Taxanes represent some of the most common chemotherapeutic agents for ovarian cancer treatment. However, they are only effective in approximately 40% of patients. As such, novel therapeutic strategies are required to potentiate their effect in ovarian cancer to improve patient outcome and prevent chemotherapy resistance. A hallmark of many cancers is the constitutive activation of the PI3K/AKT pathway, which drives cell survival and metabolism. In this thesis, I identified a potential vulnerability in ovarian cancer cell lines during paclitaxel-induced mitotic arrest, comprising of a striking decrease in AKT activity coupled with a significant reduction in glucose 6-phosphate and ATP levels. The reanalysis of a high content siRNA screen to discover metabolic enzymes important for ovarian cancer cell survival during paclitaxel-induced mitotic arrest, identified the metabolic enzyme PFKFB4. PFKFB4 depletion followed by paclitaxel treatment resulted in a significant decrease in mitotically arrested cells. This was accompanied by a significant increase in caspase-3/7 activity and the levels of reactive oxygen species only in mitotically arrested cells, and a significant enhancement in mitotic cell death and mitotic slippage. Depletion of the related family member, PFKFB3, demonstrated a similar phenotype. The exogenous expression of constitutively active AKT or siRNA-resistant PFKFB4 did not confer resistance to PFKFB4 depletion and paclitaxel treatment, indicating that the mechanism of mitotic cell reduction is complex. Nonetheless, the observation that some ovarian cancer cells lose AKT activity during mitotic arrest and could become vulnerable to metabolic targeting is a new concept in cancer therapy. In addition, I have identified previously unrecognised roles of PFKFB3 and PFKFB4 in mitotically arrested ovarian cancer cells. This work supports the notion that combining mitotic-targeted therapies with metabolic inhibitors may act to potentiate the effects of antimitotics in ovarian cancer cells.

Le CD95 appartient à la famille du TNF-R. Il est capable de déclencher un signal apoptotique et joue un rôle prépondérant dans le maintien de l’homéostasie du système immunitaire et dans l’élimination de cellules infectées ou transformées. Suite à la fixation de son ligand le CD95L ou d’un anticorps agoniste, le CD95 recrute la protéine FADD, qui à son tour agrège les caspases initiatrices (i.e., caspase -8 et -10). Le complexe formé par le CD95, FADD et les caspases -8/10 est appelé DISC, pour Deah Inducing Signaling Complex. Une fois le DISC formé, l’agrégation des caspases initiatrices entraîne leur activation, et la mort de la cellule par apoptose. Ce signal médié par CD95, peut être modulé par la distribution ou l’exclusion du récepteur vis à vis des microdomaines membranaires ou radeaux lipidiques. Ces domaines de la membrane plasmique sont des structures membranaires enrichies en sphingolipides et cholestérol. La relocalisation de CD95 dans ces radeaux lipidiques permet d’amplifier le signal de mort. L’activation de la voie phosphatidylinositol 3-kinase (PI3K)/Akt, est connue pour sa capacité à protéger les cellules tumorales du signal apoptotique médié par CD95, et à induire des mouvements latéraux de CD95 à la membrane. Nos travaux montrent que l’inhibition de la voie PI3K/Akt induit i) la relocalisation du CD95 dans les microdomaines et ii) l’induction du signal apoptotique CD95 indépendamment de la présence du ligand CD95L. Ainsi, nous mettons en évidence que la voie PI3K/Akt est capable d’augmenter le seuil d’activation du signal CD95 en agissant en amont de la formation du DISC ou de l’interaction CD95/CD95L, en maintenant le récepteur exclu des microdomaines. / CD95 belongs to the TNF-R superfamily, and it triggers an apoptotic signal. CD95 plays a key role in homeostasis of the immune system and in the elimination of infected and transformed cells. Upon CD95L binding, CD95 recruits FADD, which in turn aggregates initiator caspases (i.e., caspase-8 and -10). The complex CD95, FADD and caspase-8/10 is called DISC for Death Inducing Signaling Complex. At the DISC level, caspase aggregation leads to their activation and death of the cells through apoptosis. The CD95-mediated apoptotic signal is modulated by microdomains, or lipid rafts, which are plasma membrane sub-domains enriched in sphygolipids and cholesterol. Thereby, partition of CD95 into lipid rafts promotes the apoptotic signal. Activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is known to prevent the CD95-mediated apoptotic signal in malignant cells, and to control lateral mobility of CD95 at the plasma membrane. Herein, we showed that inhibition of PI3K signal induced i) the distribution of CD95 into lipid rafts and ii) the subsequent induction of the CD95-mediated apoptotic signal through a CD95L independent manner. In conclusion, we pinpointed that PI3K/Akt signaling pathway inhibits the CD95 signal by acting upstream DISC formation and even upstream the CD95-CD95L interaction through the exclusion of the death receptor from the microdomains.