# Intermediate-conductance Ca2+-activated K+ channels (IKCa1) regulate human prostate cancer cell proliferation through a close control of calcium entry

H Lallet-Daher, M Roudbaraki, A Bavencoffe, P Mariot, F Gackière, Gabriel Bidaux, R Urbain, P Gosset, P Delcourt, L Fleurisse, et al.

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## To cite this version:

H Lallet-Daher, M Roudbaraki, A Bavencoffe, P Mariot, F Gackière, et al.. Intermediate-conductance Ca2+-activated K+ channels (IKCa1) regulate human prostate cancer cell proliferation through a close control of calcium entry. Oncogene, 2009, 28 (15), pp.1792-1806. 10.1038/onc.2009.25 . hal-03060326

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Submitted on 13 Dec 2020

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ORIGINAL ARTICLE

# Intermediate-conductance Ca²⁺-activated K⁺ channels (IK<sub>Ca1</sub>) regulate human prostate cancer cell proliferation through a close control of calcium entry

H Lallet-Daher<sup>1,2,6</sup>, M Roudbaraki<sup>1,2,6</sup>, A Bavencoffe<sup>1,2</sup>, P Mariot<sup>1,2</sup>, F Gackière<sup>1,2</sup>, G Bidaux<sup>1,2</sup>, R Urbain<sup>3</sup>, P Gosset<sup>4</sup>, P Delcourt<sup>1,2</sup>, L Fleurisse<sup>4</sup>, C Slomianny<sup>1,2</sup>, E Dewailly<sup>1,2</sup>, B Mauroy<sup>5</sup>, JL Bonnal<sup>5</sup>, R Skryma<sup>1,2</sup> and N Prevarskaya<sup>1,2</sup>

<sup>1</sup>INSERM U800, Equipe labellisée par la Ligue Nationale contre le cancer, Villeneuve d'Ascq, France; <sup>2</sup>Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France; <sup>3</sup>Institut de Recherche Pierre Fabre, Paris, France; <sup>4</sup>Faculté Libre de Médecine, Laboratoire d'Anatomie et de Cytologie Pathologique du groupement hospitalier de l'Institut Catholique de Lille, Lille, France and <sup>5</sup>Service d'Urologie de l'hôpital St-Philibert, Lomme, France

Accumulating data point to K⁺ channels as relevant players in controlling cell cycle progression and proliferation of human cancer cells, including prostate cancer (PCa) cells. However, the mechanism(s) by which K⁺ channels control PCa cell proliferation remain illusive. In this study, using the techniques of molecular biology, biochemistry, electrophysiology and calcium imaging, we studied the expression and functionality of intermediate-conductance calcium-activated potassium channels (IK<sub>Ca1</sub>) in human PCa as well as their involvement in cell proliferation. We showed that IK<sub>Ca1</sub> mRNA and protein were preferentially expressed in human PCa tissues, and inhibition of the IK<sub>Ca1</sub> potassium channel suppressed PCa cell proliferation. The activation of IK<sub>Ca1</sub> hyperpolarizes membrane potential and, by promoting the driving force for calcium, induces calcium entry through TRPV6, a cation channel of the TRP (Transient Receptor Potential) family. Thus, the overexpression of the IK<sub>Ca1</sub> channel is likely to promote carcinogenesis in human prostate tissue.

Oncogene (2009) 28, 1792–1806; doi:10.1038/onc.2009.25; published online 9 March 2009

**Keywords:** intermediate-conductance potassium channels; TRPV6; prostate cancer; proliferation; capacitative calcium entry; patch clamp

## Introduction

Prostate cancer (PCa) is a leading cause of cancer-related deaths in western countries (Woolf, 1995). Standard therapies include androgen ablation, which

causes tumor regression. However, the disease often relapses, in the form of a hormone-refractory tumor that no longer responds to conventional therapies (Isaacs, 1994). In the context of this need for new approaches and drug targets, we have investigated the role of the intermediate-conductance Ca²⁺-activated K⁺ channels (IK<sub>Ca1</sub>) in PCa.

Accumulating data from a variety of cell types have shown that potassium channels are essential for cell proliferation and appear to play a role in carcinogenesis (Kunzelmann, 2005; Pardo *et al.*, 2005). Among the known K⁺ channels, EAG1, a member of the ether-a-go-go (EAG) subfamily, has been studied in greatest detail, as it has been found to have an oncogenic potential in several human cancers (Pardo *et al.*, 1999; Pardo and van Duijn, 2005; Hemmerlein *et al.*, 2006). However, Ca²⁺-dependent potassium channels (K<sub>Ca</sub>) are also involved in cancer-related mechanisms and are attracting increasing attention (Ghanshani *et al.*, 2000; Ouadid-Ahidouch *et al.*, 2001). Intermediate-conductance Ca²⁺-activated K⁺ channels, IK<sub>Ca1</sub> (also called IK1, SK4, hSK4, hKCa4 and KCa3.1), are of particular interest, as their expression has been shown to increase in activated T lymphocytes (Ghanshani *et al.*, 2000) and pancreatic cancer cells (Jager *et al.*, 2004) and undergo modulation during the cell cycle in breast cancer cells (Ouadid-Ahidouch *et al.*, 2004). Pharmacological data also indicate the presence of IK<sub>Ca1</sub> in PCa cells. Indeed, <sup>86</sup>Rb⁺ efflux and enhanced cell proliferation induced in LNCaP and PC-3 cells by an IK<sub>Ca1</sub> channel activator (1-ethyl-2-benzimidazolinone (1-EBIO)) was inhibited by IK<sub>Ca1</sub> channel blockers (Parihar *et al.*, 2003). In this study, we investigated the expression of IK<sub>Ca1</sub> in PCa and the mechanism by which it regulates the intracellular calcium concentration and cell proliferation.

## Results

*IK<sub>Ca1</sub> potassium channel is expressed in human PCa cells*
The reverse transcription–PCR (RT–PCR) technique was initially used to check the mRNA expression of the

Correspondence: Professor M Roudbaraki or Professor N Prevarskaya, Laboratoire de Physiologie Cellulaire, INSERM U800, USTL, Bâtiment SN3, 2e étage, Villeneuve d'Ascq, Nord 59 655, France.

E-mails: morad.roudbaraki@univ-lille1.fr or natacha.prevarskaya@univ-lille1.fr

<sup>6</sup>These authors contributed equally to this work.

Received 27 May 2008; accepted 11 December 2008; published online 9 March 2009
IK<sub>Ca1</sub> potassium channel in human PCa cell lines (LNCaP, DU-145 and PC-3) and cultured primary human PCa epithelial (hPCE) cells from tissue specimens. Analysis of RT-PCR products revealed the amplification of a single fragment of the expected size (867 bp) from the cDNA of all cell lines and hPCE cells (Figure 1a). Further cloning and sequencing of the amplified PCR product confirmed its specificity and correspondence to the published sequence for human IK<sub>Ca1</sub> channel mRNA.

Further studies focused on two PCa cell lines, an androgen-sensitive LNCaP and an androgen-insensitive PC-3. First, we examined IK<sub>Ca1</sub> channel protein expression by western blot analysis of the total protein preparations of LNCaP and PC-3 cells, using a specific antibody directed against the N-terminal end of the IK<sub>Ca1</sub> channel. To validate the specificity of the anti-IK<sub>Ca1</sub> antibody, western blotting experiments were performed on 10 µg of the total protein extracts from HEK-293 cell lines stably transfected either by empty vector (HEK-Neo) or bearing the IK<sub>Ca1</sub> coding cDNA (HEK-IK<sub>Ca1</sub>). In these experiments, the anti-IK<sub>Ca1</sub> antibody revealed a band of about 52 ± 2 kDa only in protein extracts from HEK-IK<sub>Ca1</sub> cells (Figure 1b). The expected size, according to amino-acid sequence of the channel, being 47 kDa, the differences in protein size observed may be due to post-translational modifications, for example, glycosylation and/or phosphorylations of the channel protein.

