Role Of PI3K / Akt / GSK3β / P190ARhoGAP / RhoA Signaling Pathway In Wnt5a - Induced Gastric Cancer Cell Migration

Gastric cancer is one of the most frequently occurring malignancies in China. Metastasis remains the major cause of gastric cancer-related death. Wnt5a is a well known non-transforming Wnt family member and its expression is correlated with aggressiveness and poor prognosis of gastric cancer. Wnt5a has ample opportunities to influence cancer cell signaling, resulting in functional promiscuity on cancer development. However, the molecular mechanisms underlying the effect of Wnt5a on gastric cancer cell migration and metastasis are still largely unknown. In this study, we established the cell migration model of SGC-7901 gastric cancer cells induced by Wnt5a in vitro. The function of PI3K/Akt/GSK3β/p190ARhoGAP/RhoA signaling pathway in stress fibers formation and cell migration were studied systematically in those gastric cancer cells.In this study, we found that1. Wnt5a stimulated the migration of SGC-7901 gastric cancer cells in a dose-dependent manner.2. Wnt5a enhanced phosphorylation of Akt (Ser473 and Thr308 sites) in SGC-7901 gastric cancer cells. Pretreatment with LY294002, a broad spectrum PI3K inhibitor, blocked Wnt5a-induced Akt phosphorylation (Ser473 and Thr308 sites) and cell migration. Additionally, depletion of Akt by siRNA resulted in a remarkable inhibition of Wnt5a-induced cell migration. Thus, Wnt5a-induced cell migration requires PI3K/Akt activation. Analogously, LY294002 inhibited Wnt5a induced MDA-MB-231 breast cancer cell migration significantly. These results demonstrated that PI3K not only played an important role in Wnt5a induced cell migration in SGC-7901, but also in MDA-MB-231.3. Wnt5a stimulated GSK3β phosphorylation at Ser9 in SGC-7901 gastric cancer cells. Pretreatment with LiCl, which induced phosphorylation of GSK3β at Ser9 site, increased Wnt5a-induced cell migration. However, depletion of GSK3β by siRNA reduced Wnt5a-induced migration of SGC-7901 cells significantly. These experiments demonstrated that GSK3β was required for Wnt5a-induced SGC-7901 cell migration.4. Reduction of PI3K by LY294002 and knockdown of Akt expression by siRNA blocked Wnt5a-induced GSK3β phosphorylation in SGC-7901 cells. However, there was no change in Akt phosphorylations (Ser 473 and Thr308 sites) after transfection of GSK3β siRNA or LiCl pretreatment. These results showed that GSK3P acted as a downstream effector of PI3K/Akt in response to Wnt5a in SGC-7901 cells.5. The activation of RhoA was increased by Wnt5a treatment in SGC-7901 cells. Wnt5a treatment significantly increased cell migration in cells expressing the vector, it failed to do so in cells expressing DN-RhoA (pCB6-GFP-RhoA-N19) mutant. Additionally, decreased GSK3β expression by transfecting with siRNA blocked Wnt5a-induced RhoA activation. Consequently, RhoA activation was required for Wnt5a mediated SGC-7901 cell migration.6. The formation of stress fibers was enhanced by Wnt5a treatment or overexpression of Wnt5a. Expression of DN-RhoA by transfecting, blocking PI3K by treatment with LY294002, Akt or GSK3β by siRNA-mediated knockdown disrupted the formation of actin stress fibers in SGC-7901 cells. In contrast, increasing GSK3β phosphorylation by the addition of LiCl enhanced the formation/maintenance of actin stress fibers in SGC-7901 cells. Thus, the findings from a cell biology perspective are consistent with the biochemical evidence that RhoA activation is required for PI3K/Akt/GSK3β activity.7. Wnt5a increased SGC-7901 cell adhesive ability. Wnt5a induced cell adhesion was decreased by LY294002, and increased by LiCl treatment. Consequently, PI3K and GSK3β were required for Wnt5a induced formation of adhesion in SGC-7901 cells.8. Wnt5a stimulated the phosphorylation and inactivation of p190ARhoGAP in SGC-7901 gastric cancer cells. Overexpression of Wnt5a blocked the activation of p190ARhoGAP in GES-1 gastric epithelium cells, BGC-823 and SGC-7901 gastric cancer cells. Additionally, depletion of p190ARhoGAP by siRNA resulted in a remarkable promotion of Wnt5a-induced cell migration and RhoA activation. Consequently, p190ARhoGAP plays an important role in Wnt5a induced cell migration.Taken together, we demonstrated that Wnt5a promoted gastric cancer cell stress fibers formation and migration via the PI3K/Akt/GSK3β/p190ARhoGAP/RhoA signaling pathway systematically for the first time. These findings could provide a basis for understanding the mechanism of gastric cancer metastasis comprehensively and deeply.