The Hippo Pathway Targets Rae1 to Regulate Mitosis and Organ Size and to Feed Back to Regulate Upstream Components Merlin, Hippo, and Warts.

Hippo signaling acts as a master regulatory pathway controlling growth, proliferation, and apoptosis and also ensures that variations in proliferation do not alter organ size. How the pathway coordinates restricting proliferation with organ size control remains a major unanswered question. Here we identify Rae1 as a highly-conserved target of the Hippo Pathway integrating proliferation and organ size. Genetic and biochemical studies in Drosophila cells and tissues and in mammalian cells indicate that Hippo signaling promotes Rae1 degradation downstream of Warts/Lats. In proliferating cells, Rae1 loss restricts cyclin B levels and organ size while Rae1 over-expression increases cyclin B levels and organ size, similar to Hippo Pathway over-activation or loss-of-function, respectively. Importantly, Rae1 regulation by the Hippo Pathway is crucial for its regulation of cyclin B and organ size; reducing Rae1 blocks cyclin B accumulation and suppresses overgrowth caused by Hippo Pathway loss. Surprisingly, in addition to suppressing overgrowth, reducing Rae1 also compromises survival of epithelial tissue overgrowing due to loss of Hippo signaling leading to a tissue "synthetic lethality" phenotype. Excitingly, Rae1 plays a highly conserved role to reduce the levels and activity of the Yki/YAP oncogene. Rae1 increases activation of the core kinases Hippo and Warts and plays a post-transcriptional role to increase the protein levels of the Merlin, Hippo, and Warts components of the pathway; therefore, in addition to Rae1 coordinating organ size regulation with proliferative control, we propose that Rae1 also acts in a feedback circuit to regulate pathway homeostasis.

Missense mutations in the NF2 gene result in the quantitative loss of merlin protein and minimally affect protein intrinsic function.

Neurofibromatosis type 2 (NF2) is a multiple neoplasia syndrome and is caused by a mutation of the NF2 tumor suppressor gene that encodes for the tumor suppressor protein merlin. Biallelic NF2 gene inactivation results in the development of central nervous system tumors, including schwannomas, meningiomas, ependymomas, and astrocytomas. Although a wide variety of missense germline mutations in the coding sequences of the NF2 gene can cause loss of merlin function, the mechanism of this functional loss is unknown. To gain insight into the mechanisms underlying loss of merlin function in NF2, we investigated mutated merlin homeostasis and function in NF2-associated tumors and cell lines. Quantitative protein and RT-PCR analysis revealed that whereas merlin protein expression was significantly reduced in NF2-associated tumors, mRNA expression levels were unchanged. Transfection of genetic constructs of common NF2 missense mutations into NF2 gene-deficient meningioma cell lines revealed that merlin loss of function is due to a reduction in mutant protein half-life and increased protein degradation. Transfection analysis also demonstrated that recovery of tumor suppressor protein function is possible, indicating that these mutants maintain intrinsic functional capacity. Further, increased expression of mutant protein is possible after treatment with specific proteostasis regulators, implicating protein quality control systems in the degradative fate of mutant tumor suppressor proteins. These findings provide direct insight into protein function and tumorigenesis in NF2 and indicate a unique treatment paradigm for this disorder.

Tumor suppressor schwannomin/merlin is critical for the organization of Schwann cell contacts in peripheral nerves.

