Karine Laud

Common variants near TARDBP and EGR2 are associated with susceptibility to Ewing sarcoma

Ewing sarcoma, a pediatric tumor characterized by EWSR1-ETS fusions, is predominantly observed in populations of European ancestry. We performed a genome-wide association study (GWAS) of 401 French individuals with Ewing sarcoma, 684 unaffected French individuals and 3,668 unaffected individuals of European descent and living in the United States. We identified candidate risk loci at 1p36.22, 10q21 and 15q15. We replicated these loci in two independent sets of cases and controls. Joint analysis identified associations with rs9430161 (P = 1.4 × 10−20; odds ratio (OR) = 2.2) located 25 kb upstream of TARDBP, rs224278 (P = 4.0 × 10−17; OR = 1.7) located 5 kb upstream of EGR2 and, to a lesser extent, rs4924410 at 15q15 (P = 6.6 × 10−9; OR = 1.5). The major risk haplotypes were less prevalent in Africans, suggesting that these loci could contribute to geographical differences in Ewing sarcoma incidence. TARDBP shares structural similarities with EWSR1 and FUS, which encode RNA binding proteins, and EGR2 is a target gene of EWSR1-ETS. Variants at these loci were associated with expression levels of TARDBP, ADO (encoding cysteamine dioxygenase) and EGR2.

Alteration of cyclin D1 transcript elongation by a mutated transcription factor up-regulates the oncogenic D1b splice isoform in cancer

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.

Zoledronic Acid as a New Adjuvant Therapeutic Strategy for Ewing's Sarcoma Patients

Ewings sarcoma (ES) is the second most frequent pediatric bone tumor also arising in soft tissues (15% of cases). The prognosis of patients with clinically detectable metastases at diagnosis, not responding to therapy or with disease relapse, is still very poor. Among new therapeutic approaches, bisphosphonates represent promising adjuvant molecules to chemotherapy to limit the osteolytic component of bone tumors and to protect from bone metastases. The combined effects of zoledronic acid and mafosfamide were investigated on cell proliferation, viability, apoptosis, and cell cycle distribution of human ES cell lines differing in their p53 and p16/ink4 status. ES models were developed to reproduce both soft tissue and intraosseous tumor development. Mice were treated with 100 μg/kg zoledronic acid (two or four times per week) and/or ifosfamide (30 mg/kg, one to three cycles of three injections). ES cell lines showed different sensitivities to zoledronic acid and mafosfamide at the cell proliferation level, with no correlation with their molecular status. Both drugs induced cell cycle arrest, but in the S or G(2)M phase, respectively. In vivo, zoledronic acid had no effect on soft tissue tumor progression, although it dramatically inhibited ES development in bone. When combined with ifosfamide, zoledronic acid exerted synergistic effects in the soft tissue model: Its combination with one cycle of ifosfamide resulted in an inhibitory effect similar to three cycles of ifosfamide alone. This very promising result could allow clinicians to diminish the doses of chemotherapy.

Oncostatin M Is a Growth Factor for Ewing Sarcoma

Primary bone tumors, osteosarcomas and chondrosarcomas, derive from mesenchymal stem cells committed into osteoblasts and chondrocytes; in Ewing sarcomas (ESs), the oncogenic fusion protein EWS-FLI1 prevents mesenchymal differentiation and induces neuroectodermic features. Oncostatin M (OSM) is a cytokine from the IL-6 family that modulates proliferation and differentiation in numerous cells. The basis for inhibition versus induction of proliferation by this cytokine is obscure, although MYC was described as a potent molecular switch in OSM signaling. We show herein that, in contrast to osteosarcomas and chondrosarcomas, for which OSM was cytostatic, OSM induced proliferation of ES cell lines. Knockdown experiments demonstrated that growth induction by OSM depends on both types I [leukemia inhibitory factor receptor (LIFR)] and II [OSM receptor (OSMR)] receptors, high STAT3 activation, and induction of MYC to a high expression level. Indeed, ES cell lines, mice xenografts, and patient biopsy specimens poorly expressed LIF, precluding LIFR lysosomal degradation and OSMR transcriptional induction, thus leading to a high LIFR/OSMR ratio. Because other neuroectodermic tumors (ie, glioma, medulloblastoma, and neuroblastoma) had a similar expression profile, the main role of EWS-FLI1 could be through maintenance of stemness and neuroectodermic features, characterized by a low LIF, a high LIFR/OSMR ratio, and high MYC expression. Thus, this study on rare bone malignancies gives valuable insights on more common cancer regulatory mechanisms and could provide new therapeutic opportunities.

Systems biology of Ewing sarcoma: a network model of EWS-FLI1 effect on proliferation and apoptosis

Ewing sarcoma is the second most frequent pediatric bone tumor. In most of the patients, a chromosomal translocation leads to the expression of the EWS-FLI1 chimeric transcription factor that is the major oncogene in this pathology. Relative genetic simplicity of Ewing sarcoma makes it particularly attractive for studying cancer in a systemic manner. Silencing EWS-FLI1 induces cell cycle alteration and ultimately leads to apoptosis, but the exact molecular mechanisms underlying this phenotype are unclear. In this study, a network linking EWS-FLI1 to cell cycle and apoptosis phenotypes was constructed through an original method of network reconstruction. Transcriptome time-series after EWS-FLI1 silencing were used to identify core modulated genes by an original scoring method based on fitting expression profile dynamics curves. Literature data mining was then used to connect these modulated genes into a network. The validity of a subpart of this network was assessed by siRNA/RT-QPCR experiments on four additional Ewing cell lines and confirmed most of the links. Based on the network and the transcriptome data, CUL1 was identified as a new potential target of EWS-FLI1. Altogether, using an original methodology of data integration, we provide the first version of EWS-FLI1 network model of cell cycle and apoptosis regulation.

