Src signaling plays an important role in prostate cancer (PrCa) progression. It has previously been shown that Src interacts with androgen receptor (AR) and enhances AR transactivation. Although it has been shown that Src promotes AR activity, the underlying pathway has not been defined. To help characterize the Src-AR pathway, the cellular localizations of Src, p-Src, AR, pAR, and Prostate Specific Antigen (PSA, an AR target gene) were analyzed in androgen-dependent (AD) LNCaP cells and in androgen-independent (AI) castration-resistant C4-2B cells. Using sub-cellular fractionation, the data showed that treatment of AD cells with synthetic androgen R1881 increased p-Src, AR, pAR, and PSA in the nucleus, while the levels of c-Src remained unchanged. Treatment of AI cells with R1881 increased pSrc and AR in the nucleus, while the levels of c-Src and PSA remained unchanged. When using immunofluorescence microscopy, R1881 did not appear to increase the nuclear levels of p-Src or c-Src, so perhaps this technique is not as sensitive or quantitative as subcellular fractionation immunoblots. The presence of PSA in the nucleus was unexpected given its well proven role as a secreted protein. Nuclear PSA was observed upon androgen stimulation in AD and AI cells, and in the nucleus of AI cells upon androgen deprivation. Given PSA's ability to induce cell division and decrease apoptosis when transfected into cells, its presence in the nucleus may imply that PSA acts there to help induce tumorigenesis. The effect of Src on AR activity was further studied by transfection of a dominant negative src (SrcK298M) in AD and AI cells. Transfection with SrcK298M did not affect PSA expression in LNCaP cells, but strongly inhibited PSA levels in AI cells. Integrin signaling through Src was investigated in PrCa by ligand binding assay in AD and AI cells. The data showed that alpha v beta 3 integrin (but not alpha v beta 6) upon attachment to fibronectin or TGF-beta-latency associated peptide (TGF- beta-LAP) increases p-Src levels in AD and AI cells, while the levels of c-Src, PSA, and AKT remain unchanged. Thus, alpha v beta 3 integrin facilitates Src signaling, but the activation does not appear to affect AR transactivation. In conclusion, these data show that Src is required for AR activity and, consequently, PSA expression in AI prostate cancer cells, but not in AD cells. These data also suggest that the nuclear co-localization of p-Src, AR and PSA might allow macromolecular interactions, which can further enhance AR transactivation and promote disease progression. With respect to the switch in tumor progression from an AD to AI state, the data indicate that the integrin-Src pathway does not include AKT or PSA (and not AR by deduction), so perhaps other non-AR pathways help facilitate tumor growth at the AI state.

No description available.

Traditionally, drug discovery programs have focused on prioritising compounds by their affinity to a specific target in isolation, which was hypothesised to be the cause of a particular disease. Through chemical inhibition, the disease could, thus, be prevented or at the very least, controlled. These hypotheses require significant validation before drug screening can begin which relates to lengthy and expensive programs. Furthermore, drug screening against a single target in isolation is not a realistic model of cellular behaviour and is not appropriately tailored to more complex diseases such as cancer. Phenotypic drug discovery, on the other hand, bypasses any involvement of known targets, instead focusing on the desired outcome – the phenotype. In this way, drugs are biased by their potency on the phenotype and not against any particular targets. The molecular mechanism of action need not be known at all, however, it can be useful to later reveal the target(s) involved by various deconvolution methods. This thesis describes a cooperative ligand based phenotypic drug discovery approach, undertaken in order to develop more suitable small molecule drugs for cancer treatment. For this purpose, the promiscuous pyrazolopyrimidine inhibitor PP1 was chosen as a starting model compound. Modification of PP1 on the N1 position allowed a series of water solubilising groups to be incorporated into the pyrazolopyrimidine scaffold which created an initial 12-membered library. Testing against MCF7 breast cancer cells and looking at phenotypic end points such as cell proliferation, cell mobility and cell cycle, generated early target-agnostic structure/anti-proliferative activity relationships. These early results, along with compounds published in recent literature, were used to generate further libraries. Profiling lead compounds against a selection of 18 kinases known to be targeted by PP1, showed the compounds were inhibiting either SRC family or mTOR kinases which enabled the creation of two, structure specific, groups of inhibitors. Further lead optimisation led to the rapid discovery of preclinical candidates with excellent drug-like properties and potencies in both cellular assays and against their respective targets. Compounds also showed improved selectivity profiles compared to PP1 and commonly known inhibitors of SRC and mTOR kinases. Reported, herein, is the discovery of the first sub-nanomolar SRC inhibitor which does not inhibit the kinase ABL and shows excellent properties suitable for further preclinical development.

