Functional expression of the voltage-gated Na channel Nav1 ...current (I Na) (Fig. 1A). Two cell...
Transcript of Functional expression of the voltage-gated Na channel Nav1 ...current (I Na) (Fig. 1A). Two cell...
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Functional expression of the voltage-gated Na+-channel Nav1.7 is necessary for EGF-mediatedinvasion in human non-small cell lung cancer cells
Thomas M. Campbell1, Martin J. Main2 and Elizabeth M. Fitzgerald1,*1Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK2Discovery Sciences, AstraZeneca, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK
*Author for correspondence ([email protected])
Accepted 13 July 2013Journal of Cell Science 126, 4939–4949� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.130013
SummaryVarious ion channels are expressed in human cancers where they are intimately involved in proliferation, angiogenesis, invasion andmetastasis. Expression of functional voltage-gated Na+ channels (Nav) is implicated in the metastatic potential of breast, prostate, lung
and colon cancer cells. However, the cellular mechanisms that regulate Nav expression in cancer remain largely unknown. Growthfactors are attractive candidates; they not only play crucial roles in cancer progression but are also key regulators of ion channelexpression and activity in non-cancerous cells. Here, we examine the role of epidermal growth factor receptor (EGFR) signalling and
Nav in non-small cell lung carcinoma (NSCLC) cell lines. We show unequivocally, that functional expression of the a subunit Nav1.7promotes invasion in H460 NSCLC cells. Inhibition of Nav1.7 activity (using tetrodotoxin) or expression (by using small interferingRNA), reduces H460 cell invasion by up to 50%. Crucially, non-invasive wild type A549 cells lack functional Nav, whereas exogenous
overexpression of the Nav1.7 a subunit is sufficient to promote TTX-sensitive invasion of these cells. EGF/EGFR signalling enhancesproliferation, migration and invasion of H460 cells but we find that, specifically, EGFR-mediated upregulation of Nav1.7 is necessaryfor invasive behaviour in these cells. Examination of Nav1.7 expression at mRNA, protein and functional levels further reveals that EGF/EGFR signalling via the ERK1/2 pathway controls transcriptional regulation of channel expression to promote cellular invasion.
Immunohistochemistry of patient biopsies confirms the clinical relevance of Nav1.7 expression in NSCLC. Thus, Nav1.7 has significantpotential as a new target for therapeutic intervention and/or as a diagnostic or prognostic marker in NSCLC.
Key words: Ion channel, Voltage-gated sodium channel, Epidermal growth factor receptor, Invasion, Metastasis, NSCLC
IntroductionLung cancer is the most prevalent cancer world-wide. Small cell
lung carcinoma (SCLC) and non-small cell lung carcinoma
(NSCLC) occur with a frequency of ,20% and ,80%,
respectively. NSCLC has a low, average 5-year, survival rate
(14%) and the development of metastases results in extremely poor
prognosis (Jemal et al., 2011). The epidermal growth factor receptor
(EGFR) plays key roles in the progression of various cancers
(Araujo et al., 2007; Cohen, 2002; Mendelsohn, 2003), and in
NSCLC its overexpression in ,40–80% of patients is also
associated with poor prognosis (Mendelsohn, 2003). Small-
molecule EGFR kinase inhibitors (gefitinib and erlotinib) have
recently shown significant therapeutic benefit in many patients with
advanced NSCLC, particularly those with somatic mutations of the
EGFR (Araujo et al., 2007). However, drug resistance, whether
inherent or acquired, remains a main cause of chemotherapy failure
(Cataldo et al., 2011; Domingo et al., 2010; Lynch et al., 2004; Paez
et al., 2004). Consequently, there is a pressing need for improved
treatments based upon improved understanding of the effector
pathways downstream of EGFR signalling.
Voltage-gated Na+ channels (Nav), normally associated with
cellular excitability, have been found to play crucial roles inmetastatic behaviours of several cancers, including breast
(Brackenbury et al., 2007; Fraser et al., 2005; Gao et al., 2009;
Gillet et al., 2009; Roger et al., 2003), prostate (Abdul andHoosein, 2002; Bennett et al., 2004; Diss et al., 2001; Diss et al.,
2005; Grimes et al., 1995; Laniado et al., 1997; Nakajima et al.,2009; Smith et al., 1998), cervical (Diaz et al., 2007), colon
(House et al., 2010), ovarian (Gao et al., 2010), SCLC (Blandino
et al., 1995; Onganer and Djamgoz, 2005; Onganer et al., 2005;Pancrazio et al., 1989) and NSCLC (Roger et al., 2007). In
prostate cancer, functional expression of Nav enhances cellular
migration, invasion and endocytosis (Ding et al., 2008; Uysal-Onganer and Djamgoz, 2007). In NSCLC, Nav activity reportedly
promotes invasion but not migration (Roger et al., 2007). Theacquisition of functional Nav, thus appears to accelerate
development of cancer metastases in a variety of carcinomas.
However, details of the downstream effects of Nav (Brisson et al.,2011; Gillet et al., 2009) and the cellular mechanism(s) that
regulate their expression and activity in metastatic cells remain
unclear (Ding et al., 2008; Uysal-Onganer and Djamgoz, 2007).
In addition to their roles in cell growth and cancer progression,
growth factors also regulate the expression and activity ofnumerous ion channels (Akopian et al., 1999; Brackenbury and
This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distributionand reproduction in any medium provided that the original work is properly attributed.
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Djamgoz, 2007; Goetz et al., 2009; Goldfarb et al., 2007; Lei
et al., 2001; Lou et al., 2005; Toledo-Aral et al., 1995). EGF
enhances Nav currents in non-cancerous cells, including cardiac
muscle (Liu et al., 2007). In human and rat prostate cancer,
functional upregulation of Nav is implicated in the pro-invasive
response of cells to EGF, although the cellular mechanism(s)
involved are unknown (Ding et al., 2008; Uysal-Onganer and
Djamgoz, 2007). In the present study, we conducted a detailed
characterisation of Nav in NSCLC and examined the effects of
EGF/EGFR signalling on expression and activity of Nav. Briefly,
we show that functional expression of the Nav1.7 a subunit
isoform specifically promotes invasion of strongly invasive H460
NSCLC cells. Moreover, non-invasive A549 NSCLC cells that
lack Nav, can be induced to invade simply by overexpression of
Nav1.7. In strongly invasive H460 cells, EGF/EGFR signalling
through the ERK1/2 pathway controls transcriptional regulation
of Nav1.7 to promote invasion. Finally, immunohistochemistry of
patient biopsies provides evidence that supports the clinical
relevance of Nav1.7 expression.
ResultsTo assess the role of Nav in NSCLC it was first necessary to
identify suitable model cell lines for comparison of Nav activity
and expression in weakly versus strongly invasive cells. We,
therefore, tested 22 NSCLC cell lines for functional Nav
expression using a high-throughput IonWorksH QuattroTM
Automated Patch Clamp System to record the inward Nav
current (INa) (Fig. 1A). Two cell lines were selected for further
study: A549 cells that lacked INa and H460 cells that exhibited
the most-robust INa both in terms of current amplitude and
proportion of cells with current (,45%, n589). Conventional
patch-clamp recordings confirmed the IonWorksH data
(Fig. 1B,C). H460 cells were more proliferative (Fig. 1D) and
more invasive (Fig. 1F) than A549 cells. However, migration was
greater in A549 cells (Fig. 1E). H460 cells were, thus, chosen as
a strongly invasive model, whereas A549 cells were used as a
model of weak invasive potential. These preliminary findings
agree with those described by Roger et al. (Roger et al., 2007).
