Critical roles of Wnt5a-Ror2 signaling in aggressiveness of tongue squamous cell carcinoma and production of matrix metalloproteinase-2 via DeltaNp63beta-mediated epithelial-mesenchymal transition.

OBJECTIVES: We previously showed that DeltaNp63beta, a splicing variant of DeltaNp63, mediated EMT and affected cell motility. DNA microarray was thus performed to elucidate the mechanism that DeltaNp63beta affects cell motility. As the results, Wnt5a was significantly down-regulated by DeltaNp63beta overexpression in tongue SCC cell line (SQUU-B) with EMT phenotype. MATERIALS AND METHODS: Seven OSCC cell lines were used. Expression of DeltaNp63, Wnt5a, its receptor Ror2, and matrix metalloproteinases (MMPs) were analyzed by RT-PCR, real-time PCR, and western blotting, and gelatin zymography. Furthermore, we examined the effects of siRNA for Wnt5a or Ror2 and recombinant human Wnt5a (rhWnt5a) on motility of tongue SCC cells. Biopsy specimens from tongue SCC patients were used for immunohistochemical staining of Wnt5a and Ror2. RESULTS: Wnt5a and Ror2 were expressed only in SQUU-B cells without DeltaNp63 expression, and negatively associated with DeltaNp63 expression in other cells. DeltaNp63beta overexpression in SQUU-B cells decreased Wnt5a and Ror2 expression. By Wnt5a or Ror2 knockdown, cell motility was remarkably inhibited, but EMT markers expression was unaffected. MMP-2 expression and the activities inversely correlated with DeltaNp63 expression, and were inhibited by Wnt5a or Ror2 knockdown. Cell motility and MMP-2 activities were recovered by adding rhWnt5a in the cells with Wnt5a knockdown, but not in those with Ror2 knockdown. Moreover, immunohistochemical analyses in tongue SCC specimens found that high expression of Wnt5a or Ror2 was associated with poorer prognosis. CONCLUSION: Wnt5a-Ror2 signaling enhanced tongue SCC cell aggressiveness and promoted production of MMP-2 following DeltaNp63beta-mediated EMT.