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O câncer colorretal (CCR) é o terceiro tipo de câncer mais comum no mundo. Apesar dos avanços nos tratamentos convencionais, aproximadamente dois terços dos pacientes com CCR são submetidos à cirurgia potencialmente curativa. Entretanto, grande parte desses pacientes evolui mal, apresentando recidivas e/ou metástases. A busca de novos alvos moleculares para a terapia do CCR revelou a proteína celular Prion (PrPC) como um possível candidato. Trabalhos recentes sugerem participação direta ou indireta de PrPC no crescimento de tumores, na formação de metástases, na composição de complexos multiproteicos e na indução de vias de sinalização envolvidas em diversos processos biológicos, como proliferação. Além disso, PrPC foi descrito como um importante modulador do crescimento de tumor colorretal. Resultados prévios mostraram que a interação da proteína PrPC com a proteína HSP70/HSP90 Organizing Protein (HOP) induz proliferação em glioblastomas. HOP é uma proteína predominantemente citoplasmática, podendo também ser secretada associada às vesículas extracelulares. Assim, o presente estudo objetivou avaliar o papel do complexo PrPC-HOP e das vesículas extracelulares no desenvolvimento e progressão dos tumores colorretais. Os nossos resultados mostram que HOP induziu migração e invasão em linhagens de CCR de maneira dependente de PrPC, uma vez que o uso do peptídeo sem atividade que compete pelo sítio ligação de HOP a PrPC inibiu estes processos. Além disso, nossos dados apontaram que o aumento de migração e invasão das células de CCR induzida pela interação PrPC-HOP é mediada pela ativação da via ERK1/2. Os achados in vitro estimularam a avaliação do perfil de expressão de PrPC e HOP por imuno-histoquímica em tecidos de pacientes com diferentes tipos de tumores colorretais. Nossos resultados sugeriram que essas proteínas são importantes no início do desenvolvimento tumoral e na transição de adenomas para adenocarcinomas, não havendo correlação entre a presença de HOP e/ou PrPC com metástase, linfonodos acometidos, estadiamento, sobrevida ou região tumoral versus tecido normal. Em relação ao papel das vesículas extracelulares na progressão dos tumores colorretais, nossos resultados mostraram que linhagens celulares que apresentam padrões parecidos de agressividade tumoral podem ter perfis de secreção de proteínas e vesículas extracelulares bastante diferentes, induzindo, portanto, processos biológicos com intensidades distintas. O meio condicionado e as vesículas extracelulares da linhagem WiDr apresentaram maior potencial de indução de migração quando comparado com a linhagem HCT8. Além disso, a modulação negativa da proteína VPS4, uma das responsáveis pela formação dos corpos multivesiculares, mostrou-se uma abordagem interessante no estudo da secreção de vesículas por células de CCR, uma vez que o dominante negativo de VPS4 promoveu diminuição do cargo proteico e da secreção de vesículas extracelulares, redução da proliferação celular e do efeito indutor do processo de migração na linhagem WiDr. Assim, em conjunto, o presente trabalho indicou que o complexo PrPC-HOP pode ser um bom alvo terapêutico nos processos de migração e invasão em CCR. Ainda, essas proteínas se mostraram importantes nos estágios iniciais da formação dos tumores. A modulação da secreção de vesículas extracelulares pode contribuir para retardar a progressão dos tumores colorretais. / Colorectal cancer (CRC) is the third most common type of cancer in the world. Despite improvements in conventional treatments, approximately two-thirds of CRC patients undergo potentially curative surgery. However, most of these patients evolve poorly, showing recurrence and/or metastasis. Search of new molecular targets for CRC therapy revealed the cellular protein Prion (PrPC) as a putative candidate. Recent studies have shown that PrPC exhibit direct or indirect participation in tumor growth, formation of metastasis, composition of multiprotein complexes and induction of signaling pathways involved in many biological processes such as proliferation. Moreover, PrPC has been described as an important modulator of colorectal tumor growth. Previous findings showed that the interaction between PrPC and its ligand HSP70/90 heat shock organizing protein (HOP) induces gliobastoma proliferation. It is well known that HOP localizes mainly in the cytoplasm but HOP is also secreted associated with extracellular vesicles. In this way, the present study sought to evaluate the role of PrPC-HOP complex and extracellular vesicles in the development and progression of CRC. We demonstrate that HOP induces the migration and invasion of CRC cell lines in a PrPC-dependent manner because the use of HOP peptide, which is able to bind to PrPC, blocking PrPC-HOP complex formation, inhibited the migration and invasion processes. In addition, our data showed that the enhancement of migration and invasion induced by PrPC-HOP interaction is mediated