ONECUT2 acts as a lineage plasticity driver in adenocarcinoma as well as neuroendocrine variants of prostate cancer.

Androgen receptor- (AR-) indifference is a mechanism of resistance to hormonal therapy in prostate cancer (PC). Here we demonstrate that ONECUT2 (OC2) activates resistance through multiple drivers associated with adenocarcinoma, stem-like and neuroendocrine (NE) variants. Direct OC2 gene targets include the glucocorticoid receptor (GR; NR3C1) and the NE splicing factor SRRM4, which are key drivers of lineage plasticity. Thus, OC2, despite its previously described NEPC driver function, can indirectly activate a portion of the AR cistrome through epigenetic activation of GR. Mechanisms by which OC2 regulates gene expression include promoter binding, enhancement of genome-wide chromatin accessibility, and super-enhancer reprogramming. Pharmacologic inhibition of OC2 suppresses lineage plasticity reprogramming induced by the AR signaling inhibitor enzalutamide. These results demonstrate that OC2 activation promotes a range of drug resistance mechanisms associated with treatment-emergent lineage variation in PC and support enhanced efforts to therapeutically target OC2 as a means of suppressing treatment-resistant disease.

Reproducible preclinical models of androgen receptor driven human prostate cancer bone metastasis.

BACKGROUND: Preclinical models recapitulating the metastatic phenotypes are essential for developing the next-generation therapies for metastatic prostate cancer (mPC). We aimed to establish a cohort of clinically relevant mPC models, particularly androgen receptor positive (AR+) bone metastasis models, from LuCaP patient-derived xenografts (PDX) that reflect the heterogeneity and complexity of mPC. METHODS: PDX tumors were dissociated into single cells, modified to express luciferase, and were inoculated into NSG mice via intracardiac injection. The progression of metastases was monitored by bioluminescent imaging. Histological phenotypes of metastases were characterized by immunohistochemistry and immunofluorescence staining. Castration responses were further investigated in two AR-positive models. RESULTS: Our PDX-derived metastasis (PDM) model collection comprises three AR+ adenocarcinomas (ARPC) and one AR- neuroendocrine carcinoma (NEPC). All ARPC models developed bone metastases with either an osteoblastic, osteolytic, or mixed phenotype, while the NEPC model mainly developed brain metastasis. Different mechanisms of castration resistance were observed in two AR+ PDM models with distinct genotypes, such as combined loss of TP53 and RB1 in one model and expression of AR splice variant 7 (AR-V7) expression in another model. Intriguingly, the castration-resistant tumors displayed inter- and intra-tumor as well as organ-specific heterogeneity in lineage specification. CONCLUSION: Genetically diverse PDM models provide a clinically relevant system for biomarker identification and personalized medicine in metastatic castration-resistant prostate cancer.

Sigma1 Regulates Lipid Droplet-mediated Redox Homeostasis Required for Prostate Cancer Proliferation.

Lipid droplets (LD) are dynamic organelles that serve as hubs of cellular metabolic processes. Emerging evidence shows that LDs also play a critical role in maintaining redox homeostasis and can mitigate lipid oxidative stress. In multiple cancers, including prostate cancer, LD accumulation is associated with cancer aggressiveness, therapy resistance, and poor clinical outcome. Prostate cancer arises as an androgen receptor (AR)-driven disease. Among its myriad roles, AR mediates the biosynthesis of LDs, induces autophagy, and modulates cellular oxidative stress in a tightly regulated cycle that promotes cell proliferation. The factors regulating the interplay of these metabolic processes downstream of AR remain unclear. Here, we show that Sigma1/SIGMAR1, a unique ligand-operated scaffolding protein, regulates LD metabolism in prostate cancer cells. Sigma1 inhibition triggers lipophagy, an LD selective form of autophagy, to prevent accumulation of LDs which normally act to sequester toxic levels of reactive oxygen species (ROS). This disrupts the interplay between LDs, autophagy, buffering of oxidative stress and redox homeostasis, and results in the suppression of cell proliferation in vitro and tumor growth in vivo. Consistent with these experimental results, SIGMAR1 transcripts are strongly associated with lipid metabolism and ROS pathways in prostate tumors. Altogether, these data reveal a novel, pharmacologically responsive role for Sigma1 in regulating the redox homeostasis required by oncogenic metabolic programs that drive prostate cancer proliferation. SIGNIFICANCE: To proliferate, cancer cells must maintain productive metabolic and oxidative stress (eustress) while mitigating destructive, uncontrolled oxidative stress (distress). LDs are metabolic hubs that enable adaptive responses to promote eustress. Targeting the unique Sigma1 protein can trigger distress by disrupting the LD-mediated homeostasis required for proliferation.

ONECUT2 Activates Diverse Resistance Drivers of Androgen Receptor-Independent Heterogeneity in Prostate Cancer.

Androgen receptor- (AR-) indifference is a mechanism of resistance to hormonal therapy in prostate cancer (PC). Here we demonstrate that the HOX/CUT transcription factor ONECUT2 (OC2) activates resistance through multiple drivers associated with adenocarcinoma, stem-like and neuroendocrine (NE) variants. Direct OC2 targets include the glucocorticoid receptor and the NE splicing factor SRRM4, among others. OC2 regulates gene expression by promoter binding, enhancement of chromatin accessibility, and formation of novel super-enhancers. OC2 also activates glucuronidation genes that irreversibly disable androgen, thereby evoking phenotypic heterogeneity indirectly by hormone depletion. Pharmacologic inhibition of OC2 suppresses lineage plasticity reprogramming induced by the AR signaling inhibitor enzalutamide. These results demonstrate that OC2 activation promotes a range of drug resistance mechanisms associated with treatment-emergent lineage variation in PC. Our findings support enhanced efforts to therapeutically target this protein as a means of suppressing treatment-resistant disease.

Supraphysiological Androgens Promote the Tumor Suppressive Activity of the Androgen Receptor through cMYC Repression and Recruitment of the DREAM Complex.

The androgen receptor (AR) pathway regulates key cell survival programs in prostate epithelium. The AR represents a near-universal driver and therapeutic vulnerability in metastatic prostate cancer, and targeting AR has a remarkable therapeutic index. Though most approaches directed toward AR focus on inhibiting AR signaling, laboratory and now clinical data have shown that high dose, supraphysiological androgen treatment (SPA) results in growth repression and improved outcomes in subsets of patients with prostate cancer. A better understanding of the mechanisms contributing to SPA response and resistance could help guide patient selection and combination therapies to improve efficacy. To characterize SPA signaling, we integrated metrics of gene expression changes induced by SPA together with cistrome data and protein-interactomes. These analyses indicated that the dimerization partner, RB-like, E2F, and multivulval class B (DREAM) complex mediates growth repression and downregulation of E2F targets in response to SPA. Notably, prostate cancers with complete genomic loss of RB1 responded to SPA treatment, whereas loss of DREAM complex components such as RBL1/2 promoted resistance. Overexpression of MYC resulted in complete resistance to SPA and attenuated the SPA/AR-mediated repression of E2F target genes. These findings support a model of SPA-mediated growth repression that relies on the negative regulation of MYC by AR leading to repression of E2F1 signaling via the DREAM complex. The integrity of MYC signaling and DREAM complex assembly may consequently serve as determinants of SPA responses and as pathways mediating SPA resistance. SIGNIFICANCE: Determining the molecular pathways by which supraphysiological androgens promote growth arrest and treatment responses in prostate cancer provides opportunities for biomarker-selected clinical trials and the development of strategies to augment responses.

A Phase 1/2 Study of Rapamycin and Cisplatin/Gemcitabine for Treatment of Patients With Muscle-Invasive Bladder Cancer.

INTRODUCTION: Cisplatin-based neoadjuvant chemotherapy (NAC) followed by cystectomy is the standard for muscle-invasive bladder cancer (MIBC), however, NAC confers only a small survival benefit and new strategies are needed to increase its efficacy. Pre-clinical data suggest that in response to DNA damage the tumor microenvironment (TME) adopts a paracrine secretory phenotype dependent on mTOR signaling which may provide an escape mechanism for tumor resistance, thus offering an opportunity to increase NAC effectiveness with mTOR blockade. PATIENTS & METHODS: We conducted a phase I/II clinical trial to assess the safety and efficacy of gemcitabine-cisplatin-rapamycin combination. Grapefruit juice was administered to enhance rapamycin pharmacokinetics by inhibiting intestinal enzymatic degradation. Phase I was a dose determination/safety study followed by a single arm Phase II study of NAC prior to radical cystectomy evaluating pathologic response with a 26% pCR rate target. RESULTS: In phase I, 6 patients enrolled, and the phase 2 dose of 35 mg rapamycin established. Fifteen patients enrolled in phase II; 13 were evaluable. Rapamycin was tolerated without serious adverse events. At the preplanned analysis, the complete response rate (23%) did not meet the prespecified level for continuing and the study was stopped due to futility. With immunohistochemistry, successful suppression of the mTOR signaling pathway in the tumor was achieved while limited mTOR activity was seen in the TME. CONCLUSION: Adding rapamycin to gemcitabine-cisplatin therapy for patients with MIBC was well tolerated but failed to improve therapeutic efficacy despite evidence of mTOR blockade in tumor cells. Further efforts to understand the role of the tumor microenvironment in chemotherapy resistance is needed.

Bipolar androgen therapy plus olaparib in men with metastatic castration-resistant prostate cancer.

BACKGROUND: Bipolar androgen therapy (BAT) results in rapid fluctuation of testosterone (T) between near-castrate and supraphysiological levels and has shown promise in metastatic castration-resistant prostate cancer (mCRPC). Its clinical effects may be mediated through induction of DNA damage, and preclinical studies suggest synergy with PARP inhibitors. PATIENTS AND METHODS: This was a single-center, Phase II trial testing olaparib plus BAT (T cypionate/enanthate 400 mg every 28 days) with ongoing androgen deprivation. Planned recruitment was 30 subjects (equal proportions with/without homologous recombination repair [HRR] gene mutations) with mCRPC post abiraterone and/or enzalutamide. The primary objective was to determine PSA50 response (PSA decline ≥50% from baseline) rate at 12-weeks. The primary analysis utilized the entire (intent-to-treat [ITT]) cohort, with those dropping out early counted as non-responders. Secondary/exploratory analyses were in those treated beyond 12-weeks (response-evaluable cohort). RESULTS: Thirty-six patients enrolled and 6 discontinued prior to response assessment. In the ITT cohort, PSA50 response rate at 12-weeks was 11/36 (31%; 95% CI 17-48%), and 16/36 (44%, 95% CI 28-62%) had a PSA50 response at any time on-study. After a median follow-up of 19 months, the median clinical/radiographic progression-free survival in the ITT cohort was 13.0 months (95% CI 7-17). Clinical outcomes were similar regardless of HRR gene mutational status. CONCLUSIONS: BAT plus olaparib is associated with high response rates and long PFS. Clinical benefit was observed regardless of HRR gene mutational status.

Circulating and Intratumoral Adrenal Androgens Correlate with Response to Abiraterone in Men with Castration-Resistant Prostate Cancer.

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.

Hormonal intervention for the treatment of veterans with COVID-19 requiring hospitalization (HITCH): a multicenter, phase 2 randomized controlled trial of best supportive care vs best supportive care plus degarelix: study protocol for a randomized controlled trial.

BACKGROUND: Therapeutic targeting of host-cell factors required for SARS-CoV-2 entry is an alternative strategy to ameliorate COVID-19 severity. SARS-CoV-2 entry into lung epithelium requires the TMPRSS2 cell surface protease. Pre-clinical and correlative data in humans suggest that anti-androgenic therapies can reduce the expression of TMPRSS2 on lung epithelium. Accordingly, we hypothesize that therapeutic targeting of androgen receptor signaling via degarelix, a luteinizing hormone-releasing hormone (LHRH) antagonist, will suppress COVID-19 infection and ameliorate symptom severity. METHODS: This is a randomized phase 2, placebo-controlled, double-blind clinical trial in 198 patients to compare efficacy of degarelix plus best supportive care versus placebo plus best supportive care on improving the clinical outcomes of male Veterans who have been hospitalized due to COVID-19. Enrolled patients must have documented infection with SARS-CoV-2 based on a positive reverse transcriptase polymerase chain reaction result performed on a nasopharyngeal swab and have a severity of illness of level 3-5 (hospitalized but not requiring invasive mechanical ventilation). Patients stratified by age, history of hypertension, and severity are centrally randomized 2:1 (degarelix: placebo). The composite primary endpoint is mortality, ongoing need for hospitalization, or requirement for mechanical ventilation at 15 after randomization. Important secondary endpoints include time to clinical improvement, inpatient mortality, length of hospitalization, duration of mechanical ventilation, time to achieve a normal temperature, and the maximum severity of COVID-19 illness. Exploratory analyses aim to assess the association of cytokines, viral load, and various comorbidities with outcome. In addition, TMPRSS2 expression in target tissue and development of anti-viral antibodies will also be investigated. DISCUSSION: In this trial, we repurpose the FDA approved LHRH antagonist degarelix, commonly used for prostate cancer, to suppress TMPRSS2, a host cell surface protease required for SARS-CoV-2 cell entry. The objective is to determine if temporary androgen suppression with a single dose of degarelix improves the clinical outcomes of patients hospitalized due to COVID-19. TRIAL REGISTRATION: ClinicalTrials.gov NCT04397718. Registered on May 21, 2020.

