Voltage-gated sodium channel expression and potentiation of human breast cancer metastasis.

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 VGSCalpha 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.

A novel polyclonal antibody specific for the Na(v)1.5 voltage-gated Na(+) channel 'neonatal' splice form.

Voltage-gated Na(+) channel (VGSC) diversity is achieved through a number of mechanisms: multiple subunits, multiple genes encoding the pore-forming VGSC alpha-subunit and multiple gene isoforms generated by alternative splicing. A major type of VGSCalpha alternative splicing is in D1:S3, which has been proposed to be developmentally regulated. We recently reported a D1:S3 spliced form of Na(v)1.5 in human metastatic breast cancer cells. This novel 'neonatal' isoform differs from the counterpart 'adult' form at seven amino acids (in the extracellular loop between S3-S4 of D1). Here, we generated an anti-peptide polyclonal antibody, named NESOpAb, which specifically recognised 'neonatal' but not 'adult' Na(v)1.5 when tested on cells specifically over-expressing one or other of these Na(v)1.5 spliced forms. The antibody was used to investigate developmental expression of 'neonatal' Na(v)1.5 (nNa(v)1.5) in a range of mouse tissues by immunohistochemistry. Overall, the results were consistent with nNa(v)1.5 protein being more abundantly expressed in selected tissues (particularly heart and brain) from neonate as compared to adult animals. Importantly, NESOpAb blocked functional nNa(v)1.5 ion conductance when applied extracellularly at concentrations as low as 0.05 ng/ml. Possible biological and clinical applications of NESOpAb are discussed.

T-lymphocyte invasiveness: control by voltage-gated Na+ channel activity.

Whole-cell patch-clamp recordings showed that a sub-population (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. Semi-quantitative PCR analysis revealed that mRNAs for the alpha-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 microM 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.

Sequence of a conserved region of a new sea urchin homeobox gene from the NK family.

The partial sequence of a novel homeobox-containing gene from Paracentrotus lividus is described. Both cDNA and genomic DNA were screened using probes from the vnd/NK-2 homeobox gene found in Drosophila melanogaster. The new DNA sequence found in P. lividus encodes a protein fragment that is closely related to the NK family of homeodomain transcriptional regulators originally discovered in the fruit fly. This study thus represents the first finding of a homeobox gene from the NK family in sea urchin. The DNA that was sequenced includes the most highly conserved region of the NK genes and contains the 180 basepair homeobox (i.e. the DNA segment that encodes the homeodomain), the NK-2 box that encodes the NK-2-specific domain (NK-2 SD), and the acidic box that encodes an acidic domain, but which is found only in a limited subset of the NK genes. In this deduced sequence, the 60 amino acid residue homeodomain contains tyrosine in position 54 and leucine in position 7, which implies that the protein will bind to an unusual sequence of DNA that contains 5'-CAAGTG-3' as its core. The presence of tyrosine in position 54 identifies the gene as a member of the NK-2 class of homeobox genes. Positions 37 and 56 of the homeodomain contain isoleucine and leucine, respectively, which is the first finding in the NK family of homeodomains of these particular amino acid residues in those positions. The presence of the NK-2 box is consistent with identification of the gene as a member of the NK-2 class, and suggests an important role for the C-terminal portion of the protein in transcriptional activation. The sequence homology of the NK-2 box and the spacing between it and the homeobox further suggest that this gene is a member of the NKx-2.2 subclass, whose genes typically are expressed in brain and play a role in axonal guidance, and whose full lengths often are of the order of 900 bases. Homologous NK genes have been found in such diverse invertebrate and vertebrate species, such as Amphioxus sp., Xenopus sp., Caenorhabditis elegans, zebra fish, chicken, hamster, mouse and humans. The finding of this new gene together with sequence comparisons suggests possible evolutionary relationships between sea urchins and vertebrates in the developmental pathways of their body plans.