Deletion of Interstitial Genes between TMPRSS2and ERG Promotes Prostate Cancer ProgressionDouglasE. Linn1,KathrynL.Penney2,3, RoderickT.Bronson4, LoreleiA.Mucci2,3, andZheLi1
Abstract
TMPRSS2–ERG gene fusions that occur frequently in humanprostate cancers can be generated either through insertionalchromosomal rearrangement or by intrachromosomal deletion.Genetically, a key difference between these two mechanisms isthat the latter results in deletion of a �3-Mb interstitial regioncontaining genes with unexplored roles in prostate cancer. In thisstudy, we characterized two mouse models recapitulatingTMPRSS2–ERG insertion or deletion events in the backgroundof prostate-specific PTEN deficiency. We found that only themicethat lacked the interstitial region developed prostate adenocarci-nomas marked by poor differentiation and epithelial-to-mesen-
chymal transition. Mechanistic investigations identified severalinterstitial genes, including Ets2 andBace2, whose reduced expres-sion correlated in the gene homologs in human prostate cancerwith biochemical relapse and lethal disease. Accordingly, PTEN-deficient mice with prostate-specific knockout of Ets2 exhibitedmarked progression of prostate adenocarcinomas that was partlyattributed to activation of MAPK signaling. Collectively, ourfindings established that Ets2 is a tumor suppressor gene inprostate cancer, and its loss along with other genes within theTMPRSS2–ERG interstitial region contributes to disease progres-sion. Cancer Res; 76(7); 1869–81. �2016 AACR.
IntroductionOver half of prostate-specific antigen (PSA)–screened prostate
cancer patients possess gene fusions involving members of theETS transcription factor family (1, 2). In the United States, thistranslates tomore than 120,000men diagnosed with ETS fusion–positive prostate cancer each year. The most common subtype ofETS fusion juxtaposes the androgen-regulated promoter of theserine protease gene TMPRSS2 with the coding region of an ETSgene, ERG, leading to ERG overexpression. Both TMPRSS2 andERG genes are located on human chromosome 21q22 and areseparated by a 3-Mb interstitial region. The TMPRSS2–ERG genefusion is predominantly generated either through intrachromo-somal deletion of this interstitial region (referred to as "deletion")or via chromosomal rearrangement through insertion of theinterstitial region (referred to as "insertion"; refs. 3–6). As awhole,ERG rearrangements are believed to be a prostate cancer–specificevent (7, 8), yet their prognostic value to date remains contro-versial (9). Numerous studies found no association betweenTMPRSS2–ERG fusions and tumor grade, stage, Gleason score,PSA recurrence or mortality (9–12). A recent review and meta-analysis both have exhaustively summarized literature to date
demonstrating the overall lack of consistent prognostic value forTMPRSS2–ERG fusion in prostate cancer (9, 12). Differences inpatient cohorts, diagnosis method, and fusion detection methodmay account for these conflicting results. Nonetheless, it isimportant to note that because TMPRSS2–ERG fusions arethought to represent an early event in prostate tumorigenesis(2, 8), other oncogenic events that drive prostate cancer progres-sion to later stages may also contribute to differential clinicaloutcomes for patients with fusion-positive prostate cancer. Inaddition, another important consideration is that TMPRSS2–ERGrearrangements are genetically distinct and may have differentbiological consequences. Various subclasses of TMPRSS2–ERGfusions have largely been grouped together in the literature, thuspotentially masking their true prognostic value. For instance, ithas been suggested that prostate cancers with TMPRSS2–ERGfusions generated through deletion represent more aggressivecases than those with fusions formed via insertion (5). Further-more, it was reported that patients with duplicated copies ofdeletion-generated fusions exhibited extremely poor survival(13). Lastly, in a rapid autopsy cohort, TMPRSS2–ERG fusionsin fusion-positive metastases from patients with hormone-refrac-tory prostate cancer were uniformly generated through deletion(14). Collectively, it remains unclear whether interstitial deletion,which deletes one copy of the intervening genes betweenTMPRSS2 and ERG, represents a separate genetic event that con-tributes to prostate tumorigenesis, and whether deletion alone orin conjunction with TMPRSS2–ERG fusion expression correlateswith amore aggressive subtype of fusion-positive prostate cancer.A better understanding of this may reveal novel tumor suppressor(s) in the interstitial region and enhance our understanding of theunderlying biology of aggressive prostate cancer.
The TMPRSS2–ERG interstitial region contains �16 knownprotein-encoding genes (Supplementary Fig. S1), several of whichhave reported tumor-suppressive roles. A gene encoding anotherETS factor,ETS2, is located in this region and its ectopic expression
1DivisionofGenetics, BrighamandWomen'sHospital andDepartmentof Medicine, Harvard Medical School, Boston, Massachusetts. 2Chan-ning Division of Network Medicine, Brigham and Women's Hospitaland Department of Medicine, Harvard Medical School, Boston, Mas-sachusetts. 3Department of Epidemiology, Harvard School of PublicHealth, Boston, Massachusetts. 4Rodent Histopathology, HarvardMedical School, Boston, Massachusetts.
Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).
Corresponding Author: Zhe Li, Division of Genetics, Brigham and Women'sHospital, 77 Avenue Louis Pasteur, Room 458E, Boston, MA 02115. Phone:617-525-4740; Fax: 617-525-4705; E-mail: [email protected]
decreased proliferation and invasion of prostate cancer cell lines(15, 16). HMGN1, which binds to and maintains an openchromatin configuration, plays a critical role in DNA repair; itsloss led to increased N-cadherin expression (17), a gene asso-ciated with high-grade prostate cancer (18). In addition, MX1encodes an interferon-inducible GTPase MxA and its ectopicexpression drastically reduced the motility and invasiveness ofan aggressive subline of PC3 prostate cancer cells (19). Together,these studies suggest that deletion of one or more genes withinthis region may lead to haploinsufficiency or loss of expression,which may promote prostate cancer progression.
