Androgen Receptor Splice Variant AR-V7 Research Articles

Abstract 590: Multiplex liquid biopsy for AR pathway activity in metastatic androgen receptor-positive triple negative breast cancer

Abstract Introduction: The androgen receptor (AR) is expressed in up to 50% of patients with triple negative breast cancer (TNBC), and a luminal androgen receptor (LAR) subtype has been identified that is dependent on androgen receptor signaling, but AR protein expression by immunohistochemistry (IHC) has been a suboptimal biomarker for response to anti-androgen therapies in AR-TNBC and there remains an unmet need for biomarkers to identify patients likely to benefit from anti-androgens. We have developed a multiplex liquid biopsy encompassing AR protein expression and downstream gene expression analysis in circulating tumor cells (CTCs). We report here the longitudinal evaluation of this liquid biopsy as part of a phase I trial (NCT03090165) of the anti-androgen bicalutamide with selective CDK4/6 inhibitor ribociclib in metastatic AR-TNBC. Methods: Peripheral blood was collected (n=11) prior to treatment, after two cycles of treatment, and at progression and processed using the VERSA (Versatile Exclusion-based Rare Sample Analysis) microfluidic chip that integrates CTC capture and downstream analysis. For protein expression, fixed CTCs were captured immunomagnetically and stained on chip for AR followed by quantitative fluorescent microscopy. For gene expression analysis, live CTCs were captured immunomagnetically followed by RNA isolation on chip, reverse transcription, and RT-qPCR for a panel of 40 luminal AR genes. Results: AR protein and transcript were detected in CTCs from 10/11 patients, and AR target gene expression in 8/11 patients. The pathologic ligand independent AR splice variant AR-V7 was detected in 2/11 patients. Pretreatment CTC number was higher in patients with anti-androgen resistant (range 3-753) than anti-androgen sensitive (range 0-30) disease. Among patients with low AR IHC (<10%) on biopsy, high AR protein and transcript expression in pre-treatment CTCs was associated with anti-androgen sensitivity. Among patients with high AR IHC (>50%) on biopsy, AR-V7 was detected in CTCs of 2/3 of patients with resistant disease and 0/2 of patients with sensitive disease. Conclusions: This study demonstrates the feasibility of a longitudinal multiplex liquid biopsy as a pharmacodynamic biomarker of AR pathway activity in AR-TNBC, and suggests that AR pathway activity in CTCs may provide additional information to solid tumor biopsy AR expression in predicting sensitivity to anti-androgens. This work also demonstrates for the first time the detection of AR-V7 in CTCs in anti-androgen resistant AR-TNBC, suggesting that AR-V7 expression may be a mechanism of anti-androgen resistance in AR-TNBC as it is in advanced prostate cancer. Citation Format: Marina N. Sharifi, Serena K. Wolfe, Jamie M. Sperger, Jennifer L. Schehr, Saswati Bhattacharya, Kari B. Wisinski, Joshua M. Lang, Ruth M. O'Regan. Multiplex liquid biopsy for AR pathway activity in metastatic androgen receptor-positive triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 590.

Upregulation of potential regulatory signaling molecules correlate with androgen receptor splice variants AR-V7 and AR-V567es in prostate cancer metastasis.

Androgen receptor splice variants (AR-Vs) produced by alternative splicing of the AR play an important role in the treatment resistance and progression of prostate cancer (PCa). In this study, two most common AR variants and how they associate with the inflammatory response (NF-Kβ) and regulatory transcriptional activity (HSP-27) genes were investigated in patients with PCa and metastatic PCa (Met-PCa). Our study was carried out with the whole blood obtained from 25 healthy control subjects, 25 PCa patients and 39 Met-PCa patients. We examined the expression levels of AR, AR-V7 and AR-V567es genes via Real-time PCR and those of HSP-27 and NF-Kβ via ELISA method. AR, AR-V7 and AR-V567es expressions were observed in 84.61%, 64.1%, 23.07% of Met-PCa patients respectively. The expression levels of full-length AR and variants (AR-V7 and AR-V567es) were associated with the prostate cancer stage. In the Met-PCa, the expression levels of AR, AR-V7 and AR-V567es were associated with the Gleason Scores but not with the PSA levels. AR-V7 expression levels in stage T4 patients significantly increased. NF-Kβ and HSP-27 protein levels were significantly higher in Met-PCa patients. Our findings highlight the targeting of the proteostasis and inflammation pathways through inhibiting HSP-27 and NF-Kβ. This might be a valuable strategy to overcome anti-androgen resistance and improve drug therapy in Met-PCa patients whose gene expression levels of AR-V7 and AR-V567es variants are high.

ACK1-AR and AR-HOXB13 signaling axes: epigenetic regulation of lethal prostate cancers.

