Notch1 regulates Orai1 and Orai3 expression in breast cancer cells

Abstract

Store-operated Ca²⁺ entry (SOCE) is a major pathway for Ca²⁺ entry that regulates several cellular functions. SOCE remodeling mediated by changes in the expression and/or function of the Orai channels results in the reorganization of intracellular Ca²⁺ homeostasis leading to a variety of pathologies, including cancer. Notably, a significant alteration of Orai function has been reported in breast cancer cells, where the dysregulation of the Notch1 signaling pathway plays a role in the development and progression of cancer hallmarks. Here, we have investigated the possible role of Notch1 in the regulation of the expression of Orai1 and Orai3 in different breast cancer cell lines. Expression of the active form of Notch1, as well as cell stimulation with the Notch1 agonist Jagged-1 (Jag-1), demonstrates a differential role of Notch1 in the regulation of Orai expression in non-tumoral breast epithelial cells and triple negative or luminal breast cancer cells. The role of Notch1 was confirmed using DAPT, a γ-secretase inhibitor that prevents activation of the Notch pathway. Modulation of Orai1 and Orai3 expression by Notch1 was paralleled by changes in SOCE. The effect in Orai expression mediated by activation of Notch1 signaling pathway was mimicked by the expression of HEY1 or the non-phosphorylatable HEY1-S68A mutant; by contrast, expression of the phosphomimetic HEY1-S68D mutant was without effect on Orai expression. Understanding the Notch1-HEY1-Orai axis might provide insights into the development of subtype-specific therapeutic strategies targeting breast cancer.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-33071-x.

Introduction

Calcium (Ca²⁺) signaling plays a key role in a variety of physiological processes, including gene expression, secretion, metabolism, and cell proliferation. One of the main mechanisms for Ca²⁺ entry into non-excitable cells is store-operated Ca²⁺ entry (SOCE), a tightly regulated process that is activated upon depletion of the intracellular Ca²⁺ stores, mainly the endoplasmic reticulum (ER)¹. A fundamental component of SOCE is the Orai channels, a family of highly selective Ca²⁺ channels located in the plasma membrane. The Orai family comprises three homologs—Orai1, Orai2, and Orai3—among which Orai1 is the best characterized and essential for the function of the Ca²⁺ release-activated Ca²⁺ (CRAC) channels², with Orai3 also playing a crucial role in SOCE in certain cell types including B cells³, cells of the luminal subtype of breast cancer cells^4–6 and pancreatic adenocarcinoma cells⁷. Activation of Orai channels is mediated by stromal interaction molecule (STIM) proteins, which sense ER Ca²⁺ levels and physically interact with Orai proteins to trigger CRAC channel opening. Given their critical role in Ca²⁺ homeostasis and signaling, dysregulation of Orai channels has been implicated in various pathophysiological conditions, including immune deficiencies, cardiovascular diseases and cancer.

Aberrant intracellular Ca²⁺ signaling has emerged as a hallmark of cancer, contributing to processes such as proliferation, migration, invasion, and resistance to cell death. In breast cancer cells, where SOCE is dysregulated, Orai1 is often overexpressed in triple negative breast cancer (TNBC) cells which has been linked to enhanced cell proliferation, migration and metastatic potential^4,8–10. In triple negative breast cancer cells, SOCE is mediated by the canonical STIM1/Orai1 pathway⁴. Orai3, in particular, is frequently upregulated in luminal, estrogen receptor-positive (ER⁺), breast cancer cells and has been implicated in SOCE, as well as the regulation of cell survival and resistance to oxidative stress^4,11–13. The altered expression profiles and functional roles of Orai channels suggest their involvement in the reprogramming of Ca²⁺ homeostasis that supports oncogenic signaling in breast cancer.

Notch1 signaling pathway plays a critical role in cell fate determination, proliferation, and survival, and its dysregulation has been strongly associated with breast cancer development and progression^14,15. Activation of Notch1 begins when a membrane-bound Notch ligand, such as Delta-like (DLL) or Jagged, is presented by a neighboring cell and binds to the Notch1 receptor. This interaction induces a series of proteolytic cleavages, mediated by ADAM-family metalloproteases and subsequently by the γ-secretase complex, resulting in the release of the Notch1 intracellular domain (N1ICD). The N1ICD translocates into the nucleus, where it associates with the DNA-binding protein RBP-Jκ and coactivators such as Mastermind-like (MAML) to drive the transcription of target genes, including HES and HEY family members¹⁶. Elevated Notch1 activity has been observed in various breast cancer subtypes, particularly in TNBC, where it contributes to increased tumor aggressiveness, stemness, and resistance to therapy^17,18. Recent studies suggest a functional interplay between Notch1 signaling and Ca²⁺ homeostasis¹⁹. For instance, hypoxia has been reported to up-regulate Orai1 and Notch1 expression, and the enhanced expression of Orai1 has been shown to be dependent on Notch1 signaling²⁰. Understanding the molecular interplay between Notch1 and Orai channels in TNBC and luminal breast cancer subtypes could open new avenues for targeted therapies in breast cancer.

Materials and methods

Cell culture

Estrogen receptor-positive (ER⁺) breast cancer cell lines MCF7 (RRID: CVCL_0031) and T47D (RRID: CVCL_0553), as well as triple-negative breast cancer (TNBC) cell lines MDA-MB-231 (RRID: CVCL_0062) and BT20 (RRID: CVCL_0178), were maintained in high-glucose Dulbecco’s Modified Eagle Medium (DMEM; ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; ThermoFisher Scientific), 100 U/mL penicillin (ThermoFisher Scientific), and 100 µg/mL streptomycin (ThermoFisher Scientific). The non-tumorigenic mammary epithelial cell line MCF10A (RRID: CVCL_0598) was maintained in Dulbecco’s Modified Eagle Medium F12 (DMEM/F12; ThermoFisher Scientific) supplemented with 5% (v/v) horse serum (ThermoFisher Scientific), 0.5 µg/mL hydrocortisone (ThermoFisher Scientific), 10 µg/mL insulin (ThermoFisher Scientific), 20 ng/mL epidermal growth factor (EGF; (ThermoFisher Scientific), and 100 ng/mL cholera toxin (ThermoFisher Scientific). All cell types were incubated at 37 °C in a humidified atmosphere containing 5% CO₂, as previously described¹⁰. All cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) during the last 5 years. Cell line authentication was performed routinely at least once per year using the analysis of Short Tandem Repeat (STR), obtaining a percentage match surpassing 90% in comparison with established reference values. Cells were routinely monitored for contamination and the absence of mycoplasma was confirmed using confocal microscopy-based detection methods.

