Cancer is one of the leading causes of morbidity and mortality worldwide, with more than 19 million new cases and near to 10 million cancer-related deaths in 2020, according to the GLOBOCAN 2020 report and, unfortunately, the number of new cases is expected to rise by about 56% over the next 2 decades (Sung et al., 2021).

Metastasis formation, which defines the ability of cancer cells to spread to distant organs in the body, is responsible for more than 90% of cancer-associated deaths (Spano et al., 2012). The complex process of metastasis is a result of a succession of cell-biology changes, beginning with uncontrolled proliferation and local invasion of surrounding normal tissue, then intravasation of cancer cells into vessels (blood and lymphatic), transit and survival through the lymphatic or hematogenous systems, arrest in capillary beds of distant tissues, followed by extravasation, attachment and proliferation of cancer cells at secondary organs (micrometastasis), and finally the growth of new colonies into macroscopic tumors (Hanahan and Weinberg, 2011) (Figure 1). This multistep process, often called the invasion-metastasis cascade, involves the dysregulation of numerous proteins including several members of the ion channel superfamily (Talmadge and Fidler, 2010; Litan and Langhans, 2015). In particular, voltage-gated sodium channels (VGSCs or Na_V channels) are functionally expressed in many types of cancer, comprising astrocytoma, breast, cervix, colon, gastric, leukemia, lung, melanoma, mesothelioma, neuroblastoma, ovary, prostate, and thyroid cancer (Blandino et al., 1995; Allen et al., 1997; Gu et al., 1997; Laniado et al., 1997; Bordey and Sontheimer, 1998; Smith et al., 1998; Diss et al., 2001; Roger et al., 2003; Fraser et al., 2004; Fraser et al., 2005; Onganer and Djamgoz, 2005; Ou et al., 2005; Fulgenzi et al., 2006; Diaz et al., 2007; Roger et al., 2007; Carrithers et al., 2009; Gao et al., 2010; House et al., 2010; Hernandez-Plata et al., 2012; Campbell et al., 2013; Xing et al., 2014; Huang et al., 2015; Xia et al., 2016; Li et al., 2022), where their upregulation has been correlated with the potentiation of cellular migration and invasiveness, as well as with other metastatic cell behaviors (MCBs) including proliferation, invadopodia formation, endocytosis, angiogenesis, and resisting apoptosis (Mycielska et al., 2003; Krasowska et al., 2004; Onganer and Djamgoz, 2005; Carrithers et al., 2009; Andrikopoulos et al., 2011; Xing et al., 2014).

The family of VGSCs consists of transmembrane protein complexes conformed by a pore-forming functional α-subunit (∼230 kDa) typically associated with one or more non-pore forming β-subunits (∼25–45 kDa) (Isom, 2001; Catterall et al., 2005); and they are responsible for the action potential initiation and propagation in excitable cells, including nerves, muscles, and neuroendocrine cells (Hille, 2001). The VGSCs α-subunits are formed by approximately 2000 amino acid residues organized in four homologous domains, each one comprising six transmembrane segments (S1-S6) (Figure 2A). So far, nine mammalian VGSCs α-subunits, Na_V1.1 to Na_V1.9, encoded by the genes SCN1A-SCN5A and SCN8A-SCN11A, have been identified, cloned, and characterized (Goldin et al., 2000; Alexander et al., 2021) (Table 1). The SCN7A and SCN6A genes code for the 10th sodium channel protein (named Na_X) that is not voltage-gated and is involved in sodium-level sensing (Noda and Hiyama, 2015; Noland et al., 2022). During the last 12 years the use of X-ray crystallography, but mainly the cryo-electron microscopy techniques have provided amazing atomic resolution about the structural architecture, activation, inactivation, voltage-sensing, and pharmacology of VGSCs (Payandeh et al., 2011; Pan et al., 2018; Jiang et al., 2020; Jiang et al., 2022; Fan et al., 2023), (Figure 2B). These different sodium channels have similar structural and functional properties, but their expression is cell type-specific, and they have distinct regulatory and pharmacological characteristics (Goldin, 2001; Catterall et al., 2005; Catterall, 2012; Shen et al., 2019; Catterall et al., 2020). According to their sensitivity to the neurotoxin tetrodotoxin (TTX), the VGSCs can be classified into two main groups: the TTX-sensitive channels, including Na_V1.1-Na_V1.4, Na_V1.6, and Na_V1.7, which are inhibited by low nanomolar concentrations of TTX; and the TTX-resistant channels Na_V1.5, Na_V1.8, and Na_V1.9, that are inhibited by TTX at micromolar concentrations (Table 1).

Sodium current through VGSCs displays two main components: the characteristic “transient” component (I _NaT), responsible for the fast sodium influx into the cells during action potentials, and the “persistent” component (I _NaP), which is a small fraction of the corresponding I _NaT (∼10%) that shows little or no inactivation at all (Crill, 1996; Djamgoz and Onkal, 2013). The persistent current has a crucial relevance in functionality of neurons with repetitive firing activity, where low but constant sodium influx maintains the membrane potential subtly depolarized facilitating the action potential generation (Osorio et al., 2010; Nakamura and Jang, 2022).

Among the great diversity of proteins related to VGSCs, it has been shown that the C-terminal region of several VGSCs channels interacts with the fibroblast growth factor homologous factor (FHF)-calmodulin, regulating current density, availability, frequency -dependent inhibition, and resurgent currents (Goldfarb, 2005; Pitt and Lee, 2016; Mahling et al., 2021). In addition, the clustering of VGSCs in the membrane is functionally relevant (Nirenberg and Yifrach, 2019); this property can be regulated by interactions with the ubiquitin ligases nedd4 and nedd4-2 (van Bemmelen et al., 2004), and other cytoskeletal proteins such as ankyrin, neurofascin, and spectrin (Liu et al., 2020). Evidence from central nervous system research have shown that Na_V1.2 channels interact with the anchoring protein AKAP, which in turn recruits the kinases PKA and PKC. Serine residues phosphorylation made by PKA in the intracellular loop joining DI and DII domains of the α-subunit reduces the sodium currents transported by VGSCs, and the PKC activity increases this effect (Cantrell et al., 2002).

Similarly, tyrosine phosphorylation by the Src family kinase Fyn produces a depolarizing shift of the steady-state inactivation on Na_V1.5 channels. Single amino acid substitution assays have revealed the importance of the Y1495 located in the intracellular linker of domains III and IV for this effect (Ahern et al., 2005). On Na_V1.2 channel, the Fyn kinase accelerates the inactivation which reduces the sodium current and shifts the voltage dependence of the inactivation to more negative potentials (Ahn et al., 2007).

On the other hand, β subunits of VGSCs are composed by a prominent N-terminal extracellular immunoglobulin-like fold related to the L1 family of cell adhesion molecules, a single transmembrane segment, and a short intracellular carboxyl-terminal region. These proteins modulate the voltage sensitivity, gating kinetics, and trafficking of VGSCs, but they also play functional roles independently of α subunits in a variety of cellular processes in both excitable and non-excitable cells, including cancer cells (Brackenbury and Isom, 2011). Four genes (SCNB1-SCNB4) encode for five VGSCs β-subunits: β1a and β1b (generated by alternative splicing), β2, β3, and β4 (Table 2). β1 and β3 resemble each other in amino acid sequence and are associated non-covalently with α subunits. In contrast, β2 and β4 form disulfide bonds with α subunits and have similar amino acid sequences (Isom et al., 1992; Isom et al., 1995; Morgan et al., 2000; Yu et al., 2003). Additionally, the VGSCs β-subunits are able to interact with intracellular molecules (e.g., ankyrin_G and ankyrin_B), and also act as cell adhesion molecules (CAMs) through their immunoglobulin extracellular domain, interacting with each other (homophilic interaction) or other CAMs (heterophilic interaction, e.g., contactin, NF-155 and NrCAMs), either expressed in the same cell (cis-interaction) or with CAMs from other cells (trans-interaction), as well as with components of the extracellular matrix (e.g., tenascin-C and tenascin-R; Figure 2A) (Brackenbury and Isom, 2011). In addition, abnormalities in expression of VGSCs β-subunits, such as mutations or gene expression dysregulations are implicated in several inherited pathologies, including some epilepsies and heart arrhythmias and syndromes (O'Malley and Isom, 2015; Edokobi and Isom, 2018).

