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. 2012 Jul;14(7):600–611. doi: 10.1593/neo.12600

Molecular Subtyping of Primary Prostate Cancer Reveals Specific and Shared Target Genes of Different ETS Rearrangements1,2

Paula Paulo *,†,‡,§, Franclim R Ribeiro *,†,‡,§, Joana Santos *,†, Diana Mesquita *,†, Mafalda Almeida *,, João D Barros-Silva *,†, Harri Itkonen #, Rui Henrique ¶,**,††, Carmen Jerónimo *,¶,††, Anita Sveen ‡,§, Ian G Mills ‡,#,‡‡, Rolf I Skotheim ‡,§, Ragnhild A Lothe ‡,§, Manuel R Teixeira *,†,§,††
PMCID: PMC3421956  PMID: 22904677

Abstract

This work aimed to evaluate whether ETS transcription factors frequently involved in rearrangements in prostate carcinomas (PCa), namely ERG and ETV1, regulate specific or shared target genes. We performed differential expression analysis on nine normal prostate tissues and 50 PCa enriched for different ETS rearrangements using exon-level expression microarrays, followed by in vitro validation using cell line models. We found specific deregulation of 57 genes in ERG-positive PCa and 15 genes in ETV1-positive PCa, whereas deregulation of 27 genes was shared in both tumor subtypes. We further showed that the expression of seven tumor-associated ERG target genes (PLA1A, CACNA1D, ATP8A2, HLA-DMB, PDE3B, TDRD1, and TMBIM1) and two tumor-associated ETV1 target genes (FKBP10 and GLYATL2) was significantly affected by specific ETS silencing in VCaP and LNCaP cell line models, respectively, whereas the expression of three candidate ERG and ETV1 shared targets (GRPR, KCNH8, and TMEM45B) was significantly affected by silencing of either ETS. Interestingly, we demonstrate that the expression of TDRD1, the topmost overexpressed gene of our list of ERG-specific candidate targets, is inversely correlated with the methylation levels of a CpG island found at -66 bp of the transcription start site in PCa and that TDRD1 expression is regulated by direct binding of ERG to the CpG island in VCaP cells. We conclude that ETS transcription factors regulate specific and shared target genes and that TDRD1, FKBP10, and GRPR are promising therapeutic targets and can serve as diagnostic markers for molecular subtypes of PCa harboring specific fusion gene rearrangements.

Introduction

Genomic rearrangements involving five members of the ETS family of transcription factors have been found in prostate carcinomas (PCa). Rearrangements of ERG and ETV1 were first described by Tomlins et al. [1] and are found in approximately 50% and 5% to 10% of PCa, respectively [2,3]. Rearrangements of ETV4 and ETV5 were later identified in a small proportion of PCa, representing less than 5% of all rearranged cases [4–7]. Recently, we identified FLI1 as the fifth member of the ETS family of transcription factors involved in gene fusions in PCa, being fused to the SLC45A3 gene [8].

The products of specific chimeric genes could be ideal therapy targets, but the nuclear localization of the aberrant ETS proteins makes them a difficult therapy target in vivo [9]. Therefore, it is important to characterize in detail the downstream molecular targets of each of the aberrant transcription factors, not only to understand the deregulated signaling pathways but also because some of them may turn out to be more amenable to targeted therapy. In vitro studies revealed that ERG activates plasminogen and Wnt pathways to promote degradation of the extracellular matrix and decrease cell adhesion, but very few genes have been validated as direct ERG targets [10–12]. Because ETV1 rearrangements are considerably less frequent than those of ERG, reports focusing on the oncogenic effectors of ETV1 overexpression are scarce and not based in the expression profile observed in ETV1 rearrangement-positive tumors, with some in vitro and in vivo models linking overexpression of ETV1 with the invasion potential of cancer cells by activation of matrix metalloproteinases and integrins [13–15].

Despite the apparently overlapping oncogenic potential of ERG and ETV1 gene fusions, it has not been established whether different ETS transcription factors have shared or specific downstream targets. We addressed this issue by using exon-level expression arrays in a series of 50 PCa enriched for different ETS rearrangements and validated the findings using in vitro cell line models.

Materials and Methods

Prostate Tissue Samples

We used a series of 50 tumor samples selected from a consecutive series of 200 clinically localized PCa that were previously typed for ETS rearrangements [8]. The 50 prostatectomy samples were selected to represent the various molecular subtypes of PCa, namely 21 samples with ERG rearrangement, 13 samples with ETV1 rearrangement, 2 samples with other ETS rearrangements (one with ETV4 and one with ETV5 rearrangements), and 14 samples without known ETS rearrangement. For control purposes, nine normal prostate tissues (NPTs) were collected from cystoprostatectomy specimens of bladder cancer patients. This study was approved by the institutional review board, and informed consent was obtained from all subjects.

Prostate Cell Lines

VCaP and PNT2 cells were acquired from the European Collection of Cell Cultures (Sigma-Aldrich, St Louis, MO). LNCaP, PC3, and DU145 cells were acquired from the German Resource Centre for Biological Material (DSMZ, Braunschweig, Germany). 22Rv1 cells were kindly provided by Dr David Sidransky from the Johns Hopkins University School of Medicine. The virus packaging Retro-Pack PT67 cell line was acquired from Clontech Laboratories, Inc (Saint-Germain-en-Laye, France). All prostate cell lines were cultured under the recommended conditions, being karyotyped by G banding for validation purposes and tested for Mycoplasma spp. contamination (PCR Mycoplasma Detection Set; Clontech Laboratories). After transfection, cells were grown in medium supplemented with G418 (300 µg/ml; GIBCO by Life Technologies, Carlsbad, CA) or puromycin (5 µg/ml, Clontech Laboratories), as appropriate.

Gene Expression Microarrays

RNA was extracted from tissue samples using TRIzol (Invitrogen by Life Technologies, Carlsbad, CA), as previously described [8], and 1 µg of RNA was processed into complementary DNA (cDNA) and hybridized to GeneChip Human Exon 1.0 ST arrays, following the manufacturer's recommendations. The Affymetrix Expression Console v1.1 software was used to obtain gene-level RMA-normalized expression values for the core probe sets only. We used analysis of variance in Partek Genomics Suite 6.4 (Partek, Inc, St Louis, MO) to identify differentially expressed genes among the different sample groups. The two PCa with ETV4 and ETV5 rearrangements were not included in this analysis. Specific ERG target genes were identified from genes differentially expressed between each of the three group comparisons: NPT versus ERG-positive PCa, ETS-negative PCa versus ERG-positive PCa and ETV1-positive PCa versus ERG-positive PCa. To select specific ETV1 target genes, the same approach was applied comparing NPT versus ETV1-positive PCa, ETS-negative PCa versus ETV1-positive PCa and ERG-positive PCa versus ETV1-positive PCa. Targets common to ERG and ETV1 rearrangements were identified from the differentially expressed genes in each of the four group comparisons: NPT versus ERG-positive PCa, NPT versus ETV1-positive PCa, ETS-negative PCa versus ERG-positive PCa and ETS-negative PCa versus ETV1-positive PCa. Only differentially expressed genes with a false discovery rate less than 5% and P < .02 between each two-group comparison were retained for further analyses. Principal components analysis (PCA) was performed with Partek Genomics Suite 6.4 and hierarchical clustering with MultiExperiment Viewer 4.6.0. Hierarchical clustering was performed using Spearman rank correlation and average linkage optimized for gene leaf order.

Transient Silencing of ERG Expression in VCaP Cells

To induce down-regulation of ERG expression in VCaP cells we used small interfering RNAs (siRNAs) as described by others [12]. The SMART-pool siRNA directed to ERG (M-003886-01; Dharmacon, Thermo Fisher Scientific, Rockford, IL) and the siCONTROL Non-Targeting RNA (D-001210-01; Dharmacon) were transfected into VCaP cells using Oligofectamine (Invitrogen). After 48 and 72 hours, RNA and protein extractions were performed using the TriplePrep Kit (GE Healthcare, Cleveland, OH). Expression data of VCaP-siERG cells were normalized to VCaP-siCont for each time point.

Generation of Plasmid Constructs for Stable Silencing and De Novo Overexpression

To generate constructs for stable silencing of ETV1, specific short hairpin RNA (shRNA) sequences were selected and designed using the RNAi Target Sequence Selector and the shRNA Sequence Designer, respectively (both from Clontech Laboratories). shETV1-553 (5′-GATCCGCTCATACACCGAAACCTGATTCAAGA GATCAGGTTTCGGTGTATGAGTTTTTTACGCGTG-3′) and shETV1-1037 (5′-GATCCACAAGAGCCAGGAATGTATTTCAAGAGAATACATTCCTGGCTCTTGTTTTTTTACGCGTG-3′) oligonucleotides were acquired from Sigma-Aldrich, annealed, and cloned into the pSIREN-Retro-Q vector (Clontech Laboratories) at BamHI and EcoRI restriction sites, together with the negative control shNeg (Clontech Laboratories). To generate constructs for de novo overexpression of ETV1 and ERG, full-length ETV1 CDS and truncated ERG CDS (ΔERG and ΔERGΔ8) were amplified from LNCaP and VCaP cells, respectively, using In-fusion primers (Table W1) and the Phusion Taq DNA polymerase (Finnzymes, Vantaa, Finland). Polymerase chain reaction (PCR) products were cloned into the pMSCVneo vector (Clontech Laboratories) at BglII and EcoRI restriction sites using the In-Fusion Advantage PCR cloning kit (Clontech Laboratories), according to instructions. The pMSCVneo-ETV1 construct contains the ETV1 full-length CDS (ENST00000242066), as expected; the pMSCVneo-ΔERG contains the expected CDS derived from the type III TMPRSS2-ERG transcript [16] and pMSCVneo-ΔERGΔ8 contains the alternatively spliced transcript lacking 72 bp (exon 8) [17,18].

