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Balakrishnan, Sanjeevi. "Establishing a biological role for class II phosphoinositide 3-kinase (PI3K) enzyme PI3K-C2??" Thesis, Imperial College London, 2012. http://hdl.handle.net/10044/1/39135.
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Phosphoinositide 3-Kinase (PI3K) enzymes control a variety of cellular processes and their deregulation has been extensively investigated in disease conditions including renal disease, autoimmunity and chronic inflammation. Whilst PI3K enzymes have been the subject of intense research activity, the precise biological role of PI3K-C2?? enzyme remains unclear. PI3K-C2??-/- mice developed focal segmental glomerulosclerosis, damaging podocyte morphology and affecting function. My thesis explored the contribution of the PI3K-C2?? enzyme in the pathology of chronic diseases. PI3K-C2??flx/+ mice were mated with ??-actin cre mice, and the resulting F1 progeny, PI3K-C2??flx/- mice were backcrossed to produce PI3K-C2??-/- mice. Analysis of PI3K-C2??-/- mice revealed slightly elevated proteinuria and mild hyperglycaemia. Histological examination of their kidneys showed an increase in glomerular volume, intraglomerular nuclei, mesangial matrix expansion and an infiltration of T cell subsets in the glomeruli compared to control mice. In vitro, mesangial cells derived from PI3K-C2??-/- mice proliferated twice as fast as mesangial cells from control animals, without any stimulus. PI3K-C2??-/- mice exhibited an increase in the levels of leukocytes and displayed augmented proliferation, in presence of mitogenic stimuli. Wild type (WT) mice mesangial cells cultured in PI3K-C2??-/- mice splenocyte conditioned medium, exhibited a dose dependent increase in their proliferation. Induction of glomerulonephritis (GN) using a sub-nephritogenic dose of nephrotoxic serum (NTS) in PI3K-C2??-/- mice, resulted in impaired renal function and aggravated renal tissue damage. PI3K-C2??-/- mice had a greater influx of T cells and macrophages in the diseased glomeruli, but also of activated macrophage functions compared to WT mice. During GN, PI3K-C2??-/- mice developed splenomegaly leading to extramedullary haematopoiesis and increased splenocyte proliferation. PI3K-C2??-/- mice may mediate GN by polarising their cytokine effects via a Th1 pathway. PI3K-C2??-/- mice exhibit a delay in the repair of dermal injury during cutaneous wound healing. PI3K-C2??-/- mice recruit fewer macrophages to the wound site which affects re-epithelialisation, myofibroblast differentiation, angiogenesis and fibroblast mediated remodelling. Delayed wound healing in PI3K-C2??-/- mice might be a consequence of the failure to upregulate pro-inflammatory cytokines at the wound site, which are potential modulators for the recruitment of various cell types to the wound site to accelerate the healing process. To complement these in vivo studies, biochemical analysis on the putative Ras binding domain present in PI3K-C2?? and PI3K-C2?? enzymes was performed by expressing as recombinant proteins. The binding ability of the recombinant proteins to Ras family GTPases was tested. Although this approach initially proved unsuccessful, two putative binding partners, eEF1?? and eEF1?? were identified and have been shown to immunoprecipitate with full length enzymes. My data is the first study to demonstrate a role for the intracellular signalling enzyme, PI3K-C2?? in pathological conditions involving chronic inflammation. Agonist mediated activation of PI3K-C2?? enzyme might serve as a potential therapeutic target to treat chronic diseases.
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Hale, Benjamin G. "Influenza A viruses and PI3K signalling." Thesis, University of St Andrews, 2007. http://hdl.handle.net/10023/483.
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The influenza A virus non-structural (NS1) protein is multifunctional, and during virus-infection NS1 interacts with several factors in order to manipulate host-cell processes. This study reports that NS1 binds directly to p85β, a regulatory subunit of phosphoinositide 3-kinase (PI3K), but not to the related p85α. Expression of NS1 was sufficient to activate PI3K and cause the phosphorylation of a downstream mediator of PI3K signalling, Akt. However, in virus-infected MDCK cells, the kinetics of Akt phosphorylation did not correlate with NS1 expression, and suggested that negative regulation of this signalling pathway occurs subsequent to ~8h post-infection. Mapping studies showed that the NS1:p85β interaction is primarily mediated by the NS1 C-terminal domain and the p85β inter-SH2 (Src homology 2) domain. Additionally, the highly conserved tyrosine at residue 89 (Y89) of NS1 was found to be important for binding and activating PI3K in a phosphorylation-independent manner. The inter-SH2 domain of p85β is a coiled-coil structure that acts as a scaffold for the p110 catalytic subunit of PI3K. As NS1 does not displace p110 from the inter-SH2 domain, a model is proposed whereby NS1 forms an active heterotrimeric complex with PI3K, and disrupts the ability of p85β to control p110 function. Biological studies revealed that a mutant influenza A virus (Udorn/72) expressing NS1 with phenylalanine substituted for tyrosine-89 (Y89F) exhibited a small-plaque phenotype, and grew more slowly in MDCK cells than wild-type virus. Unexpectedly, another mutant influenza A virus strain (WSN/33) expressing NS1-Y89F was not attenuated in MDCK cells, yet appeared to be less pathogenic than wild-type in vivo. Overall, these data indicate a role for NS1-mediated PI3K activation in efficient influenza A virus replication. The potential application of this work to the design of novel anti-influenza drugs and vaccine production is discussed.
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Hale, Benjamin G. "Influenza A viruses and PI3K signalling /." St Andrews, 2008. http://hdl.handle.net/10023/483.
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White, Angela R. "Shared PI3K signaling abnormalities in brain tumors and epilepsy: PI3K inhibition in PTEN-deficient disorders of the brain." University of Cincinnati / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1603712831970142.
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Castel, Morales Pau. "Molecular mechanisms of resistance to PI3K inhibitors." Doctoral thesis, Universitat de Barcelona, 2016. http://hdl.handle.net/10803/396640.
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The development of high-throughput sequencing technologies has prompted the evaluation of large cohorts of different tumor types. The results from these comprehensive genomic studies have revealed the most commonly mutated genes across human cancers, providing further understanding of the pathogenesis, molecular classification, and therapeutic strategies for this disease.
PIK3CA, the gene encoding the PI3Kα isoform, is among the most frequently mutated genes in breast, head and neck, colorectal, and lung cancer, among others. Activating mutations in PIK3CA promote hyperactivation of the PI3K/AKT pathway, leading to increased proliferation, cell growth, survival, and metabolism.
Current efforts are aimed to develop PI3K inhibitors as an effective therapy for PIK3CA mutated cancers and, despite promising clinical responses, the emergence of drug resistance is a clear limitation. In this doctoral thesis, we have explored these mechanisms of resistance in order to provide a better understanding of tumor evolution upon therapy, define subpopulations of patients that are likely to respond to PI3K inhibitors, and provide novel pharmacological combinations to overcome therapy refractoriness.
The loss of the tumor suppressor PTEN was found to play an important role in the resistance of PI3Kα inhibitors in preclinical models and patients, mainly by reactivating the PI3K/AKT pathway as a result of an increased dependency on the PI3Kβ isoform. Our work also demonstrated the notion of tumor evolution and phenotypic convergent evolution in response to therapeutic pressure.
Moreover, we have established that intrinsic resistance to PI3Kα inhibitors occurs as a result of incomplete inhibition of the mammalian target of rapamycin complex 1 (mTORC1), a downstream effector of the PI3K/AKT pathway. PI3Kα inhibitor-resistant cells could be re-sensitized through the blockade of phosphoinositide-dependent kinase (PDK1), a constitutively active kinase, using genetic or pharmacologic inhibition. Further experiments showed that the downstream effector of PDK1 is the serum and glucocorticoid-induced kinase 1 (SGK1), which promotes cell survival through the
phosphorylation of key proteins such as FOXO3 and TSC2. Accordingly, the resistant phenotype could be also reverted by inhibiting SGK1, a novel pharmacological approach that has revealed interesting roles of this kinase in tumor biology.
Genetically engineered mouse models represent reliable tools for investigating the etiology, biology, and progression of human diseases, as well as for exploring novel therapeutic approaches. By serendipity, we discovered the role of PIK3CA mutations in the genesis of venous malformations, an aberration of normal venous development that currently lacks effective treatments. Our mouse models recapitulated the histopathologic features of the disease and provided an experimental platform to test novel pharmacological approaches. PI3Kα inhibitors were effective at reducing the morbidity and mortality of mice carrying venous malformations.
The results from this thesis highlight the importance of defining the molecular determinants of sensitivity and resistance to PI3K inhibitors, a therapy that will most likely benefit PIK3CA mutant patients.El desenvolupament de noves tècniques de seqüenciació massiva ha fomentat l’estudi d’un gran nombre de mostres de diversos tipus tumorals. Els resultats d’aquests estudis genòmics exhaustius ha revelat els gens que es troben mutats en major prevalença, contribuint a una millor comprensió dels processos de patogènesis, classificació molecular i estratègies terapèutiques per a aquesta malaltia. PIK3CA, el gen que codifica per a la isoforma PI3Kα, es troba entre els gens mes freqüentment mutats en el carcinoma de mama, cap i coll, colorectal, pulmó, entre d’altres. Les mutacions activadores a PIK3CA promouen la hiperactivació de la via de senyalització de PI3K/AKT, donant lloc a un increment en la proliferació, la supervivència, i el metabolisme de les cèl·lules tumorals. Els esforços actuals es centren en el desenvolupament d’inhibidors de l’enzim PI3K com a una possible teràpia efectiva en tumors que presenten mutacions a PIK3CA. Tot i que els assajos clínics inicials son prometedors, l’emergència de resistència a aquestes teràpies és una clara limitació. En aquesta tesis doctoral s’han explorat els possibles mecanismes de resistència per intentar entendre com els tumors evolucionen enfront d’aquest fàrmacs, poder definir les subpoblacions de pacients que respondran als inhibidors de PI3K i proporcionar noves combinacions farmacològiques per combatre el fenomen de la resistència. Hem demostrat que la pèrdua del supressor tumoral PTEN juga un paper important en la resistència als inhibidors de PI3Kα, tant en models preclínics com en pacients, mitjançant la reactivació de la via de PI3K/AKT que és resultat d’un increment en la dependència de la isoforma PI3Kβ. El nostre treball també ha evidenciat la noció d’evolució tumoral i ha demostrat el concepte d’evolució convergent fenotípica en resposta a la pressió terapèutica. També s’ha demostrat que la resistència intrínseca als inhibidors de PI3Kα es pot donar com a resultat d’una inhibició incompleta del complex 1 de mTOR (mTORC1), un efector clau de la via de PI3K/AKT. Cèl·lules resistents a inhibidors de PI3Kα es van poder sensibilitzar amb el bloqueig genètic o farmacològic de PDK1, una quinasa constitutivament activa. Experiments addicionals van poder demostrar que l’efector molecular de PDK1 era la quinasa SGK1, la qual promou la supervivència cel·lular a través de la fosforilació de proteïnes clau com FOXO3 i TSC2. El fenotip resistent es va poder revertir mitjançant la inhibició farmacològica d’aquesta proteïna, una aproximació terapèutica que ha revelat un rol interessant en la biologia tumoral. Els models murins modificats genèticament representen una eina segura per a l’estudi de la etiologia, biologia i progressió de malalties humanes, així com per explorar noves aproximacions terapèutiques. Com a resultat d’un descobriment imprevist, també hem pogut revelar el rol de les mutacions de PIK3CA en la formació de malformacions venoses, una aberració del desenvolupament normal de les venes que actualment no tenen un tractament específic. El nostre model animal de malformació venosa recapitula les característiques histopatològigues de la malaltia i proporciona una plataforma experimental única per a l’estudi de noves teràpies. En aquests models animals, els inhibidors de PI3Kα han demostrat ser efectius en la reducció de la morbiditat de les malformacions venoses.
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Figueiredo, Ana Raquel Martins. "Role of PI3K in pericytes during angiogenesis." Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/404276.
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Pericytes (PCs) are important regulators of the vascular development promoting vessel growth and stabilization. They have recently emerged as a therapeutic target to promote or inhibit angiogenesis. Therefore, advancing our basic understanding on PCs biology and its molecular regulators during physiological angiogenesis is essential to develop PC-related therapies. One of the major pathways leading to cellular proliferation, migration, and survival is relayed by PI3K. Data from our laboratory and others have demonstrated isoform selectivity stimulating PI3K signalling in the regulation of both, the ECs and vSMCs. Interestingly, in these two cell populations’ p110α seems to be the major isoform.
By using pharmacological and genetic tools together with cultured PCs and retinal systems we have found that PCs pass through different states during vessel development, and that PI3K signalling regulates these cells in an isoform-specific manner. We have seen that immature and active PCs promote vessel growth while, mature and quiescent PCs result in vascular stabilization and remodelling. Moreover and unexpectedly, our results have identified p110β as the key regulator of PCs proliferation and growth. Inactivation of p110β in PCs, but not p110α, results in PC proliferation arrest and in several morphological changes, which resemble a more mature PC. Lack of p110β in PCs also leads to a more mature vascular plexus. We observed reduced vessel density, EC proliferation arrest and increased deposition of collagen IV. Furthermore, PTEN seems to be the regulator of PI3K in PCs, opposing the p110β effects and, leading to a more mature and stable PC and subsequently also more mature vasculature.
Since p110β-PI3K controls PC growth and proliferation, it suggests that target therapy directed to p110β in cancer could affect PCs. Using a pancreatic neuroendocrine tumour mouse model (RIP1-Tag2) we have seen that pharmacological inhibition of p110β impacts on tumour progression and in PCs growth. However, it also results in a slight reduction in the overall survival of the animal, without affecting their metastatic potential. Therefore, other studies are needed to further investigate p110β as a possible PC-specific target therapy.
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Fedrigo, Carlos Alexandre. "Inibição da via PI3K-Akt em gliomas." Pontifícia Universidade Católica do Rio Grande do Sul, 2012. http://hdl.handle.net/10923/4518.
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Previous issue date: 2012Glioblastoma multiforme (GMB) is the most malignant and common type of all astrocytic tumours. Current standard treatment for GBM patients involves maximum surgical resection of the tumour, followed by radiotherapy and chemotherapy, usually containing the alkylating agent Temozolomide (TMZ). Despite this aggressive combination therapy, the survival rate of GBM patients is still low. This work consisted in investigating the cytotoxic effects of Akt-inhibition by MK-2206 with irradiation (RT) and TMZ on in vitro human malignant glioma. Seven malignant glioma cell lines were cultured and tested for clonogenic survival, invasion inhibition, tumour spheroid growth and proliferation. The Akt-inhibitor MK-2206 and TMZ were added at different time treatments and in varying doses. Cultures were irradiated with single dose and with fractionated γ-irradiation. Cellular modulation of Akt and p-Akt were assessed by Western blot analysis. MK-2206 reduced the levels of phospho- Akt key protein in the PI3Kinase-Akt pathway, decreased cell survival, and inhibited invasion, proliferation and cell growth. The combination of MK-2206 and RT lead to enhanced inhibition of cell proliferation and invasion, which is not observed with RT alone. The radioenhancing effect of MK-2206 was most striking in inhibition of spheroid volume growth by fractionated RT; the radiosensitizing effect of MK-2206 was stronger than that of TMZ. MK-2206 enhanced the in vitro effects of RT and TMZ in terms of decreased cell survival, invasion, proliferation and growth in malignant glioma. Effects could be ascribed to inhibition of PI3K-Akt pathway.
O Glioblastoma multiforme (GBM) é o tipo mais maligno e mais comum de todos tumores astrocíticos. O tratamento atual para pacientes de GBM envolve máxima remoção cirúrgica, seguida de radio e quimioterapia, normalmente com o agente alquilante Temozolamida (TMZ). Apesar da agressividade da terapia combinada, o tempo de sobrevivência dos pacientes ainda é baixo. Este trabalho procurou investigar os efeitos citotóxicos do inibidor de Akt MK-2206 em combinação com irradiação (RT) e TMZ em um painel de células de gliomas humanos. Sete linhagens de glioma foram cultivadas e testadas em ensaio de sobrevivência clonogênica, inibição de invasão, e modelos de proliferação e crescimento de volume em esferóides. O inibidor MK-2206 e TMZ foram adicionados em diferentes tempos de tratamento e diferentes doses. As culturas foram irradiadas com doses únicas ou em terapias fracionadas com irradiação γ. A modulação celular de Akt e fosfo-Akt foi checada via Western Blot. O composto MK-2206 reduziu a fosforilação da proteína chave Akt na via PI3K, diminuindo a sobrevivência celular e inibindo invasão, proliferação e crescimento celular. A combinação de MK-2206 com RT levou a uma maior inibição de invasão e proliferação, o que não é observado somente com a RT. O efeito radiosensível de MK-2206 foi ainda maior na inibição do volume dos esferóides em terapia combinada com RT fracionada, sendo ainda maior do que o efeito combinado com TMZ. MK-2206 aumentou os efeitos in vitro de RT e TMZ em termos de redução de sobrevivência celular, invasão, proliferação e crescimento celular em gliomas malignos. Os efeitos podem ser atribuídos a inibição da via PI3KAkt.
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Ramos, Delgado Carmen Fernanda. "Exploring PI3K signalling dynamics in pancreatic cancer." Thesis, Toulouse 3, 2020. http://www.theses.fr/2020TOU30152.
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Les PI3K sont des enzymes qui phosphorylent le groupe hydroxyl en position 3 des phosphatidylinositols comme le PIP2 ou le PI. Ces lipides sont impliqués dans de nombreux processus cellulaires tels que la croissance, la prolifération, la motilité, l'autophagie et le trafic cellulaire. Chez les mammifères, il y a 8 isoformes de PI3K et elles sont regroupées en trois classes (I, II et III) en fonction de leur structure et spécificité du substrat. Les PI3K de classe I sont les mieux caractérisées et impliquées dans le cancer. Les rôles oncogéniques des PI3K de classe II et III restent méconnus. La voie PI3K/Akt est fréquemment suractivée dans les cancers et est corrélée à un mauvais pronostic, particulièrement dans l'adénocarcinome canalaire pancréatique (PDAC). La mutation oncogénique de Kras est détectée dans plus de 90% des cas de PDAC et induit l'activation des voies effectrices, y compris de la voie PI3K. Le PDAC est l'un des cancers les plus mortels, caractérisé par un diagnostic tardif, une progression rapide et des options thérapeutiques limitées. Des études antérieures de l'équipe ont démontré que la PI3Kalpha, une PI3K de classe I, est cruciale dans les étapes précoces du PDAC. Néanmoins, son rôle dans la progression du PDAC reste inconnu. Ma thèse vise à élucider la dynamique de signalisation des PI3K dans le PDAC. J'ai commencé par caractériser le rôle de la PI3Kalpha dans la progression du PDAC et j'ai déterminé sa pertinence en tant que cible thérapeutique. Enfin, je montre des données préliminaires sur le rôle de Vps34, une PI3K de classe III, dans la physiologie des cellules acineuses et son rôle éventuel dans la cancérogenèse pancréatique. L'inactivation pharmacologique et génétique de PI3Kalpha in vitro démontre que cette PI3K contrôle les paramètres cellulaires qui régulent la progression des cellules tumorales pancréatiques, et ce quelles que soient leurs mutations génétiques. Ces résultats ont été validés in vivo dans le modèle murin appelé KPC. Ainsi, des souris KPC avec des taux élevés de cfDNA (cell free DNA, marqueur d'inflammation) et une tumeur détectée par échographie ont été traitées avec l'inhibiteur spécifique de PI3Kalpha, BYL-719. J'ai également comparé l'inhibition pharmacologique de PI3Kalpha avec l'inactivation génétique de PI3Kalpha dans l'épithélium pancréatique de souris KPC (réalisé avec des souris génétiquement modifiées). L'inhibition de la PI3Kalpha in vivo, diminue le volume tumoral, prolonge la survie et retarde la dissémination métastatique. L'effet anti-métastatique des inhibiteurs de PI3Kalpha a été validé par une injection de cellules cancéreuse pancréatiques dans la veine caudale avec ou sans traitement avec du BYL-719. L'inhibition de la PI3Kalpha a également diminué l'infiltration des macrophages protumoraux, suggérant un rôle dans la réponse immunitaire, facteur connu de progression du PDAC. [...]PI3Ks are enzymes that catalyse the phosphorylation of inositol phospholipids in the 3-position of the inositol ring. These substrates and products are involved in multiple cellular processes such as cell growth, proliferation, cell motility and cellular trafficking. In mammals, there are 8 isoforms of PI3Ks and they are grouped into three classes (class I, II and III) depending on their structure and substrate specificity. Class I PI3Ks are the best characterised and the most commonly implicated in cancer. Current evidence on the oncogenic roles of class II and class III PI3Ks is limited. The PI3K/Akt signalling pathway is frequently hyperactivated in cancers and is usually correlated to a poor prognosis, particularly in pancreatic ductal adenocarcinoma (PDAC). More than 90% of PDAC cases are driven by activating mutations in Kras, which then activate downstream effector-signalling pathways, including the PI3K pathway. PDAC is one of the most lethal cancers, characterised by a late-stage diagnosis, a rapid progression and limited therapeutic options. There is a dire need to find new biomarkers and to design novel therapeutics for PDAC management. Previous studies from the team demonstrated that PI3Kalpha, a class I PI3K, is crucial in the initial stages of PDAC. Nonetheless, its role during PDAC progression remains unknown. My PhD aims to elucidate PI3K signalling dynamics in PDAC. I focused on characterising the role of PI3Kalpha in PDAC progression and on determining its suitability as a therapeutic target. Additionally, I show preliminary data on the role of Vps34, a class III PI3K, in acinar cell physiology and its possible role in pancreatic carcinogenesis. The pharmacological and genetic inactivation of PI3Kalpha in vitro demonstrate that this PI3K isoform regulates parameters that drive pancreatic tumour cell progression regardless of oncogenic mutations. These effects are organ-specific; depending on the organ context, another class I PI3K isoform could drive the cancer progression. These results were then validated in vivo in the KPC mouse model used for preclinical testing of PDAC. KPC mice with high levels of cfDNA and a detected tumour via ultrasound imaging were treated with the PI3Kalpha-specific inhibitor, BYL-719. Likewise, I compared the pharmacological inhibition of PI3Kalpha with the genetic inactivation of PI3Kalpha in the pancreatic epithelium of KPC mice. Targeting PI3Kalpha in vivo, pharmacologically and genetically, decreases tumour volume, increases life expectancy and delays metastatic dissemination. To further support the anti-metastatic effect of PI3Kalpha, a tail vein assay was performed and the mice were also given BYL-719. This last experiment reproduced the previous results obtained with the other mouse models, reinforcing the role of PI3Kalpha in decreasing metastatic dissemination. Besides delaying metastatic dissemination, PI3Kalpha also decreased the infiltration of protumoral macrophages, suggesting a role for this isoform in shaping the immune response. [...]
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Karnes, Jonathan Burgess. "PI3K Class IIalpha Is Required for Autophagy." Thesis, Van Andel Research Institute, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10268645.
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Autophagy is a cellular recycling process in which cytoplasmic proteins and organelles are sequestered in a double membrane vesicle, delivered to the lysosome, and degraded following fusion of the two vesicles. A key part of the initiation signaling for autophagy is the generation of phosphoinositol 3-phosphate (P13P) by class III phosphoinositol 3-kinase also knows as Vps 34. In humans there are eight P13K isoforms divided into three classes, four class I enzymes, three class II enzymes, and a single class III enzyme. Of these eight enzymes, only the class III isoform is thought to participate directly in autophagic signaling. A quantitative microscopy based, loss-of-function survey of all eight P13K isoforms was used to determine their relative contribution to autophagic signaling, as measured by LC3 positive autophagic vesicles. As predicted, knockdown of P13K-class III reduced the number of autophagic vesicles in cells. Interestingly, knockdown of the P13K-class IIα isoform had an even more potent effect on reducing the number of autophagic vesicles than knockdown of P13K-class III. In follow up studies, knockdown of P13K-class IIα reduced endogenous LC3 conversion, caused the accumulation of p62 and lipid droplets, and colocalized with endosomal markers. These results suggest P13K-class IIα may act to promote autophagy through the shuttling of endosomal vesicles into the autophagic pathway and approaches to test this hypothesis will be discussed. The requirement of P13K-class IIα for autophagy is an important finding as it indicates a role for class II P13Ks in autophagy.
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Zunder, Eli Richard. "A yeast screen for PI3K inhibitor resistance." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3339211.
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Rampanarivo, Hariniaina. "Contrôle du signal apoptotique CD95 par la voie PI3K / AKT et caractérisation de nouveaux inhibiteurs de PI3Ks." Rennes 1, 2012. http://www.theses.fr/2012REN1S086.
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Le récepteur CD95 appartient à la famille du récepteur au TNF. Son ligand, le CD95L retrouvé à la surface des cellules NK et des lymphocytes T activés, joue un rôle dans l'élimination des cellules infectées et cancéreuses. La liaison CD95L/CD95, entraîne le recrutement de FADD, qui fixe les procaspases-8/-10, pour former le DISC, nécessaire à l'induction du signal de mort. Selon la transmission du signal apoptotique CD95, 2 types cellulaires existent: les type I forment le DISC efficacement et les type II forment un DISC inefficace. Ce signal CD95 est modulé par la distribution ou l'exclusion de CD95 dans les radeaux lipidiques, des structures compactes flottant dans un environnement membranaire plus fluide, qui servent de plateforme de signalisation pour de nombreux récepteurs. La PI3K phosphoryle le PtdIns(4,5)P2 en PtdIns(3,4,5)P3 permettant le recrutement et l'activationd'effecteurs à la membrane comme la kinase AKT. La voie PI3K/AKT déclenche des signauxde survie permettant aux cellules tumorales d'être protégées de l'apoptose par CD95. Nousdémontrons que chez les cellules de type II, l'inhibition de la voie PI3K/AKT induit la redistribution de CD95 dans les radeaux lipidiques indépendamment du CD95L, la formation du DISC et la transmission du signal apoptotique CD95 qui ne fait intervenir ni le remodelage du cytosquelette d'actine ni l'ezrine, une protéine reliant CD95 aux filaments d'actine. Ainsi, la voie PI3K/AKT bloque la transmission du signal apoptotique en excluant CD95 des radeaux lipidiques, par un mécanisme moléculaire à définir. En collaboration avec des chimistes, nous avons aussi caractérisé de nouveaux inhibiteurs de la PI3K de classe IAApoptosis is a physiological process that controls immune response through the death receptor CD95 that belongs to the TNF-Receptor family. In the presence of CD95L, CD95 is aggregated and forms the DISC, which is necessary to transmit the apoptotic signal andconsists of the adaptor protein FADD and procaspases-8/-10. Depending on DISC formation,cells are classified as type I cells, in which the DISC is efficiently formed, and type II cells,where the DISC formation is impaired. DISC formation is enhanced when CD95 is redistributed into plasma membrane sub-domains designated lipid rafts, compact structures floating in the more fluid lipid bilayer. The lipid kinase PI3K catalyzes the synthesis of thePtdIns(3,4,5)P3, which serves as a docking lipid recruiting the kinase AKT, which in turn participates in the induction of several anti-apoptotic processes. We have shown that inhibition of the PI3K/AKT pathway in type II leukemic cells induces the redistribution of CD95 into lipid rafts, eliciting the DISC formation and the CD95-mediated apoptotic signal,through a CD95L-independent mechanism. Ultimately, this complex transmits an apoptoticsignal and the death of the malignant cell. In addition, we have observed that neither ezrin, a protein connecting transmembrane receptors with actin cytoskeleton, nor actin remodeling,contributed to this CD95-driven apoptotic signal. In conclusion, we have demonstrated that the PI3K/AKT pathway inhibits the CD95-induced apoptotic-signaling pathway by preventing the redistribution of CD95 into lipid rafts, through a yet unknown mechanism. We have also generated new PI3K class IA inhibitors and characterized their biologic activity
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Shibuya, Hideyuki. "TNFα, PDGF and TGFβ synergistically induce synovial lining hyperplasia via inducible PI3Kδ." Kyoto University, 2015. http://hdl.handle.net/2433/199195.
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Killip, Marian J. "RNA virus modulation of IFN, PI3K and apoptosis." Thesis, St Andrews, 2009. http://hdl.handle.net/10023/777.
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Marshall, Andrew James. "The development of PI3K inhibitors as anticancer drugs." Thesis, University of Auckland, 2010. http://hdl.handle.net/2292/6440.
