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Purpose: Abiraterone is a potent inhibitor of the steroidogenic enzyme CYP17A1 and suppresses tumor growth in patients with castration-resistant prostate cancer (CRPC). The effectiveness of abiraterone in reducing tumor androgens is not known, nor have mechanisms contributing to abiraterone resistance been established.Experimental Design: We treated human CRPC xenografts with abiraterone and measured tumor growth, tissue androgens, androgen receptor (AR) levels, and steroidogenic gene expression versus controls.Results: Abiraterone suppressed serum PSA levels and improved survival in two distinct CRPC xenografts: median survival of LuCaP35CR improved from 17 to 39 days (HR = 3.6, P = 0.0014) and LuCaP23CR from 14 to 24 days (HR = 2.5, P = 0.0048). Abiraterone strongly suppressed tumor androgens, with testosterone (T) decreasing from 0.49 ± 0.22 to 0.03 ± 0.01 pg/mg (P < 0.0001), and from 0.69 ± 0.36 to 0.03 ± 0.01 pg/mg (P = 0.002) in abiraterone-treated 23CR and 35CR, respectively, with comparable decreases in tissue DHT. Treatment was associated with increased expression of full-length AR (ARFL) and truncated AR variants (ARFL 2.3-fold, P = 0.008 and ARdel567es 2.7-fold, P = 0.036 in 23 CR; ARFL 3.4-fold, P = 0.001 and ARV7 3.1-fold, P = 0.0003 in 35CR), and increased expression of the abiraterone target CYP17A1 (∼2.1-fold, P = 0.0001 and P = 0.028 in 23CR and 35CR, respectively) and transcript changes in other enzymes modulating steroid metabolism.Conclusions: These studies indicate that abiraterone reduces CRPC growth via suppression of intratumoral androgens and that resistance to abiraterone may occur through mechanisms that include upregulation of CYP17A1, and/or induction of AR and AR splice variants that confer ligand-independent AR transactivation. Clin Cancer Res; 17(18); 5913–25. ©2011 AACR.

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Purpose: Prostate cancer is dependent on androgen receptor (AR) activation. Optimal AR antagonism may effectively cytoreduce local disease and suppress or eliminate micrometastases. We evaluated neoadjuvant therapy prior to prostatectomy with the potent AR antagonist enzalutamide (enza) either alone or in combination with dutasteride (dut) and leuprolide (enza/dut/luteinizing hormone-releasing hormone analogues [LHRHa]).Experimental Design: Forty-eight of 52 men with intermediate or high-risk localized prostate cancer proceeded to prostatectomy after neoadjuvant enzalutamide or enza/dut/LHRHa for 6 months. We assessed pathologic complete response (pCR), minimal residual disease (MRD; ≤3 mm maximum diameter of residual disease), residual cancer burden (RCB), and expression of PSA and serum and tissue androgen concentrations. We compared the proportion of patients with pCR in each treatment arm with a historical control rate of 5%, based on previous reports of flutamide with LHRHa.Results: In the enzalutamide arm, none of the 25 patients achieved pCR or MRD. In the enza/dut/LHRHa arm, one of 23 patients (4.3%) achieved pCR and 3 of 23 (13.0%) achieved MRD. Median RCB was higher in the enzalutamide arm than in the enza/dut/LHRHa arm (0.41 cm3 vs. 0.06 cm3, respectively). Tissue testosterone and dihydrotestosterone levels correlated with RCB. No adverse events leading to study drug discontinuation were reported.Conclusions: Combination therapy with enza/dut/LHRHa resulted in pCR and MRD rates comparable with historical controls. Evidence of continued AR activity in residual tumor suggests that AR signaling may contribute to survival. Strategies to more effectively ablate AR activity are warranted to determine whether more substantial antitumor effects are observed. Clin Cancer Res; 23(9); 2169–76. ©2016 AACR.

Purpose: Despite the efficacy of abiraterone, a CYP17A1 inhibitor, in metastatic castration-resistant prostate cancer (CRPC), nearly all patients develop resistance. The purpose of this phase II study was to evaluate mechanisms of resistance to more complete androgen synthesis inhibition with abiraterone and dutasteride.Experimental Design: Eligible patients with metastatic CRPC underwent a baseline metastasis biopsy. Patients received abiraterone and prednisone for two 4-week cycles. After this time, high-dose dutasteride (3.5 mg daily) was added. Patients continued therapy until study withdrawal or radiographic progression. Repeat metastasis biopsy was obtained at progression. The primary endpoint was to assess mechanisms of resistance. Serum hormone and abiraterone levels were assessed. Tissue was assessed for androgen receptor (AR) and AR splice variant-7 (ARV7) expression.Results: Forty patients were enrolled. Sixty percent (n = 24) achieved a ≥50% reduction in prostate-specific antigen (PSA). The median time to radiographic progression was 11 months. Nearly all baseline (n = 29 of 31) and posttreatment (n = 16 of 16) tumors tested for AR nuclear expression were positive. Of those tested, ARV7 expression was present in 48% (n = 10 of 21) of baseline and 42% (n = 5 of 12) of treatment discontinuation specimens. Compared with patients with higher serum abiraterone levels at treatment discontinuation, patients with lower levels had higher circulating androgens.Conclusions: Despite increased androgen synthesis inhibition, we demonstrate that tumor AR axis remains important in disease progression. We highlight that abiraterone metabolism and pharmacokinetics may play a role in resistance. The noncomparative design limits conclusions on the efficacy of dual therapy with abiraterone and dutasteride, but the results support development of further multifaceted approaches toward AR inhibition. Clin Cancer Res; 23(4); 935–45. ©2016 AACR.