Over the last 25 years, numerous studies have documented the novo expression/overexpression of multiple VGSC subunits in several types of cancer and their contribution to different malignant behaviors, remarkably migration and invasion. In addition, the predominantly upregulated α subunit appears to be cancer-type specific (Table 3), and some reports have evidenced the preferential expression of variants generated by alternative splicing of the corresponding coding mRNAs. Importantly, neonatal splice variants of VGSCs are abundantly expressed in breast cancer (BCa), cervical cancer, colon cancer, neuroblastoma, non-small-cell lung cancer, and prostate cancer (PCa) (Diss et al., 2001; Fraser et al., 2005; Ou et al., 2005; Roger et al., 2007; Guzel et al., 2018; Lopez-Charcas et al., 2018). This is consistent with the large amount of re-expression of embryonic genes during oncogenesis (Monk and Holding, 2001).

The neonatal Na_V1.5 isoform has been characterized in BCa and colon cancer cells and found to have distinct electrophysiological properties compared to the adult variant; e.g., it is more active and allows a higher influx of sodium ions into the cell (Onkal et al., 2008; Guzel et al., 2018). The neonatal Na_V1.5 variant differs from the adult isoform by 7 amino acids in the S3 and the S3/S4 linker from domain I, and is also found in neuroblastoma (Ou et al., 2005), where another splice variant of Na_V1.5 lacking the full exon 18 (Na_V1.5-Δ18) has been shown to generate a functional sodium channel, even though this exon encodes 54 amino acids in the intracellular loop between domains II and III (Ou et al., 2005). On the other hand, in cervical cancer, leukemia, and melanoma, the expression of the Na_V1.6-Δ18 splice variant is promoted (in this case, exon 18 encodes a 41-amino acid portion of the S3 and S4 segments of domain III). This isoform has been reported to have an intracellular localization, rather than being expressed in the plasma membrane, and it appears to contribute to the invadopodia formation and the invasive potential of these cells by a different mechanism than the sole influx of sodium through the plasma membrane (Carrithers et al., 2009; Lopez-Charcas et al., 2018).

Nowadays it is well known that cancer cells generally possess a more depolarized membrane potential with respect to normal healthy tissue, which has been correlated with cancer progression, as the depolarized membrane potential itself plays important roles in cell cycle progression, DNA synthesis, mitosis, proliferation, migration, and differentiation (Lobikin et al., 2012; Yang and Brackenbury, 2013). In this context, several studies have demonstrated that intracellular sodium concentration is higher in cancer cells compared with normal cells, and such increased concentration is related to mitogenesis and oncogenesis (Cameron et al., 1980; Ouwerkerk et al., 2007). Moreover, it has been proposed that the I _NaP is the main responsible for the elevation of the intracellular sodium concentration and the consequent potentiation of several MCBs, as I _NaP significantly manifests itself under a variety of physiological and pathophysiological conditions, including cancer (Djamgoz and Onkal, 2013). In addition, almost all the reported studies have revealed the role of VGSCs in metastatic behaviors of cancer cells by means of the modulation of their activity (Roger et al., 2003; Fraser et al., 2005; Brackenbury and Djamgoz, 2006; Gillet et al., 2009), employing pharmacological drugs or natural toxins to modulate the channel opening or closing, and the corresponding sodium ion flow. More recently, the regulation of gene expression using small interference RNAs (siRNAs) has allowed to modulate the amount of functionally expressed channels and characterize their role more precisely (Brisson et al., 2011; Driffort et al., 2014). The role of VGSC β-subunits have also been studied, although less extensively, in breast, cervical, prostate, and thyroid cancer (Diss et al., 2008; Chioni et al., 2009; Jansson et al., 2012; Jansson et al., 2014; Nelson et al., 2014; Bon et al., 2016; Gong et al., 2018; Sanchez-Sandoval and Gomora, 2019; Haworth et al., 2022), in which they have an important role in several MCBs as well (Table 4).

Despite all the new evidence of the VGSCs overexpression and functional contribution to numerous MCBs in different types of cancer, the entire mechanisms by which the sodium channel upregulation enhances this phenotype has not been completely elucidated (Lopez-Charcas et al., 2021). However, the evidence compiled hitherto provide an approach to understand the pathobiology of cancer and could be useful in a near future to fight the metastatic phenotype of cancer.

The aim of the present review is to collect the newest information that conforms to the state of the art in this field of knowledge, focusing on the role of VGSCs in different metastatic cell behaviors including migration, invasion, proliferation, angiogenesis, endocytosis, and evasion of apoptosis, as well as the latest revealed molecular mechanisms of action. Most evidence included in this review come from studies in cancer, nevertheless some non-cancer examples that could explain phenomena observed in cancer cells are also reviewed.

One of the most fundamental characteristics of cancer cells is their ability to maintain uncontrolled proliferation, which is carefully controlled in normal cells by different signals, including growth factors and hormones, in order to ensure the homeostasis of tissue architecture and function (Hanahan and Weinberg, 2011). While several studies have reported no effect on cell proliferation by VGSCs (e.g., Fraser et al., 1999; Fulgenzi et al., 2006; Roger et al., 2007; Gao et al., 2010; Hernandez-Plata et al., 2012; Huang et al., 2015), a few others have shown the opposite, demonstrating once more the tissue-specific functionality of the channels, as well as the variability given by the study model.

By inhibiting the sodium currents with VGSCs-blockers, including phenytoin analogues and riluzole, the proliferation of PCa cell lines decreases without affecting cell viability (Anderson et al., 2003; Driffort et al., 2014; Rizaner et al., 2020). Also, cell proliferation is inhibited in U251 astrocytoma cells when Na_V1.5 expression is downregulated by siRNAs (Xing et al., 2014). In the same sense, in vitro proliferation of gastric cancer cell lines is markedly slower when they are incubated in the presence of TTX, EIPA (5-(N-ethyl-N-isopropyl) amiloride; a Na⁺/H⁺ exchanger 1 inhibitor), or when they are transfected with siRNAs against Na_V1.7, consistent with a reduced xenograft tumor growth of stable Na_V1.7 knockdown BGC-823 cells compared to scramble-transfected control cells (Xia et al., 2016), and smaller tumor size generated by MDA-MB-231 cells in phenytoin-treated animals (Nelson et al., 2015). Very recently, it has been shown that knocking down Na_V1.6 expression significantly inhibited the proliferation, epithelial–mesenchymal transition, and invasion of follicular thyroid carcinoma (FTC), through the JAK/STAT signaling pathway (Li et al., 2022), which is associated with crucial pathological processes of tumors including cell proliferation, invasion, and apoptosis (Hu et al., 2021). Remarkably, in this work the migration behavior of FTC cells was not explored and the proposed Na_V1.6-activated signaling pathway seems to be the same for the proliferation and invasiveness of FTC. Since tumor cell proliferation mechanisms can be different in vitro versus in vivo, the effect of different drugs, toxins, and gene expression regulation on Na_V channels in this cell function should be considered carefully, as several cross-effects involving other proteins may be present.

Proliferation of non-cancer cells has also been demonstrated to be regulated by VGSCs activity. Vascular endothelial growth factor-induced proliferation in human umbilical vein endothelial cells (HUVECs) is reduced when VGSCs activity is blocked by TTX (Andrikopoulos et al., 2011). Similarly, proliferation of embryonic cardiomyocytes in zebrafish is dependent of the cardiac sodium channel ortholog scn5Lab (Bennett et al., 2013).

