Background: Clinical and epidemiologic studies have investigated the effects of diet, physical activity, and inflammation on the risk of colorectal adenoma occurrence and recurrence. Inflammation has been proposed as a mechanism of action for the development of colorectal adenoma and cancer. Research indicates that fruit phenolic exposure may attenuate the inflammatory response and some data suggest that berries are effective in mitigating this process. Inflammatory cytokines such as interleukin-6 (IL-6), C-reactive protein (CRP), and tumor necrosis factor alpha (TNF-α) are of particular interest due to their role in adenoma development. Epidemiological investigations have studied the association between bioactive fruit phenolic compounds and colorectal neoplasia; however, epidemiological data for the association between consumption of berries, which contain high concentrations of these compounds, and colorectal adenoma recurrence are limited. In addition to a potential role of phenolics in reducing inflammation, physical activity has also been proposed as a mitigator of this process. Numerous studies have investigated the association between physical activity and colorectal neoplasia, yet data on sedentary behavior and colorectal adenoma recurrence are limited. This dissertation was designed to further elucidate the role of fruit phenolics and sedentary behavior on colorectal adenoma recurrence and to specifically highlight the potential role of black raspberries in mitigating the postprandial inflammatory response among overweight and obese individuals. Methods: Ten overweight or obese males (BMI>25 kg/m²), ages 55-72, participated in an open-label, randomized, 14-day, pilot crossover study. Subjects consumed a high- fat, high- calorie (HFHC) meal, with (Group 1) or without (Group 2) a 5 day regimen of 45 g of black raspberry powder in the form of a slurry. The study included a two-day washout period before Group 1 and Group 2 were crossed over. The two-day washout period was based on a pharmacokinetic study conducted with black raspberry powder (1); peak plasma concentrations of ellagic acid and anthocyanin metabolites peaked at 1 to 2 hours following consumption of 45 grams of black raspberry powder and by 12 hours, plasma concentrations of these metabolites were almost fully washed out, with plasma concentrations returning to near baseline levels. Blood samples were obtained prior to consumption of the HFHC breakfast and at 1, 2, 4, 8, and 12 hours afterwards, during two 14-hour clinic visits. The primary study outcomes were changes in areas under the curves (AUCs) of serum biomarkers of TNF-α, CRP, and IL-6. A secondary pooled analysis was conducted among participants from two randomized, double blind, placebo-controlled Phase III clinical trials to investigate the association between berry consumption and colorectal adenoma recurrence, and the association between sedentary behavior and colorectal adenoma recurrence. Analyses included 2,502 subjects who had completed the baseline Arizona Food Frequency Questionnaire to ascertain berry consumption history in the past year and 1,730 men and women who had completed the baseline Arizona Activity Frequency Questionnaire to ascertain sedentary behavior. All subjects had a follow-up colonoscopy during the trial. Logistic regression modeling was employed to estimate the effect of sedentary behavior or berry consumption on colorectal adenoma recurrence. Results: The mean AUC of serum IL-6 was significantly lower (p=0.03) with black raspberry (BRB) feeding (45.5±36.3 pg/mL; mean±SD), compared to high fat, high calorie meal alone (56.7±50.0 pg/mL). No statistically significant differences were observed in the mean AUC of serum TNF-α or CRP. In the pooled analysis, no significant associations were observed between berry consumption and adenoma recurrence in the pooled population or when stratified by sex. In the evaluation of association between sedentary behavior and adenoma recurrence, subjects in the second, third, and fourth quartiles of sedentary behavior experienced higher odds of adenoma recurrence; however, the difference was only statistically significant for the third quartile. Sex-stratified analyses revealed that in men, sedentary activity was statistically significantly associated with 45% higher odds of adenoma recurrence. Compared to the lowest quartile of sedentary activity, the ORs (95% CIs) for the second, third, and fourth quartiles among men were 1.31 (0.93, 1.84), 1.47 (1.04, 2.09), and 1.45 (1.02, 2.06) respectively (P trend=0.03). In contrast, no association with sedentary activity was observed in women. Conclusion: Polyphenol exposure in the form of a black raspberry slurry significantly decreased post-prandial IL-6 in a clinical trial among ten older overweight and obese men. These findings suggest short-term attenuation of an inflammatory maker may not translate to decreased adenoma recurrence, however, long term randomized clinical trials with black raspberries are needed to evaluate this further. However, in an epidemiological analysis, consumption of up to 1 cup per week of whole berries was not associated with lower odds for adenoma recurrence among a pooled population of participants in the Wheat Bran Fiber and Ursodeoxycholic Acid Phase III clinical trials. While the epidemiological results indicated that berry consumption are not associated with the development of early colorectal neoplasia, the effects on later stages of carcinogenesis are unknown. Higher levels of berry consumption may be required in order to reach a cancer inhibitory effect. Finally, results of the physical activity study suggest that sedentary behavior is associated with a higher risk of adenoma recurrence among men, providing evidence of detrimental effects of a sedentary lifestyle early in the carcinogenesis pathway. Efforts to further evaluate these findings in other cohorts or in an intervention trial should be considered.

