AR-V7 and prostate cancer: The watershed for treatment selection?

Cancer Treatment Reviews 43 (2016) 27–35

Contents lists available at ScienceDirect

Cancer Treatment Reviews journal homepage: www.elsevierhealth.com/journals/ctrv

Anti-Tumour Treatment

AR-V7 and prostate cancer: The watershed for treatment selection? Chiara Ciccarese a,1, Matteo Santoni b,1, Matteo Brunelli c, Sebastiano Buti d, Alessandra Modena a, Massimo Nabissi e, Walter Artibani f, Guido Martignoni c, Rodolfo Montironi g, Giampaolo Tortora a, Francesco Massari a,⇑ a

Medical Oncology, Azienda Ospedaliera Universitaria Integrata, University of Verona, Verona, Italy Medical Oncology, AOU Ospedali Riuniti, Polytechnic University of the Marche Region, Ancona, Italy Department of Pathology and Diagnostic, A.O.U.I., University of Verona, Verona, Italy d Medical Oncology Unit, University Hospital of Parma, Italy e School of Pharmacy, Experimental Medicine Section, University of Camerino, Camerino, Italy f Urologic Clinic, Department of Oncological and Surgical Sciences, Azienda Ospedaliera Universitaria Integrata, University of Verona, Verona, Italy g Section of Pathological Anatomy, Polytechnic University of the Marche Region, School of Medicine, AOU Ospedali Riuniti, Ancona, Italy b c

a r t i c l e

i n f o

Article history: Received 17 October 2015 Received in revised form 6 December 2015 Accepted 11 December 2015

Keywords: Prostate cancer Androgen receptor Splice variant AR-V7 Prognostic role Anti-androgens sensitivity Taxane sensitivity

a b s t r a c t The androgen receptor (AR) plays a key role in progression to metastatic castration-resistant prostate cancer (mCRPC). Despite the recent progress in targeting persistent AR activity with the nextgeneration hormonal therapies (abiraterone acetate and enzalutamide), resistance to these agents limits therapeutic efficacy for many patients. Several explanations for response and/or resistance to abiraterone acetate and enzalutamide are emerging, but growing interest is focusing on importance of AR splice variants (AR-Vs) and in particular of AR-V7. Increasing evidences highlight the concept that variant expression could be used as a potential predictive biomarker and a therapeutic target in advanced prostate cancer. Therefore, understanding the mechanisms of treatment resistance or sensitivity can help to achieve a more effective management of mCRPC, increasing clinical outcomes and representing a promising and engaging area of prostate cancer research. Ó 2015 Elsevier Ltd. All rights reserved.

Introduction Androgen-deprivation therapy (ADT) represents the gold standard treatment for relapsed or advanced prostate cancer patients, given the androgen receptor-dependent nature of this tumor. However, disease is usually only temporarily controlled and progression typically occurs within 12–24 months of initial response, a state named castration-resistant prostate cancer (CRPC), and defined as a prostate cancer that has progressed despite castrate levels of serum testosterone (650 ng/dL) [1]. The evidence that many genes known to be under androgen receptor (AR) transcriptional control in prostate cancer are re-expressed in CRPC shows that, despite ADT, AR pathway remains active and provides the source for the growth and survival of tumor cells. Based on these assumptions, AR may be considered the first example of a ⇑ Corresponding author at: Medical Oncology, Azienda Ospedaliera Universitaria Integrata, University of Verona, P.le L.A. Scuro n.10, 37134 Verona, Italy. Tel.: +39 0458128115; fax: +39 045 8027410. E-mail address: [email protected] (F. Massari). 1 Equally contributors. http://dx.doi.org/10.1016/j.ctrv.2015.12.003 0305-7372/Ó 2015 Elsevier Ltd. All rights reserved.

lineage oncogene [2], representing a critical and functionally important therapeutic target. Owing to this new understanding, two next-generation AR-targeting therapies (abiraterone acetate and enzalutamide) have been recently approved by the FDA for the treatment of metastatic CRPC (mCRPC) [3–6]. Abiraterone acetate is a potent and highly selective irreversible inhibitor of cytochrome P450 17A1, which impairs androgen synthesis in the adrenal glands, testes and in prostate tumor itself. Enzalutamide (formerly MDV3100) acts as an AR antagonist, binding competitively to the AR and therefore displacing the natural ligands and preventing nuclear translocation of the ligand–receptor complex. Although these agents represent breakthroughs in the treatment of mCRPC, almost all patients develop innate or acquired resistance with a widely variable duration of response. Constitutively active AR splice variants (AR-Vs), which lack the AR ligand-binding domain as a result of alternative splicing of the human AR gene, represent an emerging crucial mechanism responsible for castration resistance prostate cancer progression. Among several truncated AR-Vs identified, AR-V7 is the major clinically meaningful receptor variant characterized in prostate cancer samples, being involved in tumor progression and anti-cancer treatments

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resistance. In this review we analyzed the clinical relevance of AR-Vs, focusing on the prognostic value of AR-V7 and its emerging predictive role as a biomarker of resistance/sensitivity to nextgeneration hormonal therapies and taxane chemotherapy.

The androgen receptor structure The human androgen receptor (AR) gene, mapped on chromosome Xq11-12, belongs to the steroid hormone receptor genes family. The structure of the AR gene, mature spliced mRNA, and protein domains resembles that of the family members estrogen receptors and progesterone receptor. The AR acts as ligandactivated transcription factor, by regulating the expression of definite genes. Eight different exons of the AR gene encode for corresponding functional regions forming the AR multidomain protein [7]. The N-terminal domain (NTD, also called activation function 1 [AF1]), coded by the first exon, is the main effector region of the AR, controlling its transactivation. It contributes in regulating the transcription of the AR gene, which is tissue-specific, steroid hormones-related and age-dependent [7,8]. The NTD can include a variable number of polyglutamine (17–29) and polyglycine repeats, encoded by polymorphic (CAG)n and (GGN)n repeat units, whose length influences the AR transcription activity. In particular, the receptor activity is augmented in case of shorter polyglutamine repeats, while the loss of amino acids 141–338 (especially residues 210–337) markedly impairs the AR function [9]. The DNA-binding domain (DBD), encoded by exons 2 and 3 and highly conserved among the steroid hormone receptors, consists of a globular body and two coordination zinc finger complexes, with four cysteine residues and a Zn2+ ion each [10]. The AR binds to the androgen-response element (ARE) of the major groove of DNA through these two zinc fingers, which also contribute to the binding complex stabilization [11] and the AR dimerization [12]. Exons 4–8 encode for the COOH-terminal region, which represents the ligand-binding domain (LBD). The LBD contains a ligand-binding pocket formed by 12 folded helices, which is responsible for hormone recognition and guarantees the specificity and selectivity of the signaling pathway. According to the nature of the bound ligand – agonist or antagonist – the C-terminal helix 12 acquires two different conformations. The agonists (dihydrotestosterone and testosterone) binding leads helix 12 to bend over and closes the pocket describing a groove that binds a region of NTD, which acts as an AR co-activator. Conversely, when an antagonist is bound to the AR, helix 12 opens the entrance to the LBD, interfering with co-activators binding [13]. Specifically, after agonist binding, the conformational change of helices 3, 4, 5 and 12 of the LBD constitute the activating factor (AF)-2, also involved in controlling the receptor transcription activity. AF-2 indeed recruits p160 coactivator proteins (members of the steroid receptor coactivator family) in a hormone-dependent way [14]. The portion between the DBD and the LBD is called the hinge region. The hinge region has a key role in controlling AR activity, regulating the nuclear translocation signal, DNA binding, coactivator recruitment, and the transcriptional activity of the receptor [15]. In the presence of intracellular androgen exposure, full-length AR (FL-AR) can dimerize, thereby overcoming inhibition due to the AR N-terminal domain [16]. After ligand binding and dimerization, the AR translocates from the cytoplasm to the nucleus, where it binds to the promoter of specific DNA regions (AREs), induces the assembly of a co-activator protein transcriptional complex [17], and stimulate transcription of genes (including PSA, TMPRSS2, and hK2) involved in cell growth, differentiation and survival [18].

The AR and prostate cancer Similarly to the healthy prostate glandular epithelium, prostate cancer cells are strictly dependent on androgens. To support this reliance, virtually all prostate tumors express the AR. Androgen deprivation therapy (ADT), dropping serum testosterone levels under castration value (via surgical or pharmacological castration, or through AR-antagonists, both used alone or in combination) represents the mainstream therapeutic strategy for prostate cancer. Tumor progression despite androgen depletion defines an advanced stage of the disease, defined castration-resistant prostate cancer (CRPC). Interestingly, CRPC cells continue to be largely androgen-dependent and AR signaling-guided [19]. Indirect evidence of the persistent role of AR signaling in driving late-stage disease is the remarkable clinical efficacy of the secondgeneration anti-androgens (enzalutamide [3,4] and abiraterone [5,6]) in CRPC patients. Abiraterone, an inhibitor of the Cytochrome P450 17alpha-hydroxylase/17,20-lyase (CYP17) enzyme, avoids the tumor conversion of progesterone and adrenal androgens to dihydrotestosterone (DHT). Enzalutamide, a non-steroidal antiandrogen, blocks the receptor even in case of AR overexpression. A deep understanding of mechanisms underlying castration resistance and sustained AR-signaling activity is still lacking. Several hypothesis, not mutually exclusive, have been proposed: (1) The amplification and over-expression of wild-type AR gene, allowing androgen-dependent cancer cells to proliferate despite the low serum androgens values, has been described as a driving mechanism supporting CRPC progression [20,21]. (2) Somatic point mutations in the AR gene provide a growth advantage after androgen ablation therapy, both with firstgeneration [22–24] and second-generation [25–27] antiandrogen molecules. Moreover, gross deletions in the LBD of the AR leads to constitutively active receptors [28]. (3) Proliferative stimuli triggered by alternative pathways converge to modulate and stimulate the AR signaling cascade [29–31]. (4) The intratumoral de novo over-production of testosterone and DHT from adrenal androgens or cholesterol also represents a possible cause for androgen deprivation therapy failure [32,33]. (5) The activation of non-conventional pathways involved in DHT biosynthesis, as a result of mutations in steroid metabolism enzymes, can contribute to tumor progression [34]. Therefore, despite the absence of a thorough knowledge of the etiopathogenesis, targeting AR signaling remains an important treatment option for CRPC.