Western blot and RT-qPCR analyses were performed using approximately 5 × 10⁶ cells seeded in 100-mm culture dishes. For calcium imaging experiments involved 4 × 10⁵ cells were seeded in 35-mm six-well multidishes. In preparation for the chorioallantoic membrane (CAM) assay, MCF7 cells were maintained in T175 tissue culture flasks equipped with canted necks and filter caps, suitable for adherent cell growth. The culture medium was replaced every 3 to 4 days, and subculturing was performed at 80% confluency using a 1:4 split ratio. To generate the CAM onplants, 1 × 10⁶ cells per embryo were counted and collected into a 50 mL centrifuge tube. After gentle centrifugation at 200 × g for 5 min, the pellet was resuspended in 15 µL of PBS and combined with 5 µL of Geltrex (ThermoFisher Scientific), yielding a final volume of 20 µL. The resulting cell–matrix suspension was kept on ice until application to the CAM.

Cell transfections and treatments

Transient transfections were performed when cells reached 60–80% confluency using Lipofectamine 3000 (ThermoFisher Scientific) in BT20 and T47D cells and DharmaFECT kb transfection reagent (Horizon Discovery, Waterbeach, UK) in MCF10A, MCF7 and MDA-MB-231 cells. All subsequent analyses were performed 48 h post-transfection. The following plasmids were used in this study: pSG5-flag, pSG5-flag-HEY1, pSG5-flag-HEY1 S68A, and pSG5-flag-HEY1 S68D (kindly provided by Dr. Borja Belandia, Department of Cancer Biology, Instituto de Investigaciones Biomédicas Alberto Sols, CSIC-UAM, Madrid, Spain), as well as the myc-tagged Notch-1 intracellular domain (Notch-1-ICD) plasmid (a generous gift from Dr. Marco A. Calzado, Maimonides Biomedical Research Institute of Córdoba (IMIBIC), Córdoba, Spain). Cell viability post-transfection was evaluated using the Live/Dead Viability/Cytotoxicity Kit (ThermoFisher Scientific), consistently ranged between 92% and 95% throughout the study. All experimental protocols were reviewed and approved by the Ethics Committee of the University of Extremadura and the Servicio Extremeño de Salud.

To investigate the functional role of Notch1 signaling, cells were treated with 50 µM recombinant Jagged1 protein (Jagged-1 (188–204), Anaspec Inc, Freemont, CA, USA) for 48 h to activate the pathway, and with the γ-secretase inhibitor DAPT (MedChem Express, Monmouth Junction, NJ, USA) to inhibit Notch1 activation for 24 h, 48 h, 72 h or once daily for five consecutive days, depending of experimental procedures.

Western blotting and antibodies

Cells cultured in 100-mm Petri dishes (5 × 10⁶ cells) were lysed in ice-cold NP-40 lysis buffer (137 mM NaCl, 20 mM Tris, 2 mM EDTA, 10% glycerol, 1% Nonidet P-40) supplemented with 1 mM sodium orthovanadate (Na₃VO₄), and a complete EDTA-free protease inhibitor cocktail (Reference name: COEDTAF-RO; Roche Diagnostics GmbH, Mannheim, Germany). Expression of Orai1α and Orai1β variants was evaluated by enzymatic deglycosylation of whole-cell lysates using N-glycosidase F (PNGase F) from Elizabethkingia miricola (New England Biolabs, Ipswich, MA, USA), following the manufacturer’s protocol. Equal amounts (25 µg) of proteins extracted from cell lysates, quantified using Pierce™ BCA protein assay kit (ThermoFisher Scientific), were resolved by SDS-PAGE using 10% or 12% polyacrylamide gels and subsequently transferred onto nitrocellulose membranes. Later, membranes were blocked overnight at 4 °C with EveryBlot Blocking Buffer (Catalog number #12010020; Bio-Rad Laboratories, Inc, Hercules, CA, USA) to reduce nonspecific binding. Immunodetection of Orai1, Orai3, Notch1, myc-tagged proteins, flag-tagged proteins, and β-actin was performed by incubating the membranes with primary antibodies diluted in EveryBlot Blocking Buffer for 1 h at room temperature, with the following dilutions: 1–500 for anti-Notch1 antibody (rabbit polyclonal Notch1 antibody, catalog number: 100–401−407, Rockland Immunochemicals, Inc, Pottstown, PA, USA), anti-myc antibody (mouse monoclonal anti-c-Myc antibody (Clone 9E10; RRID: AB_2533008; catalog number 13–2500, ThermoFisher Scientific) and anti-flag antibody (mouse monoclonal anti-DYKDDDDK (flag) antibody (Clone FG4R; RRID: AB_2537626; catalog number MA1-91878-HRP, ThermoFisher Scientific). 1:1000 for anti-Orai1 antibody (rabbit polyclonal anti-Orai1 C-terminal antibody; epitope: amino acids 288–301 of human Orai1; RRID: AB_1078883; catalog number O8264, MilliporeSigma, Burlington, MA, USA), 1:1000 for anti-STIM1 antibody (mouse monoclonal anti-GOK/STIM1 antibody, clone 44/GOK; catalog number 610954, epitope: amino acids: 25–139 of human STIM1; RRID: AB_398267, BD Biosciences, San Jose, CA, USA) and anti-Orai3 antibody (mouse monoclonal anti-Orai3 antibody (Clone EPR22575-17; RRID: AB_2530307; catalog number ab254260, Abcam, Cambridge, UK). 1:2000 for anti-β-actin antibody (rabbit polyclonal anti-β-actin antibody, epitope: amino acids 365–375 of human β-actin; RRID: AB_2816311; catalog number A2066, MilliporeSigma). Fluorescent secondary antibodies goat anti-rabbit IgG StarBright Blue 700 (RRID: AB_2721073; Catalog number #12004161, Bio-Rad Laboratories, Inc) and goat anti-mouse IgG StarBright Blue 520 (RRID: AB_2934034; Catalog number #12005866, Bio-Rad Laboratories, Inc) were applied to membranes at a 1:3000 dilution in Tris-buffered saline with 0.1% Tween-20 (TBST) and incubated for 1 h to facilitate primary antibody detection. Alternatively, membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse antibody (RRID: AB_10015289, Jackson laboratories, West Grove, PA, USA) and goat anti-rabbit IgG antibody (RRID: AB_2337913, Jackson laboratories) at a 1:10,000 dilution in TBST, followed by SuperSignal^® West Dura extended duration substrate reagent (ThermoFisher Scientific) exposure for 5 min. Signal detection for both modalities was executed using a ChemiDoc MP Imaging System (Bio-Rad Laboratories, Hercules, CA, USA), with densitometric quantification performed using Image Lab 6.1 software (Bio-Rad Laboratories).

Determination of cytosolic free-Ca²⁺ concentration ([Ca²⁺]_i)

Cytosolic Ca²⁺ concentration ([Ca²⁺]_i) was measured using the ratiometric fluorescent indicator fura-2, as previously described²¹. Cells were incubated with 2 µM fura-2 acetoxymethyl ester (fura-2/AM; Molecular Probes, Leiden, The Netherlands) for 30 min at 37 °C to facilitate dye loading. Post-incubation, cells cultured on glass coverslips positioned within a perfusion chamber on an inverted epifluorescence microscope platform (Nikon Eclipse Ti2) equipped with a videomicroscopy image acquisition and image analysis software (NIS-Elements Imaging Software v5.02.00). At the time of experiment, cells were superfused at ambient temperature with HEPES-buffered saline (HBS), composed of 125 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 5 mM glucose, and 25 mM HEPES, adjusted to pH 7.4, supplemented with 0.1% (w/v) bovine serum albumin containing 100 µM EGTA to chelate residual extracellular Ca²⁺, and 100 s later, ER Ca²⁺ depletion was triggered by stimulation with 2 µM thapsigargin (TG; MilliporeSigma), a sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor, followed by the readdition of extracellular Ca²⁺ to a final concentration of 1.8 mM to initiate SOCE. Alternatively, cells were stimulated with 50 µM 2-APB (MilliporeSigma) in a medium containing 1.8 mM Ca²⁺. Imaging was conducted at 40× magnification using a Nikon CFI S Fluor 40× Oil objective. Fluorescence excitation alternated between 340 nm and 380 nm wavelengths using a xenon arc lamp filtered through a high-speed monochromator (Optoscan ELE 450, Cairn Research, Faversham, UK). Fluorescence emission was detected at 510 nm using a cooled sCMOS digital camera (PCO Panda 4.2, Excelitas PCO GmbH, Germany) processed using NIS-Elements AR software (Nikon). Ratiometric fluorescence values (F₃₄₀/F₃₈₀) were calculated on a per-pixel basis, with data expressed as changes in ratio (ΔF₃₄₀/F₃₈₀). TG-evoked Ca²⁺ release and Ca²⁺ entry was estimated as the area under the curve measured as the integral of the rise in fura-2 fluorescence ratio 4 min after the addition of TG (for Ca²⁺ release) or Ca²⁺ (for Ca²⁺ entry), respectively and taking a sample every second.

Quantitative real-time PCR (RT-qPCR)

Total RNA was isolated from cultured cells using TRIzol reagent (ThermoFisher Scientific), followed by purification employing the Direct-zol RNA Kit (Zymo Research, Irvine, CA, USA), strictly adhering to the manufacturer’s protocols to ensure RNA integrity and high purity. Complementary DNA (cDNA) synthesis was performed through reverse transcription using High Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific), according to the manufacturer’s guidelines. Quantitative assessment of RNA and cDNA concentrations was conducted using the Qubit RNA HS Assay Kit (ThermoFisher Scientific) and Qubit dsDNA HS Assay Kit (ThermoFisher Scientific), respectively, with fluorescence intensities measured on a Qubit 3.0 Fluorometer (ThermoFisher Scientific) Quantitative real-time PCR (RT-qPCR) amplification was carried out on a QuantStudio 6 Real-Time PCR System (ThermoFisher Scientific, Waltham, MA, USA) using PowerTrack SYBR Green Master Mix (ThermoFisher Scientific, Waltham, MA, USA) using the following primers sequences: HEY1 (forward primer: 5’-CGAGGTGGAGAAGGAGAGTG-3’; reverse primer: 5’-CTGGGTACCAGCCTTCTCAG-3’) and GAPDH (forward primer: 5’-GTCTCCTCTGACTTCAACAGCG-3’; reverse primer: 5’-ACCACCCTGTTGCTGTAGCCAA-3’). Amplification was carried out under the following cycling conditions: initial denaturation at 96 °C for 2 min, followed by 35 cycles of denaturation at 96 °C for 15 s, annealing at 60 °C for 25 s and a final extension step at 72 °C for 10 min. Relative quantification of mRNA expression levels was performed applying the comparative threshold cycle (ΔΔCT) method, with expression fold changes calculated using the equation RQ = 2^−ΔΔCT. The amount of HEY mRNA transcripts were normalized to GAPDH mRNA levels and represented as mean expression relative to mock-treated cells ± standard error of the mean (SEM).

Chicken embryo chorioallantoic membrane (CAM) assay

The ex-ovo CAM assay was conducted as previously described by Handl et al.²². Fertilized white Lohmann eggs, sourced from accredited local farms, were washed, disinfected, and labeled with the date of collection, designated as embryonic development day 0 (EDD0). Eggs were incubated at 37.6 °C with 40–60% relative humidity for 3 days to initiate embryonic development. Subsequently, under sterile conditions, a small incision was made in the eggshell using an electric blade to weaken the shell structure, allowing for the complete transfer of the egg contents into anti-static trays. The embryos were then incubated under the same conditions for an additional 6–7 days. On embryonic development day 10 (EDD10), autoclaved silicone rings (diameter = 5 mm; thickness = 1 mm) were placed onto the CAM. Then, 20 µL of a cell suspension (10⁶ cells), composed of 15 µL of PBS and 5 µL of Geltrex (ThermoFisher Scientific), were pipetted into each ring. On EDD11, tumors were treated with 20 µM DAPT, administered once daily over a period of five consecutive days. Following the treatment period, tumors were imaged microscopically, excised from the pre-cooled eggs, and transferred into phosphate-buffered saline (PBS). Embryos were maintained on ice and euthanized via decapitation. Tumors were subsequently fixed in 4% paraformaldehyde (PFA). Excised tumors were fixed in 4% paraformaldehyde (PFA) at 4 °C. Fixed tissues were then subjected to a graded ethanol dehydration protocol, progressing from 70% to 100% ethanol, followed by toluene treatment. Subsequently, tumors were embedded in paraffin for subsequent histological and immunohistochemical analyses.

Caspase 3 activation assay

Caspase-3 activation was assessed using the NucView^® 530 Caspase-3 substrate kit (Biotium™, Fremont, CA, USA), following the manufacturer’s protocol as previously described²³. Briefly, cells were transfected with c-myc-Notch1 for 24 h or empty vector; alternatively, cells was treated with 1 µM of TG for 24 h, as a positive control of apoptosis in MDA-MB-231 cells²⁴. Cells were detached and re-suspended in fresh-medium supplemented with 2 µM of NunView^® 530 and incubated at room temperature for 30 min. Cells were washed and re-suspended in PBS and fluorescence resulting of caspase-3 degradation of the substrate was measured using a fluorescence microplate reader VarioSKAN LUX multimode microplate reader (Thermo Fisher Scientific) by using 528/563 nm (Ex/Em) wavelengths. NunView^®530 fluorescence was presented as mean ± SEM of non-transfected cells.

Statistical analysis

All data are expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism software (version 8.4.3; GraphPad Software, San Diego, CA, USA). Normality of data distribution was assessed using the Kolmogorov–Smirnov test. For comparisons among multiple groups, the non-parametric Kruskal–Wallis test followed by Dunn’s post hoc test was applied. For comparisons between two groups, either an unpaired Student’s t-test (for normally distributed data) or the Mann–Whitney U test (for non-normally distributed data) was employed. Differences were considered statistically significant at p < 0.05.

Results

Notch1 differentially modulates Orai1 and Orai3 expression in breast cancer cells

As mentioned above, the N1ICD is the active, signaling fragment of the Notch1 receptor, which plays a crucial role in the transcription of target genes such as HEY1 ¹⁶. Hence, we have now investigated the possible role of Notch1 in the expression of Orai1, the predominant Orai channel in non-tumoral breast epithelial cells and most breast cancer cells^4,25 by expressing N1ICD. Non-tumoral breast epithelial MCF10A cells as well as breast cancer cells of the triple negative subtype (MDA-MB-231 and BT20 cells) or the luminal (estrogen receptor positive) subtype (MCF7 and T47D) were transfected with N1ICD or empty vector and the Orai1 expression was analyzed by Western blotting. Detection of Orai1 by Western blotting leads to several diffuse bands, as previously described^26–28, likely due to N-linked glycosylation of Orai1 (Fig. 1), as treatment of cell lysates with N-glycosidase F (PNGaseF) that remove N-linked glycosylation leads to two bands corresponding to the molecular sizes of Orai1α and Orai1β (Fig. 1; specificity of the anti-Orai1 C-terminal antibody used has been previously demonstrated in Orai1-knockout MCF7 and HEK-293 cells^29,30. As shown in Fig. 1, N1ICD expression did not significantly alter the total Orai1 protein level or the expression of the individual Orai1 variants in MCF10A. In contrast, in TNBC cells, expression of N1ICD significantly attenuated the expression of total Orai1 or both variants in MDA-MB-231 cells (p < 0.001) while enhancing the expression of Orai1 (both variants; p < 0.05) in BT20 cells at the protein level (Fig. 1). Despite MDA-MB-231 and BT20 are two TNBC cell lines, they have been reported to differ in several aspects including that MDA-MB-231 cells are highly tumorigenic and metastatic, while BT20 cells are intermediate tumorigenic and less metastatic³¹. Similarly, we have found differences in the role of Notch1 in the luminal breast cancer cell lines MCF7 and T47D, which show a different progesterone receptor expression and glycolytic rate, among other differences^32,33. Our results indicate that the active domain of Notch1 has no significant effect on the expression of Orai1 in MCF7 cells but significantly reduced Orai1 expression in T47D cells (p < 0.01). Altogether, these findings indicate that Notch1 plays a differential role in the expression of Orai1 in distinct breast cancer cells. It should be noted that these experiments are not intended to compare the expression of Orai1 or its variants in different breast cancer cells.

Fig. 1.

Effect of Notch1 intracellular domain (N1ICD) expression on the expression of total Orai1 and Orai1α and Orai1β isoforms in non-tumoral breast epithelial cells and breast cancer cells. (A–E) Non-tumoral breast epithelial MCF10A cells (A), TNBC MDA-MB-231 (B) and BT20 cells (C), as well as luminal breast cancer MCF7 (D) and T47D cells (E) were transfected with myc-N1ICD fragment (myc-N1ICD) or empty vector (Control) and 24 h later cells were lysed. Cell lysates were subjected to 10% SDS-PAGE and Western blotting with anti-myc or anti-Orai1 antibody, as described in Materials and Methods. Membranes were reprobed with anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. n = 10, 13,10, 7 and 11 separate experiments for MCF10A, MDA-MB-231, BT20, MCF-7 and T47D, respectively. (F–H) Bar graphs represent the quantification of Orai1 (F), Orai1α (G) or Orai1β (H) protein expression as fold change over the level in mock-transfected cells (Control) and presented as means ± S.E.M. Data were statistically analyzed using Mann–Whitney U test. *p < 0.05 and **p < 0.01.

As SOCE is strongly dependent on Orai1 and STIM1 in non-tumoral breast epithelial cells and TNBC cells, but, in contrast, luminal breast cancer cells are more dependent on Orai3^4,34. Hence, we investigated whether the expression of the active domain of Notch1 influences the expression of Orai3 as well as STIM1 in non-tumoral epithelial cells and breast cancer cells or the luminal and triple negative subtypes. Concerning Orai3 expression, N1ICD did not alter Orai3 protein level in MCF10A cells (Fig. 2A,F). In contrast, in TNBC cells, expression of N1ICD significantly attenuated Orai3 protein content (p < 0.0001) while enhancing its expression in BT20 cells (Fig. 2; p < 0.05). Furthermore, we have not detected differences in the protein content of Orai3 in luminal MCF7 cells upon expression of N1ICD, which significantly reduced Orai3 expression in T47D cells (p < 0.01). N1ICD expression significantly reduced STIM1 protein level in MCF10A cells (p < 0.05) but did not significantly alter STIM1 expression in breast cancer cells (Fig. 2A–E,G). Altogether, these findings indicate that, as for Orai1, Notch1 plays a differential role in the expression of STIM1 and Orai3 in distinct breast cancer cells and non-tumoral breast epithelial cells.

Fig. 2.

Effect of Notch1 intracellular domain (N1ICD) expression on the expression of STIM1 and Orai3 in non-tumoral breast epithelial cells and breast cancer cells. (A–E) Non-tumoral breast epithelial MCF10A cells (A), TNBC MDA-MB-231 (B) and BT20 cells (C), as well as luminal breast cancer MCF7 (D) and T47D cells (E) were transfected with myc-N1ICD fragment (myc-N1ICD) or empty vector (Control) and 24 h later cells were lysed. Cell lysates were subjected to 10% SDS-PAGE and Western blotting with anti-myc, anti-STIM1 or anti-Orai3 antibody, as described in Materials and Methods. Membranes were reprobed with anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. * Non-specific band. n = 9, 13, 8, 11 and 10 separate experiments for Orai3 and 4, 6, 4, 3 and 5 separate experiments for STIM1 in MCF10A, MDA-MB-231, BT20, MCF-7 and T47D, respectively. (F, G) Bar graphs represent the quantification of Orai3 (F) or STIM1 (G) protein expression as fold change over the level in mock-transfected cells (Control) and presented as means ± S.E.M. Data were statistically analyzed using Mann–Whitney U test. *p < 0.05 and **p < 0.01.

As Orai1 has been reported to be essential for the development of triple negative breast cancer hallmarks^13,25, we have explored whether expression of the active form of Notch1 induces apoptosis in MDA-MB-231 cells by looking for the activation of caspase 3. For comparison we tested caspase 3 activity in non-tumoral MCF10A cells, which show no changes in Orai1 or Orai3 expression associated to the expression of N1ICD (see Fig. 1). Cell treatment with TG was used as a positive control of apoptosis. As shown in supplementary Fig. 1, MDA-MB-231 cells expressing N1ICD exhibited a significantly greater caspase 3 activity than mock-transfected cells (p < 0.05). By contrast, in MCF10A, expression of the active form of Notch1 did not significantly increase caspase 3 activity as compared to control. These findings indicate that attenuation of Orai1 in MDA-MB-231 cells upon expression of N1ICD results in caspase 3 activation, which is an indicator of apoptosis.

The effect of N1ICD on Orai1/Orai3 expression in non-tumoral breast epithelial and breast cancer cells was confirmed by cell treatment with the Notch1 receptor ligand Jagged-1 (Jag-1). Non-tumoral breast epithelial MCF10A cells, TNBC (MDA-MB-231 and BT20) cells as well as luminal breast cancer (MCF7 and T47D) cells were stimulated with Jag-1 (50 µM, as previously reported³⁵ or the vehicle for 48 h and Orai1 or Orai3 expression were estimated by Western blotting. Activation of Notch1 was estimated by detection of endogenous N1ICD (Fig. 3A–E,H). As shown in Fig. 3, Jag-1 reduced Orai1 expression in MDA-MB-231 and T47D cells, while it increased Orai1 expression in BT20 cells and had no significant effect in MCF10A and MCF7 cells. In addition, treatment with Jag-1 reduced Orai3 protein level in MDA-MB-231 and T47D cells, while enhancing its expression in BT20 and had no detectable effect in MCF10A and MCF7 cells. Altogether, these findings are consistent with those observed upon expression of the N1ICD (Figs. 1 and 2).

Fig. 3.

Effect of Jagged-1 in the expression of Orai1 and Orai3 in non-tumoral breast epithelial cells and breast cancer cells. (A–E) Non-tumoral breast epithelial MCF10A cells (A), TNBC MDA-MB-231 (B) and BT20 cells (C), as well as luminal breast cancer MCF7 (D) and T47D cells (E) were stimulated with 50 µM Jagged-1 (Jag-1) or the vehicle (Mock) for 48 h and lysed. Cell lysates were subjected to 10% SDS-PAGE and Western blotting with the anti-Notch1, anti-Orai1 or anti-Orai3 antibody, as described in Materials and Methods. Membranes were reprobed with the anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. n = 5–9 separate experiments. (F–H) Bar graphs represent the quantification of protein expression as fold change over the level in vehicle-transfected cells and presented as means ± S.E.M. Data were statistically analyzed using Mann–Whitney U test. *p < 0.05 and **p < 0.01.

The functional role of Notch1 in Orai1 and Orai3 expression was further assessed by using the γ-secretase inhibitor, DAPT, which prevents cleaving and activation of Notch receptors³⁶. Non-tumoral breast epithelial MCF10A cells as well as TNBC (MDA-MB-231 and BT20) cells and luminal breast cancer (MCF7 and T47D) cells were pretreated with 20 µM DAPT for 24, 48 and 72 h and Orai1 and Orai3 expression was estimated by Western blotting. Inhibition of Notch1 was estimated by detection of endogenous N1ICD (Fig. 4). As shown in Fig. 4A, pretreatment of MCF10A with DAPT did not modify Orai1 expression but significantly enhanced Orai3 protein level, thus suggesting that basal Notch signaling is crucial for maintaining Orai3 at its normal low levels, however, Orai3 is not sensitive to further repression when the Notch pathway is further activated (Figs. 1 and 3), suggesting that basal Notch repression is already near complete on the Orai3 gene. The enhancement of Orai3 expression by DAPT was confirmed by treating the cells with the Orai channel modulator 2-APB, which at high concentrations has been reported to stimulate Orai3 channel activity³⁷. As shown in supplementary Fig. 2, stimulation of MCF-10 A with 50 µM 2-APB in a medium containing 1.8 mM Ca²⁺ induced a transient increase in cytosolic free-Ca²⁺ concentration ([Ca²⁺]_i) that was significantly enhanced in cells treated with 20 µM DAPT for 72 h. In MDA-MB-231 cells, treatment with DAPT did not significantly alter the expression of Orai1 or Orai3 (Fig. 4B,C), which contrast with the results obtained upon expression of exogenous N1ICD or treatment with Jag-1, which might suggest that under basal conditions, the level of NICD generated by the endogenous Notch pathway is too low to exert significant repression on Orai1. Alternatively, Orai1/Orai3 might already be expressed at a maximum level, and Notch inhibition by DAPT does not cause a detectable increase. However, BT20 cells, treatment with DAPT modifies the expression of Orai1 and Orai3, resulting in effects consistent with those observed with N1ICD overexpression or stimulation with Jag-1 (Fig. 4C). In luminal MCF7 cells DAPT did not induce any modification in Orai1 or Orai3 protein content at the times investigated (Fig. 4D), which is consistent with the lack of effect of N1ICD overexpression or Jag-1 stimulation. Finally, in T47D cells, treatment with DAPT did not significantly modify Orai1 expression (Fig. 4E) in contrast to the effect of N1ICD overexpression or stimulation with Jag-1 (see Figs. 1 and 3), which might be attributed to the mechanisms mentioned above for MDA-MB-231 cells. However, consistent with the results obtained with the expression of N1ICD or stimulation with Jag-1, treatment with DAPT significantly enhanced Orai3 expression (Fig. 4E; p < 0.05), thus suggesting that Orai3 expression is differentially regulated by Notch1 in luminal breast cancer cells.

