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TMPRSS2:ERG Fusion by Translocation or Interstitial Deletion Is Highly Relevant in Androgen-Dependent Prostate Cancer, But Is Bypassed in Late-Stage Androgen Receptor–Negative Prostate Cancer

Departments of ¹ Pathology and ² Urology, Josephine Nefkens Institute, Erasmus University Medical Center, Rotterdam, the Netherlands

Requests for reprints: Jan Trapman, Department of Pathology, Josephine Nefkens Institute, Erasmus University Medical Center, PO Box 2040, 3000 CA Rotterdam, the Netherlands. Phone: 31-104087933; Fax: 31-104089487; E-mail: j.trapman{at}erasmusmc.nl.

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Recently, a unique fusion between the prostate-specific, androgen-regulated TMPRSS2 gene and the ETS genes ERG, ETV1, or ETV4 has been described in clinical prostate cancer. We investigated mechanisms of expression of four ETS genes, ERG, ETV1, ETV4, and FLI1, in 11 xenografts representing different stages of prostate cancer. All five androgen-dependent xenografts showed as major transcript overexpression of two splice variants of TMPRSS2:ERG, linking TMPRSS2 exon 1 or 2 sequences to ERG exon 4. In one of two androgen-sensitive xenografts, fusion transcripts of TMPRSS2 and ETV1 were detected. Array-based comparative genomic hybridization and interphase fluorescence in situ hybridization indicated both interstitial deletions and translocations as mechanisms of TMPRSS2:ERG gene fusion. Importantly, TMPRSS2 to ERG fusions were also observed in three of four androgen-independent, androgen receptor (AR)–negative xenografts and in two AR-negative clinical prostate cancer specimens; however, the fusion gene was not expressed. In almost all AR-negative tumor samples, overexpression of wild-type ETV4 or FLI1 was detected. Combined, our observations indicate a key role of fusion of TMPRSS2 and ETS genes in most androgen-regulated prostate cancers, which might be bypassed by androgen-independent expression of wild-type ETS factors in late-stage disease. (Cancer Res 2006; 66(22): 10658-63)

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Prostate cancer is the most frequent cancer in men in countries with a Western lifestyle and the second cause of male cancer death (1). Surgery and radiation are standard therapy of localized prostate cancer. Palliative therapy of metastatic prostate cancer aims at blocking androgen receptor (AR) function. A better understanding of the molecular mechanisms of tumorigenesis is essential for the development of novel therapies. Additionally, a knowledge of the mechanism of prostate cancer development will improve prediction of the clinical course of the disease.

Recently, overexpression of the ETS gene ERG has been described in clinical prostate cancer (2). Subsequently, it was shown that overexpression of ERG and related ETV1 was due to fusion of the TMPRSS2 gene to either ERG or ETV1 (3). This important finding adds gene fusion to the mechanisms of gene overexpression in epithelial tumors. At low frequency, TMPRSS2 might also be fused to ETV4 in prostate cancer (4). Expression of TMPRSS2 that maps to 21q22 is androgen regulated and prostate specific (5). ERG is also located on 21q22, 3 Mbp proximal to TMPRSS2. ETV1 maps to 7p21 and ETV4 to 17q21. Together with FLI1, modified ERG, ETV1, and ETV4 are well-known oncogenes involved in translocations in Ewing sarcoma and acute myeloid leukemia (6).

We investigated the ETS genes ERG, ETV1, ETV4, and FLI1 in human prostate cancers transplanted on nude mice. Xenografts are powerful models for dedicated genetic and molecular studies because they lack normal cells of human origin. The xenografts used represent a variety of clinical stages of prostate cancer, ranging from primary tumors and local metastases to recurrent disease and distant metastases, and from androgen-dependent to androgen-independent cancers (7–9). Our data reveal both interstitial deletion and gene translocation as mechanisms of fusion between TMPRSS2 and ERG. Further, our results show high overexpression of two splice variants of the TMPRSS2:ERG fusion gene in all androgen-dependent xenografts and absence of ERG overexpression in late-stage, AR-negative xenografts, even if they contain a TMPRSS2:ERG fusion gene. The latter observation was also made in AR-negative clinical prostate cancer. In almost all late-stage, AR-negative prostate cancer samples, apparently androgen-independent expression of wild-type ETV4 and FLI1 is detected. These findings show a key role of TMPRSS2:ERG in androgen-dependent prostate cancer, which might be bypassed by other ETS factors in late-stage, AR-negative disease.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Prostate cancer samples. The in vivo growing xenografts PCEW, PC82, PC133, PC135, PC295, PC310, PC324, PC329, PC339, PC346, and PC374 were propagated by serial transplantation on male nude mice as described (7–9). PCEW, PC82, PC295, PC310, and PC329, derived from primary tumors or local metastases, are androgen-dependent. PC133, PC324, PC339, PC346, and PC376 are derived from distant metastases or local progressive disease and are androgen-independent (PC133, PC324, and PC339) or androgen-sensitive (PC346 and PC374). PC135 is androgen independent and is derived from a lymph node metastasis (see Supplementary Table S1).

