RESUMO
Vestibular schwannoma (VS) is intracranial tumor arising from neoplastic Schwann cells, causing hearing loss in about 95% of patients. The traditional belief that hearing deficit is caused by physical expansion of the VS, compressing the auditory nerve, does not explain the common clinical finding that patients with small tumors can have profound hearing loss, suggesting that tumor-secreted factors could influence hearing ability in VS patients. Here, we conducted profiling of patients' plasma for 67 immune-related factors on a large cohort of VS patients (N>120) and identified candidate biomarkers associated with tumor growth (IL-16 and S100B) and hearing (MDC). We identified the 7-biomarker panel composed of MCP-3, BLC, S100B, FGF-2, MMP-14, eotaxin, and TWEAK that showed outstanding discriminatory ability for VS. These findings revealed possible therapeutic targets for VS-induced hearing loss and provided a unique diagnostic tool that may predict hearing change and tumor growth in VS patients and may help inform the ideal timing of tumor resection to preserve hearing.
RESUMO
Vestibular schwannoma (VS) is an intracranial tumor arising from neoplastic Schwann cells and typically presenting with hearing loss. The traditional belief that hearing deficit is caused by physical expansion of the VS, compressing the auditory nerve, does not explain the common clinical finding that patients with small tumors can have profound hearing loss, suggesting that tumor-secreted factors could influence hearing ability in VS patients. We conducted profiling of patients' plasma for 66 immune-related factors in patients with sporadic VS (N > 170) and identified and validated candidate biomarkers associated with tumor size (S100B) and hearing (MCP-3). We further identified a nine-biomarker panel (TNR-R2, MIF, CD30, MCP-3, IL-2R, BLC, TWEAK, eotaxin, and S100B) with outstanding discriminatory ability for VS. These findings revealed possible therapeutic targets for VS, providing a unique diagnostic tool that may predict hearing change and tumor growth in VS patients, and may inform the timing of tumor resection to preserve hearing.
Assuntos
Surdez , Perda Auditiva , Neuroma Acústico , Humanos , Neuroma Acústico/diagnóstico , Neuroma Acústico/patologia , Neuroma Acústico/cirurgia , Perda Auditiva/etiologia , Audição , BiomarcadoresRESUMO
Hearing loss in patients with vestibular schwannoma (VS) is commonly attributed to mechanical compression of the auditory nerve, though recent studies suggest that this retrocochlear pathology may be augmented by cochlear damage. Although VS-associated loss of inner hair cells, outer hair cells, and spiral ganglion cells has been reported, it is unclear to what extent auditory-nerve peripheral axons are damaged in VS patients. Understanding the degree of damage VSs cause to auditory nerve fibers (ANFs) is important for accurately modeling clinical outcomes of cochlear implantation, which is a therapeutic option to rehabilitate hearing in VS-affected ears. A retrospective analysis of human temporal-bone histopathology was performed on archival specimens from the Massachusetts Eye and Ear collection. Seven patients met our inclusion criteria based on the presence of sporadic, unilateral, untreated VS. Tangential sections of five cochlear regions were stained with hematoxylin and eosin, and adjacent sections were stained to visualize myelinated ANFs and efferent fibers. Following confocal microscopy, peripheral axons of ANFs within the osseous spiral lamina were quantified manually, where feasible, and with a "pixel counting" method, applicable to all sections. ANF density was substantially reduced on the VS side compared to the unaffected contralateral side. In the upper basal turn, a significant difference between the VS side and unaffected contralateral side was found using both counting methods, corresponding to the region tuned to 2000 Hz. Even spiral ganglion cells (SGCs) contralateral to VS were affected by the tumor as the majority of contralateral SGC counts were below average for age. This observation provides histological insight into the clinical observation that unilateral vestibular schwannomas pose a long-term risk of progression of hearing loss in the contralateral ear as well. Our pixel counting method for ANF quantification in the osseous spiral lamina is applicable to other pathologies involving sensorineural hearing loss. Future research is needed to classify ANFs into morphological categories, accurately predict their electrical properties, and use this knowledge to inform optimal cochlear implant programming strategies.