Purpose: Germline variation in solute carrier organic anion (SLCO) genes influences cellular steroid uptake and is associated with prostate cancer outcomes. We hypothesized that, due to its steroidal structure, the CYP17A inhibitor abiraterone may undergo transport by SLCO-encoded transporters and that SLCO gene variation may influence intracellular abiraterone levels and outcomes.Experimental Design: Steroid and abiraterone levels were measured in serum and tissue from 58 men with localized prostate cancer in a clinical trial of LHRH agonist plus abiraterone acetate plus prednisone for 24 weeks prior to prostatectomy. Germline DNA was genotyped for 13 SNPs in six SLCO genes.Results: Abiraterone levels spanned a broad range (serum median 28 ng/mL, 108 nmol/L; tissue median 77 ng/mL, 271 nmol/L) and were correlated (r = 0.355, P = 0.001). Levels correlated positively with steroids upstream of CYP17A (pregnenolone, progesterone), and inversely with steroids downstream of CYP17A (DHEA, AED, testosterone). Serum PSA and tumor volumes were higher in men with undetectable versus detectable tissue abiraterone at prostatectomy (median 0.10 vs. 0.03 ng/dL, P = 0.02; 1.28 vs. 0.44 cc, P = 0.09, respectively). SNPs in SLCO2B1 associated with significant differences in tissue abiraterone (rs1789693, P = 0.0008; rs12422149, P = 0.03) and higher rates of minimal residual disease (tumor volume < 0.5 cc; rs1789693, 67% vs. 27%, P = 0.009; rs1077858, 46% vs. 0%, P = 0.03). LNCaP cells expressing SLCO2B1 showed two- to fourfold higher abiraterone levels compared with vector controls (P < 0.05).Conclusions: Intraprostatic abiraterone levels and genetic variation in SLCO genes are associated with pathologic responses in high-risk localized prostate cancer. Variation in SLCO genes may serve as predictors of response to abiraterone treatment. Clin Cancer Res; 23(16); 4592–601. ©2017 AACR.

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Purpose:Tumor androgens in castration-resistant prostate cancer (CRPC) reflect de novo intratumoral synthesis or adrenal androgens. We used C.B.-17 SCID mice in which we observed adrenal CYP17A activity to isolate the impact of adrenal steroids on CRPC tumors in vivo.Experimental Design:We evaluated tumor growth and androgens in LuCaP35CR and LuCaP96CR xenografts in response to adrenalectomy (ADX). We assessed protein expression of key steroidogenic enzymes in 185 CRPC metastases from 42 patients.Results:Adrenal glands of intact and castrated mice expressed CYP17A. Serum DHEA, androstenedione (AED), and testosterone (T) in castrated mice became undetectable after ADX (all P < 0.05). ADX prolonged median survival (days) in both CRPC models (33 vs. 179; 25 vs. 301) and suppressed tumor steroids versus castration alone (T 0.64 pg/mg vs. 0.03 pg/mg; DHT 2.3 pg/mg vs. 0.23 pg/mg; and T 0.81 pg/mg vs. 0.03 pg/mg, DHT 1.3 pg/mg vs. 0.04 pg/mg; all P ≤ 0.001). A subset of tumors recurred with increased steroid levels, and/or induction of androgen receptor (AR), truncated AR variants, and glucocorticoid receptor (GR). Metastases from 19 of 35 patients with AR positive tumors concurrently expressed enzymes for adrenal androgen utilization and nine expressed enzymes for de novo steroidogenesis (HSD3B1, CYP17A, AKR1C3, and HSD17B3).Conclusions:Mice are appropriate for evaluating adrenal impact of steroidogenesis inhibitors. A subset of ADX-resistant CRPC tumors demonstrate de novo androgen synthesis. Tumor growth and androgens were suppressed more strongly by surgical ADX than prior studies using abiraterone, suggesting reduction in adrenally-derived androgens beyond that achieved by abiraterone may have clinical benefit. Proof-of-concept studies with agents capable of achieving true “nonsurgical ADX” are warranted.

Supplementary Figs S1-S6

Prostate androgen levels after enza or enza/dut/LHRHa. Tissue androgens from prostatectomy specimens after 6 months of therapy were measured by mass spectroscopy to determine levels of (A) pregnenolone, (B) progesterone, (C) DHEA, (D) AED, (E) testosterone, and (F) DHT. P values were calculated using the nonparametric Mann-Whitney test. Limits of quantification were 0.015 pg/mg for AED, progesterone, and testosterone, 0.03 pg/mg for DHT and pregnenolone, and 0.49 pg/mg for DHEA.

Figure S3. Change in serum androgen levels after enza or enza/dut/LHRHa. Box plots of serum androgens for each cohort at initiation of therapy (day 0) and at prostatectomy (day 180) are depicted for each treatment arm. Levels of (A) pregnenolone, (B) progesterone, (C) DHEA, (D) AED, (E) testosterone, and (F) DHT were measured by mass spectroscopy. P values were calculated using the nonparametric Mann-Whitney test. Limits of quantification were 0.005 ng/mL for AED, progesterone and testosterone, 0.001 ng/mL for DHT and pregnenolone, and 0.15 ng/mL for DHEA.

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Figure S4. Correlation of immunohistochemical expression of the glucocorticoid receptor (GR) after enza or enza/dut/LHRHa with residual cancer burden (RCB). A tissue microarray comprising cores of cancer tissue from each patient was analyzed for RCB and nuclear expression of GR expressed as total proportion of GR-positive tumor nuclei in (A) enza-treated and (B) enza/dut/LHRHa-treated patients. P values were calculated using the nonparametric Mann-Whitney test.

