The androgen receptor (AR) is a key prostate cancer drug target. Suppression of AR signaling mediated by the full-length AR (AR-FL) is the therapeutic goal of all existing AR-directed therapies. AR-targeting agents impart therapeutic benefit, but lead to AR aberrations that underlie disease progression and therapeutic resistance. Among the AR aberrations specific to castration-resistant prostate cancer (CRPC), AR variants (AR-Vs) have emerged as important indicators of disease progression and therapeutic resistance.
We conducted a systemic review of the literature focusing on recent laboratory studies on AR-Vs following our last review article published in 2016. Topics ranged from measurement and detection, molecular origin, regulation, genomic function, and preclinical therapeutic targeting of AR-Vs. We provide expert opinions and perspectives on these topics.
Transcript sequences for 22 AR-Vs have been reported in the literature. Different AR-Vs may arise through different mechanisms, and can be regulated by splicing factors and dictated by genomic rearrangements, but a low-androgen environment is a prerequisite for generation of AR-Vs. The unique transcript structures allowed development of in situ and in-solution measurement and detection methods, including mRNA and protein detection, in both tissue and blood specimens. AR-V7 remains the main measurement target and the most extensively characterized AR-V. Although AR-V7 coexists with AR-FL, genomic functions mediated by AR-V7 do not require the presence of AR-FL. The distinct cistromes and transcriptional programs directed by AR-V7 and their coregulators are consistent with genomic features of progressive disease in a low-androgen environment. Preclinical development of AR-V-directed agents currently focuses on suppression of mRNA expression and protein degradation as well as targeting of the amino-terminal domain.
Current literature continues to support AR-Vs as biomarkers and therapeutic targets in prostate cancer. Laboratory investigations reveal both challenges and opportunities in targeting AR-Vs to overcome resistance to current AR-directed therapies.
Prostate cancer is an androgen-dependent disease. Management of patients with advanced prostate cancer often involves androgen-deprivation therapies (ADT) established in 1941 . Under ADT, castrate levels of androgens indicated by circulating testosterone (T) less than 50 ng/dL are achieved. Castration-resistant prostate cancer (CRPC) defines disease progression under castrate levels of T. In CRPC, expression level of the androgen receptor (AR) is often elevated, leading to AR activity under reduced androgen levels. In addition, the AR gene on the X chromosome may undergo genomic alterations including structural changes and point mutations. These CRPC-specific AR alterations provided a mechanistic explanation for continued dependence of CRPC on AR signaling [2,3,4]. This important concept in CRPC biology has guided and resulted in successful clinical development of second-generation AR-targeting therapies to treat CRPC, including agents that antagonize AR (enzalutamide, apalutamide, darolutamide) or further suppress extragonadal androgen synthesis (abiraterone, orteronel) [5,6,7,8,9,10,11,12,13,14,15,16,17]. The next-generation AR antagonists bind to the AR ligand-binding domain (LBD) with higher affinity than first-generation antiandrogens [6, 8], while abiraterone inhibits CYP17A1, a rate-limiting enzyme in the synthesis of adrenal and intratumoral androgens [5, 7]. Recently, clinical use of these next-generation AR-targeting therapies has been extended to castration-sensitive prostate cancer (CSPC) [9, 18, 19] and nonmetastatic CRPC [10,11,12, 20,21,22].
AR variants (AR-Vs) have mRNA sequences that are structurally different from the canonical full-length AR (AR-FL). A total of 22 AR-Vs have been cloned and reported in the literature (Fig. 1). The majority of these AR-Vs lack the LBD, the therapeutic target of all existing AR-targeting agents. In preclinical models, some but not all of these AR-Vs mediate constitutively active AR signaling, i.e., their activity is not dependent of the presence of androgens or AR-FL . Among the AR-Vs described to date, AR-V7 remains to be the most extensively evaluated and characterized, and several blood-based tests for AR-V7 have been developed (see companion review). General topics on AR-Vs have been reviewed extensively in the past [23,24,25,26]. The intent of the current review is to provide a sequel to a previous review article published in 2016 . Specifically, we will highlight recent preclinical studies covering topics ranging from measurement and detection, molecular origin, regulation, genomic function, and preclinical therapeutic targeting of AR-Vs. We will provide expert opinions and perspectives on these topics. Readers are directed to a companion review focusing on clinical studies related to AR-Vs.
