Evaluationof ERGResponsive Proteomein ProstateCancer

Shyh-Han Tan,1* Bungo Furusato,1 Xueping Fang,2 Fang He,2 Ahmed A. Mohamed,1

Nicholas B. Griner,1 Kaneeka Sood,1 Sadhvi Saxena,1 Shilpa Katta,1 Denise Young,1

Yongmei Chen,1 Taduru Sreenath,1 Gyorgy Petrovics,1 Albert Dobi,1 David G. McLeod,1

Isabell A. Sesterhenn,3 Satya Saxena,2 and Shiv Srivastava1

1Center for ProstateDisease Research,Departmentof Surgery,Uniformed ServicesUniversityoftheHealth Sciences, Rockville,Maryland

2Calibrant Biosystems, Inc.,Gaithersburg,Maryland3The Joint PathologyCenter, Silver Spring,Maryland

BACKGROUND. Gene fusion between TMPRSS2 promoter and the ERG proto-oncogene is amajor genomic alteration found in over half of prostate cancers (CaP), which leads to aberrantandrogen dependent ERG expression. Despite extensive analysis for the biological functions ofERG in CaP, there is no systematic evaluation of the ERG responsive proteome (ERP). ERP hasthe potential to define new biomarkers and therapeutic targets for prostate tumors stratifiedby ERG expression.METHODS. Global proteome analysis was performed by using ERG (þ) and ERG (�) CaPcells isolated by ERG immunohistochemistry defined laser capture microdissection and byusing TMPRSS2-ERG positive VCaP cells treated with ERG and control siRNA.RESULTS. We identified 1,196 and 2,190 unique proteins stratified by ERG status fromprostate tumors and VCaP cells, respectively. Comparative analysis of these two proteomesidentified 330 concordantly regulated proteins characterizing enrichment of pathwaysmodulating cytoskeletal and actin reorganization, cell migration, protein biosynthesis, andproteasome and ER-associated protein degradation. ERPs unique for ERG (þ) tumors revealenrichment for cell growth and survival pathways while proteasome and redox functionpathways were enriched in ERPs unique for ERG (�) tumors. Meta-analysis of ERPs againstCaP gene expression data revealed that Myosin VI and Monoamine oxidase A were positivelyand negatively correlated to ERG expression, respectively.CONCLUSIONS. This study delineates the global proteome for prostate tumors stratified byERG expression status. The ERP data confirm the functions of ERG in inhibiting celldifferentiation and activating cell growth, and identify potentially novel biomarkers andtherapeutic targets. Prostate 74: 70–89, 2014. # 2013 Wiley Periodicals, Inc.

KEY WORDS: ERG; proteomics; myosin VI; MAOA; actin and cytoskeletalreorganization

INTRODUCTIONCarcinoma of prostate is the most frequently diag-

nosed non-skin cancer in the United States with anestimated 238,590 newly diagnosed cases and 29,720deaths in 2013 [1]. Rapidly increasing understandingof the molecular basis of CaP is providing new insightsinto the etiology and improved prognosis of thedisease [2–4]. Prevalent gene rearrangements in CaPinvolve the fusion promoter region of AR regulatedgenes (predominantly, serine 2 trans-membrane prote-

ase: TMPRSS2) and protein coding sequence of an ETSrelated gene (primarily ERG). While TMPRSS2-ERG is

Grant sponsor: National Cancer Institute; Grant number:R01CA162383.�Correspondence to: Dr. Shiv Srivastava, 1530 East Jefferson St.,Rockville, MD 20852. E-mail: ssrivastava@cpdr.orgReceived 4 August 2013; Accepted 27 August 2013DOI 10.1002/pros.22731Published online 21 September 2013 in Wiley Online Library(wileyonlinelibrary.com).

The Prostate 74:70^89 (2014)

� 2013 Wiley Periodicals, Inc.

detected in 40–65% of patients, SLC45A3 and NDRG1serve as fusion partners for approximately 10% of thetumors with ERG rearrangements [5–7].

Despite the high prevalence of TMPRSS2-ERG genefusions detected in CaPs of Western populations, thefrequency is lower in African Americans (31–43%)compared to Caucasian Americans (50–66%), and it iseven lower in Asian populations (5–24.4%) [8–10]. Wehave recently reported that ERG frequency is strikinglyless in the index tumors of African American patients(28.6%) compared to Caucasian Americans (63.3%),suggesting that the ERG based stratification of CaP mayhelp distinguish the biologic differences of CaP betweenthe ethnic groups [10]. Studies comparing ERG (þ) andERG (�) CaP have also suggested the expression ofgenes unique to ERG (þ) or ERG (�) tumors [11,12].

Multiple studies on the ERG regulated transcriptomehave investigated the function of ERG in the context ofprostate epithelial cells and its effect on tumor cellinvasion or prostate epithelial differentiation [13–16].However, the underlying mechanisms of ERG functionremain to be better elucidated. Although there havebeen considerable efforts to characterize the CaP prote-ome [17–21], a systematic evaluation of ERG responsiveproteome (ERP) has not been carried out. Since ERGoncoprotein is a nuclear transcription factor, it is neitheran optimal biomarker nor an ideal cancer therapeutictarget. The evaluation of ERG responsive proteins(ERPs) may identify surrogate biomarkers from secret-ed or cell surface proteins or druggable targets such asgrowth factor receptors or kinases in the ERG network.Furthermore, differential expression of proteins in ERG(þ) and ERG (�) CaP may delineate the biochemicaldifferences and identify potential biomarkers and thera-peutic targets of specific for these two tumor types.

Until recently, the lack of reliable ERG antibodieshas restricted the analysis of ERG aberrations in CaPspecimens to fluorescence in situ hybridization (FISH)or reverse transcriptase polymerase chain reaction (RT-PCR) assays [22,23]. We have adopted a novelapproach to study the ERG modulated proteome byidentifying tumor cells positive or negative for ERGprotein expression using ERG-MAb-based immuno-histochemistry (IHC) staining of prostate tumor speci-mens [24], followed by the isolation of cells using lasercapture microdissection (LCM) [25]. Using ERGsiRNA, we also inhibited the expression of the ERGprotein in VCaP cells, which enabled us to compareERP in the presence or absence of ERG.

The application of sensitive and quantitative meth-ods in shotgun proteomics has significantly improvedthe resolution proteomic of analysis. In this study, weused a unique platform based on capillary isotacho-phoresis (CITP) and capillary zone electrophoresis(CZE) coupled with electrospray ionization (ESI) linear

ion trap tandem mass spectrometry (MS/MS). Thecombined CITP/CZE-nano-ESI-MS/MS system hasbeen demonstrated to be at least one to two orders ofmagnitude more sensitive than that found in conven-tional electrophoresis and column-chromatographybased proteome technology, covering a much widerconcentration range, necessary for increasing the rangeof protein profiling [26]. This improvement is achievedby the selective analyte enrichment through electroki-netic stacking of CITP, and the excellent resolvingpower of CZE [27], which results in diluting the majorcomponents while concentrating the trace compounds.The on-column transition from CITP to CZE alsominimizes additional band broadening with superioranalyte resolution.

We defined the differentially expressed ERP bothfrom prostate tumor specimens and from VCaP cellline and revealed a total of 330 overlapping proteinsthat concordantly respond to ERG expression. Litera-ture-based evaluation for functionally interacting sig-naling pathways revealed networks regulatingmultiple cellular functions including AR signaling,protein synthesis and trafficking, and cell growth andmigration. By sorting for ERPs that were detected athigher MS ratios in ERG (þ) and ERG (�) tumorsrelative to benign tissues, we sought to distinguishthese tumors based on their specific signal transductionpathway signatures. ERPs unique for ERG (þ) andERG (�) tumors were examined for potential surrogatebiomarkers or therapeutic targets based on their cellu-lar localization or enzymatic activity. Consistent withprevious reports, we observed the effect of ERG onstimulating cell growth and inhibiting cell differentia-tion. This is evident in ERG silenced VCaP cells, wherewe observed increased expression of markers of pros-tate luminal epithelial differentiation and regulators ofcell polarity concomitant with reduced expression ofEGFR signaling pathway proteins. Furthermore, toidentify correlation to ERG expression at the level ofboth protein and mRNA expressions, ERPs werecompared against the CPDR 80-GeneChip/40-patienttumor versus benign gene expression dataset [5].Myosin VI and MAOAwere found to be positively andinversely correlated to ERG expression, respectively.Combined detection of ERG, Myosin VI, and MAOA todistinguish ERG (þ) and Myosin VI (þ) tumors fromERG (�) and MAOA (þ) tumors may facilitate thediagnosis and stratification of CaP patients.

MATERIALSANDMETHODS

Cell Culture and ERGsiRNAKnock-Down

Human prostate tumor cell lines, VCaP, LNCaP,CWR22Rv1, DU145, PC-3, RWPE-1, and RWPE-2 werepurchased from American Type Culture Collection

ERGResponsive Proteome 71

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(ATCC, Rockville, MD) and maintained as recom-mended. The LNCaP subline, C4-2B was purchasedfrom Urocor (Oklahoma, OK) and cultured as recom-mended. LAPC-4 cells were kindly provided by Dr.Charles L. Sawyers. RC170N cells were established inour laboratory and cultured in Keratinocyte serum-free medium, supplemented with bovine pituitaryextract and recombinant epidermal growth factor (LifeTechnologies, Inc., Carlsbad, CA) [28]. ERG (50-CGA-CAUCCUUCUCUCACAUAU-30) and non-targeting(NT; D-001206-13-20) small interference RNA (siRNA)oligo duplexes were from Thermo Scientific (Lafayette,CO) [13]. VCaP cells were seeded in 10 cm tissueculture dishes at 2� 106 cells per dish in DMEM(ATCC), supplemented with 10% charcoal:dextranstripped fetal bovine serum (cFBS; Gemini Bioprod-ucts, West Sacramento, CA) and propagated for 3days. Cells were transfected with 25 or 50 nM of ERGor non-targeting (NT) siRNAs using Lipofectamine2000 (Life Technologies, Inc.) [29]. Twelve hours aftertransfection, VCaP cells were treated with 0.1 nM ofthe synthetic androgen analogue R1881. A near com-plete ERG knock-down was achieved by growing thecells for 4 days following transfection, which wasconfirmed by immunoblot analysis of the cell lysates.

