The NADPH oxidase homolog, dual oxidase 1 (DUOX1), is an H2O2 producing transmembrane enzyme highly expressed in the airway epithelium. DUOX1-dependent redox signaling has been characterized to regulate many homeostatic processes in the lung epithelium, such as host defense, wound healing, and type II immune responses. Intriguingly, DUOX1 has been found to be suppressed in many epithelial cancers, including lung cancer, by hypermethylation of its promoter. Epigenetic silencing of DUOX1 in cancer is paradoxical to the understanding that tumors harbor elevated levels of reactive oxygen species (ROS), suggesting that DUOX1 may be a tumor suppressor. Since DUOX1 loss occurs in many forms of lung cancer, we aimed to characterize the functional importance of DUOX1 suppression. RNAi-mediated knockdown of DUOX1 in lung epithelial cells induced features of the epithelial-to-mesenchymal transition (EMT), a characteristic of aggressive or invasive tumor cells. Indeed, DUOX1 suppression promoted the acquisition of molecular signatures associated with EMT, such as the loss of E-cadherin, and induced expression of vimentin and smooth muscle actin. Additionally, we find that DUOX1 suppression promotes the acquisition of other EMT-related features, such as enhanced levels of cancer stem cell molecular markers, cellular invasiveness, and critically, resistance to epidermal growth factor receptor (EGFR) inhibition. Importantly, overexpression of DUOX1 in DUOX1-lacking lung cancer cells promoted the recovery of epithelial characteristics, pinning DUOX1 as a critical mediator of the epithelial phenotype. Based on prior studies demonstrating DUOX1 as an important regulator of EGFR signaling in the lung epithelium, we hypothesized that DUOX1 loss in lung cancer may impact EGFR regulation. EGFR belongs to a larger family of ErbB receptor tyrosine kinases, which are often overexpressed or mutated in many forms of lung cancer. Surprisingly, we find that lung cancer cells lacking DUOX1 have significantly altered EGFR redox regulation, specifically, kinetically enhanced cysteine oxidation-reduction dynamics. Additionally, our results demonstrate DUOX1-lacking cancer cells have altered intracellular EGFR trafficking with enhanced nuclear targeting. Indeed, we observe many oncogenic features of nuclear EGFR e.g. enhanced migratory capacity, resistance to EGFR blocking antibodies. Finally, we have uncovered that EGFR cysteine redox dynamics may regulate intracellular trafficking and/or nuclear transport, offering potentially novel avenues in the design of therapeutics. Proper DUOX1 localization and enzymatic function in the plasma membrane requires partnership with its maturation factor, dual oxidase maturation factor 1 (DUOXA1). Preliminary findings from a newly designed DUOX1-DUOXA1 co-expression system suggests that following enzymatic activation of DUOX1, DUOXA1 dissociates from DUOX1 and potentially translocates to the nucleus, a feature not previously described in lung epithelial or cancer cells. While these preliminary results require additional experimentation, this could be a unique regulatory feature of DUOX1 and a novel role for DUOXA1. Collectively, the research demonstrated in this dissertation characterizes the functional and mechanistic importance of DUOX1 suppression in cancer. Indeed, loss of DUOX1 expression may be an indicator of tumor aggressiveness and responsiveness to EGFR-targeted therapies, warranting its potential for use as a clinical biomarker in lung cancer.

The use of stereotactic body radiation therapy employing few large fractions of radiation dose for the treatment of non-small cell lung cancer has been proven very successful, high values of tumour control probability (TCP) being clinically achieved. In spite of the success of the fractionation schedules currently used, there is a tendency towards reducing the number of fractions for economical and practical reasons, and also for maximizing the comfort of the patients. It is therefore the main aim of this thesis to investigate the impact of a severely reduced number of fractions on the tumour control probability for tumours that contain hypoxic areas. The impact on TCP of other factors such as hypoxic fraction, distribution of the oxygen partial pressure and location of the hypoxic volume within the tumour were also investigated. The effect of tumour motion due to breathing was included and evaluated using Cone Beam Computed Tomography (CBCT) data from patients imaged with internal markers in the liver and pancreas. The results clearly showed that in the presence of hypoxia, TCP is seriously compromised if there is not enough time for reoxygenation between fractions. A reduction in the number of fractions of just one fraction may require an increase of several Gy per fraction to obtain a similar TCP. The diaphragmatic tumour motion range showed little influence on TCP provided that the PTV encompassed all tumour positions. The dose delivered to the PTV margin was found not to be the only factor that is significant for local control, the average dose correlated better with TCP. The agreement of the results of this work with clinical results also serve as a strong indicator that inter-fraction reoxygenation is an important process in real-life patients treated with stereotactic body radiotherapy.

