Previously, we reported ETV1
overexpression in eight out of 84 prostate cancer samples. In four cases this was caused by a gene fusion, in the other four a full-length ETV1
transcript was overexpressed 
. In the present study we investigated overexpression of ETV1
by quantitative reverse transcriptase reaction (QPCR) in a novel cohort of 66 prostate cancers. In six RNAs ETV1
overexpression was detected. Samples G51, G59, G233, G270 and G268 were derived from primary tumors and G210 was derived from a recurrent tumor. The six samples were further studied by QPCR with primer pairs at the 5′-end of ETV1
mRNA (exon 1F and exon 4R) and at the 3′-end of the mRNA (exon 11F and exon 12R) (). A high exon 11–12 to exon 1–4 ratio is indicative for a fusion gene; a 1
1 ratio indicated overexpression of full-length ETV1
mRNA. Based on these criteria, tumors G51, G59, G233 and G270 expressed full-length ETV1
whereas G210 and G268 expressed a fusion transcript (see also control PC374 that expresses TMPRSS2-ETV1
was found as the fusion transcript in sample G210 (data not shown); the fusion transcript in G268 has not been identified as yet. These two samples were not investigated further in this study.
Full-length ETV1 overexpression in clinical prostate cancers correlates with genomic rearrangement of the complete gene.
In the next experiments we focused on the elucidation of the mechanism of overexpression of full-length ETV1
. Recently, it has been shown that in two prostate cancer cell lines overexpressing full-length ETV1
, LNCaP and MDA PCa2b, ETV1
is translocated 
. We now addressed the question whether in clinical samples translocation of the complete ETV1
gene might occur. To detect genomic rearrangements tissue slides of all four prostate cancer samples with full-length ETV1
overexpression were analyzed by break-apart interphase FISH with two labeled BAC probes, one BAC recognized ETV1
and the second one the flanking gene DGKB
(). The series was supplemented with two samples harboring full-length ETV1
overexpression from our previous study, G89 and G308 
, for which frozen tissues of sufficient morphological quality were available. shows the results of the FISH experiments. Interestingly, we found split signals in all samples except for G59, indicating frequent ETV1
rearrangements in prostate cancers that overexpress full-length ETV1
. Absence of a split signal in G59 suggests absence of translocation, although we cannot exclude a breakpoint outside the investigated region. Its observed high frequency indicates genomic rearrangement as an important mechanism of full-length ETV1
overexpression in clinical prostate cancer. Obviously, the chromosomal region to which ETV1
is translocated can contribute to elucidation of the mechanism of ETV1
overexpression. We were able to identify more details of the rearrangements in samples G270 and G89.
It has been shown in LNCaP cells that ETV1
is translocated to 14q13.3-q21.1. The whole gene is integrated in the last intron of MIPOL1
. In MDA PCa2b, ETV1
is translocated to the same region, although the precise position is unknown 
. Moreover, we previously described insertion of truncated ETV1
in the intron of a two exon gene encoding a ncRNA, denoted EST14
, giving rise to an EST14-ETV1
fusion transcript that contains ETV1
exon 5–12 sequences (sample G342 in ref. 10; ). Importantly, EST14
maps directly adjacent to MIPOL1
on 14q. Like most ETV1
fusion partners, EST14
is an androgen-regulated prostate-specific gene. To investigate whether the same chromosomal region was involved in full-length ETV1
translocation in our novel cohort, interphase FISH was performed with the ETV1
BAC () in combination with a MIPOL1
BAC (). A merging yellow signal was detected in sample G270 () but in none of the other tumors. These data indicate that although there seems a preference for chromosome 14q13.3-21.1, other genomic regions will also contribute to rearrangement and overexpression of full-length ETV1
Characterization of the EST14 to ETV1 gene fusion in prostate cancer G270.
Additional information of ETV1 rearrangement in G270 came from 5′-RACE of tumor cDNA (data not shown). Remarkably, we did not only detect as expected the full-length ETV1 transcript but also a fusion transcript (). Such a result was not found for any of the other tumors overexpressing full-length ETV1. Sequencing showed that the fusion transcript in G270 was composed of ETV1 exon 1–12 preceded by the first exon of EST14 (). Scanning the EST14 intron and ETV1 flanking region by long-range PCR and sequencing mapped the breakpoints in G270 ~1.9 Kbp upstream of ETV1 exon 1 and ~5 Kbp downstream of EST14 exon 1. The breakpoint in EST14 is only 180 bp apart from the breakpoint in G342 ( and ref. 10).
Further information of ETV1
rearrangement was also collected for sample G89. Previously, a xenograft propagated on male nude mice had been generated from this tumor (PC135). Like tumor G89, PC135 overexpressed full-length ETV1 
. The availability of the xenograft allowed the preparation of metaphase chromosome spreads. In multicolor FISH a complex chromosomal rearrangement pattern was found (data not shown). Individual chromosome paints were used to validate the multicolor FISH data. Painting of chromosome 7 indicated the presence of multiple chromosome 7 fragments (). Hybridization with an ETV1
BAC identified the presence of three gene copies: two in apparently normal chromosomes 7 and one in a complex marker chromosome. Follow-up experiments showed that the marker chromosome contained fragments of chromosomes 4, 7 and 10, as first indicated by multicolor FISH (). The ETV1
BAC hybridized at the junction of the chromosome 7 and the chromosome 4 fragment, strongly suggesting that the 4;7 translocation was instrumental in overexpression of ETV1
. The precise positions of the breakpoints in 4 and 7 remain to be determined. Our data predict that multiple chromosomal regions are involved in overexpression of full-length ETV1
. At least one of these regions is on chromosome 14 and a second one on chromosome 4. The chromosome 14 region is also involved in ETV1
gene fusion. Deep sequencing technology could be instrumental in identification of other ETV1
A complex ETV1 translocation in xenograft PC135 involves chromosomes 4, 7 and 10.
Detailed characterization of the full-length ETV1
transcripts in the various tumors by 5′-RACE and sequencing showed that not only ETV1
exon 1- exon 12 transcripts were present but also various other full-length ETV1
transcripts, resulting from alternative promoter usage. These transcripts were denoted as ETV1
and Supplementary Figure S1
show the positions of the different first exons in the gene and indicate the various ATG start codons. Of both ETV1-1a
two splice variants were found (data not shown). QPCR experiments using transcript-specific primers on RNA from all six clinical prostate cancer samples that overexpressed ETV1
showed that the level of expression of the different transcripts was variable in the various tumors (see Figure S2
was hardly expressed in control benign prostate hyperplasia sample G277. schematically represents the predicted composition of the various ETV1 protein isoforms that will be produced. Note that ETV1-1c is by far the shortest, lacking the N-terminal 60 amino acids, including the major part of the conserved acidic TAD. In dETV1 that is expressed by most fusion genes, the N-termimal 131 amino acids are absent. ETV1, ETV1-1b1 and -1b2 were of similar size, as shown in Western blots of lysates from transfected HEK293T cells ().
Alternative full-length ETV1 transcripts give rise to proteins that all induce in vitro anchorage-independent growth.
Previously, we have shown that ETV1 and dETV1 differed in stimulation of in vitro
anchorage-independent growth 
. PNT2C2 cells infected with all novel ETV1 constructs induced anchorage-independent growth in a similar manner as ETV1 (). Remarkably, ETV1-1c, although expressed at a lower level and much smaller, is as active as the longer ETV1 isoforms. Thus, the full N-terminal TAD was not needed but amino acids 61–131 seem essential for biological activity of ETV1 ().
In summary, the data presented reveal two important novel aspects of the role of ETV1 in prostate cancer. First, it is shown that in clinical prostate cancers a subgroup of ETV1 positive patients show full-length ETV1 overexpression due to translocations of the whole gene to different chromosomes. This novel observation complements the well-described mechanism of overexpression of truncated ETV1 caused by gene fusions where expression regulation is determined by the promoter and enhancers of the fusion partners. Secondly, in contrast to dETV1 produced by gene fusions, a short isoform of full-length ETV1, ETV1-1c, lacking most of the N-terminal TAD, is as active as longer ETV1 isoforms, containing the complete N-terminal acidic TAD. This finding pinpoints the anchorage-independent growth to a small region that is absent in truncated ETV1 expressed by fusion genes.
It is highly relevant to extend the number of clinical samples in order to be able to compare tumor progression in the two subgroups of prostate cancers showing overexpression of truncated vs. full-length ETV1, and to determine the molecular mechanisms involved in their different biological behavior.