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First published online 25 September 2007
doi: 10.1242/jcs.010488

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EGF-receptor-mediated mammary epithelial cell migration is driven by sustained ERK signaling from autocrine stimulation

¹ Department of Biological Engineering, MIT, Cambridge, MA 02139, USA
² Systems Biology Program, Pacific Northwest National Laboratory, PO Box 999, Richland, WA 99352, USA
³ Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA 15261, USA
⁴ Center for Cancer Research, MIT, Cambridge, MA 02139, USA

View larger version (16K): [in this window] [in a new window]   Fig. 1. EGF ligand chimera constructs. (A) EGFR ligands are synthesized as integral membrane proteins that have variable membrane anchoring domains. Chimeras containing the receptor-binding domain of EGF (core) attached to the stem, transmembrane domain (TM) and cytoplasmic domain of either EGF or TGF- were constructed as shown. (B) Amino acid sequence of each chimera showing the published cleavage site (arrowheads).

View larger version (46K): [in this window] [in a new window]   Fig. 2. Sparse-labeled monolayer cell migration assay. Fluorescently labeled cells were plated 1:10 with unlabeled cells to form a confluent monolayer in a 96-well plate. Images were acquired at 15-minute intervals for 6-8 hours using a Cellomics KineticScan. Labeled cells within the monolayer were tracked using Imaris. Cell paths from a sample field for a single condition are shown. Individual cell paths are displayed as a function of time.

View larger version (13K): [in this window] [in a new window]   Fig. 3. EGF ligand production leads to proportional receptor binding and uptake. HMECs were treated with increasing amounts of adenovirus containing TCT and ECT expression constructs. (A) At approximately 40 hours after infection, the rate of EGF appearance in the extracellular medium was measured by ELISA over a 3 hour period in either the presence (white squares/circles) or absence (black squares/circles) of 10 µg/ml EGFR-blocking antibody 225. The amount of ligand observed in the presence of 225 is considered total EGF production, whereas the difference between the presence and absence of 225 reflects the amount of EGF consumed at that production level. (B) The relationship between the amount of EGF produced and consumed at each adenovirus MOI is shown both for TCT constructs (squares) and ECT constructs (circles).

View larger version (18K): [in this window] [in a new window]   Fig. 4. EGF ligand release can be quantitatively tuned across a wide dynamic range. (A) Ligand release rate is measured from the slope of EGF accumulation over time. After 16 hours in serum-free media, cells were switched to fresh serum-free media or to media containing an inhibitor and samples were collected at 2-hour time intervals from triplicate wells. EGF concentration was measured using an EGF ELISA. The approximate ligand release rates (molecules of EGF released/cell/minute), verified in independent experiments, are shown next to each linear fit. (B) TCT release into the media over an 8 hour time period is inhibited using increasing concentrations of batimastat. Fitting the conditioned EGF concentration to a dose-response curve reveals a median inhibition concentration of 0.33 µM batimastat. (C) Adding increasing concentrations of batimastat down-modulates the linear ligand release of TCT cells. Error bars represent one standard deviation from the mean of triplicate samples.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Autocrine signaling leads to surface EGFR downregulation and tyrosine phosphorylation in a ligand-release-rate-dependant manner. (A) Surface receptor numbers were quantified using an equilibrium-binding assay with 125I-labeled 13A9 mAb. Cells were serum-starved overnight before incubating the cells in fresh serum-free media containing labeled 13A9 alone (black bars) or with 2 nM exogenous EGF (gray bars) and allowed to reach steady state (5 hours), as described in the Materials and Methods. Surface-bound antibody was determined by counting acid strip solutions on a gamma counter. Receptor numbers were normalized to the average cell number counted from parallel plates. Error bars represent one standard deviation from the mean of triplicate wells. (B) Phospho-EGFR levels were measured using a quantitative immunoprecipitation bead-based tyrosine EGFR BioRad Bio-Plex phosphoprotein detection assay. Parental, ECT or TCT cells were switched to fresh serum-free media for 2 hours and then lysed. In addition, TCT cells were incubated for 2 hours with increasing concentrations of batimastat (0.12, 0.33 and 0.86 µM) to achieve additional release rates prior to lysis. Error bars represent standard deviation from triplicate lysates.

View larger version (29K): [in this window] [in a new window]   Fig. 6. Autocrine stimulation leads to sustained ERK phosphorylation. (A) Phosphorylation of ERK1/2 measured over an 8-hour time course. Cells were incubated in serum-free media (SFM) and lysed at the indicated time points (16, 20, 22 or 24 hours). Blots were stripped and re-probed for total ERK1/2. (B) Phospho-ERK (pERK1/2) dynamics measured using a quantitative immunoprecipitation bead-based ERK1/2 BioRad Bio-Plex phosphoprotein detection assay. Cells were incubated in serum-free media for 16 hours and then spiked with 2 nM exogenous EGF (black circles, squares and diamonds) or blocked with 10 µg/ml of 225 for 2 hours prior to three PBS washes and serum-free media replacement (white circles and squares). Error bars represent one standard deviation from the mean of triplicate lysates. (C) ERK phosphorylation was measured from triplicate lysates after 2 hours in serum-free media. In addition, TCT cells were incubated for 2 hours in batimastat (0.12, 0.33, 0.86 and 10.00 µM) to achieve additional release rates before lysis. Error bars represent standard deviation of triplicate lysates.

View larger version (6K): [in this window] [in a new window]   Fig. 7. Autocrine presentation stimulates normal cell proliferation. Cell growth rates were measured by cell counting with a Vi Cell XR. Cell number was measured over 3 days in regular DFCI-1 media or serum-free media (SFM). Error bars represent standard deviation of triplicate measurements.

View larger version (59K): [in this window] [in a new window]   Fig. 8. Autocrine stimulation leads to elevated cell migration speed. Cell migration was measured in a sparse-labeled monolayer high-throughput assay. Parental, ECT and TCT cells were starved for 15 hours and then switched to fresh serum-free media, 0.2 nM EGF, 2 nM EGF, 10 µg/ml mAb225 or batimastat (0.12, 0.33, 0.86 or 10.00 µM) 1 hour before imaging. Fluorescence images were acquired at 15-minute intervals for 8 hours and imported in Imaris for single-cell tracking of the labeled cells. Cell paths were analyzed in Matlab. (A) Cell paths are shown starting at each origin. (B) Average cell migration speed under each condition. (C) The average cell speed was measured for all cell types when stimulated with exogenous EGF while ligand cleavage at the cell surface was inhibited with batimastat. After 14 hours of starvation in serum-free media, the cells were treated for 2 hours with 10 µM batimastat and then switched to fresh batimastat alone or with 2 nM EGF. Cell speed is compared to that measured under serum-free media alone for each cell type. Error bars represent s.e.m., and n >200 cells per condition.

View larger version (23K): [in this window] [in a new window]   Fig. 9. Cell migration speed is a function of ligand release rate and ERK phosphorylation. ERK phosphorylation was measured from triplicate lysates using a BioPlex assay (see supplementary material Fig. S5 for ERK data alone). The parental cells were lysed after a 2-hour incubation with serum-free media (SFM) or exogenous EGF (0.2 and 2.0 nM). ECT cells were lysed after a 2-hour incubation in fresh serum-free media. In addition, TCT cells were lysed after a 2-hour incubation in serum-free media or increasing concentrations of batimastat (0.12, 0.33, 0.86 and 10.00 µM Bat). (A) Cell migration speed (shown in Fig. 7, error represents s.e.m., and n >200 cells) is plotted as a function of ERK phosphorylation (error represents standard deviation from triplicate lysates). (B) Parental and TCT cells were pre-incubated with multiple concentrations of the MEK inhibitor PD98059 (0.04, 0.20, 1.00, 5.00 and 25.00 µM PD) for 30 minutes. Parental cells were then stimulated with 2 nM of EGF under the same PD conditions. Cell migration speed is shown as a function of ERK1/2 phosphorylation measured at 2 hours after exogenous stimulation (see supplementary material Fig. S6 for ERK and migration data separately).

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