CRMP5 appartient à la famille des Collapsin Response Mediator Protein. Ces protéines sont très exprimées dans le cerveau en développement et les zones de neurogénèse chez l'adulte. Dans un contexte tumoral, l'expression des messagers de CRMP5 émergent dans un cluster de gènes associés à la plus faible survie des 20 patients suivis, et à la prolifération (Liang et al., 2005). Nous avons confirmé ces résultats dans une série rétrospective de 183 GBM où la forte expression protéique de CRMP5 est corrélée à une plus faible survie des patients (7.14 mois vs 10 mois) ; de plus les tumeurs exprimant fortement CRMP5 présentent un index mitotique 2 fois plus important (p = 0.0009) que les tumeurs exprimant faiblement CRMP5. Dans des cultures primaires ou lignées cellulaires de GBM nous montrons que la prolifération des glioblastomes est dépendante de l'expression de CRMP5 et de la voie de signalisation Notch. Des analyses en western blot démontrent que CRMP5 protège les récepteurs Notch de la dégradation lysosomale. Nous avons approfondi ce mécanisme et montré une nouvelle voie de régulation de Notch par CRMP5 qui par une interaction protéique avec Numb, l'inhibiteur de Notch empêche la dégradation du récepteur. Parallèlement, l'analyse en immunohistochimie sur les biopsies de GBM montrent une forte expression Notch et sa cible Hes1 dans les tumeurs exprimant fortement CRMP5. Ces résultats montrent la corrélation entre l'expression de CRMP5 dans les GBM, l'activation de la voie Notch et la faible survie des patients. Le ciblage de l'interaction CRMP5-Numb est une stratégie potentielle pour un traitement ciblé des glioblastomes / CRMP5 belongs to the Collapsin Response Mediator Protein family, highly expressed in the developing brain and in adult brain neurogenesis areas. In pathology, we identified CRMP5 as a marker of aggressivity in neuroendocrine lung tumors (Meyronet et al, 2008) while Liang et al (2005) using transcriptomic analysis of 20 Glioblastomas, revealed CRMP5 in a cluster of genes related to proliferation correlated with a poor overall survival. We confirmed these results at the protein level in a retrospective serie of 183 GBMs and correlated higher CRMP5 expression with a significantly lower median survival (7.14 months) than those with negative expression (10 months) (p=0,026).Furthermore, GBM with higher CRMP5 expression were characterized by a higher mitotic index. CRMP5 knockdown (siRNA) in primary culture from GBM xenograft and in the GBM cell line GL15 showed a dependence of GBM cell proliferation for CRMP5 expression. Notch signaling pathway expression and activation was found post-translationally dependent of CRMP5 expression. These results are enlightened by a clinical study showing a poor expression of Notch 1 and Hes1 in CRMP5-negative tumours compared to CRMP5-positive GBM. Mechanistically, we show a novel regulation of the Notch signaling pathway in GBM by CRMP5 counteracting Numb-dependent Notch receptors degradation by a direct protein interaction. These results show that CRMP5 expression in GBM activates Notch signalling pathway to promote proliferation and poor survival. Targeting CRMP5-Numb interaction is a promising strategy for future glioblastoma treatment

Les intégrines, protéines transmembranaires hétérodimériques de type αβ, sont impliquées dans un grand nombre de phénomènes physiologiques et pathologiques. L’intégrine α5β1 est considérée à l’heure actuelle comme une cible thérapeutique pertinente en oncologie, en particulier pour le traitement des glioblastomes. Ces tumeurs cérébrales très agressives résistent aux traitements actuels, en partie par leur capacité à envahir le tissu cérébral sain. Nos résultats mettent en évidence, in vitro, le rôle de l’intégrine α5β1 dans la migration de cellules de glioblastome. Ils ont permis également de caractériser les effets inhibiteurs de la migration d’antagonistes sélectifs de l’intégrine α5β1 non reproduits par des antagonistes de l’intégrine αvβ3. Pour caractériser des antagonistes originaux de l’intégrine α5β1, nous avons combiné des techniques in silico et un test fonctionnel de migration in vitro. Cette démarche a permis la sélection de 3 molécules intéressantes, antagonistes allostériques de l’intégrine α5β1, se démarquant des antagonistes de référence par leur capacité à inhiber la migration cellulaire sans affecter la liaison du ligand endogène de l’intégrine, la fibronectine. / Integrins are αβ heterodimeric transmembrane proteins implicated in various physiological and pathological processes. Currently, α5β1 integrin is considered as a relevant therapeutic target in oncology, particularly for the treatment of glioblastomas. These highly aggressive brain tumours are resistant to current therapies, notably by their ability to invade healthy brain tissues. Our results highlight the role of the α5β1 integrin in the in vitro migration of glioblastoma cells. We characterized the inhibitory effects of selective α5β1 integrin antagonists in cell migration, which are not reproduced by αVβ3 integrin antagonists. To identify original and selective α5β1 integrin antagonists, we combined in silico screening and in vitro functional cell migration assays. This allowed the selection of 3 interesting molecules, behaving as allosteric α5β1 integrin antagonists. Contrarily to known α5β1 antagonists, our three hits inhibit cell migration without interfering with the binding of fibronectin, the endogenous ligand of this integrin.