Nav promotes H460 cell invasion
To explore the link between functional expression of Nav and
invasion of H460 cells, we used the Nav-specific inhibitor
(tetrodotoxin, TTX) or activator (veratridine) that, respectively,
block or enhance the opening of Nav channels. TTX (0.5 mM)
abolished INa, whereas 50 mM veratridine induced a modest increase
in the persistent inward Na+ current (INa,P) (Fig. 2A). Neither TTX
nor veratridine affected proliferation or migration (Fig. 2B,C).
However, as predicted, blocking Nav channels (using TTX) reduced
invasion of H460 cells by 39.767.2%, whereas enhanced opening
of Nav (using veratridine) promoted invasion (19.764.7%)
(Fig. 2D). Thus, Nav activity is specifically associated with
invasion in these cells. We next generated two H460 dilution
clones on the basis of high versus low Nav activity (Fig. 2E). In
agreement with the pharmacology, invasion was enhanced in the
‘high’ compared with the ‘low’ clone cells (Fig. 2D). The relatively
modest enhancement of invasion seen in WT H460 cells treated with
veratridine, or, in the high versus low clone cells, reflects the
similarly modest increases in current density.
Nav are usually expressed in excitable cells (neurons and
muscle), where they are activated by depolarisation of the cell
membrane in response to action potentials (Catterall, 2000).
However, cancerous cells are most commonly of epithelial origin
(non-excitable), raising the question; how are Nav activated? In
other invasive cancer cells, a small INa window current at the
resting membrane potential (Em) of cells has been postulated to
produce sufficient Na+ influx to promote invasion (Gillet et al.,
Fig. 1. Characterisation of H460 and A549 NSCLC cell lines. (A) Histogram showing average maximum peak current amplitude Imax for inward Na+ current and
uncharacterised outward currents in NSCLC cell lines at 210, 0 and +10 mV. Currents were recorded using the IonWorksH QuattroTM Automated Patch Clamp
System (n$40 for all cell lines). (B) Average current-density–voltage (I–V) curves for H460 (n517) and A549 (n516) cells. Continuous lines indicate Boltzmann curve
fits using the Boltzmann function described in Materials and Methods. Currents were evoked using 30-millisecond depolarising voltage steps in 5-mV intervals
(290 to +70 mV) from a holding potential Vh5 290 mV. (C) Representative current traces from H460 and A549 cells. (D) Proliferation of H460 and A549 cells,
recorded over 144 hours, measured as percent change in confluence from time 0 hours (n516 wells per cell line and at least two separate experiments). (E) H460 and
A549 cell migration, recorded over 58 hours using the scratch-wound assay, measured as percent change in wound confluence (n58 wells per cell line, two separate
experiments). (F) MatrigelTM invasion of H460 and A549 cells, recorded after 48 hours (n59 for both cell lines, ***P,0.001, Student’s t-test).
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2009; Roger et al., 2007). As shown in Fig. 2F, H460 cells mayalso possess a small INa window current (between 240 and210 mV), where Nav are partially activated but not fully
inactivated. Notably, the Em of these cells (22764 mV) lieswithin this voltage range, thus, allowing Na+ influx (Fig. 2G).However, the more hyperpolarised Em of weakly invasive A549
cells (23663 mV) is consistent with the lack of Nav channelactivity in these cells. TTX-induced Nav block hyperpolarised theEm of H460 cells to 23761 mV (similar to that of A549 cells),
where Na+ influx is predicted to be negligible. By contrast, when thecell membrane was depolarised to 22361 mV by using veratridine,INa,P was enhanced. Consistent with these findings, the intracellularNa+ concentration [Na+]i, was ,2-fold higher in H460 than A549
cells (22.364.3 mM versus 9.763.2 mM; Fig. 2H). Moreover, inTTX-treated H460 cells, [Na+]i was reduced to 10.862.8 mM,although veratridine had no significant effect (28.864.0 mM).
Nav1.7 underlies Nav current and promotes the invasivecapability of H460 cellsNav channels typically comprise one pore-forming a subunit andone or two auxiliary b subunits that modulate channel gating and
cell surface expression (Catterall, 2000; Joho et al., 1990). b
subunits, particularly b1, also function as cell adhesion molecules
(CAMs) (Brackenbury and Isom, 2008; Chioni et al., 2009; Isom,
2001; Isom, 2002; Patino and Isom, 2010). Nine a- and four b-
subunit isoforms have been identified (Catterall et al., 2005). Of
these, Nav1.5, Nav1.8 and Nav1.9 are TTX-resistant (EC5055.7–
60 mM), whereas the remainder are TTX-sensitive (EC5054–
25 nM). Since 0.5 mM TTX abolished INa in H460 cells, we
measured mRNA levels for all TTX-sensitive Nav a subunits, using
quantitative PCR (qPCR) (Fig. 3A). We also assessed mRNA
expression of the four known b subunits (Fig. 3A). In strongly
invasive H460 cells, Nav1.7 mRNA expression was ,4-fold higher
compared with other a subunit isoforms, at levels comparable with
functional expression of Nav in SH-SY5Y neuroblastoma cells. Low
mRNA expression of all Nav a subunits was seen in A549 cells,
consistent with their lack of INa. Western immunoblotting (Fig. 3B)
and immunocytochemistry (Fig. 3C) also confirmed the presence of
Nav1.7 protein in H460 but not in A549 cells. Furthermore, Nav1.7
mRNA expression was ,60% lower in the ‘low’ versus ‘high’ H460
dilution clone, consistent with the difference in INa (Fig. 3D).
Transfection of H460 cells with small interfering RNA (siRNA)
against Nav1.7 (SCN9A) completely abolished INa (Fig. 3E,F) and
reduced the invasive capability of the cells by 50.261.8% (Fig. 3G),
indicating that the functional expression of Nav1.7 is necessary for
Nav-mediated invasion of H460 cells.
H460 cells exhibited very low expression of all Navb subunit
mRNAs (Fig. 3A), whereas A549 cells expressed high levels of
b1 mRNA (,8-fold more than H460 cells; Fig. 3A). Consistent
with a role for the Navb subunit in cell adhesion (Brackenbury
and Isom, 2008; Chioni et al., 2009; Isom, 2001; Isom, 2002;
Patino and Isom, 2010), in A549 cells, adhesion was
Fig. 2. Effects of tetrodotoxin and veratridine on Nav channel activity,
proliferation, migration and on invasion of H460 cells. (A) Representative
traces showing the effects of tetrodotoxin (TTX, 0.5 mM, n55) and
veratridine (Ver, 50 mM, n56) on the Nav current at +10 mV.
(B) Lack of effect of 0.5 mM TTX and 50 mM veratridine on cell proliferation
over 120 hours (n58 wells for each condition, two separate experiments).
(C) Lack of effect of 0.5 mM TTX (closed squares) and 50 mM veratridine
(open circles) on migration, over 58 hours (n58 wells for each condition, two
separate experiments). (D) Effect of 0.5 mM TTX and 50 mM veratridine
(Ver) on MatrigelTM invasion, after 48 hours. Invasion in two H460 dilution
clones with high versus low Nav expression and activity are shown for
comparison. n59 for all conditions; *P,0.05, **P,0.01, one-way ANOVA
and SNK correction. (E) Average current-density–voltage
(I–V) relationships for Nav currents in high (open square, n59) and low
(closed square, n510) H460-cell dilution clones, recorded using conventional
patch clamp technique (*P,0.05, Student’s t-test). Continuous lines indicate
Boltzmann curve fits using as described in Materials and Methods. Inset:
representative current traces from high versus low clone cells. Currents were
evoked using 30-millisecond depolarising voltage steps in 5-mV intervals
(290 to +70 mV), from Vh5 290 mV. (F) Average conductance-voltage
(conductance; squares, n517) and steady-state inactivation-voltage
(availability; circles, n510) curves for INa in H460 cells. Continuous lines
indicate Boltzmann curve fits using the functions described in Materials
and Methods. (G) Average resting membrane potential Em in H460 cells,
A549 cells and H460 cells treated with 0.5 mM TTX or 50 mM veratridine
(Ver) for 2–3 minutes. n510 for all conditions (*P,0.05, **P,0.01,
***P,0.001, one-way ANOVA and SNK correction). (H) Intracellular Na+
concentration [Na+]i, in control versus TTX-treated (0.5 mM) or veratridine-
treated (50 mM, Ver) H460 cells and control A549 cells. [Na+]i was monitored
using the ratiometric fluorescent dye SBFI (n55 for all conditions,
**P,0.01, one-way ANOVA and SNK correction). CTL, untreated control
H460 cells.
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approximately double than that of H460 cells (Fig. 4B).
Moreover, inhibiting the expression of the b1 subunit by using
siRNA against SCN1B resulted in a modest decrease in adhesion
and a concurrent increase in invasion compared with wild-type
A549 cells (Fig. 4B,C). Notably, the invasive capability of H460
cells was reduced simply by overexpressing b1-GFP (Fig. 4D,F).
Conversely, in A549 cells, TTX-sensitive invasion was enhanced
by overexpression of Nav1.7-DsRed alone (Fig. 4E,F). Thus,
functional expression of Nav1.7 provides a driving force for
NSCLC cell invasion, whereas the b1 subunit promotes cell
adhesion and reduced invasion.
Nav1.7 expression is enhanced in cancerous lung tissue
The clinical relevance of our findings was assessed by comparing
Nav1.7 protein expression in cancerous versus normal-matched
lung tissue from patient biopsies. Nav1.7 immunostaining was
markedly higher in tumour versus normal lung tissue for all
patient samples tested (Fig. 5A–C), consistent with Nav1.7
expression in H460 versus A549 cells (Fig. 5D). Notably,
Fig. 5C clearly shows disorganised tumour tissue bordering
healthy lung tissue, with a marked difference in the levels of
immunostaining for Nav1.7. Increased Nav1.7 protein expression
may thus accompany the transition from low- to high-grade
tumours. Differently staged lung cancer samples could not be
obtained, but comparison of prostate cancer tumour samples
showed negligible immunostaining for Nav1.7 in low-grade
tumours, whereas in high-grade tumours Nav1.7 protein levels
were high (Fig. 5E). Like NSCLC, metastatic prostate cancer is
also associated with functional expression of Nav1.7 (Diss et al.,
2001; Diss et al., 2005; Nakajima et al., 2009; Yildirim et al.,
2012). Together, these data support a role for Nav1.7 in the
development of an invasive cancer phenotype in vivo.
EGF upregulates Nav1.7 expression and H460 cell invasion
EGF/EGFR signalling plays key roles in cancer progression
(Araujo et al., 2007; Cohen, 2002; Mendelsohn, 2003). In
Fig. 3. Nav1.7 is necessary for Navchannel-mediated invasion in H460 cells. (A) Relative mRNA expression for all TTX-sensitive Nav a subunits and all
auxiliary Nav b subunits in A549 and H460 NSCLC cells, and in positive control SH-SY5Y cells. All expression data were normalised to 18S rRNA expression
(n56 for all primer sets, *P,0.05, Student’s t-test). (B) Representative western immunoblots showing expression of Nav1.7 (upper) and b-actin (lower)
proteins in Nav1.7-negative (HEK-293) and Nav1.7-positive (HEK-293 cells stably transfected with Nav1.7, HEK-Nav1.7) control cells, A549 cells, H460 cells
and H460 cells transfected with 5 nM siRNA directed against Nav1.7 (n53 for all blots). (C) Representative fluorescence images showing cellular distribution of
Nav1.7 protein in Nav1.7-positive (HEK-Nav1.7) and Nav1.7-negative (HEK-293) control cells, and in H460 versus A549 cells. Nav1.7 was stained using a
Nav1.7-specific antibody recognising a C-terminal intracellular epitope (green). CTL 1, no primary antibody incubation; CTL 2, no secondary antibody incubation
(n$10 for all cell lines; scale bars: 10 mm; blue, DAPI nuclear staining. (D) Relative Nav1.7 mRNA expression in high and low H460 cell dilution clones
(n56 for both clones, **P ,0.01, Student’s t-test). Inset: representative western immunoblots showing Nav1.7 (upper) and b-actin (lower) protein expression in
high and low dilution clones (n53 for both blots). (E) Relative Nav1.7 mRNA expression for control H460 cells (CTL), H460 cells transfected with 5 nM
scrambled siRNA (Scrambled) and H460 cells transfected with 5 nM siNav1.7 (n56 for all conditions, ***P,0.001, one-way ANOVA and SNK correction).
(F) Average current-density–voltage (I–V) plots for control untransfected (open squares, n510), and H460 cells transfected with 5 nM scrambled siRNA (closed
squares, n510) or 5 nM siNav1.7 (open circles, n58). Continuous lines indicate Boltzmann curve fits as described in Materials and Methods. Inset: representative
current traces from untransfected and 5 nM siNav1.7-transfected H460 cells. Currents were evoked using 30-millisecond depolarising steps in 5-mV intervals
(290 to +70 mV), from Vh5 290 mV. (G) Relative MatrigelTM invasion in H460 cells; untransfected (CTL), transfected with 5 nM scrambled siRNA
(Scrambled) or 5 nM siNav1.7. n59 for all conditions, ***P,0.001, one-way ANOVA and SNK correction.
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NSCLC, EGFR overexpression is associated with particularlyaggressive malignancies (Mendelsohn, 2003). To assess the role
of EGFR signalling in the upregulation of Nav1.7 and the
subsequent invasion in NSCLC, we initially compared Nav1.7
mRNA expression in H460 cells following treatments with EGFor EGFR signalling modulators (Fig. 6A). Serum starvation
(48 hours) reduced Nav1.7 mRNA levels by 81.261.1%
compared with control cells maintained in complete medium.
In contrast, addition of EGF (100 ng.ml21, 24 hours) following24 hour serum starvation, fully restored Nav1.7 mRNA
expression. Conversely, the EGFR inhibitor gefitinib (1 mM,
24 hours), in complete medium reduced Nav1.7 mRNAexpression to serum-starved levels. Moreover, gefitinib blocked
EGF-mediated enhancement of Nav1.7 mRNA expression in
these cells. The Nav1.7 protein levels mirrored these changes in
mRNA expression (inset Fig. 6A).