Fig. 4.

Effect of DAPT in the expression of Orai1 and Orai3 in non-tumoral breast epithelial cells and breast cancer cells. (A–E) Non-tumoral breast epithelial MCF10A cells (A), TNBC MDA-MB-231 (B) and BT20 cells (C), as well as luminal breast cancer MCF7 (D) and T47D cells (E) were treated with 20 µM DAPT or the vehicle (DMSO) for 24, 48–72 h and lysed. Cell lysates were subjected to 10% SDS-PAGE and Western blotting with the anti-Notch1, anti-Orai1 or anti-Orai3 antibody, as described in Materials and Methods. Membranes were reprobed with the anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. n = 4–6 separate experiments. Bar graphs represent the quantification of protein expression as fold change over the level in vehicle-treated cells and presented as means ± S.E.M. Data were statistically analyzed using Mann–Whitney U test. *p < 0.05, **p < 0.01 and ***p < 0.001.

Our results indicate that the Notch1 pathway does not significantly alter the Orai3 expression in luminal MCF7 breast cancer cells (Fig. 4). On the other hand, Orai3 plays a major role in SOCE and supports cancer hallmarks in these cells^4,5,34. Hence, we have further explored whether Notch1 supports tumor growth in this cell model by using the chorioallantoic membrane (CAM) assay. Cells were engrafted at embryonic development day 9 (EDD9). Onplantation of MCF7 tumor cells onto the CAM of a developing chick embryo led to rapid growth and strong neovascularization (supplementary Fig. 3) as previously described²². On EDD11, tumors were treated with 20 µM DAPT for five consecutive days (according to the procedures for animal research). Topical treatment with DAPT was administered by applying a 10 µL drop of 20 µM DAPT within the silicone ring, following a protocol designed to mimic the dosing profile of daily chemotherapy administration. Tumor diameters of both control and DAPT-treated groups were measured at EDD15, as indicated by the dashed lines (supplementary Fig. 3). Treatment for 120 h with 20 µM DAPT slightly but not significantly reduced the tumor growth (the average diameter of control and DAPT-treated tumors were 1,51 × 10⁵ ± 0.31 × 10⁵ µm² and 1.11 × 10⁵ ± 0.18 × 10⁵ µm², respectively). This finding is consistent with the lack of effect of Notch1 on the Orai3 expression and SOCE in MCF7 cells, and further supports a relevant role for Orai3 in the development of MCF7 cancer hallmarks^4,5,34.

Effect of Notch1 activity in SOCE in non-tumoral breast epithelial and breast cancer cells

As Notch1 modulates the expression of Orai1 and Orai3, we have further explored its role in SOCE in non-tumoral breast epithelial and breast cancer cells. As shown in Fig. 5A, treatment of non-tumoral MCF10A cells with TG in the absence of extracellular Ca²⁺ results in a transient increase in [Ca²⁺]_i, as a result of inhibition of the sarco/endoplasmic Ca²⁺ ATPase (SERCA) and subsequent passive Ca²⁺ release from the intracellular Ca²⁺ stores. Subsequent addition of 1.8 mM Ca²⁺ to the extracellular medium led to a more sustained increase in [Ca²⁺]_i, which is indicative of SOCE. Expression of N1ICD did not significantly alter either TG-induced Ca²⁺ release or SOCE as compared to mock-treated MCF10A cells (Fig. 5A). Moreover, the expression of the active form of Notch1 in TNBC MDA-MB-231 and BT20 cells had no significant effect on SOCE in MDA-MB-231 cells while enhancing SOCE in BT20 cells, the latter mirroring the effect of N1ICD expression on Orai1 protein content in BT20 cells (Fig. 5B and C). In luminal MCF7 cells, and consistent with the lack of effect of N1ICD expression on the protein level of Orai1 and Orai3, TG-evoked Ca²⁺ efflux from the intracellular Ca²⁺ stores as well as SOCE were found to be similar in cells expressing N1ICD and their corresponding controls (Fig. 5D). By contrast, T47D cells expressing the active form of Notch1 show smaller TG-induced SOCE, as well as Ca²⁺ release from the intracellular stores, the latter might be attributed to the reduced SOCE. Therefore, the analysis of the role of Notch1 in SOCE mostly aligns with our earlier findings regarding its influence on Orai1 and Orai3 expression.

Fig. 5.

Role of the expression of N1ICD in TG-induced Ca²⁺ release and entry in non-tumoral breast epithelial cells and breast cancer cells. Non-tumoral breast epithelial MCF10A cells (A), TNBC MDA-MB-231 (B) and BT20 cells (C), as well as luminal breast cancer MCF7 (D) and T47D cells (E) were transfected with myc-N1ICD fragment (Myc-N1ICD) or empty vector (Control). Twenty-four hours later cells were loaded with fura-2. Cells were then suspended in a Ca²⁺-free (100 µM EGTA) HBS and stimulated with 2 µM TG followed by reintroduction of external Ca²⁺ (final concentration 1.8 mM) to initiate Ca²⁺ entry. Representative traces are shown in the left panel. Scatter plots represent quantification of TG-evoked Ca²⁺ release and Ca²⁺ entry determined as described in Materials and Methods. Dots represent single cell data for 2–3 separate experiments. n = 44 and 45 (for Control and N1ICD-expressing MCF10A cells, respectively), 48 and 47 (for Control and N1ICD- expressing MDA-MB-231 cells, respectively), 78 and 83 (for Control and N1ICD-expressing BT20 cells), 84 and 94 (for Control and N1ICD-expressing MCF7 cells) and 85 and 86 (for Control and N1ICD-expressing T47D cells, respectively). Data are presented as means ± SEM and are statistically analyzed using Student´s t-test. *p < 0.05, **p < 0.01 and ****p < 0.0001.

