Vestibular Schwannomas Cell Proliferation Research Articles

Prospects of Drug Therapy of Vestibular Schwannoma

Vestibular schwannoma (VS) is one of the most common types of benign tumors of the central nervous system. At present, the prevailing treatment methods of VS include surgery, stereotactic radiotherapy, and follow-up observation, etc. However, there is still no drug therapy available for treating VS. Although the surgical technique is relatively mature, the complications cannot be completely avoided. Furthermore, both the growth rate of different cases and patients' sensitivity to radiotherapy vary greatly. With the constant progress made in molecular biology research, most of the studies on the growth mechanism of VS focus on the upstream and downstream of neurofibromin 2 ( NF2) gene and merlin protein, and a number of corresponding targets, including receptor protein tyrosine kinase (RTK), vascular endothelial growth factor receptor (VEGFR), mammalian target of rapamycin complex 1 (mTORC1) and platelet derived growth factor receptor (PDGFR). It has been reported in some studies that quite a few drugs could inhibit the proliferation of VS cells. Most of the studies are still in the stage of in vitro cell experiment and/or animal experiment. A small number of studies have entered phase Ⅰ and phase Ⅱ clinical trials, but have not led to any clinical treatment yet. This paper provides a comprehensive understanding of the current status and the prospects of drug therapies of VS, which is conducive to the development of subsequent research.

Long non-coding RNA BRCAT54 sponges microRNA-21 in vestibular schwannoma to suppress cell proliferation

ABSTRACT BRCAT54 (also known as MRPS30 divergent transcript) is an anti-tumor long non-coding RNA (lncRNA) in lung cancer, while its role in vestibular schwannoma (VS) is unclear. We predicted that BRCAT54 could interact with microRNA (miR)-21, which suppresses VS cell proliferation. This study was then carried out to study the interaction between BRCAT54 and miR-21 in VS. A total of 56 VS samples and 42 normal vestibular nerve (VN) samples were included in this study. The expression of BRCAT54 and miR-21 in these samples were analyzed with RT-qPCR. Subcellular location of BRCAT54 in primary VS cells was analyzed by subcellular fractionation assay. The direct interaction between BRCAT54 and miR-21 was analyzed through RNA pull-down assay. Overexpression assay was performed to explore the interaction between BRCAT54 and miR-21. The role of BRCAT54 and miR-21 in primary VS cell proliferation was analyzed using BrdU assay. We found that BRCAT54 was downregulated in VS samples than that in VN samples, while miR-21 was upregulated in VS samples. BRCAT54 and miR-21 were not closely correlated. BRCAT54 was detected in both nuclear and cytoplasm samples, and BRCAT54 directly interacted with miR-21. However, BRCAT54 and miR-21 did not affect the expression of each other. BRCAT54 suppressed primary VS cell proliferation and inhibited the role of miR-21 in promoting cell proliferation. Therefore, BRCAT54 may sponge miR-21 to suppress cell proliferation in VS.

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.

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.

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.

Preclinical validation of anti-nuclear factor-kappa B therapy to inhibit human vestibular schwannoma growth

Vestibular schwannomas (VSs), the most common tumors of the cerebellopontine angle, arise from Schwann cells lining the vestibular nerve. Pharmacotherapies against VS are almost non-existent. Although the therapeutic inhibition of inflammatory modulators has been established for other neoplasms, it has not been explored in VS. A bioinformatic network analysis of all genes reported to be differentially expressed in human VS revealed a pro-inflammatory transcription factor nuclear factor-kappa B (NF-κB) as a central molecule in VS pathobiology. Assessed at the transcriptional and translational level, canonical NF-κB complex was aberrantly activated in human VS and derived VS cultures in comparison to control nerves and Schwann cells, respectively. Cultured primary VS cells and VS-derived human cell line HEI-193 were treated with specific NF-κB siRNAs, experimental NF-κB inhibitor BAY11-7082 (BAY11) and clinically relevant NF-κB inhibitor curcumin. Healthy human control Schwann cells from the great auricular nerve were also treated with BAY11 and curcumin to assess toxicity. All three treatments significantly reduced proliferation in primary VS cultures and HEI-193 cells, with siRNA, 5 μM BAY11 and 50 μM curcumin reducing average proliferation (±standard error of mean) to 62.33% ± 10.59%, 14.3 ± 9.7%, and 23.0 ± 20.9% of control primary VS cells, respectively. These treatments also induced substantial cell death. Curcumin, unlike BAY11, also affected primary Schwann cells. This work highlights NF-κB as a key modulator in VS cell proliferation and survival and demonstrates therapeutic efficacy of directly targeting NF-κB in VS.

Nonsteroidal anti-inflammatory medications are cytostatic against human vestibular schwannomas

Vestibular schwannomas (VSs) are the most common tumors of the cerebellopontine angle. Significant clinical need exists for pharmacotherapies against VSs. Motivated by previous findings that immunohistochemical expression of cyclooxygenase 2 (COX-2) correlates with VS growth rate, we investigated the role of COX-2 in VSs and tested COX-2 inhibiting salicylates against VSs. COX-2 was found to be aberrantly expressed in human VS and primary human VS cells in comparison with control human nerve specimens and primary Schwann cells (SCs), respectively. Furthermore, levels of prostaglandin E2, the downstream enzymatic product of COX-2, were correlated with primary VS culture proliferation rate. Because COX-2 inhibiting salicylates such as aspirin are well tolerated and frequently clinically used, we assessed their repurposing for VS. Changes in proliferation, cell death, and cell viability were analyzed in primary VS cultures treated with aspirin, sodium salicylate, or 5-aminosalicylic acid. These drugs neither increased VS cell death nor affected healthy SCs. The cytostatic effect of aspirin in vitro was in concurrence with our previous clinical finding that patients with VS taking aspirin demonstrate reduced tumor growth. Overall, this work suggests that COX-2 is a key modulator in VS cell proliferation and survival and highlights salicylates as promising pharmacotherapies against VS.

Contribution of persistent C-Jun N-terminal kinase activity to the survival of human vestibular schwannoma cells by suppression of accumulation of mitochondrial superoxides

Vestibular schwannomas (VSs) result from inactivating mutations in the merlin tumor suppressor gene. The merlin protein suppresses a variety of progrowth kinase-signaling cascades, including extracellular regulated kinase/mitogen-activated protein kinase (ERK/MAPK), c-Jun N-terminal kinase (JNK), and phosphatidyl-inositol 3-kinase (PI3-K)/Akt. Recent studies indicate that ERKs and Akt are active in human VSs, and here we show that JNKs are also persistently active in human VS cells. With use of cultures of human VSs, we investigated the contribution of each of these signals to the proliferative and survival response of VS cells. Inhibition of ERK or Akt signaling reduced VS cell proliferation but did not increase apoptosis, whereas inhibition of JNK with SP600125, I-JIP, or siRNA knock-down reduced VS cell proliferation and survival by inducing apoptosis. By contrast, JNK activity promotes apoptosis in normal Schwann cells. Inhibition of JNK increased the fluorescence intensity of VS cells loaded with 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA), a fluorescent probe for reactive oxygen species (ROS). Furthermore, ebselen, a ROS scavenger, rescued VS cells with suppressed JNK from apoptosis, suggesting that JNK activity protects VS cells from apoptosis by limiting accumulation of ROS. VS cultures treated with JNK inhibitors demonstrated significantly higher levels of MitoSOX Red fluorescence, implying that persistent JNK activity specifically suppresses superoxide production in the mitochondria. Overexpression of superoxide dismutase 2 (MnSOD; mitochondrial SOD) prevented apoptosis in VS cells with suppressed JNK signaling. Taken together, these results indicate that persistent JNK activity enhances VS cell survival, at least in part, by suppressing accumulation of mitochondrial superoxides.

MicroRNA-21 Overexpression Contributes to Vestibular Schwannoma Cell Proliferation and Survival

Elevated levels of hsa-microRNA-21 (miR-21) in vestibular schwannomas (VSs) may contribute to tumor growth by downregulating the tumor suppressor phosphatase and tensin homolog (PTEN) and consequent hyperactivation of protein kinase B (AKT), a key signaling protein in the cellular pathways that lead to tumor growth. Vestibular schwannomas are benign tumors that arise from the vestibular nerve. Left untreated, VSs can result in hearing loss, tinnitus, vestibular dysfunction, trigeminal nerve dysfunction, and can even become life threatening. Despite efforts to characterize the VS transcriptome, the molecular pathways that lead to tumorigenesis are not completely understood. MicroRNAs are small RNA molecules that regulate gene expression posttranscriptionally by blocking the production of specific target proteins. We examined miR-21 expression in VSs. To determine the functional significance of miR-21 expression in VS cells, we transfected primary human VS cultures with anti-miR-21 or control, scrambled oligonucleotides. We found consistent overexpression of miR-21 when compared with normal vestibular nerve tissue. Furthermore, elevated levels of miR-21 correlated with decreased levels of PTEN, a known molecular target of miR-21. Anti-miR-21 decreased VS cell proliferation in response to platelet-derived growth factor stimulation and increased apoptosis, suggesting that increased miR-21 levels contributes to VS growth. Because PTEN regulates signaling through the growth-promoting phosphoinositide 3-kinase/AKT pathway, our findings suggest that miR-21 may be a suitable molecular target for therapies aimed specifically at reducing VS growth.