Assuntos
Surdez , Perda Auditiva , Neuroma Acústico , Humanos , Nervo Coclear/patologia , Surdez/patologia , Perda Auditiva/patologia , Neuroma Acústico/patologia , Estudos Retrospectivos , Gânglio Espiral da Cóclea/patologia , Lâmina EspiralRESUMO
Hearing loss is one of the most common symptoms of neurofibromatosis type 2 (NF2) caused by vestibular schwannomas (VSs). Fibrosis in the VS tumor microenvironment (TME) is associated with hearing loss in patients with NF2. We hypothesized that reducing the fibrosis using losartan, an FDA-approved antihypertensive drug that blocks fibrotic and inflammatory signaling, could improve hearing. Using NF2 mouse models, we found that losartan treatment normalized the TME by (i) reducing neuroinflammatory IL-6/STAT3 signaling and preventing hearing loss, (ii) normalizing tumor vasculature and alleviating neuro-edema, and (iii) increasing oxygen delivery and enhancing efficacy of radiation therapy. In preparation to translate these exciting findings into the clinic, we used patient samples and data and demonstrated that IL-6/STAT3 signaling inversely associated with hearing function, that elevated production of tumor-derived IL-6 was associated with reduced viability of cochlear sensory cells and neurons in ex vivo organotypic cochlear cultures, and that patients receiving angiotensin receptor blockers have no progression in VS-induced hearing loss compared with patients on other or no antihypertensives based on a retrospective analysis of patients with VS and hypertension. Our study provides the rationale and critical data for a prospective clinical trial of losartan in patients with VS.
Assuntos
Perda Auditiva , Neurilemoma , Neurofibromatose 2 , Animais , Humanos , Losartan/farmacologia , Losartan/uso terapêutico , Camundongos , Estudos Prospectivos , Estudos Retrospectivos , Roedores , Resultado do Tratamento , Microambiente TumoralRESUMO
Improved biomarkers are needed for vestibular schwannoma (VS), the most common tumor of the cerebellopontine angle, as existing clinical biomarkers have poor predictive value. Factors such as tumor size or growth rate do not shed light on the pathophysiology of associated sensorineural hearing loss (SNHL) and suffer from low specificity and sensitivity, whereas histological markers only sample a fraction of the tumor and are difficult to ascertain before tumor treatment or surgical intervention. Proteases play diverse and critical roles in tumorigenesis and could be leveraged as a new class of VS biomarkers. Using a combination of in silico, in vitro, and ex vivo approaches, we identified matrixmetalloprotease 14 (MMP-14; also known as MT1-MMP), from a panel of candidate proteases that were differentially expressed through the largest meta-analysis of human VS transcriptomes. The abundance and proteolytic activity of MMP-14 in the plasma and tumor secretions from VS patients correlated with clinical parameters and the degree of SNHL. Further, MMP-14 plasma levels correlated with surgical outcomes such as the extent of resection. Finally, the application of MMP-14 at physiologic concentrations to cochlear explant cultures led to damage to spiral ganglion neuronal fibers and synapses, thereby providing mechanistic insight into VS-associated SNHL. Taken together, MMP-14 represents a novel molecular biomarker that merits further validation in both diagnostic and prognostic applications for VS.
RESUMO
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.
Assuntos
Benzamidas/farmacologia , Dasatinibe/farmacologia , Modelos Animais de Doenças , Morfolinas/farmacologia , Neurofibromina 2/fisiologia , Neuroma Acústico/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Quimioterapia Combinada , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurofibromina 2/genética , Neurofibromina 2/metabolismo , Neuroma Acústico/metabolismo , Neuroma Acústico/patologia , Receptor EphA1/metabolismoRESUMO
Vestibular schwannoma (VS) is the fourth most common intracranial tumor, arising from neoplastic Schwann cells of the vestibular nerve and often causing debilitating sensorineural hearing loss (SNHL) and tinnitus. Previous research suggests that the abnormal upregulation of inflammatory pathways plays a highly significant, though infrequently described role in VS pathobiology, and that VS-associated SNHL is due not only to mechanical compression of the auditory nerve but also to differences in the intrinsic biology of these tumors. We hypothesize that patients who present with poor hearing associated with VS experience a more robust inflammatory response to this tumor than VS patients who present with good hearing. To investigate this hypothesis, we conducted a comprehensive pathway analysis using gene expression data from the largest meta-analysis of vestibular schwannoma microarray data, comprising 80 tumors and 16 healthy peripheral nerves. We identified the NLRP3 inflammasome as a novel target worthy of further exploration in VS research and validated this finding at the gene and protein expression level in human VS tissue using qRT-PCR and immunohistochemistry. To date, NLRP3 inflammasome activation has not been reported in VS, and this finding may represent a new and potentially significant therapeutic avenue. Notably, after analysis of 30 VSs, we observe that overexpression of key components of the NLRP3 inflammasome is preferentially associated with tumors that produce increased hearing loss in VS patients. Therefore, therapeutic development for VS should include considerations for minimizing NLRP3-associated inflammation to best preserve hearing.
