Oncogenic K-Ras decouples glucose and glutamine metabolism to support cancer cell growth

Abstract

• A systems approach using 13C metabolic flux analysis (MFA), non-targeted tracer fate detection (NTFD), and transcriptional profiling was applied to investigate the role of oncogenic K-Ras in metabolic transformation.

• K-Ras transformed cells exhibit an increased glycolytic rate and lower flux through the oxidative tricarboxylic acid (TCA) cycle.

• K-Ras transformed cells show a relative increase in glutamine anaplerosis and reductive TCA metabolism.

• Transcriptional changes driven by oncogenic K-Ras suggest control nodes associated with the metabolic reprogramming of cancer cells.

Abstract

The ras and myc oncogenes drive pleiotropic changes in cell signaling, nutrient uptake, and intracellular metabolism ( Chiaradonna et al, 2006b; Yuneva et al, 2007; Wise et al, 2008; Vander Heiden et al, 2009). Mutated ras proteins, identified in 25% of human cancers ( Bos, 1989; Downward, 2003), correlate with an increased rate of glucose consumption, lactate accumulation, altered expression of mitochondrial genes, increased ROS production, and reduced mitochondrial activity ( Bos, 1989; Downward, 2003; Vizan et al, 2005; Chiaradonna et al, 2006a; Yun et al, 2009; Baracca et al, 2010; Weinberg et al, 2010). Furthermore, K-Ras transformed cancer cells are dependent upon glucose and glutamine availability, since their withdrawal induces apoptosis and cell-cycle arrest, respectively ( Ramanathan et al, 2005; Telang et al, 2006; Yun et al, 2009). However, the precise metabolic effects downstream of oncogenic Ras signaling as well as the mechanisms by which intracellular glucose and glutamine metabolism change have not been completely elucidated.

In this report, we have investigated the reprogramming of central carbon metabolism in cancer cells and its regulation by the K- ras oncogene, applying a systems level approach using ¹³C metabolic flux analysis (MFA), non-targeted tracer fate detection (NTFD), and transcriptional profiling. These data reveal a coordinated decoupling of glycolysis and the tricarboxylic acid (TCA) cycle. K-Ras transformed mouse and human cells exhibited a high glucose to lactate flux and relatively lower oxidative metabolism of pyruvate. Such changes were supported by increased expression of glycolytic genes as well as several pyruvate dehydrogenase kinases. In contrast to glucose, the contribution of glutamine carbon to TCA cycle intermediates through both oxidative and reductive metabolism was significantly increased upon K-Ras transformation. Despite this increase in glutamine anaplerosis, oxidative TCA flux was significantly decreased. Additionally, we observed elevated levels of glutamine-derived nitrogen in various biosynthetic metabolites in transformed cells, including amino acids, 5-oxoproline, and the nucleobase adenine. Consistent with these changes, we detected increased transcription of genes associated with glutamine metabolism and nucleotide biosynthesis in cells expressing oncogenic K-Ras.

Taken together, these findings indicate an important role of oncogenic K-Ras in cancer cell metabolism. The observed decoupling of glucose and glutamine metabolism enables the efficient utilization of both carbon and nitrogen from glutamine for biosynthetic processes. In accord with these alterations, oncogenic K-Ras induces gene expression changes that may drive this metabolic reprogramming. Finally, these results may enable the identification of metabolic and transcriptional targets throughout the network and allow more effective cancer therapies.

Abstract

Oncogenes such as K- ras mediate cellular and metabolic transformation during tumorigenesis. To analyze K-Ras-dependent metabolic alterations, we employed ¹³C metabolic flux analysis (MFA), non-targeted tracer fate detection (NTFD) of ¹⁵N-labeled glutamine, and transcriptomic profiling in mouse fibroblast and human carcinoma cell lines. Stable isotope-labeled glucose and glutamine tracers and computational determination of intracellular fluxes indicated that cells expressing oncogenic K-Ras exhibited enhanced glycolytic activity, decreased oxidative flux through the tricarboxylic acid (TCA) cycle, and increased utilization of glutamine for anabolic synthesis. Surprisingly, a non-canonical labeling of TCA cycle-associated metabolites was detected in both transformed cell lines. Transcriptional profiling detected elevated expression of several genes associated with glycolysis, glutamine metabolism, and nucleotide biosynthesis upon transformation with oncogenic K-Ras. Chemical perturbation of enzymes along these pathways further supports the decoupling of glycolysis and TCA metabolism, with glutamine supplying increased carbon to drive the TCA cycle. These results provide evidence for a role of oncogenic K-Ras in the metabolic reprogramming of cancer cells.