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

Human MG-63 and U2OS osteosarcoma cell lines were purchased from Cells Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). These cells were cultured in Dulbecco-modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT), at 37 °C in a humidified atmosphere with 5% CO₂. MG-63 and U2OS cells were plated onto 6-well cell culture clusters (Costar) and grown to 80% confluence, and then serum-starved for 24 h. These cells were subsequently treated with HS-173 (PI3Kα inhibitor), MK-2206 (Akt inhibitor), A-674563 (Akt1 inhibitor), or CCT128930 (Akt2 inhibitor) (Selleck, Houston, TX) before RhoA activation assays and wound healing assays.

The construct ROR2-Flag was purchased from Addgene (Cambridge, MA). The constructs GFP-RhoA-N19, GFP-RhoA-V14 and vectors were kindly provided by Dr. Zhu (Nanjing Medical University, China). SiRNA duplexes specific for ROR2 or RhoA (Santa Cruz Biotechnology, Santa Cruz, CA) were transiently transfected into MG-63 and U2OS cells by using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) in serum-free OPTI-MEM according to the manufacturer’s instructions. The commercial siRNAs are a pool of 3 siRNAs specifically targeting ROR2 or RhoA, respectively (Santa Cruz Biotechnology). The cells were switched to fresh medium containing 10% FBS 6 h after the transfection and cultured for 48 h. The cells transfected with indicated constructs or siRNAs were used for analyzing the protein expression and cell migration.

ROR2 shRNA Plasmid (Santa Cruz Biotechnology) is a pool of three target-specific lentiviral vector plasmids each encoding 19–25 nt (plus hairpin) shRNAs designed to knock down gene expression. Each plasmid contains a puromycin resistance gene for the selection of cells stably expressing shRNA. ShRNAs specific for ROR2 or scrambled shRNAs were transfected into MG-63 cells using Lipofectamine 2000 reagent (Invitrogen). The cells were switched to fresh medium containing 10% FBS 6 h after the transfection and cultured for 48 h. After selection with puromycin (1 μg/mL) and serial limit dilutions, the ROR2 expression was controlled by Western blotting assays. Four selected clones of control and positive cells were pooled in order to avoid clonal variation. All cells were maintained in a 37 °C incubator with 5% CO₂ and cultured as the parental cells.

MG-63 and U2OS cells transfected with indicated constructs or siRNA and stable ROR2 knockdown MG-63 cells were plated onto 96-well cell culture clusters (Costar) and grown to confluence, and then serum starved for 24 h. The monolayer cells were scratched manually with a plastic pipette tip, and after two washes with PBS, the wounded cellular monolayer was allowed to heal for 12 h in DMEM containing 100 ng/mL recombinant Wnt5a (rWnt5a) (R&D Systems). Photographs of central wound edges per condition were taken at time 0 and 12 h after scratched using digital camera (Nikon, Tokyo, Japan).

MG-63 and U2OS cells transfected with ROR2-Flag or ROR2-siRNA and stable ROR2 knockdown MG-63 cells were seeded into 6-well plates and treated with 100 ng/mL Wnt5a. The experiments were then performed according to the manufacturer’s protocol (Cytoskeleton Inc., Denver, CO) [15]. G-LISA small GTPase activation assays offer a fast and sensitive method for performing small G-protein activation assays. Briefly, equal protein concentration in all samples is a prerequisite for accurate comparison between samples in GTPase activation assays. Cell extracts were equalized with ice-cold Lysis Buffer containing protease inhibitor cocktail to give identical protein concentrations. The Precision Red Advanced Protein Assay Reagent is a simple one step procedure that results in a red to purple/blue color change characterized by an increase in absorbance at 600 nm. Add 10 µL of each lysate or Lysis Buffer into the well of a 96 well plate. Add 290 µL of Precision Red Advanced Protein Assay Reagent to each well. Incubate for 1 min at room temperature. Blank spectrophotometer with 290 µL of Precision Red plus 10 µL of lysis buffer at 600 nm. Read absorbance of lysate samples. The activation of RhoA was normalized to the control group. RhoA activation assays were performed in triplicate.