Residual cancer burden (RCB) from each patient in the (A) enza and (B) enza/dut/LHRHa arms.

Supplementary Methods and Supplementary Tables 1-3

Supplementary Figures and Tables Supplementary Table S1. Baseline Patient Demographic and Clinical Characteristics Supplementary Table S2. Observed and Expected Hardy Weinberg (HW) Frequency of SLCO Genotypes Supplementary Table S3. Distribution of Men with Undetectable Tissue ABI Levels Supplementary Table S4. Characteristics of SLCO Genotypes and Potential Prostate Cancer Associations Supplementary Table S5. Association of SLCO Genotype with Tissue Abiraterone, Estimated Tumor Volume and Minimal Residual Disease (MRD) Supplementary Figure S1. Representative chromatograms showing retention time for abiraterone standard and tissue samples. Supplementary Figure S2. Serum steroid levels in samples with undetectable vs detectable prostate abiraterone levels. Supplementary Figure S3. Association of SLCO single nucleotide polymorphisms with serum or tissue steroid levels. Supplementary Figure S4. Absolute abiraterone levels measured in LNCaP cells overexpressing SLCO2B1 or SLCO1B3.

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Purpose: In metastatic castration-resistant prostate cancer (mCRPC) low serum androgens prior to starting abiraterone acetate (AA) is associated with more rapid progression. We evaluated the effect of AA on androgens in castration-resistant prostate cancer (CRPC) metastases and associations of intratumoral androgens with response. Experimental Design: We performed a phase II study of AA plus prednisone in mCRPC. The primary outcome was tissue testosterone at 4 weeks. Exploratory outcomes were association of steroid levels and genomic alterations with response, and escalating AA to 2,000 mg at progression. Results: Twenty-nine of 30 men were evaluable. Testosterone in metastatic biopsies became undetectable at 4 weeks (P < 0.001). Serum and tissue dehydroepiandrosterone sulfate (DHEAS) remained detectable in many patients and was not increased at progression. Serum and tissue DHEAS in the lowest quartile (pretreatment), serum DHEAS in the lowest quartile (4 weeks), and undetectable tissue DHEAS (on-therapy) associated with rapid progression (20 vs. 48 weeks, P = 0.0018; 20 vs. 52 weeks, P = 0.0003; 14 vs. 40 weeks, P = 0.0001; 20 vs. 56 weeks, P = 0.02, respectively). One of 16 men escalating to 2,000 mg had a 30% PSA decline; 13 developed radiographic progression by 12 weeks. Among patients with high serum DHEAS at baseline, wild-type (WT) PTEN status associated with longer response (61 vs. 33 weeks, P = 0.02). Conclusions: Low-circulating adrenal androgen levels are strongly associated with an androgen-poor tumor microenvironment and with poor response to AA. Patients with CRPC with higher serum DHEAS levels may benefit from dual androgen receptor (AR)-pathway inhibition, while those in the lowest quartile may require combinations with non–AR-directed therapy.

OBJECTIVE Ageing in male adults is typically accompanied by adiposity accumulation and changes in circulating sex hormone concentrations. We hypothesized that an ageing-associated increase in oestrogens and decrease in androgens would correlate with an increase in adiposity. DESIGN 10-year prospective, observational study. STUDY SUBJECTS A total of 190, community-dwelling men in the Japanese American Community Diabetes Study. MEASUREMENTS At 0 and 10 years, CT scanning quantified intra-abdominal fat (IAF) and subcutaneous fat (SCF) areas while plasma concentrations of oestradiol, oestrone, testosterone and dihydrotestosterone were measured by liquid chromatography-tandem-mass spectrometry at each time point. Multivariate linear regression analyses assessed correlations between 10-year changes in hormone concentrations and IAF or SCF, adjusting for age and baseline fat depot area. RESULTS Participants were middle-aged [median 54.8 years, interquartile range (IQR) 39.9-62.8] men and mostly overweight by Asian criterion (median BMI 24.9, IQR 23.3-27.1) and with few exceptions had normal sex-steroid concentrations. Median oestradiol and dihydrotestosterone did not change significantly between 0 and 10 years (P = .084 and P = .596, respectively) while median oestrone increased (P

Background Androgen deprivation therapy improves the survival of castration-resistant prostate cancer (CRPC) patients, yet ultimately fails with debilitating side effects. Supraphysiological testosterone (SPT)-based therapy produces clinical responses with improved quality of life in a subset of patients. Currently, no information defines a durable response to SPT. Objective To identify key molecular phenotypes underlying SPT response to improve patient selection and guide combination treatment to achieve a durable response. Design, setting, and participants A patient-derived xenograft (PDX) preclinical trial was performed with 13 CRPC PDXs to identify molecular features associated with SPT response. Comprehensive intratumoral androgen, tumor growth, and integrated transcriptomic and protein analyses were performed in three PDXs resistant to the newer androgen receptor (AR) pathway inhibitor enzalutamide (ENZ) to define SPT response and resistance. Intervention Testosterone cypionate. Outcome measurements and statistical analysis SPT efficacy was evaluated by PDX growth, prostate-specific antigen (PSA) change, and survival. Intratumoral androgens were analyzed using mass spectrometry. Global transcriptome analysis was performed using RNA sequencing, and confirmed by quantitative real-time polymerase chain reaction and immunohistochemistry. Log-rank and Mann-Whitney tests were used for survival and molecular analyses, respectively. Results and limitations A durable SPT responder was identified, presenting robust repressions of ARv7 and E2F transcriptional outputs, and a DNA damage response (DDR) transcriptomic program that were altogether restored upon SPT resistance in the transient responder. ENZ rechallenge of SPT-relapsed PDXs resulted in PSA decreases but tumor progression. Conclusions SPT produces a durable response in AR-pathway inhibitor ENZ CRPC that is associated with sustained suppression of ARv7 and E2F transcriptional outputs, and the DDR transcriptome, highlighting the potential of combination treatments that maintain suppression of these programs to drive a durable response to SPT. Patient summary Patients with ENZ-resistant prostate cancer have very limited treatment options. Supraphysiological testosterone presents a prominent option for improved quality of life and a potential durable response in patients with sustained suppression on ARv7/E2F transcriptional outputs and DNA repair program.

Background Localized prostate cancers (PCs) may resist neoadjuvant androgen receptor (AR)-targeted therapies as a result of persistent intraprostatic androgens arising through upregulation of steroidogenic enzymes. Therefore, we sought to evaluate clinical effects of neoadjuvant indomethacin (Indo), which inhibits the steroidogenic enzyme AKR1C3, in addition to combinatorial anti-androgen blockade, in men with high-risk PC undergoing radical prostatectomy (RP). Methods This was an open label, single-site, Phase II neoadjuvant trial in men with high to very-high-risk PC, as defined by NCCN criteria. Patients received 12 weeks of apalutamide (Apa), abiraterone acetate plus prednisone (AAP), degarelix, and Indo followed by RP. Primary objective was to determine the pathologic complete response (pCR) rate. Secondary objectives included minimal residual disease (MRD) rate, defined as residual cancer burden (RCB) ≤ 0.25cm3 (tumor volume multiplied by tumor cellularity) and elucidation of molecular features of resistance. Results Twenty patients were evaluable for the primary endpoint. Baseline median prostate-specific antigen (PSA) was 10.1 ng/ml, 4 (20%) patients had Gleason grade group (GG) 4 disease and 16 had GG 5 disease. At RP, 1 (5%) patient had pCR and 6 (30%) had MRD. Therapy was well tolerated. Over a median follow-up of 23.8 months, 1 of 7 (14%) men with pathologic response and 6 of 13 (46%) men without pathologic response had a PSA relapse. There was no association between prostate hormone levels or HSD3B1 genotype with pathologic response. Conclusions In men with high-risk PC, pCR rates remained low even with combinatorial AR-directed therapy, although rates of MRD were higher. Ongoing follow-up is needed to validate clinical outcomes of men who achieve MRD.

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)

Purpose: Tumor androgens in castration-resistant prostate cancer (CRPC) reflect de novo intratumoral synthesis or adrenal androgens. We used C.B.-17 SCID mice in which we observed adrenal CYP17A activity to isolate the impact of adrenal steroids on CRPC tumors in vivo. Experimental Design: We evaluated tumor growth and androgens in LuCaP35CR and LuCaP96CR xenografts in response to adrenalectomy (ADX). We assessed protein expression of key steroidogenic enzymes in 185 CRPC metastases from 42 patients. Results: Adrenal glands of intact and castrated mice expressed CYP17A. Serum DHEA, androstenedione (AED), and testosterone (T) in castrated mice became undetectable after ADX (all P < 0.05). ADX prolonged median survival (days) in both CRPC models (33 vs. 179; 25 vs. 301) and suppressed tumor steroids versus castration alone (T 0.64 pg/mg vs. 0.03 pg/mg; DHT 2.3 pg/mg vs. 0.23 pg/mg; and T 0.81 pg/mg vs. 0.03 pg/mg, DHT 1.3 pg/mg vs. 0.04 pg/mg; all P ≤ 0.001). A subset of tumors recurred with increased steroid levels, and/or induction of androgen receptor (AR), truncated AR variants, and glucocorticoid receptor (GR). Metastases from 19 of 35 patients with AR positive tumors concurrently expressed enzymes for adrenal androgen utilization and nine expressed enzymes for de novo steroidogenesis (HSD3B1, CYP17A, AKR1C3, and HSD17B3). Conclusions: Mice are appropriate for evaluating adrenal impact of steroidogenesis inhibitors. A subset of ADX-resistant CRPC tumors demonstrate de novo androgen synthesis. Tumor growth and androgens were suppressed more strongly by surgical ADX than prior studies using abiraterone, suggesting reduction in adrenally-derived androgens beyond that achieved by abiraterone may have clinical benefit. Proof-of-concept studies with agents capable of achieving true “nonsurgical ADX” are warranted.

OBJECTIVE This study aimed to determine whether a relationship was evident between gliosis in the mediobasal hypothalamus (MBH) and plasma testosterone concentrations in men. METHODS A total of 41 adult men (aged 18-50 years) from 23 twin pairs underwent fasting morning blood draw and brain magnetic resonance imaging. T2 relaxation time was used to quantify gliosis in the MBH and control areas in the putamen and amygdala. Plasma concentrations of testosterone and 17β-estradiol were measured by liquid chromatography-tandem mass spectrometry. Body composition including visceral adiposity was measured by dual x-ray absorptiometry. RESULTS A negative association was found between MBH T2 relaxation time and plasma concentrations of both free and total testosterone (r = -0.29, P

