La progression tumorale en cancer métastatique implique la perte de fonctions oncosuppressives, comme c'est le cas dans le mélanome. Une migration cellulaire aberrante est caractéristique de la progression du mélanome. SYK (Spleen tyrosine kinase) est une tyrosine kinase cytoplasmique impliquée dans la suppression tumorale du cancer du sein et du mélanome. Dans la peau, SYK est exprimée dans les mélanocytes mais est fréquemment réprimée épigénétiquement dans le mélanome. Nous avions pu montré que cette perte était associée à un échappement de la sénescence. Qu'elle puisse réguler la migration des cellules tumorales et la formation des métastases reste peu connu. Dans mes travaux j'ai utilisé des approches gain et perte de fonction pour analyser l'effet de SYK sur les mélanomes humains et murins. Respectivement, la réexpression et l'extinction de SYK diminue et augmente la migration, l'invasion et les métastases des cellules de mélanome. L'extinction de SYK induit notamment un phénotype et une signature mésenchymateuse. Notre étude dévoile ce rôle pour SYK dans la répression d'une adhérence dépendante des intégrines, points de tractions et plateforme de signalisation de la migration, et souligne l'importance la perte de SYK dans la formation de métastases. Pour clarifier le rôle de SYK dans la progression du mélanome, j'ai généré un modèle murin de KO conditionnel de SYK spécifique des mélanocytes concomitants à une perte de Pten et de l'activation de BrafV600E. Des résultats préliminaires suggèrent que la perte de SYK n'accélère pas la formation de mélanome dans ce contexte mutationnel mais mène à une invasion plus profonde des cellules tumorales dans le derme. / The progression of tumors to metastatic disease involves the loss of metastatic suppressor functions, as it is the case in melanoma. Thus, aberrant cell migration is a key feature of melanoma progression, and is required for metastasis. SYK (Spleen tyrosine kinase) is a cytoplasmic tyrosine kinase that has been implicated in tumor suppression of breast cancer and melanoma. In skin cells, SYK is found expressed in melanocytes but SYK is frequently downregulated in melanoma by epigenetic silencing. We showed previously that its loss has been associated with senescence escape. Whether it also regulates tumor cell migration and subsequent metastasis remains poorly understood. In this work we used gain- and loss-of-function approaches to analyze SYK’s effects on metastatic abilities of human and murine melanoma cells. Respectively, the reexpression or knockdown of SYK results in decreased or increased migration, invasion and metastasis of melanoma cells. Notably, SYK knockdown cells displayed a mesenchymal-like phenotype with upregulation of mesenchymal markers. Our study unveils a novel role for SYK in suppressing integrin-mediated adhesion, both a points of traction and a signaling platform during cell migration, and outlines the importance of SYK inactivation in acquisition of a metastatic phenotype. To clarify the role of SYK in melanoma formation and progression, we have generated a conditional Syk KO mouse model in melanoma based on melanocyte-specific Pten loss and BrafV600E activating mutation. Preliminary results suggest that Syk loss does not accelerate Pten/Braf-driven melanoma formation but leads to deep invasion of Braf/Pten tumor cells into the dermis.

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.

Airway hyperresponsiveness (AHR) is a cardinal feature of asthma that is aggravated by environmental air pollution (EAP). Splenocyte tyrosine kinase Syk has been associated with asthma pathogenesis. Therefore, we sought to investigate the effect of Syk inhibition on AHR and its exacerbation by EAP. For this purpose, we examined Syk protein expression in lung homogenates from three murine models of ovalbumin (OVA)-induced asthma expressing different pathophysiological features of the disease: airway inflammation, AHR and remodeling. Increased Syk expression was observed only in the chronic model of airway inflammation and remodeling. In vivo Syk inhibition attenuates AHR in this model, and further augmentation induced by EAP without affecting the underlying airway inflammation. We demonstrated, for the first time, that Syk inhibition effectively reverted AHR in an already established chronic model of asthma. These findings highlight the therapeutic potential of targeting Syk for the treatment of asthma and its exacerbations by EAP.

Airway hyperresponsiveness (AHR) is a cardinal feature of asthma that is aggravated by environmental air pollution (EAP). Splenocyte tyrosine kinase Syk has been associated with asthma pathogenesis. Therefore, we sought to investigate the effect of Syk inhibition on AHR and its exacerbation by EAP. For this purpose, we examined Syk protein expression in lung homogenates from three murine models of ovalbumin (OVA)-induced asthma expressing different pathophysiological features of the disease: airway inflammation, AHR and remodeling. Increased Syk expression was observed only in the chronic model of airway inflammation and remodeling. In vivo Syk inhibition attenuates AHR in this model, and further augmentation induced by EAP without affecting the underlying airway inflammation. We demonstrated, for the first time, that Syk inhibition effectively reverted AHR in an already established chronic model of asthma. These findings highlight the therapeutic potential of targeting Syk for the treatment of asthma and its exacerbations by EAP.

