Phd in CELL AND DEVELOPMENTAL BIOLOGY

Thesis title: Interaction between M2 muscarinic receptor and β-Arrestin1 in human glioblastoma: implication in cell proliferation and migration.

Glioblastoma multiforme (GBM) is an incurable form of primary brain tumour, associated with a very poor prognosis correlated to its drug resistance capability. The standard therapy is based on surgery followed by radio/chemotherapy with Temozolomide, but more than half of patients do not respond to this treatment. For this reason, from years, in our laboratory we are working on new potential therapeutic target for GBM, demonstrating that the M2 muscarinic receptor subtype (M2R) can negatively modulate cell proliferation and survival in different kind of cancer, including Glioblastoma. In this work, I firstly characterized the drugs-receptor interactions and the dynamics resulting from the selective activation of M2R mediated by two molecules: the orthosteric agonist Arecaidine Propargyl Ester (APE) and the dualsteric Iper-8-Naphthalimide (N8). After assessing the capability of N8 to inhibit GBM cell proliferation upon low-doses treatment, I deeper investigated its biased agonism capability, which allows N8 to activate one pathway in preference to another downstream M2R activation. To do this, I introduced in my project the study of the β-Arrestin1, a protein from the Arrestins family known as a key factor in G-protein-coupled receptors (GPCRs) desensitization and recycling but also in promoting non-canonical signaling pathways G-protein independent. Interestingly, the data show that not only the β-Arrestin1 can exert its role in receptor internalization in our reference model, but it is also essential in mediating intracellular signaling pathway downstream M2R selective activation with dualsteric agonist N8. In particular, the interaction between the M2R and the β-Arrestin1 seems to be crucial in PI3K/Akt and AMPK pathway activation as well as in GBM cell proliferation and migration. Finally, thanks to the collaboration with the University of Artois, I was able to assess the ability of our agonists to cross the Blood-Brain-Tumour Barrier (BBTB) without damaging its integrity.