RESUMEN
Benign neurofibromas and malignant peripheral nerve sheath tumors are serious complications of neurofibromatosis type 1. The epidermal growth factor receptor is not expressed by normal Schwann cells, yet is overexpressed in subpopulations of Nf1 mutant Schwann cells. We evaluated the role of EGFR in Schwann cell tumorigenesis. Expression of EGFR in transgenic mouse Schwann cells elicited features of neurofibromas: Schwann cell hyperplasia, excess collagen, mast cell accumulation, and progressive dissociation of non-myelin-forming Schwann cells from axons. Mating EGFR transgenic mice to Nf1 hemizygotes did not enhance this phenotype. Genetic reduction of EGFR in Nf1(+/-);p53(+/-) mice that develop sarcomas significantly improved survival. Thus, gain- and loss-of-function experiments support the relevance of EGFR to peripheral nerve tumor formation.
Asunto(s)
Receptores ErbB/metabolismo , Neurofibroma/metabolismo , Neurofibromatosis 1/metabolismo , Neurofibromatosis 1/patología , Nervios Periféricos/metabolismo , Nervios Periféricos/patología , Células de Schwann/fisiología , 2',3'-Nucleótido Cíclico Fosfodiesterasas/genética , 2',3'-Nucleótido Cíclico Fosfodiesterasas/metabolismo , Animales , Células Cultivadas , Receptores ErbB/genética , Fibrosis , Humanos , Mastocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias de Tejido Nervioso/genética , Neoplasias de Tejido Nervioso/metabolismo , Neoplasias de Tejido Nervioso/patología , Neurofibroma/genética , Neurofibroma/patología , Neurofibromatosis 1/genética , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Nervios Periféricos/ultraestructura , Células de Schwann/citología , Transducción de Señal/fisiología , Tasa de Supervivencia , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Malignant peripheral nerve sheath tumors (MPNST) are highly invasive soft tissue sarcomas that arise within the peripheral nerve and frequently metastasize. To identify molecular events contributing to malignant transformation in peripheral nerve, we compared eight cell lines derived from MPNSTs and seven normal human Schwann cell samples. We found that MPNST lines are heterogeneous in their in vitro growth rates and exhibit diverse alterations in expression of pRb, p53, p14(Arf), and p16(INK4a) proteins. All MPNST cell lines express the epidermal growth factor receptor and lack S100beta protein. Global gene expression profiling using Affymetrix oligonucleotide microarrays identified a 159-gene molecular signature distinguishing MPNST cell lines from normal Schwann cells, which was validated in Affymetrix microarray data generated from 45 primary MPNSTs. Expression of Schwann cell differentiation markers (SOX10, CNP, PMP22, and NGFR) was down-regulated in MPNSTs whereas neural crest stem cell markers, SOX9 and TWIST1, were overexpressed in MPNSTs. Previous studies have implicated TWIST1 in apoptosis inhibition, resistance to chemotherapy, and metastasis. Reducing TWIST1 expression in MPNST cells using small interfering RNA did not affect apoptosis or chemoresistance but inhibited cell chemotaxis. Our results highlight the use of gene expression profiling in identifying genes and molecular pathways that are potential biomarkers and/or therapeutic targets for treatment of MPNST and support the use of the MPNST cell lines as a primary analytic tool.
Asunto(s)
Neoplasias de la Vaina del Nervio/genética , Células de Schwann/fisiología , Apoptosis/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Resistencia a Antineoplásicos , Perfilación de la Expresión Génica , Humanos , Neoplasias de la Vaina del Nervio/metabolismo , Neoplasias de la Vaina del Nervio/patología , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Interferente Pequeño/genética , Células de Schwann/metabolismo , Transfección , Proteína 1 Relacionada con Twist/biosíntesis , Proteína 1 Relacionada con Twist/genéticaRESUMEN
To investigate the role of erbB signaling in the interactions between peripheral axons and myelinating Schwann cells, we generated transgenic mice expressing a dominant-negative erbB receptor in these glial cells. Mutant mice have delayed onset of myelination, thinner myelin, shorter internodal length, and smaller axonal caliber in adulthood. Consistent with the morphological defects, transgenic mice also have slower nerve conduction velocity and defects in their responses to mechanical stimulation. Molecular analysis indicates that erbB signaling may contribute to myelin formation by regulating transcription of myelin genes. Analysis of sciatic nerves showed a reduction in the levels of expression of myelin genes in mutant mice. In vitro assays revealed that neuregulin-1 (NRG1) induces expression of myelin protein zero (P0). Furthermore, we found that the effects of NRG1 on P0 expression depend on the NRG1 isoform used. When NRG1 is presented to Schwann cells in the context of cell-cell contact, type III but not type I NRG1 regulates P0 gene expression. These results suggest that disruption of the NRG1-erbB signaling pathway could contribute to the pathogenesis of peripheral neuropathies with hypomyelination and neuropathic pain.