Dectin-1 is a pattern-recognition receptor recognizing β-(1,3)-glucans found on fungal cell walls. Dectin-1 plays an important role in immunity to fungi by mediating phagocytic clearance of fungal particles and inducing transcription of innate response genes. We show here that the two processes are linked and that Dectin-1 signalling for inflammation is attenuated by phagocytosis. Blocking Dectin-1 ligand-dependent internalization using either actin polymerization or dynamin inhibitors, large non-phagocytosable β-glucan particles or poorly phagocytic cells leads in all cases to enhanced and sustained activation of downstream signalling pathways and culminates in production of high levels of proinflammatory cytokines. These findings establish the importance of phagocytosis not only in the clearance of pathogens, but also in the modulation of pattern-recognition receptor signalling and strongly suggest that internalization is the first step to attenuation of Dectin-1-mediated pro-inflammatory responses.

Spleen tyrosine kinase (SYK) regulates mitogenic signaling, inflammatory responses and cell fate in a number of diverse cell types. KRAS is a proto-oncogene that controls cell growth and proliferation through several mitogenic pathways. In pancreatic cancer, KRAS is frequently mutated, resulting in constitutive activation in 90% of pancreatic cancer cell lines. We previously showed that SYK is highly expressed in a subset of KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) cell lines. We demonstrated that SYK kinase inhibition with PRT062607 (SYKi) causes decreased cell proliferation of PDAC cell lines. Furthermore, combined SYKi and MEK inhibitor (MEKi) treatment promotes additive effects on suppression of PDAC cell proliferation and clonogenic growth. Mechanistically, SYK activates the mTORC1 kinase complex as shown by reduced phosphorylation of ribosomal S6 protein and its upstream kinase p70 S6 kinase (p70S6K) following SYKi treatment in PDAC cell lines. SYK-mediated mTORC1 activation occurs independently of MEK/ERK and PI3K/AKT effector signaling pathways. The mTORC1 complex suppresses lysosome biogenesis and macroautophagy (autophagy). Consequently, mTORC1 suppression via SYK inhibition or shRNA-mediated depletion causes accumulation of autolysosomes. These effects are mediated by the enhanced nuclear localization of MITF, a key transcriptional regulator of genes involved in lysosome biogenesis and autophagy pathway activation. In summary, SYK positively regulates mTORC1 activation in a subset of PDAC cell lines to suppress hyperactivation of autophagy. These findings open new avenues for further exploration of SYK as a critical regulator of the autophagy pathway in KRAS/mTORC1-dependent PDAC, and how this may be exploited for therapeutic benefit.

Recognition of microbial components by germ-line encoded pattern recognition receptors (PRR) initiates immune responses to infectious agents. We and others have proposed that pairs or sets of PRR mediate host immunity. One such pair comprises the fungal β-glucan receptor, Dectin-1, which collaborates through an undefined mechanism with Toll-like receptor 2 (TLR2) to induce optimal cytokine responses in macrophages. We show here that Dectin-1 signaling through the spleen tyrosine kinase (Syk) pathway is required for this collaboration, which can also occur with TLR4, 5, 7 and 9. Deficiency of either Syk or the TLR adaptor MyD88 abolished collaborative responses, which include TNF,MIP-1α andMIP-2 production, and which are comparable to the previously described synergy between TLR2 and TLR4. Collaboration of the Syk and TLR/MyD88 pathways results in sustained degradation of the inhibitor of kB (IkB), enhancing NFkB nuclear translocation. These findings establish the first example of Syk-and MyD88-coupled PRR collaboration, further supporting the concept that paired receptors collaborate to control infectious agents.

No description available.

