Philosophiae Doctor - PhD / This thesis consists of a brief description on cancer, carcinogenesis, the changes in the type and level of dietary fat available in our diets over time and association with the development of certain diseases. The main focus of this research was on omega 6 and omega 3 essential fatty acids (EFA) and their interaction with regards to carcinogenesis. / South Africa

A l`heure où le cancer a supplanté les maladies cardiovasculaires en tant que première cause de mortalité en France, le CCR représente la deuxième cause de mortalité par cancer. Longtemps dominé par la génétique, le paradigme du cancer colorectal a récemment évolué laissant une place prépondérante aux facteurs environnementaux. Il est néanmoins difficile d’étudier l’impact de l’environnement sur la carcinogénèse colorectale de façon exhaustive compte tenu de la multiplicité de ces facteurs environnementaux. Dans la présente étude, nous avons essayé d’appréhender la contribution de la composition du microbiote intestinal, de la composition métabolomique des eaux fécales et des altérations épigénétiques de l’hôte comme témoin de ces facteurs environnementaux au cours de la carcinogénèse colorectale et d’en évaluer le bénéfice en tant que marqueur diagnostique non invasif. Nous avons ainsi pu montrer au sein d’une population de patients à risque moyen de cancer colorectal qu’il existait une signature microbiologique, métabolomique et épigénétique spécifique du cancer colorectal. Nous avons également pu montrer que ces marqueurs présentaient des performances diagnostiques supérieures au test colorimétrique au guaiac utilisé dans le dépistage organisé du cancer colorectal. / Colorectal cancer (CRC) is a significant cause of morbidity and mortality in developed countries. The majority of CRC are called sporadic, meaning they are due to environmental factors rather than constitutional genetic alterations. Indeed, the role of environment, i.e. western lifestyle, is also underlined by dramatic geographic variations in CRC incidence in both sexes. However, it is difficult to take into account the totality of human environmental exposures for a better understanding of the colorectal cancer pathogenesis. In the present work, we tried to highlight the contribution of the environment in the development of colorectal cancer by studying the role of the intestinal microbiota together with the role of the fecal metabolites and the presence of epigenetic alterations of the host. We also investigated the performance accuracy of the latter changes for colorectal cancer diagnosis as compared to the guaiac fecal occult blood test which is widely used as a non-invasive test in several screening program. We demonstrated a specific signature associated with advanced colorectal neoplasia for the intestinal microbiota and the fecal metabolite profile for colorectal cancer as well as a link between colorectal cancer and Wif-1 gene methylation in urine and/or fecal samples. Those specific signatures disclosed higher diagnostic accuracy compared to guaiac fecal occult blood test as colorectal cancer screening test.

Cigarette smoke condensates from all cigarettes tested were found to be potent inducers of AHH enzyme in cultured human lymphocytes and, with the exception of Kent Lights and Carlton CSC's, all were found to be toxic under the experiment conditions. Most of the AHH inducing activity was found in basic and neutral fractions of the lAl standard cigarettes. A radiometric assay of BP metabolites in cultured human lymphocytes was developed in which we were able to separate the primary metabolites and the secondary metabolites from the parent compound (BP) by neutral alumnia HPLC. The primary metabolites were further separated by a selective enzyme hydrolysis and/or reverse phase HPLC.

The human papillomavirus (HPV) has been a “companion” of humanity for as long as humanity has existed. The migration of peoples around the globe has given rise to more than 170 different types of the virus, which cause a variety of conditions. All five genera of HPV infect epithelial cells in the body, but only the Alphapapillomaviruses infect the genital mucosa. Most infections are benign and typically regress to subclinical within two years, but persistent infections can cause precancerous lesions. HPV types 16 and 18 are among the highest risk and account for the majority of cervical cancer, and more than 90% of all other HPV-related cancers. While the two vaccines, Gardasil and Cervarix, have been successfully implemented in the US market and some European and Asian countries, complete world penetrance has been burdened by multiple factors, including financial constraints and social norms. Treatments for established papillomas are invasive (cryosurgery, conization, etc.) and advanced malignant HPV-related tumors have been targeted with chemo- and radiotherapy with varied success. The high morbidity and long-term effects of current treatment options make clear the need for easy-to-administer, low-cost therapies, which can specifically treat both early and advanced HPV-associated cancers.<div><br></div><div>The hallmark of HPV tumors is the inactivation of p53, an evasion strategy key to the progression of HPV- derived cancers. Through an interaction between the viral protein E6 and the E3 ubiquitin ligase E6AP, p53 is polyubiquitinated and targeted for proteasomal degradation, allowing infected cells to bypass their own defense mechanisms. This work explores interruption of the association between E6 and E6AP as an opportunity to combat the infection and resulting malignancies.<br></div><div><br></div><div>In the first part of this project, disruption of the E6-E6AP interactions is pursued through the development of helical stabilized peptidomimetics of the LxxLL motif, which E6AP uses for E6 recognition and binding. Several reports have indicated that targeting the E6 binding groove is a viable means for disrupting the interaction. However, reported peptides were not cell permeable or optimized for α-helicity and proteolytic resistance (for reference, the LxxLL motif is an α-helix when bound). To address this challenge a peptide stabilization strategy was applied, which uses an all-hydrocarbon chain to connect two non-adjacent residues and enforce α-helicity. Results from in silico simulations and biochemical assay with these new stapled peptides showed that affinity for E6, α-helicity, and cell permeability can all be improved with the installment of the proper staple.<br></div><div><br></div><div>The second question examined by this work is whether fragment-based drug design can be successfully employed to derive new small-molecule inhibitors of the formation of the E6-E6AP complex. From a design perspective, the significant challenge was to define discreet binding hot-spots capable of accommodating fragments with reasonable affinity, which can then be linked together into a complete ligand. Using existing structural knowledge of the E6 protein and computational hot-spot searching tools, three previously-unidentified regions (sub-pockets) on E6 were discovered, which are near but not directly engaged by either the E6AP motif or p53. Using high-throughput in silico and biochemical screening, three sets of sub-pocket specific fragments were defined and elaborated into larger molecules with two different scaffolds. As a result, the work herein presents a stepwise approach to targeting the E6-E6AP protein-protein interaction – the discovery of new binding hot spots, the identification of site-specific fragments, and the design of complete molecules with versatile scaffold.<br></div>

Les cancers papillaires de la thyroïde (PTC) sont les tumeurs endocrines les plus courantes et représentent 2-3% de tous les cancers humains. Les altérations génétiques les plus pertinentes trouvées dans ces tumeurs sont des mutations dans les gènes BRAF et RAS, et des translocations du gène RET. Ces translocations oncogéniques, connues sous le nom de RET/PTC, résultent de la fusion de RET avec des gènes partenaires non-apparentés. L’exposition aux radiations ionisantes est le facteur de risque le plus important pour la formation de RET/PTC. Durant ces dernières années, notre groupe a mis en évidence un rôle crucial des espèces réactives de l'oxygène (ROS) dans la formation de RET/PTC dans des cellules thyroïdiennes in vitro et a notamment montré que l'irradiation (IR) induit l’établissement d’un stress oxydatif persistant du aux ROS produites par la NADPH Oxydase DUOX1, laquelle est induite à post-IR. Cela conduit à des dommages à l'ADN. Les enfants présentent un risque significativement plus élevé de développer des cancers radio-induits de la thyroïde exprimant RET/PTC, probablement en raison du taux de prolifération élevé des cellules. Ceci suggère que la dynamique de réplication pourrait être impliquée dans la formation de la translocation RET/PTC1. En effet, il a été montré que l'induction pharmacologique d’un stress réplicatif peut favoriser la formation de RET/PTC in vitro dans les cellules thyroïdiennes. Ainsi, pour déterminer si un stress réplicatif peut contribuer aux effets à long terme de l'irradiation: à savoir une persistance des lésions de l'ADN et la formation de RET/PTC1, nous avons analysé les effets à post-IR dans les cellules NTHY-ori3.1. Nos résultats confirment qu’une irradiation des cellules aux rayons X à la dose de 5 Gy induit deux vagues de stress oxydatif: une première vague forte mais transitoire qui se produit dans les minutes qui suivent l'irradiation et une deuxième vague dont l’ augmentation débute 2 jours après l'irradiation pour persister ensuite. Ces deux pics de stress oxydatif conduisent à deux pics de dommages à l'ADN. L'irradiation des cellules à cette dose n’a aucun effet sur la prolifération et sur la progression du cycle cellulaire. Cependant, plusieurs marqueurs de stress réplicatif sont exprimés trois jours après l'irradiation. Par ailleurs, l'analyse de la dynamique de réplication révèle une diminution de la vitesse de réplication à post-IR qui est contrecarrée par les antioxydants, suggérant qu’un stress oxydatif peut contribuer à un stress réplicatif. Enfin, par ChIP-QPCR, nous observons que les gènes impliqués dans RET/PTC1 présentent plus de cassures double brin que des gènes endogènes, et ce, trois jours après l'irradiation. Ainsi, nous proposons qu’un stress réplicatif induit par un stress oxydatif pourrait être potentiellement impliqué dans l'étiologie des tumeurs RET/PTC-positives. / Papillary thyroid cancers (PTC) are the most common endocrine tumors and account for 2-3% of all human cancers. The most relevant genetic alterations found in these tumors are mutations in the genes BRAF and RAS, and chromosomal translocations in RET, a proto-oncogene activated in 15-20% of PTCs. These oncogenic translocations, known as RET/PTCs, result from the fusion of RET with unrelated partner genes. Ionizing radiation is a major risk factor for RET/PTC formation, however, the molecular mechanisms involved in these radioinduced translocations just begun to be unveiled. In the past few years, our group has reported a critical role for reactive oxygen species (ROS) in the formation of RET/PTC in thyroid cells in vitro and has also shown that irradiation can elicit a persistent oxidative stress caused by the upregulation of the NADPH Oxidase DUOX1 that leads to DNA damage, mediating at least part of the effects of radiation. However, how could ROS lead to the formation of RET/PTC is not fully understood. Children are at significantly higher risk of developing radio-induced thyroid tumors, specially RET/PTC positive, probably due to the intense proliferation rate of their follicular thyroid cells. This epidemiological observation prompts the assumption that replication dynamics may be involved in RET/PTC formation. Indeed, it has been shown that the pharmacological induction of replicative stress can stimulate the in vitro formation of RET/PTC in thyroid cells. Thus, to investigate whether replicative stress might contribute for the long-term effects of irradiation on DNA damage and RET/PTC formation, we analyzed the effects of radiation in NTHY-ori3.1 thyroid cell lineage in terms of oxidative and replicative stress and replication dynamics. Our results confirm that irradiation triggers two waves of oxidative stress: first, a strong but transient oxidative burst takes place minutes after irradiation and next, a persistently increased oxidative stress that starts only 2 days after irradiation. These two peaks of oxidative stress lead to two peaks of DNA damage. Irradiation caused little or no effect on proliferation nor on cell cycle progression. However, several protein markers of replicative stress, such as pATR, pATM, pChk1 and pRPA are induced three days after irradiation. Moreover, replication dynamics analysis revealed a diminished replication speed that has been reversed by antioxidants, suggesting that oxidative stress may contribute to replication defects. Finally, using ChIP-qPCR, we observed that the genes involved in RET/PTC1 translocation present more double-stranded breaks than RET/PTC-unrelated genes 3 days after irradiation. Hence, we propose that replicative stress is potentially involved in the etiology of RET/PTC-positive tumors. / HECHT, Fabio. The role of reactive oxygen species in thyroid radio-carcinogenesis. Rio de Janeiro, 2018. Doctoral thesis - Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil and Université Paris-Saclay, Orsay, France, 2018.O câncer papilífero de tireoide é o tumor endócrino mais comum e corresponde a 2-3% de todos os cânceres humanos. As alterações genéticas mais relevantes relacionadas a esse tumor são mutações nos genes BRAF e RAS e translocações do gene RET, um proto-oncogene ativado em 15-20% dos tumores papilíferos. Essas translocações, conhecidas como RET/PTC, resultam da fusão de RET com diversos outros genes. A radiação ionizante é um importante fator de risco para a formação de RET/PTC, no entanto, o mecanismo molecular responsável por essa translocação radioinduzida ainda não foi elucidado. Nos últimos anos, nosso grupo demonstrou um papel crítico exercido pelas espécies reativas de oxigênio na formação de RET/PTC em células tireoidianas in vitro e também mostrou que a irradiação promove um estresse oxidativo persistente causado pelo aumento de expressão da NADPH Oxidase DUOX1, levando à dano ao DNA, mediando assim parte dos efeitos da radiação. No entanto, como o ROS leva à formação de RET/PTC ainda não é compreendido. Crianças possuem um risco significativamente mais alto de desenvolver tumores tireodianos após a irradiação, especialmente RET/PTC positivos, provavelmente em função da intensa proliferação das células tireodianas. Essa