La mitochondrie est une organelle très importante vu son implication dans plusieurs processus cellulaires. Elle produit notamment la majeure partie de l'énergie qui est consommée par la cellule, grâce aux processus d'oxydation phosphorylante (OXPHOS). La phosphorylation des enzymes impliquées dans les OXPHOS apparait comme une voie de régulation importante de la production énergétique. L'objectif de ce thèse était donc de comprendre comment les phosphorylations, et plus particulièrement, les phosphorylations sur tyrosine induites par la Src kinase influencent les OXPHOS. Il a donc été démontré qu'il existe, à l'intérieur des mitochondries, des voies de régulation de ces processus de phosphorylation induits par la Src kinase. Ces processus pouvant induire la phosphorylation de plusieurs enzymes mitochondriales, notamment plusieurs sous-unités des complexes du système des électrons et ainsi, grandement influencer les OXPHOS. Il a aussi été démontré que la Src kinase semble aussi présente dans les mitochondries de cellules cancéreuses, induisant la phosphorylation d'une sous-unité de la NADH-oxidoréductase et une augmentation du métabolisme énergétique mitochondrial. Cette régulation des OXPHOS dans les cellules cancéreuses par la Src kinase pourrait participer à l'établissement du phénotype hautement prolifératif de ces cellules. / Mitochondria are implicated in several key cellular processes. They are producing most part of the energy that is consumed by the cell via oxidative phosphorylation processes (OXPHOS). Phosphorylation of different components implicated in OXPHOS are known to constitute an important regulation pathway of energetic production. The objective of this thesis was to understand how tyrosine phosphorylation induced by the Src kinase could influence OXPHOS. First, it was shown that Src kinase mediated phosphorylation can be regulated directly in mitochondria, inducing phosphorylation of several mitochondrial proteins and different effects on OXPHOS. I also demonstrated that Src kinase is also present in mitochondria of cancer cells where it can lead to phosphorylation of NADH-oxidoreductase. This phosphorylation site is associated with increase of OXPHOS which could be implicated in the establishment of global phenotype of cancer cells.

iv Abstract Crosstalk of integrin and mTOR signalling is an essential process that monitors cellular interaction with extracellular matrix and transmits these inputs to cell growth signalling. Although adhesion status of the cell monitored by integrin signalling is clearly important for regulation of cellular growth, a little is known about the crosstalk of integrin and mTOR signalling. In this study, we employed two different approaches to describe and elucidate character of this crosstalk. p130Cas is an adaptor protein phosphorylated by Src kinase and focal adhesion kinase upon integrin ligand binding and implicated in cell adhesion, motility and survival in both Src-transformed and untransformed cells. Recently, p130Cas was also described in cellular pathology, mainly by its ability to stimulate cell invasion and metastasis. In this study, we described that p130Cas affects mTOR signalling in Src-transformed cells. Substrate domain of p130Cas was found to be indispensable for this effect and is also responsible for serum-induced activation of mTOR signalling. In addition, we prepared cell lines overexpressing various Rheb protein versions and characterized them in context of mTOR signalling, integrin signalling and cell cycle progression. Interestingly, a cell line overexpressing constitutively active...

