BAG3 Directly Stabilizes Hexokinase 2 MRNA And Promotes Aerobic Glycolysis In PDAC Cells

Objective:In contrast to normal differentiated cells,which rely primarily on mitochondrial oxidative phosphorylation to generate the energy needed for cellular processes,most cancer cells instead rely on aerobic glycolysis,a phenomenon termed“the Warburg effect.”Malignant cells prefer to convert glucose to lactate even in the presence of oxygen to coordinate their macromolecule synthesis demands.It has been widely recognized that reprogramming cellular metabolism is one of characteristic hallmarks of cancer cells and contributes to tumor development.Hexokinase 2(HK2)is the first key enzyme involved in glycolysis,which phosphorylates glucose to 6-phosphate.Most normal mammalian tissues express very little HK2,whereas its expression is highly up-regulated in various types of tumors.Therefore,HK2 plays an important role in reprogramming cellular metabolism of cancer cells.Human BAG(Bcl-2-associated athanogene)proteins form an evolutionarily conserved family of antiapoptotic proteins.BAG3 is a member of BAG family which is constitutively present in a very few cell types,including cardiomyocytes and skeletal muscle cells.A growing body of evidence indicate that BAG3 is overexpressed in several tumor types to sustain cell survival.BAG3 appears to have multiple regulation of major biological processes,that is,apoptosis,oxidative stress and autophagy.Currently,we have found that BAG3 significantly regulates glucose metabolism in many tumor cells.Both transcriptional and posttranscriptional mechanisms are implicated in altering gene expression in cells.Recruitment of protein is implicated in every aspect of RNA life,from biosynthesis to degradation.In eukaryotic cells,the interaction of RNA-binding proteins(RBPs)with their cognate RNAs leads to the formation of ribonucleoprotein particles,thereby regulating multiple posttranscriptional processes.In our previous study,we have found that BAG3 has a definite regulation of pancreatic cancer proliferation and glucose metabolism,and it has been found that the expression of HK2 is down-regulated with the down-regulation of BAG3.BAG3 can bind to many proteins as a chaperone.We found that many classical RNA-binding proteins can interact with BAG3.Given the extensive participation of RNA-binding proteins in post-transcriptional regulation,this study is aim to determining BAG3's regulation on the proliferation and glucose metabolism of pancreatic cancer cells,and also further exploring the mechanism of the complex posttranscriptional regulation of HK2 by BAG3 in pancreatic ductal adenocarcinomas(PDACs).Methods:?.BAG3 expression was evaluated by immunohistochemical analysis in 90pancreatic cancer specimens.?.PDAC cells BxPC3,SW1990 were cultured.BxPC3,SW1990 cell lines that stably overexpressing or knocking down BAG3 were constructed by lentivirus infection.1.Cell counting,EdU and RTCA were used to detect cell proliferation.Nude mice xenograft experiments were used to detect primary tumor growth.2.XF~e Seahorse cellular analyzer was used to detect OCR and ECAR.Assay kits were used to analyze consumption of glucose and production of lactate in medium.3.qRT-PCR measured HK2 mRNA expression,novel HK2 m RNA synthesis and degradation of the HK2 mRNA.HK2 expression was confirmed using Western blot.4.HK2 activity was inhibited using 3-bromopyruvic acid(3-BrPA),RTCA was used to detect cell proliferation.5.Luciferase construct containing HK2 5'UTR,CR and 3'UTR were generated,luciferase activity was measured 2 d after transfection.6.RIP was performed using NCL,GAPDH,AUF1,IMP1,IMP2,IMP3,Roquin and BAG3 antibody,enrichment of HK2 mRNA was measured using qRT-PCR.Luciferase construct containing HK2 3'UTR with core CDE mutation was generated,luciferase activity was measured 2 d after transfection.7.BAG3protein contains three potential RNA-binding domains located at 67-76 aa,261-294 aa,and 473–485 aa,constructs containing BAG3 with the indicated deletion were then generated.HK2 expression was confirmed using qRT-PCR and Western blot.8.Co-IP and Duolink PLA were used to detect interaction of BAG3 and IMP3.9.Roquin and IMP3were knocked down using lentivirus containing shRNA,HK2 mRNA expression was measured using qRT-PCR.?.HK2 expression was evaluated by immunohistochemical analysis in pancreatic cancer specimens.Statistical analyzed the correlation between HK2and BAG3.HK2 expression of the tissues from Bag3 KI mice was detected by Western blot.MEF cells were isolated from a control or Bag3 KI embryo.XF~e Seahorse cellular analyzer was used to detect OCR and ECAR.Results:1.BAG3 expression was significantly increased in most tumor specimens relative to peritumor tissues.2.Knockdown of BAG3 inhibited proliferation of Bx PC3 and SW1990 cells,and also suppressed primary tumor growth in humanized mice.Up-regulation of BAG3 promoted proliferation of Bx PC3 and SW1990 cells.3.Forced overexpression of BAG3 decreased OCR,and increased ECAR in BxPC3 and SW1990cells.Glucose consumption and extracellular lactate secretion were accelerated by forced BAG3 expression in BxPC3 and SW1990 cells.On the contrary,down-regulation of BAG3 decreased glycolysis of both BxPC3 and SW1990 cells.4.BAG3 knockdown significantly decreased HK2 expression in both Bx PC3 and SW1990 cells.No obvious alteration of novel HK2 mRNA synthesis was observed in Bx PC3 and SW1990 cells with BAG3 knockdown.BAG3 knockdown promoted degradation of HK2 mRNA in Bx PC3and SW1990 cells.BAG3 overexpression increased HK2 expression in both BxPC3 and SW1990 cells.5.3-BrPA suppressed cell index in both BxPC3 and SW1990 cells.The suppressive effect of 3-BrPA was compromised in BAG3-knockdown cells,whereas it was enhanced in forced BAG3 expression cells.6.BAG3 knockdown reduced the luciferase activity of construct with insertion of a WT 3'UTR,whereas it did not alter the luciferase activity of the construct with the insertion of a 3'UTR containing a CDE mutation.BAG3directly interacts with 3'UTR of HK2 mRNA via its 67-76 aa and 473-485 aa.7.RIP using Roquin antibody demonstrated that Roquin was recruited to HK2 mRNA in BxPC3 or SW1990 cells with BAG3 knockdown,whereas it was not recruited in control BxPC3 or SW1990 cells.Forced BAG3 expression markedly increased IMP3 recruitment to HK2mRNA in Bx PC3 and SW1990 cells.8.Knockdown of Roquin increased HK2 expression in Bx PC3 cells with BAG3 knockdown.IMP3 knockdown significantly decreased HK2expression in Bx PC3 cells with forced BAG3 expression.9.There were interaction of endogenous BAG3 and IMP3 in BxPC3 and SW1990 cells.10.BAG3 expression was positively correlated with HK2 expression in pancreatic cancer tissues and Bag3 KI mice.11.HK2 expression was increased in MEF cells isolatesd from BAG3 KI embryo,and BAG3 decreased OCR and increased ECAR in MEF cells isolated from Bag3 KI embryo.Conclusion:BAG3 facilitates proliferation of PDACs and promotes reprograming of glucose metabolism by stabilization of HK2 mRNA via competition with Roquin and cooperation with IMP3 to interact with the HK2 transcript.Our results indicate that BAG3might serve as a potential clinic target for cancer treatment.