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Glucocorticoids have been used in the treatment of prostate cancer to slow disease progression, improve pain control and offset side effects of chemo- and hormonal therapy. However, they may also have the potential to drive prostate cancer growth via mutated androgen receptors or glucocorticoid receptors (GRs). In this review we examine historical and contemporary use of glucocorticoids in the treatment of prostate cancer, review potential mechanisms by which they may inhibit or drive prostate cancer growth, and describe potential means of defining their contribution to the biology of prostate cancer.

Purpose: Mechanisms mediating androgen receptor (AR) reactivation in prostate cancer that progresses after castration (castration-resistant prostate cancer; CRPC) and subsequent treatment with abiraterone (CYP17A1 inhibitor that further suppresses androgen synthesis) remain unclear. Experimental Design: Prostate cancer xenografts were examined to identify mechanism of progression after castration and abiraterone. Results: AR reactivation in abiraterone-resistant VCaP xenografts was not associated with restoration of intratumoral androgens or alterations in AR coregulators. In contrast, mRNA encoding full-length AR (AR-FL) and a constitutively active splice variant (AR-V7) were increased compared with xenografts before castration, with an increase in AR-V7 relative to AR-FL. This shift toward AR-V7 was due to a feedback mechanism whereby the androgen-liganded AR stimulates expression of proteins that suppress generation of AR-V7 relative to AR-FL transcripts. However, despite the increases in AR-V7 mRNA, it remained a minor transcript ( Conclusions: AR-V7 at these low levels is not adequate to restore AR activity, but its rapid induction after androgen deprivation allows tumors to retain basal AR activity that may be needed for survival until more potent mechanisms emerge to activate AR. Agents targeting AR splice variants may be most effective when used very early in conjunction with therapies targeting the AR ligand-binding domain. Clin Cancer Res; 20(6); 1590–600. ©2014 AACR.

BACKGROUND: Mantle cell lymphoma (MCL) demonstrates a male to female ratio of 4:1. While the pathobiology underlying this gender-distribution imbalance is unknown, anecdotal clinical observations, as well as in vitro evidence of relative androgen receptor (AR) overexpression in MCL cell lines and response to androgen blockade, implicate the AR signaling axis as a potential driver of lymphomagenesis in MCL (Mostaghel et al. Experimental Hematology 2017). Thus, we hypothesized that blocking the AR axis could be a potential treatment strategy for MCL. Here, we report results from our pilot study using the second-generation AR antagonist enzalutamide for treatment of MCL. METHODS: A single arm pilot study was carried out at the Fred Hutchinson Cancer Research Center/University of Washington/Seattle Cancer Care Alliance. Patients ≥ 18 years, with relapsed/refractory or untreated MCL, metabolically active disease > 1.5 cm in long axis, ECOG ≤ 2, ANC ≥ 1000/mm3 & platelet ≥ 50,000/mm3, and acceptable liver and renal function were eligible. Enzalutamide 160 mg was administered daily until disease progression, intolerance, or decision of patient or physician to stop study drug. The primary endpoint was overall response rate (ORR) including complete response (CR) and partial response (PR). The secondary endpoints were time to treatment failure (TTF), progression free survival (PFS), overall survival (OS), adverse events (AEs) as measured by NCI-CTCAE version 4.0, and disease control rate. Correlative studies included impact of AR expression and changes in sex hormone levels on clinical outcomes. RESULTS: Eight patients were enrolled between September 2015 and January 2018. Median age at enrollment was 60 (range 47-79), six were male, median number of prior treatments was 2.5 (range 0-5), three received prior Bruton's tyrosine kinase (BTK) inhibitor therapy, and four had a prior autologous stem cell transplant. Mantle cell lymphoma international prognostic index (MIPI) scores were low (1), intermediate (4) and high (3). The median Ki-67 was 28% (range < 5 to 60). Patients were treated for a median of 3.5 28-day cycles. Reasons for discontinuation included progressive disease (6) and development of rash at day 10 (1). One patient remains on study and without disease progression at 16 months. Best responses in the six response-evaluable patients included three with stable disease (ranging from 5 to 8 months) and three with progressive disease. Low response rates led to closure of the study. Median PFS and OS were 157 days (range 38 to 248) and 1168 days (range 187 to 1289), respectively (figure 1). All patients had tumor AR expression available for review and stained positive (6 variably positive, 1 uniformly positive, and 1 weakly focally positive). No clinical factor, AR staining nor baseline or changes in hormone levels were correlated with response or PFS, though the single patient with ongoing disease control >15 months is the only evaluable female on study who is receiving enzalutamide as first-line therapy. Two grade 3 (abdominal pain, agitation) and one grade 4 (leukocytosis) AEs were reported. CONCLUSIONS: Blocking the AR axis with enzalutamide in the treatment of MCL was well tolerated but did not appear to translate into a meaningful ORR despite supportive preclinical data. This study was approved and funded in part by the National Comprehensive Cancer Network (NCCN) Oncology Research Program from general research support provided by Astellas. Disclosures Smith: Merck Sharp & Dohme Corp: Consultancy, Research Funding; Pharmacyclics: Research Funding; Incyte Corporation: Research Funding; Genentech: Research Funding; Denovo Biopharma: Research Funding; Portola Pharmaceuticals: Research Funding; Ayala (spouse): Research Funding; Bristol-Myers Squibb (spouse): Research Funding; Seattle Genetics: Research Funding; Ignyta (spouse): Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma BV: Research Funding. Till:Mustang Bio: Patents & Royalties, Research Funding. Graf:AstraZeneca: Research Funding; TG Therapeutics: Research Funding; BeiGene: Research Funding. Gopal:Teva, Bristol-Myers Squibb, Merck, Takeda, Seattle Genetics, Pfizer, Janssen, Takeda, and Effector: Research Funding; Seattle Genetics, Pfizer, Janssen, Gilead, Sanofi, Spectrum, Amgen, Aptevo, BRIM bio, Acerta, I-Mab-pharma, Takeda, Compliment, Asana Bio, and Incyte: Honoraria; Seattle Genetics, Pfizer, Janssen, Gilead, Sanofi, Spectrum, Amgen, Aptevo, BRIM bio, Acerta, I-Mab-pharma, Takeda, Compliment, Asana Bio, and Incyte.: Consultancy. OffLabel Disclosure: Enzalutamide is FDA approved for metastatic and non-metastatic castration-resistant prostate cancer.

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.

5081 Background: Studies have shown that localized PCs may resist neoadjuvant androgen receptor (AR)-targeted therapies as a result of persistent intraprostatic androgens, likely arising through upregulation of steroidogenic enzymes. Therefore, we sought to evaluate clinical effects of combinatorial AR-targeted therapy, including indomethacin (Indo) to inhibit the steroidogenic enzyme AKR1C3, 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 localized high to very-high risk PC, as defined by NCCN criteria. Patients received 12 weeks of neoadjuvant apalutamide (Apa), abiraterone (Abi) plus prednisone, degarelix, and Indo at their respective FDA-approved doses followed by RP. The primary objective was to determine the pathologic complete response (pCR) rate. Secondary objectives included assessing for minimal residual disease (MRD) (i.e. ≤0.25 cm3 tumor volume corrected for cellularity), measuring intraprostatic androgens and assessing molecular features associated with drug resistance. Twenty evaluable patients provided 91% power (one-sided alpha = 7.5%) to detect a difference in pCR rate of 5% (H0) vs. 25% (H1). Results: Twenty-two patients enrolled and 20 were evaluable for the primary endpoint (1 patient came off to pursue stereotactic radiosurgery; 1 was removed after developing grade 2 transaminitis). At baseline, the median PSA was 10.1 ng/mL (4.4-159.4), 4 (20%) patients had Gleason grade group (GG) 4 disease and 16 had GG 5 disease. At RP, 1 (5%) patient had a pCR, 6 (30%) had MRD, 18 (90%) had ypT3 disease and 7 (35%) had lymph node (LN) metastases. Treatment was generally well tolerated and adverse events were consistent with each individual drug’s known safety profile. Additional follow up data and correlative work will be presented at the meeting. Conclusions: In our cohort of men with high-risk PC, pCR rates remained low even with combinatorial AR-directed therapy. Ongoing pharmacodynamic studies aimed at determining if Indo effectively inhibited AKR1C3 will provide important insights regarding the utility of targeting this steroidogenic enzyme. Clinical trial information: NCT02849990.

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.

Purpose Ligand-mediated activation of the androgen receptor (AR) is critical for prostate cancer (PCa) survival and proliferation. The failure to completely ablate tissue androgens may limit suppression of PCa growth. We evaluated combinations of CYP17A and 5-α-reductase inhibitors for reducing prostate androgen levels, AR signaling, and PCa volumes. Patients and Methods Thirty-five men with intermediate/high-risk clinically localized PCa were randomly assigned to goserelin combined with dutasteride (ZD), bicalutamide and dutasteride (ZBD), or bicalutamide, dutasteride, and ketoconazole (ZBDK) for 3 months before prostatectomy. Controls included patients receiving combined androgen blockade with luteinizing hormone-releasing hormone agonist and bicalutamide. The primary outcome measure was tissue dihydrotestosterone (DHT) concentration. Results Prostate DHT levels were substantially lower in all experimental arms (0.02 to 0.04 ng/g v 0.92 ng/g in controls; P < .001). The ZBDK group demonstrated the greatest percentage decline in serum testosterone, androsterone, and dehydroepiandrosterone sulfate (P < .05 for all). Staining for AR and the androgen-regulated genes prostate-specific antigen and TMPRSS2 was strongly suppressed in benign glands and moderately in malignant glands (P < .05 for all). Two patients had pathologic complete response, and nine had ≤ 0.2 cm3 of residual tumor (defined as a near-complete response), with the largest numbers of complete and near-complete responses in the ZBDK group. Conclusion Addition of androgen synthesis inhibitors lowers prostate androgens below that achieved with standard therapy, but significant AR signaling remains. Tissue-based analysis of steroids and AR signaling is critical to informing the search for optimal local and systemic control of high-risk prostate cancer.

While androgen deprivation therapy (ADT) remains the primary treatment for metastatic prostate cancer (PCa), castration does not eliminate androgens from the prostate tumor microenvironment, and residual intratumoral androgens are implicated in nearly every mechanism by which androgen receptor (AR)-mediated signaling promotes castration-resistant disease. The uptake and intratumoral (intracrine) conversion of circulating adrenal androgens such as dehydroepiandrosterone sulfate (DHEA-S) to steroids capable of activating the wild type AR is a recognized driver of castration resistant prostate cancer (CRPC). However, less well-characterized adrenal steroids, including 11-deoxcorticosterone (DOC) and 11beta-hydroxyandrostenedione (11OH-AED) may also play a previously unrecognized role in promoting AR activation. In particular, recent data demonstrate that the 5α-reduced metabolites of DOC and 11OH-AED are activators of the wild type AR. Given the well-recognized presence of SRD5A activity in CRPC tissue, these observations suggest that in the low androgen environment of CRPC, alternative sources of 5α-reduced ligands may supplement AR activation normally mediated by the canonical 5α-reduced agonist, 5α-DHT. Herein we review the emerging data that suggests a role for these alternative steroids of adrenal origin in activating the AR, and discuss the enzymatic pathways and novel downstream metabolites mediating these effects. We conclude by discussing the potential implications of these findings for CRPC progression, particularly in context of new agents such as abiraterone and enzalutamide which target the AR-axis for prostate cancer therapy.

Prostate tumors develop resistance to androgen deprivation therapy (ADT) by multiple mechanisms, one of which is to express constitutively active androgen receptor (AR) splice variants lacking the ligand binding domain. AR splice variant 7 (AR-V7, also termed AR3) is the most abundantly expressed variant that drives prostate tumor progression under ADT conditions. However, the molecular mechanism by which AR-V7 is generated remains unclear. In this manuscript, we demonstrated that RNA splicing of AR-V7 in response to ADT was closely associated with AR gene transcription initiation and elongation rates. Enhanced AR gene transcription by ADT provides a pre-requisite condition that further increases the interactions between AR pre-mRNA and splicing factors. Under ADT conditions, recruitment of several RNA splicing factors to the 3’ splicing site for AR-V7 was increased. We identified two RNA splicing enhancers and their binding proteins (U2AF65 and ASF/SF2) that played critical roles in splicing AR pre-mRNA into AR-V7. These data indicate that ADT-induced AR gene transcription rate and splicing factor recruitment to AR pre-mRNA contribute to the enhanced AR-V7 levels in prostate cancer cells.

Solute Carrier (SLC) transporters are a large superfamily of transmembrane carriers involved in the regulated transport of metabolites, nutrients, ions and drugs across cellular membranes. A subset of these solute carriers play a significant role in the cellular uptake of many cancer therapeutics, ranging from chemotherapeutics such as antimetabolites, topoisomerase inhibitors, platinum-based drugs and taxanes to targeted therapies such as tyrosine kinase inhibitors. SLC transporters are co-expressed in groups and patterns across normal tissues, suggesting they may comprise a coordinated regulatory circuit serving to mediate normal tissue functions. In cancer however, there are dramatic changes in expression patterns of SLC transporters. This frequently serves to feed the increased metabolic demands of the tumor cell for amino acids, nucleotides and other metabolites, but also presents a therapeutic opportunity, as increased transporter expression may serve to increase intracellular concentrations of substrate drugs. In this review, we examine the regulation of drug transporters in cancer and how this impacts therapy response, and discuss novel approaches to targeting therapies to specific cancers via tumor-specific aberrations in transporter expression. We propose that among the oncogenic changes in SLC transporter expression there exist emergent vulnerabilities that can be exploited therapeutically, extending the application of precision medicine from tumor-specific drug targets to tumor-specific determinants of drug uptake.

Background Although men on active surveillance for prostate cancer (PCa) may benefit from intervention with 5α-reductase inhibitors (5-ARIs), it has not been resolved whether 5-ARIs are effective for delaying disease progression and, if so, whether specific patients are more likely to benefit. Objective To identify molecular features predictive of patient response to 5-ARIs. Design, setting, and participants Nkx3.1 mutant mice, a model of early-stage PCa, were treated with the 5-ARI finasteride, and histopathological and molecular analyses were performed. Cross-species computational analyses were used to compare expression profiles for treated mice with those of patients who had received 5-ARIs before prostatectomy. Intervention Finasteride administered to Nkx3.1 mutant mice. 5-ARI-treated patient specimens obtained retrospectively. Outcome measurements and statistical analysis Endpoints in mice included histopathology, immunohistochemistry, and molecular profiling. GraphPad Prism software, R-studio, and Matlab were used for statistical and data analyses. Results and limitations Finasteride treatment of Nkx3.1 mutant mice resulted in a significant reduction in prostatic intraepithelial neoplasia (PIN), as evident from histopathological and expression profiling analyses. Cross-species computational analysis comparing finasteride-treated mice with two independent 5-ARI–treated patient cohorts showed that reduced NKX3.1 expression is predictive of response to 5-ARI. A limitation of the study is that these retrospective human cohorts have relatively few patients with limited clinical outcome data. Future prospective clinical trials are needed to validate whether stratifying patients on the basis of NKX3.1 expression improves the benefit of 5-ARIs during active surveillance. Conclusions This co-clinical study implicates NKX3.1 status as a predictor of response to 5-ARIs, and suggests that molecular features, including NKX3.1 expression, may help to identify PCa patients most likely to benefit from 5-ARIs during active surveillance. Patient summary The aim of precision cancer prevention is to tailor interventions on the basis of individualized patient characteristics. We propose that patients with low NKX3.1 expression are optimal candidates for intervention with 5α-reductase inhibitors as an adjunct to active surveillance.

Current treatments for castration-resistant prostate cancer (CRPC) that target androgen receptor (AR) signaling improve patient survival, yet ultimately fail. Here, we provide novel insights into treatment response for the antiandrogen abiraterone by analyses of a genetically engineered mouse (GEM) model with combined inactivation of Trp53 and Pten, which are frequently comutated in human CRPC. These NPp53 mice fail to respond to abiraterone and display accelerated progression to tumors resembling treatment-related CRPC with neuroendocrine differentiation (CRPC-NE) in humans. Cross-species computational analyses identify master regulators of adverse response that are conserved with human CRPC-NE, including the neural differentiation factor SOX11, which promotes neuroendocrine differentiation in cells derived from NPp53 tumors. Furthermore, abiraterone-treated NPp53 prostate tumors contain regions of focal and/or overt neuroendocrine differentiation, distinguished by their proliferative potential. Notably, lineage tracing in vivo provides definitive and quantitative evidence that focal and overt neuroendocrine regions arise by transdifferentiation of luminal adenocarcinoma cells. These findings underscore principal roles for TP53 and PTEN inactivation in abiraterone resistance and progression from adenocarcinoma to CRPC-NE by transdifferentiation. Significance: Understanding adverse treatment response and identifying patients likely to fail treatment represent fundamental clinical challenges. By integrating analyses of GEM models and human clinical data, we provide direct genetic evidence for transdifferentiation as a mechanism of drug resistance as well as for stratifying patients for treatment with antiandrogens. Cancer Discov; 7(7); 736–49. ©2017 AACR. See related commentary by Sinha and Nelson, p. 673. This article is highlighted in the In This Issue feature, p. 653

Mantle cell lymphoma (MCL) affects approximately 4500 patients/year in the US and demonstrates a male to female ratio of approximately 4:1. While the pathobiology underlying this ratio is unknown, the hematopoietic system is characterized by sex-related differences in androgen receptor (AR) expression, leading us to hypothesize that the male-biased incidence of MCL may reflect sex-related differences in AR signaling during MCL lymphomagenesis. To explore the AR axis in MCL, we evaluated AR expression in MCL cell lines and human tumors, and tested the impact of androgen pathway inhibition on MCL proliferation. AR transcript levels ranged up to ~26 fold higher in MCL lines vs non-MCL NHL lines (p = 0.006) and were correlated with expression of the canonical AR-regulated gene, prostate-specific antigen (PSA; r = 0.715, p = 0.001), consistent with functional AR activity. Patient-derived MCL samples demonstrated a range of AR expression. Treatment of four different MCL lines with the potent AR antagonist enzalutamide demonstrated suppression of proliferation across both male and female-derived cell lines. These data suggest androgen-axis blockade may represent a novel therapeutic modality in MCL. This novel treatment approach is currently under investigation in a phase II clinical trial of AR inhibition in patients with relapsed/refractory MCL.

Docetaxel plus androgen deprivation therapy (ADT) offers a survival benefit in metastatic hormone-sensitive prostate cancer (mHSPC). However, one trial evaluating docetaxel in mHSPC (GETUG-AFU15) showed unexpected toxicity; raising concerns that docetaxel may carry increased toxicity when used to treat mHSPC compared to metastatic castration-resistant prostate cancer (mCRPC). We conducted a retrospective analysis evaluating differences in toxicity based on the clinical state (i.e., mHSPC vs. mCRPC) that docetaxel was used. Patients initiating docetaxel between 1/1/2014 and 7/15/2015 were included, with the former date chosen to coincide with the press release for the first mHSPC study that showed a survival benefit with early docetaxel; ensuring contemporary docetaxel-treated cohorts. Thirty-nine mCRPC and 22 mHSPC patients were included. Compared to mCRPC, mHSPC patients were younger (median years: 66.3 vs. 71.8, P = 0.007); had better performance status (ECOG 0-1: 100 vs. 62 %, P

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.

Targeted next generation sequencing (tNGS) is increasingly used in oncology for therapeutic decision-making, but is not yet widely used for prostate cancer. The objective of this study was to determine current clinical utility of tNGS for prostate cancer management.Seven academic genitourinary medical oncologists recruited and consented patients with prostate cancer, largely with unusual clinical and/or pathologic features, from 2013 to 2015. UW-OncoPlex was performed on formalin-fixed, paraffin-embedded (FFPE) primary tumors and/or metastatic biopsies. Results were discussed at a multidisciplinary precision tumor board prior to communicating to patients. FFPE tumor DNA was extracted for tNGS analysis of 194 cancer-associated genes. Results, multidisciplinary discussion, and treatment changes were recorded.Forty-five patients consented and 42 had reportable results. Findings included mutations in genes frequently observed in prostate cancer. We also found alterations in genes where targeted treatments were available and/or in clinical trials. 4/42 (10%) cases, change in treatment directly resulted from tNGS and multidisciplinary discussion. In 30/42 (71%) cases additional options were available but not pursued and/or were pending. Notably, 10/42 (24%) of patients harbored suspected germline mutations in moderate or high-penetrance cancer risk genes, including BRCA2, TP53, ATM, and CHEK2. One patient's tumor had bi-allelic MSH6 mutation and microsatellite instability. In total, 34/42 (81%) cases resulted in some measure of treatment actionability. Limitations include small size and limited clinical outcomes.Targeted NGS tumor sequencing may help guide immediate and future treatment options for men with prostate cancer. A substantial subset had germline mutations in cancer predisposition genes with potential clinical management implications for men and their relatives. Prostate 76:1303-1311, 2016. © 2016 Wiley Periodicals, Inc.

