Introdução: o câncer de mama que apresenta amplificação/superexpressão do HER2 apresenta-se com pior prognóstico e seu amplicon se localiza no locus 17q12-q21. O gene WIPF2, relacionado a motilidade celular, localiza-se no mesmo locus e também se encontra presente no amplicon do HER2. Objetivo: avaliar a superexpressão simultânea do HER2 e WIPF2 através de marcação imunohistoquímica. Métodos: foram selecionados 94 casos de carcinoma ductal invasor de mama, sendo possível a obtenção de material em 87. Foi realizada técnica de microarranjo tecidual e as lâminas foram analisadas. Informações clínicas foram obtidas de prontuário médico. Resultados: após microscopia óptica das lâminas de TMA obtidas e comparação com lâminas convencionais arquivadas, validou-se o método, com bom índice de correlação kappa (0,83 para receptores de estrógeno e 0,84 para receptores de progesterona). Houve associação entre a superexpressão do WIPF2 com casos Ki-67 positivos e em pacientes nuligestas. O grupo HER2 apresentou maior porcentagem de casos WIPF2 positivos em relação ao grupo triplo-negativo à microscopia óptica (66,7% vs. 22,7%, respectivamente; p=0,03). Na análise digital, o perfil molecular HER2 apresentou maior expressão do WIPF2 em relação ao perfil luminal A e ao triplo negativo (23,9 ± 9,0% vs. 14,6 ± 9,0% e 14,2 ± 9,0%, respectivamente; p=0,01). Conclusão: O WIPF2 é mais expresso por tumores com o perfil HER2, assim como se associa a tumores com maior proliferação e tumores em pacientes nulíparas. / Introduction: the breast cancer with amplification/overexpression of HER2 shows worse prognosis and its amplicon is located in the 17q12-21 locus. The gene WIPF2, related to cellular mobility, is located in the same locus and is also present in the HER2 amplicon. Objective: to evaluate HER2 and WIPF2 simultaneous overexpression using immunohistochemistry technique. Methods: we selected 94 invasive ductal carcinoma samples and it was possible to evaluate 87. Tissue microarray has been done and slides analyzed. Clinical informations were obtained from clinical files. Results: after optical microscopy of TMA slides and comparison with conventional slides, the method was validated with a high correlation kappa index (0.83 for estrogen receptors and 0.84 for progesterone receprtors). The WIPF2 overexpression was associated with positive Ki-67 tumors and nuliparous women. The HER2 group presented greater percentage of WIPF2 positive cases when compared with triple-negative group using optical microscopy (66.7% vs. 22.7%, respectively, p=0.03). With the digital analysis, the HER2 group presented greater expression of WIPF2 when compared with luminal A and triple-negative groups (23.9 ± 9.0% vs. 14.6 ± 9.0% e 14.2 ± 9.0%, respectively; p=0.01). Conclusion: WIPF2 is more expressed in the HER2 group tumors. It is also related to high proliferation index and nuliparous women´s tumors.

