Proliferation Of Schwannoma Cells Research Articles

Selective nitration of Hsp90 acts as a metabolic switch promoting tumor cell proliferation

Tumors develop in an oxidative environment characterized by peroxynitrite production and downstream protein tyrosine (Y) nitration. We showed that tyrosine nitration supports schwannoma cell proliferation and regulates cell metabolism in the inheritable tumor disorder NF2-related Schwannomatosis (NF2-SWN). Here, we identified the chaperone Heat shock protein 90 (Hsp90) as the first nitrated protein that acts as a metabolic switch to promote schwannoma cell proliferation. Doubling the endogenous levels of nitrated Hsp90 in schwannoma cells or supplementing nitrated Hsp90 into normal Schwann cells increased their proliferation. Metabolically, nitration on either Y33 or Y56 conferred Hsp90 distinct functions; nitration at Y33 (Hsp90NY33) down-regulated mitochondrial oxidative phosphorylation, while nitration at Y56 (Hsp90NY56) increased glycolysis by activating the purinergic receptor P2X7 in both schwannoma and normal Schwann cells. Hsp90NY33 and Hsp90NY56 showed differential subcellular and spatial distribution corresponding with their metabolic and proliferative functions in schwannoma three-dimensional cell culture models. Collectively, these results underscore the role of tyrosine nitration as a post-translational modification regulating critical cellular processes. Nitrated proteins, particularly nitrated Hsp90, emerge as a novel category of tumor-directed therapeutic targets.

Site-Specific Nitration of Hsp90 Alters Cell Metabolism and Supports Schwannoma Cell Proliferation

Site-Specific Nitration of Hsp90 Alters Cell Metabolism and Supports Schwannoma Cell Proliferation

Nitration of Hsp90 Affects its Spatial Distribution and Promotes Schwannoma Cell Proliferation

Neurofibromatosis Type 2 is a genetic disorder characterized by the development of schwannomas throughout the nervous system caused by loss of merlin tumor suppressor activity. Solid tumors develop in an oxidative environment due to production of oxidants such as peroxynitrite by tumor and infiltrating immune cells. We discovered peroxynitrite production and subsequent protein tyrosine (Y) nitration support schwannoma cell survival/proliferation. Further, Y nitration induces a metabolic reprogramming characterized by decreased oxidative phosphorylation activity, and increased glycolysis and glutaminolysis. Here, we identified the first nitrated protein supporting schwannoma cell proliferation, the molecular chaperone heat shock protein 90 (Hsp90). Out of five Y residues prone to nitration in Hsp90, we showed nitration at Y33 down‐regulates mitochondrial metabolism, while nitration at Y56 activates the ATP‐gated receptor P2X7, which increases glycolysis in tumor cells. We found schwannomas contain endogenous levels of Hsp90 nitrated at either position. Therefore, we hypothesized that nitrated Hsp90 (Hsp90NY) promotes schwannoma cell proliferation by regulating their metabolism. To investigate the role of Hsp90NY in proliferation, we increased the intracellular concentration of Hsp90NY in schwannoma cells by delivering Hsp90NY intracellularly at concentrations like those endogenously present in these cells (~6.5% of cellular Hsp90, calculated by quantitative dot blot). Doubling the intracellular concentration of Hsp90NYbut not wild‐type Hsp90 significantly increased cell proliferation 24 and 48 h post‐delivery. To establish the metabolic role of Hsp90NY, we delivered wild‐type or Hsp90NY into wild‐type Schwann cells (WT‐SC) and studied the metabolic changes by extracellular flux analysis 24 h post‐delivery. Hsp90NY decreased all parameters of mitochondrial activity, with no effect on the glycolytic rate. To test if Y33 and/or Y56 were responsible for the tumorigenic activity of Hsp90NY, we intracellularly delivered to WT‐SC either wild‐type Hsp90, or different site‐specific nitrated Hsp90 proteins produced by genetic code expansion: Hsp90 nitrated at Y33 (Hsp90NY33), at Y56 (Hsp90NY56), or at Y33 and Y56 simultaneously (Hsp90NY33+56). All three forms of Hsp90NY significantly increased WT‐SC proliferation at 24 and 48 h post‐delivery, suggesting Y33 and Y56 induce schwannoma cell proliferation independently. Using a three‐dimensional schwannoma cell culture model (tumoroids), we discovered Hsp90NY33colocalized with mitochondria and remained in the tumoroid periphery, while Hsp90NY56was also found in nuclei with a homogenous distribution throughout the tumoroid. Collectively, our results show that Hsp90, when nitrated at Y33 or Y56, gains a tumorigenic activity that supports schwannoma cell proliferation. In addition, residue‐specific nitration results in distinct localization, and potentially function, of the protein. This is the first nitrated protein shown to support tumor cell proliferation, and a potential tumor‐directed target for therapeutic development.

