Pivotal Role of the Lipid Raft SK3–Orai1 Complex in Human Cancer Cell Migration and Bone Metastases

Published OnlineFirst June 17, 2013; DOI: 10.1158/0008-5472.CAN-12-4572

Cancer Therapeutics, Targets, and Chemical Biology Research

Aurelie Chantome^ 1, Marie Potier-Cartereau1, Lucie Clarysse1,Gaelle€ Fromont5,6, Severine Marionneau-Lambot7, Maxime Gueguinou 1, Jean-Christophe Pages 2,3, Christine Collin3, Thibauld Oullier7, Alban Girault1, Flavie Arbion3, Jean-Pierre Haelters8, Paul-Alain Jaffres 8, Michelle Pinault1, Pierre Besson1, Virginie Joulin9, Philippe Bougnoux1,4, and Christophe Vandier1

Abstract The SK3 channel, a potassium channel, was recently shown to control cancer cell migration, a critical step in metastasis outgrowth. Here, we report that expression of the SK3 channel was markedly associated with þ bone metastasis. The SK3 channel was shown to control constitutive Ca2 entry and cancer cell migration þ through an interaction with the Ca2 channel Orai1. We found that the SK3 channel triggers an association with the Orai1 channel within lipid rafts. This localization of an SK3–Orai1 complex seemed essential to control cancer cell migration. This suggests that the formation of this complex in lipid rafts is a gain-of- function, because we showed that none of the individual proteins were able to promote the complete phenotype. We identiﬁed the alkyl-lipid Ohmline as a disrupting agent for SK3–Orai1 lipid raft localization. þ Upon Ohmline treatment, the SK3–Orai1 complex moved away from lipid rafts, and SK3-dependent Ca2 entry, migration, and bone metastases were subsequently impaired. The colocalization of SK3 and Orai1 in primary human tumors and bone metastases further emphasized the clinical relevance of our observations. Targeting SK3–Orai1 in lipid rafts may inaugurate innovative approaches to inhibit bone metastases. Cancer Res; 73(15); 4852–61. 2013 AACR.

Introduction role in regulating neuronal excitability (7). This channel is not The emerging concept of ion channels as key regulators of restricted to neuronal tissues (8), and was found to be cancer expansion (for review; 1–3) has several implications, expressed in smooth muscle, where it regulates smooth muscle – including the potential of their chemical targeting for cancer tone (9 11). Interestingly, the SK3 channel is expressed in treatment. Therefore, a precise understanding of the mechan- tumor breast biopsies and melanoma cells, but its expression isms underlying the role of ion channels in cancer cells is was not observed in nontumor breast tissues and primary paramount. We have recently shown that SK3 (KCNN3 gene), a cultures of melanocytes (6, 12). The lack of effect of SK3 þ potassium channel of the small conductance Ca2 -activated channel expression on cell proliferation (12) led us to inves- fi potassium (KCa) channel family (4), is a mediator of cancer cell tigate whether this speci c role in cell migration conferred this migration (5, 6). The physiologic expression of the SK3 channel channel a role in metastases development. Indeed, the forma- was first studied in central neurons where it has a fundamental tion of secondary tumors from primary sites seems to be a multistep process in which tumor cell migration is a critical event. Authors' Affiliations: 1Inserm, UMR1069; Universite Francois¸ Rabelais; 2 3 4 In this report, we show a role for SK3 in bone metastases, Inserm, U966; Universite Francois¸ Rabelais; CHRU de Tours; Centre HS fi Kaplan, CHRU Tours, Tours, France; 5CHRU de Poitiers; 6Universitede which is the rst report establishing an ion channel as a control Poitiers, Poitiers; 7Cancerop ole^ du Grand Ouest, Nantes; 8Universite factor for bone metastases development. SK3 action proved to Europeenne de Bretagne, Universite de Brest, CNRS UMR 6521, CEMCA, be mediated through an association with Orai1, a voltage- SFR 148 ScInBios, Brest; and 9Inserm, U1009; Institut Gustave Roussy, þ independent Ca2 channel. The SK3–Orai1 complex regulates Villejuif, France þ a constitutive Ca2 entry, calpain activation, and cell migra- Note: Supplementary data for this article are available at Cancer Research – Online (http://cancerres.aacrjournals.org/). tion. At the cellular level, the SK3 Orai complex was localized in lipid rafts. The alkyl-lipid Ohmline disrupted SK3–Orai1 þ A. Chantome^ and M. Potier-Cartereau contributed equally to this work. complexes from lipid rafts and impaired SK3-dependent Ca2 Current address for A. Girault: CRCHUM, Hotel-Dieu,^ 3840, rue Saint- entry, migration, and bone metastases, qualifying this lipid as a Urbain, Montreal (Quebec) H2W 1T8, Canada. potential platform for drug development. Finally, the coloca- Corresponding Authors: ChristopheVandier,Inserm, UMR1069,Universite lization of SK3 and Orai1 in primary human tumors and bone Francois¸ Rabelais, Tours, F-37032 France. Phone: 33-247366024; Fax: 33- metastases from clinical samples emphasized the clinical 247366226; E-mail: [email protected] consistency of these observations. This is the first report doi: 10.1158/0008-5472.CAN-12-4572 showing that the deregulation of an ion channel complex by 2013 American Association for Cancer Research. a lipid could control metastases.

