O câncer de pulmão está entre os tipos de câncer com maiores índices de mortalidade no mundo. Na década de 90, houve a descoberta de populações de células tumorais com características de células-tronco e implicou na hipótese das células-tronco cancerosas (CTCs). Se a hipótese das CTCs for correta, a recorrência/recidiva dos cânceres é gerada por uma porcentagem de células (CTCs) que prolifera pouco e é resistente a quimioterapia convencional. Desta forma, a busca por novos alvos terapêuticos que tenham como alvo estas CTCs tem enorme implicação no desenvolvimento de novos fármacos ou modalidades terapêuticas contra o câncer. O guaraná (Paullinia cupana Mart var. sorbilis) tem demonstrado efeitos promissores sobre o câncer, em especial efeitos quimiopreventivos e antineoplásicos. Portanto, objetivando-se neste projeto avaliar a presença de CTCs a partir de células tumorais de pulmão de camundongo, onde se possa conseguir rapidamente uma alta porcentagem deste tipo celular para posteriores estudos, além de avaliar os efeitos do extrato etanólico do guaraná e de suas frações butanólica e aquosa, sobre as células tumorais e em especial verificar os efeitos sobre a população rica em CTCs, onde foi possível observar a presença de possíveis CTCs, a partir de cultivo de células tumorais de pulmão de camundongo, como também que o guaraná apresentou um efeito sobre estas possíveis CTCs devido a diminuição da expressão dos genes ABCG2 e ALDH1a1. / Lung cancer is among the cancers with the highest mortality rates in the world. In the 90\'s, there was the discovery of tumor cell populations with stem cell characteristics implied in the case of cancer stem cells (CSCs). If the hypothesis is correct CSCs of the recurrence / relapse of cancers is generated by a percentage of cells (CSCs) that growth low and are more resistant to conventional chemotherapy. Thus, the search for new therapeutic targets that target these CSCs has huge implications in developing new drugs or therapeutic approaches against cancer. Guarana (Paullinia cupana Mart var. Sorbilis) has shown promising effects on cancer, especially chemopreventive and anticancer effects. Therefore, the objective in this project is to evaluate the presence of CSCs from tumor cells of mouse lung, where it can quickly get a high percentage of this cell type for further studies and to evaluate the effects of ethanol extract of guarana and its butanol and aqueous fractions on tumor cells and in particular to verify the effect on the population rich in CSCs, where it was possible to observe the possible presence of CSCs from cultivation of tumor cells from mouse lung, as well as guarana presented an effect on these CSCs possible to decreased expression of ABCG2 and ALDH1a1 genes.

Células dendríticas (DCs) são centrais na indução da resposta imune e o desvio de sua diferenciação para macrófagos, no microambiente tumoral pode ser um mecanismo de escape do tumor frente à resposta imune. Assim, o objetivo deste projeto foi comparar, entre tecido neoplásico e não-neoplásico, a freqüência de DCs, macrófagos e mastócitos e da produção in situ das citocinas IL-4 e TNF-<font face=\"symbol\">a. Nesta análise observou-se maior quantidade de DCs (CD1a+ ou S100+), macrófagos (CD68+), mastócitos (azul de toluidina+) e células produtoras de TNF-<font face=\"symbol\">a no tumor do que no tecido pulmonar. No tecido pulmonar encontrou-se maior freqüência de células positivas para IL-4, assim como, entre células dissociadas de tecido fresco, observou-se maior freqüência de DCs maduras. Identificou-se também a expressão, por células tumorais ou do estroma pulmonar, do marcador CD83, característico de DCs ativadas, em 10 de 11 pacientes. O sobrenadante de cultura destas células CD83+ inibiu a ativação de linfócitos por DCs alogenêicas, mas esta atividade foi suprimida por anticorpos anti-CD83. / Dendritic cells (DCs) play a crucial role in the immune response and the deviation of its differentiation to macrophages in the tumor microenvironment may be a mechanism of escape of tumor from immune response. Thus, the aim of this project was to compare between affected and non-affected lung, the frequency of DCs, macrophages and mast cells and production in situ of IL-4 and TNF-<font face=\"symbol\">a. In this analysis, we observe higher amount of DCs (CD1a+ and S-100+), macrophages (CD68+), mast cells (toluidine blue+) and cells producing TNF-<font face=\"symbol\">a in the tumor than non-affected lung. In lung, we found higher frequency of cells IL-4+ and in lung digests we observed higher amount of mature DCs. We also identified the expression by tumor or stroma cells the CD83, present in activated DCs, in 10 of 11 patients. The supernatant of such cells CD83+ inhibited the activation of lymphocyte by alogeneic DCs, but this effect was decreased by anti-CD83.

As alterações genéticas mais frequentes em câncer de pulmão são mutações pontuais que ativam o oncogene KRAS. Embora estas mutações estejam causalmente relacionadas à oncogênese, até hoje diferentes abordagens para inibir as proteínas RAS diretamente não obtiveram sucesso. Portanto, para que melhores alvos terapêuticos para o câncer de pulmão se tornem disponíveis é necessário identificar os mecanismos moleculares ativados por KRAS que estão diretamente envolvidos com a aquisição de propriedades malignas importantes, como o desenvolvimento e a manutenção de um fenótipo tronco-tumoral pelas células iniciadoras de tumor (CITs). CITs, também conhecidas como células tronco-tumorais, são definidas como uma subpopulação de células tumorais capazes de se autorrenovar, iniciar a formação de tumores e sustentar o crescimento tumoral. O desenvolvimento de estratégias terapêuticas dirigidas a estas células é imprescindível para melhorar a eficácia da terapia antitumoral. Uma vez que KRAS está associada a manutenção de um fenótipo tronco-tumoral e ativa o fator de transcrição NF-kB através da quinase IKK&#946; para promover a tumorigênese pulmonar, nós hipotetizamos que a quinase IKK&#946; contribui para o fenótipo tronco-tumoral induzido por KRAS em câncer de pulmão. Nós utilizamos ensaios de formação de tumoresferas para enriquecer e avaliar a função de CITs das linhagens pulmonares positivas para KRAS A549 e H358. As células A549 e H358 formaram tumoresferas em cultura de baixa aderência e, quando comparadas às células derivadas da cultura aderente, as células oriundas da cultura de tumoresferas apresentaram maior crescimento clonogênico, maior expressão de genes associados ao fenótipo tronco por qPCR e maior atividade da quinase IKK&#946;. A inibição da atividade de IKK&#946; através de um inibidor farmacológico altamente específico (Composto A) diminuiu levemente a proliferação de células A549 e H358, sem resultar em morte celular significativa. Entretanto, a inibição da atividade ou da expressão de IKK&#946; por interferência de RNA reduziu a expressão de genes associados ao fenótipo tronco e diminuiu a formação de tumoresferas. A inibição da expressão de IKK&#946; em células A549 reduziu também a capacidade de autorrenovação de CITs. Estes resultados sugerem que IKK&#946; desempenha um papel importante na manutenção do fenótipo tronco-tumoral de CITs pulmonares induzidas por KRAS. Em seguida, nós demonstramos que a inibição da atividade de IKK&#946; afetou preferencialmente a proliferação celular e o crescimento clonogênico de células oriundas da cultura de tumoresfera, sugerindo que IKK&#946; desempenha um papel mais importante em CITs do que em células derivadas da cultura aderente. A análise por citometria de fluxo identificou que células derivadas da cultura de tumoresfera apresentam um enriquecimento para células CD24+ na linhagem A549 e células CD44+ na linhagem H358, sugerindo que estes possam ser marcadores promissores para purificação de CITs nestas linhagens. Adicionalmente, demonstramos, por ensaios de wound-healing de células A549 e H358, que a inibição da atividade de IKK&#946; reduziu a migração celular, uma outra uma propriedade aumentada em CITs. Além disso, mostramos que a atividade da quinase IKK&#946; em células A549 e H358 não depende das vias da MAPK ou PI3K/Akt. Interessantemente, a inibição combinada de IKK (um efetor downstream de KRAS) e de EGFR/ERRB2 (reguladores upstream de KRAS que ativam as vias MAPK e PI3K/Akt) reduziu de forma aditiva a formação de tumoresferas, proliferação e migração celular. Quando avaliados em conjunto, nossos resultados sugerem que a quinase IKK&#946; desempenha um papel importante na biologia de CITs pulmonares portadoras de KRAS oncogênica e que a inibição desta quinase sozinha ou em combinação com a inibição de outras vias pode representar uma estratégia terapêutica promissora a ser explorada para reduzir a recidiva e metástase no câncer de pulmão induzido por KRAS. / The most frequent genetic alterations in lung cancer are point mutations that activate the KRAS oncogene. Although these mutations are causally related to oncogenesis, different approaches to inhibit RAS proteins directly have not been successful to date. Therefore, for better therapeutic targets for lung cancer to become