<p>During oncogenesis, cancer cells go through metabolic reprogramming to maintain their high growth rates and adapt to changes in the microenvironment and the lack of essential nutrients. Several types of cancer are dependent on de novo fatty acid synthesis to sustain their growth rates by providing precursors to construct membranes and produce vital signaling lipids. Fatty acid synthase (FASN) catalyze the terminal step of de novo fatty acid synthesis and it is highly expressed in many types of cancers where it’s up-regulation is correlated with cancer aggressiveness and low therapeutic outcome. Many FASN inhibitors were developed and showed potent anticancer activity however, only one inhibitor advanced to early stage clinical trials with some dose limiting toxicities. Using a modified fluorescence-linked enzyme chemoproteomic strategy (FLECS) screen, we identified HS-106, a thiophenopyrimiden FASN inhibitor that has anti-neoplastic activity against breast cancer in vitro and in vivo. HS-106 was able to inhibit both; purified human FASN activity and cellular fatty acid synthesis activity as evaluated by radioactive tracers incorporation into lipids experiments. In proliferation and apoptosis assays, HS-106 was able to block proliferation and induce apoptosis in several breast cancer cell lines. Several rescue experiment and global lipidome analysis were performed to probe the mechanism by which HS-106 induces apoptosis. HS-106 was found to induce several changes in lipids metabolism: (i) inhibit fatty acids synthesis. (ii) Inhibit fatty acids oxidation as indicated by the ability of inhibiting Malonyl CoA accumulation to block HS-106 induced apoptosis and the increase in the abundance of ceramides. (iii) Increase fatty acids uptake and neutral lipids formation as confirmed 14C Palmitate uptake assay and neutral lipids staining. (iv)Inhibit the formation of phospholipids by inhibiting de novo fatty acid synthesis and diverting exogenous fatty acids to neutral lipids. All of these events would lead to disruption in membranes structure and function. HS-106 was also tested in Lapatinib resistant cell lines and it was able to induce apoptosis and synergizes Lapatinib activity in these cell lines. This may be due the disruption of lipid rafts based on the observation that HS-106 reduces the expression of both HER2 and HER3. HS-106 was found to be well tolerated and bioavailable in mice with high elimination rate. HS-106 efficacy was tested in MMTV neu mouse model. Although did not significantly reduced tumor size (alone), HS-106 was able to double the median survival of the mice and showed potent antitumor activity when combined with Carboplatin. Similar results were obtained when same combinations and dosing schedule was used in C3Tag mouse model except for the inability of HS-106 affect mice survival.</p><p>From the above, HS-106 represent a novel FASN inhibitor that has anticancer activity both in vivo and in vitro. Being a chemically tractable molecule, the synthetic route to HS-106 is readily adaptable for the preparation of analogs that are similar in structure, suggesting that, the pharmacological properties of HS-106 can be improved.</p> / Dissertation

This research investigated the changes in body adipocyte size and density as well as parameters relating to adipogenesis in growing cobia, Rachycentron canadum. The cobia from a same batch were sampled 5 , 7 , 9 , 13 , 17 , 21 , 26 and 31 weeks after hatching, Liver activities of fatty acid synthase, (FAS), mailc enzyme (ME), and glucose-6-phosphate dehydrogenase (G6PDH) as well as serum total triacylglycerol (TG), total cholesterol (TC), phospholipid (PL), non-esterified fatty acid (NEFA) were assayed. Adipocyte abundance and size spectrum in liver, ventral muscle and dorsal muscle were determined. The cobia attained an average weight of 13.6 ¡Ó 0.5 g at week 5, and were cultured in nursery outdoor ponds until an average weight 87.5 ¡Ó 3.8 g when moved to open ocean cages. They grew to an average weight 1690.8 ¡Ó 106.6 g at week 31. The crude lipid content in liver, ventral and dorsal muscle increased significantly with fish age, and were 36.1 ¡Ó 3.2 g, 14.8 ¡Ó 0.2g, 4.6 ¡Ó 0.0 g at week 31. No significant change was found in hepatic FAS activity. Hepatic ME activity increased significantly after the fish were stocked in the cages. Whilr hepatic ME activity reduced with age, TG and PL were both increased significantly with age. While adipocyte diameter in liver showed a decreasing trend as the fish grew its abundance increased significantly at week 13 and maintained between 3070¡ã3356 x 104 cell/g tissue during week 17-31. Similarly, adipocyte abundance in ventral muscle increased significantly at week 13, and maintained between 255¡ã269 x 104 cell/g tissue afterwards. There was no significant change in size of ventral muscle adipocyte as fish grew. These results indicate that the adipocyte hyperplasia occurred during week 7- 13, when body weight was 100- 400 g. Adipocyte hypertrophy was observed when the fish reached week 31, there was a sign of adipocyte hypertrophy in liver when the fish were at the last phase of nursery outdoor ponds before being moved to the offshore cages.

