Critical roles of Wnt5a-Ror2 signaling in aggressiveness of tongue squamous cell carcinoma and production of matrix metalloproteinase-2 via ΔNp63β-mediated epithelial-mesenchymal transition

Objectives: We previously showed that Delta Np63 beta, a splicing variant of Delta Np63, mediated EMT and affected cell motility. DNA microarray was thus performed to elucidate the mechanism that Delta Np63 beta affects cell motility. As the results, Wnt5a was significantly down-regulated by Delta Np63 beta overexpression in tongue SCC cell line (SQUU-B) with EMT phenotype. Materials and methods: Seven OSCC cell lines were used. Expression of Delta Np6 beta, 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 Delta Np63 beta expression, and negatively associated with Delta Np63 expression in other cells. Delta Np63 beta 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 Delta Np63 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 Delta Np63 beta-mediated EMT. (C) 2017 The Authors. Published by Elsevier Ltd.