Neurofibromin 2 Research Articles

NF2 Activates Hippo Signaling and Promotes Ischemia/Reperfusion Injury in the Heart.

NF2 (neurofibromin 2) is an established tumor suppressor that promotes apoptosis and inhibits growth in a variety of cell types, yet its function in cardiomyocytes remains largely unknown. We sought to determine the role of NF2 in cardiomyocyte apoptosis and ischemia/reperfusion (I/R) injury in the heart. We investigated the function of NF2 in isolated cardiomyocytes and mouse myocardium at baseline and in response to oxidative stress. NF2 was activated in cardiomyocytes subjected to H2O2 and in murine hearts subjected to I/R. Increased NF2 expression promoted the activation of Mst1 (mammalian sterile 20-like kinase 1) and the inhibition of Yap (Yes-associated protein), whereas knockdown of NF2 attenuated these responses after oxidative stress. NF2 increased the apoptosis of cardiomyocytes that appeared dependent on Mst1 activity. Mice deficient for NF2 in cardiomyocytes, NF2 cardiomyocyte-specific knockout (CKO), were protected against global I/R ex vivo and showed improved cardiac functional recovery. Moreover, NF2 cardiomyocyte-specific knockout mice were protected against I/R injury in vivo and showed the upregulation of Yap target gene expression. Mechanistically, we observed nuclear association between NF2 and its activator MYPT-1 (myosin phosphatase target subunit 1) in cardiomyocytes, and a subpopulation of stress-induced nuclear Mst1 was diminished in NF2 CKO hearts. Finally, mice deficient for both NF2 and Yap failed to show protection against I/R indicating that Yap is an important target of NF2 in the adult heart. NF2 is activated by oxidative stress in cardiomyocytes and mouse myocardium and facilitates apoptosis. NF2 promotes I/R injury through the activation of Mst1 and inhibition of Yap, thereby regulating Hippo signaling in the adult heart.

Clinical impact of targeted amplicon sequencing for meningioma as a practical clinical-sequencing system

Recent genetic analyses using next-generation sequencers have revealed numerous genetic alterations in various tumors including meningioma, which is the most common primary brain tumor. However, their use as routine laboratory examinations in clinical applications for tumor genotyping is not cost effective. To establish a clinical sequencing system for meningioma and investigate the clinical significance of genotype, we retrospectively performed targeted amplicon sequencing on 103 meningiomas and evaluated the association with clinicopathological features. We designed amplicon-sequencing panels targeting eight genes including NF2 (neurofibromin 2), TRAF7, KLF4, AKT1, and SMO. Libraries prepared with genomic DNA extracted from PAXgene-fixed paraffin-embedded tissues of 103 meningioma specimens were sequenced using the Illumina MiSeq. NF2 loss in some cases was also confirmed by interphase-fluorescent in situ hybridization. We identified NF2 loss and/or at least one mutation in NF2, TRAF7, KLF4, AKT1, and SMO in 81 out of 103 cases (79%) by targeted amplicon sequencing. On the basis of genetic status, we categorized meningiomas into three genotype groups: NF2 type, TRAKLS type harboring mutation in TRAF7, AKT1, KLF4, and/or SMO, and ‘not otherwise classified' type. Genotype significantly correlated with tumor volume, tumor location, and magnetic resonance imaging findings such as adjacent bone change and heterogeneous gadolinium enhancement, as well as histopathological subtypes. In addition, multivariate analysis revealed that genotype was independently associated with risk of recurrence. In conclusion, we established a rapid clinical sequencing system that enables final confirmation of meningioma genotype within 7 days turnaround time. Our method will bring multiple benefits to neuropathologists and neurosurgeons for accurate diagnosis and appropriate postoperative management.

A novel alternative splicing isoform of NF2 identified in human Schwann cells.