by ERK1/2 pathway activation. These in vitro results lead us to evaluate the PrPC and HOP expression by immunohistochemistry in tissues from patients with different tumor types. Our data showed that these proteins could be important for the initial steps of tumor development, represented by the transition from adenoma to adenocarcinoma. No correlation was found among HOP and/or PrPC expression and metastasis, lymph node involvement, staging, survival or tumor area versus normal tissue. Regarding the role of extracellular vesicles in the progression of colorectal tumors, our results showed that cell lines exhibiting similar aggressive tumor behavior can have a different protein secretion pattern and a distinct profile of extracellular vesicles release, which could induce biological process with different intensities. The conditioned medium and the extracellular vesicles derived from WiDr cell line showed a higher potential to induce migration than HCT8 cell line. Moreover, the negative modulation of VPS4, one of the proteins responsible for multivesicular body formation, showed to be an interesting approach in the study of extracellular vesicles secretion secreted by CRC cells; the negative dominant of VPS4 promoted in the WiDr cell line a reduction in the protein cargo and secretion of the extracellular vesicles, a decrease of cell proliferation and induction of migration process. Therefore, taken together, our data highlights that PrPC-HOP complex can be considered a new therapeutic target in migration and invasion processes of CRC. Moreover, these proteins appeared to be important at onset of tumor formation. The modulation of extracellular vesicles secretion may contribute for delaying the progression of colorectal tumors.

En France, en 2012, les sujets âgés d’au moins 65 ans représentaient 71 % de l’incidence du Cancer ColoRectal (CCR). Depuis 2005, des thérapies ciblées ont été autorisées dans le CCR métastatique (CCRm) et sont recommandées en 1ère ligne en association à une chimiothérapie. Face à un manque d’évaluation de ces médicaments chez le sujet âgé, l’objectif de ce travail était l’étude, en situation réelle de soins, de l’utilisation, des bénéfices et de la sécurité d'emploi des médicaments anticancéreux, dont les thérapies ciblées, chez le sujet âgé et/ou fragile atteint de CCRm. Une revue systématique de la littérature a confirmé la faible inclusion des sujets âgés et fragiles dans les essais cliniques évaluant les thérapies ciblées dans le CCRm. D’après une étude sur des données mondiales de pharmacovigilance et la réunion de deux cohortes de terrain de patients traités par thérapies ciblées, nous avons montré que leur effectivité et sécurité d’emploi chez le sujet âgé étaient équivalentes à celles du sujet plus jeune. Dans la cohorte, la fragilité vis-à-vis des effets indésirables graves et du décès était liée aux caractéristiques du CCRm. Enfin, d’après une étude de terrain pilote et une cohorte dans les données de l’Assurance Maladie française, nous avons montré que, chez le sujet âgé, le traitement par médicaments anticancéreux, dont les thérapies ciblées, n’était pas optimal et encore très lié à l’âge des patients. Le rapport bénéfices/risques des thérapies ciblées semble donc positif dans la population âgée atteinte de CCRm actuellement traitée. Cependant, la population âgée traitée ne semble pas encore totalement correspondre à celle qui pourrait bénéficier du traitement. / In France, in 2012, patients aged at least 65 years accounted for 71% of the incidence of ColoRectal Cancer (CCR). Since 2005, targeted therapies have been authorized in metastatic CRC (mCRC) and are recommended in first-line in combination with conventional chemotherapy. Given this lack of evaluation of these drugs in the elderly, the objective of this work was the study, in real-life setting, of the use, the benefits and the safety of anticancer drugs, including targeted therapies in elderly and/or frail mCRC patients. A systematic review of the literature confirmed the low inclusion of elderly and frail patients in clinical trials evaluating targeted therapies in the mCRC. According to a study on international pharmacovigilance data and the pooling of two field cohorts of patients treated by targeted therapies, we showed that their effectiveness and safety in the elderly were equivalent to those of the younger. In the cohort, frailty regarding serious adverse events and death was related to the characteristics of the mCRC. Finally, according to a pilot field study and a cohort in the French health insurance data, we have shown that, in the elderly, the treatment with anticancer drugs, including targeted therapies, was not optimal and still very related to the age of the patients. The benefit/risk ratio of targeted therapies therefore seems positive in the elderly population with mCRC currently treated. However, the elderly population treated does not seem to be fully matching with those who would benefit from treatment.