Abstract Pulmonary adenocarcinoma is the most common and most heterogeneous form of lung cancer, and its histological and biological diversity is well recognized. On its publication in 2011, the IASLC/ATS/ERS lung adenocarcinoma classification drew attention to the prognostic value of adenocarcinoma subtypes, and it has been anticipated to provide a novel architecture based grading system. The prognostic role of other tumor-associated features in lung adenocarcinoma is less established. MUC1 overexpression has been demonstrated in many carcinomas, and in lung adenocarcinoma, depolarized MUC1 expression has been associated with poor outcome. The role of MUC4 in lung cancer, however, is somewhat conflicting. Moreover, there is no published data on either MUC1 or MUC4 expression with regard to the different subtypes of lung adenocarcinoma. This study aimed to investigate the correlation between the IASLC/ATS/ERS classification, prognosis, and clinical characteristics in a series of 112 surgically resected lung adenocarcinoma patients. The analysis of tumor architecture aimed also at the discovery of new morphological biomarkers for lung cancer. Additionally, the study focused on the expression of MUC1, MUC4, and EGFR in lung adenocarcinoma, evaluating their relationship with tumor architecture, patient outcome, and smoking. The study applied the methods of light microscopy, immunohistochemistry, and cell culture with experimental cigarette smoke exposure combined with real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunoelectron microscopy. The study demonstrated that the prognostic value of the current adenocarcinoma classification is not limited to predominant growth patterns as a more favorable clinical outcome was associated with minor lepidic pattern. Significant associations were observed between adenocarcinoma subtypes and smoking history. Classic histological features of malignancy correlated with tumor architecture and survival, further confirming the prognostic value of semiquantitative growth pattern analysis and identifying potential prognostic biomarkers such as mitotic activity and tumor necrosis. Depolarized MUC1 expression correlated with histology and patient outcome, and moreover, with smoking both in vivo and in vitro, suggesting a pathogenetic relationship between cigarette smoke exposure and MUC1 in lung adenocarcinoma. / Tiivistelmä Keuhkon adenokarsinooma on maailmanlaajuisesti yleisin ja monella tapaa monimuotoisin keuhkosyöpätyyppi. Vuonna 2011 uusi kansainvälinen keuhkosyöpäluokitus nosti esille adenokarsinooman histologisten alatyyppien ennustemerkityksen, ja luokituksen on odotettu muodostavan pohjan uudelle kasvutapoihin perustuvalle gradeerausmenetelmälle. Kasvaimen muiden histopatologisten piirteiden ennusteellinen merkitys keuhkon adenokarsinoomassa on vähemmän tunnettu. MUC1-proteiinin yli-ilmentymistä on kuvattu monissa karsinoomatyypeissä, ja keuhkon adenokarsinoomassa MUC1:n poikkeava eli depolarisoitunut ilmentyminen on liitetty huonoon ennusteeseen. MUC4:n merkitys keuhkosyövässä on puolestaan ristiriitainen. Toisaalta MUC1- tai MUC4-ilmentymistä ei ole tutkittu tarkemmin keuhkon adenokarsinooman eri alatyypeissä. Väitöskirjatutkimuksessa pyrittiin selvittämään uuden adenokarsinoomaluokituksen yhteyttä ennusteeseen ja muihin kliinisiin muuttujiin aineistossa, joka käsitti 112 Oulun yliopistosairaalassa leikkaushoidettua keuhkon adenokarsinoomapotilasta. Kasvainten histopatologisten ominaispiirteiden kartoittamisen toivottiin tuovan ilmi myös uusia morfologisia ennustetekijöitä. Lisäksi tutkimus keskittyi MUC1-, MUC4- ja EGFR-proteiinien ilmentymiseen keuhkon adenokarsinoomassa, arvioiden niiden suhdetta kasvaimen histologiaan, potilaiden ennusteeseen