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Background: Highly infiltrative growth and resistance to radiation as well as chemotherapy contribute to a poor prognosis of glioblastoma. To improve the survival of patients with glioblastoma, further research to uncover the complex signaling network is essential. Due to the central role of the signaling adaptor lamellipodin in nervous system development and cell migration, a function of lamellipodin in glioblastoma is conceivable. However, the specific function of lamellipodin in the invasion and radioresistance of glioblastoma is so far entirely unknown. Therefore, the present work investigates the invasion and radioresistance of glioblastoma cells and the underlying signaling mechanism under the influence of lamellipodin. Material and Methods: Expression of lamellipodin was evaluated in human astrocytes and nine glioblastoma cell lines by Western blot analysis. Localization of lamellipodin in glioblastoma cells was analyzed by applying immunofluorescence staining. The effects of lamellipodin silencing by siRNA on the glioblastoma characteristics invasion, survival, and residual DNA double strand breaks (DSB) upon X-ray irradiation, proliferation, apoptosis, and senescence were investigated. Alterations in molecular signaling were analyzed by phosphoproteome analysis upon control and lamellipodin depletion combined with/-out X-ray irradiation in A172 cells. Colony formation assays were performed after single and double knockdown of lamellipodin and the affected proteins to connect latter findings to clonogenic survival. Direct lamellipodin binding partners were explored using mass spectrometry analysis. Validation of protein-protein interaction was determined by immunoprecipitation and proximity ligation assay. Clonogenic survival assay was performed after triple knockdown of lamellipodin, previous proteins identified by phosphoproteome, and the direct interaction partner of lamellipodin. Hierarchical analysis was performed by analyzing the expression and phosphorylation of the determined proteins by immunoblot. Results: Lamellipodin was shown to be expressed and phosphorylated in varying amounts in the glioblastoma cell culture panel, with a preferential localization in membrane protrusions and the cytoplasm. The siRNA-specific depletion of lamellipodin tremendously decreased glioblastoma invasion and proliferation while exerting no impact on apoptosis or senescence. Moreover, seven of the nine studied glioblastoma cell lines were radiosensitized by lamellipodin silencing without affecting the number of residual DNA double strand breaks, while its overexpression improved radiation survival. Mechanistically, the loss of lamellipodin impaired the phosphorylation of nine proteins (EIF2A, EGFR, FOS, MKK6, NFKBIA, PRKAA2, RSK2, SRC, and TAK1), which are mostly implicated in the EGFR-MAPK signaling. The combinational silencing of lamellipodin and the relevant proteins achieved overall radiosensitization in A172 and U343MG cells. Furthermore, mass spectrometric analysis of lamellipodin immunoprecipitates demonstrated that the lamellipodin interactome alters in response to X ray irradiation conditions. RICTOR was confirmed as a direct linker of lamellipodin to the EGFR-MAPK signaling by immunoprecipitation and proximity ligation assay. In addition, triple depletion of lamellipodin, RICTOR, and EGFR resulted in similar degrees of radiosensitization as reported for lamellipodin knockdown, highlighting their superimposable role in glioblastoma radiation response. In line, lamellipodin silencing increased EGFR expression and phosphorylation, while lamellipodin phosphorylation was decreased upon EGFR deficiency. Conclusion: The results uncover the crucial function of lamellipodin for invasion, proliferation, and radiosensitivity of glioblastoma cells. Based on molecular analyses, lamellipodin was discovered as a determinant of EGFR signaling by interacting with RICTOR. Based on these data, the complexity of the signaling networks conducting radiation survival is broadened by adding the adaptor protein lamellipodin. Hintergrund: Ein stark infiltrierendes Wachstum und Resistenzen gegenüber Bestrahlung und Chemotherapie tragen zu einer schlechten Prognose des Glioblastoms bei. Um die Therapie des Glioblastoms zu verbessern, ist die weitere Erforschung des komplexen Signalnetzwerkes notwendig. Aufgrund der essentiellen Rolle des Signaladaptors Lamellipodin für die Entwicklung des Nervensystems und der Migration von Zellen ist eine Funktion von Lamellipodin im Glioblastom vorstellbar. Die spezifische Funktion von Lamellipodin in der Invasion und Strahlenresistenz des Glioblastoms ist allerdings bisher völlig unbekannt. Die vorliegende Arbeit untersucht daher die Invasion und Strahlenresistenz von Glioblastomzellen sowie den zugrundliegenden molekularen Mechanismus im Hinblick auf Lamellipodin. Material und Methoden: Die Expression von Lamellipodin wurde in humanen Astrozyten und neun Glioblastom-Zelllinien mittels Western Blot Analyse untersucht. Die Lokalisierung von Lamellipodin in Glioblastomzellen wurde mit Immunfluoreszenzfärbung analysiert. Die Auswirkungen eines Lamellipodin-Knockdown mittels siRNA auf die Glioblastom-Eigenschaften Invasion, Überleben und residuale DNA-Doppelstrangbrüche (DSB) bei Röntgenbestrahlung, Proliferation, Apoptose und Seneszenz wurden untersucht. Veränderungen in molekularen Signalwegen wurden durch Phosphoproteomanalyse nach Kontroll- und Lamellipodin-Knockdown in Kombination mit und ohne Röntgenbestrahlung in A172 Zellen analysiert. Koloniebildungsassays wurden nach einfachem und doppeltem Knockdown von Lamellipodin und den betroffenen Proteinen durchgeführt, um letztere Ergebnisse mit dem klonogenen Überleben in Verbindung zu bringen. Direkte Lamellipodin-Bindungspartner wurden mittels massenspektrometrischer Analyse untersucht. Die Validierung der Protein-Protein-Interaktion wurde durch Immunpräzipitation und Proximity Ligation Assay bestimmt. Ein klonogenes Überlebensassay wurde nach dreifachem Knockdown von Lamellipodin, durch das Phosphoproteom identifizierten Proteinen und dem direkten Interaktionspartner von Lamellipodin durchgeführt. Die hierarchische Analyse erfolgte durch Analyse der Expression und Phosphorylierung der ermittelten Proteine mittels Immunblot. Ergebnisse: Die Expression und Phosphorylierung von Lamellipodin war unterschiedlich in den Glioblastomzelllinien. Die siRNA-vermittelte Deletion von Lamellipodin verringerte die Invasion und Proliferation von Glioblastomzellen enorm, während die Reduktion von Lamellipodin keine Auswirkungen auf Apoptose oder Seneszenz hatte. Darüber hinaus wurden sieben der neun untersuchten Glioblastomzelllinien durch das Ausschalten von Lamellipodin radiosensibilisiert, ohne dass sich dies auf die Anzahl der residualen DNA-Doppelstrangbrüche auswirkte, während die Überexpression von Lamellipodin das Überleben nach Bestrahlung verbesserte. Mechanistisch beeinträchtigte der Verlust von Lamellipodin die Phosphorylierung von neun Proteinen (EIF2A, EGFR, FOS, MKK6, NFKBIA, PRKAA2, RSK2, SRC und TAK1), die hauptsächlich an der EGFR-MAPK-Signalübertragung beteiligt sind. Die kombinierte Ausschaltung von Lamellipodin und den relevanten Proteinen führte zu einer allgemeinen Radiosensibilisierung in den Zelllinien A172 und U343MG. Darüber hinaus zeigte die massenspektrometrische Analyse von Lamellipodin-Immunpräzipitaten, dass sich das Lamellipodin-Interaktom als Reaktion auf Röntgenbestrahlung verändert. RICTOR wurde durch Immunpräzipitation und Proximity Ligation Assay als direktes Bindeglied von Lamellipodin zum EGFR-MAPK-Signalweg identifiziert. Darüber hinaus führte die dreifache Deletion von Lamellipodin, RICTOR und EGFR zu einem ähnlichen Grad an Radiosensibilisierung wie der Knockdown von Lamellipodin, was ihre gemeinsame Rolle bei der Strahlenreaktion von Glioblastomen unterstreicht. Darüber hinaus erhöhte die Ausschaltung von Lamellipodin die EGFR-Expression und -Phosphorylierung, während die Lamellipodin-Phosphorylierung bei EGFR-Mangel verringert wurde. Schlussfolgerung: Die Ergebnisse decken die entscheidende Funktion von Lamellipodin für die Invasion, Proliferation und Radiosensitivität von Glioblastomzellen auf. Basierend auf molekularen Analysen wurde Lamellipodin als eine Determinante der EGFR-Signalübertragung durch Interaktion mit RICTOR identifiziert. Auf der Grundlage dieser Daten wird die Komplexität des Signalnetzwerks, welches das Überleben durch Strahlung reguliert, durch das Adaptorprotein Lamellipodin erweitert.

Regulated cell death (RCD) is indispensable for homeostasis and plays a crucial role in the pathophysiology of numerous diseases. Adrenocortical carcinomas (ACCs) represent a rare and highly malignant type of cancer. Currently, the most common therapeutic options include the complete surgical removal of the adrenal gland and/or the administration of mitotane, a derivative of the pesticide DDT. Yet patient survival remains poor and the mechanism of action of mitotane remains elusive. In this thesis it is demonstrated that the human ACC cell line NCI-H295R is sensitive to mitotane-induced cell death. In the first part, the involvement of three different RCD pathways, namely apoptosis, necroptosis and ferroptosis, in mitotane induced necrosis was investigated. To this end, different inhibitors were used, which were not able to block mitotane-induced cell death. When the medium was supplemented with insulin, transferrin, sodium selenite and linoleic acid (ITS+1) no cell death of the ACC cells was observed. This phenomenon was attributed to the presence of linoleic acid, since ITS supplementation lacking this component was not able to reverse mitotane-induced necrosis. Identification of new drug targets for alternative options of ACC treatment led to the investigation of key molecules involved in the pathways of necroptosis and ferroptosis. The receptor-interacting protein kinase 1 and 3 (RIPK1 and 3) and the mixed lineage kinase domain-like protein (MLKL) were considered as interesting targets given their crucial role in the execution of necroptosis. A western blot analysis of those molecules revealed the presence only of RIPK1, suggesting that the necroptosis machinery is not present in the NCI-H295R cells. Of interest, evaluation of the expression levels of glutathione peroxidase four (GPX4), one of the main inhibitory molecules of ferroptosis, showed a much higher expression in the ACC cells compared to the standard cell line used for studying ferroptosis, the human fibrosarcoma HT1080 cells. A hypothesis that the NCI-H295R cells are susceptible to ferroptosis induction was formed based on this finding. Compounds representative of all the four classes of ferroptosis inducers (FINs) were tested. Direct inhibition of GPX4 using the small compound RSL3, a type II FIN, led to high necrotic populations. Co-treatment with the ferroptosis inhibitor ferrostatin-1 (Fer-1) completely reversed RSL3-induced ferroptosis. Type IV FIN FINO2, that causes indirect loss of the enzymatic activity of GPX4, lead also to high necrotic populations, while Fer-1 prevented FINO2-induced ferroptosis. Data from public databases concerning gene methylation or mutation status of ACC tissues and normal human adrenal tissues was used to investigate potential key players of ferroptosis that might be either mutated or silenced in ACCs. Of note, glutathione peroxidases 3 and 5 (GPX3 and 5) were highly methylated, while the enzyme cystathionine gamma-lyase (CSE) involved in the transsulfuration pathway via the break down of cystathionine into cysteine and α-ketobutyrate and ammonia was found to be highly mutated. Collectively, these data point towards a high sensitivity of ACCs to ferroptosis induction. This could provide a new chapter for the therapeutic approaches of ACCs. Additionally, these findings provide a better understanding of the biology of this type of cancer that highly mutates or silences ferroptosis-related genes. The second part of this thesis focuses on the involvement of RCD in spontaneous cell death in isolated murine tubules. Existing literature points towards an involvement of necroptosis and ferroptosis pathways in the kidney in models of acute kidney injury (AKI). Acute tubular necrosis (ATN) represents a hallmark of AKI. While the work in the Linkermann lab has shown that isolated tubules perfused with type I FIN erastin undergo cell death in a “wave-of-death” manner, no deeper insights into the propagation of tubular necrotic injury exist. A protocol for isolation of murine kidney tubules was established, providing an ex-vivo model for investigation of tubular death. The absence of potentially confounding blood cells as well as immune cells was ensured by extensive washing steps as well as the use of collagenase. Visual observation and staining of isolated tubules with the nucleic acid stain SYTOX green revealed a spontaneous cell death in a “wave-of-death” manner. This wave was running in parallel with a calcium concentration change, indicating its involvement in the spontaneous necrosis. To investigate the potential involvement of mitochondria in this process, electron microscopy images were obtained from parts of the tubules with different levels of damage which revealed highly damaged and ballooned mitochondria. These data provided with a phenotypic characterisation of the spontaneous tubular necrosis. Aiming to approach this type of death genetically, necroptosis and pyroptosis deficient mice (MLKL/GSDMDDKO) were used. Comparison of the LDH release, used as a measure of necrosis, from isolated kidney tubules of the MLKL/GSDMDDKO mice and wild type (WT) mice showed no difference. This indicated that neither necroptosis nor pyroptosis are involved in the tubular necrosis. Therefore, the next step was to investigate the effects of Fer-1 at the levels of LDH of isolated tubules from WT mice. A significantly lower LDH release was observed in tubules treated with Fer-1 compared to the ones treated with vehicle. However, this reduction in the LDH release was not complete, suggesting that ferroptosis is only partially responsible for the spontaneous death of isolated tubules. The difference of male and female mice towards AKI sensitivity has been noted in the literature in that female mice are less susceptible compared to the male mice. Therefore, the next step was to investigate whether this protection of females can be observed at the level of isolated tubules. Indeed, the LDH release from tubules isolated from female mice was significantly less compared to the LDH release of tubules isolated from male mice. Based on the data obtained from isolated tubules from WT male mice treated with Fer-1, a similar experiment was performed with tubules isolated from WT female mice. No difference in the LDH release was observed between the Fer-1-treated tubules and the vehicle-treated ones, indicating that another cell death pathway might be involved. The most obvious difference between male and female organisms is the sex hormones. Whether testosterone or β-estradiol are responsible for the higher susceptibility or protection against cell death has been a debate over the last years. To test this hypothesis, three different cell lines were utilised. A pre-treatment of 16 h with either testosterone or β-estradiol was performed. Treatment with either type I FIN erastin or type II FIN RSL3 followed, and cells were analysed via flow cytometry. Data revealed protective effects of β-estradiol against ferroptosis induction. Next, the effects of β-estradiol in a simultaneous treatment with RSL3 were investigated. Interestingly the protective effects of the hormone were still observed. Among the metabolites of β-estradiol, 2-hydroxyestradiol (2-OHE2) has been reported to exert antioxidant effects. Therefore, 2-OHE2 was used in a simultaneous treatment with RSL3, and the obtained data showed that it was a much more potent inhibitor of necrotic cell death than β-estradiol even at lower concentrations. Collectively these data indicate that the lower susceptibility of female organisms towards cell death might be explained by the presence of β-estradiol and its more potent antioxidant metabolites. Such findings could change the way the two sexes are approached scientifically, while providing new insights on different therapeutic strategies between male and female organisms.