Open Access

Marco Reis

• The canonical Wnt pathway, also known as Wnt/β-­‐catenin pathway, comprises a network of proteins which control diverse developmental and adult processes in all metazoan organisms. The binding of canonical Wnt ligands to a cell surface receptor complex, consisting of frizzled family members and low density lipoprotein receptor-­‐ related protein 5 or 6 co‐receptors, triggers a signaling cascade which results in a β-­catenin-­‐mediated transcriptional activation of different target genes, implicated in cellular proliferation, apoptosis, migration and differentiation. A couple of years ago, several groups including us, iden2fied transient activation of the canonical Wnt-pathway in endothelial cells (ECs) of the developing central nervous system (CNS). In this context, Wnt/β-­‐catenin signaling could be demonstrated to be crucial for brain angio genesis as well as for the establishment of the blood-­brain barrier (BBB) phenotype in the newly formed vessels. Gliomas, in particular the glioblastoma (GBM), belong to the group of highly vascularized solid tumors which gain their vascularization due to an angiogenic switch occurring during tumor progression. Interestingly, nuclear localized β-­‐catenin could be exclusively detected in the activated endothelium of induced rat gliomas and of human GBM, suggesting a so far unknown and not further characterized involvement of the canonical Wnt pathway in pathological angiogenesis. In order to systematically decipher the precise role of endothelial Wnt/β-­‐catenin signaling in tumor angiogenesis, I established murine GL261 glioma cell lines overexpressing either Wnt1 or Dickkopf (Dkk) 1 in a doxycycline-­‐dependent manner, an activator and potent inhibitor of Wnt/β-­‐catenin signaling, respectively. In subcutaneous and intracranial transplantations, tumor-­derived Wnt1 reduced, while Dkk1 increased GL261 tumor growth without affecting in vitro proliferation, cell cycle or cell death of the established cell lines. Nowadays, it is well accepted that solid tumors are dependent on vascular support allowing them to grow beyond a certain size. In my work I could show that tumor-­‐derived Wnt1 targets the tumor vasculature by increasing endothelial Wnt/β-­‐catenin signaling, which reduced tumor vessel density and resulted in a more quiescent tumor vasculature. Furthermore, Wnt1-­‐expression mediated tight association of smooth muscle cells (SMCs) and pericytes to the tumor endothelium, a phenotype which is unusual for tumor vessels and a described hallmark of tumor vessel normalization. In contrast, inhibition of endothelial Wnt/β-­‐catenin signaling by Dkk1 mediated an opposing effect, characterized by endothelial hyper-­proliferation and a tumor vasculature with a rough basal lamina distribution and loosely anached mural cells, indicative of a strong angiogenic activity. The described vascular effects in Wnt1-­expressing GL261 tumors could be verified by subcutaneous transplantations of a rat glioma cell line constitutively expressing Wnt1. Furthermore, an applied in vivo MatrigelTM plug assay uncovered the reduction in vessel density upon Wnt1 simulation to be tumor cell independent, suggesting an EC-­‐autonomous effect. This hypothesis was confirmed by subcutaneous transplantations of parental GL261 cells into mice with genetically generated endothelial β-­‐catenin gain-­of-­function (GOF). The derived GOF tumor from this experiment comprised a quiescent and normalized tumor vasculature and phenocopied the vascular effects observed in Wnt1-­expressing tumors. Our previous work provided evidence that Wnt/β-­‐catenin signaling contributes to the BBB phenotype of the developing CNS through the transcriptional regulation of the tight junction protein claudin-­‐3. Furthermore, the coverage of pericytes to brain vessels has been described to correlate with BBB integrity. In agreement with these publications, vessels of intracranial Wnt1-­‐expressing GL261 tumors retained or regained barrier properties, indicated by a reduced leakage of the tracer Evans blue and endogenous mouse immunoglobulin G and increased junctional localiza2on of the tight junction proteins claudin-­‐3, -­‐5 and zonula occludens-­‐1. Overall, we detected sustained endothelial Wnt/β-­‐catenin signaling to induce a quiescent and normalized tumor vascularization. Interestingly, the Notch signaling pathway has been shown to inhibit the angiogenic tip cell and to promote the quiescent stalk cell phenotype via its ligand Delta-­like ligand 4 (Dll4) and the receptors Notch1 and 4. Mechanistically, my work demonstrated for the first time that overactivation of endothelial Wnt/β-­‐catenin signaling reactivated expression of Dll4 in the tumor endothelium, which could be shown in vitro to increase Notch signaling and to favor a stalk cell-­like gene signature. Furthermore, we uncovered the platelet-­derived growth factor subunit B (pdgm) as a novel transcriptional target of Wnt/β-­catenin signaling in ECs. Hence endothelial-­‐derived PDGF-­‐B is known to promote the recruitment of mural cells, the upregulation of this factor might explain the increased SMC/pericyte coverage observed in the tumor vasculature upon sustained endothelial Wnt/β-­‐catenin signaling which additionally might promote a cycle of vascular normalization. Taken together, my work reveals several vascular effects, being mediated by reinforced endothelial Wnt/β-­‐catenin signaling during tumor angiogenesis. While a moderate level of canonical Wnt signaling, observed in vessels of human astrocytomas and murine control tumors, is considered to be associated with tumor angiogenesis, dominant activation of this pathway in ECs is shown to limit angiogenesis and to promote a quiescent and normalized tumor vasculature with increased barrier properties. Furthermore, my work discovers pdgm as a novel target of canonical Wnt signaling in ECs. The work presented in this dissertation therefore not only uncovers the role of endothelial Wnt/β-­‐catenin signaling in tumor angiogenesis but additionally reveals this pathway to be a novel modulator in pathological vessel development which might proof to be a valuable therapeutic target for anti-angiogenic and edema glioma therapy.
• Der kanonische Wnt-Signalweg, der auch als Wnt/β-­‐catenin-Signalweg bezeichnet wird, ist eine hoch konservierte und komplexe zelluläre Signalkaskade, die inallen mehrzelligen Organismen vorkommt. Die Aktivierung dieser Signaltransduktion erfolgt durch die Sekretion Cystein-­‐reicher Glykoproteine, welche als kanonische Wnt-Proteine bezeichnet werden und denen beispielsweise Wnt1 angehört. Die Liganden binden an einen Zellrezeptorkomplex, bestehend aus Rezeptoren der Frizzled-­‐Familie und dem Korezeptor low density lipoprotein receptor-­‐related protein 5 beziehungsweise 6. Der dadurch gebildete ternäre Komplex verminelt die Stabilisierung und nukleäre Translokation von β-­‐catenin, welches seinerseits die transkriptionelle Aktivierung von Zielgenen induziert. Auf diese Weise steuert der kanonische Wnt-Signalweg während der Embryonalentwicklung zelluläres Verhalten wie zum Bespiel Proliferation, Migration, Apoptose und Differenzierung. Erst kürzlich konnten mehrere Forschergruppen, darunter auch unsere Arbeitsgruppe, eine transiente endotheliale Aktivierung des Wnt/β-­‐catenin Signalweges während der Vaskularisierung des sich entwickelnden zentralen Nervensystems beschreiben. In diesem Zusammenhang konnte gezeigt werden, dass aktives Wnt-Signaling in Endothelzellen für die Gehirnangiogenese essenziell ist und darüber hinaus die Etablierung der Blut-­‐Hirn-­‐Schranke verminelt. Gliome, und insbesondere das am häufigsten vorkommende hoch-­‐maligne Glioblastom, gehören zu der Gruppe der stark vaskularisierten, soliden Tumore. Der Prozess der Tumorvaskularisierung, auch als Tumorangiogenese bezeichnet, wird durch die tumorale Sezernierung angiogener Faktoren induziert. Der wohl bekannteste angiogene Faktor ist der vaskuläre endotheliale Wachstumsfaktor (VEGF), welcher das umliegende quieszente Gefäßsystem aktiviert und die Sprossung neuer Gefäße in den Tumor induziert. Die auf diese Weise neu entstehenden pathologischen Gefässe versorgen die Tumorzellen mit Sauerstoff und Nährstoffen und fördern dadurch maßgeblich die Tumorprogression...