Wnt5a promotes ewing sarcoma cell migration through upregulating CXCR4 expression.
BACKGROUND
As one of the malignant tumors most often affecting children and young adults, Ewing sarcoma (ES) is characterized by early metastasis contributing to unfavorable prognosis. However, the molecular mechanisms responsible for ES metastasis remain poorly understood. In this study, we aimed to explore whether Wnt5a, a putative pro-metastatic factor, plays a role in ES metastasis.


METHODS
Expression of Wnt5a and CXCR4 was determined by real-time PCR or Western blot in 15 ES specimens and 4 ES cell lines, A-673, RD-ES, SK-N-MC and SK-ES-1. Expression of Wnt antagonists, SFRP1, SFRP2 and SFRP5, and some components in noncanonical Wnt pathway (p-JNK, p-cJUN and p-PKC) was also analyzed in this study. Methylation status of SFRP1, SFRP2 and SFRP5 was detected by Methylation-specific PCR (MSP). Wnt5a shRNA and pcDNA3.1 SFRP5 vector were used to abrogate Wnt5a expression and overexpress SFRP5 in ES cells, respectively.


RESULTS
Wnt5a expression was positively correlated with CXCR4 expression in ES specimens. Levels of both Wnt5a mRNA and CXCR4 mRNA were significantly higher in specimens from ES patients with metastasis at diagnosis compared with specimens from those without metastasis. Recombinant Wnt5a enhanced CXCR4 expression in ES cells, which was accompanied by increased ES cell migration, whereas Wnt5a shRNA has opposite effects. SFRP5 was methylated and silenced in ES cells, and both recombinant SFRP5 and pcDNA3.1 SFRP5 vector suppressed CXCR4 expression as well as ES cell migration. Wnt5a shRNA and recombinant SFRP5 inhibited phosphorylation of JNK and cJUN, and JNK inhibitor also reduced CXCR4 expression and cell migration in ES cells.


CONCLUSIONS
Wnt5a increases ES cell migration via upregulating CXCR4 expression in the absence of Wnt antagonist SFRP5, suggesting that Wnt5a overexpression and SFRP5 deficiency may jointly promote ES metastasis.
Department of Orthopedics, The First Hospital of China Medical University, Shenyang, China. jinzhecmu@yahoo.cn
Zhe
Z
Jin
Chenghai
C
Zhao
Xiaorui
X
Han
Yaxin
Y
Han
D048868:Adaptor Proteins, Signal Transducing; D000873:Anthracenes / Q000494:pharmacology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects / Q000235:genetics*; D019175:DNA Methylation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D019718:Receptors, CXCR4 / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012512:Sarcoma, Ewing / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D015854:Up-Regulation; D051153:Wnt Proteins / Q000235:genetics* / Q000378:metabolism; D000071818:Wnt-5a Protein; D000094742:Secreted Frizzled-Related Proteins
D048868:Adaptor Proteins, Signal Transducing; D000873:Anthracenes; D004791:Enzyme Inhibitors; D005136:Eye Proteins; D007211:Indoles; D048031:JNK Mitogen-Activated Protein Kinases; D008301:Maleimides; D008565:Membrane Proteins; D011493:Protein Kinase C; D011518:Proto-Oncogene Proteins; D019718:Receptors, CXCR4; D011994:Recombinant Proteins; D051153:Wnt Proteins; D000071818:Wnt-5a Protein; C118511:SFRP5 protein, human; D000094742:Secreted Frizzled-Related Proteins; C085389:WNT5A protein, human; C432165:pyrazolanthrone; C070515:bisindolylmaleimide I