DKK1Promotes Hepatocellular Carcinoma Cell Migration And Invasion Throughβ-catenin/MMP7Signaling Pathway

Background:Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed cancers, ranking as the third leading cause of all cancer-related death in the world. Despite some advances in early detection and the use of modern surgical techniques in combination with various treatment modalities, such as radiotherapy and chemotherapy, to date, the prognosis of the patients with HCC still remains poor. Recently several reports have indicated that elevated expression of DKK1is tightly associated with the progression of HCC. However, the biological function of DKK1in HCC has not yet been well documented.Objective:In this study, we will examine the expression of DKK1in both HCC cell lines and clinical samples, and try to determine the potential role of DKK1in HCC.Methods:RT-PCR and Western blotting analysis were used to examine the mRNA and protein level of DKK1in both normal liver cell line (QSG-7701) and HCC cell lines (Hep G2, SMMC-7721, HuH-7and BEL-7402). Immunohistochemical staining was used to detect the expression of DKK1and β-catenin in HCC tissues. The role of DKK1in tumor cell proliferation was performed using MTT and colony formation assay; cell migration and invasion were investigated using wound scratch and transwell assays. In order to determine the biological function of β-catenin in DKKl-mediated migration and invasion of HCC cells, GSK3β inhibitor LiCl and β-catenin inhibitor IWP-2were used. RT-PCR and Western Blotting analysis were also used to detect the mRNA and protein level of β-catenin, MMP7, LRP6. In addition, the protein level of β-catenin, MMP7, LRP6, GSK3β and c-Myc was detected using Western blotting analysis in HCC cells.Results:1. We observed that the mRNA and protein level of DKK1in HuH-7, SMMC-7721, and BEL-7402cell lines were much higher in comparison with that in control normal QSG-7701hepatocytes. Of note, HCC BEL-7402cells exhibited the highest level of DKK1in both mRNA and protein levels. In contrast, the level of DKK1mRNA and protein in Hep G2cells was comparable to that in the normal liver cell line (QSG-7701).2. When DKK1was up-regulated or down-regulated, no change of proliferation rates occurred compared with that without transfection or transfected with Vector. Using colony formation assay, although DKK1-transfectants displayed a tendency of increased colonies, while shDKKl-transfectants showed a reduced tendency compared with their control cells respectively, the difference did not reach statistical significance.3. Enforced expression of DKKl significantly promoted wound closure compared with that without transfection or transfected with Vector in Hep G2cells, vice versa, down-regulation of DKK1remarkably attenuated the wound closure. By using transwell assay, we found that tumor cells derived from DKK1-transfectants displayed a higher ability of migration and invasion, compared with that derived from Vector or nontransfected cells. In contrast, tumor cells derived from shDKKl-transfectants displayed a lower ability of migration and invasion. Consistently, in the human HCC tissues, the primary tumors showed slightly higher DKK1expression, compared to that in the adjacent non-tumor liver tissues, moreover, the metastatic tumors showed much higher DKK1expression in comparison with that in,the primary tumors.4. By using Immunohistochemical staining in71cases of human HCC tissues, our results showed that the DKK1level was positively correlated with β-catenin expression. Consistently, introduction of DKK1did not inhibit β-catenin expression, but remarkably increased the mRNA and protein level of β-catenin in Hep G2cells, compared with those of control cells. In addition, by using immunofluorescence staining, we also observed that up-regulation of DKK1expression noticeably promoted the cytoplasmic/nuclear β-catenin accumulation in Hep G2cells. Furthermore, dual-luciferase reporter analysis also showed that restoration of DKK1significantly promoted the activity of firefly luciferase that carried wildtype but not mutant TCF binding sites. In contrast, the blockage of DKK1dramatically attenuated the mRNA and protein level of P-catenin in BEL-7402cells. Moreover, the activity of firefly luciferase that carried wildtype but not mutant TCF binding sites was dramatically attenuated when DKK1was silenced in BEL-7402cells.5. Upon treatment with β-catenin inhibitor IWP-2, the promotive effect of DKK1on migration and invasion was significantly attenuated in DKK1-transfectants, whereas there were no statistically significant differences in those of Vector-transfectants. On the other hand, overexpression of β-catenin in shDKKl-transfectants restored the transcription of β-catenin, and attenuated the inhibitory effect of shDKK1on invasion. Upon treatment with GSK3β inhibitor LiCl, the inhibitory effect of shDKKl on migration and invasion was dramatically released.6. Neither the extracellular domain nor the intracellular domain of LRP6were changed, although the expression of β-catenin was dramatically altered upon DKK1up-regulation or knockdown. In addition, Western blotting analysis also displayed that the protein level of LRP6and GSK3β was not changed in comparison with those of control groups. Whereas restoration of DKK1significantly enhanced the expression of β-catenin, and the protein level of its downstream target c-Myc was also increased in Hep G2cells. Vice versa, the blockage of DKK1reduced the expression of β-catenin and c-Myc in BEL-7402cells. Furthermore, upon β-catenin inhibitor IWP-2treatment, the expression of β-catenin was attenuated, which is consistent with the above observation on migration and invasion, compared with the control. On the other hand, GSK3β inhibitor LiCl indeed recovered the expression of β-catenin in shDKKl-transfectants.7. Introduction of DKK1significantly enhanced the mRNA and protein levels of MMP7in Hep G2cells. Vice versa, when DKK1was silenced by shRNA, the expression of MMP7mRNA and protein were remarkably down-regulated in BEL-7402cells. Moreover, upon treatment with β-catenin inhibitor IWP-2, the protein level of MMP7was reduced in a dose-dependent manner. Hep G2cells transiently transfeeted with MMP7plasmids displayed a much invasive activity, which phenocopied those of enhanced DKK1expression.Conclusions:1. DKK1is overexpressed in HCC cell lines.2. DKK1does not influence the proliferation of HCC cells.3. DKK1promotes HCC cell migration and invasion, moreover, DKK1is manifest overexpression in HCC tissues, especially in the metastatic tumors.4. Both gain-and loss-of-function showed that DKK1promotes β-catenin expression through a non-canonical Wnt signaling pathway, which in turn, stimulates the migration and invasion of HCC cells.5. MMP7is an important target downstream of DKK1/β-catenin signaling pathway, which plays a key role in DKK1-mediated invasion of HCC cells.