Plakoglobin is differentially expressed in ARMS and ERMS rhabdomyosarcoma cells

Plakoglobin (γ-catenin) and β-catenin are pivotal components of cell-cell adherent junctions, linking cadherin receptors to the actin cytoskeleton. Unlike β-catenin overexpression, which is implicated in proliferation and tumor formation, high levels of plakoglobin suppress cell growth and tumorigenicity, whereas reduction of plakoglobin expression was found in highly invasive and metastatic tumors. We studied the expression of β-catenin and plakoglobin in 5 alveolar (ARMS) and 4 embryonal (ERMS) rhabdomyosarcoma (RMS) cell lines and 11 RMS tumor biopsies, 4 ARMS carrying the translocation t(2;13)(q35;q14), 2 ARMS without translocation and 3 ERMS. We found a consistent and homogeneous β-catenin expression in all of the cell lines and tumors tested, while plakoglobin, detectable in ERMS, was absent or almost undetectable in ARMS. These findings were confirmed by semi-quantitative RT-PCR assay at the RNA level. Immunocytochemical analysis of RMS cell lines showed membrane and cytoplasmic localization of β-catenin in all of them, whereas staining of plakoglobin was restricted to the cytoplasm of the ERMS cells. We did not detect any nuclear staining of both β-catenin and plakoglobin in fixed cells due, perhaps, to sensitivity limitations of the technique. However, cell fractionation analyses demonstrated that both catenins localized in the cytoplasm and in the nucleus, where they may exert transcriptional activity. Because gene silencing can be mediated by aberrant methylation and/or deacetylation of promoter regions, we assessed the effects of the DNA demethylating agent 5-Aza-2’-deoxycytidine (5AzadC) and of the histone deacetylase inhibitor Trichostatin-A (TSA). Our results suggest that plakoglobin expression is inhibited by both hypermethylation and deacetylation mechanisms in ARMS. We showed a partial restoration of plakoglobin expression in 5AzadC treated RH30 cells in a dose-dependent manner, whereas time course analysis of plakoglobin expression in TSA-treated RH30 cells showed a partial restoration in a time-dependent fashion. β-catenin expression was not increased by 5AzadC or by TSA treatments. We showed a synergistic increase in plakoglobin expression in the ARMS cells RH4 and RH30 treated with both agents. To further investigate this aspect, methylation-specific PCR of bisulfite-modified DNA was applied to assess the methylation status of promoter-associated CpG islands. RH4 and RH30 ARMS cells displayed methylated or emi-methylated alleles, respectively, whereas RD ERMS cells, which were 5AzadC unresponsive and endogenously expressed plakoglobin, contained only unmethylated alleles. These results may explain some of the biological and clinical differences between ARMS and ERMS and set the basis for a wider functional characterization of catenins in RMS and other solid tumors of childhood.