Supplementary Material for: β-Catenin Expression and Activation in Conjunctival Melanoma

Purpose: To assess the role of the canonical Wnt pathway via activation of β-catenin in tumor progression of conjunctival melanoma. Methods: β-Catenin localization was assessed by immunohistochemistry in 43 conjunctival nevi, 48 primary acquired melanoses (PAM; conjunctival melanocytic intraepithelial neoplasia), and 44 conjunctival melanomas. Activation of the canonical or the noncanonical Wnt pathway was tested in vitro in 4 conjunctival melanoma cell lines with stimulation of either Wnt5a or Wnt3a. Wound healing assays were performed with Wnt5a. Results: Nuclear β-catenin expression was found in 16% of the nevi, in 15% of the melanomas, and in 4% of the PAM. Membranous β-catenin expression was identified in all the nevi and PAM and in 97.7% of the melanomas. In vitro, Wnt5a stimulation in the 4 conjunctival melanomas and in 1 skin melanoma cell line did not induce nuclear translocation of β-catenin, nor did it increase cell motility in the wound healing assays. Wnt3a stimulation did not induce nuclear localization of β-catenin in any of the cell lines tested. Conclusions: In conjunctival melanoma, nuclear localization and activation of β-catenin appear to be limited, suggesting that inhibition of ARF6, responsible for β-catenin activation, in subsets of skin melanoma may not represent a treatment option for this tumor. In vitro, Wnt3a or Wnt5a did not induce nuclear β-catenin localization.