The role of ERK in the crosstalk between Smad4 and ERα signaling in ERα positive breast carcinoma cells Free

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It was reported that 23% of breast tumors were Smad4 negative, and 41% expressed low levels of smad4. Overexpression of Smad4 in estrogen receptor alpha (ERα) positive breast carcinoma cells has been shown to induce apoptosis. However, the mechanism by which Smad4 induces apoptosis in ERα positive breast cancer cells is not clear. MCF-7 breast carcinoma cells express endogenous Smad4 and a low level of ERα. In contrast, ZR-75-1 breast carcinoma cells do not express Smad4, but do express a high level of ERα. We have developed MCF-7 cells that stably express either a non-specific shRNA or Smad4 shRNA. We have also developed ZR-75-1 cells that were stably transfected with either a control vector or a tetracycline inducible Smad4 expression vector. Knockdown of Smad4 in MCF-7 cells decreased the protein and mRNA levels of ERK1/2 and increased the protein and mRNA levels of ERα. In contrast, overexpression of Smad4 in ZR-75-1 cells increased the protein and mRNA levels of ERK1/2, and decreased the protein and mRNA levels of ERα. Smad4 expression was associated with increased apoptosis in both model systems. Transient overexpression of ERα blocked Smad4-induced apoptosis. Conversely, when ERα was knocked down by siRNA, apoptosis increased significantly. More interestingly, when ZR-75-1 Smad4-Tet-on cells were treated both with tetracycline for the induction of Smad4 and with an ERK inhibitor, PD98059, ERα expression was no longer repressed and apoptosis was inhibited. Our data for the first time indicates that Smad4 expression induces apoptosis by positively regulating ERK1/2 expression, which negatively regulates ERα, resulting in apoptosis. This is a novel mechanism by which the crosstalk among Smad, ERK, and ERα pathways regulates the survival of ERα breast cancer cells.