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by the selective loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc). PD affects ~1% of the population over 65, as demonstrated by characteristic symptoms such as tremor, rigidity, and bradykinesia. While the majority of PD cases are idiopathic, some cases are familial, including those caused by homozygous loss-of-function mutations in DJ-1 (PARK7), which lead to early onset PD. Although the physiological role of DJ-1 is not fully understood, DJ-1’s neuroprotective role against oxidative stress is well documented. DJ-1 is required for AKT-mediated neuroprotective effects, however the mechanism by which DJ-1 affects membrane localization/activation of AKT is unknown and is likely a critical aspect of DJ-1 function. In this thesis we explore the mechanism through which DJ-1 confers neuroprotection through AKT membrane recruitment, particularly in the case of oxidative stress insult. We demonstrate here that DJ-1 interacts with PHLDA3, a negative regulator of AKT, and loss of DJ-1 leads to hypersensitivity of neurons to PHLDA3-mediated death. Additionally, we demonstrate that in the absence of DJ-1, PHLDA3 localization at the membrane is increased, and overexpression of PHLDA3 causes reduced AKT phosphorylation in DJ-1 KO MEFs in response to oxidative stress. Taken together, these studies provide a potential novel mechanism by which DJ-1 regulates the activity of AKT, a critical neuronal survival pathway. Elucidation of these mechanisms may provide insight into the design of neuroprotective therapies for PD.

Cisplatin (CDDP; cis-diamminedichloroplatinum) resistance presents a major impediment in the treatment of several gynecologic solid tumors, including ovarian and cervical tumors. p53, a critical regulator of cellular apoptosis, is a determinant of CDDP sensitivity. In our study, we have observed that the dysregulation of p53 regulators, checkpoint kinase 1 (Chk1) and protein phosphatase magnesium-dependent 1 (PPM1D), significantly reduced CDDP responsiveness in human cancer cells. Isogenic wt-p53 CDDP-sensitive (OV2008) and -resistant (C13*) cervical cancer cells, and isogenic wt-p53 CDDP-sensitive (A2780s) and p53 mutant resistant (A2780cp) ovarian cancer cells, along with CDDP-resistant ovarian cancer cell lines (OCC-1 and OVCAR-3, mutant p53; SKOV-3, p53 null) were used to elucidate the mechanisms of p53 regulation in human gynecologic cancer cells. We have complemented our study with a xenograft model (A2780s) and a tissue microarray of human ovarian tumors to validate our in vitro observations. We have demonstrated that CDDP differentially regulated the p53 activator Chk1 in sensitive and resistant cancer cells; it enhances Chk1 activation in sensitive but not resistant cells. This differential regulation also extended to PPM1D, whereby CDDP enhanced PPM1D content in resistant but not sensitive cells. PPM1D knockdown sensitized resistant cells to CDDP, which was associated with up-regulation of Chk1 and p53 activations, while PPM1D over-expression had the opposite effect. We have also shown that CDDP sensitivity in response to PPM1D down-regulation was p53-dependent. Moreover, CDDP promotes PPM1D nuclear localization in resistant cells and nuclear exclusion in sensitive cells and xenograft tumors. Enhanced PPM1D expression in human ovarian tumors is significantly associated with tumor aggression. Dysregulation of the oncogene Akt has been implicated in a variety of human malignancies, including ovarian cancer. We have demonstrated that Akt regulates PPM1D stability, since activated Akt over-expression in sensitive cells rescued PPM1D from CDDP-induced proteasomal degradation and Akt down-regulation in resistant cells lead to PPM1D de-stabilization and down-regulation. We have shown for the first time that PPM1D is downstream of Akt through which it can modulate CDDP sensitivity in human cancer cells. These findings extend the current knowledge on the molecular basis of CDDP resistance in gynecological cancers and may help in developing effective therapeutic strategies.

