Le microbiote intestinal, c'est-à-dire l'ensemble des micro-organismes résidant dans l'intestin, a un impact majeur sur la santé de l'hôte. Certains d'entre eux sont notamment impliqués dans l'apparition et la progression du cancer colorectal (CCR). Le CCR est responsable de 700 000 décès par an dans le monde, ce qui en fait la quatrième cause de mortalité par cancer : un Canadien sur 13 et une Canadienne sur 16 seront touchés par le CCR au cours de leur vie. Il a été démontré que l'apparition du cancer colorectal peut être favorisée par des facteurs alimentaires, en raison de leur influence sur la composition du microbiote intestinal. En outre, l'utilisation fréquente d'antibiotiques est une source majeure de perturbation de l'écosystème intestinal. Le rôle du microbiote intestinal dans l'apparition possible et la promotion du CCR est une question dont les mécanismes ne sont encore que partiellement compris. Il a été démontré que les bactéries pathogènes se développent dans un environnement riche en fer via l’augmentation de leur virulence. Les patients atteints de CCR reçoivent souvent des suppléments de fer par voie orale pour traiter l'anémie liée au CCR, une affection très répandue dans cette population. En outre, lors de l'ablation chirurgicale du CCR, des antibiotiques oraux sont administrés pour réduire le risque d'infection, ce qui entraîne une réduction de la quantité de micro-organismes dans l'intestin. Plus généralement, une proportion importante de la population mondiale reçoit des suppléments de fer et l'utilisation d'antibiotiques est fréquente. Par conséquent, le microbiote intestinal est fréquemment soumis à des perturbations qui peuvent être délétères. L'objectif de ce travail était d'étudier le rôle du fer sur la cancérogénicité du côlon à travers l'hypothèse d'un effet médié par le microbiote intestinal. Le travail présenté démontre dans un modèle de souris, qu'en cas de supplémentation orale en fer après un traitement antibiotique, il y a une augmentation significative de 3 espèces bactériennes et de 4 fonctions microbiennes connues pour favoriser l'inflammation intestinale et/ou le CCR. En outre, il y a eu une diminution de la concentration fécale de butyrate, un produit de la fermentation microbienne, essentiel pour la santé intestinale et pour l'inhibition de la prolifération des cellules du CCR. Dans une deuxième série d'expériences, des souris ApcMin/+, un modèle murin de polypose intestinale, ont été soumises à un régime riche ou pauvre en fer. Il a été démontré que la carcinogenèse colorectale dépendait du microbiote intestinal. Plus précisément, les souris ApcMin/+ précolonisées avec le microbiote intestinal de patients atteints de CCR, mais pas avec des contrôles sains ou murin, ont montré une progression accélérée du CCR dans le groupe recevant un supplément de fer par voie orale. En particulier, les souris précolonisées avec le microbiote intestinal de patients atteints de CCR ont développé davantage de tumeurs coliques de plus grande taille, une prolifération accélérée des cellules épithéliales et des carcinomes invasifs lorsqu'elles étaient supplémentées en fer. Ces effets s'accompagnent d'une diminution unique de l'abondance des bactéries bénéfiques. Ces modifications de la composition du microbiote intestinal en réponse au fer ont entraîné des changements délétères spécifiques dans les fonctions microbiennes intestinales liées à la progression et à l'initiation du CCR, ainsi qu'une diminution de la concentration de butyrate dans les selles. De plus, afin de démontrer un lien de cause à effet entre la promotion du CCR observée lors de la supplémentation orale en fer et l'abondance réduite des espèces bactériennes bénéfiques précédemment identifiées, des souris ApcMin/+précolonisées avec le microbiote intestinal de patients atteints de CCR ont été hebdomadairement supplémentées par voie orale. Ces interventions ont permis de réduire l'effet cancérigène sur le côlon d'une supplémentation orale en fer, par rapport à des souris non supplémentées nourries avec le même régime riche en fer, via l’augmentation de la concentration fécale en acides gras à chaine courte. Enfin, ces travaux ont permis de développer un outil bio-informatique, destiné à tous les chercheurs voulant représenter et analyser les données du microbiote. Il répond au besoin de permettre aux chercheurs, même ceux qui ne sont pas formés à la bio-informatique ou à l'analyse du microbiote, de produire des représentations graphiques de la composition du microbiote. Ces graphiques sont faciles à produire, prêts à être publiés et contiennent des informations utiles pour comprendre la dynamique du microbiote. De plus, un outil statistique spécialisé dans l'analyse des variations de l'abondance des communautés