BBA - Molecular and Cell Biology of Lipids 1866 (2021) 158906

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BBA - Molecular and Cell Biology of Lipids

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TMEM97 facilitates the activation of SOCE by downregulating the

association of cholesterol to Orai1 in MDA-MB-231 cells
C. Cantonero a, P.J. Camello b, G.M. Salido a, J.A. Rosado a, P.C. Redondo a, *
a
Department of Physiology (Phycell group), University of Extremadura, Caceres 10003, Spain
b
Department of Physiology (FIMUL group), University of Extremadura, Caceres 10003, Spain

A R T I C L E I N F O A B S T R A C T

Keywords: The expression of TMEM97, a regulator of cholesterol transport, has been reported to be enhanced in some
TMEM97 tumour cells. We have recently shown that TMEM97 is involved in the proliferation of the breast cancer cell line
SOCE MDA-MB-231, probably through changes in store-operated calcium entry (SOCE). By using silencing and over­
Orai1
expression of TMEM97 in MDA-MB-231 cells (two manoeuvres that either reduce or increase the calcium influx,
Cholesterol
MDA-MB-231 cells
respectively), we show enhanced cholesterol uptake in these cells as compared to the non-tumoral breast cell
line, MCF10A. The enhanced cholesterol uptake in MDA-MB-231 cells was inhibited by silencing TMEM97, while
overexpression of this protein increased cholesterol uptake in MCF10A cells and, therefore, indicating that this
protein plays a role in the enhanced cholesterol uptake in MDA-MB-231 cancer cell line. TMEM97 silencing and
overexpression resulted in an increase and decrease in the association of cholesterol to the SOCE calcium channel
Orai1, respectively. Interestingly, silencing of TMEM97 in MDA-MB-231 cells significantly reduced the co-
localization of Orai1 with the SOCE regulatory protein STIM1. Finally, neither silencing nor overexpression of
TMEM97 altered SOCE in MDA-MB-231 cells transfected with the cholesterol insensible mutant of Orai1(Y80E).
Our results reveal a novel regulatory mechanism of SOCE that relies on TMEM97 activity that courses through
the reduction of the cholesterol content in the plasma membrane, and subsequently, by impairing its interaction
with Orai1.

1. Introduction (σ-2R/TMEM97), reduced SOCE and, subsequently, impaired the

Influx of extracellular calcium ion (Ca2+) through the activation of
the store operated Ca2+ entry (SOCE) requires the association between
the plasma membrane selective Ca2+ channel, Orai1, and the endo­
plasmic reticulum Ca2+ sensor, STIM1 [1–3]. In addition, the Ca2+ entry
through Orai1 regulates the activation of transcription factors, such as
NAFT1, which has been involved in the exacerbated proliferation of
certain cancer cell lines like, the triple negative breast cancer cells,
MDA-MB-231 [4,5]. Nowadays, searching for Orai1 antagonists or drugs
that may target its regulatory proteins has gained great interest within
the scientific community, because its potential for preventing the
development of different types of cancer [6–8]. Very recently, we have
tested the effects of NO1 in the activation of SOCE in MDA-MB-231 cells
[9]. This novel antagonist of σ-2 receptor/Transmembrane protein 97
contribution of the Ca2+ entry in the cancer hallmarks of these cells
[9,10]. Our results revealed a plausible positive role of σ-2R/TMEM97 in
cancer progression, since NO1 decreased proliferation in MDA-MB-231
and MDA-MB-468 cells [9]. Additionally, the link between TMEM97
and development of different cancer types has been well stablished.
Many studies can be found in the literature pointing the involvement of
TMEM97 (also referred as meningioma-associated protein 30, MAC30)
in colorectal, gastric, ovarian and breast cancers [11–15]. Although the
nature of σ-2 receptor has been subjected to an intense debate, its ge­
netic characterization as TMEM97 [16], has prompted a revisitation of
the physiological roles previously attributed to σ-2 receptor [17].
TMEM97 has been described to act as a regulator of the cholesterol
metabolism [18,19]. In this sense, cholesterol depletion from the culture
medium exacerbated the expression of TMEM97, among other genes in

Abbreviations: SOCE, store operated calcium entry; TMEM97, transmembrane protein 97; LDLDR, LDL receptor; Orai1, calcium release-activated calcium channel
protein 1; STIM1, stromal interaction molecule 1; TG, thapsigargin; OverTMEM97, GFP-overexpression plasmid of TMEM97; siTMEM97, small interference RNA
against TMEM97.
* Corresponding author at: Department of Physiology, University of Extremadura, 10003 Cáceres, Spain.
E-mail address: pcr@unex.es (P.C. Redondo).

