La migration cellulaire est un processus fondamental au maintien des fonctions physiologiques de l’organisme. Elle est également centrale dans de nombreuses pathologies et entre notamment en jeu lors de la dissémination métastatique. Lorsqu’elles migrent, les cellules utilisent des structures d’adhésion afin de s’appuyer sur leur environnement. Nous avons récemment montré que les puits recouverts de clathrine, plus connus pour leur rôle dans l’endocytose, peuvent également servir de structures d’adhésion. Dans ce manuscrit, je démontre que certains ligands internalisés par la voie d’endocytose clathrine peuvent également se lier à la matrice et orienter la migration cellulaire en régulant les structures adhésives de clathrine.J’ai commencé par montrer que le collagène est associé à plus de structures de clathrine et a plus de protrusions lorsqu’il est recouvert par des ligands. J’ai ensuite montré que les cellules appliquaient plus de forces sur des fibres de collagènes décorées par des ligands et que ce surplus de force nécessite la présence de structures de clathrine. Enfin j’ai montré que les cellules suivent les ligands liés à des réseaux de collagène en 3D et que cette migration dirigée nécessite également la présence de structures de clathrine. Ce mécanisme de migration pourrait notamment permettre aux cellules de suivre des gradients de ligands liés à la matrice in vivo et ainsi de s’orienter dans l’organisme. / Cell migration is a fundamental process in the development and homeostasis of multicellular organisms. It is also central to many pathologies and it is especially important for metastatic dissemination. When migrating, cells use adhesion structures to push on their substrate in order to move forward. We recently showed that clathrin coated structures, primarily known as endocytic structures, can also serve as adhesion structures. In this manuscript, I show that some ligands internalized through clathrin mediated endocytosis can also bind to the extracellular matrix and orient cell migration using adhesive clathrin structures.I first showed that ligand-decorated collagen fibers are associated with more clathrin structures and more protrusions. I then showed that cells applied more forces to the ligand-decorated collagen fibers and this extra amount of forces requires the presence of clathrin structures. Finally, I showed that cells can migrate following collagen-bound ligands in 3D, this directed migration also requiring the presence of clathrin structures. Such migration mechanism could be used by cells to follow in vivo gradient of matrix-bound ligands and thus find their way when migrating inside the body.

Signalling through the EGF receptor is subject to a complex and multilayered regulation. One such mode of regulation is through control of ligand production which plays an important role in fine- tuning EGF receptor activation. In mammals, the production of soluble, biologically active forms of EGF receptor ligands relies on ADAM metalloproteases, predominantly ADAM10 and ADAM17. Recently, a pseudoprotease from the rhomboid-like family of intramembrane proteases, iRhom, emerged as a key positive regulator of ADAM17. However, Drosophila iRhom has also been implicated in the negative regulation of EGF receptor signalling by promoting the degradation of precursors of its ligands. Cell culture based assays suggest that mammalian iRhoms might also be involved in a similar process. In this thesis, the effect of mammalian iRhom overexpression on the levels of EGF receptor ligands has been investigated. Contrary to previous findings, the data presented in this thesis suggest that the observed effect might not be entirely iRhom specific, for the inactive mutants of rhomboid proteases also diminish the levels of EGF receptor ligands. Nor do we find the effect to be specific to EGF receptor ligands, as unrelated transmembrane proteins were also depleted by iRhom overexpression. The coexpression of ADAM17 was...

The Notch signalling pathway is conserved in all metazoan species and plays a crucial role in development and tissue maintenance. Canonical Notch signalling requires cell-cell contact to allow the interaction between Drosophila Notch receptor and its ligands, Serrate and Delta. The Notch Abruptex (Ax) region comprises 24-29 of the 36 epidermal growth factor-like (EGF) repeats in the Notch extracellular domain. Mutations in the Ax region give rise to three distinct phenotypes in Drosophila. Notch EGF repeats 11-12 form the ligand binding region (LBR). The recently solved structure of the module at the N-terminal of Notch ligands (MNNL) of a human Notch ligand, Jagged1, revealed that the domain was a calcium-binding C2 domain with Ca²⁺-dependent lipid binding. This study aimed to investigate the intra- and intermolecular properties of Drosophila Notch Ax region, LBR, and the MNNL of Notch ligand Serrate. In WT Drosophila Notch EGF23-25, all three EGF domains were found to be Ca²⁺-binding, and a previously unknown Ca²⁺ binding consensus sequence was identified. Ax^N-suppressor mutations D948V and N986I were shown to impair the Ca²⁺-binding properties of the mutant EGF domain without affecting the neighbouring domains, suggesting a mechanism to explain the signalling phenotype associated with this mutation type. Notch EGF11-13 showed Ca²⁺-binding in each EGF domain and binding to ligand-expressing cells. Its C-terminal tag was found to influence the Ca²⁺-dependent fold of EGF13, suggesting a future strategy for protein expression. A Serrate fragment MNNL-EGF3 showed Ca²⁺-dependent lipid binding, which was not observed in a construct lacking MNNL. The lipid binding could be reduced by a substitution D197A in MNNL, suggesting this mutant could be used to probe functional importance of MNNL in model organism studies. Binding between Serrate and Notch was assessed with a new cell aggregation assay method using flow cytometry, and agreed with previously published studies. Binding to Delta was subsequently measured, which suggested ligand specific differences although Notch residue L504 was important for both Serrate- and Delta-binding. Collectively these studies establish that Drosophila Notch and its ligand Serrate has similar properties to mammalian homologues, which will facilitate future structural and functional studies.

<p>To further develop cancer therapy, targeted delivery of cell killing agents directly to tumor cells is an interesting approach. This thesis describes the development of PEG-stabilized liposome conjugates targeting either epidermal growth factor receptor (EGFR) using its natural ligand EGF, or human epidermal growth factor receptor 2 (HER-2) using the antibody trastuzumab. Both receptors are known to be overexpressed on a variety of tumors. The liposomes were loaded with the boronated compounds water soluble boronated acridine (WSA) or water soluble boronated phenantridine (WSP), compounds primarily developed for boron neutron capture therapy, BNCT. </p><p>The liposome conjugates bound specifically to their receptors in cell culture. Because the WSA conjugates exhibited the most favorable boron uptake this compound was chosen for further study. The WSA-loaded liposome conjugates was internalized, an important characteristic for BNCT, and had a long retention inside the cells. The cellular localization of WSA, studied using fluorescence was found to be mainly cytoplasmic. </p><p>To increase the boron uptake studies comparing different incubation methods was performed. It was shown for both EGF and trastuzumab targeted liposomes the uptake could be increased over 10 times by changing from incubation in monolayer culture to incubation in cell suspension in roller flasks. With this treatment the boron concentrations reached after 24 h incubation time was 90 ppm for EGF-liposomes and 132 ppm for trastuzumab-liposomes, levels that are clinically interesting. </p><p>To study the cell-killing efficacy of the liposome-conjugates an experimental BNCT study was performed using EGF-liposome-WSA on cultured glioma cells. About half the number of thermal neutron was needed to inactivate 90% of the cells if the cells had been incubated with EGF-liposome-WSA compared to control cells. When comparing the survival to dose it was shown that to inactivate 90% of the cells 2.9 Gy was needed for EGF-liposome-WSA and neutrons compared to 5.6 Gy with ¹³⁷Cs gamma. </p><p>The biodistribution of EGF-liposomes was also studied in mice. It was compared to EGF and it was found that the addition of a PEG-stabilized liposome to EGF significantly reduced EGF uptake in liver and kidneys, the circulation time in blood was prolonged as well. The reduced liver uptake might be due to inability of the 100 nm liposomes to pass the sinusoidal fenestrations of the liver and bind to the EGFR-rich hepatocytes. The reduced liver uptake potentates the use of EGF-liposome conjugates for systemic injection.</p>

