Intracrine Androgen Research Articles

Prostate cancer androgen biosynthesis relies solely on CYP17A1 downstream metabolites

Prostate cancer (PC) is dependent on androgen receptor (AR) activation by testosterone and 5α-dihydrotestosterone (DHT). Intratumoral androgen accumulation and activation despite systemic androgen deprivation therapy underlies the development of castration-resistant PC (CRPC), but the precise pathways involved remain controversial. Here we investigated the differential contributions of de novo androgen biosynthesis and androgen precursor conversion to androgen accumulation. Steroid flux analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed on (CR)PC cell lines and fresh patient PC tissue slices after incubation with classic and alternative biosynthesis intermediates, alongside quantitative PCR analysis for steroidogenic enzyme expression. Activity of CYP17A1 was undetectable in all PC cell lines and patient PC tissue slices. Instead, steroid flux analysis confirmed the generation of testosterone and DHT from adrenal precursors and reactivation of androgen metabolites. Precursor steroids upstream of DHEA were converted down the first steps of the alternative DHT biosynthesis pathway, but did not proceed through to active androgen generation. Comprehensive steroid flux analysis of (CR)PC cells provides strong evidence against intratumoral de novo androgen biosynthesis and demonstrates that androgen precursor steroids downstream of CYP17A1 activities constitute the major source of intracrine androgen generation.

An androgen receptor regulated gene score is associated with epithelial to mesenchymal transition features in triple negative breast cancers

BackgroundAndrogen receptor (AR) is considered a marker of better prognosis in hormone receptor positive breast cancers (BC), however, its role in triple negative breast cancer (TNBC) is controversial. This may be attributed to intrinsic molecular differences or scoring methods for AR positivity. We derived AR regulated gene score and examined its utility in BC subtypes. MethodsAR regulated genes were derived by applying a bioinformatic pipeline on publicly available microarray data sets of AR+ BC cell lines and gene score was calculated as average expression of six AR regulated genes. Tumors were divided into AR high and low based on gene score and associations with clinical parameters, circulating androgens, survival and epithelial to mesenchymal transition (EMT) markers were examined, further evaluated in invitro models and public datasets. Results53% (133/249) tumors were classified as AR gene score high and were associated with significantly better clinical parameters, disease-free survival (86.13 vs 72.69 months, log rank p = 0.032) when compared to AR low tumors. 36% of TNBC (N = 66) were AR gene score high with higher expression of EMT markers (p = 0.024) and had high intratumoral levels of 5α-reductase, enzyme involved in intracrine androgen metabolism. In MDA-MB-453 treated with dihydrotestosterone, SLUG expression increased, E-cadherin decreased with increase in migration and these changes were reversed with bicalutamide. Similar results were obtained in public datasets. ConclusionDeciphering the role of AR in BC is difficult based on AR protein levels alone. Our results support the context dependent function of AR in driving better prognosis in ER positive tumors and EMT features in TNBC tumors.

A role for steroid 5 alpha-reductase 1 in vascular remodeling during endometrial decidualization.

Decidualization is the hormone-dependent process of endometrial remodeling that is essential for fertility and reproductive health. It is characterized by dynamic changes in the endometrial stromal compartment including differentiation of fibroblasts, immune cell trafficking and vascular remodeling. Deficits in decidualization are implicated in disorders of pregnancy such as implantation failure, intra-uterine growth restriction, and pre-eclampsia. Androgens are key regulators of decidualization that promote optimal differentiation of stromal fibroblasts and activation of downstream signaling pathways required for endometrial remodeling. We have shown that androgen biosynthesis, via 5α-reductase-dependent production of dihydrotestosterone, is required for optimal decidualization of human stromal fibroblasts in vitro, but whether this is required for decidualization in vivo has not been tested. In the current study we used steroid 5α-reductase type 1 (SRD5A1) deficient mice (Srd5a1-/- mice) and a validated model of induced decidualization to investigate the role of SRD5A1 and intracrine androgen signaling in endometrial decidualization. We measured decidualization response (weight/proportion), transcriptomic changes, and morphological and functional parameters of vascular development. These investigations revealed a striking effect of 5α-reductase deficiency on the decidualization response. Furthermore, vessel permeability and transcriptional regulation of angiogenesis signaling pathways, particularly those that involved vascular endothelial growth factor (VEGF), were disrupted in the absence of 5α-reductase. In Srd5a1-/- mice, injection of dihydrotestosterone co-incident with decidualization restored decidualization responses, vessel permeability, and expression of angiogenesis genes to wild type levels. Androgen availability declines with age which may contribute to age-related risk of pregnancy disorders. These findings show that intracrine androgen signaling is required for optimal decidualization in vivo and confirm a major role for androgens in the development of the vasculature during decidualization through regulation of the VEGF pathway. These findings highlight new opportunities for improving age-related deficits in fertility and pregnancy health by targeting androgen-dependent signaling in the endometrium.

