IJC

International Journal of Cancer

Epigenetic dysregulation of KCa3.1 channels induces poor

prognosis in lung cancer
Etmar Bulk1, Anne-Sophie Ay2, Mehdi Hammadi2,3, Halima Ouadid-Ahidouch2, Sonja Schelhaas4, Antje Hascher5,
Christian Rohde5,6, Nils H. Thoennissen5,7, Rainer Wiewrodt5, Eva Schmidt5, Alessandro Marra8, Ludger Hillejan8,
Andreas H. Jacobs4,9, Hans-Ulrich Klein10, Martin Dugas10, Wolfgang E. Berdel5, Carsten Mu
€ller-Tidow5,6*
1
and Albrecht Schwab *
1
Institute of Physiology II, University of M€
unster, M€
unster, Germany
2
Laboratory of Cellular Physiology, EA 4667, SFR CAP-SANTE (FED4231), UFR Sciences, University of Picardie Jules Verne, Amiens, 80039, France
3
Inserm U916, Institut Bergonie, Bordeaux, 33076, France
4
European Institute for Molecular Imaging (EIMI), University of Mu €nster, Mu
€nster, Germany
5
Department of Medicine, Hematology, Oncology and Pneumology, University of Mu €nster, Mu
€nster, Germany
6
Department of Medicine, Hematology and Oncology, University Hospital of Halle (Saale), Halle (Saale), Germany
7
Department of Internal Medicine II and Clinic (Oncology Center), University Hospital Hamburg-Eppendorf, Hamburg, Germany
8
Department of Thoracic Surgery, Niels-Stensen Clinics, Ostercappeln, Germany
9
Department of Geriatric Medicine, Johanniter Hospital, Bonn, Germany
10
Institute of Medical Informatics, University of Mu€nster, Mu
€nster, Germany
Cancer Cell Biology

Epigenomic changes are an important feature of malignant tumors. How tumor aggressiveness is affected by DNA methylation
of specific loci is largely unexplored. In genome-wide DNA methylation analyses, we identified the KCa3.1 channel gene
(KCNN4) promoter to be hypomethylated in an aggressive non–small-cell lung carcinoma (NSCLC) cell line and in patient sam-
ples. Accordingly, KCa3.1 expression was increased in more aggressive NSCLC cells. Both findings were strong predictors for
poor prognosis in lung adenocarcinoma. Increased KCa3.1 expression was associated with aggressive features of NSCLC cells.
Proliferation and migration of pro-metastatic NSCLC cells depended on KCa3.1 activity. Mechanistically, elevated KCa3.1 expres-
sion hyperpolarized the membrane potential, thereby augmenting the driving force for Ca21 influx. KCa3.1 blockade strongly
reduced the growth of xenografted NSCLC cells in mice as measured by positron emission tomography–computed tomography.
Thus, loss of DNA methylation of the KCNN4 promoter and increased KCa3.1 channel expression and function are mechanisti-
cally linked to poor survival of NSCLC patients.

Lung cancer is a leading cause of cancer-related death.1 The 5-
year survival rate of patients with non–small-cell lung cancer
(NSCLC), which accounts for 80% of lung cancers, may be as
Key words: KCa3.1, lung cancer, aggressiveness
Additional Supporting Information may be found in the online
version of this article.
*A.S. contributed equally to this work
Grant sponsor: Deutsche Krebshilfe; Grant number: 110261,
110262 and 109666; Grant sponsor: Wilhelm-Sander Stiftung;
Grant number: 2009.041.2; Grant sponsor: Else-Kr€oner Fresenius
Stiftung; Grant number: 2012_A76; Grant sponsor: DAAD
(PROCOPE) (to A.S. and H.O.A.); Grant sponsor: Interdisciplinary
Centre for Clinical Research (IZKF, core unit PIX); Grant sponsor:
Cells-in-Motion Cluster of Excellence (EXC 1003–CiM), University
of M€ unster, Germany
DOI: 10.1002/ijc.29490
History: Received 21 July 2014; Accepted 26 Jan 2015; Online 20
Feb 2015
Correspondence to: Albrecht Schwab, Institute of Physiology II,
University of M€ unster, M€
unster, Germany, Tel.: 149 251 835 5329,
E-mail: aschwab@unimunester.de
low as 10%.2–4 Unfortunately, many patients develop local or
distant metastasis relapse even after complete resection. Thus,
high aggressiveness is an intrinsic feature of many NSCLC
tumors. Adjuvant and palliative chemotherapy are of limited
benefit in NSCLC, and novel therapeutic targets are needed.
Individual NSCLC cancers harbor multiple mutations in
protein-coding genes, most of which cannot be targeted ther-
apeutically.5 In contrast, epigenetic changes might be target-
able. Effector mechanisms, e.g., for metastatic spread, could
be promising therapeutic targets. Metastasis development
originates from subpopulations of tumor cells that acquire
additional features for increased aggressiveness. These fea-
tures depend on changes in gene expression, including that
of ion channels,6–8 presumably often by epigenetic mecha-
nisms rather than metastasis-specific mutations.9–11 Altered
DNA methylation of specific loci as a stable epigenetic mark
is a likely culprit in many instances. DNA hypermethylation
and gene silencing occur in CpG islands and promoters of
tumor suppressors.9 DNA hypomethylation is thought to
occur mainly in intergenic regions, with effects mainly on
genome stability.9 However, the potential pathophysiological
and clinical relevance of DNA hypomethylation of specific

