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Endocrine therapy remains the most used systemic treatment in hormone-dependent breast cancer. The estrogen receptor-α (ER-α) is used to predict those patients who will benefit from hormone therapy. However, most patients develop antiestrogen resistance and growth factors play a critical role in this process. We identified a new pathway in the cross talk between ER-α and growth factor signaling in MCF-7 cells through the ErbB2/PI 3-K/Akt pathway (signalome). Estradiol and growth factors activate ER-α, mediated by PI3-K, Akt1, and ErbB2. In cells stably transfected with the dominant negative K179M-Akt1 or R25C-Akt1, the effect of estradiol was significantly blocked. In cells transfected with constitutively active myr-Akt1, progesterone receptor induction, cell growth, and colony formation was observed, and tamoxifen could not fully abolish these or the estradiol effects. Inoculation of parental cells into ovariectomized female nude mice led to tumor appearance 4 weeks after estradiol supplementation and AG825 blocked this effect. Tumor volumes were ∼80% smaller upon inoculation of K179M-Akt1 or R25C-Akt1 cells. Western blots from tumor extracts from animals inoculated with MCF-7 cells with an estradiol pellet or myr-Akt1 cells without estradiol showed high Akt activity that was blocked by AG825, suggesting that loss of Akt1 activity was associated with tumor growth inhibition. To determine the role of this signalome in the development of hormone resistance, the MCF-7/LCC model was used. In LCC1, LCC2, and LCC9 cells, expression and activity of ErbB2, expression of ErbB3, and Akt activity were elevated compared to MCF-7 cells. Estradiol rapidly activated Akt in MCF-7 and LCC cells. However, another peak of high Akt phosphorylation was observed at long-term treatment, paralleled by phosphorylation of GSK3. Growth of LCC cells treated with estradiol or antiestrogens was blocked by AG825 and LY 294,002, suggesting that activation of the ErbB2/PI 3-K/Akt pathway can override the growth inhibitory antiestrogen effects. To determine the physiological relevance of Akt, immunohistochemical analyses were performed in 31 pairs of high grade, ductal human breast tumors and adjacent normal tissue. Akt and pAkt staining occurred in ductal areas. Akt expression and activity was higher in the cytoplasm (97%) than in the nucleus (64.5%, 35.5%). In normal tissue, hyperplastic ducts and scattered end units were positive for p-Akt and Akt expression and activity was lower. These data were confirmed by western blots. pAkt correlated with AKT1 expression, Tyr1248 phosphorylation of ErbB2, an increase in ErbB2 and ErbB3 expression, and GSK3 phosphorylation. Taken together, these results suggest that both nuclear actions and membrane signaling by ER-α via intracellular cascades are likely to contribute to antiestrogen resistance. Therefore, this multi-protein-based signalome may lead to developing combined therapeutic manipulations.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]