Introduction: There is evidence to suggest that common genetic polymorphisms can modify the effect of environmental risk factors on colorectal neoplasia. Methods: Data on 1430 individuals were obtained from two chemoprevention trials, the Wheat Bran Fiber Trial (WBF) (1) and the Effects of Ursodeoxycholic Acid on Adenomatous Polyp Recurrence Trial (URSO) (2). Data were analyzed to test for gene-environment interactions between allelic variation in PPAR-γ (Pro12Ala, C1431T), body mass index (BMI) and waist circumference, and the biochemical biomarkers of metabolic syndrome. Simulated data were then used to determine if the sample size required to formally test the relationship between gene-exposure interactions could be reduced by using a genetically enriched study population. For this simulation aspect of the work, an established gene-drug interaction (i.e.: flavin monooxygenase 3 (FMO3) and sulindac) was used as a model system. Results: There was a borderline significant interaction between BMI and PPAR-γ for the Pro12Ala genotype (p(inter)=0.11) and significant interactions between BMI and the C1431T genotype (p(inter)=0.09). Results from the recursive partition model identified BMI (p = 0.007) and baseline fasting glucose levels (p =0.033) as significant predictors of colorectal adenoma recurrence for carriers of Ala12 and waist circumference as a significant predictor for the Pro12Pro12 carriers (p=0.002). Results from the simulated studies indicated that using genetically pre-screened and enriched populations can result in a 50% savings in the number of subjects required to test the candidate gene-drug interaction. Conclusions: These findings provide evidence that the effect of allelic variation in PPAR-γ on colorectal adenoma recurrence is modified by BMI and that component traits of metabolic syndrome differentially affect the risk of colorectal adenoma recurrence depending on genotype. In addition, using genotype as an inclusion criterion in future studies of adenoma recurrence will result in a smaller sample size required to test gene-environment interactions.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / O objetivo deste estudo, à avaliar a expressÃo das proteÃnas p53 e Ki-67 em adenomas colorretais, suas relaÃÃes com caracterÃsticas clinico-patolÃgicas e avaliar a relaÃÃo entre as duas proteÃnas. A amostra consistiu de 50 pÃlipos adenomatosos encontrados em pacientes submetidos a exames colonoscÃpicos. ApÃs a realizaÃÃo de polipectomia, os pÃlipos eram conservados em soluÃÃo tamponada de formalina a 10% e submetidos à rotina de preparo de cortes e lÃminas e coloraÃÃo pela hematoxilina-eosina para confirmaÃÃo da natureza adenomatosa. Realizou-se imunohistoquÃmica especÃfica para as proteÃnas p53 e Ki-67 pelo mÃtodo imunoenzimÃtico da streptoavidina-biotina-peroxidase para cada adenoma. A proteÃna p53 foi positiva em 18% dos adenomas e a proteÃna Ki-67, expresso como Ãndice (i.Ki-67), obteve mÃdia de 0,49. Houve diferenÃa estatisticamente significante na expressÃo de p53 (p=0,0003) e Ki-67(p=0,02) entre os adenomas com alto e baixo grau de displasia, sendo maior no primeiro grupo. Encontrou-se, ainda maior expressÃo da proteÃna Ki-67 nos adenomas retais em relaÃÃo aos de localizaÃÃo cÃlica (p= 0,02). NÃo houve relaÃÃo entre a expressÃo das duas proteÃnas, na amostra / Ki-67 (p=0,02) expression between adenomas with high and low grade dysplasia, greater in the first group. There was greater expression of Ki-67 protein in the rectal adenomas than colic adenomas (p=0,02). There was no relation between the expression of the two proteins in the sample.

