Lung cancer is the leading cause of cancer related deaths in the United States, with an estimated 158, 040 deaths in 2015, accounting for 27% of all cancer deaths. Recent research has identified several important molecular driver oncogenes, including epidermal growth factor receptor (EGFR). EGFR is encoded by exons 18-21, each of which harbor specific mutations within the tyrosine kinase domain. These mutations can drive cell growth, proliferation, and survival, resulting in the formation of non-small cell lung cancer. The development of EGFR tyrosine kinase inhibitors, allows the targeting of these specific mutations without the toxicity normally associated with standard chemotherapy. Unfortunately, inevitably resistance to therapy manifests, requiring a change in therapy and adding complexity to treatment decision making for clinicians and patients alike. Through a comprehensive examination of current literature, this review will establish a standard for first line, targeted treatment for specific genetic mutations within the EGFR gene, as well as address treatment options once resistance to first-line therapy inevitably develops.

Des mutations du gène STK11 furent initialement décrites comme responsable du syndrome Peutz-Jeghers, dont la gravité est lliée à une incidence accrue d’apparition de tumeurs. Le produit de ce gène, la sérine/thréonine kinase LKB1, a une expression ou une activité catalytique réduite, voir perdue, consécutivement à des mutations somatiques dans plusieurs types de cancer mais principalement du poumon (30% des NSCLC). Cette kinase est considérée de ce fait comme un suppresseur de tumeur d’importance. Les mécanismes moléculaires responsables de sa propriété suppresseur de tumeur restent à identifier. En effet, alors que sa fonction dans le métabolisme cellulaire, au travers de l’activation de la kinase AMPK, fut longtemps privilégiée, elle est actuellement remise en cause au profit de sa fonction de régulatrice de la signalisation Wnt canonique. Mes travaux de thèse confortent cette éventualité dans le cas des tumeurs pulmonaires (NSCLC). En effet, parmi les deux complexes fonctionnels que forme LKB1 avec les pseudokinases STRADα ou β, mes résultats démontrent que seul celui impliquant STRADβ intervient dans la régulation de la voie Wnt. Pour cela, le complexe LKB1/STRADβ se localise au niveau du cil primaire et participe à l’activation de la kinase MARK3. Ces résultats, étayés par un modèle murin invalidé pour STRADβ ainsi que l’analyse, a posteriori, de bases de données transcriptomiques adossées aux données cliniques de patients atteints de NSCLC, suggèrent que l’activité suppresseur de tumeur de LKB1 est associée à sa localisation et à sa fonction au niveau du cil primaire en participant à l’activation de MARK3 et à la régulation de la signalisation Wnt canonique. / STK11 gene mutations were originally identified as responsible for the Peutz-Jeghers syndrome of which severity is mainly related to an increase incidence of tumor development. The product of this gene the serine/threonine kinase LKB1 gets its activity or its expression reduced, sometimes even lost, following somatic mutations in several types of cancer such as pancreas, liver but mainly from lung. Indeed, almost 30% of non-small cell lung carcinoma (NSCLC) does not express anymore or only an inactive form, has led to consider this kinase as tumor suppressor of importance. While there is no doubt of the involvement of its catalytic activity molecular mechanisms responsible for its tumor suppressor properties remain to be identified. Indeed, whereas its function as regulator of cellular metabolism through AMPK has been favor for a while, it is currently re-assess to benefit to its regulator function on canonical Wnt signaling. My thesis work, reinforce this eventuality in NSCLC. Indeed, among the two functional complexes formed by LKB1 through its association with STRADα or β pseudokinases, my results show that only the complex related to STRADβ is involved in the canonical Wnt pathway regulation. For that, LKB1/STRADβ complex localizes at primary cilia and participates to MARK3 kinase activation. These results strengthened by a STRADβ knockout mouse model and an a posteriori transcriptomic analysis of lung adenocarcinoma patient datasets related to their clinical records, suggest that LKB1 tumour suppressor activity is associated with its localization and its function at primary cilia participating in the activation of MARK3 and thus regulation of canonical Wnt signaling.

Lung cancer is the leading cause of cancer-related death and the second most diagnosed cancer in the United States. Unfortunately, many patients either do not have any common mutations for which there are already targetable agents, or they eventually become resistant to these compounds. As such, there is a high demand for new, effective methods of treating this disease as well as predicting patient prognosis and potential benefit from chemotherapy. In this work, numerous strategies for treating lung cancer are explored. The first method described here is through the use of a pan-early 2 factor (E2F) inhibitor, HLM006474, which is shown to synergize with paclitaxel in non-small cell lung cancer (NSCLC). Next, we explored the creation and utilization of an E2F signature that is prognostic and predictive of early-stage NSCLC patient benefit from adjuvant chemotherapy (ACT). The third project examined possible targets to enhance sensitivity to cisplatin in NSCLC lacking Kirsten rat sarcoma viral oncogene homolog (KRAS) and epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma receptor tyrosine kinase (ALK) fusions (triple-negative), for which cisplatin is one of the few treatment options. These studies led to the identification of a kinase that is overexpressed in NSCLC and whose knockdown sensitizes cells to platinum agents.

The gene PTPN11 was identified in the early 1990s, and encodes the non-transmembrane protein tyrosine phosphatase SHP2. SHP2 is expressed ubiquitously in cells, and plays an important role in cancer. Unlike most phosphatases, SHP2 positively regulates several signaling pathways including the Ras/MAPK and Src signaling pathways and acts as a proto-oncogene. SHP2 is also a cancer essential gene in certain types of carcinomas, and promotes growth, survival, and epithelial to mesenchymal transformation. Gain of function (GOF) SHP2 mutations are known leukemic oncogenes, and have been identified to a smaller extent in solid tumors as well. Currently, the roles of SHP2 in lung carcinoma are not fully understood. While GOF SHP2 mutations have been detected in lung cancer, their contributions to cellular transformation had not been established. In addition, SHP2 is known to promote EGF growth factor receptor (EGFR) signaling. Since GOF EGFR mutations induce transformation of lung epithelial cells, it is possible that SHP2 plays a role in promoting GOF EGFR mutant driven tumorigenesis. The objective of this dissertation is to determine whether SHP2 can act as an oncogene in lung epithelial cells and whether SHP2 inhibition can affect GOF EGFR mutant induced lung cancer. To achieve these aims, we generated two novel doxycycline (Dox) inducible transgenic mouse models which express either the GOF SHP2E76K leukemic oncogene or the dominant negative SHP2CSDA mutant under the control of the Clara cell secretory protein (CCSP) promoter to regulate transgene expression to type II pneumocytes. To determine whether SHP2 plays a role in promoting GOF EGFR mutant signaling, we started by disrupting SHP2 function in vitro. Two non small lung cancer cell lines were used for this project: HCC827 carries the LREA deletion in exon 19, and H1975 co-expresses the EGFRL858R point mutation and the EGFRT790M gatekeeper mutation. After SHP2 PTP inhibition or knock-down by shRNA and siRNA, both cell lines exhibited decreased cell proliferation and reduced levels of pErk1/2 and c-Myc. Based on these results, we acquired a transgenic mouse line which expresses the EGFRL858R mutant under the control of the tet-O promoter and generated bitransgenic CCSP-rtTA/tetO-EGFRL858R and tritransgenic CCSP-rtTA/tetO-EGFRL858R/tetO-SHP2CSDA mice to study the effects of the dominant negative SHP2CDSA mutant on EGFRL858R mediated carcinogenesis in vivo. After 4, 6, and 8 weeks of Dox induction, pErk1/2 and pSrc levels were increased in the lungs of bitransgenic mice compared to wild type controls. Both kinases were suppressed by SHP2CSDA expression in tritransgenic mice. In addition, SHP2CSDA expression delayed tumor onset and prevented progression to a more aggressive phenotype. Tritransgenic mice also developed a smaller tumor burden compared to bitransgenic animals. These results suggest that SHP2 is critical for GOF EGFR mutant mediated lung tumorigenesis and describe a new role of SHP2 as a potential therapeutic target for the development of novel NSCLC drugs. Once we generated our CCSP-rtTA/tetO-SHP2E76K transgenic mouse model, we administered Dox for one month and found that SHP2E76K expression upregulates pErk1/2, pSrc, pGab1, c-Myc and Mdm2 levels in the lungs of bitransgenic mice compared to controls. After six to nine months of Dox induction, SHP2E76K expression caused formation of lung adenomas and adenocarcinoma. We then took advantage of the reversible feature of our mouse model to test whether lung tumors are dependent on sustained SHP2E76K expression for survival. MRI analysis of lung adenocarcinomas showed full regression of the lung tumors after Dox withdrawal. Histological evaluation of lung tissues revealed residual hyperplastic lesions as well as evidence of necrosis, while biochemical analysis showed that pGab1, pErk1/2, pSrc and c-Myc returned to basal levels. These results demonstrate that sustained SHP2E76K expression is required for lung tumor maintenance. Moreover, this data describe a novel function of SHP2E76K as an oncogene in lung carcinoma.

