Martin Gleave

Although systemic androgen deprivation prolongs life in advanced prostate cancer, remissions are temporary because patients almost uniformly progress to a state of a castration-resistant prostate cancer (CRPC) as indicated by recurring PSA. This complex process of progression does not seem to be stochastic as the timing and phenotype are highly predictable, including the observation that most androgen-regulated genes are reactivated despite castrate levels of serum androgens. Recent evidence indicates that intraprostatic levels of androgens remain moderately high following systemic androgen deprivation therapy, whereas the androgen receptor (AR) remains functional, and silencing the AR expression following castration suppresses tumor growth and blocks the expression of genes known to be regulated by androgens. From these observations, we hypothesized that CRPC progression is not independent of androgen-driven activity and that androgens may be synthesized de novo in CRPC tumors leading to AR activation. Using the LNCaP xenograft model, we showed that tumor androgens increase during CRPC progression in correlation to PSA up-regulation. We show here that all enzymes necessary for androgen synthesis are expressed in prostate cancer tumors and some seem to be up-regulated during CRPC progression. Using an ex vivo radiotracing assays coupled to high-performance liquid chromatography-radiometric/mass spectrometry detection, we show that tumor explants isolated from CRPC progression are capable of de novo conversion of [(14)C]acetic acid to dihydrotestosterone and uptake of [(3)H]progesterone allows detection of the production of six other steroids upstream of dihydrotestosterone. This evidence suggests that de novo androgen synthesis may be a driving mechanism leading to CRPC progression following castration.

Article20 May 2011free access The androgen receptor fuels prostate cancer by regulating central metabolism and biosynthesis Charles E Massie Charles E Massie CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Andy Lynch Andy Lynch CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Antonio Ramos-Montoya Antonio Ramos-Montoya CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Joan Boren Joan Boren CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Rory Stark Rory Stark CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Ladan Fazli Ladan Fazli The Vancouver Prostate Centre, Vancouver, British Columbia, Canada Search for more papers by this author Anne Warren Anne Warren Department of Pathology, Addenbrookes Hospital, Cambridge, UK Search for more papers by this author Helen Scott Helen Scott CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Basetti Madhu Basetti Madhu CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Naomi Sharma Naomi Sharma CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Helene Bon Helene Bon CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Vinny Zecchini Vinny Zecchini CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Donna-Michelle Smith Donna-Michelle Smith CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Gina M DeNicola Gina M DeNicola CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Nik Mathews Nik Mathews CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Michelle Osborne Michelle Osborne CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author James Hadfield James Hadfield CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Stewart MacArthur Stewart MacArthur CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Boris Adryan Boris Adryan Cambridge Systems Biology Centre and Department of Genetics, University of Cambridge, Cambridge, UK Search for more papers by this author Scott K Lyons Scott K Lyons CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Kevin M Brindle Kevin M Brindle CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author John Griffiths John Griffiths CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Martin E Gleave Martin E Gleave The Vancouver Prostate Centre, Vancouver, British Columbia, Canada Search for more papers by this author Paul S Rennie Paul S Rennie The Vancouver Prostate Centre, Vancouver, British Columbia, Canada Search for more papers by this author David E Neal David E Neal CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Ian G Mills Corresponding Author Ian G Mills CRUK Cambridge Research Institute, Cambridge, UK Centre for Molecular Medicine Norway, Nordic European Molecular Biology Laboratory Partnership, University of Oslo, Oslo, Norway Search for more papers by this author Charles E Massie Charles E Massie CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Andy Lynch Andy Lynch CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Antonio Ramos-Montoya Antonio Ramos-Montoya CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Joan Boren Joan Boren CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Rory Stark Rory Stark CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Ladan Fazli Ladan Fazli The Vancouver Prostate Centre, Vancouver, British Columbia, Canada Search for more papers by this author Anne Warren Anne Warren Department of Pathology, Addenbrookes Hospital, Cambridge, UK Search for more papers by this author Helen Scott Helen Scott CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Basetti Madhu Basetti Madhu CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Naomi Sharma Naomi Sharma CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Helene Bon Helene Bon CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Vinny Zecchini Vinny Zecchini CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Donna-Michelle Smith Donna-Michelle Smith CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Gina M DeNicola Gina M DeNicola CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Nik Mathews Nik Mathews CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Michelle Osborne Michelle Osborne CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author James Hadfield James Hadfield CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Stewart MacArthur Stewart MacArthur CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Boris Adryan Boris Adryan Cambridge Systems Biology Centre and Department of Genetics, University of Cambridge, Cambridge, UK Search for more papers by this author Scott K Lyons Scott K Lyons CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Kevin M Brindle Kevin M Brindle CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author John Griffiths John Griffiths CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Martin E Gleave Martin E Gleave The Vancouver Prostate Centre, Vancouver, British Columbia, Canada Search for more papers by this author Paul S Rennie Paul S Rennie The Vancouver Prostate Centre, Vancouver, British Columbia, Canada Search for more papers by this author David E Neal David E Neal CRUK Cambridge Research Institute, Cambridge, UK Search for more papers by this author Ian G Mills Corresponding Author Ian G Mills CRUK Cambridge Research Institute, Cambridge, UK Centre for Molecular Medicine Norway, Nordic European Molecular Biology Laboratory Partnership, University of Oslo, Oslo, Norway Search for more papers by this author Author Information Charles E Massie1, Andy Lynch1, Antonio Ramos-Montoya1, Joan Boren1, Rory Stark1, Ladan Fazli2, Anne Warren3, Helen Scott1, Basetti Madhu1, Naomi Sharma1, Helene Bon1, Vinny Zecchini1, Donna-Michelle Smith1, Gina M DeNicola1, Nik Mathews1, Michelle Osborne1, James Hadfield1, Stewart MacArthur1, Boris Adryan4, Scott K Lyons1, Kevin M Brindle1, John Griffiths1, Martin E Gleave2, Paul S Rennie2, David E Neal1,‡ and Ian G Mills 1,5,‡ 1CRUK Cambridge Research Institute, Cambridge, UK 2The Vancouver Prostate Centre, Vancouver, British Columbia, Canada 3Department of Pathology, Addenbrookes Hospital, Cambridge, UK 4Cambridge Systems Biology Centre and Department of Genetics, University of Cambridge, Cambridge, UK 5Centre for Molecular Medicine Norway, Nordic European Molecular Biology Laboratory Partnership, University of Oslo, Oslo, Norway ‡These authors contributed equally to this work *Corresponding author. Department of Uro-Oncology, CRUK Cambridge Research Institute, Robinson Way, Cambridge CB2 0RE, UK. Tel.: +44 122 340 4463; Fax: +44 122 340 4199; E-mail: [email protected] or [email protected] The EMBO Journal (2011)30:2719-2733https://doi.org/10.1038/emboj.2011.158 There is a Have you seen? (July 2011) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The androgen receptor (AR) is a key regulator of prostate growth and the principal drug target for the treatment of prostate cancer. Previous studies have mapped AR targets and identified some candidates which may contribute to cancer progression, but did not characterize AR biology in an integrated manner. In this study, we took an interdisciplinary approach, integrating detailed genomic studies with metabolomic profiling and identify an anabolic transcriptional network involving AR as the core regulator. Restricting flux through anabolic pathways is an attractive approach to deprive tumours of the building blocks needed to sustain tumour growth. Therefore, we searched for targets of the AR that may contribute to these anabolic processes and could be amenable to therapeutic intervention by virtue of differential expression in prostate tumours. This highlighted calcium/calmodulin-dependent protein kinase kinase 2, which we show is overexpressed in prostate cancer and regulates cancer cell growth via its unexpected role as a hormone-dependent modulator of anabolic metabolism. In conclusion, it is possible to progress from transcriptional studies to a promising therapeutic target by taking an unbiased interdisciplinary approach. Introduction The androgen receptor (AR) is a ligand activated transcription factor and is the main therapeutic target in prostate cancer. Androgen deprivation therapy (chemical castration) is an effective first-line therapy for prostate cancer, but despite good initial responses the recurrence of castrate-resistant disease is common and ultimately fatal. Functional studies have shown that the AR is essential for cell viability, proliferation and invasion in both hormone-sensitive and castrate-resistant prostate cancer (Haag et al, 2005; Hara et al, 2008; Snoek et al, 2009). These findings are supported by clinical studies reporting the sensitivity of castrate-resistant prostate cancer to second-generation AR antagonists and hormone synthesis blockade (Attar et al, 2009; Attard et al, 2009; Tran et al, 2009). In castrate-resistant disease, where tumours are less sensitive to androgen depletion, AR activity is maintained by gene amplification (Visakorpi et al, 1995), activating mutations (Veldscholte et al, 1990; Steinkamp et al, 2009) or signalling cross talk with other oncogenic pathways (Craft et al, 1999). All of these mechanisms suggest a strong selective pressure to maintain AR-regulated signalling pathways in castrate-resistant disease, although the nature of these important pathways has remained unclear. Previous studies have aimed to identify androgen-regulated genes or AR genomic binding sites (Wang et al, 2007; Jia et al, 2008; Yu et al, 2010). However, no single study has identified the transcriptional networks, which underlie AR dependency in both hormone naive and castrate-resistant prostate cancer. Individual studies have focussed on the use of a single model of prostate cancer (LNCaP or its in vitro-derived subclones) (Velasco et al, 2004; Massie et al, 2007; Wang et al, 2007, 2009), been limited by genome coverage on microarrays (Massie et al, 2007; Wang et al, 2007; Jia et al, 2008), did not map sites of transcriptional activity (Massie et al, 2007; Wang et al, 2009) or assessed only a limited number of time points following androgen stimulation (Velasco et al, 2004). These studies have provided important insights into the upstream mechanisms which direct the transcriptional activities of the AR and identified a number of transcription factors which cooperate with or antagonize AR activity (Wang et al, 2007; Jia et al, 2008; Yu et al, 2010). However, efforts to identify the key downstream targets of the AR in prostate cancer have identified indirect links to anabolic pathways (Heemers et al, 2001, 2004; Xu et al, 2006) or focussed on cell-cycle regulators (Knudsen et al, 1998; Wang et al, 2009), some of which appear only to be AR targets in models of castrate-resistant prostate cancer (Wang et al, 2009). Therefore, a detailed map of AR-regulated genes in diverse models of prostate cancer is needed to better understand the essential signalling pathways downstream of the AR in both hormone-sensitive and castrate-resistant stages of the disease. Results Identifying a core set of direct AR-regulated genes To identify direct AR-regulated genes, we combined genome-wide AR binding profiles with detailed transcript profiling. We also integrated androgen-stimulated recruitment of the transcriptional machinery to identify a core set of AR binding sites, which regulate gene expression in prostate cancer cells. First, we mapped AR binding profiles in two cell lines which represent distinct molecular subtypes of prostate cancer: one harbouring an AR ligand binding domain mutation (LNCaP); one harbouring an AR gene amplification (VCaP). Using chromatin immunoprecipitation with direct Solexa sequencing (ChIP-seq), we identified 11 053 AR binding sites in LNCaP cells and 51 811 androgen-dependent AR binding sites in VCaP cells (Supplementary Tables S1 and S2). VCaP cells harbour a copy number gain of the AR gene locus resulting in elevated AR expression (Supplementary Figure S1; Makkonen et al, 2011), which may at least in part explain the larger number of AR binding sites found in VCaP compared with LNCaP cells. Despite the difference in total number of identified binding sites, over 90% of the LNCaP AR binding sites were also found in the VCaP cells (Figure 1A; Supplementary Figure S2; Supplementary Table S3), suggesting that these core binding sites are commonly occupied by the AR even in distinct molecular subtypes of prostate cancer. The common AR binding sites between LNCaP and VCaP cells included all established AR target genes (Figure 1A; Supplementary Figure S2), had significant correlations with other published data sets (Supplementary Figure S3) and identified thousands of AR targets not identified in previous AR ChIP studies (Supplementary Table S3). As a resource we have compiled all published AR ChIP-chip studies together with our data in Supplementary Table S3. Figure 1.Mapping transcriptional targets of the AR. (A) ChIP-seq enrichment profiles for AR and RNAP II with or without androgen treatment and in LNCaP or VCaP cells, as indicated (cells cultured in steroid depleted media and treated with 1 nM R1881 or 0.01% ethanol for 4 h). Location of the PSA gene is indicated below enrichment plots, arrow indicates the direction of transcription. Androgen-stimulated expression is shown at the bottom, represented as a heatmap showing expression changes with time following androgen stimulation (1 nM R1881, data from Illumina beadarray gene expression time course). (B) Venn diagram showing the overlap between AR binding sites and androgen-dependent RNAP II enriched genomic regions (1 nM R1881, 4 h; data from intersects of all peaks overlapping by >1 bp). (C) Gene set enrichment analysis (GSEA) of androgen-regulated genes (Illumina beadarray androgen stimulation time course), using gene sets identified within genomic windows from 1 to 500 kb from AR binding sites. (D) Combined analysis of AR binding sites, androgen-dependent RNAP II enriched regions and androgen-regulated genes. Genes are grouped by expression changes early (<4 h), late (>4 h), up, down or no change in response to androgen stimulation (left). Pie charts indicate the proportion of genes with adjacent AR, RNAP II or overlapping AR-RNAP II sites in each set (<25 kb from gene boundaries; groups indicated above). (E) Sequence logos for 15 and 6 bp AR/GR binding motifs (P-values indicate enrichment in AR-RNAP II overlapping regions). (F) Venn diagram showing the occurrence of 15 and 6 bp AR binding motifs in androgen-dependent AR-RNAP II overlapping sites. Download figure Download PowerPoint Next, we integrated the location of the transcriptional machinery on the prostate cancer genome together with detailed expression profiling. We identified 15 761 androgen-dependent RNAP II (serine 5 phosphorylated RNA polymerase II) regions in LNCaP cells using ChIP-seq (Supplementary Table S4), 1283 of which overlapped with androgen-stimulated AR binding sites (Figure 1B). The regions enriched for RNAP II alone (n=14 478) represent sites of paused, primed or active transcription (Bernstein et al, 2006; Core et al, 2008); however, sites to which the AR and RNAP II are dynamically co-recruited (n=1283) are candidate regions for androgen-stimulated transcriptional initiation (explored in detail below). Using Illumina BeadArrays, we made a detailed study of androgen-regulated gene expression with samples taken every 30 min for 4 h and then every hour up to 24 h following androgen stimulation of LNCaP cells. This detailed expression array time course made it possible to identify androgen-regulated gene expression changes based on trends with time following androgen stimulation (using autocorrelation), allowing detection of early and even small gene expression changes (see Materials and methods for details). In total, we found 3319 transcripts with altered expression in response to androgens, 1556 (47%) transcripts were upregulated and 1763 (53%) were downregulated (Supplementary Table S5). We made a combined analysis of our treatment contrast ChIP and detailed gene expression profiling to identify AR binding sites which recruit the transcriptional machinery and direct AR-regulated genes. Such combined analyses require a predefined genomic distance between transcript factor binding sites and genes, which is often set arbitrarily. To address this issue, we took a more empirical approach using gene set enrichment analysis (GSEA) to define the optimal genomic distance between AR binding sites (peaks) and androgen-regulated genes (gene boundaries). We found that genes located within 25 kb of an AR binding site were the most significantly enriched for androgen-regulated genes; the maximal enrichment score at 25 kb suggests that smaller genomic windows would include a greater proportion of false negatives and that larger genomic windows would include a greater proportion of false positives (Figure 1C; Supplementary Figure S3; Supplementary data). Therefore, using this 25 kb window we integrated the genomic locations of AR binding sites, androgen-stimulated recruitment of the core transcriptional machinery and detailed gene expression profiling (Figure 1D). This integrated analysis revealed that loci bound by either the AR or RNAP II alone contain genes which are upregulated, downregulated or not affected by AR signalling (Figure 1D). This finding is consistent with previous studies showing that the AR can directly activate or repress transcription (Margiotti et al, 2007; Prescott et al, 2007), that only a proportion of transcription factor binding sites are active in a given cellular context (Carroll et al, 2006) and that RNAP II enriched regions of the genome include both sites of active transcription and also sites where transcription has paused or stalled (Bernstein et al, 2006; Core et al, 2008). In contrast, the core overlap of androgen-stimulated AR binding sites and RNAP II recruitment showed specific enrichment for androgen upregulated transcripts (Figure 1D). Therefore, our combined analysis has identified a core set of AR binding sites that recruit the transcriptional machinery and upregulate the transcription of adjacent genes. These core AR targets may help to identify the features of transcriptionally active AR binding sites. Analysis of the genomic sequences underlying all AR binding sites found in LNCaP, VCaP or those identified in both cell lines revealed evolutionary conservation, enrichment of AR binding sequences and motifs for previously reported AR interacting transcription factors, including forkhead and NF-1 (Supplementary Figure S1; Supplementary Table S6) (Wang et al, 2007; Jia et al, 2008). The overlapping AR and RNAP II sites showed significant enrichment of 6 bp and 15 bp AR binding motifs (Figure 1E and F), but also showed enrichment for CREB and AHR binding motifs, in contrast to the full set of AR binding sites (Supplementary Figure S1; Supplementary Table S6). De novo motif analysis revealed an inverted-repeat 15 bp ARE similar to the in vitro-derived consensus binding motif (Roche et al, 1992) and also a 6 bp motif consisting of one half of the consensus 15 bp element, as previously reported in other AR ChIP studies (Massie et al, 2007; Wang et al, 2007) (Figure 1E; Supplementary Figure S1). Identifying cellular processes regulated by the AR Our combined analysis of AR, RNAP II and detailed expression profiling also identified a core set of direct androgen-regulated genes for further investigation. We defined direct AR targets as those genes induced by androgen treatment and for which there were overlapping hormone-induced AR binding sites within 25 kb of the genes. This definition identified a sufficient number of genes to allow pathway enrichment analysis and as a resource we have included a table noting these genes together with their androgen-stimulated expression profile, annotated with the location of adjacent AR and RNAP II binding sites (Supplementary Table S7). Interestingly, this core set of direct AR-regulated genes gave overlapping but distinct functional enrichment by gene ontology (GO) analysis compared with gene expression data alone (Figure 2A; Supplementary Tables S8 and S9). Figure 2.Functional annotation of direct AR-regulated genes. (A) Gene ontology (GO) network of direct AR-regulated genes (androgen upregulated genes within 25 kb of AR binding site, Cytoscape BiNGO analysis). (B) Gene set enrichment analysis (GSEA) plots for direct AR-regulated genes, showing enrichment for carbohydrate metabolism GO and the curated peroxisome proliferator-activated receptor γ co-activator 1-α (PPARGC1A) metabolic gene set. (C) Gene expression heatmaps, showing androgen-regulated genes within 25 kb of an AR binding site, grouped by functional categories (indicated above; data from Illumina beadarray time course in LNCaP cells; pathway annotations from GO annotations, KEGG pathways and literature reviews). (D) Schematic showing the locations of direct AR-regulated genes in metabolic and cell-cycle pathways. Red boxes indicate direct AR upregulated genes, blue boxes represent direct AR downregulated genes and yellow boxes indicate proteins not found to be regulated by the AR. Dashed lines indicate intermediate steps not shown. (E–I) Levels of (E) glucose, (F) lactate, (G) citrate, (H) succinate and (I) oxygen consumption rates were measured following growth of LNCaP prostate cancer cells in steroid depleted media with and without androgen stimulation (1 nM R1881). Expressed as μM lactate, μM citrate, μM succinate and % glucose consumption in cell culture media and nmol/ml/min oxygen consumption rate, all normalized to cell number (represented as mean±s.e.m.; each data point represents triplicate measurements). Download figure Download PowerPoint While many established classes of AR target gene were represented within the data sets as a whole, including cell-cycle regulators (e.g., CDC25, CDK6 and E2F1) and signalling molecules which have been implicated in prostate cancer (e.g., WWP1, ERBB2, MEK5, SGK1 and IGF1R) (Figure 2C; Craft et al, 1999; Hellawell et al, 2002; Mehta et al, 2003; Chen et al, 2007; Sherk et al, 2008), the combined analysis of direct AR targets and androgen-regulated genes also revealed a significant enrichment of metabolic targets (Figure 2A–C; Supplementary Table S9). GO analysis highlighted central metabolism and biosynthetic pathways among the direct AR targets (Figure 2A) and GSEA also revealed significant enrichment of central metabolism and metabolic gene signatures (Mootha et al, 2003) among direct AR-regulated genes (Figure 2B). Unlike other biological processes, metabolism has been subjected to decades of detailed biochemical study and consists of a series of interlinked and well-characterized processing steps. This makes the functional consequences of changes in enzyme expression uniquely predictable for metabolic pathways and made these pathways an ideal testing ground for the linkages established by our genomics data. We found that the AR directly upregulated expression of key steps in glucose uptake and glycolysis including glucose transporter 1 (GLUT1/SLC2A1), hexokinase I and II (HK1 and HK2), phosphofructokinase (PFK2/PFKFB2) and also many anabolic enzymes at both the transcript and protein level (Figure 2C and D; Supplementary Figures S4 and S5). Based on the annotation of these direct metabolic AR target genes within well-defined metabolic pathways we predicted that the AR may facilitate cell growth by promoting glucose uptake and anabolic metabolism (Figure 2D). To test these predictions from our genomics data, we undertook comprehensive metabolomic profiling to assess the effects of AR signalling on glucose consumption and lactate production, O2 consumption and detailed metabolite profiling of intracellular and extracellular metabolites using proton nuclear magnetic resonance (1H NMR) and glucose flux using carbon-13-labelled glucose with gas chromatography–mass spectrometry (GC/MS). The AR regulates aerobic glycolysis and anabolism in prostate cancer cells Androgen stimulation of prostate cancer cells increased glucose uptake and increased lactate production in normoxia, but had no effect on oxygen consumption, showing that AR signalling does indeed stimulate aerobic glycolysis (Christofk et al, 2008; Vander Heiden et al, 2009) (Figure 2E–I; Supplementary Figure S4F). Citrate levels were also increased following androgen stimulation (Figure 2G) while the levels of the tricarboxylic acid (TCA) cycle metabolite succinate remained unchanged (Figure 2H), further underscoring androgen stimulation of glycolysis and highlighting the truncated TCA cycle in prostate epithelial cells (Costello et al, 1997). We also found that AR signalling significantly stimulated anabolic synthesis, by measuring the flux from carbon-13-labelled glucose to amino acids (e.g., glutamine) and RNA (ribose) in response to androgen stimulation (Supplementary Figure S6; Figure 4F and G). Therefore by specifically upregulating these rate-limiting steps in glycolysis, the AR stimulates energy production and provides carbon needed for macromolecule synthesis. In addition, we found that the AR stimulated the expression of key anabolic enzymes, which utilize glucose metabolites (e.g., fatty acid synthase (FASN) and acetyl-CoA carboxylase α (ACACA)) and master regulators of biosynthesis (e.g., MTOR, encoded by FRAP1; Supplementary Figure S5). Therefore, AR signalling appears to coordinately regulate energy production and biosynthesis at multiple levels (Figure 2D), highlighting these metabolic pathways as potential targets to inhibit the growth of prostate cancer cells (Migita et al, 2009). CAMKK2 is a metabolic master regulator downstream of the AR in prostate cancer cells Identifying cancer-specific alterations that affect central metabolism is a major challenge for drug discovery in cancer biology. Therefore, we used clinical gene expression data (Rhodes et al, 2004) to assess cancer-specific expression of AR targets (Supplementary Table S7). Among the direct AR target genes, we identified calcium/calmodulin-dependent kinase kinase 2 (CAMKK2, Figure 3A) as consistently overexpressed in prostate cancer in nine independent clinical gene expression studies (Rhodes et al, 2004; Varambally et al, 2005), showing a similar pattern to the established prostate cancer marker AMACR (Jiang et al, 2001, 2002) (Figure 3B; Supplementary Figure S5). CAMKK2 was only highlighted by integrating AR genomic targets with clinical expression data, since we identified a core set of 1164 direct AR-regulated genes and on average CAMKK2 was ranked only 40th over the nine clinical studies (Figure 3B). CAMKK2 has previously been linked to central metabolism as an essential regulator of the metabolic sensor AMP kinase (AMPK) in the hypothalamus (Anderson et al, 2008), implicating CAMKK2 also as a regulator of cellular metabolism. The AR was recruited to the CAMKK2 promoter in both androgen-dependent and castrate-resistant prostate cancer cell lines (Supplementary Figure S7), suggesting that CAMKK2 is an AR target in both stages of the disease. CAMKK2 transcript and protein were upregulated early (<4 and <12 h, respectively) in response to androgen stimulation and downregulated in response to the AR antagonist bicalutamide, underscoring the direct regulation of CAMKK2 by the AR (Figure 3C and D; Supplementary Figure S7i). In clinical samples, we found CAMKK2 protein overexpression in two independent prostate cancer cohorts (Figure 3E; Supplementary Figure S7; Supplementary Table S10) and confirmed that CAMKK2 was a direct AR target in a panel of clinical prostate cancer samples using ChIP from human tissue (Figure 3F). These data highlight CAMKK2 as an AR-regulated gene in prostate cancer and in combination with the published role of CAMKK2 as a metabolic regulator (Anderson et al, 2008), prompted us to investigate the functional effects of AR-CAMKK2 signalling. Figure 3.The regulation and expression of CAMKK2 in prostate cancer. (A) ChIP-seq enrichment profiles for the AR (LNCaP and VCaP) and RNAP II (LNCaP), as indicated. The CAMKK2 gene position is indicated below, arrow indicates the direction of transcription. Androgen-regulated expression of CAMKK2 is sh

In advanced prostate cancer (APC), successful drug development as well as advances in imaging and molecular characterisation have resulted in multiple areas where there is lack of evidence or low level of evidence. The Advanced Prostate Cancer Consensus Conference (APCCC) 2017 addressed some of these topics. To present the report of APCCC 2017. Ten important areas of controversy in APC management were identified: high-risk localised and locally advanced prostate cancer; “oligometastatic” prostate cancer; castration-naïve and castration-resistant prostate cancer; the role of imaging in APC; osteoclast-targeted therapy; molecular characterisation of blood and tissue; genetic counselling/testing; side effects of systemic treatment(s); global access to prostate cancer drugs. A panel of 60 international prostate cancer experts developed the program and the consensus questions. The panel voted publicly but anonymously on 150 predefined questions, which have been developed following a modified Delphi process. Voting is based on panellist opinion, and thus is not based on a standard literature review or meta-analysis. The outcomes of the voting had varying degrees of support, as reflected in the wording of this article, as well as in the detailed voting results recorded in Supplementary data. The presented expert voting results can be used for support in areas of management of men with APC where there is no high-level evidence, but individualised treatment decisions should as always be based on all of the data available, including disease extent and location, prior therapies regardless of type, host factors including comorbidities, as well as patient preferences, current and emerging evidence, and logistical and economic constraints. Inclusion of men with APC in clinical trials should be strongly encouraged. Importantly, APCCC 2017 again identified important areas in need of trials specifically designed to address them. The second Advanced Prostate Cancer Consensus Conference APCCC 2017 did provide a forum for discussion and debates on current treatment options for men with advanced prostate cancer. The aim of the conference is to bring the expertise of world experts to care givers around the world who see less patients with prostate cancer. The conference concluded with a discussion and voting of the expert panel on predefined consensus questions, targeting areas of primary clinical relevance. The results of these expert opinion votes are embedded in the clinical context of current treatment of men with advanced prostate cancer and provide a practical guide to clinicians to assist in the discussions with men with prostate cancer as part of a shared and multidisciplinary decision-making process.