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Phosphatidylinositol 3-kinases (PI3Ks) are a lipid enzyme family that are vitally important
regulators of intracellular signalling pathways which control cellular activities including cell
survival, growth and proliferation. Deregulation of the PI3K signalling cascade has been
observed in a broad range of human diseases including cancer, diabetes, thrombosis, immunity
and inflammatory disorders. With the discovery of PI3K’s link to a variety of diseases, there has
been a race to produce ATP competitive inhibitors as therapeutic agents against the Class I PI3K
isozymes. Herein, compounds from two structurally distinct chemotypes were synthesised and
their activity and specificity characterized against isolated Class PI3K enzymes and two cellular
lines.
The aryl morpholine containing pyrido[1,2-a]pyrimidines probed the requirements of the Class
IA PI3K active sites through modification of the pendant C9 position. Interestingly, no
compound synthesised exhibited superior activity towards the p110β enzyme than TGX-221
(1.14). The second series of compounds probed the requirements of the thiazole-linked
pyrazolo[1,5-a]pyridine 4.41B, identified through scaffold hopping studies using the novel
p110α selective inhibitor PIK-75 (1.34). Although 4.41B was not synthetically accessible,
analogues explored alternative linkers and substitution of the 2-methyl-5-nitrobenzene ring, to
investigate the effect on p110α selectivity and potency. The sulfone-pyrazole linker group in
(5.5) was found to be critical, with alternative linker groups in the thiazole series SO2CH2 4.123,
CH2 4.122, CHOH 4.114 and linker absent 4.108 ablating activity, while activity was retained by
thiazole-CH2SO2 4.124.
As the complexes between the pyrido[1,2-a]pyrimidine and pyrazolo[1,5-a]pyridine chemotypes
with the active sites of p110β and p110α respectively are not known, docking simulations were
performed using structural p110β models and p110α (pdb:2RD0) respectively to understand the
molecular basis for the isoform selectivity exhibited by the two chemotypes. Suitable docking
methods were obtained by first investigating the ability of three docking protocols GOLD,
SURFLEX and AutoDock to find and correctly rank an experimentally derived conformation
both retrospectively (rescoring), where the compounds were docked back into the p110γ crystal,
and prospectively, where the ligands were docked into the apo p110α (2RD0).
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Patton, Daniel Timothy. "The role of PI3K p110δ in T cells." Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612110.
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Fedrigo, Carlos Alexandre. "Inibi??o da via PI3K-Akt em gliomas." Pontif?cia Universidade Cat?lica do Rio Grande do Sul, 2012. http://tede2.pucrs.br/tede2/handle/tede/1696.
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Previous issue date: 2012-05-29Glioblastoma multiforme (GMB) is the most malignant and common type of all astrocytic tumours. Current standard treatment for GBM patients involves maximum surgical resection of the tumour, followed by radiotherapy and chemotherapy, usually containing the alkylating agent Temozolomide (TMZ). Despite this aggressive combination therapy, the survival rate of GBM patients is still low. This work consisted in investigating the cytotoxic effects of Akt-inhibition by MK-2206 with irradiation (RT) and TMZ on in vitro human malignant glioma. Seven malignant glioma cell lines were cultured and tested for clonogenic survival, invasion inhibition, tumour spheroid growth and proliferation. The Akt-inhibitor MK-2206 and TMZ were added at different time treatments and in varying doses. Cultures were irradiated with single dose and with fractionated γ-irradiation. Cellular modulation of Akt and p-Akt were assessed by Western blot analysis. MK-2206 reduced the levels of phospho- Akt key protein in the PI3Kinase-Akt pathway, decreased cell survival, and inhibited invasion, proliferation and cell growth. The combination of MK-2206 and RT lead to enhanced inhibition of cell proliferation and invasion, which is not observed with RT alone. The radioenhancing effect of MK-2206 was most striking in inhibition of spheroid volume growth by fractionated RT; the radiosensitizing effect of MK-2206 was stronger than that of TMZ. MK-2206 enhanced the in vitro effects of RT and TMZ in terms of decreased cell survival, invasion, proliferation and growth in malignant glioma. Effects could be ascribed to inhibition of PI3K-Akt pathway
O Glioblastoma multiforme (GBM) ? o tipo mais maligno e mais comum de todos tumores astroc?ticos. O tratamento atual para pacientes de GBM envolve m?xima remo??o cir?rgica, seguida de radio e quimioterapia, normalmente com o agente alquilante Temozolamida (TMZ). Apesar da agressividade da terapia combinada, o tempo de sobreviv?ncia dos pacientes ainda ? baixo. Este trabalho procurou investigar os efeitos citot?xicos do inibidor de Akt MK-2206 em combina??o com irradia??o (RT) e TMZ em um painel de c?lulas de gliomas humanos. Sete linhagens de glioma foram cultivadas e testadas em ensaio de sobreviv?ncia clonog?nica, inibi??o de invas?o, e modelos de prolifera??o e crescimento de volume em esfer?ides. O inibidor MK-2206 e TMZ foram adicionados em diferentes tempos de tratamento e diferentes doses. As culturas foram irradiadas com doses ?nicas ou em terapias fracionadas com irradia??o γ. A modula??o celular de Akt e fosfo-Akt foi checada via Western Blot. O composto MK-2206 reduziu a fosforila??o da prote?na chave Akt na via PI3K, diminuindo a sobreviv?ncia celular e inibindo invas?o, prolifera??o e crescimento celular. A combina??o de MK-2206 com RT levou a uma maior inibi??o de invas?o e prolifera??o, o que n?o ? observado somente com a RT. O efeito radiosens?vel de MK-2206 foi ainda maior na inibi??o do volume dos esfer?ides em terapia combinada com RT fracionada, sendo ainda maior do que o efeito combinado com TMZ. MK-2206 aumentou os efeitos in vitro de RT e TMZ em termos de redu??o de sobreviv?ncia celular, invas?o, prolifera??o e crescimento celular em gliomas malignos. Os efeitos podem ser atribu?dos a inibi??o da via PI3KAkt
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López, Fauqued Marta. "Preclinical Study of PI3K and BRAF Inhibitors in Malignant Melanoma / Estudio preclínico de inhibidores de PI3K y BRAF en melanoma maligno." Doctoral thesis, Universitat de Barcelona, 2010. http://hdl.handle.net/10803/1040.
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Malignant melanoma is the most lethal skin cancer with no effective therapeutic treatment in its metastatic stages. RAS and PI3K pathways have been shown to play a critical role in melanoma development and progression. In this study, we assessed the in vitro and in vivo inhibition potential of a BRAF inhibitor (Sorafenib, Bayer) and a PI3K/mTOR inhibitor (PI-103, PIramed-Genentech) in primary melanoma cell lines. We used primary cell lines isolated from spontaneous melanomas obtained in the UV induced HGF transgenic melanoma mouse model.Although PI-103 and sorafenib inhibited melanoma in vitro cell proliferation and viability, the inhibition of RAS pathway was more effective. The combination of the two drugs showed a synergistic effect inhibiting RAS and PI3K pathways and in vitro melanoma cell proliferation in a cell line dependent manner. However, the combined treatment of orthotopic xenographs in immunocompetent FVB mice did not cooperate blocking tumor growth. Surprisingly, the in vivo treatment with PI-103 enhanced tumor growth. Our results also revealed that PI-103 caused immunosuppression inducing thymus atrophy and upregulating the intratumoral transcriptional levels of inmunosuppressors. In addition, PI-103 induced the antiapoptotic BH3 family proteins Mcl-1, Bcl-2 and BclXL, which correlated with the lower apoptotic rate observed within the PI-103 treated tumors.
These data indicates that due to melanoma heterogeneity, some precautions should be taken when using these inhibitors for treatment. Moreover, these results certainly make an argument for investigating unexpected effects of rational drug combinations on immunocompetent animal models before conducting clinical studies.
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Duarte, Andressa. "Participação da via PI3K/AKT na produção de óxido nítrico por macrófagos peritoneais." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/17/17147/tde-21102013-112320/.
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A imunidade inata é responsável pela resposta inicial aos microrganismos, uma vez que impede, controla ou elimina a infecção. Esse sistema consiste em barreiras epiteliais, proteínas plasmáticas e células circulantes e teciduais. Dentre esses componentes, os macrófagos possuem grande importância, sendo capazes de controlar e eliminar agentes patogênicos através da fagocitose e produção de espécies reativas de oxigênio e nitrogênio. A ativação de PRRs por constituintes oriundos dos patógenos em macrófagos desencadeia eventos da resposta imune inata, ativados por diversas vias de sinalização intracelular. A via das PI3Ks é conhecida por regular várias funções nas células, como a regulação do ciclo celular, migração e produção de espécies reativas de oxigênio e nitrogênio. O NO é um mediador central na imunidade inata que, após estímulos inflamatórios, é produzido em altas quantidades através da iNOS. Macrófagos deficientes em PI3K produzem menos NO e apresentam prejudicado controle da infecção quando infectados por T. cruzi. O objetivo do presente trabalho foi investigar o papel da via PI3K na produção de NO por macrófagos peritoneais estimulados com LPS. Os macrófagos empregados no estudo, WT e PI3K-/-, possuem o mesmo fenótipo. Observamos que macrófagos PI3K-/- possuem uma menor produção de NO e expressam menos iNOS. A reduzida expressão de iNOS, após estímulo com LPS, é também observada quando macrófagos WT são tratados com inibidores seletivos da PI3K e AKT. Além disso, demonstramos que, concomitantemente à menor expressão da iNOS, ocorre deficiência na fosforilação da AKT e diminuição da ativação do fator de transcrição NF-kB, sugerindo que a PI3K participa da ativação do NF-kB. Foi observado ainda que o tratamento com PTX também diminui a expressão da iNOS. No entanto, macrófagos PAFR-/- expostos ao LPS presentam maior expressão da iNOS, enquanto os macrófagos CCR2-/- apresentam menor expressão dessa enzima nessas condições. Para investigar a implicação da via PI3K in vivo foi administrado LPS i.v., como modelo de choque endotoxemico, no qual observamos maior sobrevida em animais PI3K-/- comparado aos animais WT e menores níveis de nitrito no soro. Nossos dados sugerem que a enzima PI3K é crítica para expressão de iNOS e produção de NO pelos macrófagos, possivelmente através da ativação do receptor CCR2, estando envolvida na fisiopatologia do choque induzido por LPS.Innate immunity is the initial response to microorganisms, since it prevents, controls and eliminates infection. This system consists in epithelial barriers, plasma proteins and circulating and tissue cells. Among these components, macrophages have great importance, being capable of control and eliminate pathogen agents through phagocytosis and production of reactive oxygen and nitrogen species. Activation of PRRs by pathogens constituents in macrophages triggers events of the innate immune response, activated by various intracellular signaling pathways. PI3Ks pathway is known to regulate several functions in the cell, such as regulation of the cell cycle, migration and production of reactive oxygen and nitrogen species. NO is a central mediator in innate immunity, which after inflammatory stimuli, is produced in high levels by iNOS. PI3K-deficient macrophages produce less NO and exhibit impaired control of infection when infected by T. cruzi. The aim of the present study is to investigate the role of PI3K pathway in NO production by LPS-estimulated peritoneal macrophages. The macrophages used in this study, WT and PI3K- / -, have the same phenotype. We observed that PI3K- / - macrophages have a lower NO production and express less iNOS. The low expression of iNOS after stimulation with LPS was also observed in WT macrophages treated with selective inhibitors of PI3K and AKT. Furthermore, we demonstrate that, along to lower iNOS expression, there is deficiency in AKT phosphorylation and decreased activation of the transcription factor NF-kB, suggesting that PI3K participates of the NF-kB activation. It was also observed that PTX treatment has decreased iNOS expression. However, LPS-exposed PFAR-/- macrophages present greater expression of iNOS, while CCR2-/- macrophage exhibit lower expression of this enzyme under these conditions. To investigate involvement of the PI3K pathway has \"in vivo\",LPS was administered i.v., as an endotoxic model, in which we observed a higher survival in PI3K- / - animals compared to WT animals and lower nitrite levels in serum. Our data suggest that PI3K enzyme is critical to iNOS expression and NO production by macrophages, possibly through activation of the CCR2 receptor, being involved in the LPS-induced shock pathophysiology
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Chu, Ying Ying Julia. "Apoptosis is promoted by unconventional FcγR-PI3KCdc42-Pak-Mek-Erk signalling in the human neutrophil." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28813.
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Neutrophils form a first line of defence against infections. These short-lived, terminally differentiated cells perform many important functions, including chemotaxis, degranulation, reactive oxygen species (ROS) release and cytokine production. Whilst neutrophils are essential for host immunity, their inappropriate recruitment, activation and/or removal can contribute to excessive inflammation and host damage, as exemplified in autoimmune diseases such as rheumatoid arthritis. It is therefore essential that neutrophil function is tightly regulated. Neutrophils are activated by a range of stimuli, including immune complexes. Neutrophil functions are tightly regulated by intracellular signalling events that are induced by the ligation of cell surface receptors, for example, the binding of immune complexes to Fc receptors. Phosphoinositide 3-kinase (PI3K) and extracellular signal-regulated kinase (Erk) are key signalling intermediates that act downstream of many cell surface receptors. They are involved in the regulation of numerous biological processes in the neutrophil. Using pharmacological interventions, I analysed PI3K signalling in immune complex-stimulated human neutrophils and uncovered a previously uncharacterised, noncanonical signalling pathway, PI3K-Cdc42-Pak-Mek-Erk. This represents an unusual situation where Pak acts as the MAP3K downstream of Cdc42 in a PI3K-dependent fashion. By performing a range of functional experiments, I showed that this unconventional signalling pathway promotes apoptosis in human neutrophils by regulating the ratio between anti- and pro-apoptotic members of the Bcl-2 family proteins. No other immune complex-induced, PI3K-dependent neutrophil function tested depended on PI3K-Cdc42-Pak-Mek-Erk signalling. Mouse knock-outs of all components of this signalling pathway have been described. Immune complex-induced apoptosis was also PI3K-dependent in mouse neutrophils, but experiments performed with inhibitors showed that, in contrast to human neutrophils, this was not dependent on PI3K-Cdc42-Pak-Mek-Erk signalling. The myeloid leukaemia cell line, PLB-985 is amenable to knock-down and can be differentiated to become neutrophil-like. These cells are not notably activated by immune complexes, perhaps because they do not express the major Fcγ receptor, CD16. Since retroviral expression of CD16 in PLB985 cells did not improve their response to activation by immune complexes, I was not able to confirm my observations with human neutrophils genetically. Collectively, I showed that a novel, pro-apoptotic signalling pathway operates downstream of Fcγ receptors in the human neutrophil. The fact that this signalling pathway appears to regulate apoptosis specifically suggests uncoupling pro- and anti-inflammatory effects induced by immune complexes might be possible. This may be helpful in the design of improved therapies of autoimmune diseases such as rheumatoid arthritis, in which immune complex-driven neutrophilic inflammation contributes to disease pathogenesis and where neutrophil apoptosis is disturbed.
