Reduced apoptosis rates in human schwannomas

Schwannomas, tumors originating from Schwann cells, represent a frequent neurological tumor and can occur both in a genetic disorder called neurofibromatosis type 2 (NF2) and sporadically. In both cases the genetic background is identical as all schwannomas are caused by biallelic mutations in the tumor suppressor gene NF2 coding for merlin. Mutations in this gene have also been found to be responsible for 50% to 60% of spontaneous and 100% of the NF2 associated meningiomas. The NF2 gene product, merlin, links transmembrane proteins to the cytoskeleton and is involved in intracellular signaling processes. It has previously been shown that reexpression of wild-type merlin in primary human schwannoma cells leads to an increase in the number of apoptotic cells. Here, we report in vivo and in vitro evidence that the basal apoptosis rate of primary human schwannoma cells is reduced in comparison to that of normal Schwann cells, supporting the idea that in this benign tumor type, apoptosis has a role in tumorigenesis.     

Functional analysis of the relationship between the neurofibromatosis 2 tumor suppressor and its binding partner,hepatocyte growth factor-regulated tyrosine kinase substrate

Individuals with the neurofibromatosis 2 (NF2) inherited tumor predisposition syndrome are prone to the development of nervous system tumors, including schwannomas and meningiomas. The NF2 tumor suppressor protein, merlin or schwannomin, inhibits cell growth and motility as well as affects actin cytoskeleton-mediated processes. Merlin interacts with several proteins that might mediate merlin growth suppression, including hepatocyte growth factor-regulated tyrosine kinase substrate (HRS or HGS). Previously, we demonstrated that regulated overexpression of HRS in RT4 rat schwannoma cells had the same functional consequences as regulated overexpression of merlin. To determine the functional significance of this interaction, we generated a series of HRS truncation mutants and defined the regions of HRS required for merlin binding and HRS growth suppression. The HRS domain required for merlin binding was narrowed to a region (residues 470-497) containing the predicted coiled-coil domain whereas the major domain responsible for HRS growth suppression was distinct (residues 498-550). To determine whether merlin growth suppression required HRS, we demonstrated that merlin inhibited growth in HRS (+/+), but not HRS( -/-) mouse embryonic fibroblast cells. In contrast, HRS could suppress cell growth in the absence of Nf2 expression. These results suggest that merlin growth suppression requires HRS expression and that the binding of merlin to HRS may facilitate its ability to function as a tumor suppressor.     

Defects in neurofibromatosis 2 protein function can arise at multiple levels

Neurofibromatosis 2 (NF2) is an inherited cancer syndrome resulting from mutations in the NF2 tumor suppressor gene. Analysis of NF2 mutations has revealed some general genotype-phenotype correlations. Severe disease has been associated with mutations that produce a premature termination while more mild disease has been associated with missense mutations. Here, we provide experimental proof for these genotype-phenotype correlations by demonstrating that nonsense mutations fail to produce stable merlin protein while missense mutations result in the generation of merlin proteins defective in negative growth regulation. This inability to suppress cell growth may result from defects in the function of merlin at several levels, including failure to form an intramolecular complex. Based on these findings, we propose a model for merlin growth suppression that provides a framework for analyzing NF2 patient mutations and merlin function.      

Increased expression of the NF2 tumor suppressor gene product, merlin, impairs cell motility, adhesionand spreading

The neurofibromatosis 2 ( NF2 ) gene product, merlin, is a tumor suppressor protein mutated in schwanno-mas and several other tumors. Merlin, which shares significant homology with the actin-associated proteins ezrin, radixin and moesin (ERM proteins), inhibits cell growth when overexpressed in cell lines. The similarities between merlin and ERM proteins suggest that merlin's growth-regulatory capabilities may be due to alterations in cytoskeletal function. We examined this possibility in rat schwannoma cell lines overexpressing wild-type merlin isoforms and mutant merlin proteins. We found that overexpression of wild-type merlin resulted in transient alterations in F-actin organization, cell spreading and cell attachment. Merlin overexpression also impaired cell motility as measured in an in vitro motility assay. These effects were only observed in cells overexpressing a merlin isoform capable of inhibiting cell growth and not with mutant merlin molecules (NF2 patient mutations) or a merlin splice variant (isoform II) lacking growth-inhibitory activity. These data indicate that merlin may function to maintain normal cytoskeletal organization, and suggest that merlin's influence on cell growth depends on specific cytoskeletal rearrangements.      