AR splice variants Constitutively active ARs, resulted from alternative splicing of the human AR gene leading to truncated AR isoforms lacking the LBD, represent an emerging key mechanism responsible for castration resistance tumor progression. Truncated AR variants (AR-Vs) wanting the LBD can be the result of somatic nonsense mutations that introduce stop codons in the AR gene [35], AR gene rearrangements, or posttranslational AR proteolysis [36]. Furthermore, alternative splicing of the primary transcript, considering the complexity of the AR protein multi-domain structure, can actually impair the AR transcriptional activity by generating functionally distinct receptors from the same AR gene. Structurally, these AR variants lack the open reading frame of the LBD as a result of insertions of ‘‘intronic”

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cryptic exons downstream of the exons coding the DBD or deletions of exons encoding the COOH terminal domain [37–39]. The LBD-truncated AR proteins usually localize to the nucleus (despite the loss of the nuclear localization signal) [40] and retain the NTD with the AF1 transactivation domain and the DBD, being therefore constitutively activated and capable of sustain gene transcription independently from ligand (androgens) binding [41] (Figs. 1 and S1). Interestingly, the AR-Vs can induce a sustained AR signaling pathway either through an enhanced transcriptional activity or via a non-genomic mechanism (as in the case of the AR8 isoform) [42]. Therefore, these truncated receptor forms drive an androgenindependent transcription of AR target genes, thereby supporting a hormone-independent prostate cancer cells growth. They represent an interesting mechanism supporting the resistance and tumor progression to androgen deprivation therapy. Among the 15 different AR-Vs that have been recognized until now, AR-V7 (also known as AR3) and ARv567es (or AR-V12) are the two main clinically meaningful receptor variants identified in prostate cancer samples [37,38,43]. Future perspectives concern also the role of AR-V1, which was reported to be overexpressed in prostate tumor tissues, especially in CRPC compared to hormone-naïve prostate cancer specimens [37]; but solid clinical evidence that support the prognostic value of this AR variant are still lacking. The ARv567es isoform preserves the exons 1–4 and the exon 8, while lacking the skipped exons 5–7 (Figs. 1 and S1). It maintains the second and most important part of AR’s bipartite nuclear localization signal (NLS) within the hinge region of the exon 4, thus facilitating the nuclear localization. This variant acts as a constitutively active receptor, augments the expression of full-length AR, reinforcing the AR transcriptional activity independently from the ligand binding and thus contributing to the development of prostate cancer resistance to castration. In fact, although the ARv567es receptor form can be expressed in both benign, malign and metastatic prostate cancer tissues, its levels tend to increase in case of androgen suppression as a consequence of a dynamic process influenced by environmental issues and aimed at supporting cell proliferation and progression [43]. AR-V7 is the best-characterized AR variant, encoded by contiguously spliced AR exons 1, 2, 3, and the cryptic exon 3 (CE3 – originally discovered thanks to bioinformatic identification of an expressed sequence tag mapping to AR intron 3) [37,38]. It contains the NLS in its cryptic exon, allowing an intra-nuclear localization and an enhanced ligand-independent transcriptional activity (Figs. 1 and S1). It is important to underline that: – The AR-V7 mRNA can be detected through RT-PCR in normal prostate tissue, hormone-naïve prostate cancer and CRPC specimens, with a raising gradient (20-fold higher expression in CRPC when compared to hormone-naïve; P < 0.0001) [37]. – The AR-V7 truncated protein (identified by an immunohistochemical assay that uses specific polyclonal antibodies against the 16aa COOH-terminal extension encoded by exon CE3 of the AR-V7 isoform) can be expressed rarely in benign prostate tissue, seldom in hormone-naive prostate cancer specimens, and commonly in CRPC samples [37,38]. – Of note, a close correlation between AR-V7 mRNA transcript and protein expression not always exists in clinical specimens. AR-Vs mRNAs levels are lower than that of the FL-AR, while the AR-Vs and FL-AR proteins expression are similar [44]. The cause of this discrepancy can be probably due to the use of different methods not adequately accurate. – Noteworthy, the sub-cellular localization of AR-V7 protein seems to represent an accurate mechanism for regulating the receptor functionality. In fact, the AR-V7 protein is localized

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in the basal and stromal cells (not in the luminal epithelial cells) in healthy prostate glands, hypothesizing a role in hormoneinsensitive glandular homeostasis regulation and justifying the insensitivity of the prostatic stroma to androgen deprivation. In malignant prostate tissues, before androgen suppression therapy the AR-V7 protein is expressed at increased levels in the cytoplasm – therefore in a transcriptionally inactive form. The AR-V7 localization in the nucleus, where the receptor carries out its transcriptional activity, usually occurs in case of disease progression as a result of a selective pressure of androgen-deprived conditions [38]. – Functionally, the truncated AR-V7 isoform is constitutively active in transcription, irrespective of androgens binding, thereby representing a cancer way to escape from ADT. In particular AR-V7, by forming homodimers and heterodimers with ARv567es or with FL-AR [45], can regulates the expression of a panel of genes that partially recapitulate those controlled by ligand-induced full-length AR even in the absence of androgens (canonical AR targets like IGFBP3, FKBP5, and PSA genes), and partly differ from the FL-AR gene expression program (a unique set of target genes, including AKT1 gene – which is associated to biochemical disease progression [46]) [38]. Therefore, the constitutively active AR-V7 variant, which still requires FL-AR [47], has a key role in supporting the androgen-independent growth and progression, favoring the CRPC phenotype acting as a driver of castration or antiandrogen resistance. – AR splice variants (and AR-V7 in particular) can also be detected in circulating tumor cells (CTCs). This represents an interesting, timely, non-invasive and easily available method to monitor changes in peripheral blood concentrations of the receptor isoform during different therapies. Interestingly, performing serial blood-based AR-V7 sampling across different hormonal or cytotoxic treatments for metastatic prostate cancer, temporal changes in AR-V7 status have been identified. Transitions in AR-V7 status (conversion from AR-V7 negative to positive during AR-targeted therapies and reversions from AR-V7 positive to negative with taxanes) support the potential role of AR-V7 as a dynamic marker [48]. Further studies are required to clarify the clinical significance of these AR-V7 modifications, thanks to the availability of liquid biopsies by monitoring CTCs or maybe circulating-DNA (cDNA) changes. Prognostic role of AR-V7 The clinical relevance of AR-V7 constitutes one of the current major topics in research on prostate cancer. To date, the knowledge reached through multiple searches allows to state that: – AR-V7 plays a crucial role in prostate cancer development and progression. It supports epithelial-to-mesenchymal transition (EMT), which contributes to cancer metastatic wide spreading and treatment resistance [49]. The mesenchymal biomarker N-cadherin (along with other EMT markers Vimentin, Snail and Zeb1) hyper-expression was observed in the cells of the human prostate tumor LNCaP cell line overexpressing constitutively active AR variants (AR-V7 or ARQ640X), but not in cells expressing the FL-AR [50]. To further support the role of ARV7 in promoting prostate cancer, a transgenic mouse model (AR3Tg) over-expressing AR-V7 showed to positively modulates the expression of tumor-promoting autocrine/paracrine growth factors (transforming growth factor b2 and insulin-like growth factor 1) and EMT-related genes (Vimentin, Snail, CDH2, TWIST) [51]. – High AR-V7 mRNA levels (identified by semiquantitative reverse transcription-PCR) significantly correlate with an increased risk of biochemical disease recurrence after radical

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Fig. 1. Schematic structures of AR splice variants.

prostatectomy in 82 hormone-naïve prostate cancer patients (P = 0.012) [37], suggesting its potential poor prognostic value in early stage disease. Interestingly, for the first time, a polyclonal variant-specific antibody against the LBD-truncated ARV7 was performed, allowing to detect the AR-V7 protein commonly in CRPC samples but infrequently in hormone-naive PCa specimens [37]. – The expression of very high transcript levels of AR-V7 (or the detectable expression of ARv567es) in prostate cancer bone metastases correlates with particularly poor prognosis (significantly shorter survival), as demonstrated by Hörnberg and Colleagues [52]. They analyzed the mRNA levels of FL-AR and ARVs (AR-V1, AR-V7, and AR-V567es) with RT-PCR and wholegenome transcription profiles with an Illumina Beadchip array in hormone-naïve (n = 10) and CRPC bone metastases samples (n = 30) compared to the expression in primary PC and in non-malignant prostate samples (n = 13). Interestingly, the AR-V7 expression is higher in CRPC compare to hormonesensitive disease, emphasizing a correlation with the development of castration resistance and with a more aggressive prostate cancer behavior [52]. – AR-V7 displays a peculiar pattern of expression in different stages of prostate cancer disease (significantly higher in mCRPC patients than in newly diagnosed metastatic or localized prostate cancer patients). Moreover, a recently published analysis of the immunohistochemical expression of AR-V7 in different stages of prostate cancer (100 localized prostate cancer [cohort 1], 104 newly diagnosed metastatic prostate cancer [cohort 2], and 46 CRPC [cohort 3] patients) revealed that AR-V7 expression correlates with CRPC development (AR-V7 expression rate in cohort 3 was significantly higher than the other two cohorts, and AR-V7 was an independent predictive factor for CRPC development at the multivariate analysis – P = 0.001), and is associated with shorter survival in CRPC patients, thus representing a interesting prognostic biomarker of aggressive cancer behavior, and a promising predictive marker for progression to CRPC [53].

