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Gab2 regulates cytoskeletal organization and migration of mammary epithelial cells by modulating RhoA activation.

ABSTRACT: The docking protein Gab2 is overexpressed in several human malignancies, including breast cancer, and is associated with increased metastatic potential. Here we report that Gab2 overexpression in MCF-10A mammary epithelial cells led to delayed cell spreading, a decrease in stress fibers and mature focal adhesions, and enhanced cell migration. Expression of a Gab2 mutant uncoupled from 14-3-3-mediated negative feedback (Gab2(2xA)) led to a more mesenchymal morphology and acquisition of invasive potential. Expression of either Gab2 or Gab2(2xA) led to decreased activation of RhoA, but only the latter increased levels of Rac-GTP. Expression of constitutively active RhoA in MCF-10A/Gab2 cells restored stress fibers and focal adhesions, indicating that Gab2 signals upstream of RhoA to suppress these structures. Mutation of the two Shp2-binding sites to phenylalanine (Gab2(ΔShp2)) markedly reduced the effects of Gab2 on cellular phenotype and RhoA activation. Expression of Gab2 or Gab2(2xA), but not Gab2(ΔShp2), promoted Vav2 phosphorylation and plasma membrane recruitment of p190A RhoGAP. Knockdown of p190A RhoGAP reversed Gab2-mediated effects on stress fibers and focal adhesions. The identification of a novel pathway downstream of Gab2 involving negative regulation of RhoA by p190A RhoGAP sheds new light on the role of Gab2 in cancer progression.

SUBMITTER: Herrera Abreu MT

PROVIDER: S-EPMC3016968 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:To identify novel proteins that control melanoma development and progression, we integrated high-throughput proteomics, mRNA and miRNAs datasets from isogenic primary (WM115) and metastatic (WM266-4) melanoma cell-lines. We focused on differentially expressed proteins that had non-differential mRNA levels and were putatively targeted by a high number of miRNAs. SNX18, a sub-family member of sorting-nexins that participates in endocytosis and autophagy was down-regulated by 6-fold in WM266-4, and predicted to be targeted by 17 miRNAs. Over-expression of SNX18 in several melanoma cell-lines significantly reduced migration rate, while silencing of SNX18 resulted in the opposite effect. Notably, progression tissue microarrays demonstrated a trend for SNX18 downregulation in early metastasis. Luciferase assays confirmed the direct regulation of SNX18 by multiple miRNAs. Indeed, over-expression of these miRNAs repressed endogenous SNX18 expression at the protein but not mRNA level. Further, protein but not mRNA expression levels correlated with better overall survival as determined in a cohort of 48 metastatic melanoma patients and in the TCGA database. Mechanistically, SNX18-mediated reduced migration coincided with RhoA activation and Cdc42 inactivation. Both play a role, as RhoA inhibitor and constitutively activated Cdc42 mutants abrogated this effect. Moreover, phosphorylation-defective SNX18 mutants (S233A,S233D) migrated faster, with S233A inactivating RhoA and activating Cdc42, while S233D activated both. The downstream phosphorylation of the p38>MAPKAPK2>HSP27 signaling pathway was concordant. Both mutants were more abundant in the plasma membrane. Taken together, phosphorylation of S233 is crucial for SNX18 activation, but de-phosphorylation dynamics, which may occur at the membrane, is required for functional effect. In this study we compared the proteome, transcriptome and miRnome of primary and metastatic melanoma isogenic cell-lines and identified SNX18 as a novel inhibitor of melanoma migration.

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Gab2 regulates cytoskeletal organization and migration of mammary epithelial cells by modulating RhoA activation.

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