Pemetrexed (PTX), an antifolate drug, has been approved by the US FDA for first line therapy of mesothelioma and non-small cell lung cancer. In addition to its primary site of action on thymidylate synthase (TS), PTX also inhibits the second folate-dependent enzyme of purine biosynthesis aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART). The accumulation of the substrate for AICART, ZMP, in PTX-inhibited cancer cells leads to activation of AMP-activated protein kinase (AMPK) with subsequent inhibition of mammalian target of rapamycin (mTOR) and hypophosphorylation of its downstream targets responsible for protein synthesis and cell proliferation. Inhibitors of mTORC1 like Rapamycin and its analogs (rapalogs) have only partial effects on tumor cells as they do not inhibit mTORC2, which phosphorylates Akt subsequently relieving the inhibition of mTORC1, thus leading to poor cytotoxicity by rapalogs. AMPK exerts control on mTORC1 kinase activity and PTX mediated activation of AMPK leads to its subsequent downregulation and hence, would be expected to have a therapeutic interaction with direct mTOR inhibitors. AZD8055, an ATP-competitive inhibitor of mTOR kinase, potently inhibits both mTORC1 and mTORC2 and therefore, can overcome the feedback mechanism(s) limiting the action of rapalogs to cytostatic effects. To study the effects of AMPK activation and mTOR inhibition pharmacologically, we performed growth suppression assays using pemetrexed, AICAR, RAD001, and AZD8055. The effect of inhibition of mTOR with these drugs was assessed by examining the dephosphorylation of mTORC1 substrates S6K1 and 4E-BP1, as single agents and in combination, at their 50% inhibitory concentrations (IC50) by western blotting. Our data suggested that AMPK activation via PTX mediated AICART inhibition in combination with direct mTOR inhibition by AZD8055 has a synergistic interaction on the proliferation of NSCLC cells in culture. Inhibition of mTOR endogenously by pemetrexed, along with direct pharmacological inhibition of mTOR prevents the feedback circuit which may compromise the therapeutic efficacy of rapamycin analogs. Pemetrexed and AZD8055, as single agents, demonstrated inhibitory activity on phosphorylation events of mTORC1 substrates. This activity was markedly increased by combining both the drugs. Our findings suggest that direct inhibitors of mTOR enhance the effects of activators of AMPK. These effects appear to be mediated via combined effects on mTORC1. Taken together, the combination of catalytic site mTOR inhibitors and pemetrexed is a promising therapeutic strategy and calls for further preclinical and clinical investigations.

Lung cancer is the leading cause of cancer-related deaths worldwide. Radiotherapy is one of the main treatment modalities of lung cancer. However, the achievable accuracy of radiotherapy treatment is limited for lung-based tumors due to respiratory motion. Four-dimensional radiotherapy explicitly accounts for anatomic motion by characterizing the motion, creating a treatment plan that accounts for this motion, and delivering this plan to the moving anatomy. This thesis focuses on the current problems and solutions throughout the course of four-dimensional radiotherapy. For characterization of respiratory-induced motion, patient tumor motion data were analyzed. It is shown that tumor motion can be significant during radiotherapy treatment, and its extent, direction, and linearity vary considerably between patients, between treatment fractions, and between respiratory cycles. After this, approaches to four-dimensional intensity-modulated radiation therapy treatment planning were developed and investigated. Among the techniques to manage respiratory motion, tumor tracking using a dynamic multileaf collimator delivery technique was chosen as a promising method. A formalism to solve a general four-dimensional intensity-modulated radiation therapy treatment-planning problem was developed. Specific solutions to this problem accounting for tumor motion initially in one dimension and extending this to three dimensions were developed and investigated using four-dimensional computed tomography planning scans of lung cancer patients. For four-dimensional radiotherapy treatment delivery, accuracy of two-dimensional projection imaging methods was investigated. Geometric uncertainty due to the limitation of two-dimensional imaging in monitoring three-dimensional tumor motion during treatment delivery was quantified. This geometric uncertainty can be used to estimate proper margins when a single two-dimensional projection imager is used for four-dimensional treatment delivery. Lastly, tumor-tracking delivery using a moving average algorithm was investigated as an alternative delivery technique that reduces mechanical motion constraints of a multileaf collimator. Moving average tracking provides an approximate solution that can be immediately implemented for delivery of four-dimensional intensity-modulated radiation therapy treatment. The clinical implementation of four-dimensional guidance, intensity-modulated radiation therapy treatment planning, and dynamic multileaf collimator tracking delivery may have a positive impact on the treatment of lung cancer.

