Une transition épithélio-mésenchymateuse (TEM) et une expression élevée de la sphingosine kinase 1 (SPHK1) sont souvent observées dans les cancers. Notre étude du génome et du transcriptome d'adénocarcinomes pulmonaires (AP) montre que l'expression élevée de SPHK1 est en rapport, d'une part, avec des gains de la région incluant le locus SPHK1 et, d'autre part, avec une signature d'expression génique de TEM dans des tumeurs invasives. L'expression de SPHK1 est restreinte aux cellules tumorales. La surexpression de SPHK1 dans des cellules d'AP et l'exposition à son produit, la sphingosine-1-phosphate (S1P), entraînent une TEM, de manière réversible pour la S1P. La surexpression de SPHK1 active aussi NF-kB. La surexpression du facteur anti-apoptotique FLIP active NF-kB, induit une TEM et augmente l'expression de SPHK1, suggérant une boucle d'amplification entre NF-kB et SPHK1. Une TEM et la surexpression de FLIP ont été impliquées dans la résistance primaire aux inhibiteurs pharmacologiques de l'EGFR (EGFR TKI). Nous montrons que la surexpression de SPHK1 dans des cellules A549 diminue modestement la sensibilité au gefitinib, alors que l'inhibition de SPHK1 ou la déplétion du sérum en S1P l'augmentent modestement. L'invalidation de SPHK1 entraîne l'apoptose d'A549 y compris quand FLIP est surexprimé. L'activation et le maintien d'une TEM sont généralement attribués à des signaux contextuels du stroma. Cette thèse montre que les cellules tumorales elles-mêmes favorisent la surexpression de SPHK1 qui peut induire une TEM de façon autonome. De plus, la surexpression de FLIP impliquée dans la résistance aux EGFR TKI, n'empêche pas l'apoptose induite par l'invalidation de SPHK1. / Epithelial-mesenchymal transition (EMT) and sphingosine kinase 1 (SPHK1) high expression are often seen in cancers. Our study of genomic and gene expression data in pulmonary adenocarcinomas (AP) shows that SPHK1 high expression correlates with both gains in the region encompassing the SPHK1 locus, and an EMT gene expression signature in invasive tumors. SPHK1 expression is restricted to tumors cells. SPHK1 overexpression in AP cells, as well as exposure to its productsphingosine-1-phosphate (S1P),induce an EMT -in a reversible manner for S1P. SPHK1 overexpression also activates NF-kB. Overexpression of FLIP – an antiapoptotic factor - activates NF-kB, induces an EMT, and increases SPHK1 expression, suggesting an amplification loop between NF-kB and SPHK1. EMT and FLIP overexpression are known to favor primary resistance to EGFR pharmacological inhibitors (EGFR TKI). We show that SPHK1 overexpression in A549 cells slightly decreases cell sensitivity to gefitinib, while pharmacologic inhibition of SPHK1 or serum S1P depletionincrease it. Downregulation of SPHK1 expression induces apoptosis of A549 cells even when FLIP is overexpressed. Activation and maintenance of EMT are generally attributed to contextual signals from the stroma. Here, we show that tumor cells themselves favor SPHK1 overexpression, which can led to EMT in cell-autonomous manner. In addition, FLIP overexpression which is implicated in EGFR TKI resistance, cannot prevent apoptosis that is induced by SPHK1 invalidation.

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.

Doctor of Philosophy / In healthy individuals, the autoimmune system is the body's natural defense against foreign materials and organisms. The main tools utilized for this defense mechanism are immune cells. However, in patients suffering from autoimmune diseases, the autoimmune system is overactive resulting in its attack on healthy cells, which leads to reduced or eliminated function of the targeted organs. To suppress these overreactive immune responses, pharmaceutical intervention is needed. An integral part of autoimmune response is the lipid sphingosine-1-phosphate (S1P). Interactions of S1P with its response-inducing receptors prompts the release of immune cells, lymphocytes in particular, from lymph tissue to migrate and participate in the invoked immune response. The pharmaceutical industry has produced five FDA approved drugs that disrupt this S1P-receptor interaction by blocking the receptor to reduce the autoimmune response in patients suffering from autoimmune diseases such as multiple sclerosis and ulcerative colitis. However, these treatments had adverse side effects on the cardiovascular system due to the presence of S1P receptors in the heart. Due to this, there is attraction to target a different node of the S1P signaling pathway to avoid these side effects while still suppressing the immune response. A node that is a viable target for therapeutic target that has recently become the focus of medicinal chemistry campaigns is the transporter protein spinster homolog 2 (Spns2). This protein is responsible for the transport of S1P from intracellular space to extracellular space to interact with its receptors and induce the immune response. Recently, our group has developed several effective inhibitors of Spns2. In this dissertation, several improvements of previously reported inhibitors are revealed. The pinnacle of this work is the development of 4.22v that is optimized to have drug-like properties for testing in mice. Administration of 4.22v to mice resulted in reduced circulating lymphocytes and without showing signs of toxicity following chronic dosing for 14 days. These results suggest that 4.22v is a potential drug candidate and is currently undergoing further biological evaluation.

