Identification of KvLQT1 K+ channels as new regulators of non-small cell lung cancer cell proliferation and migration
- Authors:
- Published online on: December 23, 2013 https://doi.org/10.3892/ijo.2013.2228
- Pages: 838-848
Abstract
Introduction
After injury, cell migration and proliferation are important mechanisms of tissue repair to restore airway and alveolar epithelia in several respiratory diseases (1–4). However, uncontrolled cell growth, dysregulation of cell motility and acquisition of an invasive phenotype are some of the hallmarks of cancer development and propagation (5,6). In fact, cell proliferation and motility are regulated by many actors, including growth factors and down-stream signalling effectors. However, a growing body of evidence demonstrates that K+ channels also participate in the control of cell motility and growth of various cell types (7–13). Indeed, inhibiting or silencing of voltage-dependent (Kv), calcium-activated (KCa), inward-rectifying (Kir) and 2-pore (K2P) channels reduces the proliferation of numerous normal and cancer cells in vitro (14–22) and animal models (20,23,24). The mechanisms underlying the regulation of cellular proliferation by K+ channels may be manifold, but evidence points to cell cycle control (10,25–27). In addition, K+ channels could also be involved in tumor cell invasion and metastasis propagation via regulation of cell migration (11,14,28–31).
The role of K+ channels in lung cancer has not been studied extensively. It has been shown that the inhibition of Girk channels (21) in small-cell lung cancer (SCLC) cells and Kv1.3 channels in non-small-cell lung cancer (NSCLC) A549 cells (20) has anti-proliferative effects. We previously reported the involvement of different types of K+ channels, including a member of the Kv channel subfamily, KvLQT1, in the control of normal alveolar and bronchial epithelial cell proliferation, migration and epithelial repair (18,19). However, the participation of this channel in lung tumor cells has not been reported. Growing interest in K+ channels as therapeutic targets in cancer is not only due to their role in cell growth and motility, but also because they have been found to be overexpressed in various tumor tissues, including in lung cancer (16,23,25,31–36). However, to the best of our knowledge, possible KvLQT1 overexpression in lung adenocarcinoma (AD) has never been observed.
In the present study, we first examined the mRNA expression of 3 Kv channels; KvLQT1, EAG1 and ERG1. The impact of pharmacological inhibition of these channels, with various drugs, was then evaluated in scratch assays. Because chromanol and clofilium reduced wound-healing rates more efficiently than astemizole and ergtoxin, we verified the involvement of KvLQT1 via a molecular approach with specific siRNA. The impact of KvLQT1 inhibitors on A549 cell motility and proliferation mechanisms was then assessed. KvLQT1 was also investigated in wound healing, migration and growth of another lung cancer cell line: H460. Finally, KvLQT1 expression levels were measured in tumor tissues from patients with lung AD.
Materials and methods
Lung tissue samples
Our protocol on human tissues has been approved by the Ethics Committee of our Institution (Comité d’éthique à la recherche du CHUM). All participants provided their written informed consent to participate in this study and all clinical investigation has been conducted according to the principles expressed in the Declaration of Helsinki. Tumor and matched, adjacent, non-neoplastic lung tissues were sourced from 26 patients with lung adenocarcinoma (AD), the most common subtype of NSCLC. Tissues were collected immediately after surgical resection at the CHUM Hôpital Notre-Dame (Montréal, Québec, Canada) or obtained from the tissue bank of the Respiratory Health Network of the FRQS. Tissues were dissected from surgical samples by a pathologist, who confirmed the diagnosis of primary lung cancer and established clinical and histological parameters. The different growth patterns were quantified using a 5% increment by a thoracic pathologist, who classified the AD tumor samples based on the latest IASLC classification (37), into the following sub-categories: i) AD in situ (1 patient), ii) lepidic-predominant AD [4 patients, including formely non-mucinous bronchioloalveolar carcinoma (BAC) class], iii) acinar-predominant AD (8 patients), iv) papillary-predominant AD (3 patients), v) micropapillary-predominant AD (2 patients) and vi) solid-predominant AD (4 patients) as well as vii) invasive mucinous AD (4 patients, formely mucinous BAC). As reported in Table I, among the 26 patients included in our study, 46% were men and 54% were women, with a median age of 66 years (range 39–77 years); 76% had a smoking history. No patients in our cohort had metastasis at the time of lung resection.
