Castration-recurrent Prostate Cancer Research Articles

Abstract 2398: The tobacco cembranoid β-cembrenediol as promising prostate cancer recurrence suppressor lead

Abstract Prostate cancer (PC) is the second leading cause of men death in the US, annually killing >34,000 men. Despite progress in early PC diagnosis and treatment, many patients have locally and distant disease recurrence. Therapeutic androgens deprivation have limited curative potential and associated with severe side effects and rapid resistance development justifying the dire need for discovery of novel recurrence inhibitors. (1S,2E,4R,6R,-7E,11E)-2,7,11-cembratriene-4,6-diol (β-CBT) and its 4-epi-analog (α-CBT) are the precursors to key flavor ingredients in leaves of most Nicotiana species. Nearly 40-60% of β- and α-CBT are purposely degraded during the commercial tobacco fermentation. Earlier, we reported the lack of β-CBT cytotoxicity below 50 µM while at 50 nM it reversed the calcitonin-stimulated decrease in transepithelial resistance and increased paracellular permeability in PC-3M PC cells. β-CBT also proven to stimulate tight junction formation between PC-3M cells, producing a tighter intercellular barrier. β-CBT 10-50 nM potently inhibited the calcitonin-stimulated PC-3M cells invasion in Matrigel assay. This study demonstrates the in vitro β-CBT anti-migratory (wound-healing assay) and anti-clonogenicity (colony-formation assay) activities against five PC cell lines, including the metastatic castration-recurrent human prostate cancer (mCRPC) CWR-R1ca. Meanwhile, β-CBT potently suppressed the in vivo locoregional and distant recurrences after the primary tumor surgical excision of the tumor formed by engrafting the human metastatic prostate PC-3M-Luc cells at the suprascupular region in male nude mice model. β-CBT treatments prevented organ and significantly suppressed bone metastasis and lacked any major toxicity over the 60-day study course. Therefore, β-CBT emerges as an effective promising PC recurrence and metastasis suppressive lead appropriate for near future use as a prospective nutraceutical. Citation Format: Ethar A. Mudhish, Abu Bakar Siddique, Khaldoun S. Abdelwahed, Judy A. King, Khalid A. El Sayed. The tobacco cembranoid β-cembrenediol as promising prostate cancer recurrence suppressor lead [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2398.

2019 Canadian Urological Association (CUA)-Canadian Uro Oncology Group (CUOG) guideline: Management of castration-resistant prostate cancer (CRPC).

Castration-resistant prostate cancer (CRPC) is defined by disease progression despite castrate levels of testosterone and may present as either a continuous rise in serum prostate-specific antigen (PSA) levels, the progression of pre-existing disease, and/or the appearance of new metastases. Advanced prostate cancer has been known under a few names over the years, including hormone-resistant prostate cancer (HRPC) and androgen-insensitive prostate cancer (AIPC). Most recently, the terms castration-resistant prostate cancer or castration-recurrent prostate cancer were introduced with the realization that extra-testicular androgen production plays a significant role in the resistance of prostate cancer cells to medical or surgical castration therapy.1 In their second publication, the Prostate Cancer Working Group defined CRPC as a continuum on the basis of whether metastases are detectable (clinically or by imaging) and whether the serum testosterone is in the castrate range by surgical orchidectomy or medical therapy.2 This definition creates a clinical-states model, where patients can be classified. The rising PSA states (castrate and non-castrate) signify that no detectable (measurable or non-measurable) disease has ever been found. The clinical metastases states (castrate and non-castrate) signify that disease was detectable at some point in the past, regardless of whether it is detectable now.3 Prognosis is associated with several factors that go beyond PSA levels. These include performance status, presence of visceral metastases, presence of bone pain, extent of disease on bone scan, and serum lactate dehydrogenase and alkaline phosphatase levels. Bone metastases will occur in 90% of men with CRPC and can produce significant morbidity, including pain, pathological fractures, spinal cord compression, and bone marrow failure. Paraneoplastic effects, including anemia, weight loss, fatigue, hypercoagulability, and increased susceptibility to infection, are also common. CRPC includes patients without metastases or symptoms with rising PSA levels despite androgen-deprivation therapy (ADT) to patients with metastases and significant debilitation due to cancer symptoms.

Protein Kinase N1 control of androgen-responsive serum response factor action provides rationale for novel prostate cancer treatment strategy.

