The Molecular Mechanism Of Sunitinib Induced-autophagic Degradation Of P53

Objectives:Our previous study found that WT p53 get degraded through autophagy-lysosome pathway upon sunitnib stimulation.p53 is an important transcription factor that participates in many signaling pathways including apoptosis,cell cycle arrest and tumor cell metastasis.So far,p53 level is critically controlled by E3 ubiquitin ligases which promote p53 degradation through the ubiquitination-26S proteasome pathway in a normal cell.Recent studies showed that mutant p53 can be degraded through chaperone-mediated autophagy(CMA).Yet,to date,it has not been established whether the autophagy-lysosome pathway plays a role in degradation of WT p53.Based on this,we further confirmed that WT p53 get degraded through autophagy-lysosome pathway upon sunitnib stimulation and discover the key protein participates in this events.We aimed to find new regulation mode of p53 expression and supply a original strategy to increase the stability of p53.Methods:1.Effects of sunitinib on p53 stability(1)Western blot,Real-Time PCR and Immunofluorescence were applied to detect the effects of sunitinib on p53 protein stability;(2)primary colorectal cancer cells were acquired and Western blot was applied to detect the effects of sunitinib on p53 protein stability2.Effects of autophagy-lysosome pathway on sunitinib induced p53 degradationWestern blot was used to detect the effects of autophagy-lysosome and ubiquitin-proteasme pathway on sunitinib induced p53 degradation;(2)Atg5-/-or Atg7-/-MEF cells were treated with sunitinib and p53 protein was detected using Western blot;(3)Immunoprecipitation assay was applied to inspect the interation between p53 and p62,the effects of sunitinib on p53 aggregation formation.3.Effects of nuclear export signals of p53 on sunitinib induced p53 degradation(1)Subcellular distribution of p53 was detected using Western blot and Immunofluorescence assay;(2)lipofectin transfection was used to overexpression the p53-NES-Mut and WT plasmid into p53-/-cells and Western blot and Immunofluorescence assay were used to detect the sunitinib induced subcellular distribution and degradation of p53.4.Effects of HMGB1 on sunitinib induced degradation and nuclear export of p53(1)shRNA or CRISPR/Cas9 technology were applied to knockout of HMGB1 protein and then p53 was detected using Western blot and Immunofluorescence assay;(2)lipofectin transfection was used to overexpress the HGMB1 plasmid and effects of HMGB1 overexpression on sunitinib induced p53 degradation were inspected by Western blot;(3)Immunoprecipitation assay was applied to inspect the interation between p53 and HMGB1;(4)lipofectin transfection was used to overexpress the WT and NES-Mut HMGB1 plasmids into HMGB1-/-cells and Western blot and Immunofluorescence assay were applied to detect the effects of HMGB1 NES on sunitinib induced degradation of p53.5.HMGB1 mediated autophagy dependent degradation of p53 antagonized the antitumor activity of sunitinib and promoted the prometastasis effects of sunitinib(1)SRB method was used to detect anti-proliferation activity of sunitinib plus autophagy inhibitor,and HMGB1 knockdown respectively;(2)the nude mice xenograft model was introduced to detect the effects of HMGB1 knockdown on anti-tumor activity of sunitinib in vivo;(3)Western blot was applied to inspect the effects of sunitinib on tumor metastasis markers like Fibronectin and E-cadherin.Results:1.Sunitinib promoted autophagy dependent degradation of p53(1)We demonstrated that sunitinib apparently activated autophagy and promoted degradation of p53 in a time and dose dependent manner;(2)we confirmed that sunitinib induced degradation of p53 was autophagy dependent;(3)we found that sunitinib facilitated the aggregation of p53 and promoted the interaction between p53 and p62;2.Sunitinib induced p53 nuclear export and degradation in a p53 NES independent manner(1)we found that p53 was translocated to cytoplasm after sunitinib treatment;(2)We transfected p53 NES-Mut plasmid into p53-/-cells and demonstrated that sunitinib induced p53 nuclear export and degradation was p53 NES independent.3.HMGB1 nuclear export defines p53 nuclear export and autophagic degradation(1)We found that HMGB1 was translocated to cytoplasm after sunitinib treatment;(2)we demonstrated that sunitinib promoted the interaction between HMGBland p53;(3)We confirmed that sunitinib induced p53 nuclear export and degradation could be reversed by HMGB1 silence or knockout,while HMGB1 overexpression could enhance such effects;(4)we transfected HMGB1 NES-Mut plasmid into HMGB1-/=cells and demonstrated that sunitinib induced p53 degradation was HMGB1 NES dependent.Conclusions:1.Sunitinib promoted autophagy-lysosome pathway dependent degradation of p53.2.HMGB1 defines p53 nuclear export and degradation.3.HMGB1-induced p53 autophagy-dependent degradation reduced the antitumor activity and promoted the metastasis of sunitinib,and HMGB1-loss rescued the proapoptosis of p53 and inhibited prometastasis potential of sunitinib.