Stable Silencing of ETV1 in LNCaP Cells

To silence the expression of ETV1 in LNCaP cells, PT67 cells were transfected with each construct and also with the control vector pSIREN-shNeg using the CaPO4-based transfection method, following Clontech's recommendations (protocol no. PT3132-1, version no. PR631543). Transfected cells were selected with puromycin and expanded. LNCaP cells were exposed to viral medium for 8 hours and allowed to recover for 24 hours in regular growth conditions. Stable LNCaP-shETV1 and LNCaP-shNeg populations were obtained with puromycin-selective pressure. Two independent, low ETV1 expression clones (LNCaP-shETV1-C1 and LNCaP-shETV1-C2) were isolated and used for further analyses.

Stable Overexpression of ETV1 and ΔERG Isoforms in PNT2 Cells

For stable expression of the ETV1 and ΔERG isoforms described, PT67 cells were transfected with pMSCV constructs and with the empty vector pMSCVneo as previously described. Transfected cells were selected with G418 and expanded. Transduction of PNT2 cells was carried out as previously described for LNCaP cells. A control population (PNT2-Neo) and two independent populations showing overexpression of either ETV1 (PNT2-ETV1-A and PNT2-ETV1-B) or ΔERG isoforms (PNT2-ΔERG-A, PNT2-ΔERG-B, PNT2-ΔERGΔ8-A and PNT2-ΔERGΔ8-B) were obtained with G418-selective pressure.

In Silico Selection of Target Genes for Further Validation

We used the expression data of VCaP, LNCaP, PC3, and DU145 cell lines available from Taylor et al. that can be accessed from the Gene Expression Omnibus (GSE21034) to select the candidate target genes where differential expression was specific of the cell line models harboring ETS rearrangements, taking into consideration the candidate target genes resulting from the microarray analysis of the prostate tumor samples. Using the RMA-normalized signal intensity values, ERG-associated genes were selected as those differentially upregulated or down-regulated at least 1.5-fold in VCaP cells comparing with the others. Similarly, ETV1-associated genes were those differentially upregulated or downregulated at least 1.5-fold in LNCaP cells comparing with the others, and target genes shared by ERG and ETV1 rearrangements were those differentially upregulated or downregulated at least 1.5-fold in VCaP or LNCaP cells comparing with PC3 and DU145 cell lines.

Quantitative Real-time Polymerase Chain Reaction

RNA was extracted from subconfluent cell lines using the RNeasy mini kit (Qiagen, GmbH, Hilden, Germany). cDNA was obtained from 1 µg of RNA using oligo-dT primers and the H-minus RevertAid cDNA synthesis kit (Fermentas, Ontario, Canada), according to the manufacturer's instructions. Expression of target genes was quantified using pre-developed TaqMan assays from Applied Biosystems (Life Technologies, Foster City, CA) (Table W1) and normalized to the expression of the GUSB housekeeping gene using the comparative Ct method [19].

Western Blot Analysis

Protein was extracted from subconfluent cells using RIPA lysis buffer in the presence of protease inhibitors (Santa Cruz Biotechnology, Inc, Heidelberg, Germany), and concentration was determined by the BCA protein assay (Thermo Fisher Scientific), following the manufacturer's recommendations. Specific detection of ERG and ETV1 was achieved by incubation with rabbit anti-ERG (1:1000; Epitomics, Burlingame, CA) and mouse anti-ETV1 (1:500; Sigma-Aldrich) monoclonal antibodies, respectively. An anti-β-actin monoclonal antibody (1:8000; Sigma-Aldrich) was used to control protein loading.

Bisulfite Treatment and Quantitative Methylation-Specific PCR of TDRD1

Genomic DNA was extracted from prostate tissues and cell lines using a standard technique comprising digestion with proteinase K (20 mg/ml) in the presence of 10% SDS at 55°C, followed by phenol-chloroform extraction and precipitation with 100% ethanol [20]. In 4 of the 50 PCa samples (1 ERG-positive, 1 ETV1-positive, and the 2 samples with other ETS rearrangements), it was not possible to obtain DNA. One microgram of DNA was submitted to bisulfite modification using the EZ DNA Methylation Gold Kit (Zymo Research, Orange, CA) following the manufacturer's instructions. Bisulfite-modified DNA was amplified by quantitative methylation-specific PCR (qMSP) using TaqMan technology [21]. Specific TDRD1 primers and TaqMan probe were designed using the Methyl Primer Express Software v1.0 (Applied Biosystems; Table W1). β-Actin (ACTB) was used as an internal reference gene to normalize for DNA input and qMSP reaction was performed as previously described [22]. Methylation levels for each sample were obtained from calibration curves constructed using serial dilutions of bisulfite-modified CpGenome Universal Methylated DNA (Millipore, Billerica, MA). TDRD1 methylation levels were obtained after normalization to ACTB.

