School of Biomedical Sciences and Pharmacy, The University of Newcastle, NSW, 2308, Australia.
Translational Research Institute, Henan Provincial People's Hospital and People's Hospital of Zhengzhou University, Henan Provincial and Zhengzhou City Key laboratory of Long Non-coding RNA and Cancer Metabolism, Henan, 450053, China.
Theranostics. 2021 Sep 21;11(19):9605-9622. doi: 10.7150/thno.63763. eCollection 2021.
Recurrent and metastatic cancers often undergo a period of dormancy, which is closely associated with cellular quiescence, a state whereby cells exit the cell cycle and are reversibly arrested in G0 phase. Curative cancer treatment thus requires therapies that either sustain the dormant state of quiescent cancer cells, or preferentially, eliminate them. However, the mechanisms responsible for the survival of quiescent cancer cells remain obscure. Dual genome-editing was carried out using a CRISPR/Cas9-based system to label endogenous p27 and Ki67 with the green and red fluorescent proteins EGFP and mCherry, respectively, in melanoma cells. Analysis of transcriptomes of isolated EGFP-p27mCherry-Ki67 quiescent cells was conducted at bulk and single cell levels using RNA-sequencing. The extracellular acidification rate and oxygen consumption rate were measured to define metabolic phenotypes. SiRNA and inducible shRNA knockdown, chromatin immunoprecipitation and luciferase reporter assays were employed to elucidate mechanisms of the metabolic switch in quiescent cells. Dual labelling of endogenous p27 and Ki67 with differentiable fluorescent probes allowed for visualization, isolation, and analysis of viable p27Ki67 quiescent cells. Paradoxically, the proto-oncoprotein c-Myc, which commonly drives malignant cell cycle progression, was expressed at relatively high levels in p27Ki67 quiescent cells and supported their survival through promoting mitochondrial oxidative phosphorylation (OXPHOS). In this context, c-Myc selectively transactivated genes encoding OXPHOS enzymes, including subunits of isocitric dehydrogenase 3 (IDH3), whereas its binding to cell cycle progression gene promoters was decreased in quiescent cells. Silencing of c-Myc or the catalytic subunit of IDH3, IDH3α, preferentially killed quiescent cells, recapitulating the effect of treatment with OXPHOS inhibitors. These results establish a rigorous experimental system for investigating cellular quiescence, uncover the high selectivity of c-Myc in activating OXPHOS genes in quiescent cells, and propose OXPHOS targeting as a potential therapeutic avenue to counter cancer cells in quiescence.
复发性和转移性癌症经常经历一段休眠期，这与细胞静止密切相关，细胞静止是指细胞退出细胞周期并可逆地停留在 G0 期。因此，治愈癌症的治疗需要维持静止癌细胞休眠状态的治疗方法，或者更优选地，消除它们。然而，负责静止癌细胞存活的机制仍然不清楚。
使用基于 CRISPR/Cas9 的系统进行双基因组编辑，用绿色和红色荧光蛋白 EGFP 和 mCherry 分别标记黑色素瘤细胞内源性 p27 和 Ki67。使用 RNA 测序在批量和单细胞水平上分析分离的 EGFP-p27mCherry-Ki67 静止细胞的转录组。测量细胞外酸化率和耗氧量来定义代谢表型。使用 siRNA 和诱导性 shRNA 敲低、染色质免疫沉淀和荧光素酶报告基因测定来阐明静止细胞代谢转换的机制。
用可区分的荧光探针对内源性 p27 和 Ki67 进行双重标记，允许可视化、分离和分析有活力的 p27Ki67 静止细胞。矛盾的是，原癌蛋白 c-Myc 通常驱动恶性细胞周期进展，在 p27Ki67 静止细胞中表达水平相对较高，并通过促进线粒体氧化磷酸化 (OXPHOS) 来支持其存活。在这种情况下，c-Myc 选择性地转录激活编码 OXPHOS 酶的基因，包括异柠檬酸脱氢酶 3 (IDH3) 的亚基，而其与细胞周期进展基因启动子的结合在静止细胞中减少。沉默 c-Myc 或 IDH3 的催化亚基 IDH3α，优先杀死静止细胞，再现 OXPHOS 抑制剂治疗的效果。
这些结果建立了一个严格的实验系统来研究细胞静止，揭示了 c-Myc 在激活静止细胞中的 OXPHOS 基因方面的高选择性，并提出靶向 OXPHOS 作为对抗静止癌细胞的潜在治疗途径。
