This gene encodes a transcription factor containing an ETS DNA-binding domain. The gene can undergo a t(11;22)(q24;q12) translocation with the Ewing sarcoma gene on chromosome 22, which results in a fusion gene that is present in the majority of Ewing sarcoma cases. An acute lymphoblastic leukemia-associated t(4;11)(q21;q23) translocation involving this gene has also been identified. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Aug 2012]
FLI1 is implicated in: - blood circulation
- DNA binding
- organ morphogenesis
- RNA polymerase II distal enhancer sequence-specific DNA binding
- sequence-specific DNA binding
- sequence-specific DNA binding transcription factor activity
- transcription, DNA-dependent
Data from Gene Ontology via CGAP [Hide]
t(11;22)(q24;q12) EWSR1-FLI1 Translocation in Ewing's Sarcoma The t(11;22)(q24;q12) translocation is present in over 90% of Ewing's sarcoma cases. The resulting EWS-FLI1 fusion gene has been demonstrated to have oncogenic potential. Many alternative forms of the translocation exist, corresponding to variations in the locations of the EWS and FLI1 breakpoints. The most common form, "Type 1", accounts for approximately 60% of cases and consists of the first seven exons of EWS joined to exons 6-9 of FLI1. "Type 2", accounts for approximately 25% of cases and also includes FLI1 exon 5. The type of translocation has been related to prognosis.
EWSR1-FLI1 Fusion Transcript Structure and Prognosis?Prognostic There is wide variation in EWS-FLI1 transcripts. 'Type 1' are created as a result of fusion between exons 7 of EWS and 6 of FLI1, and have reported to be associated with an improved outcome compared to other types of EWS-FLI1 fusions (Zoubek, 1996 and de Alava, 1998). However, in a subsequent COG study of 132 patients (van Doorninck, 2010) concluded that current intensive treatment protocols for localized ESFT have erased the clinical disadvantage that was formerly observed in patients with non-type 1 fusions.
Atlas of Genetics and Cytogenetics in Oncology and Haematology
FLI1 OMIM, Johns Hopkin University Referenced article focusing on the relationship between phenotype and genotype.
FLI1 International Cancer Genome Consortium. Summary of gene and mutations by cancer type from ICGC
FLI1 Cancer Genome Anatomy Project, NCI Gene Summary
FLI1 COSMIC, Sanger Institute Somatic mutation information and related details
FLI1 TICdb, Universidad de Navarra Search the database of Translocation breakpoints In Cancer for "FLI1"
FLI1 GEO Profiles, NCBI Search the gene expression profiles from curated DataSets in the Gene Expression Omnibus (GEO) repository.
Latest Publications: FLI1 (cancer-related)
He T, Surdez D, Rantala JK, et al. High-throughput RNAi screen in Ewing sarcoma cells identifies leucine rich repeats and WD repeat domain containing 1 (LRWD1) as a regulator of EWS-FLI1 driven cell viability. Gene. 2017; 596:137-146 [PubMed] Related Publications
A translocation leading to the formation of an oncogenic EWS-ETS fusion protein defines Ewing sarcoma. The most frequent gene fusion, present in 85 percent of Ewing sarcomas, is EWS-FLI1. Here, a high-throughput RNA interference screen was performed to identify genes whose function is critical for EWS-FLI1 driven cell viability. In total, 6781 genes were targeted by siRNA molecules and the screen was performed both in presence and absence of doxycycline-inducible expression of the EWS-FLI1 shRNA in A673/TR/shEF Ewing sarcoma cells. The Leucine rich repeats and WD repeat Domain containing 1 (LRWD1) targeting siRNA pool was the strongest hit reducing cell viability only in EWS-FLI1 expressing Ewing sarcoma cells. LRWD1 had been previously described as a testis specific gene with only limited information on its function. Analysis of LRWD1 mRNA levels in patient samples indicated that high expression associated with poor overall survival in Ewing sarcoma. Gene ontology analysis of LRWD1 co-expressed genes in Ewing tumors revealed association with DNA replication and analysis of differentially expressed genes in LRWD1 depleted Ewing sarcoma cells indicated a role in connective tissue development and cellular morphogenesis. Moreover, EWS-FLI1 repressed genes with repressive H3K27me3 chromatin marks were highly enriched among LRWD1 target genes in A673/TR/shEF Ewing sarcoma cells, suggesting that LRWD1 contributes to EWS-FLI1 driven transcriptional regulation. Taken together, we have identified LRWD1 as a novel regulator of EWS-FLI1 driven cell viability in A673/TR/shEF Ewing sarcoma cells, shown association between high LRWD1 mRNA expression and aggressive disease and identified processes by which LRWD1 may promote oncogenesis in Ewing sarcoma.
Ishiguro M, Yuki M, Fukushige T, et al. Molecular cytogenetic characterization of two established ESFT cell lines. Hum Cell. 2017; 30(1):41-48 [PubMed] Related Publications
Ewing's sarcoma/primitive neuroectodermal tumor/Askin's tumor (Ewing`s sarcoma family of tumors: ESFT) is the most common type of malignant tumor of bone and soft tissue in children and young adults, and morphologically is a member of a group of small round cell tumors. We report, here, on the establishment of two human ESFT cell lines, FU-PNET-3 and FU-PNET-4, from the iliac and the chest wall, respectively, the cells of both cell lines were tumorigenic in immunodeficient mice. Histologically, both original and xenograft tumors and cultured cells were composed of small round cells with positive immunoreactivity for CD99 and Nkx2.2. Molecular biological examination demonstrated chimeric transcripts of EWSR1 exon 7 to FLI1 exon 6 in FU-PNET-3 cells, and EWSR1 exon 10 to FLI1 exon 6 in FU-PNET-4 cells. Cytogenetic analysis revealed chromosome translocation t(11;22)(q24;q12) and some secondary changes in both cultured cells. These histological, molecular biological, and cytogenetical findings indicate ESFT in both cell lines. ESFT is well studied, but its recurrent fusion genes are heterogeneous and its biological behaviors are unclear. The FU-PNET-3 and FU-PNET-4 cell lines have been well examined and may become useful tools for studying the genetic and biological behavioral properties of ESFT.
