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Despite ongoing advancement in detection and treatment, breast cancer remains a major clinical challenge worldwide. Cancer has traditionally been conceptualized as a ‘disease of the genes’ by virtue of the mutagenic events necessary for its inception. It is now clear, however, that complex interactions take place between cancer cells and the array of non-cancerous cells and molecules in their immediate surroundings, known generally as the tumour microenvironment. Cancer-microenvironment interactions are increasingly recognized as processes that critically influence the outcome of disease. Cells of the host immune system are major components of the breast tumour microenvironment. While their presence in tumours is thought to reflect an attempt at disease eradication or containment, cancer cells can exploit the immune system through a variety of means, including the recognition of leukocyte-derived cytokines. As such, intratumoral leukocytes and high cytokine content are frequently associated with aggressive subtypes of breast cancer and poor prognosis. This dissertation explores the influence of one such cytokine, oncostatin-M (OSM), on the behaviour of breast cancer cells. Our results collectively demonstrate that OSM can rapidly and potently induce aggressive features in well-characterized cell models of luminal, well-differentiated breast cancer. These features include suppression of the important biomarker estrogen receptor-α (the key molecular target of endocrine therapy), gain of the breast cancer oncogene S100A7, loss of luminal epithelial differentiation and gain of mesenchymal features, and induction of a phenotype consistent with breast cancer stem cells. Each of these changes can potentially influence treatment responsiveness, the metastatic process, or both. Along with high levels of intratumoural leukocytes, the OSM-induced features listed above are known to associate with one another in human breast cancer. Tumours that display such characteristics have a poor prognosis and present the greatest challenges for modern breast cancer therapy, both because they are inherently prone to rapid metastasis and because targeted therapies for such tumours are lacking. The etiology of these aggressive disease subsets is largely unknown, and resolution of this issue would represent a major advancement in our understanding of breast cancer. Importantly, we found that expression of OSM and/or its receptor OSMR was reproducibly associated with these features in multiple breast cancer cohorts, largely confirming our experimental results. OSMR, in particular, was associated with poor clinical outcome. OSM signalling may thus provide a novel mechanistic explanation for the development of aggressive forms of breast cancer. If our findings are validated and expanded upon in future studies, OSM signalling could serve as a novel therapeutic target and may be an important consideration in the design and deployment of breast cancer immunotherapies. / Graduate

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

Aggrecan is one of the most important structural components of the extracellular matrix (ECM) of articular cartilage, where it contributes to the hydration of the tissue and its ability to resist compressive loads during joint movement. Increased aggrecan degradation and loss occurs in joint diseases and is thought to be mediated by enzymes such as the matrix metalloproteinases (MMPs) and aggrecanases (ADAMTS). It has also been proposed that aggrecan release from the cartilage can be mediated by a non-proteolytic mechanism which involves the degradation of hyaluronan (RA) to which the aggrecan is bound. As aggrecan degradation and loss is known to be induced by pro-inflammatory cytokines, IL-1, TNFalpha, IL-6, IL-17 and OSM were used to investigate the mechanisms involved in proteoglycan catabolism in organ cultures of bovine articular cartilage. Irrespective of the cytokine, all aggrecan fragments generated were characteristic of aggrecanase action, and no additional aggrecan-degrading enzymatic activity was detected. In the presence of OSM, more rapid aggrecan release was observed, due to both proteolysis and fragmentation of HA by hyaluronidase activity. Moreover, addition of OSM resulted in the cleavage of aggrecan at a non-canonical aggrecanase site near its carboxy-terminal globular domain. Such cleavage could be reproduced in vitro by the action of either ADAMTS-4 or ADAMTS-5. Gene expression analysis revealed that both aggrecanases were highly induced by the cytokines, and while ADAMTS-4 was the major aggrecanase to be stimulated in all conditions, ADAMTS-5 remains the predominant aggrecanase to be expressed in cartilage. Thus, the present study shows that aggrecanase activity is primarily responsible for aggrecan degradation in the early stages of cytokine stimulation, and that in the presence of OSM, aggrecanase substrate specificity can be differentially modulated and hyaluronidase-mediated RA degradation can be induced.

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Indiana University-Purdue University Indianapolis (IUPUI) / Pancreatic Ductal Adenocarcinoma (PDAC) is the third leading cause of cancerrelated deaths with a five-year survival rate of 11%. PDAC tumors are characterized by a dense desmoplastic stromal microenvironment, mediated in part through local cytokine production. PDAC tumors also elicit a systemic inflammatory response in the host; this, combined with a loss of body weight due to muscle and fat wasting, is characteristic of cachexia. Understanding the molecular mechanisms that drive malignant inflammation is critical to improve PDAC therapy and increase patient survival. Oncostatin M (OSM) belongs to the IL-6/GP130 family of cytokines, members of which have been shown to promote PDAC tumor development, inflammation, and cachexia. Much less is known of OSM. My central hypothesis was that OSM promotes pancreatic cancer and cachexia by inducing local and systemic inflammation, fibrosis, and wasting via OSM signaling through the receptor, OSM receptor (OSMR). We investigated effects of exogenous OSM administration in wildtype and IL-6 null mice without cancer. OSM induced systemic fibrosis, bone loss, local muscle wasting, and cardiac dysfunction in presence and absence of IL-6. We further defined the roles of OSM/OSMR in the pancreatic cancer microenvironment and macroenvironment. OSM activated genes involved in inflammation, fibrosis, and tumor progression in both tumor cells and fibroblasts and altered the tumor microenvironment, promoting a dense compaction of tumor cells and cancer associated fibroblasts. Loss of systemic OSM signaling altered tumor metabolism and reduced the stromal compartment without affecting tumor size. Loss of OSMR signaling in tumor cells reduced tumor size and promoted survival. However, systemic loss of OSM or OSMR in host cells did not halt effects of cachexia including muscle dysfunction, atrophy, or inflammation/anemia. Overall, OSM/OSMR signaling in the microenvironment is necessary in modulating tumor phenotype and promoting survival in PDAC but may not be necessary for pancreatic cancer cachexia. / 2024-08-02

<p>Allergic atopic asthma is a respiratory condition that involves immune responses to specific allergens resulting in coughing, wheezing, shortness of breath and tightness in the chest. During an atopic asthmatic attack, the immune system initiates cellular infiltration of lymphocytes and eosinophils, airway hyper-responsiveness and ECM remodeling, which manifests in lung dysfunction in chronic disease. ASMC have recently been shown to play a role in the inflammatory processes of asthma through the production of inflammatory mediators. Various cytokines and chemokines serve as stimulants for these pathways and therefore require further attention to examine inflammatory signaling. OSM, a member of the gp130 family of cytokines, is secreted by inflammatory cells and has been detected in the sputum of asthmatics. Previous findings have established the potential of OSM in induction of lung inflammation, its role in increasing ECM, and its potential role in asthma. Viral or bacterial infections cause asthma exacerbations which result in increased severity of symptoms. The innate immune system relies on pattern recognition receptors including the TLRs to recognize invading pathogens and activate cells such as macrophages and natural killer cells. Although there are a number of these TLRs, this project will focus on the role of TLR3 and TLR4 in ASMC. I generally hypothesized that OSM markedly increases lung cell airway smooth muscle cell responses to external stimulae, such as products of bacteria or viruses that activate toll-like receptors. This exacerbates inflammation and extracellular matrix remodeling which contributes to pathology in asthmatic patients. Findings in this thesis have demonstrated that OSM stimulation increases the production of various cytokines and chemokines and growth factors seen in asthma. Co-stimulations with OSM and TLR-ligands augmented the production of a variety of these inflammatory mediators in comparison to ligands alone. TLR responses were shown to be associated with TLR expression, at both the mRNA and protein level, as well through the activation of the JAK-STAT and NFκB pathways. These findings implicate ASMC in immunomodulatory roles in response to TLR-ligands and OSM, and could play a role in the increased severity of asthma seen during exacerbations.</p> / Master of Science (MSc)

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal. / Des souris transgéniques ont été créées chez lesquelles l'expression de l'oncostatin M (OM), une cytokine de la famille de l'interleukin-6 (IL-6), est restreinte aux lymphocytes T immatures par l'emploi du promoteur proximal de la tyrosine kinase p561ck Ces souris, nommées LckOM, dévoilent des particularités fascinantes du système . immunitaire que nos connaissances actuelles ne permettent pas de bien définir. organes lymphoïdes des souris LckOM sont étrangement affectés par la surexpression d'OM, de telle sorte que le thymus perd sa capacité de générer des nouveaux lymphocytes T, et les ganglions acquièrent cette fonction thymique et se mettent à produire un quantité phénoménale de lymphocytes T. Les mécanismes qui contrôlent cette voie de différentiation donnent naissance à des populations polyclonales de lymphocytes T matures CD4 + et CD8+ indépendamment de la fonction thymique. Une littérature abondante démontre l'existence de populations lymphocytaires extrathymiques dans le foie, les intestins et la moelle. En revanche, très peu d'études observent directement une thymopoïèse extrathmique de telle envergure dans les ganglions La présente étude a pour but de déterminer les caractéristiques des lymphocytes T issus de la différentiation extrathymique modulée par l 'OM. Les travaux menés au cours de ce projet de maîtrise visaient à caractériser phénotypiquement, par cytométrie en flux, ces lymphocytes extrathymiques et à établir la topographie et la cinétique de leur développement ainsi qu'à examiner leur comportement migratoire. Le modèle choisi pour l'étude de ces lymphocytes T d'origine extrathymique est un modèle de chimère hématopoïétique thymectomisée et reconstituée à l'aide d'une source de cellules souches hématopoïétiques (CSH) provenant de moelle osseuse transgénique (LckOM) et de foie foetal nom1al (C57BL/6). Ce modèle permet d'étudier spécifiquement le développement extrathymique de lymphocytes T normaux dans un environnement dépourvu d'influences thymiques. La prolifération et la demi-vie des lymphocytes extrathymiques sont établies grâce au marqueur de division cellulaire bromo-2'-deoxyuridine (BrdU) qui est incorporé dans l'ADN lors de sa synthèse, tandis que leur migration est suivie à l'aide de la molécule fluorescente carboxy-fluorescein diacete succinimidyl ester (CFSE). Les résultats de ces travaux illustrent l'étonnant pouvoir de modulation qu'exerce l'OM sur le développement et l'homéostasie du système immunitaire. Ils démontrent premièrement que les lymphocytes T extrathymiques possèdent un répertoire Vp polyclonal et un phénotype indiquant qu'ils ont vécu une expérience antigénique antérieure. Deuxièmement, ils démontrent que le niveau de prolifération de ces lymphocytes extrathymiques est supérieur à celui des lymphocytes normaux. Troisièmement, ils démontrent que les lymphocytes T matures LckOM sont fortement attirés vers les ganglions. Ces travaux laissent sous-entendre que l'OM a des effets pléiotropiques qui engendrent des changements morphologiques et fonctionnels dans les organes lymphoïdes et des changements dans le comportement de circulation et de proliferation des lymphocytes. Ce phénomène hors du commun de thymopoïèse extrathymique est digne d'intérêt puisqu'il fait surgir des faits importants à propos de l'organisation globale et de l'homéostasie du système immunitaire qui suscite actuellement beaucoup d'intérêt dans la communauté scientifique.

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