Table I.KvLQT1 expression ratio [in tumor vs non-tumor (non-neoplastic) tissue], with individual demographic and histopathological characteristics for the 26 patients included in the study. |
Cell culture
A549 and H460 lung cancer cells were purchased from the ATCC and maintained in culture medium (A549: DMEM, Gibco, Invitrogen, Burlington, ON, Canada; H460: RPMI, Gibco, Invitrogen) supplemented with 10% of fetal bovine serum (FBS, Gibco, Invitrogen), 2 mM L-glutamine (Gibco, Invitrogen) and 50 U/ml penicillin and 50 μg/ml streptomycin (Gibco, Invitrogen).
Wound-healing assays
The commonly-employed ‘wound-healing assay’, which gives an estimation of the capacity of cells to migrate and proliferate (Fig. 1A) after mechanical injury, was used. Briefly, A549 and H460 cell monolayers were injured mechanically with a pipette tip (6 wounds/Petri dish), then washed to remove detached/injured cells, before photography with a Nikon camera under light microscopy (×4 enlargement). Marks on the Petri dishes allowed us to photograph the wounds exactly at the same place at various times, with measurement of the wound areas initially and after repair, by ImageJ software (National Institutes of Health), as described previously (38,39). The results, reported as the rates of wound closure in μm2/h, are compared to the controls and in the presence of K+ channel inhibitors, i.e., clofilium (Sigma-Aldrich, Oakville, ON, Canada), chromanol (Tocris, Bristol, UK), ergtoxin (Alomone Labs Ltd., Jerusalem, Israel) and astemizole (Tocris). Wound-healing rates were also evaluated in the presence of negative controls or KvLQT1 siRNAs (Invitrogen). For Ki67 staining during wound healing (Fig. 1A), confluent A549 cell monolayers, cultured on glass slides in Flexiperm (Dako Denmark, Glostrup, Denmark), were fixed in cold methanol at time 0 after injury and after 18 h of repair before staining with anti-Ki67 antibody (Dako), then Alexa Fluor 488 goat anti-mouse secondary antibody (proliferating cells stained in green) and finally counterstaining with DAPI (staining of nuclei in blue, Sigma).
Video-microscopy time-lapse experiments
The 2D cell migration rates (μm/h) of non-confluent A549 cells were evaluated by single-cell tracking with Axiovision software (Carl Zeiss, Toronto, ON, Canada) on sequence images captured at 5-min intervals over an 18-h period by digital camera connected to a Carl Zeiss microscope (Carl Zeiss, ×10 enlargement).
Cell migration in Boyden-type chamber
The 3D migratory capacities of A549 and H460 cells were evaluated by Boyden-type chamber migration assay (18,19). Briefly, cells obtained after trypsinization were counted, and cell viability was verified by trypan blue assay. The cell suspensions were then placed in the upper compartment of 8-μm pore filters (0.33 cm2, ThinCerts-TC inserts, Greiner Bio-one; MJS Biolynx, Brockville, ON, Canada) coated on the lower side with a collagen matrix. After cell migration, the filters were washed with PBS, and the cells were fixed with paraformaldehyde-acetone solution, then stained with hematoxylin. Non-migrating cells of the upper compartment were scrapped-off with cotton-tipped applicators (Fisher, Napean, ON, Canada), whereas migrating cells that passed through the lower face of the filters were counted in 5 different randomly-chosen fields by a light-inverted microscope at ×20 magnification (18,19).
