ROR2 receptor promotes the migration of osteosarcoma cells in response to Wnt5a
Dai et al. Cancer Cell Int
ROR2 receptor promotes the migration of osteosarcoma cells in response to Wnt5a
Bin Dai 2
Ting Yan 1
Ailiang Zhang 0
0 Spine Surgery, Third Affiliated Hospital of Soochow University , Changzhou 213003, Jiangsu , China
1 Safety Assessment and Research Center for Drug, Pesticide and Veterinary Drug of Jiangsu Province, School of Public Health, Nanjing Medical University , Nanjing 211166, Jiangsu , China
2 Department of Orthopedics, Binhai County People's Hospital , Binhai 224500, Jiangsu , China
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.
ROR2; Wnt5a; Osteosarcoma; Migration
Wnt5a, a non-transforming Wnt family member, plays
complicated roles in oncogenesis and cancer metastasis,
exerting both oncogenic and tumor suppressive effects
]. Wnt5a functions as a promoter in osteosarcoma
]. Our previous study illuminates that
Wnt5a mediates the migration of osteosarcoma cells
via elevating the phosphatidylinositol-3 kinase (PI3K)/
Akt and RhoA signaling [
]. However, there is still
unknown which receptors responds to Wnt5a signaling
and participates in osteosarcoma metastasis.
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
]. Wnt5a competes with Wnt3a for binding
to Fzd2 and thereby inhibits Wnt3a-dependent LRP6
phosphorylation and β-catenin-dependent Wnt
]. Wnt5a also can activate β-catenin-dependent
pathway and induce secondary axis formation in
Xenopus embryos that express the Fzd5 receptor [
Wnt5a induces heterooligermization of ROR1/ROR2,
which activates RhoA and Rac1 and then enhances
leukemia-cell chemotaxis and proliferation . Purified
Wnt5a protein inhibits canonical Wnt/β-catenin
signaling in ROR2-expressed cells, but also induces canonical
Wnt/β-catenin signaling in cells that expressed Fzd4 and
Although there are substantial evidences given that
Wnt5a binds to diverse receptors and promotes cellular
behaviors (e.g. cell chemotaxis, cell proliferation), it is
still much uncertainty regarding the receptor responds
to Wnt5a and regulates metastatic behavior of
osteosarcoma cells. Here, we demonstrates that ROR2 receptor
activates PI3K/Akt/RhoA signaling and mediates
Wnt5ainduced the migration of osteosarcoma cells.
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.
Plasmids, small interfering RNA (siRNA) and short hairpin
The construct ROR2-Flag was purchased from Addgene
(Cambridge, MA). The constructs GFP-RhoA-N19,
GFPRhoA-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
OPTIMEM 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.
Wound healing assay
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,
RhoA activation assay (G‑LISA small GTPase activation assays)
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) [
]. 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 [
] 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,
MG-63 cells transfected with ROR2-Flag or
ROR2siRNA 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
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.
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 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.
ROR2 is essential for Wnt5a‑induced RhoA activity
The finding that Wnt5a elevates RhoA activation in
MG-63 cells [
] 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
(RhoACA, 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.
PI3Kα/Akt signaling acts as the downstream of Wnt5a/ROR2
Given that PI3Kα/Akt signaling (specific PI3Kα, Akt1
and Akt2 isoforms) mediate Wnt5a-induced the
migration of osteosarcoma cells [
], 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 does not alter osteosarcoma cell proliferation
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 [
], 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.
Receptor tyrosine kinase-like orphan receptor is a receptor
family consisting of two closely related type I
transmembrane proteins ROR1 and ROR2. Owing to mutations in
their canonical motifs required for proper kinase
activity, RORs are classified as pseudokinases lacking
detectable catalytic activity [
]. ROR2 is up-regulated in a lot
of human tumors including osteosarcoma, melanoma,
renal cell carcinoma, prostate carcinoma, colorectal
cancer, squamous cell carcinomas of the head and neck,
stromal tumors, and breast cancers [
]. Wnt5a is a
prototypic ligand which activates a β-catenin
independent pathway in Wnt signaling [
]. Owing to the
synchronous highly expression pattern of Wnt5a in breast
cancer, gastric cancer, non-small-cell lung cancer, prostate
], we predict that Wnt5a and ROR2 may
act as co-effector in certain specific tumors. Wnt5a/ROR2
signaling elevates expression and secretion of CXCL16 in
mesenchymal stem cells (MSCs), leading to the promotion
of its proliferation [
]. Here, we demonstrate that ROR2
mediates Wnt5a-induced cell migration of osteosarcoma.
Our previous study finds that Wnt5a mediates the
migration of osteosarcoma cells via elevating the PI3K/
Akt and RhoA signaling [
]. Down-regulation of PI3K/
Akt/GSK3β signaling in gastric cancer cells suppresses
Wnt5a-induced activation of RhoA and cell migration
. In this study, overexpression of constitute active
RhoA rescues Wnt5a-induced cell migration blocked
by shRNA or siRNA against ROR2 in osteosarcoma
cells. Specific inhibitors targeting PI3K and Akt retard
Wnt5a-induced cell migration in ROR2-overexpressed
osteosarcoma cells. These results indicates that the
potential role of Wnt5a/ROR2/PI3K/Akt/RhoA signaling
is an accelerator in osteosarcoma metastatic behavior.
Wnt5a and its receptor ROR2 act synergistically to
increase autocrine signaling and inhibits canonical Wnt
signaling in myeloid leukemia cells [
]. A large
number of studies demonstrate that canonical Wnt signaling
facilitates the proliferation in both embryo development
and tumor progression [
]. Finally, we find that
Wnt5a/ROR2 signaling does not affect the proliferation
of osteosarcoma cells.
We present the evidence here that ROR2 mediates
Wnt5a-induced osteosarcoma cell migration via PI3K/Akt
and RhoA signaling. These findings elucidate a molecular
pathway linking ROR2 signaling to Wnt5a ligand in cell
motility. This result will contribute to further
understanding of biological roles of Wnt5a/ROR2/PI3K/Akt/RhoA in
cell migration of osteosarcoma and other cancers.
PI3K: phosphatidylinositol-3 kinase; Fzd: frizzled family receptors; LRP:
lowdensity lipoprotein receptor-related protein; ROR: receptor tyrosine kinase-like
orphan receptor; MSC: mesenchymal stem cell; DMEM: Dulbecco-modified
Eagle’s medium; FBS: fetal bovine serum.
Conceived and designed the experiments: AZ. Performed the experiments:
BD, TY. Analyzed the data: BD, TY. Contributed reagents/materials/analysis
tools: BD, TY. Wrote the paper: AZ. All authors read and approved the final
We kindly thank Dr. Zhu (Nanjing Medical University, China) to provide the
constructs GFP-RhoA-V14 and vectors.
The authors declare that they have no competing interests.
Availability of data and materials
The dataset(s) supporting the conclusions of this article are included within
Consent for publication
Ethics approval and consent to participate
This study was supported by grants from Jiangsu Provincial Commission of
Health and Family Planning to Ailiang Zhang (Z201614), Jiangsu Province’s 333
research project to Ailiang Zhang (BRA2016208) and the Science and
Technology Bureau of Changzhou to Ailiang Zhang (No. CJ20130029).
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
1. Zhou Y , Kipps TJ , Zhang S. Wnt5a signaling in normal and cancer stem cells . Stem Cells Int . 2017 ; 2017 : 5295286 .
2. Enomoto M , Hayakawa S , Itsukushima S , Ren DY , Matsuo M , Tamada K , Oneyama C , Okada M , Takumi T , Nishita M , et al. Autonomous regulation of osteosarcoma cell invasiveness by Wnt5a/Ror2 signaling . Oncogene . 2009 ; 28 ( 36 ): 3197 - 208 .
3. Wei R , Deng Z , Su J . miR -217 targeting Wnt5a in osteosarcoma functions as a potential tumor suppressor . Biomed Pharmacother . 2015 ; 72 : 158 - 64 .
4. Zhou H , Zhang M , Yuan H , Zheng W , Meng C , Zhao D . MicroRNA-154 functions as a tumor suppressor in osteosarcoma by targeting Wnt5a . Oncol Rep . 2016 ; 35 ( 3 ): 1851 - 8 .
5. Zhang A , He S , Sun X , Ding L , Bao X , Wang N. Wnt5a promotes migration of human osteosarcoma cells by triggering a phosphatidylinositol-3 kinase/Akt signals . Cancer Cell Int . 2014 ; 14 ( 1 ): 15 .
6. Zhang A , Yan T , Wang K , Huang Z , Liu J. PI3Kalpha isoform-dependent activation of RhoA regulates Wnt5a-induced osteosarcoma cell migration . Cancer Cell Int . 2017 ; 17 : 27 .
7. Nile AH , Mukund S , Stanger K , Wang W , Hannoush RN . Unsaturated fatty acyl recognition by Frizzled receptors mediates dimerization upon Wnt ligand binding . Proc Natl Acad Sci USA . 2017 ; 114 ( 16 ): 4147 - 52 .
8. Gilardoni MB , Remedi MM , Oviedo M , Dellavedova T , Sarria JP , Racca L , Dominguez M , Pellizas CG , Donadio AC . Differential expression of low density lipoprotein receptor-related protein 1 (LRP-1) and matrix metalloproteinase-9 (MMP-9) in prostate gland: from normal to malignant lesions . Pathol Res Pract . 2017 ; 213 ( 1 ): 66 - 71 .
9. Craft TR , Forrester WC . The Caenorhabditis elegans matrix non-peptidase MNP-1 is required for neuronal cell migration and interacts with the Ror receptor tyrosine kinase CAM-1 . Dev Biol . 2017 ; 424 ( 1 ): 18 - 27 .
10. Sato A , Yamamoto H , Sakane H , Koyama H , Kikuchi A . Wnt5a regulates distinct signalling pathways by binding to Frizzled2 . EMBO J. 2010 ; 29 ( 1 ): 41 - 54 .
11. He X , Saint-Jeannet JP , Wang Y , Nathans J , Dawid I , Varmus H. A member of the Frizzled protein family mediating axis induction by Wnt-5A . Science . 1997 ; 275 ( 5306 ): 1652 - 4 .
12. Ishikawa T , Tamai Y , Zorn AM , Yoshida H , Seldin MF , Nishikawa S , Taketo MM . Mouse Wnt receptor gene Fzd5 is essential for yolk sac and placental angiogenesis . Development . 2001 ; 128 ( 1 ): 25 - 33 .
13. Yu J , Chen LG , Cui B , Widhopf GF , Shen ZX , Wu RR , Zhang L , Zhang SP , Briggs SP , Kipps TJ . Wnt5a induces ROR1/ROR2 heterooligomerization to enhance leukemia chemotaxis and proliferation . J Clin Investig . 2016 ; 126 ( 2 ): 585 - 98 .
14. Mikels AJ , Nusse R. Purified Wnt5a protein activates or inhibits betacatenin-TCF signaling depending on receptor context . PLoS Biol . 2006 ; 4 ( 4 ): 570 - 82 .
15. Lu M , Wang T , He M , Cheng W , Yan T , Huang Z , Zhang L , Zhang H , Zhu W , Zhu Y , et al. Tumor suppressor role of miR-3622b-5p in ERBB2-positive cancer . Oncotarget . 2017 ; 8 ( 14 ): 23008 - 19 .
16. Shan X , Wen W , Zhu D , Yan T , Cheng W , Huang Z , Zhang L , Zhang H , Wang T , Zhu W , et al. miR 1296 - 5p inhibits the migration and invasion of gastric cancer cells by repressing ERBB2 expression . PLoS ONE . 2017 ; 12 ( 1 ): e0170298 .
17. Yuan Y , Niu CC , Deng G , Li ZQ , Pan J , Zhao C , Yang ZL , Si WK . The Wnt5a/ Ror2 noncanonical signaling pathway inhibits canonical Wnt signaling in K562 cells . Int J Mol Med . 2011 ; 27 ( 1 ): 63 - 9 .
18. Karvonen H , Niininen W , Murumagi A , Ungureanu D . Targeting ROR1 identifies new treatment strategies in hematological cancers . Biochem Soc Trans . 2017 ; 45 ( 2 ): 457 - 64 .
19. Morioka K , Tanikawa C , Ochi K , Daigo Y , Katagiri T , Kawano H , Kawaguchi H , Myoui A , Yoshikawa H , Naka N , et al. Orphan receptor tyrosine kinase ROR2 as a potential therapeutic target for osteosarcoma . Cancer Sci . 2009 ; 100 ( 7 ): 1227 - 33 .
20. O 'Connell MP , Fiori JL , Xu M , Carter AD , Frank BP , Camilli TC , French AD , Dissanayake SK , Indig FE , Bernier M , et al. The orphan tyrosine kinase receptor, ROR2, mediates Wnt5A signaling in metastatic melanoma . Oncogene . 2010 ; 29 ( 1 ): 34 - 44 .
21. Wright TM , Brannon AR , Gordan JD , Mikels AJ , Mitchell C , Chen S , Espinosa I , van de Rijn M , Pruthi R , Wallen E , et al. Ror2, a developmentally regulated kinase, promotes tumor growth potential in renal cell carcinoma . Oncogene . 2009 ; 28 ( 27 ): 2513 - 23 .
22. Yamamoto H , Oue N , Sato A , Hasegawa Y , Yamamoto H , Matsubara A , Yasui W , Kikuchi A . Wnt5a signaling is involved in the aggressiveness of prostate cancer and expression of metalloproteinase . Oncogene . 2010 ; 29 ( 14 ): 2036 - 46 .
23. Mei HJ , Lian SJ , Zhang S , Wang W , Mao QS , Wang H . High expression of ROR2 in cancer cell correlates with unfavorable prognosis in colorectal cancer . Biochem Biophys Res Commun . 2014 ; 453 ( 4 ): 703 - 9 .
