Linkage of Rapid Estrogen Action to MAPK Activation by ERα-Shc Association and Shc Pathway Activation

Abstract

E2 rapidly activates MAPK in breast cancer cells, and the mechanism for this effect has not been fully identified. Since growth factor-induced MAPK activation involves signaling via the adapter protein Shc (Src-homology and collagen homology) and its association with membrane receptors, we hypothesized that breast cancer cells utilize similar signaling mechanisms in response to E2. In the present study, we demonstrated that E2 rapidly induced Shc phosphorylation and Shc-Grb2 (growth factor receptor binding protein 2)-Sos (son of sevenless) complex formation in MCF-7 cells. Overexpression of dominant negative Shc blocked the effect of E2 on MAPK, indicating a critical role of Shc in E2 action. Using selective inhibitors, we also demonstrated that ERalpha and Src are upstream regulators of Shc. A rapid physical association between ERalpha and Shc upon E2 stimulation further evidenced the role of ERalpha on Shc activation. Mutagenesis studies showed that the phosphotyrosine binding and SH2 domains of Shc are required to interact with the activation function 1, but not activation function 2, domain of ERalpha. Using a glutathione-S-transferase-Shc pull-down assay, we demonstrated that this ERalpha-Shc association was direct. Biological consequences of this pathway were further investigated at the genomic and nongenomic levels. E2 stimulated MAPK-mediated Elk-1 transcriptional activity. Confocal microscopy studies showed that E2 rapidly induced formation of membrane ruffles, pseudopodia, and ERalpha membrane translocation. The E2-induced morphological changes were prevented by antiestrogen. Together our results demonstrate that ERalpha can mediate the rapid effects of E2 on Shc, MAPK, Elk-1, and morphological changes in breast cancer cells

Nuclear insulin receptor substrate 1 interacts with estrogen receptor α at ERE promoters

“…ERa association with cytoplasmic signaling molecules is not unusual. Recently, ERa has been shown to bind the PI-3K/Akt complex (Simoncini et al, 2000;Sun et al, 2001), and to interact with growth factor receptor docking protein Shc (Song et al, 2002) as well as with IGF-IR (Kahlert et al, 2000). Similarly, we reported that unliganded ERa can associate with cytoplasmic IRS-1 in MDA-MB-231/ER cells (Morelli et al, 2003).…”

MAP kinase/estrogen receptor cross-talk enhances estrogen-mediated signaling and tumor growth but does not confer tamoxifen resistance

“…In this report, we demonstrated that MAPK induced ERa activation (cross-talk) in MEK/MCF-7 cells leads to estrogenindependent ER phosphorylation and ERE-reporter transcriptional activity. ER transcriptional activity can also be enhanced by non-genomic E 2 -mediated signaling in some cell lines (Castoria et al, 1999;Song et al, 2002). When MCF-7/MEK and MCF-7 control cells were treated with membrane impermeable BSA conjugated E 2 , no ERE transcriptional activity was detected ruling out the possible non-genomic e ects of E 2 in these cells.…”

Crosstalk between steroid receptors and the c-Src-receptor tyrosine kinase pathways: implications for cell proliferation

“…ERs can interact with caveolin-1 and are localized to caveolae raft domains isolated from plasma membranes of endothelial cells (Schlegel et al, 1999;Razandi et al, 2002). Imaging analysis of endogenous ERa in MCF7 breast cancer cells shows a pool of receptors near the plasma membrane (Song et al, 2002). In the presence of E2, there is increased membrane ruffling and increased association of ERa on the extending filipodia.…”

“…ERs have also been proposed to couple directly or functionally with G proteins Gaq and Gai, leading to downstream signaling through PKC, PLC, and c-Src (reviewed in Levin, 2003). ERa association with the adaptor molecule Shc occurs within 3 min of E2 treatment in MCF7 and long-term estrogen-deprived (LTED) MCF7 cells, resulting in Shc and MAPK phosphorylation (Song et al, 2002). The phosphorylation responses to E2 were inhibited by antiestrogens and by pharmacological inhibitors of c-Src.…”