The sodium channel β1 subunit mediates outgrowth of neurite-like processes on breast cancer cells and promotes tumour growth and metastasis

Voltage-gated Na+ channels (VGSCs) are heteromeric proteins composed of pore-forming α subunits and smaller β subunits. The β subunits are multifunctional channel modulators and are members of the immunoglobulin superfamily of cell adhesion molecules (CAMs). β1, encoded by SCN1B, is best characterized in the central nervous system (CNS), where it plays a critical role in regulating electrical excitability, neurite outgrowth and migration during development. β1 is also expressed in breast cancer (BCa) cell lines, where it regulates adhesion and migration in vitro. In the present study, we found that SCN1B mRNA/β1 protein were up-regulated in BCa specimens, compared with normal breast tissue. β1 upregulation substantially increased tumour growth and metastasis in a xenograft model of BCa. β1 over-expression also increased vascularization and reduced apoptosis in the primary tumours, and β1 over-expressing tumour cells had an elongate morphology. In vitro, β1 potentiated outgrowth of processes from BCa cells co-cultured with fibroblasts, via trans-homophilic adhesion. β1-mediated process outgrowth in BCa cells required the presence and activity of fyn kinase, and Na+ current, thus replicating the mechanism by which β1 regulates neurite outgrowth in CNS neurons. We conclude that when present in breast tumours, β1 enhances pathological growth and cellular dissemination. This study is the first demonstration of a functional role for β1 in tumour growth and metastasis in vivo. We propose that β1 warrants further study as a potential biomarker and targeting β1-mediated adhesion interactions may have value as a novel anti-cancer therapy.

Although the death rate from breast cancer (BCa) is falling in many countries, it is still the leading cause of cancerrelated deaths in women, due to metastasis. 1,2 To metastasize, tumour cells undergo a complex sequence of events, including adhesion/detachment, migration, and invasion. Given that treatment options for metastatic BCa are mainly restricted to palliation, it is necessary to better understand the mechanism(s) involved in order to identify new targets and develop new therapies. 3 Voltage-gated Na 1 channels (VGSCs) contain one poreforming a subunit with smaller b subunits. 4 There are nine a subunits, Na v 1.1-Na v 1.9, and four b subunits, b1-b4. The b subunits are members of the immunoglobulin superfamily of cell adhesion molecules (CAMs). They modulate channel gating, and can function as CAMs both in the presence and absence of a subunits. 5 They are substrates for secretase cleavage, releasing soluble intracellular domains that may regulate gene expression. 6 The b1 subunit (gene: SCN1B) participates in trans-homophilic adhesion, resulting in cellular aggregation and cytoskeleton recruitment. 7,8 b1 also interacts heterophilically with other CAMs, including b2, contactin, neurofascins, NrCAM, N-cadherin [9][10][11][12] and the extracellular matrix protein, tenascin-R. 13 b1 mediates neurite outgrowth by a trans-homophilic adhesion mechanism that requires fyn kinase, contactin and g-secretase activity. 5,14,15 b1 plays a critical role during central nervous system (CNS) development, regulating electrical excitability, proliferation, fasciculation, pathfinding and migration. [15][16][17] VGSCs are widely expressed in cancers, and contribute to cellular behaviours associated with metastasis. 18,19 In BCa, the predominant a subunit, Na v 1.5 (gene: SCN5A), is expressed in MDA-MB-231 cells, where Na 1 current potentiates invasion by enhancing cysteine cathepsin activity. [20][21][22] SCN5A is up-regulated in tumours, associating with recurrence, metastasis and reduced survival. 20,23 b1 is the predominant b subunit in MCF-7 cells, where it enhances cell-substrate adhesion, but slows transwell migration. 24 Over-expression of b1 in MDA-MB-231 cells increases cellcell adhesion and Na 1 current. 24 Both a and b1 subunits are expressed in lamellipodia of MCF-7 and MDA-MB-231 cells, suggesting that their expression and function are not mutually exclusive. 23 Thus, VGSC a and b subunits appear to play complex, dynamic roles in metastatic BCa cells. However, the functional significance of b1-dependent adhesion, and its contribution to tumour growth and metastasis, are unknown.
Our aim here was to study the involvement of b1 in BCa progression in vivo. We show that SCN1B mRNA/b1 protein are upregulated in BCa specimens, compared with normal breast tissue. Up-regulation of b1 potentiates tumour growth and metastasis in vivo. In addition, b1 increases process outgrowth on BCa cells via a trans-homophilic adhesion mechanism that requires fyn kinase and Na 1 current. We propose that b1 enhances metastatic behaviour of BCa cells by recapitulating mechanism(s) that are critical for neuronal migration during CNS development. These findings suggest that b1 warrants further study as a potential biomarker/therapeutic target.