Les domaines SH3 (Src Homology domain) représentent l'un des modules protéiques le plus largement répandu dans la nature. Ils participent à des interactions intra- et intermoléculaires avec d’autre partenaires au travers de la formation et de la dissociation de complexes multi-protéiques. Le gène nef du Virus d'Immunodéficience Humain (VIH-1) code pour la protéine nef, importante pour la réplication du virus et le développement optimal du SIDA (Syndrome d’Immunodéficience Acquise) chez les personnes infectées. De précédentes études ont mis en évidence que la protéine nef utilise un mode « tertiaire » d’interaction pour mettre en place une affinité et une sélectivité élevées envers les domaines SH3 des kinases de la famille Src (SFKs). Savoir si cette stratégie de reconnaissance tertiaire des domaines SH3 peut être retrouvée dans des protéines cellulaires humaines est donc une question importante pour évaluer le degré de spécificité de la protéine nef comme cible anti-HIV. Nous avons identifié Alix (ALG-2 [apoptosis-linked gene 2]-interacting protein X) comme protéine originale interagissant avec le domaine SH3 de la kinase de cellules Hématopoïétique (Hck). Alix possède une sélectivité comparable à nef envers les domaines SH3 de SFKs. Nous avons combiné une analyse biophysique et structurale, alliant des méthodes telles que la microcalorimetrie à titration isotherme(‘ITC’), la Résonance Plasmonique de Surface (‘SPR’), des méthodes in vitro dites de ‘GST pulldown’, l'interférométrie (‘NPOI’), la Résonance Magnétique Nucléaire (‘NMR’ - HSQC) et la diffusion des rayons X aux petits angles (SAXS) pour explorer les caractéristiques définissant le mode d’interaction entre Alix et le domaine SH3 de la kinase Hck. Cette étude démontre que la protéine cellulaire Alix est unique, structurellement différente mais fonctionnellement semblable à nef. / Src homology (SH) 3 domains is one of the most wide-spreaded protein modules found in nature. They mediate both inter- and intra-molecular protein-protein interactions (PPIs) through the formation and dissociation of multi-protein complexes. These SH3-mediated interactions are responsible for signal transduction, cytoskeleton organization and other cellular processes. The nef gene of Human immunodeficiency virus (HIV-1) encodes the HIV-1 Nef protein, which is important for optimal virus replication and development of AIDS (acquired immunize deficiency syndrome) in HIV-1 infected persons. Previous studies show that the HIV-1 Nef protein uses a “tertiary” binding mode to achieve high affinity and selectivity toward SH3 domains of Src-family kinases (SFKs). Whether this strategy of ‘tertiary’ binding mode of SH3 domains can be found in human cellular proteins, besides HIV-1 Nef, is an important question in the specificity of the HIV-1 Nef protein as an anti-HIV target. We identified Alix (ALG-2 [apoptosis-linked gene 2]-interacting protein X) as a novel protein interacting with Hemopoietic cell kinase (Hck) SH3 domain. Alix has similar selectivity towards SH3 domains of SFKs as the HIV-1 Nef. We have combined biophysical and structural biology analysis, including ITC (isothermal titration calorimetry), SPR (surface Plasmon resonance), GST (glutathione S-transferase) pull-down, interferometry, HSQC (heteronuclear single quantum coherence) and SAXS (small-angle X-ray scattering) to explore the characteristics of Alix-SH3 recognition mode. This study shows that Alix as a unique cellular protein, which is structurally different but functionally similar in recognizing HIV-1 Nef. The structural information of the Alix-Hck association facilitates the understanding of how Hck and Alix assist viral budding and cell surface receptor regulation.

The aryl hydrocarbon receptor (AhR) has been reported to interact with multiple signaling pathways during prostate development including the androgen receptor. AhR was overexpressed in LNCaP using PLNCX2 retrovirus vector containing AhR cDNA to determine if ectopic overexpression induces castrate resistant phenotype. The highly overexpressed AhR clone illustrated further increase in transcriptional and promotor activity for AhR and AR compared to the moderately overexpressed AhR clone and control. Western blot analysis showed more AhR, AR, cSrc, and pSrc protein expression in clones. AhR overexpression was found to induce several biological properties such as migration, invasion, proliferation, and promotion of G1 to S phase during the cell cycle. Bicalutamide treatment had no effect on AR transcriptional activity in either clone, proving resistance to anti-androgen therapy. Our results confirm that overexpression of AhR induces constitutive activity and stimulates androgen receptor signaling. This suggests a role for AhR in the development of CRPC.

No description available.

No description available.

