Purpose: Retinoblastoma (RB) is a progressive eye cancer of infancy and childhood. Hypermethylation, epigenetic silencing of genes is one of the key events in tumorigenesis. The purpose of this study is to investigate hypermethylation of adenomatosis polyposis coli homologue, APC-2 and possible interaction of APC-2 with Wnt signaling β-catenin protein in Retinoblastoma.
Methodology: Primary RB tumor samples and cell line were used for the study. DNA isolation, bisulfite conversion, methylation specific PCR and DNA sequencing analysis of PCR products were performed to identify CpG islands and methylation in primary RB tumor samples (n = 30). Chemical demethylation and retrieval of APC-2 expression was studied using 5-Azacytidine (5'-AZC). Flow cytometry, immunofluorescence, western blot were performed for APC-2 expression analysis in demethylated Y79 cells. Co-localization study was conducted to understand the interaction between APC-2 and β-catenin.
Results: APC-2 gene was methylated and down regulated in primary RB tumors. We observed that 70% of RB tumors (21/30) showed positivity with APC-2 methylation. The RB Y79 cells after treatment with demethylating agent 5'-AZC retrieved APC-2 expression, which was confirmed by immunofluorescence and Western blot. Flow cytometry showed APC-2 expression of 29.22% in 5'-AZC treated cells. Co-localization study showed interaction of APC-2 and RB upregulated β-catenin in Y79 cells.
Conclusion: We report that APC-2 gene is hypermethylated in both RB tumor samples and Y79 cells. Reduced APC-2 lead to increased Wnt signaling pathway protein, β-catenin suggesting tumor suppressive role of APC-2 gene.
Keywords: APC-2; Y-79 cell line; methylation; retinoblastoma; tumor suppressors.