ENDOCYTIC CONTROL OF CELL-AUTONOMOUS AND NON-CELL-AUTONOMOUS FUNCTIONS OF P53

NUMB is a multifunctional adaptor protein historically recognized for its role in determining cell fate decisions through inhibition of NOTCH signaling, a process achieved via endocytic trafficking. Subsequently, NUMB has emerged as a significant tumor suppressor, particularly in the context of breast cancer, where it stabilizes the critical tumor suppressor protein p53 by inhibiting its targeting to proteasomal degradation by the E3 ubiquitin ligase MDM2. This role of NUMB is isoform-specific, mediated by alternatively spliced variants containing a short insert encoded by exon 3 (Ex3) – NUMB-1 and NUMB-2. Loss of expression of these NUMB isoforms, but not of isoforms lacking Ex3 (NUMB-3 and NUMB-4), is a frequent alteration in breast cancer that correlates with reduced p53 activity, increased chemoresistance, and poor clinical outcomes. Despite the importance of isoform-specific functions of NUMB to cancer, little is known about their differential biological roles in physiological or pathological contexts. To bridge this gap, we identified the interactomes of the four major mammalian NUMB isoforms using an affinity purification-mass spectrometry approach. Among these interactomes, the endocytic adaptor protein Sorting Nexin 9 (SNX9) was found to be the most abundant specific interactor of NUMB-1 and NUMB-2. We validated this interaction through in vitro binding, GST-pulldown and co-immunoprecipitation assays, which revealed that NUMB-1/2 and SNX9 participate in tripartite complex with p53. We conducted a systematic analysis of the molecular determinants mediating the formation of the NUMB-SNX9-p53 complex by in vitro binding assays with a series of recombinant full-length proteins and domain-specific fragments, alongside in vivo co-immunoprecipitation experiments with wild-type (WT) and mutant versions of the proteins. Our results revealed that formation of the NUMB-SNX9-p53 complex critically depends on the interaction of both NUMB-1/2 and SNX9 with acidic phospholipids (primarily PIP2) enriched in the plasma membrane. The polybasic Ex3-encoded sequence of NUMB-1/2 is critical for mediating this protein-phospholipid interaction, while Ex3-lacking isoforms (NUMB-3/4) are defective in this binding. In SNX9, its PX domain serves as the crucial lipid binding surface, and this interaction likely induces a conformational change unmasking a p53 binding site. Membrane tethered SNX9 and NUMB-1/2 can bind through a bidentate interaction involving distinct binding sites in SNX9’s low complexity (LC) domain and NUMB’s phosphotyrosine-binding (PTB) domain, specifically the PTB-binding groove and Ex3 insert. Through this interaction, SNX9 recruits p53 to the complex, serving as an intermediary scaffold bridging or facilitating the interaction between p53 and NUMB-1/2 at the plasma membrane. The biological role of the NUMB-SNX9-p53 complex is to mediate vesicular trafficking of p53 via the exosome pathway. By characterizing the molecular composition of purified EVs derived from various normal-like and cancer cell lines, in which the complex proteins or exosome biogenesis pathways were inhibited (genetically or pharmacologically), we demonstrated that the sorting of p53 into intraluminal vesicles (ILVs), intermediates in exosome biogenesis, was critically dependent on both SNX9 and NUMB-1/2. The exosomal secretion of p53 has biological consequences for both the donor and recipient cells. In the donor cell, exosomal export of p53 significantly diminishes the intracellular pool under basal conditions, suggesting that this mechanism could function alongside the well-established p53 regulatory mechanisms based on post-translational modifications and protein-protein interactions, to control p53 homeostasis in a cell-autonomous manner. In the recipient cells, p53 delivered by exosomes remains functionally active, capable of mediating classical p53-dependent responses to DNA damage, including activation of transcriptional targets and growth arrest. This finding points to a previously unrecognized role of vesicular p53 trafficking in mediating non-cell-autonomous paracrine-like tumor suppressive signaling to the surrounding microenvironment, with implications for both tissue homeostasis and cancer development. Given that the Ex3-encoded sequence is also critical for mediating NUMB’s interaction with MDM2 and p53 in a tripartite complex that leads p53 stabilization, we explored the relationship between this complex and the NUMB-SNX9-p53 complex in regulating p53. Through in vitro binding assays, size-exclusion chromatography analysis, liposome binding assays, and in vivo immunofluorescence and co-immunoprecipitation assays, we established that the formation of these two Ex3-dependent complexes is mutually exclusive, with MDM2 and SNX9/PIP2 competing for binding to NUMB’s Ex3-encoded sequence. The result of this competition at the cellular level is the formation of two spatially restricted NUMB-p53 complexes that have opposing effects on p53: NUMB-MDM2-p53 stabilizes p53 leading to an increase in intracellular levels while NUMB-SNX9-p53 traffics p53 to exosomal export thereby reducing the intracellular pool. These findings reveal an additional layer of complexity in the regulation of p53’s tumor suppressor function, where endocytic trafficking complements canonical p53 regulatory mechanisms to maintain p53 homeostasis. In summary, this thesis describes for the first time the regulation of WT p53 through vesicular trafficking to the surrounding microenvironment, via a mechanism that is dependent on two endocytic proteins, SNX9 and NUMB. It establishes NUMB-1 and NUMB-2 as central players in both the intracellular stabilization and extracellular dissemination of p53 and define a mechanistic basis for NUMB’s isoform-specific tumor suppressor role. These findings represent a paradigm shift in our understanding of non-cell-autonomous tumor suppression and the maintenance of tissue homeostasis. The deregulation of this process is likely to play a critical role in tumor development and progression, opening new avenues in the development of anti-cancer therapies.