cyclin-d1 and Sarcoma--Ewing

In cancer cells, gene expression is altered at the levels of transcription and mRNA maturation, with many splice variants being associated with cancer. Splicing is tightly connected to transcription and can be affected by transcription elongation dynamics. Moreover, various transcriptional coregulators that are altered in cancer, such as the proto-oncogene EWS, are thought to play a role in splicing. A recent study shows that an alteration of EWS in Ewing sarcoma alters the dynamics of RNA polymerase II over the CCND1 proto-oncogene encoding cyclin D1, leading to an increase in its transcription and to an alteration of splicing that results in high levels of the oncogenic cyclin D1b splice isoform. The cyclin D1b isoform is highly expressed in Ewing sarcoma cells and tumors and stimulates Ewing sarcoma cell growth. Thus, alterations of transcriptional regulators in disease may lead to splicing alterations. We review these data and discuss how this concept may apply to various factors that are altered in cancer.

Topics: Animals; Bone Neoplasms; Cyclin D1; Humans; Models, Biological; Neoplasms; Oncogene Proteins, Fusion; Oncogenes; Proto-Oncogene Mas; Proto-Oncogene Protein c-fli-1; RNA Splicing; RNA-Binding Protein EWS; Sarcoma, Ewing; Trans-Activators; Transcription, Genetic

Primary superficial Ewing sarcoma (psES) cases are exceedingly rare, with fewer than 150 cases reported in the literature. Small case series have suggested differences between psES and Ewing sarcoma (ES) of bone or deep soft tissues: psES appears to have a more indolent course and a higher 5-year overall survival rate. PsES is more common in older adolescent females as opposed to younger males in their peak growth velocity years in bone or deep soft tissue ES. We present a case report of a 17-year-old female with a relatively static nodule on her left thigh for 4 years. Morphologic, immunohistochemical, and molecular evaluations confirmed ES. Patient underwent a gross-total resection and a shortened course of adjuvant chemotherapy without radiation. Cancer gene panel testing found three gene abnormalities (in addition to EWSR1-FLI1 fusion): CCND1 copy number gain, ELF3 copy number loss, and TNFRSF14 copy number loss. To our knowledge, this is the first published case report of psES to include genomic sequencing and the first to report ELF3 and TNFRSF14 abnormalities in ES. Larger series of psES cases with genomic profiling are needed to elucidate a possible genetic etiology for its more indolent clinical course and favorable outcomes.

Topics: Adolescent; Chemotherapy, Adjuvant; Cyclin D1; DNA Copy Number Variations; DNA-Binding Proteins; Female; Humans; Immunohistochemistry; Magnetic Resonance Imaging; Proto-Oncogene Protein c-fli-1; Proto-Oncogene Proteins c-ets; Receptors, Tumor Necrosis Factor, Member 14; RNA-Binding Protein EWS; Sarcoma, Ewing; Skin Neoplasms; Transcription Factors; Treatment Outcome; Ultrasonography, Doppler, Color

Ewing sarcoma (ES) is a type of childhood cancer probably arising from stem mesenchymal or neural crest cells. The epidermal growth factor receptor (EGFR) acts as a driver oncogene in many types of solid tumors. However, its involvement in ES remains poorly understood.. Human SK-ES-1 and RD-ES ES cells were treated with EGF, the EGFR inhibitor tyrphostin (AG1478), or phosphoinositide 3-kinase (PI3K) or extracellular-regulated kinase (ERK)/mitogen-activated kinase (MAPK) inhibitors. Cell proliferation survival, cycle, and senescence were analyzed. The protein content of possible targets of EGFR manipulation was measured by Western blot.. Cell proliferation and survival were increased by EGF and inhibited by AG1478. The EGFR inhibitor also altered the cell cycle, inducing arrest in G1 and increasing the sub-G1 population, reduced polyploidy and increased the population of senescent cells. In addition, AG1478 reduced the levels of phosphorylated AKT (p-AKT), ERK, p-ERK, cyclin D1, and brain-derived neurotrophic factor (BDNF), while enhancing p53 levels. Cell proliferation was also impaired by inhibitors of PI3K or ERK, alone or combined with AG1478.. Our findings reveal novel aspects of EGFR regulation of ES cells and provide early evidence for antitumor activities of EGFR inhibitors in ES.