Advances in AR-V measurement and detection methods
Accurate, reliable, and reproducible measurement of AR-Vs is a key requirement for inferring functional and clinical relevance. A variety of detection methods have been developed for the measurement of AR-Vs. These methods differ according to the method of sampling and specific measurement target. Some methods developed for blood-based AR-V7 detection have been analytically validated and implemented for clinical use (see companion review).
Detection by CTC mRNA
Blood-based detection of AR-V7 in the treatment setting was first reported in 2014 . In this initial report, CTC enrichment was achieved by AdnaTest, followed by RT-PCR-based detection of AR-FL and AR-V7. Following analytical validation, a slightly modified version of the laboratory-developed test was implemented in a CLIA- and CAP-certified laboratory for clinical use [28, 29], and another modified version was implemented as a clinical trial test . The AdnaTest employs a simple workflow enabling fast turnaround time at low cost, but with the drawback of requiring sample processing within 24 h of blood collection. Nevertheless, with careful management this drawback can be overcome. For example, a global biomarker selection trial was conducted by Tokai after implementing three central laboratories in three continents . A recent prospective study further validated the feasibility of conducting the test in the multi-institutional setting involving overnight shipping . While analytical and clinical validity are the two key requirements for clinical implementation, further improvement of the CTC-based test should take into consideration many factors that may also impede or facilitate clinical implementation, including cost and ease of use. Because the number of CTCs detected is always the limiting factor, new technologies to improve efficiency/sensitivity of CTC enrichment may further improve the test. For example, novel microfluidic apparatus or in vivo CTC collection methods were designed for CTC isolation and molecular analysis. Using negative depletion microfluidics (CTC-iChip)  (ClinicalTrials.gov NCT01961843) or positive selection microfluidic chip (e.g., IsoFlux) , CTC mRNA analysis including analysis of AR-FL/AR-V7 were conducted with digital droplet PCR (ddPCR). An intravascular CTC collection rod with antibody-coated surface (CellCollector, Gilupi, Germany) [33,34,35], as well as antibody-coated magnetic wire designed for CTC collection directly from blood flow , may also help to address the limitation of low CTC numbers. With regard to the limitation posed by low amount of CTC RNA, multiplexing may be the solution. For example, a 27-gene panel (iGene panel) was tested with high-throughput qPCR on Biomark platform (Fluidigm, San Francisco, CA) from CRPC patients receiving docetaxel treatment . These technologies may be used for biomarker development, including the detection of AR-Vs. However, it is challenging to conduct direct comparison of various CTC-based mRNA detection platforms.
Detection in whole blood
AR-Vs may be detected in whole blood without CTC enrichment. Using blood collection tube containing additives for the purpose of stabilizing intracellular RNA (e.g., PAXgene Blood RNA tube), total RNA from peripheral blood were prepared for AR-V7 detection [38,39,40,41]. However, mainly due to RNA contamination from large amount of leukocytes, the tumor-cell origin of AR-V7 will need to be validated especially when the measured signals are low. In addition, it is important to note that measurement of AR-FL is no longer possible in whole blood samples because non-CTC cells such as regulatory T cells and macrophages have low level of AR expression [42, 43].
Detection in exosomes
Exosomal RNA from extracellular vesicles in plasma represents another source for AR-V detection. Using exosomes, AR-V7 was detected by ddPCR using TaqMan probe with high sensitivity (at 2 copies/mL blood) and shown to be a strong predictive marker for enzalutamide/abiraterone resistance in mCRPC patients . In another report, extracellular vehicles from urine were used in detecting AR-V7 . Again, studies designed to conduct head-to-head comparisons are important but challenging to implement in the treatment setting.
Tissue detection by RNA in situ hybridization (RISH)
PCR-based detection methods described above are in-solution methods that generally do not capture cellular heterogeneity and the morphological context in tumors. In situ detection methods such as RISH and immunohistochemistry (IHC) (see below) have been developed. The use of RISH for AR-V7 detection was first reported in 2014 . Subsequently, different types of probes, including padlock probes, modified branched DNA probe, or junction-specific probes with enhanced sensitivity and specificity were developed to evaluate AR-FL and AR-Vs in FFPE tissue biopsy and isolated CTCs [34, 46,47,48]. In these studies, higher AR-V7 levels were associated with poorer response to AR-targeting therapies in mCRPC [47, 48].