Bisulfite Sequencing

To obtain detailed information about the methylation status of CpG sites in the CpG island found in the TDRD1 promoter, bisulfite-sequencing PCR primers (Table W1) that span the region of interest were tested in bisulfite-modified DNA from VCaP, LNCaP, PC3, DU145, 22Rv1, and PNT2 cells. PCR was performed as previously described [23].

Chromatin Immunoprecipitation and Quantitative PCR

Transcription factor-dependent gene expression is associated with the recruitment of transcription factors to regulatory sequences. This is detected commonly through the use of chromatin immunoprecipitation and, where sites are known or predicted, through quantitative PCR (qPCR) for the bound regions. We used VCaP cells and the rabbit anti-ERG monoclonal antibody (Epitomics) to detect ERG binding to the promoter of candidate target genes. For each immunoprecipitation with the EZ-Magna chromatin immunoprecipitation (ChIP) G kit (Millipore), 2 x 106 cells were used, following the manufacturer'sinstructions [24,25]. We used two approaches to identify candidate sites within the promoter regions. One approach was to mine the ChIP-Seq data set available from Yu et al. [26] for ERG generated in the VCaP cell line (GSE14092, sample GSM353647). Processed sequencing files were uploaded into the UCSC genome browser and aligned to build hg18. Gene proximal sites were then selected. In addition, we also used in silico prediction by uploading promoter sequences into ConSite—http://asp.ii.uib.no:8090/cgi-bin/CONSITE/consite/ [27]. Overall, four promoter regions were analyzed for TDRD1 (-1207, -3196, -7686, and -8768) and three for GRPR (-583, -1386, and -4858), KCNH8 (-797, -1472, and -3506), and TMEM45B (-260, -2847, and -5687). Primers were designed using the Primer3 online software and acquired from Metabion (Martinsried, Germany). Primers for a negative control region were also included to correct for unspecific binding (Table W1) [28]. qPCR was performed using Power SYBR Green (Applied Biosystems), according to the manufacturer's recommendations. Results are shown as a fold enrichment of ERG bound chromatin relative to IgG and corrected to the negative control region [29].

Cell Line Treatment with Epigenetic Modulating Drugs

To evaluate whether TDRD1 promoter demethylation was regulated by ERG, we treated PNT2 cells with de novo expression of ΔERG and ETV1 with 1 µM of the DNA methyltransferases inhibitor 5-aza-2′ deoxycytidine (DAC; Sigma-Aldrich) and with 0.5 µM of the histone deacetylase inhibitor Trichostatin A (TSA; Sigma-Aldrich), both individually and in combination, as described by Costa et al. [23]. After 72 and 24 hours of treatment with DAC and TSA, respectively, DNA and RNA were extracted using the TriplePrep Kit (GE Healthcare), according to the manufacturer's recommendations.

Statistical Analysis

To compare gene expression data between the different sample groups, the Mann-Whitney nonparametric test was applied on RMA-normalized data using the Statistical Package for Social Sciences, version 15.0 (SPSS, Inc, Chicago, IL). Data from qMSP and mRNA expression of TDRD1 were compared with the Spearman nonparametric correlation test. Student's t test was applied to evaluate differences in the expression data obtained by quantitative real-time PCR (qRT-PCR). P < .05 was considered statistically significant.

Results

Differential Expression Analysis in Primary Tumors

Expression array data allowed the identification of both specific and shared ERG and ETV1 expression-associated genes (Figure 1). Distribution of samples according to the expression profile of the 22,000 genes shows that the PCa samples form a unique cluster that deviates from the expression profile found in the NPT control samples (Figure 2A). Combined analysis of genes differentially expressed among the different sample groups (NPT, ETS-negative PCa, ERG-positive PCa and ETV1-positive PCa) led to the identification of 57 genes specifically associated with PCa with ERG rearrangement (Table W2) and 15 genes specifically associated with PCa with ETV1 rearrangement (Table W3), with 27 genes being differentially expressed in both PCa subgroups comparing to PCa without ETS rearrangements and with NPT (Table W4). PCA using the expression data of the 99 genes thus selected shows four completely independent sample clusters: NPT controls, ETS-negative PCa, ERG-positive PCa, and ETV1-positive PCa (Figure 2B). Hierarchical clustering of the samples according to expression of the 99 genes and of ERG and ETV1 shows clear stratification according to the ETS rearrangement status (Figure 2C). Interestingly, the two PCa with other ETS rearrangements (involving ETV4 and ETV5) cluster in close proximity with ETV1-positive PCa samples.

