Team Publications Diversity and Plasticity of Childhood Tumors (DePiCT)

Year of publication 2005

Franck Bourdeaut, Delphine Trochet, Isabelle Janoueix-Lerosey, Agnès Ribeiro, Anne Deville, Carole Coz, Jean-François Michiels, Stanislas Lyonnet, Jeanne Amiel, Olivier Delattre (2005 Jun 14) Germline mutations of the paired-like homeobox 2B (PHOX2B) gene in neuroblastoma. Cancer letters : 51-8

Summary

Hereditary predisposition to neuroblastoma accounts for less than 5% of neuroblastomas and is probably heterogeneous. Recently, a predisposition gene has been mapped to 16p12-p13, but has not yet been identified. Occurrence of neuroblastoma in association with congenital central hypoventilation and Hirschsprung’s disease suggests that genes, involved in the development of neural-crest-derived cells, may be altered in these conditions. The recent identification of PHOX2B as the major disease-causing gene in congenital central hypoventilation prompted us to test it as a candidate gene in familial neuroblastoma. We report a family with three first-degree relatives with neuroblastic tumours (namely two ganglioneuromas and one neuroblastoma) in one branch and two siblings with Hirschsprung’s disease in another branch. A constitutional R100L PHOX2B mutation was identified in all three patients affected with tumours. We also report a germline PHOX2B mutation in one patient treated for Hirschsprung’s disease who subsequently developed a multifocal neuroblastoma in infancy. Both mutations disrupt the homeodomain of the PHOX2B protein. No loss of heterozygosity at the PHOX2B locus was observed in the tumour, suggesting that haplo-insufficiency, gain of function or dominant negative effects may account for the oncogenic effects of these mutations. These observations identify PHOX2B as the first predisposing gene to hereditary neuroblastic tumours.

Gudrun Schleiermacher, Franck Bourdeaut, Valérie Combaret, Gaelle Picrron, Virginie Raynal, Alain Aurias, Agnes Ribeiro, Isabelle Janoueix-Lerosey, Olivier Delattre (2005 Mar 1) Stepwise occurrence of a complex unbalanced translocation in neuroblastoma leading to insertion of a telomere sequence and late chromosome 17q gain. Oncogene : 3377-84

Summary

In neuroblastoma, the most frequent genetic alterations are unbalanced translocations involving chromosome 17. To gain insights into these rearrangements, we have characterized a previously identified der(1)t(1;17) of the CLB-Bar cell line. The 17q breakpoint was mapped by FISH. Subsequently, a rearranged fragment was identified by Southern analysis, cloned in a lambda vector and sequenced. The chromosome rearrangement is more complex than expected due to the presence of an interstitial 4p telomeric sequence between chromosome 1p and 17q. Three different genes, which may play a role in neuroblastoma development, are disrupted by the translocation breakpoints.

INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 1 Team Publications Diversity and Plasticity of Childhood Tumors (DePiCT)

Indeed, the 3’UTR of the PIP5K2B gene on chromosome 17q is directly fused to the (TTAGGG)n repeat of the chromosome 4p telomere, and the (1;4) fusion disrupts the MACF1 (microtubule-actin crosslinking factor 1) and POLN genes, respectively. Interestingly, the (1;4) fusion was present at diagnosis and at relapse, whereas the (4;17) fusion was detected at relapse only, leading to a secondary 17q gain confirmed by array CGH therefore indicating that 17q gain may not be a primary event in neuroblastoma. Finally, screening of a panel of neuroblastoma cell lines identified interstitial telomeric sequences in three other cases, suggesting that this may be a recurrent mechanism leading to unbalanced translocations in neuroblastoma.

Marie-Françoise Rousseau-Merck, Laurence Fiette, Agnès Klochendler-Yeivin, Olivier Delattre, Alain Aurias (2005 Feb 22) Chromosome mechanisms and INI1 inactivation in human and mouse rhabdoid tumors. Cancer genetics and cytogenetics : 127-33

Summary

The human rhabdoid tumorigenesis orchestrated by INI1 inactivation is associated with specific rearrangements of chromosome 22 that correlate with preferential anatomic tumor locations. A literature review revealed significant correlations between an apparently normal karyotype and kidney tumors, monosomy 22 and cerebral tumors, and chromosome 22 translocations and tumors at other anatomic sites. In the mouse rhabdoid tumor model, specifically in the four tumors that we tested for loss of heterozygosity, neither partial deletion nor monosomy of chromosome 10 could be detected. In contrast to the human data, the only chromosome mechanism involved in the 18 mouse tumors studied appears to be a mitotic recombination or a nondisjunction-duplication. Additionally, and despite mouse tumor incidence across a variety of sites, no rhabdoid tumor could be observed in the mouse kidney. These data suggest that the chromosome mechanisms for INI1 inactivation and the selective cell survival pressure differ in human and mouse.

