RASSF1A is a tumour suppressor, the silencing of which occurs through promoter methylation in a variety of human cancers. Loss of RASSF1A is associated with decreased sensitivity to DNA damaging agents and worse prognosis in breast, colon and lung cancers amongst others. RASSF1A functions in a number of cellular processes, promoting apoptosis in response to DNA damage or death receptor signalling, or cell cycle arrest at both G1/S and pro-metaphase checkpoints. As a scaffold protein, RASSF1A imparts these functions through direct interaction with target proteins. We have identified a novel interaction between RASSF1A and the SRC activator, OSSA. Further studies identify a role for RASSF1 in SRC signalling. We find that a second isoform of RASSF1, RASSF1C, the expression of which is maintained in cancers, is able to activate SRC. We also identify a novel tumour suppressor role for RASSF1A inhibiting SRC activation through binding of RASSF1C. SRC activation by RASSF1C expression promotes internalisation of adherens junctions leading to subsequent loss of tight junctions and cell polarity markers from sites of cell-cell contact. -catenin is also found to be re-localised throughout the cells from where it is hypothesised to be able to upregulate pro-proliferative genes. In addition, we find that RASSF1C expression promotes cell motility in both scratch wound and transwell assays. Finally, we show that RASSF1C expression enhances tumour cell aggressiveness using a mammosphere growth assay. We conclude that RASSF1C is an oncogene that can promote EMT through the activation of SRC family kinases. This function is inhibited by the tumour suppressor RASSF1A. This work highlights why RASSF1A is lost through epigenetic mechanisms and not mutation and why loss of RASSF1A is associated with more aggressive, metastatic cancers.

Breast cancer (BC) is one of the leading causes of cancer-related deaths in Canadian women. Aggressive BCs (e.g. triple-negative subtype; TN) present a clinical challenge as defined biomarkers, particularly those indicative of unique cancer-associated signaling pathways, are needed to improve prognostication and prediction of therapeutic response. Overexpression of Src and its substrates, Ezrin and Tks5, have been associated with poor prognosis in many cancers. We have previously shown that Ezrin regulates proteolytic-independent invasion, while others have shown that Tks5 is associated with proteolytic-dependent invasion. Thus, expression of Ezrin versus Tks5 in BC cases may represent different invasion modalities. Additionally, immunofluorescence (IF)-based technologies may provide a more quantitative and objective approach for analysis of biomarker expression and subcellular compartmentalization compared to immunohistochemistry (IHC). In this study, I hypothesize that expression and subcellular localization of Src, Ezrin and Tks5, have improved prognostic significance in BCs, compared to current clinico-pathological parameters. To assess this, I optimized an IF-based automated quantification analysis (AQUA) system to measure subcellular expression in a 63-patient BC cohort and tested associations with clinico-pathological data. This thesis presents that: 1) Expression of Src and Ezrin increased, but that of Tks5 decreased in breast tumours compared to normal breast. 2) Src and Ezrin localized to the apical regions of normal breast epithelia but shifted to the cytoplasm in breast tumours. Tks5 exhibited a granular basal expression in normal breast epithelia, and is weakly expressed in tumour cellular compartments. 3) In our 63-patient cohort, Src and Ezrin had significant correlations with multiple clinico-pathological parameters, including TN status and lymphovascular invasion. 4) Clinico-pathological associations with IF-based AQUA scoring are directly comparable to conventional manual IHC scoring. Our study supports the role of Src and Ezrin as potential prognostic biomarkers for BC. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2013-07-09 12:51:09.527

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

ABSTRACT Phosphorylation is a post-translational modification important for regulating protein activity and protein binding capacity. It is used in many different signaling pathways within the cell. Src Family Kinases and Protein Kinase A (PKA) are two prototyptical non-receptor tyrosine and serine/ threonine kinases, respectively, which are found in canonical signaling pathways. These two kinases are critical for signaling in essentially every cell of a multicellular organism, and are particularly important in development, cell migration and proliferation. Although both proteins have been intensely studied for many decades, an understanding of the molecular mechanisms which govern their regulation and the regulation that they effect on other proteins are still being elucidated. Fyn, like its related Src Family Kinase members, has previously been shown to be regulated by two tyrosine phosphorylation events at residues Y420 and Y531. Y420 is located in the kinase (Src Homology 1(SH1)) domain and it is a highly-characterized intermolecular autophosphorylation site that increases the activity of the kinase. Y531 is located near the C-terminus and is phosphorylated by C-terminal Src kinase (Csk). Phosphorylation of Y531 allows it to bind to R176 in the SH2 domain in an intramolecular fashion. In this conformation Fyn has only basal activity. Since these sites are essential for regulating the activity of the kinase, we hypothesized that four novel sites of tyrosine phosphorylation in Fyn could also importantly regulate the protein. Three of the novel sites lie in the SH2 domain, and one is located in the kinase domain. Mass spectrometry, in vitro kinase assays, as well as western blot analysis aided in uncovering that these novel Fyn phosphorylation sites fine tune the activity and substrate binding of the protein. PKA has been implicated in a multitude of signaling pathways and is particularly important in cell growth, proliferation, and migration. Fyn and PKA have classically been considered to be in separate signaling pathways. However, research over the past several decades has provided evidence that there is crosstalk that exists between the two pathways. The SFK Fyn and PKA can phosphorylate each other, thereby regulating each other's activity. Based on these data, we hypothesized the existence of downstream effectors of this relatively uncharacterized pathway. It was hypothesized that the presence of Fyn could lead to PKA activation and to differences in PKA binding partners. Through the use of co-immunoprecipitations, Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC) and quantitative mass spectrometry, many proteins were found to increase their binding to PKA in the presence of Fyn. Several proteins were selected and further biochemically validated. These data suggest that the presence of Fyn could allow for PKA to more importantly interact with discrete pools of proteins within the cell to effectuate its signal transduction. Together these studies provide understanding on critical and fundamental processes by which all cells function.

La perméabilité vasculaire est une caractéristique cruciale de l’angiogenèse. Les acteurs principaux sont les cellules endothéliales qui la régulent en réponse à divers facteurs perméabilisant, tels que le « Vascular Endothelial Growth Factor » (VEGF). Dans le contexte pathologique du cancer, les cellules tumorales produisent de grandes quantités de VEGF qui stimulent la perméabilité, ce qui leur permet d’infiltrer le réseau vasculaire. Il est connu que la tyrosine kinase Src contrôle cette modulation de la perméabilité. Puisque notre laboratoire a préalablement démontré que la phosphatase de type récepteur (PTP) DEP-1 est impliquée dans l’activation de Src en réponse au VEGF, nous avons émis l'hypothèse que DEP-1 pourrait aussi jouer un rôle dans la perméabilité des cellules endothéliales. Grâce à des expériences de transfections d’ARN interférant, nous démontrons que DEP-1 est important pour la régulation de la phosphorylation de la VE-Cadhérine, un médiateur critique de la perméabilité. L’impact de DEP-1 sur la dissociation de jonctions intercellulaires est également démontré par microscopie à immunofluorescence de cellules endothéliales. DEP-1 est également nécessaire à l’augmentation de la perméabilité induite par VEGF in vitro. Deux résidus tyrosine retrouvés dans la queue carboxy-terminale de DEP-1 sont essentiels à l’activation de Src en réponse au VEGF. Suite à la transfection d’un plasmide encodant DEP-1 muté pour ces deux résidus, nous démontrons aussi leur implication dans la régulation de la perméabilité in vitro par DEP-1. Ces travaux permettent ainsi d’approfondir nos connaissances sur un nouveau régulateur potentiel de la perméabilité vasculaire. / Endothelial cell permeability is a crucial step of angiogenesis. The main actors behind permeability are endothelial cells who accomplish this in response to permeabilizing factors, most notably Vascular Endothelial Growth Factor (VEGF). In a pathological context, migrating tumor cells produce great quantities of VEGF that stimulate an increase of vascular permeability, which allows them to intravasate into the vasculature. Src has been shown to mediate this process. Our laboratory has previously shown that the protein tyrosine phosphatase DEP-1 is involved in the regulation of VEGF-dependant activation of Src. These data thus suggested that DEP-1 might play a role in endothelial cell permeability. Here, we show through siRNA experiments that DEP-1 is important for the regulatory phosphorylation of VE-Cadherin which is critical for the induction of permeability. The impact of DEP-1 on intercellular junction dissociation is also demonstrated through immunofluorescence microscopy of endothelial cells. We further show that DEP-1 is absolutely required for the VEGF-dependent increase of permeability as illustrated by in vitro permeability assay on siRNA-transfected endothelial cells. Finally, we show that tyrosine residues in DEP-1’s carboxy-terminal tail, which are crucial for mediating Src activity in response to VEGF, are implicated in VEGF-dependant increase in permeability by transfecting plasmids coding for DEP-1 mutants of these tyrosine residues. These findings shed light on a novel potential key regulator of in vivo permeability.