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.

Relapse of castration-resistant prostate cancer (CRPC) that occurs after androgen deprivation therapy of primary prostate cancer can be mediated by reactivation of the androgen receptor (AR). One important mechanism mediating this AR reactivation is intratumoral conversion of the weak adrenal androgens DHEA and androstenedione into the AR ligands testosterone and dihydrotestosterone. DHEA and androstenedione are synthesized by the adrenals through the sequential actions of the cytochrome P450 enzymes CYP11A1 and CYP17A1, so that CYP17A1 inhibitors such as abiraterone are effective therapies for CRPC. However, the significance of intratumoral CYP17A1 and de novo androgen synthesis from cholesterol in CRPC, and the mechanisms contributing to CYP17A1 inhibitor resistance/relapse, remain to be determined. We report that AR activity in castration-resistant VCaP tumor xenografts can be restored through CYP17A1-dependent de novo androgen synthesis, and that abiraterone treatment of these xenografts imposes selective pressure for increased intratumoral expression of CYP17A1, thereby generating a mechanism for development of resistance to CYP17A1 inhibitors. Supporting the clinical relevance of this mechanism, we found that intratumoral expression of CYP17A1 was markedly increased in tumor biopsies from CRPC patients after CYP17A1 inhibitor therapy. We further show that CRPC cells expressing a progesterone responsive T877A mutant AR are not CYP17A1 dependent, but that AR activity in these cells is still steroid dependent and mediated by upstream CYP11A1-dependent intraturmoral pregnenolone/progesterone synthesis. Together, our results indicate that CRPCs resistant to CYP17A1 inhibition may remain steroid dependent and therefore responsive to therapies that can further suppress de novo intratumoral steroid synthesis. Cancer Res; 71(20); 6503–13. ©2011 AACR.

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.

Continued activation of the androgen receptor (AR) axis despite castration remains a critical force in the development of castration-resistant prostate cancer (CRPC). Therapeutic strategies designed to more effectively ablate tumoral androgen activity are required to improve clinical efficacy and prevent disease progression. Tumor-based alterations in expression and activity of the AR and in steroidogenic pathways mediating ligand generation facilitate the development of CRPC. This article reviews AR and ligand-dependent mechanisms underlying CRPC progression and the status of novel hormonal therapies targeting the AR axis that are currently in clinical and preclinical development.

Context: Concern exists that androgen treatment might adversely impact prostate health in older men. Dihydrotestosterone (DHT), derived from local conversion of testosterone to DHT by 5α-reductase enzymes, is the principal androgen within the prostate. Exogenous androgens raise serum DHT concentrations, but their effects on the prostate are not clear. Objective: To determine the impact of large increases in serum DHT concentrations on intraprostatic androgen concentrations and androgen action within the prostate. Design: Double-blind, randomized, placebo-controlled. Setting: Single academic medical center. Participants: 31 healthy men ages 35–55. Intervention: Daily transdermal DHT or placebo gel. Main Outcome Measures: Serum and prostate tissue androgen concentrations and prostate epithelial cell gene expression after 4 wk of treatment. Results: Twenty-seven men completed all study procedures. Serum DHT levels increased nearly sevenfold, while testosterone levels decreased in men treated with daily transdermal DHT gel but were unchanged in the placebo-treated group (P < 0.01 between groups). In contrast, intraprostatic DHT and testosterone concentrations on d 28 were not different between groups (DHT: placebo = 2.8 ± 0.2 vs. DHT gel = 3.1 ± 0.5 ng/g; T: placebo = 0.6 ± 0.2 vs. DHT gel = 0.4 ± 0.1, mean ± se). Similarly, prostate volume, prostate-specific antigen, epithelial cell proliferation, and androgen-regulated gene expression were not different between groups. Conclusions: Robust supraphysiologic increases in serum DHT, associated with decreased serum T, do not significantly alter intraprostatic levels of DHT, testosterone, or prostate epithelial cell androgen–regulated gene expression in healthy men. Changes in circulating androgen concentrations are not necessarily mimicked within the prostate microenvironment, a finding with implications for understanding the impact of androgen therapies in men.

Testosterone and inflammation have been linked to the development of common age-associated diseases affecting the prostate gland including prostate cancer, prostatitis, and benign prostatic hypertrophy. We hypothesized that testosterone regulates components of prostate tight junctions which serve as a barrier to inflammation, thus providing a connection between age- and treatment-associated testosterone declines and prostatic pathology. We examined the expression and distribution of tight junction proteins in prostate biospecimens from mouse models and a clinical study of chemical castration, using transcript profiling, immunohistochemistry and electron microscopy. We determined that low serum testosterone is associated with reduced transcript and protein levels of Claudin 4 and Claudin 8, resulting in defective tight junction ultrastructure in benign prostate glands. Expression of Claudin 4 and Claudin 8 was negatively correlated with the mononuclear inflammatory infiltrate caused by testosterone deprivation. Testosterone suppression also induced an auto-immune humoral response directed toward prostatic proteins. Testosterone supplementation in castrate mice resulted in re-expression of tight junction components in prostate epithelium and significantly reduced prostate inflammatory cell numbers. These data demonstrate that tight junction architecture in the prostate is related to changes in serum testosterone levels, and identify an androgen-regulated mechanism that potentially contributes to the development of prostate inflammation and consequent pathology.