BACKGROUND: Numerous epidemiologic studies have identified obesity as a vital risk factor for the development of colorectal cancer (CRC). More recently, obesity has been linked to the development of colorectal adenomatous polyps (adenomas), the precursor lesion of up to 80% of CRCs. The extent to which weight loss could reduce risk in obese patients is unclear. PROPOSED PROJECT: The proposed study is a randomized clinical trial that aims to evaluate the relationship between weight reduction and the prevalence of recurrent (metachronous) adenomas among obese patients in a safety-net health care setting. The intervention group will participate in a comprehensive, individually structured weight loss program in order to achieve successful long-term weight loss. The control group will receive no special recommendations about weight loss other than as part of “usual care”. Anthropometric measures (weight in kilograms [kg], height in meters squared [m2] and body mass index [BMI]) will be monitored annually until the time of surveillance colonoscopy which will occur in accordance with the U.S. Multi-Society Task Force recommendations. Statistical methods will be used to compare rates of recurrent adenomas among the two study groups after adjustments for duration of follow-up and potential confounders. CONCLUSION/SIGNIFICANCE: The results of this study will provide new evidence to support weight reduction as a preventive strategy for reducing CRC risk among obese patients.

Background: Many studies have reported that adherence to health promotion guidelines for diet, physical activity, and maintenance of healthy body weight may decrease cancer incidence and mortality, including site-specific cancers such as colorectal cancer. To date, there have been no studies investigating adherence to the American Cancer Society's (ACS) Nutrition and Physical Activity Cancer Prevention Guidelines and the development and characteristics of premalignant lesions. Several individual lifestyle factors targeted by the ACS guidelines have also been associated with circulating concentrations of vitamin D metabolites. These associations suggest that adherence to the ACS guidelines may be related to improved vitamin D status. This dissertation sought to 1) synthesize the evidence from published prospective cohort studies regarding adherence to the ACS and World Cancer Research Fund/American Institute for Cancer Research (WCRF/AICR) nutrition and physical activity cancer prevention guidelines and the risk of overall cancer incidence and/or cancer mortality and 2) to further explore the role of adhering to a healthy lifestyle pattern as outlined by the ACS guidelines on a) colorectal adenoma occurrence and b) circulating concentrations of vitamin D metabolites using secondary data analyses from completed large prevention trials. Methods: A systematic review was performed to examine associations between adherence to established cancer prevention guidelines for diet and physical activity and overall cancer incidence and mortality. PubMed, Google Scholar, and Cochrane Reviews databases were searched following the current recommendations of Preferred Reporting Items for Systematic Reviews and Meta-analysis Approach (PRISMA). Cross-sectional and prospective analyses of pooled participants were also conducted from the Wheat Bran Fiber (n=503) and Ursodeoxycholic Acid (n=854) trials. A cumulative adherence score was constructed using baseline data regarding body size, diet, physical activity, and alcohol consumption. Continuous vitamin D metabolite concentrations and clinically significant vitamin D categories were evaluated with adherence score category using multiple linear and logistic regression models, respectively. Baseline adenoma characteristics and new colorectal adenomas were evaluated by adherence score category using multivariate logistic regression models. Results: Twelve studies met inclusion criteria for the systematic review. High versus low adherence to established nutrition and physical activity cancer prevention guidelines was consistently and significantly associated with decreases of 10-61% in overall cancer incidence and mortality. Consistent significant reductions were also shown for breast cancer incidence (19-60%), endometrial cancer incidence (23-60%), and colorectal cancer incidence in both men and women (27-52%). Findings for lung cancer incidence were equivocal and no significant relationships were found between adherence and ovarian or prostate cancer. In the pooled analyses, concentrations of circulating 25-hydroxycholecalciferol [25(OH)D] were statistically significantly higher among participants with high versus low adherence to guidelines (31.4 ± 0.8 and 26.3 ± 0.8 ng/ml, respectively; p<0.001). For 1,25(OH)₂D concentrations, high adherence was again significantly related to greater metabolite levels, with mean concentrations of 36.4 ± 1.1 and 31.2 ± 1.2 pg/mL for high- and low-adherers, respectively (p<0.001). Furthermore, the odds of attaining sufficient 25(OH)D status were 4.30 times higher for those most adherent versus those least adherent (95% CI: 2.43-7.60). Significantly reduced odds of having three or more adenomas at baseline were shown for moderate (odds ratio [OR]=0.67, 95% confidence intervals [CI]: 0.46-0.99) and highly adherent (OR=0.50, 95% CI: 0.31-0.81) participants compared to those with low adherence (p-trend=0.005). Conversely, guideline adherence was not associated with the development of a new colorectal adenoma (moderate adherence OR=1.16, 95% CI: 0.85-1.59, high adherence OR=1.23, 95% CI: 0.85-1.79). Conclusion: From the systematic review, greater adherence to cancer prevention guidelines for diet and physical activity was consistently associated with lower risks of overall cancer incidence and mortality, including for some site-specific cancers. In addition, adherence to the ACS guidelines was associated with higher concentrations of both of 25(OH)D and 1,25(OH)₂D. Following the ACS guidelines could potentially increase 25(OH)D levels as much as that observed by a supplement of 1000 IU/d in a population similar to ours, and therefore may be a viable strategy for increasing both 25(OH)D and 1,25(OH)₂D concentrations. Further, our findings suggest that following the ACS Nutrition and Physical Activity guidelines may lead to a lower odds of multiple adenomas when at least one adenoma is detected. Finally, these guidelines and recommendations are consistent with strategies for the prevention of major diseases, and if followed, will ultimately lead to healthier lives overall.