The most common malignant glioma, glioblastoma multiforme (GBM), remains an intractable tumor despite advances in therapy. Its proclivity to infiltrate surrounding brain tissue contributes greatly to its treatment failure and the grim prognosis of patients. Radiation is a staple in modern therapeutic regimens, though cells surviving radiation become more aggressive and invasive. Consequently, it is imperative to define further the cellular mechanisms that control GBM invasion and identify promising novel therapeutic targets. Melanoma differentiation associated gene-9 (MDA-9/Syntenin) is a highly conserved PDZ domain-containing scaffolding protein that promotes invasion and metastasis in human melanoma models. We show that MDA-9/Syntenin is robustly expressed in GBM cell lines and patient samples, and expression increases by tumor grade. These findings are confirmed through database analysis, which revealed MDA-9/Syntenin expression correlates with shorter survival times and patient tumors high in MDA-9/Syntenin have a worse prognosis when undergoing radiotherapy. Modulating MDA-9/Syntenin levels produced changes in invasion, angiogenesis, and signaling, indicating MDA-9/Syntenin enhances glioma pathogenesis. Overexpression of MDA-9/Syntenin enhances invasion, while knockdown inhibits invasion, migration, and anchorage-independent growth in soft agar. MDA-9/Syntenin increases activation of c-Src, P38MAPK, and NF-kB, leading to elevated MMP2 expression and IL-8 secretion. Through an orthotopic tumor model, we show that shmda-9 tumor cells formed smaller tumors and had a less invasive phenotype in vivo. Knockdown of MDA-9/Syntenin radiosensitizes GBM cells and significantly reduces post-radiation invasion gains through abrogation of radiation-induced Src and EphA2 activity. In efforts to pharmacologically inhibit MDA-9/Syntenin, we describe the effects of a novel small molecule, PDZ1i, which targets the PDZ1 domain of MDA-9/Syntenin and successfully reduces invasion gains in GBM cells following radiation. While it does not effect astrocyte radiosensitivity, PDZ1i radiosensitizes GBM cells. PDZ1i inhibits crucial GBM signaling including FAK and mutant EGFR, EGFRvIII, and can negate gains in secreted proteases, such as MMP2 and MMP9, following radiation. In a model of glioma, PDZ1i treatment combined with radiation results in less invasive tumors and extends survival. Our findings indicate that MDA-9/Syntenin is a novel and important mediator of GBM pathogenesis, and further identify it as a targetable protein that enhances radiotherapy for treatment in glioma.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Protein p130Cas is the major tyrosine phosphorylated protein in cells transformed by v-crk and v-src oncogenes. P130Cas plays an important role in invasiveness and metastasis of Src-transformed cells. In breast cancer patients, high p130Cas levels are associated with higher recurrence of disease, poor response to tamoxifen treatment and lower overall survival. In non-transformed cells, after the stimulation of integrins, protein p130Cas is phosphorylated in substrate domain affecting cell migration and cytoskeletal dynamics. For this signalling is the SH3 domain of p130Cas indispensable. In this thesis, was for the first time using the Phage display method analysed and subsequently characterized the binding motif of SH3 domain of p130Cas. Based on this high-affinity motif [AP]-P-[APMS]-K-P-[LPST]-[LR]- [LPST], we predicted new interaction partners of protein p130Cas and subsequently confirmed the interaction with the Ser/Thr kinase PKN3. This kinase colocalizes with p130Cas in the nucleus and perinuclear region and could phosphorylate p130Cas. In this thesis, we also analysed the effect of phosphomimicking mutation of tyrosine from sequence ALYD, which is conserved in the sequence of SH3 domains, on ability of these domains to bind ligands. This mutation reduced binding by about 3 orders of...

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are exploited by human-specific pathogens to anchor themselves to or invade host cells. Interestingly, human granulocytes express a specific isoform, CEACAM3, that can direct efficient, opsonin-independent phagocytosis of CEACAM-binding Neisseria, Moraxella and Haemophilus species. As opsonin-independent phagocytosis of CEACAM-binding Neisseria depends on Src-family protein tyrosine kinase (PTK) phosphorylation of the CEACAM3 cytoplasmic domain, we hypothesized that an SH2-containing protein might be involved in CEACAM3-initiated, phagocytosis-promoting signals. Accordingly, we screened glutathione-S-transferase (GST) fusion proteins containing SH2 domains derived from a panel of signaling and adapter molecules for their ability to associate with CEACAM3. In vitro pull-down assays demonstrated that the SH2 domain of the adapter molecule Nck (GST-Nck SH2), but not other SH2 domains such as the Grb2 SH2 domain, interact with CEACAM3 in a phosphotyrosine-dependent manner. Either deletion of the cytoplasmic tail of CEACAM3, or point-mutation of a critical arginine residue in the SH2 domain of Nck (GST-NckSH2R308K) that disrupts phosphotyrosine binding, both abolished CEACAM3-Nck-SH2 interaction. Upon infection of human cells with CEACAM-binding Neisseria, full-length Nck comprising an SH2 and three SH3 domains co-localized with tyrosine phosphorylated CEACAM3 and associated bacteria as analyzed by immunofluorescence staining and confocal microscopy. In addition, Nck could be detected in CEACAM3 immunoprecipitates confirming the interaction in vivo. Importantly, overexpression of a GFP-fusion protein of the isolated Nck SH2 domain (GFP-Nck-SH2), but not GFP or GFP-Nck SH2 R308K reduced CEACAM3-mediated phagocytosis of CEACAM-binding Neisseria suggesting that the adaptor molecule Nck plays an important role in CEACAM3-initiated signaling leading to internalization and elimination of human-specific pathogens.