PI3K\u03b1 isoform-dependent activation of RhoA regulates Wnt5a-induced osteosarcoma cell migration

Ailiang Zhang,Zhihui Huang,Jinbo Liu,Ting Yan,Kun Wang

doi:10.1186/s12935-017-0396-8

Abstract

BackgroundWe have reported that the phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway mediated Wnt5a-induced osteosarcoma cell migration. However, the signaling pathways regulating Wnt5a/PI3K/Akt-mediated cell migration remains poorly defined in osteosarcoma cells.MethodsWe evaluated the activations of RhoA, Rac1 and Cdc42 in osteosarcoma MG-63 and U2OS cells with small G-protein activation assay. Boyden chamber assays were used to confirm the migration of cells transfected indicated constructs or siRNA specific against RhoA. A panel of inhibitors of PI3K and Akt treated osteosarcoma cells and blocked kinase activity. Western blotting and RhoA activation assay were employed to measure the effect of kinase inhibitors and activations of RhoA and Akt.ResultsWe found that Wnt5a had a potent stimulatory effect on RhoA activation, but not on Rac1 and Cdc42 activations. Wnt5a-induced cell migration was largely abolished by siRNA specific against RhoA. DN-RhoA (GFP-RhoA-N19) was also capable of retarding Wnt5a-induced cell migration, but the overexpression of CA-RhoA (GFP-RhoA-V14) was not able to accelerate cell migration. The Wnt5a-induced activation of RhoA was mostly blocked by pretreatment of LY294002 (PI3K inhibitor) and MK-2206 (Akt inhibitor). Furthermore, we found that the Wnt5a-induced activation of RhoA was mostly blocked by pretreatment of HS-173 (PI3Kα inhibitor). Lastly, the phosphorylation of Akt (p-Ser473) was not altered by transfection with siRNA specific against RhoA or DN-RhoA (GFP-RhoA-N19).ConclusionsTaken together, we demonstrate that RhoA acts as the downstream of PI3K/Akt signaling (specific PI3Kα, Akt1 and Akt2 isoforms) and mediated Wnt5a-induced the migration of osteosarcoma cells.

Highlights

We have reported that the phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway mediated Wnt5a-induced osteosarcoma cell migration
RhoA activation was stimulated by Wnt5a treatment in osteosarcoma cells To assess the effect of Wnt5a on Rho activation in human osteosarcoma cells, we treated MG-63 and U2OS cells with 100 or 200 ng/mL Wnt5a, and measured the Rho activation by small G-protein activation assay
Wnt5a-induced cell migration was largely abolished by small interfering RNA (siRNA) specific against RhoA (Fig. 2a, d), suggesting that RhoA activation is required for Wnt5a-induced migration of MG-63 and U2OS cells

Summary

Introduction

We have reported that the phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway mediated Wnt5a-induced osteosarcoma cell migration. The signaling pathways regulating Wnt5a/PI3K/Akt-mediated cell migration remains poorly defined in osteosarcoma cells. The integrity and dynamics of the actin cytoskeleton is a key point for cancer cell migrating into adjacent tissues and leading to the cancer metastasis in the distance [1, 2]. Regulation of the dynamic behavior and assembly of the actin filaments allows cancer cells to build an enormous range. The small GTPase Rho regulates the formation and rearrangement of actin filaments, and have been implicated in the control of cell motility and invasion [5,6,7]. Cdc and RhoA have been shown to play vital roles in growth factor- or cytokine-induced chemotaxis in fibroblasts, macrophages and neutrophils [10,11,12].

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