tmprss2-erg controls luminal epithelial … · within one tumor is essential to combat...

Translational Cancer Mechanisms and Therapy

TMPRSS2-ERG Controls Luminal EpithelialLineage andAntiandrogen Sensitivity inPTEN andTP53-Mutated Prostate CancerAlexandra M. Blee1,2, Yundong He1, Yinhui Yang1,3, Zhenqing Ye4, Yuqian Yan1,Yunqian Pan1, Tao Ma4, Joseph Dugdale1, Emily Kuehn1, Manish Kohli5,Rafael Jimenez6, Yu Chen7,Wanhai Xu3, Liguo Wang4, and Haojie Huang1,8,9

Abstract

Purpose: Deletions or mutations in PTEN and TP53 tumorsuppressor genes have been linked to lineage plasticity intherapy-resistant prostate cancer. Fusion-driven overexpres-sion of the oncogenic transcription factor ERG is observed inapproximately 50%of all prostate cancers,many ofwhich alsoharbor PTEN and TP53 alterations. However, the role of ERGin lineage plasticity of PTEN/TP53–altered tumors is unclear.Understanding the collective effect of multiple mutationswithin one tumor is essential to combat plasticity-driventherapy resistance.

Experimental Design:We generated a Pten-negative/Trp53-mutated/ERG-overexpressing mouse model of prostate cancerand integrated RNA-sequencing with ERG chromatin immu-noprecipitation-sequencing (ChIP-seq) to identify pathwaysregulated by ERG in the context of Pten/Trp53 alteration. Weinvestigated ERG-dependent sensitivity to the antiandrogenenzalutamide and cyclin-dependent kinase 4 and 6 (CDK4/6)inhibitor palbociclib in human prostate cancer cell lines,

xenografts, and allografted mouse tumors. Trends were eval-uated in TCGA, SU2C, and Beltran 2016 published patientcohorts and a human tissue microarray.

Results: Transgenic ERG expression in mice blocked Pten/Trp53 alteration–induced decrease of AR expression anddownstream luminal epithelial genes. ERGdirectly suppressedexpression of cell cycle–related genes, which induced RBhypophosphorylation and repressed E2F1-mediated expres-sion of mesenchymal lineage regulators, thereby restrictingadenocarcinomaplasticity andmaintaining antiandrogen sen-sitivity. In ERG-negative tumors, CDK4/6 inhibition delayedtumor growth.

Conclusions: Our studies identify a previously undefinedfunction of ERG to restrict lineage plasticity and maintainantiandrogen sensitivity in PTEN/TP53–altered prostate can-cer. Our findings suggest ERG fusion as a biomarker to guidetreatment of PTEN/TP53-altered, RB1-intact prostate cancer.Clin Cancer Res; 24(18); 4551–65. �2018 AACR.

IntroductionCastration-resistant prostate cancers respond to current anti-

androgen therapies with variable levels of success (1), in part, dueto extensive genetic heterogeneity (2–4). While mechanisms ofandrogen receptor (AR) pathway restoration and compensationare well documented, adenocarcinoma cell lineage plasticity andreprogramming to AR independence represents an additionalresistance mechanism (5). Interestingly, the incidence of AR-independent tumor progression after castration and antiandrogentreatment has increased since the advent of enzalutamide andabiraterone use in the clinic, highlighting that prostate cancerlineage plasticity is an increasingly important barrier to overcome(6). Recent studies have identified a few key molecular eventsinvolved in AR-independent tumor progression, such as RB1/PTEN/TP53 loss, MYCN/AURKA amplification, and altered epi-genetic regulators including EZH2 (7). However, the molecularbasis underlying prostate cancer lineage plasticity and antiandro-gen resistance remains poorly understooddue to extensive patienttumor heterogeneity and model limitations.

PTEN loss frequently overlaps with TP53 mutation or loss indrug-resistant, morphologically distinct, reprogrammed tumors(8–11). A significant proportion of both primary and castration-resistant tumors with PTEN/TP53 alteration also have AR-depen-dent, TMPRSS2 fusion–driven overexpression of the ETS family

1Department of Biochemistry and Molecular Biology, Mayo Clinic College ofMedicine, Rochester, Minnesota. 2Biochemistry and Molecular Biology GraduateProgram, Mayo Clinic Graduate School of Biomedical Sciences, Rochester,Minnesota. 3Department of Urology, the Fourth Hospital of Harbin MedicalUniversity, Harbin, Heilongjiang, China. 4Division of Biomedical Statistics andInformatics, Department of Health Sciences Research, Mayo Clinic College ofMedicine, Rochester, Minnesota. 5Department of Oncology, Mayo Clinic Collegeof Medicine, Rochester, Minnesota. 6Department of Laboratory Medicine andPathology, Mayo Clinic College of Medicine, Rochester, Minnesota. 7HumanOncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center,New York, New York. 8Department of Urology, Mayo Clinic College of Medicine,Rochester, Minnesota. 9Mayo Clinic Cancer Center, Mayo Clinic College ofMedicine, Rochester, Minnesota.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

A.M. Blee, Y. He, and Y. Yang contributed equally to this article.

CorrespondingAuthors:Haojie Huang, Department of Biochemistry andMolec-ular Biology, Mayo Clinic College of Medicine, 200 First Street Southwest,Rochester, MN 55905. Phone: 507-293-1712; Fax: 507-293-3071; E-mail:[email protected]; Liguo Wang, Department of Health SciencesResearch, Mayo Clinic College of Medicine, Rochester, MN 55905. E-mail:[email protected]; and Wanhai Xu, Department of Urology, the FourthHospital of HarbinMedical University, Harbin, Heilongjiang 150001, China. E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-18-0653

�2018 American Association for Cancer Research.

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transcription factor ERG (2–4). ERG alone has been shown torepress a neural gene expression signature (12) as well as partiallyrescue the AR pathway under PTEN loss conditions (13), but themechanistic role of ERG in the clinically relevant context of bothPTEN/TP53 alteration remains uncharacterized.

To address these gaps in the field, we generated a mousemodel of prostate cancer that encompasses Pten deletion, Trp53mutation, and ERG overexpression. Notably, we revealed anovel function of ERG to repress expression of a subset of cellcycle–related genes and block RB hyperphosphorylation inPten/Trp53-altered, Rb1-intact tumors. As a result, ERG-positive,Pten/Trp53-altered tumors had minimal expression of E2F1downstream targets involved in a mesenchymal cell lineageswitch. We extended these findings to both preclinical xenograftand allograft models of tumor progression and demonstratedthat ERG overexpression maintained AR positivity and sensi-tivity to enzalutamide. In stark contrast, ERG-negative, Pten/Trp53–altered tumors were resistant to enzalutamide treatmentand instead developed a reliance on the RB/E2F1 pathway,which was effectively targeted with a CDK4/6 inhibitor, palbo-ciclib. This study emphasizes the importance of evaluating theindividual genetic profile of tumors when designing therapeuticstrategies, with particular emphasis on ERG fusion, RB1, andPTEN/TP53 status.

Materials and MethodsCell lines, cell culture, and drug treatment

LNCaP, HEK293T, VCaP, and PC-3 cells were obtained fromthe ATCC. C4-2 cells were purchased from Uro Corporation.LNCaP-RF cells were described previously (14). HEK293T cellswere maintained in DMEM supplemented with 10% FBS. VCaPcellsweremaintained inDMEMsupplementedwith13%FBS.C4-2, LNCaP, LNCaP-RF, and PC-3 cells were maintained inRPMI1640 medium supplemented with 10% FBS. All cell lineswere authenticated (karyotyping, mutations in p53 and ERGfusions, and AR, PTEN, p53, and ERG protein expression) and

used within 6 months of thawing. No mycoplasma contamina-tion was detected in these cell lines by testing with the LookoutMycoplasma PCR Detection Kit (Sigma-Aldrich). Charcoal-stripped serum (CSS) was purchased from Thermo Fisher Scien-tific-Gibco (#12676029). Enzalutamide was kindly provided byMedivation. LNCaP-RF cells were treated with 10 mmol/L ofenzalutamide for 72 hours unless otherwise noted. Palbociclib(PD-0332991) was obtained from ApexBio. LNCaP-RF cells weretreatedwith 1mmol/L of palbociclib for 72hours unless otherwisenoted. For combination treatment, LNCaP-RF cells were treatedwith 10 mmol/L enzalutamide and 1 mmol/L palbociclib for72 hours.

