O mecanismo de interferência por RNA (RNAi) é um evento de silenciamento gênico através da degradação do RNA mensageiro. Desta forma, a administração de siRNA (molécula efetora do RNAi) é uma terapia promissora para o tratamento de diversas doenças como o câncer. Porém, para a sua efetiva aplicação terapêutica é necessário o desenvolvimento de sistemas de liberação capazes de liberar o siRNA nas células alvo, promover a sua internalização celular e evitar a sua degradação. Dentre os sistemas de liberação, os de liberação localizada, como os sistemas de geleificação in situ, apresentam vantagens sobre administração sistêmica. Formulações fluidas precursoras (FFP) que formam sistemas líquido cristalinos viscosos in situ podem ser obtidas a partir de lipídeos anfifílicos que absorvem água do meio e se rearranjam. Neste contexto, a presente pesquisa teve como objetivo avaliar o gel formado in situ a partir da FFP (G-FFP), composta por monoglicerídeos (MO), polietilenoimina (PEI), propilenoglicol (PG) e tampão tris, como sistema de liberação localizada de siRNA na terapia do câncer de pele. Os resultados mostraram que o G-FFP é uma mistura de fase cúbica e fase hexagonal. O G-FFP liberou o siRNA de maneira sustentada e complexado ao PEI. A FFP pode ser esterilizada por filtração em membrana e foi capaz de complexar altas concentrações de siRNA (15 mM) e de proteger o siRNA da degradação. A citotoxicidade foi dependente da concentração de FFP e esta quando complexada com siRNA teve a toxicidade diminuída. O siRNA liberado do G-FFP foi internalizado pelas células A431, FaDu, HeLa, A549, WM35/DLC2-GFP e MCF-7/DLC2-GFP. Além disto, siRNAs específicos liberados pelo G-FFP foram capazes de reduzir a expressão da proteína Firefly luciferase em células HeLa e FaDu, pórem não foram capazes de reduzir a expressão do receptor do fator de crescimento epidérmico (EGFR) nas células A431, HeLa, A549 e FaDu. A redução da expressão de EGFR em células A549 foi observado quando a terapia com siRNA foi combinada com internalização fotoquímica. Destes resultados, pode-se inferir que a transfecção celular do siRNA e o silenciamento gênico promovido por ele foi dependente tanto do tipo de linhagem celular como do alvo desejado. Os estudos in vivo mostraram que ocorre a formação de gel intratumoral e após 3 dias da administração intratumoral da FFP contendo siRNA específico para EGFR houve redução de 30% no tamanho dos tumores comparados aos tumores tratados com FFP sem siRNA. Diante destes resultados, pode-se concluir que o sistema desenvolvido tem potencial como sistema de liberação localizada de siRNA quando aplicado subcutaneamente ou intratumoral, uma vez que complexa o siRNA, promove a sua internalização celular e o siRNA liberado no citoplasma das células pode reduzir a expressão de proteínas de interesse. / RNA interference (RNAi) is a mechanism in which small interfering RNA molecules (siRNA) inhibit gene expression, by causing the messenger RNA degradation. Thus, siRNA is a promising therapy for the treatment of several diseases such as cancer. However, the development of delivery systems able to protect the siRNA from degradation and promote its cell uptake is essential for therapeutic use of siRNA. Among the delivery systems, the localized delivery system such as in situ gelling delivery system, have advantages over systemic administration. Precursor fluid formulations (FFP), which forms in situ viscous liquid crystalline systems, can be obtained from amphiphilic lipids that absorb water from the environment and self-assembling. In this context, the present study aimed to evaluate the in situ gel formed from the FFP (G-FFP), composed of monoglycerides (MO), polyethyleneimine (PEI), propylene glycol (PG) and Tris buffer, as localized delivery system for siRNA in skin cancer therapy. The results showed that the G-FFP is a mixture of cubic and hexagonal phase. The G-FFP sustained release of siRNA and the siRNA is released complexed with PEI. The FFP can be sterilized by membrane filtration at 0.22 ?m. FFP was able to complex high siRNA concentration (15 mM) and protect the siRNA from degradation. The cytotoxicity was dependent on the FFP concentration, when FFP was complexed with siRNA it was observed a decreased in toxicity. The siRNA released from G-FFP was uptake by A431, FaDu, HeLa, A549, WM35/DLC2-GFP and MCF-7/DLC2-GFP cells. In addition, specific siRNAs released from G-FFP were able to reduce the expression of Firefly luciferase in HeLa and FaDu cells, but they were unable to reduce the expression of the epidermal growth factor receptor (EGFR) in A431, HeLa, A549 and FaDu cells. The reduction of expression of EGFR in A549 cells was observed when siRNA therapy was combined with photochemical internalization. From these results, can be inferred that the efficiency of siRNA transfection and knockdown was dependent on both the type of cell line and the desired target protein. In vivo studies showed that the gel was formed in situ after intratumoral injection. 3 days after intratumoral administration of FFP containing EGFR specific siRNA, 30% of reduction in the tumors size compared to tumors treated with FFP without siRNA was observed. Based on these results, could be concluded that the developed system is a potential siRNA delivery system when administered subcutaneously and intratumoral, because it was able to complex siRNA, promoted its cell uptake and the siRNA released into the cytoplasm of the cell may reduce the expression of target proteins

