RESUMEN
The Drosophila testis is a model organism stem cell niche in which two stem cell populations coordinate together to produce sperm; thus, these stem cells must be balanced in the niche. Merlin, a tumor-suppressor and human disease gene required for contact inhibition of proliferation, is known to limit the proliferation of the somatic cyst stem cells in the testis niche. Expanded encodes a protein that is structurally similar to Merlin in Drosophila, and is semi-redundant with Merlin in multiple tissues. We found that expanded depletion caused similar cyst lineage cell over-proliferation as observed with Merlin, and double mutants showed more severe phenotypes than either gene individually. Thus, these genes have partially redundant functions in the cyst lineage cells of this niche. We also expressed non-phosphorylatable constitutively "tumor suppressing" alleles of Merlin in cyst lineage cells, and surprisingly, we observed a similar cyst lineage over-proliferation phenotype. Merlin is known to impact multiple different signaling pathways to exert its effect on proliferation. We found that the Merlin loss of function phenotype was associated with an increase in MAPK/ERK signaling, consistent with Merlin's established role in transmembrane receptor inhibition. Constitutive Merlin displayed a reduction in both MAPK/ERK signaling and PI3K/Tor signaling. PI3K/Tor signaling is required for cyst cell differentiation, and inhibition of this pathway by Merlin activation phenocopied the Tor cyst lineage loss of function phenotype. Thus, Merlin impacts and integrates the activity of multiple signaling pathways in the testis niche. The ability of Merlin to dynamically change its activity via phosphorylation in response to local contact cues provides an intriguing mechanism whereby the signaling pathways that control these stem cells might be dynamically regulated in response to the division of a neighboring germ cell.
Asunto(s)
Células Madre Adultas/fisiología , Proliferación Celular/fisiología , Proteínas de Drosophila/fisiología , Drosophila/citología , Proteínas de la Membrana/fisiología , Neurofibromina 2/fisiología , Transducción de Señal , Testículo/citología , Animales , Linaje de la Célula , Drosophila/embriología , Proteínas de Drosophila/metabolismo , Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Masculino , Modelos Biológicos , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Péptidos de Invertebrados/metabolismo , Testículo/embriologíaRESUMEN
Uveal coloboma represents one of the most common congenital ocular malformations accounting for up to 10% of childhood blindness (~1 in 5000 live birth). Coloboma originates from defective fusion of the optic fissure (OF), a transient gap that forms during eye morphogenesis by asymmetric, ventral invagination. Genetic heterogeneity combined with the activity of developmentally regulated genes suggests multiple mechanisms regulating OF closure. The tumor suppressor and FERM domain protein Neurofibromin 2 (NF2) controls diverse processes in cancer, development and regeneration, via Hippo pathway and cytoskeleton regulation. In humans, NF2 mutations can cause ocular abnormalities, including coloboma, however, its actual role in OF closure is unknown. Using conditional inactivation in the embryonic mouse eye, our data indicate that loss of Nf2 function results in a novel underlying cause for coloboma. In particular, mutant eyes show substantially increased retinal pigmented epithelium (RPE) proliferation in the fissure region with concomitant acquisition of RPE cell fate. Cells lining the OF margin can maintain RPE fate ectopically and fail to transition from neuroepithelial to cuboidal shape. In the dorsal RPE of the optic cup, Nf2 inactivation leads to a robust increase in cell number, with local disorganization of the cytoskeleton components F-actin and pMLC2. We propose that RPE hyperproliferation is the primary cause for the observed defects causing insufficient alignment of the OF margins in Nf2 mutants and failure to fuse properly, resulting in persistent coloboma. Our findings indicate that limiting proliferation particularly in the RPE layer is a critical mechanism during OF closure.