available, it is necessary to identify the molecular mechanisms activated by KRAS that are directly involved with important malignant features, such as the development and maintenance of a cancer stem-like phenotype by the tumour-initiating cells (TICs). TICs, also known as cancer stem cells, are defined as a subpopulation of tumour cells able to self-renew, promote tumour initiation, and sustain tumour growth. The development of therapeutic strategies to target these cells is imperative to improve the efficacy of antitumor therapy. Since KRAS is associated with the maintenance of a cancer stem-like phenotype and activates the transcription factor NF-kB through the IKK&#946; kinase to promote lung tumourigenesis, we hypothesised that IKK&#946; kinase contributes to the cancer stem-like phenotype induced by KRAS in lung cancer. We used tumoursphere formation assays to enrich and evaluate the function of TICs of KRAS-mutant cell lines A549 and H358. A549 and H358 cells formed tumourspheres in low adhesion culture and, when compared to cells grown in adherent culture, sphere-derived cells displayed increased clonogenic growth, higher expression of stemness genes by qPCR, and increased IKK&#946; kinase activity . Inhibition of IKK&#946; activity through a highly specific pharmacological inhibitor (Compound A) slightly decreased proliferation of A549 and H358 cells without inducing significant cell death. On the other hand, inhibition of IKK&#946; activity or expression by RNA interference reduced the expression of stemness genes and decreased tumoursphere formation. Inhibition of IKK&#946; expression in A549 cells also reduced TICs self-renewal . These results suggest that IKK&#946; plays an important role in maintaining the cancer stem-like phenotype of KRAS-driven lung TICs. Next, we demonstrated that IKK&#946; inhibition preferentially reduced cell proliferation and clonogenic growth of sphere-derived cells, suggesting that IKK&#946; plays a more important role in TICs than in adherent culture-derived cells. Flow cytometry analysis identified that sphere-derived cells display an enrichment for the surface marker CD24 in A549 cells and CD44 in H358 cells, indicating that these could be promising markers for the purification of TICs in these cell lines. Furthermore, we have shown by wound-healing assays of A549 and H358 cells that IKK&#946; inhibition reduced cell migration , another feature increased in TICs. In addition, we have shown that IKK&#946; activity in A549 and H358 cells does not depend on the MAPK or PI3K/Akt pathways. Interestingly, combined inhibition of IKK&#946; (a downstream effector of KRAS) and EGFR/ERBB2 (upstream regulators of KRAS that activate the MAPK and PI3K/Akt pathways) additively reduced tumoursphere formation, cell proliferation and migration. Taken together, our results suggest that IKK&#946; kinase plays an important role in the biology of KRAS-driven lung TICs, and that inhibition of this kinase alone or in combination with inhibition of other signalling pathways may represent a promising therapeutic strategy to be explored in order to reduce tumour recurrence and metastasis in KRAS-driven lung cancer.

As alterações genéticas mais frequentes em tumores de pulmão são mutações pontuais que ativam o oncogene KRAS. Apesar destas mutações estarem ligadas à oncogênese de forma causal, diferentes abordagens para inibir as proteínas RAS diretamente fracassaram na clínica. Portanto, para que melhores alvos terapêuticos para o câncer de pulmão se tornem disponíveis, será necessário identificar as vias sinalizadoras ativadas pela proteína KRAS, que são críticas para a oncogênese. O objetivo deste projeto foi identificar novos alvos terapêuticos na oncogênese pulmonar induzida pela KRAS. Este projeto se baseou na seguinte hipótese: (1) a KRAS oncogênica leva à ativação das quinases mitóticas Aurora A e/ou B e (2) que as quinases Aurora A e/ou B são alvos terapêuticos relevantes no câncer de pulmão induzido pelo oncogene KRAS. Esta hipótese foi formulada com base em estudos anteriores mostrando que a quinase Aurora A fosforila diretamente componentes das vias efetoras de RAS, e que a Aurora A e Aurora B cooperam com a RAS oncogênica na transformação maligna. Para testar esta hipótese, nós inicialmente determinamos se a forma