Ethanol‐induced in vitro invasion of breast cancer cells: The contribution of MMP‐2 by fibroblasts

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Abstract
Ethanol is a tumor promoter and may promote metastasis of breast cancer. However, the underlying cellular/molecular mechanisms remain unknown. Overexpression and high activity of matrix metalloproteinase‐2 (MMP‐2) are frequently associated with metastatic breast cancers and serve as a prognostic indicator of clinical outcome. MMP‐2 is predominantly expressed in stromal fibroblasts and plays a pivotal role in regulating the invasive behavior of breast tumor cells. We hypothesized that ethanol may enhance the invasion of breast tumor cells by modulating the activity of fibroblastic MMP‐2. With in vitro models (HS68 and CCD1056SK human fibroblasts), we showed that ethanol at physiologically relevant concentrations (50–200 mg/dl) activated MMP‐2; conversely, at a higher concentration (400 mg/dl), it inhibited the MMP‐2 activity. Consistently, conditioned medium collected from ethanol (50–200 mg/dl)‐exposed fibroblasts markedly enhanced the invasive potential of breast cancer cells and mammary epithelial cells overexpressing ErbB2/HER2 (BT474, SKBR‐3 and HB2ErbB2 cells) but had little effect on cells with low ErbB2 levels (BT20 and HB2 cells). In contrast, conditioned medium obtained from ethanol (400 mg/dl)‐treated fibroblasts inhibited cell invasion. Selective inhibitors of MMP‐2 (SB‐3CT and OA‐Hy) eliminated ethanol‐stimulated invasion, indicating that the effect of ethanol was mediated by MMP‐2. Ethanol activated conventional PKCs and JNKs in fibroblasts; inhibitors of PKC (Go6850 and Go6976) and JNKs (SP600125) significantly inhibited ethanol‐mediated MMP‐2 activation as well as cell invasion, indicating that PKCs and JNKs play a role in ethanol‐induced MMP‐2 activation and cell invasion in vitro. Thus, ethanol‐promoted breast cancer cell invasion may be mediated by the modulation of fibroblastic MMP‐2.