Asunto(s)
Fibras Nerviosas Mielínicas/metabolismo , Neurregulina-1/metabolismo , Proteínas Oncogénicas v-erbB/genética , Nervios Periféricos/crecimiento & desarrollo , Células de Schwann/metabolismo , Sensación/genética , Animales , Axones/metabolismo , Axones/patología , Comunicación Celular/genética , Diferenciación Celular/genética , Modelos Animales de Enfermedad , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/fisiología , Ratones , Ratones Transgénicos , Proteína P0 de la Mielina/biosíntesis , Proteína P0 de la Mielina/genética , Proteínas de la Mielina/biosíntesis , Proteínas de la Mielina/genética , Vaina de Mielina/genética , Vaina de Mielina/metabolismo , Fibras Nerviosas Mielínicas/patología , Conducción Nerviosa/genética , Neurregulina-1/genética , Neurregulina-1/farmacología , Nervios Periféricos/metabolismo , Nervios Periféricos/patología , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedades del Sistema Nervioso Periférico/metabolismo , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacología , Células de Schwann/patología , Nervio Ciático/crecimiento & desarrollo , Nervio Ciático/metabolismo , Nervio Ciático/patologíaRESUMEN
Loss of axonal contact characterizes Schwann cells in benign and malignant peripheral nerve sheath tumors (MPNST) from neurofibromatosis type 1 (NF1) patients. Tumor Schwann cells demonstrate NF1 mutations, elevated Ras activity, and aberrant epidermal growth factor receptor (EGFR) expression. Using cDNA microarrays, we found that brain lipid binding protein (BLBP) is elevated in an EGFR-positive subpopulation of Nf1 mutant mouse Schwann cells (Nf1(-/-) TXF) that grows away from axons; BLBP expression was not affected by farnesyltransferase inhibitor, an inhibitor of H-Ras. BLBP was also detected in EGFR-positive cell lines derived from Nf1:p53 double mutant mice and human MPNST. BLBP expression was induced in normal Schwann cells following transfection with EGFR but not H-Ras12V. Furthermore, EGFR-mediated BLBP expression was not inhibited by dominant-negative H-Ras, indicating that BLBP expression is downstream of Ras-independent EGFR signaling. BLBP-blocking antibodies enabled process outgrowth from Nf1(-/-) TXF cells and restored interaction with axons, without affecting cell proliferation or migration. Following injury, BLBP expression was induced in normal sciatic nerves when nonmyelinating Schwann cells remodeled their processes. These data suggest that BLBP, stimulated by Ras-independent pathways, regulates Schwann cell-axon interactions in normal peripheral nerve and peripheral nerve tumors.
Asunto(s)
Axones/metabolismo , Proteínas Portadoras/fisiología , Neoplasias de la Vaina del Nervio/etiología , Proteínas del Tejido Nervioso/fisiología , Células de Schwann/metabolismo , Proteínas Supresoras de Tumor , Animales , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Movimiento Celular , Células Cultivadas/citología , Células Cultivadas/metabolismo , Citoplasma/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes Dominantes , Genes de Neurofibromatosis 1 , Genes ras , Humanos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Compresión Nerviosa , Regeneración Nerviosa , Neoplasias de la Vaina del Nervio/metabolismo , Neoplasias de la Vaina del Nervio/patología , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Cresta Neural/citología , Neurofibromina 1/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Proteínas Recombinantes de Fusión/fisiología , Células de Schwann/citología , Nervio Ciático/lesiones , Transducción de Señal , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/metabolismoRESUMEN
Neurofibromatosis type 1 (NF1) is a common genetic disorder that predisposes affected individuals to tumours. The NF1 gene encodes a RAS GTPase-activating protein called neurofibromin and is one of several genes that (when mutant) affect RAS-MAPK signalling, causing related diseases collectively known as RASopathies. Several RASopathies, beyond NF1, are cancer predisposition syndromes. Somatic NF1 mutations also occur in 5-10% of human sporadic cancers and may contribute to resistance to therapy. To highlight areas for investigation in RASopathies and sporadic tumours with NF1 mutations, we summarize current knowledge of NF1 disease, the NF1 gene and neurofibromin, neurofibromin signalling pathways and recent developments in NF1 therapeutics.