oncogênica da KRAS induz a expressão das quinases Aurora A e B. Para tanto, nós usamos 3 modelos celulares: (1) uma linhagem primária epitelial pulmonar imortalizada e seu par isogênico transformado pela KRAS oncogênica; (2) células tumorais pulmonares H1703 manipuladas geneticamente para expressar a forma oncogênica da KRAS de forma induzível; e (3) células de adenocarcinoma pulmonar portadoras de mutações oncogênicas em KRAS H358 e A549 manipuladas geneticamente para expressar short hairpin RNAs (shRNAs) para KRAS de forma induzível. Em todos os casos, a expressão da forma oncogênica da KRAS se correlacionou positivamente com a expressão de Aurora A e B. Para validar as quinases Aurora A e B como alvos relevantes do ponto de vista terapêutico, nós usamos, nas células mencionadas acima, abordagens genéticas ou farmacológicas para inibir a expressão ou atividade das quinases Aurora A e B. Nas células A549 e H358, portadoras da forma oncogênica da KRAS, a inibição da expressão das quinases Aurora A ou B por interferência de RNA de forma induzível, bem como o tratamento com um inibidor dual destas quinases, reduziu o crescimento, viabilidade e tumorigenicidade celulares in vitro. Mais importante do que isso, no modelo celular primário isogênico, bem como na linhagem H1703 com expressão induzível de KRAS oncogênica, a inibição farmacológica dual das quinases Aurora A e B levou a uma redução no crescimento, viabilidade e tumorigenicidade celulares de forma dependente da presença da KRAS oncogênica, sugerindo que a inibição das quinases Aurora A e B afeta especificamente células transformadas pela KRAS. Em conclusão, nossos resultados apoiam a nossa hipótese de que as quinases Aurora são alvos da KRAS oncogênica no pulmão, e sugerem a inibição das quinases Aurora como uma nova abordagem para a terapia do câncer de pulmão induzido pela forma oncogênica da KRAS. / The most frequent genetic change found in lung tumors are activating point mutations in the KRAS gene, which have been causally linked to the oncogenic process. Unfortunately, different approaches to target RAS proteins for therapy have been unsuccessful. Therefore, in order to select better targets for lung cancer therapy, key cancer-relevant KRAS downstream pathways will need to be identified. The overall objective of this study was to identify novel therapeutic targets in KRAS-mediated lung cancer. This project was based on the following hypothesis: (1) KRAS activates mitotic kinases Aurora A and/or B; and (2) Aurora A and/or B are relevant therapeutic targets in KRAS-induced lung cancer. This hypothesis was formulated on the basis of published studies showing that Aurora A directly phosphorylates RAS effector pathway components, and Aurora A and B both cooperate with oncogenic RAS to promote malignant transformation. In order to test this hypothesis, we first determined whether oncogenic KRAS induces Aurora kinase expression. For that purpose, we used three different cell-based models: (1) an immortalized primary lung epithelial cell line and its isogenic KRAS-transformed counterpart, (2) H1703 lung cancer cell line engineered to express oncogenic KRAS inducibly, and (3) KRAS positive lung cancer cell lines H358 and A549 stably expressing inducible shRNAs targeting KRAS. In all cases, KRAS expression positively correlated with Aurora A and Aurora B expression. In order to validate Aurora A and/or B as therapeutically relevant KRAS targets in lung cancer, we used genetic and/or pharmacological approaches in the abovementioned cells to inactivate Aurora A or B. In KRAS positive H358 and A549 cell lines, inducible shRNA-mediated knockdown of Aurora A or B, as well as treatment with a dual Aurora A and B inhibitor, decreased growth, viability and tumorigenicity in vitro. More importantly, in the primary isogenic model and in the H1703 KRAS-inducible cell line, dual pharmacological inhibiton of Aurora A and B reduced growth, viability and tumorigenicity in an oncogenic KRAS-dependent manner. This suggests that Aurora kinase inhibition therapy can specifically target KRAS transformed cells. In conclusion, our results support our hypothesis that Aurora kinases are important KRAS targets in lung cancer and suggest Aurora kinase inhibition as a novel approach for KRAS-induced lung cancer therapy.

