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.
CONCLUSIONS: 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.
方法:使用高效的3D软纤维蛋白凝胶系统来富集ER乳腺癌细胞系MCF7和MDA-MB-415的BCSC样细胞。然后,我们研究了两个Ca2可渗透离子通道Orai1和Orai3在体外BCSC样细胞的生长和干性中的作用,和在雌性NOD/SCID小鼠体内的致瘤性。
结果:Orai1RNA沉默和药物抑制降低了肿瘤球体中BCSC样细胞的生长,降低BCSC标志物的表达水平,并减少NOD/SCID小鼠中肿瘤异种移植物的生长。Orai3RNA沉默在体外对BCSC样细胞的生长和干性也有类似的抑制作用,和体内肿瘤异种移植生长。机械上,Orai1和SPCA2介导存储操作的Ca2输入。敲除Orai1或SPCA2抑制糖酵解途径,而Orai3或STIM1的敲除对糖酵解没有影响。
结论:我们发现Orai1与SPCA2相互作用,以介导与存储无关的Ca2进入,随后通过糖酵解途径促进BCSC样细胞的生长和致瘤性。相比之下,Orai3和STIM1调解商店操作的Ca2+进入,通过糖酵解非依赖性途径促进BCSC样细胞的生长和致瘤性。一起,我们的研究揭示了一种精心策划的机制,通过该机制,两种Ca2+进入途径通过不同的信号轴作用,精细地控制BCSC的生长和致瘤性.