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Clinical Cancer Research Vol. 11, 5381-5389, August 1, 2005
© 2005 American Association for Cancer Research

Human Cancer Biology

Voltage-Gated Sodium Channel Expression and Potentiation of Human Breast Cancer Metastasis

Scott P. Fraser1, James K.J. Diss1,2, Athina-Myrto Chioni1, Maria E. Mycielska1, Huiyan Pan1, Rezan F. Yamaci4, Filippo Pani1, Zuzanna Siwy7, Monika Krasowska7, Zbigniew Grzywna7, William J. Brackenbury1, Dimis Theodorou1, Meral Koyutürk5, Handan Kaya6, Esra Battaloglu4, Manuela Tamburo De Bella3, Martin J. Slade3, Robert Tolhurst3, Carlo Palmieri3, Jie Jiang3, David S. Latchman2, R. Charles Coombes3 and Mustafa B.A. Djamgoz1

Authors' Affiliations: 1 Neuroscience Solutions to Cancer Research Group, Department of Biological Sciences, Imperial College London; 2 Medical Molecular Biology Unit, Institute of Child Health, University College; 3 Department of Cancer Medicine, CRC Laboratories, Medical Research Council Cyclotron Building, Imperial College School of Medicine, London, United Kingdom; 4 Department of Molecular Biology and Genetics, Bogazici University; 5 Department of Histology and Embryology, Kadir Has University; 6 Department of Pathology, Marmara University, Medical School, Istanbul, Turkey; and 7 Department of Physical Chemistry and Technology of Polymers, Silesian Technical University, Gliwice, Poland

Requests for reprints: Mustafa B.A. Djamgoz, Department of Biological Sciences, Imperial College London, Sir Alexander Fleming Building, South Kensington Campus, London SW7 2AZ, United Kingdom. Phone: 20-7594-54370; Fax: 20-7584-2056; E-mail: m.djamgoz{at}imperial.ac.uk.


   Abstract
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Materials and Methods
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 Discussion
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Purpose: Ion channel activity is involved in several basic cellular behaviors that are integral to metastasis (e.g., proliferation, motility, secretion, and invasion), although their contribution to cancer progression has largely been ignored. The purpose of this study was to investigate voltage-gated Na+ channel (VGSC) expression and its possible role in human breast cancer.

Experimental Design: Functional VGSC expression was investigated in human breast cancer cell lines by patch clamp recording. The contribution of VGSC activity to directional motility, endocytosis, and invasion was evaluated by in vitro assays. Subsequent identification of the VGSC {alpha}-subunit(s) expressed in vitro was achieved using reverse transcription-PCR, immunocytochemistry, and Western blot techniques and used to investigate VGSC{alpha} expression and its association with metastasis in vivo.

Results: VGSC expression was significantly up-regulated in metastatic human breast cancer cells and tissues, and VGSC activity potentiated cellular directional motility, endocytosis, and invasion. Reverse transcription-PCR revealed that Nav1.5, in its newly identified "neonatal" splice form, was specifically associated with strong metastatic potential in vitro and breast cancer progression in vivo. An antibody specific for this form confirmed up-regulation of neonatal Nav1.5 protein in breast cancer cells and tissues. Furthermore, a strong correlation was found between neonatal Nav1.5 expression and clinically assessed lymph node metastasis.

Conclusions: Up-regulation of neonatal Nav1.5 occurs as an integral part of the metastatic process in human breast cancer and could serve both as a novel marker of the metastatic phenotype and a therapeutic target.

Breast cancer is the most common cancer of women and the second leading cause of female cancer mortality, accounting for about 10% of all cancer deaths in the western world (1, 2). To date, several breast cancer metastasis–associated genes have been identified both individually and in combination in microarray analyses (3, 4). These include oncogenes (e.g., ras and c-myc), cell cycle–associated markers (e.g., Ki67), adhesion molecules (e.g., E-cadherins), motility factors (e.g., hepatic growth factor), growth factors and their receptors (e.g., epidermal growth factor/Her-2 and fibroblast growth factor), and the well-established steroid hormones (e.g., estrogen and progesterone; refs. 3, 4). However, indirect measures of metastatic progression (including size of primary carcinoma, assessment of intratumoral vascular invasion, and lymph node involvement) remain the most widely used methods in clinical management. At present, although it is possible to detect micrometastases, approximately one third of women who seem disease-free at primary diagnosis eventually develop overt metastases (5, 6). Clinicians, therefore, require a more accurate diagnosis to predict the development of metastatic disease.