O câncer colorretal está entre as principais neoplasias malignas sendo a quarta causa de morte por câncer no mundo e a terceira no Brasil. Mutações nos genes APC, DCC, K-RAS e TP53 foram originalmente associadas com a progressão do câncer colorretal (CCR) esporádico. Entretanto, estudos de genoma completo e exoma têm revelado outros genes relacionados com o CCR. Como consequência dessas mutações, um conjunto de genes alteram sua expressão modulando vias gênicas em cada estágio da progressão tumoral. Nesse sentido, há um grande esforço para definir assinaturas gênicas que auxiliem na classificação dos tumores quanto ao diagnóstico e prognóstico dos pacientes. Portanto, o objetivo deste projeto foi analisar a expressão gênica em escala genômica de amostras de adenoma e adenocarcinoma colorretal visando identificar novos marcadores genéticos ligados a transição adenoma-adenocarcinoma. Para isso, dez amostras pareadas de adenoma e adenocarcinoma do mesmo paciente foram submetidas à análise de expressão gênica pela técnica de microarranjos. Análises de bioinformática, revelaram uma assinatura de 689 genes diferencialmente expressos (fold-change>2, p<0.05), que permitiram a classificação genética entre o adenoma e o adenocarcinoma. Oitos genes (IL6, IL8, OSM, SFRP4, ETV4, ESM1, SIM2 e RETNLB) foram escolhidos para validação com base na sua função e valor de expressão no tecido tumoral. A análise in silico dos genes hiperexpressos realizada no programa MetaCore (análise de dados e vias gênicas) destacou diversas vias gênicas ligadas à tumorigênese, incluindo as de adesão celular e Transição Epitélio-Mesenquimal (TEM), importantes na fase avaçada da progressão tumoral. O gene ETV4 foi selecionado para realização dos ensaios funcionais em virtude de seus altos níveis de expressão nas dez amostras de adenocarcinoma e participação nos mecanismos de proliferação celular e no TEM. Ensaios in vitro de siRNA para o gene ETV4 resultou na diminuição da proliferação celular e no potencial clonogênico da linhagem HT29. Adicionalmente, foram investigadas mutações nos genes APC, K-RAS e TP53, nas amostras pareadas de adenoma, adenocarcinoma, tecido normal e sangue periférico dos dez pacientes. Todos os pacientes apresentaram mutação germinativas nos três genes. No entanto, apenas os genes K-RAS (40%) e TP53 (30%) apresentaram mutações somáticas e patogênicas, exclusivamente nos adenocarcinomas. Esses resultados demonstraram que, na nossa coorte, mutações nos genes TP53 e K-RAS podem estar contribuindo para a progressão em uma parcela do câncer colorretal do tipo esporádico. Em resumo, o presente estudo aponta que o gene ETV4 pode contribuir para ativar o mecanismo de proliferação celular em adenocarcinoma colorretal. Além disso, o estudo demonstra a importância da combinação da análise de mutação com o perfil de expressão para melhor compreensão da base molecular do câncer colorretal. / Colorectal cancer is among the main malignant neoplasia, it is the fourth leading cause of death in the world and the third in Brazil. Mutations in APC, DCC, KRAS and TP53 genes have been originally associated with the progression of sporadic colorectal cancer (CRC). However genome wide and exome studies have revealed other genes related to CRC. As a consequence of these mutations, a set of genes alters their expression modulating gene pathways in every stage of tumor progression. In this regard, there is great effort to define gene signatures that help to classify tumors in relation to patients diagnosis and prognosis. Therefore, the objective of this project was to analyze gene expression in genomic scale of colorectal adenoma and adenocarcinoma samples aiming to identify new genetic markers linked to adenoma- adenocarcinoma transition. For this purpose, ten paired adenoma and adenocarcinoma samples of the same patient were subjected to gene expression analysis by microarrays technique. Bioinformatics analyses revealed a signature of 689 genes differentially expressed (fold-change>2, p<0.05), which allowed the genetic classification between adenoma and adenocarcinoma. Eight genes (IL6, IL8, OSM, SFRP4, ETV4, ESM1, SIM2 and RETNLB) were chosen for validation based on their function and expression value in tumor tissue. In silico analysis of hyperexpressed genes, done in the program MetaCore (data analysis and gene pathways), highlighted diverse gene pathways linked to tumorigenesis, including the ones of cell adhesion and Epithelial-Mesenchymal Transition (EMT), important in the advanced phase of tumor progression. ETV4 gene was selected for functional assays due to its high expression levels in the ten samples of adenocarcinoma and due to its participation in cell proliferation mechanisms and in EMT. In vitro siRNA assays for ETV4 gene resulted in the decrease of cell proliferation and in the clonogenic potential of HT29 line. In addition, mutations in APC, KRAS and TP53 genes were investigated in paired samples of adenoma, adenocarcinoma, normal tissue, and peripheral blood from ten patients. All patients showed germline mutations in the three genes. However, only KRAS (40%) and TP53 (30%) genes showed somatic and pathogenic mutations, exclusively in adenocarcinomas. These results demonstrated that, in our cohort, mutations in TP53 and KRAS genes might be contributing to progression in a portion of sporadic-type colorectal cancer. In summary, the present study points out that ETV4 gene might contribute to activate cell proliferation mechanism in colorectal adenocarcinoma. Moreover, the study demonstrates the importance of combining the mutation analysis with expression profile in order to better understanding the molecular basis of colorectal cancer.

A mitocôndria é a organela citoplasmática responsável pelo maior sistema produtor de energia, a fosforilação oxidativa (OXPHOS). Foi proposto que em células tumorais a hiper-regulação da glicólise em condições normais de oxigênio (Efeito Warburg), está associada a defeitos na OXPHOS e pode regular o fenótipo tumoral, por exemplo, o potencial metastático da célula por meio da indução de vias pseudohipóxicas durante a normóxia. Estudos recentes mostraram que vários tipos de tumores possuem mutações somáticas em seu genoma mitocondrial, o que pode alterar as funções da OXPHOS levando a troca de metabolismo energético nas células tumorais e induzindo a tumorigênese. Diante disto, o presente trabalho avaliou a instabilidade do genoma mitocondrial em etapas bem definidas da progressão do câncer colorretal. O DNA genômico foi extraído de amostras de adenoma, adenocarcinoma, tecido adjacente e sangue periférico de nove pacientes diagnosticados com Câncer colorretal. O genoma mitocondrial foi amplificado e sequenciado para que fossem feitas as buscas por mutações nas amostras de sangue periférico, adenomas e adenocarcinoma. Foi também medido o número de cópias relativas do mtDNA. Foram encontradas um total de 233 mutações, das quais 162 foram em comum entre os três tecidos avaliados. As amostras de adenocarcinoma foram as que apresentaram uma maior média de mutações por amostra (44,6), seguidas dos adenoma (40,2) e do sangue periférico (34). As amostras de adenocarcinoma apresentaram uma maior instabilidade do mtDNA refletidas a partir de um maior número de mutações somáticas (tanto do tipo InDel como mutações de uma única base), mutações não sinônimas com maior patogenicidade, maior número de mutações em heteroplasmia e com taxa de heteroplasmia elevada. Já as amostras de adenoma apresentaram instabilidade dos seus mtDNA intermediários entre o tecido não tumoral e tumoral, refletindo bem a etapa de modificação celular no qual esses tecidos se encontram. Na análise do número de cópias relativas, as amostras de adenocarcinoma tiveram diminuição no número de cópias relativas quando comparadas com tecido adjacente (p= 0,01) e com adenomas (p= 0,04). Em síntese, o presente trabalho sugere que a instabilidade do genoma mitocondrial parece ter um papel importante no desenvolvimento de tumores colorretais. / The mitochondrion is a cytoplasmic organelle responsible for the major energy producing system, which is the oxidative phosphorylation enzyme pathway (OXPHOS). It was proposed that glycolysis up-regulation during normal oxygen conditions (Warburg effect) may induce defects in the mitochondrial respiration and regulate tumoral phenotypes, for example, metastatic potential through the induction of pseudohipoxic pathways during normoxia. Recent studies have shown that many kinds of tumors have mtDNA somatic mutations, which could alter the OXPHOS functions, leading to changes in glucose metabolismo and improvind tumorigenesis. This study analyzed the mitochondrial genome instability of well defined stages of colorectal cancer. Genomic DNA was extracted from adenoma, adenocarcinoma, adjacente tissue and peripheral blood of patients diagnosed with Colorectal cancer. The mitochondrial genome was amplified and sequenced for mutations screening in adenoma, adenocarcinoma e blood samples. It was also analyzed the relative mtDNA copy number. It was find a total of 233 mutations, which 162 were in common among the three analyzed tissues. The adenocarcinoma samples presented a greater mutation mean per sample (44.6) followed by adenomas samples (40.2) and blood samples (34). The adenocarcinoma samples also shown a greater mitochondrial genome instability refleted by increased of somatic mutations (InDels and single nucleotide variation), non sinonimous mutations with higher patogenicity, increased number of heteroplasmatic mutations and higher heteroplasmatic levels. The adenoma samples showed intermadiate instability of its mtDNA, which well reflects the intermediate stage of cellular modifications of this tissue. The mtDN copy number analysis shown that the adenocarcinoma samples presented decreased number of mtDNA content when compared with adjacente tissue (p= 0.01) and adenoma samples (p= 0.04). In summary the presente study suggests that the mitochondrial genomic instability seems to play an importante role in colorectal tumorigenesis.

Putrescine, spermidine and spermine are the polyamines biosynthesized by human cells via ornithine decarboxylase (ODC) and are also sourced from the diet. Polyamines are required for malignant and normal cell growth and development. Pharmacological suppression of polyamine biosynthesis, by difluoromethylornithine, and inflammation, via sulindac, has demonstrated ~70% efficacy in preventing premalignant colorectal adenomas (CRA) in a clinical trial; however, high polyamine intakes mitigated this preventative action. Further, dietary polyamines increase the dysplasia of CRA in initiated animal models of colorectal cancer (CRC) and are hypothesized to function as tumor promoters. Human research on dietary polyamines was limited until the development of a dietary database in 2007 but, continues to be limited by the lack of a biomarker of exposure. Chapter 1 of this dissertation tests the hypothesis that dietary polyamines increase risk of CRA in polyp-formers (n = 1164) and found evidence to support this hypothesis. However, only women, younger participants and certain genotypes experienced more risk of CRA with high polyamine exposure. Chapter II tests the hypothesis that dietary polyamines increase the risk for CRC in an average risk cohort of post-menopausal women (n = 87,620) and did not find evidence to support this hypothesis in the whole population. Rather, dietary polyamines were non-significantly protective against CRC and significantly protective when paired with aspirin use and against CRC-specific death. There was some evidence to support an increase in risk of CRC in younger participants with high polyamine exposure. Overall, the first two chapters suggest that dietary polyamines protect the colorectum in normal risk individuals but promote carcinogenesis in high risk individuals. Chapter III tests the hypothesis that dietary polyamine intake correlates with urinary polyamine output in a group of overweight/obese, older men (n = 36) and Chapter IV tests the hypothesis that intake of highly ripe sweet cherries will increase urinary polyamine output in a subgroup of 10 men from Chapter III. The findings from these chapters suggest there may be a positive correlation, but that a better measure of dietary polyamine intake is needed to determine if urinary polyamines are biomarkers of exposure to polyamines.

A mitocôndria é a organela citoplasmática responsável pelo maior sistema produtor de energia, a fosforilação oxidativa (OXPHOS). Foi proposto que em células tumorais a hiper-regulação da glicólise em condições normais de oxigênio (Efeito Warburg), está associada a defeitos na OXPHOS e pode regular o fenótipo tumoral, por exemplo, o potencial metastático da célula por meio da indução de vias pseudohipóxicas durante a normóxia. Estudos recentes mostraram que vários tipos de tumores possuem mutações somáticas em seu genoma mitocondrial, o que pode alterar as funções da OXPHOS levando a troca de metabolismo energético nas células tumorais e induzindo a tumorigênese. Diante disto, o presente trabalho avaliou a instabilidade do genoma mitocondrial em etapas bem definidas da progressão do câncer colorretal. O DNA genômico foi extraído de amostras de adenoma, adenocarcinoma, tecido adjacente e sangue periférico de nove pacientes diagnosticados com Câncer colorretal. O genoma mitocondrial foi amplificado e sequenciado para que fossem feitas as buscas por mutações nas amostras de sangue periférico, adenomas e adenocarcinoma. Foi também medido o número de cópias relativas do mtDNA. Foram encontradas um total de 233 mutações, das quais 162 foram em comum entre os três tecidos avaliados. As amostras de adenocarcinoma foram as que apresentaram uma maior média de mutações por amostra (44,6), seguidas dos adenoma (40,2) e do sangue periférico (34). As amostras de adenocarcinoma apresentaram uma maior instabilidade do mtDNA refletidas a partir de um maior número de mutações somáticas (tanto do tipo InDel como mutações de uma única base), mutações não sinônimas com maior patogenicidade, maior número de mutações em heteroplasmia e com taxa de heteroplasmia elevada. Já as amostras de adenoma apresentaram instabilidade dos seus mtDNA intermediários entre o tecido não tumoral e tumoral, refletindo bem a etapa de modificação celular no qual esses tecidos se encontram. Na análise do número de cópias relativas, as amostras de adenocarcinoma tiveram diminuição no número de cópias relativas quando comparadas com tecido adjacente (p= 0,01) e com adenomas (p= 0,04). Em síntese, o presente trabalho sugere que a instabilidade do genoma mitocondrial parece ter um papel importante no desenvolvimento de tumores colorretais. / The mitochondrion is a cytoplasmic organelle responsible for the major energy producing system, which is the oxidative phosphorylation enzyme pathway (OXPHOS). It was proposed that glycolysis up-regulation during normal oxygen conditions (Warburg effect) may induce defects in the mitochondrial respiration and regulate tumoral phenotypes, for example, metastatic potential through the induction of pseudohipoxic pathways during normoxia. Recent studies have shown that many kinds of tumors have mtDNA somatic mutations, which could alter the OXPHOS functions, leading to changes in glucose metabolismo and improvind tumorigenesis. This study analyzed the mitochondrial genome instability of well defined stages of colorectal cancer. Genomic DNA was extracted from adenoma, adenocarcinoma, adjacente tissue and peripheral blood of patients diagnosed with Colorectal cancer. The mitochondrial genome was amplified and sequenced for mutations screening in adenoma, adenocarcinoma e blood samples. It was also analyzed the relative mtDNA copy number. It was find a total of 233 mutations, which 162 were in common among the three analyzed tissues. The adenocarcinoma samples presented a greater mutation mean per sample (44.6) followed by adenomas samples (40.2) and blood samples (34). The adenocarcinoma samples also shown a greater mitochondrial genome instability refleted by increased of somatic mutations (InDels and single nucleotide variation), non sinonimous mutations with higher patogenicity, increased number of heteroplasmatic mutations and higher heteroplasmatic levels. The adenoma samples showed intermadiate instability of its mtDNA, which well reflects the intermediate stage of cellular modifications of this tissue. The mtDN copy number analysis shown that the adenocarcinoma samples presented decreased number of mtDNA content when compared with adjacente tissue (p= 0.01) and adenoma samples (p= 0.04). In summary the presente study suggests that the mitochondrial genomic instability seems to play an importante role in colorectal tumorigenesis.

