ROR2 inhibits the proliferation of gastric carcinoma cells via activation of non-canonical Wnt signaling

Abstract

Gastric carcinoma is one of the most common human cancers and has a poor prognosis. Receptor tyrosine kinase-like orphan receptor 2 (ROR2), which is a non-canonical receptor of the Wnt signaling pathway, has been reported to be deregulated in numerous types of human cancers, including gastric carcinoma. However, the exact role of ROR2 in the regulation of the malignant phenotypes of gastric carcinoma, as well as the underlying molecular mechanism, remains largely unclear. The present study demonstrated that ROR2 was recurrently downregulated in gastric carcinoma tissues, as compared with their matched adjacent normal tissues. Furthermore, the expression levels of ROR2 were reduced in several common gastric carcinoma cell lines, as compared with normal gastric epithelial cells. Gastric carcinoma cells were transfected with ROR2 plasmids, and it was demonstrated that restoration of ROR2 expression significantly inhibited the proliferation and induced the apoptosis of gastric carcinoma cells by a Wnt5a-independent mechanism. In addition, it was observed that ROR2-overexpressing cells accumulated in the G0/G1 phase; thus suggesting that overexpression of ROR2 induced cell cycle arrest at the G0/G1 phase. An investigation of the underlying mechanism demonstrated that activation of the non-canonical Wnt signaling pathway inhibited canonical Wnt signal transduction, as demonstrated by the decreased level of β-catenin in nuclei, as well as the reduced expression levels of c-Myc. The results of the present study indicated a tumor suppressive role for ROR2 in gastric carcinoma growth in vitro, and suggested that ROR2 may be used as a molecular target for the treatment of gastric carcinoma.

Keywords: Wnt signaling; gastric carcinoma; proliferation; receptor tyrosine kinase-like orphan receptor 2.

Figure 1.

ROR2 expression was recurrently downregulated in gastric carcinoma tissues and cell lines. (A) The mRNA expression levels of ROR2 in gastric carcinoma tissues and their matched adjacent normal tissues were examined by RT-qPCR. The (B) mRNA and (C and D) protein expression levels of ROR2 in four common human gastric cancer cell lines (BGC823, SGC7901, HGC27 and AGS) and a normal gastric mucosa epithelial cell line (GES-1) were examined by RT-qPCR and western blotting, respectively. **P<0.01, vs. GES-1. ROR2, receptor tyrosine kinase-like orphan receptor 2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Figure 2.

ROR2 overexpression inhibited the proliferation of AGS gastric carcinoma cells. The (A) mRNA and (B) protein expression levels of ROR2 in AGS cells transfected with the pcDNA3.1-ROR2 plasmid were examined by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. (C) MTT assays were performed to determine the proliferation capacity of AGS cells transfected with the pcDNA3.1-ROR2 plasmid. AGS cells transfected with the blank vector and non-transfected AGS cells were used as controls. (D) MTT assays were performed to determine the proliferation capacity of AGS cells transfected with the pcDNA3.1-ROR2 plasmid in the presence or absence of the ROR2 ligand, Wnt5a. **P<0.01, vs. AGS. ROR2, receptor tyrosine kinase-like orphan receptor 2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; OD, optical density.

Figure 3.

ROR2 overexpression induced the apoptosis of AGS gastric cancer cells. (A) Cell apoptosis assays were conducted for AGS cells transfected with the pcDNA3.1-ROR2 plasmid. AGS cells transfected with the blank vector and non-transfected AGS cells were used as controls. (B) Cell apoptosis assays were conducted for AGS cells transfected with the pcDNA3.1-ROR2 plasmid in the presence or absence of the ROR2 ligand, Wnt5a. **P<0.01, vs. AGS. ROR2, receptor tyrosine kinase-like orphan receptor 2.

Figure 4.

ROR2 overexpressing AGS cells accumulated at the G1 phase. Cell cycle distribution assays were conducted for AGS cells transfected with the pcDNA3.1-ROR2 plasmid. AGS cells transfected with the blank vector and non-transfected AGS cells were used as controls. **P<0.01, vs. AGS. ROR2, receptor tyrosine kinase-like orphan receptor 2.

Figure 5.

ROR2 overexpression suppresses canonical Wnt signaling. Western blotting was performed to examine the protein expression levels of (A) β-catenin in the nucleus and (B) c-Myc in AGS cells transfected with the pcDNA3.1-ROR2 plasmid. GAPDH was used as an internal reference. AGS cells transfected with the blank vector and non-transfected AGS cells were used as controls. **P<0.01, vs. AGS. ROR2, receptor tyrosine kinase-like orphan receptor 2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.