Christensen Anne G, Ehmsen Sidse, Terp Mikkel G, Batra Richa, Alcaraz Nicolas, Baumbach Jan, Noer Julie B, Moreira José, Leth-Larsen Rikke, Larsen Martin R, Ditzel Henrik J
Department of Cancer and Inflammation Research, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark.
Department of Mathematics and Computer Science, Faculty of Science, University of Southern Denmark, Odense, Denmark.
Stem Cells. 2017 Aug;35(8):1898-1912. doi: 10.1002/stem.2654. Epub 2017 Jun 23.
A limited number of cancer cells within a tumor are thought to have self-renewing and tumor-initiating capabilities that produce the remaining cancer cells in a heterogeneous tumor mass. Elucidation of central pathways preferentially used by tumor-initiating cells/cancer stem cells (CSCs) may allow their exploitation as potential cancer therapy targets. We used single cell cloning to isolate and characterize four isogenic cell clones from a triple-negative breast cancer cell line; two exhibited mesenchymal-like and two epithelial-like characteristics. Within these pairs, one, but not the other, resulted in tumors in immunodeficient NOD/Shi-scid/IL-2 Rγ null mice and efficiently formed mammospheres. Quantitative proteomics and phosphoproteomics were used to map signaling pathways associated with the tumor-initiating ability. Signaling associated with apoptosis was suppressed in tumor-initiating versus nontumorigenic counterparts with pro-apoptotic proteins, such as Bcl2-associated agonist of cell death (BAD), FAS-associated death domain protein (FADD), and myeloid differentiation primary response protein (MYD88), downregulated in tumor-initiating epithelial-like cells. Functional studies confirmed significantly lower apoptosis in tumor-initiating versus nontumorigenic cells. Moreover, central pathways, including β-catenin and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-related signaling, exhibited increased activation in the tumor-initiating cells. To evaluate the CSC model as a tool for drug screening, we assessed the effect of separately blocking NF-κB and Wnt/β-catenin signaling and found markedly reduced mammosphere formation, particularly for tumor-initiating cells. Similar reduction was also observed using patient-derived primary cancer cells. Furthermore, blocking NF-κB signaling in mice transplanted with tumor-initiating cells significantly reduced tumor outgrowth. Our study demonstrates that suppressed apoptosis, activation of pathways associated with cell viability, and CSCs are the major differences between tumor-initiating and nontumorigenic cells independent of their epithelial-like/mesenchymal-like phenotype. These altered pathways may provide targets for future drug development to eliminate CSCs, and the cell model may be a useful tool in such drug screenings. Stem Cells 2017;35:1898-1912.
肿瘤内数量有限的癌细胞被认为具有自我更新和启动肿瘤的能力，可产生异质性肿瘤块中的其余癌细胞。阐明肿瘤起始细胞/癌症干细胞（CSCs）优先使用的核心通路，可能有助于将其作为潜在的癌症治疗靶点加以利用。我们使用单细胞克隆从三阴性乳腺癌细胞系中分离并鉴定了四个同基因细胞克隆；其中两个表现出间充质样特征，两个表现出上皮样特征。在这些配对中，其中一对而非另一对在免疫缺陷的NOD/Shi-scid/IL-2Rγnull小鼠中形成肿瘤，并能高效形成乳腺球。定量蛋白质组学和磷酸化蛋白质组学被用于绘制与肿瘤起始能力相关的信号通路。与凋亡相关的信号在肿瘤起始细胞与非致瘤性对应细胞中受到抑制，促凋亡蛋白如细胞死亡的Bcl2相关激动剂（BAD）、FAS相关死亡结构域蛋白（FADD）和髓样分化初级反应蛋白（MYD88）在肿瘤起始上皮样细胞中下调。功能研究证实，肿瘤起始细胞与非致瘤性细胞相比凋亡明显减少。此外，包括β-连环蛋白和活化B细胞的核因子κ轻链增强子（NF-κB）相关信号在内的核心通路在肿瘤起始细胞中表现出增强的激活。为了评估CSC模型作为药物筛选工具的作用，我们评估了分别阻断NF-κB和Wnt/β-连环蛋白信号的效果，发现乳腺球形成明显减少，尤其是对于肿瘤起始细胞。使用患者来源的原发性癌细胞也观察到类似的减少。此外，在移植了肿瘤起始细胞的小鼠中阻断NF-κB信号显著减少了肿瘤生长。我们的研究表明，凋亡受抑制、与细胞活力相关通路的激活以及CSCs是肿瘤起始细胞与非致瘤性细胞之间的主要差异，与它们的上皮样/间充质样表型无关。这些改变的通路可能为未来消除CSCs的药物开发提供靶点，并且该细胞模型可能是此类药物筛选中的有用工具。《干细胞》2017年；35:1898 - 1912。