O glioblastoma multiforme (GBM) é uma neoplasia do sistema nervoso central (SNC), caracterizada por uma elevada capacidade proliferativa e migratória. O desenvolvimento do tumor provoca uma remodelação da matriz extracelular (MEC) que facilita a migração tumoral. Eicosanóides são moléculas lipídicas importantes na carcinogênese e a sua síntese está correlacionada com o grau de desenvolvimento do tumor. As prostaglandinas são eicosanóides envolvidas na estimulação da angiogênese, na adesão celular e proliferação celular. Este estudo tem por objetivo avaliar in vitro o efeito da PGE2 na expressão moléculas da MEC e das moléculas de adesão envolvidas na migração, em células U87MG e U251MG. As células U251MG e U87MG foram tratadas com PGE2 (10µM) e Ibuprofeno (25µM), por um período 48hs. As proteínas da MEC foram analisadas por RT-qPCR após o tratamento. Foram realizadas reações de imunohistoquímica para as moléculas da MEC. As alterações foram encontradas na expressão de laminina, fibronectina, colágeno tipo IV e as integrinas αv , α3 e α5 para células U87MG . Observamos imunomarcação nas linhas celulares para colágeno tipo IV, laminina e fibronectina. Concluímos que o tratamento com IBU e PGE2, afeta a expressão gênica de moléculas de MEC. / Glioblastoma Multiforme (GBM) is a neoplasm of the central nervous system (CNS), characterized by a high proliferative and migratory capacity. Tumor development leads to extracellular matrix (ECM) remodeling and facilitating the migration of these cells. Eicosanoids are important lipid molecules in carcinogenesis, and their synthesis often correlates with the degree of tumor development. Prostaglandins are eicosanoids involved in the stimulation of angiogenesis, cell adhesion and cell proliferation. This study is aimed to evaluate the expression of several ECM molecules involved in migration after altering the concentration of prostaglandins, using human glioma cell lines as an in vitro model. The cell lines U87MG and U251MG were treated with PGE2 (10µM) and Ibuprofen (25µM), for a predetermined period of 48hs. Proteins involved in extracellular matrix were analyzed by RT-qPCR after treatment in vitro. Immunohistochemical reactions were also performed for the ECM molecules. Changes were found in the expression of laminin, fibronectin, type IV collagen and αv, α3 and α5 integrins in cells U87MG. We observed immunostaining in cell lines to type IV collagen, laminin and fibronectin. In conclusion, Ibuprofen and PGE2, affects gene expression of ECM molecules.

Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2017-10-11T12:59:22Z No. of bitstreams: 1 PEDRO_VARGAS_DIS.pdf: 3468332 bytes, checksum: 563022a7fa710bed810c652069a0777c (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-10-11T20:09:27Z (GMT) No. of bitstreams: 1 PEDRO_VARGAS_DIS.pdf: 3468332 bytes, checksum: 563022a7fa710bed810c652069a0777c (MD5) / Made available in DSpace on 2017-10-11T20:19:08Z (GMT). No. of bitstreams: 1 PEDRO_VARGAS_DIS.pdf: 3468332 bytes, checksum: 563022a7fa710bed810c652069a0777c (MD5) Previous issue date: 2017-03-10 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Glioblastoma multiform (GBM) is considered the most aggressive tumors of central nervous system (CNS), and the most lethal among primary tumors presenting low survival prognosis, resistance to radiotherapy and chemotherapy. P2Y12 is considered a chemoreceptor for adenosine diphosphate (ADP). Its expression is documented in some cancer types, such as the C6 lineage of rat glioma, renal carcinoma and colon carcinoma. However, its role in the development of tumour progression and the resistance mechanism in chemotherapy are not well elucidated. Currently, it is well established that normal platelet function is an important factor for the progression of tumors, with thrombocytopenic rats demonstrating a significant reduction of metastases. Clopidogrel bisulfate is a drug, which belongs to the class of antiplatelet agents, being a P2Y12 receptor antagonist whose endogenous ligand is ADP. The aim of the present study was to determine the effects of tumor cells exposed to the treatment with clopidogrel. The C6 rat cell line was sensitive to the drug tested (150 ?M) in the viability and cell counts (69.63 ? 8.70) and (53.05 ? 20.06). Both cell lines showed a significant reduction in the number of colonies after 10 days of treatment with clopidogrel. The U251-MG strain demonstrated a significant increase in the G1 phase and a significant reduction in the S phase in the cell cycle (20.32 ? 3.05) and (19.45 ? 