Topics: Bone Neoplasms; Brain-Derived Neurotrophic Factor; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; G1 Phase Cell Cycle Checkpoints; Humans; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Quinazolines; Sarcoma, Ewing; Signal Transduction; Tumor Suppressor Protein p53; Tyrphostins

Ewing sarcoma (ES) is a rare and highly malignant cancer that occurs in the bone and surrounding tissue of children and adolescents. The. Using Western blot, high-content imaging, flow cytometry, ELISA, RNA sequencing, and CpG methylation assays, we characterized the. Abemaciclib induced quiescence in ES cell lines via a G. Collectively, our data demonstrate that the antitumor effects of abemaciclib in preclinical ES models are multifaceted and include cell-cycle inhibition, DNA demethylation, and immunogenic changes.

Topics: Aminopyridines; Animals; Benzimidazoles; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Models, Animal; DNA (Cytosine-5-)-Methyltransferase 1; DNA Methylation; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Humans; Interferons; Mice; Sarcoma, Ewing; Signal Transduction; Xenograft Model Antitumor Assays

Topics: Adolescent; Child; Cyclin D1; Humans; Sarcoma, Clear Cell; Sarcoma, Ewing

Topics: Adolescent; Child; Cyclin D1; Humans; Sarcoma, Clear Cell; Sarcoma, Ewing

Small round blue cell tumors (SRBCTs) of children and adolescents are often diagnostically challenging lesions. With the increasing diagnostic approach based on small biopsies, there is the need of specific immunomarkers that can help in the differential diagnosis among the different tumor histotypes to assure the patient a correct diagnosis for proper treatment. Based on our recent studies showing cyclin D1 overexpression in both Ewing sarcoma/primitive peripheral neuroectodermal tumor (EWS/pPNET) and peripheral neuroblastic tumors (neuroblastoma and ganglioneuroblastoma), we immunohistochemically assessed cyclin D1 immunoreactivity in 128 cases of SRBCTs in children and adolescents to establish its potential utility in the differential diagnosis. All cases of EWS/pPNET and the undifferentiated/poorly differentiated neuroblastomatous component of all peripheral neuroblastic tumors exhibited strong and diffuse nuclear staining (>50% of neoplastic cells) for cyclin D1. In contrast, this marker was absent from rhabdomyosarcoma (regardless of subtype) and lymphoblastic lymphoma (either B- or T-cell precursors), whereas it was only focally detected (<5% of neoplastic cells) in some cases of Wilms tumor (blastemal component) and desmoplastic small round cell tumor. Our findings suggest that cyclin D1 can be exploitable as a diagnostic adjunct to conventional markers in confirming the diagnosis of EWS/pPNET or neuroblastoma/ganglioneuroblastoma. Its use in routine practice may also be helpful for those cases of SRBCT with undifferentiated morphology that are difficult to diagnose after application of the conventional markers.

Topics: Adolescent; Biomarkers, Tumor; Biopsy; Bone Neoplasms; Cell Differentiation; Child; Child, Preschool; Cyclin D1; Desmoplastic Small Round Cell Tumor; Diagnosis, Differential; Female; Ganglioneuroblastoma; Humans; Immunohistochemistry; Infant; Kidney Neoplasms; Male; Neuroblastoma; Neuroectodermal Tumors, Primitive, Peripheral; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Predictive Value of Tests; Retrospective Studies; Rhabdomyosarcoma; Sarcoma, Ewing; Wilms Tumor; Young Adult

Distinguishing clear cell sarcoma of the kidney (CCSK) from other paediatric malignancies, particularly blastema-rich Wilms tumour (WT) and congenital mesoblastic nephroma (CMN), is challenging. Specific immunohistochemistry for CCSK does not exist, and diagnosis rests upon histopa thology. Recently, the YWHAE-FAM22 rearrange ment, identical to that in endometrial stromal sarcoma (ESS), has been identified in CCSKs. As this fusion results in overexpression of cyclin D1 in ESS, we postulated that overexpression would also occur in CCSK; cyclin D1 immunohistochemistry could then be used to differentiate CCSK from other tumours. The goal of this study was therefore to evaluate the utility of cyclin D1 immunohistochemistry in identifying CCSK and helping to differentiate it from its mimics.. Cyclin D1 expression was evaluated in 59 renal tumours-CCSK (14), WT (25), rhabdoid tumour (four), Ewing sarcoma (five), and CMN (11)-and four neuroblastomas. All 14 CCSKs showed diffuse and strong reactivity. In contrast, the blastematous component of most WTs showed only rare positive nuclei, that of rhabdoid tumours showed rare to focal immunoreactivity, and that of more than half of CMNs showed weak or focal immunoreactivity. Most Ewing sarcomas and all neuroblastomas showed diffuse moderate to strong staining.. Cyclin D1 is most helpful in distinguishing CCSK from WT, rhabdoid tumour, and some CMNs, but not from neuroblastoma or Ewing sarcomas.