Clinical prostate tumor samples were obtained from recurrent disease by transurethral resection (TUR-P) after informed consent, following approval of the institutional ethical committee.

DNA and RNA preparation. Genomic DNA was isolated using the Puregene system from Gentra Systems (Minneapolis, MN) according to the procedure described by the manufacturer. Xenograft RNA was isolated according to the LiCl protocol (10). For isolation of RNA from clinical samples, the Illustra mini RNA kit (General Electric Healthcare, Fairfield, CT) was used.

Array-based comparative genomic hybridization. Arrays were produced from the human 3600 bacterial artificial chromosome (BAC)/P1-derived artificial chromosome genomic clone set of the Welcome Trust Sanger Institute, covering the full genome at 1 Mb spacing. Degenerated oligonucleotide PCR products were prepared for spotting on CodeLink slides (General Electric Healthcare) according to published protocols (11) with some modifications (12). DNA labeling and hybridization were done essentially as described (11) with minor modifications (13). After hybridization arrays were scanned in a ScanArray Express HT (Perkin-Elmer, Freemont, CA). The resulting images were analyzed with GenePix Pro 5.0 software (Axon Instruments, Foster City, CA) and subsequently visualized with an excel macro (12).

Quantitative PCR. Analysis of mRNA expression was done by quantitative PCR (QPCR). Two micrograms RNA were reverse transcribed using 400 units M-MLV RT (Invitrogen Life Technologies, Carlsbad, CA) and an oligo-dT12 primer. QPCR was done in an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). QPCR reactions were done in Power SYBR Green PCR Master Mix (Applied Biosystems) containing 330 nmol/L forward and reverse primer in a total volume of 25 µL. Thermocycling conditions were according to the recommendations of the manufacturer. Amounts of specific RNAs for each sample were determined relative to posphobilinogen deaminase (PBDG) by the standard curve method (Applied Biosystems). Primer combinations used were as follows: PBGD-F (5'-CATGTCTGGTAACGGCAATG-3') and PBGD-R (5'-GTACGAGGCTTTCAATGTTG-3'). PSA-4A (5'-ACGTGTGTGCAAGTTCACC-3') and PSA-5B (5'-TGTACAGGGAAGGCCTTTCG-3'). TMPRSS2-F (5'-CCTCTGGTCACTTCGAAGAAC-3') and TMPRSS2-R (5'-GTAAAACGACGTCAAGGACG-3'). AR-7/8A (5'-TGACTCCGTGCAGCCTATTG-3') and AR-8B (5'-ATGGGAAGCAAAGTCTGAAG-3'). TMPRSS2-E1:ERG-E4F (5'-AGCGCGGCAGGAAGCCTTA-3') and ERG-E4/5R (5'-CATCAGGAGAGTTCCTTGAG-3'). TMPRSS2-E2:ERG-E4F (5'-GATGGCTTTGAACTCAGAAGC-3') and ERG-E4/5R. TMPRSS2-E3F (5'-CCACCAGCTATTGGACCTTA-3') and ERG-E4/5R (5'-CATCAGGAGAGTTCCTTGAG-3'). TMPRSS2-E1F (5'-GAGCTAAGCAGGAGGCGGA-3') and ETV1-E5R (5'-TGACTGCAGGCAGAGCTGAT-3'). TMPRSS2-E2F (5'-CCTATCACTCGATGCTGT-3') and ETV1-E5R. ERG-F (5'-TGCTCAACCATCTCCTTCCA-3') and ERG-R (5'-TGGGTTTGCTCTTCCGCTCT-3'). ETV1-F (5'-CATACCAACGGCGAGGATCA-3') and ETV1-R (5'-TGGAGAAAAGGGCTTCTGGA-3'). ETV4-F (5'-ACCGGCCAGCCATGAATTAC-3') and ETV4-R (5'-GAGAGCTGGACGCTGATTG-3'). FLI1-F (5'-GAGGAGCTTGGGGCAATAAC-3') and FLI1-R (5'-AGAGCAGCTCCAGGAGGAAT-3').

Interphase fluorescent in situ hybridization. Nuclear suspensions of the prostate cancer xenografts were prepared essentially as described by Vindelov et al. (14). Interphase fluorescent in situ hybridization (FISH) was done as described (15). BAC clones RP11-164E1, RP5-1031F17 (both flanking ERG, see Fig. 2A), RP11-113F1 (TMPRSS2, see Fig. 2A), RP11-79G16 (ETV1), RP11-268E15 (ETV4) and RP11-44O2 (FLI1) were purchased from BacPac Resources (Oakland, CA). Specificity of BACs is shown in Supplementary Figs. S1 and S2. BAC DNA clones were biotin-16-dUTP or digoxigenin-11-dUTP labeled using a nick translation reagent kit (Vysis, Downers Grove, IL) according to the directions of the manufacturer. Biotin-labeled probes were visualized with FITC-conjugated avidin (Vector Laboratory, Burlingame, CA) and digoxigenin-labeled probes with rhodamine-conjugated antidigoxigenin antibody (Roche, Mannheim, Germany). Cells were 4',6-diamidino-2-phenylindole counterstained. Images of the three fluorochromes were collected on an epifluorescence microscope (Leica DM, Rijswijk, the Netherlands) equipped with appropriate filter sets (Leica) and a CCD cooled camera (Photometrics, Tucson, AZ).

View larger version (28K): [in this window] [in a new window]   Figure 2. Array-based CGH analysis of chromosome 21 and interphase FISH of nuclei of prostate cancer xenografts PC295 and PC82. A, chromosome 21 region indicating the position of ERG, TMPRSS2, and flanking genes in Mbp from the top of the p arm. Positions of BAC clones in this region are indicated above the map. Genes mapping in this chromosomal region are indicated below the map. BACs used in interphase FISH are in red or green. B, PC295: array-CGH of 21q and representative interphase FISH (two green spots and one red spot) of a nucleus, using BACs RP11-164E1 (green) and RP5-1031P17 (red). C, PC82: array-CGH of 21q and representative interphase FISH of a nucleus, using the same BACs as above. Ratios of green to red spots in interphase FISH, as calculated from over 30 nuclei, are indicated below the pictures. White arrows, separate spots in the PC82 nucleus. Black arrows, positions of ERG and TMPRSS2 in the array-CGH figures. X axis, log2 ratio of normalized hybridization signal of tumor DNA versus reference DNA (T/R).

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Eleven xenografts derived from various stages of clinical prostate cancer (Supplementary Table S1; refs. 7–9) were used to decipher the role of ETS genes. PCEW, PC82, PC295, PC310, PC329, PC346, and PC374 are AR positive. PCEW, PC82, PC295, PC310, and PC329 grow androgen dependent on male nude mice; PC346 and PC374 are, to a varying extent, androgen sensitive. PC133, PC135, PC324, and PC339 are androgen independent.

First, expression of AR, PSA, and TMPRSS2 in xenografts was assessed by QPCR. There is a good correlation between the expression of AR and the well-known, androgen-regulated PSA and TMPRSS2 genes in androgen-dependent and androgen-sensitive xenografts (Fig. 1A ; refs. 5, 9, 16). An exception is PC310, which shows AR and PSA expression, but clearly is TMPRSS2 negative (see below). All androgen-independent xenografts are AR negative or express an inactive AR, as deduced from lack of PSA and TMPRSS2 expression (see also ref. 17).

View larger version (33K): [in this window] [in a new window]   Figure 1. Expression of AR, PSA, TMPRSS2, and ERG and ETV1 fusion transcripts in prostate cancer xenografts. A, QPCR of AR, PSA, and TMPRSS2 mRNAs. B, QPCR of ERG and TMPRSS2:ERG fusion transcripts. C, QPCR of ETV1 and TMPRSS2:ETV1 fusion transcripts and interphase FISH with TMPRSS2 (green spots) and ETV1 (red spots) specific BACs of nuclei from PC135 and PC374. D, composition of TMPRSS2:ERG and TMPRSS2:ETV1 fusion transcripts. Details of methods, FISH BACs, and QPCR primers are described in Materials and Methods. PBGD expression was used as a QPCR reference.

Androgen-sensitive PC374 showed high ETV1 overexpression (Fig. 1C). High ETV1 expression correlated with TMPRSS2:ETV1 gene fusion, as shown by interphase FISH on nuclear suspensions using ETV1 and TMPRSS2 specific BACs (Fig. 1C), and the presence of two splice variants of TMPRSS2:ETV1 (Fig. 1C and D). ERG and ETV1 were not overexpressed in PC346 or in late-stage, AR-negative prostate cancer xenografts. Interphase FISH indicated that low-level ETV1 expression in PC135 could not be correlated with TMPRSS2 gene fusion (Fig. 1C). 5'-Rapid amplification of cDNA ends (5'-RACE) confirmed that ETV1 expressed in PC135 was wild type (data not shown).

TMPRSS2 and ERG map in the same orientation at short distance on chromosome band 21q22.2-q22.3 (Fig. 2A ). To determine the mechanism of TMPRSS2:ERG gene fusion, genomic DNA from the five xenografts overexpressing the fusion gene was investigated by 1-Mbp-spaced array-based comparative genomic hybridization (array-CGH). Two different representative array-CGH profiles of chromosome 21 are depicted in Fig. 2B and C. In PC295, the region between ERG and TMPRSS2 was lost, as indicated by the low T/R ratio of the four BACs mapping in this chromosomal region (Fig. 2B). A similar profile was present in PC329 (data not shown). Although PC82 contains the fusion transcript, the region between ERG and TMPRSS2 was largely present (Fig. 2C). A comparable profile was found in PCEW and PC310 (data not shown). In PC310, the profile was accompanied by a small homozygous deletion of the last exons of TMPRSS2 (data not shown), explaining total absence of TMPRSS2 transcripts in this xenograft. We extended the array-CGH data by interphase FISH of PC82, PC295, and PC310 nuclei. We used as hybridization probes BACs RP11-164E1 and RP5-1031P17, which map at a distance of 1 Mbp, flanking ERG at positions indicated in Fig. 2A. Both BACs exclusively stained chromosome band 21q22.2 and showed two spots on interphase nuclei from normal cells (Supplementary Fig. S1A and B). Representative nuclei of PC295 and PC82 are presented in Fig. 2B and C, respectively. In nuclei from PC295 cells, we found a higher number of green spots than red spots (average ratio 1.6), indicative of loss of the region between ERG and TMPRSS2 in one copy of chromosome 21. In PC82 and PC310, we observed an identical number of green and red spots (average ratios 1.0 and 1.1, respectively). In PC82 that contains four copies of chromosome 21, three pairs of red and green spots were always closely linked and one pair was clearly separated, as illustrated in Fig. 2C. Both array-CGH and FISH data strongly suggest two different mechanisms of TMPRSS2:ERG fusion: one by an 3 Mbp interstitial deletion of one copy of chromosome 21, and a second more complex mechanism by chromosomal translocation.

Array-CGH of genomic DNA from two of the six xenografts that did not overexpress TMPRSS2:ERG showed a remarkable pattern. In the androgen-independent, AR-negative xenografts PC133 and PC339, we detected a similar interstitial deletion as in PC295 and PC329. The chromosome 21 profile of PC339 is depicted in Fig. 3A . Long-range PCR followed by sequencing confirmed the fusion between TMPRSS2 and ERG in PC133, mapping the chromosomal breakpoints in ERG intron 3 and in TMPRSS2 intron 1, respectively (Fig. 3B). Similarly, PCR plus sequencing identified TMPRSS2:ERG fusion in PC324 that does not show a 21q22 interstitial deletion. This adds a third AR-negative xenograft to those with TMPRSS2:ERG gene fusion without expression of the fusion gene. Like in PC133, the breakpoint in PC324 is in TMPRSS2 intron 1. We also mapped the breakpoints in AR-positive PC82, PC295, PC310, and PC329 (Fig. 3B). As expected from expression data (Fig. 1B), these breakpoints were in intron 2 of TMPRSS2 and in intron 3 of ERG. All six breakpoints in ERG were in the last part of intron 3, suggesting a preferred region of recombination in this part of the gene. It remains to be investigated whether the difference in TMPRSS2 introns involved in ERG fusion between AR-positive and AR-negative xenografts, introns 2 and 1, respectively, is coincidental or of functional importance. The absence of TMPRSS2:ERG expression in PC133, PC324, and PC339 (Fig. 1B) indicates that it is not involved in the androgen-independent growth of these xenografts. Importantly, however, the presence of TMPRSS2:ERG in genomic DNA strongly suggests that the fusion gene has been instrumental in an earlier androgen-dependent stage of tumor growth.

View larger version (19K): [in this window] [in a new window]   Figure 3. Genetic and expression analysis of AR-negative prostate cancer xenografts. A, array-CGH of chromosome 21q of androgen-independent, AR-negative xenograft PC339 (for details, see Fig. 2 and Materials and Methods). B, positions of breakpoints in ERG and TMPRSS2 in xenograft DNAs. Breakpoints were mapped by standard long-range PCR using a mixture of Taq and Proofstart DNA polymerase, as indicated by the manufacturer (Qiagen, Valencia, CA). Above the genes, the positions of breakpoints in AR-positive, androgen-dependent xenografts are shown; below the genes, the breakpoints in AR-negative xenografts are indicated. C, QPCR of ETV4 expression in prostate cancer xenografts. D, QPCR of FLI1 expression in prostate cancer xenografts. Details are described in the legend to Fig. 1 and in Materials and Methods. Standard 5'-RACE, using the Generacer kit from Invitrogen, indicated that ETV4 and FLI1 transcripts in AR-negative PC133, PC135, PC324, or PC399 were wild type. Insets (C and D), representative interphase FISH pictures of nuclei from indicated xenografts, showing the absence of fusion of TMPRSS2 to ETV4 or FLI1. Green spots, TMPRSS2 (C and D); red spots, ETV4 (C) and FLI1 (D).

In clinical prostate cancer, many recurrent tumors still express a functional AR. However, a substantial proportion of recurrent tumors is heterogeneous for AR expression or is AR negative (18, 19). We investigated whether, like in xenografts, TMPRSS2:ERG gene fusion without expression of the fusion gene was present in clinical samples. Array-CGH showed in 4 of 11 recurrent tumors the interstitial deletion at 21q22 indicative of TMPRSS2:ERG fusion (see, e.g., Fig. 4A and B ). From three of these tumors (T1-1, T1-8, and T3-7), RNA was available. Importantly, we also had available DNA and RNA from recurrent tumor T1-7 of which AR-negative xenograft PC324 was derived. These four tumors and a control recurrent tumor without TMPRSS2:ERG fusion (T6-9) were investigated by QPCR for specific gene expression patterns (Fig. 4C and D). T1-1, T3-7, and T6-9 expressed AR and its target genes PSA and TMPRSS2, although expression in T1-1 was low. In contrast, T1-8 and, as expected, T1-7 showed hardly any AR, PSA, and TMPRSS2 expression. All tumor samples had a basal level of ERG expression. ERG overexpression, combined with TMPRSS2:ERG fusion transcripts, was clearly detected in T3-7 and absent in T1-8 and T1-7. T1-1 showed a low level of TMPRSS2:ERG expression, which might indicate tumor heterogeneity. The data confirm and extend the findings in xenografts showing that AR-negative tumors can carry a TMPRSS2:ERG fusion gene without expression of the gene. Background expression levels and presumed heterogeneity of tumors hampered accurate investigation of ETV4 and FLI1 in the clinical samples (Supplementary Fig. S3). ETV4 expression was highest in AR-negative T1-8 and T1-7, but differences with other tumor samples were small. Like in PC324, expression of FLI1 was high in T1-7, but T1-1 also showed high expression of FLI1. The latter might be explained by tumor heterogeneity, as proposed previously (Fig. 4C and D). Obviously, more detailed immunohistochemical studies, including AR and ETV4 or FLI1 double staining, are needed to substantiate the latter observation.

View larger version (17K): [in this window] [in a new window]   Figure 4. Array-based CGH of chromosome 21 and specific gene expression patterns of clinical recurrent prostate cancer specimens. A and B, array-CGH of chromosome 21 of recurrent tumors T1-8 and T3-7. Black arrows, positions of ERG and TMPRSS2. X axis, log2 ratio of normalized hybridization signal of tumor DNA versus reference DNA. C, QPCR of AR, PSA, and TMPRSS2 mRNAs. D, QPCR of ERG and TMPRSS2:ERG fusion transcripts. Details of methods and QPCR primers are described in Materials and Methods. PBGD expression was used as a QPCR reference.

	Acknowledgments

 
Grant support: Erasmus University Medical Center and the Dutch Cancer Society (Koningin Wilhelmina Fonds).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	Footnotes

 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 5/24/06. Revised 9/13/06. Accepted 10/ 5/06.

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