Abstract A35: Cytosolic reductive carboxylation is required for mitochondrial redox homeostasis during anchorage-independent cell growth
Authors: Jiang Lei , Shestov Alexander , Terada Lance S. , Adams Nicholas D. , McCabe Michael T. , Pietrak Beth , Schimidt Stan J. , Schwartz Benjamin , DeBerardinis Ralph J. 
Published: May 2016

Abstract
Epithelial 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, an essential hallmark of cells capable of metastasis2. Previous study showed that detachment from the ECM is associated with enhanced reactive oxygen species (ROS) levels due to suppression of glucose metabolism in the pentose phosphate pathway3. Here we used metabolic flux analysis to identify an unconventional metabolic pathway that supports redox homeostasis and growth during adaptation to anchorage independence. We observed that detachment from monolayer culture and growth as anchorage-independent tumor spheroids was accompanied by changes in both glucose and glutamine metabolism. Specifically, oxidative metabolism of both glucose and glutamine was suppressed in the spheroids, whereas reductive formation of citrate from glutamine was enhanced. Enhanced reductive glutamine metabolism was highly dependent on cytosolic isocitrate dehydrogenase-1 (IDH1) rather than mitochondrial IDH2, because this activity was eliminated in cells homozygous null for IDH1 or treated with an IDH1 inhibitor. Reductive carboxylation occurred in the absence of hypoxia, a well-known inducer of reductive glutamine metabolism4, 5. IDH1dependent reductive carboxylation mitigated mitochondrial ROS during spheroid growth, and IDH1 deletion or inhibition blunted spheroid growth in a ROS-dependent manner. Ablating expression of IDH2 or the mitochondrial citrate transporter did not substantially alter reductive labeling, but suppressed spheroid growth. Together, the data indicate that adaptation to anchorage independence requires a fundamental change in citrate metabolism. During anchorage-independent culture, reductive carboxylation produces cytosolic citrate, some of which is then imported into the mitochondria and metabolized in the TCA cycle. This results in the net transfer of NADPH from the cytosol to the mitochondria, mitigating mitochondrial ROS and maximizing cell growth.
References:
1. Bhowmick NA, Neilson EG, Moses HL. Stromal fibroblasts in cancer initiation and progression. Nature 2004; 432:332-7.
2. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011; 144:646-74.
3. Schafer ZT, Grassian AR, Song L, Jiang Z, Gerhart-Hines Z, Irie HY, Gao S, Puigserver P, Brugge JS. Antioxidant and oncogene rescue of metabolic defects caused by loss of matrix attachment. Nature 2009; 461:109-13.
4. Wise DR, Ward PS, Shay JE, Cross JR, Gruber JJ, Sachdeva UM, Platt JM, DeMatteo RG, Simon MC, Thompson CB. Hypoxia promotes isocitrate dehydrogenase-dependent carboxylation of alpha-ketoglutarate to citrate to support cell growth and viability. Proceedings of the National Academy of Sciences of the United States of America 2011; 108:19611-6.
5. Metallo CM, Gameiro PA, Bell EL, Mattaini KR, Yang J, Hiller K, Jewell CM, Johnson ZR, Irvine DJ, Guarente L, et al. Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia. Nature 2012; 481:380-4.
Citation Format: Lei Jiang, Alexander Shestov, Lance S. Terada, Nicholas D. Adams, Michael T. McCabe, Beth Pietrak, Stan J. Schimidt, Benjamin Schwartz, Ralph J. DeBerardinis. Cytosolic reductive carboxylation is required for mitochondrial redox homeostasis during anchorage-independent cell growth. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A35.