microbiennes est directement inclus, et les différences sont mises en évidence dans le graphique. En résumé, nous avons démontré qu'une supplémentation orale en fer chez des modèles de souris induit un changement délétère, pro-cancérigène, de la composition et de la fonction du microbiote intestinal qui dépendent du microbiote intestinal initial. Ces changements induits par le fer conduisent à une diminution des bactéries protectrices et des métabolites bactériens, induisant une augmentation de la carcinogenèse colorectale dans le modèle de souris ApcMin/+ précolonisées avec le microbiote intestinal CCR. Enfin, des outils d'analyse et de représentation du microbiote intestinal ont été développés et rendus accessibles. / The gut microbiota, i.e. all the microorganisms residing in the gut, have a major impact on host health. In particular, some of them are implicated in the onset and progression of colorectal cancer (CRC). CRC is responsible for 700,000 deaths a year worldwide, making it the 4th leading cause of cancer mortality: one out of 13 Canadian men and one out of 16 Canadian women will be affected by CRC during their lifetime. It has been shown that the onset of CRC can be promoted by dietary factors, through their influences on the composition of the gut microbiota. Furthermore, the frequent use of antibiotics is a major source of disturbance on the intestinal ecosystem. The mechanisms by which the gut microbiota may contribute to the onset and progression of the CRC remain only partially understood. It has been shown that pathogenic bacteria thrive in an iron-rich environment by increasing their virulence. CRC patients are often given oral iron supplementation to treat the CRC-related anemia, a highly prevalent condition in this population. Moreover, when CRC is surgically removed, oral antibiotics are given to reduce the risk of infection, leading to a reduction in the amount of microorganisms within the gut. More generally, a significant proportion of the world's population receives oral iron supplementation, and antibiotic use is frequent. As a result, the gut microbiota is frequently subject to perturbations that may be deleterious. By this work, the aim was to investigate the role of iron on colonic carcinogenicity through the hypothesis of a gut microbiota mediated effect. The presented work demonstrated, in a wild-type (WT) mouse model, that in the occurrence of oral iron supplementation after antibiotic treatment, there was a significant increase in 3 bacterial species and 4 microbial functions known to promote intestinal inflammation and/or CRC. Furthermore, there was a decrease in the fecal concentration of butyrate, a product of microbial fermentation, essential for intestinal health and for the inhibition of the CRC cell proliferation. In a second set of experiments, ApcMin/+ mice, a murine model of intestinal polyposis, were subjected to an iron-rich or iron-sufficient diet. A gut microbiota-dependent response in colonic carcinogenesis was demonstrated. Specifically, ApcMin/+ mice pre-colonized with gut microbiota from CRC patients, but not with healthy controls or murine gut microbiota, showed an accelerated cancer progression among the oral iron supplementation group. In particular, the mice pre-colonized with the CRC gut microbiota developed more colonic tumors of greater size, accelerated epithelial cell proliferation and invasive carcinomas when iron supplemented. These effects were accompanied with a unique decrease in the abundance of beneficial bacteria. These gut microbiota dependant changes in gut microbiota composition in response to iron led to specific deleterious shifts in gut microbial functions related to CRC progression and initiation, as well as a decrease in fecal butyrate concentration. Moreover, in order to demonstrate a causal link between the promotion of CRC observed during oral iron supplementation and the reduced abundance of the beneficial bacterial species previously identified, pre-colonized ApcMin/+ mice with gut microbiota from CRC patients were weekly orally supplemented. These interventions were able to reduce the colonic carcinogenicity effect of oral iron supplementation, comparatively to unsupplemented mice fed the same iron rich diet, through increased fecal short-chain-fatty-acids concentrations. Finally, these works allowed to develop a bioinformatic tool, intended for all researchers looking for representing and analyzing microbiota data. It fills the gap of allowing researchers, even the one not trained on bioinformatics or microbiota analysis, to produce graphical representations of microbiota composition. These graphs are easy to produce, publication-ready, and contain information useful for understanding the dynamics of the microbiota. In addition, a statistical tool specialized in the analysis of variations in microbial community abundance is directly included, and differences are highlighted within the graph. In summary, we have demonstrated that oral iron supplementation in mice models induces a deleterious, pro-carcinogenic, shift of the gut microbiota composition and function that are gut microbiota dependant. These iron-induced changes lead to decreased of protective bacteria and bacterial metabolites, inducing an increase in colorectal carcinogenesis in the ApcMin/+ mouse model pre-colonized with CRC gut microbiota. Finally, tools for analysis and representation of the gut microbiota have been developed and made accessible.

