Segmental chromosomal alterations lead to a higher risk of relapse in infants with MYCN-non-amplified localised unresectable/disseminated neuroblastoma (a SIOPEN collaborative study).

BACKGROUND: In neuroblastoma (NB), the presence of segmental chromosome alterations (SCAs) is associated with a higher risk of relapse. METHODS: In order to analyse the role of SCAs in infants with localised unresectable/disseminated NB without MYCN amplification, we have performed an array CGH analysis of tumours from infants enrolled in the prospective European INES trials. RESULTS: Tumour samples from 218 out of 300 enroled patients could be analysed. Segmental chromosome alterations were observed in 11%, 20% and 59% of infants enroled in trials INES99.1 (localised unresectable NB), INES99.2 (stage 4s) and INES99.3 (stage 4) (P<0.0001). Progression-free survival was poorer in patients whose tumours harboured SCA, in the whole population and in trials INES99.1 and INES99.2, in the absence of clinical symptoms (log-rank test, P=0.0001, P=0.04 and P=0.0003, respectively). In multivariate analysis, a SCA genomic profile was the strongest predictor of poorer progression-free survival. CONCLUSION: In infants with stage 4s MYCN-non-amplified NB, a SCA genomic profile identifies patients who will require upfront treatment even in the absence of other clinical indication for therapy, whereas in infants with localised unresectable NB, a genomic profile characterised by the absence of SCA identifies patients in whom treatment reduction might be possible. These findings will be implemented in a future international trial.

The constitutive activity of the ALK mutated at positions F1174 or R1275 impairs receptor trafficking.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK), which is transiently expressed during development of the central and peripheral nervous system. ALK has been recently identified as a major neuroblastoma predisposition gene and activating mutations have also been identified in a subset of sporadic neuroblastoma tumors. Two hot spots of ALK mutations have been observed at positions F1174 and R1275. Here, we studied stably transfected cell lines expressing wild-type or F1174L- or R1275Q-mutated ALK in parallel with a neuroblastoma cell line (CLB-GE) in which the allele mutated at position F1174 is amplified. We observed that the mutated ALK variants were essentially intracellular and were largely retained in the reticulum/Golgi compartments. This localization was corroborated by a defect of N-linked glycosylation. Although the mutated receptors exhibited a constitutive activation, the minor pool of receptor addressed to the plasma membrane was much more tyrosine phosphorylated than the intracellular pool. The use of antagonist monoclonal antibodies suggested that the constitutive activity of the mutated receptors did not require the dimerization of the receptor, whereas adequate dimerization triggered by agonist monoclonal antibodies increased this activity. Finally, kinase inactivation of the mutated receptors restored maturation and cell-surface localization. Our results show that constitutive activation of ALK results in its impaired maturation and intracellular retention. Furthermore, they provide a rationale for the potential use of kinase inhibitors and antibodies in ALK-dependent tumors.

An integrative genomics screen uncovers ncRNA T-UCR functions in neuroblastoma tumours.

Different classes of non-coding RNAs, including microRNAs, have recently been implicated in the process of tumourigenesis. In this study, we examined the expression and putative functions of a novel class of non-coding RNAs known as transcribed ultraconserved regions (T-UCRs) in neuroblastoma. Genome-wide expression profiling revealed correlations between specific T-UCR expression levels and important clinicogenetic parameters such as MYCN amplification status. A functional genomics approach based on the integration of multi-level transcriptome data was adapted to gain insights into T-UCR functions. Assignments of T-UCRs to cellular processes such as TP53 response, differentiation and proliferation were verified using various cellular model systems. For the first time, our results define a T-UCR expression landscape in neuroblastoma and suggest widespread T-UCR involvement in diverse cellular processes that are deregulated in the process of tumourigenesis.

Molecular pathogenesis of peripheral neuroblastic tumors.

