B-346 Serum ERG Protein as a Potential Biomarker for Prostate Cancer

The erythroblast transformation-specific (ETS) family of transcription factors plays important roles in both physiological and pathological conditions. Even though many studies have focused on single ETS factors within a single tissue and within the context of specific promoters, the functional impact of multiple ETS members present within a specific cell type has not yet been investigated, especially in prostate cancer (PCa). As the most prominent gene rearrangement in PCa leads to the overexpression of the ETS-related gene (ERG), the aim of this study was to investigate whether ERG is part of a complex integrated transcriptional network that involves other ETS factors. More specifically, as the ETS family consists of 27 members, we focused our efforts initially on investigating whether ERG is associated with the three family members, ETS-1, ETS-2 and ETS variant gene-4 (ETV-4), in PCa as a proof of principle. Using western blot analysis, we show that ERG, ETS-1, ETS-2 and ETV-4 are expressed in PC3 cell nuclear extracts and in protein lysates prepared from human PCa prostatectomy specimens. Immunoprecipitations using an anti-ERG antibody were used with PC3 cell nuclear extracts as well as with a pooled protein lysate sample prepared from the PCa tissue samples of five patients. Importantly, our results revealed that ERG is specifically associated with ETS-2 and ETV-4, but not with ETS-1, in PC3 cell nuclear extracts and PCa tissue protein lysates. Our findings strongly support the notion that ERG is part of a complex integrated transcriptional network that involves other ETS factors, which are likely to cooperate or influence the activity of ERG in PCa. The functional impact of multiple ETS factors being associated with ERG in PCa requires further study, as it may provide insights into the mechanism by which ERG exerts its influence in PCa and may subsequently contribute to our understanding of the molecular basis of PCa.

Potential for targeted therapy in prostate cancers with ERG abnormalities

Objective To study the expressions of transmembrane protease serine 2-E26 transformation specific (TMPRSS2-ETS) fusion gene and ETS related gene (ERG), ETS variant 1 (ETV1), phosphatase and tensin homolog deleted on chromosome 10 (PTEN), androgen receptor (AR) in prostate cancer. Methods A total of 50 cases of prostate cancer paraffin specimens and 30 cases of benign prostatic hyperplasia as the controls in the same period from the Third People's Hospital of Datong between December 2016 and October 2017 were collected. TMPRSS2-ERG, TMPRSS2-ETV1 and TMPRSS2-ETV4 gene fusion status were detected by using fluorescence in situ hybridization. Immunohistochemistry was used to detect the expressions of ERG, ETV1, PTEN and AR protein. Results In prostate cancer tissues, TMPRSS2-ETS fusion gene positive rate was 70% (35/50), and TMPRSS2-ETS fusion gene was not detected in benign prostatic hyperplasia. The expression of TMPRSS2-ETS fusion gene in prostate cancer patients with different age, serum prostate specific antigen level and whether distant metastasis had no statistically significant differences (all P > 0.05). The expression of TMPRSS2-ETS fusion gene in Gleason score > 7 patients was higher than that in Gleason score≤7 patients (P 0.05). The expressions of TMPRSS2-ETS fusion gene and ERG in prostate cancer were positively correlated (r=0.302, P 0.05). Conclusions The expressions of TMPRSS2-ETS fusion gene and ERG, ETV1 protein are upregulated in prostate cancer, which may involve in the occurrence and development of prostate cancer. The detection of TMPRSS2-ETS fusion genes and ERG protein can provide a reference for the diagnosis of prostate cancer. Key words: Prostatic neoplasms; Transmembrane protease serine 2; E26 transformation specific; Gene fusion; Fluorescence in situ hybridization

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. 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. 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. 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.

