Dickkopf-1 induces angiogenesis via VEGF receptor 2 regulation independent of the Wnt signaling pathway

Abstract

Tumor angiogenesis is essential for invasive tumor growth and metastasis. Dickkopf-1 (DKK-1), an antagonist of Wnt signaling, participates in tumor development and progression. We evaluated whether DKK-1 stimulation induces angiogenesis and the endothelial-mesenchymal transition (EnMT). Human umbilical vein endothelial cells (HUVECs) were stimulated with recombinant DKK-1 (rDDK-1) or conditioned medium from a culture of DKK-1-transfected 293 cells. Following stimulation, the expression levels of angiogenesis-related factors and EnMT related markers were determined by immunoblot assays. In addition, the effects of exogenous DKK-1 on angiogenesis and EnMT were assessed by tube-formation, cell invasion, and wound-healing assays. Human hepatoma cells, such as Hep3B and Huh-7, showed high levels of DKK-1 expression, whereas 293 cells and HUVECs showed little or no DKK-1 expression. Increased endothelial cell tube formation and invasiveness were observed in HUVECs treated with concentrated conditioned medium from DKK-1-overexpressing 293 cells or rDKK-1. DKK-1-stimulated HUVECs also exhibited increased motility in wound-healing assays. Furthermore, the expression levels of angiogenesis-related factors, including vascular endothelial growth factor receptor 2 and vascular endothelial-cadherin, were increased in DKK-1-stimulated HUVECs. The expression of EnMT markers, such as vimentin and Twist, was also increased in DKK-1-stimulated HUVECs. However, no significant change in β-catenin or GSK3β expression was observed. Our in vitro data suggest that DKK-1 can enhance angiogenesis and EnMT by HUVECs independent of the Wnt signaling pathway. Modulation of DKK-1 expression may facilitate development of novel strategies to control tumor angiogenesis and metastasis.

Keywords: HUVEC; Hepatocellular carcinoma; angiogenesis; dickkopf-1; vascular endothelial growth factor receptor.

Figure 1. Dickkopf-1 (DKK-1) expression in hepatocellular carcinoma (HCC)

DKK-1 expression in c-myc-overexpressing HCC mice tissue (A) and xenograft mice using Hep3B cells (B). DKK-1 mRNA (C) protein (D) and secreted protein (E) levels were high in HepG2, Hep3B, and Huh7 cells, but lower in SNU 449 and SNU 475 cells.

Figure 2. DKK-1 increases the motility, invasion, and tube formation of human umbilical vein endothelial cells (HUVECs)

DKK-1 conditioned medium was manufactured by transfecting DKK-1 into 293 cells and confirmed by Western blot and ELISA (A). HUVECs cultured in DKK-1-conditioned medium exhibited increased motility (B), invasion (C), and tube formation (D) compared to the controls. NT, non-treated.

Figure 3. DKK-1 increases the expression of endothelial–mesenchymal transition (EnMT) markers in HUVECs

DKK-1 stimulation resulted in increased N-cadherin, twist, and vimentin expression, and decreased VE-cadherin expression, compared to the controls by western blot (A) and mRNA level (B). NT, non-treated.

Figure 4. Effect of DKK-1 on gene expression

DKK-1 stimulation influenced the expression of genes related to angiogenesis and cell morphology (A). A protein-protein interaction analysis indicated that VEGFR2 and N-cadherin are involved in the DKK1mediated enhancement of the EnMT potential of HUVECs (B).

Figure 5. DKK-1 induces angiogenesis via the VEGFR2 signaling cascade

Treatment of HUVECs with DKK-1 conditioned medium resulted in increased expression of VEGFR2, and phosphorylation of downstream molecules such as Akt and Erk, compared to the controls (A–C). However, β-catenin and GSK3β expression was unaffected (D).

Figure 6. Conclusive figure summarizing the results