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Ma, Kewei. "Investigation of the phosphatidylinositol 3-kinase pathway in B cells." Thesis, University of British Columbia, 2009. http://hdl.handle.net/2429/3818.
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There is hardly a cellular process that is not regulated in some way by phosphoinositides, which makes biochemical and physiological studies of these lipids extremely important. PI 3-kinases are key regulators of phosphoinositide metabolism and have been shown to affect a large variety of cellular responses. The key products of PI 3-kinases that have functional activity in higher eukaryotic cells are PI(3,4,5)P₃ and PI(3,4)P₂. PI(3,4,5)P₃ is universally accepted as one of the most important second messengers in signal transduction. However, our knowledge of the functions of PI(3,4)P₂ as a lipid second messenger is much less precise. In this dissertation, work was undertaken to elucidate the regulation of PI(3,4,5)P₃ and PI(3,4)P₂ production and downstream signaling in B cells. Cells with membrane targeted exogenous SHIP were utilized to manipulate phosphoinositide levels. The relationship of PI(3,4,5)P₃ and PI(3,4)P₂ levels to downstream PKB phosphorylation and activation was studied. PI(3,4,5)P₃ and PI(3,4)P₂ levels were found to closely correlate with PKB phosphorylation levels at Thr308 and Ser473, respectively. In addition, PI(3,4)P₂ levels determine the PKB activity in the cytosol; while PI(3,4,5)P₃ levels determine PKB activity at the plasma membrane. Different doses and different forms of B cell receptor (BCR) agonists were used for stimulation. PI 3-kinase activation was studied carefully following stimulation with low doses of anti-BCR antibody and F(ab')₂ fragments. Low concentrations of F(ab')₂ fragments produced higher levels of PI(3,4,5)P₃ than did a high concentration of F(ab')₂ fragments. Downstream PKB signaling was studied in these models. Similar conclusions were drawn from both SHIP over-expressing BJAB cells and dose-dependent BCR stimulations. We speculated that phosphoinositides’ regulation of the kinetics of PKB phosphorylation at Ser473 and Thr308 might be mediated by additional proteins. Investigation of plasma membrane-associated PKB showed that it formed a protein complex of around 400KD, which we attempted to characterize further with respect to PKB phosphorylation and association with lipids. In conclusion, phosphoinositide production is intricately regulated in vivo to control downstream signaling. The levels of PI(3,4)P₂ and PI(3,4,5)P₃ have precise and profound effects on PKB and other molecules such as TAPP and Bam32. This study has contributed new insight into the PI 3-kinase signaling pathway from the aspect of phosphoinositide lipid function.
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Boller, Danielle. "The role of PI3K signaling in neuroblastoma and glioblastoma /." Zürich, 2008. http://opac.nebis.ch/cgi-bin/showAbstract.pl?sys=000253047.
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Sears, Daniel. "Identification of PI3K/Akt targets in chronic myeloid leukaemia." Thesis, Imperial College London, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.505451.
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Hervieu, Vilches Alexia. "Understanding and targeting PI3K downstream of oncogenic Met mutant." Thesis, Queen Mary, University of London, 2015. http://qmro.qmul.ac.uk/xmlui/handle/123456789/33935.
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The Receptor Tyrosine Kinase (RTK) Met, overexpressed or mutated in cancer, plays a major role in cancer progression and represents an attractive target for cancer therapy. This study aimed to investigate whether PI3K plays a role in Met oncogenicity. Three cell models were used: (i) NIH3T3 cells expressing WT Met or the constitutively active mutant M1268T Met; (ii) U87MG glioblastoma cells, with endogenous WT Met constitutively activated due to an autocrine loop; (iii) A549 lung cancer cells expressing endogenous WT Met, activated upon binding exogenous HGF. Met dependent Rac1 translocation to the plasma membrane, actin cytoskeleton organisation, cell migration, anchorage independent growth in soft agar and tumour growth were studied in the presence of inhibitors of pan-PI3K / mTOR, various PI3K Class I isoforms, mTOR or Akt, or following siRNA knock-down of PI3K isoforms. We report that PI3K class I (but not class III) regulates Met dependent cell migration. The PI3K class I isoforms required varies among the cell models. Interestingly, the combined inhibition of all p110 Class I isoforms lead to the strongest reduction of Met dependent cell migration. Met dependent phosphorylation of Akt, an effector of PI3K class I, is reduced upon endocytosis inhibition, suggesting that Met signals to PI3K Class I on endosomes. Our results indicate that mTOR is responsible for Met dependent anchorage independent growth and tumour growth in vivo. Surprisingly, PI3K class I (and class III) are not required. Moreover, Rac1 is required for Met dependent mTOR activation, (phosphorylation of mTORC1's effector, p70 S6K) subcellular translocation of mTOR and anchorage independent growth. Finally, our results suggest that this Met-Rac1- mTOR pathway occurs on endosomes. Thus while PI3K class I regulates Met dependent cell migration, mTOR regulates Met driven anchorage independent growth and in vivo tumorigenesis. Thus PI3K Class I / mTOR may be targeted in Met driven cancers.
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Nunes, Tinoco Nunes. "Estudo funcional da via PI3K/AKT em Aedes aegypti." Botucatu, 2018. http://hdl.handle.net/11449/157112.
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Orientador: Jayme Augusto de Souza NetoResumo: 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
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Pothiraju, Deepika [Verfasser]. "Targeting PI3K/mTOR pathway in hepatocellular carcinoma / Deepika Pothiraju." Hannover : Technische Informationsbibliothek und Universitätsbibliothek Hannover (TIB), 2011. http://d-nb.info/1013286243/34.
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Torroba, Balmori Mª Blanca. "The Multiple Tasks Endured by PI3K during neural tube development." Doctoral thesis, Universitat de Barcelona, 2016. http://hdl.handle.net/10803/399595.
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Development of the spinal cord involves coordination between exposure to localized extracellular signals and controlled activation of intracellular signaling pathways. This way, neuroepithelial cells firstly proliferate apically to increase the progenitor pool and, later on, initiate neurogenic divisions giving rise to a variety of neuronal cell types. Class IA PI3Ks are heterodimeric enzymes (catalytic+regulatory subunits) activated by receptors tyrosine kinase (RTKs) or G protein coupled receptors (GPCRs) that, upon extracellular stimuli, modulate diverse target proteins through local production of PtdIns(3,4,5)P3 lipids. Vertebrates express three Class IA catalytic subunits (p110-alpha, p110-beta, and p110-delta), all important for the development of the central nervous system. However, it is unclear to what extent these p110-alpha isoforms have overlapping or distinct biological roles, and what exact functions they hold in neural development.
Analysis of PI3Kalpha (p110-alpha-alpha+regulatory subunit) expression in the embryonic spinal cord revealed abundant mRNA and protein levels in cycling progenitors followed by restriction of PI3K-alpha exclusively to differentiating neurons. To examine the role of PI3K-alpha in progenitors and neurons, we interfered with normal PI3K-alpha regulation by expressing active mutants or knocking down of p110-alpha-alpha in the chicken neural tube. Loss of p110-alpha resulted in high apoptotic rates in both progenitors and neurons, sustaining a role for PI3K-alpha in neural survival as seen in other studies. Instead, uncontrolled upregulation of PI3K-alpha activity resulted in severely disrupted neural tubes, with abnormal cell masses in the luminal face of the neuroepithelium and ectopic mitosis. Additionally, we observed alterations in the neural lamination characterized by basement membrane breaches followed by enhanced neural migration and misoriented axonal growth. A thorough analysis of the tissue unveiled loss of polarity as the main cellular mechanism driving the luminal structural aberrations, suggesting a major role of PI3K-alpha in neuroepithelial apico basal polarity. Moreover, the rescue of the depolarization phenotype with a dominant negative form of RhoA proposes local regulation of the Rho family of small GTPases as the molecular mechanism responsible for the PIP3 dependent regulation of adherens junction dynamics. Alternatively, we found the neural overmigration caused by excess of PI3K-alpha activity explained by increased basal accumulation of PIP3, leading to actin based membrane protrusions and basement membrane breaches. Coherently, when we assessed the neural positioning after p110-alpha knock down, we detected neurons inserted in the proliferative layer and reduction of the neuronal cytoskeletal component beta III tubulin, suggesting that PI3K-alpha also modulates morphological maturation and apico basal positioning of differentiating neurons. Interestingly, PIP3 induced overmigration seemed to be carried out through local activation of other two members of the Rho GTPases, Cdc42 and Rac1. These results shed some light upon the PI3K-alpha/PIP3 specific roles during early neural tube development, stressing out its function in cell polarity. Furthermore, we propose a mechanism that may partially explain how the PI3K-alpha /PIP3 signaling is able to control different types of polarity corresponding to different developmental moments. This could help to understand the initial events leading to some neurodevelopmental disorders caused by hyperactivation of PI3K signaling.El desarrollo de la médula espinal requiere una fina coordinación entre señales extracelulares y la activación de vías intracelulares específicas. De este modo se da una primera fase de proliferación de las células neuroepiteliales en la zona apical para aumentar el número de progenitores y una segunda fase de neurogénesis, a partir de la cual se originan diferentes tipos de neuronas. La clase IA de las PI3Ks se encuentra implicada en la transducción de señales a través de receptores tirosina quinasa (RTKs) o receptores acoplados a proteínas G (GPCRs). En respuesta a estímulos extracelulares, controlan la actividad de distintas proteínas diana a través de la producción local de lípidos PtdIns(3,4,5)P3. La clase IA de las PI3Ks, formada por enzimas heterodiméricas, consta de tres tipos de subunidades catalíticas (p110-alfa, p110-beta y p110-delta). Todas ellas son importantes para el desarrollo del sistema nervioso, sin embargo no están claras las funciones específicas de cada isoforma. El análisis de la expresión de PI3K a nivel de RNAm y proteína en la médula espinal embrionaria reveló una expresión diferencial según el estadío, siendo alta en progenitores antes de la neurogénesis y restringida a neuronas en estadíos más tardíos. Para estudiar su función en progenitores y neuronas, transfectamos formas activas de PI3K-alfa o suprimimos transitoriamente la p110-alfa en el tubo neural de embriones de pollo. La pérdida de p110-alfa provocó una alta tasa de apoptosis en ambas poblaciones, revelando su importancia en supervivencia. La sobreexpresión de la PI3K-alfa activa, en cambio, generó disrupciones muy severas del tejido neural caracterizadas por la presencia de masas celulares en la pared ventricular y mitosis ectópicas. En el lado basal, se observaron alteraciones en la laminación neuronal con células atravesando la lámina basal y crecimiento axonal aberrante. Nuestros resultados apuntan hacia la pérdida de polaridad como la principal causa de las aberraciones estructurales apicales, indicando que la PI3K-alfa tiene una función en la regulación de la polaridad apico basal. Asimismo, la PI3K-alfa parece implicada en la maduración del citoesqueleto neural y en el posicionamiento de las neuronas en el eje apico basal, funciones parcialmente mediadas por miembros de las Rho GTPasas.
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Guerrero, Hernández Martina. "Targeting tumor microenvironment crosstalk through GPCR receptors and PI3K pathway." Doctoral thesis, Universitat de Barcelona, 2019. http://hdl.handle.net/10803/667975.
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The tumor microenvironment (TME) is gaining momentum due to its contribution to cancer progression and therapy resistance. This TME has a direct crosstalk with tumor cells that involves the activation of different pathways.
Follicular lymphoma (FL) is the most common indolent non-Hodgkin lymphoma. Although FL is generally characterized by slow progression and high response rates to therapy, it is still considered incurable, because almost virtually all the patients relapse. FL is probably the NHL with the highest dependence on microenvironment. PI3K is a common denominator transducing the signaling from FL crosstalk with the TME and plays an important role in multiple cellular functions, and also contributes to cancer promoting aspects of the TME, such as angiogenesis and inflammatory cell recruitment. Idelalisib is a first-in-class δ isoform- specific PI3K inhibitor that receive regulatory approval for relapsed CLL, SLL and FL in 2014. Idelalisib blocks PI3K δ which is restricted to leukocytes. BCL2 deregulation is paramount in the pathogenesis of FL, as a consequence of the t(14;18), and therefore it is an attractive target for novel therapeutic approaches. Venetoclax (ABT-199, AbbVie) is a small BCL-2 inhibitors. Even though 85% of FL patients harbor the t(14;18), the results of the first clinical trial with venetoclax were not satisfactory (overall response 38%). From this first study we conclude that Idelalisib modulates key pathways in the germinal center and shapes the FL immune microenvironment by decreasing the recruitment of TFH and Treg to the tumor site leading to less immunesuppresive phenotype. Furthermore Idelalisib induces a moderate cytotoxic effect on FL cells in co-cultures. This co-culture decrease FL dependence on BCL-2 and consequently, venetoclax cytotoxicity, but Idelalisib sensitizes FL co-cultures to venetoclax.
In summary, Idelalisib interferes with the crosstalk of FL and its immune microenvironment and potentiates the activity of venetoclax targeting the tumor cells, thus representing a promising combination therapy that may improve FL outcome.