Ion channels are major signaling molecules expressed in a wide range of tissues where they have significant involvement in determining a variety of cellular functions: proliferation, solute transport, volume control, enzyme activity, secretion, invasion, gene expression, excitation-contraction coupling, intercellular communication, etc. (7). Consequently, ion channel defects (both genetic and epigenetic) are frequently an underlying cause of disease states (e.g., refs. 8–10). Ion channels, including voltage-gated ion channels (i.e., those activated by a change in membrane potential), could similarly have a significant role in cancer. Interestingly, electrodiagnosis has been practiced clinically, although its cellular/molecular basis remains unknown (11). We have shown previously that strongly metastatic human and rat prostate cancer cells express functional voltage-gated Na+ channels (VGSC; refs. 12, 13). Importantly, VGSC activity contributes to many cellular behaviors integral to metastasis, including cellular process extension (14), lateral motility and galvanotaxis (15, 16), transverse invasion (12, 13, 17), and secretory membrane activity (18, 19). Consistent with this, (i) endogenous VGSC levels/activity were increased in a subline of the weakly metastatic LNCaP cells that exhibited significantly greater invasiveness and (ii) overexpression of VGSC alone was sufficient to increase in vitro cellular invasive potential, leading to the conclusion that VGSC activity is necessary and sufficient for cancer cell invasiveness (20).

Carcinomas of the breast and prostate share a number of similar features, including hormone sensitivity, a pronounced tropism for metastasis to bone and tendency for cooccurrence in families (21). A recent in vitro study has shown that the human MDA-MB-231 breast cancer cell line expressed functional VGSCs (22). However, both the molecular nature of the VGSC and its functional relevance to breast cancer in vivo are currently unknown. The present study aimed to determine (i) functional VGSC expression in breast cancer cell lines with a range of metastatic potential, (ii) whether VGSC activity contributed to cellular behaviors integral to metastasis, (iii) the molecular nature of the "culprit" VGSC(s), and (iv) whether VGSC{alpha} expression also occurred in breast cancer in vivo and correlated with metastasis.


   Materials and Methods
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 Materials and Methods
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 Discussion
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Cell culture. MDA-MB-231, MDA-MB-468, and MCF-7 cells were grown in DMEM supplemented with 4 mmol/L L-glutamine and 5% to 10% fetal bovine serum. MCF-10A cells were grown in DMEM/Nut Mix F-12 supplemented with 4 mmol/L L-glutamine, 5% horse serum, 10 µg/mL insulin, 5 µg/mL hydrocortisone, 20 ng/mL epidermal growth factor, and 100 ng/mL cholera toxin.

Electrophysiology and pharmacology. Details of the patch pipettes, solutions, and the whole cell recording protocols were as described previously (12, 13, 23). Experiments on the cell lines were done on at least three separate dishes that had been in culture for 1 to 3 days. Further details are given in Fig. 1 legend. Tetrodotoxin was applied locally to individual cells by a puff pipette. All other compounds were bath applied.



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  		Fig. 1. Voltage-gated membrane currents in a human breast epithelial cell line and human breast cancer cells. A, voltage-gated membrane currents recorded in (left to right) MCF-10A, MCF-7, MDA-MB-468, and MDA-MB-231 cells. The currents were generated by pulsing the membrane potential from a holding voltage of –100 mV, in 5 mV steps, from –60 to +60 mV for 200 ms. Voltage pulses were applied with a repeat interval of 20 seconds. Every second current trace generated is displayed. B, dose-response curve for the effects of tetrodotoxin (TTX) on the VGSC current in MDA-MB-231 cells. The percentage reduction of the peak current at the fourth pulse (to –10 mV) following drug application was plotted as a function of drug concentration. Points, means of >5 different cells; bars, SE. Inset, a typical effect (and recovery) of one concentration of the drug on the inward current. B, the holding potential was –100 mV; the cell was pulsed repeatedly to –10 mV for 40 milliseconds every 20 seconds. The effect of tetrodotoxin was recorded from the fourth pulse following application.

