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Histiocytic sarcoma (HS) is an exceptionally rare malignant neoplasm derived from dendritic cells and histiocytes, with no available effective treatment options. Akt signaling and proteasome dysfunction have been implicated in the pathogenesis of the disease, both in humans and dogs. Our work aims to investigate the importance of the Akt signaling pathway and evaluate the potential of Akt-targeted therapy in a canine model of histiocytic sarcoma. We demonstrated Akt signaling to be active in 9 out of 10 canine HS tumor samples, regardless the presence of PTEN. Moreover, the Akt signaling pathway appears to be constitutively active in DH82 cells — a cell line model of canine HS, when compared to control canine dendritic cells. Pharmacologic Akt inhibition resulted in significant decrease in Akt S473 phosphorylation, GSK-3β S9 phosphorylation, Akt activity, cell viability, increased apoptosis, and resulted in sensitization to proteasome inhibition-depended cell death in a synergistic manner. Proteasome inhibition using carfilzomib, an irreversible proteasome inhibitor, induced dose-depended/caspase-3 independent cell death, at clinically relevant drug concentrations. The therapeutic effect of Akt inhibition was validated in vivo using a DH82 xenograft murine model. Akt inhibition lead to reduced tumor growth, prolonged overall survival, and ameliorated splenomegaly, but not affected the lung metastasis. Moreover, the therapeutic effect of Akt inhibition was potentiated in combination with carfilzomib. In conclusion, targeting Akt signaling may represent an attractive potential therapeutic target for the HS. Future studies are required to examine the clinical efficacy of Akt-targeted therapy in dogs with HS using novel selective Akt inhibitors. / Ph. D. / Histiocytic sarcoma (HS) is an exceptionally rare cancer of the immune system, with no effective treatment options available. Canine histiocytic sarcoma (cHS) is an aggressive tumor of the same cellular lineage, identified at increased relative frequency in specific dog breeds, with significant translational value. Akt signaling and proteasome dysfunction have been implicated in the pathogenesis of the disease, both in humans and dogs. Our study aims to investigate the importance of the Akt signaling pathway in the dog model of the disease and evaluate the potential of Akt-targeted therapy in a translational of histiocytic sarcoma. The work presented here demonstrates that Akt signaling appears aberrantly and constitutively activated in the canine model of HS. Importantly, Akt inhibition significantly reduced the tumor growth and prolonged the overall survival of the experimental animals. Moreover, Akt inhibition potentiated the anti-cancer activities of other anticancer drugs. Collectively, these findings provide an attractive therapeutic approach for the treatment of HS.

Devant l’incidence de la suractivation de la voie PI3K/Akt/mTOR sur les cancers, nous avons choisi d’inhiber cette voie de signalisation. Etant donné la forte analogie structurale qui existe entre les enzymes PI3K et mTOR, nous avons conçu des inhibiteurs doubles ciblant deux kinases majeures de la voie. Ces inhibiteurs possèdent un noyau original pyrido[3,2-d]pyrimidinique. Afin d’apporter de la diversité fonctionnelle et d’engendrer un effet thérapeutique, les sommets C-4, C-2 et C-7 furent fonctionnalisé séquentiellement selon l’ordre suivant. Tout d’abord, la position C-4 fut fonctionnalisée par des hétérocycles par substitution nucléophile aromatique. Puis divers cycles hétéroaromatiques furent introduits en position C-2 par couplage de type Suzuki-Miyaura. Enfin, des groupements variés furent insérés en position C-7 par différentes réactions. Dans l’étude de l’influence du squelette, l’isomère de position pyrido[2,3-d]pyrimidine fut également synthétisé et fonctionnalisé. Afin de tester ces inhibiteurs originaux, une plateforme de tests d’activités in vitro a été mise en place où cinq kits enzymatiques ont été optimisés sur la kinase PI3K, et un test sur mTOR. Ces tests exploitant la méthode de TR-FRET et de bioluminescence ont été validés avec des inhibiteurs de référence