To further develop cancer therapy, targeted delivery of cell killing agents directly to tumor cells is an interesting approach. This thesis describes the development of PEG-stabilized liposome conjugates targeting either epidermal growth factor receptor (EGFR) using its natural ligand EGF, or human epidermal growth factor receptor 2 (HER-2) using the antibody trastuzumab. Both receptors are known to be overexpressed on a variety of tumors. The liposomes were loaded with the boronated compounds water soluble boronated acridine (WSA) or water soluble boronated phenantridine (WSP), compounds primarily developed for boron neutron capture therapy, BNCT. The liposome conjugates bound specifically to their receptors in cell culture. Because the WSA conjugates exhibited the most favorable boron uptake this compound was chosen for further study. The WSA-loaded liposome conjugates was internalized, an important characteristic for BNCT, and had a long retention inside the cells. The cellular localization of WSA, studied using fluorescence was found to be mainly cytoplasmic. To increase the boron uptake studies comparing different incubation methods was performed. It was shown for both EGF and trastuzumab targeted liposomes the uptake could be increased over 10 times by changing from incubation in monolayer culture to incubation in cell suspension in roller flasks. With this treatment the boron concentrations reached after 24 h incubation time was 90 ppm for EGF-liposomes and 132 ppm for trastuzumab-liposomes, levels that are clinically interesting. To study the cell-killing efficacy of the liposome-conjugates an experimental BNCT study was performed using EGF-liposome-WSA on cultured glioma cells. About half the number of thermal neutron was needed to inactivate 90% of the cells if the cells had been incubated with EGF-liposome-WSA compared to control cells. When comparing the survival to dose it was shown that to inactivate 90% of the cells 2.9 Gy was needed for EGF-liposome-WSA and neutrons compared to 5.6 Gy with 137Cs gamma. The biodistribution of EGF-liposomes was also studied in mice. It was compared to EGF and it was found that the addition of a PEG-stabilized liposome to EGF significantly reduced EGF uptake in liver and kidneys, the circulation time in blood was prolonged as well. The reduced liver uptake might be due to inability of the 100 nm liposomes to pass the sinusoidal fenestrations of the liver and bind to the EGFR-rich hepatocytes. The reduced liver uptake potentates the use of EGF-liposome conjugates for systemic injection.

LIV-1, a zinc transporter, is an effector molecule downstream from soluble growth factors. This protein has been shown to promote epithelial-to-mesenchymal transition (EMT) in human pancreatic, breast, and prostate cancer cells. Despite the implication of LIV-1 in cancer growth and metastasis, there has been no study to determine the role of LIV-1 in prostate cancer progression. Moreover, there is no clear delineation of the molecular mechanism underlying LIV-1 function in cancer cells. In this study, we found increased LIV-1 expression in a progresssive manner in benign, PIN, primary and bone metastatic human prostate cancer. We characterized the mechanism by which LIV-1 drives prostate cancer EMT in an androgen-refractory human prostate cancer cell (ARCaP) bone metastasis model. LIV-1, when overexpressed in ARCaPE cells (derivative cells of ARCaP with epithelial phenotype), promoted EMT irreversibly. LIV-1 overexpressed ARCaPE cells had elevated levels of HB-EGF and matrix metalloproteinase (MMP) 2 and MMP 9 proteolytic enzyme activities, without affecting intracellular zinc concentration. The activation of MMPs resulted in the shedding of heparin binding-epidermal growth factor (HB-EGF) from ARCaPE cells, eliciting constitutive epidermal growth factor receptor (EGFR) phosphorylation and its downstream extracellular signal regulated kinase (ERK) signaling. Further investigation of the HB-EGF promoter revealed that both Stat3 and AP-1 controlled HB-EGF promoter activity. Ectopic LIV-1 overexpression induced AP-1 and Stat3 activation. Blockade of both Stat3 and AP-1 by specific inhibitors or dominant negative expression vectors diminished the HB-EGF promoter activity induced by LIV-1 overexpression. These results suggest that LIV-1 is involved in prostate cancer progression as an intracellular target of growth factor receptor signaling which promotes EMT and cancer metastasis. LIV-1 could be an attractive therapeutic target for the eradication of pre-existing human prostate cancer and bone and soft tissue metastases.

Myeloid Cell Leukemia-1 (Mcl-1) is a widely expressed anti-apoptotic member of the Bcl-2 family that is elevated in a variety of tumour types including breast cancer. Mcl-1 promotes tumour cell survival and drug resistance and was a mechanism of resistance to first generation Bcl-2 family inhibitors. To determine the significance of Mcl-1 in breast cancer, we evaluated the regulation of Mcl-1 by signalling via the epidermal growth factor receptors (EGFRs). EGFR signalling is frequently deregulated in breast cancer and leads to increased proliferation and survival of tumour cells. We aimed to determine whether Mcl-1 is a critical downstream effector of this pathway and therefore an important therapeutic target. We found that Mcl-1 protein and messenger RNA levels were rapidly induced upon stimulation of breast cancer cells with epidermal growth factor. This induction was blocked by inhibitors of the Ras/Raf/Mek/Erk signalling cascade and was dependent upon activation of the transcription factor Elk-1. We found Mcl-1 to be an essential survival protein, as targeted knock-down with small interfering RNA alone was sufficient to induce apoptosis. Mcl-1 may be critical for the survival advantage conferred by EGFR activation, as prevention of its up-regulation by Mek/Erk inhibitors significantly reduced the drug resistance conferred by EGF. Furthermore, we found a correlation between phosphorylated Elk-1 and Mcl-1 protein levels in breast tumour samples. Therefore, we conclude that Mcl-1 is an important downstream effector of survival and drug resistance mediated by elevated EGF signalling, making it an important therapeutic target in breast cancer.