Cell transfection and lentivirus transductionFor lentiviral shRNA or stable plasmid expression, HEK293T

cells were transiently transfected with pTsin-HA-ERG FL, pTsin-HA-ERG-T1-E4, pTsin-EV, pLKO-shNT, pLKO-shRB, pLKO-shERG, pLKO-shPTEN, or pLKO-shE2F1 as indicated using Lipo-fectamine 2000 (Thermo Fisher Scientific) following manufac-turer's instructions. Virus-containing supernatant was collected48 hours posttransfection and indicated cells were infected withvirus-containing supernatant and 8 mg/mL polybrene. Selectionwas performed with 1.5 mg/mL puromycin. Sequences of gene-specific shRNAs are listed in Supplementary Table S1. TwoshRNAs per gene were tested.

Coimmunoprecipitation and Western blottingCoimmunoprecipitation and subsequent Western blotting

was performed as described previously (15). The followingantibodies were used: anti-ERG (ab92513, Abcam; CM421C,Biocare Medical), anti-PTEN (CST9559L, Cell Signaling Tech-nology), anti-p53 (sc126, Santa Cruz Biotechnology), anti-AR(sc816, Santa Cruz Biotechnology), anti-NKX3.1 (NB100-1828,Novus Biologicals), anti-RB (554136, BD Biosciences), anti-pRB S795 (CST9301S, Cell Signaling Technology), anti-SKP2(32-3300, Life Technologies), anti-CCND1 (sc718, Santa CruzBiotechnology), anti-CDK1 (sc54, Santa Cruz Biotechnology),anti-TWIST (sc6269, Santa Cruz Biotechnology), anti-CDH1(610181, BD Biosciences), anti-VIM (sc73258, Santa CruzBiotechnology), anti-ERK2 (sc1647, Santa Cruz Biotechnology),anti-CDK2 (sc6248, Santa Cruz Biotechnology), anti-E2F1(sc193, Santa Cruz Biotechnology), anti-pAKT S473 (CST4060L,Cell Signaling Technology), and anti-AKT (CST9272, Cell Signal-ing Technology).

qRT-PCRqRT-PCR was performed as described previously (15). All

quantifications were normalized to the level of endogenousGAPDH gene. Primers used are listed in Supplementary Table S2.

Chromatin immunoprecipitation and qPCRChromatin immunoprecipitation (ChIP) was performed as

described previously (16). DNA was pulled down with indicatedprimary antibodies (anti-ERG, ab92513; anti-E2F1, sc193) ornonspecific IgG. Primers to amplify DNA by real-time qPCR arelisted in Supplementary Table S3.

Cell proliferation assaysLNCaP-RF, VCaP, or PC-3 cells were seeded in 96-well plates

(�3,000 cells/wells) and treated as indicated. Cells were fixed atindicated timepoints (day 0–5) and cell growth was measured

Translational Relevance

Prostate cancer resistance to androgen deprivation and AR-targeted therapies remains a pressing clinical obstacle, partlyexplained by lineage plasticity and transition to AR-indepen-dent tumor types in response to these therapies. A compre-hensive understanding of genetic prostate tumor subtypes andthe unique response of each mutational subtype to AR-tar-geted therapies is necessary to develop new, subtype-specifictherapeutic strategies that overcome therapy-induced lineageplasticity. Our results demonstrate that E-twenty-six transfor-mation specific (ETS)-related gene (ERG) prevents PTEN- andtumor protein 53 (TP53)-negative tumor cell lineage plasticityand antiandrogen resistance by blocking E2F1-mediatedexpression of lineage switch genes. These findings also revealthe efficacy of targeting retinoblastoma (RB)/E2F1 activitywith palbociclib in ERG-negative, PTEN/TP53-altered tumors.This study redefines the role of ERG in a specific tumor subtypeand may guide evaluation of the status of concomitant ERGfusion, PTEN/TP53 alteration, and RB1 when selecting ther-apeutic strategies.

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using sulfohodamine B (SRB) assay (n ¼ 5) as described previ-ously (17).

Hematoxylin and eosin stainingFour micron–thick sections were cut from formalin-fixed par-

affin-embedded (FFPE) tumor samples from indicated samples.Xylene washes were used to deparaffinize the tissue, followed bygraded ethanolwashes to rehydrate tissue. Tissuewas stainedwithhematoxylin, washed, and counterstained with 1% eosin. Stainedtissue was dehydrated with graded ethanol washes and a finalxylene wash before mounting and sealing with coverslips.

IHC and immunofluorescent cytochemistryFour micron–thick sections were cut from FFPE tumor samples

from indicated mice, xenografts, or human tissue microarrays.Tissue was deparaffinized with xylene and rehydrated throughgraded ethanol washes. Antigen retrieval and immunostainingwas performed as described previously (18, 19). Antibodies forIHC and IFC include: anti-AR (ab108341), anti-ERG (ab92513),anti-CD31 (ab28364), anti-CKAE1/3 (ab27988), anti-RB pS795(ab85607), anti-Ki67 (ab15580), anti-pAKT S473 (CST4060L),anti-CK8/18 (ab531826), anti-CK5 (ab52635), anti-Vimentin(CST5741S). Ki67 and pRB S795 staining ofmouse and xenografttissueswasquantifiedby counting thenumber of positive cells outof 100 cells in five random fields of view at 400� per mouse.Staining intensity and percentage for ERG and AR staining ofhuman tissue microarrays were graded using a set of criteria.Intensity was graded 0–3: 0 no staining, 1 low staining, 2mediumstaining, and 3 strong staining. A staining index score for eachtissue biopsy was obtained by multiplying the staining intensityand percentage values, and used for Pearson product–momentcorrelation analysis.

Gene set enrichment analysisGene set enrichment analysis (GSEA) was performed with

a preranked list of the target genes identified by integrated analy-sis of RNA-seq and ChIP-seq data against curated datasets includ-ing HALLMARK_E2F_TARGETS, HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION, CHARAFE_BREAST_CANCER_LUMINAL_VS_MESENCHYMAL_DOWN, CHARAFE_BREAST_CANCER_LUMINAL_VS_MESENCHYMAL_UP from the BroadInstitute (20).

Samples from patients with prostate cancerThe advanced prostate cancer dataset was generated from

patients undergoing standard-of-care clinical biopsies at MayoClinic (Rochester, MN). A tissuemicroarray was constructed fromthe FFPE samples of metastatic prostate cancer, identified after asearch of pathologic and clinical databases of archival tissues. TheMayo Clinic institutional review board approved the experimen-tal protocols for retrieving pathology blocks/slides and for acces-sing electronic medical records. The human tissue microarraycontained 157 cores (16 0.6mm and 141 1.0mm cores) resultingfrom 53 samples (20 bone metastases and 33 nonbone metasta-ses) from 51 patients. Cores in which greater than 50% of thetissue was lost during IHC were excluded from analysis.

Meta-analysis of publicly available datasetsERG fusion and genetic alterations of PTEN and TP53 for TCGA

(n ¼ 333) and SU2C (n ¼ 150) cohorts were downloaded fromcBioPortal (http://www.cbioportal.org/; refs. 21, 22). ERG fusion

status for Beltran cohort (metastatic tumor specimens¼ 114) wasdownloaded from Supplementary Table S5 of the originalstudy (23). Only ERG fusions with RNA-seq or NanoStringevidence were included into the analysis. ORs were calculated incBioPortal where OR > 1 indicates cooccurrence and OR < 1indicates mutual exclusivity, followed by two-tailed Fisher exacttests to determine significance of the cooccurrence or mutualexclusivity, as described previously (24).