To study the role of TMPRSS2–ERG fusions in prostate cancer,we recently generated two knockin mouse models that recapitu-late either TMPRSS2–ERG insertion or deletion events (20).Notably, prostate-specific activation of both conditional knockinalleles leads to expression of the same fusion transcript as thepredominant TMPRSS2–ERGa subtype originally described byTomlins and colleagues (1), and the only genetic difference is thatone model has deletion of the interstitial region, whereas theother has no deletion. Importantly, these interstitial regions aresyntenic between human and mouse with almost all knownprotein-encoding genes being conserved (Supplementary Fig.S1). Thus, these models offer a unique opportunity to studywhether the interstitial deletion also represents a genetic eventthat contributes to prostate tumorigenesis. In this study, wecompared the prostate phenotypes of these two models underthe background of Probasin-Cre (Pb-Cre)-mediated biallelic Pteninactivation, and provided genetic evidence that reduced expres-sion of the interstitial genes, in particular, ETS2, promotes pros-tate cancer progression. We also explored associations of theinterstitial genes in prostate cancer cohorts to test their clinicalrelevance.
Materials and MethodsMouse strains, procedures, and tissue preparation
T-ERG and T-3Mb-Erg knockin mice were generated previously(20). Pb-Cre (Pb-Cre4) transgenic mice were obtained from theMouse Models of Human Cancers Consortium repository.Rosa26-STOP-YFP (R26YFP) conditional Cre-reporter mice andPten and Ets2 conditional knockout mice (PtenL/L, Ets2L/L) wereobtained from The Jackson Laboratory. All mice were maintainedon a mixed genetic background and housed in a pathogen-freebarrier environment. All mouse studies were approved by theInstitutional Animal Care and Use Committee. Prostate tissuesused for IHC were fixed for 16 hours in 10% formalin (Fisher),dehydrated, and embedded in paraffin. Tissues used for immu-nofluorescent staining were fixed in 10% formalin for 1 hour,washed in PBS, then saturated in 30% sucrose overnight at 4�C.Tissues were then embedded in OCT compound (Sakura) andstored at �80�C prior to cryosectioning.
Histology, immunohistochemistry, and immunofluorescentstaining
Paraffin-embedded tissues were stained with hematoxylin andeosin (H&E) and reviewed by a trained rodent histopathologist.Pathology was defined as previously described (21, 22). IHC wascarried out by rehydrating sections, performing antigen unmask-ing with Tris-EDTA buffer. Sections were blocked with 2.5% goatserum for 1 hour at room temperature and incubated withprimary antibodies overnight at 4�C. Antibodies for IHC wereused todetect ERG(Epitomics 2805), AR (SantaCruz sc816), SMA
(Sigma A2547), p63 (Chemicon MAB4135), E-cadherin (CellSignaling 3195), vimentin (Abcam 92547), ETS2 (SantaCruz sc351), HMGN1 (Abcam 5212), or BACE2 (Abcam5670). Staining was visualized using DAB substrate (Vector) andcounter-stained with hematoxylin. Slides were dehydrated andsealed using Permount mounting media (Fisher). For immuno-fluorescent staining, cryosections of prostate tissues were sec-tioned at 8 mm, blocked in 2.5% goat serum, and incubated withprimary antibodies overnight at 4�C. Antibodies for immunoflu-orescence were used to detect YFP (Abcam 13970), vimentin(Abcam 92547), K5 (Covance PRB-160P) or K8 (CovanceMMS-162P). Alexa Fluor–conjugated secondary antibodies (LifeTechnologies) were incubated for 1 hour at room temperature.Nuclei were counterstained with DAPI, and slides were sealedwith Vectashield mounting media (Vector).
Laser capture microdissection and gene expression profilingParaffin-embedded prostates were stained with hematoxylin
and excised using the ArcturusXT Laser Capture Microdissectionsystem to collect genomic DNA or total RNA. DNA was isolatedusing the Arcturus PicoPure kit (Life Technologies) and RNAwas isolated using the RNeasy FFPE Kit (Qiagen). Nucleic acidquality was validated on a BioAnalyzer (Agilent) and sampleswere processed using the Ovation RNA Amplification System(NuGEN) prior to gene expression profiling with the AffymetrixMouse Gene 2.0 ST chip.
Prostate regeneration assaysIn vivo prostate regeneration assays were performed as previ-
ously described (23). Briefly, prostate epithelial cells were dis-sociated and MACS-sorted (Miltenyi Biotec) to removed Lineagepositive cells (CD31, CD45, and Ter119). Cre-na€�ve cells usedfor prostate cancer initiation studies were infected with CMV-Creadenovirus (50 MOI, from University of Iowa Gene TransferCore) for 1 hour at 37�C. Approximately 2.5 � 105 sorted cellswere mixed with 2.5 � 105 Urogenital sinus mesenchymecells and subcutaneously injected with Matrigel into flanks ofRag2-deficient mice. Outgrowths were collected 8 weeks afterinjection.
Cell culture, shRNA knockdown, and Western blotVCaP cell line was purchased from ATCC (<5 years ago) and
was authenticated again internally by short tandem repeat (STR)profiling test; it was free frommycoplasma contamination and wascultured in DMEMmediumwith 10%FBS. VCaP cells were stablytransduced by pGIPZ lentiviruses with shRNAs for ETS2[shETS2_1: V3LHS_646035; shETS2_2: V3LHS_642164, or withcontrol shRNA (empty vector pGIPZ shRNA, #RHS4351), fromOpen Biosystems]. Western blot was performed as previouslydescribed (24) on lysates were collected 72 hours after infection.Antibodies used included, from Cell Signaling: pMET (3077),totalMET (3127), pERK1/2 (4370), total ERK1/2 (9102),GAPDH(2118), and from Santa Cruz: ETS2 (sc351).