Schwannomin/merlin is the product of a tumor suppressor gene mutated in neurofibromatosis type 2 (NF2). Although the consequences of NF2 mutations on Schwann cell proliferation are well established, the physiological role of schwannomin in differentiated cells is not known. To unravel this role, we studied peripheral nerves in mice overexpressing in Schwann cells schwannomin with a deletion occurring in NF2 patients (P0-SCH-Delta39-121) or a C-terminal deletion. The myelin sheath and nodes of Ranvier were essentially preserved in both lines. In contrast, the ultrastructural and molecular organization of contacts between Schwann cells and axons in paranodal and juxtaparanodal regions were altered, with irregular juxtaposition of normal and abnormal areas of contact. Similar but more severe alterations were observed in mice with conditional deletion of the Nf2 gene in Schwann cells. The number of Schmidt-Lanterman incisures, which are cytoplasmic channels interrupting the compact myelin and characterized by distinct autotypic contacts, was increased in the three mutant lines. P0-SCH-Delta39-121 and conditionally deleted mice displayed exuberant wrapping of nonmyelinated fibers and short internodes, an abnormality possibly related to altered control of Schwann cell proliferation. In support of this hypothesis, Schwann cell number was increased along fibers before myelination in P0-SCH-Delta39-121 mice but not in those with C-terminal deletion. Schwann cell numbers were also more numerous in mice with conditional deletion. Thus, schwannomin plays an important role in the control of Schwann cell number and is necessary for the correct organization and regulation of axoglial heterotypic and glio-glial autotypic contacts.

Expression of c-Myc, neurofibromatosis Type 2, somatostatin receptor 2 and erb-B2 in human meningiomas: relation to grades or histotypes.

Meningiomas, which originate from arachnoid cells, represent one of the largest subgroups of intracranial tumors. They are generally benign, but can progress to malignancy. The aim of our study was to determine the expression of 4 genes, c-Myc, neurofibromatosis Type 2 (NF2), somatostatin receptor isoform 2 (sst2) and erb-B2, that have been associated with tumorogenesis or, possibly, with aggressive behavior or recurrence of meningiomas. We measured levels of mRNAs coding for these genes by qRT-PCR in 51 cases and levels ofc-Myc protooncogene and sst2 protein by immunohistochemistry in 26 cases of meningiomas of various grades and histotypes. C-Myc mRNA and protein levels were not grade-related, but validated subdivision of the 36 benign meningiomas into two groups, Groups IA and IB, based on histological and clinical features (Ki-67-proliferative index, absence or presence of mitoses, rate of recurrence and incidence of perilesional edema). In addition to histopathological grading, c-Myc expression may be useful in predicting tumor recurrence in patients with low-grade meningiomas. NF2 mRNA levels and sst2 mRNA and receptor levels were not grade-related, but were histotype-related, with significantly higher levels in the meningothelial subtype than in the fibroblastic subtype. Erb-B2 mRNA levels were not grade- or histotype-related. Furthermore, the high expression of sst2 in meningothelial meningioma suggests the possibility of a different tumorigenesis process in this meningioma subtype and may open perspectives for the diagnosis and therapy of this subtype using somatostatin as an antiproliferative agent.

Is NF2 a Key Player of the Differentially Expressed Gene Between Spinal Cord Ependymoma and Intracranial Ependymoma?

BACKGROUND: Although intracranial and spinal ependymomas are histopathologically similar, the molecular landscape is heterogeneous. An urgent need exists to identify differences in the genomic profiles to tailor treatment strategies. In the present study, we delineated differential gene expression patterns between intracranial and spinal ependymomas. METHODS: We searched the Gene Expression Omnibus database using the term "ependymoma" and analyzed the raw gene expression profiles of 292 ependymomas (31 spinal and 261 intracranial). The gene expression data were analyzed to find differentially expressed genes (DEGs) between 2 regions. The fold change (FC) and false discovery rate (FDR) were used to assess DEGs after gene integration (|log2FC|>2; FDR P < 0.01). Enrichment and pathway analysis was also performed. RESULTS: A total of 201 genes (105 upregulated and 96 downregulated) were significant DEGs in the data sets. The underexpression of NF2 in spinal ependymomas was statistically significant (FDR P = 7.91 × 10-9). However, the FC of NF2 did not exceed the cutoff value (log2FC, -1.2). The top 5 ranked upregulated genes were ARX, HOXC6, HOXA9, HOXA5, and HOXA3, which indicated that spinal ependymomas frequently demonstrate overexpression of HOX family genes, which play fundamental roles in specifying anterior/posterior body patterning. Moreover, the gene ontology enrichment analysis specified "anterior/posterior pattern specification" and "neuron migration" in spinal and intracranial ependymomas, respectively. CONCLUSIONS: The most substantial magnitude of DEGs in ependymoma might be HOX genes. However, whether the differential expression of these genes is the cause or consequence of the disease remains to be elucidated in a larger prospective study.