Selective androgen receptor modulators activate the canonical prostate cancer androgen receptor program and repress cancer growth.

Prostate cancer (PC) is driven by androgen receptor (AR) activity, a master regulator of prostate development and homeostasis. Frontline therapies for metastatic PC deprive the AR of the activating ligands testosterone (T) and dihydrotestosterone (DHT) by limiting their biosynthesis or blocking AR binding. Notably, AR signaling is dichotomous, inducing growth at lower activity levels, while suppressing growth at higher levels. Recent clinical studies have exploited this effect by administration of supraphysiological concentrations of T, resulting in clinical responses and improvements in quality of life. However, the use of T as a therapeutic agent in oncology is limited by poor drug-like properties as well as rapid and variable metabolism. Here, we investigated the antitumor effects of selective AR modulators (SARMs), which are small-molecule nonsteroidal AR agonists developed to treat muscle wasting and cachexia. Several orally administered SARMs activated the AR program in PC models. AR cistromes regulated by steroidal androgens and SARMs were superimposable. Coregulatory proteins including HOXB13 and GRHL2 comprised AR complexes assembled by both androgens and SARMs. At bioavailable concentrations, SARMs repressed MYC oncoprotein expression and inhibited the growth of castration-sensitive and castration-resistant PC in vitro and in vivo. These results support further clinical investigation of SARMs for treating advanced PC.

Targeting backdoor androgen synthesis through AKR1C3 inhibition: A presurgical hormonal ablative neoadjuvant trial in high-risk localized prostate cancer.

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.

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 3ß-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.

Durable Response of Enzalutamide-resistant Prostate Cancer to Supraphysiological Testosterone Is Associated with a Multifaceted Growth Suppression and Impaired DNA Damage Response Transcriptomic Program in Patient-derived Xenografts.

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.

The role of adrenal derived androgens in castration resistant prostate cancer.

Castration resistant prostate cancer (CRPC) remains androgen dependant despite castrate levels of circulating testosterone following androgen deprivation therapy, the first line of treatment for advanced metstatic prostate cancer. CRPC is characterized by alterations in the expression levels of steroidgenic enzymes that enable the tumour to derive potent androgens from circulating adrenal androgen precursors. Intratumoral androgen biosynthesis leads to the localized production of both canonical androgens such as 5α-dihydrotestosterone (DHT) as well as less well characterized 11-oxygenated androgens, which until recently have been overlooked in the context of CRPC. In this review we discuss the contribution of both canonical and 11-oxygenated androgen precursors to the intratumoral androgen pool in CRPC. We present evidence that CRPC remains androgen dependent and discuss the alterations in steroidogenic enzyme expression and how these affect the various pathways to intratumoral androgen biosynthesis. Finally we summarize the current treatment strategies for targeting adrenal derived androgen biosynthesis.

Molecular determinants of response to high-dose androgen therapy in prostate cancer.

Clinical trials of high-dose androgen (HDA) therapy for prostate cancer (PC) have shown promising efficacy but are limited by lack of criteria to identify likely responders. To elucidate factors that govern the growth-repressive effects of HDAs, we applied an unbiased integrative approach using genetic screens and transcriptional profiling of PC cells with or without demonstrated phenotypic sensitivity to androgen-mediated growth repression. Through this comprehensive analysis, we identified genetic events and related signaling networks that determine the response to both HDA and androgen withdrawal. We applied these findings to develop a gene signature that may serve as an early indicator of treatment response and identify men with tumors that are amenable to HDA therapy.

Testosterone accumulation in prostate cancer cells is enhanced by facilitated diffusion.

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 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 vs 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 nonautonomous mechanism for testosterone signaling in CRPC.