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. Cancer Res; 73(2); 483–9. ©2012 AACR.

Androgen depletion therapy (ADT) achieves clinical regression or disease stabilization for men with advanced prostate cancer (PCa). However, castration-resistant or -recurrent prostate cancer (CRPC) invariably develops due to aberrant reactivation of the androgen/androgen receptor (AR) signaling axis (1). This has been confirmed clinically through the recent retargeting of persistent androgen/AR activity in CRPC patients with next generation AR-axis inhibitors such as the potent antiandrogen enzalutamide (2, 3). Despite an increase in overall survival, de novo resistance to enzalutamide precludes responses for many patients, and resistance can develop rapidly in initial responders (4). However, mechanisms that could mediate resistance to enzalutamide are poorly understood.

Altered AR mRNA splicing and synthesis of COOH-terminally truncated AR variant (AR-V) proteins lacking the AR ligand-binding domain (LBD) is one mechanism that has been postulated to drive an overall resistance to conventional and next generation ADT (5). Indeed, the transcriptionally active NH2-terminal domain (NTD) and central DNA binding domain are sufficient for AR-Vs to function as ligand-independent transcription factors (6). Moreover, AR-Vs are frequently expressed in CRPC metastases (7, 8), and high mRNA and/or protein expression levels in PCa tissues predict disease progression and shorter survival (7, 9, 10). However, despite being able to induce a CRPC growth phenotype, ectopically expressed AR-Vs were shown to remain dependent on activity of endogenous full-length AR (11). Therefore, it has been concluded that constitutive AR-V activity in CRPC tissues could be overcome by targeting full-length AR with antiandrogens.

Recent studies showing AR gene rearrangements in CRPC that underlie functional AR-V expression and activity have revealed scenarios where full-length AR activity may not be required in CRPC cells (12, 13). Foremost, the LuCaP 86.2 xenograft derived from CRPC bladder metastasis harbors an 8.5-kb intragenic deletion of AR exons 5 to 7, which prevents full-length AR synthesis but favors expression of a truncated AR-V species encoded by mRNA lacking exons 5 to 7 (12). The 22Rv1 and CWR-R1 models of CRPC also harbor underlying AR gene rearrangements, leading to coexpression of full-length AR and AR-Vs (12, 13). The effects of targeting full-length AR on AR-V function have not been evaluated in these rearrangement-driven models. Therefore, the purpose of this study was to test the roles of full-length and AR-V species in supporting the CRPC phenotype and mediating responsiveness to enzalutamide in the context of rearrangement-driven changes in AR splicing.

Cell culture

The 22Rv1 (#CRL-2505) and LNCaP (#CRL-1740) cell lines were obtained from the American Type Culture Collection (ATCC) and cultured according to the ATCC protocol. The ATCC ensures authenticity of these human cell lines using short tandem repeat (STR) analyses. All experiments with these cells were conducted within 4 months of resuscitation of frozen cell stocks prepared within 3 passages of receipt from the ATCC. CWR-R1 cells (14) were a kind gift from Dr. Elizabeth Wilson (University of North Carolina at Chapel Hill, Chapel Hill, NC) and cultured in RPMI-1640 with 10% FBS. Authentication of the CWR-R1 cell line was conducted by sequence-based validation of 2 signature AR gene alterations: an AR H874Y point mutation and a 50-kb intragenic deletion within AR intron 1 (13). Sequence-based authentication of CWR-R1 was carried out every 5 to 10 passages, and cells were kept in culture no longer than 3 months after authentication unless otherwise indicated. Details of cell treatment with bicalutamide (AstraZeneca) or enzalutamide (a kind gift from Dr. Michael Jung, University of California, Los Angeles, Los Angeles, CA) are provided in the Supplementary Material.

Transient transfections

Cells were electroporated with siRNAs targeted to AR Exon 7 (15), AR Exon 1 (15), AR Exon 2b (15), AR Exon CE3 (10), or a mouse mammary tumor virus-luciferase (MMTV-LUC) reporter as described previously (15). Growth of electroporated cells was monitored by crystal violet staining as described earlier (12). Luciferase activity was measured as previously described (15).

Lentiviral infections

LNCaP cells were infected with increasing titers of lentivirus encoding AR 1/2/3/CE3 and AR Δ5/6/7 as described previously (6). Infected cells were maintained in RPMI1640 with 10% charcoal-stripped, steroid-depleted serum (CSS) for 48 hours and then switched to serum-free medium for 24 hours before lysis.

