Metastasis, the cardinal feature of malignant tumors, is an important clinical variable in patient prognosis. To understand the basis for metastasis, we systematically selected for highly invasive cells from breast cancer cell lines, MCF7 and MDA-MB-453, with moderate to low invasive ability using Boyden chamber invasion assay. The four-cycle selected invasive lines, named MCF7-I4 and MDA-MB-453-I4, respectively, displayed epithelial-mesenchymal transition (EMT) and dramatically enhanced invasive ability. EMT changes were corroborated with decreased level of E-cadherin and increased vimentin, fibronectin, and β₁ integrin. Twist, a basic helix-loop-helix transcription factor, and AKT2, a known proto-oncogene, were found to be elevated in the invasive cells compared with the parental. Ectopic expression and knockdown of Twist by short interference RNA resulted in significant increase and reduction, respectively, of AKT2 protein and mRNA expression. Twist bound to E-box elements on AKT2 promoter and enhanced its transcriptional activity. Moreover, silencing AKT2 decreased Twist-promoted migration, invasion, and paclitaxel resistance. Reintroducing AKT2 largely rescued the phenotype resulted from knockdown of Twist in I4 cells, suggesting that AKT2 is a downstream target and functional mediator of Twist. Finally, we observed a 68.8% correlation of elevated Twist and AKT2 expression in late-stage breast cancers as oppose to 13% in early-stage breast cancers. Our study identifies Twist as a positive transcriptional regulator of AKT2 expression, and Twist-AKT2 signaling is involved in promoting invasive ability and survival of breast cancer cells. [Cancer Res 2007;67(5):1979–87]