A number of complex biological forces drive the mutation and selection of cancer cells – competitive growth, angiogenesis, immune system evasion, etc. A recent review in the journal Science has shed light on another key aspect of tumor development – the hijacking of the metabolism.

Over 80 years ago, Otto Warburg observed that tumor cells exhibit a different metabolic pattern than normal (adult) cells: increased glucose uptake and aerobic glycolysis coupled with decreased oxidative phosphorylation. The pyruvate generated by glycolysis is converted to lactate and secreted from the cell. He suggested that this metabolic switch explained the cancer phenotype and possibly represented a causal event in tumorigenesis.

Warburg’s observations seemed to yield more questions than answers. Why would tumor cells favor a metabolism pathway that was energentically inefficient? How did it contribute to cancer development and growth? And how was the metabolic switch made? Remarkably, Warburg’s findings did not spur additional research, and understanding of the implications eluded scientists for decades. Yet research in the last 10 years has begun to answer some of these questions, and generated new interest in the “Warburg effect.”

Glucose, Glutamine, and the Metabolism

In this review, Levine and Puzio-Kuter provide an excellent overview of the signaling pathways and related components of cellular metabolism. It begins with the uptake of glucose and glutamine, which serve as substrates for a number of tasks in proliferating cells:

1. Generating energy, in the form of ATP
2. Regulating the redox potential (the ratio of oxidized to reduced molecules in the cell)
3. Control of reactive oxygen species (ROS) and their adverse effects on cellular components
4. Synthesis of the substrates for membranes, nucleic acids (purines and pyrimidines), and proteins.

In normal differentiated cells, the need for cell division is low. Glucose metabolism is therefore optimized for efficient energy generation: glycolysis in the cell linked to the tricarboxylic acid (TCA) cycle (also known as the “Krebs cycle”) in mitochondria.

The Switch for Rapid Growth

In times when rapid cell division is required, however – during development, wound healing, or for immune responses – cells need to synthesize substrates for membranes, nucleic acids, and proteins; in other words, the intermediates for cell growth. This means not metabolizing all glucose to carbon dioxide and water. One way that cells accomplish this by slowing the entry of pyruvate into the mitochondria. The last step of glycolysis is catalyzed by pyruvate kinase, which receives input about the energy status of the cell and the presence of these intermediates. This enzyme is normally stimulated in a feed-forward loop by glycolysis. However, cancer cells make a splice variant, the M2 (fetal) isoform of pyruvate kinase in which several amino acids are added, including a tyrosine. This gets phosphorylated in tumors with activated tyrosine kinase signaling, inhibiting the feed-forward loop. Thus the last step of glycolysis is slowed in cancer cells, and keeps those intermediates around for growth.

A side-effect of delaying the TCGA cycle is the accumulation of pyruvate in the cell. As it builds up, pyruvate is converted to lactate and secreted, in order to keep glycolysis active. The secreted lactate lowers the pH of the cellular matrix; in tumors, this may influence remodeling of the matrix and help stimulate blood vessel invasion (angiogenesis). The use of glycolysis by tumors can make them acidotic, encouraging the selection of motile cells that can break free of the basement membrane and metastasize.

Glucose and Glutamine Uptake by Cancer Cells

Proliferating cells, including cancerous ones, also require favorable energetics – that is, higher ATP/ADP and ATP/AMP ratios. Many tumors satisfy this need by increasing glucose uptake. Glucose is brought into the cell by several transmembrane glucose transporters (GLUT1 to GLUT9), and then phosphorylated by one of several hexokinase enzymes to keep it in the cell. MYC and HIF-1, transcription factors and oncogenes that are often amplified in tumors, stimulate transcription of GLUT transporters and hexokinase 2, thereby ensuring that more glucose is brought into and kept in the cell. Many but not all tumors exhibit increasd glucose uptake in this fashion, which can be observed by PET scans of radioactive F-19-2-deoxyglucose.

Cancer cells also take up excessive amounts of glutamine (via glutamine transporters), which is converted to glutamate by glutaminase-1 before entering the mitochondria. There, the TCA cycle produces malate and citrate, both of which leave the mitochondria. In the cytosol, malate is converted to pyruvate and NADPH, while citrate is processed to or alpha-ketoglutarate (producing NADPH), acetyl-CoA (for lipid synthesis), or oxaloacetate (for amino acid synthesis).

