Dottorato in MEDICINA MOLECOLARE

Titolo della tesi: Deciphering the role of AMBRA1 in the control of the Hedgehog signaling pathway and in the tumorigenesis of medulloblastoma

Medulloblastoma (MB) is the most common childhood brain tumor that originates from cerebellar development alterations. Multi-omics analysis has defined four MB molecular subgroups - Wingless (WNT), Sonic-Hedgehog (SHH), Group 3 (G3) and Group 4 (G4) - each with distinct molecular signatures and clinical features. Among them, SHH is the most abundant and genetically characterized. Given its crucial role in tumorigenesis, SHH signaling has emerged as druggable target for MB, offering a valuable opportunity to overcome the pitfalls of the current anti-cancer therapies. Unveiling the molecular mechanisms underlying the control of this important developmental pathway could lead to the identification of new drivers and promising therapeutic strategies for cancer treatment. To address this aim, my research project has been focused on the characterization of novel interactors of RENKCTD11 (here REN), a substrate receptor subunit of the Cullin3-RING ubiquitin ligase complex (CRL3REN) and tumor suppressor lost in ~30% of human SHH-MBs. Starting from the mass spectrometry analysis we identified AMBRA1, a well-known pro-autophagic regulators, as potential interactor of CRL3REN. We demonstrate that CRL3REN binds and induces polyubiquitylation and proteasome-mediated degradation of AMBRA1. Interestingly, we demonstrated that AMBRA1 acts as a positive regulator of the SHH pathway enhancing Gli1 protein stability, the final effector of SHH signalling. We found that AMBRA1 interacts with βTrCP, the substrate receptor of SCFβTrCP ubiquitin ligase known to promote proteolysis of Gli1, and impairs βTrCP-associated Gli1. This event results in the accumulation of Gli1, and the enhancement of SHH activity. Remarkably, genetic depletion of AMBRA1 strongly reduces SHH-dependent MB growth both in vitro and in vivo. In conclusion, results obtained by my Ph.D. project identify AMBRA1 as a previously unknown regulator of the SHH pathway and unveil a novel molecular mechanism responsible of SHH-MB tumorigenesis. Our findings highlight AMBRA1 as a promising therapeutic target for the development of more effective intervention for SHH-dependent tumors care.