ProstateTissues and Laser Capture-Microdissection

Under an IRB approved protocol (Protocol No.20405-28), prostate tumor cells and benign cells (dis-tant to tumor focus) from the same tissue section wereisolated by LCM from whole-mounted FFPE prostatesections of five patients that were matched for age (50–65 years), race (Caucasian American), and tumor celldifferentiation (well to moderate), Gleason grade(3þ 3 or 3þ 4) and nuclear grade (grade II). Whole-mounted prostate tissue sections of 8mm thicknessplaced on uncharged glass slides were analyzed formalignant and benign cells by hematoxylin and eosinstaining and for ERG oncoprotein expression status byIHC with the CPDR anti-ERG monoclonal antibody,ERG-MAb, 9FY [24]. Two ERG (þ) and three ERG (�)specimens were selected. Approximately 100,000 tu-mor cells and an equivalent number of matchingbenign cells were isolated using the Arcturus PixCell IIsystem on LCM caps from each of the sections. Thecaps were placed into micro-centrifuge tubes with50ml of ultra-pure water, immediately frozen on dryice and stored in �80°C until proteomic analysis.

ProteomicAnalysis of ERGResponsive Proteome

ERG (þ) tumor cells were pooled together from twospecimens; ERG (�) tumor cells, from three specimens;

and benign cell, from five specimens. The workflowfor proteomic analysis is outlined in Figure 1A.Proteins extracted from the cell pellets were denatured,reduced, and alkylated before trypsinization. Digestedpeptides were desalted, purified, and lyophilized.Peptides were then stacked, resolved, and fractionatedusing CITP and CZE-based multidimensional separa-tions [26]. Peptides fractions were analyzed by nano-reversed-phase liquid chromatography and eluantswere monitored by a linear ion-trap mass spectrometerequipped with an ESI interface. Raw LTQ data wereconverted to peak list files, which were searchedagainst the UniProt sequence library (www.uniprot.org). A 1% false discovery rate (FDR) for total peptideidentifications, which correlates with the maximumsensitivity versus specificity, was chosen as a cutoff.Only proteins identified with at least two peptides andone unique peptide were included in the final list ofidentified proteins. The complete description of theseprocedures is described in detail in the supplementarymaterials.

GeneOntologyAnnotation andComparison ofDatasets

The classification and clustering of proteins datasetwere performed using ProteinCenter, v3.2 (ThermoScientific, West Palm Beach, FL). The differentiallyexpressed proteins detected from the NT siRNA andERG siRNA experiments were analyzed using theGenomatix (Ann Arbor, MI) GeneRanker and GenomatixPathway System (GePS) programs. The over-representa-tions of different biological terms (literature associa-tion-based or curated canonical pathways) within theinput protein list were ranked by their P-values byGeneRanker. Functional interaction networks were gen-erated from these ranked lists based on co-citationswithin the same sentence in PubMed abstracts linkedby a function word. The interaction of ERPs wasrepresented by a network layout that emphasizes highco-citations connectivity and interactions.

Western Blot and ImmunofluorescenceAssays

VCaP cells were lysed in mammalian proteinextraction reagent (M-PER) (Pierce, Rockford, IL) con-taining protease and phosphatase inhibitors (Sigma, StLouis, MO). Cell lysates equivalent to 20mg of proteinwere separated on 4–12% Bis-Tris Gel (Life Technolo-gies, Inc.) and transferred to PVDF membrane. Mem-branes were incubated overnight at 4°C with primaryantibodies and washed before treated with goat anti-Mouse IRDye 800CW or goat anti-Rabbit IRDye680CW secondary antibodies (Li-Cor Biosciences, Lin-coln, NE) at 25°C. Bands were visualized and signal

72 Tan et al.

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intensities of the bands were quantitatively measuredusing the Odyssey infra-red imaging scanner (Li-CorBiosciences).

VCaP cells were seeded onto poly-L-lysine coatedcoverglass (BD Bioscience; San Jose, CA) in 10% CSS 2days prior to siRNA transfection. Cells were inducedwith 0.1 nM R1881 1 day after transfection andincubated for 48 hr. Cells were fixed with PBS buffered4% paraformaldehdye before permeabilization in 1�PBS with 0.1% Triton X-100. Prior to incubation inprimary antibody, cells were blocked in 1% normalhorse serum (Vector Laboratories; Burlingame, CA)in PBS. Cells were incubated with a species specificsecondary antibody (Alexa-Fluor-594 goat anti-mouse,Alexa-Fluor-488 goat anti-rabbit; Life Technologies,Inc.), and with DAPI (40,6-Diamidino-2-Phenylindole)as a nuclear counterstain.

The antibodies used in for immunoblot and immu-nofluorescence analysis were acquired from the fol-lowing sources: ERG-MAb (9FY) from BiocareMedical, Concord, CA; GAPDH (sc-25778) from SantaCruz Biotechnology, (Santa Cruz, CA); SHC (610082)

from BD BioSciences; PAP (2906–1), and ERG(EPR3864(2)) from Epitomics (Burlingame, CA); PSA(A056201-2) and SLC45A3/Prostein (Clone 5E10,M3615) from Dako (Carpinteria, CA); p44/ERK1(#4372), a-tubulin (11H10, #2125), Cool1/bpix/ARH-GEF7 (#4515), and Myosin VI (#9200) from CellSignaling Technology (Beverly, MA); RAC1 (ARCO3)from Cytoskeleton (Denver, CO); MSMB (TA501072)from Origene (Rockville, MD); Myosin VI (ab126751)and MAOA (EPR7101; ab11096) from Abcam (Cam-bridge, MA).

Comparison of ERGResponsive Proteome toGene ExpressionDatasets

The CPDR 40 patient/80-gene-chip gene expressiondataset (GSE32448) was acquired on Affymetrix Hu-man Genome U133 Plus 2.0 arrays using RNA derivedfrom LCM isolated prostate tumors and matchingbenign tissue specimens. The ERG expression statusof the specimens, which were equally representedby moderately-differentiated tumors and poorly-

Fig. 1. Analysis of ERGResponsive Proteome (A) Outline of the strategy for analysis of ERG responsive proteome from ERG (þ), ERG(�), andbenign cells isolatedby LCM fromprostate cancer specimens and fromTMPRSS2-ERG positiveVCaPcells. (B) IHCofrepresentativetumor specimens used for LCMwith ERG (�) (a & b) and ERG (þ) (c & d) expression, stained with H & E and with ERGMAb. (C) Qualitycontrol of VCaP cell lysates used in proteomic analysis.Cell lysates were prepared from 50nMNTsiRNA (lane1) and ERG siRNA (lane 2)transfectedVCaPcells andERGproteinwas analyzedbyimmuno-blotanalysiswithERGMAb.

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differentiated tumors, were confirmed by IHC andFISH as 14 ERG (�) and 26 ERG (þ). Gene expressiondata of probesets that match ERPs from ERG (þ) andERG (�) tumors were fitted by linear regression modelfitting using the lmfit function in the Limma package[30] within the R program. Probesets with mostsignificant correlation or inverse correlation to ERGexpression were ranked by eBayes according to theBayes test statistics in the order differential expression.Statistical significance of a data set was computedusing two-tailed t-tests after excluding outliers definedby data-points that are greater than 2.5 standarddeviations. Genes and proteins were then comparedfor concordance in similar up- or down-regulation oftumor versus normal gene expression ratios to relativeMS ratio in ERG (þ) and ERG (�) tumors.

RESULTS

Isolation of ERGResponsive Proteins (ERPs)

The strategy to analyze the ERPs in ERG (þ) andERG (�) CaP cells is outlined in Figure 1A. Proteinswere isolated from pooled ERG (þ) or ERG (�) tumorcells and benign cells from whole-mounted sections offive prostatectomy specimens from patients matchedfor pathologic stage, age and race (Fig. 1B). Proteinswere also isolated from NT siRNA and ERG siRNAtreated VCaP cells (Fig. 1C). Trypsin digested proteinswere fractionated by using a CITP/CZE- based multi-dimensional separations. Peptide fragments weredetected with nano-electrospray ionization linear iontrap-tandem mass spectrometry (nano-ESI-MS/MS).The near complete silencing of ERG protein expressionwas confirmed by immunoblot analysis of ERG siRNAtreated VCaP cell lysates (Fig. 1C) using ERG-MAb(9FY), which detects ERG protein of 52 kDa in VCaPcells [24].

