ABSTRACT
Ewing sarcoma (EWS) is a paediatric bone cancer with high metastatic potential. Cellular plasticity resulting from dynamic cytoskeletal reorganization, typically regulated via the Rho pathway, is a prerequisite for metastasis initiation. Here, we interrogated the role of the Ewing sarcoma driver oncogene EWS-FLI1 in cytoskeletal reprogramming. We report that EWS-FLI1 strongly represses the activity of the Rho-F-actin signal pathway transcriptional effector MRTFB, affecting the expression of a large number of EWS-FLI1-anticorrelated genes including structural and regulatory cytoskeletal genes. Consistent with this finding, chromatin immunoprecipitation sequencing (ChIP-seq) revealed strong overlaps in myocardin-related transcription factor B (MRTFB) and EWS-FLI1 chromatin occupation, especially for EWS-FLI1-anticorrelated genes. Binding of the transcriptional co-activator Yes-associated protein (YAP)-1, enrichment of TEAD-binding motifs in these shared genomic binding regions and overlapping transcriptional footprints of MRTFB and TEAD factors led us to propose synergy between MRTFB and the YAP/TEAD complex in the regulation of EWS-FLI1-anticorrelated genes. We propose that EWS-FLI1 suppresses the Rho-actin pathway by perturbation of a MRTFB/YAP-1/TEAD transcriptional module, which directly affects the actin-autoregulatory feedback loop. As spontaneous fluctuations in EWS-FLI1 levels of Ewing sarcoma cells in vitro and in vivo, associated with a switch between a proliferative, non-migratory EWS-FLI1-high and a non-proliferative highly migratory EWS-FLI1-low state, were recently described, our data provide a mechanistic basis for the underlying EWS-FLI1-dependent reversible cytoskeletal reprogramming of Ewing sarcoma cells.
Subject(s)
Cellular Reprogramming/genetics , Cytoskeleton/genetics , Oncogene Proteins, Fusion/genetics , Proto-Oncogene Protein c-fli-1/genetics , RNA-Binding Protein EWS/genetics , Sarcoma, Ewing/genetics , Actins/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Chromatin/genetics , Gene Expression Regulation, Neoplastic , Humans , Sarcoma, Ewing/pathology , Signal Transduction/geneticsABSTRACT
BACKGROUND: Despite the effectiveness of current treatment protocols for Ewing sarcoma (ES), many patients still experience relapse, and survival following recurrence is <15%. We aimed to identify genetic variants that predict treatment outcome in children diagnosed with ES. PATIENTS AND METHODS: We carried out a pharmacogenetic study of 384 single-nucleotide polymorphisms (SNPs) in 24 key transport or metabolism genes relevant to drugs used to treat in pediatric patients (<30 years) with histologically confirmed ES. We studied the association of genotypes with tumor response and overall survival (OS) in a discovery cohort of 106 Spanish children, with replication in a second cohort of 389 pediatric patients from across Europe. RESULTS: We identified associations with OS (P < 0.05) for three SNPs in the Spanish cohort that were replicated in the European cohort. The strongest association observed was with rs7190447, located in the ATP-binding cassette subfamily C member 6 (ABCC6) gene [discovery: hazard ratio (HR) = 14.30, 95% confidence interval (CI) = 1.53-134, P = 0.020; replication: HR = 9.28, 95% CI = 2.20-39.2, P = 0.0024] and its correlated SNP rs7192303, which was predicted to have a plausible regulatory function. We also replicated associations with rs4148737 in the ATP-binding cassette subfamily B member 1 (ABCB1) gene (discovery: HR = 2.96, 95% CI = 1.08-8.10, P = 0.034; replication: HR = 1.60, 95% CI = 1.05-2.44, P = 0.029), which we have previously found to be associated with poorer OS in pediatric osteosarcoma patients, and rs11188147 in cytochrome P450 family 2 subfamily C member 8 gene (CYP2C8) (discovery : HR = 2.49, 95% CI = 1.06-5.87, P = 0.037; replication: HR = 1.77, 95% CI = 1.06-2.96, P = 0.030), an enzyme involved in the oxidative metabolism of the ES chemotherapeutic agents cyclophosphamide and ifosfamide. None of the associations with tumor response were replicated. CONCLUSION: Using an integrated pathway-based approach, we identified polymorphisms in ABCC6, ABCB1 and CYP2C8 associated with OS. These associations were replicated in a large independent cohort, highlighting the importance of pharmacokinetic genes as prognostic markers in ES.
Subject(s)
Cytochrome P-450 CYP2C8/genetics , Multidrug Resistance-Associated Proteins/genetics , Sarcoma, Ewing/genetics , ATP Binding Cassette Transporter, Subfamily B/genetics , Adolescent , Adult , Child , Child, Preschool , Female , Genetic Association Studies , Genotype , Humans , Infant , Male , Neoplasm Recurrence, Local/drug therapy , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/pathology , Polymorphism, Single Nucleotide , Sarcoma, Ewing/drug therapy , Sarcoma, Ewing/pathology , Survival Analysis , Treatment Outcome , Young AdultABSTRACT
Sarcomas are mesenchymal tumors characterized by blocked differentiation process. In Ewing sarcoma (EWS) both CD99 and EWS-FLI1 concur to oncogenesis and inhibition of differentiation. Here, we demonstrate that uncoupling CD99 from EWS-FLI1 by silencing the former, nuclear factor-κB (NF-κB) signaling is inhibited and the neural differentiation program is re-established. NF-κB inhibition passes through miR-34a-mediated repression of Notch pathway. CD99 counteracts EWS-FLI1 in controlling NF-κB signaling through the miR-34a, which is increased and secreted into exosomes released by CD99-silenced EWS cells. Delivery of exosomes from CD99-silenced cells was sufficient to induce neural differentiation in recipient EWS cells through miR-34a inhibition of Notch-NF-κB signaling. Notably, even the partial delivery of CD99 small interfering RNA may have a broad effect on the entire tumor cell population owing to the spread operated by their miR-34a-enriched exosomes, a feature opening to a new therapeutic option.
Subject(s)
12E7 Antigen/physiology , MicroRNAs/physiology , NF-kappa B/physiology , Receptors, Notch/physiology , Sarcoma, Ewing/pathology , Signal Transduction/physiology , Cell Differentiation , Humans , Oncogene Proteins, Fusion/physiology , Proto-Oncogene Protein c-fli-1/physiology , RNA, Small Interfering/genetics , RNA-Binding Protein EWS/physiologyABSTRACT
Curative therapies for Ewing sarcoma have been developed within cooperative groups. Consecutive clinical trials have systematically assessed the impact and timing of local therapy and the activity of cytotoxic drugs and their combinations. They have led to an increase of long-term disease-free survival to around 70% in patients with localized disease. Translational research in ES remains an area in which interdisciplinary and international cooperation is essential for future progress. This article reviews current state-of-the art therapy, with a focus on trials performed in Europe, and summarizes novel strategies to further advance both the cure rates and quality of survival.
Subject(s)
Bone Neoplasms/therapy , Cooperative Behavior , Interdisciplinary Communication , Sarcoma, Ewing/therapy , Soft Tissue Neoplasms/therapy , Antineoplastic Combined Chemotherapy Protocols , Bone Neoplasms/mortality , Child , Clinical Trials as Topic , Combined Modality Therapy , Disease Progression , Humans , Neoadjuvant Therapy , Osteotomy , Radiotherapy, Adjuvant , Sarcoma, Ewing/mortality , Soft Tissue Neoplasms/mortality , Survival RateABSTRACT
The Ewing sarcoma (ES) EWS-FLI1 chimeric oncoprotein is a prototypic aberrant ETS transcription factor with activating and repressive regulatory functions. We report that EWS-FLI1-repressed promoters are enriched in forkhead box (FOX) recognition motifs, and identify FOXO1 as a EWS-FLI1-suppressed regulator orchestrating a major subset of EWS-FLI1-repressed genes. In addition to FOXO1 regulation by direct promoter binding of EWS-FLI1, its subcellular localization and activity is regulated by cyclin-dependent kinase 2- and AKT-mediated phosphorylation downstream of EWS-FLI1. Restoration of nuclear FOXO1 expression in ES cells impaired proliferation and significantly reduced clonogenicity. Gene-expression profiling revealed a significant overlap between EWS-FLI1-repressed and FOXO1-activated genes. As a proof of principle for a potential therapeutic application of our findings, the treatment of ES cell lines with methylseleninic acid (MSA) reactivated endogenous FOXO1 in the presence of EWS-FLI1 in a dose- and time-dependent manner and induced massive cell death dependent on FOXO1. In an orthotopic xenograft mouse model, MSA increased FOXO1 expression in the tumor paralleled by a significant decrease in ES tumor growth. FOXO1 reactivation by small molecules may therefore serve as a promising strategy for a future ES-specific therapy.
Subject(s)
Bone Neoplasms/metabolism , Forkhead Transcription Factors/metabolism , Gene Expression Regulation, Neoplastic , Oncogene Proteins, Fusion/metabolism , Proto-Oncogene Protein c-fli-1/metabolism , RNA-Binding Protein EWS/metabolism , Sarcoma, Ewing/metabolism , Animals , Antineoplastic Agents/pharmacology , Base Sequence , Binding Sites , Bone Neoplasms/drug therapy , Bone Neoplasms/genetics , Cell Line, Tumor , Cell Proliferation , Consensus Sequence , Cyclin-Dependent Kinase 2/metabolism , Forkhead Box Protein O1 , Forkhead Box Protein O3 , Forkhead Transcription Factors/genetics , Gene Silencing , Humans , Mice , Oncogene Proteins, Fusion/genetics , Organoselenium Compounds/pharmacology , Phosphorylation , Promoter Regions, Genetic , Protein Processing, Post-Translational , Protein Transport , Proto-Oncogene Protein c-fli-1/genetics , Proto-Oncogene Proteins c-akt/metabolism , RNA-Binding Protein EWS/genetics , Sarcoma, Ewing/drug therapy , Sarcoma, Ewing/genetics , Transcription, Genetic , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Though p53 mutations are rare in ES, there is a strong indication that p53 mutant tumours form a particularly bad prognostic group. As such, novel treatment strategies are warranted that would specifically target and eradicate tumour cells containing mutant p53 in this subset of ES patients. METHODS: PRIMA-1(Met), also known as APR-246, is a small organic molecule that has been shown to restore tumour-suppressor function primarily to mutant p53 and also to induce cell death in various cancer types. In this study, we interrogated the ability of APR-246 to induce apoptosis and inhibit tumour growth in ES cells with different p53 mutations. RESULTS: APR-246 variably induced apoptosis, associated with Noxa, Puma or p21(WAF1) upregulation, in both mutant and wild-type p53 harbouring cells. The apoptosis-inducing capability of APR-246 was markedly reduced in ES cell lines transfected with p53 siRNA. Three ES cell lines established from the same patient at different stages of the disease and two cell lines of different patients with identical p53 mutations all exhibited different sensitivities to APR-246, indicating cellular context dependency. Comparative transcriptome analysis on the three cell lines established from the same patient identified differential expression levels of several TP53 and apoptosis-associated genes such as APOL6, PENK, PCDH7 and MST4 in the APR-246-sensitive cell line relative to the less APR-246-sensitive cell lines. CONCLUSION: This is the first study reporting the biological response of Ewing sarcoma cells to APR-246 exposure and shows gross variability in responses. Our study also proposes candidate genes whose expression might be associated with ES cells' sensitivity to APR-246. With APR-246 currently in early-phase clinical trials, our findings call for caution in considering it as a potential adjuvant to conventional ES-specific chemotherapeutics.
Subject(s)
Antineoplastic Agents/pharmacology , Bone Neoplasms/genetics , Gene Expression Regulation, Neoplastic/drug effects , Quinuclidines/pharmacology , Sarcoma, Ewing/genetics , Tumor Suppressor Protein p53/genetics , Apoptosis/drug effects , Apoptosis/genetics , Bone Neoplasms/pathology , Gene Knockdown Techniques , Humans , Microarray Analysis , Mutation/physiology , RNA, Small Interfering/pharmacology , Sarcoma, Ewing/pathology , Transcriptome , Tumor Cells, Cultured , Tumor Suppressor Protein p53/antagonists & inhibitorsABSTRACT
EWS-FLI1 is a chromosome translocation-derived chimeric transcription factor that has a central and rate-limiting role in the pathogenesis of Ewing's sarcoma. Although the EWS-FLI1 transcriptomic signature has been extensively characterized on the mRNA level, information on its impact on non-coding RNA expression is lacking. We have performed a genome-wide analysis of microRNAs affected by RNAi-mediated silencing of EWS-FLI1 in Ewing's sarcoma cell lines, and differentially expressed between primary Ewing's sarcoma and mesenchymal progenitor cells. Here, we report on the identification of hsa-mir-145 as the top EWS-FLI1-repressed microRNA. Upon knockdown of EWS-FLI1, hsa-mir-145 expression dramatically increases in all Ewing's sarcoma cell lines tested. Vice versa, ectopic expression of the microRNA in Ewing's sarcoma cell lines strongly reduced EWS-FLI1 protein, whereas transfection of an anti-mir to hsa-mir-145 increased the EWS-FLI1 levels. Reporter gene assays revealed that this modulation of EWS-FLI1 protein was mediated by the microRNA targeting the FLI1 3'-untranslated region. Mutual regulations of EWS-FLI1 and hsa-mir-145 were mirrored by an inverse correlation between their expression levels in four of the Ewing's sarcoma cell lines tested. Consistent with the role of EWS-FLI1 in Ewing's sarcoma growth regulation, forced hsa-mir-145 expression halted Ewing's sarcoma cell line growth. These results identify feedback regulation between EWS-FLI1 and hsa-mir-145 as an important component of the EWS-FLI1-mediated Ewing's sarcomagenesis that may open a new avenue to future microRNA-mediated therapy of this devastating malignant disease.
Subject(s)
MicroRNAs/genetics , Oncogene Proteins, Fusion/genetics , Proto-Oncogene Protein c-fli-1/genetics , RNA-Binding Protein EWS/genetics , Sarcoma, Ewing/genetics , Base Sequence , DNA Primers , HumansABSTRACT
The treatment of sarcoma urgently requires new, innovative therapeutic strategies. The most recent improvements in the cure of patients with localized disease have been achieved by dose-intensification, in turn paying the price of acute severe toxicity and secondary malignancies. Keeping side-effects to a minimum is an important goal for pediatric patients and this may be achieved by combining standard cytotoxic chemotherapy with targeted approaches. In addition, after first-line therapy, very limited treatment options remain for patients with disease progression, who, like patients with metastasis at diagnosis, are in urgent need of more effective drugs. The present review highlights key examples of target identification in bone sarcomas, including chimeric oncoproteins, insulin-like growth factor receptor (IGF-IR), and tumor/microenvironment interactions. The review identifies questions and concerns that still need to be addressed before proceeding to safe clinical trials with agents against these promising new targets.
Subject(s)
Bone Neoplasms/drug therapy , Osteosarcoma/drug therapy , Signal Transduction/drug effects , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Bone Neoplasms/genetics , Drug Delivery Systems , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Humans , Models, Biological , Osteosarcoma/genetics , Sarcoma/geneticsABSTRACT
The oncogene EWS-FLI1 encodes a chimeric transcription factor expressed in Ewing's sarcoma family tumors (ESFTs). EWS-FLI1 target gene expression is thought to drive ESFT pathogenesis and, therefore, inhibition of EWS-FLI1 activity holds high therapeutic promise. As the activity of many transcription factors is regulated by post-translational modifications, we studied the presence of modifications on EWS-FLI1. The immuno-purified fusion-protein was recognized by an antibody specific for O-linked beta-N-acetylglucosaminylation, and bound readily to a phosphoprotein-specific dye. Inhibition of Ser/Thr-specific phophatases increased EWS-FLI1 molecular weight and reduced its O-GlcNAc content, suggesting that phosphorylation and O-GlcNAcylation of EWS-FLI1 interact dynamically. By mutation analysis, O-GlcNAcylation was delineated to Ser/Thr residues of the amino-terminal EWS transcriptional-activation domain. Metabolic inhibition of the hexosamine biosynthetic pathway abrogated O-GlcNAcylation of EWS-FLI1 and interfered specifically with transcriptional activation of the EWS-FLI1 target Id2. These results suggest that drugs modulating glycosylation of EWS-FLI1 interfere functionally with its activity and might, therefore, constitute promising additions to the current ESFT chemotherapy.
