The Role Of DKK1 In Vasculogenic Mimicry Formation Of Non-small Cell Lung Cancer

ObjectiveVasculogenic mimicry(VM),a special blood supply mode,has been reported in those tumors that showed more malignant behavior and poor prognosis.Theories of epithelial-mesenchymal transition(EMT)and cancer stem cells(CSC)are believed to contribute to VM formation.Moreover,Wnt signaling pathway is involved in mechanisms of both EMT and C.SC.However,the relationship between DKK1,an important molecule of Wnt signaling pathway,and VM formation in non-small cell lung cancer(NSCLC),which is the first maglignant tumor,has not been reported.This study sought to investigate the role of DKK1 in VM formation of NSCLC and its molecular mechanism.Methods1.Tissue specimens were obtained from 205 patients with NSCLC.HE staining and CD31/PAS double staining were performed to investigate the existence of VM in these 205 NSCLC specimens.SPSS was used to analyze the relationship between VM and clinicopathological parameters.2.Immunohistochemistry was used to detect the expression of DKK1 in NSCLC tissues,and then the relationship between DKK1 and VM and clinicopathological parameters was analyzed.3.The expressions of(3-catenin,EMT-related proteins(E-cadherin,vimentin,Twist,Slug),CSC-related proteins(EpCAM,CD34,CD44,nestin),VM-related proteins(VE-cadherin,MMP2,MMP9)and microvessel density(MVD)marked with CD34 were detected by immunohistochemistry and analyzed with VM and DKK1.4.Western blot was used to choose the NSCLC line which DKK1 expression was high or low.Then stable H460 cell lines with DKK1 overexpression and stable A549 cell lines with DKK1 downexpression were selected and validated by plasmid transfection and western blot.Changes in cell morphology,expression of DKK1,β-catenin,EMT-related proteins,CSC-related proteins,and VM-related proteins were observed after transfection by western blot and immunofluorescence.5.MTT assay,wound healing assay,invasion assay and 3D culture assay were performed to detect the influence of DKK1 on proliferation,migration,invasion and tube structure formation abilities of NSCLC cells.6.Xenograft model of nude mouse by subcutaneous injection with stable transfected cells was made to investigate the effect of DKK1 on tumorigenic abilities of NSCLC cells.Then,the existence of VM and the expressions of DKK1,β-catenin,EMT-associated proteins,CSC-associated proteins,and VM-associated proteins were detected by immunohistochemistry and endomucin/PAS dual staining to identify the effect of DKK1 on VM formation in NSCLC.Results1.The presence of VM was found in NSCLC tissues,it was often found in those specimens with metastasis,poor dif-ferentiation,high stage and poor prognosis(P<0.05);VM was positively correlated with β-catenin,EMT-associated proteins(vimentin,Twist,Slug),CSC-associated proteins(CD34,CD44,nestin),VM-associated proteins(VE-cadherin,MMP2,MMP9),while negatively related with E-cadherin(P<0.05);The relationship between VM and EpCAM was not statistically significant(P>0.05).2.Most of NSCLC tissues showed enhanced expression of DKK1 and DKK1 was often positively expressed in those cases with poor differentiation and poor prognosis(P<0.05);VM was frequently present in NSCLC tissues with positive expression of DKK1(P<0.05);The overexpression of DKK1 was positively related with β-catenin,EMT-associated proteins(vimentin,Twist,Slug),CSC-associated proteins(CD44,nestin),and VM-associated proteins(VE-cadherin,MMP2,MMP9),while negatively correlated with E-cadherin(P<0.05);The relationship between EpCAM,CD34 and DKK1 had not statistical significance(P>0.05).3.DKK1 overexpression could enhance the proliferation,migration,invasion and tube structure formation abilities of H460 cells(P<0.05);DKK1 reduced expression could suppress the proliferation,migration,invasion and tube structure formation abilities of A549 cells(P<0.05).4.The expression of DKK1,β-catenin,EMT-associated proteins(vimentin,Twist,Slug),CSC-associated proteins(CD44,nestin)and VM-associated proteins(VE-cadherin,MMP2,MMP9)were elevated in H460-DKK1 cells,while E-cadherin expression was reduced(P<0.05);A549-shDKKl cells showed downexpression of DKK1,β-catenin,EMT-associated proteins(vimentin,Twist,Slug),CSC-associated proteins(CD44,nestin)and VM-associated proteins(VE-cadherin,MMP2,MMP9)while upexpression of E-cadherin(P<0.05).5.H460-DKK1 transplanted tumor grew more quickly and showed larger than the control’s,it was also showed more often the presence of VM and LPPCN,as well as the increased expression of DKK1,β-catenin,EMT-associated proteins(vimentin,Twist,Slug),CSC-associated proteins(CD44,nestin),VM-associated proteins(VE-cadherin,MMP2,MMP9)and decreased expression of E-cadherin(P<0.05);Compared with the control,A549-shDKK1 transplanted tumor grew more slowly and exhibited smaller,and it was closely associated with the absence of VM and LPPCN,elevated expression of E-cadherin and reduced expression of DKK1(3-catenin,EMT-associated proteins(vimentin,Twist,Slug),CSC-associated proteins(CD44,nestin)and VM-associated proteins(VE-cadherin,MMP2,MMP9)(P<0.05).Conclusions1.VM exists in NSCLC,contributes to progression of NSCLC and indicates unfavorable prognosis.2.DKK1 is overexpressed in NSCLC and may participate in VM formation.3.DKK1 promotes proliferation,migration,invasion and then VM formation of NSCLC cells by stimulating EMT and acquiring CSC.