Figure 1.

Figure 1

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Workflow applied to 9 NPTs and 50 PCa previously characterized for the presence of known ETS rearrangements to identify both specific and shared ERG and ETV1 target genes by differential expression analysis. The two PCa with other ETS rearrangements were not included in Partek Genomics Suite analysis.

Figure 2.

Figure 2

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PCA and hierarchical clustering of the expression profile obtained for normal controls (NPT) and PCa samples. (A and B) Sample distribution obtained by PCA in Partek Genomics Suite using the full gene expression profile and the 99 gene panel obtained by differential expression analysis, respectively. (C) Hierarchical clustering of the 9 NPT samples and the 50 PCa obtained with the RMA-normalized expression data of the panel of 99 genes selected with Partek's differential expression analysis. The nine NPTs are shown in black, the ETS-negative PCa in gray, the ERG-positive PCa in red, the ETV1-positive PCa in dark purple, and the other ETS-positive PCa in light purple.

Selection of Target Genes for Validation in VCaP and LNCaP Cell Line Models

Using the expression profile of VCaP, LNCaP, PC3, and DU145 cell lines available from Taylor et al. [30] (GSE21034), of our list of 57 ERG candidate target genes, only 7 (ATP8A2, CACNA1D, HLADMB, PDE3B, PLA1A, SH3RF1, and TDRD1) were significantly upregulated and 1 (TMBIM1) was significantly downregulated in VCaP cells compared with the other cell lines (Figure W1). Following the same approach, only 2 (FKBP10 and GLYATL2) of the 15 candidate ETV1 target genes were significantly upregulated in LNCaP cells comparing with the other cell lines, and only 7 (CDC2L6, GRPR, KCNH8, NCALD, PLA2G7, TMEM45B, and ZNF385B) of the 27 target genes shared by ERG and ETV1 rearrangements were overexpressed at least in one of the two ETS-positive cell lines comparing with PC3 and DU145 (Figure W1). In silico analysis of the ChIP-Seq data set available from Yu et al. [26] confirmed ERG binding to the promoter of both the eight specific and the seven shared ERG candidate target genes in VCaP cells (Table W5).

ERG-Dependent Deregulation of Tumor-Associated ERG Target Genes in VCaP Cells

Quantitative expression analysis of the eight ERG candidate target genes after siRNA-mediated ERG silencing in VCaP cells shows that expression of all genes but SH3RF1 is significantly affected by ERG knockdown (Figure 3, A and B). De novo overexpression of the most common ERG truncated isoforms (ΔERG and ΔERGΔ8) in the benign prostate cell line PNT2, however, did not show the reverse effect (Figure 3, C and D). Expression levels of the seven deregulated ERG candidate target genes were not affected by ETV1 silencing in LNCaP cells (data not shown), thus confirming that the observed ERG-dependent regulation is specific of tumor cells harboring ERG overexpression.

Figure 3.

Figure 3

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Validation of tumor-associated ERG target genes in cell line models. (A) Using siRNAs directed to ERG, a significant down-regulation of ERG expression was achieved in VCaP cells, as evaluated both by qRT-PCR and by Western blot analyses at 48 and 72 hours after transient transfection. (B) Of the eight tumor-associated ERG candidate target genes, seven were significantly deregulated after ERG silencing, as evaluated by qRT-PCR. (C) De novo expression of two ΔERG isoforms was stably induced in the benign PNT2 prostate cells, as shown both by qRT-PCR and Western blot analyses. (D) De novo expression of ΔERG isoforms was not sufficient to induce expression of the tumor-associated ERG target genes in PNT2 cells. *P < .05. **P < .01. NS indicates not significant (P > .05).

ETV1 Overexpression Drives Up-regulation of the Tumor-Associated Target FKBP10

A significant down-regulation of FKBP10 and GLYATL2 was observed in the LNCaP-shETV1 clones (Figure 4, A and B). Interestingly, de novo expression of full-length ETV1 in PNT2 cells showed significant up-regulation of FKBP10 in the PNT2-ETV1-B population (Figure 4C and 4D). These observations were not found in VCaP-siERG cells neither in any of the PNT2-ERG cells (data not shown), thus suggesting that ETV1 overexpression specifically drives FKBP10 up-regulation in prostate cells.

Figure 4.

Figure 4

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Validation of tumor-associated ETV1 target genes in cell line models. (A) Using shRNAs directed to ETV1, a significant down-regulation of ETV1 expression was achieved in LNCaP cells, as evaluated both by qRT-PCR and by Western blot analyses. (B) FKBP10 and GLYATL2, two tumor-associated ETV1 candidate target genes, were shown to be significantly decreased after ETV1 silencing. (C) De novo expression of ETV1 was stably induced in the benign PNT2 prostate cells, as shown by qRT-PCR and Western blot analyses. (D) De novo expression of ETV1 induced significant up-regulation of FKBP10 expression in the PNT2-ETV1-B cells, which show higher expression of ETV1. (E) Box plot distribution of the expression of FKBP10 among the different sample groups shows a significant up-regulation of FKBP10 in ETV1-positive PCa comparing with other PCa and with NPT samples. **P < .01. NS indicates not significant (P > .05).

