Androgen-targeted Therapies Research Articles

Evaluating Changes in Immune Function and Bone Microenvironment During Radium-223 Treatment of Patients with Castration-Resistant Prostate Cancer.

The effects of radium-223 on the immune system and the bone tumor microenvironment are incompletely understood. The authors describe mechanisms by which radium-223 may interact with the immune system, specifically through STAT-3 and impact on tumor and circulating lymphocyte populations. They review mechanisms through which effects of radium-223 and androgen-targeted therapy on bone microenvironment could be better elucidated. These knowledge gaps currently limit development of optimal combination therapy approaches for radium-223. Tissue based studies are currently underway in a prospective clinical trial to enhance therapeutic perspective on radium-223 treatment in the prostate cancer landscape.

Transient Sox9 Expression Facilitates Resistance to Androgen-Targeted Therapy in Prostate Cancer.

Patients with metastatic prostate cancer are increasingly presenting with treatment-resistant, androgen receptor-negative/low (AR-/Low) tumors, with or without neuroendocrine characteristics, in processes attributed to tumor cell plasticity. This plasticity has been modeled by Rb1/p53 knockdown/knockout and is accompanied by overexpression of the pluripotency factor, Sox2. Here, we explore the role of the developmental transcription factor Sox9 in the process of prostate cancer therapy response and tumor progression. Unique prostate cancer cell models that capture AR-/Low stem cell-like intermediates were analyzed for features of plasticity and the functional role of Sox9. Human prostate cancer xenografts and tissue microarrays were evaluated for temporal alterations in Sox9 expression. The role of NF-κB pathway activity in Sox9 overexpression was explored. Prostate cancer stem cell-like intermediates have reduced Rb1 and p53 protein expression and overexpress Sox2 as well as Sox9. Sox9 was required for spheroid growth, and overexpression increased invasiveness and neural features of prostate cancer cells. Sox9 was transiently upregulated in castration-induced progression of prostate cancer xenografts and was specifically overexpressed in neoadjuvant hormone therapy (NHT)-treated patient tumors. High Sox9 expression in NHT-treated patients predicts biochemical recurrence. Finally, we link Sox9 induction to NF-κB dimer activation in prostate cancer cells. Developmentally reprogrammed prostate cancer cell models recapitulate features of clinically advanced prostate tumors, including downregulated Rb1/p53 and overexpression of Sox2 with Sox9. Sox9 is a marker of a transitional state that identifies prostate cancer cells under the stress of therapeutic assault and facilitates progression to therapy resistance. Its expression may index the relative activity of the NF-κB pathway.

Evaluation of potential surrogate endpoints for prediction of overall survival in patients with castration-resistant prostate cancer: trial-level meta-analysis.

Overall survival (OS) has traditionally been the primary endpoint to evaluate drug efficiency in oncology but is often limited by the long observation period and high cost. The aims of this study were to perform a comprehensive meta-analysis at a clinical trial level to investigate the potential surrogate endpoints of OS in patients with castration-resistant prostate cancer (CRPC), and to predict OS based on the relationships associated with the potential surrogate endpoints. A systematic literature search was conducted in the PubMed database up to August 2018. Correlations between OS and potential surrogate endpoints were determined by linear regression analysis weighted by the square roots of sample size. Simulations were conducted to assess the effect of covariates on the relationships between OS and surrogate endpoints. A total of 233 studies including clinical trials and real-world data were included in our dataset. The correlations between median OS and potential surrogate endpoints for androgen-targeting therapy (R2 = 0.58-0.92) were generally stronger than those for taxane chemotherapy (R2 = 0.37-0.71). Median radiographic progression-free survival (rPFS) showed the strongest correlations with median OS (R2 = 0.94) in patients treated with novel androgen-targeting therapy. The meta-analysis demonstrated that rPFS might serve as a potential surrogate endpoint of OS and offer opportunity to facilitate the interim analyses and decision-making during the early stage of clinical trials for androgen-targeting agents.

Limited short-term effects on human prostate cancer xenograft growth and epidermal growth factor receptor gene expression by the ghrelin receptor antagonist [D-Lys3]-GHRP-6.

The ghrelin axis regulates many physiological functions (including appetite, metabolism, and energy balance) and plays a role in disease processes. As ghrelin stimulates prostate cancer proliferation, the ghrelin receptor antagonist [D-Lys3]-GHRP-6 is a potential treatment for castrate-resistant prostate cancer and for preventing the metabolic consequences of androgen-targeted therapies. We therefore explored the effect of [D-Lys3]-GHRP-6 on PC3 prostate cancer xenograft growth. NOD/SCID mice with PC3 prostate cancer xenografts were administered 20 nmoles/mouse [D-Lys3]-GHRP-6 daily by intraperitoneal injection for 14 days and tumour volume and weight were measured. RNA sequencing of tumours was conducted to investigate expression changes following [D-Lys3]-GHRP-6 treatment. A second experiment, extending treatment time to 18 days and including a higher dose of [D-Lys3]-GHRP-6 (200 nmoles/mouse/day), was undertaken to ensure repeatability. We demonstrate here that daily intraperitoneal injection of 20 nmoles/mouse [D-Lys3]-GHRP-6 reduces PC3 prostate cancer xenograft tumour volume and weight in NOD/SCID mice at two weeks post treatment initiation. RNA-sequencing revealed reduced expression of epidermal growth factor receptor (EGFR) in these tumours. Further experiments demonstrated that the effects of [D-Lys3]-GHRP-6 are transitory and lost after 18 days of treatment. We show that [D-Lys3]-GHRP-6 has transitory effects on prostate xenograft tumours in mice, which rapidly develop an apparent resistance to the antagonist. Although further studies on [D-Lys3]-GHRP-6 are warranted, we suggest that daily treatment with the antagonist is not a suitable treatment for advanced prostate cancer.