The androgen receptor (AR) is a critical transcription factor in prostate cancer (PC) pathogenesis. Its activity in malignant cells is dependent on interactions with a diverse set of co-regulators. These interactions fluctuate depending on androgen availability. For example, the androgen depletion increases the dependence of castration-resistant PCs (CRPCs) on the ACK1 and HOXB13 cell survival pathways. Activated ACK1, an oncogenic tyrosine kinase, phosphorylates cytosolic and nuclear proteins, thereby avoiding the inhibitory growth consequences of androgen depletion. Notably, ACK1-mediated phosphorylation of histone H4, which leads to epigenetic upregulation of AR expression, has emerged as a critical mechanism of CRPC resistance to anti-androgens. This resistance can be targeted using the ACK1-selective small-molecule kinase inhibitor (R)-9b. CRPCs also deploy the bromodomain and extra-terminal domain protein BRD4 to epigenetically increase HOXB13 gene expression, which in turn activates the MYC target genes AURKA/AURKB. HOXB13 also facilitates ligand-independent recruitment of the AR splice variant AR-V7 to chromatin, compensating for the loss of the chromatin remodeling protein, CHD1, and restricting expression of the mitosis control gene HSPB8. These studies highlight the crosstalk between AR–ACK1 and AR–HOXB13 pathways as key mediators of CRPC recurrence.

Abstract 5384: Analytic validation of an assay to detect androgen receptor splice variant ARv7 protein expression on circulating tumor cells from prostate cancer patients

Abstract Circulating tumor cells (CTCs) can provide information on drug target expression, response to therapy, and disease prognosis from a non-invasive blood draw. Currently, investigating biomarkers on CTCs is difficult due to challenges of developing multiplexed assays that also identify rare cells. Presence of the androgen receptor splice variant ARv7 in prostate cancer cells is associated with resistance to second generation anti-androgen therapies. We report here the analytical validation of an immunofluorescence assay for characterization of ARv7 protein expression on CTCs using the RareCyte platform - an end-to-end platform that combines CTC sample preparation, multiparameter fluorescence staining, digital imaging, and single cell retrieval. Blood samples spiked with positive and negative cell lines for ARv7 expression were processed using the AccuCyte Sample Preparation System. Slides were auto-stained by immunofluorescence with the RarePlex ARv7 CTC Panel Kit comprised of a three-channel CTC detection base plus an ARv7 biomarker channel. The detection base consists of a nuclear dye, anti-CD45 antibody to exclude white blood cells, and cocktailed antibodies to cytokeratin (CK) and epithelial cell adhesion molecule (EpCAM). Stained slides were imaged with the CyteFinder Instrument. CTCs were identified using machine learning-based algorithms and confirmed by user review. Mean fluorescence intensity (MFI) measurements were used as a metric for ARv7 expression on confirmed CTCs. Analytic validation studies of the AR-V7 CTC assay were performed using 22RV1 (high), LNCaP (low), and BT-474 (negative) cell lines. Performance characteristics tested for ARv7 included accuracy, sensitivity, specificity, repeatability, and inter-stainer run coefficient of variation. Performance metrics for CTC recovery were calculated on spike-in and clinical samples. For recovery calculations, the number of CTCs found with the ARv7 assay was compared to the number of CTCs found with the CTC detection base assay. An ARv7 MFI threshold that segregated negative and positive cell lines was statistically defined. This threshold identified 80% of 22RV1 cells as positive for ARv7, 97% of BT-474 cells as negative, with an overall accuracy of 90%. When the assay was applied to clinical prostate cancer samples, staining with proper nuclear localization was observed. CTC recovery was at least as high with the ARv7 assay as with the base CTC detection assay. Citation Format: Daniel Campton, Edward Lo, Lillian Costandy, Brady Gardner, Ryan Houston, Jeffery L. Werbin, Kyla Teplitz, Daniel E. Sabath, Alisa C. Clein, Celestia S. Higano, Tanisha M. Mojica, Kristin Province, Eric P. Kaldjian, Arturo B. Ramirez, Tad George. Analytic validation of an assay to detect androgen receptor splice variant ARv7 protein expression on circulating tumor cells from prostate cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5384.