Methods
In silico analysis SCN1B expression in microarrays was studied using Oncomine. 25 Meta-analysis of correlations between SCN1B expression and histoclinical characteristics across multiple datasets was as described. 26 Datasets, patients, specimen characteristics and assay methods are detailed/referenced at www.oncomine.org.

Cell culture
Molecular identity of all BCa cell lines was confirmed by short tandem repeat analysis. All cell lines were grown in Dulbecco's modified eagle medium (DMEM) supplemented with 5% fetal bovine serum and 4 mM L-glutamine. 23 BT474 and SKBR3 cells were a gift from J. Rae, University of Michigan. MCF-7 and MDA-MB-231 cells were a gift from M. Djamgoz, Imperial College London. "Control" MDA-MB-231 cells stably expressing enhanced green fluorescent protein (GFP) or MDA-MB-231 cells over-expressing b1-GFP Cterminal fusion 24 (hereafter called "MDA-MB-231-b1" cells) were maintained in medium containing selective antibiotics. MCF-10A cells were a gift from N. Maitland, University of York. R1610 Chinese hamster lung (CHL) fibroblasts and CHL fibroblasts stably expressing b1 were gifts from L. Isom, University of Michigan. MDA-MB-231-GFP and MDA-MB-231 b1-GFP cells were stably transduced with recombinant lentivirus for firefly luciferase (AMS Biotechnology). For experiments using estrogen, MCF-7 cells were maintained in phenol red-free DMEM supplemented with 5% charcoalstripped fetal bovine serum and 4 mM L-glutamine. Cells were confirmed as mycoplasma-free using the DAPI method.

Pharmacology
Tetrodotoxin (TTX) was diluted in culture medium to 30 mM. Staurosporine, PP2, estradiol and fulvestrant were prepared as stocks in DMSO and then diluted in culture medium to 10 nM230 mM. In assays that exceeded 24 h, treatments were replaced daily. The effect of TTX on invasion was determined using Matrigel assays. 23 The effect of staurosporine on apoptosis was determined using DeadEnd fluorometric TUNEL assays (Promega). The effect of PP2 on cell viability and proliferation was determined using trypan blue and MTT assays. 23 RNA isolation and RT-qPCR RNA extraction and cDNA synthesis were as described. 27 QPCR was carried out using triplicate 12-ml reactions containing 20ng cDNA. Amplification conditions were: 95 C for 30 s followed by 35 cycles of 95 C for 5 s and 60 C for 10 s on a Bio-Rad thermal cycler. Relative gene expression was quantitated using the comparative C T method. Primers are in Supporting Information Table S1.

