ARTICLE ABSTRACTTo understand the mechanisms leading to trastuzumab resistance in HER2-overexpressing breast tumors, we created trastuzumab-insensitive cell lines (SKBR3/100-8 and BT474/100-2). The cell lines maintain HER2 receptor overexpression and show increase in EGF receptor (EGFR). Upon trastuzumab treatment, SKBR3/100-8 and BT474/100-2 cell lines displayed increased growth rate and invasiveness. The trastuzumab resistance in SKBR3/100-8 and BT474/100-2 was accompanied with activation of the Wnt/β-catenin signaling pathway. Further investigation found that Wnt3 overexpression played a key role toward the development of trastuzumab resistance. The expression of Wnt3 in trastuzumab-resistant cells increased nuclear expression of β-catenin and transactivated expression of EGFR. The increased Wnt3 in the trastuzumab-resistant cells also promoted a partial EMT-like transition (epithelial-to-mesenchymal transition); increased N-cadherin, Twist, Slug; and decreased E-cadherin. Knockdown of Wnt3 by siRNA restored cytoplasmic expression of β-catenin and decreased EGFR expression in trastuzumab-resistant cells. Furthermore, the EMT markers were decreased, E-cadherin was increased, and the cell invasiveness was inhibited in response to the Wnt3 downregulation. Conversely, SKBR3 cells which had been stably transfected with full-length Wnt3 exhibited EMT-like transition. The Wnt3 transfectants, SKBR3/Wnt3-7 and SKBR3/Wnt3-9, showed a significant decrease in E-cadherin and increase in N-cadherin, Twist, and Slug. The cells were less sensitive to trastuzumab than parental SKBR3 and vector-transfected cells. In summary, our data suggest that Wnt3 overexpression activates Wnt/β-catenin signaling pathway that leads to transactivation of EGFR and promotes EMT-like transition. This could be an important mechanism leading to trastuzumab resistance in HER2-overexpressing breast cancer cells. Mol Cancer Res; 10(12); 1597–606. ©2012 AACR.