Altered EGFR localization and degradation in human breast cancer cells with an amphiregulin/EGFR autocrine loop

Abstract

The epidermal growth factor receptor (EGFR) and its ligand amphiregulin (AR) have been shown to be co-over expressed in breast cancer. We have previously shown that an AR/EGFR autocrine loop is required for SUM149 human breast cancer cell proliferation, motility and invasion. We also demonstrated that AR can induce these altered phenotypes when expressed in the normal mammary epithelial cell line MCF10A, or by exposure of these cells to AR in the medium. In the present studies, we demonstrate 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 than MCF10A cells cultured in EGF. Pulse chase analysis showed that EGFR protein remained stable in the presence of AR, yet was degraded in the presence of EGF. Consistent with this observation, tyrosine 1045 on the EGFR, the c-cbl binding site, exhibited less phosphorylation following stimulation with AR than following stimulation with EGF. Ubiquitination of the receptor was also dramatically less following stimulation with AR than following stimulation with EGF. Flow cytometry analysis showed that EGFR remained on the cell surface following stimulation with AR but was rapidly internalized following stimulation with EGF. Immunofluorescence and confocal microscopy confirmed the flow cytometry results. EGFR in MCF10A cells cultured in the presence of EGF exhibited a predominantly intracellular, punctate localization. In stark contrast, SUM149 cells and MCF10A cells growing in the presence of AR expressed EGFR predominantly on the membrane and at cell-cell junctions. We propose that AR alters EGFR internalization and degradation in a way that favors accumulation of EGFR at the cell surface and ultimately leads to changes in EGFR signaling.

RIN1 orchestrates the activation of RAB5 GTPases and ABL tyrosine kinases to determine the fate of EGFR

“…Compared to EGF, AREG produces less EGFR phosphorylation (Willmarth et al, 2009). AREG treatment activates RAS signaling pathways but is not normally accompanied by EGFR degradation, leading instead to the accumulation of cell surface EGFR (Willmarth et al, 2009).…”

EGFR-Ligand Signaling in Breast Cancer Metastasis: Recurring Developmental Themes

“…The differential impact on breast cancer cell behavior that AREG exhibits compared to EGF has drawn considerable attention to the concept that various EGFR ligands have discrete functions (41)(42)(43). Among the ErbB receptors, AREG appears to exclusively bind and activate the EGFR.…”

“…More recent studies have focused on the distinct downstream signaling and cellular behavior induced by AREG. Unlike exogenous EGF treatment, AREG stimulation of model cell lines and breast cancer cell lines is unable to induce efficient phosphorylation of many of the tyrosine residues in the C-terminal tail of the EGFR (22,43,51,52) and (Fig. 1).…”

“…In addition, AREG binding to the EGFR is very resistant to acidic pH suggesting that the ligand does not disengage in the endosome as does TGF (22). It appears that AREG may be unique among the ligands in that it induces EGFR trafficking through Rab 4 and Rab 11 containing endosomes (22,43). AREG induces prolonged phosphorylation of ERK relative to EGF (41,52).…”

Combinatorial intervention of prostaglandin E2 receptor and calcium sensing receptor to attenuate breast cancer cell proliferation, migration and bone metastasis

“…EP2 activates matrix metalloproteinases (such as MMP1 and MMP2) that in turn act to release the EGFR ligands amphiregulin (AR) and transforming growth factor α (TGF-α), which then bind to EGFR and initiate EGFR-signaling ( Fig. 1) (17)(18)(19).…”