Reductive carboxylation supports redox homeostasis during anchorage-independent growth

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From: Nature(Vol. 532, Issue 7598)
Publisher: Nature Publishing Group
Document Type: Report
Length: 5,001 words
Lexile Measure: 1440L

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Cells receive growth and survival stimuli through their attachment to an extracellular matrix (ECM) (1). Overcoming the addiction to ECM-induced signals is required for anchorage-independent growth, a property of most malignant cells (2). Detachment from ECM is associated with enhanced production of reactive oxygen species (ROS) owing to altered glucose metabolism (2). Here we identify an unconventional pathway that supports redox homeostasis and growth during adaptation to anchorage independence. We observed that detachment from monolayer culture and growth as anchorage-independent tumour spheroids was accompanied by changes in both glucose and glutamine metabolism. Specifically, oxidation of both nutrients was suppressed in spheroids, whereas reductive formation of citrate from glutamine was enhanced. Reductive glutamine metabolism was highly dependent on cytosolic isocitrate dehydrogenase-1 (IDH1), because the activity was suppressed in cells homozygous null for IDH1 or treated with an IDH1 inhibitor. This activity occurred in absence of hypoxia, a wellknown inducer of reductive metabolism. Rather, IDH1 mitigated mitochondrial ROS in spheroids, and suppressing IDH1 reduced spheroid growth through a mechanism requiring mitochondrial ROS. Isotope tracing revealed that in spheroids, isocitrate/citrate produced reductively in the cytosol could enter the mitochondria and participate in oxidative metabolism, including oxidation by IDH2. This generates NADPH in the mitochondria, enabling cells to mitigate mitochondrial ROS and maximize growth. Neither IDH1 nor IDH2 was necessary for monolayer growth, but deleting either one enhanced mitochondrial ROS and reduced spheroid size, as did deletion of the mitochondrial citrate transporter protein. Together, the data indicate that adaptation to anchorage independence requires a fundamental change in citrate metabolism, initiated by IDH1-dependent reductive carboxylation and culminating in suppression of mitochondrial ROS.

In monolayer cultures, growth factors direct cells to take up glucose and glutamine and use them to produce macromolecules. Both nutrients are used to produce the lipogenic precursor citrate (Extended Data Fig. 1a). To identify metabolic alterations during anchorage independence, H460 lung cancer cells were detached from monolayers and aggregated into spheroids. Cells within spheroids proliferated at a reduced rate (Extended Data Fig. 2a). Although growth in both conditions required glucose and glutamine (Extended Data Fig. 2b), spheroids consumed less of both and secreted less lactate, glutamate and ammonia (Extended Data Fig. 2c, d). The ratio of ammonia released to glutamine consumed was comparable between conditions (Extended Data Fig. 2d). Spheroids displayed reduced entry of glucose-derived carbon into citrate (Fig. 1a) and consumed less oxygen per cell (Fig. 1b). These findings implied reduced pyruvate dehydrogenase (PDH) activity, as demonstrated previously during matrix detachment (3). Indeed, inhibitory PDH phosphorylation and expression of PDH kinase-1 (PDK1) were elevated in spheroids (Fig. 1c). Citrate labelling from [U-[sup.13]C]glutamine persisted in spheroids, but the [sup.13]C distribution was altered, particularly in that the m+5 fraction (the fraction containing five [sup.13]C nuclei) exceeded m+4 (Fig. 1d). This persisted when cells were disaggregated and permitted to reform spheroids (Extended Data Fig. 2e). The m+5 fraction appeared rapidly and endured as the most prominent labelled form (Fig. 1e), regardless of the type of culture medium (Supplementary Table 1; this Table contains all...

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Gale Document Number: GALE|A449661961