BACKGROUND: Ewing sarcoma is the second most common bone tumor, occurring mainly in children and young adults. It shows a typical primitive, small round cell morphology and a characteristic fusion oncogene involving EWSR1 and members of the ETS family in most of the cases. Neuronal maturation after chemotherapy is a rare phenomenon and we herein describe such an exceptional case. CASE PRESENTATION: An 8-year old boy was diagnosed with a Ewing sarcoma in the left femur. On biopsy the morphology was typical and there was an EWSR1-FLI1 gene fusion. He underwent neo-adjuvant chemotherapy and resection of the tumor. On microscopic evaluation, part of the tumor showed ganglioneuroblastoma-like differentiation with expression of neuronal markers. The continued presence of EWSR1 rearrangement in both the blue round cell component and the ganglioneuroblastoma-like component was shown by FISH analysis. CONCLUSIONS: In conclusion, this case describes the possibility of a Ewing sarcoma to differentiate into a ganglioneuroblastoma-like lesion after neo-adjuvant chemotherapy treatment; the prognostic value of this phenomenon remains questionable.
EWS-FLI1, a multi-functional fusion oncogene, is exclusively detected in Ewing sarcomas. However, previous studies reported that rare varieties of osteosarcomas also harbor EWS-ETS family fusion. Here, using the doxycycline-inducible EWS-FLI1 system, we established an EWS-FLI1-dependent osteosarcoma model from murine bone marrow stromal cells. We revealed that the withdrawal of EWS-FLI1 expression enhances the osteogenic differentiation of sarcoma cells, leading to mature bone formation. Taking advantage of induced pluripotent stem cell (iPSC) technology, we also show that sarcoma-derived iPSCs with cancer-related genetic abnormalities exhibited an impaired differentiation program of osteogenic lineage irrespective of the EWS-FLI1 expression. Finally, we demonstrate that EWS-FLI1 contributed to secondary sarcoma development from the sarcoma iPSCs after osteogenic differentiation. These findings demonstrate that modulating cellular differentiation is a fundamental principle of EWS-FLI1-induced osteosarcoma development. This in vitro cancer model using sarcoma iPSCs should provide a unique platform for dissecting relationships between the cancer genome and cellular differentiation.
BACKGROUND: Ewing's sarcoma is an aggressive bone and soft tissue tumor with a high incidence in children and adolescents. Due to its high malignancy and poor prognosis, identification of novel biomarkers for intervention therapies is necessary to improve outcome. The EWS/FLI1 fusion gene is a characteristic of Ewing's sarcoma in most cases. Sex determining region Y-box 2 (SOX2) is a primary target of EWS/FLI1. It has been identified as an oncogene and linked to apoptotic resistance in several types of cancer. However, its role and regulatory mechanisms in Ewing's sarcoma are largely unknown. METHODS: We systematically investigated the role of SOX2 in Ewing's sarcoma cell lines, human tissue samples and xenograft models. The expression of SOX2 was detected in Ewing's sarcoma samples by WB and IHC. siRNAs were used to knockdown EWS/FLI1 and SOX2 in A673 and RD-ES cell lines with the efficiencies tested by qRT-PCR and WB. The effect of SOX2 on cell cycle and apoptosis was determined by Flow cytometric and TUNEL assays. Akt overexpression was performed with plasmid. The protein expression of the corresponding factors was examined by WB analysis. Inhibition of SOX2 in vivo was performed by siRNA against SOX2 in xenograft models, and the protein expression of the regulators testified in vitro was examined in xenograft tumors by IHC and WB. RESULTS: The results confirmed that SOX2 was highly expressed in Ewing's sarcoma and was the target of EWS/FLI1. SOX2 advanced Ewing's sarcoma cell survival and proliferation by regulating p21, p27 and cyclin-E to facilitate G1/S phase transition and mediating caspase-3, PARP via both extrinsic (Fas and caspase-8) and intrinsic (caspase-9, Bad, Bcl-2 and XIAP) apoptotic pathways to restrain cell apoptosis. Additionally, SOX2 regulated the cell-cycle progression and apoptosis via activation of the PI3K/Akt signaling pathway. The mechanisms were proved both in vitro and in vivo. CONCLUSIONS: The results demonstrate that SOX2 played a central role in promoting Ewing's sarcoma cell proliferation in vitro and in vivo with the underlying mechanisms expounded. These findings suggest that SOX2 may serve as a potential biomarker for targeted intervention in Ewing's sarcoma.
Duren RP, Boudreaux SP, Conneely OM Genome Wide Mapping of NR4A Binding Reveals Cooperativity with ETS Factors to Promote Epigenetic Activation of Distal Enhancers in Acute Myeloid Leukemia Cells. PLoS One. 2016; 11(3):e0150450 [PubMed] Free Access to Full ArticleRelated Publications
Members of the NR4A subfamily of orphan nuclear receptors regulate cell fate decisions via both genomic and non-genomic mechanisms in a cell and tissue selective manner. NR4As play a key role in maintenance of hematopoietic stem cell homeostasis and are critical tumor suppressors of acute myeloid leukemia (AML). Expression of NR4As is broadly silenced in leukemia initiating cell enriched populations from human patients relative to normal hematopoietic stem/progenitor cells. Rescue of NR4A expression in human AML cells inhibits proliferation and reprograms AML gene signatures via transcriptional mechanisms that remain to be elucidated. By intersecting an acutely regulated NR4A1 dependent transcriptional profile with genome wide NR4A binding distribution, we now identify an NR4A targetome of 685 genes that are directly regulated by NR4A1. We show that NR4As regulate gene transcription primarily through interaction with distal enhancers that are co-enriched for NR4A1 and ETS transcription factor motifs. Using a subset of NR4A activated genes, we demonstrate that the ETS factors ERG and FLI-1 are required for activation of NR4A bound enhancers and NR4A target gene induction. NR4A1 dependent recruitment of ERG and FLI-1 promotes binding of p300 histone acetyltransferase to epigenetically activate NR4A bound enhancers via acetylation at histone H3K27. These findings disclose novel epigenetic mechanisms by which NR4As and ETS factors cooperate to drive NR4A dependent gene transcription in human AML cells.
Ewing sarcoma is an aggressive primary pediatric bone tumor, often diagnosed in adolescents and young adults. A pathognomonic reciprocal chromosomal translocation results in a fusion gene coding for a protein which derives its N-terminus from a FUS/EWS/TAF15 (FET) protein family member, commonly EWS, and C-terminus containing the DNA-binding domain of an ETS transcription factor, commonly FLI1. Nearly 85% of cases express the EWS-FLI protein which functions as a transcription factor and drives oncogenesis. As the primary genomic lesion and a protein which is not expressed in normal cells, disrupting EWS-FLI function is an attractive therapeutic strategy for Ewing sarcoma. However, transcription factors are notoriously difficult targets for the development of small molecules. Improved understanding of the oncogenic mechanisms employed by EWS-FLI to hijack normal cellular programming has uncovered potential novel approaches to pharmacologically block EWS-FLI function. In this review we examine targeting the chromatin regulatory enzymes recruited to conspire in oncogenesis with a focus on the histone lysine specific demethylase 1 (LSD1). LSD1 inhibitors are being aggressively investigated in acute myeloid leukemia and the results of early clinical trials will help inform the future use of LSD1 inhibitors in sarcoma. High LSD1 expression is observed in Ewing sarcoma patient samples and mechanistic and preclinical data suggest LSD1 inhibition globally disrupts the function of EWS-ETS proteins.
Kovar H, Amatruda J, Brunet E, et al. The second European interdisciplinary Ewing sarcoma research summit--A joint effort to deconstructing the multiple layers of a complex disease. Oncotarget. 2016; 7(8):8613-24 [PubMed] Free Access to Full ArticleRelated Publications
Despite multimodal treatment, long term outcome for patients with Ewing sarcoma is still poor. The second "European interdisciplinary Ewing sarcoma research summit" assembled a large group of scientific experts in the field to discuss their latest unpublished findings on the way to the identification of novel therapeutic targets and strategies. Ewing sarcoma is characterized by a quiet genome with presence of an EWSR1-ETS gene rearrangement as the only and defining genetic aberration. RNA-sequencing of recently described Ewing-like sarcomas with variant translocations identified them as biologically distinct diseases. Various presentations adressed mechanisms of EWS-ETS fusion protein activities with a focus on EWS-FLI1. Data were presented shedding light on the molecular underpinnings of genetic permissiveness to this disease uncovering interaction of EWS-FLI1 with recently discovered susceptibility loci. Epigenetic context as a consequence of the interaction between the oncoprotein, cell type, developmental stage, and tissue microenvironment emerged as dominant theme in the discussion of the molecular pathogenesis and inter- and intra-tumor heterogeneity of Ewing sarcoma, and the difficulty to generate animal models faithfully recapitulating the human disease. The problem of preclinical development of biologically targeted therapeutics was discussed and promising perspectives were offered from the study of novel in vitro models. Finally, it was concluded that in order to facilitate rapid pre-clinical and clinical development of novel therapies in Ewing sarcoma, the community needs a platform to maintain knowledge of unpublished results, systems and models used in drug testing and to continue the open dialogue initiated at the first two Ewing sarcoma summits.
Cancer metastasis is the most deadly stage in cancer progression. Despite significant efforts over the past decades, it remains elusive why only a very small fraction of cancer cells is able to generate micrometastasis and metastatic colonization. Recently we have shown that tumor-repopulating cells (TRCs), a highly tumorigenic subpopulation of mouse melanoma cells, can be selected by being cultured and grown in 3D soft fibrin gels. Here we show that when injected into the yolk of a 2 day-post-fertilization (dpf) embryo of Tg (fli1:EGFP or kdrl:mCherry) zebrafish, TRCs are much more efficient in surviving and growing at various secondary sites to generate micrometastasis and metastatic colonization than control melanoma cells that are grown on rigid plastic. The metastasis of TRCs is dependent on the presence of Sox2, a self-renewal gene, and silencing Sox2 leads to the inhibition of TRC metastasis. High-resolution of 3D confocal images of the TRCs at the secondary sites show that extravasation and formation of micrometastases by TRCs are more efficient than by the control cells. Remarkably, efficient extravasation of TRCs in vivo and transmigration in vitro are determined by TRC deformability, as a result of low Cdc42 and high Sox2. Our findings suggest that tumor cell deformability is a key factor in controlling extravasation dynamics during metastasis.
Ewing sarcoma cells depend on the EWS-FLI1 fusion transcription factor for cell survival. Using an assay of EWS-FLI1 activity and genome-wide RNAi screening, we have identified proteins required for the processing of the EWS-FLI1 pre-mRNA. We show that Ewing sarcoma cells harboring a genomic breakpoint that retains exon 8 of EWSR1 require the RNA-binding protein HNRNPH1 to express in-frame EWS-FLI1. We also demonstrate the sensitivity of EWS-FLI1 fusion transcripts to the loss of function of the U2 snRNP component, SF3B1. Disrupted splicing of the EWS-FLI1 transcript alters EWS-FLI1 protein expression and EWS-FLI1-driven expression. Our results show that the processing of the EWS-FLI1 fusion RNA is a potentially targetable vulnerability in Ewing sarcoma cells.
Sepulveda JL, Gutierrez-Pajares JL, Luna A, et al. High-definition CpG methylation of novel genes in gastric carcinogenesis identified by next-generation sequencing. Mod Pathol. 2016; 29(2):182-93 [PubMed] Related Publications
Gastric cancers are the most frequent gastric malignancy and usually arise in the sequence of Helicobacter pylori-associated chronic gastritis. CpG methylation is a central mechanism of epigenetic gene regulation affecting cancer-related genes, and occurs early in gastric carcinogenesis. DNA samples from non-metaplastic gastric mucosa with variable levels of gastritis (non-metaplastic mucosa), intestinal metaplasia, or gastric cancer were screened with methylation arrays for CpG methylation of cancer-related genes and 30 gene targets were further characterized by high-definition bisulfite next-generation sequencing. In addition, data from The Cancer Genome Atlas were analyzed for correlation of methylation with gene expression. Overall, 13 genes had significantly increased CpG methylation in gastric cancer vs non-metaplastic mucosa (BRINP1, CDH11, CHFR, EPHA5, EPHA7, FGF2, FLI1, GALR1, HS3ST2, PDGFRA, SEZ6L, SGCE, and SNRPN). Further, most of these genes had corresponding reduced expression levels in gastric cancer compared with intestinal metaplasia, including novel hypermethylated genes in gastric cancer (FLI1, GALR1, SGCE, and SNRPN), suggesting that they may regulate neoplastic transformation from non-malignant intestinal metaplasia to cancer. Our data suggest a tumor-suppressor role for FLI1 in gastric cancer, consistent with recently reported data in breast cancer. For the genes with strongest methylation/expression correlation, namely FLI1, the expression was lowest in microsatellite-unstable tumors compared with other gastric cancer molecular subtypes. Importantly, reduced expression of hypermethylated BRINP1 and SGCE was significantly associated with favorable survival in gastric cancer. In summary, we report novel methylation gene targets that may have functional roles in discrete stages of gastric carcinogenesis and may serve as biomarkers for diagnosis and prognosis of gastric cancer.
Chen S, Deniz K, Sung YS, et al. Ewing sarcoma with ERG gene rearrangements: A molecular study focusing on the prevalence of FUS-ERG and common pitfalls in detecting EWSR1-ERG fusions by FISH. Genes Chromosomes Cancer. 2016; 55(4):340-9 [PubMed] Free Access to Full ArticleRelated Publications
The genetics of Ewing sarcoma (ES) are characterized by a canonical fusion involving EWSR1 gene and a member of the ETS family of transcription factors, such as FLI1 and ERG. In fact, ERG gene rearrangements represent the second most common molecular alteration, with EWSR1-ERG being identified in 5-10% of cases, while only a handful of reports document a FUS-ERG fusion. In this study, we focus on ES with ERG gene abnormalities, specifically to investigate the prevalence and clinicopathologic features of FUS-ERG fusions in a large cohort of small blue round cell tumors (SBRCTs) and compare to the eight reported FUS-positive ES. Among the 85 SBRCTs tested, seven (8.2%) cases harbored FUS gene rearrangements; six fused to ERG and one with FEV. During this investigation we came across a number of ERG-rearranged ES lacking both EWSR1 and FUS abnormalities by FISH. In one case, RNA sequencing identified an EWSR1-ERG transcript despite the negative EWSR1 rearrangements by FISH. Additional 3-color FISH fusion assay demonstrated the fusion of EWSR1 and ERG signals in all four cases negative for break-apart EWSR1 FISH. These results emphasize a potential pitfall of relying on EWSR1 FISH assay alone for diagnosis of ES. In cases with classic morphology and/or strong CD99 and ERG immunoreactivity, additional molecular testing should be applied, such as ERG FISH or RT-PCR/next generation sequencing, for a more definitive diagnosis. Although our study group is small, there were no differences noted between the clinical, morphologic features and immunoprofile of the different subsets of ERG-rearranged SBRCTs.
PURPOSE: Primary Ewing sarcoma of the jaw is rare. The aim of this study was to describe new cases of primary Ewing sarcoma of the jaw and investigate reported prognostic factors of Ewing sarcoma in this series and treatment outcome. MATERIALS AND METHODS: Six patients with primary Ewing sarcoma of the jaw were treated at the Memorial Sloan Kettering Cancer Center (MSKCC) from 1992 through 2013. Clinical data, pathology reports, treatment prescribed, treatment regimens, outcome, and follow-up information were reviewed. RESULTS: Five of 6 patients were female and 5 cases were in the mandible. No patient presented with metastatic disease at diagnosis. All cases were positive for CD99, and 3 patients with genetic confirmation were positive for EWS-FLI1 fusion or EWSR1 gene rearrangement. All patients received induction multiagent chemotherapy and surgical resection and 2 patients received adjuvant radiotherapy. Total (grade IV) or nearly total (grade III) tumor necrosis in 3 of 5 patients (60%) assessed for histologic response to chemotherapy indicated intense sensitivity. All patients were alive and free of disease, with no history of local recurrence, at a median follow-up period of 6.5 years. CONCLUSION: Patients with primary Ewing sarcoma of the jaw have a good prognosis and metastasis is an uncommon occurrence at initial presentation.
MicroRNA molecules have a variety of roles in cellular development and proliferation processes, including normal osteogenesis. These effects are exerted through post-translational inhibition of target genes. Altered miRNA expression has been demonstrated in several cancers, both in the tumor tissue and in the peripheral circulation. This may influence carcinogenesis if the specific miRNA targets are encoded by tumor suppressor genes or oncogenes. To date, most research investigating the role of microRNAs and primary bone tumors has focused on osteosarcoma and Ewing sarcoma. Several microRNAs including the miR-34 family have been implicated in osteosarcoma tumorigenesis via effects on the Notch signaling pathway. Progression, invasion, and metastasis of osteosarcoma tumor cells is also influenced by microRNA expression. In addition, microRNA expression may affect the response to chemotherapy in osteosarcoma and thus hold potential for future use as either a prognostic indicator or a therapeutic target. The EWS-FLI1 fusion protein produced in Ewing sarcoma has been shown to induce changes in miRNA expression. MicroRNA expression profiling may have some potential for prediction of disease progression and survival in Ewing sarcoma. There is limited evidence to support a role for microRNAs in other primary bone tumors, either malignant or benign; however, early work is suggestive of involvement in chondrosarcoma, multiple osteochondromatosis, and giant cell tumors of bone.
Ewing sarcomas (ES) are highly malignant bone or soft tissue tumors. Genetically, ES are defined by balanced chromosomal EWS/ETS translocations, which give rise to chimeric proteins (EWS-ETS) that generate an oncogenic transcriptional program associated with altered epigenetic marks throughout the genome. By use of an inhibitor (JQ1) blocking BET bromodomain binding proteins (BRDs) we strikingly observed a strong down-regulation of the predominant EWS-ETS protein EWS-FLI1 in a dose dependent manner. This was further enhanced by co-treatment with an inhibitor of the PI3K pathway. Microarray analysis further revealed JQ1 treatment to block a typical ES associated expression program. The effect on this expression program was mimicked by RNA interference with BRD3 or BRD4 expression, indicating that the EWS-FLI1 mediated expression profile is at least in part mediated via such epigenetic readers. Consequently, contact dependent and independent proliferation of different ES lines was strongly inhibited. Mechanistically, treatment of ES resulted in a partial arrest of the cell cycle as well as induction of apoptosis. Tumor development was suppressed dose dependently in a xeno-transplant model in immune deficient mice, overall indicating that ES may be susceptible to treatment with epigenetic inhibitors blocking BET bromodomain activity and the associated pathognomonic EWS-ETS transcriptional program.
Genes occupy preferred spatial positions within interphase cell nuclei. However, positioning patterns are not an innate feature of a locus, and genes can alter their localization in response to physiological and pathological changes. Here we screen the radial positioning patterns of 40 genes in normal, hyperplasic, and malignant human prostate tissues. We find that the overall spatial organization of the genome in prostate tissue is largely conserved among individuals. We identify three genes whose nuclear positions are robustly altered in neoplastic prostate tissues. FLI1 and MMP9 position differently in prostate cancer than in normal tissue and prostate hyperplasia, whereas MMP2 is repositioned in both prostate cancer and hyperplasia. Our data point to locus-specific reorganization of the genome during prostate disease.
There is substantial heterogeneity among primary prostate cancers, evident in the spectrum of molecular abnormalities and its variable clinical course. As part of The Cancer Genome Atlas (TCGA), we present a comprehensive molecular analysis of 333 primary prostate carcinomas. Our results revealed a molecular taxonomy in which 74% of these tumors fell into one of seven subtypes defined by specific gene fusions (ERG, ETV1/4, and FLI1) or mutations (SPOP, FOXA1, and IDH1). Epigenetic profiles showed substantial heterogeneity, including an IDH1 mutant subset with a methylator phenotype. Androgen receptor (AR) activity varied widely and in a subtype-specific manner, with SPOP and FOXA1 mutant tumors having the highest levels of AR-induced transcripts. 25% of the prostate cancers had a presumed actionable lesion in the PI3K or MAPK signaling pathways, and DNA repair genes were inactivated in 19%. Our analysis reveals molecular heterogeneity among primary prostate cancers, as well as potentially actionable molecular defects.
Ewing sarcoma is an aggressive tumor of bone and soft tissue affecting predominantly children and young adults. Tumor-specific chromosomal translocations create EWS-FLI1 and similar aberrant ETS fusion proteins that drive sarcoma development in patients. ETS family fusion proteins and over-expressed ETS proteins are also found in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients. Transgenic expression of EWS-FLI1 in mice promotes high penetrance erythroid leukemia with dense hepatic and splenic infiltrations. We identified a small molecule, YK-4-279, that directly binds to EWS-FLI1 and inhibits its oncogenic activity in Ewing sarcoma cell lines and xenograft mouse models. Herein, we tested in vivo therapeutic efficacy and potential side effects of YK-4-279 in the transgenic mouse model with EWS-FLI1 induced leukemia. A two-week course of treatment with YK-4-279 significantly reduced white blood cell count, nucleated erythroblasts in the peripheral blood, splenomegaly, and hepatomegaly of erythroleukemic mice. YK-4-279 inhibited EWS-FLI1 target gene expression in neoplastic cells. Treated animals showed significantly better overall survival compared to control mice that rapidly succumbed to leukemia. YK-4-279 treated mice did not show overt toxicity in liver, spleen, or bone marrow. In conclusion, this in vivo study highlights the efficacy of YK-4-279 to treat EWS-FLI1 expressing neoplasms and support its therapeutic potential for patients with Ewing sarcoma and other ETS-driven malignancies.
Alternative splicing plays a key role in the DNA damage response and in cancer. Ewing Sarcomas (ES) are aggressive tumors caused by different chromosomal translocations that yield in-frame fusion proteins driving transformation. RNA profiling reveals genes differentially regulated by UV light irradiation in two ES cell lines exhibiting different sensitivity to genotoxic stress. In particular, irradiation induces a new isoform of the RNA helicase DHX9 in the more sensitive SK-N-MC cells, which is targeted to nonsense-mediated decay (NMD), causing its downregulation. DHX9 protein forms a complex with RNA polymerase II (RNAPII) and EWS-FLI1 to enhance transcription. Silencing of DHX9 in ES cells sensitizes them to UV treatment and impairs recruitment of EWS-FLI1 to target genes, whereas DHX9 overexpression protects ES cells from genotoxic stress. Mechanistically, we found that UV light irradiation leads to enhanced phosphorylation and decreased processivity of RNAPII in SK-N-MC cells, which in turn causes inclusion of DHX9 exon 6A. A similar effect on DHX9 splicing was also elicited by treatment with the chemotherapeutic drug etoposide, indicating a more general mechanism of regulation in response to DNA damage. Our data identify a new NMD-linked splicing event in DHX9 with impact on EWS-FLI1 oncogenic activity and ES cell viability.
Ewing Sarcoma (ES) is the second most common primary malignant bone tumor in children and adolescents. microRNAs (miRNAs) are involved in cancer as tumor suppressors or oncogenes. We studied the involvement of miRNAs located on chromosomes 11q and 22q that participate in the most common translocation in ES. Of these, we focused on 3 that belong to the let-7 family.We studied the expression levels of let-7a, and let-7b and detected a significant correlation between low expression of let-7b and increased risk of relapse. let-7 is known to be a negative regulator of the RAS oncogene. Indeed, we detected an inverse association between the expression of let-7 and RAS protein levels and its downstream target p-ERK, following transfection of let-7 mimics and inhibitors. Furthermore, we identified let-7 as a negative regulator of HIF-1α and EWS-FLI-1. Moreover, we were able to show that HIF-1α directly binds to the EWS-FLI-1 promoter. Salirasib treatment in-vitro resulted in the reduction of cell viability, migration ability, and in the decrease of cells in S-phase. A significant reduction in tumor burden and in the expression levels of both HIF-1α and EWS-FLI-1 proteins were observed in mice after treatment.Our results support the hypothesis that let-7 is a tumor suppressor that negatively regulates RAS, also in ES, and that HIF-1α may contribute to the aggressive metastatic behavior of ES. Moreover, the reduction in the tumor burden in a mouse model of ES following Salirasib treatment, suggests therapeutic potential for this RAS inhibitor in ES.
Ewing sarcoma (ES) is a highly aggressive bone and soft-tissue tumor with peak incidence among adolescents and young adults. Despite advances in local control and systemic chemotherapy, metastatic relapse after an initial clinical remission remains a significant clinical problem. In addition, metastasis at the time of presentation or at relapse continues to be the leading cause of death for patients diagnosed with ES. Since the discovery of the pathognomonic EWS-FLI1 fusion gene more than 20 years ago, much about the molecular and cellular biology of ES pathogenesis has been learned. In addition, more recent exploitation of advances in stem cell and developmental biology has provided key insights into the cellular origins of ES and the role of epigenetic deregulation in tumor initiation and maintenance. Nevertheless, the mechanisms that drive tumor relapse and metastasis remain largely unknown. These gaps in our knowledge continue to hamper the development of novel therapeutic strategies that may improve outcomes for patients with relapsed and metastatic disease. In this article we review the current status of ES biology research, highlighting areas of investigation that we consider to have the greatest potential to yield findings that will translate into clinically significant advances.
Kennedy AL, Vallurupalli M, Chen L, et al. Functional, chemical genomic, and super-enhancer screening identify sensitivity to cyclin D1/CDK4 pathway inhibition in Ewing sarcoma. Oncotarget. 2015; 6(30):30178-93 [PubMed] Free Access to Full ArticleRelated Publications
Ewing sarcoma is an aggressive bone and soft tissue tumor in children and adolescents, with treatment remaining a clinical challenge. This disease is mediated by somatic chromosomal translocations of the EWS gene and a gene encoding an ETS transcription factor, most commonly, FLI1. While direct targeting of aberrant transcription factors remains a pharmacological challenge, identification of dependencies incurred by EWS/FLI1 expression would offer a new therapeutic avenue. We used a combination of super-enhancer profiling, near-whole genome shRNA-based and small-molecule screening to identify cyclin D1 and CDK4 as Ewing sarcoma-selective dependencies. We revealed that super-enhancers mark Ewing sarcoma specific expression signatures and EWS/FLI1 target genes in human Ewing sarcoma cell lines. Particularly, a super-enhancer regulates cyclin D1 and promotes its expression in Ewing sarcoma. We demonstrated that Ewing sarcoma cells require CDK4 and cyclin D1 for survival and anchorage-independent growth. Additionally, pharmacologic inhibition of CDK4 with selective CDK4/6 inhibitors led to cytostasis and cell death of Ewing sarcoma cell lines in vitro and growth delay in an in vivo Ewing sarcoma xenograft model. These results demonstrated a dependency in Ewing sarcoma on CDK4 and cyclin D1 and support exploration of CDK4/6 inhibitors as a therapeutic approach for patients with this disease.