Neuropathology and Molecular Genetics of Neurofibromatosis 2 and Related Tumors

Neurofibromatosis 2 (NF2) is an uncommon, autosomal dominant disorder in which patients are predisposed to neoplastic and dysplastic lesions of Schwann cells (schwannomas and schwannosis), meningeal cells (meningiomas and meningioan-giomatosis) and glial cells (gliomas and glial hamar-tomas). Clinical and genetic criteria that distinguish NF2 from neurofibromatosis 1 have allowed more accurate assignment of specific pathological features to NF2. The NF2 tumor suppressor gene on chromosome 22q12 encodes a widely expressed protein, named merlin, which may link the cytoskeleton and cell membrane. Germline NF2 mutations in NF2 patients and somatic NF2 mutations in sporadic schwannomas and meningiomas have different mutational spectra, but most NF2 alterations result in a truncated, inactivated merlin protein. In NF2 patients, specific mutations do not necessarily correlate with phenotypic severity, although grossly truncating alterations may result in a more severe phenotype. In schwannomas, NF2 mutations are common and may be necessary for tumorigenesis. In meningiomas, NF2 mutations occur more commonly in fibroblastic than meningothelial subtypes, and may cluster in the first half of the gene. In addition, in meningiomas, a second, non- NF2 meningioma locus is probably also involved. Future efforts in NF2 research will be directed toward elucidating the role of merlin in the normal cell and the sequelae of its inactivation in human tumors.     

Characterization of chicken Nf2/merlin indicates regulatory roles in cell proliferation and migration.

The neurofibromatosis 2 (NF2) tumor suppressor protein merlin, or schwannomin, functions as a negative growth regulator such that inactivating mutations in Nf2 predispose humans to tumors. In addition, merlin has a critical role during embryonic development. Nf2-deficient mice die early during embryogenesis, with defects in gastrulation and extraembryonic tissues. To investigate the function of Nf2/merlin during embryonic development, we first identified the homologous Nf2 gene in chicken (cNf2) and examined the distribution of chicken merlin (c-merlin) during myogenesis. cNf2 encoded a full-length mRNA of 1,770 nucleotides and a protein of 589 residues. C-merlin shared high sequence homology and common protein motifs with vertebrate and Drosophila merlins. In addition, cNF2 functions as a negative growth regulator similar to human and Drosophila merlin in vitro. In vivo, c-merlin was expressed diffusely in the forming dermomyotome but down-regulated in migratory muscle precursors in the forelimb. As muscle formed in the limb, c-merlin expression was up-regulated. As an initial examination of c-merlin function during myogenesis, c-merlin was ectopically expressed in muscle precursors and the effects on muscle development were examined. We show that ectopic merlin expression reduces the proliferation of muscle precursors as well as their ability to migrate effectively in limb mesoderm. Collectively, these results demonstrate that c-merlin is developmentally regulated in migrating and differentiating myogenic cells, where it functions as a negative regulator of both muscle growth and motility.     

Transduction of wild-type merlin into human schwannoma cells decreases schwannoma cell growth and induces apoptosis.

Mutations in both alleles of the tumour suppressor gene coding for merlin/schwannomin, an ERM family protein, cause the hereditary disease neurofibromatosis type 2 (NF2). NF2 is characterized by the development of multiple nervous system tumours especially vestibular schwannomas. Efficient oncoretrovirus-mediated gene transfer of different merlin constructs was used to stably re-express wild-type merlin in primary cells derived from human schwannomas. Using two-parameter FACS analysis we show that expression of wild-type merlin in NF2 cells led to significant reduction of proliferation and G0/G1 arrest in transduced schwannoma cells. In addition, we show increased apoptosis of schwannoma cells transduced with wild-type merlin. Our findings in primary schwannoma cells from NF2 patients strongly support the hypothesis of merlin acting as a tumour suppressor and may help in understanding development of human schwannomas in NF2.     

