Skip to main content
NCBI home page
The Journal of Physiology logoLink to The Journal of Physiology
. 2011 Sep 12;589(Pt 22):5349–5359. doi: 10.1113/jphysiol.2011.214635

The potassium–chloride cotransporter 2 promotes cervical cancer cell migration and invasion by an ion transport-independent mechanism

Wei-Chun Wei 1, Colin J Akerman 2, Sarah E Newey 2, Jiliu Pan 1, Nicholas W V Clinch 1, Yves Jacob 3, Meng-Ru Shen 4, Robert J Wilkins 1, J Clive Ellory 1
PMCID: PMC3240877  PMID: 21911617

Non-technical summary

K+–Cl− cotransporters (KCCs) play a fundamental role in epithelial cell function, both in the context of ionic homeostasis and also in cell morphology, cell division and locomotion. Unlike other ubiquitously expressed KCC isoforms, expression of KCC2 is widely considered to be restricted to neurons. Here we report a novel finding that KCC2 is widely expressed in several human cancer cell lines including the cervical cancer cell line (SiHa). Our data establish that KCC2 expression and function is not restricted to neurons and that KCC2 serves to increase cervical cancer progression via an ion transport-independent mechanism.

Abstract

Abstract

K+–Cl− cotransporters (KCCs) play a fundamental role in epithelial cell function, both in the context of ionic homeostasis and also in cell morphology, cell division and locomotion. Unlike other ubiquitously expressed KCC isoforms, expression of KCC2 is widely considered to be restricted to neurons, where it is responsible for maintaining a low intracellular chloride concentration to drive hyperpolarising postsynaptic responses to the inhibitory neurotransmitters GABA and glycine. Here we report a novel finding that KCC2 is widely expressed in several human cancer cell lines including the cervical cancer cell line (SiHa). Membrane biotinylation assays and immunostaining showed that endogenous KCC2 is located on the cell membrane of SiHa cells. To elucidate the role of KCC2 in cervical tumuorigenesis, SiHa cells with stable overexpression or knockdown of KCC2 were employed. Overexpression of KCC2 had no significant effect on cell proliferation but dramatically suppressed cell spreading and stress fibre organization, while knockdown of KCC2 showed opposite effects. In addition, insulin-like growth factor 1 (IGF-1)-induced cell migration and invasiveness were significantly increased by overexpression of KCC2. KCC2-induced cell migration and invasion were not dependent on KCC2 transport function since overexpression of an activity-deficient mutant KCC2 still increased IGF-1-induced cell migration and invasion. Moreover, overexpression of KCC2 significantly diminished the number of focal adhesions, while knockdown of KCC2 increased their number. Taken together, our data establish that KCC2 expression and function are not restricted to neurons and that KCC2 serves to increase cervical tumourigenesis via an ion transport-independent mechanism.

Introduction

K+–Cl− cotransporters (KCCs) mediate the electroneutral transport of Cl− coupled to K+ across the plasma membrane. Four KCC isoforms (KCC1–KCC4) have been identified and are encoded by the SLC12 gene family, specifically SLC12A4 to SLC12A7. KCCs have been implicated in several cellular functions, such as cell volume regulation, epithelial ion transport, and osmotic homeostasis (Lauf & Adragna, 2000; Song et al. 2002; Adragna et al. 2004). Recent studies reveal the emerging roles of KCCs in tumour biology. It has been shown that cervical carcinogenesis is accompanied by the up-regulation of mRNA transcripts of KCC1, KCC3 and KCC4 and KCC transport activity (Shen et al. 2000, 2003). KCC1, the ‘housekeeper’ isoform, is considered to be osmotically sensitive and involved in volume regulation, whereas KCC3 promotes cell growth by modulating cell cycle progression (Shen et al. 2001). KCC3 also promotes epithelial–mesenchymal transition (EMT), a key event in carcinogenesis, by down-regulating E-cadherin/β-catenin complex formation (Hsu et al. 2007). Motor-dependent membrane trafficking of KCC4 contributes to cell invasion. In addition to ion transport, KCC4 functions as a plasma membrane scaffold protein with ezrin in the assembly of the cytoskeletal reorganization complexes, which also facilitates cancer cell invasion (Chen et al. 2009).

Unlike other widely expressed KCC isoforms, KCC2 has been considered as neuron-specific. The expression of KCC2 appears to be tightly restricted to the central nervous system (Payne et al. 1996; Lu et al. 1999; Williams et al. 1999). Within neurons, KCC2 is essential in permitting fast hyperpolarising post-synaptic responses to inhibitory neurotransmitters GABA and glycine (Rivera et al. 1999). However, there is some evidence to suggest that KCC2 is expressed in non-neuronal cells as well. Using RT-PCR, KCC2 mRNA has been detected in rat vascular smooth muscle cells, mouse testis, human osteoblasts and human lens epithelial cells (Di Fulvio et al. 2001; Brauer et al. 2003; Uvarov et al. 2007; Lauf et al. 2008). These studies imply that KCC2 is not simply a neuronal-specific KCC isoform.

The aberrant differentiation programmes in cancer cells allow them to express differentiation markers beyond their tissue of origin (Miyoshi et al. 2002; Motel et al. 2009; Quirós et al. 2009). One recent study shows that human breast cancer cell lines co-express epithelial and neuronal differentiation markers (Zhang et al. 2010). Moreover, some potassium channels such as Eag1 and TREK-1 have been found aberrantly expressed in tumour cells and act as oncological targets (Pardo & Stühmer 2008; Voloshyna et al. 2008). Thus, the aim of the present study was to investigate whether KCC2 is expressed in human epithelial cancer cells. Here, we report that KCC2 is widely expressed in the membrane of human cervical cancer cells. Overexpression of KCC2 enhanced cell migration and invasiveness induced by insulin-like growth factor 1 (IGF-1). Furthermore, IGF-1-induced cell migration and invasion was still apparent following overexpression of an activity-deficient mutant form of KCC2. This study demonstrates that KCC2 is expressed in non-neuronal cancer cells and regulates cancer migration and invasion through a mechanism that is independent of ion transport.

