published_or_final_version / Paediatrics / Master / Master of Philosophy

The fps proto-oncogene encodes a 92 kDa cytoplasmic tyrosine kinase. Previous studies have shown that Fps expression in the mammary gland changes with development, and Fps has a suppressor function in mammary tumorigenesis. The aim of my thesis was to elucidate the role of the Fps tyrosine kinase in regulating mammary gland development and function. We have shown that the expression of the Fps kinase in the mammary gland increased during pregnancy and reached its maximum during lactation. The level of Fps tyrosine phosphorylation paralleled the expression pattern. Pups reared by fps-null females gained weight more slowly than those reared by wild-type females. Epithelial cells were the primary source of Fps expression. Milk protein and fat content were not affected by the absence of Fps. Similarly, no differences in mammary gland structure were observed with whole mount or histological analysis. Fps was shown to be in a multi-protein complex with E-cadherin, β-catenin and p120-catenin. A strong co-localization signal was observed for Fps and E-cadherin. Immunofluorescence analysis indicated that the localization of E-cadherin and β-catenin was disorganized and less concentrated at sites of cell-cell contacts in the fps-null glands. The interactions between the different adherens junction components were altered in the fps-null tissue. Specifically, less E-cadherin and β-catenin was associated with p120-catenin in the fps-null glands. Suprisingly, no phosphotyrosine differences were detected for the adherens junction components. Conditions were established to grow primary murine epithelial cell cultures that could be used to investigate the function of Fps. Fps expression was up-regulated in these cells in response to lactogenic hormones. A lentiviral system encoding a murine p53 shRNA sequence was used to increase the growth potential of the primary cells. Continual growth of the infected and uninfected primary epithelial cell mixture resulted in the establishment of an immortalized cell line. Immunofluorescent and immunoblot analyses revealed that the cells have undergone an epithelial-to-mesenchymal transition. With the transduction of a myc-epitope tagged Fps into the cells, we have generated cell lines with the appropriate genetic backgrounds to study the function of the Fps kinase in the mammary gland, specifically as it relates to tumorigenesis. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2008-07-03 11:53:01.135

FES protein-tyrosine kinase (PTK) activation downstream of the KIT receptor in mast cells (MC) promotes cell polarization and migration towards the KIT ligand Stem cell factor (SCF). A variety of tumours secrete SCF to promote MC recruitment and release of mediators that enhance tumour vascularization and growth. This study investigates whether FES promotes MC migration via regulation of microtubules (MTs), and if FES is required for MC recruitment to the tumour microenvironment. MT binding assays showed that FES has at least two MT binding sites, which likely contribute to the partial co-localization of FES with MTs in polarized bone marrow-derived mast cells (BMMCs). Live cell imaging revealed a significant defect in chemotaxis of FES-deficient BMMCs towards SCF embedded within an agarose drop, which correlated with less MT organization compared to control cells. To extend these results to a tumour model, mouse mammary carcinoma AC2M2 cells were engrafted under the skin and into the mammary fat pads of immune compromised control (nu/nu) or FES-deficient (nu/nu:fes-/-) mice. A drastic reduction in tumour-associated MCs was observed in FES-deficient mice compared to control in both mammary and skin tissue sections. This correlated with a trend towards reduced tumour volumes in FES-deficient mice. These results implicate FES signaling downstream of KIT, in promoting MT reorganization during cell polarization and for chemotaxis of MCs towards tumour-derived SCF. Thus, FES is a potential therapeutic target to limit recruitment of stromal mast cells or macrophages to solid tumours that enhance tumour progression. / Thesis (Master, Biochemistry) -- Queen's University, 2011-09-14 11:49:32.871

No description available.

Agrin triggers the subsynaptic aggregation of acetylcholine receptor (AChR) via activation of the receptor tyrosine kinase MuSK (muscle-specific kinase). At present, the intracellular mechanisms utilized by MuSK to initiate such a complex process remain unknown. In the present study, I first tested if H-ras was involved in the process of synaptogenesis induced by agrin. The data presented suggest that ras could have a role in this process because a dominant inhibitory ras mutant (ras-N17) partially blocked the inductive effects of agrin while two activated ras mutants (ras-V12 and ras-V12-D38) induced agrin-independent AChR clusters. These effects were not due to major alterations in the levels of AChR, though more experiments are required to confirm these preliminary findings. / Second, I investigated whether the adaptor protein Shc was a downstream effector of activated MuSK. MuSK and Shc could be co-immunoprecipitated, but this association was not consistently observed nor was it modulated by agrin at all times. Generally, no alteration in Shc phosphotyrosine content was observed in response to agrin, and when an increase was detected, it was modest. Finally, agrin did not modulate the interaction between Shc and Grb2. Based on these results, I conclude that Shc interaction with MuSK is not regulated by agrin.

SRMS (Src-Related tyrosine kinases lacking C-terminal Regulatory tyrosine and N terminal Myristoylation Sites) belongs to a family of non-receptor tyrosine kinases, which also includes breast tumor kinase (BRK). SRMS was first identified in 1994 in a screen for the genes that regulate the growth and differentiation of neuroepithelial cells. This 54 kDa protein spanning 488 amino acids, consists of the prototypical Src homology 3 (SH3), Src homology 2 (SH2) and a tyrosine kinase domain. While BRK has been documented for its expression in over 60 % of breast carcinomas, information on SRMS on similar grounds remains absent from the literature. Furthermore, unlike BRK, knowledge of how SRMS regulates its enzymatic activity as well as the identification of its substrates remains unknown. The work in this thesis demonstrates that SRMS is potentially expressed in the majority of breast carcinomas. To understand the biochemical and cellular functions of SRMS, a series of mutants comprising point mutations as well as the deletion of the N-terminal region and the functional, SH3 and SH2 domains, were generated and assessed for enzymatic activity in cells. This study demonstrates for the first time that the wild type protein is apparently constitutively active and that its N-terminal region regulates its enzymatic activity. As well, three critical amino acid residues in the protein namely, lysine 258 (ATP binding site), tyrosine 380 (auto-phosphorylation site) and tryptophan 223 (intramolecular interaction) have been characterized. All three residues have been determined to be essential for the enzymatic activity of SRMS. Finally, the adapter protein Dok1 has been characterized as a novel substrate of SRMS. The results from the present study underscore the potential significance of the catalytically active non-receptor tyrosine kinase, SRMS that should serve as a foundation upon which further research may ensue in the context of breast tumorigenesis.

