Vav3 and Rho GTPases, involved in the regulation of cell transformation, migration and invasion in NIH 3T3 fibroblasts and prostate cancer cells.

摘要

Vav3 is a member of the Vav family of guanine nucleotide exchange factors (GEF). In this study, we constructed a series of site-specific mutants in different domains of Vav3 and demonstrated that ZF and SH2 domains are essential for promoting cell migration and invasion. The abilities of Vav3 and its mutants to promote migration and invasion were closely correlated with their ability to activate Rac1 and Akt in NIH 3T3 cells. Dominant negative mutants of Rac1 and Akt most significantly inhibited an activated variant of Vav3, Vav3-6-10-mediated cell migration and invasion. STAT3 signaling was also involved in Vav3-6-10-promoted invasion. Constitutively active forms of Akt and Rac1 were able to rescue ZF and SH2 domain mutants to significantly regain the ability to promote migration and invasion. Over-expression of Vav3 or Vav3-6-10 enhanced migration and invasion in LNCaP prostate cancer cells, whereas knock down of the endogenous Vav3 by siRNA or shRNA in PC3 prostate cancer cells decreased their migration and invasion ability. Rac1 acted upstream of Akt in mediating Vav3-promoted migration/invasion in NIH 3T3 and prostate cancer cells.;Focus formation is a phenomenon on which cells evade contact inhibition of growth and a property usually associated with abnormal growth state and tumorigenesis. To explore the molecular basics underlying Vav3-6-10 induced focus formation, these site-specific point mutations were analyzed. There is a correlation between their focus inducing ability and RhoA activity. RhoA plays an important role mediating Vav3-6-10-induced focus formation. Using ROCK inhibitor Y27632 and dominant negative mutants of ROCK, mDia and PLDs, it was demonstrated that RhoA downstream signaling is important for Vav3-6-10 induced focus formation. The effect of Vav3 on cell cycle regulators and its relationship with Vav3-6-10 mediated evasion of contact inhibition was examined. Cyclin D1 and E were downregulated while the cyclin inhibitor p27 was upregulated in dense NIH 3T3 cells, and the expression of constitutively activated RhoA reverted this phenomenon. Upregulation of cyclin D1 and downregulation of p27 were found to correlate with the focus forming ability of Vav3, Vav3-6-10 and its mutants.;In conclusion, our results show that Vav3 is important for migration and invasion in fibroblast and epithelial cancer cells, and this Vav3 function depends on the activation of the Vav3- Rac1- PI3K/Akt and STAT3 pathway. Activated Vav3 evades contact growth inhibition via RhoA/ROCK/mDia/PLD pathway and Vav3-mediated regulation of cyclin D1 and p27 in high density cells depends on this signaling pathway.