Proteogenomic Characterization of Patient- Derived Xenografts

Published OnlineFirst October 1, 2018; DOI: 10.1158/1078-0432.CCR-18-0729

Precision Medicine and Imaging Clinical Cancer Research Proteogenomic Characterization of Patient- Derived Xenografts Highlights the Role of REST in Neuroendocrine Differentiation of Castration- Resistant Prostate Cancer Amilcar Flores-Morales1,2,3, Tobias B. Bergmann1,2, Charlotte Lavallee1,2, Tanveer S. Batth3, Dong Lin4, Mads Lerdrup5, Stine Friis1,2, Anette Bartels1,2, Gitte Kristensen6, Agnieszka Krzyzanowska7, Hui Xue4, Ladan Fazli4, Klaus H. Hansen5, Martin A. Røder6, Klaus Brasso6, Jose M. Moreira1,2, Anders Bjartell7, Yuzhuo Wang4, Jesper V. Olsen3, Colin C. Collins4, and Diego Iglesias-Gato1,2

Abstract

Purpose: An increasing number of castration-resistant and elevated in NEPC, while the reduced levels of proteins prostate cancer (CRPC) tumors exhibit neuroendocrine (NE) involved in mitochondrial functions suggested a prevalent features. NE prostate cancer (NEPC) has poor prognosis, and glycolytic metabolism of NEPC tumors. Integration of the its development is poorly understood. REST chromatin bound regions with expression changes Experimental Design: We applied mass spectrometry– indicated a direct role of REST in regulating neuronal gene based proteomics to a unique set of 17 prostate cancer expression in prostate cancer cells. Mechanistically, deple- patient–derived xenografts (PDX) to characterize the effects tion of REST led to cell-cycle arrest in G1, which could be of castration in vivo, and the proteome differences between rescued by p53 knockdown. Finally, the expression of the NEPC and prostate adenocarcinomas. Genome-wide profiling REST-regulated gene secretagogin (SCGN) correlated with of REST-occupied regions in prostate cancer cells was corre- an increased risk of suffering disease relapse after radical lated to the expression changes in vivo to investigate the role of prostatectomy. the transcriptional repressor REST in castration-induced NEPC Conclusions: This study presents the first deep characteri- differentiation. zation of the proteome of NEPC and suggests that concom- Results: An average of 4,881 proteins were identified and itant inhibition of REST and the p53 pathway would promote quantified from each PDX. Proteins related to neurogenesis, NEPC. We also identify SCGN as a novel prognostic marker in cell-cycle regulation, and DNA repair were found upregulated prostate cancer.

Introduction androgen receptor (AR) blockage and are driven by complex mechanisms that involve genetic, epigenetic, and posttranscrip- Death in prostate cancer patients mostly occurs after the devel- tional changes (1, 2). Extensive studies have been carried out to opment of castration-resistant metastatic disease (CRPC). Castra- identify tumor driving genetic alterations in CRPC, but a unified tion-resistant tumors develop after androgen ablation therapy or genetic model of disease progression is yet to emerge. Amplifi- cation of the AR gene locus or point mutations in the AR gene occurs in 60% of tumors that have been subjected to androgen 1Department of Drug Design and Pharmacology, Faculty of Health and Medical ablation therapy (3). The appearance of ligand-independent AR Sciences, University of Copenhagen, Copenhagen, Denmark. 2The Danish Can- fi 3 splice variants or AR posttranslational modi cation may also cer Society, Copenhagen, Denmark. Novo Nordisk Foundation Center for contribute to continued AR activation following castration (4). Protein Research, Faculty of Health and Medical Sciences, University of Copen- hagen, Copenhagen, Denmark. 4Vancouver Prostate Centre, University of British Other common genetic alterations in AR-expressing CRPC Columbia, Vancouver, British Columbia, Canada. 5Biotech Research and Inno- include heterozygous deletions of PTEN and, to a lesser extent, vation Center, University of Copenhagen, Copenhagen, Denmark. 6Copenhagen p53 inactivation or loss of Rb1 expression (5–8). Prostate Cancer Center, Department of Urology, Rigshospitalet, University of One recurrent cellular feature of CRPC is the presence of 7 Copenhagen, Copenhagen, Denmark. Department of Translational Medicine, carcinoma cells that exhibit a neuroendocrine phenotype. Neu- Division of Urological Cancers, Lund University, Lund, Sweden. roendocrine differentiation (NED) of prostate cells is frequently Note: Supplementary data for this article are available at Clinical Cancer associated with highly proliferative potential of tumors and Research Online (http://clincancerres.aacrjournals.org/). increased chemoresistance, and as a consequence understanding Corresponding Author: Diego Iglesias-Gato, University of Copenhagen, The the development of NED in CRCP can lead to the development of Danish Cancer Society. Strandboulevarden 49Strand, Copenhagen 2100, new therapeutic strategies for CRPC. Neuroendocrine prostate Denmark. Phone: 4535330640; Fax: 4535325001; E-mail: cancer (NEPC) constitutes a distinct class of castration-resistant [email protected] tumors characterized by the expression of neuronal markers such doi: 10.1158/1078-0432.CCR-18-0729 as Chromogranin A (CHGA), Synaptophysin (SYP), and neural 2018 American Association for Cancer Research. cell adhesion molecule (CD56; ref. 9). NEPCs show similarities to

