The Expression Of SGK3 On The Antitumor Effects Of PI3K Inhibitors And Its Molecular Mechanism

AimsTo explore the changes of cell sensitivity to PI3K inhibitor GDC-0941 after downregulation of SGK3 and potential molecular mechanisms in ESCC.Methods1.Effects of GDC-0941 on the proliferation of ESCC cells and expression of PI3K and SGK3Effects of GDC-0941 on the proliferation of ESCC cells and the expression of PI3K and SGK3 were detected by CCK-8 assay and Western blot,respectively.2.Effects of SGK3 on the anti-ESCC activity of GDC-0941(1)EC9706 and ECa109 cell lines were infected with lentiviral vector carrying SGK3-shRNA,cells and monoclonal cells stably expressed LV3-SGK3 were screened using puromycin.Cells and monoclonal cells screened with the highest interfering efficiency detected by Western blot were used to further exploration.(2)Effects of GDC-0941 on proliferation,colony formation,migration and cell cycle phase of ESCC cells with downregulation of SGK3 were investigated by CCK-8,colony formation assay,wounding healing assay and flow cytometry,respectively.3.Molecular mechanism of expression of SGK3 affecting anti-ESCC activity of GDC-0941(1)The expression of relevant proteins of GSK3? and ?-catenin was detected by Western blot after treated with GDC-0941 for 72 h.(2)The expression of relevant proteins of SGK3 and PI3K/Akt/p70S6K was detected by Western blot after screened and un-transfected cells were treated with GDC-0941 for 0 h,3 h,24 h,72 h and 120 h.(3)GDC-0941 alone or combined with MEK inhibitor AS703026 treated EC9706 and ECa109 cells for 72 h,the expression of ERK and Akt/p70S6K-related proteins was detected by Western blot.4.In vivo evaluation and molecular mechanism of the effect of SGK3 on the anti-tumor activity of GDC-0941 in ESCC(1)The monoclonal ECa109-LV3-SGK3 cells were used to establish the xenografts of nude mice,which were treated with GDC-0941 for 15 d.The tumor growth curve was graphed and the tumor suppression rate and relative tumor growth rate were calculated.Cell morphology and apoptosis in tumor tissues were investigated by H&E staining and TUNEL staining.Body weight of nude mice,blood routine parameters,toxicity markers of liver and kidney were investigated and cell morphology of liver and kidney detected by H&E staining to evaluate the potential drug toxicity.(2)Expression of GSK-3? and ?-catenin in tumor tissues was detected by Western blot.Results1.GDC-0941 inhibited the proliferation of ESCC cells by regulating PI3K while activating SGK3(1)GDC-0941 inhibited the proliferation of ESCC cells.The IC50 of GDC-0941 on EC9706 cells for 24 and 48 h was 18.67± 1.27 ?M and 11.05± 1.04 ?M,respectively.The IC50 of GDC-0941 on ECa109 cells for 24 and 48 h was 17.96± 1.25 ?M and 8.07±0.91 ?M,respectively.(2)The expression of PI3K decreased in a time-dependent manner while the expression of p-SGK3 and SGK3 increased after ESCC cells were treated with GDC-0941.2.Downregulation of SGK3 could enhance sensitivity of ESCC cells to GDC-0941(1)LV3-SGK3-1701-EC9706,LV3-SGK3-1077-ECa109 cells and the monoclonal cells with the highest interfering efficiency were succeed screened.(2)Downregulation of SGK3 could enhance the inhibiting effects of GDC-0941 on proliferation,clone formation,migration and retarding effects to G1 phase of GDC-0941to ESCC cells.3.Molecular mechanism of downregulation of SGK3 enhancing sensitivity of ESCC cells to GDC-0941.(1)Down-regulation of SGK3 enhancing inhibition of GDC-0941 to GSK3?/?-catenin pathway.GDC-0941 enhanced the expression of Active-?-catenin and inhibited the expression of p-GSK3?.Downregulation of SGK3 decreased the expression of Active-?-catenin and p-GSK3?,and inhibiting the increase of Active-?-catenin caused by GDC-0941.(2)Down-regulation of SGK3 affecting the regulation of GDC-0941 to Akt/p70S6K pathwayDownregulation of SGK3 inhibited the expression of p-p70S6K.When cells with down-regulated SGK3 were treated with GDC-0941 for a short time,Akt/p70S6K pathway was inhibited,while it was activated after a long-term treatment.(3)Prolonged treatment with GDC-0941 induced the re-activation of Akt/p70S6K by activating ERK.GDC-0941 activated ERK by increasing the phosphorylation of ERK.MEK inhibitor AS703026 eliminated the increase of p-ERK caused by GDC-0941 and enhanced the inhibition of GDC-0941 to p-Akt(Ser473),p-Akt(Thr308),p-p70S6K.The results indicated that inhibition at short time and activation at long time of GDC-0941 to Akt/p70S6K were related with the compensatory activation of ERK.4.Downregulation of SGK3 could enhance the inhibitory effects of GDC-0941 on the tumor growth of ESCC.(1)The tumor growth curve,tumor suppression rate and relative tumor proliferation rate showed that down-regulation of SGK3 could enhance the inhibitory effects of GDC-0941 on ESCC xenografts.H&E staining results showed that down-regulation of SGK3 or GDC-0941 alone caused the changes of apoptosis-related cell morphology like cell and nuclear shrinkage,the apoptosis phenomenon especially obvious in LV3-SGK3+GDC-0941 group.TUNEL results showed that the cell apoptosis rates obviously increased compared to the control group,especially in LV3-SGK3+GDC-0941 group.Nude mice body weight,blood routine parameters,toxicity markers of liver and kidney as well as cell morphology in every group had no obvious differences.(2)The expression of relevant proteins in ESCC xenografts was consistent with that in vitro.GDC-0941 decreased expression of p-GSK3? while increased expression of Active-?-catenin.Downregulation of SGK3 decreased expression of Active-?-catenin and p-GSK3?,thus eliminated the activation of GDC-0941 to Active-?-catenin.ConclusionsDownregulation of SGK3 could enhance sensitivity of ESCC cells to GDC-0941 by regulating SGK3/GSK3?/?-catenin pathway.