Pharmacological and nutritional targeting of voltage-gated sodium channels in the treatment of cancers

Summary Voltage-gated sodium (NaV) channels, initially characterized in excitable cells, have been shown to be aberrantly expressed in non-excitable cancer tissues and cells from epithelial origins such as in breast, lung, prostate, colon, and cervix, whereas they are not expressed in cognate non-cancer tissues. Their activity was demonstrated to promote aggressive and invasive potencies of cancer cells, both in vitro and in vivo, whereas their deregulated expression in cancer tissues has been associated with metastatic progression and cancer-related death. This review proposes NaV channels as pharmacological targets for anticancer treatments providing opportunities for repurposing existing NaV-inhibitors or developing new pharmacological and nutritional interventions.


INTRODUCTION
Voltage-gated sodium (Na V ) channels, composed of pore-forming Na V a and auxiliary Na V b subunits, were initially characterized in excitable cells in which they are responsible for the triggering and the propagation of action potentials. Their physiological activity, through a transient depolarizing inward sodium current in cell types such as cardiomyocytes, skeletal muscle cells, or neurons, is well characterized as being responsible for the initiation of excitation-contraction, excitation-secretion, or excitation-expression couplings. As such, these ion channels are critical in numerous physiological functions and mutations in their encoding genes, as well as dysregulation of their activity may lead to serious pathologies called ''sodium channelopathies.'' Na V channels are targets for multiple inhibitory molecules that are FDA approved and clinically used in the treatment of pathologies such as cardiac angina or arrhythmias, epilepsies, chronic pain, or in anesthesiology.
Although the activity of Na V channels has been characterized about 70 years ago, recent data obtained in the past 5 years have shed light on the protein structure, arrangement, and functioning at the molecular level. Indeed, the activity of Na V channels, i.e. sodium currents (I Na ), was first recorded by Hodgkin and Huxley in 1952 from the squid giant axon, using the voltage-clamp technique. These pioneering experiments led to the ionic theory of membrane excitability (Hodgkin and Huxley, 1952). However, at that time the structure of Na V channels was not known and the first evidence of molecular properties came to light at the beginning of the 1980s with the identification of the channel proteins using radiolabeled-neurotoxins highly selective for Na V channels in combination with protein solubilization and purification methods (Agnew et al., 1980;Beneski and Catterall, 1980). Further structural insights into Na V channels were obtained by cloning and screening of cDNA libraries leading to the discovery of the amino acid sequence for these proteins and allowing for modeling of secondary structures based on aliphatic profiles (Noda et al., 1984(Noda et al., , 1986. These seminal studies allowed development of a model in which the pore-forming Na V principal subunit in eukaryotes, later called Na V a-subunit, is composed of a single polypeptide chain of approximately 260 kDa containing four repeated homologous domains (I-IV) of six transmembrane segments (S1-S6). This protein was identified to interact with one or two single-span transmembrane auxiliary subunits, called Na V b-subunits (30-40 kDa), initially characterized as bringing regulatory functions (Isom et al., 1992(Isom et al., , 1994 to the macromolecular complex of eukaryotic Na V channels (Brackenbury and Isom, 2011;Catterall, 2000).
Interestingly, understanding of protein organization and function at the molecular level substantially progressed very recently with the use of X-ray protein crystallography and cryo-electron microscopy, first Na V a and Na V b subunits are differentially and developmentally expressed in several tissues and cell types (Black and Waxman, 2013;Roger et al., 2015). Initial studies identified Na V to be distributed in excitable tissues such as in mammalian central and peripheral nervous systems as well as in skeletal and cardiac muscle. The central nervous system channels mainly comprise the Na V 1.1, Na V 1.2, Na V 1.3, and Na V 1.6 isoforms, whereas the peripheral nervous system channels include Na V 1.7, Na V 1.8, and Na V 1.9. Na V 1.4 and Na V 1.5 were characterized as being the main skeletal and heart muscle isoforms, respectively (Goldin, 2001), whereas they also have been identified to be expressed in the brain and in the dorsal root ganglion (Wang et al., 2008(Wang et al., , 2009(Wang et al., , 2018a(Wang et al., , 2018bBergareche et al., 2015). Therefore, the expression of Na V has long been considered as the hallmark of excitable cells. Again, this paradigm has recently changed with the identification of expression (mRNA and protein) and sometimes activity at the plasma membrane (transient sodium currents) in non-excitable tissues and cells such as in chondrocytes, endothelial cells, microglia, astrocytes, fibroblasts, keratinocytes, islet b-cells, red blood cells, T-lymphocytes, dendritic cells, and macrophages, among others, in which the biological role and subcellular distribution of Na v s is still elusive (Black and Waxman, 2013;Roger et al., 2015).
Recently, it has emerged that Na V a channels, as well as auxiliary Na V b subunits, are aberrantly expressed in non-excitable cancer tissues and cells from different epithelial origins such as breast, lung, prostate, colon, and cervix, whereas they are not expressed in cognate non-cancer tissues. Their expression in carcinoma cells has been associated with cancer progression, suggesting that they could serve as cancer markers and prognostic factors. The expression and activity of Na V channels was shown to promote pro-cancerous properties and, importantly, to contribute to disease progression, in both in vitro and in vivo models. Recent studies shed light on the signaling pathways that are under the control of these channel proteins, coupling membrane activity to cellular properties. Furthermore, the inhibition of Na V channel activity, using small synthetic compounds and FDA-approved drugs as well as with natural dietary compounds potentially opens up new therapeutic strategies. In this review, we summarize current knowledge on the expression of Na V channels in cancers, highlight the signaling pathways involved, and discuss pharmacological and nutritional strategies that represent opportunities for novel anticancer treatments.

The aberrant expression of Na V a and Na V b subunits in cancers
The expression of voltage-gated sodium channel subunits, both Na V a and Na V b, has been reported to be altered in several types of cancer (Table 1). The channel subunits have been detected by molecular biology and biochemical techniques, and in multiple cancer types Na V activity at the plasma membrane, i.e. I Na , could be recorded. Most subunits have been shown to be upregulated in cancers, whereas some of them appear to be downregulated. This aberrant expression has been correlated with oncogenic properties in both in vitro and in vivo models of cancer, mostly in solid tumors including carcinomas (Figure 1).

Na V a and Na V b subunits in prostate cancer
Although early works assessing ion channel activity identified sodium currents in small-cell lung cancer cells (Pancrazio et al., 1989), the first report of the direct contribution of Na V channels in cancer properties came from work performed in prostate cancer (PCa) cells by Prof. M. Djamgoz' group almost 25 years ago. This first study was performed in two rat prostatic tumor cell lines in which they found a differential expression of voltage-activated Na + currents: highly metastatic Mat Ly-Lu cells expressed Na + currents, whereas weakly metastatic AT-2 cell did not express this type of current. For the first time, the functional relevance of the Na + current was demonstrated: blocking I Na with nanomolar concentrations of TTX reduced by $30% the invasiveness of the highly metastatic Mat-Ly-Lu cells (Grimes et al., 1995). Further evidence showed that the Na V 1.7 pore-forming subunit, encoded by the SCN9A gene, was overexpressed in highly metastatic human and rat prostate cell lines in comparison with weakly metastatic cell lines (Diss et al., 2001). Later, this observation was confirmed in vivo by showing that Na V 1.7 was overexpressed approximately 20 times in PCa biopsies versus non-cancer samples . Finally, in a rat model, inoculation with highly metastatic Mat-Ly-Lu cells promotes prostate cancer metastasis in vivo, and blockade of Na V in primary tumors with TTX  or ranolazine (Bugan et al., 2019) reduced lung metastases by 40% and 63%, respectively. However, most of these studies were performed in cell lines, and there are no systematic studies that show a positive correlation between mRNA/protein of Na V 1.7 upregulation and human PCa patient samples.   the Na V b2 subunit may mediate metastatic behavior through association with neural substrates (Jansson et al., 2014).