Subconfluent cells were washed twice with PBS, and then lysed with ice-cold RIPA lysis buffer (50 mmol/L Tris, 150 mmol/L NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mmol/L sodium orthovanadate, 1 mmol/L sodium fluoride, 1 mmol/L EDTA, 1 mmol/L PMSF, and 1% cocktail of protease inhibitors) (pH7.4). The lysates were then clarified by centrifugating at 12,000g for 20 min at 4 °C. The protein extracts were separated by SDS-PAGE. The immunoblotting procedure was performed as described [16] and the following antibodies were used: rabbit anti-ROR2 antibody, mouse anti-GAPDH antibody (Proteintech, Wuhan, China), rabbit anti-Akt antibody, rabbit anti-phospho-Akt (p-Ser473) antibody (Cell Signaling Technology, Danvers, MA). Protein bands were detected by incubating with horseradish peroxidase-conjugated antibodies (Santa Cruz Biotechnology) and visualized with ECL reagent (Thermo Scientific, Rockford, IL). The gray values were taken by Tanon imaging analysis system (Tanon, Shanghai, China).

MG-63 cells transfected with ROR2-Flag or ROR2-siRNA and stable ROR2 knockdown MG-63 cells were placed into 6-well plates (5000 cells/well), incubated with 100 ng/mL Wnt5a at 37 °C for 2 weeks, fixed and stained with crystal violet. The mean ± SD number of colonies was counted under a microscope from three independent replicates.

All experiments here were repeated at least three times, with independent treatments, each of which showed essentially the same results. The data were analyzed using Student’s t test by SPSS statistical software package. All the results were expressed as mean ± SD. For all analyses a two-sided p < 0.05 was deemed statistically significant.

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 siRNA targeting ROR2 and measured the cell migration by wound healing assays. The 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). Thus, ROR2 participates in Wnt5a-induced osteosarcoma cell migration.

The finding that Wnt5a elevates RhoA activation in MG-63 cells [6] prompted us to determine whether ROR2 receptor was required for Wnt5a-induced RhoA activity. Wnt5a-induced RhoA activity was largely abolished by shRNA or siRNA specific against ROR2 (Fig. 2a), and significantly increased by ROR2 overexpression in MG-63 and U2OS cells (Fig. 3a). These results suggest that ROR2 is required for Wnt5a-induced RhoA activity of MG-63 and U2OS cells.

Next, we used constitute activity constructs (RhoA-CA, RhoA-V14) to elevate RhoA activity in osteosarcoma cells and checked whether the reductive migration rate by ROR2 knockdown could be rescued. RhoA-CA (RhoA-V14) was capable of increasing the cell migration in ROR2-knockdown MG-63 and U2OS cells (Fig. 2b, c and d). Moreover, siRNA specific against RhoA retarded Wnt5a/ROR2-mediated cell migration of MG-63 and U2OS cells (Fig. 3b and c). These findings suggest that ROR2/RhoA signaling mediates the Wnt5a-induced cell migration of osteosarcoma cells.

Given that PI3Kα/Akt signaling (specific PI3Kα, Akt1 and Akt2 isoforms) mediate Wnt5a-induced the migration of osteosarcoma cells [5, 6], we propose that PI3Kα/Akt act as the downstream of Wnt5a and ROR2. MG-63 cells, transfected with ROR2-Flag or ROR2-siRNA, were treated with 100 ng/mL of Wnt5a. The cells were harvested at 15 min after the start of Wnt5a treatment, followed by SDS-PAGE and Western blotting analyses (Fig. 4a and b). Akt showed the significantly increased or decreased signs of phosphorylation at Ser473 after ROR2-Flag or ROR2-siRNA transfection, respectively (Fig. 4a and b).

Moreover, we want to know whether inhibitors of PI3Kα/Akt signaling block Wnt5a/ROR2-mediated cell migration. MG-63 cells, transfected with ROR2-Flag, were pretreated 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), respectively, then were incubated with 100 ng/mL Wnt5a. The Wnt5a/ROR2-mediated cell migration was largely blocked by pretreatment of HS-173, MK-2206, A-674563 and CCT128930 in MG-63 cells, respectively (Fig. 4c and d). These data indicate that PI3Kα/Akt signaling acts as the downstream of Wnt5a/ROR2 and regulates the migration of osteosarcoma cells.

Wnt5a/ROR2 signaling is associated with suppression of β-catenin/TCF-dependent transcriptional activity and down-regulated the expression of cyclin D1 in erythroleukemia cells [17], suggesting its anti-tumor role on cell proliferation. Here, we transfected osteosarcoma MG-63 cells with ROR2-Flag or stable ROR2 knockdown MG-63 cells, then were incubated with 100 ng/mL of Wnt5a and subjected to clonogenic assays. Neither overexpression nor knockdown of ROR2 did not alter the proliferation of osteosarcoma cells (Fig. 5a and b). In conclusion, ROR2 receptor, acting as the upstream of PI3Kα/Akt/RhoA signaling, is required for Wnt5a-induced the migration, not the proliferation of osteosarcoma cells.