Sustained reliance on androgen receptor (AR) after failure of AR-targeting androgen deprivation therapy (ADT) prevents effective treatment of castration-recurrent (CR) prostate cancer (CaP). Interfering with the molecular machinery by which AR drives CaP progression may be an alternative therapeutic strategy but its feasibility remains to be tested. Here, we explore targeting the mechanism by which AR, via RhoA, conveys androgen-responsiveness to serum response factor (SRF), which controls aggressive CaP behavior and is maintained in CR-CaP. Following a siRNA screen and candidate gene approach, RNA-Seq studies confirmed that the RhoA effector Protein Kinase N1 (PKN1) transduces androgen-responsiveness to SRF. Androgen treatment induced SRF-PKN1 interaction, and PKN1 knockdown or overexpression severely impaired or stimulated, respectively, androgen regulation of SRF target genes. PKN1 overexpression occurred during clinical CR-CaP progression, and hastened CaP growth and shortened CR-CaP survival in orthotopic CaP xenografts. PKN1's effects on SRF relied on its kinase domain. The multikinase inhibitor lestaurtinib inhibited PKN1 action and preferentially affected androgen regulation of SRF over direct AR target genes. In a CR-CaP patient-derived xenograft, expression of SRF target genes was maintained while AR target gene expression declined and proliferative gene expression increased. PKN1 inhibition decreased viability of CaP cells before and after ADT. In patient-derived CaP explants, lestaurtinib increased AR target gene expression but did not significantly alter SRF target gene or proliferative gene expression. These results provide proof-of-principle for selective forms of ADT that preferentially target different fractions of AR's transcriptional output to inhibit CaP growth.

Abstract 3745: Inhibition of Protein Kinase N1 prevents control of AR over SRF action in advanced prostate cancer

Abstract A major limitation in the management of castration-recurrent prostate cancer (CR-CaP) is the lack of treatments to inhibit androgen receptor (AR) action that is driving CaP growth when androgen-deprivation therapy (ADT) has failed. AR action breaks down in fractions that are regulated differently at the molecular level, control diverse aspects of CaP biology and contribute differentially to CaP progression. Blocking a specific fraction(s) of AR action that mediates CaP progression may serve as alternative treatment strategy, yet evidence for such an approach and drugs that render this approach feasible are not known. Our laboratory has identified a mechanism of AR action in which AR conveys androgen-responsiveness to Serum Response Factor (SRF) target genes via androgen activation of RhoA. Androgen-responsive SRF action mediates cell migration and is associated with aggressive CaP behavior and recurrence. Importantly, control of AR over RhoA-SRF action is maintained in CR-CaP, which renders this signaling a potentially attractive novel therapeutic target. Using an siRNA screen, we identified Protein Kinase N1 (PKN1) as the Rho effector that transduces androgen-responsiveness from RhoA to SRF. In promoter-reporter, qRT-PCR and oligoarray assays, knockdown of PKN1 severely impaired the androgen-regulation of SRF target genes, but affected androgen response of only a few (12 of 452) direct AR target genes. In contrast, transient overexpression of PKN1 preferentially stimulated androgen-responsive SRF target gene expression. Stable overexpression of PKN1 hastened growth and shortened survival in CaP orthotopic xenografts. Site-directed mutagenesis showed that the effect of PKN1 on SRF target genes relied on its kinase activity. Treatment with lestaurtinib, a multikinase inhibitor that is also a potent inhibitor of PKN1, mimicked the effects of PKN1 knockdown on expression of SRF and AR target genes in several CaP cell lines and ex vivo CaP explants. Lestaurtinib also inhibited the proliferation of CaP cells that are either ADT-naïve or -resistant, express only the constitutively active AR variant ARv567es, or are growth-stimulated by the AR-activating ADT drug metabolite 5α-abiraterone. RNA-Seq followed by MSigDB analyses confirmed that lestaurtinib impairs androgen-dependent PKN1 activity and revealed that 100 of the 127 MgSigDB gene sets isolated after PKN1 silencing overlapped with 123 gene sets derived after lestaurtinib treatment. Overlapping gene sets included several related to hormonal carcinogenesis and cancer progression, prostate development, signaling cascades that have been implicated in SRF and RhoA function and/or are relevant to CaP. PKN1 inhibition via lestaurtinib, which is used already to treat human hematologic malignancies and is well tolerated by carcinoma patients, may thus serve as novel alternative treatment strategy to target AR-dependent SRF action in CR-CaP. Citation Format: Varadha Balaji Venkadarkrishnan, Adam DePriest, Yixue Su, Giridhar Mudduluru, Salma Ben-Salem, Sangeeta Kumari, Qiang Hu, Eduardo Cortes, Scott Dehm, Cristina Magi-Galluzzi, Eric Klein, Nima Sharifi, Song Liu, Hannelore Heemers. Inhibition of Protein Kinase N1 prevents control of AR over SRF action in advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3745.

