[Show abstract][Hide abstract] ABSTRACT: The anaplastic lymphoma kinase (ALK) gene is overexpressed, mutated or amplified in most neuroblastoma (NB), a pediatric neural crest-derived embryonal tumor. The two most frequent mutations, ALK-F1174L and ALK-R1275Q, contribute to NB tumorigenesis in mouse models, and cooperate with MYCN in the oncogenic process. However, the precise role of activating ALK mutations or ALK-wt overexpression in NB tumor initiation needs further clarification. Human ALK-wt, ALK-F1174L, or ALK-R1275Q were stably expressed in murine neural crest progenitor cells (NCPC), MONC-1 or JoMa1, immortalized with v-Myc or Tamoxifen-inducible Myc-ERT, respectively. While orthotopic implantations of MONC-1 parental cells in nude mice generated various tumor types, such as NB, osteo/chondrosarcoma, and undifferentiated tumors, due to v-Myc oncogenic activity, MONC-1-ALK-F1174L cells only produced undifferentiated tumors. Furthermore, our data represent the first demonstration of ALK-wt transforming capacity, as ALK-wt expression in JoMa1 cells, likewise ALK-F1174L, or ALK-R1275Q, in absence of exogenous Myc-ERT activity, was sufficient to induce the formation of aggressive and undifferentiated neural crest cell-derived tumors, but not to drive NB development. Interestingly, JoMa1-ALK tumors and their derived cell lines upregulated Myc endogenous expression, resulting from ALK activation, and both ALK and Myc activities were necessary to confer tumorigenic properties on tumor-derived JoMa1 cells in vitro.
[Show abstract][Hide abstract] ABSTRACT: Pure invasive micropapillary carcinoma (IMPC) is a special type of breast carcinoma characterized by clusters of cells presenting polarity abnormalities. The biological alterations underlying this pattern remain unknown.
Breast cancer research: BCR 05/2014; 16(3):R46. · 5.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The ALK (Anaplastic Lymphoma Kinase) gene encodes a tyrosine kinase receptor preferentially expressed in the central and peripheral nervous systems. A syndromic presentation associating congenital neuroblastoma with severe encephalopathy and an abnormal shape of the brainstem has been described in patients harbouring de novo germline F1174V and F1245V ALK mutations. Here, we investigated the phenotype of knock-in (KI) mice bearing the AlkF1178L mutation (F1174L in human). Although heterozygous KI mice did not reproduce the severe breathing and feeding difficulties observed in human patients, behavioral tests documented a reduced activity during dark phases and an increased anxiety of mutated mice. Matings of heterozygotes yielded the expected proportions of wild-type, heterozygotes and homozygotes at birth but a high neonatal lethality was noticed for homozygotes. We documented Alk expression in several motor nuclei of the brainstem involved in the control of sucking and swallowing. Evaluation of basic physiological functions 12 hours after birth revealed slightly more apneas but a dramatic reduced milk intake for homozygotes compared to control littermates. Overall, our data demonstrate that Alk activation above a critical threshold is not compatible with survival in mice, in agreement with the extremely severe phenotype of patients carrying aggressive de novo ALK germline mutations.
[Show abstract][Hide abstract] ABSTRACT: Activating mutations of the ALK (Anaplastic lymphoma Kinase) gene have been identified in sporadic and familial cases of neuroblastoma, a cancer of early childhood arising from the sympathetic nervous system (SNS). To decipher ALK function in neuroblastoma predisposition and oncogenesis, we have characterized knock-in (KI) mice bearing the two most frequent mutations observed in neuroblastoma patients. A dramatic enlargement of sympathetic ganglia is observed in AlkF1178L mice from embryonic to adult stages associated with an increased proliferation of sympathetic neuroblasts from E14.5 to birth. In a MYCN transgenic context, the F1178L mutation displays a higher oncogenic potential than the R1279Q mutation as evident from a shorter latency of tumor onset. We show that tumors expressing the R1279Q mutation are sensitive to ALK inhibition upon crizotinib treatment. Furthermore, our data provide evidence that activated ALK triggers RET upregulation in mouse sympathetic ganglia at birth as well as in murine and human neuroblastoma. Using vandetanib, we show that RET inhibition strongly impairs tumor growth in vivo in both MYCN/KI AlkR1279Q and MYCN/KI AlkF1178L mice. Altogether, our findings demonstrate the critical role of activated ALK in SNS development and pathogenesis and identify RET as a therapeutic target in ALK mutated neuroblastoma.