Shared Tumor-Associated ETS Target Genes Are Regulated by Both ERG and ETV1 in Prostate Cancer Cell Lines

Quantification of the expression levels of the seven candidate target genes shared by ERG and ETV1 rearrangements showed that GRPR, KCNH8, and TMEM45B are significantly downregulated after silencing of both ETS transcription factors (Figure 5, A and B). Interestingly, GRPR and KCNH8 were the topmost overexpressed genes of our list of ERG and ETV1 shared candidate target genes (Figure 5C). qPCR on the ERG-immunoprecipitated chromatin from VCaP cells showed direct binding of ERG to the promoters of GRPR (region -583), KCNH8 (region -1472), and TMEM45B (region -260) (Figure 5D).

Figure 5.

Figure 5

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Validation of shared ERG and ETV1 tumor-associated target genes in cell line models. (A and B) Quantitative expression analysis of the seven ETS shared candidate target genes in ERG-downregulated VCaP cells and ETV1-downregulated LNCaP cells, respectively, shows that GRPR, KCNH8, and TMEM45B are significantly downregulated after silencing of either ERG or ETV1. (C) Box plot distribution of the expression of GRPR (left panel) and KCNH8 (right panel) among the different prostate sample groups shows significant up-regulation of both genes in ERG- and ETV1-positive PCa comparing with ETS-negative PCa and with NPT samples. (D) qPCR of ERG-immunoprecipitated chromatin from VCaP cells shows that ERG binds to the GRPR, KCNH8, and TMEM45B promoters. Only the promoter regions that gave a relative enrichment value above the negative control are shown. (E) De novo expression of two ΔERG isoforms and of ETV1 was not sufficient to induce expression of these genes in PNT2 cells. *P < .05. **P < .01. NS indicates not significant (P > .05).

TDRD1 Expression Is Regulated by ERG in Prostate Tumors Harboring ERG Rearrangement

Considering the highly significant association of our topmost over-expressed gene, TDRD1, with PCa harboring ERG rearrangements (Figure 6A), we first questioned whether TDRD1 was a direct target of ERG. qPCR on the ERG-immunoprecipitated chromatin from VCaP cells showed that ERG binds to the TDRD1 promoter in two promoter regions (Figure 6B).

Figure 6.

Figure 6

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Analysis of the topmost tumor-associated ERG target gene - TDRD1. (A) Box plot distribution of the nine prostate controls (NPT) and the 48 PCa according to the expression of TDRD1 shows a significant P value comparing ERG-positive PCa with other PCa and with NPT. (B) qPCR of ERG-immunoprecipitated chromatin from VCaP cells shows that ERG binds to two regions of the TDRD1 promoter. Only the promoter regions that gave a relative enrichment value above the negative control are shown. (C) Quantitative expression and methylation levels of TDRD1 in prostate controls and tumors show an inverse correlation (rs = -0.417, P = .0015). NPTs are underlined in black, ETS-negative PCa in gray, ERG-positive PCa in red, and ETV1-positive PCa in purple. (D) Schematic representation of the CpG island found in the TDRD1 promoter and of the methylation status of each CG dinucleotide in prostate cell lines. The location of the primers used for bisulfite sequencing is shown by black arrows and the location of the primers and the TaqMan probe used for qMSP is shown in blue. (E) Quantitative expression and methylation levels of TDRD1 in PNT2 cells show that de novo expression of two ΔERG isoforms and of ETV1 does not increase the demethylation-induced expression of TDRD1 in the presence of DAC.

Because TDRD1 is described as a cancer germ line gene regulated by methylation [31], we questioned whether methylation levels of TDRD1 promoter differ among NPT control samples and the different subgroups of PCa of our series. A CpG island with 28 CpG dinucleotides was found starting at -66 bp of the transcription start site (Ensembl gene ID ENSG00000095627) and covering 330 bp (Figure 6, C and D). As expected, a significant inverse correlation was obtained between TDRD1 mRNA expression (exon array data, linear values) and TDRD1 methylation levels (rs = -0.417, P = .0015). A significant decrease in TDRD1 methylation was found between tumors harboring ERG rearrangements and both NPT (P =.004) and tumors without ETS rearrangements (P = .0001), whereas methylation levels of NPT and ETS-negative PCa were not statistically different (P = .124).