Tissue detection by IHC
Recent studies on in situ detection of AR-V7 protein by IHC focused on newly developed and validated antibodies [48,49,50,51,52,53]. Using matched CSPC and CRPC tissue samples, nuclear AR-V7 protein detected by a rabbit AR-V7 monoclonal antibody (EPR15656; Abcam, Burlingame, CA) was associated with poor prognosis of CRPC patients after abiraterone or enzalutamide treatment . Following detailed characterization in clinical cohorts [13, 49, 54], the EPIC CTC AR-V7 IHC test (Epic Sciences, San Diego, CA) initially developed using the same anti-AR-V7 antibody was recently implemented for clinical use (see companion review). In tissue-based studies, the use of the RM7 (RevMab) antibody further established CRPC-specific expression AR-V7 [48, 50, 55]. Interestingly, AR-V7 protein expression was detected using this well-validated antibody in a subset of cells in small cell prostate carcinoma and some salivary ductal carcinoma specimens from untreated female patients [52, 56]. These recent studies on AR-V7 protein expression further supported that AR-V7 expression arises specifically in a low-androgen environment.
Tissue detection by NGS
AR-Vs can be detected in clinical specimens by RNA-seq . In Antonarakis et al., two autopsy specimens from mCRPC patients with positive CTC AR-V7 underwent RNA-seq analysis. Using number of reads spanning the exon 3/cryptic exon 3 (CE3) and exon 7/exon 8 junctions as surrogate expression values for AR-V7 and AR-FL, respectively, the AR-V7/AR-FL ratios in the two samples were 25.8% (139/539) and 12.1% (151/1248), in line with AR-V7/AR-FL ratios (median 21%, range 1.8–208%) estimated from RT-PCR-based CTC AR-V7 test reported in the same study. Using a slightly different method, the median AR-V7/AR-FL ratio from a larger-scale study of CRPC tissues was estimated at ~5% . The lower % reported in tissue-based studies is expected and does not contradict with our initial report in unselected CRPC tissues  or the ratios reported in CTC AR-V7 positive cases, because higher ratios reported in the Antonarakis study  excluded CTC AR-V7 negative cases (no ratios can be calculated in AR-V7 negative cases). In addition, RNA-seq in primary hormone-naive prostate cancers also detected AR-V7 at slightly lower AR-V7/AR-FL ratio . This is not unexpected either, as primary PC tissues are known to have low levels of AR-V7 detected by RT-PCR . However, we previously posited  that since AR-FL is also overexpressed in CRPC, a ratio of AR-V7/AR-FL at 10%, under the assumption of tenfold overexpression of AR-FL in CRPC tissues, would bring the absolute level of AR-V7 equivalent to the level of AR-FL detected in primary hormone-naive prostate cancers tissues. This interpretation also explained why AR-V7 protein is not detected in primary tissues .
Recent AR-V studies using whole-genome sequencing
Recent studies have used whole-genome NGS to examine AR for DNA structural alterations and RNA expression. Henzler et al.  evaluated 30 soft tissue metastases from 15 rapid autopsies for AR structural changes and AR-V expression. In this study, diverse AR-genomic structural rearrangements (AR-GSRs) including deletion, duplication, inversion, and translocation were observed in 10/30 metastases (6/15 patients). This study discovered many novel AR-Vs driven by AR-GSR. Cloned AR-Vs are depicted in Fig. 1. A pilot study by De Laere et al.  applied NGS to CTC and cell-free DNA (cfDNA) from mCRPC patients. In this study, cfDNA was evaluated by low-pass whole-genome sequencing and targeted sequencing of 112 genes including all coding exons and nonrepetitive intronic regions of AR, and expression levels of AR-FL and AR-Vs (AR45, AR-V1, AR-V2, AR-V3, AR-V5, AR-V7, and AR-V9) were interrogated by amplicon sequencing from multiplex junction-specific PCR using cDNA derived from CTCs. In 30 CTC samples from 26 mCRPC patients, 15/26 (57.7%) patients were AR-V-positive with AR-V7 being the most frequently detected variant (12/15 patients), followed by AR-V3 (11/15), AR45 (10/15), AR-V9 (6/15), AR-V1 (5/15), AR-V2 (3/15), and AR-V5 (3/15). In this study, 50% (15/30) of cfDNA samples had an intra-AR structural variation, and 14 of these 15 samples were positive for AR-Vs. Within the small cohort of patients treated with abiraterone or enzalutamide, 15/26 (57.7%) were AR-V-positive, and positive patients were either resistant (13/15) or moderately responsive (2/15) to abiraterone or enzalutamide.
