Cellular Senescence Induced by Oncogenic H-Ras Prompts Exosome Release

Cell proliferation induced by oncogene activation is restrained by cellular senescence, which act as barriers in pre-neoplastic lesions. Both the p53/p21 and p16/Rb pathways have been implicated in normal cells protection against neoplastic transformation in response to the expression of activated oncogenes such as RAS. Exosomes, 30 - 100 nm extracellular vesicles derived from the endosomal system, have been recently implicated in cell-to-cell communication in a variety of biological processes; in particular exosomes secreted by senescent cells have been hypothesized to transfer senescence signals to surrounding cells. We investigated the role of exosomes in an experimental model represented by human fibroblasts transfected with H-RasV12, in order to unravel their role in the induction of senescent vs tumoral phenotypes. In our cell model, we observed cell growth arrest followed by cell death, induced by autophagy and apoptosis. We purified exosomes from cell medium by ultracentrifugation and precipitation, and observed that in H-RasV12 expressing cells oncogene-induced senescence is associated with a significantly enhanced release of exosome-like microvesicles. In addition, immunoblotting analysis demonstrated that proteins involved in exosome biogenesis, such as Alix, or in exosome interaction with recipient cells, like the tetraspanins CD9 and CD63, are notably enriched not only in microvesicles secreted by senescent cells but also in H-RasV12 expressing cells. These data strongly suggest that H-RasV12 induces an increase in the biogenesis of exosome-like vesicles and affects their composition. These changes could in turn alter exosome-mediated senescence signals to surrounding cells. It remains to be elucidated whether this modification of intercellular signals could prevent or prompt tumorigenesis, in particular affecting tumor niche formation, and the role of the p53/p21 and p16/Rb pathways in this context.