Dans les cancers du poumon, une des altérations les plus souvent observées est la perte ou l’atténuation de l’expression du gène FHIT (Fragile Histidine Triad). Nous avons précédemment montré que FHIT est un suppresseur d’invasion tumorale. En effet, FHIT contrôle l’invasion des cellules tumorales bronchiques en régulant négativement l'expression de gènes associés à la transition épithélio-mésenchymateuse (TEM), en particulier la vimentine et la MMP-9 via l’inhibition d’une voie orchestrée par l’EGFR. Un intérêt particulier a donc été porté aux relations entre FHIT et un autre membre de la famille de l’EGFR : HER2. Nous avons non seulement mis en évidence, in vivo et in vitro, une corrélation inverse entre les taux de FHIT et l’activité du récepteur HER2 dans les CBNPC mais également montré que FHIT est capable de réguler l’activité du récepteur HER2 dans les cellules tumorales pulmonaires et ce grâce à sa dimérisation avec HER3. De plus, l’utilisation de deux inhibiteurs spécifiques d’HER2, le Trastuzumab et l’Irbinitinib, nous a permis de mettre en évidence, que l’activation du récepteur HER2 lors de l’inhibition de FHIT, participe à l’acquisition par les cellules tumorales bronchiques de caractéristiques invasives via la régulation de certaines cibles de la TEM, telles la vimentine, la MMP-14 ou encore le facteur de transcription TWIST-1. Ces résultats montrent que FHIT régule l’activité d’HER2 dans les cellules tumorales pulmonaires et que les inhibiteurs d’HER2 sont capables de limiter l’invasion induite par l’inhibition de FHIT. Cette étude laisse envisager de nouvelles perspectives thérapeutiques pour le cancer du poumon. / The lack or decrease of FHIT (fragile histidine triad) expression is a common event in lung cancer. We recently showed that FHIT acts as a suppressor of tumor invasion. Indeed, FHIT controls the invasive phenotype of lung tumor cells by regulating the expression of genes associated with epithelial-mesenchymal transition (EMT) such as vimentin or MMP-9 through an EGFR signaling pathway. Accordingly, we focused on the relationships between FHIT and another member of this tyrosine kinase receptor family: HER2. First, we observed in vivo and in vitro a negative correlation between FHIT expression and the activated form of HER2 in lung tumor cells. Moreover, FHIT controls HER2 activation through its dimerization with HER3. The use of HER2 specific inhibitors, Trastuzumab and Irbinitinib, allowed to demonstrate that the in vitro invasion induced by FHIT inhibition is HER2-dependent. Furthermore, FHIT controls the HER2-dependent invasion by regulating genes associated with EMT such as vimentin, MMP-14 or TWIST-1. In conclusion, we showed that FHIT regulates HER2 activity in lung tumor cells and that HER2 inhibitors reduce invasion induced by FHIT inhibition. This study would allow for the identification of new therapeutic leads for lung cancer.

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.

Glioblastoma Multiforme (GBM) is the most common lethal brain tumour associated with dismal survival rate. GBM is considered to be an incurable malignancy as these tumours evade all intricate attempts of therapy and no contemporary chemotherapeutic regimen is effective. Although the existence of cancer stem cells (CSCs) is still debatable, it is widely accepted that GBM has a small population of cells expressing CSC markers (~1%) that are highly resistant to chemo-radiation therapy. Recent evidence indicates that hypoxia induces cancer stem cell (CSC) phenotypes via epithelial-to-mesenchymal transition (EMT) that promote therapeutic resistance in solid tumours. Given that GBMs are extensively hypooxygenated heterogenous tumours, understanding the molecular relationship between hypoxia, biology of CSCs, EMT and chemoresistance would be invaluable for development of drugs that can target CSCs. Evidence suggests that hypoxia inducible factors (HIFs), NF-B and aldehyde dehydrogenase (ALDH) together orchestrate the stemness and chemoresistance in hypoxia induced CSCs. But the insights on the mechanisms still remain obscure. In this study we used an in vitro GBM CSC and hypoxia model along with NF-B-p65 and HIF transfected GBM cell lines to investigate the relationship between HIFs, NF-B activation and ALDH activity and their role in chemoresistance. The findings of this study demonstrated that GBM cells grown as spheres consist of a vast proportion of hypoxic cells with elevated CSC and EMT markers suggesting hypoxia induced EMT. GBM-CSCs are chemoresistant and displayed increased levels of HIFs, NF-B and ALDH activity. It was also observed that stable transfection of GBM cells with NF-B-p65 or HIFs induced CSC and EMT markers indicating their essential role in maintaining CSC phenotypes. The study also highlighted the importance of NF-B and ALDH in driving chemoresistance and the potential role of NF-B as the master regulator of hypoxia induced stemness in GBM cells. In this study, we used Disulfiram (DS), an anti-alcoholism drug, in combination with copper (Cu) to target the hypoxia-NF-B axis and inhibit ALDH activity to reverse chemoresistance in GBM CSCs. We showed that DS/Cu is cytotoxic to GBM cells and completely eradicated the resistant CSC population at low nanomolar levels in vitro. We also demonstrated that DS/Cu effectively inhibited GBM in vivo using newly formulated PLGA-DS nanoparticles. DS is an FDA approved drug with low/no toxicity to normal tissues and can freely pass through the blood brain barrier (BBB). Further study may lead to quick translation of DS into clinical trials.