https://doi.org/10.1016/j.bbalip.2021.158906
Received 6 November 2020; Received in revised form 2 February 2021; Accepted 11 February 2021
Available online 20 February 2021
1388-1981/© 2021 Published by Elsevier B.V.
C. Cantonero et al.
charge of the metabolism of cholesterol [18,20]. TMEM97 silencing
allowed the authors to conclude that it is involved in the cellular
cholesterol homeostasis and, thus, they claimed that TMEM97 is a
SREBP protein (sterol regulatory element binding protein) that may
regulate the LDL cholesterol transport-regulating protein Niemann-Pick
C1 (NPC1) [21]. Later, it was described that TMEM97 together with the
progesterone receptor membrane complex 1 (PGRMC1) and LDL re­
ceptor (LDLDR) may constitute a protein complex responsible of LDL
uptake by the cells [22]. Interestingly, MDA-MB-231 cells and other
triple negative breast cancer cells, presented enhanced LDLR, which has
been linked to their elevated proliferation rates and, as a consequence,
the exacerbated migration rates presented by these cells that was also
promoted by the exogenous lipids administration and cholesterol
esterification [23,24]. Moreover, the concentration of LDL-cholesterol
in the plasma of the patients has been proposed as predictor of breast
cancer progression during its diagnosis [25].
SOCE components, such as Orai1 channels and STIM1, present
cholesterol binding regions and both molecules were strongly colo­
calized within cholesterol patches in the plasma membrane, kwon as
lipid-raft [26–29]. These cholesterol rich regions provide the physical
stability to SOCE activation, since they promote the generation of the
regulatory protein complexes [30]. It has been demonstrated that the
disruption of lipid-raft structure by using chemical agents decreased
SOCE [31,32]. In keeping with this, the association of cholesterol to
Orai1 was also reported to limit SOCE activation [33]. Mutation of the
L74I and Y80S within the N-terminal domain of Orai1 resulted in an
increased TG-evoked SOCE, while reduced the association of cholesterol
to Orai1 [33]. Therefore, a strict balance in the cholesterol content
within the plasma membrane would be required for controlling SOCE. In
the present study, we have analysed whether TMEM97 may regulate
SOCE by modifying the cholesterol association to Orai1 in MDA-MB-231
cells.
2. Material and methods
2.1. Materials
TopFluor-cholesterol was from Avanti Polar Lipids (Alabaster, AL,
USA). SiRNA against TMEM97 (siTMEM97), the GFP-overexpression
plasmid of TMEM97 (overTMEM97) and the rabbit polyclonal anti-
TMEM97 antibody (Catalogue number TA309912, epitope: amino acid
105–155) were from Origene© (Rockville, MD, USA)). DharmaFECT Kb
transfection reagent was purchased by Dharmacon Inc. (Lafayette, CO,
USA). G-GECO1.2-Orai1Y80E was a gift from Michael Cahalan (Addg­
ene plasmid # 73565; http://n2t.net/addgene:73565; RRID:Addg­
ene_73565) [35].Orai1-CFP was a gift from Anjana Rao (Addgene
plasmid # 19757; http://n2t.net/addgene: 19757; RRID:Addg­
ene_19757) [51]. pENTR1a-mCherry-STIM1 was a gift from Nicolas
Demaurex (Addgene plasmid # 114176; http://n2t.net/addgene:
114176; RRID:Addgene_114176) [52]. Horseradish peroxidase-
conjugated goat anti-mouse immunoglobulin G (IgG) antibody and
goat anti-rabbit IgG antibody were from Jackson laboratories (West
Grove, PA, USA). SuperSignal® West Dura extended duration substrate
reagent was from ThermoFisher Scientific (Waltham, MA, USA). Fura-2
acetoxymethyl ester (fura-2/AM) was from Molecular Probes (Leiden,
The Netherlands). Anti-STIM1 antibody (Catalogue number 6140954,
epitope: amino acid 25-139) was purchased from BD-Bioscience®
(Madrid, Spain). SiRNAA, rabbit polyclonal anti-Orai1 antibody (cata­
logue number O8264, epitope: amino acids 288–301 of human Orai1),
rabbit polyclonal anti-β-actin antibody (catalogue number A2066,
epitope: amino acids 365–375 of human β-actin), thapsigargin (TG),
EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N′ ,N′ -tetraacetic
acid), HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid),
and bovine serum albumin (BSA) as well as other reagents of analytical
grade were from Sigma (Madrid, Spain).
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BBA - Molecular and Cell Biology of Lipids 1866 (2021) 158906
2.2. Cell culture
MDA-MB-231 cells were ordered to ATCC® collection (Manassas,
VA, USA) and MCF10A cells were gently provided by Dr. Potier-
Cartereau (Université Francois Rabelais, Tours France). DMEN, supple­
mented with fetal bovine serum (FBS) and penicillin/streptomycin, was
used for culturing the MDA-MB-231 cells; meanwhile, MCF10A cells
were cultured using DMEN-F12 that was supplemented with horse
serum, insulin, hydrocortisone, EGF, cholera toxin and penicillin/
streptomycin. Regular cultured conditions were used, which consisted
on to maintain the cells at 37 ◦ C, 95% of humidity and 5% of CO2, and
then, replacement of the culture medium was done twice a week as
required due to the cell growth.
2.3. Monitorization of the changes in the cytosolic calcium concentration
([Ca2+]c)
Changes in the cytosolic Ca2+ concentration ([Ca2+]C) were deter­
mined in MDA-MB-231 and MCF10A loaded with the Ca2+ dye fura-2, as
widely described elsewhere [9,31,34]. Briefly, MDA-MB-231 and
MCF10A cells were allowed to spread onto coverslips imbibed in the
culture medium for 24 h. Untreated cells or cells transfected for 24 h
either with siRNA A or siTMEM97 or overTMEM97, were incubated with
fura-2/AM for 30 min at 37 ◦ C. Coverslips containing the fura-2 loaded
cells were mounted in a perfusion chamber and maintained in a HEPES
Buffered Saline (HBS)-rich Ca2+ buffer (in mM: 125 NaCl, 5 KCl, 1 MgCl2,
5 glucose, 25 HEPES and pH 7.3, supplemented with 0.1% (w/v) BSA
and 50 μM of CaCl2). The perfusion chamber was placed under an epi­
fluorescence microscope in order to monitor the changes in the fluo­
rescence of fura-2 at 505 nm, while it was alternatively excited at 340/
380 nm. Changes in the [Ca2+]C evoked by blocking the activity of the
sarcoendoplasmic reticulum-Ca2+-ATPase (SERCA) with thapsigargin
(TG) were monitored for 5 min. Subsequently, 1 mM of CaCl2 was added
to the extracellular medium in order to monitor the Ca2+ entry through
the SOCE mechanism that has been previously activated as result of the
addition of TG to the cells, which was recorded for additional 2 min.
Areas under the curves were estimated in order to compare the changes
in the Ca2+ homeostasis between the different cell types or treatments.
Alternatively, MDA-MB-231 cells were transfected for 24 h with
either the empty plasmids (Mock) or the overexpression plasmids of the
GECO-Orai1(Y80E) + RFP-STIM1 (Control) and GECO-Orai1(Y80E) +
RFP-STIM1 together with either GFP-overTMEM97 (overTMEM97) or
the siTMEM97. According to the literature, GECO fused to a Ca2+
channel can be used to monitor the Ca2+ entry that is actually passing
through the tagged Ca2+ channels [35]. Additionally, transfected cells
were also loaded with fura-2/AM in order to simultaneously monitoring
the changes in the [Ca2+]C and the amount of Ca2+ actually passing
across the pore of the Orai1 channel. The experimental protocol per­
formed with these transfected MDA-MB-231 cells was similar to the one
explained in the previous paragraph.
2.4. Protein immunoprecipitation and Western blotting
MDA-MB-231 cells were kept under resting conditions or were
stimulated for 1 min with TG, upon which, cells were lysed using NP-40
buffer. Anti-STIM1 or anti-Orai1 antibodies (2 μg/mL) and agarose-
beads (25 μL) were added to the cell lysates for immunoprecipitation
of the proteins of interest. Alternatively, protein content in the cell ly­
sates were determined with the BCA protocol, and following, upon
protein samples were normalized, they were mixed with an equal vol­
ume of Laemmli’s buffer (LB) containing 5% of dithiothreitol (DTT).
Similarly, immunoprecipitated proteins were washed several times with
ice-cold PBS buffer (containing in mM: 137 NaCl, 2.7 KCl, 8 Na2HPO4,
and 2 KH2PO4, and adjusted to pH 7.4), and finally, they were denatu­