Pancreatic cancer is a disease with an extremely poor prognosis. Tumor protein 53-induced nuclear protein 1 ( TP53INP1 ) is a proapoptotic stress-induced p53 target gene. In this article, we show by immunohistochemical analysis that TP53INP1 expression is dramatically reduced in pancreatic ductal adenocarcinoma (PDAC) and this decrease occurs early during pancreatic cancer development. TP53INP1 reexpression in the pancreatic cancer-derived cell line MiaPaCa2 strongly reduced its capacity to form s.c., i.p., and intrapancreatic tumors in nude mice. This anti-tumoral capacity is, at least in part, due to the induction of caspase 3-mediated apoptosis. In addition, TP53INP1 −/− mouse embryonic fibroblasts (MEFs) transformed with a retrovirus expressing E1A/ras V12 oncoproteins developed bigger tumors than TP53INP1 +/+ transformed MEFs or TP53INP1 −/− transformed MEFs with restored TP53INP1 expression. Finally, TP53INP1 expression is repressed by the oncogenic micro RNA miR-155, which is overexpressed in PDAC cells. TP53INP1 is a previously unknown miR-155 target presenting anti-tumoral activity.

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

Purpose The prevalence and features of treatment-emergent small-cell neuroendocrine prostate cancer (t-SCNC) are not well characterized in the era of modern androgen receptor (AR)–targeting therapy. We sought to characterize the clinical and genomic features of t-SCNC in a multi-institutional prospective study. Methods Patients with progressive, metastatic castration-resistant prostate cancer (mCRPC) underwent metastatic tumor biopsy and were followed for survival. Metastatic biopsy specimens underwent independent, blinded pathology review along with RNA/DNA sequencing. Results A total of 202 consecutive patients were enrolled. One hundred forty-eight (73%) had prior disease progression on abiraterone and/or enzalutamide. The biopsy evaluable rate was 79%. The overall incidence of t-SCNC detection was 17%. AR amplification and protein expression were present in 67% and 75%, respectively, of t-SCNC biopsy specimens. t-SCNC was detected at similar proportions in bone, node, and visceral organ biopsy specimens. Genomic alterations in the DNA repair pathway were nearly mutually exclusive with t-SCNC differentiation ( P = .035). Detection of t-SCNC was associated with shortened overall survival among patients with prior AR-targeting therapy for mCRPC (hazard ratio, 2.02; 95% CI, 1.07 to 3.82). Unsupervised hierarchical clustering of the transcriptome identified a small-cell–like cluster that further enriched for adverse survival outcomes (hazard ratio, 3.00; 95% CI, 1.25 to 7.19). A t-SCNC transcriptional signature was developed and validated in multiple external data sets with &gt; 90% accuracy. Multiple transcriptional regulators of t-SCNC were identified, including the pancreatic neuroendocrine marker PDX1. Conclusion t-SCNC is present in nearly one fifth of patients with mCRPC and is associated with shortened survival. The near-mutual exclusivity with DNA repair alterations suggests t-SCNC may be a distinct subset of mCRPC. Transcriptional profiling facilitates the identification of t-SCNC and novel therapeutic targets.

While mutations affecting protein-coding regions have been examined across many cancers, structural variants at the genome-wide level are still poorly defined. Through integrative deep whole-genome and -transcriptome analysis of 101 castration-resistant prostate cancer metastases (109X tumor/38X normal coverage), we identified structural variants altering critical regulators of tumorigenesis and progression not detectable by exome approaches. Notably, we observed amplification of an intergenic enhancer region 624 kb upstream of the androgen receptor (AR) in 81% of patients, correlating with increased AR expression. Tandem duplication hotspots also occur near MYC, in lncRNAs associated with post-translational MYC regulation. Classes of structural variations were linked to distinct DNA repair deficiencies, suggesting their etiology, including associations of CDK12 mutation with tandem duplications, TP53 inactivation with inverted rearrangements and chromothripsis, and BRCA2 inactivation with deletions. Together, these observations provide a comprehensive view of how structural variations affect critical regulators in metastatic prostate cancer.

Abstract A model of human prostate cancer was established to study cellular interaction between prostate cancer and bone stroma in vivo . In this model, subcutaneous co‐injection of 2 non‐tumorigenic human cell lines‐LNCaP, a prostate cancer cell line, and MS, a bone stromal cell line‐into intact adult male mice resulted in formation of carcinomas that secreted prostate‐specific antigen (PSA), a clinically useful human serum prostate cancer marker. In castrated hosts, upon cellular interaction with bone fibroblasts, we observed the progression of these tumors from an androgen‐dependent (AD) to an androgen‐independent state (AI). We derived 4 LNCaP cell sublines from the chimeric LNCaP/MS tumors: the M subline from intact hosts and the C4, C4‐2 and C5 sublines from castrated hosts. The LNCaP sublines had chromosomal markers similar to those of the parental LNCaP cells and distinctly different from those of the MS bone stromal cell line. Although the parental and derived cell lines expressed similar steady‐state levels of orni‐thine decarboxylase transcript, the sublines expressed 5‐ to 10‐fold higher basal steady‐state levels of PSA transcript than did the parental LNCaP cell line. The LNCaP sublines formed 13‐ to 26‐fold more soft‐agar colonies than the parental LNCaP cell line. The sublines became tumorigenic, yielding an incidence of tumors in intact athymic mice of 7‐75%. The LNCaP sublines C4 and C5 (but not the parental and M cell line) formed tumors in castrated hosts when co‐injected with bone fibroblasts. A second‐generation LNCaP subline, C4‐2, was derived from a chimeric tumor induced by co‐inoculating castrated mouse with C4 cells and MS cells. We found that C4‐2 subline was tumorigenic when inoculated into castrated hosts in the absence of inductive fibroblasts. Moreover, C4‐2 was the only subline capable of forming soft‐agar colonies when cultured in serum‐free medium. In comparison with the parental LNCaP cells, the C4‐2 subline expressed lower steady‐state levels of androgen receptor (AR) protein and mRNA transcript and lost its androgen responsiveness in vitro . Our results suggest that certain genetic traits of prostate cancer cells may be selected or altered through an “adaptive” mechanism that involves cellular interaction with the bone stromal cells. © 1994 Wiley‐Liss, Inc.

Most early stage kidney cancers are renal cell carcinomas (RCCs), and most are diagnosed incidentally by imaging as small renal masses (SRMs). Indirect evidence suggests that most small RCCs grow slowly and rarely metastasize.To determine the progression and growth rates for newly diagnosed SRMs stratified by needle core biopsy pathology.A multicenter prospective phase 2 clinical trial of active surveillance of 209 SRMs in 178 elderly and/or infirm patients was conducted from 2004 until 2009 with treatment delayed until progression.Patients underwent serial imaging and needle core biopsies.We measured rates of change in tumor diameter (growth measured by imaging) and progression to ≥ 4 cm, doubling of tumor volume, or metastasis with histology on biopsy.Local progression occurred in 25 patients (12%), plus 2 progressed with metastases (1.1%). Of the 178 subjects with 209 SRMs, 127 with 151 SRMs had>12 mo of follow-up with two or more images, with a mean follow-up of 28 mo. Their tumor diameters increased by an average of 0.13 cm/yr. Needle core biopsy in 101 SRMs demonstrated that the presence of RCC did not significantly change growth rate. Limitations included no central review of imaging and pathology and a short follow-up.This is the first SRM active surveillance study to correlate growth with histology prospectively. In the first 2 yr, the rate of local progression to higher stage is low, and metastases are rare. SRMs appear to grow very slowly, even if biopsy proven to be RCC. Many patients with SRMs can therefore be initially managed conservatively with serial imaging, avoiding the morbidity of surgical or ablative treatment.

Abstract PTEN loss or PI3K/AKT signaling pathway activation correlates with human prostate cancer progression and metastasis. However, in preclinical murine models, deletion of Pten alone fails to mimic the significant metastatic burden that frequently accompanies the end stage of human disease. To identify additional pathway alterations that cooperate with PTEN loss in prostate cancer progression, we surveyed human prostate cancer tissue microarrays and found that the RAS/MAPK pathway is significantly elevated in both primary and metastatic lesions. In an attempt to model this event, we crossed conditional activatable K-rasG12D/WT mice with the prostate conditional Pten deletion model. Although RAS activation alone cannot initiate prostate cancer development, it significantly accelerated progression caused by PTEN loss, accompanied by epithelial-to-mesenchymal transition (EMT) and macrometastasis with 100% penetrance. A novel stem/progenitor subpopulation with mesenchymal characteristics was isolated from the compound mutant prostates, which was highly metastatic upon orthotopic transplantation. Importantly, inhibition of RAS/MAPK signaling by PD325901, a mitogen–activated protein (MAP)–extracellular signal–regulated (ER) kinase (MEK) inhibitor, significantly reduced the metastatic progression initiated from transplanted stem/progenitor cells. Collectively, our findings indicate that activation of RAS/MAPK signaling serves as a potentiating second hit to alteration of the PTEN/PI3K/AKT axis, and cotargeting both the pathways is highly effective in preventing the development of metastatic prostate cancers. Cancer Res; 72(7); 1878–89. ©2012 AACR.

Abstract Purpose: Although novel agents targeting the androgen–androgen receptor (AR) axis have altered the treatment paradigm of metastatic castration-resistant prostate cancer (mCRPC), development of therapeutic resistance is inevitable. In this study, we examined whether AR gene aberrations detectable in circulating cell-free DNA (cfDNA) are associated with resistance to abiraterone acetate and enzalutamide in mCRPC patients. Experimental Design: Plasma was collected from 62 mCRPC patients ceasing abiraterone acetate (n = 29), enzalutamide (n = 19), or other agents (n = 14) due to disease progression. DNA was extracted and subjected to array comparative genomic hybridization (aCGH) for chromosome copy number analysis, and Roche 454 targeted next-generation sequencing of exon 8 in the AR. Results: On aCGH, AR amplification was significantly more common in patients progressing on enzalutamide than on abiraterone or other agents (53% vs. 17% vs. 21%, P = 0.02, χ2). Missense AR exon 8 mutations were detected in 11 of 62 patients (18%), including the first reported case of an F876L mutation in an enzalutamide-resistant patient and H874Y and T877A mutations in 7 abiraterone-resistant patients. In patients switched onto enzalutamide after cfDNA collection (n = 39), an AR gene aberration (copy number increase and/or an exon 8 mutation) in pretreatment cfDNA was associated with adverse outcomes, including lower rates of PSA decline ≥ 30% (P = 0.013, χ2) and shorter time to radiographic/clinical progression (P = 0.010, Cox proportional hazards regression). Conclusions: AR gene aberrations in cfDNA are associated with resistance to enzalutamide and abiraterone in mCRPC. Our data illustrate that genomic analysis of cfDNA is a minimally invasive method for interrogating mechanisms of therapeutic resistance in mCRPC. Clin Cancer Res; 21(10); 2315–24. ©2015 AACR.

Abstract Primary resistance to androgen receptor (AR)–directed therapies in metastatic castration-resistant prostate cancer (mCRPC) is poorly understood. We randomized 202 patients with treatment-naïve mCRPC to abiraterone or enzalutamide and performed whole-exome and deep targeted 72-gene sequencing of plasma cell-free DNA prior to therapy. For these agents, which have never been directly compared, time to progression was similar. Defects in BRCA2 and ATM were strongly associated with poor clinical outcomes independently of clinical prognostic factors and circulating tumor DNA abundance. Somatic alterations in TP53, previously linked to reduced tumor dependency on AR signaling, were also independently associated with rapid resistance. Although detection of AR amplifications did not outperform standard prognostic biomarkers, AR gene structural rearrangements truncating the ligand binding domain were identified in several patients with primary resistance. These findings establish genomic drivers of resistance to first-line AR-directed therapy in mCRPC and identify potential minimally invasive biomarkers. Significance: Leveraging plasma specimens collected in a large randomized phase II trial, we report the relative impact of common circulating tumor DNA alterations on patient response to the most widely used therapies for advanced prostate cancer. Our findings suggest that liquid biopsy analysis can guide the use of AR-targeted therapy in general practice. Cancer Discov; 8(4); 444–57. ©2018 AACR. See related commentary by Jayaram et al., p. 392. This article is highlighted in the In This Issue feature, p. 371

<h2>Summary</h2> Metabolic adaptation is essential for cell survival during nutrient deprivation. We report that eukaryotic elongation factor 2 kinase (eEF2K), which is activated by AMP-kinase (AMPK), confers cell survival under acute nutrient depletion by blocking translation elongation. Tumor cells exploit this pathway to adapt to nutrient deprivation by reactivating the AMPK-eEF2K axis. Adaptation of transformed cells to nutrient withdrawal is severely compromised in cells lacking eEF2K. Moreover, eEF2K knockdown restored sensitivity to acute nutrient deprivation in highly resistant human tumor cell lines. In vivo, overexpression of eEF2K rendered murine tumors remarkably resistant to caloric restriction. Expression of <i>eEF2K</i> strongly correlated with overall survival in human medulloblastoma and glioblastoma multiforme. Finally, <i>C. elegans</i> strains deficient in <i>efk-1</i>, the <i>eEF2K</i> ortholog, were severely compromised in their response to nutrient depletion. Our data highlight a conserved role for eEF2K in protecting cells from nutrient deprivation and in conferring tumor cell adaptation to metabolic stress. <h3>PaperClip</h3>

A subset of patients with advanced castration-resistant prostate cancer may eventually evolve into an androgen receptor (AR)-independent phenotype, with a clinical picture associated with the development of rapidly progressive disease involving visceral sites and hormone refractoriness, often in the setting of a low or modestly rising serum prostate-specific antigen level. Biopsies performed in such patients may vary, ranging from poorly differentiated carcinomas to mixed adenocarcinoma-small cell carcinomas to pure small cell carcinomas. These aggressive tumors often demonstrate low or absent AR protein expression and, in some cases, express markers of neuroendocrine differentiation. Because tumor morphology is not always predicted by clinical behavior, the terms "anaplastic prostate cancer" or "neuroendocrine prostate cancer" have been used descriptively to describe these rapidly growing clinical features. Patients meeting clinical criteria of anaplastic prostate cancer have been shown to predict for poor prognosis, and these patients may be considered for platinum-based chemotherapy treatment regimens. Therefore, understanding variants within the spectrum of advanced prostate cancer has important diagnostic and treatment implications.

Background: Real-time knowledge of the somatic genome can influence management of patients with metastatic castration-resistant prostate cancer (mCRPC). While routine metastatic tissue biopsy is challenging in mCRPC, plasma circulating tumor DNA (ctDNA) has emerged as a minimally invasive tool to sample the tumor genome. However, no systematic comparisons of matched “liquid” and “solid” biopsies have been performed that would enable ctDNA profiling to replace the need for direct tissue sampling. Methods: We performed targeted sequencing across 72 clinically relevant genes in 45 plasma cell-free DNA (cfDNA) samples collected at time of metastatic tissue biopsy. We compared ctDNA alterations with exome sequencing data generated from matched tissue and quantified the concordance of mutations and copy number alterations using the Fisher exact test and Pearson correlations. Results: Seventy-five point six percent of cfDNA samples had a ctDNA proportion greater than 2% of total cfDNA. In these patients, all somatic mutations identified in matched metastatic tissue biopsies were concurrently present in ctDNA. Furthermore, the hierarchy of variant allele fractions for shared mutations was remarkably similar between ctDNA and tissue. Copy number profiles between matched liquid and solid biopsy were highly correlated, and individual copy number calls in clinically actionable genes were 88.9% concordant. Detected alterations included AR amplifications in 22 (64.7%) samples, SPOP mutations in three (8.8%) samples, and inactivating alterations in tumor suppressors TP53, PTEN, RB1, APC, CDKN1B, BRCA2, and PIK3R1. In several patients, ctDNA sequencing revealed robust changes not present in paired solid biopsy, including clinically relevant alterations in the AR, WNT, and PI3K pathways. Conclusions: Our study shows that, in the majority of patients, a ctDNA assay is sufficient to identify all driver DNA alterations present in matched metastatic tissue and supports development of DNA biomarkers to guide mCRPC patient management based on ctDNA alone.

Standardized and reproducible preclinical models that recapitulate the dynamics of prostate cancer are urgently needed. We established a bank of transplantable patient-derived prostate cancer xenografts that capture the biologic and molecular heterogeneity currently confounding prognostication and therapy development. Xenografts preserved the histopathology, genome architecture, and global gene expression of donor tumors. Moreover, their aggressiveness matched patient observations, and their response to androgen withdrawal correlated with tumor subtype. The panel includes the first xenografts generated from needle biopsy tissue obtained at diagnosis. This advance was exploited to generate independent xenografts from different sites of a primary site, enabling functional dissection of tumor heterogeneity. Prolonged exposure of adenocarcinoma xenografts to androgen withdrawal led to castration-resistant prostate cancer, including the first-in-field model of complete transdifferentiation into lethal neuroendocrine prostate cancer. Further analysis of this model supports the hypothesis that neuroendocrine prostate cancer can evolve directly from adenocarcinoma via an adaptive response and yielded a set of genes potentially involved in neuroendocrine transdifferentiation. We predict that these next-generation models will be transformative for advancing mechanistic understanding of disease progression, response to therapy, and personalized oncology.

Abstract Mechanisms controlling the emergence of lethal neuroendocrine prostate cancer (NEPC), especially those that are consequences of treatment-induced suppression of the androgen receptor (AR), remain elusive. Using a unique model of AR pathway inhibitor–resistant prostate cancer, we identified AR-dependent control of the neural transcription factor BRN2 (encoded by POU3F2) as a major driver of NEPC and aggressive tumor growth, both in vitro and in vivo. Mechanistic studies showed that AR directly suppresses BRN2 transcription, which is required for NEPC, and BRN2-dependent regulation of the NEPC marker SOX2. Underscoring its inverse correlation with classic AR activity in clinical samples, BRN2 expression was highest in NEPC tumors and was significantly increased in castration-resistant prostate cancer compared with adenocarcinoma, especially in patients with low serum PSA. These data reveal a novel mechanism of AR-dependent control of NEPC and suggest that targeting BRN2 is a strategy to treat or prevent neuroendocrine differentiation in prostate tumors. Significance: Understanding the contribution of the AR to the emergence of highly lethal, drug-resistant NEPC is critical for better implementation of current standard-of-care therapies and novel drug design. Our first-in-field data underscore the consequences of potent AR inhibition in prostate tumors, revealing a novel mechanism of AR-dependent control of neuroendocrine differentiation, and uncover BRN2 as a potential therapeutic target to prevent emergence of NEPC. Cancer Discov; 7(1); 54–71. ©2016 AACR. This article is highlighted in the In This Issue feature, p. 1

The molecular landscape underpinning response to the androgen receptor (AR) antagonist enzalutamide in patients with metastatic castration-resistant prostate cancer (mCRPC) is undefined. Consequently, there is an urgent need for practical biomarkers to guide therapy selection and elucidate resistance. Although tissue biopsies are impractical to perform routinely in the majority of patients with mCRPC, the analysis of plasma cell-free DNA (cfDNA) has recently emerged as a minimally invasive method to explore tumor characteristics.To reveal genomic characteristics from cfDNA associated with clinical outcomes during enzalutamide treatment.Plasma samples were obtained from August 4, 2013, to July 31, 2015, at a single academic institution (British Columbia Cancer Agency) from 65 patients with mCRPC. We collected temporal plasma samples (at baseline, 12 weeks, end of treatment) for circulating cfDNA and performed array comparative genomic hybridization copy number profiling and deep AR gene sequencing. Samples collected at end of treatment were also subjected to targeted sequencing of 19 prostate cancer-associated genes.Enzalutamide, 160 mg, daily orally.Prostate-specific antigen response rate (decline ≥50% from baseline confirmed ≥3 weeks later). Radiographic (as per Prostate Cancer Working Group 2 Criteria) and/or clinical progression (defined as worsening disease-related symptoms necessitating a change in anticancer therapy and/or deterioration in Eastern Cooperative Group performance status ≥2 levels).The 65 patients had a median (interquartile range) age of 74 (68-79) years. Prostate-specific antigen response rate to enzalutamide treatment was 38% (25 of 65), while median clinical/radiographic progression-free survival was 3.5 (95% CI, 2.1-5.0) months. Cell-free DNA was isolated from 122 of 125 plasma samples, and targeted sequencing was successful in 119 of 122. AR mutations and/or copy number alterations were robustly detected in 48% (31 of 65) and 60% (18 of 30) of baseline and progression samples, respectively. Detection of AR amplification, heavily mutated AR (≥2 mutations), and RB1 loss were associated with worse progression-free survival, with hazard ratios of 2.92 (95% CI, 1.59-5.37), 3.94 (95% CI, 1.46-10.64), and 4.46 (95% CI, 2.28-8.74), respectively. AR mutations exhibited clonal selection during treatment, including an increase in glucocorticoid-sensitive AR L702H and promiscuous AR T878A in patients with prior abiraterone treatment. At the time of progression, cfDNA sequencing revealed mutations or copy number changes in all patients tested, including clinically actionable alterations in DNA damage repair genes and PI3K pathway genes, and a high frequency (4 of 14) of activating CTNNB1 mutations.Clinically informative genomic profiling of cfDNA was feasible in nearly all patients with mCRPC and can provide important insights into enzalutamide response and resistance.

The transition from androgen-dependent to castration-resistant prostate cancer is a lethal event. N-cadherin seems to be a major cause underlying this transition, and targeting this adhesion molecule may have positive clinical benefit. The transition from androgen-dependent to castration-resistant prostate cancer (CRPC) is a lethal event of uncertain molecular etiology. Comparing gene expression in isogenic androgen-dependent and CRPC xenografts, we found a reproducible increase in N-cadherin expression, which was also elevated in primary and metastatic tumors of individuals with CRPC. Ectopic expression of N-cadherin in nonmetastatic, androgen-dependent prostate cancer models caused castration resistance, invasion and metastasis. Monoclonal antibodies against the ectodomain of N-cadherin reduced proliferation, adhesion and invasion of prostate cancer cells in vitro. In vivo, these antibodies slowed the growth of multiple established CRPC xenografts, blocked local invasion and metastasis and, at higher doses, led to complete regression. N-cadherin–specific antibodies markedly delayed the time to emergence of castration resistance, markedly affected tumor histology and angiogenesis, and reduced both AKT serine-threonine kinase activity and serum interleukin-8 (IL-8) secretion. These data indicate that N-cadherin is a major cause of both prostate cancer metastasis and castration resistance. Therapeutic targeting of this factor with monoclonal antibodies may have considerable clinical benefit.

Large oncosomes (LO) are atypically large (1-10 µm diameter) cancer-derived extracellular vesicles (EVs), originating from the shedding of membrane blebs and associated with advanced disease. We report that 25% of the proteins, identified by a quantitative proteomics analysis, are differentially represented in large and nano-sized EVs from prostate cancer cells. Proteins enriched in large EVs included enzymes involved in glucose, glutamine and amino acid metabolism, all metabolic processes relevant to cancer. Glutamine metabolism was altered in cancer cells exposed to large EVs, an effect that was not observed upon treatment with exosomes. Large EVs exhibited discrete buoyant densities in iodixanol (OptiPrep(TM)) gradients. Fluorescent microscopy of large EVs revealed an appearance consistent with LO morphology, indicating that these structures can be categorized as LO. Among the proteins enriched in LO, cytokeratin 18 (CK18) was one of the most abundant (within the top 5th percentile) and was used to develop an assay to detect LO in the circulation and tissues of mice and patients with prostate cancer. These observations indicate that LO represent a discrete EV type that may play a distinct role in tumor progression and that may be a source of cancer-specific markers.

The first St Gallen Advanced Prostate Cancer Consensus Conference (APCCC) Expert Panel identified and reviewed the available evidence for the ten most important areas of controversy in advanced prostate cancer (APC) management. The successful registration of several drugs for castration-resistant prostate cancer and the recent studies of chemo-hormonal therapy in men with castration-naïve prostate cancer have led to considerable uncertainty as to the best treatment choices, sequence of treatment options and appropriate patient selection. Management recommendations based on expert opinion, and not based on a critical review of the available evidence, are presented. The various recommendations carried differing degrees of support, as reflected in the wording of the article text and in the detailed voting results recorded in supplementary Material, available at Annals of Oncology online. Detailed decisions on treatment as always will involve consideration of disease extent and location, prior treatments, host factors, patient preferences as well as logistical and economic constraints. Inclusion of men with APC in clinical trials should be encouraged.

