By 2030, the global colorectal cancer burden is projected to approximately double. This highlights the immediate need to expand our understanding of the etiological origins of colorectal cancer, so that novel therapeutic strategies can be identified and validated. The putative tumor suppressor gene RNF20 encodes a histone H2B mono-ubiquitin ligase and has been found altered/mutated in colorectal and numerous other cancer types. Several studies suggest that RNF20, and by extension mono-ubiquitinated histone H2B (H2Bub1), play important roles in maintaining genome stability in human cells. Indeed, hypomorphic RNF20 expression and/or function have been shown to underlie several phenotypes consistent with genome instability, making aberrant RNF20 biology a potential driver in oncogenesis. Through an evolutionarily conserved trans-histone pathway, RNF20 and H2Bub1 have been shown to modulate downstream di-methylation events at lysines 4 (H3K4me2) and 79 (H3K79me2) of histone H3. Accordingly, understanding the biology associated with RNF20, H2Bub1, H3K4me2, and H3K79me2 is an essential preliminary step towards understanding the etiological origins of cancer-associated RNF20 alterations and identifying a novel therapeutic strategy to selectively kill RNF20-deficient cancers. In this thesis, I employ single-cell imaging, and multiple biochemical techniques to investigate the spatial and temporal patterning and characterize the biology of RNF20, H2Bub1, H3K4me2 and H3K79me2 throughout the cell cycle. In addition, I employ the CRISPR-Cas9 genome editing system to generate RNF20-deficient HCT116 cells. Finally, I employ synthetic lethal strategies to selectively kill RNF20-depleted cells. In conclusion, the research chapters contained within this thesis have characterized putative drivers in cancer (Chapter 3), generated a valuable research reagent for CRISPR-Cas9 ii genome editing experiments (Chapter 4), and identified a novel therapeutic strategy to selectively kill certain cancer cells (Chapter 5). This thesis has increased our understanding of the etiological origins of cancer and generated novel reagents and treatments strategies that after further validation and clinical studies, could be employed to reduce morbidity and mortality rates associated with cancer. / October 2016

Despite strong evidence that screening for Colorectal cancer (CRC) can reduce cancer incidence and mortality, screening adherence remains low. Racial differences in CRC incidence and mortality are well documented in the literature. Racial differences in CRC screening use remain mixed with most studies using race as an independent variable and focusing on racial differences in CRC screening rates. Few studies have examined correlates of CRC screening use, stratifying by race. The purpose of this study was to determine if there are racial differences in correlates of CRC screening, using the Health Belief Model as the theoretical framework. Data analyzed in this study came from the 2003 Health Information National Trends Survey (HINTS) of the National Cancer Institute. White (n=1988) and non-white (562) respondents age ≥50 years, without a history of cancer were interviewed by phone. Multivariable logistic regression was used to identify correlates of FOBT and endoscopy adherence stratified by race and screening test. Independent variables included age, gender, education, income, insurance status, regular care visit frequency, perceived risk of CRC, family history of cancer, CRC knowledge, cancer worry, perceptions of screening benefits, and perceptions of expense as a barrier. Predictors of adherence to FOBT for whites included being older and having at least 1 regular car visit. Predictors of FOBT adherence for non-whites included having health insurance. Endoscopy adherence for whites was significantly associated with being older, being female, and agreeing with perceptions of benefits to CRC screening. Predictors of endoscopy adherence for non-whites included being older, and disagreeing with perceptions of benefits to CRC screening. Such differences, if confirmed in future studies, may inform race-specific interventions to increase CRC screening utilization.

Combined intakes of specific dietary fiber and fat subtypes protect against colon cancer in animal models. We evaluated associations between self-reported individual and combinations of fiber (insoluble, soluble, and pectins, specifically) and fat (omega-6, omega-3, and docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), specifically) and colorectal cancer (CRC) risk in the Women's Health Initiative prospective cohort (n = 134,017). During a mean 11.7 years (1993-2010), 1952 incident CRC cases were identified. Cox regression models computed multivariate adjusted hazard ratios to estimate the association between dietary factors and CRC risk. Assessing fiber and fat individually, there was a modest trend for lower CRC risk with increasing intakes of total and insoluble fiber (p-trend 0.09 and 0.08). An interaction (p = 0.01) was observed between soluble fiber and DHA + EPA, with protective effects of DHA + EPA with lower intakes of soluble fiber and an attenuation at higher intakes, however this association was no longer significant after correction for multiple testing. These results suggest a modest protective effect of higher fiber intake on CRC risk, but not in combination with dietary fat subtypes. Given the robust results in preclinical models and mixed results in observational studies, controlled dietary interventions with standardized intakes are needed to better understand the interaction of specific fat and fiber subtypes on colon biology and ultimately CRC susceptibility in humans.