The underlying molecular mechanism linking sodium channels to cell proliferation is not completely clear yet, however it appears to be related to a higher response to growth factors and/or hormones. A strong relationship between several of these molecules and the VGSCs activity has been reported, including the epidermal growth factor (EGF) (Liu et al., 2007; Uysal-Onganer and Djamgoz, 2007; Ding et al., 2008; Campbell et al., 2013), the vascular endothelial growth factor (VEGF) (Andrikopoulos et al., 2011; Pan et al., 2012; Sun and Ma, 2013), the insulin-like growth factor (IGF) (Yanagita et al., 2011), the nerve growth factor (NGF) (Brackenbury and Djamgoz, 2007), β-estradiol (Fraser et al., 2010; Hu et al., 2012), and dihydrotestosterone (Diss et al., 2008).

Additionally, the role of VGSCs β-subunits in the proliferation of different cancer cells has also been studied. Overexpressing β2 in stably transfected LNCaP cells in vitro has no effect on the proliferation rate in the first days, however, it suppresses the contact inhibition of proliferation, allowing the cells to keep proliferating even in saturation density (Jansson et al., 2012; Jansson et al., 2014). Likewise, overexpression of β1 led to an increased proliferation of SiHa cervical cancer cells, but no effects are observed in HeLa and CaSki cells, evidencing the importance of specific cellular characteristics of one type of cells and not a general behavior in cervical cancer (Sanchez-Sandoval and Gomora, 2019). In BCa, the role of β-subunits is even more controversial. For instance, the proliferation of MDA-MB-231 cells overexpressing β1 is reduced in vitro compared to control cells (Chioni et al., 2009), on the other hand, the tumors produced by these cells in an orthotopic model in vivo are bigger than those produced by control cells, suggesting a promoted proliferation exerted by β1 (Nelson et al., 2014).

Tumor cells, like normal tissue, require oxygen and nutrients as well as the ability to evacuate metabolic wastes. Given their uncontrolled proliferation, malignant tumoral cells address these needs throughout the activation of angiogenesis, promoting tumor-associated neovasculature, which is induced very early during the development of invasive cancers. These new vessels are typically anomalous, with an excessive vessel branching, leakiness, distorted, and enlarged structures and irregular blood flow (Hanahan and Weinberg, 2011). The formation of new vessels is triggered by factors produced and secreted by tumoral cells; most types of human cancers express elevated levels of VEGF, the major mediator of tumor angiogenesis (Kerbel, 2008), whose expression is induced by several environmental and molecular factors, including hypoxia and VGSCs’ activity, among others. For example, it has been proved that Na_V1.5 channels potentiate the angiogenesis process induced by VEGF through the activation of the PKCα-B-RAF-ERK1/2 signaling pathway, in HUVECs; the tubular differentiation on Matrigel (a commercial basement membrane matrix), is significantly inhibited by the exposure of these cells to TTX (Andrikopoulos et al., 2011).

On the other hand, in epithelial BCa cells, the induced expression of modulatory β1 subunit promotes the VEGF secretion and increases the formation of new vascular vessels, suggesting a contributing role of β1 proteins through the modulation of cellular adhesion as CAMs (Nelson et al., 2014).

