The standard systemic treatment for prostate cancer (PCa) is androgen ablation, which causes tumor regression by inhibiting activity of the androgen receptor (AR). Invariably, PCa recurs with a fatal androgen-refractory phenotype. Importantly, the growth of androgen-refractory PCa remains dependent on the AR through various mechanisms of aberrant AR activation. Here, we studied the 22Rv1 PCa cell line, which was derived from a CWR22 xenograft that relapsed during androgen ablation. Three AR isoforms are expressed in 22Rv1 cells: a full-length version with duplicated exon 3 and two truncated versions lacking the COOH terminal domain (CTD). We found that CTD-truncated AR isoforms are encoded by mRNAs that have a novel exon 2b at their 3′ end. Functionally, these AR isoforms are constitutively active and promote the expression of endogenous AR-dependent genes, as well as the proliferation of 22Rv1 cells in a ligand-independent manner. AR mRNAs containing exon 2b and their protein products are expressed in commonly studied PCa cell lines. Moreover, exon 2b–derived species are enriched in xenograft-based models of therapy-resistant PCa. Together, our data describe a simple and effective mechanism by which PCa cells can synthesize a constitutively active AR and thus circumvent androgen ablation. [Cancer Res 2008;68(13):5469–77]

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.

Alternative splicing is an important mechanism for increasing functional diversity from a limited set of genes. Deregulation of this process is common in diverse pathologic conditions. The androgen receptor (AR) is a steroid receptor transcription factor with functions critical for normal male development as well as the growth and survival of normal and cancerous prostate tissue. Studies of AR function in androgen insensitivity syndrome (AIS) and prostate cancer (PCa) have demonstrated loss-of-function AR alterations in AIS and gain-of-function AR alterations in PCa. Over the past two decades, AR gene alterations have been identified in various individuals with AIS, which disrupt normal AR splicing patterns and yield dysfunctional AR protein variants. Recently, altered AR splicing patterns have been identified as a mechanism of PCa progression and resistance to androgen depletion therapy. Several studies have described the synthesis of alternatively spliced transcripts encoding truncated AR isoforms that lack the ligand-binding domain, which is the ultimate target of androgen depletion. Many of these truncated AR isoforms function as constitutively active, ligand-independent transcription factors that can support androgen-independent expression of AR target genes, as well as the androgen-independent growth of PCa cells. In this review, we will summarize the various alternatively spliced AR variants that have been discovered, with a focus on their role and origin in the pathologic conditions of AIS and PCa.

Synthesis of truncated androgen receptor (AR) splice variants has emerged as an important mechanism of prostate cancer (PCa) resistance to AR-targeted therapy and progression to a lethal castration-resistant phenotype. However, the precise role of these factors at this stage of the disease is not clear due to loss of multiple COOH-terminal AR protein domains, including the canonical nuclear localization signal (NLS) in the AR hinge region. Despite loss of this NLS, we show that diverse truncated AR variant species have a basal level of nuclear localization sufficient for ligand-independent transcriptional activity. Whereas full-length AR requires Hsp90 and importin-β for active nuclear translocation, basal nuclear localization of truncated AR variants is independent of these classical signals. For a subset of truncated AR variants, this basal level of nuclear import can be augmented by unique COOH-terminal sequences that reconstitute classical AR NLS activity. However, this property is separable from ligand-independent transcriptional activity. Therefore, the AR splice variant core consisting of the AR NH(2)-terminal domain and DNA binding domain is sufficient for nuclear localization and androgen-independent transcriptional activation of endogenous AR target genes. Indeed, we show that truncated AR variants with nuclear as well as nuclear/cytoplasmic localization patterns can drive androgen-independent growth of PCa cells. Together, our data demonstrate that diverse truncated AR species with varying efficiencies of nuclear localization can contribute to castration-resistant PCa pathology by driving persistent ligand-independent AR transcriptional activity.

Reactivation of the androgen receptor (AR) during androgen depletion therapy (ADT) underlies castration-resistant prostate cancer (CRPCa). Alternative splicing of the AR gene and synthesis of constitutively active COOH-terminally truncated AR variants lacking the AR ligand-binding domain has emerged as an important mechanism of ADT resistance in CRPCa. In a previous study, we demonstrated that altered AR splicing in CRPCa 22Rv1 cells was linked to a 35-kb intragenic tandem duplication of AR exon 3 and flanking sequences. In this study, we demonstrate that complex patterns of AR gene copy number imbalances occur in PCa cell lines, xenografts and clinical specimens. To investigate whether these copy number imbalances reflect AR gene rearrangements that could be linked to splicing disruptions, we carried out a detailed analysis of AR gene structure in the LuCaP 86.2 and CWR-R1 models of CRPCa. By deletion-spanning PCR, we discovered a 8579-bp deletion of AR exons 5, 6 and 7 in the LuCaP 86.2 xenograft, which provides a rational explanation for synthesis of the truncated AR v567es AR variant in this model. Similarly, targeted resequencing of the AR gene in CWR-R1 cells led to the discovery of a 48-kb deletion in AR intron 1. This intragenic deletion marked a specific CWR-R1 cell population with enhanced expression of the truncated AR-V7/AR3 variant, a high level of androgen-independent AR transcriptional activity and rapid androgen independent growth. Together, these data demonstrate that structural alterations in the AR gene are linked to stable gain-of-function splicing alterations in CRPCa.