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SK3–Orai1 Channel Complex Promotes Bone Metastasis

Materials and Methods Cell proliferation and migration assays Cell lines Cell proliferation and cell migration were determined as Human breast cancer cell line MDA-MB-435s was purchased described elsewhere (6, 12, 28) and are specified in Supple- from the American Type Culture Collection (LGC Promochem) mentary Methods. and was grown as already described (6). A recent study suggested that the MDA-MB-435s cell line originated from Experimental and spontaneous metastasis models breast tissue (26). This cell line was transduced by a retrovector Mice (Janvier laboratories) were bred and housed at Inserm containing the luciferase gene and with a lentivector contain- U892 (Nantes-University) under the animal care license no. ing either an interfering short hairpin RNA (shRNA) specific 44565. For experimental metastases, 6-week-old female NMRI þ to SK3 (SK3 cells) or a nontargeting shRNA (SK3 cells) as nude mice were used. Unanesthetized mice were placed into previously validated (13). No difference of luciferase expres- a plastic restraining device, and 0.75 106 MDA-MB-435s þ þ sion and activity has been observed between SK3 and SK3 (SK3 /SK3 ) cells were injected into the lateral tail vein in cells (see Supplementary Fig. S1BC). HEK293 and 518A2 cells 100 mL of serum-free Dulbecco's Modified Eagle Medium are described in Supplementary Methods. (DMEM) through a 25-gauge needle. For the mammary fat- pad (MFP) model, female NMRI/nude mice, 3- to 4-weeks old, Immunohistochemistry were used. Mice were anesthetized by intraperitoneal 100 Cells were fixed in 10% formalin, included in gel, and mg/kg ketamine plus 10 mg/kg xylazine administration and þ embedded in paraffin. Murine tissues were fixed in 10% a right fat pad was cleared. Subconfluent SK3 and SK3 cells formalin and embedded in paraffin, with a mild decalcification were harvested, washed in PBS, and 2 106 cells were injected for bone tissues. Tissue microarrays (TMA) were constructed in a volume of 50 mL of DMEM without serum into the cleared from human formalin-fixed tissues obtained from 177 primary fat-pad. Tumor volumes were calculated using the formula: prostate cancers and 37 bone metastases specimens, including length width depth. For MFP-metastases in vivo assays þ 15 prostate cancer metastases and 22 breast cancer metasta- with Ohmline, SK3 cells were incubated with 1 mmol/L ses. Normal prostate and breast tissues were also included in Ohmline or with vehicle (0.6‰ ethanol/0.4‰ dimethyl sulf- the TMAs. All primary prostate cancers were of the acinar type, oxide) for 24 hours and injected into the cleared fat pad. with 59 having a Gleason score of 6, 106 with a Gleason score of Mice were treated 3 times a week for 15 weeks with Ohmline 7, and 12 with a Gleason score of 8 and more; 137 tumors were at 15 mg/kg or with vehicle administered intravenously. Primary pT2 and 40 pT3. Among the 20 breast cancer bone metastases, tumors were removed when the volume reached 400 mm3.In 16 expressed estrogen receptors, 11 progesterone receptors, control animals, we have not observed adverse effects upon and 3 were positive for Her2; 5 tumors were triple negative. For Ohmline administration (no compartmental, weight growth each tumor, 4 cores (0.6-mm diameter) were included in the abnormalities, or liver and heart toxicities were observed after TMA, as previously described (27). Immunohistochemical necropsy; ref. 13). This absence of side effects is explained staining was conducted on 3-mm slides from embedded cell by the low and noncytotoxic concentration of Ohmline used and lines, xenografts and TMA, using anti-Ki67 (DakoCytomation), the selective effect of this lipid on SK3 channel. The materials anti-SK3 channel (Sigma, P0608, dilution 1/50), and anti-Orai1 and methods used for bioluminescence imaging (BLI) are de- (Life Span Bioscience, dilution 1/4,000). scribed in Supplementary Methods.

Electrophysiology Membrane fractionation, immunoﬂuorescence, and All experiments were conducted using whole-cell recording incorporation of Omhline in tissues conﬁguration of the patch-clamp technique, as previously The materials and methods used are described in Supple- described (6, 12) and as described in Supplementary Methods. mentary Methods (29–31).

þ Intracellular Ca2 measurements Statistics Cells were loaded in Petri dishes for 45 minutes at 37 C with Data were expressed as median with quartile or mean the ratiometric dye Fura2-AM (5 mmol/L). Then, cells were SEM (N, number of experiments; n, number of cells). Statistical trypsinized, washed with Opti-MEM Reduced Serum Medium, analyses were made using the unpaired Student t test or the GlutaMax (Life-Technologies), and centrifuged (800 g for 5 Mann–Whitney test. For comparison between more than minutes). Immediately after centrifugation, cells were resus- 2 means, we used Kruskal–Wallis one-way ANOVA followed þ pended at 1 106 cells in 2 mL PSS Ca2 -free solution. Fluores- by Dunn test. Differences were considered signiﬁcant when cence emission was measured at 510 nm with an excitation light P < 0.05 (SigmaStat, Systat Software and Minitab software, at 340 and380 nm(Hitachi FL-2500). See SupplementaryFig. S3A Minitab Inc.) þ for the validation of constitutive Ca2 entry protocol used. Results and Discussion Western blot, reverse transcriptase qPCR, and calpain The SK3 channel controls bone metastasis development activity assay To investigate the role of SK3 in metastases development, Western blot experiments were conducted as described (6). we engineered luciferase SK3-positive, MDA-MB-435s breast The antibodies and the materials and methods used are cancer-derived cells. Using speciﬁc shRNA knockdown of the described in Supplementary Methods. KCNN3 gene product, we obtained SK3 cells; control cells