Asunto(s)
Proliferación Celular , Coloboma/patología , Ojo/patología , Regulación del Desarrollo de la Expresión Génica , Neurofibromina 2/fisiología , Organogénesis , Epitelio Pigmentado de la Retina/patología , Animales , Coloboma/etiología , Coloboma/metabolismo , Ojo/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
BACKGROUND: In this review, we describe how the cytoskeletal protein Merlin, encoded by the Neurofibromin 2 (NF2) gene, orchestrates developmental signaling to ensure normal ontogeny, and we discuss how Merlin deficiency leads to aberrant activation of developmental pathways that enable tumor development and malignant progression. MAIN BODY: Parallels between embryonic development and cancer have underscored the activation of developmental signaling pathways. Hippo, WNT/ß-catenin, TGF-ß, receptor tyrosine kinase (RTK), Notch, and Hedgehog pathways are key players in normal developmental biology. Unrestrained activity or loss of activity of these pathways causes adverse effects in developing tissues manifesting as developmental syndromes. Interestingly, these detrimental events also impact differentiated and functional tissues. By promoting cell proliferation, migration, and stem-cell like phenotypes, deregulated activity of these pathways promotes carcinogenesis and cancer progression. The NF2 gene product, Merlin, is a tumor suppressor classically known for its ability to induce contact-dependent growth inhibition. Merlin plays a role in different stages of an organism development, ranging from embryonic to mature states. While homozygous deletion of Nf2 in murine embryos causes embryonic lethality, Merlin loss in adult tissue is implicated in Neurofibromatosis type 2 disorder and cancer. These manifestations, cumulatively, are reminiscent of dysregulated developmental signaling. CONCLUSION: Understanding the molecular and cellular repercussions of Merlin loss provides fundamental insights into the etiology of developmental disorders and cancer and has the potential, in the long term, to identify new therapeutic strategies. Video Abstract.
Asunto(s)
Desarrollo Embrionario , Neoplasias/metabolismo , Neurofibromina 2 , Animales , Humanos , Neurofibromina 2/deficiencia , Neurofibromina 2/fisiología , Transducción de SeñalRESUMEN
Neurofibromatosis type 2 (NF2) is an inherited disorder characterized by bilateral vestibular schwannomas (VS) that arise from neoplastic Schwann cells (SCs). NF2-associated VSs are often accompanied by meningioma (MN), and the majority of NF2 patients show loss of the NF2 tumor suppressor. mTORC1 and mTORC2-specific serum/glucocorticoid-regulated kinase 1 (SGK1) are constitutively activated in MN with loss of NF2. In a recent high-throughput kinome screen in NF2-null human arachnoidal and meningioma cells, we showed activation of EPH RTKs, c-KIT, and SFK members independent of mTORC1/2 activation. Subsequently, we demonstrated in vitro and in vivo efficacy of combination therapy with the dual mTORC1/2 inhibitor AZD2014 and the multi-kinase inhibitor dasatinib. For these reasons, we investigated activated mTORC1/2 and EPH receptor-mediated signaling in sporadic and NF2-associated VS. Using primary human VS cells and a mouse allograft model of schwannoma, we evaluated the dual mTORC1/2 inhibitor AZD2014 and the tyrosine kinase inhibitor dasatinib as monotherapies and in combination. Escalating dose-response experiments on primary VS cells grown from 15 human tumors show that combination therapy with AZD2014 and dasatinib is more effective at reducing metabolic activity than either drug alone and exhibits a therapeutic effect at a physiologically reasonable concentration (~0.1 µM). In vivo, while AZD2014 and dasatinib each inhibit tumor growth alone, the effect of combination therapy exceeds that of either drug. Co-targeting the mTOR and EPH receptor pathways with these or similar compounds may constitute a novel therapeutic strategy for VS, a condition for which there is no FDA-approved pharmacotherapy.
