In Canada, colorectal cancer is the second leading cause of cancer death for both men and women. There are many different factors that contribute to the progression and spread of the disease. However, increasing evidence now suggests that the tumor microenvironment plays a paramount role in these processes. CD26 is a multifunctional, cell-surface glycoprotein that has intrinsic enzyme activity, binds adenosine deaminase and interacts with the extracellular matrix. Through its various functions it serves to constrain cancer progression. For example, it is known to cleave CXCL12, the ligand for CXCR4. The CXCL12:CXCR4 axis is normally involved in cancer metastasis by promoting cancer cell migration, invasion and proliferation. Down-regulation of CD26 is observed in certain cancers - this has been shown in vitro to occur in response to certain soluble mediators. The first part of this study looked at the effects of glucose and its metabolic product lactate on CD26 expression in colorectal carcinoma cells. Our study showed that CD26 expression is lower in cancer cells that are grown in low-glucose, high-lactate conditions, which replicates the situation within a tumor. The second part of this study examined the effect of adenosine, a purine nucleoside, on colorectal carcinoma cells and supportive stromal cells - cancer-associated HS675.T fibroblasts (CAFs) and Met-5a mesothelial cells. Adenosine increased the proliferation of CAFs and increased CXCL12 mRNA in both stromal cell lines. It also increased MMP-13 mRNA in stromal cells as well as colorectal cancer cells, suggesting that adenosine may promote progression and metastasis through various mechanisms. The last section focused on the ability of cellular products and 3-dimensional tissue topology to coordinate and affect the behaviour of the different cell populations. Here we show that secretory products from colorectal cancer cells promote CAF proliferation but inhibit mesothelial cell proliferation, and are also able to modulate MMP-13 expression. Finally, certain responses are enhanced in multicellular spheroids. In conclusion, the tumor microenvironment represents a major consideration in the treatment of solid tumors. Our data suggest that various soluble mediators, such as adenosine, may have therapeutic implications in cancer treatment and might represent novel targets for future research.

Loss of genomic stability is associated with a variety of diseases, particularly cancer. Of the many proteins which maintain genomic integrity, two of the most important are MLH1 and MSH2, which participate in DNA mismatch repair. Previous work established derivatives of the CaCo2 human colorectal cancer cell line with siRNA-mediated knockdown of these proteins. When xenografted into mice, tumors with reduced MLH1 or MSH2 expression grew faster than controls. Following growth in vivo, clonal cell lines were established from the tumors and used to examine the effects that knockdown of MSH2 had on other members of the DNA mismatch repair system. Clonal survival following exposure to 5-fluorouracil was also evaluated, and those cells with reduced MLH1 and MSH2 levels were found to be resistant. This study has implications for the importance of knowing the MMR status of a given tumor when deciding on a course of treatment, and of the compounding effects of the loss of one MMR protein on others in the family. / Canadian Cancer Society Research Institute

No description available.

No description available.

L'épithélium intestinal est en constant renouvellement et les mécanismes qui contrôlent la prolifération y sont parfaitement orchestrés. Le facteur de transcription E2F4 est un régulateur clé de la transition G1/S, de la prolifération et de l'homéostasie des cellules épithéliales intestinales. Contrairement à E2F1, la localisation cellulaire d'E2F4 varie en fonction de l'état prolifératif des cellules : il est exprimé majoritairement au cytoplasme des cellules quiescentes ou différenciées et au noyau des cellules prolifératives. Dans cette thèse, les mécanismes de contrôle de la localisation d'E2F4, de sa phosphorylation et de son expression, de même que les mécanismes qui contrôlent l'entrée en phase S des cellules épithéliales intestinales normales humaines (HIEC) ont été analysés. Nos résultats démontrent que l'activation de la signalisation MEK/ERK par le sérum est requise pour la translocation nucléaire d'E2F4 et la transition G 1/S des HIEC. Par contre, la stimulation du sentier MEK/ERK par l'EGF n'est pas suffisante à induire ces événements: l'inhibition concomitante des GSK3?/? ou des p38?/? est aussi requise. En effet, la combinaison de l'EGF ou du FGF9 avec un inhibiteur pharmacologique des GSK3 entraîne la translocation nucléaire d'E2F4 et l'entrée en phase S. De manière analogue, l'inhibition des p38 en combinaison avec l'EGF cause aussi la translocation nucléaire d'E2F4 et l'entrée en phase S des HIEC. Par ailleurs, l'activation des IKK?/? semble aussi requise pour la translocation nucléaire d'E2F4 et la transition G1/S des HIEC induites par le sérum. Ensuite, nos résultats indiquent qu'E2F4 est rapidement phosphorylé suivant la stimulation par le sérum de manière dépendante du sentier MEK/ERK. Ainsi, des essais kinases in vitro démontrent qu'ERK1 phosphoryle efficacement E2F4, potentiellement sur les S244 et S384. Nos résultats suggèrent aussi que GSK3? interagit avec E2F4, principalement dans les cellules quiescentes, et pourrait alors le phosphoryler. Par ailleurs, E2F4 est phosphorylé, surexprimé et localisé au noyau des adénomes colorectaux humains. De plus, les mutants d'E2F4 retrouvés dans les cancers colorectaux avec instabilité des microsatellites, E2F4(Ser) 12 et E2F4(Ser)14 , sont plus fortement exprimés en raison d'une stabilité accrue et ont une meilleure activité transcriptionnelle. Nous démontrons aussi l'existence de 2 formes principales du partenaire d'interaction d'E2F4, DP-2, exprimées dans les HIEC: DP-2 40 , dont l'expression augmente avec l'entrée en phase S et DP-266 , dont l'expression diminue. De plus, DP-2 40 est surexprimée dans les cancers colorectaux humains et pourrait alors y favoriser la localisation nucléaire d'E2F4. En conclusion, nous avons identifié des mécanismes de régulation du facteur de transcription E2F4 et de l'entrée en phase S des HIEC. Cependant, ces mécanismes sont altérés lors de la carcinogenèse. D'ailleurs, la surexpression et la localisation aberrante d'E2F4 de même que la surexpression de DP-2 40 dans les cancers colorectaux pourraient contribuer à l'hyperprolifération et à la formation de cancers dans le côlon et le rectum. [symboles non conformes]

Abstract: Advances in curing rectal cancer came from successful chemoradiotherapy. Platinum-based drugs such as oxaliplatin have also been studied and integrated in treatment strategies against rectal cancer. Although platinum-based drugs can act as radiosensitizers, their radiosensitizing activity is limited by their narrow therapeutic index which avoids the dose escalation. In addition, it is important also to optimize the schedule of drug administration with radiation treatment to gain advantage of drug-radiation interactions and maximize tumor response. We evaluated the new liposomal formulation of cisplatin and oxaliplatin (Lipoplatin and Lipoxal, respectively) that should increase the anticancer effectiveness while minimizing the side effects. We investigated different chemoradiation schedules to assess the best antitumor efficacy with regard to our hypothesis of the "true" concomitant chemoradiotherapy which consist in the addition of radiation at the time of maximum accumulation of platinum in the DNA of cancer cells. We performed in vitro studies using human colorectal carcinoma HCT116, and in vivo using nude mice HCT116 xenograft. Pharmacokinetic studies on platinum accumulation were measured by inductively coupled plasma mass spectrometry. Regarding the results of DNA-platinum concentrations, the synergy with radiation was assessed for in vitro and in vivo studies. Cytotoxicity was determined by a colony formation assay, while the resulting tumor growth delay in animal model was correlated to induction of apoptosis and histophatology analyses. The synergism of combined treatments was evaluated using the combination index method. In this study, a radiosensitizing enhancement was observed with combining radiation treatment with cisplatin, oxaliplatin and their liposomal formulations in both in vitro and in vivo studies. Variations of platinum accumulation with incubation time in normal and tumor tissues and in different cell compartments, as well as platinum-DNA were measured. A higher level of synergism was observed when radiotherapy was performed in vitro at 8 h of exposure and in vivo at 4 and 48 h after drug administration, which corresponded to the times of maximal platinum binding to tumor DNA. These results were correlated to a highest induction of apoptosis and a low mitotic activity. In conclusion, the optimal treatment schedule of chemoradiotherapy is dependent on the time interval between drug administration and radiation, which was closely associated to the kinetics of platinum accumulation to DNA and the intracellular concentration of the platinum drugs. Regarding our hypothesis, administered radiotherapy to the time intervals of maximum synergism could improve efficacy of chemoradiation treatment. This should be confirmed in clinical trials. //Résumé: Les progrès des traitements du cancer colorectal proviennent principalement des succès en chimio-radiothérapie. Toutefois, l'augmentation de l'activité radiosensibilisante du cisplatine et de l'oxaliplatine est principalement limitée par leur toxicité ce qui limite la dose administrée. Par conséquent, l’optimisation de la séquence et de la cédule d’administration entre la chimiothérapie et la radiothérapie devient essentielle pour maximiser l’interaction du rayonnement ionisant et de la chimiothérapie et ainsi optimiser la réponse tumorale. Notre objectif est de développer une cédule optimale de la chimio-radiothérapie pour obtenir la meilleure efficacité anti-tumorale tout en minimisant les effets secondaires aux tissus sains. Pour atteindre ce dernier objectif, nous avons également évalué la nouvelle formulation liposomale de cisplatine et d’oxaliplatine (Lipoplatin™ et Lipoxal™, respectivement) qui ont pour but d'accroitre l'efficacité anticancéreuse tout en réduisant les effets secondaires. Nous avons étudié la séquence optimale de radio-chimiothérapie en lien avec notre hypoThèse de "vraie" concomitance qui favorise l'ajout de rayonnement au moment de l'accumulation maximale de platine dans l'ADN des cellules cancéreuses. Nous avons effectué des études in vitro utilisant le carcinome colorectal humain HCT116, et in vivo sur des souris nue HCT116 xénogreffe. Les études pharmacocinétiques sur l'accumulation de platine ont été mesurées par spectrométrie de masse couplée au plasma induit. La cytotoxicité a été déterminée par un essai de formation de colonie, tandis que le retard de croissance tumorale obtenue en modèle animal est corrélé à l'apoptose et analyses histopathologiques. La synergie des traitements combinés a été évaluée en utilisant l'indice de combinaison. Dans cette étude, nous avons observé une amélioration de la radiothérapie combinée avec le cisplatine, l'oxaliplatine et leurs formulations de liposomes à la fois in vitro et in vivo. Des variations entre l'accumulation du platine dans les cellules cancéreuses, de tissus normaux et tumoraux, ainsi que des adduits du platine à ADN en fonction de la cédule d'administration du médicament ont été observées. Un fort effet concomitant in vitro a été observé lorsque la radiothérapie a été délivrée à 8 h et in vivo à 4 et 48 h après l'administration du médicament, ce qui correspondait au temps de liaison maximale du platine à l'ADN tumoral. L'augmentation de la radiosensibilisation a été corrélée à une élévation de l’apoptose et une réduction de l’activité mitotique. La cédule de traitement optimal de la chimio-radiothérapie dépend de l'intervalle de temps entre l'administration de la radiation et de la drogue, ce qui a été étroitement associé à la cinétique d’accumulation de platine à l’ADN et de leurs concentrations intracellulaires. En conclusion, les meilleurs résultats in vitro et in vivo pourraient être ultérieurement confirmés en essai clinique pour valider ces concepts.