AR-V7 and anti-androgens sensitivity AR-V7 is involved in prostate cancer treatments resistance. First of all, the constitutively active receptor isoform, lacking the LBD, can resist androgen deprivation therapies [39,54]. As reported above, AR-V7 is over-expressed in CRPC compared to hormonenaïve prostate cancer, probably as a consequence of the selective pressure of anti-AR therapies. In fact, the observation in xenograft models of a inverse linear correlation between the AR-V7 (and ARV1) mRNA levels and the serum androgen concentrations (prompt increase of AR-V7 after castration, and vice versa) [47] supports the role of androgens in negatively regulating AR-V7 expression and highlights the potential contribute of AR-V7 in circumvent androgen ablation. Increased levels of AR-V7 in LNCaP cells stimulates cell proliferation, while knocking down AR-V7 in 22Rv1 cells attenuates cell growth in androgen-deprived status in vivo and in vitro [38,39,43,44,46]. Therefore, AR splice variants are common in castration resistant PCa tissue/cells and have been implicated as a potential mechanism of resistance to ADT. More recently, AR-V7 and ARv567es variants have been associated to AR-driven tumor progression in CRPC, conferring resistance also to novel anti-androgen therapies, abiraterone and enzalutamide. Under enzalutamide or abiraterone treatment, prostate cancer cells and CRPC xenografts show an increased expression of truncated AR-V7 and ARv567es (but not FL-AR), leading to over-expression of the androgen receptor–driven cell-cycle gene UBE2C and therefore contributing to treatment resistance [55]. Preclinical studies have demonstrated that AR-Vs can mediate resistance to therapies targeting full-length AR, such as enzalutamide. Knockdown of AR-Vs (AR-V7 and ARv567es) augments the sensitivity of 22Rv1 cells and NFjB p52-transfected LNCaP cells to enzalutamide antitumor activity, thus supporting their role in driving resistance to enzalutamide by persistently and independently activating the specific AR-transcriptional program [56,57]. In addition to trans-activating peculiar target genes independent of FL-AR, another mechanism of resistance to enzalutamide has been proposed. AR-V7 and ARv567es can also attenuate the ability

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of enzalutamide to inhibit AR-signaling by favoring FL-AR nuclear localization in an androgen-independent way, therefore acting as ‘‘rheostats” to control the degree of response of FL-AR to androgen-directed therapy [58]. Similarly, hyper-expression of AR splice variants represents a potential mechanism of resistance also to abiraterone therapy in CRPC xenografts, along with steroidogenic genes up-regulation (including CYP17A1) [59]. In 2014, Antonarakis et al. firstly prospectively evaluated AR-V7 messenger-RNA expression in circulating tumor cells from CRPC patients receiving enzalutamide or abiraterone [60]. A total of 31 enzalutamide-treated patients and 31 patients receiving abiraterone were enrolled, of whom 39% and 19%, respectively, had detectable AR-V7 in CTCs. They showed that AR-V7 in CTCs from patients with castration resistant PCa was associated with resistance to enzalutamide and abiraterone (Fig. 2). AR-V7-positive patients had significantly lower PSA response rates (0% vs 53%, P = 0.004 among enzalutamide-treated patients; 0% vs 68%, P = 0.004 among abiraterone-treated men), and shorter PSA progression-free survival (median 1.4 vs 6.0 months, P < 0.001; and 1.3 months vs not reached, P < 0.001 in enzalutamide- and abiraterone-treated patients respectively), clinical or radiographic progression-free survival (median 2.1 vs 6.1 months, P < 0.001; and 2.3 months vs not reached, P < 0.001) and overall survival (mOS 5.5 months vs not reached, P = 0.002; and 10.6 months vs not reached, P = 0.006) compared with their AR-V7-negative counterparts. The association between AR-V7 detection and therapeutic resistance was maintained after adjustment for expression of FLAR mRNA. Similarly, a recent German study prospectively demonstrated that the development of AR-V7 in CTCs of patients under therapy correlates with limited efficacy of sequential therapies (enzaluta mide–abiraterone or abiraterone-enzalutamide) [61]. The potential predictive role of resistance to next-generation anti-androgen agents arouses the collective interest for useful implications in the difficult choice of the best treatment algorithm in daily clinical practice. Taken together, these promising results suggest that AR-V7 detection may become an interesting strategy in future years, given its potential role as a biomarker of CRPC poor prognosis, and as a predictive marker of enzalutamide or abiraterone resistance. However several questions should be answered in order to introduce this technique into daily clinical practice: (1) Based on these findings, AR-V7 seems to be not associated with primary or acquired resistance to enzalutamide and abiraterone, but only with reduced sensitivity to these agents. (2) The identification of AR-V7 has been performed by the ‘‘Adnatest” antibody, assessing a rate of 19–39% of incidence [60]. Nevertheless, this technique should be validated and compared with other viable methods in order to optimize the detection of AR-V7 prevalence in CRPC patients. Notably, these data are different from those presented by Hu et al. [37], who found AR-V7 in 70% of patients with CRPC. (3) Is the detection of AR-V7 in CTC a routinely-applicable and cost-effective strategy? Based on present data, the translation of this approach into clinical practice on a large scale seems to be still far away. (4) Only 6 (14%) of the 42 AR-V7 negative patients became positive during treatment (4 with enzalutamide and 2 with abiraterone) [60]. The small number (6 patients) does not allow to compare the conversion rate of abiraterone and enzalutamide during treatment and the role of AR-V7 conversion in these patients. Currently, several novel AR inhibitors are in development (including galeterone, EPI-001, ARN-509, VT-464, AZD3514, and

