The vascular endothelium continually senses and responds to both biochemical and mechanical stimuli to regulate vascular function in health and disease. The purpose of this dissertation was to understand the molecular mechanisms by which endothelial cells (ECs) respond to sphingosine 1-phosphate (S1P) and fluid wall shear stress (WSS) to initiate angiogenesis. To accomplish this, a novel cell culture system was developed to study the combined effects of S1P and WSS on inducing EC invasion into three-dimensional (3-D) collagen matrices. EC invasion required the presence of S1P, with the effects of S1P being enhanced by WSS to an extent comparable with S1P combined with pro-angiogenic growth factor stimulation. The extent of EC invasion depended on the magnitude of WSS in a biphasic manner, with the greatest induction occurring at 5.3 dyn/cm2 WSS. Several proteins have been implicated in EC invasion, including calpain, Akt, vimentin, p21-activated kinase (PAK), and membrane type 1-matrix metalloproteinase (MT1-MMP). Interestingly, activations of calpain and MT1-MMP and phosphorylations of Akt, PAK, and vimentin coincided with, and were required for, S1P- and WSS- induced EC invasion. Further, inhibitors of calpain, MT1-MMP, Akt and PAK all attenuated invasion induced by WSS and S1P. Calpain inhibition reduced Akt phosphorylation, vimentin cleavage, and MT1-MMP membrane translocation, suggesting that calpain regulates MT1-MMP via Akt phosphorylation and vimentin remodeling. Akt inhibition also completely blocked MT1-MMP membrane translocation and decreased phosphorylation of PAK and vimentin. In summary, these results suggest a new molecular pathway by which the combination of S1P and WSS stimulates EC invasion through calpain, Akt, PAK and vimentin to regulate activation and membrane translocation of MT1-MMP in 3-D collagen matrices.

Pancreatic ductal adenocarcinoma is an extremely lethal cancer that is difficult to treat. A better understanding of the biology of pancreatic ductal cancer will help to develop targeted therapies that may improve clinical outcomes. Recently, the lipid signaling molecule sphingosine-1-phosphate (S1P) has emerged as a driver of malignant behavior in many types of cancer. Its role in pancreatic cancer remains unknown. Pancreatic cancer cells express high levels of the S1P receptor known as S1PR1, which is the receptor most important for mediating growth and migration through S1P signaling. In addition, the subcellular expression of the sphingosine kinases is altered in pancreatic cancer cells, which may contribute to their malignant behavior. Exogenous S1P increases pancreatic cancer cell migration, while inhibition of S1P signaling decreases the metabolic activity of pancreatic cancer cells as well as their ability to invade and migrate. Taken together, these results demonstrate the importance of S1P signaling in maintaining malignant behavior in pancreatic cancer cells. In addition, inhibition of S1P signaling represents a potential therapeutic target in pancreatic ductal cancer.

Les maladies inflammatoires du système nerveux central (SNC) sont caractérisées par l'altération de la barrière hémato-encéphalique induite par les cellules immunitaires et les cellules tumorales. Il est reconnu que les macrophages peuvent induire l'inflammation en infiltrant la BHE. Lors d'un dommage ou d'une infection du SNC, les macrophages dérivés du sang sont activés. Une fois activés, ceux-ci migrent au site infecté ou endommagé et libèrent des cytokines et médiateurs inflammatoires tels que IL1, TNFα, VEGF. Ces cytokines jouent un rôle essentiel dans l'inflammation du SNC puisqu'elles induisent des chimiokines tels que la sphingosine-1-phosphate (S1P), un sphingolipide fortement exprimé dans les glioblastomes et qui joue un rôle important dans la chimiotaxie et le trafic des cellules immunitaires. Nous avons étudié l'efficacité d'une molécule dérivée de notre diète possédant des propriétés chimiopréventives et anti-inflammatoires, l'épigallocatéchine gallate (EGCG), sur la régulation transcriptionnelle des récepteurs de la S1P à divers stades de différenciation des cellules promyélomonocytaires HL-60. Nous avons d'abord différencié les cellules promyélomonocytaires HL-60 en « macrophages-like » en utilisant un promoteur tumorigène et activateur de la protéine kinase C-Phorbol 12-myristate 13-acétate (PMA). Nous avons démontré que le PMA induit l'adhésion cellulaire et augmente l'expression des transcrits S1P1, S1P2 et S1P5. Nous avons ensuite constaté que les cellules adhérentes semblaient être sensibles à la S1P en induisant la phosphorylation d'ERK, de JNK et de P38 MAPK. Cependant, l'inclusion de l'EGCG avant la différenciation par le PMA inhibe l'induction de ces trois voies MAPK par la S1P. D'autre part, un traitement par l'EGCG au cours de la différenciation, c'est-à-dire simultanément avec le PMA, affecte seulement la voie P38 MAPK. De plus, les cellules différenciées « macrophages-like » devenaient insensibles à l'EGCG. Enfin, nous démontrons que seul le récepteur S1P2, parmi les récepteurs fortement induits par le PMA, diminue lors du prétraitement par l'EGCG. Nos résultats suggèrent que l'EGCG antagonise la réponse à la S1P dans les cellules prédifférenciées via l'inhibition de la signalisation induite par le PMA. Par conséquent, une réponse réduite à la S1P pourrait abroger la migration transendothéliale des monocytes vers le SNC, et prévenir la neuroinflammation, les infections cérébrales secondaires ou certaines pathologies cérébrales conséquentes à l'infiltration de cellules immunitaires. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Neuroinflammation, S1P, EGCG, macrophages, chimiotactisme, chimioprévention

