RESUMEN
Neurofibromatosis type 2 (NF2) is an autosomal-dominant disorder characterized by the development of bilateral vestibular schwannomas. The NF2 gene encodes the tumor suppressor merlin, and loss of merlin activity promotes tumorigenesis and causes NF2. Cellular redox signaling has been implicated in different stages of tumor development. Among reactive nitrogen species, peroxynitrite is the most powerful oxidant produced by cells. We recently showed that peroxynitrite-mediated tyrosine nitration down-regulates mitochondrial metabolism in tumor cells. However, whether peroxynitrite supports a metabolic shift that could be exploited for therapeutic development is unknown. Here, we show that vestibular schwannomas from NF2 patients and human, merlin-deficient (MD) Schwann cells have high levels of endogenous tyrosine nitration, indicating production of peroxynitrite. Furthermore, scavenging or inhibiting peroxynitrite formation significantly and selectively decreased survival of human and mouse MD-Schwann cells. Using multiple complementary methods, we also found that merlin deficiency leads to a reprogramming of energy metabolism characterized by a peroxynitrite-dependent decrease of oxidative phosphorylation and increased glycolysis and glutaminolysis. In MD-Schwann cells, scavenging of peroxynitrite increased mitochondrial oxygen consumption and membrane potential, mediated by the up-regulation of the levels and activity of mitochondrial complex IV. This increase in mitochondrial activity correlated with a decrease in the glycolytic rate and glutamine dependence. This is the first demonstration of a peroxynitrite-dependent reprogramming of energy metabolism in tumor cells. Oxidized proteins constitute a novel target for therapeutic development not only for the treatment of NF2 schwannomas but also other tumors in which peroxynitrite plays a regulatory role.
Asunto(s)
Supervivencia Celular/fisiología , Genes Supresores de Tumor , Ácido Peroxinitroso/fisiología , Células de Schwann/metabolismo , Animales , Células Cultivadas , Glutamina/metabolismo , Glucólisis , Humanos , Ratones , Mitocondrias/metabolismo , Neurofibromatosis 2/genética , Fosforilación Oxidativa , Consumo de OxígenoRESUMEN
The neurofibromatoses (NF) are autosomal dominant genetic disorders that encompass the rare diseases NF1, NF2, and schwannomatosis. The NFs affect more people worldwide than Duchenne muscular dystrophy and Huntington's disease combined. NF1 and NF2 are caused by mutations of known tumor suppressor genes (NF1 and NF2, respectively). For schwannomatosis, although mutations in SMARCB1 were identified in a subpopulation of schwannomatosis patients, additional causative gene mutations are still to be discovered. Individuals with NF1 may demonstrate manifestations in multiple organ systems, including tumors of the nervous system, learning disabilities, and physical disfigurement. NF2 ultimately can cause deafness, cranial nerve deficits, and additional severe morbidities caused by tumors of the nervous system. Unmanageable pain is a key finding in patients with schwannomatosis. Although today there is no marketed treatment for NF-related tumors, a significant number of clinical trials have become available. In addition, significant preclinical efforts have led to a more rational selection of potential drug candidates for NF trials. An important element in fueling this progress is the sharing of knowledge. For over 20 years the Children's Tumor Foundation has convened an annual NF Conference, bringing together NF professionals to share novel findings, ideas, and build collaborations. The 2012 NF Conference held in New Orleans hosted over 350 NF researchers and clinicians. This article provides a synthesis of the highlights presented at the conference and as such, is a "state-of-the-field" for NF research in 2012.