HSP60 IS A NOVEL TARGET IN LETHAL PROSTATE CANCER

Currently, various therapeutic agents exist in the clinic to treat aggressive prostate cancer (PCa) by modulating androgen receptor (AR) activity, which is one of the main drivers of prostate cancer. However, resistance to these agents nearly always occurs and results in the production of a more aggressive form of cancer, typically referred to as castrate‐recurrent prostate cancer (CRPC). Patients within a subset of CRPC have tumors that undergo neuroendocrine (NE) transdifferentiation (NE‐CRPC). NE‐CRPC is poorly differentiated, readily metastasizes and is resistant to androgen deprivation therapy (ADT). Therefore, it is essential that alternative therapeutic targets that do not solely rely on AR modulation be better characterized for patients with this advanced disease.The mitochondrial unfolded protein response (mitoUPR) is responsible for maintaining mitochondrial quality control by preserving the integrity of the mitochondria in response to numerous stressors including hypoxia and chemotherapeutic agents. MitoUPR activation is a hallmark sign of mitochondrial dysfunction. Heat Shock Protein60 (Hsp60) is a mitochondrial chaperone and is a key player of the mitoUPR. Thus, we hypothesize that inhibition of Hsp60 renders aggressive NE‐like cells susceptible to stress, thereby alleviating tumorigenicity. We observed that protein expression of Hsp60 was upregulated in tumors derived from NE models such as the transgenic adenocarcinoma of mouse prostate tumor (TRAMP), DKO (pten−/−/rb1−/−) and TKO (rb1−/−/pten−/−/p53−/−) when compared to wild‐type controls. Transcript levels of HSPD1 positively correlated to Gleason Score and are upregulated in patients with NE‐like phenotypes from TCGA. Additionally, Hsp60 expression associated with severe mitochondrial dysfunction in TRAMP tumors. Specifically, mito‐nuclear imbalances and defects in oxidative phosphorylation activity were observed. Lastly, Inhibition of Hsp60 significantly increased sensitivity of NE cells to mitotoxic stressors such as hypoxia and the chemotherapeutic agent cisplatin.Together, our data indicate that inactivation of Hsp60 sensitizes NE cells to mitotoxic stress and has clinical and therapeutic relevance. Specifically, the use of cisplatin in small cell CRPC has been shown to have minimal effect, yet we determined that inhibition of Hsp60 sensitizes PCa cells to cisplatin. We envision that inactivation of Hsp60 will alleviate tumor burden by itself, but will also enhance sensitivity of chemo‐resistant PCa cells. Additionally, inhibition of Hsp60 may enhance therapeutic potential of anticancer agents in other types of cancers that overexpress Hsp60 including liver, lung and colon cancers. Thus, Hsp60 is a novel target in prostate cancers with NE‐like differentiation.Support or Funding InformationThis work was supported in part by the National Cancer Institute of the National Institutes of Health under Award no. R01CA160685This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Novel Use of Olaparib for the Treatment of Metastatic Castration-Recurrent Prostate Cancer.

Although mortality from prostate cancer has declined over the past 20years as a result of early detection and treatment, the 5-year survival rate for men with prostate cancer who develop metastatic disease is only 29%. Current treatment options for metastatic castration-recurrent prostate cancer (mCRPC) are associated with toxicity and a limited durable response; therefore, additional lines of efficacious and minimally toxic therapy are needed. Olaparib, a poly(adenosine 5'-diphosphate) ribose polymerase (PARP) inhibitor, received a U.S. Food and Drug Administration breakthrough therapy designation in January 2016 for the treatment of patients with BRCA1/2 or ATM gene-mutated mCRPC based on results of a compelling phase II trial of olaparib in patients with advanced castration-resistant prostate cancer (TOPARP-A). This study found that men with mCRPC and genetic mutations in DNA damage repair genes had an overall response rate of nearly 90% with olaparib treatment. In this review, we describe current therapies for mCRPC, the rationale for anti-PARP therapies, the pharmacology of olaparib for prostate cancer, clinical trials of olaparib for mCRPC, our clinical experience with olaparib for prostate cancer at a comprehensive cancer center, and future directions of olaparib for the treatment of mCRPC. Olaparib may constitute a promising treatment to prolong survival in patients with mCRPC, with an acceptable adverse effect profile. As the role of PARP inhibition in prostate cancer and other malignancies becomes further elucidated, olaparib may be shown to be beneficial for other patient populations.

