P23 Inhibits Ferroptosis In Lung Cancer By Reversing GPX4 Degradation In CMA Pathway

Objective:p23 is a transcription factor and an important molecular chaperone that is ubiquitous and highly conserved from yeast to humans.It is highly expressed in tumors and promotes tumor growth and drug resistance.Based on the fact that p23 plays an important role in tumors,it may have additional functions that warrant further investigation.In our study,we found that knockdown of p23 in human non-small cell lung cancer cells（A549）resulted in cell death,which was different from apoptosis.The morphological features of cell death were smaller mitochondria,increased membrane density and outer membrane rupture.The cell morphology was highly similar to that of ferroptosis.Therefore,in this thesis,we investigated the molecular mechanism by which p23 regulates ferroptosis in lung cancer cells.Methods:In A549 cells,the effect of p23 gene editing on ferroptosis was detected by electron microscopy,flow cytometry,MDA,Fe²⁺and GSH kits.The ferroptosis-related protein GPX4 was identified by mass spectrometry,and the effect of p23 on GPX4 protein was verified by Western Blot and qPCR.The recombinant expression vectors of p23 and GPX4 were constructed,and the effect of p23 on lung tumors through GPX4 was determined by real-time label-free,xenograft tumor formation in mice,and immunohistochemistry.Protein synthesis inhibitor and autophagy inhibitor were added,and the effect of p23 on GPX4 protein degradation was detected by laser confocal imaging.The effect of p23 on chaperone-mediated autophagy（CMA）was also determined by lysosomal separation and native gel electrophoresis.The interaction between p23 and HSC70 was simulated by molecular docking assay,and verified by co-immunoprecipitation and laser confocal immunofluorescence.In addition,the effect of succinylated p23 on CMA and ferroptosis was detected by double fluorescent protein Western blot.Results:Knockdown of p23 significantly promoted ferroptosis in A549 and H1299 cells.It has been found that p23 can affect the expression of ferroptosis key factor GPX4 at the protein level,and p23 knockdown can inhibit tumor growth by reducing GPX4 protein level.Further mechanistic studies showed that knockdown of p23 promoted the protein degradation of GPX4 by promoting CMA pathway activation.By mass spectrometry and molecular simulation docking,we found that p23 could interact with HSC70,an important molecular chaperone in CMA pathway,and the binding sites were R93,R12and E81 of p23.By binding to HSC70,p23 inhibits the binding of HSC70 to CMA substrate proteins,thereby inhibiting the occurrence of CMA,at which time the protein degradation of GPX4 is inhibited,further inhibiting the occurrence of ferroptosis.In addition,p23 succinylation promotes the binding of p23 to HSC70,which in turn inhibits CMA activation.Conclusions:p23 inhibits the degradation of GPX4 by binding to HSC70 to inhibit the activation of CMA pathway,thereby inhibiting the ferroptosis of lung cancer cells and promoting the occurrence and development of human lung cancer cells.Our study suggests that p23 may be a novel anti-tumor gene target and knockdown of p23 may have potential application in the treatment of lung cancer.