Hypoxic Tumor Microenvironment Induces Invasive Growth And Angiogenesis In NSCLC Via The Akt-PDK1-YKL40 Pathway

ObjectiveMalignant metastasis and recurrence of lung cancer are difficult problems in clinical treatment.It’s a great significance to explore the inducement and mechanism of systemic metastasis of lung cancer for clinical treatment of lung cancer and improvement of prognosis of patients.Hypoxic microenvironment is an important regulator of tumor proliferation,invasion and metastasis,while tumor neovascularization is the basis.The purposes of this study are to clarify the role of hypoxia-induced Akt2-PDK1 pathway in regulating YKL-40 in lung cancer angiogenesis and to provide new targets and ideas for clinical treatment of malignant lung cancer.Methods1.Strongly invasive lung cancer cell line CL1-5 were cultured in normoxic and hypoxic environment,to compare the enrichment and phosphorylation of Akt2 and PDK1 in mitochondria,the expression and secretion of YKL-40,VEGF,TGF-β and other proteins in mitochondria.2.The lentiviral vectors overexpressing Akt2 sh RNA and YKL-40 were constructed and transfected into CL1-5 cell line to compare the effects of Akt2 interference and / or YKL-40 overexpression on the expression and secretion of PDK1 phosphorylation,YKL-40,VEGF,and TGF-β in CL1-5 cells.Meanwhile,the cell activity,migration.invasion and angiogenesis were detected by CCK8,transwell and tube formation assay.3.The subcutaneous tumors of nude mice were established by the above-mentioned treated cell lines,and the tumor-forming ability of CL1-5 cells with Akt2 interference and /or YKL-40 overexpression was observed.4.The tumor tissues of each group were collected on the 32 nd day.The enrichment and phosphorylation of Akt2 and PDK1 and the expression of YKL-40,VEGF,TGF-βwere detected by immunoblotting.The proliferative activity and angiogenic ability of tumor cells in each group were compared by immunohistochemistry.Results1.Hypoxia treatment of CL1-5 cells significantly increased the phosphorylation level of Akt2 and PDK1 in mitochondria,the expression of p-Smad3,YKL-40 and HIF-1αprotein,and the secretion of TGF-β,YKL-40 and VEGF.2.After CL1-5 cells were transfected with Akt2 sh RNA vector and treated in hypoxia for 24 hours,the expression of Akt2,p-Akt2,YKL-40,p-Smad3,HIF-1α protein,N-cadherin and Vimentin dropped significantly,while the expression of E-cadherin protein increased.After transfection with YKL-40 overexpression vector,the expression of YKL-40,N-cadherin,and Vimentin protein increased,while the expression of E-cadherin protein decreased significantly.There was no significant change in Akt2 and p-Akt2,p-Smad3 and HIF-1α protein.The secretion of cytokines in the supernatant of Akt2 sh RNA stable transformants decreased,and that of TGF-β,YKL-40 and VEGF increased after transfection with YKL-40 overexpression vector.3.Akt2 interference inhibits the proliferation,migration,invasion and tubule formation of CL1-5 cells under hypoxia,while the overexpression of YLK-40 can promote the proliferation,migration,invasion and tubule formation of CL1-5 cells.4.After subcutaneous injection of CL1-5 cells in each group,the tumor volume growth rate of mice treated with Akt2 sh RNA stable strain was significantly lower than that of the control group,while that infected with YKL-40 overexpression virus was significantly increased after injection of Akt2 sh RNA stable strain.On the 32 nd day,the tumor weight of mice after injection of Akt2 sh RNA stable strain was significantly lower than that of the control group,while the tumor weight of mice infected with YKL-40 overexpression virus was significantly increased after injection of Akt2 sh RNA stable strain.5.Compared with Scramble mice,the expression of p-PDK1,HIF-1α,TGF-β,p-Smad3,YKL-40,N-cadherin,vimentin and VEGF protein in Akt2 sh RNA mice tumor was significantly decreased.However,the expression of E-cadherin protein was increased.There was no significant change in Akt2,p-Akt2,p-PDK1,HIF-1α,p-Smad3,PDK1 and Smad3 protein in tumors infected with YKL-40 overexpression virus.The expression of TGF-β,YKL-40,N-cadherin,vimentin,and VEGF protein increased significantly,while the expression of E-cadherin protein dropped.The positive expression rate of Ki67 and v WF,the proliferation of cells,and angiogenesis in Akt2 sh RNA-treated mice were significantly lower than those in control group.Compared with the non-carrier virus infection group,the tumor Ki67 positive expression rate,cell proliferation,v WF positive expression and angiogenesis were significantly increased in the mice infected with YKL-40 overexpression virus.ConclusionThis study found that lung cancer cells increased the expression of YKL-40 through Akt2-PDK1 aggregation and phosphorylation in mitochondria under hypoxic microenvironment,which promoted cancer cell proliferation,invasion,migration and angiogenesis.In this study,interference RNA and overexpression vectors were constructed to verify the existence of Akt2-PDK1-YKL-40 pathway and promote the malignant phenotype of lung cancer by cell and animal experiments.