2.35) in the concentration of 500 ?M. Other results demonstrate important antiproliferative activity in both tumor lines, suggesting an important participation of the P2Y12 receptor in this process. / O glioblastoma multiforma (GBM) ? considerado o mais agressivo dos tumores do sistema nervoso central (SNC) e o mais letal entre os tumores prim?rios apresentando baixo progn?stico de sobrevida, resist?ncia ? radioterapia e a quimioterapia. O P2Y12 ? considerado um quimiorreceptor para a adenosina difosfato (ADP). Sua express?o ? documentada em algumas linhagens de c?ncer, como a linhagem C6 de glioma de rato, carcinoma renal e carcinoma de c?lon. Contudo, seu papel no desenvolvimento da sinaliza??o e resposta ? quimioterapia n?o est? bem elucidado. Atualmente, est? bem estabelecido que o funcionamento normal das plaquetas ? um importante fator para a progress?o de tumores, sendo que ratos com trombocitopenia demonstraram uma redu??o significativa de met?stases. O bissulfato de clopidogrel ? um f?rmaco pertencente ? classe dos antiagregantes plaquet?rios, sendo um antagonista do receptor P2Y12, cujo ligante end?geno ? o ADP. O presente trabalho teve como objetivo averiguar quais os efeitos ocasionados ?s c?lulas de gliomas quando expostas ao clopidogrel. Para isto, foram utilizadas duas linhagens de gliomas, U251-MG de glioma humano e C6 de ratos, e o tratamento com clopidogrel (150, 300, e 500 ?M), foi avaliado nos par?metros de viabilidade celular, contagem de c?lulas, RT-PCR e citometria de fluxo. A linhagem de rato C6 mostrou-se sens?vel ao f?rmaco testado (150 ?M) com redu??o da viabilidade e contagem celular (69.63?8,70) e (53,05 ? 20.06), respectivamente. Ambas as linhagens tapresentaram uma redu??o significativa no n?mero de col?nias ap?s 10 dias tratamento com clopidogrel. A linhagem U251-MG demonstrou um aumento significativo na fase G1 e uma redu??o significativa na fase S e do ciclo celular (20,32 ? 3.05) e (19.45? 2.35 ) na concentra??o de 500 ?M, por?m nos ensaios de viabilidade e contagem n?o houve diferen?a significativa. Nossos resultados demonstram atividade antiproliferativa importante em ambas as linhagens tumorais, sugerindo uma participa??o importante do receptor P2Y12 neste processo.

Background Glioblastoma Multiforme (GBM) is the most commonly occurring tumour of the central nervous system (CNS). Currently GBM is considered an incurable malignancy with patients experiencing abysmal life expectancies. Lack of progress in the discovery of novel treatments has led to the repurposing of existing licenced medication as a possible alternative option. Metformin is from the biguanide family of drugs and is the most common medication used in the treatment of type 2 diabetes. Clinical studies have reported that, in type 2 diabetic patients, metformin might reduce cancer incidence and severity. Currently, metformin is being assessed in clinical trials as a treatment for a range of cancer types including GBM. The antineoplastic mechanisms utilized by metformin and other biguanides have not been fully elucidated. Methods The effects of metformin were evaluated, alone and in combination with other agents, on a panel of GBM cell cultures. Functional analysis of metformin mechanism of action was assessed through measurement of apoptosis, depolarisation of the mitochondria membrane, caspase pathway activation, cell cycle progression and the expression levels of micoRNAs. Results Analysis of fourteen GBM cell cultures showed a cytotoxic response to metformin that was significantly linked to the P53 status (p=0.0024). In combination drug testing, one of the four drugs showed a synergistic pairing with metformin. The kinase inhibitor sorafenib, showed synergism (CI ≤ 1) in eight GBM cell cultures. Flow cytometry of metformin treated GBM cells showed no significant increase (p > 0.005) in apoptotic cell populations. Caspase 3/7 levels showed no significant increase post metformin treatment (p > 0.005). Metformin caused depolarisation of the mitochondrial membrane in six GBM cell cultures. Four microRNAs were shown to have expression levels changes post-metformin treatment. Upregulation of expression was identified in miR-140, miR-192, let-7c. Downregulation was identified in miR-222. Conclusions Metformin was shown to have cytotoxic effect on a GBM cell cultures and has potential as GBM therapeutic agent and possible treatment synergy with sorafenib. The significance of P53 status to metformin sensitivity may suggest that its use should be directed to a sub-set of GBM patients. Mechanism for cell death by metformin was shown not to rely on apoptotic pathways but caspase 3/7 independent depolarization of mitochondrial cell membranes and cell cycle arrest. Investigations into autophagy may help to further define the pathways metformin is utilising to promote cell death. The impact of metformin on the expression profile of miR-222, miR-192 and let-7c is in line with clinical studies of other cancer types. This shows possible insight into the cancer independent actions of metformin. The interplay recorded between glucose availability and cell death indicates a possible key factor in the utilisation of metformin as a therapeutic agent. This finding may warrant the addition of dietary control regimes in clinical trials to maximise metformin efficacy. This work highlights the strong potential for biguanides in the development of new drug treatments and in expanding our knowledge of cancer metabolism.

