Numb prevents a complete EMT by modulating Notch signalling

Abstract

Epithelial-Mesenchymal Transition (EMT) plays key roles during embryonic development, wound healing, and cancer metastasis. Cells in a partial EMT or hybrid epithelial/mesenchymal (E/M) phenotype tend to exhibit collective cell migration, forming clusters of circulating tumour cells - the primary drivers of metastasis. Activation of cell-cell signalling pathways such as Notch fosters a partial or complete EMT, yet the mechanisms enabling cluster formation remain poorly understood. Using an integrated computational-experimental approach, we examine the role of Numb - an inhibitor of Notch intercellular signalling - in mediating EMT and clusters formation of hybrid E/M cells. Knockdown of Numb in stable hybrid E/M cells H1975 results in a full EMT, thereby showing that Numb acts as a brake for a full EMT. Consistent with this observation, we show via a mathematical model that Numb inhibits a full EMT by stabilizing a hybrid E/M phenotype. Thus, Numb can behave as a phenotypic stability factor by modulating Notch-driven EMT. By generalizing the mathematical model to a multi-cell level, Numb is predicted to alter the balance of hybrid E/M versus mesenchymal cells in clusters, potentially resulting in a higher tumour-initiation ability. Finally, Numb correlates with a poor survival in multiple independent lung and ovarian cancer datasets, hence confirming its relationship with increased cancer aggressiveness.\n\nMajor Findings: we adopt an integrative computational-experimental approach to identify that Numb, an inhibitor of Notch signalling, can stabilize a hybrid epithelial/mesenchymal (E/M) phenotype. We show that knockdown of Numb in H1975 cells that display a stable hybrid E/M state is sufficient to destabilize a hybrid E/M state and push them to a full EMT phenotype. Next, we develop a mechanism-based mathematical model that recapitulates this ability of Numb in maintaining a hybrid E/M state, and predicts that Numb can alter the relative frequency of hybrid E/M and mesenchymal cells at a tissue level or in clusters of circulating tumor cells (CTCs) - the primary drivers of metastasis. Finally, we show that across cancer types, Numb correlates with worse patient survival, thus reinforcing the emerging notion that a hybrid E/M, but not necessarily a completely mesenchymal, phenotype associates with elevated tumour progression.