basés sur 4 facteurs : la corrélation entre IC50(littérature) et IC50(mesurée), Z’, R², et S/B.Au final, plus de soixante produits finaux ont été évalués in vitro sur PI3K et mTOR. La moitié présentent une IC50 inférieure à 100 nM et 5 ont des IC50 inférieures à 10 nM. Dans le cadre des échanges du Cancéropôle Grand Ouest, les produits ont été testés sur 6 lignées de cellules cancéreuses. / Considering the impact of overactivation of the PI3K/Akt/mTOR pathway in cancer, we chose to inhibit this signaling pathway. Given the high structural similarity between the PI3K and mTOR enzymes, we designed dual inhibitors targeting two of the three major kinases of the pathway. These inhibitors posses an original pyrido[3,2-d] pyrimidine scaffold. In order to provide a functional diversity and generate a therapeutic effect, the peaks C-4, C-2 and C-7 were functionalized sequentially in the following order. Position C-4 was first functionalized with aliphatic heterocycles by nucleophilic aromatic substitution. Then, various heteroaromatic rings were introduced at the C-2 position by Suzuki-Miyaura coupling. Finally, different functions were inserted at the C-7 peak by different reactions. In order to study the influence of the scaffold, the pyrido[2,3-d]pyrimidine isomer was also synthesized and functionalized. To test these original inhibitors a platform testing in vitro activities was set up in which five assay kits were optimized for the kinase PI3K, and one kit for mTOR. These tests exploit the TR-FRET and bioluminescence methods and were validated with commercially available inhibitors based on four factors: the correlation between IC50(literature) and IC50(measured), Z’, R², and S/B. In the end, more than sixty final products were evaluated in vitro on PI3K and mTOR. Half present an IC50 below 100 nM and 5 of them show an IC50 under 10 nM. As part of collaboration within the Cancéropôle Grand Ouest, the products were also tested on six cancer cell lines.

Orientadores: José Andrés Yunes, Nilson Ivo Tonin Zanchin / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T06:00:33Z (GMT). No. of bitstreams: 1 Silveira_AndreBortolini_D.pdf: 16883235 bytes, checksum: e0759c48520d471791a5272349e1a837 (MD5) Previous issue date: 2013 / Resumo: A via PI3K está frequentemente hiperativada em células primárias de leucemia linfoide aguda T (LLA-T) pediátrica, característica previamente associada à resistência a glucocorticoides. Pacientes cujas células leucêmicas apresentam mutações em PTEN, o principal regulador negativo de PI3K, podem apresentar maior risco de falha na terapia de indução e recaída. Neste estudo, uma assinatura baseada em expressão gênica foi utilizada para acessar o nível de ativação da via PI3K em amostras diagnósticas de LLA-T. Nós identificamos Myc como um importante integrador da atividade de sinalização por PI3K e observamos que maior atividade da via está associada à resistência a glucocorticoides e pior prognóstico. O inibidor de PI3K AS605240 mostrou atividade antileucêmica e forte sinergismo com glucocorticoides tanto in vitro como em um modelo xenográfico de LLA-T em camundongos NOD/SCID. Em contraste, a inibição de PI3K resultou em antagonismo com metotrexato e daunorrubicina, drogas que atuam preferencialmente em células em divisão. Esta interação antagonística, no entanto, pôde ser revertida pelo uso de um esquema temporal específico de administração das drogas. Nossos dados indicam os potenciais benefícios e limitações para a incorporação de inibidores de PI3K na terapia da LLA-T / Abstract: The PI3K pathway is frequently hyperactivated in primary T-cell acute lymphoblastic leukemia (T-ALL) cells. Activation of the PI3K pathway has been suggested as one mechanism of glucocorticoid resistance in T-ALL, and patients harboring mutations in the PI3K negative regulator PTEN may be at increased risk of induction failure and relapse. In this study, a PI3K gene expression signature was used as readout of PI3K activity in diagnostic T-ALL samples. We identified Myc as an important downstream integrator of PI3K pathway activity in T-ALL and found that higher PI3K activity is associated with glucocorticoid resistance and worse clinical outcome. The