Abstract
Background
One of major challenges in breast tumor therapy is the existence of
breast cancer stem cells (BCSCs). BCSCs are a small subpopulation of
tumor cells that exhibit characteristics of stem cells. BCSCs are
responsible for progression, recurrence, chemoresistance and metastasis
of breast cancer. Ca^2+ signalling plays an important role in diverse
processes in cancer development. However, the role of Ca^2+ signalling
in BCSCs is still poorly understood.
Methods
A highly effective 3D soft fibrin gel system was used to enrich
BCSC-like cells from ER+ breast cancer lines MCF7 and MDA-MB-415. We
then investigated the role of two Ca^2+-permeable ion channels Orai1
and Orai3 in the growth and stemness of BCSC-like cells in vitro, and
tumorigenicity in female NOD/SCID mice in vivo.
Results
Orai1 RNA silencing and pharmacological inhibition reduced the growth
of BCSC-like cells in tumor spheroids, decreased the expression levels
of BCSC markers, and reduced the growth of tumor xenografts in NOD/SCID
mice. Orai3 RNA silencing also had similar inhibitory effect on the
growth and stemness of BCSC-like cells in vitro, and tumor xenograft
growth in vivo. Mechanistically, Orai1 and SPCA2 mediate store-operated
Ca^2+ entry. Knockdown of Orai1 or SPCA2 inhibited glycolysis pathway,
whereas knockdown of Orai3 or STIM1 had no effect on glycolysis.
Conclusion
We found that Orai1 interacts with SPCA2 to mediate store-independent
Ca^2+ entry, subsequently promoting the growth and tumorigenicity of
BCSC-like cells via glycolysis pathway. In contrast, Orai3 and STIM1
mediate store-operated Ca^2+ entry, promoting the growth and
tumorigenicity of BCSC-like cells via a glycolysis-independent pathway.
Together, our study uncovered a well-orchestrated mechanism through
which two Ca^2+ entry pathways act through distinct signalling axes to
finely control the growth and tumorigenicity of BCSCs.
Supplementary Information
The online version contains supplementary material available at
10.1186/s13287-024-03875-1.
Keywords: Breast cancer stem cells, Orai1, Orai3, Glycolysis
Introduction
Breast cancer is the most frequently happened malignancy in women
[[42]1]. Although there has been great improvement in breast cancer
treatment, breast cancer mortality is still high partly due to the
existence of breast cancer stem cells (BCSCs) that are resistant to
current therapies. BCSCs are the highly tumorigenic cancer cells, and
they also possess self-renewal, metastatic and drug-resistant
properties [[43]2]. Therefore, advancing our understanding on BCSCs is
crucial for developing new breast cancer therapies.
Orai proteins, including Orai1 to Orai3, are Ca^2+-selective ion
channels mediating Ca^2+ entry [[44]3]. Orai1 proteins contain
functional domains in N- and C-termini that can interact with other
proteins such as stromal interaction molecule 1 (STIM1) and the
secretary pathway Ca^2+-ATPase 2 (SPCA2) [[45]4]. In most cell types,
Orai1 mediates store-operated Ca^2+ entry (SOCE), in which depletion of
intracellular stores is sensed by Ca^2+ sensor STIM1 located in the
endoplasmic reticulum to activate Orai1 in the plasma membrane [[46]5].
However, in estrogen receptor-positive (ER+) breast cancer cells, SOCE
is mediated by Oria3-STIM1 [[47]6], whereas Orai1 interacts with SPCA2
to form a signaling complex mediating store-independent Ca^2+ influx
(SICE) [[48]7]. Intriguingly, Orai1-SPCA2-mediated SICE in mammary
acinar cells is important for high-throughput Ca^2+ transport from
maternal blood to milk during lactation [[49]4, [50]7].
Orai1 and Orai3 also play important functional roles in breast cancer
[[51]3, [52]8]. The expression levels of Orai1 and/or Orai3 are often
upregulated in human breast cancer [[53]3, [54]9]. Mechanistically,
Orai1 and Orai3 promote breast cancer cell proliferation [[55]7,
[56]10], migration and invasion in vitro [[57]6, [58]11], and tumor
growth and metastasis in vivo [[59]3, [60]7–[61]11]. However, none of
these previous studies have investigated Orai1 and Orai3 in the context
of BCSCs. The role of Orai1 and Orai3 in BCSCs is poorly understood.
Metabolic phenotype is a crucial determinant of BCSC properties,
influencing tumorigenesis, metastasis, drug resistance and tumor
relapse [[62]12]. Therefore, targeting BCSC metabolism has been a
recent hot strategy for breast cancer therapy [[63]12, [64]13]. One
unique characteristic of BCSCs is their metabolic flexibility. BCSCs
can switch between oxidative phosphorylation and glycolysis dependent
on cancer microenvironment [[65]12]. However, in general, BCSCs
strongly favor the glycolysis pathway [[66]12, [67]14]. Ca^2+ signaling
is a well-known regulator of cancer cell metabolism [[68]15]. However,
up to the present there is still lack of information about the role of
Ca^2+ signaling in the regulation of glycolysis in cancer stem cells
(CSCs).
In the present study, we employed a highly effective 3D soft fibrin gel
method [[69]16, [70]17] to enrich BCSC-like cells from ER+ MCF7 cell
line and MDA-MB-415 cell line. When cultured within these gels, single
cancer cells from human cancer cell lines can grow within a few days
into individual tumor spheroids that resembled stem cell embryonic
bodies [[71]16, [72]17]. This method offers great advantage over
conventional stem cell marker-based CSC enrichment methods, because CSC
markers are often unreliable [[73]16, [74]17]. Indeed, the CSCs
enriched by 3D soft fibrin gel demonstrate remarkable tumorigenicity
[[75]16, [76]17]. As few as 10 such selected murine B16-F1 melanoma
cells are already capable of forming a tumor and metastasizing to lung
[[77]16, [78]17]. Herein, we utilized the BCSC-like cells enriched by
this method to investigate the role of Orai1 and Orai3 in BCSCs. We
hypothesized that Orai1-SPCA2 mediates SICE to promote the growth and
tumorigenicity of BCSC-like cells via glycolysis pathway, whereas
Orai3-STIM1 mediates SOCE to promote the growth and tumorigenicity of
BCSC-like cells via a glycolysis-independent pathway. A variety of
methodologies, including tumor spheroid growth in 3D soft fibrin gel,
cytosolic Ca^2+ measurement, tumor xenograft growth in NOD/SCID mice,
RT-qPCR and immunoblots, were used to test these hypotheses.
Materials and methods
Reagents
Paclitaxel, fluorouracil (5-fu), collagenase, dispase II, β-Estradiol
and cyclopiazonic acid (CPA) were from Sigma-Aldrich. 2-deoxy-d-glucose
(2-DG) was from Alfa Aesar. Puromycin and blasticidin were from
InvivoGen. Polybrene was from Santa Cruz. Fluo-4/AM were from
Invitrogen. AnCoA4 was from Merck Millipore. RO 2959 were from AOBIOUS.
Salmon fibrinogen and thrombin were from Searun Holdings.
Animals and cell lines
The work has been reported in line with the ARRIVE guidelines 2.0. All
experimental mice were kept in SPF-grade animal facilities in the
Laboratory Animal Service Center of The Chinese University of Hong
Kong. Female NOD/SCID mice were housed in SPF-level mouse facilities
and raised until 4–5 weeks of age for experimental use. The experiment
on mice was approved by the Animal Experimental Ethics Committee of The
Chinese University of Hong Kong.
Human breast cancer cell lines MCF7, MDA-MB-415 and human embryonic
kidney (HEK) 293 FT cells were purchased from ATCC and were cultured in
DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and
1% penicillin–streptomycin (PS; Gibco).
Cell culture in 3D soft fibrin gel
3D soft fibrin gels were prepared as described previously [[79]16].
Briefly, salmon fibrinogen (Searun Holdings) was diluted to 2 mg/ml
using T7 buffer (50 mM Tris, 150 mM NaCl, pH7.4). Then, fibrinogen was
mixed with the cell suspension at a ratio of 1:1, resulting in 1 mg/ml
(90 Pa) fibrinogen. Next, 50 µl fibrinogen/cell mixture was seeded in
one well of 96-well plate and mixed with pre-added 1 µl thrombin (100
U/ml, Searun Holdings). The plate was incubated in a humidified
incubator (37 °C, 95% O[2] and 5% CO[2]) for at least 15 min to
solidify before adding 150 µl DMEM (with FBS and PS). Culture medium
was changed every two days. Starting from day2, at least 30 colonies
and two wells were recorded per condition every two days by Olympus
IX83 Inverted Microscope.
siRNA transfection, RNA isolation and RT-qPCR
siRNA transfection was performed using lipofectamine RNAiMAX
transfection reagent (Thermofisher) following the manufacturer’s
protocol. To improve the transfection efficacy, we performed
transfection when cell confluency reached to 60%-80%. Meanwhile, PS was
removed from the culture medium during transfection and re-introduced
72 h post-transfection.
Total RNA was isolated using RNeasy Micro Kit (Qiagen) following the
manufacturer’s protocol. The RNA concentration was measured using a
NanoDrop 2000 Spectrophotometer. 1 µg RNA was used for cDNA synthesis
using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems)
at the condition of 25 ℃ for 10 min, 37 ℃ for 120 min and 85 ℃ for
5 min in a PCR machine.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
was conducted using a quantitative SYBR Green Select Master Mix
(Applied Biosystems) in 7900HT Fast Real-Time PCR System (Applied
Biosystems). The RT-qPCR reactions were run under the following
conditions: initial denaturation at 95 ℃ for 10 min, followed by 40
cycles of denaturation at 95 ℃ for 15 s, annealing and extension at
60 ℃ for 1 min. A melting curve analysis was subsequently performed,
consisting of 95 ℃ for 15 s, 60 ℃ for 15 s and 95 ℃ for 15 s. Triple
wells were included for each sample. The mRNA levels of the targeted
genes were normalized to β-actin and expressed with the
[MATH: 2-ΔΔCT :MATH]
method [[80]18, [81]19]. All primer sequences are listed in Table S1.
Plasmids transfection, shRNA lenti-viral vector packaging and infection
Plasmid transfection was performed using lipofectamine 2000
(Invitrogen) according to the manufacturer’s protocol. shRNA constructs
were co-transfected with psPAX2 and pMD2.G at a ratio of 4:3:1 to
HEK293 FT cells to generate lentivirus. After 48 h and 72 h, the
culture medium was harvested, centrifuged and added to MCF7 cells in
the presence of 10 μg/ml polybrene (Santa Cruz). 2 μg/ml puromycin
(Orai1 and STIM1) or 16 μg/ml blasticdin (Orai3) were used to select
stably knockdown cell lines [[82]20]. Targeted sequences are listed in
Table S2.
Glucose uptake assay, pyruvate and lactate level measurement
Cells were starved for 1 h in glucose-free medium, followed by
incubating with 300 µM 2-(N-nitrobenz-2-oxa-1,3-diazol-4-yl)amino
(2-NBDG; ThermoFisher) for 30 min. Then, fibrin gel was dissolved by
collagenase (Sigma) and dispase II (Sigma) [[83]21]. Colonies were
collected and pipetted into single cells. Finally, 2-NBDG uptake level
was measured by flow cytometry.
Cell culture medium was collected. Pyruvate production and lactic acid
level were measured using pyruvate assay kit (Nanjing Jiancheng
Bioengineering Institute) and lactic assay kit (Nanjing Jiancheng
Bioengineering Institute) following the manufacturer’s protocol. All
values were normalized to protein concentration.
Western blotting
Detailed methods were described elsewhere [[84]22, [85]23]. Briefly,
cells were lysed by RIPA lysis buffer (Beyotime) containing 1 mM PMSF
(Beyotime) on ice for 30 min. The protein concentration was measured
using an Enhanced BCA Protein Assay Kit (Beyotime). The protein
supernatant was directly boiled in SDS loading buffer for 5 min at
95 °C.
Equal amounts (25–50 μg/lane) of protein were separated by 10% SDS-PAGE
gel (Bio-Rad) at 80 V for 30 min and then at 120 V for 90–120 min. The
protein on the gel was transferred to polyvinylidene fluoride membranes
(PVDF; Bio-Rad) at 300 mA for 90 min. The membranes were then blocked
with 4% BSA, incubated with diluted primary antibodies to Orai1
(1:1000, Proteintech), GAPDH (1:5000, Proteintech), β-actin (1:5000,
Proteintech), STIM1 (1:1000, Alomone Labs) and SPCA2 (1:200, Santa
Cruz) overnight at 4 °C. Proteins were visualized using an
HPR-conjugated secondary antibody (GE Healthcare) and imaged using ECL
Western Blotting Detection Reagents (Amersham). Quantification was
performed using ImageJ.
Cytosolic Ca^2+ measurement
Cytosolic Ca^2+ measurement was performed as described previously
[[86]24]. Briefly, cells were loaded with fluo-4/AM and incubated for
30 min. For SICE (or basal Ca^2+ influx) measurement, the cells loaded
with Fluo-4/AM were buffered in a calcium-free physiological saline
solution (calcium-free PSS), which contained 140 mM NaCl, 5 mM KCl,
2 mM MgCl[2], 5 mM HEPES, 10 mM glucose, PH 7.4, followed by adding
2 mM Ca^2+ to induced Ca^2+ entry. For SOCE measurement, cells loaded
with Fluo-4/AM were buffered in calcium-free PSS. Then, 10 µM CPA was
added to deplete Ca^2+ stores, followed by adding back of 2 mM Ca^2+ to
trigger SOCE. The fluo-4 fluorescence signals were recorded using
FV1000 laser scanning confocal imaging system at excitation/emission of
488/515 nm at room temperature, and the data were analyzed using
META-FLOUR software. Each experiment had at least 20 cells. Real-time
changes in cytosolic Ca^2+ were displayed as a ratio of fluorescence
relative to the average intensity before stimulation (F[t]/F[0]).
Xenograft experiment
Single cells were isolated from tumor spheroids in soft fibrin gel by
collagenase (Sigma) and dispase II (Sigma). The cells were mixed with
Matrigel (BD Biosciences) at 1:1 ratio. Xenograft procedures were
similar to previous publications with modification [[87]25, [88]26].
Briefly, mice were randomly assigned to either control group or
experimental groups. Ketamine (80 mg/kg) and xylazine (5 mg/kg) were
given to mice for anesthesia before tumor cell inoculation. Each mouse
was given in one flank a subcutaneous injection of 100 µl cell/Matrigel
mixture containing certain numbers of BCSC-like cells (10^2, 10^3 or
10^4) with or without Orai1 or Orai3 knockdown. Following inoculation,
100 µl of 100 µM estradiol solution (Sigma) was intraperitoneally
injected to each mouse every two days and tumor growth was measured at
the same days. Control and treatment mice were placed in nearby cages.
Any expected and unexpected adverse evented were monitored daily by a
veterinarian. Tumor volume was calculated based on the formula
[MATH:
V=len<
/mi>gth×(width2)2.
:MATH]
Mice were sacrificed until the diameter (width/length) of tumors
reached near 2 cm and endpoint tumor weight values were measured. The
tumor size was limited to no more than 2 cm in diameter to reduce
animal suffering. At the experimental endpoint, mice were euthanized by
CO[2] to minimize suffering. The number of mice used in each
experimental group was 5–6, as specified in relevant Figure legends.
The data from all mice were included.
Bioinformatics
RNA-Seq data for bioinformatics analysis were sourced from The Cancer
Genome Atlas (TCGA) breast cancer database and The Genotype-Tissue
Expression (GTEx) database with 420 Luminal A (LumA) and 572
Normal-Like (N-Like). Normal-Like samples from TCGA and GTEx database
were unified using an established database incorporation method
[[89]27].
Patient samples and immunostaining
Human tissue experiments were approved by Ethics Committee of
Affiliated Hospital of Shandong First Medical University (No. 2021001).
Luminal A type breast cancer specimens along with the corresponding
clinical pathologic data from 30 patients were collected from
Affiliated Hospital of Shandong First Medical University (Shandong
Academy of Medical Sciences) with informed consent of patients.
Pathological diagnosis was performed by haematoxylin and eosin (H&E)
staining and immunohistochemistry index based on PR, ER, HER2 and
Ki-67. The histological grades were evaluated by Elston-Ellis
modification of Scarff-Bloom-Richardson grading system which is based
on the degree of duct formation, nuclear size, nuclear pleomorphism and
mitotic rate [[90]28].
For Orai1 and Orai3 immunostaining of breast samples, tissues were
fixed using 10% formalin, paraffin embedded and sliced into
cross-sections of 5 µm. After rehydrating in a graded alcohol series to
80% ethanol and antigen retrieval (HC ST02 Antigen Retriever—PT Module,
Thermo), MXB UltraSensitive TM SP (Mice/Rabbits), IHC Kit were used to
inhibit endogenous peroxidase activity and blocking. Sections were
incubated at 4 °C temperature overnight with primary polyclonal
antibodies to Orai1 (1:150, Proteintech) or Orai3 (1:150, Proteintech).
MXB IHC Kit was consequently applied for the following
immunoreactivity. The sections were then developed using DAB and the
pictures were captured by microscopy (HC U02—Nikon Ni-U Eclipse Upright
Microscope).
Statistics
All data are presented as means ± SEM or mean ± SD of at least three
independent experiments. Statistical analyses were performed using
GraphPad Prism 8.0 software. Comparisons between two groups were
measured by Student’s unpaired two-tailed t-test. Differences among
three or more groups were examined by one-way analysis of variance
(ANOVA) test followed by Tukey’s multiple comparisons test. Comparisons
among multiple growth curves of tumors or tumor spheroids were
performed by two-way ANOVA followed by Bonferroni post-test. P
value < 0.05 was considered as significantly different. Conclusions
would not be drawn if relevant comparisons could not reach statistical
significance.
