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Diffuse midline glioma (DMG) is the most aggressive primary brain tumor in children. All previous studies examining the role of systemic agents have failed to demonstrate a survival benefit; the only standard of care is radiation therapy (RT), which provides transient symptomatic relief and limited survival advantage. Successful implementation of radiosensitization strategies in DMG remains elusive. In this project, we identify Napabucasin, an NAD(P)H Quinone Dehydrogenase 1 (NQO1)-bioactivatable reactive oxygen species (ROS) inducer, as a potent radiosensitizer in DMG both in vitro and in vivo. We show Napabucasin-mediated ROS production and cytotoxicity are dependent on NQO1, and establish the novel safety, feasibility, and survival benefit of convection-enhanced drug delivery (CED) of Napabucasin to circumvent the blood-brain barrier (BBB) concurrent with RT in an orthotopic DMG mouse model. Using this multi-modality strategy, we identify a promising treatment paradigm in DMG that may also be utilized to develop novel therapeutic treatments for other brain tumors.

Οι όγκοι εγκεφάλου αποτελούν μια ομάδα όγκων με ιδιαίτερα χαρακτηριστικά. Η ογκογένεση έχει συσχετισθεί σε αρκετούς όγκους, ανάμεσά τους και ορισμένοι όγκοι εγκεφάλου, με την πρωτείνη θερμικού σοκ 90 (Hsp90). Σε πρόσφατες μελέτες, έχει αποδειχθεί η η συμμετοχή της Hsp90 στην αποδόμηση της ογκοκατασταλτικής πρωτείνης pVHL, η οποία είναι απαραίτητητη για την αποδόμηση του μεταγραφικού παράγοντα που ενεργοποιείται στην υποξία (HIF-1α). Επιπλέον, έχει βρεθεί πως οι ανταγωνιστές της Hsp90 μειώνουν τα επίπεδα έκφρασης του VEGFR-3. Στόχος της παρούσας μελέτης ήταν η εκτίμηση των επιπέδων έκφρασης των μορίων Hsp90, pVHL, HIF-1α και VEGFR-3 στους όγκους εγκεφάλου και οι πιθανές συσχετίσεις μεταξύ τους. Εξετάσθηκαν συνολικά 89 óγκοι εγκεφάλου με την ανοσοϊστοχημική μέθοδο. Στους αστροκυτταρικούς όγκους, η Ηsp90 φαίνεται πως οδηγεί στη σταθεροποίηση του HIF-1α μέσω της σύνδεσής της με τη pVHL. Στα μυελοβλαστώματα από την άλλη, ο HIF-1α ρυθμίζεται από τη Hsp90 με τρόπο ανεξάρτητο από τη pVHL. Βρέθηκε συσχέτιση της έκφρασης του VEGFR-3 με τη Hsp90 αλλά και τον HIF-1α σε ομάδα όγκων του εγκεφάλου, η βιολογική συμπεριφορά των οποίων θα πρέπει να διερευνηθεί. / Brain tumors constitute a special group of tumors. In many tumors, including brain tumors, tumorigenesis has been associated with heat shock protein 90 (Hsp90). ). Recent studies have demonstrated that Hsp90 is essential for the degradation of tumor suppressor protein, pVHL, which is essential for the degradation of Hypoxia Induced Factor 1α (HIF-1α). In addition,it has been proved that Hsp90 antagonists, reduce the expression VEGFR-3. the aim of the current study was to estimate the levels of expression of Hsp90, pVHL, HIF-1α and VEGFR-3 in brain tumors and the possible correlations among them.89 human brain tumors were studied immunohistochemically. In astrocytic tumors, Hsp90 seems to stabilize HIF-1α, through binding to pVHL. On the other hand, in medulloblastomas, HIF-1α seems to be regulated by Hsp90, in a way that seems to be independent from pVHL. We found a correlation between the expression of VEGFR-3 and Hsp90 and HIF-1α in a group of brain tumors, the biological behavior of which must be studied.

published_or_final_version / Electrical and Electronic Engineering / Doctoral / Doctor of Philosophy

This dissertation elucidates several independent molecular mechanisms that function in astrocytes and glial tumor cells, and suggest that developmental and inflammatory signals may contribute to the development of brain tumors. First, we analyzed the mechanism of TIMP-1 activation in astrocytes and glioblastoma cells. TIMP-1 expression is activated by IL-1, which is the major neuroinflammatory cytokine, via simultaneous activation of IKK/NF-kB and MEK3/6/p38/ATF-2 pathways in primary human astrocytes. In contrast to astrocytes, TIMP-1 is expressed at lower levels in glioblastomas, and is not regulated by IL-1 due to either dysfunctional IKK/NF-kB or MEK3/6/p38/ATF-2 activation. Thus, we propose a novel mechanism of TIMP-1 regulation, which ensures an increased supply of the inhibitor after tissue injury to limit the ECM degradation. This mechanism does not operate in gliomas, and may in part explain the increased invasiveness of glioma cells.Inflammation has been associated with the development of several cancers, including glioblastoma multiforme. However, it has not been linked to other brain tumors. Here we show for the first time that inflammation is associated with oligodendroglioma tumors as pro-inflammatory cytokines, such as OSM, IL-6, MCP1, MIP1α, and MIP1β and inflammatory markers, such as ACT and COX-2, were expressed at higher levels in oligodendroglioma samples. In addition, cytokine-induced STAT3 signaling, but not NF-kB, is highly activated in the oligodendroglioma patients. Moreover, OSM promotes oligodendroglioma cell proliferation in vitro, and this effect is mediated through STAT3. In summary, oligodendroglioma tumors secrete and respond to inflammatory mediators, with OSM being the major cytokine that activates STAT3 to promote the growth of tumor cells, and express ACT and COX-2 as a hallmark of ongoing inflammation. Since STAT3 promotes the growth of oligodendroglioma, as well as glioblastoma cells, and also regulates gliogenesis, we studied molecular mechanisms of this process in an in vitro differentiation model. We turn our attention to the NFI family of transcription factors since they have recently emerged as novel regulators of the development of vertebral neocortex. We developed a stem cell-neural progenitor-astrocyte differentiation model, in which the generated astrocytes were characterized by proper morphology, increased glutamate uptake, and expression of early and late astrocyte markers. Moreover, we found that NFI-X and NFI-C but not NFI-A or NFI-B, control the expression of GFAP and SPARCL1, the markers of terminal differentiation of astrocytes.In summary, the three mechanisms of gene regulation we studied, provided new insights into astrocyte biology, with the important implications for understanding the basis leading to the development and progression of brain tumors.

A epilepsia associada a tumor cerebral é uma condição debilitante, causadora de importante prejuízo sobre a qualidade de vida dos que sofrem desta condição. Relacionada à grande refratariedade ao tratamento medicamentoso, tanto a epilepsia quanto o uso de drogas antiepilépticas (DAEs) predispõem à deterioração das funções cognitivas. Em casos raros, a epilepsia secundária a tumor cerebral pode ser devastadora, aumentando os riscos de morte súbita. Buscando auxiliar a tomada de decisões e enfatizando os benefícios de uma discussão ampla entre equipes de oncologia, neurologia infantil, epilepsia e neurocirurgia, este trabalho objetiva descrever as características clínicas gerais, eletroencefalográficas, histopatológicas dos pacientes, verificar o impacto do tratamento cirúrgico sobre a epilepsia quanto ao desfecho clínico relacionado ao controle das crises, comparar os resultados da avaliação cognitiva nos períodos pré e pós-operatórios e descrever a ocorrência de complicações cirúrgicas intra-operatórias, pós-operatórias e óbitos. Trata-se de estudo observacional transversal retrospectivo, por revisão de prontuários de pacientes com idade inferior a 19 anos quando submetidos à cirurgia para tratamento de epilepsia refratária secundária a tumores cerebrais entre 1996 e 2013, pela equipe do Centro de Cirurgia de Epilepsia do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto. Para análise dos desfechos quanto à incidência de crises, utilizou-se a classificação de Engel, adaptada para uso após o primeiro ano da cirurgia. Procedeu-se à análise descritiva dos dados. Foram incluídas 27 crianças, com média de 6,0 anos (3 meses a 15,8 anos) no início dos sintomas. O intervalo entre o início das crises epilépticas e o diagnóstico do tumor foi de 3,6 anos (um mês a 14,5 anos). A média da quantidade de drogas antiepilépticas (DAEs) utilizadas antes da cirurgia foi 3,6. A média da idade no diagnóstico do tumor cerebral foi 9,7 anos (10 meses a 16,8 anos). A localização do tumor foi lobo temporal em 59,2%, sendo ganglioglioma e DNET os mais frequentes, em igual proporção, 33,3%. Envolvimento de área eloquente ocorreu em 18,5%. A vídeomonitorização eletrográfica evidenciou descargas focais na área tumoral em 85,2%. O intervalo entre o diagnóstico tumoral e a realização da cirurgia foi de 1,5 anos (dias a 7 anos). A média de idade dos pacientes no momento da cirurgia foi 11,3 anos (3 a 17, 4 anos). A ressecção tumoral foi completa em 88,8% dos pacientes. Complicação pós-cirúrgica, osteomielite, ocorreu em 1 (3,7%). Um paciente (3,7%) com oligodendroglioma anaplásico foi a óbito após 2 anos da cirurgia. Os desfechos clínicos relacionados ao controle de crises dos pacientes submetidos à cirurgia foram satisfatórios, com Engel I correspondendo a 92,6% no primeiro ano pós-operatório. Apenas 14,8% apresentaram Engel III - IV durante todo o período de seguimento. A média do tempo para retirada das DAEs após a cirurgia foi de 3,2 anos (1,7 a 7 anos). Alterações neurológicas após a cirurgia ocorreram em 18,5%, sendo os déficits neurológicos focais transitórios. Evoluíram com melhora do perfil intelectual 31,3%, inalterado 50% e piora 18,7%. A cirurgia para tratamento da epilepsia secundária a tumor cerebral evidenciou-se uma modalidade terapêutica potencialmente curativa e segura, portanto, o diagnóstico tumoral não pode ser postergado / Tumor-associated epilepsy is a debilitating condition causing injury to the quality of life of those who suffer from a brain tumor. It has been shown to have a greater refractivity to antiepileptic drug therapy. Both epilepsy and the use of antiepileptic drugs have a predisposition to the deterioration of cognitive functions. In rare cases tumor-associated epilepsy can be devastating, increasing the risk of sudden death. Seeking help with decisionmaking and emphasizing the benefits of a broad discussion among oncology teams, child neurology, epilepsy and neurosurgery, this paper describes the general, clinical, electroencephalographic, and histopathological patient characteristics, verifies the impact of surgical treatment of epilepsy as the clinical outcome related to the control of seizures, compares the result of cognitive assessment in the pre to the postoperative and describes the occurrence of intraoperative surgical complications and postoperative deaths. It is a retrospective cross-sectional observational study, by review of medical records of patients under the age of 19 who underwent surgery to treat tumor-associated epilepsy between 1996 and 2013, by the Epilepsy Surgery Center of the Hospital of School of Medicine of Ribeirão Preto, São Paulo University. For analysis of outcomes in the incidence of crises, the Engel classification was used and adapted for use after the first year of surgery. It was used with the descriptive analysis of the data. Twenty seven children were included, with a mean of 6.0 years (3 months to 15.8 years) at the beginning of symptoms. The interval between the onset of seizures and the diagnosis of the tumor was 3.6 years (1 month to 14.5 years). The average number of antiepileptic drugs (AEDs) used before surgery was 3.6. The average age at diagnosis of brain tumor was 9.7 years (10 months to 16.8 years). The tumor site was the temporal lobe in 59.2% of patients and ganglioglioma and DNET were the most common, in equal proportion, 33.3%. Eloquent area of involvement occurred in 18.5%. The electrographic video monitoring showed focal discharges at the tumor site in 85.2% of patients. The interval between tumor diagnosis and the surgery was 1.5 years (days to 7 years). The average patient age at surgery was 11.3 years (3-17, 4 years). Tumor resection was complete in 88.8% of patients. Post-surgical complication, osteomyelitis, occurred in 1 (3.7%) of patients. Only one patient (3,7%) had anaplastic oligodendroglioma and dead two years after surgery. Clinical outcomes related to the control of seizures in patients undergoing surgery were satisfactory, with Engel I corresponding to 92,6% in the first year of follow up. Only 14.8% had Engel III - IV during the follow-up period. The average time for withdrawal of AEDs after surgery was 3.2 years (1.7 to 7 years). Neurological changes after surgery occurred in 18.5%, and were transient focal neurological deficits. The improvement of the intellectual profile occurred in 31.3%, unchanged in 50% and 18.7% worsened. Surgery to treat tumor-associated epilepsy showed up a potentially curative and safe therapeutic modality, therefore, tumor diagnosis cannot be postponed

Numerous studies have suggested rapid weight gain following diagnosis and initial treatment of childhood craniopharyngioma (CP) due to the damage sustained by the hypothalamus. Hypothalamic lesions formed by the treatment of the tumor and/or by invasiveness of the tumor itself are known to cause intractable weight gain, known as hypothalamic obesity. In contrast, hypothalamic obesity manifested in pediatric low-grade glioma (PLGG) patients is not as prominently addressed in literature; likely due to the expansive set of histological tumor subtypes that makes generalization challenging. Specifically, there is a lack of analysis that examines the difference in treatment, endocrinopathies, and weight gain between CP and PLGG patients. The purpose of this study was to compare hypothalamic damage in subjects diagnosed with pediatric hypothalamic low-grade glioma versus subjects diagnosed with childhood craniopharyngioma. We hypothesized that CP patients will have a more rapid post diagnosis weight gain and a greater degree of obesity compared with PLGG patients due to the more invasive nature of the tumor and the aggressive surgical treatments involved. We performed a retrospective review of the clinical records of patients who received a diagnosis of childhood craniopharyngioma or pediatric low-grade glioma at Dana-Farber Cancer Institute between 1980 and 2009. We identified 45 patients, who met criteria for evaluation, 28 were previously diagnosed with childhood craniopharyngioma and 17 were diagnosed with hypothalamic pediatric low-grade glioma. We analyzed the impact of treatment, the presence of endocrinopathies, and weight gain after diagnosis. We concluded that there was no statistically significant difference in the rate or magnitude of post diagnosis weight gain, disproving our initial hypotheses.

Introdução: Gliomas são os tumores cerebrais mais frequentes em pacientes com neoplasias de Sistema Nervoso Central (SNC), sendo o Glioblastoma Multiforme (GBM) o mais agressivo e letal deles. Apesar dos esforços na melhoria dos tratamentos atuais, o prognóstico para os pacientes com GBM continua sendo incerto. Sendo necessário o uso de novas estratégias terapêuticas que visem melhorar o manejo dos gliomas malignos. A combinação de terapias que agem nas principais vias de sinalização celular envolvidas na progressão do câncer poderia potencializar o efeito antitumoral das monoterapias. Métodos: As linhagens celulares U-87 e A-172 foram tratadas com o anti-EGFR tyrphostin AG1478, o inibidor de histonas deacetilases butirato de sódio (NaB) ou a combinação de ambos, por 72 horas. Tanto a viabilidade avaliada em 72 horas quanto a proliferação celular a longo prazo foram medidas através do ensaio de exclusão com azul de tripan em câmara de Neubauer. A influência do tratamento no ciclo celular e a capacidade de formar colônias foram avaliadas através da marcação com iodeto de propídeo e ensaio clonogênico, respectivamente. Resultados: Foi possível observar que o tratamento combinado com AG1478 e NaB foi capaz de reduzir a viabilidade e a proliferação celular na linhagem U-87 de GBM. Conclusão: Nosso trabalho mostrou que a inibição da via do receptor do fator de crescimento epidérmico (EGFR) combinada com a inibição das histonas deacetilases foi mais efetiva que as monoterapias na inibição da viabilidade e a proliferação celular. Esta redução foi significativa na linhagem U-87. Futuros estudos devem ser feitos para descobrir as possíveis interações entre as duas vias de sinalização em GBM. / Introduction: Gliomas are the most frequent brain tumors, in patients with Central Nervous system (NCS) malignancies, being the Glioblastoma Multiforme the most aggressive and lethal of all. Despite current multimodality treatment efforts, the prognosis for GBM patients remains poor. New therapeutic strategies that target these pathways to improve the treatment of malignant gliomas are needed. Combination of therapies with synergistic effects in the cellular signaling pathways of cancer could potentiate the anti-tumor effect of monotherapy alone. Methods: U87 and A172 cell lines were treated with the anti-EGFR Thyrphostin AG1478, the Histone Deacetylase inhibitor (HDACi) Sodyum Butyrate (NaB), or combination of both, for 72 hours. The cellular proliferation in short and in a long time was measured through the trypan-blue assay on neubauer chamber, the influence on the cell cycle and the capability of form colonies was evaluated by nuclear staining with propidium iodide and clonogenic assay respectively. Results: We found that combined treatment with AG1478 and NaB, are able to reduce the viability and proliferation in U-87. Conclusion: Our work show that combined inhibition of both epidermal growth factor receptor and histone deacetylases was able to reduce cell proliferation in GBM cell lines. This reduction was considerably significant in U-87 cell lines when compared with individual treatments. Further studies should be performed to discover the possible crosstalk between the signaling pathways of both targets in GBM.

Glioblastoma multiforme (GBM) is the most common malignant central nervous system tumor. The ability of glioma cells to rapidly disperse and invade healthy brain tissue, coupled with their high resistance to chemotherapy and radiation have resulted in extremely poor prognoses among patients. In recent years, nitric oxide (NO) has been discovered to play a ubiquitous of role in human physiology and studies have shown that, at sufficient concentrations, NO is able to induce apoptosis as well as chemosensitization in tumor cells. This thesis discusses the synthesis and characterization of targeted NO donors for the treatment of GBM. Two glioma targeting biomolecules, Chlorotoxin (CTX) and VTWTPQAWFQWVGGGSKKKKK (VTW) were reacted with NO gas to synthesize NO donors. These NO donors, CTX-NO and VTW-NO, released NO for over 3 days and were able to induce cytotoxicity in a dose dependent manner in glioma cells. The biggest advantage, a result of the targeted delivery of NO, was that the NO donors did not have toxic effects on astrocytes and endothelial cells. To characterize the chemosensitizing effects of CTX-NO, cells were incubated with CTX-NO prior to exposure to temozolomide (TMZ) or carmustine (BCNU). These drugs are the most popular chemotherapeutics used in the treatment of GBM, but have only shown modest improvements in patient survival. Viability studies showed that CTX-NO selectively elicited chemosensitivity in glioma cells, whereas the chemosensitivty of astrocytes and endothelial cells remained unaffected. Further investigation showed that CTX-NO pretreatment decreased O6-methylguanine DNA methyltransferase (MGMT) and p53 levels, suggesting that a decrease in DNA repair ability may be the mechanism by which chemosensitivity is induced. Lastly, the effects of CTX-NO on glioma cell invasion and migration were studied using Boyden chamber and modified scratch assays. Non-toxic doses of CTX-NO decreased glioma cell invasion in a dose dependent manner. Studies quantifying matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) surface expression demonstrated that while MMP-2 expression was decreased by both CTX and CTX-NO, MMP-9 expression was decreased only by CTX-NO. Furthermore quantifying MMP-2 and MMP-9 activity levels showed that NO and CTX work synergistically to decrease the activity of the enzymes. These studies demonstrate that the decrease in glioma invasion resulting from CTX-NO treatment was partially a consequence of decreased levels of surface and activated MMP-2 and MMP-9. The work presented in this thesis describes a novel approach to treating GBM that can be modified to develop treatments for various other tumors. Furthermore this is the first study to develop glioma-targeting NO donors.