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

Abstract

Background: We 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.

Methods: Wound 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.

Results: Wnt5a-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.

Conclusions: Taken together, we demonstrate that ROR2 receptor responding to Wnt5a ligand activates PI3K/Akt/RhoA signaling and promotes the migration of osteosarcoma cells.

Keywords: Migration; Osteosarcoma; ROR2; Wnt5a.

Fig. 1

ROR2 participates in Wnt5a-induced osteosarcoma cell migration. a Stable ROR2 knockdown MG-63 cells and osteosarcoma U2OS cells transfected with ROR2-siRNA or scrambled siRNA were subjected to Western blotting assays. The expression of ROR2 in MG-63 and U2OS cells was significantly knockdown by specific shRNAs or siRNAs targeting ROR2. GAPDH was used as an internal control. b and c Stable ROR2 knockdown MG-63 cells and osteosarcoma U2OS cells transfected with ROR2-siRNA or scrambled siRNA were subjected to wound healing assays. Cells incubated with 100 ng/mL Wnt5a were allowed to migrate for 12 h. Data were presented as mean ± SD of 5 determinations. The relative migration distance was normalized to the average value of scrambled group

Fig. 2

Knockdown of ROR2 decreases Wnt5a-induced RhoA activity and cell migration. a Stable ROR2 knockdown MG-63 cells and osteosarcoma U2OS cells transfected with ROR2-siRNA or scrambled siRNA were incubated with 100 ng/mL Wnt5a and harvested at 30 min after the start of Wnt5a treatment. The determination of RhoA activity was described in “Methods”. Data were presented as mean ± SD of 5 determinations. The relative RhoA activity was normalized to the average value of scrambled group. b, c, and d Stable ROR2 knockdown MG-63 cells (b, c) and osteosarcoma U2OS cells (b, d) transfected with ROR2-siRNA and/or RhoA-V14 (RhoA-CA), were subjected to wound healing assays. Cells incubated with 100 ng/mL Wnt5a were allowed to migrate for 12 h. Data were presented as mean ± SD of 5 determinations. The relative migration distance was normalized to the average value of the control group

Fig. 3

Overexpression of ROR2 increases Wnt5a-induced RhoA activity and cell migration. a Human osteosarcoma MG-63 and U2OS cells, transfected with ROR2-Flag or vectors (Ctrl), were incubated with 100 ng/mL Wnt5a and harvested at 30 min after the start of Wnt5a treatment. The determination of RhoA activity was described in “Methods”. Data were presented as mean ± SD of 5 determinations. The relative RhoA activity was normalized to the average value of scrambled group. b and c MG-63 and U2OS cells, transfected with ROR2-Flag and/or RhoA-siRNA, were subjected to wound healing assays. Cells incubated with 100 ng/mL Wnt5a were allowed to migrate for 12 h. Data were presented as mean ± SD of 5 determinations. The relative migration distance was normalized to the average value of the control group. Ns no significance

Fig. 4

PI3Kα and Akt acts as the downstream of Wnt5a/ROR2 signaling. a and b Human osteosarcoma MG-63 cells, transfected with ROR2-Flag or ROR2-siRNA, were incubated with 100 ng/mL Wnt5a and harvested at 15 min after the start of Wnt5a treatment. The expression and activity of Akt were determined by Western blotting assays. Data were presented as mean ± SD of 3 determinations. The ratio of p-Akt and total Akt was normalized to the average value of control group. c and d MG-63 cells, transfected with ROR2-Flag or vectors, were treated with 1 nmol/L HS-173 (PI3Kα inhibitor), 10 nmol/L MK-2206 (Akt inhibitor), 10 nmol/L A-674563 (Akt1 inhibitor), or 10 nmol/L CCT128930 (Akt2 inhibitor) for 1 h, then subjected to wound healing assays. Cells incubated with 100 ng/mL Wnt5a were allowed to migrate for 12 h. Data were presented as mean ± SD of 5 determinations. The relative migration distance was normalized to the average value of control group

Fig. 5

Wnt5a/ROR2 signaling does not alter osteosarcoma cell proliferation. a Human osteosarcoma MG-63 cells transfected with ROR2-Flag or stable ROR2 knockdown MG-63 cells were treated with 100 ng/mL of Wnt5a and subjected to the clonogenic assay. b The number of colonies were counted under a microscope. Data were presented as mean ± SD of 3 determinations. Ns no significance