Kallio et al. also applied targeted AR DNA-seq and RNA-seq to examine AR-Vs and other AR aberrations in CSPC, CRPC, mCRPC, BPH, and noncancerous tissues . AR-V3, AR-V7, and AR-V9 were most frequently detected and coexpressed AR mRNA variants in mCRPC, and these variants were also detected in BPH and hormone-naive primary tumors with lower abundance and frequency, although corresponding protein expression (in BPH and hormone-naive samples) was not shown. AR mutations and copy number changes were only detected in locally recurrent and metastatic CRPC specimens but not in untreated patients. Nonrecurring AR-GSRs (i.e., breakpoints are unique to each sample) were also specifically detected in 5/30 mCRPC. In this study, no AR-GSR coexisted with AR missense mutations in mCRPC . In addition, no definitive association was found between these AR-GSRs and AR-Vs, in line with the finding by Henzler et al. .
Due to limitation of short-read NGS sequencing by Illumina, the contribution of AR-GSR to AR-V generation remains to be thoroughly investigated. To date, only one study used a long-read sequencing method (PacBio sequencing platform) to characterize AR-Vs. In this prospective biomarker study (PROMOTE trial, NCT01953640), Kohli et al. performed targeted AR RNA-seq, whole transcriptome sequence, and long-read sequencing of AR 3′ rapid amplification of cDNA ends products in cell lines, patient-derived xenograft, and metastatic CRPC biopsy tissue samples . Expression of AR and AR-Vs was also measured by qRT-PCR in CTC samples. This study found that the 3′ UTR of AR-V9 included CE3 of AR-V7, and high-level of baseline AR-V9 was associated with resistance to abiraterone .
Molecular origin and regulation of AR-V expression
It is well established that expression of AR-Vs are regulated by androgens [64,65,66,67,68,69]. Since our last review in 2016 , many studies have been published that are relevant to the topic of molecular origin and regulation of AR-V expression. We will summarize the current literature on this topic in the following three general categories: (1) studies focusing on the role of genomic alterations; (2) studies focusing on the role of RNA splicing; and (3) studies demonstrating the role of other cofactors and signaling pathways on regulating AR-V expression. The findings from studies focusing on splicing factors and related regulators are summarized in Fig. 2. Readers are directed to a recent review  for further reading on the specific topic of AR-V regulation, the role of genomic structural alterations, and the requirement of low androgens for AR-V7 generation.
Role of genomic alterations
AR-GSRs underlie the generation of some of the AR-Vs. As discussed in “Recent AR-V studies using whole-genome sequencing”, AR-GSRs may also contribute to the generation of AR-Vs [60,61,62,63]. In an interesting model proposed by Henzler et al., successive structural alterations may occur on the same AR allele, leading to generation of AR-Vs. The study presented a case in which tumors from multiple metastatic sites in the same patient showed similar complex patterns of deletion and duplication within the AR LBD that explained the dominant expression of ARv567es . However, due to the limitation of short-read sequencing and the involvement of target enrichment, this model may need further validation with long-read genomic sequencing.
Role of RNA splicing
Although CRPC-specific splicing factors involved in the process have not been definitively identified, studies focusing on factors that regulate AR splicing have identified several key factors related to the RNA spliceosome. On the basis of predicted intronic/exonic splicing enhancer sequences in CE3 of the AR gene, ChIP and RNA-pull down assays were performed and identified U2AF65 (splicing factor U2AF65 kDa subunit, aka U2AF2, U2 small nuclear RNA auxiliary factor 2) and ASF/SF2 (alternative splicing factor 1/splicing factor 2) as two key factors mediating AR-V7 splicing . A general splicing factor HNRNPA1 (heterogeneous nuclear ribonucleoprotein A1) was also reported to regulate AR-V7 expression in prostate cancer cells. Activation of NFκB2/p52 and c-Myc signaling resulted in recruitment of HNRNPA1 to the splice site of AR pre-mRNA to promote AR-V7 expression .
The ADT-induced lncRNA prostate cancer gene expression marker 1 (PCGEM1) was also implicated in AR-V7 generation. PCGEM1 regulates the binding of splice factors HNRNPA1 and U2AF65 to AR pre-mRNA . In addition, p54/nrb (nuclear RNA-binding protein, aka NONO, non-POU domain-containing octamer-binding protein), a nuclear protein with multiple functions in RNA splicing and gene regulation, positively regulates PCGEM1 expression, as revealed by DNA-pull down assay using PCGEM1 gene promoter in prostate cancer cells . Suppression of p54/nrb by siRNA or a natural compound from cruciferous vegetables that interferes with p54/nrb-DNA binding resulted in reduced expression of PCGEM1 and AR-V7 .
Natural spliceosome inhibitors, thailanstatins (TST-A or TST-D), were used to investigate the relationship between AR-V7 and splice factors SAP155 (spliceosome-associated protein 155, aka SF3B1, splicing factor 3b subunit 1) and U2AF65. TSTs interrupt the interaction between U2AF65 and SAP155 leading to reduced binding to the polypyrimidine tract located between the branch point and the 3′ splice site, and consequentially reduced AR-V7 expression .
AR-V7 may be regulated by Jumonji domain containing 1A (JMJD1A, aka KDM3A), a histone demethylase that removes the repressive H3K9 methylation marks (H3K9me1 or H3K9me2) . KDM3A also interacts with AR as an AR coactivator to regulate prostate cancer cell proliferation and survival by altering AR transcriptional program and elevating c-Myc levels [76,77,78]. KDM3A promotes alternative splicing of AR-V7 by binding to guanosine (G)-tract sequences in CE3 of the AR gene leading to recruitment of heterogeneous nuclear ribonucleoprotein F (HNRNPF) and other splice factors such as U2AF265 .
DDX39 [DEAD (Asp-Glu-Ala-Asp) box polypeptide 39] is an ATP-dependent RNA helicase implicated in RNA splicing, mRNA export, and telomere structure integrity. Using a shRNA library focused on 88 spliceosome-related genes, DDX39B was found to be a regulator of AR-V7 expression . After knockdown of DDX39B and its paralogue DDX39A in AR-V-expressed cells, the AR-V7 mRNA was selectively downregulated .
LIN28 is an RNA-binding protein involved in AR and c-Myc signaling in prostate cancer. Overexpression of LIN28 in PCa cell lines resulted in increased AR splice variant expression and resistance to antiandrogens. Downregulation of LIN28 can resensitize PCa cells to enzalutamide treatment .
The long noncoding RNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) was also implicated in regulating AR-V7 activity. Using enzalutamide-resistant prostate cancer cell lines, AR-V7 and MALAT1 were both elevated when AR signaling is suppressed. AR negatively regulates MALAT1 by direct binding to androgen response elements (AREs) in the promoter of MALAT1. Increased MALAT1 during AR suppression by enzalutamide could elevate AR-V7 transcription through interaction between MALAT1 and serine/arginine rich splicing factor 1 (SRSF1, aka ASF/SF2). CRPC progression in animal models was suppressed following MALAT1 knockdown .
Other cofactors regulating AR-V expression
Protein kinase pathways may regulate AR-V expression. The most relevant is the AKT pathway, which modulates AR function and mediates survival signaling in CRPC. In a screen of kinase inhibitors involving a 145 small‐molecule compound library and a high-throughput siRNA-kinome library, several kinases including Akt, Abl, and Src family kinases were found to regulate AR-V7 mRNA expression and protein nuclear translocation. Following treatment of CWR22Rv1 cells with a Src/Abl dual kinase inhibitor PD180970, AR-V7 protein was decreased and cell proliferation inhibited in the absence of DHT. In the presence of DHT (when AR-FL is activated), the potency of PD180970 was decreased. Pretreatment with AR-FL antagonist bicalutamide resensitized CWR22Rv1 cells to PD180970 with a substantial EC50 drop (from 184.2 to 12.6 nM) in the presence of DHT , suggesting that PD180970 reduced AR-V7 protein level and CWR22Rv1 growth in AR-independent manner. Tyrosine kinases such as ACK1/TNK2 regulate AR gene transcription through epigenetic modification. ACK1/TNK2 phosphorylates histone H4 at tyrosine 88 upstream of the AR transcription start site, and treatment with an ACK1 inhibitor (R)-9bMS reduced AR and AR-V7 levels and suppressed CRPC tumor growth .