Abstract 1836: ONC201 shows synergistic effect with the androgen receptor AR-inhibitor darotulamide in prostate cancer models

Abstract Prostate cancer (PC) is the most frequently diagnosed cancer among men in the United States and is the 3rd cause of cancer mortality. Despite advances in the treatment and understanding of pathogenesis, patients with metastatic PC invariably progress to a lethal stage of castration-resistant prostate cancer (CRPC). Inhibition of androgen signaling remains crucial for the treatment of CRPC, but novel treatment strategies are urgently needed. ONC201 is a first-in-class, selective inhibitor of dopamine receptor D2 that upregulates death-receptor 5 and induces apoptosis. ONC201 induced apoptosis in PC cell lines and showed synergy with enzalutamide, docetaxel and everolimus (Lev A Mol Cancer Res 2018). Darolutamide (DARO) is a novel androgen receptor (AR) antagonist approved for treatment of patients with non-metastatic CRPC. DARO has higher affinity to AR and preclinical activity against enzalutamide-resistant PC cell lines including AR variants associated with enzalutamide agonism (Borgmann H Eur Urol 2018). We treated human PC cell lines (LNCAP [castration sensitive, AR wild-type], 22Rv1 [castration-resistant cell line that expresses AR splice variant AR-V7], DU145 [AR negative], PC3 [AR negative]) with DARO alone and combined with ONC201. Single-agent DARO decreased PC cell viability in Cell-Titer-Glo assays (IC50s of 10 and 693 nM for LNCAP and DU145, respectively). ONC201 showed strong synergism with DARO in LNCAP cells (combination indices < 1 at concentrations of 156, 312, 625 nM, and 1.25 µM of DARO with 2.5 μM ONC201). ONC201 induced robust apoptosis in 22Rv1 as measured by PARP cleavage, which was partially amplified by DARO. PARP cleavage was also observed in LNCAP cells with less intensity and not potentiated by DARO. ONC201 reduced the expression of phospho-AR (p-AR) in both 22Rv1 and LNCAP. The combination of ONC201 and DARO had a significant additive effect in reducing p-AR in 22Rv1, but not in LNCAP. ONC201 reduced PSA protein levels in LNCAP cells while DARO alone did not cause any significant change in PSA. Immunofluorescence experiments showed that DARO caused significant reduction of AR nuclear translocation in both 22rV1 and LNCAP cells. The combination with ONC201 potentiated this inhibition of AR translocation in both cell lines. ONC201 showed strong antiproliferative activity against PC cells lines independent of AR status. Combination of ONC201 with DARO demonstrated synergy in enzalutamide resistant 22Rv1 cells expressing AR-V7 with marked reduction of nuclear localization of AR. Our studies provide preclinical rationale for combination of ONC201 with DARO as a novel therapy of PC. Citation Format: Fabio Tavora, Lanlan Zhou, Ali Amin, Safran Howard, Navaraj Arunasalam, Andre de Souza, Anthony Mega, Dragan Golijanin, Wafik El-Deiry, Benedito Carneiro. ONC201 shows synergistic effect with the androgen receptor AR-inhibitor darotulamide in prostate cancer models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1836.

Abstract 1016: The androgen receptor splice variant AR-V7 associates with the glucocorticoid receptor following androgen blockade

Abstract Expression of the constitutively active androgen receptor splice variant AR-V7 is associated with resistance to anti-androgen therapies and worse clinical outcomes in prostate cancer. Dimerization is a requirement for AR-V7 transcriptional activity, but whether that occurs through homodimerization or heterodimerization remains poorly understood. Because NR3C-family nuclear hormone receptors rely on a highly conserved D-box motif within the DNA-binding domain to mediate dimerization, we hypothesized that AR-V7 can heterodimerize with other NR3C-family nuclear hormone receptors, with a particular interest in the glucocorticoid receptor (GR). Like AR-V7, GR is upregulated in prostate cancer following androgen blockade, regulates canonical AR target genes, and is associated with resistance to anti-androgen therapies. Using NanoBRET technology in HEK293 cells, we found that AR-V7 interacts with liganded NR3C-family nuclear hormone receptors, including the full-length androgen receptor, the progesterone receptor, the mineralocorticoid receptor, and, most notably, GR. Using co-immunoprecipitation and proximity ligation assays, we demonstrate that AR-V7 associates with GR following androgen blockade in both the VCaP cell line and the CWR-22RV1 cell line, two models of prostate cancer that express endogenous AR-V7 and GR. Preliminary ChIP-Seq experiments in these cell lines suggest AR-V7 and GR chromatin occupancy at the same genomic sites in a ligand-dependent manner. Taken altogether, this data suggests AR-V7 can utilize GR as an alternative binding partner to initiate heterodimerization and transcriptional activation. Citation Format: Eric Bueter, David Hosfield, Erin McAuley, Phillip Selman, Geoffrey Greene, Suzanne D. Conzen, Russell Z. Szmulewitz. The androgen receptor splice variant AR-V7 associates with the glucocorticoid receptor following androgen blockade [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 1016.

Castration-resistant prostate cancer: Androgen receptor inactivation induces telomere DNA damage, and damage response inhibition leads to cell death.

Telomere stability is important for cell viability, as cells with telomere DNA damage that is not repaired do not survive. We reported previously that androgen receptor (AR) antagonist induces telomere DNA damage in androgen-sensitive LNCaP prostate cancer cells; this triggers a DNA damage response (DDR) at telomeres that includes activation of ATM, and blocking ATM activation prevents telomere DNA repair and leads to cell death. Remarkably, AR antagonist induces telomere DNA damage and triggers ATM activation at telomeres also in 22Rv1 castration-resistant prostate cancer (CRPC) cells that are not growth inhibited by AR antagonist. Treatment with AR antagonist enzalutamide (ENZ) or ATM inhibitor (ATMi) by itself had no effect on growth in vitro or in vivo, but combined treatment with ENZ plus ATMi significantly inhibited cell survival in vitro and tumor growth in vivo. By inducing telomere DNA damage and activating a telomere DDR, an opportunity to inhibit DNA repair and promote cell death was created, even in CRPC cells. 22Rv1 cells express both full-length AR and AR splice variant AR-V7, but full-length AR was found to be the predominant form of AR associated with telomeres and required for telomere stability. Although 22Rv1 growth of untreated 22Rv1 cells appears to be driven by AR-V7, it is, ironically, expression of full-length AR that makes them sensitive to growth inhibition by combined treatment with ENZ plus ATMi. Notably, this combined treatment approach to induce telomere DNA damage and inhibit the DDR was effective in inducing cell death also in other CRPC cell lines (LNCaP/AR and C4-2B). Thus, the use of ENZ in combination with a DDR inhibitor, such as ATMi, may be effective in prolonging disease-free survival of patients with AR-positive metastatic CRPC, even those that co-express AR splice variant.

Abstract B076: Osteoclasts influence androgen-related gene expression in prostate cancer cells

Abstract Background: The majority of progressive prostate cancer will develop metastases in the skeleton. In bone environment, the regulated destruction of bone matrix by osteoclasts is closely connected to the formation of new bone by osteoblasts. Although prostate cancer often forms sclerotic bone lesions, evidence from recent research show that prostate cancer cells also produce factors to promote osteoclastic action. However, little is known on how osteoclasts regulate the prostate cancer growth in bone. The aim of the present study was to investigate the role of osteoclasts in the crosstalk of bone microenvironment and prostate cancer cells. Methods: The interactions between prostate cancer cells and osteoclasts were studied in a coculture model using the transwell permeable supports system. Two castration-resistant prostate cancer cell lines (LNCaP-19 and PC3) were used together with osteoclasts that were transformed from the RAW 264.7 cells by stimulation with RANKL. After 48 hours coculturing, analysis on gene expression level was carried out with qRT-PCR. Results: The results show that osteoclasst directly influence gene expression patterns in prostate cancer cells, affecting genes generally involved in tumor progression, mainly in the osteolytic cell line PC3. In LNCaP-19 cells, which are sensitive to androgens, osteoclast stimulation upregulated expression of androgen receptor (AR), as well as the AR splice-variant ARV7. In contrast, in androgen-insensitive PC3 cells no such changes in AR and AR-V7 mRNA were detected. Instead, the expression of the steroidogenic enzymes CYP11A1 (converting cholesterol into pregnenolone) and CYP19A1 (converting testosterone into estrogen) were increased by osteoclast stimulation. In addition, osteoclast stimulation differentially affects expression of genes related to bone formation in the tumor cells. LNCaP-19, which gives osteoblastic lesions in mouse tibia, responds with increased expression of genes related to TGF-b signaling, osteogenesis, and bone matrix formation. In contrast, the osteolytic cell line PC3 downregulates expression of osteogenic genes in response to osteoclast stimulation. Conclusion: This study shows that there is a direct crosstalk between osteoclasts and prostate cancer cells, where the osteoclasts influence gene expression related to androgen signaling, steroidogenesis, and osteogenesis in different ways based on the androgen and bone-related characteristics of the prostate cancer cells. Since upregulated AR and induced ARV7 expression have been shown to assist the progression of castration-resistant prostate cancer cells, inhibiting this crosstalk by targeting osteoclasts may improve efficacy of androgen-targeting drugs. Citation Format: Junchi Huang, Jan-Erik Damber, Karin Welen. Osteoclasts influence androgen-related gene expression in prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B076.

