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First published online September 19, 2007
doi: 10.1242/10.1242/jcs.002774

Journal of Cell Science 120, 3395-3403 (2007)
Published by The Company of Biologists 2007
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EGF receptor signalling is essential for electric-field-directed migration of breast cancer cells

Jin Pu1, Colin D. McCaig1, Lin Cao2, Zhiqiang Zhao1, Jeffrey E. Segall3 and Min Zhao1,*,{ddagger}

1 School of Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, UK
2 School of Medicine, University of Aberdeen, Aberdeen, AB25 2ZD, UK
3 Department of Anatomy and Structural Biology, Program in Motility and Invasion, Albert Einstein College of Medicine, Bronx, NY 10461, USA


Figure 1
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  		Fig. 1. Breast cancer cells migrate anodally in a small physiological electric field. (A) Human breast cancer cells (MDA-MB-231) migrate to the anode (left) (see supplementary material Movie 1). The cells were starved in serum-free medium containing 0.35% HSA overnight before exposure to a direct current EF of 1.5 V/cm. White lines with blue arrowheads represent trajectories and direction of cell movement. (B) Directedness of cell migration shows voltage dependence of the directional migration with the threshold voltage inducing directional migration below 1 V/cm. (C) Small electric fields significantly increased the migration speed. *P<0.001 compared to no EF control, ^P<0.01 compared to 1-3V/cm EF. (D) Rat mammary cancer cells (MTLn3) migrate to the anode in an electric field of 1.5 V/cm (see supplementary material Movie 2). White lines and blue arrowheads represent trajectories and direction of cell movement. (E,F) Voltage dependence of the migration directedness and speed. Data are mean ± s.e.m. of three independent experiments. *P<0.01 compared with no EF control. Bar, 50 µm. (G) MTLn3 cells migrate toward the anode in EF. After 40 minutes, the polarity of electric field was reversed. Cells of the same field continued to be recorded for the indicated period. White arrows represent displacement distances and direction of cell movement (see supplementary material Movie 3). EF=1.5 V/cm.

 

Figure 2
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  		Fig. 2. Strong correlation between electrotactic migration and EGFR (ErbB1) expression levels. (A) A small EF of 0.5 V/cm was applied to cells for 2 hours. Non-metastatic rat mammary adenocarcinoma MTC clone (13762NF) showed weak electrotaxis, whereas the high metastatic clone, MTLn3 showed robust electrotaxis. There were significant quantitative differences in migration speeds and in cell directedness. *P<0.001 compared with MTC cells. Data are mean ± s.e.m. of three independent experiments. (B) Significantly varied expression levels of ErbB1 in five breast cancer cell lines were detected by western blot. {alpha}-tubulin is loading control. (C) ErbB1 expression level (upper histogram) correlates strongly with the directionality of EF-directed migration (lower histogram). Cells were starved for 3 hours, and then a small EF was applied for 2 hours (0.5 V/cm for rat cell lines, 1.0 V/cm for human cell lines).

 

Figure 3
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  		Fig. 3. ErbB1 expression enhances electrotactic responses. MTC and MTC-B1 cell lines were starved for 3 hours, and then 0.5 V/cm EF was applied for 2 hours. (A) Non-metastatic MTC cell line did not show electrotaxis at 0.5 V/cm (upper panel), after ErbB1 transfection (MTC-B1), cells showed significant strong electrotaxis (lower panel). Red lines and yellow arrowheads represent trajectories and direction of cell movement. (B) Bar graphs showing that ErbB1-transfected cells (MTC-B1) move faster and with greater anodal directedness than MTC cells. *P<0.01 compared with MTC parental cells. Data are mean ± s.e.m. from three independent experiments.

 

Figure 4
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  		Fig. 4. ErbB1 dependence of electrotactic migration. (A) Inhibition of EGF receptor signalling with AG1478 abolished electrotactic response in mammary cancer cells (see supplementary material Movie 5). Cells were plated on collagen-1 coated dishes overnight, starved in serum-free {alpha}-MEM for 3 hours before the experiment, with or without 2 µM AG1478. An EF of 1 V/cm was applied for 2 hours. Red lines and blue arrowheads represent trajectories and direction of cell movement. (B) Trajectories of cells over 2 hours with the starting points positioned at the origin. x- and y-axes give distance in µm. (C) AG1478 inhibited displacement speed in EF-induced MTLn3 electrotaxis (*P<0.001 compared with EF alone), but cells in AG1478 group still moved faster than no EF control, (^P<0.01 compared with no EF control). There are no differences in trajectory speed between the two groups. (D) Anodal directedness was largely suppressed by AG1478 to the same value as no EF control *P<0.001 compared with EF alone. Results were calculated from the means of three independent experiments.

 

Figure 5
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  		Fig. 5. ErbB2/ErbB3 expression enhances directed migration in tumour cell electotaxis. MTLn3 and MDA-MB-435 cells were transduced with either empty vector (PLXSN alone) or PLXSN containing the cDNAs for ErbB1, ErbB2 or ErbB3. Cells were plated on collagen-1-coated dishes overnight and starved in serum-free {alpha}-MEM for 2-3 hours before EF exposure. (A) An EF of 0.5 V/cm was applied to MTLn3 cells for 2 hours. *P<0.05, **P<0.01 compared with MTLn3-PL cells. (B) An EF of 2 V/cm was applied to MDA-MB-435 cells for 2 hours. Directedness of cell migration in an EF was increased for all three lines transduced with ErbB1, ErbB2 or ErbB3. *P<0.05; **P<0.01 compared with 435-PL cells. Data are mean ± s.e.m from three independent experiments on more than 200 cells.

 

Figure 6
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  		Fig. 6. PI3K and Rho family small GTPases are partly involved in the directional migration of tumour cells in electrotaxis. (A,B) MTLn3 cells were pretreated with Ly294002 (50 µM) or Genistein (100 µM; Geni) for 2 hours before EF application. PI3K inhibitor Ly294002 (Ly) only slightly (but statistically significantly) inhibited displacement speed and anodal directedness (*P<0.01 compared with EF alone), but Genistein can significantly block migration speeds and directedness in electrotaxis (**P<0.001 compared with EF alone). (C,D) Cells were pretreated with Toxin B (10 ng/ml), or the peptide LS201 (RhoA inhibitor, 100 ng/ml), LS202 (Rac inhibitor, 100 ng/ml) or LS203 (Cdc42 inhibitor, 100 ng/ml) for 2 hours before EF stimulation. *P<0.01 compared with no inhibitor control; ^P< 0.05 compared with LS201, LS202; ^^P<0.01 compared with LS202. Data are presented as mean ± s.e.m. Data shown are from three independent experiments. EF=1.5 V/cm, 2 hours.

 

Figure 7
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  		Fig. 7. Phosphorylation of ERK1/2 is required in EF-induced tumour cell migration. (A,B) An EF of 1.5 V/cm enhanced activation of ERK 1/2 in mammary breast cancer cells (MTLn3). The expression of active ERK1/2 increased within 15 minutes (m) of EF application, reached a maximum at 30 minutes that lasted for at least 1 hour, whereas the total level of ERK remained unchanged. Each membrane was a representative of two to three repeated experiments. (C) Inhibition of ERK partly inhibited EF-induced MTLn3 cell migration. Cells were pretreated with 50 µM U0126 (ERK-specific inhibitor) for 2 hours before application of 0.5 V/cm EF. *P<0.01 compared with EF alone; ^P<0.01 compared with no EF control.

 

Figure 8
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  		Fig. 8. Schematic diagram of the electrical gradient in the breast duct. A 30 mV transepithelial electrical potential difference (TEP) (the difference between the apical and the basolateral potentials) exists, with the lumen side positive (Faupel et al., 1997Go). This would generate a voltage of 6 V/cm across the epithelium layer (30 mV over 50 µm), which is up to ten times greater than the EF strengths used in our experiments. The vector of this EF (minus to plus) with the anode in the lumen coincides with the direction of the first metastasis of breast cancer cells, in which the cells migrate into the lumen (Wellings and Jensen, 1973Go).

 

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