Search for germline alterations in CDKN2A/ARF and CDK4 of 42 Jewish melanoma families with or without neural system tumours.

To gain insight into the molecular mechanisms involved in the inherited predisposition to melanoma and associated neural system tumours, 42 Jewish, mainly Ashkenazi, melanoma families with or without neural system tumours were genotyped for germline point mutations and genomic deletions at the CDKN2A/ARF and CDK4 loci. CDKN2A/ARF deletion detection was performed using D9S1748, an intragenic microsatellite marker. Allele dosage at the p14ARF locus was analysed by quantitative real-time PCR employing a TaqMan probe that anneals specifically to exon 1β of the p14ARF gene. For detecting point mutations, dHPLC and direct sequencing of the coding sequences of CDKN2A/ARF and CDK4 was used. No germline alterations in any of the tested genes were detected among the families under study. We conclude that in the majority of Ashkenazi Jewish families, the genes tested are unlikely to be implicated in the predisposition to melanoma and associated neural system tumours.

EWS-FLI1 inhibits TNFα-induced NFκB-dependent transcription in Ewing sarcoma cells

Ewing sarcoma is primarily caused by a t(11;22) chromosomal translocation encoding the EWS-FLI1 fusion protein. To exert its oncogenic function, EWS-FLI1 acts as an aberrant transcription factor, broadly altering the gene expression profile of tumor cells. Nuclear factor-kappaB (NFkappaB) is a tightly regulated transcription factor controlling cell survival, proliferation and differentiation, as well as tumorigenesis. NFkappaB activity is very low in unstimulated Ewing sarcoma cells, but can be induced in response to tumor necrosis factor (TNF). We wondered whether NFkappaB activity could be modulated by EWS-FLI1 in Ewing sarcoma. Using a knockdown approach in Ewing sarcoma cells, we demonstrated that EWS-FLI1 has no influence on NFkappaB basal activity, but impairs TNF-induced NFkappaB-driven transcription, at least in part through inhibition of NFkappaB binding to DNA. We detected an in vivo physical interaction between the fusion protein and NFkappaB p65, which could mediate these effects. Our findings suggest that, besides directly controlling the activity of its primary target promoters, EWS-FLI1 can also indirectly influence gene expression in tumor cells by modulating the activity of key transcription factors such as NFkappaB.

Abstract 3989: High throughput screening highlights NFkB signaling in Ewing sarcoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Ewing sarcoma (ES) is the second most frequent pediatric bone tumor and still remains of poor prognosis especially for metastatic patients. Genetically, ES is characterized by a chromosomal translocation between EWSR1 and ETS family members (FLI1 in 85% of cases). This leads to the expression of EWS-FLI1 chimeric oncogene transcription factor. Aiming at identifying EWS-FLI1 regulated genes with potential therapeutic targets, a genome wide method was developed to rank these potential hits by combining Ewing sarcoma transcriptome and ChIPSeq data. Accordingly, 273 selected genes were further investigated using a siRNA approach for their impact on cell cycle phases and apoptosis using high throughput imaging methods. 133 of these genes displayed a phenotype and bioinformatics tools were used to connect these genes. NFkB turned out to be a major hub in this network and upstream activation pathways were further investigated. Interleukin-1 pathway may account for this observed effect and in vitro and in vivo experiments are currently on going to validate this hypothesis. Interestingly, preliminary results indicate that both tumor and tumor microenvironment are of prime importance for the activation of this pathway. Citation Format: Didier Surdez, Gautier Stoll, Franck Tirode, Karine Laud, Emmanuel Barillot, Olivier Delattre. High throughput screening highlights NFkB signaling in Ewing sarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3989. doi:10.1158/1538-7445.AM2014-3989

Abstract B71: A systems biology approach to study Ewing's sarcoma

Ewing9s sarcoma is the second most frequent pediatric bone tumor. It is characterized in 85% of cases by a chromosomal translocation which leads to the expression of a chimeric transcription factor: EWS‐FLI1. This oncogene is transforming in various cell and animal models. Although important EWS‐FLI1 transcription targets and altered pathways have been described, a comprehensive understanding of the disease remains to be achieved. For this purpose, a systems biology approach was used. In this project, gene profiling of (1) primary tumors and (2) of EWS‐FLI1‐inducible cell models were performed. Based on these experimental data and on literature knowledge, we constructed an annotated influence network of EWS‐FLI1 effects that was validated using small scale silencing/qPCR experiments and home‐made software. In a second step, in order to better understand the global impact of EWS‐FLI1 in Ewing sarcoma context, EWS‐FLI1 ChIP‐seq experiments were performed. These results were combined to the gene expression profile data using rational approaches and/or PCA based analyses. This resulted in a genome wide analysis in which the genes were ranked upon their implication/significance in Ewing sarcoma. In the future, we intend to integrate these results to an extended version of our annotated influence network and confront high‐throughput experimental results (siRNA screen) with predictive computational models of this network. This should allow identifying novel crucial genes/pathways in Ewing9s sarcoma. Citation Information: Cancer Res 2009;69(23 Suppl):B71.