Colorectal cancer (CRC) is the third most common cancer in males and the second in females, and the fourth most common cause of cancer-related death worldwide. Patients with advanced and distal metastatic disease (stage IV), the survival rate drops to 10%, which accounts for approximately 18% of cases. The TME in CRC, is a complex structure composed by different type of cells, which are interacting each other’s and secreting a variety of growth factors and other molecules, such as cytokines and chemokines. Tumor development is based on the crosstalk between tumor cells and their surrounding microenvironment, and this crosstalk is mediated by the receptors and its ligand expression in both types of cells. G
protein-coupled receptors (GPCRs) are an important family of membrane signaling receptors, which have an important role in cancer growth and development. Originally, GPCRs were considered as monomeric functional entities, nevertheless, in recent years has become evident that GPCRs form dimers and this dimers formation may modify the cellular response. In cancer, CXCR4 (has been studied extensively) plays an important role at different stages of cancer development, and is involved in the metastasis process of tumor cells. The up- regulation of CXCR4 in CRC correlates with a poor prognosis. Another GPCR, CB2 receptor modulates the downstream signaling and it is able to activate a wide range of signaling pathways, including extracellular signal-regulated kinases 1/2 (ERK1/2). In CRC, it has been described an up-regulation of CB2 receptor expression. GPCRs show differential expression in cancer cells and tissues, and they are highly druggable sites. From this second study we concluded that CXCR4 and CB2 expression is increased in primary colon tumor cells and in metastasis cells compared to normal epithelial cells from colon mucosa, and they formed heterodimers in colon tumoral cells and are associated with more aggressive phenotypes. Moreover, a bidirectional cross-antagonism crosstalk is established between these receptors. These heterodimers regulate in vitro CXCL12-induced migration, and in vivo, the simultaneous inhibition of both receptors shows superior anti-tumoral and anti-metastatic activities than the single agent inhibition.
In summary, targeting the heterodimerization of CXCR4 and CB2 that are biologically relevant in cancer can be an effective way to reduce proliferation and dissemination in CRC.El estudio del microambiente tumoral está ganando importancia en las últimas décadas debido a su contribución en la formación y desarrollo del cáncer, además de contribuir en la resistencia de las células tumorales a diferentes terapias. Este microambiente interactúa con las células tumorales y activa diferentes vías. El linfoma folicular (FL), es el linfoma no Hodgkin indolente más común y con mayor dependencia del microambiente tumoral, además es considerado incurable. PI3K desempeña un papel importante en la comunicación con el microambiente, y es importante en múltiples funciones celulares, además de contribuir en la angiogénesis, reclutamiento de células inflamatorias y promover el crecimiento tumoral. Idelalisib es un inhibidor de PI3K (específicamente de la isoforma δ), que se aprobó en 2014 por la FDA. Paralelamente la desregulación de BCL2 es primordial en la patogénesis de FL, como consecuencia de la t (14; 18), presente en un 85% de los pacientes, y por lo tanto es un objetivo atractivo para novedosos enfoques terapéuticos. Venetoclax (ABT-199, AbbVie) es un pequeño inhibidor de BCL2, que mostró unos resultados del primer ensayo clínico no satisfactorios (respuesta global del 38%). De este primer estudio concluimos que Idelalisib interfiere en la comunicación de FL y su microambiente inmune, además potencia la actividad de venetoclax atacando a las células tumorales, lo que representa una terapia de combinación prometedora que puede mejorar el resultado del tratamiento de FL.
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Körtl, Thomas [Verfasser], and Sven A. [Akademischer Betreuer] Lang. "PI3K-Inhibition im Pankreaskarzinommodell / Thomas Körtl ; Betreuer: Sven A. Lang." Regensburg : Universitätsbibliothek Regensburg, 2019. http://d-nb.info/1199104353/34.
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Dobler, Melanie. "Analyse des PI3K-Signalwegs bei der Entstehung des duktalen Pankreaskarzinoms." Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-172239.
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Cizkova, Magdalena. "Pronostic and Predictive Markers in Breast Cancer - PI3K Signaling Pathway." Thesis, Paris 11, 2013. http://www.theses.fr/2013PA11T021.
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Les résultats des projets actuels apportent une information, sur différents aspects des rôles de la voie PI3K, dans le développement du cancer du sein, et la réponse au traitement. Les projets particuliers couvrent des sujets liés à la voie aux niveaux concernant les récepteurs de la famille HER, activant la voie PI3K, ainsi que PI3K et les effecteurs en découlant. Les effets pronostic et prédictif de la dérégulation de PI3K sont les sujets centraux de la recherche décrite ici. Une baisse d’expression de PI3KR1 est associée à une survie réduite dans notre cohorte de patients. Une attention particulière a été portée aux mutations de PIK3CA communes dans le cancer du sein. Tandis que les mutations de PIK3CA agissent comme des marqueurs de bon pronostic chez les patients anti-HER2-naïfs, ces mutations agissent au contraire comme prédicteurs négatifs de la réponse au traitement par trastuzumab. Les résultats décrits mènent un peu plus vers l’implication de plusieurs voies moléculaires altérées, en particulier la voie de signalisation Wnt, dans la tumorigénèse des cancers du sein PIK3CA mutés. De plus, nous avons testé les taux de lapatinib plasmatique montrant une augmentation pertinente dans les périodes d’état d’équilibre du traitement. Par ailleurs, nous avons démontré des incohérences dans l’évaluation de l’EGFR et proposé des approches pour l’interprétation des comptages d’immunohistochimie et de FISH. Tous ces sujets sont connectés par la 170 voie PI3K, et le besoin d’approfondir les connaissances actuelles, et d’apporter de nouvelles informations utiles applicables dans le futur dans les pratiques cliniquesResults of the presented research projects bring information about several aspects of the PI3K signaling pathway roles in breast cancer development and treatment response. The particular projects covered the subjects connected with the signaling pathway, ranging from the HER family receptors activating the pathway, and PI3K to the downstream levels of signalisation. The prognostic and predictive effect of PI3K deregulation was the central subject of the described research. The decreased expression of PIK3R1 associated with reduced survival of our patients. A special focus was put on the PIK3CA mutations which are common in breast cancer. Whereas the PIK3CA mutations act as a good prognostic marker in patients non-treated with the HER2 inhibitors, these mutations predict a negative response to trastuzumab treatment. The described results, furthermore, draw attention to the role of several altered molecular signaling pathways in breast cancer development, especially to the Wnt signaling pathway. The lapatinib plasma levels showing the relevant increase in comparison with the already described efficient steady-state levels were also described in one of the projects. Moreover, various modifications to EGFR status assessment were compared and showed that EGFR FISH and IHC count interpretation depended significantly on method and thresholds used. All these subjects are connected by the PI3K pathway, the need to deepen current knowledge and bring new useful information applicable in future clinical practice
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Stamatkin, Christopher W. "PHOSPHATIDYLINOSITOL 3-KINASE (PI3K) AS A THERAPEUTIC TARGET IN NSCLC." UKnowledge, 2014. http://uknowledge.uky.edu/pharmacy_etds/58.
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Deregulated activation of phosphatidylinositol 3-kinase (PI3K) pathway is central to many human malignancies. The functions of this pathway are critical for normal cell metabolism, proliferation, and survival. In lung cancers, the PI3K pathway activity is often aberrantly driven by multiple mutations, including EGFR, KRAS, and PIK3CA. Molecules targeting the PI3K pathway are intensely investigated as potential anti-cancer agents. Although inhibitors of the pathway are currently in clinical trials, rational and targeted use of these compounds, alone or in combination, requires an understanding of isoform-specific activity in context. We sought to identify class IA PI3K enzyme (p110a/PIK3CA, p110b/PIK3CB, p110d/PIK3CD) activities using isoform-specific inhibitors in a lung cancer model system. Treatment of non-small cell lung cancer (NSCLC) cell lines with PIK3CA, PIK3CB, PIK3CD or PIK3CB/D inhibitors resulted in pharmacokinetic and pharmacodynamic responses that frequently tracked with a specific mutation status. Activation of PIK3CA dictated response to the PIK3CA-specific inhibitor while deletion of PTEN phosphatase indicated response to the PIK3CB inhibitor. The PIK3CD isoform-specific inhibitors lacked efficacy in all NSCLC cell lines tested, however treatment at increased concentrations likely provide concurrent inhibition of both PIK3CB/D isoforms improving activity of either agent alone but did not track with a single biomarker. The observed pharmacodynamic and proliferation responses to isoform-specific inhibitors suggested that PI3K isoforms may functionally compensate for loss of another in certain genetic backgrounds. These studies demonstrate unanticipated cellular responses to PI3K isoform inhibition in NSCLC, suggesting that patient populations with specific mutations can benefit from certain isoform-selective inhibitors, or combinations, allowing for rational and targeted clinical use of these agents.
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Wang, Ling, and Shunbin Ning. "LMP1 Signaling Pathway Activates IRF4 through the PI3K-Src Axis." Digital Commons @ East Tennessee State University, 2017. https://dc.etsu.edu/etsu-works/6544.
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Tavelis, Christodoulos. "Investigating the potential role of PIP4Ks in PI3K/Akt signalling." Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/investigating-the-potential-role-of-pip4ks-in-pi3kakt-signalling(e70d9473-5932-468a-bdad-01668a68db58).html.
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Class I phosphoinositide 3-kinases (PI3Ks) generate the essential lipid second messenger PtdIns(3,4,5)P3 which plays a key role in the regulation of numerous cellular processes including cell growth and survival, gene transcription, cytoskeletal organisation and glucose metabolism through its downstream effectors and in particular the serine/threonine protein kinase Akt. Therefore, the PI3K/Akt signalling plays a critical regulatory role in diverse cellular processes and its dysregulation is implicated in the pathogenesis of many human diseases including cancer and type 2 diabetes. Overexpression of phosphatidylinositol-5-phosphate 4-kinase β (PIP4Kβ), a lipid kinase which phosphorylates the poorly understood phosphoinositide phospholipid PtdIns5P to produce PtdIns(4,5)P2, has been demonstrated to lead to the attenuation of PtdIns(3,4,5)P3 levels and decreased Akt phosphorylation in insulin-stimulated cells. Conversely, mice lacking PIP4Kβ exhibit increased insulin-induced Akt phosphorylation, suggesting a potential role of PIP4Ks in the regulation of PI3K/Akt signalling. The aim of this project was to investigate the potential role of PIP4Kα, the most active isoform among PIP4Ks, in the regulation of PI3K/Akt signalling and whether its substrate, PtdIns5P, is involved in this regulation.While YM201636, an inhibitor of PIKfyve an enzyme believed to be involved in PtdIns5P production, markedly reduced Akt phosphorylation in PDGF-stimulated NIH/3T3 cells, contrary to expectations, overexpression of PIP4Kα in NIH/3T3 and HeLa S3 cells had no effect on PtdIns(3,4,5)P3 levels or Akt phosphorylation upon stimulation with PDGF and IGF-1 respectively. However, PtdIns(3,4,5)P3 levels were significantly decreased in insulin-stimulated L6 myotubes overexpressing PIP4Kα, indicating a possible cell type specific modulation of PI3K signalling by PIP4Kα. Interestingly, despite the decreased PtdIns(3,4,5)P3 levels, insulin-stimulated Akt phosphorylation remained unaffected in PIP4Kα expressing L6 myotubes. PIP4Kα overexpression also resulted in increased levels of PtdIns(3,4)P2. This suggests an increased 5-dephosphorylation of PtdIns(3,4,5)P3 through the action of one or more 5-phosphatases. Although the precise 5-phosphatase(s) are not known, the data indicate that this cannot be SHIP2 which has previously been implicated in the regulation of PtdIns(3,4,5)P3 levels in insulin signalling. Taken together, the data presented in this thesis indicate a role for PIP4Kα in PI3K signalling in a cell type specific manner. This might have important physiological implications.
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Fero, Daniel James. "The role of PI3K in Ephrin-A1 induced cell retraction." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2008. http://wwwlib.umi.com/cr/ucsd/fullcit?p3315045.
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Thesis (Ph. D.)--University of California, San Diego, 2008.Title from first page of PDF file (viewed August 4, 2008). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 104-113).
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Mammana, Santa. "Evaluation of the PI3K/Akt/mTOR pathway in Multiple Sclerosis." Doctoral thesis, Università di Catania, 2017. http://hdl.handle.net/10761/3951.
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Background
The PI3K/AKT/mTOR pathway is an intracellular signaling pathway that regulates cell activation. proliferation, metabolism and apoptosis. Increasing body of data suggests that alterations in the PI3K/AKT/mTOR pathway may result in an enhanced susceptibility to autoimmunity. Aim of our work was to evaluate the involvement of the mTOR pathway in the pathogenesis of autoimmune diseases, with particular focus on Multiple Sclerosis (MS). Multiple sclerosis (MS) is one of the most common chronic inflammatory diseases of the central nervous system leading to demyelination and neurodegeneration. Drugs targeting the PI3K/AKT/mTOR pathway are currently under extensive investigation for their possible use as cancer chemotherapics and as immunosuppressive agents, but no clinical trial has been so far approved for the evaluation of their efficacy in the context of immunological disorders.
Methods
In the current study, we have firstly evaluated in silico the involvement of the mTOR network on the generation and progression of MS, making use of currently available whole-genome transcriptomic data. Also, the involvement of the mTOR network on oligondendrocyte function was studied, in order to ascertain whether treatment with drugs targeting the PI3K/Akt/mTOR pathway may be useful to promote the remyelination process, so to reverse disability in MS patients.
Then, the data generated in silico were subjected to an ex-vivo evaluation. To this aims the involvement of mTOR was validated on a well-known animal model of MS and in vitro on Th17 cells.
Results
Our data indicate that there is a significant involvement of the mTOR network in the ethiopathogenesis of MS and that Rapamycin treatment may represent a useful therapeutic approach in the clinical setting. Ex vivo analysis showed that treatment of MOG-specific T cells from EAE affected mice with the mTOR inhibitor, Rapamycin, and the dual PI3K/mTOR inhibitor, BEZ-235, was able to significantly reduce antigen-specific proliferation. In addition, we show that Rapamycin treatment of murine T cell stimulated under Th17 conditions, was able to significantly inhibit the expression of some of the genes previously identified in the in silico analysis. On the other hand, our data showed that a significant involvement of the mTOR network could be observed only in the early phases of oligodendrocyte maturation, but not in the maturation process of adult oligodendrocytes and in the process of remyelination following demyelinating injury.
Conclusions
Overall, our study suggest that targeting the PI3K/mTOR pathway, although it may not be a useful therapeutic approach to promote remyelination in MS patients, it can be exploited to exert immunomodulation, preventing/delaying relapses, and to treat MS patients in order to slow down the progression of disability.
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Balista, Priscila Alves. "Inibidores de fosfatidilinositol-3-cinase (PI3K) e neuroproteção mediada pela cascata de sinalização da Akt na fase aguda do modelo de Pilocarpina." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/17/17140/tde-17022011-144706/.