MP83-06 COMBINATION THERAPY WITH EPI-002 AND PARP INHIBITOR FOR CASTRATION-RESISTANT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II1 Apr 2017MP83-06 COMBINATION THERAPY WITH EPI-002 AND PARP INHIBITOR FOR CASTRATION-RESISTANT PROSTATE CANCER Yusuke Ito, C. Adriana Banuelos, and Marianne D. Sadar Yusuke ItoYusuke Ito More articles by this author , C. Adriana BanuelosC. Adriana Banuelos More articles by this author , and Marianne D. SadarMarianne D. Sadar More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2574AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Following the results of the TOPARP-A Phase II trial, Olaparib, an oral PARP inhibitor was recently recognized as a FDA breakthrough therapy for metastatic castration recurrent prostate cancer (mCRPC) patients who have germline mutations in DNA repair genes. Although these results are noteworthy, the problem remains of how to treat the remaining mCRPC patients who do not have detectable germline mutations of DNA repair genes. Androgen receptor (AR) signaling regulates DNA repair in prostate cancer and AR modulating drugs induce DNA damage. Thus a combination approach using AR modulating drugs with a PARP inhibitor could be a promising option for the treatment of mCRPC. All currently approved AR modulating drugs, such as enzalutamide and abiraterone, either directly or indirectly target the AR C-terminus ligand-binding domain (LBD). Such drugs are often unsuccessful due to the emergence of AR splice variants (ARV-7, ARv567es) that are constitutively active and lack a LBD. EPI-002 is a first-in-class AR antagonist that targets both full-length AR and AR splice variants. Here we present data to support that a combination of Olaparib, a PARP inhibitor, and EPI-002 have beneficial effects in vitro. METHODS Combination therapy using EPI-002 and Olaparib were evaluated in vitro using human prostate cancer cells, LNCaP (androgen sensitive and expresses full-length AR) and LNCaP95, an androgen-independent cell line that expresses full-length AR and AR-V7 and is resistant to enzalutamide. The effects of monotherapy and combination therapy on cell cycle and DNA damage were analysed using FACS and Western blot. RESULTS Unexpectedly, EPI-002 caused an enormous decrease of Checkpoint kinase 1 (Chk1) protein levels in LNCaP and LNCaP95 cells. Chk1 is one of the important mediators in cell cycle checkpoint during the DNA damage response. Whereas, enzalutamide also decreased the expression of Chk1 in LNCaP, it had no effect on Chk1 levels in LNCaP95 cells. Consistent with these data, AR knockdown in LNCaP cells also decreased Chk1 levels. The PARP inhibitor, Olaparib, induced phosphorylation of Chk1. EPI-002 induced G1 cell cycle arrest whereas Olaparib induced G2/M cell cycle arrest. FACS analysis of γH2AX showed increased DNA damage with combination therapy compared to monotherapies. CONCLUSIONS EPI-002 decreased the expression of Chk1 in prostate cancer cells that expressed both full-length AR and AR-V7. Combination therapy of EPI-002 plus Olaparib may provide a therapeutic approach for prostate cancer that expresses AR-V7. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1108 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Yusuke Ito More articles by this author C. Adriana Banuelos More articles by this author Marianne D. Sadar More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Inhibition of androgen receptor by decoy molecules delays progression to castration-recurrent prostate cancer.

Androgen receptor (AR) is a member of the steroid receptor family and a therapeutic target for all stages of prostate cancer. AR is activated by ligand binding within its C-terminus ligand-binding domain (LBD). Here we show that overexpression of the AR NTD to generate decoy molecules inhibited both the growth and progression of prostate cancer in castrated hosts. Specifically, it was shown that lentivirus delivery of decoys delayed hormonal progression in castrated hosts as indicated by increased doubling time of tumor volume, prolonged time to achieve pre-castrate levels of serum prostate-specific antigen (PSA) and PSA nadir. These clinical parameters are indicative of delayed hormonal progression and improved therapeutic response and prognosis. Decoys reduced the expression of androgen-regulated genes that correlated with reduced in situ interaction of the AR with androgen response elements. Decoys did not reduce levels of AR protein or prevent nuclear localization of the AR. Nor did decoys interact directly with the AR. Thus decoys did not inhibit AR transactivation by a dominant negative mechanism. This work provides evidence that the AR NTD plays an important role in the hormonal progression of prostate cancer and supports the development of AR antagonists that target the AR NTD.

Highlights of This Issue

MYC is a pleiotropic transcription factor that plays an important role in promoting the development and progression of human tumors. In the current study, Posternak and colleagues expand on a global role for MYC in regulating mRNA cap methylation. MYC specifically targets genes involved in Wnt/β-catenin signaling leading to augmented translational capacity and increased Wnt signaling activity. Mechanistically, MYC promotes recruitment of RNA methyltransferase to Wnt signaling gene promoters in part through an interaction between the TRRAP/TIP60 acetyltransferase complex and TFIIH. These data provide additional avenues to pursue in the development of targeted therapies for MYC-driven cancers.Prostate stem cells and prostate cancer stem cells have low or no androgen receptor (AR) expression. The ABC transporter, ABCG2, effluxes dihydrotestosterone (DHT) and is a prostate stem cell marker. Sabnis and colleagues demonstrate that inhibition of the ABCG2-mediated DHT efflux, promotes AR nuclear translocation, activation of AR target genes, and AR-dependent luminal differentiation. ABCG2 inhibition in the presence of DHT reduced the prostate cancer stem cell population and reduced growth rates in the CWR-R1 castration recurrent prostate cancer cell line in vitro and in vivo xenografts. Thus, ABCG2 inhibition is a possible differentiation therapeutic option in prostate cancer.The estrogen receptor (ER) is well known for its ability to integrate hormonal signals that promote the expression of critical genes involved in breast cancer differentiation, proliferation, and survival. Evidence indicates that ER levels correlate with CpG methylation status suggesting that ER controls expression through pathways that involve DNA methylation. Comprehensive profiling was used to identify ER-silenced genes that could be re-activated using demethylating agents, therapeutic loss of ER, or re-suppressed by gain of ER activity. Importantly, ER-dependent DNA methylation targets were identified in key biological pathways and a subset associated with patient outcomes suggesting the potential for new biomarkers.Patients with glioblastoma multiforme (GBM), a common malignant brain tumor, display poor survival. Glioma stem-like cells (GSC) enhance GBM escape from chemotherapy and promote tumor relapse. Hu and colleagues demonstrate that astrocyte elevated gene 1 (AEG-1) plays a crucial role in glioma stem cell biology. The expression of AEG-1 is elevated in GBM neurospheres, facilitating β-catenin nuclear translocation and elevating expression of stem cell markers CD133 and SOX2 and activating downstream Wnt signaling genes. Accordingly, this study identifies a novel AEG-1 signaling axis in GSCs and establishes AEG-1 as a therapeutic target for glioblastoma multiforme.

Src promotes castration-recurrent prostate cancer through androgen receptor-dependent canonical and non-canonical transcriptional signatures.

Progression of prostate cancer (PC) to castration-recurrent growth (CRPC) remains dependent on sustained expression and transcriptional activity of the androgen receptor (AR). A major mechanism contributing to CRPC progression is through the direct phosphorylation and activation of AR by Src-family (SFK) and ACK1 tyrosine kinases. However, the AR-dependent transcriptional networks activated by Src during CRPC progression have not been elucidated. Here, we show that activated Src (Src527F) induces androgen-independent growth in human LNCaP cells, concomitant with its ability to induce proliferation/survival genes normally induced by dihydrotestosterone (DHT) in androgen-dependent LNCaP and VCaP cells. Src induces additional gene signatures unique to CRPC cell lines, LNCaP-C4-2 and CWR22Rv1, and to CRPC LuCaP35.1 xenografts. By comparing the Src-induced AR-cistrome and/or transcriptome in LNCaP to those in CRPC and LuCaP35.1 tumors, we identified an 11-gene Src-regulated CRPC signature consisting of AR-dependent, AR binding site (ARBS)-associated genes whose expression is altered by DHT in LNCaP[Src527F] but not in LNCaP cells. The differential expression of a subset (DPP4, BCAT1, CNTNAP4, CDH3) correlates with earlier PC metastasis onset and poorer survival, with the expression of BCAT1 required for Src-induced androgen-independent proliferation. Lastly, Src enhances AR binding to non-canonical ARBS enriched for FOXO1, TOP2B and ZNF217 binding motifs; cooperative AR/TOP2B binding to a non-canonical ARBS was both Src- and DHT-sensitive and correlated with increased levels of Src-induced phosphotyrosyl-TOP2B. These data suggest that CRPC progression is facilitated via Src-induced sensitization of AR to intracrine androgen levels, resulting in the engagement of canonical and non-canonical ARBS-dependent gene signatures.

Molecular mechanisms underlying resistance to androgen deprivation therapy in prostate cancer.

Prostate cancer (PCa) is the most widely diagnosed male cancer in the Western World and while low- and intermediate-risk PCa patients have a variety of treatment options, metastatic patients are limited to androgen deprivation therapy (ADT). This treatment paradigm has been in place for 75 years due to the unique role of androgens in promoting growth of prostatic epithelial cells via the transcription factor androgen receptor (AR) and downstream signaling pathways. Within 2 to 3 years of ADT, disease recurs—at which time, patients are considered to have castration-recurrent PCa (CR-PCa). A universal mechanism by which PCa becomes resistant to ADT has yet to be discovered. In this review article, we discuss underlying molecular mechanisms by which PCa evades ADT. Several major resistance pathways center on androgen signaling, including intratumoral and adrenal androgen production, AR-overexpression and amplification, expression of AR mutants, and constitutively-active AR splice variants. Other ADT resistance mechanisms, including activation of glucocorticoid receptor and impairment of DNA repair pathways are also discussed. New therapies have been approved for treatment of CR-PCa, but increase median survival by only 2-8 months. We discuss possible mechanisms of resistance to these new ADT agents. Finally, the practicality of the application of “precision oncology” to this continuing challenge of therapy resistance in metastatic or CR-PCa is examined. Empirical validation and clinical-based evidence are definitely needed to prove the superiority of “precision” treatment in providing a more targeted approach and curative therapies over the existing practices that are based on biological “cause-and-effect” relationship.

