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Background: Mechanism of castration-resistant prostate cancer (CRPC) is still unclear. Our objective is to investigate the association between genes expression and CRPC through the genome-wide approach and functional researches. Methods: Differentially expressed genes (DEGs) between PCa and CRPC tissues were identified using expression profile obtained from Gene Expression Omnibus database (GEO). Survival analysis was performed using online database Gene Expression Profiling Interactive Analysis (GEPIA). Oncomine database was further used to explore the relationship between DEGs expression levels with clinical parameters. After in silico study, SEC14L2-knockdown CRPC cells and normal prostatic epithelial cells were used for in vitro study to verify its biological functions. Results: A total of 3 consistently changed DEGs (SEC14L2, DMD, SEL1L) were identified correlating with CRPC after cross validation in three independent datasets. Low expression of SEC14L2 was associated with poorer disease-free survival and higher Gleason score than normal/high expression of SEC14L2. SEC14L2 knockdown promoted cell proliferation, migration, invasion as well as cell cycle progression in CRPC cells (all P

The treatment for metastatic castration-resistant prostate cancer patients remains a great challenge in the clinic and continuously demands discoveries of new targets and therapies. Here, we assess the function and therapeutic value of SIRT6 in metastatic castration-resistant prostate cancer. Methods: The expression of SIRT6 was examined in prostate cancer tissue microarray by immunohistochemistry staining. The functions of SIRT6 and underlying mechanisms were elucidated by in vitro and in vivo experiments. We also developed an efficient method to silence SIRT6 by aptamer-modified exosomes carrying small interfering RNA and tested the therapeutic effect in the xenograft mice models. Results: SIRT6 expression is positively correlated with prostate cancer progression. Loss of SIRT6 significantly suppressed proliferation and metastasis of prostate cancer cell lines both in vitro and in vivo. SIRT6-driven prostate cancer displays activation of multiple cancer-related signaling pathways, especially the Notch pathway. Silencing SIRT6 by siRNA delivered through engineered exosomes inhibited tumor growth and metastasis. Conclusions: SIRT6 is identified as a driver and therapeutic target for metastatic prostate cancer in our findings, and inhibition of SIRT6 by engineered exosomes can serve as a promising therapeutic tool for clinical application.

Prostate cancer (PCa) is one of the most common epithelial malignant tumors and the fifth leading cause of cancer death in men. An increasing number of studies have demonstrated that N6-methyladenosine (m6A) plays a crucial role in tumorigenesis and tumor development. However, little is known about the role and levels of common m6A regulators and m6A levels in PCa. In this study, we analyzed the characteristic expression of m6A regulators in PCa and castration-resistant prostate cancer (CRPC). UALCAN and cBioPortal were used to estimate the clinical value and genetic alterations of m6A regulators, respectively. The correlation between m6A regulators and androgen receptor (AR) was assessed using Gene Expression Profiling Interactive Analysis (GEPIA) by Pearson correlation statistics. Total m6A levels were detected in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice and PCa cell lines. Results showed that the expression of methyltransferase-like 3 (METTL3) and YTH domain family members, namely, YTHDC2, YTHDF1, and YTHDF2 were generally upregulated in PCa, whereas those of fat mass and obesity-associated protein (FTO), AlkB homolog 5 (ALKBH5), and methyltransferase-like 14 (METTL14) were downregulated. The expression of METTL3, METTL14, Wilms' tumor 1-associating protein (WTAP), YTHDC2, YTHDF1, and YTHDF2 were remarkably higher in CRPC with lymph node metastasis than that in CRPC with bone metastasis, whereas ALKBH5, FTO, and YTHDF3 significantly decreased in CRPC with lymph node metastasis tissues. YTHDF1, YTHDF2, and YTHDC2 were positively correlated with the Gleason grades of PCa, and METTL14, FTO, and ALKBH5 were negatively associated with the Gleason classification. M6A regulators were positively correlated with AR. Patients with a genomic alteration of m6A were associated with poor disease-free survival (DFS). The total m6A levels in TRAMP mice increased dramatically compared with those in tumor-free mice, and m6A levels in LNCaP cell lines were higher than DU145 and PC3 cell lines. In summary, METTL3, METTL14, ALKBH5, FTO, YTHDC2, YTHDF1, and YTHDF2 were abnormally expressed in PCa and related to Gleason classification. Changes in m6A levels maybe contributed to the development and progression of PCa.

Rationale: Aberrant androgen receptor (AR) signaling via full-length AR (AR-FL) and constitutively active AR variant 7 (AR-V7) plays a key role in the development of castration-resistant prostate cancer (CRPC) and resistance to hormone therapies. Simultaneous targeting of AR-FL and AR-V7 may be a promising strategy to overcome resistance to hormone therapy. This study aimed to identify novel drug candidates co-targeting AR-FL and AR-V7 activities and elucidate their molecular mechanism of anti-CRPC activities. Methods: Using a CRPC cell-based reporter assay system, we screened a small library of antimalarial agents to explore the possibility of repositioning them for CRPC treatment and identified bruceantin (BCT) as a potent anti-CRPC drug candidate. A series of cell-based, molecular, biochemical, and in vivo approaches were performed to evaluate the therapeutic potential and molecular mechanism of BCT in CRPC. These approaches include reporter gene assays, cell proliferation, RNA-seq, qRT-PCR, mouse xenografts, co-immunoprecipitation, GST pull-down, immobilized BCT pull-down, molecular modeling, and bioinformatic analyses. Results: We identified BCT as a highly potent inhibitor co-targeting AR-FL and AR-V7 activity. BCT inhibits the transcriptional activity of AR-FL/AR-V7 and downregulates their target genes in CRPC cells. In addition, BCT efficiently suppresses tumor growth and metastasis of CRPC cells. Mechanistically, BCT disrupts the interaction of HSP90 with AR-FL/AR-V7 by directly binding to HSP90 and inhibits HSP90 chaperone function, leading to degradation of AR-FL/AR-V7 through the ubiquitin-proteasome system. Clinically, HSP90 expression is upregulated and correlated with AR/AR-V7 levels in CRPC. Conclusion: Our findings suggest that BCT could serve as a promising therapeutic candidate against CRPC and highlight the potential benefit of targeting AR-FL/AR-V7-HSP90 axis to overcome resistance caused by aberrant AR-FL/AR-V7 signaling.