Re-expression of the tumor suppressor NF2/merlin inhibits invasiveness in mesothelioma cells and negatively regulates FAK.

The neurofibromatosis type 2 NF2 gene product, merlin, is a tumor suppressor frequently inactivated in malignant mesothelioma (MM). To investigate a possible correlation between merlin inactivation and MM invasiveness, we restored merlin expression in NF2-deficient MM cells. Re-expression of merlin markedly inhibited cell motility, spreading and invasiveness, properties connected with the malignant phenotype of MM cells. To test directly whether merlin inactivation promotes invasion in a nonmalignant system, we used small interfering RNA to silence Nf2 in mouse embryonic fibroblasts (MEFs) and found that downregulation of merlin resulted in enhanced cell spreading and invasion. To delineate signaling events connected with this phenotype, we investigated the effect of merlin expression on focal adhesion kinase (FAK), a key component of cellular pathways affecting migration and invasion. Expression of merlin attenuated FAK phosphorylation at the critical phosphorylation site Tyr397 and disrupted the interaction of FAK with its binding partners Src and p85, the regulatory subunit of phosphatidylinositol-3-kinase. In addition, NF2-null MM cells stably overexpressing FAK showed increased invasiveness, which decreased significantly when merlin expression was restored. Collectively, these findings suggest that merlin inactivation is a critical step in MM pathogenesis and is related, at least in part, with upregulation of FAK activity.

A functional association between merlin and HEI10, a cell cycle regulator.

Merlin and ezrin are homologous proteins with opposite effects on neoplastic growth. Merlin is a tumor suppressor inactivated in the neurofibromatosis 2 disease, whereas upregulated ezrin expression is associated with increased malignancy. Merlin's tumor suppressor mechanism is not known, although participation in cell cycle regulation has been suggested. To characterize merlin's biological activities, we screened for molecules that would interact with merlin but not ezrin. We identified the cyclin B-binding protein and cell cycle regulator HEI10 as a novel merlin-binding partner. The interaction is mediated by the alpha-helical domain in merlin and the coiled-coil domain in HEI10 and requires conformational opening of merlin. The two proteins show partial subcellular colocalization, which depends on cell cycle stage and cell adhesion. Comparison of Schwann cells and schwannoma cultures demonstrated that the distribution of HEI10 depends on merlin expression. In transfected cells, a constitutively open merlin construct affected HEI10 protein integrity. These results link merlin to the cell cycle control machinery and may help to understand its tumor suppressor function.

[Expression of Merlin in cortex of temporal lobe and in hippocampal CA1 region of the Kindling Model of Epilepsy induced by corciaria lactone in rats].

OBJECTIVE: To explore the relationship between merlin and hippocampal sclerosis of temporal epilepsy. METHODS: The kindling model of epilepsy induced by corciaria lactone (CL) in rats was used. The expression of merlin in neuron of cortex of temporal lobe and hippocampal CA1 region was observed using immunohistochemistry method. Comparison of the amount of neuron with expression of merlin in the two locations was made between the kindled group, non-kindled group and control group. RESULTS: The expression of merlin in neuron of cortex of temporal lobe and hippocampal CA1 region of the kindled group was higher than the expression of the other two groups (P < 0.05), and there was no significant difference between the expression in the non-kindled group and that in the control group (P > 0.05). The expression of merlin in glial cell of the same region of all groups was seldom seen. CONCLUSION: The super-expression of merlin in neuron of cortex of temporal lobe and hippocampal CA1 region of the kindled rats may be involved in the process of neuronal apoptosis and hippocampal sclerosis.

Biallelic Alteration and Dysregulation of the Hippo Pathway in Mucinous Tubular and Spindle Cell Carcinoma of the Kidney.

Mucinous tubular and spindle cell carcinoma (MTSCC) is a relatively rare subtype of renal cell carcinoma (RCC) with distinctive morphologic and cytogenetic features. Here, we carry out whole-exome and transcriptome sequencing of a multi-institutional cohort of MTSCC (n = 22). We demonstrate the presence of either biallelic loss of Hippo pathway tumor suppressor genes (TSG) and/or evidence of alteration of Hippo pathway genes in 85% of samples. PTPN14 (31%) and NF2 (22%) were the most commonly implicated Hippo pathway genes, whereas other genes such as SAV1 and HIPK2 were also involved in a mutually exclusive fashion. Mutations in the context of recurrent chromosomal losses amounted to biallelic alterations in these TSGs. As a readout of Hippo pathway inactivation, a majority of cases (90%) exhibited increased nuclear YAP1 protein expression. Taken together, nearly all cases of MTSCC exhibit some evidence of Hippo pathway dysregulation. SIGNIFICANCE: MTSCC is a rare and relatively recently described subtype of RCC. Next-generation sequencing of a multi-institutional MTSCC cohort revealed recurrent chromosomal losses and somatic mutations in the Hippo signaling pathway genes leading to potential YAP1 activation. In virtually all cases of MTSCC, there was evidence of Hippo pathway dysregulation, suggesting a common mechanistic basis for this disease. Cancer Discov; 6(11); 1258-66. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 1197.

Merlin Regulates Epithelial-to-Mesenchymal Transition of ARPE-19 Cells via TAK1-p38MAPK-Mediated Activation.

PURPOSE: The purpose of this study was to investigate the function of merlin, which is a binding partner of the hyaluronan receptor CD44, during the epithelial-to-mesenchymal transition (EMT) of human retinal pigment epithelium (ARPE-19) cells. METHODS: Human retinal pigment epithelium cells were stimulated with TNF-α and treated using epithelial-to-mesenchymal-transition inhibitors (a dynamin inhibitor or transforming growth factor-ß-activated kinase 1 [TAK1] inhibitor). Levels of protein expression were assessed by immunoblot analysis, and localization of the relevant proteins was determined by immunofluorescence microscopy. Cell proliferation was evaluated by bromodeoxyuridine (BrdU) incorporation assays. All experiments were performed in serum-free medium. RESULTS: Tumor necrosis factor-α treatments downregulated the expression of merlin and led to the dissociation of CD44 and merlin. The ezrin-radixin-moesin (ERM) proteins were phosphorylated, and hyaluronan endocytosis was accelerated in merlin small interfering RNA (siMerlin)-transfected cells. Treatment with the endocytosis inhibitor dynasore blocked hyaluronan endocytosis, whereas treatment with TNF-α induced mesenchymal phenotypes and downregulation of merlin. Additionally, siMerlin transfection promoted p38MAPK phosphorylation, which was inhibited not only by TAK1 inhibitor treatment but also by TAK1 small interfering RNA (siRNA, siTAK1) transfection. The increased level of BrdU incorporation in siMerlin cells was reduced by additional siTAK1 transfection. Furthermore, TNF-α-induced mesenchymal differentiation and high motility were also inhibited by TAK1 inhibitor treatment and by siTAK1 transfection. CONCLUSIONS: Our findings demonstrated that merlin exerts inhibitory effects on TNF-α-induced EMT by regulating hyaluronan endocytosis and the TAK1-p38MAPK signaling pathway. The proliferative and mesenchymal characteristics of RPE cells play important roles in the development of intraocular fibrotic disorders, such as proliferative vitreoretinopathy (PVR), and our findings provide new therapeutic strategies to prevent the development of PVR.

NF2 blocks Snail-mediated p53 suppression in mesothelioma.

