The Functional Role of Voltage-Gated Sodium Channel Nav1.5 in Metastatic Breast Cancer.
ABSTRACT: Voltage-gated sodium channels (VGSCs), which are abnormally expressed in various types of cancers such as breast cancer, prostate cancer, lung cancer, and cervical cancer, are involved in the metastatic process of invasion and migration. Nav1.5 is a pore-forming ? subunit of VGSC encoded by SCN5A. Various studies have demonstrated that Nav1.5, often as its neonatal splice form, is highly expressed in metastatic breast cancer cells. Abnormal activation and expression of Nav1.5 trigger a variety of cellular mechanisms, including changing H+ efflux, promoting epithelial-to-mesenchymal transition (EMT) and the expression of cysteine cathepsin, to potentiate the metastasis and invasiveness of breast cancer cells in vitro and in vivo. Here, we systematically review the latest available data on the pro-metastatic effect of Nav1.5 and its underlying mechanisms in breast cancer. We summarize the factors affecting Nav1.5 expression in breast cancer cells, and discuss the potential of Nav1.5 blockers serving as candidates for breast cancer treatment.
Project description:Voltage-gated Na+ channels (VGSCs) mediate action potential firing and regulate adhesion and migration in excitable cells. VGSCs are also expressed in cancer cells. In metastatic breast cancer (BCa) cells, the Nav1.5 ? subunit potentiates migration and invasion. In addition, the VGSC-inhibiting antiepileptic drug phenytoin inhibits tumor growth and metastasis. However, the functional activity of Nav1.5 and its specific contribution to tumor progression in vivo has not been delineated. Here, we found that Nav1.5 is up-regulated at the protein level in BCa compared with matched normal breast tissue. Na+ current, reversibly blocked by tetrodotoxin, was retained in cancer cells in tumor tissue slices, thus directly confirming functional VGSC activity in vivo. Stable down-regulation of Nav1.5 expression significantly reduced tumor growth, local invasion into surrounding tissue, and metastasis to liver, lungs and spleen in an orthotopic BCa model. Nav1.5 down-regulation had no effect on cell proliferation or angiogenesis within the in tumors, but increased apoptosis. In vitro, Nav1.5 down-regulation altered cell morphology and reduced CD44 expression, suggesting that VGSC activity may regulate cellular invasion via the CD44-src-cortactin signaling axis. We conclude that Nav1.5 is functionally active in cancer cells in breast tumors, enhancing growth and metastatic dissemination. These findings support the notion that compounds targeting Nav1.5 may be useful for reducing metastasis.
Project description:A wide body of evidence suggests that voltage-gated sodium channels (VGSCs) are expressed de novo in several human carcinomas where channel activity promotes a variety of cellular behaviours integral to the metastatic cascade. These include directional motility (including galvanotaxis), pH balance, extracellular proteolysis, and invasion. Contrary to the substantial in vitro data, however, evidence for VGSC involvement in the cancer process in vivo is limited. Here, we critically assess, for the first time, the available in vivo evidence, hierarchically from mRNA level to emerging clinical aspects, including protein-level studies, electrolyte content, animal tests, and clinical imaging. The evidence strongly suggests that different VGSC subtypes (mainly Nav1.5 and Nav1.7) are expressed de novo in human carcinoma tissues and generally parallel the situation in vitro. Consistent with this, tissue electrolyte (sodium) levels, quantified by clinical imaging, are significantly higher in cancer vs. matched non-cancer tissues. These are early events in the acquisition of metastatic potential by the cancer cells. Taken together, the multi-faceted evidence suggests that the VGSC expression has clinical (diagnostic and therapeutic) potential as a prognostic marker, as well as an anti-metastatic target. The distinct advantages offered by the VGSC include especially (1) its embryonic nature, demonstrated most clearly for the predominant neonatal Nav1.5 expression in breast and colon cancer, and (2) the specifically druggable persistent current that VGSCs develop under hypoxic conditions, as in growing tumours, which promotes invasiveness and metastasis.
Project description:Voltage-gated Na(+) channels (VGSC) have been implicated in the metastatic potential of human breast, prostate, and lung cancer cells. Specifically, the SCN5A gene encoding the VGSC isotype Na(v)1.5 has been defined as a key driver of human cancer cell invasion. In this study, we examined the expression and function of VGSCs in a panel of colon cancer cell lines by electrophysiologic recordings. Na(+) channel activity and invasive potential were inhibited pharmacologically by tetrodotoxin or genetically by small interfering RNAs (siRNA) specifically targeting SCN5A. Clinical relevance was established by immunohistochemistry of patient biopsies, with strong Na(v)1.5 protein staining found in colon cancer specimens but little to no staining in matched-paired normal colon tissues. We explored the mechanism of VGSC-mediated invasive potential on the basis of reported links between VGSC activity and gene expression in excitable cells. Probabilistic modeling of loss-of-function screens and microarray data established an unequivocal role of VGSC SCN5A as a high level regulator of a colon cancer invasion network, involving genes that encompass Wnt signaling, cell migration, ectoderm development, response to biotic stimulus, steroid metabolic process, and cell cycle control. siRNA-mediated knockdown of predicted downstream network components caused a loss of invasive behavior, demonstrating network connectivity and its function in driving colon cancer invasion.
Project description:Although survival rates of breast, colon, and prostate cancers are improving, deaths from these tumors frequently occur due to metastasis. Voltage-gated Na(+) channels (VGSCs) are membrane proteins, which regulate membrane current and cellular migration during nervous system organogenesis. VGSCs are also expressed in fibroblasts, immune cells, glia, and metastatic cancer cells. VGSCs regulate migration and invasion of breast, bowel, and prostate cancer cells, suggesting that they may be novel anti-metastatic targets. We conducted a systematic review of clinical and preclinical studies testing the effects of VGSC-inhibiting drugs in cancer. Two-hundred and four publications were identified, of which two human, two mouse, and 20 in vitro publications were included. In the clinical studies, the effect of these drugs on survival and metastatic relapse is not clear. The 22 preclinical studies collectively suggest that several VGSC-inhibiting drugs inhibit cancer proliferation, migration, and invasion. None of the human and only six of the preclinical studies directly investigated the effect of the drugs on VGSC activity. Studies were difficult to compare due to lack of standardized methodology and outcome measures. We conclude that the benefits of VGSC inhibitors require further investigation. Standardization of future studies and outcome measures should enable meaningful study comparisons.
Project description:Eslicarbazepine acetate (ESL) is a dibenzazepine anticonvulsant approved as adjunctive treatment for partial-onset epileptic seizures. Following first pass hydrolysis of ESL, S-licarbazepine (S-Lic) represents around 95% of circulating active metabolites. S-Lic is the main enantiomer responsible for anticonvulsant activity and this is proposed to be through the blockade of voltage-gated Na+ channels (VGSCs). ESL and S-Lic both have a voltage-dependent inhibitory effect on the Na+ current in N1E-115 neuroblastoma cells expressing neuronal VGSC subtypes including Nav1.1, Nav1.2, Nav1.3, Nav1.6, and Nav1.7. ESL has not been associated with cardiotoxicity in healthy volunteers, although a prolongation of the electrocardiographic PR interval has been observed, suggesting that ESL may also inhibit cardiac Nav1.5 isoform. However, this has not previously been studied. Here, we investigated the electrophysiological effects of ESL and S-Lic on Nav1.5 using whole-cell patch clamp recording. We interrogated two model systems: (1) MDA-MB-231 metastatic breast carcinoma cells, which endogenously express the "neonatal" Nav1.5 splice variant, and (2) HEK-293 cells stably over-expressing the "adult" Nav1.5 splice variant. We show that both ESL and S-Lic inhibit transient and persistent Na+ current, hyperpolarise the voltage-dependence of fast inactivation, and slow the recovery from channel inactivation. These findings highlight, for the first time, the potent inhibitory effects of ESL and S-Lic on the Nav1.5 isoform, suggesting a possible explanation for the prolonged PR interval observed in patients on ESL treatment. Given that numerous cancer cells have also been shown to express Nav1.5, and that VGSCs potentiate invasion and metastasis, this study also paves the way for future investigations into ESL and S-Lic as potential invasion inhibitors.
Project description:Functional expression of voltage-gated Na(+) channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes.
Project description:Voltage-gated sodium channel (VGSC) activity has previously been reported in endothelial cells (ECs). However, the exact isoforms of VGSCs present, their mode(s) of action, and potential role(s) in angiogenesis have not been investigated. The main aims of this study were to determine the role of VGSC activity in angiogenic functions and to elucidate the potentially associated signaling mechanisms using human umbilical vein endothelial cells (HUVECs) as a model system. Real-time PCR showed that the primary functional VGSC ?- and ?-subunit isoforms in HUVECs were Nav1.5, Nav1.7, VGSC?1, and VGSC?3. Western blots verified that VGSC? proteins were expressed in HUVECs, and immunohistochemistry revealed VGSC? expression in mouse aortic ECs in vivo. Electrophysiological recordings showed that the channels were functional and suppressed by tetrodotoxin (TTX). VGSC activity modulated the following angiogenic properties of HUVECs: VEGF-induced proliferation or chemotaxis, tubular differentiation, and substrate adhesion. Interestingly, different aspects of angiogenesis were controlled by the different VGSC isoforms based on TTX sensitivity and effects of siRNA-mediated gene silencing. Additionally, we show for the first time that TTX-resistant (TTX-R) VGSCs (Nav1.5) potentiate VEGF-induced ERK1/2 activation through the PKC?-B-RAF signaling axis. We postulate that this potentiation occurs through modulation of VEGF-induced HUVEC depolarization and [Ca(2+)](i). We conclude that VGSCs regulate multiple angiogenic functions and VEGF signaling in HUVECs. Our results imply that targeting VGSC expression/activity could be a novel strategy for controlling angiogenesis.
Project description:Voltage-gated Na(+) channels (VGSCs) are heteromeric protein complexes containing pore-forming ? subunits together with non-pore-forming ? subunits. There are nine ? subunits, Nav1.1-Nav1.9, and four ? subunits, ?1-?4. The ? subunits are multifunctional, modulating channel activity, cell surface expression, and are members of the immunoglobulin superfamily of cell adhesion molecules. VGSCs are classically responsible for action potential initiation and conduction in electrically excitable cells, including neurons and muscle cells. In addition, through the ?1 subunit, VGSCs regulate neurite outgrowth and pathfinding in the developing central nervous system. Reciprocal signalling through Nav1.6 and ?1 collectively regulates Na(+) current, electrical excitability and neurite outgrowth in cerebellar granule neurons. Thus, ? and ? subunits may have diverse interacting roles dependent on cell/tissue type. VGSCs are also expressed in non-excitable cells, including cells derived from a number of types of cancer. In cancer cells, VGSC ? and ? subunits regulate cellular morphology, migration, invasion and metastasis. VGSC expression associates with poor prognosis in several studies. It is hypothesised that VGSCs are up-regulated in metastatic tumours, favouring an invasive phenotype. Thus, VGSCs may have utility as prognostic markers, and/or as novel therapeutic targets for reducing/preventing metastatic disease burden. VGSCs appear to regulate a number of key cellular processes, both during normal postnatal development of the CNS and during cancer metastasis, by a combination of conducting (i.e. via Na(+) current) and non-conducting mechanisms.
Project description:SCN5A encodes the ?-subunit of the voltage-gated sodium channel NaV1.5. Many patients with cardiac arrhythmias caused by mutations in SCN5A also have symptoms of irritable bowel syndrome (IBS). We investigated whether patients with IBS have SCN5A variants that affect the function of NaV1.5.We performed genotype analysis of SCN5A in 584 persons with IBS and 1380 without IBS (controls). Mutant forms of SCN5A were expressed in human embryonic kidney-293 cells, and functions were assessed by voltage clamp analysis. A genome-wide association study was analyzed for an association signal for the SCN5A gene, and replicated in 1745 patients in 4 independent cohorts of IBS patients and controls.Missense mutations were found in SCN5A in 13 of 584 patients (2.2%, probands). Diarrhea-predominant IBS was the most prevalent form of IBS in the overall study population (25%). However, a greater percentage of individuals with SCN5A mutations had constipation-predominant IBS (31%) than diarrhea-predominant IBS (10%; P < .05). Electrophysiologic analysis showed that 10 of 13 detected mutations disrupted NaV1.5 function (9 loss-of-function and 1 gain-of-function function). The p. A997T-NaV1.5 had the greatest effect in reducing NaV1.5 function. Incubation of cells that expressed this variant with mexiletine restored their sodium current and administration of mexiletine to 1 carrier of this mutation (who had constipation-predominant IBS) normalized their bowel habits. In the genome-wide association study and 4 replicated studies, the SCN5A locus was strongly associated with IBS.About 2% of patients with IBS carry mutations in SCN5A. Most of these are loss-of-function mutations that disrupt NaV1.5 channel function. These findings provide a new pathogenic mechanism for IBS and possible treatment options.
Project description:BACKGROUND AND PURPOSE: The cardiovascular benefits of red wine consumption are often attributed to the antioxidant effects of its polyphenolic constituents, including quercetin, catechin and resveratrol. Inhibition of cardiac voltage-gated sodium channels (VGSCs) is antiarrhythmic and cardioprotective. As polyphenols may also modulate ion channels, and possess structural similarities to several antiarrhythmic VGSC inhibitors, we hypothesised that VGSC inhibition may contribute to cardioprotection by these polyphenols. EXPERIMENTAL APPROACH: The whole-cell voltage-clamp technique was used to record peak and late VGSC currents (INa) from recombinant human heart NaV1.5 channels expressed in tsA201 cells. Right ventricular myocytes from rat heart were isolated and single myocytes were field-stimulated. Either calcium transients or contractility were measured using the calcium-sensitive dye Calcium-Green 1AM or video edge detection, respectively. KEY RESULTS: The red grape polyphenols quercetin, catechin and resveratrol blocked peak INa with IC50s of 19.4 microM, 76.8 microM and 77.3 microM, respectively. In contrast to lidocaine, resveratrol did not exhibit any frequency-dependence of peak INa block. Late INa induced by the VGSC long QT mutant R1623Q was reduced by resveratrol and quercetin. Resveratrol and quercetin also blocked late INa induced by the toxin, ATX II, with IC50s of 26.1 microM and 24.9 microM, respectively. In field-stimulated myocytes, ATXII-induced increases in diastolic calcium were prevented and reversed by resveratrol. ATXII-induced contractile dysfunction was delayed and reduced by resveratrol. CONCLUSIONS AND IMPLICATIONS: Our results indicate that several red grape polyphenols inhibit cardiac VGSCs and that this effect may contribute to the documented cardioprotective efficacy of red grape products.