Pattern Recognition Receptors (PRRs) are a group of evolutionarily conserved and germline-encoded cellular receptors of the innate immune system that are responsible for recognizing and responding to the entirety of the pathogens a host encounters. The ingenuity of the innate immune system is that with a comparatively miniscule pool of receptors, these receptors are capable of responding to a diverse and large array of pathogens and damage signals. Two highly relevant subsets of PRRs include nucleotide binding domain leucine rich repeat containing (NOD-like) receptors (NLRs) and Toll-like receptors (TLRs). Both NLRs and TLRs have been implicated in several diseases, including autoimmune disorders, inflammatory conditions, and cancer. Mice lacking a specific NLR, NLRP1, are more susceptible to chemically induced colitis and colitis-associated tumorigenesis. We investigated whether the absence of NLRP1 in the gastrointestinal tract influenced the composition of the microbiome, and whether it was responsible for the predisposition of these animals to colitis-associated cancer. By carefully controlling for non-genotype influences, we found that in fact maternal and housing factors were greater predictors over genotype of gut flora composition. This study concluded with a clearer understanding of NLRP1. We next investigated the effectiveness of a novel tumor ablation therapy, termed High-Frequency Irreversible Electroporation (H-FIRE) in a murine model of triple negative breast cancer. The chosen 4T1 model closely mimics aggressive human metastatic triple negative breast cancer, and metastasizes to the same organs. After ablation of the primary mammary tumor, we saw significant improvements in disease burden and metastases, both of which were accompanied by PRR activation within the tumor microenvironment, implicating PRRs in the successful treatment outcome following H-FIRE ablation. Lastly, we generated novel CRISPR-Cas9 plasmids to genetically manipulate the Tlr4 gene of wild type C57Bl/6 mice in order to recapitulate the LPS-hyporesponsive TLR4 protein of C3H/HeJ mice. This proof-of-concept study successfully demonstrated that PRRs can be targets for gene editing purposes, and that nanoparticle delivery leads to enhanced and improved delivery. Collectively, this work attempts to better appreciate the role of PRRs in understanding, treating, and targeting cancer. / Doctor of Philosophy / The work presented here focuses on the role of the immune system in the progression of cancer. Put simply, the properly functioning immune system of a healthy individual should recognize and eliminate mutated or cancerous cells prior to the development of a tumor, thereby implying that the progression to a tumor is due to some dysfunction of the immune system. The immune system is made up of two arms: the innate and adaptive. A key difference between the innate and adaptive immune systems is that upon an infection, the adaptive response is slow and specific while the innate response is rapid and broad. Pattern Recognition Receptors (PRRs) are a group of cellular receptors of the innate immune system that are responsible for recognizing and responding to the entirety of the pathogens a host encounters. The ingenuity of the innate immune system is that with a comparatively miniscule pool of receptors, these receptors are capable of responding to a diverse and large array of pathogens. Two highly relevant PRR families are nucleotide binding domain leucine rich repeat containing (NOD-like) receptors (NLRs) and Toll-like receptors (TLRs). Both NLRs and TLRs have been implicated in several diseases, including autoimmune disorders, inflammatory conditions, and cancer. In this work, we investigated whether the absence of an NLR protein influenced the composition of the microbes that reside within the gastrointestinal tract, and whether this absence was responsible for the predisposition of these animals to colitis-associated cancer. By carefully controlling for all additional influences, we found that in our mice, the other animals with which they shared a cage were more influential on the microbes within the gut, rather than the NLR deficiency. We next investigated a novel tumor ablation therapy in an animal model of breast cancer, which closely mimics human metastatic triple negative breast cancer and metastasizes to the same organs. After treatment of the mammary tumor, we saw significant improvements in disease burden and metastases, both of which were accompanied by PRR activation. Lastly, we manipulated a TLR gene in mice to demonstrate that PRRs can be targeted for therapeutic gene editing. Collectively, this work provides evidence that PRRs are a highly useful tool for improving our understanding of cancer.