associação sugere que a replicação esteja envolvida na formação de RET/PTC. De fato, foi observado que a indução farmacológica de estresse replicativo pode estimular a formação in vitro de RET/PTC em células tireodianas. Portanto, para investigar se o estresse replicativo contribui com os efeitos da irradiação no longo prazo sobre o dano ao DNA e formação de RET/PTC, nós investigamos o papel da radiação sobre o estresse oxidativo e replicativo, além da dinâmica de replicação de linhagem de células tireodianas NTHY-ori 3.1. Nossos resultados confirmam que a irradiação desencadeia duas ondas de estresse oxidativo: primeiramente, um forte, mas transitório pico de espécies reativas de oxigênio é observado minutos após a irradiação, seguido por um novo e persistente pico que só é observado a partir de dois dias após a irradiação. Esses dois picos de estresse oxidativo resultam em dois picos de dano ao DNA. A irradiação causou pouco ou nenhum efeito na proliferação ou na progressão do ciclo celular. No entanto, vários marcadores de estresse replicativo foram observados três dias após a irradiação, como pATR, pATM, pChk1 e pRPA. Além disso, a análise da dinâmica de replicação mostrou uma diminuição na velocidade da replicação que foi revertida por antioxidantes, sugerindo que o estresse oxidativo contribui para distúrbios dos mecanismos replicativos. Por fim, utilizando ChIP-qPCR, nós observamos que os genes envolvidos na translocação RET/PTC possuem mais quebras duplas do que genes endógenos, dias após a irradiação. Portanto, propomos que o estresse replicativo está potencialmente envolvido na etiologia dos tumores RET/PTC positivos.

Colorectal carcinoma (CRC) ranks high in mortality and morbidity in most developed countries. Following theses focus on specific aspects of colorectal carcinoma pathogenesis including the issue of screening. The goal of the first study was assessment of expression of epithelial markers of colorectal carcinogenesis p53, COX-2, bcl-2. The study included patients with active ulcerative colitis (UCA), ulcerative colitis in remission (UCR), primary sclerosing cholangitis with ulcerative colitis (PSC-UC) (PSC), patients after liver transplantation for PSC (OLT) and a control group (N). We found significantly increased expression of tumour suppressor gene p53 in non-dysplastic mucosae in PSC-UC compared with UCA, UCR, OLT, and N, which may indicate higher neoplastic potential of PSC. Statistically significant correlation was found between PSC incidence and p53 expression. Surprisingly, OLT showed no p53 expression in non-dysplastic mucosa compared with PSC-UC. This indicates that PSC may contribute to increased expression of p53 and p53-induced colorectal carcinogenesis. Furthermore, a correlation between expression of p53 and COX-2 together with the increased expression of bcl-2 in UCA compared to N can support the role of inflammation in colorectal carcinogenesis. The goal of the second study was...

Acute Myeloid Leukemia (AML) is a potentially fatal disease, more common in an elderly population. The American Cancer Society estimates 21,450 new cases of AML and 10,920 deaths from AML in the United States in 2019. This malignancy originating in the Bone Marrow (BM), usually presents with peripheral blood (PB) abnormalities. Rarely, AML, particularly monoblastic variants can present with extramedullary disease. Here we describe a case of AML presenting with diffuse lymphadenopathy and a biopsy revealing myeloid sarcoma. A 53 years old male developed diffuse lymphadenopathy. Failure of outpatient empiric antibiotic treatment prompted right cervical lymph node biopsy. Lymph node architecture was distorted by the presence of malignant monocytic myeloid cells. Both the peripheral blood and bone marrow were involved by AML with monocytic features. The monoblasts count was 14% in PB and 24% in BM and the promonocyte count was 12% in PB and 26% in BM. Complete Blood Count showed total white blood cell count of 31,700, hemoglobin 11.8, monocytes 22.5% and platelets 122,000. Flow cytometry of the bone marrow demonstrated a blast population with positive expression of cMPO, CD33, CD13, CD11b, HLA-DR, CD64, CD14 and CD4; and negative for CD34, CD117, nTdT, cCD3, cCD79a. Fluorescence in situ hybridization study was positive for MLL gene rearrangement. Molecular study was positive for IDH1 mutation, and negative for IDH2, RUNX1, FLT3 mutations. Further laboratory analysis was significant for lactate dehydrogenase 346, uric acid 8.6, prothrombin time 13.6, INR 1.2, partial thromboplastin time 33.5 and fibrinogen 293. Computed tomography of chest, abdomen, pelvis with contrast revealed extensive adenopathy with enlarged bilateral supraclavicular, bilateral axillary, mediastinal, bilateral hilar, upper abdominal, periaortic retroperitoneal, pelvic and inguinal lymph nodes. Hepatosplenomegaly was also reported. The term Myeloid Sarcoma (MS) is used when leukemic cells are present outside the bone marrow and peripheral blood. MS tends to oocur more commonly in middle aged males (male-to-female ratio, 2:1, median age, 56 years). The Mayo Clinic Experience of 96 cases demonstrated 27% of patients had no bone marrow involvement, and 69% of patients had primary bone marrow disease. Extramedullary involvement can occur prior to, simultaneously, or after bone marrow involvement. Just as in our case this is an important feature for clinicians to remember so that they may recognize this rare entity early.