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Benzamidas/farmacología , Dasatinib/farmacología , Modelos Animales de Enfermedad , Morfolinas/farmacología , Neurofibromina 2/fisiología , Neuroma Acústico/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Quimioterapia Combinada , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neurofibromina 2/genética , Neurofibromina 2/metabolismo , Neuroma Acústico/metabolismo , Neuroma Acústico/patología , Receptor EphA1/metabolismoRESUMEN
Inactivation of the tumor suppressor NF2/merlin underlies neurofibromatosis type 2 (NF2) and some sporadic tumors. Previous studies have established that merlin mediates contact inhibition of proliferation; however, the exact mechanisms remain obscure and multiple pathways have been implicated. We have previously reported that merlin inhibits Ras and Rac activity during contact inhibition, but how merlin regulates Ras activity has remained elusive. Here we demonstrate that merlin can directly interact with both Ras and p120RasGAP (also named RasGAP). While merlin does not increase the catalytic activity of RasGAP, the interactions with Ras and RasGAP may fine-tune Ras signaling. In vivo, loss of RasGAP in Schwann cells, unlike the loss of merlin, failed to promote tumorigenic growth in an orthotopic model. Therefore, modulation of Ras signaling through RasGAP likely contributes to, but is not sufficient to account for, merlin's tumor suppressor activity. Our study provides new insight into the mechanisms of merlin-dependent Ras regulation and may have additional implications for merlin-dependent regulation of other small GTPases.
Asunto(s)
Neurofibromina 2/fisiología , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Animales , Células Cultivadas , Proteínas Activadoras de GTPasa/metabolismo , Genes Supresores de Tumor , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Neurofibromatosis 2/genética , Neurofibromatosis 2/metabolismo , Neurofibromina 2/metabolismo , Unión Proteica , Transducción de Señal/genéticaRESUMEN
Disruption of endometrial gland formation or function can cause female infertility. Formation of endometrial glands via tubulogenesis of luminal epithelial cells requires the establishment and maintenance of cell polarity and cell adhesion. The FERM domain-containing protein Merlin coordinates epithelial cell polarity and cell adhesion and is critical for epithelial tissue function in the skin and kidney. We now demonstrate a requirement for Merlin in endometrial gland development. Conditional deletion of Merlin in the endometrium results in female infertility caused by the absence of gland formation. Interestingly, we observed glandular epithelial markers within discrete groups of cells in the Merlin-deficient luminal epithelium. Wnt signaling, a pathway necessary for endometrial gland development is maintained in Merlin-deficient endometrium, suggesting the glandular fate program is active. Instead, we observe increased levels of apical actin and markers indicative of high membrane tension on the basal surface of the Merlin-deficient luminal epithelium. These findings suggest that the structural integrity of the luminal epithelium during gland formation is required for appropriate endometrial tubulogenesis and tissue function. Moreover, our work implicates Merlin-dependent regulation of mechanical tension in the proper formation of endometrial gland architecture and function.
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Endometrio/crecimiento & desarrollo , Neurofibromina 2/fisiología , Animales , Adhesión Celular/fisiología , Polaridad Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Endometrio/citología , Endometrio/metabolismo , Endometrio/patología , Células Epiteliales/metabolismo , Femenino , Hibridación in Situ , Infertilidad Femenina , Ratones , Ratones Endogámicos C57BL , Morfogénesis/fisiología , Neurofibromina 2/deficiencia , Neurofibromina 2/genética , Neurofibromina 2/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiologíaRESUMEN
The architectural and biochemical features of the plasma membrane are governed by its intimate association with the underlying cortical cytoskeleton. The neurofibromatosis type 2 (NF2) tumor suppressor merlin and closely related membrane:cytoskeleton-linking protein ezrin organize the membrane:cytoskeleton interface, a critical cellular compartment that both regulates and is regulated by growth factor receptors. An example of this poorly understood interrelationship is macropinocytosis, an ancient process of nutrient uptake and membrane remodeling that can both be triggered by growth factors and manage receptor availability. We show that merlin deficiency primes the membrane:cytoskeleton interface for epidermal growth factor (EGF)-induced macropinocytosis via a mechanism involving increased cortical ezrin, altered actomyosin, and stabilized cholesterol-rich membranes. These changes profoundly alter EGF receptor (EGFR) trafficking in merlin-deficient cells, favoring increased membrane levels of its heterodimerization partner, ErbB2; clathrin-independent internalization; and recycling. Our work suggests that, unlike Ras transformed cells, merlin-deficient cells do not depend on macropinocytic protein scavenging and instead exploit macropinocytosis for receptor recycling. Finally, we provide evidence that the macropinocytic proficiency of NF2-deficient cells can be used for therapeutic uptake. This work provides new insight into fundamental mechanisms of macropinocytic uptake and processing and suggests new ways to interfere with or exploit macropinocytosis in NF2 mutant and other tumors.
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Membrana Celular/metabolismo , Factor de Crecimiento Epidérmico/fisiología , Receptores ErbB/metabolismo , Neurofibromina 2/fisiología , Pinocitosis , Actomiosina/metabolismo , Animales , Células Cultivadas , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Humanos , Ratones , Neurofibromina 2/genética , Biosíntesis de ProteínasRESUMEN
The optic neuroepithelial continuum of vertebrate eye develops into three differentially growing compartments: the retina, the ciliary margin (CM), and the retinal pigment epithelium (RPE). Neurofibromin 2 (Nf2) is strongly expressed in slowly expanding RPE and CM compartments, and the loss of mouse Nf2 causes hyperplasia in these compartments, replicating the ocular abnormalities seen in human NF2 patients. The hyperplastic ocular phenotypes were largely suppressed by heterozygous deletion of Yap and Taz, key targets of the Nf2-Hippo signaling pathway. We also found that, in addition to feedback transcriptional regulation of Nf2 by Yap/Taz in the CM, activation of Nf2 expression by Mitf in the RPE and suppression by Sox2 in retinal progenitor cells are necessary for the differential growth of the corresponding cell populations. Together, our findings reveal that Nf2 is a key player that orchestrates the differential growth of optic neuroepithelial compartments during vertebrate eye development.
Asunto(s)
Cilios/fisiología , Hiperplasia/patología , Células-Madre Neurales/citología , Neurofibromina 2/fisiología , Organogénesis/fisiología , Epitelio Pigmentado de la Retina/citología , Aciltransferasas , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Ciclo Celular , Linaje de la Célula , Polaridad Celular , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica , Vía de Señalización Hippo , Humanos , Hiperplasia/metabolismo , Ratones , Ratones Noqueados , Células-Madre Neurales/fisiología , Fenotipo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Epitelio Pigmentado de la Retina/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Señalizadoras YAPRESUMEN
Neurofibromatosis type 2 (NF2) is a dominantly inherited autosomal disease characterized by schwannomas of the 8th cranial nerve. The NF2 tumor suppressor gene encodes for Merlin, a protein implicated as a suppressor of multiple cellular signaling pathways. To identify potential drug targets in NF2-associated malignancies we assessed the consequences of inhibiting the tyrosine kinase receptor MET. We identified crizotinib, a MET and ALK inhibitor, as a potent inhibitor of NF2-null Schwann cell proliferation in vitro and tumor growth in vivo. To identify the target/s of crizotnib we employed activity-based protein profiling (ABPP), leading to identification of FAK1 (PTK2) as the relevant target of crizotinib inhibition in NF2-null schwannoma cells. Subsequent studies confirm that inhibition of FAK1 is sufficient to suppress tumorigenesis in animal models of NF2 and that crizotinib-resistant forms of FAK1 can rescue the effects of treatment. These studies identify a FDA approved drug as a potential treatment for NF2 and delineate the mechanism of action in NF2-null Schwann cells.
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Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Neurilemoma/tratamiento farmacológico , Neurofibromina 2/fisiología , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Piridinas/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Crizotinib , Humanos , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The NF2 gene product Merlin is a FERM-domain protein possessing a broad tumor-suppressing function. NF2/Merlin has been implicated in regulating multiple signaling pathways critical for cell growth and survival. However, it remains unknown whether NF2/Merlin regulates Wnt/ß-catenin signaling during vertebrate embryogenesis. Here we demonstrate that NF2/Merlin is required for body pattern formation in the Xenopus laevis embryo. Depletion of the maternal NF2/Merlin enhances organizer gene expression dependent on the presence of ß-catenin, and causes dorsanteriorized development; Morpholino antisense oligo-mediated knockdown of the zygotic NF2/Merlin shifts posterior genes anteriorwards and reduces the anterior development. We further demonstrate that targeted depletion of NF2 in the presumptive dorsal tissues increases the levels of nuclear ß-catenin in the neural epithelial cells. Biochemical analyses reveal that NF2 depletion promotes the production of active ß-catenin and concurrently decreases the level of N-terminally phosphorylated ß-catenin under the stimulation of the endogenous Wnt signaling. Our findings suggest that NF2/Merlin negatively regulates the Wnt/ß-catenin signaling activity during the pattern formation in early X. laevis embryos.