O câncer colorretal está entre as principais neoplasias malignas sendo a quarta causa de morte por câncer no mundo e a terceira no Brasil. Mutações nos genes APC, DCC, K-RAS e TP53 foram originalmente associadas com a progressão do câncer colorretal (CCR) esporádico. Entretanto, estudos de genoma completo e exoma têm revelado outros genes relacionados com o CCR. Como consequência dessas mutações, um conjunto de genes alteram sua expressão modulando vias gênicas em cada estágio da progressão tumoral. Nesse sentido, há um grande esforço para definir assinaturas gênicas que auxiliem na classificação dos tumores quanto ao diagnóstico e prognóstico dos pacientes. Portanto, o objetivo deste projeto foi analisar a expressão gênica em escala genômica de amostras de adenoma e adenocarcinoma colorretal visando identificar novos marcadores genéticos ligados a transição adenoma-adenocarcinoma. Para isso, dez amostras pareadas de adenoma e adenocarcinoma do mesmo paciente foram submetidas à análise de expressão gênica pela técnica de microarranjos. Análises de bioinformática, revelaram uma assinatura de 689 genes diferencialmente expressos (fold-change>2, p<0.05), que permitiram a classificação genética entre o adenoma e o adenocarcinoma. Oitos genes (IL6, IL8, OSM, SFRP4, ETV4, ESM1, SIM2 e RETNLB) foram escolhidos para validação com base na sua função e valor de expressão no tecido tumoral. A análise in silico dos genes hiperexpressos realizada no programa MetaCore (análise de dados e vias gênicas) destacou diversas vias gênicas ligadas à tumorigênese, incluindo as de adesão celular e Transição Epitélio-Mesenquimal (TEM), importantes na fase avaçada da progressão tumoral. O gene ETV4 foi selecionado para realização dos ensaios funcionais em virtude de seus altos níveis de expressão nas dez amostras de adenocarcinoma e participação nos mecanismos de proliferação celular e no TEM. Ensaios in vitro de siRNA para o gene ETV4 resultou na diminuição da proliferação celular e no potencial clonogênico da linhagem HT29. Adicionalmente, foram investigadas mutações nos genes APC, K-RAS e TP53, nas amostras pareadas de adenoma, adenocarcinoma, tecido normal e sangue periférico dos dez pacientes. Todos os pacientes apresentaram mutação germinativas nos três genes. No entanto, apenas os genes K-RAS (40%) e TP53 (30%) apresentaram mutações somáticas e patogênicas, exclusivamente nos adenocarcinomas. Esses resultados demonstraram que, na nossa coorte, mutações nos genes TP53 e K-RAS podem estar contribuindo para a progressão em uma parcela do câncer colorretal do tipo esporádico. Em resumo, o presente estudo aponta que o gene ETV4 pode contribuir para ativar o mecanismo de proliferação celular em adenocarcinoma colorretal. Além disso, o estudo demonstra a importância da combinação da análise de mutação com o perfil de expressão para melhor compreensão da base molecular do câncer colorretal. / Colorectal cancer is among the main malignant neoplasia, it is the fourth leading cause of death in the world and the third in Brazil. Mutations in APC, DCC, KRAS and TP53 genes have been originally associated with the progression of sporadic colorectal cancer (CRC). However genome wide and exome studies have revealed other genes related to CRC. As a consequence of these mutations, a set of genes alters their expression modulating gene pathways in every stage of tumor progression. In this regard, there is great effort to define gene signatures that help to classify tumors in relation to patients diagnosis and prognosis. Therefore, the objective of this project was to analyze gene expression in genomic scale of colorectal adenoma and adenocarcinoma samples aiming to identify new genetic markers linked to adenoma- adenocarcinoma transition. For this purpose, ten paired adenoma and adenocarcinoma samples of the same patient were subjected to gene expression analysis by microarrays technique. Bioinformatics analyses revealed a signature of 689 genes differentially expressed (fold-change>2, p<0.05), which allowed the genetic classification between adenoma and adenocarcinoma. Eight genes (IL6, IL8, OSM, SFRP4, ETV4, ESM1, SIM2 and RETNLB) were chosen for validation based on their function and expression value in tumor tissue. In silico analysis of hyperexpressed genes, done in the program MetaCore (data analysis and gene pathways), highlighted diverse gene pathways linked to tumorigenesis, including the ones of cell adhesion and Epithelial-Mesenchymal Transition (EMT), important in the advanced phase of tumor progression. ETV4 gene was selected for functional assays due to its high expression levels in the ten samples of adenocarcinoma and due to its participation in cell proliferation mechanisms and in EMT. In vitro siRNA assays for ETV4 gene resulted in the decrease of cell proliferation and in the clonogenic potential of HT29 line. In addition, mutations in APC, KRAS and TP53 genes were investigated in paired samples of adenoma, adenocarcinoma, normal tissue, and peripheral blood from ten patients. All patients showed germline mutations in the three genes. However, only KRAS (40%) and TP53 (30%) genes showed somatic and pathogenic mutations, exclusively in adenocarcinomas. These results demonstrated that, in our cohort, mutations in TP53 and KRAS genes might be contributing to progression in a portion of sporadic-type colorectal cancer. In summary, the present study points out that ETV4 gene might contribute to activate cell proliferation mechanism in colorectal adenocarcinoma. Moreover, the study demonstrates the importance of combining the mutation analysis with expression profile in order to better understanding the molecular basis of colorectal cancer.