SYMPTOM CLUSTERS IN LUNG CANCER PATIENTS By Debra Rattican, PhD, RN A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. Virginia Commonwealth University, 2012 Major Director: Debra E. Lyon, PhD. Professor and Chair Family and Community Health Nursing The purpose of the study was to examine selected relationships among symptoms common to individuals with lung cancer. The specific aims were: 1) To examine the relationship between the symptoms of dyspnea and anxiety in patients with lung cancer. 2) To examine the relationships among the symptoms of dyspnea, anxiety, and symptom cluster components (depressive symptoms, fatigue, pain) in patients with lung cancer. 3) To examine the correlation between functional ability and quality of life in patients with lung cancer. 4) To explore the relationships among the symptoms of dyspnea, anxiety, and symptom cluster components (depressive symptoms, fatigue, pain) in patients with lung cancer and patients’ functional ability. 5) To explore the relationships among the symptoms of dyspnea, anxiety, and symptom cluster components (depressive symptoms, fatigue, pain) in patients with lung cancer and patients’ quality of life. Data were gathered through online survey and analyzed using descriptive, correlation, principal component analysis, exploratory factor analysis, and forward stepwise regression techniques. A strong positive correlation was found between dyspnea and anxiety (both anxiety in general and anxiety at the time the survey was completed. While results of this study cannot provide conclusive evidence of the existence of a symptom cluster composed of depressive symptoms, fatigue, and pain, the results are consistent with other studies in this area. Significant positive correlations among these three symptoms indicate that this is a possible symptom cluster experienced by lung cancer patients in general. This study provides preliminary data on how these symptoms are related and how they affect functional ability, or the ability to perform routine activities of daily living (ADLS) and instrumental activities of daily living (IADLS), and quality of life in patients with lung cancer. Further study is needed on to better understand the symptom experience of these individuals in order to develop robust interventions targeting effective symptom management.

Lung cancer arises from epithelial cells that line the air passages of the lungs. It is the second most common malignancy in the United States; trends suggest that over 228,000 new patients will be diagnosed with lung cancer in 2013. Due to the fact that lung cancer is highly aggressive, it has proven difficult to control. The 5-year survival rate has been shown to be only 15.9%, despite the advances made in terms of diagnosis and treatment. Therefore, we are faced with the problem of finding more effective methods that allow for an earlier diagnosis and the improved treatment of lung cancer. This study attempts to address these issues by investigating Tarsh, a novel molecule that is involved in the regulation of cellular senescence. Previous studies have shown that Tarsh is expressed in normal lung cells, but is significantly downregulated in lung tumors. These studies also determined that Tarsh is likely dependent upon the expression of p53, a tumor suppressor gene. The current study investigated these results, in addition to the biological effects of ectopically increasing Tarsh and/or knocking down p53 expression in two lung cancer cell lines: A549 and H1299 cell lines. It was determined that increasing the expression of Tarsh decreased the rate of proliferation in both cell lines. Additionally, it was shown that the knockdown of p53 increased proliferation in A549 cells. In regards to the migration rate of these cell lines, the overexpression of Tarsh decreased migration in A549 cells, but had no effect on H1299 cells. However, the role of p53 in migration is still unclear. The results of this study suggest that the knockdown of p53 decreases cell migration in A549 cells. This contradicts the fact that H1299 cells do not express p53, yet was found to have the highest migration rate. It is evident that a further investigation is needed to make more concrete conclusions. Nevertheless, the suppressive features of Tarsh on cell proliferation, and possibly migration, make it a promising target of research for lung cancer therapy.