ralized by mixing with equal volume of LB + 5% of DTT. Following,
denaturalized protein samples were suggested to Western blotting (WB)
C. Cantonero et al.
using an anti-Orai1 (1:500 in TBST), anti-STIM1 antibody (1:1000 in
TBST) and anti-TMEM97 (1:500 in TBST) antibodies for 2 h at room
temperature. Upon incubation with the adequate HRP-conjugated anti-
IgG secondary antibody, the membranes were exposed to the chem­
ioluminiscence solution (Dura ECL from Pierce ®, Thermofisher,
Madrid, Spain) for 10 min and the optic densitometry images were ac­
quired using the C-digit device (Licor® Biosciences, Lincoln, NE, USA).
Reprobing of the membranes with either anti-STIM1 antibody and anti-
Orai1 antibody (in case of the IP samples) or anti-β-actin antibody (in
case of the WB samples) was done as protein loading control.
2.5. Cholesterol visualization
TopFluor-cholesterol was used to monitorization of the cholesterol
uptake by the cells, as well as to visualize its intracellular redistribution
and co-localization with other proteins, being this later more deeply
explained in the following section [33,53,54]. Briefly, MCF10A and
MDA-MB-231 cells were cultured under resting conditions or were
transfected as indicated, either with the overTMEM97 or siTMEM97,
respectively. At the day of the experiment, these cells were further
incubated for additional 30 min at 37 ◦ C with 2.5 μM of TopFluor-
cholesterol inside the cell incubator [33]. Fluorescence images were
acquired at 488/505 (Ex/Em wavelengths) before (time 0) and after the
cells were incubated with the TopFluor-cholesterol (time 30 min) by
using an epifluorescence microscope (Nikon Eclipse Ti2, Amsterdam,
The Netherlands), a 40× oil-fluorescence objective and a cooled digital
sCMOS camera (Zyla 4.2, Andor, Belfast, UK). Images were analysed
using the NIS-Elements AR software (Nikon, Amsterdam, The
Netherlands).
2.6. Co-localization assays
MDA-MB-231 cells were transfected for 24 h with the overexpression
plasmid of CFP-Orai1 and RFP-STIM1 (control) and, additionally, with
either siTMEM97 or overTMEM97. Next day, upon positive cell trans­
fection was confirmed, the cells were incubated in the culture medium
with TopFluor-Cholesterol for 30 min (2.5 μM at 37 ◦ C). Once incubation
time was over, the culture medium was replaced with a HBS solution
containing 1 mM CaCl2 and the coverslips containing the cells were
placed at the stage of a confocal microscope (Nikon A1, Eclipse Ti, Nikon
Corporation, Tokyo, Japan). Images were acquired with a 60× oil-
objective (n.a. 1.49) at rest and 4 min after stimulation with TG (1
μM). The cells were excited in sequential line scan mode at 405 nm (CFP-
Orai1), 488 nm (TopFluor Cholesterol) and 561 nm (RFP-STIM1) using a
2× average and a nominal z resolution of 1 μm. NIS Elements AR soft­
ware (Nikon) was used to calculate the Pearson’s colocalization index.
The colocalization index (Pearson’s correlation) was analysed in the
captured images using the NIS-Elements AR software (Nikon, Amster­
dam, The Netherlands). Data were presented in the histograms as the
percentage of cells that presented positive increase in Pearson’s index
upon stimulating with TG, and further, we also determined the fold
increase in the colocalization evoked by TG addition to the cells.
2.7. Statistical analysis
Analysis of significance between two data groups was done using the
Student’s t-test. One way ANOVA and subsequent Dunnett’s post-hoc
test were used for multiple comparisons. Only p < 0.05 was consid­
ered as significative.
3. Results
3.1. Analysis of the TMEM97 expression and its involvement in the Ca2+
homeostasis in MDA-MB-231 cells
MCF10A and MDA-MB-231 cells loaded with fura-2 were stimulated
3
BBA - Molecular and Cell Biology of Lipids 1866 (2021) 158906
in a HBS-poor Ca2+ buffer (75 μM of EGTA was added as indicated by the
arrowheads) with TG (1 μM for 5 min) which evoked a similar Ca2+
release from the intracellular stores in both cells types, as shown in
Fig. 1A (beside histogram; p < 0.001; n = 6). Interestingly, TG-evoked
Ca2+ entry through activation of SOCE was much smaller in MCF10A
cells than in MDA-MB-231 cells, as evidenced during the addition of 1
mM of CaCl2 to the extracellular medium, which is in agreement with
previous results [10,34]. Analysis of TMEM97 expression in both cells
types by Western blotting revealed a significant elevated expression of
TMEM97 in MDA-MB-231 cells respect to MCF10A cells, where it was
almost undetected (Fig. 1B; p < 0.01; n = 4). In order to confirm the
involvement of TMEM97 in the exacerbated SOCE described in the
MDA-MB-231 cells, we used siTMEM97 or overTMEM97 to efficiently
reduce the expression of TMEM97 (Fig. 1C; n = 4) and overexpress
TMEM97 in MDA-MB-231 cells, respectively (Fig. 1D, n = 3). As
depicted in Fig. 1E, TMEM97 silencing evoked a significant reduction in
TG-evoked SOCE (24 ± 15%; see the insert, p < 0.01; n = 6), while,
overTMEM97 induced an increase in TG-evoked SOCE (56 ± 18%; see
the insert, p < 0.001, n = 6). Interestingly, none of these genetic mod­
ifications affected to the apparent amount of Ca2+ mobilized from the
Ca2+ stores of the cells in response to the TG administration. These data
agrees with previous reports from our laboratory and reinforce the
positive role of TMEM97 in SOCE activation in these cells [9].
3.2. TMEM97 regulates the cholesterol transport in MDA-MB-231 cells
TMEM97 has been reported to be involved in the lipid uptake and
transport to the endoplasmic reticulum of the cells [18–21]. Therefore,
we analysed the involvement of TMEM97 in the cholesterol uptake by
both MCF10A (Fig. 1F-H) and MDA-MB-231 cells (Fig. 1I-L) using the
fluorescent conjugated Top-Fluor cholesterol [33]. So, cell incubation
with TopFluor-cholesterol (2.5 μM) was recorded for 30 min and
revealed that the cholesterol uptake by MCF10A was lower than that
observed in MDA-MB-231 cells (Compare Fig. 1F vs 1I). Interestingly,
MCF10A transfected with overTMEM97 exhibited an enhanced
cholesterol-derived fluorescence than those transfected with the empty
vector (compared Fig. 1F vs 1G and see the beside histogram, Fig. 1H, p
< 0.001, n = 4). In keeping with this, MDA-MB-231 cells transfected
with siTMEM97, presented significantly less cholesterol-derived stain
than those transfected with the siRNAA after 30 min of incubation with
TopFluor-cholesterol (see the Fig. 1J, and the beside histogram, Fig. 1L;
p < 0.001, n = 4). These data reinforce the previously described role of
TMEM97 in cholesterol metabolisms. However, although a slight in­
crease in the overall YFP-cholesterol uptake in the MDA-MB-231 cells
was found in some cell preparation that were previously transfected
with overTMEM97, the statistical analysis did not reported statistical
significance respect mock MDA-MB-231 cells (see the Fig. 1K, and the
above histogram in Fig. 1L; p < 0.001, n = 4); which may be explained
by the exacerbated amount of TMEM97 expressed in these cells, so to
overexpress more protein may affect to the redistribution cholesterol
within the cells, as previously demonstrated by others [21].
3.3. TMEM97 interacts with the SOCE components
SOCE activation requires the interaction of STIM1 and Orai1 in the
plasma membrane [4,30], and considering the stimulatory effect of
TMEM97 on SOCE described above, we analysed its possible interaction
with the components of SOCE, STIM1 and Orai1. As depicted in Fig. 2A,
STIM1 was immunoprecipitated in samples of MDA-MB-231 cells kept
under resting conditions or stimulated with TG for 1 min. We found that
TMEM97 interacted with STIM1 under resting conditions, and this
coupling was reduced to 0.7 ± 0.1 fold increase upon stimulating the
cells with TG (white bars in the beside histogram; p < 0.05, n = 6). A
reproduction of the immunoprecipitation protocols using anti-Orai1
antibody also revealed an Orai1/TMEM97 association in MDA-MB-231
cells under resting conditions, which seems to be greater than that
C. Cantonero et al. BBA - Molecular and Cell Biology of Lipids 1866 (2021) 158906