Assuntos
Perda Auditiva Neurossensorial/etiologia , Audição , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neuroma Acústico/complicações , Nervo Vestibulococlear/metabolismo , Estudos de Casos e Controles , Regulação da Expressão Gênica , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/metabolismo , Perda Auditiva Neurossensorial/fisiopatologia , Humanos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Neuroma Acústico/patologia , Nervo Vestibulococlear/patologia , Nervo Vestibulococlear/fisiopatologiaRESUMO
BACKGROUND: We evaluated apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) as a schwannoma tumor suppressor and explored its utilization in a schwannoma gene therapy strategy that may be translated to clinical use. METHODS: ASC protein expression and mRNA level were assessed in human schwannoma by immunohistochemistry and quantitative PCR, respectively. Methylation- specific PCR was used to assess ASC promoter methylation. The effect of ASC overexpression in schwannoma cells was evaluated through ATP-based viability, lactate dehydrogenase release, and apoptosis staining. Western blotting and colorimetric assay were used to test the effect of ASC overexpression on endogenous pro-apoptotic pathways. Bioluminescence imaging, behavioral testing, and immunohistochemistry in human xenograft and murine allograft schwannoma models were used to examine the efficacy and toxicity of intratumoral injection of adeno-associated virus (AAV) vector encoding ASC. RESULTS: ASC expression was suppressed via promoter methylation in over 80% of the human schwannomas tested. ASC overexpression in schwannoma cells results in cell death and is associated with activation of endogenous caspase-9, caspase-3, and upregulation of BH3 interacting-domain death agonist. In a human xenograft schwannoma model, AAV1-mediated ASC delivery reduced tumor growth and resolved tumor-associated pain without detectable toxicity, and tumor control was associated with reduced Ki67 mitotic index and increased tumor-cell apoptosis. Efficacy of this schwannoma gene therapy strategy was confirmed in a murine schwannoma model. CONCLUSION: We have identified ASC as a putative schwannoma tumor suppressor with high potential clinical utility for schwannoma gene therapy and generated a vector that treats schwannomas via a novel mechanism that does not overlap with current treatments.
Assuntos
Apoptose , Biomarcadores Tumorais/genética , Proteínas Adaptadoras de Sinalização CARD/administração & dosagem , Dor do Câncer/prevenção & controle , Terapia Genética , Neurilemoma/terapia , Animais , Proteínas Adaptadoras de Sinalização CARD/genética , Dor do Câncer/etiologia , Proliferação de Células , Metilação de DNA , Dependovirus/genética , Humanos , Masculino , Camundongos , Neurilemoma/genética , Neurilemoma/patologia , Prognóstico , Regiões Promotoras Genéticas , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
RESUMO
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.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Reposicionamento de Medicamentos/métodos , Mifepristona/farmacologia , Neuroma Acústico/patologia , Algoritmos , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , HumanosRESUMO
Vestibular schwannomas are the most common neoplasms of the cerebellopontine angle, making up 6-8% percent of all intracranial growths. Though these tumors cause sensorineural hearing loss in up to 95% of affected individuals, the molecular mechanisms underlying this hearing loss remain elusive. This article outlines the steps established in our laboratory to facilitate the collection and processing of various primary human tissue samples for downstream research applications integral to the study of vestibular schwannomas. Specifically, this work describes a unified methodological framework for the collection, processing, and culture of Schwann and schwannoma cells from surgical samples. This is integrated with parallel processing steps now considered essential for current research: the collection of tumor and nerve secretions, the preservation of RNA and the extraction of protein from collected tissues, the fixation of tissue for the preparation of sections, and the exposure of primary human cells to adeno-associated viruses for application to gene therapy. Additionally, this work highlights the translabyrinthine surgical approach to collect this tumor as a unique opportunity to obtain human sensory epithelium from the inner ear and perilymph. Tips to improve experimental quality are provided and common pitfalls highlighted.