Androgen-targeted therapy in men with prostate cancer: evolving practice and future considerations

BackgroundAndrogen deprivation therapy (ADT) is foundational in the management of advanced prostate cancer (PCa) and has benefitted from a recent explosion in scientific advances. These include approval of new therapies that suppress testosterone (T) levels or inactivate its function, improvements in diagnostic and assay technologies, identification of lower therapeutic targets for T, discovery of the relevance of germline genetic mutations and identification of the benefits of sequential and combination therapies.MethodsThis review discusses the clinical profiles of the most up-to-date options for ADT, best practices for managing patients with advanced PCa and future directions in therapy.Results and conclusionsModern assay technologies reveal that bilateral orchiectomy results in a serum T level of approximately 15 ng/dL as compared to the historical definition of castration of T < 50 ng/dL. Evidence shows that lowering T levels to <20 ng/dL improves patient survival and delays disease progression. Routine monitoring of T in addition to prostate-specific antigen throughout treatment is important to ensure continuing efficacy of T suppression. New drugs that inhibit androgen signaling in combination with traditional ADT suppress T activity to near zero and have significantly improved patient survival. When personalizing ADT regimens physicians should consider a number of factors including initiation and duration of ADT, monitoring of T levels and PSA, the possibility of switching monotherapies if a patient does not achieve adequate T suppression, and consideration of intermittent vs. continuous ADT according to patients’ lifestyles, comorbidities, risk factors and tolerance to treatment.

Sequencing of Taxanes and New Androgen-targeted Therapies in Metastatic Castration-resistant Prostate Cancer: Results of the International Multicentre Retrospective CATS Database

BackgroundThe optimal sequence of life-extending therapies in metastatic castration-resistant prostate cancer (mCRPC) is unknown. ObjectiveTo evaluate outcomes among mCRPC patients treated with docetaxel (DOC), cabazitaxel (CABA), and a novel androgen receptor–targeted agent (ART; abiraterone acetate or enzalutamide) according to three different sequences. Design, setting, and participantsData from 669 consecutive mCRPC patients were retrospectively collected between November 2012 and October 2016. Outcome measurements and statistical analysisThe primary endpoint was the prostate-specific antigen (PSA) response (decrease ≥50% from baseline) to each therapy. Secondary endpoints included best clinical benefit, time to PSA progression, radiological progression-free survival (rPFS), overall survival (OS), and toxicity. Results and limitationsA total of 158 patients received DOC→CABA→ART (group 1), 456 received DOC→ART→CABA (group 2), and 55 received ART→DOC→CABA (group 3). At baseline, PSA progression only and Gleason <8 were more common in group 3. PSA response on DOC was lower in group 3 than in other groups (p=0.02) and PSA response on CABA was higher in the second than in the third line (p=0.001). In Group 3, rPFS on ART (6.6 mo) and DOC (9.2 mo) was also shorter than in the other groups. OS calculated from the first life-extending therapy reached 34.8, 35.8, and 28.9 mo in groups 1, 2 and 3, respectively (p=0.007). Toxicity was comparable between the arms. The main limitations of the trial are its retrospective design and the low number of patients in group 3. ConclusionsIn this retrospective trial, sequencing of DOC, CABA, and one ART, was associated with median OS of up to 35.8 mo. CABA seemed to retain its activity regardless of treatment sequence. DOC activity after ART appeared to be reduced, but the data are insufficient to conclude that cross-resistance occurs. Patient summaryThe order of drugs administered to patients with metastatic castration-resistant prostate cancer could impact their efficacy, with cabazitaxel appearing to retain its activity whatever the therapeutic sequence.

Neoadjuvant enzalutamide and androgen deprivation therapy for high-risk prostate cancer: Early results from a feasibility trial.