Data analysisHierarchical clustering and heatmaps were generated using
MultiExperiment Viewer. Statistical significance was calculatedusing the Student t test. Gene set enrichment analysis (GSEA) wasperformed as described previously (25). Analysis of human datawas performed using cbio-portal.
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For the PHS and HPFS analysis, we utilized data from a geneexpression profiling study. Briefly, men were sampled from theHPFS and PHS Prostate Tumor Cohort using an extreme casedesign, which includes 119 men who died of their cancer ordeveloped bony or distant metastases ("lethal") and 282 menwho lived at least 8 years after prostate cancer diagnosis andwere not diagnosed with metastases through 2012 ("indolent").Briefly, RNA was extracted from tissue, then amplified using theWT-Ovation FFPE System V2 (NuGEN), a whole transcriptomeamplification system that allows for complete gene expressionanalysis from archives of FFPE samples. After reverse transcriptionand fragmentation, the cDNA was hybridized to the AffymetrixGeneChipHumanGene1.0 STmicroarray. After normalization ofthe data, we mapped gene names to Affymetrix transcript clusterIDs using the NetAffx annotations as implemented in Biocon-ductor annotationpackagepd.hugene.1.0.st.v1.We compared theexpression of the interstitial genes across the lethal and indolentcases using linear regression.
Accession numbersThe microarray expression profiling data set generated in this
article has been deposited to the GEO database under the acces-sion number GSE63070.
ResultsTmprss2–Erg fusion with interstitial deletion more stronglycooperates with Pten loss
We previously reported the generation and characterizationof two knockin mouse models of Tmprss2–Erg fusions forprostate cancer (20). In the insertion model, an N-terminustruncated human ERG cDNA was knocked-in to the mouseTmprss2 locus directly (Tmprss2–STOP-ERG, Cre-mediated exci-sion of the floxed STOP cassette leads to activation of theTmprss2–ERG fusion allele; these are collectively referred to as"T-ERG" knockin). The second model utilized sequential genetargeting to introduce loxP sites into the mouse Tmprss2 and Ergloci, respectively, thus allowing deletion of the entire interstitialregion upon Cre recombinase expression (i.e., referred to as "T-3Mb-Erg" before Cre-mediated excision of the interstitialregion, and "T-D-Erg" after excision). In the presence of a singlecopy of Pten-null allele, both lines expedited formation of low-grade prostatic intraepithelial neoplasia (LG-PIN) at similarrates and exhibited indistinguishable phenotypes that rarelydeveloped into higher grade lesions, but never to frank ade-nocarcinoma (20). In the presence of biallelic Pten inactivationmediated by Pb-Cre, however, most Pb-Cre;T-3Mb-Erg;PtenL/L
male mice developed large poorly differentiated prostatetumors (i.e., T-D-Erg/Pten-null tumors) in dorsolateral andventral lobes by 12 months of age (20). To determine whetherthis advanced prostate cancer phenotype is caused by Tmprss2–Erg fusion expression or interstitial deletion, or both, wesimilarly generated Pb-Cre;T-ERG;PtenL/L males and character-ized their prostate phenotype at 12 months of age. Intriguingly,none of the Pb-Cre;T-ERG;PtenL/L males developed poorly dif-ferentiated prostate adenocarcinoma at this age (Fig. 1A and B;compared with almost 100% penetrance for Pb-Cre;T-3Mb-Erg;PtenL/L males to develop poorly and/or moderately differenti-ated adenocarcinomas, Fig. 1B). The prostate lesions developedin these males were quite similar to those high-grade PIN (HG-PIN) lesions often observed in Pb-Cre;PtenL/L control males atthis age (Fig. 1A). Furthermore, control Pb-Cre;PtenL/L males
and Pb-Cre;T-ERG;PtenL/L males only displayed signs of localinvasion, which was confirmed through IHC staining ofsmooth muscle actin (SMA; Fig. 1A). In stark contrast, Pb-Cre;T-3Mb-Erg;PtenL/L males developed invasive prostatetumors that lacked expression of SMA and basal marker p63(Fig. 1A). Such T-D-Erg/Pten-null tumors were positive for theprostate luminal epithelial marker Keratin 8 (K8), but werenegative for the basal epithelial marker Keratin 5 (K5; Fig. 1C).These Pb-Cre;T-3Mb-Erg;PtenL/L males also developed typicalHG-PIN lesions composed of K8þ luminal cells surrounded byK5þ basal cells that are often observed in the control Pb-Cre;PtenL/L males (Fig. 1C), as well as localized invasive cancerswith microducts mainly composed of K8þ prostate luminalcells [with almost no K5þ basal cells (Fig. 1C), and almost noSMA and p63 expression (Supplementary Fig. S2A)]. Poorlydifferentiated adenocarcinomas were never observed in controlPb-Cre;PtenL/L or Pb-Cre;T-ERG;PtenL/L males, whereas presenceof invasive microducts consistent with moderately differenti-ated adenocarcinoma was only infrequently detected in thesecontrols (Fig. 1B).