Yes / The naturally-occurring omega (ω)-3 polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (EPA) reduces colorectal adenoma (polyp) number and size in patients with familial adenomatous polyposis. The safety profile and potential cardiovascular benefits associated with ω-3 PUFAs make EPA a strong candidate for colorectal cancer (CRC) chemoprevention, alone or in combination with aspirin, which itself has recognized anti-CRC activity. Colorectal adenoma number and size are recognized as biomarkers of future CRC risk and are established as surrogate end-points in CRC chemoprevention trials. The seAFOod Polyp Prevention Trial is a randomized, double-blind, placebo-controlled, 2 × 2 factorial ‘efficacy’ study, which will determine whether EPA prevents colorectal adenomas, either alone or in combination with aspirin. Participants are 55–73 year-old patients, who have been identified as ‘high risk’ (detection of ≥5 small adenomas or ≥3 adenomas with at least one being ≥10 mm in diameter) at screening colonoscopy in the English Bowel Cancer Screening Programme (BCSP). Exclusion criteria include the need for more than one repeat endoscopy within the three-month BCSP screening period, malignant change in an adenoma, regular use of aspirin or non-aspirin non-steroidal anti-inflammatory drugs, regular use of fish oil supplements and concomitant warfarin or anti-platelet agent therapy. Patients are randomized to either EPA-free fatty acid 1 g twice daily or identical placebo AND aspirin 300 mg once daily or identical placebo, for approximately 12 months. The primary end-point is the number of participants with one or more adenomas detected at routine one-year BCSP surveillance colonoscopy. Secondary end-points include the number of adenomas (total and ‘advanced’) per patient, the location (left versus right colon) of colorectal adenomas and the number of participants re-classified as ‘intermediate risk’ for future surveillance. Exploratory end-points include levels of bioactive lipid mediators such as ω-3 PUFAs, resolvin E1 and PGE-M in plasma, urine, erythrocytes and rectal mucosa in order to gain insights into the mechanism(s) of action of EPA and aspirin, alone and in combination, as well as to discover predictive biomarkers of chemopreventive efficacy. The recruitment target is 904 patients. / Medical Research Council (MRC) and managed by the National Institute for Health Research (NIHR) on behalf of the MRC-NIHR partnership

No / Inflammatory bowel diseases are associated with increased risk of developing colitis-associated colorectal cancer (CAC). Epidemiological data show that the consumption of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) decreases the risk of sporadic colorectal cancer (CRC). Importantly, recent data have shown that eicosapentaenoic acid-free fatty acid (EPA-FFA) reduces polyp formation and growth in models of familial adenomatous polyposis. However, the effects of dietary EPA-FFA are unknown in CAC. We tested the effectiveness of substituting EPA-FFA, for other dietary fats, in preventing inflammation and cancer in the AOM-DSS model of CAC. The AOM-DSS protocols were designed to evaluate the effect of EPA-FFA on both initiation and promotion of carcinogenesis. We found that EPA-FFA diet strongly decreased tumor multiplicity, incidence and maximum tumor size in the promotion and initiation arms. Moreover EPA–FFA, in particular in the initiation arm, led to reduced cell proliferation and nuclear β-catenin expression, whilst it increased apoptosis. In both arms, EPA-FFA treatment led to increased membrane switch from ω-6 to ω-3 PUFAs and a concomitant reduction in PGE2 production. We observed no significant changes in intestinal inflammation between EPA-FFA treated arms and AOM-DSS controls. Importantly, we found that EPA-FFA treatment restored the loss of Notch signaling found in the AOM-DSS control and resulted in the enrichment of Lactobacillus species in the gut microbiota. Taken together, our data suggest that EPA-FFA is an excellent candidate for CRC chemoprevention in CAC.