<p>Tumor angiogenesis is critical to tumor growth and metastasis, yet much is unknown about the role vascular cells play in the tumor microenvironment. A major outstanding challenge associated with studying tumor angiogenesis is that existing preclinical models are limited in their recapitulation of in vivo cellular organization in 3D. This disparity highlights the need for better approaches to study the dynamic interplay of relevant cells and signaling molecules as they are organized in the tumor microenvironment. In this thesis, we combined 3D culture of lung adenocarcinoma cells with adjacent 3D microvascular cell culture in 2-layer cell-adhesive, proteolytically-degradable poly(ethylene glycol) (PEG)-based hydrogels to study tumor angiogenesis and the impacts of neovascularization on tumor cell behavior. </p><p>In initial studies, 344SQ cells, a highly metastatic, murine lung adenocarcinoma cell line, were characterized alone in 3D in PEG hydrogels. 344SQ cells formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media that significantly increased with exposure to transforming growth factor beta 1 (TGF-β1), a potent tumor progression-promoting factor. Vascular cells alone in hydrogels formed tubule networks with localized activated TGF-β1. To study cancer cell-vascular cell interactions, the engineered 2-layer tumor angiogenesis model with 344SQ and vascular cell layers was employed. Large, invasive 344SQ clusters developed at the interface between the layers, and were not evident further from the interface or in control hydrogels without vascular cells. A modified model with spatially restricted 344SQ and vascular cell layers confirmed that observed 344SQ cluster morphological changes required close proximity to vascular cells. Additionally, TGF-β1 inhibition blocked endothelial cell-driven 344SQ migration. </p><p>Two other lung adenocarcinoma cell lines were also explored in the tumor angiogenesis model: primary tumor-derived metastasis-incompetent, murine 393P cells and primary tumor-derived metastasis-capable human A549 cells. These lung cancer cells also formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media. Epithelial morphogenesis varied for the primary tumor-derived cell lines compared to 344SQ cells, with far less epithelial organization present in A549 spheroids. Additionally, 344SQ cells secreted the highest concentration of two of the three angiogenic growth factors assessed. This finding correlated to 344SQ exhibiting the most pronounced morphological response in the tumor angiogenesis model compared to the 393P and A549 cell lines. </p><p>Overall, this dissertation demonstrates the development of a novel 3D tumor angiogenesis model that was used to study vascular cell-cancer cell interactions in lung adenocarcinoma cell lines with varying metastatic capacities. Findings in this thesis have helped to elucidate the role of vascular cells in tumor progression and have identified differences in cancer cell behavior in vitro that correlate to metastatic capacity, thus highlighting the usefulness of this model platform for future discovery of novel tumor angiogenesis and tumor progression-promoting targets.</p> / Dissertation

Dans de nombreux cancers, l’expression du gène Fhit (Fragile Histidine Triad) estfréquemment altérée. Fhit est décrit comme un important gène suppresseur de tumeur de parson rôle pro-apoptotique et anti-prolifératif. Nous avons mis en évidence que, in vivo et invitro, la diminution de l’expression de Fhit est associée au caractère infiltrant des cellulestumorales bronchiques, ce qui suggère que Fhit pourrait être impliqué dans le processusd’invasion tumorale. Nous avons en effet montré que la surexpression et l’inhibition de Fhitinduisent respectivement une diminution et une augmentation des capacités migratoires etinvasives des cellules bronchiques. Nous avons aussi mis en évidence que Fhit contrôlel’invasion des cellules tumorales bronchiques en régulant l’expression d’éléments clés de latransition épithélio-mésenchymateuse (TEM) tels que l’organisation des jonctions serrées etadhérentes, l’expression des métalloprotéinases matricielles et de la vimentine. De plus, Fhitrégule la TEM via une cascade de signalisation impliquant le récepteur au TGF-β, lesrécepteurs à tyrosine kinase (RTK), Src, Erk et Slug. Le double rôle de Fhit en tant quesuppresseur de tumeur et d’invasion renforce l’idée que Fhit pourrait représenter un nouveaubiomarqueur d’agressivité tumorale et pourrait constituer une nouvelle cible thérapeutiquedans le traitement des cancers broncho-pulmonaires. / In many types of cancers, Fhit (Fragile histidine triad) expression is frequentlydecreased or lost. Fhit is described as a tumor suppressor gene by its ability to induceapoptosis and to inhibit proliferation of tumor cells. We have demonstrated that a low Fhitexpression is associated with in vivo and in vitro invasiveness of lung tumor cells. Then, wehave shown that Fhit controls the invasive phenotype of lung tumor cells by regulating keyelements of epithelial-mesenchymal transition (EMT) such as cell-cell adhesion molecules,matrix metalloproteinase and vimentin expression. Our results provide also evidence that Fhitcontrols EMT by regulating several signaling pathways implying TGF-βR, RTK, Src, ERKand Slug. The dual function of Fhit as a tumor and invasion suppressor gene strengthens theidea that Fhit could represent a new biomarker of aggressiveness of lung cancer and couldconstitute a new therapeutic target to limit tumor progression.

O câncer de pulmão é o tipo de câncer que apresenta o maior índice de mortalidade em todo o mundo. As alterações genéticas mais frequentes em câncer de pulmão são as mutações pontuais no oncogene que codifica a GTPase KRAS. Apesar destas mutações estarem diretamente ligadas à oncogênese, terapias que visam inibir diretamente a proteína Ras falharam em ensaios clínicos. Uma das propriedades mais importantes na oncogênese é a aquisição de capacidade metastática tumoral. Desta forma, o objetivo deste projeto é identificar alvos terapêuticos que inibam as metástases tumorais induzidas pelo oncogene KRAS no pulmão. Com base em relatos recentes mostrando que a forma oncogênica de KRAS promove, não só a iniciação tumoral, mas também promove a aquisição de um fenótipo metastático, a hipótese deste projeto é que (1) a capacidade mestastática tumoral induzida por KRAS no pulmão é potencializada pela quinase IKK&#946;; e (2) que a inibição desta quinase reduzirá a capacidade invasiva celular e metastática tumoral. Esta hipótese foi formulada com base em estudos anteriores, os quais demonstraram que o principal substrato da IKK&#946;, o fator de transcrição NF-&#954;B, é ativado por KRAS em tumores pulmonares in situ de forma dependente da IKK&#946;, que o NF-&#954;B é capaz de promover metástase em diferentes modelos tumorais, e que a inibição da atividade da IKK&#946; com um inibidor farmacológico em um modelo animal de câncer de pulmão induzido por KRAS, diminui o crescimento tumoral e a progressão tumoral para graus histológicos mais avançados. Nosso objetivo era avaliar se a inibição de IKK&#946; é capaz de afetar a migração e invasão de células portadoras de mutação em KRAS in vitro e se a inibição de IKK&#946; é capaz de afetar a capacidade metatática dessas células in vivo. Primeiramente, avaliamos a expressão de enzimas relacionadas ao fenótipo metastático, as metaloproteinases de matriz 2 e 9 (MMP-2 e MMP-9) e, também uma molécula intimamente relacionada ao processo de adesão mediado por integrinas, FAK (quinase de adesão focal), frente a inibição de IKK&#946; através de um inibidor farmacológico altamente especifico (Composto A) e frente a inibição genética de IKK&#946; por interferência de RNA (siRNA) em células A549 e H358. Avaliamos também a atividade das MMPs frente inibição genética de KRAS (siKRAS) e IKK&#946; (siIKK&#946;) e vimos que IKK&#946; parece modular a expressão ou atividade de MMP-9 e reduz a expressão de FAK. Já a expressão de MMP-2 não apresentou alteração. Posteriormente avaliamos migração na célula A549 e invasão nas células A549 e H358 com inibição de IKK&#946;, por ensaios Transwell, e observamos uma redução da migração e invasão celular in vitro. Em seguida, fomos gerar linhagens celulares paraa expressar luciferase, as linhagens A549 pLUC e H358 pLUC. Os clones A549 pLUC B4 e H358 pLUC F1 com inibição de KRAS e IKK&#946; por interferência de RNA, foram injetados pela veia da cauda nesses camundongos e as metástases foram monitoradas por imageamento in vivo. Houve metástases em 20% dos animais com siIKK&#946; na região anatômica da boca. Os animais que receberam siControle e siKRAS não apresentaram nenhuma metástase visível no equipamento, mas foi observado micrometástases nas análises histológicas dos pulmões. O resultado do experimento de metástase in vivo é inesperado, não só pelo fato de ocorrer no grupo experimental siIKK&#946;, mas também pelo local anatômico do tumor, sendo necessária uma maior investigação do papel de IKK&#946; nesse processo, podendo ser um resultado aleatório. Quando avaliamos em conjunto, nossos resultados sugerem que a quinase IKK&#946; desempenha um papel importante no fenótipo migratório e invasivo de células pulmonares portadoras de KRAS oncogênica, contribuindo para a capacidade metastática. / Lung cancer is the leading cause of cancer deaths worldwide. The most frequent genetic changes found in lung cancer are driver mutations in the KRAS proto-oncogene. Even though KRAS mutations have been causally linked to the oncogenic process, therapies targeted to oncogenic RAS have failed in clinical trials. One of the main characteristics in oncogenesis is the ability of tumors to acquire metastatic capability. The objective of this project is to identify therapeutic targets that reduce KRASinduced lung cancer metastasis. Based on previous reports that oncogenic KRAS, drives not only tumor initiation, but also promotes a metastatic phenotype, the hypothesis of this project is that (1) the acquisition of metastatic ability induced by KRAS in the lung is potentiated by the IKK kinase; and (2) that IKK&#946; inhibition will reduce KRAS-induced cell invasive properties and KRAS-induced tumor metastasis. This hypothesis has been formulated on the basis of previous studies showing that the main IKK&#946; substrate, the transcription factor NF-&#954;B, is activated by KRAS in lung tumors in situ in an IKK&#946;-dependent manner, that NF-&#954;B is known to promote metastasis in different tumor models, and that pharmacological IKK&#946; inhibition in a KRAS-induced lung cancer mouse model reduces tumor growth and progression to higher histological tumor grades. Our goal was evaluate how inhibition of IKK&#946; affects migration and invasion of KRAS-positive lung cells in vitro and whether inhibition of IKK&#946; is capable of affecting the metatactic capacity of these cells in vivo. First, we evaluated the expression of enzymes involved in the metastatic phenotype, matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) and also a molecule involved in the integrinmediated adhesion, FAK (focal adhesion kinase), we targeted IKK&#946; by a highly specific IKK inhibitor (Compound A) or with RNA interference in A549 and H358 cells. We also used colorimetric Matrix Biotrak Activity Assay System to measure the activity of MMPs with RNA interference for KRAS (siKRAS) and IKK&#946; (IKK&#946;) and we have seen that IKK&#946; appears to modulate the expression or activity of MMP-9 and decreases the expression of FAK. The expression of MMP-2 did not change. Then we evaluated migration in A549 cell and invasion in A549 and H358 cells with inhibition of IKK by RNA interference or with Compound A treatment in Transwell assays, and observed a significantly reduced cell migration and invasion in vitro. We then generated cell lines to express luciferase, the A549 pLUC and H358 pLUC lines. A549 pLUC B4 and H358 pLUC F1 cells with RNA interference for KRAS and IKK&#946; were injected in the tail vein in nude (balb/c) mice and metastases were monitored by in vivo imaging. There were metastases in 20% of IKK&#946; animals in the anatomical region of the mouth. Animals that received siControl and siKRAS had no visible metastasis in the live imaging, but micrometastases were observed in the histological analyzes of the lungs. The result of this experiment is unexpected, not only due to the fact that it occurs in the IKK&#946; experimental group, but also due to the anatomical site of the tumor, and a further investigation of the role of IKK&#946; in this process, can be a random result. When evaluated together, our results suggest that the IKK&#946; kinase plays an important role in the migratory and invasive phenotype of in KRAS positive lung cancer cells, contributing to metastatic capacity.

Introdução: O câncer de pulmão é associado a campanhas antitabaco que, se por um lado ajudaram a reduzir a incidência de fumo e câncer de pulmão, por outro contribuíram para a culpabilização e estigmatização do doente em relação à sua doença, além associá-la fortemente a morte e sofrimento. Estigma é um rótulo socialmente negativo imposto a um indivíduo que acarreta sofrimento a quem o recebe. Existem poucos instrumentos que mensuram o estigma associado ao câncer de pulmão e nenhum deles foi validado no Brasil. Objetivo: Adaptar e validar para a língua portuguesa brasileira a Cataldo Lung Cancer Stigma Scale (CLCSS) em pacientes com câncer de pulmão. Método: Trata-se de um estudo metodológico desenvolvido com 188 pacientes em tratamento ambulatorial por câncer de pulmão. Os dados foram coletados nos anos de 2014 e 2015 em dois serviços privados de oncologia na cidade de São Paulo. Os pacientes foram caracterizados nos aspectos sociodemográficos e clínicos. Para testar as validades convergentes e divergentes verificou-se a associação entre estigma e ansiedade, estigma e depressão e estigma e autoestima. A validade do construto foi avaliada por meio da análise fatorial exploratória. A consistência interna e o teste-reteste (37 pacientes) foram utilizados para análise da confiabilidade. Os dados foram analisados por meio do programa estatístico SPSS® v. 22.0. Resultados: A maioria dos pacientes era composta por homens (55,3%), brancos (86,7%), viviam com companheiros (72,9%), tinham nível de escolaridade superior (43,6%), idade média de 63 anos e renda familiar média de R$13.747,90. O tipo de câncer de pulmão prevalente foi o de células não pequenas (94,1%), o estágio foi o avançado (82,5%) e a média de tempo do diagnóstico foi de 22,9 meses. Tinham história prévia de uso do tabaco 68,6% dos pacientes. Os pacientes apresentaram baixos escores de ansiedade e depressão, a autopercepção de ser estigmatizado quase não foi observada e demonstraram altos escores de autoestima. A CLCSS versão brasileira foi validada com 24 itens e a análise fatorial exploratória confirmou os quatro fatores da escala original. Os coeficientes alfa de Cronbach variaram entre 0,63-0,89 para as subescalas (0,77 para estigma e vergonha, 0,88 para isolamento social, 0,89 para discriminação e 0,63 para tabagismo) e o alfa total foi de 0,85. Houve fraca correlação positiva entre estigma e depressão (r=0,25; p=0,000) e estigma e ansiedade (r=0,30; p=0,000) e fraca correlação negativa entre estigma e autoestima (r=-0,44; p= 0,000). A CLCSS apresentou-se estável nas duas avaliações (CCI= 0,7). Conclusões: Os testes psicométricos apontaram a validade e a confiabilidade da CLCSS-versão brasileira. Novas pesquisas devem ser realizadas em amostras com características diversas. / Introduction: Lung cancer is associated with tobacco campaigns, that helped to reduce the incidence of smoking and lung cancer but, on the other hand, contributed to the blame and stigmatization of the patient about the disease, as well as strongly associate it with death and suffering. Stigma is a socially negative label imposed on a person who causes suffering to the recipient. There are few instruments that measure the stigma associated with lung cancer and none have been validated in Brazil. Objective: To validate a Brazilian Portuguese language Cataldo Lung Cancer Stigma Scale (CLCSS) in patients with lung cancer. Methods: This is a methodological study conducted with 188 patients receiving outpatient treatment for lung cancer. Data were collected in 2014 and 2015 in two private oncology services in the city of São Paulo. Patients were characterized in sociodemographic and clinical aspects. To test the convergent and divergent validity it has been found the association between stigma and anxiety, stigma and depression and stigma and self-esteem. The construct validity was evaluated by exploratory factor analysis. Internal consistency and test-retest (37 patients) were used to analyze the reliability. Data were analyzed through the statistical program SPSS v. 22.0. Results: Most patients were men (55.3%), white (86.7%), living with partners (72.9%), had higher level of education (43.6%), mean age 63 years and average household income of R$ 13,747.90. The prevalent type of lung cancer is the non-small cell (94.1%), the stage was advanced (82.5%) and the mean time from diagnosis was 22.9 months. 68.6% of patients had a history of tobacco use. The patients had low scores of anxiety and depression, self-perception of being stigmatized almost was not observed and demonstrated high scores of self-esteem. The CLCSS - Brazilian version was validated with 24 items and exploratory factor analysis confirmed the four factors of the original scale. The Cronbach alpha coefficients ranged from 0.63 to 0.89 for the subscales (0.77 to stigma and shame, 0.88 for social isolation, discrimination and 0.89 to 0.63 for smoking) and the total alpha was 0.85. There was a weak positive correlation between stigma and depression (r=0.25; p=0.000) and stigma and anxiety (r=0.30; p=0.000) and a weak negative correlation between stigma and self-esteem (r=-0.44; p=0.000). The CLCSS remained stable in both assessments (ICC=0.7). Conclusions: The psychometric tests showed the validity and reliability of the Brazilian CLCSS-version. Further studies should be performed on samples with different characteristics

O câncer de pulmão é a principal causa de morte relacionada ao câncer no mundo. Mutações em KRAS são altamente prevalentes no câncer e têm sido diretamente associadas ao processo tumorigênico. Apesar disso, até hoje todas as terapias visando inibir KRAS diretamente falharam e a caracterização de alvos indiretos, importantes para a oncogênese mediada por KRAS, é fundamental para o desenvolvimento de novas terapias contra o câncer de pulmão. Nós mostramos previamente que as quinases Aurora A (AURKA) e B (AURKB) são alvos a jusante de KRAS, importantes para o crescimento, viabilidade e oncogenicidade de linhagens celulares derivadas de tumores pulmonares mediados por KRAS. Aqui, nós aprofundamos os nossos estudos para melhor caracterizar AURKA e AURKB como potenciais alvos terapêuticos no câncer de pulmão. Os objetivos deste trabalho foram (1) investigar o mecanismo de perda de viabilidade induzido pela inibição de AURKA e/ou AURKB; (2) avaliar como a inibição de AURKA e/ou AURKB afeta propriedades oncogênicas relacionadas à agressividade tumoral; e (3) como a inibição destas quinases afeta o crescimento tumoral in vivo. Para tanto, nós utilizamos dois modelos celulares: (1) células A549 e H358, que apresentam mutações em KRAS, geneticamente modificadas para a expressão estável e induzível de shRNAs contra AURKA ou AURKB, e (2) células tumorais H1703, que não apresentam mutações em KRAS, geneticamente modificadas para a expressão induzível de KRASG12V, tratadas ou não com inibidores farmacológicos das quinases Aurora. A inibição farmacológica ou por interferência de RNA de AURKA e/ou AURKB em células H358 e A549 reduziu a proliferação celular, sendo esta inibição acompanhada de anomalias mitóticas, além de aneuploidia e poliploidia. A inibição destas quinases também induziu morte celular in vitro, tanto em mitose, quanto em interfase. Mais interessantemente, a inibição farmacológica dual de AURKA e AURKB induziu morte celular in vitro em células H1703, somente na presença de KRASG12V, indicando que a inibição das quinases Aurora afeta preferencialmente células portadoras de mutações em KRAS. Além disso, a inibição de AURKA e/ou AURKB reduziu propriedades malignas celulares relacionadas à agressividade tumoral, como migração, invasão e adesão. Finalmente, a inibição de AURKA por RNA de interferência em células A549 também reduziu a formação de tumores in vivo. Entretanto, como a inibição destas quinases levou a anomalias mitóticas e à instabilidade genética, nós resolvemos investigar se a inibição de TPX2, um substrato e ativador de AURKA, poderia ser uma abordagem alternativa para inibir esta via em câncer de pulmão induzido por KRAS. Primeiramente, nós observamos nos nossos modelos celulares que KRAS regula positivamente a expressão de TPX2. Além disso, a inibição de TPX2 em células pulmonares portadoras de KRAS oncogênica reduziu a viabilidade e proliferação celulares e induziu morte celular. Mais interessantemente, esses efeitos ocorreram preferencialmente em células que expressam KRAS oncogênica. Em conclusão, nossos resultados apoiam a hipótese de que a ativação de AURKA/TPX2 e AURKB por KRAS são eventos importantes no câncer de pulmão e sugerem a inibição destas vias, possivelmente em combinação com outras terapias citotóxicas, como uma nova abordagem terapêutica para o câncer de pulmão induzido por KRAS. / Lung cancer is the leading cause of cancer-related deaths worldwide. KRAS mutations are widespread in lung cancer and have been causally linked to tumorigenesis. Nonetheless, therapies targeting KRAS directly have so far failed and characterization of indirect KRAS targets, which play important roles in KRAS-mediated oncogenesis, is crucial for the development of new therapies for lung cancer. We have previously shown that mitotic kinases Aurora A (AURKA) and B (AURKB) are downstream targets of oncogenic KRAS, important for the growth, viability, and oncogenicity of KRAS-transformed lung cancer cell lines. Here, we studied these kinases more in depth in order to better characterize them as potential therapeutical targets for KRAS-induced lung cancer. The aims of this study were (1) to investigate the mechanism leading to loss of viability upon AURKA and/or AURKB targeting; (2) to evaluate how AURKA and/or AURKB inhibition affects malignant properties associated with tumor aggressiveness; and (3) to determine whether AURKA and/or AURKB inhibition reduces KRAS-induced tumor growth in vivo. For that purpose, we used two cell-based models: (1) KRAS mutant A549 and H358 cells with stable and inducible shRNA-mediated knockdown of AURKA or AURKB, and (2) KRAS wildtype H1703 tumor cell lines, genetically engineered to inducibly express oncogenic KRASG12V treated or not with Aurora kinase pharmacological inhibitors. Targeting AURKA and/or AURKB pharmacologically or by RNA interference in H358 and A549 cells led to decreased cell proliferation, which was accompanied by mitotic abnormalities, leading to aneuploidy and hyperploidy. Aurora kinase targeting also induced cell death in vitro, both during mitosis and interphase. More importantly, AURKA and AURKB inhibition with a dual pharmacological inhibitor in H1703 cells induced cell death in vitro, but only in the presence of KRASG12V, indicating that Aurora kinase targeting affects preferentially lung cells harboring oncogenic KRAS. Furthermore, AURKA and/or AURKB targeting reduced malignant properties associated with tumor aggressiveness, such as cell migration, invasion and adhesion. Finally, AURKA targeting by RNA interference in A549 cells also reduced growth of xenograft tumors in vivo. Nonetheless, since Aurora targeting was associated with mitotic abnormalities and genetic instability, we decided to investigate if targeting TPX2, a substrate and an activator of AURKA, could constitute an alternative approach to targeting this pathway in KRAS-induced lung cancer. First, using our cell-based models, we determined that KRAS positively regulates TPX2 expression. In addition, TPX2 inhibition by RNA interference in KRAS-positive lung cells reduced cell viability and proliferation and induced cell death. Finally, these effects occurred preferentially in cells harboring oncogenic KRAS. In conclusion, our results support the hypothesis that activation of AURKA/TPX2 and AURKB by KRAS are important events in lung cancer and suggest inhibition of these pathways, possibly in combination with other cytotoxic therapies, as a new approach for KRAS-induced lung cancer therapy.