La transcription pervasive génère des milliers de lncRNA pouvant réguler l'expression des gènes et jouer un rôle important dans le développement et dans différentes maladies. Parmi eux, les asRNA restent mal caractérisés malgré l'importance de leurs rôles régulateurs. Durant ma thèse, nous avons utilisé des données de séquençage à haut débit directionnelles de diverses lignées cellulaires et tissus humains pour définir de nouveaux asRNA. L'analyse bioinformatique nous a permis de classer les nouveaux asRNA selon 3 critères: niveau d'expression, la présence d'EST épissée et de jonction prédite. Nous avons défini 4 catégories: la classe1 répond aux 3 critères (la plus robuste), la classe2a satisfait les critères d'expression et de jonction, la classe2b satisfait les critères d'expression et d'EST et la classe3 satisfait le critère d'expression. Le niveau d'expression de ces asRNA est très faible et comparable à celui des lncRNA. Ils sont exprimés de manière spécifique, enrichis dans le noyau, coiffés et polyadénylés. Deuxièmement, j'ai posé la question de savoir s'ils sont impliqués dans le cancer et dans l'EMT. Nous avons analysé l'expression des gènes dans les tumeurs de la prostate, du sein et d'angiosarcomes. Nous avons comparé le tissu tumoral au sain et avons obtenu une liste de gènes dérégulés incluant des asRNA. Cette analyse nous a permis de découvrir 3 nouveaux asRNA spécifiques aux tumeurs de la prostate pouvant être utilisés comme outil de diagnostic. L'analyse de l'EMT a permis de définir des ARN spécifiques au système étudié. Cette analyse a révélé l'importance de la transcription antisens chez l'Homme et leur rôle potentiel dans la régulation des gènes. / Pervasive transcription generates thousands of lncRNAs that can regulate gene expression and play an important role in development and in different diseases. Among them, asRNAs remain poorly characterized despite the importance of their regulatory roles in cells. During my PhD, we used high-throughput sequencing directional data from multiple human cell lines and tissues to define new asRNAs. Bioinformatics analysis allowed us to classify new asRNAs according to 3 criteria: level of expression, presence of spliced EST and predicted junction. We defined 4 categories: class1 responds to the 3 criteria (strongest class), class2a satisfies the criteria of expression and splice junction, class2b satisfies the criteria of expression and spliced EST and class3 responds to criterion of expression. The level of expression of these asRNAs is lower than mRNAs but is comparable to lncRNAs. They are specifically expressed in a tissue or cell line, enriched in the nucleus, capped and polyadenylated. Secondly, I posed the question of whether this asRNAs are expressed in cancer and in the EMT. We analyzed gene expression in prostate tumors, breast and angiosarcoma. We compared tumor tissue and healthy one and we obtained a list of genes differentially expressed, among them asRNAs. We defined for each tissue a specific transcriptome signature. This analysis allowed us to discover 3 new asRNAs specific to prostate tumors that can be used as a diagnostic tool. Analysis of the EMT helped to define specific markers in the studied system including mRNAs, lncRNAs and asRNAs. This analysis revealed the importance of antisense transcription in humans and their potential role in gene regulation.