Analysis of theDifferential Expression of ERPsbetween LCMDerived ERG (þ) Versus ERG (�)

ProstateTumorCells

The analysis of ERG responsive proteins isolatedfrom LCM derived ERG (þ) and ERG (�) prostatetumor cells and from matched benign cells detected, at5% FDR threshold for total peptide identifications, acombined global proteome of 6171 proteins (Supple-mentary Fig. 1A, Supplementary Table IA), of whicha total of 4,684 were ERPs (Supplementary Fig. 1B,Supplementary Table IB). At stringent threshold fortotal peptide identifications of 1% FDR, a total of 1,196ERPs were detected, of which 518 and 500 wereunique to ERG (þ) and ERG (�) tumor cells, respec-tively (Fig. 2A).

Analysis of theDifferential Expressionof ERPsbetween ERGsiRNAVersusNTsiRNA

TransfectedVCaPCells

VCaP cells transfected with control NT siRNA oligosshowed a robust expression of ERG protein and ERGexpression was successfully depleted in the ERG siRNAtransfected VCaP cells (Fig. 1C). At 5% FDR thresholdfor total peptide identifications, a total number of11,416 proteins detected in NT siRNA and ERG siRNAtreated VCaP cells (Supplementary Fig. 1C, Supplemen-tary Table IC). At stringent threshold for total peptideidentifications of 1% FDR, a combined ERG responsiveglobal proteome of 2,190 proteins was detected. Thisproteome consisted of 562 proteins detected exclusivelyin control NT siRNA transfected VCaP cells, 59 proteinsexclusively in ERG silenced VCaP cells, and 1,569differentially expressed proteins common in both NTsiRNA and ERG siRNA transfected cells (Fig. 2B).

The technical reproducibility of the methodsapplied for proteomic analyses was verified by per-forming two independent runs through sequentialfractionations by CITP and CZE coupled with LC MS/MS using tryptic digests from VCaP cells that weretransfected with NT siRNA and expressing ERG. Thereproducibility of the methods used was confirmed bythe detection of 80% proteins that were commonto two independent runs (Supplementary Fig. 1D,Supplementary Table ID).

The comparative distribution of the ERG responsiveproteins from both NT siRNA and ERG siRNA treatedVCaP cells, according to Gene Ontology (GO) instan-ces of defined physiochemical characteristics, includ-ing molecular functions, biological processes, andcellular compartments, are shown in SupplementaryFigure 2. The overall results showed a broad similarityin the range and distribution of proteins from bothcells transfected with NT siRNA and with ERG siRNAin the different sub-categories of the GO instances,suggesting robust coverage in the isolation and detec-tion of cellular proteome by the methods employed.

Comparisonof ERGResponsive Proteome andERGResponsiveTranscriptome inVCaPCells

We have previously evaluated the transcriptome ofVCaP cells in response to ERG knock-down by siRNAusing GeneChip microarray analysis [13]. Normalizedgene expression data from 48 hr post transfection weredenoted as NT siRNA/ERG siRNA ratios. Comparisonof the present set of 2,190 ERPs from VCaP cells(Fig. 2B) against probe-sets representing 1,052 distinctgenes revealed 250 genes and proteins with concor-dance response to ERG expression. This represents23.8% (250/1,052) of the ERG responsive genes from

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the gene-chip experiments and 11.4% (250/2,190) ofthe ERG responsive proteins (Supplementary Fig. 3).

High StringencyAnalysis of ERGResponsiveProteomes of LCMIsolated TumorCells and

VCaPCells Showa Strong Concordancein Regulationby ERG

We compared the proteome of ERPs detected in theLCM isolated prostate tumor specimens (Fig. 2A) andin VCaP cells (Fig. 2B) to determine the extent ofcorrelation between these two sets of proteome. Thisevaluation showed an overlap of 489 ERPs, of which330 ERPs show concordance in their response to up- ordown-regulation of ERG protein levels (Fig. 3A). The330 proteins account for 15.1% (330/2,190) of ERPs inVCaP cells and 27.6% (330/1,196) of ERPs in LCMisolated ERG (þ) and ERG (�) tumors. The differentiallevels of detection in ERG (þ) versus ERG (�) tumorsand NT siRNA versus ERG siRNA VCaP cells areshown in Figure 3B.

Signal Transduction Pathways of LCMIsolatedTumorCells andVCaPCells

To evaluate the overall impact of down-streamtargets that respond to ERG expression, ERPs fromERG (þ) versus ERG (�) prostate tumor specimensand from VCaP cells were further analyzed usingGeneRanker and GePS. ERG responsive proteome net-works derived from 1,196 ERPs isolated from tumorsand 2,190 ERPs from VCaP cells, as revealed by GePSanalysis tool are shown in Figure 4A and B, respective-ly. Proteins that were detected at positive and negativeMS ratios for ERG (þ) versus ERG (�) tumor and NT

siRNA versus ERG siRNA in VCaP cells are shown asred and green nodes, respectively. By inference, the redand green nodes represent potential up-and down-regulation by ERG. These networks reflect the impactof ERG expression on protein biosynthesis, chaperoneand redox functions, protein trafficking, AR signaling,cell survival and apoptosis, DNA replication, cell cyclecontrol, cell polarity, and cell migration. For example,in both ERG (þ) versus ERG (�) tumors and in NTsiRNA versus ERG siRNA treated VCaP cells prolife-rating cell nuclear antigen (PCNA) is upregulated, incontrast to prostate specific antigen (PSA/KLK3),which is downregulated.

To highlight the conservation of function in prostatetumors and in the cell culture model, the 330 over-lapping ERPs with concordant response to ERGexpression in both tumor specimens and cell culturemodel were analyzed by using GeneRanker and GePSsoftware. The set of 330 overlapping ERPs show, aslisted according to P-value rankings in Table I andmapped in the resulting network in Figure 4C, anenrichment of pathways regulating cytoskeletal andactin reorganization, represented by the CDC42-RAC1, the P21 activated protein kinase (PAK) and theactin filaments Y-branching pathways.

ERGKnock-Down Induces the ExpressionofProstateDifferentiationMarkersAssociatedwithits Secretory Function and Impacts the Epidermal

Growth Factor Receptor (EGFR)Signaling Pathway

In our earlier publication, we have noted thatERG interferes with prostate epithelial differentiationby inhibiting a number of genes including KLK3,

Fig. 2. Global ERGResponsive Proteome detectedwith at least two uniquepeptide hits and at1% FDR from LCMisolated tumors (A) andfromNTsiRNAversusERG siRNAtransfectedVCaPcells (B).

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SLC45A3 (Prostein), C15ORF21 (Dresden prostate carci-noma 2 protein (D-PCa-2)), and MSMB (b-microsemino-protein/PSP94) [13]. In the current study, we detected aconsistent expression pattern of these proteins inrelation to ERG expression in both the LCM isolatedtumor cells and VCaP cells with the ERG knock-down.The protein expression and sub-cellular localization ofseveral ERG responsive downstream targets werevalidated in VCaP cells following ERG siRNA treat-ment. In response to ERG knock-down the expression

of cytoplasmic SLC45A3 and prostatic acid phospha-tase (PAP/ACPP) were dramatically upregulated (Fig.5A and B), but MSMB expression showed a moresubtle increase (Fig. 5C), consistent with the resultsof ERG responsive transcriptome. The upregulatedexpressions of Prostein, PSA, and PAP/ACPP inresponse to the ERG siRNA in VCaP cells were alsovalidated by immunoblot analysis (Fig. 5D).

Markers of cell growth and proliferations from theepidermal growth factor receptor (EGFR) signaling

Fig. 3. Overlapping ERGResponsive Proteome of LCM isolated tumors and VCaP cells. (A) Pie-chart showing 489 ERPs common to ERG(þ) vs. ERG (�) tumors and VCaP cells. (B) 330 ERPs concordantly regulated by ERG.Dark red and dark green colors represent proteinsunique to ERG (þ) tumors orNTsiRNA treated VCaPcells, and ERG (�) tumors or ERG siRNA transfectedVCaPcells, respectively.Lightershades of red and green represent proteins differentially upregulated or downregulated in these cells.The number of peptides detected foreachproteinis shownadjacent to eachgroupofERPs.