Estudos anteriores (Zayas, 1990) tem sugerido a existência de uma melhor transportabilidade por cílio do muco respiratório de pacientes fumantes que não apresentam câncer de pulmão em comparação com pacientes fumantes com câncer de pulmão e semelhante carga tabágica. Nosso principal objetivo foi verificar esta hipótese. Nós estudamos 16 tabagistas com câncer de pulmão (média carga tabágica = 58,78), 16 tabagistas com câncer extra-pulmonar (média carga tabágica = 53,87) e 11 não Tabagistas com Metástase Pulmonar com indicação de broncoscopia diagnóstica. O muco respiratório foi coletado durante a broncoscopia, usando um pequeno cateter através do canal de aspiração do broncoscopio. A transportabilidade por cílio no palato de rã, ângulo de contato (wetabilidade), transportabilidade pela tosse e viscosidade (cone-plate) e análise morfológica do epitélio respiratório foi realizado. Não foi encontrada diferença estatística entre os pacientes Tabagistas (Câncer de pulmão e Câncer extra-Pulmonar) para os parâmetros de muco estudos. Da mesma forma não foi encontrada diferença estatística nas análises do muco coletado de um lado do tumor comparado com o lado contralateral. Entretanto, encontramos diferença estatística entre os grupos não tabagistas com Metástase Pulmonar e Tabagistas com câncer Pulmonar e Extra-Pulmonar para os parâmetros de Transportabilidade pela Tosse (p = 0,018), Viscosidade 10 rpm (p= 0,021), FEV 1 (L) (p= 0,028) e FEV 1 (%) (p= 0,042) e diferença estatística nos Tabagistas para Correlação entre carga tabágica e idade (p=0,038) e Viscosidade (p= 0,029). Na análise histologica observamos 10 Tabagistas (60% alteração, sendo, 30% metaplasia escamosa; 20% hyperplasia e 10% epitélio com ausência de cílios) e 15 não Tabagistas (40% com alteração histológica sendo 20% destes com metaplasia escamosa e em pacientes com Câncer pulmonar ou Câncer extra-pulmonar). Não teve diferença na composição das mucinas entre os tabagistas. Concluímos que não há diferença entre as propriedades físicas do muco respiratório de Tabagistas com Câncer de Pulmão e Câncer Extra-Pulmonar com similar carga tabágica e que essas alterações das propriedades físicas do muco respiratório e alterações morfológicas, devem-se mais à exposição dose-tempo da fumaça do cigarro ao epitélio respiratório / Previous study (Zayas 1990) has suggested the existence of a better transportability by cilia in respiratory mucus of smoking patients who did not present lung cancer in comparison to lung cancer patients smoking similar packages/year. Our aim was to verify this hypothesis. We studied 16 smoking patients with lung cancer (mean packages/year = 58,78), 16 smoking patients with extra pulmonary cancer (esophagus and head and neck), (mean packages/year = 53,87), and 11 non-smoking patients (metastasis) that underwent diagnostic bronchoscopy. Respiratory mucus was collected during bronchoscopy, using a small catheter passed through the aspiration channel of the bronchoscope. Mucus transportability in frog palate, contact angle (wettability), transportability by cough and viscosity (cone-plate) as well as morphological analysis the respiratory epithelium were performed. No statistical differences were found between smoking patients (lung and extra pulmonary cancer) in the mucus parameters studied. In the same way, no difference was found in the analysis of mucus samples collected from the tumor side compared to contra lateral samples. Nevertheless, statistical difference between Smoking (Lung Cancer and Extra-Pulmonary Cancer) and non Smoking (metastasis Pulmononary) for valous Clearance by cough (p = 0,018), viscosity 10rpm (p=0,021) FEV 1 (L) (p= 0,028) and FEV1 (%) (p=0,042) and statistical difference for correlation between smoking history and age (p=0,038) and viscosity 10 rpm (p= 0,029). The analysis histological of the 10 smoking, observed 60% of cases with alteration histological (30% with squamous metaplasia; 20% with hyperplasia and 10% with epithelium with cilia absence) and 15 non smoking presented 40% of cases with alteration histological (205 with squamous metaplasia in patients with lung cancer or exra-pulmonary cancer. We conclude that there is no difference between the physical properties of the respiratory mucus of smokers with lung cancer and extra-pulmonary cancer with similar packages/year and that changes in physical properties of respiratory mucus and morphological changes, due to more exposure to the dose-time of cigarette smoke in the respiratory epithelium