Pyruvate Carboxylase Supports MCF10A-Ras Cell Survival in Extracellular Matrix Detached Conditions
Abstract Background: Throughout metastatic progression, cancer cells acquire anchorage independence, or the ability to survive detached from the extracellular matrix (ECM). While untransformed epithelial cells reduce energy metabolism when detached, cancer cells display metabolic flexibility to continue important metabolic processes. Glucose and glutamine are predominant nutrients utilized for energy as well as other purposes, and their metabolism is regulated by cancer cells.Methods: The purpose of the current studies was to determine the effects of detachment on glucose and glutamine metabolism in human breast epithelial MCF10A cells transfected with the Harvey-ras oncogene (MCF10A-ras), a model of early-stage cancer. Detachment was simulated with poly-HEMA coated plates, and intracellular metabolic flux was determined using stably labeled 13C5-glutamine and 13C6-glucose tracers.Results: Results show reduced glutamine flux in detached cells as determined by reduced accumulation of label in glutamate (21%), malate (30%), and aspartate (23%) from 13C5-glutamine. Detachment also reduced flux of 13C6-glucose to pyruvate and lactate pools by 51% and 29%, respectively. Similarly, detachment reduced total intracellular pool sizes of pyruvate (51%), lactate (49%), α-ketoglutarate (43%), fumarate (32%), malate (19%), alanine (35%), serine (35%), and glutamate (28%) compared to attached cells, but citrate and aspartate pool sizes were unchanged. Compared to attached cells, detachment increased pyruvate carboxylase (PC) mRNA abundance and protein expression by 131% and 190%, respectively. In detachment, PC activity, determined by 13C6-glucose derived M + 3 isotopomers, was shown to preferentially replenish malate and aspartate, but not citrate pools. In addition, doxycycline-inducible shRNA depletion of PC significantly decreased, while doxycycline-inducible PC overexpression significantly increased, detached cell viability. Further, a switch from glutamine to PC activity for anaplerosis was demonstrated, as supplementation with the cell permeable analog of the tricarboxylic acid cycle intermediate, α-ketoglutarate, a downstream metabolite of glutamine, decreased PC mRNA abundance in detached cells.Conclusion: Collectively, these results suggest that detached breast cancer cells increase PC activity in response to decreased glutamine-derived anaplerosis to promote cell survival.