The ErbB Inhibitors Trastuzumab and Erlotinib Inhibit Growth of Vestibular Schwannoma Xenografts in Nude Mice

To analyze the ability of ErbB inhibitors to reduce the growth of vestibular schwannoma (VS) xenografts. Vestibular schwannoma xenografts were established in the interscapular fat pad in nude mice for 4 weeks. Initially, a small cohort of animals was treated with the ErbB2 inhibitor trastuzumab or saline for 2 weeks. Animals also received bromodeoxyuridine injections to label proliferating cells. In a longer-term experiment, animals were randomized to receive trastuzumab, erlotinib (an ErbB kinase inhibitor), or placebo for 12 weeks. Tumor growth was monitored by magnetic resonance imaging during the treatment period. Cell death was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling of fragmented DNA. Tumors can be distinguished with T2-weighted magnetic resonance imaging sequences. Trastuzumab significantly reduced the proliferation of VS cells compared with control (p < 0.01) as analyzed by bromodeoxyuridine uptake. Control tumors demonstrated slight growth during the 12-week treatment period. Both trastuzumab and erlotinib significantly reduced the growth of VS xenografts (p < 0.05). Erlotinib, but not trastuzumab, resulted in a significant increase in the percentage of terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling of fragmented DNA-positive VS cells (p < 0.01). In this preliminary study, the ErbB inhibitors trastuzumab and erlotinib decreased growth of VS xenografts in nude mice, raising the possibility of using ErbB inhibitors in the management of patients with schwannomas, particularly those with neurofibromatosis Type 2.

Fowler Award Presentation: Effects of ErbB2 Signaling on the Response of Vestibular Schwannoma Cells to γ‐Irradiation

AbstractObjective: For vestibular schwannomas (VSs) that require treatment, options are limited to microsurgery or irradiation (IR). Development of alternative therapies that augment or replace microsurgery or IR would benefit patients not suitable for current therapies. This study explored the ability of ErbB2 inhibitors to modulate the effects of IR on VS cells.Study Design: Prospective study using primary cultures derived from human VSs.Methods: Primary cultures of VS cells were derived from acutely resected tumors. Cultures received single escalating doses (15–40 Gy) of γ‐irradiation from a 137Cs γ‐irradiation source. Cell proliferation was determined by BrdU uptake and apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Trastuzumab (Herceptin) and PD158780 were independently used to inhibit ErbB2 signaling while neuregulin‐1β (NRG‐1) was used to activate ErbB2.Results: IR induces VS cell cycle arrest and apoptosis in doses greater than 20 Gy, demonstrating that VS cells are relatively radioresistant. This radioresistance likely arises from their low proliferative capacity as a sublethal dose of IR (10 Gy) strongly induces deoxyribonucleic acid (DNA) damage evidenced by histone H2AX phosphorylation. Inhibition of ErbB2, which decreases VS cell proliferation, protects VS cells from radiation‐induced apoptosis, while NRG‐1, an ErbB2 ligand and VS cell mitogen, increases radiation‐induced VS cell apoptosis.Conclusions: Compared with many neoplastic conditions, VS cells are relatively radioresistant. The radio‐protective effect of ErbB2 inhibitors implies that the sensitivity of VS cells to IR depends on their proliferative capacity. These results hold important implications for current and future treatment strategies.

Constitutive neuregulin‐1/ErbB signaling contributes to human vestibular schwannoma proliferation

Vestibular schwannomas (VSs) are benign tumors that arise from the Schwann cells (SCs) lining the vestibular nerve. VS cells survive and proliferate far from neurons and axonally derived growth factors. We have previously shown that VSs produce the glial growth factor, neuregulin-1 (NRG1), and its receptors, ErbB2 and ErbB3. In the present work, we explore the contribution of constitutive NRG1:ErbB signaling to human VS cell proliferation. We confirm that human VSs, which express markers of immature and denervated SCs, also express endogenous NRG1 and activated ErbB2. We find that a blocking anti-NRG1 antibody and trastuzumab (Herceptin, HCN), a humanized anti-ErbB2 inhibitory monoclonal antibody, effectively inhibit NRG1 induced SC proliferation. Treatment of primary VS cultures with anti-NRG1 or HCN reduces cell proliferation in the absence of exogenous NRG1. Furthermore, conditioned medium from VS cell cultures contains NRG1 and stimulates SC proliferation in SC cultures, an effect that is inhibited by anti-NRG1 and HCN. These data suggest an autocrine pathway of VS growth stimulation involving NRG and ErbB receptors. Inhibition of constitutive NRG:ErbB signaling reduces VS cell proliferation in vitro and may have therapeutic potential for patients with VSs.