NanoLuc Binary Technology as a methodological approach: an important new tool for studying the localization of androgen receptor and androgen receptor splice variant V7 homo and heterodimers.

BACKGROUND: The androgen/androgen receptor (AR)-signaling axis plays a central role in prostate cancer (PCa). Upon androgen-binding the AR dimerizes with another AR, and translocates into the nucleus where the AR-dimer activates/inactivates androgen-dependent genes. Consequently, treatments for PCa are commonly based on androgen deprivation therapy (ADT). The clinical benefits of ADT are only transitory and most tumors develop mechanisms allowing the AR to bypass its need for physiological levels of circulating androgens. Clinical failure of ADT is often characterized by the synthesis of a constitutively active AR splice variant, termed AR-V7. AR-V7 mRNA expression is considered as a resistance mechanism following ADT. AR-V7 no longer needs androgenic stimuli for nuclear entry and/or dimerization. METHODS: Our goal was to mechanistically decipher the interaction between full-length AR (AR-FL) and AR-V7 in AR-null HEK-293 cells using the NanoLuc Binary Technology under androgen stimulation and deprivation conditions. RESULTS: Our data point toward a hypothesis that AR-FL/AR-FL homodimers form in the cytoplasm, whereas AR-V7/AR-V7 homodimers localize in the nucleus. However, after androgen stimulation, all the AR-FL/AR-FL, AR-FL/AR-V7 and AR-V7/AR-V7 dimers were localized in the nucleus. CONCLUSIONS: We showed that AR-FL and AR-V7 form heterodimers that localize to the nucleus, whereas AR-V7/AR-V7 dimers were found to localize in the absence of androgens in the nucleus.

Androgen receptor variant-7 regulation by tenascin-c induced src activation.

BACKGROUND: Bone metastatic prostate cancer does not completely respond to androgen-targeted therapy and generally evolves into lethal castration resistant prostate cancer (CRPC). Expression of AR-V7- a constitutively active, ligand independent splice variant of AR is one of the critical resistant mechanisms regulating metastatic CRPC. TNC is an extracellular matrix glycoprotein, crucial for prostate cancer progression, and associated with prostate cancer bone metastases. In this study, we investigated the mechanisms that regulate AR-V7 expression in prostate cancer cells interacting with osteogenic microenvironment including TNC. METHODS: Prostate cancer/preosteoblast heterotypical organoids were evaluated via immunofluorescence imaging and gene expression analysis using RT-qPCR to assess cellular compartmentalization, TNC localization, and to investigate regulation of AR-V7 in prostate cancer cells by preosteoblasts and hormone or antiandrogen action. Prostate cancer cells cultured on TNC were assessed using RT-qPCR, Western blotting, cycloheximide chase assay, and immunofluorescence imaging to evaluate (1) regulation of AR-V7, and (2) signaling pathways activated by TNC. Identified signaling pathway induced by TNC was targeted using siRNA and a small molecular inhibitor to investigate the role of TNC-induced signaling activation in regulation of AR-V7. Both AR-V7- and TNC-induced signaling effectors were targeted using siRNA, and TNC expression assessed to evaluate potential feedback regulation. RESULTS: Utilizing heterotypical organoids, we show that TNC is an integral component of prostate cancer interaction with preosteoblasts. Interaction with preosteoblasts upregulated both TNC and AR-V7 expression in prostate cancer cells which was suppressed by testosterone but elevated by antiandrogen enzalutamide. Interestingly, the results demonstrate that TNC-induced Src activation regulated AR-V7 expression, post-translational stability, and nuclear localization in prostate cancer cells. Treatment with TNC neutralizing antibody, Src knockdown, and inhibition of Src kinase activity repressed AR-V7 transcript and protein. Reciprocally, both activated Src and AR-V7 were observed to upregulate autocrine TNC gene expression in prostate cancer cells. CONCLUSION: Overall, the findings reveal that prostate cancer cell interactions with the cellular and ECM components in the osteogenic microenvironment plays critical role in regulating AR-V7 associated with metastatic CRPC. Video Abstract.