RNA-seq and data analysisTotal RNA was isolated from mouse prostates by homogeni-

zation of frozen tissue and purified using the RNeasy PlusMini Kit(Qiagen). Two-hundred micrograms of high-quality total RNAwas used to generate the RNA sequencing library. cDNA synthesis,end-repair, A-base addition, and ligation of the Illumina indexedadapters were performed according to the TruSeq RNA SamplePrep Kit v2 (Illumina). The concentration and size distribution ofthe completed libraries was determined using an Agilent Bioa-nalyzer DNA 1000 chip and Qubit fluorometry (Invitrogen).Paired-end libraries were sequenced on an Illumina HiSeq4000 following Illumina's standard protocol using the IlluminacBot and HiSeq 3000/4000 PE Cluster Kit. Samples weresequenced in biological triplicates and each sample yielded60–90 million paired-end reads (2 � 50 nucleotide read length).Base calling was performed using Illumina's RTA software (ver-sion 2.5.2). Paired-end RNA-seq reads were aligned to the mousereference genome (GRCm38/mm10) using RNA-seq spliced readmapper Tophat2 (v2.0.6; ref. 25). Pre- and postalignment qualitycontrols, gene-level raw read count, and normalized read count (i.e., FPKM) were performed using RSeQC package (v2.3.6) withNCBI mouse RefSeq gene model (26). Differential gene expres-sion analyses were conducted using edgeR (version 3.6.8) and thebuilt-in "TMM" (trimmed mean of M values) normalizationmethod were used (27). Differentially expressed genes weredetermined on the basis of the false discovery rate (FDR) thresh-old 0.01.

ChIP-seq data analysisERG, H3K4me1, and H3K4me3 ChIP-seq data in mouse pros-

tate tissue was downloaded from NCBI Gene Expression Omni-bus (GEO) with accession number GSE47119 (13). To be com-patible with our RNA-seq analysis results, raw reads were rea-ligned to the mouse reference genome (GRCm38/mm10) usingbowtie2 (version 2.2.9; ref. 28).MACS2 (version 2.0.10)was usedto identify peaks with input samples used as background and aP value cutoff 1E�5 (macs2 callpeak –bdg–SPMR -f BAM; ref. 29).ChIP-seq tag intensity tracks (bedGraph files) were generated byMACS2, and then were converted into bigWig files using UCSC"wigToBigWig" tool. The association of peaks to target genes wasperformed by Genomic Regions Enrichment of Annotations Tool(GREAT; ref. 30). ERG ChIP-seq data in VCaP cells (GSE14092;ref. 31), E2F1ChIP-seqdata inPC-3 cells (GSE77448; ref. 32), andH3K4me3ChIP-seq data in LNCaP cells (GSE43791; ref. 33)weredownloaded from GEO. ChIP-seq analysis procedure was thesame as described above after mapping reads to the humanreference genome (GRCh37/hg19).

Generation of Pten/Trp53/ERG-altered mouse model andgenotyping

All animal studies were approved by the Mayo Clinic Institu-tional Animal Care and Use Committee (IACUC). All mice were

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housed in standard conditions with a 12-hour light/12-hour darkcycle and access to food andwater ad libitum. The indicated groupsof mice were generated by crossing the following mice: Probasin(Pb)-driven Cre4 recombinase transgenic mice, acquired from theNational Cancer Institute (NCI) Mouse Repository and originallygenerated in the laboratory of Dr. Pradip Roy-Burnam at Univer-sity of Southern California (Los Angeles, CA; ref. 34); transgenicERG mice purchased from the Jackson Laboratory (010929),originally generated in the laboratory of Dr. Valeri Vasioukhinat Fred Hutchinson Cancer Research Center (Seattle, WA; ref. 35);Pten loxp/loxp conditional mice, acquired from Jackson Labora-tory (004597) and originally generated in the laboratory of Dr.Hong Wu at University of California (Los Angeles, CA; ref. 36);Trp53 loxp/loxp conditional mice, acquired from the NCI MouseRepository and originally generated in the laboratory of Dr. TylerJacks at Massachusetts Institute of Technology (Cambridge, MA;ref. 37); and Trp53 loxp-STOP-loxp-R172H conditional mice,acquired from theNCIMouseRepository andoriginally generatedin the laboratory of Dr. Tyler Jacks at Massachusetts Institute ofTechnology (Cambridge, MA; ref. 37). PCR genotyping primersare listed in Supplementary Table S4.

Generation and treatment of prostate cancer cell line xenograftsand mouse-derived allografts

All animal studies were approved by the Mayo Clinic IACUC.Allmicewere housed in standard conditionswith a 12-hour light/12-hour dark cycle and access to food and water ad libitum. NOD-SCID IL2 receptor g-null (NSG)micewere generated in house andat 6 weeks of age, were randomly divided into different experi-mental treatment groups as indicated (six mice per group). Forprostate cancer cell line xenografts, 5 � 106 LNCaP-RF cells perinjectionwere suspended in 0.1mLof 50%PBS and 50%CorningMatrigelMatrix and implanted by subcutaneous injection into theleftflankof eachNSGmouse (one implantation permouse) usinga 16 gauge needle. LNCaP-RF cells were tested and ensured to bemycoplasma-free prior to injection using the Lookout Mycoplas-ma PCR Detection Kit purchased from Sigma-Aldrich, and werestably expressing either pTsin-EV or pTsin-HA-ERG-T1-E4. Formouse-derived allografts, three ARlow/KRTlow DMT prostatetumors and three ARhigh/KRThigh TMT prostate tumors werehomogenized and 200mL of tissue perNSGmousewas implantedby subcutaneous injection into the left flank of each NSG mouse(one implantation per mouse) using a 16 gauge needle. Once theimplanted cells grew to reach a size of 100 mm3 measuredexternally with calipers (approximately 4–5 weeks posttransplan-tation), drug treatment began.Micewere treatedwith vehicle (100mL sodium lactate), enzalutamide (30 mg/kg/day), palbociclib(100 mg/kg/day), or combination by oral gavage, once daily fivedays per week for three weeks. Mouse weight and tumor size wasmeasured every three days by measuring tumor length (L) andwidth (W) using a caliper, and tumor volume (TV) was calculatedwith the following formula: TV ¼ (L � W2)/2. Posttreatment,xenografted tissue was harvested and collected for subsequentstudy.

Data availabilityThe datasets generated and/or analyzed during the current

study are available in the following repositories. The CancerGenome Atlas (TCGA) and Stand Up To Cancer (SU2C) datasetsanalyzed in Fig. 1 and Supplementary Fig. S1 were accessed fromcBioPortal (http://www.cbioportal.org/; refs. 21, 22). The Beltran

cohort analyzed in Fig. 5 was downloaded from SupplementaryTable S5 of the original study (23). The ERG, H3K4me1, andH3K4me3ChIP-seq datasets analyzed in Fig. 3were accessed fromthe NCBI Gene Expression Omnibus (GEO) with accession num-ber GSE47119 (13), the ERG and H3K4me3 ChIP-seq datasetsanalyzed in Fig. 4 were accessed from the NCBI GEO with theaccession numbers GSE14092 (31) and GSE43791 (33), andthe E2F1 and H3K4me3 ChIP-seq datasets analyzed in Supple-mentary Fig. S5 were accessed from the NCBI GEO with theaccession numbers GSE77448 (32) and GSE43791 (https://www.ncbi.nlm.nih.gov/geo/; ref. 33). The HALLMARK_E2F,HALLMARK_EPITHELIAL_TO_MESENCHYMAL, and CHARA-FE_BREAST_CANCER datasets for GSEA in Fig. 3 were accessedfrom the Broad Institute (http://software.broadinstitute.org/gsea/index.jsp; ref. 20). The RNA-seq data generated from mouseprostate tissues in Fig. 3 is accessible from the NCBI GEO withthe accession number GSE103871.

Statistical analysisAll data are shown as mean values � SE for experiments

performed with at least three replicates. Differences between twogroupswere analyzed using paired Student t tests unless otherwisenoted. P values < 0.05 were considered significant.