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Bulk et al.
What’s new?
Another possible avenue for thwarting metastasis has been found: the potassium channel KCa3.1. Aggressiveness in lung can-
cer (NSCLC) is linked to methylation of the potassium channel promoter gene. When the promoter is under-methylated, more
of the potassium channel protein is produced, and the cancer cells proliferate and spread more aggressively. The authors also
showed that blocking KCa3.1 stops the cells from dividing and spreading, both in vitro and in mice, suggesting that KCa3.1
could make a promising therapeutic target.
genes and promoters and their mechanistic link to aggres-
siveness of lung cancer cells remain unresolved.
Here, we describe the epigenetic dysregulation of the
KCNN4 gene coding for the calcium-activated KCa3.1 channel
in lung cancer. High-level expression of KCa3.1 channels fre-
quently occurs in aggressive NSCLC cells. KCa3.1 channels
increase migratory activity by elevating the driving force for
Ca21 influx. Their inhibition strongly impairs growth of tumor
xenografts. KCNN4 DNA hypomethylation and KCa3.1 overex-
pression are intimately linked to poor prognosis in NSCLC.
Material and Methods
Cell culture
A549 lung adenocarcinoma cells were cultured at 37  C
under 5% CO2 in Dulbecco’s modified Eagle’s medium (Invi-
trogen, Carlsbad, CA) supplemented with 10% fetal calf
serum. The generation of the highly aggressive A549 cell line,
designated as A549-3R, was described previously.12
Gene expression analyses of published microarray
expression data
Published gene expression data from Stage I and II lung can-
cer patients and normal lung tissue were analyzed
(GSE31210).13 The robust multiarray average normalized
data were analyzed for KCNN4 expression (Affymetrix probe:
204401_at) and its relation to clinical parameters. Prognostic
analysis for overall survival was carried out by log-rank test
or Cox proportional hazard models for univariate and multi-
variate analyses, respectively.
Microarray gene expression analyses of A549 cells
For gene expression analyses of the parental and highly meta-
static A549 cell lines, we used the human Gene 1.0 ST Array
(Affymetrix, Santa Clara, CA). In brief, total RNA was iso-
lated from both A549 cell lines using RNeasyV R Mini Kit
(QIAgen, Hilden, Germany). We used a class comparison
tool in BRB ArrayTools (v4.1.0) to examine differential
expression in both A549 cell lines.
NSCLC patient cohorts
Fresh frozen lung cancer tissue and matching normal lung
tissue were obtained from patients at the time of surgery fol-
lowing informed consent. Samples were snap-frozen in liquid
nitrogen. Tumor cell content of >70% was verified before
samples were prepared for DNA and RNA extraction. Ano-
nymized paraffin sections from NSCLC patients with related
Int. J. Cancer: 137, 1306–1317 (2015) V
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1307
clinical information were obtained from a pathology program
affiliated with the Osterkappeln Hospital. Ethics committee
approval was obtained (4XM€ uller1 and 2006-150-f-S).
Gene expression analyses by quantitative real-time
reverse transcription polymerase chain reaction
Quantitative real-time reverse transcription polymerase chain
reaction (RT-PCR) was carried out to determine gene expres-
sion in A549 cell lines and in fresh frozen patient samples as
described.14 KCa3.1 was detected with the following primers:
forward 50 -CCTTTCAGACACACTTTGGCTGATCC-30 and
reverse 50 -CAGTGCTAAGCAGCTCAGTCAGG-30 . KCa3.1
Cancer Cell Biology
channel expression was normalized to that of GAPDH.
Western blot analysis
Cells were lysed in radioimmunoprecipitation assay buffer (50
mmol/l Tris, 150 mmol/l NaCl, 0.1% sodium dodecyl sulfate,
0.5% sodium deoxycholate, 1% NP-40 and protease inhibitors
from Roche, Germany). Proteins were detected using the follow-
ing antibodies: rabbit anti-KCa3.1 (# AV35098; Sigma, St. Louis,
MO), mouse anti-GAPDH (# MA1-16757; Thermo Scientific,
Waltham, MA) and mouse anti-b-actin (# A2228, Sigma) as
first antibodies, and goat anti-mouse and goat anti-rabbit
(Sigma, St. Louis, MO) antibodies as secondary antibodies.
Immunofluorescence
A549 cells were fixed in 3.5% paraformaldehyde (in
phosphate-buffered saline) and blocked for 30 min with goat
serum. After incubation with the first antibody (anti-KCa3.1
1:1,000) for 1 hr at room temperature, cells were incubated
with the secondary antibody (1:200; goat anti-rabbit labeled
with Alexa Fluor 488, Invitrogen) for 45 min. Cells were cov-
ered in Vectashield (Vector Laboratories, Germany), and
images were taken as described.15
DNA methylation analysis
A549 and A549-3R were analyzed for DNA methylation dif-
ferences by using reduced representation bisulfite sequencing
(RRBS), which was performed and analyzed as described.12
DNA methylation in paraffin-embedded and fresh frozen
matched tumor/normal primary lung cancer specimens was
analyzed using the Infinium HumanMethylation450 K Bead-
Chip (RevB, Illumina, San Diego, CA). DNA was extracted
from paraffin-embedded specimens using 10-mm sections.
Every tenth section was stained with hematoxylin and eosin
and analyzed for tumor cell content. Methylation levels
 1308
(termed b-values) were calculated using the Methylation
Module v1.0 of the GenomeStudio software package (Illu-
mina). Fresh frozen specimens from matched normal and
tumor (n 5 16 pairs) and paraffin-embedded specimens
(n 5 186) were analyzed separately. Methylation levels for
CpG sites located at KCNN4 gene of matched normal and