Bisulfite sequencing of the TDRD1 promoter in VCaP, LNCaP, PC3, DU145, 22Rv1, and PNT2 cells showed that the TDRD1 promoter is completely methylated in all cell lines except in VCaP cells (Figure 6D), the only cell line that shows expression of TDRD1 by qRT-PCR (data not shown). Interestingly, the CpG island completely overlaps with the promoter region at -8768 bp shown to be bound by ERG using ChIP. To evaluate whether ERG overexpression modulates the levels of methylation-controlled TDRD1 expression in prostate cancer cells, we treated PNT2 cell populations (PNT2-Neo, PNT2-ERG, and PNT2-ETV1) with epigenetic modulating drugs. Treatment with DAC induced TDRD1 expression in all PNT2 cells, and this reexpression was associated with decreased methylation levels of the TDRD1 promoter (Figure 6E). These effects were not observed when cells were treated with TSA alone, and neither were they increased with the combination of TSA and DAC (data not shown).

Discussion

We have analyzed a clinical series of PCa enriched for ERG and ETV1 rearrangements with a genome-scale and exon-level expression microarray platform that ensures robust gene-level expression measures. Of 57 ERG-associated genes in primary PCa, 8 were also deregulated in VCaP cells with the TMPRSS2-ERG fusion. In fact, seven of these genes were shown to be significantly affected by ERG knockdown. Six of these genes (PLA1A, CACNA1D, ATP8A2, HLA-DMB, PDE3B, and TDRD1) have been previously described as coexpressed with ERG in prostate cancer, but only PLA1A and CACNA1D are validated as direct ERG target genes [12,18,32–35]. The top-ranked tumor-associated ERG target gene in our study was TDRD1, and we showed not only that TDRD1 expression is regulated by methylation of a CpG island located at -66 bp of the transcription start site [31] but also that ERG binds to the unmethylated CpG island of the TDRD1 promoter in VCaP cells. Although ERG silencing in VCaP cells resulted in down-regulation of TDRD1, our data on de novo overexpression of ERG in PNT2 cells suggest that another regulatory mechanism acting upstream of ERG actively leads to demethylation of TDRD1 promoter or that other cofactors may be required for ERG-mediated TDRD1 demethylation. TDRD1 encodes the tudor domain-containing protein 1 described as involved in male germ cell differentiation and in the small RNAs pathway [36–38]. Although the biologic consequence of over-expressed or reexpressed TDRD1 is not known, loss of TDRD1 in germ line cells is associated with changes in small RNA profile and with loss of methylation of L1 transposons [39] and may thus establish a link between ERG overexpression and the epigenetic reprogramming described by others [26,40,41].

Of the 15 genes highly associated with tumors harboring ETV1 rearrangements, only 2 genes were shown to have the expected over-expression in the LNCaP cell line harboring an ETV1 rearrangement. Both FKBP10 and GLYATL2 were significantly downregulated after ETV1 knockdown, but only FKBP10 seemed to be upregulated in PNT2 cells with de novo expression of ETV1. FKBP10 (FK506-binding protein 10) encodes a member of the highly conserved family of intracellular receptors called immunophilins, which acts as a molecular chaperone in the endoplasmic reticulum [42]. We found no reports on FKBP10 involvement in prostate carcinogenesis, but other immunophilins, namely FKBP51 and FKBP52, have been described to be androgen regulated and their interaction with androgen receptor (AR) seems to be necessary for AR-mediated proliferation of LNCaP cells [43]. In the same cells, the presence of its ligand, FK506, was sufficient to block several stages of the AR signaling [44]. Taken together, these observations suggest that inhibition of FKBP10 by FK506 may be a good therapy approach for the treatment of PCa harboring ETV1 rearrangements. Interestingly, when the expression profiles of the two PCa with ETV4 and ETV5 rearrangements were included in the hierarchical clustering, they clustered among the ETV1-positive PCa samples. This suggests that the ETV4 and ETV5 tumor-associated target genes might be, at least in part, shared with ETV1, which, altogether, represent the PEA3 subfamily of ETS transcription factors [45].

Although the identification of specific target genes of ERG and ETV1 rearrangements in PCa is a major finding in this work, the existence of shared target genes was expected because both genes belong to the same family of transcription factors [46]. In fact, we report a list of 27 target genes shared by ERG and ETV1 rearrangements. KCNH8 and NCALD have been previously associated with tumors harboring ERG rearrangements [32–34], but no biologic validation of their ERG dependence had been shown. Our results, using the VCaP and LNCaP knockdown cell line models, clearly validate KCNH8, GRPR, and TMEM45B as downstream targets of both ERG and ETV1, as also indicated by our demonstration of direct binding of ERG to the promoter of these genes using ERG-immunoprecipitated chromatin from VCaP cells. TMEM45B encodes a putative membrane protein with unknown function, so its role in prostate carcinogenesis might be worth exploring. Nevertheless, GRPR, which encodes the gastrin-releasing peptide receptor, has been described as overexpressed in several cancer types, including PCa [47–51]. Overexpression of GRPR was found in androgen-dependent prostate cancer xenografts [52], and it seems to be dependent on AR activation [53]. Recently, Beer et al. [50] described that combined overexpression of GRPR and AR was associated with a favorable prognosis in patients with PCa. These observations, together with our findings showing GRPR overexpression in a high proportion of PCa harboring either ERG or ETV1 rearrangements, warrant further investigation on the cooperation of ETS transcription factors and AR signaling in regulating the expression of GRPR in PCa.