The NF2 interactor, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), associates with merlin in the "open" conformation and suppresses cell growth and motility

The neurofibromatosis 2 tumor suppressor protein, merlin or schwannomin, functions as a negative growth regulator; however, its mechanism of action is not known. In an effort to determine how merlin regulates cell growth, we analyzed a recently identified novel merlin interactor, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS). We demonstrate that regulated overexpression of HRS in rat schwannoma cells results in similar effects as overexpression of merlin, including growth inhibition, decreased motility and abnormalities in cell spreading. Previously, we showed that merlin forms an intramolecular association between the N- and C-termini and exists in "open" and "closed" conformations. Merlin interacts with HRS in the unfolded, or open, conformation. This HRS binding domain maps to merlin residues 453-557. Overexpression of C-terminal merlin has no effect on HRS function, arguing that merlin binding to HRS does not negatively regulate HRS growth suppressor activity. These results suggest the possibility that merlin and HRS may regulate cell growth in schwannoma cells through interacting pathways.     

Mutations of the neurofibromatosis type 2 gene and lack of the gene product in vestibular schwannomas

Schwannomas are common tumors of the nervous system and arefrequently found in patients with neurofibromatosis (NF) 2.Although loss of heterozygosity in NF2 tumors suggests thatthe NF2 gene functions as a tumor suppressor gene, the NF2 geneshows amino acid sequence homology to structural proteins inone of which dominantly acting mutations have been described.We performed a mutational analysis in 30 vestibular schwannomasand examined the effect of mutations on the NF2 protein. Wedetected 18 mutations in 30 vestibular schwannomas of whichseven contained loss or mutation of both NF2 alleles. Most mutationswere predicted to result in a truncated protein. Mutationalhot spots were not identified. Immunocytochemical studies usingantibodies to the NF2 protein showed complete absence of stainingin tumor Schwann cells, whereas staining was observed in normalvestibular nerve. These data indicate that loss of NF2 proteinfunction is a necessary step in schwannoma pathogenesis andthat the NF2 gene functions as a recessive tumor suppressorgene.     

Schwann cell hyperplasia and tumors in transgenic mice expressing a naturally occurring mutant NF2 protein

Specific mutations in some tumor suppressor genes such as p53 can act in a dominant fashion. We tested whether this mechanism may also apply for the neurofibromatosis type-2 gene (NF2) which, when mutated, leads to schwannoma development. Transgenic mice were generated that express, in Schwann cells, mutant NF2 proteins prototypic of natural mutants observed in humans. Mice expressing a NF2 protein with an interstitial deletion in the amino-terminal domain showed high prevalence of Schwann cell-derived tumors and Schwann cell hyperplasia, whereas those expressing a carboxy-terminally truncated protein were normal. Our results indicate that a subset of mutant NF2 alleles observed in patients may encode products with dominant properties when overexpressed in specific cell lineages.     

Universal absence of merlin, but not other ERM family members, in schwannomas.

NF2 (neurofibromatosis 2, encoding the merlin protein) gene mutations and chromosome 22q loss have been demonstrated in the majority of sporadic and NF2-associated schwannomas, but many schwannomas fail to demonstrate genetic evidence of biallelic NF2 gene inactivation. In addition, the role of the merlin-related ERM family members (ezrin, radixin, and moesin) remains unclear in these tumors. We therefore studied expression of NF2-encoded merlin as well as ezrin, radixin, and moesin in 22 vestibular and peripheral schwannomas that had been evaluated for NF2 mutations and chromosome 22q loss. Western blotting and immunohistochemistry with antibodies directed against the amino and carboxy termini of merlin demonstrated loss of merlin expression in all studied schwannomas, including 12 tumors lacking genetic evidence of biallelic NF2 gene inactivation. Western blotting with antibodies directed against ezrin, radixin, and moesin, however, showed expression of these proteins in all schwannomas. In addition, immunohistochemistry with an antibody to moesin revealed widespread expression in tumor and endothelial cells. These data indicate that the specific loss of merlin is universal to schwannomas and is not linked to loss of ezrin, radixin, or moesin expression.     

