| Background and ObjectiveColon cancer is one of the most malignancies worldwide. Metastasis and recurrence of colon cancer was considered as the main cause of lethality. As we know, tumor growth and metastasis depend on the angiogenesis to some extent. Vasculogenic mimicry is a novel kind blood supply patterns. VM is one tumor microcirculation pattern which does not rely on endothelial cells. Many studies have reported that VM exists in a variety of malignant tumors such as hepatocellular carcinoma, prostate cancer, ovarian cancer etc. Our previous work has concluded that VM also exists in colon cancer and VM was associated with poor prognosis. The elaborate mechanism of the formation of VM is still not fully understood. Aberrant activation of canonical Wnt/β-catenin signaling pathway has been observed in colon cancer and related with epithelial-mesenchymal transition. Our previous research has found that canonical Wnt/β-catenin signaling pathway plays an important role in VM formation in colon cancer. On the other hand, non-canonical Wnt signaling cascade was considered to be responsible for the development of many malignancies, for example gastric cancer, breast cancer and prostate cancer. Wnt5a is one of non-canonical Wnts family. Wnt5a is a typical representative Wnt protein which signals via non-canonical Wnt pathway. There is evidence indicating that increasing Wnt5a expression is involved in the aggressiveness and initiation of EMT by activating transcription factors in breast cancer and prostate cancer. However, the non-canonical Wnt signaling functions remain obscure in colon cancer. Herein, we select Wnt5a as a representative non-canonical Wnt signaling pathway member and explore the roles of Wnt5a in colon cancer.The aim of this study is exploring the underlying molecular mechanism involving the EMT and formation of VM roles of Wnt5a in colon cancer. We evaluated the Wnt5a expression in a cohort of217colon cancers by using immunohistochemical staining technique. Moreover, combined with the previous results analyzed the relationship between EMT associated protein and VM. We constructed ectopic genetic manipulating Wnt5a HCT116cells model by cells transfection technique and verified the role of Wnt5a in colon cancer cells in vitro. Furthermore, in vivo assay, we also compared with the different roles of overexpression and knock-down Wnt5a expression in transplantation tumor formation in immunodeficiency mice models. Taken together, our work is willing to clarify the potential role of Wnt5a in colon cancer.Methods) Immunohistochemical staining method was used to detect the expression of Wnt5a in217cases of colon cancer tissue samples, and combined with clinical and follow-up data, investigating the relationship between Wnt5a expression and clinical pathological characteristics as well as survival condition. Combined with our previous results, analysed the expression of Wnt5a and EMT related proteins in colon cancer and the relationship with canonical Wnt/β-catenin signaling pathway2) Construction stable Wnt5a gene expression colon cancer HCT116cells through validating the Wnt5a expression respectively.3) Western Blot and immunofluorescence were performed to clarify the effect of Wnt5a in EMT and canonical Wnt/β-catenin signaling pathway in colon cancer.4) MTT assay and soft agar cloning formation experiment were used to detect the role of Wnt5a in cell proliferation. Invasion and motility assay was performed to detect the influence of Wnt5a on cell migration and invasion ability. Adhesion test was used for Wnt5a role in cell adhesion.5) Mouse xenograft experiments were performed to detect the Wnt5a effect on tumor formation and growth in vivo. Immunohistochemical staining was used to examine EMT related protein.6) Levels of intracellular calcium concentration in ectopic Wnt5a HCT116cells were measured by flow cytometry.7) Three-dimension cell culture was used to evaluate the influence of Wnt5a on tube-like structure formation. The elements of angiogenesis factors were also detected by Real-time PCR and Western Blot.Results1) Expression of Wnt5a was significantly diminished in majority of colon cancer. Wnt5a positive expression was associated with good prognosis in colon cancer patients. The expression of Wnt5a was negatively correlated with EMT related proteins in colon cancer. Wnt5a could inhibit the canonical Wnt/β-catenin signaling pathway and suppress the EMT process to some extent. VM positive colon cancers were negatively related with Wnt5a expression.2) Wnt5a was greatly down-regulated in colon cancer cell lines and construction of stable overexpression and knock-down Wnt5a HCT116cell line.3) Wnt5a restoration antagonized canonical Wnt/β-catenin signaling pathway and inhibited EMT in colon cancer cells.4) Wnt5a can obviously inhibit HCT116cell proliferation and clonogenicity. Wnt5a impaired colon cancer cell motility and invasive ability. Wnt5a can enhance cell adhesion ability.5) Wnt5a restoration diminished tumorigenicity in mice, and inhibited EMT related biomarkers in the tumor samples.6) Wnt5a enhanced the intracellular calcium levels in colon cancer cells and activated the Wnt/Ca2+signaling pathway.7) Wnt5a impaired tube-like structure formation ability, and inhibited angiogenesis factors expression in protein levels.ConclusionsOur findings provide that Wnt5a acts as tumor suppressor gene was diminished in majority of colon cancer. Wnt5a was down-regulated in colon cancer, but Wnt5a was associated with good prognosis in colon cancer. Wnt5a antagonized canonical Wnt/β-catenin signaling pathway and EMT process. Interestingly, Wnt5a increased the intracellular calcium and mediated non-canonical Wnt/Ca2+signaling pathway. Through this pathway, Wnt5a inhibited cell proliferation was partly dependent on Bax induced apoptosis and eventually diminished tumorigenicity in vivo. Furthermore, Wnt5a suppressed cell migration and the ability of VM formation. |
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