Obesity is a disorder of energy imbalance and the most prevalent nutritional diseases in developed countries. Besides, obesity is also strongly associated with health problems such as type 2 diabetes (NIDDM), hypertension, hyperlipidaemia, cardiovascular diseases and cancers. However, the defects in lipid metabolism underlying obesity-related disorders are extremely complicated. Thus, extensive studies on the mechanism of endogenous fatty acids synthesis would be one of the keys to elucidate molecular pathogenesis of obesity. In liver or adipose, fatty acid synthase (FAS) utilizes acetyl-CoA, malonyl-CoA and NADPH to synthesize long-chain fatty acids (C16 or C18), which can be converted to triglycerides and stored as fat. During lactation, thioesterase II (TE II) expresses in mammary glands and interacts with FAS to produce medium-chain fatty acid (primarily C10) in milk, which provides immune protection and energy for the newborn. TE II causes premature termination of fatty acid synthesis catalyzed by FAS and releases medium-chain fatty acids. Unlike long-chain fatty acids, medium-chain fatty acids can enter mitochondria directly for beta-oxidation to generate ATP, thus provide energy more efficiently. Since TE II gene expression is under strict regulation, we utilized adenovirus gene transfer techniques to deliver and express TE II in hepatocytes. It was postulated that expression of TE II in hepatocytes might result in the increase of ATP and reduction of long-chain fatty acids, subsequently decrease the fat production. Recombinant adenovirus was used as gene delivery system for TE II because of its high titer, wide host range, and transduction efficiency. In the present study, we have generated and characterized the recombinant Ad-TE II by PCR, western blot analysis, and enzymatic assay, respectively. By using Ad-GFP, we have determined the optimal multiplicity of infection (MOI) for adenovirus to infect HepG2 cells is about 100-200. Adenovirus-mediated TE II expression in hepatocytes was demonstrated by western blot as well as TE II enzymatic assay. We have demonstrated that the adenovirus-mediated TE II expression was slightly cytotoxic to hepatocytes. Besides, an increase of free fatty acids, asparate transaminase, lactate dehydrogenase levels, as well as ATP synthesis was also noted in the TE II-expressed hepatocytes. The enhanced the release of asparate transaminase (AST/GOT) and lactate dehydrogenase (LDH) after TE II expression in the hepatocytes further supported its cytotoxcity to hepatocytes. In the future, we will carry out experiments to further characterize the effects of TE II expression on cellular lipid metabolism through adenovirus gene delivery. We hope that the present studies will not only provide further insights into mammalian lipid metabolism, but also enable us to evaluate the therapeutic potential of TE II on the treatment of obesity and its related disorders.