Methods

Cell cultures, plasmids and transfection

C20/A4, DAOY, SiHa, HeLa, HCT116 and MCF7 cell lines were maintained in Dulbecco's modified Eagle's medium (Gibco-BRL) supplemented with 10% fetal bovine serum, 2 mm l-glutamine, 100 units ml−1 penicillin and 100 μg ml−1 streptomycin under 5% CO2 at 37°C. Plasmids containing the full-length cDNA of human KCC2 (Origene), Y1087D mutant KCC2 (a generous gift from Dr Eric Delpire, Vanderbilt University, Nashville, TN, USA), KCC2 shRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) or control scrambled shRNA (Santa Cruz) were transfected into SiHa cells by Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Stable clones were selected by 750 μg ml−1 G418 (Sigma) or 1 μg ml−1 puromycin (Invitrogen) for 14 days. The Y1087D mutant KCC2 was cloned in a bidirectional pCS2 vector (containing two CMV promoters, one on each strand). One multiple cloning site contained EGFP and the other multiple cloning site contained the Y1087D mutation of KCC2.

RNA interference

The REST (repressor element 1-silencing transcription factor) siRNA pool of three duplexes (5′-CGAGUCUACAAGUGUAUCATT-3′ (sense) and 5′-UGAUACACUUGUAGACUCGTT-3′ (antisense); 5′-GCAGACAGGAAGCAAUUCATT-3′ (sense) and 5′-UGAAUUGCUUCCUGUAUGCTT-3′ (antisense) and 5′-GAAGGAACCUGUUAAGAUATT-3′ (sense) and 5′-UAUCUUAACAGGUUCCUUCTT-3′ (antisense)) targeting exons 2 and 5 of human REST (NM_005612) were purchased from Santa Cruz Biotechnology. Scrambled siRNA was used as a negative control. Different dosages (50 nm or 100 nm) of REST or scrambled siRNA were transfected into SiHa cells by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

RNA extraction and RT-PCR

Total RNA from cultured cells was extracted using RNeasy MiniKit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocols. For RT-PCR, first-strand cDNA was synthesized from 1 μg of total RNA with an oligo-dT primer and the moloney murine leukaemia virus reverse transcriptase (Promega Corp., Madison, WI, USA). For KCC1, forward (5′-TGGGACCATTTTCCTGACC-3′) and reverse (5′-CATGCTTCTCCACGATGTCAC-3′) primers were used to amplify a 422 bp fragment from human KCC1. For KCC2, forward (5′-CTACCTGGGCACTACCTTTG-3′) and reverse (5′-CCAGGAAGACAAGGGCAAAC-3′) primers were used to amplify a 230 bp fragment from human KCC2. For KCC3, forward (5′-CTATCCTTGCCATCCTGACC-3′) and reverse (5′-GCAGCAGT00TGTCACTCGAAC-3′) primers were used to amplify a 1099 bp fragment from human KCC3. For KCC4, forward (5′-GACTCGTTTCCGCAAAACC-3′) and reverse (5′-AGAGTGCCGTGATGCTGTTGG-3′) primers were used to amplify a 783 bp fragment from human KCC4. For REST, forward (5′-GAATCTGAAGAACAGTTTGTGCAT-3′) and reverse (5′-TTTGAAGTTGCTTCTATCTGCTGT-3′) primers were used to amplify a 627 bp fragment from human REST. For GAPDH, forward (5′-GTGAAGGTCGGAGTCAACGGATTT-3′) and reverse (5′-CACAGTCTTCTGGGTGGCAGTGAT-3′) primers were used to amplify a 555 bp fragment from human GAPDH.

Surface biotinylation, collection of cell lysates and Western blotting

Cells were rinsed three times with ice-cold phosphate-buffered saline (PBS). Cell surface proteins were biotinylated for 30 min with 100 μM biotin (EZ-link NHS-SS-biotin; Pierce) diluted in PBS. Unbound biotin was quenched by incubation (5 min at 4°C) with PBS containing 100 μm lysine. Total cell lysates were harvested in RIPA lysis buffer (Sigma) containing protease inhibitor cocktail (Roche). One milligram of total protein was precipitated with 70 μl of UltraLink immobilized NeutrAvidin beads (Pierce) overnight at 4°C by rotation. The beads were washed three times in cold RIPA buffer supplemented with protease. Biotinylated proteins (membrane proteins) were eluted from the beads by boiling in sample buffer. The lysates were analysed by Western blot with antibodies against KCC1 (Everest Biotech Ltd, Upper Heyford, UK), KCC3 (Abnova Corp., Taipei City, Taiwan), KCC4 (was generated with the epitope of AERTEEPESPESVDQTSPT), KCC2 (Millipore, Billerica, MA, USA), GLUT-1 (Chemicon, Temecula, CA, USA), REST (Chemicon), α-tubulin (Santa Cruz) and visualized using enhanced chemiluminescence (ECL) system (GE Healthcare, Little Chalfont, UK).

Immunofluorescence

Cells were washed three times with ice-cold PBS, fixed with 4% buffered paraformaldehyde for 10 min at room temperature and then permeabilized with 0.5% Triton X-100 in PBS for 10 min at room temperature. Fixed cells were blocked with blocking solution (Thermal Scientific, Welwyn Garden City, UK) for 30 min at room temperature and then incubated with primary antibody overnight at 4°C. Cells were then washed and incubated with the proper fluorescence-conjugated secondary antibody (Molecular Probes), phalloidin-tetramethylrhodamine isothiocyanate (Sigma) and Hoechst 33258 (Invitrogen) for 1 h at room temperature. The immunofluorescence images were taken with a confocal microscope (Zeiss, LSM 510).

