pivotal role of the lipid raft sk3-orai1 complex in human...
TRANSCRIPT
1
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
2
Number of figures: 6
+These authors contributed equally to this work.
*Present address: CRCHUM, Hôtel-Dieu, 3840, rue Saint-Urbain, Montréal (Québec) H2W
1T8.
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
3
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.
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
4
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
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
5
observations. This is the first report showing that the deregulation of an ion channel complex
by a lipid could control metastases.
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
6
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).
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
7
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
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
8
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
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
9
considered significant when p < 0.05 (SigmaStat, Systat Software and Minitab software,
Minitab Inc.)
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
10
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
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
11
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).
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
12
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
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
13
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
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
14
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
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
15
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.
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
16
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.
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
17
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”.
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
18
REFERENCES
1. Arcangeli A, Crociani O, Lastraioli E, Masi A, Pillozzi S, Becchetti A. Targeting
ion channels in cancer: a novel frontier in antineoplastic therapy. Curr Med Chem
2009;16:66-93.
2. Cuddapah VA, Sontheimer H. Ion channels and transporters [corrected] in cancer.2.
Ion channels and the control of cancer cell migration. Am J Physiol Cell Physiol
2011;301:C541-9.
3. Schwab A, Fabian A, Hanley PJ, Stock C. Role of ion channels and transporters in
cell migration. Physiological reviews 2012;92:1865-913.
4. Kohler M, Hirschberg B, Bond CT, Kinzie JM, Marrion NV, Maylie J, et al. Small-
conductance, calcium-activated potassium channels from mammalian brain. Science
1996;273:1709-14.
5. Girault A, Haelters JP, Potier-Cartereau M, Chantome A, Jaffres PA, Bougnoux P,
et al. Targeting SKCa channels in cancer: potential new therapeutic approaches. Curr Med
Chem 2012;19:697-713.
6. Potier M, Joulin V, Roger S, Besson P, Jourdan ML, Leguennec JY, et al.
Identification of SK3 channel as a new mediator of breast cancer cell migration. Mol Cancer
Ther 2006;5:2946-53.
7. Hosseini R, Benton DC, Dunn PM, Jenkinson DH, Moss GW. SK3 is an important
component of K(+) channels mediating the afterhyperpolarization in cultured rat SCG
neurones. J Physiol 2001;535:323-34.
8. Chen MX, Gorman SA, Benson B, Singh K, Hieble JP, Michel MC, et al. Small and
intermediate conductance Ca(2+)-activated K+ channels confer distinctive patterns of
distribution in human tissues and differential cellular localisation in the colon and corpus
cavernosum. Naunyn Schmiedebergs Arch Pharmacol 2004;369:602-15.
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
19
9. Burnham MP, Bychkov R, Feletou M, Richards GR, Vanhoutte PM, Weston AH, et
al. Characterization of an apamin-sensitive small-conductance Ca(2+)-activated K(+) channel
in porcine coronary artery endothelium: relevance to EDHF. Br J Pharmacol 2002;135:1133-
43.
10. Herrera GM, Pozo MJ, Zvara P, Petkov GV, Bond CT, Adelman JP, et al. Urinary
bladder instability induced by selective suppression of the murine small conductance
calcium-activated potassium (SK3) channel. J Physiol 2003;551:893-903.
11. Taylor MS, Bonev AD, Gross TP, Eckman DM, Brayden JE, Bond CT, et al.
Altered expression of small-conductance Ca2+-activated K+ (SK3) channels modulates
arterial tone and blood pressure. Circ Res 2003;93:124-31.
12. Chantome A, Girault A, Potier M, Collin C, Vaudin P, Pages JC, et al. KCa2.3
channel-dependent hyperpolarization increases melanoma cell motility. Exp Cell Res
2009;315:3620-30.
13. Girault A, Haelters JP, Potier M, Chantome A, Pinault M, Marionneau-Lambot S, et
al. New alkyl-lipid blockers of SK3 channels reduce cancer-cell migration and occurrence of
metastasis. Curr Cancer Drug Targets 2011;11:1111-25.
14. Wang ZH, Shen B, Yao HL, Jia YC, Ren J, Feng YJ, et al. Blockage of
intermediate-conductance-Ca(2+) -activated K(+) channels inhibits progression of human
endometrial cancer. Oncogene 2007;26:5107-14.
15. Gomez-Varela D, Zwick-Wallasch E, Knotgen H, Sanchez A, Hettmann T, Ossipov
D, et al. Monoclonal antibody blockade of the human Eag1 potassium channel function
exerts antitumor activity. Cancer Res 2007;67:7343-9.
16. Monet M, Lehen'kyi V, Gackiere F, Firlej V, Vandenberghe M, Roudbaraki M, et
al. Role of cationic channel TRPV2 in promoting prostate cancer migration and progression
to androgen resistance. Cancer Res 2010;70:1225-35.
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
20
17. Gu B, Espana L, Mendez O, Torregrosa A, Sierra A. Organ-selective
chemoresistance in metastasis from human breast cancer cells: inhibition of apoptosis,
genetic variability and microenvironment at the metastatic focus. Carcinogenesis
2004;25:2293-301.
18. Zhang C, Yan Z, Arango ME, Painter CL, Anderes K. Advancing bioluminescence
imaging technology for the evaluation of anticancer agents in the MDA-MB-435-HAL-Luc
mammary fat pad and subrenal capsule tumor models. Clin Cancer Res 2009;15(1):238-46.
19. Dvorak MM, Riccardi D. Ca2+ as an extracellular signal in bone. Cell Calcium
2004;35:249-55.
20. Franco SJ, Huttenlocher A. Regulating cell migration: calpains make the cut. J Cell
Sci 2005;118:3829-38.
21. Yang S, Zhang JJ, Huang XY. Orai1 and STIM1 are critical for breast tumor cell
migration and metastasis. Cancer Cell 2009;15:124-34.
22. Chen YF, Chiu WT, Chen YT, Lin PY, Huang HJ, Chou CY, et al. Calcium store
sensor stromal-interaction molecule 1-dependent signaling plays an important role in cervical
cancer growth, migration, and angiogenesis. Proc Natl Acad Sci U S A 2011;108:15225-30.
23. Pani B, Singh BB. Lipid rafts/caveolae as microdomains of calcium signaling. Cell
Calcium 2009;45:625-33.
24. Gajate C, Mollinedo F. Lipid rafts and Fas/CD95 signaling in cancer chemotherapy.
Recent Pat Anticancer Drug Discov 2011;6:274-83.
25. Fedida-Metula S, Feldman B, Koshelev V, Levin-Gromiko U, Voronov E, Fishman
D. Lipid rafts couple store-operated Ca2+ entry to constitutive activation of PKB/Akt in a
Ca2+/calmodulin-, Src- and PP2A-mediated pathway and promote melanoma tumor growth.
Carcinogenesis 2012;33:740-50.
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
21
26. Chambers AF. MDA-MB-435 and M14 cell lines: identical but not M14
melanoma? Cancer Res 2009;69:5292-3.
27. Kallioniemi OP, Wagner U, Kononen J, Sauter G. Tissue microarray technology for
high-throughput molecular profiling of cancer. Hum Mol Genet 2001;10:657-62.
28. Calaghan S, Kozera L, White E. Compartmentalisation of cAMP-dependent
signalling by caveolae in the adult cardiac myocyte. Journal of molecular and cellular
cardiology 2008;45:88-92.
29. Barascu A, Besson P, Le Floch O, Bougnoux P, Jourdan ML. CDK1-cyclin B1
mediates the inhibition of proliferation induced by omega-3 fatty acids in MDA-MB-231
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.
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
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
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
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.
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
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
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
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.
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
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.
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
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.