In cells undergoing stress, the p53 transcription factor is stabilized and activates the expression of numerous genes contributing to p53-mediated tumor suppression. One p53 target gene is Mdm2, which encodes an oncoprotein that binds and ubiquitinates p53 for proteasomal degradation, thus limiting the amplitude and duration of the p53-mediated stress response. Our lab recently discovered that Mdm2 phosphorylation by ATM and c-Abl regulates the DNA damage response and tumorigenesis in mice. AKT has also been found in transfection studies to phosphorylate Mdm2 at serine residues 166 and 186 (mouse S163 and S183) to alter p53 activity. However, the physiological significance of Mdm2 phosphorylation by Akt remains unknown. Therefore, I generated Mdm2S163A or Mdm2S183A mice expressing mutant Mdm2 incapable of being phosphorylated by Akt. In contrast with our previous studies, Akt phosphorylation of Mdm2 does not alter spontaneous tumorigenesis or the DNA damage response to ionizing radiation. However, Akt phosphorylation of Mdm2-S183 (but not -S163) upregulates nuclear localization of Mdm2, destabilizes p53, and reduces p53-mediated senescence in response to elevated levels of reactive oxygen species (ROS). To examine the effects of Mdm2-S183 phosphorylation on p53 tumor suppression, I utilized three different mouse models of ROS-induced cancer. Increased levels of p53 and senescence in Mdm2S183A mice yielded reduced tumorigenesis in an activated Ras model of lung cancer, a phorbal ester-induced skin cancer model, and a diethylnitrosamine-induced model of hepatocellular carcinoma. Since AKT is also important regulator of cell metabolism, I explored the impact of the Mdm2-S183 allele on metabolic functions. Mdm2 phosphorylation by Akt reduced glucose metabolism via glycolysis in vitro, and reduced insulin tolerance in mice, without altering glucose tolerance and glucose-stimulated insulin secretion. Collectively, these findings document a unique physiologic role for the AKT-Mdm2-p53 signaling axis in regulating cell growth and tumorigenesis.

Bupivacaine is one of the amide type local anesthetics and is widely used for epidural anesthesia and blockade of nerves. Bupivacaine administration locally could result in neuron injury showing transient neurologic symptoms. Dexamethasone is a synthetic glucocorticoid and may exert cytoprotective properties against damage induced by some stimuli. In the present study, we evaluated the effects of dexamethasone on bupivacaine-induced toxicity in mouse neuroblastoma N2a cells. N2a cells were exposed to bupivacaine in the presence or absence of dexamethasone. After treatment, the cell viability, nuclear condensation, and lactate dehydrogenase levels were evaluated. Mitochondrial potential and Akt (threonine-serine protein kinase B) activation were also examined. In a separate experiment, we examined the effect of Akt inhibition by triciribine on cell viability following dexamethasone treatment. We also investigated whether dexamethasone could prevent lidocaine-induced neurotoxicity. Treatment of N2a cells with bupivacaine resulted in significant cell injury as evidenced by morphological changes, LDH leakage, and nuclear condensation. Pretreatment of the cells with dexamethasone significantly attenuated bupivacaine- and lidocaine-induced cell injury. Dexamethasone treatment prevented the decline of mitochondrial potential caused by bupivacaine and increased the levels of Akt phosphorylation. Importantly, pharmacological inhibition of Akt abolished the protective effect of dexamethasone against bupivacaine-induced cell injury. Our data suggest that pretreatment of neuroblastoma cells with dexamethasone exerts a protective effect on bupivacaine-induced neuronal cell injury. The mechanisms involve activating the Akt signaling pathway.

The nerve-derived trophic factor neuregulin (NRG) is a prime candidate molecule for modulating muscle fiber growth. NRG regulates signal transduction in skeletal muscle through activation of ErbB receptors present at the neuromuscular junction. In this study, we hypothesize that NRG increases protein synthesis in maturing muscle via a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. NRG signal transduction and its ability to stimulate protein synthesis (measured by incorporation of [3H]phenylalanine into the protein pool) were investigated in differentiated C2C 12 myotubes and rat diaphragm muscle (DIAm). In C2C 12 myotubes, NRG dose dependently increased phosphorylation of ErbB3 and recruitment of the p85 subunit of PI3K. NRG also increased phosphorylation of Akt, a downstream effector of PI3K. NRG treatment increased total protein synthesis by 35% compared with untreated control myotubes. This NRG-induced increase in Akt phosphorylation and protein synthesis was completely blocked by wortmannin, an inhibitor of PI3K but was unaffected by PD-98059, an inhibitor of MEK. In DIAm obtained from 3-day-old rat pups, Akt phosphorylation increased ∼30-fold with NRG treatment (vs. untreated DIAm). NRG treatment also significantly increased protein synthesis in the DIAm by 29% after 3 h of incubation with [3H]phenylalanine (vs. untreated DIAm). Pretreatment with wortmannin abolished the NRG-induced increase in protein synthesis, suggesting a critical role for PI3K in this response. The results of the present study support the hypothesis that nerve-derived NRG contributes to the regulation of skeletal muscle mass by increasing protein synthesis via activation of PI3K.