Abstract 1164: Human endogenous retrovirus type K promotes proliferation of Merlin negative schwannoma and meningioma which can be inhibited by anti-retroviral and anti-TEAD drugs

Abstract Deficiency of the tumor suppressor Merlin causes the development of schwannoma, meningioma and ependymoma tumors. These can occur spontaneously or in the hereditary disease Neurofibromatosis type 2 (NF2). Merlin mutations are also relevant in a variety of other tumors. Currently available treatments are surgery or radiosurgery which are only partially effective, with tumors frequently reoccurring and in case of meningioma often as a higher grade. There is an urgent need for effective drug treatments.In this study, we investigated the role of Human Endogenous Retrovirus Type K (HERV-K) and its targetability in Merlin negative schwannoma and meningioma tumors by using tissues and primary cells isolated form patients employing immunohistochemistry, immunocytochemistry, western blotting and proliferation assays. We demonstrate that HERV-K proteins are overexpressed in Merlin negative schwannoma and all meningioma grades. Furthermore, we implicate CRL4DCAF1 and YAPT/EAD Hippo pathway in this overexpression and suggest that exosome transport of the HERV-K Envelope (Env) protein might contribute to schwannoma development. We also show that: (i) Ectopic overexpression of HERV-K Env in normal Schwann cells increased proliferation and upregulated the transcription factor c-Jun. Env overexpression also increased activity of the extracellular signal-regulated kinase 1/2 (pERK1/2), a known mitogenic pathway in schwannoma. (ii) An anti-Env monoclonal antibody decreased pERK1/2 and reduced proliferation of schwannoma cells. iii) FDA-approved retroviral protease inhibitors Ritonavir, Atazanavir and Lopinavir decreased pERK1/2 and cyclin D1 and reduced proliferation of schwannoma and grade I meningioma cells. We provide evidence for HERV-K Env contributing to the development of NF2-associated schwannomas and meningiomas. Considering the urgent need of drugs to treat NF2, we suggest the trialling of antiretroviral protease inhibitors, and consideration of anti-HERV-K Env immunotherapy, as well as TEAD-specific inhibitors in these patients. The above treatments would also be potentially beneficial for patients with other tumors caused by Merlin deficiency. Citation Format: Sylwia Ammoun, Emmanuel A. Maze, Bora Agit, Robert Belshaw, C Oliver Hanemann. Human endogenous retrovirus type K promotes proliferation of Merlin negative schwannoma and meningioma which can be inhibited by anti-retroviral and anti-TEAD drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1164.

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.

Synergistic effect of Nutlin-3 combined with MG-132 on schwannoma cells through restoration of merlin and p53 tumour suppressors.

BackgroundThe great majority of sporadic vestibular schwannomas (VSs) are due to the mutations of the NF2 gene encoding merlin. Sporadic VSs exhibit variable growth patterns and only a small fraction of the tumours are fast-growing; however, the underlying mechanisms remain undefined.MethodsDNA sequencing and dosage analysis were used to identify the NF2 mutation status in sporadic schwannomas. The expression and sub-cellular localization of merlin and p53-MDM2 were assessed by immunoblotting, qRT-PCR and immunofluorescence. In vitro and in vivo studies were performed to reveal the effects of Nutlin-3 (a MDM2 inhibitor) and/or MG-132(a proteasome inhibitor) on schwannomas. The proliferation of schwannoma cells was assessed by CCK-8 assay, EdU staining and Flow cytometry analysis.FindingsDouble genetic hits of NF2 tended to occur in fast-growing tumours, characterized by the absence of merlin. The deregulation of p53-MDM2 was demonstrated to mediate merlin-deficient tumour growth, characterized by a nuclear accumulation of stabilized MDM2, contributing to a nuclear export of p53 for degradation. Nutlin-3 blocked the proliferation of schwannoma cells via a cooperative recovery of merlin and p53, accompanied by the shuttling of both proteins from the cytoplasm to the nucleus. We further demonstrated a difference in the sensitivity to Nutlin-3 between schwannoma cells with and without merlin expression. Nutlin-3 combined with MG-132 narrowed this between-group difference and triggered stronger inhibitory effects on the growth of schwannomas through coordinated reactivation of p53.InterpretationThese findings present treatment strategies directed on the pathogenesis of sporadic schwannomas.FundNational Natural Science Foundation of China.