Asunto(s)
Neurilemoma/etiología , Neurofibromatosis/etiología , Neurofibromatosis 1/etiología , Neurofibromatosis 2/etiología , Neoplasias Cutáneas/etiología , Humanos , Neurilemoma/genética , Neurilemoma/terapia , Neurofibromatosis/genética , Neurofibromatosis/terapia , Neurofibromatosis 1/genética , Neurofibromatosis 1/terapia , Neurofibromatosis 2/genética , Neurofibromatosis 2/terapia , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/terapiaRESUMEN
Neurofibromatosis Type 2 (NF2)-related schwannomatosis is a genetic disorder that causes development of multiple types of nervous system tumors. The primary and diagnostic tumor type is bilateral vestibular schwannoma. There is no cure or drug therapy for NF2. Recommended treatments include surgical resection and radiation, both of which can leave patients with severe neurological deficits or increase the risk of future malignant tumors. Results of our previous pilot high-throughput drug screen identified phosphoinositide 3-kinase (PI3K) inhibitors as strong candidates based on loss of viability of mouse merlin-deficient Schwann cells (MD-SCs). Here we used novel human schwannoma model cells to conduct combination drug screens. We identified a class I PI3K inhibitor, pictilisib and p21 activated kinase (PAK) inhibitor, PF-3758309 as the top combination due to high synergy in cell viability assays. Both single and combination therapies significantly reduced growth of mouse MD-SCs in an orthotopic allograft mouse model. The inhibitor combination promoted cell cycle arrest and apoptosis in mouse merlin-deficient Schwann (MD-SCs) cells and cell cycle arrest in human MD-SCs. This study identifies the PI3K and PAK pathways as potential targets for combination drug treatment of NF2-related schwannomatosis.
Asunto(s)
Indazoles , Neurilemoma , Neurofibromatosis , Neurofibromatosis 2 , Neoplasias Cutáneas , Sulfonamidas , Humanos , Animales , Ratones , Neurofibromatosis 2/tratamiento farmacológico , Neurofibromatosis 2/genética , Neurofibromatosis 2/metabolismo , Neurofibromina 2/genética , Neurofibromina 2/metabolismo , Fosfatidilinositol 3-Quinasas , Quinasas p21 Activadas/genética , Fosfatidilinositol 3-Quinasa/uso terapéutico , Neurilemoma/tratamiento farmacológico , Neurilemoma/genéticaRESUMEN
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.
Asunto(s)
Proliferación Celular , Proteínas HSP90 de Choque Térmico , Neurilemoma , Células de Schwann , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Neurilemoma/metabolismo , Neurilemoma/patología , Células de Schwann/metabolismo , Línea Celular Tumoral , Animales , Tirosina/metabolismo , Procesamiento Proteico-Postraduccional , Fosforilación OxidativaRESUMEN
Myelination is a complex process requiring coordination of directional motility and an increase in glial cell size to generate a multilamellar myelin sheath. Regulation of actin dynamics during myelination is poorly understood. However, it is known that myelin thickness is related to the abundance of neuregulin-1 (NRG1) expressed on the axon surface. Here we identify cofilin1, an actin depolymerizing and severing protein, as a downstream target of NRG1 signaling in rat Schwann cells (SCs). In isolated SCs, NRG1 promotes dephosphorylation of cofilin1 and its upstream regulators, LIM kinase (LIMK) and Slingshot-1 phosphatase (SSH1), leading to cofilin1 activation and recruitment to the leading edge of the plasma membrane. These changes are associated with rapid membrane expansion yielding a 35-50% increase in SC size within 30 min. Cofilin1-deficient SCs increase phosphorylation of ErbB2, ERK, focal adhesion kinase, and paxillin in response to NRG1, but fail to increase in size possibly due to stabilization of unusually long focal adhesions. Cofilin1-deficient SCs cocultured with sensory neurons do not myelinate. Ultrastructural analysis reveals that they unsuccessfully segregate or engage axons and form only patchy basal lamina. After 48 h of coculturing with neurons, cofilin1-deficient SCs do not align or elongate on axons and often form adhesions with the underlying substrate. This study identifies cofilin1 and its upstream regulators, LIMK and SSH1, as end targets of a NRG1 signaling pathway and demonstrates that cofilin1 is necessary for dynamic changes in the cytoskeleton needed for axon engagement and myelination by SCs.