Through endocrine and exocrine functioning, physiological needs are communicated to body systems. Physiological need is met through the actions of intracellular signaling cascades and calibrated through an extensive network of regulatory cross talk within the cells of a given tissue. The insulin receptor belongs to a family of perhaps one of the most well studied family of dual receptor and tyrosine kinases (RTK). The signaling cascade downstream of the insulin RTK can be initiated through Insulin or growth factor ligand binding and bears growing relevance to the projected epidemic of obesity related illness and associated cancers. The primary function of the post-prandial insulin response is to support nutrient uptake and storage. Insulin (IS), Insulin-Like Growth Factor (IGF), and Epidermal Growth Factors (EGF) contribute to glucose metabolism, energetic homeostasis, and anabolic applications through effector kinases downstream of activated (phosphorylated) insulin receptor substrates (IRS). Protein Kinase B (AKT) kinase is one such cytosolic effector known to be of critical importance to anabolic metabolism and general cell survival. Under normal circumstances, AKT activity is dependent upon dual phosphorylation events known to occur at the plasma membrane. In an attempt to better understand the mechanism of AKT recruitment to the plasma membrane, earlier experiments reported that IRS stimulation by Insulin-Like Growth Factors (IGF) and Epidermal Growth Factors (EGF) resulted in downstream poly-ubiquitination and subsequent activation of the AKT kinase. This sequence of post-translational modification events suggested that non-proteolytic AKT ubiquitination, accomplished by the E2 Ubiquitin Conjugating enzyme (UBC13), was an important mediator of AKT activation. Through subsequent experimentation, it was determined that non-proteolytic ubiquitination was a necessary step for AKT activation following IRS activation by Insulin. Furthermore, the same two sites previously described in the context of IGF/EGF signaling were exploited through targeted mutagenesis and shown to synergistically regulate AKT translocation to the plasma membrane. Mutant AKT variants with a single mutation to either ubiquitination site resulted in partial knock down of phosphorylated AKT (pAKT), while variants with double mutations resulted in a complete loss of pAKT detection. Under physiologic conditions UBC13 activity can be antagonized by a small multifunctional protein called G-Protein Pathway Suppressor 2 (GPS2). Bearing the kinetics of an endogenous inhibitor, GPS2-mediated regulation directly inhibits the ubiquitin conjugating activity of the enzyme; thereby restricting AKT non-proteolytic poly-ubiquitination and antagonizing the insulin signaling network through a conserved mechanism. In accordance with this role, we have previously shown that GPS2 presence in adipocytes modulates systemic metabolism by restricting the activation of insulin signaling during the fasted state, whereas in absence of GPS2, the adipose tissue is more efficient at lipid storage, and obesity becomes uncoupled from inflammation and insulin resistance. As we are just beginning to unravel the regulatory network governing the cellular response to nutrient excess and pro-growth signaling, it remains unclear whether UBC13 activity is universally engaged in AKT translocation and activation. Here we have focused on the mitochondrial pool of AKT and investigated its regulation. Our findings add to the growing body of knowledge by demonstrating that in pre-adipocytes mitochondrial AKT is activated, in a UBC13-dependent fashion, following insulin stimulation. We also show that GPS2-mediated inhibition of UBC13 equally antagonizes AKT activation in different subcellular compartments, and that mitochondrial AKT activation is partially Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) dependent.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18848号 / 医博第3959号 / 新制||医||1007(附属図書館) / 31799 / 京都大学大学院医学研究科医学専攻 / (主査)教授 岩田 想, 教授 岩井 一宏, 教授 楠見 明弘 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