(caption on next page)

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C. Cantonero et al. BBA - Molecular and Cell Biology of Lipids 1866 (2021) 158906

Fig. 1. Role of TMEM97 in Ca2+ and cholesterol homeostasis. a) MCF10A and MDA-MB-231 cells were loaded with fura-2. Single cells imaging experiments were
done and we monitored for 5 min the Ca2+ mobilization evoked by the administration TG (1 μM) to the cells in absence of extracellular Ca2+ (EGTA 75 μM was added
as indicated by the arrowhead). SOCE evoked by TG was visualized for additional 2 min after the addition of 1 mM of CaCl2 to the extracellular medium (n = 6).
Areas under the curves as result of the changes in the fluorescence of fura-2 upon the addition of TG and CaCl2 to the cells were used for comparing between the
different cell types as depicted in the beside graph-bar. b) MCF10A and MDA-MB-231 cells cultured under regular conditions or were transfected for 24 h with
siTMEM97 (c), at the day of the experiment, the cells were count and lysed by mixing with NP40 buffer. After proteins normalization, the different samples were
mixed with Laemmlis’s buffer to evoke protein denaturalization and finally, the amount of TMEM97 expression in the different samples was determined by WB using
the specific anti-TMEM97 antibody, as detailed in Material and Methods (n = 4). Presented images belong to the same gel where protein were transferred to the
nitrocellulose membrane, cut according to the molecular weight of the proteins of interest, and subsequently, developed with the indicated antibodies. d) MDA-MB-
231 cells were transfected with overTMEM97 and after 24 h, positive transfected cells were observed under an epifluorescence microscope at the middle plane (40×
oil-fluorescence objective was used) (n = 3). e) MDA-MB-231 cells were cultured under control conditions (Mock) or were transfected with either siTMEM97 or
overTMEM97. The next day, cells were loaded with fura-2 and the changes in the TG-evoked SOCE as result of the modification of the TMEM97 expression were
analysed by using a similar protocol that the one in the Fig. A; bar-graph in the inset represent the mean ± S.E.M. of TG-evoked Ca2+ entry of transfected cells with
either siTMEM97 or overTMEM97 as compared to the non-genetically modified mock cells. f & g) MCF10A and MDA-MB-231 cells (i-k) were cultured under control
conditions or were transfected with overTMEM97 (g & k) or siTMEM97 (j), as indicated. 24 h later, cells were further incubated with 2.5 μM of TopFluor-cholesterol
for around 30 min at 37 ◦ C inside the incubator. Images at the middle plane of the cells were captured before and after cell treatment with TopFluor-cholesterol by
using an epifluorescence microscope (40×-oil fluorescence objective was used). In those cells transfected with overTMEM97, the fluorescence emitted by the GFP-tag
was estimated before the incubation with TopFluor-cholesterol and it was used for correcting the fluorescence emitted by the cells upon the incubation with the
cholesterol. Fluorescence emitted by the TopFluor-cholesterol (488/505 nm (Ex/Em)) was determined using the NIS-images software and it was shown in (h) and (l),
where the histograms shown the means of the percentage ± S.E.M. of the arbitrary units the TopFluor-cholesterol fluorescence found in the genetically modified cells
respect the fluorescence emitted by their respective mock cells, and are representative of four independent transfections. Bars in the images represent 50 μm. *, ***:
represents p < 0.05 and 0.001, respectively, respect mock cells non-genetically modified cells.