Based on the hypothesis that early use of androgen deprivation therapy (ADT) may improve outcomes, we conducted a phase 2 trial of neoadjuvant leuprolide for 24 weeks in combination with abiraterone acetate plus prednisone (referred to subsequently as leuprolide plus AAP) for 12 or 24 weeks prior to radical prostatectomy (RP). As reported recently, we confirmed that the addition of AAP further markedly reduced intraprostatic androgen levels and appeared to improve responses relative to historical controls using single-agent GnRH agonists. Nonetheless, residual prostate cancer (PCa) was found in the majority of patients, with only a small number of patients demonstrating complete pathologic responses. Moreover, substantial nuclear and cytoplasmic AR expression was detected by immunohistochemistry in most cases, suggesting that AR activity still may persist and contribute to residual disease. Residual PCa foci in RPs from 18 men treated with neoadjuvant intensive androgen deprivation therapy (leuprolide plus AAP) were microdissected and analyzed for resistance mechanisms. Transcriptome profiling showed reduced but persistent androgen receptor (AR) activity in residual tumors, with no increase in neuroendocrine differentiation. Unexpectedly, proliferation was negatively correlated with AR activity, but positively correlated with decreased RB1 expression, and whole-exome sequencing (WES) further showed enrichment for RB1 genomic loss. In 14 cases where two tumor foci were microdissected, WES confirmed a common origin, but identified multiple oncogenic alterations unique to one focus. Primary PCa can have extensive microheterogeneity, but its contribution to the later emergence of metastatic castration-resistant PCa (mCRPC) has not been clear. These findings indicate that neoadjuvant intense androgen deprivation therapy selects for subclonal genomic alterations, including RB1 loss, which may be the origin for metastatic castration-resistant PCa. This study indicates that subclonal RB1 loss may be more common than previously appreciated in intermediate- to high-risk primary PCa, and may be an early event, independent of neuroendocrine differentiation, in the development of mCRPC. Comprehensive molecular analyses of primary PCa may detect aggressive subclones, and possibly inform on adjuvant strategies to prevent the emergence of mCRPC. Citation Format: Adam G. Sowalsky, Huihui Ye, Manoj Bhasin, Eliezer M. Van Allen, Massimo Loda, Rosina T. Lis, Laleh Montaser, Carla Calagua, Fen Ma, Joshua W. Russo, Rachel J. Schaefer, Olga S. Voznesensky, Zhenwei Zhang, Glenn J. Bubley, Robert B. Montgomery, Elahe A. Mostaghel, Peter S. Nelson, Mary-Ellen Taplin, Steven P. Balk. Prostate cancer neoadjuvant intensive androgen deprivation therapy selects for tumor foci with diverse oncogenic alterations [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A024.

5030Background: Ductal prostate cancer (dPC) is a rare prostate cancer variant associated with poor outcomes. Prior small case series have documented that dPCs may be enriched for alterations in DN...

Androgens and the androgen receptor (AR) influence prostate carcinogenesis. Lowering intraprostatic dihydrotestosterone (DHT) by inhibiting 5-alpha-reductase (SRD5A) reduces prostate cancer (PCa) incidence, but is not uniformly effective. Mechanisms by which SRD5A inhibition influences PCa initiation and/or progression among different individuals have not been established. We sought to identify molecular alterations underlying the differential chemo-preventive activity of SRD5A inhibition. Men with clinically-localized PCa were randomized to prostatectomy alone (n=25) or 4 months treatment with the SRD5A-inhibitor dutasteride (0.5mg (n=26) or 3.5mg (n=24)) preceding prostatectomy. Serum and prostate androgens were measured using mass spectrometry. We evaluated benign epithelial gene expression using expression profiling and immunohistochemistry, and characterized tumor TMPRSS2-ERG fusion status using FISH. Dutasteride at 0.5 or 3.5mg decreased prostatic DHT by 93% (0.23ng/g; p

By using LNCaP and its derivative cell lines, we first observed an association between tumor necrosis factor-α (TNF-α) resistance and hormone independence. Moreover, we found that the expression of tumor necrosis factor receptor–associated death domain (TRADD) was reduced in androgen deprivation–independent cells compared with that in androgen deprivation–dependent cells. TRADD is a crucial transducer for TNF-α–induced nuclear factor-κB (NF-κB) activation. Knocking down TRADD expression in LNCaP cells impaired TNF-α–induced NF-κB activation and androgen receptor repression, whereas overexpression of TRADD in C4-2B cells restored their sensitivity to TNF-α. Finally, we found that androgen deprivation reduces TRADD expression in vitro and in vivo, suggesting that androgen deprivation therapy may promote the development of TNF-α resistance by reducing TRADD expression during prostate cancer progression. [Cancer Res 2009;69(24):9448–56]

Emerging evidence suggests that despite testicular androgen ablation, residual androgens, likely of adrenal--though potentially of prostatic--origin, play a critical role in the progression of prostate cancer to recurrent "castration-resistant" disease. Thus, a reassessment of the concept of total androgen deprivation is warranted. Current treatment strategies may not only lack optimal efficacy, but may actually contribute to the selection of neoplastic clones adapted to exist and proliferate in a low (but not zero) androgen environment. Moreover, the adequacy of androgen receptor (AR) pathway inhibition cannot be surmised from serum or plasma androgen levels, but must be ascertained at the tissue and molecular level prior to drawing conclusions regarding clinical efficacy or failure. Recent studies by our group and others indicate that prostate cancers undergo an adaptive response to castration that is associated with the up-regulation of transcripts encoding enzymes involved in the biosynthesis of androgens. Targeting these metabolic enzymes either individually or using combinations of agents to inhibit testicular, adrenal, and intracrine sources may provide enhanced clinical responses in the setting of both localized and metastatic disease.

Purpose Despite treatments which lower circulating androgens, advanced prostate cancers often maintain androgen receptor (AR) signaling. The variable response to secondary hormonal manipulations in men with castrate-resistant prostate cancer (CRPC) creates a compelling need for strategies to individualize therapy based on the molecular features of each patient's tumor. Methods A transcription-based AR activity signature was developed from an androgen-sensitive prostate cancer cell (LNCaP) and tested on independent data sets of prostate cancer cell lines and human tumors to assess its precision and accuracy in detecting AR activity. The AR signature was applied to multiple sets of prostate specimens to determine how AR activity changes with hormone therapy and progression and oncogenic pathway analysis was used to identify biologic pathways correlating with AR activity. Results A robust AR signature accurately predicts AR activity in multiple prostate cancer cell lines, has minimal variation between replicate samples, and accurately reflects an individual's hormone status and intraprostatic dihydrotestosterone levels. The AR signature finds AR activity to be high in local, untreated prostate tumors and decreased in prostate tissue after neoadjuvant hormone therapy and in CRPC. Heterogeneity of AR activity exists along the spectrum of prostate cancer progression and decreasing predicted AR activity correlates with increasing predicted Src activity and sensitivity to dasatinib (Src-targeting kinase inhibitor). Conclusion A transcription-based AR signature can detect AR activity within individual prostate cancer specimens and has the potential to help individualize and improve care for patients with CRPC.

Androgen deprivation therapy remains a critical component of treatment for men with advanced prostate cancer, and data supports its use in metastatic disease and in conjunction with surgery or radiation in specific settings. Alternatives to standard androgen deprivation therapy, such as intermittent androgen suppression and estrogen therapy, hold the potential to improve toxicity profiles while maintaining clinical benefit. Current androgen deprivation strategies seem to incompletely suppress androgen levels and androgen-receptor-mediated effects at the tissue level. Advances in the understanding of mechanisms that contribute to castration-resistant prostate cancer are leading to rationally designed therapies targeting androgen metabolism and the androgen receptor. The results of large trials investigating the optimization of primary androgen deprivation therapy, including evaluation of intermittent androgen suppression and estrogen patch assessment, as well as phase III studies of novel androgen synthesis inhibitors, such as abiraterone acetate, are eagerly awaited.