94 Background: Intensive androgen-targeted therapy improves survival for patients with androgen naive metastatic prostate cancer. Neoadjuvant androgen deprivation therapy (nADT) with abiraterone for localized disease leads to complete responses (pCR) rates of up to 10%. In this study we tested the efficacy of nADT with enzalutamide for patients with high risk, localized disease and the ability of post-treatment multiparametric MRI (mpMRI) to evaluate response. Methods: A single institution trial (NCT02430480) evaluated nADT and enzalutamide 160 mg daily in patients with high risk non-metastatic prostate cancer. Patients underwent MRI-TRUS-guided fusion biopsy at screening and repeat mpMRI followed by radical prostatectomy (RP) after completing six months of therapy. Results: Twenty patients have completed therapy and undergone radical prostatectomy. Clinical characteristics were typical of those with high risk disease: median PSA 8.96 ng/ml (range 2.07-985.9) and clinical stage by mpMRI cT2 (one patient), cT3 (17 patients), or cT4 (2 patients); 7 patients had enlarged pelvic lymph nodes. Following 6 months of therapy, median pre-RP PSA was &lt; 0.02 ng/ml (range &lt; 0.02 – 0.35). On final pathology, 0 patients were upstaged, 5 (25%) were unchanged, and 15 (75%) were downstaged, including 3 (16%) pCR. Four patients were downstaged from N1 to N0. There was little correlation between post-treatment mpMRI tumor volume and pathologic tumor volume. Two patients had radiographic complete responses, one of whom had extensive residual disease. Conclusions: Six months of nADT with enzalutamide has activity in high risk, localized prostate cancer, with a small number of patients having exceptional responses. Standard analysis of mpMRI does not reliably characterize depth of response. Evaluation of molecular characteristics that predict exceptional response or intrinsic resistance is on-going. Patients are being followed for recurrence. Clinical trial information: NCT02430480.

Abstract 818: Unravelling the role of androgen targeted therapies in epithelial plasticity during prostate cancer progression

Abstract Recent evidence suggests that the use of potent androgen receptor (AR) antagonists have contributed to the increased prevalence of prostate cancer (PCa) tumors exhibiting therapy resistance. Androgen targeted therapies (ATT) inhibit the androgen/AR axis which PCa cells rely on for growth. ATT are initially effective however most men progress to metastatic castration resistant prostate cancer (mCRPC). Mechanisms of resistance include programs of epithelial plasticity (EP) such as epithelial-to-mesenchymal transition (EMT) and neuroendocrine transdifferentiation (NEtD). EMT enables PCa cells to become more motile and invasive. PCa cells that undergo NEtD gain neuroendocrine (NE)-like traits. Patients with NE prostate cancer (NEPC) usually present with visceral metastasis, are refractory to ATT and have short response to chemotherapies. We aim to characterize models of therapy-induced NEtD (tNEPC) and to investigate the role of tumor plasticity in the progression to mCRPC. To recapitulate ATT in vitro, androgen-dependent LNCaP cells were treated with either enzalutamide (Enz) (10μM) or shRNA targeting AR in culture medium containing fetal bovine serum (FBS) or charcoal stripped serum (CSS) with or without 10nM dihydrotestosterone (DHT). To model EMT, LNCaP cells were engineered to express doxycycline (Dox) inducible overexpression of EMT transcription factor Snail. Androgen deprivation in vitro induced expression of NE markers in LNCaP cells compared with FBS treated cells (mean fold increase ± SEM): neuron specific enolase (ENO2) (6.0 ± 2.5), neural cell adhesion molecule (NCAM) (10.4 ± 6.8) and midkine (MDK) (13.2 ± 2.4). Cells co-treated with Enz and DHT had increased expression of NE markers compared to DHT alone: ENO2 (2.7 ± 0.6), NCAM (2.9 ± 0.3) and MDK (2.3 ± 1.8). AR knockdown resulted in a potent induction of NE markers compared with control cells: ENO2 (8.3 ± 1.4), NCAM (8.3 ± 2.4) and MDK (9.0 ± 2.2). Dox-induced Snail enhanced androgen deprivation-mediated upregulation of NE markers compared with no-Dox cells: ENO2 (1.3 ± 0.1), NCAM (8.3 ± 2.4) and MDK (9.0 ± 2.2). To more accurately model a preclinical setting, LNCaP tumors were grown subcutaneously in severe combined immune deficiency mice. Once tumors grew to 200mm3, mice were castrated and upon tumor recurrence mice were treated with Enz (10mg/kg). At the ethical endpoint, expression of EMT and NE markers will be assessed. Patients treated with ATTs show an increase in NE-like traits. Thus, there is a need to identify molecular mechanisms involved in this adaptive response. In vitro, we have modelled the acquisition of NE-like features in PCa cells using ATTs, EMT drivers and AR knockdown. Further investigation using these models may lead to the development of biomarkers and therapeutic targets for the clinical management of advanced PCa. Citation Format: Ellca Ratther, Katrina G. Sweeney, Nataly Stylianou, Qiuhua Hu, Gregor Tevz, Colleen C. Nelson, Elizabeth D. Williams, Brett G. Hollier. Unravelling the role of androgen targeted therapies in epithelial plasticity during prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 818. doi:10.1158/1538-7445.AM2017-818

Abstract 2855: Monitoring tumor growth and oxygen distribution of prostate cancer xenografts in vivo using ultrasound and photoacoustic imaging

Abstract Introduction Prostate cancer (PCa) is the second leading cause of cancer-related death in men. Initially, PCa growth and progression is primarily driven by androgen stimulation of the androgen receptor (AR). As such, the standard treatments for PCa target the androgen/AR axis and are collectively termed androgen targeted therapies (ATTs). In spite of initial tumor regression, tumor cells ultimately adapt and become resistant to ATTs and progress to castration resistant PCa (CRPC) or metastatic CRPC. The tumor microenvironment (TME) has long been recognized to modulate the response of tumors to therapies, however, the impact of ATTs on the TME and their contribution to the development of resistance has not been investigated. This project aims to evaluate the potential of non-invasive photoacoustic imaging combined with ultrasound imaging to assess the alterations of the TME in response to ATTs in vivo. Methods PCa cell lines, PC-3 (androgen-independent) and LNCaP (androgen-sensitive), were subcutaneously implanted into male severe combined immune deficiency mice. When the tumor size reached 200-300 mm3, mice were castrated and subsequently treated with enzalutamide once serum PSA had reached pre-castration levels. Ultrasound and photoacoustic (US-PA) scanning (Vevo LAZR) was performed at intervals during PCa progression to simultaneously assess changes in tumor growth, anatomical structure, vasculature, and intratumoral oxygen saturation. Results We provide evidence that US-PA imaging is a feasible approach to monitor TME and the response of subcutaneous prostate tumors to ATTs in vivo. PC-3 tumors (nPC-3=11) showed no response to castration in volume, but slight changes in total hemoglobin and oxygen saturation. Growth of LNCaP tumors and their subsequent responses to castration were variable (nLNCaP=10). We identified a strong correlation between ultrasound and caliper-derived tumor volumes in these models and in the majority of tumors the tumor center was more hypoxic than the peripheral tumor area, which is likely due to low oxygen and nutrient transmission and/or cell apoptosis. However, in a subset of tumors higher oxygen levels were observed in the center compared with regions nearer to the skin surface, a pattern associated with a higher rate of tumor growth compared to the rest of the cohort. Importantly, we were able to detect a relatively increase in oxygen levels between pre- and post-castration tumors in individual mice. Conclusion We have successfully used a non-invasive imaging method to simultaneously assess multiple biological parameters in PCa xenografts in vivo. The information gained in this study will provide a broader view of the prostate tumor response to ATTs, which has the potential to reveal novel mechanisms of therapy resistance. Citation Format: Qiuhua Hu, Ellca Ratther, Katrina Sweeney, Pamela J. Russell, Colleen C. Nelson, Brian W. Tse, Elizabeth D. Williams, Brett G. Hollier. Monitoring tumor growth and oxygen distribution of prostate cancer xenografts in vivo using ultrasound and photoacoustic imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2855. doi:10.1158/1538-7445.AM2017-2855

Abstract 4908: Neuropilin-1 is up-regulated in the adaptive response of prostate tumors to androgen targeted therapies and is prognostic of metastatic progression and patient mortality

Abstract Aims: Androgen-targeted therapies (ATTs) are the mainstay treatment for metastatic prostate cancer (PCa). However, ATTs promote adaptation of tumour cells and lead to castration resistant disease (CRPC). We have recently identified the cell surface receptor, Neuropilin-1 (NRP1) as increased during EMT and in CRPC. However, the role of NRP1 in the prostate epithelium is poorly understood. This study aims to determine whether the inhibition of NRP1 will be a feasible therapeutic strategy for blocking PCa metastasis and therapy resistance. Methods: qPCR and western blotting were used to assess NRP1 expression in PCa cell lines. NRP1 expression in CRPC was assessed using a murine LNCaP xenograft model of castration. NRP1 was knocked down with shRNA sequences from the pLKO.1 lentiviral construct. For metastasis assays, PC3 cells were microinjected into the zebrafish yolk sac and metastatic dissemination imaged 5 days later. NRP1 expression in radical prostatectomy (RP) samples from Mayo Clinic (545 patients) and Johns Hopkins Medical Institutions (JHMI; 188 patients) cohorts was quantified by Affymetrix exon arrays and multivariable analysis performed. Wound scratch migration and invasion assays were performed with the WoundMaker™ tool and IncuCyte™ FLR imaging systems. Results: NRP1 levels were elevated in humanCRPC xenografts, metastatic and castrate resistant clinical PCa samples (p &amp;lt;0.0001), and PCa cell lines. NRP1 suppression significantly reduced metastasis of human xenografts in zebrafish and the migratory and invasive behaviour of metastatic PCa cells (p=0.0002). Multivariable analysis identified NRP1 as a significant independent prognostic indicator of metastasis and prostate cancer specific mortality in two large clinical cohorts (Mayo Clinic and JHMI; p=0.008/0.048 and 0.013/0,034 respectively). We show that NRP1 knockdown promotes E-Cadherin expression and loss of vimentin in mesenchymal PCa cells. Conclusion: These results will provide the preclinical data necessary to rationalise the use of anti-NRP1 directed adjuvant therapies for clinical use in PCa patients receiving ATTs, and will pave the way for larger scale preclinical and clinical trials in the PCa setting. Citation Format: Marianna Volpert, Brian Tse, Ellca Ratther, Nataly Stylianou, Mannan Nouri, Melanie Lehman, Stephen McPherson, Mani Roshan-Moniri, Mandeep Takhar, Nicholas Erho, Mohamed Alshalafa, Elai Davicioni, Robert Jenkins, Ashley Ross, Jeffrey Karnes, Robert Den, Ladan Fazli, Martin Gleave, Elizabeth Williams, Paul Rennie, Ralph Buttyan, Pamela Russell, Colleen Nelson, Brett Hollier. Neuropilin-1 is up-regulated in the adaptive response of prostate tumors to androgen targeted therapies and is prognostic of metastatic progression and patient mortality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4908. doi:10.1158/1538-7445.AM2017-4908

Abstract 4909: Androgen targeted therapy induces ZEB1 expression and is associated with suppression of androgen signalling and therapy resistance

Abstract Reactivation of the embryonic developmental pathway, epithelial-to-mesenchymal transition (EMT) is associated with prostate cancer (PCa) metastasis and therapy resistance. Recent evidence has demonstrated EMT is stimulated following androgen targeted therapy (ATT). In the present study we investigated the role of EMT transcription factor ZEB1 in ATT-driven EMT, PCa metastasis and drug resistance. Immunohistochemistry (IHC) staining of ZEB1 on tissue microarrays of primary tumors from clinical samples demonstrated ZEB1 expression correlated with increased tumor aggressiveness and Gleason score. Upon stratification of patient data (n=198), high ZEB1 protein expression correlated with a shorter time to biochemical recurrence (BCR). Furthermore, IHC staining of primary tumours from 148 treatment-naïve patients with and without metastasis demonstrated high ZEB1 levels and correlated to a reduced time to metastasis. To delineate the role of ZEB1 as a molecular driver of late-stage PCa, microarray analysis of an in vivo LNCaP progression model demonstrated ZEB1 levels increased following castration. Significantly, high ZEB1 levels were associated with patients who had undergone neoadjuvant hormone therapy with or without docetaxel and high ZEB1 levels were associated with a shorter time to BCR and metastasis. Inhibition of the androgen/androgen receptor (AR) axis in LNCaP cells using antiandrogen treatment or shRNA targeting AR resulted in upregulation of ZEB1 in LNCaP cells suggesting enhanced levels of ZEB1 may drive the early adaptive response of PCa after hormone therapy. Interestingly, in models where ZEB1 expression was elevated this was accompanied with repression of classical androgen-regulated genes suggesting ZEB1 is able to regulate the AR transcriptional pathway. We also investigated the role of ZEB1 in cancer invasion and chemoresistance. An inducible model of ZEB1 overexpression in LNCaP cells produced a robust EMT upon ZEB1 expression and was accompanied by an invasive phenotype in 3D cultures. ZEB1 overexpression also conferred resistance to docetaxel (IC50 of 8.17±2.45nM in ZEB1-expressing cells vs. 3.35±0.23nM in control cells) potentially as a result of a reduced apoptotic cell death response mediated by ZEB1. At suboptimal doses of docetaxel, the percentage of apoptotic cells (annexin-V+/propidium iodide-) decreased 4-fold when ZEB1 was expressed compared with control cells and was accompanied by a reduction in PARP cleavage and cleaved caspase-7 expression. In summary, we provide evidence that ZEB1 expression is increased in response to ATTs and correlates with disease progression, metastasis and therapy resistance. We also show ZEB1 is a transcriptional regulator of AR signalling in PCa, Together this provides the rationale to target ZEB1 for the development of novel therapies for the treatment of CRPC. Note: This abstract was not presented at the meeting. Citation Format: Katrina G. Sweeney, Nataly Stylianou, Gregor Tevz, Atefeh Taherianfard, Katrina Pirlo, Akanksha Upadhyaya, Ellca Ratther, Melanie Lehman, Martin Gleave, Jennifer Gunter, Elizabeth D. Williams, Colleen C. Nelson, Brett Hollier. Androgen targeted therapy induces ZEB1 expression and is associated with suppression of androgen signalling and therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4909. doi:10.1158/1538-7445.AM2017-4909

Abstract 851: Reversion of epithelial to mesenchymal transition under androgen deprived conditions promotes chemoresistance in prostate cancer

Abstract Introduction: Metastatic prostate cancer (PC) is one of the leading causes of cancer related male deaths in Australia. Radical prostatectomy followed by androgen deprivation therapy (ADT) upon recurrence of disease is the conventional form of treatment. Upon relapse, administration of androgen targeted therapy (ATT) and subsequent chemotherapy improves quality of life and overall survival of patients. However, it is not curative and the disease ultimately progresses. Epithelial to mesenchymal transition (EMT) is a process that can facilitate tumour growth and progression to metastasis and has also been associated with chemoresistance. We therefore aim to determine how EMT and resultant reversal of this state (MET) influences the chemoresistance profile of PC cells and further determine if and how specific genetic regulators are involved in this process. Methods: Doxycycline hyclate (Dox) inducible Snai1 and Zeb1, LNCaP EMT models were used to determine the effect of the EMT status in response to a clinically relevant panel of drugs using cellular and molecular assays. All assays were performed in both fetal bovine serum (FBS) and charcoal stripped serum (CSS) supplemented culture media. CSS media was used to mimic an ADT-like environment. Using 2D monolayer and 3D spheroid [Happy Cell©] formats. Cells were induced for 5 days of EMT in the presence of Dox and were allowed to revert for 14 days of MET (absence of Dox) before the drug assays were carried out over a period of 72 hours. Live/dead cell staining was used to determine viability percentage in both models. Flow cytometry was used for 2D assays and 3D assays were analysed using either a GE IN Cell Analyzer live cell imaging system or confocal microscopy. Results: Induction of Snai1 and Zeb1 induced an elongated cell shape and increased mRNA and protein expression of mesenchymal markers such as vimentin during EMT. This was associated with a decrease in expression of epithelial markers E-cadherin and EpCam,. An eventual drop in the expression levels of vimentin at around 14 days of dox removal along with an increase in epithelial markers was observed. These changes were also associated with a gradual reversal in the phenotype of these cells. However, this reversal in phenotype and expression of EMT markers was not as striking in cells grown in CSS compared to cells in FBS media (both 2D and 3D). Mitoxantrone concentration-response assays in a 2D format showed that cells that undergo a subsequent MET are more chemoresistant than uninduced or EMT cells when grown in CSS media. This was however, not observed in cells undergoing similar transitions in the FBS media. Conclusions: The chemoresistance profile of prostate cancer cells is altered by their EMT status. This effect is enhanced in the context of ADT. Cells that revert back to a more epithelial phenotype after undergoing an EMT exhibit higher resistance towards chemotherapy drugs. Citation Format: Akanksha Upadhyaya, Nataly Stylianou, Anthony M. Davies, Sarah-Louise Ryan, Brett G. Hollier, Elizabeth D. Williams. Reversion of epithelial to mesenchymal transition under androgen deprived conditions promotes chemoresistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 851. doi:10.1158/1538-7445.AM2017-851

Abstract 3623: Preclinical efficacy of the selective GR antagonist, CORT125134

Abstract The role of activation of the glucocorticoid receptor (GR) in the response of several solid tumor types to chemotherapy has been reported by several groups. Skor et al1 have demonstrated that the non-selective GR antagonist mifepristone can potentiate the effect of paclitaxel in a mouse xenograft derived from a human triple negative breast cancer (TNBC) cell line. GR activation is thought to play a role in the regulation of pro- and anti-apoptotic genes. Isikbay et al2 have demonstrated the benefits of combining androgen deprivation therapy with GR antagonism in prostate cancer xenograft model. Signaling via GR appears to be able to compensate for lack of androgen receptor signaling, and to contribute to castrate resistance. CORT125134 is a novel, selective GR antagonist being developed for the treatment of a variety of solid tumors, in combination with an appropriate standard of care agent. CORT125134 is currently being studied in a Phase1/2 study in solid tumors, in combination with nab-paclitaxel. The efficacy of CORT125134 in combination with standard of care agents has been investigated in mouse xenograft models of several tumor types. Previous reports of the benefit of adding a GR antagonist to chemotherapy in a TNBC model and an ovarian cancer model have been confirmed. An enhancement of the anti-tumor effect of castration by the addition of a GR antagonist has been confirmed in a model of prostate cancer. In addition, CORT125134 has been shown to provide a significant improvement in the efficacy of paclitaxel in a pancreatic cancer model. In a MIAPaca-2 xenograft model, paclitaxel was given i.v. every 4 days either alone or in combination with CORT125134. The combination provided significantly better tumor group inhibition than paclitaxel alone (p&amp;lt;0.0001). These encouraging results in a pancreatic cancer model extend the potential utility of GR antagonism in a variety of solid tumor types. In the TNBC model, alternative dosing regimens have been investigated, and suggest that stress during dosing is a confounding factor in these studies. 1. Skor MN, Wonder E, Kocherginsky M, et al. Glucocorticoid receptor antagonism as a novel therapy for triple negative breast cancer. Clinical Cancer Res. 2013;19(22):6163-6172. 2. Isikbay M, Otto K, Kregel S, et al. Glucocorticoid receptor activity contributes to resistance to androgen-targeted therapy in prostate cancer. Horm Cancer. 2014;5(2):72-89. Citation Format: Hazel Hunt, Joseph Belanoff, Thad Block, Stacie P. Shepherd. Preclinical efficacy of the selective GR antagonist, CORT125134 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3623. doi:10.1158/1538-7445.AM2017-3623

Evaluating the impact of bone-targeted agents in the era of novel androgen targeted therapy for metastatic castration-resistant prostate cancer.