Mesoporous silica nanoparticles combined with AKR1C3 siRNA inhibited the growth of castration-resistant prostate cancer by suppressing androgen synthesis in vitro and in vivo

Intracrine androgen synthesis plays a critical role in the development of castration-resistant prostate cancer (CRPC). Aldo-keto reductase family 1 member C3 (AKR1C3) is a vital enzyme in the intracrine androgen synthesis pathway. In this study, mesoporous silica nanoparticles (MSNs) were employed to deliver small interfering RNA targeting AKR1C3 (siAKR1C3) to downregulate AKR1C3 expression in CPRC cells. The optimal weight ratio of MSNs/siAKR1C3 was determined by a gel retardation assay. Prostate cancer cells such as VCaP cells, which intracrinally express AKR1C3, and LNCaP-AKR1C3 cells stably transfected with AKR1C3 were used to investigate the antitumour effect of MSNs-siAKR1C3. Fluorescence detection and Western blot analyses were applied to confirm the entrance of MSNs-siAKR1C3 into the cells. A SRB (Sulforhodamine B) assay was employed to assess the cell viability, and a radioimmunoassay was used to measure the androgen concentration. Moreover, real-time PCR (RT-PCR), Western blot analysis and ELISA were used to determine the transcription and expression of prostate-specific antigen (PSA), AKR1C3 and androgen receptor (AR). Meanwhile, a reporter gene assay was performed to determine the AR activity. Additionally, a castrated nude mouse xenograft tumour model was produced to verify the inhibitory effect of MSNs-siAKR1C3 in vivo. The results showed that the optimal weight ratio of MSNs/siAKR1C3 was 140:1, and the complex could effectively enter cells, downregulate AKR1C3 expression, reduce the androgen concentration, inhibit AR activation, and inhibit CRPC development both in vitro and in vivo. These results indicate that decreasing intracrine androgen synthesis and inactivating AR signals by MSNs-siAKR1C3 may be a potential effective method for CRPC treatment.

Steroid Sulfatase Stimulates Intracrine Androgen Synthesis and is a Therapeutic Target for Advanced Prostate Cancer.

Most patients with prostate cancer receiving enzalutamide or abiraterone develop resistance. Clinical evidence indicates that serum levels of dehydroepiandrosterone sulfate (DHEAS) and biologically active DHEA remain in the high range despite antiandrogen treatment. The conversion of DHEAS into DHEA by steroid sulfatase (STS) may contribute to sustained intracrine androgen synthesis. Here, we determine the contribution of STS to treatment resistance and explore the potential of targeting STS to overcome resistance in prostate cancer. STS expression was examined in patients and cell lines. In vitro, STS activity and expression were modulated using STS-specific siRNA or novel STS inhibitors (STSi). Cell growth, colony formation, androgen production, and gene expression were examined. RNA-sequencing analysis was conducted on VCaP cells treated with STSi. Mice were treated with STSis with or without enzalutamide to determine their effects in vivo. STS is overexpressed in patients with castration-resistant prostate cancer (CRPC) and resistant cells. STS overexpression increases intracrine androgen synthesis, cell proliferation, and confers resistance to enzalutamide and abiraterone. Inhibition of STS using siRNA suppresses prostate cancer cell growth. Targeting STS activity using STSi inhibits STS activity, suppresses androgen receptor transcriptional activity, and reduces the growth of resistant C4-2B and VCaP prostate cancer cells. STSis significantly suppress resistant VCaP tumor growth, decrease serum PSA levels, and enhance enzalutamide treatment in vitro and in vivo. These studies suggest that STS drives intracrine androgen synthesis and prostate cancer proliferation. Targeting STS represents a therapeutic strategy to treat CRPC and improve second-generation antiandrogen therapy.

Genistein Combined Polysaccharide (GCP) Can Inhibit Intracrine Androgen Synthesis in Prostate Cancer Cells.

Our group and others have previously shown that genistein combined polysaccharide (GCP), an aglycone isoflavone-rich extract with high bioavailability and low toxicity, can inhibit prostate cancer (CaP) cell growth and survival as well as androgen receptor (AR) activity. We now elucidate the mechanism by which this may occur using LNCaP and PC-346C CaP cell lines; GCP can inhibit intracrine androgen synthesis in CaP cells. UPLC-MS/MS and qPCR analyses demonstrated that GCP can mediate a ~3-fold decrease in testosterone levels (p < 0.001) and cause decreased expression of intracrine androgen synthesis pathway enzymes (~2.5-fold decrease of 3βHSD (p < 0.001), 17βHSD (p < 0.001), CYP17A (p < 0.01), SRB1 (p < 0.0001), and StAR (p < 0.01)), respectively. Reverse-phase HPLC fractionation and bioassay identified three active GCP fractions. Subsequent NMR and LC-MS analysis of the fraction with the highest level of activity, fraction 40, identified genistein as the primary active component of GCP responsible for its anti-proliferative, pro-apoptotic, and anti-AR activity. GCP, fraction 40, and genistein all mediated at least a ~2-fold change in these biological activities relative to vehicle control (p < 0.001). Genistein caused similar decreases in the expression of 17βHSD and CYP17A (2.5-fold (p < 0.001) and 1.5-fold decrease (p < 0.01), respectively) compared to GCP, however it did not cause altered expression of the other intracrine androgen synthesis pathway enzymes; 3βHSD, SRB1, and StAR. Our combined data indicate that GCP and/or genistein may have clinical utility and that further pre-clinical studies are warranted.

SAT-114 Loss of DHEA-Targeting SULT2b1b Sulfotransferase Exacerbates Aggressive Traits of Prostate Cancer

The prostate-expressed sulfotransferase SULT2B1b (SULT2B) regulates intracrine androgen homeostasis by mediating 3β-sulfation of DHEA, thus reducing the precursor pool in the androgen biosynthesis pathway. We explored how loss of SULT2B might influence prostate cancer progression. Results show that SULT2B ablation in castration-resistant prostate cancer (CRPC) cells, generated by stable RNA interference or gene knockout, led to robust activation of the ERK1/2 Map kinase survival signal and induction of epithelial to mesenchymal transition (EMT). EMT activation was concluded on the basis of increased levels of vimentin (a mesenchymal protein) and the EMT-activating transcription factors SNAI1 (Snail) and TWIST1, shown by Western blotting, mass spectrometry and single-cell mass cytometry. Loss of SULT2B was associated with enhanced motility and invasive activity of CRPC cells in vitro and their growth escalation in vivo as xenografts. Higher invasion and metastasis potential of SULT2B-ablated CRPC cells was further indicated by results that these cells are less adhesive (i.e. easily detachable) and less stiff (i.e. more pliable) based on atomic force microscopy analysis of individual cells. Notably, AKR1C3, an aldo-keto reductase, which is elevated frequently in advanced prostate cancer, showed marked upregulation in SULT2B-deficient cells. AKR1C3 regulates androgen receptor (AR) signaling by promoting androgen biosynthesis and functioning as an AR-selective coactivator. While levels of AR and DHT did not change, AR activity was elevated, since PSA and FKBP5 mRNA induction by DHT-activated AR was several fold higher in SULT2B-silenced cells. The DHT-metabolizing AKR1C2 aldo-keto reductase was also upregulated, which likely accounts for a steady-state androgen level despite elevated AKR1C3 expression. Phosphorylation of ERK decreased in AKR1C3-silenced cells, signifying a causal link between AKR1C3 upregulation and ERK1/2 activation. SULT2B was undetectable immunohistochemically in tissue microarrays of clinical CRPC metastases, while SULT2B-negative samples showed AKR1C3-positive immunostaining. Primary prostate cancer exhibited variable, Gleason score independent SULT2B levels -- varying from strong positive to significantly reduced or undetectable. The reciprocal expression pattern for SULT2B and AKR1C3 in clinical CRPC suggests that AKR1C3 upregulation, ERK1/2 activation and increased aggressive traits of SULT2B-ablated cells, observed in vitro in cell models, may be clinically significant. Pathways regulating the inhibitory SULT2B-AKR1C3 axis may inform new avenue(s) for delaying disease progression in SULT2B-deficient prostate cancer.Funding Support: 1I01BX000280, VA (BC); W81XWH-14-1-0606, DOD (BC); IK6 BX004207, VA (BC); P50 CA97186, NIH & W81XWH-12-1-0208, DOD (EAM)

Inhibitory Interplay of SULT2B1b Sulfotransferase with AKR1C3 Aldo-keto Reductase in Prostate Cancer.

SULT2B1b (SULT2B) is a prostate-expressed hydroxysteroid sulfotransferase, which may regulate intracrine androgen homeostasis by mediating 3β-sulfation of dehydroepiandrosterone (DHEA), the precursor for 5α-dihydrotestosterone (DHT) biosynthesis. The aldo-keto reductase (AKR)1C3 regulates androgen receptor (AR) activity in castration-resistant prostate cancer (CRPC) by promoting tumor tissue androgen biosynthesis from adrenal DHEA and also by functioning as an AR-selective coactivator. Herein we report that SULT2B-depleted CRPC cells, arising from stable RNA interference or gene knockout (KO), are markedly upregulated for AKR1C3, activated for ERK1/2 survival signal, and induced for epithelial-to-mesenchymal (EMT)-like changes. EMT was evident from increased mesenchymal proteins and elevated EMT-inducing transcription factors SNAI1 and TWIST1 in immunoblot and single-cell mass cytometry analyses. SULT2B KO cells showed greater motility and invasion in vitro; growth escalation in xenograft study; and enhanced metastatic potential predicted on the basis of decreased cell stiffness and adhesion revealed from atomic force microscopy analysis. While AR and androgen levels were unchanged, AR activity was elevated, since PSA and FKBP5 mRNA induction by DHT-activated AR was several-fold higher in SULT2B-silenced cells. AKR1C3 silencing prevented ERK1/2 activation and SNAI1 induction in SULT2B-depleted cells. SULT2B was undetectable in nearly all CRPC metastases from 50 autopsy cases. Primary tumors showed variable and Gleason score (GS)-independent SULT2B levels. CRPC metastases lacking SULT2B expressed AKR1C3. Since AKR1C3 is frequently elevated in advanced prostate cancer, the inhibitory influence of SULT2B on AKR1C3 upregulation, ERK1/2 activation, EMT-like induction, and on cell motility and invasiveness may be clinically significant. Pathways regulating the inhibitory SULT2B-AKR1C3 axis may inform new avenue(s) for targeting SULT2B-deficient prostate cancer.

Intracrine androgen biosynthesis and drug resistance.

Castration-resistant prostate cancer is the lethal form of prostate cancer and most commonly remains dependent on androgen receptor (AR) signaling. Current therapies use AR signaling inhibitors (ARSI) exemplified by abiraterone acetate, a P450c17 inhibitor, and enzalutamide, a potent AR antagonist. However, drug resistance to these agents occurs within 12-18 months and they only prolong overall survival by 3-4 months. Multiple mechanisms can contribute to ARSI drug resistance. These mechanisms can include but are not limited to germline mutations in the AR, post-transcriptional alterations in AR structure, and adaptive expression of genes involved in the intracrine biosynthesis and metabolism of androgens within the tumor. This review focuses on intracrine androgen biosynthesis, how this can contribute to ARSI drug resistance, and therapeutic strategies that can be used to surmount these resistance mechanisms.

GnRH Antagonists Have Direct Inhibitory Effects On Castration-Resistant Prostate Cancer Via Intracrine Androgen and AR-V7 Expression.

Hormone therapy is currently the mainstay in the management of locally advanced and metastatic prostate cancer. Degarelix (Firmagon), a gonadotropin-releasing hormone (GnRH) receptor antagonist differs from luteinizing hormone-releasing hormone (LHRH) agonists by avoiding "testosterone flare" and lower follicle-stimulating hormone (FSH) levels. The direct effect of degarelix and leuprolide on human prostate cancer cells was evaluated. In LNCaP, C4-2BMDVR, and CWR22Rv1 cells, degarelix significantly reduced cell viability compared with the controls (P ≤ 0.01). Leuprolide was stimulatory in the same cell lines. In C4-2B MDVR cells, degarelix alone or combined with abiraterone or enzalutamide reduced the AR-V7 protein expression compared with the control group. SCID mice bearing VCaP xenograft tumors were divided into 4 groups and treated with surgical castration, degarelix, leuprolide, or buffer alone for 4 weeks. Leuprolide slightly suppressed tumor growth compared with the vehicle control group (P > 0.05). Tumors in degarelix-treated mice were 67% of those in the leuprolide-treatment group but 170% larger than in surgically castrated ones. Measurements of intratumoral steroids in serum, tumor samples, or treated cell pellets by LC/MS confirmed that degarelix better decreased the levels of testosterone and steroidogenesis pathway intermediates, comparable to surgical castration, whereas leuprolide had no inhibitory effect. Collectively, our results suggested a selective mechanism of action of degarelix against androgen steroidogenesis and AR-variants. This study provides additional molecular insights regarding the mechanism of degarelix compared with GnRH agonist therapy, which may have clinical implications.

Abstract 1018: GnRH antagonists have direct inhibitory effects on castration-resistant prostate cancer via intracrine androgen and AR-V7 expression

Abstract Hormone therapy is currently the mainstay in the management of locally advanced and metastatic prostate cancer (PCa). Degarelix (Firmagon, Deg), a GnRH receptor antagonist demonstrated some advantages over the LHRH agonist Leuprolide (Leup) by avoiding “testosterone flare” and lower FSH levels. We compared the effect of Deg and Leup on prostate cancer (PC) cell progression in vitro and in vivo. GnRHR2 was readily detectable in PC cells. AR transcriptional activity reported by PSA-Luc assay was modulated by both Leup and Deg. In LNCaP and C4-2B MDVR cells, 20µM Deg significantly reduced the cell viability (p&amp;lt;0.01). GnRH-antagonist (alone or in combination with AA or Enza) counteracted the transactivation activity of AR by reducing AR-FL and AR variants at the protein level. In C4-2BMDVR cells, Deg reduced AR-V7 protein expression by 26 to 40% alone or combined with AA and Enza compared to control. Leup, however, enhanced variant expression. Deg reduced AR-variant levels from 17 to 41% in monotherapy or combinations compared to control. In mice, Leup slightly suppressed tumor growth compared to controls (p&amp;gt;0.05). However, tumors in Deg-treated group were 1.5-fold smaller than Leup-treated group but 1.7-fold bigger than surgical castration-group. Ki67 IHC staining confirmed the difference in tumor proliferation among groups. Measurements of intratumoral steroids by LC-MS from tumors, serum samples or cell pellets confirmed that Deg decreased the levels of testosterone and steroidogenesis pathway intermediates, comparable to surgical castration; while Leup had no inhibitory effect. Our results suggest a selective mechanism of action of Deg against AR-variants. The present study provides additional molecular insights regarding the mechanism of Deg compared to GnRH agonist therapy which may have clinical implications. Citation Format: Vito Cucchiara, Joy C. Yang, Chengfei Liu, Hans Adomat, Emma Guns, Martin E. Gleave, Allen C. Gao, Christopher P. Evans. GnRH antagonists have direct inhibitory effects on castration-resistant prostate cancer via intracrine androgen and AR-V7 expression [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 1018.

Abstract 3030: HSD3B1 mediated anti-androgen resistance in prostate cancer requires specific androgen precursors

Abstract Resistance to anti-androgens, such as enzalutamide (Enza) or abiraterone (Abi), develops within 24 months of initial exposure in most prostate cancer (PCa) patients. Commonly, dysregulated androgen signaling is a key feature of resistant disease. Previous studies demonstrate that androgen synthesis is upregulated in Enza and Abi resistance. HSD3B1 is steroidogenic enzyme which contributes to androgen synthesis and is associated with PCa progression. This study aims to determine the role of HSD3B1 in promoting Enza and Abi resistance in PCa. Enza resistant (C4-2B MDVR) and Abi resistant (C4-2B AbiR) C4-2B PCa cells were generated by chronically exposing parental C4-2B cells to increasing Enza or Abi concentrations for &amp;gt;12 months. Cells were maintained in 20 µM Enza or 10 µM Abi thereafter. Microarray, RNA-seq, and rtPCR were used to determine differences in gene expression between parental and anti-androgen resistant cells and confirmed by Western blot. HSD3B1 expression was knocked down in C4-2B MDVR and C4-2B AbiR cells using shRNA and cell number was determined in media containing FBS, charcoal dextran stripped FBS (CDFBS), or CDFBS supplemented with 100 nM pregnenolone (P5), 100 nM DHEA, or 10 nM DHT in the presence and absence of 20 µM Enza or 10 µM Abi. PSA secretion was determined by ELISA and PSA-luciferase activity was measured by reporter assay. C4-2B MDVR and C4-2B AbiR cells have increased HSD3B1 expression compared to parental C4-2B cells. This correlates to an increase in intracrine androgens in C4-2B MDVR cells as determined by LC-MS. Knockdown of HSD3B1 in C4-2B MDVR resensitized cells to Enza in FBS, CDFBS+DHT and CDFBS+P5 conditions as determined by a reduction in cell number and PSA secretion and/or PSA-luciferase activity in response to Enza. In the C4-2B AbiR cells, inhibition of HSD3B1 re-sensitized cells to treatment with Abi in FBS, CDFBS, and CDFBS+P5 conditions with the greatest effects seen in the FBS and CDFBS+P5 conditions. Supplementation with P5, but not DHEA, was able to induce PSA-luciferase activity and cell growth in C4-2B MDVR and C4-2B AbiR cells and this could be blocked by knockdown of HSD3B1. HSD3B1 overexpression in C4-2B MDVR and C4-2B AbiR cells contributes to Enza and Abi resistance and targeting this enzyme could be a viable strategy to improve anti-androgen response in PCa cells. HSD3B1 activity is reliant on select androgen precursors, such as pregnenolone, indicating preference towards a specific androgen synthesis pathway by HSD3B1 in mediating anti-androgen resistance. Citation Format: Cameron M. Armstrong, Chengfei Liu, Wei Lou, Alan P. Lombard, Christopher Evans, Allen C. Gao. HSD3B1 mediated anti-androgen resistance in prostate cancer requires specific androgen precursors [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 3030.

The value of AKR1C3 in predicting the therapeutic efficacy of abiraterone for patients with metastatic castration-resistant prostate cancer.

313 Background: AKR1C3 is a multifunctional enzyme playing a significant role in androgen synthesis and metabolism. Previous studies have proved that the activation of AKR1C3 was associated with resistance to abiraterone through increasing the intracrine androgen synthesis. However, clinical validation is still lacking as to the prognostic value of AKR1C3 in metastatic castration-resistant prostate cancer (mCRPC) patients treated with abiraterone. Methods: Data of 117 patients with mCRPC between 2016-2018 in our center were retrospectively analyzed. AKR1C3 was detected by the immunohistochemical staining from the 12-core prostate biopsy. Kaplan-Meier curves and COX regression were used to analyze the association between AKR1C3 and the treatment outcomes of abiraterone. The endpoints of this study were PSA progression-free survival (PSA-PFS) and radiograph progression-free survival (rPFS). Results: In total, AKR1V3 was detected in 40/117 (34.2%) cases. The positive AKR1C3 was significantly associated with shorter PSA-PFS for mCRPC patients treated with abiraterone (median PSA-PFS: 6.2 Mo vs. 11.1 Mo, p &lt; 0.001). Those with positive AKR1C3 were also accompanied with obviously poorer rPFS compared to those with negative AKR1C3 staining, despite that the difference was not statistically significant (median rPFS: 11.1 Mo vs. 21.8 Mo, p = 0.250). Multivariate COX regression indicated that, AKR1C3 was an independent prognosticator of rapid PSA progression for the abiraterone treatment (HR,95%CI: 2.612, 1.54-4.44, p &lt; 0.001). Conclusions: This is the first study verifying the adverse prognostic significance of AKR1C3 for mCRPC patients receiving abiraterone treatment. Our results suggested that, AKR1C3 was closely related to early treatment failure of abiraterone, and thus, was worthwhile to be routinely described in pathological report.

Mathematical modeling of intracrine androgen metabolism in prostate cancer: Methodological aspects.