Vestibular schwannoma (VS) is a benign, slow-growing cranial tumor that originates from the hypertrophy of Schwann cells. The majority of sporadic VS are unilateral, and the mechanisms underlying VS tumorigenesis are not fully understood. The human neurofibromin 2 (NF2) gene encodes the tumor suppressor protein merlin and the NF2 transcript can be alternatively spliced to form numerous isoforms. The present study investigated human Schwann cells (HSCs) at the mRNA and protein level to understand the function of the alternative splicing (AS) isoform of NF2. The total RNA of HSCs was isolated and the full-length coding sequence of NF2 was amplified. The amplified products were excised from agarose gels, purified and sequenced. NF2 at a protein level was assayed by immunoprecipitation and western blot analysis. The full-length and spliced NF2 forms were amplified by polymerase chain reaction (PCR) from the HSC complementary DNA and ligated into eukaryotic expression vector pcDNA3.1(+). The plasmids were transfected into the HSC HEI-193 cell line and cell proliferation assays were performed using Cell Counting Kit-8. PCR analysis using HSC total RNA as a template revealed the presence of a shortened NF2 transcript, which was due to splicing at the 3'-end of the NF2 mRNA. Sequence analysis confirmed that this AS isoform omitted exons 11, 12, 13, 14, 15 and 16. Immunoprecipitation and western blot analysis demonstrated that the AS isoform was highly expressed in the HSCs at 38 kDa, while the wild-type (WT) isoform, which was expected at 66 kDa, was undetectable. Transfection and cell proliferation assays revealed that the WT isoform exhibited significant growth inhibition, while the AS isoform did not suppress cell growth. In conclusion, the present study detected AS NF2 isoforms in HSC for the first time, and investigated the function of the principle AS isoform. The present study suggests that although HSCs have an undetectable level of WT isoform of the NF2 protein merlin, they are not merlin-null, since they express the AS isoform. Although the AS merlin isoform has no suppressive effect on cell growth, certain mechanisms may exist that underlie this phenomenon, and this may be associated with the genesis and development of VS.

MNGO-21GENOTYPING OF MENINGIOMA BY TARGETED AMPLICON SEQUENCING USING MiSeq

BACKGROUND: Meningiomas are the most common primary brain tumors, accounting for about 27% of all intracranial tumors. Loss of Neurofibromin 2 (NF2) is found in about half of sporadic meningiomas, and recently, mutations in TRAF7, KLF4, AKT1, and SMO are reported in non-NF2 meningioma. To establish practical genotyping methods, we performed targeted amplicon sequencing with 45 meningioma specimens and evaluated the association with clinicopathological features. METHODS: Frozen samples of 45 meningiomas were fixed with PAXgene Tissue System (QIAGEN) and embedded in paraffin (PFPE). Amplicon sequencing panel targeted NF2, TRAF7, KLF4, AKT1 and SMO were designed by GeneRead Mix-n-Match panel and Custom panel (QIAGEN). Genomic DNA was extracted from PFPE tissues and libraries were prepared using GeneRead DNAseq Targeted Panels V2 (QIAGEN). The libraries were sequenced by the MiSeq (Illumina). The raw read data obtained from amplicon sequencing were processed by GeneRead Panel Variant Calling service for analysis of mutations and copy number alterations (CNA). RESULTS: Thirty three cases (73%) harbored NF2 loss, in which 20 cases (61%) simultaneously carried NF2 mutation. Mutations in TRAF7, KLF4, AKT1, and SMO were observed in 6 (13 %), 4 (9 %), 2 (4 %) and 0 (0%) cases, respectively. Altogether, 40 of 45 case (89 %) carried at least one or more gene alteration including mutations in 5 genes listed above and NF2 loss. Result of genotyping was correlated with histological subtypes as follows. All of nine fibrous meningiomas carried NF2 loss, while all of 4 meningiomas with secretory component carried both TRAF7 and KLF4 mutations. A case of chordoid meningioma harbored only AKT1-E17K mutation. CONCLUSIONS: Here we established the clinical sequence system for meningioma by targeted amplicon sequencing with desktop sequencer in which we demonstrated favorable results for CNAs as well as mutations.