Synergistic Effect of Nutlin-3 Combined with MG-132 on Schwannoma Cells Through Restoration of Merlin and p53 Tumour Suppressors

The growth patterns of sporadic vestibular schwannomas are highly variable, and only a small fraction of the tumours are fast-growing; however, the underlying mechanisms remain undefined. In this study, we observed differential NF2 gene alterations in correlation to clinical characteristics in sporadic schwannomas. The two genetic hits tended to occur in fast-growing tumours, which were characterized by the absence of the NF2 product merlin. The deregulation of p53-MDM2 axis was demonstrated to mediate merlin-deficient tumour growth, characterized by a nuclear accumulation of stabilized MDM2, contributing to a nuclear export of p53 for degradation. Inhibition of MDM2 by Nutlin-3 blocked the proliferation of schwannoma cells via a cooperative recovery of merlin and p53, accompanied by the shuttling of both proteins from the cytoplasm to the nucleus. We further demonstrate a difference in the sensitivity to Nutlin-3 between schwannoma cells with and without merlin expression. A combination of Nutlin-3 and MG-132 (a proteasome inhibitor), narrows this between-group difference and triggers stronger inhibitory effects on the growth of schwannomas through coordinated reactivation of p53. Taken together, we present treatment strategies directed on pathogenesis of sporadic schwannomas. Funding: This research was funded by the National Natural Science Foundation of China (Grant No. 81600815 to Yongchuan Chai, Grant No. 81371086 to Zhaoyan Wang, Grant No. 81570906 to Hao Wu). Declaration of Interest: The authors disclose no conflict of interest. Ethical Approval: All experimental protocols were approved by the Research Ethics Review Committee of Shanghai Jiao Tong University. Methods used in the present study were carried out in accordance with approved guidelines and regulations. It conformed to the provisions of the Declaration of Helsinki.

PP03. HUMAN ENDOGENOUS RETROVIRUS TYPE K (HERVK) PROTEINS ARE OVEREXPRESSED AND RELEASED FROM HUMAN PRIMARY SCHWANNOMA CELLS CONTRIBUTING TO TUMOUR DEVELOPMENT VIA ERK, AKT, FAK PATHWAYS ACTIVATION AND P53 DOWNREGULATION

Loss of function mutations in the gene coding for the tumour suppressor Merlin causes development of multiple tumours of the nervous system such as schwannomas, meningiomas and ependymomas. These tumours may occur spontaneously or as a part of the hereditary disease Neurofibromatosis type 2 (NF2). Current therapies, surgery and radiosurgery, are not fully effective and new drug treatments are urgently needed. Schwannoma is the most common Merlin-deficient tumour, a hallmark for NF2 and a model for all other Merlin-deficient tumours. Therefore, to study Merlin-deficient tumours and to test different drugs we have developed a schwannoma in vitro cell model. Using this model we have previously identified several targets and tested various drugs including Sorafenib, which we have in phase 0 clinical trials on NF2 patients. Our preliminary data suggest that a promising target for Merlin-deficient tumours is one group of endogenous retroviruses called HERVK (Human Endogenous Retrovirus type K). Endogenous retroviruses are viruses that have integrated themselves into germ-line chromosomes and thereby become part of human genome (8%). HERVK are overexpressed in several cancers and activate pathways known to be involved in schwannoma development such as NFkB, β-catenin, ERK and AKT. HERVK proteins are currently being investigated as potential immunotherapy targets for cancer and HIV. We find that HERVK envelope, capsid/gag, Rec and Np9 proteins are overexpressed in human primary schwannoma cells and tissues. Additionally, HERVK envelope and capsid proteins are released from schwannoma cells. HERVK envelope expression is Merlin dependent and both HERVK envelope and capcid/gag expression are transcriptionally and translationally regulated. Anti-HERVK antibodies reduced schwannoma proliferation, pERK/pAKT/ pFAK and cyclin D1 levels and increased expression of p53. Also, pre-incubation of schwannoma cells with anti-HERVK antibodies prior to treatment with Selumetinib potentiated drug’s efficiency. An HIV protease inhibitor with some potency for HERVK protease, Ritonavir, also decreased proliferation of schwannoma cells and increased the efficiency of Selumetinib, Sorafenib and BEZ235 when combined. We suggest that HERVK plays a relevant role in schwannoma and other Merlin-deficient tumours development.