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As Photodynamic Therapy (PDT) becomes increasingly popular as a treatment modality for some solid tumours, the need for a better understanding of the mechanism(s) of action and resistance are paramount. To this end we have generated Photofrin® PDT-induced resistant variants to numerous cell lines including the colon cancer cell line HT29. There is significant evidence indicating that stress proteins play an important role in determining the outcome of PDT on a cell. In this thesis the roles of the mitochondrial Heat Shock Protein 60 (Hsp60) as well as the endoplasmic Glucose Related Protein 78 (GRP78) were examined in the HT29 cells and their Photofrin induced resistant variant HT29-P14. The expression and role of these two stress proteins were also examined in T24 Bladder carcinoma cells and their GRP 78 stable-overexpressing clones Hsp60 protein was expressed at slightly higher basal levels in the resistant HT29-P14 cells relative to the parental HT29 cells. After incubation alone or PDT action, a temporal and dose dependent induction of Hsp60 was observed and this too was found to be significantly greater in the resistant cells. In the T24 model, no Hsp60 induction was observed following drug incubation or PDT. GRP78 protein levels were increased by PDT action but not by Photofrin® incubation alone in all cell lines tested. In the T24 model, GRP78 transfection resulted in a stable 2-fold increase in protein levels and a 10-20-fold increase in cell survival after PDT at the highest dose tested. A temporal and dose dependent response was noted in all cells and induction of GRP78 protein was lower in the stable overexpresser such that all cell lines had similar post induction levels. In the HT29 and HT29-P14 resistant cells, GRP78 protein levels were similar at basal level, and, both cell lines exhibited the same temporal and dose dependent increases in expression post PDT. Finally, broad scale expression profiling using a "stress" microarray in the HT29 and HT29-P14 resistant variants revealed a very similar expression profile for the 168 of the 169 stress proteins tested with the exception of the small Heat Shock Protein 27 (Hsp27). As confirmed by northern and western blot analysis, Hsp27 is over 20 fold greater at the transcriptional level and 10-15 fold greater at the translational level in the HT29-P14 resistant variant. These findings implicate Hsp27, Hsp60 and GRP78 as possible mediators of cellular sensitivity to Photofrin-mediated PDT. Specifically, Hsp27 appears to play a role in the increased resistance of our induced resistant HT29-P14 cells. / Thesis / Master of Science (MS)

BACKGROUND:The aim of this study was to evaluate clinico-pathologic specific predictors of recurrence for stage II/III disease. Improving recurrence prediction for resected stage II/III colon cancer patients could alter surveillance strategies, providing opportunities for more informed use of chemotherapy for high risk individuals.METHODS:871 stage II and 265 stage III patients with colon cancers were included. Features studied included surgery date, age, gender, chemotherapy, tumor location, number of positive lymph nodes, tumor differentiation, and lymphovascular and perineural invasion. Time to recurrence was evaluated, using Cox's proportional hazards models. The predictive ability of the multivariable models was evaluated using the concordance (c) index.RESULTS:For stage II cancer patients, estimated recurrence-free survival rates at one, three, five, and seven years following surgery were 98%, 92%, 90%, and 89%. Only T stage was significantly associated with recurrence. Estimated recurrence-free survival rates for stage III patients at one, three, five, and seven years following surgery were 94%, 78%, 70%, and 66%. Higher recurrence rates were seen in patients who didn't receive chemotherapy (p=0.023), with a higher number of positive nodes (p<0.001). The c-index for the stage II model was 0.55 and 0.68 for stage III.CONCLUSIONS:Current clinic-pathologic information is inadequate for prediction of colon cancer recurrence after resection for stage II and IIII patients. Identification and clinical use of molecular markers to identify the earlier stage II and III colon cancer patients at elevated risk of recurrence are needed to improve prognostication of early stage colon cancers.

The objective of this study was to investigate food consumption frequency patterns, defecation habits, and incidence of disease states associated with colon cancer by active LDS adult males, residing in Texas, which may help explain the lower incidence of colon cancer observed in the religious group. To accomplish this objective, a sample of 50 was randomly selected and administered a questionnaire, designed to gather information covering personal and demographic characteristics, defecation habits, incidence of associated disease states, and frequency of consumption of 132 selected foods. Data was analyzed by comparison of percentages, means, and frequencies, and a Pearson Product Moment Correlation. Results reported LDS males chose a wide variety of foods with a high frequency of fruits, vegetables, and cereals. A low incidence of problems associated with colon cancer and "western" or refined diets was also reported. Defecation habits were more frequent than general population and compared favorably to another low-risk population, rural Scandinavians.

Les modifications post-traductionnelles des microtubules sont accumulées sur la queue carboxy-terminale des tubulines α et β, situées à l'extérieur des microtubules. Ces modifications peuvent réguler sélectivement les interactions avec les moteurs moléculaires et les protéines associées aux microtubules (MAPs). Ces interactions sont essentiels pour les fonctions cellulaires et demandent une régulation stricte. La glycylation est une modification que génère des chaînes latérales glycine sur les protéines. Jusqu'à présent, la glycylation a été à peine étudiée, et la plupart des travaux se sont concentrés sur son rôle potentiel dans les cils et les flagelles. Peu est connu sur le rôle de la glycylation des microtubules dans les cils primaires. Les cils primaires sont des organelles sensorielles, impliquées dans la transduction des signaux et dans la progression du cycle cellulaire. Récemment, une étude approfondie de 13 023 gènes dans le cancer colorectal a révélé que les tumeurs individuelles accumulent une moyenne d'environ 90 gènes mutés. Un de ces gènes potentiels de cancer est la glycylase TTLL3. Notre équipe a testé les deux mutations décrites pour TTLL3 et a pu constaté que chacun d'entre eux conduit à une perte complète de l'activité de cette glycylase in vivo et in vitro. Mon travail vise donc à élucider le rôle de glycylation de protéines dans la signalisation cellulaire et ses conséquences pour la formation du cancer du côlon. J'ai analysé des échantillons provenant de patients par RT-PCR quantitative et j'ai trouvé une diminution des niveaux d'expression de TTLL3 dans les cancers. Les souris TTLL3-knockout ont été soumises à un modèle murin de carcinome du côlon, induit chimiquement à la base de l'azoxyméthane (AOM) et du sulfate de dextran de sodium (DSS). Mes données montrent une formation tumorale élevée dans le groupe TTLL3-KO, ce qui suggère que la perte de la glycylation est liée au développement du cancer du côlon. La glycylation se trouve dans les cils primaires, et les défauts ciliaires ont été décrits dans différents types de tumeurs solides. La présence de cils primaires et l'importance de la glycylation dans le côlon n'étaient pas encore connues au début de mes travaux. Collectivement, mes résultats indiquent que la glycylation est nécessaire, mais pas indispensable pour les cils primaires. Remarquablement, j'ai pu démontrer la présence de cils primaires sur les cellules épithéliales du côlon pour la première fois, et j'ai mis en évidence un défet de ces cils dans les souris TTLL3-KO in vitro et in vivo. Par ailleurs, j'ai démontré que le dysfonctionnement des cils coliques dans les souris KO TTLL3 est associé à une augmentation de l'activité proliférative des cellules épithéliales. Par conséquent, la glycylation pourrait être importante pour la genèse et le fonctionnement des cils primaires. Dans le côlon, l'absence de la glycylase TTLL3 peut entraîner un manque de la glycylation qui favorise la formation de tumeurs. / Tubulin posttranslational modifications are involved in the regulation of many microtubule functions. Glycylation has been related to the stability and maintenance of motile cilia in different organisms including mammals. We had previously shown that some colon-cancer related mutations in the glycylating enzyme TTLL3 lead to a complete loss of enzymatic activity, which brought up a surprising link between this rather cilia-specific tubulin modification and cancer. To evaluate potential role of glycylation in colon carcinoma formation we first confirmed the link between TTLL3 and colon cancer in a greater cohort of patients. We next studied TTLL3-knockout mice, which strikingly did not show any obvious phenotypic alterations or spontaneous cancer development. However, when submitted to a murine model of chemically induced colon carcinoma, TTLL3-knockout mice show a higher level of tumor formation, pointing towards an acceleration of colon cancer development. Because glycylation of microtubules has been specifically detected on ciliary tubulin, we next analysed the presence of primary cilia in colon epithelium. While in most organs and tissues a second glycylating enzyme, TTLL8, is expressed, TTLL3 is the unique enzyme in colon. We found a significantly reduced number of primary cilia in TTLL3-KO colon epithelium, suggesting that similar to motile cilia, primary cilia are maintained by glycylation of the axonemal tubulin. Moreover, we measured a strongly increased mitotic index in colon epithelial cells isolated from TTLL3-KO mice, indicating that his loss of cilia is accompanied by decreased level of cell cycle control. Thus we have demonstrated for the first time a tight link between the posttranslational glycylation of the microtubule cytoskeleton, the control of cell cycle and the acceleration of cancer development.

