Abstract A35: Cytosolic reductive carboxylation is required for mitochondrial redox homeostasis during anchorage-independent cell growth

Abstract

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.