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Introdução: A epilepsia do lobo temporal (ELT) é a forma mais frequente de epilepsia em adultos. Um modelo experimental de ELT consiste na indução de status epilepticus (SE) em animais por administração de Pilocarpina. Este modelo induz mudanças patofisiológicas e comportamentais em ratos muito semelhantes às observadas em seres humanos com ELT. Apesar da literatura apresentar dados relacionados às respostas celulares, pouco se conhece a respeito do envolvimento de cascatas de sinalização com insultos epileptogênicos no sistema nervoso. A enzima fosfatidilinositol-3-cinase (PI3K) está envolvida na ativação da cascata de sinalização intracelular da Akt. A Akt é uma proteína cinase especifíca de serina/treonina cuja forma ativa proporciona um controle no crescimento e proliferação celular, bem como induz um sinal de sobrevivência para a proteção de células contra a apoptose. Alguns estudos mostram que a ativação da PI3K é inibida por potentes drogas, tais como a LY294002 e a Wortmanina. A PI3K ativa a Akt e a cascata de sinais extra e intracelulares neuroprotetores pós-insultos. O estudo da ação de inibidores da PI3K em modelos de epilepsia pode fornecer dados sobre o envolvimento da Akt em sinais neuroprotetores. Objetivos: Avaliar o efeito do SE por injeção intra-hipocampal de Pilocarpina na ativação da Akt, bem como os efeitos do bloqueio desta cascata sobre as alterações patológicas observadas no hipocampo de ratos na fase aguda pós-SE. Metodologia: Ratos da cepa Wistar machos (250-300 g) foram divididos em grupos de tratamento, sendo tratados com injeções ipsilaterais na região posterior do hipocampo, com uma hora de intervalo entre drogas, das drogas: Salina e Pilocarpina (grupo Sal+Pilo), LY294002 e Pilocarpina (LY+Pilo) e Wortmanina e Pilocarpina (Wort+Pilo). No grupo considerado como controle foram injetadas as seguintes drogas: Salina e Salina (grupo Sal+Sal) ou Dimetilsulfóxido e Salina (grupo DMSO+Sal). O tempo de SE induzido por Pilocarpina foi fixado em 2 horas. Grupos de animais foram sacrificados nos instantes de 1 dia e 7 dias após o SE e seus encéfalos foram processados objetivando a imuno-histoquímica para NeuN, GFAP e Akt (pan). Resultados: A densidade neuronial na região do hilo do hipocampo foi menor no grupo Sal+Pilo, seguido dos grupos LY+Pilo e Wort+Pilo, avaliando-se os vários níveis de formação hipocampal e região posterior. Para a análise da astrogliose, na camada granular, o grupo LY+Pilo apresentou maior número de astrócitos positivos; na região do hilo hipocampal, os grupos LY+Pilo e Wort+Pilo apresentaram baixa expressão para a proteína Akt, comparados aos grupos Controles; assim como o grupo Wort+Pilo, em CA2, apresentou baixa expressão da proteína Akt somente na região posterior. Os animais Sal+Pilo sobrevida-7dias pós-SE revelaram maior expressão da Akt quando comparados com o grupo Sal+Pilo sobrevida-1dia pós-SE. Quanto à análise comportamental, o grupo Wort+Pilo apresentou maior latência para o início do SE que o grupo Sal+Pilo, não sendo observado diferença de severidade, uma vez atingido o SE. Conclusões: Na hipótese de trabalho, o uso de inibidores da fosforilação de Akt resultaria em maior morte neuronial, astrogliose e expressão inalterada da Akt. Ao contrário do esperado, os grupos que receberam injeções intra-hipocampais de inibidores de Akt, antes da indução de SE, exibiram menor perda neuronial, menor astrogliose e menor expressão da Akt para os vários níveis de formação hipocampal e no hipocampo posterior. Porém, o grupo Sal+Pilo sobrevida-7 dias pós-SE exibiu maior expressão para Akt quando comparado com o grupo Sal+Pilo de sobrevida-1 dia pós-SE. Além disso, o pré-tratamento com Wortmanina demonstrou um maior tempo de latência para o início do SE, o que nos sugere uma maior neuroproteção do que LY294002.Introduction: Temporal lobe epilepsy (TLE) is the most frequent type of adult human epilepsy. An experimental model of TLE, the Pilocarpine induced epilepsy, followed by pathofisiologic and behavioural alterations in rats resembling human diagnosis with TLE. Although there are several data on cell response in literature, few information exist on the cascade of signals involved in epileptogenesis process of central nervous system. The phosphatidylinositol-3-kinase (PI3K) is involved in activation of Akt intracellular signaling cascade. The serine/threonine kinase Akt, that in active form promote a control of growth and cell proliferation, such as survival sign protecting cells from apoptosis. Some studies have shown that activation of PI3K is blocked by potents pharmacological inhibitors, for example the LY294002 and Wortmannin. PI3K activate Akt and both extra and intracellular cascade signals involved in neuroprotection after seizures. The study of inhibitors mechanism in model of epilepsy can provide information on the involvement of Akt in signals of neuroprotection. Objectives: To evaluate the effects of SE by the intrahippocampal injection of Pilocarpine in Akt activation, and the effects of the blockade of this cascade on pathologic alterations observed in hippocampus of rats in acute phase after SE as well. Methods: Male Wistar rats (weighing 250 to 300 g) were divided in groups treated ipsilaterally with injections in the posterior region of hippocampus with an elapsed time of one hour subsequently after each injection of following substances: Physiological Saline and Pilocarpine (group Sal+Pilo), LY294002 and Pilocarpine (LY+Pilo), and Wortmannin and Pilocarpine (Wort+Pilo). Control groups were treated with the following drugs Saline + Saline (group Sal+Sal) or Dimethylsulphoxide + Saline (DMSO+Sal). A fixed time of 2 hours was considered for evaluating the SE induced by Pilocarpine. Animals were sacrificed 1 day and 7 days after SE, and their brains were processed imunohistochemically for NeuN, GFAP and Akt (pan) detecting. Results: The neuronal density in the hippocampal hilus was lower in the Sal+Pilo group, followed by LY+Pilo and Wort+Pilo groups, this evaluate various levels of hippocampal formation and posterior region. For the analysis of reactive astrogliosis of the granular cell layer, the LY+Pilo group presented a great number of GFAP-positive astrocytes. In the hilar region, the LY+Pilo and Wort+Pilo groups presented a reduced Akt expression compared to the Control group, such as the group Wort+Pilo in CA2, presented a reduced Akt expression only in posterior region. The Sal+Pilo animals with a survival time of 7 days after SE revealed higher Akt expression when compared to the Sal+Pilo animals with a survival time of 1 day. In behavioural analysis, the Wort+Pilo group presented major time of latency to SE than Sal+Pilo group, without differences in the disease severity, once reached the SE. Conclusions: On the contrary to the original hypothesis of this work, the use of inhibitors of Akt phosphorylating resulted in an unaltered neuronal death, astrogliosis and Akt expression. The groups that received intrahippocampal injection of Akt inhibitors before inducing SE exhibited a reduced neuronal loss, astrogliosis and Akt expression to the various levels of hippocampal formation and posterior region. But, to the Sal+Pilo group with a survival time of 7 days after SE induction exhibited greater Akt expression than Sal+Pilo group with a survival time of 1 day after SE induction. However, the pretreatment with Wortmannin displayed major time of latency to SE induction, suggesting this substance as a better neuroprotector than LY294002, according to the methodology applied in this study.
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Proctor, Victoria Kate. "Signalling pathways linking interleukin 13 receptor activation to lung epithelial cell function." Thesis, University of Bath, 2013. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.589658.
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The passage of fluid, ions and macromolecules across the epithelium is controlled primarily by epithelial tight junctions. Altered epithelial permeability is associated with lung disease, and barrier function is impaired by the Th2 cytokine IL-13. This thesis investigates the signalling pathways involved in the modulation of the epithelial barrier by IL-13 stimulation. Initial experiments demonstrated that the human sub-bronchial epithelial cell line Calu-3 could be easily manipulated when grown using an air-liquid culture system. Expression of various key tight junction proteins was demonstrated, as well as a high trans-epithelial resistance (TER) for up to 7 days. Stimulation with IL-13 resulted in a decrease in TER compared with controls and this decrease was shown to be prevented with the PI3K inhibitor ZSTK474. IL-13 did not increase paracellular permeability of the epithelial monolayer to FITC-dextran from the apical to the basolateral chamber and ZSTK474 did not influence FITC-dextran flux. Immunocytochemistry showed that the expression of the tight junction protein claudin 2 was increased by IL-13 stimulation and this change in expression was shown to be PI3K dependent with the PI3K inhibitor ZSKT474 preventing the increase. Further studies were carried out in an attempt to uncover the PI3K isoform responsible for the effects seen on both the TER and the TJ expression. It was shown that inhibition of the p110α isoform with PIK75 mimicked the result observed with the pan-PI3K inhibitor ZSTK474 and prevented the IL-13-induced claudin 2 upregulation. However none of the PI3K isoform inhibitiors showed the prevention of TER, as shown by the pan PI3K inhibitor ZSTK474. The role of STAT6 in TJ modulation was shown to be similar to that of PI3K, in that inhibition of STAT6 had a positive effect on the epithelial barrier by preventing the IL-13-induced TER decrease and the increase in the expression of claudin 2. In addition, both PI3K inhibition and STAT6 inhibition demonstrated effects on basal TER and claudin 2 expression, indicating that both pathways are involved in maintenance of epithelial barrier integrity.
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Levade, Marie. "Mécanismes moléculaires de la production et des fonctions plaquettaires : rôle de Vps34 et impact des inhibiteurs ciblés de kinases." Thesis, Toulouse 3, 2017. http://www.theses.fr/2017TOU30036.
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Les plaquettes sanguines jouent un rôle essentiel dans le maintien de l'intégrité des vaisseaux sanguins. En cas de brèche vasculaire, elles conduisent à la formation d'un clou hémostatique via des étapes successives d'adhésion, sécrétion et agrégation finement régulées et préviennent alors un saignement excessif. Les plaquettes jouent également un rôle critique dans les pathologies thrombotiques comme l'athérothrombose, ce qui en fait des cibles pharmacologiques majeures dans ces situations. Au cours de ma thèse, deux axes de recherche ont été abordés : (i) l'étude du rôle de la PI3-kinase de classe III (Vps34) dans la formation et l'activation des plaquettes et (ii) l'étude de l'impact de nouvelles drogues ciblant les kinases en thérapie anti-cancéreuse sur les fonctions plaquettaires et l'hémostase. Dans un premier temps, je me suis concentrée sur la caractérisation du rôle de Vps34 et de son produit lipidique, le phosphatidylinositol 3 monophosphate (PtdIns3P), dans la physiologie plaquettaire à l'aide d'un modèle de souris présentant une délétion de Vps34 spécifiquement dans la lignée mégacaryocyte/plaquette (PF4-Cre/Vps34lox/lox). Nous avons observé une microthrombopénie modérée associée à un défaut de migration des mégacaryocytes ainsi que des anomalies morphologiques des granules de sécrétion. Ce phénotype apparaît lié à une diminution de taux de PtdIns3P associé à un trafic vésiculaire perturbé. De plus, nous avons mis en évidence une altération des fonctions prothrombotiques des plaquettes, ex vivo en conditions de flux mais aussi in vivo en conférant aux souris une protection contre la thrombose induite à la carotide par lésion au chlorure ferrique. La contribution de Vps34 dans les mécanismes d'activation plaquettaire, indépendamment de son rôle dans le mégacaryocyte, a été montrée ex vivo via l'utilisation de nouveaux inhibiteurs spécifiques de Vps34 (SAR405 et INH1) récemment développés pour une application en oncologie, notamment pour réduire la résistance de certains cancers aux chimiothérapies. Dans un second temps, je me suis intéressée à l'impact des nouvelles thérapies ciblées anticancéreuses sur les plaquettes afin de comprendre la majoration du risque hémorragique associée à ces molécules. Nous avons étudié l'effet de l'ibrutinib, un inhibiteur des tyrosine-kinases de la famille BTK activées en aval des PI3-kinases de classe I, utilisé en clinique dans le traitement des hémopathies lymphoïdes B (lymphomes et leucémie lymphoïde chronique). L'exploration des fonctions plaquettaires au sein d'une cohorte de patients du service d'hématologie du CHU-Toulouse a permis de corréler les signes hémorragiques de certains patients traités par ibrutinib avec un défaut de signalisation plaquettaire en aval des récepteurs GPVI et GPIb, se traduisant par une diminution de l'agrégation au collagène et au CRP et par un défaut d'adhésion sur matrice de facteur von Willebrand en conditions de flux. En conclusion, mes travaux de thèse (i) apportent de nouvelles données fondamentales sur la participation de Vps34 dans les mécanismes de production et d'activation plaquettaires et (ii) ont permis de proposer des recommandations quant à l'utilisation clinique des nouvelles thérapies ciblées anti-cancéreusesBlood platelets play an essential role in the maintenance of vascular integrity. They prevent excessive blood loss after vessel injury by orchestrating haemostatic clot formation through successive steps of adhesion, secretion and aggregation. Platelets are also major pharmacologic targets as they participate in thrombotic pathologies such as atherosclerosis. My thesis work was focused on two axis: (i) the role of class III PI3-kinase (Vps34) in platelet formation and activation and (ii) the impact of new anticancer drugs targeting kinases on platelet functions and haemostasis. First, I studied the role of Vps34 and its lipid product, phosphatidylinositol 3 monophosphate (PtdIns3P), in platelet physiology using a unique mouse model of Vps34 deletion specifically in megakaryocyte lineage (PF4-Cre/Vps34lox/lox). We observed a moderate microthrombocytopenia associated to a defect in megakaryocyte migration and morphologic abnormalities in secretion granules. This phenotype is linked to a decrease in PtdIns3P level associated with defective vesicular trafficking. Moreover, PF4-Cre/Vps34lox/lox mice exhibit altered prothrombotic functions, ex vivo in shear conditions and in vivo by conferring a protection against ferric chloride-induced carotid thrombosis. A role for Vps34 in platelet activation, independently from its role in megakaryocyte, was shown ex vivo using two specific Vps34 inhibitors (SAR405 and INH1) recently developed to reduce autophagy-mediated resistance to chemotherapy. Secondly, I assessed the impact of new targeted drugs used in cancer therapy on platelets in order to understand the increased bleeding risk associated to these molecules. We studied the effect of ibrutinib, a specific inhibitor of BTK family tyrosine kinases recently approved for the treatment of B malignancies (mantle cell lymphoma, chronic lymphocytic leukemia). By exploring platelet functions of ibrutinib-treated patients treated from Hematology department of Toulouse, we correlated bleeding symptoms to a defective platelet signaling downstream GPVI and GPIb receptors as shown by a strongly reduced platelet aggregation in response to collagen and CRP and by a defect in platelet adhesion on von Willebrand matrix under flow conditions. In conclusion, my thesis work (i) brings fundamental insights about Vps34 contribution in mechanisms of platelet production and functions and (ii) allows recommendations about clinical use of new targeted molecules in cancer therapy
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Galleiske, Hanne. "Experimentelle Evaluation der Bedeutung des PI3K/AKT-Signalweges für die Tumorstrahlenempfindlichkeit." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2017. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-227334.