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Cofilina 1/genética , Cofilina 1/fisiología , Vaina de Mielina/fisiología , Neurregulina-1/genética , Neurregulina-1/fisiología , Células de Schwann/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Animales , Axones/fisiología , Axones/ultraestructura , Western Blotting , Polaridad Celular/genética , Proliferación Celular , Tamaño de la Célula , Técnicas de Cocultivo , Colorantes , Femenino , Técnica del Anticuerpo Fluorescente , Adhesiones Focales/genética , Ganglios Espinales/citología , Quinasas Lim/genética , Quinasas Lim/fisiología , Masculino , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/fisiología , Microscopía Electrónica , Vaina de Mielina/ultraestructura , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/fisiología , Fosforilación , ARN Interferente Pequeño/genética , Ratas , Ratas Sprague-Dawley , Células de Schwann/ultraestructuraRESUMEN
Neurofibromatosis type 2 is an autosomal dominant disorder characterized by tumors, predominantly schwannomas, in the nervous system. It is caused by mutations in the gene NF2, encoding the growth regulator schwannomin (also known as merlin). Mutations occur throughout the 17-exon gene, with most resulting in protein truncation and undetectable amounts of schwannomin protein. Pathogenic mutations that result in production of defective schwannomin include in-frame deletions of exon 2 and three independent missense mutations within this same exon. Mice with conditional deletion of exon 2 in Schwann cells develop schwannomas, which confirms the crucial nature of exon 2 for growth control. Here we report that the molecular adaptor paxillin binds directly to schwannomin at residues 50-70, which are encoded by exon 2. This interaction mediates the membrane localization of schwannomin to the plasma membrane, where it associates with beta 1 integrin and erbB2. It defines a pathogenic mechanism for the development of NF2 in humans with mutations in exon 2 of NF2.
Asunto(s)
Membrana Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Neurofibromina 2/genética , Neurofibromina 2/metabolismo , Fosfoproteínas/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Exones , Integrina beta1/metabolismo , Ratones , Mutación , Neurofibromatosis 2/genética , Neurofibromatosis 2/fisiopatología , Paxillin , Isoformas de Proteínas , Ratas , Receptor ErbB-2/metabolismo , Células de Schwann/citología , Células de Schwann/metabolismoRESUMEN
Neurofibromatosis Type 2 (NF2)-related schwannomatosis is a genetic disorder that causes development of multiple types of nervous system tumors. The primary and diagnostic tumor type is bilateral vestibular schwannoma. There is no cure or drug therapy for NF2. Recommended treatments include surgical resection and radiation, both of which can leave patients with severe neurological deficits or increase the risk of future malignant tumors. Results of our previous pilot high-throughput drug screen identified phosphoinositide 3-kinase (PI3K) inhibitors as strong candidates based on loss of viability of mouse merlin-deficient Schwann cells (MD-SCs). Here we used novel human schwannoma model cells to conduct combination drug screens. We identified a class I PI3K inhibitor, pictilisib and p21 activated kinase (PAK) inhibitor, PF-3758309 as the top combination due to high synergy in cell viability assays. Both single and combination therapies significantly reduced growth of mouse MD-SCs in an orthotopic allograft mouse model. The inhibitor combination promoted cell cycle arrest and apoptosis in mouse merlin-deficient Schwann (MD-SCs) cells and cell cycle arrest in human MD-SCs. This study identifies the PI3K and PAK pathways as potential targets for combination drug treatment of NF2-related schwannomatosis.
RESUMEN
BACKGROUND: Vestibular schwannomas (VS) are benign intracranial tumors caused by loss of function of the merlin tumor suppressor. We tested three hypotheses related to radiation, hearing loss (HL), and VS cell survival: (1) radiation causes HL by injuring auditory hair cells (AHC), (2) fractionation reduces radiation-induced HL, and (3) single fraction and equivalent appropriately dosed multi-fractions are equally effective at controlling VS growth. We investigated the effects of single fraction and hypofractionated radiation on hearing thresholds in rats, cell death pathways in rat cochleae, and viability of human merlin-deficient Schwann cells (MD-SC). METHODS: Adult rats received cochlear irradiation with single fraction (0 to 18 Gray [Gy]) or hypofractionated radiation. Auditory brainstem response (ABR) testing was performed for 24 weeks. AHC viabilities were determined using immunohistochemistry. Neonatal rat cochleae were harvested after irradiation, and gene- and cell-based assays were conducted. MD-SCs were irradiated, and viability assays and immunofluorescence for DNA damage and cell cycle markers were performed. RESULTS: Radiation caused dose-dependent and progressive HL in rats and AHC losses by promoting expression of apoptosis-associated genes and proteins. When compared to 12 Gy single fraction, hypofractionation caused smaller ABR threshold and pure tone average shifts and was more effective at reducing MD-SC viability. CONCLUSIONS: Investigations into the mechanisms of radiation ototoxicity and VS radiobiology will help determine optimal radiation regimens and identify potential therapies to mitigate radiation-induced HL and improve VS tumor control.
