Transcriptional regulation of Warburg effect in 3D cultured breast cancer cells by Orai1

Ph.D.Cancer stem cells (CSCs) are a subpopulation of tumor cells that have high tumorigenic ability in severe combined immunodeficiency (SCID) mice, meaning that a very small number of CSCs are capable of generating solid tumors. CSCs usually contribute to relapse and metastasis after chemotherapy or radiotherapy, which represents a therapeutic challenge. Selection of CSCs is sometimes based on conventional stem cell markers. However, this selection method is often unreliable. Here, I used a soft fibrin gel-cultured system to select cancer stem cells. Compared to other 3D culture systems, such as collagen scaffold, or stem cell marker-based method, soft fibrin gel selected CSCs are of higher tumorigenicity. Reports indicated that only 10 of such selected melanoma cancer stem cells were enough to induce tumor growth and metastasis in vivo.Breast cancer is the most common cancer in women and the second most common cancer overall. The treatment strategies for breast cancer include surgery, chemotherapy, radiotherapy and hormonal therapy. However, many treated patients experience recurrence and metastasis, which is mainly due to the presence of breast cancer stem cells (BCSCs).Ca²⁺ is an important second messenger in tumor growth and metastasis. Orai1 is a store operated calcium channel. It is well documented that Orai1 can interact with an ER protein STIM1 to regulate Ca²⁺ entry through a store-operated mechanism. In this project, I found that the Ca2+-selective channel Orai1 plays a crucial role in the growth of BCSCs both in vitro and in vivo. Orai1 mRNA and protein expression levels were significantly higher in fibrin gel selected MCF7 cells compared with 2D cultured MCF7 cells. Knockdown of Orai1 dramatically reduced the size of spheroids derived from MCF7 cells grown in fibrin gel. Therefore, soft fibrin gel selected MCF7 with Orai1 knockdown have a delayed tumor formation and smaller tumor size compared to those without Orai1 knockdown.RNA sequencing results between 2D cultured- and 3D cultured-MCF7 cells were also compared. KEGG pathway enrichment analysis showed that fibrin gel selection of MCF-7 cells significantly altered glycolysis pathway. Interestingly, I also found that knockdown of Orai1 downregulated glycolytic genes in fibrin gel selected MCF7 CSCs but had no effect on 2D cultured MCF7 cells. Moreover, current results suggested that Orai1 may impact spheroid size through a Ca²⁺-regulated transcriptional factor NFATc2. NFATc2 translocated to nucleus in 3D cultured MCF7 cells, which was blocked as a result of Orai1 deficiency.It is well documented that Orai1 is a store operated calcium channel. However, I found that, in MCF7 cells, Orai3 but not Orai1 was responsible for store operated calcium entry. Instead, Orai1 was responsible for store independent calcium entry in MCF7 cells. Therefore, I hypothesized that Orai1 may regulate spheroid growth and glycolytic pathway through a store