The Mechanism Of Stress Granules Regulating Mitochondrial Unfolded Protein Response To Antagonize Dual PI3K/mTOR Inhibitors

Background:Drug resistance in ovarian cancer is a challenge that remains unresolved.It is now believed that in addition to tumor cell genetic mutations,the adaptive stress response induced by chemotherapeutic drugs may also be one of the major causes of drug resistance.Phosphorylated eukaryotic initiation factor 2α（eIF2α）is a key nodal factor in the stress response,maintaining the adaptive stress response and re-establishing proteostasis leading to reduced sensitivity to chemotherapeutic agents.Phosphorylated eIF2α also rapidly inhibits mRNA translation by disrupting cap-dependent translation initiation,resulting in the accumulation of large amounts of untranslated mRNA,RNAbinding proteins and messenger ribonucleoprotein（m RNP）complexes in the cytoplasm to form stress granules（SGs）.SGs are now thought to play an important role in sustaining adaptive stress responses by rapidly regulating intracellular mRNA translation changes and by recruiting signalling molecules.The PI3K/mTOR pathway is often overactivated in ovarian cancer and singletargeted inhibitors are ineffective,suggesting that there is a more complex molecular network mechanism involved in regulating this pathway.After complete inhibition of the mTOR pathway with a PI3K/mTOR dual-targeted inhibitor,the overall translation in the cell was arrested,leading to the formation of SGs and becoming new translation centers,allowing cells to adapt to the new environment.Notably,inhibition of the mTOR pathway also affected mitochondria-related protein translation,causing mitochondrial stress,allowing activating transcription factor 5（ATF5）to enter the nucleus to initiate mitochondrial unfolded protein responses.Therefore,the translocation of ATF5 is a key factor in the activation of mt UPR.Although there is no experimental study on the relationship between ATF5 and SG,it has been found that when Hela cells and H293 cells are subjected to oxidative stress,stress granules are formed near mitochondria.Therefore,we speculate that SGs,as a cellular defense mechanism,may intercept ATF5,resulting in changes in ATF5 localization,activation of adaptive stress responses in cells,and decreased responsiveness to some antitumor drugs.This suggests that it is more important for us to explore the mechanism of action of stress granule molecular targeting drugs from a new perspective.Objective:Our objectives are to analyze the effect of stress granules on the formation of mt UPR,and to clarify the mechanism of stress granules regulating mt UPR to antagonize PI3K/mTOR dual-targeting inhibitors.Methods:1.Ovarian cancer cells A2780 and SKOV3 were treated with different concentrations of PI3K/mTOR dual-targeting inhibitor PKI-402 for 24 h.The sensitivity of both cells to the dual-targeting inhibitor was measured by MTT;Western blot assays were performed to detect the relevant apoptotic proteins.2.After treatment with PKI-402（2.5 μM）for 12 h and 24 h,the differences in the expression of ATF5,the stress-critical protein p-eIF2α in A2780 and SKOV3 cells and verify the inhibitory effect of PI3K/mTOR dual-targeted inhibitors were detected by Western blot.3.After treatment with PKI-402（2.5 μM）for 12 hours,immunofluorescence was performed to detect the co-localization of stress granule-associated proteins YB-1 and G3BP1 and to observe whether stress granules were formed in the cells.4.Treated with PKI-402（2.5μM）for 12 hours,mitochondrial proteins and cytoplasmic proteins were isolated,and mitochondrial unfolded protein responserelated protein expression was detected by Western blot.Mitochondrial function was evaluated by flow cytometry by detecting the mitochondrial membrane potential as well as intracellular reactive oxygen species（ROS）.5.Immunoprecipitation assays were performed after treatment with PKI-402（2.5μM）for 12 hours to detect the binding of ATF5 and G3BP1 proteins;nuclear proteins were separated from cytoplasmic proteins and Western blot was used to detect whether ATF5 entered the nucleus.6.Inhibit the formation of stress granules by adding cycloheximide（5μg/ml）for45 minutes,then treat with different concentrations of PI3K/mTOR dual-targeting inhibitor PKI-402 for 24 hours and again test the sensitivity of the cells to the dualtargeting inhibitor.7.Cycloheximide（5 μg/ml）was added for 45 min,followed by PKI-402（2.5 μM）.Western blot was performed to detect changes in the expression of ATF5,the stress key protein p-eIF2α and downstream proteins of PI3K/mTOR in the cells;mitochondrial proteins and cytoplasmic proteins were isolated and Western blot was performed to detect changes in mitochondrial unfolded proteins The mitochondrial membrane potential and intracellular reactive oxygen species（ROS）were measured by flow cytometry to observe changes in mitochondrial function.Results:1.A2780 cells were insensitive to PI3K/mTOR dual-targeting inhibitor PKI-402 compared to SKOV3 cells,and there was significant anti-apoptotic phenomenon in A2780 cells.2.In the presence of PKI-402,the expression levels of stress-related proteins were all significantly increased in A2780 cells.3.In the presence of PKI-402,stress granules were found to be formed in A2780 cells.4.In the presence of PKI-402,A2780 mitochondrial proteins and cytoplasmic proteins were isolated,mitochondrial unfolded protein responses were activated,mitochondria-associated protease and molecular chaperone expression were increased,and mitochondrial membrane potential was elevated,reactive oxygen species were reduced,and mitochondrial function was enhanced.5.Further studies on the presence of stress granules in A2780 cells revealed that ATF5 interacted with the stress granules marker protein G3BP1;nuclear and cytoplasmic proteins were isolated from A2780 cells,and ATF5 was clearly incorporated into the nucleus.6.The sensitivity of A2780 cells to PI3K/mTOR dual-targeting inhibitors was increased after stress granule formation was inhibited.7.Stress-related protein expression decreased in response to PKI-402 after stress granule inhibition.Further isolation of mitochondrial and cytoplasmic proteins,decreased expression of proteins associated with the mitochondrial unfolded protein response,decreased mitochondrial membrane potential,increased reactive oxygen species and reduced mitochondrial function.Conclusion:1.The insensitivity of A2780 cells to PI3K/mTOR dual-targeted inhibitors is due to the adaptive stress response that occurs in A2780 cells,causing the formation of stress granules,which reshape intracellular protein translation and increase resistance to chemotherapeutic agents.2.The stress granule formation was accompanied by stress on mitochondria within A2780 cells,activation of the mitochondrial unfolded protein response,increased expression levels of proteases and molecular chaperone proteins within mitochondria,increased mitochondrial membrane potential,reduced intracellular ROS and enhanced mitochondrial function.3.The stress granules in A2780 cells activate the mitochondrial unfolded protein response by intercepting ATF5,altering its localization and allowing it to enter the nucleus,thus establishing a signaling exchange network with stress granules as the centre and mitochondria as effectors,which in turn reshapes proteostasis and increases the resistance of A2780 cells to chemotherapeutic drugs.