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þ þ receiving a random shRNA remained SK3 . Compared to SK3 channels. In active bone resorptive lacunae, osteolysis arose cells, SK3 cells displayed almost no outward current, their in legs and rachis of the 2 metastases models (Fig. 2A), þ plasma membrane was more depolarized, and they exhibited a extracellular Ca2 concentrations could be as high as 8 to lower migration capacity although their proliferation was not 40 mmol/L, whereas in the vicinity of unaltered bone surface, it affected (Fig. 1A and Supplementary Fig. S1). Next, we inves- is normally closer to 2 mmol/L (19). In vitro, changing the þ tigated SK3 function using a cancer cell xenograft model in extracellular Ca2 concentration from 2 to 5 mmol/L led to an þ NMRI/nude mice (Fig. 1B). Silencing of the SK3 channel led to a increase in the migration of SK3 cells, an effect not observed lower composite metastatic score, based on the number of for SK3 cells (Fig. 2B). Because we showed that SK3 channel metastases per mouse and on the intensity of the biolumines- control cancer cell migration by hyperpolarizing the plasma cent signal per metastasis (Supplementary Fig. S2A; ref. 13). membrane of cancer cells (12), we tested the effect of increas- þ Interestingly, this lower score essentially reflected a lower ing external Ca2 concentration on the membrane potential of bone metastases development in SK3 -grafted mice compared wild-type MDA-MB-435s cells. Figure 2C shows the outward þ to SK3 -grafted mice (Fig. 1B). Conversely, the lung biolumi- potassium currents recorded using a ramp protocol from 70 nescent signal intensity was not significantly different between to þ70 mV: within 2 minutes, the amplitude of potassium þ SK3 - and SK3 -grafted mice (Supplementary Fig. S2B). At currents increased, leading to a shift of the membrane poten- week 9, bone metastases were detected in 83% (10 of 12) of tial toward more negative values (membrane hyperpolariza- þ the mice injected with SK3 cells but only in 36% (4 of 11) of tion). The apamin-sensitive current carried by the SK3 channel the mice injected with SK3 cells (Fig. 1B, middle). Moreover, was assessed in cells incubated in PSS solution with 2 or 5 þ þ the intensity of the bioluminescent signal was significantly mmol/L extracellular Ca2 (Fig. 2C). Increasing external Ca2 þ different between SK3 - and SK3 -grafted mice (Fig. 1B, concentrations more than doubled the amplitude of SK3 bottom). Consistent with in vivo observations, both the fre- currents, leading to a 20 mV membrane hyperpolarization quency of bone metastases (100% vs. 54%) and the intensity of (Fig. 2C). Interestingly, we noticed that SK3 hyperpolarization þ þ the bioluminescent signal, detected ex vivo at necropsy, were promoted Ca2 entry and, thus, elevated intracellular Ca2 þ þ lower in mice injected with SK3 as compared to SK3 cells concentration by increasing the Ca2 -driving force (Fig. 2D). þ (Supplementary Fig. S2C). Hence, a physiologic 2 mmol/L extracellular Ca2 concentra- These observations did not examine the impact of SK3 tion would activate the SK3 channel, which could be over- þ channel on the primary tumors in relation to metastatic activated by higher extracellular Ca2 concentrations. Of note, þ development. Other channels, such as hEag1 (voltage-gated activated SK3 channels increased the activity of the Ca2 potassium channel), IKCa (intermediate conductance KCa -sensitive protease calpain (Fig. 2E), a factor contributing to channel) or TRPV2 (transient receptor potential V2) have been many aspects of cell migration, such as cell spreading, mem- reported to influence the volume of subcutaneously xeno- brane protrusion, chemotaxis, and adhesion complex forma- grafted tumors, by acting on their proliferation and/or migration and turnover (20). In addition, the proteolysis of the tion capacities (14–16). Because ectopic tumor models could calpain target talin is promoted by SK3 expression and is þ not accurately reflect the metastatic potential of tumor cells, increased by A23187 and/or by high external Ca2 concentra- þ we used an orthotopic mammary-tumor model known to tions (Fig. 2E), conditions that increase intracellular Ca2 support the development of metastases in several tissues. We concentrations. Because calpain activation is a critical step þ grafted SK3 or SK3 cells into the mammary fat pad (MFP) of leading to adhesion complex turnover and cell migration (20), NMRI/nude mice (17, 18). SK3 channel suppression did not we can hypothesize that at least part of the role of SK3 in influence primary tumor growth, and the proliferation index migration could be attributed to by calpain activation. (Ki67 staining) was identical in the 2 groups of mice (Fig. 1C). þ Importantly, SK3 tumors were still positive for SK3 staining, SK3 action is mediated through its association with the whereas SK3 tumors remained negative (Fig. 1C), confirming Orai1 channel, forming a lipid-raft SK3–Orai1 complex þ the stability of the SK3 phenotype following grafting. Metas- We next aimed at identifying the Ca2 channel involved in þ þ tases occurred in both groups and were mainly observed in Ca2 entry. The voltage-independent Ca2 channel Orai1, and bones and lungs. However, the bioluminescent signal was weak its regulator STIM1, have been shown to be store-operated in bones (Fig. 1C) but not in lungs of SK3 -grafted mice channels (SOC) in breast cancer cells and have been implicated (Supplementary Fig. S2D). This suggested that SK3 channel in cancer cell migration (21) and calpain activation (22). Orai1 expression in cancer cells affected their ability to form metas- knockdown totally abolished SK3-dependent cell migration tases in bone but not in lung. (Fig. 3A). The suppression of STIM1 had no effect on MDA-MB- 435s cell migration (Fig. 3A) in contrast to the MDA-MB231 þ External Ca2 elevation upregulates SK3 channel activity breast cancer cell line (21) that did not express SK3 protein (6). þ and activates Ca2 entry promoting calpain activation These results suggest a role for Orai1 channels in constitutive þ and cell migration SK3-dependent Ca2 entry, independently of STIM1 (see Sup- These findings suggest that SK3 channel might contribute plementary Fig. S3A for the validation of the constitutive þ to/or facilitate bone metastases. As an interaction with the Ca2 entry protocol used). Consistently, the inhibition of bone microenvironment could influence SK3 activity and Orai1, either by siRNA, shRNA (with 2 different sequences) or þ because this channel is Ca2 sensitive (5), we evaluated the by using 2-APB, totally abolished SK3-dependent constitutive þ þ effect of external Ca2 concentrations in modulating SK3 Ca2 entry and the increase of cancer cell migration observed