A YAP/TAZ-induced feedback mechanism regulates Hippo pathway homeostasis.

YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) are major downstream effectors of the Hippo pathway that influences tissue homeostasis, organ size, and cancer development. Aberrant hyperactivation of YAP/TAZ causes tissue overgrowth and tumorigenesis, whereas their inactivation impairs tissue development and regeneration. Dynamic and precise control of YAP/TAZ activity is thus important to ensure proper physiological regulation and homeostasis of the cells. Here, we show that YAP/TAZ activation results in activation of their negative regulators, LATS1/2 (large tumor suppressor 1/2) kinases, to constitute a negative feedback loop of the Hippo pathway in both cultured cells and mouse tissues. YAP/TAZ in complex with the transcription factor TEAD (TEA domain family member) directly induce LATS2 expression. Furthermore, YAP/TAZ also stimulate the kinase activity of LATS1/2 through inducing NF2 (neurofibromin 2). This feedback regulation is responsible for the transient activation of YAP upon lysophosphatidic acid (LPA) stimulation and the inhibition of YAP-induced cell migration. Thus, this LATS-mediated feedback loop provides an efficient mechanism to establish the robustness and homeostasis of YAP/TAZ regulation.

Abstract 1178: Gene and protein expression profiling identifies molecular signature of resistance to anti-VEGF therapy in ovarian cancer

Abstract Purpose: Angiogenesis plays an important role in ovarian cancer progression, and the addition of anti-angiogenic agents targeting the vascular endothelial growth factor (VEGF) signaling pathways to conventional chemotherapy improves progression-free survival in relapsed, platinum-resistant ovarian cancer when bevacizumab is given as concurrent or maintenance therapy. Unfortunately, the long-term clinical benefits of anti-VEGF therapy have been consistently limited by the rapid development of tumor resistance. Understanding of tumor's functional adaptations to anti-VEGF therapy and identifying early indicators of resistance are crucial for better exploiting anti-angiogenic therapies. The objective of this study was to perform gene and protein expression profiling of ovarian cancer to identify tumor and stroma signature of anti-VEGF treatment-resistance using preclinical resistance models. Methods: We developed phenotypic resistance xenograft models of anti-VEGF therapy that mimic the clinical feature of resistance. SKOV3 xenografted mice were treated with sorafenib or bevacizumab over two months until there were phenotypic differences between tumors that remained responsive to the treatment and that regained growth after initial response. We conducted Illumina next generation sequencing to compare the gene expression in resistant and sensitive tumors. Sequencing analysis was performed using Perkin Elmer's Genesifter software and alignment to the H. sapiens and M. musculus reference genomes. Further, Reverse phase protein array (RPPA) analysis was done to identify differentially expressed proteins between resistant and sensitive tumors. Results: Our sequencing analysis revealed significant differences in expression of various genes between sensitive and resistant tumors. Based on their role in angiogenesis and angiogenesis resistance, we chose a set of genes for follow-up studies. In sorafenib treatment group amphiregulin and heparanase expression was ≥4 fold higher (P ≤ 0.05) in resistant group compared to sensitive group. In bevacizumab resistant group prostaglandin E receptor 1 and keratin 24 ≥3 fold higher expression ((P ≤ 0.05) was observed. RPPA analysis indicated that proteins such as ER alpha, VEGFR-2, elF4G, E-cadherin, mTOR and beta-catenin expressed ≥ 2.8 fold (P ≤ 0.0001) higher in sorafenib resistant tumors compared to sensitive tumors. Neurofibromin 2 (NF2) protein is expressed 2 fold (P ≤ 0.0001) higher in bevacizumab resistant tumors compared to sensitive tumors. Conclusion: Using gene and protein profiling, we identified important tumor and stroma biomarkers of resistance to anti-VEGF therapy in ovarian cancer that may provide insight into ovarian tumor's adaption principles to VEGF inhibition. Citation Format: Bharat Kumar Devapatla, Pharavee Jaiprasart, Sukyung Woo. Gene and protein expression profiling identifies molecular signature of resistance to anti-VEGF therapy in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1178. doi:10.1158/1538-7445.AM2014-1178

Hippo/YAP pathway for targeted therapy.

Malignant pleural mesothelioma (MPM) is molecularly characterized by loss of function or mutations in the neurofibromin 2 (NF2) and the cyclin-dependent kinase inhibitor 2 genes. NF2 activates a cascade of kinases, called Hippo pathway, which downregulates Yes associated protein (YAP) function as transcription co-activator for TEA domain transcription factors (TEAD). In the absence of functional NF2, the expression of genes essential for cell cycling such as survivin is increased. New therapeutic strategies aimed at interfering with YAP activity include inhibition of hedgehog pathway, which downregulates the YAP protein, verteporfin, which inhibits the assembly of a functional YAP-TEAD transcription factor, and interference with thrombin and lysophosphatidic acid (LPA) receptors downstream signalling, since upon agonist binding they activate YAP.

Hippo Pathway (HP) Activity In Acute Myelogenous Leukemia (AML): Different Prognostic Implications Of TAZ versus YAP Inactivation By Phosphorylation

BackgroundThe highly evolutionarily conserved Hippo pathway (HP) regulates proliferation, apoptosis polarity and stem cell maintenance. In Humans, TAZ (transcriptional co-activator with PDZ-binding motif) and YAP (Yes associated protein) are the functional effectors, regulating gene expression by co-activating several transcription factors (RUNX, TEADS, SMADS). Phosphorylation inactivates both, causing expulsion from the nucleus and either proteosomal degradation or sequestration by 14-3-3 or membrane protein complexes. A 4 protein complex of LATS 1/2 kinase, MST1/2, MOB and SAV1, phosphorylates YAP and TAZ, and is regulated by many pathways including Neurofibromin 2 (NF2) , and WNT, and by cell-cell contact. YAP and TAZ share many binding and interaction partners, sharing overlapping functions, but they also have exclusive interactions leading to some exclusive functions, notably in SMAD family and TGFβ regulation. Deregulation of HP is associated with numerous solid tumors (breast, lung, colorectal, ovary, liver), with upregulation conferring an adverse prognosis, but mutations of HP components is rare, suggesting other mechanism for deregulation. The role of the HP pathway in AML is undefined and similar to most solid tumors, mutations in AML were not observed by TCGA. MethodsTo define the role of the HP in AML we used reverse phase protein array (RPPA) technology to print protein from leukemia enriched cells from 511 newly diagnosed AML patients. The RPPA was probed with 231 antibodies, including antibodies against both total and phosphorylated HP components NF2, YAP (p-Serine 127) and TAZ (p-Serine 89). We could not validate antibodies for LATS 1 or 2. ResultsLevels of total NP2, TAZ YAP were often above that of normal CD34+ cells in 30%, 15% and 17% of cases. Levels of phosphorylated NF2 (p=0.07, 23%) and YAP (p=0.009, 10%) were often above, and those of pTAZ (p= 0.008, 29%) below that of normal CD34+ cells. Higher levels of pTAZ were associated with FAB MO and M1 subtypes, higher CD7 and CD34 expression and higher % marrow blasts. Higher levels of pYAP were associated with lower % marrow and peripheral blood blasts and with cases transformed from RAEBT and CMML. The optimum number of principal components for these 6 antibodies was two, depending on the phosphorylation state of YAP and TAZ. Another statistical method determined the optimal number of patient groups to be 6 (figure 1) with expression 1) Pan Off (19% n=79, Yellow in Fig.) 2) High Total (2%, n =8, Dark Pink.) with very high levels of NF2, TAZ and YAP and high pNF2, 3) Active HP (30% n=123, Blue), with moderate total levels but scant phosphorylation, 4) YAP inactive (19%, n=78, Light Pink) and 5) TAZ inactive (22.5%, n=93, Purple) where only one is phosphorylated and 6) Both inactivated (9% n=37, Green) with both heavily phosphorylated. The TAZ inactive motif was prognostically adverse for overall survival (OS) (Median 30 weeks p=0.009) compared to active, pYAP, both inactivated (median 48-60 weeks), while the High Total pattern had longer OS (median 224 wks). Notably, high pTAZ alone or with pYAP was associated with inferior response and OS in patients treated with demethylating and histone deacetylating drugs. Favorable cytogenetics patients with active HP, High pTAZ or both inactive, treated with fludarabine-HDAC had shorter OS and significantly shorter remission duration (52-70 weeks vs. not reached). High pTAZ was associated with higher SMAD family levels and with unfavorable cytogenetics as well as with higher levels of phosphorylated (inactivated) β-Catenin, likely due to cytoplasmic localization. [Display omitted] ConclusionsHP proteins are heterogeneously expressed in AML and key effectors YAP and TAZ are differentially phosphorylated. Differential phosphorylation of YAP and TAZ has not been previously reported. This suggests that active YAP in the presence of inactive TAZ is inducing expression of genes that provide proliferative or survival advantages to leukemic blasts. The observation that the Highly active HP group did very well may be identifying a high proliferative group that is more susceptible to cycle specific chemotherapy, similar to Burkitt's leukemia. In summary, this data suggest that the HP pathway is active in AML and has a previously undescribed pattern of separate phosphorylation of YAP and TAZ. Disclosures:No relevant conflicts of interest to declare.

