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An Investigation of Molecular Pathways to Aid in Therapeutic Development for Neurofibromatosis Type 2Hawley, Eric Thomas 05 1900 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Neurofibromatosis type 2 (NF2) is an autosomal dominant cancer predisposition
in which loss of heterozygosity at the NF2 gene locus leads to the development of tumors
of neural crest derived origin, most commonly bilateral vestibular schwannomas. There
are currently no FDA approved chemotherapeutic agents for treatment in patients with
NF2. Development of therapeutic agents has been hampered by our incomplete
knowledge of how Merlin, the protein product of the NF2 gene, functions as a tumor
suppressor. In order develop a deeper understanding for how loss of Merlin leads to
oncogenic transformation in Schwann cells we have developed a genetically engineered
mouse model (GEMM) of Neurofibromatosis Type 2 in which functional expression of
Merlin is lost in Schwann cell precursors. In parallel studies utilizing these mice, we have
sought to understand the pathophysiology driving tumor formation in Merlin deficient
Schwann cells.
In Chapter 1, we explore the role of Merlin as a negative regulator of the Group A
p21 activated kinases, PAK1 and PAK2. We demonstrate that PAK1, a previously well
established oncogene in solid tumors and Merlin binding partner, is hyperactivated in
Merlin deficient schwannomas. Through therapeutic interventions and genetic
manipulations we demonstrate that inhibition of PAK1 was capable of reducing tumor
formation and alleviating sensorineural hearing loss in our NF2 GEMM.
In Chapter 2, we investigate the role of NF-kB inducing kinase (NIK) and NF-kB
signaling in the formation and growth of Merlin deficient Schwann cell tumors. Prior work in our lab as well as by others demonstrated elevated NF-kB signaling in Merlin
deficient Schwann cell tumors. We observed accumulation of a catalytically active
fragment of NF-kB inducing kinase and present data that accumulation of a 55Kd
constitutively active fragment of NIK is sufficient trigger wild type Schwann cells to
form tumors. In vivo however, Schwann cell intrinsic expression of NIK is not required
for tumor formation or growth. / 2 years (2021-05-24)
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Experiences from Cochlear Implantation and Auditory Brainstem Implantation in Adults and Children : Electrophysiological Measurements, Hearing Outcomes and Patient SatisfactionLundin, Karin January 2016 (has links)
Cochlear implants (CIs) and auditory brainstem implants (ABIs) are prostheses for hearing used in patients with profound hearing impairment. A CI requires an operational cochlear nerve to function in contrast to an ABI. ABIs were initially designed for adult patients with neurofibromatosis type 2 (NF2), suffering from bilateral vestibular schwannomas. Now ABIs are also used for patients, both adults and children, with congenital cochlear malformations, cochlear nerve hypoplasia/aplasia, and cochlear ossification. The aims of this thesis are to evaluate hearing outcome in patients implanted with a CI after long-term deafness. An extended period of deafness has earlier been considered as a contraindication for CI surgery. Further, we analyzed if electrically evoked auditory brainstem responses (eABRs) can predict CI outcome and pinpoint the optimal selection of treatment such as CI or ABI. We also disclose our experiences from ABI surgery in Uppsala, such as implant use, hearing outcome, complications, and satisfaction among the patients. Finally, we evaluated the results and benefits of ABIs in non-NF2 pediatric patients. Results show that patients with an extended deafness period and durations over 20 years can achieve speech understanding and benefit from CIs. Patients with long-term deafness and limited years of hearing before deafness did not perform as well as those with shorter deafness duration and longer hearing experience did. eABR seems to have a definite role in the diagnostic armamentarium, to better consider alternative surgical strategies such as ABI. No eABR waveform predicted a poor CI outcome. There was no correlation between speech perception and eABR waveform latencies or eABR waveform quality. A majority of the ABI patients used their ABIs and benefited from them for at least some period. ABI assisted voice control in a majority of the full-time users and they reported improved understanding of speech with the implant switched on. No severe complications from ABI surgery or ABI stimulation were noted. The patients were generally satisfied, even if their hearing remained very limited. All pediatric patients but one used the implant continuously and benefited from it.
