Androgen Receptor Splice Variant 7 Expression Research Articles

Recruitment of miR-8080 by luteolin inhibits androgen receptor splice variant 7 expression in castration-resistant prostate cancer.

A need exists for seeking effective treatments for castration-resistant prostate cancer (CRPC) in response to its emergence following androgen deprivation therapy as a major clinical problem. In the present study, we investigated the chemopreventive and chemotherapeutic potential of luteolin, a flavonoid with antioxidative properties, on prostate cancer, including CRPC. Luteolin inhibited the progression of rat prostate carcinogenesis by induction of apoptosis in a transgenic rat for adenocarcinoma of prostate (TRAP) model. Luteolin decreased cell proliferation in a dose-dependent manner and induced apoptosis with the activation of caspases 3 and 7 in both rat (PCai1, established from a TRAP prostate tumor) and human (22Rv1) CRPC cells. Dietary luteolin also suppressed tumor growth via an increase in apoptosis and inhibition of angiogenesis in PCai1 and 22Rv1 xenografts implanted in castrated nude mice. We also focused on androgen receptor splice variant 7 (AR-V7), which contributes to cell proliferation and therapeutic resistance in CRPC. Luteolin dramatically suppressed AR-V7 protein expression in 22Rv1 cells in vitro and ex vivo. Microarray analysis identified MiR-8080, which contains a possible target sequence for AR-V7 3′-UTR, as a gene upregulated by luteolin. MiR-8080 transfection decreased the AR-V7 expression level and the induction of apoptosis in 22Rv1 cells. Furthermore, miR-8080 knockdown canceled luteolin decreasing AR-V7 and the cell growth of 22Rv1. MiR-8080 induced by luteolin intake enhanced the therapeutic effect of enzalutamide on 22Rv1 xenografts under castration conditions. These results indicate luteolin inhibits CRPC by AR-V7 suppression through miR-8080, highlighting luteolin and miR-8080 as promising therapeutic agents for this disease.

MRNA expressions of androgen receptor and its variants in matched hormone-sensitive and castration-resistant prostate cancer

Objectives: Androgen receptor splice variants (AR-Vs), especially androgen receptor splice variant 7 (AR-V7), are considered as important factors in developing castration-resistance of prostate cancer and also as candidate predictive factors. Our aim was to evaluate changes in the mRNA expression of full-length AR (AR-FL) and AR-Vs in the primary prostate cancers from the same patients before and after ADT.Methods: We compared morphologic differences and evaluated AR-FL, AR-V7, AR-V4, ARv567es, AR-V3 and AR8 mRNA expression in matched samples of primary hormone-sensitive and castration-resistant prostate cancer (CRPC) from 19 patients.Results: mRNA expression of AR-FL, AR-V7, ARv567es and AR-V3 was present in hormone sensitive prostate cancer (HSPC) and was significantly increased in CRPC in 81.2% (13/16). There were strong positive correlations between AR-FL and AR-V7 (r = 0.93, p < .001), ARv567es (r = 0.72, p < .001) and AR-V3 (r = 0.81, p < .001) mRNA expression. AR-V7/AR-FL ratio was more significantly (>30%) increased after ADT in 25% (4/16) of the patients, who showed significantly (p < .001) worse overall survival. Neuroendocrine differentiation was seen in one patient (5.3%) and the Gleason score was increased in 10 (52.6%) patients.Conclusion: We demonstrated that the expression of AR-V7 is present at low levels in HSPC and is increased in CRPC and the increase is an active process possibly related to aggressive clinical course.

Androgen Receptor Splice Variant, AR-V7, as a Biomarker of Resistance to Androgen Axis-Targeted Therapies in Advanced Prostate Cancer.

Many therapeutic options are now available for men with metastatic castration-resistant prostate cancer (mCRPC), including next-generation androgen receptor axis-targeted therapies (AATTs), immunotherapy, chemotherapy, and radioisotope therapies. No clear consensus has been reached for the optimal sequencing of treatments for patients with mCRPC, and few well-validated molecular markers exist to guide the treatment decisions for individual patients. The androgen receptor splice variant 7 (AR-V7), a splice variant of the androgen receptor mRNA resulting in the truncation of the ligand-binding domain, has emerged as a biomarker for resistance to AATT. AR-V7 expression in circulating tumor cells has been associated with poor outcomes in patients treated with second- and third-line AATTs. Clinically validated assays are now commercially available for the AR-V7 biomarker. In the present review of the current literature, we have summarized the biology of resistance to AATT, with a focus on the AR-V7; and the clinical studies that have validated AR-V7 expression as a strong independent predictor of a lack of clinical benefit from AATTs. Existing evidence has indicated that patients with AR-V7-positive mCRPC will have better outcomes if treated with taxane chemotherapy regimens rather than additional AATTs.

Incidence of androgen receptor and androgen receptor variant 7 coexpression in prostate cancer.