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Radical prostatectomy tissue microarray Translational Relevance The study cohort is composed of a consecutive series of men The number of castration-resistant prostate tumors exhibit- (n ¼ 336) with clinically localized prostate cancer who underwent ing neuroendocrine features is increasing, due in part to the radical prostatectomy (RP) with curative intent from January 1, improvement of drugs targeting the androgen receptor. Neu- 2002, until December 31, 2005, at the Department of Urology, roendocrine prostate cancer has poor prognosis due to the Rigshospitalet, Copenhagen, Denmark. This study was approved limited efficacy of currently available therapies. In this study, by the Danish National Committee on Health Research Ethics for we provide the first deep characterization of the proteome of the Capital Region (Journal no.: H-6-2014-111). Biochemical castration-resistant neuroendocrine prostate cancer. Proteins failure (BF) was defined as PSA 0.2 mg/L. The collection of are the ultimate targets of most anticancer drugs; therefore, our data was approved by The Danish Data Protection Agency work constitutes a valuable resource for the development of (file#2006-1-6256). The validation cohort (Malmo)€ was previ- novel compounds targeting proteins with increased expression ously described (18, 21). All studies were conducted following the in these tumors. Moreover, we provide evidence that early principles of the Declaration of Helsinki. All participants gave expression of the neuron-specific protein secretagogin in early written or verbal consent. Verbal consent was documented by the stages of prostate cancer correlates with increased risk of physician in the patient journal. biochemical recurrence after radical prostatectomy and could be accounted for in patients managed with conservative pro- IHC and statistics tocols such as active surveillance. TMA sections were deparaffinized in xylene and rehydrated through graded ethanol- Antigen retrieval performed at pH6. IHC staining was performed using anti SCGN antibody (HPA006641, Sigma-Aldrich, diluted 1:250). The SCGN staining was quantified small-cell carcinoma of the prostate (SCCP), which accounts for by scoring the intensity and the percentage of stained tumor cells 0.5% to 2% of all primary prostatic malignancies (10). Patients (0 ¼ negative or positive only in scattered neuroendocrine pros- diagnosed with SCCP have poor prognosis, with a median sur- tate cells; 1 ¼ weak staining in less than 30% for the tumor cells; vival time ranging from 5 to 17.5 months and the 5-year survival 2 ¼ intense staining in less than 20% of the cells on a localized rate of 14.3% (11). Moreover, in patients with locally advanced focus; 3 ¼ moderated staining in more than 50% of the cells or tumors treated with androgen ablation therapy (alone or in a intense staining in less than 50%; 4 ¼ intense staining in more coadjuvant setting), and in patients with CRPC, the extent of NED than 50% of the cells). Of all cores obtained from the same has been shown to be an independent prognostic factor (12). specimen, the highest expression was selected. Scores 2 were The molecular mechanisms driving the development of NE considered as high SCGN expression. The association between tumors after castration therapy are yet to be fully understood. The SCGN expression and clinicopathologic variables was analyzed loss of AR expression, the identification of recurrent mutations in using c2 test or Fisher exact test for categorical variables and the tumor suppressor genes such as Rb1 and p53, and the amplifi- Mann–Whitney U test for continuous variables. The median time cation of genes such as Aurora Kinase A (AURKA) and MYCN are of follow-up was calculated using the reverse Kaplan–Meier thought to play important roles in NE transdifferentiation and the method (22). Follow-up for BF was calculated until the latest aggressiveness of prostate cancer (13–17). Additionally, we PSA measurement, whereas time to death was calculated until the recently demonstrated that inactivation of the transcriptional latest follow-up date. Cumulative incidences of study endpoints repressor REST in prostate cancer cells results in the enhanced were analyzed using the Aalen–Johansen method for competing expression of a cluster of neuronal-specific genes including the NE risks. Death before BF was treated as competing event when markers CHGA and SYP. AR signaling enhances REST stability analyzing risk of BF. Other causes of mortality were treated as through the inhibition of the E3 ubiquitin ligase b-TrcP, which competing events when analyzing risk of prostate cancer–specific might explain why androgen ablation therapy can, in some cases, death. Gray test was used to assess differences in the cumulative promote NE transdifferentiation (18). Recently, REST has been incidences between biomarker subgroups (23). suggested as regulator of the epithelial-to-mesenchymal transi- Univariate and multivariate cause-specific Cox proportional tion of hormone-refractory prostate cancer (19). Whether REST hazard regression models were performed for risk of BF contributes to other aspects of the NE phenotype in prostate and prostate cancer–specific death. All tests were two-sided, and cancer, other than regulating the expression of neuron-specific P < 0.05 was considered to be statistically significant. Statistical genes, has not been studied in depth. analyses were performed using SPSS (software version 22; IBM), R In this study, we characterized the proteome changes occurring (R Development Core Team, Vienna, Austria), or GraphPad during the differentiation from adenocarcinoma to NEPC in PRISM. patient-derived xenograft (PDX) models and investigated the role For the evaluation of the public MCKCC cohort (24), high of REST in this process by matching in vivo expression changes to SCGN tumors were defined as those with expression above the REST-occupied chromatin regions. Functional studies also mean expression level of SCGN in nonmalignant neighboring revealed an interplay between REST and the tumor suppressor tissue plus 2 times the standard deviation. p53 in regulating cell-cycle progression of prostate cancer cells. Chromatin immunoprecipitation analysis of REST-occupied Materials and Methods regions Patient-derived xenografts LNCaP cells were grown in normal medium to 80% confluency. Detailed information about the tissues of origin and generation Cells were crosslinked in 1% cold formaldehyde for 10 minutes of patient-derived primary xenografts has been published else- and the fixation was stopped with 0.125 M glycine for 5 minutes. where (20). Cells were harvested in SDS buffer and then pelleted further to be

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resuspended in a cold IP-buffer supplemented with phosphatase expressed as well (Supplementary Table S3). Raw proteomic files and protease inhibitors. The DNA complexes were further soni- are available at ProteomeXchange (PXD009636). cated to an average size of 300 to 500 bp using a Bioruptor plus sonication device (Diagenode; with settings 30 seconds on, Other bioinformatic tools and statistics 30 seconds off 7 times). Anti-rabbit IgG (10 mg; Millipore) or 5 To compare gene-expression variation between mRNA and mg of anti-REST (Millipore) was added to 50 mg of DNA lysate. protein, tumors were ranked according to the expression Magnetic Dynabeads (Invitrogen) were added into the mix and levels for each gene and distributions were compared using incubated overnight at 4C. After washes, the DNA was decros- Spearman correlation. The overall correlation to different slinked at 65C for 12 hours and purified using a Minelute PCR ontological processes was performed using the 1D analysis purification kit (Qiagen). The DNA was sequenced at the National tool implemented in the Perseus software as described else- High-Throughput DNA Sequencing Centre at Copenhagen Uni- where (35). Briefly, the score compares the mean RANK of versity. Raw reads were mapped to the human genome (hg19 the correlation values of given group (e.g., Ribosome) and the assembly) using bowtie (25) and converted to bam- and bed-files mean RANK of the correlation values of the rest of the proteins using Samtools and Bedtools (26, 27). Only one read per chro- under the formula s ¼ 2 (R1 R2)/n,whereR1andR2are mosomal position per strand was allowed for downstream visu- the average ranks within the group under consideration and its alization and analysis. For peak finding, measurements of dis- complement (all remaining proteins in the experiment), tances to TSSs, as well as visualization of tracks, superimposed respectively, and n is the total number of data points. Gene tracks, and heat maps, data were imported into EaSeq (28) using ontology classification and enrichment analysis were per- default settings and an extension of each read from its 50-end to a formed using DAVID (36), and results were graphically repre- total length of 250 bases. Raw ChIP files are available at the Gene sented with Cytoscape (37). Expression Omnibus database (GSE119385). Cell-cycle analysis Quantitative proteomic profiling C4-2B and PNT2 cells were transfected with two siRNAs Whole-protein extracts were purified from frozen specimens as targeting REST or a siRNA control, pulsed with BrdUrd (Life previously described (29). Twenty-five to 40 g of protein extracts Technologies) for 30 minutes, collected by trypsinization, and was trypsin digested following the Filter-Aid Sample Preparation fixed in methanol. Cells were permeabilized in 0.2 mol/L HCl (FASP) methodology (30). The resulting peptides were fraction- containing 0.25% Triton X-100 for 30 minutes. Anti-BrdUrd ated by Strong Anion Exchange chromatography (SAX) into five antibody (Santa Cruz Biotechnology) was incubated at 1:200 fractions to reduce sample complexity and maximize depth of dilution. As secondary antibody Alexa 488-anti-mouse (Life tech- proteome coverage (30). Each fraction was then analyzed by LC- nologies) at 1:500 dilution was used. Cells were washed in PBS MS/MS using the Q-Exactive mass spectrometer (Thermo scien- and resuspended in propidium iodide solution containing tific). Peptides were separated using a 4-hour gradient of water: RNaseA for 30 minutes at 37C. Samples were analyzed on a acetonitrile, on a 30 cm C18 column. MS spectra were acquired in FACSVerse instrument (BD Biosciences) using appropriate the Orbitrap with 70,000 resolution. MS/MS spectra were settings. acquired in data-dependent mode, after Higher Energy Collision- al Dissociation fragmentation, at a resolution of 17,500 (31). The Transfections, cell viability, RT-PCR, and Western blot analysis obtained mass spectrometric raw data were analyzed in the The Neon transfection system (Invitrogen) and Lipofectamine MaxQuant environment, version 1.3.7.1 (32), with the inte- 2000 (Invitrogen) were used for transfection of siRNA into grated Andromeda searching engine and false discovery rate prostate cancer cells following the manufacturer's instructions. (FDR) cutoff for peptide identification of 0.1 (ref. 33; Fig. 1B). The preparation of cell lysates and Western blot has been Proteins were identified by searching MS/MS data against the described (38). Viability analyses were performed using the cell human proteome sequences from UniProt (UniprotKB, 2012). proliferation kit I (Roche), following the manufacture's guide- To compare NEPC PDXs and androgen-sensitive (AS) adeno- lines. For evaluation of cell death, transfected cells were incubated carcinoma PDXs, the differences between the mean protein with propidium iodide (Sigma-Aldrich) and Hoechst-33342 expressiononeachgroup,defined as label-free quantification (Invitrogen) for 15 minutes at 37C in the dark and of the number (LFQ) intensity (34), were evaluated by the Student t test of dead and live cells estimated using the Celigo Imaging followed by Benjamini–Hochberg correction for multiple test- Cytometer (Nexcelom Bioscience). RT-PCR was performed as ing. FDR values smaller than 0.05 were considered statistically previously described (39). Primers used in this study: b-actin significant. Additionally, proteins with valid LFQ values for (ACTB), 50-CTGGCTGCTGA-CCGAGG-30 and 50-GAAGGTCT- each of the NEPC tumors but 25% values in the adenocar- CAAACATGATCTGGGT-30; TP53,50-ACCTACCAGGGCAGC- cinoma group were considered upregulated in NEPC. Proteins TACGGTTTC-30 and 50-GCCGCCCATGCAGGAACTGTTACA-30; with no detected in NEPC tumors but with LFQ values in REST,50-GGAGGAGGAGGGCTGTTTAC-30 and 50-ACCGACCA- >60% of adenocarcinoma tumors were defined as downregu- GGTAATCACAGC-30; p21 (CDKN1A), 50-CCTGTCACTGTCTTG- lated in NEPC (Supplementary Table S2). TACCCT-30 and 50-GCGTTTGGAGTGGTAGAAATCT-30; MDM2, To compare expression of proteins between PDX on castrated 50-TGTTGTGAAAGAAGCAGTAGCA-30 and 50-CCTGATCCAAC- and intact hosts, the difference between the mean LFQ of each CAATCACCTG-30; FAS,50-GGGCATCTGGACCCTCCTAC-30 and group was evaluated by the Student t test (P < 0.05 was considered 50-GATAATCTAGCAACAGACGTAAGAACCA-30; CHGA,50GCG- statistically significant). Additionally, proteins with no valid LFQ GTGGAAGAGCCATCAT-30 and 50-TCTGTGGCTTCACCACTT- value on any of the tumors of one group but at least on 75% of the TTCTC-30; SYN1,50-GCACGTCCTGGCTGGGTTTCTGGG-30 and tumors of the other group were considered differentially 50-AGGCTACCCGTCAGACATCCGTCTC-30.