Neuroblastoma (NB) is an embryonal cancer of the sympathetic nervous system observed in early childhood, characterized by a broad spectrum of clinical behaviors, ranging from spontaneous regression to fatal outcome despite aggressive therapies. NB accounts for 8-10% of pediatric cancers and 15% of the deaths attributable to malignant conditions in children. Interestingly, NB may occur in various contexts, being mostly sporadic but also familial or syndromic. This review focuses on recent advances in the identification of the genes and mechanisms implicated in NB pathogenesis. Although the extensive characterization of the genomic aberrations recurrently observed in sporadic NBs provides important insights into the understanding of the clinical heterogeneity of this neoplasm, analysis of familial and syndromic cases also unravels essential clues on the genetic bases of NB. Recently, the ALK gene emerged as an important NB gene, being implicated both in sporadic and familial cases. The identification of gene expression signatures associated with patient's outcome points out the potential of using gene expression profiling to improve clinical management of patients suffering from NB. Finally, based on recent observations integrating genomic analyses, biological data and clinical information, we discuss possible evolution/progression schemes in NB.

RGS14 is a novel Rap effector that preferentially regulates the GTPase activity of galphao.

In an attempt to elucidate the physiological function(s) of the Ras-related Rap proteins, we used the yeast two-hybrid system and isolated a cDNA encoding a protein that interacts with both Rap1 and Rap2, but not with Ras; the use of Rap2 mutants showed that this interaction is characteristic of a potential Rap effector. This protein was identified as RGS14, a member of the recently discovered family of RGS ('regulators of G-protein signalling') proteins that stimulate the GTPase activity of the GTP-binding alpha subunit of heterotrimeric G-proteins (Galpha). Deletion analysis, as well as in vitro binding experiments, revealed that RGS14 binds Rap proteins through a domain distinct from that carrying the RGS identity, and that this domain shares sequence identity with the Ras/Rap binding domain of B-Raf and Raf-1 kinases. RGS14 is distinguished from other RGS proteins by its marked preference for Galpha(o) over other Galpha subunits: RGS14 binds preferentially to Galpha(o) in isolated brain membranes, and also interacts preferentially with Galpha(o) (as compared with Galpha(i1)) to stimulate its GTPase activity. In adult mice, RGS14 expression is restricted to spleen and brain. In situ hybridization studies showed that it is highly expressed only in certain areas of mouse brain (such as the CA1 and CA2 regions of the hippocampus), and that this pattern closely resembles that of Rap2, but not Rap1, expression. Double in situ hybridization experiments revealed that certain cells in the hippocampus express both RGS14 and Galpha(o), as well as both RGS14 and Rap2, showing that the interaction of RGS14 with Galpha(o) and Rap2 is physiologically possible. Taken together, these results suggest that RGS14 could constitute a bridging molecule that allows cross-regulation of signalling pathways downstream from G-protein-coupled receptors involving heterotrimeric proteins of the G(i/o) family and those involving the Ras-related GTPase Rap2.

Molecular analysis of chromosome arm 17q gain in neuroblastoma.

Complete or partial gain of the long arm of chromosome 17 (17q) has been shown recently by molecular cytogenetic techniques to be the most frequent chromosomal change in neuroblastoma and to be associated with adverse prognosis. Few reports, however, have focused on the precise mapping of the commonly overrepresented region. We have investigated 17q gain by the analysis of allelic imbalances at microsatellite loci dispersed along chromosome 17 in a series of 69 neuroblastomas. Allelic imbalances for at least two consecutive loci were observed in 39/59 informative cases, that is in agreement with previously reported frequencies of 17q gain. In a subset of the cases, comparative genomic hybridization analysis established the relationship between these allelic imbalances and the gain of 17q material. A partial 17q gain was observed in 9 cases, delineating a common region of 17q gain between the marker D17S787 (75 cM, 360 cR) and the telomere. In most cases, molecular results were suggestive of partial tri- or tetrasomy, whereas in 4 cases a higher copy number was documented. Our results also confirm that the presence of additional 17q material is closely associated with 1p36 deletion, MYCN amplification, and diploid or tetraploid chromosomal content. Genes Chromosomes Cancer 28:276-284, 2000.

Use of the two-hybrid system to identify Rab-interacting proteins.

The yeast two-hybrid system has been useful for identifying many partners and effectors of small GTPases of the Rab family. We describe here such a screen using Rab6, a protein involved in the regulation of intracellular transport at the level of the Golgi apparatus, as bait.

Characterization of GAPCenA, a GTPase activating protein for Rab6, part of which associates with the centrosome.

The Rab6 GTPase regulates intracellular transport at the level of the Golgi apparatus, probably in a retrograde direction. Here, we report the identification and characterization of a novel human Rab6-interacting protein named human GAPCenA (for 'GAP and centrosome-associated'). Primary sequence analysis indicates that GAPCenA displays similarities, within a central 200 amino acids domain, to both the yeast Rab GTPase activating proteins (GAPs) and to the spindle checkpoint proteins Saccharomyces cerevisiae Bub2p and Schizosaccharomyces pombe Cdc16p. We demonstrate that GAPCenA is indeed a GAP, specifically active in vitro on Rab6 and, to a lesser extent, on Rab4 and Rab2 proteins. Immunofluorescence and cell fractionation experiments showed that GAPCenA is mainly cytosolic but that a minor pool is associated with the centrosome. Moreover, GAPCenA was found to form complexes with cytosolic gamma-tubulin and to play a role in microtubule nucleation. Therefore, GAPCenA may be involved in the coordination of microtubule and Golgi dynamics during the cell cycle.

Identification and characterization of potential effector molecules of the Ras-related GTPase Rap2.

In search for effectors of the Ras-related GTPase Rap2, we used the yeast two-hybrid method and identified the C-terminal Ras/Rap interaction domain of the Ral exchange factors (RalGEFs) Ral GDP dissociation stimulator (RalGDS), RalGDS-like (RGL), and RalGDS-like factor (Rlf). These proteins, which also interact with activated Ras and Rap1, are effectors of Ras and mediate the activation of Ral in response to the activation of Ras. Here we show that the full-length RalGEFs interact with the GTP-bound form of Rap2 in the two-hybrid system as well as in vitro. When co-transfected in HeLa cells, an activated Rap2 mutant (Rap2Val-12) but not an inactive protein (Rap2Ala-35) co-immunoprecipitates with RalGDS and Rlf; moreover, Rap2-RalGEF complexes can be isolated from the particulate fraction of transfected cells and were localized by confocal microscopy to the resident compartment of Rap2, i.e. the endoplasmic reticulum. However, the overexpression of activated Rap2 neither leads to the activation of the Ral GTPase via RalGEFs nor inhibits Ras-dependent Ral activation in vivo. Several hypotheses that could explain these results, including compartmentalization of proteins involved in signal transduction, are discussed. Our results suggest that in cells, the interaction of Rap2 with RalGEFs might trigger other cellular responses than activation of the Ral GTPase.

Identification of a specific effector of the small GTP-binding protein Rap2.

Rap2 is a small GTP-binding protein that belongs to the Ras superfamily and whose function is still unknown. To elucidate Rap2 function, we searched for potential effectors by screening a mouse brain cDNA library in a yeast two-hybrid system using as a bait a Rap2A protein bearing a mutation of Gly to Val at position 12. This strategy lead to the identification of a protein that interacts specifically with Rap2A complexed with GTP, and requires an intact effector domain of Rap2A for interaction; we designated this protein Rap2-interacting protein 8 (RPIP8). Biochemical data obtained from in vitro studies with purified proteins confirmed the genetic results. Mouse RPIP8 consists of 446 amino acids, bears a coiled-coil domain between residues 265 and 313, and is expressed principally in brain. Its human counterpart, of 400 amino acids, exhibits 93.7% identity in their common region. A search for similar sequences in expressed-sequence-tags databanks revealed the presence in human and rodents of mRNAs encoding the 400-residue and 446-residue forms of RPIP8. Furthermore a doublet of 45-50 kDa, corresponding to the 400-residue and 446-residue forms of the protein, was detected by western blotting of mouse brain extracts and lysates from pheochromocytoma PC12 cells and the pancreatic beta-cell lines HIT-T15 and RIN-m5F. Using transient transfections of HIT-T15 cells it was possible to demonstrate that [Val12]Rap2 and wild-type Rap2 could be immunoprecipitated with RPIP8. These data therefore argue for RPIP8 being a specific effector of the Rap2 protein in cells exhibiting neuronal properties.