[Show abstract][Hide abstract] ABSTRACT: Smoothened (SMO) inhibitors recently entered clinical trials for sonic-hedgehog-driven medulloblastoma (SHH-MB). Clinical response is highly variable. To understand the mechanism(s) of primary resistance and identify pathways cooperating with aberrant SHH signaling, we sequenced and profiled a large cohort of SHH-MBs (n = 133). SHH pathway mutations involved PTCH1 (across all age groups), SUFU (infants, including germline), and SMO (adults). Children >3 years old harbored an excess of downstream MYCN and GLI2 amplifications and frequent TP53 mutations, often in the germline, all of which were rare in infants and adults. Functional assays in different SHH-MB xenograft models demonstrated that SHH-MBs harboring a PTCH1 mutation were responsive to SMO inhibition, whereas tumors harboring an SUFU mutation or MYCN amplification were primarily resistant.
Cancer cell 03/2014; 25(3):393-405. · 25.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Medulloblastoma comprises four distinct molecular subgroups: WNT, SHH, Group 3, and Group 4. Current medulloblastoma protocols stratify patients based on clinical features: patient age, metastatic stage, extent of resection, and histologic variant. Stark prognostic and genetic differences among the four subgroups suggest that subgroup-specific molecular biomarkers could improve patient prognostication.
Molecular biomarkers were identified from a discovery set of 673 medulloblastomas from 43 cities around the world. Combined risk stratification models were designed based on clinical and cytogenetic biomarkers identified by multivariable Cox proportional hazards analyses. Identified biomarkers were tested using fluorescent in situ hybridization (FISH) on a nonoverlapping medulloblastoma tissue microarray (n = 453), with subsequent validation of the risk stratification models.
Subgroup information improves the predictive accuracy of a multivariable survival model compared with clinical biomarkers alone. Most previously published cytogenetic biomarkers are only prognostic within a single medulloblastoma subgroup. Profiling six FISH biomarkers (GLI2, MYC, chromosome 11 [chr11], chr14, 17p, and 17q) on formalin-fixed paraffin-embedded tissues, we can reliably and reproducibly identify very low-risk and very high-risk patients within SHH, Group 3, and Group 4 medulloblastomas.
Combining subgroup and cytogenetic biomarkers with established clinical biomarkers substantially improves patient prognostication, even in the context of heterogeneous clinical therapies. The prognostic significance of most molecular biomarkers is restricted to a specific subgroup. We have identified a small panel of cytogenetic biomarkers that reliably identifies very high-risk and very low-risk groups of patients, making it an excellent tool for selecting patients for therapy intensification and therapy de-escalation in future clinical trials.
Journal of Clinical Oncology 02/2014; · 18.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: DNA copy number profiles characterize regions of chromosome gains, losses, and breakpoints in tumor genomes. Although many models have been proposed to detect these alterations, it is not clear which model is appropriate before visual inspection the signal, noise, and models for a particular profile.
We propose SegAnnDB, a web-based computer vision system for genomic segmentation: first visually inspect the profiles and manually annotate altered regions, then SegAnnDB determines the precise alteration locations using a mathemetical model of the data and annotations. SegAnnDB facilitates collaboration between biologists and bioinformaticians, and uses the UCSC genome browser to visualize copy number alterations alongside known genes.
The breakpoints project on INRIA GForge hosts the source code, an Amazon Machine Image can be launched, and a demonstration web site is http://bioviz.rocq.inria.fr.
[Show abstract][Hide abstract] ABSTRACT: Somatically acquired genomic alterations with MYCN amplification (MNA) are key features of neuroblastoma (NB), the most common extra-cranial malignant tumour of childhood. Little is known about the frequency, clinical characteristics and outcome of NBs harbouring genomic amplification(s) distinct from MYCN.
PLoS ONE 01/2014; 9(7):e101990. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The present study describes a new case of EWSR1-negative undifferentiated sarcoma with CIC/DUX4 gene fusion. This case is similar to tumors described as primitive undifferentiated round cell sarcomas that occur mainly in the trunk and display an aggressive behavior. To our knowledge, this is the first report of such a tumor presenting locoregional lymph node metastasis. In view of previous studies that prove the existence of a particular variant of undifferentiated sarcoma with Ewing-like morphology and CIC/DUX-4 gene fusion, a search for this gene fusion in all undifferentiated round cell sarcomas should be considered if a conclusive diagnosis cannot be reached following other conventional studies. Although additional cases with more extensive follow-up studies are needed, we believe that EWSR1-negative undifferentiated small round cell sarcoma with CIC/DUX4 gene fusion should be added to the list of new sarcoma variants with the possibility of lymph node metastasis.
