MMP-2 mediates ethanol-induced invasion of mammary epithelial cells over-expressing ErbB2

Ethanol is a tumor promoter and may enhance the metastasis of breast cancer. We have previously demonstrated that over-expression of ErbB2 promoted ethanol-mediated invasion of mammary epithelial cells and breast cancer cells. However, the underlying cellular/molecular mechanisms remain unknown. By gelatin zymography, we showed that over-expression of ErbB2 increased the production of matrix metalloproteinase-2 (MMP-2) and MMP-9 in human mammary epithelial cells (HB2). Transient or stable transfection of ErbB2 cDNA to HB2 cells upregulated the transcripts and the activity of the MMP-2/-9 gene promoter; the upregulation of MMP-2/-9 expression was mediated by p38 mitogen-activated protein kinase (p38 MAPK) and phosphatidylinositol 3-kinase (PI3K). Although ethanol, at physiologically relevant concentrations (100-400 mg/dl), did not affect the production of MMP-2/-9, it activated MMP-2 in HB2 cells over-expressing ErbB2 (HB2^ErbB2), but not HB2 cells; it enhanced the cleavage of proform MMP-2 (72 kDa) to an active form (62 kDa). The activation was dependent on c-jun N-terminal kinases (JNKs) and reactive oxygen species (ROS). On the other hand, ethanol affected neither the expression nor the activation of MMP-9. Selective inhibitors of MMP-2 (SB-3CT and OA-Hy) and antioxidants significantly inhibited ethanol-stimulated invasion of HB2^ErbB2 cells. Furthermore, knocking down MMP-2 by small interference RNA also induced a partial blockage on ethanol-promoted invasion of HB2^ErbB2 cells. Thus, ethanol-stimulated invasion of cells over-expressing ErbB2 was mediated, at least partially, by MMP-2 activation.