JUSTIFICATIVA E OBJETIVO: Muitos estudos reportaram o benefício dos métodos avançados de ressonância magnética (RM) para a avaliação da resposta ao tratamento do tumor cerebral, a fim de distinguir entre recidiva tumoral e radionecrose. No entanto, o tamanho da amostra em cada estudo é relativamente pequeno, o que torna difícil avaliar a validade externa. Nós realizamos uma revisão sistemática e metanálise de dados publicados afim de avaliar a acurácia dos métodos avançados de RM para diferenciação entre a recidiva e a radionecrose. Nosso objetivo foi determinar o valor diagnóstico da difusão (DWI), da perfusão Dynamic Susceptibility Contrast (PWI DSC), perfusão Dynamic Contrast Enhancement (PWI DCE) e espectroscopia (MRS). MATERIAL E MÉTODOS: A revisão sistemática incluiu todos os estudos que usaram métodos avançados de RM para detectar recidiva ou radionecrose em pacientes em segmento por tratamento radioterápico para tumor cerebral. A pequisa foi realizada nas bases de dados MEDLINE e Embase, das publicações até 31 de Julho de 2015. As sensibilidades e especificidades de cada estudo foram calculadas e a acurácia diagnóstica foi metanalisada, com um intervalo de confiança (IC) de 95%, em um modelo de efeito randômico. Foram realizados testes de heterogeneidade, sobre o efeito dos pontos de corte e modelos de metarregressão. Análises de subgrupos foram feitas com base em conjuntos de estudos com características homogêneas. RESULTADOS: 49 artigos foram incluídos na análise quantitativa, englobando um total de 1.508 pacientes. Cinco estudos avaliaram DWI, 32 avaliaram PWI e 21 avaliaram MRS. A sensibilidade (SEN) e especificidade (ESP) geral da DWI foi 81,0% (IC 95%: 71,0 a 89,0%) e 68,0% (IC 95%: 52,0 a 82,0%), respectivamente. A SEN e ESP da PWI DSC foi de 83,0% (IC 95%: 80,0 a 86,0%) e 81,0% (IC 95%: 76,0 a 85,0%) e da PWI DCE foi 76,0% (IC 95%: 66,0 a 85,0%) e 85,0% (IC 95%: 74,0 a 93,0%), respectivamente. A SEN e ESP da MRS foi de 76,0% (IC 95%: 71,0 a 80,0%) e 83,0% (IC 95%: 77,0 a 88,0%), respectivamente. O odds ratio diagnóstico (DOR) da DWI, PWI DSC, PWI DCE e MRS foi 14,83; 25,81; 14,45 e 27,39; respectivamente. O maior valor do DOR nos estudos com PWI DSC foi quando o ponto de corte do Volume Sanguíneo Cerebral relativo (rCBV) foi maior ou igual a 1,8 e o maior valor do DOR nos estudos com MRS foi quando o ponto de corte da relação Cho/Cr foi maior ou igual a 1,3. O valor do DOR nos estudos com MRS foi muito maior nos trabalhos realizados com equipamento de 3T (DOR = 40,07; IC 95%: 15,44 a 104,03) quando comparado aos trabalhos realizados em equipamento com 1,5T (18,69; IC95%: 8,32 a 42,02). CONCLUSÃO: Esta metanálise demonstrou que os métodos avançados de RM tem uma moderada e alta acurácia na diferenciação da recidiva tumoral da radionecrose usando os métodos de DWI, PWI DSC, PWI DCE e MRS. Algumas análises de subgrupos e testes de efeito do ponto de corte mostraram que alguns cenários têm uma tendência de melhor acurácia. / BACKGROUND AND PURPOSE: Several studies reported the benefit of magnetic resonance (MR) advanced methods for the treatment response of brain tumor assessment, for distinguishing tumor recurrence from radionecrosis in gliomas and other brain tumors. However, the sample size in each study is relatively small, which becomes difficult to draw conclusions about external validity. We performed a systematic review and meta-analysis of published data to evaluate the accuracy of the advanced MR methods for differentiating recurrence from radionecrosis. Our objective was to determine the diagnostic value of Diffusion (DWI), Dynamic Susceptibility Contrast Perfusion (PWI DSC), Dynamic Contrast Enhancement Perfusion (PWI DCE) and Spectroscopy (MRS), and compare the results between the methods MATERIALS AND METHODS: The systematic review included all studies that used MR advanced methods to detect recurrence or radionecrosis in patients followed by brain tumor radiotherapy. The databases selected were MEDLINE and Embase, for published data prior to July 31, 2015. The sensitivities and specificities of individual studies were calculated and the pooled diagnostic accuracies, with 95% confidence intervals (CI), were assessed under a random-effects model. It was also performed heterogeneity test, threshold effect test and meta-regression models for each MR method. A subgroup analysis was performed based on homogeneous subsets of the studies. RESULTS: 49 articles were included in the quantitative analysis, compromising 1,508 patients (919 with recurrence and 589 with radionecrosis). Five studies assessed DWI, 32 assessed PWI, and 21 assessed MRS. Overall sensitivity (SEN) and specificity (SPE) of DWI were 81.0% (95% CI: 71.0 to 89.0%) and 68.0% (95% CI: 52.0 to 82.0%), respectively. The SEN and SPE of PWI DSC were 83.0% (95% CI: 80.0 to 86.0%) and 81.0% (95% CI: 76.0 to 85.0%) and PWI DCE were 76.0% (95% CI: 66.0 to 85.0%) and 85.0% (95% CI: 74.0 to 93.0%), respectively. The SEN and SPE of MRS were 76.0% (95% CI: 71.0 to 80.0%) and 83.0% (95% CI: 77.0 to 88.0%), respectively. The overall diagnostic odds ratio (DOR) of DWI, PWI DSC, PWI DCE, and MRS were 14.83, 25.81, 14.45, and 27.39, respectively. The point with the highest DOR in the PWI DSC studies was when the relative Cerebral Blood Volume (rCBV) threshold was equal or higher than 1.8, and the point with the highest DOR in the MRS studies was when the Cho/Cr threshold was equal or higher than 1.3. The MRS DOR value is much higher in the 3T subgroup (40.07, 95% IC: 15.44 to 104.03), compared to the 1.5T subgroup (18.69, 95% CI: 8.32 to 42.02). CONCLUSIONS: This meta-analysis showed that MR advanced methods have moderate to high accuracy in differentiating tumor recurrence from radiation necrosis using DWI, PWI DSC, PWI DCE and MRS. Some subgroup analysis and threshold effect tests demonstrated subsets that have a better accuracy trend.

Glioblastoma multiforme é o tumor maligno mais comum do sistema nervoso central em adultos e a sobrevida média é de apenas 12 a 15 meses após o diagnóstico. Por isso, é extremamente importante desenvolver tratamentos mais eficazes e específicos contra essa neoplasia. A presença do sistema imune, incluindo macrófagos associados ao tumor, promove a proliferação tumoral e está associada com um pior prognóstico em pacientes com essa doença maligna. A sinalização purinérgica e o receptor purinérgico P2X7, um canal iônico, têm sido implicados na progressão de diferentes tipos de tumores tanto in vitro como in vivo. A ciclooxigenase 2 (COX-2) desempenha um papel importante na regulação da proliferação celular, diferenciação e na tumorigênese. O ácido ursólico é um triterpeno pentacíclico encontrado em uma variedade de plantas e exibe diversas atividades biológicas e farmacológicas. O objetivo dessa tese é verificar a participação do sistema purinérgico, sistema imune e COX-2 no desenvolvimento e progressão do glioblastoma multiforme, e também investigar os efeitos citotóxicos do ácido ursólico. Primeiramente, verificamos que macrófagos expostos ao meio condicionado de glioma (GL-CM) foram modulados para um fenótipo do tipo M2 e houve um aumento da liberação de IL-10, IL-6 e MCP-1. Esses efeitos foram diminuídos na presença de antagonistas dos receptores P2X7 e A2A. Portanto, os resultados apresentados contribuem para o melhor entendimento da interação entre inflamação e câncer e demonstram que os receptores purinérgicos são importantes para a progressão do glioma. Após, analisamos o papel do receptor P2X7 na proliferação de células de glioma. Surpreendentemente, in vitro, não se observou nenhuma diferença no crescimento das células quando houve a transfecção com o P2X7. Entretanto, in vivo, essas células geraram tumores maiores quando comparado com o controle. Os nossos resultados demonstram que, como em outros tipos de cânceres, o P2X7 tem um papel importante no desenvolvimento e progressão tumoral. Uma vez verificado o importante papel do receptor P2X7 nos macrófagos associados ao tumor e nas células de glioma, investigamos se esse receptor poderia interagir com a enzima COX-2 em células de glioma. Porém, não houve diferença na expressão do P2X7 ou da COX-2 tanto in vitro como in vivo. E também não houve nenhum efeito adicional entre o antagonista de P2X7 e o inibidor seletivo de COX-2. Esse trabalho fornece evidências de que não há relação entre o P2X7 e COX-2 em células de glioma. Em conclusão, todos esses resultados reforçam a hipótese do envolvimento da sinalização purinérgica na progressão do glioblastoma multiforme e tornam o P2X7 como um interessante alvo terapêutico. Finalmente, também investigamos a possível atividade anticâncer do ácido ursólico contra as células de glioma. Essa molécula foi capaz de diminuir o número de células e induziu parada no ciclo celular. In vivo, o ácido ursólico reduziu ligeiramente o tamanho do tumor, mas não alterou as características malignas. Em conclusão, o ácido ursólico pode ser um potencial candidato como adjuvante para o tratamento do glioblastoma multiforme. Em conjunto, todos os resultados apresentados nessa tese indicam possíveis novas abordagens terapêuticas no tratamento e novos conhecimentos em relação a esse maligno câncer cerebral. / Glioblastoma multiforme is the most common malignant tumor of central nervous system in adults and the median survival is only 12 to 15 months after diagnosis. Therefore, it is extremely important to develop more effective and specific treatments. The presence of an inflammatory environment, including tumor-associated macrophages, promotes tumor proliferation and is associated with a poor prognosis in patients with this malignancy. Disruption of purinergic signaling has also been implicated in cancer progression. P2X7R is an ion channel receptor, whose participation in tumor progression has been demonstrated in in vitro and in vivo studies. Cyclooxygenase 2 (COX-2) plays an important role in regulating cell proliferation, differentiation, and tumorigenesis. Ursolic acid is a pentacyclic triterpenoid found in a variety of plants that exhibits several biological and pharmacological activities. The aim of this study is to verify the participation of the purinergic system, immune system and COX-2 in the glioblastoma multiforme development and progression, and also to investigate the anti-proliferative effects of ursolic acid. We first verified that macrophages exposed to glioma conditioned medium (GL-CM) were modulated to an M2-like phenotype and there was an increased IL-10, IL-6 and MCP-1 secretion and these effects were diminished by P2X7 and A2A receptors antagonists. Therefore, the results presented contribute to advancing in the field of cancer-related inflammation and point specific purinergic receptors as targets for glioma progression. After that, we analyzed the role of P2X7 receptor in glioma cell proliferation. Surprisingly, in vitro, no difference in cell growth was observed when the cells were transfected with P2X7R but in vivo these cells generated larger tumors when compared to the control. Our data demonstrate that, as in other type of cancers, P2X7R has an important role in sustaining the development of glioma. Once verified the important role of P2X7 receptor in tumorassociated macrophages and glioma cells, we verified whether this receptor could interact with the COX-2 enzyme in glioma cells. No differences in mRNA expression of P2X7R or COX-2 were verified both in vitro and in vivo experiments. And any additional effect with selective P2X7R antagonist and COX-2 inhibitor were observed in in vitro and in vivo experiments. This work provides evidence that there is no relationship between the P2X7R and COX-2 in glioma. In conclusion, all these results reinforce the hypothesis of purinergic signaling involvement in glioma progression and point to P2X7R as an interesting target for glioma treatment. Finally, we also investigated the potential anticancer activity of ursolic acid against to glioma cells. Ursolic acid decreased the cell number and induced an arrest in the cell cycle in glioma cells. In vivo, ursolic acid slightly reduced the glioma tumor size but did not alter the malignant features. In conclusion, the ursolic acid may be a potential candidate as adjuvant for glioblastoma therapy. Taken together, the results presented herein indicate new adjuvant treatment approaches and new knowledge regarding to this deadliest brain tumor.

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.

O glioblastoma (GBM) é o mais comum dos tumores primários malignos que afetam o Sistema Nervoso Central (SNC), sendo um dos cânceres mais letais. A ressecção cirúrgica desse tumor é o tratamento de intervenção inicial mais utilizado nos pacientes. Embora a radioterapia e a quimioterapia aumente a sobrevida, a maioria dos pacientes chega ao óbito em até um ano após o diagnóstico. Além disso, os GBM estão entre os tumores mais resistentes à radiação e à quimioterapia. Dessa forma, torna-se imprescindível a busca de novas estratégias terapêuticas que visem à melhora da qualidade de vida dos pacientes e ao aumento do tempo de sobrevida. O glutamato (L-Glu) é o aminoácido encontrado em maior concentração no SNC e ele exerce seus papéis fisiológicos e patológicos através da ativação de receptores de membrana metabotrópicos e ionotrópicos. Diversos estudos in vitro e in vivo têm demonstrado que células de GBM liberam altos níveis de L-Glu para o meio extracelular, o que promove a sua proliferação e migração, contribuindo para a malignidade deste tipo de tumor. De fato, é provável que a ligação desse aminoácido aos receptores metabotrópicos de glutamato (mGluR) relacione-se com a agressividade dos GBM, visto que eles são amplamente expressos nestas células. Dessa forma, o objetivo desta tese foi investigar o papel dos mGluR sobre a agressividade de GBM, buscando uma assinatura gênica que tenha valor como potencial biomarcador prognóstico e preditivo de tratamento complementar adjuvante. Através da uma meta-análise de duas coortes de amostras de GBM humanos foi possível identificar uma assinatura gênica de mGluR com valor prognóstico, na qual biópsias com alta expressão gênica de mGluR3 e baixa de mGluR4 e mGluR6 predizem um desfecho antecipado