e16500 Background: Patients with metastatic castrate resistant prostate cancer (mCRPC) often develop bone metastases, resulting in a risk of symptomatic skeletal events (SSE). Bone-targeted agent (BTA) has been integrated into the overall treatment strategy, but its role in the era of novel androgen-targeted therapy (ATT) is still unclear. Methods: A retrospective analysis of real-world practice from 2010 to 2015 was conducted. Patients diagnosed with mCRPC and bone metastases who received systemic therapy (docetaxel/abiraterone/enzalutamide) with or without BTA (zoledronic acid/denosumab/alendronate) were included. Results: We obtained data from 299 patients with a median follow-up of 75.5 months. Compared with no BTA, concomitant BTA was associated with decreased incident of SSE (3.8% vs. 19.2%, p&lt; 0.001), especially in the need for bone radiation (1.9% vs. 15.8%, p&lt; 0.001). Compared to patients without SSE, patients with SSE were more likely to have previous fracture history (10.8% vs. 9.7%, p= 0.047), steroid use history (2.4% vs. 1.4%, p= 0.019), smoking history (53.0% vs. 38.9%, p= 0.009), diabetes (19.3% vs. 8.8%, p= 0.012) and reduced baseline mobility (9.6% vs. 4.2%, p= 0.002). Among those who received first-line novel ATT (n = 152), concurrent ATT+BTA significantly decreased the incident of SSE (2.8% vs. 27.3%, p= 0.004) and prolonged the time to SSE (24.3 vs. 3.2 mo, p&lt; 0.001), when compared to ATT alone. But there was no difference in overall survival between two groups (134.2 vs. 138.9 mo, p= 0.99). In patients who responded to systemic treatment with PSA declining, the SSE rate (2.4% vs. 14.3%, p= 0.037) was lower and the time to SSE was longer (49.6 vs. 32.3 mo, p= 0.026) than those who did not respond. Smoking history (adjusted HR = 1.46, p= 0.013) and PSA response (adjusted HR = 0.19, p&lt; 0.001) are independent risk factors for SSE. Conclusions: This real-world database suggests that concomitant BTA with novel ATT was associated with reducing SSEs. PSA decrease might be used as a predictive factor for those under BTA treatment. Further studies are warranted to validate the role of PSA response.

Neuropilin-1 is upregulated in the adaptive response of prostate tumors to androgen-targeted therapies and is prognostic of metastatic progression and patient mortality

Recent evidence has implicated the transmembrane co-receptor neuropilin-1 (NRP1) in cancer progression. Primarily known as a regulator of neuronal guidance and angiogenesis, NRP1 is also expressed in multiple human malignancies, where it promotes tumor angiogenesis. However, non-angiogenic roles of NRP1 in tumor progression remain poorly characterized. In this study, we define NRP1 as an androgen-repressed gene whose expression is elevated during the adaptation of prostate tumors to androgen-targeted therapies (ATTs), and subsequent progression to metastatic castration-resistant prostate cancer (mCRPC). Using short hairpin RNA (shRNA)-mediated suppression of NRP1, we demonstrate that NRP1 regulates the mesenchymal phenotype of mCRPC cell models and the invasive and metastatic dissemination of tumor cells in vivo. In patients, immunohistochemical staining of tissue microarrays and mRNA expression analyses revealed a positive association between NRP1 expression and increasing Gleason grade, pathological T score, positive lymph node status and primary therapy failure. Furthermore, multivariate analysis of several large clinical prostate cancer (PCa) cohorts identified NRP1 expression at radical prostatectomy as an independent prognostic biomarker of biochemical recurrence after radiation therapy, metastasis and cancer-specific mortality. This study identifies NRP1 for the first time as a novel androgen-suppressed gene upregulated during the adaptive response of prostate tumors to ATTs and a prognostic biomarker of clinical metastasis and lethal PCa.

Sequencing Treatment for Castration-Resistant Prostate Cancer.

Prostate cancer is the most common non-cutaneous cancer diagnosed in men and the second leading cause of male cancer deaths in the USA. While most cases are diagnosed in early stages, some will present as or progress to metastatic disease and eventually castration-resistant prostate cancer (mCRPC) which has a mortality rate exceeding 50%. There are currently six approved systemic life-prolonging therapies for use in mCRPC, yet little data to guide sequencing. Clinical factors, including the presence or absence of symptoms and the presence or absence of visceral metastases, should help determine the best therapeutic choice at each treatment node. Those with asymptomatic bone-only disease could be considered for sipuleucel-T, abiraterone, enzalutamide, or docetaxel in the first-line setting. For symptomatic disease, docetaxel could be used or radium-223 if disease is only present in the bone. In the second-line setting, sipuleucel-T or radium-223 can be used in the appropriate clinical setting. Taxane chemotherapy could be used if a novel androgen-directed therapy was used in the first-line setting. Cabazitaxel, if docetaxel was previously used, should be considered. There is scarce data on best treatment options in the third-line setting. In general, we recommend alternating between androgen-targeting agents and taxane chemotherapy. Finally, consideration should be given to testing for the androgen receptor splice variant AR-V7, which may be a relevant treatment-specific biomarker to aid in the selection of androgen-targeting therapy versus chemotherapy at each treatment juncture. Mutation testing for DNA damage repair defects can also be considered, as such patients may benefit from investigational poly ADP ribose polymerase(PARP) inhibitors or platinum-based chemotherapies. Several ongoing studies have been designed to answer some of these sequencing questions, including the biomarker questions, and will hopefully continue to inform us about rational therapy selection in mCRPC.