Results
Orai1 promotes the growth of tumor spheroids and increases cancer cell
stemness in vitro
3D soft fibrin gel was used to enrich BCSC-like cells from ER + breast
cancer cells MCF7 and MDA-MB-415 using the method described elsewhere
[[91]16]. MCF7 and MDA-MB-415 cells were seeded in 90 Pa soft fibrin
gel. Within a few days, they grew to form tumor spheroids [[92]16].
Western blot showed that the 3D gel-enriched BCSC-like cells in tumor
spheroids had a substantially higher expression of Orai1 than that of
2D cultured ordinary cancer cells (Fig. [93]1A, S1A). Three
lentiviral-based Orai1-shRNAs were constructed, all of which could
effectively knockdown the expression of Orai1 in MCF7 cells by western
blots (Fig. [94]1B) and RT-qPCRs (Fig. S2A). RT-qPCRs further confirmed
that overexpression of exogenous Orai1 could reverse the knockdown
effect of Orai1-shRNAs, significantly increasing Orai1 expression (Fig.
S2B). Similar results were obtained using Orai1-siRNA in MDA-MB-415
cells (S1B, S2C-D).
Fig. 1.
[95]Fig. 1
[96]Open in a new tab
Orai1 promotes the growth of tumor spheroids and increases stemness of
MCF7 BCSC-like cells. A The protein expression of Orai1 in MCF7 cells
grown in 2D culture or in 3D 90 Pa soft fibrin gel with representative
western blot images (left) and summary data normalized to β-actin
(right) (n = 4). B The protein expression of Orai1 in MCF7 cells
treated with lenti-scrambled-shRNA or lenti-Orai1-shRNAs. Shown are
representative western blot images (left) and summary data normalized
to β-actin (right) (n = 4). C and D The growth of tumor spheroids
(colonies) formed by MCF7 cells in 3D 90 Pa soft fibrin gel with or
without Orai1 knockdown, shown photographically (C) and graphically
(D). Bar, 50 µm (n = 4–10). E Orai1 overexpressing plasmid was
re-introduced to the Orai1 knockdown MCF7 cells. Shown was the growth
of MCF7 tumor spheroids with lenti-scrambled-shRNA or lenti-Orai1-shRNA
or lenti-Orai1-shRNA + Orai1 plasmid (n = 4). F MCF7 cells were
pre-treated with Orai1 inhibitor (AnCoA4, 50 µM) for two days before
seeded in 90 Pa fibrin gel for spheroid growth. AnCoA4 was maintained
in the culture medium. DMSO was used as control. The spheroid size was
shown graphically (n = 4). G RT-qPCR analysis of cancer stem cell
markers in MCF7 cells, showing the effect of 3D culture,
lenti-Orai1-shRNA, and lenti-Orai1-shRNA plus Orai1 plasmid (n = 4–8).
H and I Summary of flow cytometric results showing the percentage of
CD133-positive (H, n = 5–12) and Nanog-positive (I, n = 5–11) cells.
The cells were treated with lenti-scrambled-shRNA or lenti-Orai1-shRNA
or lenti-Orai1-shRNA plus Orai1 plasmid. Mean ± SEM. ns, not
significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001, by Student t
test in A, or one-way ANOVA in B and G–I or two-way ANOVA in D–F.
Original western blot images are provided as Additional File #2.
A key feature of BCSCs is self-renewal capacity, which is the driving
force of tumorigenic initiation [[97]29]. Growth kinetics of tumor
spheroids is an indicator of BCSC renewal capacity. Interestingly,
Orai1 RNA silencing reduced the growth of BCSC-like cells in tumor
spheroids (Fig. [98]1C–D, S1C–D), the effect of which could be rescued
by overexpression of exogenous Orai1 (Fig. [99]1E, S1D). An Orai1
inhibitor AnCoA4 at 50 µM [[100]30] also inhibited the growth of
BCSC-like cells in tumor spheroids (Fig. [101]1F, S1E). However, Orai1
RNA silencing had no effect on the growth of 2D cultured cancer cells
(Figs. S3A, S3C), suggesting that Orai1 knockdown preferentially
inhibited the growth of BCSC-like cells in tumor spheroids but not that
of ordinary breast cancer cells (non-CSCs) grown in conventional 2D
culture.
Importantly, the cells in tumor spheroids were CSC-like, displaying
higher expression levels of multiple CSC markers (Fig. [102]1G, S1F)
and showing higher chemoresistance than 2D cultured cells (Fig. S2E,
F). Based on flow cytometer analysis of BCSC markers (CD133 and Nanog
for MCF7, CD133 and CD44 for MDA-MB-415), 3D tumor spheroids contained
about 34–70% BCSCs, whereas 2D cultured cells only contained 3–5% BCSCs
(Fig. [103]1H–I, S1G–H). Importantly, RT-qPCR showed that Orai1 RNA
silencing reduced the expression levels of multiple BCSC markers
(CD133, Nanog, Sox2, and Oct3/4) (Fig. [104]1G, S1F), whereas
overexpression of exogenous Orai1 reversed the effect of Orai1 RNA
silencing and elevated the expression of several BCSC markers
(Fig. [105]1G, S1F). Flow cytometry analysis further confirmed that
Orai1 RNA silencing reduced the expression of CD133 and Nanog/CD44, the
effect of which was reversed by overexpression of exogenous Orai1
(Fig. [106]1H–I, S1G–H).
Orai1 promotes tumorigenicity in vivo
A gold standard method of studying CSCs is to determine their
capability of tumor formation in NOD/SCID mice. We isolated BCSC-like
cells from 3D MCF7 tumor spheroids. Subcutaneously injection of only
100 such BCSC-like cells to NOD/SCID mice could successfully induce
tumor formation in all mice (5 out of 5 mice) (Fig. [107]2A),
indicating remarkable tumorigenic capability of these BCSC-like cells.