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ODM-201), trying to overcome resistance to enzalutamide and abiraterone [62]. As we explained above, since the expression of AR splice variants (such as the AR-V7 isoform) represents a potential mechanism of escape, some of these new agents act by recognizing and blocking AR-Vs. In particular, two small molecules antagonists of the AR NTD, EPI-001 and EPI-506, inhibit the AR-Vs transcriptional activity by binding to the AF1 region in the NTD domain, blocking protein– protein interactions between AR and co-regulators p300/CBP necessary for AR transcriptional activity and reducing AR interaction with androgen-response elements on target genes, and therefore outflanking resistance mechanisms associated with the loss of the AR LBD [63,64]. These compounds demonstrated to selectively inhibit AR-dependent growth of prostate cancer cells [63], leading to their evaluation in clinical trials. A phase 1/2 study is assessing the safety and antitumor activity of EPI-506 in mCRPC patients (NCT02606123). Galeterone (TOK-001) is a novel anti-androgen inhibitor that acts with a trimodal mechanism: (1) it selectively and irreversibly inhibits CYP17A1 (like abiraterone) through the specific inhibition of C17,20-lyase (but not 17a-hydroxylase), therefore preventing intratumoral androgen synthesis avoiding the secondary mineralocorticoid excess; (2) it blocks the androgenic ligand binding (similarly to the AR antagonist enzalutamide); (3) it has the peculiar property to degrade the AR and decrease AR levels [65]. Preclinical evidence of persistent activity of galeterone also in prostate cancer cells carrying AR-Vs and point mutations has been subsequently reinforced in the clinical setting. At the 2014 EORTC-NCI-AACR, Taplin and colleagues reported the findings of the phase 2 ARMOR2 trial of galeterone in CRPC patients (NCT01709734). This study demonstrated among abiraterone-refractory mCRPC patients a PSA reduction in 27% of cases, and a 30% PSA reduction in 13% of patients [66]. Moreover, galeterone seems to induce AR-V7 degradation [67]. It is still unclear if this AR-V7 destruction (and subsequently inhibition of AR-V7 transcriptional activity) represents a direct effect of the drug, or the indirect consequence of galeterone binding to the full-length AR/AR-V7 hetero-dimer (being AR-V7 frequently co-expressed with the full-length receptor in CTCs) [45]. The ongoing ARMOR3-SV phase 3 trial, which randomizes treatment-naïve mCRPC patients with AR-V7-positive circulating prostate cancer cells to galeterone or enzalutamide, will clarify the predictive role of AR-V7 in mediating primary resistance (NCT02438007). Testing galeterone in mCRC patients after progression to enzalutamide or abiraterone would be of great interest to investigate the role of AR-V7 as a biomarker of secondary resistance [65]. VT-464 is a new, non-steroidal, small-molecule CYP17A117,20-lyase selective inhibitor. Preclinical results indicate superior suppression of the AR axis with VT-464 compared to abiraterone in in castration-resistant prostate cancer cell lines and xenograft models enzalutamide-responsive or resistant, probably due to both greater selective suppression of androgen synthesis and AR antagonism [68]. Several phase 1–2 trials are currently testing the safety and anti-tumor activity of VT-646, both in mCRPC patients abiraterone and/or enzalutamide naïve (NCT02361086, NCT02012920) or resistant (NCT02130700, NCT02445976). In the latter context it would be interesting to investigate the predictive role of AR-V7. Other next-generation anti-androgen compounds seem to overcome resistance to abiraterone and/or enzalutamide, with mechanisms that are far from being entirely understood. ARN-509 is a competitive, highly selective, fully AR antagonist (with no ARagonist activity), which inhibits AR nuclear translocation and AR binding to androgen response elements. It demonstrated a manageable safety profile and an interesting antitumor activity in mCRPC patients not previously treated with enzalutamide or abiraterone [69]. ARN-509 is under evaluation in mCRPC patients in

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Fig. 2. Resistance to abiraterone and enzalutamide: the role of AR-V7. A: abiraterone acetate; AR-V7: androgen receptor splice variant 7; E: enzalutamide; PCa: prostate cancer cells.