Bakgrund: Lungcancer är den cancerform med högst mortalitet. Att drabbas av lungcancer kan leda till att personen upplever olika känslor som rädsla, uppgivenhet och hopp vilket är viktigt för sjuksköterskor att ha kunskap och förståelse om. Sjuksköterskors uppgift är att främja den fysiska, psykiska, sociala och existentiella hälsan hos personer de vårdar genom ett personcentrerat arbetssätt. Syfte: Att beskriva upplevelsen av hopp hos personer med lungcancer. Metod: En litteraturöversikt med kvalitativ design och deduktiv ansats. Tretton artiklar inkluderades i resultatet. Polit och Becks modell användes i analysprocessen. Resultat: Analysen resulterade i två teman som beskrev upplevelsen av hopp hos personer med lungcancer. Det första temat var hälso- och sjukvårdens betydelse med tillhörande underteman att ha tilltro till medicinsk behandling och att få ett gott bemötande. Det andra temat var inneboende resurser med underteman att ha en religiös tro, att ha en meningsfull resterande tid och att tänka positivt. Slutsatser: Upplevelsen av hopp är betydelsefull hos personer med lungcancer. Det är en positiv känsla och en önskan kring vad personen vill ska ske i framtiden. Sjuksköterskor har därmed en betydande roll för att stärka hoppet hos personer med lungcancer genom sitt bemötande och förmåga till att arbeta personcentrerat. / Background: Lung cancer is the type of cancer with the highest mortality. When receiving a lung cancer diagnosis, feelings like fear, despair and hope can arise, which is important for nurses to know and understand. The role of the nurses is to promote the physical, psychological, social and spiritual well-being of the people they care for by providing person-centered care. Aim: To describe the experience of hope among people with lung cancer. Method: A literature review with qualitative design and a deductive approach. Thirteen qualitative articles were included in the result. Polit and Beck’s model was used in the analysis process. Result: The analysis resulted in two themes which described the experience of hope among people with lung cancer. The first theme was the significance of healthcare with the associated subthemes to have faith in medical treatment and to receive a good encounter. The second theme was intrinsic resources with the following subthemes: to have a religious belief, to have a meaningful remaining time and to think positively. Conclusions: The experience of hope is meaningful among people with lung cancer. Hope is a positive feeling and a wish for the individual’s future. Nurses have a significant role in empowering the hope of people with lung cancer through their communication and ability to remain person-centered.

An improved method is introduced for prediction of local tumor control following lung stereotactic body radiation therapy (SBRT) for early stage non-small cell lung cancer (NSCLC) patients using 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET). A normalized background-corrected tumor maximum Standard Uptake Value (SUVcmax) is introduced using the mean uptake of adjacent aorta (SUVref), instead of the maximum uptake of lung tumor (SUVmax). This method minimizes the variations associated with SUVmax and objectively demonstrates a strong correlation between the low SUVcmax (< 2.5-3.0) and local control of post lung SBRT. The false positive rates of both SUVmax and SUVcmax increase with inclusion of early (<6 months) PET scans, therefore such inclusion is not recommended for assessing local tumor control of post lung SBRT. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2013.

Les expositions professionnelles représentent la 2nde étiologie de cancer du poumon. Parmi elles, les laines minérales (LM) utilisées comme substitut à l’amiante sont suspectées d’augmenter le risque de cancer du poumon en raison de leur structure proche de celle de l’amiante. Dans ce contexte, ce travail a pour objectif d'évaluer le rôle de l'histoire professionnelle et en particulier celui de l'exposition professionnelle aux LM dans la survenue de cancer du poumon en tenant compte des expositions à l'amiante, à la silice, autres substances très prévalentes dans le secteur de la construction. Une dernière partie a consisté à étudier le risque de cancer du poumon associé à l'exposition professionnelle aux poussières de ciment.Nous avons analysés les histoires professionnelles complètes des cas et témoins inclus dans l'étude cas-témoins en population générale ICARE, identifiés dans 10 départements français abritant un registre général de cancer. Au total, 2 276 cas de cancer du poumon chez les hommes et 2 780 témoins ont été inclus ainsi que 650 cas de cancers du poumon féminins et 775 témoins. Deux stratégies d'évaluation des expositions aux LM et à l'amiante ont été utilisées: une matrice