4854 Cancer Res; 73(15) August 1, 2013 Cancer Research

SK3–Orai1 Channel Complex Promotes Bone Metastasis

AB C + – SK3 SK3 Tail vein injection SK3+ SK3– a 1342 a

515.0516.01.00.0 msm 22.621.4-1.20.0 pApA

516.0515.01.00.0 msms -32.7-29.3-3.40.0 pApA 200 pA 100 ms SK3

0 0 Lung X 5 X 40 X 40 b SK3+ SK3–

) 2 3 SK3 + (N = 12) SK3 75 KDa 1.5 SK3 – (N = 13)

1 luc 61 KDa Bone X 4 0.5 Actin

43 KDa Tumor (cm volume b 0 Lungs + 100 SK3 0246 81012141618

1.0 ) Weeks post-graft 6

10 –

0.8 X SK3 SK3+ SK3– Mann–Whitney c 0.6 P < 0.0001 Mann–Whitney 0.4 50 P = 0.372 Ki 67 0.2 X 20 X 20

Cell normalized migration, 0.0 SK3+ SK3- (N = 8) (N = 8) in Photons flux lungss (

0 SK3+ SK3– 12 SK3+ (N = 3) c (N = 12) (N = 11) metastasis SK3– (N = 3) 10 Bone 100 Orthotopic tumour 8 SK3+ 6 80 (N = 12) SK3+ SK3– 4 60

Proliferation rate d Photon flux x Photon flux 10 2 SK3– Forelegs 40 4 )

Mice with Mice with Log rank test (N = 11) 0 3

01234 P = 0.032 10 X

bone metastases, % bone metastases, 20 Days 3 3 0 Hind legs 0 246810 Weeks 2 d SK3+ SK3- Mann–Whitney P = 0.008 1 3 ) 6 Photons flux in Photons flux bones ( 10 X 0 2 SK3+ SK3– (N = 24) (N = 26)

Mann–Whitney 1 P = 0.002 SK3+

Photons flux in Photons flux bones ( 0 X 20 SK3+ SK3– Bone metastasis Photon flux 106 (N = 12) (N = 11)

Figure 1. SK3 suppression inhibited bone metastases. A, validation of the MDA-MB-435s cell system expressing the luciferase gene and expressing or þ not the KCNN3 gene. Whole-cell SK3-current recorded on MDA-MD-435s-shRD (SK3 ) and MDA-MD-435s-shSK3 (SK3 ) (top). Representative recordings from at least 5 cells in each group. Validation of SK3 protein extinction in SK3 cells and luciferase expression in SK3þ and SK3 cells (middle). Representative immunoblots from at least 3 different experiments. Cell migration and proliferation in SK3þ and SK3 cells (bottom). Histograms showing analyses of migration 24 hours after seeding. Data were normalized to results obtained with SK3þ cells. Columns, mean; bars, SEM. Graph showing proliferation rates evaluated by MTT assays, daily, for 4 days. Points, mean; bars, SEM. N, the number of independent experiments. B, SK3 knockdown inhibits bone metastases. Lung and bone metastases observed 9 weeks after tail vein injection of SK3þ cells assessed by BLI in vivo and by hematoxylin and eosin staining (a); BLI quantification of excised lungs (b); BLI assessment of bone metastases likelihood in mice (c); intensity of the bioluminescence signal monitored 9 weeks postinjection (d, right); and BLI of representative mice with spinal column metastases (d, left). N, the number of mice. Box plots indicate the first quartile, the median, and the third quartile, squares indicate the mean. C, breast primary tumor growth is not influenced by SK3 channel. Representative SK3 immunostaining in the primary tumor tissues from mice orthotopically grafted with SK3þ and SK3 cells (a). Graph showing mammary tumor growth in SK3þ- and SK3-grafted mice (b). Ki67 staining of primary tumor tissue sections from mice grafted with SK3þ or SK3 cancer cells, 16 weeks post-graft (c). Hematoxylin and eosin sections of bone metastases and BLI quantification of excised legs (d). Box plots indicate the first quartile, the median, and the third quartile, squares indicate the mean. N indicates the number of mice.

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Mann–Whitney A B P < 0.0001 X-ray scanner X-ray radiography 1.6 1.6

SK3+ cells SK3– cells 1.4 (N = 6) 1.4 (N = 3)

1.2 1.2 Mann–Whitney P = 0.4799 1.0 1.0 Cell migration, normalized Cellmigration, normalized 0.8 0.8 0.0 0.0

2 mmol/L Ca2+ 5 mmol/L Ca2+ 2 mmol/L Ca2+ 5 mmol/L Ca2+

C Current amplitude (pA) D Ca2+ free 2+ 2 mmol/L external [Ca ] 1.2 Mann–Whitney 8 P = 0.018 1,500 5 mmol/L Ca2+ 7 1.0 12 SK3+ 6 8 0.8 5Ca 5 1,000 2 mmol/L Ca2+ 4 (n = 5) 4 0.6 Mann–Whitney 2 Ca P = 0.0051 pA/pF at +25 mV (n = 7) 3

0 F340/F380 500 SK3– 0.4 Apamin-sensitive current 2

1 normalized F340/F380, 0.2 Em (n = 5) N = 5 N = 4 0 -60 -40 -20 0 20 40 60 04080120160 0.0 SK3+ SK3– Em Membrane potential (mV) Time (sec)

E Kruskal-Wallis P = 0.005 1,2 and post hoc Mann– HEKSK3– cells HEK SK3+ cells Whitney (compared to 518A2 cells control) SK3 + (N = 4) – + + – + + 5 µmol/L A23187 1,0 SK3 – (N = 4) ––+ ––+ 10 mmol/L Ca2+ 0,8 P = 0.034 0,6 235 KDa Talin 200 KDa talin cleavage product P = 0.034 0,4 P = 0.034 Calpain activity 0,2

fluoresence, normalized) Hsc70

( 81 KDa 0,0

Z-LLY-FSK – + – + calpain inhibitor

þ þ Figure 2. External Ca2 elevation upregulated SK3 channel activity and activated Ca2 entry promoting calpain activation and cell migration. A, osteolytic lesions in mice receiving SK3þ cells. Representative X-ray scanner of a vertebrae 9 weeks after the injection of cells in the tail vein and X-ray radiography of the hind limbs 16 weeks after the injection of cells in MFP. Osteolytic lesions are indicated by the arrows. B, external Ca2þ elevation promoted SK3-dependent cell migration. SK3þ and SK3 cell migration recorded with 2 and 5 mmol/L external Ca2þ concentration. Data were normalized to conditions obtained with a 2 mmol/L external Ca2þ concentration. C, external Ca2þ elevation increased the amplitude of SK3 currents leading to membrane hyperpolarization. Representative SK3þ whole-cell currents recorded in the presence of 2 mmol/L external Ca2þ concentrations and following the addition of 3 mmol/L external Ca2þ concentrations after 2 minutes (final external Ca2þ concentration ¼ 5 mmol/L). To maintain a constant surface charge, the same concentration of divalent ions in both PSS solutions was used (see Supplementary Methods). Currents were generated by ramp protocol from 100 to þ70 mV in 500 ms from a constant holding of 70 mV and with a pCa7. The arrows indicate membrane potential (Em) values. The inset showing apamin-sensitive current amplitude at þ25 mV in 2 and 5 mmol/L external Ca2þ concentrations. The amplitude of the apamin-sensitive current was obtained by subtraction of the amplitude of the current before and after application of 50 nmol/L apamin (a specific SKCa blocker) in 2 and 5 mmol/L external calcium concentration. D, the SK3 channel promoted Ca2þ entry. Fluorescence measurement (left) and relative fluorescence to Ca2þ entry (right) in SK3þ and SK3 cells. Data were normalized to conditions obtained with SK3þ cells. E, the SK3 channel promoted calpain activity and talin cleavage. Relative fluorescent analyses of calpain activities, measured with a fluorogenic calpain substrate Ac-LLY-AFC, with or without the calpain inhibitor Z-LLY-FMK, in 518A2 cells expressing or not SK3 (left). Data were normalized to results obtained in SK3þ cells without calpain inhibitor. Immunoblots of talin cleavage characteristics of calpain activation in HEK293 cells expressing or not SK3. Cells were preincubated or not for 5 minutes with the Ca2þ ionophore A23187 and treated or not with Ca2þ for 30 minutes. Representative immunoblots from 3 different experiments are shown. Columns, means; bars, SEM. N, the number of independent experiments; n, number of cells.

þ þ at 5 mmol/L Ca2 concentration (Fig. 3B and Supplementary tutive and store-independent Ca2 -signaling that promoted Fig. S3BC). Thus, our ﬁndings revealed a novel signaling cell migration. Having shown that Orai1 was necessary for pathway in which the SK3–Orai1 complex elicited a consti- cancer cell migration, we assessed its cellular localization.

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A SK3+ cells B SK3+ cells

2 mmol/L external [Ca2+ ] 1.2 1.2 P = 0.3314 5 1.0 1 4 Kruskal–Wallis siControl P = 0.001 and post 0.8 0.8 hoc Mann–Whitney Orai 1 38 KDa 3 Mann-Whitney (compared to P = 0.0051 0.6 0.6 control) siOrai1 STIM 1 70 KDa 2

0.4 F340/F380 0.4 1 P = 0.0027 Hsc 70 81 KDa 0.2 0.2 0 F340/F380, normalized N = 4 N = 4 N = 3 N = 3 N = 3 Cell migration, normalized 04080120160 0.0 0 siControl siSTIM 1 siOrai 1 Time (sec) siControl siOrai1

SK3– cells SK3– cells

2 mmol/L external [Ca2+ ] 1.2 Mann–Whitney 1.2 Kruskal–Wallis P = 0.089 5 P = 0.3827 100 1 1.0 4 80 siControl 0.8 0.8 3 60 0.6 0.6 2 siOrai1 40 F340/F380 0.4 1 0.4 (% of SiControl)