Asunto(s)
Neurofibromina 2/fisiología , Proteínas de Xenopus/fisiología , Xenopus laevis/embriología , Xenopus laevis/fisiología , Animales , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Genes de la Neurofibromatosis 2 , Hibridación in Situ , Masculino , Neurofibromina 2/deficiencia , Neurofibromina 2/genética , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/genética , Oocitos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Vía de Señalización Wnt , Proteínas de Xenopus/deficiencia , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Cigoto/metabolismo , beta Catenina/metabolismoRESUMEN
The NF2 gene product Merlin is a protein containing ezrin, radixin, and moesin domains; it is a member of the 4.1 protein superfamily associated with the membrane cytoskeleton and also interacts with cell surface molecules. The mammalian Hippo cascade, a downstream signaling cascade of merlin, inactivates the Yes-associated protein (YAP). Yes-associated protein is activated by loss of the NF2 gene and functions as an oncogene in meningioma cells; however, the factors controlling YAP expression, phosphorylation, and subcellular localization in meningiomas have not been fully elucidated. Here, we demonstrate that merlin expression is heterogeneous in 1 NF2 gene-negative and 3 NF2 gene-positive World Health Organization grade I meningiomas. In the NF2 gene-positive meningiomas, regions with low levels of merlin (tumor rims) had greater numbers of cells with nuclear YAP versus regions with high merlin levels (tumor cores). Merlin expression and YAP phosphorylation were also affected by cell density in the IOMM-Lee and HKBMM human meningioma cell lines; nuclear localization of YAP was regulated by cell density and extracellular matrix (ECM) stiffness in IOMM-Lee cells. These results suggest that cell density and ECM stiffness may contribute to the heterogeneous loss of merlin and increased nuclear YAP expression in human meningiomas.
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Regulación Neoplásica de la Expresión Génica/fisiología , Neoplasias Meníngeas/metabolismo , Meningioma/metabolismo , Neurofibromatosis 2/metabolismo , Neurofibromina 2/fisiología , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Recuento de Células , Proteínas de Ciclo Celular , Línea Celular Tumoral , Humanos , Antígeno Ki-67/metabolismo , Neoplasias Meníngeas/patología , Meningioma/patología , Proteínas Nucleares/genética , Factores de Transcripción/genéticaRESUMEN
UNLABELLED: The intercellular adhesion molecule 1 (ICAM-1) is induced in mouse liver after bile duct ligation (BDL) and plays a key role in neutrophil-mediated liver injury in BDL mice. ICAM-1 has been shown to interact with cytoskeletal ezrin-radixin-moesin (ERM) proteins that also interact with the PDZ protein, Na(+) /H(+) exchanger regulatory factor 1 (NHERF-1/EBP50). In NHERF-1(-/-) mice, ERM proteins are significantly reduced in brush-border membranes from kidney and small intestine. ERM knockdown reduces ICAM-1 expression in response to tumor necrosis factor alpha. Here we show that NHERF-1 assembles ERM proteins, ICAM-1 and F-actin into a macromolecule complex that is increased in mouse liver after BDL. Compared to wild-type (WT) mice, both sham-operated and BDL NHERF-1(-/-) mice have lower levels of activated ERM and ICAM-1 protein in the liver accompanied by significantly reduced hepatic neutrophil accumulation, serum alanine aminotransferase, and attenuated liver injury after BDL. However, total bile acid concentrations in serum and liver of sham and BDL NHERF-1(-/-) mice were not significantly different from WT controls, although hepatic tetrahydroxylated bile acids and Cyp3a11 messenger RNA levels were higher in NHERF-1(-/-) BDL mice. CONCLUSION: NHERF-1 participates in the inflammatory response that is associated with BDL-induced liver injury. Deletion of NHERF-1 in mice leads to disruption of the formation of ICAM-1/ERM/NHERF-1 complex and reduction of hepatic ERM proteins and ICAM-1, molecules that are up-regulated and are essential for neutrophil-mediated liver injury in cholestasis. Further study of the role of NHERF-1 in the inflammatory response in cholestasis and other forms of liver injury should lead to discovery of new therapeutic targets in hepatic inflammatory diseases.