Archiv für Pathologische Anatomie und Physiologie und für Klinische Medicin 11/2013; · 2.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: RNA interference has boosted the field of functional genomics, by making it possible to carry out "loss of function" screens in cultured cells. Here, we performed a siRNA screening, in three breast cancer cell lines, for 101 candidate driver genes overexpressed in amplified breast tumors and belonging to eight amplicons on chromosomes 8q and 17q, investigating their role in cell survival/proliferation. This screening identified eight driver genes that were amplified, overexpressed and critical for breast tumor cell proliferation or survival. They included the well described oncogenic driver genes for the 17q12 amplicon, ERBB2 and GRB7. Four of six other candidate driver genes - RAD21 and EIF3H, both on chromosome 8q23, CHRAC1 on chromosome 8q24.3 and TANC2 on chromosome 17q23 -were confirmed to be driver genes regulating the proliferation/survival of clonogenic breast cancer cells presenting an amplification of the corresponding region. Indeed, knockdown of the expression of these genes decreased cell viability, through both cell cycle arrest and apoptosis induction, and inhibited the formation of colonies in anchorage-independent conditions, in soft agar. Strategies for inhibiting the expression of these genes or the function of the proteins they encode are therefore of potential value for the treatment of breast cancers presenting amplifications of the corresponding genomic region.
[Show abstract][Hide abstract] ABSTRACT: Amplification of the 8p11-12 chromosomal region is a common genetic event in many epithelial cancers. In breast cancer, several genes within this region have been shown to display oncogenic activity. Among these genes, the enzyme-encoding genes, PPAPDC1B and WHSC1L1, have been identified as potential therapeutic targets. We investigated whether PPAPDC1B and WHSC1L1 acted as general driver genes, thereby serving as therapeutic targets in other tumors with 8p11-12 amplification. By using publicly available genomic data from a panel of 883 cell lines derived from different cancers, we identified the cell lines presenting amplification of both WHSC1L1 and PPAPDC1B. In particular, we focused on cell lines derived from lung cancer and pancreatic adenocarcinoma and found a correlation between the amplification of PPAPDC1B and WHSC1L1 with their overexpression. Loss-of-function studies based on the use of siRNA and shRNA demonstrated that PPAPDC1B and WHSC1L1 played a major role in regulating the survival of pancreatic adenocarcinoma and small-cell lung cancer-derived cell lines, both in anchorage-dependent and anchorage-independent conditions, displaying amplification and overexpression of these genes. We also demonstrated that PPAPDC1B and WHSC1L1 regulated xenograft growth in these cell lines. Finally, quantitative RT-PCR experiments after PPAPDC1B and WHSC1L1 knockdown revealed exclusive PPAPDC1B and WHSC1L1 gene targets in small-cell lung cancer and pancreatic adenocarcinoma-derived cell lines compared with breast cancer.
American Journal Of Pathology 09/2013; · 4.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Nucleic Acids Research 08/2013; · 8.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Embryonal tumor with multilayered rosettes (ETMR), including embryonal tumor with abundant neuropil and true rosettes (ETANTR), and ependymoblastoma (EBL) constitute a distinct entity of the primitive neuroectodermal tumor (PNET) family. The presence of a focal amplification at chromosome region 19q13.42 associated with an up-regulation of the oncogenic miRNA cluster C19MC suggests that they may represent a histological spectrum of a single biological entity. Their histopathological spectrum is wide, including medulloepithelioma, their location may be supra- or infra-tentorial, their prognosis is poor. Recent data on molecular subgroups of PNETs have led to new insights on diagnosis and treatment of these tumors. Subsequently, LIN28A immunoexpression was identified as a highly specific marker for ETMR. In this study, we report 4 cases diagnosed initially as ETANTR with CGH-array data, including 19q13.42 gain with absence of other amplicons, particularly of the MYC gene family, and inconstant gain of whole chromosome 2. Immunohistochemical positive expression of LIN28A and absence of Olig2 expression were observed. We summarize the literature on ETMR, pointing out on the nosological evolution of this entity and the findings on genetic hallmarks of this particular tumor. Our results emphasize the usefulness of immunohistochemistry as a highly sensitive and fast diagnostic tool for ETMR and for genetic data, especially for 19q13.42 locus. Biological features may offer new therapeutic options for these embryonal tumors that do not usually respond to conventional treatments of PNETs.