Relative mRNA level 20

0.2 0 F340/F380, normalized 0.2 N = 3 N = 3 N = 3 N = 4 N = 3 N = 3 04080120160200 N = 4

Cell migration, normalized 0 0.0 Time (sec) 0 siControl siSTIM 1 siOrai 1 siOrai 1 siSTIM1 siControl siOrai1

C SK3- ATTO-594 T Orai1-FITC D

SK3+ cells SK3– cells Membrane fractions Membrane fractions

Lipid rafts Non-Lipid rafts Lipid rafts Non-Lipid rafts

12 3 45 6 7 8 12 3 45 6 7 8 Caveolin-1 20 kDa

Merge B-adaptin 109 kDa

Orai 1 37 kDa

SK3 75 kDa

þ Figure 3. Lipids raft SK3–Orai1 complex elicited a constitutive and store-independent Ca2 -signaling that promoted MDA-MB-435s cell migration. A, the Orai1 channel was involved in SK3-dependent cell migration independently of STIM1. Histograms showing SK3þ and SK3 cell migration when transfected for 48 hours with siOrai1 or siSTIM1 (left). Validation of Orai1 and STIM1 protein extinction by immunoblots 48 hours after transfection (top, right). Representative immunoblots from 3 different experiments. Validation of Orai1 and STIM1 mRNA extinction by qPCR 48 hours after transfection (bottom, right). B, Orai1 channel controlled a constitutive SK3-dependent Ca2þ entry. Fluorescence measurement (left) and relative ﬂuorescence to Ca2þ entry (right) in SK3þ and SK3 cells transfected for 48 hours with siControl or siOrai1. Data were normalized to results obtained in cells transfected with the siControl. The constitutive Ca2þ entry protocol has been validated in Supplementary Fig. S3A. C, immunocolocalization of SK3 and Orai1 channels. Representative confocal images of SK3 and Orai1 staining conducted in SK3þ cells. Scale bars, 10 mm. D, SK3 and Orai1 channels were colocalized in lipid rafts, and SK3 knockdown moved Orai1 outside of lipid rafts. Immunoblots of Orai1 and SK3 proteins after membrane fractionation of SK3þ and SK3 cells on a sucrose gradient. Caveolin and B-adaptin are markers of lipid rafts and non-lipid rafts, respectively. Representative immunoblots from at least 3 different experiments. Columns, means; bars, SEM. N, the number of independent experiments.

Immunoﬂuorescence analysis showed that SK3 and Orai1 were The alkyl-lipid Ohmline moved the SK3–Orai1 complex þ localized at the plasma membrane (Fig. 3C), and membrane- outside of lipid rafts and impaired SK3-dependent Ca2 fractionation experiments speciﬁed this localization to lipid entry, migration, and bone metastases rafts (Fig. 3D). Although the SK3–Orai1 complex was always To challenge these observations, we used a lipid inhibitor detected in lipid rafts, SK3-silencing experiments totally dis- of SK3 channels called Ohmline (13). We previously showed placed Orai1 outside of lipid rafts (Fig. 3D). Thus, we concluded that Ohmline does not displace pore-binding compounds that the SK3–Orai1 complex is one of the components of the (13) but, like edelfosine and owing to its phospholipid þ Ca2 -signaling microdomain constituted by lipid rafts (23). structure, could act on SK3 channels by being incorporated

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Vehicle A 1 µmol/L Ohmline, 24 h Ohmline Lipid raft Non-Lipid raft Lipid raft Non-Lipid raft

1 2 3 4 567 8 12 3 45678 β-adaptin 109 KDa Orai1 SK3 Caveolin-1 out 20 kDa

SK3 75 kDa in Lipid raft Orai1 31 kDa

B Constitutive Ca2+ entry C Ca2+ free 2 mmol/L external [Ca2+ ] 1.2 Migration, 5 mmol/L Ca2+ 4.5 Vehicle 1.0 1.0 4

3.5 0.8 0.8 3 Mann–Whitney 0.6 2.5 P = 0.0122 0.6 2 Ohmline 0.4

F340/F380 0.4 1.5 1 µmol/L, 24 h F340/F380, normalized 1 0.2 0.2 N = 5 N = 5 normalized migration, Cell 0.5 N = 2 N = 2 0.0 0.0 0 Vehicle Ohmline Vehicle Ohmline 0 20406080100 1 µmol/L, 24 h 1 µmol/L, 24 h Time (sec)

D E Vehicle Ohmline 15 mg/kg or vehicle, 3 times a week/i.v. 80 50 Vehicle (N = 6) Week 1 Week 6–7 Week 15 60 Ohmline (N = 9) 40

4 30 40

Ohmline 20 Graft Removal BLI of 20

2M cells Primary tumor metastases Photon flux10 x 10 Occurrence of metastases (%) metastases Occurrence of Not ≤400 mm3 Ohmline (µg/total lipids) detected 0 0 Lung Bone Lung Bone

F 400

) Vehicle (N = 19)

3 N = 16 300 Ohmline (N = 16) 30

200 20

100 Ohmline 10 (µg/total lipids) Tumor volume volume (mm Tumor 0 0 23456 Week post-graft

þ Figure 4. The alkyl-lipid Ohmline moved the SK3–Orai1 complex outside of lipid rafts and impaired SK3-dependent Ca2 entry, migration, and bone metastases. A, Ohmline treatment moved the SK3–Orai1 complex outside of lipid rafts. Immunoblots representing membrane fractionation on a sucrose gradient of cells treated or not with 1 mmol/L Ohmline for 24 hours (left). Representative immunoblots from 2 different experiments. Hypothetical scheme of Ohmline effects on Orai1 and SK3 (right). B, Ohmline treatment reduced the constitutive Ca2þ entry. Fluorescence measurement (left) and relative ﬂuorescence (right) of constitutive Ca2þ entry in cells treated or not with 1 mmol/L Ohmline for 24 hours. Data were normalized to results obtained in cells treated with vehicle. The constitutive Ca2þ entry protocol has been validated in Supplementary Fig. S3A. Columns, means; bars, SEM. N indicates the number of experiments. C, Ohmline treatment reduced the migration of MDA-MD-435s cells. Histograms showing migration of cells treated or not with 1 mmol/L Ohmline for 24 hours in 5 mmol/L external Ca2þconditions. Columns, mean; bars, SEM. N indicates the number of experiments. D, MFP-tumor model protocol used for Ohmline injections. E, Ohmline treatment abolished bone metastases in MFP-tumor model. Images of nude mice 15 weeks after SK3þ cell injections in MFP and treated either with vehicle or Ohmline at 15 mg/kg (left). Occurrence of lung and bone metastases in mice treated with either Ohmline or vehicle and representative bioluminescent images ex vivo of lung and bone metastases (vehicle condition; middle). N indicates the number of mice. Measurements of Ohmline incorporation in lung and bone tissues (tissues were pooled from 4 different samples) at week 15 (right). F, Ohmline incorporation in the primary tumors has no effect on their growth. Time course of tumor growth recorded in vehicle and Ohmline-treated mice postgraft (left). Measurement of Ohmline incorporation in tumors from treated mice (right). N indicates the number of mice.

4858 Cancer Res; 73(15) August 1, 2013 Cancer Research

SK3–Orai1 Channel Complex Promotes Bone Metastasis

into lipid rafts (24). Addition of Ohmline for 24 hours at 1 mmol/L had no effect on SK3 or Orai1 protein expression, but A Primary tumor Bone metastasis totally delocalized SK3 and Orai1 channels from lipid-raft fractions (Fig. 4A and Supplementary Fig. S4A). Functionally, Prostate þ Ohmline reduced the constitutive Ca2 entry and thus cancer cell migration (Fig. 4B and C), as observed when the X 20 X 40 X 40 SK3 channel is knocked down (see Figs. 2 and 3; ref. 6). Interestingly, identical results were obtained when using 10 Breast times less Ohmline (Supplementary Fig. S4B). As SK3 activity is abolished shortly after Ohmline application (120 seconds; X 40 X 40 ref. 13), we hypothesized that Ohmline is incorporated in SK3 Orai1 lipid rafts and acts by dissociating or preventing SK3–Orai1 B complex cauterization. This indicates that the SK3–Orai1 complex might only function when localized in rafts and that Normal prostate a delocalization of one of the 2 partners is sufficient to þ X 40 X 40 suppress SK3-dependent Ca2 entry and SK3-dependent migration. We next tested Ohmline potency to reduce metastases Normal breast development in the MFP model (see protocol Fig. 4D). Ohmline X 40 X 40 incorporation was measured in primary tumors and in bone and lung metastases (Fig. 4E and F and Supplementary Fig. C SK3 Orai1 SK3 + Orai1 S4C). Despite incorporation, Ohmline had no effect on primary a tumor development (Fig. 4F), strengthening the observation that the SK3–Orai1 complex has no role in primary tumor X 20 X 20 X 20 growth (Fig. 1C). Mice treated with Ohmline did not present any sign of bone metastases confirming the crucial role of the b SK3–Orai1 complex in bone metastases development (Fig. 4E). Unexpectedly, an effect of Ohmline was also observed on lung metastases (Fig. 4E). Ohmline was shown to inhibit the SK1 X 100 X 100 X 100 channel (13) and might increase SOCs channel activity (21); this might be the mechanism of decreased lung metastases by Figure 5. Expression of SK3 and Orai1 proteins in breast and prostate Ohmline. As SK3 is expressed in the central nervous system, tissues. A, SK3 protein was expressed in human breast and prostate and despite the high concentration of Ohmline in this tissue, cancers. Representative images of cancer cells detected by SK3 we observed no neurological effects. This can be explained by: immunostaining in primary tumor and bone metastases from human (i) the absence of lipid rafts SK3–Orai1 complexes in the brain prostate and breast cancer. B, SK3 protein was not expressed in normal human prostate and breast tissues in contrast to Orai1. (Orai1 expression being low) or (ii) the organisation of SK3 Representative images of normal epithelial cells from human prostate channels as heteromultimeric complexes involving SK1 or SK2, and breast without SK3 immunostaining (left) and in contrast with in contrast to cancer cells where SK1 is not expressed. Orai1 expression (right). C, coexpression of SK3 and Orai1 complexes by human cancer cells. a, representative SK3 (brown) and Orai1 (red) SK3 and Orai1 are expressed in breast/prostate cancer immunoperoxydase staining in prostate cancer bone metastasis, with double staining (right). b, representative SK3 (green), Orai1 (red), clinical samples and Orai1–SK3 (yellow) immunofluorescence staining on the same Because bone is a privileged site for metastases in prostate sample of primary prostate cancer. cancer, we assessed SK3 epithelial expression in clinical samples. Many (60%) of the prostate cancer samples, both from primary tumors (113 of 177) or bone metastases (9 of 15), Taken together, our results reveal a hitherto unknown þ showed positive epithelial SK3 staining, with a granular and function for SK3 channels in regulating Ca2 entry through predominantly membranous profile (Fig. 5A). Identical results Orai1 channels (Fig. 6). In vivo data further suggest a were obtained with breast cancer clinical samples (Fig. 5A). To participation of the SK3–Orai1 complex in the migration of evaluate the clinical value of these observations we analyzed cancer cells and their establishment at permissive secondary the coexpression of SK3 with Orai1. In human cancer samples, sites. Intriguingly, the Orai1 partner STIM1 seems not to be including primary tumors and bone metastases, the expression involved in this effect, which could reflect a differential role þ þ of SK3 and Orai1 were significantly associated (Qui2 test, P < for Ca2 signaling in tumors, one connecting Ca2 entry to 0.0001; Fig. 5C). SK3 protein was not expressed in normal proliferation (25) and the other to metastases. Finally, by tissues in contrast to Orai1 (Fig. 5B), supporting that this is the detecting SK3 channels in human samples, we confirmed the expression of SK3 in tumor cells that triggers Orai1 to associate clinical relevance of SK3–Orai1 expression in bone metas- with SK3 as a complex in lipid rafts. Note that it is well known tases. Hence, the in vivo efficacy of Omhline in preventing that Orai1 protein expression at the cellular level reveals near and/or treating bone metastases could have a therapeutic ubiquitous distribution. application.

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Chantome^ et al.

A Lipid-rafts Orai1 No SK3 : No cancer cell Orai1 outside lipid-rafts migration Plasma membrane Cytosol

Ca2+ No consititutive calcium enty

B K+ K+ Hyperpolarization + K+ Orai1 K+ SK3 Figure 6. Proposed mechanism SK3 expression : for SK3–Orai1 role in bone SK3–Orai1 complex Cancer cell metastases. A, in the absence of within lipid-rafts migration SK3, Orai1 is not embedded within lipid rafts and does not promote 2þ Ca2+ Ca2+ constitutive Ca inﬂux. B, the Ca2+ Ca2+ Ca2+ + Calpaïn/Talin presence of SK3 triggers SK3– No consititutive Orai1 to associate within lipid rafts, calcium enty resulting in plasma membrane hyperpolarization and constitutive Osteolytic lesions Ca2þ entry. C, increased external Ca2þ concentration observed in osteolytic metastatic sites ﬁ 2þ C K+ Hyperpolarization ampli es Ca entry, leading K+ + K+ to a positive feedback loop. D, Orai1 K+ SK3 Cancer cell disrupting lipid rafts with the alkyl- migration lipid Ohmline allows Orai1–SK3 to move away from lipid rafts and abolishes SK3-dependent 2þ Ca2+ Ca2+ constitutive Ca entry. Thus, SK3- 2+ 2+ + Ca2+ Ca Ca Calpaïn/Talin dependent cancer cell migration Consititutive and bone metastases are calcium enty counteracted.

D K+ Orai1 SK3 Ohmline No cancer cell Lipid rafts disturbance migration SK3–Orai1 complex splited

2+ Ca No consititutive calcium enty

Disclosure of Potential Conflicts of Interest Acknowledgments No potential conflicts of interest were disclosed. The authors thank Dr. F. Redini, Dr. P. Pilet for radiography and scanner expertise, Pr. P.-M. Martin for technical assistance in setting the MFP model, Pr. G. Lalmanach for assistance in conducting calpain activity measurements, Ms J. Authors' Contributions Godet and Dr. B. Constantin for assistance in immunofluorescence experiments. Conception and design: A. Chantome,^ M. Potier-Cartereau, J.-P. Haelters, P.-A. The authors also thank A. Douaud-Lecaille and I. Domingo for technical Jaffres, V. Joulin, P. Bougnoux, C. Vandier assistance and C. Leroy for secretarial support. Development of methodology: A. Chantome,^ J.-C. Pages, M. Pinault, P. Besson, C. Vandier Grant Support Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A. Chantome,^ L. Clarysse, G. Fromont, S. Marionneau- This work was funded by INCa, ANR; N ANR-08-EBIO-020-01, Ligue Contre le Cancer, Region Centre, INSERM, and Canceropole^ Grand Ouest. A. Girault Lambot, M. Gueguinou, T. Oullier, A. Girault, F. Arbion, M. Pinault ^ Analysis and interpretation of data (e.g., statistical analysis, biostatistics, held fellowships from the Region Centre and ARC, A. Chantome from INCa computational analysis): A. Chantome,^ M. Potier-Cartereau, L. Clarysse, and ANR. G. Fromont, S. Marionneau-Lambot, M. Gueguinou, A. Girault The costs of publication of this article were defrayed in part by the Writing, review, and/or revision of the manuscript: A. Chantome,^ M. Potier- payment of page charges. This article must therefore be hereby marked Cartereau, G. Fromont, J.-C. Pages, J.-P. Haelters, P.-A. Jaffres, P. Besson, V. Joulin, advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this P. Bougnoux, C. Vandier fact. Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): A. Chantome,^ C. Vandier Received December 18, 2012; revised April 15, 2013; accepted May 20, 2013; Study supervision: V. Joulin, P. Bougnoux, C. Vandier published OnlineFirst June 17, 2013.

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SK3–Orai1 Channel Complex Promotes Bone Metastasis

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Pivotal Role of the Lipid Raft SK3−Orai1 Complex in Human Cancer Cell Migration and Bone Metastases

Aurélie Chantôme, Marie Potier-Cartereau, Lucie Clarysse, et al.

Cancer Res 2013;73:4852-4861. Published OnlineFirst June 17, 2013.

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