Expression profiles of erythroblast transformation-specific (ETS)-related gene fusions and serine protease inhibitor Kazal-type 1 (SPINK1) in early onset prostate cancer have not been thoroughly explored. We retrieved 151 radical prostatectomy specimens from young men with prostate cancer (<55 years) and characterized the expression of ETS-related gene (ERG), SPINK1, ETS Variant 1 (ETV1), and ETV4 by dual immunohistochemistry and dual RNA in situ hybridization. Age, race, family history, preoperative prostate-specific antigen, biochemical recurrence, and pathological variables using whole-mount radical prostatectomy tissue were collected. A total of 313 tumor nodules from 151 men including 68 (45%) Caucasians and 61 (40%) African Americans were included in the analysis. Positive family history of prostate cancer was seen in 65 (43%) patients. Preoperative prostate-specific antigen ranged from 0.3 to 52.7 ng/mL (mean = 7.04). The follow-up period ranged from 1 to 123.7 months (mean = 30.3). Biochemical recurrence was encountered in 8 of 151 (5%). ERG overexpression was observed in 85 of 151 (56%) cases, followed by SPINK1 in 61 of 151 (40%), ETV1 in 9 of 149 (6%), and ETV4 in 4 of 141 (3%). There were 25 of 151 (17%) cases showing both ERG and SPINK1 overexpression within different regions of either the same tumor focus or different foci. Higher frequency of ERG overexpression was seen in younger patients (≤45 years old; 76% vs 49%, P = .002), Caucasian men (71% vs 41% P = .0007), organ-confined tumors (64% vs 33%, P = .0008), and tumors of Gleason Grade groups 1 and 2 (62% vs 26%, P = .009). SPINK1 overexpression was more in African American men (68% vs 26%, P = .00008), in tumors with high tumor volume (>20%) and with anterior located tumors. ETV1 and ETV4 demonstrated rare overexpression in these tumors, particularly in the higher-grade tumors. This study expands the knowledge of the clonal evolution of multifocal cancer in young patients and support differences in relation to racial background and genetics of prostate cancer.

Title: SNP Microarray Reveals Predicted Outcomes of a Novel High Risk AML Subgroup with ERG Amplification

Increasing evidence indicates that inflammation may play important roles in tumorigenesis and progression, and an elevated peripheral monocyte count predicts a poor prognosis in various types of malignancies. Here, we evaluate the roles of peripheral monocyte count in the diagnosis and prognosis for prostate cancer in Chinese patients. A total of 1107 consecutive patients who had undergone prostate biopsy and 290 prostate cancer patients receiving androgen deprivation therapy as first-line therapy were retrospectively analyzed. The parameters were measured at the time of diagnosis. Univariate and multivariate logistic regression analyses were performed to identify the independent predictors of a positive biopsy. Patients were categorized in two groups using a cutoff point of 0.425 × 109 l−1 as calculated by the receiver-operating curve analysis for prognosis. Univariate and multivariate Cox regression analyses were performed to determine the associations of monocyte count with progression-free survival, cancer-specific survival, and overall survival. Multivariate logistic regression analyses showed that monocyte count, age, prostate-specific antigen (PSA), free/total PSA, and prostate volume were independent predictors for prostate cancer. Multivariate Cox regression analyses identified an elevated monocyte count as an independent prognostic factor for worse cancer-specific survival (hazard ratio = 2.244, P < 0.05) and overall survival (hazard ratio = 1.995, P < 0.05), but not progression-free survival (P = 0.117). Our results indicated that an elevated monocyte count was an independent diagnostic biomarker for prostate cancer, and pretreatment peripheral monocyte count might play a significant role in the prognosis of prostate cancer patients treated with androgen deprivation therapy.

An ETS family member, ETS Related Gene (ERG) is involved in the Ewing family of tumors as well as leukemias. Rearrangement of the ERG gene with the TMPRSS2 gene has been identified in the majority of prostate cancer patients. Additionally, overexpression of ERG is associated with unfavorable prognosis in prostate cancer patients similar to leukemia patients. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) regulate transcription as well as epigenetic status of genes through acetylation of both histones and transcription factors. Deregulation of HATs and HDACs is frequently seen in various cancers, including prostate cancer. Many cellular oncogenes as well as tumor viral proteins are known to target either or both HATs and HDACs. Several studies have demonstrated that there are alterations of HDAC activity in prostate cancer cells. Recently, we found that ERG binds and inhibits HATs, which suggests that ERG is involved in deregulation of protein acetylation. Additionally, it has been shown that ERG is associated with a higher expression of HDACs. In this study, we tested the effect of the HDAC inhibitors valproic acid (VPA) and trichostatin-A (TSA) on ERG-positive prostate cancer cells (VCaP). We found that VPA and TSA induce apoptosis, upregulate p21/Waf1/CIP1, repress TMPRSS2-ERG expression and affect acetylation status of p53 in VCaP cells. These results suggest that HDAC inhibitors might restore HAT activity through two different ways: by inhibiting HDAC activity and by repressing HAT targeting oncoproteins such as ERG.

Prostate Cancer (CaP) is the most common non-cutaneous form of cancer in men. It is also the second leading cause of cancer mortality in men in the USA. Oncogenic activation of the ETS Related Gene (ERG) as a result of gene fusions is the most common genomic alteration in prostate cancer reported to date. ERG has a milieu of transcriptional targets that regulate genes involved in various cellular processes including oncogenesis, inflammation, cell invasion, and DNA damage. Moreover, knockdown (KD) of ERG in TMPRSS2-ERG expressing CaP cells (VCaP) suppresses the growth and invasiveness of VCaP cells and in xenograft models. ERG protein has been implicated as a key player in the progression from pre-invasive to invasive disease status of CaP. Therefore, understanding mechanisms by which ERG promotes CaP progression is essential for developing novel prognostic/therapeutic targets for CaP. Our analyses of miRNA expressions in the human TMPRSS2-ERG fusion harboring VCaP cells compared to ERG-suppressed (with siRNA against ERG) VCaP controls indicate altered expression of miRNAs. Interestingly, we find that ERG suppresses the expression of miR-449a, which has been shown to regulate proliferation of CaP cells. In silico analyses (IPA and Genomatix) of cellular pathways targeted by these miRs focus on regulatory networks that are indicative of EMT and include molecules like DES (desmin), PDGFRA (platelet-derived growth factor receptor, alpha polypeptide) and TGFBR1 (transforming growth factor beta receptor 1). These pathways are potential candidate therapeutic targets. These data form the basis of new insights into the miRNA alterations in the context of major oncogenic activation in prostate cancer. Moreover, these miRNAs will provide novel prognostic marker and therapeutic targets for prostate cancer and will ultimately define CaP associated miRNAs in the context of ERG positive and ERG negative prostate cancers. Supported by: United States Military Cancer Institute [RB] and [SS]. Citation Format: Parameet Kumar, Albert Dobi, George Petrovics, Ahmed Mohamed, Shiv Srivastava, Roopa Biswas. Role of microRNAs in the development of prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5342. doi:10.1158/1538-7445.AM2013-5342

TMPRSS2-ERG fusion resulting in strong Ets-related gene (ERG) overexpression occurs in about 50% of prostate cancers. This study was undertaken to determine the prevalence of ERG overexpression in other tumour types as well as in normal tissues. A total of 11 483 tumours and 72 different normal tissue types were analysed in a tissue microarray format. Strong nuclear ERG overexpression was found in 36.7% of prostate carcinomas as well as in various vascular tumours, including Kaposi sarcomas (91.7%), angiosarcomas (100%) and haemangiomas (90.9%). Moderate to strong nuclear ERG immunostaining was also observed in thymoma (6.1%). Weak to moderate ERG staining was found in a small number of squamous cell carcinomas of the skin, squamous carcinomas of the lung, malignant mesotheliomas, carcinosarcomas of the uterus, gastrointestinal stromal tumours, hepatocellular carcinomas, teratomas of the testis, anaplastic carcinomas of the thyroid, giant cell tumours of the tendon sheath and benign fibrous histiocytomas of the skin. ERG overexpression was not seen in 8886 samples from 132 other tumour types and subtypes. Within normal tissues, immunohistochemically detectable ERG overexpression was restricted to endothelial cells and subsets of lymphocytes. The high specificity of ERG expression in both normal and neoplastic tissues suggests a very narrow biological role for ERG in highly selected tissues.

ERG (ETS-related gene) is a member of the ETS (Erythroblast-transformation specific) family of transcription factors abundantly present in vascular endothelial cells. Recent studies demonstrate that ERG has important roles in blood vessel stability and angiogenesis. However, it is unclear how ERG is potentially involved in microvascular barrier functions and permeability. A wide variety of diseases and clinical conditions including trauma-hemorrhagic shock and burn injury are associated with microvascular dysfunctions, which causes excessive microvascular permeability, tissue edema and eventually, multiple organ dysfunction and death. The main purpose of this study was to determine the specific role of ERG in regulating microvascular permeability in human lung microvascular endothelial cells (HLMEC) and to evaluate if exogenous ERG will protect the barrier. The HLMECs were grown on Transwell inserts as monolayers and were transfected with ERG CRISPR/cas9 knockdown plasmid, ERG CRISPR activation plasmid, recombinant ERG protein or their respective controls. Recombinant vascular endothelial growth factor (VEGF) was used as an inducer of permeability for evaluating the effect of ERG activation on permeability. Changes in barrier integrity and permeability were studied using monolayer permeability assay and immunofluorescence of adherens junction proteins (VE-cadherin and β-catenin) respectively. CRISPR/cas9-based ERG knockdown as well as VEGF treatment induced monolayer hyperpermeability, VE-cadherin, and β-catenin junctional relocation and cytoskeletal F-actin stress fiber formation. CRISPR based ERG activation and recombinant ERG transfection attenuated VEGF-induced monolayer hyperpermeability. ERG activation preserved the adherens junctions and cytoskeleton. These results demonstrate that ERG is a potent regulator of barrier integrity and permeability in human lung microvascular endothelial cells and endogenously or exogenously enhancing ERG provides protection against barrier dysfunction and hyperpermeability.

Spondin-2, a Secreted Extracellular Matrix Protein, is a Novel Diagnostic Biomarker for Prostate Cancer

ERG3 (ETS-related gene) is a member of the ETS (erythroblast transformation-specific) family of transcription factors, which contain a highly conserved DNA-binding domain. The ETS family of transcription factors differ in their binding to promoter DNA sequences, and the mechanism of their DNA-sequence discrimination is little known. In the current study, crystals of the ETSi domain (the ETS domain of ERG3 containing a CID motif) in space group P41212 and of its complex with the E74 DNA sequence (DNA9) in space group C2221 were obtained and their structures were determined. Comparative structure analysis of the ETSi domain and its complex with DNA9 with previously determined structures of the ERGi domain (the ETS domain of ERG containing inhibitory motifs) in space group P65212 and of the ERGi-DNA12 complex in space group P41212 were performed. The ETSi domain is observed as a homodimer in solution as well as in the crystallographic asymmetric unit. Superposition of the structure of the ETSi domain on that of the ERGi domain showed a major conformational change at the C-terminal DNA-binding autoinhibitory (CID) motif, while minor changes are observed in the loop regions of the ETSi-domain structure. The ETSi-DNA9 complex in space group C2221 forms a structure that is quite similar to that of the ERG-DNA12 complex in space group P41212. Upon superposition of the complexes, major conformational changes are observed at the 5' and 3' ends of DNA9, while the conformation of the core GGA nucleotides was quite conserved. Comparison of the ETSi-DNA9 structure with known structures of ETS class 1 protein-DNA complexes shows the similarities and differences in the promoter DNA binding and specificity of the class 1 ETS proteins.

ERG (ETS-related gene) is a member of the ETS (erythroblast transformation-specific) family of transcription factors. Overexpression of the ERG transcription factor is observed in half of all prostate tumors and is an underlying cause of this disease. However, the mechanisms involved in the functions of ERG are still not fully understood. In the present study, we showed that ERG can directly bind to KDM4A (also known as JMJD2A), a histone demethylase that particularly demethylates lysine 9 on histone H3. ERG and KDM4A cooperated in upregulating the promoter of Yes-associated protein 1 (YAP1), a downstream effector in the Hippo signaling pathway and crucial growth regulator. Multiple ERG binding sites within the human YAP1 gene promoter were identified and their impact on transcription was determined through mutational analysis. Furthermore, we found that ERG expression reduced histone H3 lysine 9 trimethylation at the YAP1 gene promoter, consistent with its epigenetic regulation through the ERG interaction partner, KDM4A. Finally, downregulation of YAP1 phenocopied the growth-retarding effect of ERG or KDM4A depletion in human VCaP prostate cancer cells. Collectively, these results elucidated a novel mechanism - ERG promotes prostate tumorigenesis together with KDM4A through the upregulation of YAP1. A corollary is that KDM4A as well as YAP1 inhibitors may prove beneficial for the therapy of ERG-overexpressing prostate tumors.

Chromosomal rearrangements that result in high expression levels of the ETS-related gene (ERG) present in approximately 50% of prostate cancer (PCa) patients, making this one of the most common oncogenic alterations in PCa. However, ERG overexpression at the protein level has not been rigorously evaluated in Japanese PCa patients. In this study, we evaluated ERG expression using antibody-based detection in 230 prostate specimens in a Japanese PCa cohort. Overall, we identified 20.1% ERG-positive PCa cases. ERG was not detected in benign glands. The specificity of ERG staining for detecting PCa was almost 100%; all of the ERG-positive samples were also diagnosed as PCa. The expression level of the ERG protein correlated with clinicopathological variables, including grade (P= 0.038), stage (P= 0.005), and metastatic status (P= 0.014). No correlation was observed with age (P= 0.196) or with preoperative prostate-specific antigen level (P= 0.322). Although the frequency of ERG-positive cases in Japanese PCa patients (20.1%) was lower than that reported in a PCa cohort in Western countries (approximately 50%), our study demonstrates that the clinical utility of ERG detection at the protein level can serve as an ancillary tool for diagnosing PCa in the Japanese population.