PI3K inhibitor AS605240 showed anti-leukemic activity and strong synergism with glucocorticoids both in vitro and in a NOD/SCID xenograft model of T-ALL. In contrast, PI3K inhibition showed antagonism with methotrexate and daunorubicin, drugs that preferentially target dividing cells. This antagonistic interaction, however, could be circumvented by the use of correct drug scheduling schemes. Our data indicate the potential benefits and difficulties for the incorporation of PI3K inhibitors in T-ALL therapy. OBSERVAÇÃOArquivo pdf com capa, página de rosto, folha de assinatura da banca examinadora, resumo e abstract foi editado segundo informação CCPG/002/2013 / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

La voie PI3Kinase/AKT/mTOR, est une cible thérapeutique du temsirolimus, un inhibiteur de mTORC1. Dans le but d'obtenir une inhibition plus importante de cette voie j’utilise dans ce projet deux nouvelles molécules :- le NVP-BEZ 235 (BEZ) qui inhibe à la fois mTORC1 et la PI3kinase- l'AZD8055 (AZD), un inhibiteur des complexes mTORC1 et mTORC2. En utilisant différentes lignées de LCM, j’ai démontré que l'effet de ces nouveaux inhibiteurs sur la survie cellulaire est plus important que celui du temsirolimus. Cela est probablement dû à l'inhibition de la phosphorylation de l'AKT et la 4EBP. La deuxième partie de ce projet étudie la synergie entre les inhibiteurs de m-TOR kinase et l'aracytine. Un effet additif important a été démontré. J’ai trouvé en western blot que l’aracytine inhibe la phosphorylation des substrats de la voie Akt –mTOR notamment le 4EBP. L’ibrutinib (un inhibiteur de la voie Btk) a un effet modeste mais j’ai pu démontrer qu'il est capable à induire une inhibition plus importante de la survie cellulaire lorsqu'il est associé à l’aracytine. Cependant il s'est révélé antagoniste aux inhibiteurs de la voie PI3K-AKT-mTOR, cela reste difficile à décortiquer. Enfin, j’ai trouvé un effet additif de l’ibrutinib en combinaison avec la doxorubicine. Cependant les inhibiteurs de m-TOR n'ont pas le même effet. Afin d’expliquer ces résultats, j’ai étudié l’effet de ces molécules sur l’expression de GSTPi, enzyme de détoxification connue pour avoir un rôle important dans la résistance de LCM à l’anthracycline. J’ai mis en évidence une diminution de l’expression de cet enzyme par l’Ibrutinib. En revanche, les inhibiteurs de mTOR n’ont pas un effet sur l’expression de GSTPi. L’ibrutinib pourrait donc sensibiliser le LCM à l’anthracycline en diminuant l’expression de GSTPi. / The PI3K / AKT / mTOR pathway is the target of Temsirolimus. However, important resistance is observed. We tried to obtain a more important inhibition of PI3K / AKT pathway using two new molecules :- NVP-BEZ 235 (BEZ) which inhibits both mTORC1 and PI3K- AZD8055 (AZD) an inhibitor of mTORC1 and mTORC2 complexes. Using different cell lines of MCL, we have shown that the effect of these new inhibitors on cell survival was more important than that of Temsirolimus. This is probably because contrary to Temsirolimus, the two new molecules can inhibit AKT and 4EBP phosphorylation. In the second part of this project we studied the synergy between the m-TOR kinase inhibitors and aracytine (conventional treatment of MCL). We revealed a significant additive effect in MCL cell lines. We demonstrated by Western blot analysis that aracytine inhibits S6 and 4EBP phosphorylation. This may explain the results obtained from this drug association. We then showed that Ibrutinib (an inhibitor of Btk pathway) can induce a significant inhibition of cell survival when combined with aracytine. In this study, Ibrutinib proved antagonist effect to PI3K-AKT-mTOR inhibitors. The mechanisms of these results remain unclear. Finally, we demonstrated an additive effect of Ibrutinib in combination with doxorubicin. We did not obtain the same results when we combined m-TOR inhibitors with doxorubicin. To explain these data, we studied the effect of these drugs on the expression of GSTPi by western blot. This enzyme is known to have an important role in MCL resistance to anthracycline. Importantly, Ibrutinib induced a decrease in the expression of GSTPi but AZD8055, Temsirolimus and NVP-BEZ235 had no effect.