RESUMEN
BACKGROUND: The vestibular schwannoma (VS) secretome can initiate monocyte recruitment and macrophage polarization to M1 (proinflammatory) and/or M2 (protumorigenic) phenotypes, which in turn secrete additional cytokines that contribute to the tumor microenvironment. Profiling cyst fluid and cerebrospinal fluid (CSF) in cystic VS provides a unique opportunity to understand mechanisms that may contribute to tumor progression and cyst formation. HYPOTHESIS: Cystic VSs secrete high levels of cytokines into cyst fluid and express abundant M1 and M2 macrophages. METHODS: Tumor, CSF, and cyst fluid were prospectively collected from 10 cystic VS patients. Eighty cytokines were measured in fluid samples using cytokine arrays and compared with normal CSF from normal donors. Immunofluorescence was performed for CD80 + M1 and CD163 + M2 macrophage markers. Demographic, audiometric, and radiographic information was obtained through retrospective chart review. RESULTS: Cyst fluid expressed more osteopontin and monocyte chemotactic protein-1 (MCP-1; p < 0.0001), when compared with normal CSF. Cyst fluid also expressed more protein ( p = 0.0020), particularly MCP-1 ( p < 0.0001), than paired CSF from the same subjects. MCP-1 expression in cyst fluid correlated with CD80 + staining in VS tissue ( r = 0.8852; p = 0.0015) but not CD163 + staining. CONCLUSION: Cyst fluid from cystic VS harbored high levels of osteopontin and MCP-1, which are cytokines important in monocyte recruitment and macrophage polarization. MCP-1 may have a significant role in molding the tumor microenvironment, by polarizing monocytes to CD80 + M1 macrophages in cystic VS. Further investigations into the role of cytokines and macrophages in VS may lead to new avenues for therapeutic intervention.
Asunto(s)
Neuroma Acústico , Osteopontina , Humanos , Macrófagos Asociados a Tumores/metabolismo , Líquido Quístico/metabolismo , Estudios Retrospectivos , Citocinas/metabolismo , Microambiente TumoralRESUMEN
Neurofibromatosis Type 2 (NF2) is a tumor predisposition syndrome caused by germline inactivating mutations in the NF2 gene encoding the merlin tumor suppressor. Patients develop multiple benign tumor types in the nervous system including bilateral vestibular schwannomas (VS). Standard treatments include surgery and radiation therapy, which may lead to loss of hearing, impaired facial nerve function, and other complications. Kinase inhibitor monotherapies have been evaluated clinically for NF2 patients with limited success, and more effective nonsurgical therapies are urgently needed. Schwannoma model cells treated with PI3K inhibitors upregulate activity of the focal adhesion kinase (FAK) family as a compensatory survival pathway. We screened combinations of 13 clinically relevant PI3K and FAK inhibitors using human isogenic normal and merlin-deficient Schwann cell lines. The most efficacious combination was PI3K/mTOR inhibitor omipalisib with SRC/FAK inhibitor dasatinib. Sub-GI50 doses of the single drugs blocked phosphorylation of their major target proteins. The combination was superior to either single agent in promoting a G1 cell-cycle arrest and produced a 44% decrease in tumor growth over a 2-week period in a pilot orthotopic allograft model. Evaluation of single and combination drugs in six human primary VS cell models revealed the combination was superior to the monotherapies in 3 of 6 VS samples, highlighting inter-tumor variability between patients consistent with observations from clinical trials with other molecular targeted agents. Dasatinib alone performed as well as the combination in the remaining three samples. Preclinically validated combination therapies hold promise for NF2 patients and warrants further study in clinical trials.
