| Summary: | Currently, androgen ablation therapy for advanced prostate cancer (PCa) attempts to inactivate the androgen receptor (AR) by diminishing circulating androgen levels and/or inhibiting AR ligand-binding. Although patient response is initially positive, the cancer ultimately recurs in a more aggressive and therapy-resistant form, termed ‘castration- resistant’ PCa (CRPC). Importantly, the majority of CRPCs retain AR dependency, supported by the continued expression of functional AR as well as androgen-dependent genes, suggesting new AR targeting therapies will be beneficial in this disease. One method of overcoming castration resistance is the selection of mutations residing in the AR ligand binding domain (LBD) which allows receptor activation in response to promiscuous ligands such as estrogens, progesterone, cortisol and anti-androgens. Recently, a novel AR-targeting compound, AZD3514, has been developed by AstraZeneca that modulates AR signalling by inhibiting ligand driven activity in addition to initiating AR protein down-regulation. However, given that this and other next-generation anti-androgens are to be used in advanced PCa, an important consideration in future treatment is to determine whether these targeted therapies will be effective in forms of CRPC and AR mutations. Here we outline the mechanism of AZD3514 action before determining the efficacy of next-generation anti-androgens in models of CRPC using three distinct approaches. Firstly, compound efficacy was determined in a panel of in vitro cell line models reflecting aspects of CRPC including anti-androgen therapy resistance, AR amplification and AR-variants. Secondly, the compounds were tested in the background of an AR negative PCa cell line transiently expressing AR mutants, which was coupled to a luciferase based reporter assay to measure AR transcriptional activity. Thirdly, a physiologically relevant AR rescue/replacement approach was generated in the background of LNCaP cells using targeted siRNA oligos to effectively knockdown endogenous AR without impacting on the expression of an ectopically expressed mutant. This approach permitted the functional interrogation of any given AR mutant, however we focussed on the casodex activating ARw74ii. mutant to demonstrate the models effectiveness and ability to be targeted by next generation anti-androgens. Finally, following validation of the AR\v74il rescue model, we performed microarray analysis to interrogate the casodex/ARw74iL transcriptional program to identify casodex-induced genes that could be of potential as future biomarkers or therapeutic targets.
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