Au sein d’une tumeur des régions hypoxiques se forment ce qui conduit à l’activation du facteur de transcription HIF1. HIF1, composé des deux protéines HIF1a et HIF1b, permet l’adaptation des cellules à des faibles concentrations d’oxygène en activant la transcription de gène cible. Un de ces gènes est celui codant pour la lysyl oxydase (LOX). Cette enzyme structure la matrice extracellulaire et est impliquée dans la tumorigénèse. Pour comprendre les liens entre LOX et HIF1a, nous avons modulé leurs expressions dans des lignées humaines de carcinome du côlon. En condition hypoxique, HIF1a contrôle l’expression de LOX et réciproquement, LOX régule la synthèse protéique d’HIF1a via l’activation de la voie de signalisation PI3K/AKT. Nous avons donc mis en évidence l’existence d’une boucle de régulation positive entre LOX et HIF1 en conditions hypoxique. Sachant que ces deux protéines sont des acteurs majeurs de la progression tumorale, nous avons cherché à comprendre le rôle de cette régulation mutuelle dans ce processus. Nos résultats démontrent que l’activité enzymatique de LOX promeut la croissance tumorale in vitro et in vivo et que son action est potentialisée par la présence de son partenaire HIF1a. De plus, LOX et HIF1a agissent en synergie afin d’augmenter la potentiel métastatiques des cellules tumorale de côlon in vitro. Ainsi, ce travail de thèse a permis de mettre en évidence l’existence d’une boucle de régulation entre HIF1a et LOX qui est critique dans la progression tumorale et semble également être impliquée dans le processus métastatiques / The microenvironment of solid tumors is exposed to hypoxic conditions which lead to the activation of Hypoxia‐Inducible Factor 1 (HIF1). HIF1, composed by a heterodimer of HIF1a and HIF1b protein, is a key transcription factor involved in cellular adaptation to changes in oxygen level, inducing the expression of several transcriptional targets such as Lysyl Oxidase (LOX). LOX is an amine oxidase that catalyzes crosslinking of fibrillar collagens and elastin in the extracellular matrix. Furthermore, LOX is implied in tumor progression. To clarify, the link between LOX and HIF1a, their expression were modulated in human colorectal carcinoma cell lines. We pointed out that besides HIF1‐dependant regulation of LOX, LOX can also act on the HIF1 pathway under hypoxic conditions. Indeed, LOX enzymatic activity upregulates HIF1a protein synthesis, and this action is mediated by the PI3K/AKT pathway. Thus, these results emphasize the existence of a regulation loop between HIF‐1a and LOX, which represent two main actors of tumoral progression. Thus, we wanted to determine the implication of this amplification loop in tumor progression. Our results show that LOX enzymatic activity increase tumor growth in vitro and in vivo, and this role is partially dependant of its partner HIF1a. Furthermore, we established that that LOX and HIF1a act in synergy to foster metastatic potential in colorectal carcinoma cell lines. Taken together, our results demonstrate a regulation loop between LOX and HIF1a with is critical for tumor progression and metastasis formation

Le comportement et le devenir des lymphocytes T (LT) est conditionné par l’intégration de nombreux signaux solubles et cellulaires. Lorsque les LT ne sont pas stimulés, les facteurs de transcription FoxO orchestrent un réseau moléculaire important participant au maintien de la quiescence et à la capacité migratoire des LT. Longtemps considéré comme un état par défaut, le maintien des LT dans cet état quiescent est hautement régulé par un ensemble de signaux parmi lesquels la signalisation via le récepteur à l’interleukine 7 (IL7) et le récepteur à l’antigène (TCR) activé par des molécules du complexe majeur d’histocompatibilité (CMH) chargées avec des peptides du soi. Etonnamment, ces mêmes signaux sont nécessaires pour induire l’entrée des cellules dans le cycle cellulaire. L’inhibition de la prolifération des LT est donc un mécanisme actif qui peut être levé par des