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Background: The established methods for detecting prostate cancer (CaP) are based on tests using PSA (blood), PCA3 (urine), and AMACR (tissue) as biomarkers in patient samples. The demonstration of ERG oncoprotein overexpression due to gene fusion in CaP has thus provided ERG as an additional biomarker. Based on this, we hypothesized that ERG protein quantification methods can be of use in the diagnosis of prostate cancer., Methods: An antibody-free assay for ERG3 protein detection was developed based on PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing)-SRM (selected reaction monitoring) mass spectrometry. We utilized TMPRSS2-ERG positive VCaP and TMPRSS2-ERG negative LNCaP cells to simulate three different sample types (cells, tissue, and post-DRE urine sediment). Enzyme-linked immunosorbent assay (ELISA), western blot, NanoString, and qRT-PCR were also used in the analysis of these samples., Results: Recombinant ERG3 protein spiked into LNCaP cell lysates could be detected at levels as low as 20 pg by PRISM-SRM analysis. The sensitivity of the PRISM-SRM assay was approximately 10,000 VCaP cells in a mixed cell population model of VCaP and LNCaP cells. Interestingly, ERG protein could be detected in as few as 600 VCaP cells spiked into female urine. The sensitivity of the in-house ELISA was similar to the PRISM-SRM assay, with detection of 30 pg of purified recombinant ERG3 protein and 10,000 VCaP cells. On the other hand, qRT-PCR exhibited a higher sensitivity, as TMPRSS2-ERG transcripts were detected in as few as 100 VCaP cells, in comparison to NanoString methodologies which detected ERG from 10,000 cells., Conclusions: Based on this data, we propose that the detection of both ERG transcriptional products with RNA-based assays, as well as protein products of ERG using PRISM-SRM assays, may be of clinical value in developing diagnostic and prognostic assays for prostate cancer given their sensitivity, specificity, and reproducibility.

Unlabelled: Overexpression of ERG in the prostate epithelium, due to chromosomal translocations, contributes to prostate tumorigenesis. Here, genomic analysis of ERG siRNA-treated prostate cells harboring the endogenous TMPRSS2-ERG fusion revealed an inverse relationship between ERG and Annexin A2 (ANXA2) expression at both the RNA and protein level. ANXA2, a Ca(2+)-dependent and phospholipid-binding protein, is involved in various cellular functions, including maintenance of epithelial cell polarity. Mechanistic studies defined the prostate-specific transcription start site of ANXA2 and showed that the recruitment of ERG to the ANXA2 promoter is required for transcriptional repression by ERG. Knockdown of ERG enhanced the apical localization of ANXA2, the bundling of actin filaments at cell-cell junctions and formation of a polarized epithelial phenotype. ERG overexpression disrupted ANXA2-mediated cell polarity and promoted epithelial-mesenchymal transition (EMT) by inhibiting CDC42 and RHOA, and by activating cofilin. Immunohistochemistry demonstrated a reciprocal relationship of ANXA2 and ERG expression in a large fraction of primary prostate cancer clinical specimens. ANXA2 was absent or markedly reduced in ERG(+) tumors, which were mostly well differentiated. ERG(-) tumors, meanwhile, expressed moderate to high levels of ANXA2, and were either poorly differentiated or displayed subsets of poorly differentiated cells. Taken together, the transcriptional repression of ANXA2 by ERG in prostate epithelial cells plays a critical role in abrogating differentiation, promoting EMT, and in the reciprocal correlation of ERG and ANXA2 expression observed in human prostate cancer., Implications: ANXA2 is a new component of the ERG network with potential to enhance biologic stratification and therapeutic targeting of ERG-stratified prostate cancers., (©2014 American Association for Cancer Research.)

Background: Gene fusion between TMPRSS2 promoter and the ERG proto-oncogene is a major genomic alteration found in over half of prostate cancers (CaP), which leads to aberrant androgen dependent ERG expression. Despite extensive analysis for the biological functions of ERG in CaP, there is no systematic evaluation of the ERG responsive proteome (ERP). ERP has the potential to define new biomarkers and therapeutic targets for prostate tumors stratified by ERG expression., Methods: Global proteome analysis was performed by using ERG (+) and ERG (-) CaP cells isolated by ERG immunohistochemistry defined laser capture microdissection and by using TMPRSS2-ERG positive VCaP cells treated with ERG and control siRNA., Results: We identified 1,196 and 2,190 unique proteins stratified by ERG status from prostate tumors and VCaP cells, respectively. Comparative analysis of these two proteomes identified 330 concordantly regulated proteins characterizing enrichment of pathways modulating cytoskeletal and actin reorganization, cell migration, protein biosynthesis, and proteasome and ER-associated protein degradation. ERPs unique for ERG (+) tumors reveal enrichment for cell growth and survival pathways while proteasome and redox function pathways were enriched in ERPs unique for ERG (-) tumors. Meta-analysis of ERPs against CaP gene expression data revealed that Myosin VI and Monoamine oxidase A were positively and negatively correlated to ERG expression, respectively., Conclusions: This study delineates the global proteome for prostate tumors stratified by ERG expression status. The ERP data confirm the functions of ERG in inhibiting cell differentiation and activating cell growth, and identify potentially novel biomarkers and therapeutic targets., (© 2013 Wiley Periodicals, Inc.)

