No description available.

Breast cancer is the most frequently diagnosed form of cancer in women and the second leading cause of cancer-related deaths. Breast cancer is a heterogeneous disease consisting of many types of tissue neoplasia, and there appears to be no model of how a particular lesion develops into an aggressive, malignant, invasive carcinoma. Genetic mutation and aberrant epigenetic regulation are among the most common events that lead to neoplasia. In breast cancer, p53 mutation is the most common genetic defect related to a single gene. Therefore, this dissertation focuses on the mechanisms and consequences of p53 mutation during breast tumorigenesis. Genome-wide analysis of gene expression and epigenetic modifications in a panel of breast cancer cell lines suggested that p53 mutation and aberrant epigenetic silencing were cooperating mechanisms in the silencing of wild-type p53 target genes during cancer progression. Therefore, models of p53 inactivation were created in non-malignant human mammary epithelial cells to determine the role of p53 mutation on the epigenetic status of its target genes and the acquisition of malignant phenotypes. Comparisons of each model demonstrated that differing modes of p53 inactivation produced different functional consequences. Loss of wild-type p53 function alone ablated the normal cellular response to external stress stimuli, but had no affect on the expression of genes or epigenetic status in untreated cells. Introduction of missense mutant p53 protein caused very few changes when the protein was expressed at low levels. However, accumulation of mutant p53 caused a variety of gene expression changes and interfered with endogenous wild-type p53. The accumulation of mutant p53 also caused an increase in migration and invasion of the cells that expressed it. Interestingly, epigenetic aberrations were not detected in response to any of the p53 manipulations. These data suggest that accumulation of missense mutation is particularly dangerous to normal cells. They also suggest that p53 mutation and epigenetic aberration are two distinct mechanisms, which overlap and cooperate during tumorigenesis. These data suggest that treatment strategies for human breast cancer should include modalities to target both defects for increased efficacy.

L'activation du plasminogène est importante dans les phénomènes liés au cancer. Ma thèse démontre que la mélanotransferrine agit comme accélérateur dans l'activation du plasminogène avec ses deux activateurs : l'uPA, lié au cancer et le tPA, lié à la fibrinolyse. Son action ambivalente, à la fois protumorale et antitumorale, est caractérisée par son site d'action au niveau cellulaire. La mélanotransferrine membranaire agit comme un catalyseur pour amplifier l'activation du plasminogène à la membrane, tandis que la forme soluble de la mélanotransferrine entre en compétition avec la mélanotransferrine membranaire pour inhiber son effet. Dans la présente étude, nous avons découvert que deux composantes du système plasminolytique : le pro-uPA, précurseur de l'uPA et le plasminogène interagissent avec la mélanotransferrine. L'interaction de la mélanotransferrine avec le système plasminolytique stimule l'activation du plasminogène par ses deux activateurs. Nous démontrons également avec un anticorps dirigé contre la mélanotransferrine ou par l'inhibition de son expression par un siRNA que la mélanotransferrine intervient dans le processus menant aux métastases par l'inhibition de la migration cellulaire. De plus, nous démontrons que la mélanotransferrine module la dissolution de caillots de fibrine dépendante du tPA ou de l'uPA, une composante importante de la matrice provisoire extracellulaire impliquée dans la dispersion des métastases. Dans un modèle où le dépôt de fibrine provoqué par le facteur tissulaire (TF) stimule l'invasion des cellules de mélanome (SK-Mel-28), nous démontrons une inhibition de l'invasion induite par le TF dans le poumon lorsque l'expression de la mélanotransferrine est inhibée par un siRNA. Ces résultats suggèrent que la mélanotransferrine est directement impliquée dans la propagation des métastases des mélanomes qui l'expriment fortement. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Activation du plasminogène, Invasion tumorale, Migration, Fibrinolyse, uPA, tPA.

Metastasis is the leading cause of mortality in human breast cancer. However, there are few predictive, prognostic, or therapeutic targets of breast cancer metastasis. Ezrin, a membrane cytoskeletal cross-linker, is frequently over-expressed in human breast cancer and is required for motility and invasion by cultured epithelial cells. Our group has recently shown that ezrin acts co-operatively with the non-receptor tyrosine kinase, Src, in the transformation of epithelial cells, in which ezrin is phosphorylated on specific tyrosines, such as Y477, by Src (91, 93). We therefore examined whether Src/ezrin interaction also regulates invasion and metastasis of breast cancer. This thesis presents the following results: 1) In a murine system, ezrin and Src are differentially localized in nulliparous, lactating mammary glands and PyMT-induced tumours, with pronounced apical expression in nulliparous mammary glands but non-polarized strong cytoplasmic expression in PyMT-induced tumours. 2) Increased expression and activation of ezrin, Src and Met in PyMT-induced tumours compared to normal breast tissues was observed. A concomitant increased expression of activated Stat3 and HGF was also observed in PyMT-induced tumours, consistent with the establishment of an HGF/Met autocrine loop. 3) In invasive human breast tumours, from a premenopausal patient cohort, ezrin showed significantly greater cytoplasmic localization compared to non-neoplastic epithelial ducts in normal mammoplasties. 4) In a mouse breast carcinoma xenograft model, a Y477F ezrin mutant (not phosphorylatable by Src), significantly reduced local invasion of primary tumours and spreading into visceral organs, yet, it did not significantly affect primary tumour growth rate. 5) Y477F ezrin-expressing tumours exhibited focal areas of incomplete membranous ezrin staining which was absent in control tumours. Moderate/strong cytoplasmic ezrin staining was evident in both tumour groups. Thus, ezrin is differentially localized in non-invasive versus invasive mammary tumours. Our study implicates a role of the Src/ezrin pathway in regulating local invasion and metastasis of breast carcinoma cells and provides a clinically relevant model for assessing the Src/ezrin pathway as a potential prognostic marker and treatment target for invasive breast cancer. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2010-04-28 12:24:25.286

Lactoferrin (Lf) is an iron binding protein produced in mammals. It has antimicrobial and immunomodulatory properties. Some bacteria that regularly colonize mammalian hosts have adapted to living in high Lf environments. Helicobacter pylori, which inhabits the human gut, was chosen as a model organism to investigate how bacteria may adapt to Lf. H. pylori was able to use iron from fully saturated human Lf (hLf) in various low iron media, achieving growth levels similar to the ironreplete control. Partially saturated hLf decreased growth, yet both partially saturated bovine Lf (bLf) and hLf were able to increase internalization of bacteria into mammalian tissue culture cells. A substantially larger increase in internalization was seen when bacteria were supplemented with hLf in low iron conditions, possibly mediated by iron-regulated cellular receptors or bacterial lactoferrin binding proteins. In eukaryotes, Lf is known to bind and facilitate internalization of DNA into cells and sometimes the nucleus, and upregulate gene expression. Here, one hundred bacterial genomes were surveyed for known Lf binding sites as an indication that Lf had similar functions using bacterial DNA. While the frequency and location of Lf binding sites suggest they occur at random, their presence in all genomes suggests that Lf may be able to act as a vector for bacterial DNA, and facilitate the movement of genes between species. Lf is being widely considered for commercial and therapeutic uses, with significant interest in producing it in genetically modified organisms (GMO). Widespread production and use of Lf could increase the number of bacteria that are adapted to it. How Lf interacts with bacteria adapted to it, and the ability of it to act as a DNA vector, may have relevance for GMO risk assessment.