Chromosomal structural variations during progression of a prostate epithelial cell line to a malignant metastatic state inactivate the NF2, NIPSNAP1, UGT2B17, and LPIN2 genes

Prostate cancer is the second highest cause of male cancer deaths in the United States. A significant number of tumors advance to a highly invasive and metastatic stage, which is typically resistant to traditional cancer therapeutics. In order to identify chromosomal structural variants that may contribute to prostate cancer progression we sequenced the genomes of a HPV-18 immortalized nonmalignant human prostate epithelial cell line, RWPE1, and compared it to its malignant, metastatic derivative, WPE1-NB26. There were a total of 34 large (>1 Mbp) and 38 small copy number variants (<100 kbp) in WPE1-NB26 that were not present in the precursor cell line. We also identified and validated 46 structural variants present in the two cell lines, of which 23 were unique to WPE1-NB26. Structural variants unique to the malignant cell line inactivated: (1) the neurofibromin2 (NF2) gene, a known tumor suppressor; (2) its neighboring gene NIPSNAP1, another putative tumor suppressor that inhibits TRPV6, an anti-apoptotic oncogene implicated in prostate cancer progression; (3) UGT2B17, a gene that inactivates dihydrotestosterone, a known activator of prostate cancer progression; and (4) LPIN2, a phosphatidic acid phosphatase and a co-factor of PGC1a that is important for lipid metabolism and for suppressing autoinflammation. Our results illustrate the value of comparing the genomes of defined related pairs of cell lines to discover chromosomal structural variants that may contribute to cancer progression.

Merlin, the NF2 Gene Product

Merlin, the protein product of NF2 gene, is one of the most versatile tumor suppressors capable of integrating different mechanisms that regulate cell proliferation, motility, survival and signaling pathways underlying and governing those mechanisms. Merlin is considered a member of the band 4.1 families of cytoskeleton-associated proteins also called ERM family and acts as tumor suppressor. The main cause for transformation of Schwann cells into schwannomas is credited to the inactivation of the neurofibromin 2 (NF2) gene and the consecutive loss of its protein merlin. Recent scientific advances improved our understanding of pathogenic mechanisms involving NF2 gene. The present review brings genetic properties of NF2 gene, molecular characteristics of merlin, summarizes mutational spectra and explains merlin's multifunctional roles regarding its involvement in neurofibromatosis associated tumorigenesis.

The Hippo pathway and human cancer

The Hippo pathway controls organ size in diverse species, whereas pathway deregulation can induce tumours in model organisms and occurs in a broad range of human carcinomas, including lung, colorectal, ovarian and liver cancer. Despite this, somatic or germline mutations in Hippo pathway genes are uncommon, with only the upstream pathway gene neurofibromin 2 (NF2) recognized as a bona fide tumour suppressor gene. In this Review, we appraise the evidence for the Hippo pathway as a cancer signalling network, and discuss cancer-relevant biological functions, potential mechanisms by which Hippo pathway activity is altered in cancer and emerging therapeutic strategies.

Microarray analysis of gene expression in vestibular schwannomas reveals SPP1/MET signaling pathway and androgen receptor deregulation

Vestibular schwannomas are benign neoplasms that arise from the vestibular nerve. The hallmark of these tumors is the biallelic inactivation of neurofibromin 2 (NF2). Transcriptomic alterations, such as the neuregulin 1 (NRG1)/ErbB2 pathway, have been described in schwannomas. In this study, we performed a whole transcriptome analysis in 31 vestibular schwannomas and 9 control nerves in the Affymetrix Gene 1.0 ST platform, validated by quantitative real-time PCR (qRT-PCR) using TaqMan Low Density arrays. We performed a mutational analysis of NF2 by PCR/denaturing high-performance liquid chromatography (dHPLC) and multiplex ligation-dependent probe amplification (MLPA), as well as a microsatellite marker analysis of the loss of heterozygosity (LOH) of chromosome 22q. The microarray analysis demonstrated that 1,516 genes were deregulated and 48 of the genes were validated by qRT-PCR. At least 2 genetic hits (allelic loss and/or gene mutation) in NF2 were found in 16 tumors, seven cases showed 1 hit and 8 tumors showed no NF2 alteration. MET and associated genes, such as integrin, alpha 4 (ITGA4)/B6, PLEXNB3/SEMA5 and caveolin-1 (CAV1) showed a clear deregulation in vestibular schwannomas. In addition, androgen receptor (AR) downregulation may denote a hormonal effect or cause in this tumor. Furthermore, the osteopontin gene (SPP1), which is involved in merlin protein degradation, was upregulated, which suggests that this mechanism may also exert a pivotal role in schwannoma merlin depletion. Finally, no major differences were observed among tumors of different size, histological type or NF2 status, which suggests that, at the mRNA level, all schwannomas, regardless of their molecular and clinical characteristics, may share common features that can be used in their treatment.

Genetic and pharmacological disruption of the TEAD–YAP complex suppresses the oncogenic activity of YAP

The Drosophila TEAD ortholog Scalloped is required for Yki-mediated overgrowth but is largely dispensable for normal tissue growth, suggesting that its mammalian counterpart may be exploited for selective inhibition of oncogenic growth driven by YAP hyperactivation. Here we test this hypothesis genetically and pharmacologically. We show that a dominant-negative TEAD molecule does not perturb normal liver growth but potently suppresses hepatomegaly/tumorigenesis resulting from YAP overexpression or Neurofibromin 2 (NF2)/Merlin inactivation. We further identify verteporfin as a small molecule that inhibits TEAD-YAP association and YAP-induced liver overgrowth. These findings provide proof of principle that inhibiting TEAD-YAP interactions is a pharmacologically viable strategy against the YAP oncoprotein.

Clinicopathologic and Molecular Analysis of a Choroidal Pigmented Schwannoma in the Context of a PTEN Hamartoma Tumor Syndrome

To report the first case of choroidal schwannoma in a patient affected by PTEN hamartoma tumor syndrome (PHTS) and investigate the molecular involvement of the phosphatase and tensin homolog (PTEN) and neurofibromin 2 (NF2) genes in this rare intraocular tumor. Observational case report. A 10-year-old girl diagnosed with PHTS. The enucleated specimen underwent histologic, immunohistochemical, and transmission electronic microscopy. The expression of PTEN and NF2 and their protein products were evaluated by reverse transcription-polymerase chain reaction and immunohistochemistry. Somatic mutations of PTEN and NF2, as well as allelic loss, were investigated by direct sequencing of DNA extracted from the tumor. PTEN epigenetic silencing was investigated by pyrosequencing. Histopathologic and molecular characterization of a choroidal pigmented schwannoma. Histopathologic, immunohistochemical, and electron microscopic analysis demonstrated features consistent with a pigmented cellular schwannoma of the choroid. We found no loss of heterozygosity at the genomic level for the PTEN germline mutation and no promoter hypermethylation or other somatic intragenic mutations. However, we observed an approximate 40% reduction of PTEN expression at both the mRNA and the protein level, indicating that the tumor was nonetheless functionally deficient for PTEN. Although DNA sequencing of NF2 failed to identify any pathologic variants, its expression was abolished within the tumor. We report the first description of a pigmented choroidal schwannoma in the context of a PHTS. This rare tumor showed a unique combination of reduction of PTEN and absence of NF2 expression.

Abstract 17445: NF2 Regulates Mst1 Activation and Ischemia Reperfusion Injury

Neurofibromin 2 (NF2)/merlin is a tumor suppressor that can regulate activation of the hippo pathway in Drosophila and the mammalian liver, thereby inhibiting cell growth and promoting apoptosis. We have shown previously that the mammalian homologue of hippo, Mst1, has a similar function in the heart. Therefore, we sought to determine whether NF2 can mediate activation of Mst1 in the heart and modulate cardiac ischemia/reperfusion (I/R) injury. Both NF2 and Mst1 are activated by oxidative stress in isolated cardiomyocytes in vitro and in the heart following I/R. Increased expression of NF2 in cardiomyocytes increased Mst1 phosphorylation (2.9±0.2-fold vs LacZ, p&lt;0.05) and elicited apoptosis (4.8±1.7-fold vs LacZ, p&lt;0.05) that was prevented by co-expression of kinase inactive Mst1 (61±12% decrease vs. NF2, p&lt;0.05). To examine the importance of endogenous NF2 in I/R injury, we employed two different genetic models - systemic heterozygous nf2 deletion ( nf2 +/- ) and cardiomyocyte-specific disruption using nf2 floxed mice crossed with αMHC-Cre transgenic mice ( nf2 +/flox Cre). We performed global I/R (30/60min) injury on isolated perfused hearts using the Langendorff method. Functional parameters and infarct size were determined following the reperfusion phase. While nf2 +/- hearts showed no difference in function and infarct size (45±5% vs 48±7%, NS) compared to WT, nf2 +/flox Cre hearts had significantly smaller infarcts (22±5% vs 53±8%, p&lt;0.05) and improved function versus nf2 +/flox controls. nf2 +/- hearts also failed to protect against I/R injury in vivo as Infarct/AAR (34±3% vs 32±2%, NS) and myocyte apoptosis (1.5±0.2% vs 1.7±0.3%, NS) were unchanged despite significant attenuation of Mst1 activation in ventricular homogenates (71±8% reduced vs WT, p&lt;0.05). Preliminary experiments to determine the effect of cardiomyocyte nf2 deletion show a trend toward protection (Infarct/AAR, 23±6% vs 33±6%, n=4) in nf2 +/flox Cre versus nf2 +/flox control mice. Based on these data we conclude that nf2 disruption in cardiomyocytes is protective against I/R injury through inhibition of Mst1 activity. However, NF2 signaling in residential non-myocytes appears beneficial for the heart and obscures the detrimental function of NF2 in cardiomyocytes during I/R.