76 Tan et al.

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A B

C

TXN

MBP

ACACA

KLK2

CLUPCNA

YWHAZ

SDHA

FLNB

ENO1

FLNA KLK3

VIMGLUL

GLUD1BCAT2

ENG

PAK2

LMNA

NUMA1

VCL

GSN

CALD1

TAGLN

TPM1

ALB

COL1A2

HBB

AHSG

B2M

AKR1A1

ADIPOQ

ADH5

CPT1A

DPP7

CSTBCTSB

HLA-A C4A

TGM2

CRYZ

GSTK1

TXNRD1

HSPA1A

MPST

SOD2

TXNL1MIF

P4HB

CANX

RPL26NCL

UBTF

DNAJB1

HSPA8

Cell Polarityand Migration

Protein Traffickingand Secretion

AR Signaling

Cell Survival and Apoptosis

DNA Replication, Repair and Cell Cycle Control

Protein Biosynthesis, Processing, Chaperone

and Redox Functions

AR Signaling

PCK2PCK2

A2MA2M

ABCB7ABCB7

ABL1ABL1

ACACAACACA

MAOAMAOA

ACADSACADS

ACADVLACADVL

ACACBACACB

MAPTMAPT

USP9XUSP9X

RBM10RBM10

SMC1ASMC1A

MBNL1MBNL1

MBPMBP

MCM2MCM2

MCM5MCM5

MCM7MCM7

MDH2MDH2

ARID1AARID1A

RAB8ARAB8A

ADH5ADH5

ARID1BARID1B

MGST2MGST2

MIFMIF

AHCYAHCY

AHSGAHSG

MLLMLL

MLLT4MLLT4

ALBALB

ALCAMALCAM

MMEMME

ALDH2ALDH2

EEA1EEA1

GNPATGNPAT

CUL4ACUL4A

CUL1CUL1

MRE11AMRE11A

ABCC1ABCC1

CUL2CUL2

IKBKAPIKBKAP

APLP2APLP2

APOBAPOBAGPSAGPS

PIRPIR

APRTAPRT

KLK3KLK3

GADD45GIP1GADD45GIP1

ARAFARAF

ARF1ARF1

CASKCASK

ARF6ARF6

RHOGRHOG

ARHGAP1ARHGAP1

ARHGAP6ARHGAP6

RHOCRHOC

ASAH1ASAH1

MTHFRMTHFR

GSTK1GSTK1

BRD7BRD7

EIF3AEIF3A

EIF3BEIF3B

EIF3GEIF3GEIF3IEIF3I

ATP2B1ATP2B1

ATP2B4ATP2B4

EIF3JEIF3J

ATP2B2ATP2B2

ATP2B3ATP2B3

DGAT1DGAT1

EEDEED

ATRXATRX

MYO5AMYO5A

MYO5BMYO5B

MYO6MYO6

GBF1GBF1

B2MB2M

BADBAD

NCLNCL

NDUFA5NDUFA5

NDUFA6NDUFA6

SUCLG2SUCLG2

NDUFB4NDUFB4

CCNKCCNK

NCK1NCK1

LIN7ALIN7A

SEPT2SEPT2

GGHGGH

BIDBID

NEFHNEFH

NF1NF1

PELOPELO

BRAFBRAF

NFIANFIA

BSGBSG

NFIBNFIB

BTDBTD

NFIXNFIX

NAE1NAE1

SQSTM1SQSTM1

C1QBPC1QBP

ARHGEF7ARHGEF7

NFICNFIC

C4AC4A

NME1NME1

MBD2MBD2

NPYNPYPNPPNP

CALD1CALD1

NOL3NOL3

PTBP2PTBP2

CAMK2GCAMK2G

CANXCANX

CASTCAST

NUMA1NUMA1

BAZ1BBAZ1B

UBA3UBA3

UBE2MUBE2M

SPAG9SPAG9

CATCAT

CBSCBS

ASH2LASH2L

OPA1OPA1

CCNT1CCNT1

OXCT1OXCT1

CD9CD9

P4HBP4HB

AIFM1AIFM1

PEBP1PEBP1

SCARB1SCARB1

HGSHGS

CD47CD47

PAK1PAK1

PAK3PAK3

CD81CD81

PDCD5PDCD5

CDK1CDK1

PAK2PAK2

PCPC

PCBP2PCBP2

PCK2PCK2

PCNAPCNA

LRRFIP1LRRFIP1

LRRFIP2LRRFIP2

NOLC1NOLC1

CDK2CDK2

CENPBCENPB

CENPC1CENPC1

PEX1PEX1

PEX6PEX6

PEX14PEX14

CHGACHGA

PFKMPFKM

PFKPPFKP

TRIP12TRIP12

TRIP4TRIP4

PGK2PGK2

SLC9A3R2SLC9A3R2

TXNL1TXNL1PNPT1PNPT1

RAB9ARAB9A

PHKA1PHKA1

PHKA2PHKA2

ADIPOQADIPOQ

MID1IP1MID1IP1

DNAJC10DNAJC10

SLC9A3R1SLC9A3R1

CLUCLU

TPP1TPP1

PKLRPKLR

QPCTQPCT

LPXNLPXN

PLCB3PLCB3

PRDX5PRDX5

TXN2TXN2

ITM2BITM2B

MLPHMLPH

WDR77WDR77

COL1A2COL1A2

DPP7DPP7

ROCK2ROCK2

COL6A1COL6A1COL6A2COL6A2

COL6A3COL6A3

STRN3STRN3

EXOSC10EXOSC10

POLD1POLD1

EI24EI24

MYRIPMYRIP

ERO1LERO1L

CPECPE

CLIC4CLIC4

PON2PON2

CPT1ACPT1A

CPT2CPT2

CRATCRAT

CPDCPD

PPM1BPPM1B

CRMP1CRMP1

PPP1CAPPP1CA

CRKLCRKL

PPP1R2PPP1R2

PDIA4PDIA4

NCOR1NCOR1

PPP2R1APPP2R1A

PPP2R1BPPP2R1B

PPP2R4PPP2R4

CRYZCRYZ

PPP1R8PPP1R8

PPP6CPPP6C

PREPPREP

GCC2GCC2

MDC1MDC1

PRKAA1PRKAA1

CSNK2BCSNK2B

CST3CST3

ARAR

APPAPP

BAXBAX

CDC42CDC42

A2MA2M

ABCB7ABCB7

ABL1ABL1

ACACAACACA

MAOAMAOA

ACADSACADS

ACADVLACADVL

ACACBACACB

MAPTMAPT

USP9XUSP9X

RBM10RBM10

SMC1ASMC1A

MBNL1MBNL1

MBPMBP

MCM2MCM2

MCM5MCM5

MCM7MCM7

MDH2MDH2

ARID1AARID1A

ADH5ADH5

ARID1BARID1B

MGST2MGST2

MIFMIF

AHCYAHCY

AHSGAHSG

MLLMLL

MLLT4MLLT4

ALBALB

ALCAMALCAM

MMEMME

ALDH2ALDH2

EEA1EEA1

GNPATGNPAT

CUL4ACUL4A

CUL1CUL1

MRE11AMRE11A

ABCC1ABCC1

CUL2CUL2

IKBKAPIKBKAP

APLP2APLP2

APOBAPOBAGPSAGPS

APPAPP

PIRPIR

APRTAPRT

KLK3

GADD45GIP1GADD45GIP1

ARAFARAF

ARF1ARF1

CASKCASK

ARF6ARF6

RHOGRHOG

ARHGAP1ARHGAP1

ARHGAP6ARHGAP6

RHOCRHOC

ASAH1ASAH1

MTHFRMTHFR

GSTK1GSTK1

BRD7BRD7

EIF3AEIF3A

EIF3BEIF3B

EIF3GEIF3GEIF3IEIF3I

ATP2B1ATP2B1

ATP2B4ATP2B4

EIF3JEIF3J

ATP2B2ATP2B2

ATP2B3ATP2B3

DGAT1DGAT1

EEDEED

ATRXATRX

GBF1GBF1

B2MB2M

BADBAD

BAXBAX

NCLNCL

NDUFA5NDUFA5

NDUFA6NDUFA6

SUCLG2SUCLG2

NDUFB4NDUFB4

CCNKCCNK

NCK1NCK1

LIN7ALIN7A

SEPT2SEPT2

GGHGGH

BIDBID

NEFHNEFH

NF1NF1

PELOPELO

BRAFBRAF

NFIANFIA

BSGBSG

NFIBNFIB

BTDBTD

NFIXNFIX

NAE1NAE1

SQSTM1SQSTM1

C1QBPC1QBP

ARHGEF7ARHGEF7

NFICNFIC

C4AC4A

NME1NME1

MBD2MBD2

NPYNPYPNPPNP

CALD1CALD1

NOL3NOL3

PTBP2PTBP2

CAMK2GCAMK2G

CANXCANX

CASTCAST

NUMA1NUMA1

BAZ1BBAZ1B

UBA3UBA3

UBE2MUBE2M

SPAG9SPAG9

CATCAT

CBSCBS

ASH2LASH2L

OPA1OPA1

CCNT1CCNT1

OXCT1OXCT1

CD9CD9

P4HBP4HB

AIFM1AIFM1

PEBP1PEBP1

SCARB1SCARB1

HGSHGS

CD47CD47

PAK1PAK1

PAK3PAK3

CD81CD81

PDCD5PDCD5

CDK1CDK1

PAK2PAK2

PCPC

PCBP2PCBP2

PCK2PCK2

LRRFIP1LRRFIP1

LRRFIP2LRRFIP2

NOLC1NOLC1

CDK2CDK2

CENPBCENPB

CENPC1CENPC1

PEX1PEX1

PEX6PEX6

PEX14PEX14

CHGACHGA

PFKMPFKM

PFKPPFKP

TRIP12TRIP12

TRIP4TRIP4

PGK2PGK2

SLC9A3R2SLC9A3R2

TXNL1TXNL1PNPT1PNPT1

PHKA1PHKA1

PHKA2PHKA2

ADIPOQADIPOQ

MID1IP1MID1IP1

DNAJC10DNAJC10

SLC9A3R1SLC9A3R1

CLUCLU

TPP1TPP1

PKLRPKLR

QPCTQPCT

LPXNLPXN

PLCB3PLCB3

PRDX5PRDX5

TXN2TXN2

ITM2BITM2B

WDR77WDR77

COL1A2COL1A2

DPP7DPP7

ROCK2ROCK2

COL6A1COL6A1COL6A2COL6A2

COL6A3COL6A3

STRN3STRN3

EXOSC10EXOSC10

POLD1POLD1

EI24EI24

ERO1LERO1L

CPECPE

CLIC4CLIC4

PON2PON2

CPT1ACPT1A

CPT2CPT2

CRATCRAT

CPDCPD

PPM1BPPM1B

CRMP1CRMP1

PPP1CAPPP1CA

CRKLCRKL

PPP1R2PPP1R2

PDIA4PDIA4

NCOR1NCOR1

PPP2R1APPP2R1A

PPP2R1BPPP2R1B

PPP2R4PPP2R4

CRYZCRYZ

PPP1R8PPP1R8

PPP6CPPP6C

PREPPREP

MDC1MDC1

PRKAA1PRKAA1

CSNK2BCSNK2B

CST3CST3

MYO5AMYO5A MYO6MYO6

MLPHMLPH

MYRIPMYRIP

RAB9ARAB9A

GCC2GCC2

RAB8ARAB8A

MYO5BMYO5B

ARAR

CDC42CDC42

PCNAPCNA

EPHX1

MAP1A

MAP1B

FBL

ACPP MBP

ACTB

ACACA

AKR1A1

F2

F7

ADH1B

ADH5

FABP3

S100A10

S100A11

S100A6

S100A8

FBLN1

FBLN2

FBN1

FBN2

F13A1

FEN1

AHSG

ALAD

AKR1B1

FKBP1A

FKBP5

SDHA

MPO

AMBP

SEMG1

MRE11A

FLNA

FLNB

FBLN5

SRSF1

FMOD

SRSF3

SRSF5

FKBP4

NOP56

FOLH1

ANXA1

ANXA6

ANXA2

APOA1

APOA2

APOA4

APOD

SKP1

SLC2A1

SLC1A3

POSTN

GSTK1

MTOR

SERPINC1

EIF3A

EIF3B

SNRNP70

EIF3J

ATP2B1

G6PD

ATP2A2

XRCC6

CACYBP

SORD

SOD2

SPARC

MYH11

MYLK

SNRPA

GAPDH

SNRPE

PPP1R12A

AZU1

NACA

B2M

NCAM1

GC

SSBTROVE2

BGN

NEDD8

STX3

STXBP1

SQSTM1

GLO1

VAMP2

GLUL

C1QBP

SYN1

SERPING1

NID1

C3

ERP29

C4A

C4B

