Vestibular Schwannoma Cells Research Articles

MiR-205 Inhibits Sporadic Vestibular Schwannoma Cell Proliferation by Targeting Cyclin-Dependent Kinase 14

Sporadic vestibular schwannoma (VS) is a benign primary tumor that arises from the vestibular nerve. Growing VS can negatively compress the brain stem, which can lead to death. MicroRNAs (miRNAs) can negatively regulate target genes at the post-transcriptional level and are critical in tumorigenesis. Studies have demonstrated the tumor suppressive function of microRNA-205-5p (miR-205) across many cancers, but no studies have evaluated the role of miR-205 in sporadic VS. We conducted this study to examine the role of miR-205 in sporadic VS cell proliferation. We evaluated miR-205 expression in sporadic VS tissues and normal great auricular nerve by real-time quantitative polymerase chain reaction. Then, we transfected miR-205 mimics and control oligonucleotides into sporadic VS primary cells to examine the functional significance of miR-205 expression at a cellular level by CCK8 and colony formation and used dual-luciferase reporter assays to find the target gene of miR-205. We determined that miR-205 levels were downregulated in sporadic VS tissues in comparison to normal controls. In functional assays, miR-205 suppressed proliferation and colony formation ability of sporadic VS cells. CDK14 (cyclin-dependent kinase 14) was identified as a target gene of miR-205 by bioinformatics, and validated using dual-luciferase reporter assays. Moreover, miR-205 overexpression inhibited levels of phosphorylated PI3K and Akt. These findings suggested that miR-205 suppressed sporadic VS proliferation by targeting CDK14 and may be considered as a potential drug therapy for sporadic VS treatment in the future.

Effects of Neurod1 Expression on Mouse and Human Schwannoma Cells.

The objective was to explore the effect of the proneuronal transcription factor neurogenic differentiation 1 (Neurod1, ND1) on Schwann cells (SC) and schwannoma cell proliferation. Using a variety of transgenic mouse lines, we investigated how expression of Neurod1 effects medulloblastoma (MB) growth, schwannoma tumor progression, vestibular function, and SC cell proliferation. Primary human vestibular schwannoma (VS) cell cultures were transduced with adenoviral vectors expressing Neurod1. Cell proliferation was assessed by 5-ethynyl-2'-deoxyuridine (EdU) uptake. Basic science investigation. Expression of Neurod1 reduced the growth of slow-growing but not fast-growing MB models. Gene transfer of Neurod1 in human schwannoma cultures significantly reduced cell proliferation in dose-dependent way. Deletion of the neurofibromatosis type 2 (Nf2) tumor-suppressor gene via Cre expression in SCs led to increased intraganglionic SC proliferation and mildly reduced vestibular sensory-evoked potentials (VsEP) responses compared to age-matched wild-type littermates. The effect of Neurod1-induced expression on intraganglionic SC proliferation in animals lacking Nf2 was mild and highly variable. Sciatic nerve axotomy significantly increased SC proliferation in wild-type and Nf2-null animals, and expression of Neurod1 reduced the proliferative capacity of both wild-type and Nf2-null SCs following nerve injury. Expression of Neurod1 reduces slow-growing MB progression and reduces human SC proliferation in primary VS cultures. In a genetic mouse model of schwannomas, we find some effects of Neurod1 expression; however, the high variability indicates that more tightly regulated Neurod1 expression levels that mimic our in vitro data are needed to fully validate Neurod1 effects on schwannoma progression. NA Laryngoscope, 131:E259-E270, 2021.