Na V a and Na V b subunits in breast and colorectal cancer
Breast cancer (BCa) is the most lethal female cancer worldwide (Bray et al., 2018), and colorectal cancer (CRCa) is the third most commonly diagnosed cancer (Arnold et al., 2017). The incidence of these cancers is gradually increasing, thus representing a serious global health problem. The main cause of patient Figure 1. Expression of Na V a in carcinoma and role in invadopodial activity and invasion of extracellular matrices Progression of precancerous into cancer cells is illustrated in the context in the malignant transformation of colon epithelium. Transformed cells have lost cell polarity, replication control, and cell-cell adherent junctions, and they acquired a mesenchymal pro-invasive phenotype. Migrating cancer cells develop a specialized actin-based membrane protrusions called ''invadopodia'' that facilitate cell invasion by providing a coupling of focal extracellular matrix (ECM) degradation together with a directional cell movement. Na V channels are expressed in invadopodial structures, colocalizing with the Na + /H + exchanger type 1 (NHE1). Activity of Na V channels enhances the extrusion of protons by NHE1 and therefore the acidification of the peri-invadopodial microenvironment, thus favoring both secretion and activity of ECM proteases such as cysteine cathepsins and matrix metalloproteinases (MMPs). Cancer cell resting potential (V m ) is around À40 mV, in a window of voltage of Na V channels (overlap between activation and steady-state inactivation curves) in which a small proportion of channels are activated but non-inactivated, thus generating a small but continuous Na + influx through a so-called ''window sodium current.'' Na V channels are also proposed to increase the intracellular levels of Ca 2+ ions by the functioning of Na + /Ca 2+ exchanger (NCX) in a ''reverse mode.'' Thus, the increase in the intracellular concentration of Na + and Ca 2+ , sustains SRC kinase activity, leading to the polymerization of acting filaments and the formation of invadopodial structure. mortality from these two types of cancers, as for the majority of carcinomas, is the development of metastases in distant organs, following the dissemination of cancer cells from the primary tumor ( Figure 1).
Multiple studies have investigated the expression of Na V channels and their contribution to tumor progression and metastasis in BCa and CRCa. Knowledge about signaling pathways and cellular mechanisms induced by Na V channels have been mostly acquired from these cancers. In both cases, the major isoform identified was Na V 1.5, encoded by the SCN5A gene. In BCa samples, Na V 1.5 is overexpressed as compared with normal tissues (Fraser et al., 2005). A high expression was correlated with cancer recurrence, metastasis development, and reduced patient survival (Yang et al., 2012). Most of the mechanistic studies in BCa have been performed in human cancer cell lines such as MDA-MB-231 (highly metastatic) and compared with weakly metastatic cell lines such as MCF-7. It has been shown initially that MDA-MB-231 expresses a TTX-resistant Na + current, lacking in MCF-7 cells , which is encoded by a neonatal splice variant of the SCN5A gene (Fraser et al., 2005). This neonatal variant is due to a switch from adult exon 6B to fetal exon 6A, which are mutually exclusive and encode for a part of the voltage sensor, segments 3 and 4 located in the domain I of the channel. Therefore, these two variants, called hNa V 1.5 and hNa V 1.5e for the adult and the neonatal channels respectively, show different electrophysiological properties in terms of voltage sensitivity and current kinetics (Murphy et al., 2012;Onkal et al., 2008). These changes result in a greater Na + influx for neonatal hNa V 1.5e (Onkal et al., 2008). In the heart, splicing of SCN5A is developmentally regulated, such that the neonatal exon 6A is rapidly replaced by the ''adult'' exon 6B after birth (Murphy et al., 2012) and molecular determinants explaining the abnormal expression of hNaV1.5e in cancer cells have not been identified so far. Nevertheless, the inhibition of channel activity, by either pharmacological (TTX, ranolazine and phenytoin) or molecular (siRNA and inhibitory antibody) approaches, has shown its contribution to migration and invasion of BCa cell lines Fraser et al., 2005;Brackenbury et al., 2007;Driffort et al., 2014;Yang et al., 2012). There has also been some significant progress into uncovering the mechanisms underlying the promotion of invasiveness behavior by Na V 1.5. Experimental evidence suggested that Na V 1.5 induces pro-migratory and pro-invasive properties through a persistent activity at the membrane potential called ''window current,'' and a correlated depolarization of the membrane voltage of breast cancer cells. Particularly, Na V 1.5 activity induced the allosteric modulation of the Na + -H + exchanger type 1 (NHE-1), resulting in an increased activity, leading to the acidification of the extracellular space, thus favoring the pH-dependent activity of proteolytic cysteine cathepsins (Gillet et al., 2009;Brisson et al., 2011). In addition, Na V 1.5 expression and activity were demonstrated to increase Src kinase activity, which promotes the acquisition of an invasive morphology (invadopodia) in MDA-MB-231 cells. Taken together, these observations indicate that Na V 1.5 promotes invadopodia activity of breast cancer cells and the invasion of the surrounding ECM (Brisson et al., 2013) (Figure 1). Recently, Na V 1.5 was identified as importantly promoting the epithelial-to-mesenchymal transition (EMT) and cancer cell invasiveness through the regulation of the salt-inducible kinase 1 (SIK1) (Gradek et al., 2019). Furthermore, Na V 1.5 activates the small GTPase Rac1 by sustaining a plasma membrane depolarization, which as a regulator of activation, induces cytoskeletal reorganization and cellular migration . In addition, in vivo experiments have shown that Na V 1.5 activity promotes metastasis in immunodeficient mice (Driffort et al., 2014;Nelson et al., 2015aNelson et al., , 2015b. Na V 1.5 activity also increases MMP9 expression and reduces apoptosis in primary tumors in vivo (Nelson et al., 2015b).
The SCN5A gene and its protein product the Na V 1.5 channel have also been shown to be overexpressed in colorectal cancer biopsies, as compared with non-cancer samples (House et al., 2010). Na V 1.5 was found to be expressed at the plasma membrane of tumor cells, and its activity (I Na ) was recorded in several carcinoma cell lines (mainly SW-480, SW-620, and HT-29) (House et al., 2010). In colon cancer cells, Na V 1.5 activity promotes cancer cell invasion in vitro, in both 2-and 3-dimensional models, and regulates a network of invasion-promoting genes via modulation of the PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway (House et al., 2010(House et al., , 2015Poisson et al., 2020) (Figure 2). It was shown that, similar to BCa, the neonatal exon 6A splice variant of the Na V 1.5 isoform has a predominant contribution to the invasiveness of CRCa cell lines (Guzel et al., 2019), even though both adult hNa V 1.5 and neonatal hNa V 1.5e splice variants could be detected (Baptista-Hon et al., 2014).
A study reported the downregulation of the SCN9A gene, encoding for Na V 1.7, in CRCa . In this study, authors analyzed genes differentially expressed in CRCa utilizing three Gene Expression Omnibus (GEO) datasets. By screening 46 biomarkers associated with cancer proliferation, drug-resistance, and metastasis, i.e., genes closely associated to patient overall survival, they proposed a risk score with high prognostic value based Figure 2. Participation of Na V a and/or Na V b in pro-metastatic signaling pathways Na V a subunit overexpression and activity in cancer cells trigger biochemical or an electro-biochemical cascades, leading to the acquisition of a pro-invasive cell phenotype. Na V is co-localized with NHE1 in caveolin-1 (Cav-1)-containing lipid rafts and promotes the efflux of protons. Na V activity can be further stimulated by the use of pharmacological activators such as veratridine (inhibitor of the inactivation phase). Activity of Na V a subunits leads to a cAMP-independent activation of protein kinase A (PKA) that activates the cytosolic small GTPase Ras-related protein 1 (Rap1A/B) and the extracellularsignal-regulated kinases (ERK1/2). The transcription factor (TF) metastasis associated in colon cancer 1 (MACC1) is activated by the p38/NF-kb signaling, whereas the TFs c-jun, ELK1, and ETS1 are activated by ERK1/2 and the zinc finger protein SNAI1 is activated through a Na V a-dependent mechanism regulating the expression of genes associated with cytoskeleton reorganization, cell motility, extracellular matrix degradation, and cell invasiveness. It has been demonstrated that MACC1 upregulates the expression of the SLC9A1 gene, encoding for NHE1, thus enhancing its activity at plasma membrane. On the other hand, the electro-biochemical triggering begins with a resting potential depolarization due to the activity of Na V a subunits promoting the activation and recruitment of the small GTPase Rasrelated C3 botulinum toxin substrate 1 (Rac1) at the leading edge of migrating cells. Transforming growth factor b 1 on the expression of five genes: MET (MET proto-oncogene and receptor tyrosine kinase), CPM (carboxypeptidase M), SHMT2 (serine hydroxymethyltransferase 2), GUCA2B (guanylate cyclase activator 2B), and SCN9A. MET and SHMT2 were upregulated whereas CPM, GUCA2B, and SCN9A were downregulated. Interestingly, this observation was confirmed in the human protein atlas immunohistochemistry database (www.proteinatlas. org), as the staining for Na V 1.7 was lower in some CRCa sample tissues . There are also reports indicating the downregulation of Na V 1.6, encoded by the SCN8A gene, in CRCa (Igci et al., 2015). Tumor samples from CRCa patients exhibited reduced expression of Na V 1.6 compared with paired tumour-surrounding normal tissues. SCN8A mRNA levels, analyzed by real-time qPCR, were significantly lower in tumor tissues and in patients younger than 45 years. Results also reveal a relationship between SCN8A expression, gender, grade of CRCa, tumor location, and histopathological classification (Igci et al., 2015). On the contrary, Na V 1.6 protein was highly expressed in metastatic lymph nodes from CRCa patients (Lin et al., 2019). Although the reduced expression of SCN8A, encoding for Na V 1.6, and SCN9A, encoding for Na V 1.7, might harbor predictive values in CRCa, we are still missing clear information to assert whether they have a role, either causative or consecutive, in the carcinogenesis or whether their expression dysregulation is only correlative to cancer transformation or progression. Indeed, the functional activity of Na V 1.6 and/or Na V 1.7 at the plasma membrane of colorectal noncancer or cancer cells has not been demonstrated so far. Furthermore, it cannot be excluded that these channels might be expressed in intracellular compartments, in which they might play diverse functions. Eventually, it is not clear at the moment whether these changes in expression levels concern epithelial cells, or non-epithelial cells in the colorectal tract, such as immune cells, which are key protagonists in colorectal carcinogenesis. As such, the participation of SCN8A and SCN9A in CRCa biology will require further studies.
The expression of Na V b subunits has been studied in BCa and in CRCa. Some Na V b have been shown to be upregulated, whereas others are downregulated in cancer tissues, and mostly these changes appear to correlate with the metastatic behavior of cancer cells, in particular with cell migration and invasion. Most research performed so far studying the role of Na V b subunits in metastatic behavior has been undertaken in BCa. Originally, it was shown that Na V b1 was more abundantly expressed in the weakly metastatic MCF-7 than in the highly metastatic MDA-MB-231 cell line. Interestingly, when MCF-7 cells were transfected with specific siRNA directed against Na V b1, cell adhesion was reduced by 35%, whereas migration was increased by 121%. In contrast, stable expression of Na V b1 in MDA-MB-231 cells increased process length and adhesion while reducing lateral motility and proliferation. Thus, Na V b1 was proposed to act as a cell adhesion molecule in BCa cells, negatively controlling cellular migration (Chioni et al., 2009). Later, it was found that Na V b1 was the Na V b subunit most expressed in BCa and was upregulated (both mRNA and protein) in BCa biopsies, compared with normal breast tissue (Nelson et al., 2014;Bon et al., 2016). More importantly, by using a xenograft model of BCa, it was shown that Na V b1 overexpression increased tumor growth, metastasis, and vascularization, while decreasing apoptosis in the primary tumors. Therefore, this study was the first showing the functional role for Na V b1 in tumor growth and metastasis in vivo (Nelson et al., 2014). Consistent with these results, the use of siRNA to specifically target Na V b1 expression in MDA-MB-231 cells inhibited cancer cell invasion (Bon et al., 2016).
The participation of Na V b3 in tumorigenesis process is poorly understood. Two missense mutations have been identified in the SCN3B gene in high-grade metastatic colorectal cancer biopsies (Sjoblom et al., 2006). The first report suggested that non-mutated Na V b3 mediates a p53-dependent apoptotic pathway in Saos-2, a bone osteosarcoma cell line, after DNA damage (Adachi et al., 2004). In agreement with this, the SCN3B gene is not expressed in highly invasive MDA-MB-231 breast cancer cells (Gillet et al., 2009) or weakly invasive Continued (TGF-b1) increases the expression levels of Na V channels genes (SCNxA), whereas ring finger protein 1 (RING1B), RE1 silencing transcription factor (REST), histone deacetylase 2 (HDAC2) and salt inducible kinase 1 (SIK-1), as well as the n-3 polyunsaturated fatty acids n-3 (PUFA) repress their expression. SIK-1 also impairs the functioning of NHE1 exchanger. The ''auxiliary subunit'' Na V b4 is expressed in normal epithelial cells but is importantly downregulated in invasive cells and high-grade metastatic tumors. The absence of this protein, but specifically the lack of the intracellular C-terminus domain, triggers the acquisition of an amoeboid-mesenchymal hybrid phenotype dependent of the small GTPase Ras homolog family member A (RhoA). Na V b1 proteins have a dual role in cancer cells acting as cell adhesion molecules (CAMs), reducing cell migration and proliferation. However, it has also been demonstrated that Na V b1 promotes tumor growth, metastasis, and vascularization via the proto-oncogene tyrosine-protein kinase Fyn. The Rho-associated protein kinases (ROCK1/2) negatively regulate the expression of Na V a subunits, therefore, silencing or inhibition of these repressors restore Na V channels activity promoting an aggressive cell phenotype.
Pharmacological intervention with FDA-approved drugs or new-design small-molecule lead compounds against Na V channels represents a promising strategy to decrease sodium-channel-associated metastases.
MCF-7 cells (Chioni et al., 2009). In non-tumour breast samples, SCN3B expression was the lowest among all Na V b encoding genes and was still significantly reduced in cancer samples (Bon et al., 2016).
The SCN4B gene was shown to play a critical role as a metastasis-suppressor gene in BCa (Bon et al., 2016). In this study SCN4B mRNA appeared to be significantly expressed in normal breast, colon, rectum, lung, and prostate but consistently downregulated in cancer samples. Furthermore, Na V b4 protein was expressed in normal epithelial cells but significantly reduced in BCa biopsies, especially in high-grade primary and metastatic tumors.
In vitro experiments showed that reducing Na V b4 expression potentiates cell migration and invasiveness though an increase in RhoA activity and the acquisition of a hybrid mesenchymal-amoeboid aggressive phenotype. This effect was independent of Na V a channel activity and was prevented by overexpression of the intracellular C-terminus of Na V b4. On the contrary, SCN4B overexpression reduced cancer cell invasiveness and tumor progression. The findings are in line with previous observations showing decreased levels of SCN4B in invasive versus non-invasive PCa cells . Interestingly, a recent study identified dysregulated miRNA in CRCa and reported an increased miR-424-5p expression in tumor samples that was associated with poor prognosis (Dai et al., 2020). miR-424-5p was found to be elevated in the peripheral blood of CRCa patients, most probably secreted in tumor exosomes. In this study, it was demonstrated that overexpression of SCN4B inhibited HT-29 CRCa cell proliferation, migration, and invasion, and expression of SCN4B was directly inhibited by miR-424-5p (Dai et al., 2020). These results support the tumour-suppressor role of SCN4B in CRCa and identified miR-424-5p as a regulator of its expression in tumors.

Na V a and Na V b subunits in lung cancer
There are two subtypes of lung cancer, small-cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Early works assessing ion channel activity have been undertaken in small-cell lung cancer cells.
In these pioneering works, I Na was initially recorded in human H146, H69, and H128 small-cell lung cancer cell lines (Pancrazio et al., 1989), although this study did not relate the presence of Na V currents to lung tumorigenesis process itself. These currents were most probably due to TTX-sensitive channels, because they were fully inhibited by 5 mM TTX. Although neither the molecular characterization of the channels nor their biological role was described, they were proposed to participate in a ''neuroendocrine-like'' tumor cell phenotype (Pancrazio et al., 1989). Later, it was shown that these sodium currents were actually able to participate in the generation of action potentials in H146 SCLC cells, thus supporting this idea. Interestingly, 5 mM TTX abolished these action potentials, which implies the contribution of a TTX-resistant current (Blandino et al., 1995). In fact, H146 cell sodium currents demonstrated an IC 50 to TTX of 215 nM, leading the authors to indicate that Na V channels were weakly TTX-sensitive. However, this concentration is too high to consider the channels to belong to the TTX-S category but too low to belong to TTX-R isoforms. The most likely explanation for this would be the expression of a population of different isoforms of Na V at the plasma membrane of cells, thus leading to an apparent IC 50 that would be intermediate between TTX-S and TTX-R. Nevertheless, the possibility that Na V channels expressed in these cells are variants (splice-variants or polymorphism variants) showing specific pharmacological properties cannot be excluded.
The hypothesis of the role of Na V a in the acquisition of a neuroendocrine phenotype was also proposed by M. Djamgoz's team (Onganer et al., 2005). Unexpectedly, in this later study, the endocytic activity of SCLC cells was inhibited by using lower nanomolar concentrations of TTX, suggesting the participation of TTXsensitive sodium channels in these SCLC cells. In addition, they found mRNA encoding for Na V 1.3, Na V 1.5, and Na V 1.6 in H69, H209, and H510 cell lines. The latter also showed the additional presence of Na V 1.9 mRNA. Thus, it remains to be elucidated which Na V subunit is responsible for the generation of TTX-resistant action potentials in H146 cells (Blandino et al., 1995) and whether a TTX-resistant Na V channels contribute to migration, invasion, or some other metastatic component, other than endocytic activity (Onganer et al., 2005), in SCLC cell lines. More studies are needed investigating the expression profile and role of Na V channels in SCLC biopsy tissue.
Later analyses were also performed in non-small cell lung cancer (NSCLC) in which the Na V 1.7 isoform was shown to potentiate cancer cell invasion (Roger et al., 2007;Campbell et al., 2013). Although different NSCLC cell lines (H23, H460, and Calu-1) express mRNA for several Na + channel isoforms (Roger et al., 2007), the selective inhibition of Na V 1.7 activity (using TTX) or reduction of expression (by using small interfering RNA) reduced H460 cell invasion by up to 50%. On the contrary, weakly invasive A549 cells showed no evidence of functional Na V channels (Roger et al., 2007). In addition, exogenous overexpression of the Na V 1.7 subunit was sufficient to promote TTX-sensitive invasion of these cells. Interestingly, Na V 1.7 protein ll OPEN ACCESS iScience 24, 102270, April 23, 2021 11 iScience Review expression was found to be higher in cancerous compared with normal-matched human lung tissue (Campbell et al., 2013). It is worth noting that at least in one NSCLC cell line (Calu-1), expression of a TTX-resistant Na V channel significantly contributes to the invasion capacity of this strongly metastatic cell line. However, the molecular identity of the molecular mediator of I Na has not been fully characterized. Non-quantitative PCR results suggested that mRNAs encoding the three known TTX-resistant Na V channels (Na V 1.5, Na V 1.8, and Na V 1.9) may be more abundantly expressed in Calu-1 than in H23 and H460 cells (Roger et al., 2007). Although kinetics and TTX-sensitivity of currents recorded in Calu-1 cells suggest Na V 1.5 activity, more studies are needed to properly identify the molecular identity of the Na V channels mediating the Na + current in these cells. In addition, there are currently no data correlating Na V a expression in lung cancer tissue with clinical outcome.
The expression of Na V b in LCa has been assessed in several studies but so far it is difficult to conclude a general pattern. SCN1B mRNA was found to be expressed in H460, Calu-1, and A549 but not in the H23 NSCLC cell lines. It was also expressed in non-cancer NL-20 and BEAS-2B cells. SCN2B appeared to be weakly expressed in A549 cancer and NL-20 non-cancer cells, whereas not expressed at all in H23, H460, and Calu-1 cancer cell lines. SCN3B mRNA was found to be expressed in all these cell types with the only exception of H460. SCN4B mRNA was expressed in cancer H23 and non-cancer NL-20 and BEAS-2B but not in H460, Calu-1, and A549 (Roger et al., 2007). In patient samples, SCN4B expression levels were downregulated in lung cancer compared with normal lung tissue, and preliminary immunohistochemical analyses in lung cancer tissue microarrays showed a tendency toward decreased protein expression in high-grade primary lung tumors and metastases (Bon et al., 2016). The role of the Na V b1 protein in cell adhesion was also proposed in human non-small cell lung cancer cell lines (Campbell et al., 2013). In this study, it was shown that the highly invasive H460 cells exhibited very low expression of all Na V b subunit mRNAs, confirming previous results (Roger et al., 2007), whereas A549 cells expressed 8-fold higher levels of Na V b1 mRNA. Accordingly, cell adhesion was 2-fold higher in A549 cells compared with H460 cells (Campbell et al., 2013). Moreover, manipulation of Na V b1 mRNA expression by using siRNA or cDNA targeting SCN1B in these two cell lines confirmed the contribution of this subunit in the promotion of cell adhesion and reduced invasion (Campbell et al., 2013).