Molecular Origin, Expression Regulation, and Biological Function of Androgen Receptor Splicing Variant 7 in Prostate Cancer.

The problem of resistance to therapy in prostate cancer (PCa) is multifaceted. Key determinants of drug resistance include tumor burden and growth kinetics, tumor heterogeneity, physical barriers, immune system and microenvironment, undruggable cancer drivers, and consequences of therapeutic pressures. With regard to the fundamental importance of the androgen receptor (AR) in all stages of PCa from tumorigenesis to progression, AR is postulated to have a continued critical role in castration-resistant prostate cancer (CRPC). Suppression of AR signaling mediated by the full-length AR (AR-FL) is the therapeutic goal of all AR-directed therapies. However, AR-targeting agents ultimately lead to AR aberrations that promote PCa progression and drug resistance. Among these AR aberrations, androgen receptor variant 7 (AR-V7) is gaining attention as a potential predictive marker for as well as one of the resistance mechanisms to the most current anti-AR therapies in CRPC. Meanwhile, development of next-generation drugs that directly or indirectly target AR-V7 signaling is urgently needed. In the present review of the current literature, we have summarized the origin, alternative splicing, expression induction, protein conformation, interaction with coregulators, relationship with AR-FL, transcriptional activity, and biological function of AR-V7 in PCa development and therapeutic resistance. We hope this review will help further understand the molecular origin, expression regulation, and role of AR-V7 in the progression of PCa and provide insight into the design of novel selective inhibitors of AR-V7 in PCa treatment.

miR-103a-2-5p/miR-30c-1-3p inhibits the progression of prostate cancer resistance to androgen ablation therapy via targeting androgen receptor variant 7.

Androgens and androgen receptors are vital factors involved in prostate cancer progression, and androgen ablation therapies are commonly used to treat advanced prostate cancer. However, the acquisition of androgen ablation therapy resistance remains a challenge. Recently, androgen receptor splicing variants lacking the ligand-binding domain have been reported to play a critical role in the acquisition of androgen ablation therapy resistance. In the present study, we revealed that the messenger RNA expression and the protein levels of an androgen receptor variant 7 (AR-V7) were higher in prostate cancer tissue samples and in the AR-positive prostate cancer cell line, VCaP. In contrast, microRNA (miR)-30c-1-3p/miR-103a-2-5p expression was significantly downregulated in tumor tissues and cells. miR-30c-1-3p/miR-103a-2-5p overexpression could inhibit AR-V7 expression, suppress VCaP cell growth, and inhibit AR-V7 downstream factor expression by directly targeting the 3'-untranslated region of AR-V7. Under enzalutamide (Enza) treatment, the effects of AR-V7 overexpression were the opposite of those of miR-103a-2-5p/miR-30c-1-3p overexpression; more importantly, the effects of miR-103a-2-5p/miR-30c-1-3p overexpression could be significantly reversed by AR-V7 overexpression under Enza. In summary, we demonstrated a novel mechanism of the miR-30c-1-3p/miR-103a-2-5p/AR-V7 axis modulating the cell proliferation of AR-positive prostate cancer cells via AR downstream targets. The clinical application of miR-30c-1-3p/miR-103a-2-5p needs further in vivo validation.

Ethanolic Extracts from Azadirachta indica Leaves Modulate Transcriptional Levels of Hormone Receptor Variant in Breast Cancer Cell Lines.