We further examined the effect of DAPT on SOCE across the cell lines investigated and found that, a 48-hour pretreatment with 20 µM DAPT enhanced SOCE in MCF10A cells (Fig. 6A), which might be attributed to the enhanced Orai3 expression (Fig. 4). In addition, DAPT attenuated SOCE in BT20 cells (Fig. 6C), which is likely due to the reduction in Orai1 and Orai3 protein content (Fig. 4). By contrast, DAPT did not significantly alter SOCE in the remaining breast cancer cell lines investigated (Fig. 6). Despite not having been fully investigated in T47D cells, our findings might suggest a predominant role for Orai1, whose expression is not significantly modified by DAPT, in SOCE in this cell line.

Fig. 6.

Effect of DAPT in TG-induced Ca²⁺ release and entry in non-tumoral breast epithelial cells and breast cancer cells. Non-tumoral breast epithelial MCF10A cells (A), TNBC MDA-MB-231 (B) and BT20 cells (C), as well as luminal breast cancer MCF7 (D) and T47D cells (E) were treated with 20 µM DAPT or the vehicle (DMSO; Control). Fourty-eight hours later cells were loaded with fura-2. Cells were then suspended in a Ca²⁺-free (100 µM EGTA) HBS and stimulated with 2 µM TG followed by reintroduction of external Ca²⁺ (final concentration 1.8 mM) to initiate Ca²⁺ entry. Representative traces are shown in the left panel. Scatter plots represent quantification of TG-evoked Ca²⁺ release and Ca²⁺ entry determined as described in Materials and Methods. Dots represent single cell data for 2–3 separate experiments. n = 85 and 85 (for Control and DAPT-treated MCF10A cells, respectively), 90 and 90 (for Control and DAPT-treated MDA-MB-231 cells), 99 and 83 (for Control and DAPT-treated BT20 cells), 80 and 90 (for Control and DAPT-treated MCF7 cells) and 80 and 90 (for Control and DAPT-treated T47D cells, respectively). Data are presented as means ± SEM and are statistically analyzed using Student´s t-test. **p < 0.01.

Functional role of HEY1 in Orai1 and Orai3 expression in breast cancer cells

HEY1 has long been demonstrated as a downstream Notch1 target gene in different cell types, including breast cancer cells³⁸. Using qRT-PCR we have demonstrated that HEY1 mRNA expression in enhanced upon transfection of N1ICD in all the cell lines investigated, with a significant effect in TNBC MDA-MB-231 and BT20 cells and luminal MCF7 cells (Fig. 7; p < 0.05). Furthermore, we have investigated the possible role of HEY1 in Orai1 and Orai3 expression in non-tumoral breast epithelial cells and breast cancer cells of the TNBC and luminal subtypes, focusing on MDA-MB-231 and MCF7 cells, where Notch1 activation significantly enhanced HEY1 transcript (see Fig. 7). Analysis of Orai1 and Orai3 expression in MCF10A, MDA-MB-231 and MCF7 cells transiently expressing HEY1 revealed a reduction in both Orai1 and Orai3 solely in MDA-MB-231 cells (p < 0.0001) while it had no significant effect on the expression of Orai1 and Orai3 in MCF10A and MCF7 cells (Fig. 8). These findings are consistent with the effect of N1ICD expression and Jag-1 stimulation presented in Figs. 1, 2 and 3. HEY1 has been reported to be phosphorylated at Ser-68, which increases HEY1 protein stability but attenuates its ability to enhance the tumor suppressor p53 transcriptional activity³⁹. Hence, we have further assessed whether HEY1 Ser-68 phosphorylation is necessary for the regulation of Orai expression by transfecting cells with the non-phosphorylatable HEY1-S68A or the phosphomimetic HEY1-S68D expression plasmids. As shown in Fig. 8, the Orai1 and Orai3 expression was similar in cells expressing HEY1 or HEY1-S68A, reporting a significant attenuation in Orai1 and Orai3 levels in MDA-MB-231 cells (Fig. 8B; p < 0.0001); by contrast, in MDA-MB-231 cells expressing the phosphomimetic HEY1 mutant, the Orai1 and Orai3 expression was similar to that in cells transfected with empty vector, which strongly suggests that phosphorylation of HEY1 at S68 impairs its functional role in the regulation of Orai1/Orai3 protein content.

Fig. 7.

Role of the expression of N1ICD in the mRNA expression of HEY1 in non-tumoral breast epithelial cells and breast cancer cells. Non-tumoral breast epithelial MCF10A cells, TNBC MDA-MB-231 and BT20 cells, as well as luminal breast cancer MCF7 and T47D cells were transfected with myc-N1ICD fragment or empty vector and 24 h later cells were lysed. Total RNA isolation and RT-qPCR analysis was performed as described in Material and Methods. Values were normalized to GAPDH expression. Bar graphs represent the quantification of HEY1 mRNA expression as fold change over the level in mock-transfected cells (Control) and presented as means ± S.E.M. n = 6 except for MCF7 (n = 3). Data were statistically analyzed using Mann–Whitney U test. *p < 0.05.

Fig. 8.

Role of HEY1 in the expression of Orai1 and Orai3 in non-tumoral breast epithelial cells and breast cancer cells. Non-tumoral breast epithelial MCF10A cells (A), TNBC MDA-MB-231 (B) and luminal breast cancer MCF7 cells (C) were transfected with Flag-HEY1, Flag-HEY1 S68A or Flag-HEY1 S68D expression plasmids or empty vector and 24 h later cells were lysed. Cell lysates were subjected to 10% SDS-PAGE and Western blotting with the anti-Flag, anti-Orai1 or anti-Orai3 antibody, as described in Materials and Methods. Membranes were reprobed with the anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. n = 6–8 separate experiments. Bar graphs represent the quantification of protein expression as fold change over the level in mock-transfected cells (Control) and presented as means ± S.E.M. Data were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn’s test). *p < 0.05.

Discussion

Our findings reveal a differential regulatory role of Notch1 in the protein expression of Orai1 and Orai3 Ca²⁺ channels across non-tumoral breast epithelial cells and different subtypes of breast cancer cells (see supplementary Table 1). Through the expression of the active Notch1 intracellular domain, N1ICD, we demonstrate that Notch1 activity can either suppress or enhance Orai1 and Orai3 expression in a cell type–specific manner, ultimately influencing SOCE.

In non-tumoral MCF10A cells, N1ICD expression did not induce any detectable modification in Orai1 or Orai3 protein levels, although we detected a reduction in STIM1 expression. Similar results were observed after treatment with the Notch ligand Jag-1. However, γ-secretase inhibition with DAPT, at preincubation times that attenuated endogenous N1ICD expression, enhanced Orai3 level and SOCE, but failed to significantly alter Orai1 expression, suggesting that endogenous Notch activity at rest might play a relevant role in the protein content of Orai3. It should be noted that the use of DAPT has certain limitations, such as its lack of specificity for Notch1 over other Notch isoforms and its possible side effects on other γ-secretase-regulated pathways.