MACC1, ein Hauptregulator von Metastasen, ist an zahlreichen Kennzeichen von Krebs beteiligt, einschließlich dereguliertem Metabolismus. Dennoch ist seine Rolle im Krebsstoffwechsel unklar. In der vorliegenden Arbeit wurde eine systematische Analyse von MACC1-getriebenen metabolischen Netzwerken durchgeführt. MACC1 erhöhte die GLUT1 auf Zellmembrane, was zu einer erhöhten Glukoseanreicherung, einem erhöhten Glukosefluss und somit zu einer erhöhten Zellproliferation führte. Außerdem, reduzierte MACC1 den Glutaminfluss unabhängig von der Nährstoffverfügbarkeit. Bei Glucoseentzug erhöhte MACC1 die Pyruvataufnahme und zeigte aber die geringe Auswirkungen auf den Pyruvatfluss. In vivo, MACC1 zeigte erhöhte Aufnahme von 18F-FDG und 18F-Glutamat in Lebermetastasen. Zusammengefasst zeigen diese Ergebnisse, dass MACC1 mehrere Wirkungen auf den Krebs-Metabolismus zeigt, was es attraktiv macht, seine Wirkungen in Krebsmodellen weiter zu untersuchen. Metastasierung ist die Haupttodesursache bei Darmkrebs. Fünfzehn bis zwanzig Prozent der Darmkrebs Patienten im Stadium II entwickeln im Verlauf der Erkrankung Metastasen, jedoch bleiben die Kriterien der Wahrscheinlichkeit mit welcher Patienten von Chemotherapie profitieren werden ungenau. Hier wurde das Potenzial der Plasma-Metabolomik zur Vorhersage von Metachronmetastasen untersucht. Plasma metabolische Profile wurden wesentlich unterschiedlich zwischen nicht-metastasierten und metachron metastasierten Patienten gefunden. Wie Klassifikationsmodelle aus Entscheidungsbäumen und Support-Vektor-Maschinen gezeigt haben die Plasmametaboliten haben die Fähigkeit nicht-metastasierte von metachron metastasierten Darmkrebs Patienten zu unterscheiden, mit einer durschnittlichen Vorhersagegenauigkeit von 0,75 bzw. 0,82 für jede der Methoden, angemessen. Zusammen, zeigen diese Ergebnisse, dass Plasmametaboliten das Potenzial haben, Darmkrebs Patienten gemäß ihrem Metastasierungsrisiko nichtinvasiv zu stratifizieren. / MACC1, a master regulator of metastasis, is involved in most hallmarks of cancer, including deregulated metabolism. Yet, fragmentary data on its role in cancer metabolism exist. Here, a systematic analysis of MACC1-driven metabolic networks by elucidation of cell nutrient preferences, environment dependent alterations of nutrient utilization, metabolic pathway functionality and metabolic tracing using 13C-labeled metabolic substrates had been performed. MACC1 was found to enhance surface GLUT1 thus leading to increased glucose depletion, glucose flux and hence increased cell proliferation. Besides, MACC1 was found to reduce glutamine flux independent of nutrient availability. Upon glucose deprivation MACC1 was found to enhance pyruvate uptake exhibiting minor effects on pyruvate flux. In vivo, MACC1 increased uptakes of 18F-FDG and 18F-glutamate in liver metastatic lesions. Together, these findings demonstrate that MACC1 exhibits multiple effects on cancer metabolism, thus making it attractive to further study its effects in cancer models. Metastasis is the main cause of death from colorectal cancer (CRC). Fifteen to twenty percent of stage II CRC patients develop metastasis during the course of disease, however the criteria of likely benefitting patients from chemotherapy remain imprecise. Here, the potential of plasma metabolomics to predict metachronous metastasis was assessed. Plasma metabolic profiles were shown to be significantly different between non-metastasized and metachronously metastasized CRC patients. As demonstrated by supervised classifications using decision trees and support vector machines plasma metabolites have the power to distinguish non-metastasized from metachronously metastasized CRC patients giving average prediction accuracy of 0.75 and 0.82 for each of the methods, respectively. Together, these results demonstrate that plasma metabolites have the potential to non-invasively stratify CRC patients according to their metastasis risk.

De par leur incidence et un diagnostic tardif, les cancers du sein et du colon restent parmi les plus meurtriers en France. L’identification de marqueur précoce semble primordiale pour détecter rapidement ces maladies et ainsi améliorer la survie des patients. Le protéome de chaque stade du cancer du côlon a été identifié et quantifié par spectrométrie de masse. L’Olfactomedine-4 (OLFM4) a été définie comme un biomarqueur potentiel par sa présence dans le sérum et ses variations d’expression. Alors que cette protéine est surexprimée, au niveau du site tumoral primaire,uniquement dans les stades précoces du cancer colorectal, sa surexpression est maintenue dans le sang des patients tous stades confondus. Cette variabilité d’expression est également retrouvée dans le cancer du sein. L’OLFM4 est décrite comme un marqueur des cellules souches colorectales mais ses fonctions restent contradictoires. Selon le contexte cellulaire, nos travaux associent cette protéine sécrétée à l’établissement de propriétés de cellule souche cancéreuse comme la prolifération en faible adhésion et la formation de mammosphères. Elle facilite également le processus migratoire et la résistance à la chimiothérapie. De plus, des expériences in vivo ont confirmé le caractère protumoral de cette protéine. Nous avons également mis en évidence son implication dans la régulation de l’expression du facteur de transcription GLI1 de la voie Sonic Hedgehog et de protéines d’adhésion telle que l’E-Cadherine. Cette étude décrit le lien étroit entre l’induction d’un phénotype agressif et l’OLFM4 dont le dosage sérique permettrait une détection précoce de ces maladies. / Through their high frequencies and the lack of early diagnosis, breast and colorectal cancer remain poor prognosis’ diseases. Therefor, the identification of early markers appears as crucial. The proteomic approach isone of the potential tools to identify these biomarkers as it enables the study of tumour cell lines or tissues amples. Indeed, proteins enriched from a shotgun proteomic approach can be identified and quantified by mass spectrometry. In a previous study, we have analysed the proteome of colorectal tumour at different stages and defined Olfactomedine-4 (OLFM4) as a potential biomarker. While OLFM4 expression is increased at primary tumoral site only in non-invasive stages, we have observed that OLFM4 isover expressed in the blood of patients regardless of the cancer stage. The same analysis was made on breast cancer patients. Although OLFM4 has been described as a stem cell marker, its functions remain unclear. In this study, we found that OLFM4 confers cancer stem cell properties. It acts as a regulator of proliferation in low adhesion conditions, migration, mammosphere formation and tumor growth. These abilities could be dependent of Sonic Hedgehog signalling pathway, especially of transcriptional factor GLI1, and regulation of adhesion proteins like E-cadherin. According to the cellular background, all these features highlight a close relationship between a potential biomarker and its involvement in the acquisition of an aggressive phenotype.

O câncer colorretal (CCR) é o terceiro tipo de câncer mais comum no mundo. Apesar dos avanços nos tratamentos convencionais, aproximadamente dois terços dos pacientes com CCR são submetidos à cirurgia potencialmente curativa. Entretanto, grande parte desses pacientes evolui mal, apresentando recidivas e/ou metástases. A busca de novos alvos moleculares para a terapia do CCR revelou a proteína celular Prion (PrPC) como um possível candidato. Trabalhos recentes sugerem participação direta ou indireta de PrPC no crescimento de tumores, na formação de metástases, na composição de complexos multiproteicos e na indução de vias de sinalização envolvidas em diversos processos biológicos, como proliferação. Além disso, PrPC foi descrito como um importante modulador do crescimento de tumor colorretal. Resultados prévios mostraram que a interação da proteína PrPC com a proteína HSP70/HSP90 Organizing Protein (HOP) induz proliferação em glioblastomas. HOP é uma proteína predominantemente citoplasmática, podendo também ser secretada associada às vesículas extracelulares. Assim, o presente estudo objetivou avaliar o papel do complexo PrPC-HOP e das vesículas extracelulares no desenvolvimento e progressão dos tumores colorretais. Os nossos resultados mostram que HOP induziu migração e invasão em linhagens de CCR de maneira dependente de PrPC, uma vez que o uso do peptídeo sem atividade que compete pelo sítio ligação de HOP a PrPC inibiu estes processos. Além disso, nossos dados apontaram que o aumento de migração e invasão das células de CCR induzida pela interação PrPC-HOP é mediada pela ativação da via ERK1/2. Os achados in vitro estimularam a avaliação do perfil de expressão de PrPC e HOP por imuno-histoquímica em tecidos de pacientes com diferentes tipos de tumores