About 90% of cancers originate from epithelial tissues, thus designed as carcinomas. In order to invade surrounding tissues, primary tumor cells must undergo dedifferentiation or transdifferentiation processes. The epithelial-to-mesenchymal transition (EMT) takes place through gradual changes in which multiple tumoral cell subpopulations proliferate to increase the tumor size. From an epithelial to a complete mesenchymal phenotype, several steps modulated by different specific cellular niches must occur (Pastushenko et al., 2018). This phenomenon, which is normal in embryonic morphogenesis and wound healing, can be activated transiently, stably, or gradually by carcinoma cells. The EMT process is characterized by loss of cell-cell and cell-matrix adherent junctions and it is typically associated with the transformation from a polygonal apical-basal polarized morphology to a more spindle-like shape capable of degrading and invading the extracellular matrix of surrounding tissues firstly, and secondary distant sites latter (Davis et al., 2014; Ye and Weinberg, 2015). At the molecular level, it has been shown that EMT is under the control of transcription factors such as SNAIL, bHLH and ZEB factors (Lamouille et al., 2014). In this regard, it has been shown that the activity of Na_V1.5 channels can induce the expression of SNAI1 and ZEB1 transcription factors in breast cancer cells; and the downregulation of this channel reverts the mesenchymal phenotype (Gradek et al., 2019).

The EMT involves the downregulation of characteristic epithelial proteins such as E-cadherin, cytokeratin, and laminin-1, while increasing the expression of mesenchymal phenotype molecules, including N-cadherin, vimentin, and fibronectin (Christofori, 2006), which in turn leads to a diminished affinity for epithelial tissue and higher affinity for mesenchymal cells. As a result, cell detachment, migration and invasion are facilitated (Yilmaz and Christofori, 2009). Moreover, as mentioned before, the EMT process can be progressive, as some cancer cells conforming the tumoral mass in transition can express protein markers from both epithelial and mesenchymal phenotypes; this plasticity in protein expression is directly related to the response to pharmacological treatments, and development of drug resistance (Navas et al., 2020).

Generated changes from the benign phenotype can progress to an invasive cancer and then dissemination of malignant cells to distant organs resolving the selection pressure imposed by the tumor environment, leading to the generation of metastasis (Reeves et al., 2018). In these processes, several VGSCs (both α and β subunits) have been shown to play important roles, which are described below.

A crucial step in the progression of metastasis rest on the capacity of cancer cells to degrade the extracellular matrix, enabling the invasion through the adjacent tissue (Linder, 2007). This proteolytic activity, mediated by different enzymes, is highly dependent on the acidification of the peri-membrane extracellular environment, and is regulated by complex mechanisms in which VGSCs play important roles, which are discussed in the next 3 subsections.

Entering to the bloodstream demands changes in shape and cellular volume, including variations in intracellular osmolarity. As exposed previously, VGSCs are activated with V _m depolarization, so they also participate in this cellular property, contributing to cytoskeletal reorganization, changing the ionic balance, and stablishing a V _m shifted to depolarized values compared to normal cells. Functional expression of VGSCs (both, sensitive and resistant to TTX) has been identified in leukemic cells, including cell lines (Fraser et al., 2004), and circulating cells in peripheral blood samples (Lo et al., 2012). Importantly, the blockade of sodium current decreased the invasion capacity of this cells, bolstering the relevant role of VGSCs in the founding of favorable molecular conditions to potentiate invasion (Huang et al., 2015). Also, VGSCs promote the degradation of extracellular matrix surrounding endothelial cells, facilitating the intravasation of cancer cells into the blood or lymph circulatory systems to reach distant organs and produce secondary tumors (Gillet et al., 2009; Brisson et al., 2011; Bon et al., 2016). This occurs as a response to chemotaxis exerted by growth factors, hormones, chemokines, and nutrients, as well as oxygen gradients or galvanotaxis, as has been shown in breast, cervical, lung, and prostate cancer (Djamgoz et al., 2001; Roger et al., 2003; Yan et al., 2009; Pan et al., 2012; Campbell et al., 2013; Lee et al., 2019; Alassaf and Mueller, 2020).

