Polysialic acid (polySia) is a carbohydrate polymer highly expressed during embryonic development but rarely expressed during postnatal development. Two polysialyltransferase (polyST) enzymes are responsible for the synthesis of polySia: ST8SiaII and ST8SiaIV. During oncogenesis polySia is re-expressed and it modulates cell-cell and cell-matrix adhesion, migration, invasion and metastasis. PolySia expression is strongly associated with poor clinical prognosis and correlates with aggressive and invasive disease in neuroblastoma and many other tumours. PolyST inhibition thus presents a novel, selective and largely unexplored therapeutic opportunity to reduce tumour dissemination. Progress towards development of polyST inhibitors has been limited by lack of an efficient technique for quantitative assessment of enzyme activity. We have validated a highly sensitive cell-based and cell-free high throughput HPLC-based inhibition assays. Using isogenic cell lines (C6-STX: polySia+/ST8SiaII+ and C6-WT: polySia-/ST8SiaII-) and naturally polySia expressing human neuroblastoma cells (SH-SY5Y), a set of ST8SiaII inhibitors designed and synthesised in house were evaluated for their ability to reduce polySia expression and to modulate cell migration in vitro. We have identified CMP-sialic acid precursors, including ICT-3176, which reduced polySia expression and tumour cell migration by up to 70%. These effects were only found in cell lines expressing ST8SiaII and polySia. Furthermore, we have investigated the possible additive anti-migratory effect of combining polyST inhibition with the inhibition of certain signalling pathways that have been previously suggested to be modulated by polySia expression. Out of these combinations it was found that combining ST8SiaII and C-MET/ALK inhibition had a synergistic effect on inhibiting cancer cell migration. Additionally, the effect of polySia expression on cancer cell behaviour under hypoxic conditions was examined, where it was found that polySia expression enhanced cell migration and survival and inhibits cell adhesion. In summary, polyST inhibitors which dramatically decrease cell migration in vitro through modulation of polySia assembly were identified, using optimised cell-free and cell-based assays. Initial investigations into the role of polySia in hypoxia were also accomplished. This work paves the way for development of a novel therapeutic for the treatment of neuroblastoma.

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Polysialic acid (polySia) is a carbohydrate polymer highly expressed during embryonic development but rarely expressed during postnatal development. Two polysialyltransferase (polyST) enzymes are responsible for the synthesis of polySia: ST8SiaII and ST8SiaIV. During oncogenesis polySia is re-expressed and it modulates cell-cell and cell-matrix adhesion, migration, invasion and metastasis. PolySia expression is strongly associated with poor clinical prognosis and correlates with aggressive and invasive disease in neuroblastoma and many other tumours. PolyST inhibition thus presents a novel, selective and largely unexplored therapeutic opportunity to reduce tumour dissemination. Progress towards development of polyST inhibitors has been limited by lack of an efficient technique for quantitative assessment of enzyme activity. We have validated a highly sensitive cell-based and cell-free high throughput HPLC-based inhibition assays. Using isogenic cell lines (C6-STX: polySia+/ST8SiaII+ and C6-WT: polySia-/ST8SiaII-) and naturally polySia expressing human neuroblastoma cells (SH-SY5Y), a set of ST8SiaII inhibitors designed and synthesised in house were evaluated for their ability to reduce polySia expression and to modulate cell migration in vitro. We have identified CMP-sialic acid precursors, including ICT-3176, which reduced polySia expression and tumour cell migration by up to 70%. These effects were only found in cell lines expressing ST8SiaII and polySia. Furthermore, we have investigated the possible additive anti-migratory effect of combining polyST inhibition with the inhibition of certain signalling pathways that have been previously suggested to be modulated by polySia expression. Out of these combinations it was found that combining ST8SiaII and C-MET/ALK inhibition had a synergistic effect on inhibiting cancer cell migration. Additionally, the effect of polySia expression on cancer cell behaviour under hypoxic conditions was examined, where it was found that polySia expression enhanced cell migration and survival and inhibits cell adhesion. In summary, polyST inhibitors which dramatically decrease cell migration in vitro through modulation of polySia assembly were identified, using optimised cell-free and cell-based assays. Initial investigations into the role of polySia in hypoxia were also accomplished. This work paves the way for development of a novel therapeutic for the treatment of neuroblastoma.