[Show abstract][Hide abstract] ABSTRACT: Metabolic adaptation is essential for cell survival during nutrient deprivation. We report that eukaryotic elongation factor 2 kinase (eEF2K), which is activated by AMP-kinase (AMPK), confers cell survival under acute nutrient depletion by blocking translation elongation. Tumor cells exploit this pathway to adapt to nutrient deprivation by reactivating the AMPK-eEF2K axis. Adaptation of transformed cells to nutrient withdrawal is severely compromised in cells lacking eEF2K. Moreover, eEF2K knockdown restored sensitivity to acute nutrient deprivation in highly resistant human tumor cell lines. In vivo, overexpression of eEF2K rendered murine tumors remarkably resistant to caloric restriction. Expression of eEF2K strongly correlated with overall survival in human medulloblastoma and glioblastoma multiforme. Finally, C. elegans strains deficient in efk-1, the eEF2K ortholog, were severely compromised in their response to nutrient depletion. Our data highlight a conserved role for eEF2K in protecting cells from nutrient deprivation and in conferring tumor cell adaptation to metabolic stress. PAPERCLIP:
[Show abstract][Hide abstract] ABSTRACT: Background; Many models have been proposed to detect copy number alterations in chromosomal copynumber profiles, but it is usually not obvious to decide which is most effective for a givendata set. Furthermore, most methods have a smoothing parameter that determines the numberof breakpoints and must be chosen using various heuristics.Results; We present three contributions for copy number profile smoothing model selection. First, wepropose to select the model and degree of smoothness that maximizes agreement with visualbreakpoint region annotations. Second, we develop cross-validation procedures to estimatethe error of the trained models. Third, we apply these methods to compare 17 smoothingmodels on a new database of 575 annotated neuroblastoma copy number profiles, which wemake available as a public benchmark for testing new algorithms.Conclusions; Whereas previous studies have been qualitative or limited to simulated data, our annotation-guided approach is quantitative and suggests which algorithms are fastest and most accuratein practice on real data. In the neuroblastoma data, the equivalent pelt.n and cghseg.k meth-ods were the best breakpoint detectors, and exhibited reasonable computation times.
[Show abstract][Hide abstract] ABSTRACT: The genetic cause of some familial nonsyndromic renal cell carcinomas (RCC) defined by at least two affected first-degree relatives is unknown. By combining whole-exome sequencing and tumor profiling in a family prone to cases of RCC, we identified a germline BAP1 mutation c.277A>G (p.Thr93Ala) as the probable genetic basis of RCC predisposition. This mutation segregated with all four RCC-affected relatives. Furthermore, BAP1 was found to be inactivated in RCC-affected individuals from this family. No BAP1 mutations were identified in 32 familial cases presenting with only RCC. We then screened for germline BAP1 deleterious mutations in familial aggregations of cancers within the spectrum of the recently described BAP1-associated tumor predisposition syndrome, including uveal melanoma, malignant pleural mesothelioma, and cutaneous melanoma. Among the 11 families that included individuals identified as carrying germline deleterious BAP1 mutations, 6 families presented with 9 RCC-affected individuals, demonstrating a significantly increased risk for RCC. This strongly argues that RCC belongs to the BAP1 syndrome and that BAP1 is a RCC-predisposition gene.
The American Journal of Human Genetics 05/2013; · 11.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: As prognostic factors, MYC and MYCN amplifications are routinely assessed in medulloblastomas. Fluorescence in situ hybridization (FISH) is currently considered as the technique of reference. Recently, array comparative genomic hybridization (aCGH) has been developed as an alternative technique to evaluate genomic abnormalities in other tumor types; however, this technique has not been widely adopted as a replacement for FISH in medulloblastoma. In this study, 34 tumors were screened by both FISH and aCGH. In all cases showing amplification by FISH, aCGH also unambiguously revealed the abnormality. The aCGH technique was also performed on tumors showing no amplification by FISH, and the absence of amplification was confirmed in all cases. Interestingly, one tumor showed a subclonal MYC amplification by FISH. This subclonal amplification was observed in approximately 20% of tumor cells and was clearly evident on aCGH. In conclusion, our analysis confirms that aCGH is as safe as FISH for the detection of MYC/MYCN gene amplification. Given its cost efficiency in comparison to two FISH tests and the global genomic information additionally provided by an aCGH experiment, this reproducible technique can be safely retained as an alternative to FISH for routine investigation of medulloblastoma.
[Show abstract][Hide abstract] ABSTRACT: Metastasis, a fatal complication of breast cancer, does not fully benefit from available therapies. In this study we investigated whether ATIP3, the major product of 8p22 MTUS1 gene, may be a novel biomarker and therapeutic target for metastatic breast tumors. We show that ATIP3 is a prognostic marker for overall survival among patients with breast cancer. Notably, among metastatic tumors, low ATIP3 levels associate with decreased survival of the patients. By using a well-defined experimental mouse model of cancer metastasis we show that ATIP3 expression delays the time-course of metastatic progression and limits the number and size of metastases in vivo. In functional studies, ATIP3 silencing increases breast cancer cell migration whereas ATIP3 expression significantly reduces cell motility and directionality. We report here that ATIP3 is a potent microtubule stabilizing protein whose depletion increases microtubule dynamics. Our data support the notion that by decreasing microtubule dynamics, ATIP3 controls the ability of microtubule tips to reach the cell cortex during migration, a mechanism that may account for reduced cancer cell motility and metastasis. Of interest, we identify a functional ATIP3 domain that associates with microtubules and recapitulates the effects of ATIP3 on microtubule dynamics, cell proliferation and migration. Our study is a major step toward the development of new personalized treatments against metastatic breast tumors that have lost ATIP3 expression.