tumor samples were analyzed using beta regression models
(R package betareg). Methylation levels of paraffin-embedded
samples were imported into SPSS (SPSS, Chicago, IL) and
analyzed with regard to clinical parameters and survival.
siRNA transfection
Transfection of A549 cells was performed using the Amax-
aTM NucleofectorTM technology. The following sequences for
siRNA were used to diminish KCa3.1 expression in both
A549 cell lines: sense 50 -UGUAAAGCUUGGCCACGAAC-30 ,
antisense 50 -GUUCGUGGCCAAGCUUUACA-30 . A nontar-
geting siRNA (siGENOME #1, Dharmacon, Lafayette, CO)
was used as a negative control.
Cancer Cell Biology
Patch clamp experiments
Untransfected or transfected A549 cells were seeded overnight
at a density of 6,000 cells per cm2. Whole cell currents or
membrane potential were recorded with an Axopatch 200 B
patch-clamp amplifier and a Digidata 1322 interface (Molecu-
lar Devices, Sunnyvale, CA). PClamp 9 software (Molecular
Devices) was used for voltage control, data acquisition and
analysis. Resistance of the borosilicate fire-polished pipettes
(HirschmannV R Laborger€
ate) was 3–5 MX. Whole cell currents
were allowed to stabilize for 5 min before being measured.
Currents were recorded during ramps of 250 ms duration
from 2120 to 1 40 mV, starting from a holding potential of
240 mV. The bath and pipette solutions contained (in mmol/
l): bath: NaCl 145, KCl 5, CaCl2 2, MgCl2 2, HEPES 10 and
glucose 5 at pH 7.4 (NaOH); pipette: KCl 150, HEPES 10,
EGTA 0.1 and MgCl2 2 at pH 7.2 (KOH). The free Ca21 con-
centration of the pipette solution amounted to 100 nmol/l.
The cells were continuously superfused with control or test
solutions at room temperature.
Proliferation assay
The proliferation rate of A549 cells was determined after 24,
48 and 72 hrs. A549 cells at a concentration of 3 3 105 cells
were seeded on 35-mm dishes and starved in serum-free
medium for 24 hrs. The next day, growth medium was added
(including different amounts of TRAM-34 or its solvent dime-
thylsulfoxide (DMSO)). Media were replaced after 24 hrs and
48 hrs, and proliferation was analyzed by counting, after the
cells were harvested. All experiments were repeated four times.
Single cell migration analysis
A549 cells were seeded on a collagen matrix (containing 13
RPMI, 10 mmol/l HEPES, 0.02 mg/ml laminin, 0.04 mg/ml
fibronectin, 0.01 mg/ml collagen IV, 0.01 mg/ml collagen III
and 0.8 mg/ml collagen I at pH 7.4) and were allowed to
KCa3.1 channel and poor lung cancer prognosis
adhere for 3 hrs under growth conditions. When applicable,
cells were stimulated with 100 ng/ml epidermal growth factor
(EGF) and/or treated with DMSO or TRAM-34 (10 mmol/l) 10
min before starting the experiment. Images were captured in
10-min intervals for 10 hrs using the ZEISS microscope Axio-
vert 40C (Carl Zeiss, Oberkochen, Germany), linked to a video
camera (Hamamatsu, Germany). The analysis of single cells was
performed as described previously.15 At least three independent
experiments were performed for each experimental condition.
Measurements of the intracellular Ca21 concentration
Measurements of the intracellular calcium concentration
([Ca21]i) of A549 cells were performed by means of ratio imag-
ing with the fluorescent Ca21 indicator fura-2-AM. Cells were
loaded with 3 mmol/l fura-2-AM for 45 min at 37  C. The
experiment started with superfusion of Ringer’s solution (in
mmol/l: NaCl 122.5, KCl 5.4, CaCl2 1.2, MgCl2 0.8, HEPES 10
and D-glucose 5.5 at pH 7.4) on an inverse microscope (ZEISS
Axiovert TV 100, Carl Zeiss; equipped with a 340 objective),
followed by Ca21-free solution (in mmol/l: NaCl 122.5, KCl 5.4,
MgCl2 0.8, HEPES 10 and EGTA 5 at pH 7.4). To release Ca21
from intracellular stores, 1 mmol/l thapsigargin was added until
the basal steady state ratio was reached. Then, the cells were
superfused with Ringer’s solution containing 5 mmol/l Ca21,
followed by Ringer’s solution containing 5 mmol/l Ca21 supple-
mented with 10 mmol/l TRAM-34. To test the contribution of
other K1 channels, cells were depolarized with modified Ring-
er’s solution containing 50 mmol/l KCl (substituting NaCl). Flu-
orescence was excited alternately at 340 nm and 380 nm, using
a monochromator. Data acquisition was controlled by Metafluor
software (Visitron Systems, Puchheim, Germany). All experi-
ments were carried out at room temperature.
Animal experiments
All animal procedures were approved by the animal care com-
mittee of the local government (North Rhine-Westphalia State
Agency for Nature, Environment and Consumer Protection).
A549-3R cells at a concentration of 23 106 cells were
subcutaneously inoculated at three positions into the
shoulder region of 8-weeks-old female NMRI nu/nu mice
(Janvier Labs, Paris, France). Xenograft growth was deter-
mined by caliper measurements. When tumor size reached
100 mm3, treatment was started. 120 mg/kg senicapoc or
DMSO (total volume of 50 ml) was injected intraperitoneally
every second day. During the experimental procedures, body
weight and general health of the mice were monitored.
Positron emission tomography–computed tomography
imaging and analysis
Proliferation of tumors was assessed by 18F-fluorodeoxy thy-
midine (FLT) positron emission tomography (PET) before
and 7 days after treatment initiation. Radiotracer synthesis
and PET and computed tomography (CT) image acquisition
and coregistration were performed as described previously.16
The anatomical information of the CT images was used to
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Bulk et al. 1309