Year of publication 2004

Alexandre Prieur, Franck Tirode, Pinchas Cohen, Olivier Delattre (2004 Jul 30) EWS/FLI-1 silencing and gene profiling of Ewing cells reveal downstream oncogenic pathways and a crucial role for repression of insulin-like growth factor binding protein 3. Molecular and cellular biology : 7275-83

Summary

Ewing tumors are characterized by abnormal transcription factors resulting from the oncogenic fusion of EWS with members of the ETS family, most commonly FLI-1. RNA interference targeted to the junction between EWS and FLI-1 sequences was used to

INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 2 Team Publications Diversity and Plasticity of Childhood Tumors (DePiCT)

inactivate the EWS/FLI-1 fusion gene in Ewing cells and to explore the resulting phenotype and alteration of the gene expression profile. Loss of expression of EWS/FLI-1 resulted in the complete arrest of growth and was associated with a dramatic increase in the number of apoptotic cells. Gene profiling of Ewing cells in which the EWS/FLI-1 fusion gene had been inactivated identified downstream targets which could be grouped in two major functional clusters related to extracellular matrix structure or remodeling and regulation of signal transduction pathways. Among these targets, the insulin-like growth factor binding protein 3 gene (IGFBP-3), a major regulator of insulin-like growth factor 1 (IGF-1) proliferation and survival signaling, was strongly induced upon treating Ewing cells with EWS/FLI-1-specific small interfering RNAs. We show that EWS/FLI-1 can bind the IGFBP-3 promoter in vitro and in vivo and can repress its activity. Moreover, IGFBP-3 silencing can partially rescue the apoptotic phenotype caused by EWS/FLI-1 inactivation. Finally, IGFBP-3-induced Ewing cell apoptosis relies on both IGF-1-dependent and -independent pathways. These findings therefore identify the repression of IGFBP-3 as a key event in the development of Ewing’s sarcoma.

Isabelle Janoueix-Lerosey, Eugene Novikov, Marta Monteiro, Nadège Gruel, Gudrun Schleiermacher, Béatrice Loriod, Catherine Nguyen, Olivier Delattre (2004 Jun 15) Gene expression profiling of 1p35-36 genes in neuroblastoma. Oncogene : 5912-22

Summary

Deletion of the chromosome 1p36 region is a frequent abnormality in neuroblastoma. To gain further insights into the role of this alteration in oncogenesis, we have constructed a specific cDNA microarray representing most known genes and ESTs from the 1p35-36 region and analysed the expression profiles of 15 neuroblastoma cell lines and 28 neuroblastoma tumours. Hierarchical clustering using expression levels of 320 cDNAs from 1p35-36 separated localized or 4S cases without 1p deletion from advanced stages and cell lines. Supervised learning classification enabled to predict reliably the status of chromosome 1p according to its expression profile. Around 15% of the genes or ESTs presented a significantly decreased expression in samples with 1p deletion as compared to 1p-normal samples suggesting that 1p deletion results in a gene dosage effect on a subset of genes critical for the development of 1p-deleted neuroblastoma. Several genes presumed to have functions in neural differentiation (CDC42, VAMP3, CLSTN1), signal transduction in neural cells (GNB1) and cell cycle regulation (STMN1, RPA2, RBAF600, FBXO6, MAD2L2) exhibited a decreased expression in samples presenting 1p deletion. The identification of such genes provides baseline information for further studies to elucidate how these genes could individually or collectively play a critical role in neuroblastoma tumorigenesis.