Note that MCF7 cells are low aggressive cancer cells. A few millions of
MCF7 cells are often needed to be injected for tumor formation in
NOD/SCID mice [[108]31]. Therefore, our 3D soft fibrin gel platform is
highly robust in enriching tumorigenic cells.
Fig. 2.
[109]Fig. 2
[110]Open in a new tab
Orai1 promotes tumorigenicity of MCF7 BCSC-like cells in NOD/SCID mice.
A Tumorigenic capability of MCF7 BCSC-like cells derived from 3D fibrin
gel tumor spheroids in NOD/SCID mice (n = 5). B and C The mice were
injected with MCF7 BCSC-like cells carrying lenti-scrambled shRNA or
lenti-Orai1-shRNA (n = 6). Shown are images of endpoint primary tumor
size (B) and summary of endpoint tumor weight (C) in NOD/SCID mice.
Bar, 1 cm. D Tumor growth curve in NOD/SCID mice injected with 1000
BCSC-like cells carrying lenti-scrambled shRNA or lenti-Orai1-shRNA
over 28 days post injection (n = 5). E Tumor incidence in NOD/SCID mice
injected with 100 MCF7 BCSC-like cells carrying lenti-scrambled shRNA
or lenti-Orai1-shRNA (n = 5). F Immunohistochemical analysis of
expression of Orai1, CD133, Nanog and Ki67 in primary tumors with
lenti-scrambled shRNA or lenti-Orai1-shRNA (n = 3). A total of 52 mice
were used for expriments in this Figure. Bar, 50 µm. Mean ± SD. *,
P < 0.05; **, P < 0.01, by Student t test in B, or two-way ANOVA in D
Importantly, Orai1 RNA silencing with lenti-Orai1-shRNA reduced tumor
xenograft growth in NOD/SCID mice, with smaller endpoint tumor size
(Fig. [111]2B) and less endpoint tumor weight on day 30 after injection
(Fig. [112]2C), and slower tumor growth rate (Fig. [113]2D). Orai1 RNA
silencing also prolonged tumor free duration (Fig. [114]2E).
Immunostaining on tumor sections confirmed that lenti-Orai1-shRNA could
effectively knockdown the expression of Orai1 and reduce the expression
of CD133, Nanog and Ki-67 in xenograft tumors (Fig. [115]2F).
Orai1 stimulates glycolysis pathway
We examined the metabolic profile of 3D gel-enriched BCSC-like cells.
RNA seq experiments followed by KEGG pathway enrichment analysis
demonstrated that glycolysis was the most significantly upregulated
pathway in 3D gel-enriched BCSC-like cells compared with 2D cultured
cells (Fig. [116]3A). RT-qPCR analysis confirmed that 3D culture indeed
caused upregulation of multiple glycolytic genes (Fig. [117]3B, S4A).
Furthermore, a glycolysis inhibitor 2-DG at 5 mM dramatically
suppressed the growth of tumor spheroids (Fig. [118]3C, S4B),
suggesting that glycolysis is a key player in BCSC growth.
Fig. 3.
[119]Fig. 3
[120]Open in a new tab
Orai1 stimulates glycolysis pathway in MCF7 BCSC-like cells. A and B
Shown were the KEGG pathway enrichment analysis of upregulated genes
(A) in 3D 90 Pa soft fibrin gel-enriched MCF7 BCSC-like cells compared
to 2D cultured MCF7 cells, and confirmation of glycolysis pathway by
RT-qPCR (B, n = 3). C The growth of MCF7 tumor spheroids (colonies) in
3D soft fibrin gel treated with or without glycolysis inhibitor, 2-DG,
over 8 days (n = 3). D Relative fold change of glycolytic genes in 3D
gel-enriched MCF7 BCSC-like cells treated with or without 50 µM AnCoA4,
as detected by RT-qPCR. The data were normalized to β-actin (n = 3). E
Relative fold change of glycolytic genes in 3D gel-enriched MCF7
BCSC-like cells and 2D cultured MCF7 cells with or without Orai1
knockdown as detected by RT-qPCR (n = 3–5). F Glucose uptake shown as
mean fluorescent intensity (MFI) of 2-NBDG in 3D gel-enriched MCF7
BCSC-like cells with or without Orai1 knockdown (n = 5). G and H
Relative fold change of pyruvate production (G) and lactate levels (H)
in 3D gel-enriched MCF7 BCSC-like cells with or without Orai1 knockdown
(n = 5), Mean ± SEM. ns, not significant; *, P < 0.05; **, P < 0.01;
***, P < 0.001, by Student t test in B, D, F, G and H, or two-way ANOVA
in C and E
Interestingly, Orai1 inhibitor AnCoA4 at 50 µM or Orai1 RNA silencing
decreased the expression of multiple glycolytic genes in 3D
gel-enriched BCSC-like cells (Fig. [121]3D–E, S4C–D). Orai1 RNA
silencing also reduced glucose uptake, pyruvate level and lactate
production in 3D gel-enriched BCSC-like cells (Fig. [122]3F–H).
However, Orai1 RNA silencing did not affect the expression of
glycolytic genes in 2D cultured cells (Fig. [123]3E, S4D).
Together, these results identified a novel function of Orai1 in
regulating glycolysis pathway in BCSCs.
Orai1 mediates SICE whereas Orai3 mediates SOCE
It was previously reported that in ER + breast cancer cells,
Orai1-SPCA2 mediates SICE whereas Orai3-STIM1 mediates SOCE [[124]6,
[125]7]. Indeed, we found that in MCF7 cells and MDA-MB-415 cells,
small molecule inhibitors of Orai1 AnCoA4 and RO2959 (Figs. S5A–B,
S6A–B), and Orai1 RNA silencing (Figs. S5C–D, S6A–B) all reduced the
basal Ca^2+ influx (or SICE) initiated by Ca^2+ addback to the bath. In
contrast, Orai3 RNA silencing had no effect on SICE (Figs. S5D, S6A–B).