INTRODUÇAO: O câncer de cólon é a terceira neoplasia mais prevalente no país, com aumento progressivo da incidência associada ao envelhecimento populacional. Os avanços nos tratamentos local e sistêmico do câncer de cólon metastático tem aumentado significativamente o tempo de sobrevida global. Entretanto, ainda não existem biomarcadores consolidados na literatura, capazes de predizer resposta a estes tratamentos ou o prognóstico. No processo da carcinogênese, uma das importantes vias que se encontra alterada é a via de reparo do DNA. A Chk2 é uma proteína quinase com atividade no reparo celular atuando de forma supressora no processo da carcinogênese, sendo que alterações em sua expressão e/ou função têm sido associadas à progressão tumoral em outras neoplasias como no câncer de mama, pulmão, vulva, bexiga, cólon, ovário, osteossarcoma e linfomas. OBJETIVO: Avaliar a expressão imunohistoquímica do Chk2 no câncer de cólon metastático e correlacionar sua expressão com características clínico-patológicas e sobrevida. PACIENTES E MÉTODOS: Foram incluídos 58 pacientes com diagnóstico confirmado de câncer de cólon metastático, tratados em primeira linha com quimioterapia baseada em fluorouracila e oxaliplatina. O tempo mínimo de seguimento foram de 2 anos pós-diagnóstico. Para análise da expressão do Chk2 foram utilizadas as técnicas de tissue microarray e imunohistoquímica. Estes resultados foram correlacionados com características clínicas, patológicas e de sobrevida. Para análise estatística, foi utilizado o programa SPSS17 e o valor de p<0,05 foi considerado estatisticamente significativo. RESULTADOS: A expressão de Chk2 foi positiva em 69% dos pacientes. Houve associação entre a expressão de Chk2 e o status linfonodal (p = 0,012) e entre a sobrevivência (p=0,034). A expressão negativa de Chk2 aumentaram as chances de envolvimento linfonodal (OR:10,2, p=0,03). O tempo de sobrevida global de pacientes Chk2 negativo foi maior (72 versus 59 meses, p=0,155), o mesmo foi observado com o tempo sobrevida livre de progressão (19 versus 13 meses, p=0,293). As curvas de sobrevida foram diferentes de acordo com a expressão do Chk2 em pacientes com ou sem envolvimento linfonodal, sendo menor nos pacientes com Chk2 positivo, p=0,028. Houveram mais óbitos em pacientes com Chk2 positivo. Análise multivariada identificou o performance status segundo a escala de ECOG (p=0,001 ); metástase sincrônica (p=0,037); diferenciação das células tumorais (p=0,029) e expressão de Chk2 (p=0,020) como fatores independentes para sobrevida global. CONCLUSÃO: A expressão positiva do Chk2 no adenocarcinoma de cólon metastático foi indicativa de maior agressividade e disseminação tumoral, impactando de forma negativa na sobrevida e desfecho dos pacientes. / INTRODUCTION: The DNA damage checkpoint pathway has been of interest to the field of cancer biology, since checkpoint defects result in the accumulation of altered genetic information, a central feature of carcinogenesis. Little is known about the role of Chk2 in colorectal cancer tumorigenesis. OBJECTIVE: The purpose of this study was to evaluate Chk2 expression in metastatic colon cancer and correlate this with clinicopathological features and patient survival. PATIENTS AND METHODS: Tissues were obtained from 58 patients with confirmed metastatic colon cancer diagnosis, treated with capecitabine and oxaliplatin chemotherapy as standard doses. Patients included had, at least, 2 years post diagnosis of clinical following. The tissue microarray immunohistochemistry was the technic to evaluate Chk2 expression. Statistics analysis used SPSS 17. A p-value <0,050 was considered to be statistically significant. Immunohistochemical expression of Chk2 and its relationship with clinical and pathological characteristics and survival data was reported. RESULTS: The expression of Chk2 was positive in 69%. There was association between expression of Chk2 and Iymph node status (p=0.012) and between survival (p=0.034). The negative expression of Chk2 enhanced the chances of linfonodal involvement (OR:10,2, p=0.03). The global survival time of Chk2 negative patients was higher (72 versus 59 months, p= 0.155); the same was observed with progression-free survival time (19 versus 13 months, p=0.293). The survival curves were different according to Chk2 expression in patients with or without Iymph node involvement, being lower in patients with Chk2 positive, p=0.028. There were more deaths in patients with Chk2 positive. Multivariate regression analysis identified performance status ECOG (p=0.001), synchronous metastasis (p=0.037), tumor cell differentiation (p=0.029) and expression of Chk2 (p=0.020) as independent factors to overall survival. CONCLUSION: This study demonstrated that the Chk2 positive expression in colon cancer indicates increased tumor spread and tumoral aggressiveness, impacting negatively on survival and outcome of patients.

O presente estudo teve como objetivo investigar se existe uma correlação entre ansiedade, depressão e níveis séricos de citocinas de pacientes com câncer colorretal em diferentes estágios da terapia antitumoral. Para tanto, utilizamos um total de 100 indivíduos, divididos em cinco grupos amostrais. Grupo 1. Voluntários saudáveis livres de qualquer doença psiquiátrica ou associada com alterações do sistema imune (n=20); Grupo 2. Pacientes com diagnóstico de adenocarcinoma colorretal confirmado e que não foram submetidos à ressecção cirúrgica (n=20); Grupo 3. Pacientes submetidos à ressecção cirúrgica e que não iniciaram a terapia adjuvante (n=20); Grupo 4. Pacientes em tratamento quimioterápico há cerca de 3 meses, independentemente do esquema de quimioterapia aplicado (n=20); Grupo 5. Pacientes que concluíram o esquema de quimioterapia adjuvante há cerca de 6 meses (n=20). Depressão e ansiedade foram analisados utilizando Hospital Anxiety and Depression Scale (HADS) e níveis séricos de IL-1, IL-6, IL-8, IL-10, IL-12, TNF-?, TGF-? e Fractalcina foram mensurados por CBA. Níveis clinicamente relevantes de ansiedade e/ou depressão foram verificados em todos os pacientes com CCR em diferentes estágios da terapia antitumoral. Um padrão de produção semelhante foi observado para as citocinas pró-inflamatórias avaliadas. IL-1, IL-6, IL-8, TNF-? e Fractalcina foram encontradas em níveis elevados nos pacientes com CCR em estágio pré (G2) e pós-operatório (G3). Estes níveis foram, contudo, reduzidos durante (G4) e após (G5) o tratamento quimioterápico. Além disso, verificamos níveis diminuidos de IL-10 no soro dos pacientes dos grupos 2 e 3 (pré e pós-operatório). Ao analisarmos a correlação entre a pontuação HADS e os níveis séricos de citocinas, observamos uma associação positiva de ansiedade e/ou depressão com as concentrações de IL-1, IL-6, IL-8, TNF-? e Fractalcina e negativa com IL-10 em pacientes nos diferentes estágios da terapia antitumoral. Estes resultados indicam haver uma importante correlação entre os níveis séricos de citocinas pró-inflamatórias, ansiedade e depressão em pacientes com câncer colorretal, sugerindo que tais citocinas estão envolvidas na patofisiologia dessas comorbidades / This study aimed to investigate whether there is a correlation between anxiety, depression and serum cytokine levels in colorectal cancer in different stages of the antitumor therapy. A sample of 100 subjects was divided in 5 groups. Group 1: Healthy volunteers free of any psychiatric or immune system disease (n=20); Group 2: Patients with colorectal cancer who did not undergo surgical resection (n=20); Group 3: Patients who underwent surgical resection and who did not start adjuvant therapy (n=20); Group 4: Patients undergoing chemotherapy for about 3 months, regardless of chemotherapy protocols applied (n=20); Group 5: Patients who have completed adjuvant chemotherapy regimen for about 6 months (n=20). Depression and anxiety were analyzed using the Hospital Anxiety and Depression Scale (HADS), and serum levels of IL-1, IL-6, IL-8, IL-10, IL-12, TNF-?, TGF-?, and Fractalkine were measured by CBA. Clinically relevant levels of anxiety and/or depression were found in all CRC patients in different stages of the antitumor therapy. A similar pattern of production was observed for proinflammatory cytokines. Elevated levels of IL-1, IL-6, IL- 8, TNF-?, and Fractalkine were found in CRC patients in pre (G2) and postoperative (G3) stages. However, these levels were reduced during (G4) and after (G5) chemotherapy. Furthermore, we found decreased levels of IL-10 in serum of patients in CRC patients in pre and postoperative stages. By analyzing the correlation between HADS scores and serum cytokine levels, we observed a positive association of anxiety and/or depression with the concentrations of IL-1, IL-6, IL-8, TNF-?, and Fractalkine, and negative with IL-10 in patients in different stages of the antitumor therapy. These results indicate an important link between serum levels of proinflammatory cytokines, anxiety and depression in CRC patients, suggesting that such cytokines are involved in the pathophysiology of these comorbidities