To avoid adverse effects of serum starvation on the integrity of
cell membranes, electrophysiology was performed on H460 cells
maintained in the presence of serum. Under these conditions,
EGF (24 hours) had a negligible effect on INa (Fig. 6B),
presumably because EGF-mediated signalling is already close
to its maximum under control conditions. However, in the
presence of either gefitinib or anti-EGF function-blocking
antibody (10 mg.ml21), INa was almost completely abolished.
The concurrent changes in Nav1.7 mRNA, protein and INa
(current density, pA.pF21) suggest that EGF/EGFR signalling
controls transcriptional regulation of Nav1.7 and, hence, numbers
of functional channels at the cell surface. Growth factor
signalling can, however, induce rapid post-translational
regulation of ion channels within minutes, e.g. phosphorylation
of channels at the cell surface (Martin et al., 2006; Woodall et al.,
2008) and/or rapid trafficking of channels to the cell surface
(Kanzaki et al., 1999). However, because acute (10-minute)
application of gefitinib had no effect on INa (Fig. 6C), we
conclude that endogenous EGF/EGFR signalling controls
transcriptional rather than post-translational regulation of Nav1.7.
Fig. 4. Reciprocal expression of Nav1.7 and b1 subunits
regulates invasion and adhesion. (A) Relative expression of Nav
b1 subunit mRNA in control A549 cells versus A549 cells
transfected with 5 nM siRNA against Nav b1 (siSCN1B), or
scrambled siRNA (n56 for all conditions, ***P,0.001, one-way
ANOVA and SNK correction). (B) Effect of b1-siRNA on A549 cell
adhesion, analysed by Crystal Violet staining (n58 for all
conditions, **P,0.01, ***P,0.001, one-way ANOVA and SNK
correction). Adhesion in H460 cells is shown for comparison.
(C) Effect of siSCN1B on A549 cell MatrigelTM invasion after
48 hours (n59 for both conditions, *P,0.05, Student’s t-test).
(D) Relative invasion into MatrigelTM of H460 cells transfected with
either control GFP (CTL) or b1-GFP (H460 + b1-GFP) (n54 for all
conditions, **P ,0.01, Student’s t-test). (E) Relative invasion into
MatrigelTM of A549 cells transfected with GFP control (CTL) or
Nav1.7-DsRed in the absence (A549 + Nav1.7-DsRed) or presence
of 0.5 mM TTX (A549 + Nav1.7-DsRed + TTX). n54 for all
conditions, **P ,0.01, one-way ANOVA and SNK correction).
(F) Fluorescence images for H460 cells (left) and A549 cells (right)
transfected with b1-GFP and Nav1.7-DsRed, respectively (scale
bars: 50 mm).
Fig. 5. Nav1.7 protein expression is higher in
cancerous compared with normal-matched human
lung tissue. Representative images showing Nav1.7-
immunostaining (DAB+-stained areas, brown) from
sections of the following formalin-fixed, paraffin-
embedded specimens: (A–C) normal versus cancerous
lung tissue from three patients (P1–P3), (D) weakly
invasive A549 cells compared with strongly invasive
H460 cells, (E) low- versus high-grade prostate tumour
tissue microarray preparations. (F) Control
untransfected HEK-293 cells (negative control) and
HEK-293 cells stably transfected with Nav1.7 (positive
control) (n53 for all preparations, scale bars: 200 mm).
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H460 cell proliferation, migration and invasion were all
upregulated by EGF/EGFR signalling, whereas Nav contributed
only to invasion (Fig. 6D–F). Since inhibition of Nav (TTX), EGFR
(gefitinib), or combined EGF/TTX treatments all result in ,40%
inhibition of invasion compared with control untreated cells, we
infer that the pro-invasive response to EGF is mediated exclusively
via Nav activity. Notably, however, neither gefitinib nor TTX
completely suppressed invasion. Thus, although EGF/EGFR/Nav
signalling accounts for ,40–50% of invasion, other cellular factors
must also contribute to the pro-invasive capabilities of these cells.
Finally, we examined the involvement of the signalling
pathways downstream of EGFR. Four main pathways are
associated with EGFR signalling: Jak/Stat, PLCc, PI3-K/Akt
and ERK1/2 (Oda et al., 2005). Of these, we focused on the PI3-
K/Akt and ERK1/2 signalling cascades, which modulate ion
channels in excitable cells (Adams et al., 2000; Black et al., 2008;
Persson et al., 2011; Qiu et al., 2003; Smani et al., 2010;
Stamboulian et al., 2010; Woodall et al., 2008) and are intimately
involved in cancer progression (Fremin and Meloche, 2010;
Keshet and Seger, 2010; Li et al., 2005; Stahl et al., 2004).
Western immunoblotting confirmed basal activity of both
phospho-ERK1/2 and phospho-Akt in H460 cells (Fig. 7A).
U0126 inhibited Nav1.7 mRNA expression but wortmannin had
no effect, implying that ERK1/2 (not PI3-K) controls Nav1.7
expression (Fig. 7B). Effects on INa were initially screened using
IonWorksH Automated Patch Clamp (Fig. 7C). As with Nav1.7
mRNA expression, U0126 inhibited mean INa by 44% from
0.3960.05 nA (control) to 0.2260.02 nA, whereas wortmannin
application had no effect. These data were further supported by
conventional patch clamp recordings (Fig. 7D). Consistent with
these effects on Nav1.7 expression and activity, H460 cell
invasion was unaffected by wortmannin but inhibited by U0126
(Fig. 7E). No additive effect of combining U0126 with TTX was
observed, suggesting that regulation via ERK1/2 is crucial for
Nav1.7-mediated invasion of H460 cells.
DiscussionWe have shown that EGF/EGFR signalling via a U0126-sensitive
ERK1/2 pathway controls transcriptional upregulation of Nav1.7
to promote cellular invasion in NSCLC cell lines. Moreover,
immunohistochemistry of patient biopsies provides evidence
supporting the clinical relevance of Nav1.7 protein expression in
NSCLC. Thus, we identify Nav1.7 as a new downstream effector
of the EGF/EGFR pathway with potential as a target for
therapeutic intervention and/or as a diagnostic or prognostic
marker in NSCLC.
The necessity of Nav1.7 functional expression for invasion of
NSCLC cells agrees with other work showing the crucial role of
Fig. 6. Modulation of EGF/EGFR signalling regulates functional expression of Nav1.7 and invasion in H460 cells. (A) Relative Nav1.7 mRNA expression
showing the effect of 48-hour serum starvation, stimulation with 100 ng.ml21 EGF for 24 hours post serum starvation, treatment with 1 mM gefitinib for 24 hours
in the presence of serum, and co-application of 100 ng.ml21 EGF and 1 mM gefitinib for 24 hours post serum starvation (n56 for all conditions, ***P,0.001,
one-way ANOVA and SNK correction). Inset: representative western immunoblots showing expression of Nav1.7 (upper) and b-actin (lower) in H460 cells
subjected to the following treatments: serum starved for 48 hours (SS); 100 ng.ml21 EGF for 24 hours post serum starvation (EGF); 1 mM gefitinib (Gef) for
24 hours in the presence of serum (n53 for both blots). (B) Average current-density–voltage (I–V) plots for control untreated H460 cells (open squares, n510) and
H460 cells treated with 100 ng.ml21 EGF (closed squares, n511), 1 mM gefitinib (open circles, n512) or 10 mg.ml21 anti-EGF function blocking antibody
(closed circles, n510) for 24 hours in the presence of serum (***P,0.001, one-way ANOVA and SNK correction). Inset: representative current traces from
untreated and gefitinib-treated H460 cells. (C) Average current-density–voltage (I–V) plots for control untreated (open squares, n511), and acutely treated
(10-minute pre-incubation) with 1 mM gefitinib (closed squares, n512) H460 cells. Inset: representative current traces from untreated and acute gefitinib-treated
H460 cells. Continuous lines indicate Boltzmann curve fits as described in Materials and Methods. Currents were evoked using 30-millisecond depolarising steps
in 5-mV intervals (290 to +70 mV) from Vh5290 mV. (D) Cell proliferation over 120 hours in control untreated (CTL), TTX-treated (0.5 mM), EGF-treated
(100 ng.ml21), EGF/TTX co-treated, and gefitinib-treated (1 mM) H460 cells (n$8 wells for each condition, at least two separate experiments). (E) Migration
over 58 hours in control untreated (CTL), TTX-treated (0.5 mM), EGF-treated (100 ng.ml21), EGF/TTX co-treated, and gefitinib-treated (1 mM) H460 cells (n58
wells for each condition, two separate experiments). (F) H460 cell MatrigelTM invasion after 48 hours in control untreated (CTL), 0.5 mM TTX-treated,
100 ng.ml21 EGF-treated, 1 mM gefitinib-treated (Gef) and EGF/TTX co-treated cells (n59 for all conditions, *P,0.05, **P,0.01, ***P ,0.001, one-way
ANOVA and SNK correction).
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Nav – particularly Nav1.5 and Nav1.7 – in metastatic behaviours
of several cancers, including NSCLC. Nav1.7 mRNA expression
in H460 cells has been reported (Roger et al., 2007) but its
predominant role in Nav-dependent invasion of these cells has not
been shown before. In breast cancer, the Nav b1 subunit is
reported to act as a CAM, being highly expressed in non-invasive
cells but not in strongly invasive cells that exhibit reduced cell
adhesion (Brackenbury and Isom, 2008; Chioni et al., 2009). We
see similar trends in the reciprocal expression of the Nav b1 and
Nav1.7 subunits in non-invasive A549 versus highly invasive
H460 NSCLC cells. Moreover, regulation of Nav b1 and Nav1.7
expression alone is sufficient to alter significantly the invasive
properties of H460 and A549 cells. Thus, the balance of Nav b1
versus a subunit expression also appears to regulate the invasive
potential of NSCLC cells.
To date, there has been little detailed characterisation of the
mechanisms that drive functional expression of Nav in cancerous
cells (Ding et al., 2008; Fraser et al., 2010; Uysal-Onganer and
Fig. 7. EGF upregulates Nav1.7 expression and the subsequent H460 cell invasion through ERK1/2 signalling. (A) Representative western immunoblots
showing the levels of active phospho-ERK1/2 (pERK1/2) versus total ERK (top), and active phospho-Akt (pAkt) versus total Akt (bottom) proteins in
control untreated (CTL), U0126- (10 mM) and wortmannin-treated (100 nM, Wort) H460 cells after 24 hours (n53 for all blots). (B) Relative Nav1.7 mRNA
expression in H460 cells showing the effect of treatment with U0126 (10 mM) or wortmannin (100 nM, Wort) for 24 hours compared with control untreated
(CTL) H460 cells (n56 for all conditions, *P,0.05, one-way ANOVA and SNK correction). (C) Maximum current amplitudes (Imax) from individual H460 cells
that had been left untreated (CTL), or had been treated with U0126 (10 mM) or wortmannin (100 nM, Wort), were recorded using the IonWorksH QuattroTM
Automated Patch Clamp System (n5number of cells with TTX-inhibitable INa/total number of cells tested). (D) Average current-density–voltage (I–V) plots for
control untreated (open squares, n59) and 10 mM U0126-treated (closed squares, n511) H460 cells (**P,0.01, Student’s t-test). Continuous lines indicate
Boltzmann curve fits as described in Materials and Methods. Inset: representative current traces from untreated (CTL) and U0126-treated H460 cells. Currents
were evoked using 30-millisecond depolarising steps in 5-mV intervals (290 to +70 mV), from Vh5290 mV. (E) Relative MatrigelTM invasion of H460 cells
after 48 hours in control untreated conditions (CTL) and following treatment with 10 mM U0126, 10 mM U0126/1 mM TTX, 100 nM wortmannin (Wort)
and 100 nM wortmannin/1 mM TTX (Wort + TTX) (n59 for all conditions, *P,0.05, **P,0.01, one-way ANOVA and SNK correction).
Table 1. Primers used in qPCR to determine mRNA expression of TTX-sensitive Nav channel a and b subunits in human
NSCLC cell lines
Sense primer (59-39) Anti-sense primer (59-39)
Nav1 a subunits
Gene symbol Subunit
SCN1A Nav1.1 ATGATCTGCCTATTCCAAATTACAA GGTTCCCACAGTCTCCCTTASCN2A Nav1.2 CGTGATGGTGATGTGTTTGTG TCTCTGTCTTGTTATAGGCACTGSCN3A Nav1.3 GAATAGTTGTTCCACTTTCTGCTG ACATGCACCACAGGATAAAATAACSCN4A Nav1.4 TGTCTGCTTGTATGCTTTTATAGG CTCCAGTACACAGTGGTTCACSCN8A Nav1.6 GCGTTGAGGCACTACTACTTC TCCCACAATGGAGAGGATGACSCN9A Nav1.7 AGACCTCTCTTTCCATGTAGATTAC TGTAACTGCCTTTCTGTATTGTTG
Nav1 b subunits
Gene symbol Subunit
SCN1B b1 GTATGTGCTCATTGTGGTGTTG CCAGGTATTCCGAGGCATTCSCN2B b2 AGGTTCCAACTGACAGAAACTG ATCATAGACCAGGACACAGAGGSCN3B b3 CACCAGCCTACTTCCAATCTC TCTAAGCCCTTTAATAAGCAACATCSCN4B b4 CCAAGTCTGTCCTGTCTTCATC GGTGGGATGGAAAGAAAGAGAG
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Djamgoz, 2007). To address this crucial issue, we focused on the
role of EGF/EGFR signalling, which not only plays key roles in
NSCLC (Fujimoto et al., 2005; Mendelsohn, 2003; Volante et al.,
2007) but also regulates transcription and activity of various ion
channels, including Nav (Adams et al., 2000; Black et al., 2008;
Ding et al., 2008; Liu et al., 2007; Persson et al., 2011; Qiu et al.,
2003; Smani et al., 2010; Stamboulian et al., 2010; Uysal-Onganer
and Djamgoz, 2007; Woodall et al., 2008). In H460 cells, our data
suggest that EGF/EGFR signalling via ERK1/2 tonically
upregulates the functional expression of Nav1.7 to enhance Na+
influx and, hence, invasion. Whereas proliferation, migration and
invasion of H460 cells are all promoted via the EGF/EGFR
pathway, only invasion is sensitive to Nav1.7 inhibition. Since
blocking of EGFR, ERK1/2 or Nav1.7 inhibits invasion by ,40–
50% – with no additive effects of combined treatments – we infer
that EGF/EGFR–ERK-mediated invasion in these cells is most
likely to occur via upregulation of Nav1.7. However, since this
pathway promotes only 40–50% of H460 cell invasion, other
cellular factors must also be involved. Vascular endothelial,
fibroblast and platelet-derived growth factors all play roles in
NSCLC (Ballas and Chachoua, 2011) and are linked with Nav
function (Andrikopoulos et al., 2011; Goldfarb et al., 2007;
Hilborn et al., 1998). Although further work is required to establish
the relevance of such growth factors, it is conceivable that Nav1.7
is a convergence point in several cancer signalling pathways.