Prostate cancer is the most common newly diagnosed non-skin cancer and the second leading cause of cancer death in men. It is a unique neoplasm because of the large discrepancy between its clinical incidence and the much higher incidence of latent cancer. Predicting the prognosis of prostate cancer, especially the cancers detected incidentally or by screening, remains a clinically important problem. Immunoreactivity for Onco-antigen 519 (OA- 519), a recently described fatty acid synthase (FAS), has been associated with poor prognosis in breast cancers. The authors have previously shown that its detection in prostate cancer correlated with high-grade, large volume, and advanced stage tumors. This study examines the association between OA- 519 immunoreactivity in primary prostate cancer and disease progression. The authors used immunohistochemistry with an affinity-purified anti-OA-519 antibody and examined primary prostate cancers (stages A1 to D1) from 99 men with a mean follow-up of 4 years (range= 2 to 9.3). Survival analysis was used to evaluate differences in progression-free survival. OA-519 immunoreactivity was seen in 56 (57%) of the 99 primary prostate cancers examined. OA-519-positive cancers were more likely to progress than the OA- 519-negative cancers (P < .04). Univariate survival analysis showed that OA- 519 (FAS), histological grade (Gleason score), and clinical stage were significant predictors of disease progression. Multivariate analyses of all cases showed that only histological grade was significant. However, multivariate analysis of the 85 cancers with Gleason scores 2-7 (ie, low to intermediate grade) showed OA-519 (FAS) immunoreactivity to be the only statistically significant predictor of cancer progression (P<.02). Expression of the fatty acid synthase OA-519 by prostate cancers is potentially a clinically useful predictor of disease progression. It appears to be independent of histological grade (Gleason score), at least in cancers with low to intermediate grades. Further studies are needed to evaluate the role of fatty acid synthase in malignancy and the potential therapeutic implications of enzyme blockers.

Mit dem Beginn der Industrialisierung stieg in den westlichen Kulturen rasant die Prävalenz von Krankheitsbildern wie Adipositas, arterieller Hypertonie, Typ-2-Diabetes Mellitus und Hyperlipidämie, die als Cluster eines multifaktoriellen Krankheitsbildes namens „Metabolisches Syndrom“ (MTS) verstanden werden. Tierstudien, in denen durch die Inhibition der Fettsäuresynthase (FASN) ein rapider Abfall des Körpergewichts in Mäusen erzeilt wurden, bestätigen zunehmend genetische Prädispositionen als ursächlich für die Ausbildung des MTS. Um herauszufinden ob und in welchem Ausmaß das FASN-Gen mit humanen Merkmalen des MTS assoziiert ist, wurde das Gen in 48 nicht verwandten ostdeutschen Probanden sequenziert. Acht repräsentative tagging-SNPs wurden dabei identifiziert, anschließend in 1311 deutschen Probanden (Erwachsene) genotypisiert und in Fall-Kontroll-Studien zwischen 389 schlanken Probanden (BMI ≤ 25kg/m²) vs. 446 adipösen Teilnehmern (BMI ≥ 30kg/m²) sowie zwischen 502 glukosetoleranten Probanden (NGT) vs. 640 Probanden mit Typ-2-Diabetes (T2D) miteinader verglichen. Für den Polymorphismus rs2229422 (P = 1.3x10-5 adjustiert auf Alter, Geschlecht und Diabetes-status) konnten die stärksten Assoziationen mit BMI und weiteren Merkmalen der Fettleibigkeit identifiziert werden (adjustiert P < 0.05). Des Weiteren wurde der zuvor in der Literatur beschriebene protektive Einfluss der Val1483Ile Substitution (rs2228305) gegenüber Adipositas, sowie der geschlechts-spezifische Effekt auf den BMI bestätigt (adjustiert, P = 0.03).

The study was to investigate the changes in (1) triacylglycerol (TAG) contents and its relationship to (2) lipid synthesis- and metabolism-related enzyme activity and (3) their gene expression in cobia (Rachycentron canadum) during the fast growth period (from October 2006 to April 2007) in ventral muscle and liver in Hsiao-Lu-Chiao island in southwestern Taiwan. The crude lipid was 12% for fed diet, 30-40% for liver while 13% in February and 11% to 9% in other month for muscle. The TAG content of crude lipid was 36 % for fed diets, and from 22% (December) to 40% (February) for muscle, and from 63% (October to February) to 47% (March) for liver. Oil red-O (ORO) staining showed that TAG accumulated in muscle in February but in December in liver. Muscle TAG contents and enzyme activities and mRNA levels of GPDH and FAS increased in February. A decrease in GPDH enzyme activity and mRNA levels but an increase in PEPCK enzyme activity and mRNA levels indicate the increased supply of acetyl-CoA for fatty acid synthesis is in muscle. An increase in FATP2 mRNA levels suggest the influx of fatty acid also contributes to increased fatty acid accumulation in muscle.In liver, TAG and fatty acid contents decreased in March April but increased FAS and PEPCK enzyme activity and mRNA levels. It is possible that fatty acid synthesis is enhanced in March, but a fast transport to other organs results in a net decline in liver fatty acid contents and subsequently a decrease in TAG contents. FATP contents decreased in March-April mRNA, indicating that the influx of fatty acid in decreasing in liver in adult fish. GPDH and GAPDH were not related to lipid metabolism in liver. These data from enzyme activity and mRNA level, demonstrated that a potentially increase in acetyl-CoA via PEPCK contributes to fatty acid synthesis and GPDH-mediated synthesis of G-3-P provide the C skeleton for TAG synthesis.