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FASP/Trypsin SAX A B digestion fractionation

pH3 Primary adenocarcinoma LN met. NEPC met. pH11 pH5 pH8 pH6

Primary 313C 313D 313H 418 412 352 370 xenografts 6,000 311 313A 313B 331 MaxQuant LC-MS/MS analysis Castration 311-X 313A-X 313B-X 331-X 5,000 (3 weeks) Castration 313BR 331R 4,000 resistant Retention time

# of proteins (LFQ) 3,000 Samples m/z C D 0.8 0.4 Mean: 0.28 0 370 352 331-X 331R 418 331 412 311-X 311 313A-X 313B-X 313A 313C 313D 313H 313BR 313B -0.4 -0.8 Spearman correlation Score FDR Tryptophan metabolism 0.45 0.008 Arg and Pro metabolism 0.43 0.001 Propanoate metabolism 0.41 0.005 Val, Leu and Iso degradation 0.36 0.002 Pyruvate metab. 0.4 0.005 Resp. elec. transport chain −0.41 1.6×10−6 Mitochondria resp. chain C-I −0.5 0.0002 Ribosome −0.37 3.8×10−6 Translation termination −0.37 1.3×10−5 0.7 0.85 1 Translation initiation −0.3 0.0001 Translation elongation −0.28 0.001 Oxidative phosphorylation −0.35 1.6×10−5 Cell cycle Lipid oxidation E Chromosome F Lipid and amino acid segregation Chromatin catabolism organization Lipid catabolism Response Resp to oxidative to ROS Chromatin modification Mitosis Branched stress AA catabolism FA oxidation Chromatin remodeling Mitotic spindle Cellular response organization to oxidative stress AA catabolism Acetyl-CoA catabolism NER Aerobic Cell cycle respiration DNA repair regulation TCA cycle DNA damage Carbohydrate response signal Alcohol catabolism Neuron DNA Neuron biosynthesis differentiation replication development Lysosome Carboxylic acid Cell projection Glucose Acetyl-CoA organization biosynthesis morphogenesis metabolism catabolism DNA repair and replication Neuron projection dev Lysosome Organic acid biosynthesis Carbohydrate Carbohydrate Neuronal biosynthesis development metab.

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Results These findings demonstrate that label-free quantitative proteomic profiling of PDXs can precisely segregate tumor samples Quantitative proteomic data can cluster PDXs by origin and based on patient/genetic origin. In addition, it can also provide hormone status unbiased information on effects of treatment and tumor progres- We analyzed the proteome of 17 prostate cancer PDX tumors, sion that cannot be obtained through genetic profiling. of which 4 tumors were obtained 3 weeks after castration of the host and in 2 cases tumors relapsed after castration and were Proteomic profiling provides additional information to collected (Fig. 1A; ref. 20). On average, 4,881 proteins were gene-expression studies in prostate cancer identified in each of the tumors (Fig. 1B). Unsupervised hierar- Transcriptomic profiling is widely used to estimate protein chical cluster analysis revealed a high positive correlation between expression in biological samples under the presumption that all samples (0.85 0.05; Fig. 1C). Untreated tumors derived from changes in mRNA levels would translate into changes in protein the same patient such as PDXs in the LTL-313 series (0.93 0.02) abundance. To test this hypothesis, specifically in prostate tissue, or PDXs LTL-352 and LTL-370 (0.96) are grouped together, and to identify concordant and discordant mRNA/protein pairs, reflecting a common genetic origin. On the other hand, the we compared how changes in mRNA levels correlate to protein response to castration therapy shows a larger degree of variability. expression changes for gene product across all the samples ana- PDX LTL-311 shows little alteration of proteome expression lyzed. We found a positive but limited correlation (average after therapy (LTL-311-X), while PDX LTL-331 shows a more of 0.28) between mRNA and protein variation across samples pronounced response. The proteome of PDX LTL-331 after 3 (Fig. 1D). The correlation between protein/mRNA pairs variation weeks of castration (LTL-331-X) shows higher similarity to tumors measured across the different experiments does not seem to be a with NEPC PDXs (LTL-370 and LTL-352), than to the parental function of abundance of the mRNA or the protein, indicating that untreated PDX (LTL-331), suggesting that a castration-induced the calculated correlation coefficients are not likely to be an transdifferentiation process occurred in this case (Fig. 1C). artifact of the measurement methodologies (Supplementary Indeed, once castration resistant, the LTL-331R PDX shows a Fig. S1A). Overall, genes involved in intermediary metabolism neuroendocrine phenotype characterized by low expression of of lipids and carbohydrates show positive mRNA/protein corre- AR-regulated genes and elevated expression of neuronal-specific lation, while genes related to ribosomal biogenesis, translational genes (Supplementary Tables S1 and S2; ref. 20). In contrast, control, as well as mitochondrial proteins show close to random castration-resistant LTL-313BR PDX exhibits a proteome profile correlation (Fig. 1D). Therefore, transcriptomic studies involving similar to the parental LTL-313B PDX, reflecting both the genetic the latest functional categories must by cautiously interpreted. similarity between LTL-313B and LTL-313BR and the AR-driven phenotype (Supplementary Table S2; ref. 20). Protein-driven mechanisms of NE transdifferentiation Interestingly, despite the efficacy of androgen ablation therapy Androgen deprivation therapy (ADT) can induce regression of in limiting tumor growth of advanced prostate cancer, limited most advanced prostate cancer, at least temporarily, before CRPC proteome alterations were observed only in tumors collected arises. Primary or therapy-induced NEPCs are highly aggressive, 3 weeks after castration of the host compared with tumors but their proteome is poorly characterized, beyond the fact that propagated in intact mice (Supplementary Table S3). Tumors on they express high levels of neurosecretory proteins and have castrated hosts showed signs of reduced androgen signaling, as reduced expression of AR-regulated proteins (14). Therefore, we suggested by the lower expression of androgen-regulated proteins compared the protein expression profiles of NEPCs to those from such as SARG, DHCR24, and FASN and of proteins involved in AR-dependent adenocarcinomas. We identified 861 proteins fatty acid biosynthesis (FASN, PCCB, and ACSM3). Castration (FDR < 0.05; Supplementary Table S2) differentially expressed also resulted in reduced expression of proteins involved in rRNA between these tumor types. Among the proteins with differential processing and DNA metabolism (DDX56, HEATR1, NOB1, expression, we found some previously validated by IHC (e.g., AR, RPS27A, PCNA, and ATR), while leading to elevated expression PSA), as well as some of bona fide markers of NED (e.g., SYN1, SYP, of proapoptotic proteins such as BCL2L1. Moreover, proteins and CHGA), thereby supporting the validity of our proteomic related to axon guidance (DPYSL2, MATN2, SPTAN1, and data (20). Gene ontology enrichment analysis of the biological SPTBN1) increased their expression, although the canonical NE processes connected to differentially expressed proteins showed markers did not consistently show upregulation at this point after that NEPCs have increased expression of proteins that are castration (Supplementary Table S3).

