DATE ( DD - MM - YYYY ) September 14 , 2012 2 . REPORT TYPE : Final 3 . DATES COVERED ( From - To ) Aug 15 , 2008 - Sept 14 , 2012

Purpose: The expression of the ETS-related gene (ERG) is low or undetectable in benign prostate epithelial cells. High prevalence of ERG overexpression in prostate cancer cells due to TMPRSS2-ERG fusions suggest for causal roles of ERG protein in the neoplastic process. TMPRSS2-ERG fusion junctions have been extensively studied in prostate cancer. However, virtually nothing is known about the nature of full-length transcripts and encoded proteins. This study focuses on qualitative and quantitative features of full-lengthTMPRSS2-ERG transcripts in prostate cancer. Experimental Design: Full-lengthTMPRSS2-ERG transcripts were cloned and sequenced from a cDNA library generated from pooled RNA of sixTMPRSS2-ERG fusion ^ positive prostate tumors.The encoded ERG proteins were analyzed in HEK293 cells. Copy numbers ofTMPRSS2ERG splice variants were determined by quantitative reverse transcription-PCR in laser capture microdissectedprostate cancer cells. Results:Two types ofTMPRSS2-ERG cDNAs were identified: type I, which encodes full-length prototypical ERG protein (ERG1, ERG2, ERG3), and type II, encoding truncated ERG proteins lacking the ETS domain (ERG8 and a new variant,TEPC). In microdissected prostate tumor cells from122 patients, relative abundance of these variantswas in the followingorder: ERG8 > TEPC > ERG 3 > ERG1/2 with combined overexpression rate of 62.3% in prostate cancer. Increased ratio of type I over type II splice forms showed a trend of correlationwith less favorable pathology and outcome. Conclusions: Qualitative and quantitative features of specific ERG splice variants defined here promise to enhance the utility of ERG as a biomarker and therapeutic target in prostate cancer. Molecular genetic evaluations of prostate cancer are defining mutational and expression alterations of critical oncogenes involved in disease onset and/or progression (reviewed in refs. 1–3). Discovery of prevalent chromosomal rearrangements/ translocations leading to the activation of ETS transcription factors (predominantly ERG) through the androgen receptor– regulated TMPRSS2 gene promoter underscore the critical roles of ERG-encoded protein in prostate cancer (4–7). Because ERG represents the majority of TMPRSS2-ETS factor alterations described thus far (6, 7), we have focused on the expression and regulation of TMPRSS2-ERG in prostate cancer. Oncogenic functions of ETS factors, including ERG, have also been implicated in diverse cancers (8). Structure and function of ERG-encoded proteins remain to be defined in prostate cancer. ERG consists of 17 exons spanning about 300 kb and generates at least nine alternate splice forms, seven of them coding for protein products of varying sizes (9). These ERG splice variants have been primarily described in nonprostate tissues. Despite the large body of data on the TMPRSS2-ERG fusion junctions in prostate cancer (reviewed in refs. 6, 7), virtually nothing is known about the full-length TMPRSS2-ERG transcripts in prostate cancer, including the existence and relative abundance of specific splice variants. In this context, it is important to note that the cancer-associated splice variants of numerous genes, e.g., androgen receptor, fibroblast growth factor receptor, survivin , and MDM2 , have functional implications (10, 11). Thus, characterization of full-length TMPRSS2-ERG transcripts is essential to better understand ERG function(s) in prostate cancer and to further enhance its utility as biomarker and therapeutic target. Human Cancer Biology Authors’ Affiliations: Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences, Rockville, Maryland; Armed Forces Institute of Pathology, Department of Genitourinary Pathology and Urology Service, Department of Surgery,Walter Reed Army Medical Center,Washington, District of Columbia; and U.S. Military Cancer Institute,UniformedServicesUniversityof theHealthSciences, Bethesda,Maryland Received 2/26/08; revised 4/25/08; accepted 4/28/08. Grant support: Center for Prostate Disease Research and NIH grants RO1 DK065977 (S. Srivastava and G. Petrovics) and RO1CA106653 (S. Srivastava and