Therapeutic potential for phenytoin: targeting Na(v)1.5 sodium channels to reduce migration and invasion in metastatic breast cancer

Abstract

Voltage-gated Na(+) channels (VGSCs) are heteromeric membrane protein complexes containing pore-forming α subunits and smaller, non-pore-forming β subunits. VGSCs are classically expressed in excitable cells, including neurons and muscle cells, where they mediate action potential firing, neurite outgrowth, pathfinding, and migration. VGSCs are also expressed in metastatic cells from a number of cancers. The Na(v)1.5 α subunit (encoded by SCN5A) is expressed in breast cancer (BCa) cell lines, where it enhances migration and invasion. We studied the expression of SCN5A in BCa array data, and tested the effect of the VGSC-blocking anticonvulsant phenytoin (5,5-diphenylhydantoin) on Na(+) current, migration, and invasion in BCa cells. SCN5A was up-regulated in BCa samples in several datasets, and was more highly expressed in samples from patients who had a recurrence, metastasis, or died within 5 years. SCN5A was also overexpressed as an outlier in a subset of samples, and associated with increased odds of developing metastasis. Phenytoin inhibited transient and persistent Na(+) current recorded from strongly metastatic MDA-MB-231 cells, and this effect was more potent at depolarized holding voltages. It may thus be an effective VGSC-blocking drug in cancer cells, which typically have depolarized membrane potentials. At a concentration within the therapeutic range used to treat epilepsy, phenytoin significantly inhibited the migration and invasion of MDA-MB-231 cells, but had no effect on weakly metastatic MCF-7 cells, which do not express Na(+) currents. We conclude that phenytoin suppresses Na(+) current in VGSC-expressing metastatic BCa cells, thus inhibiting VGSC-dependent migration and invasion. Together, our data support the hypothesis that SCN5A is up-regulated in BCa, favoring an invasive/metastatic phenotype. We therefore propose that repurposing existing VGSC-blocking therapeutic drugs should be further investigated as a potential new strategy to improve patient outcomes in metastatic BCa.

Fig. 1

SCN5A is up-regulated in breast tumors and associates with poor prognosis. a Expression of SCN5A in invasive breast cancer (IBCa), ductal carcinoma in situ (DCIS), invasive ductal breast carcinoma (IDBCa), or invasive mixed breast carcinoma (IMBCa), versus normal breast in three datasets analyzed in Oncomine: [41] (n = 59); [42] (n = 7); and The Cancer Genome Atlas (TCGA; n = 371). b Receiver operating characteristic (ROC) curve analysis of prediction of metastasis at five years in [43] (n = 181). c Comparison of SCN5A expression between those with/without recurrence at five years in [44] (n = 8). d Comparison of SCN5A expression between patients with invasive breast carcinoma alive or dead at five years in TCGA dataset (n = 6). e Kaplan–Meier survival analysis comparing overall survival of those with high versus low SCN5A expression in Ref. [45] (n = 40). Box plot dots maximum and minimum values; whiskers 90th and 10th percentile values; and horizontal lines 75th, 50th, and 25th percentile values. *P < 0.05; ***P < 0.001

Fig. 2

SCN5A expression as an outlier associates with metastasis. a SCN5A (normalized expression units) is shown for all profiled samples in [70]. Horizontal line 95th percentile cut-off, above which extend the top 5th percentile samples. b Percentage of those that developed metastasis within five years is shown for the 10 % most highly expressing, and bottom 90 % of samples in [43]. *P < 0.05 (n = 181)

Fig. 3

Subcellular distribution of voltage-gated Na⁺ channel α and β1 subunits. a MCF-7 and MDA-MB-231 cells labeled with pan-VGSC α subunit and β1 antibodies (magenta and green, respectively), phalloidin to label actin cytoskeleton (red), and DAPI to label nucleus (blue). b MCF-7 and MDA-MB-231 cells labeled with pan-VGSC α subunit antibody (magenta) and GM130 antibody (Golgi marker; green), phalloidin (red), and DAPI (blue). Arrows indicate co-expression of α and β1 at the cell edge. Arrowheads indicate perinuclear expression of α and β1, colocalizing with GM130 (b). Intensity profiles (normalized to maximum signal) for pan-VGSC α subunit and phalloidin across representative lamellipodia indicated by lines in (a) are shown for MCF-7 (c) and MDA-MB-231 (d) cells. e VGSC α subunit intensity in lamellipodia relative to internal signal, for MCF-7 and MDA-MB-231 cells. Data are mean ± SEM (n ≥ 36). ***P < 0.001

Fig. 4

Effects of phenytoin on Na⁺ current. a Typical whole-cell recordings from MCF-7 cell (top) and MDA-MB-231 cell (bottom) following depolarization to −10 mV (black arrows) from a holding potential of −80 mV. Na⁺ current in MDA-MB-231 cell is shown in control solution, following perfusion with 50 μM phenytoin, and drug washout. b Tonic block (%) of transient and persistent current in MDA-MB-231 cells (activated by depolarization to −10 mV from a holding potential of −120 mV) following perfusion with 50 μM phenytoin, and drug washout. c Tonic block (%) of transient and persistent current in MDA-MB-231 cells (activated by depolarization to −10 mV from a holding potential of −80 mV) following perfusion with 50 μM phenytoin, and drug washout. d Steady-state inactivation in MDA-MB-231 cells. Normalized current, elicited by 60 ms test pulses at −10 mV following 250 ms conditioning pulses between −120 and −10 mV, applied from a holding potential of −80 mV, plotted as a function of the prepulse voltage for cells in control and following perfusion with 50 μM phenytoin. Data are fit with Boltzmann functions. e Use-dependent block of transient current in MDA-MB-231 cells, elicited by 50 Hz pulse trains to 0 mV, applied from a holding potential of −120 mV, normalized to the current evoked by the first pulse plotted as a function of the pulse number for cells in control and following perfusion with 50 μM phenytoin. Data are fit with single exponential functions, which are significantly different between control and phenytoin (P < 0.001). Data are mean ± SEM (n ≥ 7). *P < 0.05; ***P < 0.001

Fig. 5

Effect of phenytoin on viability and proliferation. a Viability (%) of MCF-7 and MDA-MB-231 cells following treatment with phenytoin (5, 50, 200 μM) or vehicle for 24 h, normalized to control (n = 60). b Proliferation of MCF-7 and MDA-MB-231 cells following treatment with phenytoin (5, 50, 200 μM) or vehicle for 24 h, normalized to control (n ≥ 9). Data are mean ± SEM

Fig. 6

Effect of phenytoin on migration and invasion. a Representative images of MCF-7 and MDA-MB-231 cells in a wound healing assay at 0 h, and 24 h following treatment with phenytoin (5, 50, 200 μM) or vehicle. b Migration of MCF-7 and MDA-MB-231 cells treated with phenytoin (5, 50, 200 μM) or vehicle for 24 h in wound healing assay, normalized to control (n = 135 measurements per condition). c Invasion of MCF-7 and MDA-MB-231 cells ± phenytoin (50 μM) for 48 h, normalized to control (n = 9). Data are mean ± SEM. **P < 0.01; ***P < 0.001