Cell proliferation and cell cycle analyses
A549 and H460 cell growth was evaluated by counting cell number over a 72-h period. Briefly, the cells were cultured at low density (15,000/cm2) in 35-mm Petri dishes for 24 h in the absence of inhibitor. At this time (T0), they were exposed or not to K+ channel inhibitors for 24–72 h, then separated with trypsin-EDTA (0.05%, Gibco, Invitrogen) at times 0, 24, 48 and 72 h, before counting by hemacytometer. The absence of drug cytotoxicity was verified by trypan-blue exclusion assay. At the end of growth assays, A549 cells were fixed in ethanol (70%) for cell cycle analysis. After centrifugation, they were re-suspended in PBS supplemented with pancreatic RNase A (Roche, Mannheim, Germany). After 30 min, propidium iodide solution (1 mg/ml, Sigma-Aldrich) was added to each sample, and flow cytometry performed with a flow Coulter EpicsXL cytometer (Beckman Coulter, Inc., Mississauga, ON, Canada). Data were recorded for at least 10,000 events, and signals were analyzed by Expo32 software (Applied Cytometry Systems, Sheffield, UK).
KvLQT1 channel silencing with siRNAs
As described previously (40), A549 cells (40–50% confluence) were exposed to control siRNAs (Stealth siRNA-negative control, 12935-400, 100 pmol, Invitrogen) or siRNA directed against KvLQT1 (Stealth™ siRNA duplex oligonucleotides; Kcnq1-HSS142716, Invitrogen, 100 pmol), mixed with Lipofectamine™ RNAiMax transfection reagent in OptiMEM (Invitrogen). Fresh, complete DMEM was added onto cells after 5-h exposure to siRNAs, and then replaced every 24 h for 72–96 h. Transfection efficiency >90% was observed under these experimental conditions (40).
Reverse transcription-polymerase chain reaction (RT-PCR) amplification
Total RNA purified from A549 cells with TRIzol reagent (Invitrogen) was reverse-transcribed with MMLV reverse transcriptase (Invitrogen) in the presence of oligodT primers. cDNAs were amplified with Taq polymerase (Invitrogen), and specific primers designed from sequences of human KvLQT1 [Kv7.1 (41), NM_000218, 5′-taaggaagagccc aacactg-3′, 5′-cgatccttgctcttttctga-3′, 1 μM, 355-bp product], human ether-a-go-go 1 (hEAG1, Kv10.1, NM_002238.3, 5′-gag aacgtggatgagggcat-3′, 5′-gtagtggtccagcttacggg-3′, 1 μM, 96-bp product), human ether-a-go-go-related gene (hERG1, Kv11.1, NM_000238.3, NM_172056.2, 5′-ggccagagccgtaagttcat-3′, 5′-aagccgtcgttgcagtagat-3′, 1 μM, 74-bp product) and β-actin (hβ-actin, 5′-agagctacgagctgcctgac-3′, 5′-aaagccatgccaatctcatc-3′, 0.25 μM, 499-bp product), for normalization. Primer pairs were designed in distinct exons to avoid genomic DNA amplification. KvLQT1, EAG and ERG products were amplified for 30 cycles, while β-actin amplification was stopped after 18 cycles. RT-PCR products were finally separated on 1% agarose gel and stained with SYBR® Safe (Invitrogen). Signals were detected by Typhoon Gel Imager and analyzed by ImageQuant software (Molecular Dynamics, Baie d’Urfe, QC, Canada). KvLQT1 silencing with siRNA was also verified by RT-PCR (40).
Immunoblotting
Immunoblotting was undertaken according to a well-established laboratory protocol (18,40). Total A549 proteins were solubilized in lysis buffer [150 mM NaCl, 50 mM Tris-HCl, pH 7.6, 1% Triton X-100, 0.1% SDS, protease inhibitor cocktail (Complete Mini EDTA-free protease inhibitor cocktail, Roche)] on ice for 30 min. Proteins from lung tissues were extracted by homogenization, with a glass putter for 2 min in lysis buffer (150 mM NaCl, 25 mM Tris-HCl, 1 mM EDTA, leupeptin, aprotinin, PMSF protease and orthovana-date phosphatase inhibitors). Cell lysates were centrifuged, supernatants collected, and protein concentration measured by Bradford assay. Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. After blocking, the membranes were incubated with anti-KvLQT1 (sc-10645, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and anti-β-actin antibodies (CLT9001, Cedarlane Laboratory Ltd., Burlington, ON, Canada) on the same blot, to ensure equivalent loading. KvLQT1 antibody specificity was verified with its blocking peptide [sc10645P, Santa Cruz, (18)]. After washing, the membranes were incubated with donkey anti-goat (for KvLQT1, Santa Cruz Biotechnology, Inc.) and horse anti-mouse (for β-actin; Cell Signaling Technology, Boston, MA, USA) IgG linked to horseradish peroxidase for 1 h. The intensity of each specific band was quantified with ChemiDoc XRS+ Molecular Imager and Image Lab software (Bio-Rad, Mississauga, ON, Canada), then normalized to the β-actin signal.