As shown in Figure 1c, the IK<sub>Ca1</sub> antibody revealed a protein band of approximately 52 kDa in LNCaP and PC-3 cells similar to that observed in HEK-IK<sub>Ca1</sub> cells. IK<sub>Ca1</sub> expression at the protein level was also confirmed in both LNCaP and PC-3 PCa cell lines by immunofluorescence studies using the anti-IK<sub>Ca1</sub>-specific antibody (data not shown).

### IK<sub>Ca1</sub> is overexpressed in PCa tissues

IK<sub>Ca1</sub> expression was also studied at the mRNA level by RT-PCR and at the protein level by immunofluorescence in human tissue specimens from benign prostate hyperplasia (BPH) and PCa. Of a total of 19 PCa tissues and 22 BPH tissues used in these studies, the IK<sub>Ca1</sub> mRNA was expressed in 89.5% (17 out of 19) of PCa tissues and in 18% (4 out of 22) of BPH tissues, suggesting a preferential expression of the IK<sub>Ca1</sub> in PCa tissues. To ensure that the absence of the IK<sub>Ca1</sub> expression in BPH tissues used in this study is not due to the absence of the epithelial cells, we also searched for the expression of the apical epithelial cell marker, the cytokeratin 18. As shown in Supplementary information, Figure 1b, cytokeratin 18 is expressed at a comparable level in all samples used for this study, whereas IK<sub>Ca1</sub> is expressed preferentially in PCa tissues. To confirm these observations, we studied the expression of the channel protein in three BPH and five PCa formalin-fixed, paraffin-embedded tissues using the IK<sub>Ca1</sub>-specific antibody. As shown in Figure 1e, the IK<sub>Ca1</sub> protein was highly expressed in epithelial cells of grade 3 (Figure 1e) and grade 4 (Figure 1f) PCa tissues, whereas a very faint

labeling was observed in the stromal cells. In BPH samples, the expression of the IK<sub>Ca1</sub> was either absent or very low compared with that in PCa tissues. However, in some BPH sections, a clear IK<sub>Ca1</sub> labeling was observed in the pluri-stratified epithelial cells (Figure 1e, BPH), an aspect caused due to the high proliferation activity of the acinar cells in which IK<sub>Ca1</sub> may play a major role. These studies show clearly the overexpression of IK<sub>Ca1</sub> in PCa and confirm the data obtained in the RT-PCR studies presented in Figure 1d.

### Ca<sup>2+</sup>-induced activation of IK<sub>Ca1</sub> channels hyperpolarizes human PCa cell membrane potential

The patch-clamp technique in whole-cell configuration was used to study the functionality of IK<sub>Ca1</sub> and its involvement in the modulation of the membrane potential in LNCaP cells. First, we examined the impact of an increase in intracellular free calcium concentration on the membrane potential in these cells. To address this aspect, combined current-clamp and calcium-imaging experiments in perforated patch-clamp configurations were performed. To artificially increase the intracellular free calcium concentration ([Ca<sup>2+</sup>]<sub>i</sub>), we used thapsigargin (TG), which inhibits the endoplasmic reticulum SERCA Ca<sup>2+</sup> pump, causing calcium mobilization from intracellular stores followed by store-operated calcium entry in PCa cells (Skryma *et al.*, 2000).

As shown in Figure 2a, the TG-induced increase in [Ca<sup>2+</sup>]<sub>i</sub> was accompanied by a large hyperpolarization of membrane potential. The average resting membrane potential of LNCaP cells, -47 ± 8 mV, shifted to -71 ± 2.7 mV following TG application. To study the involvement of the IK<sub>Ca1</sub> functionality in the TG-induced membrane hyperpolarization, the following experiments were performed. Using electrophysiological techniques of patch clamp in whole-cell configuration, we examined the impact of an increase in intracellular calcium on potassium current activation. A voltage ramp of -150 to +100 mV was applied to the LNCaP cells and the resulting currents were recorded. Figure 2b shows typical recording acquired in an LNCaP cell under control conditions (CTL) and after the application of TG (100 nM). In control conditions, only outward and voltage-dependent currents were observed at all membrane potentials above -30 mV as described by Skryma *et al.* (1997, 1999). An increase in intracellular free calcium concentration by the application of TG (100 nM) induced the development of an additional potassium current due to the activation of K<sub>Ca</sub> channels. The currents recorded after TG applications were K<sup>+</sup> selective, as could be seen from their mean reversal potential (E<sub>rev</sub>) of -80 ± 5 mV (n = 32) (Nernst potential of K<sup>+</sup> (E<sub>K</sub>) = -80 mV). To study the involvement of the IK<sub>Ca1</sub> channels in the calcium-activated potassium currents (I<sub>KCa</sub>) observed in response to TG, we used two known inhibitors of this channel, clotrimazole (CLT) and a modified CLT molecule, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34), developed to inhibit IK<sub>Ca1</sub> channels selectively without blocking the cytochrome P450 enzyme (Wulff *et al.*,
<figure><img src="image_3.png" /></figure>