// Marcus Moses 1 , Ulkuhan Koksal 1 , Elisa Ledet 1 , Charlotte Manogue 1 , Patrick Cotogno 1 , Brian Lewis 1 , Jodi Layton 1 , A. Oliver Sartor 1 and Pedro Barata 1 1 Tulane Cancer Center, School of Medicine, Tulane University, New Orleans, LA, USA Correspondence to: Pedro Barata, email: pbarata@tulane.edu A. Oliver Sartor, email: osartor@tulane.edu Keywords: castration-resistant prostate cancer; high-dose testosterone; next-generation sequencing; androgen receptor Received: October 29, 2019 Accepted: December 16, 2019 Published: January 07, 2020 ABSTRACT Introduction: Castration resistant prostate cancer (CRPC) has been characterized by a reactivation of the androgen receptor (AR) signaling pathway via alterations in androgen metabolism and AR aberrations. High-dose testosterone (HDT) is emerging as an active treatment in metastatic CRPC, however, biomarkers of response are unknown. We hypothesized that responses to HDT might impact the genomic expression of AR alterations found in circulating-tumor DNA (ctDNA). Methods: Retrospective analysis of mCRPC patients treated with HDT (testosterone cypionate q 2–4 weeks) with available clinical and somatic genomic data using a commercially available assay (Guardant360, Redwood City, CA). Clinical outcomes included PSA response (PSA50), time to PSA progression (TPP) and safety. Results: A total of 33 mCRPC patients were treated with ≥2 testosterone cypionate injections. ctDNA testing revealed alterations in AR (39%), TP53 (48%), and DNA repair genes (12%). HDT was given for median of 4.0 months (95% CI, 2.6–5.3) with 24% of PSA50. Twenty patients were re-challenged with abiraterone ( n = 2) or enzalutamide ( n = 18) with 30% PSA50. Significant (grade ≥3) adverse events were observed in 5% of patients (grade 4 thrombocytopenia and asthenia). Patients with median baseline ctDNA% of ≥1.10 had numerically worse TPP outcomes and all patients with AR alterations exhibited decreased AR expression post-HDT ( n = 9), yet no association between clinical outcomes and ctDNA findings was observed. Conclusions: HDT led to a decrease in AR copy number and mutations which was independent from responses to therapy. Further understanding of the genomic alterations as potential predictor of response to HDT is needed.

Anomalous epidermal growth factor receptor (EGFR) signaling plays an important role in the progression of prostate cancer (PCa) and the transformation to castration-resistant PCa (CRPC). A novel tumor suppressor CKLF-like MARVEL transmembrane domain-containing member 5(CMTM5) has a MARVEL domain and may regulate transmembrane signaling. Thus, we postulated that CMTM5 could regulate EGFR and its downstream molecules to affect the biological behaviors of PCa cells. In this study, we found that CMTM5 was expressed in benign prostatic hyperplasia (BPH) tissues but was undetectable in PCa cells. However, the EGFR was upregulated in PCa cells, especially in two metastatic CRPC cell lines, PC3 and DU145. Furthermore, ectopic expression of CMTM5-v1 suppressed cell proliferation and migration and p-EGFR levels. Further investigation revealed that restoration of CMTM5-v1 inhibited not only EGF-mediated proliferation but also chemotactic migration by EGF in PC3 and DU145 cells. Moreover, mechanistic studies showed that CMTM5-v1 attenuated EGF-induced receptor signaling by repressing EGFR and Akt phosphorylation in PCa cells, which were essential for malignant features. Finally, CMTM5-v1can promote the sensitivity of PC3 cells to Gefetinib, a tyrosine kinase inhibitor (TKI) targeting the EGFR. These observations indicate that CMTM5-v1 suppressed PCa cells through EGFR signaling. The loss of CMTM5 may participate in the progression of PCa resulting from deregulated EGFR, and CMTM5 might be associated with the efficacy of TKIs in terms of their potent inhibition of EGFR and human epidermal growth factor-2 (HER2) activation.

Epigenetic alterations that lead to dysregulated gene expression in the progression of castration-resistant prostate cancer (CRPC) remain elusive. Here, we investigated the role of histone deubiquitinase MYSM1 in the pathogenesis of prostate cancer (PCa). Tissues and public datasets of PCa were evaluated for MYSM1 levels. We explored the effects of MYSM1 on cell proliferation, senescence and viability both in vitro and in vivo. Integrative database analyses and co-immunoprecipitation assays were performed to elucidate genomic association of MYSM1 and MYSM1-involved biological interaction network in PCa. We observed that MYSM1 were downregulated in CRPC compared to localized prostate tumors. Knockdown of MYSM1 promoted cell proliferation and suppressed senescence of CRPC cells under condition of androgen ablation. MYSM1 downregulation enhanced the tumorigenic ability in nude mice. Integrative bioinformatic analyses of the significantly associated genes with MYSM1 revealed MYSM1-correlated pathways, providing substantial clues as to the role of MYSM1 in PCa. MYSM1 was able to bind to androgen receptor instead of increasing its expression and knockdown of MYSM1 resulted in activation of Akt/c-Raf/GSK-3β signaling. Together, our findings indicate that MYSM1 is pivotal in CRPC pathogenesis and may be established as a potential target for future treatment.

Purpose: To investigate the risk factors for progression to castration-resistant prostate cancer (CRPC) in metastatic prostate cancer (mPCa) patients who underwent androgen deprivation therapy (ADT). Methods: We analyzed 216 patients with mPCa who underwent ADT between January 2006 and December 2015 at the First Affiliated Hospital of Fujian Medical University. Univariate and multivariate Cox regression analysis were used to explore the risk factors for progression to CRPC. Kaplan-Meier analysis and log-rank test were used to evaluate the difference in progression-free survival (PFS). Results: A total of 121 (56.0%) patients who underwent ADT showed progression to CRPC. Multivariate Cox regression analysis demonstrated that Gleason grade group, prostate-specific antigen nadir (nPSA), and time to PSA nadir (TTN) were risk factors for progression to CRPC in mPCa patients. Kaplan-Meier analysis demonstrated that patients in Gleason grade group ≥3, nPSA >0.2 ng/ml and TTN

