RESUMEN
Atypical teratoid/rhabdoid tumors (ATRT) are highly malignant brain tumors arising in young children. The majority of ATRT is characterized by inactivation of the chromatin remodeling complex member SMARCB1 (INI1/hSNF5). Little is known, however, on downstream pathways involved in the detrimental effects of SMARCB1 deficiency which might also represent targets for treatment. Using Drosophila melanogaster and the Gal4-UAS system, modifier screens were performed in order to identify the role of SMAD dependent signaling in the lethal phenotype associated with knockdown of snr1, the fly homolog of SMARCB1. Expression and functional role of human homologs was next investigated in ATRT tumor samples and SMARCB1-deficient rhabdoid tumor cells. The lethal phenotype associated with snr1 knockdown in Drosophila melanogaster could be shifted to later stages of development upon additional knockdown of several decapentaplegic pathway members including Smox, and Med. Similarly, the transforming growth factor beta (TGFbeta) receptor type I kinase inhibitor SB431542 ameliorated the detrimental effect of snr1 knockdown in the fruit fly. Examination of homologs of candidate decapentaplegic pathway members in human SMARCB1-deficent ATRT samples revealed SMAD3 and SMAD6 to be over-expressed. In SMARCB1-deficent rhabdoid tumor cells, siRNA-mediated silencing of SMAD3 or SMAD6 expression reduced TGFbeta signaling activity and resulted in decreased proliferation. Similar results were obtained upon pharmacological inhibition of TGFbeta signaling using SB431542. Our data suggest that SMAD dependent signaling is involved in the detrimental effects of SMARCB1-deficiency and provide a rationale for the investigation of TGFbeta targeted treatments in ATRT.
Asunto(s)
Proteínas de Drosophila/metabolismo , Tumor Rabdoide/metabolismo , Proteína SMARCB1/metabolismo , Transducción de Señal , Proteínas Smad/metabolismo , Teratoma/metabolismo , Factores de Transcripción/metabolismo , Animales , Benzamidas/administración & dosificación , Dioxoles/administración & dosificación , Modelos Animales de Enfermedad , Proteínas de Drosophila/genética , Drosophila melanogaster , Femenino , Humanos , Masculino , ARN Mensajero/metabolismo , Tumor Rabdoide/genética , Proteína SMARCB1/genética , Proteína smad3/metabolismo , Proteína smad6/metabolismo , Teratoma/genética , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Platelet-derived growth factor receptor (PDGFR) signaling plays an important role in the biology of malignant gliomas. To investigate mechanisms modulating PDGFR signaling in gliomagenesis, we employed a Drosophila glioma model and genetic screen to identify genes interacting with Pvr, the fly homolog of PDGFRs. Glial expression of constitutively activated Pvr (λPvr) led to glial over migration and lethality at late larval stage. Among 3316 dsRNA strains crossed against the tester strain, 128 genes shifted lethality to pupal stage, including tetraspanin 2A (tsp2A). In a second step knockdown of all Drosophila tetraspanins was investigated. Of all tetraspanin dsRNA strains only knockdown of tsp2A partially rescued the Pvr-induced phenotype. Human CD9 (TSPAN29/MRP-1), a close homolog of tsp2A, was found to be expressed in glioma cell lines A172 and U343MG as well as in the majority of glioblastoma samples (16/22, 73 %). Furthermore, in situ proximity ligation assay revealed close association of CD9 with PDGFR α and ß. In U343MG cells, knockdown of CD9 blocked PDGF-BB stimulated migration. In conclusion, modulation of PDGFR signaling by CD9 is evolutionarily conserved from Drosophila glia to human glioma and plays a role in glia migration.
Asunto(s)
Movimiento Celular/fisiología , Glioma/patología , Neuroglía/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Tetraspanina 29/metabolismo , Análisis de Varianza , Animales , Animales Modificados Genéticamente , Evolución Biológica , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Relación Dosis-Respuesta a Droga , Drosophila , Proteínas de Drosophila/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Neuroglía/patología , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Receptores del Factor de Crecimiento Derivado de Plaquetas/farmacología , Transducción de SeñalRESUMEN
Atypical teratoid/rhabdoid tumours (AT/RT) are malignant brain tumours. Unlike most other human brain tumours, AT/RT are characterized by inactivation of one single gene, SMARCB1. SMARCB1 is a member of the evolutionarily conserved SWI/SNF chromatin remodelling complex, which has an important role in the control of cell differentiation and proliferation. Little is known, however, about the pathways involved in the oncogenic effects of SMARCB1 inactivation, which might also represent targets for treatment. Here we report a comprehensive genetic screen in the fruit fly that revealed several genes not yet associated with loss of snr1, the Drosophila homologue of SMARCB1. We confirm the functional role of identified genes (including merlin, kibra and expanded, known to regulate hippo signalling pathway activity) in human rhabdoid tumour cell lines and AT/RT tumour samples. These results demonstrate that fly models can be employed for the identification of clinically relevant pathways in human cancer.
Asunto(s)
Neoplasias Encefálicas/genética , Proteínas de Drosophila/genética , Tumor Rabdoide/genética , Teratoma/genética , Factores de Transcripción/genética , Animales , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Drosophila melanogaster , Técnicas de Silenciamiento del Gen , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Neurofibromina 2/genética , Proteínas Serina-Treonina Quinasas/genética , Proteína SMARCB1 , Transducción de Señal , Proteínas Supresoras de Tumor/genéticaRESUMEN
Atypical teratoid/rhabdoid tumor (AT/RT) is a rare malignant pediatric brain tumor characterized by genetic alterations affecting the SMARCB1 (hSNF5/INI1) locus in chromosome band 22q11.2. To identify potential additional genetic alterations, high-resolution genome-wide analysis was performed using a molecular inversion probe single-nucleotide polymorphism (MIP SNP) assay (Affymetrix OncoScan formalin-fixed paraffin-embedded express) on DNA isolated from 18 formalin-fixed paraffin-embedded archival samples. Alterations affecting the SMARCB1 locus could be demonstrated by MIP SNP in 15 out of 16 evaluable cases (94%). These comprised five tumors with homozygous deletions, six tumors with heterozygous deletions, and four tumors with copy number neutral loss of heterozygosity (LOH) involving chromosome band 22q11.2. Remarkably, MIB SNP analysis did not yield any further recurrent chromosomal gains, losses, or copy neutral LOH. On MIP SNP screening for somatic mutations, the presence of a SMARCB1 mutation (c.472C>T p.R158X) was confirmed, but no recurrent mutations of other cancer relevant genes could be identified. Results of fluorescence in situ hybridization, multiplex ligation-dependent probe amplification, and SMARCB1 sequencing were highly congruent with that of the MIP SNP assay. In conclusion, these data further suggest the absence of recurrent genomic alterations other than SMARCB1 in AT/RT.