Abstract 5230: Development of a multi-parameter immunofluorescence assay for simultaneous detection of androgen receptor and androgen receptor variant 7 in prostate cancer circulating tumor cells

Abstract Background. Androgen receptor (AR) signaling is the primary driver of prostate cancer. Expression of the AR splice variant AR-V7, is predictive of resistance to anti-androgen therapies enzalutimide and abiratirone. There is great interest to non-invasively identify circulating tumor cells (CTCs) and investigate their expression of AR and AR-V7 and monitor them over time. RareCyte has developed a platform for automated visual identification of CTCs by immunofluorescence (IF) that allows up to 6-parameter assessment. We have developed a set of prototype assays using tyramide amplification to detect AR and AR-V7 individually and in combination with prostate membrane-specific antigen (PSMA). Using a novel method, we also developed an assay to identify AR and AR-V7 simultaneously. Methods. Normal human whole blood samples were spiked with prostate cancer lines serving as model CTCs (mCTCs). Prostate cancer blood from a patient with advanced and known high CTC count were collected under an IRB-approved protocol. Blood was processed onto microscope slides and stained on an automated stainer with CTC detection assays incorporating 4 core parameters (nuclear dye, CD45, pan-cytokeratin, EpCAM) plus the following markers: (1) AR; (2) AR-V7; (3) AR and PSMA; (4) AR-V7 and PSMA; (5) AR and AR-V7. Each assay was applied to the cancer cell lines PC3, LNCaP, and 22RV1 and to the patient sample. An antibody denaturing process was used to sequentially amplify two rabbit monoclonals for the assay containing both AR and AR-V7. No-primary (diluent only) staining controls were run to confirm success of the denaturing step. Results. Staining of spike-in CTC models confirmed the reported AR and AR-V7 phenotype of the cell lines, supporting the specificity of the assays. When applied to an equivalent volume of the patient sample, the assays identified a mean (SD) of 36 (11) CTCs. In the assays staining AR, the mean percent of AR+ CTCs was 70% (range 61 - 86) and for the assays staining AR-V7, the percent of AR-V7+ CTCs was 30% (range 26 - 39). In the assay staining both AR and AR-V7, 10 of 38 CTCs were AR-V7+ (26%); all of these were AR+; 15 CTCs were only AR+ (39%). Conclusions. We have developed multi-parameter IF assays for detection of AR and AR-V7 in prostate CTCs, including the first assay we are aware of to simultaneously visualize AR and AR-V7. It has been validated in prostate cancer CTC models. The percentages of AR and AR-V7-positive CTCs identified in a patient sample with the assay are consistent with the percentages observed when AR and AR-V7 are detected in independent assays, supporting the accuracy of the simultaneous detection assay. All AR-V7-positive CTCs were also AR-positive, supporting specificity of AR-V7 staining. Citation Format: Yao Sun, Daniel Campton, Arturo Ramirez, Tanisha Mojica, Alisa Clein, Celestia Higano, Daniel Sabath, Stephen Plymate, Eric Kaldjian. Development of a multi-parameter immunofluorescence assay for simultaneous detection of androgen receptor and androgen receptor variant 7 in prostate cancer circulating tumor cells [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 5230.

The impact of the androgen receptor splice variant AR-V7 on the prognosis and treatment of advanced prostate cancer

A recently discovered mechanism enabling prostate cancer cells to escape the effects of endocrine therapies consists in the synthesis of C-terminally truncated, constitutively active androgen receptor (AR) splice variants (AR-V). Devoid of a functional C-terminal hormone/ligand binding domain, various AR-Vs are insensitive to therapies targeting the androgen/AR signalling axis. Preliminary studies suggest that AR-V7, the most common AR-V, is a promising predictive tumour marker and a relevant selection marker for the treatment of advanced prostate cancer. This review critically outlines recent advances in AR-V7 diagnostics and presents an overview of current AR-V7 targeted therapies.

Branched Chain RNA In Situ Hybridization for Androgen Receptor Splice Variant AR-V7 as a Prognostic Biomarker for Metastatic Castration-Sensitive Prostate Cancer.

The androgen receptor (AR) mRNA splice variant AR-V7 has emerged as a predictive biomarker for response to AR-targeted therapies. There are currently no commercially available assays to detect AR splice variants. The branched chain RNA in situ hybridization (ISH) platform enables the highly sensitive detection of RNA transcripts in formalin-fixed, paraffin-embedded (FFPE) tissues. We designed a branched chain RNA ISH probe to target the unique cryptic exon CE3 of AR-V7 using multiple tiling probes. This automated ISH assay was applied to tumor tissue from two distinct clinical cohorts that we hypothesized would differ in AR-V7 status. We detected AR-V7 in all tumor samples from men with metastatic castration-resistant prostate cancer with tissue obtained after disease progression despite at least one subsequent line of hormonal therapy (abiraterone, enzalutamide, or bicalutamide; n = 12). We detected AR-V7 in just one tumor from men who had undergone prostatectomy for localized adenocarcinoma (n = 30; Gleason 4 + 5 = 9 in the AR-V7-positive sample). Given the apparent distinction between the above groups by AR-V7 signal, we analyzed pretreatment AR-V7 status as a predictive and prognostic biomarker in men with treatment-naïve metastatic disease. Patients with metastases but without detectable AR-V7 RNA at baseline had significantly longer overall survival (log-rank P = 0.044) and a trend toward superior progression-free survival (log-rank P = 0.055). Within an institutional cohort, the RNA ISH assay identified AR-V7 within FFPE tissue and may have prognostic value in metastatic castration-sensitive prostate cancer. These preliminary findings warrant further study in larger cohorts. Clin Cancer Res; 23(2); 363-9. ©2016 AACR.

