The impact of autophagy regulation by p38α in cell fate determination

Apoptosis and senescence are two mutually exclusive cell fate programs that can be activated as a consequence of persistent stress. Apoptosis results in cell elimination, while senescence is an irreversible cell cycle arrest in a metabolically active state with the possibility to persist long-term. The factors that instruct cells to enter into either senescence or apoptosis are not totally clear, but both programs can be regulated by the stress kinase p38. Using an inducible system to express an active form of MKK6 that specifically activates p38 signalling, we showed that sustained p38 activation triggers autophagosome production with subsequent increase of the autophagic flux in U2OS and Saos-2 cells. In addition, we presented evidence that p38mediated autophagy is mTOR-independent, based on the absence of detectable changes in the phosphorylation of the mTOR target p70 S6K. Since we observed increased p38mediated autophagic flux in p53 null Saos-2 cells, we concluded that p38mediated autophagy is p53 independent. Furthermore, we revealed that sustained activation of p38leads to the production of mitochondrial ROS via MK2, a direct target of p38 We also presented evidence that p38 can phosphorylate ULK1 on the serine 555, which requires that the levels of mitochondrial ROS reach a critical threshold. We confirmed the direct phosphorylation of ULK1 by p38 in vitro. Consistent with the idea that mitochondrial ROS enables p38mediated autophagy, we showed that chemical inhibitors of MK2 rescued the autophagy induced by sustained p38activation. Moreover, we showed that p38activation suffices to induce apoptosis and increase the number of -galactosidase+ cells. We confirmed the enhanced -galactosidase activity correlated with elevated levels of p21 protein and mRNA, as well as with augmented mRNA levels for IL1, IL8 and IL24 cytokines, involved in the senescence associated secretory phenotype (SASP). Interestingly, autophagy inhibition increased cell death-induced by sustained p38activation while decreasing the number of -galactosidase+ cells. Moreover, apoptotic cells showed a lower autophagic flux in comparison to senescent cells. Taken together, these results indicate that increased autophagic flux triggered by specific and sustained activation of the p38 pathway favors that U2OS cells enter the senescent programme instead of undergoing to apoptosis. To verify the implications of our results in pathophysiological conditions, we used doxorubicin and cisplatin, two drugs that are broadly used for cancer chemotherapy and that can induce both senescence and apoptosis. We confirmed the importance of p38 in the regulation of cisplatin-induced autophagy, but could find no evidence that this affects whether cisplatin-treated cells enter into senescence or apoptosis. In contrast, we verified that p38-mediated autophagy controls the switch between apoptosis and senescence in doxorubicin-treated cells. Further work is required to validate the hypothesis that the level of p38-controlled autophagic flux is a key factor for cancer cells to choose their fate under stress conditions. Overall, unveiling the molecular basis for these processes and the cellular scenarios in which they applied would contribute to the development of new strategies for cancer treatment.