Asunto(s)
Genes Supresores de Tumor , Mutación , Neurofibromatosis 1/genética , Neurofibromina 1/genética , Proteínas Activadoras de GTPasa/genética , Genes ras/genética , Predisposición Genética a la Enfermedad , Humanos , Transducción de Señal/genéticaRESUMEN
Neurofibromatosis type 1 (NF1) patients develop benign neurofibromas and malignant peripheral nerve sheath tumors (MPNST). These incurable peripheral nerve tumors result from loss of NF1 tumor suppressor gene function, causing hyperactive Ras signaling. Activated Ras controls numerous downstream effectors, but specific pathways mediating the effects of hyperactive Ras in NF1 tumors are unknown. We performed cross-species transcriptome analyses of mouse and human neurofibromas and MPNSTs and identified global negative feedback of genes that regulate Ras/Raf/MEK/ERK signaling in both species. Nonetheless, ERK activation was sustained in mouse and human neurofibromas and MPNST. We used a highly selective pharmacological inhibitor of MEK, PD0325901, to test whether sustained Ras/Raf/MEK/ERK signaling contributes to neurofibroma growth in a neurofibromatosis mouse model (Nf1(fl/fl);Dhh-Cre) or in NF1 patient MPNST cell xenografts. PD0325901 treatment reduced aberrantly proliferating cells in neurofibroma and MPNST, prolonged survival of mice implanted with human MPNST cells, and shrank neurofibromas in more than 80% of mice tested. Our data demonstrate that deregulated Ras/ERK signaling is critical for the growth of NF1 peripheral nerve tumors and provide a strong rationale for testing MEK inhibitors in NF1 clinical trials.
Asunto(s)
Benzamidas/farmacología , Difenilamina/análogos & derivados , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Neurofibromatosis 1/tratamiento farmacológico , Neurofibromatosis 1/enzimología , Neoplasias del Sistema Nervioso Periférico/tratamiento farmacológico , Neoplasias del Sistema Nervioso Periférico/enzimología , Animales , Niño , Preescolar , Difenilamina/farmacología , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Mutantes , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Trasplante de Neoplasias , Neurofibromatosis 1/genética , Neurofibromatosis 1/patología , Proteína Oncogénica p21(ras)/genética , Proteína Oncogénica p21(ras)/metabolismo , Neoplasias del Sistema Nervioso Periférico/genética , Neoplasias del Sistema Nervioso Periférico/patología , Transcriptoma/efectos de los fármacos , Transcriptoma/genética , Trasplante Heterólogo , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasas raf/genética , Quinasas raf/metabolismoRESUMEN
PURPOSE: Patients with neurofibromatosis type 1 (NF1) develop malignant peripheral nerve sheath tumors (MPNST), which are often inoperable and do not respond well to current chemotherapies or radiation. The goal of this study was to use comprehensive gene expression analysis to identify novel therapeutic targets. EXPERIMENTAL DESIGN: Nerve Schwann cells and/or their precursors are the tumorigenic cell types in MPNST because of the loss of the NF1 gene, which encodes the RasGAP protein neurofibromin. Therefore, we created a transgenic mouse model, CNP-HRas12V, expressing constitutively active HRas in Schwann cells and defined a Ras-induced gene expression signature to drive a Bayesian factor regression model analysis of differentially expressed genes in mouse and human neurofibromas and MPNSTs. We tested functional significance of Aurora kinase overexpression in MPNST in vitro and in vivo using Aurora kinase short hairpin RNAs (shRNA) and compounds that inhibit Aurora kinase. RESULTS: We identified 2,000 genes with probability of linkage to nerve Ras signaling of which 339 were significantly differentially expressed in mouse and human NF1-related tumor samples relative to normal nerves, including Aurora kinase A (AURKA). AURKA was dramatically overexpressed and genomically amplified in MPNSTs but not neurofibromas. Aurora kinase shRNAs and Aurora kinase inhibitors blocked MPNST cell growth in vitro. Furthermore, an AURKA selective inhibitor, MLN8237, stabilized tumor volume and significantly increased survival of mice with MPNST xenografts. CONCLUSION: Integrative cross-species transcriptome analyses combined with preclinical testing has provided an effective method for identifying candidates for molecular-targeted therapeutics. Blocking Aurora kinases may be a viable treatment platform for MPNST.