Prostate cancer (PRCA) is an androgen-driven disease, where androgens act through the androgen receptor (AR) to induce proliferation and survival of tumor cells. Recently, AR splice variant 7 (ARv7) has been implicated in advanced stages of PRCA and clinical recurrence. With the widespread use of AR-targeted therapies, there has been a rising interest in the expression of full-length AR and ARv7 in PRCA progression and how these receptors, both independently and together, contribute to adverse clinicopathologic outcomes. Despite a multitude of studies measuring the expression levels of AR and ARv7 in PRCA progression, the results have been inconsistent and sometimes contradictory due to technical and analytical discrepancies. To circumvent these inconsistencies, we used an automated multiplexed immunostaining platform for full-length AR and ARv7 in human PRCA samples and objectively quantified expression changes with machine learning-based software. With this technology, we can assess receptor prevalence both independently, and coexpressed, within specific tissue and cellular compartments. Full-length AR and ARv7 expression increased in epithelial nuclei of metastatic samples compared to benign. Interestingly, a population of cells with undetectable AR persisted through all stages of PRCA progression. Coexpression analyses showed an increase of the double-positive (AR+ /ARv7+ ) population in metastases compared to benign, and an increase of the double-negative population in PRCA samples compared to benign. Importantly, analysis of clinicopathologic outcomes associated with AR/ARv7 coexpression showed a significant decrease in the double-positive population with higher Gleason score (GS), as well as in samples with recurrence in under 5 years. Conversely, the double-negative population was significantly increased in samples with higher GS and in samples with recurrence in under 5 years. Changes in AR and ARv7 coexpression may have prognostic value in PRCA progression and recurrence. A better understanding of the prevalence and clinicopathologic outcomes associated with changes in these receptors' coexpression may provide a foundation for improved diagnosis and therapy for men with PRCA.

Analysis of AR/ARV7 Expression in Isolated Circulating Tumor Cells of Patients with Metastatic Castration-Resistant Prostate Cancer (SAKK 08/14 IMPROVE Trial).

Despite several treatment options and an initial high response rate to androgen deprivation therapy, the majority of prostate cancers will eventually become castration-resistant in the metastatic stage (mCRPC). Androgen receptor splice variant 7 (ARV7) is one of the best-characterized androgen receptor (AR) variants whose expression in circulating tumor cells (CTCs) has been associated with enzalutamide resistance. ARV7 expression analysis before and during enzalutamide treatment could identify patients requiring alternative systemic therapies. However, a robust test for the assessment of the ARV7 status in patient samples is still missing. Here, we implemented an RT-qPCR-based assay for detection of AR full length (ARFL)/ARV7 expression in CTCs for clinical use. Additionally, as a proof-of-principle, we validated a cohort of 95 mCRPC patients initiating first line treatment with enzalutamide or enzalutamide/metformin within a clinical trial. A total of 95 mCRPC patients were analyzed at baseline of whom 27.3% (26/95) had ARFL+ARV7+, 23.1% (22/95) had ARFL+ARV7−, 23.1% (22/95) had ARFL−ARV7−, and 1.1% (1/95) had ARFL−ARV7+ CTCs. In 11.6% (11/95), no CTCs could be isolated. A total of 25/95 patients had another CTC analysis at progressive disease, of whom 48% (12/25) were ARV7+. Of those, 50% (6/12) were ARV7− and 50% (6/12) were ARV7+ at baseline. Our results show that mRNA analysis of isolated CTCs in mCRPC is feasible and allows for longitudinal endocrine agent response monitoring and hence could contribute to treatment optimization in mCRPC.

Abstract 1335: Digital droplet PCR (ddPCR)-based detection of androgen receptor splice variant 7 (AR-v7) in non-metastatic castration resistant prostate cancer