Rationale: The progression of prostate cancer (PCa) to castration-resistant PCa (CRPC) despite continuous androgen deprivation therapy is a major clinical challenge. Over 90% of patients with CRPC exhibit sustained androgen receptor (AR) signaling. KDM4B that removes the repressive mark H3K9me3/2 is a transcriptional activator of AR and has been implicated in the development of CRPC. However, the mechanisms of KDM4B involvement in CRPC remain largely unknown. Here, we sought to demonstrate the molecular pathway mediated by KDM4B in CRPC and to provide proof-of-concept evidence that KDM4B is a potential CRPC target. Methods: CRPC cells (C4-2B or CWR22Rv1) depleted with KDM4B followed by cell proliferation (in vitro and xenograft), microarray, qRT-PCR, Seahorse Flux, and metabolomic analyses were employed to identify the expression and metabolic profiles mediated by KDM4B. Immunoprecipitation was used to determine the KDM4B-c-Myc interaction region. Reporter activity assay and ChIP analysis were used to characterize the KDM4B-c-Myc complex-mediated mechanistic actions. The clinical relevance between KDM4B and c-Myc was determined using UCSC Xena analysis and immunohistochemistry. Results: We showed that KDM4B knockdown impaired CRPC proliferation, switched Warburg to OXPHOS metabolism, and suppressed gene expressions including those targeted by c-Myc. We further demonstrated that KDM4B physically interacted with c-Myc and they were co-recruited to the c-Myc-binding sequence on the promoters of metabolic genes (LDHA, ENO1, and PFK). Importantly, KDM4B and c-Myc synergistically promoted the transactivation of the LDHA promoter in a demethylase-dependent manner. We also provided evidence that KDM4B and c-Myc are co-expressed in PCa tissue and that high expression of both is associated with worse clinical outcome. Conclusions: KDM4B partners with c-Myc and serves as a coactivator of c-Myc to directly enhance c-Myc-mediated metabolism, hence promoting CRPC progression. Targeting KDM4B is thus an alternative therapeutic strategy for advanced prostate cancers driven by c-Myc and AR.

Long non-coding RNAs (lncRNAs) have been found essential for tumorigenesis of prostate cancer (PC), but its role in the regulation of castration-resistant prostate cancer (CRPC) is poorly identified. Here, we showed that a lncRNA, Breast-Cancer Anti-Estrogen Resistance 4 (BCAR4), which plays a pivotal role in the tamoxifen-resistance of breast cancer, was significantly upregulated in CRPC, but not in castration-sensitive prostate cancer (CSPC), compared to normal prostate tissue. High BCAR4 levels in CRPC were correlated with poor patients’ overall survival. Androgen increased growth and migration of androgen receptor (AR)-positive PC346 cells, which was abolished by the antagonist of androgen. Overexpression of BCAR4 in PC346 cells increased cell growth and migration, but turned the cells insensitive to androgen. On the other hand, growth and migration of AR-negative DU145 cells are insensitive to androgen, while depletion of BCAR4 in DU145 cells not only decreased cell growth, but also turned the cells sensitive again to androgen. Moreover, BCAR4 activated GLI2 downstream genes, and correlated with the levels of these GLI2-target genes in CRPC. Depletion of GLI2 abolished the effects of BCAR4 on cell growth and migration. Together, our data suggest that BCAR4 may activate GLI2 signaling in PC to contribute to castration resistance.

// Satoshi Tamada 1 , Taro Iguchi 1 , Minoru Kato 1 , Jumpei Asakawa 1 , Kazuaki Kita 1 , Sayaka Yasuda 1 , Takeshi Yamasaki 1 , Yudai Matsuoka 2 , Kazuyuki Yamaguchi 2 , Kentaro Matsumura 2 , Ishun Go 2 , Tetsuji Ohmachi 2 and Tatsuya Nakatani 1 1 Department of Urology, Osaka City University Graduate School of Medicine, Abeno-ku, Osaka 545–8585, Japan 2 Department of Urology, Bell Land General Hospital, Naka-ku, Sakai City, Osaka 599–8247, Japan Correspondence to: Satoshi Tamada, email: s-tamada@med.osaka-cu.ac.jp Keywords: castration-resistant prostate cancer; combined androgen blockade; hormone-sensitive prostate cancer; progression; survival Received: September 19, 2018 Accepted: November 26, 2018 Published: December 11, 2018 ABSTRACT Purpose: The aim of our retrospective study was to determine the time to progression to castration-resistant prostate cancer (CRPC) in prostate cancer patients who undergo combined androgen blockade (CAB), as well as their prognoses. Materials and Methods: We examined the overall survival (OS) and disease-specific survival rates, as well as the time to CRPC development, in 387 patients who were treated with CAB for prostate cancer. The disease-specific survival rate and time to CRPC were stratified by prostate-specific antigen (PSA) levels, Gleason score (GS), and presence of metastasis at diagnosis. We designated high-risk patients as those satisfying at least two of the following three criteria: extent of disease of bone metastasis grade ≥2, presence of metastasis at diagnosis, and a GS ≥8. Results: The 10- and 15-year OS rates were 74.0% and 50.4%, respectively, while the corresponding disease-specific survival rates were both 86.8%. Metastasis at diagnosis was an independent prognostic factor for disease-specific survival. The median time to CRPC development was 140.7 months. A PSA level ≥20 ng/mL, a GS ≥8, and the presence of metastasis at diagnosis were independent predictors of a shorter time to CRPC development. The 10-year disease-specific survival rate in the high-risk group was significantly lower than that in the low-risk group (approximately 74% vs. 98%), and the time to CRPC development was significantly shorter (median: 20.5 months vs. not reached). Conclusions: The time to CRPC development was shorter in high-risk prostate cancer patients with metastases. Such patients require alternative novel treatment modalities.

// Takeo Kosaka 1 , Hiroshi Hongo 1 , Yasumasa Miyazaki 1 , Koshiro Nishimoto 2 , Akira Miyajima 3 and Mototsugu Oya 1 1 Department of Urology, Keio University School of Medicine, Tokyo, Japan 2 Department of Uro-Oncology, Saitama Medical University International Medical Center, Hidaka, Japan 3 Department of Urology, Tokai University School of Medicine, Hiratsuka-shi, Japan Correspondence to: Takeo Kosaka, email: takemduro@gmail.com Keywords: castration-resistant prostate cancer, cabazitaxel, ROS, SESN3 Received: April 04, 2017 Accepted: August 23, 2017 Published: September 21, 2017 ABSTRACT Reactive oxygen species (ROS) production induced by taxanes in cancer cells may influence the taxane-induced cell death or the drug resistance. We investigated the correlation between the cytotoxic effect of taxanes and ROS production in human castration-resistant prostate cancer (CRPC) cell lines. Three human prostate cancer cell lines were treated with increasing concentrations of docetaxel or cabazitaxel in vitro . Cabazitaxel showed significantly higher cytotoxic efficacy than docetaxel in human CRPC cells, accompanied by elevated ROS production detected by FACS analysis. To investigate whether cabazitaxel-mediated cell death was caused by the ROS generation induced by cabazitaxel, we treated CRPC cells in the presence of antioxidant NAC. NAC reduced the cytotoxic effect induced by cabazitaxel. We found that ROS elimination by Sestrin-3 ( SESN3 ) was significantly inhibited by cabazitaxel, but not by docetaxel. These results indicate higher sensitivity of human CRPC to cabazitaxel compared to docetaxel involves ROS production through inhibiting the expression of antioxidant enzyme SESN3.

// Anne Offermann 1, * , Ignacija Vlasic 1, * , Isabella Syring 2, 6, 7 , Wenzel Vogel 1 , Christian Ruiz 3 , Tobias Zellweger 4 , Cyrill A. Rentsch 5 , Susanne Hagedorn 1 , Jochen Behrends 8 , Michael Nowak 6, 7 , Axel Merseburger 9 , Lukas Bubendorf 3 , Jutta Kirfel 7 , Stefan Duensing 10 , Zaki Shaikhibrahim 1, * , Sven Perner 1, * 1 Pathology of the University Medical Center Schleswig-Holstein, Campus Luebeck and Research Center Borstel, Leibniz Center for Medicine and Biosciences, Borstel, Germany 2 Department of Urology, University Hospital Bonn, Bonn, Germany 3 Institute for Pathology, University Hospital Basel, Basel, Switzerland 4 Department of Urology, St. Claraspital, Basel, Switzerland 5 Department of Urology, University Hospital Basel, Basel, Switzerland 6 Section of Prostate Cancer Research, Center for Integrated Oncology Cologne/Bonn, University Hospital of Bonn, Bonn, Germany 7 Institute of Pathology, Center for Integrated Oncology Cologne/Bonn, University Hospital of Bonn, Bonn, Germany 8 Core Facility Fluorescence Cytometry, Research Center Borstel, Leibniz Center for Medicine and Biosciences, Borstel, Germany 9 Department of Urology, University Hospital Luebeck, Luebeck, Germany 10 Section of Molecular Urooncology, Department of Urology, University of Heidelberg School of Medicine, Heidelberg, Germany * These authors have contributed equally to this work Correspondence to: Sven Perner, email: sven.perner@uksh.de Keywords: mediator complex, MED15, castration-resistant prostate cancer, androgen deprivation, PI3 kinase Received: April 15, 2016 Accepted: November 21, 2016 Published: December 10, 2016 ABSTRACT Androgen deprivation therapy (ADT) is the main therapeutic option for advanced prostate cancer (PCa). After initial regression, most tumors develop into castration-resistant PCa (CRPC). Previously, we found the Mediator complex subunit MED15 to be overexpressed in CRPC and to correlate with clinical outcome. Therefore, we investigated whether MED15 is implicated in the signaling changes taking place during progression to CRPC. Immunohistochemistry (IHC) for MED15 on matched samples from the same patients before and after ADT reveals significantly increased MED15 expression after ADT in 72%. A validation cohort comprising samples before and after therapy confirmed our observations. Protein analysis for pAKT and pSMAD3 shows that MED15 correlates with PI3K and TGFs activities, respectively, and that hyper-activation of both pathways simultaneously correlates with highest levels of MED15. We further show that MED15 protein expression increases in LNCaP cells under androgen deprivation, and via EGF mediated PI3K activation. PI3K/mTOR and TGFs-receptor inhibition results in decreased MED15 expression. MED15 knockdown reduces LNCaP cell viability and induces apoptosis during androgen deprivation, while cell cycle is not affected. Collectively, MED15 overexpression arises during ADT via hyper-activation of PI3K/mTOR signaling, thus MED15 may serve as a predictive marker for response to PI3K/mTOR inhibitors. Furthermore, MED15 is potentially a therapeutic target for the treatment of CRPC.

Purpose: We examined the effect of GV1001 in castration castration-resistant prostate cancer (CRPC) cell growth and invasion and explored the potential molecular mechanisms of action. Materials and Methods: The in vitro anti-cancer effects of GV1001 in CRPC cells were examined using cell viability assay, TUNEL assay, and flow cytometry analysis. To evaluate the effects of GV1001 on different steps of angiogenesis, wound healing assay, transwell invasion assay, endothelial cell tube formation assay, and western blot analysis were performed. Finally, the anti-cancer effects of GV1001 on tumor growth in vivo were examined in a CRPC xenograft model. Results: GV1001 inhibited cell viability and induced apoptosis in CRPC cells in vitro, accompanied by down-regulation of Bcl-2 and caspase-3. GV1001 also inhibited different steps of angiogenesis, such as migration, invasion, and endothelial tube formation, along with decreased expression of MMP-2, MMP-9, and CD31 and increased expression of TIMP-1 and TIMP-2. Mechanistically, GV1001 significantly decreased the levels of phosphorylated AKT, phosphorylated p65, and VEGF in CRPC cells in a dose-dependent manner. GV1001 was effective in suppressing tumor growth and inducing apoptosis in a CRPC xenograft mouse model. Conclusions: Our data demonstrated that GV1001 inhibited cell viability, induced apoptosis, and inhibited angiogenesis in CRPC cells by inhibition of the AKT/NF-κB/VEGF signaling pathway.