Myeloid Cell Leukemia-1 (Mcl-1) is a widely expressed anti-apoptotic member of the Bcl-2 family that is elevated in a variety of tumour types including breast cancer. Mcl-1 promotes tumour cell survival and drug resistance and was a mechanism of resistance to first generation Bcl-2 family inhibitors. To determine the significance of Mcl-1 in breast cancer, we evaluated the regulation of Mcl-1 by signalling via the epidermal growth factor receptors (EGFRs). EGFR signalling is frequently deregulated in breast cancer and leads to increased proliferation and survival of tumour cells. We aimed to determine whether Mcl-1 is a critical downstream effector of this pathway and therefore an important therapeutic target. We found that Mcl-1 protein and messenger RNA levels were rapidly induced upon stimulation of breast cancer cells with epidermal growth factor. This induction was blocked by inhibitors of the Ras/Raf/Mek/Erk signalling cascade and was dependent upon activation of the transcription factor Elk-1. We found Mcl-1 to be an essential survival protein, as targeted knock-down with small interfering RNA alone was sufficient to induce apoptosis. Mcl-1 may be critical for the survival advantage conferred by EGFR activation, as prevention of its up-regulation by Mek/Erk inhibitors significantly reduced the drug resistance conferred by EGF. Furthermore, we found a correlation between phosphorylated Elk-1 and Mcl-1 protein levels in breast tumour samples. Therefore, we conclude that Mcl-1 is an important downstream effector of survival and drug resistance mediated by elevated EGF signalling, making it an important therapeutic target in breast cancer.

The Rab family of proteins are low molecular weight GTPases that have the ability to switch between GTP- (active) and GDP- (inactive) bound form, and in that sense act as molecular switches. Through distinct localization on various vesicles and organelles and by cycling through GTP/GDP bound forms, Rabs are able to recruit and activate numerous effector proteins, both spatially and temporally, and hence behave as key regulators of trafficking in both endocytic and biosynhtetic pathways. The Rab5 protein has been shown to regulate transport from plasma membrane to the early endosome as well as activate signaling pathways from the early endosome. This dissertation focused on understanding Rab5 activation via endocytosis of receptor tyrosine kinases (RTKs). First, tyrosine kinase activity of RTKs was linked to endosome fusion by demonstrating that tyrosine kinase inhibitors block endosome fusion and activation of Rab5, and a constitutively active form of Rab5 is able to rescue endosome fusion. However, depending on how much ligand is available at the cell surface, the receptor-ligand complexes can be internalized via a number of distinct pathways. Similarly, Rab5 was activated in a ligand-dependent concentration dependent manner via clathrin- and caveolin-mediated pathways, as well as a pathway independent of both. However, overexpression Rabex-5, a nucleotide exchange factor for Rab5, is able to rescue activation even when all of the pathways of EGF-receptor internalization were blocked. Next, the three naturally occurring splice variants of Rabex-5 selectively activated Rab5. Lastly, Rabex-5 inhibits differentiation of 3T3-L1 and PC12 cells through 1) degradation of signaling endosome via Rab5-dependent fusion with the early endosome, 2) and inhibition of signaling cascade via ubiquitination of Ras through the ZnF domain at the N-terminus of Rabex-5. In conclusion, these data shed light on complexity of the endosomal trafficking system where tyrosine kinase activity of the receptor is able to affect endosome fusion; how different endocytic pathways affect activation of one of the key regulators of early endocytic events; and how selective activation of Rab5 via Rabex-5 can control adipogenesis and neurogenesis.

This thesis deals with the determination of biological activity of adiponectin, a recombinant protein using cell culture. First it was important to acquire the working skills for the cell culture of cell line 3T3-L1. An optimal concentration of inactivated fetal bovine serum in cell culture media was determined. A stimulation of the cell proliferation by HB-EGF, PDGF-BB and bFGF growth factors was observed at various concentration levels. Afterwards the biological activity of adiponectin was determined as an inhibition of growth stimulation with 5 ng/ml PDGF-BB. This biological activity assay for adiponectin was also conducted with lyophilized adiponectin and a growth factor bFGF (0.1 ng/ml). The lyophilization did not affect the biological activity of adiponectin.