Overexpression of HER2 (erbB2) in human breast epithelial cells unmasks transforming growth factor β-induced cell motility

We have examined overexpression of the human epidermal growth factor receptor 2 (HER2) to determine if it modifies the anti-proliferative effect of transforming growth factor (TGF)-β against MCF-10A human mammary epithelial cells. Exogenous TGF-β inhibited cell proliferation and induced Smad-dependent transcriptional reporter activity in both MCF-10A/HER2 and MCF-10A/vector control cells. Ligand-induced reporter activity was 7-fold higher in HER2-overexpressing cells. In wound closure and transwell assays, TGF-β induced motility of HER2-transduced, but not control cells. The HER2-blocking antibody trastuzumab (Herceptin) prevented TGF-β-induced cell motility. Expression of a constitutively active TGF-β type I receptor (ALK5^T204D) induced motility of MCF-10A/HER2 but not MCF-10A/vector cells. TGF-β-induced motility was blocked by coincubation with either the phosphatidylinositol 3-kinase inhibitor LY294002, the mitogen-activated protein kinase (MAPK) inhibitor U0126, the p38 MAPK inhibitor SB202190, and an integrin β₁ blocking antibody. Rac1 activity was higher in HER2-overexpressing cells, where both Rac1 and Pak1 proteins were constitutively associated with HER2. Both exogenous TGF-β and transduction with constitutively active ALK5 enhanced this association. TGF-β induced actin stress fibers as well as lamellipodia within the leading edge of wounds. Herceptin blocked basal and TGF-β-stimulated Rac1 activity but did not repress TGF-β-stimulated transcriptional reporter activity. These data suggest that 1) overexpression of HER2 in nontumorigenic mammary epithelial is permissive for the ability of TGF-β to induce cell motility and Rac1 activity, and 2) HER2 and TGF-β signaling cooperate in the induction of cellular events associated with tumor progression.