Western blot

Western blotting with AR NTD (N-20; Santa Cruz Biotechnology), AR CTD (C-19; Santa Cruz Biotechnology), and ERK-2 (D-2; Santa Cruz Biotechnology) antibodies was carried out as described previously (12).

Quantitative reverse transcription-PCR

Total RNA was extracted from 22Rv1, CWR-R1, and LNCaP cells as described previously (16). Primers and quantitative reverse transcription PCR (qRT-PCR) conditions for assessment of prostate specific antigen (PSA), hK2, and TMPRSS2 mRNA expression have been described previously (15). Androgen- and AR variant-responses of M–phase-specific genes were assessed using specific primers with sequences provided in Supplementary Material. Fold change in mRNA expression levels was calculated by the comparative Ct method, using the formula 2−(ΔΔCt) and GAPDH as calibrator, as described previously (15).

Genomic PCR

Primers and PCR conditions for deletion-spanning PCR of the AR intron 1 deletion in CWR-R1 cells have been described previously (13).

Gene expression analysis with Illumina Beadchips

CWR-R1 cells that had been maintained in long-term culture in RPMI1640 with 10% CSS were used for global gene expression profiling. These CWR-R1 cells were electroporated with siRNAs targeting AR exon 1, 7, or CE3, and seeded in RPMI with 10% CSS. Following 48-hour recovery, cells were switched to serum-free RPMI1640 and treated for 24 hours with 1 nmol/L dihydrotestosterone (DHT) or 0.01% (v/v) vehicle control (ethanol). Details of RNA isolation, Illumina Beadchip hybridization, and data analysis are provided as Supplementary Material. Data are available through NCBI's Gene Expression Omnibus (GSE41784).

AR-Vs are sufficient for resistance to enzalutamide in 22Rv1 cells

The CRPC 22Rv1 cell line is characterized by a 35-kb tandem duplication encompassing AR exon 3 (12). This rearrangement is associated with enhanced mRNA and protein expression of truncated AR-Vs AR 1/2/3/2b and AR 1/2/3/CE3 (also referred to as AR-V7/AR3; refs. 6, 9, 10, 12). The 22Rv1 cells display robust growth under castrated conditions, which was unaffected by the antiandrogens bicalutamide or enzalutamide (Fig. 1A). However, both bicalutamide and enzalutamide were able to antagonize androgen-mediated activation of the AR target genes PSA and hK2 (Supplementary Fig. S1A) as well as an AR-responsive MMTV-LUC reporter (Fig. 1B), showing that these drugs can achieve on-target inhibition of full-length AR in these cells. Similarly, androgen-induced MMTV activity was blocked following selective knockdown of full-length AR (Fig. 1B). However, knockdown of AR-Vs resulted in robust inhibition of constitutive, androgen-independent MMTV-LUC activity. Similarly, constitutive, androgen-independent expression of PSA and hK2 was blocked by AR-V knockdown, but not by manipulations that block full-length AR (bicalutamide, enzalutamide, full-length AR knockdown, or AR knockdown combined with antiandrogens, Fig. 1C). Interestingly, constitutive AR-V activity appeared to maintain TMPRSS2 expression at a maximal AR-inducible level, as there was no response to androgens (Supplementary Fig. S1B), but expression was inhibited by AR-V knockdown (Fig. 1C). Together, these data show that AR-Vs are independent effectors of constitutive AR transcriptional activity in these cells. To test the biological implications of this, we assessed the effects of androgens and antiandrogens on the growth of 22Rv1 cells under conditions of full-length versus AR-V knockdown. Remarkably, knockdown of AR-Vs, but not full-length AR, reduced the androgen-independent growth rate of these cells and restored robust growth-responsiveness to androgens (Fig. 1D). Perhaps more importantly, AR-V knockdown restored the ability of antiandrogens to inhibit this newly acquired androgen-dependent growth phenotype (Fig. 1D). On the basis of this finding, we conclude that AR-Vs are sufficient for resistance of 22Rv1 cells to therapies targeting full-length AR, including enzalutamide.

Figure 1.