**Figure 1** Expression of IK<sub>Ca1</sub> in human prostate cancer cell lines and tissues. (a) RT-PCR studies of IK<sub>Ca1</sub> mRNA expression in androgen-sensitive (LNCaP), androgen-insensitive (PC-3 and DU-145) and primary cultured human prostate cancer epithelial (hPCE) cells. Two micrograms of total RNA were reverse transcribed and 150 ng RNA equivalents used in PCRs to study IK<sub>Ca1</sub> (867 bp) mRNA expression, using specific IK<sub>Ca1</sub> (867 bp) primers, as described in Materials and methods. H<sub>2</sub>O and samples without reverse transcriptase (not shown) were used as negative controls. Amplified fragments were resolved on 1.5% agarose gel by electrophoresis and visualized by EtBr staining. M, 1-kb DNA ladder, a molecular weight marker. (b) Western blotting experiments performed on 10 µg of the total protein extracts from HEK-293 cell lines stably transfected either by empty vector (HEK-Neo) or bearing the IK<sub>Ca1</sub>-coding cDNA (HEK-IK<sub>Ca1</sub>). A polyclonal IK<sub>Ca1</sub> antibody raised against the first 16 amino acids of the N-terminal end of the protein (kindly provided by Dr Mao Xiang Chen, GlaxoSmithKline R&D, UK) was used in the following IK<sub>Ca1</sub> protein expression studies. The anti-IK<sub>Ca1</sub> antibody revealed a band of about 52 ± 2 kDa only in protein extracts from HEK-IK<sub>Ca1</sub> cells. (c) IK<sub>Ca1</sub> protein expression in human prostate cancer cells (LNCaP and PC-3) in western blotting experiments using the anti-IK<sub>Ca1</sub> antibody. The RT-PCR studies of IK<sub>Ca1</sub> expression in human tissues from benign prostate hyperplasia (BPH) and prostate cancer biopsies are presented in (d). The panel presents an agarose gel electrophoresis image of IK<sub>Ca1</sub> gene expression in six prostate cancer (lanes 10–15) and nine BPH (non-cancerous) biopsies (lanes 1–9). β-Actin mRNA expression (212 bp) was used as a control for the amount of RNA equivalents used in the RT-PCR experiments. (e) IK<sub>Ca1</sub> protein expression studies in grade 3 PCa and BPH formalin-fixed, paraffin-embedded tissues using the IK<sub>Ca1</sub>-specific antibody. After an overnight incubation with IK<sub>Ca1</sub> antibody, sections were then incubated with the secondary Alexa fluor 488-labeled anti-rabbit IgG (in green). The tissues were then incubated in phosphate-buffered saline containing 1 µM propidium iodide (IP, in red) for 10 min to stain the nuclei. (f) The expression of the IK<sub>Ca1</sub> protein in grade 4 PCa (left panel) with characteristic fusioned acini and in a grade 3 PCa (middle panel) in a region containing cancerous (PCa) and BPH acini. A control experiment was performed on a PCa tissue (right panel) by omitting the first antibody (anti-IK<sub>Ca1</sub>). After incubation with the secondary Alexa fluor 488-labeled anti-rabbit IgG (in green), the nuclei were stained by 1 µM propidium iodide (IP, in red). RT-PCR, reverse transcription-PCR; IK<sub>Ca1</sub>, intermediate-conductance calcium-activated potassium channel; PCa, prostate cancer.
<figure><img src="image_4.png" /><figcaption>**Figure 2** Functional expression of IK<sub>Ca1</sub> and involvement in membrane potential in LNCAP prostate cancer cells. (a) A simultaneous recording of the free cytoplasmic Ca<sup>2+</sup> concentration ([Ca<sup>2+</sup>]<i>i</i>) (a) and membrane potential (V<sub>m</sub>) (b) in LNCaP cells is presented. TG (100 nM) induced a hyperpolarization consecutive to the increase in [Ca<sup>2+</sup>]. Typical traces obtained using the patch-clamp technique in whole-cell configuration, showing the involvement of IK<sub>Ca1</sub> in the calcium-activated K<sup>+</sup> currents (b), 1-EBIO-induced IK<sub>Ca1</sub> currents (c and d) and the involvement of the IK<sub>Ca1</sub> in the calcium-induced membrane hyperpolarization (d) in LNCaP cells. A voltage ramp of –150 to +100 mV was applied to the LNCaP cells and the resulting current (I) recorded under control (CTL) conditions and after the increase in intracellular free calcium concentration triggered by TG application (100 nM). To study the involvement of IK<sub>Ca1</sub> in the calcium-induced potassium currents in LNCaP cells, following TG-induced potassium current developments, the effects of CLT (10 µM), an IK<sub>Ca1</sub> inhibitor, were studied. To confirm the functionality of the IK<sub>Ca1</sub> channels in LNCaP cells, IK<sub>Ca1</sub> currents were elicited by the application of the IK<sub>Ca1</sub> activator 1-EBIO (200 µM) (c and d) and then pharmacological inhibitors, CLT (10 µM) (e) or TRAM-34 (5 and 10 µM) (f), were used to block the IK<sub>Ca1</sub> current. To study the involvement of IK<sub>Ca1</sub> in the calcium-induced hyperpolarization of membrane potential in LNCaP cells, membrane potential was measured before and after the application of either TG (100 nM) (e) or 1-EBIO (200 µM) (f). Following TG- or 1-EBIO-induced hyperpolarization, CLT (10 µM) was applied. In both cases, hyperpolarization was completely reversed (n=9) when CLT (10 µM) was applied. CLT, clotrimazole; 1-EBIO, 1-ethyl-2-benzimidazolinone; IK<sub>Ca1</sub>, intermediate-conductance calcium-activated potassium channel; TG, thapsigargin.</figcaption></figure>

2004). As shown in Figure 2b, the application of CLT (10 µM) inhibited the TG-induced I<sub>KCa</sub> current by about 72% at +100 mV and 100% at –150 mV. The residual CLT-insensitive I<sub>KCa</sub> was probably mediated by the large-conductance potassium channel (BK<sub>Ca</sub>), as the mRNA and the protein for this channel were also detected in LNCaP cells (data not shown).

To confirm the functionality of IK<sub>Ca1</sub> in LNCaP cells in electrophysiological studies, we used a specific pharmacological tool, 1-EBIO, known to activate both heterologously expressed (Pedersen *et al.*, 1999; von Hahn *et al.*, 2001) and native IK<sub>Ca1</sub> channels (Devor *et al.*, 1996; Khanna *et al.*, 1999). In these studies, the concentration of the free calcium in the pipette solution was 200 nM and a voltage ramp of –150 to +40 mV was applied. The resulting currents were

recorded in the absence (CTL) or in the presence of 1-EBIO in the extracellular medium. As shown in Figure 2c and d, 1-EBIO (200 µM) induced K⁺ currents similar to those developed in the presence of TG in LNCaP cells. Moreover, the 1-EBIO-induced currents were blocked by CLT (10 µM) (Figure 2c) and dose dependently by 5 and 10 µM TRAM-34 (Figure 2d), whereas the apamin (500 nM), an inhibitor of the SK family of the K<sub>Ca</sub> channels, and TEA (5 mM), an inhibitor known to block BK<sub>Ca</sub> channel, were without effect on the 1-EBIO-elicited K⁺ currents (data not shown). In addition to these studies conducted using the pharmacological tools (CLT and TRAM-34), the specificity of 1-EBIO effects to activate IK<sub>Ca1</sub> was verified by using small interfering RNA (siRNA) targeting IK<sub>Ca1</sub> as presented in Supplementary information (Figure 3b).
<figure><img src="image_5.png" /></figure>

**Figure 3** Functional expression of IK<sub>Ca1</sub> and involvement in membrane potential in primary cultured prostate cancer hPCE cells. Similar experiments to those described in Figure 2 were performed on primary cultured cells from human prostate cancer tissue (hPCE cells) to study the involvement of IK<sub>Ca1</sub> in Ca<sup>2+</sup>-activated K<sup>+</sup> current and plasma membrane hyperpolarization. Typical traces obtained using the patch-clamp technique in whole-cell configuration as described in Materials and methods and in Figure 2 and showing the involvement of IK<sub>Ca1</sub> in the calcium-activated K<sup>+</sup> currents (a), 1-EBIO-induced IK<sub>Ca1</sub> currents (b) and the involvement of the IK<sub>Ca1</sub> in the calcium-induced membrane hyperpolarization (c) in primary cultured hPCE cells. Following TG- or 1-EBIO-induced potassium current development, CLT (10 µM) (a) or TRAM-34 (1 and 10 µM) (b) was applied. To study the involvement of IK<sub>Ca1</sub> in the calcium-induced hyperpolarization of membrane potential in hPCE cells, membrane potential was measured before and after TG application (100 nM). Following TG-induced hyperpolarization, CLT (10 µM) was applied. TG-induced hyperpolarization was completely reversed (n=6) when CLT (10 µM) was applied. CLT, clotrimazole; 1-EBIO, 1-ethyl-2-benzimidazolinone; IK<sub>Ca1</sub>, intermediate-conductance calcium-activated potassium channel; TG, thapsigargin.

To characterize the functional role of IK<sub>Ca1</sub> channels
in the control of membrane potential, we performed
current-clamp experiments. To test whether IK<sub>Ca1</sub>
activation was involved in the Ca<sup>2+</sup>-induced hyperpo-
larization, membrane potential was measured before
and after the TG-induced increase in [Ca<sup>2+</sup>]<sub>i</sub>. Applica-
tion of TG (100 nM) induced sustained hyperpolariza-
tion of the membrane potential (Figure 2e). In the
presence of 10 µM CLT (n=9), membrane hyperpolar-
ization was completely reversed. In the same manner, an
application of IK<sub>Ca1</sub> activator, 1-EBIO (200 µM),
induced sustained hyperpolarization of the membrane
potential, which could be prevented by 10 µM CLT
(Figure 2f). These data show that IK<sub>Ca1</sub> is functional in
LNCaP cells, assures the majority of the IK<sub>Ca</sub> currents
in these cells and is involved in the calcium-induced
modulation of the membrane potential.