// Georges Daoud 1, * , Alissar Monzer 1, * , Hisham Bahmad 1, * , Farah Chamaa 1 , Layal Hamdar 1 , Tarek H. Mouhieddine 1 , Sami Shayya 1 , Assaad Eid 1 , Firas Kobeissy 2 , Yen-Nien Liu 3 , Wassim Abou-Kheir 1 1 Department of Anatomy, Cell Biology and Physiological Sciences, Faculty of Medicine, American University of Beirut, Beirut, Lebanon 2 Department of Biochemistry and Molecular Genetics, Faculty of Medicine, American University of Beirut, Beirut, Lebanon 3 Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan * These authors contributed equally to this work Correspondence to: Wassim Abou-Kheir, email: wa12@aub.edu.lb Yen-Nien Liu, email: liuy@tmu.edu.tw Firas Kobeissy, email: fk02@aub.edu.lb Keywords: prostate cancer, castration-resistant prostate cancer, cancer stem cells, Pten, TP53 Received: September 21, 2015 Accepted: March 04, 2016 Published: March 28, 2016 ABSTRACT Cell lines representing the progression of prostate cancer (PC) from an androgen-dependent to an androgen-independent state are scarce. In this study, we used previously characterized prostate luminal epithelial cell line (Plum), under androgen influence, to establish cellular models of PC progression. Cells derived from orthotopic tumors have been isolated to develop an androgen-dependent (PLum-AD) versus an androgen-independent (PLum-AI) model. Upon immunofluorescent, qRT-PCR and Western blot analyses, PLum-AD cells mostly expressed prostate epithelial markers while PLum-AI cells expressed mesenchymal cell markers. Interestingly, both cell lines maintained a population of stem/progenitor cells. Furthermore, our data suggest that both cell lines are tumorigenic; PLum-AD resulted in an adenocarcinoma whereas PLum-AI resulted in a sarcomatoid carcinoma when transplanted subcutaneously in NOD-SCID mice. Finally, gene expression profiles showed enrichment in functions involved in cell migration, apoptosis, as well as neoplasm invasiveness and metastasis in PLum-AI cells. In conclusion, these data suggest that the newly isolated cell lines represent a new in vitro model of androgen-dependent and –independent PC.

Androgen deprivation therapy is the mainstay of treatment of advanced prostate cancer (PCa). However, a significant portion of patients experience disease relapse and tumors ultimately evolve into castration resistant prostate cancer (CRPC), for which there is no cure in the clinic. The Polycomb protein enhancer of zeste homolog 2 (EZH2) is frequently overexpressed in CRPC. It is unclear whether EZH2 can be a therapeutic target in CRPC. Here, we demonstrated that chemo- and radiotherapy agents such as camptothecin (CPT) and γ irradiation decrease EZH2 expression in various PCa cell lines. We provided evidence that functional p53 and RB proteins are required for CPT- and irradiation-induced downregulation of EZH2 in CRPC cells. We demonstrated that EZH2-specific small molecule inhibitors mitigate CRPC cell growth. We further showed that the EZH2 inhibitor GSK126 inhibits both Polycomb-dependent and -independent functions of EZH2 in PCa cells. Importantly, we found that inhibition of EZH2 by genetic and pharmacological means sensitizes CRPC cells to CPT-induced apoptotic death and growth inhibition in culture and in mice. Our data suggest that concomitant administration of small molecule inhibitors of EZH2 may significantly increase the anti-tumor efficacy of conventional chemo- and radiotherapies in CRPC.

// Jin Zhu 1, * , Sugui Wang 1, 2, * , Wenyu Zhang 3, * , Junyi Qiu 4 , Yuxi Shan 1 , Dongrong Yang 1 , Bairong Shen 3 1 Department of Urology, Second Affiliated Hospital of Soochow University, Suzhou, China 2 Department of Urology, Huai’an Hospital Affiliated to Xuzhou Medical College and Second People’s Hospital of Huai’an, Huai’an, China 3 Center for Systems Biology, Soochow University, Suzhou, China 4 Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou, China * These authors have contributed equally to this work Correspondence to: Dongrong Yang, e-mail: doc_ydr@163.com Bairong Shen, e-mail: bairong.shen@suda.edu.cn Keywords: castration-resistant prostate cancer, microRNA biomarker, microRNA-mRNA interaction, bioinformatics model, pathway analysis Received: May 04, 2015 Accepted: October 17, 2015 Published: October 31, 2015 ABSTRACT High-throughput methods have been used to explore the mechanisms by which androgen-sensitive prostate cancer (ASPC) develops into castration-resistant prostate cancer (CRPC). However, it is difficult to interpret cryptic results by routine experimental methods. In this study, we performed systematic and integrative analysis to detect key miRNAs that contribute to CRPC development. From three DNA microarray datasets, we retrieved 11 outlier microRNAs (miRNAs) that had expression discrepancies between ASPC and CRPC using a specific algorithm. Two of the miRNAs (miR-125b and miR-124) have previously been shown to be related to CRPC. Seven out of the other nine miRNAs were confirmed by quantitative PCR (Q-PCR) analysis. MiR-210, miR-218, miR-346, miR-197, and miR-149 were found to be over-expressed, while miR-122, miR-145, and let-7b were under-expressed in CRPC cell lines. GO and KEGG pathway analyses revealed that miR-218, miR-197, miR-145, miR-122, and let-7b, along with their target genes, were found to be involved in the PI3K and AKT3 signaling network, which is known to contribute to CRPC development. We then chose five miRNAs to verify the accuracy of the analysis. The target genes of each miRNA were altered significantly upon transfection of specific miRNA mimics in the C4–2 CRPC cell line, which was consistent with our pathway analysis results. Finally, we hypothesized that miR-218, miR-145, miR-197, miR-149, miR-122, and let-7b may contribute to the development of CRPC through the influence of Ras, Rho proteins, and the SCF complex. Further investigation is needed to verify the functions of the identified novel pathways in CRPC development.

SRC kinase is activated in castration resistant prostate cancer (CRPC), phosphorylates the androgen receptor (AR), and causes its ligand-independent activation as a transcription factor. However, activating SRC mutations are exceedingly rare in human tumors, and mechanisms of ectopic SRC activation therefore remain largely unknown. Performing a functional genomics screen, we found that downregulation of SRC inhibitory kinase CSK is sufficient to overcome growth arrest induced by depriving human prostate cancer cells of androgen. CSK knockdown led to ectopic SRC activation, increased AR signaling, and resistance to anti-androgens. Consistent with the in vitro observations, stable knockdown of CSK conferred castration resistance in mouse xenograft models, while sensitivity to the tyrosine kinase inhibitor dasatinib was retained. Finally, CSK was found downregulated in a distinct subset of CRPCs marked by AR amplification and ETS2 deletion but lacking PTEN and RB1 mutations. These results identify CSK downregulation as a principal driver of SRC activation and castration resistance and validate SRC as a drug target in a molecularly defined subclass of CRPCs.