Yes / Although the ability of bioactive lipid sphingosine-1-phosphate (S1P) to positively regulate anti-apoptotic/pro-survival responses by binding to S1P1 is well known, the molecular mechanisms remain unclear. Here we demonstrate that expression of S1P1 renders CCL39 lung fibroblasts resistant to apoptosis following growth factor withdrawal. Resistance to apoptosis was associated with attenuated accumulation of pro-apoptotic BH3-only protein Bim. However, although blockade of extracellular signal-regulated kinase (ERK) activation could reverse S1P1-mediated suppression of Bim accumulation, inhibition of caspase-3 cleavage was unaffected. Instead S1P1-mediated inhibition of caspase-3 cleavage was reversed by inhibition of phosphatidylinositol-3-kinase (PI3K) and protein kinase C (PKC), which had no effect on S1P1 regulation of Bim. However, S1P1 suppression of caspase-3 was associated with increased expression of anti-apoptotic protein Mcl-1, the expression of which was also reduced by inhibition of PI3K and PKC. A role for the induction of Mcl-1 in regulating endogenous S1P receptordependent pro-survival responses in human umbilical vein endothelial cells was confirmed using S1P receptor agonist FTY720- phosphate (FTY720P). FTY720P induced a transient accumulation of Mcl-1 that was associated with a delayed onset of caspase-3 cleavage following growth factor withdrawal, whereas Mcl-1 knockdown was sufficient to enhance caspase-3 cleavage even in the presence of FTY720P. Consistent with a pro-survival role of S1P1 in disease, analysis of tissue microarrays from ER þ breast cancer patients revealed a significant correlation between S1P1 expression and tumour cell survival. In these tumours, S1P1 expression and cancer cell survival were correlated with increased activation of ERK, but not the PI3K/PKB pathway. In summary, pro-survival/anti-apoptotic signalling from S1P1 is intimately linked to its ability to promote the accumulation of pro-survival protein Mcl-1 and downregulation of pro-apoptotic BH3-only protein Bim via distinct signalling pathways. However, the functional importance of each pathway is dependent on the specific cellular context. / Diabetes UK; British Heart Foundation

Atherosclerosis is a chronic inflammatory disease affecting large- and medium-sized arteries and is considered the major cause behind cardiovascular diseases (CVD). Elevated low-density lipoprotein (LDL)-cholesterol and low high-density lipoprotein (HDL)-cholesterol are considered major risk factors for the CVD. HDL mediates a variety of atheroprotective actions, many of them involving the interaction with the scavenger receptor class B, type 1 (SR-B1). Despite the efforts placed in raising HDL-cholesterol, no improvement has been achieved in reducing CVD risk, suggesting that other components of the HDL particles may be responsible for HDL-mediated atheroprotection. One of these may be sphingosine-1-phospate (S1P). In this thesis, the role of S1P receptors (S1PRs) in atherosclerosis is explored, with emphasis in macrophage apoptosis. In particular, the importance of the macrophage S1PR1 and its role in apoptosis and atherosclerosis was evaluated. We demonstrated that diabetes exacerbates atherosclerosis development and myocardial infarction in SR-B1 KO/apoE-hypomorphic mice and that treatment with FTY720, a S1PR agonist, protects against diabetes pro-atherogenic effects. We also show that S1PR1 agonists protected macrophages against apoptosis through phosphoinositide 3-kinase (PI3K)/AKT, and that HDL failed to protect S1PR1 deficient macrophages against apoptosis. In vivo, macrophage S1PR1 deficiency translated into increased atherosclerosis, necrotic core formation and numbers of apoptotic cells in the atherosclerotic plaque. BIM deficiency in BM cells was protective against atherosclerosis development and HDL treatment reduced BIM protein levels in cells exposed to ER stressors, suggesting that the pro-apoptotic protein may be an important target for HDL in macrophages. We conclude that signaling through the S1PRs, in particular S1PR1 is important in controlling macrophage apoptosis and atherosclerosis development. Our data suggests that S1PR1 signaling axis and the pro-apoptotic protein BIM play an important role in mediating HDL anti-apoptotic signaling, however further studies are required to clarify the interaction between all of these factors. / Thesis / Doctor of Philosophy (PhD)

