Introduction
It has been well established that a tug-of-war exists between the tumor suppression barrier senescence and the tumor promoting Wnt signaling pathway. However, the implications of this complex feedback loop in the context of small extracellular vesicles (sEV) biogenesis and secretion are unclear.
Methods
sEVs were isolated using differential centrifugation coupled with ultracentrifugation and characterised using nanoparticle tracking analysis, transmission electron microscopy and western blotting analysis. To investigate the role of Wnt signaling and senescence in sEV secretion, CRISPR/Cas9-based knockout, luciferase assay, quantitative proteomic analysis, co-localization assays, immunofluorescence, and RNAi-based knockdown and cellular survivability assays were performed using isogenic cells that are both addicted and non-addicted to Wnt.
Results
CRISPR/Cas9-based knockout of β-catenin attenuated Wnt signalling activity, reduced proliferation and induced senescence via AKT signalling pathway in colorectal cancer cells. Induction of senescence increased the secretion of sEVs and altered their protein cargo. Confocal microscopy, proteomic analysis and Western blotting highlighted the perturbation of several EV regulators including CD63, Rab27a and Cortactin upon induction of senescence. RNAi-based knockdown of multiple regulators rescued the secretion of sEVs. Utility of P53 and P21 knockout and exogenous expression of P21 or P16 highlighted the role of senescence in sEV biogenesis and secretion in Wnt addicted cells.
Conclusion
Overall, this study, for the first time, reveals the mechanism by which senescent cells regulate the increased secretion of sEVs in Wnt regulated manner.