Na V a and Na V b subunits in gastric cancer
In gastric cancer (GCa) tissue samples and in two human GCa cell lines (BGC-823 and MKN-28 cells), it was shown that the SCN9A gene, encoding Na V 1.7, is the most abundantly expressed Na V a isoform (Xia et al., 2016). A systematic evaluation of 319 GCa tumor tissue samples by immunohistochemistry revealed a correlation of Na V 1.7 expression with poor prognosis, as well as correlation with the expression of the NHE1 exchanger type 1 and the oncoprotein metastasis associated in colon cancer-1 (MACC1). In addition, Na V 1.7 suppression resulted in reduced invasion and proliferation rates of GC cells and growth of GC xenografts in nude mice (Xia et al., 2016). In brief, results of this study indicate that Na V 1.7 promotes GCa progression through MACC1-mediated upregulation of NHE1.

Na V a and Na V b subunits in cervical cancer
The product of the SCN8A gene, the Na V 1.6 channel, has been shown to be upregulated in cervical cancer (CeCa). In a study performed by using primary cultures derived from three different patient CeCa biopsies, the presence of functional Na V channels has been identified and I Na recorded. Primary cells from CeCa biopsies expressed mRNA for different TTX-sensitive Na V a subunits: Na V 1.1-1.4, Na V 1.6, and Na V 1.7 (Diaz et al., 2007). Among these, only the SCN8A gene encoding for Na V 1.6 was shown to be overexpressed by about 40-fold at the mRNA level in CeCa primary cultures and biopsies in comparison with noncancerous cervical tissue. The functional relevance of this Na V channel was demonstrated by blocking its activity with TTX as well as with the Cn2 specific toxin, which in both cases led to a significant decrease in the invasion capacity of CeCa primary culture cells, without affecting proliferative or migratory cell behavior (Hernandez-Plata et al., 2012). This suggested a role for Na V 1.6 in extracellular matrix degradation, and indeed Na V 1.6-mediated invasiveness of CeCa cells specifically involved MMP-2 activity along with increased expression of the NHE1 exchanger . In addition, CeCa cell lines more abundantly express the mRNA for the Na V 1.6 variant, which has exon 18 deleted (D18 variant) rather than the neonatal and adult splice variants. This variant appeared to be distributed in intracellular compartments . However, the functional relevance of the D18 variant to the metastatic behavior of CeCa cells remains to be elucidated. Another interesting question regarding the expression of this D18 variant of Na V 1.6 in CeCa cells is whether it has the same function in intracellular compartments as observed in macrophages and melanoma cells in which the channel has a role in podosome formation and activity (Carrithers et al., 2009).
The role of Na V b1 as a migration suppressor gene was demonstrated in three different CeCa cell lines (HeLa, SiHa, and CaSki) in which SCN1B mRNA levels were around 3-to 6-fold higher than those of Na V b2, Na V b3, or Na V b4. However, differences in protein levels among the four Na V b subunits were more discrete; Na V b1 was again the most highly expressed in HeLa and CaSki cells, whereas in SiHa cells, protein levels for all Na V b were more uniform (Sanchez-Sandoval and Gomora, 2019). Previously, the same group had demonstrated that Na V b1 mRNA levels were also slightly higher in CeCa biopsies than in non-CeCa tissue (Hernandez-Plata et al., 2012). In addition, it was demonstrated that Na V b1 regulated SiHa cell proliferation, specifically by affecting the proportion of cells in the G0/G1 phase of cell cycle (Sanchez-Sandoval and Gomora, 2019). Because Na V b3 was proposed to have anti-cancer properties (Adachi et al., 2004), the effect of its expression in CeCa cells was tested. However, neither its overexpression nor its downregulation affected proliferation in CeCa cell lines, suggesting that the likely pro-apoptotic activity of Na V b3 might not be a generalized mechanism in all cancer types or cells. In this regard, it has been suggested that the p53 protein status in CeCa cell lines is under the control of the E6 protein, the main oncogene expressed as a result of human papillomavirus (HPV) infection (the most frequent risk factor for CeCa incidence) of cervical epithelial cells. The early expression of E6 protein leads to the specific ubiquitination and degradation of p53 (Scheffner et al., 1993), thereby inactivating any pro-apoptotic effect due to the Na V b3 expression in basal conditions. In line with this interpretation, SCN3B expression was increased almost 2-fold in CeCa biopsies when compared with non-cancer samples (Hernandez-Plata et al., 2012). Further studies are needed to fully understand the potential role of SCN3B as well as the mechanism involved in the pro-apoptotic effect in cancer cells. More recent observations in CeCa cell lines confirm the contribution of Na V b4 to cell invasive potential, as the downregulation of SCN4B leads to an increase in the percentage of invading cells in three CeCa cell lines (Sanchez-Sandoval and Gomora, 2019). However, a previous study indicated that mRNA levels for Na V b4 were not significantly different between CeCa and non-CeCa biopsies tissues (Hernandez-Plata et al., 2012).