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
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
0.2
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)
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
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
5 Ca
Apamin-sensitive current
(n=5)
2
3
4
5
6
7
8
SK3+
SK3-
F340
/F38
0Mann-Whitney
p=0.018
0.4
0.6
0.8
1.0
1.2
Mann-Whitneyp=0.0051
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
Calp
ain
activ
ityre
senc
e, n
orm
aliz
ed)
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
Author m
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
1.0
1.2
B
4
52 mM external [Ca2+ ]
A
1
1.2
zed
Figure 3SK3+ cells
zed
SK3+ cells
p=0.3314
0 0
0.2
0.4
0.6
0.8
0
1
2
3
4
0 40 80 120 160
F340
/F38
0
siControl
siOrai1
0
0.2
0.4
0.6
0.8
N=4 N=4F340
/F38
0, n
orm
aliz
Mann-Whitneyp=0.0051
Cell
mig
ratio
n, n
orm
aliz
Orai 1
Hsc 70
STIM 1
N=3N=3N=3
38 KDa
70 KDa
81 KDa
Kruskal-Wallis p=0.001 and post
hoc Mann-Whitney (compared to
control)
p=0.0027
0.0
1 0
1.2
Time (sec)
0siControl siOrai1
4
5
2 mM external [Ca2+ ]
1
1.2
zed
SK3- cells
zed
siControl siSTIM 1 siOrai 1
SK3- cells
100
Mann-Whitneyp=0.3827Kruskal-Wallis p=0.089
0 0
0.2
0.4
0.6
0.8
1.0
0
1
2
3
4
0 40 80 120 160 200
siControl
siOrai1
0
0.2
0.4
0.6
0.8
N=4 N=4
F340
/F38
0, n
orm
aliz
Cell
mig
ratio
n, n
orm
aliz
N=3 N=3 N=3
F340
/F38
0
0
20
40
60
80R
elat
ive
mR
NA
leve
l(%
of S
iCon
trol)
N=3 N=30.0
SK3- ATTO-594 T Orai1-FITC
Membrane fractions
SK3+ cells
C
Membrane fractions
SK3- cells
Time (sec) 0siControl siOrai1
D
siControl siSTIM 1 siOrai 1 siOrai 1 siSTIM1
20 µm20 µm
Β-adaptin109 kDa
Caveolin-120 kDa
1 2 3 4 5 6 7 8
Lipid-rafts Non Lipid-rafts
1 2 3 4 5 6 7 8
Lipid-rafts Non Lipid-rafts
Merge
Orai 137 kDa
SK375 kDa
on April 26, 2018. ©
2013 Am
erican Association for C
ancer Research.
cancerres.aacrjournals.org D
ownloaded from
Author m
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
lize
d
Migration, 5 mM Ca2+
0 6
0.8
1.0
1.2Ca2+ free 2 mM external [Ca2+ ]
0/F
38
0
Vehicle
no
rma
lize
dConstitutive Ca2+ entry
Mann-Whitney0 0122
3
3.5
4
4.5
0.0
0.2
0.4
0.6
EVehicle
Ohmline 1 µM, 24h
Ce
ll m
igra
tion
, n
N=2 N=20.0
0.2
0.4
0.6
Time (sec)
F34
0
Ohmline 1µM, 24 h
F3
40
/F3
80
, n
Vehicle Ohmline 1 µM, 24h
N=5 N=5
D
p=0.0122
0
0.5
1
1.5
2
2.5
0 20 40 60 80 100
40
60
80Vehicle
Ohmline
n flu
x x
104
20
30
40
50
ne
(µ
g/to
tal l
ipid
s)
ren
ce o
f me
tast
ase
s (%
)
Vehicle (N=6)
Ohmline (N=9)
0
20
Pho
ton
0
10
Oh
lmlin
Occ
urr
lung bone
Not
de
tect
ed
100
200
300
400vehicle
Ohmline
or
volu
me
(m
m3
)
10
20
30
Ohl
mlin
e g/
tota
l lip
ids)
F
(N=19)
(N=16)
0
100
2 3 4 5 6
Week post-graft
Tu
mo
0
10O(µ
g
N=16
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
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
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
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
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
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
Updated version
10.1158/0008-5472.CAN-12-4572doi:
Access the most recent version of this article at:
Material
Supplementary
http://cancerres.aacrjournals.org/content/suppl/2013/06/19/0008-5472.CAN-12-4572.DC1
Access the most recent supplemental material at:
Manuscript
Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
.http://cancerres.aacrjournals.org/content/early/2013/06/15/0008-5472.CAN-12-4572To request permission to re-use all or part of this article, use this link
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