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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.
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Ferreira, Marília Gabriele Prado Albuquerque [UNESP]. "Expressão proteica da via PI3K/AKT/mTOR em mastocitomas cutâneos caninos." Universidade Estadual Paulista (UNESP), 2014. http://hdl.handle.net/11449/122032.
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000813877.pdf: 1968360 bytes, checksum: ba893fb51a41ad4d26fafd388f7a43d2 (MD5)A via PI3K/AKT/mTOR está relacionada com a proliferação, síntese proteica, sobrevivência e motilidade das células. Mutações nesta via tem sido associadas com o processo carcinogênico de diferentes tipos tumorais. Atualmente, inibidores específicos de proteínas como a PI3K, AKT, mTOR, vem sendo testados com o intuito de otimizar a terapia antitumoral em seres humanos e animais. Além de potenciais alvos terapêuticos, o aumento da expressão de algumas destas proteínas tem sido associado a tumores de pior prognóstico em seres humanos. Em cães, o mastocitoma (MCT) é um tumor cutâneo extremamente comum, de comportamento bastante variável e etiologia ainda não esclarecida. Diante disso, o objetivo deste estudo, foi avaliar a imunomarcação dos componentes proteicos da via PI3K/AKT/mTOR, por meio da técnica de imuno-histoquímica e em um segundo momento, associar esses resultados com os parâmetros clínicos dos pacientes, com as características morfológicas e biológicas dos tumores e com o tempo de sobrevida dos pacientes, visando determinar possíveis marcadores prognósticos para esta neoplasia. Para tal 46 MCTs caninos foram utilizados. Todos os tumores apresentaram positividade na avaliação imuno-histoquímica para as proteínas analisadas. As proteínas p-AKT Thr 308 e p-S6K1 apresentaram forte intensidade de imunomarcação, quando comparadas a parâmetros associados a piores prognósticos. Esses resultados indicam que a forte intensidade de imunomarcação de p- AKT Thr 308 e p-S6K1 está relacionada a um pior prognóstico para os cães acometidos pelo MCT cutâneo
The PI3K/AKT/mTOR pathway is related to proliferation, protein synthesis, cell motility and survival. Mutations in this pathway have been associated with the carcinogenic process in diferent tumor types. Currently, specific inhibitors of some proteins such as PI3K, AKT, mTOR, are being tested in order to optimize antitumor therapy in humans and animals. Apart from potential therapeutic targets, increased in immunostaining of some of these proteins have been associated with poorer prognostic in humans tumors. In dogs, mast cell tumor (MCT) is an extremely common skin tumor, which presents a highly variable behavior and unclear etiology. Therefore, the aim of this study was to evaluate the immunostaining of the protein components of PI3K/AKT/mTOR pathway, through the technique of immunohistochemistry, and in a second stage, correlate this results with clinical parameters of patients, with morphological and biological tumor characteristics and the survival time of patients to determine possible prognostic markers for this disease. For this, 46 canine MCTs were used. All tumors were positive in immunohistochemical analysis for the proteins analyzed. Proteins p-AKT Thr 308 and p-S6K1 showed increases in the intensity of immunostaining compared to parameters associated with worse prognosis. These results suggest that the increase in the intensity of immunostaining of p-AKT Thr 308 and p-S6K1 are related to a worse prognosis for dogs affected by cutaneous MCT
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McCarragher, Leeza Sarah Marie. "PI3K signalling blockade : a target for chemotherapeutic enhancement in breast cancer." Thesis, University of Sheffield, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.401117.
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Walsh, Michael Hartley. "Phosphoproteomic investigation of differential signalling downstream of class IA PI3K isoforms." Thesis, Queen Mary, University of London, 2014. http://qmro.qmul.ac.uk/xmlui/handle/123456789/8917.
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The PI3K family is central to numerous cellular processes in both health and disease. The class IA isoforms of PI3K control such outputs as proliferation, metabolism and survival through their well-characterised function as lipid kinases, with their signalling thought to predominantly mediated by the Akt/PKB protein kinase. However there exist other signalling routes, including from the lipid kinase activity through other effectors, but also through a protein kinase function of the class IA isoforms themselves. Mass spectrometry is a powerful tool which has been central to the recent advances in phosphoproteomic techniques. It is now possible to use mass-spectrometry to probe the phosphoproteome of any number of systems in an unbiased and global manner. In this project, we aimed to advance our understanding of two aspects of class IA PI3K signalling which are relatively poorly understood. We used phosphoproteomic techniques which allowed us to provide answers to some old questions which have up to now proved elusive. First, we investigated the protein kinase activity of p110α. We used an in vitro protein kinase assay and coupled this to mass spectrometry techniques to identify direct substrates of p110α. We proposed two novel protein substrates and attempted to characterise them further, although we were hampered by lack of available biochemical tools. Second, we investigated the differential phosphoproteomes of the ubiquitously expressed class IA PI3K isoforms p110α and p110β in a panel of breast cancer cell lines. We used mass spectrometry-based phosphoproteomics and found significant differences in signalling between p110α and p110β in 4T1 cells, including differential regulation of previously described PI3K effectors, amongst them the Akt substrate PRAS40, and potential novel targets. Additionally, we found that some of these effects were conserved between cell lines.
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Ferreira, Marília Gabriele Prado Albuquerque. "Expressão proteica da via PI3K/AKT/mTOR em mastocitomas cutâneos caninos /." Jaboticabal, 2014. http://hdl.handle.net/11449/122032.
Full textAbstract:
Orientador: Andrigo Barboza De NardiCoorientador: Mirela Tinucci Costa
Coorientador: Reneé Laufer Amorim
Banca: Rosemeri de Oliveira Vasconcelos
Banca: Daniel Guimarães Gerardi
Resumo: A via PI3K/AKT/mTOR está relacionada com a proliferação, síntese proteica, sobrevivência e motilidade das células. Mutações nesta via tem sido associadas com o processo carcinogênico de diferentes tipos tumorais. Atualmente, inibidores específicos de proteínas como a PI3K, AKT, mTOR, vem sendo testados com o intuito de otimizar a terapia antitumoral em seres humanos e animais. Além de potenciais alvos terapêuticos, o aumento da expressão de algumas destas proteínas tem sido associado a tumores de pior prognóstico em seres humanos. Em cães, o mastocitoma (MCT) é um tumor cutâneo extremamente comum, de comportamento bastante variável e etiologia ainda não esclarecida. Diante disso, o objetivo deste estudo, foi avaliar a imunomarcação dos componentes proteicos da via PI3K/AKT/mTOR, por meio da técnica de imuno-histoquímica e em um segundo momento, associar esses resultados com os parâmetros clínicos dos pacientes, com as características morfológicas e biológicas dos tumores e com o tempo de sobrevida dos pacientes, visando determinar possíveis marcadores prognósticos para esta neoplasia. Para tal 46 MCTs caninos foram utilizados. Todos os tumores apresentaram positividade na avaliação imuno-histoquímica para as proteínas analisadas. As proteínas p-AKT Thr 308 e p-S6K1 apresentaram forte intensidade de imunomarcação, quando comparadas a parâmetros associados a piores prognósticos. Esses resultados indicam que a forte intensidade de imunomarcação de p- AKT Thr 308 e p-S6K1 está relacionada a um pior prognóstico para os cães acometidos pelo MCT cutâneo
Abstract: The PI3K/AKT/mTOR pathway is related to proliferation, protein synthesis, cell motility and survival. Mutations in this pathway have been associated with the carcinogenic process in diferent tumor types. Currently, specific inhibitors of some proteins such as PI3K, AKT, mTOR, are being tested in order to optimize antitumor therapy in humans and animals. Apart from potential therapeutic targets, increased in immunostaining of some of these proteins have been associated with poorer prognostic in humans tumors. In dogs, mast cell tumor (MCT) is an extremely common skin tumor, which presents a highly variable behavior and unclear etiology. Therefore, the aim of this study was to evaluate the immunostaining of the protein components of PI3K/AKT/mTOR pathway, through the technique of immunohistochemistry, and in a second stage, correlate this results with clinical parameters of patients, with morphological and biological tumor characteristics and the survival time of patients to determine possible prognostic markers for this disease. For this, 46 canine MCTs were used. All tumors were positive in immunohistochemical analysis for the proteins analyzed. Proteins p-AKT Thr 308 and p-S6K1 showed increases in the intensity of immunostaining compared to parameters associated with worse prognosis. These results suggest that the increase in the intensity of immunostaining of p-AKT Thr 308 and p-S6K1 are related to a worse prognosis for dogs affected by cutaneous MCT
Mestre
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Liao, Wenjing. "Targeting PI3K pathway to enhance cisplatin cytotoxicity in lung cancer models." Thesis, University of Leicester, 2017. http://hdl.handle.net/2381/39671.
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Lung cancer is a leading cause of cancer deaths and is commonly diagnosed in both males and females. The cause of lung cancer is mainly attributable to cigarette smoking and aging, accounting for more than half of all lung cancer deaths. Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer (about 80%) compared to small cell lung cancer (SCLC) and it is normally subclassified into squamous cell carcinoma, adenocarcinoma and large cell carcinoma. Cisplatin-based treatments are the main first-line regimens for any stage of NSCLC, although platinum has over a 40-year history of use in clinical practice. Recently, molecularly targeted agents have been developed for improvement of treatment outcomes in patients with advanced lung cancers, such as EGFR-and ALK-targeted drugs. However, the acquired resistance to these agents eventually impairs sensitivity to treatment. Studies have shown that PI3K/Akt signaling activation is associated with resistance to the targeted treatments, and highlights a role for targeting PI3K/Akt signaling to restore sensitivity to targeted therapies. Moreover, Akt inhibition has been shown to promote sensitivity to cisplatin treatment. In this project, both cisplatin and various agents which target Akt directly or indirectly, were tested on 2D NSCLC cell cultures for assessment of potencies. Single-agent treatment showed that the mutant P53 cell line (H596) showed the greatest sensitivity to cisplatin treatment compared to two WT P53 cell lines (A549 and H460). This was similarly observed in a patient-derived tissue explant model which showed that P53 mutant cases were more sensitive to cisplatin treatment than those WT P53 cases. In 2D adherent cell culture and 3D organotypic co-culture of cancer cells with fibroblasts, Akt activity was activated in response to cisplatin treatment, which was similarly observed in one explant case. Moreover, decreased Akt phosphorylation was significantly correlated with increased PARP cleavage in WT P53 cases only. Both PI3K- and Akt-targeted treatments were shown to be highly potent on 2D culture and 3D spheroid models compared to other cisplatin-based regimens. Further analysis of molecular biomarkers by In-Cell western assay corroborated this, with induction of caspase-3-dependent cell death significantly higher when cisplatin treatment was combined with PI3K- or Akt-targeting drugs. The increase in apoptosis was concurrently observed with decreases in pAkt levels. P53 and PTEN levels were not induced by combination treatments compared to cisplatin alone, suggesting that functional P53 might not be required for induction of cell death.
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Dong, Shuai. "Pharmacologic and Genetic Investigation of PI3K p110delta in Chronic Lymphocytic Leukemia." The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1503066319794882.
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Brennan, Tracy A. "Abrogation of Cbl-PI3K Interaction Increases Bone Volume and Osteoblast Proliferation." Diss., Temple University Libraries, 2011. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/107475.
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Cell BiologyPh.D.
Cbl is a multivalent protein that interacts with a number of signaling molecules that affect cell proliferation, migration and apoptosis. Although it is a downstream effector of growth factors, cytokines and integrin signaling all of which influence bone mass, very few studies have examined the role of Cbl in osteoblast proliferation and differentiation. To examine the role(s) of Cbl in the skeletal system we have focused specifically on phosphorylation of CblY737 since it is a unique to Cbl (not present on other family members) and upon phosphorylation by Src family kinases it provides a binding site for the p85 subunit of PI3K which regulates signaling events that modulate apoptosis and survival. To determine the role of tyrosine 737 we are using CblYF/YF knock-in mice (YF) where tyrosine 737 has been substituted to phenylalanine. YF mice had increased bone volume (WT 9%; YF 14%; p= 0.05 vs WT), trabecular thickness, and trabecular numbers. Although the increased bone volume is partly attributed to the decreased bone resorption, static and dynamic parameters of bone formation indicated that numbers of osteoblasts (WT 13 N.OB/BS; YF 20 N.OB/BS; p=0.05 vs WT) and bone formation rates were also upregulated in the CblYF/YF mice. To investigate the role of osteoblast differentiation in increased bone formation, we differentiated osteoblast and assessed ALP activity and Alizarin Red S staining. Both WT and YF osteoblasts had similar levels of ALP activity and mineral deposition during differentiation. To determine if the increased numbers of osteoblasts were due to increased survival and/or proliferation, we performed in vitro experiments with calvarial osteoblasts from age-matched WT and YF pups. MTT assay and TUNEL-staining, for cell viability, showed abrogation of Cbl-PI3K interaction did not affect osteoblast survival. Interestingly, inhibition of PI3K activity with LY294002 showed comparable survival between the WT and YF osteoblasts. We next examined proliferation and found that there was a 2-fold increase in the rate of the proliferation for the YF osteoblasts. This result was further substantiated by colony forming unit assay using bone marrow stromal cells. To establish the role of extracellular factors on osteoblast increased proliferation, various growth factors were assessed (EGF, FGF, IGF, PDGF). Treatment with the growth factors has no differential effects on the WT versus YF osteoblasts. We next used conditioned media from differentiated osteoclasts and bone marrow cells to treat MC3T3-E1, preosteoblast cell line. The osteoclast media from YF osteoclasts did not increase osteoblast proliferation. However, YF bone marrow conditioned media increased proliferation of the MC3T3-E1. Cytokine assays were done to determine the factor(s) that were increased in the YF conditioned media compared to the WT conditioned media. SDF-1 was found to be increased in the YF conditioned media compared to the WT conditioned media. Taken together, this suggests that the abrogation of Cbl-PI3K interaction leads to increased bone formation due to osteoclast resorption deficiency and increased osteoblast proliferation, which may be caused in part by increased SDF-1 expression in the bone marrow niche.