MERLIN EXPRESSION IN PEDIATRIC ANAPLASTIC EPENDYMOMAS REAL TIME PCR STUDY

The most common genetic abnormalities of ependymomas involve the chromosome 22 where there is the oncosuppressor gene neurofibromin 2 (NF2). NF2 mutations are primarily encountered in spinal lesions. In contrast, NF2 alterations do not seem related to tumor grade. We studied the NF2 expression through a real-time polymerase chain reaction in 25 pediatric anaplastic ependymomas. We compared the NF2 expression in neoplastic and non-neoplastic tissues, in supratentorial and infratentorial ependymomas and in primitive and non-primitive tumors (recurrences and metastases). Statistical analysis did not prove significant differences. Our results suggest that NF2 alterations are not typical of intracranial anaplastic ependymomas.

Meningioma of the cerebellopontine angle in identical twins: a case report

Meningiomas in identical twins are extremely rare. To our knowledge, only one previous report of meningiomas in identical twins has been published. We present identical twin sisters with meningiomas. The tumors were located at a similar, but not a common, position (the cerebellopontine angle) in both twins. Histologically, both tumors were diagnosed as meningothelial meningiomas with an angiomatous component. Immunohistochemically, the Ki-67 indices in the two cases were 1.0 and 1.1, and the p53 positive rates were 0.2 and 0.9. The specimens in both cases were reactive to neurofibromin 2 (NF2). A comparative genomic hybridization (CGH) assay revealed an aberration in the long arm of chromosome X, but no aberrations in the long arm of chromosome 22 in either case. These results strongly suggest that genetic aberrations other than NF2 are associated with tumorigenesis in some types of sporadic meningiomas.

Molecular biology of familial and sporadic vestibular schwannomas: implications for novel therapeutics

Vestibular schwannomas (VSs) are benign tumors arising from the sheath of cranial nerve VIII. The pathogenesis underlying most familial and sporadic VSs has been linked to a mutation in a single gene, the neurofibromin 2 (NF2) gene located on chromosome 22, band q11-13.1. In this review, the authors summarized what is known about the epidemiology of NF2 mutations and patients with VSs. The authors also discuss the function of the NF2 gene product, merlin, and describe the known and hypothetical effects of genetic mutations that lead to merlin dysfunction on a broad variety of cellular and histological end points. A better understanding of the molecular pathobiology of VSs may lead to novel therapeutics to augment current modalities of treatment while minimizing morbidity.

BCR expression is decreased in meningiomas showing loss of heterozygosity of 22q within a new minimal deletion region

Neurofibromin 2 ( NF2), located on chromosome arm 22q, has been established as a tumor suppressor gene involved in meningioma pathogenesis. In our study, we investigated 149 meningiomas to determine whether there are additional tumor suppressor genes localized on chromosome 22q, apart from NF2, that might be involved in meningioma pathogenesis. The LOH analysis on chromosome 22q identified two regions of deletion: the first one, which is limited to the NF2 gene locus, and the second one, which is outside this location. The new minimal deletion region (MDR) included the following genes: BCR (breakpoint cluster region), RAB36 (a member of RAS oncogene family), GNAZ [guanine nucleotide binding protein (G protein), α-z polypeptide], and RTDR1 (rhabdoid tumor deletion region gene 1). The expression levels of all these genes, including NF2, were subsequently analyzed by quantitative real-time polymerase chain reaction. We observed a significantly lowered expression level of NF2 in meningiomas with 22q loss of heterozygosity (LOH) within NF2 region compared to the one in meningiomas with 22q retention of heterozygosity (ROH, P<0.05). Similarly, BCR showed a significantly lowered expression in meningiomas with 22q LOH within the new MDR compared to cases with 22q ROH ( P<0.05). Our data, together with the already published information considering BCR function suggest that BCR can be considered as a candidate tumor suppressor gene localized on chromosome 22q which may be involved in meningioma pathogenesis.