Rationale: 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. Methods: 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-p27highmCherry-Ki67low 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. Results: Dual labelling of endogenous p27 and Ki67 with differentiable
fluorescent probes allowed for visualization, isolation, and analysis of viable p27highKi67low quiescent cells. Paradoxically, the proto-
oncoprotein c-Myc, which commonly drives malignant cell cycle progression, was expressed at relatively high levels in p27highKi67low 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. Conclusion: 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.