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      Functional analysis of androgen receptor mutations that confer anti-androgen resistance identified in circulating cell-free DNA from prostate cancer patients

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          Abstract

          Background

          The androgen receptor (AR) is a pivotal drug target for the treatment of prostate cancer, including its lethal castration-resistant (CRPC) form. All current non-steroidal AR antagonists, such as hydroxyflutamide, bicalutamide, and enzalutamide, target the androgen binding site of the receptor, competing with endogenous androgenic steroids. Several AR mutations in this binding site have been associated with poor prognosis and resistance to conventional prostate cancer drugs. In order to develop an effective CRPC therapy, it is crucial to understand the effects of these mutations on the functionality of the AR and its ability to interact with endogenous steroids and conventional AR inhibitors.

          Results

          We previously utilized circulating cell-free DNA (cfDNA) sequencing technology to examine the AR gene for the presence of mutations in CRPC patients. By modifying our sequencing and data analysis approaches, we identify four additional single AR mutations and five mutation combinations associated with CRPC. Importantly, we conduct experimental functionalization of all the AR mutations identified by the current and previous cfDNA sequencing to reveal novel gain-of-function scenarios. Finally, we evaluate the effect of a novel class of AR inhibitors targeting the binding function 3 (BF3) site on the activity of CRPC-associated AR mutants.

          Conclusions

          This work demonstrates the feasibility of a prognostic and/or diagnostic platform combining the direct identification of AR mutants from patients’ serum, and the functional characterization of these mutants in order to provide personalized recommendations regarding the best future therapy.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s13059-015-0864-1) contains supplementary material, which is available to authorized users.

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          Most cited references 39

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          Maintenance of intratumoral androgens in metastatic prostate cancer: a mechanism for castration-resistant tumor growth.

          Therapy for advanced prostate cancer centers on suppressing systemic androgens and blocking activation of the androgen receptor (AR). Despite anorchid serum androgen levels, nearly all patients develop castration-resistant disease. We hypothesized that ongoing steroidogenesis within prostate tumors and the maintenance of intratumoral androgens may contribute to castration-resistant growth. Using mass spectrometry and quantitative reverse transcription-PCR, we evaluated androgen levels and transcripts encoding steroidogenic enzymes in benign prostate tissue, untreated primary prostate cancer, metastases from patients with castration-resistant prostate cancer, and xenografts derived from castration-resistant metastases. Testosterone levels within metastases from anorchid men [0.74 ng/g; 95% confidence interval (95% CI), 0.59-0.89] were significantly higher than levels within primary prostate cancers from untreated eugonadal men (0.23 ng/g; 95% CI, 0.03-0.44; P < 0.0001). Compared with primary prostate tumors, castration-resistant metastases displayed alterations in genes encoding steroidogenic enzymes, including up-regulated expression of FASN, CYP17A1, HSD3B1, HSD17B3, CYP19A1, and UGT2B17 and down-regulated expression of SRD5A2 (P < 0.001 for all). Prostate cancer xenografts derived from castration-resistant tumors maintained similar intratumoral androgen levels when passaged in castrate compared with eugonadal animals. Metastatic prostate cancers from anorchid men express transcripts encoding androgen-synthesizing enzymes and maintain intratumoral androgens at concentrations capable of activating AR target genes and maintaining tumor cell survival. We conclude that intracrine steroidogenesis may permit tumors to circumvent low levels of circulating androgens. Maximal therapeutic efficacy in the treatment of castration-resistant prostate cancer will require novel agents capable of inhibiting intracrine steroidogenic pathways within the prostate tumor microenvironment.
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            Antitumour activity of MDV3100 in castration-resistant prostate cancer: a phase 1-2 study.

            MDV3100 is an androgen-receptor antagonist that blocks androgens from binding to the androgen receptor and prevents nuclear translocation and co-activator recruitment of the ligand-receptor complex. It also induces tumour cell apoptosis, and has no agonist activity. Because growth of castration-resistant prostate cancer is dependent on continued androgen-receptor signalling, we assessed the antitumour activity and safety of MDV3100 in men with this disease. This phase 1-2 study was undertaken in five US centres in 140 patients. Patients with progressive, metastatic, castration-resistant prostate cancer were enrolled in dose-escalation cohorts of three to six patients and given an oral daily starting dose of MDV3100 30 mg. The final daily doses studied were 30 mg (n=3), 60 mg (27), 150 mg (28), 240 mg (29), 360 mg (28), 480 mg (22), and 600 mg (3). The primary objective was to identify the safety and tolerability profile of MDV3100 and to establish the maximum tolerated dose. The trial is registered with ClinicalTrials.gov, number NCT00510718. We noted antitumour effects at all doses, including decreases in serum prostate-specific antigen of 50% or more in 78 (56%) patients, responses in soft tissue in 13 (22%) of 59 patients, stabilised bone disease in 61 (56%) of 109 patients, and conversion from unfavourable to favourable circulating tumour cell counts in 25 (49%) of the 51 patients. PET imaging of 22 patients to assess androgen-receptor blockade showed decreased (18)F-fluoro-5alpha-dihydrotestosterone binding at doses from 60 mg to 480 mg per day (range 20-100%). The median time to progression was 47 weeks (95% CI 34-not reached) for radiological progression. The maximum tolerated dose for sustained treatment (>28 days) was 240 mg. The most common grade 3-4 adverse event was dose-dependent fatigue (16 [11%] patients), which generally resolved after dose reduction. We recorded encouraging antitumour activity with MDV3100 in patients with castration-resistant prostate cancer. The results of this phase 1-2 trial validate in man preclinical studies implicating sustained androgen-receptor signalling as a driver in this disease. Medivation, the Prostate Cancer Foundation, National Cancer Institute, the Howard Hughes Medical Institute, Doris Duke Charitable Foundation, and Department of Defense Prostate Cancer Clinical Trials Consortium. Copyright 2010 Elsevier Ltd. All rights reserved.
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              Mutation of the androgen-receptor gene in metastatic androgen-independent prostate cancer.

              Metastatic prostate cancer is a leading cause of cancer-related death in men. The rate of response to androgen ablation is high, but most patients relapse as a result of the outgrowth of androgen-independent tumor cells. The androgen receptor, which binds testosterone and stimulates the transcription of androgen-responsive genes, regulates the growth of prostate cells. We analyzed the androgen-receptor genes from samples of metastatic androgen-independent prostate cancers to determine whether mutations in the gene have a role in androgen independence. Complementary DNA was synthesized from metastatic prostate cancers in 10 patients with androgen-independent prostate cancer, and the expression of the androgen-receptor gene was estimated by amplification with the polymerase chain reaction. Exons B through H of the gene were cloned, and mutations were identified by DNA sequencing. The functional effects of the mutations were assessed in cells transfected with mutant genes. All androgen-independent tumors expressed high levels of androgen-receptor gene transcripts, relative to the levels expressed by an androgen-independent prostate-cancer cell line (LNCaP). Point mutations in the androgen-receptor gene were identified in metastatic cells from 5 of the 10 patients examined. One mutation was in the same codon as the mutation found previously in the androgen-independent prostate-cancer cell line. The mutations were not detected in the primary tumors from of the two patients. Functional studies of two of the mutant androgen receptors demonstrated that they could be activated by progesterone and estrogen. Most metastatic androgen-independent prostate cancers express high levels of androgen-receptor gene transcripts. Mutations in androgen-receptor genes are not uncommon and may provide a selective growth advantage after androgen ablation.
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                Author and article information

                Contributors
                nlallous@prostatecentre.com
                svolik@prostatecentre.com
                sawrey@prostatecentre.com
                eleblanc@prostatecentre.com
                rst2@sfu.ca
                jmurillo@prostatecentre.com
                ksingh@prostatecentre.com
                Arun.Azad@bccancer.bc.ca
                awyatt@prostatecentre.com
                slebihan@prostatecentre.com
                kchi@bccancer.bc.ca
                mgleave@prostatecentre.com
                prennie@prostatecentre.com
                ccollins@prostatecentre.com
                604-875-4818 , artc@interchange.ubc.ca
                Journal
                Genome Biol
                Genome Biol
                Genome Biology
                BioMed Central (London )
                1474-7596
                1474-760X
                26 January 2016
                26 January 2016
                2016
                : 17
                Affiliations
                [ ]Vancouver Prostate Centre, University of British Columbia, 2660 Oak St., Vancouver, BC V6H 3Z6 Canada
                [ ]Department of Medical Oncology, BC Cancer Agency, 600 West 10th Avenue, Vancouver, BC V5Z 4E6 Canada
                [ ]Laboratory for Advanced Genome Analysis (LAGA), Vancouver Prostate Centre, 2660 Oak St., Vancouver, BC V6H 3Z6 Canada
                Article
                864
                10.1186/s13059-015-0864-1
                4729137
                26813233
                © Lallous et al. 2016

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100000109, Prostate Cancer Canada, Canada Safeway;
                Award ID: SP2013-02
                Award Recipient :
                Funded by: Movember
                Award ID: T2013-1
                Award Recipient :
                Funded by: CIHR/Terry Fox Foundation
                Award ID: #TFF-116129
                Award Recipient :
                Funded by: Canadian Institutes of Health Research and a Movember Discovery Program award
                Award ID: #272111
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000054, National Cancer Institute;
                Award ID: P50CA097186
                Award Recipient :
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                Research
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                © The Author(s) 2016

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