No description available.

The primary treatment for colorectal cancer is surgery. Radiotherapy and chemotherapy, sometimes combined, are also frequently used to diminish recurrence risk. In response to radiation exposure, several cellular signaling cascades are activated to repair DNA breaks, prevent apoptosis and to keep the cells proliferating. Several proteins in the radiation response and cell survival pathways are potential targets to enhance the effects of radiation. The epidermal growth factor receptor (EGFR), which is frequently upregulated in colorectal cancer and exhibits a radiation protective function, is an attractive target for treatment. EGFR is activated by radiation which in turn activates numerous signaling pathways such as the PI3 kinase/AKT cascade, the RAS/RAF/ERK pathway and STAT leading to tumor cell proliferation. EGFR is also believed to interact with proteins in the DNA repair process, such as DNA-PKcs and MRE11. The cytotoxic effect of an affibody molecule (ZEGFR:1907)2, with high affinity to EGFR,  in combination with radiation produced a small, but significant, reduction in survival in a KRAS mutated cell line. However, not in the BRAF mutated cell line. The next step was therefore to target proteins downstream of EGFR such as AKT. There was an interaction between AKT and the DNA repair proteins DNA-PKcs and MRE11 and both AKT1 and AKT2 were involved in the radiation response. The knockout of both AKT isoforms impaired the DNA double strand break rejoining after radiation and suppression of DNA-PKcs increased the radiations sensitivity and decreased the DNA repair further. The AKT isoforms also affected the expression of cancer stem cell markers CD133 and CD44 which are associated with the formation of metastasis as well as radiation and drug resistance. The CD133 expression was associated with AKT1 but not AKT2, whereas the CD44 expression was influenced by the presence of either AKT1 or AKT2. AKT was also involved in cell migration, cell-adhesion and metabolism. Overall, these results illustrate the complexity in response to radiation and drugs in cells with different mutations and the need for combining inhibitors against several targets such as EGFR, AKT, DNA-PKcs, CD133 or CD44.

In this thesis, I chose to identify and evaluate different monitoring methods on surgeon specific outcomes in colorectal surgery. An initial literature search identified different methods that were applied to a cohort of colorectal patients operated on by general surgeons using an electronic hospital database. Surgeon specific complications were validated with a chart review. General surgeons at The Ottawa Hospital were surveyed on their opinions regarding monitoring outcomes. We can conclude that different methods may be needed as they are dependent heavily on specified target limits. With our derived cohort, we had adequate risk adjustment using a modified Escobar model for 30 day mortality and morbidity. These complications were derived from electronic algorithms and had excellent specificity and sensitivity. General surgeons at The Ottawa Hospital have different opinions regarding monitoring their outcomes and surgeon engagement is necessary to make monitoring fruitful for patients, public, hospital administration, and surgeons.

No description available.