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The transition from proliferation to quiescence in glioblastoma stem-like cells requires Ca2+ signaling and mitochondria remodeling

ABSTRACT: Quiescence is a reversible cell-cycle arrest used by Cancer Stem Cells (CSCs) to evade killing following conventional therapies. Quiescent CSCs are therefore one of the main cause of cancer recurrence. In glioblastoma, the most common and aggressive primary brain tumors, the quiescent glioblastoma stem-like cells (GSCs) are localized in hypoxic and acidic microenvironments and microenvironmental changes can control cell cycle re-entering of the GSCs. Here, we show that proliferating GSCs isolated from patients can be induced and maintained in a quiescent state by lowering the extracellular pH. Through RNA-seq analysis we characterized the RNA signatures of quiescent versus proliferating GSCs. We identified genes involved in the control of Ca2+ signaling and differentially expressed between the two states. Using the bioluminescent Ca2+ reporter EGFP-aequorin targeted to the mitochondria or the cytosol we explored the changes in Ca2+ homeostasis occurring during the switch from proliferation to quiescence. This remodeling is controlled through store-operated channels (SOCs). SOCs play a causal role since the inhibition of the Ca2+ influx through SOC drives proliferating GSCs to quiescence. We showed that the switch to quiescence is characterized by both an increased capacity of GSCs’ mitochondria to capture Ca2+ and a dramatic and reversible change of mitochondrial morphology from a tubular to a donut shape. Our data suggest that the remodeling of the Ca2+ homeostasis and the reshaping of mitochondria during the transition from proliferation to quiescence constitute a protective mechanism that favors cancer stem-like cells’ survival and their aggressiveness in glioblastoma.

ORGANISM(S): Homo sapiens

PROVIDER: GSE93991 | GEO | 2018/05/31

REPOSITORIES: GEO

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Project description:Background: Although quiescence—reversible cell-cycle arrest—is a key part in the life history and fate of many mammalian cell types, the mechanisms of gene regulation in quiescent cells are poorly understood. We sought to clarify the role of microRNAs as regulators of the cellular functions of quiescent human fibroblasts. Results: Using microarrays, we discovered that the expression of the majority of profiled microRNAs differed between proliferating and quiescent fibroblasts. Fibroblasts induced into quiescence by contact inhibition or serum starvation had similar microRNA profiles, indicating common changes induced by distinct quiescence signals. By analyzing the gene expression patterns of microRNA target genes with quiescence, we discovered a strong regulatory function for miR-29, which is downregulated with quiescence. Using microarrays and immunoblotting, we confirmed that miR-29 targets genes encoding collagen and other extracellular matrix proteins and that those target genes are induced in quiescence. In addition, overexpression of miR-29 resulted in more rapid cell cycle re-entry from quiescence. We also found that let-7 and miR-125 were upregulated in quiescent cells. Overexpression of either one alone resulted in slower cell cycle re-entry from quiescence, while the combination of both together slowed cell cycle re-entry even further. Conclusions: microRNAs regulate key aspects of fibroblast quiescence including the proliferative state of the cells as well as their gene expression profiles, in particular, the induction of extracellular matrix proteins in quiescent fibroblasts. microRNAs in human neonatal dermal fibroblasts in 3 cell cycle conditions (proliferating, 4 days serum starved, 7 days contact inhibited) were analyzed by one color microarray. 3 separate fibroblast cell lines from different human donors were analyzed for each condition, with two separate labeling and hybridization samples for each cell line. In total, 18 samples were hybridized to the microRNA microarray.

Project description:Many cells in mammals exist in the state of quiescence, which is characterized by reversible exit from the cell cycle. Quiescent cells are widely reported to exhibit reduced size, nucleotide synthesis, and metabolic activity. Much lower glycolytic rates have been reported in quiescent compared with proliferating lymphocytes. In contrast, we show here that primary human fibroblasts continue to exhibit high metabolic rates when induced into quiescence via contact inhibition. By monitoring isotope labeling through metabolic pathways and quantitatively identifying fluxes from the data, we show that contact-inhibited fibroblasts utilize glucose in all branches of central carbon metabolism at rates similar to those of proliferating cells, with greater overflow flux from the pentose phosphate pathway back to glycolysis. Inhibition of the pentose phosphate pathway resulted in apoptosis preferentially in quiescent fibroblasts. By feeding the cells labeled glutamine, we also detected a “backwards” flux in the tricarboxylic acid cycle from α-ketoglutarate to citrate that was enhanced in contact-inhibited fibroblasts; this flux likely contributes to shuttling of NADPH from the mitochondrion to cytosol for redox defense or fatty acid synthesis. The high metabolic activity of the fibroblasts was directed in part toward breakdown and resynthesis of protein and lipid, and in part toward excretion of extracellular matrix proteins. Thus, reduced metabolic activity is not a hallmark of the quiescent state. Quiescent fibroblasts, relieved of the biosynthetic requirements associated with generating progeny, direct their metabolic activity to preservation of self integrity and alternative functions beneficial to the organism as a whole. mRNAs were analyzed by two color microarray from two separate human neonatal dermal fibroblasts cell lines in proliferating, 7 days contact inhibition, or 14 days contact inhibition. Contact inhibited samples were co-hybridized to proliferating samples as a control, while an additional array co-hybridized the two proliferating samples to analyze reproducibility.

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The transition from proliferation to quiescence in glioblastoma stem-like cells requires Ca2+ signaling and mitochondria remodeling

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