Cancer Cell Biology

Figure 1. DNA hypomethylation of the KCNN4 promoter is associated with increased KCa3.1 expression in aggressive A549 cells. (a) RRBS of A549
cells. The three major regions covered by RRBS, phylogenetic conservation and major regulatory tracks from the UCSC browser are depicted. Transcrip-
tional start site (TSS) of the KCNN4 gene and the start of the first exon are indicated. The relevant CpGs are indicated below the tracks. (b) mRNA
expression of KCa3.1 channels in A549 cells was analyzed by real-time RT-PCR. (c, d) Western blot of KCa3.1 protein expression in A549 cells and its
quantification (n 5 5). *p<0.05. (e) Immunofluorescence of KCa3.1 channels in A549 cells. Each (green) dot indicates the localization of a single KCa3.1
channel protein. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

delineate the contours of the tumors and determine the
respective tumor volumes. 18F-FLT uptake was calculated as
maximal percentage injected dose (%IDmax/ml).
Statistical analysis
Experimental data are shown as mean 6 standard error of
mean. The mean values of two groups were compared by the
Student’s t-test. Mean values of more than two groups were
tested using one-way analysis of variance. A p-value <0.05
was considered significant.
Results
Hypomethylation of the KCNN4 promoter and increased
expression of the KCa3.1 channel in highly aggressive lung
adenocarcinoma cells
We recently developed a model of human lung adenocarci-
noma with increased aggressiveness.12 Genome-wide DNA
methylation analysis revealed that many of the altered pro-
moters and genes showed marked DNA hypomethylation.12
The comparison of DNA methylation with microarray gene
expression data revealed several genes with decreased pro-
moter methylation and increased mRNA expression. One of
the top-ranking genes was KCNN4 that encodes the Ca21-
activated K1 channel KCa3.1, which was expressed 1.95 times
higher in A549-3R than in A549-0R (p 5 0.00019; GSE52143).
Three regions upstream and downstream of its transcriptional
start site showed significant loss of DNA methylation in the
more aggressive A549-3R cell line (Fig. 1a). The most striking
difference in DNA methylation was observed within a 100-bp
region 2,000 bp upstream of the transcriptional start site.
This highly conserved and CpG-rich region bears important
DNA regulatory activity as evidenced by the presence of
DNAse clusters, frequent transcription factor binding sites
and H3K27 acetylation in the Encode project (Fig. 1a).

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 1310 KCa3.1 channel and poor lung cancer prognosis
Cancer Cell Biology

Figure 2. KCa3.1 expression is increased in aggressive A549 cells and in tumor samples of NSCLC patients. (a) Boxplot of microarray mRNA
expression data from GSE31210 from 226 NSCLC patients and 20 normal lung samples. (b) mRNA expression levels of KCa3.1 channels
from 15 primary NSCLC tumor samples (matched to normal tissue samples from the same patients) were analyzed by real-time RT-PCR. The
ratios of tumor/matched normal lung tissues are indicated. (c) Methylation status of the KCNN4 gene for 16 matched (tumor vs. normal)
DNA samples from NSCLC. The seven CpGs within the promoter region (indicated in Fig. 1a) are numbered. (d) Methylation levels (CpG#3)
of 16 matched (tumor vs. normal) DNA samples from NSCLC patients with regard to the clinical stage. [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]

Real-time RT-PCR (Fig. 1b) confirmed that DNA hypome-
thylation was associated with the induction of KCa3.1 mRNA
expression in the more aggressive A549-3R cell line. This cor-
responded with increased protein expression as revealed by
immunoblot (Figs. 1cd) and by immunofluorescence staining
(Fig. 1e). Importantly, KCa3.1 channel expression was also sig-
nificantly increased in samples from NSCLC patients. Figure
2a compares mRNA expression data from published microar-
ray analyses (GSE31210) in normal lung and NSCLC tissue.
Similar findings were obtained by real-time RT-PCR in our
own cohort of primary NSCLC tumors (Fig. 2b). In most of
the 15 primary tumor samples from NSCLC patients, the
KCa3.1 mRNA expression was higher than in the matched
normal tissue samples from the same patients.
KCNN4 hypomethylation and increased Kca3.1 channel
expression predict poor prognosis of NSCLC patients
We next analyzed DNA methylation of the KCNN4 gene in
16 matched NSCLC tumor and normal lung specimens. The
KCNN4 promoter region (Fig. 2c) was significantly DNA
hypomethylated in tumor samples (p < 0.000001 for all seven
CpG sites within promoter/first exon). In contrast, increased
DNA methylation in NSCLC samples was observed in the
gene body, a situation which is often associated with increased
mRNA expression.17 In Figure 2d, we present the differences
of the methylation of CpG#3 (CG14066757) in normal and
tumor tissue samples. A significant hypomethylation was
observed in tumor samples (p < 0.001; beta regression), which
tended to be more pronounced in advanced stages. An inverse
correlation between methylation and expression was observed
in matched (normal/tumor) specimens from NSCLC patients
(Pearson’s correlation coefficient r 5 20.70).
We next analyzed 186 samples of patients with NSCLC
using the 450K DNA methylation arrays, focusing on the
seven CpGs located within the KCNN4 promoter region
(Supporting Information Tables S1 and S2). CpG locations
are indicated in Figure 1a. Gender (145 male and 41 female
patients), clinical performance status (Eastern Cooperative
Oncology Group performance status), age or smoking status
was not associated with altered DNA methylation of the
CpGs located in the KCNN4 promoter. This also applied for
the clinical stage and the histological type. The clinical and

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Bulk et al. 1311

Cancer Cell Biology

Figure 3. Loss of methylation in the KCNN4 gene and increased KCa3.1 channel expression predict a poor outcome for NSCLC patients. (a)
DNA methylation of the KCNN4 promoter. Patients were dichotomized into two groups with low and high DNA methylation levels of the
KCNN4 promoter. The Kaplan–Meier plot shows progression-free survival for lung adenocarcinoma patients who underwent complete resec-
tion (n 5 118). (b) Overall survival analysis for patients with low versus high DNA methylation levels of the KCNN4 gene promoter. (c) A multi-
variate analysis (Cox-regression) for overall survival of lung cancer patients was performed that included KCNN4 promoter DNA methylation.
Hazard ratios with 95% confidence intervals are indicated as well as p-values. (d) Kaplan–Meier survival chart for progression-free survival of
204 lung adenocarcinoma patients with regard to KCa3.1 expression. mRNA levels were dichotomized, and high- versus low-level expressions
were analyzed. Microarray data were obtained from GSE31210. (e) Overall survival analysis with regard to low/high KCa3.1 (KCNN4) mRNA
expression in the same patient cohort. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

pathological characteristics of the patients are listed in the
Supporting Information Table S3.
We next investigated potential association of CpG methyl-
ation level and progression-free survival. The survival analysis
included all patients with a non-squamous cell histology
(n 5 118) because the A549 cells were also of adenocarci-
noma origin. The relative DNA methylation level of the
KCNN4 region in lung adenocarcinoma was highly variable,
ranging from 7% to 93% (Supporting Information Table S4).
Methylation levels of CpG2 (CG01424145) positively corre-
lated with that of other neighboring CpGs (Supporting Infor-
mation Table S5), and thus, this CpG2 was chosen for
further analyses. Methylation levels of CpG2 (CG01424145)
were dichotomized using the median methylation level (30%)
as threshold. The methylation status was strongly associated
with progression-free survival. Patients experienced a median
progression-free survival of 756 days (2.1 years) when the
DNA methylation of CpG2 in tumor biopsies was low,
whereas 60% of the patients were disease-free even after 8
years, when DNA methylation was high (p 5 0.002; Fig. 3a).
The corresponding values after 5 years were 37% and 66%,
respectively. Accordingly, overall survival was significantly
decreased for patients whose tumor samples showed
decreased DNA methylation at CpG2 (p 5 0.001). Median
survival was 923 days (2.5 years) for those with low DNA
methylation, whereas 80% of patients whose tumor samples
had high DNA methylation were still alive at this time point
(Fig. 3b). The corresponding 5-year survival rates were 37%
and 72% for patients with low and high DNA methylation of
their tumors, respectively. The association of CpG
(cg01424145) with progression-free and overall survival was
also evident when only patients with Stage I and II cancer
were analyzed (Supporting Information Fig. S1).
The association of CpG2 (cg01424145) with survival was
evident in univariate and in multivariate analyses. A Cox-
regression analysis included CpG2 (cg01424145) as well as
Eastern Cooperative Oncology Group performance status,
smoking status, grading, age, tumor stage, resection status
and gender. Loss of DNA methylation of the CpG2
(cg01424145) was an independent predictor of shortened

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 1312
progression-free survival, with a hazard ratio of 2.24
(p 5 0.018). In a similar Cox-regression analysis for overall
survival, DNA hypomethylation predicted increased risk of
poor survival (Fig. 3c hazard ratio 2.19, p 5 0.031, Wald test).
Analysis of published mRNA expression data from another
patient cohort (GSE31210) also revealed an association of
KCa3.1 with survival in NSCLC. We focused on patients with
Stage I or II adenocarcinoma who had undergone complete
resection with negative margins (n 5 204). Tumor samples of
patients with high level of KCa3.1 mRNA expression (high and
low expression dichotomized at the median) experienced
shortened time to first progression (p 5 0.00001; Fig. 3d) and
reduced overall survival (p 5 0.003; Fig. 3e). This was evident
in both univariate and multivariate analyses. The multivariate
analyses included KCNN4 gene expression (dichotomized at
median), stage, gender and smoking history.
Thus, DNA hypomethylation correlated with increased
KCa3.1 channel mRNA and protein expression in cell lines
and in primary tumor samples of NSCLC patients. DNA
hypomethylation of the KCa3.1 channel promoter and
Cancer Cell Biology
increased mRNA expression determined in independent
patient cohorts were associated with a poor prognosis in
NSCLC. We next assessed the functional impact of elevated
KCa3.1 channel expression on NSCLC cell aggressiveness.
KCa3.1 channels are functionally active in highly
metastatic A549 cells
We performed patch clamp experiments to identify functional
KCa3.1 channel expression (Fig. 4a, upper panels). Using the
KCa3.1channel activator 1-EBIO (200 mmol/l), KCa3.1 currents
were activated in both A549 cell strains that were transfected
with the scrambled control siRNA. KCa3.1 currents were more
than twice as high in aggressive A549-3R cells than in the
parental A549-0R cells. The activated KCa3.1 current had a
reversal potential of 279.1 6 1.6 mV (n 5 20) in A549-3R and
276.0 6 2.4 mV (n 5 15) in A549-0R cells, respectively. The
activation of KCa3.1 currents was completely blocked with the
KCa3.1 channel blocker TRAM-34. In cells transfected with
siRNA against KCa3.1 channels, 1-EBIO essentially failed to
activate KCa3.1-mediated currents (Fig. 4a, lower panels and
Fig. 4b). At 140 mV, the amplitude of 1-EBIO-sensitive cur-
rents was 1,605 6 228 pA in highly metastatic A549-3R cells
(n 5 20) and 778 6 152 pA in A549-0R cells with low meta-
static potential (n 5 15; Fig. 4b). siRNA against KCa3.1 reduced
the amplitude to 542 6 111 pA in the A549-3R (n 5 20) and
to 170 6 30 pA in the A549-0R cells (n 5 15).
Figure 4c demonstrates that the resting membrane poten-
tial of the pro-metastatic A549-3R cells was more negative
than that of the parental cells. Hyperpolarization of the rest-
ing membrane potential with 200 mmol/l 1-EBIO was largely
blunted in cells transfected with siRNA against KCa3.1 chan-
nels (Fig. 4c). Taken together, highly metastatic A549-3R cells
expressed more functional KCa3.1 channels in the plasma
membrane than the parental A549-0R cells, leading to a
hyperpolarized membrane potential in the former ones.
KCa3.1 channel and poor lung cancer prognosis
KCa channel-dependent hyperpolarization of the mem-
brane potential can have an important impact on the intra-
cellular Ca21 concentration of nonexcitable cells by
increasing the electrical driving force for Ca21 entry.18
Because activation of KCa3.1 channels by 1-EBIO hyperpolar-
ized the membrane potential of A549-3R cells (Fig. 4c), we
investigated whether KCa3.1 channel activity also impacted
on [Ca21]i of A549 cells. Initially, we measured [Ca21]i
under baseline conditions. Consistent with the differences of
the membrane potential, [Ca21]i was higher in prometastatic
A549-3R than in parental A549-0R cells (43.2 6 0.5 nmol/l
versus 30.3 6 0.3 nmol/l; n 5 20 cells each; data not shown).
We exposed the cells to thapsigargin (1 mmol/l) in Ca21-free
solution to deplete intracellular Ca21 stores. On adding 5
mmol/l Ca21, [Ca21]i rose immediately in both cell lines and
remained almost stable (Figs. 4de). At this point, the 5
mmol/l Ca21-containing solution was supplemented with 10
mmol/l TRAM-34. In both A549 cell lines, the [Ca21]i ratio
dropped by 22% (Figs. 4df A549-0R: Dratio 5 21.15 6 0.10;
A549-3R: Dratio 5 21.25 6 0.13; n > 34 cells). Finally, we
superfused the cells with a modified Ringer’s solution con-
taining 50 mmol/l KCl. This led to a further reduction in
[Ca21]i (Figs. 4df A549-0R Dratio 5 21.27 6 0.11 and A549-
3R Dratio 5 21.17 6 0.08), indicating that A549 cells express
other K1 channels as well.19
KCa3.1 channel blockade reduces A549 cell proliferation
and migration
Next, we determined the role of KCa3.1 channels for tumor
cell growth. Proliferation of A549 cells was dose-dependently
inhibited by TRAM-34. Both cell strains responded with a
slight inhibition of their growth rate when exposed to 10
mmol/l TRAM-34 (Fig. 5a). The treatment with 30 mmol/l
TRAM-34 inhibited proliferation of both cell lines more
effectively. Overall, DMSO-exposed A549-0R and A549-3R
cells grew three times and four times faster, respectively,
compared with cells treated with TRAM-34.
We applied live cell imaging to monitor migration of
A549 cells. As shown in Figures 5ef, the maximum speed was
reached after 7 hrs of stimulation. The cell paths of single
cells, normalized to common starting points are presented in
Figure 5b. The more aggressive A549-3R cells migrated over
a longer distance compared with their parental cells
(117 6 14 versus 75 6 8 mm, Figure 5c, Supporting Informa-
tion Videos S1 and S2). When treated with the KCa3.1 inhibi-
tor TRAM-34 (10 mmol/l), the migration of A549-3R cells
resembled that of A549-0R cells (Supporting Information
Video S3). TRAM-34 reduced the translocation of A549-3R
cells by 33% (78 6 9 mm), whereas that of the parental A549-
0R cells was reduced only by 25% (56 6 6 mm). Furthermore,
during the last 3 hrs of the experiment, the speed of highly
metastatic A549-3R cells was reduced from 0.55 6 0.03 to
0.42 6 0.04 mm/min when treated with 10 mmol/l TRAM-34
(224%, Figs. 1f). The speed of A549-0R cells was only mar-
ginally reduced from 0.47 6 0.03 to 0.42 6 0.03 mm/min with
Int. J. Cancer: 137, 1306–1317 (2015) V
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Cancer Cell Biology