para os pacientes. O potencial desses receptores sobre a malignidade desses tumores foi avaliado por experimentos in vitro utilizando o tratamento de linhagens de GBM com ligantes de mGluR. O bloqueio farmacológico de mGluR do grupo II pelo antagonista LY341495 e a ativação farmacológica de mGluR III por L-AP4 diminuíram a porcentagem de células de linhagem de GBM em 25-28 %. A combinação desses tratamentos não apresentou efeito sinérgico. O potencial da assinatura gênica também foi avaliado in vivo utilizando ratos implantados ortotopicamente com células da linhagem C6 e tratados intracisternalmente com os ligantes de mGluR e intraperitonealmente com quimioterápico padrão, a temozolamida. Os resultados obtidos nos experimentos in vivo, apesar de preliminares, em relação ao tratamento com os ligantes de mGluR, são muito promissores, permitindo várias perspectivas em relação a adaptações de protocolo e a realização de experimentos complementares. Este estudo demonstrou que a avaliação da expressão gênica de mGluR3, 4 e 6 em biópsias de GBM humanos possui um grande potencial prognóstico. A diminuição do número de células da linhagem C6 após o tratamento in vitro com ligantes de mGluR (LY341495 e L-AP4) está de acordo com o comportamento de agressividade previsto nos estudos in silico. Embora mais experimentos in vitro e in vivo sejam necessários para melhor avaliação do potencial dessa assinatura gênica de mGluR, os resultados obtidos indicam que a avaliação da expressão dos oito subtipos de mGluR em biópsias de GBM pode ser considerada em âmbito clínico para guiar futuras intervenções quimioterápicas. / Glioblastoma (GBM) is the most common malignant primary tumor of Central Nervous System (CNS) and one of the most lethal cancers. Surgical resection of this tumor is the most commonly initial intervention treatment used in patients. Although radiotherapy and chemotherapy increase survival, it is expected that the majority of patients will die within a year after diagnosis. In addition, GBM are among the most radiation- and chemotherapy-resistant tumors. Thus, it is imperative to search for new therapeutic strategies that aim to improve patients' quality of life and to increase survival time. Glutamate (L-Glu) is the amino acid found in higher concentration in CNS and it exerts its physiological and pathological roles through the activation of metabotropic and ionotropic membrane receptors. Several studies have demonstrated in vitro and in vivo that GBM cells release high levels of L-Glu into extracellular medium. This event was shown to promote GBM proliferation and migration, contributing to the malignancy of this type of tumor. Indeed, it is likely that the binding of that amino acid to metabotropic glutamate receptors (mGluR) relates to GBM aggressiveness, since they are widely expressed in these cells. Thus, the objective of this work was to investigate the role of mGluR on GBM aggressiveness, searching for a gene signature that has potential value as biomarker to predict the prognosis and adjuvant complementary treatment. Through the meta-analysis of two GBM human samples cohorts, it was possible to identify an mGluR gene signature with prognostic value, in which biopsies with high mGluR3 and low mGluR4 and mGluR6 gene expression predict an early outcome for patients. The potential of these receptors on the malignancy of these tumors was assessed by in vitro experiments through the treatment of GBM lineages with mGluR ligands. Pharmacological blockade of group II mGluR by LY341495 and pharmacological activation of group III mGluR by L-AP4 decreased the amount of C6 cells in 25-28 %. The combination of these treatments had no synergistic effect. The potential of the gene signature was also assessed in vitro using GBM-implanted rats treated intracisternally with mGluR ligands and intraperitoneally with standard chemotherapy, temozolomide. The results obtained in in vivo experiments, although very preliminary in relation to treatment with the mGluR ligands, are very promising, allowing several perspectives regarding protocol adaptations and the accomplishment of complementary experiments. This study demonstrated that evaluation of mRNA levels of mGluR3, 4, and 6 in human GBM biopsies has a great prognostic potential. The decrease in number of C6 cells after in vitro treatment with mGluR ligands (LY341495 and L-AP4) is in accordance with the predicted aggressiveness behavior proposed in silico. Although further in vitro and in vivo experiments are required for better evaluation of mGluR gene signature potential, these results indicate that evaluation of the eight mGluR subtypes in GBM biopsies may be considered in clinical scope to guide future chemotherapeutic interventions.