 
Proliferation and toxicity assays. Proliferation was determined using the colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (12). Results were obtained from eight separate experiments (each done in triplicate) with or without 10 µmol/L tetrodotoxin applied for 48 hours. Determination of tetrodotoxin toxicity was as described previously (14).

In vitro assays. Transwell assays were done with cells plated onto a 24-well cell insert with 12-µm pores at a density of 1.5 x 105 cells/mL, according to the manufacturer's instructions (BD Labware, Franklin Lakes, NJ). Cells were allowed to settle for 3 hours and treated appropriately for 7 hours. The number of cells migrating over 7 hours was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (12). Results were compiled as the mean of eight repeats of drug versus control readings from individual dishes. Galvanotaxis was studied and variables determined as described previously (16). Endocytosis, employing horseradish peroxidase as a tracer, was done and effects quantified as described previously (18). Invasion assays were as before (12, 13) with cells plated at 2.5 x 105 cells per well in a chemotactic gradient of 1:10% fetal bovine serum. After 48 hours, invaded cells were quantified using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide.

Reverse transcription-PCRs on breast cancer cells in vitro. Total cellular RNA was isolated from two batches of each of the cell lines by the acid guanidium thiocyanate-phenol-chloroform method (24). VGSC{alpha} degenerate primer screens were then done, as described previously (25) to identify the major VGSC{alpha}s expressed. Reactions designed to amplify specific VGSC{alpha}s were subsequently done on both strongly and weakly metastatic cell line extracts, using primer sequences and reaction annealing temperatures as described previously (25). VGSC{alpha} sequences were submitted to Genbank (accession nos. AJ310882-AJ310887 and AJ310896-AJ310900). Finally, semiquantitative PCRs based on kinetic observation of reactions were carried out as described previously (25) to determine relative VGSC{alpha} expression levels. NADH/cytochrome b5 reductase (hCytb5R) was used to control for the effects of variations in quality and quantity of the initial RNA, efficiency of the reverse transcription, and amplification between samples (25, 26).

"Neonatal" Nav1.5 antibody. A polyclonal antibody (NESOpAb) was generated against a synthetic peptide with an amino acid sequence contained within the extracellular D1:S3 of neonatal Nav1.5/VSENIKLGNLSALRC-NH2. Four rabbits were immunized and antibody purified as described previously (27). The specificity of the antibody for the neonatal splice form of Nav1.5 was validated on cell lines transfected with either neonatal or "adult" Nav1.5 expression plasmids, by Western blotting, immunocytochemistry, and electrophysiology (28).

Immunocytochemistry and immunohistochemistry. Cells were plated on poly-L-lysine-coated coverslips for 48 hours. Paraformaldehyde fixation protocol was standard procedure. NESOpAb was used as the primary antibody. The secondary antibody was swine anti-rabbit conjugated to FITC (DAKO, Glostrup, Denmark). For immunohistochemistry, fresh-frozen or wax-embedded breast biopsies were prepared according to standard protocols. Primary antibody was NESOpAb. Secondary antibody was biotinylated swine anti-rabbit (DAKO). Avidin-biotin complex (DAKO) was then applied according to manufacturer's recommendation and the colour reaction was developed with a diaminobenzidine kit (Vector Laboratories, Burlingame, CA). Digital images were captured using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD) and exported without further manipulation.

Reverse transcription-PCRs on breast biopsy tissues. Total cellular RNA was isolated from 0.1 to 0.5 g pieces of frozen tissue and single-stranded cDNA synthesized as above. Expression of Nav1.5, Nav1.6, and Nav1.7 was then investigated by reverse transcription-PCR (RT-PCR), with hCytb5R reactions also done to control for the quality of the extracted RNA; samples which did not yield evident hCytb5R products were rejected unless UGSC expression was evident. RT-PCRs were carried out on each of at least two cDNA templates, manufactured independently from the same RNA extract. Sequences obtained from the human biopsies were submitted to Genbank (accession nos. AJ310888-AJ310895).

Epithelial cell purification. Epithelial cells were purified as described previously (29). Briefly, tissue was minced and digested in type IV collagenase in RPMI 1640 and 5% FCS, 2 mmol/L L-glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin, 50 units/mL polymixin B, and 2.5 mg/mL amphotericin B until a single cell suspension was achieved. Undigested material was removed and redigested. Epithelial cells were purified and cultured in BCM [DMEM/F-12 (1:1) supplemented with 15 mmol/L HEPES, 2 mmol/L L-glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin, 50 units/mL polymixin B, 2.5 mg/mL amphotericin B, 5 mg/mL insulin, 10 mg/mL apo-transferrin, 100 mmol/L ethanolamine, 1 mg/mL hydrocortisone, and 10 ng/mL epidermal growth factor] containing 10% FCS.

Data analysis. All quantitative data were determined to be normally distributed and are presented as means ± SEs. Statistical significance was determined with Student's t test or {chi}2 test, as appropriate. Results were considered significant at P < 0.05 (*).


   Results
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 Materials and Methods
 Results
Discussion
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Functional voltage-gated Na+ channel expression in breast cancer in vitro: electrophysiology and pharmacology. The essential electrophysiologic characteristics of a normal human breast epithelial cell line and three human breast cancer cell lines with a range of metastatic potentials were determined. Importantly, 70% of the strongly metastatic MDA-MB-231 cells tested (n = 69 of 99) expressed inward currents (representing influx of positive charge) activated by membrane depolarization (Fig. 1A). The inward currents were abolished in Na+-free medium (data not shown), consistent with functional VGSC expression. In contrast, the normal breast epithelial cell line MCF-10A and the weakly metastatic MCF-7 and MDA-MB-468 breast cancer cells (n = 19-72) showed no inward currents (Fig. 1A). Membrane depolarization also activated outward currents (representing efflux of positive charge), which were reduced in line with increased metastatic potential in the cell lines studied (Fig. 1A, left to right). These currents were nearly completely (97%) abolished upon substituting Cs+ for intracellular K+ in MCF-7 cells, consistent with functional voltage-gated K+ channel expression. Resting potentials in the normal extracellular bath medium were also inversely correlated with metastatic potential: MDA-MB-231 (–18.9 ± 2.1 mV), MDA-MB-468 (–31.1 ± 2.2 mV), MCF-7 (–39.9 ± 2.9 mV), and MCF-10A (–49.8 ± 2.6 mV; n = 9-27).

The VGSC currents in the MDA-MB-231 cells were suppressed by tetrodotoxin in a concentration-dependent manner with a concentration for half-blockage (IC50) of 2.7 ± 0.5 µmol/L (n = 6; Fig. 1B), in agreement with functional expression of tetrodotoxin-resistant VGSCs. However, there was a small but consistent significant reduction (9 ± 3%; P < 0.05) in peak current with 100 nmol/L tetrodotoxin, indicating that a tetrodotoxin-sensitive VGSC was also present as a minor component (Fig. 1B). In addition, several clinically relevant antiarrhythmics and local anesthetics, as follows, blocked the VGSC currents with a range of potencies (IC50 values): flecainide (8.2 ± 1.3 µmol/L), mexiletine (11.0 ± 4.4 µmol/L), lidocaine (20.3 ± 3.0 µmol/L), procainamide (911 ± 163 µmol/L), and disopyramide (4,100 ± 200 µmol/L; n = 3-5).

Contribution of voltage-gated Na+ channel activity to metastatic cell behaviors in vitro. The possibility that functional VGSCs found in MDA-MB-231 cells contributed directly to metastatic behavior was examined using assays of (A) motility, (B) endocytosis, and (C) invasion (Fig. 2). These were measured in the presence and absence of tetrodotoxin (10 µmol/L) that would significantly (~80%) block VGSC activity but was nontoxic and did not affect cell proliferation. (A) Directional motility of the MDA-MB-231 cells was suppressed by tetrodotoxin (10 µmol/L). Transwell migration was reduced by 52% (P < 0.01; Fig. 2A1). A lower (200 nmol/L) concentration of tetrodotoxin had no effect (data not shown). In addition, in a direct current electric field, the cells had an anodal occupancy of 94% and this was reduced to 56% following tetrodotoxin treatment, similar to control (i.e., nonfield) conditions (57%; Fig. 2A2). (B) Endocytosis, a measure of secretion and plasma membrane protein internalization, was also reduced by tetrodotoxin (47%) as well as by the removal of extracellular Na+ (53%; P < 0.05 for both). However, the VGSC "opener" aconitine increased endocytosis by 14% (P < 0.05; Fig. 2B). (C) Finally, in a widely used in vitro assay of metastatic cell behavior, tetrodotoxin application inhibited Matrigel invasion of MDA-MB-231 cells by 49% (P < 0.001; Fig. 2C).



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  		Fig. 2. In vitro evidence for VGSC involvement in metastatic MDA-MB-231 cell behaviors. A1, transwell motility data, normalized with respect to the control condition (CON, 100%) and following a 10-hour treatment with 10 µmol/L tetrodotoxin (TTX). A2, galvanotaxis. Superimposed trajectories of 50 cells are shown in each panel, the starting point being at the origin. i, control (no applied electric field); ii, electric field of 3 V/cm; iii, electric field of 3 V/cm with 10 µmol/L tetrodotoxin. B, endocytosis. Histobars, control (CON); 10 µmol/L tetrodotoxin (TTX); Na+-free (SF); 400 µmol/L aconitine (ACN) treatments. C, Matrigel invasion. Each part details control conditions or following treatment with 10 µmol/L tetrodotoxin. *, P < 0.05; **, P < 0.01; and ***, P < 0.001, statistically significant differences.

 
In contrast to the MDA-MB-231 cells, weakly metastatic MCF-7 cells were unable to migrate across transwell filters or invade through Matrigel and their galvanotactic motility and endocytic activity (both significantly weaker, compared with MDA-MB-231 cells) were unaffected by 10 µmol/L tetrodotoxin treatment (data not shown).

Molecular identity of breast cancer voltage-gated Na+ channels in vitro. Using RT-PCR techniques, three VGSC{alpha}s were identified in both MDA-MB-231 and MCF-7 cells: Nav1.5 (tetrodotoxin resistant), Nav1.6 and Nav1.7 (both tetrodotoxin sensitive; Fig. 3A). The overall level of VGSC{alpha} expression was much higher (>100-fold) in MDA-MB-231 compared with MCF-7 cells (Fig. 3B). This higher expression level was primarily due to Nav1.5 (~1,800-fold greater expression in MDA-MB-231 cells), which constituted ~82% of the overall VGSC{alpha} mRNA expression in strongly metastatic cells. Nav1.7 levels, making up most of the remaining ~18%, were also relatively higher in MDA-MB-231 cells. This agrees with the functional VGSC expression specifically in MDA-MB-231 cells being mainly tetrodotoxin resistant. Nav1.6 was expressed at relatively low levels, which were similar in both cell lines.



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  		Fig. 3. Expression of VGSC{alpha} isoforms in breast cancer cells. A, semiquantitative PCR electrophoresis results for Nav1.5, Nav1.6, Nav1.7, and hCytb5R controls done on MDA-MB-231 and MCF-7 cells. Representative PCR cycle numbers for given bands are indicated above the gels. Top, derived from MDA-MB-231 cell extracts; bottom, from MCF-7 extracts. B, proposed relative (%) expression levels of the three VGSC{alpha}s found to occur in the strongly (white columns) and weakly (black columns) metastatic cell lines. In each case, the vertical axis denotes the approximate level of expression with respect to total levels of expression of these three VGSC{alpha}s in the strongly metastatic MDA-MB-231 cells. Relative expression levels were estimated from degenerate screens and semiquantitative PCR data, taken together. C, details of the VGSC{alpha} splice forms expressed in the breast cancer cells. Gel images (left) and idealized bands representing each PCR product are indicated (side, right). 5' and 3' denote D1:S3 5' (neonatal) and D1:S3 3' (adult) alternatively spliced exons. {Delta} denotes forms with both alternatively spliced exons missing. D, Nav1.5 D1:S3 5' splice form amino acid data compared with the 3' form and a VGSC{alpha} consensus sequence for this alternatively spliced exon. The 10 residue "neonatal-specific" sequence to which the neonatal Nav1.5-specific antibody was generated is boxed. Location of adult/neonatal Nav1.5 alternative splicing in the extracellular S3-4 linker of domain 1 is shown.

 
All three VGSC{alpha}s were present in multiple splice forms (Fig. 3C). Importantly, DNA sequencing revealed that Nav1.5 and Nav1.7 were present predominantly in their D1:S3 5'-splice forms characterized by the absence at exon residue 7 of an aspartate (10). This form has previously been found in Nav1.1-Nav1.3, Nav1.6, and Nav1.7. The present study is the first to identify the existence of a D1:S3 5'-splice form of Nav1.5. This differs from the known D1:S3 3'-splice form at 31 nucleotides, resulting in seven-amino-acid substitutions in an extracellular region of the VGSC{alpha} protein (Fig. 3D). All other VGSC{alpha} D1:S3 5'-splice forms differ from their D1:S3 3' counterparts at just one to two amino acids.

Where examined, VGSC{alpha} D1:S3 5'-splice forms have previously been found to be expressed specifically in neonatal tissues (30, 31). We generated a novel D1:S3 5'-splice form–specific antibody and used it to verify that the D1:S3 5'-splice variant of Nav1.5 was indeed neonatal (28). This was shown both by immunohistochemistry and Western blotting, comparing expression in neonatal and adult mouse cardiac muscle (where Nav1.5 is abundant; Fig. 4A and B). Furthermore, application of this antibody to the MDA-MB-231 and MCF-7 cells confirmed expression of the Nav1.5 neonatal D1:S3 5'-splice form protein in the strongly metastatic cells specifically (Fig. 4C and D). Importantly, neonatal Nav1.5 was present in the plasma membrane of the MDA-MB-231 cells, confirmed by Western blots on membrane fractions containing Glut-1, a specific marker of plasma membrane (Fig. 4D).



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  		Fig. 4. Characterization of neonatal Nav1.5 expression. A, immunohistochemical comparison of neonatal Nav1.5 expression (as detected with the neonatal Nav1.5-specific voltage-gated Na+ channel antibody, NESOpAb) to "total" VGSC expression (as detected with a pan-specific VGSC antibody) in mouse heart from neonatal or adult tissue. B, Western blot data showing expression of neonatal Nav1.5 in membrane fractions of neonatal but not adult mouse heart tissue. Membrane fractionation was confirmed through expression of the Glut-1 plasma membrane marker. C, representative images of the plasma membrane staining of (1) MDA-MB-231 but not (2) MCF-7 cells with NESOpAb. Negative controls gave no reaction (not illustrated). The cells were not permeabilized, because the antibody was raised to an extracellular epitope. D, Western blot data showing expression of neonatal Nav1.5 (using NESOpAb) in membrane fractions of (1) MDA-MB-231 but not (2) MCF-7 cells. Membrane fractionation was confirmed through expression of the Glut-1 plasma membrane marker.

 
In vivo expression of neonatal Nav1.5 in human breast biopsy tissues. Neonatal Nav1.5 protein expression was markedly up-regulated in human breast cancer biopsy sections (n = 6), in comparison with normal human breast tissues (n = 4; Fig. 5A). Stained cells were of epithelial origin, as determined by MUC-1 immunoreactivity (not illustrated). Thus, the high level of neonatal Nav1.5 protein expression found earlier in breast cancer in vitro also occurred in vivo. Expression of neonatal Nav1.5 in vivo was further investigated by RT-PCR. In a "double-blind" test, expression of Nav1.5 mRNA (but not Nav1.6 nor Nav1.7) in primary tumors was found to be strongly related to lymph node metastasis (LNM; Fig. 5B). The two characteristics were directly correlated in 14 of the 20 (70%) cases examined, being Nav1.5+/LNM+ (n = 8) or Nav1.5–/LNM– (n = 6; {chi}2 = 8.0; degree of freedom = 3; 0.05 > P > 0.01). There was no case of Nav1.5–/LNM+; that is, metastasis to lymph nodes did not occur when Nav1.5 was not detectable in the primary tumor. In a further case of a patient with bilateral breast cancer, Nav1.5 expression matched the occurrence of respective LNM: Nav1.5 was present in breast cancer with LNM (10 of 12) but absent from the contralateral breast with no LNM. Importantly, Nav1.5 products were sequenced for 11 of the 14 Nav1.5+ cases and 10 (91%) were found to be the neonatal splice form. In addition, we were also able to readily detect neonatal Nav1.5 mRNA expression in three of five epithelial cell populations purified from primary breast tumors (data not shown).



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  		Fig. 5. Correlation of neonatal Nav1.5 expression and breast cancer progression. A, immunohistochemical staining of human breast tissues with NESOpAb. Little staining was detected in normal human breast tissue as illustrated in (i and ii), whereas strong heterogeneous staining was detected in the corresponding image from breast cancer tissue (iii and iv). Bright field images of the sections (i and iii); corresponding phase-contrast images (ii and iv) to show the epithelial structure. The epithelial nature of the stained tissue was verified using an antibody raised against the epithelial marker MUC-1 (not illustrated). Controls done on H&E-stained breast biopsies by preabsorbing the primary antibody with the immunizing peptide did not yield evident staining (vi), in contrast to sections stained with NESOpAb (Av). Bar, 50 µm. B, electrophoresis results of Nav1.5, Nav1.6, Nav1.7, and hCytb5R control RT-PCRs done on 20 breast cancer tissue samples. LNM data for each sample are indicated above the gel images. Multiple bands corresponding to the evident splice form products (as previously described in Fig. 3 and ref. 25; left). PCRs were done for 55, 40, 40, and 30 cycles for Nav1.5, Nav1.6, Nav1.7, and hCytb5R tests, respectively. (+), LNM was present; (–), LNM was not clinically evident.

 

   Discussion
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 Materials and Methods
 Results
 Discussion
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The present study shows (i) that strongly but not weakly nor nonmetastatic breast cancer cells, displayed VGSC currents, mainly composed of a tetrodotoxin-resistant component; (ii) that blockage of the VGSC suppressed several metastatic cell behaviors in vitro; (iii) that a particular tetrodotoxin-resistant VGSC{alpha}, Nav1.5, in its newly characterized neonatal splice form, was predominant in strongly metastatic cells; and (iv) that neonatal Nav1.5 protein was markedly up-regulated in clinical breast cancer samples and that Nav1.5 mRNA expression in biopsy samples correlated strongly with clinically assessed lymph node metastasis.

Up-regulation of voltage-gated Na+ channel activity and enhancement of metastatic cell behaviors in vitro. MDA-MB-231 cells expressed a functional VGSC that was predominantly tetrodotoxin resistant. Weakly metastatic/nontumorigenic cell lines did not express functional VGSCs. These results agree with the basic findings of Roger et al. (22). Importantly, the high-level VGSC expression was accompanied by much reduced outward currents in the MDA-MB-231 cell line. Although outward currents are known to play a role during the cell cycle in breast cancer cells (e.g., ref. 32), any significance of the reduction of the outward currents with increased metastatic potential in the cell lines studied remains to be investigated. Nevertheless, the specific combination of reduced outward and emergent VGSC inward currents would render these cells potentially more excitable in line with their "hyperactive" metastatic character.

The effectiveness of tetrodotoxin under resting conditions (in recordings and in vitro assays) would be consistent with VGSCs being tonically active in these cells. Indeed, Roger et al. showed there to be a "window current" between more than –60 and less than –20 mV, covering the prevailing resting membrane potential of approximately –19 mV. Furthermore, the concentration of tetrodotoxin (10 µmol/L) required to produce a functional effect was consistent with (a) Nav1.5 being the VGSC underlying this behavior (at least in the in vitro migration assay where this was specifically tested) and (b) the patch-clamp pharmacology. The molecular mechanisms through which VGSC activity could potentiate directional motility, endocytosis, and invasion, could be direct and/or indirect. Direct effects could involve protein-protein interactions with cytoskeletal or extracellular matrix elements. Indeed, VGSCs physically associate, either via protein-binding domains in the major VGSC{alpha} or the auxillary VGSCßs, to ankyrin, contactin, neurofascin, and tenascin (33–37). In addition, Nav1.5 is one of only two VGSC{alpha}s that has PDZ domains that could also enable cytoskeletal interactions. Indirect effects could involve a number of intracellular signaling mechanisms. In particular, changes in intracellular Na+, Ca2+, and/or H+ could occur locally as a result of VGSC activity and lead to a variety of cellular effects that could contribute to metastasis. As well as effects upon motility and secretion, such changes could underlie more complex interactive functions such as gene expression, possibly in a feedback fashion (e.g., ref. 38).

Up-regulation of neonatal Nav1.5 voltage-gated Na+ channel in metastatic breast cancer in vitro. RT-PCR showed that the predominant VGSC{alpha} expressed was Nav1.5, in agreement with the mainly tetrodotoxin-resistant nature of the VGSC currents recorded. In fact, Nav1.5 was expressed at ~1,000-fold higher levels in these strongly versus weakly metastatic cells. Regarding the other two minor VGSC{alpha}s expressed, Nav1.6 was mainly present in its highly truncated "fail-safe" form and would not be functional (39). On the other hand, Nav1.7 may account for the minor tetrodotoxin-sensitive component of the VGSC currents but its functional relevance, if any, it is not yet known.

Sequencing of Nav1.5 PCR products revealed that Nav1.5 transcripts predominantly possessed the D1:S3 5' rather than the 3' exon, being described here for the first time. For other VGSC{alpha}s with alternate D1:S3 forms, the 5' exon is classically associated with neonatal expression (30, 31). Indeed, this was confirmed to also be the case for Nav1.5 using a novel splice form-specific polyclonal antibody. Expression of the neonatal form of the culprit VGSC is consistent with the concept of oncofetal gene expression (e.g., refs. 40, 41). Nevertheless, it is not clear at this stage whether neonatal Nav1.5 specifically is required for the proposed role of VGSC activity in breast cancer metastasis. Bennett et al. (20) have shown that invasion of human prostate cancer cells can be potentiated by the overexpression of a VGSC{alpha} (Nav1.4) other than that normally predominant (Nav1.7) in prostate cancer (25). Further work is required to elucidate whether neonatal Nav1.5 is the only VGSC{alpha} subtype that can enhance metastastic cell behavior in breast cancer.

At present, the mechanism(s) responsible for the up-regulation of Nav1.5 are not clear. Steroid hormones, especially estrogen, and growth factors (e.g., epidermal growth factor and fibroblast growth factor) are possible candidates, because breast epithelial tissue homeostasis and breast cancer onset/progression are under their strong influence. Epidermal growth factor has been shown to up-regulate VGSC functional expression (42–44). A functional association between fibroblast growth factor and Nav1.5 has also been described (45). Importantly, the Nav1.5 gene (SCN5A) core promoter has been characterized (46) and contains two putative estrogen receptor–binding ERE half-sites.8

Expression of neonatal Nav1.5 in vivo: clinical implications. Taken together, the in vivo data were highly consistent with the in vitro findings regarding both increased VGSC expression with breast cancer progression (metastasis) and the molecular identity (neonatal Nav1.5) of the candidate underlying VGSC{alpha}. The strong positive correlation between VGSC{alpha} expression and LNM in breast cancer biopsy tissue would suggest that VGSCs could act as an independent prognostic variable in a multivariant approach to this problem. Furthermore, the nature of involvement of VGSC activity in metastatic cell behavior is such as to make it likely that VGSC expression/up-regulation is an early event in the progression of breast cancer to the metastatic mode. The neonatal Nav1.5 may also have therapeutic potential, in two main respects. First, the pharmacologic data indicated that neonatal Nav1.5 was blocked by clinically important antiarrhythmics and local anesthetics; consistent with this, flecainide and mexiletine significantly inhibited endocytic activity in MDA-MB-231 but not MCF-7 cells (data not shown). Although not specifically tested here, it is possible that some such agents would block the neonatal form of the channel more than the adult and could thus be used clinically against metastatic breast cancer, with minimized side effects. Second, the antibody to the neonatal splice form of Nav1.5 (recognizing an extracellular epitope) might itself be a novel, specific mechanism for targeting metastatic breast cancer in the adult (28). Interestingly, tamoxifen, a major anti-breast cancer drug, has been shown to strongly reduce VGSC activity (47, 48).

In conclusion, our results show that a novel neonatal splice form of Nav1.5 is significantly up-regulated during breast cancer progression and potentiates a series of cell behaviors integral to the metastatic cascade. Accordingly, neonatal Nav1.5 may have diagnostic and therapeutic potential in the clinical management of breast cancer.


   Footnotes
 
Grant support: Cancer Research UK (M.B.A. Djamgoz and R.C. Coombes); Breast Cancer Research Trust (M.J. Slade); Cancer Research Trust, KAV (M. Koyutürk); Medical Research Council, UK (W.J. Brackenbury); Pro Cancer Research Fund (M.B.A. Djamgoz, S.P. Fraser, and F. Pani); and Pro Cancer Research Fund Amber Fellowships (A-M. Chioni and H. Pan).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: S.P. Fraser and J.K.J. Diss contributed equally to this work.

8 J.K.J. Diss, unpublished analysis. Back

Received 2/11/05; revised 4/13/05; accepted 4/18/05.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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