A degradação lisossomal de proteínas de membrana endocitadas ocorre por meio do direcionamento destas proteínas para vesículas intralumenais (ILVs), formadas no lúmen dos corpos multivesiculares (MVBs), e subsequente fusão dos MVBs com lisossomos. Apesar de sua importância na degradação de proteínas transmembrana, os MVBs possuem outra importante função, a de produzir e liberar vesículas extracelulares (EVs). Neste processo os MVBs não se fundem com lisossomos, mas sim com a membrana plasmática o que resulta na liberação das vesículas residentes no interior dos MVBs para o meio extracelular. Diversas proteínas participam do direcionamento de cargas para os MVBs. Os estudos que delinearam a via de tráfego mediada por AP1 concentraram-se nos complexos contendo a subunidade ?1 que medeia o transporte de proteínas entre a rede trans-Golgi (TGN) e endossomos. Contudo, o genoma humano codifica uma segunda isoforma desta subunidade, denominada ?2, e evidências presentes na literatura e também observadas por nosso grupo indicam que AP1?2 pode regular uma via de tráfego distinta da via classicamente atribuída a AP1. Utilizando ensaios de uptake de EGF em condições onde foi realizado o KD de ?1 ou ?2, foi observado que o silenciamento de ?2 prejudica a degradação de EGF internalizado por seu receptor. Efeito também observado para o próprio receptor de EGF (EGFR) em ensaios de biotinilação da superfície celular. Demonstrando que a degradação lisossomal do complexo EGF-EGFR pela via canônica dos MVBs requer o complexo AP1?2, mas não AP1?1. Em conjunto com este estudo também foi analisado o mecanismo molecular de direcionamento da proteína Nef do HIV-1 para os MVBs associados a liberação de EVs. A proteína Nef do HIV é determinante na modulação do ambiente intracelular favorecendo a replicação do vírus e progressão à AIDS. Nef é ativamente secretado em EVs e sua liberação pode levar a apoptose de células vizinhas aceptoras dessas vesículas. Nef também medeia a redução dos níveis de CD4 e moléculas de MHC-I em EVs. Ainda não é conhecido o mecanismo molecular utilizado por Nef para ser exportado em EVs, mas sabe-se que Nef interage fisicamente com a proteína II acessória da maquinaria ESCRT, Alix, importante no processo de formação das ILVs e seleção das cargas que serão internalizadas nos MVBs. EVs coletadas de células HeLa e linfócitos T CD4+ silenciados para Alix demostraram reduções significativas na liberação de Nef. Estes resultados indicam que Nef requer Alix para sua eficiente liberação em EVs. / Lysosomal degradation of endocytosed membrane proteins occurs through the targeting of these proteins to intraluminal vesicles (ILVs), formed in the multivesicular bodies (MVBs) lumen, and the subsequent fusion of MVBs with lysosomes. Despite its importance in the degradation of transmembrane proteins, MVBs have another important function, the production and release of extracellular vesicles (EVs). In this process, the MVBs do not fuse with lysosomes, but fuse with the plasma membrane resulting in the release of these vesicles that reside within MVBs to the extracellular environment. Several proteins regulate the targeting of cargo to MVBs. Studies that delineated the functions of AP1 in protein trafficking, focused on complexes containing the ?1 subunit, which mediates transport between trans-Golgi network (TGN) and endosomes. However, the human genome encodes a second isoform of this subunit, named ?2. Evidences from the literature, as well as results from our research group, indicate that AP1?2 regulates transport pathways that are distinct from the pathways classically attributed to AP1. By performing EGF-uptake assays under ?1 or ?2 knockdown (KD) conditions, it was observed that ?2 is required for degradation of internalized EGF, effect also observed for the EGF receptor (EGFR) using cell surface biotinylation assays. These results demonstrate that the lysosomal degradation of the EGFEGFR complexes via the canonical MVBs pathway requires the AP1?2 complex, but not AP1?1. In parallel with this study, we also analyzed the molecular mechanism of HIV-1 Nef targeting to MVBs associated with the EVs release. Nef is an important determinant in the modulation of the intracellular environment for efficient HIV replication and progression to AIDS. Nef is actively secreted via EVs and its release may lead to apoptosis of bystander acceptor cells. Moreover, Nef reduces the levels of CD4 and MHC-I molecules in EVs. Despite the importance of Nef release in EVs, the molecular mechanism used by Nef to be exported via EVs is unknown. Nef physically interacts with the ESCRT machinery accessory protein Alix, an important player in the process of ILVs formation and cargo selection. EVs released from HeLa cells and CD4+ T lymphocytes under Alix KD conditions demonstrated a significant IV reduction in Nef release via EVs. These results indicate that Nef requires Alix for its efficient release in EVs.

