Knockdown of receptor tyrosine kinase‑like orphan receptor 2 inhibits cell proliferation and colony formation in osteosarcoma cells by inducing arrest in cell cycle progression

Huang,Jianjun; Shi,Ying; Li,Hui; Tan,Dunyong; Yang,Meisongzhu; Wu,Xiang

doi:10.3892/ol.2015.3797

Knockdown of receptor tyrosine kinase‑like orphan receptor 2 inhibits cell proliferation and colony formation in osteosarcoma cells by inducing arrest in cell cycle progression

Authors:
- Jianjun Huang
- Ying Shi
- Hui Li
- Dunyong Tan
- Meisongzhu Yang
- Xiang Wu
View Affiliations

Affiliations: The Second Department of Orthopedics, The First Affiliated Hospital of Jishou University, Jishou, Hunan 416000, P.R. China, Teaching and Research Department of Pathology and Pathophysiology, Medical School of Jishou University, Jishou, Hunan 416000, P.R. China, Department of Immunology and Microbiology, Medical School of Jishou University, Jishou, Hunan 416000, P.R. China
Published online on: October 12, 2015 https://doi.org/10.3892/ol.2015.3797
Pages: 3705-3711

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Osteosarcoma (OS) is the most common malignant tumor of the bone, with a high mortality rate and poor prognosis. Receptor tyrosine kinase‑like orphan receptor 2 (ROR2) has been reported to be dysregulated in human malignancies. More recently, ROR2 has been demonstrated to promote OS cell migration and invasion. However, the role of ROR2 in the regulation of OS cell proliferation, as well as the underlying molecular mechanism, remains unclear. The present study aimed to investigate the underlying mechanism of ROR2 in osteosarcoma growth. Reverse transcription‑quantitative polymerase chain reaction analysis and western blot analysis were used to examine the mRNA and protein expression. MTT assay, colony formation assay and cell cycle analysis were conducted to explore the function of ROR2 in osteosarcoma cells. In the present study, the expression of ROR2 was found to be frequently upregulated in OS tissues compared with matched adjacent normal tissues. It was also upregulated in the OS cell lines Saos‑2, MG‑63 and U‑2 OS, relative to normal osteoblast hFOB 1.19 cells. Knockdown of ROR2 expression by transfection with ROR2‑specific siRNA markedly inhibited the proliferation and colony formation of OS cells. Data from the cell cycle distribution assay revealed an accumulation of ROR2‑knockdown cells in the G0/G1 phase, indicating that knockdown of ROR2 leads to an arrest in cell cycle progression. Mechanistic investigation revealed that the protein levels of c‑myc, a target gene of the Wnt signaling, as well as cyclin D1, cyclin E and cyclin‑dependent kinase 4 were markedly reduced in the ROR2‑knockdown OS cells, suggesting that the inhibitory effect of ROR2 knockdown on OS cell proliferation is associated with the Wnt signaling pathway. In summary, the current study indicates an important role for ROR2 in the proliferation of OS cells. Therefore, ROR2 may be a promising therapeutic target in OS.