Epigenetic changes represent chemical modifications of the DNA molecule and histone proteins by which gene expression is altered. Among them, DNA methylation is a known mechanism of silencing of tumor-suppressor and DNA repair genes, with an important role in carcinogenesis. Many studies have been done in order to identify the methylation signatures of these genes in different types of cancer. In our study, we investigated the methylation status of promoter regions of eight mismatch repair genes (MLH1, MSH2, MSH3, MLH3, PMS1, PMS2, MSH6 and EXO1) in 45 sporadic colorectal cancer cases and 12 head and neck cancer patients. Two out of eight genes, MLH1 and MLH3, exhibited promoter methylation. The results from both groups of patients were concordant. We summarize that the methylation profiles of MLH1 and MLH3 promoters could be potential candidates for epigenetic biomarkers in colorectal cancer, and eventually in head and neck cancer. Further investigations, which would confirm this theory, should be carried out.

The development of RNAi based technologies has given researchers the tools to interrogate processes as diverse as cancer biology, metabolism and organ development. Here I employ genome-wide shRNA screens to discover the genes involved in two different processes in carcinogenesis, oncogene-induced senescence [OIS] and epigenetic silencing of tumor suppressor genes [TSGs]. OIS is a poorly studied yet significant tumor suppressing mechanism in normal cells where they enter cell cycle arrest [senescence] or programmed cell death [apoptosis] in the presence of an activated oncogene. Here I employ a genomewide shRNA screen and identify a secreted protein, IGFBP7, that induces senescence and apoptosis in melanocytes upon introduction of the oncogene BRAFV600E. Expression of BRAFV600E in primary cells leads to synthesis and secretion of IGFBP7, which acts through autocrine/paracrine pathways to inhibit BRAF-MEK-ERK signaling and induce senescence and apoptosis. Apoptosis results from IGFBP7-mediated upregulation of BNIP3L, a proapoptotic BCL2 family protein. Recombinant IGFBP7 has potent pro-apoptotic and anti-tumor activity in mouse xenograft models using BRAFV600E-postive melanoma cell lines. Finally, IGFBP7 is epigenetically silenced in human melanoma samples suggesting IGFBP7 expression is a key barrier to melanoma formation. Next I investigated the factors involved in epigenetic silencing in cancer. The TSG p14ARFis inactivated in a wide range of cancers by promoter hypermethylation through unknown mechanisms. To discover p14ARF epigenetic silencing factors, I performed a genome-wide shRNA screen and identified ZNF304, a zinc finger transcription factor that contains a Krüppel-associated box [KRAB] repressor domain. I show that ZNF304 binds to the p14ARF promoter and recruits a KRAB co-repressor complex containing KAP1, SETDB1 and DNMT1 for silencing. We find oncogenic RAS signaling to promote the silencing of p14ARF by USP28-mediated stabilization of ZNF304. In addition I find ZNF304 to be overexpressed in human colorectal cancers and responsible for hypermethylation of over 50 TSGs known as Group 2 CIMP marker genes. My findings establish ZNF304 as a novel oncogene that directs epigenetic silencing and facilitates tumorigenicity in colorectal cancer.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19177号 / 医博第4019号 / 新制||医||1010(附属図書館) / 32169 / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 武田 俊一, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM