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

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.