Abstract
Epithelial cells are dependent on extracellular matrix (ECM) attachment for maintenance of metabolic activity and suppression of apoptosis. Here we show that loss of ECM attachment causes down-regulation of epidermal growth factor receptor (EGFR) and β1 integrin protein and mRNA expression and that ErbB2, which is amplified in 25% of breast tumors, reverses these effects of ECM deprivation. ErbB2 rescue of β1 integrin mRNA and protein in suspended cells is dependent on EGFR, however, the rescue of EGFR expression does not require β1 integrin. We show that there is a significant decrease in the stability of EGFR in ECM-detached cells that is reversed by ErbB2 overexpression. Rescue of both EGFR and β1 integrin protein by ErbB2 is dependent on Erk activity and induction of its downstream target Sprouty2, a protein known to regulate EGFR protein stability. Interestingly, expression of EGFR and β1 integrin protein is more dependent on Erk/Sprouty2 in ECM-detached ErbB2-overexpressing cells when compared with ECM-attached cells. These results provide further insight into the ErbB2-driven anchorage independence of tumor cells and provide a new mechanism for regulation of EGFR and β1 integrin expression in ECM-detached cells.
Highlights
Pendence is an important aspect of the metastatic process, both for survival in the vasculature and lymphatic system and for survival in distant sites with altered matrix environments [2]
ErbB2 Prevents the Decrease of epidermal growth factor receptor (EGFR) and 1 Integrin after extracellular matrix (ECM) Detachment—We have previously shown that EGFR expression as well as activation of its downstream targets Erk and Akt are dramatically decreased when mammary epithelial cells are plated in suspension and that this decrease can be blocked by overexpression of ErbB2 [4, 7]
The ErbB2 Maintenance of EGFR and 1 Integrin Protein Levels Is Erk-dependent in ECM-detached Cells—To investigate the mechanism involved in ErbB2 regulation of EGFR and 1 integrin expression in ECM-detached cells, we examined whether ErbB2 induction of Erk or PI3K is required for the maintenance of EGFR and 1 integrin
Summary
MCF-10A cells expressing ErbB2 or MekDD were generated as described previously [24]. Assays on ECM-detached cells were performed on cells grown on tissue culture plates coated with poly(2-hydroxy methacrylate) (poly-HEMA) for 24 h except where indicated. MCF-10A or MCF10A ErbB2 cells were grown for 24 h on poly-HEMA plates. Reagents—The following reagents were used at the doses indicated and as described under “Results” and in the figure legends 1–5: poly-HEMA (Sigma-Aldrich), LY294002 (EMD Biosciences), U0126 (EMD Biosciences), PD98059 (Calbiochem), cell dissociation buffer (Invitrogen), cycloheximide (Sigma-Aldrich), chloroquine diphosphate salt (Sigma-Aldrich), bafilomycin A1 (Sigma-Aldrich), and concanamycin A (Sigma-Aldrich). For experiments involving siRNA in detached cells, cells were plated on poly-HEMA-coated plates 24 h after siRNA transfection, and assays were conducted 48 h after siRNA transfection. Data are presented as an average of three or more independent experiments
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