ROR2 receptor promotes the migration of osteosarcoma cells in response to Wnt5a

Bin Dai,Ting Yan,Ailiang Zhang

doi:10.1186/s12935-017-0482-y

Abstract

BackgroundWe have reported that the phosphatidylinositol-3 kinase (PI3K)/Akt/RhoA signaling pathway mediates Wnt5a-induced cell migration of osteosarcoma cells. However, the specific receptors responding to Wnt5a ligand remain poorly defined in osteosarcoma metastasis.MethodsWound healing assays were used to measure the migration rate of osteosarcoma cells transfected with shRNA or siRNA specific against ROR2 or indicated constructs. We evaluated the RhoA activation in osteosarcoma MG-63 and U2OS cells with RhoA activation assay. A panel of inhibitors of PI3K and Akt treated osteosarcoma cells and blocked kinase activity. Western blotting assays were employed to measure the expression and activation of Akt. Clonogenic assays were used to measure the cell proliferation of ROR2-knockdown or ROR2-overexpressed osteosarcoma cells.ResultsWnt5a-induced osteosarcoma cell migration was largely abolished by shRNA or siRNA specific against ROR2. Overexpression of RhoA-CA (GFP-RhoA-V14) was able to rescue the Wnt5a-induced cell migration blocked by ROR2 knockdown. The Wnt5a-induced activation of RhoA was mostly blocked by ROR2 knockdown, and elevated by ROR2 overexpression, respectively. Furthermore, we found that Wnt5a-induced cell migration was significantly retarded by RhoA-siRNA transfection or pretreatment of HS-173 (PI3Kα inhibitor), MK-2206 (Akt inhibitor), A-674563 (Akt1 inhibitor), or CCT128930 (Akt2 inhibitor). The activation of Akt was upregulated or downregulated by transfected with ROR2-Flag or ROR2-siRNA, respectively. Lastly, Wnt5a/ROR2 signaling does not alter the cell proliferation of MG-63 osteosarcoma cells.ConclusionsTaken together, we demonstrate that ROR2 receptor responding to Wnt5a ligand activates PI3K/Akt/RhoA signaling and promotes the migration of osteosarcoma cells.

Highlights

We have reported that the phosphatidylinositol-3 kinase (PI3K)/Akt/RhoA signaling pathway mediates Wnt5a-induced cell migration of osteosarcoma cells
ROR2 participates in Wnt5a‐induced osteosarcoma cell migration To assess the effect of ROR2 receptors on Wnt5a-induced osteosarcoma cell migration, we generated the stable ROR2 knockdown MG-63 cells and transfected U2OS cells with specific small interfering RNA (siRNA) targeting ROR2 and measured the cell migration by wound healing assays
The short hairpin RNA (shRNA) or siRNA against human ROR2 knocked down ROR2 expression by approximately 50% as assessed by Western blotting in MG-63 and U2OS cells (Fig. 1a), which resulted in a significant reduction of Wnt5a-induced cell migration (Fig. 1b and c)

Summary

Introduction

We have reported that the phosphatidylinositol-3 kinase (PI3K)/Akt/RhoA signaling pathway mediates Wnt5a-induced cell migration of osteosarcoma cells. Our previous study illuminates that Wnt5a mediates the migration of osteosarcoma cells via elevating the phosphatidylinositol-3 kinase (PI3K)/ Akt and RhoA signaling [5, 6]. Wnt factors can bind to three types of receptors, which are identified as frizzled family receptors (Fzd), low-density lipoprotein receptor-related protein (LRP), and receptor tyrosine kinase-like orphan receptor (ROR) [7,8,9]. Wnt5a competes with Wnt3a for binding to Fzd and thereby inhibits Wnt3a-dependent LRP6 phosphorylation and β-catenin-dependent Wnt signaling [10]. Wnt5a can activate β-catenin-dependent pathway and induce secondary axis formation in Xenopus embryos that express the Fzd receptor [11, 12]. Wnt5a induces heterooligermization of ROR1/ROR2, which activates RhoA and Rac and enhances

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Discussion

Conclusion