Glutamine is conditionally essential in cancer cells, being utilized as a carbon and nitrogen source for macromolecule production, as well as for anaplerotic reactions fuelling the tricarboxylic acid (TCA) cycle. In this study, we demonstrated that the glutamine transporter ASCT2 (SLC1A5) is highly expressed in prostate cancer patient samples. Using LNCaP and PC-3 prostate cancer cell lines, we showed that chemical or shRNA-mediated inhibition of ASCT2 function in vitro decreases glutamine uptake, cell cycle progression through E2F transcription factors, mTORC1 pathway activation and cell growth. Chemical inhibition also reduces basal oxygen consumption and fatty acid synthesis, showing that downstream metabolic function is reliant on ASCT2-mediated glutamine uptake. Furthermore, shRNA knockdown of ASCT2 in PC-3 cell xenografts significantly inhibits tumour growth and metastasis in vivo, associated with the down-regulation of E2F cell cycle pathway proteins. In conclusion, ASCT2-mediated glutamine uptake is essential for multiple pathways regulating the cell cycle and cell growth, and is therefore a putative therapeutic target in prostate cancer.

Innovations in treatments, imaging, and molecular characterisation in advanced prostate cancer have improved outcomes, but there are still many aspects of management that lack high-level evidence to inform clinical practice. The Advanced Prostate Cancer Consensus Conference (APCCC) 2019 addressed some of these topics to supplement guidelines that are based on level 1 evidence. To present the results from the APCCC 2019. Similar to prior conferences, experts identified 10 important areas of controversy regarding the management of advanced prostate cancer: locally advanced disease, biochemical recurrence after local therapy, treating the primary tumour in the metastatic setting, metastatic hormone-sensitive/naïve prostate cancer, nonmetastatic castration-resistant prostate cancer, metastatic castration-resistant prostate cancer, bone health and bone metastases, molecular characterisation of tissue and blood, inter- and intrapatient heterogeneity, and adverse effects of hormonal therapy and their management. A panel of 72 international prostate cancer experts developed the programme and the consensus questions. The panel voted publicly but anonymously on 123 predefined questions, which were developed by both voting and nonvoting panel members prior to the conference following a modified Delphi process. Panellists voted based on their opinions rather than a standard literature review or formal meta-analysis. The answer options for the consensus questions had varying degrees of support by the panel, as reflected in this article and the detailed voting results reported in the Supplementary material. These voting results from a panel of prostate cancer experts can help clinicians and patients navigate controversial areas of advanced prostate management for which high-level evidence is sparse. However, diagnostic and treatment decisions should always be individualised based on patient-specific factors, such as disease extent and location, prior lines of therapy, comorbidities, and treatment preferences, together with current and emerging clinical evidence and logistic and economic constraints. Clinical trial enrolment for men with advanced prostate cancer should be strongly encouraged. Importantly, APCCC 2019 once again identified important questions that merit assessment in specifically designed trials. The Advanced Prostate Cancer Consensus Conference provides a forum to discuss and debate current diagnostic and treatment options for patients with advanced prostate cancer. The conference, which has been held three times since 2015, aims to share the knowledge of world experts in prostate cancer management with health care providers worldwide. At the end of the conference, an expert panel discusses and votes on predefined consensus questions that target the most clinically relevant areas of advanced prostate cancer treatment. The results of the voting provide a practical guide to help clinicians discuss therapeutic options with patients as part of shared and multidisciplinary decision making.

<h3>Importance</h3> Understanding molecular mechanisms of response and resistance to anticancer therapies requires prospective patient follow-up and clinical and functional validation of both common and low-frequency mutations. We describe a whole-exome sequencing (WES) precision medicine trial focused on patients with advanced cancer. <h3>Objective</h3> To understand how WES data affect therapeutic decision making in patients with advanced cancer and to identify novel biomarkers of response. <h3>Design, Setting, and Patients</h3> Patients with metastatic and treatment-resistant cancer were prospectively enrolled at a single academic center for paired metastatic tumor and normal tissue WES during a 19-month period (February 2013 through September 2014). A comprehensive computational pipeline was used to detect point mutations, indels, and copy number alterations. Mutations were categorized as category 1, 2, or 3 on the basis of actionability; clinical reports were generated and discussed in precision tumor board. Patients were observed for 7 to 25 months for correlation of molecular information with clinical response. <h3>Main Outcomes and Measures</h3> Feasibility, use of WES for decision making, and identification of novel biomarkers. <h3>Results</h3> A total of 154 tumor-normal pairs from 97 patients with a range of metastatic cancers were sequenced, with a mean coverage of 95X and 16 somatic alterations detected per patient. In total, 16 mutations were category 1 (targeted therapy available), 98 were category 2 (biologically relevant), and 1474 were category 3 (unknown significance). Overall, WES provided informative results in 91 cases (94%), including alterations for which there is an approved drug, there are therapies in clinical or preclinical development, or they are considered drivers and potentially actionable (category 1-2); however, treatment was guided in only 5 patients (5%) on the basis of these recommendations because of access to clinical trials and/or off-label use of drugs. Among unexpected findings, a patient with prostate cancer with exceptional response to treatment was identified who harbored a somatic hemizygous deletion of the DNA repair gene<i>FANCA</i>and putative partial loss of function of the second allele through germline missense variant. Follow-up experiments established that loss of FANCA function was associated with platinum hypersensitivity both in vitro and in patient-derived xenografts, thus providing biologic rationale and functional evidence for his extreme clinical response. <h3>Conclusions and Relevance</h3> The majority of advanced, treatment-resistant tumors across tumor types harbor biologically informative alterations. The establishment of a clinical trial for WES of metastatic tumors with prospective follow-up of patients can help identify candidate predictive biomarkers of response.

Background Abiraterone acetate plus prednisone and enzalutamide are both used for the treatment of metastatic castration-resistant prostate cancer. We aimed to determine the best sequence in which to use both drugs, as well as their second-line efficacy. Methods In this multicentre, randomised, open-label, phase 2, crossover trial done in six cancer centres in British Columbia, Canada, we recruited patients aged 18 years or older with newly-diagnosed metastatic castration-resistant prostate cancer without neuroendocrine differentiation and Eastern Cooperative Oncology Group performance status 2 or less. Patients were randomly assigned (1:1) using a computer-generated random number table to receive either abiraterone acetate 1000 mg orally once daily plus prednisone 5 mg orally twice daily until PSA progression followed by crossover to enzalutamide 160 mg orally once daily (group A), or the opposite sequence (group B). Treatment was not masked to investigators or participants. Primary endpoints were time to second PSA progression and PSA response (≥30% decline from baseline) on second-line therapy, analysed by intention-to-treat in all randomly assigned patients and in patients who crossed over, respectively. The trial is registered with ClinicalTrials.gov, NCT02125357. Findings Between Oct 21, 2014, and Dec 13, 2016, 202 patients were enrolled and randomly assigned to either group A (n=101) or group B (n=101). At the time of data cutoff, 73 (72%) patients in group A and 75 (74%) patients in group B had crossed over. Time to second PSA progression was longer in group A than in group B (median 19·3 months [95% CI 16·0–30·5] vs 15·2 months [95% CI 11·9–19·8] months; hazard ratio 0·66, 95% CI 0·45–0·97, p=0·036), at a median follow-up of 22·8 months (IQR 10·3–33·4). PSA responses to second-line therapy were seen in 26 (36%) of 73 patients for enzalutamide and three (4%) of 75 for abiraterone (χ2 p<0·0001). The most common grade 3–4 adverse events throughout the trial were hypertension (27 [27%] of 101 patients in group A vs 18 [18%] of 101 patients in group B) and fatigue (six [10%] vs four [4%]). Serious adverse events were reported in 15 (15%) of 101 patients in group A and 20 (20%) of 101 patients in group B. There were no treatment-related deaths. Interpretation Enzalutamide showed activity as a second-line novel androgen receptor pathway inhibitor, whereas abiraterone acetate did not, leading to a longer time to second PSA progression for the sequence of abiraterone followed by enzalutamide than with the opposite treatment sequence. Our data suggest that using a sequencing strategy of abiraterone acetate followed by enzalutamide provides the greatest clinical benefit. Funding Canadian Cancer Society Research Institute, Prostate Cancer Canada, Movember Foundation, Prostate Cancer Foundation, Terry Fox New Frontiers Program, BC Cancer Foundation, Jane and Aatos Erkko Foundation, Janssen, and Astellas.

Purpose To determine the clinical activity of OGX-011, an antisense inhibitor of clusterin, in combination with docetaxel/prednisone in patients with metastatic castration-resistant prostate cancer. Patients and Methods Patients were randomly assigned 1:1 to receive docetaxel/prednisone either with (arm A) or without (arm B) OGX-011 640 mg intravenously weekly. The primary end point was the proportion of patients with a prostate-specific antigen (PSA) decline of ≥ 50% from baseline, with the experimental therapy being considered of interest if the proportion of patients with a PSA decline was more than 60%. Secondary end points were objective response rate, progression-free survival (PFS), overall survival (OS), and changes in serum clusterin. Results Eighty-two patients were accrued, 41 to each arm. OGX-011 adverse effects included rigors and fevers. After cycle 1, median serum clusterin decreased by 26% in arm A and increased by 0.9% in arm B (P &lt; .001). PSA declined by ≥ 50% in 58% of patients in arm A and 54% in arm B. Partial response occurred in 19% and 25% of patients in arms A and B, respectively. Median PFS and OS times were 7.3 months (95% CI, 5.3 to 8.8 months) and 23.8 months (95% CI, 16.2 months to not reached), respectively, in arm A and 6.1 months (95% CI, 3.7 to 8.6 months) and 16.9 months (95% CI, 12.8 to 25.8 months), respectively, in arm B. Baseline factors associated with improved OS on exploratory multivariate analysis were an Eastern Cooperative Oncology Group performance status of 0 (hazard ratio [HR], 0.27; 95% CI, 0.14 to 0.51), presence of bone or lymph node metastases only (HR, 0.45; 95% CI, 0.25 to 0.79), and treatment assignment to OGX-011 (HR, 0.50; 95% CI, 0.29 to 0.87). Conclusion Treatment with OGX-011 and docetaxel was well tolerated with evidence of biologic effect and was associated with improved survival. Further evaluation is warranted.

Under cell stress, global protein synthesis is inhibited to preserve energy. One mechanism is to sequester and silence mRNAs in ribonucleoprotein complexes known as stress granules (SGs), which contain translationally silent mRNAs, preinitiation factors, and RNA-binding proteins. Y-box binding protein 1 (YB-1) localizes to SGs, but its role in SG biology is unknown. We now report that YB-1 directly binds to and translationally activates the 5' untranslated region (UTR) of G3BP1 mRNAs, thereby controlling the availability of the G3BP1 SG nucleator for SG assembly. YB-1 inactivation in human sarcoma cells dramatically reduces G3BP1 and SG formation in vitro. YB-1 and G3BP1 expression are highly correlated in human sarcomas, and elevated G3BP1 expression correlates with poor survival. Finally, G3BP1 down-regulation in sarcoma xenografts prevents in vivo SG formation and tumor invasion, and completely blocks lung metastasis in mouse models. Together, these findings demonstrate a critical role for YB-1 in SG formation through translational activation of G3BP1, and highlight novel functions for SGs in tumor progression.

More potent targeting of the androgen receptor (AR) in advanced prostate cancer is driving an increased incidence of neuroendocrine prostate cancer (NEPC), an aggressive and treatment-resistant AR-negative variant. Its molecular pathogenesis remains poorly understood but appears to require TP53 and RB1 aberration. We modeled the development of NEPC from conventional prostatic adenocarcinoma using a patient-derived xenograft and found that the placental gene PEG10 is de-repressed during the adaptive response to AR interference and subsequently highly upregulated in clinical NEPC. We found that the AR and the E2F/RB pathway dynamically regulate distinct post-transcriptional and post-translational isoforms of PEG10 at distinct stages of NEPC development. In vitro, PEG10 promoted cell-cycle progression from G0/G1 in the context of TP53 loss and regulated Snail expression via TGF-β signaling to promote invasion. Taken together, these findings show the mechanistic relevance of RB1 and TP53 loss in NEPC and suggest PEG10 as a NEPC-specific target.

Drug discovery is a rigorous process that requires billion dollars of investments and decades of research to bring a molecule "from bench to a bedside". While virtual docking can significantly accelerate the process of drug discovery, it ultimately lags the current rate of expansion of chemical databases that already exceed billions of molecular records. This recent surge of small molecules availability presents great drug discovery opportunities, but also demands much faster screening protocols. In order to address this challenge, we herein introduce Deep Docking (DD), a novel deep learning platform that is suitable for docking billions of molecular structures in a rapid, yet accurate fashion. The DD approach utilizes quantitative structure-activity relationship (QSAR) deep models trained on docking scores of subsets of a chemical library to approximate the docking outcome for yet unprocessed entries and, therefore, to remove unfavorable molecules in an iterative manner. The use of DD methodology in conjunction with the FRED docking program allowed rapid and accurate calculation of docking scores for 1.36 billion molecules from the ZINC15 library against 12 prominent target proteins and demonstrated up to 100-fold data reduction and 6000-fold enrichment of high scoring molecules (without notable loss of favorably docked entities). The DD protocol can readily be used in conjunction with any docking program and was made publicly available.

Although DNA methylation is a key regulator of gene expression, the comprehensive methylation landscape of metastatic cancer has never been defined. Through whole-genome bisulfite sequencing paired with deep whole-genome and transcriptome sequencing of 100 castration-resistant prostate metastases, we discovered alterations affecting driver genes that were detectable only with integrated whole-genome approaches. Notably, we observed that 22% of tumors exhibited a novel epigenomic subtype associated with hypermethylation and somatic mutations in TET2, DNMT3B, IDH1 and BRAF. We also identified intergenic regions where methylation is associated with RNA expression of the oncogenic driver genes AR, MYC and ERG. Finally, we showed that differential methylation during progression preferentially occurs at somatic mutational hotspots and putative regulatory regions. This study is a large integrated study of whole-genome, whole-methylome and whole-transcriptome sequencing in metastatic cancer that provides a comprehensive overview of the important regulatory role of methylation in metastatic castration-resistant prostate cancer.

Exosomes are emerging as a source of biomarkers with putative prognostic and diagnostic value. However, little is known about the efficiency, reproducibility and reliability of the protocols routinely used to quantify exosomes in the human serum. We used increasing amounts of the same serum sample to isolate exosomes using two different methods: ultracentrifugation onto a sucrose cushion and ExoQuick™. Quantitative analysis of serum-derived exosomes was performed by determining protein concentration (BCA assay) and the number of nanoparticles (Nanosight™ technology). Exosome quality was assessed by Coomassie staining and Western blotting for CD9, LAMP2 exosomal markers and a negative marker Grp94. Correlation between serum volume and the number of isolated exosomes is significant for both methods when exosomes are quantified using protein concentration. However, when the number of nanoparticles is used to quantify exosomes, ExoQuick™ is the only reproducible and efficient method. CD9, LAMP2 and Grp94 exosomal markers are equivalently expressed in both methods. However, exosomes isolated using ultracentrifuge method are strongly contaminated with albumin and IgG. ExoQuick™ is an efficient and reproducible method to isolate exosomes for quantitative studies, whereas ultracentrifugation is not. Moreover, high albumin contamination of ultracentrifuged-derived exosomes impairs the use of protein concentration as a mean to quantify serum-derived exosomes.

Metastasis is the most common cause of death of prostate cancer patients. Identification of specific metastasis biomarkers and novel therapeutic targets is considered essential for improved prognosis and management of the disease. MicroRNAs (miRNAs) form a class of non-coding small RNA molecules considered to be key regulators of gene expression. Their dysregulation has been shown to play a role in cancer onset, progression and metastasis, and miRNAs represent a promising new class of cancer biomarkers. The objective of this study was to identify down- and up-regulated miRNAs in prostate cancer that could provide potential biomarkers and/or therapeutic targets for prostate cancer metastasis.Next generation sequencing technology was applied to identify differentially expressed miRNAs in a transplantable metastatic versus a non-metastatic prostate cancer xenograft line, both derived from one patient's primary cancer. The xenografts were developed via subrenal capsule grafting of cancer tissue into NOD/SCID mice, a methodology that tends to preserve properties of the original cancers (e.g., tumor heterogeneity, genetic profiles).Differentially expressed known miRNAs, isomiRs and 36 novel miRNAs were identified. A number of these miRNAs (21/104) have previously been reported to show similar down- or up-regulation in prostate cancers relative to normal prostate tissue, and some of them (e.g., miR-16, miR-34a, miR-126*, miR-145, miR-205) have been linked to prostate cancer metastasis, supporting the validity of the analytical approach.The use of metastatic and non-metastatic prostate cancer subrenal capsule xenografts derived from one patient's cancer makes it likely that the differentially expressed miRNAs identified in this study include potential biomarkers and/or therapeutic targets for human prostate cancer metastasis.

PURPOSE As part of the ENTHUSE (Endothelin A Use) program, the efficacy and safety of zibotentan (ZD4054), an oral specific endothelin A receptor antagonist, has been investigated in combination with docetaxel in patients with metastatic castration-resistant prostate cancer (CRPC). PATIENTS AND METHODS In this randomized, double-blind, placebo-controlled, phase III study, patients received intravenous docetaxel 75 mg/m(2) on day 1 of 21-day cycles plus oral zibotentan 10 mg or placebo once daily. The primary end point was overall survival (OS). Secondary end points included time to pain and prostate-specific antigen (PSA) progression, pain and PSA response, progression-free survival, health-related quality of life, and safety. Results A total of 1,052 patients received study treatment (docetaxel-zibotentan, n = 524; docetaxel-placebo, n = 528). At the time of data cutoff, there had been 277 and 280 deaths, respectively. There was no difference in OS for patients receiving docetaxel-zibotentan compared with those receiving docetaxel-placebo (hazard ratio, 1.00; 95% CI, 0.84 to 1.18; P = .963). No significant differences were observed on secondary end points, including time to pain progression (median 9.3 v 10.0 months, respectively) or pain response (odds ratio, 0.84; 95% CI, 0.61 to 1.16; P = .283). The median time to death was 20.0 and 19.2 months for the zibotentan and placebo groups, respectively. The most commonly reported adverse events in zibotentan-treated patients were peripheral edema (52.7%), diarrhea (35.4%), alopecia (33.9%), and nausea (33.3%). CONCLUSION Docetaxel plus zibotentan 10 mg/d did not result in a significant improvement in OS compared with docetaxel plus placebo in patients with metastatic CRPC.

Germline mutations in DNA repair genes were recently reported in 8–12% of patients with metastatic castration-resistant prostate cancer (mCRPC). It is unknown whether these mutations associate with differential response to androgen receptor (AR)-directed therapy. To determine the clinical response of mCRPC patients with germline DNA repair defects to AR-directed therapies and to establish whether biallelic DNA repair gene loss is detectable in matched circulating tumor DNA (ctDNA). We recruited 319 mCRPC patients and performed targeted germline sequencing of 22 DNA repair genes. In patients with deleterious germline mutations, plasma cell-free DNA was also sequenced. Prostate-specific antigen response and progression were assessed in relation to initial androgen deprivation therapy (ADT) and subsequent therapy for mCRPC using Kaplan–Meier analysis. Of the 319 patients, 24 (7.5%) had deleterious germline mutations, with BRCA2 (n = 16) being the most frequent. Patients (n = 22) with mutations in genes linked to homologous recombination were heterogeneous at initial presentation but, after starting ADT, progressed to mCRPC with a median time of 11.8 mo (95% confidence interval [CI] 5.1–18.4). The median time to prostate-specific antigen progression on first-line AR-targeted therapy in the mCRPC setting was 3.3 mo (95% CI 2.7-3.9). Ten out of 11 evaluable patients with germline BRCA2 mutations had somatic deletion of the intact allele in ctDNA. A limitation of this study is absence of a formal control cohort for comparison of clinical outcomes. Patients with mCRPC who have germline DNA repair defects exhibit attenuated responses to AR-targeted therapy. Biallelic gene loss was robustly detected in ctDNA, suggesting that this patient subset could be prioritized for therapies exploiting defective DNA repair using a liquid biopsy. Patients with metastatic prostate cancer and germline DNA repair defects exhibit a poor response to standard hormonal therapies, but may be prioritized for potentially more effective therapies using a blood test.

Defining the mechanisms underlying metastatic progression of prostate cancer may lead to insights into how to decrease morbidity and mortality in this disease. An important determinant of metastasis is epithelial-to-mesenchymal transition (EMT), and the mechanisms that control the process of EMT in cancer cells are still emerging. Here, we report that the molecular chaperone Hsp27 (HSPB1) drives EMT in prostate cancer, whereas its attenuation reverses EMT and decreases cell migration, invasion, and matrix metalloproteinase activity. Mechanistically, silencing Hsp27 decreased IL-6-dependent STAT3 phosphorylation, nuclear translocation, and STAT3 binding to the Twist promoter, suggesting that Hsp27 is required for IL-6-mediated EMT via modulation of STAT3/Twist signaling. We observed a correlation between Hsp27 and Twist in patients with prostate cancer, with Hsp27 and Twist expression each elevated in high-grade prostate cancer tumors. Hsp27 inhibition by OGX-427, an antisense therapy currently in phase II trials, reduced tumor metastasis in a murine model of prostate cancer. More importantly, OGX-427 treatment decreased the number of circulating tumor cells in patients with metastatic castration-resistant prostate cancer in a phase I clinical trial. Overall, this study defines Hsp27 as a critical regulator of IL-6-dependent and IL-6-independent EMT, validating this chaperone as a therapeutic target to treat metastatic prostate cancer.

Metastatic biopsy programmes combined with advances in genomic sequencing have provided new insights into the molecular landscape of castration-resistant prostate cancer (CRPC), identifying actionable targets, and emerging resistance mechanisms. The detection of DNA repair aberrations, such as mutation of BRCA2, could help select patients for poly(ADP-ribose) polymerase (PARP) inhibitor or platinum chemotherapy, and mismatch repair gene defects and microsatellite instability have been associated with responses to checkpoint inhibitor immunotherapy. Poor prognostic features, such as the presence of RB1 deletion, might help guide future therapeutic strategies. Our understanding of the molecular features of CRPC is now being translated into the clinic in the form of increased molecular testing for use of these agents and for clinical trial eligibility. Genomic testing offers opportunities for improving patient selection for systemic therapies and, ultimately, patient outcomes. However, challenges for precision oncology in advanced prostate cancer still remain, including the contribution of tumour heterogeneity, the timing and potential cooperation of multiple driver gene aberrations, and diverse resistant mechanisms. Defining the optimal use of molecular biomarkers in the clinic, including tissue-based and liquid biopsies, is a rapidly evolving field. A number of targetable molecular alterations and resistance mechanisms have been identified in metastatic castration-resistant prostate cancer (mCRPC). As our understanding of the genomic landscape of mCRPC increases, biomarker-driven clinical trials investigating targeted therapies will enable an increasingly personalized approach to its treatment.

The androgen receptor (AR) is a pivotal drug target for the treatment of prostate cancer, including its lethal castration-resistant (CRPC) form. All current non-steroidal AR antagonists, such as hydroxyflutamide, bicalutamide, and enzalutamide, target the androgen binding site of the receptor, competing with endogenous androgenic steroids. Several AR mutations in this binding site have been associated with poor prognosis and resistance to conventional prostate cancer drugs. In order to develop an effective CRPC therapy, it is crucial to understand the effects of these mutations on the functionality of the AR and its ability to interact with endogenous steroids and conventional AR inhibitors. We previously utilized circulating cell-free DNA (cfDNA) sequencing technology to examine the AR gene for the presence of mutations in CRPC patients. By modifying our sequencing and data analysis approaches, we identify four additional single AR mutations and five mutation combinations associated with CRPC. Importantly, we conduct experimental functionalization of all the AR mutations identified by the current and previous cfDNA sequencing to reveal novel gain-of-function scenarios. Finally, we evaluate the effect of a novel class of AR inhibitors targeting the binding function 3 (BF3) site on the activity of CRPC-associated AR mutants. This work demonstrates the feasibility of a prognostic and/or diagnostic platform combining the direct identification of AR mutants from patients’ serum, and the functional characterization of these mutants in order to provide personalized recommendations regarding the best future therapy.

Neuroendocrine prostate cancer (NEPC), a lethal form of the disease, is characterized by loss of androgen receptor (AR) signaling during neuroendocrine transdifferentiation, which results in resistance to AR-targeted therapy. Clinically, genomically and epigenetically, NEPC resembles other types of poorly differentiated neuroendocrine tumors (NETs). Through pan-NET analyses, we identified ONECUT2 as a candidate master transcriptional regulator of poorly differentiated NETs. ONECUT2 ectopic expression in prostate adenocarcinoma synergizes with hypoxia to suppress androgen signaling and induce neuroendocrine plasticity. ONEUCT2 drives tumor aggressiveness in NEPC, partially through regulating hypoxia signaling and tumor hypoxia. Specifically, ONECUT2 activates SMAD3, which regulates hypoxia signaling through modulating HIF1α chromatin-binding, leading NEPC to exhibit higher degrees of hypoxia compared to prostate adenocarcinomas. Treatment with hypoxia-activated prodrug TH-302 potently reduces NEPC tumor growth. Collectively, these results highlight the synergy between ONECUT2 and hypoxia in driving NEPC, and emphasize the potential of hypoxia-directed therapy for NEPC patients.

The incidence of aggressive neuroendocrine prostate cancers (NEPC) related to androgen-deprivation therapy (ADT) is rising. NEPC is still poorly understood, such as its neuroendocrine differentiation (NED) and angiogenic phenotypes. Here we reveal that NED and angiogenesis are molecularly connected through EZH2 (enhancer of zeste homolog 2). NED and angiogenesis are both regulated by ADT-activated CREB (cAMP response element-binding protein) that in turn enhances EZH2 activity. We also uncover anti-angiogenic factor TSP1 (thrombospondin-1, THBS1) as a direct target of EZH2 epigenetic repression. TSP1 is downregulated in advanced prostate cancer patient samples and negatively correlates with NE markers and EZH2. Furthermore, castration activates the CREB/EZH2 axis, concordantly affecting TSP1, angiogenesis and NE phenotypes in tumor xenografts. Notably, repressing CREB inhibits the CREB/EZH2 axis, tumor growth, NED, and angiogenesis in vivo. Taken together, we elucidate a new critical pathway, consisting of CREB/EZH2/TSP1, underlying ADT-enhanced NED and angiogenesis during prostate cancer progression.