Abstract Background: The androgen receptor (AR) is a clinically important driver in prostate cancer. In metastatic castration resistant prostate cancer (mCRPC), increased expression of the ligand-independent AR variant 7 (AR-v7) is a biomarker of hormonal therapy resistance. However, the prevalence and clinical importance of AR-v7 in non-metastatic CRPC (nmCRPC) is not yet known. Low circulating tumor cell (CTC) frequencies in these patients make emergence of AR-v7 during anti-androgen therapy difficult to study. We report blood-based detection of AR-v7 using digital droplet PCR (ddPCR) in nmCRPC patients enrolled in the SPARTAN trial, a randomized phase 3 study testing ADT vs apalutamide (APA)+ADT. Method: To investigate simultaneous and quantitative expression of AR-v7 and AR, we utilized ddPCR to measure individual mRNA transcripts in blood samples collected in PAXgene tubes. AR-v7 positivity was calculated as the normalized fraction of AR-v7 vs total AR transcripts (AR-v7/AR). Normalized AR-v7/AR frequency in healthy volunteers (HV) and mCRPC was measured to determine an expression cutoff to separate normal and prostate cancer blood samples. The ddPCR AR-v7 biomarker assay was then used to measure AR-v7 expression in ADT (N=47) and APA+ADT (N=53) SPARTAN samples taken at time of study initiation and correlated with clinical outcome. Results: By setting a cutoff at 0.3 AR-v7/AR normalized fraction we could differentiate HV from mCRPC patients. In nmCRPC, mean AR-v7 and AR-FL expression were calculated as 1.2 and 349.3 transcripts, respectively. The 0.3 AR-v7/AR normalized fraction cutoff could not differentiate AR-v7 expression between HV and nmCRPC. Using this assay, we detected AR-v7 transcripts in 47% of nmCRPC SPARTAN patients analyzed. However, results of AR-v7 expression as a continuous and discretized variable were inconclusive when correlated with clinical outcome. Conclusion: This study reports ddPCR-based detection of whole blood mRNA as a sensitive assay to detect simultaneously low and high expressing AR transcripts in nmCRPC. Our technical analysis demonstrates that unlike in mCRPC, low level transcript counts of AR-v7 in nmCRPC may not distinguish expression from baseline in healthy patients. Data from this limited cohort suggest that while AR-v7 is detected in 47% of patients, a higher threshold of expression may be biologically important for driving treatment resistance. Further analysis of this assay in mCRPC and APA refractory samples sequenced with other therapies are needed to confirm the clinical and biological utility of AR-v7 detection by ddPCR assay and inform disease continuum management. Citation Format: Mel Pilar Espaillat, Yashoda Rajpurohit, Mike Gormley, Denis Smirnov, Ian McCaffery, Angela Lopez-Gitlitz, Deborah Ricci, Shibu Thomas. Digital droplet PCR (ddPCR)-based detection of androgen receptor splice variant 7 (AR-v7) in non-metastatic castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1335.

The effect of androgen receptor splice variant 7 on the growth of castration-resistant prostate cancer and the efficacy of taxane chemotherapy.

302 Background: Expression of androgen receptor (AR) splice variant 7 (AR-V7) has been identified as a mechanism associated with the development of castration-resistant prostate cancer (CRPC), but a potential link between AR-V7 expression and resistance to taxanes, such as docetaxel or cabazitaxel, has not been unequivocally demonstrated. We clarify the relationship between AR-V7 expression and resistance to taxanes. Furthermore, we assess the inhibitory effect of EPI-002, an antagonist of AR amino-terminal domain (NTD), to an AR-V7 driven CRPC cell line with acquired resistance to docetaxel. Methods: LNCaP95-DR cells, which express AR-V7 and exhibit resistance to enzalutamide and docetaxel, was developed. WST-1 assay or BrdU ELISA assay was performed to evaluate the inhibitory effect of drugs. We examined alterations of AR and AR-V7 signaling using Western blot analyses, real-time RT-qPCR and reporter gene assay. Results: LNCaP95-DR cells showed cross-resistance to cabazitaxel. Although the LNCaP95-DR cells had increased expression of AR-V7 and its target genes (UBE2C, CDC20), knockdown of AR-V7 did not restore sensitivity to docetaxel or cabazitaxel. However, despite resistance to docetaxel and carbazitaxel, EPI-002 had an inhibitory effect on the proliferation of LNCaP95-DR cells that was similar to that achieved with the parental LNCaP95 cells, whereas enzalutamide had no effect on the proliferation of either cell line. Conclusions: Our results suggest that EPI-002 may be an option for the treatment of AR-V7-driven CRPC that is resistant to taxanes.

Effect of Monoamine oxidase A (MAOA) inhibitors on androgen-sensitive and castration-resistant prostate cancer cells.

Monoamine oxidase A (MAOA) is best known for its role in neuro-transmitter regulation. Monoamine oxidase inhibitors are used to treat atypical depression. MAOA is highly expressed in high grade prostate cancer and modulates tumorigenesis and progression in prostate cancer. Here, we investigated the potential role of MAOA inhibitors (MAOAIs) in relation to the androgen receptor (AR) pathway and resistance to antiandrogen treatment in prostate cancer. We examined MAOA expression and the effect of MAOI treatment in relation to AR-targeted treatments using the LNCaP, C4-2B, and 22Rv1 human prostate cancer cell lines. MAOA, AR-full length (AR-FL), AR splice variant 7 (AR-V7), and PSA expression was evaluated in the presence of MAOAIs (clorgyline, phenelzine), androgenic ligand (R1881), and antiandrogen (enzalutamide) treatments. An enzalutamide resistance cell line was generated to test the effect of MAOAI treatment in this model. We observed that MAOAIs, particularly clorgyline and phenelzine, were effective at decreasing MAOA activity in human prostate cancer cells. MAOAIs significantly decreased growth of LNCaP, C4-2B, and 22Rv1 cells and produced additive growth inhibitory effects when combined with enzalutamide. Clorgyline decreased expression of AR-FL and AR-V7 in 22Rv1 cells and was effective at decreasing growth of an enzalutamide-resistant C4-2B cell line with increased AR-V7 expression. MAOAIs decrease growth and proliferation of androgen-sensitive and castration-resistant prostate cancer cells. Clorgyline, in particular, decreases expression of AR-FL and AR-V7 expression and decreases growth of an enzalutamide-resistant cell line. These findings provide preclinical validation of MAOA inhibitors either alone or in combination with antiandrogens for therapeutic intent in patients with advanced forms of prostate cancer.