The UK Government has identified that nearly 15% of the UK’s electricity generation must come from offshore wind by 2020. The reliability of the offshore windfarms and their electrical transmission systems is critical for their feasibility. Offshore windfarms located more than 50-100km from shore, including most Round 3 offshore windfarms, are likely to employ Voltage Source Converter (VSC) High Voltage Direct Current (HVDC) transmission schemes. This thesis studies factors which affect the reliability of VSC-HVDC transmission schemes, in respect to availability, protection, and system modelling. The expected availability of VSC-HVDC systems is a key factor in determining if Round 3 offshore windfarms are technically and economically viable. Due to the lack of publications in this area, this thesis analyses the energy availability of a radial and a Multi-Terminal (MT) VSC-HVDC system, using component reliability indices derived from academic and industrial documentation, and examining the influence of each component on the system’s energy availability. An economic assessment of different VSC-HVDC schemes is undertaken, highlighting the overall potential cost savings of HVDC grids. The connection of offshore windfarms to a MT HVDC system offers other potential benefits, in comparison to an equivalent radial system, including a reduction in the volume of assets and enhanced operational flexibility. However, without suitable HVDC circuit breakers, a large MT HVDC system would be unviable. In this thesis, a review of potential HVDC circuit breaker topologies and HVDC protection strategies is conducted. A HVDC circuit breaker topology, which addresses some of the limitations of the existing designs, was developed in this thesis, for which a UK patent application was filed. Accurate simulation models are required to give a high degree of confidence in the expected system behaviour. Modular Multi-level Converters (MMCs) are the preferred HVDC converter topology, however modelling MMCs in Electromagnetic Transient (EMT) simulation programs has presented a number of challenges. This has resulted in the development of new modelling techniques, for which the published validating literature is limited. In this thesis these techniques are compared in terms of accuracy and simulation speed and a set of modelling recommendations are presented. Cable models are the other main DC component which, upon analysis, is found to have a significant impact on the overall model’s simulation results and simulation time. A set of modelling recommendations are also presented for the leading cable models. Using the modelling recommendations to select suitable MMC models, radial and MT EMT MMC-HVDC models for the connection of typical Round 3 windfarms are developed in this thesis. These models are used to analyse the steady-state and transient performance of the connections, including their compliance to the GB grid code for AC disturbances and reactive power requirements. Furthermore, the MT model is used to investigate the effect of MT control strategies on the internal MMC quantities.

Bakgrund: Sjukdomar i cirkulationsorganen är den vanligaste dödsorsaken i Sverige följt av tumörsjukdomar. År 2019 stod tumörsjukdomarna för hela 27% av dödsfallen. Epithelial-mesenchymal transition (EMT) är en process då epitelceller de-deffinerentierar till celler med en mesenkymal fenotyp. Vid denna process mister cellerna sin polaritet och cell-cell-kontakt. Cellerna får även en ändrad morfologi, från en rektangulär form till mer utsträckta och oregelbundna former. Processen möjliggör att cellerna får migrerande och invasiva egenskaper. EMT behövs för att cancercellerna ska kunna sprida sig från den primära tumören och bilda metastaser/dottertumörer på andra ställen i kroppen. Det är välkänt att Transforming Growth Factor beta (TGF-b) kan framkalla EMT. TGF-b kan stimulera tumörutvecklingen på många olika sätt, där ett av dessa sätt är genom att stimulera EMT. Andra studier har visat att kalcitrol, som är den aktiva formen av vitamin D, kan hämma TGF-b förmåga att driva tumörutveckling.   Syfte: Syftet är att undersöka om vitamin D kan motverka TGF-b orsakat EMT.    Metod: Data hämtades från databasen PubMed. För att finna relevanta studier användes sökorden Vitamin D, TGF-beta, EMT, Cancer tillsammans med sökkriterier. Inklusionskriterna var vetenskapliga orginalartiklar, artiklar i full text samt skrivna på språket engelska. Exklusionskriterierna vara artiklar äldre än 10 år.   Resultat: När humana cancerceller behandlades med vitamin D resulterade det i minskad EMT. Förändrat EMT kunde påvisas genom minskad migration och invasion samt ökning av epitela egenskaper. Vitamin D minskade cellmigrationen på ett tids- och dosberoende sätt. In vitro-testerna indikerade på att i cellkultur av cancerceller verkar vitamin D genom att motverka EMT, både i närvaro och i frånvaro av TGF-b. När EMT-programmet däremot redan initerats m.h.a. TGF-b kan inte längre vitamin D motverka att cellerna genomgår EMT.   Slutsats: Intag av vitamin D före och under TGF-b-signalering har ett positivt resultat på EMT-programmet.