Proliferation, invasion and migration assays

To assess proliferation, cells were plated at the density of 1 × 105 per dish on 60 mm dishes and the medium supplemented with 10% fetal bovine serum was changed every 2 days. Cell counts were performed with the aid of a hemocytometer by using trypan blue exclusion (0.08%) to monitor cell viability. To assess migration and invasion, cells were serum-starved overnight and then incubated in the presence or absence of 100 ng ml−1 IGF-1 for 24 h. The cells were trypsinized, resuspended and applied onto a Transwell Insert (8 μm pore size, Corning) for migration assays or Matrigel invasion chamber (BD Biosciences, Franklin Lakes, NJ, USA) for invasion assays. Cells were added into the upper chamber and allowed to migrate across the membrane towards medium containing 10 μg ml−1 fibronectin for 6 h (migration assay) or 12 h (invasion assay). Cells that migrated through the membrane were then fixed with methanol, stained with GIEMSA stain, and counted immediately. The results represent the mean ± SD of three independent experiments.

Measurement of intracellular chloride concentration

Intracellular chloride concentration was measured by a CFP-YFP-based probe (Cl-sensor) as described previously (Markova et al. 2008). In brief, Cl-sensor was transiently transfected into SiHa cells for 48 h. Excitation light was supplied to the preparation via a light pipe transmitting light from a xenon burner. A galvanometer-driven mirror and dichroic cube were used to produce alternating excitation at 440 nm and 480 nm (HyperSwitch; IonOptix, Milton, MA, USA). Emission (>510 nm) at 440 nm and 480 nm was recorded using a photomultiplier (Electron Tubes, Middlesex, UK) and the ratio was analysed using IonWizard software (IonOptix). The mean background fluorescence (measured from a non-fluorescent area) was subtracted.

Measurement of cell area

Cells were cultured on a dish for 24 h. Five random fields were photographed under light microscopy for each condition. The cell area was measured by the software of Image Tool (from The University of Texas Health Science Center at San Antonio).

Statistical analysis

The results of experiments were expressed as means ± standard deviation (SD), and data were collected from at least three independent experiments. Student's t test was used to test for statistical differences. P < 40.05 was taken to be statistically significant.

Results

Atypical expression of KCC2 in human cervical cancer cells

To examine whether KCC2 is expressed in non-neuronal cancer cells, several human epithelial cancer cell lines, including cervical cancer cells (SiHa and HeLa), colon cancer cells (HCT116) and breast cancer cells (MCF7), were employed. Figure 1A shows that KCC2 mRNA is expressed in all four cell lines investigated, although to a variable extent. Localization of endogenous KCC2 in SiHa cells was then examined using surface biotinylation assays, which revealed that endogenous KCC2 is present in the cell membrane (Fig. 1B). Furthermore, immunostaining for KCC2 was consistent with the membrane localization of endogenous KCC2 (Fig. 1C). The KCC2 gene encodes two isoforms, KCC2a and KCC2b, which differ in their N termini (Uvarov et al. 2007). Although the primers and antibody employed in the experiments reported in Fig. 1 cannot distinguish between KCC2 and KCC2b, further examination with specific primers for KCC2a and KCC2b revealed that KCC2b is the dominant isoform (data not shown). Together, these data suggest a novel expression of KCC2 in human epithelial cancer cells.

Figure 1. Expression and localization of KCC2 in human cervical cancer cell lines.

Figure 1

Open in a new tab

A, the mRNA levels of KCC2 in C-20/A4 (human chondrocytes, negative control), DAOY (medulloblastoma cells, positive control), SiHa and HeLa (cervical cancer) cells, HCT116 (colon cancer) cells and MCF7 (breast cancer) cells were examined by RT-PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. B, surface biotinylated proteins of SiHa cells (‘Biotinylated’) were captured by neutravidin-conjugated beads and processed for Western blotting using antibodies against KCC2, GLUT1 (glucose transporter 1, marker for membrane protein) and α-tubulin (marker for cytosolic protein). SiHa cell lysate (‘Total’) was run in parallel and lysate of postnatal day 21 rat hippocampus was used as a positive control (‘+’). C, immunofluorescence confocal image of a SiHa cell stained for endogenous KCC2. Scale bar = 10 μm. A–C, results are representative of three independent experiments.

Previous studies showed that repressor element 1-silencing transcription factor (REST) restricts KCC2 expression to neurons by binding to the RE-1 element in the KCC2 gene in non-neuronal cells (Karadsheh & Delpire 2001; Yeo et al. 2009). We therefore examined whether REST is involved in the regulation of KCC2 expression in SiHa cells. Supplemental Fig. S1A shows that SiHa cells indeed possess REST mRNA. The expression level of REST mRNA in SiHa cells is similar to that in C20/A4 cells, which don't express KCC2 mRNA (Fig. S1B). This result shows that the atypical expression of KCC2 in SiHa cells is not due to insufficient expression of REST mRNA. However, knockdown of REST did not alter KCC2 expression in SiHa cells (Fig. S1C and D). Thus, these data suggest that the expression of KCC2 in SiHa cells is not dependent on the level of REST.