Androgen receptor splice variant-7 expression emerges with castration resistance in prostate cancer.

BACKGROUND. Liquid biopsies have demonstrated that the constitutively active androgen receptor splice variant-7 (AR-V7) associates with reduced response and overall survival from endocrine therapies in castration-resistant prostate cancer (CRPC). However, these studies provide little information pertaining to AR-V7 expression in prostate cancer (PC) tissue.METHODS. Following generation and validation of a potentially novel AR-V7 antibody for IHC, AR-V7 protein expression was determined for 358 primary prostate samples and 293 metastatic biopsies. Associations with disease progression, full-length androgen receptor (AR-FL) expression, response to therapy, and gene expression were determined.RESULTS. We demonstrated that AR-V7 protein is rarely expressed (<1%) in primary PC but is frequently detected (75% of cases) following androgen deprivation therapy, with further significant (P = 0.020) increase in expression following abiraterone acetate or enzalutamide therapy. In CRPC, AR-V7 expression is predominantly (94% of cases) nuclear and correlates with AR-FL expression (P ≤ 0.001) and AR copy number (P = 0.026). However, dissociation of expression was observed, suggesting that mRNA splicing remains crucial for AR-V7 generation. AR-V7 expression was heterogeneous between different metastases from a patient, although AR-V7 expression was similar within a metastasis. Moreover, AR-V7 expression correlated with a unique 59-gene signature in CRPC, including HOXB13, a critical coregulator of AR-V7 function. Finally, AR-V7–negative disease associated with better prostate-specific antigen (PSA) responses (100% vs. 54%, P = 0.03) and overall survival (74.3 vs. 25.2 months, hazard ratio 0.23 [0.07–0.79], P = 0.02) from endocrine therapies (pre-chemotherapy).CONCLUSION. This study provides impetus to develop therapies that abrogate AR-V7 signaling to improve our understanding of AR-V7 biology and to confirm the clinical significance of AR-V7.FUNDING. Work at the University of Washington and in the Plymate and Nelson laboratories is supported by the Department of Defense Prostate Cancer Research Program (W81XWH-14-2-0183, W81XWH-12-PCRP-TIA, W81XWH-15-1-0430, and W81XWH-13-2-0070), the Pacific Northwest Prostate Cancer SPORE (P50CA97186), the Institute for Prostate Cancer Research, the Veterans Affairs Research Program, the NIH/National Cancer Institute (P01CA163227), and the Prostate Cancer Foundation. Work in the de Bono laboratory was supported by funding from the Movember Foundation/Prostate Cancer UK (CEO13-2-002), the US Department of Defense (W81XWH-13-2-0093), the Prostate Cancer Foundation (20131017 and 20131017-1), Stand Up To Cancer (SU2C-AACR-DT0712), Cancer Research UK (CRM108X-A25144), and the UK Department of Health through an Experimental Cancer Medicine Centre grant (ECMC-CRM064X).

Androgen Receptor Splice Variant 7 Drives the Growth of Castration Resistant Prostate Cancer without Being Involved in the Efficacy of Taxane Chemotherapy.

Expression of androgen receptor (AR) splice variant 7 (AR-V7) has been identified as the mechanism associated with the development of castration-resistant prostate cancer (CRPC). However, a potential link between AR-V7 expression and resistance to taxanes, such as docetaxel or cabazitaxel, has not been unequivocally demonstrated. To address this, we used LNCaP95-DR cells, which express AR-V7 and exhibit resistance to enzalutamide and docetaxel. Interestingly, LNCaP95-DR cells showed cross-resistance to cabazitaxel. Furthermore, these cells had increased levels of P-glycoprotein (P-gp) and their sensitivity to both docetaxel and cabazitaxel was restored through treatment with tariquidar, a P-gp antagonist. Results generated demonstrated that P-gp mediated cross-resistance between docetaxel and cabazitaxel. Although the LNCaP95-DR cells had increased expression of AR-V7 and its target genes (UBE2C, CDC20), the knockdown of AR-V7 did not restore sensitivity to docetaxel or cabazitaxel. However, despite resistance to docetaxel and carbazitaxel, EPI-002, an antagonist of the AR amino-terminal domain (NTD), had an inhibitory effect on the proliferation of LNCaP95-DR cells, which was similar to that achieved with the parental LNCaP95 cells. On the other hand, enzalutamide had no effect on the proliferation of either cell line. In conclusion, our results suggested that EPI-002 may be an option for the treatment of AR-V7-driven CRPC, which is resistant to taxanes.