Souhila Medjkane, Eugene Novikov, Isabella Versteege, Olivier Delattre (2004 May 20) The tumor suppressor hSNF5/INI1 modulates cell growth and actin cytoskeleton organization. Cancer research : 3406-13

INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 3 Team Publications Diversity and Plasticity of Childhood Tumors (DePiCT)

Summary

hSNF5/INI1, which encodes a component of the ATP-dependent chromatin remodeling hSWI- SNF complex, is a tumor suppressor gene mutated in malignant rhabdoid tumors. We have developed a tetracycline-based hSNF5/INI1-inducible system in a hSNF5/INI1-deficient malignant rhabdoid tumor cell line and studied time course variation of 22,000 genes/expressed sequence tags upon hSNF5/INI1 induction. A total of 482 responsive genes were identified and further clustered into 9 groups of coregulated genes. Among genes with early and strong inductions, the use of a fusion protein with the hormone-binding domain of the estrogen receptor enabled the identification of a subset of direct targets regulated independently of de novo protein synthesis. We show that the G(1) arrest induced by hSNF5/INI1 is reversible and associated with the down-regulation of components of the DNA replication complex. We also identify an unsuspected role of hSNF5/INI1 in cytoskeleton organization. Indeed, induction of hSNF5/INI1 induces dramatic modifications of the cell shape including complete disruption of the actin stress fiber network and disappearance of focal adhesions associated with up-regulation of genes involved in the organization of the actin cytoskeleton. We document a strong decrease of Rho activity upon hSNF5/INI1 expression, suggesting that the regulation of this activity constitutes a crucial step of the hSNF5/INI1-induced reorganization of the actin network. This study identifies hSNF5/INI1 target genes and provides evidence that hSNF5/INI1 may modulate the cell cycle control and cytoskeleton organization through the regulation of the retinoblastoma protein-E2F and Rho pathways.

Anne Fix, Martine Peter, Gaëlle Pierron, Alain Aurias, Olivier Delattre, Isabelle Janoueix-Lerosey (2004 May 13) High-resolution mapping of amplicons of the short arm of chromosome 1 in two neuroblastoma tumors by microarray-based comparative genomic hybridization. Genes, chromosomes & cancer : 266-70

Summary

Deletion of chromosome arm 1p is one of the most frequent genetic alterations in neuroblastoma. However, using conventional comparative genomic hybridization, we have observed amplifications on 1p in 2 neuroblastoma tumors at bands 1p34.2 and 1p36.3, respectively. Using a medium-resolution genomic array containing 178 PACs/BACs from 1p and then 2 high-resolution arrays containing contigs of overlapping PACs/BACs from the amplified regions, we could precisely map and delineate both amplicons. The 1p34.2 amplicon appeared as a homogeneous amplification unit, whereas the 1p36.3 amplicon had a more complex structure, with 2 noncontiguous, highly amplified regions and several moderate amplification units. In this case, fluorescence in situ hybridization analysis confirmed the amplification of several clones and indicated that the 2 highest amplification units corresponded to 2 populations of double minute chromosomes, one of which also contained the MYCN locus. This is the first report of 1p amplifications in primary neuroblastomas.

INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 4 Team Publications Diversity and Plasticity of Childhood Tumors (DePiCT)

Year of publication 2003

Gudrun Schleiermacher, Virginie Raynal, Isabelle Janoueix-Lerosey, Valérie Combaret, Alain Aurias, Olivier Delattre (2003 Dec 30) Variety and complexity of chromosome 17 translocations in neuroblastoma. Genes, chromosomes & cancer : 143-50

Summary

In neuroblastoma, the most frequent genetic alteration is gain of chromosome arm 17q, which arises from unbalanced translocations. To document these genetic events more precisely, we performed an extensive study of chromosome 17 breakpoints in 27 neuroblastoma cell lines by using a combination of fluorescence in situ hybridization mapping with BAC/PAC clones and allele analysis with polymorphic markers. All cases exhibited one or more unbalanced chromosome 17 translocations, and 15 distinct breakpoint regions could be mapped. This high variability indicates that gene fusion or disruption events are extremely unlikely to account for the underlying oncogenic role of these translocations. However, breakpoints were not randomly distributed, most of them mapping to the proximal part of 17q. As a result of translocations, all cell lines but one exhibited gain of the 53.5 Mb–>qter fragment, bordered proximally by the clone CTC-462L7. The most telomeric breakpoint, flanked by the clone RP11-443M10, defined the 70.9 Mb–>qter fragment as a region of additional gain. In addition to chromosome gains, loss of heterozygosity for the short arm of chromosome 17 was observed in close to half the cases. It was either related to a chromosome 17 monosomy or to a uniparental isodisomy. Finally, in cases with a single normal chromosome 17, we show that the parental origin of the translocated chromosome 17 can be either distinct or identical to that of the normal chromosome. Similarly, multiple translocations within the same cell line can either involve the same or different chromosome 17 homologues, indicating the likely absence of parental origin bias in the generation of these alterations.

Gudrun Schleiermacher, Isabelle Janoueix-Lerosey, Valérie Combaret, Josette Derré, Jérome Couturier, Alain Aurias, Olivier Delattre (2003 Feb 13) Combined 24-color karyotyping and comparative genomic hybridization analysis indicates predominant rearrangements of early replicating chromosome regions in neuroblastoma. Cancer genetics and cytogenetics : 32-42

Summary

Neuroblastoma is characterized by several distinct genetic alterations including MYCN amplification, chromosome 1p deletion and gain of chromosome 17. Although these alterations are thought to play a crucial role in oncogenesis, to date little is known about their underlying mechanisms. In order to more precisely document these genetic alterations, we have performed a combined study of 27 neuroblastoma cell lines using 24-color karyotyping (24-CK) and comparative genomic hybridization (CGH). 24-CK detected balanced

INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 5 Team Publications Diversity and Plasticity of Childhood Tumors (DePiCT)

translocations in 13 cases with recurrent involvement of chromosome 8. More importantly, 144 nonreciprocal translocations were observed in the 27 cell lines, with chromosome 1 as the most frequent recipient and chromosome 17 the most frequent donor. Each cell line exhibited at least one unbalanced translocation involving 17q, with 14 cell lines demonstrating more than one such translocation. Other recurrent alterations were amplification of the 2p24 chromosome region, which encodes the MYCN oncogene, losses of 1p, 3p and 11q, and gains of 1q and 7. In most cases, CGH profiles were directly linked to the presence of unbalanced translocations with gain of the donor fragment and loss of the replaced region on the recipient chromosome. Strikingly, over 60% of the chromosome breakpoints mapped to early replicating chromosome bands, which represent around 13% of the genome. Altogether these data suggest that neuroblastoma is characterized by rearrangements that predominantly involve chromosome fragments replicating early in the S-phase.

Year of publication 2002

Josiane Sancéau, Marie-France Poupon, Olivier Delattre, Xavier Sastre-Garau, Juana Wietzerbin (2002 Oct 26) Strong inhibition of Ewing tumor xenograft growth by combination of human interferon-alpha or interferon-beta with ifosfamide. Oncogene : 7700-9

Summary

Ewing sarcoma is the second most common bone tumor in childhood. Despite aggressive chemotherapy and radiotherapy strategies, the prognosis of patients with metastatic disease remains poor. We have recently reported that Ewing tumor cell proliferation was strongly inhibited by IFN-beta and to a lesser degree by IFN-alpha. Moreover, under IFN-beta treatment, some cell lines undergo apoptosis. Since the possibility of using IFNs for Ewing tumor treatments may be of interest, we have evaluated the efficacy of Hu-IFNs in a nude mice model of Ewing tumor xenografts. The results reported here show that human type I IFNs, Hu-IFN-alpha and Hu-IFN-beta impaired tumor xenograft take and displayed an anti- growth effect toward established xenografts. Furthermore, we have also shown that combined therapy with Hu-IFNs and ifosfamide (IFO), an alkylating agent widely used in high- dose chemotherapy of Ewing tumors, results in a strong antitumor effect. Pathological analysis showed that Hu-IFN-alpha/IFO and Hu-IFN-beta/IFO were characterized by a dramatic decrease in the mitotic index and marked necrosis, as well as extensive fibrosis associated with numerous calcifications. To our knowledge, this is the first demonstration of a potential antitumor effect of human type I IFNs and IFO on Ewing tumors, providing a rational foundation for a promising therapeutic approach to Ewing sarcoma.

