Hintergrund und Fragestellung/Hypothese Trotz verbesserter Diagnose und Therapiestrategien in der Behandlung von Plattenepithelkarzinomen des Kopf-Hals-Bereichs (HNSCC) in den letzten Jahrzehnten liegt das 5-Jahres-Überleben der Patienten bei nur etwa 60 %. Die hohe Mortalität ist zurückzuführen auf eine hohe Rezidivrate nach Radio- und Radiochemotherapie. Experimentelle und klinische Daten zeigen in dieser Tumorentität eine häufige Dysregulation des PI3K/AKT-Signalweges, der an verschiedenen Resistenzmechanismen beteiligt ist. Die Zielmoleküle des Signalweges sind involviert in DNA-Reparatur, Reoxygenierung und Repopulierung. Aufgrund der bekannten Beteiligung des PI3K/AKT-Signalweges an diesen wichtigen radiobiologischen Resistenzmechanismen beschäftigt sich die vorliegende Arbeit mit folgenden Fragestellungen: 1. Charakterisierung des Aktivierungsstatus des PI3K/AKT-Signalweges in Korrelation mit radiobiologischen Parametern in HNSCC-Experimentaltumoren 2. Bestimmung der Wirksamkeit des selektiven PI3K-Inhibitors Bay 80-6946 in HNSCC-Zelllinien in vitro 3. Untersuchung der Wirksamkeit von Bay 80-6946 in einem Panel von HNSCC-Experimentaltumoren in vivo mit und ohne Bestrahlung sowie des Einflusses der PI3K-Inhibition auf radiobiologische Parameter Material und Methode/Ergebnisse 1. Charakterisierung des Aktivierungsstatus des PI3K/AKT-Signalweges in Korrelation mit radiobiologischen Parametern in HNSCC-Experimentaltumoren Für die vorliegende Untersuchung wurde auf Tumormaterial von sechs verschiedenen Plattenepithelkarzinomzelllinien (FaDu, SAS, XF354, UT-SCC-5, UT-SCC-14, UT-SCC-15) aus einer Biobank zurückgegriffen. Analysiert wurden Xenografttumoren, die mit 0, 3, 5 oder 10 Fraktionen bestrahlt worden waren. Die Aktivierung des PI3K/AKT-Signalweges wurde mittels Western Blot-Analyse untersucht. Die publizierten Daten der lokalen Tumorkontrolle nach 30 Fraktionen in sechs Wochen, der Pimonidazol hypoxischen Fraktion und der HIF-1α-Proteinexpression wurden mit der pAKT-Expression korreliert. In allen unbehandelten Tumoren konnte pAKT nachgewiesen werden, wobei sich eine intratumorale Heterogenität in der Expression zeigte. Unter fraktionierter Strahlentherapie konnte in dem Panel von sechs Tumorlinien keine einheitliche Aktivierung des PI3K/AKT-Signalweges nachgewiesen werden. Die Expression des endogenen Hypoxiemarkers HIF 1α korrelierte in unbehandelten Tumoren mit der pAKT-Proteinexpression. Kein Zusammenhang wurde zwischen dem Gehalt von pAKT und der Pimonidazol hypoxischen Fraktion sowie der lokalen Tumorkontrolle nach 30 Fraktionen in sechs Wochen gefunden. 2. Bestimmung der Wirksamkeit des selektiven PI3K-Inhibitors Bay 80-6946 in HNSCC-Zelllinien in vitro Die Experimente wurden an den humanen Plattenepithelkarzinomzelllinien Cal33, FaDu, UT-SCC-5 und UT-SCC-14 durchgeführt. In Proliferationsassays und weiteren Zellkultur-experimenten wurde der Effekt des selektiven PI3K-Inhibitors Bay 80-6946 auf die Proliferation, die Phosphorylierung von AKT sowie die Dauer des inhibitorischen Effektes untersucht. Das klonogene Zellüberleben wurde bestimmt, indem die Tumorzellen mit 0, 2, 4, 6 oder 8 Gy bestrahlt, 24 Stunden mit dem Inhibitor inkubiert und die entstandenen Kolonien nach 14 Tagen ausgezählt wurden. Eine deutliche Hemmung der Proliferation konnte an den Plattenepithelkarzinomzelllinien schon bei geringen Konzentrationen von Bay 80-6946 gezeigt werden. Die Zelllinie Cal33, welche eine bekannte PI3K Mutation besitzt, reagierte am empfindlichsten auf die Inhibition von PI3K, gefolgt von UT-SCC-14 und FaDu. Nach Inkubation mit 1 µM Bay 80 6946 konnte in keiner der drei Tumorlinien phosphoryliertes AKT nachgewiesen werden. Die Dauer der Inhibition hielt allerdings nur für die Dauer der Inkubation mit Bay 80-6946 an. Das klonogene Zellüberleben nach Bestrahlung wurde durch die Behandlung mit dem Inhibitor nicht signifikant verändert. 3. Untersuchung der Wirksamkeit von Bay 80-6946 in einem Panel von HNSCC-Experimentaltumoren in vivo mit und ohne Bestrahlung sowie des Einflusses der PI3K-Inhibition auf radiobiologische Parameter Die Wirksamkeit von Bay 80-6946 wurde in einer Gruppe von neun verschiedenen Kopf-Hals-Tumorlinien sowie zwei NSCLC-Tumorlinien untersucht (A549, Cal33, FaDu, GLF, H460, SAS, SAT, UT-SCC-5, UT-SCC-8, UT-SCC-14 und XF354). Experimenteller Endpunkt war die Tumorwachstumsverzögerung. Bay 80-6946 wurde insgesamt fünfmal im Abstand von 48 Stunden in einer Konzentration von 20 mg/kg KG intravenös injiziert. Die Behandlung begann bei einem Tumordurchmesser von 7 mm. In den drei Tumorlinien Cal33, FaDu und UT-SCC-5 wurde Bay 80-6946 zusätzlich zur Monotherapie auch in Kombination mit fraktionierter Bestrahlung untersucht. Diese Tumormodelle wurden ausgewählt, da sie die intertumorale Heterogenität im Ansprechen auf die alleinige PI3K-Inhibition widerspiegeln. Die Tiere wurden mit 5 x 2 Gy täglich (Gesamtdosis 10 Gy) bestrahlt. Bay 80-6946 wurde einmal wöchentlich in einer Konzentration von 20 mg/kg KG über einen Zeitraum von 29 Tagen verabreicht. Die Behandlung begann eine Woche vor der ersten Bestrahlungsfraktion. Aufgrund des verlängerten Gesamtbehandlungs-zeitraums begann die Behandlung bereits bei einem Tumordurchmesser von 4 mm. In allen Experimentalarmen wurden zu verschiedenen Zeitpunkten Tumoren entnommen und immunhistochemisch auf die radiobiologischen Parameter Hypoxie, Anzahl der Gefäße und Perfusion untersucht. Hinsichtlich der Wirksamkeit von Bay 80-6946 auf unbehandelte Plattenepithelkarzinome lassen sich drei Gruppen abgrenzen: starkes Ansprechen bei Cal33, intermediäres Ansprechen (SAS, UT-SCC-8, SAT, FaDu) und fehlendes Ansprechen (XF354, GLF, UT SCC-5, UT-SCC-14). Die NSCLC-Modelle A549 und H460 sprachen moderat auf Bay 80 6946 an. Bei der Kombinationstherapie von Bay 80-6946 mit fraktionierter Bestrahlung deutet sich bei Cal33 ein additiver und bei FaDu ein supraadditiver Effekt an. Bei UT-SCC-5 war in der Kombinationstherapie kein signifikanter Effekt auf das Tumorwachstum nachweisbar. Die immunhistochemischen Analysen zeigten in allen drei Tumorlinien weder bei der Mono- noch bei der Kombinationstherapie einen Einfluss des Inhibitors auf die Parameter Hypoxie, Anzahl der Gefäße und Perfusion im Vergleich zur Kontrolle. Schlussfolgerung Die in einem Panel von Experimentaltumoren gewonnen Daten unterstützen derzeit nicht den Einsatz von pAKT als Biomarker, um das Ansprechen auf eine fraktionierte Strahlentherapie vorherzusagen. Vielmehr scheinen die gewonnenen Ergebnisse im Einklang zu den kontroversen und teilweise inkonsistenten Daten anderer Arbeitsgruppen zu stehen. Somit sind trotz klarer radiobiologischer Rationale weitere methodische und translationale Untersuchungen zum potenziellen Stellenwert des AKT-Signalweges als prognostischer Biomarker für die Strahlentherapie von Kopf-Hals-Tumoren notwendig. Durch die Kombination einer fraktionierten Strahlentherapie mit einem selektiven PI3K-Inhibitior konnte in der vorliegenden experimentellen Arbeit ein deutlicher Effekt auf die Tumorwachstumsverzögerung von Kopf-Hals-Tumoren gezeigt werden. Das Ausmaß des Ansprechens unterschied sich jedoch zwischen den drei untersuchten Tumorlinien und wirft die Frage nach der Ursache dieser Variabilität auf. Vor dem Einsatz eines PI3K-Inhibitors unter fraktionierter Strahlentherapie sollte in weiteren Experimenten der zugrundeliegende Mechanismus der Strahlensensibilisierung geklärt werden. Als zentrale Mechanismen sollten dabei vertiefend der Einfluss der selektiven PI3K-Inhibition auf das Tumormikromilieu sowie die Beteiligung des PI3K-Inhibitors an der Reparatur strahleninduzierter Schäden untersucht werden.