Fatty acid synthase (FAS) is over-expressed in many human cancers and its activity is required for cancer cell survival. To understand why FAS is over-expressed, we compared in breast cancer cells the utilization of fatty acids synthesized endogenously by FAS to those supplied exogenously in the culture medium. We found that endogenously synthesized fatty acids are esterified to the same lipid and phospholipid classes in the same proportions as those derived exogenously and that some endogenous fatty acids are excreted. Thus, FAS over-expression in cancer does not fulfill a specific requirement for endogenously synthesized fatty acids. We next investigated whether lipogenic activity mediated by FAS was, instead, involved in the maintenance of high glycolytic activity in cancer cells. By culturing breast cancer and non-cancer cells in anoxic conditions, we increased glycolysis 2-3 fold but observed no concomitant increase in lipogenesis. More research is needed to understand why FAS is over-expressed in cancer.

Fatty acid synthase (FAS) is over-expressed in many human cancers and its activity is required for cancer cell survival. To understand why FAS is over-expressed, we compared in breast cancer cells the utilization of fatty acids synthesized endogenously by FAS to those supplied exogenously in the culture medium. We found that endogenously synthesized fatty acids are esterified to the same lipid and phospholipid classes in the same proportions as those derived exogenously and that some endogenous fatty acids are excreted. Thus, FAS over-expression in cancer does not fulfill a specific requirement for endogenously synthesized fatty acids. We next investigated whether lipogenic activity mediated by FAS was, instead, involved in the maintenance of high glycolytic activity in cancer cells. By culturing breast cancer and non-cancer cells in anoxic conditions, we increased glycolysis 2-3 fold but observed no concomitant increase in lipogenesis. More research is needed to understand why FAS is over-expressed in cancer.

No description available.