Fusobacterium nucleatum is an anaerobic, Gram-negative, oral bacterium that disseminates from the mouth, and contributes to preterm birth, tissue infections, and acceleration of multiple cancers including colorectal and pancreatic. It is well-established that most Fusobacterium species exhibit genetic recalcitrance, which has led to hindrance in the understanding of their biology and molecular pathogenesis. Though the association of Fusobacterium in diseases is well-established, the majority of our experimental work stems from the strain F. nucleatum ATCC 23726 because it is genetically tractable. Here, in this dissertation, we show that we are able to enhance our existing molecular tools for genome editing to introduce the first mutants in a clinically relevant strain, F. nucleatum ATCC 25586, a feat that was never accomplished in decades of trying. Furthermore, we created a deletion library of genes predicted to be involved in host cellular invasion and survival. In this work, we identified a novel small adhesin, FadA2, that played a significant role in the invasive ability of F. nucleatum ATCC 25586 to colorectal cancer cells. This dissertation also sheds the first insight into the roles of the type 5a autotransporters. Using a deletion library of genes encoding for the type 5a autotransporter proteins in F. nucleatum ATCC 23726, we systemically characterized altogether 12 type 5a proteins with a focus on the invasion of colorectal cancer cells. Most notably, we found that a wide assortment of type 5a proteins contributing to binding and invasion of F. nucleatum to HCT116 cancer cells. Furthermore, we identified that RadD was not directly involved in inducing secretions of the cytokines IL-8 and CXCL1 while confirmed the specific association of Fap2 in bacterial-induced cytokine secretion. Thus, our findings provided the first comparative and functional analysis of Fusobacterium type 5a autotransporter proteins in colorectal cancer cells which will be crucial to the understanding of Fusobacterium involvement in cancer progression. Finally, this dissertation reported on the first ever observation on the survival strategy of Fusobacterium inside the host cells. We uncovered a novel protein that contributed to enhanced survival of Fusobacterium residing in colorectal cancer cells. This work undoubtedly helps expand the current Fusobacterium genetic toolkit to study proteins and mechanisms relevant to Fusobacterium-accelerated diseases. By identifying and characterizing novel virulence strategies that Fusobacterium can take advantage of, we can increase our comprehension on this opportunistic microbe while devising innovative therapeutic treatments. / Doctor of Philosophy / Fusobacterium, a member of the microbial community in our mouth, has been a captivating study target due to its association with human health and diseases. By nature, Fusobacterium lives in oxygen-free pockets between our teeth and gumline in which this organism has been correlated with a multitude of complications and diseases including periodontitis, inflammatory bowel disease, preterm birth, and most importantly colorectal cancer. Though the connection to human health is established, we still have to learn more about the mechanisms utilized by Fusobacterium to exacerbate diseases. This challenge is mainly hindered by the lack of efficient tools and resources to systematically investigate the relationship between the bacterium and its human host. Therefore, the work in this dissertation focuses on expanding the existing molecular toolkit to study clinically relevant Fusobacterium strain, which provides the power and convenience to discover novel mechanisms that Fusobacterium can take advantage of to be a successful pathogen. Accordingly, we first enhanced our ability to work with a wider range of Fusobacterium species. We successfully introduced exogenous genetic materials into a clinical strain of Fusobacterium, Fusobacterium nucleatum ATCC 25586. This breakthrough was built on the success of our current toolkit to make genetic modifications to a sister strain, Fusobacterium nucleatum ATCC 23726. With this newfound capacity to modify F. nucleatum ATCC 25586, we have described the importance of a novel protein aiding in the invasion of Fusobacterium to colorectal cancer. Furthermore, we have determined that certain proteins within the fusobacterial type 5a protein family can play a key role in governing binding and invasion of colorectal cancer cells in this study. Concurrently, for the first time, we provided the snapshot of a small protein and its role in fusobacterial long-term survival inside its targeted host cells. Altogether, the findings in this dissertation will bring forth an innovative framework to better the comprehension of current Fusobacterium-induced disease implications, while exploring alternative treatments for enhanced patient health.

MACC1, ein Hauptregulator von Metastasen, ist an zahlreichen Kennzeichen von Krebs beteiligt, einschließlich dereguliertem Metabolismus. Dennoch ist seine Rolle im Krebsstoffwechsel unklar. In der vorliegenden Arbeit wurde eine systematische Analyse von MACC1-getriebenen metabolischen Netzwerken durchgeführt. MACC1 erhöhte die GLUT1 auf Zellmembrane, was zu einer erhöhten Glukoseanreicherung, einem erhöhten Glukosefluss und somit zu einer erhöhten Zellproliferation führte. Außerdem, reduzierte MACC1 den Glutaminfluss unabhängig von der Nährstoffverfügbarkeit. Bei Glucoseentzug erhöhte MACC1 die Pyruvataufnahme und zeigte aber die geringe Auswirkungen auf den Pyruvatfluss. In vivo, MACC1 zeigte erhöhte Aufnahme von 18F-FDG und 18F-Glutamat in Lebermetastasen. Zusammengefasst zeigen diese Ergebnisse, dass MACC1 mehrere Wirkungen auf den Krebs-Metabolismus zeigt, was es attraktiv macht, seine Wirkungen in Krebsmodellen weiter zu untersuchen. Metastasierung ist die Haupttodesursache bei Darmkrebs. Fünfzehn bis zwanzig Prozent der Darmkrebs Patienten im Stadium II entwickeln im Verlauf der Erkrankung Metastasen, jedoch bleiben die Kriterien der Wahrscheinlichkeit mit welcher Patienten von Chemotherapie profitieren werden ungenau. Hier wurde das Potenzial der Plasma-Metabolomik zur Vorhersage von Metachronmetastasen untersucht. Plasma metabolische Profile wurden wesentlich unterschiedlich zwischen nicht-metastasierten und metachron metastasierten Patienten gefunden. Wie Klassifikationsmodelle aus Entscheidungsbäumen und Support-Vektor-Maschinen gezeigt haben die Plasmametaboliten haben die Fähigkeit nicht-metastasierte von metachron metastasierten Darmkrebs Patienten zu unterscheiden, mit einer durschnittlichen Vorhersagegenauigkeit von 0,75 bzw. 0,82 für jede der Methoden, angemessen. Zusammen, zeigen diese Ergebnisse, dass Plasmametaboliten das Potenzial haben, Darmkrebs Patienten gemäß ihrem Metastasierungsrisiko nichtinvasiv zu stratifizieren. / MACC1, a master regulator of metastasis, is involved in most hallmarks of cancer, including deregulated metabolism. Yet, fragmentary data on its role in cancer metabolism exist. Here, a systematic analysis of MACC1-driven metabolic networks by elucidation of cell nutrient preferences, environment dependent alterations of nutrient utilization, metabolic pathway functionality and metabolic tracing using 13C-labeled