24. Kobayashi M , Shibuya Y , Takeuchi J , Murata M , Suzuki H , Yokoo S , Umeda M , Minami Y , Komori T. Ror2 expression in squamous cell carcinoma and epithelial dysplasia of the oral cavity . Oral Surg Oral Med Oral Pathol Oral Radiol Endodontol . 2009 ; 107 ( 3 ): 398 - 406 .
25. Edris B , Espinosa I , Muhlenberg T , Mikels A , Lee CH , Steigen SE , Zhu S , Montgomery KD , Lazar AJF , Lev D , et al. ROR2 is a novel prognostic biomarker and a potential therapeutic target in leiomyosarcoma and gastrointestinal stromal tumour . J Pathol . 2012 ; 227 ( 2 ): 223 - 33 .
26. Henry C , Quadir A , Hawkins NJ , Jary E , Llamosas E , Kumar D , Daniels B , Ward RL , Ford CE . Expression of the novel Wnt receptor ROR2 is increased in breast cancer and may regulate both beta-catenin dependent and independent Wnt signalling . J Cancer Res Clin . 2015 ; 141 ( 2 ): 243 - 54 .
27. Asem MS , Buechler S , Wates RB , Miller DL , Stack MS . Wnt5a signaling in cancer . Cancers (Basel) . 2016 ; 8 ( 9 ): 79 .
28. Leris AC , Roberts TR , Jiang WG , Newbold RF , Mokbel K. WNT5A expression in human breast cancer . Anticancer Res . 2005 ; 25 ( 2A ): 731 - 4 .
29. Prasad CP , Chaurasiya SK , Guilmain W , Andersson T. WNT5A signaling impairs breast cancer cell migration and invasion via mechanisms independent of the epithelial-mesenchymal transition . J Exp Clin Cancer Res . 2016 ; 35 : 144 .
30. Takiguchi G , Nishita M , Kurita K , Kakeji Y , Minami Y. Wnt5a-Ror2 signaling in mesenchymal stem cells promotes proliferation of gastric cancer cells by activating CXCL16-CXCR6 axis . Cancer Sci . 2016 ; 107 ( 3 ): 290 - 7 .
31. Huang CL , Liu D , Nakano J , Ishikawa S , Kontani K , Yokomise H , Ueno M. Wnt5a expression is associated with the tumor proliferation and the stromal vascular endothelial growth factor-an expression in non-smallcell lung cancer . J Clin Oncol . 2005 ; 23 ( 34 ): 8765 - 73 .
32. Zhu YC , Tian YH , Du J , Hu ZZ , Yang L , Liu JJ , Gu L . Dvl2 -dependent activation of Daam1 and RhoA regulates Wnt5a-induced breast cancer cell migration . PLoS ONE . 2012 ; 7 ( 5 ): e37823 .
33. Liu J , Zhang Y , Xu R , Du J , Hu Z , Yang L , Chen Y , Zhu Y , Gu L. PI3K/ Akt-dependent phosphorylation of GSK3beta and activation of RhoA regulate Wnt5a-induced gastric cancer cell migration . Cell Signal . 2013 ; 25 ( 2 ): 447 - 56 .
34. Yang XT , Bi YY , Chen ET , Feng DF . Overexpression of Wnt3a facilitates the proliferation and neural differentiation of neural stem cells in vitro and after transplantation into an injured rat retina . J Neurosci Res . 2014 ; 92 ( 2 ): 148 - 61 .
35. Du Y , Zhang S , Yu T , Du G , Zhang H , Yin Z. Wnt3a is critical for endothelial progenitor cell-mediated neural stem cell proliferation and differentiation . Mol Med Rep . 2016 ; 14 ( 3 ): 2473 - 82 .
36. Lin SY , Chen CL , Wu YL , Yang YC , Hwu YM . Ratio of Wnt3a to BMP4 doses is critical to their synergistic effects on proliferation of differentiating mouse embryonic stem cells . Cell Prolif . 2008 ; 41 ( 3 ): 492 - 505 .
37. Nygren MK , Dosen G , Hystad ME , Stubberud H , Funderud S , Rian E. Wnt3A activates canonical Wnt signalling in acute lymphoblastic leukaemia (ALL) cells and inhibits the proliferation of B-ALL cell lines . Br J Haematol . 2007 ; 136 ( 3 ): 400 - 13 .
38. Yun MS , Kim SE , Jeon SH , Lee JS , Choi KY . Both ERK and Wnt/betacatenin pathways are involved in Wnt3a-induced proliferation . J Cell Sci . 2005 ; 118 (Pt 2): 313 - 22 .