Abstract 5208: PDGFRα stimulates glioma cell invasion through tyrosine phosphorylation of Dock180 at Y1811 and activation of the Dock180-CrkII-p130cas-Rac1 pathway

Abstract

Abstract Platelet-derived growth factor receptor (PDGFR)α ranks third among the top 11 amplified genes in high-grade glioblastoma multiforme (GBM) and is frequently overexpressed in low-grade gliomas. However, the molecular mechanisms by which PDGFRα promotes glioma growth and invasion are largely unknown. Here we report that PDGFRα induces glioma cell invasion through tyrosine phosphorylation of Dock180, a Rac1 guanine nucleotide exchange factor (GEF). By immunohistochemical analysis of primary glioma tissue specimens, we found that PDGFRα, PDGF-A and Dock180 are co-expressed in invasive areas but not the central regions of the clinical biopsies. Ectopic expression of PDGF-A by human glioma LN444 cells with endogenous PDGFRα significantly promotes glioma growth and invasion in the brain of mice. Cellular depletion of Dock180 by RNAi in LN444 and LN443 cells inhibits PDGF-A-promoted cell migration in vitro and suppressed PDGF-A-expressing LN444 glioma infiltration and growth in the brain. In vitro, PDGF-A stimulates cell migration, invasion and induces tyrosine phosphorylation of Dock180, whereas AG1296, an inhibitor of PDGFRα inhibits the PDGF-A-induced phosphorylation of Dock180 and glioma cell migration. By co-expression of Dock180, its various deletion mutants and an oncogenic mutant PDGFRαΔ8,9, we identified tyrosine residue (Y) 1811 of Dock180 as a major phosphorylation (p-Y) site of Dock180. Y1811 is located within the CrkII-binding domain at the C-terminus of Dock180 and a potential tyrosine phosphorylation site for Src kinase. Y1811 is also highly conserved in Dock180 protein from various species. Mutation of the Y to a phenylalanine (F) at Y1811 of Dock180 significantly attenuates PDGFRα-induced phosphorylation and subsequently decreases Rac1 GTPase activity. Co-expression of Dock180 and PDGFRα and CrkII promotes PDGF-A-induced association of Dock180 with CrkII and p130Cas whereas the Y1811F mutant markedly disrupts their association. Inhibition of Src by its inhibitors and a dominant negative Src mutant attenuates PDGF-A-stimulated p-Y of wild-type Dock180, Rac1 activity and cell migration while a constitutively active Src mutant induces p-Y of wild-type Dock180 but not Dock180 Y1811F mutant. Finally, re-introduction of wild-type Dock180 into LN444/PDGF-A/shRNA-Dock180 cells that endogenous Dock180 was depleted rescued PDGFRα-promoted CrkII association, Rac1 activity and glioma cell motility in vitro, and tumor growth and invasion the brain whereas re-expression of Dock180 Y1811F mutant in these cells failed to restore PDGFRα-stimulated cellular behaviors. Taken together, this data reveals a molecular mechanism by which PDGFRα stimulates glioma cell invasion through tyrosine phosphorylation at Y1811 of a Rac1 GEF, Dock180, leading to increases in Rac1 activity and glioma cell invasion in the brain. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5208.