Inflammatory bowel disease (IBD) confers a high risk of development of colitis-associated colorectal cancer in patients with extensive colitis. Crypt fission is a mechanism of clonal expansion in the intestinal epithelium. Although fission is rare in the normal colon, many crypts in IBD patients are in the process of fission. Protumourigenic mutations can spread through the entire inflamed colon relatively quickly indicating that stem cell dynamics are altered in IBD. Some patients with IBD develop pseudopolyps as a result of mucosal ulceration and epithelial regeneration. The aim of this PhD was to investigate the effect of inflammation on niche succession, the crypt cycle and the expansion of clones in the IBD intestine. Pseudopolyps were examined as potential sites for clonal expansion by determining the frequency of mutated pseudopolyps and proliferative potential, and examining their microRNA (miRNA) profile relative to inactive, active and dysplastic mucosa, and adenoma and cancerous tissue. This thesis will show that crypt fission cycles in inflammatory bowel diseased colon are protracted and that each stage of crypt fission appears to be slow. Overall, clonally related adjacent IBD crypts seem to share a more recent common ancestor than non-related IBD crypts, supporting increased crypt fission rates in IBD. The proliferative drive induced by continuous inflammation and mucosal repair in ulcerative colitis (UC) appears to promote the expansion of CCO-deficient patches. Furthermore, niche succession appears to be faster in active IBD. Pseudopolyps are a source of regeneration within the epithelium and, as shown here, have a faster proliferative drive than background mucosa in IBD patients. Pseudopolyps are not genetically inert and are a potential source of protumourigenic mutations in UC. Hence, pseudopolyps are a potential reservoir within the inflamed epithelium where mutations are harboured and where there is no competition from neighbouring epithelium, as it has been denuded following previous inflammation. MiRNA expression in pseudopolyps differs from that of UC-dysplasia and mucosa. In particular, the MiR-29 family was downregulated in pseudopolyps, a miRNA family that has been implicated in intestinal fibrosis formation in stricturing Crohn’s disease. Pseudopolyps have been traditionally thought of as benign, genetically inert and incidental findings characteristic of chronic inflammation. My research runs counter to this view indicating an exciting paradigm shift in the way we consider pseudopolyps, which may eventually alter the endoscopic management of these lesions in the future.

Mutations in the tumour suppressor gene Adenomatous polyposis coli (APC) are found in 80 % of sporadic colorectal cancer (CRC) tumours and are also responsible for the inherited form of CRC, Familial adenomatous polyposis (FAP). In order to identify novel therapeutic targets for the treatment of APC mutated CRC, we have generated an in vitro model of APC mutant CRC using CRISPR-cas9 gene editing. Using the APC wildtype colorectal carcinoma cell line RKO, we targeted the cells with guide RNA (gRNA) targeting exon 2 or exon 15 (encodes 80 % of APC) of the APC gene. We generated isogenic cell lines which differed in the expression of APC, the controls were APC wildtype and the APC mutant (APC Lys736fs) cell lines expressed a truncated ~80 kDa APC protein. We used these cell lines to perform an siRNA screen against 720 kinases and kinase-related genes. We selected seven genes to investigate further, unfortunately none of the potential hits validated. Additionally, we performed an FDA-approved compound screen targeting over 1000 compounds. From this, we identified a group of HMG-CoA reductase (HMGCR) inhibitors known as statins, which selectively cause a greater loss in cell viability in the APC mutated cell lines, compared to the APC wildtype cells. Mechanistically, our data suggests this synthetic lethal relationship is due to a greater decrease in the anti-apoptotic protein survivin. We propose this is due to statins altering the localisation of Rac1, reducing Pak1 activation and reducing the level of Wnt signalling. This results in the reduction of the Wnt target gene survivin. We have successfully identified an FDA-approved family of compounds, which show synthetic lethality with the APC mutation in our in vitro model.

With 50,310 related deaths this year, colorectal cancer (CRC) has emerged as the second largest cause of cancer related deaths among Americans. While 70 million Americans are considered at-risk of developing CRC, it is highly curable if detected early. Cohesin proteins, which hold sister chromatids together during replication, have emerged as a potential biomarker in multiple cancer lines. Because of their probable role in DNA replication, DNA repair, chromatin nano-architecture, and gene expression, this paper assessed whether cohesion proteins could be used as a potential biomarker for colorectal cancer risk stratification. While cohesin protein mutations have been reported in different cancers and involved in chromosomal instability, its role in early cancer formation has yet to be observed. Using immunohistochemical and Quantitative Real Time PCR analysis, this thesis assessed the protein and RNA expression levels of cohesin proteins SA-1, NIPBL, and SMC3 from human biopsies at different stages and locations of colorectal cancer development. The results showed that SA-1, a structural cohesion subunit, was significantly (p<0.01) down regulated in cancerous compared to normal tissue. The SA-1 protein was also down regulated in the involved mucosa adjacent to CRC polyps. The cohesion loading protein, NIPBL, was also significantly (p<0.01) under expressed in cancerous versus normal tissue. The RNA expression analysis of rectal mucosa showed that SMC3 and SA-1 was over expressed two fold in patients harboring hyperplastic and adenomous polyps, giving evidence that cohesin proteins are differentially expressed throughout the field of carcinogenesis. Our results demonstrate for the first time that cohesion dysregulation is an early event in human colorectal cancer development and may serve as an important biomarker of field carcinogenesis.

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.