Interaction of a Golgi-associated kinesin-like protein with Rab6.

Rab guanosine triphosphatases regulate vesicular transport and membrane traffic within eukaryotic cells. Here, a kinesin-like protein that interacts with guanosine triphosphate (GTP)-bound forms of Rab6 was identified. This protein, termed Rabkinesin-6, was localized to the Golgi apparatus and shown to play a role in the dynamics of this organelle. The carboxyl-terminal domain of Rabkinesin-6, which contains the Rab6-interacting domain, inhibited the effects of Rab6-GTP on intracellular transport. Thus, a molecular motor is a potential effector of a Rab protein, and coordinated action between members of these two families of proteins could control membrane dynamics and directional vesicular traffic.

Crystal structures of the small G protein Rap2A in complex with its substrate GTP, with GDP and with GTPgammaS.

The small G protein Rap2A has been crystallized in complex with GDP, GTP and GTPgammaS. The Rap2A-GTP complex is the first structure of a small G protein with its natural ligand GTP. It shows that the hydroxyl group of Tyr32 forms a hydrogen bond with the gamma-phosphate of GTP and with Gly13. This interaction does not exist in the Rap2A-GTPgammaS complex. Tyr32 is conserved in many small G proteins, which probably also form this hydrogen bond with GTP. In addition, Tyr32 is structurally equivalent to a conserved arginine that binds GTP in trimeric G proteins. The actual participation of Tyr32 in GTP hydrolysis is not yet clear, but several possible roles are discussed. The conformational changes between the GDP and GTP complexes are located essentially in the switch I and II regions as described for the related oncoprotein H-Ras. However, the mobile segments vary in length and in the amplitude of movement. This suggests that even though similar regions might be involved in the GDP-GTP cycle of small G proteins, the details of the changes will be different for each G protein and will ensure the specificity of its interaction with a given set of cellular proteins.

Two-hybrid system screen with the small GTP-binding protein Rab6. Identification of a novel mouse GDP dissociation inhibitor isoform and two other potential partners of Rab6.

Rab6 is a small GTP-binding protein that belongs to the Ras superfamily and is involved in intra-Golgi transport. Using a two-hybrid system screen of a mouse brain cDNA library, we have isolated several clones encoding proteins that interact with Rab6. Approximately 60% of the clones identified encoded a new mouse Rab GDP dissociation inhibitor (GDI) isoform. This GDI isoform is distinct from mouse mGDI-1 and mGDI-2, which have been characterized previously, and most likely represents the mouse counterpart of the rat Rab GDI beta isoform. In the two-hybrid system, GDI beta interacts with wild-type Rab6 and Rab5, but not with a GTP-bound Rab6 mutant, or a Rab6 mutant that cannot be post-translationally processed. We further examined whether mouse GDI beta is functional; we show that recombinant mouse GDI beta is able to remove several Rab proteins, including Rab1, Rab2, Rab4, and Rab6, from membranes. The identification of a third GDI isoform in mouse raised the question whether GDI genes belong to a larger multigenic family. We have shown, by Southern blot analysis of genomic DNA, that at least five GDI gene copies exist in both the mouse and rat genomes. In our two-hybrid screen, we have also characterized another clone that specifically interacts with Rab6. This clone was partially sequenced but shows no homology to known sequences. Finally, a third clone, interacting with both Rab5 and Rab6, also appears to encode a novel protein.

Phosphorylation of Rap1GAP during the cell cycle.

Rap1GAP (for Rap1 GTPase Activating Protein) is an 89 kD protein that highly stimulates the intrinsic GTPase activity of the small GTP binding protein Rap1. It has been shown that Rap1GAP is phosphorylated in vitro by purified p34cdc2 kinase, which regulates the G2/M transition of the cell cycle. In this work, we have studied the phosphorylation of Rap1GAP during the cell cycle and showed that Rap1GAP is phosphorylated in vivo in interphasic and mitotic Hela cells; the electrophoretic mobility of Rap1GAP from mitotic cells is reduced compared with that from interphasic cells, suggesting that the mitotic form of the protein is hyperphosphorylated. As the cdc2 kinase is specifically active during mitosis, we sought to investigate whether it actually phosphorylates Rap1GAP during this phase of the cell cycle. We show that p34cdc2 co-immunoprecipitated from mitotic Hela cell lysates with an anti human cyclin B1 antibody, but not from interphasic cell lysates, is able to phosphorylate efficiently wild-type Rap1GAP, but not a mutant in which the putative consensus site for phosphorylation by the cdc2 kinase (serine 484) has been altered. Moreover, depletion of p34cdc2 from mitotic extracts abolishes the phosphorylation of Rap1GAP by such lysates. These results therefore strongly suggest that Rap1GAP is indeed a substrate of the cdc2 kinase during mitosis. This phosphorylation does not affect the stimulation of the GTPase activity of Rap1 by Rap1GAP but may play a role in regulating the interaction of Rap1GAP with other proteins involved in the cellular functions regulated by Rap1 and Rap1GAP.

Characterization of the Ras-related RAP2A protein expressed in the baculovirus-insect cell system: processing of the protein in insect cells and comparison with the bacterially produced unprocessed form.

The Ras-related protein Rap2A is a 21kD GTP-binding protein that exhibits 46% identity with Ras proteins and is similarly post-translationally modified by farnesyl and palmitate groups. Using a recombinant baculovirus, we expressed Rap2A in Sf9 insect cells. The protein is initially synthesized as a soluble hydrophilic precursor, that is post-translationally processed to a hydrophobic membrane-bound form (Rap2Am) that contains both isoprenoid and palmitate groups. The processed form of the protein was purified from the membranes of infected Sf9 cells, and its biochemical properties were compared with those of the unprocessed form produced in recombinant bacteria (Rap2Ab). Both proteins exhibited similar kinetics of GDP dissociation and GTP binding and displayed a weak intrinsic GTPase activity that was stimulated to the same extent by a factor present in bovine brain cytosol. We conclude that Rap2A is correctly processed in insect cells and that maturation does not alter its biochemical properties.

Regulation of the GTPase activity of the ras-related rap2 protein.

The small GTP-binding protein rap2A exhibits a high level of identity with rap1 and ras proteins (60% and 46%, respectively). Nevertheless, its intrinsic GTPase activity is not stimulated by ras-GAP, and unlike the rap1A protein, it cannot compete with ras proteins for their interaction with ras-GAP. In addition, rap1-GAPm that is highly active on the GTPase activity of the rap1A product, also stimulates the GTPase activity of the rap2A protein but with a 30-40-fold lower efficiency. An activity that greatly stimulated the GTPase activity of the rap2 protein (rap2-GAP) was found in bovine brain cytosol and purified. However, it copurified with the cytosolic form of rap1-GAP and was more efficient at stimulating the GTPase activity of the rap1 protein; this 55 kD polypeptide, that is recognized by an antibody raised against rap1-GAPm, likely represents a degraded and soluble form of the full size 89 kD molecule. In bovine brain membranes, a weak GAP activity toward the rap2A protein was also detected; however, it was also attributable to the membrane-associated rap1-GAPm. Thus, it appears that a single rap-GAP protein, complete or degraded, is able to stimulate the GTPase activity of both rap1 and rap2 proteins.