Detection of androgen receptor (AR) and AR-V7 in small cell prostate carcinoma: Diagnostic and therapeutic implications

ObjectiveSmall cell prostate carcinoma (SCPC) is a rare and highly malignant subtype of prostate cancer. SCPC frequently lacks androgen receptor (AR) and prostate-specific antigen (PSA) expression, and often responds poorly to androgen deprivation therapy (ADT). AR splice variant-7 (AR-V7) is a truncated AR protein implicated in resistance to AR-targeting therapies. AR-V7 expression in castration-resistant prostate cancers has been evaluated extensively, and blood-based detection of AR-V7 has been associated with lack of response to abiraterone and enzalutamide. However, whether AR-V7 is expressed in SCPC is not known. MethodsUsing validated antibodies, we performed immunohistochemistry (IHC) assay for the full-length AR (AR-FL) and (AR-V7) on post-ADT surgical SCPC specimens. ResultsSeventy-five percent (9/12) of the specimens showed positive staining for the AR-FL with various intensities. Thirty-three percent (4/12) of the specimens showed positive staining for AR-V7. Among the specimens with positive AR-V7 staining, two samples displayed very weak staining, one sample showed weak-to-moderate staining, and one sample showed strong staining. All positive specimens displayed a heterogeneous pattern of AR-FL/AR-V7 staining. All specimens positive for AR-V7 were also positive for AR-FL. ConclusionThe study findings support the existence of measurable AR-FL and AR-V7 proteins in SCPC specimens. The results also have implications in detection of AR-V7 in specimens obtained through systemic sampling approaches such as circulating tumor cells. A positive AR-V7 finding by blood-based tests is not impossible in patients with SCPC who often demonstrate low PSA values.

ZFX Mediates Non-canonical Oncogenic Functions of the Androgen Receptor Splice Variant 7 in Castrate-Resistant Prostate Cancer

Androgen receptor splice variant 7 (AR-V7) is crucial for prostate cancer progression and therapeutic resistance. We show that, independent of ligand, AR-V7 binds both androgen-responsive elements (AREs) and non-canonical sites distinct from full-length AR (AR-FL) targets. Consequently, AR-V7 not only recapitulates AR-FL's partial functions but also regulates an additional gene expression program uniquely via binding to gene promoters rather than ARE enhancers. AR-V7 binding and AR-V7-mediated activation at these unique targets do not require FOXA1 but rely on ZFX and BRD4. Knockdown of ZFX or select unique targets of AR-V7/ZFX, or BRD4 inhibition, suppresses growth of castration-resistant prostate cancer cells. We also define an AR-V7 direct target gene signature that correlateswith AR-V7 expression in primary tumors, differentiates metastatic prostate cancer from normal, and predicts poor prognosis. Thus, AR-V7 has both ARE/FOXA1 canonical and ZFX-directed non-canonical regulatory functions in the evolution of anti-androgen therapeutic resistance, providing information to guide effective therapeutic strategies.

Abstract 5777: Ormeloxifene augments the therapeutic response of enzalutamide via targeting androgen receptor splice variant 7

Abstract Enzalutamide (ENZ) is a second generation anti-androgen drug in clinical use for the treatment of advanced prostate cancer (PrCa). Use of ENZ has some limitations because of its toxic side effect and developing chemoresistance. It has been shown that expression of androgen receptor splice variant 7 (ARV7) in advanced PrCa is involved in ENZ therapy resistance. It is noteworthy that ENZ does not target AR splice variants because these lack the ligand binding domain of AR. Thus, identification of non-toxic agents which can target ARV7 can improve the enzalutamide therapy response and could be used for the management of advanced stage PrCa. Herein, we identified a clinically approved selective estrogen receptor modulator ormeloxifene (ORM), which augments ENZ therapy response via suppression of ARV7 expression in PrCa cells. We used androgen-independent human prostate cancer cells (22Rv1), which express ARV7 as a model system to study the effect of ORM alone or in combination with ENZ. ORM treatment showed a more potent effect in 22Rv1 cells as compared to ENZ alone. ORM treatment also sensitized the effects of ENZ in 22Rv1 cells. ORM treatment effectively inhibited colony formation ability of 22Rv1 cells; this effect was additive in combination with ENZ. We next performed Western blot analysis of ARV7 in control and ORM treated 22Rv1 cells. ORM effectively inhibited the protein level of AR V7 at 12 hrs post-treatment in 22Rv1 cells; however, no effect was observed with the treatment of ENZ. ORM treatment also inhibited the promoter activity of the AR target gene PSA as determined by luciferase assay. To translate these finding to an in vivo model system, we investigated the chemo-sensitization potential of ORM in a 22Rv1 cell-derived xenograft mouse model. ORM treatment (200 µg/mouse three times a week for 8 consecutive weeks) significantly (P&amp;lt;0.01) inhibited prostate tumor growth. However, we did not observe any significant tumor growth inhibition with oral administration of ENZ (10 mg/kg body weight). ORM treatment improved the therapeutic response of ENZ in 22Rv1 cells derived xenograft mouse model as monitored by significant (P&amp;lt;0.01) decreases in both tumor volume and tumor weight as compared to ENZ treatment alone. Our findings indicate that ORM alone or in combination with ENZ could be an effective therapeutic approach for the treatment of advanced PrCa. Citation Format: Bilal B. Hafeez, Andrew E. Massey, Vivek K. Kashyap, Mohammed Sikander, Advit Shetty, Mehdi Chaib, Hassan Mandil, Shabnam Malik, Mohan M. Yallapu, Meena Jaggi, Subhash C. Chauhan. Ormeloxifene augments the therapeutic response of enzalutamide via targeting androgen receptor splice variant 7 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5777.