Activation of platelets is essential for hemostasis. Following damage to the vascular endothelium collagen is exposed, to which platelets stably adhere. After adhesion on collagen, a signaling cascade is initiated, mediated by Glycoprotein VI (GPVI), which results in platelet activation. A major signaling protein in GPVI signaling is Spleen tyrosine kinase (Syk). It undergoes phosphorylation and activation following GPVI stimulation. Syk's central role in this physiological process suggests regulation of its activity is required to maintain the platelets response to collagen within physiological limits. The regulation of Syk activation is the focus of this work. Previously published reports implicate the phosphatases SHP-1, SHP-2 and TULA-2 in the negative regulation of Syk. Therefore, we tested these phosphatases possible role in platelets. We show that SHP-1 can dephosphorylate Syk in vitro, but is unable to bind Syk. Also, Syk is hypophosphorylated in GPVI-stimulated SHP-1 deficient platelets and platelet functional responses are minimally affected compared to wild-type platelets. SHP-2 is unable to bind Syk and Syk is not a good substrate for SHP-2 in vitro. TULA-2 dephosphorylated Syk in vitro and associated with Syk in platelets. In TULA-2 deficient platelets, Syk and PLCγ2 were hyperphosphorylated compared to wild-type platelets. Deletion of TULA-2 resulted in enhanced GPVI-dependent platelet functional responses and a prothrombotic phenotype. c-Cbl has been shown to be a negative regulator of GPVI signaling, possibly by regulating Syk phosphorylation. Thus, SHP-1, SHP-2 and TULA-2’s role in c-Cbl regulation of GPVI was also investigated. We show that TULA-2 is able to bind c-Cbl in platelets. SHP-1 and SHP-2 do not. Furthermore, we show a striking similarity between the phenotype of TULA-2 and c-Cbl deficient platelets. However, in vitro binding studies show TULA-2 is able to bind Syk independently of c-Cbl. Thus, the exact role of c-Cbl in regulating Syk dephosphorylation is unclear. In conclusion, we show SHP-1 and SHP-2 are probably not involved in the negative regulation of Syk. However, TULA-2 is the major phosphatase responsible for the negative regulation of Syk in GPVI signaling. This serves to negatively regulate GPVI-mediated platelet function and prevent uncontrolled platelet activation that could lead to thrombosis. / Pharmacology

Die Langlebigkeit reifer periphärer B Zellen ist abhängig von mindestens zwei Überlebenssignalen, einem tonischen Signal, welches vom B Zellrezeptor ausgeht und dem Zytokin B Zell aktivierender Faktor der TNF-Familie (BAFF). BAFF fördert nicht nur das Überleben von reifen B Zellen, sondern kontrolliert auch deren Funktionstüchtigkeit, indem es vielschichtige physiologische Prozesse wie Zellwachstum und –metabolismus, Energiehaushalt und Eintritt in den Zellzyklus reguliert. Zwei BAFF-induzierte molekulare Mechanismen, zum einen die Aktivierung des Akt Signaltransduktionsweges sowie die erhöhte Expression der onkogenen kinase Pim-2 zum anderen, führen zu Veränderungen in Effektorproteinen welche in der Lage sind diese physiologischen Zellveränderungen auszulösen. Die BAFF-induzierte Aktivierung von Akt hängt von der klassischen Proteinkinase C (PKC) beta ab und sowohl PKC beta-defiziente B Zellen als auch Mäuse zeigen Anzeichen von Unsensitivität gegenüber BAFF-Stimulation. Die Proteintyrosinkinase Syk spielt eine Rolle während der frühen B Zellentwicklung und wird in reifen B Zellen durch Stimulation des B Zellrezeptors aktiviert. Induzierbare Inaktivierung von Syk in Mäusen führt zum Verschwinden reifer B Zellen aus den periphären lymphoiden Organen, was auf eine unverzichtbare Funktion von Syk in der Vermittlung des tonischen B Zellrezeptorsignals schliessen läßt. / The maintenance of mature peripheral B cells depends on at least two survival cues, tonic signaling from the B cell receptor (BCR) complex and the extracellular cytokine B cell activating factor of the TNF family (BAFF). In addition to enhancing viability, BAFF controls the functional efficiency of the peripheral B cell pool by regulating complex physiological processes including cell growth, metabolism, energy homeostasis and entry into the cell cycle. BAFF-mediated induction of two molecular mechanisms, namely activation of the Akt signal transduction pathway and upregulation of the oncogenic kinase Pim-2 results in the modification of effector proteins including transcription factors and regulators of protein synthesis which are capable of executing the observed cellular physiological changes. The classic protein kinase C beta is instrumental in BAFF-induced Akt-activation and PKC beta-deficient B cells and mice show signs of partial refractiveness to BAFF. The protein tyrosine kinase Syk plays a role in early B cell development and is activated in mature B cells by immunogenic BCR-stimulation. Inducible ablation of Syk in mice results in the loss mature B cells from the peripheral lymphoid organs and reveals an indispensable function for Syk in tonic BCR survival signaling.