Cell survival is contingent upon signals from the extracellular matrix (ECM) and the loss of this signals typically causes cell death, or apoptosis. The ability to survive without these ECM signals, anchorage-independent growth, is what allows cancer cells to metastasize and continue to grow and divide. This is a particularly hot topic in cancer, as movement of cells means the ability for tumors to develop throughout the body. Because of the crosstalk between the human cell’s many growth and survival pathways, there are many places at which a cancer cell could mutate to promote cell movement. Previously, evidence for detachment was related to an increase of reactive oxygen species (ROS) via altered glucose metabolism, and these enhanced levels should compromise the cell. Jiang et al. expand upon this knowledge with the discovery of a surprising pathway that shows evidence of change in metabolism allows for the ability to maintain redox homeostasis.
In order to identify the specific changes in metabolism, the authors studied both lung cancer cells in culture and lung cancer cells aggregated into spheroids, which they saw to proliferate at a slower rate than in culture. It is known that cells use glucose and glutamine to create citrate, so it was not surprising that both conditions required these, however they saw the spheroid cells to consume less and secrete less product in the forms of lactate, glutamate, and ammonia. Coupled with this, they saw the spheroid cells also consumed less oxygen per cell and incorporated less glucose-derived carbon into citrate, indicating lower pyruvate dehydrogenase (PDH) activity, which they confirmed to be a result of inhibitory PDH phosphorylation and increased expression of PDH kinsase-1. Using labeled carbons, they were able to track if citrate was produced through reductive or oxidative measures (see figure below). Labeled carbons in citrate would indicate a reductive pathway was used. This reductive citrate was observed in detached spheroid cells from various cancer lines, however nonmalignant cells did not show these trends when detached.
Because reductive metabolism is a known result of a lack of oxygen, they performed tests in low oxygen environments and found that changes in low oxygen environments were distinct from changes due to anchorage. They further analyzed these metabolic changes by creating a model (see below), and found that incorporating by forcing isocitrate/citrate produced reductively in the cytosol to enter the mitochondria showed the best fit for the data. This model indicated an unexpected role of isocitrate dehydrogenase (IDH) flux. IDH has three forms in mammals. IDH1 and IDH2 participate in NADP+/NADPH dependent reductive carboxylation in the cytosol and mitochondria respectively, while IDH3 is only NAD+ dependent. They produced cells with knockouts of one of the three isoforms and found that IDH3 depletion caused a build-up of citrate, while knockouts of the other two isoforms caused little impact, which lead to the conclusion that IDH3 likely performed citrate oxidation in the citric acid cycle, so the authors turned their focus to the other isoforms.
Knowing that lower pentose phosphate pathway activity increased ROS, they looked at IDH1 knockout or inhibited cells and saw increased ROS in the mitochondria with lower ROS in the cytosol. IDH2 knockout cells or pentose phosphate pathway inhibited cells also showed higher mitochondrial ROS. The authors interpreted this data to suggest that NADPH from the pentose phosphate pathway was used to reduce ROS levels. They used further labeling in conjunction with IDH1 and IDH2 knockout cell lines and found spheroids contained citrate labelling that would use IDH1 activity, but not IDH2, which was consistent with their proposed pathway.
Overall, their data indicate that this alternative pathway, using IDH1, is used to reduce oxidative stress in the mitochondria of detached cells, which was a surprising and previously unknown role. This pathway provides new insight on ROS regulation as it showed that the regulation of mitochondrial ROS, rather than cytosolic ROS was the key to anchorage independent growth as their findings indicate that IDH1 knockout caused an increase in mitochondrial ROS, but lowered cytosolic ROS. Their pathway gives a target for the inhibition of anchorage-dependent growth by inhibiting the cell’s ability to control ROS levels.
Jiang et al. Reductive carboxylation supports redox homeostasis during anchorage-independent growth. Nature 532, 255-258 (2016).
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