Introduction: Over the past decades, considerable advances in understanding of cell biology at genetic, epigenetic and proteomic levels led to development of new strategies for better outcome of cancer therapy. One of these new strategies is targeting the class I PI3K/Akt/mTOR signaling pathway, in that this pathway plays a key role in regulation of many cellular functions, including proliferation, survival, metabolism, autophagy and motility. Dysregulation of the class I PI3K/Akt/mTOR pathway has been documented in a variety of human tumours and inhibition of this pathway has been observed to hamper tumour proliferation in vitro and prevent tumour progression in vivo and in clinic. More recently, emerging evidence suggests that the class I PI3K/Akt/mTOR pathway is associated with Cancer Stem Cell (CSC) biology, in light of maintenance, viability and conventional therapy resistance of CSCs. The CSC theory conceptualizes that a subset of tumour cells with Stem Cell-like properties, including self-renewal, multipotency, differentiation, and resistance to chemotherapy and radiotherapy, can recapitulate new tumours and resistance to cancer therapy. Materials and Methods: To explore class I PI3K/Akt/mTOR signaling pathway and CSCs as therapeutic targets in canine oncology, in one series of experiments, smallmolecular inhibitors Wortmannin, ZSTK474, KP372-1 and Rapamycin, which selectively target pan-class I PI3K, pan-class I PI3K, Akt and mTOR, respectively, were utilized to treat canine cancer cell lines using inhibitors alone or in combination with conventional therapeutic drugs. The human acute lymphoblastic leukaemia of T-cell origin cell line (Jurkat T cell line) was used as a comparative control. In another, a stem cell culture system was performed to isolate CSCs from canine glioma J3T cell line. Subsequently, microarray analysis of transcriptional expression profiles of J3T spheres (the putative CSCs) versus J3T parental cells was performed. Results: In this study, small molecules ZSTK474 and KP372-1 were found to significantly decrease cell viability at lower micromolar and nanomolar ranges, respectively. Rapamycin decreased cell viability at lower micromolar concentrations. However, the efficacy of Wortmannin varied from one cell line to another. Dissection of the mechanism of these inhibitors using Western Blot analysis and annexin V staining showed that all inhibitors functioned by decreasing phosphorylation of class I PI3K pathway members. Notably, the efficacy of Wortmannin for this pathway inhibition is confined to certain cell lines. In addition, Wortmannin had shorter drug duration than the other three inhibitors. Annexin V staining showed that KP372-1 was a potent inducer of apoptosis, with decreasing potency in hierarchy order, Rapamycin, Wortmannin and ZSTK474. The data obtained from the combination of pan-class I PI3K inhibitor (Wortmannin or ZSTK474) and mTOR inhibitor (Rapamycin) suggested that additive/synergistic effects were, in part, due to inactivation of Akt. The class I PI3K pathway inhibitors enhanced the efficacy of Doxorubicin in SB cells but not in canine REM, 3132 and J3T cells. The CSC colonies of canine glioma J3T cells were successfully isolated and expanded in the neurosphere formation assay. By microarray analysis, several class I PI3K signaling network-associated genes, particularly IGFBP2 (27-fold), FYN (9.3- fold), and DDIT4 (8.5-fold), were found to be highly up-regulated in the J3T CSCs. However, the genes encoding components, such as Akt1 and eIF4E, of class I PI3K/Akt/mTOR axis signaling were either unchanged or down-regulated in the CSCs. The majority of the genes encoding translation initiation factors were also downregulated in the CSCs. Conclusions: This study demonstrates that class I PI3K/Akt/mTOR signaling pathway is critical for proliferation and survival of cell lines derived from human acute lymphoblastic leukemia of T cell origin (Jurkat T cell line) and a variety of canine tumours. However, it appears that this pathway is dispensible for maintainence and viability of the CSCs isolated from canine gloma J3T cell line. This study suggests that the strategy of dual inhibition of class I PI3K and mTOR kinases may have better outcomes than the combination inhibitors of this pathway (such as ZSTK474 and KP372-1) with Doxorubicin in canine oncology.