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The potential of CRL4-DCAF1 and KSR1 as therapeutic targets in low-grade Merlin-deficient tumoursLyons Rimmer, Jade January 2018 (has links)
Merlin is a tumour suppressor protein that is frequently mutated or downregulated in cancer. Biallelic Merlin inactivation is causative of tumour formation, including schwannoma, meningioma and ependymoma. These tumours can occur sporadically or as part of the genetic condition Neurofibromatosis type 2 (NF2) and cause significant morbidity. The current treatment options are restricted to surgery and radiotherapy, which are invasive and may cause further tumour development. The activity of both the E3 ubiquitin ligase complex Cullin 4 really interesting new gene (RING) E3 ubiquitin ligase- DNA damage binding protein (DDB1) and Cullin 4 associated factor 1 (CRL4-DCAF1) and Kinase suppressor of RAS 1 (KSR1) have been shown to be upregulated in schwannoma to drive tumour growth. KSR1 has also been shown to interact with components of the CRL4-DCAF1 complex. We investigated the expression, interaction and therapeutic potential of targeting these proteins in Merlin deficient schwannoma and meningioma using a primary human cell model and relevant cell lines. We found that DCAF1 and KSR1 protein were overexpressed in schwannoma and meningioma and confirmed that targeting both DCAF1 and KSR1 in meningioma had additive effects on proliferation. We also identified that CRL4-DCAF1 facilitates KSR1 dependent RAF/Mitogen-activated protein kinase (MAPK)/ Extracellular signal regulated kinase (ERK) kinase (MEK)/ERK pathway activity. We showed MLN3651, a neddylation inhibitor that targets ubiquitin ligase activity, reduced proliferation and activated apoptosis in Merlin-deficient tumours. We also showed that Merlin-positive tumours were less sensitive to MLN3651 than Merlin-deficient tumours; therefore, MLN3651 sensitivity may be CRL4-DCAF1-dependent. Finally, combination of MLN3651 and the MEK1/2 inhibitor AZD6244 had additive effects, particularly in meningioma. Combinatorial therapy activated the Hippo pathway, inhibited RAF/MEK/ERK pathway activity and proliferation demonstrating that targeting the activity and downstream pathways of both DCAF1 and KSR1 represents an attractive novel therapeutic strategy in Merlin-deficient tumours.
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Modèles précliniques de schwannomes vestibulaires pour l'évaluation d’une stratégie de réduction de dose d’irradiation par combinaison avec des thérapies ciblées / Preclinical models of vestibular schwannomas for the evaluation of radiation dose reduction in combination with targeted therapiesBonne, Nicolas-Xavier 31 October 2018 (has links)
Contexte : Le schwannome vestibulaire (SV) est une tumeur bénigne de la gaine du nerf vestibulaire. La plupart des SV présentent une inactivation somatique bi-allèlique du gène suppresseur de tumeur NF2. L’inactivation congénitale du gène NF2 est impliquée dans le développement de la Neurofibromatose de type 2, une maladie génétique autosomique dominante prédisposant au développement de tumeurs multiples du système nerveux central et en particulier de schwannomes vestibulaires bilatéraux. Le traitement des schwannomes vestibulaires repose sur la chirurgie ou la radiothérapie délivrée en conditions stéréotaxiques. La réduction de la dose d’irradiation des schwannomes vestibulaires a permis d’améliorer le pronostic fonctionnel auditif tout en garantissant un taux de réponse satisfaisant. Pourtant de nombreux patients présenteront une surdité neurosensorielle progressive. Afin de poursuivre cette réduction de dose d’irradiation, des modèles biologiques fidèles récapitulant le statut d’inactivation du gène NF2 et la surdité neurosensorielle sont nécessaires à l’élaboration d’une approche préclinique.Problématique : Nous avons proposé de développer des systèmes modèles in-vitro et in-vivo compatibles avec l’étude de la radiosensibilité des schwannomes vestibulaires en combinaison avec des thérapies ciblant les voies de signalisation spécifiquement activées par la perte de fonction NF2.Méthodes : Les lignées cellulaires humaines de schwannomes vestibulaires NF2 (HEI_193, HEI_182), et de cellules de Schwann vestibulaire contrôle (HEI_286) ont été cultivées en essai clonogénique afin de déterminer le nombre d’unité formatrices de colonies à doses croissantes d’inhibiteur mTOR (Rapamycine), PI3K (GDC_0941), mTOR et PI3K (BEZ_235) pour déterminer le 50% d’inhibition de croissance (GI50%) puis en combinaison à doses croissantes de radiation gamma (Co60). La lignée cellulaire murine inactivée pour nf2 (SC4#9) a été utilisée pour réaliser des greffes syngéniques orthotopiques. La croissance des tumeurs a été suivie par IRM et bioluminescence et l’audition déterminée par potentiels évoqués auditifs. L’analyse histologique des cochlées a été réalisée par coloration en hématoxyline et éosine puis par fluorescence après clarification cochléaire. Des volumes complets ont été obtenus par microscopie confocale à balayage laser.Résultats : Les essais clonogéniques réalisés en Agarose ont identifié une radiorésistance relative des lignées humaines de schwannomes mutées pour NF2 par comparaison au contrôle humain non muté. Cette résistance identifiée en réponse à l’exposition à une dose unique d’irradiation gamma peut être contournée par l’inhibition de la voie mTOR au moment de l’irradiation restituant une sensibilité comparable au contrôle humain non muté. Une tendance à un bénéfice de l’association d’une inhibition mTOR à un inhibiteur PI3 kinase a été retrouvée à une dose maximum d’irradiation. Un modèle murin de schwannome vestibulaire qui récapitule la croissance dans l’angle ponto-cérébelleux et la perte d’audition a été développé par injection stéréotaxique dans le paquet acoustico faciale. Le suivi de croissance de ce schwannome a été caractérisé par IRM et bio-luminescence in-vivo. Enfin un protocole de clarification cochléaire a été adapté aux mammifères murins pour permettre l’étude histologique de cochlées intactes compatible avec l’étude de l’otoxicité des schwannomes et/ou de leur traitement .Conclusion : Les modèles décrits dans cette thèse permettent l’évaluation pré-clinique de stratégies thérapeutiques combinant thérapie ciblée et irradiation gamma en dose unique. L’amélioration des connaissances des mécanismes participant à l’ototoxicité des schwannomes et de leur traitement permettra d’améliorer le ciblage moléculaire afin de réduire les effets auditifs secondaires de la radiochirurgie. / Context: Vestibular schwannomas (VS) are benign neoplasm arising from the Schwann cells of the vestibular nerve. Most of sporadic VS carry a bi-allelic inactivation of the tumor suppressor gene NF2. Congenital inactivation of the NF2 gene is linked to the onset of Neurofibromatosis type 2 (NF2), a genetic condition predisposing to the development of multiple benign tumor of the central nervous system with bilateral VS as a hallmark. Treatment of VS is either surgical or by use of radiation therapy delivered in stereotactic condition. A significant dose reduction has led to improving the hearing outcomes while maintaining good tumor control. Meanwhile a significant number of treated patients will develop a progressive sensorineural hearing loss (SNHL). Laboratory models that faithfully recapitulate NF2 gene inactivation and SNHL are needed to pursue the reduction of the dose delivered.Aim: We aimed at developing new models in-vitro and in-vivo for the study of vestibular schwannoma radio sensitivity in combination with selected compounds that selectively target the pathways activated secondary to NF2 loss of function.Methodes: Human vestibular schwannoma cell lines (HEI_193, HEI_182) and control human Schwann cell line (HEI_286) were used in clonogenic assay to determine the number of colony forming unit (CFU) spontaneously and at increasing dosing of mTOR inhibitor (Rapamycin), PI3 kinase inhibitor (GDC_0941), PI3K-mTOR dual inhibitor (BEZ_235) to determine the 50% growth inhibitory threshold (GI50%) then in combination with increasing radiation regimen of gamma radiation emitted by a source of Co60. The mouse cell line inactivated for nf2 (SC4#9) was used to generate orthotropic syngrafts. The growth of the tumor was monitored using MRI and bioluminescence imaging and hearing was tested by recording auditory brainstem responses. Pathology of the cochlea were obtained from paraffin embedded sections and then using fluorescence confocal microscopy of whole mounted transparent cochleae.Results: Soft agar clonogenic assays were used and identified a resistance to radiation therapy in human cell lines of VS inactivated for NF2 when compared to the non-mutated control. This radiation resistance could be overcome by pre-exposure to the mTOR inhibitor Rapamycin allowing a return to the radiosensibility of non-mutated control. There was a tendency toward a beneficial effect when using a dual inhibition of the mTOR and PI3 kinase at a maximum dose of exposure to radiation. A mouse model of VS has been developed by stereotactic seeding of nf2 deficient cell line SC4#9 targeting the cochleo-vestibular nerve complex. It recapitulates the growth in the suitable micro-environment and secondary SNHL. The growth has been characterized using MRI and in-vivo bioluminescence imaging. Hearing loss was confirmed using sequential ABR. Last a protocol for the clarification of whole mounted cochleae has been adapted to species of rodents suitable for the pathological study of ototoxic change secondary to VS and/or its treatment.Conclusion: The models presented in this thesis may be used for the preclinical evaluation of combined therapeutic approaches with single dose gamma radiation. A better understanding of the mechanisms involved in ototoxicity secondary to VS and of its treatment would improve the molecular targeting in order to warrant better auditory outcomes.
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The role of cellular prion protein in the development of schwannomas and other Merlin-deficient tumoursProvenzano, Lucy January 2018 (has links)
Neurofibromatosis type 2 (NF2) is an inherited, multiple tumour disease caused by loss of the tumour suppressor protein, Merlin. There are several tumours associated with NF2 including; ependymomas, meningiomas and schwannomas. Merlin loss can also occur sporadically in all of these tumours and is associated with upregulation of various growth factor receptors and their relevant signalling pathways. At present the only treatment options for NF2 are surgery or radiosurgery, both of which incur serious morbidity and are unable to prevent recurrence of tumours. Either new drug treatments, or re-profiling of other drugs already commercially available, are urgently needed to improve outcome for NF2 patients. Cellular prion protein (PrPC), encoded by PRNP gene, is involved in tumour development by altering proliferation, adhesion, and survival in some cancers via focal adhesion kinase (FAK) /Src/ NFκB, cyclin D1 and p53 -proteins. Our group previously showed a strong elevation of PRNP gene activity in schwannoma. I hypothesise that PrPC may contribute to schwannoma development. To study the role of PrPC in schwannoma development I have used the well-established in vitro model of schwannoma that comprises primary human Schwann and schwannoma cells. I show that PrPC is upregulated in schwannoma as well as in Merlin-deficient meningiomas and human malignant mesotheliomas. In schwannoma PrPC is released both via exosomes and by α-cleavage which forms biologically active N- and C-terminal portions of the protein. PrPC contributes to pathological proliferation, adhesion and survival of schwannoma cells by activating ERK1/2, PI3K/AKT, cyclin D1, FAK, p53 pathways via the 37/67kDa non-integrin laminin receptor (LR/37/67kDa) and CD44. Furthermore, schwannoma cells appear to be intrinsically drug-resistant due to upregulation of MDR1 protein p-glycoprotein (p-gp) expression. P-gp expression is dependent on PrPC thus, inhibiting PrPC may be a good potential new therapeutic option for schwannoma patients, either alone or in combination with Sorafenib and p-gp inhibitor Valspodar (PSC833). An inhibitor of LR/37/67kDa/PrP interaction, NSC47924, or Bortezomib, a proteasome/NFκB inhibitor which has been approved for the treatment of multiple myeloma, could also be of beneficial therapeutic effect and is something to investigate in future work. I conclude that PrPC is an interesting new therapeutic target through its involvement with schwannoma patholgenesis and resistance to drug treatments PrPC may prove to be a good therapeutic target in other NF2-related tumours like meningiomas and schwannomas.
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Infrared neural stimulation of the cochlear nucleus : towards a new generation of auditory brainstem implantsVerma, Rohit January 2014 (has links)
In an effort to improve the auditory brainstem implant, a prosthesis in which user outcomesare modest, infrared neural stimulation (INS) was applied to the cochlear nucleus in a ratanimal model. Pulsed INS, delivered to the surface of the cochlear nucleus via an opticalfibre, evoked auditory brainstem responses (ABR) and generated broad neural activation inthe inferior Colliculus (IC). Varying the parameters of the laser stimulation revealed laserpeak power to be the dominating parameter for both ABR and IC responses. Strongestresponses were recorded when the fibre was placed at lateral positions on the cochlearnucleus, close to the temporal bone. After deafening by auditory nerve section, ABR andIC responses to INS disappeared, consistent with a reported "optophonic" effect, a laser-inducedacoustic artifact. Thus, for deaf individuals who use the auditory brainstemimplant, INS alone does not appear promising as a new approach.
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Target Validation For Neurofibromatosis Type 2 Therapeutics.Guinart, Alejandra 01 January 2013 (has links)
Neurofibromatosis type 2 (NF2) is a benign tumor disease of the nervous system. Development of bilateral vestibular schwannomas is characteristic of NF2; however patients frequently present schwannomas on other nerves, as well as meningiomas and ependymomas. Currently, there are no drug therapies for NF2. There is an urgent need for development of NF2 therapeutics and this dissertation presents two independent potential therapeutic targets. The disease is caused by mutations in the NF2 gene that encodes a tumor suppressor called merlin. Loss of merlin function is associated with increased activity of Rac and p21-activated kinases (PAK) and deregulation of cytoskeletal organization. LIM domain kinases (LIMK1 and 2) are substrates for Cdc42/Rac-PAK, and modulate actin dynamics by phosphorylating cofilin, an actin severing and depolymerizing agent. LIMKs also translocate into the nucleus and regulate cell cycle progression. Here we report that mouse Schwann cells (MSCs) in which merlin function is lost as a result of Nf2 exon2 deletion (Nf2ΔEx2) exhibited increased levels of LIMK1, LIMK2, and active phospho-Thr508/505-LIMK1/2, as well as phospho-Ser3-cofilin, compared to wild-type normal MSCs. Similarly, levels of LIMK1 and 2 total protein and active phosphorylated forms were elevated in human vestibular schwannomas compared to normal human Schwann cells (SCs). Reintroduction of wild-type NF2 into Nf2ΔEx2 MSC reduced LIMK1 and LIMK2 levels. Pharmacological inhibition of LIMK with BMS-5, decreased the viability of Nf2ΔEx2 MSCs in a dose-dependent manner, but did not affect viability of iv control MSCs. Similarly, LIMK knockdown decreased viability of Nf2ΔEx2 MSCs. The decreased viability of Nf2ΔEx2 MSCs was due to inhibition of cell cycle progression as evidenced by accumulation of cells in G2/M phase. Inhibition of LIMKs arrest cells in early mitosis by decreasing Aurora A activation and cofilin phosphorylation. To increase the search for NF2 therapeutics, we applied an alternative approach to drug discovery with an unbiased pilot high-throughput screen of the Library of Pharmacologically Active Compounds. We assayed for compounds capable of reducing viability of Nf2ΔEx2 MSC as a cellular model for human NF2 schwannomas. AGK2, a SIRT2 (sirtuin 2) inhibitor, was identified as a candidate compound. SIRT2, a mammalian sirtuin, is a NAD+ -dependent protein deacetylase. We show that Nf2ΔEx2 MSC have higher expression levels of SIRT2 and lower levels of overall lysine acetylation than wild-type control MSC. Pharmacological inhibition of SIRT2 decreases Nf2ΔEx2 MSC viability in a dose dependent manner without substantially reducing wildtype MSC viability. Inhibition of SIRT2 activity in Nf2ΔEx2 MSC causes cell death accompanied by release of the necrotic markers lactate dehydrogenase and high mobility group box 1 protein into the medium in the absence of significant apoptosis, autophagy, or cell cycle arrest. Overall this work uncovered two novel potential therapeutic targets, LIMK and SIRT2 for NF2 and tumors associated with merlin deficiency.
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The Presence of Pain Related Cytokines and Chemokines in Schwannomas and Their Potential Association with Chronic Pain in SchwannomatosisNagamoto, Jackson D 01 January 2019 (has links)
Schwannomatosis (SWN) is a genetic disorder that predisposes affected individuals to develop multiple Schwannomas anywhere in the peripheral nervous system. This can be due to a mutation in the LZTR1 or SMARCB1 genes on chromosome 22. SWN has the defining clinical symptom of chronic pain and a lack of vestibular schwannomas, which sets it apart from other, related disorders such as Neurofibromatosis Type II (NF2). Currently, it is unknown what causes the chronic pain of SWN patients but it is hypothesized that cytokines may have promote the neuropathic pain experienced by patients. This study investigates the presence of the chemokine CCL2 and the cytokine IL6 in human SWN schwannomas and non-SWN schwannomas to determine if there is a difference in the presence of these cytokines between the two tumor types. It was demonstrated that all of the SWN schwannomas expressed both CCL2 and IL6 whereas the non-SWN schwannomas expressed only one or the other protein if either. These results indicate that the presence of these cytokines within the SWN schwannomas is different from non-SWN schwannomas and could be a potential contributing factor in the occurrence of neuropathic pain experienced by SWN which is part of the differential diagnosis for NF2 and SWN.