Statistical analyses
The data are presented as mean ± standard error of the mean (SEM) and were compared with Statview software (SAS Institute, Cary, NC, USA). Statistical analyses were made using paired Student’s t-test, Wilcoxon signed rank test and one sample sign test. For comparison between more than two means, we used Friedman analysis followed by Dunn’s test. Differences were considered significant when p<0.05.
Results
Detection of Kv channels and impact of their inhibition on wound healing in A549 cell monolayers
The presence of human KvLQT1, EAG1 and ERG1 mRNA was first detected by RT-PCR in lung AD A549 cells (Fig. 1B). To evaluate the contribution of these channels, A549 cells were exposed to various pharmacological Kv channel inhibitors in scratch-assays, which engages cell migration and proliferation processes (Fig. 1A). Neither astemizole (EAG and ERG channel inhibitor, 5 and 20 μM), nor ergtoxin (specifically blocking ERG channels, 100 nM) significantly affected A549 wound-healing rates (Fig. 1C and D). On the contrary, clofilium, which is frequently used to study KvLQT1 function in respiratory epithelia (42–44), dose-dependently decreased wound-healing rates of A549 cell monolayers (11, 21 and 31% inhibition in the presence of 5, 10 and 25 μM clofilium, respectively, Fig. 1E). Similarly, the KvLQT1 blocker chromanol reduced A549 wound healing (Fig. 1F).
To confirm the role of KvLQT1, we adopted a complementary approach with stealth KvLQT1 siRNAs to test if its specific silencing would subsequently affect wound-healing rates. First, the efficiency of KvLQT1 siRNA in downregulating KvLQT1 mRNA (Fig. 2A) and protein (Fig. 2B) expression was verified. We then found that partial KvLQT1 silencing elicited a significant decrease in wound-healing (28.9±6.9×103 μm2/h, Fig. 2C), compared to A549 cells exposed to negative control siRNA (44.3±8.9×103 μm2/h, i.e., 35% inhibition). Data from both pharmacological and molecular approaches thus point toward KvLQT1 involvement in the regulation of A549 wound healing. We then undertook complementary assays to dissect its implication in both cell migration and proliferation.
Impact of clofilium on A549 cell motility
The effect of clofilium on 2D cell migration dynamics was assessed by single-cell tracking of sub-confluent A549 cells in time-lapse video-microscopy experiments. As illustrated in Fig. 3A, cell migration rates were reduced by 42% in the presence of 5 μM clofilium; no further inhibition (45%) was elicited by higher (10 μM) clofilium concentration. 3D cell motility, an important determinant of cancer propagation, was then estimated by Boyden-type chamber assay. It was observed that the number of cells migrating through the chambers was significantly reduced by clofilium (25, 31 and 42% inhibition at 5, 10 and 25 μM, respectively, Fig. 3B). Altogether, these data indicate that clofilium could depress 2D and 3D A549 cell motility.
Anti-proliferative effect of KvLQT1 inhibition through A549 cell cycle control
Because cell proliferation is a crucial component of tumor growth, we then evaluated KvLQT1’s contribution to cell proliferation, by counting the number of A549 cells after a 24- to 72-h period in the presence of KvLQT1 inhibitors. We noted that 20 μM chromanol significantly reduced cell growth, compared to untreated cells (Fig. 4A). Similarly, increasing clofilium concentrations (from 5 to 25 μM) decreased A549 cell proliferation in a dose- and time-dependent manner (Fig. 4B). At the 72-h post-treatment time-point, we estimated that <5 μM of clofilium was required to inhibit 50% of cell growth.
