Role Of Wnt5a In The Vasculogenic Mimicry Of Ovanan Cancer

Ovarian cancer (OC) is one of the most common causes of cancer-related death in women. Metastasis had occurred in70%of patients when they were diagnosed. The mortality rate of ovarian cancer patients has not improved although diagnosis and treatments have advanced significantly in recent years. Therefore, advances in ovarian cancer treatment must be supported by a deep understanding of its biological features. Vasculogenic Mimicry (VM) describes the ability of aggressive cancer cells to form vasculogenic-like networks in vivo. These networks lack endothelial cells or fibroblasts, and this lack is concomitant with the expression of vascular cell-associated molecules. The prognosis of patients with VM is significantly worse than that of patients without VM. EMT (Epithelial Mesenchymal Transition) is pivotal in malignant tumor progression and VM formation. However, the molecular mechanism of VM remains unclear. Wnt5a is an important noncanonical Wnt pathway that plays key role in VM and EMT. Wnt5a may active the canonical Wnt/β-catenin pathway and noncanonical Wnt pathway. So Wnt5a plays different roles in tumor. Investigating the role and mechanism of Wnt5a in VM and EMT will contribute the theory of microcirculation of tumor.ObjectiveThe study aims to investigate the function of Wnt5a in VM and EMT and analyze its mechanism in ovarian cancer. Exploring the mechanism of Wnt5a in VM and EMT will enhance our understanding of cancer biology and may identify new therapeutic approaches.Methods1. Immunohistochemistry was used to evaluate the expression of Wnt5a, PKCa and CD44in79ovarian cancer cases through Tianjin Cancer Hospital. SPSS was used to analyze the relationship between Wnt5a and clinicopathologic ovarian cancer characteristics. And we analyze the relationship between Wnt5a and VM, Wnt5a and PKCa, Wnt5a and CD44.2. We established a stablely transfected cell lines overexpression or knock-down Wnt5a expression according to the basical expression of ovarian cancer cell lines.3. A well-established model of Three-dimensional Matrigel culture in vitro was used to test the change of vasculogenic mimicry formation affer overexpression or knock-down the Wnt5a expression of ovarian cancer cells.4. The effect of Wnt5a on the motility and invasion of ovarian cancer cells in vitro was investigated. Cell migration was evaluated by wound healing assay, also known as the "scratch" assay, and cell invasion was examined by transwell assay.5. Immunofluorescence microscopy staining was performed to confirm the effect of Wnt5a on EMT assoctiated proteins.6. The expression levels of Wnt5a, PKCa, Snail, P13K, MMP-2,β-catenin were assessed using Western Blotting.Results1. Wnt5a expression was significantly associated with VM in ovarian cancer and its clinicopathologic characteristics.Of the79ovarian cancer cases in this study,25(31.6%) exhibited positive Wnt5a expression and54(68.4%) exhibited negative Wnt5a expression. The result showed that Wnt5a expression was significant relationship with cancer metastasis (P<0.05). The expression of Wnt5a was shown to be significantly correlated with VM (P<0.01). The survival time of79ovarian cancer patients was analyzed based on Wnt5a expression, and Wnt5a-positive patients were revealed to survive longer than Wnt5a-negative patients.2. Wnt5a expression was significantly associated with PKCa in ovarian cancer. Of the79ovarian cancer cases,35(44.3%) exhibited positive PKCa expression and44(55.6%) exhibited negative expression. The Wnt5a staining was found to be significantly correlated with PKCa (P<0.01).3. CD44expression was significantly associated with VM in ovarian cancer. Of the79ovarian cancer cases,29(36.7%) exhibited positive CD44expression and50(63.3%) exhibited negative expression. The Wnt5a staining was found to be significantly correlated with CD44(P<0.00).4. Wnt5a enhanced the vasculogenic capacity of ovarian cancer cells in vitro. Ovcar3, a poorly differentiated ovarian cancer line, displayed higher vasculogenic capacity than Skov3, a well-differentiated cell line. However, Ovcar3transfected with Wnt5a shRNA displayed low vasculogenic capacity, and Skov3transfected with Wnt5a cDNA exhibited high vasculogenic capacity.5. Wnt5a enhanced EMT in ovarian cancer cells in vitro.Immunofluorescence and western blot were performed to investigate the changes in the expressions of these two proteins after in vitro Wnt5a transfection. The result revealed that vimentin expression increased and E-cadherin expression decreased in Skov3after Wnt5a upregulation. By contrast, vimentin expression decreased and E-cadherin expression increased in Ovcar3after shWnt5a transfection.6. Wnt5a increased the motility and invasion of ovarian cancer cells in vitro.The results showed that Wnt5a overexpression enhanced cell migration and invasion in Skov3cells with pWnt5a (P<0.05). These findings corresponded with the weakened motility and invasion in Ovcar3cells with shWnt5a as a result of Wnt5a downregulation (P<0.05).7. Wnt5a promoted ovarian cancer via the PKCa pathway in vitro.The effect of Wnt5a expression on PKCa was investigated via western blot analysis. The results showed that changes in Wnt5a expression impaired changes in PKCa expression. The addition of PKCa inhibitor (1μM) to Skov3-pWnt5a cells significantly reduced cell motility. This result indicated that PKCa is critical to the effect of Wnt5a on ovarian cancer cells in vitro. Finally, Wnt5a and PKCa mechanisms were determined by investigating the changes in the two ovarian cancer cell lines at the protein level and analyzing the proteins associated with VM, EMT, and ECM remodeling. The findings showed that PI3K, Snail, and MMP2expression increased with Wnt5a upregulation. However, β-catenin was not affected.Conclusions1. Wnt5a expression was correlated with metastasis in ovarian cancer and was associated with poor prognosis.2. Wnt5a promoted EMT and VM in ovarian cancer, via PKCa pathway but not the (3-catenin pathway. Snail, PI3K, MMP-2play important roles in this way. 3. Wnt5a increased the metastasis and invasion of ovarian cancer cells in vitro.4. Wn5a was correlated with CD44expression in ovarian cancer, indicating that Wnt5a may be involved in cancer stem like phenotype.