Exploitation of Castration-Resistant Prostate Cancer Transcription Factor Dependencies by the Novel BET Inhibitor ABBV-075.

The ability of BET family inhibitor ABBV-075 to inhibit transcription activation downstream of the initiating events of transcription factors like AR and TMPRSS2:ETS fusion proteins provides a promising therapeutic option for CRPC patients who have developed resistance to second-generation antiandrogens. Mol Cancer Res; 15(1); 35-44. ©2016 AACR.

The RNA helicase DDX39B and its paralog DDX39A regulate androgen receptor splice variant AR-V7 generation

Mounting evidence suggests that constitutively active androgen receptor (AR) splice variants, typified by AR-V7, are associated with poor prognosis and resistance to androgen deprivation therapy in prostate cancer patients. However, mechanisms governing the generation of AR splice variants are not fully understood. In this study, we aimed to investigate the dynamics of AR splice variant generation using the JDCaP prostate cancer model that expresses AR splice variants under androgen depletion. Microarray analysis of JDCaP xenografts before and after expression of AR splice variants suggested that dysregulation of RNA processing pathways is likely involved in AR splice variant generation. To explore factors contributing to generation of AR-V7 mRNA, we conducted a focused RNA interference screen in AR-V7-positive JDCaP-hr cells using an shRNA library targeting spliceosome-related genes. This screen identified DDX39B as a regulator of AR-V7 mRNA expression. Simultaneous knockdown of DDX39B and its paralog DDX39A drastically and selectively downregulated AR-V7 mRNA expression in multiple AR-V7-positive prostate cancer cell lines. DDX39B was upregulated in relapsed JDCaP xenografts expressing AR splice variants, suggesting its role in expression of AR splice variants. Taken together, our findings offer insight into the mechanisms of AR splice variant generation and identify DDX39 as a potential drug target for the treatment of AR splice variant-positive prostate cancer.

Sequencing Treatment for Castration-Resistant Prostate Cancer.

Prostate cancer is the most common non-cutaneous cancer diagnosed in men and the second leading cause of male cancer deaths in the USA. While most cases are diagnosed in early stages, some will present as or progress to metastatic disease and eventually castration-resistant prostate cancer (mCRPC) which has a mortality rate exceeding 50%. There are currently six approved systemic life-prolonging therapies for use in mCRPC, yet little data to guide sequencing. Clinical factors, including the presence or absence of symptoms and the presence or absence of visceral metastases, should help determine the best therapeutic choice at each treatment node. Those with asymptomatic bone-only disease could be considered for sipuleucel-T, abiraterone, enzalutamide, or docetaxel in the first-line setting. For symptomatic disease, docetaxel could be used or radium-223 if disease is only present in the bone. In the second-line setting, sipuleucel-T or radium-223 can be used in the appropriate clinical setting. Taxane chemotherapy could be used if a novel androgen-directed therapy was used in the first-line setting. Cabazitaxel, if docetaxel was previously used, should be considered. There is scarce data on best treatment options in the third-line setting. In general, we recommend alternating between androgen-targeting agents and taxane chemotherapy. Finally, consideration should be given to testing for the androgen receptor splice variant AR-V7, which may be a relevant treatment-specific biomarker to aid in the selection of androgen-targeting therapy versus chemotherapy at each treatment juncture. Mutation testing for DNA damage repair defects can also be considered, as such patients may benefit from investigational poly ADP ribose polymerase(PARP) inhibitors or platinum-based chemotherapies. Several ongoing studies have been designed to answer some of these sequencing questions, including the biomarker questions, and will hopefully continue to inform us about rational therapy selection in mCRPC.

Expression of AR-V7 in Circulating Tumour Cells Does Not Preclude Response to Next Generation Androgen Deprivation Therapy in Patients with Castration Resistant Prostate Cancer

The androgen receptor splice variant AR-V7 has recently been discussed as a predictive biomarker for nonresponse to next-generation androgen deprivation therapy (ADT) in patients with castration-resistant prostate cancer. However, we recently identified one patient showing a response from abiraterone despite expression of AR-V7 in his circulating tumour cells (CTC).Therefore, we precisely assessed the response in a cohort of 21 AR-V7 positive castration-resistant prostate cancer patients who had received therapy with abiraterone or enzalutamide. We detected a subgroup of six AR-V7 positive patients showing benefit from either abiraterone or enzalutamide. Their progression free survival was 26 d (censored) to 188 d. Four patients displayed a prostate-specific antigen decrease of >50%. When analysing prior therapies, we noticed that only one of the six patients had received next-generation ADT prior to CTC collection.As a result, we conclude that AR-V7 status in CTC cannot entirely predict nonresponse to next generation ADT and AR-V7-positive patients should not be systematically denied abiraterone or enzalutamide treatment, especially as effective alternative treatment options are still limited. Patient summaryA subgroup of patients can benefit from abiraterone and/or enzalutamide despite detection of AR-V7 splice variants in their circulating tumour cells.