Abstract 4627: The role of human endogenous retroviral proteins in the development of Merlin-deficient tumors and as potential drug targets

Abstract Neurofibromatosis Type 2 (NF2) is a genetic disorder caused by loss of the tumour suppressor Merlin leading to the development of multiple tumours of the nervous system such as schwannomas, meningiomas and ependymomas. Merlin-deficient tumours may also occur sporadically including all schwannomas, 50-60% meningiomas and 29-38% ependymomas. Loss of functional Merlin have been also observed in other tumours e.g. mesothelioma, melanoma, and a portion of glioma, prostate and breast cancers. Current therapies for Merlin-deficient tumours, comprising surgery and radiosurgery are invasive and not fully effective therefore the emphasis has to be made on the development of new effective drug-based treatments. Schwannoma is the most common Merlin-deficient tumour, a hallmark for NF2 and a model for all other Merlin-deficient tumours. We have developed the human in vitro model for schwannoma to study tumour pathobiology and for drug testing. Using this model we have shown strong overexpression and activation of ErbB2/ErbB32, platelet-derived growth factor receptor (PDGFR), Insulin like growth factor receptor-I (IGF-IR) and activation of Raf/MEK/ERK, PI3K/AKT and Wnt/β-catenin signalling pathways. Following, we have successfully tested new drugs such as AZD6244, Sorafenib, BEZ235, Lapatinib, Imatinib and Nilotinib. These studies revealed that schwannomas’ pathological proliferation, survival and adhesion is regulated by highly complex networks interacting and compensating each other resulting in increased tumour pathology. We suggest that the inhibition of a single target will not be sufficient to stop tumour growth and that multi-targeted therapy would be needed. Endogenous retroviruses are viruses that have integrated themselves into germ-line chromosomes and thereby become part of the human genome sequence (8%). One group, HERVK, has been found to be overexpressed in several cancers and is currently being investigated as potential immunotherapy targets. Our preliminary data demonstrate that HERVK envelope, capsid, Rec and Np9 proteins are overexpressed in human primary schwannoma cells and tissues. Additionally, HERVK envelope and capsid proteins are released from schwannoma cells. HERVK overexpression is regulated at both the transcriptional and translational level and is partially Merlin dependent. Anti-HERVK antibodies reduced schwannoma proliferation, pERK/pAKT/ pFAK and cyclin D1 levels and increased expression of p53. Also, pre-incubation of schwannoma cells with anti-HERVK antibodies prior to treatment with AZD6244 potentiated drug's efficiency. An HIV/HERVK protease inhibitor, Ritonavir, also decreased proliferation of schwannoma cells and increased the efficiency of AZD6244, Sorafenib and BEZ235 when combined. We suggest that HERVK plays a relevant role in schwannoma and possibly other Merlin-deficient tumours’ development and is a promising therapeutic target. Citation Format: Emmanuel Maze, Shona Reeves, David Hilton, Lucy Provenzano, Robert Belshaw, Sylwia Ammoun. The role of human endogenous retroviral proteins in the development of Merlin-deficient tumors and as potential drug targets. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4627.

Tanshinone IIA exerts antitumor activity against vestibular schwannoma cells by inhibiting the expression of hypoxia-inducible factor-1α.

The aim of the present study was to evaluate the effect of the herbal medicine, tanshinone IIA (Tan IIA), on vestibular schwannoma (VS) cells and assess the functional targets of Tan IIA. HEI‑193 cells and Nf2‑/‑mouse Schwann (SC4) cells were used to investigate the inhibitory effects of Tan IIA on VS. Cell viability was measured using an MTT assay and apoptosis was assessed by flow cytometry. Western blot analysis and reverse transcription quantitative polymerase chain reaction (RT‑qPCR) were performed to assess the expression of hypoxia‑inducible factor‑1α (HIF‑1α) and its signaling pathways. In addition, the effect of Tan IIA on HIF‑1α transcription was determined using a luciferase reporter assay. Schwannoma cell proliferation was observed to be inhibited as the Tan IIA concentration increased under normoxic and hypoxic conditions. Furthermore, Tan IIA induced apoptosis in the HEI‑193 cells and inhibited the protein expression of HIF‑1α in the HEI‑193 cells under hypoxia, thus repressing the transcriptional activity of HIF‑1α. The present study demonstrated that HIF‑1α is expressed in hypoxic VS cells and Tan IIA inhibits VS cells by suppressing the activity of HIF‑1α. In conclusion, these results indicate that Tan IIA is a potential chemotherapeutic agent for the treatment of VS.