Neuroendocrine prostate cancer (NEPC) is an aggressive subtype of castration-resistant prostate cancer that typically does not respond to androgen receptor pathway inhibition (ARPI), and its diagnosis is increasing.To understand how NEPC develops and to identify driver genes to inform therapy for NEPC prevention.Whole-transcriptome sequencing data were extracted from prostate tumors from two independent cohorts: The Beltran cohort contained 27 adenocarcinoma and five NEPC patient samples, and the Vancouver Prostate Centre cohort contained three patient samples and nine patient-derived xenografts.A novel bioinformatics tool, comparative alternative splicing detection (COMPAS), was invented to analyze alternative RNA splicing on RNA-sequencing data.COMPAS identified potential driver genes for NEPC development. Biochemical and biological validations were performed in both prostate cell and tumor models.More than 66% of the splice events were predicted to be regulated by the RNA splicing factor serine/arginine repetitive matrix 4 (SRRM4). In vitro and in vivo evidence confirmed that one SRRM4 target gene was the RE1 silencing transcription factor (REST), a master regulator of neurogenesis. Moreover, SRRM4 strongly stimulated adenocarcinoma cells to express NEPC biomarkers, and this effect was exacerbated by ARPI. ARPI combined with a gain of SRRM4-induced adenocarcinoma cells to assume multicellular spheroid morphology and was essential in establishing progressive NEPC xenografts. These SRRM4 actions were further enhanced by loss of function of TP53.SRRM4 drives NEPC progression. This knowledge may guide the development of novel therapeutics aimed at NEPC.Using next-generation RNA sequencing and our newly developed bioinformatics tool, we identified a neuroendocrine prostate cancer (NEPC)-specific RNA splicing signature that is predominantly controlled by serine/arginine repetitive matrix 4 (SRRM4). We confirmed that SRRM4 drives NEPC progression, and we propose SRRM4 as a potential therapeutic target for NEPC.

Several systemic therapeutic options exist for metastatic castrate-sensitive prostate cancer (mCSPC). Circulating tumor DNA (ctDNA) can molecularly profile metastatic castration-resistant prostate cancer and can influence decision-making, but remains untested in mCSPC.To determine ctDNA abundance at de novo mCSPC diagnosis and whether ctDNA provides complementary clinically relevant information to a prostate biopsy.We collected plasma cell-free DNA (cfDNA) from 53 patients newly diagnosed with mCSPC and, where possible, during treatment. Targeted sequencing was performed on cfDNA and DNA from diagnostic prostate tissue.The median ctDNA fraction was 11% (range 0-84%) among untreated patients but was lower (1.0%, range 0-51%) among patients after short-term (median 22d) androgen deprivation therapy (ADT). TP53 mutations and DNA repair defects were identified in 47% and 21% of the cohort, respectively. The concordance for mutation detection in matched samples was 80%. Combined ctDNA and tissue analysis identified potential driver alterations in 94% of patients, whereas ctDNA or prostate biopsy alone was insufficient in 19 cases (36%). Limitations include the use of a narrow gene panel and undersampling of primary disease by prostate biopsy.ctDNA provides additional information to a prostate biopsy in men with de novo mCSPC, but ADT rapidly reduces ctDNA availability. Primary tissue and ctDNA share relevant somatic alterations, suggesting that either is suitable for molecular subtyping in de novo mCSPC. The optimal approach for biomarker development should utilize both a tissue and liquid biopsy at diagnosis, as neither captures clinically relevant somatic alterations in all patients.In men with advanced prostate cancer, tumor DNA shed into the bloodstream can be measured via a blood test. The information from this test provides complementary information to a prostate needle biopsy and could be used to guide management strategies. Sequencing data were deposited in the European Genome-phenome Archive (EGA) under study identifier EGAS00001003351.

Cancers adapt to increasingly potent targeted therapies by reprogramming their phenotype. Here we investigated such a phenomenon in prostate cancer, in which tumours can escape epithelial lineage confinement and transition to a high-plasticity state as an adaptive response to potent androgen receptor (AR) antagonism. We found that AR activity can be maintained as tumours adopt alternative lineage identities, with changes in chromatin architecture guiding AR transcriptional rerouting. The epigenetic regulator enhancer of zeste homologue 2 (EZH2) co-occupies the reprogrammed AR cistrome to transcriptionally modulate stem cell and neuronal gene networks-granting privileges associated with both fates. This function of EZH2 was associated with T350 phosphorylation and establishment of a non-canonical polycomb subcomplex. Our study provides mechanistic insights into the plasticity of the lineage-infidelity state governed by AR reprogramming that enabled us to redirect cell fate by modulating EZH2 and AR, highlighting the clinical potential of reversing resistance phenotypes.

Innovations in treatments, imaging, and molecular characterisation in advanced prostate cancer have improved outcomes, but various areas of management still lack high-level evidence to inform clinical practice. The 2021 Advanced Prostate Cancer Consensus Conference (APCCC) addressed some of these questions to supplement guidelines that are based on level 1 evidence.To present the voting results from APCCC 2021.The experts identified three major areas of controversy related to management of advanced prostate cancer: newly diagnosed metastatic hormone-sensitive prostate cancer (mHSPC), the use of prostate-specific membrane antigen ligands in diagnostics and therapy, and molecular characterisation of tissue and blood. A panel of 86 international prostate cancer experts developed the programme and the consensus questions.The panel voted publicly but anonymously on 107 pre-defined questions, which were developed by both voting and non-voting panel members prior to the conference following a modified Delphi process.The voting reflected the opinions of panellists and did not incorporate a standard literature review or formal meta-analysis. The answer options for the consensus questions received varying degrees of support from panellists, as reflected in this article and the detailed voting results reported in the Supplementary material.These voting results from a panel of experts in advanced prostate cancer can help clinicians and patients to navigate controversial areas of management for which high-level evidence is scant. However, diagnostic and treatment decisions should always be individualised according to patient characteristics, such as the extent and location of disease, prior treatment(s), comorbidities, patient preferences, and treatment recommendations, and should also incorporate current and emerging clinical evidence and logistic and economic constraints. Enrolment in clinical trials should be strongly encouraged. Importantly, APCCC 2021 once again identified salient questions that merit evaluation in specifically designed trials.The Advanced Prostate Cancer Consensus Conference is a forum for discussing current diagnosis and treatment options for patients with advanced prostate cancer. An expert panel votes on predefined questions focused on the most clinically relevant areas for treatment of advanced prostate cancer for which there are gaps in knowledge. The voting results provide a practical guide to help clinicians in discussing treatment options with patients as part of shared decision-making.

Innovations in imaging and molecular characterisation and the evolution of new therapies have improved outcomes in advanced prostate cancer. Nonetheless, we continue to lack high-level evidence on a variety of clinical topics that greatly impact daily practice. To supplement evidence-based guidelines, the 2022 Advanced Prostate Cancer Consensus Conference (APCCC 2022) surveyed experts about key dilemmas in clinical management.To present consensus voting results for select questions from APCCC 2022.Before the conference, a panel of 117 international prostate cancer experts used a modified Delphi process to develop 198 multiple-choice consensus questions on (1) intermediate- and high-risk and locally advanced prostate cancer, (2) biochemical recurrence after local treatment, (3) side effects from hormonal therapies, (4) metastatic hormone-sensitive prostate cancer, (5) nonmetastatic castration-resistant prostate cancer, (6) metastatic castration-resistant prostate cancer, and (7) oligometastatic and oligoprogressive prostate cancer. Before the conference, these questions were administered via a web-based survey to the 105 physician panel members ("panellists") who directly engage in prostate cancer treatment decision-making. Herein, we present results for the 82 questions on topics 1-3.Consensus was defined as ≥75% agreement, with strong consensus defined as ≥90% agreement.The voting results reveal varying degrees of consensus, as is discussed in this article and shown in the detailed results in the Supplementary material. The findings reflect the opinions of an international panel of experts and did not incorporate a formal literature review and meta-analysis.These voting results by a panel of international experts in advanced prostate cancer can help physicians and patients navigate controversial areas of clinical management for which high-level evidence is scant or conflicting. The findings can also help funders and policymakers prioritise areas for future research. Diagnostic and treatment decisions should always be individualised based on patient and cancer characteristics (disease extent and location, treatment history, comorbidities, and patient preferences) and should incorporate current and emerging clinical evidence, therapeutic guidelines, and logistic and economic factors. Enrolment in clinical trials is always strongly encouraged. Importantly, APCCC 2022 once again identified important gaps (areas of nonconsensus) that merit evaluation in specifically designed trials.The Advanced Prostate Cancer Consensus Conference (APCCC) provides a forum to discuss and debate current diagnostic and treatment options for patients with advanced prostate cancer. The conference aims to share the knowledge of international experts in prostate cancer with health care providers and patients worldwide. At each APCCC, a panel of physician experts vote in response to multiple-choice questions about their clinical opinions and approaches to managing advanced prostate cancer. This report presents voting results for the subset of questions pertaining to intermediate- and high-risk and locally advanced prostate cancer, biochemical relapse after definitive treatment, advanced (next-generation) imaging, and management of side effects caused by hormonal therapies. The results provide a practical guide to help clinicians and patients discuss treatment options as part of shared multidisciplinary decision-making. The findings may be especially useful when there is little or no high-level evidence to guide treatment decisions.

Innovations in imaging and molecular characterisation together with novel treatment options have improved outcomes in advanced prostate cancer. However, we still lack high-level evidence in many areas relevant to making management decisions in daily clinical practise. The 2022 Advanced Prostate Cancer Consensus Conference (APCCC 2022) addressed some questions in these areas to supplement guidelines that mostly are based on level 1 evidence.To present the voting results of the APCCC 2022.The experts voted on controversial questions where high-level evidence is mostly lacking: locally advanced prostate cancer; biochemical recurrence after local treatment; metastatic hormone-sensitive, non-metastatic, and metastatic castration-resistant prostate cancer; oligometastatic prostate cancer; and managing side effects of hormonal therapy. A panel of 105 international prostate cancer experts voted on the consensus questions.The panel voted on 198 pre-defined questions, which were developed by 117 voting and non-voting panel members prior to the conference following a modified Delphi process. A total of 116 questions on metastatic and/or castration-resistant prostate cancer are discussed in this manuscript. In 2022, the voting was done by a web-based survey because of COVID-19 restrictions.The voting reflects the expert opinion of these panellists and did not incorporate a standard literature review or formal meta-analysis. The answer options for the consensus questions received varying degrees of support from panellists, as reflected in this article and the detailed voting results are reported in the supplementary material. We report here on topics in metastatic, hormone-sensitive prostate cancer (mHSPC), non-metastatic, castration-resistant prostate cancer (nmCRPC), metastatic castration-resistant prostate cancer (mCRPC), and oligometastatic and oligoprogressive prostate cancer.These voting results in four specific areas from a panel of experts in advanced prostate cancer can help clinicians and patients navigate controversial areas of management for which high-level evidence is scant or conflicting and can help research funders and policy makers identify information gaps and consider what areas to explore further. However, diagnostic and treatment decisions always have to be individualised based on patient characteristics, including the extent and location of disease, prior treatment(s), co-morbidities, patient preferences, and treatment recommendations and should also incorporate current and emerging clinical evidence and logistic and economic factors. Enrolment in clinical trials is strongly encouraged. Importantly, APCCC 2022 once again identified important gaps where there is non-consensus and that merit evaluation in specifically designed trials.The Advanced Prostate Cancer Consensus Conference (APCCC) provides a forum to discuss and debate current diagnostic and treatment options for patients with advanced prostate cancer. The conference aims to share the knowledge of international experts in prostate cancer with healthcare providers worldwide. At each APCCC, an expert panel votes on pre-defined questions that target the most clinically relevant areas of advanced prostate cancer treatment for which there are gaps in knowledge. The results of the voting provide a practical guide to help clinicians discuss therapeutic options with patients and their relatives as part of shared and multidisciplinary decision-making. This report focuses on the advanced setting, covering metastatic hormone-sensitive prostate cancer and both non-metastatic and metastatic castration-resistant prostate cancer.Report of the results of APCCC 2022 for the following topics: mHSPC, nmCRPC, mCRPC, and oligometastatic prostate cancer.At APCCC 2022, clinically important questions in the management of advanced prostate cancer management were identified and discussed, and experts voted on pre-defined consensus questions. The report of the results for metastatic and/or castration-resistant prostate cancer is summarised here.

Patients with prostate cancer who have high-risk biochemical recurrence have an increased risk of progression. The efficacy and safety of enzalutamide plus androgen-deprivation therapy and enzalutamide monotherapy, as compared with androgen-deprivation therapy alone, are unknown.In this phase 3 trial, we enrolled patients with prostate cancer who had high-risk biochemical recurrence with a prostate-specific antigen doubling time of 9 months or less. Patients were randomly assigned, in a 1:1:1 ratio, to receive enzalutamide (160 mg) daily plus leuprolide every 12 weeks (combination group), placebo plus leuprolide (leuprolide-alone group), or enzalutamide monotherapy (monotherapy group). The primary end point was metastasis-free survival, as assessed by blinded independent central review, in the combination group as compared with the leuprolide-alone group. A key secondary end point was metastasis-free survival in the monotherapy group as compared with the leuprolide-alone group. Other secondary end points were patient-reported outcomes and safety.A total of 1068 patients underwent randomization: 355 were assigned to the combination group, 358 to the leuprolide-alone group, and 355 to the monotherapy group. The patients were followed for a median of 60.7 months. At 5 years, metastasis-free survival was 87.3% (95% confidence interval [CI], 83.0 to 90.6) in the combination group, 71.4% (95% CI, 65.7 to 76.3) in the leuprolide-alone group, and 80.0% (95% CI, 75.0 to 84.1) in the monotherapy group. With respect to metastasis-free survival, enzalutamide plus leuprolide was superior to leuprolide alone (hazard ratio for metastasis or death, 0.42; 95% CI, 0.30 to 0.61; P<0.001); enzalutamide monotherapy was also superior to leuprolide alone (hazard ratio for metastasis or death, 0.63; 95% CI, 0.46 to 0.87; P = 0.005). No new safety signals were observed, with no substantial between-group differences in quality-of-life measures.In patients with prostate cancer with high-risk biochemical recurrence, enzalutamide plus leuprolide was superior to leuprolide alone with respect to metastasis-free survival; enzalutamide monotherapy was also superior to leuprolide alone. The safety profile of enzalutamide was consistent with that shown in previous clinical studies, with no apparent detrimental effect on quality of life. (Funded by Pfizer and Astellas Pharma; EMBARK ClinicalTrials.gov number, NCT02319837.).

Most trials of immunomodulators in metastatic renal-cell carcinoma have been uncontrolled and subject to selection bias. The objective of this blinded, placebo-controlled study was to compare overall response rates, time to disease progression, and survival of patients with metastatic renal-cell carcinoma treated with recombinant human interferon gamma-1b or placebo.Patients with biopsy-proved metastatic renal-cell carcinoma were randomly assigned to receive interferon gamma-1b (60 microg per square meter of body-surface area subcutaneously once weekly) or placebo. The primary tumor had been treated by nephrectomy or angioinfarction at least three weeks previously. Patients were evaluated for radiologic evidence of progression, and all responses were independently reviewed by a committee that was unaware of the treatment.A total of 197 patients with metastatic renal-cell carcinoma were enrolled at 17 centers in Canada. One hundred eighty-one patients could be evaluated; of these, 91 were assigned to receive interferon gamma-1b and 90 were given placebo. The groups were well balanced in terms of prognostic factors. Two thirds of all patients had Karnofsky scores of 90 or 100, and more than half had two or more metastatic sites. Grade I and II toxicity, mostly chills, fever, asthenia, or headaches, was reported in 91 percent and 61 percent, respectively, of the patients in the interferon group, as compared with 76 percent and 63 percent in the placebo group. Life-threatening drug-related events were rare, occurring in 1 percent of patients in the interferon group. No significant differences between groups were observed in overall response rates, time to disease progression, or survival. The overall response rate was 4.4 percent (3.3 percent complete response and 1.1 percent partial response) in the interferon group and 6.6 percent (3.3 percent complete response and 3.3 percent partial response) in the placebo group (P=0.54), with a rate of durable complete response of 1 percent in both groups. The median time to disease progression was 1.9 months in both groups (P=0.49), and there was no significant difference in median survival (12.2 months with interferon vs. 15.7 months with placebo, P=0.52).No difference in outcome was observed in patients with metastatic renal-cell carcinoma who were treated with interferon gamma-1b as compared with placebo. These results emphasize the necessity of testing the efficacy of immunomodulators in randomized studies.

Abstract Heat shock protein 27 (Hsp27) is a chaperone implicated as an independent predictor of clinical outcome in prostate cancer. Our aim was to characterize changes in Hsp27 after androgen withdrawal and during androgen-independent progression in prostate xenografts and human prostate cancer to assess the functional significance of these changes using antisense inhibition of Hsp27. A tissue microarray was used to measure changes in Hsp27 protein expression in 232 specimens from hormone naive and posthormone-treated cancers. Hsp27 expression was low or absent in untreated human prostate cancers but increased beginning 4 weeks after androgen-ablation to become uniformly highly expressed in androgen-independent tumors. Androgen-independent human prostate cancer PC-3 cells express higher levels of Hsp27 mRNA in vitro and in vivo, compared with androgen-sensitive LNCaP cells. Phosphorothioate Hsp27 antisense oligonucleotides (ASOs) and small interference RNA potently inhibit Hsp27 expression, with increased caspase-3 cleavage and PC3 cell apoptosis and 87% decreased PC3 cell growth. Hsp27 ASO and small interference RNA also enhanced paclitaxel chemosensitivity in vitro, whereas in vivo, systemic administration of Hsp27 ASO in athymic mice decreased PC-3 tumor progression and also significantly enhanced paclitaxel chemosensitivity. These findings suggest that increased levels of Hsp27 after androgen withdrawal provide a cytoprotective role during development of androgen independence and that ASO-induced silencing can enhance apoptosis and delay tumor progression.

A prospective phase 3 trial was initiated to determine whether 8 compared with 3-month neoadjuvant hormonal therapy reduces prostate specific antigen (PSA) recurrence rates after radical prostatectomy. Our interim analysis includes secondary end points of differences in biochemistry, pathology and adverse events between the 2 groups.Men with clinically confined prostate cancer were randomized to receive 7.5 mg. leuprolide intramuscularly monthly and 250 mg. flutamide orally 3 times daily for 3 or 8 months before radical prostatectomy. Our study was powered to detect a 35% decrease in PSA recurrence, assuming a 30% recurrence rate in the 3-month arm after 3 years.A total of 547 men were randomized between August 1995 and April 1998. Men in the 8 and 3-month groups were equally stratified for T stage (29% T1c, 70% T2), Gleason grade (68% less than 4, 32% 4 or greater) and pretreatment PSA (63% less than 10, 27% 10 to 20 and 10% greater than 20 microg./l.). Mean pretreatment PSA was slightly higher in the 8-month compared with the 3-month group (11.64 versus 9.95 microg./l., respectively, p = 0.0539). A total of 44 men withdrew from study before surgery and, therefore, were nonevaluable. Preoperative PSA nadir was less than 0.1 microg./l. in 43.3% versus 75.1% (p <0.0001), and 0.3 microg./l. or greater in 21% versus 9.2% after 3 versus 8 months, respectively (p <0.0006). Mean serum PSA decreased 98% to 0.12 microg./l. after 3 months, with a further 57% to 0.052 microg./l. from 3 to 8 months. Transrectal ultrasound determined that prostatic volume decreased 37% from a mean of 40.6 to 25.4 cc after 3-month neoadjuvant hormonal therapy (p = 0.0001) and a further 13% to 22.2 cc after 8 months (p = 0.03). Mean hemoglobin decreased 15% (148.2 to 125.4 gm./dl.) after 3-month neoadjuvant hormonal therapy but stabilized thereafter. Radical prostatectomy was completed in 500 men, while surgery was aborted intraoperatively in 3. Positive margin rates were significantly lower in the 8 than 3-month group (12% versus 23%, respectively, p = 0.0106). There were no fatal adverse events and no differences between the 2 groups in the severity or causality (p = 0.287, 0.0564) of adverse events, or incidence of increased liver enzymes or diarrhea (p = 0.691, 0.288, respectively). However, men in the 8-month group noticed a higher number of newly reported adverse events (4.5 versus 2.9, p <0.0001) and higher incidence of hot flushes than the 3-month group (87% versus 72%, respectively, p <0.0001).Ongoing biochemical and pathological regression of prostate tumors occurs between 3 and 8 months of neoadjuvant hormonal therapy, suggesting that the optimal duration of neoadjuvant hormonal therapy is longer than 3 months. Longer followup is needed to determine whether longer therapy alters PSA recurrence rates.

Objectives To test the feasibility of using intermittent androgen suppression in the treatment of prostate cancer by taking advantage of the reversible action of medical castration. Methods Observations were made on a group of 47 patients (clinical Stage D2, 14; D1, 10; C, 19; B2, 2; and A2, 2) with a mean follow-up time of 125 weeks. Treatment was initiated with combined androgen blockade and continued for at least 6 months until a serum prostate-specific antigen (PSA) nadir was observed. Medication was then withheld until the serum PSA increased to a mean value between 10 and 20 ng/mL. This cycle of treatment and no treatment was repeated until the regulation of serum PSA became androgen independent. Results The first two treatment cycles lasted 73 and 75 weeks, with a mean time off therapy of 30 and 33 weeks and an overall mean percentage time off therapy of 41% and 45%, respectively. The mean time to achieve a nadir level of serum PSA was 20 weeks in cycle 1 and 18 weeks in cycle 2. Serum testosterone returned to the normal range within 8 weeks (range, 1 to 26) of stopping treatment. The off-treatment period in both cycles was associated with an improvement in sense of well-being and the recovery of libido and potency in the men who reported normal or near-normal sexual function before the start of therapy. In 7 patients with Stage D2 disease, the cancer progressed to an androgen-independent state. The mean and median times to progression were 128 weeks and 108 weeks, respectively. Seven patients have died, 1 from a noncancer-related illness, with mean and median overall survival times of 210 weeks and 166 weeks, respectively. Conclusions Prostate cancer is amenable to control by intermittent androgen suppression. This approach-affords an improved quality of life when the patient is off therapy. It also results in reduced toxicity and cost of treatment and possibly delays tumor progression. Whether survival is affected in a beneficial or adverse way remains to be studied in a randomized, prospective study.

Androgen ablation, the most common therapeutic treatment used for advanced prostate cancer, triggers the apoptotic regression of prostate tumors. However, remissions are temporary because surviving prostate cancer cells adapt to the androgen-deprived environment and form androgen-independent (AI) tumors. We hypothesize that adaptive responses of surviving tumor cells result from dysregulated gene expression of key cell survival pathways. Therefore, we examined temporal alterations to gene expression profiles in prostate cancer during progression to androgen independence at several time points using the LNCaP xenograft tumor model. Two key genes, sterol response element-binding protein (SREBP)-1 and -2 (SREBP-1a,-1c, and -2), were consistently dysregulated. These genes are known to coordinately control the expression of the groups of enzymes responsible for lipid and cholesterol synthesis. Northern blots revealed modest increased expression of SREBP-1a, -1c, and -2 after castration, and at androgen independence (day 21-28), the expression levels of both SREBP-1a and -1c were significantly greater than precastrate levels. Changes in SREBP-1 and -2 protein expression were observed by Western analysis. SREBP-1 68-kDa protein levels were maintained throughout progression, however, SREBP-2 68-kDa protein expression increased after castration and during progression (3-fold). SREBPs are transcriptional regulators of over 20 functionally related enzymes that coordinately control the metabolic pathways of lipogenesis and cholesterol synthesis, some of which were likewise dysregulated during progression to androgen independence. RNA levels of acyl-CoA-binding protein/diazepam-binding inhibitor and fatty acid synthase decreased significantly after castration, and then, during progression, increased to levels greater than or equal to precastrate levels. Expression of farnesyl diphosphate synthase did not decrease after castration but did increase significantly during progression to androgen independence. Levels of SREBP cleavage-activating protein, a regulator of SREBP transcriptional activity, decreased after castration and increased significantly at androgen independence. In clinical prostate cancer specimens from patients with varying grades of disease, the stained tissue sections showed high levels of SREBP-1 protein compared with noncancerous prostate tissue. After hormone withdrawal therapy, tumor levels of SREBP-1 decreased significantly after 6 weeks. AI tumors expressed significantly higher levels of SREBP-1. In summary, the LNCaP xenograft model of human prostate cancer as well as clinical specimens of prostate cancer demonstrated an up-regulation of SREBPs and their downstream effector genes during progression to androgen independence. As the AI phenotype emerges, enzymes critical for lipogenesis and cholesterol synthesis are activated and likely contribute significantly to cell survival of AI prostate cancer.

Clusterin is a cytoprotective chaperone protein that promotes cell survival and confers broad-spectrum treatment resistance. OGX-011 is a 2'-methoxyethyl modified phosphorothioate antisense oligonucleotide that is complementary to clusterin mRNA and has been reported to inhibit clusterin expression and enhance drug efficacy in xenograft models. The primary objective of this clinical study was to determine a biologically effective dose of OGX-011 that would inhibit clusterin expression in human cancer.Subjects (n = 25) with localized prostate cancer with high-risk features who were candidates for prostatectomy were treated with OGX-011 by 2-hour intravenous infusion on days 1, 3, and 5 and then weekly from days 8-29 combined with androgen blockade starting on day 1; prostatectomy was performed on days 30-36. Six different doses were tested, from 40 to 640 mg. OGX-011 plasma and prostate tissue concentrations were measured by an enzyme-linked immunosorbent assay method, and the pharmacokinetics of OGX-011 were determined from these data. Prostate cancer tissue, lymph nodes, and serial samples of peripheral blood mononuclear cells were assessed for clusterin expression using quantitative real-time polymerase chain reaction and immunohistochemistry. All statistical tests were two-sided.Only grade 1 and 2 toxicities were observed. The plasma half-life of OGX-011 was approximately 2-3 hours, and the area under the concentration versus time curve and CMAX (peak plasma concentration) increased proportionally with dose (Ptrend < .001). OGX-011 in prostate tissue increased with dose (Ptrend < .001). Dose-dependent decreases in prostate cancer and lymph node clusterin expression were observed by polymerase chain reaction of greater than 90% (Ptrend = .008 and Ptrend < .001, respectively) and by immunohistochemistry (Ptrend < .001 and Ptrend = .01, respectively).OGX-011 is well tolerated and reduces clusterin expression in primary prostate tumors. The optimal biologic dose for OGX-011 at the schedule used is 640 mg.

We show that integrin-linked kinase (ILK) stimulates the expression of VEGF by stimulating HIF-1α protein expression in a PKB/Akt- and mTOR/FRAP-dependent manner. In human prostate cancer cells, knockdown of ILK expression with siRNA, or inhibition of ILK activity, results in significant inhibition of HIF-1α and VEGF expression. In endothelial cells, VEGF stimulates ILK activity, and inhibition of ILK expression or activity results in the inhibition of VEGF-mediated endothelial cell migration, capillary formation in vitro, and angiogenesis in vivo. Inhibition of ILK activity also inhibits prostate tumor angiogenesis and suppresses tumor growth. These data demonstrate an important and essential role of ILK in two key aspects of tumor angiogenesis: VEGF expression by tumor cells and VEGF-stimulated blood vessel formation.

Prostate cancer, the most prevalent cancer affecting men, frequently metastasizes to the axial skeleton where it produces osteoblastic lesions with growth rates often exceeding that of the primary tumor. To evaluate the role of tumor cell-host stromal interaction and stromal specific growth factors (GFs) in prostate cancer growth and progression, we coinoculated athymic mice with human prostate cancer cells (LNCaP) and various nontumorigenic fibroblasts s.c. LNCaP tumor formation was most consistently induced by human bone (MS) fibroblasts (62%), followed by embryonic rat urogenital sinus mesenchymal (rUGM) cells (31%) and Noble rat prostatic fibroblasts (17%), but not by NIH-3T3, normal rat kidney, or human lung CCD16 fibroblasts. Carcinomas formed preferentially in male hosts, demonstrating in vivo androgen sensitivity. The human prostate component of these tumors was confirmed with immunohistochemical staining for prostate-specific antigen (PSA), Northern analysis for PSA expression, and Southern analysis for human repetitive Alu sequences. Elevations in serum PSA paralleled the histomorphological and biochemical findings. LNCaP and fibroblast cell-conditioned media (CM) was used to determine whether autocrine and paracrine mitogenic pathways exist between LNCaP and fibroblast cells in vitro, and various defined GFs were tested to identify possible active factors. Mitogenic assays revealed a 200-300% bidirectional stimulation between LNCaP and bone or prostate fibroblast-derived CM. Lung, normal rat kidney, and 3T3 fibroblast CM were not mitogenic for LNCaP cells. Among the purified GFs tested basic fibroblast growth factor (bFGF) was the most potent mitogen, stimulating LNCaP growth 180% in a concentration-dependent manner. Transforming growth factor alpha and epidermal growth factor were both minimally mitogenic. Coinoculation of LNCaP cells with a slowly absorbed matrix (Gelfoam) absorbed with bFGF or dialyzed and concentrated rUGM or MS CM was also capable of inducing LNCaP tumor formation in vivo. These observations illustrate that fibroblasts differentially modulate prostate cancer growth through the release of paracrine-mediated GFs, possibly including bFGF, and that tumor-stromal cell interactions play an important role in prostate cancer growth and progression.

Androgen receptor (AR) transactivation is known to enhance prostate cancer cell survival. However, the precise effectors by which the prosurvival effects of androgen and AR drive prostate cancer progression are poorly defined. Here, we identify a novel feed-forward loop involving cooperative interactions between ligand-activated AR and heat-shock protein 27 (Hsp27) phospho-activation that enhance AR stability, shuttling, and transcriptional activity, thereby increasing prostate cancer cell survival. Androgen-bound AR induces rapid Hsp27 phosphorylation on Ser(78) and Ser(82) residues in an AR- and p38 kinase-dependent manner. After this androgen-induced, non-nuclear phospho-activation, Hsp27 displaces Hsp90 from a complex with AR to chaperone AR into the nucleus and interact with its response elements to enhance its genomic activity. Inhibition of Hsp27 phosphorylation, or knockdown using the antisense drug OGX-427, shifted the association of AR with Hsp90 to MDM2, increased proteasome-mediated AR degradation, decreased AR transcriptional activity, and increased prostate cancer LNCaP cell apoptotic rates. OGX-427 treatment of mice bearing LNCaP xenografts transfected with an androgen-regulated, probasin-luciferase reporter construct resulted in decreased bioluminescence and serum PSA levels as pharmacodynamic readouts of AR activity, as well as AR, Hsp27, and Hsp90 protein levels in LNCaP tumor tissue. These data identify novel nongenomic mechanisms involving androgen, AR, and Hsp27 activation that cooperatively interact to regulate the genomic activity of AR and justify further investigation of Hsp27 knockdown as an AR disrupting therapeutic strategy in prostate cancer.

The purpose of this study was to identify and compare information and decision preferences of men with prostate cancer and their partners at the time of diagnosis. A convenience sample of 80 couples was recruited from The Prostate Centre in Vancouver, Canada. Participants used a computerized version of two previously used measures with this population: Control Preferences Scale and Information Survey Questionnaire. Results showed that men had a preference to play either an active or a collaborative role in decision making with their physician (92.5%) and partners (100%). The majority (55%) of partners wanted to play a collaborative role in treatment decision making. Couples identified prognosis, stage of disease, treatment options, and side effects as the top 4 information preferences. Men ranked information on sexuality more important than partners, and partners ranked information on home self-care higher than men. Men who had sons, a positive family history, and lower levels of education ranked heredity risk significantly higher. Profiles of information categories did not differ according to role preferences of either men or partners. The computer program has been shown to be a reliable and acceptable method of assessing information and decision preferences of these couples. An individualized approach is suggested, given the high reliability of individual's profiles.

Progression of prostate cancer to androgen independence (AI) results in part from the upregulation of anti-apoptotic genes following androgen withdrawal, and androgen-independent disease remains the primary obstacle to improved survival. Testosterone-repressed prostate message-2 (TRPM-2) encodes the anti-apoptotic protein clusterin, which is upregulated in response to cellular compromise as observed in normal and malignant tissues undergoing apoptosis. Systemic administration of antisense clusterin oligonucleotides in prostate cancer xenograft models delays progression to AI and enhances chemosensitivity. The objective of this study was to define changes in clusterin expression following neoadjuvant hormone therapy (NHT) in prostate cancer patients.Archival radical prostatectomy (RP) specimens were obtained for 128 patients who received either no NHT or treatment for 2-8 weeks, 3 months, or 8 months. Paired needle biopsy specimens were acquired for 30 patients and all tissues were subjected to clusterin immunohistochemistry. Western blot analysis was performed on frozen tissue from 5 untreated and 5 treated patients.Clusterin expression in malignant prostatic tissue was significantly greater in patients who underwent preoperative NHT (P < 0.001). Needle biopsies obtained prior to NHT consistently demonstrated lower staining intensity than corresponding RP specimens (P < 0.001). Western blot analysis confirmed clusterin levels increased 17-fold beginning within 4 weeks after androgen withdrawal.Upregulation of clusterin levels following androgen ablation therapy may represent an adaptive cell survival response following apoptotic signals like androgen withdrawal. These findings support clusterin as a valid therapeutic target in strategies employing novel multimodality therapy for advanced prostate cancer.

Abstract One strategy to improve therapies in prostate cancer involves targeting cytoprotective genes activated by androgen withdrawal to delay the emergence of the androgen-independent (AI) phenotype. The objectives of this study were to define changes in Hsp27 levels after androgen ablation and to evaluate the functional relevance of these changes in AI progression. Using a tissue microarray of 232 specimens of hormone-naïve and post-hormone ablation–treated prostate cancer, we found that Hsp27 levels increase after androgen ablation to become highly expressed (&amp;gt;4-fold, P ≤ 0.01) in AI tumors. Hsp27 overexpression rendered LNCaP cells highly resistant to androgen withdrawal both in vitro and in vivo. Tumor volume and serum prostate–specific antigen levels increased 4.3- and 10-fold faster after castration when Hsp27 was overexpressed. Treatment of LNCaP tumor cells in vitro with Hsp27 antisense oligonucleotides (ASO) or short-interfering RNA suppressed Hsp27 levels in a dose-dependent and sequence-specific manner increased the apoptotic sub–G0-G1 fraction and caspase-3 cleavage &amp;gt;2-fold, as well as decreased signal transducers and activators of transcription 3 (Stat3) levels and its downstream genes, c-fos and sPLA-2. The cytoprotection afforded by Hsp27 overexpression was attenuated by Stat3 knockdown using specific Stat3 ASO. Coimmunoprecipitation and immunofluorescence confirmed that Hsp27 interacts with Stat3 and that Stat3 levels correlated directly with Hsp27 levels. Hsp27 ASO treatment in athymic mice bearing LNCaP tumors significantly delayed LNCaP tumor growth after castration, decreasing mean tumor volume and serum prostate–specific antigen levels by 57% and 69%, respectively. These findings identify Hsp27 as a modulator of Stat3-regulated apoptosis after androgen ablation and as a potential therapeutic target in advanced prostate cancer.

Although recent laboratory and population studies suggest that prostate cancer may be responsive to insulin, there is a gap in knowledge concerning the expression of insulin receptors on benign or malignant prostate tissue.We immunostained 644 cores on tissue microarrays prepared from 29 prostate tissue samples without malignancies, 78 Gleason grade 3 cancers, 21 Gleason grade 4 cancers and 33 Gleason grade 5 cancers with antibodies against the insulin-like growth factor I receptor and the insulin receptor.We observed immunoreactivity with both antibodies, which implies the presence of hybrid receptors as well as IGF-I receptors and insulin receptors. Insulin receptor staining intensity was significantly (P < 0.001) higher on malignant than benign prostate epithelial cells. Analysis of information from public gene expression databases confirmed that co-expression of insulin receptor mRNA and IGF-I receptor mRNA is common in prostate cancer specimens. RT-PCR methods provided evidence for the presence of mRNA for both IR-A and IR-B insulin receptor isoforms.These observations document the presence of insulin receptors on primary human prostate cancers. The findings are relevant not only to ongoing clinical trials of drug candidates that target IGF-I and/or insulin receptors, but also to the hypothesis that obesity-associated hyperinsulinemia mediates the adverse effect of obesity on prostate cancer prognosis.

More than one-third of men may experience erectile dysfunction (ED) after nerve sparing radical retropubic prostatectomy. The efficacy and safety of vardenafil, a potent, selective, phosphodiesterase 5 inhibitor, was assessed for the treatment of ED after radical prostatectomy.In this double-blind study 440 men with ED after nerve sparing radical prostatectomy were randomized to take placebo, or 10 or 20 mg vardenafil. Efficacy was measured after 12 weeks using the erectile function domain of the International Index of Erectile Function, diary questions measuring vaginal penetration and intercourse success rates, and a global assessment question (GAQ) on erection.Of the intent to treat population 70% had severe ED (erectile function less than 11) at baseline. After 12 weeks both vardenafil doses were significantly superior to placebo (p <0.0001) for all efficacy variables. Improved erections (based on GAQ) were reported by 65.2% and 59.4% of patients on 20 and 10 mg vardenafil, respectively, and by only 12.5% of patients on placebo (p <0.0001). Among men with bilateral neurovascular bundle sparing, positive GAQ responses were reported by 71.1% and 59.7% of patients on 20 and 10 mg vardenafil, respectively, versus 11.5% of those on placebo (p <0.0001). The average intercourse success rate per patient receiving 20 mg vardenafil was 74% in men with mild to moderate ED and 28% in men with severe ED, compared to 49% and 4% for placebo, respectively. Few adverse events were observed. They were generally mild to moderate headache, flushing and rhinitis.In men with severe ED after nerve sparing radical retropubic prostatectomy, vardenafil significantly improved key indices of erectile function.

Abstract Clusterin/Apolipoprotein J (CLU) is a heterodimeric ubiquitously expressed secreted glycoprotein that is implicated in several physiological processes and is differentially expressed in many severe physiological disturbances, including tumor formation and in vivo cancer progression. Despite extensive efforts, clarification of CLU’s biological role has been exceptionally difficult and its precise function remains elusive. Short RNA duplexes, referred to as small interfering RNAs (siRNAs), provide a new approach for the elucidation of gene function in human cells. Here, we describe siRNA-mediated CLU gene silencing in osteosarcoma and prostate human cancer cells and illustrate that CLU mRNA is amenable to siRNA-mediated degradation. Our data demonstrate that CLU knockdown in human cancer cells induces significant reduction of cellular growth and higher rates of spontaneous endogenous apoptosis. Moreover, CLU knockdown cancer cells were significantly sensitized to both genotoxic and oxidative stress induced by chemotherapeutic drugs and H2O2, respectively. These effects were more pronounced in cell lines that express high endogenous steady-state levels of the CLU protein and occur through hyperactivation of the cellular apoptotic machinery. Overall, our results reveal that, in the distinct cellular contexts of the osteosarcoma and prostate cancer cells assayed, CLU is a central molecule in cell homeostasis that exerts a cytoprotective function. The described CLU-specific siRNA oligonucleotides that can potently silence CLU gene expression may thus prove valuable agents during antitumor therapy or at other pathological conditions where CLU has been implicated.

Purpose Pediatric glioblastoma (pGBM) is a rare, but devastating brain tumor. In contrast to GBM in adults (aGBM), little is known about the mechanisms underlying its development. Our aim is to gain insight into the molecular pathways of pGBM. Materials and Methods Thirty-two pGBM and seven aGBM samples were investigated using biochemical and transcriptional profiling. Ras and Akt pathway activation was assessed through the phosphorylation of downstream effectors, and gene expression profiles were generated using the University Health Network Human 19K cDNA arrays. Results were validated using real-time polymerase chain reaction and immunohistochemistry and compared with existing data sets on aGBM. Results There are at least two subsets of pGBM. One subset, associated with Ras and Akt pathway activation, has very poor prognosis and exhibits increased expression of genes related to proliferation and to a neural stem-cell phenotype, similar to findings in aggressive aGBM. This subset was still molecularly distinguishable from aGBM after unsupervised and supervised analysis of expression profiles. A second subset, with better prognosis, is not associated with activation of Akt and Ras pathways, may originate from astroglial progenitors, and does not express gene signatures and markers shown to be associated with long-term survival in aGBM. Both subsets of pGBM show overexpression of Y-box-protein-1 that may help drive oncogenesis in this tumor. Conclusion Our work, the first study of gene expression profiles in pGBM, provides valuable insight into active pathways and targets in a cancer with minimal survival, and suggests that these tumors cannot be understood exclusively through studies of aGBM.

In exploring the possible mechanisms of androgen independence of prostate-specific antigen (PSA) gene expression, we investigated the effect of elevating AP-1 by both 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment and transfection of the c-Jun expression vector in LNCaP cells. Transcription of PSA is initiated when ligand-activated androgen receptor (AR) binds to a region in the PSA promoter that contains an androgen-responsive element (ARE). It was found that TPA inhibited androgen-induced PSA gene expression by a mechanism that did not alter nuclear levels of AR protein. Overexpression of AP-1 (junand fos proteins) also inhibited androgen-induced PSA promoter activity. These observations were apparently related to the disruption of AR·ARE complexes as demonstrated by the results of electrophoretic mobility shift assays. Specifically, c-Jun inhibited the formation of AR·ARE complexes and conversely that AR-glutathioneS-transferase proteins inhibited the formation of c-Jun·TPA-responsive element (TRE) complexes. Consistent with the inhibitory effect of both proteins, anti-c-Jun antibody blocked the inhibition of AR·ARE complex formation by c-Jun. A similar, but less marked, effect was obtained when anti-AR antibody was used to prevent AR inhibition of c-Jun·TRE complex formation. These findings together with results obtained from co-immunoprecipitation experiments strongly suggest that mutual repression of DNA binding activity is due to direct interaction between the two proteins and that the degree of repression may be determined by the ratio of AR to c-Jun. The mechanism of repression studied in mutant analysis experiments yielded evidence of an interaction between the DNA- and ligand-binding domains of AR and the leucine zipper region of c-Jun. Thus, the AR is similar to other nuclear receptors in its ability to interact with AP-1. This association provides a link between AP-1 and AR signal transduction pathways and may play a role in the regulation of the androgen-responsive PSA gene. In exploring the possible mechanisms of androgen independence of prostate-specific antigen (PSA) gene expression, we investigated the effect of elevating AP-1 by both 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment and transfection of the c-Jun expression vector in LNCaP cells. Transcription of PSA is initiated when ligand-activated androgen receptor (AR) binds to a region in the PSA promoter that contains an androgen-responsive element (ARE). It was found that TPA inhibited androgen-induced PSA gene expression by a mechanism that did not alter nuclear levels of AR protein. Overexpression of AP-1 (junand fos proteins) also inhibited androgen-induced PSA promoter activity. These observations were apparently related to the disruption of AR·ARE complexes as demonstrated by the results of electrophoretic mobility shift assays. Specifically, c-Jun inhibited the formation of AR·ARE complexes and conversely that AR-glutathioneS-transferase proteins inhibited the formation of c-Jun·TPA-responsive element (TRE) complexes. Consistent with the inhibitory effect of both proteins, anti-c-Jun antibody blocked the inhibition of AR·ARE complex formation by c-Jun. A similar, but less marked, effect was obtained when anti-AR antibody was used to prevent AR inhibition of c-Jun·TRE complex formation. These findings together with results obtained from co-immunoprecipitation experiments strongly suggest that mutual repression of DNA binding activity is due to direct interaction between the two proteins and that the degree of repression may be determined by the ratio of AR to c-Jun. The mechanism of repression studied in mutant analysis experiments yielded evidence of an interaction between the DNA- and ligand-binding domains of AR and the leucine zipper region of c-Jun. Thus, the AR is similar to other nuclear receptors in its ability to interact with AP-1. This association provides a link between AP-1 and AR signal transduction pathways and may play a role in the regulation of the androgen-responsive PSA gene. Prostate-specific antigen (PSA) 1The abbreviations used are: PSA, prostate-specific antigen; AR, androgen receptor; ARE, androgen-responsive element; TPA, 12-O-tetradecanoylphorbol 13-acetate; PKC, protein kinase C; TRE, TPA-responsive element; FBS, fetal bovine serum; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; EMSA, electrophoretic mobility shift assay; GR, glucocorticoid receptor. belongs to the family of kallikrien-like serine proteases (for a review, see Ref. 1MacDonald R.J. Margolius H.S. Erdös E.G. Biochem. J. 1988; 253: 313-321Crossref PubMed Scopus (130) Google Scholar). In males, expression of PSA occurs exclusively in the prostate with serum levels of PSA glycoprotein being an important marker in the diagnosis and progression of prostate cancer (2Catalona W.J. Smith D.S. Ratliff T.L. Basler J.W. J. Am. Med. Assoc. 1993; 270: 948-954Crossref PubMed Scopus (866) Google Scholar, 3Oesterling J.E. J. Urol. 1991; 145: 907-923Crossref PubMed Scopus (1174) Google Scholar). PSA is an androgen-induced gene that contains androgen response elements (AREs) to which the androgen receptor (AR) binds (4Riegman P.H.J. Vlietstra R.J. van der Korput J.A.G.M. Brinkmann A.O. Trapman J. Mol. Endocrinol. 1991; 5: 1921-1930Crossref PubMed Scopus (397) Google Scholar, 5Cleutjens K.B.J.M. van Eekelen C.C.E.M. van der Korput H.A.G.M. Brinkmann A.O. Trapman J. J. Biol. Chem. 1996; 271: 6379-6388Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). The AR belongs to the superfamily of nuclear receptors that mediate the responses of lipophilic ligands, including steroids, retinoids, vitamin D3, and thyroid hormones (6Zilliacus J. Wright A.P.H. Carlstedt-Duke J. Gustafsson J.-Å. Mol. Endocrinol. 1995; 9: 389-400Crossref PubMed Google Scholar). These receptors contain a highly conserved DNA-binding domain comprised of zinc finger-like motifs responsible for sequence-specific DNA binding, as well as protein-protein interactions. There is evidence to suggest that the DNA-binding domain of a nuclear receptor may interact with the leucine zipper of AP-1 to result in mutual transrepression (7Yang-Yen H.-F. Chambard J.-C. Sun Y.-L. Smeal T. Schmidt T.J. Drouin J. Karin M. Cell. 1990; 62: 1205-1215Abstract Full Text PDF PubMed Scopus (1319) Google Scholar, 8Schüle R. Rangarajan P. Kliewer S. Ransone L.J. Bolado J. Yang N. Verma I.M. Evans R.M. Cell. 1990; 62: 1217-1226Abstract Full Text PDF PubMed Scopus (1038) Google Scholar, 9Lucibello F.C. Slater E.P. Jooss K.U. Beato M. Müller R. EMBO J. 1990; 9: 2827-2834Crossref PubMed Scopus (225) Google Scholar). However, this interaction between nuclear receptors and AP-1 may be cell-specific, gene-specific, and may involve various mechanisms, including protein-protein interaction and/or adjacent or overlapping binding sites (10Pfahl M. Endocr. Rev. 1993; 14: 651-658Crossref PubMed Scopus (447) Google Scholar). AP-1 is a transcriptional factor whose components are nuclear proteins encoded by c-fos and c-jun proto-oncogenes induced by 12-O-tetradecanoylphorbol 13-acetate (TPA). AP-1 has been implicated in cell growth, differentiation, and development with its activity modulated by growth factors, cytokines, oncogenes, and tumor promoters activating protein kinase C (PKC) (10Pfahl M. Endocr. Rev. 1993; 14: 651-658Crossref PubMed Scopus (447) Google Scholar). AP-1 induces transcriptional activation through interaction with the TPA-responsive element (TRE or AP-1 DNA-binding site) (11Angel P. Imagawa M. Chiu R. Stein B. Imbra R.J. Rahmsdort H.J. Jonat C. Herrlich P. Karin M. Cell. 1987; 49: 729-739Abstract Full Text PDF PubMed Scopus (2155) Google Scholar, 12Lee W. Mitchell P. Tjian R. Cell. 1987; 49: 741-752Abstract Full Text PDF PubMed Scopus (1364) Google Scholar). TREs are recognized by Jun homodimers and Jun/Fos heterodimers that are formed through the leucine zipper domain of both proteins (11Angel P. Imagawa M. Chiu R. Stein B. Imbra R.J. Rahmsdort H.J. Jonat C. Herrlich P. Karin M. Cell. 1987; 49: 729-739Abstract Full Text PDF PubMed Scopus (2155) Google Scholar). The basic region adjacent to the leucine zipper on Jun and Fos proteins mediates AP-1 DNA binding activity (13Kouzarides T. Ziff E. Cancer Cells. 1989; 1: 71-76PubMed Google Scholar, 14Verma I.M. Ransone L.J. Curr. Opin. Cell Biol. 1989; 1: 536-540Crossref PubMed Scopus (7) Google Scholar, 15Vogt P.K. Morgan I. Cancer Biol. 1990; 1: 27-36PubMed Google Scholar). Since the zinc finger motifs of the AR share a high degree of homology with the same regions of other nuclear receptors and in the face of mounting evidence of AP-1 interaction with such motifs, the question arose whether AR function could also be affected by interaction with AP-1. Accordingly, in this study we examined the direct interaction of AP-1 with AR protein and the effects of elevated AP-1 on androgen-stimulated PSA gene expression. All chemicals were purchased from Sigma, unless stated otherwise. PC3 and CV-1 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% FBS (Life Technologies, Inc., Burlington, Ontario, Canada). LNCaP cells were maintained in RPMI 1640 supplemented with 5% FBS. Cells between the 37th and 49th generation were used in the these experiments. For Northern blot analyses, LNCaP cells were down-shifted to RPMI 1640 containing 2% FBS plus 2% TCM™ (serum replacement obtained from Celox Corp., Hopkins, MN), for 10–14 days and 5 × 105 or 3 × 105 cells were initially plated on 6-cm dishes or 6-well plates, respectively. Culture medium was changed to RPMI 1640 with 2% TCM™ (i.e. serum-free), with or without 10 nm R1881, when wells were 60–70% confluent with the cells. Total RNA was extracted with acid guanidinium thiocyanate/phenol/chloroform (16Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63190) Google Scholar) and fractionated by electrophoresis prior to blotting onto Hybond-N+ filters (Amersham, Oakville, Ontario, Canada). The 1.4-kb EcoRI fragments of the PSA cDNA (17Lundwall Å. Lilja H. FEBS Lett. 1987; 214: 317-322Crossref PubMed Scopus (363) Google Scholar), 0.7-kb EcoRI/HindIII fragments of human AR cDNA (18Chang C. Kokontis J. Liao S. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7211-7215Crossref PubMed Scopus (460) Google Scholar) and 1.9-kb PstI fragments of chicken β-actin (19Cleveland D.W. Lopata M.A. MacDonald R.J. Cowan N.J. Rutter W.J. Kirschner M.W. Cell. 1980; 20: 95-105Abstract Full Text PDF PubMed Scopus (1312) Google Scholar) were labeled with [α-32P]dCTP by Random Primers DNA labeling kit (Life Technologies, Inc.). The 40-base oligonucleotides for either c-Jun or c-Fos (Ciderlane, Toronto, Ontario, Canada) were end-labeled by T4 polynucleotide kinase with [γ-32P]ATP. Hybridization was performed as reported previously (20Sato N. Gleave M.E. Bruchovsky N. Rennie P.S. Goldenberg S.L. Lange P.H. Sullivan L.D. J. Steroid Biochem. Mol. Biol. 1996; 58: 139-146Crossref PubMed Scopus (188) Google Scholar). Filters with PSA and β-actin were washed in 0.1 × SSC, 0.1% SDS for 30 min at 65 °C, while filters with AR, c-Jun and c-Fos were in 0.5 × SSC, 0.2% SDS for 30 min at 55 °C. Densitometric analyses of mRNA bands were performed using NIH image (National Institutes of Health) from scanned x-ray films. LNCaP cells were incubated in RPMI 1640 containing 2% TCM™ for 24 h prior to the addition of vehicle (0.1% ethanol), 10 nm R1881, or 1 nm TPA. After incubation with compounds, cytosolic and nuclear extracts were prepared as described by Antras et al. (21Antras J. Lasnier F. Pairault J. J. Biol. Chem. 1991; 266: 1157-1161Abstract Full Text PDF PubMed Google Scholar). Western blots were performed with approximately 40 μg of protein in each lane. Immunoblots were blocked overnight in 5% dry milk (w/v) in 20 mm Tris-HCl, pH 7.4, containing 500 nm NaCl (TBS). Blots were incubated for 4 h with antibodies to AR (PG-21) (22Prins G.S. Birch L. Greene G.L. Endocrinology. 1991; 129: 3187-3199Crossref PubMed Scopus (385) Google Scholar), c-Jun (sc-045, Santa Cruz Biotechnology, Inc, Santa Cruz, CA), or c-Fos (sc-052, Santa Cruz Biotechnology, Inc.). The blots were then washed and incubated for 1 h with the secondary antibody (1:10,000). Antibodies were diluted in 5% milk/TBS solution. AR, c-Jun, and c-Fos proteins were detected using the ECL luminescence kit (Amersham Corp.). Densitometric analyses of protein bands were performed using NIH image from scanned x-ray films. LNCaP and PC3 cells were incubated in RPMI 1640 and Dulbecco's modified Eagle's medium, respectively, containing 5% dextran-coated stripped serum, 10 nm R1881 for 24 h, and 10 nm TPA for 6 h. The cells were harvested and nuclear extracts were prepared as described by Antraset al. (21Antras J. Lasnier F. Pairault J. J. Biol. Chem. 1991; 266: 1157-1161Abstract Full Text PDF PubMed Google Scholar). The nuclei were solubilized in mild Nonidet P-40 buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 5 mm EDTA, 0.1% Nonidet P-40, 5 μg/ml leupeptin 5 μg/ml aprotinin, 5 μg/ml trypsin inhibitor, 5 μg/ml bacitracin, and 1 mm phenylmethylsulfonyl fluoride) for 15 min at 4 °C. The nuclear extracts were precleared with protein A-Sepharose for 30 min and incubated with anti-AR antibody PG-21 (22Prins G.S. Birch L. Greene G.L. Endocrinology. 1991; 129: 3187-3199Crossref PubMed Scopus (385) Google Scholar) or 15071A (Pharmingen, San Diego, CA) for 1 h at 4 °C. The antigen antibody complexes were collected by the addition of protein A-Sepharose. Immune complexes were washed once with mild Nonidet P-40 buffer prior to separating on a 10% SDS-PAGE. Western blots analyses were carried out with anti c-Fos antibody and anti c-Jun antibody as described above. PSA 5′-flanking DNA was obtained by PCR-mediated amplification of human genomic DNA using oligonucleotide primers corresponding to the PSA gene. The sequences for primers were 5′-CATTGTTTGCTGCACGTTGGAT-3′ and 5′-TCCGGGTGCAGGTGGTAAGCTTGG-3′. The PCR fragments were purified by gel electrophoresis, blunt-ended, and ligated withEcoRV-digested pBluescript (pBS) SK(−) (Stratagene, La Jolla, CA). pBS containing the PSA 5′-flanking DNA (designated as pBS-PSA-630) was amplified and purified from transformedEscherichia coli DH-5α and sequenced by dideoxynucleotide chain termination method using double-stranded DNA cycle sequencing system kit (Life Technologies, Inc.). DNA fragments corresponding to −630/+12 of the PSA 5′-flanking region were excised from pBS-PSA-630 with HindIII and inserted into the HindIII site of promoterless plasmid, pGL-2 basic (Promega, Madison, WI), which contains firefly luciferase as a reporter gene. This pGL-2 basic containing the −630/+12 fragment of the PSA 5′-flanking DNA was referred to as pPSA-630. LNCaP cells (2–2.5 × 105) were plated on 6-well plates and incubated in RPMI 1640 with 5% FBS for 3 days, resulting in 50–60% confluence. Plasmid DNA was mixed with 5 μl of Lipofectin agent (Life Technologies, Inc.) and incubated for 15–20 min at room temperature. The total amount of plasmid DNA used was normalized to 3 μg/well by the addition of empty plasmid. Medium was replaced after 24 h by RPMI 1640 with 2% TCM™ with or without R1881 (i.e. serum-free media). Cells were collected after 48-h incubation using cell lysis buffer (100 mm potassium phosphate (pH 7.8), 0.2% Triton X-100, and 1 mmdithiothreitol). Luciferase activities were measured using a commercial kit from Promega according to the manufacturer's protocol, and activities were normalized by either protein concentration determined by the method of Bradford (23Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar) or β-galactosidase activities measured with Galacto-Light (Tropix Inc.). Luciferase activities are expressed as relative luminescent units/mg of protein/min. All transfection experiments were carried out in triplicate wells and repeated two to eight times using at least two sets of plasmids prepared separately. Full-length rat AR cDNA (amino acid 1–902) was cloned into pRc-CMV whose transcription is driven by cytomegalovirus promoter and this plasmid was referred to as pAR6. In our previous report, transcriptional efficacy of pAR6 was determined by expression of mRNA and binding assay in AR-negative PC3 cells when stably transfected with pAR6 (24Rennie P.S. Bruchovsky N. Leco K.J. Sheppard P.C. McQueen S.A. Cheng H. Snoek R. Hamel A. Bock M.E. MacDonald B.S. Nickel B.E. Chang C. Liao S. Cattini P.A. Matusik R.J. Mol. Endocrinol. 1993; 7: 23-26Crossref PubMed Scopus (215) Google Scholar). Several mutant rat AR expression plasmids were constructed in our laboratory. Regions of amino acids encoded by these mutant AR expression plasmids are as follows: pAR4 (232–649), pAR5 (390–649), and pAR7 (232–902). pRSV-c-Jun and pRSV-c-Fos are wild type c-Jun and c-Fos expression plasmids, respectively. pRSV-c-Jun-Δ1 is lacking a small portion of the N-terminal activator domain, -c-Jun-Δ3 lacks the N-terminal domain, and -c-Jun-ΔLZ lacks the leucine zipper. pRSV-Jun B, -Jun D, -Fra 1, and -Fra 2 are Jun B, Jun D, Fra 1, and Fra 2 expression plasmids, respectively. pRSV-0 is the empty plasmid for pRSV series of plasmids. ARR3-tk-luciferase reporter construct consists of three congruent rat probasin AREs (−244 to −96) ligated in tandem into theHindIII site of the pT81 luciferase vector (ATCC, Rockville, MD) as described by us previously (25Snoek R. Rennie P.S. Kasper S. Matusik R.J. Bruchovsky N. J. Steroid Biochem. Mol. Biol. 1996; 59: 243-250Crossref PubMed Scopus (37) Google Scholar). pCH110 (Amersham), a β-galactosidase expression plasmid, was co-transfected as an internal marker for normalizing efficacy of transient transfection. The prokaryotic expression vector pET-8c/c-Jun coding for the full-length c-Jun was transformed into E. coli BL21(DE3)pLysS. The recombinant protein was induced with 0.1 mm isopropyl-β-d-thiogalactopyranoside and extracted from inclusion bodies according to the protocol of Lin and Cheng (26Lin K.-H. Cheng S.-Y. BioTechniques. 1991; 11: 752-753Google Scholar) with slight modifications. Briefly, the inclusion bodies were isolated and subjected to several rounds of sonication in the appropriate buffers. The insoluble pellet was solubilized in 5m guanidinium HCl, followed by stepwise dialysis against buffers containing 2 m, 1 m, and 0.5m guanidinium HCl. Yields were typically 14 mg of c-Jun protein per 200 ml of bacterial culture with 90% purity. AR1 and AR2 were expressed in E. coli as isopropyl-β-d-thiogalactopyranoside-induced fusion protein with glutathione S-transferase (GST), purified through glutathione affinity chromatography, and calculated to have greater than 90% purity when assayed by Coomassie Blue staining of polyacrylamide gels (25Snoek R. Rennie P.S. Kasper S. Matusik R.J. Bruchovsky N. J. Steroid Biochem. Mol. Biol. 1996; 59: 243-250Crossref PubMed Scopus (37) Google Scholar). AR1 encodes amino acids 524–902 (rat AR) encompassing the DNA-binding domain, hinge region, and ligand-binding domain. AR2 encodes amino acids 524–649 (rat AR) encompassing the DNA-binding domain and hinge region. Nuclear extracts from LNCaP cells or purified proteins were used for EMSA studies. Nuclear extracts were prepared from cells treated with vehicle (0.1% ethanol), 10 nm R1881, or TPA (1 and 10 nm) for 6 h before harvesting. DNA binding reactions were carried out in a total volume of 25 μl, containing DNA binding buffer (20 mm HEPES, pH 7.9, 20% (v/v) glycerol, 100 mm KCl, 0.2 mm EDTA, 1 mmdithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and 500 ng of poly(dI-dC) (Pharmacia Biotech Inc.)). Approximately 1.5 fmol of double-stranded 32P-labeled TRE oligonucleotide (5′-CGCTTGATGAGTCAGCCGGAA-3′), PSA ARE oligonucleotide (5′-TTGCAGAACAGCAAGTGCTAGCTC-3′), PSA mutant ARE (5′-TTGCAAAAAAGCAAGTGCTAGCTC-3′) were used. SP-1 oligonucleotide (5′-ATTCGATCGGGGCGGGGCGAG-3′) and the TRE oligonucleotide were obtained from Promega. All lanes were normalized for additions of purified proteins by correction for the amount of buffer and total protein. For competition experiments 100-fold excess unlabeled oligonucleotide was used. DNA-protein complexes were separated under nondenaturing conditions in a 8% polyacrylamide gel (29:1) containing 2.5% glycerol and run in 0.5 × TBE (1 × = 89 mm Tris borate, 89 mm boric acid, and 2 mm EDTA, pH 8.3) at 200 V. Bands from dried EMSA gels were quantified by the STORM 860 PhosphorImager (Molecular Dynamics). Experiments employing antibodies were first preincubated for 30 min at room temperature with the antigen and antibody before the probe was added. The Student's t test was used for statistical analysis. The significance levels were set at: ***,p < 0.001; **, p < 0.01; and *,p < 0.05, unless stated otherwise in the figure legend. TPA alters the transcriptional activity of numerous nuclear receptors. Therefore, to examine the biological effects of TPA upon androgen regulation of PSA gene expression, we exposed LNCaP cells to TPA and performed Northern blot analyses. Cells exposed to the synthetic androgen, R1881 (10 nm), showed a 5-fold increase in accumulated PSA mRNA as compared with control levels (Fig.1 A). Androgen-stimulated cells exposed to TPA for 9 h showed a dose-dependent decrease in PSA mRNA levels. However, TPA concentrations of 10 and 100 nm induced “apoptosis-like” cell death of LNCaP cells consistent with the observations of Day et al. (27Day M.L. Zhao X. Wu S. Swanson P.E. Humphrey P.A. Cell Growth Differ. 1994; 5: 735-741PubMed Google Scholar) and Young et al. (28Young C.Y.F. Murtha P.E. Zhang J. Oncol. Res. 1994; 6: 203-210PubMed Google Scholar). TPA concentrations of 1 nmand less did not alter morphology nor reduce cell viability (data not shown). Therefore, all subsequent experiments employed a TPA concentration of 1 nm. β-Actin mRNA levels did not appear to be altered in cells undergoing apoptosis, as compared with untreated cell levels, after 9 h of treatment with 10 and 100 nm TPA, which is consistent to observations with HeLa cells undergoing programmed cell death (29French L.E. Wohlwend A. Sappino A.-P. Tschopp J. Schifferli J.A. J. Clin. Invest. 1994; 93: 877-884Crossref PubMed Scopus (164) Google Scholar). TPA is thought to increase AP-1 transactivation through the PKC signal transduction pathway (30Angel P. Karin M. Biochim. Biophys. Acta. 1991; 1072: 129-157Crossref PubMed Scopus (3270) Google Scholar). Thus, inhibition of PKC activity by staurosporine, a PKC inhibitor, should block TPA induction of AP-1. In Fig. 1 B, a 50 nm concentration of staurosporine completely blocked the TPA-associated decrease of androgen-induced PSA mRNA levels. Staurosporine (50 nm) alone had no effect on androgen-induced PSA mRNA levels. A time-dependent decrease of androgen-induced levels of PSA mRNA was observed when cells were exposed to 1 nm TPA (Fig. 1 C). These decreases were apparent after 6 h of TPA treatment and levels continued to decline for the duration of the experiment at 24 h. β-Actin mRNA levels remained consistent, and androgen-induced levels of PSA mRNA remained elevated in the absence of TPA for the duration of the study. TPA has been shown to induce c-jun and c-fosmRNA levels in LNCaP cells (27Day M.L. Zhao X. Wu S. Swanson P.E. Humphrey P.A. Cell Growth Differ. 1994; 5: 735-741PubMed Google Scholar). In agreement, TPA (1 nm) caused a transient increase in both c-junand c-fos mRNA levels (Fig. 1 D), regardless of the presence of androgen (R1881). This suggests that androgen stimulation and the subsequent activation of AR does not interfere with the signal transduction pathway leading from PKC to c-junand c-fos induction in LNCaP cells. A time course study of TPA induction of c-Jun and c-Fos proteins in LNCaP cells showed that maximum levels were achieved after 4.5 h of exposure (Fig. 2). At this time point, c-Jun levels were 6.6-fold higher and c-Fos levels 49-fold higher than levels in untreated cells. c-Jun levels remained 3–4-fold higher in TPA-treated cells, as compared with levels in untreated cells, for the duration of the experiment (32 h). Androgen induction of PSA mRNA has been shown to be mediated by the AR which binds to AREs on the PSA promoter (4Riegman P.H.J. Vlietstra R.J. van der Korput J.A.G.M. Brinkmann A.O. Trapman J. Mol. Endocrinol. 1991; 5: 1921-1930Crossref PubMed Scopus (397) Google Scholar, 5Cleutjens K.B.J.M. van Eekelen C.C.E.M. van der Korput H.A.G.M. Brinkmann A.O. Trapman J. J. Biol. Chem. 1996; 271: 6379-6388Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). To determine whether the TPA-associated decreases in androgen-induced PSA mRNA demonstrated in Fig. 1 A were due to reduced expression of AR, we examined levels of AR protein. AR protein was detected as a band at approximately 110 kDa in both nuclear and cytosolic extracts (Fig. 3). Nuclear extracts prepared from cells exposed to R1881 had increased levels of nuclear AR, as compared with cells not exposed to androgen. Cytosolic and nuclear levels of AR were not altered by treatment with TPA (1 nm) for 24 h. Therefore, TPA does not appear to decrease PSA gene expression by a mechanism that involves decreasing the nuclear levels of AR protein. PSA promoter activity was examined by transient transfection of LNCaP cells with a PSA promoter-luciferase reporter plasmid (pPSA-630). LNCaP cells express endogenous AR, and the addition of 10 nm R1881 to cells resulted in a 5-fold increase of PSA promoter activity (data not shown). Co-transfection of cells with the rat wild type AR (pAR6) expression plasmid (0.5 μg/well) resulted in a 34-fold increase in androgen-induced PSA promoter activity in the presence of 10 nm R1881 (Fig. 4 A). Therefore, all subsequent studies measuring androgen-induced PSA promoter activities were performed with cells transiently transfected with pAR6. In Fig. 1, A and C, it was demonstrated that TPA decreased androgen-induced PSA mRNA. In agreement with these data, androgen-induced PSA promoter activity was also inhibited by 39% in LNCaP cells exposed to 1 nm TPA for 24 h (data not shown). Numerous nuclear receptors have been shown to interact with TPA-inducible proteins, c-Jun and/or c-Fos (10Pfahl M. Endocr. Rev. 1993; 14: 651-658Crossref PubMed Scopus (447) Google Scholar). Therefore, to determine whether c-Jun and/or c-Fos are involved in TPA repression of androgen-induced PSA promoter activity, pPSA-630, pAR6, and increasing amounts of c-Jun and/or c-Fos expression plasmids were transfected into LNCaP cells. Androgen-induced PSA promoter activity was inhibited in a dose-dependent manner with transfection of increasing amounts of c-Jun and c-Fos, c-Jun, or c-Fos expression plasmids (in order of descending potency) (Fig. 4 B). Overexpression of c-Jun and c-Fos had negligible effects on the empty plasmid GL-2 basic (data not shown) that originates from pUC18 plasmid reported to contain a TRE (31Kushner P. Baxter J.D. Duncan K.G. Lopez G.N. Schaufele F. Uht R.M. Webb P. West B.L. Endocrinology. 1994; 8: 405-407Google Scholar). In addition, overexpression of c-Jun and c-Fos also had no effect on the pGL2-Control vector (Promega), which contains the SV40 promoter inserted into the same HindIII site as the PSA promoter (data not shown). To investigate the specificity of c-Jun and c-Fos inhibition, we examined the effects of other members of thejun and fos families on androgen-induced PSA promoter activity by transfection of the respective expression vectors into LNCaP cells. All members of the jun family examined significantly attenuated androgen-induced PSA promoter activity relative to RSV-0 values (Fig. 5). However, for members of the fos family investigated, only c-Fos significantly inhibited androgen-induced PSA promoter activity in the absence ofjun members. Maximum inhibition of androgen-induced PSA promoter activity was generally observed in cells co-transfected with expression plasmids from both jun and fosfamilies (lanes 8–16). EMSA with radiolabeled synthetic oligonucleotide containing a TRE consensus site and nuclear extracts prepared from LNCaP cells exposed to TPA for 6 h showed comparable DNA binding activities in vehicle-treated (0.1% ethanol) and 10 nmR1881-treated cells (Fig. 6 A). Cells treated with 1 nm TPA (lane 3) showed a 3.7-fold increase in DNA binding activity (compared with vehicle-treated levels,lane 1), which was similar to levels obtained with nuclear extracts from cells treated with 1 nm TPA and R1881 (lane 4). These data are comparable with the 4-fold increase in cellular levels of TPA-induced c-Jun protein for this time point as shown in Fig. 2. In Fig. 6 A, TPA (10 nm) was the more potent of the two concentrations of TPA examined and resulted in a 11.7-fold increase in DNA binding activity of nuclear extracts to the TRE oligonucleotide (lane 5). The addition of R1881 to 10 nm TPA-treated cells consistently resulted in approximately a 33% reduction in AP-1 DNA-binding activity (lane 6). Specificity of DNA binding activity was shown by competition experiments using 100-fold excess unlabeled SP-1 oligonucleotide (nonspecific competitor) (lane 7) and TRE oligonucleotide (specific competitor) (lane 8). Thus, nuclear extracts from TPA-treated LNCaP cells are characterized by enhanced AP-1 DNA-binding activity which is reduced by R1881. The work of Kallio et al. (32Kallio P.J. Poukka H. Moilanen A. Jänne O.A. Palvimo J.J. Mol. Endocrinol. 1995; 9: 1017-1028Crossref PubMed Google Scholar) indicated that AR inhibits c-Jun DNA binding activity, while c-Jun does not affect AR DNA binding to the C3 ARE. Similarly, the c-Jun DNA binding activity seen in our experiments (Fig. 6 B, lane 3) was also inhibited by peptide fragments of the AR. AR1-GST, containing both the ligand- and DNA-binding domains, was more potent in the inhibition of c-Jun DNA binding activity (lanes

In most patients with prostate cancer, continuous androgen suppression (CAS) therapy causes tumour regression and an accompanying decrease in serum prostate specific antigen (PSA). However, with tumour progression, regulation of both tumour growth and PSA gene expression becomes androgen-independent. Because androgen resistance develops, in part, from adaptive cell survival mechanisms activated by androgen withdrawal, we hypothesize that intermittent re-exposure to androgens may prolong time to androgen-independent progression. The objective of this study was to determine whether intermittent androgen suppression (IAS) could delay the onset of androgen-independent PSA gene regulation in LNCaP prostate tumour model when compared to CAS. Five or six cycles of IAS were possible before progression developed. IAS prolonged time to androgen-independent PSA gene regulation from an average of 26 days in CAS to 77 days in IAS. Serum PSA increased above pre-castrate levels in all mice treated with CAS by 28 days post-castration, but remained below pre-castrate levels in 75% of IAS-treated mice by 60 days post-castration. By 15 weeks post-castration, serum PSA levels increased 7-fold above pre-castrate levels in CAS-treated mice compared to 1.9-fold increase in IAS-treated mice. PSA mRNA expression levels highly correlated with serum PSA levels in both groups. Maintenance of androgen dependency through IAS may be due to androgen-induced differentiation and/or down-regulation of androgen-suppressed gene expression.

Oncologic outcomes in men with radiation-recurrent prostate cancer (PCa) treated with salvage radical prostatectomy (SRP) are poorly defined.To identify predictors of biochemical recurrence (BCR), metastasis, and death following SRP to help select patients who may benefit from SRP.This is a retrospective, international, multi-institutional cohort analysis. There was a median follow-up of 4.4 yr following SRP performed on 404 men with radiation-recurrent PCa from 1985 to 2009 in tertiary centers.Open SRP.BCR after SRP was defined as a serum prostate-specific antigen (PSA) ≥ 0.1 or ≥ 0.2 ng/ml (depending on the institution). Secondary end points included progression to metastasis and cancer-specific death.Median age at SRP was 65 yr of age, and median pre-SRP PSA was 4.5 ng/ml. Following SRP, 195 patients experienced BCR, 64 developed metastases, and 40 died from PCa. At 10 yr after SRP, BCR-free survival, metastasis-free survival, and cancer-specific survival (CSS) probabilities were 37% (95% confidence interval [CI], 31-43), 77% (95% CI, 71-82), and 83% (95% CI, 76-88), respectively. On preoperative multivariable analysis, pre-SRP PSA and Gleason score at postradiation prostate biopsy predicted BCR (p = 0.022; global p < 0.001) and metastasis (p = 0.022; global p < 0.001). On postoperative multivariable analysis, pre-SRP PSA and pathologic Gleason score at SRP predicted BCR (p = 0.014; global p < 0.001) and metastasis (p < 0.001; global p < 0.001). Lymph node involvement (LNI) also predicted metastasis (p = 0.017). The main limitations of this study are its retrospective design and the follow-up period.In a select group of patients who underwent SRP for radiation-recurrent PCa, freedom from clinical metastasis was observed in >75% of patients 10 yr after surgery. Patients with lower pre-SRP PSA levels and lower postradiation prostate biopsy Gleason score have the highest probability of cure from SRP.

Increased expression of the bcl-2 gene has been observed in prostate cancer cells after androgen withdrawal and has been associated with the development of androgen independence and chemoresistance. The objective of this study was to determine whether antisense Bcl-2 oligodeoxynucleotides could enhance paclitaxel cytotoxicity and delay androgen-independent progression.Northern and western blot analyses were used to measure changes in Bcl-2 expression in mouse Shionogi tumor cells after treatment with antisense Bcl-2 oligodeoxynucleotides and/or paclitaxel. Growth inhibition and induction of apoptotic cell death were assessed with the use of standard methods. All P values are two-sided.Treatment of Shionogi tumor cells with 500 nM antisense Bcl-2 oligodeoxynucleotides decreased expression of Bcl-2 messenger RNA (mRNA) by approximately 85%. Paclitaxel treatment induced Bcl-2 protein phosphorylation but did not alter Bcl-2 mRNA expression. Antisense Bcl-2 oligodeoxynucleotide treatment substantially enhanced paclitaxel chemosensitivity in a dose-dependent manner. Characteristic apoptotic DNA laddering and cleavage of poly(adenosine diphosphate-ribose) polymerase were demonstrated only after combined treatment. Adjuvant in vivo administration of antisense Bcl-2 oligodeoxynucleotides and micellar paclitaxel following castration resulted in a statistically significant delay of androgen-independent, recurrent tumors compared with administration of either agent alone (P<.001, Mantel-Cox log-rank test). Combination therapy also statistically significantly inhibited the growth of established hormone-refractory tumors compared with treatment with either agent alone (P<.001, Student's t test). CONCLUSIONS. Combined treatment with antisense Bcl-2 oligodeoxynucleotides and paclitaxel could be a novel and attractive strategy to inhibit progression to androgen-independent disease as well as growth of hormone-refractory prostate cancer through deprivation of Bcl-2 function.

No AccessJournal of Urology1 Apr 1992Prostate and Bone Fibroblasts Induce Human Prostate Cancer Growth in Vivo: Implications for Bidirectional Tumor-Stromal Cell Interaction in Prostate Carcinoma Growth and Metastasis M.E. Gleave, J.T. Hsieh, A.C. Von Eschenbach, and L.W.K. Chung M.E. GleaveM.E. Gleave More articles by this author , J.T. HsiehJ.T. Hsieh More articles by this author , A.C. Von EschenbachA.C. Von Eschenbach More articles by this author , and L.W.K. ChungL.W.K. Chung More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(17)37506-7AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Prostate cancer selectively metastasizes to the axial skeleton to produce osteoblastic lesions, which suggests that bidirectional paracrine interactions exist between prostate cancer and bone cells. To evaluate the role of tumor-stromal cell interaction and stromal-specific growth factors in prostate cancer growth and dissemination, we coinoculated nontumorigenic human prostate cancer cells (LNCaP) and various tissue-specific fibroblasts subcutaneously in athymic mice. LNCaP tumors were induced most consistently by human bone fibroblasts (62%), followed by two prostate fibroblast cell lines (31% and 17%), but not by lung, kidney, or embryonic 3T3 fibroblasts. Carcinomas formed preferentially in male hosts, demonstrating in vivo androgen sensitivity. Immunohistochemical and biochemical techniques confirmed the human prostate component of these tumors and were paralleled by elevations in serum prostate specific antigen. In vitro mitogenic assays revealed a two-to three-fold bidirectional stimulation between LNCaP and bone or prostate fibroblast conditioned media, but not lung, kidney, or 3T3 fibroblast conditioned media. A novel method developed to deliver concentrated bone or prostate fibroblast conditioned media in vivo using a slowly absorbed matrix (gelfoam) also induced tumor formation, emphasizing the importance of fibroblast growth factors in LNCaP tumor formation. Northern analysis identified the stromal compartment as the primary source of extracellular matrix (collagen, fibronectin), while only LNCaP cells expressed transforming growth factor a. Although LNCaP and stromal cells express basic fibroblast growth factor (bFGF), the bidirectional paracrine-mediated mitogenic activity between these cells is not inhibited by anti-bFGF antibodies, suggesting that other undefined growth factors may be involved in stimulating LNCaP growth. These observations illustrate the importance of stromal-epithelial interaction in prostate tumor growth and suggest that extracellular matrix and paracrine-mediated growth factors play a role in prostate cancer growth and metastasis. © 1992 by The American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetailsCited byGOTOH A, KO S, SHIRAKAWA T, CHEON J, KAO C, MIYAMOTO T, GARDNER T, HO L, CLEUTJENS C, TRAPMAN J, GRAHAM F and CHUNG L (2018) DEVELOPMENT OF PROSTATE-SPECIFIC ANTIGEN PROMOTER-BASED GENE THERAPY FOR ANDROGEN-INDEPENDENT HUMAN PROSTATE CANCERJournal of Urology, VOL. 160, NO. 1, (220-229), Online publication date: 1-Jul-1998.Campbell S (2018) ADVANCES IN ANGIOGENESIS RESEARCH: RELEVANCE TO UROLOGICAL ONCOLOGYJournal of Urology, VOL. 158, NO. 5, (1663-1674), Online publication date: 1-Nov-1997.Gleave M, Sato N, Goldenberg S, Stothers L, Bruchovsky N and Sullivan L (2018) Neoadjuvant Androgen Withdrawal Therapy Decreases Local Recurrence Rates Following Tumor Excision in the Shionogi Tumor ModelJournal of Urology, VOL. 157, NO. 5, (1727-1730), Online publication date: 1-May-1997.Pisters L, Troncoso P, Zhau H, Li W, von Eschenbach A and Chung L (2018) C-met Proto-Oncogene Expression in Benign and Malignant Human Prostate TissuesJournal of Urology, VOL. 154, NO. 1, (293-298), Online publication date: 1-Jul-1995.Cohen D, Simak R, Fair W, Melamed J, Scher H and Cordon-Cardo C (2018) Expression of Transforming Growth Factor-α and the Epidermal Growth Factor Receptor in Human Prostate TissuesJournal of Urology, VOL. 152, NO. 6 Part 1, (2120-2124), Online publication date: 1-Dec-1994. Volume 147Issue 4April 1992Page: 1151-1159 Advertisement Copyright & Permissions© 1992 by The American Urological Association Education and Research, Inc.Keywordsgrowth factorsprostatic neoplasmsMetricsAuthor Information M.E. Gleave More articles by this author J.T. Hsieh More articles by this author A.C. Von Eschenbach More articles by this author L.W.K. Chung More articles by this author Expand All Advertisement PDF downloadLoading ...

No AccessJournal of UrologyAdult Urology: Oncology: Renal/Upper Tract/Bladder1 Jul 2004GUIDELINES FOR THE SURVEILLANCE OF LOCALIZED RENAL CELL CARCINOMA BASED ON THE PATTERNS OF RELAPSE AFTER NEPHRECTOMY ANDREW J. STEPHENSON, MICHAEL P. CHETNER, KEITH ROURKE, MARTIN E. GLEAVE, M. SIGNAEVSKY, BRUCE PALMER, JAMES KUAN, GERALD B. BROCK, and SIMON TANGUAY ANDREW J. STEPHENSONANDREW J. STEPHENSON Current address: Department of Urology, Sidney Kimmel Center for Prostate and Urologic Cancers, Memorial Sloan-Kettering Cancer Center, New York, New York. More articles by this author , MICHAEL P. CHETNERMICHAEL P. CHETNER More articles by this author , KEITH ROURKEKEITH ROURKE More articles by this author , MARTIN E. GLEAVEMARTIN E. GLEAVE More articles by this author , M. SIGNAEVSKYM. SIGNAEVSKY More articles by this author , BRUCE PALMERBRUCE PALMER More articles by this author , JAMES KUANJAMES KUAN More articles by this author , GERALD B. BROCKGERALD B. BROCK More articles by this author , and SIMON TANGUAYSIMON TANGUAY More articles by this author View All Author Informationhttps://doi.org/10.1097/01.ju.0000132126.85812.7dAboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract Purpose: We characterized relapse patterns in patients with sporadic renal cell carcinoma (RCC) following radical and partial nephrectomy, and developed surveillance guidelines. Materials and Methods: Between 1989 and 2000, 495 patients underwent nephrectomy for RCC at 1 of 5 Canadian referral centers. Median followup was 42 months. Results: The rate of relapse, time to relapse and site of relapse were associated with pathological stage. Five-year progression-free probability was 93% for pT1, 81% for pT2, 67% for pT3A and 57% for pT3B (p <0.001). Compared to patients with pT1–2 those with pT3A–B lesions had earlier relapse after nephrectomy (median 12 vs 26 months, p = 0.001) and were at higher risk for relapse at abdominal sites (14% vs 1.8%, p < 0.001). Abdominal relapse was detected in the absence of symptoms, abnormal biochemical profile or thoracic metastases detectable by chest x-ray in 7 patients (1.4%) overall, including 3 (0.9%) with pT1, 3 (4%) with pT3A and 1 (3%) with pT3B. Conclusions: The risk and the pattern of relapse of RCC after nephrectomy are associated with pathological stage. For the surveillance of recurrent disease after nephrectomy we recommend annual clinical assessment and chest x-ray in pT1–2 cases. Patients with pT3A–B should be followed every 6 months for the first 3 years with clinical assessment and chest x-ray, and annual followup thereafter. The higher risk of abdominal relapse in patients with pT3A–B indicates that they should receive surveillance abdominal imaging. We recommend abdominal computerized tomography 6, 12, 24 and 36 months postoperatively. References 1 : The changing natural history of renal cell carcinoma. J Urol2001; 166: 1611. Link, Google Scholar 2 : Cancer statistics, 2003. CA Cancer J Clin2003; 53: 5. Google Scholar 3 National Cancer Institute of Canada: Canadian Cancer Statistics 2002. Toronto, 2002 Google Scholar 4 : Renal cell carcinoma: management of advanced disease. J Urol1999; 161: 381. Link, Google Scholar 5 : Resection of metastatic renal cell carcinoma. J Clin Oncol1998; 16: 2261. Google Scholar 6 : A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer. N Engl J Med2003; 349: 427. Google Scholar 7 : Follow-up after partial or total nephrectomy for renal cell carcinoma. Urol Clin North Am1994; 21: 589. Google Scholar 8 : A new protocol for the followup of renal cell carcinoma based on pathological stage. J Urol1995; 154: 28. 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Mayo Clin Proc2000; 75: 1236. Google Scholar 16 : Long-term results of nephron sparing surgery for localized renal cell carcinoma: 10-year followup. J Urol2000; 163: 442. Link, Google Scholar 17 : Temporal change in risk of metachronous contralateral renal cell carcinoma: influence of tumor characteristics and demographic factors. J Clin Oncol2002; 20: 2370. Google Scholar 18 : Prognostic factors of survival and rapid progression in 782 patients with metastatic renal carcinomas treated by cytokines: a report from the Groupe Francais d'Immunotherapie. Ann Oncol2002; 13: 1460. Google Scholar 19 : Therapy of locally recurrent renal cell carcinoma after nephrectomy. J Urol1996; 155: 26. Link, Google Scholar 20 : Outcome of isolated renal cell carcinoma fossa recurrence after nephrectomy. J Urol2000; 164: 322. Link, Google Scholar From the Divisions of Urology, McGill University (AJS, ST), Montréal, Québec, University of Alberta (MPC, KR), Edmonton, Alberta, University of British Columbia (MEG, MS), Vancouver, British Columbia, Valley Regional Hospital (BP), Kentville, Nova Scotia, and University of Western Ontario (JK, GBB), London, Ontario, Canada© 2004 by American Urological Association, Inc.FiguresReferencesRelatedDetailsCited byEl-Arabi A, Sherman G, Kennon C, Chase B, Jones J, Mirza M, Parker W, Holzbeierlein J, Duchene D and Lee E (2019) The Limited Usefulness of Ultrasound Surveillance after Radical and Partial Nephrectomy for Renal Cell CarcinomaUrology Practice, VOL. 7, NO. 1, (34-40), Online publication date: 1-Jan-2020.Narayan V, Puligandla M, Haas N, Subramanian P, DiPaola R and Uzzo R (2018) Patterns of Relapse and Implications for Post-Nephrectomy Surveillance in Patients with High Risk Nonclear Cell Renal Cell Carcinoma: Subgroup Analysis of the Phase 3 ECOG-ACRIN E2805 TrialJournal of Urology, VOL. 201, NO. 1, (62-69), Online publication date: 1-Jan-2019.Mouracade P, Kara O, Maurice M, Dagenais J, Malkoc E, Nelson R and Kaouk J (2018) Patterns and Predictors of Recurrence after Partial Nephrectomy for Kidney TumorsJournal of Urology, VOL. 197, NO. 6, (1403-1409), Online publication date: 1-Jun-2017.Laguna M (2018) Re: A Prospective Risk-Stratified Follow-up Programme for Radically Treated Renal Cell Carcinoma Patients: Evaluation after Eight Years of Clinical UseJournal of Urology, VOL. 197, NO. 4, (999-1000), Online publication date: 1-Apr-2017.Canvasser N, Stouder K, Lay A, Gahan J, Lotan Y, Margulis V, Raj G, Sagalowsky A and Cadeddu J (2018) The Usefulness of Chest X-Rays for T1a Renal Cell Carcinoma SurveillanceJournal of Urology, VOL. 196, NO. 2, (321-326), Online publication date: 1-Aug-2016.Laguna M (2018) Re: Oncologic Surveillance after Surgical Resection for Renal Cell Carcinoma: A Novel Risk-Based ApproachJournal of Urology, VOL. 196, NO. 1, (58-59), Online publication date: 1-Jul-2016.Laguna M (2018) Re: Guideline of Guidelines: Follow-up after Nephrectomy for Renal Cell CarcinomaJournal of Urology, VOL. 196, NO. 2, (349-350), Online publication date: 1-Aug-2016.Laguna M (2018) Re: Evaluation of the National Comprehensive Cancer Network and American Urological Association Renal Cell Carcinoma Surveillance GuidelinesJournal of Urology, VOL. 194, NO. 1, (43-43), Online publication date: 1-Jul-2015.Laguna M (2018) Re: Features Associated with Recurrence beyond 5 Years after Nephrectomy and Nephron-Sparing Surgery for Renal Cell Carcinoma: Development and Internal Validation of a Risk Model (PRELANE Score) to Predict Late Recurrence Based on a Large Multicenter Database (CORONA/SATURN Project)Journal of Urology, VOL. 191, NO. 4, (949-949), Online publication date: 1-Apr-2014.Laguna M (2018) Re: Significant Variability in 10-Year Cumulative Radiation Exposure Incurred on Different Surveillance Regimens after Surgery for pT1 Renal Cancers: Yet Another Reason to Standardize Protocols?Journal of Urology, VOL. 190, NO. 5, (1711-1711), Online publication date: 1-Nov-2013.Verhoest G, Avakian R, Bensalah K, Thuret R, Ficarra V, Artibani W, Tostain J, Guille F, Cindolo L, De La Taille A, Abbou C, Salomon L, Rioux-Leclercq N and Patard J (2018) Urinary Collecting System Invasion is an Independent Prognostic Factor of Organ Confined Renal Cell CarcinomaJournal of Urology, VOL. 182, NO. 3, (854-859), Online publication date: 1-Sep-2009.Eggener S, Yossepowitch O, Kundu S, Motzer R and Russo P (2018) Risk Score and Metastasectomy Independently Impact Prognosis of Patients With Recurrent Renal Cell CarcinomaJournal of Urology, VOL. 180, NO. 3, (873-878), Online publication date: 1-Sep-2008.Spiess P, Brown G, Liu P, Tu S, Tannir N, Evans J, Kamat A, Kassouf W and Pisters L (2018) Recurrence Pattern and Proposed Surveillance Protocol Following Post-Chemotherapy Retroperitoneal Lymph Node DissectionJournal of Urology, VOL. 177, NO. 1, (131-138), Online publication date: 1-Jan-2007.LAM J, SHVARTS O, LEPPERT J, PANTUCK A, FIGLIN R and BELLDEGRUN A (2018) POSTOPERATIVE SURVEILLANCE PROTOCOL FOR PATIENTS WITH LOCALIZED AND LOCALLY ADVANCED RENAL CELL CARCINOMA BASED ON A VALIDATED PROGNOSTIC NOMOGRAM AND RISK GROUP STRATIFICATION SYSTEMJournal of Urology, VOL. 174, NO. 2, (466-472), Online publication date: 1-Aug-2005. Volume 172Issue 1July 2004Page: 58-62 Advertisement Copyright & Permissions© 2004 by American Urological Association, Inc.Keywordsrenal cellkidneycarcinomanephrectomyx-ray computedtomographyepidemiologyMetricsAuthor Information ANDREW J. STEPHENSON Current address: Department of Urology, Sidney Kimmel Center for Prostate and Urologic Cancers, Memorial Sloan-Kettering Cancer Center, New York, New York. More articles by this author MICHAEL P. CHETNER More articles by this author KEITH ROURKE More articles by this author MARTIN E. GLEAVE More articles by this author M. SIGNAEVSKY More articles by this author BRUCE PALMER More articles by this author JAMES KUAN More articles by this author GERALD B. BROCK More articles by this author SIMON TANGUAY More articles by this author Expand All Advertisement PDF downloadLoading ...

We previously showed that the 44-kDa serine/threonine kinase Pim-1 (Pim-1L) can protect prostate cancer cells from apoptosis induced by chemotherapeutic drugs (Xie, Y., Xu, K., Dai, B., Guo, Z., Jiang, T., Chen, H., and Qiu, Y. (2006) Oncogene 25, 70-78). To further explore the mechanisms of Pim-1L-mediated resistance to chemotherapeutic drugs in prostate cancer cells, we employed a yeast two-hybrid screening to identify cellular proteins that were associated with Pim-1L, and we found the ABC transporter BCRP/ABCG2 as one of the potential interacting partners of Pim-1L. We also showed that the expression level of Pim-1L and BCRP was up-regulated in mitoxantrone and docetaxel-resistant prostate cancer cell lines. Pim-1L was co-localized with BCRP on the plasma membrane and induced phosphorylation of BCRP at threonine 362. Knocking-down Pim-1L expression in the drug-resistant prostate cancer cells abolished multimer formation of endogenous BCRP and resensitized the resistant cells to chemotherapeutic drugs suggesting that BCRP phosphorylation induced by Pim-1L was essential for its functionality. This is further corroborated by our finding that the plasma membrane localization and drug-resistant activity of BCRP were compromised by T362A mutation. Our data suggest that Pim-1L may protect prostate cancer cells from apoptosis, at least in part, through regulation of transmembrane drug efflux pump. These findings may provide a potential therapeutic approach by disrupting Pim-1 signaling to reverse BCRP-mediated multidrug resistance.

Abstract Heat shock protein 27 (Hsp27) is a cytoprotective chaperone that is phosphoactivated during cell stress that prevents aggregation and/or regulate activity and degradation of certain client proteins. Recent evidence suggests that Hsp27 may be involved in tumor progression and the development of treatment resistance in various tumors, including bladder cancer. The purpose of this study was to examine, both in vitro and in vivo, the effects of overexpression of Hsp27 and, correspondingly, the down-regulation of Hsp27 using small interfering (si) RNA and OGX-427, a second-generation antisense oligonucleotide targeting Hsp27. Hsp27 overexpression increased UMUC-3 cell growth and resistance to paclitaxel. Both OGX-427 and Hsp27 siRNA decreased Hsp27 protein and mRNA levels by &amp;gt;90% in a dose- and sequence-specific manner in human bladder cancer UMUC-3 cells. OGX-427 or Hsp27 siRNA treatment induced apoptosis and enhanced sensitivity to paclitaxel in UMUC-3 cells. In vivo, OGX-427 significantly inhibited tumor growth in mice, enhanced sensitivity to paclitaxel, and induced significantly higher levels of apoptosis compared with xenografts treated with control oligonucleotides. Collectively, these findings suggest that Hsp27 knockdown with OGX-427 and combined therapy with paclitaxel could be a novel strategy to inhibit the progression of bladder cancer. [Mol Cancer Ther 2007;6(1):299–308]

The current paradigm of cancer care relies on predictive nomograms which integrate detailed histopathology with clinical data. However, when predictions fail, the consequences for patients are often catastrophic, especially in prostate cancer where nomograms influence the decision to therapeutically intervene. We hypothesized that the high dimensional data afforded by massively parallel sequencing (MPS) is not only capable of providing biological insights, but may aid molecular pathology of prostate tumours. We assembled a cohort of six patients with high-risk disease, and performed deep RNA and shallow DNA sequencing in primary tumours and matched metastases where available. Our analysis identified copy number abnormalities, accurately profiled gene expression levels, and detected both differential splicing and expressed fusion genes. We revealed occult and potentially dormant metastases, unambiguously supporting the patients' clinical history, and implicated the REST transcriptional complex in the development of neuroendocrine prostate cancer, validating this finding in a large independent cohort. We massively expand on the number of novel fusion genes described in prostate cancer; provide fresh evidence for the growing link between fusion gene aetiology and gene expression profiles; and show the utility of fusion genes for molecular pathology. Finally, we identified chromothripsis in a patient with chronic prostatitis. Our results provide a strong foundation for further development of MPS-based molecular pathology.

We demonstrate that PTEN loss causes reduced NKX3.1 expression in both murine and human prostate cancers. Restoration of Nkx3.1 expression in vivo in Pten null epithelium leads to decreased cell proliferation, increased cell death, and prevention of tumor initiation. Whereas androgen receptor (AR) positively regulates NKX3.1 expression, NKX3.1 negatively modulates AR transcription and consequently the AR-associated signaling events. Consistent with its tumor suppressor functions, NKX3.1 engages cell cycle and cell death machinery via association with HDAC1, leading to increased p53 acetylation and half-life through MDM2-dependent mechanisms. Importantly, overexpression of Nkx3.1 has little effect on Pten wild-type epithelium, suggesting that PTEN plays a predominant role in PTEN-NKX3.1 interplay. Manipulating NKX3.1 expression may serve as a therapeutic strategy for treating PTEN-deficient prostate cancers.

Abstract Purpose: To determine the antitumor activity and safety of oblimersen sodium, a phosphorothioate antisense oligonucleotide directed to the bcl-2 mRNA, with docetaxel in patients with hormone-refractory prostate cancer (HRPC) and to determine if relevant pharmacokinetic and pharmacodynamic variables of oblimersen or docetaxel influence response to this therapy. Experimental Design: Patients with HRPC were treated with oblimersen sodium by continuous i.v. infusion on days 1 to 8 with docetaxel given i.v. over 1 hour on day 6 every 3 weeks. Plasma samples were analyzed to characterize the pharmacokinetic variables of both oblimersen and docetaxel, and paired collections of peripheral blood mononuclear cells were collected to determine Bcl-2 protein expression pretreatment and post-treatment. Results: Twenty-eight patients received 173 courses of oblimersen (7 mg/kg/d continuous i.v. infusion on days 1-8) and docetaxel (75 mg/m2 i.v. on day 6). Prostate-specific antigen responses were observed in 14 of 27 (52%) patients, whereas 4 of 12 (33%) patients with bidimensionally measurable disease had objective responses. The mean oblimersen steady-state concentration (Css) was a significant determinant of antitumor activity; mean Css values were higher in responders compared with nonresponders (6.24 ± 1.68 versus 4.27 ± 1.22; P = 0.008). The median survival of all patients was 19.8 months. Bcl-2 protein expression decreased a median of 49.9% in peripheral blood mononuclear cells post-treatment, but the individual incremental change did not correlate with either oblimersen Css or response. Conclusions: Oblimersen combined with docetaxel is an active combination in HRPC patients demonstrating both an encouraging response rate and an overall median survival. The absence of severe toxicities at this recommended dose, evidence of Bcl-2 protein inhibition, and encouraging antitumor activity in HPRC patients warrant further clinical evaluation of this combination, including studies to optimize oblimersen Css.

Currently, the main obstacle to curing advanced prostate cancer is development of androgen independence (AI), where malignant cells acquire the ability to survive in the absence of androgens. Our initial experimental approach used cDNA microarrays to characterize changes in gene expression in the LNCaP human prostate tumor model during progression to AI. The transcription factor Y-box binding protein (YB-1) was shown to be one of the genes upregulated. We focused on increased YB-1 expression during progression in clinical specimens, and further examined one of its downstream targets, P-glycoprotein (P-gp).Northern blot analysis was performed on LNCaP tumor series, as well as immunohistochemical analyses of human prostate cancer tissue samples. YB-1 was transiently transfected and transport analysis were performed to analyze P-gp efflux activity.YB-1 expression is markedly increased during benign to malignant transformation and further following androgen ablation. In addition, increased YB-1 expression after castration in the LNCaP model is linked to upregulation of P-gp. We demonstrate that YB-1 upregulates P-gp activity resulting in a 40% intracellular decrease in the P-gp substrate vinblastine. We have also found that P-gp increases the efflux of the endogenous androgen, dihydrotestosterone (DHT), from prostate cells and leads to decreased androgen regulated gene expression.We hypothesize that early in prostate cancer progression, increased expression of YB-1 may increase P-gp activity which may in turn lower androgen levels in the prostate tumor cells. Suppression of androgen levels may activate cell survival pathways and lead to an adaptive survival advantage of androgen independent prostate cancer cells following androgen ablation therapy.

The presence of the TMPRSS2-ERG fusion gene in prostate tumors has recently been associated with an aggressive phenotype, as well as recurrence and death from prostate cancer. These associations suggest the hypothesis that the gene fusion may be used as a prognostic indicator for prostate cancer.In this study, fluorescent in situ hybridization (FISH) assays were used to assess TMPRSS2-ERG fusion status in a group of 214 prostate cancer cases from two population-based studies. The FISH assays were designed to detect both fusion type (deletion vs. translocation) and the number of fusion copies (single vs. multiple). Genotyping of four ERG and one TMPRSS2 SNPs using germline DNA was also performed in a sample of the cases (n = 127).Of the 214 tumors scored for the TMPRSS2-ERG fusion, 64.5% were negative and 35.5% were positive for the fusion. Cases with the TMPRSS2-ERG fusion did not exhibit reduced prostate cancer survival (HR = 0.92, 95% CI = 0.22-3.93), nor was there a significant difference in cause-specific survival when stratifying by translocation or deletion (HR = 0.84, 95% CI = 0.23-3.12) or by the number of retained fusion copies (HR = 1.22, 95% CI = 0.45-3.34). However, evidence for reduced prostate cancer-specific survival was apparent in those cases whose tumor had multiple copies of the fusion. The variant T allele of the TMPRSS2 SNP, rs12329760, was positively associated with TMPRSS2-ERG fusion by translocation (p = 0.05) and with multiple copies of the gene fusion (p = 0.03).If replicated, the results presented here may provide insight into the mechanism by which the TMPRSS2-ERG gene fusion arises and also contribute to diagnostic evaluations for determining the subset of men who will go on to develop metastatic prostate cancer.

Abstract Apoptosis and inhibition of mitosis are primary mechanisms mediating androgen ablation therapy-induced regression of prostate cancer (PCa). However, PCa readily becomes androgen independent, leading to fatal disease. Up-regulated growth and survival signaling is implicated in development of resistance to androgen ablation therapy. We are testing the hypothesis that insulin-like growth factor (IGF) responsiveness is required for androgen-independent (AI) progression. Using the LNCaP human PCa progression model, we have determined that IGF-I–mediated protection from apoptotic stress and enhanced mitotic activity is androgen dependent in LNCaP cells but is androgen independent in lineage-derived C4-2 cells. Both cell lines exhibit androgen-responsive patterns of IGF-I receptor (IGF-IR) expression, activation, and signaling to insulin receptor substrate-2 and AKT. However, C4-2 cells express higher levels of IGF-IR mRNA and protein and exhibit enhanced IGF-I–mediated phosphorylation and downstream signaling under androgen-deprived conditions. In comparisons of naïve and AI metastatic human PCa specimens, we have confirmed that IGF-IR levels are elevated in advanced disease. Together with our LNCaP/C4-2 AI progression model data, these results indicate that increased IGF-IR expression is associated with AI antiapoptotic and promitotic IGF signaling in PCa disease progression.

Abstract Purpose: Prostate tumors express antigens that are recognized by the immune system in a significant proportion of patients; however, little is known about the effect of standard treatments on tumor-specific immunity. Radiation therapy induces expression of inflammatory and immune-stimulatory molecules, and neoadjuvant hormone therapy causes prominent T-cell infiltration of prostate tumors. We therefore hypothesized that radiation therapy and hormone therapy may initiate tumor-specific immune responses. Experimental Design: Pretreatment and posttreatment serum samples from 73 men with nonmetastatic prostate cancer and 50 cancer-free controls were evaluated by Western blotting and SEREX (serological identification of antigens by recombinant cDNA expression cloning) antigen arrays to examine whether autoantibody responses to tumor proteins arose during the course of standard treatment. Results: Western blotting revealed the development of treatment-associated autoantibody responses in patients undergoing neoadjuvant hormone therapy (7 of 24, 29.2%), external beam radiation therapy (4 of 29, 13.8%), and brachytherapy (5 of 20, 25%), compared with 0 of 14 patients undergoing radical prostatectomy and 2 of 36 (5.6%) controls. Responses were seen within 4 to 9 months of initiation of treatment and were equally prevalent across different disease risk groups. Similarly, in the murine Shionogi tumor model, hormone therapy induced tumor-associated autoantibody responses in 5 of 10 animals. In four patients, SEREX immunoscreening of a prostate cancer cDNA expression library identified several antigens recognized by treatment-associated autoantibodies, including PARP1, ZNF707 + PTMA, CEP78, SDCCAG1, and ODF2. Conclusion: We show for the first time that standard treatments induce antigen-specific immune responses in prostate cancer patients. Thus, immunologic mechanisms may contribute to clinical outcomes after hormone and radiation therapy, an effect that could potentially be exploited as a practical, personalized form of immunotherapy.

Hsp27 and clusterin (CLU are stress-activated small heat shock proteins that are up-regulated in many cancers where they play important roles in stress-induced protein homeostasis (proteostasis), inhibition of cell death pathways, and modulation of pro-survival signaling and transcriptional networks. They are associated with poor prognosis and treatment resistance in many cancers, protecting cells from many varied therapeutic stressors that induce apoptosis, including androgen or estrogen withdrawal, radiation, cytotoxic chemotherapy, and biologic agents. Both Hsp27 and sCLU are ATP-independent molecular chaperones making them less amenable to inhibition by small molecules, and so strategies to inhibit Hsp27 and sCLU at the gene-expression level are appealing. Indeed, known nucleotide sequences of cancer-relevant genes offer the possibility to rapidly design antisense oligonucleotides (ASO) for loss-of-function and preclinical proof-of-principle studies and subsequent clinical use. Here, we will review the rationale for Hsp27 and sCLU as therapeutic targets in cancer, and update the current status of pre-clinical and clinical studies using Hsp27 and CLU inhibitors, OGX-427 and OGX-011, respectively. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.

Cabazitaxel, abiraterone acetate (AA), and enzalutamide have been approved for the treatment of patients with metastatic castration-resistant prostate cancer (mCRPC) following docetaxel chemotherapy. Whether taxanes and next-generation androgen receptor (AR) axis inhibitors are cross-resistant or not is a subject of debate. To evaluate the antitumour activity of cabazitaxel in mCRPC pretreated with abiraterone or enzalutamide. The antitumour activity of cabazitaxel was assessed in patients with mCRPC and progressive disease after treatment with docetaxel and AA. In parallel, cabazitaxel antitumour activity was studied in enzalutamide-resistant models. Changes in prostate-specific antigen (PSA) levels and progression-free survival were used to determine the activity of cabazitaxel treatment. Cell proliferation, immunofluorescence, and AR transactivation assay were used in enzalutamide-resistant models. A total of 79 patients who had progressive mCRPC after docetaxel (median: 8 cycles; range: 4–12 mo), and AA (median: 4.8 mo; range:1–55 mo) received cabazitaxel 25 mg/m2 every 3 weeks (median: 6 cycles; range:1–15 cycles). A PSA decline ≥30% was achieved in 48 patients (62%; 95% confidence interval [CI], 51–73), and a decline ≥50% was achieved in 28 patients (35%; 95% CI, 25–47). The median progression-free survival and overall survival were 4.4 and 10.9 mo, respectively. In vitro, cabazitaxel decreased cell viability in both enzalutamide-sensitive and enzalutamide-resistant prostate cancer cells within the same range of concentrations. PC3, an AR-negative cell line, exhibited similar sensitivity to cabazitaxel. Cabazitaxel and AR-pathway inhibitors are not cross-resistant. Preclinical data suggest that cabazitaxel activity does not act mainly through AR axis inhibition. The antitumour activity of cabazitaxel, a chemotherapy agent, was studied in prostate cancer resistant to conventional hormonal therapy and to more recent endocrine therapies (abiraterone or enzalutamide). Cabazitaxel retained anticancer activity in more than half of the cases.

L-type amino acid transporters (LATs) uptake neutral amino acids including L-leucine into cells, stimulating mammalian target of rapamycin complex 1 signaling and protein synthesis. LAT1 and LAT3 are overexpressed at different stages of prostate cancer, and they are responsible for increasing nutrients and stimulating cell growth.We examined LAT3 protein expression in human prostate cancer tissue microarrays. LAT function was inhibited using a leucine analog (BCH) in androgen-dependent and -independent environments, with gene expression analyzed by microarray. A PC-3 xenograft mouse model was used to study the effects of inhibiting LAT1 and LAT3 expression. Results were analyzed with the Mann-Whitney U or Fisher exact tests. All statistical tests were two-sided.LAT3 protein was expressed at all stages of prostate cancer, with a statistically significant decrease in expression after 4-7 months of neoadjuvant hormone therapy (4-7 month mean = 1.571; 95% confidence interval = 1.155 to 1.987 vs 0 month = 2.098; 95% confidence interval = 1.962 to 2.235; P = .0187). Inhibition of LAT function led to activating transcription factor 4-mediated upregulation of amino acid transporters including ASCT1, ASCT2, and 4F2hc, all of which were also regulated via the androgen receptor. LAT inhibition suppressed M-phase cell cycle genes regulated by E2F family transcription factors including critical castration-resistant prostate cancer regulatory genes UBE2C, CDC20, and CDK1. In silico analysis of BCH-downregulated genes showed that 90.9% are statistically significantly upregulated in metastatic castration-resistant prostate cancer. Finally, LAT1 or LAT3 knockdown in xenografts inhibited tumor growth, cell cycle progression, and spontaneous metastasis in vivo.Inhibition of LAT transporters may provide a novel therapeutic target in metastatic castration-resistant prostate cancer, via suppression of mammalian target of rapamycin complex 1 activity and M-phase cell cycle genes.

Prostate cancer (PCa) is the most common noncutaneous malignancy and the second leading cause of cancer mortality amongst men in the Western world. Up to 40% of men diagnosed with PCa will eventually develop metastatic disease, and although most respond to initial medical or surgical castration, progression to castration resistance is universal. The average survival for patients with castration-resistant prostate cancer (CRPC) is 2-3 yr.To discuss the biologic rationale and evidence supporting current management of patients with CRPC and to review promising novel agents.Electronic databases (PubMed, ClinicalTrials.gov), relevant journals, and conference proceedings were searched manually for preclinical studies, clinical trials, and biomarker analyses focused on the treatment of CRPC. Keywords included castrate resistant prostate cancer and: targeted therapy, novel therapy, immunotherapy, androgen therapy, bone therapy, mechanisms, biomarkers, and trial endpoints; no time range was specified. Information pertaining to current studies was discussed with key opinion leaders.We focus on the efficacy and safety of approved agents, promising therapies that have proceeded to phase 3 evaluation, and those that have enhanced our understanding of the biology of CRPC. Biomarkers are considered in the context of novel targeted agents and immunotherapy.CRPC has many targets. Four new agents with different mechanisms of action have recently been shown to have positive results in large phase 3 randomized trials, and have already been approved in the United States for CRPC: cabazitaxel, sipuleucel-T, denosumab, and abiraterone acetate. With our improved understanding of tumor biology and the incorporation of new prognostic and molecular biomarkers into clinical trials, we are making progress in the management of patients with CRPC.

Abstract TGF-β promotes epithelial–mesenchymal transition (EMT) and induces clusterin (CLU) expression, linking these genes to cancer metastasis. CLU is a pleiotropic molecular chaperone that confers survival and proliferative advantage to cancer cells. However, the molecular mechanisms by which TGF-β regulates CLU expression and CLU affects metastasis remain unknown. In this study, we report that the transcription factor Twist1 mediates TGF-β–induced CLU expression. By binding to E-boxes in the distal promoter region of CLU gene, Twist1 regulated basal and TGF-β–induced CLU transcription. In addition, CLU reduction reduced TGF-β induction of the mesenchymal markers, N-cadherin and fibronectin, thereby inhibiting the migratory and invasive properties induced by TGF-β. Targeted inhibition of CLU also suppressed metastasis in an in vivo model. Taken together, our findings indicate that CLU is an important mediator of TGF-β–induced EMT, and suggest that CLU suppression may represent a promising therapeutic option for suppressing prostate cancer metastatic progression. Cancer Res; 72(20); 5261–72. ©2012 AACR.

Understanding the mechanism underlying the regulation of the androgen receptor (AR), a central player in the development of castration-resistant prostate cancer (CRPC), holds promise for overcoming the challenge of treating CRPC. We demonstrate that the ubiquitin ligase Siah2 targets a select pool of NCOR1-bound, transcriptionally-inactive AR for ubiquitin-dependent degradation, thereby promoting expression of select AR target genes implicated in lipid metabolism, cell motility, and proliferation. Siah2 is required for prostate cancer cell growth under androgen-deprivation conditions in vitro and in vivo, and Siah2 inhibition promotes prostate cancer regression upon castration. Notably, Siah2 expression is markedly increased in human CRPCs. Collectively, we find that selective regulation of AR transcriptional activity by the ubiquitin ligase Siah2 is important for CRPC development.

Progression to the castration-resistant state is the incurable and lethal end stage of prostate cancer, and there is strong evidence that androgen receptor (AR) still plays a central role in this process. We hypothesize that knocking down AR will have a major effect on inhibiting growth of castration-resistant tumors.Castration-resistant C4-2 human prostate cancer cells stably expressing a tetracycline-inducible AR-targeted short hairpin RNA (shRNA) were generated to directly test the effects of AR knockdown in C4-2 human prostate cancer cells and tumors.In vitro expression of AR shRNA resulted in decreased levels of AR mRNA and protein, decreased expression of prostate-specific antigen (PSA), reduced activation of the PSA-luciferase reporter, and growth inhibition of C4-2 cells. Gene microarray analyses revealed that AR knockdown under hormone-deprived conditions resulted in activation of genes involved in apoptosis, cell cycle regulation, protein synthesis, and tumorigenesis. To ensure that tumors were truly castration-resistant in vivo, inducible AR shRNA expressing C4-2 tumors were grown in castrated mice to an average volume of 450 mm(3). In all of the animals, serum PSA decreased, and in 50% of them, there was complete tumor regression and disappearance of serum PSA.Whereas castration is ineffective in castration-resistant prostate tumors, knockdown of AR can decrease serum PSA, inhibit tumor growth, and frequently cause tumor regression. This study is the first direct evidence that knockdown of AR is a viable therapeutic strategy for treatment of prostate tumors that have already progressed to the castration-resistant state.

One strategy to improve therapies in advanced prostate cancer (PC) involves targeting genes that are activated by androgen withdrawal to delay the emergence of the androgen-independent (AI) phenotype. Heat shock protein 27 (Hsp27) expression becomes highly upregulated in PC cells after androgen withdrawal or chemotherapy, in which it functions as a cytoprotective chaperone to confer broad-spectrum treatment resistance. The purpose of this study is to elucidate anti-apoptotic pathways regulated by Hsp27 that are activated during PC progression. Using two-hybrid experiment, we found that Hsp27 was having a major role in the protein translational initiation process. Furthermore, using complementary DNA (cDNA) microarray analysis, 4E binding protein 1 was identified as being proportionately and highly regulated by Hsp27. These data led us to analyze the protein synthesis initiation pathway, which is a prerequisite for cell growth and proliferation. Using northern and western blot analysis, we found that Hsp27 downregulation decreased eukaryotic translation initiation factor 4E (eIF4E) expression at the protein, but not mRNA, level. The cytoprotection afforded by Hsp27 overexpression was attenuated by eIF4E knockdown using specific eIF4E short interfering RNA (siRNA). Co-immunoprecipitation and co-immunofluorescence confirmed that Hsp27 colocalizes and interacts directly with eIF4E. Hsp27-eIF4E interaction decreases eIF4E ubiquitination and proteasomal degradation. By chaperoning eIF4E, Hsp27 seems to protect the protein synthesis initiation process to enhance cell survival during cell stress induced by castration or chemotherapy. Forced overexpression of eIF4E induces resistance to androgen-withdrawal and paclitaxel treatment in the prostate LNCaP cells in vitro. These findings identify Hsp27 as a modulator of eIF4E and establish a potential mechanism for the eIF4E-regulated apoptosis after androgen ablation and chemotherapy. Targeting Hsp27-eIF4E interaction may serve as a therapeutic target in advanced PC.

With treatment leading to nearly uniform cure in clinical stage I nonseminomatous testicular cancer (CSI-NSGCT), diminishing treatment-related morbidity has become the primary concern. This study examined feasibility and outcome of active surveillance as treatment in an unselected CSI patient population.All patients with CSI-NSGCT referred from 1998 to 2007 to the British Columbia Cancer Agency and the Oregon Testis Cancer Program were retrospectively reviewed. A total of 233 patients were identified, of which 223 chose active surveillance.Vascular invasion (VI) was absent, present and unknown in 66%, 27% and 7% of cases, respectively. Overall, 49% of patients had embryonal predominant disease. Fifty-nine patients (26%) relapsed, all but one with good prognosis disease. VI was present in 30 relapsed patients. Most patients relapsed within 2 years (88%). Only 7 of 223 patients (3%) relapsed beyond 2 years. All relapses were in long-term remission following chemotherapy with or without retroperitoneal lymph node dissection (RPLND). Only 17 of 223 patients (8%) required postorchiectomy surgery. Disease-specific survival is 100% after a median follow-up of 52 months (3-136). No patient has required second-line chemotherapy.Active surveillance for all CSI-NSGCT patients is associated with excellent outcomes comparable with the best results reported with primary RPLND or adjuvant chemotherapy. Nearly 75% of patients are spared any therapy after orchiectomy.

Many strategies used to kill cancer cells induce stress-responses that activate survival pathways to promote emergence of a treatment resistant phenotype. Secretory clusterin (sCLU) is a stress-activated cytoprotective chaperone up-regulated by many varied anticancer therapies to confer treatment resistance when overexpressed. sCLU levels are increased in several treatment recurrent cancers including castrate resistant prostate cancer, and therefore sCLU has become an attractive target in cancer therapy. sCLU is not druggable with small molecule inhibitors, therefore nucleotide-based strategies to inhibit sCLU at the RNA level are appealing. Preclinical studies have shown that antisense oligonucleotide (ASO) or siRNA knockdown of sCLU have preclinical activity in combination with hormone- and chemotherapy. Phase I and II clinical trial data indicate that the second generation ASO, custirsen (OGX-011), has biologic and clinical activity, suppressing sCLU expression in prostate cancer tissues by more than 90%. A randomized study comparing docetaxel-custirsen to docetaxel alone in men with castrate resistant prostate cancer reported improved survival by 7 months from 16.9 to 23.8 months. Strong preclinical and clinical proof-of-principle data provide rationale for further study of sCLU inhibitors in randomized phase III trials, which are planned to begin in 2010.

Active surveillance represents a strategy to address the overtreatment of prostate cancer, yet uncertainty regarding individual patient outcomes remains a concern. We evaluated outcomes in a prospective multicenter study of active surveillance.We studied 905 men in the prospective Canary PASS enrolled between 2008 and 2013. We collected clinical data at study entry and at prespecified intervals, and determined associations with adverse reclassification, defined as increased Gleason grade or greater cancer volume on followup biopsy. We also evaluated the relationships of clinical parameters with pathology findings in participants who underwent surgery after a period of active surveillance.At a median followup of 28 months 24% of participants experienced adverse reclassification, of whom 53% underwent treatment while 31% continued on active surveillance. Overall 19% of participants received treatment, 68% with adverse reclassification, while 32% opted for treatment without disease reclassification. In multivariate Cox proportional hazards modeling the percent of biopsy cores with cancer, body mass index and prostate specific antigen density were associated with adverse reclassification (p=0.01, 0.04, 0.04, respectively). Of 103 participants subsequently treated with radical prostatectomy 34% had adverse pathology, defined as primary pattern 4-5 or nonorgan confined disease, including 2 with positive lymph nodes, with no significant relationship between risk category at diagnosis and findings at surgery (p=0.76).Most men remain on active surveillance at 5 years without adverse reclassification or adverse pathology at surgery. However, clinical factors had only a modest association with disease reclassification, supporting the need for approaches that improve the prediction of this outcome.

The progression to castration-resistant prostate cancer (CRPC) correlates with gain-of-function of the androgen receptor (AR) and activation of AKT. However, as single agents, AR or AKT inhibitors result in a reciprocal feedback loop. Therefore, we hypothesized that combination of an AKT inhibitor with an antiandrogen might result in a more profound, long-lasting remission of CRPC. Here, we report that the AKT inhibitor AZD5363 potently inhibits proliferation and induces apoptosis in prostate cancer cell lines expressing the AR and has anticancer activity in vivo in androgen-sensitive and castration-resistant phases of the LNCaP xenograft model. However, we found that the effect of castration-resistant tumor growth inhibition and prostate-specific antigen (PSA) stabilization is transient and resistance occurs with increasing PSA after approximately 30 days of treatment. Mechanistically, we found that single agent AZD5363 induces increase of AR binding to androgen response element, AR transcriptional activity, and AR-dependent genes such as PSA and NKX3.1 expression. These effects were overcome by the combination of AZD5363 with the antiandrogen bicalutamide, resulting in synergistic inhibition of cell proliferation and induction of apoptosis in vitro, and prolongation of tumor growth inhibition and PSA stabilization in CRPC in vivo. This study provides a preclinical proof-of-concept that combination of an AKT inhibitor with antiandrogen results in prolonged disease stabilization in a model of CRPC.

Significance The transcription factor E-twenty-six related gene (ERG) is a major driver of prostate cancer, which makes this protein an interesting target for drug development. In this study, we report the discovery of an enzyme, ubiquitin-specific peptidase 9, X-linked (USP9X), which stabilizes ERG. We demonstrate that inhibition of USP9X with the small molecule WP1130 causes rapid degradation of ERG and blocked the growth of cultured prostate cancer cells and prostate tumors that express ERG. These findings suggest that inhibition of USP9X with small molecules should be explored for the development of a prostate cancer therapy that targets ERG.

Abstract Purpose: Clusterin (CLU) is an antiapoptotic, stress-induced protein conferring treatment resistance when overexpressed. This study tested custirsen, a CLU inhibitor, in patients with metastatic castration-resistant prostate cancer (mCRPC) progressing during or within 6 months of initial docetaxel therapy. Patients and Methods: Men were randomized to receive either docetaxel + prednisone + custirsen (DPC) or mitoxantrone + prednisone + custirsen (MPC). Results: Forty-two patients received study treatment. Toxicity was similar in both arms. Twenty patients treated with DPC received a median of 8 cycles; overall survival (OS) was 15.8 months. Median time to pain progression (TTPP) was 10.0 months; 10 of 13 (77%) evaluable patients had pain responses. Three of 13 (23%) evaluable patients had objective partial responses. Prostate-specific antigen (PSA) declines of 90% or more, 50% or more, and 30% or more occurred in 4 (20%), 8 (40%), and 11 (55%) patients, respectively. Twenty-two patients treated with MPC received a median of 6 cycles; OS was 11.5 months. The median TTPP was 5.2 months; 6 of 13 (46%) evaluable patients had pain responses. No objective responses were observed. PSA declines of 50% or more and 30% or more occurred in 6 (27%) and 7 (32%) patients, respectively. Low serum CLU levels during treatment showed superior survival for patients based on modeling with proportional hazard regression with a time-dependent covariate and different landmarks. Conclusions: Custirsen plus either docetaxel or mitoxantrone was feasible in patients with progressive mCRPC following first-line docetaxel therapy. Pain relief was higher than expected, with interesting correlations between serum CLU and survival. A phase III trial evaluating the pain palliation benefit of custirsen with taxane therapy is ongoing. Clin Cancer Res; 17(17); 5765–73. ©2011 AACR.

Abstract RNA interference (RNAi) holds great promise for the treatment of inherited and acquired diseases, provided that safe and efficient delivery systems are available. Herein we report that structurally flexible triethanolamine (TEA) core PAMAM dendrimers are able to deliver an Hsp27 siRNA effectively into prostate cancer (PC‐3) cells by forming stable nanoparticles with siRNA, protecting the siRNA nanoparticles from enzymatic degradation, and enhancing cellular uptake of siRNA. The Hsp27 siRNA resulted in potent and specific gene silencing of heat‐shock protein 27, an attractive therapeutic target in castrate‐resistant prostate cancer. Silencing of the hsp27 gene led to induction of caspase‐3/7‐dependent apoptosis and inhibition of PC‐3 cell growth in vitro. In addition, the siRNA–dendrimer complexes are non‐cytotoxic under the conditions used for siRNA delivery. Altogether, TEA core PAMAM dendrimer‐mediated siRNA delivery, in combination with RNAi that specifically targets Hsp27, may constitute a promising approach for combating castrate‐resistant prostate cancer, for which there is no efficacious treatment.

Secretory clusterin (sCLU) is a stress-activated, cytoprotective chaperone that confers broad-spectrum cancer treatment resistance, and its targeted inhibitor (OGX-011) is currently in phase II trials for prostate, lung, and breast cancer. However, the molecular mechanisms by which sCLU inhibits treatment-induced apoptosis in prostate cancer remain incompletely defined. We report that sCLU increases NF-kappaB nuclear translocation and transcriptional activity by serving as a ubiquitin-binding protein that enhances COMMD1 and I-kappaB proteasomal degradation by interacting with members of the SCF-betaTrCP E3 ligase family. Knockdown of sCLU in prostate cancer cells stabilizes COMMD1 and I-kappaB, thereby sequestrating NF-kappaB in the cytoplasm and decreasing NF-kappaB transcriptional activity. Comparative microarray profiling of sCLU-overexpressing and sCLU-knockdown prostate cancer cells confirmed that the expression of many NF-kappaB-regulated genes positively correlates with sCLU levels. We propose that elevated levels of sCLU promote prostate cancer cell survival by facilitating degradation of COMMD1 and I-kappaB, thereby activating the canonical NF-kappaB pathway.

MicroRNAs (miRNAs) have emerged as key regulators of numerous biological processes, and increasing evidence suggests that circulating miRNAs may be useful biomarkers of clinical disease. In this study, we sought to identify plasma miRNAs that differentiate patients with metastatic castration resistant prostate cancer (mCRPC) from those with localized prostate cancer (PCa). Pooled plasma samples from patients with localized PCa or mCRPC (25 per group) were assayed using the Exiqon miRNA qPCR panel, and the differential expression of selected candidates was validated using qRT-PCR. We identified 63 miRNAs upregulated in mCRPC versus localized PCa, while only four were downregulated. Pearson's correlation analysis revealed two highly correlated groups: one consisting of miR-141, miR375 and miR-200c and the other including miR151-3p, miR423-3p, miR-126, miR152 and miR-21. A third group, containing miR-16 and miR-205, showed less correlation. One miRNA from each group (miR-141, miR151-3p and miR-16) was used for logistic regression analysis and proved to increase the sensitivity of the prostate-specific antigen (PSA) test alone. While no miRNA alone differentiated localized PCa and mCRPC, combinations had greater sensitivity and specificity. The expression of these 10 candidates was assayed for association with clinical parameters of disease progression through the cBio portal. Our results demonstrate that plasma levels of selected miRNAs are potential biomarkers to differentiate localized PCa and mCRPC.

Abstract Purpose: Enzalutamide (ENZ) is a potent androgen receptor (AR) antagonist with activity in castration-resistant prostate cancer (CRPC); however, progression to ENZ-resistant (ENZ-R) CRPC frequently occurs with rising serum PSA levels, implicating AR full-length (ARFL) or variants (AR-Vs) in disease progression. Experimental Design: To define functional roles of ARFL and AR-Vs in ENZ-R CRPC, we designed 3 antisense oligonucleotides (ASO) targeting exon-1, intron-1, and exon-8 in AR pre-mRNA to knockdown ARFL alone or with AR-Vs, and examined their effects in three CRPC cell lines and patient-derived xenografts. Results: ENZ-R-LNCaP cells express high levels of both ARFL and AR-V7 compared with CRPC-LNCaP; in particular, ARFL levels were approximately 12-fold higher than AR-V7. Both ARFL and AR-V7 are highly expressed in the nuclear fractions of ENZ-R-LNCaP cells even in the absence of exogenous androgens. In ENZ-R-LNCaP cells, knockdown of ARFL alone, or ARFL plus AR-Vs, similarly induced apoptosis, suppressed cell growth and AR-regulated gene expression, and delayed tumor growth in vivo. In 22Rv1 cells that are inherently ENZ-resistant, knockdown of both ARFL and AR-Vs more potently suppressed cell growth, AR transcriptional activity, and AR-regulated gene expression than knockdown of ARFL alone. Exon-1 AR-ASO also inhibited tumor growth of LTL-313BR patient-derived CRPC xenografts. Conclusions: These data identify the AR as an important driver of ENZ resistance, and while the contributions of ARFL and AR-Vs can vary across cell systems, ARFL is the key driver in the ENZ-R LNCaP model. AR targeting strategies against both ARFL and AR-Vs is a rational approach for AR-dependent CRPC. Clin Cancer Res; 21(7); 1675–87. ©2015 AACR.

(Cell 174, 758–769.e1–e9; July 26, 2018) It has come to our attention that we inadvertently swapped the headings on the two columns of Table S4. From left to right, the headings should read “No AR peak amplification” and then “AR peak amplification”. Only the headings were swapped. The manuscript reports the correct result, and the statistical tests we performed on the values (two-by-two contingency table tests) are unchanged. The error has been corrected online, and we apologize for any confusion it may have caused. Genomic Hallmarks and Structural Variation in Metastatic Prostate CancerQuigley et al.CellJuly 19, 2018In BriefIntegrative whole-genome and -transcriptome sequencing provides a comprehensive view of structural variations that affect major regulators in prostate cancer and would escape detection by exome-based approaches. Full-Text PDF Open Archive