Abstract LB-256: Clonogenic growth of androgen receptor-overexpressing models of castration recurrent prostate cancer cells becomes insensitive to receptor ligands when their complete cellular secretory milieu is restored

Abstract Evidence that androgen receptor (AR) overexpression causes castration recurrent prostate cancer (CRPC) cells to thrive on minimal residual post-castrate androgen is derived from studies of in vitro colony formation in the absence of cell-secreted factors or from studies demonstrating synergy between androgen and individual signaling pathways in isolation. We examined androgen-dependence using in vivo generated CRPC model cells overexpressing full length AR (C4-2 cells) or co-expressing a splice-variant (22Rv1 cells). The cells showed robust colony formation in conditioned media lacking androgenic activity derived from the same cells or from HEK293 fibroblasts. This effect was (i) not enhanced by androgen (ii) insensitive to blocking ligand-dependent transcriptional activity of AR, (iii) insensitive to mifepristone (iv) AR-dependent, (v) associated with AR phosphorylation and (vi) similar to androgen-stimulation in sensitivity to signaling pathway inhibitors. This ligand-insensitive response of overexpressed AR to the total cellular secretory complement may limit interventions targeting hormonal factors in CRPC. Citation Format: Mugdha Patki, Yanfang Huang, Manohar Ratnam. Clonogenic growth of androgen receptor-overexpressing models of castration recurrent prostate cancer cells becomes insensitive to receptor ligands when their complete cellular secretory milieu is restored. [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 LB-256.

Characterization of fibroblast-free CWR-R1ca castration-recurrent prostate cancer cell line.

The previously established CWR-R1 cell line has been used as an in vitro model representing castration-recurrent prostate cancer. Microscopic observation of subconfluent cells demonstrated two distinct cellular morphologies: polygonal closely aggregated epithelial cells surrounded by bipolar fibroblastic cells with long processes. This study sought to establish and characterize a fibroblast-free derivative of the CWR-R1 cell line. The CWR-R1ca cell line was established from CWR-R1 cells by removing fibroblasts using multiple cycles of short-term trypsinization, cloning, and pooling single-cell colonies. Authentication of fibroblast-free CWR-R1ca cells was demonstrated by analyzing the expression of cytodifferentiation and prostate-associated markers, DNA and cytogenetic profiling, and growth pattern in the absence or presence of androgen. CWR-R1ca is an androgen-sensitive cell line that expresses the androgen receptor (AR) and its splice variant 7 and the luminal epithelia markers, CK-8, CK-18, and c-Met. CWR-R1fb fibroblasts isolated from CWR-R1 cells express AR, hepatocyte growth factor-α, and mouse β-actin but not AR-V7 or epithelial markers. Cytogenetic analysis of CWR-R1ca cells revealed a hyperdiploid male with numerical gains in chromosomes 1, 7, 8, 10, 11, and 12, deletion of one chromosome 2 allele, structural abnormalities that include der(1)t(1:4), der(4)t(2:4), der(10)t(4:10), and an unbalanced reciprocal translocation between chromosome 6 and 14. DNA-profiling revealed that CWR-R1ca cells had significant short-tandem repeat marker homology with CWR22Pc and CWR22Rv1 cell lines, which indicated lineage derivation from CWR22 prostate cancer xenografts. CWR-R1ca cells were responsive to the growth stimulatory effects of dihydrotestosterone (DHT) in the femtomolar range. This study establishes CWR-R1ca cells as a fibroblast-free derivative of the castration-recurrent CWR-R1 cell line. Prostate 76:1067-1077, 2016. © 2016 Wiley Periodicals, Inc.

Prostate Cancer, Version 1.2016.

The NCCN Guidelines for Prostate Cancer address staging and risk assessment after an initial diagnosis of prostate cancer and management options for localized, regional, and metastatic disease. Recommendations for disease monitoring, treatment of recurrent disease, and systemic therapy for metastatic castration-recurrent prostate cancer also are included. This article summarizes the NCCN Prostate Cancer Panel's most significant discussions for the 2016 update of the guidelines, which include refinement of risk stratification methods and new options for the treatment of men with high-risk and very-high-risk disease and progressive castration-naïve disease.

An Sp1 Modulated Regulatory Region Unique to Higher Primates Regulates Human Androgen Receptor Promoter Activity in Prostate Cancer Cells.

Androgen receptor (AR) mediated signalling is necessary for normal development of the prostate gland and also drives prostate cancer (PCa) cell growth and survival, with many studies showing a correlation between increased receptor levels and therapy resistance with progression to fatal castrate recurrent PCa (CRPC). Although it has been held for some time that the transcription factor Sp1 is the main stimulator of AR gene transcription, comprehensive knowledge of the regulation of the AR gene remains incomplete. Here we describe and characterise in detail two novel active regulatory elements in the 5’UTR of the human AR gene. Both of these elements contain overlapping binding sites for the positive transcription factor Sp1 and the repressor protein pur-α. Aberrant cell signalling is characteristic of PCa and the transcriptional activity of the AR promoter in PCa cells is dependent upon the relative amounts of the two transcription factors. Together with our corroboration of the dominant role of Sp1, the findings support the rationale of targeting this transcription factor to inhibit tumour progression. This should be of particular therapeutic relevance in CRPC where the levels of the repressor pur-α are reduced.