Asunto(s)
Antineoplásicos , Neurilemoma , Neurofibromatosis 2 , Humanos , Neurofibromatosis 2/tratamiento farmacológico , Neurofibromatosis 2/genética , Neurofibromina 2/genética , Neurofibromina 2/metabolismo , Fosfatidilinositol 3-Quinasas/farmacología , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Dasatinib/farmacología , Fosfatidilinositol 3-Quinasa/farmacología , Fosfatidilinositol 3-Quinasa/uso terapéutico , Neurilemoma/tratamiento farmacológico , Neurilemoma/genética , Antineoplásicos/farmacología , Proliferación CelularRESUMEN
The Neurofibromatosis type 2 tumor suppressor, schwannomin (Sch) is a plasma membrane-cytoskeleton linking protein that regulates receptor signaling and actin dynamics. We examined Sch's role in specifying morphological changes needed for Schwann cell (SC) function in vitro. Isolated Sch-GFP-expressing SCs extended bipolar processes 82% longer than those formed by GFP-expressing cells. In contrast, SCs expressing dominant negative Sch-BBA-GFP extended bipolar processes 16% shorter than controls and 64% shorter than Sch-GFP-expressing SCs. nf2 gene inactivation caused isolated mouse SCs to transition from bipolar to multipolar cells. Live imaging revealed that SCs co-expressing Sch-GFP and dominant negative RacN17 behaved similarly in dorsal root ganglion explant cultures; they quickly aligned on axons and slowly elongated bipolar processes. In contrast, SCs expressing constitutively active RacV12 underwent continuous transitions in morphology that interfered with axon alignment. When co-cultured with neurons under myelin-promoting conditions, Sch-GFP-expressing SCs elaborated longer myelin segments than GFP-expressing SCs. In contrast, Sch-BBA-GFP-expressing SCs failed to align on or myelinate axons. Together, these results demonstrate that Sch plays an essential role in inducing and/or maintaining the SC's spindle shape and suggest that the mechanism involves Sch-dependent inhibition of Rac activity. By stabilizing the bipolar morphology, Sch promotes the alignment of SCs with axons and ultimately influences myelin segment length.
Asunto(s)
Vaina de Mielina/metabolismo , Fibras Nerviosas Mielínicas/ultraestructura , Neurofibromina 2/metabolismo , Células de Schwann/citología , Células de Schwann/fisiología , Acetazolamida , Animales , Células Cultivadas , Técnicas de Cocultivo , Ganglios Espinales/citología , Ratones , Vaina de Mielina/genética , Fibras Nerviosas Mielínicas/metabolismo , Neurofibromatosis 2/metabolismo , Neurofibromina 2/genética , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Unión al GTP rac/metabolismoRESUMEN
Schwannomatosis is a rare genetic disorder that predisposes individuals to development of multiple schwannomas mainly in spinal and peripheral nerves and to debilitating chronic pain often unrelated to any schwannoma. Pathogenic variants of two genes, SMARCB1 and LZTR1, are causal in familial cases. However, many schwannomatosis patients lack mutations in these genes. Surgery is the standard treatment for schwannomas but leaves patients with increasing neurological deficits. Pain management is a daily struggle controlled by the use of multiple analgesic and anti-inflammatory drugs. There is a need for both nonsurgical treatment to manage tumor growth and nonaddictive, nonsedative pain control. Because standard clinical trials are exceedingly difficult for patients with rare disorders, precision medicine approaches offer the possibility of bespoke therapeutic regimens to control tumor growth. As a proof of principle, we obtained a bio-specimen of paraspinal schwannoma from a schwannomatosis patient with a germline point mutation in the SMARCB1/INI gene. We created an hTERT immortalized cell line and tested the ability of targeted small molecules with efficacy in neurofibromatosis type 2-related schwannomas to reduce cell viability and induce cell death. We identified WP1066, a STAT3 inhibitor, currently in phase 2 clinical trials for pediatric and adult brain tumors as a lead compound. It reduced cell viability and STAT-3 phosphorylation and induced expression of markers for both necroptosis and caspase-dependent cell death. The results demonstrate feasibility in creating patient-derived cell lines for use in precision medicine studies.
Asunto(s)
Neurilemoma , Neurofibromatosis , Piridinas , Neoplasias Cutáneas , Tirfostinos , Adulto , Muerte Celular , Línea Celular Tumoral , Niño , Humanos , Neurilemoma/genética , Neurilemoma/patología , Neurofibromatosis/genética , Neurofibromatosis/patología , Piridinas/farmacología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Factores de Transcripción/genética , Tirfostinos/farmacologíaRESUMEN
OBJECTIVE: To describe the RAD51 response (DNA repair) to radiation-induced DNA damage in patient-derived vestibular schwannoma (VS) cells and investigate the utility of RAD51 inhibitor (RI-1) in enhancing radiation toxicity. STUDY DESIGN: Basic and translational science. SETTING: Tertiary academic facility. METHODS: VS tumors (n = 10) were cultured on 96-well plates and 16-well slides, exposed to radiation (0, 6, 12, or 18 Gy), and treated with RI-1 (0, 5, or 10 µM). Immunofluorescence was performed at 6 hours for γ-H2AX (DNA damage marker), RAD51 (DNA repair protein), and p21 (cell cycle arrest protein). Viability assays were performed at 96 hours, and capillary Western blotting was utilized to determine RAD51 expression in naïve VS tumors (n = 5). RESULTS: VS tumors expressed RAD51. In cultured VS cells, radiation initiated dose-dependent increases in γ-H2AX and p21 expression. VS cells upregulated RAD51 to repair DNA damage following radiation. Addition of RI-1 reduced RAD51 expression in a dose-dependent manner and was associated with increased γ-H2AX levels and decreased viability in a majority of cultured VS tumors. CONCLUSION: VS may evade radiation injury by entering cell cycle arrest and upregulating RAD51-dependent repair of radiation-induced double-stranded breaks in DNA. Although there was variability in responses among individual primary VS cells, RAD51 inhibition with RI-1 reduced RAD51-dependent DNA repair to enhance radiation toxicity in VS cells. Further investigations are warranted to understand the mechanisms of radiation resistance in VS and determine whether RI-1 is an effective radiosensitizer in patients with VS.
Asunto(s)
Neuroma Acústico , Recombinasa Rad51 , Traumatismos por Radiación , Humanos , Línea Celular Tumoral , Daño del ADN , Reparación del ADN , Recombinasa Rad51/antagonistas & inhibidores , Células Tumorales Cultivadas/efectos de la radiaciónRESUMEN
Neurofibromatosis Type 2 (NF2) is a rare tumor disorder caused by pathogenic variants of the merlin tumor suppressor encoded by NF2. Patients develop vestibular schwannomas (VS), peripheral schwannomas, meningiomas, and ependymomas. There are no approved drug therapies for NF2. Previous work identified phosphoinositide-3 kinase (PI3K) as a druggable target. Here we screened PI3K pathway inhibitors for efficacy in reducing viability of human schwannoma cells. The lead compound, CUDC907, a dual histone deacetylase (HDAC)/PI3K inhibitor, was further evaluated for its effects on isolated and nerve-grafted schwannoma model cells, and primary VS cells. CUDC907 (3 nM IG50) reduced human merlin deficient Schwann cell (MD-SC) viability and was 5-100 fold selective for MD over WT-SCs. CUDC907 (10 nM) promoted cell cycle arrest and caspase-3/7 activation within 24 h in human MD-SCs. Western blots confirmed a dose-dependent increase in acetylated lysine and decreases in pAKT and YAP. CUDC907 decreased tumor growth rate by 44% in a 14-day treatment regimen, modulated phospho-target levels, and decreased YAP levels. In five primary VS, CUDC907 decreased viability, induced caspase-3/7 cleavage, and reduced YAP levels. Its efficacy correlated with basal phospho-HDAC2 levels. CUDC907 has cytotoxic activity in NF2 schwannoma models and primary VS cells and is a candidate for clinical trials.