AR-Vs support resistance to full-length AR targeting in 22Rv1 cells. A, 22Rv1 cells were cultured under castrate (CSS) conditions with 10 μmol/L bicalutamide (bic) or 1 μmol/L enzalutamide (enz). B, MMTV promoter activities in siRNA-transfected cells treated under castrate conditions with 1 nmol/L DHT, 10 μmol/L bicalutamide, or 1 μmol/L enzalutamide. Data represent mean ± SE from at least 3 independent experiments, each conducted 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 ± SD from 2 biological replicates, each carried out in duplicate. Western blots were carried out as in B. D, 22Rv1 cells were transfected and treated as in B. Growth was assessed at indicated time-points. Data represent mean ± SD from a quadruplicate experiment representative of 2 biological replicates. Inset, Western blots were carried out as in B.

Figure 1.

AR-Vs support resistance to full-length AR targeting in 22Rv1 cells. A, 22Rv1 cells were cultured under castrate (CSS) conditions with 10 μmol/L bicalutamide (bic) or 1 μmol/L enzalutamide (enz). B, MMTV promoter activities in siRNA-transfected cells treated under castrate conditions with 1 nmol/L DHT, 10 μmol/L bicalutamide, or 1 μmol/L enzalutamide. Data represent mean ± SE from at least 3 independent experiments, each conducted 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 ± SD from 2 biological replicates, each carried out in duplicate. Western blots were carried out as in B. D, 22Rv1 cells were transfected and treated as in B. Growth was assessed at indicated time-points. Data represent mean ± SD from a quadruplicate experiment representative of 2 biological replicates. Inset, Western blots were carried out as in B.

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AR gene rearrangements mark AR-V-driven, enzalutamide-resistant cells in heterogenous PCa cell populations

Recently, we discovered a 48-kb AR intron 1 deletion in a subset of cells in the heterogeneous CWR-R1 cell line (ref. 13; Fig. 2A). Single-cell cloning revealed that cells positive for the 48-kb deletion displayed high-level expression of the AR 1/2/3/CE3 variant (Fig. 2B). Conversely, cells that were negative for the 48-kb deletion expressed predominantly full-length AR (Fig. 2B). Sub-clones negative for the 48-kb AR intragenic deletion displayed a basal level of androgen-independent growth, which was enhanced by DHT (Fig. 2C). This basal level of androgen-independent growth was reduced by treatment with bicalutamide or enzalutamide, indicating that full-length AR was required (Fig. 2C). Conversely, sub-clones positive for the 48-kb AR intragenic deletion displayed rapid androgen-independent growth, which was unaffected by androgens (Supplementary Fig. S2) or antiandrogens (Fig. 2D). However, selective knockdown of AR 1/2/3/CE3 inhibited androgen-independent growth of these rearrangement-positive cells (Fig. 2D).

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 analysis 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 1 nmol/L DHT, 10 μmol/L bicalutamide, or 1 μmol/L enzalutamide. Growth was assessed at indicated time-points. Data represent mean ± SD from a quadruplicate experiment representative of 2 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 ± SD from a quadruplicate experiment representative of 2 biological replicates.

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 analysis 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 1 nmol/L DHT, 10 μmol/L bicalutamide, or 1 μmol/L enzalutamide. Growth was assessed at indicated time-points. Data represent mean ± SD from a quadruplicate experiment representative of 2 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 ± SD from a quadruplicate experiment representative of 2 biological replicates.

Close modal

To verify that this property of enzalutamide resistance was not restricted to a few rare cells, we tested the effects of antiandrogens on a version of the CWR-R1 cell line that had been propagated for the long term under castrated conditions. We have previously shown that long-term castration enriches for the AR intron 1 deletion-positive population (13), which AR gene copy number analysis showed was approximately 97% of the population (Supplementary Fig. S3A). Similar to 22Rv1 cells, androgen-independent growth of deletion-enriched CWR-R1 cells was insensitive to bicalutamide and enzalutamide (Supplementary Fig. S3B). Nevertheless, antiandrogens were able to achieve on-target activity and inhibit androgen-induced MMTV-LUC activation (Supplementary Fig. S3C). However, only AR-V knockdown was able to inhibit constitutive, androgen-independent MMTV activity (Supplementary Fig. S3C). This is in line with our previous work showing that androgen-independent growth of deletion-enriched CWR-R1 cells is not affected by knockdown of full-length AR, but can be blocked by knockdown of AR 1/2/3/CE3 (13). Therefore, we conclude that the 48-kb AR intron 1 deletion can discriminate between individual cells in the heterogeneous CWR-R1 cell line that are enzalutamide-responsive and cells that are driven by AR-V activity and resistant to inhibition of full-length AR.