SYT1

TAGLN

CALD1

CALR

CANX

GPX1

NUMA1

GPX3

CAV1

GSTM2

GSTM3

GSTP1

COL18A1

TGM2

TGM3

THY1

CD9

TJP1

PEBP1

TKT

TIA1

P4HB

PRDX1

PAK1

CD59

PAK3

PAK2

CD81

CD44

HSD17B10

HADH

HBA1

CD63

HBD

CDH2

SERPIND1

AGRN

HBA2

TP53BP1

HTT

HDAC1

MTA2

CFH

HBB

HLA-B

HLA-C

HMGB1

HMGB2

TTN

PGD

TTR

VAMP3

HNRNPK

TXNRD1

TXNL1

SERPINA1

SLC9A3R1

ADIPOQ

CLU

HPUBTF

PIN1

HPX

PLA2G2A

HRG

PLEC

DNAJA1

HSPA1A

HSPA8

HSPA9

UTRN

HSPB1

VCL

CNP

COL1A1

COL1A2

GSTO1

COL4A1

PML

EZR

DNAJB1

COL5A2

PLP1

COL3A1

COMT

GDF15

VTN

HSP90AA1

BAG3

POR

CP

IDH1

XPO1

FASN

PC

CPT1AALB

PLG

SOD1

HSPA4

SNAP25

CDH1

CDC42

PCNA

FN1TXN

GSK3B

NCL

NPM1

Protein Traffickingand Secretion

Cell Growth

Cell Polarity and Migration

KLK3

VIM

PARP1

Protein Biosynthesis, Processing, Chaperone

and Redox Functions

DNA Replication, Repair and Ribosome Assembly

Fig. 4. Functional interaction networks of ERG Responsive Proteome. Literature based functional interaction networks of ERPs fromLCMisolatedprostate tumor cells (A),NTsiRNA/ERG siRNA treatedVCaPcells (B), and ERPs concordantlyregulatedby ERG fromA andB(C). Red and green nodes represent proteins unique for ERG (þ) and in ERG (�) cells, in the respective samples. Shades of red and greenrepresent upregulated and downregulated ERPs, respectively. In (C), the left- and right-half of the nodes show response to ERG in the LCMisolated prostate tumor cells and inVCaP cells, respectively.Nodes are shown as polygons if the function is known: kinases as right pointedpolygons; phosphatases, left pointed polygons; receptors, inverted trapezoids; transporters, trapezoid; and cofactors, stars. Nodesare linkedbydotted lines if association is by co-citation andby solid lines if association is by expert curation. ( ) indicates protein A activatesproteinB; (^),AmodulatesB;acircleandbar,AinhibitsB; a filledarrowhead,geneBhas abinding site forAononeof itpromoters.

The Prostate

pathway, such as the Src homology 2 domain contain-ing transforming protein 1 (SHC1) and mitogen-activated protein kinase (p44/ERK1) [31], show higherlevels of expression when ERG is expressed in the cell,but becomes down-regulated when ERG expression issilenced by siRNA (Fig. 6A and B). In contrast, theexpression of regulators of cell polarity and apicaljunction assembly, such as Rho-GTPase, RAC1 [32]and Rho guanine nucleotide exchange factor 7 (ARH-GEF7/p85 Cool1/bPix) [33] is elevated in response toERG knock-down (Fig. 6C,D), which confirms theinhibition of prostate epithelial differentiation by ERG.The downregulation of SHC1 and p44/ERK1 andupregulation of ARHGEF7 were also validated byimmunoblot assays (Fig. 6E).

Signal Transduction Pathways Signatures Def|nedby ERG (þ) and ERG (�) Tumors

The identification of ERPs that are exclusive to oroverexpressed in either ERG (þ) or ERG (�) tumorscould further reveal functional roles of ERG in prostatetumor initiation and progression. Proteins that arecorrelated with ERG expression could serve as surro-gate biomarkers and/or therapeutic targets in ERG(þ) tumors. In contrast, proteins that are overex-pressed in tumors lacking ERG could be used asbiomarkers that define a separate category of tumors.The relative abundance of a protein in ERG (þ) versusERG (�) tumors, or in ERG (þ) and ERG (�) tumorsversus benign tissues was determined based on itsrelative MS ratio. Since the same protein could bedetected in these separate analyses, we sorted theproteome data again to identify proteins that weredetected at higher ratios in ERG (þ) or in ERG (�)tumors, relative to benign tissues. Five hundred eightynine proteins were detected at higher ratios in ERG(þ) tumors, of which 204 were unique for ERG (þ)tumors. Conversely, 504 of the 781 proteins detected athigher ratios in ERG (�) tumors, were exclusively forERG (�) tumors (Fig. 7).

To further identify the individual profiles thatdefine the proteome from ERG (þ) and ERG (�)prostate tumors, ERPs from each set were analyzed forpathway enrichment and associated literature-basednetworks using GeneRanker and GePS. Analysis ofERPs from ERG (þ) tumors revealed, as listed accord-ing to P-value rankings in Table IIA, enrichment forpathways that regulate cell shape and motility (PAKpathway), remodel cytoskeletal structure (CDC42pathway), promote cell survival (AKT pathway), andenhance protein synthesis and cell growth (AKT-MTOR pathway). The nodes that connect these path-ways include MTOR and GSK3B (Fig. 8A). ERPs fromERG (þ) tumors which are localized to the plasmaT

ABLEI.

Signal

Tran

sduc

tion

Pathw

aysof3

30ERPsCon

cord

antlyReg

ulated

byERG.P

athw

aysar

eRan

kedby

P-Value

sth

atRep

resent

Enr

ichm

entof

Pro

teinsofa

Pathw

ayin

theSam

ple

No

Con

cordan

tlyregu

lated

ERPpa

thway

sPa

thway

IDP-value

#Gen

es(observe

d)

#Gen

es(exp

ected)

#Gen

es(total)

Listof

observed

gene

s

1Roleof

PI3K

subu

nitP8

5in

regu

lation

ofactin

orga

nization

andcell

migration

BIO

CARTA

:CDC42

RAC

pathway

6.86E�0

66

0.53

16ACTR2,

ARPC

2,ARPC

1B,P

AK1,

ARPC

1A,A

RPC

4

3P2

1(C

DKN1A

)activa

ted

kina

sePW

_PAK_H

OMO_S

APIENS

1.98E�0

59

1.54

68ST

MN1,

ARPC

1B,C

ALD1,

FLNA,

PAK2,

PAK1,

MBP,

VIM

,PAK3

4Y

bran

chingof

actin

filamen

tsBIO

CARTA

:ACTIN

Ypa

thway

1.16E�0

45

0.53

16ACTR2,

ARPC

2,ARPC

1B,A

RP-

C1A

,ARPC

45

Proteasomecomplex

BIO

CARTA

:PROTEASO

ME

pathway

1.59E�0

47

1.22

37PS

MB2,

PSMC1,

PSMD7,

PSMA5,

PSME2,

PSMA6,

PSMD3

6Celldivisioncycle42

PW_C

DC42_H

OMO_S

APIENS

3.28E�0

49

2.20

97DNM2,

GNA13,A

RHGAP1

,INF2

,RALA,P

AK2,

PAK1,

VIM

,PAK3

7ER

associated

deg

radation

(ERAD)

BIO

CARTA

:ERAD

pathway

2.95E�0

34

1.22

19MAN2B

1,MOGS,

CANX,G

ANAB

78 Tan et al.

The Prostate

membrane or released into the extracellular compart-ments include: P21 protein activated kinase 1 (PAK1);synaptotagmin1 (SYN1), a regulator of exocytosis;components of the clathrin-mediated endocytosis,epidermal growth factor receptor pathway substrate15 (EPS15), and dynamin 1 (DMN1); S100 calciumbinding protein A13 (S100A13) and glutathione perox-idase 3 (GPX3) (Fig. 8A).

Pathways that were found to be enriched for ERPsderived from ERG (�) tumors, as listed according to P-value rankings in Table IIB, function in the proteolyticdegradation of proteins (proteasome pathway), integ-rin-mediated cell migration (mammalian calpain path-way), MAP kinase pathway activation via G proteincoupled receptors (GPCR pathway), actin remodelingand cell migration (CDC42-RAC pathway), and theprevention of oxidative damage of proteins (redoxpathway). The nodes that connect these pathwaysfunction in the control of cell motility (CDC42 and

Calpain II (CAPN2)), proteolysis of extracellular ma-trix (plasminogen (PLG)), fatty acid metabolism (adi-ponectin (ADIPOQ)), and signal transduction at thecaveolae scaffolding of plasma membrane (caveolin I(Cav1)) (Fig. 8B).

AssociationofMyosinVI (MYO6) andMonoamineOxidase A (MAOA) to ERGmRNAand Protein

Expression

In order to identify biomarkers that are tightlyregulated by ERG, both at the level of protein expres-sion and gene expression, we compared ERPs that aredetected at higher ratios in ERG (þ) or in ERG (�)tumors against the CPDR tumor versus normal 80-GeneChip gene expression dataset (GSE32448) oftumor versus normal gene expression ratios from 14ERG (�) and 26 ERG (þ) cases [5]. ERPs were matchedagainst the probesets in this dataset and linear regres-

Fig. 5. ERG knock-down induces the expression of prostate differentiation markers associated with its secretory function.Validation ofthe upregulated expression prostate differentiationmarkers, (A) SLC45A3, (B) PAP/ACPP and (C) MSMB inVCaP cells upon ERGsiRNAbyimmunofluorescenceassayandbyimmunoblotanalyses (D).

ERGResponsive Proteome 79

The Prostate

sion model fitting was used to identify genes that mostclosely correlate to or inversely correlate to ERGexpression. By ranking the probesets according to theirdifferential expression, MYO6 was identified to corre-late most closely to the gene expression profile of ERGacross 40 patients (P-value¼ 3.92E�06) (Table IIIA,Fig. 9A, B, and D). The probability of differentialexpression of MYO6 in ERG (þ) and ERG (�) tumorsrelative to ERG was 0.92. There is a 3.6-fold differencebetween the means of gene expression ratios betweenERG (þ) versus ERG (�) cases for MYO6. On thecontrary, the expression of MAOA is noted have themost statistically significant inverse correlation to ERGexpression (P-value¼ 0.0134) and a high probabilityto be mutually exclusive to ERG (0.90). (Table IIIB,Fig. 9C and D).

Verif|cationof Proteomic andGene ExpressionData forMyosinVIandMAOA

The correlation of Myosin VI and inverse correla-tion of MAOA to ERG expression were further validat-

ed in independent ERG siRNA experiments usingVCaP cells. The silencing of ERG in comparison to thecontrol experiment resulted in a reduction of MyosinVI expression by half compared to a twofold increaseof MAOA expression (Fig. 9E), as measured byquantitative evaluation of the immunoblot intensities.Immuno-fluorescence assay confirmed the diminishedexpression of Myosin VI expression and upregulationof MAOA in response to ERG siRNA (Fig. 9F). Theresponse of MAOA expression to ERG protein levelscorroborates with the data from mass spectrometricanalysis, which show MAOA to be detected exclusive-ly in ERG (�) tumors and at approximately threefoldhigher MS ratios in ERG siRNA versus NT siRNAtreated VCaP cells (Fig. 3B).

In order to find out the status of Myosin VI andMAOA expression in other CaP cells that do notexpress ERG, we examined the expression of these twoproteins in a panel of CaP cell lines (Fig. 9G). Inaddition to VCaP cells, Myosin VI was shown to bestrongly expressed in LNCaP cells, which lacks ERGexpression. Interestingly, the expression of MAOAwas

Fig. 6. ERG knock-down inhibits genes regulating cell growth and activates genes regulating prostate epithelial differentiation. EGFRpathway proteins (A) SHC1, (B) p44/ERK1 are down-regulated by ERG siRNA. The silencing of ERG increased the expression of theRho-GTPaseRAC1 (C), andARHGEF7 (D).The down-regulationof SHC1andp44/ERK1andup-regulationofARHGEF7 are confirmedbyim-munoblotanalysis.

80 Tan et al.

The Prostate

shown to be associated with the level of AR expressionin AR positive LNCaP, C4-2B, and CWR22rv1 cells.We next examined the regulation of Myosin VI andMAOA by androgen using LNCaP cell and VCaP cellsgrown under starvation conditions and induced with0.1 and 1 nM of R1881 (Fig. 9H). Myosin VI expressionlevel did not show any detectable response to R1881 ineither LNCaP or VCaP cells. However, a twofoldincrease for MAOA expression in LNCaP cells wasobserved after 48 hr induction, both at 0.1 and at 1 nM,as measured from the immunoblot intensities (Fig. 9H,right panel). In contrast, MAOA expression is down-regulated concomitant with the R1881 induced ERG

expression in VCaP cells (Fig. 9H, left panel), whichconfirms the inverse correlation gene expression be-tween MAOA and ERG.

Meta-Analysis of theAssociation of GeneExpressionbetween ERG,MYO6, andMAOA in

IndependentDatasets

The association of gene expression between ERGwith MYO6 and MAOA was further evaluated in twoindependent CaP gene expression data sets withknown TMPRSS2-ERG gene fusion status: the studyinvolving the Swedish watchful waiting cohort

204 (8.8%)

504 (21.7%)

950(41.0%)

306(13.2%)32

(1.4%)

121 (5.2%)

203 (8.7%)

Benign

ERG+

204 (8.8%)

254(10.9%)32

(1.4%)

99 (4.3%)

ERG+

ERG-

Unique Proteins(2320)

204/589 of ERG (+) ERPs are unique to ERG (+) tumors

504/781 of ERG (-) ERPs are unique to ERG (-) tumors

504 (21.7%)

52(2.2%)

121 (5.2%)

104 (4.5%)

ERG-

Fig.7. IdentificationofERPsdetectedathigherratiosinERG(þ)or inERG(�) cells.Twohundredfourof589ERPsdetectedmoreabundant-ly in ERG (þ) relative to ERG (�) tumors orbenign tissues areunique to ERG (þ) tumors.Fivehundred fourof 781ERPsdetectedmore abun-dantlyinERG(�) relative toERG(þ) tumorsorbenign tissuesareunique toERG(�) tumors.

ERGResponsive Proteome 81

The Prostate

TABLEII.Signal

Tran

sduc

tion

Pathw

aysfor20

4ERPsUniqu

eforERG

(þ)Tu

mor

san

dfor50

4ERPsUniqu

eforERG

(�)Tu

mor

s

No

ERG

(þ)ERPpa

thway

sPa

thway

IDP-value

#Gen

es(observe

d)

#Gen

es(exp

ected)

#Gen

es(total)

Listof

observed

gene

s

A.S

igna

ltran

sduc

tion

pathway

sof

ERPs

unique

forERG

(þ)tumors

1P2

1(C

DKN1A

)activa

tedkina

sePW

_PAK_H

OMO_S

APIEN

S1.55

E�0

58

1.14

68PA

K2,

SYN1,

PAK1,

WASF2,

STMN1,

PAK4,

CALD1,

PAK3

2Celldivisioncycle42

NCI-N

ATURE:C

DC42

9.36

E�0

46

1.17

74GSK3B,P

AK2,

PAK1,

MTOR,E

PS8,

PAK4

3VAKTmurinethym

omaviral

oncoge

neho

molog

1PW

_AKT1_

HOMO_S

APIE

NS

2.03

E�0

311

3.99

238

PPM1G

,GSK3B,P

DCD4,

DNAJC5,

PAK1,

HTT,

PITPN

A,P

HLDB1,

MTO

R,

UTRN,S

PARC

4AKT-MTO

RBIO

CARTA:IG

F1-M

TOR

3.85

E�0

36

1.51

90SY

N1,

HTT,

ITPR1,

STMN1,

EPS8,

RAB11

FIP5

No

ERG

(�)ERPpa

thway

sPa

thway

IDP-value

#Gen

es(observe

d)

#Gen

es(exp

ected)

#Gen

es(total)

Listof

observed

gene

s

B.S

igna

ltran

sduc

tion

pathway

sof

ERPs

unique

forERG

(�)tumors

1Proteasomecomplex

BIO

CARTA:PROTEASO

M

EPA

THWAY

5.49

E�0

711

1.75

37PS

MA3,

PSME1,

PSMA4,

PSMB2,

PSMC1,

PSMD7,

PSMA5,

PSMC4,

PSME2,

PSMD6,

PSMB4

2MCALPA

INan

dfriend

sin

cell

motility

BIO

CARTA:M

CALPA

IN

PATHWAY

7.73

E�0

69

1.46

31PR

KAR2B,M

AP2

K2,

CAST,

PRKAR1A

,CAPN2,

ITGA1,

PRKAR2A

,MAP2K

1,MAPK3

3Sign

alingpa

thway

from

G-protein

families

BIO

CARTA:G

PCR

PATHWAY

2.15

E�0

58

1.28

27PR

KAR2B,G

NAI1,P

RKAR1A

,PP

P3C

A,A

SAH1,

PRKAR2A

,MAP2K

1,MAPK3

4Roleof

PI3K

subu

nitP8

5in

regu

lation

ofactinorga

nization

andcellmigration

BIO

CARTA:C

DC42

RAC

PATHWAY

5.47

E�0

56

0.76

16ACTR2,

ARPC2,

ARPC1B,C

DC42,

ARPC1A

,ARPC4

5Red

oxPW

_REDOX_H

OMO_S

AP

IENS

8.55

E�0

514

4.28

126

HSP

A4,

H6P

D,C

SNK2A

1,AKR1B1,

MPS

T,AKR1A

1,NAMPT,T

XNDC17,A

DIPOQ,

PSME2,

SOD2,

TXNRD1,

PRDX4,

GPX1

82 Tan et al.

The Prostate

(GSE16560) [34] and the Memorial Sloan-KetteringCancer Center (MSKCC) study using primary andmetastatic tumors (GSE21032) [35]. ERG fusion statuswas available in 272 of the 281 cases from the Swedishwatchful waiting cohort by FISH analysis. In theMSKCC cohort, ERG fusion status was confirmed for128 cases by array comparative genomic hybridization(aCGH) data. Patient data were clustered according toERG fusion status and the ranges for log 2 geneexpression for ERG fusion positive and ERG fusion

negative were plotted as box and whisker plots.MYO6 expression was significantly higher in ERG (þ)tumors versus ERG (�) tumors in both the Swedishwatchful waiting cohort (P-value¼ 1.71E�6) and theMSKCC cohort (P-value¼ 1.40E�7), confirming thatMYO6 expression is significantly correlated withTMPRSS2-ERG fusion or ERG over-expression (Sup-plementary Fig. 4). However, a similar evaluation ofMAOA expression did not show any inverse correla-tion as observed in this study.

A

B

membraneextracellular

cytosolmembrane

nucleuscytosol

CFH

EPHX1 HLA-A

HLA-DRB1

HMGB1

HMGB2

AKR1A1

F2

MECP2

F13A1

ADH1A ADH1B

TXNRD1

PARP1

ADIPOQ

PIN1

UBTF

AHSG

HRG

PPME1

MIF

PLG

DNAJA1

AKR1B1

PLP1

HSPA4

HSPA8

CNP

GSTO1

FKBP4

FKBP5

MRE11A

PML

HYOU1

FOLH1POR

CP APOA2

APOA4

XPO1

APOH

H6PD

CRKL

ARHGAP1

PPP2R4

CRYZ

CSE1L

GSTK1

FOLH1B

SERPINC1

EIF3B

MAPK3

EIF3A

MAP2K1

MAP2K2

CTSG

SOD2

CTSB

B2M

AZU1

GC

PTPN11

GLO1

SERPING1 C4B

NPM1

RALB

RAN

RALA

RARA

RBBP4

CAPN2

RBP1

CASTGPX1

CAV1

GSTM3

NAMPT

EIF4B

ANP32A

CD59

SERPIND1

CDC42

HDAC1

membrane

extracellular

cytosol

membrane

nucleus

cytosol

EPS8

EPS15

PAK4

NCAM1

PSMD4

PDCD4

RYR3 STX1B

ITPR1 CADM2

S100A10

S100A13

CADM1

YAP1NUFIP2

FEN1

SYN1

AK1

DNM1

SYT1

PRRC2A

BAG6

DNM2

FXR2BAG3

GPX3

CAST

UBQLN1

SLC1A3

OPA1SLC2A1

GSK3B

CRMP1PPM1G

MTOR

WASF2

PAK1

DNM3

PAK2 DNAJC5

PAK3

ATP1B2

HTT

Fig. 8. Functional interaction networks of ERPs unique for ERG (þ) and ERG (�) tumors. Red and green nodes represent proteins up-regulatedinERG(þ) tumors(A) andERG(�) tumors(B), in therespective samples.

ERGResponsive Proteome 83

The Prostate

DISCUSSION

The discovery of ERG overexpression in prostatetumors and the fusion of genes involving TMPRSS2promoter region with the ERG coding sequences inmore than 50% of CaP has opened avenues forexploration of biomarkers useful for the detection andthe stratification of CaP. The PSA assay used in theclinical screening of CaP is known to lack bothsensitivity and specificity [36]. Therefore there is aneed to identify biomarkers that have the potential tonot only accurately detect clinically relevant CaP inasymptomatic patients but also able to differentiateindolent from aggressive CaP. Analysis of ERG (þ)and ERG (�) tumor specimens is likely to provideadditional information about novel biomarkers forpotential clinical use.

We analyzed ERP in CaP cells with the aim tounderstand the function of ERG in the etiology of CaPand to identify biomarkers that are associated withERG (þ) or ERG (�) status of the prostate tumor. Anotable feature of this study is the typing of tumors forERG expression by IHC followed by targeted selectionby LCM to overcome the challenges imposed by thepresence of ERG (þ) and ERG (�) tumor foci in thesame prostate. Hence, a straightforward comparativeanalysis involving ERG (þ) and ERG (�) tumors andnormal cells is likely to show the potential differencesat the proteome level. Unlike other CaP-proteomicsstudies [18,20,37,38], the current study focused specifi-cally on the detection of proteins that are differentiallyexpressed in relation to ERG oncoprotein status. Toaddress this, we have utilized a unique proteomicsplatform based on CITP/CZE multidimensional sepa-ration coupled with nano-ESI-MS/MS, which involveminimal front end purification prior to MS analysis.The ability to reproducibly detect over 80% of thesame peptides in consecutive runs using aliquots ofthe same protein samples demonstrated the reliabilityof the techniques used (Supplementary Fig. 1D).

In addition to normal prostate epithelial cells andERG stratified tumor cells, we have also analyzed theERP of VCaP cell line. Such an analysis revealed adistinct pattern of up and downregulation of proteinsin response to ERG that was corroborated by concor-dance to mRNA expression reported by previous geneexpression analyses [13]. The detection of similarresponses in protein and mRNA expression in proteinmarkers of prostate luminal epithelial differentiationand secretory function to ERG siRNA knock-downfurther confirmed the reliability of the methods usedin this proteomic analysis. Examples of these proteins,which are shown in Figure 3 and in SupplementaryFigure 3, include KLK3 (PSA), SLC45A3, TMPRSS2,and prostate-specific membrane antigen-like protein

TABLEIII.

Pro

bes

etsforMYO

6an

dMAOARev

ealedGen

eExp

ressionPro

f|les

that

aremost

Signif|c

antlyCor

relate

d(A

),an

dInve

rselyCor

relate

d(B

)to

ERGExp

ression

Symbo

lProb

esets

Ran

kcoeff¼

2,ERG

(þ),

Lg2

scale

B-statistic

Prob

ability

gene

isdifferen

tially

expressed

t-test

Med

ERG

(þ)-M

edERG

(�)

Ave

ERG

(þ)-Ave

ERG

(�)

A.M

YO6is

mostsign

ifican

tlycorrelated

toERG

gene

expression

ERG

213541_s_at

112.19

13.21E�1

14.11

3.51

MYO6

203215_s_at

32.49

0.92

3.92E�0

61.76

1.66

CSD

A201160_s_at

21�2

.78

0.06

2.16

E�0

30.99

1.29

UTRN

225093_at

25�3

.02

0.05

7.45

E�0

30.81

1.03

Symbo

lProb

esets

Ran

kcoeff¼

1(ERG�)

Lg2

scale

B-Statistic

Prob

ability

gene

isdifferen

tially

expressed

t-test

Med

ERG

(þ)-M

edERG

(�)

Ave

ERG

(þ)-A

veERG

(�)

B.M

AOA

ismostsign

ifican

tlyinve

rselycorrelated

toERG

gene

expression

MAOA

204389_at

32.16

0.9

1.34E�0

2�0

.446

�0.372

IMMT

242361_at

45�2

.92

0.05

1.04E�0

2� 0

.344

�0.467

NEDD4L

241396_at

83�3

.65

0.03

1.41E�0

2�0

.338

�0.321

ERG

213541_s_at

2,049

�5.93

03.21E�1

14.109

3.510

84 Tan et al.

The Prostate

Fig. 9. Correlation of gene and protein expression between ERG with Myosin VI and MAOA. Box-plots showing the range of log 2tumor versus normal expression ratio from 40-patient gene expression dataset for ERG (213541_s_at) (A), MYO6 (203215_s_at) (B), andMAOA_204389_at (C) according to ERG expression status. The line across the box and the blue spot represent the respective medianand mean values, respectively. The relative expression of ERG, MYO6, and MAOA are normalized by row or Z-score (D, top) or shownas original values (D, bottom). The correlation of Myosin VI and MAOA expression to ERG expression is validated in ERG siRNAknock-down of VCaP cells and assayed by immunoblot analysis (E ) and immunofluorescence assay (F ). Expression of Myosin VI andMAOAwere compared in CaP cell lines (G). Induction of VCaP and LNCaP cells with 0.1 and 1nM of R1881 for 12, 24, and 48hr follow-ing growth in starvation conditions for 3 days.

ERGResponsive Proteome 85

The Prostate

(FOLH1B), semenogelin-2 (SEMG2) and transgelin(TAGLN).

ERPHighlightsOverlapping Pathways in ERG (þ)and ERG (�) ProstateTumors andin

VCaPCell Line

The characterization of ERG function through anal-ysis of interaction networks based on ERP datasetscaptured a representation of previously reported ERGtarget genes [11–13,16]. The overlapping signal trans-duction networks revealed for ERG (þ) and ERG (�)prostate tumors and NT siRNA and ERG siRNAtreated VCaP cells are consistent with the activation ofcell growth and cell proliferation and the inhibition ofprostate epithelial differentiation by ERG oncoprotein(Fig. 4A and B).

A more concise signal transduction network ofliterature-based interactions of the ERP was generatedfrom the 330 proteins concordantly regulated by ERG inboth tumors and in VCaP cells. The central nodes of thisnetwork (Fig. 4C), represented by vimentin (VIM) andalbumin (ALB) highlights the role ERG plays in regulat-ing modulators of glandular prostate epithelial differen-tiation and secretory function. Vimentin is anintermediate filament protein that is expressed earlyduring cell differentiation, promotes cell invasiveness, isexpressed by motile prostate cell lines and positivelycorrelates with poorly differentiated cancers and bonemetastases [39]. Albumin acts as a carrier protein forsteroids, fatty acids, and thyroid hormones and func-tions to stabilize extracellular fluid volume in bodyfluids including prostatic fluids [40]. This networkcomplements the signal transduction pathways fromGeneRanker analysis, which show an enrichment ofpathways regulating cytoskeletal and actin reorganiza-tion, cell migration, protein biosynthesis, and protea-some and ER-associated protein degradation pathways(Table I). These pathways underscore the associationbetween structure and function during prostate epitheli-al differentiation or epithelial-mesenchymal-transition(EMT). During these events, changes in cell shape andpolarity occur simultaneously with changes in theexpression of protein markers of prostate epithelialdifferentiation or EMT. For example, the dynamic res-ponse to ERG expression by Rho-GTPase CDC42-RAC1signaling related pathways that regulate actin filamentdynamics (Fig. 6C and D) is accompanied by equallyrobust alterations to AR function evident from the dys-regulation of PSA, SLC45A3, and PAP/ACPP (Fig. 5).

ERPs fromERG (þ) and ERG (�) Tumors RevealDistinct ProteinMarkers and Signature Pathways

The disparity in ERG fusion frequency amongdifferent ethnic populations points to yet undiscovered

genetic alterations that may contribute to the initiationand progression of CaP. We have defined the ERP byERG expression status to better understand the biolog-ical features that distinguish these two classes oftumors. The different profiles of the ERP from ERG (þ)and ERG (�) tumors, while highlighting the role ERGplays in regulating diverse cellular functions, mayreveal distinctive signatures that could help to stratifyERG (þ) from ERG (�) tumors and discover newtreatment options. The identification of 204 and 504ERPs unique for ERG (þ) and ERG (�) tumors,respectively, represent a distinct and informative sub-set of the ERPs (Fig. 7). The connection of PAK,CDC42, and AKT pathways enriched in ERG (þ)tumor derived ERPs by MTOR and GSK3B, under-score the impact that ERG overexpression may haveon the PI3K/AKT/mTOR, the PI3K/AKT/GSK3B orWnt signaling pathways (Table IIA, Fig. 8A). Thepathways enriched in ERPs from ERG (�) tumorsregulate functions that include CDC42-RAC and cal-pain modulated cell motility and proteasome andredox functions. These pathways were shown to beconnected by CDC42, CAV1, SOD2, ADIPOQ, andMAPK3 (Table IIB, Fig. 8B). These links reveal that inaddition to changes in cell differentiation and migra-tion, cell survival, and apoptosis, the absence of ERGin these tumors also affect changes in endocytosis andprotein trafficking, redox, and proteasome functions,as well as fatty acid metabolism.

The PI3K/AKT/mTOR and the MAP kinase path-ways have been implicated in CaP tumorigenesis anddevelopment of castrate resistant prostate cancer, andtargeting these pathways to treat CaP using smallmolecule inhibitors is an active area of investigation[41,42]. Several of the ERG (þ) and ERG (�) specificERPs, which are identified to be secreted in bodyfluids or found localized to the plasma membranewarrant more detailed studies to evaluate their poten-tial as diagnostic protein biomarkers or as targets fortreatment for CaP.

Correlation ofMyosinVIandMonoamineOxidaseAwith ERGGene and Protein Expression

The combined analysis of proteomic and genomicdata for proteins positively correlated and inverselycorrelated to ERG expression identified Myosin VIand MAOA as potential protein and gene expressionbiomarkers for ERG (þ) and ERG (�) tumors.Myosin VI is one of the unconventional myosins,actin-based molecular motors involved in intracellu-lar vesicle and organelle transport. Although MyosinVI has previously been reported to be over-expressed in CaP [43], this is the first report of acorrelation with ERG expression in CaP. The locali-

86 Tan et al.

The Prostate

zation of Myosin VI on endosomes and the trans-Golgi network, suggest a function in regulatingprotein secretion [44,45]. Myosin VI has been impli-cated in autophagy by promoting autophagosomematuration and driving fusion with lysosomes [46].The correlation of Myosin VI gene and protein to theexpression of ERG suggests possible transcriptionalmodulation of Myosin VI by ERG.

MAOA is a mitochondrial enzyme expressed inthe brain and peripheral tissues that degrades biogen-ic amines including neurotransmitters serotonin andnorepinephrine by oxidative deamination, resulting inthe production of hydrogen peroxide [47]. In normalprostate glands MAOA is absent or found at verylow levels in the luminal secretory epithelial but iselevated in the basal epithelia [48]. Increased MAOAexpression is also found to be associated with poorlydifferentiated high grade CaP [49] while a rarepolymorphism of the MAOA promoter that conferslow expression was associated with reduced CaP risk[50]. In this study, both gene and protein expressionof MAOA are found to be expressed at higher ratiosin ERG (�) tumors compared to ERG (þ) tumors.MAOA was detected at almost threefold higher MSratios in ERG silenced VCaP cells compared to NTsiRNA control (Fig. 3B). In the context of prostateepithelium, MAOA is regulated by androgensthrough promoter-upstream glucocorticoid/androgenresponse elements [51]. We showed that althoughMAOA is upregulated by R1881 induction in ERG(�) LNCaP cells, the expression of ERG in VCaP cellsappear to interfere with this activation. The inversecorrelation of MAOA with ERG suggests that MAOAmay define a separate and distinct category of ERG(�) but androgen sensitive tumors. Although theprimary function of MAOA is the oxidative deamina-tion of monoamine neurotransmitters, whether theoverexpression of MAOA in prostate epithelium leadsto the oxidative deamination of prostatic polyaminessuch as spermine or spermidine, and the release ofreactive oxygen species that contribute to tumorigene-sis remains to be shown.

A comparison of ERG, MYO6, and MAOA expres-sion to independent gene expression datasets fromSboner et al. [34] and Taylor et al. [35] confirmed thesignificant correlation of MYO6 but not the inversecorrelation of MAOAwith ERG. This could be attribut-ed the differences in procedures used for mRNAsampling from tissues or in the sensitivity of micro-array platforms. Unlike the two larger datasets, whichused only tumor mRNA, the 80-GeneChip datasetanalyzed in this study used mRNA from both tumorand normal cells. The preparation of the mRNA fromLCM isolated cells further reduces heterogeneity orcontamination of non-tumor cells.

Potential Treatmentof CaPBasedontheStratif|cation for ERG,MyosinVI, andMAOA

Expression

The prevalence TMPRSS2-ERG fusion and its func-tion as a driver mutation in the initiation and progres-sion of CaP present a promising therapeutic target.Transcription factors such as ERG were considered“undruggable,” mainly due to its inaccessibility. Nev-ertheless inhibition of ERG function exemplified bythe use of small molecule inhibitors and TMPRSS2-ERG fusion junction specific siRNAs have been suc-cessfully carried out with varying degree of success[52]. The inhibition of the DNA dependent interactionof ERG with poly(ADP-ribose) polymerase (PARP)with PARP inhibitors [53] has advanced rapidly due tothe availability of pharmacological inhibitors. Thedevelopment of combination assays using tripleimmunostaining cocktails and/or nucleic acid detec-tion panels could help categorize tumors according totheir expression of ERG, Myosin VI or MAOA. Paralleladvances in the development of specific small mole-cule inhibitors, used either alone or in a combinatorialapproach with other drugs, could be applied tosynergistically inhibit ERG (þ) or ERG (�) tumors.The small molecule inhibitor, 2,4,6-triiodophenol hasbeen recently shown to reduce the number of MyosinVI-dependent vesicle fusion events at the plasmamembrane during constitutive secretion [54] and couldbe used to inhibit the formation of autophagosomes[46]. Since the genetic alterations that define ERG (�)tumors are not well understood, the identification ofERPs overexpressed in ERG (�) tumors or areexpressed in inverse correlation to ERG, such asMAOA, could help to uncover the mechanismsresponsible for the initiation and progression of ERG(�) CaP. The therapeutic potential of MAOA inhibitorssuch as clorgyline, which induces differentiation inprimary cultures of normal basal epithelial cells andhigh-grade CaP, is being actively investigated [55].Further developments in this direction could translateto the development of clinical treatments for CaPpatients based on the ERG expression status of thetumors.

ACKNOWLEDGMENTS

This work is supported by a research grant fromNational Cancer Institute R01CA162383 (S.S.) andCPDR Program Fund HU0001-10-2-0002 (D.G.M.). Theauthors would like to thank Alagarsamy Srinivasanfor his help with extensive editing of the manuscriptand Stephen Doyle for his assistance with the figures.The opinions and assertions contained herein repre-sent the personal views of the authors and are not tobe construed as official or as representing the views of

ERGResponsive Proteome 87

The Prostate

the Department of the Army, the Department ofDefense, or the United States Government.

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The Prostate