Whereas transcriptional upregulation is necessary to promote
sufficient functional expression of Nav protein at the cell surface,
our data suggest it is the influx of Na+ through Nav1.7 and the
consequent depolarisation of the cell membrane, which appears
to be essential for invasion. Although inhibition of EGF/EGFR,
ERK1/2 and Nav1.7 consistently reduced invasion by ,40–50%,
the effects on INa varied from complete loss of current (TTX) to
47% inhibition (U0126). This implies that not all available
channels need to be functional to enable sufficient Na+ influx for
invasion. Indeed, in agreement with others (Gillet et al., 2009;
Roger et al., 2007), we also found evidence of continuous Na+
influx in H460 cells, when Nav channels are partially activated
at resting membrane potential. Furthermore, block of Nav activity
hyperpolarises the plasma membrane and reduces [Na+]i, whereas
enhanced channel opening depolarises the membrane potential
and raises [Na+]i. Thus, a positive-feedback mechanism appears
to operate, where the partial opening of Nav at resting potential
allows Na+ influx, which – in turn – keeps the plasma membrane
sufficiently depolarised to maintain a continuous influx of Na+.
Under physiological conditions, this influx of Na+ is sufficient to
maintain the invasive capability of cells. Endogenous modulators
of Nav1.7, e.g. ERK1/2, can thus provide an exquisite mechanism
for fine-tuning of EGF/EGFR-mediated invasion in NSCLC.
Similarly, other transport proteins that allow Na+ influx are also
expected to promote invasive behaviour in NSCLC. For example,
in prostate cancer cells, overexpression of exogenous Nav1.4
enhances their invasion (Bennett et al., 2004). In MDA-MB-231
breast cancer cells, Nav1.5 is postulated to be functionally
coupled with the Na+/H+ exchanger NHE1. This exchanger
extrudes H+ to cause extracellular acidification, thereby
providing an optimal environment for pH-dependent activity of
cysteine cathepsins and proteolysis of the ECM, leading to
enhanced cell invasion (Brisson et al., 2011; Gillet et al., 2009).
Na+ influx has also been suggested to impact on intracellular
Ca2+ homeostasis, primarily through reversal of Na+/Ca2+
exchanger NCX activity (Blaustein and Lederer, 1999). Thiscould have numerous effects, including altered gene expression.
Regardless of how increased [Na+]i promotes invasion, wehave demonstrated unequivocally that functional expression ofNav1.7 is associated with increased invasive potential of NSCLC
cell lines. Whereas the use of small-molecule EGFR inhibitors,such as gefitinib and erlotinib, has substantial therapeutic benefitsfor many patients with advanced NSCLC, drug resistance
remains a significant problem (Cataldo et al., 2011; Domingoet al., 2010; Lynch et al., 2004; Paez et al., 2004). Drugs thattarget Nav1.7 activity could, thus, prove useful in conjunction
with existing therapies. Nav-specific blockers are already inclinical use as local anaesthetics and, more recently, Nav1.7-specific inhibitors are being developed for the treatment of pain
(Bregman et al., 2011; Chowdhury et al., 2011; Clare, 2010;Ghelardini et al., 2010). Given its functional significance andmarked upregulation in tumour tissue, we suggest that Nav1.7
represents an important new target for therapeutic interventionand/or as a diagnostic and/or prognostic marker in NSCLC.
Materials and MethodsMaterials
Primary antibodies were obtained as follows: anti-Nav1.7 (westernimmunoblotting; NeuroMab, CA, USA), anti-Nav1.7 (immunocyto-/histochemistry; AstraZeneca, UK), anti-phospho-ERK1/2 (Sigma-Aldrich orPromega, UK), anti-total ERK1/2 (Promega, UK), anti-phospho-Akt and anti-total Akt (Cell Signalling Technology, UK), anti-b-actin (Sigma-Aldrich, UK).Anti-EGF function-blocking antibody and EGF were from Millipore, UK.Secondary antibodies were obtained as follows: FITC-conjugated anti-rabbit IgG(Jackson Immunoresearch, UK), horseradish peroxidise (HRP)-conjugated anti-rabbit and anti-mouse IgGs (Dako, UK). All oligonucleotide primers for qPCRwere designed and manufactured by PrimerDesign, UK. Tetrodotoxin (TTX),veratridine and wortmannin were from Sigma-Aldrich, UK. U0126 was fromPromega, UK and gefitinib was from AstraZeneca, UK.
Cell culture
A549 and H460 cells were obtained from AstraZeneca, UK, were geneticallytested and authenticated using the PowerPlexH 16 System (Promega, UK) and werenot cultured for more than 6 months. A549 cells were cultured in Dulbecco’smodified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum(FBS; Gibco/Invitrogen, UK) and 50 U.ml21 penicillin/50 mg.ml21 streptomycin(Sigma-Aldrich, UK). H460 cells were cultured in RPMI-1640 medium (Gibco/Invitrogen, UK) supplemented with FBS and penicillin/streptomycin. Cells weremaintained at 37 C, 5% CO2. Cells were seeded and maintained in completemedium at least overnight, prior to any treatment. All pharmacological agents weredissolved in appropriate solvents and diluted in cell culture medium.
