Orai1 and Orai3 act through distinct signalling axes to promote stemness and tumorigenicity of breast cancer stem cells

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. Ca²⁺ signalling plays an important role in diverse processes in cancer development. However, the role of Ca²⁺ 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 Ca²⁺-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 Ca²⁺ 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 Ca²⁺ entry, subsequently promoting the growth and tumorigenicity of BCSC-like cells via glycolysis pathway. In contrast, Orai3 and STIM1 mediate store-operated Ca²⁺ 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 Ca²⁺ entry pathways act through distinct signalling axes to finely control the growth and tumorigenicity of BCSCs.

Keywords: Breast cancer stem cells; Glycolysis; Orai1; Orai3.

Fig. 1

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.

Fig. 2

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

Fig. 3

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

Fig. 4

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

Fig. 5

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 10³ 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 10³ 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

Fig. 6

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

Fig. 7

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

Fig. 8

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