Die antiangioneogenetischen Effekte in-vitro und in-vivo in Kombination mit der selektiven Inhibition der TNFalpha Produktion durch Thalidomid läßt dieses Medikament als geeignete Therapie für Krankheitszustände erscheinen, bei denen die TNFalpha Toxizität eine pathogenetische Rolle spielt, die Immunität jedoch intakt bleiben muß. Ziel dieser Monographie waren Untersuchungen der Wirkungen von Thalidomid in vitro, in vivo und klinische Studien. In einer prospektiv-randomisierten, doppel-blind Studie an 70 Patienten mit kleinzelligem Bronchialkarzinom konnte die Verlängerung der Überlebenszeit durch die additive Therapie mit Thalidomid in Kombination mit einer Standardpolychemotherapie und Strahlentherapie nachgewiesen werden. Vor dem additiven Einsatz des Angioneogenesehemmers Thalidomid in der Chirurgie muß unbedingt sichergestellt sein, daß die Therapie mit Thalidomid nicht zu einer erhöhten postoperativen Morbidität und Letalität führt. Durch in-vitro Untersuchungen konnte eine Proliferationshemmung von Kaninchenendothelzellen durch metabolisiertes Thalidomid bewiesen werden. In einer randomisierten Studie an New Zealand White Kaninchen konnte gezeigt werden, daß die intraperitoneale Gabe von Thalidomid den Berstungsdruck von Kolonanastomosen im Kaninchenmodell im Vergleich zu einer Kontrollgruppe nicht erniedrigt. Darüberhinaus zeigte sich in diesem Tiermodell, daß die Rate an postoperativen Verwachsungen durch die intraperitoneale Gabe von Thalidomid signifikant vermindert wird. Das Medikament könnte eine Rolle in der additiven Therapie von Patienten mit einem kleinzelligen Bronchialkarzinom spielen. Darüberhinaus wird die Heilung von Kolonanastomosen nicht durch die intraperitoneale Gabe von Thalidomid negativ beeinflusst. Somit könnte ein Einsatz in der perioperativen Therapie bei der Resektion gastrointestinaler Karzinome in der Zukunft erwogen werden. / The proven antiangiogenic effects in vivo and in vitro in combination with a selective inhibition of the tnf alpha production seem to predestine thalidomide as an agens for diseases with a pathological elevated tnf alpha level. The theses of this monography were in vitro, in vivo and clinical effects of thalidomide. In a prospective randomized placebo controlled study on 70 patients with proven small cell lung cancer the additive treatment with oral thalidomide lead to a significant prolonged survival in combination with radio chemotherapy. Considering the treatment of thalidomide in combination with surgical therapy of patients it is essential to prove that this treatment does not lead to a higher postoperative morbidity of the patients. In an in vitro assay we could prove the antiproliferative effect of metabolized thalidomide in rabbit endothelial cells. We were than able to demonstrate that intraperitoneal administered thalidomide does not impair anastomotic healing of colonic anastomoses in a New Zealand white rabbit model compared to a control group. In addition to this significant less postoperative adhesions were found in the thalidomide group. In conclusion did the additive treatment with thalidomide in patients with small cell lung cancer lead to a prolonged survival. The antiangiogenic treatment with thalidomide in a perioperative setting does not impair the healing of colonic anastomoses in a rabbit model and may be possible in patients undergoing gastrointestinal resections in the future.