Le gène ShcA code pour trois isoformes de protéines adaptatrices (p46, p52 et p66Shc), possédant entre eux une forte homologie structurelle. Les études antérieures ont attribué un rôle essentiel à l'isoforme p52Shc dans l'activation de la voie mitogénique des MAPK, ainsi que dans la transformation cellulaire en aval des récepteurs de type tyrosine kinase (RTK). L'activation des MAPK fait intervenir une liaison directe de p52Shc aux RTK suivant leur activation, ce qui permet la phosphorylation sur tyrosines de Shc et ainsi, le recrutement de la protéine adaptatrice Grb2. Pendant longtemps, ces fonctions ont été attribuées aux trois isoformes des protéines Shc. Cependant, il est maintenant évident que Pisciforme p66Shc joue des rôles distincts de p52Shc. En effet, l'expression de p66Shc n'induit pas la transformation cellulaire et inhibe l'activité des MAPK en aval des RTK. D'ailleurs, p66Shc induit l'apoptose en réponse aux stress oxydatifs, par le biais de la phosphorylation d'une sérine située dans son domaine unique en N-terminal. Bien que ces caractéristiques proposent un pouvoir anti-tumorigénique à p66Shc, son rôle dans la progression du cancer demeure controversé. Mes travaux de recherche démontrent que p66Shc est constitutivement associé au récepteur dii facteur de croissance des hépatocytes, le RTK Met, et diminue fortement l'interaction entre le récepteur Met actif et Grb2. Nos études in cellulo et in vivo dans un modèle de cellules épithéliales intestinales transformées par Tpr-Met (Tpr-Met-IEC-6), une forme oncogénique du récepteur Met, révèlent que l'expression de p66Shc induit l'apoptose à l'atteinte de la confluence et diminue la croissance tumorale, dépendamment de sa phosphorylation sur sérine. En revanche, p66Shc augmente la capacité de ces cellules à survivre sans ancrage à la matrice extracellulaire et favorise la formation de métastases. En conclusion, nous démontrons qu'une protéine Shc s'associe à un RTK indépendamment de son activation. Il s'agit de la première évidence que les modes d'interaction de p66Shc avec les RTK different de ceux caractérisés pour p52Shc. De plus, nos études permettent d'illustrer la dualité fonctionnelle de p66Shc, où son expression diminue la capacité tumorigénique des cellules en adhésion, mais augmente leur potentiel métastatique lorsqu'elles sont en suspension, dénotant le rôle controversé de p66Shc dans le cancer. Cette nouvelle compréhension des rôles distincts associés à p66Shc selon le contexte de la cellule cancéreuse permet de cibler différentes étapes du développement du cancer.

No description available.

Currently, the intensive chemotherapy remains the first line treatment for B cell acute lymphoblastic leukemia (B-ALL). Although these regimens have significantly improved patient outcomes, their use is associated with debilitating morbidities and fatal relapses, highlighting the great need in new agents that target essential survival signals in leukemia. Thus, the overall goal of my project was to gain insights into the signaling abnormalities that regulate aberrant proliferation and survival of B-ALL cells in an effort to identify novel targets in this malignancy. This study demonstrated that pre-B cell receptor (pre-BCR)-independent spleen tyrosine kinase (SYK) activity was required for the survival and proliferation of a p53-/-PrkdcSCID/SCID mouse model of B-ALL. I extended this discovery to human disease, demonstrating that SYK was activated in primary B-ALL, independent of the pre-BCR expression. The small molecule SYK inhibitor fostamatinib (fosta) significantly attenuated proliferation of 79 primary diagnostic B-ALL samples at clinically achievable concentrations. Importantly, fosta treatment reduced dissemination of engrafting B-ALL cells into the spleen, liver, kidney and central nervous system (CNS) in a NOD.Prkdcscid/scidIl2rgtm1Wjl/SzJ xenotransplant model of B-ALL. Analysis of signaling abnormalities using a high-throughput phospho-flow cytometry platform demonstrated that pediatric and adult B-ALL samples exhibit variable basal activation of BCR, iii PI3K/AKT/mTOR, MAPK and JAK/STAT pathways. Importantly, we identified that fosta-mediated inhibition of SYK, PLC2, CRKL and EIF4E phosphorylation in B-ALL was predictive of its anti-leukemic activity, and was distinct from the cellular actions of other small molecule inhibitors of key nodal signaling pathways. Examination of molecular mechanism of fosta action by gene expression profiling revealed transcriptional effects of fosta treatment that included, most notably, potent inhibition of pathways involved in lymphocyte activation and inflammation. In conclusion, this study demonstrates that SYK signaling is crucial for B-ALL survival and provides detailed characterization of cellular and molecular mechanisms of fosta action in B-ALL. These data argue in favor of testing small molecule SYK inhibitors in pediatric and adult B-ALL.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1995. / Includes bibliographical references (leaves 76-93). / Microfiche. / vii, 93 leaves, bound ill. (some col.) 29 cm