Objective: To systematically evaluate the ETS-related gene (ERG) alterations in the multifocal tumor context using whole-mount prostatectomy specimens from African and Caucasian American patients matched for age, pathologic grade and stage. Oncogenic activation of the ERG is the most common early genomic alteration in patients with prostate cancer (CaP) in Western countries. However, ERG alterations have not been systematically examined in African American patients with a known greater risk of CaP incidence and mortality., Methods: ERG oncoprotein expression was analyzed in 91 Caucasian and 91 African American patients with CaP, who were matched for age, Gleason score, and pathologic stage. A unique aspect of the present study was the evaluation of ERG in whole-mount prostatectomy sections, minimizing sampling bias and allowing the careful assessment of the ERG in the multifocal tumor context of CaP., Results: The frequency of ERG-positive prostate tumors was significantly greater among Caucasian Americans than among African Americans when assessed in all tumor foci (41.9% vs 23.9%, P < .0001). A markedly greater frequency of ERG oncoprotein expression was noted between the index tumors of the Caucasian Americans (63.3%) and those of the African Americans (28.6%). Also, in the African American patients, the higher grade index tumors were predominantly ERG negative., Conclusion: ERG typing of CaP established a major difference between the index tumors of Caucasian and African American patients. ERG-negative index tumors might indicate a less favorable outcome for African American patients. The results of the present study underscore that typing of CaP for the ERG could enhance our understanding of the biologic differences between the examined ethnic groups., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Genomic rearrangements commonly occur in many types of cancers and often initiate or alter the progression of disease. Here we describe an in vivo mouse model that recapitulates the most frequent rearrangement in prostate cancer, the fusion of the promoter region of TMPRSS2 with the coding region of the transcription factor, ERG. A recombinant bacterial artificial chromosome including an extended TMPRSS2 promoter driving genomic ERG was constructed and used for transgenesis in mice. TMPRSS2-ERG expression was evaluated in tissue sections and FACS-fractionated prostate cell populations. In addition to the anticipated expression in luminal cells, TMPRSS2-ERG was similarly expressed in the Sca-1(hi)/EpCAM(+) basal/progenitor fraction, where expanded numbers of clonogenic self-renewing progenitors were found, as assayed by in vitro sphere formation. These clonogenic cells increased intrinsic self renewal in subsequent generations. In addition, ERG dependent self-renewal and invasion in vitro was demonstrated in prostate cell lines derived from the model. Clinical studies have suggested that the TMPRSS2-ERG translocation occurs early in prostate cancer development. In the model described here, the presence of the TMPRSS2-ERG fusion alone was not transforming but synergized with heterozygous Pten deletion to promote PIN. Taken together, these data suggest that one function of TMPRSS2-ERG is the expansion of self-renewing cells, which may serve as targets for subsequent mutations. Primary prostate epithelial cells demonstrated increased post transcriptional turnover of ERG compared to the TMPRSS2-ERG positive VCaP cell line, originally isolated from a prostate cancer metastasis. Finally, we determined that TMPRSS2-ERG expression occurred in both castration-sensitive and resistant prostate epithelial subpopulations, suggesting the existence of androgen-independent mechanisms of TMPRSS2 expression in prostate epithelium.

Androgen dependent induction of the ETS related gene (ERG) expression in more than half of all prostate cancers results from gene fusions involving regulatory sequence of androgen regulated genes (i.e. TMPRSS2, SLC45A3 and NDRG1) and protein coding sequence of the ERG. Emerging studies in experimental models underscore the functions of ERG in prostate tumorigenesis. However, biological and biochemical functions of ERG in prostate cancer (CaP) remain to be elucidated. This study suggests that ERG activation plays a role in prostaglandin signaling because knockdown of ERG expression in TMPRSS2-ERG fusion containing CaP cells leads to altered levels of the 15-hydroxy-prostaglandin dehydrogenase (HPGD), a tumor suppressor and prostaglandin catabolizing enzyme, and prostaglandin E2 (PGE2) . We demonstrate that HPGD expression is regulated by the binding of the ERG protein to the core promoter of this gene. Moreover, prostaglandin E2 dependent cell growth and urokinase-type plasminogen activator (uPA) expression are also affected by ERG knockdown. Together, these data imply that the ERG oncoprotein in CaP cells positively influence prostaglandin mediated signaling, which may contribute to tumor progression.