AR-Vs are independent effectors of the androgen/AR transcriptional program

AR-Vs have been reported to induce unique transcriptional targets such as AKT1 (9), which may play a role in enzalutamide resistance (17). However, in AR intron 1 deletion-enriched CWR-R1 cells, we did not observe any changes in AKT1 expression following AR 1/2/3/CE3 knockdown (Supplementary Fig. S4). Therefore, to understand the mechanistic basis for AR-V–mediated resistance to enzalutamide, we carried out gene expression profiling of deletion-enriched CWR-R1 cells. Because constitutive activity of AR-Vs can mask androgen/AR induction targets (and vice versa) we assessed the androgen/AR transcriptional program following AR 1/2/3/CE3 knockdown and assessed the AR-V transcriptional program following full-length AR knockdown (Fig. 3A and Supplementary Fig. S5). Many, but not all, of the genes responsive to androgen/AR activity were similarly activated/repressed in a constitutive manner by AR 1/2/3/CE3 in these cells (Fig. 3B). This suggested that the AR-V transcriptional program represented a subset of the broader androgen/AR transcriptional program. Indeed, when we focused on AR-V responsive genes, nearly all were regulated in the exact same manner by androgen/AR activity (Fig. 3C). These data confirm that AR-Vs are constitutive and independent effectors of the AR transcriptional program, which explains why androgens and AR-Vs can support maximal growth of the same cell line in an interchangeable fashion (Fig. 1D). In line with this idea, the only knowledge-based multi-gene signaling networks identified as being associated with androgen/AR and AR-V gene signatures had AR as the prominent central hub (Supplementary Fig. S6).

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 1 nmol/L DHT under castrate conditions. Western blots were carried out with antibodies specific for the AR NTD or a loading control (ERK-2). Two additional biological replicates are provided in Supplementary Fig. S5. B, heat-map of the androgen/AR gene expression program (left 2 columns) with comparison of the responses of these genes to AR-V activity (right 2 columns). Androgen/AR targets are defined as those genes showing differential expression in variant knockdown cells (siAR exon CE3) treated with DHT versus vehicle control. C, heat-map of the AR-V gene expression program (left 2 columns) with comparison of the responses of these genes to androgen/AR activity (right 2 columns). AR-V targets are defined as those genes showing differential expression in cells transfected with siRNA targeting AR exon 7 versus AR exon 1. D, gene set enrichment analysis 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 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 1 nmol/L DHT under castrate conditions. Western blots were carried out with antibodies specific for the AR NTD or a loading control (ERK-2). Two additional biological replicates are provided in Supplementary Fig. S5. B, heat-map of the androgen/AR gene expression program (left 2 columns) with comparison of the responses of these genes to AR-V activity (right 2 columns). Androgen/AR targets are defined as those genes showing differential expression in variant knockdown cells (siAR exon CE3) treated with DHT versus vehicle control. C, heat-map of the AR-V gene expression program (left 2 columns) with comparison of the responses of these genes to androgen/AR activity (right 2 columns). AR-V targets are defined as those genes showing differential expression in cells transfected with siRNA targeting AR exon 7 versus AR exon 1. D, gene set enrichment analysis 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).

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These findings are in opposition to a recent study showing that AR-Vs have gene signatures distinct from full-length AR, including a set of genes involved in M-phase cell-cycle progression (18). To understand the basis for this discrepancy, we used gene set enrichment analysis (GSEA; ref. 19) to test the response of this AR-V-specific set of M-phase genes (18) in CWR-R1 cells. This AR-V-responsive M-phase gene set was positively enriched in both androgen/AR and AR-V gene expression datasets derived from CWR-R1 cells (Fig. 3D). Similarly, a gene set deemed to be full-length AR-specific (18) was positively enriched in both of these CWR-R1–derived gene expression datasets (Fig. 3D). Therefore, these signatures could not discriminate between AR-V and androgen/AR activity in CWR-R1 cells.