Elevated levels of KCC2 do not alter cell growth but attenuate cell spreading

To understand the role of KCC2 in cervical carcinogenesis, stably overexpressing (+4-fold) and knockdown (–90%) KCC2 cell lines were employed. Manipulation of KCC2 did not affect the mRNA and protein expression level of other KCC isoforms (Fig. 2A and B). The intracellular chloride concentration ([Cl−]i) of cells expressing different levels of KCC2 was measured using the genetically encoded chloride indicator, Cl-sensor (Fig. 2C). The inhibitor dihydro-indenyloxyalkanoic acid (DIOA), classically considered an inhibitor of KCC, but also having inhibitory actions on calcium-activated intermediate conductance K channels (Lauf et al. 2008) and anion exchangers (Garay et al. 1988), significantly increased [Cl−]i from 38.7 ± 6.1 mm to 61.1 ± 6.4 mm (P < 0.001). Overexpression of KCC2 significantly decreased [Cl−]i from 38.7 ± 6.1 mm to 30.8 ± 0.9 mm (P < 0.001). However, knockdown of KCC2 had no significant effect on intracellular chloride concentration. This could be the result of compensation by other endogenous KCC isoforms. To determine whether KCC2 regulates cell growth, the growth rate of KCC2-overexpressing cells and KCC2 knockdown cells was examined (Fig. 2D): levels of KCC2 expression did not affect SiHa cell growth.

Figure 2. The level of KCC2 expression does not alter cell proliferation.

Figure 2

Open in a new tab

A and B, the mRNA (A) and protein (B) levels of KCC1, 2, 3 and 4 were examined in SiHa cells (‘SiHa’), KCC2 overexpressing SiHa cells (‘KCC2’), control scrambled shRNA overexpressing SiHa cells (‘control shRNA’) and KCC2 knockdown SiHa cells (‘shRNA’) by RT-PCR and Western blotting, respectively. Results are representative of three independent experiments. C, intracellular chloride concentration of SiHa cells, SiHa cell treated by 50 μM DIOA, KCC2 overexpressing SiHa cells and KCC2 knockdown SiHa cells were estimated using the genetically encoded chloride indicator, Cl-sensor. Results are the mean value ± SD (n = 40). Significant differences between SiHa cells and each condition are indicated: ***P < 0.001; NS, not significant. D, growth curve of SiHa control cells, KCC2 overexpressing SiHa cells, control scrambled shRNA overexpressing SiHa cells and KCC2 knockdown SiHa cells cultured for 4 days. The relative cell number at each time point shown on the growth curves represents the mean value ± SD (n = 9) normalized to the cell number at Day 1.

The morphology of KCC2-overexpressing cells and of KCC2 knockdown cells was significantly different. As shown in Fig. 3A and B, KCC2-overexpressing cells were ‘rounded-up’ with a smaller spreading area (SiHa cells: 310 ± 80 μm2, KCC2-overexpressing cells: 149 ± 45 μm2, P < 0.001), whereas KCC2 knockdown cells exhibited a flat morphology with larger spreading area (SiHa cells: 310 ± 80 μm2, KCC2 knockdown cells: 512 ± 108 μm2, P < 0.001). Given that cell morphology is known to be highly controlled by the cytoskeleton, we examined the distribution of two major cytoskeletal components, α-tubulin and filamentous actin, in these cells. Overexpression or knockdown of KCC2 had no effect on α-tubulin network organization (Fig. 3C). However, altering KCC2 levels had dramatic effects upon actin organization in the cells. Overexpression of KCC2 induced cortical actin formation while knockdown of KCC2 induced the formation of a dense meshwork of bundled F-actin filaments (stress fibres) (Fig. 3D). These data indicate that KCC2 contributes to stabilisation and regulation of the actin cytoskeleton in cancer cells.

Figure 3. The level of KCC2 expression regulates cell spreading and stress fibre formation.

Figure 3

Open in a new tab

A, bright field images of SiHa cells, KCC2 overexpressing SiHa cells, control scrambled shRNA overexpressing SiHa cells and KCC2 knockdown SiHa cells. Scale bar = 40 μm. B, quantitative analysis of cell area in SiHa cells, KCC2 overexpressing SiHa cells, control scrambled shRNA overexpressing SiHa cells and KCC2 knockdown SiHa cells. Cell areas were assessed by Image Tool software. Data represent the mean ± SD (n = 50). Significant differences between SiHa cells and each condition are indicated: ***P < 0.001; NS, not significant. C and D, immunofluorescence confocal image of a SiHa cell stained for tubulin (panel C, green), actin (panel D, red) and nucleus (blue). Scale bar = 20 μm. Results are representative of three independent experiments.

Increased KCC2 expression increases IGF-1-induced cell migration and invasion by an ion transport-independent mechanism

Increased cell motility and invasiveness are key steps in the metastatic behaviour of cancer cells. Previous studies have shown that insulin-like growth factor-1 (IGF-1) promotes SiHa cell migration and invasion (Shen et al. 2004). We therefore examined whether KCC2 modulates IGF-1-induced cell migration and invasion. SiHa cells, KCC2-overexpressing SiHa cells and KCC2-knockdown SiHa cells were pretreated with 100 ng ml−1 IGF-1, or incubated in the absence of serum, for 24 h. Migration and invasion capacity were then respectively assessed using the Transwell cell migration assay and Matrigel-coated chamber invasion assay. As shown in Fig. 4A and B, overexpression of KCC2 significantly increased IGF-1-induced cell migration and invasion (1.7- and 1.9-fold, respectively, P < 0.001). Meanwhile, knockdown of KCC2 significantly repressed IGF-1-induced cell migration and invasion (decreased by 75% and 80%, respectively, P < 0.001). These data demonstrate that KCC2 functions to regulate the motility and invasiveness of SiHa cancer cells.

Figure 4. Increased expression of KCC2 promotes IGF-1-induced cell migration and invasiveness.

Figure 4

Open in a new tab

A and B, migration assay (A) and invasion assay (B) of SiHa cells, KCC2-overexpressing SiHa cells and KCC2-knockdown SiHa cells in the absence or presence of IGF-1 (100 ng ml−1). The relative value at each condition represents the mean ± SD (n = 9) normalized to SiHa cells. Significant differences between SiHa cells and KCC2-overexpressing SiHa cells or KCC2-knockdown SiHa cells in each condition are indicated. *P < 0.05; **P < 0.01; ***P < 0.001.

To understand whether KCC2 modulates cell spreading, migration and invasion through its cotransporter activity, the activity-deficient KCC2 mutant (Y1087D) (Strange et al. 2000; Akerman & Cline, 2006) was transiently transfected into SiHa cells. As shown in Fig. 5A and B, cells expressing activity-deficient KCC2 still exhibited rounded morphology with smaller spreading area (from 303 ± 68 μm2 to 191 ± 38 μm2, P < 0.001), which was comparable to the effect of expressing wild-type KCC2 (Fig. 3A and B). Furthermore, increased cell migration (1.6-fold, P < 0.001) and invasion (1.8-fold, P < 0.001) following treatment with IGF-1 persisted in cells expressing Y1087D (Fig. 5C and D). These data imply that the effects of KCC2-modulated cell spreading, migration and invasion are not dependent on a role for KCC2 as a K+–Cl− co-transporter.

Figure 5. The effects of KCC2 on cell spreading, migration and invasion are not dependent on its cotransporter activity.

Figure 5

Open in a new tab

SiHa cells were transiently transfected with an activity-deficient mutant KCC2 (Y1087D). A, a representative immunofluorescence image of cells stained for GFP (green), actin (red) and nucleus (blue). Scale bar = 10 μm. B, population data on mean cell areas. Results are the mean ± SD (n = 20). Significant difference between SiHa cells and mutant KCC2-transfected cells is indicated: ***P < 0.001. C and D, cells expressing activity-deficient KCC2 (Y1087D) were isolated by FACsort. Bar graph shows migration assay data (C) and invasion assay data (D) for SiHa cells and Y1087D KCC2-overexpressing SiHa cells in the absence or presence of IGF-1 (100 ng ml−1). Results are the mean ± SD (n = 9). Significant differences between SiHa cells and mutant KCC2-transfected cells in each condition are indicated: *P < 0.05; ***P < 0.001.

The level of KCC2 modulates focal adhesion formation

It has been shown that the formation and regulation of focal adhesion structures is a key step in efficient cell migration (Gardel et al. 2010). We therefore next examined whether KCC2-modulated cell migration and invasion capacity are correlated with focal adhesion formation. The formation of focal adhesions in SiHa cells, KCC2 overexpressing SiHa cells and KCC2 knockdown SiHa cells was examined by immunostaining for focal adhesion kinase (FAK). These experiments revealed a close correlation between KCC2 expression and the formation of focal adhesions. As shown in Fig. 6A and B, overexpression of KCC2 significantly diminished the number of focal adhesions (decreased by 90%, P < 0.001), while knockdown of KCC2 led to a 2.9-fold increase (P < 0.001) in the number of focal adhesions. Taken together, these results imply that KCC2 regulates SiHa cell migration and invasion through manipulating focal adhesion formation.

Figure 6. Focal adhesion formations correlate with the levels of KCC2 expression.

Figure 6

Open in a new tab

A, representative immunofluorescence image of cell showing actin (red), FAK (green), and nucleus (blue) staining. Scale bar = 10 μm. Images were acquired by confocal microscopy. Results are representative of three independent experiments. B, mean number of focal adhesions: results are the mean ± SD (n (cells) = 100). Significant differences between SiHa cells and KCC2-overexpressing cells or mutant KCC2-transfected cells are indicated: ***P < 0.001.

Discussion

The present study demonstrates for the first time that KCC2 is expressed at the membrane of human epithelial cancer cells. This is in contrast to previous reports identifying a strictly neuronal pattern of expression (Payne et al. 1996; Lu et al. 1999; Williams et al. 1999). A key finding in this study is that KCC2 has a role in cervical carcinogenesis. In particular, we demonstrate a crucial role of KCC2 in IGF-1-induced cell migration and invasion, which is independent of the well-established function of KCC2 as a K+–Cl− cotransporter. Previous studies have shown that KCCs are implicated in cytoskeletal interactions and perform structural functions. KCC4, for example, has been shown to associate with ezrin/radixin/moesin (ERM) proteins, and with actin and myosin to form a scaffold for membrane reorganization in tumour migration (Chen et al. 2009). Moreover, it has been shown that the binding of KCC2 C-terminal domain to the cytoskeleton-associated protein 4.1N influences the morphology of dendritic spines (Li et al. 2007). These findings are consistent with the conclusion that KCC2 can act as a structural protein rather than an ion cotransporter and that this aspect of the protein's function can promote cell migration and invasion during cervical carcinogenesis.

Our results show that, in cells overexpressing KCC2, there is a decreased focal adhesion number. This is consistent with the conclusion that alterations to the cytoskeleton mediate the effects. Whether KCC2 impedes focal adhesion formation or promotes focal adhesion turnover is an interesting but unresolved question. It is possible that, in SiHa cells, the endogenous KCC2 and adhesion molecules compete for binding to cytoskeleton-associated proteins such as 4.1 or the ERM proteins. This competition could decrease focal adhesion formation and actin organization and in turn decrease cell motility and invasiveness. On the other hand, KCC2 may increase adhesion turnover, since in the present study overexpression of KCC2 reduced the number of adhesion structures in SiHa cells.