Breast Cancer (BC) encompasses numerous entities with different biological and behavioral characteristics, favored by tumor molecular complexity. Azadirachta indica (neem) presents phenolic compounds, indicating its potential as an antineoplastic compound. The present study aimed to evaluate the cellular response of MCF10, MCF7, and MDA-MB-231 breast cell lines to ethanolic extracts of neem leaves (EENL) obtained by dichloromethane (DCM) and ethyl acetate (EA) solvent. Extracts' antiproliferative activities were evaluated against MCF 10A, MCF7, and MDA-MB-231 for 24 and 48 h using MTT assay. ESR1, ESR2, AR, AR-V1, AR-V4, and AR-V7 transcripts were quantified through qPCR for 0.03125 µg/mL of DCM and 1.0 µg/mL for EA for 48 h. The EENL was tested on Drosophila melanogaster as a sole treatment and then also together with doxorubicin. Antiproliferative effect on tumor cell lines without affecting MCF 10A were 1.0 µg/mL (P < 0.001) for EA, and 0.03125 µg/mL (P < 0.0001) for DCM, both after 48 h. Transcriptional levels of AR-V7 increased after treatment. In vivo assays demonstrated that EENL induced fewer tumors at a higher concentration with doxorubicin (DXR). The behavior of AR-V7 in the MDA-MB-231 tumor lineage indicates new pathways involved in tumor biology and this may have therapeutic value for cancer.

High levels of the AR-V7 Splice Variant and Co-Amplification of the Golgi Protein Coding YIPF6 in AR Amplified Prostate Cancer Bone Metastases.

BACKGROUND: The relation between androgen receptor (AR) gene amplification and other mechanisms behind castration-resistant prostate cancer (CRPC), such as expression of constitutively active AR variants and steroid-converting enzymes has been poorly examined. Specific aim was to examine AR amplification in PC bone metastases and to explore molecular and functional consequences of this, with the long-term goal of identifying novel molecular targets for treatment. METHODS: Gene amplification was assessed by fluorescence in situ hybridization in cryo-sections of clinical PC bone metastases (n = 40) and by PCR-based copy number variation analysis. Whole genome mRNA expression was analyzed using H12 Illumina Beadchip arrays and specific transcript levels were quantified by qRT-PCR. Protein localization was analyzed using immunohistochemistry and confocal microscopy. The YIPF6 mRNA expression was transiently knocked down and stably overexpressed in the 22Rv1 cell line as representative for CRPC, and effects on cell proliferation, colony formation, migration, and invasion were determined in vitro. Extracellular vesicles (EVs) were isolated from cell cultures using size-exclusion chromatography and enumerated by nanoparticle tracking analysis. Protein content was identified by LC-MS/MS analysis. Blood coagulation was measured as activated partial thromboplastin time (APTT). Functional enrichment analysis was performed using the MetaCore software. RESULTS: AR amplification was detected in 16 (53%) of the bone metastases examined from CRPC patients (n = 30), and in none from the untreated patients (n = 10). Metastases with AR amplification showed high AR and AR-V7 mRNA levels, increased nuclear AR immunostaining, and co-amplification of genes such as YIPF6 in the AR proximity at Xq12. The YIPF6 protein was localized to the Golgi apparatus. YIPF6 overexpression in 22Rv1 cells resulted in reduced cell proliferation and colony formation, and in enhanced EV secretion. EVs from YIPF6 overproducing 22Rv1 cells were enriched for proteins involved in blood coagulation and, accordingly, decreased the APTT in a dose-dependent fashion. CONCLUSIONS: AR amplified CRPC bone metastases show high AR-V7 expression that probably gives resistance to AR-targeting drugs. Co-amplification of the Golgi protein coding YIPF6 gene with the AR may enhance the secretion of pro-coagulative EVs from cancer cells and thereby stimulate tumor progression and increase the coagulopathy risk in CRPC patients. Prostate 77: 625-638, 2017. © 2017 Wiley Periodicals, Inc.

NF-κB and androgen receptor variant 7 induce expression of SRD5A isoforms and confer 5ARI resistance.

BACKGROUND: Benign prostatic hyperplasia (BPH) is treated with 5α-reductase inhibitors (5ARI). These drugs inhibit the conversion of testosterone to dihydrotestosterone resulting in apoptosis and prostate shrinkage. Most patients initially respond to 5ARIs; however, failure is common especially in inflamed prostates, and often results in surgery. This communication examines a link between activation of NF-κB and increased expression of SRD5A2 as a potential mechanism by which patients fail 5ARI therapy. METHODS: Tissue was collected from "Surgical" patients, treated specifically for lower urinary tract symptoms secondary to advanced BPH; and, cancer free transition zone from "Incidental" patients treated for low grade, localized peripheral zone prostate cancer. Clinical, molecular and histopathological profiles were analyzed. Human prostatic stromal and epithelial cell lines were genetically modified to regulate NF-κB activity, androgen receptor (AR) full length (AR-FL), and AR variant 7 (AR-V7) expression. RESULTS: SRD5A2 is upregulated in advanced BPH. SRD5A2 was significantly associated with prostate volume determined by Transrectal Ultrasound (TRUS), and with more severe lower urinary tract symptoms (LUTS) determined by American Urological Association Symptom Score (AUASS). Synthesis of androgens was seen in cells in which NF-κB was activated. AR-FL and AR-V7 expression increased SRD5A2 expression while forced activation of NF-κB increased all three SRD5A isoforms. Knockdown of SRD5A2 in the epithelial cells resulted in significant reduction in proliferation, AR target gene expression, and response to testosterone (T). In tissue recombinants, canonical NF-κB activation in prostatic epithelium elevated all three SRD5A isoforms and resulted in in vivo growth under castrated conditions. CONCLUSION: Increased BPH severity in patients correlates with SRD5A2 expression. We demonstrate that NF-κB and AR-V7 upregulate SRD5A expression providing a mechanism to explain failure of 5ARI therapy in BPH patients. Prostate 76:1004-1018, 2016. © 2016 Wiley Periodicals, Inc.

NF-κB and androgen receptor variant expression correlate with human BPH progression.

BACKGROUND: Benign prostatic hyperplasia (BPH) is a common, chronic progressive disease. Inflammation is associated with prostatic enlargement and resistance to 5α-reductase inhibitor (5ARI) therapy. Activation of the nuclear factor-kappa B (NF-κB) pathway is linked to both inflammation and ligand-independent prostate cancer progression. METHODS: NF-κB activation and androgen receptor variant (AR-V) expression were quantified in transition zone tissue samples from patients with a wide range of AUASS from incidental BPH in patients treated for low grade, localized peripheral zone prostate cancer to advanced disease requiring surgical intervention. To further investigate these pathways, human prostatic stromal and epithelial cell lines were transduced with constitutively active or kinase dead forms of IKK2 to regulate canonical NF-κB activity. The effects on AR full length (AR-FL) and androgen-independent AR-V expression as well as cellular growth and differentiation were assessed. RESULTS: Canonical NF-κB signaling was found to be upregulated in late versus early stage BPH, and to be strongly associated with non-insulin dependent diabetes mellitus. Elevated expression of AR-variant 7 (AR-V7), but not other AR variants, was found in advanced BPH samples. Expression of AR-V7 significantly correlated with the patient AUASS and TRUS volume. Forced activation of canonical NF-κB in human prostatic epithelial and stromal cells resulted in elevated expression of both AR-FL and AR-V7, with concomitant ligand-independent activation of AR reporters. Activation of NF-κB and over expression of AR-V7 in human prostatic epithelial cells maintained cell viability in the face of 5ARI treatment. CONCLUSION: Activation of NF-κB and AR-V7 in the prostate is associated with increased disease severity. AR-V7 expression is inducible in human prostate cells by forced activation of NF-κB resulting in resistance to 5ARI treatment, suggesting a potential mechanism by which patients may become resistant to 5ARI therapy.

The positive relationship between androgen receptor splice variant-7 expression and the risk of castration-resistant prostate cancer: A cumulative analysis.

Background: At present, androgen deprivation therapy (ADT) is still the standard regimen for patients with metastatic and locally advanced prostate cancer (PCa). The level of androgen receptor splice variant-7 (AR-V7) in men with castration-resistant prostate cancer (CRPC) has been reported to be elevated compared with that in patients diagnosed with hormone-sensitive prostate cancer (HSPC). Aim: Herein, we performed a systematic review and cumulative analysis to evaluate whether the expression of AR-V7 was significantly higher in patients with CRPC than in HSPC patients. Methods: The commonly used databases were searched to identify the potential studies reporting the level of AR-V7 in CRPC and HSPC patients. The association between CRPC and the positive expression of AR-V7 was pooled by using the relative risk (RR) with the corresponding 95% confidence intervals (CIs) under a random-effects model. For detecting the potential bias and the heterogeneity of the included studies, sensitivity analysis and subgroup analysis were performed. Publication bias was assessed Egger's and Begg's tests. This study was registered on PROSPERO (ID: CRD42022297014). Results: This cumulative analysis included 672 participants from seven clinical trials. The study group contained 354 CRPC patients, while the other group contained 318 HSPC patients. Pooled results from the seven eligible studies showed that the expression of positive AR-V7 was significantly higher in men with CRPC compared to those with HSPC (RR = 7.55, 95% CI: 4.61-12.35, p < 0.001). In the sensitivity analysis, the combined RRs did not change substantially, ranging from 6.85 (95% CI: 4.16-11.27, p < 0.001) to 9.84 (95% CI: 5.13-18.87, p < 0.001). In the subgroup analysis, a stronger association was detected in RNA in situ hybridization (RISH) measurement in American patients, and those studies were published before 2011 (all p < 0.001). There was no significant publication bias identified in our study. Conclusion: Evidence from the seven eligible studies demonstrated that patients with CRPC had a significantly elevated positive expression of AR-V7. More investigations are still warranted to clarify the association between CRPC and AR-V7 testing. Systematic review registration: https://www.crd.york.ac.uk/prospero/, identifier CRD42022297014.

Design, Synthesis, and Evaluation of Niclosamide Analogs as Therapeutic Agents for Enzalutamide-Resistant Prostate Cancer.

Niclosamide effectively downregulates androgen receptor variants (AR-Vs) for treating enzalutamide and abiraterone-resistant prostate cancer. However, the poor pharmaceutical properties of niclosamide due to its solubility and metabolic instability have limited its clinical utility as a systemic treatment for cancer. A novel series of niclosamide analogs was prepared to systematically explore the structure-activity relationship and identify active AR-Vs inhibitors with improved pharmaceutical properties based on the backbone chemical structure of niclosamide. Compounds were characterized using 1H NMR, 13C NMR, MS, and elemental analysis. The synthesized compounds were evaluated for antiproliferative activity and downregulation of AR and AR-V7 in two enzalutamide-resistant cell lines, LNCaP95 and 22RV1. Several of the niclosamide analogs exhibited equivalent or improved anti-proliferation effects in LNCaP95 and 22RV1 cell lines (B9, IC50 LNCaP95 and 22RV1 = 0.130 and 0.0997 µM, respectively), potent AR-V7 down-regulating activity, and improved metabolic stability. In addition, both a traditional structure-activity relationship (SAR) and 3D-QSAR analysis were performed to guide further structural optimization. The presence of two -CF3 groups of the most active B9 in the sterically favorable field and the presence of the -CN group of the least active B7 in the sterically unfavorable field seem to make B9 more potent than B7 in the antiproliferative activity.

Cell-by-cell quantification of the androgen receptor in benign and malignant prostate leads to a better understanding of changes linked to cancer initiation and progression.