Cancer, the uncontrolled growth of morphologically and genetically abnormal cells in the body, is a major worldwide public health problem and there is a great need for novel insights into this disease. The majority of tumors arise from the acquisition of somatic alterations leading to changes in gene function and expression. The clinical success of targeted therapeutics in molecularly defined subsets of patients has highlighted the need for comprehensive characterization of the somatic alterations in individual cancer types. Copy number profiling using SNP arrays is a common approach for profiling the extent of copy number variation across the cancer genome. In addition, next-generation sequencing technologies now offer researchers the ability to also systematically catalog nucleotide substitutions and structural rearrangements in dramatically less time and expense. In this thesis, we describe the application of SNP arrays and whole-exome sequencing to characterize two separate cohorts of cancer samples, as well as describe the development of a software tool to aid in the annotation of mutational data. Specifically, we detailed focal amplifications of PDGFRA and KIT in a combined set of lung adenocarcinoma and squamous cell carcinomas. Furthermore, in a cohort of small bowel neuroendocrine tumors, we characterized the global genetic landscape to show that these tumors are molecularly distinct from other neuroendocrine tumors. Lastly, we report Oncotator, a novel web application and service for comprehensive annotation of point mutations and indels found in cancer. It is hoped that the knowledge gained from these studies will fuel improvements in cancer diagnosis, prognosis, and therapy.

abstract: A tumor is a heterogeneous combination of proliferating tumor cells, infiltrating immune cells and stromal components along with a variety of associated host tissue cells, collectively termed the tumor microenvironment (TME). The constituents of the TME and their interaction with the host organ shape and define the properties of tumors and contribute towards the acquisition of hallmark traits such as hypoxia. Hypoxia imparts resistance to cancer from chemotherapy and radiotherapy due to the decreased production of reactive oxygen species and also promotes angiogenesis, malignant progression and metastasis. It also provides a powerful physiological stimulus that can be exploited as a tumor-specific condition, allowing for the rational design of anticancer hypoxia-activated pro-drugs (HAP). Accurate evaluation of tumor oxygenation in response to therapeutics interventions at various stages of growth should provide a better understanding of tumor response to therapy, potentially allowing therapy to be tailored to individual characteristics. The primary goal of this research was to investigate the utility of prospective identification of hypoxic tumors, by two different Magnetic Resonance Imaging (MRI) based oximetry approaches, in successful treatment with hypoxia activated therapy. In the present study, I report the utility of these two techniques 1) PISTOL (Proton Imaging of Siloxanes to map Tissue Oxygenation Levels) and 2) use of a hypoxia binding T1 contrast agent GdDO3NI in reporting the modulations of hypoxia pre and post hypoxia activated therapies in pre-clinical models of cancer. I have performed these studies in non-small cell lung cancer (NSCLC) and epidermoid carcinoma (NCI-H1975 and A431 cell lines, respectively) as well as in patient derived xenograft models of NSCLC. Both the oximetry techniques have the potential to differentiate between normoxic and hypoxic regions of the tumor and reveal both baseline heterogeneity and differential response to therapeutic intervention. The response of the tumor models to therapeutic interventions indicates that, in conjunction with pO2, other factors such as tumor perfusion (essential for delivering HAPs) and relative expression of nitroreductases (essential for activating HAPs) may play an important role. The long term goal of the proposed research is the clinical translation of both the MRI techniques and aiding the design and development of personalized therapy (e.g. patient stratification for novel hypoxia activated pro-drugs) particularly for cancer. / Dissertation/Thesis / Doctoral Dissertation Bioengineering 2017

Background: Lung cancer is the leading cause of cancer-related death and one of the most common cancer types worldwide. Epidermal growth factor receptor (EGFR) has been shown to be an important therapeutic target in non-small cell lung cancer. Kirsten rat sarcoma viral oncogene homologue (KRAS) is a downstream signalling molecule in the EGFR pathway. Lung cancer patients with EGFR mutations respond to tyrosine EGFR inhibitor therapy, in contrast, patients with KRAS mutations do not benefit of such treatment. Methods: This study investigates the frequency of EGFR and KRAS mutations in non-small cell lung cancer patients. Fifty-one lung cancer patients with primary non-small cell lung cancer diagnosed between 1995 and 2005 in the Uppsala-Örebro region were analysed by Sanger sequencing and Pyrosequencing to determine the mutation status of these genes. Results: Five EGFR mutations were found in four patients (8%), two deletions in exon 19, one point mutation in exon 20 and two point mutations in exon 21. KRAS mutations were found in 12 patients (24%), ten codon 12 mutations and two codon 61 mutations. Conclusions: This study confirms previous observations regarding the frequency of EGFR and KRAS mutations in non-small cell lung cancer. Mutations in EGFR and KRAS were mutually exclusive, indicating that both mutations present relevant tumorigenic genomic aberrations.

[EN] Lung cancer is the leading cause of cancer-related death worldwide, and is the third most common cancer type; it can be classified into two subgroups based on histology: non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC). The 5-year survival still remains poor and despite the existence of several distinct tumour phenotypes, therapeutic decisions are mainly based on clinical features such as stage or performance status. This highlights the need for new diagnostic and prognostic biomarkers in different types of samples (such as blood, fresh-frozen tissue or formalin-fixed, paraffin-embedded samples). The field of tumour immunology has changed in the last decade, and it is now accepted that the immune system plays a pivotal role in cancer. Although the immune cells that infiltrate the tumour microenvironment are potentially capable of eliminating tumour cells, they cannot prevent tumour development and progression. Tumours acquire mechanisms to regulate their immune microenvironment such as the release of a series of factors to subvert normal reaction mechanisms, the modulation of co-stimulatory pathways, also known as immune checkpoints, and the induction and attraction of suppressor cells (myeloid-derived suppressor cells, tumour-associated macrophages, and regulatory T cells). The potential effect of the patient's immune system on clinical outcome is important for the identification of prognostic markers as well as markers that predict treatment responses. The study of immune-related markers, especially those implicated in immunoregulatory processes, could provide valuable prognostic information that could help in many applications in future clinical practice. Thus, the objective of this thesis is to characterise cancer immunoregulation biomarkers and to evaluate the possible correlation between these biomarkers and clinicopathological and prognostic variables in patients with NSCLC by the use of well-tested and accurate techniques such as quantitative PCR and immunohistochemistry. Furthermore, this study will provide information about the immunological features of the tumour microenvironment in NSCLCs. / [ES] El cáncer de pulmón es una de las principales causas de muerte relacionada con cáncer en el mundo, siendo el tercer tipo de cáncer más común. El cáncer de pulmón no microcítico (CPNM) representa casi el 85% de todos los cánceres de pulmón y la supervivencia a los 5 años va desde el 50% en estadios IA hasta el 15% en estadios IIIA. Hasta el momento, no se han descubierto biomarcadores capaces de predecir la progresión de la enfermedad en pacientes tanto en estadios resecables como en estadios avanzados, por lo que existe una clara necesidad de realizar estudios centrados en la búsqueda de biomarcadores pronósticos y diagnósticos en los diferentes tipos de muestra disponibles, como por ejemplo sangre, tejido fresco y tejido parafinado. El campo de la inmunología tumoral ha cambiado en la última década y actualmente se sabe que el sistema inmune juega un papel clave en cáncer. Las células inmunes que infiltran el tumor son un componente más del microambiente tumoral. Pese a que son potencialmente capaces de eliminar los antígenos tumorales, estas células no pueden evitar la formación y progresión tumoral. Esto es debido a que el tumor adquiere diversos mecanismos de regulación del microambiente tumoral con el objetivo de escapar del ataque del sistema inmune, como por ejemplo liberación de factores que impiden el correcto funcionamiento de los mecanismos de reacción inmune, modulación de vías co-estimuladoras y reclutamiento y activación de células inmunoreguladoras como las células T reguladoras, las células mieloides supresoras y los macrófagos asociados a tumores. El estudio de marcadores relacionados con la respuesta inmune y concretamente con los procesos de inmunoregulación puede proporcionarnos información pronóstica y predictiva relevante sobre los pacientes con cáncer. Por todo ello, el principal objetivo de esta tesis doctoral es analizar la presencia de marcadores relacionados con la inmunoregulación y evaluar su posible correlación con las variables clínico-patológicas y pronósticas en pacientes con CPNM mediante el uso de técnicas fiables y aplicables en la práctica clínica como la PCR cuantitativa y la inmunohistoquímica. Así mismo, esto nos permitirá conocer en mayor profundidad las características inmunológicas del microambiente tumoral en pacientes con CPNM. / [CAT] El càncer de pulmó és una de les principals causes de mort relacionades amb càncer al món, sent a més a més el tercer tipus de càncer més comú. El càncer de pulmó no microcític (CPNM) representa el 85% de tots els casos de càncer de pulmó aproximadament i la supervivència als 5 anys continua sent molt baixa. Fins el moment, no s'han descobert biomarcadors capaços de predir la progressió de la malaltia tant en pacients en estadis inicials com en estadis avançats. Per aquest motiu, existeix una clara necessitat de realitzar estudis centrats en la recerca de biomarcadors pronòstics i predictius en els diferents tipus de mostres disponibles, com per exemple sang, teixit fresc i teixit parafinat. El camp de la immunologia tumoural ha canviat en l'última dècada i actualment se sap que el sistema immune exerceix un paper clau en el càncer. Les cèl¿lules immunològiques que infiltren el tumour són un component més del microambient tumoural. Malgrat que aquestes cèl¿lules són potencialment capaces d'eliminar el antígens tumourals, s'ha evidenciat que no poden previndre la formació i progressió tumoural. Una de les raons per les quals s'observa aquest fenomen és que el tumour adquireix diversos mecanismes de regulació del microambient tumoural. Aquests mecanismes es basen en l'alliberació de factors que impedeixen el correcte funcionament del sistema immune, la modulació de vies coestimuladores i el reclutament i activació de cèl¿lules immunoreguladores com poden ser les cèl¿lules T reguladores, les cèl¿lules mieloides supressores i els macròfags associats a tumour. L'estudi de marcadors relacionats amb la resposta immune i més concretament amb els processos d' immunoregulació pot proporcionar informació pronòstica i predictiva rellevant sobre els pacients amb càncer. Per tot això, el principal objectiu d'aquesta tesi doctoral és analitzar la presència de marcadors relacionats amb la immunoregulació i avaluar la seva possible correlació amb les variables clinicopatològiques i pronòstiques de pacients amb CPNM mitjançant l'ús de tècniques fiables i aplicables a la pràctica clínica com són la PCR quantitativa i la immunohistoquímica. Així mateix, aquestes anàlisis ens permetran conèixer amb major profunditat les característiques immunològiques del microambient tumoural de pacients amb CPNM. / Usó Marco, M. (2015). ANALYSIS OF IMMUNOREGULATORY BIOMARKERS IN NON-SMALL CELL LUNG CANCER [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/51283 / TESIS

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The evolutionary conserved Myb-MuvB (MMB) multiprotein complex has an essential role in transcriptional activation of mitotic genes. MMB target genes as well as the MMB associated transcription factor B-Myb and FoxM1 are highly expressed in a range of different cancer types. The elevated expression of these genes correlates with an advanced tumor state and a poor prognosis. This suggests that MMB could contribute to tumorigenesis by mediating overexpression of mitotic genes. Although MMB has been extensively characterized biochemically, the requirement for MMB to tumorigenesis in vivo remains largely unknown and has not been tested directly so far. In this study, conditional knockout of the MMB core member Lin9 inhibits tumor formation in vivo in a mouse model of lung cancer driven by oncogenic K-Ras and loss of p53. The incomplete recombination observed within tumors points towards an enormous selection pressure against the complete loss of Lin9. RNA interference (RNAi)-mediated depletion of Lin9 or the MMB associated subunit B-Myb provides evidence that MMB is required for the expression of mitotic genes in lung cancer cells. Moreover, it was demonstrated that proliferation of lung cancer cells strongly depends on MMB. Furthermore, in this study, the relationship of MMB to the p53 tumor suppressor was investigated in a primary lung cancer cell line with restorable p53 function. Expression analysis revealed that mitotic genes are downregulated after p53 re-expression. Moreover, activation of p53 induces formation of the repressive DREAM complex and results in enrichment of DREAM at mitotic gene promoters. Conversely, MMB is displaced at these promoters. Based on these findings the following model is proposed: In p53-negative cells, mitogenic stimuli foster the switch from DREAM to MMB. Thus, mitotic genes are overexpressed and may promote chromosomal instability and tumorigenesis. This study provides evidence that MMB contributes to the upregulation of G2/M phase-specific genes in p53-negative cells and suggests that inhibition of MMB (or its target genes) might be a strategy for treatment of lung cancer. / Der evolutionär konservierte Myb-MuvB (MMB) Multiproteinkomplex hat eine wesentliche Rolle in der transkriptionellen Aktivierung mitotischer Gene. Zielgene des MMB sowie die MMB assoziierten Transkriptionsfaktoren B-Myb und FoxM1 sind hoch exprimiert in einer Bandbreite verschiedener Krebsarten. Die erhöhte Expression dieser Gene korreliert mit einem fortgeschrittenen Tumorstadium und einer geringen Prognose. Das weißt auf darauf hin, dass MMB an der Tumorentstehung beteiligt sein könnte indem es die Überexpression mitotischer Gene fördert. Obwohl MMB biochemisch eingehend untersucht wurde, ist die Erfordernis von MMB zur Tumorentstehung in vivo weitestgehend unbekannt und wurde bisher nicht direkt getestet. In dieser Studie hemmt der konditionale Knockout der MMB Kerneinheit Lin9 die Tumorbildung in vivo in einem Lungenkrebs-Mausmodell angetrieben durch onkogenes K-Ras und den Verlust von p53. Die unvollständige Rekombination welche in Tumoren beobachtet wurde deutet auf einen starken Selektionsdruck gegen den kompletten Verlust von Lin9 hin. Die Verminderung von Lin9 und der MMB- assoziierten Untereinheit B-Myb durch RNAi-Interferenz (RNAi) liefert Beweise dafür, dass MMB für die Expression mitotischer Gene in Lungenkrebszellen notwendig ist. Zudem wurde gezeigt, dass das Zellwachstum von Lungenkrebszellen stark von MMB abhängig ist. Weiterhin wurde der Zusammenhang zwischen MMB und dem p53-Tumorsuppressor in einer primären Lungenkrebszelllinie mit wiederherstellbarer p53-Funktion untersucht. Expressionsanalysen zeigen, dass mitotische Gene nach Re-expression von p53 runterreguliert werden. Außerdem induziert die Aktivierung von p53 die Bildung des repressiven DREAM-Komplexes und führt zu einer Anreicherung von DREAM an Promotoren mitotischer Gene. Im Gegenzug wird MMB an den Promotoren verdrängt. Basierend auf den Ergebnissen wird das folgende Model vorgeschlagen: In p53- negativen Zellen begünstigen mitogene Reize den Wechsel von DREAM zu MMB. Dadurch werden mitotische Gene überexprimiert und können so chromosomale Instabilität und Tumorentstehung fördern Diese Studie liefert Hinweise, dass MMB an der Hochregulation G2/M- Phasenspezifischer Gene in p53-negativen Zellen beteiligt ist und dass die Hemmung von MMB (oder seiner Zielgene) eine Strategie zur Behandlung von Lungenkrebs sein könnte.