The neonatal splice variant of Nav1.5 potentiates in vitro invasive behaviour of MDA-MB-231 human breast cancer cells.
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 approximately 90%) the level of nNav1.5 (but not adult Nav1.5) mRNA, but the protein reduction was much smaller (approximately 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 dose-dependent 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.
Brackenbury
William J
WJ
Neuroscience Solutions to Cancer Research Group Division of Cell and Molecular Biology, Imperial College London, Sir Alexander Fleming Building, South Kensington Campus, London SW7 2AZ, UK.
Chioni
Athina-Myrto
AM
Diss
James K J
JK
Djamgoz
Mustafa B A
MB
D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects / Q000235:genetics; D005260:Female; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007231:Infant, Newborn; D018613:Microscopy, Confocal; D009124:Muscle Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D062554:NAV1.5 Voltage-Gated Sodium Channel; D009361:Neoplasm Invasiveness / Q000235:genetics; D018408:Patch-Clamp Techniques; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction; D026941:Sodium Channel Blockers / Q000494:pharmacology; D015222:Sodium Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013779:Tetrodotoxin / Q000494:pharmacology
D009124:Muscle Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; D020033:Protein Isoforms; D034741:RNA, Small Interfering; C568320:SCN5A protein, human; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D013779:Tetrodotoxin