Figure 1. Proteome characterization of prostate cancer PDXs. A, Schematic representation of the grafting process. Tumors were grafted from primary adenocarcinomas obtained after radical prostatectomy procedures (LTL-311, LTL-418, and LTL-331); biopsies (LTL-313 series); lymph node metastasis (LTL-412); and distant metastases (LTL-352 and LTL-370). Tumors were established and propagated through various generations of mice. "X" indicates that tumors were collected 3 weeks after castrating the host (LTL-311-X; LTL-313A-X; LTL-313B-X; LTL-331-X). LTL-313BR and LTL-331R tumors relapsed after initial response to castration (castration-resistant). B, Layout of proteomic methodology. Proteins from whole tumor extracts were digested with trypsin and peptides fractionated using Strong Anionic Exchange (SAX). Each fraction was analyzed on an Exactive-Q mass spectrometer (Thermo). The number of proteins identified and quantified by label-free quantification (LFQ) using the MaxQuant software is shown. C, Unsupervised hierarchical cluster of all tumors based on Pearson correlation coefficient. D, Limited correlation between mRNA and protein expression in prostate cancer. Tumors were ranked according to the expression levels for each gene and distributions compared using Spearman correlation. The overall correlation to different ontological processes was performed as described in the Materials and Methods section. E and F, Network representation of gene ontology terms enriched among proteins differentially regulated between NEPC and adenocarcinoma PDX tumors. Functional categories overrepresented among the proteins with elevated (E, red) or reduced (F, blue) expression in the neuroendocrine tumors compared with adenocarcinomas. On each node, the size of the red inner circle and the thickness of the red ring relates to the number of proteins upregulated and downregulated, respectively. Edges connect categories with shared proteins.

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normally associated with neuronal differentiation and of proteins portion in REST binding compared with the general distribution involved in mitotic cell-cycle and cell division associated with (P< 0.007, Fig. 3D). chromatin remodeling processes (Fig. 1E), in line with the highly In order to study the functional significance of the REST- proliferative phenotype often reported for NEPC (14). Addition- bound regions, we analyzed how the distance between REST- ally, we found a significant upregulation of proteins involved in occupied regions was related to the expression of genes in their DNA-repair mechanisms that have not been previously recognized vicinity. All genes significantly regulated between NEPC and AS to play a major role in NEPC (Fig. 1E). This includes proteins adenocarcinoma tumors were sorted according to their distance involved in homologous recombination (RAD51, PARP1, XRCC1, to the nearest REST peak. We found that having a REST- RECQL, and RPA2), nucleotide-excision repair (PCNA, POLD1, occupied region within 10 kb of a gene significantly (Fisher POLE, and RFC5) and mismatch repair (MSH2 and ERCC1) P < 0.0001) increases the chances of that gene being upregu- among others (Supplementary Table S2). Enzymes involved in lated in NEPC tumors compared with adenocarcinomas nucleotide metabolism such as the thymidylate synthetase (Fig. 3A). Thus, of the genes within the vicinity of the REST- (TYMS) were also found upregulated in NEPCs, presumably to occupied region, there were 4 times more genes upregulated support DNA synthesis. On the other hand, NEPCs expressed than were downregulated (217 vs. 51) in the NEPC tumors overall lower levels of mitochondrial proteins, including those relative to AS tumors (Fig. 3B). Gene ontology enrichment with functions in the catabolism of fatty acids, amino acids, and in analysis for the biological functions of these genes showed the tricarboxylic acid cycle (Fig. 1F) and also of lysosomal proteins that genes overexpressed in NE located close to a REST-occu- and of proteins involved in cellular response to oxidative stress. In pied region relate to neuronal differentiation and function, good agreement with the protein/mRNA expression correlation, while genes related to proliferation, cell-cycle progression, and enrichment in GO terms associated with metabolic pathways metabolism were mostly absent (Fig. 3C and D). These findings involving mitochondrial and lysosomal proteins was only obvi- suggest that loss of REST during NED in prostate cancer is a key ous in proteomic analysis and not through transcriptomics (Sup- driver of the expression of neuron-specific genes but seems to plementary Figs. S1 and S2), suggesting a higher degree of post- have limited direct impact on other processes relevant to NEPC transcriptional regulation on the expression of these genes. tumorigenesis, such as cell-cycle progression, DNA repair, cell Overall, these data suggest that rapidly growing NEPCs restrict motion, or changes in intermediary metabolism. their cellular activities to cell division. Reduced expression of mitochondria proteins suggests a greater reliance on glycolysis to Loss of REST negatively regulates cell-cycle progression fulfill their energetic needs (Fig. 1E and F). through the activation of the p53 pathway In order to functionally evaluate the effect of REST in cell REST primarily binds to chromatin in the proximity of neuron- proliferation, castration-resistant, AR-expressing C4-2B cells specific genes in prostate cancer were transfected with two independent siRNAs targeting REST Expression of neuron-specific proteins is a hallmark of NEPCs (Fig. 4A), and cell viability was measured by MTT assays and (Fig. 1). In previous studies, we identified the transcriptional compared with cells transfected with control siRNA. Surprisingly, repressor REST as an important regulator of NE transdifferentia- REST knockdown resulted in reduced C4-2B cell viability (Fig. 4B) tion in response to castration and other stimuli in prostate cancer and increased the number of dead cells over time (Fig. 4C). Cell- cells (18). In order to investigate whether REST function extends cycle profile analysis revealed that cell cultures with reduced levels to the regulation of proliferation and/or metabolism-related of REST showed an increased number of cells in G1 phase and genes, we performed an unbiased analysis of REST location on fewer cells in S phase (Fig. 4E). These results suggest a function of chromatin of prostate cancer cells using chromatin immunopre- REST in sustaining cell-cycle progression, which contrasts with the cipitation followed by DNA sequencing (ChIP-Seq), as an attempt highly proliferative phenotype of low-REST-expressing NEPC to identify gene directly repressed by REST. tumors. We then asked whether REST depletion would alter the REST-bound chromatin regions were isolated from AR- and expression of checkpoint regulators such as p53 and Rb1, thereby REST-expressing LNCaP cells and identified by next-generation resulting in cell-cycle arrest. REST knockdown in C4-2B cells did sequencing in two independent experiments. The analysis not affect total levels of Rb1 and had limited direct influence in identified 2,615 DNA regions significantly increased in REST- p53 total levels (Supplementary Fig. S3C). However, the expres- immunoprecipitated chromatin,commontobothbiological sion of the p53 downstream effectors such as p21 (CDKN1A), replicates, as compared with nonspecificIgG(Fig.2A).The FAS, and MDM2 was clearly induced (Fig. 4A, D, and E), suggest- validity of these data was confirmed by the significant enrich- ing the p53 pathway becomes activated upon REST depletion. ment of the RE-1 DNA motif within REST-occupied regions Importantly, simultaneous downregulation of REST and p53 was (P < 10 307), the bona fide recognition site for REST binding on able to rescue the cell-cycle inhibition caused by REST knockdown DNA. REST binding to the promoter regions of known target alone (Fig. 4C, D and F), suggesting that checkpoint inactivation is genes such as CHGA and SYP (Fig. 2B) as well as to BDNF, essential for cell division in the context of reduced REST expression. SYN1, SCGN,andCHGB (Supplementary Fig. S3) was also Indeed, low levels of REST and mutation of TP53 are commonly observed, further validating our experimental approach. observed in NEPC tumors (7, 40, 41). Further evidence of the We then identified which of the genes with increased expression interplay between REST and p53 was obtained by knocking down in the NE compared with adenocarcinoma PDX tumors (20) had REST in the PNT2 prostate cell line. PNT2 cells were immortalized REST binding regions in their vicinity (within 100 Kb), which by transfecting normal prostatic epithelial cells with the SV40 large occurred in 26.82% of the cases (Fig. 2C; Supplementary Table T and small t antigens (42). These viral proteins inhibit, among S4). Gene ontology enrichment analysis of the biological process others, p53 function (43). Thus, REST knockdown in PNT2 cells of these groups of genes revealed that only genes involved in resulted in increased expression of NE markers (Fig. 4G and H) but neuronal development related categories show an increased pro- had no inhibitory effects in cell-cycle progression (Fig. 4I).

