Hsa-mir-145 is the top EWS-FLI1-repressed microRNA involved in a positive feedback loop in Ewing's sarcoma.

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.

O-GlcNAcylation is involved in the transcriptional activity of EWS-FLI1 in Ewing's sarcoma.

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.

A small nuclear RNA, hdm365, is the major processing product of the human mdm2 gene.

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.

The EWS protein is dispensable for Ewing tumor growth.

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.

Characterization of distinct consecutive phases in non-genotoxic p53-induced apoptosis of Ewing tumor cells and the rate-limiting role of caspase 8.

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

Use of transcription reporters with novel p53 binding sites to target tumour cells expressing endogenous or virally transduced p53 mutants with altered sequence-specificity.

p53 triple mutants (120N/121G/277H, 120H/121G/ 277H, 120S/121G/277H and 120H/121G/277Y) have altered sequence specificity in bandshift assays in vitro and transcription assays in vivo. These mutants activate transcription from the site TTT CATG AAA but not from wild type sites. The triple mutants activate more strongly than p53 with a single 277Y mutation. The TTT site matches the wild type p53 consensus at only 4/10 positions and is not recognised by wild type p53. 277Y mutations have been described in human tumours, and Ewing tumour cells expressing this mutant from the endogenous p53 locus selectively activate transcription from transfected luciferase reporters regulated by TTT-mutant p53 binding sites. p53 mutants with altered sequence specificity have potential advantages for cancer gene therapy: if used to activate transcription of conditionally toxic genes they would allow tumour-targeting by p53, which acts as a sensor for the malignant state, but place control over cell killing in the hands of the clinician. Rare tumours expressing such mutants from the endogenous p53 locus could be targeted directly with p53-regulated suicide vectors, but for most tumours both the p53 mutant and the reporter would need to be encoded by the virus.

Among genes involved in the RB dependent cell cycle regulatory cascade, the p16 tumor suppressor gene is frequently lost in the Ewing family of tumors.

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.

EWS/FLI-1 antagonists induce growth inhibition of Ewing tumor cells in vitro.

Among primitive neuroectodermal tumors, Ewing tumors are characterized by a gene rearrangement recombining the 5'-EWS gene portion with one of two ETS-related proto-oncogenes, FLI-1 or ERG, in roughly 90 and 10% of cases, respectively. We attempted to antagonize EWS/FLI-1 function in Ewing tumor cell lines. As a control, a cell line derived from another small round cell tumor of neuroectodermal origin, neuroblastoma, was used. This cell line was found to express almost identical patterns of ETS proteins except for EWS/FLI-1 and a novel, ELF-related gene product. Stable expression of antisense EWS/FLI-1 cDNA resulted in decreased endogenous EWS/FLI-1 RNA levels and growth reduction of ET cells but not of neuroblastoma cells. DNA-binding FLI-1 derivatives localizing to the nucleus in which the 5'-EWS regulatory domain was replaced by bacterial beta-galactosidase dominantly modulated transcriptional transactivation from a degenerate ETS-binding motif. Transient transfection of these dominant-negative recombinants resulted in a significant decrease in the relative number of mitoses in four Ewing tumor cell lines tested but not in the neuroblastoma cell line. The presented evidence for modulation of tumor cell proliferation by EWS/FLI-1 antagonists suggests a causal role for EWS/FLI-1-mediated gene activation in the malignant transformation of the enigmatic Ewing tumor-precursor cell.

[The EWS gene rearrangement in Ewing tumors: key to the disease]. / Das EWS-Genrearrangement in Ewing Tumoren: Schlüssel zur Erkrankung.

The family of Ewing tumors (ET) is characterised by a unique gene rearrangement which is represented by a translocation t(11;22) (q24;q12) or a deletion del 22q12 in most cytogenetically analysable cases. The recent cloning of the underlying gene fusion provides the basis for the diagnostic detection of minimal amounts of residual tumor cells at resection margins, in blood and bone marrow. In addition, the very first steps in ET tumorigenesis can be studied on a functional basis. In this study, a variety of fusion products were identified with a sensitivity of 10(-6) by means of RT-PCR. In 20 of 22 ET, a gene rearrangement was identified which resulted in the substitution of the effector domain of one of the closely related DNA-binding oncogenes, FLI-1 or ERG, by the transactivating domain of a new gene, EWS. Presumably, the oncogene and consequently its target genes are activated by this type of translocation. If the EWS domain was replaced with a transcriptionally irrelevant domain by transfection of a recombinant gene into ET cells, competition with the endogenous chimeric oncogene-product for DNA-binding was observed resulting in a partial growth inhibition. Activation of FLI-1 has been previously shown to occur as a primary event in Friend virus induced mouse erythroleukemia. During progression of this disease, inactivating p53 mutations have been observed frequently. In contrast, such aberrations were found to be extremely rare in ET.

Narrow spectrum of infrequent p53 mutations and absence of MDM2 amplification in Ewing tumours.

The p53 and MDM2 genes are part of a physiological pathway frequently impaired in human cancer. With the exception of tumours occasionally associated with hereditary predisposition, childhood malignancies have not been studied in detail yet. This is the first report on the analysis of p53 and MDM2 in a group of non-hereditary paediatric neoplasms referred to as the Ewing tumours (ETs). Thirty-seven primary tumours and cell lines from 19 patients were screened for the presence of p53 mutations. Only 5% of the primary tumour specimens were found to carry an alteration within this gene. However, p53 mutations were 10-fold enriched in ET cell lines, thus indicating a selective growth advantage in vitro. Strikingly, five out of nine alterations detected were missense mutations within codon 273, which were previously reported to impair only partially the normal p53 function. Two single-base substitutions occurred at codons 277 and 176, and two alterations were loss-of-function mutations. Investigation of the MDM2 gene revealed neither gene amplification in the primary tumours and cell lines nor significant overexpression in any of the cell lines. Our data therefore suggest that impairment of cellular mechanisms involving p53 is rare in a distinct group of childhood malignancies.

p53 mosaicism with an exon 8 germline mutation in the founder of a cancer-prone pedigree.

Changes in the tumor-suppressor gene p53 are frequently acquired during the course of malignant development of human tumors. Recently, constitutional heterozygous mutations in p53 exon 7 have been identified as the primary cause of cancer predisposition in cases of the familial Li-Fraumeni cancer syndrome. These findings underline the need for extensive mutation screening in families with high cancer incidence. This report describes the detection and follow-up by two-dimensional single-strand conformation polymorphism analysis (2DSSCP) of a new germline mutation of p53 exon 8 in a case of suspected Li-Fraumeni syndrome. Although a high cancer incidence had been reported in the family history of the father of siblings suffering from brain tumor and rhabdomyosarcoma, a constitutional heterozygous p53 mutation was identified only in the affected children. Retrospective analysis of archival tissue of a half-sister who died several years ago from a tumor of previously uncertain diagnosis revealed the same mutation. The mutation had therefore occurred in the germ cells of the mother, who thus appears to be a mosaic. The cancer predisposition of the paternal ancestors must have been due to other factors.