Adenocarcinomas and HG-PINs developed in Pb-Cre;T-3Mb-Erg;PtenL/L mice exhibit Cre-mediated interstitial deletion
By IHC staining, we confirmed that ERG protein was robustlyexpressed in HG-PIN lesions developed in both Pb-Cre;T-ERG;PtenL/L and Pb-Cre;T-3Mb-Erg;PtenL/L males (Supplementary Fig.S2B). We previously reported that in Pb-Cre;T-3Mb-Erg;PtenL/L
malemice,moderately differentiated invasive cancers withmicro-ducts developed in their prostates often exhibited a mosaicpattern of ERG protein expression, whereas poorly differentiatedinvasive adenocarcinomas were largely negative for ERG (20). Torule out a possibility in which lack of ERG expression is due toreduced efficiency in generating Tmprss2–Erg fusion from Cre-mediated deletion of the large 3-Mb interstitial region in theconditional T-3Mb-Erg allele, we performed laser capture micro-dissection to isolate epithelial cells from well-defined regions ofeither HG-PIN (mainly ERG positive) or poorly differentiatedadenocarcinoma (tumor, mainly ERG negative; SupplementaryFig. S3). We then extracted genomic DNA from these isolatedtissues as well as from the whole prostates (i.e., containing bothERG-positive and -negative lesions) and by genomic DNA PCRanalysis (Supplementary Fig. S4A), we verified that in both typesof tissues, the Tmprss2–Erg gene fusion was generated effectivelyvia Cre-mediated interstitial deletion (Supplementary Fig. S4B).These data suggest that advanced prostate cancer lesions observedin the Pb-Cre;T-3Mb-Erg;PtenL/L model are most likely due to theinterstitial deletion between the Tmprss2 and Erg loci.
T-D-Erg/Pten-null tumors exhibit an EMT phenotypeTo investigate the biological mechanism underlying the
more aggressive T-D-Erg/Pten-null tumors, we performed geneexpression profiling using laser capture microdissected prostateepithelial cells. Only cells with round epithelial-like morphol-ogy were excised, leaving behind spindle-shaped mesenchymalcells. As an internal control for stage-specific differencesbetween cancer lesions developed in control Pb-Cre;PtenL/L
males (i.e., mainly HG-PIN) and Pb-Cre;T-3Mb-Erg;PtenL/L
males [i.e., poorly differentiated adenocarcinoma (tumor) andHG-PIN], we also excised HG-PIN lesions (in addition totumors) from Pb-Cre;T-3Mb-Erg;PtenL/L mice for analysis (Sup-plementary Fig. S3). Among genes differentially regulated
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between these three groups, GSEA (25) revealed that multiplepreviously defined epithelial-to-mesenchymal transition (EMT)gene sets were enriched in T-D-Erg/Pten-null tumors comparedwith control Pten-null HG-PIN lesions (Fig. 2A and Supple-mentary Fig. S5A). Enrichment of EMT gene sets could also befound when comparing T-D-Erg/Pten-null HG-PIN lesions withPten-null HG-PIN lesions (Supplementary Fig. S5B), or whencomparing T-D-Erg/Pten-null tumors with HG-PIN lesionsdeveloped in the same mice (Supplementary Fig. S5C). These
analyses suggested that the EMT signature in the T-D-Erg/Pten-null tumors was not simply due to a tumor stage difference (i.e.,poorly differentiated adenocarcinoma versus HG-PIN), but wasacquired progressively during prostate cancer progression whenunder the interstitial deletion background. We validatedthe EMT signature using IHC for E-cadherin and vimentin,which display epithelial and stromal compartment–restrictedexpression, respectively (Fig. 2B). E-cadherin was highlyexpressed in the epithelial compartment in HG-PIN lesions in
Figure 1.Tmprss2–Erg gene fusion generatedthrough interstitial deletion morestrongly cooperates with Pten loss.A, representative H&E staining (top),SMA IHC staining (middle), and p63IHC staining (bottom) of prostatesections from mice with the indicatedgenotypes. Green arrow,discontinuous SMA staining andemergence of epithelial cells throughbasement membrane. Loss of basalmarker p63 was used to validateadenocarcinoma. Red arrows,p63-expressing (p63þ) basal cells.Scale bars, 100 mm for H&E; 50 mm forSMA and p63 staining. B, graphicalsummary of dominant histologicallesions observed in aged mousemodels of Tmprss2–Erg fusions with(T-3Mb-Erg) or without (T-ERG)interstitial deletion. C, progressivelesions developed in Pb-Cre;T-3Mb-Erg;PtenL/L mice. Immunofluorescentstaining for luminalmarker K8 (green),basal marker K5 (red), and DAPI(blue) showing progressive lossof K5þ basal cells in moderatelyand poorly differentiatedadenocarcinomas. Scale bars, 50 mm.
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Pb-Cre;PtenL/L, Pb-Cre;T-ERG;PtenL/L and Pb-Cre;T-3Mb-Erg;PtenL/L mice, but was downregulated in T-D-Erg/Pten-nulltumors. Inversely, vimentin displayed stromal-specific expres-sion in all HG-PIN lesions yet was abundant within epithelialcells of the T-D-Erg/Pten-null tumors. In these Pb-Cre–basedmice, a conditional Cre-reporter Rosa26-STOP-YFP (R26Y, Cre-mediated excision of a floxed STOP cassette in this allele leadsto activation of the YFP reporter) was included to track Pb-Cre–expressing cells and their daughter cells (i.e., YFPþ prostateepithelial cells). The presence of EMT features was alsoverified in immunofluorescent analyses where the epithelialmarker K8 and the lineage marker YFP (for genetic marking)overlapped with vimentin only in tumor cells but not in HG-PIN lesions (Supplementary Fig. S5D). These data suggested
that although the EMT program was already upregulated in T-D-Erg/Pten-null HG-PIN lesions at the transcript level, changesin the expression of key EMT markers at the protein levelappeared at later tumor stages. Lastly, to rule out a possibilityin which the poorly differentiated tumors with mesenchymalfeatures developed in Pb-Cre;T-3Mb-Erg;PtenL/L mice were dueto a desmoplastic response in the stroma (as a response toinvasive prostate cancer developing nearby), similar to whatwas reported for the Pb-Cre;T-ETV1;PtenL/L mouse model (20),we stained these tumors for YFP expression and found that theywere indeed YFPþ (Supplementary Fig. S5E), thus confirmingthat these large poorly differentiated T-D-Erg/Pten-null tumorswere derived from Pb-Cre--mediated recombination and there-fore of epithelial origin.
Figure 2.T-D-Erg/Pten-null tumors exhibit anEMT phenotype. A, GSEA resultsshowing highly significant [falsediscovery rate (i.e., FDR q-val) < 0.25]enrichment of multiple EMT gene setsin T-D-Erg/Pten-null tumors in relationto HG-PIN lesions in Pb-Cre;PtenL/L
control males. The gene sets are fromthe c2 CGP (chemical and geneticperturbations) collection of MSigDB(http://www.broadinstitute.org/gsea/msigdb/index.jsp). B, IHCconfirmation of EMT features inT-D-Erg/Pten-null tumors usingE-cadherin (top) and vimentin(bottom) staining. Control Pten-nulland T-ERG/Pten-null HG-PINs (leftpanels), T-D-Erg/Pten-null HG-PIN(right panels, black arrows), andT-D-Erg/Pten-null tumors (right,red arrows) are shown. Scale bars,50 mm.
TMPRSS2–ERG Interstitial Deletion Promotes Prostate Cancer
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T-D-Erg/Pten-null tumors retain partial AR signalingSimilar to the Pb-Cre;T-3Mb-Erg;PtenL/L model, mice with Pb-
Cre–induced Pten-loss and RAS/MAPK activation also developpoorly differentiated prostate tumors with an EMT phenotype(i.e., Ras/Pten tumors; ref. 26). These Ras/Pten tumors exhibitedhighly heterogeneous androgen receptor (AR) expression andoverall reduced expression of AR target genes (26). To determinethe status of AR signaling in T-D-Erg/Pten-null prostate tumors, westained them for AR protein expression and found that T-D-Erg/Pten-null tumors are largely AR positive (Fig. 3A). By analyzingmicroarray expression data, we found that expression ofNkx3-1, awell-known AR target and tumor suppressor gene, was not
significantly changed in T-D-Erg/Pten-null tumors or HG-PINlesions compared with Pten-null HG-PIN lesions; Fkbp5, a re-cently described AR target gene that mediates reciprocal inhib-ition between PTEN/PI3K and AR pathways (27, 28), was slightlyupregulated in T-D-Erg/Pten-null tumors compared with Pten-null HG-PINs (Fig. 3B). In contrast, these two AR target genesexhibited a trend of downregulation in Ras/Pten tumors comparedwith Pten-null lesions (Supplementary Fig. S6A and ref. 26). How-ever, several other AR target genes related to normal prostatefunction, including Mme, Msmb, and Tmprss2, all exhibited atrend of downregulation in both T-D-Erg/Pten-null and Ras/Ptentumors in relation to Pten-null lesions (Fig. 3C; Supplementary
Figure 3.T-D-Erg/Pten-null tumors retain ARsignaling partially. A, IHC stainingdepicting AR expression in typicalprostate lesions from various prostatecancer mouse models. Scale bars,50 mm. B and C, relative expressionvalues of AR target genes, Nkx3-1and Fkbp5 (B), as well as Mme, Msmb,and Tmprss2 (C), in T-D-Erg/Pten-null tumors and HG-PINs inrelation to Pten-null HG-PINs (¼1),based on microarray expressionprofiling. Data represent mean� SEM.P (Student t test) for pairwisecomparisons that were significant(P < 0.05) or marginally significant(P < 0.1) are shown. All the othercomparisons were not statisticallysignificant.
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Fig. S6A and ref. 26). Of note, downregulation of Tmprss2 inT-D-Erg/Pten-null prostate tumors in relation to both T-D-Erg/Pten-null and Pten-null HG-PIN lesions may explain downregula-tion of Erg expression (which is under the control of the endog-enous Tmprss2 promoter) in T-D-Erg/Pten-null tumors, but not inT-D-Erg/Pten-null HG-PIN lesions (20). Lastly, by GSEA using anandrogen-driven signature we developed recently from humanprostate cancer cell lines (20), we found that this gene set exhibiteda higher level of downregulation in Ras/Pten tumors than inT-D-Erg/Pten-null tumors, when compared with their correspond-ing Pten-null controls (Supplementary Fig. S6B and S6C). Collec-tively, these data suggest that compared with the Ras/Pten EMTtumors, T-D-Erg/Pten-null tumors retain expression of both ARand some of the AR target genes, but also exhibit downregulationof other AR targets. This is consistent with the concept of ARcistrome reprogramming, in which AR binding sites change dur-ing prostate cancer progression, when its collaborating factorsare altered (e.g., due to different oncogenic events), leading tochanges in the expression of select AR target genes (29).
Multiple interstitial genes are candidate prostate tumorsuppressors
We next examined whether some interstitial genes could func-tion as tumor suppressors during prostate carcinogenesis toexplain the aggressive nature of T-D-Erg/Pten-null tumors. To dothis, wefirst generated an "Interstitial genes" gene set composed ofprotein-coding genes between TMPRSS2 and ERG and then per-formed GSEA using this gene set. We found that it was signifi-cantly downregulated in T-D-Erg/Pten-null tumors when com-pared with either Pten-null HG-PINs (Fig. 4A) or T-D-Erg/Pten-null HG-PINs (Fig. 4B). Among the downregulated interstitialgenes, Ets2 is abundantly expressed in prostate tissues [whereasseveral other ETS factor genes, such as Ets1, Erg, and Etv1, are not(Supplementary Fig. S7)] and its overexpression was shownpreviously to decrease proliferation and invasion of prostatecancer cell lines (15, 16); however, other interstitial genes, suchas Bace2 and Brwd1, have so far not been implicated in prostatecancer development. We analyzed expression of interstitial genesEts2, Hmgn1, and Bace2 in T-D-Erg/Pten-null HG-PIN and adeno-carcinoma at the protein level (Fig. 4C). Bace2was included as it isthe most significantly downregulated gene in the above GSEAanalysis (Fig. 4A and B). AlthoughHmgn1 failed to show up in theGSEA analysis (due to absence of Hmgn1 probe conversion), wealso included it in our validation, as it has been implicated inprostate cancer as a potential tumor suppressor (17, 18). Expres-sion at the protein level from these geneswas abundantly detectedin nuclei and cytoplasm of epithelial cells in HG-PIN lesions. Inadvanced tumors, expression levels were dramatically decreased,although weak cytoplasmic staining could still be observed (Fig.4C). Interestingly, in our GSEA analysis, although the "Interstitialgenes" gene set was not significantly enriched in HG-PIN lesionsfrom Pb-Cre;T-3Mb-Erg;PtenL/L mice to those from Pb-Cre;PtenL/L
control mice, the above-described interstitial genes, such as Ets2,Bace2, and Brwd1, all exhibited a trend toward slight downregula-tion in T-D-Erg/Pten-null HG-PINs (Supplementary Fig. S8 andSupplementary Table S1).
To determinewhether reduced expression of interstitial genes isassociatedwith prostate cancer patient outcomes, we analyzed thepotential association of deletion of interstitial genes with bio-chemical relapse after radical prostatectomy in a previously pub-lished human prostate cancer patient cohort (30). We found that
in this cohort, only 4 of 17 interstitial genes, including ETS2,BRWD1,HMGN1, andBACE2,were significant for prostate cancerprogression (when downregulated), regardless of patient fusionstatus (Fig. 4D). Patients possessing downregulation of theremaining set of interstitial genes did not exhibit differences inbiochemical recurrence compared with those with normal expres-sion levels. Similarly, ERG overexpressionwas not associatedwithtime to failure (Fig. 4D). Lastly, we examined the potential clinicalassociation of interstitial genes with lethal prostate cancer in alarger study with 119 lethal prostate cancer cases, defined as menwith metastatic disease or cancer death, and 282 indolent caseswith no evidence of metastatic disease [from the Physicians'Health Study (PHS) and the Health Professionals Follow-upStudy (HPFS; refs. 12, 31)]. We found that at least one-third ofthe interstitial genes, including ETS2 and BACE2, are expressed atsignificantly lower levels in lethal prostate cancer cases thanindolent cases (Table 1). Collectively, these analyses suggest thatmultiple genes within the TMPRSS2–ERG interstitial region areassociated with lethal prostate cancer (when downregulated) andmay function as tumor suppressors for prostate cancer.
Reduced gene dosage of Ets2 contributes to prostate cancerprogression
Interstitial genes exhibiting significantly lower expression levelsin advanced prostate cancer (e.g., ETS2, BACE2) can either directlycontribute to prostate tumorigenesis as a tumor suppressor orserve as a biomarker for lethal prostate cancer, or both. Todistinguish between these possibilities, we focused on ETS2,which is strongly downregulated in prostate tumors comparedwith benign tissues [(32) and in our PHS/HPFS study (P ¼ 8.4 �10�14)] and is also the most significantly downregulated inter-stitial gene in lethal prostate cancer s (Table 1). We crossed micecarrying a conditional knockout allele of Ets2 (Ets2L; ref. 33) withPtenL/L mice to generate Ets2L/þ;PtenL/L male mice. We then per-formed the regeneration assay upon ex vivo exposure of theirprostate cells to CMV-Cre adenovirus (Ad-CMV-Cre). Ad-CMV-Cre–infected PtenL/L control cells formed largely normal ducts
Table 1. Association of select interstitial genes with lethal prostate cancer,Physicians' Health Study and Health Professionals Follow-up Study
Figure 4.Multiple interstitial genes are candidate prostate tumor suppressors. A and B, GSEA for the "Interstitial genes" showing significant (FDR < 0.25) negativeenrichment (i.e., downregulation) of this gene set in T-D-Erg/Pten-null tumors in relation to Pten-null HG-PINs (A) or T-D-Erg/Pten-null HG-PINs (B). InA and B, enrichment plots are shown on the left, heatmaps are shown on the right. In the heatmap, red to pink to light blue to blue indicate highestto lowest expression levels of indicated genes. C, expression of select interstitial genes, Ets2, Hmgn1, and Bace2 were significantly lower in adenocarcinoma(bottom) compared with HG-PIN lesions (top) in Pb-Cre;T-3Mb-Erg;PtenL/L mice. Scale bars, 50 mm. D, Kaplan–Meier curves of human patient datareveal that downregulation of several interstitial genes (outlined in red) predict biochemical relapse after prostatectomy (top), whereas some do not(bottom). ERG overexpression (outlined in green) also did not predict recurrence in this cohort. Blue lines depict patients with normal expression whilepatients with deregulated expression are shown as red lines for each graph.
Linn et al.
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with occasional areas of hyperplasia (Fig. 5A). In contrast, Ad-CMV-Cre–infected Ets2L/þ;PtenL/L prostate cells formed small pro-liferating lesions invading into the lumen, consistent with HG-PIN (Fig. 5A). Next, we generated Pb-Cre;Ets2L/þ;PtenL/L, Pb-Cre;Ets2L/L;PtenL/L, andmatched control Pb-Cre;PtenL/Lmalemice. At 6to 9 months of age, we found that although the anterior lobes ofall these mice developed HG-PIN lesions of comparable severity,the phenotypes from the dorsolateral and ventral lobes of the Pb-Cre;Ets2L/þ;PtenL/L and Pb-Cre;Ets2L/L;PtenL/L males were notably
stronger. Loss of one copy of Ets2 resulted in development oflarger HG-PIN lesions in both the dorsolateral and ventral lobesand the dorsolateral lobes also contained significantly morestromal proliferation and inflammatory infiltrate (Fig. 5B).Importantly, loss of both copies of Ets2 led to the emergence ofpoorly differentiated prostate adenocarcinoma in the ventrallobes (Fig. 5C). Collectively, these data suggest that Ets2 is atumor suppressor in prostate cancer, and its reduced dosage inprostate epithelial cells promotes prostate cancer progression.
Figure 5.Reduceddosage ofEts2 contributes toprostate cancer progression under thePten-null background. A, prostateregeneration assay using PtenL/L orEts2L/þ;PtenL/L prostate cells infectedwith Ad-CMV-Cre adenovirus prior toimplantation. Red arrow, a lesionresembling HG-PIN. Scale bars, 50 mm.B, H&E staining showing enhancedHG-PIN phenotype in a 6-month-oldPb-Cre;Ets2L/þ;PtenL/L malecompared with its age-matched Pb-Cre;PtenL/L control male in thedorsolateral and ventral lobes. Scalebars, 100 mm. C, H&E staining showinginvasive prostate cancer andpoorly differentiated prostateadenocarcinoma phenotype in a9-month-old Pb-Cre;Ets2L/L;PtenL/L
male compared with its age-matchedPb-Cre;PtenL/L control male in thedorsolateral and ventral lobes. Scalebars, 100 mm.
TMPRSS2–ERG Interstitial Deletion Promotes Prostate Cancer
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ETS2 loss leads to activation of the MAPK pathwayTo determine how reduced Ets2 dosage contributes to prostate
cancer progression, we further analyzed the microarray data fromour animal models, focusing on comparing T-D-Erg/Pten-nullHG-PINs to Pten-null HG-PINs (thus stage-matched). By GSEA,we found that in addition to the above-described EMT-relatedgene sets (Supplementary Fig. S5B), gene sets related to the RAS/MAPK pathway, HGF/MET pathway, and WNT signaling, werealso significantly enriched in T-D-Erg/Pten-null HG-PIN lesions(Fig. 6A and Supplementary Table S2). Interestingly, a recentstudy examining a tumor suppressor role of ETS2 in non–smallcell lung cancer demonstrated association of ETS2 loss withactivation of both HGF/MET and MAPK signaling, as well asinduction of EMT-related genes (34). To determine whetherETS2 loss could induce similar pathway activation in prostatecancer cells, we used shRNAs to knockdown ETS2 expression inVCaP cells, which carry the TMPRSS2–ERG fusion without theinterstitial deletion (1). We found that upon ETS2 knockdown,the level of phosphorylated ERK1/2 (pERK1/2) was notably
increased (Fig. 6B). Due to a low level of phosphorylatedMET (pMET) in VCaP cells, we were unable to definitivelydetermine whether reduced ETS2 expression promotes activa-tion of the HGF/MET pathway (data not shown). Collectively,our data from both animal models and human prostate cancercells suggest that ETS2 loss causes activation of at least theMAPK pathway, which cooperates with Pten loss, leading to thedevelopment of advanced prostate cancer.
DiscussionOur unique mouse models have allowed examination and
comparison of the biological consequences of Tmprss2–Ergfusions generated by insertion or deletion. ERG overexpressionin the context of Pten loss is sufficient for early initiation events asbothmodels similarly expedited PIN frequency and severity in thecontext of a single copy of Pten loss (20). However, the differencebetween these two models became obvious in aged mice whenunder the Pten-null background. Prostate lesions in Pb-Cre;T-ERG;
Figure 6.ETS2 loss leads to activation of the MAPK pathway. A,GSEA results showing significant [false discoveryrate (FDR) < 0.25] enrichment of gene sets related toRAS/MAPK, HGF/MET, and WNT signaling in T-D-Erg/Pten-null HG-PIN lesions in relation to HG-PIN lesionsin Pb-Cre;PtenL/L control males. Also see SupplementaryTable S2 for a complete list of significantly enrichedpathway-related gene sets. B, Western blot showingincreased levels of pERK1/2 in VCaP cells uponknockdown of ETS2 by two independent shRNAs.Levels of total ERK1/2 and GAPDH are shown asloading controls.
Cancer Res; 76(7) April 1, 2016 Cancer Research1878
PtenL/L males (i.e., no deletion) were histologically similar tothose in control Pb-Cre;PtenL/L males, and development of inva-sive prostate cancer in either line was a relatively rare event. Instark contrast, Pb-Cre;T-3Mb-Erg;PtenL/L mice (i.e., with deletion)consistently developed poorly differentiated adenocarcinomasthat exhibited EMT features and partial AR signaling. Together,these data suggest that interstitial deletion may program prostatecells for the development of more aggressive prostate cancer thanERG overexpression alone. However, an important considerationis the prolonged delay for the phenotypic difference between Pb-Cre;T-ERG;PtenL/L and Pb-Cre;T-3Mb-Erg;PtenL/L males to emerge.This suggests that additional mutations or epigenetic events maybe required for the full T-D-Erg/Pten-null phenotype to manifest.Another consideration is that ERG protein expression is largelylost in the poorly differentiated T-D-Erg/Pten-null tumors (20),whereas the majority of human prostate cancer s with TMPRSS2–ERG fusions retain ERG overexpression (35–38). However, asmall subset of human fusion–positive castration-resistant cases(e.g., small cell/neuroendocrine prostate cancer) were also foundto be negative for ERGprotein staining (39).Whether our T-D-Erg/Pten-null model recapitulates this subset of human prostatecancer requires further investigation. Of note, a recent mousemodel of prostate-specific ERG overexpression resulted in aggres-sive prostate cancer (40), rather than HG-PIN lesions as observedin our Pb-Cre;T-ERG;PtenL/L model. This phenotypic differencemay be due to a difference in how ectopic ERG expression iscontrolled: ERGoverexpression in thatmousemodel is controlledby the constitutive Rosa26 locus, whereas ERG overexpression inour T-ERG mouse model is driven by the endogenous Tmprss2control region, which is under regulation by both androgen andestrogen (20, 41, 42).
Our study provided definitive genetic evidence to supportETS2 as a prostate tumor suppressor gene in the interstitialregion. This is consistent with our epidemiologic study dem-onstrating a significant association of ETS2 downregulationwith lethal prostate cancer (Table 1), as well as findings fromprostate cancer genomes, in which both focal deletion andpoint mutation of ETS2 were identified in lethal castration-resistant prostate cancer (16). Of note, ETS2 has also beenshown as a tumor suppressor in other cancer types (e.g., coloncancer, ref. 43; lung cancer, ref. 34). Because both ERG andETS2 belong to the ETS family of transcription factors that sharethe same or similar DNA-binding motif (44), it is possible thatectopic ERG expression may interfere with the normal functionof ETS2 in prostate epithelial cells by competing with ETS2 forDNA binding. Thus, ETS factors (e.g., ETS2) normally expressedin prostate epithelial cells may serve as prostate tumor sup-pressors, whereas those ETS factors (e.g., ERG and ETV1; Sup-plementary Fig. S7) that are not expressed in prostate epithelialcells may serve as oncogenes (once ectopically expressed), bypotentially interfering with functions of ETS factors normallyexpressed in prostate epithelial cells. This notion is consistentwith the idea of an ETS transcriptional network in prostatetumorigenesis (45). Mechanistically, we showed that ETS2 lossmight promote prostate cancer progression in part via activa-tion of the MAPK pathway. This is supported by the similaraggressive prostate cancer phenotype (e.g., EMT) observed inboth the T-D-Erg/Pten-null model and other mouse models withboth Pten-loss and RAS/MAPK activation (26, 46).
Although our T-D-Erg/Pten-null model and the Ras/Ptenmodel (26) both involve activation of the MAPK pathway,
leading to development of prostate tumors with features ofEMT, the EMT tumors developed in these models also exhibitdifference at the molecular level (e.g., different AR activity andtargets expression). Other deregulated pathways (e.g., due toETS2 loss and/or loss of other tumor suppressor genes in theinterstitial region) may contribute to this difference. In addi-tion, although our Pb-Cre;T-3Mb-Erg;PtenL/L mice had only onecopy of each interstitial gene deleted by Cre-mediated excision,the remaining alleles might also be additionally silencedthrough unidentified mechanisms. This might explain the lostexpression of select interstitial genes (e.g., Ets2, Hmgn1, andBace2) in advanced prostate cancer. Furthermore, recent studysuggested that combinations of hemizygous deletions of mul-tiple tumor suppressors might be required to produce maximalcancer phenotypes (47). This provides another potential mech-anism for loss of multiple interstitial genes to contribute toprostate tumorigenesis jointly. Overall, although we have val-idated ETS2 as a major tumor suppressor in this region, wecannot exclude that other interstitial genes (e.g., BACE2) mayhave tumor suppressor roles as well. Future characterization ofclinical samples with careful dissection of the individual andcombinatorial contribution of the loss of these candidateinterstitial genes, coupled with mouse modeling, may revealadditional tumor suppressor(s) in this interstitial region. Lastly,our study also suggests that clinical prognostic studies mayneed to consider more carefully distinguishing TMPRSS2–ERGfusions based on deletion or insertion, as they may contributeto distinct clinical outcomes.
Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.
Authors' ContributionsConception and design: D.E. Linn, Z. LiDevelopment of methodology: D.E. Linn, Z. LiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D.E. Linn, Z. LiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): D.E. Linn, K.L. Penney, R.T. Bronson, Z. LiWriting, review, and/or revision of the manuscript: D.E. Linn, K.L. Penney,L.A. Mucci, Z. LiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): D.E. LinnStudy supervision: D.E. Linn, Z. LiOther (performed pathology): R.T. Bronson
AcknowledgmentsThe authors thank Xin Zhang for her assistance in maintaining and genotyp-
ing some of the mouse models and Drs. Esther Baena and Ying Xie for technicalassistance with experimental assays.
Grant SupportThis research was supported by NIH grant R01 CA136578 (L.A. Mucci), and
by a Career Development Award from Dana-Farber/Harvard Cancer CenterProstate Cancer SPORE (P50 CA090381) and Idea Development Awards fromthe Department of Defense (W81XWH-11-1-0329 andW81XWH-15-1-0546 toZ. Li).
The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received July 13, 2015; revised January 12, 2016; accepted January 23, 2016;published OnlineFirst February 15, 2016.
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TMPRSS2–ERG Interstitial Deletion Promotes Prostate Cancer
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TMPRSS2–ERG Interstitial Deletion Promotes Prostate Cancer
2016;76:1869-1881. Published OnlineFirst February 15, 2016.Cancer Res Douglas E. Linn, Kathryn L. Penney, Roderick T. Bronson, et al. Prostate Cancer Progression
PromotesERG and TMPRSS2Deletion of Interstitial Genes between
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