La compréhension des mécanismes impliqués dans la formation des métastases est l’un des défis majeurs de la recherche sur le cancer. En effet, la formation de métastases est la cause principale de mortalité chez les patients atteints du cancer. L'influence du microenvironnement tumoral fait partie intégrante de la recherche et plusieurs études démontrent qu’il joue un rôle primordial dans l’invasion des cellules tumorales. L’une des caractéristiques du microenvironnement tumoral est l’hypoxie. Les cellules cancéreuses ont développé différentes stratégies afin de survivre dans ce microenvironnement. Des études récentes rapportent que des mécanismes posttranscriptionnels sont induits par l’hypoxie tels que le routage intracellulaire de molécules d'adhésion, de protéases et l’activation de facteurs de croissance, et qu’ils influencent le phénotype métastatique des cellules cancéreuses. L’étape importante dans l’initiation de la formation des métastases est la dégradation de la membrane basale de la tumeur et de la matrice extracellulaire. Les cellules cancéreuses ont développé des stratégies afin de faciliter leur migration dont l’une est la formation d'invadopodes. Malgré plusieurs études sur la biogenèse et les fonctions de ces structures, peu de travaux ont été accomplis concernant l’influence du microenvironnement tumoral hypoxique sur la formation et les fonctions des invadopodes. Les travaux présentés dans cette thèse portent sur l’étude des mécanismes induits par l’hypoxie dans l’invasion des cellules cancéreuses. Dans le premier chapitre de la section résultats, nous démontrons que l’hypoxie induit une relocalisation stratégique de la furine, une convertase de pro-protéines, dans une boucle de recyclage en périphérie cellulaire. Nous démontrons que la redistribution de la furine favorise l’invasion cellulaire en condition hypoxique. Dans le deuxième chapitre, nous avons étudié l'impact de la relocalisation de la furine en hypoxie sur la maturation de substrats tumorigéniques. Nos résultats indiquent que l’hypoxie favorise la maturation du TGFß par la furine dans des vésicules acides. Nous démontrons que la présence d’une histidine, sensible au pH, située au site de clivage du pro-TGFß par la furine influence la capacité de cette dernière à cliver la pro-protéine. Enfin, dans le dernier chapitre, nous démontrons que l’induction hypoxique de la formation des invadopodes est principalement causée par la signalisation dépendante de Smad3 du TGFß. Nous identifions la HDAC6 comme étant un régulateur de la signalisation du TGFß en hypoxie. La HDAC6 permettrait la libération de Smad3 du réseau de tubuline, et suite à la liaison du TGFß sécrété à son récepteur, permettrait la phosphorylation et la translocation nucléaire de Smad3 afin d'induire ses gènes cibles. L'ensemble de ces travaux a permis d'identifier des molécules clés impliquées dans la formation des invadopodes en condition hypoxique. Nos travaux ont contribué de manière substantielle à nos connaissances des mécanismes impliqués dans l’invasion cellulaire dans le microenvironnement hypoxique. Nos résultats permettront en outre l’identification de cibles thérapeutiques potentielles qui pourraient servir à inhiber l’invasion cellulaire et la formation de métastases.

No description available.

Lactoferrin (Lf) is an iron binding protein produced in mammals. It has antimicrobial and immunomodulatory properties. Some bacteria that regularly colonize mammalian hosts have adapted to living in high Lf environments. Helicobacter pylori, which inhabits the human gut, was chosen as a model organism to investigate how bacteria may adapt to Lf. H. pylori was able to use iron from fully saturated human Lf (hLf) in various low iron media, achieving growth levels similar to the ironreplete control. Partially saturated hLf decreased growth, yet both partially saturated bovine Lf (bLf) and hLf were able to increase internalization of bacteria into mammalian tissue culture cells. A substantially larger increase in internalization was seen when bacteria were supplemented with hLf in low iron conditions, possibly mediated by iron-regulated cellular receptors or bacterial lactoferrin binding proteins. In eukaryotes, Lf is known to bind and facilitate internalization of DNA into cells and sometimes the nucleus, and upregulate gene expression. Here, one hundred bacterial genomes were surveyed for known Lf binding sites as an indication that Lf had similar functions using bacterial DNA. While the frequency and location of Lf binding sites suggest they occur at random, their presence in all genomes suggests that Lf may be able to act as a vector for bacterial DNA, and facilitate the movement of genes between species. Lf is being widely considered for commercial and therapeutic uses, with significant interest in producing it in genetically modified organisms (GMO). Widespread production and use of Lf could increase the number of bacteria that are adapted to it. How Lf interacts with bacteria adapted to it, and the ability of it to act as a DNA vector, may have relevance for GMO risk assessment.

Zugl.: Marburg, Univ., Diss., 2008

Ulm, Univ., Diss., 2002.