Direct determination of androgen levels in prostate tissue provides a perspective on the organ that is not available via androgen serum levels. The principle prostatic androgens, primarily dihydrotestosterone (DHT) and secondarily testosterone, can be readily assayed in quick-frozen prostate biopsy cores or surgical specimens. Such assays have proved important in establishing (1) that DHT is a permissive factor in BPH pathogenesis, (2) a mechanism for the treatment of BPH, (3) an understanding of prostate cancer chemoprevention, (4) an explanation for the 'escape' of prostate cancer from castration therapy, (5) prostate safety of testosterone replacement therapy, and (6) insights into the cause of racial differences of prostate cancer. Future opportunities include clarification of new drug mechanisms for BPH and prostate cancer, as well as a better understanding of the pathogenesis of both, and as an aid in individual patient management. Determination of prostate tissue androgens may soon transition from research tool to clinical test.

Therapy for advanced prostate cancer centers on suppressing systemic androgens and blocking activation of the androgen receptor (AR). Despite anorchid serum androgen levels, nearly all patients develop castration-resistant disease. We hypothesized that ongoing steroidogenesis within prostate tumors and the maintenance of intratumoral androgens may contribute to castration-resistant growth. Using mass spectrometry and quantitative reverse transcription–PCR, we evaluated androgen levels and transcripts encoding steroidogenic enzymes in benign prostate tissue, untreated primary prostate cancer, metastases from patients with castration-resistant prostate cancer, and xenografts derived from castration-resistant metastases. Testosterone levels within metastases from anorchid men [0.74 ng/g; 95% confidence interval (95% CI), 0.59–0.89] were significantly higher than levels within primary prostate cancers from untreated eugonadal men (0.23 ng/g; 95% CI, 0.03–0.44; P < 0.0001). Compared with primary prostate tumors, castration-resistant metastases displayed alterations in genes encoding steroidogenic enzymes, including up-regulated expression of FASN, CYP17A1, HSD3B1, HSD17B3, CYP19A1, and UGT2B17 and down-regulated expression of SRD5A2 (P < 0.001 for all). Prostate cancer xenografts derived from castration-resistant tumors maintained similar intratumoral androgen levels when passaged in castrate compared with eugonadal animals. Metastatic prostate cancers from anorchid men express transcripts encoding androgen-synthesizing enzymes and maintain intratumoral androgens at concentrations capable of activating AR target genes and maintaining tumor cell survival. We conclude that intracrine steroidogenesis may permit tumors to circumvent low levels of circulating androgens. Maximal therapeutic efficacy in the treatment of castration-resistant prostate cancer will require novel agents capable of inhibiting intracrine steroidogenic pathways within the prostate tumor microenvironment. [Cancer Res 2008;68(11):4447–54]

Data regarding the molecular response of prostate cancer to hormone therapy continue to emerge, identifying a complex network of autocrine and paracrine signaling events mediating the tumor response to androgen suppression. Emerging data provide insight into cellular pathways important in the apoptotic response to therapy, including the transforming growth factor-beta, insulin-like growth factor-1, and vascular endothelial growth factor signaling axes. They also reveal mechanisms of direct antitumor cytotoxicity mediated by various hormonal agents and highlight the importance of developing antiandrogens capable of irreversibly inhibiting the androgen receptor. Accumulated data emphasize the presence of residual androgens and persistent activation of androgen receptor signaling in advanced prostate tumors despite castration. These factors suggest that a multitargeted treatment approach designed to ablate all contributions to the androgen receptor signaling axis within the prostate tumor microenvironment will be required in order for hormonal therapy to achieve optimal antitumor efficacy.

Androgen deprivation therapy (ADT) remains the primary treatment for advanced prostate cancer. The efficacy of ADT has not been rigorously evaluated by demonstrating suppression of prostatic androgen activity at the target tissue and molecular level. We determined the efficacy and consistency of medical castration in suppressing prostatic androgen levels and androgen-regulated gene expression. Androgen levels and androgen-regulated gene expression (by microarray profiling, quantitative reverse transcription-PCR, and immunohistochemistry) were measured in prostate samples from a clinical trial of short-term castration (1 month) using the gonadotropin-releasing hormone antagonist, Acyline, versus placebo in healthy men. To assess the effects of long-term ADT, gene expression measurements were evaluated at baseline and after 3, 6, and 9 months of neoadjuvant ADT in prostatectomy samples from men with localized prostate cancer. Medical castration reduced tissue androgens by 75% and reduced the expression of several androgen-regulated genes (NDRG1, FKBP5, and TMPRSS2). However, many androgen-responsive genes, including the androgen receptor (AR) and prostate-specific antigen (PSA), were not suppressed after short-term castration or after 9 months of neoadjuvant ADT. Significant heterogeneity in PSA and AR protein expression was observed in prostate cancer samples at each time point of ADT. Medical castration based on serum testosterone levels cannot be equated with androgen ablation in the prostate microenvironment. Standard androgen deprivation does not consistently suppress androgen-dependent gene expression. Suboptimal suppression of tumoral androgen activity may lead to adaptive cellular changes allowing prostate cancer cell survival in a low androgen environment. Optimal clinical efficacy will require testing of novel approaches targeting complete suppression of systemic and intracrine contributions to the prostatic androgen microenvironment. [Cancer Res 2007;67(10):5033–41]

A sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for the simultaneous quantitative analysis of dihydrotestosterone (DHT) and testosterone (T) from biological fluids has been developed. Commercially available deuterated analogues were used as internal standards. Steroids were extracted from serum or testicular fluid with hexane/ethyl acetate, evaporated to dryness, and treated with hydroxylamine to form their oxime derivatives. Upon chromatographic separation, the compounds were quantified using selected reaction monitoring (SRM). For T, the [M+H](+) ion at m/z 304 and the fragment ion at m/z 124 were used as the precursor and product ions. For DHT the ion cluster [M+H+ACN](+) at m/z 347 and the dissociated ion [M+H](+) at m/z 306 were used as the precursor and product ions, respectively. The limits of detectability on-column were in the sub-femtomole range for both compounds and the intra-day coefficient of variation (CV) for analysis from serum was less than 7% for both compounds. Given its high reproducibility, sensitivity, and relative simplicity, this assay should be of use in determining androgen levels in biospecimens, particularly in settings where sample quantity or steroid concentration are low.

Purpose: ErbB2 signaling appears to be increased and may enhance androgen receptor (AR) activity in a subset of patients with castration-resistant prostate cancer (CRPC), but agents targeting ErbB2 have not been effective. This study was undertaken to assess ErbB2 activity in abiraterone-resistant prostate cancer and to determine whether it may contribute to AR signaling in these tumors. Experimental Design: AR activity and ErbB2 signaling were examined in the radical prostatectomy specimens from a neoadjuvant clinical trial of leuprolide plus abiraterone and in the specimens from abiraterone-resistant CRPC xenograft models. The effect of ErbB2 signaling on AR activity was determined in two CRPC cell lines. Moreover, the effect of combination treatment with abiraterone and an ErbB2 inhibitor was assessed in a CRPC xenograft model. Results: We found that ErbB2 signaling was elevated in residual tumor following abiraterone treatment in a subset of patients and was associated with higher nuclear AR expression. In xenograft models, we similarly demonstrated that ErbB2 signaling was increased and associated with AR reactivation in abiraterone-resistant tumors. Mechanistically, we show that ErbB2 signaling and subsequent activation of the PI3K/AKT signaling stabilizes AR protein. Furthermore, concomitantly treating CRPC cells with abiraterone and an ErbB2 inhibitor, lapatinib, blocked AR reactivation and suppressed tumor progression. Conclusions: ErbB2 signaling is elevated in a subset of patients with abiraterone-resistant prostate cancer and stabilizes AR protein. Combination therapy with abiraterone and ErbB2 antagonists may be effective for treating the subset of CRPC with elevated ErbB2 activity. Clin Cancer Res; 22(14); 3672–82. ©2016 AACR.

Context: The impact of serum androgen manipulation on prostate tissue hormone levels in normal men is unknown. Studies of men with prostate cancer have suggested that prostatic androgens are preserved in the setting of castration. Tissue androgens might stimulate prostate growth, producing adverse clinical consequences. Objective: The objective of the study was to determine the effect of serum androgen manipulation on intraprostatic androgens in normal men. Design: Thirteen male volunteers ages 35–55 yr (prostate-specific antigen < 2.0 ng/ml; normal transrectal ultrasound) were randomly assigned to: 1) a long-acting GnRH-antagonist, acyline, every 2 wk; 2) acyline plus testosterone (T) gel (10 mg/d); or 3) placebo for 28 d. Serum hormones were assessed weekly. Prostate biopsies were obtained on d 28. Extracted androgens were measured by RIA, and immunohistochemistry for androgen-regulated proteins was performed. Results: The mean decrease in serum T was 94%, whereas prostatic T and dihydrotestosterone levels were 70 and 80% lower, respectively, in subjects receiving acyline alone compared with controls (P < 0.05). Despite this decrease in prostate androgens, there were no detectable differences in prostate epithelial proliferation, apoptosis, prostate-specific antigen, and androgen receptor expression. Conclusion: In this small study of healthy subjects, despite a 94% decrease in serum T with medical castration, intraprostatic T and dihydrotestosterone levels remained 20–30% of control values, and prostate cell proliferation, apoptosis, and androgen-regulated protein expression were unaffected. Our data highlight the importance of assessing tissue hormone levels. The source of persistent prostate androgens associated with medical castration and their potential role in supporting prostate metabolism deserves further study.

103 Background: Optimizing androgen suppression may provide better treatment outcomes for localized prostate cancer (PCa). Numerous trials have supported the benefit of combining androgen deprivation (ADT) with definitive radiotherapy in men with locally advanced or high-grade disease. LHRH agonist (LHRHa) is most commonly used. The LHRH antagonist degarelix may provide more robust androgen suppression. The impact on tissue androgens following use of degarelix prior to radiation has not been reported. We examined the impact on androgens in serum and tissue after 12 weeks of degarelix in this phase II study. Methods: A prospective, phase II study was conducted in men with localized PCa treated with 6 months of neoadjuvant and concurrent degarelix with radiation. Prostate biopsies were obtained at the time of fiducial placement before radiotherapy. Serum and tissue androgen levels were measured by liquid chromatography-tandem mass spectrometry. Needle biopsies from a separate analysis of untreated men or those receiving LHRHa prior to prostatectomy were used as tissue androgen level controls. Results: 16 men with intermediate (4) and high-risk (12) PCa received study therapy. 14 men completed degarelix and planned radiation to 77.4-81 Gy. Serum and tissue androgens after 12 weeks of therapy are compared to untreated control and LHRHa treated patients (12 weeks). Serum levels of dihydrotestosterone (DHT) and testosterone were similarly suppressed by LHRHa or degarelix compared to untreated controls, without statistically significant differences. Degarelix provided statistically greater reduction in androsterone than LHRHa. Conclusions: In this phase II study degarelix and LHRHa achieved similar serum and tissue androgen levels at 12 weeks; however, there was a greater suppression of tissue androsterone with degarelix. The clinical significance of this difference remains uncertain. Clinical trial information: NCT01731912. [Table: see text]

Pulmonary surfactant is a lipoprotein complex that lowers surface tension at the air-liquid interface of the lung and participates in pulmonary host defense. Surfactant proteins (SP), SP-A and SP-D, modulate a variety of immune cell functions, including the production of cytokines and free radicals. Previous studies showed that SP-A and SP-D inhibit lymphocyte proliferation in the presence of accessory cells. The goal of this study was to determine whether SP-A and SP-D directly suppress Th cell function. Both proteins inhibited CD3+/CD4+ lymphocyte proliferation induced by PMA and ionomycin in an IL-2-independent manner. Both proteins decreased the number of cells entering the S and mitotic phases of the cell cycle. Neither SP-A nor SP-D altered cell viability, apoptosis, or secretion of IL-2, IL-4, or IFN-γ when Th cells were treated with PMA and ionomycin. However, both proteins attenuated ionomycin-induced cytosolic free calcium ([Ca2+ ]i), but not thapsigargin-induced changes in [Ca2+]i. In summary, inhibition of T cell proliferation by SP-A and SP-D occurs via two mechanisms, an IL-2-dependent mechanism observed with accessory cell-dependent T cell mitogens and specific Ag, as well as an IL-2-independent mechanism of suppression that potentially involves attenuation of [Ca2+]i.

Since Huggins defined the androgen-sensitive nature of prostate cancer (PCa), suppression of systemic testosterone (T) has remained the most effective initial therapy for advanced disease although progression inevitably occurs. From the inception of clinical efforts to suppress androgen receptor (AR) signaling by reducing AR ligands, it was also recognized that administration of T in men with castration-resistant prostate cancer (CRPC) could result in substantial clinical responses. Data from preclinical models have reproducibly shown biphasic responses to T administration, with proliferation at low androgen concentrations and growth inhibition at supraphysiological T concentrations. Many questions regarding the biphasic response of PCa to androgen treatment remain, primarily regarding the mechanisms driving these responses and how best to exploit the biphasic phenomenon clinically. Here we review the preclinical and clinical data on high dose androgen growth repression and discuss cellular pathways and mechanisms likely to be involved in mediating this response. Although meaningful clinical responses have now been observed in men with PCa treated with high dose T, not all men respond, leading to questions regarding which tumor characteristics promote response or resistance, and highlighting the need for studies designed to determine the molecular mechanism(s) driving these responses and identify predictive biomarkers.

Summary Androgen receptor (AR) signaling is a distinctive feature of prostate carcinoma (PC) and represents the major therapeutic target for treating metastatic prostate cancer (mPC). Though highly effective, AR antagonism can produce tumors that bypass a functional requirement for AR, often through neuroendocrine (NE) transdifferentiation. Through the molecular assessment of mPCs over two decades, we find a phenotypic shift has occurred in mPC with the emergence of an AR-null NE-null phenotype. These "double-negative" PCs are notable for elevated FGF and MAPK pathway activity, which can bypass AR dependence. Pharmacological inhibitors of MAPK or FGFR repressed the growth of double-negative PCs in vitro and in vivo . Our results indicate that FGF/MAPK blockade may be particularly efficacious against mPCs with an AR-null phenotype.