ja tupakointihistoriaan. Tutkimusmenetelminä käytettiin valomikroskopiaa, immunohistokemiaa sekä kokeellista tupakka-altistusta soluviljelymallissa yhdistettynä kvantitatiiviseen reaaliaikaiseen käänteistranskriptiopolymeraasiketjureaktio-tekniikkaan (RT-qPCR) ja immunoelektronimikroskopiaan. Tutkimus osoitti, ettei nykyisen adenokarsinoomaluokituksen ennustearvo rajoitu hallitseviin kasvutapoihin, vaan myös väistyvä lepidinen kasvutapa vaikutti ennusteeseen suotuisasti. Keuhkon adenokarsinooman alatyyppien ja tupakoinnin välillä todettiin merkittävä yhteys. Pahanlaatuisten kasvainten klassiset histologiset piirteet liittyivät adenokarsinooman kasvutapoihin ja ennusteeseen korostaen semikvantitatiivisen kasvutapa-analyysin ennustemerkitystä ja tarjoten myös mahdollisia uusia ennustetekijöitä. Depolarisoituneen MUC1:n ilmentyminen liittyi histologiaan, ennusteeseen ja erityisesti tupakka-altistukseen sekä in vivo että in vitro. Tämä löydös viittaa mahdolliseen patogeneettiseen yhteyteen tupakoinnin ja MUC1:n välillä.

Abstract Reactive nitrogen species (RNS) and reactive oxygen species (ROS) have been linked with the pathogenesis of lung malignancies and chronic obstructive pulmonary disease (COPD). In vitro studies indicated that mesothelioma and lung carcinoma cell lines synthesize nitric oxide synthases (NOS) mRNA. The Comet-assay indicated that asbestos fibers caused DNA single -strand breaks in mesothelial cells, and this effect was enhanced by glutathione depletion. The use of FPG in the Comet assay indicated that the asbestos induced DNA strand breaks were oxidant mediated. In vivo non-neoplastic pleura was mostly negative for inducible NOS (iNOS), while inflamed pleura was positive. The immunohistochemical expression of iNOS was detected in 74% and 96% of malignant mesotheliomas and metastatic pleural adenocarcinomas, respectively. Epithelial and mixed mesotheliomas expressed more often intense iNOS immunoreactivity compared to the sarcomatoid subtype. Normal mesothelial cells showed occasional positivity for endothelial NOS (eNOS), but reactive mesothelial cells were strongly stained. eNOS was found in 89% of mesotheliomas. Vascular endothelial growth factor (VEGF) was identified in 47%, a VEGF receptor FLK1 in 69% and the VEGF receptor, FLT1, in 71% of mesotheliomas. FLK1 or FLT1 immunoreactivities were more often seen in epithelioid and biphasic mesotheliomas than in sarcomatoid mesotheliomas. In lung samples of non-smokers, smokers and COPD patients, the levels of nitrotyrosine were higher in alveolar macrophages of smokers and COPD patients than in the non-smokers and in the alveolar epithelium of smokers and COPD patients than in the non-smokers. The iNOS expression was weak in the bronchial and alveolar epithelium in all groups but eNOS was most prominently expressed in alveolar macrophages while neuronal NOS (nNOS) was negative in all of the major cell types of the lung. Bronchial metaplasia-dysplasia-sequence was clearly positive for iNOS, nNOS and nitrotyrosine. Thus, smoking can cause protein nitration also in normal lung. Prominent iNOS and nNOS immunoreactivity in metaplasia-dysplasia-lesions suggests a divergent role of NOSs in carcinogenesis and destruction of alveolar epithelium in emphysematous lung. In lung cancer samples, iNOS was detected in 40% cases, while 89% and 81% cases were positive for eNOS and nNOS, respectively. Intense eNOS staining was seen more often in adenocarcinomas than in squamous cells carcinomas, and iNOS immunoreactivity was seen more often in grade I-II tumors than in grade III tumors. The patients with tumors showing high expression of iNOS, eNOS and nNOS, exhibited better survival, but this was not an independent prognostic factor.