ResultsGeneration and characterization of a clinically relevant Pten/Trp53/ERG triple-mutant mouse model

By mining the whole-exome sequencing data from TCGApatients with primary prostate cancer (PRPC; N ¼ 333; ref. 3),we revealed significant cooccurrence (P¼1.11�10�6,OR¼3.01;95% CI¼ 1.89–4.84) of PTEN/TP53 deletions or mutations withERG gene fusions, one of the most frequent genetic alterations inprostate cancer (ref. 38; Fig. 1A and B). In contrast, while a similartrend was observed in the SU2C metastatic castration-resistantprostate cancer (mCRPC) patients (N ¼ 150; ref. 4), the correla-tion (P ¼ 0.043, OR ¼ 2.04; 95% confidence interval ¼ 0.98–4.33) wasmuch weaker than that in TCGA PRPC patients (Fig. 1Aand B). Given that AR is more commonly expressed in PRPCcomparedwithmCRPC, especially neuroendocrineCRPC (NEPC;refs. 23, 39), these data suggest that ERG fusions are prone tocooperate with PTEN and TP53 gene alterations in the pathogen-esis of AR-positive prostate cancer. It is important to note that inthe mCRPC SU2C cohort, only 3.6% of samples displayed neu-roendocrine (ARlow/KRTlow) features (4), which may partlyexplain the apparent under-representation of AR loss samples inthe SU2C dataset (Fig. 1A). To genetically test this hypothesis invivo, we generated four cohorts of mice recapitulating the geneticalterations most frequently occurring in human prostate cancers(such as R175H in TP53; refs. 3, 4, 40; Supplementary Fig. S1): (i)"wild-type" (Cre-negative littermates); (ii) ERG transgenic alone,with Met33 N-terminally truncated ERG driven by the AR-depen-dent probasin (Pb) promoter (hereafter termed Pb-ERG); (iii)prostate-specific Pten deletion and Trp53 deletion and mutation(Ptenpc�/�;Trp53pcR172H/�, hereafter termed double mutant orDMT) where Trp53 R172H is the mouse equivalent to humanTP53R175H; and (iv)Ptenpc�/�;Trp53pcR172H/�;Pb-ERG (hereaftertermed triple mutant or TMT; Supplementary Fig. S2A). Wegenerated these four groups of mice by using Pb-driven Crerecombinase (Pb-Cre4; ref. 34), Pb-ERG (35), Ptenloxp/loxp (36),and Trp53loxp-stop-loxp-R172H/loxp (37) as breeders. For comparison,

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Figure 1.

ERG tempers PTEN/TP53 alteration–induced loss of ARhigh luminal epithelial cells. A, Oncoprint image with percentage of ERG, PTEN, TP53, and AR geneticalterations in 333 primary prostate cancer patient samples (top, TCGA cohort; ref. 3) and 150 advanced mCRPC patient samples (bottom, SU2C cohort; ref. 4). B,Contingency tables used by Fisher exact test (two-tailed) to examine association between ERG fusion and PTEN/TP53 alterations in primary TCGA (left) andmCRPCSU2C (right) cohorts. C, Histologic characterization of mouse prostate tissue from 16–20 weeks of age. Wild-type n ¼ 8, Pb-ERG n ¼ 9, DMT n ¼ 10, TMTn ¼ 12. Top, hematoxylin and eosin (H&E) staining. Subsequent rows, IHC for AR, ERG, CD31, Pan-KRT, KRT8/18, KRT5, and Vimentin. CD31 as an endothelial cellmarker to distinguish between endogenous endothelial versus transgenic ERG. Asterisk in Vimentin IHC tissue indicates a stromal compartment that is distinct fromthe Vimentinlow adenocarcinoma.






we also generated prostate-specific Pten deletion (Ptenpc�/�), Ptenand Trp53 double deletion (Ptenpc�/�;Trp53pc�/�) (hereaftertermed double knockout or DKO) mice, as well as prostate-specific Pten and Trp53 double deletion plus ERG transgenic(Ptenpc�/�;Trp53pc�/�;Pb-ERG) mice (Supplementary Fig. S2A–S2C). Pten/Trp53DKOmice have been shown to develop plastic,dedifferentiated tumors (8–10, 41) and served as controls forcomparison purposes with the Trp53-mutant lines, which repre-sent an unstudied portion of patients with PTEN deletion/TP53mutation.

At the age of 8–10 weeks, 100% of both Ptenpc�/�;Trp53pc�/�

(DKO) and Ptenpc�/�;Trp53pcR172H/� (DMT) mice developedwell-differentiated adenocarcinomas with high expression of ARproteins (ARhigh; Supplementary Fig. S2B). In contrast, AR expres-sion was dramatically reduced (ARlow) in prostate tumors inapproximately 90% of DKO and DMT mice at the age of 16–20 weeks (Fig. 1C; Supplementary Fig. S2B). Consistent with thereduced AR expression, the level of pan-keratin (pan-KRT) intumors, used as an indicator of epithelial cells as opposed tomesenchymal cells, was also markedly reduced (KRTlow) in bothDKO and DMT mice at the age of 16–20 weeks compared withmice 8–10 weeks younger (Supplementary Fig. S2B). In addition,DMT tumor cells from mice at the age of 16–20 weeks were alsonegative for both luminal epithelial cell marker KRT8/18 andbasal epithelial cellmarker KRT5, but positive for vimentin (VIM),a mesenchymal cell marker (Fig. 1C). These results suggest thattumors in DKO and DMT mice at the age of 16–20 weekstransitioned to minimal luminal epithelial phenotypes and wereless differentiated compared with tumors inmice at younger ages,as indicated by the comparativelyweak but detectable pan-keratinand AR levels. These data provide support to the previous obser-vation that loss of Pten and Trp53 induces lineage plasticity inmouse prostate cancer (8–10, 41).

In striking contrast, at the same age (16–20 weeks), approxi-mately 50% of Ptenpc�/�;Trp53pcR172H/�;Pb-ERG (TMT) micedeveloped well-differentiated ARhigh/KRThigh adenocarcinomaswhile the other 50%developed ARlow/KRTlow tumors reminiscentof those in DMTmice (Fig. 1C). Notably, ARlow/KRTlow tumors inTMT mice lacked transgenic ERG expression in the majority oftumor cells, but as expected, endogenous ERG was highlyexpressed in CD31-positive endothelial cells of blood vessels(Fig. 1C). Importantly, lack of epithelial expression of transgenicERG correlated with decreased expression of AR proteins in thesePten/Trp53–altered tumors (Fig. 1C; Supplementary Fig. S2C).This observation is supported by the previous report that ERGknockdown decreases the AR-positive luminal cell population inTMPRSS2-ERG–expressing VCaP prostate cancer cells (12).Together, these findings reveal that PTEN/TP53 alteration inducesloss of the ARhigh luminal epithelial cell lineage in prostate cancerand this phenomenon is disrupted in the presence of ERGexpression.

Compared with Pten wild-type prostate tissues ("wild-type"and Pb-ERG genotypes), Pten-null PIN lesions in Ptenpc�/�mice ortumors in DMT and TMT mice had increased, but comparablelevels of phosphorylated AKT (pAKT S473; Fig. 2A and B; Sup-plementary Fig. S3A), reinforcing the concept that PTEN loss is akey driver of initial tumorigenesis in these models (42–44).Intriguingly, plasma membrane expression of pAKT S473 wasdetected in the luminal epithelial cells of ARhigh/KRThigh tumors inTMTmice,whereas no typical plasmamembrane staining of pAKTS473 was detected in ARlow/KRTlow tumor cells in DMT and TMT

mice (Fig. 2A; Supplementary Fig. S3A and S3B), a phenomenonreminiscent of prostate-specific Pten/Rb1 double KO tumors (8).At present, the exact cause-and-effect of altered cellular localiza-tion of phosphorylated AKT remains to be elucidated. Previousstudy has shown that in the presence of PTEN loss, ERG partiallyrescues AR function (13). However, further analyses showed thatARhigh/KRThigh tumors in TMT mice had lower levels of phos-phorylated RB (pRB S795) and lower expression of a subset of cellcycle–promoting genes compared with ARlow/KRTlow tumors inDMT and TMT mice (Fig. 2C and D, see Fig. 3 below). Further-more, expression of cell lineage regulators commonly associatedwith epithelial-to-mesenchymal transition (EMT) and neuroen-docrine cell lineage was also much lower in ARhigh/KRThigh TMTtumors than that in ARlow/KRTlow tumors (Fig. 1C; see Fig. 3below). These data argue that ERG-induced preservation of thelate-stage ARhigh/KRThigh phenotype was not solely mediated byrestored AR activity in the PTEN loss context, but may requireadditional drivers.

Loss of RB function has been implicated in development ofplastic, antiandrogen-resistant prostate tumors in Pten and Trp53-deficient mice (8, 10). In agreement with functional loss of RB asreflected by increased RB phosphorylation (Fig. 2C and D), cellproliferation as indicated by Ki67 staining was much higher inARlow/KRTlow dedifferentiated tumors in both DMT and TMTmice compared with that in ARhigh/KRThigh tumors in TMT mice(Fig. 2E and F). It is worth noting that proliferation in ARhigh/KRThigh tumors in TMT mice was still much higher than that inmalignant and nonmalignant prostate tissues in Pten KO aloneand ERG transgenic alone mice, respectively (Fig. 2E and F),reinforcing the concept that ERG is an oncogenic protein thatpromotes prostate tumorigenesis by cooperating with otherlesions. Nevertheless, these data suggest that ERG may regulatethe cell cycle and subsequently, RB activity.

ERG downregulates a subset of cell cycle–promoting genes inPten/Trp53–altered mouse prostate tumors

To define the molecular mechanisms by which ERG mod-ulates prostate cancer cell lineage plasticity, we performed RNAsequencing (RNA-seq) analysis in three ARlow/KRTlow tumorsfrom DMT mice and three ARhigh/KRThigh tumors in TMT mice.We selected DMT ARlow/KRTlow tissues rather than TMT ARlow/KRTlow tissues for this analysis to ensure no possible contam-ination from any tumor cells that may have low levels of ERGexpression. Although we did not observe strongly positive ERG-expressing tumor cells by IHC in the TMT ARlow/KRTlow tissues(Fig. 1C), the presence of the Pb-ERG transgene in these micewould not allow us to eliminate that possibility. RNA-seq datafor one DMT tumor was excluded from further analysis due toits poor correlation with the other two biological replicates(Supplementary Fig. S4A). Differential gene expression analy-ses revealed 1,281 and 1,598 genes that were significantlydown- and upregulated by ERG, respectively, in ARhigh/KRThigh

TMT prostate tumors in comparison with ARlow/KRTlow DMTtumors (Fig. 3A; Supplementary Fig. S4B). After integratingRNA-seq data with ERG ChIP-coupled sequencing (ChIP-seq)data obtained from prostate tumors in Rosa26 TMPRSS2-ERGmice (13), we found 76% (972 of 1,281) of ERG-downregu-lated genes and 82% (1,314 out of 1,598) of ERG-upregulatedgenes contained ERG ChIP-seq peaks in their promoter and/orenhancer regions (Fig. 3A), suggesting that they are putativeERG target genes.

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Gene ontology (GO) analysis of the 972 ERG-downregulatedgenes demonstrated a significant enrichment of genes thatregulate the cell cycle (Fig. 3B), in agreement with our findingthat ARhigh/KRThigh tumors in TMT mice display decreased RBphosphorylation and cell proliferation in comparison withARlow/KRTlow tumors in DMT and TMT mice (Fig. 2C–F). GSEArevealed that ERG-downregulated genes significantly overlapwith hallmark E2F targets and EMT genes (Fig. 3C). Additionalcomparison demonstrated that ERG-downregulated genes alsocorrelated with luminal epithelial-to-mesenchymal changes inother cancer types. For examples, these genes were also signif-icantly overlapped with genes downregulated in the luminal

breast cancer cell type as compared with the mesenchymal-likebreast cancer cell type, while ERG-upregulated genes weresignificantly overlapped with genes upregulated in the luminalbreast cancer cell type (ref. 45; Fig. 3C). Further analysis ofRNA-seq profiles between ARlow/KRTlow tumors in DMT miceand ARhigh/KRThigh tumors in TMT mice revealed that ERGexpression resulted in drastic upregulation of AR pathway genes(e.g., Ar and Nkx3.1) and luminal epithelial lineage genes (e.g.,Cdh1 and Krt8), and robust downregulation of cell-cycle genes(e.g., Ccnd1 and Cdk1) and nonluminal epithelial (mesenchy-mal and neuroendocrine) lineage-regulatory genes (ref. 11; e.g.,Twist1 and Sox11; Fig. 3D).

Figure 2.

ERG prevents Pten/Trp53 alteration–induced proliferation and loss of membrane-localized phosphorylated AKT in mouse prostate tumors. A, IHC for pAKT S473 inmouse prostate tissues from 16–20 weeks of age. Wild-type n ¼ 8, Pb-ERG n ¼ 9, DMT n ¼ 10, TMT n ¼ 12, Ptenpc�/� n ¼ 8. B, Protein levels of pAKT S473,total AKT, and AR in mouse prostate tissues at 16–20 weeks of age. Both blots for each protein of interest were exposed and developed on the same piece of film.ERK2 as a loading control. Band intensity was quantified and normalized to ERK2 for each lane. Asterisk ¼ outlier samples with significantly low levels oftotal protein.C, IHC for pRB S795 inmouse prostate tissues from 16–20weeks of age as described inA.D,Quantification of pRB S795 staining as shown inC.E, IHC forKi67 in mouse prostate tissues from 16–20 weeks of age as described in A. F, Quantification of Ki67 IHC as shown in E.






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ERG-downregulated genes are exemplified in Fig. 3E and werefurther confirmed by reverse transcription–coupled quantitativePCR (qRT-PCR; Fig. 3F). qRT-PCR analysis of key cell-cycle andEMT genes and Western blot analysis of AR proteins furtherconfirmed that ARlow/KRTlow tumors in DMT and TMT miceshared similar molecular traits (Fig. 2B; Supplementary Fig.S4C). It is important to note that although TMT ARlow/KRTlow

tissues were not analyzed by RNA-seq, the trends in gene expres-sion observed in DMT ARlow/KRTlow tissues were seeminglyconserved in the TMT ARlow/KRTlow tissues (Fig. 2B; Supplemen-tary Fig. S4C). ERG ChIP-seq data clearly showed ERG-bindingpeaks in thepromoter regionof cell-cycle genes such asCcnd1, andCdk1, but not cell lineage–regulatory genes such as Twist1 andSox11 (Fig. 3E), suggesting that cell-cycle–related genes are likelydirect targets of ERG while Twist1 and Sox11 are not. These datasuggest that in the context of Pten/Trp53 alteration, ERG tran-scriptionally downregulates a subset of key cell-cycle–promotinggenes and maintains AR signaling.

VCaP cells harbor intact PTEN, one allele loss of TP53 and again-of-function mutation R248W, which is a hotspot mutationin CRPC (40) (Supplementary Fig. S1). ERG is also overexpressedin this cell line due to TMPRSS2-ERG fusion (TMPRSS2 exon 1fused with ERG exon 4 or termed T1-E4 ERG). Importantly,knockdown of ERG in the presence of PTEN depletion increasedCCND1, CDK1, and SKP2 protein levels in VCaP cells (Supple-mentary Fig. S5A). Thus, these data provide futher support to thehypothesis and the validation of ERG regulation of a few repre-sentative gene targets defined by the integrated RNA-seq andChIP-seq analyses. Our data that ERG bound to the promoter ofCcnd1 and Cdk1 genes and repressed their expression suggestsERG is a potent upstream regulator of RB hypophosphorylationand activation. This notion is further supported by a recent reportthat in spite of the androgen-stimulating effect of RB hyperpho-sphorylation in TMPRSS2-ERG–negative LNCaP cells, RB remainshypophosphorylated in TMPRSS2-ERG–positive VCaP cells evenafter androgen stimulation (46).

Human prostate cancer cell lines recapitulate ERG-mediatedrepression of the cell cycle through the RB pathway

To delineate the relationship between ERG expression andPTEN/TP53 alteration in tumor cell proliferation, cellular iden-tity, and antiandrogen resistance, we surveyed human prostatecancer cell lines including VCaP, C4-2, LNCaP, LNCaP-RF, andPC-3 (Fig. 4A). Among the cell lines surveyed, VCaP cells had thehighest level of AR protein, hypophosphorylated RB, minimalexpression of cell-cycle–promoting proteins CCND1, CDK1, andSKP2, and low levels of mesenchymal-related proteins TWIST1

and VIM (Fig. 4A), similar to ARhigh/KRThigh tumors in TMTmice(Fig. 1C). Consistent with ARlow/KRTlow DMT or DKO tumors,PTEN- and ERG-negative CRPC cell lines PC-3 and LNCaP-RF,which lack or express very low levels of functional p53, respec-tively, displayed little to no expression of AR, but increasedhyperphosphorylated RB and augmented expression of cell-cycle–driven proteins and mesenchymal-specific proteins (Fig.4A). Overexpression of full-length or fusion (T1-E4) ERG inLNCaP-RF and PC-3 cells partially reversed these trends in adose-dependent manner (Fig. 4B; Supplementary Fig. S5B) anddecreased cell proliferation (Fig. 4C), as detected in ERG-positive,Pten/Trp53-mutated mouse prostate tumors (Fig. 2E and F).Conversely, concomitant knockdown of endogenousTMPRSS2-ERG and PTEN inTP53-mutatedVCaP cells,mimickingthe situation in ARlow/KRTlow tumors in DMT or TMT mice,resulted in increased expression of cell-cycle–related proteins,hyperphosphorylation of RB, upregulation of nonepithelial cellmarkers TWIST1 andVIM, anddecreased expression of AR and theepithelial cell markers CDH1 and NKX3.1 (Supplementary Fig.S5A and S5C).

Previous study has indicated that hypophosphorylated RB canbe recruited by AR to repress cell-cycle genes (46). Coimmuno-precipitation assay in VCaP cells demonstrated that similar toprevious study (31), ERG interacted with AR (Fig. 4D). However,no interaction was detected between ERG and RB, and similarresults were obtained in PC-3 cells stably expressing T1-E4 ERG(Fig. 4D), excluding the possibility that ERG may recruit RB torepress cell-cycle genes in a manner similar to AR (46). However,because key cell-cycle regulators such as CCND1 and CDK1 wereidentified as transcriptionally repressed target genes of ERG, it ispossible that ERG causes a reduction in RB phosphorylation andcell-cycle progression by directly downregulating cell-cycle genes.This hypothesis is consistent with decreased expression of a subsetof cell-cycle genes inARhigh/KRThigh adenocarcinomas in TMTmice(Fig. 3) and in human LNCaP-RF and PC-3 cells stably expressingERG (Fig. 4B). Moreover, ERG-mediated upregulation of AR anddownregulation of EMT genes were reversed by depletion of RB inERG-expressing LNCaP-RF cells (Fig. 4E). Similar effects wereobserved in PC-3 cells (Fig. 4E). These results along with the ERGChIP-seq data (Fig. 3) suggest that ERG functions as an upstreamactivator of RB by specifically binding to the promoter and repres-sing expression of a subset of cell-cycle–driving genes.

E2F1 activates expression of EMT-promoting factors in ERG-negative, PTEN/TP53–altered tumor cells

It has been reported recently that E2F1 promotes prostatecancer cell metastasis and enhanced mesenchymal-like

Figure 3.ERG expression downregulates key cell-cycle–driving genes and maintains both AR pathway and epithelial gene expression in mouse prostate tumors. A, Venndiagram indicating overlap between up- or downregulated genes in DMT ARlow/KRTlow (n ¼ 2) versus TMT ARhigh/KRThigh (n ¼ 3) tumors and ERG target genesidentified by ChIP-sequencing (13). Fisher exact test (assuming human genome code for 27,000 genes, estimated from RefSeq) for ERG ChIP-seq versusdownregulated genes: P < 0.001; for ERG ChIP-seq versus upregulated genes: P < 9.088e�23. B,Gene Ontology analysis of 972 ERG target genes downregulated inTMT ARhigh/KRThigh tumors, ranked by P value. C, GSEA for all 2,595 up- or downregulated ERG target genes (mouse gene names converted to humanhomologs using NCBI Homology Map). D, Heatmap showing differentially expressed genes between two DMT ARlow/KRTlow and three TMT ARhigh/KRThigh tumors,highlighting a subset of genes involved in cell cycle, AR pathway, and epithelial-to-mesenchymal transition (EMT). E, RNA-seq and ChIP-seq track viewsfrom UCSC Genome Browser for two ERG predicted target genes (Ccnd1, Cdk1) with ERG-binding peaks and two predicted passenger genes (Sox11, Twist1) withoutERG-binding peaks. Peaks underlined with black bars and boxed with a dashed red line indicate significant ERG ChIP-seq peaks with P ¼ 1e�5 or lower asdetermined by MACS. ERG ChIP-seq input tracks shown as a control for true ERG peaks. H3K4me3 peaks shown to indicate gene promoter regions. H3K4me1 peaksshown to indicate gene enhancer regions. F, qRT-PCR of n ¼ 2 DMT ARlow/KRTlow and n ¼ 3 TMT ARhigh/KRThigh tumors for Ccnd1, Cdk1, Twist1, and Sox11.Relative to Gapdh.






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phenotypes (increasedmigration and invasion) by binding to thepromoter and upregulating expression of the RHAMM gene(HMMR; ref. 47). By analyzing E2F1 ChIP-seq data obtained inPC-3 cells (32), we found robust binding of E2F1 proteins in theloci of ERG-suppressed mesenchymal lineage-driving genesincluding SNAI1, TGFB2, TWIST1, TWIST2, and HMMR (Sup-plementary Fig. S5D). This observation was further confirmed byChIP-qPCR (Supplementary Fig. S5E). Most importantly, theeffect of ERG and PTEN double knockdown on expression ofEMT-promoting genes and epithelial and mesenchymal cell mar-kers in VCaP cells was abrogated by concomitant knockdown ofE2F1 by two independent shRNAs (Supplementary Fig. S5C).These data suggest that E2F1 mediates repression of downstreammesenchymal lineage genes in the context of ERGþ/PTEN�/p53null/mutant cells. In further support of the transcriptome resultsinDMT and TMTmouse tumors, ectopic expression of T1-E4 ERGin PC-3 cells reduced expression of CCND1, CDK1, TWIST1, andSOX11, as well as other key cell cycle, EMT, and neuroendocrine-related genes (Fig. 4F; Supplementary Fig. S6A–S6C). ERG ChIP-seq in VCaP cells and ChIP-qPCR in PC-3 cells stably expressingT1-E4 ERG confirmed cell-cycle genes such as CCND1 and CDK1as direct ERG targets in human prostate cancer cells, but E2F1ChIP-seq andChIP-qPCR inPC-3 cells demonstrated TWIST1 andother cell lineage–regulatory factors asdownstreamgene targets ofE2F1 (Fig. 4G and H; Supplementary Fig. S5D and S5E; Supple-mentary Fig. S6D and S6E). It should be noted that there was anobserved ERG ChIP-seq peak at the TGFB2 locus, although thisbinding could not be validated by ChIP-qPCR (Fig. 4G and H).Thus, our data cannot completely rule out the possibility that ERGmay also potentially regulate expression of this locus. Together,these data suggest that ERG directly binds to and regulatesexpression of a subset of cell-cycle genes in human PTEN/TP53–mutated prostate cancer cells, which in turn leads to RBhypophosphorylation and inhibition of E2F1-mediated tran-scription of mesenchymal-promoting genes.

ERG and AR expression is positively associated in humanprostate tumors

In agreement with our observations in mouse models andhuman prostate cancer cell lines, genome analysis of CRPCadenocarcinomas (CRPC-Ad) and neuroendocrine tumors(CRPC-NE; ref. 23) revealed a significant association of ERGfusion gene expression with CRPC-Ad (ARhigh), but not CRPC-NE tumors (ARlow; P ¼ 0.0485; OR ¼ 0.14; 95% CI ¼ 0.003–1.06; Fig. 5A). Because CRPC-NE tumors have low or absent ARexpression and TMPRSS2-ERG gene fusion expression is driven by

AR, our analyses were only focused on those samples withexpression of ERG gene fusion as demonstrated by RNA-seq orNanoString data (23, 39). In addition, we performed IHC analysison a human tissuemicroarray with 157 cores constructed from51patients with metastatic CRPC undergoing standard-of-care clin-ical biopsies at Mayo Clinic (Rochester, MN). This analysisconfirmed a strong association between AR and ERG expression(P¼ 2.98e�7, correlation¼ 0.41; 95%CI¼ 0.263–0.536; Fig. 5Band C; Supplementary Table S5).

ERG expression in PTEN/TP53 tumors regulates prostate tumorresponse to antiandrogen and anti-RB/E2F1 pathway drugs

The above findings prompted us to hypothesize that ERGþ/ARhigh/KRThigh (TMT) adenocarcinoma cells would respond toenzalutamide treatment, but ERG�/ARlow/KRTlow (DMT) tumorcells would not. Instead, ERG�/ARlow/KRTlow (DMT) tumor cellsmay rely heavily on RB hyperphosphorylation to maintain cellproliferation, dedifferentiation, and antiandrogen-resistant phe-notypes, and therefore this type of tumor may be highly respon-sive toRB-targeted therapy such asCDK4/6 inhibitors. Palbociclib(PD-0332991) is a CDK4/6 inhibitor that has been shown to beeffective in preclinical models of prostate and other cancer types,and was recently approved by the FDA for treatment of breastcancer (46–50). Treatment of control LNCaP-RF cells (ERG�/ARlow) with enzalutamide alone had no overt effect on expressionof cell-cycle genes, RB phosphorylation, and cell proliferation(Fig. 6A and B; Supplementary Fig. S7A and S7B), confirming theantiandrogen-resistant nature of ERG�/ARlow cells. However,palbociclib treatment, either alone or in combination withenzalutamide, significantly decreased expression of cell-cyclegenes and inhibited proliferation in ERG�/ARlow cells (Fig. 6Aand B; Supplementary Fig. S7A and S7B). It is interesting tonote that combination of enzalutamide and palbociclib signif-icantly inhibited proliferation of the ERG�/ARlow LNCaP-RFcells compared with palbociclib treatment alone (Fig. 6B),suggesting that palbociclib treatment may resensitize these cellsto enzalutamide treatment. As expected, LNCaP-RF cells stablyexpressing ERG (ERGþ/ARhigh) responded favorably to enzalu-tamide alone, and such effect was not enhanced in combina-tion with palbociclib (Fig. 6A and B; Supplementary Fig. S7Aand S7B). In further support of the finding that RB1 knockdownin ERG-positive cells abrogates the subsequent downregulationof cell lineage genes (Fig. 4E), RB1 knockdown in LNCaP-RF-ERG T1-E4 cells also abolished enzalutamide sensitivity (Sup-plementary Fig. S7C and S7D). Sensitivity to enzalutamide inERGþ/ARhigh cells (LNCaP-RF-ERG T1-E4 and VCaP), but not

Figure 4.ERG binds to the promoter and regulates expression of cell-cycle genes in human PTEN/TP53–altered prostate cancer cells.A,Western blot analysis of expression ofkey AR pathway, cell-cycle, and EMT-related proteins in five prostate cancer cell lines. B, Western blot analysis of expression of key AR pathway, cell-cycle,and EMT-related proteins in LNCaP-RF and PC-3 cell lines after lentiviral-mediated expression of full-length (ERG-FL) or ERG (T1-E4). C, Cell proliferation asmeasured by SRB assay for LNCaP-RF and PC-3 cell lines with lentiviral-mediated expression of ERG-FL or ERG (T1-E4). D, Top, coimmunoprecipitation ofendogenous ERG and RB in VCaP cells. ERG and AR coimmunoprecipitation shown as positive control. Bottom, coimmunoprecipitation of ERG and RB in PC-3 cellsstably expressing ERG (T1-E4). E, Western blot analysis of expression of key AR pathway and EMT-related genes in LNCaP-RF and PC-3 cell lines afterlentiviral-mediated expression of ERG (T1-E4) with or without RB1 knockdown. F, RT-qPCR of CCND1, CDK1, SKP2, FOXM1, TWIST1, TWIST2, SOX11, and TGFB2 inPC-3 cells with or without lentiviral-mediated expression of ERG (T1-E4). Relative to GAPDH. G, ERG ChIP-seq tracks in VCaP cells (GSE14092; ref. 31) andH3K4me3 (histonemark of promoters) ChIP-seq tracks in LNCaP cells (GSE43791) (33) fromUCSC genome browser forCCND1, CDK1, SKP2, FOXM1, TWIST1, TWIST2,SOX11, and TGFB2. ERG ChIP-seq input tracks shown as a control for true ERG peaks. Peaks underlined with black bars and boxed with a dashed red lineindicate significant ERG ChIP-seq peaks with P¼ 1e�5 or lower as determined by MACS. Asterisk indicates ERG peak that could not be validated by ChIP-qPCR. H,ERG ChIP-qPCR of CCND1, CDK1, SKP2, FOXM1, TWIST1, TWIST2, SOX11, and TGFB2 in PC-3 cells with lentiviral-mediated expression of ERG (T1-E4).






ERG�/ARlow (LNCaP-RF-EV) cells, was abrogated by androgendeprivation of culture media (Supplementary Fig. S7E andS7F), confirming AR pathway dependence in ERG-positive cells.

We further examined the responsiveness of ERG-positiveprostate cancer to antiandrogen therapy using in vivo models.Similar to the findings in vitro, ERG�/ARlow LNCaP-RF xenografttumors were resistant to enzalutamide treatment (Fig. 6C;Supplementary Fig. S8A–S8C). In contrast, treatment of thesetumors with palbociclib significantly decreased tumor volume,Ki67 staining, and RB phosphorylation (Fig. 6C; Supplemen-tary Fig. S8A–S8C). Similar to the LNCaP-RF cell line study,combination of enzalutamide and palbociclib significantlydecreased ERG�/ARlow LNCaP-RF xenograft tumor volumecompared with palbociclib treatment alone (Fig. 6C; Supple-mentary Fig. S8A), which further highlights the potential effi-cacy of combination treatment in these tumors. In ERGþ/ARhigh

LNCaP-RF xenograft tumors, palbociclib treatment aloneexerted little to no effect but both enzalutamide treatmentalone and in combination with palbociclib significantlyreduced the tumor volume, Ki67 staining, and expression ofpRB S795 (Fig. 6C; Supplementary Fig. S8A–S8C).

We attempted to perform similar studies using DMT and TMTspontaneous tumor models. However, we found it was quitechalleging to crossbreed five different alleles together to simul-taneously generate large cohorts of DMT and TMT mice at thesame ages. Because of this technical difficulty, we performedsimilar drug treatment studies using allografts derived from

ERG�/ARlow/KRTlow DMT and ERGþ/ARhigh/KRThigh TMT tumorsand confirmed the findings from LNCaP-RF xenografts (Fig. 6Dand E; Supplementary Fig. S8D and S8E). Collectively, these datahighlight that PTEN/TP53–altered tumors with hyperphosphory-latedRB are resistant to enzalutamide, but are sensitive toCDK4/6inhibition alone or in combination with enzalutamide. In con-trast, ERG expression maintains antiandrogen sensitivity intumors even with PTEN/TP53 alteration and this effect is relatedto ERG-induced inhibition of cell-cycle gene expression andrestored AR signaling.

DiscussionThe findings in this study emphasize that the unique combi-

nationof geneticmutations presentwithin a single prostate tumorcan greatly affect response to androgen- and AR-targeted thera-pies. In particular, our study of the novel Pten/Trp53/ERG triple-mutant mouse model of prostate cancer recapitulates a trio ofgenetic events that cooccur in a significant subtype of prostatetumors. Previous studies demonstrated that loss of Pten and Trp53induces lineage plasticity in mouse prostate cancer, where pros-tate-specific Pten and Trp53 double KO mice develop prostateadenocarcinoma at a young age and further evolve into ARlow/KRTlow tumors (8–10, 41). These data and ours support thehypothesis that Pten/Trp53-altered tumors may transition froman ARhigh/KRThigh adenocarcinoma to an altered ARlow/KRTlow

state (Fig. 6F, left).

Figure 5.

ERG expression correlates with AR expression in human patient datasets. A, Fisher exact test to determine association between ERG fusion and CRPC-adenocarcinoma (CRPC-Ad) or CRPC-neuroendocrine (CRPC-NE) tumor subtypes from the Beltran cohort (23). B, Representative IHC images for AR and ERG fromhuman tissue microarray of metastatic CRPC obtained from Mayo Clinic. C, Pearson product–moment correlation between AR and ERG IHC staining in clinicalbiopsies of metastatic CRPC in B.


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Further analysis of the novel Pten/Trp53/ERG model revealedthat ERG binds to chromatin loci of a subset of cell-cycle–drivinggenes and decreases their expression in Pten/Trp53–alteredmouseprostate tumors, thereby preventing loss of RB activity and E2F1-

mediated cellular reprogramming (Fig. 6F, right). Studies inhuman prostate cancer cell lines also supported these findings.Most importantly, similar results were obtained through analysisof patient datasets and clinical samples. Although previous

Figure 6.

Differential responses of ERG-positive and ERG-negative human xenograft andmouse allograft tumorswith PTEN/TP53 alterations to enzalutamide and palbociclib.A, Western blot analysis of expression of key AR pathway, cell-cycle, and EMT-related proteins in LNCaP-RF cells with or without lentiviral-mediatedERG (T1-E4) expression after treatment with vehicle, enzalutamide (ENZ, 10 mmol/L), palbociclib (PD, 1 mmol/L), or combination (ENZþ PD). B, Cell proliferation asmeasured by SRB assay for LNCaP-RF cells with or without lentiviral-mediated ERG (T1-E4) expression after treatment with vehicle, ENZ (10 mmol/L), PD(1 mmol/L), or combination. C, LNCaP-RF xenograft tumor volumewith or without lentiviral-mediated ERG (T1-E4) expression during 3-week treatment with vehicle,ENZ (30mg/kg/day), PD (100 mg/kg/day), or combination. Six xenografts (n¼ 6) per cell line, per drug treatment. D, ERG�/ARlow/KRTlow DMT and ERGþ/ARhigh/KRThigh TMT allograft tumor volume during 3weeks of treatment with vehicle, ENZ (30mg/kg/day), PD (100mg/kg/day), or combination. Five allografts (n¼ 5) pergenotype, per drug treatment. E, Characterization of allograft tumors from (D) after 3 weeks of treatment. Top, H&E. Subsequent rows, IHCfor ERG, AR, pRB S795, and Ki67. F, A hypothetical model. In prostate cancer cells without the TMPRSS2-ERG fusion, PTEN deletion/mutation and TP53deletion/mutation favor cell-cycle gene expression, CDK activation, and RB inhibition (hyperphosphorylation), which in turn lead to E2F1 activation andluminal-epithelial-to-mesenchymal cell identity transition, antiandrogen resistance, and increased CDK4/6 inhibitor sensitivity. In contrast, in prostate cancer cellsharboring the TMPRSS2-ERG fusion, overexpression of ERG results in decreased expression of a subset of cell-cycle–promoting genes and RB activation(hypophosphorylation), thereby leading to E2F1 inhibition and maintenance of luminal epithelial cell identity, increased antiandrogen sensitivity, but CDK4/6inhibitor resistance.






studies have suggested a potential role for ERG in repressingneuroendocrine differentiation andpartially rescuingAR function(12, 13), this study represents the first to demonstrate ERG-mediated protection of the epithelial adenocarcinoma cell lineagein a clinically relevant mouse model with Pten/Trp53 mutations(Fig. 6F).

We further demonstrated in Pten/Trp53–mutated mouse pros-tate cancer and xenograft models that while ERG-positive tumorsare sensitive to antiandrogen treatment, ERG-negative tumorshave no overt response to antiandrogens and instead respondwell to the CDK4/6 inhibitor palbociclib. These findings wererecapitulated in human cell lines. Together, these data reveal apreviously undefined role of ERG in maintaining neoplasticepithelial cell identity and antiandrogen sensitivity in PTEN/TP53–mutated prostate cancer and highlight that different ther-apeutic strategies are needed for PTEN/TP53–altered tumors withor without ERG (Fig. 6F).

Despite a previous finding that ERG overexpression alone issufficient for focal prostatic intraepithelial neoplasia (PIN) for-mation inmice with 129/Sv background (35), we did not observeany PIN lesions in Pb-ERGmice during the course of our previousstudy (51) and this report (Fig. 1C) perhaps due to the differentgenetic (C57BL6/129) background of mice we used. ERG expres-sion levels in Pb-ERGmouse prostate tissuewere comparable withthat in the TMPRSS2-ERG fusion-positive human VCaP cell line(see Supplementary Fig. S2D). Nevertheless, our findings areconsistent with other reports that ERG alone is not sufficient topromote prostate tumorigenesis in mice within the studied timeframe (51–53). The Pb-ERG transgenic mouse model also pro-vides the unique ability to study AR-dependent transgenic expres-sion of ERG, which mimics AR-driven TMPRSS2-ERG expressionin human prostate tumors (35). For these reasons, this modelsystem is particularly relevant and analogous to human prostatecancer. However, it is important to note that the reduced ARexpression observed in approximately 50% of the tumors in TMTmice could contribute to absence of AR-dependent, Pb-promoter-driven expression of ERG. The exact underlying molecular mech-anism warrants further investigation, and future studies willexplore the exact cause-and-effect of reduced AR expression andabsence of ERG expression in the ARlow/KRTlow subset of TMTmice. Additional studies with a more robust knock-in model of

ERG (13) would be particularly useful to better characterize thismechanism, although slightly less physiologically relevant.

These findings in prostate cancer also raise the larger questionof whether the mechanism defined in the current study might beapplicable to other RB alteration–related cancer types such as lungcancer (genomic loss of RB1 promotes the transition from ade-nocarcinoma to small-cell lung cancer; ref. 54) and breast cancer(functional loss of RB due to HER2 amplification leads to for-mation of nonluminal breast cancer). Nevertheless, our findingssupport the evaluation of ERG fusion as a viable biomarker toguide antiandrogen andRBpathway–targeted therapies for PTEN/TP53–mutated, RB1-intact prostate cancer. Studies such as thesewill be essential to combat lineage plasticity-mediated therapyresistance in prostate cancer as well as other cancers.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: W. Xu, H. HuangDevelopment of methodology: A.M. Blee, Y. He, Y. ChenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.M. Blee, Y. He, Y. Yang, J. Dugdale, M. Kohli,R. JimenezAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):A.M. Blee, Y.He, Z. Ye, Y. Yan, T.Ma, Y. Chen, L.WangWriting, review, and/or revision of themanuscript:A.M. Blee, Y. He, M. Kohli,R. Jimenez, Y. Chen, L. Wang, H. HuangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.M. Blee, Y. Pan, E. KuehnStudy supervision: A.M. Blee, W. Xu, H. Huang

AcknowledgmentsThisworkwas supported in part by grants fromNIH (CA134514, CA130908,

CA203849, and CA193239; to H. Huang) and DOD (W81XWH-14-1-0486; toH. Huang).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 26, 2018; revised May 4, 2018; accepted May 23, 2018;published first May 29, 2018.

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2018;24:4551-4565. Published OnlineFirst May 29, 2018.Clin Cancer Res Alexandra M. Blee, Yundong He, Yinhui Yang, et al. Cancer

-Mutated ProstateTP53 and PTENAntiandrogen Sensitivity in Controls Luminal Epithelial Lineage andTMPRSS2-ERG

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