AR-Vs have been shown to drive biphasic AR signaling in a manner similar to androgens (6). Therefore, we hypothesized that differences previously noted between AR-V and full-length AR transcriptional programs could have risen from comparing different strengths of AR transcriptional output from AR-Vs compared with androgens. To test this, we carried out GSEA with gene expression datasets derived from LNCaP cells treated with 1 nmol/L DHT (a pro-proliferative dose) or 100 nmol/L DHT (an antiproliferative dose). As expected, the full-length AR signature displayed positive enrichment in both the 1 nmol/L DHT and 100 nmol/L DHT gene expression datasets (Supplementary Fig. S7). Conversely, the AR-V-specific signature displayed positive enrichment in the 1 nmol/L DHT dataset, but strong negative enrichment in the 100 nmol/L DHT dataset (Fig. 4A). Therefore, these data indicate that the AR-V-specific signature does not discriminate between AR-V and full-length AR signaling, but rather reflects proliferative versus growth-suppressive levels of AR signaling output. To test this further, we treated LNCaP cells with androgens at concentrations that cover the range of proliferative and growth suppressive doses (0.1–100 nmol/L DHT) and assessed expression of M-phase genes UBE2C, CDCA5, ZWINT, and CCNA2. Whereas PSA expression increased concomitant with increasing androgen concentration, all of the M–phase-specific genes displayed a biphasic response: induction at low androgen concentrations and/or repression at higher doses (Fig. 4B). Similarly, when increasing titers of lentivirus encoding the AR 1/2/3/CE3 (AR-V7/AR3) variant (Fig. 4C) or the AR Δ5/6/7 (ARv567es) variant (Fig. 4D) were used for infection, similar biphasic responses were observed for M–phase-specific genes, but not PSA. Therefore, these data challenge the notion that AR-Vs have acquired unique transcriptional targets and provide strong support for the concept that AR-Vs are independent effectors of the androgen/AR transcriptional program.

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 of AR-V–specific gene signatures in gene expression datasets derived from LNCaP cells treated with 1 nmol/L DHT (GSE26483, left) versus 100 nmol/L 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 ± SD from a triplicate experiment representative of 2 biological replicates. Western blots were carried out 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 analyses were carried out as in B. D, LNCaP cells were infected with increasing titers of lentivirus encoding AR Δ5/6/7. RNA and protein analyses were carried out as in B.

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 of AR-V–specific gene signatures in gene expression datasets derived from LNCaP cells treated with 1 nmol/L DHT (GSE26483, left) versus 100 nmol/L 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 ± SD from a triplicate experiment representative of 2 biological replicates. Western blots were carried out 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 analyses were carried out as in B. D, LNCaP cells were infected with increasing titers of lentivirus encoding AR Δ5/6/7. RNA and protein analyses were carried out as in B.

Close modal

In summary, these data provide the first demonstration that AR-V expression driven by AR gene rearrangements can mediate resistance to therapies targeting full-length AR, including the next generation antiandrogen enzalutamide/MDV3100. These studies are significant because resistance, either de novo or acquired during therapy, is a major clinical limitation for new AR-axis inhibitors (4, 20). Importantly, the majority of patients that display disease progression on enzalutamide also display rising PSA, indicating that enzalutamide-resistant tumors remain driven by persistent AR activity (3). AR-Vs are overexpressed in a subset of CRPC metastases and correlate with poor survival (7). Mechanistically, our data show that AR-Vs mediate enzalutamide resistance in CRPC through their activities as independent effectors of the AR transcriptional program, driving persistent activation of a large subset of AR target genes at a level of output sufficient to support cell proliferation. Overall, these studies with cell line models of CRPC provide proof-of-concept for reversing enzalutamide resistance through inhibition of AR-V expression and/or activity.

No potential conflicts of interest were disclosed.

Conception and design: Y. Li, S.M. Dehm

Development of methodology: Y. Li, S. Chiu Chan, S.M. Dehm

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y. Li, S. Chiu Chan, S.M. Dehm

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Y. Li, S. Chiu Chan, T. Hyun Hwang, K.A. Silverstein, S.M. Dehm

Writing, review, and/or revision of the manuscript: S. Chiu Chan, K.A. Silverstein, S.M. Dehm

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Chiu Chan, L.J. Brand, S.M. Dehm

Study supervision: S.M. Dehm

The authors are grateful to Dr. Kenny Beckman and the University of Minnesota Biomedical Genomics Center for conducting microarray expression experiments and the Minnesota Supercomputing Institute for software access, computational infrastructure, and systems administration support.

This work was financially supported by the Prostate Cancer Foundation Young Investigator Award (S.M. Dehm), the American Cancer Society Research Scholar Grant (RSG-12-031-01 to S.M. Dehm), and a Department of Defense Prostate Cancer Research Program New Investigator Award (W81XWH-10-1-0353 to S.M. Dehm). S.M. Dehm is a Masonic Scholar of the Masonic Cancer Center, University of Minnesota.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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