Malignancy is associated with a hypercoagulable state manifested clinically by an increased incidence of venous thromboembolism (VTE). Colorectal cancer (CRC) patients who develop VTE have reduced survival. This increased mortality extends beyond the acute VTE event, suggesting that VTE is associated with aggressive tumour biology. Tissue factor (TF) and other clotting factors have been implicated in this process. However, the significance of clotting factors in the tumour microenvironment (TME) remains unknown. The aim of this thesis is to i) determine if a procoagulant TME is a biomarker for poor prognosis and VTE in patients undergoing resectional surgery for CRC and ii) determine the effect of TF, thrombin and FXa on proliferation and migration in vitro in CRC and if their inhibitors have potential as anticancer therapies. In the in vitro studies, epithelial expression of TF had a modest effect on proliferation and migration when quantified using the PrestoBlue proliferation and transwell migration assays. Exogenous TF, FXa and thrombin all increased migration in DLD-1 wild type cells. In addition, exogenous thrombin increased proliferation amongst SW620 wild type cells. This suggests that coagulation factors from the TME, rather than epithelial expression, may influence tumour biology. Moreover, dabigatran, a direct thrombin inhibitor, abrogated the pro-proliferative effects of thrombin, which highlights its potential role as an anticancer therapy. In a multicentre, prospective cohort study of 159 CRC patients undergoing resectional surgery, rates of duplex screen detected deep vein thrombosis (DVT) were correlated to plasma and tumour markers of hypercoagulability. TF is upregulated in the stroma of cancer compared to normal tissue. However, stromal TF expression decreased in more advanced (T4) tumours. This suggests that a procoagulant TME has a role in early tumourigenesis. In total, 5.4%, 7.0% and 9.1% of patients had an asymptomatic DVT pre- operatively, at six weeks post-surgery and after the commencement of adjuvant chemotherapy respectively. The development of a post-operative complication was a risk factor for DVT, whilst locally advanced tumours resulted in a prolonged hypercoagulable state i.e. raised D-dimer at six weeks. This highlights a possible role for pre- and post- operative screening duplex ultrasonography and super-extended VTE prophylaxis in selected patients. In conclusion, this thesis establishes a role for exogenous coagulation factors in promoting tumour biology in CRC. VTE is more common amongst patients undergoing resectional surgery for CRC than previously estimated. The utility of tumour and plasma hypercoagulabilty as biomarkers for survival in CRC will be further analysed when long term follow-up data is available.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O câncer colorretal representa uma das maiores causas de morbidade e mortalidade relacionadas ao câncer. No Brasil, é o terceiro tipo de câncer mais frequente em homens e mulheres. Muitos estudos estão sendo desenvolvidos no sentido de esclarecer os diversos aspectos moleculares que regulam as alterações fenotípicas exibidas pelas células que constituem o câncer colorretal, no entanto, comparativamente, ainda são poucos os que são dedicados a investigar o papel de modificações co- e pós-traducionais neste processo. Entre os vários tipos destas modificações que ocorrem em proteínas, a glicosilação é a mais comum. Cogita-se que aproximadamente cinquenta por cento de todas as proteínas são glicosiladas. Durante a transformação maligna, mudanças no perfil de expressão de glicanos (carboidratos covalentemente ligados a proteínas ou lipídios) estão envolvidas em uma variedade de mecanismos celulares, tais como: perda da adesão célula-célula e célula matriz, migração, invasão e evasão da apoptose. Neste estudo, foi investigada a atividade antitumoral de inibidores da biossíntese de N-glicanos, swainsonina e tunicamicina, em células derivadas de câncer colorretal (Caco-2, HCT-116 e HT-29). Os resultados obtidos mostram que o tratamento das células HCT-116 com tunicamicina inibe mecanismos celulares relacionados ao fenótipo maligno, como formação de colônia dependente e independente de ancoragem, migração e invasão. Estes resultados sugerem que modulação da biossíntese de N-glicanos parece ser uma potencial ferramenta terapêutica para o tratamento do câncer colorretal. Em outra etapa do trabalho, foram avaliados também o impacto da estimulação com insulina e IGF-1 na expressão N-glicanos bissectados em células tumorais MDA-MB-435. Os resultados obtidos confirmaram também a existência de uma relação entre a estimulação dos receptores de insulina e IGF-1 e a regulação da expressão de N-glicanos bissectados em células tumorais MDA-MB-435, fornecendo assim informações relevantes sobre o papel desempenhado pela sinalização de insulina e IGF-1 durante a progressão de carcinomas. / Colorectal cancer is a major cause of cancer-related morbidity and mortality. In Brazil, it is the third most common cancer. Many studies have been developed to clarify several molecular features which regulate phenotypic changes exhibited by cells that constitute colorectal cancer, however, comparatively, there are few studies dedicated to investigate co- and post-translational modifications of proteins in this process. Glycosylation is the most common post-translational modification of proteins. Approximately fifty percent of all proteins are glycosylated. During malignant transformation, changes in the expression profile of glycans (carbohydrates covalently bound to proteins or lipids) may be involved in a variety of events, including the loss of cellcell and cellmatrix adhesion, migration, invasion, and evasion of apoptosis. In this study, we investigated the in vitro anticancer activity of the N-glycan biosynthesis inhibitors, swainsonine and tunicamycin, in cells derived from colorectal cancer (Caco-2, HCT-116 e HT-29). Our results show that treatment with tunicamycin inhibits cellular mechanisms related to the malignant phenotype, such as anchorage-dependent and anchorage-independent colony formation, migration and invasion, in HCT-116 colon cancer cells. Given these results, we suggest that the modulation of N-glycan biosynthesis appears to be a potential therapeutic tool for CRC treatment. Moreover, we confirmthe existence of an interplay between stimulation with insulin and IGF-1 and bisecting GlcNAc N-glycans expression in MDA-MB435 cancer cells, providing new insights into the role that Insulin/IGF-I signaling play during carcinoma progression.

Wnt signalling is a critical cellular communication pathway controlling cell fate in all metazoan organisms. Timely activation of this pathway is crucial to coordinate development, control homeostasis of adult tissues, and to avoid cancer. Wnt signal transduction depends primarily on the activities of three multiprotein complexes; the 'degradasome', which targets the central effector β-catenin for degradation in the absence of Wnt; the 'signalosome', which is assembled by Dishevelled upon Wnt-receptor binding to inactivate the degradasome, thus allowing β-catenin to accumulate; and the 'enhanceosome', which captures β-catenin, granting it access to target genes and relieving their transcriptional repression by Gro/TLE. Many of the components of these complexes have now been identified, but details of their regulation, and in particular the mechanisms by which they are switched on and off, remain poorly understood. The majority of this thesis is concerned with the mechanism by which β-catenin relieves the transcriptional repression imposed upon Wnt target genes, and thereby activates the Wnt 'transcriptional switch'. In Chapter 2, I present data showing that apposition of Gro/TLE and UBR5, a HECT E3 ubiquitin ligase, by β-catenin promotes Gro/TLE ubiquitylation, earmarking it for extraction by the VCP/p97 ATPase and ultimately leading to inactivation of its repressive function. In Chapter 3, I present the results of a different, ongoing study to identify the mechanism by which a cytoplasmic negative regulator, Naked, acts to interfere with the function of Dishevelled, promoting the switching of signalosomes and the termination of canonical Wnt signalling. These findings advance our understanding of the mechanisms by which the Wnt signalling pathway is switched on and off, and suggest new targets for therapeutic intervention in Wnt- driven cancers.

Introdução: O câncer colorretal é um dos mais frequentes no mundo ocidental. Novas medidas de prevenção, diagnóstico precoce e tratamento vêm melhorando o prognóstico para os pacientes, com novos achados biológicos inferindo relação com a evolução da doença. A PPARG é um receptor nuclear abundantemente expresso em células epiteliais do cólon, e variações na sua expressão podem ser relacionadas à evolução clínica do câncer colorretal. Objetivo: Avaliar a expressão gênica do PPARG em tumores colorretais e relacionar este dado com variáveis clínicas dos pacientes, como: idade, tipo histológico, CEA, estadiamento e a evolução clínica. Casuística e métodos: Analisamos a expressão gênica do PPARG em 50 amostras de tumores colorretais através da RT-PCR, e 20 amostras de tecido normal adjacente como controle. Os resultados destas quantificações foram correlacionados com as informações clínicas dos prontuários dos respectivos pacientes. Resultados: Houve menor expressão do PPARG no tecido tumoral em comparação ao tecido controle. Dentre os tumores, os pacientes com idade acima de 60 anos, tipo histológico com diferenciação mucinosa, estadiamento mais avançado ao diagnóstico e os pacientes que evoluíram com recidiva da doença ou óbito apresentavam maior expressão do PPARG. Discussão: Analisando os tecidos tumorais, pode-se inferir uma tendência a pior prognóstico nos pacientes com expressão mais elevada de PPARG. Esses achados, correlacionados aos demais estudos já publicados na literatura, apontam uma tendência desfavorável na evolução da doença. Estudos futuros com um maior número de pacientes e várias instituições podem inferir uma importância prognóstica e até terapêutica para a PPARG. / Introduction: Colorectal cancer is one of the most frequent neoplasm in the Western world. New strategies of prevention, early diagnosis and treatment have improved the prognosis for the patients, and new biological findings relate to the prognosis of the disease. PPARG is a nuclear receptor highly expressed in colon epithelial cells, and variations on its expression may be related to the clinical evolution of colorectal cancer. Objective: To evaluate the gene expression of PPARG in colorectal tumors and to correlate this data with clinical variables of the patients, such as: age, histological type, CEA, staging and clinical evolution. Casuistry and methods: We analyzed the gene expression of PPARG in 50 samples of colorectal tumors using RTPCR, and 20 adjacent normal tissue samples as control. The results of these quantifications were correlated with the respective patients\' medical records\' clinical information. Results: There was a lower PPARG expression in the tumor tissue compared to the control tissue. Among the tumors samples, patients older than 60 years, histological type with mucinous differentiation, more advanced staging at the time of diagnosis, and patients who evolved with recurrence of the disease or death presented higher PPARG expression. Discussion: Analyzing the tumor tissues, we can infer a tendency to worse prognosis in patients with higher PPARG expression. These findings, correlated with the other studies already published in the literature, point to na unfavorable trend in the disease\'s evolution. Future studies with a larger number of patients and several institutions may infer a prognostic and even therapeutic importance for PPARG.

The main purpose of this dissertation was to examine the statistical dependence of imputed microRNA (miRNA) data in a colorectal cancer study. The dissertation addressed three related statistical issues that were raised by this study. the first statistical issue was motivated by the fact that miRNA expression was measured in paired tumor-normal samples of hundreds of patients, but data for many normal samples were missing due to lack of tissue availability. We compared the precision and power performance of several imputation methods, and drew attention to the statistical dependence induced by K-Nearest Neighbors (KNN) imputation. The second statistical issue was raised by the necessity to address the bimodality of distributions of miRNA data along with the imputation-induced dependency among subjects. We proposed and compared the performance of three nonparametric methods to identify the dierentially expressed miRNAs in the paired tumor-normal data while accounting for the imputation-induced dependence. The third statistical issue was related to the development of a normalization method for miRNA data that would reduce not only technical variation but also the variation caused by the characteristics of subjects, while maintaining the true biological dierences between arrays.

Colorectal cancer is the 3rd leading cause of cancer-related deaths. Early screening provides the best prospects for preventing the morbidity and mortality associated with the disease. Nurses have the duty to promote health and prevent diseases. However, low rates of colorectal cancer screening continue to be reported, especially among African Americans who continue to suffer disproportionately from the disease. There is a need for a culturally-sensitive clinical practice guideline that nurses can use to educate patients appropriately on colorectal cancer. The practice focused question for this project was designed to explore whether a culturally-sensitive clinical practice guideline to increase colorectal cancer screening among African Americans could be developed using best practices. The health belief model informed the background, development, and implementation of this project. Evidence from peer-reviewed nursing literature was synthesized in a literature review matrix and then used to develop a clinical practice guideline to increase colorectal cancer screening. It is anticipated that this guideline will improve nursing practice by equipping nurses with the knowledge and skill to provide culturally-sensitive education on colorectal cancer and screening. Through the patient education and enhanced nursing practice stipulated in the clinical practice guideline, health care providers may work to eliminate disparities in colorectal cancer screening among African Americans.

<p>Both Notch and the Wnt pathways are key regulators in maintaining the homeostasis in the intestine. Defects on the key tumor suppressor adenomatous polyposis coli, APC a gene in the Wnt pathway is most frequently mutated in colorectal cancer. Previous studies have indicated that there is a crosstalk between these two pathways. We investigate if there is correlation by first using bioinformatics to find Lef1/Tcf sites in several of the Notch pathway gene promoters. Bioinformatically we found that a lot of the genes contained theses sites controlled by the APC's destruction target β-catenin. By using semi quantitative PCR and western blot we found that Hes 1, Hes 7, JAG 2, MAML 1, Notch 2, NUMB, NUMBL, RFNG and LFNG was downregulated in HT29 colon cancer cells carrying a vector containing wild type APC. All but JAG 2 contains at least one Lef1/Tcf site in their promoter region. The results were verified in HT29 cells transfected with siRNA against β-catenin. We also investigated what would happen to the Lef1/Tcf target gene program of the Wnt pathway, if the Notch pathway was inhibited with the gamma-secretase inhibitor DAPT. Results showed no downregulution of β-catenin or its target gene Cyclin D1.Taken together, these results demonstrate that the Wnt pathway can be placed upstream of the Notch pathway and regulates the latter through β-catenin and the Lef1/Tcf target gene program. However, preliminary results indicate that there is no regulation of APC/β-catenin by the Notch pathway.</p>

Background: Surgical treatment of colorectal liver metastases (CLRM) depends on resectability that is currently based on the CT scan. With the PET/CT scan, a more accurate pre-operative assessment of resectability may be possible. Methods: A Cochrane-based diagnostic test systematic review and a systematic review of cost-effectiveness studies on PET scans were conducted. Lastly, a diagnostic decision analysis model was created to assess the cost-effectiveness of the technology. Results: PET/CT scans was equally sensitive for hepatic metastases and more sensitive for extra-hepatic metastases compared to CT scans. A cost-savings of PET scans for CRLM is identified; with decision modelling demonstrating a cost-savings with the addition of PET/CT scans to the current clinical algorithm. Conclusion: There is cautious support for the addition of PET/CT scans to the pre-operative assessment in CRLM. Unnecessary surgery may be prevented, thus decreasing wait times. Future endeavours include finding, evaluating and validating methodology for appropriate effectiveness measures. / Health Technology Assessement

This dissertation explores the spatial patterns and place-based characteristics of colorectal cancer (CRC) late stage incidence and CRC-specific mortality in Miami-Dade County. Because CRC is the second leading cause of death among all cancers and is almost 90 percent preventable through medical screenings, investigations of CRC disparities across groups and communities are extremely relevant in the fight against cancer. This paper analyzes the geographic distribution of CRC cases in Miami-Dade County between two periods, 1988-1992 and 1998-2002 to: a) identify significant "hot spots" or clusters of disease; b) investigate associations of CRC patterns with neighborhood level characteristics such as socio-economic status, race/ethnicity, and poverty; and c) explore the policy implications of the spatial trends identified for the disease, with particular reference to the Welfare Reform Act of 1996. This dissertation analyzes data from the Florida Cancer Data Registry and tract level U.S. Census data, to identify the spatial distribution of CRC and study its relation to place-based variables using Geographic Information Systems (GIS) and spatial statistical modeling. Identifying spatial clusters of disease can assist in targeting public health interventions and improving social service delivery, particularly for uninsured populations. Identifying communities facing greater obstacles to screenings and quality medical care through the use of spatial analysis is an effort to mitigate these barriers while simultaneously providing empirically based evidence linking neighborhood-level social and economic conditions to health disparities.

Most human genes undergo alternative splicing, and many abnormal splicing processes are associated with human diseases. However, the molecular relationship between alternative splicing and tumorigenesis is not well understood. Here, we identified novel Krüppel-like factor 4 (KLF4) splicing variants produced by exon skipping in human cancer cell lines as well as colon tumor tissues. To elucidate the mechanism involved in KLF4 alternative splicing, we developed KLF4 minigene system and found that RNA binding motif protein 5 (RBM5) plays an important role in KLF4 splicing, as assessed by gain and loss of functional studies. Several anti-tumorigenic compounds were also tested for KLF4 splicing. Interestingly, sulindac sulfide restored wild type KLF4 (KLF4L) expression and this is mediated by dephosphorylation of RBM5. Another splicing variant, small KLF4 (KLF4S), localizes in the cytoplasm and nucleus, and antagonizes transcriptional activity of wild type KLF4. Our data suggest that RBM5 plays a pivotal role in the alternative splicing of KLF4, and these splicing variant forms may impact tumorigenesis.