pivotal role of the lipid raft sk3-orai1 complex in human...

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Pivotal role of the lipid raft SK3-Orai1 complex in human cancer cell migration and

bone metastases

Aurelie Chantôme1+, Marie Potier-Cartereau1+, Lucie Clarysse1, Gaëlle Fromont2,3, Séverine

Marionneau-Lambot4, Maxime Guéguinou1, Jean-Christophe Pagès5,6, Christine Collin6,

Thibauld Oullier4, Alban Girault1*,Flavie Arbion6, Jean-Pierre Haelters7, Paul-Alain Jaffrès7,

Michelle Pinault1, Pierre Besson1, Virginie Joulin8, Philippe Bougnoux1,9, Christophe

Vandier1#

1Inserm, UMR1069, Tours, F-37032 France; Université François Rabelais, Tours, 37032

France.

2CHRU de Poitiers, 86000 France.

3Université de Poitiers, Poitiers, 86000 France.

4Cancéropôle du Grand Ouest, Nantes, 44000 France.

5Inserm, U966, Tours, F-37032 France; Université François Rabelais, Tours, 37032 France.

6CHRU de Tours, Tours, 37032 France.

7Université Européenne de Bretagne, Université de Brest, CNRS UMR 6521, CEMCA, SFR

148 ScInBios, Brest, 29238 France.

8Inserm, U1009; Institut Gustave Roussy, Villejuif, 94805 France.

9Centre HS Kaplan, CHRU Tours, Tours, 37032 France.

Running Title: SK3-Orai1 channel complex promotes bone metastasis

Keywords: Potassium channels / Breast cancer / Calcium channels / Alkyl-lipids / Metastasis

#Correspondence should be addressed to Christophe Vandier, Phone: +(33)247366024; Fax:

+(33)247366226; E-mail: [email protected]

Word count: 4965

on April 26, 2018. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 17, 2013; DOI: 10.1158/0008-5472.CAN-12-4572

http://cancerres.aacrjournals.org/

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Number of figures: 6

+These authors contributed equally to this work.

*Present address: CRCHUM, Hôtel-Dieu, 3840, rue Saint-Urbain, Montréal (Québec) H2W

1T8.




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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 bone metastasis. The SK3 channel was shown to control

constitutive Ca2+ entry and cancer cell migration 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 appeared essential to control cancer-

cell migration. This suggests that the formation of this complex in lipid rafts is a gain-of-

function, since 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. 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 co-localization 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.




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INTRODUCTION

The emerging concept of ion channels as key regulators of cancer expansion [for review (1–

3)] has several implications, including the potential of their chemical targeting for cancer

treatment. Therefore, a precise understanding of the mechanisms underlying the role of ion

channels in cancer cells is paramount. We have recently shown that SK3 (KCNN3 gene), a

potassium channel of the small conductance Ca2+-activated potassium (KCa) channel family

(4), is a mediator of cancer cell migration (5, 6). The physiological expression of the SK3

channel was first studied in central neurons where it has a fundamental role in regulating

neuronal excitability (7). This channel is not restricted to neuronal tissues (8), and was found

to be expressed in smooth muscle, where it regulates smooth muscle tone (9–11).

Interestingly, the SK3 channel is expressed in tumor breast biopsies and melanoma cells, but

its expression was not observed in non-tumor breast tissues and primary cultures of

melanocytes (6, 12). The lack of effect of SK3 channel expression on cell proliferation (12)

led us to investigate whether this specific role in cell migration conferred this channel a role

in metastases development. Indeed, the formation of secondary tumors from primary sites

appears to be a multistep process in which tumor cell migration is a critical event.

In this report, we show a role for SK3 in bone metastases, which is the first report

establishing an ion channel as a control factor for bone metastases development. SK3 action

proved to be mediated through an association with Orai1, a voltage-independent Ca2+ channel.

The SK3-Orai1 complex regulates a constitutive Ca2+ entry, calpain activation and cell

migration. At the cellular level, the SK3-Orai complex was localized in lipid rafts. The alkyl-

lipid Ohmline disrupted SK3-Orai1 complexes from lipid rafts and impaired SK3-dependent

Ca2+ entry, migration and bone metastases, qualifying this lipid as a potential platform for

drug development. Finally, the co-localization of SK3 and Orai1 in primary human tumors

and bone metastases from clinical samples emphasized the clinical consistency of these




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observations. This is the first report showing that the deregulation of an ion channel complex

by a lipid could control metastases.




