title: Reductive carboxylation supports redox homeostasis during anchorage-independent growth.
abstract: Cells receive growth and survival stimuli through their attachment to an extracellular matrix (ECM). Overcoming the addiction to ECM-induced signals is required for anchorage-independent growth, a property of most malignant cells. Detachment from ECM is associated with enhanced production of reactive oxygen species (ROS) owing to altered glucose metabolism. 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 well-known 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.
authors.affiliation: Children's Medical Center Research Institute, UT Southwestern Medical Center, Dallas, Texas 75390-8502, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Jiang
authors.affiliation: Department of Radiology, University of Pennsylvania School of Medicine, 3620 Hamilton Walk, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Shestov
authors.affiliation: Seahorse Bioscience, 16 Esquire Road, North Billerica, Massachusetts 01862, USA.
authors.forename: Pamela
authors.initials: P
authors.lastname: Swain
authors.affiliation: Children's Medical Center Research Institute, UT Southwestern Medical Center, Dallas, Texas 75390-8502, USA.
authors.forename: Chendong
authors.initials: C
authors.lastname: Yang
authors.affiliation: Department of Bioengineering, University of California, San Diego, La Jolla, California 92093, USA.
authors.forename: Seth J
authors.initials: SJ
authors.lastname: Parker
authors.affiliation: Touchstone Diabetes Center, UT Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Qiong A
authors.initials: QA
authors.lastname: Wang
authors.affiliation: Department of Internal Medicine, UT Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Lance S
authors.initials: LS
authors.lastname: Terada
authors.affiliation: GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426, USA.
authors.forename: Nicholas D
authors.initials: ND
authors.lastname: Adams
authors.affiliation: GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426, USA.
authors.forename: Michael T
authors.initials: MT
authors.lastname: McCabe
authors.affiliation: GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426, USA.
authors.forename: Beth
authors.initials: B
authors.lastname: Pietrak
authors.affiliation: GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426, USA.
authors.forename: Stan
authors.initials: S
authors.lastname: Schmidt
authors.affiliation: Department of Bioengineering, University of California, San Diego, La Jolla, California 92093, USA.
authors.forename: Christian M
authors.initials: CM
authors.lastname: Metallo
authors.affiliation: Seahorse Bioscience, 16 Esquire Road, North Billerica, Massachusetts 01862, USA.
authors.forename: Brian P
authors.initials: BP
authors.lastname: Dranka
authors.affiliation: GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426, USA.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Schwartz
authors.affiliation: Children's Medical Center Research Institute, UT Southwestern Medical Center, Dallas, Texas 75390-8502, USA.|Department of Pediatrics, UT Southwestern Medical Center, Dallas, Texas 75390, USA.|McDermott Center for Human Growth and Development, UT Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Ralph J
authors.initials: RJ
authors.lastname: DeBerardinis
mesh_terms: D002448:Cell Adhesion; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D019343:Citric Acid / Q000378:metabolism*; D003260:Contact Inhibition; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D006706:Homeostasis; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D007523:Isocitrates / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D009249:NADP / Q000096:biosynthesis; D009369:Neoplasms / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism*; D018874:Spheroids, Cellular / Q000378:metabolism / Q000473:pathology
chemical_list: D007523:Isocitrates; D017382:Reactive Oxygen Species; D005973:Glutamine; D019343:Citric Acid; D018698:Glutamic Acid; D009249:NADP; C034219:isocitric acid; D007521:Isocitrate Dehydrogenase; C543588:IDH1 protein, human; D005947:Glucose