combination with abiraterone (NCT02123758) or associated with everolimus after failure of abiraterone (NCT02106507). Similarly, the AR-inhibitor ODM-201, which binds the AR with high affinity preventing AR nuclear translocation, showed promising antitumor activity in both chemo-resistant (phase 1–2 ARADES study) and chemotherapy- and CYP17 inhibitor-naïve mCRPC patients (phase 1 ARAFOR trial) [70,71]. Interestingly, ODM-201 suppresses AR mutants (AR F876L, AR W741L and AR T877A) known to mediate resistance to second-generation anti-androgens; there are no data available on the impact of this drug on the AR-Vs [72]. A placebo-controlled, phase 3 efficacy and safety trial of ODM-201 in high-risk non-metastatic CRPC patients is ongoing (NCT02200614 – ARAMIS study). AZD3514 is a first-in-class, orally bio-available, androgendependent and -independent AR inhibitor and selective AR downregulator (SARD). AZD3514 modulates AR signaling via two diverse mechanisms (observed in vitro and in vivo): the inhibition of ligand-driven AR nuclear translocation and the down-regulation of AR levels [73]. It showed moderate anti-tumor activity in advanced CRPC patients, with nausea and vomiting as the most frequent adverse events of low grade in a recent phase 1–2 study [74], providing the rationale for future development of SARD compounds. The ability of AZD3514 of blocking AR-Vs-induced prostate cancer cells proliferation is still unknown. Promising novel therapies and predictive biomarkers (such as AR-V7) may help in defining the correct target for therapy, the ideal patient, and the most appropriate therapeutic sequence to improve outcomes for mCRPC patients. AR-Vs and taxane sensitivity Interestingly, AR-Vs splice variants can affect not only the resistance to anti-androgens molecules, but are also involved in

regulating the prostate cancer sensibility to anti-microtubules chemotherapeutics. Taxanes (docetaxel and, more recently, cabazitaxel) are an important part of the therapeutic armamentarium for the CRPC treatment, giving a demonstrated survival advantage in this setting [75,76]. Molecularly, as spindle poison drugs, taxane bind reversibly to the polymerized form of beta-tubulin subunit, promote the tubulin polymerization and prevent the microtubule disassembly. The effect is the mitotic arrest with apoptotic cell death, as a consequence of the accumulation of a mass of microtubules without formation of a functional mitotic spindle. However, the antimitotic effect of taxane does not cover by itself the full antitumor potential of these drugs. Taxane can also impair AR trafficking and signaling. Noteworthy, taxane, by stabilizing microtubules, impede the nuclear translocation of the androgen receptor, therefore compromising its transcriptional activity. The AR signaling inhibition, as a result of taxane-mediated AR sequestration in the cytoplasm, contributes to antimicrotubules clinical cytotoxic efficacy [77,78]. Moreover, it seems that besides a common mechanism of action converging on the AR axis, taxane and AR-inhibitors share joint mechanisms of resistance [79–81]. Not surprisingly, sustained AR signaling resulting from constitutively active AR-Vs (but not FL-AR over-expression) might affect taxane sensitivity, considering the inhibitory effect on AR pathway that represent a key mechanism of action of antimicrotubules chemotherapy in prostate tumor. Thadani-Mulero and Colleagues were the first to describe the involvement of AR-Vs in resistance to taxane chemotherapy in xenograft mouse models [82]. The different androgen receptor variants displays diverse mechanism of microtubule-mediated nuclear translocation: the ARv567es, which maintains the hinge region, is microtubule- and dynein- dependent for its intra-nuclear storage and subsequent transcriptional activity, while the AR-V7, lacking the hinge region necessary for