Diversas alterações genéticas estão associadas à patogênese do carcinoma epidermoide (CE), neoplasia maligna mais comum de cabeça e pescoço. Algumas dessas alterações comprometem proteínas pertencentes à via de sinalização do Akt, envolvida em diferentes fenômenos celulares. Este trabalho teve como objetivo estudar a expressão da proteína pAkt em linhagens celulares de carcinomas epidermoides de cabeça e pescoço, de forma a verificar possíveis alterações na transcrição dessa molécula em células de CE tratadas com Curcumina. Foram utilizadas duas linhagens celulares de CE de cabeça e pescoço (FaDu e SCC9) e uma linhagem de queratinócitos normais (HaCat) divididas em dois grupos: a. Grupo controle não tratado; b. Células tratadas com Curcumina. A proliferação celular foi monitorada através do teste de viabilidade celular e a análise da expressão de proteína foi realizada através da técnica do Western Blotting que revelou supressão do pAkt na linhagem celular SCC9 nos tempos de 24 e 48 horas. Desta forma, conclui-se que a Curcumina na via do Akt em carcinomas epidermoides de cabeça e pescoço tem importante ação supressora do gene pAkt. / Several genetic alterations are associated with the pathogenesis of squamous cell carcinoma (SCC), the most common malignant neoplasm of the head and neck. Some of these changes compromise the proteins belonging to the Akt signaling pathway, involved in various cellular phenomena. The objective of this study to explores the expression of the pAkt protein in cell lines of the squamous cell carcinomas of the head and neck to verify possible changes in the transcription of this molecule in EC cells treated with curcumin. The study used two cell lines of EC head and neck (FaDu and SCC9) and a normal line of keratinocytes (HaCat), split into two groups: A. the controlled group, untreated; B. Cells treated with curcumin. The cell proliferation it was observed by cell viability test and analysis of protein expression performed through Western blotting technique revealed suppression of pAkt in SCC9 cell line at 24 and 48 hours. Thus, it is concluded that the Curcumin on the path of Akt in squamous cell carcinoma of the head and neck has a significant suppressive effect of gene Akt.

L’hématopoïèse est le processus physiologique qui permet le développement de l’ensemble des cellules sanguines matures, leur renouvellement et leur homéostasie tout au long de la vie. L’hématopoïèse est généralement décrite de façon hiérarchique avec, au sommet, les cellules souches hématopoïétiques qui s’autorenouvellent et se différencient en progéniteurs puis en cellules matures. La voie de signalisation NOTCH canonique, contrôle l’activité du facteur de transcription RBPJ. Elle joue un rôle dans le développement des lymphocytes T et la spécification de la différenciation des cellules souches hématopoïétiques normales vers la lignée mégacaryocytaire. Les protéines de la famille OTT1 (OTT1, OTT3 et SHARP) s’expriment de façon ubiquitaire et sont impliquées dans le contrôle de l’activité de RBPJ. Les modalités de régulation de ces activités et l’intégration de signaux provenant d’autres voies de signalisation sont mal caractérisées. L’utilisation d’un modèle de différenciation in vitro de cellules souches hématopoïétiques sur des cellules stromales (OP9) exprimant le ligand NOTCH Delta-like 1 (DL1) ainsi que l’utilisation de modèles murins, nous a permis de montrer un lien entre la voie NOTCH et la voie PI3K/AKT dans le développement mégacaryocytaire. Nos résultats indiquent que la différenciation mégacaryocytaire peut être engagée à partir de progéniteurs myéloïdes engagés dépendant principalement de la voie PI3K/AKT, mais également directement à partir de cellules souches hématopoïétiques pour lesquelles une activation de la voie PI3K/AKT conduit à une synergie avec la voie NOTCH, mais n’est pas essentielle à la spécification mégacaryocytaire. D’autre part, pour comprendre le mécanisme de régulation de la protéine OTT1, j’ai recherché ses partenaires protéiques par crible double hybride chez la levure, et identifié des interactions avec, entre autres, des protéines à activité tyrosine kinase de la famille SRC (dont LYN) et SHARP. La spécificité d’interaction entre OTT1 et LYN a été validée dans un modèle de surexpression ainsi que dans une lignée modélisant la leucémie aigüe mégacaryocytaire. Dans nos modèles, l’interaction avec LYN conduit à la phosphorylation d’OTT1. Les analyses fonctionnelles préliminaires n’ont pas permis à ce jour de mettre en évidence un rôle essentiel de cette interaction dans le développement mégacaryocytaire. / Hematopoiesis is generally described as a hierarchical system, with at the top hematopoietic stem cells which self-renew and differentiate in progenitors, then in mature cells. Canonical Notch signaling controls RBPJ transcriptional activity. It plays a role in T lymphocyte development and stem cell fate. OTT1 family proteins (OTT1, OTT3 and SHARP) are expressed ubiquitously and are implied in control of RBPJ activity. The regulation of these activities and signal integration are all not well characterised. The use of an in vitro model of differentiation for hematopoietic stem cells on OP9 stroma cells expressing the NOTCH Delta-like-1 (DL1) ligand and the use of murine models, allowed us to show a link between NOTCH and PI3K/AKT in megakaryocytic development. Our results indicate that megakaryocytic differentiation can be engaged from myeloid progenitors depending mostly on the PI3K pathway but also from hematopoietic stem cells for which, an activation of PI3K/AKT lead to a synergy with NOTCH, but is not essential for megakaryocytic specification. On the other hand, to understand OTT1’s mechanisms of regulation, I looked for proteic binding partners by the double hybrid screen technique. Among the candidates I identified SHARP and SRC family kinases as LYN. The specific interaction between OTT1 and LYN was validated in a overexpression model and in a cell line modeling acute megakaryoblastic leukemia. In our models, the interaction with LYN lead to the phosphorylation of OTT1. However, the first analysis did not point out an essential role of this interaction in megakaryocytic development.

Diversas alterações genéticas estão associadas à patogênese do carcinoma epidermoide (CE), neoplasia maligna mais comum de cabeça e pescoço. Algumas dessas alterações comprometem proteínas pertencentes à via de sinalização do Akt, envolvida em diferentes fenômenos celulares. Este trabalho teve como objetivo estudar a expressão da proteína pAkt em linhagens celulares de carcinomas epidermoides de cabeça e pescoço, de forma a verificar possíveis alterações na transcrição dessa molécula em células de CE tratadas com Curcumina. Foram utilizadas duas linhagens celulares de CE de cabeça e pescoço (FaDu e SCC9) e uma linhagem de queratinócitos normais (HaCat) divididas em dois grupos: a. Grupo controle não tratado; b. Células tratadas com Curcumina. A proliferação celular foi monitorada através do teste de viabilidade celular e a análise da expressão de proteína foi realizada através da técnica do Western Blotting que revelou supressão do pAkt na linhagem celular SCC9 nos tempos de 24 e 48 horas. Desta forma, conclui-se que a Curcumina na via do Akt em carcinomas epidermoides de cabeça e pescoço tem importante ação supressora do gene pAkt. / Several genetic alterations are associated with the pathogenesis of squamous cell carcinoma (SCC), the most common malignant neoplasm of the head and neck. Some of these changes compromise the proteins belonging to the Akt signaling pathway, involved in various cellular phenomena. The objective of this study to explores the expression of the pAkt protein in cell lines of the squamous cell carcinomas of the head and neck to verify possible changes in the transcription of this molecule in EC cells treated with curcumin. The study used two cell lines of EC head and neck (FaDu and SCC9) and a normal line of keratinocytes (HaCat), split into two groups: A. the controlled group, untreated; B. Cells treated with curcumin. The cell proliferation it was observed by cell viability test and analysis of protein expression performed through Western blotting technique revealed suppression of pAkt in SCC9 cell line at 24 and 48 hours. Thus, it is concluded that the Curcumin on the path of Akt in squamous cell carcinoma of the head and neck has a significant suppressive effect of gene Akt.