emplois-expositions (MEE) et une matrice tâche-exposition élaborée par des hygiénistes industriels et tenant compte des données individuelles. Les expositions à la silice et au ciment ont toujours été évaluées par MEE. Nos résultats confirment le rôle important des expositions professionnelles comme déterminant du risque de cancer du poumon en mettant en évidence des associations avec de nombreuses professions et secteurs d'activité. L'hypothèse d'une association entre le risque de cancer du poumon et l'exposition professionnelle aux LM est suggérée sans toutefois pouvoir exclure une confusion résiduelle par l'amiante car ces deux expositions sont très corrélées. Par ailleurs, nos résultats confirment le rôle cancérogène de l'amiante et de la silice et suggèrent une association entre l'exposition aux poussières de ciment et le risque de cancer du poumon. / Occupational exposures are the second main risk factor of lung cancer. Asbestos has progressively been substituted by mineral wools (MW). Because of their similarity in shape with asbestos, they have been suspected of causing cancer of the respiratory system. In this context, the objectives of this work were to assess the role of occupational history in lung cancer risk; to investigate in details lung cancer risk associated with exposure to MW, carefully taking into account occupational exposures to asbestos and crystalline silica, two common potential carcinogenic exposure among construction workers; and to study the risk of lung cancer associated with occupational exposure to cement dusts.We analysed lifetime occupational history of cases and controls included in the ICARE study, a large multi-centre, population-based, case-control study. They were recruited in 10 French départements with a general cancer registry. The study included 6481 subjects (men: 2 276 lung cancer cases and 2 780 controls, women: 650 lung cancer cases and 775 controls). Two strategies of assessment of occupational exposure to MW and asbestos were used: a job-exposure matrix (JEM) and a task-exposure matrix constructed by trained hygienist and taking into account individual information. Occupational exposures to crystalline silica and cement dusts were both assessed using specific JEMs.Our results confirm the important role of occupational exposure as a determinant of lung cancer risk by showing associations between lung cancer risk and several occupations or industries. An association between MW exposure and lung cancer risk is suggested without being able to exclude a potential residual confounding by asbestos since these two exposures are deeply correlated. Our results also confirm asbestos and silica carcinogenicity and suggest an association between cement dusts and lung cancer risk.

Les thérapies ciblées sont des agents dont le but est d’inhiber une voie oncogénique spécifiquement activée dans une tumeur. Ces thérapies peuvent donc cibler l’angiogenèse tumorale, les voies de signalisation cellulaire, la prolifération infinie, l’anti-apoptose ou le pouvoir métastatique. Outre leur effet en monothérapie, leur intérêt repose également sur leur combinaison avec les traitements standards, chimiothérapie, radiothérapie ou même chirurgie. Dans ce contexte, l’association des thérapies ciblées et de la radiothérapie paraît séduisante. En effet, les mécanismes qui sous-tendent la sensibilité ou la résistance à une irradiation sont maintenant mieux connus et permettent d’envisager des manipulations thérapeutiques afin d’améliorer encore les résultats d’une radiothérapie ou d’une radiochimiothérapie. L’objectif de cette thèse était d’exploiter les données d’histologie moléculaires des carcinomes pulmonaires pour évaluer de nouvelles approches de radiosensibilisation basée sur l’inhibition de signaux de survie en ciblant les protéines Bcl-2 et Bcl-XL et la voie IGF-1 dans les carcinomes à petites cellules (CPC) et en ciblant la voie EGFR dans les carcinomes non à petites cellules du poumon (CPNPC). L’un des objectifs était également d’intégrer une méthodologie de combinaison plus précise compte-tenu des discordances très fréquemment observées entre les effets pré-cliniques d’une association et les résultats des études cliniques de la même combinaison. Dans une première partie, nous avons cherché à inhiber les mécanismes antitapoptotiques mis en jeu dans les CPC au moyen de deux nouvelles classes de thérapies ciblées : un oligonucléotide antisens ciblant l’ARNm du gène BCL2 (oblimersen) et un « BH3 mimetic » inhibiteur des protéines Bcl-2/Bcl-XL (S44563). Nous avons démontré dans ces deux études l’intérêt du ciblage de « l’anti-apoptose » pour radiosensibliser les lignées de CPC. En utilisant le S44563, un « BH3 mimetic » qui induit une apoptose via la voie mitochondriale dans les lignées qui surexpriment les