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microtubule binding, is microtubule-independent. Therefore, tumor cells expressing the microtubule-interacting ARv567es receptor splice variant (but not AR-V7) are likely to benefit from taxane treatment as a result of the antimicrotubules-induced receptor seizure in the cytoplasm. The expression of ARv567es can be thus considered a marker of taxanes sensitivity, while the AR-V7 expression confers resistance to taxane chemotherapy [82]. In contrast to the Thadani-Mulero’s theory, Zhang et al. recently support the independence of both the AR variants (not only of ARV7) from the microtubules pathway, being the nuclear translocation of AR-V7 and AR-v567es determined by importing a/b machinery [83]. This can explain, at least in part, the ability of AR splice variants to overcome taxane-caused AR intra-nuclear translocation. Moreover, these two AR variants can impair the FLAR interaction with microtubules, thereby preventing the FL-AR cytoplasm accumulation triggered by taxane. Therefore, AR-V7 and AR-v567es might confer resistance to microtubules-targeting drugs [83]. Of note, methodological differences (in vitro and in vivo microtubule-binding assays, respectively, and different dosage of taxane) exist between the two studies, probably affecting the discrepant results. Recently Antonarakis at al. prospectively investigated the predictive role of AR-V7 mRNA expression in CTCs of 37 taxanetreated prostate cancer patients [84]. Moreover, the study evaluated the potential differential impact of the AR-V7 status on diverse kinds of treatment (taxane vs enzalutamide or abiraterone), incorporating the updated results of their previous research [60]. Clinical data contrast with mouse models reported above. AR-V7-positive patients had worse outcomes in response to taxanes, although not statically significant, as compared to ARV7-negative patients. Therefore, despite the small and underpowered study, the AR-V7 detection failed to predict the taxanes activity. Interestingly, AR-V7 detection in CTCs from mCRPC patients did not correlate with primary resistance to microtubulestargeting chemotherapy (differently from anti-androgen drugs). Furthermore, ARv-V7-positive CRPC men were more likely to benefit from taxane therapy than from AR-targeted therapies, while in AR-V7-negative patients the treatment type efficacy (chemotherapy vs abiraterone/enzalutamide) did not differ significantly. Although confirmations in large perspective clinical trials are needed, AR-V7 status may represent a reliable biomarker for prostate cancer treatment selection [84,85]. Analogously, the detection of AR-V7 splice variant gene in CTCs of 29 CRPC patients undergoing cabazitaxel treatment was not associated with a relevant clinical benefit [86]. Cabazitaxel may therefore be considered a viable therapeutic option (compared to enzalutamide/abiraterone) in AR-V7-positive patients, being the response to chemotherapy independent from the AR-V7 status. Of note, this trial utilized the CellSearch System to detect CTCs, while in the previous studies CTCs were identified by the AdnaTest, making comparisons more difficult. Assay methodology needs to be standardized. Additional investigations are warranted to shed light on these conflicting data and better delineate the role of AR-Vs in predicting the prostate cancer sensibility to taxane chemotherapy. We are awaiting the results of the ongoing phase II trial (PRIMCAB study – NCT02379390) comparing cabazitaxel to either enzalutamide or abiraterone in chemotherapy-naïve mCRPC patients primarily resistant to AR-inhibitors (abiraterone or enzalutamide). This trial will also analyze the predictive role of the biomarker AR-V7 mRNA in CTCs. Discussion The availability of several drugs in the prostate cancer therapeutic armamentarium raised the question on the most

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appropriate treatment choice. Clinicians should be able to identify patients most likely to benefit from a specific treatment, avoiding ineffective and potentially harmful therapies. Many variables must be taken into account while proposing a definite therapy, ranging from patient characteristics (performance status, age, comorbidity), to clinical cancer features (tumor burden, sites of metastases – bone vs visceral, previous treatments, duration of response), up to including molecular characteristics of the tumor with predictive significance of treatment resistance or sensitivity. Prostate cancer is an androgen-dependent tumor, relying on the AR signaling pathway both in early (hormone-sensitive) and advanced (castrationresistance) phases of metastatic disease. Therefore, it is not surprising that the AR remains a major effective therapeutic target in CRPC. The sustained AR signaling in CRPC led to the development of two next-generation androgen-directed therapies, abiraterone and enzalutamide, countering the ‘‘traditional” cytotoxic drugs (docetaxel and cabazitaxel). Translational research focuses on the identification of biomarkers predictive of treatment response. Recent advances highlighted the role of AR-Vs, mainly AR-V7, in mediating prostate cancer progression and treatment resistance. In particular, the constitutively active AR-V7 isoform could be a marker of enzalutamide and abiraterone resistance, while its role in predicting taxanes sensibility is far more ambiguous. The PRIMCAB study will evaluate the role of AR-V7 in mCRPC patients treated with enzalutamide or chemotherapy (cabazitaxel), but the receptor variant will not guide the treatment choice. In the ARMOR3 trial galeterone or enzalutamide will be evaluated in ARV7 positive CRPC patients, therefore assessing the hormonal effects in the context of AR-V7 positivity [87]. Efforts are needed to better clarify the predictive ability of ARV7, standardize sensitive and cost-sustainable clinical laboratory assays for the measurement of patients AR-Vs, and validate it in large cohorts studies. The availability of a biomarker obtained by liquid biopsy would give the revolutionary opportunity to instantly assess a predictive marker potentially dynamic over time, guiding clinicians in treatment decisions at different moments during the course of the disease. Conflicts of interest All authors declare that they have no conflicts of interest. Financial disclosures None for all authors. Acknowledgment Supported by a grant of the Italian Association for Cancer Research (AIRC-IG 11930, AIRC 5per mille 12214). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ctrv.2015.12.003. References [1] Eisenberger MA, Walsh PC. Early androgen deprivation for prostate cancer? N Engl J Med 1999;341:1837–8. [2] Garraway LA, Sellers WR. Lineage dependency and lineage-survival oncogenes in human cancer. Nat Rev Cancer 2006;6:593–602. [3] Scher HI, Fizazi K, Saad F, Taplin ME, Sternberg CN, Miller K, et al. Increased survival with enzalutamide in prostate cancer after chemotherapy. N Engl J Med 2012;367:1187–97.

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