Dihydrotestosterone (DHT) is regarded as the most potent natural androgen and is implicated in the development and progression of castration resistant prostate cancer (CRPC). AR-regulated genes as well as cellular proliferation in the androgen dependent prostate cancer cell lines LNCaP and VCaP. Proteomic analysis revealed that 11KDHT regulated the expression of more AR-regulated proteins than DHT in VCaP cells while conversion assays showed that 11KT and 11KDHT are metabolized HA14-1 at a significantly lower rate in both LNCaP and VCaP cells when compared to T and DHT respectively. Our findings show that HA14-1 11KT and 11KDHT are androgens capable of inducing androgen-dependant gene expression and cell growth and that these steroids have the potential to remain active longer than T and DHT due to the HA14-1 decreased rate at which they are metabolised. Collectively our data demonstrates that 11KT and 11KDHT likely play a vital but overlooked role in the development and progression of CRPC. Introduction Prostate cancer (PCa) is the second most common cancer among men worldwide [1] with androgen deprivation therapy (ADT) being the first line treatment for advanced PCa since androgen signalling is essential for normal and malignant growth of prostate tissue. This treatment which almost entirely eliminates circulating levels of testosterone (T) is usually initially effective. However most men experience only short term regression (2-3 years) with nearly all patients developing the more aggressive castration-resistant PCa (CRPC) which is usually associated with poor survival rates [2]. The majority of evidence suggests that CRPC develops as a result of the reactivation of androgen receptor (AR) HA14-1 signalling despite castrate levels of T (≤50 ng/dL) [3-5]. The AR and AR-regulated genes are expressed in most clinical cases of CRPC demonstrating that this AR axis is usually reactivated and drives tumour growth [4 5 HA14-1 Mechanisms proposed to be responsible for the continued AR activation include up-regulation of AR expression and/or gain-of-function mutations of the AR[6]. Recent clinical trials demonstrating beneficial clinical outcomes after treatment with the AR antagonist enzalutamide [7] and the CYP17A1 inhibitor abiraterone [8-10] have highlighted the continued androgen dependency of CRPC. Studies have confirmed that this adrenal androgen precursors dehydroepiandrosterone (DHEA) and androstenedione (A4) serve as the source of intratumoral androgen production under castrate conditions [11-15]. The potent androgen 5 (DHT) is usually produced by the alternate 5α-dione pathway which bypasses T to produce DHT via 5α-androstanedione (5α-dione) [12-15]. In addition to DHEA and A4 the human adrenal gland produces substantial amounts of the inactive C19 steroid 11β-hydroxyandrostenedione (11OHA4) [16-18] via the cytochrome P450 11β-hydroxylase (CYP11B1) catalysed hydroxylation of A4 [19 20 11 is one of the most abundant C19 steroid produced by the human adrenal both before and after adrenocorticotrophic hormone (ACTH) treatment [17]. A recent study by our laboratory identified a novel pathway for 11OHA4 metabolism in androgen dependent prostate cancer cells which leads to the production of the androgens 11-ketotestosterone (11KT) and 11keto-5α-dihydrotestosterone (11KDHT) (Fig 1). We showed that at the concentration of 1 1 nM 11 and 11KDHT have androgenic properties comparable to T and DHT respectively [21]. However further work is needed to characterize these androgens. Fig 1 Biosynthesis of 11KT and 11KDHT from the adrenal androgen precursor 11OHA4. The aim of this study DIAPH2 was therefore to compare the androgenic properties of 11KT and 11KDHT to that of T and DHT. Competitive whole cell binding assays revealed that 11KT and 11KDHT bind to the human AR with affinities comparable to that of T and DHT. Transactivation assays on a synthetic androgen response element (ARE) demonstrated that this relative agonist potencies and efficacies of 11KT and 11KDHT are comparable to that of HA14-1 T and DHT respectively. Moreover we showed that 11KT and 11KDHT treatment of two androgen dependent prostate cancer cell lines LNCaP and VCaP result in the regulation of endogenous AR-regulated genes at both the mRNA and protein level and also drive cellular proliferation. Finally we demonstrate that 11KT and 11KDHT are metabolised at a lower rate than T and DHT in both LNCaP and VCaP cells and as a result are likely able to exert prolonged androgenic effects. These findings confirm that both 11KT and 11KDHT are androgens and suggest that the 11OHA4 pathway may be a potential role player in the development and progression of CRPC. Materials.

Dihydrotestosterone (DHT) is regarded as the most potent natural androgen and
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