Subject(s)
Oncogene Proteins, Fusion/genetics , Proto-Oncogene Protein c-fli-1/genetics , Sarcoma, Ewing/genetics , Transcription, Genetic , Acylation , Humans , RNA-Binding Protein EWSABSTRACT
High CD99 expression levels and rearrangements of the EWS gene with ETS transcription factor genes characterize the Ewing's sarcoma family of tumors (ESFT). CD99 is a cell surface glycoprotein whose engagement has been implicated in cell proliferation as well as upregulation and transport of several transmembrane proteins in hematopoietic cells. In ESFT, antibody ligation of CD99 induces fast homotypic cell aggregation and cell death although its functional role in these processes remains largely unknown. Here, using an RNAi approach, we studied for the first time the consequences of modulated CD99 expression in six different ESFT cell lines, representing the most frequent variant forms of EWS gene rearrangement. CD99 suppression resulted in growth inhibition and reduced migration of ESFT cells. Among genes whose expression changes in response to CD99 modulation, the potassium-channel modulatory factor KCMF1 was consistently upregulated. In a series of 22 primary ESFT, KCMF1 expression levels inversely correlated with CD99 abundancy. Cells forced to express ectopic KCMF1 showed a similar reduction in migratory ability as CD99 silenced ESFT cells. Our results suggest that in ESFT, high CD99 expression levels contribute to the malignant properties of ESFT by promoting growth and migration of tumor cells and identify KCMF1 as a potential metastasis suppressor gene downregulated by high constitutive CD99 expression in ESFT.
Subject(s)
Antigens, CD/physiology , Bone Neoplasms/pathology , Cell Adhesion Molecules/physiology , Sarcoma, Ewing/pathology , Ubiquitin-Protein Ligases/metabolism , 12E7 Antigen , Bone Neoplasms/metabolism , Cell Movement , Cell Proliferation , Colony-Forming Units Assay , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Oligonucleotide Array Sequence Analysis , RNA, Small Interfering/pharmacology , Sarcoma, Ewing/metabolism , Tumor Cells, Cultured , Ubiquitin-Protein Ligases/genetics , Up-RegulationABSTRACT
Using reverse transcriptase polymerase chain reaction (RT-PCR) we evaluated the occurrence of tumour-cell ribonucleic acid (RNA) in the blood during surgery in patients with Ewing tumours. The patients received irradiation and chemotherapy according to the protocol of the European Intergroup Cooperative Ewing Sarcoma Study (EICESS) 92. Blood samples were taken from 15 patients. Intra-operative dissemination was found during 2/8 resections but showed no relation to patient survival. At second-look biopsy, detection of tumour-cell RNA was associated with relapse and metastases in 3/4 patients. The results suggest that pre-operative treatment did not completely prevent dissemination of tumour cells during surgery of Ewing tumours.
Subject(s)
Neoplasm Seeding , RNA, Neoplasm/analysis , Reverse Transcriptase Polymerase Chain Reaction , Sarcoma, Ewing/surgery , Adolescent , Adult , Child , Child, Preschool , Female , Humans , Infant , MaleABSTRACT
mdm2 encodes for an E3 ubiquitin ligase targeting constitutively expressed p53 for proteasomal degradation. Several protein isoforms have been described for human MDM2 (HDM2), some of which may correspond to splicing variants detectable by RT-PCR in many tumors. Upon cellular stress, p53 becomes resistant to MDM2 and, in a feedback loop, up-regulates mdm2 transcription. The physiological relevance of stress-induced mdm2 gene activity is not well understood. We describe a small nuclear RNA of 365 bases comprised of the first five hdm2 exons and lacking polyadenylation. hdm365 precedes full-length hdm2 RNA expression after induction by p53 and accumulates to significant levels in the nucleus, detectable at the site of hdm2 transcription and processing only. Considering a 10-fold lower stability and high steady-state levels of the novel RNA species, hdm365 appears to be the major processing product of hdm2 transcripts. hdm365 induction was observed after ectopic expression of p53 and after DNA damaging treatment of tumor cell lines, primary fibroblasts and lymphocytes, and was not related to apoptosis. Corresponding truncated transcripts were observed in hdm2 amplified cells. High stress-inducible expression levels, absence of a corresponding protein, and nuclear localisation of hdm365 suggest a novel RNA-based function for hdm2.
Subject(s)
Nuclear Proteins , Proto-Oncogene Proteins/genetics , RNA, Small Nuclear/metabolism , RNA/metabolism , 3T3 Cells , Animals , Base Sequence , Blotting, Northern , Blotting, Western , Cell Line , Humans , In Situ Hybridization, Fluorescence/methods , K562 Cells , Mice , Molecular Sequence Data , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-mdm2 , RNA/genetics , RNA/radiation effects , RNA Splicing , Tumor Cells, Cultured , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
EWS encodes a ubiquitously expressed RNA binding protein with largely unknown function. In Ewing sarcoma family tumors (EFT), one allele is rearranged with an ETS gene. This is the first description of an EFT with a complete EWS deficiency in the presence of two copies of a rearranged chromosome 22 carrying an interstitial EWS-FLI1 translocation. Absence of EWS protein suggested that it is dispensable for EFT growth. By sequencing of EWS cDNA from unrelated EFTs, we excluded inactivation of EWS as a general mechanism in EFT pathogenesis. Rather, EWS was found to be uniformly expressed in two splicing variants of similar abundancy, EWSalpha and EWSbeta, which differ in a single amino acid. Three EWS negative cell lines were established, which will serve as valuable models to study normal and aberrant EWS function upon reintroduction into the tumor cells.
Subject(s)
Bone Neoplasms/genetics , Bone Neoplasms/pathology , Ribonucleoproteins/genetics , Sarcoma, Ewing/genetics , Sarcoma, Ewing/pathology , Alleles , Alternative Splicing , Cell Division/genetics , Cell Division/physiology , Child, Preschool , Chromosomes, Human, Pair 22/genetics , Female , Gene Expression , Gene Expression Regulation, Neoplastic , Gene Rearrangement , Gene Silencing , Heterogeneous-Nuclear Ribonucleoproteins , Humans , Oncogene Proteins, Fusion/genetics , Proto-Oncogene Protein c-fli-1 , RNA-Binding Protein EWS , Ribonucleoproteins/physiology , Transcription Factors/genetics , Translocation, Genetic , Tumor Cells, CulturedABSTRACT
BACKGROUND: At least three genetic hallmarks identify aggressive tumour behaviour in neuroblastomas; amplification of the oncogene MYCN; deletion (loss of heterozygosity [LOH]) at the short arm of chromosome 1 (del1p36), seen in approximately 28% of the cases; and di-tetraploidy. The MYCN oncogene is amplified in approximately 23% of all neuroblastomas and becomes important for the stratification of therapy in localised and 4s tumours. Up to now, it has been believed that the genetic constellation of neuroblastic tumours is stable and does not alter during tumour evolution or during tumour progression. PROCEDURE: Using fluorescence in situ hybridisation techniques (FISH) to investigate different tumour areas on touch preparations and histological sections, we show that genetic heterogeneity can be detected in neuroblastomas, especially in tumours detected by urinary mass screening. CONCLUSION: The identification of such cell clones is important, because the MYCN amplification and/or the deletion at 1p36 appear to be responsible for aggressive local growth and development of metastases.
Subject(s)
Chromosomes, Human, Pair 1/genetics , Gene Amplification , Genes, myc , Neuroblastoma/genetics , Chromosomes, Human, Pair 1/ultrastructure , Clone Cells/ultrastructure , Disease Progression , Humans , In Situ Hybridization, Fluorescence , Loss of Heterozygosity , Neoplasm Invasiveness , Neoplasm Metastasis , Neoplasm Recurrence, Local , Neoplastic Stem Cells/ultrastructure , Neuroblastoma/pathology , PrognosisABSTRACT
BACKGROUND: Amplification of the oncogene MYCN in neuroblastoma has been found to correlate with aggressive tumour growth and is used as a predictor of clinical outcome. The MYCN amplicon is known to involve coamplification of extensive DNA regions. Therefore it is possible that other genes are coamplified in this amplicon and that they may play a role in the poor outcome of MYCN amplified tumours. PROCEDURE: We have implemented an approach for the two-dimensional separation of human genomic restriction fragments to detect and isolate as yet unknown amplified sequences in the MYCN amplicon in neuroblastoma. Using this approach we have recently cloned a novel gene referred to as NAG that is frequently coamplified with MYCN in neuroblastoma. RESULTS AND CONCLUSIONS: We report here the identification and cloning of two additional CpG islands that are amplified in neuroblastoma. One contains a sequence that is identical to the first intron of DDX1. The other represents a novel CpG island that is associated with an as yet unidentified gene. We show that the novel CpG island is located in close proximity to the MYCN locus on chromosome 2 and is as frequently coamplified with MYCN in neuroblastoma as NAG and DDX1.
Subject(s)
Chromosomes, Human, Pair 1/genetics , CpG Islands , DNA, Neoplasm/genetics , Electrophoresis, Gel, Two-Dimensional , Gene Amplification , Genes, myc , Neuroblastoma/genetics , Chromosomes, Human, Pair 1/ultrastructure , Cloning, Molecular , Humans , In Situ Hybridization, Fluorescence , Polymorphism, Restriction Fragment Length , Tumor Cells, CulturedABSTRACT
In contrast to the EWS/FLI1 fusion which is represented by a t(11;22)(q24;q12), EWS/ERG fusions are frequently cytogenetically not detectable. Three Ewing tumors (ET), two with apparently normal chromosomes 21 and 22, and one ET with a t(2;22)(p25;q12), were studied by FISH on interphase nuclei, metaphase chromosomes and on DNA fibers. EWS/ERG transcripts were detected by RT-PCR in all cases. FISH, using cosmids located proximally (F10, G9) and distally (F7) to the EWS breakpoint region, revealed no detectable separation of these probes in two cases. In contrast, co-hybridization of probe PT1526 containing the ERG breakpoint region with G9 revealed the juxtaposition of two signals per interphase nucleus in all three cases indicating the EWS/ERG fusions. Chromosome preparations displayed the juxtaposed signals on the der(22), and hybridization signals of the probes PT1526 and G9 on the non-rearranged chromosomes 21 and 22 in all cases, respectively. The PT1526 signal on the der(21) was seen only in cases 1 and 2. These results were confirmed by triple-target FISH on tumor DNA fibers. In all three cases, the hybridization pattern F10 - G9 - PT1526 indicates a centromere to telomere orientation. This finding suggests that EWS/ERG fusions in ETs may be generated by an inversion of the ERG gene or a part thereof followed by an insertion into the EWS gene on the der(22). Double-target FISH on interphase nuclei using probes flanking the EWS breakpoint region and probe PT1526 enables the detection of virtually all 22q12 rearrangements in ETs, thus providing a reliable diagnostic assay.
Subject(s)
Cation Transport Proteins , DNA, Neoplasm/genetics , DNA-Binding Proteins , Physical Chromosome Mapping , Potassium Channels, Voltage-Gated , Potassium Channels/genetics , Recombination, Genetic/genetics , Ribonucleoproteins/genetics , Sarcoma, Ewing/genetics , Trans-Activators , Translocation, Genetic/genetics , Chromosome Breakage/genetics , Chromosome Inversion , Chromosomes, Human, Pair 11/genetics , Chromosomes, Human, Pair 2/genetics , Chromosomes, Human, Pair 21/genetics , Chromosomes, Human, Pair 22/genetics , DNA Probes , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels , Heterogeneous-Nuclear Ribonucleoproteins , Humans , In Situ Hybridization, Fluorescence , Interphase , Metaphase , Mutagenesis, Insertional/genetics , RNA, Messenger/analysis , RNA, Messenger/genetics , RNA-Binding Protein EWS , Transcriptional Regulator ERG , Tumor Cells, CulturedABSTRACT
To dissect the p53-dependent apoptotic pathway, events following induction of temperature sensitive (ts) p53val138 were studied in a Ewing tumor cell line. Transcriptional deregulation of p53 targets first observable after 1 h at 32 degrees C preceded activation of caspases and the break-down of mitochondrial respiratory activity. Activation of caspases was first observed 4 h after p53 induction. Using peptide inhibitors we identified activation of caspase 8 upstream of caspases-9 and -3. Although the caspase 8 specific inhibitor z-IETD.fmk did not affect translocation of BAX to the mitochondrial membrane and cytochrome C release it almost completely blocked cleavage of the prototype caspase substrate PARP and DNA fragmentation while enforcing mitochondrial depolarization and production of reactive oxygene species (ROS). Activation of caspase 8 did not involve death-domain receptor signaling. Expression of BCL2 only partially suppressed caspase activation but blocked apoptosis. Replacement of the N-terminus of p53val138 by the related VP16 transactivation domain created a ts p53 with a tanscriptional activity indistinguishable from p53val138 until the time of caspase activation. However, the VP16 - p53 fusion failed to trigger caspases and subsequent induction of the ROS producing gene pig3 paralleled by complete loss of apoptotic activity. These results indicate that p53-dependent transcriptional deregulation, triggering of the caspase cascade and the mitochondrial break-down occur in a timely ordered sequence coordinated by the genuine p53 amino terminus and suggest caspase 8 and PIG3 as key regulatory elements in this process. Oncogene (2000) 19, 4096 - 4107