Le mésothéliome pleural malin (MPM) est une tumeur rare et agressive qui se développe au niveau de la plèvre.L'Adrénomedulline (AM) est un peptide de 52 acides aminés. Il intervient dans plusieurs processus physiologiques et physiopathologiques. Il est surexprimé dans plusieurs tumeurs où il joue un rôle important dans la croissance tumorale.Dans un premier temps nous avons montré que l'AM et ses récepteurs sont exprimés dans des biopsies de patients atteints de MPM suggérant son implication dans la croissance tumorale du MPM. In vitro l'incubation de lignées de MPM: H2452 et MSTO_211H avec des anticorps αAM ou αAMR inhibent la prolifération, l'invasion et la migration cellulaires. In vivo, le traitement avec un anticorps αAM ou l'antagoniste AM22-52, inhibe la croissance tumorale des xénogreffes de MSTO_211H par rapport au groupe contrôle. L'analyse histologique montre une augmentation significative de l'apoptose et une diminution importante de la vascularisation chez les tumeurs traitées par rapport aux tumeurs contrôles. Ces résultats démontrent le rôle important joué par l'AM dans la croissance tumorale du MPM et fait du système de l'AM une cible thérapeutique potentielle.Dans un deuxième temps, Nous avons émis l'hypothèse de la transactivation de l'EGFR par l'AM. Notre hypothèse s'est concrétisée dans la mesure où L'inhibition de l'EGFR par un inhibiteur spécifique l'AG1478 abolie l'activation de ERK par l'AM,La phosphorylation de l'EGFR par l'AM,La neutralisation de l'EGFR avec son propre anticorps inhibe la phosphorylation par l'AM suggérant une activation ligand dépendante. Ces résultats nous permettent de mieux comprendre le mécanisme d'action de l'AM. / Malignant pleural mesothelioma (MPM) grows aggressively in the thoracic cavity without curative possibilities, underlining the need for new therapeutic targets. Adrenomedullin (AM), a multifunctional peptide, is highly expressed in several tumors and plays an important role in angiogenesis and tumor. In the first part of our work, QRT-PCR showed an increase of AM mRNA levels in MPM when compared to normal pleura tissue. Immunohistochemically, AM and its receptors were localized in the carcinomatous epithelial compartment of MPM. The MPM cell lines H2452 and MSTO_211H expressed AM with a significant increase under hypoxia. The proliferation, migration and invasion of MPM cells are decreased by anti-AM and anti-AM receptors antibodies (αAM and αAMR) supporting that MPM cells can be regulated by AM. In vivo, αAM and AM22-52 antagonist therapies of MSTO_211H xenografts blocked angiogenesis and stimulated apoptosis, resulting in tumor regression. Histologic examination of treated tumors showed evidences of disruption of tumor vasculature. These findings highlight the implication of the AM pathway in the MPM growth and in neovascularization by supplying/amplifying signals essential for pathologic neoangiogenesis and lymphangiogenesis.In the second part of this work, we reported that the EGFR becomes rapidly tyrosine-phosphorylated upon stimulation of lung cancer cells lines with AM, suggesting that there is an intracellular mechanism for transactivation. Specific inhibition of EGFR function by the AG1478 or EGFR blocking antibody suppressed MAPK activation. These results suggest strongly a ligand-dependent mechanism of EGFR transactivation by AM.