AR-V7 in circulating tumor cells cluster as a predictive biomarker of abiraterone acetate and enzalutamide treatment in castration-resistant prostate cancer patients.

We examined whether androgen receptor splice variant 7 (AR-V7) in circulating tumor cell(CTC)clusters can be used to predict survival in patients with bone metastatic castration resistant-prostate cancer (mCRPC) treated with abiraterone or enzalutamide. We retrospectively enrolled 98 patients with CRPC on abiraterone or enzalutamide, and investigated the prognostic value of CTC cluster detection (+ v -) and AR-V7 detection (+ v -) using a CTC cluster detection - based AR-V7 mRNA assay. We examined ≤50% prostate-specific antigen (PSA) responses, PSA progression-free survival (PSA-PFS), clinical and radiological progression-free survival (radiologic PSF), and overall survival (OS). We then assessed whether AR-V7 expression in CTC clusters identified after On-chip multi-imaging flow cytometry was related to disease progression and survival after first-line systemic therapy. All abiraterone-treated or enzalutamide-treated patients received prior docetaxel. The median follow-up was 20.7 (range: 3.0-37.0) months in the abiraterone and enzalutamide cohorts, respectively. Forty-nine of the 98 men (50.0%) were CTC cluster (-), 23 of the 98 men (23.5%) were CTC cluster(+)/AR-V7(-), and 26 of the 98 men (26.5%) were CTC cluster(+)/AR-V7(+). CTC cluster(+)/AR-V7(+) patients were more likely to have EOD ≥3 at diagnosis (P = 0.003), pain (P = 0.023), higher alkaline phosphatase levels (P < 0.001), and visceral metastases (P < 0.001). On multivariable analysis, pretherapy CTC cluster(+), CTC cluster(+)/AR-V7(-), and ALP >UNL were independently associated with a poor PSA-PFS, radiographic PFS, and OS in abiraterone-treated patients and enzalutamide-treated patients. The CTC clusters and AR-V7-positive CTC clusters detected were important for assessing the response to abiraterone or enzalutamide therapy and for predicting disease outcome.

In Situ Detection and Quantification of AR-V7, AR-FL, PSA, and KRAS Point Mutations in Circulating Tumor Cells.

Liquid biopsies can be used in castration-resistant prostate cancer (CRPC) to detect androgen receptor splice variant 7 (AR-V7), a splicing product of the androgen receptor. Patients with AR-V7-positive circulating tumor cells (CTCs) have greater benefit of taxane chemotherapy compared with novel hormonal therapies, indicating a treatment-selection biomarker. Likewise, in those with pancreatic cancer (PaCa), KRAS mutations act as prognostic biomarkers. Thus, there is an urgent need for technology investigating the expression and mutation status of CTCs. Here, we report an approach that adds AR-V7 or KRAS status to CTC enumeration, compatible with multiple CTC-isolation platforms. We studied 3 independent CTC-isolation devices (CellCollector, Parsortix, CellSearch) for the evaluation of AR-V7 or KRAS status of CTCs with in situ padlock probe technology. Padlock probes allow highly specific detection and visualization of transcripts on a cellular level. We applied padlock probes for detecting AR-V7, androgen receptor full length (AR-FL), and prostate-specific antigen (PSA) in CRPC and KRAS wild-type (wt) and mutant (mut) transcripts in PaCa in CTCs from 46 patients. In situ analysis showed that 71% (22 of 31) of CRPC patients had detectable AR-V7 expression ranging from low to high expression [1-76 rolling circle products (RCPs)/CTC]. In PaCa patients, 40% (6 of 15) had KRAS mut expressing CTCs with 1 to 8 RCPs/CTC. In situ padlock probe analysis revealed CTCs with no detectable cytokeratin expression but positivity for AR-V7 or KRAS mut transcripts. Padlock probe technology enables quantification of AR-V7, AR-FL, PSA, and KRAS mut/wt transcripts in CTCs. The technology is easily applicable in routine laboratories and compatible with multiple CTC-isolation devices.

Assessment of immunohistochemical testing for androgen receptor splice variant 7 as a biomarker to predict resistance to androgen receptor signaling inhibitors in metastatic prostate cancer.

308 Background: Androgen receptor splice variant 7 (AR-V7) has been shown to confer resistance to androgen receptor signaling inhibitors (ARS-I) such as enzalutamide (enza) and abiraterone (abi). Resistance can be observed at the time of initial treatment, or may be acquired later in the disease course. Prior research has primarily focused on AR-V7 expression in circulating tumor cells (CTCs) but the utility of more affordable immunohistochemical (IHC) testing to predict for primary resistance to ARS-I remains unknown, which is of particular relevance as ARS-I gain use as initial therapy. Methods: We identified patients in the South Texas Veterans Health Administration Tumor Registry with metastatic, castrate-resistant prostate cancer who received ARS-I since 2011. IHC for AR-V7 staining was validated on controls and performed on tissue from the most recent tissue specimen (diagnostic biopsy, prostatectomy, or biopsy of a metastatic site) on all identified patients with adequate tissue available. Results: 25 of 42 (60%) patients receiving abi had PSA response with median duration of response (DOR) of 231 days. 14 of 26 (54%) of patients receiving enza experienced PSA response with a median DOR of 165 days. IHC is currently being interpreted on stored tissue specimens to assess predictive efficacy of AR-V7 staining. The de-novo rate of AR-V7 expression and correlation with response to ARS-I in the veteran population will be reported with final results. Conclusions: IHC testing for AR-V7 may provide a cost-effective biomarker to identify patients resistant to ARS-I, thus avoiding thus avoiding time-consuming and costly treatment with ineffective therapy. Further study is warranted to assess cost-effectiveness and reduction in unnecessary toxicity by the use of IHC testing for AR-V7 in the front-line setting to predict primary resistance for patients that would otherwise qualify for ARS-I.

Androgen-receptor splice variant-7-positive prostate cancer: a novel molecular subtype with markedly worse androgen-deprivation therapy outcomes in newly diagnosed patients

Androgen-deprivation therapy has been the standard treatment for metastatic and locally advanced prostate cancer, but the majority of patients will progress to castration-resistant prostate cancer within 2–3 years. Unlike the case in breast cancer, no clinically validated biomarker has been used to predict the outcomes of androgen-deprivation therapy. To evaluate androgen-receptor splice variant-7 (AR-V7) detection in newly diagnosed advanced prostate cancer and describe the distinctive prognosis of this novel molecular subtype, this study retrospectively enrolled 168 newly diagnosed prostate cancer patients from 2003 to 2015 who received androgen-deprivation therapy. AR-V7 immunohistochemical staining was performed with a monoclonal antibody, and AR-V7 expression was determined using Immune-Reactive Score data. The association between nuclear AR-V7 expression and prognosis was determined. Multiple cause-specific Cox regression and stratified cumulative incidences were used to analyze the prognosis risk. Among the 168 patients, 32 (19%) were AR-V7-positive. Compared with the AR-V7-negative patients, the AR-V7-positive patients had significantly lower prostate-specific antigen response rates (P<0.001) to androgen-deprivation therapy and a much shorter time to castration-resistant prostate cancer (P<0.0001). In Kaplan–Meier analysis, the AR-V7-positive group showed markedly lower castration-resistant prostate cancer progression-free survival (P<0.0001) and much lower cancer-specific (P<0.0001) and overall survival (P<0.0001) both in all enrolled patients and in patients with metastases. AR-V7 positivity was a significant predictor of castration-resistant prostate cancer progression in multiple Cox regression (hazard ratio: 4.826; 95% CI: 2.960–7.869; P<0.001). AR-V7 immunohistochemical detection in newly diagnosed prostate cancer patients who are planning to receive androgen-deprivation therapy, especially those with metastases, is necessary and valuable for prognostic assessment. AR-V7-positive prostate cancer should be considered a novel prostate cancer subtype that should be distinguished upon initial biopsy. The main limitation of this study is its observational nature.

Overexpression of nuclear AR-V7 protein in primary prostate cancer is an independent negative prognostic marker in men with high-risk disease receiving adjuvant therapy

BackgroundOverexpression of the androgen receptor (AR) splice variant 7 (AR-V7) has recently been reported to be associated with resistance to antihormonal therapy. Herein, we address the question whether tumor cells with AR-V7 expression can be detected at the time of radical prostatectomy, that is, before long-term hormonal manipulation and castration resistance, and what the potential prognostic impact on the biochemical recurrence (BCR)-free survival may be. MethodsAn anti-AR-V7 antibody was first validated in a training set of prostate cancer specimens by a comparison of AR-V7 protein to AR-V7 mRNA expression. We then analyzed nuclear AR-V7 protein expression in the primary tumors and lymph node metastases from 163 predominantly high-risk patients (cohort I) as well as the primary tumors from patients of a second, consecutive patient cohort (n = 238, cohort II) not selected for any clinicopathological features. Staining results were correlated to patient characteristics and BCR-free patient survival. ResultsHigh nuclear AR-V7 protein expression was detected in approximately 30%–40% of patients in cohort I and II at the time of radical prostatectomy. High baseline expression of nuclear AR-V7 protein was associated with an unfavorable BCR-free survival in the high-risk patient cohort I but not in the unselected consecutive cohort II. Remarkably, AR-V7 was an independent negative prognostic factor in high-risk prostate cancer patients of cohort I who were selected to receive adjuvant treatment. ConclusionsProstate cancer cells with high nuclear AR-V7 protein expression can be detected in a substantial proportion of tumors at the time of radical prostatectomy. The presence of AR-V7-positive tumor cells is associated with an unfavorable prognosis for BCR-free survival in a high-risk patient cohort including a subgroup of patients selected to receive adjuvant therapy, in which AR-V7 was an independent negative prognosticator. Overexpression of nuclear AR-V7 protein hence identifies a subset of tumors with remarkably aggressive growth characteristics among clinically and histologically high-risk patients at the time of radical prostatectomy.