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Trotz aller Fortschritte in Diagnostik und Behandlung bleibt die Therapie von BrustkrebspatientInnen – gerade bei tripelnegativen Mammakarzinomen, die nicht auf eine hormonelle Therapie ansprechen – eine Herausforderung. Eine wachsende Zahl von Belegen spricht dafür, dass Cadherine nicht nur in physiologischen Prozessen wie der Embryogenese, sondern auch in pathologischen Prozessen wie der Tumorprogression eine Rolle spielen, was diese Moleküle zu potenziellen Zielen einer targetspezifischen Tumortherapie macht. Im Mausmodell konnte bislang gezeigt werden, dass eine Herabregulierung von N-Cadherin das Mammakarzinomwachstum in vivo hemmt. N-Cadherin wird auch in humanem Brustkrebsgewebe aberrant exprimiert. In nur schwach invasiven Brustkrebszelllinien führte eine Überexpression von N-Cadherin in vitro zu einer Steigerung des migratorischen und invasiven Verhaltens der Zellen. In dieser Arbeit wurde an der humanen Mammakarzinomzelllinie SUM149PT der Einfluss einer Defizienz von N-Cadherin auf die Expression von E- und VE-Cadherin, sowie auf das Proliferations-, Migrations- und Invasionsverhalten der Zellen in vitro untersucht. Eine Charakterisierung der humanen Brustkrebszelllinien SUM149PT, der aus ihr hervorgegangenen mit shRNA transduzierten N-Cadherin-Knock-down-Klone und der Scramble-Kontrollen fand mittels Western Blot, RT-PCR, q-PCR und Immunfluoreszenzanalysen statt. Das Proliferationsverhalten der Zelllinien wurde mit Hilfe von Verdopplungszeitbestimmungen und Sphäroidversuchen analysiert. Um den Einfluss einer N-Cadherin-Defizienz auf die Migration zu untersuchen, wurden auf dem Prinzip der Boyden-Chamber basierende Versuche, sowie in vitro Wundheilungsversuche durchgeführt. Auch die Invasionsversuche basierten auf dem Prinzip der Boyden-Chamber. Eine zusätzliche Beschichtung mit Matrigel simulierte hier die extrazelluläre Matrix. Bei den Untersuchungen mittels Western Blot, RT-PCR und q-PCR wurde deutlich, dass die N-Cadherin defizienten Klone keine Veränderung in der E-Cadherin-Expression, jedoch interessanterweise eine starke Herabregulierung von VE-Cadherin aufweisen. Bei den Immunfluoreszenzanalysen wiesen SUM149PT und Scramble-Kontrollen eine Expression von N-Cadherin in der Zellmembran auf, während die N-Cadherin-defizienten Klone keine N-Cadherin-Expression zeigten. Wie schon in Western-Blot, RT-PCR und q-PCR zeigten die N-Cadherin defizienten Klone im Vergleich zu den SUM149PT und den Scramble-Kontrollen keine veränderte E-Cadherin-Expression. Bei Einsatz eines VE-Cadherin-Anitkörpers zeigten die N-Cadherin defizienten Klone keine Expression von VE-Cadherin, während SUM149PT und Scramble eine deutliche VE-Cadherin-Expression in der Zellmembran zeigten. Daraus lässt sich schließen, dass N-Cadherin möglicherweise die Expression von VE-Cadherin beeinflussen kann. In der Verdopplungszeitbestimmung ließen sich keine signifikanten Änderungen des Wachstumsverhaltens zwischen N-Cadherin defizienten Klonen und Kontrollzelllinien nachweisen. Um auch das dreidimensionale Wachstum der Zellen zu untersuchen, wurden Sphäroidversuche durchgeführt. Alle untersuchten Zelllinien bildeten stabile Sphäroide aus, die jedoch einen großen Saum an nicht integrierten Zellen aufwiesen. Es zeigte sich kein signifikanter Unterschied im Sphäroidwachstum zwischen N-Cadherin defizienten Klonen und Kontrollzelllinien. Die Herabregulierung von N-Cadherin scheint in humanen Mammakarzinomzelllinien in vitro keinen Einfluss auf das Wachstumsverhalten der Zellen zu haben. Im Boyden-Chamber-Assay zeigte sich, dass eine Herabregulierung von N-Cadherin bei der humanen Mammakarzinomzelllinie SUM149PT in vitro zu einer signifikanten Verringerung des Migrationspotenzials führt. Dieses Ergebnis wurde beim in vitro Wundheilungsversuch bestätigt. Hier zeigten die N-Cadherin defizienten Klone eine signifikant verringerte Flächenbedeckungsrate – also eine signifikant verringerte Geschwindigkeit mit der die aufeinander zuwanderndern Zellen eine freie Fläche bedecken – als die Kontrollzelllinien. Im Invasionsversuch zeigten die N-Cadherin defizienten Klone im Vergleich zu den Kontrollen eine hochsignifikant verringerte Invasivität. Eine Herabregulierung von N-Cadherin bei der humanen Mammakarzinomzelllinie SUM149PT führt also in vitro zu einer signifikanten Verringerung des Invasionspotenzials. Die Ergebnisse dieser Arbeit lassen den Schluss zu, dass N-Cadherin bei humanen Mammakarzinomzelllinien durch seinen positiven Einfluss auf Migration und Invasion der Zellen eine große Rolle beim Prozess des invasiven Wachstums und der Metastasierung spielt. Eine Anti-N-Cadherin-Therapie könnte auch bei BrustkrebspatientInnen, gerade mit tripelnegativen Mammakarzinomen, neue Möglichkeiten der Behandlung eröffnen.:Abkürzungsverzeichnis V 1 Einleitung 1 1.1 Tumorentstehung und -progression 1 1.2 Der Prozess der Metastasierung 4 1.2.1 Epithelial-mesenchymale Transition 4 1.3 Brustkrebs 7 1.3.1 Histologie und Klassifizierung 7 1.4 Cadherine 11 1.4.1 Klassische Cadherine 11 1.4.2 VE-Cadherin 13 1.4.3 N-Cadherin 14 2 Fragestellung 16 3 Material und Methoden 18 3.1 Material 18 3.1.1 Geräte 18 3.1.2 Verbrauchsmaterialien 20 3.1.3 Chemikalien, Reagenzien, Enzyme und Kits 22 3.1.4 Puffer, Ansätze und Gele 25 3.1.5 Antikörper 28 3.1.6 Primer 29 3.1.7 Zellkultur 30 3.1.8 Software 32 3.2 Methoden 33 3.2.1 Kultivierung der Zellen 33 3.2.2 Proteinisolierung und Western Blot 34 3.2.3 RNA-Isolierung und Polymerasekettenreaktion 36 3.2.4 Immunfluoreszenz 40 3.2.5 Untersuchung des Proliferationsverhaltens 41 3.2.6 Sphäroidversuch 43 3.2.7 Untersuchung des Migrationsverhaltens mittels Boyden-Chamber 44 3.2.8 Untersuchung des Migrationsverhaltens mittels in vitro Wundheilungsversuch 45 3.2.9 Untersuchung des Invasionsverhaltens 46 3.2.10 Statistische Analyse 49 4 Ergebnisse 50 4.1 Charakterisierung der N-Cadherin defizienten Klone 50 4.1.1 Untersuchung der E-, N- und VE-Cadherin-Expression auf mRNA-Ebene mittels RT-PCR und q-PCR 53 4.1.2 Untersuchung der E-, N- und VE-Cadherin-Expression auf Proteinebene mittels Western Blot 56 4.1.3 Untersuchung der E-, N- und VE-Cadherin-Expression und -Lokalisation mittels Immunfluoreszenzfärbung 57 4.2 Einfluss des N-Cadherin auf das Proliferationsverhalten 61 4.3 Einfluss des N-Cadherin auf die Sphäroidbildung 62 4.4 Einfluss des N-Cadherin auf das Migrationsverhalten 64 4.5 Einfluss des N-Cadherin auf das Invasionsverhalten 69 5 Diskussion 71 5.1 N-Cadherin-Defizienz hat keinen Einfluss auf die Expression und subzelluläre Lokalisation von E-Cadherin in der humanen Mammakarzinomzelllinie SUM149PT 72 5.2 N-Cadherin-Defizienz führt zu deutlicher Herabregulierung der VE-Cadherin-Expression in der humanen Mammakarzinomzelllinie SUM149PT 73 5.3 N-Cadherin-Defizienz hat keinen Einfluss auf die Proliferation der humanen Mammakarzinomzelllinie SUM149PT 74 5.4 N-Cadherin-Defizienz hat keinen Einfluss auf die Sphäroidbildung der humanen Mammakarzinomzelllinie SUM149PT 74 5.5 N-Cadherin-Defizienz führt zu signifikant verringerter Migration der humanen Mammakarzinomzelllinie SUM149PT 75 5.6 N-Cadherin-Defizienz führt zu signifikant verringerter Invasion der humanen Mammakarzinomzelllinie SUM149PT 76 6 Zusammenfassung 79 7 Abbildungsverzeichnis 83 8 Tabellenverzeichnis 84 9 Literaturverzeichnis 85 10 Danksagung 104 / Breast cancer is the most common cancer type for women and the most frequent type after lung cancer overall. In spite of all improvements in clinical diagnostics and treatment, the therapy of patients with breast cancer remains a difficult task. Therefore it is important to find new strategies of treatment, especially for patients with triple negative breast cancer that does not respond to hormone therapy. There is growing evidence that cadherins are not only important in physiological processes like embryogenesis but also in pathological processes like tumour progression. A so called cadherin switch has been implicated in carcinogenesis, in particular the loss of E-cadherin and the upregulation of N-cadherin. Thus, these molecules are an interesting subject of studies and a potential target for specific cancer therapy. It was shown previously that efficient silencing of N-cadherin in murine breast cancer cells inhibits tumour growth in vivo. N-cadherin is also aberrantly expressed in human breast cancer tissue. An overexpression of N-Cadherin in breast cancer cells enhances tumour cell motility, migration and invasion in vitro. In this thesis the impact of N-cadherin deficiency on the expression level of E- and VE-cadherin as well as its effect on proliferation, migration and invasion behaviour of breast cancer cells in vitro was investigated. The human breast cancer cell line SUM149PT, its N-cadherin deficient clones and scramble controls were characterised by Western Blot, RT-PCR, q-PCR and immunofluorescence staining. To analyse the impact of N-cadherin on the proliferation behaviour doubling time proliferation assays and spheroid assays were performed. A Boyden chamber assay and an in vitro wound healing assay were performed to investigate the effect of N-cadherin deficiency on cell migration. Furthermore, a Boyden chamber assay with a coating of Matrigel was used to study the invasion potential of the N-cadherin deficient cell clones and control cell lines. The results of Western Blot, RT-PCR and q-PCR show that silencing of N-cadherin expression in SUM149PT cells had no influence on E-cadherin expression, but significantly decreased VE-cadherin expression. With immunofluscence staining it was evidenced that SUM149PT and scramble controls express N-cadherin in the cell membrane, whereas N-cadherin deficient clones do not show any N-cadherin expression. There was no difference seen in E-cadherin expression between N-cadherin deficient clones and control cell lines. While SUM149PT and scramble controls expressed VE-cadherin in the cell membrane, N-cadherin deficient clones did not express VE-cadherin. These results suggest that N-cadherin may have an influence on the expression of VE-cadherin. No significant alteration in proliferation behaviour between N-cadherin deficient clones and controls could be shown with the doubling time proliferation assay. To investigate the impact of N-cadherin on the three-dimensional growth spheroid assays were performed. All cell lines formed stable speroids, however fringes of many not incorporated cells were found. There was no difference in spheroidgrowth between N-cadherin deficient clones and control cell lines. No influence of N-cadherin on cell proliferation in vitro could be seen. With the help of Boyden chamber assays it was shown that silencing of N-cadherin in the human breast cancer cell line SUM149PT results in a significant decrease of migration potential in vitro. This was confirmed with an in vitro wound healing assay. N-cadherin deficient clones showed a significantly reduced surface coverage rate than SUM149PT and scramble controls. A significant decrease of invasive cells in N-cadherin deficient clones in comparison to the controls was revealed in invasion assays. Thus silencing of N-Cadherin in the human breast cancer cell line SUM149PT leads to a significantly decreased invasion potential in vitro. The results presented this thesis provide evidence that N-cadherin is involved in invasive tumour progression and metastasis of the human breast cancer cell line SUM149PT by promoting cell migration and invasion. Anti-N-cadherin strategies could thus be a potential target specific therapy of cancer patients, particularly with triple negative breast cancer.:Abkürzungsverzeichnis V 1 Einleitung 1 1.1 Tumorentstehung und -progression 1 1.2 Der Prozess der Metastasierung 4 1.2.1 Epithelial-mesenchymale Transition 4 1.3 Brustkrebs 7 1.3.1 Histologie und Klassifizierung 7 1.4 Cadherine 11 1.4.1 Klassische Cadherine 11 1.4.2 VE-Cadherin 13 1.4.3 N-Cadherin 14 2 Fragestellung 16 3 Material und Methoden 18 3.1 Material 18 3.1.1 Geräte 18 3.1.2 Verbrauchsmaterialien 20 3.1.3 Chemikalien, Reagenzien, Enzyme und Kits 22 3.1.4 Puffer, Ansätze und Gele 25 3.1.5 Antikörper 28 3.1.6 Primer 29 3.1.7 Zellkultur 30 3.1.8 Software 32 3.2 Methoden 33 3.2.1 Kultivierung der Zellen 33 3.2.2 Proteinisolierung und Western Blot 34 3.2.3 RNA-Isolierung und Polymerasekettenreaktion 36 3.2.4 Immunfluoreszenz 40 3.2.5 Untersuchung des Proliferationsverhaltens 41 3.2.6 Sphäroidversuch 43 3.2.7 Untersuchung des Migrationsverhaltens mittels Boyden-Chamber 44 3.2.8 Untersuchung des Migrationsverhaltens mittels in vitro Wundheilungsversuch 45 3.2.9 Untersuchung des Invasionsverhaltens 46 3.2.10 Statistische Analyse 49 4 Ergebnisse 50 4.1 Charakterisierung der N-Cadherin defizienten Klone 50 4.1.1 Untersuchung der E-, N- und VE-Cadherin-Expression auf mRNA-Ebene mittels RT-PCR und q-PCR 53 4.1.2 Untersuchung der E-, N- und VE-Cadherin-Expression auf Proteinebene mittels Western Blot 56 4.1.3 Untersuchung der E-, N- und VE-Cadherin-Expression und -Lokalisation mittels Immunfluoreszenzfärbung 57 4.2 Einfluss des N-Cadherin auf das Proliferationsverhalten 61 4.3 Einfluss des N-Cadherin auf die Sphäroidbildung 62 4.4 Einfluss des N-Cadherin auf das Migrationsverhalten 64 4.5 Einfluss des N-Cadherin auf das Invasionsverhalten 69 5 Diskussion 71 5.1 N-Cadherin-Defizienz hat keinen Einfluss auf die Expression und subzelluläre Lokalisation von E-Cadherin in der humanen Mammakarzinomzelllinie SUM149PT 72 5.2 N-Cadherin-Defizienz führt zu deutlicher Herabregulierung der VE-Cadherin-Expression in der humanen Mammakarzinomzelllinie SUM149PT 73 5.3 N-Cadherin-Defizienz hat keinen Einfluss auf die Proliferation der humanen Mammakarzinomzelllinie SUM149PT 74 5.4 N-Cadherin-Defizienz hat keinen Einfluss auf die Sphäroidbildung der humanen Mammakarzinomzelllinie SUM149PT 74 5.5 N-Cadherin-Defizienz führt zu signifikant verringerter Migration der humanen Mammakarzinomzelllinie SUM149PT 75 5.6 N-Cadherin-Defizienz führt zu signifikant verringerter Invasion der humanen Mammakarzinomzelllinie SUM149PT 76 6 Zusammenfassung 79 7 Abbildungsverzeichnis 83 8 Tabellenverzeichnis 84 9 Literaturverzeichnis 85 10 Danksagung 104

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