It has been proposed that cell cycle control by K+ channels may be a possible mechanism whereby they could regulate cell proliferation (10,25–27,45). We thus decided to examine the effect of 24-, 48- and 72-h clofilium application on A549 cell cycle distribution, by flow cytometry. First, it has to be mentioned that clofilium, even at 25 μM concentration for 72 h, did not elicit cytotoxicity as indicated by the absence of significant accumulation of sub-G0 apoptotic cells (Fig. 4C, insert). Moreover, we discerned a dose-dependent effect of clofilium on the cell cycle with an increasing number of cells in the G0/G1 phase and subsequent reduction of cells in the S and G2/M phases (Fig. 4C). These results showed that KvLQT1 inhibition in A549 cells significantly curbed cell proliferation and impacted cell cycle progression.
Reduction of H460 cell proliferation, migration and wound healing by clofilium
The effect of clofilium was also assessed in a second lung cancer cell model, the H460 cell line. As depicted in Fig. 5, KvLQT1 inhibition severely reduced H460 cell growth (Fig. 5A) and migration in the Boyden-type chamber (Fig. 5B). Accordingly, healing rates, measured at 24 and 48 h after wounding, were affected by clofilium (Fig. 5C).
KvLQT1 protein overexpression in tumor tissues from patients with lung AD
KvLQT1 expression levels were then examined by immunoblotting of human lung AD tumor tissues, compared to matched, adjacent, non-neoplastic lung parenchyma, from 26 patients (Fig. 6 and Table I). An elevation (1.5- to 7-fold) of KvLQT1 protein was apparent in AD tumor tissues from 17 among 26 patients studied (i.e., 65%). More precisely, KvLQT1 overexpression (>1.5-fold) was found in 1/1 patient with AD in situ, 3/4 patients with lepidic-predominant AD, 4/8 patients with acinar-predominant AD, 3/3 patients with papillary-predominant AD, 1/2 patients with micropapillary-predominant AD, 2/4 patient with solid-predominant AD and 3/4 patients with invasive mucinous AD. KvLQT1 overexpression was detected in both males and females, with or without smoking history (Table I).
Discussion
Our data first disclosed that clofilium and chromanol as well as KvLQT1 silencing with siRNA significantly reduced the wound-healing rates of A549 monolayers. Moreover, A549 cell migration, proliferation and cell cycle were affected after KvLQT1 inhibition. The role of this channel was also verified in the lung cancer cell line H460. Finally, KvLQT1 overexpression was detected in 17 of 26 lung AD human samples.
KvLQT1, EAG1 and ERG1 mRNA was noted in A549 cells. However, application of ergtoxin failed to decrease A549 wound-healing rates. Similarly, astemizole, which blocks EAG and ERG channels, did not significantly affect wound healing, whereas clofilium, used frequently to study KvLQT1 channel function in respiratory epithelia (42–44), dose-dependently reduced wound healing. We are aware that this compound may have off-target effects, e.g., on TASK (46), EAG (47) and ERG (48) K+ channels. However, higher clofilium concentrations (∼100 μM) are necessary for TASK inhibition (46,49). Moreover, the absence of inhibitory effects of ergtoxin and astemizole suggests that the suppressive action of clofilium in our assays is mainly occurring via KvLQT1 inhibition. This hypothesis is reinforced by the observed inhibition of wound healing by chromanol and confirmed by data obtained after KvLQT1 silencing, which caused a marked decline of wound-healing rates.
The decrease in A549 and H460 cell growth with clofilium and/or chromanol that we report here is the first evidence of such a role for KvLQT1 in NSCLC cells. These results are in agreement with other studies showing that K+ channel function modulates cell proliferation both in vitro and in vivo (9,10,15,16,23). In A549 cells, an anti-proliferative effect after Kv1.3 inhibition or silencing has already been cited (20). In SCLC cells, the involvement of other K+ channels in proliferation, i.e., Girk (21) and 4-AP sensitive K+ channels (22), has been demonstrated.