Y-box-binding protein 1 (YB-1) is a transcription factor that binds Y-box (5′-ATTGG-3′), and also a RNA-binding protein involved in RNA splicing. In a study involving kinome arrays, Akt, RSK (ribosomal S6 kinase), ERK (extracellular signal-regulated kinases, p42/44 MAPK) were found to phosphorylate YB-1 and affect AR-V7 expression . Treatment of CWR22Rv1 cells with kinase inhibitors against Akt, MEK (MAPK/ERK kinase, an upstream activator of MAPK/ERK) resulted in reduced levels of phosphorylated-YB-1, AR-FL, and AR-V7. However, an inhibitor of RSK specifically downregulated AR-V7 but not AR-FL. How RSK/YB-1 signaling affects AR splicing remains largely uncharacterized .
The relationship of AKT signaling and prostate cancer was evaluated by a synthetic AKT inhibitor alkyl-lysophospholipid edelfosine (ET-18-O-CH3). Edelfosine interacts with lipid rafts on plasma membrane, and induce endoplasmic reticulum stress following inhibition of phosphatidylcholine biosynthesis. Treatment of LNCaP and VCaP cells with edelfosine resulted in suppression of AR-V7 expression and cell apoptosis that was enhanced by activating transcription factor 3, a corepressor of AR .
Other nuclear receptors may regulate AR-FL/AR-V7 expression. For example, RAR-related orphan receptor gamma (ROR-γ) recruits nuclear receptor coactivator 1 and 3 [NCOA1 and NCOA3, also known as steroid receptor coactivator (SRC)-1 and SRC-3] to an AR-ROR response element to stimulate AR gene transcription . ROR-γ antagonists suppress the expression of both AR and its variant AR-V7 in PCa cell lines and tumors . Given that many ROR-γ inhibitors are approved for treating autoimmune disease, it may be possible to repurpose these drugs to suppress AR-V7-driven prostate cancer.
Noncoding circular RNA (circRNA) or miRNA could also regulate AR-V7 expression. By in silico analysis, a circRNA 17 (circRNA17, hsa_circ_0001427) was found to interact with microRNA 181c-5p (miR-181c-5p) leading to suppression of AR-V7 expression. By interacting with and stabilizing miR-181c-5p, circRNA17 increased the relative level of miR-181c-5p, which bound to the 3′UTR of AR-V7 transcript to downregulate AR-V7 expression. Castration can suppress circRNA17 expression by inhibiting its host gene PDLIM5 (PDZ and LIM domain 5) expression via ARE in the gene promoter. In this study, the level of circRNA17 was downregulated in clinical prostate cancer specimens especially in higher Gleason score prostate cancer specimens .
Genomic functions mediated by AR-Vs
As transcription factors, AR-Vs may mediate their downstream genomic functions by DNA binding and interaction with coregulators following nuclear localization. While AR-FL and many AR-Vs may share common chromatin binding sites, genomic binding sites, and transcriptional programs specific to AR-V7 have been reported [64, 88,89,90,91]. In studies relevant to this topic, it is critical to take into consideration a few premises: (1) AR-V7 protein expression is specific to CRPC and negatively regulated by androgen signaling mediated by AR-FL [64, 68]; (2) AR-V7 expression is often associated with a progressive phenotype under low-androgen conditions [64, 90]; and (3) AR-V7 often coexists with AR-FL, and whether AR-V7/AR-FL forms heterodimer under low androgen conditions will affect interpretation of data [68, 89, 92,93,94].
Since our last review in 2016 , a few important factors mediating downstream functional effects of AR-V7 have been characterized. Chen et al. revealed diverse AR-V7 cistromes and transcriptomes in different CRPC cell lines and clinical specimens . Employing a high-resolution ChIP-exonuclease sequencing approach using an AR-V7-specific antibody, HOXB13 was found to colocalize with AR-V7 and function as an essential coactivator mediating AR-V7 function. By inhibiting HOXB13 in AR-V7-expressing cells, the oncogenic function of AR-V7 was suppressed . These results implicated that HOXB13, which is very specifically expressed in tissues of prostatic origin, could be an alternative target in suppressing the development of AR-V7-driven CRPC. Cai et al. used specific antibodies against either AR-FL or AR-V7 for ChIP-seq. In this study, 15,162 out of a total of 17,409 binding sites were shared by both ARs, and only a small proportion of binding sites (about 12.8%, 2221 out of 17,409 spots) was specifically bound by AR-V7. In these AR-V7-specific binding sites, zinc finger protein X-linked was found to exclusively colocalize with AR-V7. The AR-V7-specific binding sites are mainly located at the gene promoter and these AR-V7 targeted genes were mainly involved in MYC-bound genes or genes related to cell cycles and autophagy .