Running Too Hot: Managing Reactive Oxygen Species

The increased uptake of glucose and glutamine by cancer cells thus helps fuel cell growth and proliferation, but it comes at a cost: excessive reactive oxygen species (ROS), which damage membranes, proteins, and nucleic acids. The major eliminator of ROS in a cell is glutathionine (GSH). This molecule accepts an electron, and is converted to the oxidized form glutathionine sulfate (GSSG). The enzyme glutathionine reductase uses another molecule, NADPH, to reduce GSSG back to its “eliminator” form (GSH). NADPH can be generated either from the citrate output of the TCA cycle, or via the pentose phosphate pathway (PPP) if there’s plenty of glucose available. Thus, when a cell starts to run too hot from oxidative reactions (e.g. glycolysis), NADPH serves as a sort of cellular coolant.

Of course, high levels of ROS can be advantageous for cancer cells. Damage to nucleic acids can promote mutagenesis or genomic instability, helping stimulate cell proliferation and immune system evasion. Even so, ROS levels that are too high will induce oxidative damage-induced cell death in cancer cells. Levine and Puzio-Kuter suggest that ROS thus have therapeutic potential, as they might be exploited to selectively kill cancer cells.

The Roles of Oncogenes and Tumor Suppressor Genes

In the last decade, research has revealed a number of key oncogenes (myc, NFKB, AKT, EGFR, IGF1, etc.) that activate the Ras, MAPK, mTOR, and phosphatidylinositoal 3-kinase (PI3K) pathways. These pathways stimulate the transcription of a number of important players in the glycolysis and glutaminolysis pathways. Myc alone activates the transcription of over 1,000 genes related to cell growth and metabolism. For example, it enhances transcription of glutaminase-1, which produces glutamate from glutamine. Myc also represses miR-23a and miR-23b, which increases expression of their intended target, mitochondrial glutaminase-1.

The famous P53 tumor-suppressor protein has several important roles in the regulation of cellular metabolism:

• Reduces glucose uptake, by repressing transcription of the GLUT1 and GLUT4 transporters.
• Decreases glycolysis, by activating the TIGAR gene which leads to diversion of glucose through the PPP.
• Increases use of the TCA cycle and oxidative phosphorylation in the mitochondria, by transcribing glutaminase-2 and cytochrome C oxidase
• Controls ROS levels through regulation of genes such as Sestrins 1, 2, 3, and 4.

TP53 also stimulates transcription of PTEN, IGF1BP3, TSC2, and the beta sub-unit of AMPK, all of which are negative regulators of the PI3K-AKT and mTOR pathways. The loss of tumor suppressors TP53 and PTEN, along with concurrent activation of AKT and mTOR pathways, results in high hypoxia-induced factor (HIF) activity. As you might guess from its name, HIF is regulated by the cell’s response to hypoxia. The absence of oxygen allows HIF-1A and HIF-2A proteins to form dimers with their beta-counterparts (HIF-1B and HIF-2B), stabilizing the HIF transcription factor complex. HIF increases the transcription of hundreds of genes, promoting angiogenesis, cell migration, and survival. It also regulates genes involved in energy metabolism, including lactate dehydrogenase A and pyruvate dehydrogenase kinase, both of which keep pyruvate away from the mitochondria, and 9 of the 10 enzymes required for glycolysis.

Known Driver Mutations

Mutations in the genes that encode TCA cycle components (enzymes) have been linked to human cancers. Rare mutations in succinate dehydrogenase (SDH) and fumarate hydratase (FHY) are initiating events in familial paraganalioma and papillary renal cell cancer. In glioblastoma multiforme (GBM),  mutations in the gene encoding isocitrate dehydrogenase 1 (IDH1) block the conversion of isocitrate to alpha-ketoglutarate and generation of NADPH. IDH1/IDH2 mutations are recurrent in gliomas and leukemias, and recently have been shown to produce a new enzymatic activity: the reduction of alpha-ketoglutarate to 2-hydrozyglutarate, an “oncometabolite”.

Clearly, the metabolic switch observed by Warburg is important for the development, growth, and survival of many cancers. While some of the mechanisms by which tumors induce the “Warburg effect” have been elucidated, it’s likely that many of them remain to be discovered. Consideration of a gene’s involvement in cell metabolism or its regulation therefore will be critical as we sift through the mutations of newly sequenced cancer genomes.

References
Levine AJ, & Puzio-Kuter AM (2010). The control of the metabolic switch in cancers by oncogenes and tumor suppressor genes. Science (New York, N.Y.), 330 (6009), 1340-4 PMID: 21127244