colorretais. Nossos resultados sugeriram que essas proteínas são importantes no início do desenvolvimento tumoral e na transição de adenomas para adenocarcinomas, não havendo correlação entre a presença de HOP e/ou PrPC com metástase, linfonodos acometidos, estadiamento, sobrevida ou região tumoral versus tecido normal. Em relação ao papel das vesículas extracelulares na progressão dos tumores colorretais, nossos resultados mostraram que linhagens celulares que apresentam padrões parecidos de agressividade tumoral podem ter perfis de secreção de proteínas e vesículas extracelulares bastante diferentes, induzindo, portanto, processos biológicos com intensidades distintas. O meio condicionado e as vesículas extracelulares da linhagem WiDr apresentaram maior potencial de indução de migração quando comparado com a linhagem HCT8. Além disso, a modulação negativa da proteína VPS4, uma das responsáveis pela formação dos corpos multivesiculares, mostrou-se uma abordagem interessante no estudo da secreção de vesículas por células de CCR, uma vez que o dominante negativo de VPS4 promoveu diminuição do cargo proteico e da secreção de vesículas extracelulares, redução da proliferação celular e do efeito indutor do processo de migração na linhagem WiDr. Assim, em conjunto, o presente trabalho indicou que o complexo PrPC-HOP pode ser um bom alvo terapêutico nos processos de migração e invasão em CCR. Ainda, essas proteínas se mostraram importantes nos estágios iniciais da formação dos tumores. A modulação da secreção de vesículas extracelulares pode contribuir para retardar a progressão dos tumores colorretais. / Colorectal cancer (CRC) is the third most common type of cancer in the world. Despite improvements in conventional treatments, approximately two-thirds of CRC patients undergo potentially curative surgery. However, most of these patients evolve poorly, showing recurrence and/or metastasis. Search of new molecular targets for CRC therapy revealed the cellular protein Prion (PrPC) as a putative candidate. Recent studies have shown that PrPC exhibit direct or indirect participation in tumor growth, formation of metastasis, composition of multiprotein complexes and induction of signaling pathways involved in many biological processes such as proliferation. Moreover, PrPC has been described as an important modulator of colorectal tumor growth. Previous findings showed that the interaction between PrPC and its ligand HSP70/90 heat shock organizing protein (HOP) induces gliobastoma proliferation. It is well known that HOP localizes mainly in the cytoplasm but HOP is also secreted associated with extracellular vesicles. In this way, the present study sought to evaluate the role of PrPC-HOP complex and extracellular vesicles in the development and progression of CRC. We demonstrate that HOP induces the migration and invasion of CRC cell lines in a PrPC-dependent manner because the use of HOP peptide, which is able to bind to PrPC, blocking PrPC-HOP complex formation, inhibited the migration and invasion processes. In addition, our data showed that the enhancement of migration and invasion induced by PrPC-HOP interaction is mediated by ERK1/2 pathway activation. These in vitro results lead us to evaluate the PrPC and HOP expression by immunohistochemistry in tissues from patients with different tumor types. Our data showed that these proteins could be important for the initial steps of tumor development, represented by the transition from adenoma to adenocarcinoma. No correlation was found among HOP and/or PrPC expression and metastasis, lymph node involvement, staging, survival or tumor area versus normal tissue. Regarding the role of extracellular vesicles in the progression of colorectal tumors, our results showed that cell lines exhibiting similar aggressive tumor behavior can have a different protein secretion pattern and a distinct profile of extracellular vesicles release, which could induce biological process with different intensities. The conditioned medium and the extracellular vesicles derived from WiDr cell line showed a higher potential to induce migration than HCT8 cell line. Moreover, the negative modulation of VPS4, one of the proteins responsible for multivesicular body formation, showed to be an interesting approach in the study of extracellular vesicles secretion secreted by CRC cells; the negative dominant of VPS4 promoted in the WiDr cell line a reduction in the protein cargo and secretion of the extracellular vesicles, a decrease of cell proliferation and induction of migration process. Therefore, taken together, our data highlights that PrPC-HOP complex can be considered a new therapeutic target in migration and invasion processes of CRC. Moreover, these proteins appeared to be important at onset of tumor formation. The modulation of extracellular vesicles secretion may contribute for delaying the progression of colorectal tumors.

The human microbiome - defined as the microbial communities that live in and on our bodies - is emerging as a key factor in human diseases. The expanding research field that investigates the role of the microbiome on human cancer development, termed oncobiome, has led to important discoveries such as the role of Fusobacterium nucleatum in colorectal cancer carcinogenesis and tumor progression. Motivated by these discoveries, this thesis studied the oncobiome from different perspectives, investigating whether alterations to microbial profiles were associated with disease status or an adverse response to treatment. We used both biopsy tissue samples and 16S rRNA amplicon sequencing (N = 36), as well as privately and publicly available fecal whole metagenomes (N = 764) to investigate microbiome-colorectal cancer (CRC) associations. We observed significant increases in species richness in CRC, regardless of sample type or methodology, which was partially due to expansions of species typically from the oral cavity, as well as an overabundance of specific taxa such as Bacteroides fragilis, Fusobacterium, Desulfovibrio and Bilophila in CRC. Functional potential analysis of CRC metagenomes revealed that the choline trimethylamine-lyase (cutC) gene was over-abundant in CRC, with the strength of association dependent on four identified sequence variants, pointing at a novel potential mechanism of CRC carcinogenesis. Predictive microbiome signatures trained on the combination of multiple datasets showed very high and consistent performances on distinct cohorts (average AUC 0.83, minimum 0.81). To investigate the microbiomes role in response to treatment, we profiled microbial communities of gastric wash samples in gastric cancer patients (N = 36) before and after neoadjuvant chemotherapy through 16S rRNA amplicon sequencing. Gastric wash microbial communities presented remarkably high inter-individual variation, with significant decreases in richness and phylogenetic diversity after treatment and associations with pH, pathological response and sample collection. The most abundant genera found in patients before or after chemotherapy treatment included Streptococcus, Prevotella, Rothia and Veillonella. Despite limitations inherent to differing experimental choices, this thesis provides microbiome signatures that can be the basis for clinical prognostic tests and hypothesis-driven mechanistic studies, as well as supporting the role of the human oral microbiome in whole-body diseases. / O microbioma humano - definido como as comunidades microbianas que vivem sobre e dentro do corpo humano - está se tornando um fator cada vez mais importante em doenças humanas. O campo de estudo que investiga o papel do microbioma no desenvolvimento do câncer humano, denominado oncobioma, está crescendo e já levou a importantes descobertas como o papel da espécie Fusobacterium nucleatum na carcinogênese e progressão tumoral de tumores colorretais. Motivado por estas descobertas, esta tese de doutorado analisou o oncobioma por diferentes perspectivas, investigando se alterações nos perfis microbianos estavam associados à presença da doença ou a uma resposta adversa ao tratamento. Usamos tanto amostras de tecidos de biópsias e o sequenciamento do gene 16S rRNA (N = 36), quanto metagenomas fecais públicos e privados (N = 764), para investigar associações entre o microbioma e o câncer colorretal (CCR). Observamos um aumento significativo da riqueza microbiana no CCR, independentemente do tipo da amostra ou metodologia, que era em parte, devido ao aumento de espécies tipicamente presentes na cavidade oral. Observamos também um aumento da abundância de táxons específicos no CCR, que incluíam Bacteroides fragilis, Fusobacterium, Desulfovibrio e Bilophila. Analisando o potencial funcional dos metagenomas, encontramos um aumento significativo da enzima liase colina trimetilamina (cutC) no CCR, cuja associação era dependente de 4 variantes de sequência, demonstrando ser um possível novo mecanismo de carcinogênese no CCR. Assinaturas preditivas do microbioma treinadas na combinação dos estudos demonstraram ser altamente preditivas e consistentes nos diferentes estudos (média de AUC 0.83, mínimo de 0.81). Para investigar o possível papel do microbioma na resposta ao tratamento, analisamos os perfis microbianos do suco gástrico de pacientes com câncer gástrico (N = 36) antes e depois do tratamento quimioterápico neoadjuvante. As comunidades microbianas apresentaram uma variabilidade inter-individual notavelmente grande, com diminuições significativas na riqueza e diversidade filogenética pós tratamento, além de estarem associadas principalmente ao pH, mas também à resposta patológica e ao tempo da coleta. Os gêneros mais abundantes encontrados nos pacientes antes ou depois da quimioterapia incluíam Streptococcus, Prevotella, Rothia e Veillonella. Apesar das limitações inerentes às escolhas experimentais, esta tese proporciona assinaturas do microbioma que podem servir de base para testes clínicos prognósticos e estudos mecanísticos, além de dar mais suporte ao papel do microbioma oral em doenças humanas.

The human microbiome - defined as the microbial communities that live in and on our bodies - is emerging as a key factor in human diseases. The expanding research field that investigates the role of the microbiome on human cancer development, termed oncobiome, has led to important discoveries such as the role of Fusobacterium nucleatum in colorectal cancer carcinogenesis and tumor progression. Motivated by these discoveries, this thesis studied the oncobiome from different perspectives, investigating whether alterations to microbial profiles were associated with disease status or an adverse response to treatment. We used both biopsy tissue samples and 16S rRNA amplicon sequencing (N = 36), as well as privately and publicly available fecal whole metagenomes (N = 764) to investigate microbiome-colorectal cancer (CRC) associations. We observed significant increases in species richness in CRC, regardless of sample type or methodology, which was partially due to expansions of species typically from the oral cavity, as well as an overabundance of specific taxa such as Bacteroides fragilis, Fusobacterium, Desulfovibrio and Bilophila in CRC. Functional potential analysis of CRC metagenomes revealed that the choline trimethylamine-lyase (cutC) gene was over-abundant in CRC, with the strength of association dependent on four identified sequence variants, pointing at a novel potential mechanism of CRC carcinogenesis. Predictive microbiome signatures trained on the combination of multiple datasets showed very high and consistent performances on distinct cohorts (average AUC 0.83, minimum 0.81). To investigate the microbiomes role in response to treatment, we profiled microbial communities of gastric wash samples in gastric cancer patients (N = 36) before and after neoadjuvant chemotherapy through 16S rRNA amplicon sequencing. Gastric wash microbial communities presented remarkably high inter-individual variation, with significant decreases in richness and phylogenetic diversity after treatment and associations with pH, pathological response and sample collection. The most abundant genera found in patients before or after chemotherapy treatment included Streptococcus, Prevotella, Rothia and Veillonella. Despite limitations inherent to differing experimental choices, this thesis provides microbiome signatures that can be the basis for clinical prognostic tests and hypothesis-driven mechanistic studies, as well as supporting the role of the human oral microbiome in whole-body diseases. / O microbioma humano - definido como as comunidades microbianas que vivem sobre e dentro do corpo humano - está se tornando um fator cada vez mais importante em doenças humanas. O campo de estudo que investiga o papel do microbioma no desenvolvimento do câncer humano, denominado oncobioma, está crescendo e já levou a importantes descobertas como o papel da espécie Fusobacterium nucleatum na carcinogênese e progressão tumoral de tumores colorretais. Motivado por estas descobertas, esta tese de doutorado analisou o oncobioma por diferentes perspectivas, investigando se alterações nos perfis microbianos estavam associados à presença da doença ou a uma resposta adversa ao tratamento. Usamos tanto amostras de tecidos de biópsias e o sequenciamento do gene 16S rRNA (N = 36), quanto metagenomas fecais públicos e privados (N = 764), para investigar associações entre o microbioma e o câncer colorretal (CCR). Observamos um aumento significativo da riqueza microbiana no CCR, independentemente do tipo da amostra ou metodologia, que era em parte, devido ao aumento de espécies tipicamente presentes na cavidade oral. Observamos também um aumento da abundância de táxons específicos no CCR, que incluíam Bacteroides fragilis, Fusobacterium, Desulfovibrio e Bilophila. Analisando o potencial funcional dos metagenomas, encontramos um aumento significativo da enzima liase colina trimetilamina (cutC) no CCR, cuja associação era dependente de 4 variantes de sequência, demonstrando ser um possível novo mecanismo de carcinogênese no CCR. Assinaturas preditivas do microbioma treinadas na combinação dos estudos demonstraram ser altamente preditivas e consistentes nos diferentes estudos (média de AUC 0.83, mínimo de 0.81). Para investigar o possível papel do microbioma na resposta ao tratamento, analisamos os perfis microbianos do suco gástrico de pacientes com câncer gástrico (N = 36) antes e depois do tratamento quimioterápico neoadjuvante. As comunidades microbianas apresentaram uma variabilidade inter-individual notavelmente grande, com diminuições significativas na riqueza e diversidade filogenética pós tratamento, além de estarem associadas principalmente ao pH, mas também à resposta patológica e ao tempo da coleta. Os gêneros mais abundantes encontrados nos pacientes antes ou depois da quimioterapia incluíam Streptococcus, Prevotella, Rothia e Veillonella. Apesar das limitações inerentes às escolhas experimentais, esta tese proporciona assinaturas do microbioma que podem servir de base para testes clínicos prognósticos e estudos mecanísticos, além de dar mais suporte ao papel do microbioma oral em doenças humanas.