Base teórica: O câncer de pulmão é uma doença com alta incidência e mortalidade, cujo prognóstico permanece discreto apesar do melhor entendimento da doença nas últimas décadas. A radioterapia tem papel terapêutico em todos os estágios da doença. A expressão da cofilina-1, uma proteína relacionada à mobilidade celular, determinou maior radiossensibilidade a células de adenocarcinoma brônquico em estudos in vitro, porém pior sobrevida em estádios iniciais Objetivo: Avaliar se a expressão da cofilina-1 interfere na sobrevida e no controle local em pacientes com câncer de pulmão submetidos a tratamento com radioterapia definitiva Métodos: Foram avaliados pacientes com câncer de pulmão não pequenas células, com estádios I–IV, que receberam radioterapia exclusiva ou combinada com quimioterapia, dirigida à neoplasia brônquica, na unidade de radioterapia do HCPA, nos anos de 2009 a 2015. Todos os pacientes tiveram a expressão de cofilina-1 aferida e foram distribuídos conforme os níveis de expressão de cofilina-1 utilizando-se de protocolo específico. Os prontuários foram avaliados retrospectivamente para calcular a sobrevida mediana. A progressão foi verificada através de avaliação de tomografias de tórax de controle. Resultados: Foram avaliados 45 pacientes neste estudo. A sobrevida mediana de todos os pacientes foi de 11,3 meses e a sobrevida global em 5 anos de 17,3%. Pacientes com expressão média ou alta de cofilina-1 tiveram maior mortalidade quando comparados com pacientes com baixa expressão (respectivamente, HR 1,628, IC95 1,137-8,287 e HR 1,59 IC95 1,105-7,342). Não houve diferença significantemente estatística entre controle local e expressão de cofilina-1. Conclusão: A expressão de cofilina-1 está associada à sobrevida em pacientes com carcinoma brônquico tratados com radioterapia e existe uma tendência a melhor controle local com baixa expressão. Nossos resultados sugerem um novo campo a ser explorado no manejo do carcinoma de pulmão localmente avançado, utilizando-se dos níveis de cofilina-1. / Background: Lung cancer is a disease with high incidence and mortality, whose prognosis remains poor despite a better understanding of the disease in the last decades. Radiotherapy plays a therapeutic role in all stages of disease. The expression of cofilin-1, a protein related to cellular mobility, determined greater radiosensitivity to lung adenocarcinoma cells in in vitro studies, but worse survival at initial stages. Objective: To evaluate if the expression of cofilin-1 modified survival and local control in lung cancer patients submitted to definitive treatment with radiotherapy. Methods: Patients with non-small cell lung cancer with stage IIV who received radiotherapy alone or combined with chemotherapy for lung cancer at the HCPA radiotherapy unit from 2009 to 2015 were evaluated. All patients had the expression of measured cofilin-1 evaluated and were distributed by cofilin-1 expression according to specific protocol. The medical records were retrospectively evaluated to estimate median survival. The progression was verified through evaluation of control chest tomography. Results: 45 patients were assessed in this study. The median survival of all patients was 11.3 months and the 5-year overall survival was 17.3%. Patients with medium or high expression of cofilin-1 had higher mortality rates when compared to patients with low expression (HR, 1,628, CI95, 1,137-8,287 and HR, 1.59, CI95, 1,105-7,342). There was no statistically significant difference between local control and cofilin-1 expression. Conclusion: cofilin-1 expression is associated with survival in patients with lung cancer treated with radiotherapy and there is a tendency for better local control with low CFL1 expression. Our results suggest a new field to be explored in the management of locally advanced lung carcinoma, using cofilin-1 expression levels.

Excision Repair Cross-Complementation group 1 (ERCC1) est une enzyme de réparation de l’ADN fréquemment déficiente dans le cancer bronchique non-à-petites cellules. Bien qu’une expression faible d’ERCC1 soit prédictive de réponse aux sels de platine, l’efficacité des chimiothérapies à base de platine est limitée par leur toxicité et l’apparition de résistance, justifiant la nécessité de stratégies thérapeutiques alternatives. Par ailleurs, l’absence de test compagnon diagnostic permettant d’évaluer la fonctionnalité d’ERCC1 dans la pratique clinique empêche actuellement toute thérapie personnalisée basée sur le statut ERCC1.Afin d’identifier de nouvelles stratégies thérapeutiques pour les tumeurs ERCC1-déficientes en exploitant le concept de létalité synthétique, des screens à haut-débit , utilisant des composés pharmaceutiques ou par ARN interférence, ont été réalisés dans un modèle isogénique de CBNPC déficient en ERCC1. Cette approche a permis d’identifier plusieurs inhibiteurs de poly(ADP-ribose) polymerase 1 et 2 (PARP1/2), tels l’opalarib (AZD2281), le niraparib (MK-24827) et BMN 673 comme sélectifs pour les cellules ERCC1-déficientes. Les mécanismes sous-tendant cette