Only a fraction of the ERG and ETV1 tumor-associated genes showed the expected expression pattern in VCaP and LNCaP cell lines, the best available in vitro models of ERG- and ETV1-positive PCa. This by no means indicates that the remaining potential ETS target genes found in primary tumors are not relevant for in vivo prostate carcinogenesis; it may be that these cell lines have kept only the part of the in vivo tumor-derived gene expression signature that was advantageous for in vitro survival or the in vitro cell line-associated gene expression signature is being modulated by the environmental factors to which cells are exposed. In fact, our PCa series is derived from organ confined or locally advanced tumors [8] removed by radical prostatectomy before any other therapy, meaning that they were, most probably, androgen responsive. Nevertheless, although VCaP and LNCaP cells are androgen responsive [54,55], the gene expression signature available from Taylor et al. [30] and the ERG and ETV1 silencing experiments that were performed were obtained without androgen stimulation. This suggests that the expression of some tumor-associated ERG and ETV1 target genes might be dependent on androgen receptor activation, whereas others might be androgen independent. The same explanation may be operative with the overall absence of effect on the tested target genes that was observed with de novo expression of either ΔERG isoforms or ETV1 in the benign PNT2 cells, which are also androgen sensitive [56]. Silencing and de novo expression of ERG and ETV1 in these cell line models under androgen stimulation, together with cell line-based assays focusing in specific ERG and ETV1 targets, would be useful to clarify the cooperativity/dependence of these ETS transcription factors and/or AR signaling.

In conclusion, differential expression profile of tumors harboring either ERG or ETV1 rearrangements allowed the identification of both specific and shared ETS downstream targets. From detailed studies in prostate cancer models, we have validated ETS-dependent expression of seven ERG-specific, two ETV1-specific, and three ERG and ETV1 shared target genes. TDRD1, FKBP10, and GRPR are promising therapeutic targets and can serve as diagnostic markers for molecular subtypes of PCa harboring specific fusion gene rearrangements.

Supplementary Material

Supplementary Figures and Tables
neo1407_0600SD1.pdf (212.8KB, pdf)

Abbreviations

AR

androgen receptor

DAC

5-aza-2′deoxycytidine

NPT

normal prostate tissue

PCA

principal components analysis

PCa

prostate carcinoma

qMSP

quantitative methylation-specific PCR

qRT-PCR

quantitative real-time polymerase chain reaction

shRNA

short hairpin RNA

siRNA

small interfering RNA

TSA

Trichostatin A

Footnotes

1

This work was supported by research grant PTDC/SAU/OBD/70543/2006 awarded by Fundação para a Ciência e a Tecnologia (FCT) and by research grant CI-IPOP-8-2008 funded by the Portuguese Oncology Institute, Porto (M.T.). P.P. (SFRH/BD/27669/2006), F.R.R. (SFRH/BPD/26492/2006), J.S. (SFRH/BD/73964/2010), and J.D.B.S. (SFRH/BD/46574/2008) are research fellows from FCT. D.M. is a research fellow from Liga Portuguesa Contra o Cancro, Núcleo Regional do Norte (M.T.). A.S. has a PhD grant from the Research Council at Rikshospitalet-Radiumhospitalet Health Enterprise (R.A.L.). The study is also supported by grants from the Norwegian Cancer Society (PR-2007-0166 to R.I.S. and PR-2006-0442 to R.A.L.). H.I. is funded by an Early Stage Researcher fellowship as part of the EU FP7 Marie Curie Integrated Training Network, PRO-NEST (Prostate Research Organizations - Network Early Stage Training). I.G.M. is supported by funding from the Norwegian Research Council, Helse Sor-Ost and the University of Oslo through the Centre for Molecular Medicine (Norway), which is the part of the Nordic EMBL (European Molecular Biology Laboratory) partnership. I.G.M. is also supported by the Norwegian Cancer Society and by EU FP7 funding. I.G.M. holds a visiting scientist position with Cancer Research UK through the Cambridge Research Institute and a Senior Visiting Research Fellowship with Cambridge University through the Department of Oncology. The authors declare no conflict of interest.

2

This article refers to supplementary materials, which are designated by Tables W1 to W5 and Figure W1 and are available online at www.neoplasia.com.

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Supplementary Materials

Supplementary Figures and Tables
neo1407_0600SD1.pdf (212.8KB, pdf)

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