Les cancers sont caractérisés par une prolifération anarchique des cellules causée par une dérégulation des mécanismes de contrôle du cycle cellulaire, comme la protéine p27Kip1 (p27). Dans le noyau, p27 inhibe les complexes cycline-CDK, bloquant ainsi la progression du cycle de division cellulaire, et par conséquent, la prolifération cellulaire. Cette propriété confère à p27 un rôle de suppresseur de tumeurs. Toutefois, dans certains cancers, p27 est relocalisé dans le cytoplasme où il exerce un rôle oncogénique, cependant les mécanismes moléculaires par lesquels p27 agit comme un oncogène restent largement inconnus. Des études récentes montrent que sa localisation cytoplasmique permet à p27 d'activer l'autophagie, un processus de recyclage des constituants intracellulaires dans les cellules carencées en nutriments. Dans les cellules cancéreuses, l'autophagie est un des mécanismes d'adaptation au microenvironnement tumoral et leur permet de survivre malgré des conditions défavorables. En outre, l'autophagie peut être induite par le stress causé par les traitements anti-cancéreux, ce qui retarde l'apoptose en permettant aux cellules d'échapper aux traitements. L'autophagie induite par la localisation cytoplasmique de p27 pourrait ainsi compromettre l'efficacité des thérapeutiques anti-tumorales. Le but de mon projet de thèse était de déterminer par quels mécanismes p27 contrôle l'autophagie et participe ainsi à la survie des cellules dans des conditions de stress métabolique. Durant ma thèse, j'ai pu mettre en évidence un rôle essentiel de p27 dans la régulation de l'autophagie et de la mort cellulaire. Mes travaux indiquent que le statut de p27 cytoplasmique ou nucléaire détermine à la fois le degré d'autophagie et la susceptibilité des cellules à l'apoptose induite par la privation nutritionnelle. En utilisant des méthodes de biologie cellulaire et moléculaire, j'ai disséqué les voies de signalisation et le mécanisme moléculaire expliquant le rôle pro-autophagique de p27. De manière surprenante, il apparait que p27 régule l'autophagie par différents mécanismes en fonction de la carence infligée aux cellules. [...] / P27 controls cell cycle progression via its ability to block cyclin-CDK activity. Thus, it acts as a tumor suppressor in the nucleus. However, in certain cancers, p27 relocalizes in the cytoplasm where it may promote tumorigenesis by still largely unknown mechanisms. Recent studies have shown that the cytoplasmic localization of p27 induces autophagy, a catabolic process whereby intracellular constituents are recycled in response to nutrient depletion. In cancer cells, autophagy acts as as an adaptive response to metabolic stress in tumor tissues. Furthermore, autophagy may be induced by various cancer therapies, leading to chemotherapeutic resistance and promoting cancer cell survival. The aim of my PhD project was to determine by which mechanisms p27 controls autophagy and cell survival upon metabolic stress conditions. My results indicate that p27 plays a prominent role in the regulation of autophagy and cell death during nutrient deprivation. The status of p27 determines the rate of autophagy and the susceptibility of cells to apoptosis. Importantly, the mechanisms underlying the role of p27 in autophagy appears to be different in function of the nature of the metabolic stress. Amino acid deprivation leads to translocation of p27 to lysosomes where it participates in the inhibition of mTOR, a kinase that acts as a master regulator of cellular metabolism and autophagy. In contrast, the effect of p27 in glucose starved cells depends mostly on its role in the regulation of microtubule dynamics, which controls intracellular vesicle trafficking. Thus, in glucose starved cells, p27 promotes the fusion of autophagic vesicles and degradation of autophagy cargo. To conclude, my results show that p27 is a critical modulator of starvation-induced autophagy and its status determines the response of cells to metabolic stress. Therefore, p27 may serve as a predictive marker for treatment response targeting specific metabolic pathways and may constitute a promising target for anticancer treatment affecting these pathways.

BACKGROUND: Tuberous sclerosis complex (TSC) is a multisystem hamartomatous disease caused by inactivating mutations in the TSC1/TSC2 genes leading to hyperactivation of mammalian target of rapamycin complex 1 (mTORC1) in TSC tumors. Novel therapeutic regimens and imaging biomarkers remain critical unmet needs in TSC. Mitochondrial fatty acid oxidation (FAO) is a major cellular source of acetyl-CoA. Acetyl-CoA is a central metabolite in lipid anabolism and catabolism. The purpose of this study was to identify novel metabolic therapeutic targets, particularly involving lipid metabolism, to achieve durable responses in TSC. METHODS AND RESULTS: Fluoroacetate (FACE), an acetate derivative, is a surrogate biomarker of mitochondrial activity. It accumulates in the mitochondria without being oxidized to CO2, and it is converted to fluorocitrate, irreversibly inhibiting the TCA (tricarboxylic acid) cycle enzyme aconitase. Micro-positron emission tomography (PET) imaging of subcutaneous xenografts of TSC2-deficient Eker rat uterine leiomyoma-derived ELT3 cells showed rapid uptake of [18F]FACE that was maintained after 72-hour treatment with mTORC1 inhibitor rapamycin. This result suggests that mitochondrial oxidative metabolism is sustained in the presence of rapamycin. Consistent with this finding, treatment of TSC2-deficient cells with rapamycin led to a significant increase in FAO and a decrease in glucose oxidation in vitro as measured by a 14C-CO2 collection metabolic assay. Expression of the A isoform of carnitine palmitoyltransferase I (CPT1A; FAO rate-limiting enzyme) was selectively increased in TSC2-deficient cells. Pharmacological inhibition of CPT1A by ST1326 led to a decrease in FAO, as measured by a 14C-CO2 collection metabolic assay, and a decrease in mitochondrial ATP production, measured by the Seahorse analyzer. CONCLUSIONS: This study proposes a role for FAO in TSC tumor bioenergetics and for CPT1A as a potential therapeutic target in TSC.

The antihyperglycaemic drug metformin has become the most widely prescribed drug treatment for the management of type 2 diabetes mellitus. Despite being prescribed for over 50 years, the precise molecular mechanisms underlying metformin’s therapeutic effects remain poorly understood. Newly recognised health benefits of metformin, irrespective of diabetes status, have led to proposals of ‘re-purposing’ metformin for treatment of cancer, cardiovascular disease and ageing; conditions regularly associated with impaired regulation of the mammalian/mechanistic target of rapamycin complex 1 (mTORC1) but not safely treatable with its inhibitor, rapamycin. Here we report that in the liver, the primary target tissue of metformin, metformin regulates mTORC1 signalling by inhibiting its activation by amino acids in an AMPK-independent manner. Furthermore, we present evidence to suggest that this occurs through a reversible mechanism ‘upstream’ of the amino acid sensor involving inhibition of hepatic uptake of leucine, a potent stimulator of mTORC1 activity. Using gene expression studies, we identified a role for metformin in decoupling uptake of small and large neutral amino acids, such as glutamine and leucine, from a favourable sodium gradient, involving significant reduction in mRNA expression of SNAT2. Consistent with impaired hepatic uptake and removal from the plasma, elevations in plasma concentrations of branched chain amino acids (BCAAs) and glutamine were observed in non-diabetic humans with chronic heart failure (CHF) receiving metformin. Furthermore, elevated plasma concentrations of leucine were significantly associated with improved plasma glucose and fasting insulin resistance index parameter (FIRI). Taken together, these results suggest a role for metformin in controlling mTORC1 via amino acid transport, akin to hepatic protein restriction. This study highlights the potential for ‘re-purposing’ metformin for use as a protein restriction mimetic in treatment of age-related diseases including cardiovascular disease, cancer and diabetes.

Pancreatic ductal adenocarcinoma (PDAC) is among the most devastating of all cancers. It is responsible for only 3% of cancer cases annually but is the cause of over 7% of cancer related deaths. Despite the prevalence of this diseases there remains a scarcity of rational targeted chemotherapies. The most frequently observed driver mutation in PDAC is in the KRAS gene. KRAS is a GTPase protein in the RAS-RAF-MEK-ERK (MAPK) pathway. This pathway regulates vital functions necessary for cell proliferation, differentiation, and survival. Unfortunately, efforts to pharmacologically inhibit KRAS have been unsuccessful. PDAC can be subdivided into two classes: KRAS-dependent and KRAS-independent. KRAS-dependent cell lines acquire numerous genetic mutations yet still require sustained activity of the KRAS protein to survive. These two subtypes of PDAC have distinct genetic and morphological features. One such difference is expression of the Spleen tyrosine kinase (Syk), which is expressed at higher levels in KRAS-dependent cell lines. Syk is a non-receptor tyrosine kinase that functions downstream of KRAS and is an upstream activator of mTORC1. mTORC1 activity is associated with anabolic processes such as protein and lipid synthesis, while its suppression causes activation of the catabolic autophagy pathway. Like KRAS, mTORC1 has proven to be a poor drug target in clinical studies. This issue necessitates the discovery of other therapeutic targets in the pathway. Inhibiting Syk with the inhibitor PRT062607 (Syki) results in decreased mTORC1 activity, increased autophagy, and cell death. In this study we aim to identify compounds that act synergistically with Syki to produce an enhanced therapeutic effect. Synergy can be summarized as a combinational effect greater than the expected additive effect of each agent acting individually. We evaluated the effects of various drug combinations on cell viability and studied the impact of these compounds on the autophagy pathway. We found a synergistic killing effect when cells were treated with Syki and the iron-chelating agent Nocardimicin F (NCF). Live cell imaging assays showed that NCF is a strong activator of the autophagy pathway. Western Blot data suggest that NCF activates the autophagy pathway through a mechanism independent of mTORC1 suppression. Furthermore, our data suggest that the cytotoxicity of Nocardimicin does not result from induction of apoptosis. We hypothesize that cell death proceeds via an autophagy dependent mechanism called autosis. Autosis is a poorly understood process, however, is known to be dependent on the Na+/K+-ATPase. Our findings provide rationale for further study of the effects of iron-chelating compounds in PDAC and suggest that targeting the autophagy pathway is a viable therapeutic strategy.

The mechanisms through which a cell controls its proliferation, differentiation, metabolism, motility, and ultimate survival in response to extracellular cues are largely controlled by the Ras-extracellular signal-regulated kinase (Ras-ERK) and phosphatidylinositol 3-kinase mammalian target of rapamycin (PI3K-mTOR) signaling pathways. Originally delineated as two separate and linear signaling pathways, multitudes of evidence through experimentation have shown that these pathways can co-regulate downstream targets and cellular outcomes. Here, we provide evidence for an additional point of pathway convergence the serine/arginine protein kinase 2 (SRPK2). Originally identified as a target of the mTORC1/S6K signaling pathway, we have shown SRPK2 to be a target of the Ras-ERK-Rsk pathway, as well as the PI3K-AKT. We discovered the S6K, AKT and RSK all phosphorylate SRPK2 at serine 494 in a cell-type, stimulus dependent manner, emphasizing the redundant nature of the AGC kinases. SRPK2 regulates the phosphorylation of the constitutive and alternative splicing factors the SR proteins. This led us to question mTORC1 involvement in splice site selection, and we discovered several alternative splicing events downstream of mTORC1 signaling. We found that the protein levels of the splicing factors ASF/SF2 and hnRNPa2b1 are regulated by mTORC1 signaling, and we hypothesize this is through regulated unproductive splicing and translation (RUST). Interestingly, we found that BIN1, a target of both ASF/SF2 and hnRNPa2b1, is alternatively spliced, following modulations in mTORC1 signaling. These biochemical studies and knowledge gleaned from them will lead to a better understanding of how the cell can regulate protein expression by controlling alternative splicing.

