Androgen receptor splice variants mediate enzalutamide resistance in castration-resistant prostate cancer cell lines

Abstract

Persistent androgen receptor (AR) transcriptional activity underlies resistance to AR-targeted therapy and progression to lethal castration-resistant prostate cancer (CRPC). Recent success in retargeting persistent AR activity with next generation androgen/AR axis inhibitors such as enzalutamide (MDV3100) has validated AR as a master regulator during all stages of disease progression. However, resistance to next generation AR inhibitors limits therapeutic efficacy for many patients. One emerging mechanism of CRPC progression is AR gene rearrangement, promoting synthesis of constitutively active truncated AR splice variants (AR-V) that lack the AR ligand-binding domain. In this study, we show that cells with AR gene rearrangements expressing both full-length and AR-Vs are androgen independent and enzalutamide resistant. However, selective knock-down of AR-V expression inhibited androgen-independent growth and restored responsiveness to androgens and antiandrogens. In heterogeneous cell populations, AR gene rearrangements marked individual AR-V-dependent cells that were resistant to enzalutamide. Gene expression profiling following knock-down of full-length AR or AR-Vs showed that AR-Vs drive resistance to AR-targeted therapy by functioning as constitutive and independent effectors of the androgen/AR transcriptional program. Further, mitotic genes deemed previously to be unique AR-V targets were found to be biphasic targets associated with a proliferative level of signaling output from either AR-Vs or androgen-stimulated AR. Overall, these studies highlight AR-Vs as key mediators of persistent AR signaling and resistance to the current arsenal of conventional and next generation AR-directed therapies, advancing the concept of AR-Vs as therapeutic targets in advanced disease.

Figure 1

AR-Vs support resistance to full-length AR targeting in 22Rv1 cells. (A) 22Rv1 cells were cultured under castrate (CSS) conditions with 10μM bicalutamide (bic) or 1μM enzalutamide (enz). (B) MMTV promoter activities in siRNA-transfected cells treated under castrate conditions with 1nM DHT, 10μM bicalutamide, or 1μM enzalutamide. Data represent mean +/− S.E. from at least three independent experiments, each performed in duplicate. Inset: Western blot with antibodies targeted to the AR NTD or an internal control (ERK-2). Locations of full-length AR and truncated AR-Vs are indicated. (C) 22Rv1 cells were transfected with siRNAs under castrate conditions. Gene expression was assessed by quantitative RT-PCR. Bars represent mean +/− S.D. from two biological replicates, each performed in duplicate. Western blots were performed as in (C). (D) 22Rv1 cells were transfected and treated as in (B). Growth was assessed at indicated time-points. Data represent mean +/− S.D. from a quadruplicate experiment representative of two biological replicates. Inset: Western blots were performed as in (B).

Figure 2

Rearrangement-positive CWR-R1 cells are resistant to full-length AR targeting. (A) Schematic of the AR locus with location of PCR primers for deletion analysis. (B) CWR-R1 single-cell clones were assessed for AR-V expression by Western blot using an antibody specific for the AR NTD. Concurrently, genomic DNA was isolated and subjected to deletion-specific PCR. (C) Deletion-negative clones were cultured under castrate conditions with 1nM DHT, 10μM bicalutamide, or 1μM enzalutamide. Growth was assessed at indicated time-points. Data represent mean +/− S.D. from a quadruplicate experiment representative of two biological replicates. (D) Deletion-positive clones were cultured, treated, and subjected to growth assays as in (C). Deletion-positive clones were further transfected with siRNAs and subjected to growth assays at days 0, 4, and 6. Data represent mean +/− S.D. from a quadruplicate experiment representative of two biological replicates.

Figure 3

AR-Vs support the androgen/AR transcriptional program. (A) CWR-R1 cells transfected with siRNAs specific for full-length and/or truncated AR-Vs were treated with 1nM DHT under castrate conditions. Western blots were performed with antibodies specific for the AR NTD or a loading control (ERK-2). Two additional biological replicates are provided in Supplementary Fig. 5. (B) Heat-map of the androgen/AR gene expression program (left two columns) with comparison of the responses of these genes to AR-V activity (right two columns). Androgen/AR targets are defined as those genes demonstrating differential expression in variant knock-down cells (siAR exon CE3) treated with DHT vs. vehicle control. (C) Heat-map of the AR-V gene expression program (left two columns) with comparison of the responses of these genes to androgen/AR activity (right two columns). AR-V targets are defined as those genes demonstrating differential expression in cells transfected with siRNA targeting AR exon 7 vs. AR exon 1. (D) Gene set enrichment analysis (GSEA) of “AR-V-specific” (18) or “full-length AR-specific” (18) gene signatures in gene expression datasets supported by AR-Vs (top) or androgen/AR (bottom).

Figure 4

M-phase cell cycle genes display a biphasic response to both androgen/AR signaling and AR-V signaling. (A) Gene set enrichment analysis (GSEA) of an AR-V-specific gene signatures in gene expression datasets derived from LNCaP cells treated with 1nM DHT (GSE26483, left) vs. 100nM DHT (GSE7868, right). (B) LNCaP cells were treated with increasing concentrations of androgens and subjected to quantitative RT-PCR for indicated genes. Bars represent mean +/− S.D. from a triplicate experiment representative of two biological replicates. Western blots were performed using antibodies specific for the AR NTD or loading control (ERK-2). (C) LNCaP cells were infected with increasing titers of lentivirus encoding AR 1/2/3/CE3. RNA and protein analysis was performed as in (B). LNCaP cells were infected with increasing titers of lentivirus encoding AR Δ5/6/7. RNA and protein analysis was performed as in (B).