Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-09-22T17:36:44Z No. of bitstreams: 1 analiviadeoliveira.pdf: 855679 bytes, checksum: a8093d9f1de0ed3dc5c30c78ab17fab3 (MD5) / Approved for entry into archive by Diamantino Mayra (mayra.diamantino@ufjf.edu.br) on 2016-09-26T20:29:55Z (GMT) No. of bitstreams: 1 analiviadeoliveira.pdf: 855679 bytes, checksum: a8093d9f1de0ed3dc5c30c78ab17fab3 (MD5) / Made available in DSpace on 2016-09-26T20:29:55Z (GMT). No. of bitstreams: 1 analiviadeoliveira.pdf: 855679 bytes, checksum: a8093d9f1de0ed3dc5c30c78ab17fab3 (MD5) Previous issue date: 2010-06-28 / A depleção nutricional é observada em pacientes com câncer colorretal em estado avançado mesmo com mediadas de peso adequadas e esta perda pode estar associada a fatores de co – morbidades como: redução da imunidade, aumento de infecções, cicatrização prejudicada e fraqueza muscular. A presença de tumor maligno estimula as respostas metabólicas de fase aguda, com aumento de proteínas positivas, como a proteína-C reativa esta elevação pode simplesmente refletir um processo inflamatório inespecífico secundário a necrose tumoral ou injuria tecidual. Atualmente uma abordagem nutricional utilizada em pacientes com câncer são as dietas imunomoduladoras. O uso de prebióticos e probióticos ou a mistura dos dois os simbióticos estão entre os estudados. O objetivo deste estudo foi Avaliar o estado Nutricional e a Atividade Inflamatória Sistêmica em pacientes com câncer colorretal submetidos a suplementação com suplemento simbiótico. Foi realizado um estudo longitudinal prospectivo com pacientes com câncer colorretal .Foram coletados dados antropométricos e exames de sangue para avaliação da proteína C- reativa (PCR), Hormônio carcionombrionário (CEA), interleucina - 6 (IL6), interleucina 10(IL10) e abumina. Os índices médios da proteína C reativa eram de 11mg/dl no início do estudo, antes da administração do simbiótico, e reduziram para valore menores que 6 mg/dl no final do estudo. Os resultados sugerem que a suplementação com o simbiótico foi benéfica, pois, embora os índices de albumina e CEA se mantiveram os índices de PCR diminuíram ao longo do estudo. / Nutritional depletion, found in patients with advanced colorectal cancer even with adequate weight, may be associated with co-morbidity factors such as: reduction of immunity, increased rate of infections, impaired cicatrization and muscle weakness. The presence of a malignancy stimulates the acute phase metabolic responses, with an increase of positive proteins, such as C-reactive protein, simply reflecting a non-specific inflammatory process secondary to tumor necrosis or tissue injury. Immunomodulating diets have recently been used as a nutritional approach to cancer patients. Prebiotics, probiotics and symbiotics (a mixture of the first two) have been studied. This study assessed the Nutritional Status and Systemic Inflammatory Activity of colorectal patients on symbiotic supplementation. It was a progressive longitudinal study with colorectal cancer patients. Besides anthropometric data, the following blood components were measured: C-reactive protein (CRP), carcinoembryonic antigen (CEA), interleukin-6 (IL6), interleukin-10 (IL10) and albumin. The mean CRP level at baseline, before symbiotic administration, was 11 mg/dl, with a reduction to below 6 mg/dl at the end of the study. The results suggest a beneficial effect of symbiotic supplementation, because although albumin and CEA levels were stable during the study, there was a CRP reduction throughout the study.

No description available.

La phosphatidylinositol-3 kinase (PI3-K) est un complexe comportant une sous-unité catalytique (C) et régulatrice (R). En ce qui concerne la classe I, cinq isoformes R (p85α, p55α, p50α, p85β et p55γ) et quatre isoformes C (p110α, p110β, p110δ et p110γ) sont connues. Plusieurs études ont souligné les rôles cruciaux de la voie PI3-K/Akt dans une panoplie de processus cellulaires, dont la survie. De plus, cette voie est l’une des plus altérées dans plusieurs types de cancers, dont le cancer colorectal (CCR). Par ailleurs, il est bien établi que l’acquisition d’une résistance à l’anoïkose constitue une étape cruciale et limitante dans la progression de plusieurs types de cancers, incluant une fois de plus le CCR. Nous avons précédemment démontré que les complexes isoformes PI3-K sont engagés/recrutés par la signalisation intégrines FAK/Src de suppression d’anoïkose et, conséquemment, exercent des rôles distincts dans la survie cellulaire des entérocytes normaux selon leur état de différenciation. Ainsi, l’hypothèse de recherche de la présente étude est que les complexes isoformes PI3-K contribuent sélectivement à l’acquisition d’une résistance à l’anoïkose, et ce, chez les cellules CCR humaines. Les objectifs principaux sont les suivants : 1) Établir le profil des complexes isoformes PI3-K chez des lignées cancéreuses colorectales humaines exhibant différents degrés de résistance à l'anoïkose (modéré à fortement élevé); et 2) Déterminer fonctionnellement les contributions des complexes isoformes PI3-K identifiés dans le maintien de résistance à l'anoïkose. Nos résultats indiquent que : 1) il existe des distinctions entre les profils de complexes isoformes PI3-K prédominants chez des cellules CCR exhibant différents degrés de résistance à l’anoïkose; 2) Ces complexes PI3-K peuvent jouer ou non des rôles dans la survie cellulaire, et ce, peu importe le degré de résistance à l’anoïkose exhibé; 3) les distinctions de profils de complexes isoformes ne semblent pas corrélées avec la progression de résistance à l’anoïkose; 4) il y aurait un mécanisme anormal de compensation entre les isoformes par rapport aux complexes formés et fonctions spécifiques chez les cellules CCR; et 5) une implication de la voie MEK/Erk compliquerait d’autant plus l’investigation des rôles des complexes PI3-K chez les cellules exhibant un degré élevé de résistance à l’anoïkose. Somme toute, cela laisse entrevoir que la progression dans la résistance à l’anoïkose implique une complexité plus importante qu’anticipée, chez les cellules CCR. / Abstract : The Phosphatidylinositol-3 kinase (PI3-K) is a lipid kinase enzyme complex formed of a catalytic (C) and a regulatory (R) subunit. To date, class I PI3-K members include five R (p85α, p55α, p50α, p85β et p55γ) and four C (p110α, p110β, p110δ et p110γ). Many studies already highlighted the crucial roles of the PI3-K/Akt pathway in many, if not all, cell processes, including cell survival. Moreover, PI3-K/Akt constitutes one of the most altered pathways in cancer, including in colorectal cancer (CRC). Incidentally, it is well established that the acquisition of anoikis resistance constitutes a crucial and limiting step in the progression of many cancers, including once again, in CRC. We have already demonstrated that PI3-K isoform complexes are selectively engaged by the anoikis-suppressing integrins/FAK/Src signaling and that this translates in distinct roles in normal enterocytes survival, according to their state of differentiation. This establishes a basis for the working hypothesis of the present report which is that PI3-K isoform complexes distinctly contributes to the acquisition and maintenance of anoikis resistance in human CRC cells. The main goals were as follows: 1) To establish the expression profiles of PI3-K isoform complexes in human CRC cells displaying differing anoikis resistance degrees; and 2) To define the functional contributions of said PI3-K isoform complexes in the maintenance of anoikis resistance. Our results indicate that 1) the expression profiles of PI3-K isoform complexes are distinct between human CRC cells displaying differing anoikis resistance degrees; 2) these PI3-K isoform complexes can perform, or not, roles in cell survival regardless of the anoikis resistance degree displayed; 3) the observed distinctions of the expression profiles of PI3-K isoform complexes do not correlate with the progression of anoikis resistance; 4) there seems to be an abnormal compensation mechanism between the various PI3-K isoforms linked not only with the nature of the complexes that are formed, but to their specific functions as well; and 5) the MEK/Erk pathway seems implicated in a cross-talk signaling loop, the nature of which further complexifies the investigation of the roles of PI3-K isoform complexes in human CRC cells displaying differing anoikis resistance degrees. Taken together, these observations suggest a more complex network of interactions than previously thought in human CRC cells.

Increased understanding of the molecular mechanisms underlying cancer development has shifted drug discovery towards target driven drug development the last decades, but the development of effective cancer drugs has been hampered by the lack of predictive preclinical models. 3-D cultures, considered to more accurately reflect solid tumors in vivo, have been proposed as one way to increase the predictability of clinical efficacy in cancer drug discovery and development. The aims of this thesis were to improve preclinical models for cancer drug development, with focus on colorectal cancer (CRC) and use of multicellular tumor spheroids (MCTS), and also to mechanistically characterize some potentially new anticancer drugs (papers I – IV). The most important technical improvement was the development of direct measurement of green fluorescent protein (GFP) marked cells in spheroids, simplifying live collection of viability data and enabling high-throughput screening (HTS) in the MCTS model (paper I). In paper III and IV, the 3-D model was adapted to enable studies on the interaction between drugs and radiation. Two potentially new anticancer drugs, VLX50 and VLX60, were mechanistically characterized. VLX60, a novel copper containing thiosemicarbazone, induced reactive oxygen species (ROS) formation, was selectively active against BRAF mutated colon cancer cells and exhibited anticancer activity in vivo (paper II). Furthermore, two potentially new anticancer drugs were found suitable for further development for use in combination with radiation (papers III and IV). In paper III, synergy with radiation in spheroids compared to monolayer cultured colon cancer cells was shown with the novel iron-chelating inhibitor of oxidative phosphorylation, VLX600. In paper IV, the antiprotozoal drug nitazoxanide was shown to sensitize quiescent clonogenic colon cancer cells to radiation. In conclusion, introduction of measurement of fluorescence of GFP marked cells in spheroids makes clinically relevant 3-D models feasible for HTS experiments and characterization of candidate drugs and radiosensitizers in early cancer drug discovery and development. VLX60 has several characteristics suitable for further development into a cancer drug, notably against BRAF mutated colorectal cancer cells. VLX600 and nitazoxanide show radiosensitizing properties making them promising for further development for use as cancer drugs in combination with radiation.

L’irinotecan est un agent anticancéreux majeur utilisé dans le traitement du cancer du côlon où il agit à la fois comme un agent causant des dommages à l’ADN et un inhibiteur de l’angiogenèse. L’activité de l’irinotécan dans les cancers colorectaux est limitée par le développement d’une résistance acquise au médicament. Le but de ce travail est de caractériser l’influence d’une exposition prolongée à l’irinotécan sur la biologie des cellules cancéreuses colorectales in vivo et in vitro. Résultats majeurs : Nous avons exposé les cellules de cancer colorectal, HT-29 (LOH) et HCT-116 (MSI), au SN-38 (le métabolite actif de l'irinotécan) pendant un an. Ceci a permis de sélectionner des cellules résistances à l’irinotécan et ceci de manière stable. Une caractérisation plus approfondie a révélé que la résistance acquise à l’irinotécan était accompagnée d’altérations multiples, y compris la diminution de la formation de complexes clivables et de cassures double brins. Nos études ont montré des changements inattendus dans la distribution du cycle cellulaire et la vitesse de croissance des deux lignées résistantes. La pertinence in vivo de nos modèles cellulaires a ensuite été confirmée dans des modèles de xénogreffe.D’autres résultats basé sur l’inhibition de l’angiogenèse montrent que l'exposition prolongée à l’irinotécan est accompagnée d’une modification majeure de la voie HIF, VEGF / VEGFR dans les cellules tumorales. Ces travaux ont permis d’identifier des modifications biologiques dans les cellules résistantes susceptibles de les rendre plus résistantes ou sensibles à d’autres classes d’agents anticancéreux afin de proposer de nouvelles combinaisons thérapeutiques.Ce travail est financé par l’Institut National du Cancer / Colorectal cancer (CRC) is one of the most common tumors and a leading cause of cancer death worldwide. Until recently, fluorouracil in combination with leucovorin was the only effective systemic treatment for CRC. During the last few years, four new treatments have been approved for advanced CRC including the topoisomerase I (topo I) inhibitor irinotecan, the epidermal growth factor receptor (EGFR)-directed monoclonal antibody cetuximab and the vascular endothelial growth factor (VEGF)-directed monoclonal antibody bevacizumab. Unfortunetly their clinical activity is often limited by the development of resistance. Several lines of experimental evidence suggest a functional interaction between topo I and EGFR. Furthermore, topo I can regulate hypoxia-inducible factor 1 alpha (HIF-1alpha), a key regulator of cellular response to hypoxia, leading to VEGF production.We propose to study the EGFR-signaling pathway on the cellular response to cytotoxic agents with the topo I inhibitor irinotecan/SN-38 as model.Resistant cells were obtained by culturing HT-29 and HCT-116 cells in increasing concentrations of irinotecan. After obtaining, their drug resistance was 5 to 25 times increased compared to the parental cells. We first showed that the proliferation of the two resistant cell lines was slower than the sensitive ones. We were able to confirm it with different in vitro and in vivo experiment. It is interesting to notice that topo I expression was unchanged in our resistant cell lines. We then demonstrated that the resistant cells are less affected by the formation of DNA strand breaks (led by SN38) than the sensitive ones. Moreover, the EGFR expression is increased in the resistant cell lines. We also shown by western blot and ELISA that resistance development comes along with an increase of VEGF.Our data should provide important clues on the irinotecan acquired resistant cells and should help to set up new combinations for colorecal cancer treatment.

Fear of cancer recurrence (FCR) is regarded as one of the most common and distressing issues affecting cancer survivors. Observational studies have identified several modifiable characteristics associated with FCR. However, many of the findings are based on post-hoc analyses and come from studies in which FCR was not identified as a primary outcome. This study sought to overcome these limitations by using a model comprised of cognitive, behavioral, and social characteristics as a framework for examining modifiable characteristics associated with FCR. A sample of 120 patients who had been diagnosed with colorectal cancer and completed cancer treatment in the past 6 to 36 months was recruited during routine outpatient visits or by mail for participation in the study. Medical record reviews were conducted to assess clinical variables, and participants filled out a standard demographic questionnaire as well as self-report measures of characteristics resistant to modification (perceived risk of recurrence, neuroticism, conscientiousness), cognitive modifiable characteristics (self-efficacy, positive beliefs about worry, negative beliefs about worry, misinterpretation of symptoms, intolerance of uncertainty and rumination), behavioral modifiable characteristics (reassurance seeking and health-related reassurance seeking), and social modifiable characteristics (social support and social constraints). As hypothesized, results demonstrated that modifiable characteristics (i.e., self efficacy, positive beliefs about worry, negative beliefs about worry, intolerance of uncertainty, rumination, reassurance seeking, health-related reassurance seeking and social constraints) were associated with FCR (all p’s < .05). Multivariable regression analyses demonstrated that modifiable characteristics accounted for 13% of the variance in FCR beyond that accounted for by non-modifiable characteristics (p < .001), with self-efficacy, rumination and health-related reassurance seeking accounting for unique variance in FCR. This study has identified several modifiable characteristics that should be considered as targets for interventions seeking to reduce FCR among cancer survivors.

Access to bevacizumab, an important component of oncology treatment regimens, may be limited. This survey of oncologists in the US (n = 150), Europe (n = 230), and emerging markets (EM: Brazil, Mexico, and Turkey; n = 130) examined use of and barriers to accessing bevacizumab as treatment of advanced solid tumors. We also assessed the likelihood that physicians would prescribe a bevacizumab biosimilar, if available. Bevacizumab was frequently used as early-line therapy in metastatic colorectal cancer, metastatic non-squamous non-small-cell lung cancer, and metastatic ovarian cancer (all markets), and as a second-line therapy in glioblastoma multiforme (US, EM). A greater percentage of EM-based physicians cited access-related issues as a barrier to prescribing bevacizumab versus US and EU physicians. Lack of reimbursement and high out-of-pocket costs were cited as predominant barriers to prescribing and common reasons for reducing the number of planned cycles. Overall, similar to 50% of physicians reported they "definitely" or "probably" would prescribe a bevacizumab biosimilar, if available. Efficacy and safety data in specific tumor types and lower cost were factors cited that would increase likelihood to prescribe a bevacizumab biosimilar. A lower cost bevacizumab biosimilar could address the unmet needs of patients and physicians worldwide, and may have the greatest impact on patient outcomes in EM.

La voie de signalisation WNT/PCP, couramment associée à la polarité planaire cellulaire, joue un rôle fondamental dans la morphogenèse chez les vertébrés. Parmi les différents composants protéiques de la voie WNT/PCP, on retrouve la protéine tyrosine kinase 7 (PTK7), dont les fonctions restent encore très peu décrites. Au cours de ma thèse, j’ai montré que PTK7 interagissait avec le récepteur tyrosine kinase ROR2. Ce complexe, après fixation du ligand WNT5A, induit la migration de fibroblastes embryonnaires murins via l’activation de JNK. Au cours du développement embryonnaire du xénope, Ptk7 interagit de manière fonctionnelle avec Ror2, et contrôle l’expression de la protocadhérine Papc ainsi que la morphogénèse. De plus, en utilisant une approche de Tissue MicroArray, réalisée sur des patients atteints de cancers colorectaux, j’ai pu montrer que PTK7 était retrouvé surexprimé chez 34% des patients, et que cette surexpression était un facteur de mauvais pronostic. Dans des lignées cellulaires issues de cancers colorectaux, la suppression de PTK7 par shRNA entraine une diminution de la migration des cellules tumorales, mais n’impacte pas leur prolifération et leur résistance aux drogues anticancéreuses. Dans un modèle de xénogreffe murin, la suppression de PTK7 induit une diminution du développement tumoral et l’expression de ce dernier, dans des cellules négative pour PTK7, entraine une augmentation de l’apparition de métastases chez les animaux injectés. Ce travail apporte de nouveaux éclaircissements sur le du récepteur PTK7 dans la voie de signalisation WNT/PCP, et le définit comme potentiel biomarqueur et cible thérapeutique dans le cancer colorectal. / The non-canonical WNT/planar cell polarity (WNT/PCP) pathway plays important roles in morphogenetic processes in vertebrates. Among WNT/PCP components, protein tyrosine kinase 7 (PTK7) is a tyrosine kinase receptor with poorly defined functions lacking catalytic activity. We show that PTK7 associates with receptor tyrosine kinase-like orphan receptor 2 (ROR2) to form a heterodimeric complex in mammalian cells and physically and functionally interact with the non-canonical WNT5A ligand, leading to JNK activation and cell movements. In the Xenopus embryo, Ptk7 functionally interacts with Ror2 to regulate protocadherin papc expression and morphogenesis. Furthermore, we show that Ptk7 is required for papc activation induced by Wnt5a and that Wnt5a stimulates the release of the Ptk7 intracellular domain, which can translocate into the nucleus and activate papc expression. Moreover, using a Tissue MicroArray produced from CRC patients we correlated PTK7 expression with pathological features and patient outcome. PTK7 was significantly up-regulated in CRC tissue, and its overexpression was found in 34% of patients. In CRC cell lines, shRNA PTK7 reduced migration, but did not affect cell proliferation and resistance to drugs. In a xenograft mouse model, downregulation of PTK7 led to reduced tumor growth, whereas its overexpression in PTK7-negative cancer cells led to increased metastatic events. This work reveals novel molecular mechanisms of action of PTK7 in non-canonical WNT/PCP signaling that may promote cell and tissue movements and define PTK7 expression as a potential prognostic biomarker and a novel therapeutic target in CRC.

Thrombosis and colorectal cancer have a bi-directional relationship. The presence of a colorectal malignancy results in an increased risk of developing a thrombosis and the presence of a thrombosis results in a worse cancer prognosis. The physiology causing this is at present unclear but it is proposed that proteins from the tissue factor (TF) pathway may be the instigator of this bi-directional relationship. The in-vitro studies have shown that in colorectal cancer TF impairs that action of colorectal cancer stem cells as demonstrated by reduced cancer sphere formation and also lower expression of the stem cell marker ALDH. The ability for a colorectal cell to avoid anoikis is impaired by a reduced TF level. Proliferation is affected by the level of expression of TF with a significant increase in proliferation with additional expression of TF. The increase in proliferation is further increased by the presence of TF’s ligand factor VIIa. Paradoxically reduced expression of TF also increases colorectal cancer expression. The ERK1/2 pathway offers a possible method by which TF and factor VIIa may exert their proliferative effects. In the prospective clinical cohort study (CHAMPion) abnormal expression of TF pathway proteins (TF, PAR1, PAR2 and thrombin) by both malignant epithelial and cancer associated stromal cells has been demonstrated. The stromal expression was independent of the epithelial expression and was only in stroma in close contact (0.1mm) with epithelial cells suggesting that the TF pathway proteins may have a role in stromal/epithelial communication. There was no link between the expression of TF pathway proteins and clinicopathological markers of a poor prognosis. The plasma expression of markers of TF pathway activation did not demonstrate any role as a biomarker for colorectal cancer or prognosis. The CHAMPion study has demonstrated that 7% of patients undergoing surgery for colorectal cancer have asymptomatic pre-operative DVTs present. A further 6% who were DVT free pre-operatively developed a DVT in the peri-operative period despite receiving venous thromboprophylaxis in line with current national guidelines. Pre-operative d-dimer may have the potential to identify those patients at risk of a post-operative VTE.This thesis establishes the role that TF has in promoting proliferation and anoikis resistance. It also confirms the abnormal expression of TF pathway proteins by colorectal cancer epithelial cells and for the first time demonstrates abnormal expression by the cancer associated stroma. The interaction between the stroma and epithelial cells, combined with the cellular effects of TF suggests that targeting this interaction may have a therapeutic role. The incidence of DVTs pre-operatively suggests that screening patients for the asymptomatic presence of a DVT may have an impact on their clinical outcome. The development of DVTs despite prophylaxis suggests that the level of anticoagulation is insufficient and current guidelines need to be revisited.