sensibilité sélective ont été étudiés, et les résultats suivants ont été mis en évidence : (i) les cellules ERCC1-déficientes présentent un blocage prolongé en phase G2/M après exposition à l’olaparib ; (ii) l’isoforme 202 d’ERCC1, dont le rôle a été récemment mis en évidence dans la résistance aux sels de platine, module également la sensibilité aux inhibiteurs de PARP ; (iii) la déficience en ERCC1 est épistatique avec les défauts de recombinaison homologue (RH), malgré une capacité normale des cellules ERCC1-déficientes à former des foyers RAD51 ; ceci suggère qu’ERCC1 pourrait intervenir dans la réparation d’une lésion de l’ADN induite par l’inhibiteur de PARP1/2 en amont de l’invasion du brin d’ADN lors de la RH ; (iv) l’inhibition de l’expression de PARP1 par ARN interférence permet de restaurer la résistance aux inhibiteurs de PARP1/2, dans les cellules ERCC1-déficientes uniquement. Ces résultats suggèrent que les inhibiteurs de PARP1/2 pourraient représenter une nouvelle stratégie thérapeutique chez les patients dont la tumeur est déficiente en ERCC1 et un essai clinique va être mis en place pour évaluer cette hypothèse.Afin d’explorer la présence de biomarqueurs de la fonctionnalité d’ERCC1, quatre approches ont été entreprises en parallèle dans le modèle isogénique de CBNPC déficient en ERCC1: (i) irradiation aux UV, afin d’évaluer la voie NER (Nucleotide Excision Repair); (ii) séquençage d’exome, dans le but de rechercher une signature génomique (ADN) ; (iii) analyse du transcriptome cellulaire, pour identifier des modifications d’expression d’ARN ; et (iv) SILAC (Stable Isotope Labeling by Amino acids in Cell culture) afin de comparer le protéome des cellules ERCC1-déficientes et ERCC1-proficientes. Ces approches ont permis d’identifier une potentielle signature génomique, ainsi que de biomarqueurs d’activité – guanine deaminase (GDA) et nicotinamide phosphoribosyltransferase (NAMPT). De plus amples validations et investigations mécanistiques de ces observations préliminaires sont actuellement requises. / Excision Repair Cross-Complementation group 1 (ERCC1) is a DNA repair enzyme that is frequently deficient in non-small cell lung cancer (NSCLC). Although low ERCC1 expression correlates with platinum sensitivity, the clinical effectiveness of platinum therapy is limited - mainly by toxicities and occurrence of resistance - highlighting the need for alternative treatment strategies. In addition, the lack of a reliable assay evaluating ERCC1 functionality in the clinical setting currently precludes personalising therapy based on ERCC1 status. To discover new synthetic lethality-based therapeutic strategies for ERCC1-defective tumours, high-throughput drug and siRNA screens in an isogenic NSCLC model of ERCC1 deficiency were performed. This approach identified multiple clinical poly(ADP-ribose) polymerase 1 and 2 (PARP1/2) inhibitors such as olaparib (AZD-2281), niraparib (MK-4827) and BMN 673 as being selective for ERCC1 deficiency. The mechanism underlying ERCC1-selective effects was dissected by studying molecular biomarkers of tumour cell response, and revealed that: (i) ERCC1-deficient cells displayed a significant delay in double-strand break repair associated with a profound and prolonged G2/M arrest following PARP1/2 inhibitor treatment; (ii) ERCC1 isoform 202, which has recently been shown to mediate platinum sensitivity, also modulated PARP1/2 sensitivity; (iii) ERCC1-deficiency was epistatic with homologous recombination deficiency, although ERCC1-deficient cells did not display a defect in RAD51 foci formation. This suggests that ERCC1 might be required to process PARP1/2 inhibitor induced DNA lesions prior to DNA strand invasion; and (iv) PARP1 silencing restored PARP1/2 inhibitor resistance in ERCC1-deficient cells but had no effect in ERCC1-proficient cells, supporting the hypothesis that PARP1 might be required for the ERCC1 selectivity of PARP1/2 inhibitors. This study indicated that PARP1/2 inhibitors as a monotherapy could represent a novel therapeutic strategy for NSCLC patients with ERCC1-deficient tumours, and a clinical protocol is being written to evaluate this hypothesis.To investigate whether a surrogate biomarker of ERCC1 functionality could be developed, four parallel approaches were undertaken in the ERCC1-isogenic NSCLC model: (i) UV irradiation, to evaluate the Nucleotide Excision Repair (NER) pathway; (ii) whole exome sequencing, to look for an ERCC1-associated genomic scar at the DNA level; (iii) transcriptomic analysis, to investigate changes at the RNA expression level; and (iv) SILAC (Stable Isotope Labeling by Amino acids in Cell culture) analysis, to compare proteomic profiles between ERCC1-proficient and ERCC1-deficient cells. These approaches allowed the identification of putative genomic signature and potential metabolic surrogate biomarkers - guanine deaminase (GDA) and nicotinamide phosphoribosyltransferase (NAMPT). Further validation and mechanistic investigations of these latter preliminary observations are warranted.

No description available.

LKB1 is the second most commonly altered tumour suppressor gene in lung adenocarcinoma, the most prevalent form of lung cancer. LKB1 is a "master kinase" that has been shown to phosphorylate up to 13 downstream targets. We hypothesised that LKB1 loss is associated with an increased dependency on alternative, targetable pathways. The overall aims of this project were to better understand the role of LKB1 loss in lung cancer and to identify novel approaches to selectively target LKB1 mutated cells. We generated isogenic cells with or without LKB1 and used these to study the effect of LKB1 on cell proliferation. Importantly, we used a range of models including 2D culture, 3D spheroids and, sub-cutaneous and orthotopic xenograft models. To understand the role of LKB1 loss in lung cancer, the effect of LKB1 on mRNA expression was analysed using whole genome RNA Sequencing. To identify novel approaches to selectively target LKB1 mutated cells, we used biological screening methods and also investigated the effect of several metabolic inhibitors. We found that loss of LKB1 expression had no effect on cell proliferation in 2D culture, but was associated with increased growth in 3D spheroids, sub-cutaneous and orthotopic xenografts, as well as greater metastasis in a lung orthotopic model. Gene ontology analysis of the transcriptome identified that genes associated with cAMP signalling and cytoskeletal organisation were differentially expressed between LKB1 deficient and proficient cells. We confirmed that cAMP signalling was increased in LKB1 deficient cells, though there was no difference in sensitivity between LKB1 deficient and proficient cells to cAMP signalling modulators. The bioactive small molecule screen showed that LKB1 deficient cells underwent apoptosis more slowly and therefore, were less sensitive to many compounds, compared with LKB1 proficient cells. Screening in 3D spheroids was a novel approach that we used to identify microtubule inhibitors as potentially selective compounds acting in LKB1 deficient cells. Our RNASeq data suggests that there was a metabolic shift from oxidative phosphorylation to aerobic glycolysis in LKB1 deficient cells, although this did not affect sensitivity to complex I inhibitors. Importantly, LKB1 deficient cells were more sensitive to glucose and glutamine deprivation which suggests that targeting these metabolic pathways may hold the greatest promise to selectively inhibit proliferation in LKB1 mutated cells.

Orientador: Maria Isabel Nogueira Cano / Resumo: O câncer de pulmão é o câncer que apresenta maior mortalidade no mundo: >1,6 milhões de óbitos por ano segundo a Organização Mundial de Saúde (OMS), sendo o câncer de pulmão de células não pequenas (NSCLC) o tipo mais frequente (~ 85%) de carcinoma pulmonar. O NSCLC apresenta dois subtipos histológicos principais: adenocarcinoma (LUAD) e carcinoma de células escamosas (LUSC). Pesquisas têm sido realizadas objetivando identificar biomarcadores moleculares úteis como alvo terapêuticos em NSCLC e os telômeros têm sido considerado alvos promissores para terapias antitumoral, devido ao seu papel crucial na integridade, estabilidade e manutenção genômica. Telômeros humanos consistem em DNA repetitivo rico em G associado a proteínas do complexo shelterin e mantido pela ação da telomerase. Estudos recentes mostraram que a expressão de TERT (subunidade catalítica da telomerase) e de alguns componentes do complexo shelterin estão alterados em câncer de pulmão. No entanto, a correlação entre alterações na função telomérica e o desenvolvimento e progressão de NSCLC não foi elucidada. Portanto, o objetivo do presente estudo foi verificar alterações na expressão de genes associados aos telômeros, incluindo os ncRNAs associados à regulação e manutenção dos telômeros em NSCLC, no intuito de identificar novos biomarcadores associados ao desenvolvimento e progressão do NSCLC. Para tanto, foram realizadas análises de expressão de 50 genes associados aos telômeros em amostras de tecido tumoral e... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: According to the World Health Organization, lung cancer has a high mortality rate associated with >1.6 million deaths per year, being Non-small cell lung cancer (NSCLC) the most frequent type (~ 85%) of lung carcinoma. Lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) are the two major histological subtypes of NSCLC. Research efforts have been made to identify molecular biomarkers useful as therapeutic targets in NSCLC and telomere have been considered very promising targets for anticancer therapies due to their crucial role in genome integrity maintenance and stability. Human telomeres consist of repetitive G-rich DNA associated with proteins of the shelterin complex, maintained by the action of telomerase. Recent studies showed that expression of TERT (telomerase reverse transcriptase component) and of some of the shelterin components are altered in lung cancer. However, the mechanisms of telomere deregulation associated with NSCLC development and progression have not been elucidated. Therefore, our aim was to study expression changes affecting telomere-associated genes and ncRNAs associated with telomere regulation and maintenance in NSCLC. Such alterations may represent novel biomarkers associated with NSCLC development and progression. For this purpose, expression analyzes of 50 telomere-associated genes were performed in samples of tumor tissue and normal lung tissue, adjacent to the tumor, from patients with NSCLC. The following techniques were used: 1... (Complete abstract click electronic access below) / Mestre

Introduction : Le traitement des cancers bronchiques non à petites cellules (CBNPC) EGFR mutés repose sur les inhibiteurs de tyrosine kinase (ITK) du récepteur de l’Epidermal Growth Factor (EGFR). Cependant tous les patients traités par ITK EGFR finissent par présenter une progression tumorale, du fait de mécanismes de résistance comme l’amplification du gène codant pour le récepteur tyrosine kinase MET. Il n’existe actuellement aucune donnée sur les modifications phénotypiques induites par l’activation de MET dans ce contexte. L’objectif de cette thèse est de déterminer si l’amplification de MET, lors de la résistance aux ITK EGFR dans les CBNPC EGFR mutés, confère aux cellules tumorales un phénotype plus agressif et modifie l’histoire naturelle de la maladie.Méthodes : Les capacités de prolifération, de croissance sans ancrage, de formation de sphéroïdes, de résistance à l’anoïkis et de migration ont été étudiées in vitro dans la lignée HCC827, dérivée d’un CBNPC EGFR muté, et dans sa lignée fille HCC827-GR6 (GR6) devenue résistante aux ITK EGFR via une amplification du gène MET. L’expression de la vimentine, de ZEB1, et de la E-cadherine a également été étudiée dans les deux lignées cellulaires afin d’évaluer l’impact de l’amplification de MET sur la transition épithélio-mésenchymateuse (TEM). In vivo la croissance tumorale et le potentiel métastatique ont respectivement été analysés dans des modèles murins de xénogreffe ectopique et d’injection intracardiaque. Enfin les données cliniques de patients issus de 15 centres avec un CBNPC EGFR muté métastatique, présentant une forte surexpression de MET en immunohistochimie (score 3+) ou une amplification de MET en FISH sur une re-biopsie réalisée après la progression sous ITK EGFR ont été analysées rétrospectivement. Résultats : In vitro, l’amplification de MET induisait une augmentation significative de la prolifération, de la croissance sans ancrage, de la formation de sphéroïdes, de la résistance à l’anoïkis et de la migration. En présence d’un inhibiteur de MET, le PHA-665752, ces différentes propriétés biologiques étaient réduites de façon significative dans les cellules GR6 porteuses de l’amplification de MET. Il était également mis en évidence dans les cellules GR6 une augmentation de l’expression de la vimentine et de ZEB1. In vivo, l’amplification de MET augmentait significativement la croissance tumorale et le potentiel métastatique. Un traitement par crizotinib, ITK ciblant MET, diminuait de façon significative le potentiel métastatique des cellules porteuses de l’amplification de MET. Enfin les patients atteints d’un CBNPC EGFR muté, porteur d’une amplification de MET à la résistance à l’ITK EGFR, présentaient une durée jusqu’à apparition de nouvelles métastases plus courte après progression sous ITK EGFR que les patients avec une forte surexpression de MET sans amplification génique. Conclusion L’amplification de MET dans un contexte de résistance aux ITK EGFR est associée à un phénotype tumoral plus agressif. Ces résultats plaident en faveur d’une utilisation précoce d’inhibiteurs de MET en association avec les ITK EGFR afin d’éviter l’émergence d’un clone tumoral résistant plus agressif. / Introduction: Treatment of Epidermal Growth Factor Receptor (EGFR) mutated non-small cell lung cancers (NSCLC) relies on EGFR tyrosine kinase inhibitors (TKI). However, all patients treated with EGFR TKI eventually present tumor progression, due to mechanisms of resistance such as the MET amplification. There is currently no data on phenotypic changes induced by MET activation in this context. The objective of this thesis is to determine whether the MET amplification during EGFR TKI resistance in the EGFR mutated NSCLC induces a more aggressive phenotype in tumor cells and alters the natural history of the disease.Methods: Proliferation, anchorage independent growth, spheroid formation, anoïkis resistance and migration were studied in vitro in the HCC827 cell line, derived from an EGFR mutated NSCLC, and in its daughter cell line HCC827-GR6 (GR6) which became resistant to EGFR TKI through MET amplification. The expression of vimentin, ZEB1, and E-cadherin was evaluated in these cellular models in order to investigate an epithelial to mesenchymal transition (EMT) process induced by the MET amplification. In vivo, the tumor growth and the metastatic potential were analyzed by subcutaneous xenograft and intracardiac injection in mouse models. Finally, the clinical data of patients from 15 centers with a metastatic EGFR mutated NSCLC, exhibiting high MET overexpression in immunohistochemistry (score 3+) or MET amplification assessed by FISH on a re-biopsy performed after TKI EGFR progression were analyzed retrospectively.Results: In vitro, the MET amplification induced a significant increase in proliferation, anchorage independent growth, spheroid formation, anoïkis resistance and migration. Treatment with PHA-665752, a MET TKI, significantly reduced these biological properties in the GR6 cells harboring the MET amplification. An increase in the expression of vimentin and ZEB1 was also observed in the GR6 cells. In vivo, the MET amplification significantly increased the tumor growth and the metastatic potential. Treatment with crizotinib, another MET TKI, significantly decreased the metastatic potential of cells carrying MET amplification. Finally, patients with an EGFR mutated NSCLC, displayed a time to new metastases after TKI EGFR progression shorter than patients with high MET overexpression without MET amplification.Conclusion: The MET amplification during EGFR TKI resistance is associated in EGFR muted NSCLC with a more aggressive tumor phenotype. These results argue for the early use of MET inhibitors in combination with EGFR TKIs to avoid the emergence of a more aggressive resistant tumor clone.

Les cellules NK sont des lymphocytes de l’immunité innée, impliquées dans la reconnaissance et l’élimination de cellules infectées ou tumorales. Leur activité cytotoxique est finement régulée par un ensemble de récepteurs activateurs et inhibiteurs. Cependant, l’expression de ces récepteurs ainsi que les fonctions des cellules NK peuvent être modifiées selon l’environnement dans lequel elles se trouvent. Au cours de ma thèse, nous nous sommes intéressés à la modulation du phénotype et des fonctions des cellules NK, ainsi qu’à leur relation avec les LT CD4, dans une cohorte de patients atteints de NSCLC ou dans un contexte inflammatoire sous l’influence de l’IL-21. La première étude a mis en évidence une augmentation du taux circulant de la population de cellules NK CD56dim CD16- simultanément à une diminution du taux de la population CD56dim CD16+ chez les patients. L’analyse ex vivo du phénotype des cellules NK a mis en évidence un groupe spécifique de patients avec une altération globale de l’expression des NCR et de NKG2D par toutes les sous-populations de cellules NK. La principale modulation correspond à la diminution des cellules NK exprimant NKp46 et nous avons montré une corrélation négative entre la survie des patients et le pourcentage de cellules NK NKp46+. De manière intéressante, la neutralisation de NKp46 sur les cellules NK est associée à une meilleure réponse T CD4 anti-tumorale. La seconde étude a montré que l’IL-21 engendre la différenciation d’une population spécifique de cellules NK co-exprimant CD86/HLA-DR et CD86/CD30. Alors que l’activation des cellules NK via CD30 entraîne leur forte dégranulation et leur sécrétion d’IFN-γ, les cellules NK activées par l’IL-21 produisent également MIF. Ce facteur soluble apporte un signal de co-stimulation lors de l'initiation de l'activation des LT CD4 naïfs, induisant leur différenciation en LT centraux mémoires. De telles cellules NK MIF+ ont été identifiées dans des appendices humains inflammés suggérant qu'elles pouuraient activer des LT CD4 in vivo. Ces études ont permis de mettre en évidence une modulation différente du phénotype de cellules NK selon leur environnement, pouvant impacter le cross-talk NK-LT. Ces données supportent ainsi le rôle régulateur des cellules NK dans la mise en place des réponses immuntaires adaptatives. / NK cells are innate lymphocytes involved in the recognition and elimination of infected or tumor cells. Their cytotoxic activity is finely regulated by a set of activating and inhibitory receptors. However, receptors expression and NK cell functions may be modified according to environment. Here, we were interested in NK cell-phenotype and function modulations and their relationship with CD4 T cells, in NSCLC patients and under IL-21 influence in inflammatory context. The first study highlighted an increase circulating rate of CD56dim CD16- NK cell subset concomitantly with a decrease rate of CD56dim CD16+ NK cell subset in NSCLC patients. Ex vivo analysis of NK cell phenotype highlighted a specific group of patients with an overall altered expression of NCRs and NKG2D on NK cell subsets. The main defect was the decrease of NK cells expressing NKp46 and we showed a negative correlation between the patients’ survival and NKp46+ NK cell percentage. Interestingly, NKp46 neutralization on NK cells was associated with a better antitumor CD4 T cell response. The second study showed that IL-21 promotes the differentiation of a specific NK cell subset coexpressing CD86/HLA-DR and CD86/CD30. While NK cell activation via CD30 promotes a high degranulation and IFN-γ secretion, IL-21-activated NK cells also produce MIF. This soluble factor provide costimulatory signaling during naïve CD4 T cell priming inducing the differentiation of uncommitted central memory T cells. Such HLA-DR+ MIF+ NK cells were identified in inflammatory human appendix suggesting that they could activate CD4 T cells in vivo. Altogether, these studies highlighted a different modulation of NK cell phenotype accordingg to envoronment which could impact the crossstalk NK-T celles. thus, these findings support a regulatory role of NK celles in adaptive immune responses

Base teórica: O câncer de pulmão é uma doença com alta incidência e mortalidade, cujo prognóstico permanece discreto apesar do melhor entendimento da doença nas últimas décadas. A radioterapia tem papel terapêutico em todos os estágios da doença. A expressão da cofilina-1, uma proteína relacionada à mobilidade celular, determinou maior radiossensibilidade a células de adenocarcinoma brônquico em estudos in vitro, porém pior sobrevida em estádios iniciais Objetivo: Avaliar se a expressão da cofilina-1 interfere na sobrevida e no controle local em pacientes com câncer de pulmão submetidos a tratamento com radioterapia definitiva Métodos: Foram avaliados pacientes com câncer de pulmão não pequenas células, com estádios I–IV, que receberam radioterapia exclusiva ou combinada com quimioterapia, dirigida à neoplasia brônquica, na unidade de radioterapia do HCPA, nos anos de 2009 a 2015. Todos os pacientes tiveram a expressão de cofilina-1 aferida e foram distribuídos conforme os níveis de expressão de cofilina-1 utilizando-se de protocolo específico. Os prontuários foram avaliados retrospectivamente para calcular a sobrevida mediana. A progressão foi verificada através de avaliação de tomografias de tórax de controle. Resultados: Foram avaliados 45 pacientes neste estudo. A sobrevida mediana de todos os pacientes foi de 11,3 meses e a sobrevida global em 5 anos de 17,3%. Pacientes com expressão média ou alta de cofilina-1 tiveram maior mortalidade quando comparados com pacientes com baixa expressão (respectivamente, HR 1,628, IC95 1,137-8,287 e HR 1,59 IC95 1,105-7,342). Não houve diferença significantemente estatística entre controle local e expressão de cofilina-1. Conclusão: A expressão de cofilina-1 está associada à sobrevida em pacientes com carcinoma brônquico tratados com radioterapia e existe uma tendência a melhor controle local com baixa expressão. Nossos resultados sugerem um novo campo a ser explorado no manejo do carcinoma de pulmão localmente avançado, utilizando-se dos níveis de cofilina-1. / Background: Lung cancer is a disease with high incidence and mortality, whose prognosis remains poor despite a better understanding of the disease in the last decades. Radiotherapy plays a therapeutic role in all stages of disease. The expression of cofilin-1, a protein related to cellular mobility, determined greater radiosensitivity to lung adenocarcinoma cells in in vitro studies, but worse survival at initial stages. Objective: To evaluate if the expression of cofilin-1 modified survival and local control in lung cancer patients submitted to definitive treatment with radiotherapy. Methods: Patients with non-small cell lung cancer with stage IIV who received radiotherapy alone or combined with chemotherapy for lung cancer at the HCPA radiotherapy unit from 2009 to 2015 were evaluated. All patients had the expression of measured cofilin-1 evaluated and were distributed by cofilin-1 expression according to specific protocol. The medical records were retrospectively evaluated to estimate median survival. The progression was verified through evaluation of control chest tomography. Results: 45 patients were assessed in this study. The median survival of all patients was 11.3 months and the 5-year overall survival was 17.3%. Patients with medium or high expression of cofilin-1 had higher mortality rates when compared to patients with low expression (HR, 1,628, CI95, 1,137-8,287 and HR, 1.59, CI95, 1,105-7,342). There was no statistically significant difference between local control and cofilin-1 expression. Conclusion: cofilin-1 expression is associated with survival in patients with lung cancer treated with radiotherapy and there is a tendency for better local control with low CFL1 expression. Our results suggest a new field to be explored in the management of locally advanced lung carcinoma, using cofilin-1 expression levels.

Submitted by KELVIN FURTADO LIMA null (kelvinf.lima@hotmail.com) on 2016-05-02T17:44:44Z No. of bitstreams: 1 Dissertação - Kelvin.pdf: 3687789 bytes, checksum: d826d05398d0eb1bdb80f3a5d8770738 (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-05-04T18:32:33Z (GMT) No. of bitstreams: 1 lima_kf_me_araiq.pdf: 3687789 bytes, checksum: d826d05398d0eb1bdb80f3a5d8770738 (MD5) / Made available in DSpace on 2016-05-04T18:32:33Z (GMT). No. of bitstreams: 1 lima_kf_me_araiq.pdf: 3687789 bytes, checksum: d826d05398d0eb1bdb80f3a5d8770738 (MD5) Previous issue date: 2016-04-15 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Os diferentes tipos de cânceres estão entre as principais causas de morbidade e mortalidade em todo mundo, contabilizando 8,2 milhões de óbitos no ano de 2012. Dentre eles, o câncer de pulmão é o mais recorrente, sendo que o subtipo denominado câncer pulmonar de células não-pequenas (non-small cell lung cancer – NSCLC) é diagnosticado em 85% dos casos. Dada a importância epidemiológica do câncer pulmonar, associado ao platô atingido pelos tratamentos com quimioterapia e radioterapia, novas estratégias terapêuticas têm sido buscadas e neste contexto, o peptídeo vasoativo Angiotensina-(1-7) [Ang-(1-7)] tem se mostrado bastante promissor. Além da participação em uma gama de processo fisiológicos no organismo, a Ang-(1-7) está sendo cada vez mais exploradas nos processos patológicos devidos suas funções vasodilatadora, anti-hipertensiva, anti-proliferativa e antitumoral. Outros estudos apontam ainda que uma melhor compreensão da regulação gênica nos processo tumorais poderia direcionar o desenvolvimento de outros métodos terapêuticos para o câncer de pulmão. Neste sentido, os microRNAs assumem posição ímpar por sua capacidade regulatória pós-transcriocional de vários genes, a citar o miR-21-5p superexpresso em vários tipos tumorais, incluindo o NSCLC. Portanto, o presente trabalho investigou os efeitos bioquímicos, moleculares e fisiológicos decorrentes do tratamento da linhagem celular tumoral A549 com Ang-(1-7), concomitantemente a análises comparativas dos efeitos da superexpressão do miR-21-5p nas células tumorais. Para isso foram utilizados os grupos celulares A549 controle e tratamento [10-7 M de Ang-(1-7)], e os clones A549-pEP-miR-Controle e A549-pEP-miR-21-5p. Análises bioquímicas mostraram que a presença do heptapeptídeo no grupo tratamento, e as condições de cultura dos clones estudados, causaram um aumento na síntese de lactato pelas células A549. Foi observado também que a Ang-(1-7) retarda o crescimento celular. Análises de expressão gênica não apontaram ativação do processo apoptótico em nenhuma das condições estudadas, entretanto o peptídeo Ang-(1-7) modula positivamente a expressão do gene tjp1 e negativamente a transcrição dos genes itgb8 e mmp-8, sugerindo que o tratamento celular com o heptapeptídeo fortaleça as interações celulares e dificulte os processo de migração e invasão celular. Estes dados são corroborados pelo ensaio de cicatrização da ferida o qual evidenciou que a Ang-(1-7) diminui a taxa de migração celular em comparação ao grupo controle. Por sua vez, a superexpressão do miR-21-5p intensifica consideravelmente a capacidade invasiva das células tumorais como observado no ensaio da gota de agarose. Conjuntamente, os resultados apontam para o potencial terapêutico da Ang-(1-7) no controle dos processos de adesão, migração e invasão celular, e corrobora o papel do miR-21-5p na evolução e intensificação dos processos tumorais. / The different types of cancers are among the leading causes of morbidity and mortality worldwide, accounting for 8.2 million deaths in 2012. Among them, lung cancer is the most frequent, and the subtype called non-small cell lung cancer (NSCLC) is diagnosed in 85% of cases. Given the epidemiological importance of lung cancer, associated with the plateau reached by chemotherapy and radiation therapies, new therapeutic strategies have been pursued and in this context, the vasoactive peptide Angiotensin-(1-7) [Ang- (1-7)] has shown quite promising. Besides participating in a range of physiological process in the body, Ang- (1-7) is being increasingly exploited in pathological processes due to its vasodilator, anti-hypertensive, anti-proliferative and anti-tumor functions. Other studies also indicate that a better understanding of gene regulation in tumor process could direct the development of other therapeutic methods for lung cancer. In this sense, microRNAs assume unique position due to its post-transcriocional regulatory capacity of several genes, for exemple the miR-21-5p upregulated in several tumor types, including NSCLC. Therefore, this study investigated the biochemical, molecular and physiological effects of Ang-(1-7) treatment on tumor cell line A549, concurrently to the comparative analysis of the effects of upregulation of miR-21-5p in tumor cells. In order to do this, the cell groups A549 control and treatment [10-7 M of Ang-(1-7)], and the A549-pEP-miR-control and A549-pEP-miR-21-5p clones were used. Biochemical analyzes showed that the presence of the heptapeptide in treatment group, and the culture conditions of the studied clones, caused an increase in lactate synthesis on A549 cells. It was also observed that Ang-(1-7) slows cell growth. Gene expression analysis showed no activation of apoptotic process in any of the conditions studied, though the peptide Ang-(1-7) positively modulates the expression of tjp1 gene and negatively the transcription of itgb8 and mmp-8 genes, suggesting that the cell treatment with the heptapeptide strengthen cell interactions and prevents cell migration and invasion. These data are corroborated by wound healing assay which showed that Ang-(1-7) decreases cell migration rate compared to the control group. In turn, the upregulation of miR-21-5p considerably enhances tumor cells invasiveness as observed in the agarose spot assay. Collectively, the results point to Ang-(1-7) therapeutic potential in controling cell adhesion, migration and invasion, and supports miR-21-5p role in the evolution and enhancement of tumor processes. / CNPq: 132514/2014-1