Assuntos
Pavilhão Auricular/inervação , Neuroma Acústico/metabolismo , Neuroma Acústico/patologia , Cultura Primária de Células/métodos , Células de Schwann/metabolismo , Células de Schwann/patologia , Feminino , Perda Auditiva Neurossensorial/patologia , Humanos , Células Tumorais CultivadasRESUMO
Auditory neuropathy is a significant and understudied cause of human hearing loss, diagnosed in patients who demonstrate abnormal function of the cochlear nerve despite typical function of sensory cells. Because the human inner ear cannot be visualized during life, histopathological analysis of autopsy specimens is critical to understanding the cellular mechanisms underlying this pathology. Here we present statistical models of severe primary neuronal degeneration and its relationship to pure tone audiometric thresholds and word recognition scores in comparison to age-matched control patients, spanning every decade of life. Analysis of 30 ears from 23 patients shows that severe neuronal loss correlates with elevated audiometric thresholds and poor word recognition. For each ten percent increase in total neuronal loss, average thresholds across patients at each audiometric test frequency increase by 6.0 dB hearing level (HL). As neuronal loss increases, threshold elevation proceeds more rapidly in low audiometric test frequencies than in high frequencies. Pure tone average closely agrees with word recognition scores in the case of severe neural pathology. Histopathologic study of the human inner ear continues to emphasize the need for non- or minimally invasive clinical tools capable of establishing cellular-level diagnoses.
Assuntos
Limiar Auditivo , Biomarcadores , Cóclea/patologia , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/patologia , Transtornos Heredodegenerativos do Sistema Nervoso/diagnóstico , Transtornos Heredodegenerativos do Sistema Nervoso/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Correlação de Dados , Feminino , Histocitoquímica , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Estatísticos , Adulto JovemRESUMO
Vestibular schwannoma (VS) is the most common tumor of the cerebellopontine angle, and it typically presents with sensorineural hearing loss. The genomic landscape of schwannoma is complex and many of the molecules implicated in VS pathogenesis represent targets not amenable to antibody-based or small molecule therapeutics. Tumor-targeted delivery of small interfering RNA (siRNA) therapeutics provides a direct and effective means to interrogate targets while minimizing off-target effects. To establish a preclinical model for therapeutic inhibition of putative targets in VS, archived tumor specimens, fresh tumor cells derived from patients with sporadic VS, and an established schwannoma cell line were screened. Nanoparticles directed by the tumor-homing peptide iRGD were selectively taken up by primary VS cultures in vitro via interactions with αvß3/ß5 integrins and neuropilin-1 (NRP-1). Cellular uptake was inhibited by a neutralizing antibody against αv integrin in a dose-dependent manner. When applied to primary VS cultures, iRGD-targeted nanoparticles delivered siRNA directed against TNFα in a receptor-specific fashion to potently silence gene expression and protein secretion. Taken together, our results provide a proof of principle for tumor-targeted, nanoparticle-mediated delivery of siRNA to VS and establish a novel platform for the development and pre-clinical screening of molecular therapeutics against VS.
Assuntos
Neurilemoma/metabolismo , RNA Interferente Pequeno/administração & dosagem , Fator de Necrose Tumoral alfa/metabolismo , Vestíbulo do Labirinto/metabolismo , Adolescente , Adulto , Idoso , Linhagem Celular Tumoral , Feminino , Inativação Gênica , Humanos , Mediadores da Inflamação/metabolismo , Masculino , Pessoa de Meia-Idade , Nanopartículas/química , Oligopeptídeos/química , Receptores de Superfície Celular/metabolismo , Adulto JovemRESUMO
Here we report the methods and output of a workshop examining possible futures of speech and hearing science out to 2030. Using a design thinking approach, a range of human-centered problems in communication were identified that could provide the motivation for a wide range of research. Nine main research programs were distilled and are summarized: (a) measuring brain and other physiological parameters, (b) auditory and multimodal displays of information, (c) auditory scene analysis, (d) enabling and understanding shared auditory virtual spaces, (e) holistic approaches to health management and hearing impairment, (f) universal access to evolving and individualized technologies, (g) biological intervention for hearing dysfunction, (h) understanding the psychosocial interactions with technology and other humans as mediated by technology, and (i) the impact of changing models of security and privacy. The design thinking approach attempted to link the judged level of importance of different research areas to the "end in mind" through empathy for the real-life problems embodied in the personas created during the workshop.