View Figures

View References

1	Valery PC, Laversanne M and Bray F: Bone cancer incidence by morphological subtype: A global assessment. Cancer Causes Control. 26:1127–1139. 2015. View Article : Google Scholar : PubMed/NCBI
2	Ferrari S, Smeland S, Mercuri M, Bertoni F, Longhi A, Ruggieri P, Alvegard TA, Picci P, Capanna R, Bernini G, et al: Italian and Scandinavian Sarcoma Groups: Neoadjuvant chemotherapy with high-dose Ifosfamide, high-dose methotrexate, cisplatin, and doxorubicin for patients with localized osteosarcoma of the extremity: A joint study by the Italian and Scandinavian Sarcoma Groups. J Clin Oncol. 23:8845–8852. 2005. View Article : Google Scholar : PubMed/NCBI
3	Mialou VI, Philip T, Kalifa C, Perol D, Gentet JC, Marec-Berard P, Pacquement H, Chastagner P, Defaschelles AS and Hartmann O: Metastatic osteosarcoma at diagnosis: prognostic factors and long-term outcome - the French pediatric experience. Cancer. 104:1100–1109. 2005. View Article : Google Scholar : PubMed/NCBI
4	Thompson LD: Osteosarcoma. Ear Nose Throat J. 92:288–290. 2013.PubMed/NCBI
5	PosthumaDeBoer J, Witlox MA, Kaspers GJ and van Royen BJ: Molecular alterations as target for therapy in metastatic osteosarcoma: A review of literature. Clin Exp Metastasis. 28:493–503. 2011. View Article : Google Scholar : PubMed/NCBI
6	Sundaram MV: Canonical RTK-Ras-ERK signaling and related alternative pathways. WormBook. 1:1–38. 2013. View Article : Google Scholar
7	Jiménez G, Shvartsman SY and Paroush Z: The Capicua repressor-a general sensor of RTK signaling in development and disease. J Cell Sci. 125:1383–1391. 2012. View Article : Google Scholar : PubMed/NCBI
8	Batchu SN and Korshunov VA: Novel tyrosine kinase signaling pathways: Implications in vascular remodeling. Curr Opin Nephrol Hypertens. 21:122–127. 2012. View Article : Google Scholar : PubMed/NCBI
9	Katoh M: WNT signaling pathway and stem cell signaling network. Clin Cancer Res. 13:4042–4045. 2007. View Article : Google Scholar : PubMed/NCBI
10	Green JL, Kuntz SG and Sternberg PW: Ror receptor tyrosine kinases: Orphans no more. Trends Cell Biol. 18:536–544. 2008. View Article : Google Scholar : PubMed/NCBI
11	Liu Y, Bhat RA, Seestaller-Wehr LM, Fukayama S, Mangine A, Moran RA, Komm BS, Bodine PV and Billiard J: The orphan receptor tyrosine kinase Ror2 promotes osteoblast differentiation and enhances ex vivo bone formation. Mol Endocrinol. 21:376–387. 2007. View Article : Google Scholar : PubMed/NCBI
12	DeChiara TM, Kimble RB, Poueymirou WT, Rojas J, Masiakowski P, Valenzuela DM and Yancopoulos GD: Ror2, encoding a receptor-like tyrosine kinase, is required for cartilage and growth plate development. Nat Genet. 24:271–274. 2000. View Article : Google Scholar : PubMed/NCBI
13	Mehawej C, Chouery E, Maalouf D, Baujat G, Le Merrer M, Cormier-Daire V and Mégarbané A: Identification of a novel causative mutation in the ROR2 gene in a Lebanese family with a mild form of recessive Robinow syndrome. Eur J Med Genet. 55:103–108. 2012. View Article : Google Scholar : PubMed/NCBI
14	Debebe Z and Rathmell WK: Ror2 as a therapeutic target in cancer. Pharmacol Ther. 150:143–148. 2015. View Article : Google Scholar : PubMed/NCBI
15	Rebagay G, Yan S, Liu C and Cheung NK: ROR1 and ROR2 in human malignancies: Potentials for targeted therapy. Front Oncol. 2:342012. View Article : Google Scholar : PubMed/NCBI
16	Li L, Ying J, Tong X, Zhong L, Su X, Xiang T, Shu X, Rong R, Xiong L, Li H, et al: Epigenetic identification of receptor tyrosine kinase-like orphan receptor 2 as a functional tumor suppressor inhibiting β-catenin and AKT signaling but frequently methylated in common carcinomas. Cell Mol Life Sci. 71:2179–2192. 2014. View Article : Google Scholar : PubMed/NCBI