Figure 4. Functional expression of KCa3.1 channels in A549 cells. (a) Whole cell currents were elicited by voltage ramps from 2120 to 140 mV.
In control cells (si-ctrl, upper panels), the activation of KCa3.1 channels with 1-EBIO (200 mmol/l) is reversed by applying the KCa3.1 blocker
TRAM-34 (10 mmol/l). KCa3.1 channel activation is largely attenuated in cells transfected with siRNA against KCa3.1 channels (si-KCa3.1, lower
panel). (b) Summary of the experiments shown in (a) (n 5 15 for A549-0R and n 5 20 for A549-3R cells). (c) Membrane potential of A549 cells.
The resting cell membrane potential (Ctrl) of A549-3R cells is more hyperpolarized than that of A549-0R cells (n 5 15 for A549-0R and n 5 20 for
A549-3R cells). *p < 0.05. (d, e) Ca21 measurements with A549 cells. The pictures depict representative original recordings made with both A549
cell strains. DTRAM represents the change in the intracellular Ca21 concentration in response to the application of TRAM-34, DKCl corresponds to
the change elicited by the application of 50 mmol/l KCl. (f) Influence of TRAM-34 and 50 mmol/l KCl on the intracellular Ca21 concentration in
both A549 cell strains. The bars correspond to the mean values of DTRAM and DKCl from n 5 34 cells for A549-0R and n 5 40 for A549-3R cells.
[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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 1314 KCa3.1 channel and poor lung cancer prognosis
Cancer Cell Biology

Figure 5. Proliferation and migration of A549 cells are inhibited by KCa3.1 channel blockade. (a) A549 cells were treated with TRAM-34 (10,
20 and 30 mmol/l) for 24–72 hrs. *p < 0.05. (b) Cells were migrating on a collagen-based matrix. Ten minutes before the start of the experi-
ment, 100 ng/ml EGF and 10 mM TRAM-34 or DMSO were added. Trajectories of individual cells normalized to a common starting point.
The circle represents the mean distance covered during the course of the experiment. (c) Statistical evaluation of migration experiments for
distance of translocation. (d) Statistical evaluation of migration experiments for speed. *p<0.05. (e) Migration speed plotted as a function
of time for parental A549 cells. EGF (100 ng/ml) was added at t 5 0. Mean values of three independent experiments are shown. (f) Migra-
tion speed was plotted for the highly aggressive A549 cells.

TRAM-34 (211%, Figs. 5e). In both cell types, KCa3.1 block-
ade inhibited translocation more effectively than the speed of
migration. This is an indication for the involvement of
KCa3.1 channels in controlling the directionality of migrating
A549 cells.
KCa3.1 channel inhibition results in reduced proliferation
of A549 xenografts in vivo
Finally, we analyzed the impact of KCa3.1 channel inhibition
on tumor growth in vivo. A549-3R tumor-bearing mice were
treated with the KCa3.1 channel blocker senicapoc

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Cancer Cell Biology

Figure 6. KCa3.1 blocker senicapoc impairs proliferation of lung cancer xenografts. A549-3R tumor-bearing mice were treated with senicapoc
every day. (a) Growth of n 5 12 senicapoc-treated and n 5 14 DMSO-treated tumors was determined by caliper measurements. (b) 18F-FLT PET-
CT was performed before and 7 days after treatment initiation to assess proliferation of the tumors. CT allowed for delineation of the tumor
margins (marked with a white line) and determination of the tumor volume. Transverse slices of PET and CT images of representative mice are
depicted here. Because of nonuniform tumor growth, not all slices show three tumors. Scale bar 5 1 mm. (c) n 5 12 senicapoc-treated and
n 5 9 DMSO-treated tumors were analyzed quantitatively with respect to variations in 18F-FLT uptake and tumor size determined by CT.
*p<0.05. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

(structurally similar to TRAM-34, but with a greatly
increased metabolic stability).20,21 Tumor growth was signifi-
cantly inhibited after 4 days of treatment when compared
with DMSO-treated mice (Fig. 6a relative change in size as
assessed by caliper (day 4/day 0): senicapoc: 0.95 6 0.04;
DMSO: 1.21 6 0.07. p 5 0.005). Further, proliferation of the
tumors was reduced as assessed by PET using 18F-FLT as in
vivo proliferation marker (Figs. 6bc relative change in 18F-
FLT uptake (day 7/day 0): senicapoc: 0.85 6 004; DMSO:
1.10 6 0.07; p 5 0.0041). Accumulation of this thymidine
analog has been shown to reflect the proliferation rate of the
respective tissue.22 CT provides more precise anatomical
information on tumor volume than caliper measurements,
and it confirmed the aforementioned results: senicapoc
strongly impaired A549 xenograft growth (relative change in
size as assessed by CT (day 7/day 0): senicapoc: 1.44 6 0.04;
DMSO: 1.96 6 0.17; p 5 0.0061).
Discussion
The main findings of our study are as follows: Loss of DNA
methylation in the promoter region of the KCNN4 gene is a
frequent feature of NSCLC. Promoter hypomethylation
occurred in cells with increased aggressiveness as well as in
primary NSCLC tumors from patients. DNA hypomethyla-
tion was associated with increased expression and function of
the KCa3.1 channel as well as enhanced metastatic potential
and proliferation. Importantly, KCNN4 hypomethylation and
increased expression of the KCa3.1 channel were strong and
independent predictors for poor survival in NSCLC patients.
Functionally, DNA hypomethylation of the promoter and
increased expression of KCa3.1 channels are linked to
increased calcium influx, representing a direct mechanistic
link between epigenetic alterations and calcium-dependent
tumor aggressiveness.23 Thus, inhibition of KCa3.1 channels
in vivo in an experimental mouse model resulted in reduced
NSCLC proliferation, and hence decreased tumor volume as
measured by 18F-FLT PET/CT.
Metastatic spread is a major reason for cancer-related death.
Despite intensive genome sequencing efforts, no metastasis-
specific mutations have been discovered.11 Intriguingly, epige-
netic changes are likely to be major causal contributors for
metastatic capability. The resulting functional changes leading
to a metastatic phenotype of cancer cells might be easier to tar-
get than the mutations that provide the genetic landscape of
cancers. The molecular characterization of NSCLC has been
advanced with improved patient stratification that takes into

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 1316 KCa3.1 channel and poor lung cancer prognosis

account the presence or absence of driver mutations in impor-
tant signaling pathways.24,25 Most of these pathways, however,
cannot be targeted easily by current therapies.
In contrast, ion channels and other ion transport proteins
are well-established and validated therapeutic targets in multiple
diseases (e.g., cardiovascular, renal and neuropsychiatric dis-
eases). Ion channels could also be very attractive therapeutic
targets in oncology because they are usually blocked from their
extracellular side so that drug resistance due to the expression
of multidrug resistance proteins appears unlikely.26 Indeed,
recent data indicate that membrane proteins involved in ion
transport also play crucial roles in tumor progression because
they contribute essentially to all hallmarks of cancer.7,23,27–31
They are expressed aberrantly in almost all tumor types.32 Being
central players in proliferation, apoptosis, angiogenesis, cell–
matrix interaction and migration or invasion, ion channels
shape the aggressive phenotype of metastatic cancer cells.6,8
Thus, ion channels are potentially new therapeutic, diagnostic
and/or prognostic targets in oncology. The Ca21-sensitive K1
channel KCa3.1 is upregulated in many tumors, including pan-
Cancer Cell Biology
provided evidence that NSCLC proliferation can be inhibited
with a KCa3.1 channel blocker, senicapoc, which had already
been tested clinically.42
Our experiments indicated that KCa3.1 channels contrib-
ute to the aggressiveness of NSCLC cells by regulating prolif-
eration and migration as in other tumor cells.43 Tumor
growth of aggressive NSCLC cells in mice could be inhibited
almost completely within the first week of treatment using
the KCa3.1 blocker senicapoc. The impact of KCa3.1 channels
on [Ca21]i, by setting the membrane potential, is a likely
explanation for the dual role in proliferation and migration.
It is noteworthy that KCa3.1 channels are required for migra-
tion of EGF/fetal calf serum-stimulated cells but not for that
of unstimulated cells. This raises the question whether other
ion channels upstream to KCa3.1 channels are required to
provide them with Ca21 necessary for their activation.
TRPC1 could be a potential candidate channel. In A549 cells,
TRPC1 channels are activated by EGF, and they are central
constituents of the EGFR signaling cascade.44 Blocking
KCa3.1 channels impairs Ca21 influx and thereby EGF signal-

creas, prostate and breast cancer33–35 as well as melanoma,36
where it plays important roles in proliferation and migration.
However, so far, there is only very little information on the role
of K1 channels in NSCLC progression (e.g., Refs. [19 and 27).
KCa3.1 is one of the channels whose expression correlates
with tumor grade and metastatic spread, cell cycle progres-
sion and cell proliferation in several cancer types.34,35,37–39
Another important observation is that this channel is
expressed in nearly all migrating cells.40 Its activity is
required for efficient cell migration.41 In view of the above,
the marked dependence of NSCLC patient outcome on the
expression of KCa3.1 channels led us to hypothesize that they
contribute to disease progression and to the metastatic behav-
ior of NSCLC cells. If this were the case, its elevated expres-
sion in NSCLC would not only be a predictor of poor
outcome. Then, abundantly expressed KCa3.1 channels could
also constitute a therapeutic target with clinical potential.
This idea is also supported by our in vivo experiments that
ing. Because our experiments showed that KCa3.1 channel
blockade leads to decreased Ca21 influx into A549 cells,
impaired migration could be a consequence of a disturbed,
Ca21-dependent disassembly of focal adhesions.45–47 In such
a scenario, cells would tend to migrate “on the spot.”
Taken together, our experiments provide evidence for
increased KCa3.1 channel expression by epigenetic mecha-
nisms in NSCLC. DNA hypomethylation as well as high-level
mRNA expression are independently associated with poor
survival. We provide functional evidence that KCa3.1 chan-
nels contribute to the aggressive behavior of NSCLC cells
and, thereby, to the poor prognosis of NSCLC patients with
high channel expression.
Acknowledgements
The authors are grateful for the excellent technical assistance of Sarah Sargin,
Stefanie Bouma and Christine B€atza. TRAM-34 and Senicapoc were kind
gifts from Heike Wulff, Davis, CA, USA.

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