Cholangiocarcinoma (CCA) has a high mortality rate and its occurrence is rising. This increase prompts the need for improved CCA treatments. Studies have suggested that CCA is highly reliant on the sphingosine-1-phosphate-receptor-2 (S1PR2) and sphingosine kinase 2 (SphK2). Recently, a competitive SphK2 inhibitor, ABC294640, has been approved for clinical trial. ABC294640 has the potential to treat CCA, which is support by a phase I clinical study that was able to temporarily treat a patient suffering from metastasized CCA with ABC294640. To determine the viability of ABC294640 as a treatment for CCA, this study focused on determining the effects of ABC294640 on rat CCA cell lines. We found that ABC294640 inhibited the growth and migration of CCA and CAFs cells. The growth and count of 3-D organotypic co-culture of CCA and CAFs, which forms the “duct-like” structures, were reduced by ABC294640. The potential of inhibiting SphK2 as a treatment for CCA is supported by our finding of increased expression of S1PR2 and SphK2 in CCA patient liver samples. In conclusion, ABC294640 represents a potential therapeutic agent for CCA.

Indiana University-Purdue University Indianapolis (IUPUI) / Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that has proven to be an important signaling molecule both as an extracellular primary messenger and as an intracellular second messenger. Extracellular S1P acts through a family of five S1P receptors, S1PR1-5, all of which are G protein-coupled receptors associated with different G proteins. Previous work from our laboratory shows that externally applied S1P increases the excitability of small-diameter sensory neurons by enhancing the action potential firing. The increased neuronal excitability is mediated primarily, but not exclusively, through S1PR1. This raises the question as to which other S1PRs mediate the enhanced excitability in sensory neurons. To address this question, the expression of different S1PR subtypes in small-diameter sensory neurons was examined by single-cell quantitative PCR. The results show that sensory neurons express the mRNAs for all five S1PRs, with S1PR1 mRNA level significantly greater than the other subtypes. To investigate the functional contribution of other S1PRs in augmenting excitability, sensory neurons were treated with a pool of three individual siRNAs targeted to S1PR1, R2 and R3. This treatment prevented S1P from augmenting excitability, indicating that S1PR1, R2 and/or R3 are essential in mediating S1P-induced sensitization. To study the role of S1PR2 in S1P-induced sensitization, JTE-013, a selective antagonist at S1PR2, was used. Surprisingly, JTE-013 by itself enhanced neuronal excitability. Alternatively, sensory neurons were pretreated with FTY720, which is an agonist at S1PR1/R3/R4/R5 and presumably downregulates these receptors. FTY720 pretreatment prevented S1P from increasing neuronal excitability, suggesting that S1PR2 does not mediate the S1P-induced sensitization. To test the hypothesis that S1PR1 and R3 mediate S1P-induced sensitization, sensory neurons were pretreated with specific antagonists for S1PR1 and R3, or with siRNAs targeted to S1PR1 and R3. Both treatments blocked the capacity of S1P to enhance neuronal excitability. Therefore my results demonstrate that the enhanced excitability produced by S1P is mediated by S1PR1 and/or S1PR3. Additionally, my results indicate that S1P/S1PR1 elevates neuronal excitability through the activation of mitogen-activated protein kinase kinase. The data from antagonism at S1PR1 to regulate neuronal excitability provides insight into the importance of S1P/S1PR1 axis in modulating pain signal transduction.