Subject(s)
Apoptosis , Caspases/genetics , Tumor Suppressor Protein p53/metabolism , Blotting, Northern , Caspase 8 , Caspase 9 , Caspase Inhibitors , Caspases/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Cytochrome c Group/metabolism , DNA Fragmentation , Flow Cytometry , Herpes Simplex Virus Protein Vmw65/genetics , Humans , In Situ Nick-End Labeling , Intracellular Signaling Peptides and Proteins , Mitochondria/metabolism , Mutation , Oligopeptides/pharmacology , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism , Proteins/genetics , Proteins/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Reactive Oxygen Species/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sarcoma, Ewing , Transcriptional Activation , Tumor Cells, Cultured , Tumor Suppressor Protein p53/genetics , bcl-2-Associated X ProteinABSTRACT
Rearrangement of the EWS gene with FLI1 is thought to occur early in the pathogenesis of Ewing's sarcoma family tumors (EFTs) because the chromosomal aberration is pathognomonic for this disease. Recently, adenovirus (Ad) 5 E1A protein has been reported to induce this gene rearrangement in a variety of cell types. This finding, if generally substantiated, not only suggests an etiological role for viral agents in the generation of oncogenic chromosomal aberrations but would also significantly impact the use of adenoviral vectors for gene therapy. In contrast, we now report on the absence of EWS-FLI1 chimeric products from short- and long-term cultures of stably Ad-transformed cells lines and from transiently E1A-expressing cell lines. In addition, we demonstrate the absence of E1A from EFTs. We conclude that there is no role for Ads in EFT pathogenesis. Consequently, evidence for a viral genesis of tumor-specific gene rearrangements is not available.
Subject(s)
Adenovirus E1A Proteins/physiology , Bone Neoplasms/genetics , Oncogene Proteins, Fusion/genetics , Sarcoma, Ewing/genetics , Transcription Factors/genetics , Adenovirus E1A Proteins/genetics , Adenovirus E1A Proteins/metabolism , Blotting, Northern , Blotting, Western , Bone Neoplasms/metabolism , Cell Line , Cell Line, Transformed , DNA, Complementary/genetics , DNA, Neoplasm/genetics , Gene Expression Regulation, Neoplastic , Gene Rearrangement , HeLa Cells , Humans , Oncogene Proteins, Fusion/metabolism , Plasmids/genetics , Proto-Oncogene Protein c-fli-1 , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , RNA-Binding Protein EWS , Sarcoma, Ewing/metabolism , Transcription Factors/metabolism , Tumor Cells, CulturedABSTRACT
Type 1 and type 2 EWS-FLI1 fusion products result from variation in breakpoint locations arising from the t(11;22)(q24;q12) recurrent chromosomal translocation in Ewing's sarcoma family tumors (EFT). Previously, studies from our institution (updated in the present communication at a median follow-up of more than 6 years) and others suggested a prognostic difference for EFT patients with localized disease depending on the type of EWS-FLI1 fusion present in the tumor. It has been suggested that the observed clinical discrepancies result from different transactivation potentials of the various EWS-FLI1 fusion proteins. In an attempt to identify genes whose expression levels are differentially modulated by structurally different EWS-FLI1 transcription factors, we have used two related PCR-based subtractive approaches, cDNA representational difference analysis (cDNA-RDA) and linker-capture subtraction (LCS) to compare transcript representations in cDNA pools of type 1 versus type 2 EFT cell lines. About 800 clones obtained by the two approaches were analyzed by dot blot hybridization to cDNA pools. Eighty-six clones showing the highest variability in signal intensities on the dot blots were further hybridized to individual EFT cell line RNAs on Northern blots, and four of them were additionally studied by real-time quantitative PCR (RTQ-PCR). Although interindividual variations in gene expression patterns in the range of one- to several-fold were observed, no correlation to specific EWS-FLI1 fusion types could be identified. Among the genes differentially expressed in individual EFT cell lines are several previously implicated in tumor growth, invasion, and metastasis. Although our data may have revealed candidate genes whose composite expression pattern may be relevant for the biology of individual EFT, they do not support a role of distinct EWS-FLI1 fusion types for EFT prognosis based on different transactivation potentials.