No description available.

Sphingosine kinase 1 (SphK1) is the key enzyme catalyzing the formation of sphingosine-1-phosphate (S1P), which is an important signaling molecule that regulates multiple biological process including inflammatory responses. Elevated SphK1 activity as well as upregulated S1P levels is linked to various diseases such as cancer, fibrosis and sickle cell disease. Therefore, there is a growing interest in studying SphK1 as a potential target for these diseases. Through high-throughput screening, various SphK1 inhibitors have been discovered, among which PF-543 is the most potent and selective inhibitor reported to date (Ki=3.6 nM, >100 fold selectivity for SphK1). Previous research indicated that SphK1 inhibitor PF-543 is effective in reducing S1P levels and slowing down the development of sickle cell disease in vivo. However, the lack of in vivo stability of PF-543 still makes it necessary to develop inhibitors with an improved pharmacokinetic profile. In this study, PF-543 was employed as the lead compound, and the influence of different tails groups and head groups on binding affinity and in vivo stability were investigated. In brief, (R)-prolinol-based derivatives with various tail groups including alkyl, alkoxy and biphenyl groups were synthesized. Their inhibition potency was tested in a broken-cell assay, and hit compounds were further evaluated in a yeast cell assay to determine EC50 values. The U937 cell line and mice model were utilized for hit compounds to quantify S1P reduction in vitro and in vivo. Our preliminary results indicated compound 2.14d was the best hit discovered, with 88% SphK1 inhibition at 1 μM. In addition, compound 2.14d with a Ki of 0.68 μM and an EC50 of 0.15 μM, reduced the S1P of U937 cells by 90% at 1 μM. Its analog with a shorter tail group, 2.14a, reduced plasma S1P levels by 20% in mice (10 mg/kg, 3 h). Further modification of the head group of 2.14d produced compound 3.14c bearing a secondary benzylamine head group, with an EC50 value of 0.39 μM and less in vivo activity (14% plasma S1P reduction at 10 mg/kg, 6 h). / Doctor of Philosophy / Sphingosine-1-phosphate (S1P) is a molecule related to various diseases, such as cancers and inflammatory diseases. Elevated levels of S1P promote the development of these diseases, thus making it necessary to reduce the production of S1P in patients. Since S1P is generated in human body by an enzyme called sphingosine kinase (SphK), inhibiting the activity of SphK can be beneficial for reducing S1P levels. Developing inhibitors for SphK is also a promising strategy for curing such diseases. A very potent inhibitor has been reported, but it could be metabolized quickly into other inactive metabolites in human, which renders it ineffective. To develop better drug candidates, a series of compounds with similar structure has been synthesized and tested for their potency and metabolic stability. Based on analysis of the relationship between the compound structures and activities, several compounds with less potency and different metabolic stability has been prepared and their efficacy in reducing S1P levels has been tested.

