Cellular localization of the activated EGFR determines its effect on cell growth in MDA-MB-468 cells

Abstract

The epidermal growth factor (EGF) receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase that regulates diverse cell functions that are dependent upon cell type, the presence of downstream effectors, and receptor density. In addition to activating biochemical pathways, ligand stimulation causes the EGFR to enter the cell via clathrin-coated pits. Endocytic trafficking influences receptor signaling by controlling the duration of EGFR phosphorylation and coordinating the receptor's association with downstream effectors. To better understand the individual contributions of cell surface and cytosolic EGFRs on cell physiology, we used EGF that was conjugated to 900 nm polystyrene beads (EGF-beads). EGF-beads can stimulate the EGFR and retain the activated receptor at the plasma membrane. In MDA-MB-468 cells, a breast cancer cell line that over-expresses the EGFR, only internalized, activated EGFRs stimulate caspase-3 and induce cell death. Conversely, signaling cascades triggered from activated EGFR retained at the cell surface inhibit caspase-3 and promote cell proliferation. Thus, through endocytosis, the activated EGFR can differentially regulate cell growth in MDA-MB-468 cells.

Figure 1. EGF stimulates apoptosis in MDA-MB-468 cells

Serum starved MDA-MB-468 cells were treated with DMEM supplemented with 0-100 ng/ml EGF as indicated. Cell viability was assessed by harvesting cells and counting (A) or MTT assay (B). Data are plotted as the average ± S.E.M. (n=3-4). * Indicates p < 0.05 (student's t-test) as compared to 0 ng/ml EGF. E) Genomic DNA was collected from MDA-MB-468 cells treated for 16 hours with 0 ng/ml (DMEM), 100 ng/ml EGF, or 2 μM Taxol as indicated. DNA was run on a 1.2% agarose gel and stained with ethidium bromide.

Figure 2. EGFR endocytic trafficking is slowed in MDA-MB-468 cells

A. MDA-MB468 and HeLa cells were incubated with [¹²⁵I]-EGF for 2 hours on ice to achieve steady-state binding. After the removal of free radioligand, pre-warmed media (37°C) was added to the cells and they were placed at 37°C for the indicated amounts of time. Cell surface ligand was collected by incubation in high salt/low pH buffer (0.5M NaCl/0.2 M acetic acid pH 2.8); internalized radioligand was obtained by solubilizing the remaining cells in 1M NaOH. Radioactivity in each fraction was determined by counting on a Beckman Gamma counter. Data are plotted as the percentage of [¹²⁵I]-EGF internalized/ (internalized + cell surface). Data shown are the average ± S.E.M. (n=3) B. MDA-MB-468 and HeLa cells were incubated with [¹²⁵I]-EGF at 37°C for 7.5 minutes followed by extensive washing to remove extracellular and unbound radioligand. At the indicated time points, intact [¹²⁵I]-EGF was precipitated by trichloroacetic acid. Data are plotted as the average ± S.E.M. percentage of intact [¹²⁵I]-EGF remaining in the cell at each time point (n=3). C. MDA-MB-468 cells and HeLa cells were treated with 100 ng/ml EGF for the indicated times. Cell lysates were collected, resolved by 7.5% SDS-PAGE, and immunoblotted for the EGFR. Shown is a representative blot repeated three times. D. Quantification of three experiments as performed in B. Data are plotted as the relative level of EGF at each time point. The data are the average ± S.E.M. from three experiments.

Figure 3. EGF-beads can stimulate the EGFR

A. Serum starved MDA-MB-468 cells were treated for 1 hour with 1) DMEM, 2) ∼1 ×10⁹/ml EGF-Beads 3) ∼2 ×10⁹/ml EGF-Beads, 4) 1 ng/ml EGF, 5) 10 ng/ml EGF, 6) 100 ng/ml EGF, 7) ∼1 × 10⁹ polystyrene beads/ml (Beads), or 8) 40 μL of the final wash (Wash) in the EGF-bead synthesis (all in DMEM). Cells were lysed, cellular protein was separated by 7.5% SDS-PAGE, transferred to nitrocellulose, then probed with antibodies against phosphorylated EGFR, phosphotyrosine, EGFR, α-actin. B. Cells were treated with EGF (100 ng/ml) or ∼1 × 10⁹ EGF-beads/ml for 0, 1, 2, 4, 8, 16 hours at 37°C. Cell lysates were prepared and probed with either an anti-phosphotyrosine (PY99) or anti-EGFR antibody.

Figure 4. EGF-beads retain the activated EGFR on the cell surface

A. MDA-MB-468 cells were treated with either 100 ng/ml EGF or ∼ 1 ×10⁹ EGF-beads/ml, as indicated, for 1, 4, or 16 hours at 37°C. Cells were fixed and processed for indirect immunofluorescence using an antibody against the phosphorylated EGFR (Cell Signaling) followed by incubation with an Alexa488-conjugated goat anti-mouse secondary antibody. Images were collected using a Leica TCS NT confocal microscope. Shown are representative sections of the middle of the cell from an experiment repeated three times. Fluorescent (pEGFR) and brightfield images were collected of the same cells. The periphery of the cell was marked using Adobe Photoshop and the two images were merge to indicate the relative distribution of the the fluorescent labeling. Size bar is 20 μm. B. Serum starved MDA-MB-468 cells were treated with DMEM, 100 ng/ml EGF, or ∼1 × 10⁹ EGF-Beads/ml for the indicated times. After ligand treatment, the cells were treated with or without trypsin as indicated (see Experimental Procedures). Cell lysates were prepared, separated by 7.5% SDS-PAGE, transferred to nitrocellulose, then probed with antibodies against phosphotyrosine (PY99), transferrin, and α-tubule (upper panels). Parallel samples were treated with ligand, but not subjected to trypsinization and probed with an anti-phosphotyrosine antibody (PY99) to indicate receptor activity. Shown is a representative experiment that was repeated three times. C. Cells were treated with nothing, EGF (100 ng/ml), or EGF-beads (∼1 ×10⁹/ml) for 1 hour at 37°C and then incubated with [¹²⁵I]-transferrin at 37°C for 5-10 minutes. Cell associated [¹²⁵I]-Tfn was measured as described in Experimental Procedures. Data are plotted as the average ± S.E.M. cell associated [¹²⁵I]-Tfn (nmoles/500,000 cells) at each time point (n=3).

Figure 5. EGF-beads, but not EGF, promote cell growth and not apoptosis

A. MDA-MB-468 cells were incubated for 16 hours with 0 ng/ml EGF (DMEM), 5% FBS (Serum), or DMEM supplemented with 100 ng/ml EGF or ∼1 ×10⁹ EGF-beads/ml (EGF-beads). Images of cells were collected by light microscopy using a Nikon TE-2000 microscopy and Openlab software. Size bar = 25 μm B. MDA-MB-468 cells were incubated for 16 hours with 0 ng/ml EGF (DMEM), 5% FBS, or DMEM supplemented with 1-100 ng/ml EGF, or ∼1 ×10⁹ EGF-beads/ml (EGF-beads) for 16 hours in the presence [³H]-thymidine (described Experimental Procedures). Shown is the average ± S.E.M. fold increase in [³H]-thymidine incorporation relative to DMEM treated cells (n=3). * Indicates p < 0.05 (student's t-test) as compared to 0 ng/ml EGF.

Figure 6. EGF, but not EGF-beads, activates caspase-3

MDA-MB-468 cells were incubated for 16 hours with 0 ng/ml EGF (DMEM), or DMEM supplemented with 1-100 ng/ml EGF, or ∼1 ×10⁹ EGF-beads/ml (EGF-beads) for 16 hours and assayed for caspase-3 activity (Enz-Chek^® Caspase-3 Kit, Molecular Probes, Eugene, OR). Data were normalized to 0 ng/ml EGF and plotted as the average ± S.E.M. (n=3). * Indicates p < 0.05 (student's t-test) as compared to 0 ng/ml EGF.

Figure 7. Stimulation of caspase-3 activity by intracellular EGFRs is required to induce apoptosis

Serum starved MDA-MB-468 cells were treated with nothing or the caspase-3 inhibitor DEVDCHO (10 mM) for 1 hour prior to 16 hours of treatment with 0-100 ng/ml EGF or 1 ×10⁹ EGF-beads/ml. [³H]-thymidine incorporation was measured. Data are plotted as the average ± S.E.M. (n=3) of the [³H]-thymidine incorporation relative to DMEM. * Indicates p < 0.05 (student's t-test) as compared to 0 ng/ml EGF.

Figure 8. Differences in EGFR signaling are not due to the duration of the signal

Serum starved MDA-MB-468 cells were incubated in 1) 0 ng/ml EGF, 2) ∼1 ×10⁹ EGF-Beads 3) ∼2 ×10⁹ EGF-Beads, 4) 1 ng/ml EGF, 5) 10 ng/ml EGF, or 6) 100 ng/ml EGF. Cell lysates were prepared and resolved by 7.5% SDS-PAGE and immunoblotted using antibodies against phosphorylated EGFR, phosphotyrosine, or total EGFR. Shown in a representative experiment repeated three times.