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.

Os transtornos do espectro autista (TEA) são distúrbios neuropsiquiátricos bastante comuns, graves, e que propiciam grande impacto social e financeiro. A identificação de vias moleculares e processos celulares alterados que são compartilhados pelos pacientes, mesmo que estes apresentem causas etiológicas distintas, pode contribuir de forma significativa para o entendimento de sua patofisiologia desses transtornos. Ainda, a identificação destas vias pode propiciar o desenvolvimento de abordagens terapêuticas mais eficientes, uma vez que o uso de medicamentos nos TEA ainda é inadequado, envolvendo baixa melhora funcional e diversos efeitos colaterais, como o ganho excessivo de peso e anormalidades metabólicas associadas. Neste trabalho, selecionamos como uma primeira abordagem o estudo da via de sinalização PI3K-mTOR em pacientes com TEA não-sindrômico, via esta envolvida com diversos aspectos do desenvolvimento e funcionamento neuronal, assim como com a patofisiologia de síndromes monogênicas que apresentam alta prevalência de TEA em seu quadro clínico. Foram utilizadas como modelo experimental in vitro células-tronco mesenquimais provenientes de polpa de dente decíduo (SHEDs) de pacientes e indivíduos controles. Os resultados aqui obtidos sugerem a importância da desregulação da via PI3K/mTOR na patofisiologia de uma parcela importante dos casos de TEA não-sindrômico. Ainda, observamos que as células dos pacientes com alterações nessa via de sinalização apresentam maior capacidade proliferativa, e que a modulação deste fenótipo alterado por meio do uso concomitante de inibidores de PI3K e mTOR nas células de um destes pacientes sugere esta via como um alvo promissor para o desenvolvimento de novas abordagens terapêuticas para estes pacientes. Em seguida, na tentativa de desvendar os mecanismos subjacentes aos efeitos metabólicos adversos associados com o uso de antipsicóticos prescritos para o tratamento de pacientes com TEA, investigamos os efeitos destes psicofármacos sobre a biologia do tecido adiposo humano. Foram utilizadas como modelos in vitro células-tronco (ADSCs) e adipócitos maduros derivados de tecido adiposo humano de indivíduos controles. Os resultados obtidos sugerem que a ação direta dos antipsicóticos com alta propensão ao ganho de peso (como a olanzapina e a clozapina) sobre a proliferação, diferenciação, e o metabolismo do tecido adiposo humano parece não constituir um mecanismo importante associado ao ganho de peso apresentado pelos pacientes, e que a ação desses medicamentos sobre os sistemas centrais que regulam o peso e o metabolismo deve ser o mecanismo principal levando aos efeitos metabólicos adversos. Juntos, os resultados gerados neste trabalho podem, de certa forma, contribuir para da farmacoterapia dos TEA / Autism spectrum disorders (ASD) are common neuropsypchiatric disorders, which has serious social and economic impacts. Identification of common molecular and cellular processes altered in patients, despite the underlying genetic heterogeneity, can contribute significantly to our understanding of the disease pathophysiology and can help to develop more effective treatments, since available pharmacotherapy for ASD is inefficient and frequently associated with adverse side effects, such as weight gain and metabolic disturbances. Here, we used patient-derived Stem cells from Human Exfoliated Deciduous teeth (SHEDs) as an intro model system to investigate whether non-syndromic ASD patients show altered regulation of PI3K/mTOR signaling pathway, which is involved in multiple aspects of neuronal development and physiology, and in the pathogenesis of monogenic syndromes that share features with ASD. Our results suggest that dysregulation of PI3K/mTOR-linked networks play an important role in the pathogenesis of a subgroup of non-syndromic ASD. In addition, we found enhanced proliferative capacity in cells with altered PI3K/mTOR activity, which was rescued in one of these patients through combined pharmacological inhibition of both PI3K and mTOR kinase activity, suggesting that PI3K-mTOR signaling is a promising target for the development of new therapeutic approaches for these individuals. Next, in an attempt to better understand the mechanisms underlying the metabolic side effects of the antipsychotics prescribed for ASD treatment, we investigated the effects of some of these drugs on the biology of human adipose tissue using as in vitro model systems human adipose-derived stem cells (ADSCs) and mature adipocytes. Our results suggest that a direct and potent effect of antipsychotics with high weight gain liability (such as clozapine and olanzapine) on cell proliferation, differentiation, and metabolism of human adipose tissue is not an important mechanism by which these drugs induce metabolic disturbances. Consequently, our results suggest that these side effects may mainly reflect the action of these drugs on central pathways involved in weight control and metabolism. Together, our results can, to some extent, contribute to improving pharmacotherapy of ASD

Les cancers du sein luminaux, exprimant le récepteur aux œstrogènes (RE) représentent 65-75% des cancers du sein soit environ 35.000 nouvelles patientes par an en France. Les référentiels thérapeutiques en vigueur recommandent une prescription systématique d’hormonothérapie au stade précoce, et quasiment constante au stade avancé. Néanmoins, il est admis que plus de 20% des patientes au stade précoce, et la quasi-totalité au stade avancé, vont échapper au traitement endocrinien, rendant impératif le développement de modèles précliniques permettant d’étudier les mécanismes d’hormonorésistance. Dans un contexte de modèles cellulaires anciens et très imparfaits (MCF7, T47D), et de quasi absence de modèles murins pertinents, nous avons choisi de développer des modèles murins dérivés de tumeurs fraîches, dits PDX (patient derived xenografts). Nous avons montré que ces modèles, difficiles à obtenir, récapitulaient avec une grande fidélité les caractéristiques morphologiques et biologiques des tumeurs d’origine. Les PDX se distinguent également par une grande stabilité de ces caractéristiques lors des passages successifs, les rendant utilisables au long cours. Nous avons également évalué les modèles obtenus pour leur profil de sensibilité à diverses modalités de traitement hormonal.Dans une seconde étape, nous avons développé des modèles résistants à partir des PDX précédemment obtenues. Quatre modèles ont pu être obtenus, qui nous ont permis d’avoir à disposition des modèles rendant compte de situations cliniques variées. Ces 4 modèles ont fait l’objet d’analyses biologiques extensives visant à identifier les caractéristiques moléculaires potentiellement associées à telle modalité de résistance : nos données suggèrent fortement qu’il y a autant de mécanismes de résistance que de situations, rendant illusoire une définition biologique unifiée de l’hormonorésistance. La reprogrammation fonctionnelle du RE semble être au centre de ces mécanismes.La voie PI3K/mTOR est une des plus fréquemment associée à l’hormonorésistance. De manière originale, nous avons mis en évidence que cette voie était activée aussi bien dans les modèles sensibles que dans les modèles résistants. La troisième étape a consisté à évaluer l’efficacité de l’everolimus, agent ciblant mTORC1. Nous avons pu montrer que l’everolimus était hautement actif dans toutes les situations considérées, sans argument pour une synergie entre everolimus et tamoxifène ou exemestane. En revanche, il existe une nette tendance à la synergie avec le fulvestrant, inhibiteur hautement spécifique du RE entraînant sa dégradation, et faisant suggérer des interactions avec la voie non génomique du RE.Nous testons actuellement des inhibiteurs spécifiques de la PI3KCA grâce à diverses collaborations industrielles qui permettront également de mener des analyses génomiques approfondies. De multiples projets académiques sont en cours. / Luminal breast cancer (ER+, HER2 negative) accounts for 65-75% of all breast carcinomas. Current guidelines strongly recommend endocrine treatment at both the early and advanced stages. However, more than 20% of early stage patients, and all advanced patients will eventually develop endocrine resistance.As most preclinical models (MCF7, T47D) do not recapitulate tumor biology, we have chosen to develop murine models derived from fresh tumors, hence called patient derived xenografts (PDX). We show that these models, although difficult to generate, faithfully exhibit the morphological and biological features of their parental counterpart, with high long term stability. These models have also been evaluated for their sensitivity to various endocrine treatments.In the next step, we developed from these initially endocrine sensitive models new tumors rendered resistant to endocrine therapies. We show that there is no unique biological pattern associated with endocrine resistance, although ER functional reprogramming appears to be critical. We also show that PI3K/mTOR pathway activation, may not be always related to endocrine resistance, and suggest that fulvestrant, an ER down regulator, may be highly synergistic with everolimus in specific cases.Several PI3KCA inhibitors are currently being evaluated in this setting.

No description available.

Rab5 (Ras-related in brain) and Rab4 are small monomeric GTPases that mediate the intracellular trafficking of endocytosed growth factor receptors. Active Rab5-GTP has low intrinsic GTP hydrolysis activity that is stimulated by GTPase activating proteins (GAPs) to make inactive Rab5-GDP. GAPs provide both a catalytic arginine and switch region stabilization functions. The p85 regulatory subunit of phosphatidylinositol 3′-kinase (PI3K) has GAP activity towards Rab5 and Rab4, which is not seen in other PI3Ks. The arginine “finger” residue within p85 is R274. It is unlikely that p85 stabilizes the switch regions of Rab5, which undergo large conformational changes between activation states, because it interacts with both Rab5-GTP and Rab5-GDP. In contrast, the PI3K catalytic subunit p110β binds only Rab5-GTP, suggesting it interacts with the switch regions. Thus, the GAP functions may be provided to Rab5 by the subunits of PI3K acting together, where p85 provides the arginine finger and p110β stabilizes the switch regions. The binding interface of Rab5:p85 was sought using mutations of Rab5 residues not present in the switch regions which were conserved in p85-binding Rab proteins (S84, E106, N113, F145, E172, M175, K179, K180) in GST pull-down experiments with FLAG-p85. The p85 binding site was not resolved with these experiments, suggesting that p85 interaction may involve the contribution of multiple residues of the Rab5 protein. The p110β interaction site on Rab5 was investigated using Rab5 switch region mutants. Pull-down experiments using a stabilized p110 protein construct, where the p85-iSH2 domain was fused to p110 (alpha or beta), were performed. Rab5 mutants I53A, F57A, W74A, Q79L, E80R, Y82A, H83E, L85A, M88A, Y89A and R91E showed reduced p110β binding. All of these residues except E80 and H83 are involved in binding other Rab5 effectors. The Rab5 binding site on p110β was also resolved through mutation of p110β in its Ras binding domain, and includes residues I234, E238 and Y244. This generation of non-binding mutants of both Rab5 and p110β will be invaluable in the characterization of the importance of the p110β:Rab5-GTP interaction for receptor trafficking to endosomes in mammalian cells.

B lymphocytes are an important type of immune cell that contributes to pathogen clearance. When dysregulated, these cells contribute significantly to diseases such as autoimmunity, allergy and B cell cancers. Here we examine an important regulatory circuit that involves the lipid phosphatase SHIP, a key regulator of the PI3K signaling pathway. SHIP was first described as the major effector of inhibitory IgG receptor FcγRIIB, which downregulates B cell antigen receptor (BCR) signaling pathways when co-engaged. However, it is also known to inhibit signaling downstream of several other receptors, both activating and inhibitory. Here we examine the regulation and function of SHIP in B cells, focusing on the inter-related influences of binding partners, tyrosine phosphorylation and subcellular localization dynamics. First, we assess interaction of SHIP to selected candidate binding partners using an in vitro screening approach. The two most robust interactions were further characterized with respect to dissociation constant. These were: a novel interaction between SHIP phospho-Tyr944 and the SH2 domain of Nck, and a known interaction between the SH2 domain of SHIP and FcγRIIB phospho-Y292. Next, we perform the first examination of SHIP Tyr944. We provide evidence that it contributes to interaction with Nck after BCR engagement and is required for inhibition of actin turnover by SHIP. Finally, we perform the first detailed examination of the mechanisms controlling SHIP localization in human B cells stimulated through the BCR with and without co-engagement of FcγRIIB. We discover that SHIP is recruited to the plasma membrane equally in both stimulation contexts, however FcγRIIB co-ligation results in reduced mobility of SHIP molecules at the cell periphery. We identify a novel and essential role for Syk kinase in promoting SHIP membrane localization, tyrosine phosphorylation, and interaction with known binding partner Shc1. Together, these results provide significant and exploitable insight into the molecular control of a clinically important regulator of B cell responses. / February 2017

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.

Adaptive immune responses are highlyco-ordinated and rely upon efficient intracellular communication to orchestratecell function. Phosphoinositide 3-kinase (PI3K) signalling is a well-studiedand important positive mediator in T lymphocyte function; however the role for SH2-domaincontaining inositol phosphatase 1 (SHIP-1), a negative regulator of PI3Ksignalling, has not been so thoroughly investigated. The use of knockout mousemodels has given an insight into the role of SHIP-1 in murine T cells, butthese are compromised by loss of function during development which impingesupon mature T cell function and by the loss of non-catalytic functions ofSHIP-1. Recent work has indicated a clear rolefor reactive oxygen species (ROS), specifically hydrogen peroxide (H2O2),in immune cell signalling and functional responses including migration. Howeverlike SHIP-1, the functional roles of ROS are poorly understood in human Tlymphocytes, particularly the mechanisms by which ROS signals to alter Tlymphocyte biology. ROS has been previously shown to activate PI3K, Mitogenactivate protein kinase (MAPK) and Src family tyrosine kinase (SFK) signallingin a number of different cell types. In addition, ROS have been shown to inactivatephosphatase and tensin homology (PTEN), another negative regulator of PI3K, andare postulated to inactive SHIP-1 signalling. A pharmacological approach wasutilised to manipulate the catalytic activity of SHIP-1 and the cellularaccumulation of ROS in primary human T lymphocytes. Remarkably, it wasdetermined that both SHIP-1 activation and inhibition reduced the ligand-mediatedfunctions of human T lymphocytes, including signalling, proliferation, adhesionand migration. Furthermore, H2O2 selectively inhibited directionalmigration to chemokine CXCL11, enhanced F-actin polymerisation and decreasedactin polarisation to CXCL11. H2O2 required SFKsignalling to induce the phosphorylation/catalytic activation of SHIP-1 and todecrease the surface expression of CXCR3, both of which could be mechanismsunderlying the deficiency in migration observed with H2O2.

The PI3K/mTOR signalling pathway plays a major role in biology and disease. Therefore, effective inhibitors that target proteins of this pathway have been developed. However, acquired resistance of cancer cells is a prevalent phenomenon that limits the durable response of these compounds. It is becoming apparent that experimental approaches for comprehensive biochemical analysis contribute to understand the complex mechanisms that confer drug resistance, and advances in largescale technologies including genomic sequencing and proteomics allow unprecedented molecular coverage without being biased for specific genes/cellular pathways. Initially, the phenotypic response of sensitive and resistant cells to the absence or presence of a PI3K inhibitor (PI3Ki), as well as other kinases, was examined. This study revealed that PI3Ki-resistant cells experience extensive phenotypic changes upon withdrawal of the PI3Ki from the culture media. The regulation of the proteome and phosphoproteome of sensitive and PI3Ki-resistant cells grown with or without the PI3Ki was analysed by shotgun mass spectrometry-based label-free quantitative technology. This analysis demonstrated that the proteomes and phosphoproteomes of drug-resistant cells are remodelled conditional to the presence of PI3Ki, and that the levels of enzymes with metabolic roles are modulated in resistant cells. Functional analysis of the metabolism of cells capable to survive in absence of PI3K/mTOR activity demonstrated that the bioenergetic activity of these cells is contingent on the presence of the selection drug. The complete set of protein-coding regions of the genome (exome) of sensitive and PI3Ki-resistant cells was then sequenced. This study unveiled common alterations in exome regions across PI3Kiresistant cell lines, as well as a degree of genomic heterogeneity between them. Lastly, the impact of lactic acid, a metabolic product, on a defined signalling network of the MCF7 breast cancer cell line was analysed. This study described the capacity of this metabolite to change the activity of signalling network branches.