Overexpression of Full-Length ETV1 Transcripts in Clinical Prostate Cancer Due to Gene Translocation Delila Gasi1, Hetty A. van der Korput1, Hannie C. Douben 2 , Annelies de Klein2, Corrina M. de Ridder3, Wytske M. van Weerden3, Jan Trapman 1 * 1 Department of Pathology, Erasmus University Medical Centre, Rotterdam, The Netherlands, 2 Department of Clinical Genetics, Erasmus University Medical Centre, Rotterdam, The Netherlands, 3 Department of Urology, Erasmus University Medical Centre, Rotterdam, The Netherlands Abstract ETV1 is overexpressed in a subset of clinical prostate cancers as a fusion transcript with many different partners. However, ETV1 can also be overexpressed as a full-length transcript. Full-length ETV1 protein functions differently from truncated ETV1 produced by fusion genes. In this study we describe the genetic background of full-length ETV1 overexpression and the biological properties of different full-length ETV1 isoforms in prostate cancer. Break-apart FISH showed in five out of six patient samples with overexpression of full-length ETV1 a genomic rearrangement of the gene, indicating frequent translocation. We were able to study the rearrangements in more detail in two tumors. In the first tumor 59-RACE on cDNA showed linkage of the complete ETV1 transcript to the first exon of a prostate-specific two exon ncRNA gene that maps on chromosome 14 (EST14). This resulted in the expression of both full-length ETV1 transcripts and EST14-ETV1 fusion transcripts. In chromosome spreads of a xenograft derived from the second prostate cancer we observed a complex ETV1 translocation involving a chromosome 7 fragment that harbors ETV1 and fragments of chromosomes 4 and 10. Further studies revealed the overexpression of several different full-length transcripts, giving rise to four protein isoforms with different N-terminal regions. Even the shortest isoform synthesized by full-length ETV1 stimulated in vitro anchorage- independent growth of PNT2C2 prostate cells. This contrasts the lack of activity of even shorter N-truncated ETV1 produced by fusion transcripts. Our findings that in clinical prostate cancer overexpression of full-length ETV1 is due to genomic rearrangements involving different chromosomes and the identification of a shortened biologically active ETV1 isoform are highly relevant for understanding the mechanism of ETV1 function in prostate cancer. Citation: Gasi D, van der Korput HA, Douben HC, de Klein A, de Ridder CM, et al. (2011) Overexpression of Full-Length ETV1 Transcripts in Clinical Prostate Cancer Due to Gene Translocation. PLoS ONE 6(1): e16332. doi:10.1371/journal.pone.0016332 Editor: Andrew Wilber, Southern Illinois University School of Medicine, United States of America Received September 5, 2010; Accepted December 10, 2010; Published January 26, 2011 Copyright: 2011 Gasi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by a Marie Curie Grant (Cancure) provided by the European Union. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: j.trapman@erasmusmc.nl Introduction Gene fusions are important in the development of many hematological malignancies and sarcomas, but are rare in most other tumor types [1]. However, the frequent fusion between TMPRSS2 and the ETS gene ERG showed that gene fusion is a highly relevant event in prostate cancer [2,3]. Overexpression of the TMPRSS2-ERG fusion gene has been reported in 40–70% of prostate cancer cases [2–5]. Fusions of TMPRSS2 and three other genes encoding ETS transcription factors, ETV1, ETV4 and ETV5, which are located on different chromosomes, occur at low frequency in prostate cancer [2,6,7]. However, for ETV1 at least 10 different fusion partners have been described [8–10]. Most of these have in common that, like TMPRSS2, they are prostate-specific and androgen- regulated expressed. The properties of fusion partners are key elements in explaining the androgen-regulated overexpression of an ETS oncogene in prostate cancer. However, a unique characteristic of ETV1 is that it can not only be overexpressed in prostate cancer as a fusion transcript but also as a full-length wild-type transcript. The large family of ETS transcription factors is composed of 27 members [11–13]. All members have in common a highly homo- logous DNA binding domain, the ETS domain. The remaining regions of most ETS proteins show limited structural homology. ETV1, ETV4 and ETV5 are the members of a small subfamily of structurally related ETS proteins. These proteins contain in the N- terminal region a conserved short acidic transactivation domain (TAD) that is absent in ERG. ETS proteins regulate many target genes that modulate biological processes like cell growth, angiogenesis, migration, proliferation and differentiation. Howev- er, which of the many molecular and biological functions of ETS proteins are most important in prostate cancer is not known. Following ERG, ETV1 is the most frequently overexpressed ETS gene in prostate cancer (,10% of the tumors) [10]. The ETV1 protein translated from most fusion transcripts is truncated, lacking the 131 N-terminal amino acids (dETV1). Approx. half of the tumors with ETV1 overexpression express a fusion transcript, the others show a high level of full-length ETV1 expression. The in vitro biological and molecular properties of dETV1 seem different from those of the full-length 477 amino acid protein [10]. This observation suggests that tumors with overexpression of full-length ETV1 are different from tumors expressing dETV1. Little is known about the mechanism of full-length ETV1 overexpression and its function in clinical prostate cancer. Our present results show that overexpression of full-length ETV1 is PLoS ONE | www.plosone.org 1 January 2011 | Volume 6 | Issue 1 | e16332