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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.
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.
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.
摘要：
背景：乳腺肿瘤治疗的主要挑战之一是乳腺癌干细胞（BCSC）的存在。BCSC是表现出干细胞特征的小肿瘤细胞亚群。BCSC负责进展，复发,乳腺癌的化疗耐药和转移。Ca2+信号传导在癌症发展的不同过程中起重要作用。然而,Ca2+信号在BCSC中的作用尚不清楚。
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.
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.
