ETV1 Translocations in Prostate Cancer (C) Translocation of ETV1 to chromosome 14 in tumor G270. Tissue sections were hybridized with BAC RP11-460G19 (green) that overlaps MIPOL1 and flanks ETS14 and with the ETV1 BAC RP11-124L22 (red) (see B for details). In the left panel the position of MIPOL1 BAC on chromosome 14 is indicated. In the right panel a merging signal (yellow) shows co-localization of ETV1 and MIPOL1/EST14 in G270, as indicated by the arrow. doi:10.1371/journal.pone.0016332.g001 Previously, we have shown that ETV1 and dETV1 differed in stimulation of in vitro anchorage-independent growth [10]. PNT2C2 cells infected with all novel ETV1 constructs induced anchorage- independent growth in a similar manner as ETV1 (Figure 4C). 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 (Figure 4B, C). 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 transloca- tions 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. Materials and Methods Ethics Statement Use of the samples was approved by the Erasmus MC Medical Ethics Committee according to the Medical Research Involving Human Subjects Act in protocol MEC-2004-261, entitled ‘‘The use of human normal and cancer residual tissue from a tissue bank for characterization of DNA, RNA and protein’’. Mice were housed according to guidelines of the Erasmus Medical Centre, and procedures were carried out in compliance with standards for use of laboratory animals. Animal experiments performed in this manuscript have been approved by the animal experimental committee of the Erasmus Medical Centre (DEC- consult Erasmus MC project 102-10-01). Tissue samples, RNA and DNA isolation Snap-frozen prostate cancers were obtained by radical prosta- tectomy or transurethral resection. Hematoxilin/eosin (HE) stained tissue sections were histologically evaluated by a pathologist (G. van Leenders). All samples contained at least 50% tumor cells. Figure 2. Characterization of the EST14 to ETV1 gene fusion in prostate cancer G270. (A) Schematic representation of ETV1-EST14 fusion transcripts in prostate tumors G270 and G342 (sample G342 is from ref. 10). Arrows indicate positions of primers used in the RT-PCR experiment. Primer sequences are shown in Supplementary Table S1. (B) Sequence of the fusion point of EST14 and ETV1 in sample G270. The position of the fusion point in tumor G270 was mapped by long-range PCR on genomic DNA with a forward primer in the EST14 intron and reverse primer upstream of ETV1 exon 1. At the fusion point two T residues were lost. The breakpoint in EST14 in G270 and G342 are indicated by arrows. doi:10.1371/journal.pone.0016332.g002 PLoS ONE | www.plosone.org 4 January 2011 | Volume 6 | Issue 1 | e16332