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MATERIALS AND METHODS

Cell lines. Human breast cancer cell line MDA-MB-435s was purchased from the American

Type Culture Collection (ATCC, LGC Promochem, France) and was grown as already

described (6). A recent study suggested that the MDA-MB-435s cell line originated from

breast tissue (26). This cell line was transduced by a retrovector containing the luciferase

gene and with a lentivector containing either an interfering shRNA specific to SK3 (SK3-

cells) or a non-targeting shRNA (SK3+ cells) as previously validated (13). No difference of

luciferase expression and activity has been observed between SK3+ and SK3- cells (See

Supplementary Fig. S1BC). HEK293 and 518A2 cells are described in Supplementary

Methods.

Immunohistochemistry. Cells were fixed in 10% formalin, included in gel, and embedded in

paraffin. Murine tissues were fixed in 10% formalin and embedded in paraffin, with a mild

decalcification for bone tissues. Tissue microarrays (TMAs) were constructed from human

formalin-fixed tissues obtained from 177 primary prostate cancers and 37 bone metastases

specimens, including 15 prostate cancer metastases and 22 breast cancer metastases. Normal

prostate and breast tissues were also included in the TMAs. All primary prostate cancers were

of the acinar type, with 59 having a Gleason score of 6, 106 with a Gleason score of 7, and 12

with a Gleason score of 8 and more; 137 tumors were pT2 and 40 pT3. Among the 20 breast

cancer bone metastases, 16 expressed estrogen receptors, 11 progesterone receptors, and three

were positive for Her2; five tumors were triple negative. For each tumor, four cores (0.6-mm

diameter) were included in the TMA, as previously described (27). Immunohistochemical

staining was performed on 3-μm slides from embedded cell lines, xenografts and TMA, using

anti-Ki67 (DakoCytomation), anti-SK3 channel (Sigma, P0608, dilution 1/50), and anti-Orai1

(Life Span Bioscience, dilution 1/4,000).




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Electrophysiology. All experiments were performed using whole-cell recording

configuration of the patch-clamp technique, as previously described (6, 12) and as described

in Supplementary Methods.

Intracellular Ca2+ measurements. Cells were loaded in Petri dishes for 45 min at 37 °C

with the ratiometric dye Fura2-AM (5 µM). Then, cells were trypsinised, washed with Opti-

MEM® Reduced Serum Medium, GlutaMax (Life-Technologies) and centrifuged (800 x g

for 5 min). Immediately after centrifugation, cells were re-suspended at 1 x 106 cells in 2 mL

PSS Ca2+-free solution. Fluorescence emission was measured at 510 nm with an excitation

light at 340 and 380 nm (Hitachi FL-2500). See Supplementary Fig. S3A for the validation of

constitutive Ca2+ entry protocol used.

Western blot, RT-qPCR and calpain activity assay. Western blot experiments were

performed as described (6). The antibodies and the materials and methods used are described

in Supplementary Methods.

Cell proliferation and migration assays. Cell proliferation and cell migration were

determined as described elsewhere (6, 12, 31) and are specified in Supplementary Methods.

Experimental and spontaneous metastasis models. Mice (Janvier laboratories) were bred

and housed at Inserm U892 (Nantes-University) under the animal care license n°44565. For

experimental metastases, 6-week-old female NMRI nude mice were used. Unanesthetized

mice were placed into a plastic restraining device, and 0.75 x 106 MDA-MB-435s

(SK3+/SK3-) cells were injected into the lateral tail vein in 100 µL of serum-free DMEM




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through a 25-gauge needle. For the mammary fat-pad (MFP) model, female NMRI/Nude

mice, 3–4 weeks old, were used. Mice were anesthetized by intraperitoneal 100 mg/kg

ketamine plus 10 mg/kg xylazine administration and a right fat-pad was cleared.

Subconfluent SK3+ and SK3- cells were harvested, washed in PBS, and 2 x 106 cells were

injected in a volume of 50 µL of DMEM without serum into the cleared fat-pad. Tumor

volumes were calculated using the formula: length.width.depth. For MFP-metastases in-vivo

assays with Ohmline, SK3+ cells were incubated with 1 µM Ohmline or with vehicle (0.6‰

ethanol / 0.4‰ DMSO) for 24 h and injected into the cleared fat pad. Mice were treated three

times a week for 15 weeks with Ohmline at 15mg/kg or with vehicle administered

intravenously. Primary tumors were removed when the volume reached 400 mm3. In control

animals, we have not observed adverse effects upon Ohmline administration (no

compartmental, weight growth abnormalities or liver and heart toxicities were observed after

necropsy) (13). This absence of side effects is explained by the low and non-cytotoxic

concentration of Ohmline used and the selective effect of this lipid on SK3 channel. The

materials and methods used for Bioluminescence Imaging (BLI) are described in

Supplementary Methods.