Pathological adhesion of primary human schwannoma cells is dependent on altered expression of integrins

Mutations in the tumor suppressor gene coding for merlin cause Neurofibromatosis type 2 (NF2), all spontaneous schwannomas, and a majority of meningiomas. Merlin links transmembrane proteins to the cytoskeleton. Accordingly, primary human schwannoma cells lacking merlin show an increased number of lamellipodia and filopodia as well as increased cell spreading. We show enhanced adhesion in primary human schwannoma cells and present evidence that this is dependent on the integrin chains alpha6beta1 and alpha6beta4. We further demonstrate that the integrin chains beta1 and beta4 are upregulated in schwannomas using different complementary methods, and report higher expression of these integrins per schwannoma cell by fluorescence assisted cell sorting (FACS). Finally we report clustering of the integrin chains alpha6, beta1, and beta4 on schwannoma cells. Our findings fit well into recent data on the role of merlin in signaling cascades connected to integrins and help explain pathological ensheathment of extracellular matrix or pseudomesaxon formation which is a hallmark of schwannoma histopathology.     

Role of NF2 haploinsufficiency in NF2-associated polyneuropathy

Neurofibromatosis 2 (NF2) is a hereditary tumor disease characterized by bilateral vestibular schwannomas. Polyneuropathy seems to occur quite frequently in NF2 and in most cases, the etiology of this neuropathy is unclear, especially when the neuropathy is symmetric. NF2 is believed to follow the two-hit hypothesis. According to this, one allele is mutated in the germline, and the second hit is somatic and results in tumor formation. The second hit most frequently is a loss of the NF2 locus, often the entire chromosome 22. We set out to investigate the underlying genetics in peripheral nerve of NF2 patients with polyneuropathy. We identified NF2 patients with polyneuropathy in which we could detect the germline mutation and analyzed NF2 gene dosage in archived nerve biopsies from these patients using a newly developed method. We observed merlin haploinsufficiency in peripheral nerves of two different patients with NF2-related polyneuropathy. This finding was further supported by showing that approximately 50% merlin expression in a cell line using shRNA results in altered gene expression as previously shown in schwannomas. Thus, we suggest that reduced merlin gene dosage is relevant in NF2-associated polyneuropathy.     

NF2 deficiency promotes tumorigenesis and metastasis by destabilizing adherens junctions

Mutation of the Neurofibromatosis 2 (NF2) tumor suppressor gene leads to cancer development in humans and mice. Recent studies suggest that Nf2 loss also contributes to tumor metastasis. The Nf2-encoded protein, merlin, is related to the ERM (ezrin, radixin, and moesin) family of membrane:cytoskeleton-associated proteins. However, the cellular mechanism whereby merlin controls cell proliferation from this location is not known. Here we show that the major cellular consequence of Nf2 deficiency in primary cells is an inability to undergo contact-dependent growth arrest and to form stable cadherin-containing cell:cell junctions. Merlin colocalizes and interacts with adherens junction (AJ) components in confluent wild-type cells, suggesting that the lack of AJs and contact-dependent growth arrest in Nf2(-/-) cells directly results from the absence of merlin at sites of cell:cell contact. Our studies indicate that merlin functions as a tumor and metastasis suppressor by controlling cadherin-mediated cell:cell contact.     

Merlin expression in secretory meningiomas: evidence of an NF2-independent pathogenesis? Immunohistochemical study.

One of the most common chromosomal regions implicated in the meningiomas tumorigenesis is 22q12 where the neurofibromatosis 2 (NF2) gene resides. The NF2 tumor-suppressor gene encodes for the merlin/schwannomin protein, which is responsible for the inherited disease neurofibromatosis 2. NF2 gene mutations predominantly occur in transitional and fibroblastic meningiomas, whereas the meningothelial variant is less affected. Secretory meningioma is an infrequent meningioma subtype. Its most typical morphologic feature is the presence of intracytoplasmic or extracytoplasmic round hyaline, eosinophilic, and periodic acid Shiff-positive bodies in a lesion frequently otherwise classifiable as meningothelial meningioma. This study reviews the immunohistochemical merlin expression in 14 consecutive secretory meningiomas. Our purpose was to investigate if secretory meningiomas, analogous to meningothelial meningiomas, follow a molecular route of pathogenesis independent of the neurorofibromatosis 2 gene-associated pathway. All meningiomas showed positive immunocoloration involving the majority of the hyaline inclusions and secretory cells; in 12 (86%) meningiomas, a positive immunoreaction was also documented in nonsecretory tumoral cells. Our results may indicate a molecular, besides morphologic, similarity between secretory and meningothelial meningiomas: the almost constant merlin immunohistochemical expression in our series gives evidence for a possible NF2 gene-independent pathogenesis in secretory meningiomas.     

Conditional biallelic Nf2 mutation in the mouse promotes manifestations of human neurofibromatosis type 2

Hemizygosity for the NF2 gene in humans causes a syndromic susceptibility to schwannoma development. However, Nf2 hemizygous mice do not develop schwannomas but mainly osteosarcomas. In the tumors of both species, the second Nf2 allele is inactivated. We report that conditional homozygous Nf2 knockout mice with Cre-mediated excision of Nf2 exon 2 in Schwann cells showed characteristics of neurofibromatosis type 2. These included schwannomas, Schwann cell hyperplasia, cataract, and osseous metaplasia. Thus, the tumor suppressor function of Nf2, here revealed in murine Schwann cells, was concealed in hemizygous Nf2 mice because of insufficient rate of second allele inactivation in this cell compartment. The finding of this conserved function documents the relevance of the present approach to model the human disease.     

The NF2 tumor suppressor gene product, merlin, mediates contact inhibition of growth through interactions with CD44

The neurofibromatosis-2 (NF2) gene encodes merlin, an ezrin-radixin-moesin-(ERM)-related protein that functions as a tumor suppressor. We found that merlin mediates contact inhibition of growth through signals from the extracellular matrix. At high cell density, merlin becomes hypo-phosphorylated and inhibits cell growth in response to hyaluronate (HA), a mucopolysaccharide that surrounds cells. Merlin's growth-inhibitory activity depends on specific interaction with the cytoplasmic tail of CD44, a transmembrane HA receptor. At low cell density, merlin is phosphorylated, growth permissive, and exists in a complex with ezrin, moesin, and CD44. These data indicate that merlin and CD44 form a molecular switch that specifies cell growth arrest or proliferation.     

The tumor suppressor merlin interacts with microtubules and modulates Schwann cell microtubule cytoskeleton

The lack of neurofibromatosis 2 tumor suppressor protein merlin leads to the formation of nervous system tumors, specifically schwannomas and meningiomas. Merlin is considered to act as a tumor suppressor at the cell membrane, where it links transmembrane receptors to the actin cytoskeleton. Several tumor suppressors interact with another component of the cytoskeleton, the microtubules, in a regulated manner and control their dynamics. In this work, we identify merlin as a novel microtubule-organizing protein. We identify two tubulin-binding sites in merlin, one residing at the N-terminal FERM-domain and another at the C-terminal domain. Merlin's intramolecular association and phosphorylation of serine 518 regulate the interaction between merlin and tubulin. Analysis of cultured glioma cells indicates colocalization between merlin and microtubules especially during cell division. In primary mouse Schwann cells only minor colocalization at the cell periphery of interphase cells is seen. However, these cells drastically change their microtubule organization upon loss of merlin indicating a functional association of the proteins. Both in vitro assays and in vivo studies in Schwann cells indicate that merlin promotes tubulin polymerization. The results show that merlin plays a key role in the regulation of the Schwann cell microtubule cytoskeleton and suggest a mechanism by which loss of merlin leads to cytoskeletal defects observed in human schwannomas.