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NEUROFIBROMATOSIS TYPE 2 PROTEIN (NF2) AS A REGULATOR OF TUMOR SUPPRESSORS AND VIRAL ONCOPROTEINS IN HUMAN GLIOBLASTOMABeltrami, Sarah January 2014 (has links)
Glioblastomas are the most common brain malignancy occurring in adults with the worst prognosis. Several obstacles have prevented the development of efficacious treatment strategies. Due to the insidious nature of these malignancies, tumors are not typically detected until late in the disease. Further, the delicate nature of surrounding normal brain tissue makes surgery and treatment with cytotoxic chemotherapeutics detrimental to the patient's quality of life. Despite decades of research and aggressive therapeutic strategies, most patients will develop recurrent tumors and succumb to the disease within 1 year of diagnosis. An enhanced understanding of the molecular interplay among tumor suppressors and oncoproteins can greatly contribute to the development of novel therapeutics that will extend life expectancies. The most common abnormality in these tumors is mutation of the p53 gene (TP53). Due to the expansive network of p53-responsive genes, loss of functional p53 prohibits the cell to the ability to regain control of aberrant proliferation in response to oncogenic stresses. Accordingly, glioblastomas have developed several mechanisms to inactivate this potent tumor suppressor. Similar to oncoproteins, viral regulatory proteins utilize p53 to prevent cell cycle arrest. One such example is the protein associated with the human polyoma virus, JC Virus (JCV). JCV is the etiologic agent of the fatal demyelinating disorder, Progressive Multifocal Leukoencephalopathy (PML), seen in severely immunocompromised patients. Infection of oligodendrocytes with JCV leads to their lytic destruction and the development of white matter lesions in PML patients. Its main regulatory protein, large tumor antigen (T-antigen), targets p53 to retain cells in a virus-producing state, thereby conveying an accidental oncogenicity. JCV T-antigen transgenic mice develop a multitude of CNS tumors, including malignant peripheral nerve sheath tumors similar to patients with a form of Neurofibromatosis. Neurofibromatosis types 1 and 2 are inherited cancer disorders resulting from the inactivation of their specific tumor suppressor genes, NF1 and NF2, respectively. Inactivation of the NF2 gene, results in the development of several multiple benign nervous system tumors. Traditionally, NF2 is viewed as a scaffolding protein primarily located at the plasma membrane, where it prevents excessive signaling via several cell surface receptors and their cytoplasmic kinases. NF2 links receptors at the plasma membrane to their cytoplasmic kinases to facilitate contact inhibition. However, NF2 can also interact with an array of cytoplasmic and a few nuclear proteins. To date, little is known about the function of NF2 in tumors not associated with NF2 syndrome. Loss of functional NF2 protein has become a staple of several sporadic cancers including mesotheliomas, and meningiomas. In glial cells, NF2 depletion results in hyperproliferation and development of oncogenic features. In the only prior report addressing the role of NF2 inactivation in glioblastoma, another group demonstrated that NF2 is a potent inhibitor of glioblastoma growth. Previously, our group has identified JCV T-antigen as a nuclear binding partner for NF2 in tumors derived from JCV T-antigen transgenic mice. The association of NF2 with T-antigen in neuronal origin tumors led us to hypothesize that NF2 could regulate the expression of the JCV T-antigen. Here, we report that NF2 suppresses T-antigen protein expression in U-87 MG human glioblastoma cells, which subsequently reduces T-antigen-mediated regulation of the JCV promoter. When T-antigen mRNA was quantified, it was determined that increasing expression of NF2 correlated with an accumulation of T-antigen mRNA; however, a decrease in T-antigen at the protein level was observed. NF2 was found to promote degradation of ubiquitin-bound T-antigen protein via a proteasome dependent pathway concomitant with the accumulation of the JCV early mRNA encoding T-antigen. The interaction between T-antigen and NF2 maps to the FERM domain of NF2 domain of NF2, which has been shown previously to be responsible for its tumor suppressor activity. Co-immunoprecipitation assays performed on a glioblastoma cell line revealed a ternary complex consisting of NF2, T-antigen, and the tumor suppressor protein, p53. Furthermore, these proteins were detected in various degrees in tumor specimens from patients, suggesting that these associations may occur in vivo. Collectively, these results demonstrate that NF2 negatively regulates JCV T-antigen expression by proteasome-mediated degradation, and suggest a novel role for NF2 as a suppressor of JCV T-antigen-induced oncogenesis. Studies in mouse and human tumors have inferred a relationship between NF2 and the primary target for JCV T-antigen, p53. In mouse models of cancer, concurrent loss of NF2 and p53 genes generates a highly malignant phenotype. Other groups reported that loss of NF2 and p53 in human tumors correlated with enhanced tumor grade. In transformed fibroblasts, NF2 can enhance the expression of p53 and promote p53-mediated apoptosis. However, the molecular details of the NF2 and p53 relationship have not yet been elucidated. Based on our data and previous literature, we believed that there is a tumor suppressive synergy that exists between NF2 and p53. Contrary to our expectations, we discovered that NF2 overexpression in U-87 MG cells results in the decline in p53 expression. We observed this effect in the p53-null cell line, Saos2, and in the presence of proteasome inhibitors. Further, we determined that NF2 utilizes cysteine proteases as part of a post translational mechanism to suppress p53 expression. Mutant p53, present in many glioblastomas, was resistant to NF2-mediated degradation. Additionally, we determined that p53 can reciprocally repress NF2 expression, by a post translational mechanism, independent of the proteasome, lysosome, or cysteine proteases. NF2 conformation mutants, S518A and S518D, can both degrade p53 and localize to the cytoplasm. However, the constitutively inactive, open form of NF2, S518D is resistant to p53-mediated degradation. NF2 and p53 do not directly interact, yet we were able to detect these proteins in the same patient glioblastoma samples. Using a conformation-specific antibody, we speculate that the majority of our glioblastoma samples may contain mutated p53. This novel relationship between NF2 and p53 we believe will have strong implications for chemotherapeutic sensitization of these typically resistant tumors. Cumulatively, these studies will provide evidence for novel tumor suppressive roles for NF2 and a greater understanding of the molecular events that shape glioblastoma progression. / Biomedical Neuroscience
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Growth of Benign and Malignant Schwannoma Xenografts in Severe Combined Immunodeficiency MiceChang, Long, Abraham, Jacob, Lorenz, Mark, Rock, Jonathan, Akhmametyeva, Elena M., Mihai, Georgeta, Schmalbrock, Petra, Chaudhury, Abhik R., Lopez, Raul, Yamate, Jyoji, John, Markus R., Wickert, Hannes, Neff, Brian A., Dodson, Edward, Welling, D. Bradley 01 November 2006 (has links)
OBJECTIVES: Models for the development of new treatment options in vestibular schwannoma (VS) treatment are lacking. The purpose of this study is to establish a quantifiable human VS xenograft model in mice. STUDY DESIGN AND METHODS: Both rat malignant schwannoma cells (KE-F11 and RT4) and human malignant schwannoma (HMS-97) cells were implanted near the sciatic nerve in the thigh of severe combined immunodeficiency (SCID) mice. Additionally, human benign VS specimens were implanted in another set of SCID mice. Three-dimensional tumor volumes were calculated from magnetic resonance images over the next 6 months. RESULTS: Mice implanted with malignant schwannoma cells developed visible tumors within 2 weeks. Imaging using a 4.7-tesla magnetic resonance imaging and immunohistopathologic examination identified solid tumors in all KE-F11 and HMS-97 xenografts, whereas RT4 xenografts consistently developed cystic schwannomas. VS xenografts demonstrated variability in their growth rates similar to human VS. The majority of VS xenografts did not grow but persisted throughout the study, whereas two of 15 xenografts grew significantly. Histopathologic examination and immunohistochemistry confirmed that VS xenografts retained their original microscopic and immunohistochemical characteristics after prolonged implantation. CONCLUSIONS: This study describes the first animal model for cystic schwannomas. Also, we demonstrate the use of high-field magnetic resonance imaging to quantify VS xenograft growth over time. The VS xenografts represent a model complimentary to Nf2 transgenic and knockout mice for translational VS research.
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