Non-Cell-Autonomous Regulation of Prostate Epithelial Homeostasis by Androgen Receptor

Prostate inflammation has been suggested as an etiology for benign prostatic hyperplasia (BPH). We show that decreased expression of the androgen receptor (AR) in luminal cells of human BPH specimens correlates with a higher degree of regional prostatic inflammation. However, the cause-and-effect relationship between the two events remains unclear. We investigated specifically whether attenuating AR activity in prostate luminal cells induces inflammation. Disrupting luminal cell AR signaling in mouse models promotes cytokine production cell-autonomously, impairs epithelial barrier function, and induces immune cell infiltration, which further augments local production of cytokines and chemokinesincluding Il-1 and Ccl2. This inflammatory microenvironment promotes AR-independent prostatic epithelial proliferation, which can be abolished by ablating IL-1 signaling or depleting its major cellular source, the macrophages. This study demonstrates that disrupting luminal AR signaling promotes prostate inflammation, which may serve as amechanism for resistance to androgen-targeted therapy for prostate-related diseases.

Abstract 2533: Interlukin-1 (IL-1) may induce prostate cancer (PCa) stem-like cells

Abstract Inflammation is implicated in prostate cancer initiation and progression. Our lab found that the inflammatory cytokine, interleukin-1 beta (IL-1β) concomitantly upregulates the pro-survival scaffold protein, Sequestome-1 (SQSTM1/p62) and represses androgen receptor accumulation in a relatively small cell subpopulation within various prostate cancer cell lines. Given that cancer stem cell subpopulations have been isolated from isogenic cell lines and given that prostate cancer stem cells are androgen receptor negative, we hypothesized that the IL-1β-responsive subpopulation might be stem-like cells. Cancer stem cells are significant in prostate cancer because they are resistant to chemo-radiation and androgen-targeted therapies and can contribute to the tumor cell pool through asymmetric division. Thus, we analyzed the mRNA and protein accumulation of several stem cell markers in various prostate cancer cells lines exposed to IL-1β. We also have experiments underway to analyze stem cell biology in order to determine the functional significance of the IL-1β-responsive subpopulation. Thus far, we have discovered that IL-1β can induce stem cell marker mRNA and protein accumulation, suggesting that IL-1β alters prostate cell fate and supports the notion that inflammation contributes to prostate cancer initiation and progression. Citation Format: Shayna Thomas, Sana Merchant, Rachel Meade, Afshan Fathima Nawas, Nikki Delk. Interlukin-1 (IL-1) may induce prostate cancer (PCa) stem-like cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2533.

Snail promotes resistance to enzalutamide through regulation of androgen receptor activity in prostate cancer.

Treatment with androgen-targeted therapies can induce upregulation of epithelial plasticity pathways. Epithelial plasticity is known to be important for metastatic dissemination and therapeutic resistance. The goal of this study is to elucidate the functional consequence of induced epithelial plasticity on AR regulation during disease progression to identify factors important for treatment-resistant and metastatic prostate cancer. We pinpoint the epithelial plasticity transcription factor, Snail, at the nexus of enzalutamide resistance and prostate cancer metastasis both in preclinical models of prostate cancer and in patients. In patients, Snail expression is associated with Gleason 9-10 high-risk disease and is strongly overexpressed in metastases as compared to localized prostate cancer. Snail expression is also elevated in enzalutamide-resistant prostate cancer cells compared to enzalutamide-sensitive cells, and downregulation of Snail re-sensitizes enzalutamide-resistant cells to enzalutamide. While activation of Snail increases migration and invasion, it is also capable of promoting enzalutamide resistance in enzalutamide-sensitive cells. This Snail-mediated enzalutamide resistance is a consequence of increased full-length AR and AR-V7 expression and nuclear localization. Downregulation of either full-length AR or AR-V7 re-sensitizes cells to enzalutamide in the presence of Snail, thus connecting Snail-induced enzalutamide resistance directly to AR biology. Finally, we demonstrate that Snail is capable of mediating-resistance through AR even in the absence of AR-V7. These findings imply that increased Snail expression during progression to metastatic disease may prime cells for resistance to AR-targeted therapies by promoting AR activity in prostate cancer.

AR-v7 protein expression is regulated by protein kinase and phosphatase.

Failure of androgen-targeted therapy and progression of castration-resistant prostate cancer (CRPC) are often attributed to sustained expression of the androgen receptor (AR) and its major splice variant, AR-v7. Although the new generation of anti-androgens such as enzalutamide effectively inhibits AR activity, accumulating pre-clinical and clinical evidence indicates that AR-v7 remains constitutively active in driving CRPC progression. However, molecular mechanisms which control AR-v7 protein expression remain unclear. We apply multiple prostate cancer cell models to demonstrate that enzalutamide induces differential activation of protein phosphatase-1 (PP-1) and Akt kinase depending on the gene context of cancer cells. The balance between PP-1 and Akt activation governs AR phosphorylation status and activation of the Mdm2 ubiquitin ligase. Mdm2 recognizes phosphorylated serine 213 of AR-v7, and induces AR-v7 ubiquitination and protein degradation. These findings highlight the decisive roles of PP-1 and Akt for AR-v7 protein expression and activities when AR is functionally blocked.