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Colestasis Intrahepática/etiología , Molécula 1 de Adhesión Intercelular/fisiología , Hepatopatías/etiología , Fosfoproteínas/fisiología , Intercambiadores de Sodio-Hidrógeno/fisiología , Animales , Hepatitis/etiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neurofibromina 2/fisiología , Fosfoproteínas/genética , Intercambiadores de Sodio-Hidrógeno/genéticaAsunto(s)
Complejos Multiproteicos/antagonistas & inhibidores , Neurilemoma/prevención & control , Neurofibromatosis 2/prevención & control , Neurofibromina 2/fisiología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Humanos , Diana Mecanicista del Complejo 1 de la RapamicinaRESUMEN
BACKGROUND: Neurofibromatosis type 2 (NF2) is a rare autosomal dominant genetic disorder, resulting in a variety of neural tumors, with bilateral vestibular schwannomas as the most frequent manifestation. Recently, merlin, the NF2 tumor suppressor, has been identified as a novel negative regulator of mammalian target of rapamycin complex 1 (mTORC1); functional loss of merlin was shown to result in elevated mTORC1 signaling in NF2-related tumors. Thus, mTORC1 pathway inhibition may be a useful targeted therapeutic approach. METHODS: We studied in vitro cell models, cohorts of mice allografted with Nf2(-/-) Schwann cells, and a genetically modified mouse model of NF2 schwannoma in order to evaluate the efficacy of the proposed targeted therapy for NF2. RESULTS: We found that treatment with the mTORC1 inhibitor rapamycin reduced the severity of NF2-related Schwann cell tumorigenesis without significant toxicity. Consistent with these results, in an NF2 patient with growing vestibular schwannomas, the rapalog sirolimus induced tumor growth arrest. CONCLUSIONS: Taken together, these results constitute definitive evidence that justifies proceeding with clinical trials using mTORC1-targeted agents in selected patients with NF2 and in patients with NF2-related sporadic tumors.
Asunto(s)
Complejos Multiproteicos/antagonistas & inhibidores , Neurilemoma/prevención & control , Neurofibromatosis 2/prevención & control , Neurofibromina 2/fisiología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Proliferación Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Humanos , Técnicas para Inmunoenzimas , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Desnudos , Ratones Transgénicos , Complejos Multiproteicos/metabolismo , Neurilemoma/metabolismo , Neurilemoma/patología , Neurofibromatosis 2/metabolismo , Neurofibromatosis 2/patología , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Células Tumorales CultivadasRESUMEN
Axonal surface proteins encompass a group of heterogeneous molecules, which exert a variety of different functions in the highly interdependent relationship between axons and Schwann cells. We recently revealed that the tumour suppressor protein merlin, mutated in the hereditary tumour syndrome neurofibromatosis type 2, impacts significantly on axon structure maintenance in the peripheral nervous system. We now report on a role of neuronal merlin in the regulation of the axonal surface protein neuregulin 1 important for modulating Schwann cell differentiation and myelination. Specifically, neuregulin 1 type III expression is reduced in sciatic nerve tissue of neuron-specific knockout animals as well as in biopsies from seven patients with neurofibromatosis type 2. In vitro experiments performed on both the P19 neuronal cell line and primary dorsal root ganglion cells demonstrate the influence of merlin on neuregulin 1 type III expression. Moreover, expression of ERBB2, a Schwann cell receptor for neuregulin 1 ligands is increased in nerve tissue of both neuron-specific merlin knockout animals and patients with neurofibromatosis type 2, demonstrating for the first time that axonal merlin indirectly regulates Schwann cell behaviour. Collectively, we have identified that neuronally expressed merlin can influence Schwann cell activity in a cell-extrinsic manner.
