P62/SQSTM1 Mediates The Autophagy-Lysosome Degradation Of CDK2 Protein Undergoing PI3Kα/AKT^T308 Inhibition

Objective:The excessive activation of CDK2 in cell cycle could cause constant proliferate in many tumors.Therefore,exploring the regulation of CDK2 molecule is helpful to understand the nature of disease.In this study,we want to confirm the role of serum starvation in the downregulation of CDK2 protein and evaluate whether its downregulation requires the involvement of PI3K/AKT pathway,and explore the molecular mechanism of the autophagic degradation of CDK2 protein.Methods:1.The cell cycle was detected by flow cytometry in serum starvation-treated cells.Western Blot analysis was used to detect the protein levels of Pi-RB and CDK2 in serum starvation-treated neuroblastoma cells and colorectal cancer cells.The CDK2 of mRNA levels in serum starved cells were detected by Quantitative Real-Time PCR.2.Western Blot analysis was performed to detect the levels of PI3K/AKT signaling pathway,the expression of CDK2 in PIK3CA mutant colorectal cancer cells and PI3K inhibitor,AKT inhibitor,mTOR inhibitor,protein synthesis inhibitor CHX in neuroblastoma cells.3.Western Blot analysis was performed to detect the expression of CDK2 protein in serum starved cell treated with protease inhibitor,autophagy inhibitor and lysosome inhibitor.The expression of autophagy regulatory protein P62/SQSTM1 in cells treated with serum starvation was detected by Western Blot.The stable NB cells of knock out autophagy genes ATG7 and Beclin1 were constructed.The expression levels of CDK2,P62/SQSTM1 were detected in stable NB cells treated with serum starvation,PI3K inhibitor or AKT inhibitor.The interaction of P62 with CDK2 protein was detected by Co-immunoprecipitation.After transfection of P62,Cathepsin D,Cathepsin B siRNA,CDK2 protein was detected in serum starved neuroblastoma cells.Immunofluorescence assay was used to detect the subcellular localization of CDK2protein in serum starved neuroblastoma cells.Results:1.Serum starvation induced G0/G1 phase arrest with the downregulation of CDK2 protein.2.The repression of PI3Kα/AKT ^T308 involved in the downregulation of CDK2 protein.3.Autophagy lysosome inhibitors significantly restored the serum starvation-induced the downregulation of CDK2 protein.Blocking autophagy-related genes contributed to remarkable rescue of CDK2protein.Additionly,the inhibition of PI3K/AKT pathway induced the combination of P62/SQSTM1 with CDK2 protein into lysosomes,which subsequently degraded CDK2 protein in a Cathepsin B-dependent manner.Conclusions:P62/SQSTM1 mediated the autophagy-lysosome degradation of CDK2 protein in a Cathepsin B-dependent manner undergoing PI3Kα/AKT ^T308inhibition.This led to G0/G1 phase arrest of tumor cells.