Branched chain RNA in situ hybridization for androgen receptor splice variant AR-V7 as a prognostic biomarker for metastatic castration-sensitive prostate cancer.

e16571Background: The androgen receptor (AR) mRNA splice variant AR-V7 has emerged as a predictive biomarker for response to AR targeted therapies. There are currently no commercially available ass...

The marine triterpene glycoside frondoside A exhibits activity in vitro and in vivo in prostate cancer.

Despite recent advances in the treatment of metastatic castration-resistant prostate cancer (CRPC), outcome of patients remains poor due to the development of drug resistance. Thus, new drugs are urgently needed. We investigated efficacy, toxicity and mechanism of action of marine triterpene glycoside frondoside A (FrA) using CRPC cell lines in vitro and in vivo. FrA revealed high efficacy in human prostate cancer cells, while non-malignant cells were less sensitive. Remarkably, proliferation and colony formation of cells resistant to enzalutamide and abiraterone (due to the androgen receptor splice variant AR-V7) were also significantly inhibited by FrA. The marine compound caused cell type specific cell cycle arrest and induction of caspase-dependent or -independent apoptosis. Up-regulation or induction of several pro-apoptotic proteins (Bax, Bad, PTEN), cleavage of PARP and caspase-3 and down-regulation of anti-apoptotic proteins (survivin and Bcl-2) were detected in treated cells. Global proteome analysis revealed regulation of proteins involved in formation of metastases, tumor cell invasion, and apoptosis, like keratin 81, CrkII, IL-1β and cathepsin B. Inhibition of pro-survival autophagy was observed following FrA exposure. In vivo, FrA inhibited tumor growth of PC-3 and DU145 cells with a notable reduction of lung metastasis, as well as circulating tumor cells in the peripheral blood. Increased lymphocyte counts of treated animals might indicate an immune modulating effect of FrA. In conclusion, our results suggest that FrA is a promising new drug for the treatment of mCRPC. Induction of apoptosis, inhibition of pro-survival autophagy, and immune modulatory effects are suspected modes of actions.

The RNA-binding protein Sam68 regulates expression and transcription function of the androgen receptor splice variant AR-V7.

Castration-resistant (CR) prostate cancer (PCa) partly arises due to persistence of androgen receptor (AR) transcriptional activity in the absence of cognate ligand. An emerging mechanism underlying the CRPCa phenotype and predicting response to therapy is the expression of the constitutively-active AR-V7 splice variant generated by AR cryptic exon 3b inclusion. Here, we explore the role of the RNA-binding protein (RBP) Sam68 (encoded by KHDRBS1), which is over-expressed in clinical PCa, on AR-V7 expression and transcription function. Using a minigene reporter, we show that Sam68 controls expression of exon 3b resulting in an increase in endogenous AR-V7 mRNA and protein expression in RNA-binding-dependent manner. We identify a novel protein-protein interaction between Sam68 and AR-V7 mediated by a common domain shared with full-length AR, and observe these proteins in the cell nucleoplasm. Using a luciferase reporter, we demonstrate that Sam68 co-activates ligand-independent AR-V7 transcriptional activity in an RNA-binding-independent manner, and controls expression of the endogenous AR-V7-specific gene target UBE2C. Our data suggest that Sam68 has separable effects on the regulation of AR-V7 expression and transcriptional activity, through its RNA-binding capacity. Sam68 and other RBPs may control expression of AR-V7 and other splice variants as well as their downstream functions in CRPCa.

Abstract 2112: AR splice variants ARv7 and ARv567 utilize different mechanisms of cytoplasmic trafficking and nuclear translocation: Therapeutic implications for PC