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Neurregulina-1/fisiología , Neurofibromina 2/fisiología , Neuronas/fisiología , Receptor ErbB-2/biosíntesis , Células de Schwann/metabolismo , Transducción de Señal/fisiología , Adulto , Anciano , Animales , Línea Celular , Células Cultivadas , Femenino , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Persona de Mediana Edad , Neurofibromatosis 2/metabolismo , Neurofibromatosis 2/patología , Neuronas/patología , Células de Schwann/patologíaRESUMEN
Cancer-associated mutations in oncogene products and tumor suppressors contributing to tumor progression manifest themselves, at least in part, by deregulating microtubule-dependent cellular processes that play important roles in many cell biological pathways, including intracellular transport, cell architecture, and primary cilium and mitotic spindle organization. An essential characteristic of microtubules in the performance of these varied cell processes is their ability to continuously remodel, a phenomenon known as dynamic instability. It is therefore conceivable that part of the normal function of certain cancer-causing genes is to regulate microtubule dynamic instability. Here, we report the results of a high-resolution live-cell image-based RNA interference screen targeting a collection of 70 human tumor suppressor genes to uncover cancer genes affecting microtubule dynamic instability. Extraction and computational analysis of microtubule dynamics from EB3-GFP time-lapse image sequences identified the products of the tumor suppressor genes NF1 and NF2 as potent microtubule-stabilizing proteins. Further in-depth characterization of NF2 revealed that it binds to and stabilizes microtubules through attenuation of tubulin turnover by lowering both rates of microtubule polymerization and depolymerization as well as by reducing the frequency of microtubule catastrophes. The latter function appears to be mediated, in part, by inhibition of hydrolysis of tubulin-bound GTP on the growing microtubule plus end.
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Microtúbulos/metabolismo , Neoplasias/metabolismo , Neurofibromina 2/metabolismo , Genes Supresores de Tumor , Humanos , Microtúbulos/genética , Microtúbulos/fisiología , Neoplasias/genética , Neurofibromina 2/genética , Neurofibromina 2/fisiología , Transducción de SeñalRESUMEN
The autosomal dominant disorder neurofibromatosis type 2 (NF2) is a hereditary tumor syndrome caused by inactivation of the NF2 tumor suppressor gene, encoding merlin. Apart from tumors affecting the peripheral and central nervous systems, most NF2 patients develop peripheral neuropathies. This peripheral nerve disease can occur in the absence of nerve-damaging tumors, suggesting an etiology that is independent of gross tumor burden. We discovered that merlin isoform 2 (merlin-iso2) has a specific function in maintaining axonal integrity and propose that reduced axonal NF2 gene dosage leads to NF2-associated polyneuropathy. We identified a merlin-iso2-dependent complex that promotes activation of the GTPase RhoA, enabling downstream Rho-associated kinase to promote neurofilament heavy chain phosphorylation. Merlin-iso2-deficient mice exhibited impaired locomotor capacities, delayed sensory reactions and electrophysiological signs of axonal neuropathy. Sciatic nerves from these mice and sural nerve biopsies from NF2 patients revealed reduced phosphorylation of the neurofilament H subunit, decreased interfilament spacings and irregularly shaped axons.