independent manner. Further studies showed that a Golgi protein SPCA2 had similar effect on spheroid size and glycolytic genes as Orai1 did. The N-terminus of SPCA2 physiologically binds to Orai1, activating the downstream Ca2+ signaling. I found that introduction of SPCA2 N-terminus, which interrupted endogenous Orai1-SPCA2 interaction, greatly attenuated MCF7 spheroid size. Moreover, I found that Orai1-SPCA2 interaction was stronger in MCF7 cells under hypoxic conditions. The present results also demonstrated that the core regions of spheroids were more hypoxic than peripheral areas and Orai1 was accumulated in the core regions.Taken together, the present study demonstrated important functional roles of Orai1 and SPCA2 in determining spheroid size in MCF7 cells grown in fibrin gel, and in interfering tumor growth in athymic SCID mice. It is likely that the action of Orai1- SPCA2 was augmented in MCF7 spheroids, in turn, enhancing glycolysis through NFATc2 and HIF1A to regulate tumor growth. These findings suggested a critical role of Orai1-SPCA2 pathway in BCSC reprogramming. Orai1-SPCA2 pathway may provide a novel therapeutic target for the treatment of breast malignancy.癌症干细胞指的是在重症联合免疫缺陷小鼠（ Severe CombinedImmunodeficiency Mice, SCID）中具有高致瘤能力的肿瘤细胞亚群，只需要极少数的癌症干细胞就能够产生实体瘤。它通常是化学疗法或放射疗法后导致复发和转移的主要原因，这也为癌症的治疗带来了巨大的挑战。传统癌症干细胞的筛选方法主要基于常规干细胞标记物法，然而这种筛选方法并不是很可靠。在本课题的研究中，我使用了一种软纤维蛋白凝胶培养系统来筛选癌症干细胞。与干细胞标记物法或者其他3D 培养系统法（例如胶原蛋白支架）相比，软纤维蛋白凝胶筛选的癌症干细胞具有更高的致瘤性。目前最新的研究表明，软纤维蛋白凝胶筛选的黑素瘤癌干细胞只需10 个便足以在小鼠体内诱导肿瘤生长以及转移。乳腺癌是全球女性中最常见的癌症，也是所有人群中第二常见的癌症。乳腺癌的治疗策略包括手术，化学疗法，放射疗法和激素疗法等。然而，由于乳腺癌干细胞的存在使得许多接受治疗的患者仍会出现复发和转移。钙离子是肿瘤生长和转移的重要第二信使，它几乎参与了生物体内细胞生命活动的方方面面。Orai1 是一个非常重要的钙离子库调节通道。研究表明，Orai1 可以与内质网膜蛋白STIM1 相互作用，通过内质网钙库耗竭机制调节钙离子的内流。在本课题的研究中，我发现钙离子通道Orai1 在体外和体内对乳腺癌干细胞的生长中都起着至关重要的作用。首先，与2D 培养的MCF7 细胞相比，在软纤维蛋白凝胶筛选（3D）的MCF7 细胞中，Orai1 的mRNA 水平和蛋白表达水平都明显更高。在Orai1 基因敲低之后，软纤维蛋白凝胶中培养的MCF7 细胞会显著降低球体的大小。总的来说，与对照组细胞相比，软纤维蛋白凝胶筛选的Orai1 敲低的MCF7 细胞，其诱发肿瘤的能力较弱，形成的肿瘤较小。接着，我在2D和3D培养的MCF7细胞中进行高通量转录组测序，其中KEGG 富集通路分析结果表明，与2D 培养条件相比，软纤维蛋白凝胶筛选的MCF7 细胞最显著上调的通路是糖酵解信号通路。进一步的实验证明，当Orai1基因敲低之后，软纤维蛋白凝胶筛选的MCF7细胞的糖酵解基因的mRNA水平发生显著降低，而2D条件培养的MCF7细胞中的糖酵解基因并没有影响。此外，本研究还发现Orai1 可能通过钙离子调控的转录因子NFATc2 影响球体的大小。NFATc2 在3D 培养的MCF7 细胞中定位于细胞核中，而NFATc2 的核定位由于Orai1的敲低而被阻断在细胞质中。众所周知，Orai1 是钙库调节的钙离子通道。然而我们发现，在MCF7细胞中，Orai3才是负责钙库调节的钙离子内流，而Orai1在MCF7细胞中负责的钙离子内流与钙库无关。因此，我推测Orai1 可能通过独立于钙离子库的方式来调节软纤维蛋白凝胶筛选的MCF7 细胞的成球以及相应糖酵解通路的开启。进一步的研究表明，高尔基蛋白SPCA2 对球体大小和糖酵解基因的作用与Orai1 相似。SPCA2 的N 端与Orai1 结合，从而激活下游钙离子信号的传导。我发现通过过表达SPCA2 的N 端突变体会破坏体内Orai1-SPCA2 之间的相互作用，并且大大减弱MCF7 细胞在软纤维蛋白凝胶中产生的球体大小。此外，我发现在缺氧条件下，MCF7 细胞中的Orai1-SPCA2 的相互作用似乎更强。本研究还表明，球体的核心区域比周围区域缺氧程度更高，并且Orai1 大量聚集在球体的核心区域。综上所述，本研究阐述了Orai1-SPCA2 通路在软纤维蛋白凝胶筛选的MCF7 细胞的生长以及在重症联合免疫缺陷小鼠中肿瘤形成的重要作用。在MCF7球体中可能通过增强Orai1-SPCA2之间的相互作用，从而介导了NFATc2和HIF1A 对下游糖酵解基因的开启，最终影响了乳腺肿瘤的发生。本研究揭示了Orai1-SPCA2 通路在乳腺癌干细胞代谢中的关键作用， 从而为乳腺恶性肿瘤的治疗提供新的治疗目标。Zhuo, Duan.Thesis Ph.D. Chinese University of Hong Kong 2020.Includes bibliographical references (leaves 93-110).Abstracts also in Chinese.Title from PDF title page (viewed on February 10, 2022)