Doctor of Philosophy / Department of Human Nutrition / Weiqun Wang / Anthocyanins are flavonoid pigments that account for the purple color in many plant foods. It has been investigated that anthocyanins’ predominant occurrences in human diet and their health beneficial activities such as antioxidant, anti-inflammatory, and anti-carcinogenetic effects. Based on those scientific evidences, anthocyanins are now recognized as potential therapeutic compounds. Particularly, the chemopreventive effect of anthocyanins has been widely studied by many researchers in nutrition. However, their bioactivities are diverse due to different chemical structures of anthocyanins from different sources. In this study, we discuss the chemopreventive activity of anthocyanins from purple sweet potato. Previously, we selected a purple-fleshed sweetpotato clone, P40, crossbred seeds obtained from the International Potato Center in Lima, Peru. We hypothesized that anthocyanins enriched P40 may provide health beneficial activities in cancer prevention. For the first part of this study, we analyzed nutrient compositions, dietary fiber content, anthocyanins contents, total phenolics contents and total antioxidant activity. Even thought P40 presents similar composition and amount of nutrients with the control cultivars, white-fleshed O’Henry and yellow-fleshed NC Japanese, HPLC-MS analysis confirmed that it possesses much higher anthocyanin content even up to 7.5g/kg dry matter. Also, dietary fiber, particularly soluble dietary fiber content, total phenolics content, and total antioxidant capacity of P40 were significantly higher. For the second part of the study, we tested the potential anticancer characteristic of P40 cultivar in human colonic SW480 cancer cells and in azoxymethane-induced aberrant crypt foci in mice. Treatment with 0 – 40 μM of peonidin-3-glucoside or P40 extract containing corresponding amount of anthocyanins resulted in inhibition of cell growth in a dose-dependent manner. Interestingly, even though the patterns of growth inhibition were similar in the two treatment groups, the cells treated with P40 extract tend to survive significantly less than those treated with peonidin-3-glucoside. Cell cycle analysis confirmed that the growth inhibition was not due to cytotoxicity, but cytostatic mechanism with increased number at the G1 phase of the cell cycle. The cell cycle arrest was also significantly correlated with the anthocyanin contents in P40 cultivar when compared with the white-fleshed O’Henry and yellow-fleshed NC Japanese controls. After Azoxymethane (AOM) or saline injected mice were fed basal AIN-93M diet or diets containing 10~30% of P40, 20% O’Henry or 20% NC Japanese for 6 weeks, aberrant crypt foci (ACF) multiplicity was significantly inhibited by 10~30% P40 diet. Imunohistochemistry results of colonic mucosa showed that the expression level of apoptosis marker, caspase-3, was significantly induced in the mice treated with 10~20% P40 diet. Also, PCNA expression level, which is proliferation marker, was significantly inhibited by the 30% P40 diet. These findings indicated that consuming a purple sweet potato, P40, may prevent colon cancer by modulating antioxidant status, inducing apoptosis, and reducing cell proliferation.

