The Function Of PFKFB3 And The Molecular Mechanism Of Its Upregulation By MTOR

Objective: Mammalian target of rapamycin(mTOR)plays an important role in cell proliferation,growth,metabolism,protein synthesis and angiogenesis.Abnormal activation of mTOR is very common in tumors and has become the focus of attention.Tumor suppressor gene phosphatase and tensin homolog(PTEN),Tuberous sclerosis complex gene 1(TSC1)and Tuberous sclerosis complex gene 2(TSC2)mutation inactivation or deletion;oncogene phosphatidylinositol 3-kinase(PI3K),Protein kinase B(AKT)mutation activation,can lead to the activation of mTOR and promote tumor development.However,the specific mechanism by which mTOR causes tumorigenes remains ill-defined.Methods: Studies were carried out by using mouse embryonic fibroblasts(MEFs),including PTEN-deficient mouse embryonic fibroblasts(Phosphatase and tensin homologue null MEFs,Pten-/-MEFs)and their control cells(Pten+/+ MEFs)and TSC2-deficient mouse embryonic fibroblasts(Tuberous sclerosis complex 2 null MEFs,Tsc2-/-MEFs)and their control cells(Tsc2+/+MEFs).mRNA changes in PFKFB3 in mTOR-activated MEFs were analyzed by gene chip analysis.Western blot(WB)was used to detect changes in PFKFB3 protein levels in mTOR-activated cells.The transcriptional binding site on PFKFB3 was predicted on the JASPAR website to explore the specific mechanism by which mTOR regulates PFKFB3,and the transcriptional binding site of HIF-1? was found on the PFKFB3 promoter.Inhibits HIF-1? expression by siRNA interference assay,and observe changes of PFKFB3 by WB.The anaerobic simulated hypoxia was used to observe the change of PFKFB3 with HIF-1?,and the relationship between HIF-1? and PFKFB3 was further explored.We performed a quantitative simulation of hypoxia and observed PFKFB3 expression changed by HIF-1?.In order to confirm HIF-1? regulates PFKFB3 at the transcriptional level,we performed luciferase reporter assay.Finally,we investigated the function of PFKFB3 in mTOR-activated cells.Cell proliferation,metastasis and tumorigenicity were measured by knocking down PFKFB3 by Lentivirus and adding its inhibitor PFK15 into cells.PFKFB3 inhibitor PFK15 and the mTOR inhibitor rapamycin were used in combination to observe the difference in efficacy.Results: By gene chip analysis,we found that PFKFB3 was the highest expressed in four isoforms(PFKFB1-4)of mTOR-activated cells,and the expression of PFKFB3 was significantly increased compared with control cells(P<0.05).siRNA interference and WB experiments confirmed that HIF-1? positively regulates PFKFB3.We confirmed that HIF-1? binds to the transcriptional binding site on the PFKFB3 promoter which promotes transcription of PFKFB3 by luciferase.From these we conclude that mTOR is upregulated by PFKFB3 via HIF-1?.Through in vitro experiments,we found that the ability to inhibit PFKFB3 activity,cell proliferation,metastasis,and colony formation was significantly reduced.In vitro experiment has shown that inhibition of PFKFB3 can attenuate tumor tumorigenic ability.In vivo experiment has shown that inhibition of PFKFB3 can attenuate tumor tumorigenic ability.Using combined PFK15 and rapamycin to detect the proliferative capacity of mTOR-activated tumor cells and clone formation,it was found that the combination has a better inhibitory effect than drug alone.Conclusion: mTOR upregulated the expression of PFKFB3 by HIF-1?.Knock-down of PFKFB3 can attenuate cell proliferation,metastasis,and tumorigenic ability.And the combination of PFK15 and rapamycin in the treatment of mTOR-activated tumors may be a more effective treatment option.