Abstract Prostate cancer (PC) is one of the most prevalent cancers among men worldwide. While androgen deprivation therapy (ADT) is initially effective in the treatment of PC, resistance emerges resulting in castration-resistant prostate cancer (CRPC) disease, which is driven by continuous signaling from the androgen receptor (AR). Several new drugs designed to inhibit AR signaling (abiraterone and enzalutamide) have been recently approved for PC treatment. However resistance to these next-generation AR inhibitors inevitably occurs and is mediated by adaptive mechanisms that restore AR function. Overexpression of AR splice variants (ARv567 and ARv7), which lack the ligand-binding domain (LBD), was shown to mediate resistance to abiraterone and enzalutamide. Following ADT resistance, CRPC patients are treated with the taxanes Docetaxel (DTX) and Cabazitaxel as first and second line chemotherapy. Our recent work showed that AR binds to microtubules (MTs) utilizing them as tracks for its nuclear translocation, and that taxanes inhibit AR signaling by sequesting AR in the cytoplasm downstream of MT stabilization. We showed that MT binding is mediated by the Hinge region of AR, which is present in ARv567 but missing in ARv7 variant. In contrast to ARv567, ARv7 does not bind MTs and its nuclear localization is not affected by taxane treatment and its expression confers taxane resistance in vitro and in vivo. Since the main nuclear localization signal (NLS) of AR is in the hinge region we hypothesized that ARv7 has a different nuclear translocation mechanism than Arv567 or AR-WT. Co-immunoprecipitation experiments revealed that AR-WT and ARv567 interact with importin-α, and that DTX treatment inhibits this interaction. In contrast, ARv7 does not interact with importin-α and is insensitive to DTX. To monitor the dynamics of AR variant nuclear accumulation we performed live cell confocal microscopy using PC3 cells microinjected with GFP-AR-WT, -ARv7 or -ARv567. Our results showed that both AR-WT and AR-v567 were sensitive to inhibition of the importin pathway, using the importin-β inhibitor importazole, while ARv7 was unaffected. FRAP experiments of M12 cells stably expressing GFP-tagged AR-WT, or ARvs revealed that the recovery after photobleaching of ARv7 nuclear fraction was much faster than that of ARv567 or AR-WT, suggesting enhanced motility of ARv7. FRAP experiments in DTX or importazole treated cells further confirmed the insensitivity of ARv7 to perturbations of the MT network and importin pathway. Taken together our results show that AR-WT and ARv567 utilize MTs and, the importin-α/β pathway for nuclear translocation; while ARv7 is MTs and importin independent. Elucidation of the mechanisms of cytoplasmic trafficking and nuclear translocation of ARv7 variant will allow us to identify new druggable targets in order to revert resistance to taxanes and next generation AR targeting drugs. Citation Format: Luigi Portella, Paraskevi Giannakakou. AR splice variants ARv7 and ARv567 utilize different mechanisms of cytoplasmic trafficking and nuclear translocation: Therapeutic implications for PC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2112. doi:10.1158/1538-7445.AM2014-2112

Effect of generation 2.5 antisense inhibitor of androgen receptor on MDV3100-resistant prostate cancer cell growth in vitro and in vivo.

94 Background: MDV3100 is a potent androgen receptor (AR) antagonist with activity in castration resistant prostate cancer (CRPC); however, progression to MDV3100-resistant (MDV-R) CRPC frequently occurs with rising serum PSA levels, implicating AR full length or variants in disease progression. We studied the activity of Generation 2.5 antisense oligonucleotide (ASO) targeting the AR full length (ARfl) and splice variants in MDV-R CRPC models. Methods: and Results: ThreeASOs targeting exon 1, intron 1, or exon 8 were designed to suppress ARfl and known AR splice variants. We generated by selection MDV-R LNCaP-derived sub-lines that uniformly expressed high levels of both ARfl and AR-V7 compared to CRPC LNCaP cell lines. MDV-3100 induced time- and dose-dependent increases in ARfl and AR-V7 protein levels; ARfl levels were ~20-fold higher than AR-V7. All 3 AR-ASO decreased ARfl and PSA expression. Exon 1 ASO decreased expression of both ARfl and AR-V7 in MDV-R-LNCaP cells; in contrast, exon 8 ASO decreased ARfl without reducing AR-V7 levels. Exon 1 ASO also most potently suppressed ARfl and splice variants in M12 cells stably overexpressing AR splice variants AR-V7 and AR-V567es. Despite these differential effects on ARfl and splice variant knockdown, the AR ASO similarly inhibited cell growth and induced apoptosis and G1 cell cycle arrest in LNCaP-derived CRPC and MDV-R cell lines. In 22RV-1 cells (which express endogenous ARfl and AR-V7), exon 1 ASO more potently suppressed ARfl and AR-V7 levels, AR transcriptional activity and AR-regulated gene expression compared to exon 8 ASO, but inhibition of cell growth did not differ significantly. Exon 1 ASO was evaluated in vivo in MDV-R49F CRPC LNCaP xenografts; mean tumor volume and serum PSA levels decreased significantly by 40% and 50%, respectively, compared to controls. Conclusions: While MDV-3100 induces both ARfl and AR-V7 levels, the biologic consequences appear cell line dependent and mainly driven by ARfl. AR-ASO knockdown of ARfl and its splice variants suppresses MDV-R LNCaP tumor growth, providing pre-clinical proof of principle to support clinical evaluation in post-AR pathway inhibitor CRPC.