The mammalian target of rapamycin complex 1 (mTORC1), a master regulator of cell growth and proliferation, is aberrantly activated in cancer, genetic tumor syndromes and obesity. Much progress has been made to understand the upstream pathways that regulate mTORC1, most of which converge upon its negative regulator, the Tuberous Sclerosis Complex (TSC) 1-TSC2 complex. However, the cell intrinsic consequences of aberrant mTORC1 activation remain poorly characterized. Using systems in which mTORC1 is constitutively activated by genetic loss of TSC1 or TSC2 and pharmacologically inhibited by treatment with an mTORC1-specific inhibitor rapamycin, we have identified that mTORC1 controls specific aspects of cellular metabolism, including glycolysis, the pentose phosphate pathway, and de novo lipogenesis. Induction of the pentose phosphate pathway and de novo lipogenesis is achieved by activation of a transcriptional program affecting metabolic gene targets of sterol regulatory element-binding protein (SREBP). We have demonstrated that mTORC1 stimulates the accumulation of processed, active SREBP, although details of the molecular mechanism remain to be elucidated. To understand the physiological and pathological relevance of mTORC1-dependent activation of SREBPs and lipogenesis, we explored these findings in the liver and in cancer. While we find that the induction of hepatic SREBP1c and lipogenesis by insulin requires mTORC1, mTORC1 activation is not sufficient to stimulate hepatic SREBP1c in the absence of Akt signaling, revealing the existence of an additional downstream pathway also required for this induction. We demonstrate that this mTORC1-independent pathway involves Akt-mediated suppression of Insig2a, a liver-specific transcript encoding the SREBP1c inhibitor INSIG2. In cancer, our initial findings demonstrate that mTORC1 plays a role downstream of TSC-deficiency and oncogenic PIK3CA and K-Ras to activate lipogenic SREBP targets and de novo lipogenesis. Further studies of the connection between mTORC1 and SREBPs in disease may offer insights into novel therapeutic approaches.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24997号 / 医博第5031号 / 新制||医||1069(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 藤田 恭之, 教授 羽賀 博典, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Les Leucémies Aigües Myéloïdes (LAM) sont des hémopathies malignes hétérogènes de mauvais pronostic qui se caractérisent par une expansion clonale de progéniteurs immatures. De nombreuses dérégulations de voies de signalisation sont retrouvées dans les cellules leucémiques et leur confèrent un avantage de prolifération et de survie. La voie de signalisation mTORC1, qui contrôle la traduction protéique, l’autophagie et plusieurs voies métaboliques, est ainsi constitutivement activée dans les cellules leucémiques. La reprogrammation métabolique notamment via « l’effet Warburg » est un phénomène bien décrit dans les cellules cancéreuses. L’augmentation de l’utilisation de la glycolyse, confère aux cellules tumorales un avantage de survie en favorisant une production rapide d’ATP et d’intermédiaires métaboliques nécessaires pour les biosynthèses de nucléotides, d’acides-aminés et de lipides. C’est donc dans ce contexte que j’ai étudié le métabolisme du glucose dans les cellules de LAM et l’implication de la voie de signalisation mTORC1 dans la dérégulation de ce métabolisme. J’ai tout d’abord identifié par une étude transcriptomique dans la lignée leucémique MOLM-14 que la signalisation mTORC1 contrôle plusieurs voies métaboliques notamment celles permettant l’utilisation du glucose. Ceci a été vérifié dans plusieurs lignées de LAM puisque l’inhibition ou la sur-activation de mTORC1 entrainent respectivement une diminution ou une augmentation de la consommation de glucose et de la production de lactate. De façon intéressante, le niveau d’activation de la voie mTORC1 détermine la sensibilité des cellules leucémiques à l’inhibition de la glycolyse. En effet, lorsque mTORC1 est activé, le blocage de la glycolyse induit de l’autophagie et l’apoptose des cellules leucémiques. A l’inverse, le blocage de mTORC1 induit une reprogrammation métabolique des cellules leucémiques qui utilisent alors principalement la phosphorylation oxydative pour produire l’ATP dont elles ont besoin. Leur survie devient alors indépendante du glucose. A l’inverse des cellules primaires de LAM, les cellules hématopoïétiques immatures normales CD34+ sont moins sensibles au blocage de la glycolyse. Le ciblage du métabolisme du glucose pourrait donc constituer une stratégie thérapeutique intéressante dans les LAM. Je me suis ensuite intéressée aux effets anti-leucémiques induits par l’inhibition de la voie des pentoses phosphates (PP) et plus particulièrement au ciblage de la G6PD (glucose-6-phosphate déshydrogénase) par le composé le 6-aminonicotinamide (6-AN). En effet, une étude de flux métabolique a permis de mettre en évidence qu’une proportion importante de glucose est dirigé vers la voie des PP, laissant suggérer que l’addiction des cellules leucémiques au glucose pourrait être liée à une utilisation augmentée de cette voie annexe. J’ai alors observé que le 6-AN induit une cytotoxicité in-vitro y compris dans les cellules primaires de patients, sans avoir d’effets sur les cellules hématopoïétiques normales et in-vivo dans un modèle de xénogreffe de la lignée MOLM-14 chez la souris NUDE. Cette étude a donc permis de montrer que l’activation constitutive de mTORC1 rend la survie des cellules de LAM dépendante de la glycolyse et crée une sensibilité spécifique à l’inhibition de la G6PD. La dérégulation de la signalisation mTORC1 étant quasi-constante dans les LAM, cibler la G6PD pourrait donc représenter une stratégie thérapeutique intéressante. / Acute Myeloid Leukemia (AML) are heterogeneous hematological diseases with poor prognosis characterized by a clonal expansion of immature progenitors. Many deregulation of signaling pathways are found in leukemic cells and give them an advantage of proliferation and survival. The MTORC1 signaling pathway, which controls protein translation, autophagy and several metabolic pathways, is constitutively activated in leukemic cells. Metabolic reprogramming in particular the "Warburg effect" is a phenomenon well described in cancer cells. High rate of glycolysis has been considered to give tumour cells advantages through rapid production of ATP and intermediates for the synthesis of nucleotides, amino acids, and lipids. In this context, I studied glucose metabolism in AML cells and the involvement of the mTORC1 signaling pathway in the deregulation of this metabolism. First, I identified by a transcriptomic analysis in the MOLM-14 cell line that mTORC1 signaling controls several metabolic pathways including those for glucose utilization. This has been verified in several AML cell lines, since inhibition or over-activation of mTORC1 respectively induces a decrease or an increase in glucose consumption and lactate production. Interestingly, the level of activation of the mTORC1 signaling pathway determines the sensitivity of AML cells to the inhibition of glycolysis. Indeed, when mTORC1 is activated, the blockade of glycolysis induces autophagy and apoptosis of leukemic cells. Conversely, blocking mTORC1 induces metabolic reprogramming of leukemic cells, which then mainly use oxidative phosphorylation to produce ATP for their needs. AML cell survival become independent of glucose. Unlike primary AML cells, survival of normal immature hematopoietic cells CD34+ is only barely affected by the blockade of glycolysis. Thus, targeting the glucose metabolism may constitute an attractive therapeutic strategy in AML. I then investigated the anti-leukemic activity induced by the inhibition of the pentose phosphate pathway (PPP) and more particularly by the specific blockade of G6PD (glucose 6-phosphate dehydrogenase) with the 6-aminonicotinamide (6- AN) compound. Indeed, a metabolic flux analysis demonstrated that a significant proportion of glucose was directed towards the PPP. This result suggested that the addiction of leukemic cells toward glucose might be related to an increased use of PPP. I then observed that the 6-AN induced in vitro cytotoxicity including in primary AML cells from patients without effect on normal immature hematopoietic cells CD34+ and in vivo in a xenograft model of MOLM-14 cell line in the NUDE mouse. This study therefore demonstrated that the constitutive activation of mTORC1 makes AML cells survival dependent on glycolysis, and creates a specific vulnerability to the inhibition of G6PD. Given that deregulation of the mTORC1 signaling pathway is almost constant in AML, targeting G6PD may therefore represent an interesting therapeutic strategy.