Topics: Biomarkers, Tumor; Child, Preschool; Cyclin D1; Diagnosis, Differential; Female; Humans; Immunohistochemistry; Kidney Neoplasms; Male; Nephroma, Mesoblastic; Neuroblastoma; Rhabdoid Tumor; Sarcoma, Clear Cell; Sarcoma, Ewing; Wilms Tumor

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.

Topics: Animals; Antineoplastic Agents; Bone Neoplasms; Cell Line, Tumor; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Drug Discovery; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; High-Throughput Nucleotide Sequencing; High-Throughput Screening Assays; Humans; Mice, Inbred NOD; Mice, SCID; Molecular Targeted Therapy; Protein Kinase Inhibitors; RNA Interference; RNA-Binding Protein EWS; Sarcoma, Ewing; Signal Transduction; Time Factors; Transfection; Tumor Burden; Vascular Endothelial Growth Factor Receptor-1; Xenograft Model Antitumor Assays

Recent immunohistochemical analyses have showed that cyclin D1 is expressed in soft tissue Ewing's sarcoma/peripheral neuroectodermal tumor (PNET) of childhood and adolescents, while it is undetectable in both embryonal and alveolar rhabdomyosarcoma. In the present paper, microarray analysis provided evidence of a significant upregulation of cyclin D1 in Ewing's sarcoma as compared to normal tissues. In addition, we confirmed our previous findings of a significant over-expression of cyclin D1 in Ewing sarcoma as compared to rhabdomyosarcoma. Bioinformatic analysis also allowed to identify some other genes, strongly correlated to cyclin D1, which, although not previously studied in pediatric tumors, could represent novel markers for the diagnosis and prognosis of Ewing's sarcoma/PNET. The data herein provided support not only the use of cyclin D1 as a diagnostic marker of Ewing sarcoma/PNET but also the possibility of using drugs targeting cyclin D1 as potential therapeutic strategies.

Topics: Biomarkers, Tumor; Bone Neoplasms; Cyclin D1; Gene Expression; Humans; Oligonucleotide Array Sequence Analysis; Rhabdomyosarcoma; Sarcoma, Ewing

At diagnosis, identification of reliable biological indicators of prognosis to allow stratification of patients according to different risks is an important but still unresolved aspect in the treatment of Ewing sarcoma (EWS) patients. This study aimed to explore the role of miR-34A expression on prognosis of EWS patients.. Specimens from 109 patients with non-metastatic EWS treated at the Rizzoli Institute with neoadjuvant chemotherapy (protocols ISG/SSGIII, EW-1, EW-2, EW-REN2, EW-REN3, EW-PILOT) and 17 metastases were studied. Sixty-eight patients (62%) remained disease-free and 41 (38%) relapsed (median follow-up: 67 months, range 9-241 months). Expression of miR-34a and of some of its targets (cyclin D1, bcl-2, SIRT1 and YY1) was evaluated by qRT-PCR using TaqMan MicroRNA Assays and/or by immunohistochemistry on tissue microarrays from the same patients.. High expression of miR-34a in localized tumors was significantly related to better event-free and overall survival (P = 0.004). Relevance of miR-34a was confirmed by using different calibrators (normal mesenchymal stem cells and different normal tissues). By multivariate Cox regression analysis, low miR-34a expression as well as nontotal necrosis and high levels of lactate dehydrogenase were all confirmed as independent risk factors associated with poor outcome. Expression of miR-34a was lower in metastases than in primary tumors. It inversely correlated with expression of cyclin D1 and Ki-67.. By demonstrating its relationship with clinical outcome, we propose evaluation of miR-34a at diagnosis of EWS patients to allow early risk stratification. Validation of these results would nonetheless ultimately need a prospective assessment.

Topics: Adult; Aged, 80 and over; Cyclin D1; Disease-Free Survival; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Hydro-Lyases; Ki-67 Antigen; Male; MicroRNAs; Middle Aged; Neoadjuvant Therapy; Neoplasm Metastasis; Prognosis; Sarcoma, Ewing; Treatment Outcome

Ewing's sarcoma is a malignancy characterized by a specific 11:22 chromosomal translocation which generates a novel EWS-FLI1 fusion protein functioning as an aberrant transcription factor. In the present study, we have further characterized the junction region of the EWS-FLI1 fusion protein.. In-silico model of EWS-FLI1 fusion protein was analysed for ligand binding sites, and a putative region (amino acid (aa) 251-343 of the type 1 fusion protein) in the vicinity of the fusion junction was cloned and expressed using bacterial expression. The recombinant protein was characterized by Circular Dichroism (CD). We then expressed aa 251-280 ectopically in Ewing's sarcoma cell-line and its effect on cell proliferation, tumorigenicity and expression of EWS-FLI1 target genes were analysed.. Our modelling analysis indicated that Junction region (aa 251-343) encompasses potential ligand biding sites in the EWS-FLI1 protein and when expressed in bacteria was present as soluble form. Ectopically expressing this region in Ewing's sarcoma cells inhibited tumorigenicity, and EWS-FLI1 target genes indicating a dominant negative biological effect.. Junction region can be exploited further as target for drug development in future to specifically target EWS-FLI1 in Ewing's Sarcoma.

Topics: Amino Acid Sequence; Cell Line, Tumor; Cell Proliferation; Circular Dichroism; Cyclin D1; Enhancer of Zeste Homolog 2 Protein; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Homeobox Protein Nkx-2.2; Homeodomain Proteins; Humans; Immunoblotting; MCF-7 Cells; Molecular Sequence Data; Oncogene Proteins, Fusion; Peptides; Polycomb Repressive Complex 2; Proto-Oncogene Protein c-fli-1; Proto-Oncogene Proteins c-myc; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA-Binding Protein EWS; Sarcoma, Ewing; Transcription Factors; Transfection; Zebrafish Proteins

Pre-mRNA splicing and polyadenylation are tightly connected to transcription, and transcriptional stimuli and elongation dynamics can affect mRNA maturation. However, whether this regulatory mechanism has a physio/pathological impact is not known. In cancer, where splice variant expression is often deregulated, many mutated oncogenes are transcriptional regulators. In particular, the Ewing sarcoma (EwSa) oncogene, resulting from a fusion of the EWS and FLI1 genes, encodes a well characterized transcription factor. EWS-FLI1 directly stimulates transcription of the CCND1 protooncogene encoding cyclin D1a and a less abundant but more oncogenic splice isoform, D1b. We show that, although both EWS and EWS-FLI1 enhance cyclin D1 gene expression, they regulate the D1b/D1a transcript ratio in an opposite manner. Detailed analyses of RNA polymerase dynamics along the gene and of the effects of an inhibitor of elongation show that EWS-FLI1 favors D1b isoform expression by decreasing the elongation rate, whereas EWS has opposite effects. As a result, the D1b/D1a ratio is elevated in EwSa cell lines and tumors. The endogenous D1b protein is enriched in nuclei, where the oncogenic activity of cyclin D1 is known to occur, and depleting D1b in addition to D1a results in a stronger reduction of EwSa cell growth than depleting D1a only. These data show that elevated expression of a splice isoform in cancer can be due to an alteration of the transcription process by a mutated transcriptional regulator and provide evidence for a physio/pathological impact of the coupling between transcription and mRNA maturation.

Topics: Alternative Splicing; Bone Neoplasms; Cell Line, Tumor; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Neoplasms; Oncogene Proteins, Fusion; Protein Isoforms; Proto-Oncogene Protein c-fli-1; RNA-Binding Protein EWS; RNA, Messenger; Sarcoma, Ewing; Transcription, Genetic; Up-Regulation

Tumor markers ideally allow monitoring and prediction of disease progression. In Ewing's sarcoma, a devastating childhood cancer, only a few reliable prognostic markers have been identified. To this end, we analyzed the expression of four tumor-promoting proteins, cyclin D1, HER2/Neu, Mdm2, and vascular endothelial growth factor (VEGF), in Ewing's sarcoma.. Thirty-one tissue samples from patients with Ewing's sarcoma were stained with antibodies against cyclin D1, HER2/Neu, Mdm2, or VEGF. Whereas no significant expression of HER2/Neu and Mdm2 was detected, positive cyclin D1 and VEGF staining was observed in 42% and 55% of all tumors, respectively. Importantly, VEGF expression was found to be an independent negative predictor of survival in Ewing's sarcoma patients, whereas cyclin D1 expression did not correlate with survival in these patients. Consistently, the Ewing's sarcoma-specific EWS-ETS oncoproteins were capable of activating both the cyclin D1 and VEGF promoters in transient transfections of tissue culture cells. Furthermore, this activation was enhanced by coexpression of the Sp1 transcription factor. Using a mammalian two-hybrid system, some evidence was obtained that this may involve a physical interaction between EWS-ETS and Sp1 proteins.. Our data reveal that VEGF may serve as a prognostic marker in Ewing's sarcoma patients and provide a molecular mechanism by which VEGF and cyclin D1 expression is up-regulated in approximately half of all Ewing's sarcomas.

Topics: Antigens, Protozoan; Binding Sites; Biomarkers, Tumor; Cyclin D1; Disease Progression; Gene Deletion; Genes, Reporter; Humans; Luciferases; Nuclear Proteins; Plasmids; Prognosis; Promoter Regions, Genetic; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Receptor, ErbB-2; RNA-Binding Protein EWS; Sarcoma, Ewing; Sp1 Transcription Factor; Time Factors; Treatment Outcome; Up-Regulation; Vascular Endothelial Growth Factor A

Using Affymetrix oligonucleotide microarrays, we analyzed mRNA gene expression patterns of 12 primary pediatric rhabdomyosarcomas (RMS) and 11 Ewing's sarcomas (EWS), which belong to the small round blue cell tumors (SRBCTs). Diagnostic classification of these cancers is frequently complicated by the highly similar appearance in routine histology, and additional molecular markers could significantly improve tumor classification. A combination of three independent statistical approaches (t-test, SAM, k-nearest neighborhood analysis) resulted in 101 highly significant probe sets that clearly discriminate between EWS and RMS. We identified novel marker transcripts that have not been previously associated with either RMS or EWS yet, including CITED2, glypican 3 (GPC3), and cyclin D1 (CCND1). Expression levels for selected candidate genes were validated by quantitative real-time reverse-transcription PCR. Furthermore, to identify biologically meaningful trends, functional annotations were assigned to 946 genes differentially expressed between EWS and RMS (t-test). Genes involved in protein biosynthesis (n = 28) and complex assembly (n = 9), lipid metabolism (n = 23), energy generation (n = 22), and mRNA processing (n = 11) were expressed significantly higher in EWS. Thus, functional annotation of tumor-specific genes reveals detailed insights into tumor biology and differentiation-specific expression patterns and gives important clues related to the possible cellular origin of these pediatric tumors. Supplementary material for this article is available at the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html.

Topics: Biomarkers, Tumor; Cell Differentiation; Cell Line, Tumor; Child; Cyclin D1; Disease Progression; DNA Primers; DNA-Binding Proteins; Fibroblasts; Gene Expression Regulation, Neoplastic; Genetic Markers; Glypicans; Heparan Sulfate Proteoglycans; Humans; Lipid Metabolism; Models, Statistical; Oligonucleotide Array Sequence Analysis; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; Rhabdomyosarcoma; RNA, Complementary; RNA, Messenger; Sarcoma, Ewing; Trans-Activators

The chimeric fusion gene EWS/FLI-1 is detected in most cases of Ewing's sarcoma (ES), the second most common malignant bone tumor of childhood. Although 80% of ES tumors develop in skeletal sites, the remainder can arise in almost any soft tissue location. The lineage of the cell developing the EWS/FLI-1 gene fusion has not been fully characterized but is generally considered to be of either mesenchymal or neural crest origin. To study this oncogene in a conceptually relevant target cell, EWS/FLI-1 was introduced into the murine cell line C2C12, a myoblast cell line capable of differentiation into muscle, bone, or fat. In this cellular context, EWS/FLI-1 profoundly inhibited the myogenic differentiation program. The block in C2C12 myogenic differentiation required the nuclear localization and DNA-binding functions of EWS/FLI-1 and was mediated by transcriptional and posttranscriptional suppression of the myogenic transcription factors MyoD and myogenin. Interestingly, C2C12-EWS/FLI-1 cells constitutively expressed alkaline phosphatase, a bone lineage marker, and were alkaline phosphatase positive by histochemistry but showed no other evidence of bone lineage commitment. Consistent with recent findings in human ES tumor cell lines, C2C12-EWS/FLI-1 cells constitutively expressed cyclin D1 and demonstrated decreased expression of the cell cycle regulator p21(cip1), even under differentiation conditions and at confluent density. This C2C12-EWS/FLI-1 cell model may assist in the identification of novel differentially expressed genes relevant to ES and provide further insight into the cell(s) of origin developing ES-associated genetic fusions.

Topics: Alkaline Phosphatase; Animals; Blotting, Western; Cell Cycle; Cell Differentiation; Cell Line; Cell Lineage; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Flow Cytometry; Genetic Vectors; Immunohistochemistry; Mice; Microscopy, Fluorescence; Models, Biological; MyoD Protein; Myogenin; Oncogene Proteins, Fusion; Oncogenes; Plasmids; Proto-Oncogene Protein c-fli-1; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; RNA-Binding Protein EWS; Sarcoma, Ewing; Signal Transduction; Time Factors; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transfection

Standard and repeated fluorescence in situ hybridization and high-resolution cytometry were used to study topographical parameters of chromosome 11 and 22 territories, EWSR1 and FLI1 genes, and other genetic elements of these chromosomes in human lymphocytes and Ewing sarcoma cells. HSA 11 and its elements (BCL1, FLI1, centromere) were found, on average, more peripherally in comparison with HSA 22 and investigated elements (BCR, EWSR1, centromere). After the elimination of fluctuations of chromosome territories in nuclear volume, it was found that genetic elements in most cases adhered to their territories. The investigated genetic elements of HSA 11 were found close to each other relative to the large molecular lengths among them. This finding indicates a higher degree of chromatin condensation of at least a part of HSA 11 compared with HSA 22. In general, there is no correlation between the physical and molecular distance of two loci of the same chromosome territory. The topographical parameters of the EWSR1 and FLI1 genes do not differ substantially for G(0)-lymphocytes, stimulated lymphocytes and Ewing sarcoma cells. The fusion genes pertaining to both derivative chromosomes 11 and 22 in Ewing sarcoma cell nuclei are shifted to the midway position between the native EWSR1 and FLI1 genes. Comparing results obtained for the EWSR1/FLI1 and ABL1/BCR genes in samples of patients suffering from Ewing sarcoma or chronic myelogenous leukaemia, it can be concluded that the mean positions of the fusion genes are determined by the final structure of the chimeric chromosomes and do not depend on the location of the translocation event.

Topics: Cell Nucleus; Centromere; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 22; Cyclin D1; DNA Primers; DNA Probes; DNA-Binding Proteins; DNA, Neoplasm; Fusion Proteins, bcr-abl; Gene Expression Regulation, Neoplastic; Humans; Image Processing, Computer-Assisted; In Situ Hybridization, Fluorescence; Karyotyping; Oncogene Proteins, Fusion; Proto-Oncogene Protein c-fli-1; Proto-Oncogene Proteins; RNA-Binding Protein EWS; Sarcoma, Ewing; T-Lymphocytes; Trans-Activators; Transcription Factors; Tumor Cells, Cultured

Chromosomal translocation t(11;22)(q24:q12) is detected in approximately 90% of tumours of the Ewing family (ET). This translocation results in EWS-Fli1 gene fusion which produces a EWS-Fli1 fusion protein acting as an aberrant transcriptional activator. We previously reported that the inhibition of EWS-Fli1 expression caused the G(0)/G(1)arrest of ET cells. We, therefore, hypothesized that EWS-Fli1 may affect the expression of G(1)regulatory genes. Downregulation of EWS-Fli1 fusion proteins was observed 48 hours after the treatment with EWS-Fli1 antisense oligonucleotides. The expressions of G(1)cyclins, cyclin D1 and cyclin E, were markedly decreased in parallel with the reduction of EWS-Fli1 fusion protein. On the other hand, the expression of p21 and p27, which are important cyclin-dependent kinase inhibitors (CKIs) for G(1)--S transition, was dramatically increased after the treatment with EWS-Fli1 antisense oligonucleotides. RT-PCR analysis showed that alteration of the expressions of the cyclins and CKIs occurred at the mRNA level. Furthermore, transfection of EWS-Fli1 cDNA to NIH3T3 caused transformation of the cells and induction of the expression of cyclin D1 and E. Clinical samples of ET also showed a high level of expression of cyclin D1 mRNA, whereas mRNAs for p21 and p27 were not detected in the samples. These findings strongly suggest that the G(1)--S regulatory genes may be involved in downstream of EWS-Fli1 transcription factor, and that the unbalanced expression of G(1)--S regulatory factors caused by EWS-Fli1 may lead to the tumorigenesis of ET.