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Neurilemoma , Neurofibromatosis 2 , Humanos , Apoptosis , Caspasa 3 , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/uso terapéutico , Histona Desacetilasas , Lisina , Neurilemoma/patología , Neurofibromatosis 2/tratamiento farmacológico , Neurofibromatosis 2/metabolismo , Neurofibromatosis 2/patología , Neurofibromina 2 , Fosfatidilinositol 3-Quinasas , Fosfatidilinositoles/farmacología , Fosfatidilinositoles/uso terapéutico , Inhibidores de las Quinasa Fosfoinosítidos-3RESUMEN
Objectives Vestibular schwannomas (VS) are intracranial tumors, which are caused by NF2 gene mutations that lead to loss of merlin protein. A treatment for VS is stereotactic radiosurgery, a form of radiation. To better understand the radiobiology of VS and radiation toxicity to adjacent structures, our main objectives were (1) investigate effects of single fraction (SF) radiation on viability, cytotoxicity, and apoptosis in normal Schwann cells (SCs) and merlin-deficient Schwann cells (MD-SCs) in vitro, and (2) analyze expression of double strand DNA breaks (γ-H2AX) and DNA repair protein Rad51 following irradiation. Study Design This is a basic science study. Setting This study is conducted in a research laboratory. Participants Patients did not participate in this study. Main Outcome Measures In irradiated normal SCs and MD-SCs (0-18 Gy), we measured (1) viability, cytotoxicity, and apoptosis using cell-based assays, and (2) percentage of cells with γ-H2AX and Rad51 on immunofluorescence. Results A high percentage of irradiated MD-SCs expressed γ-H2AX, which may explain the dose-dependent losses in viability in rodent and human cell lines. In comparison, the viabilities of normal SCs were only compromised at higher doses of radiation (>12 Gy, human SCs), which may be related to less Rad51 repair. There were no further reductions in viability in human MD-SCs beyond 9 Gy, suggesting that <9 Gy may be insufficient to initiate maximal tumor control. Conclusion The MD-SCs are more susceptible to radiation than normal SCs, in part through differential expression of γ-H2AX and Rad51. Understanding the radiobiology of MD-SCs and normal SCs is important for optimizing radiation protocols to maximize tumor control while limiting radiation toxicity in VS patients.
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HYPOTHESIS: AR42, a histone deacetylase (HDAC) inhibitor, reduces viability of primary vestibular schwannoma (VS) cells and delays tumor progression and hearing loss (HL) in a xenograft model of VS. BACKGROUND: The impact of HDAC expression on AR42 response in primary VS cells is unknown, as well as the effects of AR42 on VS-associated HL and imbalance. METHODS: Primary human VS cells (n = 7) were treated with AR42 (0-3.0 µM), and viability assays were conducted. Immunohistochemistry and western blotting for phosphorylated-HDAC2 (pHDAC2) were performed on tumor chunks. Pharmacokinetic studies were conducted in Fischer rats using mass spectrometry. Merlin-deficient Schwann cells were grafted onto cochleovestibular nerves of immunodeficient rats and treated with vehicle (n=7) or AR42 (25 mg/kg/day for 4weeks; n=12). Tumor bioluminescence imaging, auditory brainstem response (ABR), and rotarod tests were conducted to 6weeks. Final tumor weight and toxicities were measured. RESULTS: AR42 caused dose-dependent reductions in viability of VS cells. Tumors with higher pHDAC2:HDAC2 ratios had greater reductions in viability with AR42. On pharmacokinetic studies, AR42 reached peak levels in nerve ~24 hours after oral administration. Although AR42-treated rats demonstrated mean ABR threshold shifts ~10 to 20 dB lower than controls, this did not persist nor reach significance. When compared to controls, AR42 did not affect tumor bioluminescence, tumor weight, and rotarod measurements. CONCLUSIONS: Response of primary VS cells to AR42 may be influenced by pHDAC2 expression in tumor. Although AR42 may delay HL in our xenograft model, it did not halt tumor growth or vestibular dysfunction. Further investigations are warranted to evaluate the AR42 effectiveness in NF2-associated VS.
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Neuroma Acústico , Animales , Modelos Animales de Enfermedad , Xenoinjertos , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Neuroma Acústico/patología , Ratas , Células de Schwann/metabolismoRESUMEN
The UMi031-A-2 hiPSC line contains a CRISPR-induced homozygous, Neurofibromatosis Type 2 (NF2) mutation (L64P (CTG > CCG)) in the NF2 gene that encodes a merlin tumor suppressor. This line was generated from an unaffected iPSC line using CRISPR technology and characterized for pluripotency and karyotypic stability. The c.191 T > C variant in NF2 is associated with a syndromic nervous system tumor disorder leading to the development of bilateral vestibular schwannomas. Once differentiated into Schwann cells, UMi031-A-2 can serve as a resource for the analysis of signaling pathways deregulated upon merlin defects and provide a pre-clinical platform for testing therapies for NF2 schwannomas.
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Neurilemoma , Neurofibromatosis 2 , Células Madre Pluripotentes , Humanos , Mutación , Neurofibromatosis 2/genética , Neurofibromina 2/genéticaRESUMEN
HYPOTHESIS: Vestibular Schwannoma (VS) can avoid cell death following radiation injury by entering cell cycle arrest and activating RAD51-related DNA repair. BACKGROUND: Although the radiobiology of various cancers is well-studied, the radiobiological effects in VS are poorly understood. In this study, we describe how VS cells enter cell cycle arrest (through p21 expression), activate DNA repair (through RAD51 upregulation), and avoid cell death after radiation-induced double-stranded breaks (DSB) in DNA (as measured by γ-H2AX). METHODS: Primary human VS cells were cultured on 96-well plates and 16-well culture slides at 10,000âcells/well and exposed to either 0 or 18 Gray of radiation. Viability assays were performed at 96âh in vitro. Immunofluorescence for γ-H2AX, RAD51, and p21 was performed at 6âh. RESULTS: Radiation (18âGy) induced the expression of γ-H2AX, p21, and RAD51 in six cultured VS, suggesting that irradiated VS acquire DSBs, enter cell cycle arrest, and initiate RAD51 DNA repair to evade cell death. However, viability studies demonstrate variable responses in individual VS cells with 3 of 6 VS showing radiation resistance to 18âGy. On further analyses, radiation-resistant VS cells expressed significantly more p21 than radiation-responsive tumors. CONCLUSIONS: In response to radiation-induced DNA damage, primary VS cells can enter cell cycle arrest and express RAD51 DNA repair mechanisms to avoid cell death. Radioresistant VS cells may mount a more robust p21 response to ensure sufficient time for DNA repair. Further investigation into DNA repair proteins and cell cycle checkpoints may provide important insight on the radiobiology of VS and mechanisms for resistance.
Asunto(s)
Neuroma Acústico , Traumatismos por Radiación , Línea Celular , Roturas del ADN de Doble Cadena , Reparación del ADN , Humanos , Neuroma Acústico/genética , Neuroma Acústico/radioterapia , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismoRESUMEN
Vestibular schwannomas (VS) are benign tumors arising from cranial nerve VIII that account for 8-10% of all intracranial tumors and are the most common tumors of the cerebellopontine angle. These tumors are typically managed with observation, radiation therapy, or microsurgical resection. Of the VS that are irradiated, there is a subset of tumors that are radioresistant and continue to grow; the mechanisms behind this phenomenon are not fully understood. In this review, the authors summarize how radiation causes cellular and DNA injury that can activate (1) checkpoints in the cell cycle to initiate cell cycle arrest and DNA repair and (2) key events that lead to cell death. In addition, we discuss the current knowledge of VS radiobiology and how it may contribute to clinical outcomes. A better understanding of VS radiobiology can help optimize existing treatment protocols and lead to new therapies to overcome radioresistance.
RESUMEN
OBJECTIVE: (1) Characterize the distribution of M1 and M2 macrophages in vestibular schwannomas by hearing status. (2) Develop assays to assess monocyte migration and macrophage polarization in cocultures with vestibular schwannoma cells. STUDY DESIGN: Basic and translational science. SETTING: Tertiary care center. METHODS: A retrospective chart review of 30 patients with vestibular schwannoma (VS) was performed. Patients were stratified into serviceable and unserviceable hearing groups. Immunohistochemistry for CD80+ M1 and CD163+ M2 macrophages was conducted. Primary VS cultures (n = 4) were developed and cocultured with monocytes. Immunohistochemistry for macrophage markers was performed to assess monocyte migration and macrophage polarization. RESULTS: Although tumors associated with unserviceable hearing had higher levels of CD80 and CD163 than those with serviceable hearing, the relationship was only significant with CD163 (P = .0161). However, CD163 level did not remain a significant predictor variable associated with unserviceable hearing on multivariate analysis when adjusted for other variables. In vitro assays show that VS cells induced monocyte migration and polarization toward CD80+ M1 or CD163+ M2 macrophage phenotypes, with qualitative differences in CD163+ macrophage morphologies between serviceable and unserviceable hearing groups. CONCLUSION: Vestibular schwannomas express varying degrees of CD80+ M1 and CD163+ M2 macrophages. We present evidence that higher expression of CD163+ may contribute to poorer hearing outcomes in patients with VS. We also describe in vitro assays in a proof-of-concept investigation that VS cells can initiate monocyte migration and macrophage polarization. Future investigations are warranted to explore the relationships between tumor, macrophages, secreted cytokines, and hearing outcomes in patients with VS.