Background: This study aimed to investigate copy number variations (CNVs) of CYP17A1 and androgen receptor (AR) genes in serum cell-free DNA collected before starting abiraterone in 53 consecutive patients with castration-resistant prostate cancer (CRPC). Methods: Serum DNA was isolated and CNVs were analysed for AR and CYP17A1 genes using Taqman copy number assays. The association between CNVs and progression-free/overall survival (PFS/OS) was evaluated by the Kaplan–Meier method and log-rank test. Results: Median PFS of patients with AR gene gain was 2.8 vs 9.5 months of non-gained cases (P

// Yi-Ping Zhu 1,2,* , Fang-Ning Wan 1,2,* , Yi-Jun Shen 1,2 , Hong-Kai Wang 1,2 , Gui-Ming Zhang 1,2 and Ding-Wei Ye 1,2 1 Department of Urology, Fudan University Shanghai Cancer Center, Fudan University, Shanghai, China 2 Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China * These authors have contributed equally to this work Correspondence to: Ding-Wei Ye, email: // Keywords : castration-resistant prostate cancer, COL6A1, reactive stroma, tumorigenesis Received : January 12, 2015 Accepted : March 01, 2015 Published : March 30, 2015 Abstract Castration-resistant prostate cancer (CRPC) remains the most critical challenge in the clinical management of prostate cancer (PCa). Reactive stromal changes in PCa are likely involved in the emergence of CRPC. In the present study, we identified a novel oncogene termed COL6A1 which was upregulated in the reactive stroma of CRPC. We established an androgen-independent LNCaP (LNCaP-AI) cell line in steroid-reduced (SR) medium within 2 months. We examined COL6A1 expression with western blot during the LNCaP-AI induction, and studied the function of COL6A1 in vitro and in vivo . Immunohistochemical staining of COL6A1 was performed in ten pairs of androgen-sensitive PCa and CRPC samples. We demonstrated that COL6A1 expression was markedly increased in LNCaP-AI cells and CRPC tissues compared with LNCaP cells and paired androgen-sensitive PCa specimens. In vitro , COL6A1 knockdown resulted in G1-S cell cycle arrest and descended vitality. Overexpression of COL6A1 was associated with accelerated S phase entry and elevated vitality in prostate cancer cells. COL6A1 also promoted tumorigenesis of LNCaP cells in vivo . Taken together, these data suggest an important role of COL6A1 in the molecular etiology of castration-resistant prostate cancer, and support the potential use of COL6A1 in CRPC therapy.

Background This study investigates the docetaxel-resistant mechanism and explores the effect of tea polyphenols (TP) on autophagy and its related mechanism in human castration-resistant prostate cancer (CRPC) cell lines PC3 and DU145. Methods Immunofluorescence assay and annexin V-FITC/PI double staining flow cytometry were used to analyze the apoptosis and autophagy of PC3 and DU145 cells. The expression of autophagy-related proteins was detected by western bolt. Results Docetaxel could induce autophagy and apoptosis, together with the expression increase in p-JNK, p-Bcl-2 and Beclin1. The level of autophagy was remarkably decreased, but apoptosis was increased after combining with TP. In addition, the expression of p-mTOR was increased after combining with TP. Conclusion Docetaxel induces protective autophagy in CRPC cells by JNK pathway activation and then Bcl-2 phosphorylation and Beclin1 dissociation. TP activates mTOR pathway, which ultimately inhibits docetaxel-induced autophagy and improves therapeutic efficacy of docetaxel in CRPC cells.

Radium-223 dichloride (Ra-223) is the first α-particle emitting radiopharmaceutical to be approved for the treatment of patients with castration-resistant prostate cancer and associated bone metastases, and the first bone-targeting agent to significantly improve patient overall survival whilst reducing pain and the symptomatic skeletal events (SSEs) associated with bone metastases. Ra-223 exhibits a favourable safety profile, with low myelosuppression rates and fewer adverse events than placebo. Compared with other approved radiopharmaceuticals, the α-particle emitting Ra-223 has a high biological efficiency and a short penetration range, potentially sparing bone marrow toxicity and limiting unwanted exposure. Ra-223 has a short half-life and decays to a stable product, reducing the problem of storage and disposal associated with radiopharmaceuticals. Ra-223 offers a new treatment option with great potential in this setting. However, concerns remain amongst patients, their families and health care professionals over the use of radiopharmaceuticals. This article, which draws on the experiences of health care workers during the ALSYMPCA (ALpharadin in SYMtomatic Prostate CAncer) study, reviews the clinical development of Ra-223, highlighting the key issues for the uro-oncology nurse who has a pivotal role within the multi-disciplinary team (MDT) to ensure safe and effective treatment to the patient. The role of the uro-oncology nurse is multifaceted, including patient pre-assessment and post-treatment monitoring and coordination of the MDT. In addition, their role in communicating with and educating those involved with Ra-223 on what to expect from the agent can alleviate fears associated with its use.

// Yusuke Goto 1,2 , Satoko Kojima 3 , Rika Nishikawa 1,2 , Hideki Enokida 4 , Takeshi Chiyomaru 4 , Takashi Kinoshita 1 , Masayuki Nakagawa 4 , Yukio Naya 3 , Tomohiko Ichikawa 2 and Naohiko Seki 1 1 Department of Functional Genomics, Chiba University Graduate School of Medicine, Chiba, Japan 2 Department of Urology, Chiba University Graduate School of Medicine, Chiba, Japan 3 Department of Urology, Teikyo University Chiba Medical Center, Chiba, Japan 4 Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan Correspondence: Naohiko Seki, email: // Keywords : microRNA, miR-23b, miR-27b, miR-24-1, castration-resistant prostate cancer Received : May 27, 2014 Accepted : July 31, 2014 Published : July 31, 2014 Abstract Our recent study of microRNA (miRNA) expression signatures in prostate cancer (PCa) has revealed that all members of the miR-23b/27b/24-1 cluster are significantly downregulated in PCa tissues. The aim of this study was to investigate the effectiveness of these clustered miRNAs as a disease progression marker and to determine the functional significance of these clustered miRNAs in PCa. Expression of the miR-23b/27b/24-1 cluster was significantly reduced in PCa tissues. Kaplan-Meier survival curves showed that low expression of miR-27b predicted a short duration of progression to castration-resistant PCa. Gain-of-function studies using mature miR-23b , miR-27b ,and miR-24-1 significantly inhibited cell proliferation, migration and invasion in PCa cells (PC3 and DU145). To identify the molecular targets of these miRNAs, we carried out gene expression and in silico database analyses. GOLM1 was directly regulated by miR-27b in PCa cells. Elucidation of the molecular targets and pathways regulated by the tumor-suppressive microRNAs should shed light on the oncogenic and metastatic processes in PCa.