Master of Science / Department of Electrical and Computer Engineering / Masaaki Tamura / Steven Warren / Lung cancer mortality is the highest among all cancer–associated deaths. Despite early detection and treatment, prognosis of this disease remains poor. Therefore, development of new therapeutic agents and effective treatment procedures are urgently needed. Endogenous Angiotensin II (Ang II) type 2 receptor (AT[subscript]2), one of two isoforms of Ang II, has been shown to mediate apoptosis. Nanoparticle delivery systems make possible targeted drug delivery and controlled release of therapeutic molecules and genes. Thus, the aim of this study was to determine the anti-cancer effect of the over-expressed AT[subscript]2 gene on lung adenocarcinoma cells in vitro using adenoviral vector (Ad-) and nanoparticle (NP-) based gene delivery systems. This study showed that over-expression of Ad-AT[subscript]2 induced cancer cell-specific apoptosis in several human lung adenocarcinoma cell lines with minimal effect on normal lung epithelial cells. Ad-AT[subscript]2 significantly attenuated multiple human lung cancers' cell growth (A549 and H358) in vitro compared to the control viral vector, Ad-[Beta]-galactosidase (Ad-LacZ) when examined by direct cell count. The growth attenuation effect was detected as early as 24 hours after Ad-AT[subscript]2 transfection and lasted 12 days. Western Blot analysis revealed the activation of the caspase pathway. Examination for Annexin V by flow cytometry also confirmed activation of the apoptotic pathway via AT[subscript]2 over-expression. Similarly, AT[subscript]2 cDNA encapsulated poly(DL-lactide-co-glycolide) (PLGA) biodegradable nanoparticles (NPs) were shown to be effectively taken up into lung cancer cells. Surface conjugation of the angiotensin II peptide significantly stimulated uptake of the particles. This PLGA vector-dependent AT[subscript]2 transfection was effective in sustained gene expression and resultant cell death. These results indicate that the AT[subscript]2 over-expression effectively attenuated growth of lung adenocarcinoma cells through activation of intrinsic apoptosis. Since PLGA safety has been proven, whereas adenoviral vectors have several drawbacks in safety, the Ang II conjugated PLGA nanoparticles may be a better therapeutic gene delivery system. Therefore, it is concluded that the discovery of AT[subscript]2 DNA encapsulated PLGA conjugated with the Ang II peptide is a potentially useful tool for lung cancer gene therapy.

This diploma thesis deals with various ways of treatment of selected oncological diseases and the effectiveness of treatment methods and evaluation of the influence of various factors influencing the survival of patients. The activity of individual healing processes is evaluated by survival analysis. The subjects of the study are patients with breast, lung and prostate cancer. The survival analysis considers the sex of the patient, the age and stage of his illness, and other factors to avoid distorted results. The aim of the work is to find out the effects of selected therapeutic procedures on patients' health and to identify factors that have a significant impact on the survival of patients. The data for the diploma thesis was provided by the Institute of Health Information and Statistics of the Czech Republic, the Statistical Office, the National Cancer Register (NOR), the US SEER database and the German Breast Cancer Study.

A common co-pathology of large lung tumors located near the central airways is collapse of portions of lung due to blockage of airflow by the tumor. Not only does the lung volume decrease as collapse occurs, but fluid from capillaries also fills the space no longer occupied by air, greatly altering tissue appearance. During radiotherapy, typically administered to the patient over multiple weeks, the tumor can dramatically shrink in response to the treatment, restoring airflow to the lung sections which were collapsed when therapy began. While return of normal lung function is a positive development, the change in anatomy presents problems for future radiation sessions since the treatment was planned on lung geometry which is no longer accurate. The treatment must be adapted to the new lung state so that the radiation continues to accurately target the tumor while safely avoiding healthy tissue. However, to account for the dose delivered previously, correspondences of anatomy between the former image when the lung was collapsed and the re-expanded lung in a current image must be obtained. This process, known as deformable image registration, is performed by registration software. Most registration algorithms assume that identical anatomy is contained in the images and that intensities of corresponding image elements are similar; both assumptions are untrue when collapsed lung re-expands. This work was to develop an algorithm which accurately registers images in the presence of lung expansion. The lung registration method matched CT images of patients aided by vessel enhancement and information of individual lobe boundaries. The algorithm was tested on eighteen patients with lung collapse using physician-specified correspondences to measure registration error. The image registration algorithm developed in this work which was designed for challenging lung patients resulted in accuracy comparable to that of other methods when large lung changes are absent.

Lung cancer is the leading cause of cancer-related death in both men and women, nationally and internationally and kills more people each year than breast, prostate, and colon cancers combined. Non-small cell lung carcinoma (NSCLC) is the most common histological subtype of lung cancer, and accounts for 85% of all cases. Cigarette smoking is the single greatest risk factor for lung cancer, and is correlated with 80-90% of all lung cancer deaths. Nicotine, the addictive component of tobacco smoke, is not a carcinogen and cannot initiate tumors itself; however, it is known to act as a tumor promoter, by enhancing the proliferation, migration, and invasion of cells in vitro, thus accelerating tumor growth and metastasis in vivo. Nicotine exerts is tumor promoting effects primarily by binding to, and activation of, nicotinic acetylcholine receptors (nAChRs), specifically the α7 subunit of nAChRs. While α7 nAChR is expressed in a wide array of cells, how its expression is regulated is not fully understood. Here we sought to elucidate the transcriptional regulation of α7 nAChR in NSCLC cells. We report that α7 nAChR expression is induced by nicotine in an autoregulatory feedforward loop, and that the α7 gene promoter is differentially regulated by E2F1 and STAT1 transcription factors at an overlapping binding site suggesting a competitive interplay. Depletion of E2F1 resulted in a reduced ability of nicotine to induce α7 nAChR, while depletion of STAT1 resulted in enhanced induction, suggesting that nicotine might use these two transcription factors to modulate the expression of α7 nAChR in a very precise fashion. More recently, nicotine has been implicated in promoting self-renewal of stem-like side-population cells from lung cancers. Cancer stem-like cells have been implicated in tumor initiation as well as the maintenance, drug resistance, dormancy, recurrence, and metastasis of various tumor types. We had previously shown that the embryonic stem cell transcription factor, Sox2, is indispensable for self-renewal of stem-like cells from lung adenocarcinoma cell lines; hence we sought to determine whether nicotine enhances stemness of lung cancer stem-like cells through Sox2. We find that nicotine can induce the expression of Sox2 at the transcriptional level and this occurs through a nAChR-Src-Yap1-E2F1 signaling axis. Over recent years, electronic cigarettes (e-cigarettes) have emerged as healthy alternatives to traditional cigarette smoking as they do not contain tobacco; however, they do still contain nicotine. Our studies show that e-cigarette components can enhance tumor promoting properties of NSCLC cells similar to that observed with nicotine alone, and find that they can induce expression of Sox2 and mesenchymal markers as well as enhance migration and stemness of NSCLC cells. Taken together, these studies reveal novel molecular mechanisms by which exposure to nicotine, via cigarette smoke or e-cigarettes, could alter the oncogenic potential of NSCLC cells.

Cancer is a serious threat to human health and is now one of major causes of death worldwide. However, the complexity of the cancer makes the development of new and specific diagnostic tools particularly challenging. A number of different strategies have been developed for biomarker discovery in cancer using microarray data. The problem that typically needs to be addressed is the scale of the data sets; we simply do not have (or are likely to obtain) sufficient data for classical machine learning approaches for biomarker discovery to be properly validated. Obtaining a biomarker that is specific to a particular cancer is also very challenging. The initial promise that was held out for gene microarray work for the development of cancer biomarkers has not yet yielded the hoped for breakthroughs. This work discusses the construction of a strategy for a systematic approach to biomarker discovery validation using lung cancer gene expression microarray data based around non-small cell cancer and in patients which either stayed disease free after surgery (a five year window) or in which the disease progressed and re-occurred. As a means of assisting the validation purposes we have therefore looked at new methodologies for using existing biological knowledge to support machine learning biomarker discovery techniques. We employ text mining strategy using previously published literature for correlating biological concepts to a given phenotype. Pathway driven approaches through the use of Web Services and workflows, enabled the large-scale dataset to be analysed systematically. The results showed that it was possible, at least using this specific data set, to clearly differentiate between progressive disease and disease free patients using a set of biomarkers implicated in neuroendocrine signaling. A validation of the biomarkers identified was attempted in three separately published data sets. This analysis showed that although there was support for some of our findings in one of these data sets, this appeared to be a function of the close similarity in experimental design followed rather than through specific of the analysis method developed.

Small cell lung cancer (SCLC) is an extremely aggressive disease characterized by early metastasis and acquired resistance to therapy. SCLC is distinguished by its neuroendocrine (NE) component; the role of which is not fully understood in metastasis and response to therapy. Patients respond exceptionally well to first round chemotherapy; however, relapse with therapy-resistant tumours is virtually inevitable. Hypoxic regions within tumours can contribute towards metastasis and therapy resistance, highlighting hypoxia-targeted therapy as a novel approach for improving treatment for SCLC patients. Tumours are highly phenotypically heterogeneous, raising debate over the roles played by each cell type. Analysis of NE and epithelial markers in SCLC cell lines highlighted this inter-tumour heterogeneity. Further heterogeneity is displayed in SCLC xenograft tumours that show areas of dual epithelial and NE marker expression as well as regions negative for both markers. Irradiating xenograft tumours enhanced heterogeneity of the NE marker, pro-opiomelanocortin (POMC), which is ectopically secreted by a subset of SCLC tumours. Examining changes in marker expression post-therapy could provide vital information regarding transitions that can serve to guide therapy. SCLC is a highly metastatic disease. The role of the NE phenotype in human SCLC is not fully understood, but is considered essential for metastasis in murine models. Sub-cutaneous, intravenous and intra-splenic injection were carried out and resulted in no metastasis, spontaneous tumour generation and peripheral liver tumour growth, respectively. POMC expression was present and extremely heterogeneous within the liver, suggesting that NE properties are maintained in metastases; however, further work is necessary to develop a more consistent metastatic model that can be used to assess responses to therapy in a more clinically relevant setting. SCLC tumours proliferate rapidly and outgrow their nutrient and oxygen supplies, resulting in hypoxic conditions. Here, carbonic anhydrase IX (CA IX) becomes up-regulated in order to maintain pH levels suitable for survival. The specific CA IX inhibitor, S4, induces hypoxia-specific cell death in vitro and impairs tumour growth in vivo. This response is further accentuated by combining S4 with single or repeated cisplatin doses. Combination treatment reduced gene expression of S-phase kinase-associated protein (Skp2), associated with cisplatin resistance. CA IX inhibition combined with cisplatin chemotherapy therefore presents a novel treatment for SCLC tumours that could reduce therapy resistance. In summary, heterogeneity is extremely important when choosing treatment options for SCLC and must be considered when basing treatment on single biopsies. NE and epithelial markers are present within sub-cutaneous and liver tumours; however, a reliable multi-organ metastatic model is necessary to fully appreciate the role of these markers in the spread of SCLC. Hypoxic regions within sub-cutaneous xenograft tumours upregulate CA IX. Inhibition of this enzyme resulted in impaired tumour growth, particularly when used together with cisplatin. Combining CA IX inhibition with cisplatin presents a much-needed novel therapy for SCLC.