Patient tissue samples
The study cohort contained tissue samples from 66 BCa cases obtained from the Breast Cancer Campaign Tissue Bank under tissue request number TR000017. Patients provided consent to the Breast Cancer Campaign Tissue Bank for their tissues to be used for research. The samples came from women aged 28-89 years, who were diagnosed between February 1992 and February 2012. For 40 cases (60%), tumour samples came with matched surrounding normal breast tissue. Clinical and histopathological data were available for all cases. Immunohistochemistry was performed on 5mm-thick sections using the EnVision1 System-HRP kit (Dako). Sections were deparaffinized in Histo-Clear (National Diagnostics) followed by antigen retrieval at 95 C for 30 min in Target Retrieval Solution (Dako). Sections were incubated with anti-b1 antibody (1:25; Abgent) for 30 mins and counterstained with dilute Mayer's hematoxylin and mounted in Faramount medium (Dako). Slides were scanned at 403 using an Aperio ScanScope. b1 immunoreactivity was scored by two independent investigators (WJB and RMS, a breast histopathologist) using the Allred method. 28 Briefly, the proportion of b1-positive cells was given a score (none: 0; <1/ 100: 1; 1/100 to 1/10: 2; 1/10 to 1/3: 3; 1/3 to 2/3: 4; >2/3: 5), followed by the intensity of staining (none: 0; weak: 1; intermediate: 2; strong: 3). For each section, the proportion and intensity scores were summed to give a total score (0-8).
A score of 0-4 was considered "low" and 5-8 was considered "high." Scoring was performed without prior knowledge of outcome data. Experiments were approved by the University of York Ethical Review Process.

Orthotopic breast tumour model
All animal procedures were carried out after approval by the University of York Ethical Review Process and under authority of a UK Home Office Project Licence. Six-weekold female Rag2 2/2 Il2rg 2/2 mice (mean weight: 16.6 6 0.2 g) were obtained from the Yorkshire Cancer Research Unit, University of York. Mice (4-5 per specific pathogen free cage) were selected at random for surgery. A 1 3 10 6 control MDA-MB-231-GFP or MDA-MB-231-b1-GFP cells expressing luciferase were suspended in Matrigel (20% v/v in saline) and injected into the left and right inguinal mammary fat pad of each animal whilst under isoflurane anaesthesia. A total of 13 mice were used (six injected with control cells and seven with b1 cells) across three independent replicated experiments. Tumour growth was monitored weekly by bioluminescence imaging. Mice were given intraperitoneal injection of D-luciferin in PBS (150 mg kg 21 ) and bioluminescence was visualized 10 min later under isoflurane anaesthesia using an IVIS100 system (Per-kinElmer). Bioluminescence from tumours was quantified within manually defined regions of interest using Living Image software (PerkinElmer) and expressed as photon flux. To quantify bioluminescence at sites of metastasis, mice were euthanized 10 min after injection with D-luciferin, primary tumours were removed and internal organs were exposed by dissection. Bioluminescence was measured from the entire mouse and then individual organs were removed for separate imaging. Measurements of the length and width of each tumour (in mm) were taken from mice daily with callipers. Tumour volume was calculated as 0.5 3 (length 3 width 2 ). Mice were euthanized when primary tumours reached 10% of starting body weight, or at the first sign of discomfort from metastatic burden. Tumours and organ sites of metastasis were fixed in 4% paraformaldehyde and frozen. 14

Image analysis
Images were exported into ImageJ for processing. Confocal Z-series projections were flattened using the maximum signal. Brightness/contrast was adjusted using the ImageJ "Auto" function. ICC colocalization was evaluated using ImageJ. Intensity correlation analysis (ICA) was performed on individual cells delineated with the freehand selection tool, and for each cell, the intensity correlation quotient (ICQ) was computed. For signal intensities that vary together, indicating colocalization, 0 < ICQ 0.5, whereas for segregated staining, 20.5 ICQ < 0. 30 Measurements were from 20 cells per line.
The following measurements were made on IHC sections, for three mice per treatment group: Density of Ki67 1 or activated caspase-3 1 cells 17 : the number of Ki67 1 nuclei or active caspase-3 1 cells was counted per 20X field of view.
Tumour vascularity 31 : the number of CD31 1 vessels was counted per 20X field of view. Metastasis to liver/lungs/spleen 32 : the number of GFP 1 metastatic foci was counted per 20X field of view.
Length of tumour cell processes and muscle fibers: The longest visible process on cancer cells and the total length of individual muscle fibers within 203 fields of view was measured using the freeform line function in ImageJ.

VEGF ELISA
Cells were cultured in 24-well plates (5 3 10 4 /well). After 1, 2, and 3 days, culture medium was removed from individual wells and stored at 220 C until analysis. VEGF secretion was determined by ELISA (Promega). Measurements were obtained from duplicate wells from three repeat experiments.

Process outgrowth assay
Process outgrowth assays were based on Ref. 14. Parental CHL fibroblasts or CHL fibroblasts expressing b1 were grown to confluence on 13mm diameter coverslips. Freshly dissociated BCa cells were plated (2 3 10 4 cells/well) on top of the monolayers and allowed to grow for 24-48 h. Cultures were fixed in 4% paraformaldehyde and the cancer cells were visualized with anti-GFP (1:1,000; NeuroMab), or anticytokeratin 18 (1:500; BioLegend) followed by Alexa-568conjugated goat anti-mouse (1:500). Images were acquired using a Nikon Eclipse TE200 fluorescent microscope with 403 objective. The longest process on each of the first 50 randomly selected, isolated cancer cells was measured using ImageJ. Measurements were obtained from three independent experiments.

RNA interference
SiGENOME SMARTpool siRNA targeting FYN and siGE-NOME Non-Targeting siRNA Pool #1 (Dharmacon) were used at 50 nM. Transfection was performed using Dharmafect 1 reagent. Transfection efficiency was confirmed to be 90% using siGENOME positive control targeting GAPDH (Supporting Information Fig. S5c). RNA extraction and process outgrowth assay were performed 96h after transfection.

Data analysis
Data are mean and SEM unless stated otherwise. Statistical analysis was performed using GraphPad Prism. Matrix data were plotted using Matrix2png software. 33 Statistical significance was determined with t tests or Mann-Whitney tests, and multiple comparisons were made using ANOVA and Tukey post hoc tests, unless stated otherwise. p values computed by Oncomine were corrected for multiple comparisons by Bonferroni method. Correlation between ESR1 and SCN1B expression was determined using Pearson's r test. Association between categorical classification criteria was determined with Fisher's exact test, or v 2 test. For meta-analysis of association between SCN1B expression and histoclinical characteristics across multiple datasets, the binomial test was used. 26 The binomial test p value indicates whether or not one criterion was associated with another in the observed number of datasets by chance, given the number of datasets studied. Kaplan-Meier curves for survival were compared by log-rank tests. Percent survival and hazard ratios are presented with 95% confidence intervals. Results were considered significant at p < 0.05.

b1 mRNA and protein are present in breast tumours
We have previously shown that b1 mRNA/protein are expressed in BCa cell lines. 24 Here, we used Oncomine to study the expression of SCN1B mRNA in normal breast and BCa specimens across multiple microarrays. SCN1B was expressed at a significantly higher level in BCa compared with normal breast in two out of eight datasets in which differential data were available (1.7-fold, p < 0.05; and >2.2fold, p < 0.05; Figs. 1a and 1b). We next performed a metaanalysis to investigate whether SCN1B expression correlates with histoclinical characteristics across multiple datasets. High SCN1B expression associated with ER status in 8/21 (38.1%) of datasets (p < 0.0001; Supporting Information Table S2; Figs. S1a and S1b). There was no significant association between SCN1B and age, pathological tumour size, grade, recurrence, progesterone receptor, or HER2 status across the same datasets. Up-regulation of SCN1B expression in ER 1 tumours correlated with several genomic neighbours on chromosome 19q (Supporting Information Fig. S1c). 26 However, mRNA levels of the two SCN1B splice variants, b1 and b1B, 34 which are both expressed across a panel of BCa cell lines (Supporting Information Figs. S1d and S1e), were unchanged in MCF-7 cells following treatment with estrogen or fulvestrant (Supporting Information Fig. S1f), suggesting that SCN1B is not estrogen-regulated.
We next studied the expression of b1 at protein level in human tissue samples by IHC. b1 immunoreactivity was mainly in the cytoplasm of epithelial and carcinoma cells, with variable expression at the plasma membrane (Figs. 1c and 1d). This pattern of expression is consistent with previous observations in neurons and cancer cell lines. 14,16,23 b1 expression was significantly higher in tumour than normal, non-cancer breast tissue samples (p < 0.001; Fig. 1e). Of the cases where tumour had matched surrounding non-cancer tissue, 27 (68%) had higher b1 in tumour than non-cancer tissue, 7 (17%) had the same level of b1 in tumour and noncancer tissue, and 6 (15%) had lower expression in tumour than non-cancer tissue. b1 expression in the tumour did not correlate with age, ER status, grade, menopausal status, or node status (Supporting Information Table S3). Similarly, there was no relationship with BCa-specific survival (Fig. 1f, Supporting Information Table S3). In agreement with the IHC data, b1 was also expressed at protein level in the noncancer mammary epithelial MCF-10A cell line and across a panel of BCa cell lines (Figs. 1g and 1h). Together, these data suggest that b1 may be up-regulated in a unique subset of breast cancers at mRNA and protein level.  Fig. S2a). By contrast, MDA-MB-231-b1 cells over-express b1-GFP by >40fold relative to endogenous b1 (Supporting Information Figs. S2a and S2b). 24 Over-expression of b1 had no effect on expression of CD44 or E-cadherin (Supporting Information   2a and 2b). To confirm the bioluminescent imaging data, we also analyzed tumour growth by daily calliper measurement. The volume of MDA-MB-231-b1 tumours increased more rapidly than MDA-MB-231 tumours, closely agreeing with the bioluminescent data (Fig. 2c). During the 5-week study period, MDA-MB-231-b1 primary tumour burden reached 10% of starting body weight in 71% of mice, compared with only 17% for control tumours (Fig. 2d). The survival of mice bearing MDA-MB-231-b1 tumours was significantly reduced compared to those bearing control tumours (p < 0.05; hazard ratio 5 6.3 [1.4-27.8] ; Fig. 2e). These data demonstrate that over-expression of b1 enhanced the growth of breast tumours, thus reducing survival.