Isabella Versteege, Souhila Medjkane, Danny Rouillard, Olivier Delattre (2002 Sep 13) A key role of the hSNF5/INI1 tumour suppressor in the control of the G1-S

INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 6 Team Publications Diversity and Plasticity of Childhood Tumors (DePiCT)

transition of the cell cycle. Oncogene : 6403-12

Summary

The hSNF5/INI1 gene encodes a member of the SWI/SNF chromatin remodelling complexes. It was recently identified as a tumour suppressor gene mutated in sporadic and hereditary Malignant Rhabdoid Tumours (MRT). However, the role of hSNF5/INI1 loss-of-function in tumour development is still unknown. Here, we show that the ectopic expression of wild-type hSNF5/INI1, but not that of truncated versions, leads to a cell cycle arrest by inhibiting the entry into S phase of MRT cells. This G1 arrest is associated with down-regulation of a subset of E2F targets including cyclin A, E2F1 and CDC6. This arrest can be reverted by coexpression of cyclin D1, cyclin E or viral E1A, whereas it cannot be counteracted by pRB- binding deficient E1A mutants. Moreover, hSNF5/INI1 is not able to arrest cells lacking a functional pRB. These observations suggest that the hSNF5/INI1-induced G1 arrest is dependent upon the presence of a functional pRB. However, the observation that a constitutively active pRB can efficiently arrest MRT cells indicates that hSNF5/INI1, at the difference of the ATPase subunits of the SWI/SNF complex, is dispensable for pRB function. Altogether, these data show that hSNF5/INI1 is a potent regulator of the entry into S phase, an effect that may account for its tumour suppressor role.

Philippe Mérel, Alexandre Prieur, Petra Pfeiffer, Olivier Delattre (2002 Aug 8) Absence of major defects in non-homologous DNA end joining in human breast cancer cell lines. Oncogene : 5654-9

Summary

Structural abnormalities of chromosomes, including translocations and deletions, are extremely frequent in human cancer cells and particularly in breast cancer cells. One hypothesis to account for these alterations is a deficiency in the repair of DNA double-strand breaks (DSB). This repair process relies on two distinct pathways, homologous recombination (HR) and non-homologous DNA end joining (NHEJ). To investigate this latter pathway, we have studied the ability of cell-free extracts from a variety of human cells to rejoin different types of DSBs. The end joining activity of eleven sporadic breast cancer cell lines (BCCLs) was compared with that of control cells including primary human fibroblasts and cells harbouring a limited number of chromosome abnormalities. In vitro rejoining activity was not detected in extracts from MO59J DNA-PKcs-deficient cells and was strongly inhibited by wortmannin in control extracts. In contrast, most sporadic BCCLs and BRCA1 or BRCA2 deficient cells demonstrated similar efficiencies and accuracies of in vitro NHEJ than control cells. Only two BCCLs, SKBR3 and MDA-MB-453 exhibited decreased in vitro NHEJ. This study therefore indicates that a major defect in the NHEJ pathway is unlikely to account for the high number of chromosomes abnormalities observed in sporadic and hereditary BCCLs.

INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 7 Team Publications Diversity and Plasticity of Childhood Tumors (DePiCT)

Isabelle Hostein, Armelle Menard, Bin Nguyen Bui, Cathy Lussan, Jean Wafflart, Olivier Delattre, Martine Peter, Jean Benhattar, Louis Guillou, Jean-Michel Coindre (2002 Feb 21) Molecular detection of the synovial sarcoma translocation t(X;18) by real-time polymerase chain reaction in paraffin-embedded material. Diagnostic molecular pathology : the American journal of surgical pathology, part B : 16-21

Summary

The t(X;18) translocation is known to be a useful marker for the diagnosis of synovial sarcoma. In this study, the authors describe a new real-time reverse transcriptase- polymerase chain reaction (RT-PCR) method to detect SYT/SSX fusion transcripts using paraffin-embedded and frozen tumor specimens. A series of 38 soft tissue sarcomas were analyzed. Diagnosis was based on clinical, histologic, and immunohistochemical examination. The fusion transcripts were detected in 16 of 17 synovial sarcoma samples (the 17th sample was not suitable for molecular analysis). No t(X;18)-fusion transcript was PCR- amplified in the 21 nonsynovial sarcoma mesenchymal tumors. Therefore, real-time PCR amplification appears to be a powerful, rapid, specific, and sensitive technique that can be used routinely to diagnose the synovial sarcoma t(X;18) translocation. In addition, the t(X;18) can be detected not only on frozen but also on paraffin-embedded tumor samples.

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