Temple University--Theses
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Butler, Dominika Ewelina. "Targeting of the PI3K/AKT/mTOR pathway in human prostate cancer." Thesis, University of York, 2014. http://etheses.whiterose.ac.uk/8531/.
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The tumour suppressor PTEN is frequently lost in advanced prostate cancer leading to over-activation of the PI3K/AKT/mTOR pathway. Therefore targeting of the PI3K signalling with pathway-specific inhibitors has been proposed as a therapeutic strategy. Inhibition of AKT and mTOR kinases in prostate cancer cell lines showed a decrease in cell viability and reduction in phospho-biomarkers expression. Although apoptosis was not induced, a decrease in cell migration and G1 cell growth arrest were observed in LNCaP cells. However, in primary prostate cultures activation of the Ras/MEK/ERK compensatory pathway was observed following treatment with AKTi. Moreover, cell viability was less affected than in cell lines and autophagy was induced following treatment with AKTi. Surprisingly, treatment with a combination of AKTi and MEK1/2 inhibitors (MEK1/2i and RO-512) did not reduce phosphorylation of ERK1/2 in primary prostate cultures, but irreversible growth arrest-senescence, was evident. Additionally, ex vivo treatment of a ‘near-patient’ prostate xenograft with a combination of AKTi and mTORi significantly reduced tumour frequency. These results demonstrate that targeting the PI3K/AKT/mTOR pathway triggers activation of the Ras/MEK/ERK compensatory pathway and therefore blockade of one pathway is not sufficient to treat prostate cancer. This study also highlights the importance of using patient-derived tumour cells in preclinical assessment of new drugs rather than relying solely on cancer cell lines.
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Müller, Anja. "Multiple outcomes for PI3K/Akt/mTOR targeting in non-Hodgkin lymphoma." Doctoral thesis, Humboldt-Universität zu Berlin, Lebenswissenschaftliche Fakultät, 2015. http://dx.doi.org/10.18452/17284.
Full textAbstract:
Wachstumsfaktor bedingte Aktivierung des PI3K/Akt/mTOR Signalweg wirkt positiv auf Vermehrung und Überleben. Konstitutive Aktivierung des Signalweges in NHL ist jedoch an Tumorprogression und Therapieresistenz beteiligt. Am Zelllinienmodell wurden zwei mögliche Therapiestrategien der PI3K/Akt/mTOR Inhibition erprobt, PI3K Inhibition mit BKM120 und horizontale Kombination von Zytostatika mit PI3K/Akt/mTOR Inhibitoren Erstens, BKM120 hat Antitumoraktivität in NHL und induziert Zelltod. Auf molekularer Ebene führt BKM120 vermittelte Dephosphorylierung von CDK1 an Y15 zur Aktivierung des M-Phase Komplex CDK1/Zyklin B und Eintritt in die Mitose. Parallel erlaubt die Degradation von Zyklin A und Hochregulation von Zyklin B Progression bis zur Metaphase, hemmt jedoch die Transition in die Anaphase. Anhaltender Metaphasearrest bewirkt programmierten Zelltod über den intrinsischen Signalweg der Apoptose durch Hochregulation der BH3-onlys Puma und Hrk, Aktivierung von Bax/Bak und proteolytische Spaltung von Caspase 9. Verlust von Bax/Bak oder Caspase Inhibition schützt vor BKM120 vermitteltem Zelltod. Bax/Bak defiziente Zellen, welche zusätzlich p53 Mutationen aufweisen, werden polyploid. Die Polyploidie ist ATM-MEK1/2 abhängig und kann mit Caffeine oder U0126blockiert werden. Zur Vermeidung von Polyploidie bedingter Tumorprogression, sollte BKM120 nur in Verbindung mit MAPK/ATM Inhibitoren verwendet werden. Zweitens. Horizontale Kombination PI3K/Akt/mTOR Inhibitoren mit cytotoxischen Substanzen schützt vor Apoptose. Der Schutzeffekt tritt auschließlich bei niedrigen Konzentration auf und ist unabhängig von der Art des Inhibitors bzw. Ebene der Inhibition. Das Onkogen und NFkB Target Pim-2 ist möglicherweise am Schutzmechanismus beteiligt. Durch die PI3K/Akt/mTOR vermittelte Pim-2 Regulation ergibt sich eine neue Rückkopplungsschleife. Im Fazit erschwert die Komplexizität des PI3K/Akt/mTOR Signalweges die Etablierung von Therapien.Growth factor mediated activation of the PI3K/Akt/mTOR pathway positively regulates proliferation and survival. Constitutive activation in NHL, however, is correlated with tumor progression and therapeutic resistance. Therefore, two possible strategies were tested in a cell line model system, Inhibition of PI3K with BKM120 and PI3K/Akt/mTOR Inhibition in addition to cytostatic drug administration. First, it is demonstrated that the pan PI3K inhibitor BKM120 has antitumor activity in NHL and induces cell death. On molecular level, BKM120 mediated dephosphorylation of CDK1 on Y15 causes activation of the M-phase complex CDK1/Cyclin B and entry into mitosis. In parallel, degradation of Cyclin A and Upregulation of Cyclin B enables progression into metaphase but inhibits transition into anaphase. Prolonged metaphase arrest induces programmed cell death via the intrinsic apoptosis pathway by upregulation of the BH3-onlys Puma and Hrk, activation of Bax/Bak and proteolytic cleavage of caspase-9. Loss of Bax/Bak or caspase inhibition protects from BKM120 induced apoptosis. Bax/Bak deficient cells with additional p53 mutation become polyploid. This polyploidy is ATM-MEK1/2 dependent and can be blocked with Caffeine or U0126. To prevent polyploidy related tumor progression, BKM120 should administered only in combination with ATM or MEK inhibitors. Second, combination of PI3K/Akt/mTOR inhibitors with cytotoxic agents protects from apoptosis. The protective effect is only detectable with low PI3K/Akt/mTOR inhibitor concentrations and independent of inhibitor type or cascade level. The oncogene and NFkB target is possibly involved in apoptosis protection and inhibition of NFkB neutralizes the protective effect. PI3K/Akt/mTOR mediated Pim-2 regulation reveals a new feedback loop within the pathway. In conclusion, the complexity of the PI3K/Akt/mTOR pathway impedes therapeutic targeting.
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Ulbar, Francesca <1983>. "Studio della via di segnale PI3K/Akt/mTOR nelle Cellule Dendritiche." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2013. http://amsdottorato.unibo.it/5401/1/Ulbar_Francesca_tesi.pdf.
Full textAbstract:
Il trapianto allogenico di cellule staminali emopoietiche è spesso l’unica soluzione per la cura di diverse malattie ematologiche. La aGVHD è la complicanza più importante che si può avere a seguito del trapianto allogenico ed è causata dai linfociti T del donatore che riconoscono gli antigeni del ricevente presentati dalle APC. Eliminare o inattivare la APC del ricevente prima del trapianto potrebbe prevenire la aGVHD. Ad oggi non esistono farmaci specifici diretti contro le APC, sono però noti i meccanismi molecolari coinvolti nella sopravvivenza cellulare come la via di segnale di PI3K. In questo lavoro abbiamo testato l’attività di due farmaci, che colpiscono target molecolari della via di PI3K, la rapamicina e la perifosina, sul differenziamento dei monociti a differenti popolazioni di cellule dendritiche (DC), in vitro. La rapamicina riduceva il recupero cellulare delle DC derivate da monociti coltivate in presenza di IL-4 aumentando l’apoptosi, mentre i monociti coltivati in presenza di GM-CSF con o senza IFN-α risultavano resistenti alla rapamicina. Inoltre la rapamicina riduceva l’espressione della molecola costimolatoria CD86 e incrementava l’espressione della molecola CD1a solo nei monociti coltivati con GM-CSF e IL-4. Nelle DC derivate dai monociti in presenza di IL-4 la rapamicina bloccava la produzione di IL-12 e TNF-α e ne alterava la capacità allostimolatoria. La rapamicina non alterava la sopravvivenza e la funzione delle DC circolanti. Il trattamento con perifosina provocava un incremento di apoptosi nei monociti coltivati sia con GM-CSF che con GM-CSF e IL-4. La perifosina bloccava la produzione di TNF-α nelle DC derivate da monociti coltivati nelle diverse condizioni. Questi risultati dimostrano che l’azione della rapamicina è strettamente dipendente dalla presenza dell’IL-4 nel terreno di coltura, in vitro, rispetto alla perifosina e suggeriscono un possibile ruolo della perifosina nella prevenzione della GVHD prima del trapianto allogenico di cellule staminali.Allogeneic transplantation of hematopoietic stem cells (HTSC) is the most effective curative option for many neoplastic hematological disease. Acute graft versus host disease (aGVHD) is the most feared complication following HTSC and is caused by donor lymphocytes recognizing recipient histocompatibility antigen presented by antigen-presenting cells (APC). Removal or inactivation of APC before transplantation prevents GVHD. Nowadays there are no drugs specifically targeting APC. The molecular mechanisms involved in cell growth of these cells are well known and mostly involve the activation of the PI3K signaling pathway. In this study we tested the effects of two drugs targeting the PI3K pathway, rapamycin and perifosine on the differentiation of monocytes to distinct DC subtypes in vitro. Rapamycin decreased the recovery of monocyte-derived DC cultured in presence of IL-4 due to increased apoptosis, while monocytes cultured in GM-CSF with or without IFN-α were not affected. Rapamycin decreased the expression of the costimulatory molecules CD86 and increased the expression of CD1a in monocyte-derived DC, only in presence of IL-4. Moreover, rapamycin blocked the secretion of IL-12 and TNF-α and altered the allostimulatory capacity only in monocytes cultured with IL-4. Rapamycin didn’t alter the survival and function of circulating DC. Treatment with perifosine was associated with increased apoptosis of monocytes cultured both with GM-CSF only or with GM-CSF and IL-4. Perifosine blocked the secretion of TNF-α by monocytes cultured with GM-CSF only and with GM-CSF and IL-4 after 3 days of culture. These results suggest that the action of rapamycin is more strictly dependent on IL-4 than perifosine, suggesting a possible use of perifosine in the prevention of GVHD before HSCT.
50
Ulbar, Francesca <1983>. "Studio della via di segnale PI3K/Akt/mTOR nelle Cellule Dendritiche." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2013. http://amsdottorato.unibo.it/5401/.
Full textAbstract:
Il trapianto allogenico di cellule staminali emopoietiche è spesso l’unica soluzione per la cura di diverse malattie ematologiche. La aGVHD è la complicanza più importante che si può avere a seguito del trapianto allogenico ed è causata dai linfociti T del donatore che riconoscono gli antigeni del ricevente presentati dalle APC. Eliminare o inattivare la APC del ricevente prima del trapianto potrebbe prevenire la aGVHD. Ad oggi non esistono farmaci specifici diretti contro le APC, sono però noti i meccanismi molecolari coinvolti nella sopravvivenza cellulare come la via di segnale di PI3K. In questo lavoro abbiamo testato l’attività di due farmaci, che colpiscono target molecolari della via di PI3K, la rapamicina e la perifosina, sul differenziamento dei monociti a differenti popolazioni di cellule dendritiche (DC), in vitro. La rapamicina riduceva il recupero cellulare delle DC derivate da monociti coltivate in presenza di IL-4 aumentando l’apoptosi, mentre i monociti coltivati in presenza di GM-CSF con o senza IFN-α risultavano resistenti alla rapamicina. Inoltre la rapamicina riduceva l’espressione della molecola costimolatoria CD86 e incrementava l’espressione della molecola CD1a solo nei monociti coltivati con GM-CSF e IL-4. Nelle DC derivate dai monociti in presenza di IL-4 la rapamicina bloccava la produzione di IL-12 e TNF-α e ne alterava la capacità allostimolatoria. La rapamicina non alterava la sopravvivenza e la funzione delle DC circolanti. Il trattamento con perifosina provocava un incremento di apoptosi nei monociti coltivati sia con GM-CSF che con GM-CSF e IL-4. La perifosina bloccava la produzione di TNF-α nelle DC derivate da monociti coltivati nelle diverse condizioni. Questi risultati dimostrano che l’azione della rapamicina è strettamente dipendente dalla presenza dell’IL-4 nel terreno di coltura, in vitro, rispetto alla perifosina e suggeriscono un possibile ruolo della perifosina nella prevenzione della GVHD prima del trapianto allogenico di cellule staminali.Allogeneic transplantation of hematopoietic stem cells (HTSC) is the most effective curative option for many neoplastic hematological disease. Acute graft versus host disease (aGVHD) is the most feared complication following HTSC and is caused by donor lymphocytes recognizing recipient histocompatibility antigen presented by antigen-presenting cells (APC). Removal or inactivation of APC before transplantation prevents GVHD. Nowadays there are no drugs specifically targeting APC. The molecular mechanisms involved in cell growth of these cells are well known and mostly involve the activation of the PI3K signaling pathway. In this study we tested the effects of two drugs targeting the PI3K pathway, rapamycin and perifosine on the differentiation of monocytes to distinct DC subtypes in vitro. Rapamycin decreased the recovery of monocyte-derived DC cultured in presence of IL-4 due to increased apoptosis, while monocytes cultured in GM-CSF with or without IFN-α were not affected. Rapamycin decreased the expression of the costimulatory molecules CD86 and increased the expression of CD1a in monocyte-derived DC, only in presence of IL-4. Moreover, rapamycin blocked the secretion of IL-12 and TNF-α and altered the allostimulatory capacity only in monocytes cultured with IL-4. Rapamycin didn’t alter the survival and function of circulating DC. Treatment with perifosine was associated with increased apoptosis of monocytes cultured both with GM-CSF only or with GM-CSF and IL-4. Perifosine blocked the secretion of TNF-α by monocytes cultured with GM-CSF only and with GM-CSF and IL-4 after 3 days of culture. These results suggest that the action of rapamycin is more strictly dependent on IL-4 than perifosine, suggesting a possible use of perifosine in the prevention of GVHD before HSCT.