Les gliomes sont les tumeurs cérébrales primitives les plus fréquentes de l’adulte. Le glioblastome (grade IV) en est la forme la plus agressive, caractérisé par sa résistance aux traitements actuels (chirurgie, chimiothérapie et radiothérapie). La mortalité de cette pathologie est quasi constante (survie médiane de 15 mois), ce qui justifie l’importance de découvrir de nouvelles cibles thérapeutiques. Le challenge est d'arriver à identifier des marqueurs spécifiques pour proposer un schéma thérapeutique alignant des stratégies de thérapies ciblées qui vont améliorer la prise en charge clinique, la survie globale et la survie sans progression des patients atteints de ces pathologies. Deux axes sont au centre des recherches fondamentales, translationnelles et cliniques. Le premier axe se définit autour du développement de molécules inhibitrices des voies de signalisation et le second autour du concept de cellules souches tumorales (CST) de glioblastomes (GBM) découvertes récemment dans le cerveau et qui révolutionnent la conception de la transformation tumorale.ASPM (Abnormal Spindle Like Microcéphaly Associated) est une cible candidate pertinente susceptible de participer au développement des gliomes (Horvath et al., 2007 ; Hagmann et al., 2008). Cette protéine régule la prolifération des neuroblastes, elle est fortement exprimée au stade embryonnaire, mais, reste faiblement exprimée dans le cerveau adulte. Par ailleurs, ASPM est impliquée dans divers processus de cancérisation (surexprimée dans les cancers du sein, du foie et du cerveau…), toute fois, le mécanisme responsable de cette dérégulation n’est pas encore bien caractérisé.Nos études menées sur une série de 169 gliomes humains, sélectionnés à partir de notre cohorte de patients, montrent que le gène ASPM est un marqueur de la progression vers la malignité, les grades les plus élevés exprimant le plus fortement ASPM. En outre, nous avons également montré que le niveau des transcrits d’ASPM est augmenté dans les récidives de gliomes et qu’en in vitro, ASPM contrôle la formation des gliomasphères (CST de GBM) avec une augmentation de l’expression de ses transcrits dans les cultures in vitro au fil des passages. En continuité de ces observations, nous avons alors développé un sh-miR-RNA spécifique d’ASPM permettant l’extinction post-transcriptionnelle de ce gène. Les résultats obtenus in vitro montrent que la perte d’expression d’ASPM conduit à un arrêt de la prolifération et aboutit à une mort cellulaire massive.Actuellement, des modèles de greffe de gliomasphères chez la souris (orthotopique) sont en cours de développement pour confirmer les effets observés in vitro et vérifier in vivo la validité de notre approche thérapeutique. En perspective, nous tenterons d’étudier les effets du silencing d’ASPM sur la voie de signalisation la plus dérégulée (pRB / E2F ou PI3K / AKT). Enfin, nous étudierons le rôle potentiel de cette protéine dans le contrôle du cycle cellulaire, et, in fine la mise en évidence de ses partenaires… / Glioblastoma (GBM) is the most frequent and aggressive form of primary brain tumors in adults; it is characterized by its resistance to current treatments (surgery, chemotherapy and radiotherapy). The prognosis is grim with a median survival of only 15 months underlining the importance to develop new therapeutic strategies. The recent development of the “tumor stem cell” (TSC) concept in hemopathies has been secondarily applied to gliomas with the identification of subpopulations of GBM cells which express neural stem cell markers and fulfill the criteria for stemness. Some evidences also suggest that this subpopulation could play a primary role in resistance to radio- and chemotherapy.ASPM (Abnormal Spindle Like Microcephaly Associated) is a protein regulating the proliferation of neuroblasts, highly expressed in the embryonic stage but weakly expressed in the adult brain. Preliminary reports suggesting that it could be involved in the development of gliomas (Horvath et al., 2007, Hagemann et al., 2008) prompted us to analyze further the role of this protein, focusing on its potential as a relevant candidate therapeutic target. In a series of 175 gliomas samples of various grades, we found that ASPM mRNA expression was strongly correlated with increasing tumor grade. We also found that ASPM expression increased at recurrence when compared to the initial lesion. Subsequently, we could demonstrate in vitro and in vivo that ASPM expression also increased over serial passages in gliomaspheres and in a mouse glioma xenograft model. In a therapeutic perspective, the effect of lentivirus-mediated shRNA post-transcriptional silencing of ASPM was evaluated in two different gliomasphere models and a dramatic proliferation arrest and cell death was observed. Taken together, these data suggest that ASPM is involved in the malignant progression of gliomas, possibly through expansion of a cancer stem cell compartment, and could be an attractive therapeutic target in glioblastoma multiforme.Another potential candidate tumor stem cell target in glioma is the sonic hedgehog pathway (hedgehog-Gli) which is required for GBM growth and stem cell expansion. In a collaborative study, it was found that NANOG, a transcription factor critically involved with self-renewal of undifferentiated embryonic stem cells, modulates gliomasphere clonogenicity, CD133+ stem cell behavior and proliferation. NANOG was regulated by hedgehog-Gli signalling and was essential for GBM tumourigenicity in orthotopic xenografts suggesting that it could also be a useful potential therapeutic target.Conclusions: Accumulating evidences suggest that tumor stem cells play an important role in the oncogenesis of gliomas and in their resistance to treatment. Our data support this concept and suggest that specific stemness markers may become useful targets to improve treatment of this devastating disease.