// Guanxiong Ding 1, * , Jianqing Wang 1, * , Chenchen Feng 1 , Haowen Jiang 1 , Jianfeng Xu 1, 2 , Qiang Ding 1 1 Department of Urology, Huashan Hospital, Fudan University, Shanghai, China 2 Department of Urology, Wake Forest University School of Medicine, Winston-Salem, North Carolina, USA * These authors have contributed equally to this work Correspondence to: Jianfeng Xu, email: drwang_shan@126.com Qiang Ding, email: dingqiang888@126.com Keywords: castration-resistant prostate cancer, lipocalin 2, androgen receptor, cell proliferation, transcriptional activity Received: April 25, 2016 Accepted: August 13, 2016 Published: September 01, 2016 ABSTRACT Background: Castration-resistant prostate cancer (CRPC) is the lethal phenotype of prostate cancer. Lipocalin 2 (LCN2) is aberrantly expressed in many cancers including primary prostate cancer (PCa), but its role in CRPC has not been reported. Results: LCN2 expression was upregulated in human primary PCa and CRPC tissues. Overexpression of LCN2 promoted C4-2B and 22RV1 cell proliferation while knockdown of LCN2 markedly inhibited C4-2B and 22RV1 cell growth. LCN2 overexpression led to increased AR downstream gene SLC45A3 without upregulating AR expression. In the xenograft model, overexpression of LCN2 significantly promoted tumor growth. Methods: LCN2 expression was detected in primary PCa and CRPC tissues and cell lines C4-2B and 22RV1 using immunohistochemistry and western blotting, respectively. Serum LCN2 level was detected vi ELISA. Lentiviruses-mediated over-expression of LCN2 and LCN2 knockdown were performed in CRPC cell lines. Expressions of androgen receptor (AR) downstream genes was examined in cell lines, in CRPC tissues, and in animal models. Conclusion: LCN2 could facilitate cell proliferation of CRPC via AR transcriptional activity. LCN2 could be a novel target in CRPC.

Currently, no therapeutic options exist for castration-resistant prostate cancer (CRPC) patients who have developed resistance to the second generation anti-androgen receptor (AR) axis therapy. Here we report that co-deletion of Pten and p53 in murine prostate epithelium, often observed in human CRPC, leads to AR-independent CRPC and thus confers de novo resistance to second generation androgen deprivation therapy (ADT) in multiple independent yet complementary preclinical mouse models. In contrast, mechanism-driven co-targeting hexokinase 2 (HK2)-mediated Warburg effect with 2-deoxyglucose (2-DG) and ULK1-dependent autophagy with chloroquine (CQ) selectively kills cancer cells through intrinsic apoptosis to cause tumor regression in xenograft, leads to a near-complete tumor suppression and remarkably extends survival in Pten −/p53-deficiency-driven CRPC mouse model. Mechanistically, 2-DG causes AMPK phosphorylation, which in turn inhibits mTORC1-S6K1 translation signaling to preferentially block anti-apoptotic protein MCL-l synthesis to prime mitochondria-dependent apoptosis while simultaneously activates ULK1-driven autophagy for cell survival to counteract the apoptotic action of anti-Warburg effect. Accordingly, inhibition of autophagy with CQ sensitizes cancer cells to apoptosis upon 2-DG challenge. Given that 2-DG is recommended for phase II clinical trials for prostate cancer and CQ has been clinically used as an anti-malaria drug for many decades, the preclinical results from our proof-of-principle studies in vivo are imminently translatable to clinical trials to evaluate the therapeutic efficacy by the combination modality for a subset of currently incurable CRPC harboring PTEN and TP53 mutations., Highlights • PTEN/TP53 loss drives HK2-dependent prostate tumor growth bypassing anti-AR axis therapy • Targeting HK2-mediated Warburg effect activates AMPK-ULK1-dependent autophagy for cell survival • Targeting HK2-mediated Warburg effect regulates AMPK-mTORC1-S6K1 translation signaling to reduce MCL-1 for cell apoptosis • Co-targeting Warburg effect and autophagy suppresses tumor growth in Pten-/p53-deficient prostate cancer models

// Anchit Khanna 1,2 , Jayant K. Rane 3 , Kati K. Kivinummi 1,4 , Alfonso Urbanucci 1,5,6 , Merja A. Helenius 1 , Teemu T. Tolonen 1 , Outi R. Saramaki 1 , Leena Latonen 1 , Visa Manni 1 , John E. Pimanda 2 , Norman J. Maitland 3 , Jukka Westermarck 7,8 and Tapio Visakorpi 1 1 Prostate Cancer Research Center (PCRC), Institute of Biosciences and Medical Technology (BioMediTech), University of Tampere and Tampere University Hospital, Tampere, Finland 2 Adult Cancer Program, The Prince of Wales Clinical School, Lowy Cancer Research Centre, UNSW Medicine, University of New South Wales, Sydney, Australia 3 YCR Cancer Research Unit, Department of Biology, The University of York, Heslington, United Kingdom 4 Department of Signal Processing, Tampere University of Technology, Tampere, Finland 5 Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, University of Oslo and Oslo University Hospital, Oslo, Norway 6 Department of Cancer Prevention, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway 7 Turku Centre for Biotechnology, University of Turku and Abo Akademi University, Turku, Finland 8 Department of Pathology, University of Turku, Turku, Finland Correspondence to: Tapio Visakorpi, email: // Anchit Khanna, email: // Keywords : CIP2A, androgen receptor, castration-resistant prostate cancer, cancer stem-like cells Received : December 30, 2014 Accepted : April 03, 2015 Published : April 19, 2015 Abstract Residual androgen receptor (AR)-signaling and presence of cancer stem-like cells (SCs) are the two emerging paradigms for clinically challenging castration-resistant prostate cancer (CRPC). Therefore, identification of AR-target proteins that are also overexpressed in the cancer SC population would be an attractive therapeutic approach. Our analysis of over three hundred clinical samples and patient-derived prostate epithelial cultures (PPECs), revealed Cancerous inhibitor of protein phosphatase 2A (CIP2A) as one such target. CIP2A is significantly overexpressed in both hormone-naive prostate cancer (HN-PC) and CRPC patients . CIP2A is also overexpressed, by 3- and 30-fold, in HN-PC and CRPC SCs respectively. In vivo binding of the AR to the intronic region of CIP2A and its functionality in the AR-moderate and AR-high expressing LNCaP cell-model systems is also demonstrated. Further, we show that AR positively regulates CIP2A expression, both at the mRNA and protein level. Finally, CIP2A depletion reduced cell viability and colony forming efficiency of AR-independent PPECs as well as AR-responsive LNCaP cells, in which anchorage-independent growth is also impaired. These findings identify CIP2A as a common denominator for AR-signaling and cancer SC functionality, highlighting its potential therapeutic significance in the most clinically challenging prostate pathology: castration-resistant prostate cancer.