cibles à savoir Bcl-2 et Bcl-XL,, nous avons montré que l’inhibition de Bcl-2 et Bcl-XL permettait d’induire une radiosensibilisation par induction de l’apoptose. Le mécanisme d’interaction reposait probablement par une induction de l’expression des protéines anti-apoptotiques, en particulier Bcl- XL à la suite d’une irradiation via l’activation de la voie NF-κB. Ceci est confirmé par les études de séquence montrant que l’administration pendant et après l’administration de la radiothérapie est plus efficace réalisant ainsi une chimiosensibilisation sous l’effet d’une irradiation rendant les cellules tumorales plus dépendantes (concept d’addiction oncogénique contextuelle) aux mécanismes de résistance à l’apoptose. Dans une seconde partie, nous avons également montré que l’inhibition d’une voie de signalisation cellulaire (la voie IGF-1) permettait également d’obtenir une radiosensibilisation tumorale validant ainsi l’intérêt de ces combinaisons. En particulier, L’anticorps monoclonal ciblant IGF-1R, le R1507 augmente l’efficacité du cisplatine et au final améliore l’efficacité de la radio-chimiothérapie dans plusieurs lignées de CPC via la diminution de l’expression de IGF-1R avec pour conséquence une diminution de l’activation de ses effecteurs, en particulier AKT, indiquant que le R1507 augmente la radiosensibilité en supprimant l’activation de IGF-1R secondaire à l’irradiation. Cependant, nous avons montré par différentes approches d’étude du transcriptome que les cellules tumorales traitées par le R1507 pendant 4 semaines s’adaptaient en ré-exprimant IGF-1R, en activant p-IRS1 et en activant différentes voies oncogéniques telles que l’angiogenèse ou d’autres voies de signalisation cellulaire. Ces résultats suggèrent donc les limites d’un schéma adapté chez l’homme lors d’une radio-chimiothérapie concomitante et plaident pour une association précoce à d’autres thérapies ciblées. / Targeted therapies are drugs that block a specific molecular target involved in following alterations in cell physiology: growth signal self-sufficiency, insensitivity to growth-inhibitory signals, evasion of apoptosis, an unlimited replicative potential, sustained angiogenesis, tissue invasion, and metastasis. Although these compounds showed efficacy when given alone, there is now a rationale to combine these agents with other antitumor therapies such as chemotherapy, radiation and surgery. In this context, there is compelling data supporting the association between targeted therapies and radiation. The better understanding of mechanisms of sensitivity or resistance to radiation may help to envision new strategies to improve its efficacy. The primary goal of this work was to assess new strategies of radiosensitization based on molecular characteristics of both small cell lung cancer (by targeting Bcl-2 and Bcl- XL proteins as well as IGF-I pathway) and non-small cell lung cancer (by targeting EGFR pathway). The second objective was also to assess new methods to better investigate combination of radiation with new targeted therapies. In the first part of the work, we evaluated the impact of the inhibition of BCL-2 in small cell lung cancer cell lines with oblimersen, an antisense BCL-2 oligodeoxynucleotide and with a small peptide BH3 mimetic, S44563 which targets both Bcl-2 and Bcl- XL proteins. We showed that inhibiting anti-apoptotic mechanisms could enhance radiosensitivity of SCLC cells. S44563 caused SCLC cells to acquire hallmarks of apoptosis through activation of the mitochondrial pathway in Bcl2 and Bcl- XL overexpressing cell lines. S44563 markedly enhanced the sensitivity of SCLC cells to radiation in both in vitro and in vivo assays through apoptosis induction. This positive interaction was explained by the induction of radiation-induced anti-apopototic proteins, mainly Bcl- XL by the NF-κB pathway. These data were confirmed by in vivo experiments showing that the radiosensitization was greater when S44563 was given after the completion of the radiation in the context of radiation-induced oncogenic addiction. In the second part of the work, we showed that IGF-1R targeting increases the antitumor effects of DNA-damaging agents in SCLC model. R1507 (a monoclonal antibody directed against IGF-1R), exhibited synergistic effects with both cisplatin and IR in SCLC cell lines through IGF-IR downregulation and reduced activation of downstream AKT. However, we observed a transient reduction of IGF-1R staining intensity in vivo, concomitant to the activation of multiple cell surface receptors and intracellular proteins involved in proliferation, angiogenesis, and survival. These data underscore the challenge of the combination of concomitant radiotherapy and chemotherapy and support the early use of targeted therapies to improve the antitumor efficacy.

