Voltage-gated sodium channels (Na V ) are functionally expressed in highly metastatic cancer cells derived from nonexcitable epithelial tissues (breast, prostate, lung, and cervix). MDA-MB-231 breast cancer cells express functional sodium channel complexes, consisting of Na V 1.5 and associated auxiliary ␤-subunits, that are responsible for a sustained inward sodium current at the membrane potential. Although these channels do not regulate cellular multiplication or migration, their inhibition by the specific blocker tetrodotoxin impairs both the extracellular gelatinolytic activity (monitored with DQ-gelatin) and cell invasiveness leading to the attenuation of colony growth and cell spreading in three-dimensional Matrigel-composed matrices. MDA-MB-231 cells express functional cysteine cathepsins, which we found play a predominant role (ϳ65%) in cancer invasiveness. Matrigel invasion is significantly decreased in the presence of specific inhibitors of cathepsins B and S (CA-074 and Z-FL-COCHO, respectively), and co-application of tetrodotoxin does not further reduce cell invasion. This suggests that cathepsins B and S are involved in invasiveness and that their proteolytic activity partly depends on Na V function. Inhibiting Na V has no consequence for cathepsins at the transcription, translation, and secretion levels. However, Na V activity leads to an intracellular alkalinization and a perimembrane acidification favorable for the extracellular activity of these acidic proteases. We propose that Na v enhance the invasiveness of cancer cells by favoring the pH-dependent activity of cysteine cathepsins. This general mechanism could lead to the identification of new targets allowing the therapeutic prevention of metastases.Breast cancer is the most common female cancer and the primary cause of death in women by cancer worldwide (1).Deaths occur primarily after the development of metastases. The invasive potential of malignant cells is mainly linked to their capacity to degrade basement membranes and extracellular matrices by various proteases. Studies have mostly focused on metalloproteases, including matrix metalloproteinases and the closely related ADAMs (a disintegrin and metalloproteinase) and ADAMTs (a disintegrin and metalloproteinase with thrombospondin motifs) (2), that are key factors in growth, invasion, and angiogenesis, and to a lesser extent on aspartyl and serine proteases. Pharmaceutical inhibitors of matrix metalloproteinases have been developed, but the results from clinical trials with these drugs have so far been disappointing (3,4

This work reports the finding of a unique fast inward sodium current (I(Na)) in MDA-MB-231 cells which is missing in MDA-MB-468 cells and in MCF-7 cells. This current is high-voltage-activated and displays a window current at the membrane potential of MDA-MB-231 cells. This current is blocked by high concentrations of tetrodotoxin (TTX). In MDA-MB-231 cells, which are the most invasive cells among the three cell lines tested, proliferation and migration were not sensitive to TTX while invasion was reduced by approximately 30%. These experiments suggest that I(Na) is involved in the invasion process, probably through its participation to the regulation of the intracellular sodium homeostasis.

The SK3 channel, a potassium channel, was recently shown to control cancer cell migration, a critical step in metastasis outgrowth. Here, we report that expression of the SK3 channel was markedly associated with bone metastasis. The SK3 channel was shown to control constitutive Ca 2þ entry and cancer cell migration through an interaction with the Ca 2þ channel Orai1. We found that the SK3 channel triggers an association with the Orai1 channel within lipid rafts. This localization of an SK3-Orai1 complex seemed essential to control cancer cell migration. This suggests that the formation of this complex in lipid rafts is a gain-offunction, because we showed that none of the individual proteins were able to promote the complete phenotype. We identified the alkyl-lipid Ohmline as a disrupting agent for SK3-Orai1 lipid raft localization. Upon Ohmline treatment, the SK3-Orai1 complex moved away from lipid rafts, and SK3-dependent Ca 2þ entry, migration, and bone metastases were subsequently impaired. The colocalization of SK3 and Orai1 in primary human tumors and bone metastases further emphasized the clinical relevance of our observations. Targeting SK3-Orai1 in lipid rafts may inaugurate innovative approaches to inhibit bone metastases. Cancer Res; 73(15); 4852-61. Ó2013 AACR.

Na V 1.5 sodium channels enhance the invasiveness of breast cancer cells through the acidic-dependent activation of cysteine cathepsins. Here, we showed that the Na þ /H þ exchanger type 1 (NHE1) was an important regulator of H þ efflux in breast cancer cells MDA-MB-231 and that its activity was increased by Na V 1.5. Na V 1.5 and NHE1 were colocalized in membrane rafts containing caveolin-1. The inhibition of Na V 1.5 or NHE1 induced a similar reduction in cell invasiveness and extracellular matrix degradation; no additive effect was observed when they were simultaneously inhibited. Our study suggests that Na V 1.5 and NHE1 are functionally coupled and enhance the invasiveness of cancer cells by increasing H þ efflux.

SummaryThe degradation of the extracellular matrix by cancer cells represents an essential step in metastatic progression and this is performed by cancer cell structures called invadopodia. Na V 1.5 (also known as SCN5A) Na + channels are overexpressed in breast cancer tumours and are associated with metastatic occurrence. It has been previously shown that Na V 1.5 activity enhances breast cancer cell invasiveness through perimembrane acidification and subsequent degradation of the extracellular matrix by cysteine cathepsins. Here, we show that Na V 1.5 colocalises with Na + /H + exchanger type 1 (NHE-1) and caveolin-1 at the sites of matrix remodelling in invadopodia of MDA-MB-231 breast cancer cells. NHE-1, Na V 1.5 and caveolin-1 co-immunoprecipitated, which indicates a close association between these proteins. We found that the expression of Na V 1.5 was responsible for the allosteric modulation of NHE-1, rendering it more active at the intracellular pH range of 6.4-7; thus, it potentially extrudes more protons into the extracellular space. Furthermore, Na V 1.5 expression increased Src kinase activity and the phosphorylation (Y421) of the actin-nucleation-promoting factor cortactin, modified F-actin polymerisation and promoted the acquisition of an invasive morphology in these cells. Taken together, our study suggests that Na V 1.5 is a central regulator of invadopodia formation and activity in breast cancer cells.

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BackgroundNaV1.5 voltage-gated sodium channels are abnormally expressed in breast tumours and their expression level is associated with metastatic occurrence and patients’ death. In breast cancer cells, NaV1.5 activity promotes the proteolytic degradation of the extracellular matrix and enhances cell invasiveness.FindingsIn this study, we showed that the extinction of NaV1.5 expression in human breast cancer cells almost completely abrogated lung colonisation in immunodepressed mice (NMRI nude). Furthermore, we demonstrated that ranolazine (50 μM) inhibited NaV1.5 currents in breast cancer cells and reduced NaV1.5-related cancer cell invasiveness in vitro. In vivo, the injection of ranolazine (50 mg/kg/day) significantly reduced lung colonisation by NaV1.5-expressing human breast cancer cells.ConclusionsTaken together, our results demonstrate the importance of NaV1.5 in the metastatic colonisation of organs by breast cancer cells and indicate that small molecules interfering with NaV activity, such as ranolazine, may represent powerful pharmacological tools to inhibit metastatic development and improve cancer treatments.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-264) contains supplementary material, which is available to authorized users.

Potassium channels have been involved in epithelial tumorigenesis but the role of small-conductance Ca 2+ -activated K + channels is unknown. We report here that small-conductance Ca 2+ -activated K + channels are expressed in a highly metastasizing mammary cancer cell line, MDA-MB-435s. Patch-clamp recordings showed typical small-conductance Ca 2+ -activated K + channelmediated currents sensitive to apamin, 4-aminopyridine, and tetraethylammonium. Moreover, the cells displayed a high intracellular calcium concentration, which was decreased after 24 hours of apamin treatment. By regulating membrane potential and intracellular calcium concentration, these channels were involved in MDA-MB435s cell migration, but not in proliferation. Only SK3 protein expression was observed in these cells in contrast to SK2, which was expressed both in cancer and noncancer cell lines. Whereas small interfering RNA directed against SK3 almost totally abolished MDA-MB435s cell migration, transient expression of SK3 increased migration of the SK3-deficient cell lines, MCF-7 and 184A1. SK3 channel was solely expressed in tumor breast biopsies and not in nontumor breast tissues. Thus, SK3 protein channel seems to be a new mediator of breast cancer cell migration and represents a potential target for a new class of anticancer agents.