Cells overexpressing KCC2 also failed to form well-organized actin filaments, while knockdown of KCC2 promoted stress fibre formation. The formation of actin filaments provides mechanical stability, maintains cell shape and regulates dynamic membrane protrusions, such as lamellipodia and filopodia, which are fundamental determinants of cell motility (Pollard & Cooper 2009). It has been shown that the Rho family of small GTPase proteins regulates cytoskeletal dynamics and participates in many different cellular processes. Numerous studies have demonstrated that the small GTPase RhoA is a critical mediator of cytoskeletal organization (Tapon & Hall 1997; Mackay & Hall 1998) and regulates ERM protein phosphorylation (Matsui et al. 1999). Stress fibre formation was the first reported cytoskeletal phenotype produced by activated RhoA (Ridley & Hall 1992). It is therefore plausible that the endogenous KCC2 plays an inhibitory role in regulating RhoA activation.

Transcriptional mechanisms underlying regulation of KCC2 gene expression are mostly undefined. The neuronal restrictive silencing element (NRSE) has been identified in the first intron of the KCC2 gene. The repressor element 1 (RE1)-silencing transcription factor (REST) is proposed to bind to NRSEs and inhibit KCC2 gene transcription in non-neuronal cells (Karadsheh & Delpire 2001; Yeo et al. 2009). This negative regulation of the KCC2 gene explains why to date KCC2 mRNA has generally only been detected in neurons. However, our data show that REST is not essential for restricting KCC2 expression since knockdown of REST did not alter KCC2 expression in SiHa cells (Fig. S1). Previous studies showed that several transcription factors such as early growth response 4 (EGR4) and upstream stimulating factor 1 and 2 (USF1, USF2) upregulate KCC2 gene expression (Uvarov et al. 2005, 2006; Markknen et al. 2008). Dysregulation of one of these – or other, as yet unidentified transcription regulatory pathways – may contribute to KCC2 transcription in cervical cancer cells.

Our data show that the expression of KCC2 in cervical cancer cells is not just circumstantial but functional: KCC2 plays an active role in the malignant properties of cervical cancer cells by increasing IGF-1-induced cell migration and invasion. This finding is important since it suggests that KCC2 is a potential biomarker for epithelial cancer. In order to determine whether aberrant KCC2 expression might also have a role in primary human cancer, we examined KCC2 gene expression in the cancer profiling database Oncomine (http://www.oncomine.org), a web-based resource of gene expression datasets for many different types of human cancer. Our examination of this database reveals increased levels of KCC2 in several epithelial cancers (breast cancer, cervical cancer, colorectal cancer, tongue squamous cell carcinoma, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer and prostate cancer) relative to normal tissues (P < 0.05). This pattern implies that KCC2 may be a biomarker for epithelial cancers. It was also apparent that not all datasets for any given epithelial cancer exhibited increased expression of KCC2. This may suggest that the elevated expression of KCC2 is a stage-dependent event, although this question awaits further investigation.

In summary, our results not only show a novel expression of KCC2 in cervical cancer cells but also indicate a structural role of KCC2 in regulating cell migration and invasion. Additional studies to determine pathological relevance, the gene regulation of KCC2 expression and the molecular mechanisms underlying KCC2-regulated cell migration and invasion in cancer cells are required. Inhibition of abnormal expression of KCC2 may provide a potential target for the prevention of IGF-1-dependent invasive metastasis in cancers of epithelial origin.

Acknowledgments

This work was supported by a grant (G0700759) from the Medical Research Council, UK S.E.N. was supported by the Royal Society.

Glossary

Abbreviations

DIOA

dihydro-indenyloxyalkanoic acid

EGR4

early growth response 4

ERM

ezrin/radixin/moesin

FAK

focal adhesion kinase

IGF-1

insulin-like growth factor 1

KCC

potassium–chloride cotransporter

NRSE

neuronal restrictive silencing element

REST

repressor element 1-silencing transcription factor

USF

upstream stimulating factor

Author contributions

W.-C.W.: conception and design of the experiments; collection, analysis and interpretation of data; drafting and revising the article. C.J.A. and S.E.N.: conception and design of the experiments; drafting and revising the article. J.P. and N.W.V.C.: collection, analysis and interpretation of data. Y.J. and M.-R.S.: conception and design of the experiments. R.J.W. and J.C.E.: conception and design of the experiments; drafting and revising the article. All authors approved the final version of this manuscript.

Supplementary material

Supplementary Fig. S1

tjp0589-5349-SD1.pdf (238.2KB, pdf)

As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors

References

  1. Adragna NC, Di Fulvio M, Lauf PK. Regulation of K-Cl cotransport: from function to genes. J Membr Biol. 2004;201:109–137. doi: 10.1007/s00232-004-0695-6. [DOI] [PubMed] [Google Scholar]
  2. Akerman CJ, Cline HT. Depolarizing GABAergic conductances regulate the balance of excitation to inhibition in the developing retinotectal circuit in vivo. J Neurosci. 2006;26:5117–5130. doi: 10.1523/JNEUROSCI.0319-06.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brauer M, Frei E, Claes L, Grissmer S, Jäger H. Influence of K-Cl cotransporter activity on activation of volume-sensitive Cl− channels in human osteoblasts. Am J Physiol Cell Physiol. 2003;285:C22–C30. doi: 10.1152/ajpcell.00289.2002. [DOI] [PubMed] [Google Scholar]
  4. Chen YF, Chou CY, Wilkins RJ, Ellory JC, Mount DB, Shen MR. Motor protein-dependent membrane trafficking of KCl cotransporter-4 is important for cancer cell invasion. Cancer Res. 2009;69:8585–8593. doi: 10.1158/0008-5472.CAN-09-2284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Di Fulvio M, Lincoln TM, Lauf PK, Adragna NC. Protein kinase G regulates potassium chloride cotransporter-3 expression in primary cultures of rat vascular smooth muscle cells. J Biol Chem. 2001;276:21046–21052. doi: 10.1074/jbc.M100901200. [DOI] [PubMed] [Google Scholar]
  6. Garay RP, Nazareth C, Hannaert PA, Cragoe EJ., Jr Demonstration of a [K+,Cl−]-cotransport system in human red cells by its sensitivity to [(dihydroindenyl)oxy]-alkanoic acids: Regulation of cell swelling and distinction from the bumetanide-sensitive [Na+,K+,Cl−]-cotransport system. Mol Pharmacol. 1988;33:696–701. [PubMed] [Google Scholar]
  7. Gardel ML, Schneider IC, Aratyn-Schaus Y, Waterman CM. Mechanical integration of actin and adhesion dynamics in cell migration. Annu Rev Cell Dev Biol. 2010;26:315–333. doi: 10.1146/annurev.cellbio.011209.122036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hsu YM, Chen YF, Chou CY, Tang MJ, Chen JH, Wilkins RJ, Ellory JC, Shen MR. KCl cotransporter-3 down-regulates E-cadhesion/β-catenin complex to promote epithelial-mesenchymal transition. Cancer Res. 2007;67:11064–11073. doi: 10.1158/0008-5472.CAN-07-2443. [DOI] [PubMed] [Google Scholar]
  9. Karadsheh MF, Delpire E. Neuronal restrictive silencing element is found in the KCC2 gene: molecular basis for KCC2-specific expression in neurons. J Neurophysiol. 2001;85:995–997. doi: 10.1152/jn.2001.85.2.995. [DOI] [PubMed] [Google Scholar]
  10. Lauf PK, Adragna NC. K-Cl cotransport: properties and molecular mechanism. Cell Physiol Biochem. 2000;10:341–354. doi: 10.1159/000016357. [DOI] [PubMed] [Google Scholar]
  11. Lauf PK, Misri S, Chimote AA, Adragna NC. Apparent intermediate K conductance channel hyposmotic activation in human lens epithelial cells. Am J Physiol Cell Physiol. 2008;294:C820–C832. doi: 10.1152/ajpcell.00375.2007. [DOI] [PubMed] [Google Scholar]
  12. Li H, Khirug S, Cai C, Ludwig A, Blaesse P, Kolikova J, Afzalov R, Coleman SK, Lauri S, Airaksinen MS, Keinänen K, Khiroug L, Saarma M, Kaila K, Rivera C. KCC2 interacts with the dendritic cytoskeleton to promote spine development. Neuron. 2007;56:1019–1033. doi: 10.1016/j.neuron.2007.10.039. [DOI] [PubMed] [Google Scholar]
  13. Lu J, Karadsheh M, Delpire E. Developmental regulation of the neuronal-specific isoform of K-Cl cotransporter KCC2 in postnatal rat brains. J Neurobiol. 1999;39:558–568. [PubMed] [Google Scholar]
  14. Mackay DJG, Hall A. Rho GTPases. J Biol Chem. 1998;273:20685–20688. doi: 10.1074/jbc.273.33.20685. [DOI] [PubMed] [Google Scholar]
  15. Markknen M, Uvarov P, Airaksinen MS. Role of upstream stimulating factors in the transcriptional regulation of the neuron-specific K-Cl cotransporter KCC2. Brain Res. 2008;1236:8–15. doi: 10.1016/j.brainres.2008.08.007. [DOI] [PubMed] [Google Scholar]
  16. Markova O, Mukhtarov M, Real E, Jacob Y, Bregestovski P. Genetically encoded chloride indicator with improved sensitivity. J Neurosci Methods. 2008;170:67–76. doi: 10.1016/j.jneumeth.2007.12.016. [DOI] [PubMed] [Google Scholar]
  17. Matsui T, Yonemura S, Tsukita S, Tsukita S. Activation of ERM proteins in vivo by Rho involves phosphatidyl-inositol 4-phosphate 5-kinase and not ROCK kinases. Curr Biol. 1999;9:1259–1262. doi: 10.1016/s0960-9822(99)80508-9. [DOI] [PubMed] [Google Scholar]
  18. Miyoshi K, Rosner A, Nozawa M, Byrd C, Morgan F, Landesman-Bollag E, Xu X, Seldin DC, Schmidt EV, Taketo MM, Robinson GW, Cardiff RD, Hennighausen L. Activation of different Wnt/β-catenin signaling components in mammary epithelium induces transdifferentiation and the formation of pilar tumors. Oncogene. 2002;21:5548–5556. doi: 10.1038/sj.onc.1205686. [DOI] [PubMed] [Google Scholar]
  19. Montel V, Suzuki M, Galloy C, Mose ES, Tarin D. Expression of melanocyte-related genes in human breast cancer and its implications. Differentiation. 2009;78:283–291. doi: 10.1016/j.diff.2009.07.007. [DOI] [PubMed] [Google Scholar]
  20. Payne JA, Stevenson TJ, Donaldson LF. Molecular characterization of a putative K-Cl cotransporter in rat brain. J Biol Chem. 1996;271:16245–16252. doi: 10.1074/jbc.271.27.16245. [DOI] [PubMed] [Google Scholar]
  21. Pardo LA, Stühmer W. Eag1: An emerging oncological target. Cancer Res. 2008;68:1611–1613. doi: 10.1158/0008-5472.CAN-07-5710. [DOI] [PubMed] [Google Scholar]
  22. Pollard TD, Cooper JA. Actin, a central player in cell shape and movement. Science. 2009;326:1208–1212. doi: 10.1126/science.1175862. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Quirós I, Sáinz RM, Hevia D, García-Suárez O, Astudillo A, Rivas M, Mayo JC. Upregulation of manganese superoxide dismutase (SOD2) is a common pathway for neuroendocrine differentiation in prostate cancer cells. Int J Cancer. 2009;125:1497–1504. doi: 10.1002/ijc.24501. [DOI] [PubMed] [Google Scholar]
  24. Ridley AJ, Hall A. The small GTP-binding protein Rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell. 1992;70:389–399. doi: 10.1016/0092-8674(92)90163-7. [DOI] [PubMed] [Google Scholar]
  25. Rivera C, Voipio J, Payne JA, Ruusuvuori E, Lahtinen H, Lamsa K, Pirvola U, Saarma M, Kai K. The K+/Cl− co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation. Nature. 1999;397:251–255. doi: 10.1038/16697. [DOI] [PubMed] [Google Scholar]
  26. Shen MR, Chou CY, Ellory JC. Volume-sensitive KCl cotransport associated with human cervical carcinogenesis. Pflugers Arch. 2000;440:751–760. doi: 10.1007/s004240000338. [DOI] [PubMed] [Google Scholar]
  27. Shen MR, Chou CY, Hsu KF, Liu HS, Dunham PB, Holtzman EJ, Ellory JC. The KCl cotransporter isoform KCC3 can play an important role in cell growth regulation. Proc Natl Acad Sci U S A. 2001;98:14714–14719. doi: 10.1073/pnas.251388798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shen MR, Chou CY, Hsu KF, Hsu YM, Chiu WT, Tang MJ, Alper SL, Ellory JC. KCl cotransport is an important modulator of human cervical cancer growth and invasion. J Biol Chem. 2003;278:39941–39950. doi: 10.1074/jbc.M308232200. [DOI] [PubMed] [Google Scholar]
  29. Shen MR, Lin AC, Hsu YM, Chang TJ, Tang MJ, Alper SL, Ellory JC, Chou CY. Insulin-like growth factor 1 stimulates KCl cotransport, which is necessary for invasion and proliferation of cervical cancer and ovarian cancer cells. J Biol Chem. 2004;279:40017–40025. doi: 10.1074/jbc.M406706200. [DOI] [PubMed] [Google Scholar]
  30. Song L, Mercado A, Vázquez N, Xie Q, Desai R, George AL, Jr, Gamba G, Mount DB. Molecular, functional, and genomic characterization of human KCC2, the neuronal K-Cl cotransporter. Mol Brain Res. 2002;103:91–105. doi: 10.1016/s0169-328x(02)00190-0. [DOI] [PubMed] [Google Scholar]
  31. Strange K, Singer TD, Morrison R, Delpire E. Dependence of KCC2 K-Cl cotransporter activity on a conserved carboxy terminus tyrosine residue. Am J Physiol Cell Physiol. 2000;279:C860–C867. doi: 10.1152/ajpcell.2000.279.3.C860. [DOI] [PubMed] [Google Scholar]
  32. Tapon N, Hall A. Rho, Rac and Cdc42 GTPases regulate the organization of the actin cytoskeleton. Curr Opin Cell Biol. 1997;9:86–92. doi: 10.1016/s0955-0674(97)80156-1. [DOI] [PubMed] [Google Scholar]
  33. Uvarov P, Pruunsild P, Timmusk T, Airaksinen MS. Neuronal K+/Cl− co-transporter (KCC2) transgenes lacking neuron restrictive silencer element recapitulate CNS neurone-specific expression and developmental up-regulation of endogenous KCC2 gene. J Neurochem. 2005;95:1144–1155. doi: 10.1111/j.1471-4159.2005.03434.x. [DOI] [PubMed] [Google Scholar]
  34. Uvarov P, Ludwig A, Markkanen M, Rivera C, Airaksinen MS. Upregulation of the neuron-specific K+/Cl− cotransporter expression by transcription factor early growth response 4. J Neurosci. 2006;26:13463–13473. doi: 10.1523/JNEUROSCI.4731-06.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Uvarov P, Ludwig A, Markkanen M, Pruunsild P, Kaila K, Delpire E, Timmush T, Rivera C, Airaksinen MS. A novel N-terminal isoform of the neuron-specific K-Cl cotransporter KCC2. J Biol Chem. 2007;282:30570–30576. doi: 10.1074/jbc.M705095200. [DOI] [PubMed] [Google Scholar]
  36. Voloshyna I, Besana A, Castillo M, Matos T, Weinstein IB, Mansukhani M, Robinson RB, Cordon-Cardo C, Feinmark SJ. TERK-1 is a novel molecular target in prostate cancer. Can Res. 2008;68:1197–1203. doi: 10.1158/0008-5472.CAN-07-5163. [DOI] [PubMed] [Google Scholar]
  37. Williams JR, Sharp JW, Kumari VG, Wilson M, Payne JA. The neuron-specific K-Cl cotransporter, KCC2. J Biol Chem. 1999;274:12656–12664. doi: 10.1074/jbc.274.18.12656. [DOI] [PubMed] [Google Scholar]
  38. Yeo M, Berglund K, Augustine G, Liedtke W. Novel repression of Kcc2 transcription by REST-RE-1 controls developmental switch in neuronal chloride. J Neurosci. 2009;29:14652–14662. doi: 10.1523/JNEUROSCI.2934-09.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zhang Q, Fan H, Shen J, Hoffman RM, Xing HR. Human breast cancer cell lines co-express neuronal, epithelial, and melanocytic differentiation markers in vitro and in vivo. PLoS ONE. 2010;5:e9712. doi: 10.1371/journal.pone.0009712. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

tjp0589-5349-SD1.pdf (238.2KB, pdf)

Articles from The Journal of Physiology are provided here courtesy of The Physiological Society

RESOURCES