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...

The voltage-gated ionic currents of two rodent prostatic cancer cell lines were investigated using the whole-cell patch clamp technique. The highly metastatic Mat-Ly-Lu cells expressed a transient, inward Na ÷ current (blocked by 600 nM tetrodotoxin), which was not found in any of the weakly metastatic AT-2 cells. Although both cell lines expressed a sustained, outward K ÷ current, this occurred at a significantly higher density in the AT-2 than in the Mat-Ly-Lu cells. Incubation of the MatLy-Lu cell line with 600 nM tetrodotoxin significantly reduced the invasive capacity of the cells in vitro. Under identical conditions, tetrodotoxin had no effect on the invasiveness of the AT-2 cells.

Previous work suggested that functional voltage-gated Na(+) channels (VGSCs) are expressed specifically in strongly metastatic cells of rat and human prostate cancer (PCa), thereby raising the possibility that VGSC activity could be involved in cellular behavior(s) related to the metastatic cascade. In the present study, the possible role of VGSCs in the lateral motility of rat PCa cells was investigated in vitro by testing the effect of modulators that either block or enhance VGSC activity. Two rat PCa cell lines of markedly different metastatic ability were used in a comparative approach: the strongly metastatic MAT-LyLu and the weakly metastatic AT-2 cell line, only the former being known to express functional VGSCs. Using both electrophysiological recording and a motility assay, the effects of two VGSC blockers (tetrodotoxin and phenytoin) and four potential openers (veratridine, aconitine, ATX II, and brevetoxin) were monitored on (a) Na(+) channel activity and (b) cell motility over 48 h. Tetrodotoxin (at 1 microM) and phenytoin (at 50 microM) both decreased the motility index of the MAT-LyLu cell line by 47 and 11%, respectively. Veratridine (at 20 microM) and brevetoxin (at 10 nM) had no effect on the motility of either cell line, whilst aconitine (at 100 microM) and ATX II (at 25 pM) significantly increased the motility of the MAT-LyLu cell line by 15 and 9%, respectively. Importantly, at the concentrations used, none of these drugs had effects on the proliferation or viability of either cell line. The results, taken together, would suggest strongly that functional VGSC expression enhances cellular motility of PCa cells. The relevance of these findings to the metastatic process in PCa is discussed.

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.