PARENT SESSION

(MS5) SIGNALING PATHWAYS ACTIVATED BY NON-GENOMIC EFFECTS OF ESTRADIOL.

Santen , Richard ¹, Song , Robert ¹, ¹ University of Virginia, Charlottesville, VA

ABSTRACT- Breast cancer cells provide a model to examine the disparate effects of estradiol (E2) acting to initiate nuclear transcription and to modulate plasma membrane growth factor signaling. To gain a better understanding of the membrane component, studies focused upon the rapid (i.e. 1-15 minute) actions of E2 on growth factor signaling pathways. E2 activates MAP kinase and Akt in breast cancer cells within 5-10 minutes, suggsting non-genomic mechanisms of ER alpha (ER). Our studies demonstrated that the adaptor protein Shc is required for MAP kinase activation. E2 causes phosphorylation of Shc through Src, binding of Shc to Grb-2 and to SOS. In the process, Shc binds to ER. E2, after binding to ER, appears to co-opt growth factor signaling pathways that activate MAP kinase and PI-3-kinase. We questioned how how ER anchors in the membrane since it does not have a membrane localization signal. It appears that Shc serves as the bus which transports ER to the membrane. After phosphorylation, Shc binds to ER but it also physically interacts with the IGF-1 receptor (IGF-1R). We reasoned that ER, when bound to Shc, would be directed to the region of the membrane by the same processes causing Shc to translocate to growth factor receptor proteins. Accordingly, we examined the role of Shc and IGF-1R in mediating ER membrane association in MCF-7 cells. We initially confirmed that E2 rapidly induced IGF-1R phosphorylation and then demonstrated that E2 induced a ternary protein complex formation among Shc, ERa and IGF-1R. The functionality of Shc in this process was demonstrated by experiments down-regulating Shc with a selective siRNA. Confocal microscopy studies provided confirmation of the functional roles of Shc and the IGF-1R in the translocation of ER to the region of the membrane. We first demonstrated that E2 caused co-localization of ER, Shc and IGF-1-R to the membrane and then showed that knock down of either Shc or IGF-1R abolished this effect. Down-regulation of Shc, ERa or IGF-1R with specific siRNAs all blocked E2-induced MAPK phosphorylation. Together our results demonstrated that Shc and IGF-1R serve as key elements in the translocation of ER to the cell membrane and in the facilitation of ER-mediated rapid E2 action. These events appear to be important in mediating the proliferative effect of E2 on breast cell growth.

KEY WORDS: non-genomic, estrogen receptor, Shc, IGF-1 recptor