L'infiltration des mélanomes par les macrophages (TAM) est souvent corrélée à un mauvais pronostic. Cependant, les mécanismes qui régulent le recrutement et la fonction de ces cellules restent encore mal compris. Des études récentes ont montré un rôle majeur de la sphingosine kinase 1 (SK1) tumorale, l'enzyme qui produit la sphingosine-1-phosphate (S1P), dans le remodelage du stroma associé à la tumeur. Le but de ce projet a été d'étudier le rôle de la SK1 tumorale sur le microenvironnement inflammatoire, et en particulier les macrophages, lors du développement des tumeurs mélaniques. In vitro, nous avons montré que l'inhibition de SK1 dans les cellules de mélanome : 1) bloque la migration des macrophages. A l'inverse, la surexpression de cette kinase dans les cellules tumorales stimule la migration des cellules inflammatoires. Cet effet est dépendant de la S1P et de sa fixation sur les récepteurs S1PR présents à la surface des macrophages ; 2) réduit la sécrétion de TGF-ß et 3) stimule la différenciation des macrophages vers un phénotype M1 antitumoral. Ce phénomène n'est pas dépendant de la S1P, ni des S1PRs, mais de la sécrétion de TGF-ß par les cellules tumorales. En effet, la différenciation macrophagique peut-être réversée par l'addition de TGF-ß recombinant dans le milieu de sécrétion des cellules tumorales. In vivo, nos résultats montrent que la greffe orthotopique, i.e. intradermique, de cellules de mélanome murin invalidées pour la SK1 à des souris syngéniques C57BL/6 est associée à une réduction de la croissance tumorale, comparée à des souris ayant reçu des cellules de mélanome contrôles. De plus, l'invalidation de la SK1 tumorale conduit à une augmentation significative de l'expression de cytokines anti-tumorales ainsi qu'à une polarisation Th1 au sein de la tumeur. Ce phénomène s'accompagne d'une réduction du pourcentage de macrophages M2 CD206+MHCIIlow, et à l'inverse, d'une augmentation du pourcentage de macrophages M1 CD206-MHCIIhigh ainsi que de lymphocytes T CD4+ et CD8+ infiltrés dans la tumeur. Ces résultats suggèrent un rôle clé de la SK1 tumorale dans le recrutement et la polarisation des macrophages dans les mélanomes. Ainsi, l'axe SK1/ TGF-ß pourrait constituer une cible thérapeutique prometteuse dans le contrôle de la croissance de cette tumeur. / Melanoma infiltration by macrophages (TAM) is often correlated with poor prognosis. However, the mechanisms that regulate the recruitment and function of these cells remain poorly understood. Recent studies have shown a major role of tumor sphingosine kinase 1 (SK1), the enzyme that produces sphingosine-1-phosphate (S1P), in tumor stroma remodeling. The aim of this project was to investigate the role of tumor SK1 on the inflammatory microenvironment, particularly macrophages, during the development of melanoma. In vitro, we showed that the inhibition of SK1 in melanoma cells: 1) blocks macrophage migration. Conversely, overexpression of this kinase in tumor cells stimulates the migration of inflammatory cells. This effect is dependent on S1P binding to its receptors (S1PR) on the macrophage surface; 2) reduces the secretion of TGF-ß and 3) stimulates macrophage differentiation towards an antitumor M1 phenotype. The latter phenomenon does not depend on S1P nor S1PRs, but on the secretion of TGF-ß by tumor cells. Indeed, macrophage differentiation can be reversed by adding recombinant TGF-ß in the tumor cell-conditioned medium. In vivo, our results showed that orthotopic injection, i.e. intradermal, of murine melanoma cells invalidated for SK1 in C57BL / 6 syngenic mice was associated with a reduction in tumor growth compared to mice having received control melanoma cells. Furthermore, the invalidation of tumor SK1 leads to a significant increase in the expression of anti-tumor cytokines and a Th1 polarization within the tumor. This phenomenon is accompanied by a reduction in the percentage of CD206+MHCIIlow M2 macrophages, and conversely, an increase in the percentage of M1 macrophages CD206-MHCIIhigh as well as CD4+ and CD8+ cells infiltrated into the tumor. These results suggest a key role of tumor SK1 in the recruitment of macrophages and their polarization in melanoma. Thus, the axis SK1 / TGF-ß could be a promising therapeutic target in controlling the growth of this tumor.