Combination therapy with mTOR kinase inhibitor and dasatinib as a novel therapeutic strategy for vestibular schwannoma

Neurofibromatosis type 2 (NF2) is an inherited disorder characterized by bilateral vestibular schwannomas (VS) that arise from neoplastic Schwann cells (SCs). NF2-associated VSs are often accompanied by meningioma (MN), and the majority of NF2 patients show loss of the NF2 tumor suppressor. mTORC1 and mTORC2-specific serum/glucocorticoid-regulated kinase 1 (SGK1) are constitutively activated in MN with loss of NF2. In a recent high-throughput kinome screen in NF2-null human arachnoidal and meningioma cells, we showed activation of EPH RTKs, c-KIT, and SFK members independent of mTORC1/2 activation. Subsequently, we demonstrated in vitro and in vivo efficacy of combination therapy with the dual mTORC1/2 inhibitor AZD2014 and the multi-kinase inhibitor dasatinib. For these reasons, we investigated activated mTORC1/2 and EPH receptor-mediated signaling in sporadic and NF2-associated VS. Using primary human VS cells and a mouse allograft model of schwannoma, we evaluated the dual mTORC1/2 inhibitor AZD2014 and the tyrosine kinase inhibitor dasatinib as monotherapies and in combination. Escalating dose-response experiments on primary VS cells grown from 15 human tumors show that combination therapy with AZD2014 and dasatinib is more effective at reducing metabolic activity than either drug alone and exhibits a therapeutic effect at a physiologically reasonable concentration (~0.1 µM). In vivo, while AZD2014 and dasatinib each inhibit tumor growth alone, the effect of combination therapy exceeds that of either drug. Co-targeting the mTOR and EPH receptor pathways with these or similar compounds may constitute a novel therapeutic strategy for VS, a condition for which there is no FDA-approved pharmacotherapy.

Ginsenoside Rh2 induces DNA damage and autophagy in vestibular schwannoma is dependent of LAMP2 transcriptional suppression

Ginsenoside Rh2 (G-Rh2), a component of ginseng extraction, exerted anti-tumor property in the occurrence and progress of human tumors. Vestibular schwannoma (VS) is a kind of benign tumor. Extraction of traditional Chinese herb has been applied to treat VS as adjuvant therapy. Nevertheless, G-Rh2-related molecular mechanisms in VS progress are not yet clear. The purpose of current study is to unveil the function and potential molecular mechanism of Rh2 in VS cellular functions. At first, the viability and apoptosis of VS cells treated with different concentrations of Rh2 were assessed. Autophagy and DNA damage response can be induced by multiple drugs. Here, we observed the changes of autophagy and DNA damage in Rh2-induced VS cells. Based on the experimental data, treatment with Rh2 contributed to cell apoptosis by inducing DNA damage and suppressing DNA damage. LAMP2 (lysosomal associated membrane protein 2), an autophagy inducer, was downregulated in Rh2-treated VS cells. Through mechanism study, we determined that Rh2 led to the transcriptional inactivation of LAMP2 by downregulating its transcription activator NR2F2 (nuclear receptor subfamily 2 group F member 2). In addition, NR2F2 overexpression recovered the role of Rh2 in cell functions, which was further rescued by the silence of LAMP2. Collectively, our study unveiled a novel NR2F2/LAMP2 axis in Rh2-mediated VS cells, which potentially contributes to the therapy for VS.

GENE-37. VESTIBULAR SCHWANNOMA IS COMPRISED OF NEURAL CREST AND IMMUNE SUBGROUPS

Abstract BACKGROUND Vestibular schwannomas (VS) are tumors arising from cranial nerve Schwann cells and show variable outcomes after treatment, including oscillation in size for many years after radiosurgery. To understand the unique biology of VS, we performed multiplatform molecular profiling to develop a single cell atlas of VS and reveal that VS exists on a molecular axis defined by neural crest and immune genes. METHODS Sixty-six sporadic VS with available tissue for molecular profiling from 59 consecutive patients at a single institution were included. 850K DNA methylation arrays and RNA sequencing were used to profile both primary (76%) and recurrent (24%) tumors. Single nuclei RNA sequencing of 7 tumors and single cell RNA sequencing of 3 tumors and cell lines were used to define the cellular composition of VS and heterogeneous changes in molecular programs following irradiation. Molecular subtyping was performed by hierarchical clustering of differentially-methylated DNA probes and validated using transcriptomic data. Mechanistic experiments were performed using cultured human schwann cells and human vestibular schwannoma cells, confocal microscopy, CRISPR interference, proteomic mass spectrometry and lymphocyte migration assays. RESULTS Multiplatform genomic profiling and machine learning revealed that VS is comprised of two distinct molecular subtypes characterized by heterogeneous cell populations. Neural crest enriched VS express primary cilia and are associated with misactivation of the Hedgehog pathway. Consistently, we find that the Hedgehog pathway antagonist vismodegib blocks the growth of human Schwann cells. Irradiation epigenetically reprograms tumors and cell lines to reduce ciliary length, attenuate Hedgehog signaling, activate senescence pathways, and express cytokines and apolipoproteins that recruit lymphocytes and macrophages to immune enriched VS. CONCLUSIONS Our data reveal novel molecular subtypes of VS and establish a framework for understanding how irradiation modifies the epigenome and tumor microenvironment.

Multiplatform Molecular Profiling of Vestibular Schwannoma Reveals Immune Enrichment after Radiosurgery and a Methylation-Based Predictor of Recurrence

Vestibular schwannomas (VS) can cause significant morbidity from cranial nerve deficits and mass effect. Broad molecular differences have been described for intracranial versus spinal axis schwannomas, but little is known about the molecular heterogeneity of VS or predictors of VS recurrence. The objective of this study was to understand VS biology and identify molecular predictors of VS recurrence. Sixty-six sporadic VS from 59 consecutive patients with available tissue who underwent initial resection (44), resection for recurrence after prior surgery (5), resection for recurrence after prior stereotactic radiosurgery (SRS) (11), or resection after both prior surgery and prior SRS (6) at a single institution from 2003-2017 were profiled using Illumina 850K DNA methylation arrays (median follow-up: 4.2 years, median volumetric resection: 91%). Twenty-four VS were further characterized using RNA sequencing (RNA-seq), and 7 were analyzed by single nuclei RNA-seq. Molecular subtyping was performed by hierarchical clustering of differentially-methylated DNA probes and validated using transcriptomic data. In vitro functional experiments were performed using human VS cells. Clustering of genome-wide DNA methylation probes revealed that VS are comprised of 2 subgroups that are delineated by enrichment of neural crest or immune genes. Fifteen of 35 immune-enriched VS (43%) had prior SRS, while only 2 of 31 neural crest-enriched VS (6%) had prior SRS (p<0.001). Analysis of 6 patients with matched primary and recurrent VS samples revealed that all recurrent VS after prior SRS exhibited similar methylation profiles and were enriched in immune genes. In contrast, the methylation profile of VS that recurred after prior surgery did not deviate from the primary tumor. Hypomethylation of G protein-coupled estrogen receptor 1 (GPER) promoter was prognostic for local failure in multivariate regression irrespective of primary treatment modality (p<0.0001). Radiation of VS cells in vitro recapitulated gene expression of immune-enriched VS. VS are comprised of 2 molecular subgroups characterized by differential enrichment of neural crest or immune genes. VS with prior SRS are associated with immune-enrichment. GPER promoter hypomethylation is prognostic for VS recurrence. These data illuminate the biology of VS and shed light on potential molecular therapies.

Preclinical assessment of MEK1/2 inhibitors for neurofibromatosis type 2-associated schwannomas reveals differences in efficacy and drug resistance development.

Neurofibromatosis type 2 (NF2) is a genetic tumor-predisposition disorder caused by NF2/merlin tumor suppressor gene inactivation. The hallmark of NF2 is formation of bilateral vestibular schwannomas (VS). Because merlin modulates activity of the Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, we investigated repurposing drugs targeting MEK1 and/or MEK2 as a treatment for NF2-associated schwannomas. Mouse and human merlin-deficient Schwann cell lines (MD-MSC/HSC) were screened against 6 MEK1/2 inhibitors. Efficacious drugs were tested in orthotopic allograft and NF2 transgenic mouse models. Pathway and proteome analyses were conducted. Drug efficacy was examined in primary human VS cells with NF2 mutations and correlated with DNA methylation patterns. Trametinib, PD0325901, and cobimetinib were most effective in reducing MD-MSC/HSC viability. Each decreased phosphorylated pERK1/2 and cyclin D1, increased p27, and induced caspase-3 cleavage in MD-MSCs. Proteomic analysis confirmed cell cycle arrest and activation of pro-apoptotic pathways in trametinib-treated MD-MSCs. The 3 inhibitors slowed allograft growth; however, decreased pERK1/2, cyclin D1, and Ki-67 levels were observed only in PD0325901 and cobimetinib-treated grafts. Tumor burden and average tumor size were reduced in trametinib-treated NF2 transgenic mice; however, tumors did not exhibit reduced pERK1/2 levels. Trametinib and PD0325901 modestly reduced viability of several primary human VS cell cultures with NF2 mutations. DNA methylation analysis of PD0325901-resistant versus -susceptible VS identified genes that could contribute to drug resistance. MEK inhibitors exhibited differences in antitumor efficacy resistance in schwannoma models with possible emergence of trametinib resistance. The results support further investigation of MEK inhibitors in combination with other targeted drugs for NF2 schwannomas.

RETRACTED: Brazilein induces apoptosis and G1/G0 phase cell cycle arrest by up-regulation of miR-133a in human vestibular schwannoma cells

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor-in-Chief. Given the comments of Dr Elisabeth Bik regarding this article “... the Western blot bands in all 400+ papers are all very regularly spaced and have a smooth appearance in the shape of a dumbbell or tadpole, without any of the usual smudges or stains. All bands are placed on similar looking backgrounds, suggesting they were copy/pasted from other sources, or computer generated”, the journal requested the authors to provide the raw data. However, the authors were not able to fulfil this request and therefore the Editor-in-Chief decided to retract the article.

Persistent Oxidative Stress in Vestibular Schwannomas After Stereotactic Radiation Therapy.

Stereotactic radiation therapy is increasingly used to treat vestibular schwannomas (VSs) primarily and to treat tumor remnants following microsurgery. Little data are available regarding the effects of radiation on VS cells. Tyrosine nitrosylation is a marker of oxidative stress following radiation in malignant tumors. It is not known how long irradiated tissue remains under oxidative stress, and if such modifications occur in benign neoplasms such as VSs treated with significantly lower doses of radiation. We immunostained sections from previously radiated VSs with an antibody that recognizes nitrosylated tyrosine residues to assess for ongoing oxidative stress. Immunohistochemical analysis. Four VSs, which recurred after excision, were treated with stereotactic radiation therapy. Ultimately each tumor required salvage reresection for regrowth. Histologic sections of each tumor before and after radiation were immunolabeled with a monoclonal antibody specific to nitrotyrosine and compared. Two VSs that underwent reresection of a growing tumor remnant without previous radiation therapy served as additional controls. Irradiated tumors enlarged in volume by 3.16 to 8.62 mL following radiation. Preradiation sections demonstrated little to no nitrotyrosine immunostaining. Three of four of irradiated VSs demonstrated increased nitrotyrosine immunostaining in the postradiation sections compared with preradiation tumor sections. Nonirradiated VSs did not label with the antinitrotyrosine antibody. VSs exhibit oxidative stress up to 7 years after radiotherapy, yet these VSs continued to enlarge. Thus, VSs that grow following radiation appear to possess mechanisms for cell survival and proliferation despite radiation-induced oxidative stress.

Fluorescent Detection of Merlin-deficient Schwann Cells and Primary Human Vestibular Schwannoma Cells Using Sodium Fluorescein.

Merlin-deficient Schwann cells (MD-SC) and primary human vestibular schwannoma (VS) cells exhibit selective uptake of sodium-fluorescein (SF), allowing for fluorescent detection and improved visualization of tumor cells, when compared with Schwann cells (SC). SF is a fluorescent compound used for fluorescence-guided resection of gliomas. The utility of SF for VS surgery has not been assessed. Mouse MD-SCs and rat SCs were cultured on 96-well plates at different cell densities and treated with SF at several drug concentrations and durations. Relative fluorescence units (RFU) were measured using a fluorometer to determine optimal treatment parameters in vitro. Subsequently, a four-point Likert scale for fluorescence visualization of pelleted cells was created and validated. Blinded observers rated SF-treated primary human VS and SC cultures, which were developed from deidentified specimens obtained from live and cadaveric donors, respectively. In contrast to SCs that showed low levels of fluorescence, MD-SCs demonstrated dose-dependent increases in RFUs when treated with incremental dosages of SF as well as longer treatment and fluorescent excitation times. In addition, RFUs were higher at greater MD-SC densities. The Likert scale for fluorescence visualization was validated using nine blinded observers and there were excellent inter- and intrarater reliabilities (intraclass coefficients of 0.989 and >0.858, respectively). Using the Likert scale, human VS treated with SF received higher scores than human SCs (p < 0.001). Mouse MD-SC and human VS cells demonstrate preferential uptake of SF when compared with normal primary SCs. Observers detected differences in fluorescence using the validated Likert scale. Further investigations into the utility of SF-guidance in VS surgery are warranted.

Ailanthone Promotes Human Vestibular Schwannoma Cell Apoptosis and Autophagy by Downregulation of miR-21.

Ailanthone (AIL) is a quassinoid isolated from the traditional Chinese medicinal herb Ailanthus altissima. The antitumor activities of AIL have been reported in several cancers. The purpose of the present study was to explore the effect of AIL on vestibular schwannomas (VSs). Various concentrations of AIL (0–1 μM) were used to treat human primary VS cells, and then cell viability, proliferation, apoptosis, and autophagy were assessed. Expression of miR-21 in VS cells was altered by miRNA transfection. The functional actions of AIL on miR-21 dysregulated cells were also assessed. AIL significantly reduced the viability of VS cells, and the IC50 value was 0.48 ± 0.023 μM. In response to 0.6 μM AIL, BrdU+ cell rate and cyclin D1 expression were reduced, apoptotic cell rate was increased, caspase 3 and caspase 9 were cleaved, Beclin-1 and LC3-II were accumulated, and p62 was downregulated. miR-21 was lowly expressed in AIL-treated cells, and AIL-induced apoptosis and autophagy were attenuated by miR-21 overexpression. In addition, AIL downregulated Ras and Raf and deactivated MEK, ERK, mTOR, and p70S6K, while the downregulation and deactivation induced by AIL were reversed by miR-21 overexpression. To conclude, AIL inhibited VS cell proliferation and induced apoptosis and autophagy. The antitumor activities of AIL in VS cells were realized possibly via downregulation of miR-21 and blocking the Ras/Raf/MEK/ERK and mTOR pathways.

Deregulation of the Hippo Pathway Promotes Tumor Cell Proliferation Through YAP Activity in Human Sporadic Vestibular Schwannoma

Vestibular schwannomas (VSs) can cause serious neurological defects including hearing loss and facial paralysis. The aim of this study is to identify whether Hippo signaling could be a potential targetable pathway for clinical treatment in VSs. Gene expression profiling was performed in 10 sporadic VSs and 4 normal nerves to identify aberrant genes expression of the Hippo pathway. Western blotting and immunohistochemical staining were used to examine the expression of Hippo core components in 20 VS samples. Neurofibromatosis type 2 (NF2) gene sequencing was also performed in all tumors using sanger sequencing. Verteporfin, inhibitor of yes-associated protein (YAP)-TEA domain family member, was used to assess the effect ofproliferation inhibition in human primary VS cells and RT4-D6P2T cell line. We found 51 differentially expressed genes of the Hippo pathway between VSs and healthy controls. Unsupervised analysis identified the 2 molecular variants that significantly related with distinct NF2 mutation status. The phosphorylation levels of large tumor suppressor 1 and YAP were significantly decreased in NF2-mutated VSs compared with wild-type VSs and normal nerves. Immunohistochemical staining showed that increased nuclear YAP expression in VSs was positively correlated with high Ki-67 index and low Merlin expression. Verteporfin reduced viability of primary VS cells and RT4-D6P2T cells. Our findings implicate that deregulation of the Hippo pathway as a molecular mechanism of pathogenesis in human VSs, and suggest inhibition of this pathway as a potential treatment strategy.

Computational repositioning and preclinical validation of mifepristone for human vestibular schwannoma

The computational repositioning of existing drugs represents an appealing avenue for identifying effective compounds to treat diseases with no FDA-approved pharmacotherapies. Here we present the largest meta-analysis to date of differential gene expression in human vestibular schwannoma (VS), a debilitating intracranial tumor, and use these data to inform the first application of algorithm-based drug repositioning for this tumor class. We apply an open-source computational drug repositioning platform to gene expression data from 80 patient tumors and identify eight promising FDA-approved drugs with potential for repurposing in VS. Of these eight, mifepristone, a progesterone and glucocorticoid receptor antagonist, consistently and adversely affects the morphology, metabolic activity, and proliferation of primary human VS cells and HEI-193 human schwannoma cells. Mifepristone treatment reduces VS cell viability more significantly than cells derived from patient meningiomas, while healthy human Schwann cells remain unaffected. Our data recommend a Phase II clinical trial of mifepristone in VS.

Anti-tumor effects of cold atmospheric pressure plasma on vestibular schwannoma demonstrate its feasibility as an intra-operative adjuvant treatment.

Vestibular schwannoma (VS), although a benign intracranial tumor, causes morbidities by brainstem compression. Since chemotherapy is not very effective in most Nf2-negative schwannomas, surgical removal or radiation therapy is required. However, depending on the size and site of the tumor, these approaches may cause loss of auditory or vestibular functions, and severely decrease the post-surgical wellbeing. Here, we examined the feasibility of cold atmospheric pressure plasma (CAP) as an intra-operative adjuvant treatment for VS after surgery. Cell death was efficiently induced in both human HEI-193 and mouse SC4 VS cell lines upon exposure to CAP for seven minutes. Interestingly, both apoptosis and necroptosis were simultaneously induced by CAP treatment, and cell death was not completely inhibited by pan-caspase and receptor-interacting serine/threonine-protein kinase 1 (RIK1) inhibitors. Upon CAP exposure, cell death phenotype was similarly observed in patient-derived primary VS cells and tumor mass. In addition, CAP exposure after the surgical removal of primary tumor efficiently inhibited tumor recurrence in SC4-grafted mouse models. Collectively, these results strongly suggest that CAP should be developed as an efficient adjuvant treatment for VS after surgery to eliminate the possible remnant tumor cells, and to minimize the surgical area in the brain for post-surgical wellbeing.

Combination Therapy with c-Met and Src Inhibitors Induces Caspase-Dependent Apoptosis of Merlin-Deficient Schwann Cells and Suppresses Growth of Schwannoma Cells.

Neurofibromatosis type 2 (NF2) is a nervous system tumor disorder caused by inactivation of the merlin tumor suppressor encoded by the NF2 gene. Bilateral vestibular schwannomas are a diagnostic hallmark of NF2. Mainstream treatment options for NF2-associated tumors have been limited to surgery and radiotherapy; however, off-label uses of targeted molecular therapies are becoming increasingly common. Here, we investigated drugs targeting two kinases activated in NF2-associated schwannomas, c-Met and Src. We demonstrated that merlin-deficient mouse Schwann cells (MD-MSC) treated with the c-Met inhibitor, cabozantinib, or the Src kinase inhibitors, dasatinib and saracatinib, underwent a G1 cell-cycle arrest. However, when MD-MSCs were treated with a combination of cabozantinib and saracatinib, they exhibited caspase-dependent apoptosis. The combination therapy also significantly reduced growth of MD-MSCs in an orthotopic allograft mouse model by greater than 80% of vehicle. Moreover, human vestibular schwannoma cells with NF2 mutations had a 40% decrease in cell viability when treated with cabozantinib and saracatinib together compared with the vehicle control. This study demonstrates that simultaneous inhibition of c-Met and Src signaling in MD-MSCs triggers apoptosis and reveals vulnerable pathways that could be exploited to develop NF2 therapies. Mol Cancer Ther; 16(11); 2387-98. ©2017 AACR.

Chr19_34670, a newly identified miRNA, represses rapid tumor growth of cystic vestibular schwannomas by targeting transforming growth factor α and inhibiting MAPK pathway

// Zirong Huo 1, 2, 3, * , Shuang Yan 1, 2, 3, * , Tao Yang 1, 2, 3 , Zhentao Wang 1, 2, 3 , Qi Huang 2, 3 , Zhaoyan Wang 1, 2, 3 , Hongsai Chen 1, 2, 3 , Penghui Chen 1, 2, 3 , Huan Jia 1, 2, 3 , Zhihua Zhang 1, 2, 3 and Hao Wu 1, 2, 3 1 Department of Otolaryngology-Head & Neck Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China 2 Ear Institute, Shanghai Jiaotong University School of Medicine, Shanghai, China 3 Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases, Shanghai, China * These authors have contributed equally to this work Correspondence to: Zhihua Zhang, email: zhihua.zhang2015@aliyun.com Hao Wu, email: wuhao622@sina.cn Keywords: miRNA; cystic vestibular schwannoma; proliferation; tumor growth; pathogenesis Received: March 16, 2017 Accepted: July 29, 2017 Published: January 02, 2018 ABSTRACT Purpose: Cystic vestibular schwannoma (CVS) is an aggressive form of vestibular schwannoma (VS) with rapid growth and poor outcomes. The molecular basis of CVS remains unclear. In this study, we aimed to investigate whether microRNAs (miRNAs) contribute to the development of CVS. Methods: We identified miRNAs that were differentially expressed between CVS and solid VS (SVS) tissues using high-throughput sequencing and quantitative real-time PCR analysis. Gene targets of the identified miRNA were predicted by bioinformatics tools and validated by luciferase reporter analysis. The effects of the miRNA and its target genes on the proliferation of primary VS cells were examined using CCK-8, flow cytometry, colony formation assay, and western-blot. Results: We identified a novel miRNA, chr19_34670, downregulated in CVS compared to SVS. chr19_34670 directly targeted the 3’ untranslated region of transforming growth factor α (TGFα) mRNA and suppressed its expression. TGFα overexpression motivated VS cell proliferation, while this effect could be attenuated by chr19_34670. We also found that chr19_34670 inhibited the function of key proteins in the MAPK pathway by suppressing phosphorylation of ERK and MEK. Conclusions: chr19_34670 suppresses the aggressive proliferation observed in CVS by inhibiting TGFα expression and phosphorylated proteins in the MAPK pathway.

Vascular hyperpermeability as a hallmark of phacomatoses: is the etiology angiogenesis related to or comparable with mechanisms seen in inflammatory pathways? Part II: angiogenesis- and inflammation-related molecular pathways, tumor-associated macrophages, and possible therapeutic implications: a comprehensive review.

Phacomatoses are a special group of familial hamartomatous syndromes with unique neurocutaneous manifestations as well as characteristic tumors. Neurofibromatosis type 2 (NF2) and tuberous sclerosis complex (TSC) are representatives of this family. A vestibular schwannoma (VS) and subependymal giant cell tumor (SGCT) are two of the most common intracranial tumors associated with these syndromes, related to NF2 and TSC, respectively. These tumors can present with an obstructive hydrocephalus due to their location adjacent to or in the ventricles. Remarkably, both tumors are also known to have a unique association with elevated protein concentrations in the cerebrospinal fluid (CSF), sometimes in association with a non-obstructive (communicating) hydrocephalus. Of the two, SGCT has been shown to be associated with a predisposition to CSF clotting, causing a debilitating recurrent shunt obstruction. However, the exact relationship between high protein levels and clotting of CSF remains unclear, nor do we understand the precise mechanism of CSF clotting observed in SGCT. 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 pathophysiological processes for that in the context of tumorigenesis are angiogenesis and inflammation. Both these processes are correlated to the phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin pathway which is tumorigenesis related in many neoplasms and nearly all phacomatoses. In this review, we discuss the influence of angiogenesis and inflammation pathways on vascular permeability in VSs and SGCTs at the phenotypic level as well as their possible genetic and molecular determinants. Part I described the historical perspectives and clinical aspects of the relationship between vascular permeability, abnormal CSF protein levels, clotting of the CSF, and communicating hydrocephalus. Part II hereafter describes the different cellular and molecular pathways involved in angiogenesis and inflammation observed in both tumors and explores the existing metabolic overlap between inflammation and coagulation. Interestingly, while increased angiogenesis can be observed in both tumors, inflammatory processes seem significantly more prominent in SGCT. Both SGCT and VS are characterized by different subgroups of tumor-associated macrophages (TAMs): the pro-inflammatory M1 type is predominating in SGCTs, while the pro-angiogenetic M2 type is predominating in VSs. We suggest that a lack of NF2 protein in VS and a lack of TSC1/TSC2 proteins in SGCT significantly influence this fundamental difference between the two tumor types by changing the dominant TAM type. Since inflammatory reactions and coagulation processes are tightly connected, the pro-inflammatory state of SGCT may also explain the associated tendency for CSF clotting. The underlying cellular and molecular differences observed can potentially serve as an access point for direct therapeutic interventions for tumors that are specific to certain phacomatoses or others that also carry such genetic changes.

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.

Sulforaphane, a natural component of broccoli, inhibits vestibular schwannoma growth in vitro and in vivo.

Vestibular schwannoma (VS) is an intracranial tumor that causes significant morbidity, including hearing loss, tinnitus, dizziness, and possibly even death from brainstem compression. However, FDA-approved pharmacologic treatments for VS do not exist. Sulforaphane (SFN) is a naturally occurring isothiocyanate found in cruciferous vegetables, such as broccoli, with potent chemoprotective effects in several cell types. Our objective was to determine whether SFN is effective against VS in vitro and in vivo. Human primary VS cells, HEI-193 schwannoma cells, and SC4 Nf2−/− Schwann cells were used to investigate the inhibitory effects of SFN in vitro. Cell proliferation was assessed by bromodeoxyuridine (BrdU) incorporation, and cell viability and metabolic activity was calculated by MTT assay. Apoptosis was measured by flow cytometry, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and Western blot for cleaved caspases. A mouse model with a murine schwannoma allograft was also used to examine the antitumor activity of SFN. SFN exhibited significant antiproliferative activity in schwannoma cells in vitro, via the inhibition of HDAC activity and the activation of ERK. SFN treatment induced apoptosis and cell cycle arrest at the G2/M phase. SFN also significantly inhibited schwannoma growth in vivo. Our preclinical studies motivate a future prospective clinical study of SFN for the treatment of VS.

Extracellular vesicles derived from human vestibular schwannomas associated with poor hearing damage cochlear cells.

Vestibular schwannoma (VS) is a tumor of the vestibular nerve that transmits balance information from the inner ear to the brain. Sensorineural hearing loss occurs in 95% of patients with these tumors, but the cause of this loss is not well understood. We posit a role of VS-secreted extracellular vesicles (EVs) as a major contributing factor in cochlear nerve damage. Using differential centrifugation, we isolated EVs from VS cell line HEI-193 and primary cultured human VS cells from patients with good hearing or poor hearing. The EVs were characterized using a Nanosight device and transmission electron microscopy and by extracting their RNA content. The EVs' effects on cultured murine spiral ganglion cells and organotypic cochlear cultures were studied using a transwell dual-culture system and by direct labeling of EVs with PKH-67 dye. EV-induced changes in cochlear cells were quantified using confocal immunohistochemistry. Transfection of VS cells with a green fluorescent protein-containing plasmid was confirmed with reverse transcription PCR. Human VS cells, from patients with poor hearing, produced EVs that could damage both cultured murine cochlear sensory cells and neurons. In contrast, EVs derived from VS cells from patients with good hearing did not damage the cultured cochlear cells. This is the first report on EVs derived from VSs and on the capacity of EVs from VSs from patients with hearing loss to selectively damage cochlear cells, thereby identifying a potential novel mechanism of VS-associated sensorineural hearing loss.