Na V a subunits in ovarian cancer and endometrial cancers
In ovarian cancer (OCa), the Na V 1.5 isoform appears to be the main Na V a subunit expressed and contributing to the migration and invasion capabilities of cancer cells (Gao et al., 2010;Liu et al., 2018); however, the splicing status of Na V 1.5 in this carcinoma is currently unknown.
In endometrial cancer tissues, a recent study identified the SCN9A gene, encoding for the Na V 1.7 channel, as being the most highly expressed Na V a subunit. Na V 1.7 expression level was associated with tumor size, local lymph node metastasis, and 5-and 10-year survival. Pharmacological inhibition using the PF-05089771 blocker selective for Na V 1.7 and Na V 1.8 induced cancer cell apoptosis and reduced cancer cell invasion .

Na V b in papillary thyroid cancer
Recent results obtained in papillary thyroid cancer (PTC) show that SCN4B is downregulated at both RNA and protein level as compared with normal thyroid tissues (Gong et al., 2018). Importantly, by using databases such as the Gene Expression Omnibus (GEO) and the Cancer Genome Atlas (TCGA)-Thyroid Cancer (THCA), the authors found that SCN4B expression was an independent indicator of favorable recurrencefree survival (RFS) in patients with classical PTC, further contributing to the notion of the SCN4B as a metastases-suppressor gene (Gong et al., 2018). So far, nothing is known about the expression of Na V a subunits in PTC.

Na V a and Na V b subunits in leukemia cells
Although most results related to Na V in cancer were obtained from solid tumors, predominantly carcinomas, there are also some indications that Na V expression might also be dysregulated in hematological disorders such as leukemia, in which they could bear oncogenic properties. In Jurkat leukemic T cell lymphoblasts, original evidence showed that a small fraction of $10% displayed I Na and mRNA encoding for Na V 1.5, Na V 1.6, and to a lesser extent Na V 1.7 and Na V 1.9, were detected (Fraser et al., 2004). I Na was likely carried mostly by a TTX-resistant Na V channel because an IC 50 of $1 mM was measured. Importantly, invasion was reduced by 93% when the cells were treated with 10 mM TTX (Fraser et al., 2004). However, more recent data have shown that Na V 1.6, Na V 1.7, and Na V 1.3 (in that order) are the most abundant Na V isoforms ll OPEN ACCESS iScience 24, 102270, April 23, 2021 13 iScience Review in three acute lymphocytic leukemia cell lines, including Jurkat, MOLT-4, and BALL-1 cells, as well as in peripheral blood mononuclear cells (PBMC). In this study, I Na recorded from approximately 20% of MOLT-4 cells was completely abolished by 2 mM TTX, indicative of TTX-sensitive channels. The same concentration of TTX decreased the invasion of MOLT-4 and Jurkat cells by 90% (Huang et al., 2015).
Interestingly, semi-quantitative PCR results indicated the presence of both the neonatal (18N) and the D18 (exon 18 skipped) isoforms of Na V 1.6 channel in the THP-1 monocytic leukemia cell line (Carrithers et al., 2009). Neither of these two variants form functional channels at the plasma membrane (Plummer et al., 1997). Instead, the D18 Na V 1.6 channel isoform is expressed in vesicular intracellular compartments and crucially contributes in the control of podosome and invadopodia formation (Carrithers et al., 2009). In addition, SCN5A (Na V 1.5) is expressed in the late endosome, rather than at the plasma membrane of the THP-1 cells. The intracellular Na V 1.5 channel was shown to enhance endosomal acidification and phagocytosis (Carrithers et al., 2007), Ca 2+ signaling, and phenotypic differentiation in human macrophages (Carrithers et al., 2011). The same group later demonstrated that SCN5A was expressed as a new splice variant lacking exon 25, resulting in a deletion of 18 amino acids in domain III (Rahgozar et al., 2013), generating non-selective outward currents and small inward currents in a heterologous expression system ).

Na V a in Ewing sarcoma
The Ewing sarcoma (ES) is the second most common primary malignant bone tumor in children and adolescents, following osteosarcoma (Choi et al., 2014). RING1B, a member of the polycomb family of epigenetic regulators, is highly expressed in primary ES tumors. Depletion of RING1B with shRNA in ES cells enriched the expression of genes involved in hematological development, without affecting cellular differentiation (Hernandez-Munoz et al., 2016). Importantly, in ES cells, RING1B directly binds to the promoter of SCN8A, and its depletion results in enhanced Na V 1.6 expression and function. In addition, the migratory speed of RING1B-depleted ES cells was attenuated, suggesting an inverse correlation between SCN8A expression and the migration capabilities of ES cells. Finally, reduced Na V 1.6 function appeared to protect ES cells from apoptosis by a mechanism that maintains low NF-kB levels (Hernandez-Munoz et al., 2016). These findings revealed striking differences in the participation of SCN8A and its product, the Na V 1.6 channel, in sarcomas compared with carcinomas and leukemia. Indeed, Na V 1.6 appeared to have anti-cancer properties in ES, whereas it has pro-invasive functions in carcinomas and leukemia. Therefore, further studies are needed to fully understand the function of SCN8A across different types of cancer.
Conclusions on the roles of Na V a and Na V b subunits in cancers Pore-forming Na V a subunits As previously indicated, the three main Na V a-encoding genes found to be upregulated in cancers are SCN5A, SCN8A, and SCN9A, which encode Na V 1.5, Na V 1.6, and Na V 1.7, respectively. On the other hand, recent reports showed the downregulation of SCN8A and SCN9A genes in some cases. The molecular determinants explaining why these specific isoforms are overexpressed in cancers are not known and might be tissue specific. However, it is tempting to consider the deregulation in tumors of transcription factors that normally restrict the expression of a suite of genes associated with specific tissue functioning in adult tissues, such as the repressor element silencing transcription factor (REST) that restrict the expression of Na V a channels in excitable cells (Bruce et al., 2004;Chong et al., 1995) or other epigenetic regulations such as histone acetylation/deacetylation (performed by Histone Acetylases HAT and Histones Deacetylases HDAC, respectively), DNA, or histone methylation. Indeed, it was recently proposed that REST and HDAC2 play important role as epigenetic regulators and their inhibition in MCF-7 breast cancer cells enhanced the expression of Na V 1.5 and promoted invasive capacities (Kamarulzaman et al., 2017). Nevertheless, it is interesting to notice that, when specifically studied in cancer cells, several neonatal splice variants of channels have been identified (Fraser et al., 2005 iScience Review perimembrane pH, and extracellular matrix degradation (Yang et al., 2012;Hernandez-Plata et al., 2012;Roger et al., 2003;Grimes et al., 1995;Fraser et al., 2005;Gillet et al., 2009;Mycielska et al., 2003;Djamgoz et al., 2001;Brisson et al., 2013). In addition, Na V a subunits promote tumor growth, invasion, and metastasis in in vivo rodent models (Nelson et al., 2015b;Driffort et al., 2014;Batcioglu et al., 2012;Yildirim et al., 2012). Comparative studies performed in different cancer types indicate the involvement of these Na V a isoforms in similar functional properties, arguing for isoform-independent signaling pathways. The activity of the channels at the plasma membrane appears to be critical. Indeed, Nava subunits are functionally active in cancer cell lines and primary tumor cells cultured in vitro, as well as in murine tumor xenograft tissue slices in vivo (Fraser et al., 2005;Roger et al., 2003;Hernandez-Plata et al., 2012;Nelson et al., 2015b), and their inhibition, using different drugs and small molecules such as TTX, ranolazine, phenytoin, Cn2 or PF-05089771, inhibits invasion (Nelson et al., 2015a(Nelson et al., , 2015bDriffort et al., 2014;Batcioglu et al., 2012;Yildirim et al., 2012;Lopez-Charcas et al., 2018;Roger et al., 2003Roger et al., , 2007Liu et al., 2019).
Importantly, the membrane potential (V m ) of cancer cells is typically relatively depolarized compared with terminally differentiated non-cancer cells (Yang and Brackenbury, 2013). At this range of Vm, Na V a channels would be expected to be predominant in the inactivated state. However, in cancer cells the V m is generally between À40 and À30 mV and is situated in a window of voltage that provides a small non-inactivating persistent Na + current flowing into the cell, locally increasing intracellular Na + concentration (Yang et al., 2012;Roger et al., 2003;Campbell et al., 2013). Recently a Na + -dependent intracellular signaling pathway, involving Salt-inducible kinase 1, has been proposed to account for pro-invasive effects of Na V a (Gradek et al., 2019).
The main role attributed to Na + is to serve as a mere mediator of the membrane potential, in excitable as well as in non-excitable cells. It is also characterized to support ion (among which Na + /K + , Na + /Ca 2+ , Na + / K + /Cl À , Na + /HCO 3 À ) exchanges and nutrient/metabolite transports across membranes (Na + /glucose for example). The role of second messenger is mostly attributed to the Ca 2+ ion, for which a lot of specific probes and tools have been developed over the last 20 years. In contrast, no direct and specific biological sensors for Na + have been identified, and tools to study Na + evolution still lack sensitivity or dynamics. Yet, there is some evidence suggesting that Na + could act as a second messenger per se and might regulate several important signaling pathways in normal cells. Indeed, recent data support a direct role of Na + in controlling kinases activity (Jaitovich and Bertorello, 2010), membrane fluidity, and protein diffusion through an interaction with phospholipids (Hernansanz-Agustin et al., 2020) or to induce inflammatory stress (Amara et al., 2016). Therefore, this raises the possibility that Na + could also serve as a second messenger in cancer cells to activate signaling pathways promoting aggressiveness. To further support this hypothesis, it is worth mentioning that early studies questioned the involvement of intracellular Na + content and the consequences on malignant cell proliferation, invasive capacities, and the development of metastases (Cone, 1974). Indeed, much higher Na + concentrations have been recorded in tumor cells, as compared with non-cancer cells by energy-dispersive X-ray microanalyses (Cameron et al., 1980) as well as by 23Na-magnetic resonance imaging (Ouwerkerk et al., 2007;Jacobs et al., 2004;Zaric et al., 2016) and was proposed to serve as an indicator of malignancy.
The inward Na + current may also further depolarize the V m , which might also participate in promoting migration. In support of this hypothesis, it was demonstrated in breast cancer cells that Na V 1.5 sustained V m depolarization, which activated the RhoGTPase Rac1, subsequently inducing cytoskeletal reorganization and cellular migration   (Figure 2). Although there is clear evidence that Na V a channels expressed at the plasma membrane are critical in the acquisition of oncogenic properties, the discovery of splice variants with expression restricted to intracellular compartments, such as endosomes, phagosomes, or lysosomes Carrithers et al., 2009), suggests a more complex role.
The Na V b1 subunit has been shown to increase cancer proliferation, cell adhesion, increase neurite-like process outgrowth formation, and promote cancer cell invasion, while slowing migration in vitro (Nelson et al., 2014; ll OPEN ACCESS iScience 24, 102270, April 23, 2021 15 iScience Review Chioni et al., 2009;Bon et al., 2016;Sanchez-Sandoval and Gomora, 2019). In vivo, Na V b1 overexpression increases angiogenesis and reduces apoptosis, thus increasing tumor growth and metastasis (Nelson et al., 2014). Taken together, these results are in favor of a pro-cancerous role of Na V b1, and the effects appear to be dependent in part on the regulation of the Na V a pore-forming subunit as well as on the extracellular CAM motif (Nelson et al., 2014). Na V b2 expression in prostate cancer cells also increases process extension, adhesion, invasion, and migration in vitro but reduces tumor take in vivo (Jansson et al., 2012(Jansson et al., , 2014. Conversely, Na V b3 and Na V b4 may function as tumor suppressors. The SCN3B gene contains p53 response elements, and Na V b3 suppresses colony formation and promotes chemotherapy-induced apoptosis in a p53-dependent manner (Adachi et al., 2004). Na V b4 expression is downregulated in breast, colorectal, lung, cervical, and prostate tumors and papillary thyroid cancer compared with normal tissue (Hernandez-Plata et al., 2012;Bon et al., 2016;Diss et al., 2008;Gong et al., 2018;Sanchez-Sandoval and Gomora, 2019). In addition, Na V b4 functions as a tumor and metastasis suppressor gene in vivo (Bon et al., 2016). This tumor-suppressing function occurs via b4-mediated control of RhoA GTPase activation (Bon et al., 2016) (Figure 2).

