Neurofibromatosis 2 Protein Research Articles

Vascular hyperpermeability as a hallmark of phacomatoses: is the etiology angiogenesis comparable with mechanisms seen in inflammatory pathways? Part I: historical observations and clinical perspectives on the etiology of increased CSF protein levels, CSF clotting, and communicating hydrocephalus: a comprehensive review.

Phacomatoses are a special group of familial hamartomatous syndromes with unique neuro-cutaneous manifestations as well as disease characteristic tumors. Neurofibromatosis 2 (NF2) and tuberous sclerosis complex (TSC) are representatives of this family. Vestibular schwannoma (VS) and subependymal giant cell tumor (SGCT) are two of the most common intracranial tumors associated with NF2 and TSC, respectively. These tumors can present with obstructive hydrocephalus due to their location adjacent to or in the ventricles. However, both tumors are also known to have a unique association with an elevated protein concentration in the cerebrospinal fluid (CSF), sometimes in association with non-obstructive (communicating) hydrocephalus (HCP), the causality of which has been unclear. Furthermore, SGCTs have repeatedly been shown to have a predisposition for CSF clotting, causing debilitating obstructions and recurrent malfunctions in shunted patients. However, the exact relation between high protein levels and spontaneous clotting of the CSF is not clear, nor is the mechanism understood by which CSF may clot in SGCTs. Elevated protein levels in the CSF are thought to be caused by increased vascular permeability and dysregulation of the blood-brain barrier. The two presumed underlying pathophysiologic mechanisms for that, in the context of tumorigenesis, are angiogenesis and inflammation. Both mechanisms are correlated to the Pi3K/Akt/mTOR pathway which is a major tumorigenesis pathway in nearly all phacomatoses. In this review, we discuss the influence of angiogenesis and inflammation on vascular permeability in VSs and SGCTs at the phenotypic level as well as their possible genetic and molecular determinants. Part I describes the historical perspectives and clinical aspects of the relationship between vascular permeability, abnormal CSF protein levels, clotting of the CSF, and communicating HCP. Part II describes different cellular and molecular pathways involved in angiogenesis and inflammation in these two tumors and the correlation between inflammation and coagulation. Interestingly, while increased angiogenesis can be observed in both VS and SGCT, inflammatory processes seem more prominent in SGCT. Both pathologies are characterized by different subgroups of tumor-associated macrophages (TAM): the pro-inflammatory, M1 type is predominating in SGCTs while pro-angiogenetic, M2 type is predominating in VSs. We suggest that lack of NF2 protein in VS and lack of TSC1/2 proteins in SGCT determine this fundamental difference between the two tumor types, by defining the predominant TAM type. Since inflammatory reactions and coagulation processes are tightly connected, a "pro-inflammatory state" of SGCT can be used to explain the observed associated enhanced CSF clotting process. These distinct cellular and molecular differences may have direct therapeutic implications on tumors that are unique to certain phacomatoses or those with similar genetics.

Regulation of mixed lineage kinase 3 is required for Neurofibromatosis-2-mediated growth suppression in human cancer

The Neurofibromatosis-2 (NF2) tumor suppressor merlin negatively regulates cell proliferation in numerous cell types. We have previously shown that the NF2 protein (merlin/schwannomin) associates with mixed lineage kinase 3 (MLK3), a mitogen-activated protein kinase (MAPK) kinase kinase that is required for the proliferation of normal and neoplastic cells. In the current study, we show that merlin inhibits MLK3 activity as well as the activation of its downstream effectors, B-Raf, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). The ability of merlin to regulate MLK3 activity requires a direct association between MLK3 and residues in the C-terminal region of merlin. Merlin integrates Rho GTPase family signaling with MAPK activity by inhibiting the binding between MLK3 and its upstream activator, Cdc42. Furthermore, we demonstrate that MLK3 is required for merlin suppression of cell proliferation and invasion. Collectively, these results establish merlin as a potent inhibitor of MLK3, ERK and JNK activation in cancer, and provide a mechanistic link between deregulated MAPK and Rho GTPase signaling in NF2 growth control.