Gene fusions prevalent in prostate cancer (CaP) lead to the elevated expression of the ERG proto-oncogene. ERG activation present in 50-70% of prostate tumors underscores one of the most common oncogenic alterations in CaP. Despite numerous reports of gene fusions and mRNA expression, ERG oncoprotein status in CaP still remains to be defined. Furthermore, development of ERG protein-based assays may provide a new dimension to evaluation of gene fusions involving diverse androgen-regulated promoters and the ERG protein-coding sequence. Through exhaustive evaluations of 132 whole-mount prostates (261 tumor foci and over 200 000 benign glands) for the ERG oncoprotein nuclear expression, we demonstrated 99.9% specificity for detecting prostate tumor cells using a highly specific anti-ERG monoclonal antibody. The ERG oncoprotein expression correlated well with fusion transcript or gene fusion in randomly selected specimens. Strong concordance of ERG-positive foci of prostatic intraepithelial neoplasia (PIN) with ERG-positive carcinoma (82 out of 85 sections with PIN, 96.5%) affirms the biological role of ERG in clonal selection of prostate tumors in 65% (86 out of 132) of patients. Conversely, ERG negative PINs were associated with ERG-negative carcinoma. Taken together, the homogeneous and strong ERG expression detected in individual tumors establishes the potential for ERG oncoprotein-based stratification of CaP.

Purpose: Prevalent gene fusions in prostate cancer involve androgen-regulated promoters (primarily TMPRSS2) and ETS transcription factors (predominantly ETS-regulated gene (ERG)], which result in tumor selective overexpression of ERG in two thirds of patients. Because diverse genomic fusion events lead to ERG overexpression in prostate cancer, we reasoned that it may be more practical to capture such alterations using an assay targeting ERG sequences retained in such gene fusions. This study evaluates the potential of an assay quantitating ERG mRNA in post-digital rectal exam (DRE) urine for improving prostate cancer detection., Experimental Design: Patients scheduled to undergo transrectal ultrasound-guided needle biopsy of the prostate were prospectively enrolled. On the day of biopsy, patients provided a urine sample immediately following a DRE. Urine ERG mRNA was measured and normalized to urine prostate-specific antigen (PSA) mRNA using the DTS 400 system. Demographic traits, clinical characteristics and biopsy results were analyzed for association with urine ERG score., Results: The study was conducted on 237 patients. Prostate cancer was shown on biopsy in 40.9% of study subjects. A higher urine ERG score associated significantly with malignancy on biopsy (P = 0.0145), but not with clinical stage or Gleason score. Urine ERG score performed best in Caucasians and in men with a PSA of

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-length TMPRSS2-ERG transcripts in prostate cancer., Experimental Design: Full-length TMPRSS2-ERG transcripts were cloned and sequenced from a cDNA library generated from pooled RNA of six TMPRSS2-ERG fusion-positive prostate tumors. The encoded ERG proteins were analyzed in HEK293 cells. Copy numbers of TMPRSS2-ERG splice variants were determined by quantitative reverse transcription-PCR in laser capture microdissected prostate cancer cells., Results: Two types of TMPRSS2-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 from 122 patients, relative abundance of these variants was in the following order: 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 correlation with 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.

Transcription factors encoded by the ETS family of genes are central in integrating signals that regulate cell growth and differentiation, stress responses, and tumorigenesis. This study, analysing laser microdissected paired benign and malignant prostate epithelial cells from prostate cancer (CaP) patients (n=114; 228 specimen) by GeneChip and quantitative real-time RT-PCR, identifies ETS-related gene (ERG), a member of the ETS transcription factor family, as the most frequently overexpressed proto-oncogene in the transcriptome of malignant prostate epithelial cells. Combined quantitative expression analysis of ERG with two other genes commonly overexpressed in CaP, AMACR and DD3, revealed overexpression of at least one of these three genes in virtually all CaP specimen (54 of 55). Comprehensive evaluation of quantitative ERG1 expression with clinicopathological features also suggested that ERG1 expression level in prostate tumor cells relative to benign epithelial cells is indicator of disease-free survival after radical prostatectomy.