The effects of Notch1 activation are more varied in breast cancer cells, which reflect the heterogeneity and complexity of tumor cells. In TNBC MDA-MB-231 cells, a cell line characterized by its high metastatic potential³¹, expression of the active form of Notch1 significantly reduced both Orai1 and Orai3 expression, but not STIM1 protein level, which resulted in no detectable change in SOCE but increased caspase-3 activation, indicating the induction of apoptosis, a pathway that might, at least partially, explain the induction of apoptosis by Notch1^40,41. By contrast, in the TNBC BT20 cell line, which exhibits lower metastatic potential³¹, N1ICD enhanced Orai1 and Orai3 protein levels and increased SOCE. These opposing effects within TNBC subtypes highlight the complexity of Notch1-regulated transcriptional regulation and underscore the heterogeneity of triple-negative breast cancers. On the other hand, in luminal breast cancer cells, N1ICD overexpression had no significant effect on Orai1, Orai3 or STIM1 levels in MCF7 cells, and SOCE as well as intracellular Ca²⁺ release were found to be unaffected by the activation of Notch1 signaling pathway. Similarly, inhibition of Notch1 by treatment with DAPT had not effect on Orai1 or Orai3 expression and did not interfere with MCF7 tumor growth. Conversely, in T47D cells, the active form of Notch1 reduced the expression of both Orai proteins and attenuated SOCE, suggesting a cell line–specific dependency on Notch1 signaling. This is consistent with previously reported phenotypic and metabolic differences between MCF7 and T47D cells, including progesterone receptor expression and glycolytic activity^32,33. These findings are mostly consistent by treatment with the ligand of the Notch1 receptor, Jag-1, or the γ-secretase inhibitor DAPT. The different effects observed upon activation or inhibition of Notch1 signaling in different cell types might be, at least partially, attributed to a different Notch1 basal activity. Thus, activation of Notch1 pathway reduces Orai1 and Orai3 expression in MDA-MB-231 and T47D cells while Notch1 signaling inhibition was without effect on the protein content of these channels in these cell types (except for Orai3 in T47D), which might reflect a low Notch1 activity in resting cells; meanwhile, DAPT enhances Orai3 expression in MCF10A while Notch1 pathway activation was without effect, which might suggest that basal Notch1 represses Orai3 expression, which might be insensitive to further repression when Notch1 pathway is activated. In the present study we have not investigated the role of the Notch1 pathway in Orai2 expression. Given the functional role of Orai2 in SOCE in BT20 and T47D⁴², the in-depth analysis of the regulation of Orai2 expression by Notch1 deserves further studies.

Importantly, our results identify HEY1 as a key downstream effector of Notch1 in regulating Orai channel expression. HEY1 overexpression mirrored the effects of Notch1 pathway activation on Orai1 and Orai3 levels in MCF10A, MDA-MB-231 and MCF7 cells, indicating its functional role downstream Notch1 receptor. Furthermore, the inability of the phosphomimetic HEY1 mutant (S68D) to downregulate Orai expression in MDA-MB-231 cells, in contrast to the wild-type and non-phosphorylatable HEY1-S68A variants, suggests that phosphorylation at Ser-68 inhibits HEY1 transcriptional repression of Orai1 and Orai3. These findings suggest that this post-translational modification might represent a regulatory switch for HEY1 activity in Ca²⁺ signaling as for other intracellular mechanisms³⁹.

In sum, our findings support a model in which Notch1 signaling exerts divergent effects on Orai1 and Orai3 channel expression and SOCE, depending on the cellular context, an observation that reflects the heterogeneity of breast tumors and has significant implications for understanding Ca²⁺ signaling dynamics in breast cancer biology. Notably, the pro-apoptotic effects of the activation of Notch1 signaling pathway in MDA-MB-231 cells via Orai1 downregulation suggest a potential therapeutic strategy that might be exploited in aggressive TNBC subtypes. At least in MDA-MB-231 cells, the underlying mechanism for the regulation of Orai channels by Notch1 involves a transcriptional pathway where Notch1 activation leads to the expression of HEY1, which in turn acts as a direct or indirect transcriptional repressor of the genes encoding Orai1 and Orai3. In other breast cancer cell types the regulation of Orai1 and Orai3 by Notch1 is more complex and might involve alternative pathways. Given the important functional role of Orai1 and SOCE in the development of breast cancer hallmarks, including proliferation, migration and metastasis, the analysis of the Notch1-HEY1-Orai axis might provide insights that may ultimately contribute to the development of subtype-specific therapeutic strategies targeting Notch-Orai signaling in breast cancer.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

J.A.R., I.J., J.J.L., T.S. conceptualization; J.N.-F., A.M.-D., A.G., P.C.R., J.J.L., V.J.-V., S.A. investigation; J.N.-F., A.M.-D., V.J.-V., A.G., P.C.R., S.A. methodology; J.N.-F., A.M.-D. A.G. formal analysis; J.A.R., I.J., J.J.L., T.S. supervision; J.A.R. writing—original draft; J.A.R., I.J., J.J.L., T.S. Writing—review and editing. J.A.R., T.S. funding acquisition; J.A.R., T.S. project administration. All the authors approved the final edited version. The degree of contribution to the design, to performance of the experiments, and to manuscript writing were used to assign authorship order among co-first authors.

Funding

This research was supported by grants PID2022-136279NB-C21 and PID2022-136279NB-C22 from MCIN/AEI/10.13039/501100011033 and by the European Regional Development Fund (ERDF) under the initiative ‘A way of making Europe,’ as well as by the Junta de Extremadura-Fondo Europeo de Desarrollo Regional (FEDER; Grant IB20007 and GR24034) awarded to JAR and TS. JN-F, AM-D, and SA are supported by contracts from the AEI/Spanish Ministry of Science and Innovation, while VJ-F is supported by a contract from the Junta de Extremadura.

Data availability

All data necessary to verify the conclusions presented in this manuscript are included within the paper and its Supplementary Materials. All data are accessible and will be made available for collaborative sharing upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

Experimental procedures were conducted in accordance with institutional guidelines and were approved by the Ethics Committee of the University of Extremadura and the Extremadura Health Service.