Notre équipe étudie les HSP, et notamment HSP110. Les HSP sont des chaperons impliqués dans le repliement des protéines nouvellement synthétisées et dénaturées. Les HSP sont surexprimées lors des stress et participent à la survie des cellules par leurs propriétés anti-apoptotiques et anti-agrégations. HSP110 est surexprimée dans le cancer colorectal et est associée à un mauvais pronostic. L’expression d’un mutant d’HSP110, nommé HSP110DE9, a été mise en évidence dans les cancers colorectaux de type MSI. Celui-ci y agit comme un dominant négatif, en se liant à HSP110 et en inhibant ses fonctions. Son expression sensibilise les cellules cancéreuses à la chimiothérapie et est associée à un bon pronostic chez les patients.Je me suis tout d’abord intéressé au rôle d’HSP110 dans la régulation de la voie oncogénique STAT3. Son activation est en effet associée à un mauvais pronostic, par l’induction de gènes impliqués dans la prolifération et la survie. La protéine HSP110 favorise la prolifération des cellules colorectales cancéreuses à travers cette voie. HSP110DE9 en revanche l’inhibe. Je me suis ensuite intéressé au rôle d’HSP110 sur la polarisation des macrophages dans le cancer colorectal. Celle-ci peut être sécrétée par les cellules cancéreuses et induit une polarisation pro-tumorale des macrophages. HSP110DE9, en bloquant la sécrétion d’HSP110, conduit en revanche à une polarisation pro-inflammatoire. L’effet d’HSP110 sur la polarisation implique le récepteur TLR4.L’ensemble de ces résultats montrent le rôle d’HSP110 dans la progression tumorale. HSP110 apparaît comme une cible thérapeutique dans le traitement du cancer colorectal. / Our team studies HSPs, including HSP110. HSPs are chaperones involved in the folding of newly synthesized and denatured proteins. HSPs are overexpressed under stress conditions and are involved in cell survival thanks to their anti-apoptotic and anti-aggregation functions. HSP110 is overexpressed in colorectal cancer and is associated with a poor prognosis. The expression of a mutant HSP110, named HSP110DE9, has been shown in MSI colorectal cancer. This one was shown to act there as a dominant negative, by binding HSP110 and inhibiting its functions. Its expression sensitizes cancer cells to chemotherapy and is associated with a better prognosis for patients.I was first interested in HSP110 role in regulating the oncogenic STAT3 pathway. Its activation is associated with a poor prognosis, as it induces the transcription of genes involved in proliferation and survival. HSP110 favors colorectal cancer cell proliferation through this pathway. Conversely, HSP110DE9 inhibited it.I then focused on the role of HSP110 on macrophage polarization in colorectal cancer. HSP110 can be secreted by cancer cells and induces a pro-tumoral macrophage polarization. In contrast, HSP110DE9, by inhibiting HSP110 release, leads to a pro-inflammatory polarization. HSP110 effect on macrophage polarization involve the TLR4 receptor.All these results show HSP110 role in tumor progression. HSP110 appear as a therapeutic target in the treatment of colorectal cancer.