Inhibition of MEK pathway in vestibular schwannoma cell culture

The purpose of this study was to evaluate the Ras GTPase (Ras) to extracellular signal-regulated kinase (ERK) pathway in vestibular schwannoma (VS) cell cultures and patient excised schwannoma tumors. Mitogen-activated protein kinase kinase (MEK) inhibitor CI-1040 (PD184352) was utilized to evaluate the effect of specific MEK inhibition on benign schwannoma cell culture proliferation and apoptosis. Prospective evaluation of human schwannoma cell lines and tumors. Western blotting was completed with phospho-antibodies for proteins in the Ras-ERK pathway. Increasing concentrations of CI-1040 were utilized in schwannoma cell cultures to evaluate cell proliferation and apoptosis. Proliferation was measured with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide proliferation assay, and apoptosis was monitored with flow cytometry of annexin V/propidium iodide-stained cells. The most consistent Ras-ERK pathway alterations were found in phospho-MEK and ERK. Phospho-MEK was not overexpressed in the schwannoma cell lines, but six out of 10 VS showed significant increases compared to benign Schwann cell controls. Similarly, nine of 10 VS tumors showed increased phospho-ERK expression. CI-1040 showed significantly reduced schwannoma cell proliferation at the 50 and 100 μM (IC(50) 20 μM and 30 μM) concentrations when compared to carrier only controls in two out three schwannoma cell lines. The remaining schwannoma cell line was relatively refractory to the antiproliferative effects of CI-1040 at doses up to 100 μM (IC(50) 58 μM). Cumulative data of four separate schwannoma cell lines demonstrated that apoptosis was increased in treated schwannoma cells at CI-1040 concentrations of 50 and 100 μM at 72 hours. There is overexpression of phosphorylated (activated) proteins in the Ras-ERK pathway in schwannoma cultures and tumors as compared to benign human Schwann cell culture controls. MEK inhibitor, CI-1040, created significantly decreased schwannoma cell proliferation and increased apoptosis in cell culture. These data justify the use of MEK inhibitors in animal treatment studies of VS.

Treatment of Vestibular Schwannoma Cells With ErbB Inhibitors

Aberrant phosphorylation of ErbB family receptor tyrosine kinases (RTK) in human vestibular schwannomas (VSs) renders them susceptible to growth suppression by RTK inhibitors. Recent evidence has implicated increased ErbB family receptor tyrosine kinase signaling in VS tumorigenesis; however, the characterization of ErbB receptor activity and the chemotherapeutic potential of RTK inhibitors in VS treatment have not been fully explored. To confirm phosphorylation of ErbB receptors in VS, protein extracts from paired VS tumor-vestibular nerve samples were examined using phospho-RTK arrays. ErbB receptor phosphorylation was similarly examined in cultured schwannoma cells, normal Schwann cells, and VS tumor tissues using Western blotting. Also, VS tumor sections were immunostained for members of the ErbB receptor family. The effects of RTK inhibitors on ErbB phosphorylation and cell proliferation were assessed in schwannoma cells after epidermal growth factor receptor (EGFR) inhibitor (Erlotinib) and EGFR/ErbB2 inhibitor (Lapatinib) treatment. VS tumor tissues consistently demonstrated higher levels of phosphorylated ErbB3 compared with paired vestibular nerves. However, cultured VS, malignant schwannoma, and normal Schwann cells demonstrated EGFR phosphorylation. Immunohistochemistry confirmed high expression of ErbB3 in a series of VS tumor sections. Erlotinib inhibited schwannoma cell proliferation with an IC50 value of 2.5 µmol/L, whereas Lapatinib was less potent for growth inhibition. Erlotinib treatment resulted in a decrease of multiple phospho-ErbB receptors in schwannoma cells. VS variably express activated ErbB receptors with consistently higher levels of phospho-ErbB3 expression relative to paired vestibular nerve samples. Chemotherapeutic targeting of ErbB3 may be a novel means of inhibiting VS growth.

The molecular biology and novel treatments of vestibular schwannomas

Vestibular schwannomas are histopathologically benign tumors arising from the Schwann cell sheath surrounding the vestibular branch of cranial nerve VIII and are related to the NF2 gene and its product merlin. Merlin acts as a tumor suppressor and as a mediator of contact inhibition. Thus, deficiencies in both NF2 genes lead to vestibular schwannoma development. Recently, there have been major advances in our knowledge of the molecular biology of vestibular schwannomas as well as the development of novel therapies for its treatment. In this article the authors comprehensively review the recent advances in the molecular biology and characterization of vestibular schwannomas as well as the development of modern treatments for vestibular schwannoma. For instance, merlin is involved with a number of receptors including the CD44 receptor, EGFR, and signaling pathways, such as the Ras/raf pathway and the canonical Wnt pathway. Recently, merlin was also shown to interact in the nucleus with E3 ubiquitin ligase CRL4(DCAF1). A greater understanding of the molecular mechanisms behind vestibular schwannoma tumorigenesis has begun to yield novel therapies. Some authors have shown that Avastin induces regression of progressive schwannomas by over 40% and improves hearing. An inhibitor of VEGF synthesis, PTC299, is currently in Phase II trials as a potential agent to treat vestibular schwannoma. Furthermore, in vitro studies have shown that trastuzumab (an ERBB2 inhibitor) reduces vestibular schwannoma cell proliferation. With further research it may be possible to significantly reduce morbidity and mortality rates by decreasing tumor burden, tumor volume, hearing loss, and cranial nerve deficits seen in vestibular schwannomas.

Dissecting and Targeting the Growth Factor–Dependent and Growth Factor–Independent Extracellular Signal-Regulated Kinase Pathway in Human Schwannoma

Schwannomas are tumors of the nervous system that occur sporadically and in patients with the cancer predisposition syndrome neurofibromatosis type 2 (NF2). Schwannomas and all NF2-related tumors are caused by loss of the tumor suppressor merlin. Using our human in vitro model for schwannoma, we analyzed extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT signaling pathways, their upstream growth factor receptors, and their role in schwannoma cell proliferation and adhesion to find new systemic therapies for these tumors that, to date, are very difficult to treat. We show here that human primary schwannoma cells show an enhanced basal Raf/mitogen-activated protein/ERK kinase/ERK1/2 pathway activity compared with healthy Schwann cells. Due to a strong and prolonged activation of platelet-derived growth factor receptor beta (PDGFRbeta), which is highly overexpressed, ERK1/2 and AKT activation was further increased in schwannoma, leading to increased proliferation. Using specific inhibitors, we discovered that ERK1/2 activation involves the integrin/focal adhesion kinase/Src/Ras signaling cascades and PDGFRbeta-mediated ERK1/2 activation is triggered through the phosphatidylinositol 3-kinase/protein kinase C/Src/c-Raf pathway. Due to the complexity of signals leading to schwannoma cell proliferation, potential new therapeutic agents should target several signaling pathways. The PDGFR and c-Raf inhibitor sorafenib (BAY 43-9006; Bayer Pharmaceuticals), currently approved for treatment of advanced renal cell cancer, inhibits both basal and PDGFRbeta-mediated ERK1/2 and AKT activity and decreases cell proliferation in human schwannoma cells, suggesting that this drug constitutes a promising tool to treat schwannomas. We conclude that our schwannoma in vitro model can be used to screen for new therapeutic targets in general and that sorafenib is possible candidate for future clinical trials.

Overexpression of the NF2 gene inhibits schwannoma cell proliferation through promoting PDGFR degradation

The loss of NF2 gene function leads to vestibular nerve schwannoma formation in humans. The NF2 gene product, Merlin/Schwannomin, has recently been found to interact with the two PDZ domains containing protein EBP50/NHE-RF, which is itself known to interact with the PDGF receptor (PDGFR) in several cell types. In this study, an up-regulation of both PDGFR and EBP50/NHE-RF, and an interaction of both proteins were found in primary human schwannoma tissue. Furthermore, using an adenoviral vector mediated gene transfer technique, changes in the phenotypic characteristics after NF2 gene restoration in a newly established NF2 gene-mutated human schwannoma cell line (HEI 193) were investigated. The overexpression of Merlin/Schwannomin in HEI 193 led to an inhibition of cell proliferation under serum-free conditions. Upon PDGF stimulation in culture, Merlin/Schwannomin appeared to inhibit the activation of the MAPK and PI3K signaling pathways, impinging on the phosphorylation of Erk 1/2 and Akt, respectively. The data also show that PDGFR is more rapidly internalized by the schwannoma cells overexpressing NF2. Therefore, this process is suggested as a model for a mechanism of Merlin/Schwannomin tumor suppressor function, which intermediates acceleration of the cell surface growth factor degradation.