signaux externes. Le mécanisme moléculaire permettant le maintien de cet état quiescent reste très peu décrit. Mon projet de thèse a consisté à étudier les conséquences fonctionnelles de l’expression de Fam65b, une nouvelle cible transcriptionnelle de FoxO, sur la prolifération. Au cours de mon travail, j’ai montré que dans des cellules transformées, ayant donc perdu la capacité de réguler leur prolifération, l’expression forcée de Fam65b perturbe la mise en place du fuseau mitotique, induisant un arrêt en phase G 2 /M et la mort des cellules. Au cours de ce processus, Fam65b agit avec deux partenaires connus pour leur implication dans le cycle cellulaire, l’histone déacétylase 6 (HDAC6) et la protéine d’échafaudage 14-3-3. J’ai également pu établir que, dans les LT primaires humains, Fam65b est un facteur de quiescence. En effet, l’engagement du TCR induit une diminution d’expression de Fam65b et le maintien de son expression bloque la prolifération des LT, suggérant que l’inhibition de son expression est un pré-requis à la prolifération. Inversement, l’inhibition de l’expression de Fam65b dans des LT naïfs diminue leur seuil d’activation. L’ensemble de ces résultats désigne donc Fam65b comme une nouvelle cible pour le contrôle de la prolifération des cellules primaires et transformées. Nous avons également développé au laboratoire un modèle murin invalidé pour Fam65b dans le lignage T afin d’étudier son rôle dans un modèle plus intégré. J’ai pu initier l’analyse du phénotype de ces souris en l’absence de toute stimulation. L’ensemble de ces travaux, en complément de ceux précédemment obtenus au laboratoire, laissent apparaître Fam65b comme un nouvel effecteur de FoxO capable d’interagir avec divers partenaires afin de contrôler conjointement des fonctions cellulaires majeures. / T cell fate is conditioned by the integration of many soluble and cellular signals. When T cells are not stimulated, FoxO transcription factors orchestrate an important molecular network involved in maintaining the quiescent state and migratory ability of the cells. Considered as a "default" state, it is now known that maintenance of T cell quiescence is a process highly regulated by a set of signals including IL7 signaling and sustained contact with MHC molecules presenting self-peptides. Surprisingly, these same signals are required to induce entry of cells into the cell cycle. Inhibition of T cell proliferation is an active mechanism that can be lifted by external signals. The molecular mechanism maintaining this quiescent state is poorly described. My thesis project was studying the functional consequences of Fam65b expression, a new transcriptional target of FoxO, on proliferation. I showed that, in transformed cells, that have lost the ability to regulate their proliferation, forced expression of Fam65b disrupts the establishment of the mitotic spindle, inducing an arrest in G 2 /M phase and cell death. During this process, Fam65b acts with two partners, known to be involved in the cell cycle process, the histone deacetylase HDAC6 and the 14-3-3 protein scaffold. I have also been able to establish that in human primary T cell, Fam65b is a quiescence factor. Indeed, the TCR stimulation induces a reduction of Fam65b expression and maintaining its expression blocks the proliferation of T cells, suggesting that inhibition of Fam65b expression is a prerequisite for proliferation. Conversely, inhibition of Fam65b expression in naive T cells reduces their activation threshold. Altogether these results show that Fam65b is a new target for the control of the proliferation of primary and transformed cells. We have also developed, in the laboratory, a mouse model invalidated for Fam65b in T cell lineage. I initiated the phenotype analysis of these mice in the absence of any stimulation. This work, in addition to the previous results obtained in the laboratory, reveal that Fam65b is a new effector of FoxO factors, able to interact with various partners to jointly control major cellular functions.