We also studied SOCE and found that Orai1 RNA silencing did not alter
the SOCE (Figs. S5E–F, S6C–D), whereas RNA silencing of Orai3 or STIM1
substantially reduced the SOCE (Figs. S5G–I, S6C, E, F). These data
support the notion that Orai1 mediates SICE while Orai3 mediates SOCE
in ER + breast cancer cells as reported by others [[126]6, [127]7].
Orai1 interacts with SPCA2 to mediate SICE and promote glycolysis
It was reported that Orai1 interacts with SPCA2 to mediate SICE
[[128]7, [129]32]. Two lentiviral-based SPCA2-specific shRNAs were
constructed, each of which could effectively knockdown the expression
of SPCA2 in western blots in MCF7 cells (Fig. [130]4A). As expected,
knockdown of SPCA2 reduced the SICE (Fig. S7A–B,D). Similar results
were obtained using SPCA2-siRNA in MDA-MB-415 cells (S6A-B). To
demonstrate the functional role of Orai1-SPCA2 physical interaction, we
employed an N-terminal fragment of SPCA2 (SPCA2-N), which was
previously reported to interfere the interaction of SPCA2 with Orai1,
consequently exerting dominant negative effect on Orai1-SPCA2-mediated
SICE [[131]7, [132]32]. Indeed, we found that SPCA2-N could reduce the
SICE (Figs. S7C–D, S6A–B). Furthermore, SPCA2-N and/or SPCA2-shRNA
reduced the growth of tumor spheroids (Fig. [133]4B, S8A) and
downregulated the expression of glycolytic genes except ENO2 and ALDOA
in MCF7 BCSC-like cells (Fig. [134]4C–D, S8B).
Fig. 4.
[135]Fig. 4
[136]Open in a new tab
SPCA2 interacts with Orai1 to promote glycolysis in MCF7 BCSC-like
cells. A Western blot analysis showing the expression of SPCA2 in MCF7
cells transfected with lenti-scrambled-shRNA or lenti-SPCA2-shRNAs
(n = 3). B The growth of tumor spheroids (colonies) formed by MCF7
cells in 3D 90 Pa soft fibrin gel with or without SPCA2 knockdown or
addition of SPCA2 N-terminal fragment (n = 4–6). C–D Relative fold
change of glycolytic genes in MCF7 cells with or without SPCA2
knockdown (C) or with or without addition of SPCA2 N-terminal fragment
(D) as detected by RT-qPCR. The results from both 2D cultured and 3D
cultured MCF7 cells were shown (n = 3–5). Mean ± SEM. ns, not
significant; *, P < 0.05, **, P < 0.01, ***, P < 0.001, by one-way
ANOVA in B, or two-way ANOVA in C–D. Original western blot images are
provided as Additional File #2
Orai3 and STIM1 promote the growth of tumor spheroids and increases cancer
cell stemness in vitro, and tumorigenicity in vivo
We next explored the role of Orai3 and STIM1 in the growth of tumor
spheroids. Western blot analysis showed that the 3D gel-enriched
BCSC-like cells had a substantially higher expression of Orai3 than
that in 2D cultured cells (Fig. [137]5A, Fig. S9A). Lentiviral-based
Orai3-shRNAs and STIM1-shRNAs were constructed, all of which could
knockdown the expression of their respective targets in MCF7 cells
(Fig. [138]5B–C). Orai3-siRNA also had similar knockdown effect in
MDA-MB-415 cells (Fig. S9B). Importantly, RNA silencing of Orai3 and
STIM1 each reduced the growth of tumor spheroids (Fig. [139]5D–E, Fig.
S9C). Furthermore, RT-qPCR analysis showed that Orai3 RNA silencing
reduced the expression levels of several BCSC markers in BCSC-like
cells (Fig. [140]5F, Fig. S9D). Flow cytometry analysis further
validated that Orai3 RNA silencing reduced the expression of CD133 and
Nanog/CD44 (Fig. [141]5G–H, Fig. S9E). These data strongly suggest that
Orai3 promotes stemness of BCSC-like cells. Interestingly, knockdown of
Orai3 had minimal effect on the growth of 2D cultured cells (Fig. S3B,
D), suggesting that Orai3 knockdown preferentially inhibited the growth
of BCSC-like cells in tumor spheroids but not that of ordinary breast
cancer cells (non-CSCs) grown in conventional 2D culture.
Fig. 5.
[142]Fig. 5
[143]Open in a new tab
Orai3 promotes the growth of tumor spheroids in vitro and
tumorigenicity of MCF7 BCSC-like cells in vivo. A The protein
expression of Orai3 in MCF7 cells grown in 2D culture or in 3D 90 Pa
soft fibrin gel with representative western blot images (up) and
summary data normalized to β-actin (bottom) (n = 3). B and C
Representative western blot images and summary data showing the
expression of Orai3 (B) and STIM1 (C) in MCF7 cells transfected with
lenti-scrambled-shRNA or lenti-Orai3-shRNA or lenti-STIM1-shRNA
(n = 3). D and E The growth of tumor spheroids formed by MCF7 cells in
3D 90 Pa fibrin gel with or without Orai3 knockdown (D) or STIM1
knockdown (E) (n = 4). F RT-qPCR analysis of cancer stemness markers in
MCF7 cells, showing the effect of 3D culture and lenti-Orai3-shRNA
(n = 4). G and H Summary of flow cytometric results showing the effect
of 3D culture and lenti-Orai3-shRNA on the percentage of CD133-positive
(G, n = 5) and Nanog-positive (H, n = 5) cells. I–L Tumorigenic
capability of BCSC-like cells in NOD/SCID mice. The mice were injected
with BCSC-like cells carrying lenti-scrambled-shRNA or
lenti-Orai3-shRNA (n = 5). Shown are images of endpoint primary tumor
size (I) and summary of endpoint tumor weight (J) in NOD/SCID mice.
Bar, 1 cm. K Tumor growth curve in NOD/SCID mice injected with 10^3
BCSC-like cells carrying lenti-scrambled-shRNA or lenti-Orai3-shRNA
over 40 days post injection (n = 5). L Tumor incidence in NOD/SCID mice
injected with 10^3 MCF7 BCSC-like cells carrying lenti-scrambled-shRNA
or lenti-Orai3-shRNA (n = 5). A total of 20 mice were used for
expriments in this Figure. Mean ± SEM. *, P < 0.05; **, P < 0.01, ***,
P < 0.001, ****, P < 0.0001, by Student t test in A and J, one way
ANOVA in B, C, F–H, or two-way ANOVA in D, E and K. Original western
blot images are provided as Additional File #2
Importantly, lenti-Orai3-shRNA nearly abolished tumor xenograft growth
in NOD/SCID mice. In control, all 5 mice grew tumors 40 days after
injection with 1000 of BCSC-like cells derived from 3D MCF7 tumor
spheroids. In contrast, with lenti-Orai3-shRNA, only 1 out of 5 mice
grew a small-sized tumor ([144]5I) with summarized data in
Fig. [145]5J–L. These results support the importance of Orai3 in
stemness, self-renewal and tumorigenicity of BCSC-like cells.
Knockdown of Orai3 and STIM1 does not affect glycolysis pathway
RT-qPCR was conducted to determine the effect of Orai3 and STIM1
knockdown on the expression of glycolytic genes in BCSC-like cells. The
results showed that RNA silencing of Orai3 (Fig. [146]6A, Fig S9F) and
STIM1 (Fig. [147]6B) had no effect on the expression of multiple
glycolytic genes. These data suggest that Orai3 and STIM1 promotes the
self-renewal of BCSC-like cells through a mechanism independent of
glycolysis pathway.
Fig. 6.
[148]Fig. 6
[149]Open in a new tab
Orai3 and STIM1 do not affect glycolysis in MCF7 BCSC-like cells. Shown
are relative fold change of glycolytic genes in MCF7 cells with or
without Orai3 knockdown (A) or STIM1 knockdown (B). The results from
both 2D cultured and 3D cultured MCF7 cells were shown. Mean ± SEM. ns,
not significant; *, P < 0.05, **, P < 0.01, ***, P < 0.001, by two-way
ANOVA
Expression levels of Orai1 and Orai3 are elevated in the tumor tissues of
luminal A type breast cancer
To explore the clinical relevance of Orai1 and Orai3 in human luminal A
type breast cancer, meta-analysis was performed in RNAseq datasets in
TCGA breast cancer database plus GTEx database. The results showed that
the expression levels of both Orai1 and Orai3 are significantly
upregulated in the breast tissues of luminal A breast cancer compared
with normal tissues (Fig. [150]7A). We further analysed the expression
levels of Orai1 and Orai3 proteins in tumor sections of 30 luminal A
type breast cancer patients. The results revealed an increased
expression levels of Orai1 and Orai3 in human breast cancer tissues
compared with normal breast tissues in a tumor histological
grade-dependent manner (Fig. [151]7B and C).
Fig. 7.
[152]Fig. 7
[153]Open in a new tab
Expression of Orai1 and Orai3 is elevated in the tumor samples of
luminal A breast cancer. A Expression analysis of Orai1 and Orai3 in
luminal A breast tumors and normal breast tissues from The Cancer
Genome Atlas (TCGA) cohort plus GTEx database. B Representative
immunohistochemical staining images comparing the expression of Orai1
and Orai3 between normal tissue and patients'samples with different
histological grades of luminal A breast cancer. Bar, 50 µm. C
Quantitative analysis of Orai1 and Orai3 expression levels as in B,
with each dot representing the sample from one patient. Mean ± SD.
****, P < 0.0001, by Student t test in A and one way ANOVA in C
Discussion
One of major challenges in breast tumor therapy is the existence of
BCSCs, which are resistant to current therapies. This is partly due to
our insufficient knowledge about the physiology of BCSCs. Up to now,
there is only very limited research on the role of Ca^2+ signaling in
BCSCs. In one study, Lu et al. [[154]33], demonstrated an important
role of RYR1, an endoplasmic reticulum Ca^2+ releasing channel, in the
BCSC enrichment. In another study, Hirata et al. [[155]34], showed that
lysophosphatidic acid can promote the expansion of BCSCs via a Ca^2+
entry channel TRPC3. With regard to Orai channels, recent studies have
reported the role of Orai1 and Orai3 in CSCs of glioblastoma, non-small
cell lung cancer and oral/oropharyngeal squamous cell carcinoma
[[156]35–[157]38]. However, none of these previous studies have
investigated Orai1 or Orai3 in the context of BCSCs. In the present
study, we explored the role of Orai1 and Orai3 in the BCSC-like cells
enriched from ER+ breast cancer cells using 3D soft fibrin gel. The
expression levels of Orai1 and Orai3 were found to be elevated in these
BCSC-like cells as well as in tissue samples from luminal A breast
cancer patients. Knockdown of Orai1 and Orai3 reduced the growth of
tumor spheroids in 3D soft fibrin gel and decreased the expression of
BCSC markers. More importantly, after knockdown of either Orai1 or
Orai3, tumorigenic ability of BCSC-like cells in NOD/SCID mice were
markedly reduced, with reduced tumor growth and prolonged tumor free
duration. These data provide strong evidence for a crucial role of
Orai1 and Orai3 in self-renewal and tumorigenicity of BCSCs.
Metabolic phenotype is a crucial determinant of BCSC properties
[[158]12, [159]13]. Compared with the differentiated breast cancer
cells, BCSCs strongly favor the glycolysis pathway, which allows BCSCs
to obtain energy faster than oxidative phosphorylation pathway
[[160]12, [161]14, [162]39]. Indeed, we found that top 3 upregulated
gene categories in KEGG analysis between 3D cultured BCSCs and 2D
cultured ordinary cancer cells are all related to the metabolic pathway
of hexose and pentose, suggesting a huge change in energy acquisition
pathway genes when cells switch from 2D-culture to 3D-culture.
Furthermore, RT-qPCR analysis found that the basal expression of
glycolytic genes is also much higher in 3D cultured BCSCs compared with
2D cultured ordinary cancer cells, confirming a switch of metabolic
pathway to more active glycolysis in 3D cultured BCSCs. Moreover, a
glycolysis inhibitor 2-DG substantially suppressed the growth of 3D
tumor spheroids. These data demonstrate a critical role of glycolysis
in the growth of BCSC-like cells.
Ca^2+ is a versatile signal that regulates diverse cellular processes
in cancer [[163]40]. Store-operated Ca^2+ entry is reported to regulate
glycolysis in T lymphocytes [[164]41]. Recently, another
Ca^2+-permeable channel TRPM7 is reported to regulate glycolytic
reprogramming in ordinary colorectal cancer cells and breast cancer
cells [[165]42]. However, up to the present, virtually nothing is known
about Ca^2+ regulation of glycolysis pathway in cancer stem cells. In
our study, Orai1 expression was found to be upregulated in 3D
gel-enriched BCSC-like cells compared with 2D cultured breast cancer
cells, which correlated with an upregulation of glycolytic pathway in
3D gel-enriched BCSC-like cells. RNA silencing of Orai1 or SPCA2, or
pharmacological inhibition of Orai1 substantially downregulated the
expression of glycolytic genes in BCSC-like cells. Orai1 knockdown also
reduced glucose uptake, pyruvate level and lactate production in 3D
gel-enriched BCSC-like cells, all of which are glycolysis-related
indexes. These data established a clear functional linkage between
Orai1-SPCA2 and the Warburg effect (glycolysis) in BCSCs. In this
regard, others have reports that hypoxic tumor microenvironment in 3D
tumor spheroids may cause upregulation of Orai1 expression, at least in
colon cancer cells [[166]43]. Therefore, a likely scenario is that
hypoxic microenvironment in 3D tumor spheroids may stimulate Orai1
expression in BCSCs, causing consequent increase in Ca^2+ entry via
Orai1-SPCA2, which in turn stimulates the glycolysis pathway to promote
self-renewal and tumorigenicity of these BCSCs. Herein, our data for
the first time establish a mechanistic linkage between two important
signalling pathways, namely Orai1-mediated Ca^2+ signalling and
glycolysis, in BCSC function.
In ER+ breast cancer cells, it has been reported that Orai1-SPCA2
mediates SICE whereas Orai3-STIM1 mediates SOCE [[167]6, [168]7], which
was confirmed by this study (Figs. S5–S6). Intriguingly, our present
study demonstrated that Orai1-SPCA2 and Orai3-STIM1 act through two
distinct downstream signaling axes to regulate self-renewal and
tumorigenicity of BCSCs. Herein, Orai1-SPCA2 mediates SICE to activate
glycolysis pathway, whereas Orai3 and STIM1 mediates SOCE to activate a
glycolysis-independent pathway, both of which subsequently regulate the
self-renewal and tumorigenicity of BCSC-like cells. This novel scheme
illustrates an elegant regulatory mechanism through which Orai1 and
Orai3 act through distinct signaling axes to finely control the
self-renewal and tumorigenicity of BCSCs. An overall scheme is depicted
Fig. [169]8.
Fig. 8.
[170]Fig. 8
[171]Open in a new tab
Schematic illustration of Orai1-SPCA2 and Orai3-STIM1 regulation on the
stemness and tumorigenicity of BCSCs. Orai1 interacts with SPCA2 to
mediate SICE, which activates glycolytic pathway to promote stemness
and tumorigenic ability of BCSCs. On the other hand, Orai3 and STIM1
mediate SOCE, which promotes stemness and tumorigenic ability of breast
cancer cells via a glycolysis-independent mechanism
An emerging strategy in breast cancer therapy is to target BCSC
metabolism, including glycolysis pathway, in order to eliminate BCSCs
[[172]12, [173]13]. In the present study, we uncovered Orai1-SPCA2-SICE
as an upstream signal to regulate glycolysis. These results highlight
an intriguing possibility of targeting Orai1-SPCA2-glycolysis pathway
as a novel strategy to eliminate BCSCs. However, literature also showed
that due to their metabolic flexibility, BCSCs can switch between
oxidative phosphorylation and glycolysis [[174]12]. Thus, targeting a
single metabolic pathway may not always be sufficient for complete
eradication of breast tumors. Dual inhibition of glycolytic and
oxidative pathways might be more effective in breast cancer therapy
[[175]12, [176]13]. Indeed, we found that silencing of Orai3 and STIM1,
which mediates SOCE, could also markedly reduce the growth of BCSC-like
cells in a glycolysis-independent manner. Therefore, we speculate that
dual inhibition of Orai1-SPCA2 and Orai3-STIM1 may have a stronger
inhibition on (or totally abolish) the growth and tumorigenicity of
BCSCs.
Note that, the BCSCs in the present study were derived from MCF7 and
MDA-MB-415 cells, both of which belong to luminal A ER+ breast cancer
[[177]44]. In clinical practice, luminal A breast cancer is often
treated with endocrine therapy. However, some patients with luminal A
breast cancer develop resistance to endocrine therapy due to existence
of BCSCs [[178]45]. Furthermore, one characteristic of luminal A breast
cancer is the long duration of cancer recurrence up to 20 years, which
is at least partly due to the existence of BCSCs [[179]46]. Therefore,
our current findings about the role of Orai in BCSCs are more relevant
to luminal A breast cancer. Further studies are needed to determine
whether similar mechanisms also exist in other types of breast cancer.
In conclusion, the present study demonstrated that Orai1 interacts with
SPCA2 to mediate SICE, subsequently promoting the growth and
tumorigenicity of BCSC-like cells via glycolysis pathway. In contrast,
Orai3 and STIM1 mediate SOCE, consequently promoting the growth and
tumorigenicity of BCSC-like cells via a glycolysis-independent pathway.
In future, it is worth to explore therapeutic potential of targeting
these pathways in breast cancer treatment.
Supplementary Information
[180]Additional file 1.^ (9.8MB, pptx)
[181]Additional file 2.^ (1.2MB, pptx)
Acknowledgements