Similar results showing the functionality of the IK<sub>Ca1</sub>
channel were obtained in primary cultured PCa cells
(hPCE cells) by using TG to induce a [Ca<sup>2+</sup>]<sub>i</sub> increase
or an IK<sub>Ca1</sub> activator (1-EBIO) and CLT (10 µM)
(Figure 3a) or TRAM-34 (1 and 10 µM) (Figure 3b) as

inhibitors of IK<sub>Ca1</sub> (Figure 3a and b). The electro-
physiological studies conducted on hPCE cells showed
that the membrane potential varied between –30 and
+20 mV with a mean value of –2.4 ± 2.2 mV (n = 45).
An application of TG (100 nM) induced hyperpolariza-
tion of the membrane potential, which was reversed in
the presence of 10 µM CLT (Figure 3c). These observa-
tions suggested that the majority of the I<sub>KCa</sub> activated in
PCa epithelial cells of various origins in response to an
increase in [Ca<sup>2+</sup>]<sub>i</sub> was due to IK<sub>Ca1</sub> channel activation,
which was responsible for the hyperpolarization induced
by an increase in [Ca<sup>2+</sup>]<sub>i</sub>.

IK<sub>Ca1</sub> controls PCa cell proliferation

To elucidate whether IK<sub>Ca1</sub> expression was functionally
important in controlling cell growth, we tested the effect
of IK<sub>Ca1</sub> inhibitors (TRAM-34 and CLT) and siIK<sub>Ca1</sub>
on the growth rate of human PCa cell lines. As shown in
Figure 4, TRAM-34 (1–30 µM) reduced the proliferation
of LNCaP (Figure 4a) and PC-3 (Figure 4e) PCa cells.
Proliferation inhibition by TRAM-34 (1–30 µM) was
<figure><img src="image_6.png" /><figcaption>**Figure 4** Blockade of IK<sub>Ca1</sub> K<sup>+</sup> channels inhibits prostate cancer cell proliferation and the accumulation of p21<sup>Cip1</sup> mRNA. Cells were trypsinized and seeded for 48 h, then treated for 4 days with either solvent (CTL) or TRAM-34 (1–30 µM), an IK<sub>Ca1</sub> K<sup>+</sup> channel blocker, in culture medium. Cell growth was then measured as described in Materials and methods. Results are presented as the percentage of CRL for (a) LNCaP and (e) PC-3 cells. In another set of experiments, the effects of cell transfection with either control siRNA (siCTL) or IK<sub>Ca1</sub>-targetted siRNA (siIK<sub>Ca1</sub>) on cell proliferation were studied for (c) LNCaP and (g) PC-3 cells. Experiments were repeated four times in independent cell cultures and a representative figure is presented for each cell type. \*P<0.001 compared with their respective controls. Typical RT-PCR analysis of p27<sup>Kip1</sup> and p21<sup>Cip1</sup> mRNA expression performed on control (CTL) and TRAM-34-treated (b) LNCaP and (f) PC-3 human prostate cancer cells. Cells were plated in culture medium in the absence (CTL) or presence of TRAM-34 (10 and 20 µM) for 3 days, and then harvested. In the same way, the effects of cell transfection with either control siRNA (siCTL) or siIK<sub>Ca1</sub> on the expression of p27<sup>Kip1</sup> (274 bp) and p21<sup>Cip1</sup> were studied for (d) LNCaP and (h) PC-3 cells. Total RNA was extracted and semi-quantitative RT-PCR experiments performed to study the expression of p27<sup>Kip1</sup> (274 bp) and p21<sup>Cip1</sup> (206 bp) mRNA using specific primers. β-Actin mRNA expression (212 bp) was used as an internal standard. PCRs were carried out using 1 µl of either a fivefold (cell cycle markers) or 10-fold (β-actin) diluted samples of the RT reaction of TRAM-34- or siRNA-treated LNCaP and PC-3 cells. The conditions were as follows: 7 min at 95 °C, then 32–34 cycles of 30-s extension at 72 °C each and a final 7-min extension step. Relative levels of p27<sup>Kip1</sup> and p21<sup>Cip1</sup> mRNA were determined by densitometric analysis of the PCR (p27<sup>Kip1</sup>, p21<sup>Cip1</sup> and β-actin) bands on 1.5% agarose gel and normalized to the β-actin expression rate in each sample. The normalized rate of p27<sup>Kip1</sup> and p21<sup>Cip1</sup> mRNA under control (CTL) conditions was considered to have a value of 1 and the expression rate for TRAM-34-treated cells was compared with that at CTL condition. A representative figure is presented for each experiment, repeated at least twice, in duplicate, and in two independent cell cultures. IK<sub>Ca1</sub>, intermediate-conductance calcium-activated potassium channel; RT-PCR, reverse transcription-PCR; siRNA, small interfering RNA.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Figure 5** IK<sub>Ca1</sub> potassium channel functionality is involved in capacitative calcium entry (CCE) in prostate cancer cells. The effects of membrane depolarization on CCE in LNCaP (a) and PC-3 (b) prostate cancer cells were studied by calcium imaging using fura-2 fluorescence. To evaluate membrane hyperpolarization dependence of the CCE in prostate cancer cells, CCE was induced by depleting endoplasmic reticulum Ca<sup>2+</sup> stores with 1 µM thapsigargin in a Ca<sup>2+</sup>-free solution. Then, Ca<sup>2+</sup> was readmitted while the cells were bathed in a solution containing 5.6 mM K<sup>+</sup>. Once CCE had been established, solutions containing 50 and 140 mM K<sup>+</sup> were applied to depolarize the membrane potential in the LNCaP and PC-3 cells. Each experiment was repeated at least six times in different cell cultures on a field of 25–40 cells and representative experiments are presented. The panels show the mean ± s.e. of 42 cells from two experiments. To study the involvement of IK<sub>Ca1</sub> potassium channel functions in passive calcium entry, CCE was induced in LNCaP (c) and PC-3 (d) prostate cancer cells, as described above, in the absence (CTL) or presence of IK<sub>Ca1</sub> inhibitor (TRAM-34, 10 µM). The effects of cell transfection with either control siRNA (siCTL) or IK<sub>Ca1</sub>-targetted siRNA (siIK<sub>Ca1</sub>) on TG-induced CCE were also studied for (e) LNCaP and (f) PC-3 cells. Each experiment was repeated at least three times in different cell cultures on a field of 25–40 cells and representative experiments are presented. A quantification of the capacitative calcium entry (CCE) for each experiment (c–f) is presented in inset (mean ± s.e., *n* = 45–50 cells), \*P\*<0.01. Periods of TG and KCl applications and extracellular Ca<sup>2+</sup> increase from 0 (0Ca) to 2 mM (2Ca) are marked by horizontal bars; see text for details. siRNA, small interfering RNA; IK<sub>Ca1</sub>, intermediate-conductance calcium-activated potassium channel; TG, thapsigargin.</figcaption></figure>
dose dependent amounting at the highest concentration used (30 µM) at 75 ± 6.5% for LNCaP cells (Figure 4a) and 50 ± 6.5% for PC-3 cells (Figure 4e). Similar results were obtained using CLT (1–30 µM), but as this drug is also known to be an antimycotic agent that inhibits fungal P450-dependent enzymes (Ayub and Levell, 1990; Maurice *et al.*, 1992), its action may not be specific to IK<sub>Ca1</sub>. The inactive TRAM-7 (30 µM) did not affect cell proliferation in our studies (data not shown), indicating that the suppressive effect of TRAM-34 was specifically related to IK<sub>Ca1</sub> inhibition. The cell arrest induced by TRAM-34 was not due to apoptosis. Indeed, TRAM-34, used at the same concentrations as in the cell proliferation assays, failed to modify the rate of LNCaP cell apoptosis (data not shown). The involvement of IK<sub>Ca1</sub> in LNCaP cell proliferation was also studied by using siRNA targeting IK<sub>Ca1</sub> in LNCaP (Figure 4c) and PC-3 (Figure 4g) cells, and the obtained results were consistent with those obtained with the IK<sub>Ca1</sub> inhibitor TRAM-34.

### IK<sub>Ca1</sub> channel inhibition induces an increase in p21<sup>CIP1</sup> levels

We then investigated the mechanism by which IK<sub>Ca1</sub> inhibition prevented the proliferation of PCa cells by assaying changes in the expression of p21<sup>CIP1</sup> and p27<sup>Kip1</sup>, cyclin-dependent kinase inhibitors (cdkis), known to act in the G1 phase of the cell cycle (Benito *et al.*, 1998; Ghiani *et al.*, 1999). In these experiments, LNCaP and PC-3 cells were incubated for 4 days in the culture medium supplemented with either the solvent (dimethyl sulfoxide, control) or various concentrations of TRAM-34, followed by semi-quantitative RT-PCR analysis of p21<sup>CIP1</sup> and p27<sup>Kip1</sup> mRNA expression. Figure 4 shows that a 4-day treatment with TRAM-34 increased p21<sup>CIP1</sup> expression by two- to fourfold (vs CTL) in both LNCaP (Figure 4b) and PC-3 (Figure 4f) cells, basically leaving the mRNA rate of p27<sup>Kip1</sup> intact. Similarly, when the expression of the IK<sub>Ca1</sub> was knocked down by using IK<sub>Ca1</sub>-targetting siRNA, the p21<sup>CIP1</sup> mRNA expression was increased by 2.5-fold (vs siCTL) in both LNCaP (Figure 4d) and PC-3 (Figure 4h) cells, without affecting the p27<sup>Kip1</sup> mRNA rate.

### IK<sub>Ca1</sub> potassium channel regulates passive calcium entry by controlling the electrochemical gradient for Ca<sup>2+</sup>

One of the mechanisms by which the IK<sub>Ca1</sub> channel may regulate PCa cell proliferation is calcium entry modulation. To investigate this possibility, we used an established experimental protocol to measure the capacitative calcium entry (CCE) activated by store depletion (Skryma *et al.*, 2000). We initially investigated whether membrane potential could affect the CCE developed in response to TG (1 µM) by exposing cells to increasing concentrations of KCl (50–140 mM) to depolarize their membrane potential during the development of TG-induced CCE in the presence of 2 mM extracellular Ca<sup>2+</sup>. As shown in Figure 5, consecutive applications of 50 and 140 mM KCl and associated membrane depolarizations induced gradual inhibition of

CCE in LNCaP (Figure 5a) and PC-3 (Figure 5b) PCa cells. To specifically address the issue of the involvement of the IK<sub>Ca1</sub> channel in CCE regulation, we used TRAM-34. The application of TG (1 µM) in 0Ca<sup>2+</sup> medium induced a calcium release from internal stores and the subsequent CCE in 2Ca<sup>2+</sup> medium in both LNCaP (Figure 5c) and PC-3 cells (Figure 5d). In these experiments, when the TRAM-34 (10 µM) was present during the whole experiment, the TG-induced calcium release from intracellular stores was not significantly modified, whereas the amplitude of the CCE was decreased by 71 ± 4.5% (n=180) in LNCaP cells (Figure 5c, inset) and by 64 ± 6% vs CTL (n=230) in PC-3 cells (Figure 5d, inset). Similar results were also obtained with CLT (10 µM) (data not shown), suggesting that IK<sub>Ca1</sub> channels regulate CCE by hyperpolarizing membrane potential, thus increasing the electrochemical gradient for calcium. The involvement of IK<sub>Ca1</sub> in TG-induced CCE was also confirmed by using siRNA targeting IK<sub>Ca1</sub> in calcium imaging experiments performed on LNCaP (Figure 5e) and PC-3 (Figure 5f) cells.

### IK<sub>Ca1</sub> activation induces an increase in [Ca<sup>2+</sup>]<sub>i</sub> in human PCa cells

We further studied the effects of cell membrane hyperpolarization, induced by a direct activation of IK<sub>Ca1</sub>, on basal [Ca<sup>2+</sup>]<sub>i</sub> in human PCa cells. [Ca<sup>2+</sup>]<sub>i</sub> was first continuously monitored in normal Hank's buffered salt solution medium containing 5.6 mM KCl, then, at the time indicated (Figure 6), the cells were superfused with a KCl-free Hank's buffered salt solution (0K<sup>+</sup>) to hyperpolarize them. Application of the 0K<sup>+</sup> solution to the cells induced a transient increase in [Ca<sup>2+</sup>]<sub>i</sub>, with a peak amplitude of 500 ± 65 nM from a baseline level of 75 ± 25 nM (n=250) in LNCaP cells (Figure 6a) and a peak amplitude of 600 ± 75 nM from 85 ± 25 baseline in PC-3 cells (Figure 6b). These observations suggested that the increase in the driving force for calcium by membrane hyperpolarization was sufficient to induce calcium entry.

To test the direct implication of IK<sub>Ca1</sub> activity in controlling basal [Ca<sup>2+</sup>]<sub>i</sub>, the pharmacological activator of the IK<sub>Ca1</sub> (1-EBIO) was used. When 1-EBIO (200 µM) was applied to the cells, a rapid, transient increase in [Ca<sup>2+</sup>]<sub>i</sub> was observed, reaching a maximal value of 550 ± 70 nM (n=140) in LNCaP cells (Figure 6c) and 450 ± 50 nM (n=165) in PC-3 cells (Figure 6d). To further confirm that the 1-EBIO-induced calcium increase was due to IK<sub>Ca1</sub> activation, the same experiments were also conducted in the presence of TRAM-34 (10 µM), an IK<sub>Ca1</sub> inhibitor, or in IK<sub>Ca1</sub>-knocked down cells using siIK<sub>Ca1</sub>. In the presence of TRAM-34 (Figure 6c and d) or in siIK<sub>Ca1</sub>-transfected cells (Figure 6e and f), 1-EBIO failed to induce an [Ca<sup>2+</sup>]<sub>i</sub> increase in either LNCaP (Figure 6c and e) or PC-3 cells (Figure 6d and f). These data show that IK<sub>Ca1</sub> activation induces a calcium increase in PCa cells, by hyperpolarizing their membrane potential as shown in Figure 2f.
<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

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<figure><img src="image_13.png" /></figure>

**Figure 6** IK<sub>Ca1</sub> activation induces an increase in [Ca<sup>2+</sup>]<sub>i</sub> in human prostate cancer cells. Calcium imaging experiments showing the effects of cell membrane hyperpolarization and direct activation of IK<sub>Ca1</sub> on the basal cytosolic free Ca<sup>2+</sup> concentration ([Ca<sup>2+</sup>]<sub>i</sub>) in human prostate cancer cells. To study the effects of cell membrane hyperpolarization on the basal calcium concentration, [Ca<sup>2+</sup>]<sub>i</sub> was first continuously monitored in normal HBSS medium containing 5.6 mM KCl, then the LNCaP (a) and PC-3 (b) cells were superfused with a KCl-free HBSS solution (0K<sup>+</sup>) to hyperpolarize the membrane, according to the Nernst potential. To study the impact of IK<sub>Ca1</sub> activity on [Ca<sup>2+</sup>]<sub>i</sub>, 1-EBIO (200 µM) was used to activate the channel in calcium imaging studies of LNCaP (c) and PC-3 (d) cells. In the presence of TRAM-34 (10 µM), 1-EBIO failed to induce a calcium increase in LNCaP or PC-3 cells. In another set of experiments, the effects of cell transfection with either control siRNA (siCTL) or IK<sub>Ca1</sub>-targeted siRNA (siIK<sub>Ca1</sub>) on 1-EBIO-induced [Ca<sup>2+</sup>]<sub>i</sub> increase were studied for (e) LNCaP and (f) PC-3 cells. A representative cell of at least 50 cells is shown for each calcium imaging experiment, each experiment was repeated at least four times with two independent cell cultures. HBSS, Hank's buffered salt solution; siRNA, small interfering RNA; 1-EBIO, 1-ethyl-2-benzimidazolinone; IK<sub>Ca1</sub>, intermediate-conductance calcium-activated potassium channel.

*IK<sub>Ca1</sub> activation induces calcium entry through TRPV6 calcium channel*

One of the constitutively active calcium-permeable channels expressed in PCa cells is Transient Receptor

Potential (TRP)V6. To assess the involvement of this channel in the calcium entry induced by 1-EBIO-mediated IK<sub>Ca1</sub> activation, we used the siRNA technology to downregulate TRPV6 in LNCaP and PC-3 cells.
<figure><img src="image_14.png" /></figure>

First, we confirmed TRPV6 expression in these cells by RT-PCR analysis. As shown on the agarose gel presented in Figure 7a, the specific primers for TRPV6 amplified a band of the expected size (255 bp), which had been sequenced earlier and identified as belonging to TRPV6 cDNA.

Next, we designed siRNA against the TRPV6 sequence (siTRPV6), specifically to downregulate its mRNA. As shown in Figure 7b, treating the cells with siTRPV6 (20 nM) for 72 h knocked down the TRPV6 mRNA expression in both LNCaP and PC-3 cells by at least 90–100% (Figure 7b for LNCaP cells), whereas non-targeting siRNA had no effect on TRPV6 mRNA expression. Moreover, the assessment of the effects of IK<sub>Ca1</sub> activator 1-EBIO (200 µM) on [Ca<sup>2+</sup>]<sub>i</sub> in LNCaP and PC-3 cells treated with either non-targeting siRNA (siCTL) or specific siTRPV6 showed that the drug was no longer able to induce a calcium increase following TRPV6 downregulation (Figure 7c and d). This result was consistent with the concept that the [Ca<sup>2+</sup>]<sub>i</sub> rise evoked by IK<sub>Ca1</sub> activation was mediated by the TRPV6 cation channel.

*IK<sub>Ca1</sub> associates with TRPV6 calcium channel*

In view of the importance of the TRPV6 calcium channel in the calcium entry evoked by IK<sub>Ca1</sub> activation in PCa cells (Figure 7c and d), we then investigated whether IK<sub>Ca1</sub> associated with TRPV6. Co-immunoprecipitation of LNCaP cell extracts by IK<sub>Ca1</sub> antibodies and western blot analysis of the immunoprecipitated proteins by TRPV6 antibodies (Figure 7e) showed that IK<sub>Ca1</sub> was associated with TRPV6. Similarly, when the LNCaP total protein extract was processed with the anti-TRPV6 antibody for co-immunoprecipitation, immunoblotting using IK<sub>Ca1</sub> antibodies identified the IK<sub>Ca1</sub> protein among the co-immunoprecipitated proteins (Figure 7e, right panel). In these experiments, negative controls consisted of incubation of the beads with the cell lysate (without any antibodies) to detect nonspecific adherence of proteins to the beads. When the extracts from siTRPV6-treated LNCaP cells were used, no TRPV6 protein band was observed in the western blot of the IK<sub>Ca1</sub>-immunoprecipitated proteins, confirming the specificity of the association between IK<sub>Ca1</sub> and TRPV6 channels (data not shown).

The involvement of the IK<sub>Ca1</sub> and TRPV6 calcium channels in PCa cell proliferation was then studied by using the siRNA targeting the mRNA of these channels (siIK<sub>Ca1</sub> and siTRPV6). When cells were incubated in the presence of either siIK<sub>Ca1</sub> (20 nM) or siTRPV6 (20 nM), the cell proliferation was inhibited by 50 ± 6% (siTRPV6) and 60 ± 11% (siIK<sub>Ca1</sub>). In these experiments, when the cells were incubated in both siRNA (siIK<sub>Ca1</sub> + siTRPV6), no additional inhibition of cell proliferation was observed (60 ± 7%) (Figure 7f) compared with using individual siRNAs. These observations suggest that these ion channels are involved in the same transduction pathway leading to the proliferation of PCa cells.

Discussion

This study reports on the expression, functionality and involvement in proliferation of the IK<sub>Ca1</sub> potassium channel in human PCa cells. For the first time, we showed IK<sub>Ca1</sub> channel expression at mRNA and protein levels in these cells. Moreover, we showed the essential upregulation of IK<sub>Ca1</sub> expression in human PCa vs non-PCa (that is, HBP) tissues and showed that this channel was functional in PCa cell lines as well as primary cultured hPCE cells. This study also showed that IK<sub>Ca1</sub> channel is critically involved in the calcium-induced hyperpolarization of the membrane potential of PCa cells.

Using TRAM-34, a specific IK<sub>Ca1</sub> inhibitor and IK<sub>Ca1</sub>-targetting siRNA, we showed that this channel appears to play an important role in PCa cell proliferation. The involvement of IK<sub>Ca1</sub> in cell proliferation has also been shown using other cancer cells, using either CLT or TRAM-34 (Jager *et al.*, 2004) or IK<sub>Ca1</sub>-targetting siRNA (Si *et al.*, 2006). There is evidence that IK<sub>Ca1</sub> is upregulated in primary pancreatic cancer tissues (Jager *et al.*, 2004) and that its expression increases in response to mitogenic activation of the Ras/extracellular signal-regulated kinase signaling pathway (Pena *et al.*, 2000). Our findings are consistent with these observations, as well as providing new evidence of enhanced IK<sub>Ca1</sub> expression in human PCa at the mRNA and protein levels. This study also brought substantial evidences in the elucidation of the mechanisms by which IK<sub>Ca1</sub> modulates PCa cell growth. The expression of another calcium-activated potassium channel (BK channel) in PCa cells was recently described by Bloch *et al.* (2007). In this study, the BK potassium channel was involved in the basal cell proliferation of PC-3 cells but not of LNCaP cells, suggesting that in the LNCaP cells, only IK<sub>Ca1</sub> calcium-activated potassium channel is involved, whereas in PC-3 cells, both IK<sub>Ca1</sub> and BK channels participate in the modulation of the cell growth.

Cells are known to be depolarized in the early G₁ phase, but hyperpolarized during the progression through G₁ into the S phase (Ghanshani *et al.*, 2000; Kunzelmann, 2005). K⁺-channel inhibition was shown to lead to membrane depolarization and cell-cycle arrest in the early G₁ phase in the MCF-7 human breast carcinoma cell line (Wang *et al.*, 1998). Conversely, K⁺-channel activation promotes cell cycle progression from the G₀/G₁ to S phases (Wonderlin and Strobl, 1996; Ouadid-Ahidouch *et al.*, 2001; Chittajallu *et al.*, 2002). Here, we showed that IK<sub>Ca1</sub> channel inhibition in human PCa cells resulted in growth arrest and an accumulation of p21<sup>Cip1</sup>, but not p27<sup>Kip1</sup> cell cycle regulator. Cell cycle proteins, in particular cdkis that regulate G₁ cyclins, are likely to be implicated in withdrawal of the cell cycle. Among these cdkis, p21<sup>Cip1</sup> is involved in cell cycle exit and terminal cell differentiation (Rodrigues *et al.*, 1987). In agreement with these findings, our results showed that the inhibition of PCa cell growth induced by the IK<sub>Ca1</sub> inhibitor was likely to involve the accumulation of
p21<sup>Cip1</sup> and cell arrest in the G1 cycle. However, the mechanism by which IK<sub>Ca1</sub> channel activity affects the accumulation of cdkis is not known. A recent study shows a correlation between CDK4 expression and activation of the calcium-dependent gene expression pathways involving calcineurin/NFAT (nuclear factor of T lymphocytes) (Baksh *et al.*, 2002). Recent data suggest the involvement of the S100 calcium-binding proteins in the link between calcium and the expression of p21. The S100 proteins are derived from the expression of 17 genes clustered on human chromosome

