rv1-EnzR cells to enzalutamide (Enz.). The clonogenic assays were repeated three times with similar results obtained each time. The results are shown as M ± SE (n = 3). *, p < 0.01, miR-124 treatment versus miRNA-negative control (miR-NC) treatment. untreat., untreated.

Figure 3

Validation of EZH2 and Src as miR-124 targets. (A) & (B) Luciferase reporter assay analyses of the 3’UTRs of EZH2 and Src in C4-2B cells. The top sequences are the miR-124 seed sequence and the predicted miR-124-binding sites in the EZH2 and Src 3′UTR target regions. The 3’UTRs of EZH2 and Src lacking the miR-124 binding site were used as controls in these experiments. The assays were repeated three times with each assay being performed in triplicate wells and similar results being obtained each time. Representative results are shown as Mean ± SD (n = 3). RLU, relative luciferase units. MBS, miR-124-binding site. miR-NC, miRNA-negative control. The numbers (25 and 50) are the concentrations (nM) of miR-124 used in these assays. (C) and (D) Western blot analyses of EZH2 and Src expression levels in C4-2B cells, treated and untreated controls.

Figure 4

Validation of AR-V4 and AR-V7 as miR-124 targets. (A) West blot analysis of AR-V7 expression level in 22Rv1 and enzalutamide-resistant 22Rv1 (22Rv1-EnzR) cells. (B) Top: Map of AR-V3, -V4 & -V7 3’UTRs (black) with exon 2 (E2) and/or exon 3 (E3) (white) plus an identical miR-124-binding site in their 3′UTRs and the miR-124 seed sequence. TGA is the stop condon. Bottom: Luciferase reporter assay analysis of the 3’UTR of AR-V7 in 22Rv1 cells. The assay was repeated three times with each assay being performed in three wells and similar results were obtained each time. Representative results are shown as Mean ± SD (n = 3). RLU, relative luciferase units. MBS, miR-124-binding site. miR-NC, miRNA-negative control. (C) Western blot analysis of AR-V7 expression in miR-124-treated 22Rv1 cells. Enz., enzalutamide. (D) Western blot analysis of AR-V7 expression in siAR-V7 (siV7)-treated 22Rv1 and VCaP cells. (E) WST proliferation analyses of 22Rv1 and VCaP cell lines that were treated with siAR-V7 for 3 days, followed by treatment with 50 nM miR-124.

Figure 5

Systemic administration of synthetic miR-124 increases the inhibitory efficacy of enzalutamide on growth of CaP xenografts. (A) Nude mice each were injected s.c. with CWR22 cell suspension (~2×106 cells). When tumor volumes reached ~50mm3, treatment was conducted by i.v. injection of 10 μg jetPEI/miR-NC or 10 μg jetPEI/miR-124 complexes (thrice weekly for 5 weeks), or by oral administration of enzalutamide (20 mg/kg/week, once weekly for 5 weeks). Tumor sizes were measured and tumor growth curves were obtained. Each time point represents mean ± SD of 8 independent values. (B) The representative results of mice treated with miR-124 and enzalutamide (Enz.), alone or in combination.

Figure 6. Systemic administration of miR-124 downregulates its targets and induces apoptosis

(A) The protein levels of AR-V7, EZH2 and Src, as well as p53, were detected by Western blot analysis in five enzalutamide (Enz.)-treated tumors (left panel) and five miR-124/enzalutamide-treated tumors (right panel). β-actin was used as a loading control. B) TUNEL assays of apoptosis in tumor sections. Apoptosis was measured by TUNEL staining in CaP tumors treated with miR-124 and enzalutamide, alone or in combination. The left panel demonstrates the representative positive TUNEL staining. For quantitation of apoptosis, TUNEL-positive areas were measured using the ImageJ program in three randomly chosen fields from three tumors and expressed as % apoptosis (mean ± SE, n=3) (right).

Figure 7. Schematic model of miR-124-regulated oncopathway in CaP cells

Data presented in this study and in our previous publications support a miR-124-AR/AR-Vs-miR-125b oncopathway in CaP cancer cells.