Albert Dobi

Frequent overexpression of ETS-related gene-1 (ERG1) in prostate cancer transcriptome.

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.

ERG Transcription Factor as an Immunohistochemical Marker for Vascular Endothelial Tumors and Prostatic Carcinoma

ERG, an ETS family transcription factor, is known to be expressed in endothelial cells, and oncogenic ERG gene fusions occur in subsets of prostatic carcinoma, acute myeloid leukemia, and Ewing sarcoma. In this study, we immunohistochemically investigated nuclear ERG expression using a new monoclonal antibody, CPDR ERG-MAb, that is highly specific for detecting ERG protein and ERG-expressing prostate carcinomas. A broad range of vascular endothelial (n=250), other mesenchymal (n=973), and epithelial tumors (n=657) was examined to determine the use of ERG immunohistochemistry in surgical pathology. Only immunostains with ERG-positive normal endothelia (internal control) were considered valid, and only nuclear staining was considered to be positive. In adult tissues, ERG was restricted to endothelial cells and to a subset of bone marrow precursors, but early fetal mesenchyme and subpopulations of fetal cartilage were also positive. In vascular tumors, ERG was expressed in endothelia of all hemangiomas and lymphangiomas, and typically extensively expressed in 96 of 100 angiosarcomas, 42 of 43 epithelioid hemangioendotheliomas, and all 26 Kaposi sarcomas. Among nonvascular mesenchymal tumors, only blastic extramedullary myeloid tumors (7 of 10) and rare Ewing sarcomas (2 of 29) were positive. Among epithelial tumors, 30 of 66 prostatic adenocarcinomas showed focal-to-extensive ERG positivity, with no immunoreactivity in the normal prostate. Other carcinomas and epithelial tumors (n=643) were ERG negative, with the exception of 1 of 42 large cell undifferentiated pulmonary carcinomas and 1 of 27 mesotheliomas, each of which showed focal nuclear ERG positivity. On the basis of the above observations, ERG is a highly specific new marker for benign and malignant vascular tumors. Among epithelial tumors, ERG shows a great promise as a marker to identify prostatic carcinoma in both primary and metastatic settings.

ERG oncoprotein expression in prostate cancer: clonal progression of ERG-positive tumor cells and potential for ERG-based stratification

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.

Mapping of TMPRSS2-ERG fusions in the context of multi-focal prostate cancer

TMPRSS2–ERG gene fusion leading to the androgenic induction of the ERG proto-oncogene expression is a highly prevalent oncogenic alteration in prostate tumor cells. Prostate cancer is a multi-focal disease, and the origins as well as biological contribution of multiple cancer foci remain unclear with respect to prostate cancer onset or progression. To assess the role of TMPRSS2–ERG alteration in prostate cancer onset and/or progression, we have evaluated the status of fusion transcripts in benign glands, prostatic intraepithelial neoplasia (PIN) and multiple cancer foci of each prostate. Quantitative expression of TMPRSS2–ERG fusion type A and C transcripts was analyzed in benign, tumor and PIN areas, selected from whole-mount radical prostatectomy slides. TMPRSS2–ERG expression was correlated with clinicopathological features. Overall, 30 of 45 (67%) patients exhibited TMPRSS2–ERG fusion transcripts in at least one tumor focus. Of 80 tumor foci analyzed, 39 had TMPRSS2–ERG fusion (type A only: 30, type C only: 2, both types A and C: 7), with predominant detection of the TMPRSS2–ERG fusion type A (27/30, 90%) in the index tumors. Of 14 PIN lesions, 2 were positive for type A fusion. Frequent presence of the TMPRSS2–ERG in index tumors suggests critical roles of ERG alterations in the onset and progression of a large subset of prostate cancer. However, heterogeneity of the TMPRSS2–ERG detection in the context of multiple cancer foci and its frequency in PIN also support the role of other genomic alterations in the origins of prostate cancer.

Prostate cancer: Diagnostic performance of the PCA3 urine test

The potential of the prostate cancer antigen 3 (PCA3) urine assay to aid prostate cancer diagnosis and minimize unnecessary biopsies has been extensively studied. Results from three recent studies that compared the performance of PCA3 with PSA underscore the advancement and future challenges for this new diagnostic biomarker.

Delineation of TMPRSS2-ERG Splice Variants in Prostate Cancer

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.

miR-30 as a tumor suppressor connects EGF/Src signal to ERG and EMT

Src tyrosine kinase (Src) is implicated in the development of bone metastasis and castration resistance of prostate cancer. Src inhibitors are currently being tested in clinical trials for such diseases. Understanding the molecular and cellular actions of Src inhibitors holds the key to future improvement of this line of therapy. Here we describe the microRNA expression profiles modulated by two Src inhibitors and demonstrate that the miR-30 family members are the most prominently induced species. Consistent with its tumor suppressor role, miR-30 is downmodulated by oncogenic signals such as epidermal growth factor (EGF) and hepatocyte growth factor, and is generally underexpressed in prostate cancer specimens. A number of epithelial-to-mesenchymal transition (EMT)-associated genes are predicted targets of miR-30. Among these genes the Ets-related gene (ERG) is the most frequently overexpressed oncogene in prostate cancer activated by genomic fusion events between promoter upstream sequences of the TMPRSS2 and coding sequences of ERG. We showed by ERG 3′ untranslated region reporter and mutagenesis assays that ERG is a direct target of miR-30. Overexpression of miR-30 in prostate cancer cells suppresses EMT phenotypes and inhibits cell migration and invasion. It also inhibits the in vitro and in vivo growth of VCaP cells, which depends on TMPRSS2-ERG for proliferation. TMPRSS2-ERG is generally regulated by androgen at the transcriptional level. Our finding reveals a new post-transcriptional mechanism of TMPRSS2-ERG regulation by Src and growth signals via miR-30 providing a rationale for targeting ERG-positive castration-resistant tumors with Src inhibitors.

Isolation and Characterization of SATB2, a Novel AT-rich DNA Binding Protein Expressed in Development- and Cell-Specific Manner in the Rat Brain

AT-rich DNA elements play an important role in regulating cell-specific gene expression. One of the AT-rich DNA binding proteins, SATB1 is a novel type of transcription factor that regulates gene expression in the hematopoietic lineage through chromatin modification. Using DNA-affinity purification followed by mass spectrometry we identified and isolated a related protein, SATB2 from the developing rat cerebral cortex. SATB2 shows homology to SATB1 and the rat protein is practically identical to the mouse and human SATB2. Using competitive EMSA, we show that recombinant SATB2 protein binds with high affinity and specificity to AT-rich dsDNA. Using RT-PCR, Western analysis and immunohistochemistry we demonstrate that SATB2 expression is restricted to a subset of postmitotic, differentiating neurons in the rat neocortex at ages E16 and P4. We suggest that similar to its homologue SATB1, SATB2 is also involved in regulating gene expression through altering chromatin structure in differentiating cortical neurons.

Androgen receptor mutation (T877A) promotes prostate cancer cell growth and cell survival.

Alteration of the AR functions due to amplification, overexpression and somatic mutation of the AR itself or altered interaction of AR with other cell growth regulatory proteins, may contribute to a significant subset of advanced prostate cancer (CaP). Very little is known about the pathways impacted by AR dysfunctions, although CaP associated AR alterations suggest the biological role of the AR dysfunction in disease progression. Comparative evaluations of wild type (wt) AR and mutant (mt) ARs in appropriate experimental models should provide a better understanding of the functional impact of AR alterations in CaP. Here, we provide direct evidence showing cell growth/cell survival promoting effects of the widely studied CaP associated AR mutation (T877A). In contrast to Ad-wtAR or Ad-control infected LNCaP or LAPC4 cells, Ad-mtAR (T877A) infected LNCaP or LAPC4 cells continued to grow in the androgen-deprived medium and exhibited an androgen independent AR-transcription factor activity. Further, Ad-mtAR (T877A) infected LNCaP or LAPC4 cells exhibited enhanced cell growth in the presence of lower concentrations of the synthetic androgen, R1881. Of note, Ad-mtAR (T877A) infected LNCaP cells showed striking resistance to cell growth inhibition/apoptosis mediated by the wt p53. Taken together, these findings provide novel insights into the AR dysfunctions resulting from the T877A mutation and functionally similar AR alterations may provide selective cell growth/survival advantage for CaP progression. These observations have important implications for developing biology-based prognostic biomarkers and therapeutic strategies for CaP showing such AR dysfunctions.

Androgen receptor and prostate cancer

Since the original observations of Huggins and Hodges that prostate cancers are androgen dependent, androgen ablation therapy has been the gold standard for the treatment of advanced prostate cancer (CaP). Androgen receptor (AR) is believed to play critical roles in the development and progression of CaP. Treatment for neoadjuvant, adjuvant and recurrent disease all center on the regulation and manipulation of the androgen pathway, in which AR plays an integral role. Recent discoveries that frequent overexpression of ETS-related proto-oncogenes may be driven by AR as a consequence of common genomic rearrangements can hold the key towards the understanding of early phases of prostate cancer. Furthermore, AR function evolves as the cell changes towards a clinically androgen depletion independent state. Comprehension of AR function, regulation and abnormalities are increasingly refined towards the understanding of the role of AR in CaP, and in therapeutic applications. Development of future therapy for CaP will be aided by improving the knowledge of dysfunctions of AR and its network in prostate cancer. This review focuses salient features of AR and on the recent advances addressing AR dysfunctions in prostate cancer.