Cancer Cell Biology
We next studied the structure and composition of the primary tumours. Both MDA-MB-231 and MDA-MB-231-b1 tumours were broadly similar, containing some invasion into surrounding fibroadipose tissue and skeletal muscle (Fig. 2f). Although the in vitro invasiveness of MDA-MB-231-b1 cells was moderately higher than control MDA-MB-231 cells, blockade of a subunits with TTX inhibited invasion of both cell lines to a similar extent (p < 0.05, Fig. 2g). Thus, asubunit-dependent invasion of MDA-MB-231 cells 20,21 appears to be unaffected by b1 over-expression. The density of Ki67 1 cycling cells was unchanged in MDA-MB-231-b1 tumours, compared to control tumours (Figs. 3a and 3b).  suggesting, together with the tumour data, that b1 overexpression enhances resistance to apoptosis. Finally, the density of vascular structures, revealed by labelling blood vessels with an antibody to the endothelial marker CD31, signifi-cantly increased by 1.5-fold in MDA-MB-231-b1 tumours, compared to control tumours, and VEGF secretion in vitro was significantly higher in MDA-MB-231-b1 cells than control MDA-MB-231 cells (p < 0.01; Figs. 3g23i). In summary, (g) A model of possible signalling mechanism underlying b1-mediated process outgrowth in BCa cells. b1 from an adjacent fibroblast or cancer cell interacts in trans with b1 on the BCa cell, initiating a signaling cascade via fyn kinase, leading to process outgrowth. 15 Na 1 conductance through the pore-forming a subunit is also required. 16 Figure was produced using Science Slides software. Bars are mean 1 SEM; ***p < 0.001. these data suggest that b1 over-expression increased the growth of MDA-MB-231 tumours, not by altering the density of cycling cells in the population, but, instead by reducing apoptosis and enhancing angiogenesis.
b1 potentiates metastasis to liver and lungs We monitored metastasis after 5 weeks by bioluminescent imaging following post mortem resection of primary tumours (Fig. 4a). Although photon flux was slightly increased in mice bearing MDA-MB-231-b1 tumours compared those bearing MDA-MB-231 tumours, and in the liver and lungs ex vivo, this difference was not statistically significant (Figs.  4b and 4c). To study metastasis to these organs in more detail at the cellular level, using a more sensitive method, we measured the density of GFP-expressing tumour cells within tissue sections. We detected GFP 1 cells in sections both in isolation, and in multicellular foci (Figs. 4d, 4f, and 4h). GFP was co-expressed in cells with HNA (Fig. S3e). HNA is present in human MDA-MB-231 cells, but absent in mouse cells, thus confirming that GFP expression was retained in the tumour cells once they had metastasized. In the spleen, the number of GFP 1 cells per field of view was unchanged between groups (Fig. 4e). However, the number of GFP 1 cells per field of view was significantly increased, by 5.9-and 3.0-fold, respectively, in the liver and lungs of MDA-MB-231-b1 tumour-bearing mice, compared to control (p < 0.05 and 0.001, respectively; Figs. 4g and 4i). Thus, b1 overexpression promoted metastasis to the liver and lungs, but not the spleen.
b1 promotes process outgrowth Enhancement of protrusions, e.g., pseudopodia, from cells is associated with increased motility in 3D cultures, invasion, and metastasis. [35][36][37] Over-expression of b1 in MDA-MB-231 cells increases the length of processes protruding from the cell body in vitro. 24 We therefore postulated that b1 might regulate cellular morphology in our tumour model. In the periphery of tumour sections, MDA-MB-231-b1 cells infiltrating surrounding skeletal muscle appeared more densely packed, and had a more elongate morphology than MDA-MB-231 cells (Fig. 5a). In these sections, processes extending from MDA-MB-231-b1 cells were significantly longer than processes on MDA-MB-231 cells (Fig. 5b). The length of muscle fibers was unchanged between tumour types (Fig. 5c).
