The Regulation Of Tumor Cell Autophagy By Glucose Regulated Protein 78 (GRP78)

Autophagy is a crucial survival mechanism for tumor cells in response to microenvironmental stress.Autophagy activation and high expression of GRP78 protein in tumor cells often occur simultaneously.Our previous study found that glucose deprivation induced GRP78 upregulation,which further induced IKK? degradation in an autophagy-lysosome-dependent manner,but the mutual regulation between GRP78 and autophagy was not clear.Thus,we intend to investigate the regulation of autophagy by GRP78 in cancer cells.The main contents of this study are as follows:1.In MCF-7 cells transfected with GFP,GFP-GRP78 expression plasmids,the expression of LC3,p62,VPS34,Beclin1 and ATG5 were detected using qRT-PCR,western blot and immunofluorescence staining,respectively.We found that overexpression of GRP78 induced autophagy and the expression of VPS34.Further,the autophagy flux induced by GRP78 was significantly reversed after autophagy inhibitors of different periods treatment.In turn,knockdown of GRP78 using GRP78 specific shRNA decreased the ratio of LC3-II /LC3-I and the autophagy induced by glucose starvation.2.Knockdown VPS34 by specific siRNAs in GRP78 overexpressing cells resulted in the suppression of autophagy that induced by GRP78,suggesting VPS34 mediated the regulation of GRP78-induced autophagy.GST-Pulldown and co-immunoprecipitation assays showed that there was an interaction between GRP78 and VPS34.Through deletion mutation analysis,it was found that the C terminal 154 amino acids of GRP78 mediated its interaction with VPS34.Analysis of point mutations showed that the K633 Q mutation near the C terminal could enhance GRP78-induced autophagy and the expression of VPS34.In addition,we found that GRP78 up-regulated the expression of VPS34 by inhibiting micro RNA-143.In conclusion,GRP78 can up-regulate the expression of VPS34 and interacts with VPS34 to activate autophagy in cancer cells.3.Interestingly,we also found that overexpression of VPS34 resulted in increased GRP78 expression.Thus we further investigated the regulation of GRP78 by VPS34 both at transcriptional and protein levels.qRT-PCR and western blot assays were used to detect the expression of transcription factors ATF6 and YY1 that regulating GRP78,and it was found that overexpression of VPS34 led to the increase of ATF6 in mRNA and protein levels.In addition,after VPS34 overexpressing cells were treated with protein synthesis inhibitor CHX,protease inhibitor MG132 or autophagic-lysosome inhibitor BAF,we found that VPS34 promoted the stability of GRP78 protein through the ubiquitin proteasome pathway,rather than the autophagy lysosomal pathway.Moreover,co-immunoprecipitation revealed that overexpression of VPS34 resulted in decreased GRP78 ubiquitination.In contrast,VPS34 knockdown reduced the expression of GRP78,accompanied by a decrease in ATF6 and an increase in the expression of miR-143.These results indicate that VPS34 can up-regulate the expression of GRP78 in tumor cells by affecting the transcription and protein stability of GRP78.Our study establishes a positive feedback regulatory loop between GRP78 and VPS34.This feedback regulation between GRP78 and autophagy may be an important mechanism of tumour drug resistance.