Le chondrosarcome (CHS) est caractérisé par une grande chimio et radiorésistance ; il y a un besoin urgent de nouvelles stratégies thérapeutiques pour cette tumeur. Parmi celles-ci, certaines approches d'immunothérapie pourraient être d'un grand intérêt. Nous étudions actuellement l'implication du système immunitaire dans la progression du CHS et la réponse thérapeutique à la fois sur des échantillons humains et dans le modèle de chondrosarcome de rat (SRC).Dans le CHS humain et de rat, des infiltrats immunitaires composés de lymphocytes et macrophages ont été identifiés dans la zone péritumorale. L’infiltration immunitaire est en corrélation avec l’évolution de la tumeur (grade, envahissement et taille). L'expression de PD1 et PDL1 ont été détectée dans les infiltrats immunitaires et cellules tumorales du CHS chez l’homme et le rat. Le niveau d'expression PD-L1 en corrélation avec la survie des patients et le taux de rechute. Dans le model SRC, la déplétion sélective de lymphocytes T a entrainé une accélération de la progression tumorale, tandis que la déplétion de macrophages l’a ralenti. Les splénocytes isolés de rats porteurs de CHS ont montré une cytotoxicité spécifique dirigée contre les cellules de chondrosarcome (27%), qui a diminué de manière significative avec des rats appauvrie en CD3 (11%). La voie de signalisation PI3K/mTOR ne peut pas être associée à une immunothérapie car elle induit une action immunosuppressive in vivo.L'environnement immunitaire contribue à la progression du CHS à la fois chez l’homme et chez le rat, ce qui suggère que une approche immunomodulatrice avec des anticorps bloquant PDL1 pourrait être testée pour le CHS / Chondrosarcoma is highly resistant to chemotherapy and radiation and there is an urgent need in developing new therapeutic strategies for this malignancy; among these, some immunotherapy approaches could be of great interest. We are currently investigating the immune system implication in chondrosarcoma progression and therapeutic response both on human samples and in rat chondrosarcoma model (SRC). In human and rat chondrosarcoma, immune infiltrates composed of lymphocytes and macrophages were identified in the peritumoral area. Immune infiltrates composition was found correlated with tumors characteristics and evolution (grade, invasiveness and size). Expression of PD-1 and PD-L1 was detected in CHS immune infiltrates, both in human and rat (and on tumor cells). PD-L1 expression level correlated with patients survival and relapse rate. In SRC, T lymphocytes depletion resulted in an accelerated tumor progression, while CD163+ macrophages depletion slowed down tumor progression. Splenocytes isolated from CHS bearing SRC showed a specific cytotoxicity directed against chondrosarcoma cells (27%), which significantly decreased in CD3 depleted SRC (11%). The immune environment contributes to CHS progression in both human and animal models, this associated with expression of immune checkpoint PD1/PDL1 suggest that immunomodulatory approaches with PD-L1 blocking antibody could be applied in CHS; this approach is currently being tested in SRC

Hepatocellular carcinoma (HCC) is the second most common cause of cancerrelated mortality worldwide. AKT pathway has been found activated in 50% of HCC cases, making it promising target. Therefore we assess efficacy of the allosteric AKT inhibitor or the combination of Sorafenib with AKT inhibitor compared to untreated control and to standard treatment, Sorafenib, in vitro and in vivo. AKT inhibitor blocked phosphorylation of AKT in vitro and strongly inhibited cell growth with significantly higher potency than Sorafenib. Similarly, apoptosis and cell migration were strongly reduced by AKT inhibitor in vitro. To mimic human advanced HCC, we used diethylnitrosamine-induced cirrhotic rat model with fully developed HCC. MRI analyses