Functional And Mechanistical Characterization Of LKB1-AMPK Axis Negatively Regulating Ferroptosis

Ferroptosis is a regulated form of cell death characterized by intracellular iron accumulation and severe lipid peroxidation,which is involved in the regulation of various tumors.Erastin activates ferroptosis by inhibiting the the cystine/glutamate antiporter(system Xc~-),leading to the depletion of cellular cysteine and glutathione(GSH),thereby breaking cellular redox homeostasis.Glutathione peroxidase 4(GPX4)is an enzyme required to reduces the toxic lipid ROS,and and its inactivation can trigger ferroptosis without the depletion of cellular cysteine or GSH.AMP-activated protein kinase(AMPK)governs glucose and lipid metabolism in response to alterations in nutrients and intracellular energy levels.Liver kinase B1(LKB1),an upstream kinase of AMPK,activates AMPK via phosphorylation of AMPKαat Thr172.Once activated,AMPK inhibits fatty acid synthesis by inhibitory phosphorylation of acetyl Co A carboxylase 1(ACC1).Deprivation of glucose elicits energy stress due to enhanced production of ROS and impaired antioxidant system,leading to redox imbalance and cell death.In this paper,we treated MEFs and non-small cell lung cancer(NSCLC)cells with ferroptosis inducers(FINs)in glucose deprived DMEM medium and found that FINs failed to induce MEFs or NSCLC cells ferroptosis in glucose free medium.Similarly,treatments with AICAR,A-769662 and 2-DG that induce or mimic energy stress also inhibit ferroptosis in MEFs and NSCLC cells.Under glucose starvation,AMPK plays a critical role in maintaining redox homeostasis and cell survival by regulating fatty acid metabolism and antioxidant response.Energy stress protects cells from ferroptosis by activating AMPK.Therefore,we studied the underlying mechanism(s)of AMPK and its upstream or downstream signaling molecules in regulating ferroptosis.In this study,CRISPR/Cas gene editing technology was used to construct LKB1,AMPK,ACC1 and fatty acid synthase(FAS)knockout cell lines.Propidium iodide staining and flow cytometry confirmed that knockout of LKB1 and AMPK promotes ferroptosis by increasing the accumulation of Lipid ROS after treatment with ferroptosis inducers(FINs).On the contrary,knockout of ACC1 and FAS inhibits ferroptosis by decreasing accumulation of Lipid ROS in MEF cells after treatment with FINs.Western blotting showed that LKB1 promotes the phosphorylation of AMPK and ACC1 during ferroptosis,However,knockdown of AMPK inhibited the phosphorylation of ACC1,indicating that the phosphorylation of ACC1,a downstream molecule of AMPK,plays an important role in the regulation of ferroptosis.Small molecule compounds ACC1 inhibitor(ND-630)and FAS inhibitor(orlistat)demonstrated that phosphorylation of ACC1 inhibits ferroptosis by inactivating ACC1 and inhibiting the activity of FAS.Together,upon induction of ferroptosis,AMPK is activated by upstream kinase LKB1,leading to the inhibi tion of cellular lipid synthesis by inhibitory phosphorylation of ACC1 and other probable substrates that are required for lipid bio-synthesis,which in turn protect cells from lipid ROS accumulation and ferroptosis.Moreover,western blotting confirmed that ferrostatin-1(Fer-1)had no effect on the activation of AMPK induced by FINs,suggesting that ferroptosis can trigger AMPK activation independent of other forms of cell death.LKB1 is an important tumor suppressor,however,its loss-of-function mutations are observed in around 15%-35%of NSCLC cases.Therefore,this study used NSCLC as a research model to examine the role of LKB1-AMPK axis in negatively regulating ferroptosis in the occurrence and development of NSCLC.In this study,we found that LKB1-mutant NSCLC cells are particularly susceptible to cell death induced by FINs.Knockout of LKB1 in LKB1-wild type NSCLC cells promoted ferroptosis,whereas reconstitution of LKB1 in LKB1-mutation NSCLC cells conferred cells resistance to ferroptosis,highlighting the importance of t he LKB1-AMPK axis for cell survival under FINs,which has important implications for the treatment of ferroptosis-associated diseases.Moreover,western blotting confirmed that the LKB1-AMPK signaling pathway inhibits ferroptosis in NSCLC cells by phosphorylation of ACC1,further indicating the universality of the phenomenon that the LKB1-AMPK signaling pathway negatively regulates ferroptosis.In summary,we revealed for the first time that LKB1-AMPK signaling pathway negatively regulates ferroptosis via inhibiting fatty acid synthesis.More importantly,this study demonstrated that NSCLC with LKB1 mutation are more sensitive to ferroptosis,suggesting that LKB1 was an effective therapeutic target for NSCLC,which laid a theoretical foundation for the development of specific drugs targeting tumors with LKB1 mutation.