Breast cancer cells are characterized by their uncontrolled proliferation, high motility and ability to escape cell death. These pathophysiological processes are driven by aberrant expression and activity of various intracellular pathways, including ER, PI3K/AKT/mTOR, MAPK and integrins. Activated by estrogen, growth factors, nutrients, and other extra- and intracellular stimuli, these signaling pathways can cooperate or compensate for each other in order to promote tumor growth, metastasis and drug resistance. In normal cells, signaling can be tightly regulated by scaffold proteins that can turn on or off pathways, according to the cellular context. One such scaffold protein is MP1, which was initially discovered within the MAPK pathway. Subsequent studies revealed its association with additional pathways and its implication in regulating spreading, migration, proliferation, and differentiation. Since the pathways MP1 interacts with regulate breast tumorigenesis, we hypothesized and MP1 can directly participate in regulating the proliferation and survival of breast cancer cells. A panel of human mammary epithelial cell lines, consisting of ER-positive and ER-negative tumorigenic and non-tumorigenic cells was used. MCF-7 ER-positive and MDA-MB-231 ER-negative cells represented the main models for our studies. We confirmed that MP1 was expressed in all the mammary cell lines investigated, with collectively higher levels in ER-positive breast cancer cells compared to ER-negative ones. We then used siRNA duplexes to investigate the role of MP1 in breast cancer cells. First, we observed that MP1 silencing specifically induced cell death of ER-positive cells, whereas ER-negative cell lines examined were largely unaffected. Next, we established that the observed cell death effect was apoptosis, as indicated by PARP cleavage and downregulation of Bcl-2 pro-survival protein. These data suggested a novel pro-survival role of MP1 protein that is specific to ER-positive breast cancer cells. After establishing the biological relevance of MP1 silencing, we undertook a mechanistic approach and investigated the effects of MP1 siRNA on several signaling pathways. Partial depletion of MP1 was correlated with significant decrease in AKT activity and ER levels and activity. In contrast, MAPK activity was not affected, whereas reduced beta1 integrin expression did not reach statistical significance. In addition, the pro-apoptotic effects triggered by MP1 silencing were not dependent on ER signaling, which is also not required for survival of MCF-7 cells. Finally, we re-confirmed that in contrast to MDA-MB-231 cells, MCF-7 cells require PI3K for survival and active AKT was able to partly rescue the cell death phenotype induced by MP1 silencing. In summary, the studies presented here indicate a novel role for MP1 scaffold protein in ER-positive breast cancer cells. MP1 silencing resulted in cell death of ER-positive breast cancer cells only, therefore, expression of MP1 appears to be a requirement for promoting survival of this subset of breast cancer cells. They further suggest that the mechanism of cell death that occurred is apoptosis and this was mediated via PI3K/AKT signaling with possible cooperation from ER and beta1 integrin.
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