Background Abiraterone acetate suppresses adrenal androgens and glucocorticoids through the inhibition of CYP17; however, given the risk of mineralocorticoid excess, it is administered with glucocorticoids. Herein, the authors performed a phase 2, single-arm study that was designed to assess the safety of abiraterone acetate without steroids in patients with castration-resistant prostate cancer. Methods Eligible patients had castration-resistant prostate cancer with controlled blood pressure and normal potassium. Patients initially received abiraterone acetate at a dose of 1000 mg daily alone. Those with persistent or severe mineralocorticoid toxicity received treatment with prednisone initiated at a dose of 5 mg twice daily. Therapy was continued until radiographic progression, toxicity, or withdrawal. The primary objective of the current study was to determine the percentage of men requiring prednisone to manage mineralocorticoid toxicity. Toxicity was graded according to Common Terminology Criteria for Adverse Events, version 4.0. Results A total of 58 patients received at least 1 dose of abiraterone acetate; the majority had metastases (53 patients; 91.4%). Sixteen patients (27.6%) received prior chemotherapy, 6 patients (10.3%) received prior enzalutamide, and 4 patients (7%) received prior ketoconazole. Grade 3 to 4 adverse events of interest included hypertension (9 patients; 15.5%) and hypokalemia (4 patients; 7%). There was no grade ≥3 edema. Seven patients (12%) initiated prednisone therapy for mineralocorticoid toxicity, 3 patients for hypertension (5%), and 4 patients for hypokalemia (7%). Two patients initiated prednisone therapy for fatigue (3%). Forty patients (68%) experienced a decline in prostate-specific antigen of ≥50% with the use of abiraterone acetate alone. Patients with lower baseline levels of androstenedione (P = .04), androsterone (P = .01), dehydroepiandrosterone (P = .03), and 17-hydroxyprogesterone (P = .03) were found to be more likely to develop mineralocorticoid toxicity. Conclusions Treatment with abiraterone acetate without steroids is feasible, although clinically significant adverse events can occur in a minority of patients. The use of abiraterone acetate without prednisone should be balanced with the potential for toxicity and requires close monitoring.

SummaryObjective Serum sex steroid concentrations may alter body composition and glucose homoeostasis in men in a dose-response manner. We evaluated these end-points in healthy men rendered medically castrate through use of a gonadotrophin-releasing hormone antagonist (acyline) with incremental doses of exogenous testosterone (T) gel. Design Subjects (n=6-9 per group) were randomly assigned to injections of acyline every 2 weeks plus transdermal T gel (1.25 g, 2.5 g, 5.0 g, 10 g or 15 g) daily or double placebo (injections and gel) for 12 weeks. Patients Healthy men, ages 25-55 years, with normal serum total T concentrations. Measurements Serum T, dihydrotestosterone (DHT) and oestradiol (E2) were measured at baseline and every 2 weeks. Body composition was analysed by dual-energy X-ray absorptiometry at baseline and week 12. Fasting serum adiponectin, leptin, glucose and insulin concentrations were measured at baseline and week 10. Results Forty-eight men completed the study. A significant treatment effect was observed for change in lean mass (ANOVAP=.01) but not fat mass (P=.14). Lean mass increased in the 15 g T group relative to all lower dose groups, except the 10 g T group. When all subjects were analysed together, changes in lean mass correlated directly and changes in fat mass correlated inversely with serum T, E2 and DHT. No changes were noted in serum glucose, insulin or adipokine levels. Conclusions In healthy men, higher serum concentrations of T, DHT and E2 were associated with greater increases in lean mass and decreases in fat mass but not with changes in serum glucose, insulin or adipokines.

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.

BACKGROUND: Testosterone is a driver of prostate cancer (PC) growth via ligand-mediated activation of the androgen receptor (AR). Tumors that have escaped systemic androgen deprivation, castration resistant prostate cancers (CRPC), have measurable intratumoral levels of testosterone, suggesting that a resistance mechanism still depends on androgen-simulated growth. However, AR activation requires an optimal intracellular concentration of androgens, a situation challenged by low circulating testosterone concentrations. Notably, PC cells may optimize their androgen levels by regulating the expression of steroid metabolism enzymes that convert androgen precursors into androgens. Here we propose that testosterone entry into the cell could be another control point. METHODS: To determine whether testosterone enters cells via a transporter, we performed in vitro (3)H-testosterone uptake assays in androgen-dependent LNCaP and androgen and AR-independent PC3 cells. To determine if the uptake mechanism depended on a concentration gradient, we modified UGT2B17 levels in LNCaP cells and measured androgen levels by LLE-MS (liquid-liquid extraction mass spectrometry). We also analyzed CRPC metastases for expression of AKR1C3 to determine whether this enzyme that converts adrenal androgens to testosterone was present in the tumor stroma (microenvironment) in addition to its expression in the tumor epithelium. RESULTS: Testosterone uptake followed a concentration gradient but unlike in passive diffusion, was saturable and temperature-dependent, thus suggesting facilitated transport. Suppression of UGT2B17 to abrogate a testosterone gradient reduced testosterone transport while overexpression of the enzyme enhanced it. The facilitated transport suggests a paracrine route of testosterone uptake for maintaining optimal intracellular levels. We found that AKR1C3 was expressed in the tumor microenvironment of CRPC metastases in addition to epithelial cells and the pattern of relative abundance of the enzyme in epithelium versus stroma varied substantially between the metastatic sites. CONCLUSIONS: Our findings suggest that in addition to testosterone transport and metabolism by tumor epithelium, testosterone could also be produced by components of the tumor microenvironment. Facilitated testosterone uptake by tumor cells supports a cell non-autonomous mechanism for testosterone signaling in CRPC.

Concern exists that T replacement therapy (TRT) might increase the risk of prostate disease. There are limited data regarding the impact of TRT on prostate androgen concentrations.Determine the dose-dependent effects of exogenous T administration on intraprostatic androgen concentrations.Twelve-week, double-blinded, randomized, placebo-controlled trial.Academic medical center.Sixty-two healthy eugonadal men, aged 25-55 years.Subjects were randomly assigned to receive injections of acyline, a GnRH antagonist (used to achieve medical castration), every 2 weeks plus transdermal T gel (1.25 g, 2.5 g, 5.0 g, 10 g, or 15 g daily), or placebo injections and transdermal gel for 12 weeks.Serum T and dihydrotestosterone (DHT) were measured at baseline and every 2 weeks during treatment. Intraprostatic T and DHT concentrations were assessed from tissue obtained through ultrasound-guided prostate needle biopsies at week 12. Androgens were quantified by liquid chromatography-tandem mass spectrometry.51 men completed the study and were included in the analysis. There were no significant adverse events. Exogenous T resulted in a dose-dependent increase in serum T and DHT concentrations (190-770 and 60-180 ng/dL, respectively). Although intraprostatic T differed among dose groups (P = .01), intraprostatic DHT was comparable regardless of T dose (P = .11) and was 10- to 20-fold greater than intraprostatic T.In healthy, medically castrate men receiving exogenous T, the total intraprostatic androgen concentration (predominantly DHT) remained stable across serum T concentrations within the physiological range. These findings further our knowledge of the relationship between serum and intraprostatic androgens and suggest that physiological serum T achieved by TRT is unlikely to alter the prostate hormonal milieu.

Purpose Optimizing androgen suppression may provide better control of localized prostate cancer (PCa). Numerous trials have supported the benefit of combining androgen deprivation therapy with definitive radiation therapy in men with locally advanced or high-grade disease. Addition of abiraterone to luteinizing hormone-releasing hormone agonist (LHRHa) with radiation has not been reported. We examined the safety of this combination as well as its impact on androgen suppression. Methods and Materials A prospective, phase 2 study was conducted in men with localized PCa treated with 6 months of neoadjuvant and concurrent abiraterone with LHRHa and radiation. Duration of adjuvant LHRHa was at the discretion of the treating clinician. Prostate biopsy assays were obtained prior to the start of therapy and prior to radiation. Sera and tissue androgen levels were measured by liquid chromatography-tandem mass spectrometry. Results A total of 22 men with intermediate- (n=3) and high-risk PCa (n=19) received study therapy. Sixteen men completed the intended course of abiraterone, and 19 men completed planned radiation to 77.4 to 81 Gy. Radiation to pelvic nodes was administered in 20 men. The following grade 3 toxicities were reported: lymphopenia (14 patients), fatigue (1 patient), transaminitis (2 patients), hypertension (2 patients), and hypokalemia (1 patient). There were no grade 4 toxicities. All 21 men who complied with at least 3 months of abiraterone therapy had a preradiation prostate-specific antigen (PSA) concentration nadir of Conclusions Addition of abiraterone to LHRHa with radiation is safe and achieves effective prostatic androgen suppression. Preliminary analysis of the clinical data is also promising, with excellent PSA nadir and no relapse to date in this high-risk population.

Purpose: Germline variation in solute carrier organic anion (SLCO) genes influences cellular steroid uptake and is associated with prostate cancer outcomes. We hypothesized that, due to its steroidal structure, the CYP17A inhibitor abiraterone may undergo transport by SLCO-encoded transporters and that SLCO gene variation may influence intracellular abiraterone levels and outcomes. Experimental Design: Steroid and abiraterone levels were measured in serum and tissue from 58 men with localized prostate cancer in a clinical trial of LHRH agonist plus abiraterone acetate plus prednisone for 24 weeks prior to prostatectomy. Germline DNA was genotyped for 13 SNPs in six SLCO genes. Results: Abiraterone levels spanned a broad range (serum median 28 ng/mL, 108 nmol/L; tissue median 77 ng/mL, 271 nmol/L) and were correlated (r = 0.355, P = 0.001). Levels correlated positively with steroids upstream of CYP17A (pregnenolone, progesterone), and inversely with steroids downstream of CYP17A (DHEA, AED, testosterone). Serum PSA and tumor volumes were higher in men with undetectable versus detectable tissue abiraterone at prostatectomy (median 0.10 vs. 0.03 ng/dL, P = 0.02; 1.28 vs. 0.44 cc, P = 0.09, respectively). SNPs in SLCO2B1 associated with significant differences in tissue abiraterone (rs1789693, P = 0.0008; rs12422149, P = 0.03) and higher rates of minimal residual disease (tumor volume < 0.5 cc; rs1789693, 67% vs. 27%, P = 0.009; rs1077858, 46% vs. 0%, P = 0.03). LNCaP cells expressing SLCO2B1 showed two- to fourfold higher abiraterone levels compared with vector controls (P < 0.05). Conclusions: Intraprostatic abiraterone levels and genetic variation in SLCO genes are associated with pathologic responses in high-risk localized prostate cancer. Variation in SLCO genes may serve as predictors of response to abiraterone treatment. Clin Cancer Res; 23(16); 4592–601. ©2017 AACR.

Background: Anti-androgen therapies suppress castration-resistant prostate cancer (CRPC) but CRPC cells develop resistance. One of the mechanisms of resistance is through overexpression of androgen receptor (AR) and AR splice variants. In contrast to AR pathway inhibition therapies, recent clinical studies using bipolar androgen therapy demonstrated CRPC inhibition using supraphysiological levels of testosterone (SPT). The objective of this study was to investigate the mechanisms driving SPT-mediated tumor growth inhibition using CRPC patient-derived xenografts (PDX). Methods: PDXs were implanted in castrated SCID mice and randomized to control or SPT arms. For enzalutamide-resistant (ENZR) PDX studies, mice with established tumors were treated with enzalutamide and randomized to control or SPT upon development of resistance. Tumors were monitored for growth and collected for analyses. Results: In a SPT preclinical trial using thirteen LuCaP CRPC PDX models, four PDXs responded to SPT treatment while nine demonstrated de novo resistance. Our analysis revealed that responding PDXs had intrinsically higher AR and ARV7 expression compared to non-responding PDXs. Moreover, ARV7 expression was negatively correlated with E2F signaling and proliferation only in responding PDXs, suggesting that the ARV7 program functions differently in responder and non-responder phenotypes. Another PDX trial using ENZR PDXs determined that SPT inhibited the growth of LuCaP 35CR ENZR and LuCaP 96CR ENZR (responders), but not LuCaP 77CR ENZR (non-responder). Serum and intratumoral T were increased in both responders and the non-responder, suggesting that differential T delivery and tumoral retention were not the cause of differential tumor responses. Tumor analyses determined that SPT decreased AR transcript levels, however, nuclear AR protein levels and canonical AR signaling remained high in both responders and the non-responder. Conversely, ARV7 transcript was consistently decreased but the ARV7 program was downregulated only in responders. Additionally, an unbiased pathway analysis of RNASeq revealed that SPT drastically decreased genes associated with E2F-mediated cell cycle progression and proliferation and the DNA damage response (DDR) exclusively in responders. Further support for these pathways driving SPT-mediated tumor inhibition was demonstrated through the resolution of the suppressed ARV7/E2F1/DDR pathways in LuCaP 35CR ENZR upon acquiring SPT resistance, whereas the pathways remained suppressed in LuCaP 96CR ENZR, which exhibited a durable response to SPT. Conclusion: Our data indicates that SPT therapy inhibits progression of a unique subset of ENZR CRPC and highlights critical roles for ARV7 signaling, DDR and E2F1-mediated proliferation in tumor inhibition. Citation Format: Hung-Ming Lam, Mark P. Labrecque, Holly M. Nguyen, Lisha G. Brown, Ilsa M. Coleman, Roman Gulati, Bryce Lakely, Daniel Sondheim, Brett Marck, Alvin M. Matsumoto, Elahe A. Mostaghel, Michael T. Schweizer, Peter S. Nelson, Eva Corey. Supraphysiological testosterone inhibits tumor growth and is associated with inhibition of ARV7 signaling and DNA damage response in preclinical models of enzalutamide-resistant prostate cancer [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 379.

208 Background: The association of serum ABI and androgen levels with clinical efficacy in men with CRPC is not well understood. Methods: We measured androgens, ABI and its key metabolites (D4A and 5α) in serum at 4, 8, and 12 weeks (wks) in 29 men with CRPC treated with ABI (for associations with PSA response and time to radiographic progression (TTP)); in 58 men from a study of ABI without prednisone; and in 22 men with localized PCa treated with neoadjuvant ABI for 3 months prior to definitive therapy (including prostate levels). Results: Median (Med) TTP was 36 mo (4-104). 52% had a PSA response (decline >30% at wk12), and 34% early progression (TTP < 6 mo). There was no difference in ABI (at wk4 or average of wks 4/8/12) in men with v without PSA response, or in men with early v late TTP. D4A and 5α at wk4 were higher in early v late progressors (2.9 v 1.5 ng/ml, p=0.05; 10.35 v 6.7 ng/ml, p=0.08). TTP was longer in the lowest v highest quartile of drug levels (ABI: 4wk 48 v 30 wks; 8wk 60 v 16 wks; D4A: 4wk 40 v 16 wks; 8wk 60 v 28 wks; 5a: 8wk 41 v 16 wks, p Med had 2-5x higher levels of all steroids (DHEAS 110 v 22 ug/dl, DHEA 184 v 49 ng/dl, AED 46 v 26 ng/dl, and T 9 v 4.9 ng/dl, p v < Med, regardless of serum or tissue ABI levels on treatment. Conclusions: In men with pre-ABI serum DHEAS < Med, androgens were suppressed even at low serum ABI levels, whereas in men with pre-ABI DHEAS > Med, levels were not completely suppressed even at high ABI levels, explaining the minimal impact of dose escalated ABI and observed noninferiority of low dose ABI previously reported in men with CRPC. The shorter TTP in the highest quartiles of ABI, D4A and 5α may reflect increased conversion to the AR agonist metabolite 5α. Men with pre-ABI DHEAS > Med may warrant stratification to more potent/combination therapy. Clinical trial information: NCT01503229.

Author(s): Harrington, LB; Marck, BT; Wiggins, KL; McKnight, B; Heckbert, SR; Woods, NF; LaCroix, AZ; Blondon, M; Psaty, BM; Rosendaal, FR; Matsumoto, AM; Smith, NL | Abstract: Essentials Endogenous hormone levels' influence on hemostatic factor levels is not fully characterized. We tested for associations of endogenous hormone with hemostatic factor levels in postmenopause. Estrone levels were inversely associated with the natural anticoagulant, protein S antigen. Dehydroepiandrosterone sulfate levels were inversely associated with thrombin generation.SummaryBackground Oral use of exogenous estrogen/progestin alters hemostatic factor levels. The influence of endogenous hormones on these levels is incompletely characterized. Objectives Our study aimed to test whether, among postmenopausal women, high levels of estradiol (E2), estrone (E1), testosterone (T), dehydroepiandrosterone sulfate (DHEAS), dehydroepiandrosterone (DHEA), and androstenedione, and low levels of sex hormone-binding globulin (SHBG), are positively associated with measures of thrombin generation (TG), a normalized activated protein C sensitivity ratio (nAPCsr), and factor VII activity (FVIIc), and negatively associated with antithrombin activity (ATc) and total protein S antigen (PSAg). Methods This Heart and Vascular Health study cross-sectional analysis included 131 postmenopausal women without a prior venous thrombosis who were not currently using hormone therapy. Adjusted mean differences in TG, nAPCsr, FVIIc, ATc and PSAg levels associated with differences in hormone levels were estimated using multiple linear regression. We measured E2, E1, total T, DHEAS, DHEA and androstenedione levels by mass spectrometry, SHBG levels by immunoassay, and calculated the level of free T. Results One picogram per milliliter higher E1 levels were associated with 0.24% lower PSAg levels (95% Confidence Interval [CI]: -0.35, -0.12) and 1 μg mL-1 higher DHEAS levels were associated with 40.8 nm lower TG peak values (95% CI: -59.5, -22.2) and 140.7 nm×min lower TG endogenous thrombin potential (ETP) (95% CI: -212.1, -69.4). After multiple comparisons correction, there was no evidence for other associations. Conclusions As hypothesized, higher E1 levels were associated with lower levels of the natural anticoagulant PSAg. Contrary to hypotheses, higher DHEAS levels were associated with differences in TG peak and ETP that suggest less generation of thrombin.

Purpose: Prostate cancer is dependent on androgen receptor (AR) activation. Optimal AR antagonism may effectively cytoreduce local disease and suppress or eliminate micrometastases. We evaluated neoadjuvant therapy prior to prostatectomy with the potent AR antagonist enzalutamide (enza) either alone or in combination with dutasteride (dut) and leuprolide (enza/dut/luteinizing hormone-releasing hormone analogues [LHRHa]). Experimental Design: Forty-eight of 52 men with intermediate or high-risk localized prostate cancer proceeded to prostatectomy after neoadjuvant enzalutamide or enza/dut/LHRHa for 6 months. We assessed pathologic complete response (pCR), minimal residual disease (MRD; ≤3 mm maximum diameter of residual disease), residual cancer burden (RCB), and expression of PSA and serum and tissue androgen concentrations. We compared the proportion of patients with pCR in each treatment arm with a historical control rate of 5%, based on previous reports of flutamide with LHRHa. Results: In the enzalutamide arm, none of the 25 patients achieved pCR or MRD. In the enza/dut/LHRHa arm, one of 23 patients (4.3%) achieved pCR and 3 of 23 (13.0%) achieved MRD. Median RCB was higher in the enzalutamide arm than in the enza/dut/LHRHa arm (0.41 cm3 vs. 0.06 cm3, respectively). Tissue testosterone and dihydrotestosterone levels correlated with RCB. No adverse events leading to study drug discontinuation were reported. Conclusions: Combination therapy with enza/dut/LHRHa resulted in pCR and MRD rates comparable with historical controls. Evidence of continued AR activity in residual tumor suggests that AR signaling may contribute to survival. Strategies to more effectively ablate AR activity are warranted to determine whether more substantial antitumor effects are observed. Clin Cancer Res; 23(9); 2169–76. ©2016 AACR.

Purpose: Despite the efficacy of abiraterone, a CYP17A1 inhibitor, in metastatic castration-resistant prostate cancer (CRPC), nearly all patients develop resistance. The purpose of this phase II study was to evaluate mechanisms of resistance to more complete androgen synthesis inhibition with abiraterone and dutasteride. Experimental Design: Eligible patients with metastatic CRPC underwent a baseline metastasis biopsy. Patients received abiraterone and prednisone for two 4-week cycles. After this time, high-dose dutasteride (3.5 mg daily) was added. Patients continued therapy until study withdrawal or radiographic progression. Repeat metastasis biopsy was obtained at progression. The primary endpoint was to assess mechanisms of resistance. Serum hormone and abiraterone levels were assessed. Tissue was assessed for androgen receptor (AR) and AR splice variant-7 (ARV7) expression. Results: Forty patients were enrolled. Sixty percent (n = 24) achieved a ≥50% reduction in prostate-specific antigen (PSA). The median time to radiographic progression was 11 months. Nearly all baseline (n = 29 of 31) and posttreatment (n = 16 of 16) tumors tested for AR nuclear expression were positive. Of those tested, ARV7 expression was present in 48% (n = 10 of 21) of baseline and 42% (n = 5 of 12) of treatment discontinuation specimens. Compared with patients with higher serum abiraterone levels at treatment discontinuation, patients with lower levels had higher circulating androgens. Conclusions: Despite increased androgen synthesis inhibition, we demonstrate that tumor AR axis remains important in disease progression. We highlight that abiraterone metabolism and pharmacokinetics may play a role in resistance. The noncomparative design limits conclusions on the efficacy of dual therapy with abiraterone and dutasteride, but the results support development of further multifaceted approaches toward AR inhibition. Clin Cancer Res; 23(4); 935–45. ©2016 AACR.

5013 Background: Germline variation in solute carrier organic anion (SLCO) transport genes influences cellular uptake of steroids and has been associated with PCa outcomes. We hypothesized that due to its steroidal structure, ABI may undergo SLCO-mediated transport, and that germline variation in SLCO genes may influence intracellular ABI levels and pathologic outcomes in men with PCa. Methods: We measured ABI levels in prostatectomy tissue from 58 men with localized PCa treated with 6 months of castration plus ABI prior to radical prostatectomy (RP). Buffy coat DNA was genotyped for13 SNPs (minimum allele frequency > 0.07) in 6 SLCO genes using TaqMan SNP assays. Transcript expression of SLCO genes was evaluated by qRT-PCR in microdissected tumor samples from a subset of patients. Results: SNPs in SLCO1B3 (rs4149177, p = 0.07) and SLCO2B1 (rs12422149, p = 0.0004; rs949069, p = 0.008; rs1789693, p = 0.01) were associated with significant differences in mean tissue ABI levels at RP. Contingency analysis sh...