showed that AKT inhibitor significantly reduced overall tumor size. Furthermore, number of tumors was decreased by AKT inhibitor, which was associated with increased apoptosis and decreased proliferation. Tumor contrast enhancement was significantly decreased in the AKT inhibitor group. Moreover, on tumor tissue sections, we observed a vascular normalization and a significant decrease in fibrosis in surrounding liver of animals treated with AKT inhibitor. Finally, pAKT/AKT levels in AKT inhibitor treated tumors were reduced, followed by down regulation of actors of AKT downstream signalling pathway: pmTOR, pPRAS40, pPLCγ1 and pS6K1. In conclusion, we demonstrated that AKT inhibitor blocks AKT phosphorylation in vitro and in vivo. In HCC-rat model, AKT inhibitor was well tolerated, showed anti-fibrotic effect and had stronger antitumor effect than Sorafenib. Our results confirm the importance of targeting AKT in HCC. / Hepatocellular carcinoma (HCC) is the second most common cause of cancerrelated mortality worldwide. AKT pathway has been found activated in 50% of HCC cases, making it promising target. Therefore we assess efficacy of the allosteric AKT inhibitor or the combination of Sorafenib with AKT inhibitor compared to untreated control and to standard treatment, Sorafenib, in vitro and in vivo. AKT inhibitor blocked phosphorylation of AKT in vitro and strongly inhibited cell growth with significantly higher potency than Sorafenib. Similarly, apoptosis and cell migration were strongly reduced by AKT inhibitor in vitro. To mimic human advanced HCC, we used diethylnitrosamine-induced cirrhotic rat model with fully developed HCC. MRI analyses showed that AKT inhibitor significantly reduced overall tumor size. Furthermore, number of tumors was decreased by AKT inhibitor, which was associated with increased apoptosis and decreased proliferation. Tumor contrast enhancement was significantly decreased in the AKT inhibitor group. Moreover, on tumor tissue sections, we observed a vascular normalization and a significant decrease in fibrosis in surrounding liver of animals treated with AKT inhibitor. Finally, pAKT/AKT levels in AKT inhibitor treated tumors were reduced, followed by down regulation of actors of AKT downstream signalling pathway: pmTOR, pPRAS40, pPLCγ1 and pS6K1. In conclusion, we demonstrated that AKT inhibitor blocks AKT phosphorylation in vitro and in vivo. In HCC-rat model, AKT inhibitor was well tolerated, showed anti-fibrotic effect and had stronger antitumor effect than Sorafenib. Our results confirm the importance of targeting AKT in HCC.

Orientador: José Andres Yunes / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T05:50:44Z (GMT). No. of bitstreams: 1 Jotta_PatriciaYoshioka_D.pdf: 8922035 bytes, checksum: 3734371a320410ba431d6e6ce6579e55 (MD5) Previous issue date: 2012 / Resumo: A leucemia linfóide aguda (LLA) é o câncer mais frequente na infância, e destas, 15% são do tipo T (LLA-T). A hiperativação da via PI3K/Akt tem sido amplamente descrita em tumores e em linhagens celulares de LLA-T. PTEN é o principal regulador negativo dessa via e frequentemente encontra-se inativado em cânceres humanos. Com frequência, pacientes com LLA-T apresentam mutações ativadoras de NOTCH1. NOTCH1 pode regular transcricionalmente PTEN, contudo ainda não está claro como as mutações ativadoras de NOTCH1 influenciariam a expressão de PTEN nas LLA-T. Nós encontramos uma ocorrência de 11 (17,7%) mutações no éxon 7 do PTEN em 62 casos de LLA-T estudados consecutivamente. Contudo, nenhuma mutação foi encontrada na análise de 71 casos de LLA-B derivada. A maioria das mutações de PTEN apresentavam inserções/deleções de mais de 3 nucleotídeos. Não encontramos associação entre mutações em PTEN e o gênero, a idade e a contagem de glóbulos brancos ao diagnóstico. Pacientes com alterações no PTEN apresentaram uma