observed between STIM1 and TMEM97. In addition, co-
immunoprecipitation between Orai1 and TMEM97 was significantly
enhanced (1.4 ± 0.2 fold increase, p < 0.05, n = 4) during SOCE stim­
ulation with 1 μM of TG (See Fig. 2A, black bars in the beside histo­
grams). As it has been widely described elsewhere, MDA-MB-231 cell
stimulation with TG (1 μM) evoked a significant increase of 1.4 ± 0.1
fold increase (p > 0.05, n = 6) in the association between STIM1 and
Orai1, which was observed independently of the antibody used for
immunoprecipation of the protein samples.
In order to corroborate whether regulation of SOCE by TMEM97
might be related to its interaction with Orai1 and STIM1, we obtained
colocalization images from MDA-MB-231 cells transfected with CFP-
Orai1, RFP-STIM1 and GFP-overTMEM97. As depicted in Fig. 2B (n =
3), in MDA-MB-231 cells kept under resting conditions, TMEM97 clearly
co-localized with Orai1 in some cell locations (see arrowheads in the top
images; mean Pearson’s coefficient = 0.35 ± 0.05, which resulted of
considering 45 cells of 3 independent cell transfections), but it was
scarce found associated to STIM1 (see asterisks in the bottom images;
mean Pearson’s coefficient = 0.1 ± 0.0, which resulted of considering 45
cells of 3 independent cell transfections).
3.4. TMEM97 regulates the cholesterol interaction with Orai1
Orai1 contains in its structure a binding region for cholesterol (CB)
[33]. In order to ascertain whether TMEM97 regulates the cholesterol
binding to the CB of Orai1, we performed the colocalization analysis
between TopFluor-cholesterol and Orai1 in MDA-MB-231 cells grown
under normal conditions (Fig. 3A, Control) or in cells that were genet­
ically modified by transfecting them with either siTMEM97 (Fig. 3B) or
overTMEM97 (Fig. 3C). Confocal images of Orai1 and TopFluor-
cholesterol acquired at the middle plane of the resting cells showed a
positive colocalization between TopFluor-cholesterol (green colour) and
Orai1 (CFP-Orai1: represented with the red colour) in control MDA-MB-
231 cells (Fig. 3A). This association was decreased by stimulation for 4
min with TG (1 μM). Interestingly, in MDA-MB-231 control cells positive
colocalization between TopFluor-cholesterol and Orai1 was observed,
and it is still evident upon stimulation of cells with TG (see yellow/or­
ange dots in Fig. 3A, which are highlighted using arrowheads).
Following, in the cells transfected with siTMEM97 (Fig. 3B), we
observed an increase in the stain with TopFluor-cholesterol within the
plasma membrane and near-plasma membrane regions, which was less
evident in control cells that also presented inclusions in the cytosol of
the cells (Fig. 3A). However, in cells transfected with overTMEM97, we
observed a more diffuse stain with cholesterol which exhibited more
enriched inclusions in the cytosol of the majority of the cells (Fig. 3C). In
line with the later, MDA-MB-231 cells transfected with overTMEM97
presented colocalization between Orai1 and TopFluor-cholesterol but
seems to be limited to the interior of the cells that remained even after
cell stimulation with TG (see yellow-orange dots highlighted in Fig. 3C).
These data suggest that TMEM97 facilitates the transport of cholesterol
to the interior of the cells as previously reported [18] and further, may
explain the fact that no statistical cholesterol differences in the choles­
terol stain was found in the overTMEM97 transfected cells (presented
above in Fig. 1K & L). These data support the hypothesis that TMEM97
cooperates in the activation of SOCE through relieving the inhibitory
interaction between cholesterol and Orai1 at the plasma membrane.
3.5. TMEM97 silencing reduces STIM1/Orai1 colocalization
Given that SOCE requires the coupling between STIM1 and Orai1
[1,4] we used confocal microscopy to ascertain whether silencing of
TMEM97 may alter the interaction between these SOCE components.
MDA-MB-231 cells were transfected with CFP-Orai1 and RFP-STIM1,
previously used to explore the interaction between both proteins dur­
ing SOCE activation by TG [33]. Once transfected the cells were kept
untreated or were additionally transfected with either siRNAA (control)
or siTMEM97 and, at day of experiment, they were also incubated with
TopFluor-cholesterol as previously described above. As shown in the
Fig. 4A, TG application increased STIM1/Orai1 colocalization as
compared to resting conditions (compare top and bottom images on the
right-hand side of the Fig. 4A (n = 4)). Additionally, we observed a
reduced colocalization between cholesterol and Orai1 in the same
evaluated cells (left-hand side of the Fig. 4A. Transfection with siT­
MEM97 reduced the STIM1/Orai1 colocalization induced by TG (see
Fig. 4B right-hand side images), but enhanced the association between
cholesterol and Orai1 (See Fig. 4B and C/D). A more detailed observa­
tion of the cell images revealed that in those cell locations where a
positive colocalization between TopFluor-cholesterol and Orai1
occurred, we did not observed colocalization between STIM1 and Orai1
(see the enlarged cell images presented in the Fig. 4E, where some points
of interest are indicated either by arrowheads (cholesterol/Orai1) or by
the asterisks (Orai1/STIM1)); therefore, reinforcing the idea that Orai1
interaction with cholesterol impairs its association with STIM1. As
presented in the bar-charts of Fig. 4C, we observed an increase in the
number of cells that upon being transfected with siTMEM97 and stim­
ulated with TG, presented enhanced colocalization between cholesterol
and Orai1, while simultaneously exhibited a reduction in the STIM1/
Orai1 colocalization index. A quantitative approximation of localization

5
C. Cantonero et al.
using Pearson’s coefficient also indicated that silencing of TMEM97
increased the association of Orai1 with cholesterol but impaired coloc­
alization with STIM, as presented in the histograms depicted in Fig. 4F.
As it is shown in the histogram, an enhanced fold increase in the
colocalization index between Orai1 and cholesterol upon TG stimulation
was evidenced in cell treated with siTMEM97 respect control cells (left
ordinate axes), while simultaneously these cells presented a reduction in
the fold increase of the Orai1/STIM1 colocalization index upon TG
stimulation (right ordinate axes).
We also studied the distribution of Orai1, cholesterol and STIM1 in
6
BBA - Molecular and Cell Biology of Lipids 1866 (2021) 158906
Fig. 2. TMEM97 interact with Orai1. a & b)
MDA-MB-231 cells were cultured under
regular conditions and at the day of the
experiment cells were kept under resting
conditions (C) or were stimulated for 1 min
with TG (TG; 1 μM), and then, the cells were
count, normalized and lysed with NP40
buffer. Cell lysates were subjected to an
immunoprecipitation protocol by adding
either 2 μg/mL of either anti-STIM1 (a) or
anti-Orai1 antibodies (b) and beads of
agarose. Next day, immunoprecipitated
proteins were subjected to WB using the
anti-TMEM97 antibody or Orai1 antibody as
indicated. Membranes were subsequently
reprobed with the same antibody used for
immunoprecipitation the protein, which was
used as input protein control. Presented
images belong to the same gel where protein
were transferred to the nitrocellulose mem­
brane, cut according to the molecular weight
of the proteins of interest, and subsequently,
developed with the indicated antibodies.
Histograms in the right hand-side represent
the mean ± S.E.M of the fold increase of TG
evoked coupling between the respective
analysed proteins. **: p < 0.05 respect the
coupling values found in control non-
stimulated cells. c) MDA-MB-231 cells were
transfected with YFP-TMEM97, CFP-Orai1
and RFP STIM1 overexpression plasmids,
and the next day cells were fixed under
resting conditions and observed under a
confocal microscope using the respective
colour channels and a 60× oil-objective.
Captures images were analysed with the
NIS-image software and Pearson’s coeffi­
cient was determined. Colocalization be­
tween cholesterol and Orai1 are indicated by
arrowheads (see light-blue/white spots);
meanwhile asterisks represents colocaliza­
tion between cholesterol and STIM1 (see
yellow/orange spots). Bars in the images
represent 50 μm.
fixed cells, both in resting condition and after TG treatment. The images
showed a pattern similar to that found in confocal images of living cells.
As observed in the Supplementary file 1, interaction between TopFluor-
cholesterol and Orai1 occurred mainly in the plasma membrane cell
plane and disappears in other cellular locations. Transfection with siT­
MEM97 inhibited STIM1/Orai1 colocalization upon TG stimulation in
parallel with a stronger interaction of Orai1 with cholesterol.
C. Cantonero et al. BBA - Molecular and Cell Biology of Lipids 1866 (2021) 158906

Fig. 3. TMEM97 controls the interaction of cholesterol to Orai1. MDA-MB-231 cells were transfected for 24 h with CFP-Orai1 and empty plasmid (Control, a) or
siTMEM97 (b) or overTMEM97 (c). At the day of the experiment, cells were further incubated for 30 min with 2.5 μM of TopFluor-cholesterol at 37 ◦ C inside the
incubator. Once incubation time was over, coverslip containing the cells were mounted in a perfusion chamber and maintained in a HBS-rich Ca2+ buffer (1 mM of
CaCl2). Perfusion chamber was placed under a confocal microscopy, and cell images in the two colour channels were taken while the cells were stimulated for 4 min
with TG (1 μM). Confocal images were used to ascertain colocalization Pearson’s coefficient between cholesterol and CFP-Orai1 and, the histogram in d, represents
the mean of the percentage ± S.E.M. of the changes in the colocalization respect control cells. Images are representative of 4 independent cells transfections where we
analysed around 1520 cells in each field. Bars in the images represent 50 μm.

3.6. Ca2+ entry through the Orai1(Y80E) was unaltered by the silencing
of TMEM97
Finally, to corroborate the involvement of TMEM97 in SOCE acti­
vation through cholesterol-Orai1 interaction, we took advantage of the
GECO-Orai1(Y80E) mutant. According to the literature mutation of the
Y80 abolishes cholesterol binding to Orai1 [35], while the fact that this
Orai1 mutant contain the GECO tag allows the detection of Ca2+ entry
through the Orai1 pore during the activation of SOCE. If the regulation
of SOCE by TMEM97 is mediated by a decrease of cholesterol binding to
Orai1, thus mitigating the inhibitory effect of cholesterol, under these
experimental conditions, changes in the TMEM97 expression should not
influence SOCE in cells expressing the mutant channel.
After transfection with GECO-Orai1(Y80E) + RFP-STIM1 to avoid
changes in the stoichiometry of the SOCE components, MDA-MB-231
cells were transfected either with siRNAA (control) or with siTMEM97
or overTMEM97 to reduce or enhance TMEM97 expression, respec­
tively. SOCE activation was studied using both fura-2 and the GECO
fluorescence of the mutant constructs. In the Panel A of Fig. 5, where
fura-2 fluorescence is presented, upon TG application we did not
observed differences in the TG-evoked Ca2+ release between the trans­
fected cells and non-transfected (mock) cells, as indicated by the small
sustained Ca2+ increase produced by TG. Interestingly, GECO fluores­
cence only changed significantly once Ca2+ entry was activated, which is
in agreement with the literature, and then it would only monitor the
Ca2+ entry through Orai1 channel pore (see Panel B of Fig. 5). Under
these experimental conditions when extracellular Ca2+ is extracellularly
applied and, subsequently, Ca2+ entry would begin, and increase in
SOCE was detected when compared between mock and controls cells,
but neither GECO fluorescence nor fura-2 fluorescence ratio showed
differences between control, silencing or overexpressing transfected
cells. The latter indicates that changes in TMEM97 only affects SOCE
when the native Y80 residue is intact and then, facilitates the
cholesterol-Orai1 interaction.
4. Discussion
Triple negative breast cancer cells, like MDA-MB-231 cells, express
elevated amount of low-density lipoprotein receptor (LDL-receptor) in
their plasma membrane, as well as an enhanced lipid metabolism
[24,36]. It was reported that the levels of LDLR mRNA expressed in
MDA-MB-231 were three- to five-fold higher than in MCF7, and simi­
larly, the elevated LDLR expression was associated with an enhanced
proliferation and migration rates in MDA-MB-231 cells [23,37]. As
result of this genetic background, MDA-MB-231 cells presented high
capability of binding, uptake, store and processing the exogenous LDL
and, subsequently, its associated cholesterol (LDL-cholesterol), which
has been reported to be the main form of circulating cholesterol and
triglycerids [37]. In order to maintain this exacerbated lipid metabolism
MDA-MB-231 cells overexpress proteins involved in the cholesterol
uptake and transport, such as TMEM97 [9,22,38]. Here, we have
corroborated these previous observations, since MDA-MB-231 cells

7
C. Cantonero et al. BBA - Molecular and Cell Biology of Lipids 1866 (2021) 158906

Fig. 4. siTMEM97 inhibits STIM1/Orai1 colocalization. MDA-MB-231 cells were transfected with RFP-STIM1, CFP-Orai1 and with either siRNAA (a; Control) or
additionally with siTMEM97 (b). Next day, cells were incubated with 2.5 μM of Top-Fluor-cholesterol for 30 min at 37 ◦ C. Confocal images for RFP-STIM1 (Ex. 561
nm, red), CFP-Orai1 (Ex. 405 nm, blue) and Top-Fluor-Cholesterol (Ex. 488 nm, green) were acquired at rest (top row images) and, consequently, in line-sequential
mode every 30 s during stimulation with TG (1 μM) for 4 min (bottom row images). Images in a & b represents the merged images between cholesterol/Orai1 (left
panels) and Orai1/STIM1 (right panels); meanwhile, images in d, are representative of the plasmids transfection in each experimental conditions. c) Histograms
represent the mean ± S.E.M of the percentage of cells where the stimulation with TG evoked a significant increase in the colocalization Pearson’s coefficient of Orai1/
cholesterol (above histogram) or Orai1/STIM1 (below histogram) as compared to control cells. e) Enlarged images of the cells presented in a & b are shown, and
positive cholesterol/Orai1 colocalization is highlighted with asterisks and Orai1/STIM1 colocalization is highlighted with arrowheads. Images are representative of 4
independent transfections where we analysed around 15–20 cells in each field. f) Histogram represents the mean ± S.E.M of the fold increase in the colocalization
Pearson’s coefficient evoked by TG stimulation respect the cells kept non-stimulated. ***: represents p < 0.001 respect non-genetically modified control cells.

exhibit enhanced TMEM97 expression respect to their non-tumoral
control cells, MCF10A, which as result, exhibited reduced capability to
uptake TopFluor-cholesterol. TMEM97 overexpression in MCF10A
increased the TopFluor-cholesterol uptake by these cells. In line with
this observation, MDA-MB-231 cell transfection with siTMEM97
reduced significantly the TopFluor-cholesterol uptake. Both results lead
us to conclude that TMEM97 is crucial for cholesterol uptake and
internalization by MDA-MB-231 cells.
In addition, MDA-MB-231 cells presented enhanced Ca2+ entry
through SOCE mechanism when compared to MCF10A that has been
previously evidenced by our research group and others [34,39]. Exac­
erbated Orai1 expression was observed in breast cancer cells [40] and,
subsequently, Orai1 would support the elevated SOCE, and subse­
quently, the enhanced proliferation rates in MDA-MB-231 cells [41,42].
In fact, the use of chemical antagonist or silencing of SOCE structural
components reduced proliferation of these cells [10,43]. Direct corre­
lation between Ca2+ entry through Orai1 and cancer cell proliferation
was linked to the promotion of NFAT1 translocation to the nucleus,
which has been involved in the mechanisms that promote a deregulated
cell proliferation [44,45]. Recently, we have demonstrated that addition
of NO1, an antagonist of the σ2R-TMEM97 activity, reduced SOCE [9],
and in line with this observation, here we demonstrate that the over­
expression of TMEM97 enhanced SOCE, while MDA-MB-231 cells
transfection with siTMEM97 reduced it. Additionally, it has been widely
accepted that SOCE structural components are strongly associated with
the cholesterol membrane patches, which were identified as lipid rafts
[46], and the structural disorganization of these lipid rafts by very
drastic approaches like the treatment with methyl-β-cyclodextrin,
resulted in a drastic reduction of SOCE [31,33,46]. Therefore, these
results suggests that SOCE requires the stability provided by the lipid
raft to accommodate the physical interaction between Orai1 and STIM1
[28,31,46]. However, Orai1 presents in its N-terminal domain a
cholesterol binding region (CB), which was recently described to
downregulate its permeability to Ca2+ [33]. Here, we have presented
evidences of TMEM97 colocalization with Orai1 in resting conditions,
and while a positive coimmunoprecitipation was also observed between
STIM1 and TMEM97, this interaction seems to be of less magnitude.
Silencing of TMEM97 increased the TopFluor-cholesterol stain in the
plasma membrane and near plasma membrane regions, and contrary,
overexpression of TMEM97 resulted in a poor stain of plasma membrane