AR-V7 in Peripheral Whole Blood of Patients with Castration-resistant Prostate Cancer: Association with Treatment-specific Outcome Under Abiraterone and Enzalutamide

BackgroundIt has been demonstrated that androgen receptor splice variant 7 (AR-V7) expression in circulating tumor cells (CTCs) predicts poor treatment response in metastatic castration-resistant prostate cancer (mCRPC) patients treated with abiraterone or enzalutamide. ObjectiveTo develop a practical and robust liquid profiling approach for direct quantification of AR-V7 in peripheral whole blood without the need for CTC capture and to determine its potential for predicting treatment response in mCRPC patients. Design, setting, and participantsWhole blood samples from a prospective biorepository of 85 mCRPC patients before treatment initiation with abiraterone (n=56) or enzalutamide (n=29) were analyzed via droplet digital polymerase chain reaction. Outcome measurements and statistical analysisThe association of AR-V7 status with prostate-specific antigen (PSA) response defined by PSA decline ≥50% and with PSA–progression-free survival (PSA-PFS), clinical PFS, and overall survival (OS) was assessed. Results and limitationsHigh AR-V7 expression levels in whole blood were detectable in 18% (15/85) of patients. No patient with high AR-V7 expression achieved a PSA response, and AR-V7 status was an independent predictor of PSA response in multivariable logistic regression analysis (p=0.03). High AR-V7 expression was associated with shorter PSA-PFS (median 2.4 vs 3.7 mo; p<0.001), shorter clinical PFS (median 2.7 vs 5.5 mo; p<0.001), and shorter OS (median 4.0 vs. 13.9 mo; p<0.001). On multivariable Cox regression analysis, high AR-V7 expression remained an independent predictor of shorter PSA-PFS (hazard ratio [HR] 7.0, 95% confidence interval [CI] 2.3–20.7; p<0.001), shorter clinical PFS (HR 2.3, 95% CI 1.1–4.9; p=0.02), and shorter OS (HR 3.0, 95% CI 1.4–6.3; p=0.005). ConclusionsTesting of AR-V7 mRNA levels in whole blood is a simple and promising approach to predict poor treatment outcome in mCRPC patients receiving abiraterone or enzalutamide. Patient summaryWe established a method for determining AR-V7 status in whole blood. This test predicted treatment resistance in patients with metastatic castration-resistant prostate cancer undergoing treatment with abiraterone or enzalutamide. Prospective validation is needed before application to clinical practice.

Melatonin Inhibits Androgen Receptor Splice Variant-7 (AR-V7)-Induced Nuclear Factor-Kappa B (NF-κB) Activation and NF-κB Activator-Induced AR-V7 Expression in Prostate Cancer Cells: Potential Implications for the Use of Melatonin in Castration-Resistant Prostate Cancer (CRPC) Therapy.

A major current challenge in the treatment of advanced prostate cancer, which can be initially controlled by medical or surgical castration, is the development of effective, safe, and affordable therapies against progression of the disease to the stage of castration resistance. Here, we showed that in LNCaP and 22Rv1 prostate cancer cells transiently overexpressing androgen receptor splice variant-7 (AR-V7), nuclear factor-kappa B (NF-κB) was activated and could result in up-regulated interleukin (IL)-6 gene expression, indicating a positive interaction between AR-V7 expression and activated NF-κB/IL-6 signaling in castration-resistant prostate cancer (CRPC) pathogenesis. Importantly, both AR-V7-induced NF-κB activation and IL-6 gene transcription in LNCaP and 22Rv1 cells could be inhibited by melatonin. Furthermore, stimulation of AR-V7 mRNA expression in LNCaP cells by betulinic acid, a pharmacological NF-κB activator, was reduced by melatonin treatment. Our data support the presence of bi-directional positive interactions between AR-V7 expression and NF-κB activation in CRPC pathogenesis. Of note, melatonin, by inhibiting NF-κB activation via the previously-reported MT1 receptor-mediated antiproliferative pathway, can disrupt these bi-directional positive interactions between AR-V7 and NF-κB and thereby delay the development of castration resistance in advanced prostate cancer. Apparently, this therapeutic potential of melatonin in advanced prostate cancer/CRPC management is worth translation in the clinic via combined androgen depletion and melatonin repletion.