tendência a pior sobrevida global (OS, p=0.07). Dentre os pacientes de LLA-T classificados como alto risco (n=56), aqueles possuindo anormalidades no PTEN mostraram-se associados significativamente a menor OS (p=0.019) e sobrevida livre de leucemia (LFS 47% vs 76%; p=0.045). As curvas de LFS foram significativamente diferentes (p=0.003), mesmo considerando apenas pacientes que atingiram a remissão no dia 28 do tratamento para a análise. Nosso estudo também mostrou que pacientes com mutações em NOTCH1 apresentavam aumento na transcrição de MYC e menor expressão de PTEN mRNA comparados a pacientes com NOTCH1 selvagem. Nós recentemente demonstramos que células de LLA-T apresentavam fosforilação de PTEN mediada por CK2, resultando na estabilização e consequentemente inativação da proteína PTEN. Corroborando ao estudo anterior, os casos de LLA-T analisados, independente do status de mutação em NOTCH1, expressam níveis significativamente mais altos de proteína PTEN do que controles normais. Para avaliar o impacto da regulação transcricional de NOTCH e a inativação postranscricional por CK2 de PTEN, nós tratamos as células de LLA-T com inibidores de gamma-secretase (DAPT e de CK2 (DRB/TBB). Nosso estudo enfatiza a relevância biológica e clínica da regulação do PTEN em LLA-T. E sugerimos o uso combinado de inibidores de gamma-secretase e CK2 devem possuir potencial terapêutico nas LLA-T / Abstract: T-cell acute lymphoblastic leukemia (T-ALL) accounts for approximately 15% of pediatric ALL. Patients with T-ALL are at increased risk of relapse compared with children treated for B-cell precursor ALL. Mutations in the phosphatase and tensin homolog (PTEN) gene leading to PTEN protein deletion and subsequent activation of the PI3K/Akt signaling pathway are common in cancer. PTEN is the main negative regulator of the PI3K/Akt survival pathway. T-ALL patients frequently display NOTCH1 activating mutations and Notch can transcriptionally down-regulate the tumor suppressor PTEN. However, it is not clear whether NOTCH1 mutations associate with decreased PTEN expression in primary T-ALL. We report that PTEN exon 7 mutations occurred in 11 (17.7%) out of 62 consecutive pediatric T-cell acute lymphoblastic leukemia (T-ALL) but in none of 71 precursor B-ALL patients. Most PTEN mutations were insertions/deletions of more than 3 nucleotides. No associations were found between PTEN mutation and age, gender, WBC at diagnosis, early response to therapy and remission rate. Patients with PTEN mutation (n=11) had a tendency toward worse overall survival (OS, p=0.07). Remarkably, PTEN mutations were significantly associated with lower OS (p=0.019) and leukemia-free survival (LFS 47% vs 76%, p=0.045) within patients classified in the high risk group (n=56). LFS curves were significantly different (p=0.003) even if only patients who reached remission on day 28 were considered for analysis. We compared patients with or without NOTCH1mutations and report that the former presented higher MYC transcript levels and decreased PTEN mRNA expression. We recently showed that T-ALL cells frequently display CK2-mediated PTEN phosphorylation, resulting in PTEN protein stabilization and concomitant functional inactivation. Accordingly, the T-ALL samples analyzed, irrespectively of their NOTCH1 mutational status, expressed significantly higher PTEN protein levels than normal controls. To evaluate the integrated functional impact of NOTCH transcriptional and CK2 post-translational inactivation of PTEN, we treated TALL cells with both the gamma-secretase inhibitor DAPT and the CK2 inhibitors DRB/TBB. Our data suggest that combined use of gamma-secretase and CK2 inhibitors may have therapeutic potential in T-ALL. And emphasize the biological and clinical relevance of PTEN regulation in pediatric T-ALL / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular