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 voltagegated 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 a-subunit(s) expressed in vitro was achieved using reverse transcription-PCR, immunocytochemistry, and Western blot techniques and used to investigate VGSCa 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 Na v 1.5, 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 Na v 1.5 protein in breast cancer cells and tissues. Furthermore, a strong correlation was found between neonatal Na v 1.5 expression and clinically assessed lymph node metastasis. Conclusions: Up-regulation of neonatal Na v 1.5 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 hav...

Upregulation of functional voltage-gated Na + channels (VGSCs) occurs in metastatic human breast cancer (BCa) in vitro and in vivo. The present study aimed to ascertain the specific involvement of the 'neonatal' splice variant of Nav1.5 (nNav1.5), thought to be predominant, in the VGSC-dependent invasive behaviour of MDA-MB-231 cells. Functional activity of nNav1.5 was suppressed by two different methods targeting nNav1.5: (i) small interfering RNA (siRNA), and (ii) a polyclonal antibody (NESO-pAb); effects upon migration and invasion were determined. nNav1.5 mRNA, protein and signalling were measured using real-time PCR, Western blotting, and patch clamp recording, respectively. Treatment with the siRNA rapidly reduced (by ~90 %) the level of nNav1.5 (but not adult Nav1.5) mRNA, but the protein reduction was much smaller (~30 %), even after 13 days. Nevertheless, the siRNA reduced peak VGSC current density by 33 %, and significantly increased the cells' sensitivity to nanomolar tetrodotoxin (TTX). Importantly, the siRNA suppressed in vitro migration by 43 %, and eliminated the normally inhibitory effect of TTX. Migrated MDA-MB-231 cells expressed more nNav1.5 protein at the plasma membrane than non-migrated cells. Furthermore, NESO-pAb reduced migration by up to 42 %, in a dosedependent manner. NESO-pAb also reduced Matrigel invasion without affecting proliferation. TTX had no effect on cells already treated with NESO-pAb. It was concluded that nNav1.5 is primarily responsible for the VGSC-dependent enhancement of invasive behaviour in MDA-MB-231 cells. Accordingly, targeting nNav1.5 expression/activity may be useful in clinical management of metastatic BCa.

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In developmentally regulated D1:S3 splicing of Nav1.5, there are 31 nucleotide differences between the 5'-exon ('neonatal') and the 3'-exon ('adult') forms, resulting in 7 amino acid differences in D1:S3-S3/S4 linker. In particular, splicing replaces a conserved negative aspartate residue in the 'adult' with a positive lysine. Here, 'neonatal' and 'adult' Nav1.5 alpha-subunit splice variants were stably transfected into EBNA-293 cells and their electrophysiological properties investigated by whole-cell patch-clamp recording. Compared with the 'adult' isoform, the 'neonatal' channel exhibited (1) a depolarized threshold of activation and voltage at which the current peaked; (2) much slower kinetics of activation and inactivation; (3) 50% greater transient charge (Na(+)) influx; (4) a stronger voltage dependence of time to peak; and (5) a slower recovery from inactivation. Tetrodotoxin sensitivity and VGSCbeta1-4 mRNA expression levels did not change. The significance of the charge-reversing aspartate to lysine substitution was investigated by mutating the lysine in the 'neonatal' channel back to aspartate. In this 'neonatal K211D' mutant, the electrophysiological parameters studied strongly shifted back towards the 'adult', that is the lysine residue was primarily responsible for the electrophysiological effects of Nav1.5 D1:S3 splicing. Taken together, these data suggest that the charge reversal in 'neonatal' Nav1.5 would (1) modify the channel kinetics and (2) prolong the resultant current, allowing greater intracellular Na(+) influx. Developmental and pathophysiological consequences of such differences are discussed.

(1) Several VGSCalpha genes and their splice variants are expressed similarly in both rat and human PC cell lines. (2) Expression levels are much higher in the strongly metastatic (MAT-LyLu/PC-3) cells. (3) Levels of SCN9A mRNA specifically are predominant in MAT-LyLu and PC-3 cells; thus, SCN9A is highly likely to be the main source of the functional VGSC detected.

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Voltage-gated Na+ channels (VGSCs) are well known for mediating regenerative cell membrane depolarization and conduction of electrical signalling in nerves and muscles. However, VGSCs may also be expressed in traditionally "non-excitable" cell types, including lymphocytes, glia, fibroblasts and metastatic cancer cells of epithelial origin. Both the diversity and modulation of VGSC expression are far more complex than was initially apparent. There are at least 10 different genes that encode the alpha-subunits of VGSCs. Since VGSCs can contribute to a range of human disease conditions, it is important to understand both the control and consequences of VGSC functioning and how these aspects may be altered under pathophysiological conditions. Such mechanisms can be at the transcriptional, pre-translational or post-translational levels. This article reviews recent literature that has contributed to our understanding of how individual VGSC subtypes can generate their unique physiological signatures within different cell types. We also highlight emerging areas of interest, in particular, the finding of multiple expression of individual VGSC subtypes within single cells, the generation of alternative splice variants and the increasingly complex set of mechanisms of plasticity through which individual VGSC subtypes may be subtly controlled, including intracellular trafficking of VGSC protein.

Whole-cell patch-clamp recordings showed that a subpopulation (10%) of Jurkat cells, a model of human T-cells, expressed a functional voltage-gated sodium channel, which was tetrodotoxin (TTX)-resistant. Expression of voltage-gated sodium channel protein was confirmed by western blots. Semiquantitative PCR analysis revealed that mRNAs for the a-subunits of multiple voltage-gated sodium channel subtypes were present but indicated that Na v 1.5 was the predominant subtype, consistent with the TTX-resistant nature of the recorded currents. Importantly, 10 lM TTX reduced the number of Jurkat cells invading a Matrigel basement membrane by 93.0 % 5.5%. Since similar sodium channels have also been detected in normal human T-lymphocytes, it is concluded that the activity of voltage-gated sodium channels could represent a novel mechanism potentiating the invasive capacity of these cells.