C., Wang Y., Yue B., Shao Z, Qiu J., Mu J., Tang X., Han T., Xiang G., Ren Supplementary Material for: Dickkopf-Related Protein 2 is Epigenetically Inactivated and Suppresses Colorectal Cancer Growth and Tumor Metastasis by Antagonizing Wnt/β-Catenin Signaling <i>Background/Aims:</i> Aberrant activation of the Wnt/β-catenin signaling pathway plays a key role in the pathogenesis of multiple tumors including digestive cancers. Recent studies have reported that Dickkopf-related protein 2 (<i>DKK2</i>) is epigenetically inactivated in numerous types of cancers and that its gene products exhibit tumor-suppressive properties. However, the biological functions and underlying molecular mechanisms of <i>DKK2</i> in colon carcinoma remains obscure. <i>Methods:</i> We examined the expression of <i>DKK2</i> in colon tumor cell lines by RT-PCR and its promoter methylation status in colon tumor cell lines and primary tumors by methylation-specific PCR (MSP). Ectopic expression of <i>DKK2</i> was measured by RT-PCR prior to the other experiments. To investigate the function of <i>DKK2</i>, we assayed colony formation and cell proliferation, utilized flow cytometric analyses of the cell cycle and acridine orange/ethidium bromide (AO/EB) fluorescence staining for apoptosis, and examined wound healing, transwell migration and tumor growth <i>in vivo</i>. Western blots were used to explore the mechanisms of <i>DKK2</i> in epithelial- mesenchymal transition and canonical Wnt/β-catenin signaling. <i>Results:</i> We show here that downregulation or silencing of <i>DKK2</i> was closely associated with the hypermethylation status of its promoter and that <i>DKK2</i> expression could be restored by demethylation treatment. Methylation of the <i>DKK2</i> promoter was detected in nearly all tumors and tumor-adjacent tissues, but not in normal colon tissues. Ectopic expression of <i>DKK2</i> in colon cell lines HCT116 and HT-29 inhibited colony formation and cell viability by inducing cell cycle G0/G1 arrest and apoptosis, and growth of stable <i>DKK2</i>-infected HCT116 cells in nude mice was decreased compared to controls. Furthermore, <i>DKK2</i> restrained cell migration through partial reversal of epithelial-to- mesenchymal transition and also by downregulating several stem cell markers. Our data further showed that restoration of <i>DKK2</i> expression resulted in downregulation of active β-catenin and its downstream target genes. <i>Conclusion:</i> <i>DKK2</i> appears to be a functional tumor suppressor regulating tumorigenesis of colorectal cancer by antagonizing Wnt/β-catenin signaling. DKK2;Colon cancer;Tumor suppressor;Methylation;Wnt/β-catenin 2017-05-30
    https://karger.figshare.com/articles/figure/Supplementary_Material_for_Dickkopf-Related_Protein_2_is_Epigenetically_Inactivated_and_Suppresses_Colorectal_Cancer_Growth_and_Tumor_Metastasis_by_Antagonizing_Wnt_-Catenin_Signaling/5048626
10.6084/m9.figshare.5048626.v1