metabolic substrates had been performed. MACC1 was found to enhance surface GLUT1 thus leading to increased glucose depletion, glucose flux and hence increased cell proliferation. Besides, MACC1 was found to reduce glutamine flux independent of nutrient availability. Upon glucose deprivation MACC1 was found to enhance pyruvate uptake exhibiting minor effects on pyruvate flux. In vivo, MACC1 increased uptakes of 18F-FDG and 18F-glutamate in liver metastatic lesions. Together, these findings demonstrate that MACC1 exhibits multiple effects on cancer metabolism, thus making it attractive to further study its effects in cancer models. Metastasis is the main cause of death from colorectal cancer (CRC). Fifteen to twenty percent of stage II CRC patients develop metastasis during the course of disease, however the criteria of likely benefitting patients from chemotherapy remain imprecise. Here, the potential of plasma metabolomics to predict metachronous metastasis was assessed. Plasma metabolic profiles were shown to be significantly different between non-metastasized and metachronously metastasized CRC patients. As demonstrated by supervised classifications using decision trees and support vector machines plasma metabolites have the power to distinguish non-metastasized from metachronously metastasized CRC patients giving average prediction accuracy of 0.75 and 0.82 for each of the methods, respectively. Together, these results demonstrate that plasma metabolites have the potential to non-invasively stratify CRC patients according to their metastasis risk.

No description available.

Growing evidence indicates that serine proteases known as kallikreins are associated with malignancy and may have potential diagnostic/prognostic applications in cancer. Kallikreins are the largest group of serine proteases. Kallikrein enzymes are often involved in proteolytic cascades through their function in degradation of extracellular matrix proteins and promotion of angiogenesis. Kallikrein 6 (KLK6) is a member of the family of fifteen highly conserved secreted trypsin- or chemotrypsin-like serine proteases. Over-expression of KLK6 has been observed in different pathophysiological states such as neurodegenerative diseases, inflammation and various cancers, including colorectal cancer. In Chapter 3 we elucidated the miRNA-based mechanism of regulation of invasion in metastatic colorectal cancer over-expressing KLK6. We developed HCT116 colon stable isogenic cell lines with knockdown of KLK6 expression using short-hairpin interference RNA (shKLK6 clones). The shKLK6 clones had decreased expression and secretion of KLK6 protein with a minimal effect on cell growth and viability in cell culture. SCID mice injected with shKLK6-3 clone 3 cells exhibited a statistically significant increase in the survival rates (P=0.005), decrease in the incidence of distant metastases and a shift in the location of the metastatic foci closer to the cell's injection site. Levels of KLK6 protein secreted into the bloodstream were significantly lower in animals injected with shKLK6-3 clone 3 compared to HCT116 control clone 1 (P < 0.04). Through bioinformatics analyses we identified and validated three miRNAs, which are important in post-translational modification of bioactive proteins, proliferation, migration and p38 MAPK signaling pathway. In Chapter 4 we developed Caco-2 colon stable isogenic cell lines with expressing enzymatically active or mutant KLK6 protein (Caco-2 stable clones). We employed these cell lines to investigate the importance of KLK6 enzymatic activity of initiation of cell invasion using in vitro and in vivo models.

Résumé : L’adénocarcinome colorectal est parmi les plus importants cancers au Canada en terme de mortalité et morbidité. Cependant, nous n’en connaissons encore que peu, entres autres sur les voies cellulaires importantes et les protéines présentant un potentiel comme biomarqueur. Cette étude fut divisée en deux sous-projets. Sous-projet A. Il n’y a présentement aucun biomarqueur permettant de prédire la réponse à la radiothérapie comme modalité de traitement pour le cancer colorectal. Le but de ce sous-projet était de mettre au point les méthodes permettant d’effectuer une étude prospective ou rétrospective par spectrométrie de masse sur la réponse à la radiothérapie en utilisant des échantillons de tissu de patient. Des échantillons de tissu de souris et de tissu humains anonymisés ont été utilisés pour évaluer la faisabilité d’une telle étude. Différentes techniques d’extraction protéique ont été évaluées. Les extraits totaux et fractionnements subcellulaires de tissu frais ont permis une analyse appropriée des protéines cellulaires. Il en était de même pour l’extraction totale de tissus fixés. Cependant, les protéines extraites suite à microdissection au laser de tissu fixé étaient inadéquates et en nombre insuffisant. Sous-projet B. Afin d’investiguer l’importance de fonctions, voies ou protéines dans différents types de cancer colorectaux, neuf lignées cellulaires de cancer colorectal et de côlon normal ont été fractionnées en quatre compartiments subcellulaires et analysées par spectrométrie de masse. Aucun groupe de recherche n’avait analysé jusqu’à présent plus de cinq lignées et plus d’un compartiment subcellulaire à la fois. Les résultats montraient que certaines voies canoniques et fonctions cellulaires étaient de haute importance dans plusieurs des lignées analysées, dont la voie de signalisation par eIF2. De plus, les régulateurs de transcription TP53, MYC et TGFB1, pouvant être responsables des caractéristiques cellulaires observées, ont été identifiés. En conclusion, ce projet nous a permis d’améliorer nos connaissances sur les caractéristiques moléculaires d’importance dans le cancer colorectal et de mettre au point des techniques qui pourraient permettre la découverte de nouveaux biomarqueurs. / Abstract : Colorectal adenocarcinoma is one of the most important cancers in Canada in terms of mortality and morbidity. However, we still know very little on its molecular features. This study was divided into two sub-projects. Sub-project A. At this time, no biomarker has the capacity of predicting a patient’s response to radiotherapy, which is a commonly used treatment of colorectal cancer. The goal of this section was to develop the methods to conduct a prospective or retrospective mass spectrometry study on the patient response to radiotherapy, through the use of human tissues. Mouse tissues and tissues of an anonymous patient were obtained in order to evaluate the feasibility of such a study. Different protein extraction techniques were evaluated. Total lysates and subcellular fractionations of fresh tissues allowed for a successful analysis of the samples. The same was true of total lysates of fixed tissues. However, proteins extracted from cells isolated through laser capture microdissection were insufficient in numbers and their types were inconsistent with the expected results. Sub-project B. In order to study the importance of proteins and cellular functions or pathways in different types of colorectal cancers. nine cell lines originating from colorectal carcinoma and from normal colon were fractionated according to four subcellular compartments and analysed through mass spectrometry. Until now, no research group had analysed, in a single study more than 5 cell lines as well as more than one subcellular compartment at once. Some cellular functions and canonical pathways were shown to be of high importance in many of the studied cell lines, such as the signalling through eIF2 pathway. Furthermore, the transcription regulators TP53, MYC and TGFB1were identified as potentially responsible for the observed proteomic characteristics. In conclusion, this study allowed for a better understanding of important molecular caracteristics of colorectal cancer and allowed for the optimization of techniques that may serve in the discovery of new biomarkers relative to the use of radiotherapy as a treatment.

Despite significant advances in cancer treatment, clinical response remains suboptimal and there is a continued requirement for improved chemotherapeutics. The attrition rate for new therapies is high, due principally to lack of in vivo efficacy and poor pharmacodynamics. Consequently better systems are required to determine in vivo preclinical efficiency and drug-target interactions. Engineering of cancer cells to express fluorescent or bioluminescent proteins, either endogenously or under the control of specific gene promoters, and their detection by noninvasive optical imaging has the potential to improve preclinical drug development. In this study, a panel of colorectal cancer cell lines were engineered to express fluorescent and luminescent proteins either constitutively or under control of gene-promoters for the DNA damage response gene p53 or the cell cycle regulator p21, both important pharmacodynamic sensors. These cell lines were characterised for their potential as in vivo models of primary and metastatic tumour therapy response, several showing significant potential. In addition to the development of these models, this study also addressed the pharmacokinetics of different luciferase substrates and identified optimal temporal and dose characteristics for each. Furthermore, a new application for bioluminescent imaging was developed and validated for use in preclinical evaluation of vascular disrupting agents, a new generation of cancer therapeutic. This study demonstrates that despite the dynamic and variable nature of fluorescent and bioluminescent imaging, reproducible results can be obtained if appropriate precautions are taken. The models developed herein will expedite cancer drug development whilst reducing and refining the use of animals in research.

Mon travail de thèse consistait à étudier la signalisation oncogénique de la tyrosine kinase (TyrK) cytoplasmique Src dans les cellules de cancer colorectal (CCR) à un stade avancé par une approche globale de phosphoprotéomique quantitative de type SILAC puis d'évaluer l'efficacité du Nilotinib, un inhibiteur de la Tyrk oncogénique BCR-Abl, sur les propriétés invasives des cellules de CCR. Dans un premier temps, nos résultats ont confirmé le rôle clé joué par Src dans l'acquisition des propriétés invasives de la tumeur. Puis, l'approche phosphoprotéomique de type SILAC a permis de mettre en évidence 136 protéines substrats de Src parmi lesquelles nous retrouvons des protéines de signalisation, des protéines associées au cytosquelette ou des protéines du trafic vésiculaire. De manière intéressante, j'ai révélé l'implication d'un réseau de TyrK dans les propriétés invasives Src-dépendantes. Nos résultats suggèrent qu'une thérapie multi-TyrK pourrait s'avérer intéressante pour traiter les CCR à un stade avancé. En complément de l'analyse SILAC, j'ai initié une approche pharmacologique pour caractériser les TyrK impliquées dans l'invasion des cellules de CCR. De manière surprenante, j'ai observé que le Nilotinib inhibe l'activité invasive des cellules de CCR avec une efficacité comparable à celle observée sur la croissance des cellules de LMC (IC50=20nM). Des approches d'invalidation génétique et de mutagénèse couplées à des tests d'invasion in vitro et in vivo ont permis de démontrer que le Nilotinib exerce son activité anti-invasive en ciblant le récepteur au collagène DDR1. Mes résultats laissent présager un intérêt thérapeutique potentiel du Nilotinib dans le traitement du cancer colorectal métastasant. / My thesis work was devoted to decipher the oncogenic signaling of the cytoplasmic tyrosine kinase (TyrK) Src in advanced colorectal cancer (CRC) cells using SILAC quantitative phosphoproteomics and to evaluate the efficiency of the oncogenic BCR-Abl inhibitor, Nilotinib, on the CRC cell invasive activity. Firstable, our results confirmed the key role of Src in the induction of cell invasion. Then, the SILAC phosphoproteomic approach revealed 136 Src substrates among which signaling proteins, cytoskeleton associated proteins or vesicular trafficking associated proteins. Interestingly, I have identified a TyrK network involved in Src-dependent invasive properties. Taken together, our results suggest that a multi-TyrK therapy may be interesting in clinic for the treatment of advanced CRC. In addition to the SILAC analysis, a pharmacological approach was set up to characterize TyrK involved in CRC cell invasion. Surprisingly, I found that Nilotinib inhibits CRC cell invasive activity with a similar efficiency to the one observed on the growth of CML (IC50 = 20nM). Knock down and mutagenesis experiments together with in vitro and in vivo invasion assay revealed the collagen receptor DDR1 as the main Nilotinib target in its anti-invasive activity. Our results suggest that Nilotinib could be of therapeutic value in metastatic CRC.