Prolactin- and testosterone-induced carboxypeptidase-D correlates with increased nitrotyrosines and Ki67 in prostate cancer.

Carboxypeptidase-D (CPD) cleaves C-terminal arginine for conversion to nitric oxide (NO) by nitric oxide synthase (NOS). Prolactin (PRL) and androgens stimulate CPD gene transcription and expression, which increases intracellular production of NO to promote viability of prostate cancer (PCa) cells in vitro. The current study evaluated whether hormonal upregulation of CPD and NO promote PCa cell viabilty in vivo, by correlating changes in expression of CPD and nitrotyrosine residues (products of NO action) with proliferation marker Ki67 and associated proteins during PCa development and progression. Fresh prostate tissues, obtained from 40 men with benign prostatic hyperplasia (BPH) or PCa, were flash-frozen at the time of surgery and used for RT-qPCR analysis of CPD, androgen receptor (AR), PRL receptor (PRLR), eNOS, and Ki67 levels. Archival paraffin-embedded tissues from 113 men with BPH or PCa were used for immunohistochemical (IHC) analysis of CPD, nitrotyrosines, phospho-Stat5 (for activated PRLR), AR, eNOS/iNOS, and Ki67. RT-qPCR and IHC analyses showed strong AR and PRLR expression in benign and malignant prostates. CPD mRNA levels increased ∼threefold in PCa compared to BPH, which corresponded to a twofold increase in Ki67 mRNA levels. IHC analysis showed a progressive increase in CPD from 11.4 ± 2.1% in benign to 21.8 ± 3.2% in low-grade (P = 0.007), 40.7 ± 4.0% in high-grade (P < 0.0001) and 50.0 ± 9.5% in castration-recurrent PCa (P < 0.0001). Immunostaining for nitrotyrosines and Ki67 mirrored these increases during PCa progression. CPD, nitrotyrosines, and Ki67 tended to co-localize, as did phospho-Stat5. CPD, nitrotyrosine, and Ki67 levels were higher in PCa than in benign and tended to co-localize, along with phospho-Stat5. The strong correlation in expression of these proteins in benign and malignant prostate tissues, combined with abundant AR and PRLR, supports in vitro evidence that the CPD-Arg-NO pathway is involved in the regulation of PCa cell proliferation. It further highlights a role for PRL in the development and progression of PCa.

Characterization of niphatenones that inhibit androgen receptor N-terminal domain.

Androgen ablation therapy causes a temporary reduction in tumor burden in patients with advanced prostate cancer. Unfortunately the malignancy will return to form lethal castration-recurrent prostate cancer (CRPC). The androgen receptor (AR) remains transcriptionally active in CRPC in spite of castrate levels of androgens in the blood. AR transcriptional activity resides in its N-terminal domain (NTD). Possible mechanisms of continued AR transcriptional activity may include, at least in part, expression of constitutively active splice variants of AR that lack the C-terminal ligand-binding domain (LBD). Current therapies that target the AR LBD, would not be effective against these AR variants. Currently no drugs are clinically available that target the AR NTD which should be effective against these AR variants as well as full-length AR. Niphatenones were originally isolated and identified in active extracts from Niphates digitalis marine sponge. Here we begin to characterize the mechanism of niphatenones in blocking AR transcriptional activity. Both enantiomers had similar IC50 values of 6 µM for inhibiting the full-length AR in a functional transcriptional assay. However, (S)-niphatenone had significantly better activity against the AR NTD compared to (R)-niphatenone. Consistent with niphatenones binding to and inhibiting transactivation of AR NTD, niphatenones inhibited AR splice variant. Niphatenone did not affect the transcriptional activity of the related progesterone receptor, but slightly decreased glucocorticoid receptor (GR) activity and covalently bound to GR activation function-1 (AF-1) region. Niphatenone blocked N/C interactions of AR without altering either AR protein levels or its intracellular localization in response to androgen. Alkylation with glutathione suggests that niphatenones are not a feasible scaffold for further drug development.

Abstract 2116: Mapping peptides critical for association of the androgen receptor with Elk1

Abstract Both early stage and advanced prostate tumors are generally dependent on the androgen receptor (AR) for growth. Indeed a major clinical approach in management of the disease is to disrupt the AR signaling in the tumors by androgen ablation and the use of anti-androgen drugs. However, these options present major drawbacks including limited efficacy in advanced disease and many undesirable side effects on non-target tissues. Therefore a more tactical therapy approach would be one that could disrupt a functional arm of AR signaling that is critical for prostate tumor growth but not for the essential physiological roles of AR in normal adult tissues. A considerable amount of evidence suggests that in prostate cancer, androgen/AR signaling is redirected to support tumor growth through the association of AR with critical growth promoting genes via tethering proteins. Our previous studies have identified Elk1, a genetically redundant DNA binding transcription factor, as an AR tethering protein essential for AR-dependent growth in established models of both hormone-dependent prostate cancer and castration recurrent prostate cancer. Elk1 recruits AR to chromatin sites to upregulate a major subset of genes that is strongly and primarily enriched for cell growth functions. Further studies in the lab demonstrate that the A/B domain of the AR alone is capable of co-activation of Elk1, suggesting that Elk1 may also mediate the hormone-independent growth supporting function of AR splice variants. Peptide or small molecule inhibitors of the Elk1-AR interaction may therefore selectively target the growth supporting function of AR in the spectrum of prostate tumors, obviating the need for androgen ablation. Toward this end we undertook to map peptide segment(s) of Elk1and AR that are essential for the Elk1-AR synergy. In this study we used deletional and mutational analysis together with a promotor-reporter assay and a mammalian two-hybrid assay. The results demonstrate that the ability to recruit AR resides largely in the C-terminal activation domain of Elk1 (amino acids 307-428) and that residues within segments 367-428 and 307-337 are essential for this interaction. They also demonstrate that the recruitment of AR by Elk1 does not require Elk1 phosphorylation. Critical sites within the AR-A/B domain required for its association with Elk1 reside in the N-terminal region that is distal to known sites of co-regulator binding. This information will guide the development of peptide inhibitors of Elk1-dependent growth induction by androgen/AR and set the stage for future development of small molecule inhibitors. Citation Format: Rayna Rosati, Mugdha Patki, Venkatesh Chari, Manohar Ratnam. Mapping peptides critical for association of the androgen receptor with Elk1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2116. doi:10.1158/1538-7445.AM2014-2116

A genome-wide RNAi screen identifies FOXO4 as a metastasis-suppressor through counteracting PI3K/AKT signal pathway in prostate cancer.

Activation of the PI3K/AKT signal pathway is a known driving force for the progression to castration-recurrent prostate cancer (CR-CaP), which constitutes the major lethal phenotype of CaP. Here, we identify using a genomic shRNA screen the PI3K/AKT-inactivating downstream target, FOXO4, as a potential CaP metastasis suppressor. FOXO4 protein levels inversely correlate with the invasive potential of a panel of human CaP cell lines, with decreased mRNA levels correlating with increased incidence of clinical metastasis. Knockdown (KD) of FOXO4 in human LNCaP cells causes increased invasion in vitro and lymph node (LN) metastasis in vivo without affecting indices of proliferation or apoptosis. Increased Matrigel invasiveness was found by KD of FOXO1 but not FOXO3. Comparison of differentially expressed genes affected by FOXO4-KD in LNCaP cells in culture, in primary tumors and in LN metastases identified a panel of upregulated genes, including PIP, CAMK2N1, PLA2G16 and PGC, which, if knocked down by siRNA, could decrease the increased invasiveness associated with FOXO4 deficiency. Although only some of these genes encode FOXO promoter binding sites, they are all RUNX2-inducible, and RUNX2 binding to the PIP promoter is increased in FOXO4-KD cells. Indeed, the forced expression of FOXO4 reversed the increased invasiveness of LNCaP/shFOXO4 cells; the forced expression of FOXO4 did not alter RUNX2 protein levels, yet it decreased RUNX2 binding to the PIP promoter, resulting in PIP downregulation. Finally, there was a correlation between FOXO4, but not FOXO1 or FOXO3, downregulation and decreased metastasis-free survival in human CaP patients. Our data strongly suggest that increased PI3K/AKT-mediated metastatic invasiveness in CaP is associated with FOXO4 loss, and that mechanisms to induce FOXO4 re-expression might suppress CaP metastatic aggressiveness.

Enzalutamide: looking back at its preclinical discovery

Introduction: In patients with metastatic prostate cancer (PCa), castration is the standard first-line therapy. However, all patients eventually develop castration-recurrent PCa (CRPC). In these patients who fail the first-line therapy, enzalutamide has emerged as a viable alternative. Enzalutamide is a second-generation small molecule androgen receptor (AR) antagonist that blocks the AR nuclear translocation and DNA binding without any known AR agonist activity.Areas covered: This review provides an overview of the history of the oral selective AR modulator, enzalutamide, and discusses its discovery and current preclinical experimental results including resistance mechanisms. The article illustrates the history of discovery based on enzalutamide’s mechanism of action. Furthermore, the authors highlight the drug’s clinical development and post-launch developments.Expert opinion: The landscape of CRPC has changed dramatically over the last few years, as new agents with proven benefit in overall survival have been approved. Compared to the average time of 10 – 15 years for a new drug to go from pre-clinical discovery to registration, enzalutamide obtained FDA approval in < 6 years after its initial characterization. Enzalutamide, a targeted agent of the androgen-AR axis, has shown promising results in CRPC and is generally well tolerated. However, drug resistance inevitably emerges is a severe limitation. The authors believe that further clinical studies that look at its use as either a combinational or sequential therapy could help to address these concerns.