The androgen receptor (AR) plays a crucial role in the development and homeostasis of the prostate and is a key therapeutic target in prostate cancer (PCa). The gold standard therapy for advanced PCa is androgen deprivation therapy (ADT), which targets androgen production and AR signaling. However, resistance to ADT develops via AR-dependent and AR-independent mechanisms. As reports on AR expression patterns in PCa have been conflicting, we performed cell-by-cell AR quantification by immunohistochemistry in the benign and malignant prostate to monitor changes with disease development, progression, and hormonal treatment. Prostates from radical prostatectomy (RP) cases, both hormone-naïve and hormone-treated, prostate tissues from patients on palliative ADT, and bone metastases were included. In the normal prostate, AR is expressed in >99% of luminal cells, 51% of basal cells, and 61% of fibroblasts. An increase in the percentage of AR negative (%AR-) cancer cells along with a gradual loss of fibroblastic AR were observed with increasing Gleason grade and hormonal treatment. This was accompanied by a parallel increase in staining intensity of AR positive (AR+) cells under ADT. Staining AR with N- and C-terminal antibodies yielded similar results. The combination of %AR- cancer cells, %AR- fibroblasts, and AR intensity score led to the definition of an AR index, which was predictive of biochemical recurrence in the RP cohort and further stratified patients of intermediate risk. Lastly, androgen receptor variant 7 (ARV7)+ cells and AR- cells expressing neuroendocrine and stem markers were interspersed among a majority of AR+ cells in ADT cases. Altogether, the comprehensive quantification of AR expression in the prostate reveals concomitant changes in tumor cell subtypes and fibroblasts, emphasizing the significance of AR- cells with disease progression and palliative ADT.

Bruceantin targets HSP90 to overcome resistance to hormone therapy in castration-resistant prostate cancer.

Rationale: Aberrant androgen receptor (AR) signaling via full-length AR (AR-FL) and constitutively active AR variant 7 (AR-V7) plays a key role in the development of castration-resistant prostate cancer (CRPC) and resistance to hormone therapies. Simultaneous targeting of AR-FL and AR-V7 may be a promising strategy to overcome resistance to hormone therapy. This study aimed to identify novel drug candidates co-targeting AR-FL and AR-V7 activities and elucidate their molecular mechanism of anti-CRPC activities. Methods: Using a CRPC cell-based reporter assay system, we screened a small library of antimalarial agents to explore the possibility of repositioning them for CRPC treatment and identified bruceantin (BCT) as a potent anti-CRPC drug candidate. A series of cell-based, molecular, biochemical, and in vivo approaches were performed to evaluate the therapeutic potential and molecular mechanism of BCT in CRPC. These approaches include reporter gene assays, cell proliferation, RNA-seq, qRT-PCR, mouse xenografts, co-immunoprecipitation, GST pull-down, immobilized BCT pull-down, molecular modeling, and bioinformatic analyses. Results: We identified BCT as a highly potent inhibitor co-targeting AR-FL and AR-V7 activity. BCT inhibits the transcriptional activity of AR-FL/AR-V7 and downregulates their target genes in CRPC cells. In addition, BCT efficiently suppresses tumor growth and metastasis of CRPC cells. Mechanistically, BCT disrupts the interaction of HSP90 with AR-FL/AR-V7 by directly binding to HSP90 and inhibits HSP90 chaperone function, leading to degradation of AR-FL/AR-V7 through the ubiquitin-proteasome system. Clinically, HSP90 expression is upregulated and correlated with AR/AR-V7 levels in CRPC. Conclusion: Our findings suggest that BCT could serve as a promising therapeutic candidate against CRPC and highlight the potential benefit of targeting AR-FL/AR-V7-HSP90 axis to overcome resistance caused by aberrant AR-FL/AR-V7 signaling.

Role of Androgen Receptor Variants in Prostate Cancer: Report from the 2017 Mission Androgen Receptor Variants Meeting.