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Neurofibromatosis 2/metabolismo , Neurofibromina 2/fisiología , Polineuropatías/metabolismo , Adulto , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Línea Celular Tumoral , Células Cultivadas , Femenino , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Datos de Secuencia Molecular , Neurofibromatosis 2/genética , Neurofibromatosis 2/patología , Neurofibromina 2/genética , Fosforilación/fisiología , Polineuropatías/genética , Polineuropatías/patología , Embarazo , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologíaRESUMEN
Neurofibromatosis type 2 (NF2), characterized by tumors of the nervous system, is a result of functional loss of the NF2 gene. The NF2 gene encodes Merlin (moesin-ezrin-radixin-like protein), an ERM (Ezrin, Radixin, Moesin) protein family member. Merlin functions as a tumor suppressor through impacting mechanisms related to proliferation, apoptosis, survival, motility, adhesion, and invasion. Several studies have summarized the tumor intrinsic mutations in Merlin. Given the fact that tumor cells are not in isolation, but rather in an intricate, mutually sustaining synergy with their surrounding stroma, the dialog between the tumor cells and the stroma can potentially impact the molecular homeostasis and promote evolution of the malignant phenotype. This review summarizes the epigenetic modifications, transcript stability, and post-translational modifications that impact Merlin. We have reviewed the role of extrinsic factors originating from the tumor milieu that influence the availability of Merlin inside the cell. Information regarding Merlin regulation could lead to novel therapeutics by stabilizing Merlin protein in tumors that have reduced Merlin protein expression without displaying any NF2 genetic alterations.
Asunto(s)
Neurofibromina 2/fisiología , Proteínas Supresoras de Tumor/fisiología , Animales , Epigénesis Genética , Humanos , Neurofibromina 2/química , Proteínas Proto-Oncogénicas c-akt/fisiología , Estabilidad del ARNRESUMEN
Recent evidence suggests that the neurofibromatosis type 2 (NF2) gene encoded protein merlin suppresses mitogenic signalling not only at the cell membrane but also in the nucleus. At the membrane, merlin inhibits signalling by integrins and tyrosine receptor kinases (RTKs) and the activation of downstream pathways, including the Ras/Raf/MEK/ERK, FAK/Src, PI3K/AKT, Rac/PAK/JNK, mTORC1, and Wnt/ß-catenin pathways. In the nucleus, merlin suppresses the E3 ubiquitin ligase CRL4(DCAF1) to inhibit proliferation. Gene expression analysis suggested that CRL4(DCAF1) could also regulate the expression of integrins and RTKs. In this review, we explore the links between merlin function at the membrane and in the nucleus, and discuss the potential of targeting the master regulator CRL4 (DCAF1) to treat NF2 and other merlin-deficient tumours.
Asunto(s)
Núcleo Celular/metabolismo , Neurofibromina 2/fisiología , Animales , Membrana Celular/metabolismo , Núcleo Celular/enzimología , Perfilación de la Expresión Génica , Humanos , Integrinas/antagonistas & inhibidores , Integrinas/metabolismo , Ratones , Neurofibromina 2/metabolismo , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal/fisiología , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Integrin-mediated activation of PAK (p21-activated kinase) causes phosphorylation and inactivation of the FERM (4.1, ezrin, radixin, moesin) domain-containing protein Merlin, which is encoded by the NF2 (neurofibromatosis type 2) tumor suppressor gene. Conversely, cadherin engagement inactivates PAK, thus leading to accumulation of unphosphorylated Merlin. Current models imply that Merlin inhibits cell proliferation by inhibiting mitogenic signaling at or near the plasma membrane. We have recently shown that the unphosphorylated, growth-inhibiting form of Merlin accumulates in the nucleus and binds to the E3 ubiquitin ligase CRL4(DCAF1) to suppress its activity. Depletion of DCAF1 blocks the hyperproliferation caused by inactivation of Merlin. Conversely, expression of a Merlin-insensitive DCAF1 mutant counteracts the antimitogenic effect of Merlin. Expression of Merlin or silencing of DCAF1 in Nf2-deficient cells induce an overlapping, tumor-suppressive program of gene expression. Mutations present in some tumors from NF2 patients disrupt Merlin's ability to interact with or inhibit CRL4(DCAF1). Lastly, depletion of DCAF1 inhibits the hyperproliferation of Schwannoma cells isolated from NF2 patients and suppresses the oncogenic potential of Merlin-deficient tumor cell lines. Current studies are aimed at identifying the substrates and mechanism of action of CRL4(DCAF1) and examining its role in NF2-dependent tumorigenesis in mouse models. We propose that Merlin mediates contact inhibition and suppresses tumorigenesis by translocating to the nucleus to inhibit CRL4(DCAF1).