The Effect Of Wnt/β-catenin Signaling In Vasculogenic Mimicry Formation Of Colon Cancer

Background and ObjectiveIn1999, Maniotis found a unique endothelial-independent microcirculation pattern and assigned it as "vasculogenic mimicry (VM)". The subsequent studies found that VM exists in hepatocellular carcinoma, prostate cancer, ovarian cancer and some bidirectional differentiated malignant tumors. VM is uaually closely associated with tumor gowth, differentiation, invasion, and is an indicator of adverse prognosis. The current opinions regard plasticity of tumor cells as the key mechanism in VM formation. However, the precise molecular mechanisms are still somewhat elusive.Some investigators found that epithelial-mesenchymal transition (EMT) exerts important effect in VM formation. EMT refers to the phenomena that epithelial cells transit to mesenchymal cells during some physical or pathological conditions. EMT is a complicated progress that is precisely regulated by many cellular signaling pathways. Wnt/β-catenin is one of the most important signalings engaged in EMT. However, the effect of Wnt/β-catenin signaling in VM formation is still vague.We sought to investigate the role of Wnt/β-catenin signaling in VM formation. On217colon cancer tissues, we analyzed the relationship between Wnt/β-catenin signaling, EMT and VM formation. By using cell transfection and tumor cell xenograft model, we studied the effect of Wnt/β-catenin signaling inhibition on EMT and VM of colon cancer cells, and further explored the relevant molecular mechanism. Taken together, our results may serve as new molecular targets for therapeutic intervention of the signaling cascade underlying VM.Methods1) H&E staining and PAS-CD34dual staining were used to detect VM existence;2) IHC staining was used to detect the expression of the Wnt/β-catenin signaling regulatory proteins and EMT markers;3) Stable HCT116cell lines with Dkkl overexpression was selected and validated;4) MTT and soft agar clone formation assays detected the influence of Dkkl on proliferation of colon cancer cells;5) Wound healing, adhesion and invision assays were performed to detect the influence of Dkkl on cell movement, adhesion and invasion of colon cancer cells;6)3D culture was uased to detect the influence of Dkkl on tube structure formation;7) EMT, VM and stem-like cell-associated proteins were detected; 8) Subcutaneous mouse xenograft model was made to validate the role of Dkkl on proliferation, invasion and VM formation in vivo.Results1) The presence of VM was found in colon cancer tissue;2) VM-positive samples showed higher expression of nuclear β-catenin and lower expression of Dkkl, showed lower expression of E-cadherin and higher expression of Vimentin;3) The Dkkl-transfected HCT116cells showed increased expression of Dkkl and decreased expression of c-myc and cyclinD1;4) Dkkl overexpression impared proliferation of HCT116cells;5) Dkkl overexpressing HCT116cells showed decresed ability to repair wound gap, increased adhesive ability and decreased invasive ability (P<0.05);6) There was a lack of vascular-like tube formation in the Dkkl-trasfected HCT116cells compare with the control cells(P<0.05);7) The Dkkl-overexpressed HCT116cells showed higher expression of E-cadherin, Keratin18and lower expression of Vimentin, Fibronectin,β-catenin, Snail, Twist, VE-cadherin, Lgr5and CD133;8) In xenograft mouse model, Dkkl inhibited tumor growth, tumor-initiating and VM formation abilities of HCT116cells and increaed the expression of E-cadherin, reduced the expression of Vimentin and P-catenin in tumor tissues.Conclusions1) In colon cancer, VM is associated with activation of Wnt/β-catenin signaling.2) Dkkl inhibits EMT and VM formation of colon cancer cells.