1q21, a region frequently rearranged in several types of cancer. Several S100 proteins (S100A2, S100A4 and S100B) are described to be involved positively (S100A2) or negatively (S100A4 and S100B) in the p53-dependent expression of p21. In low intracellular conditions, S100A2 binds p53 to form a transcription factor interacting with p21 promoter to induce the p21 expression. In high intracellular conditions, S100A4 or S100B binds to p53 to inhibit the interaction of the transcription factor with the p21 promoter leading to the suppression of the p53-dependent expression of p21.

a

<figure><img src="image_15.png" /></figure>

c

<figure><img src="image_16.png" /></figure>

e

<figure><img src="image_17.png" /></figure>

b

<figure><img src="image_18.png" /></figure>

d

<figure><img src="image_19.png" /></figure>

f

<figure><img src="image_20.png" /></figure>
It was also suggested that a complex of S100A4 with p53 and the sequestration of p53 may result in a stimulation of the cells to enter the S phase by abrogating the control functions of p53 at the G1-S checkpoint perhaps through the modulation of p21 expression (Sherbet and Lakshmi, 1998; Grigorian *et al.*, 2001). On the other hand, the expression of these S100 proteins was shown to be modulated in human PCa (Gupta *et al.*, 2003). In these studies, a progressive loss of S100A2 protein was observed in cancer specimens with increasing tumor grade, whereas S100A4 protein was upregulated. This upregulation of S100A4 would be an advantage for PCa cells to inhibit the expression of p21 in the presence of increased intracellular calcium, probably due to enhanced influx through TRPV6 promoted by the activation of IK<sub>Ca1</sub>. In this context, inhibiting IK<sub>Ca1</sub> channels may induce membrane depolarization, thus reducing the driving force for Ca²⁺ influx, leading to G₀/G₁ arrest and inhibition of cell proliferation. Therefore, IK<sub>Ca1</sub> channel inhibitors show some potential adjuvant chemotherapeutic reagents in anti-PCa therapy.

Cell cycling involves membrane potential hyperpolar-
ization and calcium entry, especially in G1 and the G1/S
transition. IK<sub>Ca1</sub> channels may be important for keeping
the cells hyperpolarized to drive a passive Ca²⁺ influx.
Calcium ions regulate virtually all cell processes,
including survival, proliferation, apoptosis and differ-
entiation (Berridge *et al.*, 2003; Kunzelmann, 2005).
Intracellular Ca²⁺ is known to play a role in cell
proliferation and division in numerous cell types
(Tomita *et al.*, 1987; Magnier-Gaubil *et al.*, 1996;
Spitzner *et al.*, 2007) including PCa cells (Legrand
*et al.*, 2001; Thebault *et al.*, 2003; Thebault *et al.*, 2006).
For example, it has also been shown that calcium
signaling is required for cell cycle progression from
the G1/S phase through to mitosis (Hazelton *et al.*, 1979;
Tupper *et al.*, 1980). Mitogenesis is accompanied by an
increase in intracellular Ca²⁺, so modifying intracellular
Ca²⁺ alters proliferation and mitogenesis (Enomoto
*et al.*, 1998; Ducret *et al.*, 2002).

Using electrophysiological and calcium imaging
techniques, we showed that activating the IK<sub>Ca1</sub>
channels hyperpolarized membrane potential and pro-
moted passive calcium entry in human PCa cell lines,

probably by providing or maintaining a sufficiently
large driving force for Ca²⁺ entry. This results in the
high [Ca²⁺]ᵢ levels necessary for cell-cycle progression,
as proposed by Nilius *et al.* (1993). Similar observations
were made in the human erythroleukemia cell line, in
which it was shown that IK<sub>Ca1</sub> channels enhanced Ca²⁺
entry by membrane hyperpolarization (Lu *et al.*, 1999)
or in colonic carcinoma cells in which the voltage-
dependent K⁺ channel, hEAG, was shown to be
implicated in the agonist-induced calcium signaling
and cell growth (Spitzner *et al.*, 2007).

We further identified the TRPV6 calcium channel as a
major provider of passive calcium influx in response to the
hyperpolarization associated with IK<sub>Ca1</sub> channel activa-
tion. In an earlier study, we showed the contribution of
endogenous TRPV6 to store-operated Ca²⁺ entry in
LNCaP cells (Vanden Abeele *et al.*, 2003). A recent study
showed the involvement of TRPV6 in the control of PCa
LNCaP cell proliferation (Lehen'kyi *et al.*, 2007). In these
studies, the authors showed that the calcium entry
through TRPV6 channel in LNCaP cells induced a
subsequent downstream activation of NFAT leading to
cell proliferation and apoptosis resistance.

Here, we show a novel role for TRPV6 in maintaining
the hyperpolarization-evoked Ca²⁺ entry required for cell
proliferation. Our data indicated that, when TRPV6 was
downregulated in PCa cells (by the use of siRNA), Ca²⁺
influx no longer occurred in response to IK<sub>Ca1</sub> activation.
Moreover, the immunoprecipitation experiments indi-
cated a close physical interaction between the IK<sub>Ca1</sub> and
TRPV6 channels in PCa cells, although the type of
coupling between them requires further elucidation.

Our immunostaining studies performed on PCa
tissues (Figure 1e) show that the IK<sub>Ca1</sub> potassium
channel appears to be more expressed in the apical side
of the cell membrane; however, the expression is present
in both apical and basal sides of the acinar cells. TRPV6
channel has been shown earlier to be expressed in the
apical membrane of intestinal epithelial cells (van de
Graaf *et al.*, 2006) in which it plays a role of the ‘Ca²⁺
uptake channel’ necessary for the calcium re-absorption.
Therefore, it is rather possible that in prostate cells,
TRPV6 channel is also expressed on the luminal apical
membrane of the secretory epithelial cells. Cell culture

**Figure 7** IK<sub>Ca1</sub> activation induces calcium entry through TRPV6 calcium channel. TRPV6 cation channel mRNA expression was studied by RT-PCR (a) in LNCaP and PC-3 human prostate cancer cell lines. RT-PCR and semi-quantitative RT-PCR were performed as described in Figure 1 and 3. Total RNA was reverse transcribed and used in PCRs to study the expression of TRPV6 mRNA (255 bp) in LNCaP and PC-3 cells (a) and in LNCaP cells treated either by a non-targeting siRNA (siCTL) for 72 h or TRPV6 targeting siRNA (siTRPV6) for 24, 48 and 72 h (b). β-Actin mRNA expression (212 bp) was considered as an internal standard. PCR products were analysed on 1.5% agarose gel containing 0.5 µg/ml ethidium bromide and visualized under ultraviolet. Calcium imaging experiments using fura-2 as a calcium probe were performed to study the effects of the IK<sub>Ca1</sub> activator, 1-EBIO (200 µM), on [Ca²⁺], in (c) LNCaP and (d) PC-3 cell lines. The involvement of the TRPV6 cation channel in the 1-EBIO-induced [Ca²⁺]ᵢ increase was assessed by pretreating cells with TRPV6 siRNA (20 nM) for 72 h. Following pre-treatment of the cells with TRPV6 siRNA, 1-EBIO failed to induce a calcium increase in (e) LNCaP and (f) PC-3 cells. A representative trace of at least 50 cells is shown for calcium imaging experiments and each experiment was carried out at least four times with two independent cell cultures. (g) IK<sub>Ca1</sub> associated with the TRPV6 channel in LNCaP cells. TRPV6 or IK<sub>Ca1</sub> western blot (WB) of co-immunoprecipitated (IP) LNCaP total protein extracts using either anti-IK<sub>Ca1</sub> (left panel) or anti-TRPV6 (Alomone Labs, Jerusalem, Israel) antibodies (right panel). (h) To study the involvement of IK<sub>Ca1</sub> and/or TRPV6 in LNCaP cell proliferation, the cells were transfected with either non-targeting siRNA (siCTL) or with siRNA targeting TRPV6 (siTRPV6) or IK<sub>Ca1</sub> (siIK<sub>Ca1</sub>) or with both of them as described above and incubated for 3–4 days. *P*<0.001 compared with siCTL. RT-PCR, reverse transcription-PCR; siRNA, small interfering RNA; 1-EBIO, 1-ethyl-2-benzimidazolinonel; IK<sub>Ca1</sub>, intermediate-conductance calcium-activated potassium channel.
models generally fail to preserve many of the characteristics, such as the formation of functional tight junctions and apical and basolateral membrane domains through cell polarization. The latter process is shown to affect the gene expression and the distribution of the membrane proteins at the cell level (Gut *et al.*, 1998; Turowski *et al.*, 2004). Further studies are needed to identify the distribution of TRPV6 and IK<sub>Ca1</sub> in polarized prostate cells and to understand the physiological role of localization of channels.

