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The epidermal growth factor receptor (EGFR) is involved in several cellular processes such as cell proliferation, differentiation, and survival. Regulated degradation of EGFR is critical to maintain normal cellular EGFR levels. EGFR can either be degraded via proteolytic pathways, or it can be recycled to the membrane and become activated again. We have shown that the inflammatory breast cancer cell line SUM149 over expresses EGFR protein without gene amplification, and an AR/EGFR autocrine loop is required for SUM149 cell proliferation. The effects of the EGF family ligand amphiregulin (AR) on EGFR degradation or recycling have not yet been examined. Therefore, we sought to investigate whether AR stabilizes EGFR by decreasing EGFR degradation in SUM149 cells and therefore might contribute to over expression of the receptor. Primarily, we observed that SUM149 cells and immortalized human mammary epithelial MCF10A cells that over express AR (MCF10A AR) or are cultured in the presence of exogenous AR, express higher levels of EGFR protein compared with MCF10A cells growing in EGF. Since the ubiquitin ligase Cbl is known to bind to phosphorylated tyrosine 1045 of the EGFR, we examined phosphorylation of this site in our cell lines. We found that phosphorylation of EGFR tyrosine 1045 is present in MCF10A cells growing in EGF but is completely absent in the SUM149 breast cancer cells and in MCF10A cells cultured in the presence of AR. This result suggests that Cbl is not able to bind to the EGFR and target it for degradation when AR is the activating ligand. We also observed that spiking EGF deprived MCF10A cells with saturating concentrations of EGF dramatically increased ubiquitination of EGFR. However, EGFR was only slightly ubiqutinated after spiking the cells with saturating concentrations of AR. To examine EGFR localization in EGF versus AR-stimulated cells, we visualized EGFR localization using immunofluorescence and confocal microscopy. We observed that in the presence of EGF, EGFR is mostly localized in endosomes. By contrast, in SUM149 cells and MCF10A cells growing in the presence of AR, EGFR is very abundant on the membrane and at cell-cell junctions. Thus, based on our results, we propose that an amphiregulin autocrine loop alters EGFR dynamics in a way that stabilizes EGFR and decreases EGFR degradation resulting in increased steady-state levels of EGFR protein, and accumulation of EGFR at the cell surface. This connection between an AR autocrine loop and EGFR over expression could explain why both factors have been shown to be predictors of aggressive breast cancer.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA