K-ras-ERK1/2 down-regulates H2A.X(Y142ph) through WSTF to promote the progress of gastric cancer

BACKGROUND: Histone H2AX phosphorylation at the site of Tyr-142 can participates in multiple biological progressions, which is including DNA repair. Ras pathway is closely involved in human cancers. Our study investigated the effects of Ras pathway via regulating H2AX.(Y142ph). METHODS: Gastric cancer cell line SNU-16 and MKN1 cells were transfected with Ras for G12D and T35S site mutation. The phosphorylation of H2A.X(Y142) and ERK1/2, WSTF and MDM2 was detected by western blot. Cell viability, cell colonies and migration was analyzed by MTT assay, soft-agar colony formation assay, and Transwell assay, respectively. The expression of Ras pathway related downstream factors, EYA3 and WSTF was detected by qRT-PCR. The relationship between Ras and downstream factors were detected by ChIP. The cell cycle progression was measured by flow cytometry. RESULTS: Ras(G12D/T35V) transection decreased the phosphorylation of H2A.X(Y142) and activated phosphorylation of ERK-1/2. H2A.X(Y142) inhibited cell viability, colonies and migration. H2A.X(Y142ph) altered the expression of Ras downstream factors. CHIP assay revealed that Ras(G12D/T35V) could bind to the promoters of these Ras pathway downstream factors. Silence of EYA3 increased H2A.X(Y142ph) and inhibited cell viability, migration and percent cells in S stage. Furthermore, silence of EYA3 also changed the downstream factors expression. WSTF and H2A.X(Y142ph) revealed the similar trend and MDM2 on the opposite. CONCLUSION: Ras/ERK signal pathway decreased H2A.X(Y142ph) and promoted cell growth and metastasis. This Ras regulation process was down-regulated by the cascade of MDM2-WSTF-EYA3 to decrease H2A.X(Y142ph) in SNU-16 cells.