Le but du projet est l’étude des régulateurs de la voie de signalisation intracellulaire de la voie Pi(3)K-Akt au travers de l’analyse du suppresseur de tumeur PTEN (Phosphatase and tenson homolog) et de la sérine/thréonine kinase mTOR (Mamalian Target of Rapamycin). Cette étude à plusieurs objectifs :1. Modulation de la voie Akt par HCV sur des foies humains, foies de souris FL-N/35 au niveau basal dans un premier temps et lors d’un boost de la voie in vitro sur des cultures primaires d’hépatocytes murins.2. Etude de l’expression ainsi que des modifications post-traductionnelles des modulateurs de la voie PI(3)K dans un modèle murin exprimant (FL-N/35) ou non l’ORF complète du virus de l’hépatite C (VHC).3. Confirmer nos précédentes données avec l’invalidation de PTEN dans un modèle de souris KO pour PTEN.4. Etendre ses données au niveau moléculaire dans l’optique d’une étude mécanistique grâce à l’analyse in vivo, ex vivo et in vitro d’un modèle murin KO pour PTEN.5. Compléter cette étude par l’analyse des déterminants viraux impliqués dans la dérégulation de la signalisation intracellulaire grâce à l’étude de souris NS5A.6. Examiner l’impact de la dérégulation de la voie PI(3)K-Akt dans le développement des Carcinomes hépatocellulaires (CHCs) induit par le VHC. / The goal of myt thesis is to study regulators of intracellular signaling pathway of Pi route (3) K-Akt through the analysis of tumor suppressor PTEN (Phosphatase and tenson homolog) and serine / threonine kinase mTOR (Target of Rapamycin Mamalian). This study has several objectives:1. Modulation of the Akt pathway by HCV in human liver, mouse livers FL-N / 35 to the basal level in a first time and at a track boost in vitro on primary cultures of mouse hepatocytes.2. Study of the expression and post-translational modifications of modulators of PI path (3) K in a murine model expressing (FL-N / 35) or not the complete ORF of hepatitis C ( HCV).3. Confirming our previous data with the invalidation of PTEN in a knockout mouse model for PTEN.4. Extending its data at the molecular level with a view to a mechanistic study through analysis in vivo, ex vivo and in vitro of a knockout mouse model for PTEN.5. Complete this study by analyzing the viral determinants involved in the dysregulation of intracellular signaling through the NS5A mouse study.6. Examine the impact of deregulation of IP route (3) K-Akt in the development of hepatocellular carcinoma (CHCs) induced by HCV.

No description available.

In an Experimental Rat model for endometrial carcinoma (EC) a minimal region of recurrent deletion/allelic loss at the neighborhood of the Tp53 gene has been identified. A similar observation of deletion at the homologous position on human chromosome 17 unassociated with TP53 mutation has been reported in several human cancer types. Thus an important tumor suppressor activity located close to, but distinct of TP53 is suggested. Detailed molecular analysis of this candidate region in a tumor model suggested Myo1c (myosin 1C) and Inpp5k (inositol polyphosphate-5-phosphatase K), also known as Skip (skeletal muscle and kidney enriched inositol polyphosphate phosphatase), as the best candidates. These two genes are suggested to be involved in glucose metabolism through PI3K/AKT signaling and neither of them has earlier been reported as a tumor suppressor gene. The present work aimed to investigate the potential correlation of MYO1C and/or INPP5K proteins with components of PI3K/AKT signaling pathway involved in cell growth and survival. Cells were transfected with increasing amounts of MYO1C- or INPP5K- gene expression constructs and protein extracts of the cells were subjected to Western Blot analysis for 13 important components of the signaling pathway: p110β\α\δ, p85, pAkt308&473, 14-3-3β, PTEN, Akt, pErk, Erk, Ras, p4EBP1 and 4EBP1. The analysis showed dose-dependent changes in the expression levels of several of these proteins, and the observed changes for the most part were directed towards negative regulation of cell proliferation and survival. The presented data further extended the initial hypothesis for potential tumor suppressor activities of MYO1C and INPP5K proteins through PI3K/AKT pathway.

