Mechanisms Of PKM2/STAT3 Regulated Metastasis In Esophageal Squamous Cell Carcinoma

Objective:To examined the expression patterns of Pyruvate Kinase M2(PKM2)in esophageal squamous cell carcinoma(ESCC)and associations between the positive expression of PKM2 and clinicopathological parameters,prognosis in ESCC patients.To exploer the correlation between the expression of PKM2 and pSTAT3 Tyr705/Ecadherin in ESCC.Meanwhile,To verify the effect of PKM2 on invasion,migration and EMT process in ESCC cells.Furthermore,to understand PKM2 regulates the EMT process,metastasis of ESCC via phosphorylating STAT3 tyr705 as a protein kinase.Methods:(1)PKM2,pSTAT3 Tyr705 and E-cadherin expression was examined in ESCC and paired normal adjacent tissues(NAT)by Immunohistochemistry(IHC).The relationship between PKM2 and clinicopathological parameters(age,sex,differentiation degree,gross classification type,T classification,lymph node metastasis and prognosis)were analyzed.The correlation between PKM2 and pSTAT3 Tyr705/E-cadherin expression were investigated concomitantly.(2)The basic expression of PKM2 was detected in ESCC cell lines by Western-blot.Specific lentiviral with PKM2 knockdown in KYSE150 cells or overexpression in Eca109 cells were constructed.To verify the effect of PKM2 on proliferation,migration,invasion and EMT process of ESCC cells,MTT,Wound healing and Transwell assays were performed after lentivirus transfection.the levels of Snail,E-cadherin and Vimentin were examined by western blot.(3)Following successful knockdown or overexpression of PKM2,protein expression of STAT3 and pSTAT3 Tyr705 were detected by western blot.Furthermore,the variations of proliferation,migration,invasion and the protein levels of Snail,E-cadherin and Vimentin,pSTAT3 Tyr705 in KYSE150-shRNA-PKM2 cells with or without overexpression of STAT3 were detected via MTT,Wound healing,Transwell assays and western blot.(4)Specific lentiviral with PKM2 mutation(R399E,K367M)in KYSE150 cells were constructed.MTT,Wound healing and Transwell assays were performed to detected the variations of proliferation,migration,invasion,Western blot were performed to detected the variations of protein levels of Snail,E-cadherin and Vimentin?(5)Following successful mutation(R399E,K367M)of PKM2,protein expression of STAT3 and pSTAT3 Tyr705 were detected by western blot.Furthermore,the variations of proliferation,migration and invasion in KYSE150-mutated-PKM2 cells with or without knockdown of STAT3 were detected via MTT,Wound healing,Transwell assays,the protein levels of Snail,E-cadherin,Vimentin and pSTAT3 Tyr705 were detected via western blot.(5)4 to 6-week-old BALB/c female nude mice were randomized into groups.PKM2 knockdown,overexpression and mutantion(R399E,K367M)KYSE150 cell lines were used to constructed xenograft model.2�107 cells/0.2m L/mouse was injected to the subcutaneous part of armpits of nude mice.Tumor volumes and body weights were measured every week.The longest diameter(A)and shortest diameter(B)were measured by vernier caliper.Tumor volumes were calculated with the using of the equation:volume=(longest diameterxshortest diameter2)/2.Paraffin embedded tissue samples were fixed and sectioned for HE staining.The expression of PKM2,pSTAT3 Tyr705,E-cadherin in mice tumor were detected by IHC.Results:(1)Compared to the adjacent non-tumor esophagus tissues in which PKM2 was undetectable or expressed at low levels,PKM2 was highly expressed in ESCC tissues(P=0.000).upregulation of PKM2 was significantly correlated with lymph node metastasis(P=0.023)while no significant correlation was observed between PKM2 expression and age,sex,or degree of cell differentiation.Kaplan-Meier survival analyses revealed a significantly shorter median survival time for patients with high PKM2 expression relative to patients with low PKM2 expression(P = 0.0036).In multivariate Cox regression analyses,overall survival time was significantly dependent on PKM2 expression,N classification,and T classification,which may serve as independent prognostic factors for patients with ESCC.PKM2 expression was negatively correlated with that of E-cadherin in clinical tissues of ESCC(P=0.032),high PKM2 expression in tumor tissues frequently coincides with high pSTAT3 Tyr705 expression(P=0.036).(2)PKM2 expressed in all ESCC cell lines(Eca109,ECa9706,TE-1,KYSE150,and T4),PKM2 levels was highest in KYSE150,while lowest in Eca109.knockdown of PKM2 markedly suppressed proliferation,migration and invasion of KYSE150 cells,PKM2 knockdown triggered significant upregulation of E-cadherin and concomitant downregulation of Snail,Vimentin in KYSE150 cells,whereas its overexpression had the opposite effects in Eca109 cells.(3)the pSTAT3 Tyr705/STAT3 ratio was reduced in PKM2-depleted KYSE150 cells and increased in PKM2-overexpressing Eca109 cells,whereas no significant changes in total STAT3 expression were observed.STAT3 was additionally overexpressed via transfection with Lv-overexpress-STAT3 in PKM2-depleted KYSE150 cells.Overexpression of STAT3 suppressed the effects of PKM2 knockdown on pSTAT3 Tyr705,Snail,Vimentin,and E-cadherin expression.Moreover,altered proliferation,migration,and invasion rates caused by PKM2 knockdown were clearly prevented in STAT3-overexpressing cells.(4)mutation(R399E,K367M)of PKM2 markedly promoted proliferation,migration and invasion of KYSE150 cells(P<0.05).meanwhile,PKM2 mutation triggered significant downregulation of E-cadherin and concomitant upregulation of Snail,Vimentin in KYSE150 cells.The effect of R399E was more significant.(5)the pSTAT3 Tyr705/STAT3 ratio was increased after mutation(R399E,K367M)of PKM2 in KYSE150 cells,whereas no significant changes in total STAT3 expression were observed.STAT3 was additionally knocked down via transfection with Lv-shRNA-STAT3 in PKM2-mutatted KYSE150 cells.knockdown of STAT3 suppressed the effects of PKM2 mutation on pSTAT3 Tyr705,Snail,Vimentin,and E-cadherin expression.Moreover,altered proliferation,migration,and invasion rates caused by PKM2 mutation were clearly prevented in STAT3-knockdown cells.The effect of R399E was more significant(P<0.05).(6)The subcutaneous xenografts of human ESCC in nude mice were formed 1 week later after injection.tumor volume was lower in the group of injected with PKM2-shRNA interference cells and was larger in PKM2 overexpression group and mutation group than that of the control group,The effect of R399E was more significant(P<0.05).These results suggested that the expression and mutation of PKM2 in vivo may promote the proliferation of ESCC cells.Expression of PKM2 and pSTAT3 Tyr705 was lower and E-cadherin was higher in the group of injected with PKM2-shRNA interference cell,while expression of PKM2 and pSTAT3 Tyr705 was higher and E-cadherin was lower in the group of injected with PKM2 overexpression and mutant cell.Conclutions:In this study,we showed that PKM2 was remarkably upregulated in ESCC,compared with paired NAT samples.The high level of PKM2 was positively correlated with lymph node metastasis and poor overall prognosis.Furthermore,PKM2 expression was negatively correlated with that of E-cadherin protein and was positively correlated with pSTAT3 Tyr705 in ESCC;PKM2 regulates proliferation,migration,invasion,and EMT via phosphorylation of STAT3 at Tyr705 in ESCC as a protein kinase.