// Min Wu 1 , Sahn-Ho Kim 1 , Indrani Datta 2 , Albert Levin 2 , Gregory Dyson 3 , Jing Li 4 , Stephanie Kaypee 5 , M. Mahadeva Swamy 5 , Nilesh Gupta 6 , Ho Jeong Kwon 7 , Mani Menon 1 , Tapas K. Kundu 5 and G. Prem-Veer Reddy 1 1 Vattikuti Urology Institute, Henry Ford Hospital, Detroit, MI, USA 2 Bioinformatics Core, Public Health Sciences, Henry Ford Hospital, Detroit, MI, USA 3 Biostatistics Core, Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA 4 Pharmacology Core, Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA 5 Transcription and Disease Laboratory, Molecular Biology and Genetics Unit, JNCASR, Bangalore, Karnataka, India 6 Department of Pathology, Henry Ford Hospital, Detroit, MI, USA 7 Department of Biotechnology, Translational Research Center for Protein Function Control, Yonsei University, Seoul, Republic of Korea Correspondence: G. Prem Veer Reddy, email: // Keywords : Androgen receptor, calmodulin, Hydrazinobenzoylcurcumin, CTK7A, castration-resistant prostate cancer Received : November 24, 2014 Accepted : January 20, 2015 Published : January 31, 2015 Abstract There is a critical need for therapeutic agents that can target the amino-terminal domain (NTD) of androgen receptor (AR) for the treatment of castration-resistant prostate cancer (CRPC). Calmodulin (CaM) binds to the AR NTD and regulates AR activity. We discovered that Hydrazinobenzoylcurcumin (HBC), which binds exclusively to CaM, inhibited AR activity. HBC abrogated AR interaction with CaM, suppressed phosphorylation of AR Serine81, and blocked the binding of AR to androgen-response elements. RNA-Seq analysis identified 57 androgen-regulated genes whose expression was significantly (p ≤ 0.002) altered in HBC treated cells as compared to controls. Oncomine analysis revealed that genes repressed by HBC are those that are usually overexpressed in prostate cancer (PCa) and genes stimulated by HBC are those that are often down-regulated in PCa, suggesting a reversing effect of HBC on androgen-regulated gene expression associated with PCa. Ingenuity Pathway Analysis revealed a role of HBC affected genes in cellular functions associated with proliferation and survival. HBC was readily absorbed into the systemic circulation and inhibited the growth of xenografted CRPC tumors in nude mice. These observations demonstrate that HBC inhibits AR activity by targeting the AR NTD and suggest potential usefulness of HBC for effective treatment of CRPC.

Castration-resistant prostate cancer (CRPC) is strongly associated with sclerotic bone metastases and poor prognosis. Models that mimic human CRPC are needed to identify the mechanisms for prostate cancer (PC) growth in bone and to develop new therapeutic strategies. We characterize a new model, LNCaP-19, and investigate the interaction between tumor cells and osteoblasts in the sclerotic tumor response of CRPC. Osteogenic profiling of PC cell lines (LNCaP-19, LNCaP, C4-2B4, and PC-3) was performed by gene expression arrays and mineral staining. Conditioned medium from MC3T3-E1 was used for osteoblast stimulation of CRPC cells. The capacity of LNCaP-19 cells to induce sclerotic lesions was assessed in intratibial xenografts and verified by serum markers, histological analysis and bone mineral density (BMD) measurements. The CRPC cell line LNCaP-19 expresses a pronounced osteogenic profile compared to its parental androgen-dependent cell line LNCaP. Osteoblast-derived factors further increase the expression of genes known to enhance metastatic progression of PC. LNCaP-19 forms sclerotic tumors in tibia of castrated mice as evident by increased total BMD (P < 0.01). There was a strong correlation between serum osteocalcin and BMD (total: R2 0.811, P < 0.01, trabecular: R2 0.673, P < 0.05). For the first time we demonstrate that a CRPC cell line generated in vitro has osteogenic capacity and that osteomimicry can be an inherent feature of these cells. Osteoblast-derived factors further promote the osteogenic and metastatic phenotype in CRPC cells. Altogether, our model demonstrates that both tumor cells and osteoblasts are mediators of the bone forming process of CRPC.

Molecular modifications of the androgen receptor (AR) can cause resistance to androgen deprivation therapy (ADT) in prostate cancer patients. Since lack of representative tumor samples hinders therapy adjustments according to emerging AR-modifications, we evaluated simultaneous detection of the two most common AR modifications (AR-V7 splice variant and AR point mutations) in circulating tumor cells (CTCs). We devised a single-tube assay to detect AR-V7 splice variants and AR point mutations in CTCs using immunomagnetic cell isolation, followed by quantitative real-time PCR and DNA pyrosequencing. We prospectively investigated 47 patients with PSA progression awaiting therapy switch. Comparison of response to newly administered therapy and CTC-AR-status allowed effect size estimation. Nineteen (51%) of 37 patients with detectable CTCs carried AR-modifications. Seventeen patients carried the AR-V7 splice variant, one harbored a p.T878A point mutation and one harbored both AR-V7 and a p.H875Y mutation. We estimated a positive predictive value for response and non-response to therapy by AR status in CTCs of ~94%. Based on a conservative calculation, we estimated the effect size for molecularly-informed therapy switches for prospective clinical trial planning to ~27%. In summary, the ability to determine key resistance-mediating AR modifications in CTCs has the potential to considerably improve prostate cancer treatment.