Reversion-inducing cysteine-rich protein with Kazal motifs and its regulation by glycogen synthase kinase 3 signaling in oral cancer

The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) and glycogen synthase kinase (GSK3) are novel tumor suppressors, and emerging evidence has suggested their active role in oral cancer pathogenesis. In the present study, 112 human samples, including 55 fresh samples of 14 adjacent normal tissues, 25 noninvasive oral tumors, and 18 invasive tumors, were included. The messenger RNA (mRNA) expression, protein expression, and promoter methylation of the RECK gene, as well as the expression of GSK3 beta, phospho/total beta-catenin, and c-myc, were measured by RT-PCR, bisulphate modification-PCR, immunohistochemistry, and Western blot analysis. Additionally, ectopic expression of in/active GSK3 beta was performed in cell culture experiments. This study provided information on the progressive silencing of RECK gene expression at the protein and mRNA levels paralleled with promoter hypermethylation at various stages of oral tumor invasion. RECK expression and the hypermethylation of the RECK gene promoter were negatively and positively correlated with pS(9)GSK3 beta/c-myc expression, respectively. Further, a negative trend of RECK protein expression with nuclear beta-catenin expression was observed. Induced expression of active GSK3 beta reversed the RECK silencing in SCC9 cells. Collectively, our results demonstrated that the silencing of the RECK gene, possibly regulated by the GSK3 beta pathway, is an important event in oral cancer invasion and this pathway could be exploited for therapeutic interventions.