In conclusion, we showed for the first time that IK<sub>Ca1</sub> channels are active players in PCa cells, probably responsible for determining their malignant potential. Upregulation of this channel in PCa vs non-PCa tissues may represent an advantage for cancer cells by promoting an intracellular calcium increase through the TRPV6 channel, also known to be upregulated in PCa (Peng *et al.*, 2001). In light of these findings, the IK<sub>Ca1</sub> channel may be added to the list of potential targets for future PCa therapies.

## Materials and methods

### Cell culture

LNCaP, PC-3 and DU-145 PCa cell lines, obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), were cultured as described by Gackiere *et al.* (2006). Primary PCa epithelial cells (hPCE) were obtained and cultured as described earlier (Bidaux *et al.*, 2005).

### Tissue specimens

Human PCa and BHP biopsies were obtained from consenting patients following the local ethical considerations. All experiments involving patient tissues were carried out under approval number 'CP 01/33', issued by the 'Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale de Lille'. For more details, see Supplementary information.

### RT-PCR analysis of mRNA expression

Total RNA isolation and RT-PCR experiments were performed as described earlier (Chomczynski and Sacchi, 1987; Roudbaraki *et al.*, 1999). The PCR primers used in this study, listed in Table 1, were designed on the basis of established GenBank sequences and synthesized by Invitrogen (Carlsbad, CA, USA). For more details, see Supplementary information.

### Immunofluorescence studies

For more details, see Supplementary information.

### siRNA cell transfection

siRNAs were synthesized by Dharmacon Inc. (Waltham, MA, USA). LNCaP and PC-3 cells were transfected overnight with 20-40 nM of control siRNA (siRNA number 1, Dharmacon Inc.) or raised against either TRPV6 mRNA (siTRPV6(1): GACUCUCUAUGACCUCACA, nt 1041-1059; siTRPV6(2): CCUGCUGCAGCAGAAGAGG, nt 327-345, accession number NM_018646) or IK<sub>Ca1</sub> mRNA (siIK<sub>Ca1</sub>(1): UGUA AAGCUUGGCCACGAAC, nt 975-994; siIK<sub>Ca1</sub>(2): CCUG UUCCUGGUAAAUGC, nt 567-585, accession number NM_002250) using GenePorter transfection reagent (Gene Therapy Systems Inc. (GTS), San Diego, CA, USA) in serum-free Dulbecco's modified Eagle's medium, according to the manufacturer's instructions. The cells were then incubated in RPMI containing 10% fetal calf serum (FCS) for 3 days to downregulate the TRPV6 or IK<sub>Ca1</sub> mRNA before use in experiments. The efficiency of both siRNA (1) and (2) to downregulate the siIK<sub>Ca1</sub> or the TRPV6 was equivalent as verified at the channel expression and functionality level (see Supplementary information, Figures 2 and 3). The siIK<sub>Ca1</sub>(1) and siTRPV6(1) were then used in the studies described in this study.

### Western blot analysis and immunoprecipitation

Western blot analysis was performed as described by Vanoverberghe *et al.* (2004). For more details, see Supplementary information.

### Cell proliferation and apoptosis assays

Cell growth and apoptosis assays were performed as described earlier by Vanoverberghe *et al.* (2003). For more details, see Supplementary information.

### [Ca²⁺]ᵢ measurements

[Ca²⁺]ᵢ imaging experiments were performed as described earlier (Gackiere *et al.*, 2006). For more details, see Supplementary information.

### Electrophysiological recordings

Patch-clamp recordings were performed in the whole-cell configuration as described earlier (Mariot *et al.*, 2002). For more details, see Supplementary information.

### Statistical analysis

Plots were produced using Origin 5.0 (Microcal Software Inc., Northampton, MA, USA). Results are expressed as mean ± s.e. Statistical analysis was performed using unpaired t-tests or analysis of variance tests followed by either Dunnett (for multiple control versus test comparisons) or Student–Newman–Keuls post-tests (for multiple comparisons).

<table><caption>**Table 1** List of primers used for RT-PCR studies</caption><thead><tr><th>Gene</th><th>5'-forward-3'</th><th>5'-reverse-3'</th><th>Expected RT-PCR product size (bp)</th><th>Accession number</th></tr></thead><tbody><tr><td>IK<sub>Ca1</sub></td><td>ACTGGTTCGTGGCCAAGCTTTACA</td><td>TCCAGCAGCCATCAGCAAGCCA</td><td>867</td><td>NM_002250</td></tr><tr><td>TRPV6</td><td>ATGGTGATGCGGCTCATCAGTG</td><td>GTAGAAGTGGCCTAGCTCCTCG</td><td>255</td><td>XM_001126541</td></tr><tr><td>P21</td><td>GATGAGTTGGGAGGAGGCAG</td><td>CGTTTGGAGTGGTAGAAATCTG</td><td>206</td><td>NM_078467</td></tr><tr><td>P27</td><td>CAGCTTGCCCGAGTTCTACTACAGA</td><td>GATTTTCTTCTGTTCTGTTGGC</td><td>274</td><td>NM_004064</td></tr><tr><td>β-Actin</td><td>CAGAGCAAGAGAGGCATCCT</td><td>GTTGAAGGTCTCAAACATGATC</td><td>212</td><td>NM_001101</td></tr></tbody></table>

Abbreviations: IK<sub>Ca1</sub>, intermediate-conductance calcium-activated potassium channel; RT-PCR, reverse transcription-PCR.
Student's *t*-test was used for statistical comparison of the
differences and *P*<0.05 was considered significant.

Abbreviations

1-EBIO, 1-ethyl-2-benzimidazolinone; BK, Ca²⁺-activated K⁺ channel with big conductance; CCE, capacitative calcium entry; IKCa1, Ca²⁺-activated K⁺ channel with intermediate conductance; RT-PCR, reverse transcription-PCR; TRAM-34, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole.

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We thank Dr Y Shuba (Bogomoletz Institute of Physiology, Kiev, Ukraine) for helpful discussions. This study was supported by grants from INSERM, the Ministère de l'Education Nationale de l'Enseignement Supérieur et de la Recherche, La Ligue Nationale Contre le Cancer. H Lallet-Daher was supported by the Association pour la Recherche sur les Tumeurs de la Prostate (ARTP). We also thank Dr Mao Xiang Chen (Laboratory of Gene Expression and Protein Biochemistry, GlaxoSmithKline R&D, UK) for the generous gift of the IK<sub>Ca1</sub> antibodies.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)