Chez les mammifères, le métabolisme énergétique exerce une influence importante sur la fertilité. Chez la femelle comme chez le mâle, une chute ou un excès dans l’apport des besoins nutritionnels induisent des modulations des synthèses hormonales et de la production de gamètes viables. L’objectif de ce travail était 1) de définir le rôle de l’AMPK, AMP-activated protein kinase, un senseur cellulaire des réserves énergétiques de l’organisme, dans la reproduction mâle ; 2) d’étudier l’implication de mTORC1, mammalian target of rapamycin complex 1, un autre indicateur du métabolisme, dans les cellules du système nerveux central régulant la reproduction. Nous avons montré une baisse de la fertilité, associée à une hyperandrogénie Leydigienne et à un dysfonctionnement des spermatozoïdes chez des souris invalidées pour le gène de l’α1 AMPK. De plus, l’exposition in utero à la metformine, un activateur de l’AMPK, induit une baisse du volume testiculaire et de la concentration en testostérone à 17jpc. Enfin, l’inactivation du complexe mTORC1, par ARN interférent ciblant Raptor, dans les cellules bordant l’hypothalamus tend à augmenter la taille de portée, associée à une hausse de la FSH et de la folliculogenèse terminale.En conclusion, ce travail confirme le rôle de ces deux complexes protéiques (AMPK et mTORC1), senseurs énergétiques, dans la fonctionnalité de l’axe hypothalamo-hypophyso-gonadique. / In mammals, the energy metabolism exerts a strong influence on fertility. In females as in males, either a drop or an excess of the nutritional supplies induce modulations of the hormonal synthesis as well as viable gametes production. Our objective was 1) to define the role of AMPK, the AMP-activated protein kinase, a cell sensor of the energy reserves, in male reproduction; 2) to study the involvement of mTORC1, the mammalian target of rapamycin complex 1, another indicator of metabolism, in the cells of the central nervous system that regulate fertility. We have shown a decrease of fertility, linked to a testicular hyperandrogenia and dysfunctional spermatozoa in α1AMPK deficient mice. Moreover, in utero exposure to an AMPK activator, the metformin, induced a decrease in testicular volume and testosterone concentration (17dpc). Finally, inactivation of mTORC1 by interferent RNA in the adjacents cells of the hypothalamus tends to increase litter size, linked to a rise of FSH and the terminal folliculogenesis. In conclusion, this study confirms the role of these two complexes, energetic sensors, on the functionality of the hypothalamo-pituitary-gonadal axis

Nous avons identifié, un ligand de Wnt, Wnt5a, comme faisant partie intégrante du complexe mTORC1, qui régule la fonction lysosomal et favorise le trafic intracellulaire du cholestérol. En diminuant l’activité de mTORC1 et en activant l’axe autophagie-lysosome, Wnt5a adapte les concentration du cholestérol intracellualire aux besoins de la cellule. Wnt5a favorise l’export du cholestérol depuis les endosomal/lysosomal (LELs) vers le réticulum endoplasmique (RE), limite l’accumulation intracellulaire du cholestérol, et protège contre l’athérosclérose. D’un point de vue mécanistique, Wnt5a se lie aux membranes riches en cholestérol et interagit spécifiquement avec la protéine membranaire Niemann-Pick C1 (NPC1), la protéine soluble Niemann-Pick C2 (NPC2), deux protéines lysosomales qui régulent l’export du cholestérol à partir des LELs. En conséquence, l’absence de Wnt5a inhibe la fonction lysosomale et l’autophagie, ainsi que la sortie du cholesterol hors des LELs. Ceci resulte en l’accumulation de larges corps d’inclusion intracellulaires, de larges LELs riches en cholestérol, d’une diminution du cholestérol au niveau du RE. / We identified the Wnt ligand, Wnt5a, as a member of the nutrient/energy/stress sensor, mTORC1 scaffolding complex, which drives lysosomal function and promotes cholesterol trafficking. By decreasing mTORC1 activity and by activating the autophagy-lysosomal axis, Wnt5a senses changes in dietary cholesterol supply, promotes endosomal/lysosomal (LELs) cholesterol egress to the endoplasmic reticulum (ER), and protects against atherosclerosis. Moreover, Wnt5a binds cholesterol-rich membranes and specifically interacts with two lysosomal proteins Niemann–Pick C1 and Niemann–Pick C2 that regulate cholesterol export from LELs. Consequently, absence of Wnt5a decoupled mTORC1 from variations in LELs sterol levels, and this resulted in accumulation of large intracellular inclusion bodies, large LELs and low ER cholesterol.