Topics: 3T3 Cells; Animals; Cyclin D1; Cyclin E; DNA-Binding Proteins; DNA, Complementary; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Mice; Oligonucleotides, Antisense; Oncogene Proteins, Fusion; Proto-Oncogene Protein c-fli-1; Proto-Oncogene Proteins; Reverse Transcriptase Polymerase Chain Reaction; Sarcoma, Ewing; Trans-Activators; Tumor Cells, Cultured

The EWS-ETS rearrangements, and their respective fusion gene products, are specifically associated with histopathologically Ewing family tumors (EFT). These translocations are implicated in generating malignant transformation of EFT, but the presence of additional genetic alterations must be considered in the pathogenesis of such tumors. We analyzed 26 samples (biopsies and/or nude mice xenotransplants) collected from 19 patients with an EFT to determine whether molecular and cytogenetic alterations of the G(1)/S checkpoint genes are implicated in the pathogenesis of EFT. We found inactivating p53 mutations in three (16%) cases, which correlated with a loss of p21(WAF1/Cip1) expression and with a monosomy of chromosome 17 in two cases. Homozygous deletion of the p16(INK4A)/p14(ARF) gene was detected in four (21%) cases, three with codeletion of the p15(INK4B) gene and with chromosome 9 abnormalities. In all of these cases, expression of the implicated genes was absent. Hypermethylation of the p16(INK4A) and p15(INK4B) genes was detected in two (10%) and three (16%) cases, respectively, and was correlated with a low level of gene expression. Neither cyclin D1, nor MDM2 and CDK4 amplification was observed. Kaplan-Meier analysis showed that patients with tumors carrying homozygous deletion of the 9p21 locus, or point mutations of the p53 gene, had poorer outcomes than those without these molecular alterations (p = 0.005). In conclusion, 58% (11 of 19) of the analyzed patients showed genetic or epigenetic alterations in either the 9p21 locus or p53 tumor suppressor genes, defining a subgroup of patients with poor clinical outcome. This fact points to an important role of the G(1)/S cell cycle checkpoint dysregulation in the pathogenesis of EFT.

Topics: Adolescent; Adult; Animals; Child; Child, Preschool; Chromosome Mapping; Chromosomes, Human, Pair 9; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; DNA Methylation; Female; Gene Amplification; Gene Deletion; Gene Expression; Genes, p53; Humans; Male; Mice; Neoplasm Transplantation; Nuclear Proteins; Point Mutation; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Sarcoma, Ewing; Survival Analysis; Transplantation, Heterologous

The pRB cell cycle regulatory cascade is frequently perturbed in neoplasia by overexpression of a component of the pRB-phosphorylating cyclin D1/CDK4 complex or by inactivation of pRB or the CDK4 inhibitors p16 and p15. We investigated the status and expression of p16, p15, CCND1, CDK4 and RB genes in the Ewing family of tumors. P16 loss was observed in 8 of 27 tumors (30%) and in 12 of 23 (52%) tumor cell lines from unrelated patients. There were no discrepancies in the p16 status between primary tumors and the corresponding cell lines and between cell lines established from consecutive tumor samples. p15 was codeleted in most cases but p15 mRNA was absent also in cell lines retaining the gene. In addition, posttranscriptional p16 inactivation was observed in two cases. Although no evidence for CDK4 or CCND1 amplification was obtained, expression of these genes varied considerably in the cell lines in a case specific manner. In wild-type p16 cell lines, pRB expression was lost in one case. Our data indicate that, despite the absence of cytogenetically detectable 9p21 chromosomal aberrations, p16 deletions constitute the most frequent secondary molecular aberration in Ewing tumors so far. These results are discussed in the context of the stage of disease and the clinical outcome of the patients. The potential prognostic impact of these findings remains to be further evaluated.

Topics: Cell Cycle; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; Gene Deletion; Gene Expression; Genes, p16; Genes, p53; Humans; Mutation; Prognosis; Proto-Oncogene Proteins; Retinoblastoma Protein; Sarcoma, Ewing; Transcription Factors; Tumor Suppressor Proteins