Although asbestos causes malignant pleural mesothelioma (MPM), rising from lung mesothelium, the molecular mechanism has not been suggested until now. Extremely low mutation rate in classical tumor suppressor genes (such as p53 and pRb) and oncogenes (including Ras or myc) indicates that there would be MPM-specific carcinogenesis pathway. To address this, we treated silica to mimic mesothelioma carcinogenesis in mesothelioma and non-small cell lung cancer cell lines (NSCLC). Treatment of silica induced p-Erk and Snail through RKIP reduction. In addition, p53 and E-cadherin were decreased by silica-treatment. Elimination of Snail restored p53 expression. We found that NF2 (frequently deleted in MPM) inhibited Snail-mediated p53 suppression and was stabilized by RKIP. Importantly, GN25, an inhibitor of p53-Snail interaction, induced p53 and apoptosis. These results indicate that MPM can be induced by reduction of RKIP/NF2, which suppresses p53 through Snail. Thus, the p53-Snail binding inhibitor such as GN25 is a drug candidate for MPM.

Merlin regulates transmembrane receptor accumulation and signaling at the plasma membrane in primary mouse Schwann cells and in human schwannomas.

The NF2 gene product, merlin/schwannomin, is a cytoskeleton organizer with unique growth-inhibiting activity in specific cell types. A narrow spectrum of tumors is associated with NF2 deficiency, mainly schwannomas and meningiomas, suggesting cell-specific mechanisms of growth control. We have investigated merlin function in mouse Schwann cells (SCs). We found that merlin regulates contact inhibition of proliferation by limiting the delivery of several growth factor receptors at the plasma membrane of primary SCs. Notably, upon cell-to-cell contact, merlin downregulates the membrane levels of ErbB2 and ErbB3, thus inhibiting the activity of the downstream mitogenic signaling pathways protein kinase B and mitogen-activated protein kinase. Consequently, loss of merlin activity is associated with elevated levels of ErbB receptors in primary SCs. We also observed accumulation of growth factor receptors such as ErbB2 and 3, insulin-like growth factor 1 receptor and platelet-derived growth factor receptor in peripheral nerves of Nf2-mutant mice and in human NF2 schwannomas, suggesting that this mechanism could play an important role in tumorigenesis.

Inactivation of the NF2 tumor suppressor protein merlin in DU145 prostate cancer cells.

BACKGROUND: The neurofibromatosis 2 (NF2) tumor suppressor gene product merlin is an important regulator of contact-dependent cell proliferation. Phosphorylation of merlin at serine 518 (Ser518) by the Rac effector p21-activated kinase (PAK) inactivates merlin's growth suppressing function, and is regulated by cell-culture conditions, including cell density, cell/substrate attachment, and growth factor availability. We examined the regulation of merlin expression and merlin phosphorylation in prostate cancer cells. METHODS: Phosphorylation of merlin in five prostate cancer cell lines (LNCaP, DU145, PC3, 22RV1, and LAPC-4) was examined by Western blotting using anti-phospho-merlin (Ser518) antibody. The activity of PAK, an upstream regulator of merlin phosphorylation, was measured by Western blotting using phospho-PAK (Ser141) antibody. The effects of various cell-culture conditions on the phosphorylation levels of merlin and PAK were analyzed. RESULTS: Both merlin expression and phosphorylation were low in LNCaP, PC3, 22RV1, and LAPC-4 prostate cancer cells. In DU145 cells, total and phosphorylated merlin were abundant, but phosphorylation was not inhibited by high cell density, serum withdrawal, the addition of hyaluronic acid or inhibition of CD44 expression, all of which are reported to inhibit merlin phosphorylation in non-neoplastic cells. PAK activation was elevated in DU145 cells and the addition of a PAK-specific inhibitor peptide but not the Rac1-specific inhibitor NSC23766 inhibited both PAK and merlin phosphorylation. CONCLUSIONS: Merlin is inactivated in DU145 prostate cancer cells by PAK-mediated constitutive phosphorylation, identifying a novel mechanism of merlin inactivation in neoplastic cells.

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.

Multiple meningiomas: Investigating the molecular basis of sporadic and familial forms.

