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First published online September 19, 2007
doi: 10.1242/10.1242/jcs.002774
Research Article |
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
Author for correspondence (e-mail: m.zhao{at}abdn.ac.uk)
Accepted 9 July 2007
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Summary |
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Key words: Breast cancer cells, Electric field, Migration, ErbBs, Tumour metastasis
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). The molecular events in breast cancer metastasis are not fully understood. To establish secondary tumours, cancer cells have to invade surrounding tissues and blood vessels and migrate away from the primary site (Banyard and Zetter, 1999; Lawrence and Steeg, 1996). The metastatic potency of tumours is associated with protrusive and motile activity of tumour cells (Chicoine and Silbergeld, 1995; Verschueren et al., 1994). Various environmental factors including extracellular matrix, cytokines, and growth factors from autocrine and paracrine sources have been demonstrated to create chemotactic signals to stimulate migration of mammary cancer cells (Segall et al., 1996; Kundra et al., 1994; Levine et al., 1995). Overexpression of epidermal growth factor (EGF) receptor in breast cancer cell results in significantly increased intravasation and metastasis from the primary tumour (Xue et al., 2006a; Hirsch et al., 2006).
Many motile cells sense naturally occurring physiological electrical gradients in extracellular spaces and show directional migration (galvanotaxis or electrotaxis) (McCaig and Zhao, 1997; McCaig et al., 2002; McCaig et al., 2005; Pu and Zhao, 2005; Robinson, 1985; Wang et al., 2003a; Wang et al., 2003b; Zhao et al., 1996; Zhao et al., 1999; Pullar and Isseroff, 2005; Pullar et al., 2006). A role for electric fields (EFs) in directing cell migration during development and in wound healing has been suggested (Nuccitelli, 2003; McCaig and Zhao, 1997; McCaig et al., 2005); however, accumulating evidence suggests that EFs may have a much more important role in wound healing than previously thought (Zhao et al., 2006). Normal breast epithelial cells maintain an asymmetric potential gradient of +30 mV between apical and basolateral surfaces (a transepithelial electrical potential difference or TEP; inside negative with respect to the lumen, outside) because of the generation of ionic gradients. As epithelial cells divide, the charge gradient across the epithelial layer is dissipated, resulting in electrical depolarisation (Faupel et al., 1997). When a cancer develops, epithelial cells in certain areas of the breast divide more rapidly than cells in normal areas, and this is accompanied by membrane depolarisation, which can produce a significantly greater electropotential difference on the skin surface over invasive cancer than benign lesions (Marino et al., 1994; Binggeli and Weinstein, 1986; Faupel et al., 1997). The electrical signal is measurable on the skin surface above the breast lesion and this non-invasive electropotential measurement has been used as a new clinical method for breast cancer diagnosis and evaluation of invasive potency (Cuzick et al., 1998; Faupel et al., 1997; Marino et al., 1994). In addition, a strongly metastatic phenotype is associated with the expression of voltage-gated Na+ channels (VGSCs) and inhibition of these channels strongly inhibits lateral motility and invasiveness of metastatic cancer cells. A voltage-gated Na+ channel opener enhances electrotaxis of highly metastatic cells, but modulation of Na+ channel activity does not affect electrotaxis in weakly metastatic cells (Grimes et al., 1995; Laniado et al., 1997; Fraser et al., 1999; Djamgoz et al., 2001). High-level VGSC expression is usually accompanied by fast inward Na+ currents in metastatic breast cancer cells. By contrast, normal breast epithelial cells and weakly metastatic breast cancer cells show no inward current (Fraser et al., 2005; Carter and Coffey, 1988).
Because there is a steady electrical gradient between tumour and normal tissues during metastasis (Cuzick et al., 1998; Marino et al., 1994), we asked whether tumour cells detect and respond to such signals. In chemotaxis, tumour cells move up a chemical gradient. Ligands bind to specific receptors, and signal transduction cascades are activated, leading to directional migration. The EGF receptor, ErbB1 is involved in the metastatic process. Overexpression of ErbB1 can increase chemotaxis to EGF (Bailly et al., 1998; Bailly et al., 2000; Gschwind et al., 2002; Dittmar et al., 2002). Activation of ErbB2/ErbB3 heterodimers can affect both proliferation and motility (Xue et al., 2006b).
To dissect the mechanisms underlying the direction of tumour cell migration in electrotaxis and explore whether ErbB1 signalling (receptor tyrosine kinase pathways) plays an important role during tumour invasion and metastasis, we compared the electrotactic responses of breast cancer cell lines with different ErbB expression levels. We show that the ErbB1 is a key element mediating tumour cell galvanotaxis and that its expression levels correlate with metastatic potential.
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Electrotaxis of tumour cells correlates with metastatic potential and ErbB1 expression levels
To assay whether tumour cells of varying metastatic potential show altered galvanotaxis in a small electric field, we compared non-metastatic (MTC) and highly metastatic (MTLn3) cells lines from a common chemically induced tumour. MTLn3 cells showed strong electrotaxis towards the anode at a low EF strength of 0.5 V/cm. The directedness of MTLn3 was three times greater than MTC cells (P<0.001). Highly metastatic cells also moved 2.5 times faster than non-metastatic cells in response to 0.5 V/cm electric field (P<0.001) (Fig. 2A). This indicates that the responsiveness of mammalian breast cancer cells to direct current EFs is correlated with the metastatic potential of the clone.
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). To test whether ErbB1 expression is also associated with the electrotactic response, we detected ErbB1 protein levels in human and rat breast cancer lines on western blot and compared them with cell directedness in EFs. We found that highly metastatic cell lines MTLn3 (rat) and MDA-MB-231 (human) have high ErbB1 levels and show strong anodal migration to an EF (0.5 V/cm for rat cell lines, 1.0 V/cm for human cell lines, t=2 hours). Rat MTC cells show a low level of ErbB1 expression and cell directedness was as low as 30% of that in MTLn3 cells. Expression of the human EGF receptor in MTC cells (MTC-B1) can enhance cell directedness to a level similar to MTLn3. ErbB1 levels in the non-metastatic human MCF7 cell line were particularly low and cells displayed a low cathodal response to the EF (Fig. 2B,C). In general, therefore, high levels of ErbB1 expression correlated with strong electrotaxis of breast cancer cells.
ErbB1 expression enhances electrotactic response
To determine whether expression of ErbB1 is involved in the signalling of tumour cell electrotaxis, two cell lines were compared: MTC cells express very low levels of ErbB1, and MTC-B1 cells are MTC cells transfected with an expression vector for the human ErbB1. MTC cells had a weak electrotactic response at 0.5 V/cm (directedness: –0.24±0.07 n=65), but MTC-B1 showed a threefold greater directedness toward the anode at the same EF strength (P<0.001). MTC-B1 cells also migrated faster than MTC cells (Tt/t 18.85±0.81; Td/t 14.10±0.56 P<0.001 n=105, compared with MTC cells) (Fig. 3A,B; supplementary material Movie 4). Transfecting cells with an expression vector for the human ErbB1 therefore markedly enhanced their responsiveness to a small EF.
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Overexpression of ErbB2 or ErbB3 increases tumour-cell-directed migration in electrotaxis
To explore further the effects of other relevant ErbB family members ErbB2 and ErbB3 on the electrotaxis of MDA-MB-435 and MTLn3 mammary tumour cells, we used a series of retroviral vectors based on the PLXSN retrovirus (Riese et al., 1995). MDA-MB-435 and MTLn3 cells were transduced with either empty vector (PLXSN alone) or PLXSN containing the cDNAs for ErbB1, ErbB2 or ErbB3 followed by selection for geneticin resistance. Cells generated by transduction of MDA-MB-435 or MTLn3 cells with PLXSN, ErbB1, ErbB2 or ErbB3 retrovirus were named 435-PL, 435-B1, 435-B2, 435-B3, or MTLn3-PL, MTLn3-B1, MTLn3-B2, MTLn3-B3, respectively (Xue et al., 2006b). We found that MTLn3-B1, -B2 and -B3 showed increased cell directedness to 0.5 V/cm EF compared with MTLn3-PL (P<0.05); MTLn3-B1 also significantly increased migration rate (P<0.01) (Fig. 5A). We also tested the electrotaxis of MDA-MB-435 transfected with different vectors. Upon exposure to an applied EF of 2 V/cm, both 435-B2 and 435-B3 increased cell directedness and displacement speed (P<0.05), but not trajectory rate (P>0.05 compared with 435-PL). 435-B1 not only markedly enhanced cell directedness, but also increased trajectory and displacement speed (P<0.01 compared with 435-PL) (Fig. 5B).
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Signalling of ErbB: involvement of tyrosine kinases, PI3K, Rho GTPases and ERK
MTLn3 cells were exposed to the tyrosine kinase inhibitor Genistein (100 µM) and an EF of 1.5 V/cm. The cells showed significant decreases in anodal directedness. The trajectory and displacement speed also were reduced by 72% and 83% to 12.6±0.6 and 5.1±0.8, respectively (n=56 in EF; n=60 in EF + Genistein, P<0.001). Directedness decreased to –0.21±0.1 (Fig. 6A,B). These results demonstrate that MTLn3 cells lost directional migration and slowed down markedly in the presence of Genistein. Compared with ErbB1 inhibition (AG1478), Genistein was more effective in reducing migration rates during tumour cell electrotaxis, indicating that tyrosine phosphorylation not mediated by the EGF receptor is important for basal cell motility.
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Treatment with 50 µM Ly294002, a PI3K inhibitor, significantly decreased EF-directed anodal migration in MTLn3 cells. However, the inhibition was not complete and the EF-directed migration largely remained (directedness value: –0.78±0.07, n=70, Fig. 6A). Displacement speed was reduced by 28% to 22.2±2.0 (n=70) (P<0.01) (Fig. 6B), indicating partial involvement of PI3K signalling.
Small GTPases contribute to cancer progression primarily through their effects on cell migration, thereby influencing invasion and metastasis (Fiordalisi et al., 2006; Jeong et al., 2005). Toxin B (10 ng/ml) as a general inhibitor of Rho, Rac and Cdc42, significantly decreased MTLn3 cells directedness and migration speed (P<0.001), but the inhibition was incomplete, and significant anodal electrotaxis still occurred (Fig. 6C). Three custom-designed, cell-permeable short peptides were used to inhibit RhoA, Rac and Cdc42 (LS201, LS202 and LS203, respectively) (Vastrik et al., 1999). Each of these peptides reduced tumour cell migration speed (P<0.01) to a similar extent as did Toxin B. LS201 and LS203, but not LS202, also decreased cell directedness (Fig. 6D), suggesting partial involvement of RhoA and Cdc42.
The MAP kinase ERK1/2 plays an important role in EF-directed migration of corneal and lens epithelial cells (Zhao et al., 2002; Wang et al., 2003b). Exposure to an EF activated ERK1/2 in MTLn3 cells (Fig. 7A,B). We observed the effect of ERK1/2 blockade on the electrotaxis of MTLn3 cells in an applied electric field of 0.5 V/cm. U0126 (50 µM) (a specific inhibitor of ERK1/2) resulted in a 32% reduction in directional migration to –0.49±0.05 (n=46) (P<0.001), but did not completely abolish MTLn3 electrotaxis (P<0.001 compared with no EF control) (Fig. 7C). These results demonstrated that ERK signalling partially contributes to tumour cell electrotaxis.
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), and EFs enhance growth of cancer spheroids (Wartenberg et al., 1997; Sauer et al., 1997). We show that breast tumour cells detect a small electrical signal and migrate anodally. The directional migration of breast epithelial cells could be influenced significantly by endogenous TEPs. Normal breast epithelium has a TEP of about +30 mV (duct lumen positive). Such a voltage gradient is larger than the applied direct current EF used to induce electrotaxis in this study (Fig. 8), because given a lumenal wall 
50 µm wide, this would represent a substantial voltage gradient of about 6 V/cm (30 mV/50 µm), around ten times greater than we studied (Faupel et al., 1997). When a cancer develops, malignant epithelial cells choose to move and grow into the lumen rather than into surrounding tissue (Olivotto and Levine, 2001). Furthermore, as metastasis progresses, the duct deforms and the charge gradient across the epithelial layer may dissipate, resulting in collapse of the EF across the epithelial layer. Cancer cells then breach the basement membrane and invade the surrounding tissue and metastasise through the blood and lymph systems (Faupel et al., 1997; Djamgoz et al., 2001).
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). This highlights the cell-specific subtlety of response to electrical cues (McCaig et al., 2005). A further indicator of this complexity and refinement is that lens epithelial cells migrate cathodally below 1.0 V/cm and anodally at and above 1.5 V/cm; both values are within the physiological range (Wang et al., 2000; Wang et al., 2003b). In epithelial cells and human keratinocyte electrotaxis, directedness generally is around 0.6-0.7 at 1.5 V/cm. However, at the same strength, over 90% of MTLn3 tumour cells show perfect migration directly along the EF vector toward the anode, giving a remarkable directedness close to 1. Some tumour cells therefore show much stronger electrotaxis than other cell types. This phenomenon correlated closely with tumour metastatic potential. The 13762NF mammary-tumour-derived clones have been widely used to examine mechanisms of breast cancer metastasis (Segall et al., 1996). The highly metastatic rat breast cancer cell line MTLn3 showed a robust anodal galvanotactic response, whereas non-metastatic MTC cells responded three times less to an electric field of the same strength (directness 0.72±0.01 compared with 0.24±0.07 at 0.5 V/cm). The migration speed of MTLn3 cells was 2.5 times faster than MTC cells. The highly metastatic human breast cancer cell line MDA-MB-231 showed strong anodal electrotaxis and a fast migration speed, whereas weakly metastatic MCF-7 cells moved slowly and surprisingly in the opposite direction – cathodally. Djamgoz et al. (Djamgoz et al., 2001) compared the galvanotactic responses of two prostate cancer cell lines with different metastatic potential. The highly metastatic MAT-lyLU cells moved toward the cathode with strong directness of 0.82±0.01. By contrast, the weakly metastatic AT-2 cells responded weakly (directness is –0.34±0.01) and moved in the opposite direction. These data accentuate the variation in electrotactic responses in different types of tumour cells with different metastatic abilities.
Mechanisms of breast cancer cell electrotaxis – the role of the EGF receptor and other ErbB members
The EGF receptor has been suggested to play a critical role in the electrotactic response of corneal epithelial cells and keratinocytes (Zhao et al., 2002; Fang et al., 1999). Application of a small EF upregulates the expression of EGF receptors and induces asymmetrical distribution of EGF receptors, and their downstream signalling element, MAP kinase ERK1/2 and filamentous actin to the cathode-facing leading lamellipodia (Zhao et al., 1999; Zhao et al., 2002). Internalisation of EGF receptors remained concentrated behind the leading edge of cells, potentially continuing to contribute to polarisation of the tumour cell (Bailly et al., 2000). Kaufmann et al. (Kaufmann et al., 1996) showed that induction of apoptosis by EGF receptors in rat mammary adenocarcinoma cells coincides with enhanced spontaneous tumour metastasis. ErbB1 signalling plays a crucial role in metastatic migration of breast cancer cells (Goswami et al., 2005; Wyckoff et al., 2004). Implantation of MTC-B1 clone intravenously into mice led to enhanced metastasis (Lichtner et al., 1995). We demonstrated that ErbB1 expression levels correlate well with the electrotactic response. MTC cells with low ErbB1 expression levels showed a weak response. Transfection of the human EGF receptor into MTC cells enhanced EF-directed migration, with both migration speed and directedness markedly increased (Fig. 3A,B; supplementary material Movie 4). This restoration and amplification of EF responsiveness indicates that the EGF receptor signal transduction pathway is involved in electrotactic responses in these cancer cells. In further support of this, MTLn3 cells treated with AG1478, a specific inhibitor of ErbB1, showed a 90% reduction of EF-directed migration, leaving directedness close to zero, although trajectory speed remained unaffected (Fig. 4; supplementary material Movie 5). Genistein – a broad spectrum receptor tyrosine kinase inhibitor – completely inhibited EF-directed cell migration. These results indicate that EGF receptor activation is required for electrotaxis of these tumour cells.
In addition to the key role of ErbB1 in tumour metastasis, a human breast cancer tumour tissue microarray revealed a significant association between ErbB2 and ErbB3 expression and metastasis. Suppression of ErbB2 or ErbB3 expression significantly reduced intravasation and metastasis (Xue et al., 2006b). To identify the potential contributions of ErbB2- or ErbB3-dependent motility responses to tumour metastasis, we evaluated the effects of overexpressing of ErbB on the electrotaxis of MDA-MB-435 and MTLn3 tumour cells. We found that enhancing ErbB2 or ErbB3 expression markedly increases tumour cell directedness and displacement speed in EFs although the effects of ErbB2 and ErbB3 are not as strong as that of ErbB1 (Fig. 4). Our results propose that ErbB2- and ErbB3-dependent signalling can contribute to metastasis through tumour cell motility in electrotaxis.
Downstream signalling of EGF receptor and ErbB family
Activation of ErbB1 stimulates several downstream pathways. The phosphatidylinositol 3-kinase (PI3K) signalling pathway plays a crucial role in the electrotactic response of epithelial cells in wound healing (Zhao et al., 2006). It is a key regulator of the actin cytoskeleton and cell migration in various cancer cells (Verbeek et al., 1998; Ellerbroek et al., 2001; Hill et al., 2000). In squamous cell carcinoma of the head and neck (HNSCC), PI3K is required for ErbB1-mediated cell movement but not for EGF-induced mitogenesis (Chen et al., 1994; O-charoenrat et al., 2002). However, PI3K inhibition only partially decreased the electrotaxis of MTLn3 cells (Fig. 6A,B). Similarly, the Rho family small GTPases Rho and Cdc42, and the MAP kinase ERK1/2 are involved, but they only played a partial role in EF-induced migration in MTLn3 cells.
In summary, we demonstrated that breast cancer cells migrate directionally in an EF; this response correlates with metastatic potential and the expression level of the EGF receptor. Highly metastatic tumour cells show robust electrotaxis and move anodally. Transfection and expression of the EGF receptor and three ErbB family members in weak metastasis tumour cells significantly enhanced the electrotactic response. Inhibition of the EGF receptor activation abolished the electrotactic response. We thus conclude that EGF receptor signalling is essential for the electrotactic responses of breast cancer cells. Identification of potential roles for endogenous or applied EFs during metastasis and their underlying molecular mechanisms could lead to novel potential therapeutic strategies.
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; Welch et al., 1983). MTC cells were transfected with retroviral expression vectors containing the human ErbB1 gene (EGFR) with a neomycin-resistance gene, termed MTC-B1. The human breast cancer cells MDA-MB-435 and MTLn3 cells were subjected to retroviral transduction of constructs containing the empty retroviral expression vector pLXSN alone or pLXSN containing the human cDNAs for ErbB1, ErbB2 and ErbB3 (Vectors came from David Stern, Yale University) (Riese et al., 1995). All experiments were conducted within 10 passages. Expression of transduced ErbBs measured by fluorescence-activated cell sorting (FACS) showed no change with passage in vitro (Xue et al., 2006b). Cells were grown in 
-MEM (Gibco), supplemented with 5% fetal bovine serum (FBS) and 50 U/ml penicillin and 50 µg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO2. For all experiments, unless otherwise mentioned, cells were plated in -MEM supplemented with 5% FBS for 16-20 hours at low density in a specially made culture chamber formed by two parallel strips of glass coverslip (4 cm long) adhering to the base of collagen 1 coated plastic dishes (Zhao et al., 1996). Cells were starved for 3 hours prior to the experiment in -MEM supplemented with 0.35% BSA (starvation medium). Human breast cancer cell lines MDA-MB-231 and MCF 7 were grown in RPMI 1640 medium supplemented with 5% FCS and antibiotics. Cells were plated on collagen-1-coated dishes and starved for 3 hours prior to experimentation. Experiments were performed with subcultured cells in passage 5. All chemicals were added to the cells 2 hours before exposure to the electric field. AG1478, LY294002 and Toxin B were purchased from Calbiochem (La Jolla, CA) and Genistein was obtained from Sigma. LS201, LS202, LS203 (which inhibit RhoA, Rac and Cdc42, respectively) are custom designed peptides synthesised and prepared by the Proteomics Facility, University of Aberdeen (Vastrik et al., 1999).
Electrotaxis assay
Cell motility was assayed using a galvanotaxis apparatus described in detail by Zhao et al. (Zhao et al., 1996). Briefly, direct current was applied through agar-salt bridges connecting silver/silver chloride electrodes in beakers of Steinberg's solution, to pools of culture medium on either side of the chamber. A roof of No. 1 coverglass was applied and sealed with silicone grease (Corning DC4). The final dimensions of the chamber, through which current was passed, were 40 mm x 10 mm x 0.3 mm. Immediately before EF application, CO2-independent medium (including 5% FBS) was transferred into the culture chambers, using a push-pull technique. Cells were exposed to 0.5-4 V/cm EF for 1-3 hours at 37°C and viewed on an inverted microscope in a temperature-controlled chamber. The serial time-lapse phase-contrast images were recorded using Metamorph (Universal Imaging Corporation, PA).
Western blot analysis
Tumour cells were rinsed with cold PBS and lysed with lysis buffer [10 mM Tris-HCl, 50 mM NaCl, 5 mM EDTA, 50 mM sodium fluoride, 1% Triton X-100, 30 mM Na4P2O7, 1 mM sodium orthovanadate and protease inhibitor cocktail (Boehringer)]. Equal amounts of protein lysates were electrophoresed in 6% SDS-PAGE for ErbB1 or 4-12% SDS-PAGE for ERK detection, followed by electroblotting onto nitrocellulose membranes (Invitrogen). The gels were stained for remaining proteins with Coomassie Blue as a loading control. Membranes were stained with Ponceau S for detection of transfer efficiency, then were blocked with 5% milk TBS (pH 7.4) with 0.1% (w/v) Tween 20 for 1 hour. Membranes were incubated with relevant primary antibodies (1:1000) in 5% BSA TBS-T overnight at 4°C (anti-ErbB1 was from BD Biosciences, anti-active and anti-total ERK 1/2 antibody was from Cell Signaling). Relevant secondary antibodies with horseradish peroxidase (1:3000, Amersham Pharmacia Biotech, Amersham, UK) were incubated for 1 hour at room temperature, and the immunoblots were detected by an enhanced chemiluminescence (ECL) detection system (Amersham).
Quantitative analysis of cell behaviour
Cell migration was analysed to determine directedness and average migration speed by tracing the position of cell nuclei at frame intervals of 2 minutes or 5 minutes using Metamorph software. The directedness of migration was assessed as cosine 
(Zhao et al., 1996), where is the angle between the EF vector and a straight line connecting the start and end position of a cell. A cell moving directly along the field lines toward the cathode would have a directedness of 1; a cell moving directly toward the anode would have a directedness of –1; a value close to 0 represents random cell movement. The cosine will provide a number between –1 and +1 and the average of all of the separate cell events yields the directedness index. The average directedness of a population of cells gives an objective quantification of the direction cells have moved. The trajectory speed (Tt/T) is the total migration distance of cells per hour, and the displacement speed (Td/T) is the straight-line distance between the start and end positions of a cell each hour. Dx/T is an x-axis displacement speed, which represents the ability of cells to migrate along the EF vector. Td/Tt, Dx/Tt and Dx/Td are a series of coefficients of movement efficiency. A value of 1 indicates cells moving persistently along one straight line in one direction, and 0 for random movement. Statistical analyses were made using unpaired, two-tailed Student's t-test. Data are expressed as mean ± s.e.m.
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* Present address: Department of Dermatology, University of California, Davis, CA 95816, USA 
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References |
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