Getting insights into the multi-faceted role of Notch3 in different tumor contexts

Notch receptor family comprises evolutionary conserved single-pass transmembrane proteins which are involved in several cellular processes during embryogenesis and in adult tissues. Given their pleiotropic effects, their deregulation is associated to the development of several diseases including cancer. Since Notch receptors are differentially implicated in essentially all of the hallmarks of cancer, dissecting every facets of each receptor in different tumor contexts could help to foster effective and specific targeted therapies. In keeping with this consideration, during my PhD project I specifically focused my attention on Notch3 receptor in three different tumor contexts: Triple-Negative Breast Cancer (Project n°1), T-cell Acute Lymphoblastic Leukemia (Project n°2), and Ovarian Cancer (Project n°3). The main objective of this work, performed in the laboratory of Prof. Isabella Screpanti (at the Department of Molecular Medicine of Sapienza University) under the direct supervision of Dr. Saula Checquolo, was to broaden the knowledge of Notch3 receptor, trying to puzzle out its role in cancer, mainly focusing on how it is specifically regulated at the post-translational level, which still represents an unknown field of Notch3 regulation process. Indeed, Project n°1 and Project n°2 report two different crosstalk between Notch3 and other signaling pathways, the EGFR signaling and the Unfolded Protein Response (UPR), respectively. Specifically, on the one hand, we demonstrate that Notch3 regulates EGFR localization in Triple-Negative Breast Cancer, making the receptor unavailable to be targeted by the anti-EGFR agents, such as tyrosine kinase inhibitors, thus highlighting how Notch3 could be crucial in promoting drug-resistance. On the other hand, we document that Notch3 is involved in the activation of pro-survival UPR by directly interacting with an UPR “effector”, thus sustaining cancer cell growth under ER stress conditions. Interestingly, in both tumor contexts Notch3 fulfils its function in a transcriptionindependent manner, paving the way for the study of a mostly untouched aspect of Notch3 function in cancer. Moreover, in Project n°3 we cover a largely unstudied but crucial layer of finetuning and regulation of Notch3: its post-translational modifications (PTMs). To date, little is known about the potential different PTMs of Notch3, mainly regarding the glycosylation of its extracellular domain and the acetylation of its intracellular domain. Here we focus our attention on the study of the phosphorylation status of Notch3 intracellular domain showing how it influences its longevity and who are the actors of this regulation, thus finally fostering a novel therapeutic approach to target Notch3-dependent tumors through the modulations of these specific Notch3 protein regulators. All in all, there are still lots of gaps in the puzzle of “Notch3 world” but some small pieces were falling neatly into place.