Energy metabolism of malignant cells

ATP as the primary form of energy is required for most cellular processes like biosynthesis, transport and signalling mechanisms, turning ATP into a limiting factor under certain growth conditions. Glycolytic degradation of monosaccharides as a source of ATP is providing only a relatively small amount due to its low energy efficiency but is oxygen independent. The rate-limiting reaction is catalysed by hexokinases. Under physiological conditions the major part of ATP is generated by oxygen-dependent mitochondrial oxidative phosphorylation. This highlights the

importance of providing sufficient availability of oxygen as well as nutrients to serve the energy demand. This also applies for tumors, since angiogenesis and increased rate of glycolysis facilitate development and growths of malignant tissue, turning these tumor-specific alterations into targets for cancer therapy.

1.9.1 Warburg effect

In the 1920s the German biochemist Otto Warburg described that one of the most characteristic phenotypes of highly malignant cancers is their increased rate of glycolysis, thereby satisfying high amounts of their energy demands by glycolysis even under normoxic conditions [87-93], which is accepted today as a key metabolic hallmark of cancers [94]. These alterations, known as the Warburg effect, are worth being reconsidered also in modern cancer research. By now we know that hexokinase II plays a central role in the Warburg effect [94, 95]. In many highly malignant rapidly growing tumors increased expression of hexokinases was detected. Since, as previously mentioned, ketohexoses such as fructose are metabolised the same way as glucose by hexokinases in most cells of the mammalian body, a potential influence of increased expression of hexokinases on fructose metabolism has to be considered. But almost none of the studies addressing Warburg’s observations discriminated between the metabolisms of these sugars.

1.9.2 Hexokinases and their role in cancer

Hexokinases catalyse the phosphorylation of certain sugars e.g. glucose or fructose at position C6, being the initial and rate-limiting step of glycolysis. Four isoforms with tissue-specific expression pattern have been described in mammalian cells [96-98].

Isoforms I-III are also called low-Km hexokinases due to their high affinity for glucose (Km ~ 0.2 mM). While these isoforms are nearly silent in the liver, isoform IV (glucokinase), characterised by a high Km (Km ~ 5 mM), is expressed and enables the energy storage function of the liver. The organ regulates blood glucose levels, by storing glucose as glycogen in phases of excessive sugar availability (resorption phase) [96-98], whereas glucose is released during post-resorption phase to ensure sufficient glucose plasma concentrations.

Enhanced glycolytic activity during cancerogenesis was shown to be due to increased expression of hexokinases [91]. Further studies of hepatomas, and

malignant cells of other origin revealed that hexokinase isoform II is the main contributor to the cancer phenotype of increased glycolytic activity [99-101].

Enhanced expression of HKII by promoter activation, gene amplification, epigenetic regulation, as well as elevated binding to mitochondria via a hydrophobic amino acid sequence in the N-terminal region of HKII are the initiators of increased glycolytic activity [102-107]. Binding of HKII to mitochondria leads to improved access of mitochondrial ATP as substrate [102], reduced product inhibition by glycose-6P [99, 108, 109], and reduced proteasomal degradation, resulting in reinforced activity of HKII. The increased rate of glycolysis ensures to cover the high energetic demand of cancer cells for cell division and growth, and warrants an advantage for survival and proliferation in tumor microenvironment. Warburg´s pioneering studies found their way into clinical applications, since upregulation of HK resulting in increased rate of glycolysis is exploited daily in the clinics for detection of tumors by positron emission tomography (PET), visualising uptake of 18F-2-deoxyglucose [110]. In this context, upregulation of HKII as adaptive mechanism to hostile hypoxic tumor environment has to be mentioned. Hypoxic microenvironment is only one inducer besides others such as certain oncogenes, like the signalling molecule Ras, Akt kinase as well as the transcription factor Myc driving changes in energy metabolism [94].

1.9.3 Hypoxia as tumor environment

The moment tumors outgrow their blood supply, regions of hypoxia develop. This leads to an oxygen gradient within malignant tissues, diminishing towards the less vascularised centre of the tumor [111, 112]. Hypoxia as a tumor environment was shown to induce an aggressive phenotype, increasing metastatic potential, promoting tumor progression, and limiting the effectiveness of radiation therapy and some chemotherapeutic agents [113, 114]. Even in case of hepatic carcinogenesis hypoxia plays an important role, since it was reported to regulate proliferation, angiogenesis, metastasis, chemo- and radioresistance, apoptosis and differentiation of HCC [115].

Additionally, hypoxia was described to stimulate growth of HCC by increased expression of HKII [116]. The pleiotropic effects of reduced oxygen availability are mainly orchestrated by the master transcription factor hypoxia-inducible factor1 (HIF1). The main targets of HIF1 signalling are induction of angiogenesis e.g. via vascular endothelial growth factor (VEGF) expression and adaption to hypoxia by

increased anaerobic glycolysis e.g. via HKII upregulation. Advanced energy supply of cancerous tissue is the effect of most HIF1 targets. It is noteworthy that cytokines, growth factors and oncogenes are also potential activators of HIF1 [117]. Influence of reduced oxygen availability itself or of its regulators, also in presence of oxygen, has been postulated to induce changes in the energy metabolism of cancers of various origins [118, 119]. However, stabilisation of HIF1 during hepatic carcinogenesis was shown in animal experiments, and its accumulation was reported in more than 50%

of human primary cancers and metastasis. Increased levels of HIF1, of hexokinase II and glycolytic activity were related to aggressiveness of HCC and patients survival time [118, 120, 121].

In context of therapeutic cancer targets, the changes in the energy metabolism are enforced, attracting attention leading to a resurrection of Warburg´s theory.

Discussions whether a shift in the energy metabolism is a requirement of transformation or a byproduct of the cell´s transformation are still going on. Whether increased rate of glycolysis according to Warburg is a cause or a consequence of cancer is a minor concern in our context, if it opens up new therapeutic approaches for cancer therapy.