Our results indicate that KvLQT1 may regulate cell growth by controlling cell cycle progression from the G0/G1 to the S phase. Indeed, clofilium blockade affected cell cycle distribution, with a dose-dependent increase of A549 cells in the G0/G1 phase (and a decrease in G2/M and S). A similar result was observed after Kv1.3 silencing in the same cell model (20). The regulation of cell proliferation by K+ channels, through cell cycle control, has also been postulated in many other cancer types (10,45,50). Moreover, it has been hypothesized that transient hyperpolarization, after K+ channel activation, could be essential for G1 phase progression (10,50,51). In fact, other mechanisms, including calcium signaling, changes in cell volume and/or activation of signaling pathways, e.g., may also be involved (10,15,20,27,50).
Our experiments provide proof that clofilium not only decreases cell proliferation but also 2D and 3D cell migration, as shown by time-lapse video-microscopy and Boyden-type chamber assays. This is the first evidence of KvLQT1 involvement in lung cancer cell motility/proliferation, although we previously described its role in non-cancer alveolar and bronchial cells (18,19). Our present study is in agreement with previous reports that the migration of other cancer cells is reduced by inhibiting or silencing many K+ channel types (11,14,29–31). Several hypotheses have been proposed to explain how these channels regulate cell migration, e.g., through the modulation of membrane potential, cell volume, intracellular calcium and signaling pathways (11). However, it has also been proposed that K+ channels may regulate cell motility through their coupling with migratory machinery proteins, such as integrins (52).
At present, it is not clear if KvLQT1 protein per se or its K+ conducting function is responsible for the observed effect of KvLQT1 modulation on cell migration/proliferation. However, the inhibitory effect of clofilium and/or chromanol indicates that K+ conductance via KvLQT1 may be involved. Similarly, K+ permeation is the central mechanism by which TASK3 exerts its oncogenic potential in vitro and in vivo, as supported by experiments on point mutations at the channel pore (53). In contrast, another study showed that ERG protein downregulation after silencing reduced SCLC cell proliferation, whereas its blockade had no impact, suggesting that its ion-conducting property was unnecessary (54).
Our western blot analysis of tissues from patients with lung AD revealed, for the first time, KvLQT1 overexpression ranging from 1.5- to 7-fold, in 17/26 of them. To the best of our knowledge, our study provides the first evidence of KvLQT1 overexpression in lung cancer tissues, although higher KvLQT1 protein levels have already been reported in testicular seminoma tumor samples (35) and in gastrointestinal cancers (36). Other K+ channels (Girk, TASK3) have also been shown to be overexpressed in SCLC tissues (21,32). In fact, higher expression levels of many K+ channels have been detected in several cancer tissue types (16,23,31–36), and it has been demonstrated that their overexpression highly increased cell proliferation/migration (14,15). It is not yet clear why K+ channels are modulated in cancer tissues but it has been postulated that they may be regulated during cell dedifferentiation or by high cytokine levels found in tumors (25).
Elevated KvLQT1 levels were detected in our study in all AD tested sub-categories, including AD in situ, lepidic-, acinar-, papillary-, micropapillary-, solid-predominant AD as well as invasive mucinous AD. However, a larger cohort would be necessary to define if the frequency and level of KvLQT1 overexpression change among these different classes. Moreover, patient number was not sufficient to establish a possible correlation between KvLQT1 expression level and cancer severity (e.g., tumor grade or the presence of lymph node metastasis). Nevertheless, correlation between clinical parameters and prognosis outcomes with K+ channel expression has already been reported in other studies (reviewed in ref. 12).
In conclusion, our data demonstrate for the first time, efficient reduction of A549 and H460 cell growth/motility after KvLQT1 inhibition, with clofilium and/or chromanol. Our study highlights increased expression of this channel in human AD samples. It would thus be interesting to further evaluate KvLQT1’s oncogenic potential promise and the potential effect of KvLQT1 inhibitors on lung cancer development in vivo.
Acknowledgements
We thank Dr P. Romeo, pathologist at the CHUM and the tissue bank of the FRQS Respiratory Health Network, for providing human tissue samples. We acknowledge the logistical assistance received from the Research Support Office, CRCHUM. This study was funded by the Canadian Institutes of Health Research (CIHR, MOP-111054), the CRCHUM and Université de Montréal (scholarship to E.B.), the CIHR training program of the Respiratory Health Network and the Fonds de Recherche du Québec - Santé (fellowships to A.G.).
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