Le traitement du mélanome métastatique a été révolutionné par le développement de thérapies ciblées, qui ont montré un bénéfice significatif sur la survie globale. En particulier, l'inhibition de la sérine-thréonine kinase BRAF, mutée dans 60% des mélanomes, par le Vémurafénib (PLX4032), a montré un gain de survie de 6 à 8 mois comparée à la chimiothérapie de référence, la Dacarbazine. Cependant, une très faible proportion de patients répond sur le long terme. En effet, la majorité des patients développent un échappement thérapeutique dans un délai médian de 6 mois. Des mécanismes cellulaires ont été mis en évidence dans l'apparition de cette résistance acquise, notamment l'implication de MITF, un facteur de transcription majeur des mélanocytes, ainsi que des modifications de l'expression de plusieurs membres de la famille de Bcl-2. Cependant, une meilleure compréhension des mécanismes de résistance aux thérapies anti-BRAF semble essentielle, tout comme l'utilisation de nouvelles approches thérapeutiques combinées afin d'optimiser l'efficacité des traitements et la durée du bénéfice clinique. Notre groupe a récemment identifié des altérations du métabolisme du céramide et de l'un de ses dérivés, la Sphingosine 1-phosphate (S1P), dans les cellules de mélanome humain comparé à des mélanocytes sains. En effet, nous avons montré que la S1P lyase (SPL), qui dégrade irréversiblement la S1P est sous exprimée. Au contraire, l'expression de la sphingosine kinase 1 (SK1), qui produit la S1P, est augmentée dans les cellules de mélanome, conséquence directe de la mutation BRAF. Ces perturbations ont pour effet d'augmenter les niveaux de S1P. Ce lysophospholipide favorise la survie cellulaire ainsi que la résistance vis-à-vis d'agents thérapeutiques dans diverses cellules tumorales. L'objectif de cette thèse a été d'évaluer si le métabolisme de la S1P peut moduler la résistance acquise des cellules de mélanome humain aux inhibiteurs de BRAF. Nous avons montré que la surexpression de la SPL ou l'inhibition pharmacologique de la SK1 (SKI-I) sensibilise les mélanomes métastatiques à l'apoptose induite par la thérapie ciblée. Ce phénomène est associé à une diminution de MITF et de l'une de ses cibles directes, la protéine anti-apoptotique Bcl-2. La diminution d'expression protéique de MITF peut être réversée par un traitement de S1P exogène. De plus, nous avons montré pour la première fois une augmentation de l'expression des récepteurs 1 et 3 à la S1P (S1PR1 et S1PR3), dans les cellules de mélanome présentant une résistance acquise au PLX4032. Ces modifications sont associées à l'expression accrue de MITF. La surexpression de la SPL, le traitement par le SKI-I ou par des inhibiteurs ciblant les S1PR1 et S1PR3, surmonte la résistance acquise de ces cellules au PLX4032 via la diminution d'expression des S1PRs, de MITF, et de Bcl-2. Par conséquent, en contrôlant l'expression de protéines clés de la survie et de la résistance, le métabolisme de la S1P représente une nouvelle approche thérapeutique pour améliorer l'efficacité des thérapies ciblées. / The treatment of metastatic melanoma has changed considerably in recent years with the development of targeted therapies, which have shown a significant benefit in overall survival. In particular, the inhibition of the frequently mutated serine-threonine kinase BRAF, by Vemurafenib (PLX4032) showed that survival rates increase by 6 to 8 months compared to standard chemotherapy, Dacarbazine. However, a very small proportion of patients will respond to the long term, and the majority of patients relapses in a median of 6 months. Cellular mechanisms have been identified in the appearance of this acquired resistance, including the involvement of MITF, a major transcription factor of melanocytes, as well as changes in the expression of several members of Bcl-2 family. However, a better understanding of these mechanisms seems essential, as is the use of new therapeutic strategies to optimize treatment efficacy and duration of clinical benefit. Our group recently showed some alterations of ceramide metabolism and its derivative sphingosine 1-phosphate (S1P) in human melanoma cells compared to healthy melanocytes. For instance, S1P lyase (SPL), which degrades S1P, is under-expressed. Conversely, sphingosine kinase 1 (SK1), which produces S1P, is over-expressed in tumor cells, as a direct result of BRAF mutation. These alterations increases the levels of S1P. This lysophospholipid promotes cell survival and the resistance to therapeutic agents in a variety of tumor cells. This PhD project aimed at defining whether S1P metabolism could modulate the resistance of human melanoma cells to PLX4032. Here, we show that SPL overexpression or pharmacological inhibition of SK1 by SKI-I sensitizes metastatic melanoma cells to PLX4032-induced apoptosis. This phenomenon is associated with a decreased expression of the master regulator of melanocyte differentiation MITF as well as its direct cellular target Bcl-2. The decrease in MITF protein can be reversed by treating cells with exogenous S1P. Interestingly, we also report for the first time an increased expression of SK1 as well as the S1P receptors, S1PR1 and S1PR3, in melanoma cells with acquired resistance to PLX4032 as compared to sensitive counterparts. These modifications are associated with high expression of MITF. Overexpression of SPL, treatment with SKI-I or antagonists of S1PR1 ans S1PR3, strongly overcomes acquired resistance to PLX4032 through a decrease in the expression of S1PR, MITF as well as Bcl-2. Thus, by controlling the expression of key proteins in melanoma cell survival and resistance, S1P metabolism could represent a new therapeutic approach to enhance the effectiveness of targeted therapies.

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(医学) / 甲第23789号 / 医博第4835号 / 新制||医||1057(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 渡邊 直樹, 教授 松田 道行, 教授 寺田 智祐 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM