An investigation into the role of mutant p53-dependent extracellular vesicles and their impact on the extracellular matrix

[摘要] Mutant forms of p53 with impaired DNA binding ability – termed mutp53 – promote cancer aggressiveness. As this occurs, in part, through mutp53’s ability to influence membrane trafficking pathways, we have investigated whether mutp53 influences extracellular vesicle (EV) production and/or function, using both cancer cell lines and mouse models of cancer.To understand whether mutp53 promotes the release of pro-invasive factors, we set up a co-culture approach, using cancer isogenic cell lines differing in their p53 status. We found that poorly invasive H1299 cells lacking p53 (H1299-p53-/- cells) displayed increased invasiveness in 3D microenvironments when cultured in the same media as mutp53-expressing H1299 cells. We then used differential centrifugation to collect EVs from H1299 cells of differing p53 status (p53-/- or mutp53-EVs). We characterised these EV preparations using a range of techniques and found that mutp53 expression in cancer cells does not influence the number or size of EV released by these cells. Nevertheless, when used to pre-treat H1299-p53-/- cells, mutp53-EVs promoted migration of recipient cells indicating that they were qualitatively different from p53-/--EVs. Further investigation determined that mutp53-EVs stimulate RCP- and DGKα-dependent recycling in recipient p53-/- cancer cells, and that inhibition of these pathways in recipient cells prevented their migratory response to mutp53-EVs. We then used quantitative proteomic approaches to characterise EVs from H1299 cells and found that mutp53 influences levels of EV-associated podocalyxin, a sialomucin with previous described roles in metastasis. Mutp53 suppressed podocalyxin levels in EVs by repressing cellular levels of podocalyxin, and we demonstrated that this likely occurs via inhibition of mutp53’s binding partner, p63. Furthermore, we showed that levels of EV-associated podocalyxin must be within a certain range to influence the behaviour of recipient cancer cells, as manipulation of EV-associated podocalyxin impaired the ability of EVs in modulating migration and integrin recycling in recipient cancer cells. Although EV release in many cell types occurs via Rab27-dependent mechanisms, we found that loss of this Rab GTPase did not compromise the ability of mutp53-EVs to influence recipient cell behaviour. Instead, we found that another Rab GTPase, Rab35 is required for the production of phenotype-altering EVs, and it does so, not by influencing EV production, but by regulating EV-associated podocalyxin levels. In particular, we found that Rab35 associates with podocalyxin in mutp53-expressing cells which promotes podocalyxin sorting to the cell surface, thereby reducing the level of podocalyxin in EVs to a range that allows non-cell autonomous communication to occur. Fibroblasts are known to be key to the acquisition of pro-invasive tumour microenvironments.We therefore evaluated the effect of EVs on fibroblasts and found that mutp53-EV promote RCP- and DGKα-dependent integrin recycling and, in turn, increased migration of these cells. Integrins are key for the deposition of ECM, and we found that mutp53-EVs encouraged fibroblasts to deposit ECM with altered (more orthogonal) morphology and that this depended on DGKα-dependent integrin recycling.We then found that cancer cells invade more efficiently in this “orthogonal” ECM, and this is likely owing to the reduced adhesiveness of this ECM.To investigate whether mutp53-expressing tumours influence ECM deposition in a non-cell autonomous fashion in vivo, we used quantitative second harmonic generation microscopy and evaluated the organisation of ECM in various organs in autochthonous models of pancreatic cancer. We found that KPC mice, which express mutp53 and develop highly metastatic tumours, display increased ECM orthogonality in the liver and lung prior to metastasis. Furthermore, we used xenograft transplantation models to demonstrate that subcutaneous tumours derived from mutp53-expressing H1299 cells also promote deposition of orthogonal ECM in the lung, and loss of podocalyxin or Rab35 in these tumours impaired their ability to do so. These data suggest that mutp53 expressing tumours promote ECM deposition in pre-metastatic niches and indicate the likelihood of this occurring via EVs and through the regulation of EV-associated podocalyxin levels. Taken together, these data provide evidence for a novel non-cell autonomous gain-of-function of mutp53 that is mediated by EVs and involves modulation of integrin recycling in recipient cells. We propose that this mechanism might contribute to the aggressiveness of mutp53-expressing cancers, by affecting both tumour cell migration and ECM deposition by fibroblasts. Thus, we have discovered an intercellular/inter-organ communication pathway comprising a number of well-characterised components, which might inform for future therapies and may also act as novel biomarkers to indicate the presence of metastatic tumours.

[发布机构] University:University of Glasgow;Department:Institute of Cancer Sciences

[关键词] extracellular matrix, exosomes, extracellular vesicles, cancer, membrane trafficking, cancer cell invasion, pre-metastatic niche.