Na V a as anticancer targets for repurposed drugs and new small inhibitory molecules
Na V a are attractive drug targets because of the broad therapeutic potential of their blockers. Considering the fact that Na V a are expressed in metastatic cells in various tumors, significant effort has been made to develop Na V a blockers as potential drugs for cancer treatment. This section of the review focuses on such efforts that took place in the past 10 years. These efforts for blocker development can broadly be classified into two sections: (1) repurposing drugs that are FDA approved for other clinical uses (local and general anesthetics, antiepileptic and anticonvulsant, antiarrhythmic drugs); (2) rational design and development of novel Na V a blockers for cancer treatment.

Repurposing of FDA-approved Na V a blockers
There are numerous existing Na V a inhibitors licensed for clinical use. In several cases Na V a inhibition is considered an off-target effect of these drugs. For example, tricyclic antidepressants, including amitriptyline, inhibit not only the serotonin transporter but also several neurotransmitter receptors and voltage-activated ion channels including Na V a. In other cases Na V a inhibition is considered the primary mechanism of the drug's intended therapeutic effect. This is true for several anti-seizure medications (e.g. phenytoin and carbamazepine) and all of the local anaesthetics, although these too have additional off-target actions. Regardless of whether it is a primary or secondary effect of a licensed medication, Na V a inhibition might be beneficial in patients with cancers associated with Na V a expression. This raises the intriguing possibility that approved Na V a-inhibiting drugs might be repurposed to treat cancer.
Benefits of repurposing approved medications include prior knowledge of their mechanisms of action and the availability of toxicology and safety data, thereby avoiding the need for drug discovery and early phase clinical trials. Drawbacks include limited potential for developing intellectual property, leading to a lack of both funding potential and industry involvement (Pushpakom et al., 2019). Nevertheless, despite the potential drawbacks, there are some notable successes, and a well-trodden pathway to repurposing is through the use of electronic health records to link prescribing data to potentially beneficial health outcomes. A good example of the impact of this type of retrospective clinical analysis is the identification of an association of aspirin use with reduced risk of colon cancer (Dube et al., 2007). Similar approaches are being used in studies exploring a possible relationship between Na V a inhibitors and outcomes in cancer patients.
Anti-seizure and class 1 antiarrhythmic Na V a inhibitors A recent study, using retrospective clinical analysis to explore the possibility of a beneficial effect of Na V inhibiting medications, examined several class 1 antiarrhythmic and antiseizure medications in patients with breast, bowel, or prostate cancer (Fairhurst et al., 2015). The combined analysis revealed that these medications (including class I antiarrhythmic drugs, lamotrigine, carbamazepine phenytoin, and valproate) were collectively associated with decreased median time to death compared with the control patient group, with significantly increased mortality in the drug group. This study clearly does not support the idea of repurposing antiseizure Na V a inhibitors in the treatment of breast, bowel, or prostate these cancers. However, as the authors pointed out, the causes of death were not available in the large primary care dataset, and co-morbidities were among the likely confounding factors. In many cases, patients treated with Na V a-inhibiting drugs will be suffering from life-threatening disorders such as epilepsy, and it is difficult to completely accommodate this confound in retrospective analyses. iScience Review

Analgesic Na V a inhibitors
There has been considerable recent interest in the idea that anesthetics and analgesics used during surgical tumor excision might influence subsequent cancer recurrence. Surgery can cause the release of tumor cells into the circulation, and the number of postsurgical circulating cancer cells is known to be a negative prognostic indicator of disease-free survival (Yu et al., 2018). The perioperative period, i.e. immediately before, during, and after surgery, may therefore be an opportune time for interventions that inhibit the potential for metastatic invasion. A variety of drugs are typically administered during surgery including general anesthetics, analgesics, and anti-muscarinic and neuromuscular blockers. Some of these, such as inhalational general anesthetics, may have the potential to worsen outcomes by suppressing the immune response (Stollings et al., 2016). By contrast, local anesthetics may provide more favorable outcomes. Local anesthetics are often administered regionally to provide blockade of afferent nociceptive fibers entering the spinal cord. Several retrospective clinical studies suggest that regional analgesia during breast and prostate cancer surgery increases disease-free survival (Forget et al., 2019). The use of regional anesthesia diminishes or abolishes the need for general anesthetic during surgery. It was therefore initially hypothesized that the general anesthetic sparing effect of regional analgesia with local anesthetics accounts for the apparent beneficial effect (Sessler et al., 2008). However, a recent large prospective multicenter trial comparing outcomes after breast cancer surgery under inhalational anesthesia with or without paravertebral analgesia by ropivacaine or levobupivacaine revealed no difference in disease-free survival (Sessler et al., 2019).
Most of the ongoing clinical trials exploring the impact of anesthetic technique on cancer outcomes are predicated on the idea that the potential benefit of local anesthetics is conferred indirectly through their inhalational anesthetic sparing effect. However, it is possible that local anesthetics such as lidocaine, ropivacaine, and levobupivacaine provide a direct beneficial effect through Na V a inhibition (Baptista-Hon et al., 2014;Elajnaf et al., 2018). If this is the case, then a more direct approach for administering these drugs directly onto the tumor may prove to be beneficial. Lidocaine, in addition to being a local anesthetic, is also used intravenously as a class 1b antiarrhythmic agent and a circulating analgesic. Ongoing clinical trials will test whether lidocaine delivered directly onto breast tumors prior to excision or intravenously during the perioperative period for colon cancer surgery will prolong postoperative disease-free survival (NCT01916317, R.A Badwe, 2013;NCT02786329, D. Ionescu, 2016). We await the outcome of these trials with interest and note that there are several other approved Na V a-inhibiting drugs that should be examined in retrospective clinical studies for potential beneficial effects in cancer outcomes.

