Dottorato in MEDICINA SPERIMENTALE

Titolo della tesi: “Effects of the tumor microenvironment in glioblastoma: the role of polysialic acid”

Glioblastoma, IDH-wild type (GBM), is a high-incidence malignant brain tumour characterized by pleomorphic cells with high mitotic activity. Hypoxia and nutrient deprivation due to restricted blood supply and inadequate vascularization represent typical features of the GBM microenvironment. These conditions significantly influence tumor biology by impairing signalling pathways that control cell proliferation and represent the main driver of glioma stem cells (GSCs) self-renewal maintenance by preventing cellular senescence. Hypersialylation observed in many tumors consists in an aberrant glycan expression on the surface of cancer cells playing an interface role in the crosstalk between glioma cells and the surrounding microenvironment, thus supporting tumor development and progression. The presence of polysialilated form of Neural Cell Adhesion Molecule (PSA-NCAM) in late life is associated with high-grade tumours characterized by cells with high invasive capacity. For this reason, polysialic acid (PSA) is considered an antigen of oncogenesis. In light of these considerations, the aim of this thesis was to investigate the role of PSA in the aggressiveness of human GBM cells influenced by the hypoxic or nutrient- deprived microenvironment. In the first part of the present work, we have demonstrated the effect of the hypoxic microenvironment in inducing the expression of PSA in GBM cells. In particular, we have shown the presence of PSA in hypoxic areas of GBM tissues and its differential expression among GBM cell lines and patient-derived primary cultures. By growing U87-MG cells under hypoxia we observed an induction of the levels of PSA, which were accompanied by a greater migratory activity that could be prevented by recurring to F-NANA, an analog of sialic acids capable of lowering the expression of PSA, never tested on GBM. These results were also confirmed in a GBM patient-derived primary culture, GL18-15. The involvement of PSA in GBM cell differentiation was then evaluated in U87-MG cells and we observed that hypoxia was able to increase the expression levels of Oct-4, Sox-2 and Nanog stemness genes and that lowering PSA levels by F-NANA administration could interfere with this process. Moreover, we described a role for PSA in maintaining an undifferentiated state of GBM stem-like cells observing an increased differentiation when U87 neurospheres (U87-NS) were treated with F-NANA. Finally, we evaluated the potential in vivo application of F-NANA, which was effective in reducing PSA levels in mouse brains by intranasal administration. Although obtained in WT animals, these findings represent a proof of concept that such a strategy is feasible. In the second part of the present work, we investigated the dynamics of PSA production in GBM cells subjected to nutrient deprivation, a typical feature of the tumor microenvironment (TME). After analyzing the expression profile of the main enzymes involved in the sialic acid pathway in GBM cells, we have observed that serum deprivation induced the up-regulation of the levels of PSA and the enzymes involved in its synthesis, both in U87-MG and U251 GBM cell lines. Cytofluorimetric analysis highlighted that serum starvation preferentially induces cell surface NCAM polysialylation while reducing other proteins sialylation in the tested GBM cells. Then, we investigated autophagy as the main process involved in PSA synthesis in GBM cells under nutrient deprivation. We confirmed the induction of autophagy by observing the reduction of the p62 protein and the increase in the levels of LC3B in U87-MG and U251 exposed to serum starvation. Then, we demonstrated the involvement of this molecular process in the control of PSA turnover by blocking autophagy with NH4Cl. In addition, to further confirm the recycling of other sialic acid in the synthesis of PSA under nutrient deprivation conditions we investigated the involvement of neuraminidases by treating GBM cells with DANA, a sialidase inhibitor. Our results highlight that neuraminidase activity inhibition affects PSA turnover in serum-deprived GBM cells. Finally, the association between PSA expression and autophagic activation was also observed directly on GBM tissues by immunohistochemical analysis, highlighting the pathophysiological relevance of our study. In conclusion, results presented in this thesis show that PSA-NCAM is highly expressed in GBM cells under stress conditions typical of the tumor microenvironment, thus underlying its role in sustaining cancer aggressiveness by maintaining stemness, escaping extreme microenvironments and avoiding immune surveillance. In light of our results, targeting PSA or the enzymes that regulate its synthesis could represent a promising strategy to help limiting the aggressive potential of a tumor such as glioblastoma to develop more effective targeted therapies. Copyright 2021-2025. Sofia Scibetta. All rights reserved