Asunto(s)
Neoplasias de la Vaina del Nervio/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Transcriptoma , Animales , Aurora Quinasa A , Aurora Quinasas , Azepinas/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Análisis por Conglomerados , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Desnudos , Neoplasias de la Vaina del Nervio/metabolismo , Neoplasias de la Vaina del Nervio/terapia , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Pirimidinas/farmacología , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Plexiform neurofibromas (pNF) are Schwann cell tumors found in a third of individuals with neurofibromatosis type 1 (NF1). pNF can undergo transformation to malignant peripheral nerve sheath tumors (MPNST). There are no identified serum biomarkers of pNF tumor burden or transformation to MPNST. Serum biomarkers would be useful to verify NF1 diagnosis, monitor tumor burden, and/or detect transformation. EXPERIMENTAL DESIGN: We used microarray gene expression analysis to define 92 genes that encode putative secreted proteins in neurofibroma Schwann cells, neurofibromas, and MPNST. We validated differential expression by quantitative reverse transcription-PCR, Western blotting, and ELISA assays in cell conditioned medium and control and NF1 patient sera. RESULTS: Of 13 candidate genes evaluated, only adrenomedullin (ADM) was confirmed as differentially expressed and elevated in serum of NF1 patients. ADM protein concentrati on was further elevated in serum of a small sampling of NF1 patients with MPNST. MPNST cell conditioned medium, containing ADM and hepatocyte growth factor, stimulated MPNST migration and endothelial cell proliferation. CONCLUSIONS: Thus, microarray analysis identifies potential serum biomarkers for disease, and ADM is a serum biomarker of NF1. ADM serum levels do not seem to correlate with the presence of pNFs but may be a biomarker of transformation to MPNST.
Asunto(s)
Adrenomedulina/genética , Biomarcadores de Tumor/genética , Neurofibromatosis 1/genética , Adolescente , Adrenomedulina/biosíntesis , Adrenomedulina/sangre , Adulto , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/sangre , Movimiento Celular/genética , Niño , Preescolar , Medios de Cultivo , Células Endoteliales/citología , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Neurofibromatosis 1/sangre , Neurofibromatosis 1/metabolismo , Neurofibromatosis 1/patología , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reproducibilidad de los Resultados , Células de Schwann/patología , Adulto JovenRESUMEN
Understanding the biological pathways critical for common neurofibromatosis type 1 (NF1) peripheral nerve tumours is essential, as there is a lack of tumour biomarkers, prognostic factors and therapeutics. We used gene expression profiling to define transcriptional changes between primary normal Schwann cells (n = 10), NF1-derived primary benign neurofibroma Schwann cells (NFSCs) (n = 22), malignant peripheral nerve sheath tumour (MPNST) cell lines (n = 13), benign neurofibromas (NF) (n = 26) and MPNST (n = 6). Dermal and plexiform NFs were indistinguishable. A prominent theme in the analysis was aberrant differentiation. NFs repressed gene programs normally active in Schwann cell precursors and immature Schwann cells. MPNST signatures strongly differed; genes up-regulated in sarcomas were significantly enriched for genes activated in neural crest cells. We validated the differential expression of 82 genes including the neural crest transcription factor SOX9 and SOX9 predicted targets. SOX9 immunoreactivity was robust in NF and MPSNT tissue sections and targeting SOX9 - strongly expressed in NF1-related tumours - caused MPNST cell death. SOX9 is a biomarker of NF and MPNST, and possibly a therapeutic target in NF1.
Asunto(s)
Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Neurofibromatosis 1/genética , Neoplasias del Sistema Nervioso Periférico/genética , Factor de Transcripción SOX9/genética , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Células Cultivadas , Humanos , Neurofibromatosis 1/diagnóstico , Neoplasias del Sistema Nervioso Periférico/diagnóstico , Células de Schwann/metabolismo , Células de Schwann/patologíaRESUMEN
Malignant peripheral nerve sheath tumors (MPNSTs), driven in part by hyperactive Ras and epidermal growth factor receptor (EGFR) signaling, are often incurable. Testing of therapeutics for MPNST has been hampered by lack of adequate xenograft models. We previously documented that human MPNST cells are permissive for lytic infection by oncolytic herpes simplex viruses (oHSV). Herein we developed and characterized a xenograft model of human MPNST and evaluated the antitumor effects of oHSV mutants (G207 and hrR3) and the EGFR inhibitor, erlotinib. Additive cytotoxicity of these agents was found in human MPNST cell lines, suggesting that EGFR signaling is not critical for virus replication. Mice bearing human MPNST tumors treated with G207 or hrR3 by intraperitoneal or intratumoral injection showed tumor-selective virus biodistribution, virus replication, and reduced tumor burden. oHSV injection demonstrated more dramatic antitumor activity than erlotinib. Combination therapies showed a trend toward an increased antiproliferative effect. Both oHSV and erlotinib were antiangiogenic as measured by proangiogenic gene expression, effect on endothelial cells and xenograft vessel density. Overall, oHSVs showed highly potent antitumor effects against MPNST xenografts, an effect not diminished by EGFR inhibition. Our data suggest that inclusion of MPNSTs in clinical trials of oHSV is warranted.