Ewing sarcoma (ES) is the second most frequent bone cancer in childhood and is characterized by the presence of the balanced translocation t(11;22)(q24;q12) in more than 85% of cases, generating a dysregulated transcription factor EWS/FLI1. This fusion protein is an essential oncogenic component of ES development which is necessary for tumor cell maintenance and represents an attractive therapeutic target. To search for modulators of EWS/FLI1 activity we screened a library of 153 targeted compounds and identified inhibitors of the PI3K pathway to directly modulate EWS/FLI1 transcription. Surprisingly, treatment of four different ES cell lines with BEZ235 resulted in down regulation of EWS/FLI1 mRNA and protein by ~50% with subsequent modulation of target gene expression. Analysis of the EWS/FLI1 promoter region (-2239/+67) using various deletion constructs identified two 14 bp minimal elements as being important for EWS/FLI1 transcription. We identified SP1 as modulator of EWS/FLI1 gene expression and demonstrated direct binding to one of these regions in the EWS/FLI1 promoter by EMSA and ChIP experiments. These results provide the first insights on the transcriptional regulation of EWS/FLI1, an area that has not been investigated so far, and offer an additional molecular explanation for the known sensitivity of ES cell lines to PI3K inhibition.
Gomez NC, Davis IJ Linking germline and somatic variation in Ewing sarcoma. Nat Genet. 2015; 47(9):964-5 [PubMed] Related Publications
The identification of gene-regulatory polymorphisms that influence cancer susceptibility can identify key oncogenic pathways. A new study links a germline variant to Ewing sarcoma disease susceptibility and EWSR1-FLI1-mediated gene activation.
Rekhi B, Chinnaswamy G, Vora T, et al. Primary Ewing sarcoma of vulva, confirmed with molecular cytogenetic analysis: A rare case report with diagnostic and treatment implications. Indian J Pathol Microbiol. 2015 Jul-Sep; 58(3):341-4 [PubMed] Related Publications
Primary vulvar Ewing sarcoma (ES)/PNET is an uncommonly documented tumor, especially with molecular results. A 10-year-old girl presented with left vulvar swelling, a year ago. Her abdominopelvic ultrasound revealed a 12 cm × 8 cm sized, mixed echogenic blood-filled lesion in the left vulva; radiologically considered as a hematoma. Vulvectomy revealed a multinodular grey-brown tumor, microscopically comprising malignant round cells. Immunohistochemically, tumor cells diffusely expressed MIC2/CD99 and Fli1 and subsequently displayed EWSR1 rearrangement, confirming diagnosis of ES/PNET. Subsequently, PET-CT scan revealed residual local lesion with lung metastases. The patient was induced on EFT 2001 chemotherapy protocol. Three months after chemotherapy completion, there was no metabolically active disease on PET scan. Four months later, MRI disclosed recurrent primary and metastatic pulmonary lesions. She was planned for scar excision and adjuvant radiotherapy, but unfortunately defaulted further treatment. This forms the eighth case of primary vulvar ES/PNET confirmed with molecular cytogenetic result, underscoring therapeutic value of objective diagnosis in such cases.
Deciphering the ways in which somatic mutations and germline susceptibility variants cooperate to promote cancer is challenging. Ewing sarcoma is characterized by fusions between EWSR1 and members of the ETS gene family, usually EWSR1-FLI1, leading to the generation of oncogenic transcription factors that bind DNA at GGAA motifs. A recent genome-wide association study identified susceptibility variants near EGR2. Here we found that EGR2 knockdown inhibited proliferation, clonogenicity and spheroidal growth in vitro and induced regression of Ewing sarcoma xenografts. Targeted germline deep sequencing of the EGR2 locus in affected subjects and controls identified 291 Ewing-associated SNPs. At rs79965208, the A risk allele connected adjacent GGAA repeats by converting an interspaced GGAT motif into a GGAA motif, thereby increasing the number of consecutive GGAA motifs and thus the EWSR1-FLI1-dependent enhancer activity of this sequence, with epigenetic characteristics of an active regulatory element. EWSR1-FLI1 preferentially bound to the A risk allele, which increased global and allele-specific EGR2 expression. Collectively, our findings establish cooperation between a dominant oncogene and a susceptibility variant that regulates a major driver of Ewing sarcomagenesis.
Breast cancer is the most prevalent malignant disease in women worldwide. In patients with breast cancer, metastasis to distant sites directly determines the survival outcome. However, the molecular mechanism underlying metastasis in breast cancer remains to be defined. In this report, we found that Friend leukemia virus integration 1 (FLI1) proto-oncogene was differentially expressed between the aggressive MDA-MB231 and the non-aggressive MCF-7 breast cancer cells. Congruently, immunohistochemical staining of clinical samples revealed that FLI1 was overexpressed in breast cancers as compared with the adjacent tissues. The abundance of FLI1 protein was strongly correlated with the advanced stage, poor differentiation, and lymph node metastasis in breast cancer patients. Knockdown of FLI1 with small interfering RNAs significantly attenuated the potential of migration and invasion in highly metastatic human breast cancer cells. FLI1 oncoprotein activated the Rho GTPase pathway that is known to play a role in tumor metastasis. This study for the first time identifies FLI1 as a clinically and functionally important target gene of metastasis, providing a rationale for developing FLI1 inhibitors in the treatment of breast cancer.
Sotoca AM, Prange KH, Reijnders B, et al. The oncofusion protein FUS-ERG targets key hematopoietic regulators and modulates the all-trans retinoic acid signaling pathway in t(16;21) acute myeloid leukemia. Oncogene. 2016; 35(15):1965-76 [PubMed] Free Access to Full ArticleRelated Publications
The ETS transcription factor ERG has been implicated as a major regulator of both normal and aberrant hematopoiesis. In acute myeloid leukemias harboring t(16;21), ERG function is deregulated due to a fusion with FUS/TLS resulting in the expression of a FUS-ERG oncofusion protein. How this oncofusion protein deregulates the normal ERG transcription program is unclear. Here, we show that FUS-ERG acts in the context of a heptad of proteins (ERG, FLI1, GATA2, LYL1, LMO2, RUNX1 and TAL1) central to proper expression of genes involved in maintaining a stem cell hematopoietic phenotype. Moreover, in t(16;21) FUS-ERG co-occupies genomic regions bound by the nuclear receptor heterodimer RXR:RARA inhibiting target gene expression and interfering with hematopoietic differentiation. All-trans retinoic acid treatment of t(16;21) cells as well as FUS-ERG knockdown alleviate the myeloid-differentiation block. Together, the results suggest that FUS-ERG acts as a transcriptional repressor of the retinoic acid signaling pathway.
Monument MJ, Grossmann AH, Baker CC, et al. Molecular Confirmation of Ewing Sarcoma in an 85-Year-Old Woman. Int J Surg Pathol. 2015; 23(6):500-4 [PubMed] Related Publications
We report a case of a Ewing sarcoma/primitive neuroectodermal tumor in an 85-year-old woman who presented with an enlarging circumscribed left flank mass. Magnetic resonance imaging revealed a 3 × 5 × 10 cm heterogeneous mass arising from the 10th rib. Computed tomography demonstrated a small nodule in the right middle lobe and bilateral pleural effusions. The patient underwent computed tomography-guided biopsy followed by open biopsy. The tumor cells were characterized by loosely cohesive sheets of tumor cells with uniform nuclei, and scant, granular, eosinophilic cytoplasm with indistinct cell membranes. Frequent mitoses, apoptosis, and necrosis were present. The cells were positive for CD99 with a strong concentric staining pattern. Epithelial, hematopoietic, and neural markers were all negative. Fluorescence in situ hybridization was performed and demonstrated EWSR1 (22q12) gene rearrangement. Sanger sequencing of the reverse transcriptase polymerase chain reaction product from the patient's tumor demonstrated the EWSR1-FLI1 type 1 fusion. Following diagnosis the patient elected to proceed with localized radiation and declined chemotherapy. She developed progressive lung disease and subsequently died of her disease a year after her initial diagnosis. Ewing sarcoma is predominantly a pediatric disease and uncommon in patients older than 40 years of age. To the best of our knowledge, this is the oldest documented case of Ewing sarcoma, diagnosed using modern molecular techniques.
Bishop JA, Alaggio R, Zhang L, et al. Adamantinoma-like Ewing family tumors of the head and neck: a pitfall in the differential diagnosis of basaloid and myoepithelial carcinomas. Am J Surg Pathol. 2015; 39(9):1267-74 [PubMed] Free Access to Full ArticleRelated Publications
Ewing sarcoma family tumors (EFTs) of the head and neck are rare and may be difficult to diagnose, as they display significant histologic overlap with other more common undifferentiated small blue round cell malignancies. Occasionally, EFTs may exhibit overt epithelial differentiation in the form of diffuse cytokeratin immunoexpression or squamous pearls, resembling the so-called adamantinoma-like EFTs and being challenging to distinguish from bona fide carcinomas. Furthermore, the presence of EWSR1 gene rearrangement correlated with strong keratin expression may suggest a myoepithelial carcinoma. Herein, we analyze a series of 7 adamantinoma-like EFTs of the head and neck, most of them being initially misdiagnosed as carcinomas because of their anatomic location and strong cytokeratin immunoexpression, and subsequently reclassified as EFT by molecular techniques. The tumors arose in the sinonasal tract (n=2), parotid gland (n=2), thyroid gland (n=2), and orbit (n=1), in patients ranging in age from 7 to 56 years (mean, 31 y). Microscopically, they departed from the typical EFT morphology by growing as nests with peripheral nuclear palisading and prominent interlobular fibrosis, imparting a distinctly basaloid appearance. Moreover, 2 cases exhibited overt keratinization in the form of squamous pearls, and 1 sinonasal tumor demonstrated areas of intraepithelial growth. All cases were positive for CD99, pancytokeratin, and p40. A subset of cases showed synaptophysin, S100 protein, and/or p16 reactivity, further confounding the diagnosis. Fluorescence in situ hybridization assays showed EWSR1 and FLI1 rearrangements in all cases. Our results reinforce that a subset of head and neck EFTs may show strong cytokeratin expression or focal keratinization, and are therefore histologically indistinguishable from more common true epithelial neoplasms. Thus, CD99 should be included in the immunopanel of a round cell malignancy regardless of strong cytokeratin expression or anatomic location, and a strong and diffuse CD99 positivity should prompt molecular testing for the presence of EWSR1 gene rearrangements.