This study investigated several somatostatin analogues labeled with copper-64 for imaging and targeted therapy of SSTr positive cancer. Among three new cross-bridged bifunctional chelators coupled to Y3-TATE, 64Cu-CB-TE2A-Y3-TATE had the most favorable tumor targeting properties. The introduction of ionizable linker groups could not remedy the slow clearance from the kidney, and other modifications will be necessary to resolve this issue. The emerging idea of using the copper-64-labeled somatostatin antagonist 64Cu-CB-TE2A-sst2-ANT as a tumor targeting agent will require further experimentation. This radiopharmaceutical showed promising initial results in a biodistribution study in male Lewis rats, however, it should be compared to 111In-DOTA-sst2-ANT in the same model. Nuclear localization of copper-64 from two somatostatin analogues differing in their chelate stability strengthened the hypothesis of copper-64 dissociation from the bifunctional chelator prior to trafficking to the nucleus. However, the increased nuclear uptake of copper-64 from the less stable 64Cu-TETA-Y3-TATE did not result in a significant effect on cell killing of A427-7 cells. In experiments with [64Cu]copper acetate and the EGFR-antibody 64Cu-DOTA-cetuximab, the tumor suppressor protein p53 was identified as a mediator of the nuclear transport of copper. 64Cu-DOTA-cetuximab was also utilized in five cervical cancer cell lines with a wide range of EGFR expression. EGFR quantification by saturation receptor binding, and EGFR function as determined via internalization of 64Cu-DOTA-cetuximab closely followed the expression pattern of these cell lines found via EGFR mRNA profiling. This constitutes a first step in the evaluation of cetuximab for the treatment, and of 64Cu-DOTA-cetuximab for the imaging of advanced cervical cancer, as EGFR expression on the tumor cell surface clearly can be quantified and visualized with this experimental system. Copper-64 has been used in this study to probe the basic biochemical process of intracellular copper trafficking, and for the targeting of cell surface receptors via radiolabeled peptides and antibodies, providing an example of the powerful combination of radiopharmaceutical chemistry and cell biology.

Introduktion: På senare tid har en ny generation av antikoagulantia (NOAK) uppkommit. NOAK kommer med fördelar då tidigare antikoagulantia behandling krävt täta läkarkontroller, reglering av dosering och hänsyn till kost. Tidigare antikoagulantia som warafarin och hepariner inhiberar flera koagulationsfaktorer. Medan Edoxaban som tillhör NOAK endast hämmar fria faktor Xa och protrombinaktivitet. Som följd av detta har Edoxaban en bättre effekt och ger ett mer säkert resultat vid korrekt ordination. Eftersom Edoxaban elimineras via njurar är det viktigt med njurfunktionskontroller som görs via estimerat glomerulär filtrationshastighet (eGFR). Kreatininbaserad eGFR gjordes med Lund-Malmö-formeln (LM) som är anpassad för den svenska populationen. Cystatin-C-baserad eGFR beräknas via Caucasian-Asian-pediatric-and-adult-cohorts-formeln (CAPA) där endast ålder tas hänsyn, till skillnad från LM-formeln där fler faktorer spelar roll. Syftet: Undersöka korrelationen mellan kreatininbaserade eGFR formeln LM och cystatin-C-baserad eGFR formeln CAPA. Metod: Blodprov togs från 43 Edoxabanbehandlade patienter i Karlstad Centralsjukhus. Patienternas eGFR beräknades via CAPA- respektive LM-formel. Faktorer som vikt, längd och ålder togs till hänsyn. Resultat: LM- och CAPA-eGFR visar en stark korrelation. Slutsats: LM-eGFR påverkas av fler faktorer och som följd fungerar bäst för patienter med balanserad längd och vikt samt stabil kost. Medan CAPA-eGFR, som påverkas av färre faktorer är anpassade för patienter som är över- och underviktiga med mycket varierad kost. / Introduction: A new generation of anticouagulants has emerged called NOAC. NOAC comes with benefits that have previously been problematic for patients treated with anticouagulants such as regular check-ups and dose regulation. Previous anticouagulants, warafarin and heparins, inhibits multiple coagulation factors. While NOAC only inhibits select few factors. For example, Edoxaban only inhibits free factor Xa and prothrombin activity. As result, Edoxaban has a better effect with proper prescription. Edoxaban eliminates through the kidneys which makes renal function tests important – eGFR. Creatinine-based eGFR is calculated via LM-formula which is adapted for the Swedish population. Cystatin-C-based eGFR is calculated via CAPA formula where only age is taken into account whereas the LM-formula relies on multiple factors. Aim: The purpose of the study is to understand the correlation between creatinine- and cystatin-C-based eGFR. Method: blood samples were taken from 43 patient treated with Edoxaban at Karlstad Hospital. eGFR were calculated via CAPA and LM formula. Weight, age and height were taken into account.  Result: A strong correlation shows in LM- and CAPA-eGFR. Conclusion: LM-eGFR is affected by several factors therefore works best for patients with balanced height and weight as well as a stable diet. Patients who are overweight, underweight or has a very varied diet is best fit for CAPA-eGFR which is unaffected by these factors.

Ziel dieser Studie war es, die Häufigkeit der Expression von EGFR, HER-2 sowie COX-2 im Zervixkarzinom zu eruieren. Dabei galt es herauszufinden, ob Unterschiede hinsichtlich des Nachweises dieser drei, möglicherweise therapeutisch relevanten Moleküle zwischen den primären, nicht vortherapierten und operierten Karzinomen und den multimodal vorbehandelten Rezidiven gab. In der vorliegenden retrospektiven Arbeit wurden 45 TMMR-operierte Primärtumoren und 28 LEER-operierte Rezidivtumoren der Universitätsfrauenklinik Leipzig (Triersches Institut) einbezogen und zusätzlich hinsichtlich der prognostischen Überlebensanalyse durch das Tumorstadium, Lymphknotenmetastasen und Rezidivauftreten sowie histologischer Charakteristika untersucht. Dazu wurden Tissue - Microarrays angefertigt mit anschließender immunhistochemischer Untersuchung dieser. Die Ergebnisse zeigten, dass die TMMR-Operation die Überlebensprognose signifikant verbessert, denn lediglich bei den LEER-therapierten Rezidivtumoren erlitten die Patientinnen sowohl Fernmetastasen als auch erneute Rezidive. Weder die Expression der drei untersuchten Moleküle noch die histopathologischen Parameter haben eine prognostische Relevanz. Es gibt keine signifikanten Zusammenhänge zwischen der Häufigkeit der Expression von EGFR, HER-2 sowie COX-2 und Primär-, bzw. Rezidivtumoren, sodass diese Moleküle keine Targets für eine individualisierte, zielgerichtete Therapie beim Zervixkarzinom darstellen.

Les inhibiteurs de tyrosine kinase (ITK) du récepteur à l’Epidermal Growth Factor (EGFR) constituent un nouveau traitement du cancer bronchique non à petites cellules (CBNPC), particulièrement efficace chez les patients porteurs d’une mutation d’EGFR (EGFR M+). Néanmoins, certains de ces patients peuvent avoir une résistance primaire à ces traitements, et les autres développent inéluctablement une rechute correspondant au phénomène de résistance acquise. L’objectif de ce travail était d’étudier les mécanismes de résistances primaire et acquise chez les patients avec CBNPC EGFR M+.Nous avons montré que le statut mutationnel de KRAS était le même dans la tumeur primitive et les métastases dans la majorité des cas de CBNPC. Dans quelques cas cependant, le statut différait entre tumeur primitive et métastase, ce qui soulève la question d’une dissociation de la réponse aux ITK, les mutations de KRAS étant associées à une résistance primaire au traitement.Nous avons par ailleurs mis en évidence la fréquente inactivation de la voie p53, soit par mutation de TP53 soit par diminution d’expression de p14arf, dans les tumeurs EGFR M+, qui pourrait être une des causes des variations de réponse aux ITK chez les patients EGFR M+. Enfin, nous avons montré que la résistance à deux ITK de nouvelle génération, PF299804 et WZ4002, passait par un phénomène en deux étapes impliquant d’une part l’activation de la voie IGF1R, via la diminution de l’expression d’IGFBP3, et d’autre part celle de la voie des MAP kinases. Ces travaux pourraient déboucher sur des stratégies thérapeutiques innovantes chez les patients présentant une résistance acquise aux ITK de nouvelle génération. / Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitors (TKI) provide clinical efficacy in Non Small Cell Lung Cancer (NSCLC) patients, especially in the presence of an EGFR mutation. However, some EGFR mutated patients display primary resistance to EGFR TKI, and the others will ultimately develop acquired resistance. We focused our work on mechanisms of primary and acquired resistance in EGFR mutated NSCLC.We first showed that KRAS mutational status is the same in primary NSCLC and matched metastases in most of the cases. However, in some patients, we found a discordant KRAS status between primary tumors and metastases, which could translate into a discordant response to EGFR TKI, since KRAS mutations are associated with resistance to EGFR TKI.We also showed that EGFR mutated tumors are associated in most of the cases with an inactivation of the p53 pathway, either through a TP53 mutation or through loss of expression of p14arf, which could account for some of the variations observed in the response to TKI in EGFR mutated patients.Last, we showed that acquired resistance to two new generation EGFR TKI, PF299804 and WZ4002, occurred through a multistep process involving activation of the IGF1R pathway through downregulation of IGFBP3 and activation of the MAP kinase pathway. These results provide new insights into the treatment of NSCLC in EGFR mutated patients with acquired resistance to new generation TKI.

La surexpression du récepteur EGFR a un rôle majeur dans le développement des carcinomes des voies aérodigestives supérieures (VADS) et son inhibition par les anticorps anti-EGFR (cetuximab) induit un effet anti-tumoral mais pourrait également avoir un effet anti-angiogénique. Cependant, les effets de ces agents sur l'angiogenèse et les cellules endothéliales (CE) n'ont pas réellement été évalués. L'objectif de ce travail est d'étudier l'angiogenèse induite par les molécules libérées par les cellules tumorales des VADS (Cal27, FaDu) dans les milieux de culture en présence ou non de cetuximab, appelés milieux conditionnés (CM). Le cetuximab n'a pas d'effet direct significatif sur les CE. Il induit une diminution de la sécrétion de VEGF par les cellules tumorales mais paradoxalement, les CM obtenus induisent un effet pro-angiogénique. L'analyse de la composition des CM ne permet pas d'identifier de molécule clé responsable de cet effet, le cetuximab diminuant à la fois la sécrétion tumorale des facteurs pro- et anti-angiogéniques. Pour expliquer ce paradoxe et en accord avec la littérature, nous avons mis en évidence une libération de microvésicules par nos cellules tumorales (TMV), TMV exprimant EGFR et TF, et une régulation de cette libération et de leur contenu par le cetuximab. Ces TMV peuvent interagir avec les CE et le cetuximab favoriserait cette interaction. La caractérisation de ces TMV et leur rôle dans le processus angiogénique en réponse ou non au cetuximab permettront dans le futur de comprendre la réelle activité des anticorps anti-EGFR sur l'angiogenèse / Overexpression of EGFR has a major role in the development of head and neck squamous cell carcinoma (HNSCC) and its inhibition by anti-EGFR antibodies (cetuximab) induced an anti-tumor effect but could also induce an anti-angiogenic effect. However, the effects of these agents onto angiogenesis and endothelial cells (EC) have not really been evaluated. The objective of this work is to study angiogenesis induced by mediators released by head and neck squamous carcinoma cells (Cal27, FaDu) in culture media with or without cetuximab exposure, known as conditioned media (CM). Cetuximab has no significant direct effect on EC. It induces a decrease in the secretion of VEGF by tumor cells but, paradoxically, the CM induces a pro-angiogenic effect. The analysis of the composition of the CM does not allow us to identify a key molecule responsible for this effect because cetuximab decreases the secretion of both pro- and anti-angiogenic factors by tumor cells. To explain this paradox and in agreement with the literature, we highlighted the release of microvesicles by our tumor cells (TMV), TMV which express EGFR and TF, and regulation of this release and the content of TMV after cetuximab exposure. These TMV may interact with the EC and cetuximab increases this interaction. Further characterization of TMV and studying their role in the angiogenic process in response to cetuximab will allow us in the future to understand the real activity of anti-EGFR antibodies onto angiogenesis

Endothelin-1 (ET-1) ist ein gefä?aktives Peptid, welches zusätzlich verschiedenste nicht kardiovaskuläre physiologische und pathophysiologische Effekte besitzt. So wurde z.B. beschrieben, dass ET-1 in der Brustdrüse während der Schwangerschaft und Stillzeit exprimiert wird. Zusätzlich zu den bekannten Nährstoffen und Wachstumsfaktoren konnte auch ET-1 in der Muttermilch nachgewiesen werden, was auf eine physiologische Rolle von ET-1 für die Laktation und den säugenden Nachwuchs hinweist. In der vorliegenden Arbeit sollte die Funktion von ET-1 in der Brustdrüsenentwicklung mit Hilfe von ET-1 transgenen Mäusen aufgeklärt werden. Die eingesetzten transgenen Tiere überexprimieren humanes ET-1 mit den entsprechenden 5''- und 3'' regulatorischen Sequenzen. Mit Hilfe dieser Strategie sollen die ET-1 spezifischen Funktionen während der Brustdrüsenentwicklung untersucht werden. Transgenes ET-1 wurde während der Tragzeit und Stillzeit in der Brustdrüse detektiert. Die Ergebnisse zeigten, dass säugende Neugeborene der ET-1 transgenen Mäuse eine geringere Gewichtszunahme und eine erhöhte Mortalität aufwiesen, welches auf einen Laktationsdefekt hinweist. Die histologische Untersuchung der Brustdrüse während der Tragzeit ergab eine reduzierte Milchkanalausbildung, kollabierte und nicht expandierende Alveoli, vermehrte Adipozytenausbildung und fortbestehende zytoplasmatische Lipidtropfen (CLDs). Zusätzlich war die Expression des Milchproteins WAP reprimiert. Interessanterweise wurde diese Repression nicht durch STAT5, einem beschriebenem Regulator der Milchproteinexpression und Alveolarexpansion, vermittelt, da dessen Aktivität unverändert war. Als Konsequenz dieses Laktationsdefekts konnte eine verfrühte Rückbildung der Brustdrüse festgestellt werden. Diese ging mit einer erhöhten Expression von STAT3 einher. Interessanterweise wies der bekannte Aktivator von STAT3, LIF, ebenfalls eine gesteigerte Aktivität auf, sowohl während der Tragzeit als auch während der Laktation. Zusätzlich zu den beschriebenen Defekten bei der Milchabgabe zeigten histologische Untersuchungen der Brustdrüse eine Laktationshyperplasie während der mittleren Laktationsphase. In diesem Zusammenhang wird darauf hingewiesen, dass ET-1 Rezeptoren, neben den klassischen Signalwegen dieser G Protein-gekoppelte Rezeptoren, auch mit Tyosinkinaserezeptoren wie z. B. dem EGFR interagieren können. Brustdrüsen von ET-1 transgenen Tieren zeigten eine erhöhte Aktivität sowohl von EGFR als auch von ERK1/2, welches im Zusammenhang mit dem hyperplastischen Phänotyp stehen könnte. Die mögliche tumorfördernde Wirkung von ET-1 wird ferner durch die erhöhte Expression von Amphiregulin, einem EGFR-Liganden, während der Tragzeit und der Laktation verstärkt. Zusammenfassend konnte festgestellt werden, dass ET-1 sowohl die Milchsekretion als auch den Milcheinschuss negativ beeinflusst, so dass eine ausreichende Versorgung säugender Jungtiere in der 1. Hälfte der Laktationsperiode nicht mehr gewährleistet ist. Zusätzlich verursachte ET-1 eine Laktationshyperplasie, welche auf die Induktion der EGFR-Achse zurückzuführen ist. Zusammenfassend kann somit festgestellt werden, dass die Ergebnisse auf eine wichtige Rolle von ET-1 in der Brüstphysiologie des Säugers hinweisen. / Endothelin-1 (ET-1) is a potent vasoactive peptide having wide physiological effects on vascular homeostasis and on a variety of pathophysiological processes unrelated to cardiovascular system. It has been noted that ET-1 is expressed in mammary glands during pregnancy and lactation periods. Furthermore, ET-1 is secreted into milk, suggesting additional physiological roles in the lactating mother and in the suckling neonate. Hence, the present study was proposed to elucidate the possible functional roles of ET-1 in mammary gland development employing ET-1 transgenic mice. ET-1 transgenic mice had been generated by using a human genomic ET-1 construct containing 5´ and 3´ regulatory sequences. This transgenic construction strategy grants to analyse the specific functions of ET-1 in normal mammary gland physiology. The transgene expression was found in mammary gland during pregnancy and lactation. ET-1 transgenic mice exhibited a lactational incompetence with reduced weight gain and increased mortality of their newborns, as a result of a secretory defect. In virtue of this defect, ET-1 transgenic mammary glands histologically revealed a reduced ductal outgrowth, collapsed alveoli with a reduced expansion capacity, increased adipocyte accumulation, and persistence of cystoplasmic lipid droplets (CLDs) during lactation. In addition, the expression of the milk protein, WAP, was found to be constantly suppressed in ET-1 mammary glands although the activity of STAT5, which is known to be a regulator of the expression of milk proteins and alveolar expansion, was found to be normal. Furthermore, as a consequence of the secretory defect, ET-1 transgenic mammary glands exhibited focal precocious involution during early stages of lactation along with an increased activity of STAT3. Consistently, the known activator of STAT3, LIF, was strongly upregulated during lactation and pregnancy. Besides the secretory defect of ET-1 transgenic mammary glands, histological analysis revealed a local lactational hyperplasia during the middle of lactation. Alternatively to the classical G protein-coupled receptors GPCR signalling pathways, endothelin receptors are able to communicate with tyrosine kinase receptors such as the epidermal growth factor receptor (EGFR) for which the term receptor transactivation was coined. Mammary glands of ET-1 transgenic animals exhibited an increased activity of the EGFR and ERK1/2, which could contribute to the observed hyperplastic phenotype. In support of the potential tumourigenicity of ET-1, one of the EGFR ligands, amphiregulin, was found significantly upregulated in ET-1 transgenic mammary glands, both during pregnancy and lactation periods. In summary, high levels of ET-1 affect the secretion and the milk let down process. Consequently the normal support of milk for the suckling neonates is severely impaired during the first half of the lactation period. In addition, ET-1 caused lactational hyperplasia in the mammary glands due to the induction of the EGFR axis. This suggests an important role for ET-1 in mammary gland physiology.

Die Behandlung von Krebserkrankungen wurde in den letzten Jahren von zwei großen Trends beeinflusst. Statt zytotoxischer Therapien, wurden zielgerichtete monoklonale Antikörper entwickelt. Zudem verstärkten sich die Bemühungen in der Entwicklung prädiktiver Biomarker, die die Stratifizierung von Patienten vor der Behandlung ermöglichen. Obwohl hunderte von zielgerichteten Krebsmedikamenten entwickelt wurden, erreichten nur wenige davon eine Zulassung. Grund hierfür ist die noch hohe Ausfallrate bei der „Übersetzung“ von vielversprechenden präklinischen Daten, die mit Krebs-Zelllinien und Xenograft-Modellen von Krebs-Zelllinien erlangt wurden, in positive klinische Phase II- und Phase III-Daten. Das Ziel der Forschung ist es, neue zielgerichtete Therapien und gleichzeitig prädiktive Marker, mit Hilfe von verbesserten präklinischen Tumormodellen zu entwickeln. Ein Panel von 133 Xenograftmodellen aller vier UICC-Stadien wurde etabliert. Zur Überprüfung dieses Modells wurde die Wirksamkeit von Cetuximab, Bevacizumab und Oxaliplatin getestet. Für dieses Experiment benutzten wir 67 Xenograft-Modelle, die aus chemonaiven CRC-Patienten entwickelt wurden. Die Behandlung mit einer Cetuximab-Monotherapie ergab eine objektive Ansprechrate von 27%. Dank dieser hohen Ansprechrate, konnten wir das verfügbare Tumorgewebe zur Vorhersage der Reaktion auf die Anti-EGFR-Antikörper in den Xenograft-Modellen verwenden. Wir untersuchten die Genauigkeit von Mutations-Markern (KRAS, BRAF und PIK3CA) kombiniert mit RNA-Expressionsdaten von Amphiregulin und Epiregulin. Weiterhin wurden neue prädiktive Marker-Kombinationen, basierend auf einer mRNA- und microRNA- Expressionsanalyse, ermittelt. Aufgrund der erfolgreichen Nachbildung der klinischen Situation in unserem Panel von Xenograft-Modellen, zeigt dieser Modellansatz vielversprechendes Potenzial für eine zukünftige Anwendung in der Erprobung neuartiger Krebsmedikamente und der Entwicklung von prädiktiven Biomarkern. / Over the last ten years two major trends have influenced drug development. Instead of cytotoxic therapies a variety of targeted monoclonal antibodies have been developed. In parallel, higher attention has been paid to the identification and validation of predictive markers, which allow the stratification of patients prior to the treatment. Hundreds of targeted cancer drugs have been developed, but only a few have been approved. Despite these two trends, there is still a high rate of failure in translating promising preclinical data obtained with cancer cell lines and xenograft models derived from cancer cell lines into positive clinical phase II and phase III data. The goal of the scientific community is to develop novel targeted therapies and predictive biomarkers using better preclinical tumour models. To bridge the gap between preclinical and clinical development a panel of 133 patients-derived CRC xenografts of all four UICC stages was established. An efficacy of cetuximab, bevacizumab and oxaliplatin was tested in the subset of 67 models. In the treatment experiment with cetuximab monotherapy an objective response rate of 27% was obtained. This high response rate allowed the use of the tumour tissues for evaluating molecular markers for predicting response to the anti-EGFR antibody in the xenograft models. The accuracy of three mutation markers including KRAS, BRAF and PIK3CA was investigated in combination with RNA expression levels of two EGFR ligands – amphiregulin and epiregulin. Novel predictive markers panels based on gene expression profiling of mRNA and microRNA were also tested and compared with the established biomarkers. Successful reconstruction of the clinical situation in the panel of xenograft models proves their potential for future use in testing of novel anti-cancer drugs. Moreover, their broad molecular characterization allows the simultaneous development of predictive biomarkers along with the testing of novel cancer drugs.