La protéine proapoptotique Bax joue un rôle fondamental au cours de la voie intrinsèque de l’apoptose. Elle participe au déclenchement de la mort en permettant la libération de facteurs apoptogéniques mitochondriaux vers le cytosol. Un des points-clé de la fonction de Bax est son activation, caractérisée par la transition entre une forme cytosolique, globulaire et inactive de la protéine et une conformation mitochondriale, membranaire et active. Les différentes étapes de l’activation de Bax sont relativement bien connues, toutefois un grand nombre de questions reste en suspens quant-à leur régulation.Ce travail s’est focalisé sur la régulation de l’activation de Bax par les kinases Akt et GSK-3β ainsi que par la protéine antiapoptotique Bcl-xL . Ces régulations ont été caractérisées en exprimant la protéine Bax humaine chez la levure Saccharomyces cerevisiae, un paradigme d’étude simplifié qui permet d’accéder aux composantes individuelles des mécanismes d’activation de Bax.Les données obtenues suggèrent qu’il existe deux étapes régulées indépendamment au cours de l’activation de Bax. Nous avons montré que la protéine kinase GSK-3β favorise l’adressage de Bax vers la mitochondrie mais qu’elle n’entraîne pas un changement de conformation suffisant à son activation complète et à la perméabilisation de la membrane mitochondriale externe. Des changements de conformations complémentaires de Bax sont requis pour conduire à une forme capable d’entraîner la libération des facteurs apoptogéniques mitochondriaux. La protéine kinase Akt est impliquée dans le contrôle de Bax via la phosphorylation de la sérine 184 et participe à l’inhibition de l’apoptose. Nous avons mis en évidence qu’une mutation phosphomimétique de la sérine 184 ou l’expression d’Akt, en l’absence de partenaires antiapoptotiques, stimulent un changement de conformation de Bax vers une forme active. Akt semble donc plus jouer un rôle sur la conformation de Bax qu’entraîner une inhibition directe. La présence de protéines antiapoptotiques serait ainsi requise pour l’inhibition de Bax en présence d’Akt.D’autre part, nous nous sommmes intéressés aux mécanismes d’action de la protéine antiapoptotique Bcl-xL . Nous avons déterminé que Bcl-xL pouvait favoriser l’adressage de Bax vers la membrane mitochondriale tout en exerçant un rôle antiapoptotique. Ceci suggère que Bcl-xL intervienne dans le contrôle des étapes tardives de l’activation de Bax. Ce contrôle est dépendant d’une interaction stable entre les deux protéines. Inversement, un variant de Bcl-xL n’interagissant que de façon transitoire avec Bax (Bcl-xL ∆C) entraîne l’activation de Bax. Cette observation est en faveur d’un modèle d’activation indirecte de Bax consécutive à la rupture de l’interaction avec Bcl-xL et dans lequel les protéines à BH3-seulement telles que Bad joueraient un rôle crucial. / Proapoptotic protein Bax plays a major role during apoptosis intrinsic pathway. Bax promotes cell death by inducing the release of apoptogenic factors from mitochondria to cytosol. Bax activation is a key step of its function which involves a change from a globular, cytosolic and inactive conformation to an active mitochondrial, membrane inserted conformation. Bax activation substeps are rather well known, however their regulation remains to be characterized.This work focuses on the study of the regulation of Bax activation by kinases Akt and GSK-3β and by antiapoptotic protein Bcl-xL . Human Bax regulations have been studied by expressing the protein in yeast Saccharomyces cerevisiae which represents a simplified paradigm for the understanding of the individual components of Bax activation mecha- nisms.Our data suggest that there are two independently regulated steps during Bax activation. We showed that GSK-3β expression led to Bax addressing to mitochondria but was not sufficent to promote a complete activation and mitochondrial outer membrane premeabilization. Further conformational changes are required to promote Bax full activation and the release of mitochondrial apoptotic factors. Protein kinase Akt is involved in Bax activation control through the phosphorylation of serine 184 and contributes to apoptosis inhibition. We observed that either a phosphomimetic mutation of serine 184 or coexpression of Akt, in the absence of antiapoptotic partners, were responsible of Bax conformational change into an active form. By itself Akt did not inhibit Bax but appeared more likely to control its conformational change. Thus, implication of antiapoptotic proteins seems to be critical in a model of Bax inhibition by Akt.Furthermore, we tried to understand the molecular mechanisms of antiapoptotic protein Bcl-xL inhibition on Bax. We determined that Bcl-xL could increase Bax mitochondrial localization while leading to its inhibition suggesting that Bcl-xL controled Bax late activation steps. Bax inhibition was dependent on a stable interaction with Bcl-xL . Conversely, a variant of Bcl-xL having a transitory interaction with Bax (Bcl-xL ∆C) was able to promote Bax activation. This supports a model of Bax indirect activation following the rupture of interaction with Bcl-xL in which BH3-only proteins like Bad would play an important role.