Le syndrome néphrotique idiopathique (SNI) est une néphropathie glomérulaire définie par une protéinurie massive associée à une hypoalbuminémie, sans lésions inflammatoires rénales, ni dépôts de complexes immuns circulants. Les travaux réalisés au cours de ma thèse concernent l’étude du rôle potentiel du gène c-mip dans la physiopathologie du SNI.Dans un premier temps, nous avons étudié la physiopathologie moléculaire de l’association maladie de Hodgkin et SNI. Nous avons démontré que cette association était liée à une forte induction de c-mip à la fois dans les cellules de Reed Sternberg (dont la présence signe le lymphome hodgkinien) et les podocytes qui sont des cellules spécialisées du glomérule rénal (Audard, et al. 2010). Nous avons montré que l’induction de c-mip résultait d’un défaut quantitatif et/ou qualitatif du gène Fyn, à la fois chez les patients et dans un modèle de souris déficiente en Fyn. Nous avons trouvé que c-mip était fortement induit dans les podocytes au cours du SNI ainsi que dans la glomérulopathie extramemenbraneuse (GEM). La surexpression de c-mip par transgénèse chez la souris déclenche une protéinurie néphrotique dont le mécanisme implique une rupture, médiée par c-mip, de la voie de signalisation de la néphrine (Science Signaling, 2010 co-auteur). L’étude de la néphrite de Heyman, le modèle expérimental de la GEM humaine, a permis de montrer que l’induction de c-mip coincidait avec l’apparition de la protéinurie et était associée à l’inhibition de l’activité RhoA, à une perte de la synaptopodine, à une diminution du VEGF tandis que l’expression de la DAPK (death-associated protein kinase) est fortement augmentée (Audard et al, manuscrit soumis 1). Nous avons recherché si l’hypogammaglobulinémie au cours du SNI était associée à des anomalies fonctionnelles des lymphocytes B (LB). Nous avons trouvé que c-mip interagit avec la sous unité régulatrice de la PI3 kinase et empêche la dissociation de la sous unité catalytique, p110, nécessaire à l’activation de la PI3 kinase. Enfin, l’expression de l’IL 21, une cytokine–clé secrétée par les lymphocytes T et intervenant dans la commutation isotypique, était fortement réduite dans le SNI (Audard et al, manuscrit en préparation 2). Ces résultats donnent un éclairage nouveau sur la physiopathologie moléculaire du SNI et suggèrent un rôle crucial de c-mip dans les anomalies lymphocytaires et podocytaires observées chez les patients / Idiopathic nephrotic syndrome comprises several podocyte diseases of unknown origin, affecting the glomerular podocyte, which plays a key role in controlling the permeability of the kidney filter to proteins. It is characterized by massive proteinuria and hypoalbuminemia, with no inflammatory lesions or cell infiltration. This works focused on the potential role of c-mip in the pathogenesis of INS. We showed that occurrence of minimal change nephrotic syndrome in the course of Hodgkin lymphoma (cHL-MCNS) is closely related to the induction of c-mip in both Hodgkin-Reed Sternberg cells and podocytes (Audard, et al. 2010), which is caused by a qualitative and/or quantitative defect in Fyn in both HRS and podocytes cells. We found that c-mip is upregulated in podocytes of patients with membranous nephropathy (MN). Transgenic mice overproducing c-mip in the podocytes developed heavy proteinuria without morphological alterations, inflammatory lesions or cell infiltrations. We showed that c-mip turned off podocyte proximal signaling by preventing the interaction between Fyn and nephrin, resulting in the inhibition of nephrin signaling pathway (Science signaling, 2010 coauthor). Moreover, the induction of c-mip in passive type Heymann nephritis (the experimental model of MN) was concomitant to proteinuria occurrence and is associated with reduction of RhoA activity, downregulation of synaptopodin and VEGF expression whereas DAPK expression is significantly increased (Audard et al manuscript submitted 1).We demonstrated that hypogammaglobulinemia, a common feature in INS patients, may result from a defect in B lymphocytes. We found that c-mip interacts with p85 regulatory subunit and prevent its dissociation from p110 catalytic subunit, resulting in inactivation of PI3 kinase. Finally, the expression of IL21, a key cytokine involved in class switching recombination, is repressed in active phases of INS, which may contribute for immunoglobulin disorders commonly observed in these patients (Audard et al manuscript in progress 2).Altogether, these results suggest that c-mip is a major player of lymphocyte and podocytes dysfunction observed in patients with INS