Membrane fractionation, Immunofluorescence and Incorporation of Omhline in tissues.

The materials and methods used are described in Supplementary Methods.

Statistics. Data were expressed as median with quartile or mean ± SEM (N, number of

experiments; n, number of cells). Statistical analyses were made using the unpaired Student’s

t-test or the Mann-Whitney test. For comparison between more than two means, we used

Kruskal-Wallis one-way analysis of variance followed by Dunn’s test. Differences were




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considered significant when p < 0.05 (SigmaStat, Systat Software and Minitab software,

Minitab Inc.)




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RESULTS AND DISCUSSION The SK3 channel controls bone metastasis development and is expressed in

breast/prostate cancer clinical samples

To investigate the role of SK3 in metastases development, we engineered luciferase SK3-

positive, MDA-MB-435s breast cancer-derived cells. Using specific shRNA knockdown of

the KCNN3 gene-product, we obtained SK3- cells; control cells receiving a random shRNA

remained SK3+. Compared to SK3+ cells, SK3- cells displayed almost no outward current,

their plasma membrane was more depolarized and they exhibited a lower migration capacity

while their proliferation was not affected (Fig. 1A and Supplementary Fig. S1). Next, we

investigated SK3 function using a cancer-cell xenograft model in NMRI/nude mice (Fig. 1B).

Silencing of the SK3 channel led to a lower composite metastatic score, based on the number

of metastases per mouse and on the intensity of the bioluminescent signal per metastasis

(13)(Supplementary Fig. S2A). Interestingly, this lower score essentially reflected a lower

bone metastases development in SK3--grafted mice compared to SK3+-grafted mice (Fig.

1B). Conversely, the lung bioluminescent signal-intensity was not significantly different

between SK3+- and SK3--grafted mice (Supplementary Fig. S2B). At week 9, bone

metastases were detected in 83% (10/12) of the mice injected with SK3+ cells but only in

36% (4/11) of the mice injected with SK3- cells (Fig. 1B middle). Moreover, the intensity of

the bioluminescent signal was significantly different between SK3+- and SK3--grafted mice

(Fig. 1B bottom). Consistent with in vivo observations, both the frequency of bone metastases

(100% versus 54%) and the intensity of the bioluminescent signal, detected ex vivo at

necropsy, were lower in mice injected with SK3- as compared to SK3+ cells (Supplementary

Fig. S2C).

These observations did not examine the impact of SK3 channel on the primary tumors

in relation to metastatic development. Other channels, such as hEag1 (voltage-gated




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potassium channel), IKCa (intermediate conductance KCa channel) or TRPV2 (transient

receptor potential V2) have been reported to influence the volume of subcutaneously

xenografted tumors, by acting on their proliferation and/or migration capacities (14–16).

Since ectopic tumor models could not accurately reflect the metastatic potential of tumor

cells, we used an orthotopicmammary-tumor model known to support the development of

metastases in several tissues. We grafted SK3+ or SK3- cells into the mammary fat pad (MFP)

of NMRI/Nude mice (17, 18). SK3 channel suppression did not influence primary tumor

growth, and the proliferation index (Ki67 staining) was identical in the two groups of mice

(Fig. 1C). Importantly, SK3+ tumors were still positive for SK3 staining, while SK3- tumors

remained negative (Fig. 1C), confirming the stability of the SK3 phenotype following

grafting. Metastases occurred in both groups and were mainly observed in bones and lungs.

However, the bioluminescent signal was weak in bones (Fig. 1C) but not in lungs of SK3--

grafted mice (Supplementary Fig. S2D). This suggested that SK3 channel expression in

cancer cells affected their ability to form metastases in bone but not in lung.

External Ca2+ elevation up-regulates SK3 channel activity and activates Ca2+entry

promoting calpain activation and cell migration

These findings suggest that SK3 channel might contribute to/or facilitate bone metastases. As

an interaction with the bone microenvironment could influence SK3 activity and since this

channel is Ca2+-sensitive (5), we evaluated the effect of external Ca2+ concentrations in

modulating SK3 channels. In active bone resorptive lacunae, osteolysis arose in legs and

rachis of the two metastases models (Fig. 2A), extracellular Ca2+ concentrations could be as

high as 8 to 40 mM, whereas in the vicinity of unaltered bone surface, it is normally closer to

2 mM (19). In vitro, changing the extracellular Ca2+ concentration from 2 mM to 5 mM led to

an increase in the migration of SK3+ cells, an effect not observed for SK3- cells (Fig. 2B).




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Since we demonstrated that SK3 channel control cancer cell migration by hyperpolarizing the

plasma membrane of cancer cells (12) we tested the effect of increasing external Ca2+

concentration on the membrane potential of wild-type MDA-MB-435s cells. Figure 2C shows

the outward potassium currents recorded using a ramp protocol from -70 mV to +70 mV:

within 2 min, the amplitude of potassium currents increased, leading to a shift of the

membrane potential toward more negative values (membrane hyperpolarization). The

apamin-sensitive current carried by the SK3 channel was assessed in cells incubated in PSS

solution with 2 or 5 mM extracellular Ca2+ (Fig. 2C). Increasing external Ca2+ concentrations

more than doubled the amplitude of SK3 currents, leading to a 20 mV membrane

hyperpolarization (Fig. 2C). Interestingly, we noticed that SK3 hyperpolarization promoted

Ca2+ entry and, thus, elevated intracellular Ca2+ concentration by increasing the Ca2+-driving

force (Fig. 2D). Hence, a physiological 2 mM extracellular Ca2+ concentration would activate

the SK3 channel, which could be over-activated by higher extracellular Ca2+ concentrations.

Of note, activated SK3 channels increased the activity of the Ca2+-sensitive protease calpain

(Fig. 2E), a factor contributing to many aspects of cell migration, such as cell spreading,

membrane protrusion, chemotaxis, and adhesion complex formation and turnover (20).

Additionally, the proteolysis of the calpain target talin is promoted by SK3 expression and is

increased by A23187 and/or by high external calcium concentrations (Fig. 2E), conditions

that increase intracellular calcium concentrations. Since calpain activation is a critical step

leading to adhesion complex turnover and cell migration (20), we can hypothesize that at

least part of the role of SK3 in migration could be attributed to by calpain activation.

SK3 action is mediated through its association with the Orai1 channel, forming a lipid-

raft SK3-Orai1 complex




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We next aimed at identifying the Ca2+ channel involved in Ca2+ entry. The voltage-

independent Ca2+ channel Orai1, and its regulator STIM1, have been shown to be Store-

Operated Channels (SOC) in breast cancer cells and have been implicated in cancer cell

migration (21) and calpain activation (22). Orai1 knockdown totally abolished SK3-

dependent cell migration (Fig. 3A). The suppression of STIM1 had no effect on MDA-MB-

435s cell migration (Fig. 3A) in contrast to the MDA-MB231 breast cancer cell line (21) that

did not express SK3 protein (6). These results suggest a role for Orai1 channels in

constitutive SK3-dependent Ca2+ entry, independently of STIM1 (see Supplementary Fig.

S3A for the validation of the constitutive Ca2+ entry protocol used). Consistently, the

inhibition of Orai1, either by siRNA, shRNA (with two different sequences) or by using 2-

APB, totally abolished SK3-dependent constitutive Ca2+ entry and the increase of cancer cell

migration observed at 5 mM Ca2+ concentration (Fig. 3B and Supplementary Fig. S3BC).

Thus, our findings revealed a novel signaling pathway in which the SK3-Orai1 complex

elicited a constitutive and store-independent Ca2+-signaling that promoted cell migration.

Having shown that Orai1 was necessary for cancer cell migration, we assessed its cellular

localization. Immunofluorescence analysis showed that SK3 and Orai1 were localized at the

plasma membrane (Fig. 3C), and membrane-fractionation experiments specified this

localization to lipid rafts (Fig. 3D). While the SK3-Orai1 complex was always detected in

lipid rafts, SK3-silencing experiments totally displaced Orai1 outside of lipid rafts (Fig. 3D).

Thus, we concluded that the SK3-Orai1 complex is one of the components of the Ca2+-

signaling microdomain constituted by lipid rafts (23).

The alkyl-lipid Ohmline moved the SK3-Orai1 complex outside of lipid rafts and

impaired SK3-dependent Ca2+ entry, migration and bone metastases




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To challenge these observations, we used a lipid inhibitor of SK3 channels called Ohmline

(5). We previously showed that Ohmline does not displace pore-binding compounds (13) but,

like edelfosine and owing to its phospholipid structure, could act on SK3 channels by being

incorporated into lipid rafts (24). Addition of Ohmline for 24 h at 1 µM had no effect on SK3

or Orai1 protein expression, but totally delocalized SK3 and Orai1 channels from lipid-raft

fractions (Fig. 4A and Supplementary Fig. S4A). Functionally, Ohmline reduced the

constitutive Ca2+ entry and thus cancer cell migration (Fig. 4BC), as observed when the SK3

channel is knocked down (see Figs. 2–3) (6). Interestingly, identical results were obtained

when using 10 times less Ohmline (Supplementary Fig. S4B). As SK3 activity is abolished

shortly after Ohmline application (120 s) (13), we hypothesized that Ohmline is incorporated

in lipid rafts and acts by dissociating or preventing SK3-Orai1 complex cauterization. This

indicates that the SK3-Orai1 complex might only function when localized in rafts and that a

delocalization of one of the two partners is sufficient to suppress SK3-dependent Ca2+ entry

and SK3-dependent migration.

We next tested Ohmline potency to reduce metastases development in the MFP model

(see protocol Fig. 4D). Ohmline incorporation was measured in primary tumors and in bone

and lung metastases (Fig. 4EF and Supplementary Fig. S4C). Despite incorporation, Ohmline

had no effect on primary tumor development (Fig. 4F), strengthening the observation that the

SK3-Orai1 complex has no role in primary tumor growth (Fig. 1C). Mice treated with

Ohmline did not present any sign of bone metastases confirming the crucial role of the 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 channel (13) and might increase SOCs channel activity (21); this might be the

mechanism of decreased lung metastases by Ohmline. As SK3 is expressed in the central

nervous system, and despite the high concentration of Ohmline in this tissue, we observed no




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neurological effects. This can be explained by: i) the absence of lipid rafts SK3-Orai1

complexes in the brain (Orai1 expression being low) or ii) the organisation of SK3 channels

as heteromultimeric complexes involving SK1 or SK2, in contrast to cancer cells where SK1

is not expressed.

Since bone is a privileged site for metastases in prostate cancer, we assessed SK3

epithelial expression in clinical samples. Many (60%) of the prostate cancer samples, both

from primary tumors (113/177) or bone metastases (9/15), showed positive epithelial SK3

staining, with a granular and predominantly membranous profile (Fig. 5A). Identical results

were obtained with breast cancer clinical samples (Fig. 5A). To evaluate the clinical value of

these observations we analyzed the co-expression of SK3 with Orai1. In human cancer

samples, including primary tumors and bone metastases, the expression of SK3 and Orai1

were significantly associated (Qui2 test, p < 0.0001) (Fig. 5C). SK3 protein was not

expressed in normal tissues in contrast to Orai1 (Fig. 5B) supporting that this is the

expression of SK3 in tumor cells that triggers Orai1 to associate with SK3 as a complex in

lipid rafts. Note that it is well known that Orai1 protein expression at the cellular level reveals

near ubiquitous distribution.

Taken together, our results reveal a hitherto unknown function for SK3 channels in

regulating Ca2+ entry through Orai1 channels (Fig. 6). In vivo data further suggest a

participation of the SK3-Orai1 complex in the migration of cancer cells and their

establishment at permissive secondary sites. Intriguingly, the Orai1 partner STIM1 appears

not to be involved in this effect, which could reflect a differential role for Ca2+ signaling in

tumors, one connecting Ca2+ entry to proliferation (25) and the other to metastases. Lastly, by

detecting SK3 channels in human samples, we confirmed the clinical relevance of SK3-Orai1

expression in bone metastases. Hence, the in vivo efficacy of Omhline in preventing and/or

treating bone metastases could have a therapeutic application.




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ACKNOWLEDGEMENTS

We thank Dr. Françoise Rédini, Dr. Paul Pilet for radiography and scanner expertise, Pr.

Pierre-Marie Martin for technical assistance in setting the MFP model, Pr. Gilles Lalmanach

for assistance in performing calpain activity measurements, Ms Julie Godet and Dr. Bruno

Constantin for assistance in immunofluorescence experiments. We also thank Aurore

Douaud-Lecaille and Isabelle Domingo for technical assistance and Catherine Leroy for

secretarial support.




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GRANT SUPPORT

This work was funded by “INCa”, “ANR; N°ANR-08-EBIO-020-01”, “Ligue Contre le

Cancer”, “Région Centre", "INSERM" and “Cancéropôle Grand Ouest”. Alban Girault held

fellowships from the “Région Centre” and ARC, Aurélie Chantôme from “INCa” and

“ANR”.




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breast cancer cells. Int J Biochem Cell Biol 2006;38:196-208.

30. Snyder F, Blank ML, Wykle RL. The enzymic synthesis of ethanolamine

plasmalogens. J Biol Chem 1971;246:3639-45.

31. Brouard T, Chantome A. Automatic nuceli cell counting in low-resolution

fluorescence images. Computational Vision and Medical Image Processing CRC Press

2009:83-8.




22

FIGURE LEGENDS

Figure 1. SK3 suppression inhibited bone metastases

A) Validation of the MDA-MB-435s cell system expressing the luciferase gene and

expressing or not 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 five cells in each group.

Validation of SK3 protein extinction in SK3- cells and luciferase expression in SK3+ and

SK3- cells (middle). Representative immunoblots from at least three different experiments.

Cell migration and proliferation in SK3+ and SK3- cells (bottom). Histograms showing

analyses of migration 24 h after seeding. Data were normalized to results obtained with SK3+

cells. Columns, mean, bars, SEM. Graph showing proliferation rates evaluated by MTT

assays, daily, for four 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 SK3+ cells assessed by BLI in vivo and by Haematoxylin and Eosin

(H&E) 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

post-injection (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 SK3+ and

SK3- cells (a). Graph showing mammary tumor growth in SK3+- and SK3--grafted mice (b).

Ki67 staining of primary tumor tissue sections from mice grafted with SK3+ or SK3- cancer

cells, 16 weeks post-graft (c). H&E sections of bone metastases and BLI quantification of




23

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.

Figure 2. External Ca2+ elevation up-regulated SK3 channel activity and activated Ca2+

entry promoting calpain activation and cell migration

A) Osteolytic lesions in mice receiving SK3+ 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 Ca2+ elevation promoted SK3-dependent cell migration. SK3+ and SK3- cell

migration recorded with 2 and 5 mM external Ca2+ concentration. Data were normalized to

conditions obtained with a 2 mM external Ca2+ concentration.

C) External Ca2+ elevation increased the amplitude of SK3 currents leading to

membrane hyperpolarization. Representative SK3+ whole-cell currents recorded in the

presence of 2 mM external Ca2+ concentrations and following the addition of 3 mM external

Ca2+ concentrations after 2 min (final external Ca2+ concentration = 5 mM). 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 mV

to +70 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 +25 mV in 2 and 5 mM external Ca2+ concentrations. The amplitude of the

apamin-sensitive current was obtained by subtraction of the amplitude of the current before

and after application of 50 nM apamin (a specific SKCa blocker) in 2 and 5 mM external

calcium concentration.




24

D) The SK3 channel promoted Ca2+ entry. Fluorescence measurement (left) and relative

fluorescence to Ca2+ entry (right) in SK3+ and SK3- cells. Data were normalized to conditions

obtained with SK3+ 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 SK3+ 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 min with the Ca2+ ionophore

A23187 and treated or not with Ca2+ for 30 min. Representative immunoblots from three

different experiments are shown.

Columns: means, bars: SEM. N: the number of independent experiments, n: number of cells.

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 SK3+ and SK3- cell migration when transfected for 48 h with

siOrai1 or siSTIM1 (left). Validation of Orai1 and STIM1 protein extinction by immunoblots

48 h after transfection (top, right). Representative immunoblots from three different

experiments. Validation of Orai1 and STIM1 mRNA extinction by qPCR 48 h after

transfection (bottom, right).

B) Orai1 channel controlled a constitutive SK3-dependent Ca2+ entry. Fluorescence

measurement (left) and relative fluorescence to Ca2+ entry (right) in SK3+ and SK3- cells

transfected for 48 h with siControl or siOrai1. Data were normalized to results obtained in




25

cells transfected with the siControl. The constitutive Ca2+ entry protocol has been validated in

Supplementary Figure S3A.

C) Immunocolocalization of SK3 and Orai1 channels. Representative confocal images of

SK3 and Orai1 staining performed in SK3+ cells. Scale bars, 10 µm.

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 SK3+ 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 three different experiments.

Columns: means, bars: SEM. N: the number of independent experiments.

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 µM

Ohmline for 24 h (left). Representative immunoblots from two different experiments.

Hypothetical scheme of Ohmline effects on Orai1 and SK3 (right).

B) Ohmline treatment reduced the constitutive Ca2+ entry. Fluorescence measurement

(left) and relative fluorescence (right) of constitutive Ca2+ entry in cells treated or not with 1

µM Ohmline for 24 h. Data were normalized to results obtained in cells treated with vehicle.

The constitutive Ca2+ entry protocol has been validated in Supplementary Figure 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 µM Ohmline for 24 h in 5 mM external

Ca2+conditions. Columns: mean; bars: SEM. N indicates the number of experiments.




26

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 SK3+ 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 four 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 post-graft (left).

Measurement of Ohmline incorporation in tumors from treated mice (right). N indicates the

number of mice.

Figure 5. Expression of SK3 and Orai1 proteins in breast and prostate tissues.

A) SK3 protein was expressed in human breast and prostate cancers. Representative images

of cancer cells detected by SK3 immunostaining in primary tumor and bone metastases from

human prostate and breast cancer. B) SK3 protein was not expressed in normal human

prostate and breast tissues in contrast to Orai1. Representative images of normal epithelial

cells from human prostate and breast without SK3 immunostaining (left) and in contrast with

Orai1 expression (right). C) Co-expression of SK3 and Orai1 complexes by human cancer

cells. a) Representative SK3 (brown) and Orai1 (red) immunoperoxydase staining in prostate

cancer bone metastasis, with double staining (right). b) Representative SK3 (green), Orai1

(red) and Orai1-SK3 (yellow) immunofluorescence staining on the same sample of primary

prostate cancer.




27

Figure 6. Proposed mechanism for SK3-Orai1 role in bone metastases.

A) In the absence of SK3, Orai1 is not embedded within lipid rafts and does not promote

constitutive Ca2+ influx.

B) The presence of SK3 triggers SK3-Orai1 to associate within lipid rafts, resulting in plasma

membrane hyperpolarization and constitutive Ca2+ entry.

C) Increased external Ca2+ concentration observed in osteolytic metastatic sites amplifies

Ca2+ entry, leading to a positive feedback loop.

D) Disrupting lipid rafts with the alkyl-lipid Ohmline allows Orai1-SK3 to move and

abolishes SK3-dependent constitutive Ca2+ entry. Thus, SK3-dependent cancer cell migration

and bone metastases are counteracted.




A BFigure 1

Tail vein injection SK3-SK3+

C

1342

SK3-SK3+

pA

aa

X 5

Bone

Lung

X 4

SK3

X 40 X 40

0 5

1

1.5

2

rvo

lum

e (

cm3 )

SK3 75 KDa

luc

515.0 ms-29.3 pA516.0 ms-32.7 pA

0.0 ms0.0 pA1.0 ms-3.4 pA

0

515.0 m22.6 pA

516.0 m21.4 pA

0.0 m0.0 pA1.0 m-1.2 pA

0

61 KDa

200

p

100 ms

SK3-SK3+

SK3 + (N=12)

SK3 - (N=13)

b

0 2

0.4

0.6

0.8

1.0

Ki 67

SK3-SK3+

0

0.5

Tu

mo

0 2 4 6 8 10 12 14 16 18

Weeks post graft

Actin 43 KDa

mig

ratio

n,

no

rma

lize

d

Mann-WhitneyP<0.0001

c

bu

x in

lun

gss

(X1

06 )

50

100 SK3+

SK3-

Mann-Whitneyp=0.372

Lungs

0.0

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80

100

%

SK3+

(N=12)

Ort

hoto

pic

tum

our

X 20 X 20

Ce

llm

SK3+ (N=3) SK3- (N=3)

atio

n r

ate

6

8

10

12

SK3-

(N=8)SK3+

(N=8)

Bon

em

etas

tasi

s

c

Ph

oto

ns

flu

0SK3-

(N=11) SK3+

(N=12)

2

3

4

10Weeks

0 2 4 6 80

20

40

60

Mic

e w

ith

bo

ne

me

tast

ase

s, (N=12)

SK3-

(N=11) Log rank testp=0.032

SK3+ SK3-

Forelegs

Photon flux x 10

3

Hind legs

SK3-SK3+

Pro

life

ra

0 1 2 3 40

2

4

6

days

ns

flux

in b

on

es

(X1

03 )

Mann-Whitneyp=0 008

d

d

0

13

Ph

oto

ns

flux

in b

on

es

(X1

06 )

2

1

0

Mann-Whitneyp=0.002

SK3-(N=26)

SK3+(N=24)

Ph

oto

n p=0.008

SK3+

Photon flux 106

0

SK3-

(N=11)

X 20

Bone metastasisSK3+

(N=12)




A

X-ray scanner X-ray radiography1.6

SK3+ cells ized

Mann-Whitneyp<0.0001

B

1.6

SK3- cells ized

Figure 2

0 8

1.0

1.2

1.4

(N=6)

Cell

mig

ratio

n, n

orm

ali

0 8

1.0

1.2

1.4 (N=3)

Cell

mig

ratio

n, n

orm

al

Mann-Whitneyp=0.4799

Current amplitude (pA)

0.00.8

2 mM Ca2+ 5 mM Ca2+

Ca2+ free 2 mM external [Ca2+ ]

C D

0.00.8

2 mM Ca2+ 5 mM Ca2+

1.21.2

500

1000

1500

2 mM Ca2+

5 mM Ca2+

(n=7)2 Ca

0

4

8

12

pA/p

F at

+25

mV

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Apamin-sensitive current

(n=5)

2

3

4

5

6

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8

SK3+

SK3-

F340

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0Mann-Whitney

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0.4

0.6

0.8

1.0

1.2


40/F

380,

nor

mal

ized

1.0

0.4

0.6

0.8

(n=5)

Membrane Potential (mV)

Em

-60 -40 -20 0 20 40 60

Em

0

1

0 40 80 120 160

Time (sec)

E Kruskal Wallis p=0 005

0.0

0.2

SK3+ SK3-

N=4N=5F34

0.0

0.2

0,4

0,6

0,8

1,0

1,2

SK3+ (N=4)

SK3- (N=4)

Talin

10 mM Ca2+- -

- + +

+ - -

- + +

+

5 µM A23187

235 KDa200 KDa talin cleavage product

HEKSK3- cells HEK SK3+ cellsE

p=0.034

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aliz

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518A2 cells

Kruskal Wallis p 0.005 and post hoc Mann-

Whitney (compared to control)

p=0.034

p=0.034

0,0

0,281KDaHsc70

- -+ +Z-LLY-FSKcalpain inhibitor

(flu

o

on April 26, 2018. ©

2013 Am

erican Association for C

ancer Research.

cancerres.aacrjournals.org D

ownloaded from

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anuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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anuscript Published O

nlineFirst on June 17, 2013; D

OI: 10.1158/0008-5472.C

AN

-12-4572


1.0

1.2

B

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A

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1.2

zed

Figure 3SK3+ cells

zed

SK3+ cells

p=0.3314

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0.2

0.4

0.6

0.8

0

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2

3

4

0 40 80 120 160

F340

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p=0.0027

0.0

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0 0

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Time (sec) 0siControl siOrai1

D

siControl siSTIM 1 siOrai 1 siOrai 1 siSTIM1

20 µm20 µm

Β-adaptin109 kDa

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1 2 3 4 5 6 7 8

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1 2 3 4 5 6 7 8

Lipid-rafts Non Lipid-rafts

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on April 26, 2018. ©

2013 Am

erican Association for C

ancer Research.

cancerres.aacrjournals.org D

ownloaded from

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anuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Author M

anuscript Published O

nlineFirst on June 17, 2013; D

OI: 10.1158/0008-5472.C

AN

-12-4572


Ohmline

Figure 4

Non Lipid raftLipid raft

Vehicle 1 µM Ohmline, 24 hA

Non Lipid raftLipid raft

out

in

Orai1 SK3

Lipid raft

Caveolin-120 kDa

1 2 3 4 5 6 7 8

Orai131 kDa

SK375 kDa

β-adaptin109 KDa

1 2 3 4 5 6 7 8

0 6

0.8

1.0

B C

no

rma

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d

Migration, 5 mM Ca2+

0 6

0.8

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Mann-Whitney0 0122

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Ce

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igra

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0.4

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N=5 N=5

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0 20 40 60 80 100

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60

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20

30

40

50

ne

(µ

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Ohmline (N=9)

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Pho

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Occ

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Not

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200

300

400vehicle

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)

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20

30

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primary tumor bone metastasisA

Figure 5

Prostate

X 20 X 40 X 40

Breast

S 3 O i1

X 40 X 40

B

Normal prostate

SK3 Orai1

X 40 X 40

Normal breast

X 40 X 40

X 40 X 40

C

SK3 + Orai1SK3 Orai1

a

b

X 20 X 20 X 20

X 100 X 100 X 100




Lipid-rafts

No SK3 :Orai1 outside lipid-rafts

No cancer cell migration

A

Plas

ma

Mem

bran

e

Figure 6

cytosol

B

+

+SK3 expression :

SK3-Orai1 complexwithin lipid-rafts

Cancer cell migration

hyperpolarizationOrai1 SK3

B

Calpaïn/Talin

Osteolytic lesions

+Cancer cell migration

hyperpolarizationOrai1 SK3

C

Calpaïn/Talin +

Ohmline

D

Lipid-rafts disturbance

SK3-Orai1 complex

No cancer cell migration

SK3 Orai1 complexsplited




Published OnlineFirst June 17, 2013.Cancer Res Aurelie Chantome, Marie Potier-Cartereau, Lucie Clarysse, et al. cell migration and bone metastasesPivotal role of the lipid raft SK3-Orai1 complex in human cancer

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