8
C. Cantonero et al. BBA - Molecular and Cell Biology of Lipids 1866 (2021) 158906

Fig. 5. TG-evoked SOCE activation in Orai1(Y80E) mutant was not affected by TMEM97 expression. a & b) MDA-MB-231 cells were non-genetically modified
(Mock) or were transfected with GECO-Orai1(Y80E) + RFP-STIM1 alone (Control) or were further transfected with siTMEM97 or overTMEM97, as indicated. At the
following day, cells were loaded with fura-2 and single cell Ca2+ imaging experiments were performed using an epifluorescence microscope. Briefly, fura-2 loaded
cells were mounted in a perfusion chamber and stimulated for 4 min with 1 μM of TG in a free-Ca2+ HBS medium (75 μM was added as indicated by the arrowheads).
Following, 1 mM of CaCl2 was added to the extracellular medium to visualized the TG-evoked SOCE that was recorded for additional 2 min. Fluorescence of fura-2 (a)
and GECO-Orai1(Y80E) (b) were alternatively recorded in order to analyse the changes in the fluorescence ratio of fura-2 and the amount of Ca2+ passing through the
channel pore in the same cells and were represented in the histograms (c) and (d), respectively. Ca2+ traces of GECO and fura-2 fluorescence are representative of 4
independent transfections. Areas under the curves of the fluorescence of the GECO-Orai1 and fura-2 that results after the additions TG and CaCl2 to the cells were
determined and were used for comparison between different genetic cell transfections. *: represent p < 0.05 respect mock non-genetically modified cells.

with TopFluor-cholesterol. In line with this observation, colocalization
between CFP-Orai1 and TopFluor-cholesterol was enhanced in cells with
siTMEM97, while it was shown to be scarce in MDA-MB-231 cells
treated with overTMEM97, where colocalization between Orai1 and
cholesterol was limited to the interior of the cells. These results would
agree with those done by others using methyl-β-cyclodextrin that shown
a drastically disorganized the lipid-raft evoking the internalization of
Orai1 [32]. Subsequently, MDA-MB-231 cells transfected with siT­
MEM97 presented reduced STIM1/Orai1 interaction as compared to
MDA-MB-231 mock cells. The later does not agree with previous ob­
servations done in HEK293 cells, where no changes in the association
between STIM1 and Orai1 were observed upon administration of
cholesterol oxidase to the cells that oxides cholesterol to cholestenone
and, therefore, according to the authors reduces the cholesterol content
in the plasma membrane [33,47]. On the other hand, mutation of the
Orai1 ETON domain (Y80S or L74I) resulted in a reduction of the as­
sociation between cholesterol and the Orai1 channel, which evokes an
enhanced activation of SOCE in response to TG stimulation [33]. Simi­
larly, other research group used the Orai1Y80E and Orai1Y80D mutants
in HEK293 finding a similar increase in the channel permeability respect
to the values found in cells transfected with the Orai1 WT [48]. Author
concluded that the increase in Orai1 activity observed in the mutants
was due to the inhibition of the fast Ca2+-dependent inactivation, but
independent of calmodulin association to Orai1, as it has been previ­
ously proposed by others [49,50]. Here, we took advantage of the GECO-
Orai1Y80E mutant to demonstrate that the regulatory mechanism of
TMEM97 over SOCE relies on its capability to metabolize the cholesterol
from the membranes, and therefore releasing its inhibition on Orai1.
Therefore, MDA-MB-231 cells expressing GECO-Orai1Y80E and RFP-
STIM1 and further transfected either with siTMEM97 or overTMEM97
were used here to perform Ca2+ experiments using single-cell imaging
configuration. TG-evoked SOCE activation was doubled checked by
monitoring the changes in the GECO-emitted fluorescence and the
changes in the fura-2 fluorescence. Both Ca2+ dyes reported similar re­
sults, and we cannot observe changes in the activation of the TG-evoked
SOCE in MDA-MB-231 cells transfected with siTMEM97 or over­
TMEM97 respect to the cells transfected with GECO-Orai1(Y80E) +
RFP-STIM1 alone. These results allow us to conclude that in cells where
the CB of Orai1 is mutated, then impairing the bind of cholesterol to
Orai1, the changes in the TMEM97 expression are unable to modify the
activation of SOCE with TG; therefore, reinforcing the idea that
TMEM97 regulates SOCE by changing the cholesterol content in the cell
membrane.
Summarizing, here for the very first time we presented a regulatory
mechanism of TMEM97 on SOCE activation, based on its capability to
decrease the cholesterol content in the plasma membrane, which may
release the cholesterol inhibition of Orai1 due to its dissociation of the
CB region. Considering the big relevance of Ca2+ entry for promoting
cancer cells proliferation, we propose that TMEM97 could be targeted in
order to prevent the exacerbated Ca2+ entry and subsequently, MDA-
MB-231 cell proliferation.
Supplementary data to this article can be found online at https://doi.

9
C. Cantonero et al.
org/10.1016/j.bbalip.2021.158906.
CRediT authorship contribution statement
Carlos Cantonero performed most of experiments. Pedro Camello
performed the confocal experiments and images processing. Gines M
Salido and Juan A Rosado contributed with the discussion and corrected
the manuscript. Pedro C Redondo contributes with the conceptual
design of the experiments and wrote the manuscript.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
We thank Mercedes Gómez and Alvarado S for their technical
assistance.
Author constribution
CC performed most of experiments. CP performed the confocal ex­
periments and images processing. SGM and RJA contributed with the
discussion and corrected the manuscript. RPC contributes with the
conceptual design of the experiments and wrote the manuscript.
Fundings
The present study was supported by MICINN (BFU-2016-74932-C2-
1-P and PID2019-104084GB-C21) and Junta de Extremadura-FEDER
(IB16046, IB18025, IB18020 and GR18061). Cantonero C was awarded
with a by a predoctoral fellowship of Junta de Extremadura-FEDER
(PD16072).
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