Yes / The delivery of anticancer agents to their subcellular sites of action is a significant challenge for effective cancer therapy. Peptides, which are integral to several oncogenic pathways, have significant potential to be utilised as cancer therapeutics due to their selectivity, high potency and lack of normal cell toxicity. Novel Ras protein-Regulator of chromosome condensation 1 (Ran-RCC1) inhibitory peptides designed to interact with Ran, a novel therapeutic target in breast cancer, were delivered by entrapment into polyethylene glycol-poly (lactic-co-glycolic acid) PEG-PLGA polymeric nanoparticles (NPs). A modified double emulsion solvent evaporation technique was used to optimise the physicochemical properties of these peptide-loaded biodegradable NPs. The anti-cancer activity of peptide-loaded NPs was studied in vitro using Ran-expressing metastatic breast (MDA-MB-231) and lung cancer (A549) cell lines, and in vivo using Solid Ehrlich Carcinoma-bearing mice. The anti-metastatic activity of peptide-loaded NPs was investigated using migration, invasion and colony formation assays in vitro. A PEG-PLGA-nanoparticle encapsulating N-terminal peptide showed a pronounced antitumor and anti-metastatic action in lung and breast cancer cells in vitro and caused a significant reduction of tumor volume and associated tumor growth inhibition of breast cancer model in vivo. These findings suggest that the novel inhibitory peptides encapsulated into PEGylated PLGA NPs are delivered effectively to interact and deactivate Ran. This novel Ran-targeting peptide construct shows significant potential for therapy of breast cancer and other cancers mediated by Ran overexpression.

L’apparition de métastases est souvent le signe d’un mauvais pronostic vital pour les personnes atteintes d’un cancer. Ce processus de formation de métastase est un phénomène complexe dans lequel la migration cellulaire est un facteur clé.De récentes études ont montré que le canal SK3 (canal potassique de faible conductance dont l’activité dépend de la concentration cytosolique en calcium) était exprimé dans des cellules cancéreuses à fort pouvoir métastatique et leur conférait des capacités de migration accrues. Cette protéine constitue donc une nouvelle cible thérapeutique très intéressante pour agir sur la dissémination de cellules cancéreuses.Les objectifs de ces travaux de thèse ont permis de mettre en oeuvre deux stratégies visant à inhiber l’activité de ce canal potassique SK3.L’édelfosine, un glycérolipide à tête phosphocholine, a rapidement été reconnue comme étant un inhibiteur efficace de l’activité de ce canal. Cependant les effets secondaires induits par cette molécule ont conduit à rechercher des analogues moins toxiques et tout aussi efficaces. Des études structures-activité menées au sein du laboratoire ont permis de développer un nouveau glycérolipide à tête lactose, l’ohmline. Dans le but de compléter cette étude, nous avons réalisé la synthèse de glyco-glycérolipides et de glycophospho-glycérolipides et avons montré leur capacité à inhiber la protéine SK3 et à réduire la migration cellulaire SK3 dépendante.Une seconde stratégie vise à l’utilisation possible d’ARN interférents pour bloquer l’expression de la protéine SK3. Dans ce but, nous nous sommes intéressés à la synthèse et à l’incorporation, dans des formulations de lipides cationiques utilisés pour la transfection, de lipides neutres portant des motifs anisamides, ligands spécifiques des récepteurs sigma surexprimés dans des lignées cellulaires de tumeurs exprimant SK3. La synthèse de lipophosphoramides comportant un motif anisamide est présentée suivie de leur utilisation dans des expériences de transfection modèles (vectorisation d’ADN plasmidique) afin d’évaluer l’efficacité du ciblage engendré par le motif anisamide. / The occurrence of metastasis in a cancer is generally associated to a bad prognostic for the patient. The formation of metastasis is the result of a complex process in which cell migration plays a key role.Recent studies have shown that the potassium calcium-dependent channel SK3 is expressed in several highly metastatic cancerous cell lines and play a direct role in the migration process. Consequently, this protein is an interesting new therapeutic target to reduce metastasis formation.This PhD thesis work aimed investigating two strategies to reduce SK3 dependent cell migration.Edelfosine, a glycerolipid with a phosphocholine head, was identified as an efficient inhibitor of the SK3 channel activity. However the side effects induced by this molecule (toxicity) led to look for efficient and less toxic analogues. Accordingly, structure-activity studies carried out in our laboratory produced new glyco-glycerolipid including one with a lactose group (ohmline). With the aim of completing this study, we report the synthesis of glyco-glycerolipids and glycophospho-glycerolipids and shown their capacity to inhibit activity of SK3 channel.The second part of this work aims to act at an early stage by using RNAi to block the expression of the SK3 protein. In this way, we have synthesized and formulated, with a cationic lipid used for the transfection, neutral co-lipids functionalized with an anisamide moiety; this motif being recognize sigma receptors which are overexpressed in tumor cell lines that also expressed SK3. First, the synthesis of the lipophosphoramides with an anisamide moiety was described followed by their use in standard transfection experiments (plasmid DNA) to evaluate the effectiveness of the targeting strategy induced by the anisamide moiety.