Colon and breast cancers are amongst the leading causes of cancer deaths in the United States, mostly attributed to metastasis and resistance to therapy. Hence, there is a critical need to identify novel biomarkers for effective prognosis and to design targeted therapies to combat the metastatic diseases. Loss of heterozygosity (LOH) at chromosome 18q and inactivation of the target gene, SMAD4, corresponds to resistance to the common chemotherapeutic agent, 5-fluorouracil (5-FU), in colon cancer. Our examination of the therapeutic resistance phenomenon in SMAD4-negative colon cancer cells with the three common agents revealed significant resistance to both 5-FU and irinotecan but not to oxaliplatin. We also followed up with the earlier findings from our group, which suggested that SMAD4 might interact with metastasis-promoting factors to suppress metastatic progression and render sensitivity to chemotherapy. Co-immunoprecipitation and mass spectrometry analysis revealed that SMAD4 interacts with and inhibits RICTOR, a component of mTORC2 that activates oncogenic AKT via phosphorylation at Serine 473. Overexpression of SMAD4, depletion of RICTOR, or inhibition of AKT signaling restores sensitivity to irinotecan in SMAD4-negative colon cancer cells in vitro. Furthermore, as expected pharmacological inhibition of AKT sensitizes these cells to irinotecan in vivo. Interestingly, high RICTOR/AKT expression correlates with worse survival in colon cancer patients, suggesting them as novel prognostic biomarkers and therapeutic targets. On the other hand, triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer due to lack of effective targeted therapies. Using miRNA expression profiling of a model for epithelial-mesenchymal transition in TNBC, we found suppression of miR-4417 during the progression from non-malignant to malignant stage. Furthermore, localization of miR-4417 to chromosome 1p36, a region corresponding to high frequency of LOH in multiple cancers and low-level expression in TNBC patients associated with poor overall survival is consistent with its likely role as a tumor suppressor. Interestingly, we found that overexpression of miR-4417 is sufficient to inhibit migration and tumorigenecity of TNBC cells in vitro. Overall, our findings suggest miR-4417 exerts a tumor-suppressive effect and could serve as a novel prognostic biomarker and therapeutic tool against TNBC. / 2021-02-20T00:00:00Z

Les tétraspanines sont des glycoprotéines membranaires impliquées dans une variété de processus physiologiques et pathologiques. En cancérologie, de nombreuses études cliniques et expérimentales ont établi un lien entre le niveau d’expression de certaines tétraspanines et la formation de métastases. Ce travail porte sur la tétraspanine Co-029/tpan8 et son rôle dans le processus métastatique dans le cancer du côlon. Afin d’appréhender ce rôle, un modèle cellulaire a été développé dans le laboratoire à partir de la lignée Isreco1 où nous avons surexprimé de manière stable le Co-029/tspan8. Sur ce modèle nous avons étudié dans un premier temps la migration cellulaire, élément capital participant à la genèse des métastases. Nous avons montré que le Co-029/tspan8 coordonne la motilité cellulaire en fonction de plusieurs signaux issus de la membrane (intégrines, complexe E-cadhérine/p120ctn, EGFR, MET). Par ailleurs, nous avons montré dans une première étude clinique que l'expression de Co-029/tspan8 dans la tumeur serait de pronostic péjoratif. Nous avons également montré que l’anticorps anti-Co029 (Ts29.2) produit dans notre laboratoire induit une réduction significative de la croissance tumorale in vivo dans la souris nude. Nos travaux suggèrent que Co-029/tspan8 serait à la fois un marqueur de pronostic et une cible potentielle pour l'utilisation thérapeutique des anticorps monoclonaux dans le cancer du côlon. / Tetraspanins are membrane glycoproteins involved in a variety of physiological and pathological processes. In cancerology, many clinical and experimental studies have established a link between expression’s level of some tetraspanins and metastasis. This work focuses on the tetraspanin Co-029/tspan8 and its implication in the metastatic process in colon carcinoma. We developed a cellular model from primary colon carcinoma cell line Isreco1 in which we stably overexpressed Co-029/tspan8. In this model we studied, first, cell migration, an essential process for metastasis formation. We have shown that Co-029/tspan8 coordinates cell motility according to several signals from the membrane (integrins, E-cadherin/p120ctn complex, EGFR, MET). Moreover, in a first clinical study the Co-029/tspan8 expression in primary tumors seems to correlate with poor prognosis. We also showed that the antibody anti-Co029 (Ts29.2) produced in our laboratory induced a significant inhibition of tumor growth in nude mice. Our study suggests Co-029/tspan8 as a marker of prognosis and a potential target for monoclonal antibody therapy in colon cancer.