The most frequently associated role of VGSCs with the metastatic cancer behavior is their participation in the enhanced capability of cancer cells to perform transversal invasion, usually analyzed in vitro by transwell assays, using inserts coated with a mixture of proteins simulating the extracellular matrix. In most of the cases where the expression of a specific type of VGSCs is correlated to the malignancy of cancer cells, the role of the channels is mainly throughout an increment of the invasiveness potential. This is the case for breast, cervical, colon, gastric, leukemia, melanoma, non-small cell lung, ovarian, and prostate cancer cells, where the presence of TTX reduced the number of cells capable to invade (Grimes et al., 1995; Laniado et al., 1997; Bennett et al., 2004; Fraser et al., 2004; Fraser et al., 2005; Brackenbury et al., 2007; Roger et al., 2007; Carrithers et al., 2009; Gao et al., 2009; Nakajima et al., 2009; Gao et al., 2010; House et al., 2010; Brisson et al., 2011; Hernandez-Plata et al., 2012; Brisson et al., 2013; Campbell et al., 2013; Huang et al., 2015; Xia et al., 2016; Guzel et al., 2018). In the same sense, the downregulation of VGSCs expression by siRNAs or shRNAs reduces the invasion capabilities of the cells, as shown in astrocytoma, breast, colon, leukemia, prostate, and thyroid cancer (Carrithers et al., 2009; Nakajima et al., 2009; House et al., 2010; Brisson et al., 2011; Brisson et al., 2013; Xing et al., 2014; Guzel et al., 2018; Li et al., 2022), as well as the incubation of colon cancer cells in presence of ranolazine, and endometrial cancer primary cultures with PF-05089771, another VGSCs blocker (Guzel et al., 2018; Liu et al., 2019). In the same line of research, heterologous overexpression of VGSCs in cervical, non-small cell lung, and prostate, cancer cells promotes a higher number of cells capable of invading (Bennett et al., 2004; Campbell et al., 2013; Lopez-Charcas et al., 2018), and the same behavior is observed when using the VGSCs positive modulator veratridine in colon, endometrial cancer cells and non-small cell lung cancer cell lines (Campbell et al., 2013; House et al., 2015; Liu et al., 2019).

Podosomes are membrane-associated structures specialized in the directed cellular movement promoted by actin polymerization at the leading edge of motile cells that respond to chemoattractants by secreting matrix metalloproteinases (MMPs), thus facilitating the cell detachment and cytoskeletal reconfiguration to drive cellular movement. Podosomes are generally associated with non-transformed cells involved with matrix remodeling events (i.e., osteoclasts, and vascular smooth-muscle cells), and not basement membrane invasion. In cancer cells, specialized protruded membrane regions are formed in the invasion process, and, due to the high similarity in structure and components, they have been identified as invadosomes or invadopodia. Both, podosomes and invadopodia, typically exhibit an actin bundle surrounded by several cell signaling regulators to promote motility, such as Arp 2/3, WASP, Src kinase, and cortactin among others. However, invadopodia are highly protrusive and matrix-degrading structures, specialized in directing cancer cell invasion through basement membrane (Murphy and Courtneidge, 2011; Lohmer et al., 2014; Angus and Ruben, 2019; Masi et al., 2020). The functional activity of VGSCs promotes changes in the cellular physiology, which activates cellular directional movement initiated by invadopodia in cancer cells at the beginning of the metastatic process (Brisson et al., 2012). The localized sodium influx permeated through VGSCs are coupled to the activity of other proteins colocalized in lipid rafts to potentiate the cytoskeletal reorganization, cell detach from the extracellular matrix, and invasion to surrounding tissues first and distant sites latter among them. It has been also demonstrated that NHE-1 and the Na⁺/Ca²⁺ exchanger (NCX) both have relevant functions in the invadopodia activity (Kemp et al., 2008; Khananshvili, 2013).

The molecular mechanisms underlying the participation of VGSCs in the metastatic process has not been totally clarified, but a significant amount of evidence has emerged during the last decade. The invasion capacity of cancer cells is a consequence of the simultaneous activation of several of the metastatic behaviors described above (e.g., differential migration, adhesion, activation of MMPs, etc.) together with the activation of other molecules involved in numerous cell signaling pathways and some of which have been correlated with the functional expression of VGSCs, including JAK/STAT, PKC, MAPK, NCX, PKA, and Src kinase (Chioni et al., 2010; House et al., 2010; Andrikopoulos et al., 2011; Brisson et al., 2013; House et al., 2015; Li et al., 2022).

The blockade of intracellular VGSCs in macrophages and melanoma cells prevents cellular invasion, whereas the activation of the channels by veratridine promotes it, apparently through a vesicular intracellular sodium release leading to a mitochondrial calcium release (by the NCX), activating the actin cytoskeleton dynamics, which in turn enables the rapid assembly and disassembly of podosomes and invadopodia (Carrithers et al., 2009). In MDA-MB-231 cells, the physical interaction of Na_V1.5, NHE-1, and caveolin-1 in invadopodia has been shown by co-immunoprecipitation assays. The activation of Na_V1.5 increases Src kinase activity, modifies F-actin polymerization and thus the regulation of the invadopodia formation (Brisson et al., 2013).

Among the hypothesis proposed to explain the molecular mechanisms by which VGSCs promote metastasis, one of them proposes the activation of cytoskeleton reorganization triggering the actin polymerization via PKA activity. Nevertheless, the precise connection between VGSCs activity and phosphorylation cascades activation was missing until a few years ago. Recent evidence has been added to clarify the molecular mechanism. The functional expression of any VGSC type in cancer cells stablish a more depolarized V _m in comparison with no cancerous cells from the same tissue; furthermore, it has been shown that the most depolarized cells in breast cancer have the highest metastatic potential (Fraser et al., 2005; Yang and Brackenbury, 2013). In the highly metastatic breast cancer cells MDA-MB-231, the membrane depolarization stablished by the Na_V1.5 activity induces a phospholipid redistribution, including phosphatidyl inositol bisphosphate (PIP2); and phosphatidylserine, which interacts with the small GTPase Rac1 (anchored to the plasma membrane) to trigger the activation of Rac1 and Src kinases which in turn prompts actin filament polymerization and cytoskeletal reorganization through phosphorylation and positive modulation of the Arp2/3 complex, cortactin and cofilin at the leading edge of the cell; thus boosting the plasma membrane protrusions, which promotes the acquisition of a cell motile and mesenchymal-like cellular phenotype (Brisson et al., 2013; Augoff et al., 2020; Yang et al., 2020). The activity of small GTPases Rac1, Rho, and cdc42 reconfigures the cytoskeleton organization promoting the directed cellular movement and participating in the assembly of specialized motility structures in the cell membrane, the lamellipodia and invadopodia (Burridge and Wennerberg, 2004; Wu et al., 2009). In the same way, the small GTPase K-Ras is also activated because of V _m depolarization and the subsequent redistribution of phosphatidylserine (Zhou et al., 2015), triggering the cell membrane reorganization and other cellular responses.

The role of β subunits of VGSCs in this behavior is less understood. β1 and β2 expression correlates with the invasive potential in prostate cancer cells. The overexpression of β2 in LNCaP cells promotes a 3.5-fold increase of transwell invasion (Diss et al., 2008; Jansson et al., 2012). On the contrary, in breast cancer cells, β1 expression is inversely correlated with the native invasiveness potential of the cells (Chioni et al., 2009), although, overexpressing this subunit in MDA-MB-231 cells enhances the in vitro transwell invasion, as well as the in vivo tumor growth, suggesting the role of β1 as CAM via a trans-homophilic adhesion mechanism that improves process outgrowth as described before, enabling the cell dissemination into surrounding tissues (Nelson et al., 2014). In the same sense, downregulation of β1 or β2 expression by siRNAs decreases invasiveness in these cells, while the treatment with siRNAs against β4 have the opposite effect, as it also does in cervical cancer cell lines, in accordance with the lower or lack of expression of β4 in breast and cervical cancer biopsies compared with normal tissue (Bon et al., 2016; Sanchez-Sandoval and Gomora, 2019). Loss of β4 expression in cancer cells seems to stimulate their invasiveness through the enhanced activation of the RhoA pathway, favoring amoeboid migration of the cells, and the amplitude of I _NaP (Bon et al., 2016).