Meningiomas are common tumors of the coverings of the central nervous system (CNS), comprising 20% of intracranial neoplasms. The only genes known to be associated with sporadic meningiomas are NF2 on chromosome 22 and the related cytoskeleton element DAL-1 on chromosome 18. Between 1 and 8% of patients with meningiomas develop multiple meningiomas, a trait transmitted occasionally in an autosomal dominant fashion. We investigated the DAL-1 and NF2 loci in 7 unrelated multiple meningioma patients without clinical evidence of NF2 by mutational and pathological analysis. Five novel intragenic microsatellite polymorphisms were developed for specific detection of loss of heterozygosity (LOH) at the DAL-1 locus. Three of 7 patients had affected relatives and all affected individuals were female. No tumors from familial patients were of a fibroblastic subtype. Truncating NF2 mutations were detected in 3 tumor specimens, but were not present in the corresponding blood samples. Two tumors showed LOH at the NF2 locus. All tumors showing mutations at the NF2 locus originated from patients without affected relatives and were of the fibroblastic subtype. Five non-truncating alterations in the DAL-1 gene were found, however, LOH of chromosome 18 markers was not seen in any tumor. In contrast to the NF2 results, all DAL-1 alterations were found in paired blood specimens. Our findings provide further evidence that the molecular basis of sporadic and familial multiple meningiomas is fundamentally different and extend this dichotomy to pathologic subtypes. DAL-1 does not function as a true tumor suppressor in these patients.

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.

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.

Differential involvement of protein 4.1 family members DAL-1 and NF2 in intracranial and intraspinal ependymomas.

Ependymomas are malignant CNS neoplasms with highly variable biologic behavior, including a generally better prognosis for intraspinal tumors. Inactivation of the NF2 gene on 22q12 and loss of its protein product, merlin, have been well documented in subsets of meningiomas and ependymomas. DAL-1, a related tumor suppressor and protein 4.1 family member on 18p11.3, has also been recently implicated in meningioma pathogenesis, though its role in ependymoma remains unknown. Therefore, we evaluated 27 ependymomas (12 intracranial and 15 spinal) using fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) to determine NF2/merlin and DAL-1/DAL-1 status at the DNA and protein levels. Demonstrable NF2 and DAL-1 gene deletions were each detected in 6 (22%) ependymomas. All 5 merlin losses by IHC occurred in spinal ependymomas (P =.047), whereas 5 (71%) DAL-1-negative cases were intracranial (P =.185). The former result is consistent with prior observations that NF2 mutations are generally limited to spinal ependymomas. In contrast to meningiomas, simultaneous merlin and DAL-1 losses were not encountered. Our findings suggest that (1) NF2 and DAL-1 losses are involved in the pathogenesis of spinal and intracranial ependymoma subsets, respectively and (2) given the number of cases with no demonstrable losses, other cellular perturbations must also be critical for tumori-genesis.

Neurofibromatosis 2 (NF2) tumor suppressor schwannomin and its interacting protein HRS regulate STAT signaling.

Mutations in the neurofibromatosis 2 (NF2) gene with the resultant loss of expression of the NF2 tumor suppressor schwannomin are one of the most common causes of benign human brain tumors, including schwannomas and meningiomas. Previously we demonstrated that the hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) strongly interacts with schwannomin. HRS is a powerful regulator of receptor tyrosine kinase trafficking to the degradation pathway and HRS also binds STAM. Both of these actions for HRS potentially inhibit STAT activation. Therefore, we hypothesized that schwannomin inhibits STAT activation through interaction with HRS. We now show that both schwannomin and HRS inhibit Stat3 activation and that schwannomin suppresses Stat3 activation mediated by IGF-I treatment in the human schwannoma cell line STS26T. We also find that schwannomin inhibits Stat3 and Stat5 phosphorylation in the rat schwannoma cell line RT4. Schwannomin with the pathogenic missense mutation Q538P fails to bind HRS and does not inhibit Stat5 phosphorylation. These data are consistent with the hypothesis that schwannomin requires HRS interaction to be fully functionally active and to inhibit STAT activation.