The Mechanism And Its Function Study Of Loss Of TSC1/TSC2 Complex-mediated Inhibition Of AKT

Objective The tuberous sclerosis complex(TSC)is caused by the activation of mammalian rapamycin target complex 1(m TORC1)due to functional inactivation mutation of two tumor suppressor genes TSC1 or TSC2.And,m TORC1hyper-activation caused by TSC1/TSC2 complex inactivation can feedback inhibition of AKT,which is considered to be one of the main causes of TSC mostly benign tumors.However,the exact mechanism of the negative feedback regulation is still unclear,and the purpose of our study is to further elaborate this mechanism.Methods In the previous study,we found that compared with control cells(Tsc2+/+MEFs),Tsc2-deficient mouse embryonic fibroblasts(Tsc2-/-MEFs)platelet-derived growth factor receptor α(PDGFRɑ)expression reduced by gene chip analysis.it was further confirmed by Western blot and real-time quantitative PCR(q RT-PCR),and can be restored by treated with rapamycin.Then we constructed Tsc2-/-MEFs or Tsc1-/-MEFs with overexpression of PDGFRα and Tsc2+/+ MEFs or Tsc1+/+ MEFs with knockdown of PDGFRα.the cell proliferation and colony formation was examined with CCK-8 and plate cloning assay.And the Tsc2-/-MEFs with overexpression of PDGFRα and control cells were inoculated subcutaneously into nude mice,followed by monitoring for tumor growth.Tumor tissues then subjected to H&E and immunohistochemical staining.The cell lines with overexpression or knockdown of PDGFRα were starved and then treated with serum or PDGF,and subjected to immunoblotting.Tsc2+/+ or MEFs,Tsc2-/-or Tsc1-/-MEFs and Tsc2-/-or Tsc1-/-MEFs treated with rapamycin were subject to WB.And Tsc2 +/+ MEFs,Tsc2-/-MEFs and Tsc2-/-MEFs treated with rapamycin were analyzed by nuclear and cytoplasmic extraction and laser confocal microscopy.The changes of FOXO3aexpression of Tsc2 deletion and rapamycin treatment were detected.Constructed Tsc2-/-MEFs or Tsc1-/-MEFs with overexpression of FOXO3 a,and depletion of FOXO3 a with RNA interference in Tsc2+/+ or Tsc1+/+ MEFs,and WB and q RT-PCR were used to detected the changes of PDGFRα.and p-AKT expression.FOXO3a-binding sites in the promoter of mouse PDGFRɑ gene was predicted on-line and were muted,then the relative luciferase activity was examined by luciferase reporter assay.Tsc2-/-MEFs or Tsc1-/-MEFs were treated with combination of rapamycin and AG1295,and the cell viability was examined with CCK-8 assay.NTC/T2-null cells were inoculated subcutaneously into the nude mice to evaluate the effects of rapamycin and AG1295 in vivo,then the tumor volumes,tumor weights and weights of the mice body were monitored.Results Herein we showed that loss of TSC1 or TSC2 down-regulation of PDGFRαexpression was mediated by activated m TORC1.In addition,ectopic expression of PDGFRα promoted AKT activation and enhanced proliferation and tumorigenic capacity of Tsc1-or Tsc2-null mouse embryonic fibroblasts(MEFs),and vice versa.Moreever,we found m TORC1 is a negative regulator of FOXO3 a,and hyperactivated m TORC1 inhibited PDGFRα expression via suppression of FOXO3a-mediated PDGFRα gene transcription.Interestingly,rapamycin in combination with AG1295,a PDGFR inhibitor,significantly inhibited cell growth of TSC1/TSC2complex-deficient cells in vitro and in vivo.Conclusion Therefore,we conclude that the TSC1/TSC2-m TORC1 pathway negative regulated PDGFRɑ by inhibits the expression of FOXO3 a,the down-regulation of PDGFRɑ inhibits AKT activation and limits the development of TSC tumor.These suggest that downregulated FOXO3a/PDGFRα/AKT pathway exerts a protective effect against hyperactivated m TORC1-induced tumorigenesis caused by loss of TSC1/TSC2 complex.And the combination of rapamycin and AG1295 may be a new effective strategy for TSC treatment.Our study will contribute to elucidating the pathogenesis of TSC and developing TSC treatment.