In the nervous system, b1 regulates neuronal morphology and neurite outgrowth via trans-homophilic adhesion. 15 We therefore set out to test the hypothesis that this neuronal function of b1 is recapitulated when it is expressed in BCa cells, enhancing process outgrowth. We examined the morphology of BCa cells plated on monolayers of control and b1-expressing CHL fibroblasts. CHL cells were chosen because they do not express endogenous b subunits. 14 When plated on CHL cells, MDA-MB-231 cells produced thin processes with foci at the tips, morphologically similar to neurites with growth cones (Fig. 5d). 15 MDA-MB-231 cells did not show any increase in process length when grown on b1expressing monolayers (Fig. 5e). However, MDA-MB-231-b1 cells did respond, such that b1-expressing monolayers increased process length by 1.6-fold (p < 0.001; Fig. 5e). A similar result was observed for MCF-7 cells, which express endogenous b1 (Supporting Information Fig. S4a and S4b). There was no increase in process length of MDA-MB-231 cells over-expressing the Ig domain-deficient mutant b1D  when grown on b1-expressing monolayers (Fig. 5f), suggesting that the adhesion domain is required for b1-mediated process outgrowth. In cerebellar granule neurons, b1mediated neurite outgrowth requires the presence of Na v 1.6 and is inhibited by the VGSC pore-blocking toxin TTX. 16 We found that TTX (30 mM) inhibited b1-mediated process outgrowth in MDA-MB-231-b1 cells (p < 0.001; Fig. 5h). However, it had no effect on process outgrowth in native MDA-MB-231 cells, which do not respond to b1-expressing fibroblasts (Fig. 5g). Together, these data suggest that b1 enhances process outgrowth on BCa cells via trans-homophilic adhesion between b1 expressed on the BCa cell, and b1 expressed on the adjacent fibroblast, similar to its function in neurons. 14 b1-mediated process outgrowth requires fyn kinase In neurons, b1 increases neurite length via fyn kinase. 15 CAM-mediated activation of fyn kinase is proposed to initiate the fyn-focal adhesion kinase (FAK) pathway, activating extracellular signal-regulated kinase 1/2, leading to neurite outgrowth. 38 Fyn is upregulated in a number of cancers, where it contributes to an invasive phenotype. 39 We found that fyn, like b1, was expressed in MCF-10A cells and BCa cell lines ( Fig. 1g and 1h, Supporting Information Fig. S2f). Given that fyn is required for b1-mediated neurite outgrowth, and fyn and b1 are coexpressed in brain membranes, 15 we hypothesized that fyn and b1 may colocalize in BCa cells. In MDA-MB-231 and MDA-MB-231-b1 cells, b1 was expressed throughout the cytoplasm, on perinuclear internal membranes and lamellipodia (Fig. 6a), consistent with previous observations, although the expression of b1 was clearly lower in the former. 23 Importantly, fyn showed a broadly similar distribution to b1, with expression strongest on F-actin-rich lamellipodia. Intensity correlation analysis gave ICQ values >0, indicating that the signals for b1 and fyn varied together 30 (Fig. 6b). This result is consistent with b1 colocalizing with fyn.
We next investigated whether fyn activity is involved in b1-mediated process outgrowth in BCa cells. Inclusion of the src family kinase inhibitor PP2 (5 mM) in the assay inhibited b1-mediated process outgrowth in MDA-MB-231-b1 cells (p < 0.001; Fig. 6d). However, it had no effect on process outgrowth in control MDA-MB-231 cells, which do not respond to b1-expressing fibroblasts (Fig. 6c). Importantly, 5 mM PP2 had no effect on cellular viability or proliferation (Supporting Information Figs. S5a and S5b). To establish whether fyn is specifically required for b1-mediated process outgrowth in BCa cells over other members of the src family that are also inhibited by PP2, we next transiently down-regulated the expression of fyn in BCa cells using siRNA, prior to plating on fibroblast monolayers (Supporting Information Fig. S5d). Down-regulation of fyn with siRNA abrogated b1-mediated process outgrowth in MDA-MB-231-b1 cells (p < 0.001; Fig.  6f). However, there was no effect on baseline process outgrowth in control MDA-MB-231 cells (Fig. 6e). PP2 and fyn siRNA also inhibited b1-mediated process outgrowth in MCF-7 cells (Supporting Information Figs. S4c and S4d). In summary, blocking fyn expression/activity with PP2 or siRNA inhibited b1-mediated process outgrowth in BCa cells. These data suggest that fyn is a critical signalling intermediary in the mechanism underlying b1-mediated process outgrowth in BCa cells (Fig. 6g), as it is in neurons. 15

Discussion
VGSCs are expressed in cells from a number of different cancers, where they are proposed to play a role in potentiating metastasis. 18 VGSCs are unique among ion channels in that their "auxiliary" b subunits not only modulate channel activity, but are also CAMs. 5 Expression of b subunits has been reported in breast, bone, cervical, colorectal, lung, and prostate cancer cell lines, and b1 is the dominant isoform in breast, cervical, lung and prostate cancer cell lines (reviewed in Ref. 19). However, in vivo evidence for b subunit expression in cancer is limited. We found that b1 was up-regulated at mRNA and protein level in BCa specimens compared with non-cancer tissue. Similarly, b1 was expressed across a panel of BCa cell lines, although the relative mRNA and protein levels differed. In addition, in tumour specimens, the positive relationship between SCN1B mRNA and ER status was not reflected at the protein level. Discrepancy between mRNA and protein levels has been reported previously for other VGSCs in other tissues. 24,40,41 Therefore, the relationship between SCN1B mRNA and b1 protein levels may be subject to complex regulation, highlighting the critical importance of studying biomarker expression at both mRNA and protein levels. We did not observe a relationship between b1 expression and outcome in patient tumour specimens. This may be due to the relatively small size of the dataset, and it would be worthwhile in the future to validate the data presented here against larger cohorts. In conclusion, our data show that SCN1B/b1 is up-regulated at the mRNA and protein level in BCa. We propose that b1 warrants further study as a potential biomarker for BCa. b1 over-expression increased tumour growth in vivo. Interestingly, this contrasts with the observation that overexpression of b1 slightly reduces proliferation in vitro, 24 suggesting that the tumour microenvironment might be critical to the in vivo tumour-promoting function of b1. In support of this, apoptosis was reduced in MDA-MB-231-b1 tumours, which may account for their increased size. Cell adhesion can promote apoptosis suppression in cancer cells via FAK activation 42 and further work is required to establish whether or not b1-dependent adhesion promotes tumour cell survival. There was increased density of vascular structures in the b1 over-expressing tumours, and VEGF secretion was increased in MDA-MB-231-b1 cells in vitro, suggesting that b1 may enhance angiogenesis. Several classes of CAMs are known to promote angiogenesis, including integrins, cadherins, and immunoglobulin superfamily CAMs, 43 raising the possibility that b1 may contribute to promoting blood vessel development through heterophilic adhesion. Interestingly, overexpression of b2 in prostate cancer cells has the reverse effect, reducing tumour growth. 44 Despite its structural similarity to b1, b2 appears to play a different role in the CNS, and is not essential for postnatal development. 5 Thus, as in the CNS, different b subunits may perform distinct functions in different cancer microenvironments.
b1 is a multifunctional molecule that plays a critical role during CNS development. 5 Although b1 is essential for regulating excitability through fine-tuning VGSC gating and kinetics, 16 its function as a CAM is required for neurite outgrowth, migration, fasciculation and synaptic connectivity. [15][16][17] In fact, b1 may function as a CAM, independent of channel activity under certain conditions. 5 Other CAMs that regulate neuronal migration and pathfinding have been reported in tumours, where they play a pathological role and associate with poor prognosis. 45 It is therefore not unreasonable to expect that b1 may do the same. We showed previously that b1 enhances cell-cell adhesion and cell-substrate adhesion in BCa cells in vitro. 24 In the present study, we found that b1 over-expression in MDA-MB-231 cells caused a more elongate cellular morphology within tumours, and enhanced process outgrowth in vitro via trans-homophilic adhesion. b1-mediated process outgrowth did not occur in control MDA-MB-231 cells, which express a low level of endogenous b1. The latter result suggests that b1 expression on the tumour cell may need to be above a threshold in order to induce process outgrowth and enhance tumour growth and metastasis. We found that, as in neurons, 15 b1-mediated process outgrowth in BCa cells required fyn kinase. b1-mediated neurite outgrowth in neurons is also activity-dependent. 16 Interestingly, Na 1 current promotes src family kinase activity and pro-invasive morphology in MDA-MB-231 cells, 46 which fits with other data showing that a subunits potentiate the invasiveness of BCa cells. [20][21][22]46,47 We found that TTX inhibited b1-mediated process outgrowth in MDA-MB-231-b1 cells, suggesting that, as in neurons, 16 a subunit function is required (Fig. 6g). Thus, Na v 1.5 and b1 may both promote mesenchymal-like elongate morphology in BCa cells, via a combination of Na 1 current and adhesion.
Our data suggest that b1 can enhance tumour growth and metastasis via a trans-homophilic adhesion mechanism that enhances process outgrowth on metastatic tumour cells, enabling their dissemination from the primary tumour and into surrounding tissues. This would fit with the observation that outgrowth of processes, e.g., pseudopodia, from cancer cells increases motility, invasion, and metastasis. [35][36][37] Thus, b1 may be involved in collective cell migration and invasion during tumour spreading, 48,49 similar to its role in pathfinding and fasciculation during CNS development. 15,17 However, we do not yet know whether b1 interactions in trans occur between adjacent tumour cells, or between tumour cells and stromal cells, or both. Further complexity is added by the possibility that b1 may interact heterophilically with other CAMs, [9][10][11][12] and/or extracellular matrix proteins 13 present in the tumour microenvironment, dependent on cell types/status within the tumour.
Our data support the hypothesis that SCN1B/b1 recapitulates its neurodevelopmental role to promote breast tumour growth and metastasis. This fits with a growing body of evi-dence implicating VGSCs as mediators of an invasive/metastatic phenotype. 19 Up-regulation of genes, e.g. SCN1B, required for normal migration and invasion processes during development, may represent a critical event in the progression towards metastasis. 50 We therefore propose that (i) b1 may represent a novel biomarker during disease development, and (ii) targeting b1-mediated adhesion interactions may have potential as novel anti-cancer therapy.