Au niveau mondial, la tuberculose est toujours l’une des dix premières causes de mortalité. L’un des principaux facteurs expliquant ce phénomène est l'émergence de souches du bacille tuberculeux, Mycobacterium tuberculosis, résistantes aux antibiotiques. Ainsi, l’OMS a déclaré prioritaire le développement d'une nouvelle génération d’antituberculeux qui soient efficaces contre les souches résistantes. Des études de criblage phénotypique récentes sur M. tuberculosis ont montré que des voies métaboliques de composés de l’enveloppe, telles que la biosynthèse des acides mycoliques, représentent des cibles thérapeutiques très pertinentes. Les acides mycoliques entrent dans la composition de facteurs de pathogénicité lipidiques du bacille tuberculeux. Ils portent divers types de fonctions chimiques déterminantes pour leurs rôles biologiques. Le système Fatty Acid Synthase de type II (FAS-II) impliqué dans leur biosynthèse constitue une cible thérapeutique validée. En effet, il est essentiel à la survie du bacille, possède des caractéristiques uniques, et a un rôle-clé dans sa virulence et sa persistance chez les organismes infectés. De plus, le système FAS-II est la cible de plusieurs médicaments antituberculeux tels que l’isoniazide et l’éthionamide. FAS-II est constitué d'un ensemble d'enzymes monofonctionnelles acyl carrier protein (ACP)-dépendantes. Une partie de ces protéines a été identifiée par des approches de génomique classique. Malgré cela, 20 ans après le séquençage du génome de M. tuberculosis, la composition complète de ce système ainsi que sa fonction précise restent encore à caractériser. L’objectif des travaux de recherche menés durant ma thèse est de caractériser le système FAS-II mycobactérien selon un nouvel angle d'attaque, à l'échelle du système entier. L'approche adoptée visait à i) développer et valider des méthodes expérimentales pour isoler le système FAS-II sous une forme la plus complète possible, ii) décrypter en profondeur la composition en protéines de FAS-II (collaboration équipe O. Schiltz, IPBS) ; iii) identifier de nouvelles enzymes partenaires potentielles. La 1ère partie de ce travail a consisté à mettre au point l'isolement du système FAS-II de deux mycobactéries, M. smegmatis, une espèce-modèle à croissance rapide, et M. bovis BCG, la souche vaccinale très proche de M. tuberculosis. Deux stratégies complémentaires ont été développées : la co-immunoprécipitation et la Single Step Affinity Purification (SSAP). Plusieurs enzymes du système FAS-II ont été utilisées alternativement comme appâts pour co-purifier des protéines partenaires. De nombreux paramètres ont été optimisés afin d'améliorer le taux d'enrichissement en FAS-II et de conserver le plus possible l’intégrité du système : nature de la protéine-appât, délétion du gène endogène (SSAP), conditions de lyse des bactéries, pontage chimique, étapes de purification... La 2ème partie de ma thèse a porté sur l'analyse par protéomique des fractions de purification obtenues et la 3ème partie a consisté à identifier de nouvelles enzymes partenaires. Le traitement des données a permis 1) de mettre en évidence des interactions physiques entre certaines enzymes du système FAS-II ; 2) de démontrer la présence d’une nouvelle enzyme partenaire du système : cette protéine, nommée HadD, est potentiellement impliquée dans l’une des étapes-clés de la biosynthèse des acides mycoliques nécessaire pour l’introduction des groupements fonctionnels sur ces lipides. / Globally, tuberculosis is still one of the top ten leading causes of death. In 2015, the World Health Organization (WHO) reported nearly 480,000 new cases of multidrug-resistant tuberculosis and called for the development of new antituberculosis agents. Recent phenotypic screening studies on Mycobacterium tuberculosis have shown that envelope metabolic pathways, such as mycolic acid biosynthesis, are highly relevant therapeutic targets. Mycolic acids constitute the lipid moiety of pathogenicity factors of the tubercle bacillus. They are essential mycobacterial fatty acids holding various chemical functions decisive for their biological roles. The Fatty Acid Synthase type II (FAS-II) system involved in their biosynthesis is a relevant and validated therapeutic target. Indeed, it is essential to the survival of the bacillus, has unique features, and plays a key role in its virulence and persistence in infected organisms. In addition, the FAS-II system is the target of several antituberculosis drugs such as isoniazid and ethionamide. FAS-II is made of discrete monofunctional acyl carrier protein (ACP)-dependent proteins. Some of these proteins have been identified by conventional genomic approaches. However, 20 years after the M. tuberculosis genome sequencing, the complete composition of this system and its precise function remain poorly known. The objective of my PhD project is to characterize the mycobacterial FAS-II multienzyme system with a completely new line of attack, at the scale of the entire system. The adopted approach was aimed at i) developing and validating experimental methods to isolate the FAS-II system in the most complete form, ii) deciphering the FAS-II protein composition (collaboration with O. Schiltz's team, IPBS); iii) identifying potential new partner enzymes. The first part of this work consisted of developing the isolation of the FAS-II system from two mycobacteria, M. smegmatis, a fast-growing model species, and M. bovis BCG, the vaccine strain very close to M. tuberculosis. Two complementary strategies were developed: co-immunoprecipitation and Single Step Affinity Purification (SSAP). Several enzymes of the FAS-II system were used alternatively as a bait in an attempt to co-purify partner proteins. To improve the level of FAS-II enrichment and to preserve as much as possible the integrity of the system, many parameters were optimized: nature of the bait protein, endogenous gene deletion (SSAP), bacteria lysis conditions, chemical cross-link, purification steps… The second part of my thesis focused on the proteomics analysis of the purification fractions obtained and the third part consisted in identifying new partner enzymes. The data processing allowed: 1) to demonstrate the presence of physical interactions between some FAS-II system enzymes; 2) to demonstrate the presence of a new partner enzyme of the system: this protein, called HadD, is potentially involved in one of the key steps of the mycolic acid biosynthesis pathway necessary for the introduction of functional groups on these lipids. The results obtained thanks to the optimized co-immunoprecipitation and SSAP protocols have contributed to the knowledge of the mycobacterial FAS-II system and bring promising leads for the discovery of new partner enzymes.