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A 50 50 2615 Count IgG_1

REST_1 0 0 0 50 50 2615 Count IgG_2

REST_2 0 0 0 −10,000 bp | 10,000 bp B 30 30 IgG_1 REST_1 0 0 30 30 IgG_2 REST_2 0 0 chr14 | 93388000 | 93395000 | 93402000 chr14 | 93388000 | 93395000 | 93402000 CHGA CHGA CHGA CHGA

12,779 bp 12,779 bp

30 REST_1 0 30

REST_2 0 chrX | 49045000 | 49050000 | 49055000 chrX | 49045000 | 49050000 | 49055000 SYP SYP

7,262 bp 7,262 bp

C D % NEPC upregulated χ2 NEPC % NEPC up P value REST binding (100 kb) up & REST binding

2,500 80 DNA replication 2.3 1.8 0.53 2,000 60 1,500 DNA repair 2.5 2.3 0.88 40 1,000 Transcrip. RNA pol II 2.9 3.6 0.42 500 20 Reg. cell cycle 0.5 1.0 0.22 − 0 0 G1 S transition 1.5 1.5 0.99 NEPC upregulated

DNA repair −S transition Reg. cell1 cycle DNA replication G

Nervous system dev.Transcrip. RNA pol II

Figure 2. REST binding to chromatin is enriched in areas near to neuron-related genes. ChIP experiments are reproducible and REST binding is enriched in the promoter region of known target genes. A, Track of the duplicated experimental replicates (REST_1 and REST_2) superimposed signal (intensity on the Y-axis) at 2,616 regions from the REST- bound regions. The X-axis represents the 10,000 bp surrounding each region and was segmented into 400 bins and smoothed for 1 bin. The Y-axis reflects signal intensity. B, REST peaks are enriched in the promoter region of known target genes such as CHGA and SYP, while absent in control IgG ChIP experiments. C, Number of genes upregulated in NEPC tumors distributed by biological function containing or not REST chromatin binding region in the proximity (100 kb) as defined in ChIP analysis from LNCaP prostate cancer cells. D, Significant enrichment of genes involved in nervous system development among those upregulated in neuroendocrine tumors as compared with adenocarcinomas with REST binding regions within 100 kb. , P < 0.05 was considered statistically significant for enrichment of genes with REST binding.

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A REST_1 REST_2 IgG_1 IgG_2 = 6,721) n NEPC vs. adeno signiﬁcant (

1,000,000 bp 1,000,000 bp 1,000,000 bp 1,000,000 bp Genes sorted according to distance nearest REST peak 50 50 50 50 -3 3 Log2 (FC) B 30 C REST (n = 268) Gene ontology REST Synaptic signaling (30) 20 Nervous system dev. (49) Reg. of memb. potential (18)

Count 10 Secretion by cell (23) Peptide hormone secretion (6) Calcium ion transport (6)

REST binding 10,000 bp 0

-10 -8-6-4-20246810 -log10(FDR) 0 5 10 15 NEPC vs. AS [log2 (fold change)]

D Synaptic signaling Nervous system development

SEZ6 CACNA1E OLFM1 CA10 SHOX2 OLFM3 NRXN1 SKOR1 SHC3 UNC5A CACNA1B FGF14 TRIM9 DAPK3 SEZ6 CELSR3 CPNE9 NPTX1 SYT5 CHRNB2 STMN3 SLC12A5 GAD1 SYT4 ACTL6B NPTX2 UNC13A SLC12A5 ELAVL3 SYNJ1 KCNB1 SNAP25 ADCYAP1 TNR PCLO SNAP25 SHC3 UNC13A DTX1 BSN RIC3 CDK5R2 MYT1 PCSK2 GRIK2 SYT4 NFASC CHRNB2 BSN POU3F1 TNR NRSN1 PCLO ADCYAP1 CRTAC1 CELF4 SCRT1 HCN1 GABBR2 GRM2 GRIN2C SRRM4 SCN3B SYNJ1 KCNC1 POU4F3 NRXN1 VGF HTR1A HPCA GNAO1 SPAG9 FGF14 DPYSL5 MYT1L GPRIN1 NFASC SLITRK1 NPTX1 MYT1L

Regulation of membrane potential Secretion by cell Peptide hormone secretion SEZ6 TRPM4 CARTPT HCN3 POU2F2 REST CELF4 CHRNB2 SYNJ1 FGF14 CDK5R2 SCN3B PTPRN CHGA VGF KCNC2 KCNH7 ATP1A3 TRIM9 CPLX3 GHRHR RAB3C PCLO KCNH6 KCNB1 KCNB1 ADCYAP1 CHRNB2 SNAP25 CACNA1B UNC13A Calcium ion CACNA1B GRIK2 CACNA1E NRXN1 GAD1 SYT5 transmembrane transport PKD2L1 GRIN3A GRIN2C HCN2 GRM2 CACNA1E TRPM8 SYT4 HTR1A HCN1 NRXN1 ADCYAP1 TRPM4 GRIN1 JPH3

Figure 3. Genes with nearby REST-occupied regions show higher expression levels in NEPC compared with adenocarcinoma PDXs. A, Heat map representing the nearest REST-occupied chromatin region relative to a significantly regulated gene in NEPC versus adenocarcinoma tumors (left) and nonsignificantly regulated genes (right). Fold change expression in NEPC tumors compared with adenocarcinomas is color coded from blue (downregulated to red, upregulated). B, Histogram display of significantly regulated genes between NEPC versus adenocarcinoma tumors with REST-binding region within 10,000 base pairs (bp). C, Gene ontology enrichment analysis of the genes in B. D, Network representation of genes in B based on their protein–protein interaction score according to the String database (http://string-db.org) and segregated by ontological categories.

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A C4-2B B C 500 48 h 96 h REST 100 *** 400 *** PHB 80 *** *** 300 *** p53 60 ** 200

p21 40 Cell death

(% of control) 100 CHGA 20

β % of Cell viabilit y 0 -Actin 0 − − − − − − siCtrl + − − siCtrl + + siCtrl + − − − − − + − siREST_1 + + siREST_1 − + − siREST_1 − − + siREST_2 − − + − − + siREST_2 − − + siREST_2

Figure 4. C4-2B D p53 p21 MDM2 FAS REST REST depletion in prostate cancer cells REST 250 l *** inhibits cell-cycle progression through 200 *** p53 ** ** activation of the p53 pathway. A, 150 **** p21 Western blot analysis of the effects of 100 * REST knockdown on REST, PHB, p53, SCGN *** ** % of Contro ** * 50 *** ** p21, CHGA, and Beta-Actin protein β-Actin *** 0 expression in C4-2B cells. B, REST − − − − − siCtrl + siCtrl + − − − knockdown reduces C4-2B cell viability sip53 − ++− − + sip53 − + − − − − − and (C) increases cell death. D, Western siREST_1 − + + siREST_1 − − + − blot analysis of the effects of REST and siREST_2 − − − + − + siREST_2 − − − + p53 knockdown on REST, p53, p21, and SCGN protein expression in C4-2B cells (left) and relative expression of p53, C4-2B (48 h) C4-2B (96 h) E •••***••• *** ** *** p21, MDM2, FAS, and REST in cell ***** *** ### ### ••• 100 ### ### # # 100 transfected with siRNAs targeting REST or p53. E, Quantification of the 80 C4-2B 80 C4-2B G G percentage of cells in the different 60 2 60 2 phases of the cell cycle upon REST and S S 40 40 p53 knockdown in C4-2B cells. F, G1 G1 C ell count (% ) Effects of REST and p53 in the viability 20 Cell count (%) 20 of C4-2B cells. G, Western blot 0 0 analysis of the effects of REST siCtrl + − − − − − siCtrl + - - - - - knockdown in PNT2 cells. H, Relative sip53 − + − − + + sip53 - + - - + + siREST_1 mRNA expression of CHGA and SYN1 in siREST_1 − − + − + − - - + - + - siREST_2 − − − + − + siREST_2 - - - + - + the same as in G. I, Quantification of the percentage of cells in the different phases of the cell cycle upon REST C4-2B (48 h) C4-2B (96 h) F ** ••• 120 ••• knockdown in PNT2 cells. Comparing 120 ## •••*** ## knockdown samples with control, 100 100 ### P < P < P < *** , 0.05; , 0.01; , 0.001; 80 *** *** ### 80 comparing samples with p53, ### ### *** 60 60 ### *** #, P < 0.05; ##, P < 0.01; ### ###, P < 0.001; comparing REST 40 40 % of Cell viability knockdown to combination of % of Cell viabilit y 20 20 REST and p53 knockdown samples, 0 0 * ** *** , P < 0.05; , P < 0.01; , P < 0.001. siCtrl + − − − − − siCtrl + − − − − − ns, nonsignificant. sip53 − + − − + + sip53 − + − − + + siREST_1 − − + − + − siREST_1 − − + − + − siREST_2 − − − + − + siREST_2 − − − + − +

G H 800 CHGA I 100 ns ns *** 80 PNT2 PNT2 600 *** SYN1 60 G2 400 S REST *** *** 40 200 G β-Actin 20 1 % of Control Cell Count (%) 0 − − 0 − − siCtrl + siCtrl + − − + − − siCtrl + − − siREST_1 + siREST_1 − + − − + − siREST_1 − + − − − siREST_2 + siREST_2 − − + − − + siREST_2 − − +

Overall, these results indicate that during NED in prostate, Expression of secretagogin (SCGN) correlates with increased loss of REST induces the expression of neuronal genes, while risk of biochemical recurrence after prostatectomy negatively regulating cell-cycle progression through activation of Prostate cancer tumors are heterogeneous and often exhibit p53. Thus, only tumors that have overcome p53 activation asso- mixed phenotypes, including the expression of neuronal proteins ciated with REST (and AR; refs. 9 and 13) loss would be able to (9). Expression of neuroendocrine proteins has been associated grow as NEPCs. with mutation-driven cell lineage switching (44) as well as

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reduced AR activity. Because both the processes are associated Discussion with increased tumor aggressiveness, we hypothesized that Patients with prostate carcinomas with NED have limited expression of REST-regulated proteins in primary tumors could therapeutic options and subsequently poor prognosis. In order have prognostic value. SCGN was the most highly upregulated to better understand the processes involved in NED in prostate protein in NEPC tumors (fold change > 150; P < 0.003) in our cancer, we have, for the first time, characterized the proteome proteomic profile among the ones with REST binding in the differences between NEPC and prostate adenocarcinoma PDXs promoter region (Supplementary Fig. S3A). We first tested the using quantitative mass spectrometry–based proteomics. NEPCs prognostic value of SCGN gene expression using data from a exhibit enhanced levels of proteins involved in the regulation of publicly available cohort of radical prostatectomies (n ¼ 131; cell proliferation and response to DNA-damage stress and express ref. 24). As expected, only a handful of tumors exhibited high lower levels of mitochondrial and lysosomal proteins, indicating SCGN expression, but most of them were associated with BF after the use of alternative pathways for energy production. Expression RP (HR log-rank: 4.82; Supplementary Fig. S3D). This prompted of neuron-specific proteins in NEPCs was directly linked to the us to validate these results using a Copenhagen Prostate Cancer reduced expression of the transcription repressor REST. Expres- cohort composed of 336 consecutive prostatectomies operated at sion of one of these proteins, SCGN, correlated with increased risk Righshospitalet (Copenhagen, Denmark) between 2002 and of BF after RP. 2005 (Fig. 5B, Supplementary Table S5). We analyzed the expres- We recently characterized the proteome of primary prostate sion of SCGN by IHC (Fig. 5A). Tumors were classified according tumors and detected increased mitochondrial activity and to the expression of SCGN as "high" and "low" (see Materials and enhanced expression of proteins involved in oxidative phosphor- Methods). High SCGN protein expression was detected in 46% ylation and TCA cycle when compared with neighboring benign of the cases (n ¼ 147), and its expression correlated with RP prostate tissue (29, 46). Here, we observe that NEPCs exhibit Gleason score (GS; P ¼ 0.02; Supplementary Table S5). Analysis reduced levels of proteins involved in these metabolic processes of the 10-year cumulative incidence of BF was 36.2% (95% CI, compared with prostate adenocarcinomas (Fig. 1). These changes 28.6–43.7) in the SCGN-low group compared with 50.1% (95% occur shortly after castration, before other NE features (increased CI, 41.8–58.4) in the SCGN-high group (P ¼ 0.0007; Fig. 5C). expression of neuronal and proliferation related proteins) were Moreover, univariate Cox regression analysis found a statistical revealed, suggesting this metabolic transition to be related to association of SCGN expression with the risk of suffering BF after reduced AR activity. This hypothesis is further supported by the RP (HR, 1.7; P ¼ 0.002; Table 1A). A trend toward this association limited chromatin association of REST to metabolic gene pro- being true was maintained after multivariate analysis (HR: 1.4; moters (Fig. 3) and the absence of changes in mitochondrial P ¼ 0.07; Table 1A). No association was found, however, with content observed in REST-depleted, AR-expressing cells (Fig. 4A). other clinical endpoints such as development of CRPC or prostate The opposite regulation of mitochondrial proteins in NEPC cancer–specific death (Supplementary Table S6). We further val- compared with prostate adenocarcinoma indicates the existence idated these results using an independent cohort of RP collected at of fundamental differences on the pathways utilized for energy the Malmo€ University Hospital (18, 21). The incidence of tumors metabolism, and suggests that NEPCs would have relatively with high SCGN expression was lower in this cohort (19%). Given higher glycolytic activity. This conclusion is supported by studies the foci pattern of SCGN expression in primary prostate tumors, using fluorodeoxyglucose (FDG) positron emission tomography this lower incidence is most likely related to the fewer cores per (PET) in patients with suspected neuroendocrine tumors. Visceral RP sampled when building the TMA: two in the Malmo€ cohort metastases, the preferred site for NEPCs, show higher uptake of compared with 4 to 12 cores in the Copenhagen cohort. In this FDG as when compared to bone metastases, the preferred site for cohort, SCGN expression also tended to correlate with increased AR–expressing castration-resistant adenocarcinomas (47). risk of suffering from BF after RP (HR ¼ 2.8; P ¼ 0.06; Supple- Interestingly, we also detect a significant downregulation of mentary Fig. S3D). multiple proteins within several cytosolic compartments in Clinical trials over the years have shown that prostate cancer NEPCs, including the lysosome, ER, and Golgi. The general patients bearing tumors with GS 3þ4 have reduced risk of reduction of these compartments would be consistent with the suffering biochemical relapse and of dying with prostate cancer reduced cytosol volume that is characteristic of small-cell carci- after RP than those with higher histologic grades (45). Conse- nomas (48). The reason for this phenotype is not clear. We detect quently, more conservative regimens such as "active surveillance" reduction in enzymes involved in lipid biosynthesis, which could are being implemented to manage these patients. In order to test influence the size of these membrane-rich organelles. The normal whether SCGN could serve as a prognostic biomarker for these prostate luminal epithelium has an important secretory function patients diagnosed with "low-risk" tumors, we studied the that is regulated by androgen signaling and, to some extent, association of SCGN expression to biochemical relapse in uni- maintained in primary tumors (49). Therefore, reduced levels of variate and multivariate Cox regression analyses on patients with AR, resulting in reduced expression of AR-regulated genes in GS 3þ4 tumors. Strikingly, both analyses retrieved statistically NEPCs (Supplementary Fig. S4A and S4B), may have widespread significant association of SCGN expression with BF (Table 1B). effects in secretory pathways, in addition to the regulation Moreover, the 10-year cumulative incidence of BF was 27.8% of mitochondria content, a hypothesis that deserves further (95% CI, 19.9–35.7) in the SCGN-low group compared exploration. with 45.2% (95% CI, 35.2–55.2) in the SCGN-high group The transcriptional regulator REST plays a key role in repressing (P ¼ 0.0007; Fig. 5D). the expression of neuronal genes in nonneuron-related tissues. These results suggest that in patients managed with conserva- Loss of REST and the expression of REST splice variants unable to tive regimens, elevated SCGN expression might be considered as a exert this repressor function have been implicated in the NED in sign of aggressiveness, suggesting the earlier application of cura- prostate cancer cells (9, 18, 50). Thus, the expression of SRRM4, a tive treatments such as RP or radiation.

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A B 0:RI 1:RI RP in the study period n = 336

Missing malignant tissue. n = 17

50 μm SCGN Low 250 μm250 μm TMA cohort n = 319 IR:2 IR:3 IR:4

Missing PSA follow-up. n = 2

Analysis of BF

SCGN High n = 317 250 μm 250 μm 052 μm C Biochemical failure Biochemical failure

BF Death before BF SCGN low Gray test: P = 0.0007 SCGN high Cumulative incidence of BF Cumulative incidence of BF Cumulative

Time from RP (years) 03691215 Time from RP (years) 0 3 691215 No. at risk 317 222 162 129 60 1 No. at risk 170 129 93 72 39 1 147 93 69 57 21 0 D Biochemical failure (GS ≤ 3 + 4) Biochemical failure (GS ≤ 3 + 4) BF SCGN low Gray test: P = 0.0007 Death before BF SCGN high Cumulative incidence of BF Cumulative Cumulative incidence of BF Cumulative

Time from RP (years) 03691215 Time from RP (years) 03691215 Not at risk 238 182 135 107 54 1 Not at risk 135 111 83 65 36 1 103 71 52 42 18 0

AR Fatty acid metabolism E CR-Neuroendocrine REST Neuronal gene expression E2F1 Cell-cycle progression (AURKB, MYCN)

ADT

Fatty acid metabolism

AR Cell-cycle progression REST AR CR-Adenocarcinoma REST p53

Neuronal gene expression

Figure 5. SCGN expression correlates with increased risk of biochemical failure after radical prostatectomy. A, Representative images of SCGN immunoreactivity (IR). IR of 0 and 1 was accounted as "low" SCGN expression and 2, 3, and 4 were considered as "high." Arrows indicate positive staining of scattered prostate neuroendocrine cells. B, Schematic description of the cohort. C, The cumulative incidence of BF for the entire cohort and in relation to SCGN expression. Death without BF was computed as competing event. D, The same as B but for patients carrying tumors with Gleason score of 3 þ 4 or below. E, Schematic representation of the proposed model of prostate cancer progression after ADT. Prostate cancer relapse after ADT can occur in several ways. Typically, prostate cancer will relapse in the form of adenocarcinoma showing metabolic features of primary tumors and with expression of AR and REST and limited expression of neuronal proteins. Alternatively, castration-resistant NE tumor with reduced AR and REST expression will express neuronal proteins and increased activity of proteins involved in cell-cycle progression, likely on a background of reduced activity of the p53 pathway.

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Table 1. Univariate and multivariate cause-specific Cox proportional hazard of BF A Univariate analysis Multivariate analysis HR (95% CI) P HR (95% CI) P SCGN Low Ref Ref High 1.7 (1.2–2.3) 0.002 1.4 (0.98–1.9) 0.07 Age at RP For 5-year differences 1.1 (0.9–1.2) 0.4 0.9 (0.8–1.1) 0.2 PSA For 2-fold difference 1.5 (1.3–1.8) <0.0001 1.3 (1.1–1.5) 0.007 Pathologic T stage pT2a/b/c Ref Ref pT3a/b 3.3 (2.4–4.6) <0.0001 2.0 (1.4–2.9) 0.0001 N stage N0/x Ref Ref N1 2.8 (1.0–7.5) 0.04 1.3 (0.5–3.8) 0.6 RP Gleason score 6 Ref Ref 3 þ 4 2.8 (1.8–4.4) <0.0001 1.8 (1.2–2.9) 0.008 4 þ 3 4.1 (2.5–6.6) <0.0001 2.8 (1.7–4.6) 0.0001 8–10 5.0 (2.9–8.8) <0.0001 2.6 (1.4–4.9) 0.002 Margin status R Ref Ref Rþ 2.3 (1.6–3.3) <0.0001 1.5 (1.0–2.2) 0.04 B Univariate analysis Multivariate analysis HR (95% CI) P HR (95% CI) P SCGN Low Ref Ref High 1.9 (1.3–2.9) 0.002 1.9 (1.2–2.9) 0.004 Age at RP For 5-year differences 1.0 (0.8–1.2) 0.9 0.8 (0.7–1.0) 0.08 PSA For 2-fold difference 1.5 (1.2–1.9) 0.0001 1.4 (1.1–1.7) 0.004 Pathologic T stage pT2a/b/c Ref Ref pT3a/b 4.0 (2.6–6.0) <0.0001 3.0 (1.9–4.9) <0.0001 N-stage N0/x Ref Ref N1 1.8 (0.2–12.6) 0.6 2.8 (0.4–21.4) 0.3 RP Gleason score 6 Ref Ref 3 þ 4 2.9 (1.9–4.5) <0.0001 1.7 (1.0–2.7) 0.03 Margin status R Ref Ref Rþ 2.2 (1.4–3.4) 0.0006 1.2 (0.7–1.9) 0.5 Abbreviations: CI, confidence interval; HR, hazard ratio; PSA, prostate-specific antigen; REF, reference; RP, radical prostatectomy.

splicing factor involved in the alternative splicing of REST, is small-cell characteristics after RP or castration therapy remains commonly upregulated in NEPCs. Interestingly, we found that to be elucidated. REST binds the chromatin regions in the vicinity of the SRRM4 The tumor suppression function of REST suggested by its gene (Supplementary Fig. S3B), suggesting a feed-forward regu- prognostic value for tumor aggressiveness (18) is challenged by latory loop that would result in promoting the expression of our experimental results. Depletion of REST in C4-2B cells REST-repressed genes. To which extent the function of neuron- resulted in decreased viability, increased cell death and cell-cycle specific proteins is critical in the development of NEPCs aggressive arrest. This phenotype can be rescued by simultaneous p53 phenotype remains to be defined. depletion (Fig. 4). This suggests that a defective p53 pathway is Similar to REST (18), the expression of SCGN, a REST- required for the survival of tumors with low REST expression. repressed gene, in prostatectomy specimens correlated with BF Because combined p53 and REST genetic inactivation in mice after surgery, while no correlation with other clinical endpoints, results in more frequent brain neuroepithelial tumors than caused such as disease-specific survival, was found (51). Strikingly, the by the inactivation of p53 alone (52), we cannot entirely exclude expression of SCGN in primary adenocarcinomas also occurs in that REST inactivation may also contribute to prostate neuroen- AR-positive cells (Supplementary Fig. S3E), suggesting that the docrine tumor growth in vivo, through mechanisms not yet expression of this neuroendocrine marker may occur indepen- fully explored here, such as the paracrine regulation of the dently or precede the loss of AR that is characteristic of NEPC. tumor microenvironment. The significance of p53 for NE prostate Whether or not SCGN and AR coexpressing tumors have malignancies is further supported by the upregulation of the increased chances to develop into full-blown NEPC with PEG10 protein in NEPCs, as the inhibition of p53 is also required

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for the cell-cycle promoting actions of PEG10 in NEPC (53). The Disclosure of Potential Conflicts of Interest fact that tumor suppressor p53 is often mutated in NE prostate No potential conflicts of interest were disclosed. tumors (13) supports our hypothesis that depletion of REST expression (as a result of ADTs) would be deleterious for the Authors' Contributions prostate cancer cells if combined with an active p53 signaling Conception and design: A. Flores-Morales, Y. Wang, D. Iglesias-Gato pathway (Fig. 4C). Development of methodology: D. Lin, A. Bartels, J.M. Moreira Expression of proteins that regulate cell-cycle progression is a Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A. Flores-Morales, T.B. Bergmann, C. Lavallee, hallmark of NEPC. Many of these proteins are controlled T.S. Batth, D. Lin, S. Friis, A. Bartels, A. Krzyzanowska, H. Xue, J.M. Moreira, through the activation of the E2F1 transcription factor (54). A. Bjartell, Y. Wang, J.V. Olsen, C.C. Collins, D. Iglesias-Gato We could validate this in the NEPC by analyzing the E2F1 Analysis and interpretation of data (e.g., statistical analysis, biostatistics, chromatin binding regions in LNCaP cells (55) in relation to computational analysis): A. Flores-Morales, T.B. Bergmann, C. Lavallee, our profile of differentially regulated genes in NEPC compared T.S. Batth, M. Lerdrup, G. Kristensen, K.H. Hansen, M.A. Røder, K. Brasso, with prostate adenocarcinoma (Supplementary Fig. S4C and J.M. Moreira, D. Iglesias-Gato Writing, review, and/or revision of the manuscript: A. Flores-Morales, S. Friis, S4D). Importantly, E2F1 also binds chromatin in the proximity G. Kristensen, M.A. Røder, K. Brasso, J.M. Moreira, A. Bjartell, Y. Wang, of two suggested driver genes of NE prostate cancer: MYCN and D. Iglesias-Gato AURKB (ref. 17; Supplementary Fig. S4E). The mechanisms Administrative, technical, or material support (i.e., reporting or organiz- leading to E2F1 activation during NED in prostate cancer ing data, constructing databases): A. Flores-Morales, G. Kristensen, remain unknown; however, these would likely be related to A. Krzyzanowska, K. Brasso, Y. Wang deletion of the Rb1 gene, an E2F1 inhibitor, commonly found Study supervision: A. Flores-Morales Other (pathology): L. Fazli mutated in NEPC (7). After integrating proteomic, transcriptomic, and ChIP-seq pro- Acknowledgments fi ling, our data suggest that relapse from ADT as NEPC requires a This work was supported by grants from the Danish Research Council series of concurrent events including loss of AR and REST protein (DFF 4004-00450, A. Flores-Morales), the Movember Foundation (A. Flores- expression, likely in a context of p53 inactivation and inhibition Morales, C.C. Collins), and the Danish Cancer Society (R90-A6060-14-S2, of RB1 function leading to E2F activation and cell-cycle progres- A. Flores-Morales). This work was also supported by The Canadian Institutes sion (Fig. 5E). Under these conditions, cells would adapt from of Health Research (Y. Wang), The Terry Fox Research Institute (Y. Wang, C.C. Collins), The Lundbeck Foundation, and The Danish Cancer Research a predominant oxidative phosphorylation-based metabolism, typ- Foundation (S. Friis). The Novo Nordisk Foundation Center for Protein ical of prostate adenocarcinomas, to a more glycolytic one. Reduc- Research, University of Copenhagen is financially supported by the Novo tion in mitochondrial content would suggest enhanced sensitivity Nordisk Foundation (grant agreement NNF14CC0001). to drugs targeting glycolytic pathways that could be exploited as fi therapeutic alternative. Finally, expression of neuron-speci cpro- The costs of publication of this article were defrayed in part by the teins, characteristic of NE tumors, is mostly a consequence of low payment of page charges. This article must therefore be hereby marked REST expression, which, as judged by our data, has limited direct advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate effect in promoting the highly proliferative phenotype of these this fact. tumors. Whether or not the expression of neuronal proteins contributes to survival of prostate cancer cells under conditions Received March 4, 2018; revised August 4, 2018; accepted September 25, of low androgen availability requires further investigation. 2018; published first October 1, 2018.

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608 Clin Cancer Res; 25(2) January 15, 2019 Clinical Cancer Research

Proteogenomic Characterization of Patient-Derived Xenografts Highlights the Role of REST in Neuroendocrine Differentiation of Castration-Resistant Prostate Cancer

Amilcar Flores-Morales, Tobias B. Bergmann, Charlotte Lavallee, et al.

Clin Cancer Res 2019;25:595-608. Published OnlineFirst October 1, 2018.

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