Survival rates in cancers have improved vastly over the years. However, some survival rates remain extremely low, as is the case for ovarian and lung cancer. The lack of robust and reliable biomarkers is strongly reflected in the absence of pre-screening programs, and as such, most patients in these cancer types are diagnosed only in advanced stages, leaving few treatment options. Moreover, relapse and resistance to therapies adds to the complexities of treating these diseases, even in the era of targeted drug development. Research has shown the presence of cancer material, in the form of circulating cancer cells (CTCs) and genomic material in the blood of patients, opening the possibility of 'liquid biopsies'. Liquid biopsies allow sampling of the disease to provide phenotypic and genomic data on the cancer in real-time and on a routine basis. Moreover, they overcome obstacles currently faced by conventional tissue biopsies. In this work we evaluate the use of a novel CTC imaging flow-cytometry platform, and report the ability to characterise and quantify these cells in blood samples. Moreover, we report significantly higher levels of CTCs in cancer patients compared to controls, and found them to be associated with a poorer prognosis. In particular, in lung cancer we observe these findings even in the early stages, suggesting a potential diagnostic use for this assay. We detect a similar trend in when analysing the ctDNA and suggest the possibility of using this technique with a prognostic value in the advanced setting. We also report on the analysis of existing microarray data by use of unique gene regulatory networks to identify biomarkers of interest. RAD51AP1 was identified by this process. Clinical validation revealed an over-expression of this gene in both tissue and blood of ovarian and lung cancers. Moreover, the gene over-expression was associated with a poor overall survival. Functional analysis in vitro revealed silencing RAD51AP1 suppressed tumour growth, in addition, various tumorigenic proteins were down-regulated, whilst apoptotic and immune genes were up-regulated. These results suggest a role for RAD51AP1 as a potential therapeutic target. In this study, we also demonstrate the ability to further exploit tumour genomic material in the blood by means of RNAseq, cancer panels, and CNI scoring to identify novel markers, that play an important role in disease genesis and evolution. RNAseq analysis identified XIST as a gene up-regulated in the blood and tissue of lung cancers. The ovarian cancer panels revealed 2 unique gene signatures in the ovarian cancer patients. With the CNI analyses also highlighting chromosomal aberrations from plasma analysis of cancer patients. Collectively, the use of all these techniques and exploitation of available blood based biomarkers could see significant improvements to survival rates in these, currently devastating diseases.

Cough in lung cancer (LC) is a significant unmet need. There are no evidence-based effective antitussives for its treatment and a lack of well-designed trials incorporating validated cough assessment tools and placebo controls. There is little research on its underlying mechanisms, perhaps with the assumption that it is simply 'due to the cancer'. Therefore, we have sought to characterise cough in terms of its severity, impact on quality of life, frequency and prevalence using LC specific subjective and objective assessment tools for the first time. We have also explored its potential mechanisms and treatment. Published preclinical data show that the substance P/neurokinin-1(NK1) pathway is implicated in cough in 5 different species. This pathway is targeted by the antiemetic aprepitant in humans. Data on the use of aprepitant as a novel antitussive are presented. To characterise cough and assess cough assessment tools in a cohort of patients with LC attending outpatient clinics, subjective and objective cough assessment tools including 24-hour ambulatory cough monitoring (ACM), were used to determine the cough severity, frequency, impact and cough- associated clinical factors in a longitudinal study. To determine cough prevalence, a cross sectional study of all patients attending thoracic oncology outpatient clinics in a single centre over a defined period were approached to determine whether they had a cough, to provide demographic and cancer related data and if applicable, to complete the Manchester Cough in Lung Cancer Scale (MCLCS) cough impact questionnaire and the cough severity visual analogue scale. To explore the role of the NK1 pathway in cough in patients with LC, a single-arm randomised placebo controlled pilot trial assessing aprepitant for the treatment of cough was conducted. The presented data demonstrated that cough affects over half of patients with LC, representing a huge unmet clinical need. Over 2/3rds of patients felt that their cough was severe enough to warrant treatment and over 1/4 described it as painful. Patients with LC suffer from a very severe and frequent cough. Its impact is considerable, with effects on physical, psychological and social domains. The longitudinal study is the first to report that cough severity and impact is predicted by gastro-intestinal co-morbidities rather than cancer related factors. The presented data demonstrate that ACM is feasible and acceptable to patients with LC. This provides researchers with an objective endpoint for use in clinical trials. The MCLCS performs well and is valid. The cough intervention trial is the first to demonstrate that aprepitant is associated with lower subjective cough scores and cough frequency using validated cough assessment tools. No antitussive therapy study has ever shown a positive antitussive effect using both types of cough assessment tools in the LC population. This suggests that the substance P/NK1 pathway is implicated in cough in LC and identifies this as a potential new therapeutic target, providing exciting data and hope for future patients with LC.