Loss of Multimerin-2 associates with YAP/TAZ activation affecting the endothelial cell barrier and impinging on immune cell transmigration

The tumor microenvironment plays a crucial role in cancer progression, influencing processes such as angiogenesis, metastasis and immune evasion. Among the constituents of the extracellular matrix, Multimerin-2 comes forward as a critical regulator of endothelial cell function and vascular stability and its expression is frequently lost in tumor-associated vessels. Mechanistically, we previously demonstrated that Multimerin-2 maintains the integrity of the endothelial cell junctions via the VEGFR2 signaling pathway. In the current thesis work, we investigated the mechanisms underlying the loss of Multimerin-2 in the tumor vasculature and its consequences in tumor angiogenesis and immune cell infiltration, in the context of ovarian cancer. Through the employment of conditioned media from different tumor cells and specific inhibitors we found that loss of Multimerin-2 is primarily due to the action of tumor cell-derived metalloproteinases, compromising endothelial cell function. To verify the molecular alterations occurring upon Multimerin-2 loss we first focused on the Notch pathway, which, in concert with VEGFR2, is a master regulator of endothelial cell behaviour. We found that the downregulation of Mutimerin-2 associated with reduced presence of the Notch Intracellular Domain in the nuclei and lower expression of Notch target genes, suggesting that Multimerin-2 may play an important role in the regulation of this pathway. Another important component affecting endothelial cell function is the basement membrane, whose composition can vary depending on the expression of adjacent extracellular matrix molecules. Interestingly, we found that Multimerin-2 loss associated with increased deposition of fibronectin, collagen IV and laminin, suggesting the engagement of a possible compensatory mechanism to restore vascular stability. Loss of Multimerin-2 was also associated with increased nuclear localization of the YAP/TAZ complex, a pathway closely tied to both Notch signalling and the basement membrane dynamics, suggesting that Multimerin-2 may represent an important molecular cue in the crosstalk between these pathways. In line with our findings, YAP/TAZ activation drives abnormal endothelial cell function, promoting vascular permeability, endothelial cell migration, and vessel dysfunction. Importantly, this result was also corroborated through the analyses of the retinal vessels exploiting the Multimerin-2 knockout model. Consistently, vessels developed in ovarian tumors grown in Multimerin-2 knockout animals displayed increased YAP/TAZ activity. Interestingly, and likely due to an altered endothelial cell barrier, these tumors also displayed an altered infiltration of immune cells, in particular CD3 and CD4 T cells. Taken together, these results highlight the importance of Multimerin-2 in regulating vascular homeostasis and its potential in cancer therapies aimed at promoting vascular normalization and restoring the immune response.