Truncated ETV1, Fused to Novel Tissue-Specific Genes, and Full-Length ETV1 in Prostate Cancer Karin G. Hermans, 1 Hetty A. van der Korput, 1 Ronald van Marion, 1 Dennis J. van de Wijngaart, 1 Angelique Ziel-van der Made, 1 Natasja F. Dits, 2 Joost L. Boormans, 2 Theo H. van der Kwast, 1 Herman van Dekken, 1 Chris H. Bangma, 2 Hanneke Korsten, 1 Robert Kraaij, 2 Guido Jenster, 2 and Jan Trapman 1 Departments of 1 Pathology and 2 Urology, Josephine Nefkens Institute, Erasmus University Medical Center, Rotterdam, the Netherlands Abstract In this study, we describe the properties of novel ETV1 fusion genes, encoding N-truncated ETV1 (dETV1), and of full-length ETV1, overexpressed in clinical prostate cancer. We detected overexpression of novel ETV1 fusion genes or of full-length ETV1 in 10% of prostate cancers. Novel ETV1 fusion partners included FOXP1 , an EST (EST14 ), and an endogenous retroviral repeat sequence (HERVK17 ). Like TMPRSS2, EST14 and HERVK17 were prostate-specific and androgen-regulated expressed. This unique expression pattern of most ETV1 fusion partners seems an important determinant in prostate cancer development. In transient reporter assays, full-length ETV1 was a strong transactivator, whereas dETV1 was not. However, several of the biological properties of dETV1 and full-length ETV1 were identical. On stable overexpression, both induced migration and invasion of immortalized non- tumorigenic PNT2C2 prostate epithelial cells. In contrast to dETV1, full-length ETV1 also induced anchorage-independent growth of these cells. PNT2C2 cells stably transfected with dETV1 or full-length ETV1 expression constructs showed small differences in induced expression of target genes. Many genes involved in tumor invasion/metastasis, including uPA/uPAR and MMPs , were up-regulated in both cell types. Integrin B3 (ITGB3 ) was clearly up-regulated by full-length ETV1 but much less by dETV1. Based on the present data and on previous findings, a novel concept of the role of dETV1 and of full-length ETV1 overexpression in prostate cancer is pro- posed. [Cancer Res 2008;68(18):7541–9] Introduction The ETS transcription factor family is composed of 27 members (1–3). Depending on the cellular context, they can function as transactivators or transrepressors. ETS transcription factors modulate many cellular functions, including proliferation, apopto- sis, differentiation, tissue remodeling, migration, invasion, and angiogenesis (1–3). Altered expression or properties of ETS trans- cription regulators affect the control of these processes. Recurrent chromosomal rearrangements are well defined in leukemias, lymphomas, and sarcomas (4). These rearrangements result in fusion genes that express oncogenic proteins with altered properties or in overexpression of wild-type oncogenes. In Ewing’s sarcoma and in acute myeloid leukemia, gene fusions of members of the ETS gene family have been found. At low frequency, gene fusions have also been described in solid tumors (4). However, recent analyses showed common gene fusions in prostate cancer, all involving members of the ETS transcription factor family (5–9). In 40% to 70% of clinical prostate cancers, ERG (21q22.1) is directly linked to androgen-regulated, prostate-specific TMPRSS2 , which is located 3 Mbp upstream of ERG . At low frequency, fusions of TMPRSS2 to ETV1, ETV4 , and ETV5 , which map on different chromosomes, have been described (5, 7, 8, 10). Considering the complexity of fusion genes in hematologic and mesenchymal malignancies, we questioned whether this would also be true for gene fusions in prostate cancer. Here, we describe overexpression of ETV1 in 8 of 84 clinical prostate cancer samples. In four samples, full-length ETV1 is overexpressed, but in the other four samples, we detected novel ETV1 fusion genes, which result in predicted N-truncated ETV1 proteins. Novel fusion partners include FOXP1 , an EST (EST14 ), and an endogenous retroviral sequence (HERVK17 , identified in two samples). Like TMPRSS2 (11), both EST14 and HERVK17 are androgen regulated and prostate specific. Transient reporter assays with full-length ETV1 and N-truncated ETV1 (dETV1) showed that these proteins possess different transcription regulation functions. However, quantitative PCR (QPCR) analysis of prostate epithelial cells with stable over- expression of full-length ETV1 or dETV1 indicated less pronounced differences in expression of candidate target genes. Biological assays showed no significant difference in migration and invasion properties between full-length ETV1 and dETV1-expressing cells. However, full-length ETV1 is capable of inducing anchorage- independent growth, whereas dETV1 is not. We propose a different role of dETV1 and full-length ETV1 in prostate cancer. Materials and Methods Tissue samples. Primary prostate cancer samples were obtained by radical prostatectomy, regional lymph node metastases were collected during surgery, and recurrences were obtained by transurethral resection. Samples were snap frozen and stored in liquid nitrogen. Use of the samples for research purposes was approved by the Erasmus MC Medical Ethics Committee according to the Medical Research Involving Human Subjects Act (MEC-2004-261). H&E-stained tissue sections were histologically evaluated by two pathologists (T.H. van der Kwast and A. van Leenders). All samples contained at least 70% tumor cells. BALB/c mouse prostate tissues were collected at different developmental stages (16.5 and 18.5 embryonal days and postnatal days 3, 9, 15, and 50). Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Jan Trapman, Department of Pathology, Josephine Nefkens Institute, Erasmus University Medical Center, P. O. Box 2040, 3000 CA Rotterdam, the Netherlands. Phone: 31-107044933; Fax: 31-107044762; E-mail: j.trapman@ erasmusmc.nl. I2008 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-07-5930 www.aacrjournals.org 7541 Cancer Res 2008; 68: (18). September 15, 2008 Research Article Research. on January 14, 2016. © 2008 American Association for Cancer cancerres.aacrjournals.org Downloaded from