Colorectal cancer (CRC) is the second most common cause of cancer-related death in the UK and Western world. More than 90% of sporadic CRCs harbour mutations in the multi-functional tumour suppressor gene Adenomatous polyposis coli (<i>Apc</i>). The most commonly studied function of APC is its role as a scaffold for the β-catenin destruction complex involved in Wnt signalling. However, APC binds many other proteins. For example, it directly binds to and stabilises microtubules and actin. These non-Wnt related functions of APC are poorly understood. My PhD examines non-Wnt functions of APC. To this end, I created degron-tagged APC in DT40 cells that allowed for the rapid, conditional degradation of endogenous APC. The aim was to identify the immediate effects on cellular processes. Then, to identify the contribution of different APC domains by measuring the ability to rescue any defects when reintroducing fragments of APC. However, creation of these degron-tagged <i>Apc </i>knock-in cell lines resulted in hypomorphic phenotypes and auxin-associated off-target effects. Nonetheless, I compared the response of APC^high, APC^low, and APC^minimal cells to DNA damaging agents and Taxol® but found no significant differences. Subsequently, I focused on the relationship between APC and apoptosis. Previous observations suggested that deficiency in <i>Apc </i>rendered cells less sensitive to low doses of Taxol®. However, <i>Apc </i>deficient cells were more readily killed when Taxol® was combined with the Bcl-2 inhibitor, ABT-737. One possible explanation is the increase in Bcl-2 protein upon <i>Apc </i>depletion. However, I found that ABT-737, Taxol® and <i>Apc </i>depletion each cause activation of the unfolded protein response. This suggests that these treatments elicit a stress response that can stimulate apoptosis. Moreover, the same treatments also cause changes in mitochondria. Importantly, all of these effects do not require an increase in the β-catenin protein. Together, my data reveal novel links between APC and apoptosis that could be exploited clinically.

O câncer colorretal (CCR) pode ocorrer em qualquer parte do cólon ou do reto e representa o terceiro câncer mais comum no mundo em ambos os sexos. As células Natural Killer (NK) fazem parte do sistema imune inato reconhecendo moléculas de HLA de classe I em células alvo, através de seus receptores de membrana killer cell immunoglobulin-like receptors (KIR). O objetivo deste estudo foi avaliar a associação entre os genes KIR e os ligantes HLA em pacientes com câncer colorretal e controles saudáveis. Examinamos o polimorfismo de 16 genes KIR e seus ligantes HLA em 154 pacientes caucasóides com CCR e 216 controles saudáveis pela técnica de PCR-SSO e PCR-SSP. Quando comparamos os dois grupos, não foram encontradas diferenças significativas para os ligantes HLA e os genes KIR após correção de Bonferroni. Entretanto, o grupo de genótipos Bx (heterozigoto e homozigoto para o haplótipo B) foi mais frequente nos controles, quando comparados com os pacientes. Estes achados sugerem que altos níveis de ativação de sinais KIR aparecem como proteção para o câncer colorretal. / Colorectal cancer (CRC) can occur anywhere in the colon or rectum and represents the third most common cancer in the world in both sexes. Natural killer cells (NK) are part of the innate immune system recognizing class I HLA molecules on target cells through their membrane receptors, called killer cell immunoglobulin-like receptors (KIR). The aim of our study was to evaluate the association between the KIR genes and HLA ligands in patients with colorectal cancer and healthy controls. We examined the polymorphism of 16 KIR genes and their HLA ligands in 154 caucasoid CRC patients and 216 healthy controls by PCR-SSO and PCR-SSP. When both groups were compared, no significant differences were found for HLA ligands and KIR genes after Bonferroni correction. However, the Bx group genotypes (heterozygous and homozygous for the haplotype B) were more frequent in controls, when compared with patients. These findings suggest that individuals with Bx genotypes could have some protection to colorectal cancer. These findings suggest that higher levels of activating KIR signals appear as protective to colorectal cancer.