Untersuchung des Wirkmechanismus von AEZS 126 auf drei triple negative Mammakarzinomzelllinien HCC1937, HCC1806 und MDA-MB468 und eine Oestrogenrezeptor positive Zelllinie MCF-7 mittels Kristallviolett assay, FACS und Western Blot. Es konnte gute Antitumorwirkung des Inhibitors in vitro gezeigt werden. / Of more than one million global cases of breastcancer diagnosed each year, a high percentage are characterized as triple-negative, lacking the oestrogen, progesterone and Her2/neu receptors. The incidence exceeds the incidence of malignancies like CML by far. Lack of effective therapies, younger age at onset and early metastatic spread have contributed to the poor prognosis and outcomes associated with these malignancies. Here, we investigate the ability of the PI3 K/AKT inhibitor AEZS 126 to selectively target the triple negative breast cancer (TNBC) cell proliferation and survival in vitro by MTT-assays and FACS-based analysis. Furthermore, the mechanism of cytotoxicity is analysed by FACS-based assays and Western blots. Results AEZS 126 showed good antitumour activity in in vitro models of TNBC as well as in MCF-7 cells. We demonstrated the highly efficient antitumour activity of AEZS 126 in in vitro models of TNBC. Due to the good anti-tumour activity and the expected favourable toxicity profile, AEZS 126 in combination with chemotherapy seems to be a promising candidate for clinical testing in TNBC especially in the basal-like subgroup of TNBC.

Les protéines subissent des modifications post-traductionnelles tout au long de leur vieillissement mais aussi dans certains états pathologiques. Parmi ces modifications progressives des protéines, on retrouve notamment la formation des résidus aspartates isomérisés. La protéine L-isoaspartyl méthyltransférase (PIMT) reconnaît et répare des résidus L-isoaspartates anormaux retrouvés dans des protéines. Il a été démontré que la PIMT joue un rôle crucial dans la formation et le maintien du système nerveux central. Ainsi, son fonctionnement s'est avéré important dans le désordre neurologique qu'est l'épilepsie, mais aussi potentiellement dans certaines maladies neurodégénératives comme l'Alzheimer. Notre laboratoire a déjà démontré que l'acide valproïque (VPA), un médicament anticonvulsant, induit l'expression de la PIMT via la voie de signalisation de ERK et également la voie de signalisation glycogènen-synthase-kinase-3 (GSK-3)/β-caténine. Le but de notre recherche a été d'identifier des nouvelles voies de signalisation qui contrôlent l'expression de la PIMT lorsque celle-ci est stimulée par le VPA. Comme modèle cellulaire nous avons utilisé des neuroblastomes SH-SY5Y qui ont été traités avec le VPA pour étudier l'implication de la voie de signalisation phosphatidylinositol 3-kinase (PI3K)/Akt dans l'induction de la PIMT. Nos résultats montrent que lors de l'induction de la PIMT par VPA, la protéine Akt est phosphorylée (Thr 308). De plus, lorsque la protéine PI3K est inhibée par des inhibiteurs pharmacologiques, wortmannin et le LY294002, la phosphorylation de Akt est bloquée et l'induction de la PIMT par VPA est arrêtée. L'inhibition de Akt par un siRNA spécifique produit le même effet. Également, lorsque la voie de signalisation PI3K/Akt est stimulée par le VPA on observe une phosphorylation de la protéine GSK-3 (Ser 21) qui est également observable lorsque les cellules sont traitées avec le lithium, un inhibiteur directe de GSK-3. Finalement, l'inhibition du facteur de transcription CREB avec un siRNA spécifique n'a pas affecté l'induction de la PIMT par VPA. En conclusion, notre étude a démontrée que j'induction de la PIMT par VPA est dépendante de la voie de signalisation PI3K/Akt. L'activation de cette voie de signalisation permet la phosphorylation et donc l'inhibition de la kinase GSK-3, mais l'induction de la PIMT par VPA est indépendante de facteur de transcription CREB. Ces résultats suggèrent plutôt que VPA en inhibant la kinase GSK-3 stabilise la β-caténine permettant ainsi l'expression de la PIMT. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : PIMT, épilepsie, VPA, PI3K/Akt

Orientador: Jayme Augusto de Souza Neto / Resumo: Aedes aegypti é a espécie de mosquito emergente em áreas urbanas com maior impacto na saúde pública, sendo o principal vetor dos arbovírus dengue, Zika e chikungunya. Por esse motivo, se faz indispensável a compreensão de mecanismos moleculares associados a processos fisiológicos em A. aegypti, como resposta imune, comportamento e homeostase intestinal. Conforme observado em outros organismos, a via PI3K/AKT tem papéis importantes no metabolismo, na reprodução, na tolerância ao estresse e na imunidade. A quinase AKT atua como um regulador negativo da via PI3K/AKT, fosforilando o fator de transcrição FOXO e impedindo sua translocação nuclear. Nosso objetivo foi avaliar o perfil transcricional em A. aegypti com o gene akt silenciado e avaliar as consequências deste silenciamento sobre a microbiota bacteriana. Além disso, investigamos uma provável ativação mitocondrial quando do silenciamento de akt. Mostramos que o silenciamento de akt resultou na ativação de genes essenciais para a manutenção da homeostase intestinal do mosquito, como peptídeos antimicrobianos (AMPs) e genes codificadores de enzimas associadas à produção de espécies reativas de oxigênio (ROS). Notavelmente, observou-se uma forte repressão de 11 profenoloxidases (PPOs), além de uma potente indução de genes codificadores de subunidades da enzima NADH desidrogenase, em comparação com o respectivo grupo controle, sugerindo que tal indução esteja associada a um provável aumento da atividade mitocondrial. No context... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Aedes aegypti is the emerging mosquito species in urban areas with the greatest impact on public health, being the main vector of arboviruses dengue, Zika and chikungunya. For this reason, it is essential to understand the molecular mechanisms associated with physiological processes in A. aegypti, such as immune response, behavior and intestinal homeostasis. As observed in other organisms, the PI3K / AKT pathway has important roles in metabolism, reproduction, stress tolerance and immunity. AKT kinase acts as a negative regulator of the PI3K / AKT pathway, phosphorylating the FOXO transcription factor and preventing its nuclear translocation. Our objective was to evaluate the transcriptional profile in A. aegypti with the silenced akt gene and to evaluate the consequences of this silencing on the bacterial microbiota. In addition, we investigated a possible mitochondrial activation during the akt silencing. We have shown that akt silencing resulted in the activation of essential genes for the maintenance of mosquito intestinal homeostasis, such as antimicrobial peptides (AMPs) and genes encoding enzymes associated with the production of reactive oxygen species (ROS). Notably, a strong repression of 11 profenoloxidases (PPOs) was observed, as well as a potent induction of genes encoding subunits of the NADH dehydrogenase enzyme, in comparison with the respective control group, suggesting that such induction is associated with a possible increase in mitochondrial activity. In t... (Complete abstract click electronic access below) / Doutor