An Interaction with Ewing's Sarcoma Breakpoint Protein EWS Defines a Specific Oncogenic Mechanism of ETS Factors Rearranged in Prostate Cancer.
ABSTRACT: More than 50% of prostate tumors have a chromosomal rearrangement resulting in aberrant expression of an oncogenic ETS family transcription factor. However, mechanisms that differentiate the function of oncogenic ETS factors expressed in prostate tumors from non-oncogenic ETS factors expressed in normal prostate are unknown. Here, we find that four oncogenic ETS (ERG, ETV1, ETV4, and ETV5), and no other ETS, interact with the Ewing's sarcoma breakpoint protein, EWS. This EWS interaction was necessary and sufficient for oncogenic ETS functions including gene activation, cell migration, clonogenic survival, and transformation. Significantly, the EWS interacting region of ERG has no homology with that of ETV1, ETV4, and ETV5. Therefore, this finding may explain how divergent ETS factors have a common oncogenic function. Strikingly, EWS is fused to various ETS factors by the chromosome translocations that cause Ewing's sarcoma. Therefore, these findings link oncogenic ETS function in both prostate cancer and Ewing's sarcoma.
Project description:Chromosomal abnormalities that give rise to elevated expression levels of the ETS genes ETV1, ETV4, ETV5, or ERG are prevalent in prostate cancer, but the function of these transcription factors in carcinogenesis is not clear. Previous work in cell lines implicates ERG, ETV1, and ETV5 as regulators of invasive growth but not transformation. Here we show that the PC3 prostate cancer cell line provides a model system to study the over-expression of ETV4. Migration assays, anchorage independent growth assays, and microarray analysis indicate that high ETV4 expression contributes to both transformation and cellular motility in PC3 cells. ETV4 directly bound the 5' and 3' MYC enhancers and modulated expression of both MYC and other cell proliferation genes, demonstrating a potential role in cell growth control. Despite this novel role for ETV4 in anchorage independent growth, ETV4 over-expression in normal prostate-derived RWPE-1 cells showed effects similar to ETV1 over-expression - increased cellular motility, and an up-regulation of genes encoding extracellular proteins as well as ones important for development, inflammation, and wound healing. Because ETV1 and ETV4 have similar roles when introduced to the same cellular background, we suggest that the requirement of high ETV4 expression for maintenance of the anchorage-independent growth in PC3 cells is due to a specific characteristic of this cell line rather than a function of ETV4 that is distinct from the other oncogenic ETS genes. Thus, the function of ETS genes in prostate cancer may differ based on other genetic alterations in a tumor.
Project description:Gene fusions involving the erythroblast transformation-specific (ETS) transcription factors ERG, ETV1, ETV4, ETV5, and FLI1 are a common feature of prostate carcinomas (PCas). The most common upstream fusion partner described is the androgen-regulated prostate-specific gene TMPRSS2, most frequently with ERG, but additional 5' fusion partners have been described. We performed 5' rapid amplification of cDNA ends in 18 PCas with ETV1, ETV4, or ETV5 outlier expression to identify the 5' fusion partners. We also evaluated the exon-level expression profile of these ETS genes in 14 cases. We identified and confirmed by fluorescent in situ hybridization (FISH) and reverse transcription-polymerase chain reaction the two novel chimeric genes OR51E2-ETV1 and UBTF-ETV4 in two PCas. OR51E2 encodes a G-protein-coupled receptor that is overexpressed in PCas, whereas UBTF is a ubiquitously expressed gene encoding an HMG-box DNA-binding protein involved in ribosome biogenesis. We additionally describe two novel gene fusion combinations of previously described genes, namely, SLC45A3-ETV4 and HERVK17-ETV4. Finally, we found one PCa with TMPRSS2-ETV1, one with C15orf21-ETV1, one with EST14-ETV1, and two with 14q133-q21.1-ETV1. In nine PCas (eight ETV1 and one ETV5), exhibiting ETS outlier expression and genomic rearrangement detected by FISH, no 5' fusion partner was found. Our findings contribute significantly to characterize the heterogeneous group of ETS gene fusions and indicate that all genes described as 5' fusion partners with one ETS gene can most likely be rearranged with any of the other ETS genes involved in prostate carcinogenesis.
Project description:Genomic rearrangements involving the ETS family of transcription factors occur in 40-70% of prostate cancer cases. ERG and ETV1 are the most common ETS members observed in these genetic alterations. The high prevalence of these rearrangements and their biological significance represents a novel therapeutic target for the treatment of prostate cancer.We recently reported the development of YK-4-279, a small molecule inhibitor of EWS-FLI1 oncoprotein in Ewing's Sarcoma. Since ERG and ETV1 belong to the same class of ETS factors as FLI1, we tested the ability of YK-4-279 to inhibit biological functions of ERG and ETV1 proteins in prostate cancer. YK-4-279 inhibited ERG and ETV1 mediated transcriptional activity in a luciferase assay. YK-4-279 also decreased ERG and ETV1 downstream target mRNA and protein expression in ETV1-fusion positive LNCaP and ERG fusion positive VCaP cells. YK-4-279 reduced the motility of LNCaP cells in a scratch assay and the invasive phenotype of both LNCaP and VCaP cells in a HUVEC invasion assay. Fusion-negative PC3 cells were unresponsive to YK-4-279. SiRNA mediated ERG knockdown in VCaP cells resulted in a loss of drug responsiveness. Concurrently, transient ERG expression in PC-3 cells resulted in increased invasive potential, which was reduced by YK-4-279.These data demonstrate that YK-4-279 inhibits ERG and ETV1 biological activity in fusion-positive prostate cancer cells leading to decreased motility and invasion. Therefore, YK-4-279 may have an impact on metastasis in prostate cancer and it may be further evaluated for its clinical applications in prostate cancer in addition to Ewing's sarcoma.
Project description:The genetic basis of 50% to 60% of prostate cancer (PCa) is attributable to rearrangements in E26 transformation-specific (ETS) (ERG, ETV1, ETV4, and ETV5), BRAF, and RAF1 genes and overexpression of SPINK1. The development and validation of reliable detection methods are warranted to classify various molecular subtypes of PCa for diagnostic and prognostic purposes. ETS gene rearrangements are typically detected by fluorescence in situ hybridization and reverse-transcription polymerase chain reaction methods. Recently, monoclonal antibodies against ERG have been developed that detect the truncated ERG protein in immunohistochemical assays where staining levels are strongly correlated with ERG rearrangement status by fluorescence in situ hybridization. However, specific antibodies for ETV1, ETV4, and ETV5 are unavailable, challenging their clinical use. We developed a novel RNA in situ hybridization-based assay for the in situ detection of ETV1, ETV4, and ETV5 in formalin-fixed paraffin-embedded tissues from prostate needle biopsies, prostatectomy, and metastatic PCa specimens using RNA probes. Further, with combined RNA in situ hybridization and immunohistochemistry we identified a rare subset of PCa with dual ETS gene rearrangements in collisions of independent tumor foci. The high specificity and sensitivity of RNA in situ hybridization provides an alternate method enabling bright-field in situ detection of ETS gene aberrations in routine clinically available PCa specimens.
Project description:Genomic rearrangements involving ETS transcription factors are found in 50-70% of prostate carcinomas. While the large majority of the rearrangements involve ERG, around 10% involve members of the PEA3 subfamily (ETV1, ETV4 and ETV5). Using a panel of prostate cancer cell lines we found co-overexpression of ETV1 and ETV4 in two cell line models of advanced prostate cancer (MDA-PCa-2b and PC3) and questioned whether these PEA3 family members would cooperate in the acquisition of oncogenic properties or show functional redundancy. Using shRNAs we found that ETV1 and ETV4 have partially overlapping functions, with ETV1 being more relevant for cell invasion and ETV4 for anchorage-independent growth. In vitro expression signatures revealed the regulation of both specific and shared candidate targets that may resemble cellular mechanisms in vivo by interaction with the same intermediate partners. By combining the phenotypic impact data and the gene expression profiles of in vitro models with clinico-pathological features and gene expression profiles of ETS-subtyped tumors, we identified a set of eight genes associated with advanced stage and a set of three genes associated with higher Gleason score, supporting an oncogenic role of ETV1 and ETV4 overexpression and revealing gene sets that may be useful as prognostic markers.
Project description:Chromosomal rearrangements resulting in the fusion of TMPRSS2, an androgen-regulated gene, and the ETS family transcription factor ERG occur in over half of prostate cancers. However, the mechanism by which ERG promotes oncogenic gene expression and proliferation remains incompletely understood. Here, we identify a binding interaction between ERG and the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complex, which is conserved among other oncogenic ETS factors, including ETV1, ETV4, and ETV5. We find that ERG drives genome-wide retargeting of BAF complexes in a manner dependent on binding of ERG to the ETS DNA motif. Moreover, ERG requires intact BAF complexes for chromatin occupancy and BAF complex ATPase activity for target gene regulation. In a prostate organoid model, BAF complexes are required for ERG-mediated basal-to-luminal transition, a hallmark of ERG activity in prostate cancer. These observations suggest a fundamental interdependence between ETS transcription factors and BAF chromatin remodeling complexes in cancer.
Project description:Recurrent gene fusions involving E26 transformation-specific (ETS) transcription factors ERG, ETV1, ETV4, or ETV5 have been identified in 40% to 70% of prostate cancers. Here, we used a comprehensive fluorescence in situ hybridization (FISH) split probe strategy interrogating all 27 ETS family members and their five known 5' fusion partners in a cohort of 110 clinically localized prostate cancer patients. Gene rearrangements were only identified in ETS genes that were previously implicated in prostate cancer gene fusions including ERG, ETV1, and ETV4 (43%, 5%, and 5%, respectively), suggesting that a substantial fraction of prostate cancers (estimated at 30-60%) cannot be attributed to an ETS gene fusion. Among the known 5' gene fusion partners, TMPRSS2 was rearranged in 47% of cases followed by SLC45A3, HNRPA2B1, and C15ORF21 in 2%, 1%, and 1% of cases, respectively. Based on this comprehensive FISH screen, we have made four noteworthy observations. First, by screening the entire ETS transcription factor family for rearrangements, we found that a large fraction of prostate cancers (44%) cannot be ascribed to an ETS gene fusion, an observation which will stimulate research into identifying recurrent non-ETS aberrations in prostate cancers. Second, we identified SLC45A3 as a novel 5' fusion partner of ERG; previously, TMPRSS2 was the only described 5' partner of ERG. Third, we identified two prostate-specific, androgen-induced genes, FLJ35294 and CANT1, as 5' partners to ETV1 and ETV4. Fourth, we identified a ubiquitously expressed, androgen-insensitive gene, DDX5, fused in frame with ETV4, leading to the expression of a DDX5-ETV4 fusion protein.
Project description:UNLABELLED:The ETS family of transcription factors has been repeatedly implicated in tumorigenesis. In prostate cancer, ETS family members, such as ERG, ETV1, ETV4, and ETV5, are frequently overexpressed due to chromosomal translocations, but the molecular mechanisms by which they promote prostate tumorigenesis remain largely undefined. Here, we show that ETS family members, such as ERG and ETV1, directly repress the expression of the checkpoint kinase 1 (CHK1), a key DNA damage response cell-cycle regulator essential for the maintenance of genome integrity. Critically, we find that ERG expression correlates with CHK1 downregulation in human patients and demonstrate that Chk1 heterozygosity promotes the progression of high-grade prostatic intraepithelial neoplasia into prostatic invasive carcinoma in Pten(+) (/-) mice. Importantly, CHK1 downregulation sensitizes prostate tumor cells to etoposide but not to docetaxel treatment. Thus, we identify CHK1 as a key functional target of the ETS proto-oncogenic family with important therapeutic implications. SIGNIFICANCE:Genetic translocation and aberrant expression of ETS family members is a common event in different types of human tumors. Here, we show that through the transcriptional repression of CHK1, ETS factors may favor DNA damage accumulation and consequent genetic instability in proliferating cells. Importantly, our findings provide a rationale for testing DNA replication inhibitor agents in ETS-positive TP53-proficient tumors.
Project description:The discovery of translocations that involve one of the genes of the ETS family (ERG, ETV1, ETV4 and ETV5) has been a major advance in understanding the molecular basis of prostate cancer (PC). Each one of these translocations results in deregulated expression of one of the ETS proteins. Here, we focus on the mechanism whereby overexpression of the ETV4 gene mediates oncogenesis in the prostate. By siRNA technology, we show that ETV4 inhibition in the PC3 cancer cell line reduces not only cell mobility and anchorage-independent growth, but also cell proliferation, cell cycle progression and tumor growth in a xenograft model. Conversely, ETV4 overexpression in the nonmalignant human prostate cell line (RWPE) increases anchorage-independent growth, cell mobility and cell proliferation, which is probably mediated by downregulation of p21, producing accelerated progression through the cell cycle. ETV4 overexpression is associated with changes in the pattern of E-cadherin and N-cadherin expression; the cells also become spindle-shaped, and these changes are characteristic of the so-called epithelial to mesenchymal transition (EMT). In RWPE cells overexpressing ETV4 EMT results from a marked increase in EMT-specific transcription factors such as TWIST1, SLUG1, ZEB1 and ZEB2. Thus, whereas ETV4 shares with the other ETS proteins (ERG, ETV5 and ETV1) a major role in invasiveness and cell migration, it emerges as unique in that it increases at the same time also the rate of proliferation of PC cells. Considering the wide spectrum in the clinical course of patients with PC, it may be highly relevant that ETV4 is capable of inducing most and perhaps all of the features that make a tumor aggressive.
Project description:The homologous ETV1, ETV4 and ETV5 proteins form the PEA3 subfamily of ETS transcription factors. In Ewing tumors, chromosomal translocations affecting ETV1 or ETV4 are an underlying cause of carcinogenesis. Likewise, chromosomal rearrangements of the ETV1, ETV4 or ETV5 gene occur in prostate tumors and are thought to be one of the major driving forces in the genesis of prostate cancer. In addition, these three ETS proteins are implicated in melanomas, breast and other types of cancer. Complex posttranslational modifications govern the activity of PEA3 factors, which can promote cell proliferation, motility and invasion. Here, we review evidence for a role of ETV1, 4 and 5 as oncoproteins and describe modes of their action. Modulation of their activation or interaction with cofactors as well as inhibiting crucial target gene products may ultimately be exploited to treat various cancers that are dependent on the PEA3 group of ETS transcription factors.