Metabolism & Cancer Group; Translational Research Laboratory; Catalan Institute of Oncology-Girona (ICO-Girona); Girona, Spain; Molecular Oncology; Girona Biomedical Research Institute (IDIBGI); Girona, Spain.
Cell Cycle. 2013 Sep 15;12(18):3109-24. doi: 10.4161/cc.26173. Epub 2013 Aug 21.
Energy metabolism plasticity enables stemness programs during the reprogramming of somatic cells to an induced pluripotent stem cell (iPSC) state. This relationship may introduce a new era in the understanding of Warburg's theory on the metabolic origin of cancer at the level of cancer stem cells (CSCs). Here, we used Yamanaka's stem cell technology in an attempt to create stable CSC research lines in which to dissect the transcriptional control of mTOR--the master switch of cellular catabolism and anabolism--in CSC-like states. The rare colonies with iPSC-like morphology, obtained following the viral transduction of the Oct4, Sox2, Klf4, and c-Myc (OSKM) stemness factors into MCF-7 luminal-like breast cancer cells (MCF-7/Rep), demonstrated an intermediate state between cancer cells and bona fide iPSCs. MCF-7/Rep cells notably overexpressed SOX2 and stage-specific embryonic antigen (SSEA)-4 proteins; however, other stemness-related markers (OCT4, NANOG, SSEA-1, TRA-1-60, and TRA-1-81) were found at low to moderate levels. The transcriptional analyses of OSKM factors confirmed the strong but unique reactivation of the endogenous Sox2 stemness gene accompanied by the silencing of the exogenous Sox2 transgene in MCF-7/Rep cells. Some but not all MCF-7/Rep cells acquired strong alkaline phosphatase (AP) activity compared with MCF-7 parental cells. SOX2-overexpressing MCF-7/Rep cells contained drastically higher percentages of CD44(+) and ALDEFLUOR-stained ALDH(bright) cells than MCF-7 parental cells. The overlap between differentially expressed mTOR signaling-related genes in 3 different SOX2-overexpressing CSC-like cell lines revealed a notable downregulation of 3 genes, PRKAA1 (which codes for the catalytic α 1 subunit of AMPK), DDIT4/REDD1 (a stress response gene that operates as a negative regulator of mTOR), and DEPTOR (a naturally occurring endogenous inhibitor of mTOR activity). The insulin-receptor gene (INSR) was differentially upregulated in MCF-7/Rep cells. Consistent with the downregulation of AMPK expression, immunoblotting procedures confirmed upregulation of p70S6K and increased phosphorylation of mTOR in Sox2-overexpressing CSC-like cell populations. Using an in vitro model of the de novo generation of CSC-like states through the nuclear reprogramming of an established breast cancer cell line, we reveal that the transcriptional suppression of mTOR repressors is an intrinsic process occurring during the acquisition of CSC-like properties by differentiated populations of luminal-like breast cancer cells. This approach may provide a new path for obtaining information about preventing the appearance of CSCs through the modulation of the AMPK/mTOR pathway.
能量代谢可塑性使体细胞在重编程为诱导多能干细胞（iPSC）状态时的干性程序成为可能。这种关系可能会在理解癌症干细胞（CSC）水平上沃伯格（Warburg）关于癌症代谢起源的理论方面引入一个新的时代。在这里，我们使用山中伸弥（Yamanaka）的干细胞技术，试图在其中创建稳定的 CSC 研究线，以剖析 mTOR 的转录控制-细胞分解代谢和合成代谢的主开关-在 CSC 样状态下。通过病毒转导将 Oct4、Sox2、Klf4 和 c-Myc（OSKM）干性因子转入 MCF-7 腔上皮样乳腺癌细胞（MCF-7/Rep）后，获得了具有 iPSC 样形态的稀有菌落，这些菌落表现出癌细胞与真正的 iPSC 之间的中间状态。MCF-7/Rep 细胞显著过表达 SOX2 和阶段特异性胚胎抗原（SSEA）-4 蛋白；然而，其他与干性相关的标记物（OCT4、NANOG、SSEA-1、TRA-1-60 和 TRA-1-81）的水平较低。OSKM 因子的转录分析证实了内源 Sox2 干性基因的强烈但独特的再激活，同时伴随着 MCF-7/Rep 细胞中外源 Sox2 转基因的沉默。与 MCF-7 亲本细胞相比，一些但不是所有 MCF-7/Rep 细胞均获得了较强的碱性磷酸酶（AP）活性。与 MCF-7 亲本细胞相比，过表达 SOX2 的 MCF-7/Rep 细胞中 CD44（+）和 ALDEFLUOR 染色的 ALDH（bright）细胞的比例明显更高。在 3 种不同的 SOX2 过表达 CSC 样细胞系中差异表达的 mTOR 信号相关基因的重叠表明，有 3 个基因的表达明显下调，PRKAA1（编码 AMPK 的催化α1亚基），DDIT4/REDD1（一种应激反应基因，作为 mTOR 的负调节剂）和 DEPTOR（一种天然存在的 mTOR 活性内源性抑制剂）。胰岛素受体基因（INSR）在 MCF-7/Rep 细胞中差异上调。与 AMPK 表达下调一致，免疫印迹程序证实，在 SOX2 过表达的 CSC 样细胞群中，p70S6K 的表达上调和 mTOR 的磷酸化增加。通过核重编程建立的乳腺癌细胞系，我们在体外建立了 CSC 样状态的从头生成模型，揭示了 mTOR 抑制剂的转录抑制是在腔上皮样乳腺癌细胞分化群体获得 CSC 样特性时发生的内在过程。这种方法可能为通过调节 AMPK/mTOR 途径获得有关预防 CSC 出现的信息提供新途径。
