Ranolazine inhibits Na_V1.5-mediated breast cancer cell invasiveness and lung colonization

Breast cancer is the primary cause of death by cancer in women worldwide and patients mostly die because of metastases appearance and development [1] which rely in part on the ability of cancer cells to degrade and migrate through extracellular matrices (ECM). Currently, there is no treatment for specifically inhibiting metastases development. Voltage-gated sodium channels (Na_V) are essential for action potential firing and as such are characteristic of excitable cells [2]. However, different Na_V isoforms have been found in non-excitable epithelial human cancer biopsies and cells, such as in breast [3, 4], lung [5‐7], prostate [8], cervix [9], ovarian [10, 11] and colon cancer [12], and their function, through persistent currents at the membrane potential, enhances degradation of ECM [5, 13‐15]. Notably, the Na_V1.5 isoform is abnormally expressed in breast cancer biopsies, while it is not in non-cancerous mammary tissues [14], and its level of expression is associated with lymph node invasion, the development of metastases and a reduced survival of patients [3, 16, 17]. In cancer cells, it is expressed as a neonatal splice variant showing a 7-amino acid substitution in the segments S3 and S4 of the domain I (DI-S3-S4) of the protein compared to the adult variant that shows a particular pharmacology [18] and was proposed to serve as a metastatic marker [16]. Na_V1.5 is functional at the plasma membrane of highly invasive breast cancer cells [3, 16, 17], and its activity maintains a pro-invasive phenotype [15], related to “mesenchymal migration” [19]. Indeed, while the complete mechanism involved is not yet elucidated, Na_V1.5 activity was shown i) to control Src kinase activity, cortactin phosphorylation (Y421) and the subsequent polymerisation of actin filaments, ii) to increase the activity of the Na⁺/H⁺ exchanger type 1 (NHE-1), thus enhancing the efflux of protons and the proteolytic activity of extracellularly-released acidic cysteine cathepsins B and S [13, 20]. Altogether, these results indicated that Na_V1.5 promotes the invadopodial activity of breast cancer cells and the invasion of the surrounding ECM [15]. Molecules reducing its activity, such as tetrodotoxin, reduce cancer cell invasiveness in vitro[3, 14, 18]. Correlatively, molecules that increase its activity, such as veratridine, enhance ECM invasion [13]. However, to the best of our knowledge, the importance of Na_V1.5 expression, and the relevance for its pharmacological inhibition, on the metastatic organ colonisation by breast cancer cells have never been reported so far. Ranolazine is an antiarrhythmic drug indicated for the treatment of chronic angina that was approved by the Food and Drug Administration (FDA) in 2006. While it is proposed to have several pharmacological actions, its best characterized one is the selective inhibition of late sodium currents [21]. This leads to a steeper Na⁺ gradient which, by increasing the activity of the Na⁺/Ca²⁺ exchanger (NCX), reduces calcium overload, and improves ventricular relaxation in pathological conditions associated with cardiac ischemia [22].

In this study we investigated how Na_V1.5 expression in human breast cancer cells affected metastatic colonisation of organs in immunodepressed mice, and whether its pharmacological inhibition by ranolazine reduced cancer cell invasiveness both in vitro and in vivo.

Highly invasive MDA-MB-231 human breast cancer cells express mRNA for Na_V1.5, Na_V1.6 and Na_V1.7 channels [16], but only Na_V1.5 channels are functional at the plasma membrane and are giving rise to transient inward sodium currents (INa) under voltage-clamp procedures [13] (see Additional file 1: Material and methods). INa-voltage (INa-V) protocols were performed using the whole-cell configuration of the patch clamp technique from MDA-MB-231-Luc cells modified to stably express a null-target small hairpin RNA (shCTL). The INa-V relationship, obtained from a holding potential of −100 mV, indicated a threshold of activation around −60 mV and maximal current of −12.1 ± 2.2 pA/pF at a voltage of −10 mV (Figure 1A). The acute application of ranolazine (50 μM) significantly reduced the maximal amplitude to −8.7 ± 1.7 pA/pF (p < 0.001). This decrease in the maximal current amplitude was associated with a significant leftward shift of the availability-voltage relationship (Figure 1B). The half (1/2)-inactivation voltage was shifted from −84.1 ± 1.4 mV to −90.3 ± 1.7 mV (p < 0.001) in absence and presence of ranolazine, respectively. The activation-voltage relationship was significantly modified (Figure 1C), and the 1/2-activation voltage was shifted from −37.1 ± 1.0 mV to −39.2 ± 0.6 mV (p < 0.01). Therefore, ranolazine reduced efficiently the activity of the neonatal Na_V1.5 isoform expressed in human breast cancer cells. This isoform is the only one to be functional in breast cancer cells [13, 16] and we selected a population of cells stably expressing a small hairpin RNA targeting its expression (shNa_V1.5) after lentiviral transduction. This led to a significant 89 ± 1% decrease of Na_V1.5 mRNA expression (Figure 2A), resulting in the complete disappearance of sodium currents in almost all cancer cells (Figure 2B), with no effect on cell viability (Figure 2C). Before assessing the effect of ranolazine in reducing cancer cell invasiveness, we addressed a possible cytotoxic effect of its application. Figure 2D indicates that ranolazine, incubated for 5 days in a range of concentration from 0.1 to 100 μM had no effect on cell viability. It was then used at 50 μM in the 24 h invasion experiment with Matrigel™-coated filters (Figure 2E). In shCTL cells, cell invasiveness was reduced by 35 ± 4% with the total inhibition of Na_V1.5 currents with 30 μM tetrodotoxin (TTX), and by 18 ± 3% with ranolazine. In comparison to shCTL cells, shNa_V1.5 cancer cells, which do not express Na_V1.5, had a reduced invasiveness of 33 ± 10%. In shNa_V1.5 cells, both TTX and ranolazine were ineffective to further reduce cell invasiveness, suggesting that ranolazine was specific in inhibiting Na_V1.5-related invasion. Na_V1.5 expression and activity was recently shown to control the acquisition of a pro-invasive phenotype, by maintaining a spindle-shape morphology and by controlling the ECM proteolysis by MDA-MB-231 cancer cells [15]. We found that ranolazine increased the circularity of shCTL cells, thus decreasing the pro-invasive morphology, to the same extent as the complete extinction of Na_V1.5 expression (Figure 2F). Furthermore, ranolazine reduced the focal ECM degradative activity of shCTL cells by 58.6 ± 10.0% (Figure 2G). This activity, which is related to the invadopodial activity, was monitored as being the release of fluorescence from DQ-gelatin at focal sites of F-actin polymerisation as previously described [15].

Because Na_V1.5 was proposed to promote metastases development from breast tumours, we assessed the importance of its expression in human breast cancer cells for the colonisation of organs. ShCTL or shNa_V1.5 cells, both expressing the luciferase gene, were injected in the tail vein of NMRI nude mice. A third experimental group was set with mice injected with shCTL cells and receiving a daily intraperitoneal injection of ranolazine (50 mg/Kg – 5 days per week). The colonisation of mice organs by human cancer cells was followed in vivo, every week for a total duration of 8 weeks, by bioluminescent imaging (BLI) after luciferin injection (Figure 3A). There was no statistical difference in the evolution of animal body weights between the three groups (Figure 3B). BLI performed in living animals indicated that shCTL cells, which express Na_V1.5, strongly colonised and developed into the chest area (which was the case for 17 out of 18 mice). In contrast, shNa_V1.5 cells led to a very weak signal (1/12 mice) or no signal at all (11/12 mice) in the chest area. Ranolazine, which inhibited Na_V1.5 currents (Figure 1), significantly reduced total BLI signal in mice injected with shCTL cells. In vitro, ranolazine treatment did not interfere with luciferase activity in shCTL cells (Additional file 2: Figure S1) indicating that the BLI signal recorded was indeed strongly correlated with the abundance of cancer cells in mice organs. In this experimental group, BLI signal was recorded in 5 out of 8 mice (Figure 3C). At completion of the study, mice were sacrificed and isolated organs (lungs, brain, liver, bones from rachis/ribs and legs) were analysed ex vivo. In the shCTL group, all mice showed lung colonisation (18/18) and a small proportion also had bioluminescent signal in rachis and ribs (2/18) and in leg bones (2/18). In the shNa_V1.5 group, 7 mice out of 12 had lung colonisation and one (1/12) had bioluminescent signal in rachis and ribs. In the ranolazine group, although 8/8 mice presented lung colonisation, bioluminescence was dramatically reduced by 77 ± 8%, at a level similar to the experimental suppression of Na_V1.5 (inhibition of lung BLI by 97 ± 2%) (Figure 3D, 3E). In the ranolazine group, mice did not show BLI signal in other organs.

While it is now well-established that Na_V channels are anomalously expressed in several epithelial tumours and are associated with metastasis occurrence and patient mortality [12, 16, 17, 23], the consequence of their expression on metastatic organ colonisation was not demonstrated so far. To our knowledge, this study is the first to clearly establish a link between Na_V1.5 expression in human breast cancer cells and the colonisation of lungs in vivo. Furthermore, this study using ranolazine, a drug that is clinically used, shows that the pharmacological inhibition of Na_V channels could be effective in reducing metastastic colonisation with no apparent toxic effect. In conclusion, this study opens a new therapeutical concept for the management of cancer disease. Inhibitors of Na_V channels, already approved for other clinical use such as antiarrhythmics, anticonvulsants [17] or anaesthetics [24], or new molecules that are even more effective in blocking neonatal variants, could be of high interest in the prevention and/or reduction of metastatic spreading of cancer cells at the diagnosis of the primary tumour.