Progression of castration-recurrent/resistant prostate cancer (CRPC) relies in part on dihydrotestosterone derived from intratumoral androgen metabolism. Mathematical modeling provides a valuable tool for studies of androgen metabolism in CRPC. This modeling approach integrates existing knowledge about complex biologic systems and provides a means of interrogating the effects of various interventions. We sought to model a single reaction in the androgen biosynthesis network, namely the oxidation of androsterone (AND) to androstanedione (5α-dione) by four 3α-oxidoreductase enzymes, as an initial effort to establish the feasibility of our modeling approach. Models were constructed for two cell culture systems, a non-prostate cancer cell line (CV-1) and a prostate cancer cell line (LAPC-4), using the SimBiology app (version 5.3) in MATLAB (version 8.6). The models included components for substrate (AND), product (5α-dione), each of the four enzymes, and each of the four enzyme-substrate complexes. Each enzymatic reaction consisted of a reversible enzyme-substrate binding step and an irreversible catalysis step. Rates of change for each component were described using ordinary differential equations. Mathematical models were developed with model parameter values derived from literature sources or from existing experimental data, which included gene expression measurements and substrate and product concentrations determined using liquid chromatography-tandem mass spectrometry. The models for both cell lines adequately described substrate and product concentrations observed after 12 h treatment with AND. This modeling approach represents an adaptable, extensible and mechanistic framework that reflects androgen metabolism. The models can be expanded systematically to describe the complex androgen metabolic pathways important for study of novel therapies for CRPC.

The Promise of Circulating Tumor Cells in Metastatic CRPC

The Promise of Circulating Tumor Cells in Metastatic CRPC

MP29-08 HSD3B1 PREFERENTIALLY UTILIZES PREGNENOLONE TO PROMOTE RESISTANCE TO ANTI-ANDROGENS IN PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I1 Apr 2018MP29-08 HSD3B1 PREFERENTIALLY UTILIZES PREGNENOLONE TO PROMOTE RESISTANCE TO ANTI-ANDROGENS IN PROSTATE CANCER Cameron Armstrong, Chengfei Liu, Wei Lou, Christopher Evans, and Allen Gao Cameron ArmstrongCameron Armstrong More articles by this author , Chengfei LiuChengfei Liu More articles by this author , Wei LouWei Lou More articles by this author , Christopher EvansChristopher Evans More articles by this author , and Allen GaoAllen Gao More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.928AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Most prostate cancer (PCa) patients receiving enzalutamide (Enza) or abiraterone (Abi) develop drug resistance within 24 months of exposure. This creates a need to better understand the underlying causes of anti-androgen resistance so that improved treatment methods can be developed. Previous studies demonstrate that uncontrolled intraprostatic androgen synthesis promotes Enza and Abi resistance. HSD3B1 is a key enzyme contributing to androgen synthesis and its expression is associated with PCa progression. The aim of this study is to determine the contribution of HSD3B1 to Enza and Abi resistance in PCa. METHODS Enza resistant (C4-2B MDVR) and Abi resistant (C4-2B AbiR) C4-2B PCa cells were generated by chronically exposing parental C4-2B cells to increasing Enza or Abi concentrations for >12 months and maintained in 20 μM Enza or 10 μM Abi. Differences in gene expression between parental and anti-androgen resistant cells was determined by microarray, RNA-seq, and rtPCR. HSD3B1 expression was knocked down in C4-2B MDVR and C4-2B AbiR cells using shRNA and cell number was determined in media containing FBS, charcoal dextran stripped FBS (CD-FBS), or CD-FBS supplemented with 100 nM pregnenolone (P5), 100 nM DHEA, or 10 nM DHT in the presence and absence of 20 μM Enza. PSA secretion was determined by ELISA and PSA-luciferase activity was measured by reporter assay. RESULTS HSD3B1 expression is higher in C4-2B MDVR and C4-2B AbiR cells compared to parental C4-2B cells. This correlates to increased intracrine androgens in C4-2B MDVR cells. Knockdown of HSD3B1 in C4-2B MDVR resensitized cells to Enza in FBS, CD-FBS+DHT and CD-FBS+P5 conditions as determined by a reduction in cell number and PSA secretion and/or PSA-luciferase activity in response to Enza. In the C4-2B AbiR cells, inhibition of HSD3B1 re-sensitized cells to treatment with Abi in FBS, CD-FBS, and CD-FBS + P5 conditions with the greatest effects seen in the FBS and CD-FBS + P5 containing media. Supplementation with P5, but not DHEA, was able to induce PSA-luciferase activity and cell growth in C4-2B AbiR cells and this could be blocked by knockdown of HSD3B1. CONCLUSIONS HSD3B1 overexpression in C4-2B MDVR and C4-2B AbiR cells contributes to Enza and Abi resistance and modulation of this enzyme could be a viable strategy to improve anti-androgen response in PCa cells. HSD3B1 activity appears to be reliant on select androgen precursors, such as pregnenolone, indicating preference towards a specific androgen synthesis pathway by HSD3B1 in mediating anti-androgen resistance. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e368-e369 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Cameron Armstrong More articles by this author Chengfei Liu More articles by this author Wei Lou More articles by this author Christopher Evans More articles by this author Allen Gao More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Modulation of AKR1C2 by curcumin decreases testosterone production in prostate cancer.

Intratumoral androgen biosynthesis has been recognized as an essential factor of castration‐resistant prostate cancer. The present study investigated the effects of curcumin on the inhibition of intracrine androgen synthesis in prostate cancer. Human prostate cancer cell lines, LNCaP and 22Rv1 cells were incubated with or without curcumin after which cell proliferation was measured at 0, 24, 48 and 72 hours, respectively. Prostate tissues from the transgenic adenocarcinoma of the mouse prostate (TRAMP) model were obtained after 1‐month oral administration of 200 mg/kg/d curcumin. Testosterone and dihydrotestosterone concentrations in LNCaP prostate cancer cells were determined through LC‐MS/MS assay. Curcumin inhibited cell proliferation and induced apoptosis of prostate cancer cells in a dose‐dependent manner. Curcumin decreased the expression of steroidogenic acute regulatory proteins, CYP11A1 and HSD3B2 in prostate cancer cell lines, supporting the decrease of testosterone production. After 1‐month oral administration of curcumin, Aldo‐Keto reductase 1C2 (AKR1C2) expression was elevated. Simultaneously, decreased testosterone levels in the prostate tissues were observed in the TRAMP mice. Meanwhile, curcumin treatments considerably increased the expression of AKR1C2 in prostate cancer cell lines, supporting the decrease of dihydrotestosterone. Taken together, these results suggest that curcumin's natural bioactive compounds could have potent anticancer properties due to suppression of androgen production, and this could have therapeutic effects on prostate cancer.

MP57-19 TARGETING AKR1C3 ACTIVATION BY INDOMETHACIN OVERCOMES RESISTANCE TO ENZALUTAMIDE AND ABIRATERONE

You have accessJournal of UrologyProstate Cancer: Advanced (including Drug Therapy) IV1 Apr 2017MP57-19 TARGETING AKR1C3 ACTIVATION BY INDOMETHACIN OVERCOMES RESISTANCE TO ENZALUTAMIDE AND ABIRATERONE Chengfei Liu, Wei Lou, Joy Yang, Chong-Xian Pan, Marc Dall'Era, Christopher Evans, and Allen Gao Chengfei LiuChengfei Liu More articles by this author , Wei LouWei Lou More articles by this author , Joy YangJoy Yang More articles by this author , Chong-Xian PanChong-Xian Pan More articles by this author , Marc Dall'EraMarc Dall'Era More articles by this author , Christopher EvansChristopher Evans More articles by this author , and Allen GaoAllen Gao More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1793AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Resistance of prostate cancer (CaP) cells to enzalutamide and abiraterone, may be mediated by a multitude of survival signaling pathways. In this study we tested whether AKR1C3 activation and intracrine androgens induce CaP cell resistance to enzalutamide and abiraterone and whether targeting these resistance mechanisms overcomes resistance to enzalutamide and abiraterone and thus improve therapy. METHODS Global gene expression analysis was analyzed by microarray and transcriptome was analyzed by RNA-seq. Steroid profile including androgens was analyzed by Liquid Chromatography-Mass Spectrometry (LC-MS). The effects of AKR1C3 expression and activation were examined by knock down of AKR1C3 expression by shRNA or inhibition of AKR1C3 enzymatic activity by indomethacin. The effects of AKR1C3 activation on enzalutamide and abiraterone sensitivity were examined in vitro and in vivo. A Phase I/II clinical trial with enzalutamide plus indomethacin has been designed. RESULTS Global gene expression analysis showed that steroid biosynthesis pathway is activated in enzalutamide and abiraterone resistant prostate cancer cells. One of the crucial steroidogenic enzymes, AKR1C3, was significantly elevated in enzalutamide/abiraterone resistant cells. In addition, AKR1C3 is highly expressed in metastatic and recurrent prostate cancer and in enzalutamide resistant prostate xenograft tumors. Liquid Chromatography-Mass Spectrometry (LC-MS) analysis of the steroid metabolites revealed that androgen precursors such as cholesterol, DHEA and progesterone, as well as androgens are highly up regulated in enzalutamide/abiraterone resistant prostate cancer cells compared to the parental cells. Knock down of AKR1C3 expression by shRNA or inhibition of AKR1C3 enzymatic activity by indomethacin resensitized enzalutamide/abiraterone resistant prostate cancer cells to enzalutamide/abiraterone treatment both in vitro and in vivo. In contrast, overexpression of AKR1C3 confers resistance to enzalutamide and abiraterone. Furthermore, the combination of indomethacin and enzalutamide/abiraterone resulted in significant inhibition of enzalutamide and abiraterone-resistant tumor growth. These results suggest that AKR1C3 activation is a critical resistance mechanism associated with enzalutamide/abiraterone resistance. In a Phase I/II combination of indomethacin with enzalutamide trial funded by Department of Defense, patients with mCRPC, good performance status (ECOG 0-2) and vital organ function, and no history of enzalutamide or indomethacin treatment will be recruited. The co-primary objective is to assess the toxicity and prostate-specific antigen (PSA) response (=50% reduction). We will also assess the overall response as determined by the Prostate Cancer Working Group 2 criteria, progression-free survival and molecular correlative studies. CONCLUSIONS AKR1C3 activation and resulting in an increase in intracrine androgens are critical resistance mechanisms confer resistance to enzalutamide and abiraterone. Targeting AKR1C3 activation may provide a potential treatment strategy for metastatic prostate cancer patients who develop resistance to enzalutamide and abiraterone. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e771 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Chengfei Liu More articles by this author Wei Lou More articles by this author Joy Yang More articles by this author Chong-Xian Pan More articles by this author Marc Dall'Era More articles by this author Christopher Evans More articles by this author Allen Gao More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP87-12 ADRENAL ANDROGENS FACILITATE PROSTATE CANCER CELL RESISTANCE TO ANDROGEN DEPRIVATION THERAPY

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III1 Apr 2017MP87-12 ADRENAL ANDROGENS FACILITATE PROSTATE CANCER CELL RESISTANCE TO ANDROGEN DEPRIVATION THERAPY Michael Fiandalo, John Stocking, Elena Pop, Krystin Mantione, John Wilton, and James Mohler Michael FiandaloMichael Fiandalo More articles by this author , John StockingJohn Stocking More articles by this author , Elena PopElena Pop More articles by this author , Krystin MantioneKrystin Mantione More articles by this author , John WiltonJohn Wilton More articles by this author , and James MohlerJames Mohler More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2716AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Prostate Cancer (CaP) growth and survival is dependent on the interaction of androgen receptor (AR) and testicular androgens, testosterone and dihydrotestosterone (DHT). Men that fail potential curative treatment are treated with androgen deprivation therapy (ADT). ADT is palliative and CaP recurs as the lethal phenotype. One mechanism that contributes to CaP recurrence is intratumoral intracrine androgen metabolism, which is the conversion of weak adrenal androgens (androstenedione [ASD] or dehydroepiandrosterone [DHEA]) to T or DHT. Mice lack adrenal androgens and therefore adrenal androgen contribute to CaP xenograft recurrence is overlooked. The hypothesis for these studies is adrenal androgen supplementation will facilitate androgen dependent human CaP CWR22 xenograft resistance to ADT. METHODS Mice were castrated, implanted with CWR22 and silastic tubes that contained T and empty silastic tubes that contained ASD or DHEA. T silastic tubes were removed after CWR22 tumor volumes reached 0.5 cc to simulate ADT. Digital calipers were used to measure changes in CWR22 tumor volume. Mouse serum, prostate and CWR22 were harvested at designated time points and liquid chromatography tandem-mass spectrometry (LC-MS/MS) was performed to measure androgen levels. RESULTS CWR22 xenografts treated with no adrenal androgen regressed and began to grow 120 days after T silastic tube removal. ASD treated CWR22 tumors did not regress; CWR22 tumor growth increased and mice were euthanized because tumors grew beyond veterinary limits. DHEA treated CWR22 did not regress and did not grow like ASD treated CWR22. However, CWR22 xenografts recurred faster than control CWR22 xenografts. Serum PSA levels correlated with tumor volume. CWR22 androgen levels decreased in all treatment groups, but T levels remained sufficient to activate AR in ASD treated CWR22, but not control or DHEA treated CWR22 xenografts. CONCLUSIONS DHEA and ASD supplementation facilitated CWR22 resistance to ADT. However, CWR22 xenografts responded differently to DHEA and ASD, which suggests that adrenal androgens may play different roles when CaP cells undergo ADT. These studies show that adrenal androgen contributes should be taken into account when in vivo studies are performed to test CaP responses to ADT or therapeutic agents. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1170 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Michael Fiandalo More articles by this author John Stocking More articles by this author Elena Pop More articles by this author Krystin Mantione More articles by this author John Wilton More articles by this author James Mohler More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Intracrine androgen biosynthesis in renal cell carcinoma

Background:Renal cell carcinoma (RCC) is one of the most lethal genitourinary cancers. The presence of androgen receptor (AR) in RCC has recently been shown to be associated with higher tumour stage irrespective of gender. Because the clinical context of androgens in female RCC patients is similar to that of prostate cancer patients undergoing androgen-deprivation therapy, mechanisms underlying the emergence of castration-resistant prostate cancer (CRPC) may be at play in AR-positive RCC cells. Therefore, we hypothesized that AR-positive RCC has intratumoral steroidogenesis and that anti-androgen therapy may result in tumour suppression.Methods:Mice were injected with an AR-positive RCC cell line. When tumours became palpable, surgical castration was performed and tumour volume was measured. Using ELISA, the levels of intracellular testosterone and dihydrotesterone were measured in AR-positive human RCC cell lines. Lastly, male mice containing xenografts were treated with enzalutamide or abiraterone acetate (AA) for 3 weeks to measure tumour volume.Results:We first observed in vivo that castration retards the growth of AR-positive RCC tumour xenograft in mice. Next, AR-positive human RCC cell lines and tissues were found to have elevated levels of testosterone and dihydrotestosterone and express key enzymes required for intracellular androgen biosynthesis. A mouse xenograft study with AR-positive RCC cell line using the commonly used anti-androgen therapies showed significant tumour suppression (P<0.01).Conclusions:Intracrine androgen biosynthesis is a potential source of androgen in AR-positive RCC and that the androgen signaling axis is a potential target of intervention in RCC.