CONTEXT: Although a number of studies have demonstrated the importance of constitutively active androgen receptor variants (AR-Vs) in prostate cancer, questions still remain about the precise role of AR-Vs in the progression of castration-resistant prostate cancer (CRPC). OBJECTIVE: Key stakeholders and opinion leaders in prostate cancer convened on May 11, 2017 in Boston to establish the current state of the field of AR-Vs. EVIDENCE ACQUISITION: The meeting "Mission Androgen Receptor Variants" was the second of its kind sponsored by the Prostate Cancer Foundation (PCF). This invitation-only event was attended by international leaders in the field and representatives from sponsoring organizations (PCF and industry sponsors). Eighteen faculty members gave short presentations, which were followed by in-depth discussions. Discussions focused on three thematic topics: (1) potential of AR-Vs as biomarkers of therapeutic resistance; (2) role of AR-Vs as functionally active CRPC progression drivers; and (3) utility of AR-Vs as therapeutic targets in CRPC. EVIDENCE SYNTHESIS: The three meeting organizers synthesized this meeting report, which is intended to summarize major data discussed at the meeting and identify key questions as well as strategies for addressing these questions. There was a critical consensus that further study of the AR-Vs is an important research focus in CRPC. Contrasting views and emphasis, each supported by data, were presented at the meeting, discussed among the participants, and synthesized in this report. CONCLUSIONS: This article highlights the state of knowledge and outlines the most pressing questions that need to be addressed to advance the AR-V field. PATIENT SUMMARY: Although further investigation is needed to delineate the role of androgen receptor (AR) variants in metastatic castration-resistant prostate cancer, advances in measurement science have enabled development of blood-based tests for treatment selection. Detection of AR variants (eg, AR-V7) identified a patient population with poor outcomes to existing AR-targeting therapies, highlighting the need for novel therapeutic agents currently under development.

Analytical Validation and Clinical Qualification of a New Immunohistochemical Assay for Androgen Receptor Splice Variant-7 Protein Expression in Metastatic Castration-resistant Prostate Cancer.

BACKGROUND: The androgen receptor splice variant-7 (AR-V7) has been implicated in the development of castration-resistant prostate cancer (CRPC) and resistance to abiraterone and enzalutamide. OBJECTIVE: To develop a validated assay for detection of AR-V7 protein in tumour tissue and determine its expression and clinical significance as patients progress from hormone-sensitive prostate cancer (HSPC) to CRPC. DESIGN, SETTING, AND PARTICIPANTS: Following monoclonal antibody generation and validation, we retrospectively identified patients who had HSPC and CRPC tissue available for AR-V7 immunohistochemical (IHC) analysis. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Nuclear AR-V7 expression was determined using IHC H score (HS) data. The change in nuclear AR-V7 expression from HSPC to CRPC and the association between nuclear AR-V7 expression and overall survival (OS) was determined. RESULTS AND LIMITATIONS: Nuclear AR-V7 expression was significantly lower in HSPC (median HS 50, interquartile range [IQR] 17.5-90) compared to CRPC (HS 135, IQR 80-157.5; p<0.0001), and in biopsy tissue taken before (HS 80, IQR 30-136.3) compared to after (HS 140, IQR 105-167.5; p=0.007) abiraterone or enzalutamide treatment. Lower nuclear AR-V7 expression at CRPC biopsy was associated with longer OS (hazard ratio 1.012, 95% confidence interval 1.004-1.020; p=0.003). While this monoclonal antibody primarily binds to AR-V7 in PC biopsy tissue, it may also bind to other proteins. CONCLUSIONS: We provide the first evidence that nuclear AR-V7 expression increases with emerging CRPC and is prognostic for OS, unlike antibody staining for the AR N-terminal domain. These data indicate that AR-V7 is important in CRPC disease biology; agents targeting AR splice variants are needed to test this hypothesis and further improve patient outcome from CRPC. PATIENT SUMMARY: In this study we found that levels of the protein AR-V7 were higher in patients with advanced prostate cancer. A higher level of AR-V7 identifies a group of patients who respond less well to certain prostate cancer treatments and live for a shorter period of time.