17	Lu BJ, Wang YQ, Wei XJ, Rong LQ, Wei D, Yan CM, Wang DJ and Sun JY: Expression of WNT-5a and ROR2 correlates with disease severity in osteosarcoma. Mol Med Rep. 5:1033–1036. 2012.PubMed/NCBI
18	Ren D, Minami Y and Nishita M: Critical role of Wnt5a-Ror2 signaling in motility and invasiveness of carcinoma cells following Snail-mediated epithelial-mesenchymal transition. Genes Cells. 16:304–315. 2011. View Article : Google Scholar : PubMed/NCBI
19	Enomoto M, Hayakawa S, Itsukushima S, Ren DY, Matsuo M, Tamada K, Oneyama C, Okada M, Takumi T, Nishita M and Minami Y: Autonomous regulation of osteosarcoma cell invasiveness by Wnt5a/Ror2 signaling. Oncogene. 28:3197–3208. 2009. View Article : Google Scholar : PubMed/NCBI
20	Stricker S and Mundlos S: FGF and ROR2 receptor tyrosine kinase signaling in human skeletal development. Curr Top Dev Biol. 97:179–206. 2011. View Article : Google Scholar : PubMed/NCBI
21	Yamagata K, Li X, Ikegaki S, Oneyama C, Okada M, Nishita M and Minami Y: Dissection of Wnt5a-Ror2 signaling leading to matrix metalloproteinase (MMP-13) expression. J Biol Chem. 287:1588–1599. 2012. View Article : Google Scholar : PubMed/NCBI
22	Chang HR, Lian JD, Lo CW, Chang YC, Yang MY and Wang CJ: Induction of urothelial proliferation in rats by aristolochic acid through cell cycle progression via activation of cyclin D1/cdk4 and cyclin E/cdk2. Food Chem Toxicol. 44:28–35. 2006. View Article : Google Scholar : PubMed/NCBI
23	Li C, Chen H, Hu L, Xing Y, Sasaki T, Villosis MF, Li J, Nishita M, Minami Y and Minoo P: Ror2 modulates the canonical Wnt signaling in lung epithelial cells through cooperation with Fzd2. BMC Mol Biol. 9:112008. View Article : Google Scholar : PubMed/NCBI
24	Lerner UH and Ohlsson C: The WNT system: Background and its role in bone. J Intern Med. 277:630–649. 2015. View Article : Google Scholar : PubMed/NCBI
25	Anastas JN and Moon RT: WNT signalling pathways as therapeutic targets in cancer. Nat Rev Cancer. 13:11–26. 2013. View Article : Google Scholar : PubMed/NCBI
26	Ford CE, Ma Qian SS, Quadir A and Ward RL: The dual role of the novel Wnt receptor tyrosine kinase, ROR2, in human carcinogenesis. Int J Cancer. 133:779–787. 2013. View Article : Google Scholar : PubMed/NCBI
27	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. 28:2513–2523. 2009. View Article : Google Scholar : PubMed/NCBI
28	Geng M, Cao YC, Chen YJ, Jiang H, Bi LQ and Liu XH: Loss of Wnt5a and Ror2 protein in hepatocellular carcinoma associated with poor prognosis. World J Gastroenterol. 18:1328–1338. 2012. View Article : Google Scholar : PubMed/NCBI
29	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. 29:34–44. 2010. View Article : Google Scholar : PubMed/NCBI
30	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. 28:2513–2523. 2009. View Article : Google Scholar : PubMed/NCBI
31	Kobayashi M, Shibuya Y, Takeuchi J, Murata M, Suzuki H, Yokoo S, Umeda M, Minami Y and Komori T: Ror2 expression in squamous cell carcinoma and epithelial dysplasia of the oral cavity. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 107:398–406. 2009. View Article : Google Scholar : PubMed/NCBI
32	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. 100:1227–1233. 2009. View Article : Google Scholar : PubMed/NCBI
33	Cheng BQ, Jiang Y, Zhu Q and Lin WG: Wnt/β-catenin aids in regulating the proliferation of hepG2 cells mediated by thy-1. Genet Mol Res. 13:5115–5127. 2014. View Article : Google Scholar : PubMed/NCBI