NF2/Merlin Is a Novel Negative Regulator of mTOR Complex 1, and Activation of mTORC1 Is Associated with Meningioma and Schwannoma Growth

Inactivating mutations of the neurofibromatosis 2 (NF2) gene, NF2, result predominantly in benign neurological tumors, schwannomas and meningiomas, in humans; however, mutations in murine Nf2 lead to a broad spectrum of cancerous tumors. The tumor-suppressive function of the NF2 protein, merlin, a membrane-cytoskeleton linker, remains unclear. Here, we identify the mammalian target of rapamycin complex 1 (mTORC1) as a novel mediator of merlin's tumor suppressor activity. Merlin-deficient human meningioma cells and merlin knockdown arachnoidal cells, the nonneoplastic cell counterparts of meningiomas, exhibit rapamycin-sensitive constitutive mTORC1 activation and increased growth. NF2 patient tumors and Nf2-deficient mouse embryonic fibroblasts demonstrate elevated mTORC1 signaling. Conversely, the exogenous expression of wild-type merlin isoforms, but not a patient-derived L64P mutant, suppresses mTORC1 signaling. Merlin does not regulate mTORC1 via the established mechanism of phosphoinositide 3-kinase-Akt or mitogen-activated protein kinase/extracellular signal-regulated kinase-mediated TSC2 inactivation and may instead regulate TSC/mTOR signaling in a novel fashion. In conclusion, the deregulation of mTORC1 activation underlies the aberrant growth and proliferation of NF2-associated tumors and may restrain the growth of these lesions through negative feedback mechanisms, suggesting that rapamycin in combination with phosphoinositide 3-kinase inhibitors may be therapeutic for NF2.

GASTRIC SCHWANNOMA WITH ADJACENT EXTERNAL PROGRESSION HARBORED ABERRANT NF2 GENE

Gastric schwannomas are rare benign mesenchymal tumors. We describe a schwannoma of gastric origin with adjacent external progression. Sections showed a spindle cell tumor arranged in interlaced bundles and fascicles that was S-100 and CD34 positive but c-KIT protein negative. Histology and immunohistochemistry revealed the typical appearance of a gastric schwannoma. Genetic evaluation revealed that the tumor harbored a point mutation in exon 6 of the tumor suppressor neurofibromatosis 2 (NF2) gene, which resulted in an amino acid substitution of NF2 protein, and no mutation in exon 4b of the NF1 gene. In conclusion, we identified a rare mutation of the NF2 gene in gastric schwannoma. A diagnosis can only be definitive when based on histological and immunohistochemical findings. Digestive tract schwannomas are rare mesenchymal tumors that are differentiated from gastrointestinal stromal tumors by the absence of KIT protein. Follow up suggested that complete resection is an effective long-term treatment strategy.

Expression of the Tumor Suppressor Genes NF2, 4.1B, and TSLC1 in Canine Meningiomas

Meningiomas are common primary brain tumors in dogs; however, little is known about the molecular genetic mechanisms involved in their tumorigenesis. Several tumor suppressor genes have been implicated in meningioma pathogenesis in humans, including the neurofibromatosis 2 (NF2), protein 4.1B (4.1 B), and tumor suppressor in lung cancer-1 (TSLC1) genes. We investigated the expression of these tumor suppressor genes in a series of spontaneous canine meningiomas using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) (NF2; n = 25) and western blotting (NF2/merlin, 4.1B, TSLC1; n = 30). Decreased expression of 4.1B and TSLC1 expression on western blotting was seen in 6/30 (20%) and in 15/30 (50%) tumors, respectively, with 18/30 (60%) of meningiomas having decreased or absent expression of one or both proteins. NF2 gene expression assessed by western blotting and RT-PCR varied considerably between individual tumors. Complete loss of NF2 protein on western blotting was not seen, unlike 4.1B and TSLC1. Incidence of TSLC1 abnormalities was similar to that seen in human meningiomas, while perturbation of NF2 and 4.1B appeared to be less common than reported for human tumors. No association was observed between tumor grade, subtype, or location and tumor suppressor gene expression based on western blot or RT-PCR. These results suggest that loss of these tumor suppressor genes is a frequent occurrence in canine meningiomas and may be an early event in tumorigenesis in some cases. In addition, it is likely that other, as yet unidentified, genes play an important role in canine meningioma formation and growth.

The distribution of constitutional and somatic mutations in the neurofibromatosis 2 gene

Constitutional heterozygous inactivating mutations in the neurofibromatosis 2 (NF2) tumor suppressor gene cause the autosomal dominant disease NF2, and biallelic inactivating somatic NF2 mutations are found in a high proportion of unilateral sporadic vestibular schwannoma (USVS) and sporadic meningioma. We surveyed the distributions of constitutional NF2 mutations in 823 NF2 families, 278 somatic NF2 mutations in USVS, and 208 somatic NF2 mutations in sporadic meningioma. Based on the available NF2 mutation data, the most dominant influence on the spectra of mutations in exons 1-15 are C>T transitions that change arginine codons (CGA) to stop codons (TGA) due to spontaneous deamination of methylcytosine to thymine in CpG dinucleotides. The paucity of reported mutations in exon 9 and the absence of reported mutations in exons 16 and 17 may be related to structure-function relationships in the NF2 protein.

Characterization of the NF2 protein merlin and the ERM protein ezrin in human, rat, and mouse central nervous system

The neurofibromatosis 2 (NF2) protein, merlin, is structurally related to the ERM (ezrin-radixin-moesin) protein family of membrane-cytoskeleton linkers and is mutated in nervous system tumors. Apart from tumor suppressor activity, merlin's functions are poorly understood. We compared the localization and expression of merlin and ezrin in developing and adult brain and in brain-derived progenitor cells. Both proteins were widely but differentially expressed in human, rat, and mouse brain. In brain tissue and neuronal progenitor cell cultures merlin was predominantly found in neurons while ezrin was expressed in astrocytes. Merlin expression was seen from E11 in mouse embryos, whereas ezrin was present earlier. Both proteins were expressed in embryonic mouse neurospheres, where ezrin was specifically localized in filopodia of adherent neuronal progenitor cells. Subcellular analysis demonstrated ezrin in fine filopodial structures in astrocytes, while merlin was detected in neuronal synaptic junctions. The widespread expression of merlin in brain and its association with protein kinase A suggest a role for merlin in brain biology.

Neurofibromatosis 2 and malignant mesothelioma

Mutations of the neurofibromatosis 2 (NF2) tumor suppressor gene cause the inherited disorder NF2 and are also common in malignant mesothelioma, which is not a characteristic feature of NF2. The authors report an asbestos-exposed person with NF2 and malignant mesothelioma. Immunohistochemical analysis of the mesothelioma confirmed loss of expression of the NF2 protein, and comparative genomic hybridization revealed losses of chromosomes 14, 15, and 22, and gain of 7. The authors propose that a person with a constitutional mutation of an NF2 allele is more susceptible to mesothelioma.

Loss of heterozygosity for the NF2 gene in retinal and optic nerve lesions of patients with neurofibromatosis 2.

Individuals affected with the neurofibromatosis 2 (NF2) cancer predisposition syndrome develop specific ocular lesions. To determine whether these lesions result from altered NF2 gene expression, microdissection and PCR were used to investigate 40 ocular lesions from seven eyes of four NF2 patients for LOH, with markers that flank the NF2 gene on chromosome 22q. NF2 protein (merlin) expression was also evaluated in these lesions, using immunohistochemistry. Retinal hamartoma was observed in all seven eyes, including one with combined pigment epithelial and retinal hamartoma (CPERH). Retinal tufts were present in four eyes (three patients), retinal dysplasia in two eyes (two patients), optic nerve neurofibroma in one eye, iris naevoid hyperplasia in two eyes (two patients) and pseudophakia in all eyes. Markers were informative in three patients (six eyes from three unrelated families). One patient was non-informative due to prolonged decalcification. All retinal and optic nerve, but not iris lesions, demonstrated consistent LOH for the NF2 gene. Merlin was not expressed in the retina, optic nerve, or iris lesions. These results suggest that inactivation of the NF2 gene is associated with the formation of a variety of retinal and optic nerve lesions in NF2 patients.

Merlin: hanging tumor suppression on the Rac

Cancer can result from any number of abnormalities in the control of cell-cycle progression, intracellular signaling and transduction of extracellular cues. Many insights into the crucial events that govern the regulation of cell growth have derived from studies of the gene products mutated in inherited cancer syndromes. Recent work on the neurofibromatosis 2 (NF2) tumor suppressor gene suggests that this negative growth regulator might function by modulating growth factor and extracellular matrix (ECM) signals that trigger Rac1-dependent cytoskeletonassociated processes. In this article, we propose a molecular model for NF2 protein (merlin) function in the light of these and related new findings. Cancer can result from any number of abnormalities in the control of cell-cycle progression, intracellular signaling and transduction of extracellular cues. Many insights into the crucial events that govern the regulation of cell growth have derived from studies of the gene products mutated in inherited cancer syndromes. Recent work on the neurofibromatosis 2 (NF2) tumor suppressor gene suggests that this negative growth regulator might function by modulating growth factor and extracellular matrix (ECM) signals that trigger Rac1-dependent cytoskeletonassociated processes. In this article, we propose a molecular model for NF2 protein (merlin) function in the light of these and related new findings.

The Protein 4.1 Tumor Suppressor, DAL-1, Impairs Cell Motility, But Regulates Proliferation in a Cell-Type-Specific Fashion

The neurofibromatosis 2 (NF2) tumor suppressor belongs to the Protein 4.1 family of molecules that link the actin cytoskeleton to cell surface glycoproteins. We have previously demonstrated that the NF2 protein, merlin, can suppress cell growth in vitro and in vivo as well as impair actin cytoskeleton-associated processes, such as cell spreading, attachment, and motility. Recently, we determined that expression of a second Protein 4.1 tumor suppressor, DAL-1, was lost in 60% of sporadic meningiomas, but not schwannomas. In this report, we demonstrate that DAL-1 suppresses cell proliferation in meningioma, but not schwannoma cells. Similar to merlin, DAL-1 interacts with other ERM proteins and βII-spectrin, but not the merlin interactor protein, SCHIP-1. In addition, we report the identification of the full-length DAL-1 tumor suppressor, termed KIAA0987. Collectively, these results suggest that the two Protein 4.1 meningioma tumor suppressors, merlin and DAL-1, may be functionally distinct proteins with different mechanisms of action.

Novel alternatively spliced isoforms of the neurofibromatosis type 2 tumor suppressor are targeted to the nucleus and cytoplasmic granules.

We cloned novel splice variants Mer150, Mer151 and Mer162 of the neurofibromatosis 2 (NF2) tumor suppressor, which demonstrate a tissue-specific and development-specific expression pattern. Isoform Mer150 is created by cryptic splicing from exon 8 to 14 and represents an N-terminal truncation of 259 residues. Mer151 is characterized by in-frame splicing out of several exons and a modified C-terminus due to a frameshift in exons 13+14 and premature termination. Mer162 represents a head-to-tail isoform resulting from in-frame skipping of exons 5-16. As a common feature, the alpha-helical domain and a variable proportion of the ERM homology domain are spliced out in these isoforms. To investigate differences in subcellular localization, we expressed epitope-tagged cDNA constructs of the wild-type NF2 as well as of the three alternatively spliced transcripts in NIH 3T3 cells by nuclear microinjection or lipid-mediated transfection. Subcellular localization of Mer151 in filopodia and ruffling membranes was similar to the wild-type NF2. Mer151, however, was targeted to the nucleus, which was not observed for wild-type NF2, Mer150 or Mer162. A putative nuclear localization signal created by alternative splicing was identified in Mer151. In contrast to Mer151, Mer150 and Mer162 were not found in regions of the plasma membrane, but localized to a granular intracellular compartment. The results suggest that the recently described actin-binding domain in exon 10, but not the presence or absence of exons 2+3, is relevant for subcellular targeting. Although the NF2 protein is known as a cytoskeletal linker, additional functions in a cytoplasmic compartment and in the nucleus may exist.

Heterogeneity of mesothelioma cell lines as defined by altered genomic structure and expression of the NF2 gene.

Germ-line mutations in the neurofibromatosis 2 (NF2) gene cause a susceptibility to the development of schwannoma and meningioma, 2 mostly benign tumors of neural crest origin. Bi-allelic inactivation of this gene has been observed in sporadic schwannomas and meningiomas. The NF2 gene may also be somatically inactivated in human malignant mesotheliomas (HMMs). Surprisingly, patients with an NF2 germ-line mutation have not been reported to be at an increased risk for this highly invasive tumor of mesodermal origin. To investigate in HMMs the silencing mechanism of the NF2 gene, we have analyzed its structure and expression in a series of 18 cell lines derived from HMMs. NF2 gene alterations were identified at a genomic level in 7 cell lines and were associated with a marked decrease in the concentration of the NF2 transcript. This decrease was also observed in 4 additional cell lines with no identified NF2 mutation. The 11 cell lines presented evidence suggesting deletion of one NF2 allele. None of these enabled the detection of normal or truncated forms of the NF2 protein by immunoprecipitational immunoblot analyses. In the 7 remaining cell lines, NF2 mRNA and NF2 protein were easily detectable. Among the latter, 4 lines were heterozygous for several chromosome 22 microsatellite loci, suggesting the presence of 2 NF2 alleles. Taken together, our data indicate that silencing of the NF2 gene is restricted to a subset of mesothelioma cell lines. The availability of established cell lines with different characterized NF2 status provides a powerful tool to explore the mechanism by which the NF2 protein exerts its tumor suppressive activity.

Neurofibromatosis 2 tumor suppressor protein colocalizes with ezrin and CD44 and associates with actin-containing cytoskeleton.

Neurofibromatosis 2 (NF2) protein (merlin; schwannomin) is a tumor suppressor involved in tumorigenesis of NF2-associated and sporadic schwannomas and meningiomas. The protein shares the domain structure of three homologous proteins: ezrin, radixin and moesin (ERM). ERM proteins function as membrane organizers and may act as linkers between plasma membrane molecules, such as CD44 and ICAM-2, and the cytoskeleton. We analyzed the distribution and effects of transfected NF2 protein in COS-1, CHO and 293 cells, and endogenous NF2 protein in U251 glioma cells. The distribution was compared to ezrin, CD44 and F-actin. Both transfected and endogenous NF2 protein localized underneath the plasma membrane in a pattern typical of an ERM protein. In COS-1 transfectants, NF2 protein typically codistributed with ezrin but, in cells with poorly developed actin cytoskeleton, it replaced ezrin in filopodia and ruffling edges. NF2 protein colocalized with CD44, which in transfected cells accumulated into restructured cell membrane protrusions. The association of CD44 and NF2 protein was further suggested by binding of CD44 from cellular lysates to recombinant NF2 protein. Interaction between NF2 protein and the actin-containing cytoskeleton was indicated by partial colocalization, by cytochalasin B-induced coclustering, and by retention of NF2 protein in the detergent-insoluble fraction. Transfected NF2 protein induced morphogenic changes. The cells contained restructured membrane extensions and blebs, and CHO cells expressing NF2 protein were more elongated than control transfectants. In conclusion, NF2 protein possesses functional properties of an ERM family member.

Loss of merlin expression in sporadic meningiomas, ependymomas and schwannomas.

Neurofibromatosis 2 (NF2) is an inherited disorder in which affected individuals develop schwannomas and meningiomas. NF2 is mapped to chromosome 22q in a region where frequent loss of heterozygosity also occurs in sporadic meningiomas, ependymomas, and schwannomas. Using NF2 protein (merlin or schwannomin)-specific antibodies, 11 of 14 sporadic schwannomas, three of eight sporadic ependymomas, and 16 of 19 sporadic meningiomas demonstrated significantly reduced or absent merlin expression, suggesting that NF2 may be involved in the pathogenesis of these sporadic tumors.

A point mutation associated with a severe phenotype of neurofibromatosis 2.

Neurofibromatosis 2 (NF2) is an autosomal dominant disease characterized by bilateral vestibular schwannomas and other nonmalignant tumors of the brain, spinal cord, and peripheral nerves. Although the average age of onset of NF2 is 20 years, some individuals may become symptomatic in childhood. We studied 5 unrelated NF2 patients who became symptomatic before age 13. All 5 had multiple tumors in addition to vestibular schwannoma, and none had a positive family history. Sequence analysis of the NF2 gene revealed identical nonsense mutation of exon 6 in 3 patients. Because this mutation destroys a restriction enzyme recognition site, genomic DNA from the 2 other children was directly tested for this change and identical alterations were detected. Although the work of our laboratory and others has not, in general, detected identical mutations in unrelated patients, this mutation seems to occur particularly frequently in the pediatric population and thus may be associated with an especially severe phenotype. Restriction analysis in children with NF2 may be a cost effective way of identifying their mutation. Further work is needed to characterize the effects of this change on the NF2 protein product and its relationship to this severe phenotype.

Neurofibromatosis 2 gene in human colorectal cancer

Colon cancers commonly have allelic losses of chromosome 22q, which suggests the presence of a tumor suppressor gene on 22q. The candidate tumor suppressor gene on 22q is the neurofibromatosis 2 (NF2) gene. Using single strand conformation polymorphism (SSCP) analysis, we screened 24 pairs of colorectal cancer and adjacent normal mucosa, as well as 10 colon cancer cell lines from non-NF2 patients, for mutations in the coding sequence of the NF2 gene. Two SSCP variants, one in exon 14 and another one in exon 16, were detected in two of the sporadic colorectal cancers, but not in adjacent normal mucosa samples. Sequencing of these variants in one tumor detected an A-to-G transition in bp 1459 of the NF2 cDNA, resulting in the change of fie to Val at codon 487 of merlin, the NF2 protein product. The other tumor showed a 2-bp (CT) deletion in the intronic sequence of the alternatively spliced exon 16. These results suggest that the NF2 gene is probably involved in some colorectal tumors, but is not the critical chromosome 22q tumor suppressor gene involved in colon tumorigenesis.