Background We sought to define the disease characteristics and outcomes of those patients presenting with urothelial carcinoma and a persistently elevated white blood cell (WBC) count. Materials and Methods We queried a prospectively maintained institutional database. Patients were included if they had had a histologic diagnosis of urothelial carcinoma and a WBC count of > 20,000 cells/μL on ≥ 2 occasions separated by 30 days. The patients' medical records were reviewed and were excluded from the analysis if an underlying cause for the leukocytosis could be identified. The clinical, histologic, and laboratory data were then collected from the remaining patient cohort. Results We identified a total of 1410 patients with a histologic diagnosis of urothelial carcinoma, 9 (0.6%) of whom met our inclusion criteria. These 9 patients had a median age of 63 years. At their presentation with leukocytosis, all 9 had muscle-invasive disease and 5 had evidence of metastatic disease. Leukocytosis was frequently associated with hypercalcemia (n = 6), thrombocytosis (n = 5), and anemia (n = 9). Chemotherapy was able to achieve a WBC response in 3 of 5 patients, although only 1 patient demonstrated a substantial reduction in tumor volume radiographically. Extirpative surgery was able to provide a response in the laboratory parameters in 2 of 4 patients. However, all studied patients ultimately developed leukocytosis recurrence after systemic or local therapy and experienced rapid disease progression, with a median interval from leukocytosis until death of 71 days. Conclusion Paraneoplastic leukocytosis in the setting of urothelial carcinoma is a rare phenomenon but confers a poor prognosis, with a rapid progression to death.

While androgen deprivation therapy (ADT) remains the primary treatment for metastatic prostate cancer (PCa) since the seminal recognition of the disease as androgen-dependent by Huggins and Hodges in 1941, therapy is uniformly marked by progression to castration-resistant prostate cancer (CRPC) over a period of about 18 months, with an ensuing median survival of 1 to 2 years. Importantly, castration does not eliminate androgens from the prostate tumor microenvironment. Castration resistant tumors are characterized by elevated tumor androgens that are well within the range capable of activating the AR and AR-mediated gene expression, and by steroid enzyme alterations which may potentiate de novo androgen synthesis or utilization of circulating adrenal androgens. The dependence of CRPC on intratumoral androgen metabolism has been modeled in vitro and in vivo, and residual intratumoral androgens are implicated in nearly every mechanism by which AR-mediated signaling promotes castration-resistant disease. These observations suggest that tissue based alterations in steroid metabolism contribute to the development of CRPC and underscore these metabolic pathways as critical targets of therapy. Herein, we review the accumulated body of evidence which strongly supports intracrine (tumoral) androgen synthesis as an important mechanism underlying PCa progression. We first discuss the presence and significance of residual prostate tumor androgens in the progression of CRPC. We review the classical and non-classical pathways of androgen metabolism, and how dysregulated expression of these enzymes is likely to potentiate tumor androgen production in the progression to CRPC. Next we review the in vitro and in vivo data in human tumors, xenografts, and cell line models which demonstrate the capacity of prostate tumors to utilize cholesterol and adrenal androgens in the production of testosterone (T) and dihydrotestosterone (DHT), and briefly review the potential role of exogenous influences on this process. Finally, we discuss the emerging data regarding mechanisms of response and resistance to potent ligand synthesis inhibitors entering clinical practice, and conclude by discussing the implications of these findings for future therapy.

TMPRSS2 is an androgen-regulated cell-surface serine protease expressed predominantly in prostate epithelium. TMPRSS2 is expressed highly in localized high-grade prostate cancers and in the majority of human prostate cancer metastases. Through the generation of mouse models with a targeted deletion of Tmprss2, we demonstrate that the activity of this protease regulates cancer cell invasion and metastasis to distant organs. By screening combinatorial peptide libraries, we identified a spectrum of TMPRSS2 substrates that include pro-hepatocyte growth factor (HGF). HGF activated by TMPRSS2 promoted c-MET receptor tyrosine kinase signaling, and initiated a proinvasive epithelial-to-mesenchymal transition phenotype. Chemical library screens identified a potent bioavailable TMPRSS2 inhibitor that suppressed prostate cancer metastasis in vivo. Together, these findings provide a mechanistic link between androgen-regulated signaling programs and prostate cancer metastasis that operate via context-dependent interactions with extracellular constituents of the tumor microenvironment. Significance: The vast majority of prostate cancer deaths are due to metastasis. Loss of TMPRSS2 activity dramatically attenuated the metastatic phenotype through mechanisms involving the HGF–c-MET axis. Therapeutic approaches directed toward inhibiting TMPRSS2 may reduce the incidence or progression of metastasis in patients with prostate cancer. Cancer Discov; 4(11); 1310–25. ©2014 AACR. See related commentary by Rubin, p. 1262 This article is highlighted in the In This Issue feature, p. 1243

While androgen deprivation therapy remains the primary treatment modality for patients with metastatic prostate cancer, treatment is uniformly marked by progression to castration resistant prostate cancer (CRPC). Abiraterone is the first new drug to enter clinical practice in a series of novel agents designed to potently target adrenal and tumor androgen production.Herein, we review the mechanism of action of abiraterone and the phase III data supporting its approval for patients with metastatic CRPC. We discuss practical treatment considerations, including the incidence and management of side effect and monitoring requirements, and conclude by discussing future directions in the use of abiraterone, including early data supporting an expanded role for abiraterone in castration sensitive disease.Accumulating data emphasize that "androgen independent" or "hormone refractory" tumors remain sensitive to hormonal activation and suggest that despite suppression of circulating testosterone (T), residual tumor androgens play a prominent role in mediating CRPC progression.Accordingly, therapeutic strategies such abiraterone that more effectively target production of intratumoral androgens are necessary.

Surfactant protein (SP) D functions as a soluble pattern recognition molecule to mediate the clearance of pathogens by phagocytes in the innate immune response. We hypothesize that SP-D may also interact with dendritic cells, the most potent antigen presenting cell, to enhance uptake and presentation of bacterial antigens. Using mouse bone marrow-derived dendritic cells, we show that SP-D binds to immature dendritic cells in a dose-, carbohydrate-, and calcium-dependent manner, whereas SP-D binding to mature dendritic cells is reduced. SP-D also binds to Escherichia coli HB101 and enhances its association with dendritic cells. Additionally, SP-D enhances the antigen presentation of an ovalbumin fusion protein expressed in E. coli HB101 to ovalbumin-specific major histocompatibility complex class II T cell hybridomas. The enhancement of antigen presentation by SP-D is dose dependent and is not shared by other collectin-like proteins tested. These studies demonstrate that SP-D augments antigen presentation by dendritic cells and suggest that innate immune molecules such as SP-D may help initiate an adaptive immune response for the purpose of resolving an infection.

We read with interest the report by Hofland and colleagues evaluating the expression of steroidogenic enzymes in castration-recurrent prostate cancer (CRPC; ref. [1][1]). Their analysis of cell lines, xenografts, normal prostate tissue, lymph node metastases, and locally recurrent CRPC tissues