Rational design of small molecule Na V a blockers
Rational designing of Na V a inhibitors has been difficult because detailed structural information of drug-binding sites for this integral membrane protein were lacking until very recently. Therefore, early effort for the discovery of Na V a blockers mainly relied on strategies such as ligand-based drug design, natural-product-based drug design, in silico screening, and similarity searches. However, recent reports on the structures of human and bacterial Na V a and bound ligands shed light on their binding site (Cervenka et al., 2018;Nguyen et al., 2019;Pan et al., 2018;Payandeh et al., 2011;Shen et al., 2017Shen et al., , 2018Shen et al., , 2019. These reports will certainly aid in the structurebased design and discovery of Na V a blockers. A summary of the available reports on the identification and evaluation of Na V a blockers for potential use in cancer therapy follows. One such effort to identify Na V a blockers used a pyrrole-imidazole marine alkaloid, clathrodin (Hodnik et al., 2013). Clathrodin was originally isolated from the Agelas clathrodes sponge. Several conformationally restricted analogues of clathrodin containing a 4,5,6,7-tetrahydrobenzo [d] thiazol-2-amine moiety are blockers of Na V 1.3, Na V 1.4, Na V 1.5, and Na V 1.7 channels. These compounds display state-dependent inhibitory activity of these channels at low micromolar concentrations. The most active compound (4e, Figure 3) identified from this study represents a novel selective blocker of Na V 1.4 channel with an IC 50 value of 8 mM. The use of clathrodin analogues as a template for ligand-based virtual screening of commercially available ZINC library of compounds using ROCS software identified two potent lead compounds 2 and 16   (Figure 3). These blocked I Na produced by Na V 1.7 with IC 50 values of 7 mM and 9 mM, respectively.
Plant-derived polyphenolic natural products have also been reported as Na V a inhibitors. For example, the plant phenolic, resveratrol (Figure 3), found at high concentrations in red grapes inhibits Na V a with an IC 50 value of 50 mM (Fraser et al., 2014). Resveratrol also suppresses lateral cell motility by up to 25%, transverse cell motility by 31%, and cell invasion by 37%, without affecting cellular proliferation or cell viability of MAT-LyLu cells. Another similar phenolic is caffeic acid phenethyl ester (CAPE, Figure 3)  Na V activity in several invasive cancer cell lines, including breast (MDA-MB-231 and MDA-MB-468), colon (SW620), and non-small cell lung cancer (H460). Motility and invasion of MDA-MB-231 cells were reduced by up to 14% and 51%, respectively by CAPE at 1 mM without affecting cell proliferative activity (Fraser et al., 2016). Shaheen et al. used an in silico approach to identify Na V a blockers with superior pharmacological profile compared with phenytoin (PHT) and carbamazepine (CBZ) in 2015 (Shaheen et al., 2015). They conducted a similarity search in the PubChem database with PHT and CBZ as query molecules using the Tanimotobased similarity search. The search was further refined by docking of these molecules into the binding site of the homology model of SCN1A using MolDock program. This study identified high-affinity compounds similar to PHT and CBZ. The lead compounds were further evaluated for toxicity profiles and biological activity. Two of the best compounds identified by this study, NSC403438 and AGN-PC-0BPCBP (Figure 3), demonstrated better binding affinity to Na V a compared with PHT and CBZ, with NSC403438 being a superior inhibitor of I Na with lower toxicity, better IC 50 value, and optimal bioactivity. Na V a inhibitors were also derived from the natural product, crambescin (Nakazaki et al., 2016). Enantiomerically pure crambescin A, B, and C carboxylic acid derivatives were synthesized and evaluated for their ability to block Na V a. Structure activity relationship studies revealed that the natural enantiomer of crambescin B, carboxylic acid (Figure 3), is the most active compound with activity comparable to TTX. The cyclic guanidinium moiety present in this molecule is indispensable for its activity.  (Dutta et al., 2018). The Na V a-binding data (IC 50 ) for 67 compounds were used to train a comprehensive CoMFA model, which effectively covered 3D space and spanned over 4 orders of magnitude in biological activity. Potency predictions by this model have been highly accurate for more than 30 compounds that were synthesized and evaluated. Five compounds shown or predicted to have low nanomolar Na V a binding were further evaluated for the inhibition of hNa V 1.5e currents in individual breast cancer MDA-MB-231 cells. Of these, two lead compounds, 1 and 4 (Figure 3), were found to be most effective in whole-cell patch-clamp studies and showed significant invasion inhibitory activities at concentrations as low as 1 mM without affecting cell viability.
Boezio et al. reported several sulfonamides with highly selective Na V 1.7 blockade activity (Boezio et al., 2018). This novel series of blockers contained a triazole sulfone, which served as a bioisostere for the acyl sulfonamide group. This work resulted in the discovery of a series of potent Na V 1.7 blockers with selectivity over Na V 1.5 and favorable pharmacokinetic properties in rodents. An example of such a blocker is compound 35 (Figure 3) Moreover, naringenin inhibited cell motility by reducing the expression of the SCN9A gene at the mRNA level. In conclusion, naringenin was found to have direct or indirect blocking activity on the SCN9A-encoded channel. Most recently, in 2019, Wang et al. has reported Na V 1.7 channel blockers using a comparative molecular field analysis (CoMFA) model for the binding of ligand to Na V a, generated based on diverse set of compounds. No channel current blockade data were presented in the paper. However, there was an extensive anticancer evaluation of the identified lead compounds S0154 and S0161 . Both showed anticancer and antimetastatic effects against PC3 prostate cancer cells and significantly inhibited cell viability, with IC 50 values in the range of 5-26 mM. Both these compounds inhibited the expression of Na V a, increased the intracellular level of Na + , and caused cell-cycle arrest in G2/M phase. The compounds also inhibited the invasion of PC3 cells. Furthermore, S0161 inhibited the PC3 tumor growth by about 51% in an in vivo xenograft model .
In conclusion, there have been major advances in Na V a-targeted drug discovery over the last decade. Specific Na V a isoforms have been implicated in the metastasis development of a variety of tumors raising the possibility of developing tumor selective drugs. Recent advances in the discovery of high-resolution crystal and cryoEM structures of Na V a should further advance the field with structure-based drug design efforts.