Preclinical and early clinical development of agents targeting AR/AR-Vs
Given the availability of blood-based detection methods and the general poor prognosis of patients having positive CTC AR-V7 testing results, recent efforts have focused on development of agents that may overcome drug resistance and poor prognosis by targeting AR-V7. Several compounds with in vitro anti-AR-V7 activity have already been evaluated in clinical trials, while many others are in various stages of preclinical evaluation and development (see companion review). A comprehensive review of the current literature on possible preclinical agents and strategies returned many studies. Due to space limitations, we summarized these studies in Fig. 3 and Table 1 (with references).
Laboratory investigations and findings reported since 2016 continue to support AR-Vs as biomarkers and therapeutic targets in prostate cancer. These new findings further validate the importance of AR-Vs, AR-V7 in particular, in CRPC. Development of mature measurement methods have enabled detection of the AR-V targets in both liquid and tissue specimens, and quantitative measurement data on AR-V7 support its functional and clinical relevance. While AR-Vs (e.g., ARv567es) driven by complex AR-GSRs, as well as dominant expression of AR-V7 in isolated cases provide compelling examples of clonal expansion consistent with a driver role for AR-Vs in castration resistance, in most cases AR-FL continue to coexist with AR-Vs, justifying further investigations to dissect the distinct roles of the different AR molecules. In this regard, distinct genomic functions mediated by AR-FL and AR-Vs have been defined in greater details now, further supporting the therapeutic relevance of AR-Vs. Given the feasibility of conducting blood-based detection for AR-V7, the hope is that clinical development of agents possessing anti-AR-V activity can be accelerated, even in the presence of many competing mechanism that may coexist. Going forward, both challenges and opportunities exist in targeting AR-Vs to overcome resistance to current AR-directed therapies. Development of agents with specific anti-AR-V7 activity remains a top priority in prostate cancer research.
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ESA has received funding from the Prostate Cancer Foundation, the Patrick C. Walsh Fund, and NIH grants R01 CA185297 and P30 CA006973. AJA has received funding from a Prostate Cancer Foundation and Movember Global Treatment Sciences Challenge Award and the NIH under a P30 CA014236 and 1R01CA233585-01 grant. JL is currently funded by a Prostate Cancer Foundation grant, NIH grant R01 CA185297, and US Department of Defense Prostate Cancer Research Program grant W81XWH-19-1-0686.
Conflict of interest
ESA has served as a paid consultant/advisor for Janssen, Pfizer, Sanofi, Dendreon, Essa, Merck, Bristol-Myers Squibb, AstraZeneca, Clovis, Eli Lilly, and Amgen; has received research funding to his institution from Janssen, Johnson & Johnson, Sanofi, Dendreon, Genentech, Novartis, Tokai, Merck, Bristol-Myers Squibb, AstraZeneca, and Constellation; and is a co-inventor of an AR-V7 biomarker technology that has been licensed to Qiagen. AJA has served as a paid consultant for AstraZeneca, Merck, Dendreon, Janssen, Clovis, Bayer, and Medivation/Astellas; is on the speaker’s bureau for Bayer and Dendreon; and receives research funding to his institution from Janssen, Medivation/Astellas, Sanofi-Aventis, Active Biotech, Bayer, Dendreon, Merck, AstraZeneca, Genentech/Roche, BMS, Constellation, Novartis, and Pfizer. JL has served as a paid consultant/advisor for Sun Pharma, Janssen, Tolero, and Sanofi; has received research funding to his institution from Orion, Mirati, Astellas, Sanofi, Constellation, Calibr, Pandomedx, and Gilead; and is a co-inventor of a technology that has been licensed to Tokai, Qiagen, and A&G. CL is a co-inventor of a technology that has been licensed to Tokai and Qiagen.
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Lu, C., Brown, L.C., Antonarakis, E.S. et al. Androgen receptor variant-driven prostate cancer II: advances in laboratory investigations. Prostate Cancer Prostatic Dis 23, 381–397 (2020). https://doi.org/10.1038/s41391-020-0217-3