Electrophysiology
Recordings of inward Nav channel current, INa, from A549 and H460 cells weremade using the whole-cell patch clamp technique. The extracellular solutioncontained (in mM): NaCl 140, CsCl 3, MgCl2 1, CaCl2 2, glucose 11, HEPES 10adjusted to pH 7.4 with NaOH and 320 mOsm.litre21 with sucrose. The internalsolution contained (in mM): CsCl 135, NaCl 5, CaCl2 0.37, MgCl2 1, HEPES 10,EGTA 5, adjusted to pH 7.2 with CsOH and 310 mOsm.litre21 with sucrose.Before recording, cells were replated onto 0.01% poly-L-lysine-coated coverslips(Sigma-Aldrich, UK) and incubated at 37 C, 5% CO2 for at least 1 hour. Patchpipettes of resistance 2–5 MV were pulled from thin-walled borosilicate glasstubing (Intracel, UK), fire-polished and coated with Sigmacote (Sigma-Aldrich,UK). An Axopatch 200B amplifier (Molecular Devices, CA, USA) was used forrecordings which were filtered at 2 kHz and digitised at 5–10 kHz using a Digidata1322A A/D converter (Molecular Devices, CA, USA). Cells were held at apotential Vh, of 290 mV, with holding current less than 250 pA and seriesresistance less than 10 MV. Currents were recorded with cell capacitancecompensated and leak currents subtracted using a P/4 subtraction protocol.Series resistance was compensated up to 80%. Standard current-voltage (I–V)protocols used 30-millisecond sweeps from 290 to +70 mV in 5 mV steps, fromVh 290 mV. I–V curves were fitted with a Boltzmann function: I5G(V-Vrev)/1+exp(-(V-V50,act)/k), where Vrev is reversal potential, V50,act is voltage for halfmaximal current activation; G is conductance and k is slope factor. Conductance–voltage relationships were derived from individual I–V plots and fitted with aBoltzmann function: G/Gmax51/1+exp((V50,act-V)/k), where Gmax is maximalconductance. Peak conductance G, at each test potential was calculated from the
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corresponding peak current I, as follows: G5I/(V-Vrev). Steady-state inactivationprotocols comprised a test pulse of +10 mV following 500-millisecond pre-pulsepotentials between 2120 and +40 mV, from Vh 290 mV. Peak current valueswere normalised to Imax and curves fitted with the following Boltzmann function:I/Imax51/1+exp((Vt-V50,inact)/k), where Imax is maximum peak current and V50,inact,the midpoint of voltage-dependent inactivation. Resting membrane potential (Em)was estimated by current clamp in physiological saline solutions (PSS), using high-resistance patch pipettes (.10 MV). Extracellular PSS contained (in mM): NaCl140, KCl 4, MgCl2 1, CaCl2 2, glucose 11, HEPES 10 adjusted to pH 7.4 withNaOH and 320 mOsm.litre21 with sucrose. Internal PSS contained (in mM): K-glutamate 125, KCl 20, CaCl2 0.37, MgCl2 1, K-ATP 1, EGTA 1, HEPES 10adjusted to pH 7.2 with KOH and 310 mOsm.litre21 with sucrose. Dataacquisition and analysis were performed using pCLAMP (v9.0, MolecularDevices) and Origin (v8.0, Microcal Software).
IonWorksH electrophysiology
High-throughput electrophysiology used the IonWorksH QuattroTM AutomatedPatch Clamp System (Molecular Devices, CA, USA). The extracellular solutionwas phosphate-buffered saline (pH 7.4). Internal solution contained (in mM): K-gluconate 100, KCl 40, MgCl2 3.2, HEPES 10, EGTA 3, adjusted to pH 7.2 withKOH and 310 mOsm/litre with sucrose. Cells were resuspended in PBS at 16106
cells.ml21. Seal resistances ranged from a user-defined lower limit of 50 MV to,250 MV. Data were rejected if the pre-compound amplitude of the Na+ currentwas less than 50 pA or if the total resistance (parallel seal plus membraneresistance) decreased by more than 50% from pre- to post-compoundmeasurement. Electrical access to the interior of cells was achieved using thepore-forming antibiotic amphotericin B (10 mg/100 ml internal solution). INa
sensitivity to TTX was determined by comparing peak INa before and after a 2-minute incubation with 1 mM TTX. All IonWorksH data are presented asmaximum current amplitude (Imax). The IonWorksH platform does not provide cellcapacitance measurements. Thus, current densities (pA.pF21) cannot be calculated.
Quantitative PCR
Total RNA extraction was performed using the RNeasy kit (Qiagen, UK). RNAyield and purity were determined by spectrophotometry and only samples with anA260:A280 ratio above 1.8 were used for experiments. Total RNA (1 mg) wasreverse transcribed using the PrecisionTM Reverse Transcription Kit(PrimerDesign, UK) and qPCR performed using cDNA obtained from 25 ng oftotal RNA. qPCR was performed using an ABI 7500 Sequence Detection Systemwith software version 1.2.3 (Applied-Biosystems, UK). All primers were specificfor each gene of interest (Table 1). Amplification and detection were carried out in96-well Optical Reaction Plates (Applied-Biosystems, UK) with SYBRH greenPrecisionTM 26qPCR Mastermix (PrimerDesign, UK). All expression data werenormalised to 18S rRNA expression, as determined by an initial reference genescreen using the geNormTM Housekeeping Gene Selection Kit (PrimerDesign,UK). Primer-specificity was confirmed at the end of each qPCR run through thegeneration of single peaks in melt-curve analyses. Data analysis was performedusing the 22DDCT method.
Western immunoblotting
Cells grown in 10-cm diameter Petri dishes were washed in PBS and lysed on icein RIPA buffer with cOmplete Mini EDTA-free protease inhibitor cocktail (Roche,UK). Cell lysates were passed through a fine-gauge syringe needle 106to sheargenomic DNA and centrifuged at 1000 g for 30 seconds. Protein samples wereseparated by 7–10% SDS-PAGE for 90 minutes at 160 V and transferred byelectrophoresis (110 V for 90 minutes) onto a nitrocellulose membrane (Whatman,UK). Membranes were ‘blocked’ at room temperature (RT) for 1 hour in 5% (w/v)BSA in Tris-buffered saline (TBS) with 0.1% Tween-20 (TTBS), washed 36withTTBS and probed with primary antibody (1:1000–5000) in 5% BSA/TTBS at 4 Covernight. Membranes were then re-washed with TTBS 36and incubated withappropriate HRP-conjugated secondary antibody (1:2000) in 5% BSA/TTBS at RTfor 90 minutes. After further washing with TTBS, blots were treated with WesternLightningH ECL reagent (PerkinElmer, UK) and immunoreactive proteins detectedby exposure to film (GE Life Sciences, UK). In all cases, loading controls of b-actin or total-protein (for phosphorylated proteins) were used in parallel.
Immunocytochemistry
Cells were seeded at 36104 onto sterile 0.01% poly-L-lysine-coated 22-mm squarecoverslips and incubated for 48 hours. After washing with PBS, cells were fixedwith 4% (w/v) paraformaldehyde at RT for 20 minutes. Paraformaldehyde wasquenched with 0.1 M glycine for 10 minutes; cells were permeabilised with 0.5%saponin (10 minutes; Sigma-Aldrich, UK) and blocked with 5% BSA/PBS for1 hour. Cells were incubated at RT for 1 hour with primary antibody (1:200 in 5%BSA/PBS), washed 36 with PBS, incubated at RT for 1 hour with a FITC-conjugated anti-rabbit secondary antibody (1:1000) in 5% BSA/PBS before finalwashing in PBS. Nuclei were stained with DAPI (100 ng.ml21), prior to mountingcoverslips onto glass slides with ProLongH Gold Antifade Reagent (Invitrogen,
UK). Images were taken on a DeltaVision RT restoration microscope (AppliedPrecision, UK) using a 606oil immersion objective lens. Appropriate wavelengthfilters were used for FITC and DAPI fluorophores. Images were collected using aCoolsnap HQ camera (Photometrics, UK) with a Z optical spacing of 0.1 mm in 12sections. Raw images were deconvolved using SoftWoRxH software and displayedas maximum projections using NIH Image J (W.S. Rasband, National Institutes ofHealth, USA). To confirm primary antibody specificity, wild-type and HEK-293cells stably transfected to express Nav1.7 were used as negative and positivecontrols, respectively. Non-specific binding of primary and secondary antibodieswas assessed using the above protocol without either the primary antibody orsecondary antibody incubation steps.
Immunohistochemistry
Cell lines (H460 and A549) and cancerous versus normal tissue (prostate and lung;Tristar Technologies LLC, MD, USA) were paraffin-embedded and cut into 4-mmsections. Sections were processed on a LabVision Autostainer 360 (Thermo FisherScientific, UK) using the ChemMate EnvisionTM+ System-HRP (DAB+) kit(Dako, UK), according to manufacturer’s instructions. Sections were incubatedwith anti-Nav1.7 antibody (1:200) for 1 hour, followed by 30 minutes with HRP-labelled polymer-conjugated anti-rabbit IgG secondary antibody. Sections werecounterstained with dilute Mayer’s haematoxylin (Dako, UK). All processing wasperformed at RT. Images were taken with a light microscope and visualcomparisons were made. Negative controls (without incubation of primaryantibody) were performed for all experiments.
Functional assays
ProliferationCells were plated at 4000 cells per well into 96-well plates and cell numbersmonitored in real time by in vitro micro-imaging using an IncuCyteTM incubator(Essen BioScience, UK), allowing for hourly monitoring of cell proliferation bydetermining cell confluence.
MigrationFor scratch-wound assay, cells were seeded into 24-well culture plates at 2.56105
cells.ml21 and then grown to 100% confluence. A linear wound was introduced inthe centre of the cell monolayer. Culture medium supplemented with 1% FBS (tominimise cell proliferation) was then replaced and cell migration (wound closure)monitored using an IncuCyteTM incubator.
InvasionInvasion was analysed in 24-well plates containing BD MatrigelTM InvasionChambers (8-mm pore size polyethylene terephthalate membrane cell cultureinserts covered with a film of MatrigelTM Basement Membrane Matrix; BDBiosciences, UK). The upper compartment was seeded with 46104 viable cells inbasal culture medium. The lower compartment contained culture medium plus20% FBS as a chemoattractant. After 48 hours at 37 C, 5% CO2, inserts werewashed in PBS for 2 minutes and cells fixed with ice-cold methanol for10 minutes. Cells were then stained with haematoxylin (Sigma-Aldrich, UK) for3 minutes and invaded cells (those attached to the lower side of the membrane)were counted in the whole insert using a light microscope at 2006magnification.
Adhesion96-well plates were coated with 20 mg.ml21 fibronectin at 37 C for 1 hour, withsome wells left uncoated as negative controls. After two washes with washingbuffer (0.1% BSA in cell culture medium without serum), wells were ‘blocked’with blocking buffer (0.5% BSA in cell culture medium without serum) at 37 C,5% CO2 for 1 hour. After two washes, cells were plated at 20,000 cells per well inserum-free medium and incubated at 37 C, 5% CO2 for 30 minutes. Plates wereleft to shake at 2000 rpm for 30 seconds before being washed 36. Remaining cellswere fixed with 4% paraformaldehyde at RT for 15 minutes, washed 36 andstained with Crystal Violet for 10 minutes. Wells were washed thoroughly withwater and left to dry completely before addition of 2% SDS (30 minutes at RT).Absorbance of the resulting SDS/Crystal Violet solution was measured at 550 nmto quantify numbers of adherent cells.
For each functional assay, cells were seeded at the same densities for control(un-treated) and treated samples. Cell proliferation, migration and invasion datawere then normalised as follows: cell proliferation to 0% confluence at 0 hours,migration to 0% wound confluence at 0 hours, and invasion to the controlcondition.
Generation of dilution clones
H460 cell dilution clones were generated using a serial dilution protocol providedby Corning, UK (http://www.level.com.tw/html/ezcatfiles/vipweb20/img/img/34963/3-2Single_cell_cloning_protocol.pdf). To increase the likelihood that cellsof a particular colony originated from a single cell, selected cells were re-clonedfor a second time. For all experiments, parental control H460 cells were also
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included to provide a direct comparison with the standard population of H460cells. To confirm that dilution clones did not revert to their original state(maintenance of the Nav phenotype), INa was routinely recorded by patch-clampelectrophysiology prior to any experiments.
Transient transfection
siRNAH460 cells were transfected with small interfering RNAs (siRNAs) directedagainst SCN9A (Nav1.7; 59-TCGGATAGTGAATACAGCAAA-39), SCN1B
(Navb1; 59-CACATTGAGGTAGTGGACAAA-39) or control scrambled siRNAs(AllStars negative control siRNA; Qiagen, UK). Cells were seeded into 24-wellplates (16105) in 0.5 ml basal culture medium. For each well, 37.5 ng of siRNAwas diluted into 100 ml of medium without serum. HiPerFect transfection reagent(3 ml per well; Qiagen, UK) was added to diluted siRNA and mixed by briefvortexing. After 10-minute incubation at RT, transfection complexes were addeddrop-wise to cells giving a final siRNA concentration of 5 nM per well. Cells wereincubated at 37 C, 5% CO2 for 24 hours before experiments were performed.
cDNATransient transfection of H460 and A549 cells was performed at 60–70% cellconfluence using jetPEITM transfection reagent (Polyplus-transfection, France),according to manufacturer’s instructions. H460 and A549 cells were transfectedwith pEGFP-N1-Navb1 (b1-GFP; L. Isom, University of Michigan/W.Brackenbury, University of York) and pcDNA3-DsRed2-Nav1.7 [Nav1.7-DsRed;J.N. Wood, UCL; (Farmer et al., 2012)], respectively. Empty pEGFP-1 was used asa negative control. After transfection, cells were maintained for 24–48 hours incomplete medium prior to conducting experiments.
Fluorescence measurement of intracellular Na+
Intracellular Na+ concentration [Na+]i, was measured using the ratiometricfluorescent dye SBFI as described by (Roger et al., 2007). Calibration of [Na+]i
was performed in each cell by comparing fluorescence in high (20 mM) and low(10 mM) Na+ calibration solutions containing (in mM): NaCl 10 or 20, KCl 150,HEPES 5, EGTA 10, monensin 0.01, adjusted to pH 7.4 with NaOH and320 mOsm/litre with sucrose.
Data analysis
All quantitative data are presented as the means 6 standard error of the mean(s.e.m.). Statistical analysis was carried out using Student’s t-test or ANOVA(One-way with Student–Newman–Keuls post hoc correction), as appropriate(GraphPad Prism v5.0), with 95% confidence limits.
AcknowledgementsWe thank Sebastien Roger (Universite Francois Rabelais, Tours,France) for initial help with in vitro invasion assays, and HelenWombwell, Roy Milner and Michael Morton (AstraZeneca, AlderleyPark, UK) for help with IHC, antibody procurement and IonWorksHelectrophysiology, respectively.
Author contributionsExperiments were conceived, designed and interpreted by E.F., T.C.and M.M. Data acquisition and analysis were performed by T.C.Overall supervision of work was by E.F. and M.M. The paper waswritten by E.F., T.C. and M.M.
FundingBiotechnology and Biological Sciences Research Council UK, CASEStudentship to T.M.C. and AstraZeneca, UK to T.M.C. Deposited inPMC for immediate release.
Conflicts of interestThe authors disclose no potential conflicts of interest.
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