Na V a as targets for nutritional management of cancers
Cancer is a metabolic disease that depends on bioenergetic parameters (Penkert et al., 2016). Cancer progression is generally associated with the survival of cancer cells under conditions of low oxygen levels and nutrient deprivation and relies on metabolic adaptations (Dumas et al., 2017;Hanahan and Weinberg, 2011). These metabolic adaptations allow cancer cells to survive the pressure of environmental conditions and to fulfill the high energy demands associated with their high anabolic activity Porporato et al., 2016). Also, this metabolic switch toward an aerobic glycolysis brings selective advantages by promoting invasive activities and metastatic properties (Brisson et al., 2012;Webb et al., 2011). Furthermore, the metabolic reprogramming concerns not only tumor cells but also multiple cell types and organs of the host, thus leading to an overall deregulation of the energetic balance in patients, called tumor cachexia (Fearon et al., 2012). This devastating syndrome, initially triggered by the release of soluble tumor factors and the participation of a systemic inflammation, is characterized by anorexia, the loss of skeletal muscle mass, in some cases with the loss of adipose tissue mass, and a general weakening of patients, impeding their quality of life and decreasing the tolerance to antineoplastic therapies (Fonseca et al., 2020;Biswas and Acharyya, 2020). In this context, bringing a nutritional support to patients is required to allow holding the most efficient possible treatment. Nutritional interventions are mostly aimed at preventing the wasting of body compartments in patients but could also be a source of active anti-cancer molecules. Furthermore, diet represents a controllable component of the environment and brings promising strategies to increase treatment efficacy in combination with conventional ll OPEN ACCESS iScience 24, 102270, April 23, 2021 19 iScience Review chemotherapeutics. Some dietary compounds have also been shown to decrease the risk of carcinoma development and may prolong the survival of patients (Dumas et al., 2017).
As pointed out in the previous section, several natural compounds in the diet, such as resveratrol and caffeic acid phenethyl esters, are effective at inhibiting Na V a subunits (Fraser et al., 2014(Fraser et al., , 2016. Dietary lipids have also been proposed to inhibit Na V a and modulate cancer progression. Indeed, dietary lipids incorporate into cellular membrane and alter Na V a or their pharmacology (Agwa et al., 2018;D'avanzo, 2016). Among dietary lipids, long-chain n-3 polyunsaturated fatty acids (n-3 PUFA) have been described in epidemiological studies to delay or prevent the appearance of breast cancer (Rose et al., 1996;Bougnoux et al., 2010). From both in vivo and in vitro studies, n-3 PUFA have been reported to induce multiple anti-tumour effects, and their dietary consumption was associated with a lower risk of cancers, such as breast or colorectal cancers (Bougnoux, 1999;Bougnoux et al., 2010;Eltweri et al., 2016). Even though n-3 PUFA were suggested to prevent prostate cancer (Moreel et al., 2014;Li et al., 2017), their beneficial effect remains to be demonstrated through more intervention trials or observational studies (Aucoin et al., 2016). A pilot study showed that fatty acid composition of breast adipose tissue differed according to breast cancer focality: low levels of the two long-chain n-3 PUFA docosahexaenoic acid (DHA, 22:6n-3) and eicosapentaenoic acid (EPA, 20:5n-3) were associated with tumor multifocality, which is considered a marker of cancer aggressiveness (Ouldamer et al., 2016a). These results could indicate that differences in adipose tissue concentration, a surrogate of past dietary uptake, may contribute to mechanisms influencing cancer progression.
Long-chain n-3 PUFA have been proposed to increase tumor sensitivity to chemotherapeutic agents with no sensitization of normal tissues and no additional side effect (Bougnoux et al., 2010). As such, DHA and EPA have generated intense interest due to their ability to reduce resistance to anthracyclines, taxanes, or radiotherapy in mammary tumor models (Bougnoux et al., 2010;Hajjaji et al., 2011Hajjaji et al., , 2012Ouldamer et al., 2016b).
N-3 PUFA have also been shown to have anti-invasive and anti-metastatic properties (Blanckaert et al., 2010;Gillet et al., 2011;Rose et al., 1994Rose et al., , 1997Bougnoux et al., 2010). On the other hand, they are capable of modulating the activity of NHE exchangers (Besson et al., 1996;Lacroix et al., 2008) and ion channels (Tillman and Cascio, 2003;Jude et al., 2003). Interestingly, in expression systems and in native rat cardiomyocytes, the activity of Na V 1.5 was initially found to be inhibited by n-3 PUFA (Kang and Leaf, 1996;Kang et al., 1997) and in initial studies have proposed that these effects could be mediated by a direct binding of n-3 PUFA to specific residues of the channel (Xiao et al., 1998(Xiao et al., , 2001. Therefore, n-3 PUFA could also exert their beneficial effects on cancers through a reduction of Na V 1.5 (Pignier et al., 2007;Gillet et al., 2011). However, contrasting results were obtained in human breast cancer cells in which I Na was not inhibited by acute applications of n-3 PUFA, even at high concentrations (30-50 mM) (Wannous et al., 2015) at which they also have anti-proliferative effects (Barascu et al., 2005). This discrepancy might be due to the fact that cancer cells mostly express the hNa V 1.5e neonatal splice variant (Fraser et al., 2005). However, growing breast cancer cells in the presence of low doses of DHA (0.5-10 mM) reduced SCN5A gene expression and levels of Na V 1.5 proteins and I Na (Wannous et al., 2015;Isbilen et al., 2006). This inhibition of SCN5A expression was mediated by the lipid-sensitive nuclear receptor peroxisome proliferator-activated receptor b (PPAR-b). Correlatively, the inhibition of Na V 1.5 activity was also responsible for a reduced activity of the downstream protagonist NHE-1, thus decreasing H + efflux, preventing extracellular matrix degradation proteolytic activity, and inhibiting breast cancer cell invasiveness (Wannous et al., 2015). A recent report also demonstrated the efficacy of EPA to reduce migration and proliferation of ovarian cancer cells by inhibiting Na V 1.5 .
Such regulations concerning other Na V a involved in cancer properties, i.e. Na V 1.6 and Na V 1.7, should be investigated. It is also of interest to note that n-3 PUFA, through the activation of PPAR-g, have been shown to downregulate the expression of NHE-1 and reduce cancer colony growth (Kumar et al., 2009). Furthermore, incorporation of n-3 PUFA into phospholipids induces changes in the physico-chemical properties of cell membranes (Yaqoob and Shaikh, 2010), which in turn affects NHE-1 activity (Dendele et al., 2014).
Further to these effects on Na V channels and downstream signaling pathways, it should be mentioned that n-3 PUFA might exert a multiplicity of actions by interfering with several signaling pathways, some of them being beneficial to the prevention or treatment of cancer. As such, a lack of specificity is not obligatorily detrimental, and pleiotropic effects might increase the efficacy of the anticancer treatment.
N-3 PUFA supplementations were proposed to have beneficial effects in reducing mammary tumor growth, by slowing down cancer cell proliferation (Barascu et al., 2005). N-3 PUFA treatment was demonstrated to inhibit cyclin B1 and the expression of the cell division cycle 25C phosphatase, which dephosphorylates cyclin-dependent kinase 1 (Barascu et al., 2005). In addition, the nuclear receptor PPARb appeared to regulate the DHA-related inhibition of MDA-MB-231 and MCF-7 cells proliferation. This allowed identifying PPARb as an important protagonist in the inhibition of breast cancer cell proliferation and mammary tumor growth under DHA-enriched diet (Wannous et al., 2013). N-3 PUFA have been proposed to regulate autophagy in cancer cells and as such could be involved in both survival and apoptosis, depending on the carcinogenetic phase and the treatment context (Ferro et al., 2020). DHA was found to induce apoptosis in cancer cells (Jing et al., 2011). DHA-induced autophagy was associated with p53 loss, with the activation of AMPK and the decrease in the activity of mTOR. Autophagy inhibition suppressed apoptosis, and autophagy induction further enhanced apoptosis in response to DHA treatment (Jing et al., 2011). There is evidence that n-3 PUFA may inhibit the expression of EMT markers and reduce associated invasive properties in cancer cells (D'eliseo et al., 2016;Yin et al., 2016). Altogether, these effects could bring beneficial values to delay the appearance/diagnosis of a primary tumor and as such be of interest in primary prevention.
N-3 PUFA, which are highly peroxidizable, were also proposed to improve the efficacy of anticancer treatments by amplifying oxidative stress generated by anthracyclines or radiotherapy . The acquisition of resistance to chemotherapeutic agents represents an important limitation in cancer.
Resistance to taxanes has been proposed to depend on the induction of signaling pathways such as PI3K/Akt and ERK1/2, which promote survival and cell growth in human cancer cells. In docetaxel-treated MDA-MB-231 cells, phosphorylated-ERK1/2 levels were increased by 60% in both membrane and nuclear compartments, compared with untreated cells, and ERK1/2 activation depended on PKCε and PKCd activation. In comparison, in cells treated with DHA, docetaxel was unable to increase PKCε and PKCd levels, thus resulting in the reduction of ERK1/2 phosphorylation and the increase in docetaxel efficacy (Chauvin et al., 2016). In addition to these effects, n-3 PUFA were proposed to increase the efficacy of the chemotherapeutic treatment by remodeling the tumor vascular network, thereby improving the delivery of anticancer drugs within the tumor (Kornfeld et al., 2012). These results support the hypothesis that n-3 PUFA, which do not induce any toxic effect, could be used as an adjuvant to cancer therapy.

Conclusions and perspective for the treatment of cancers
There is now clear evidence that the abnormal expression of Na V subunits occurs during carcinogenesis and is associated with cancer progression toward metastatic states. Several different Na V a play a role, including Na V 1.5, Na V 1.6, and Na V 1.7, but the intracellular signaling pathways they regulate appear to be the same or very similar in all studied cancer types, leading to the induction of invasive properties. The activity of such channels at the plasma membrane, and consequent I Na , appear critical. Therefore, the inhibition of Na V a in cancer cells represents a new anti-cancer strategy that could be achieved in several ways alone or in combination: repurposing existing Na V a-inhibitory drugs, developing new small inhibitory molecules, and through dietary interventions. As shown above, Na V b subunits also have important roles in cancer cell biology and in cancer progression, acting both as auxiliary subunits of Na V a and as CAMs. However, they do not exert a recordable activity per se, and their direct ''inhibition'' or ''activation'' would be challenging. Therefore, dietary strategies aiming at controlling their expression, i.e. reducing the expression of SCN1B and SCN2B, while maintaining the expression of SCN3B and SCN4B, might represent powerful strategies. For this purpose, future studies aiming at unraveling transcriptional and epigenetic regulators will be of high interest.