Apoptosis and Tumour Physiology

Mitochondria are generators of intracellular energy: in a cycle of oxidation they create the mitochondrial membrane potential that produces ATP. But mitochondria have additional life and death determining roles in cells: they produce signals that lead to apoptosis and so changes in their physiology can trigger oncogenic events. The focus of our laboratory is to study the relationship between energy metabolism and cancer. We are studying physiological changes in mitochondria that facilitate apoptosis, and biochemical signals that are elicited by mitochondria and contribute to tumourigenesis. Our aim is to develop mitochondria-related strategies for therapy.

Energy metabolism and apoptosis
Several apoptogenic factors that reside in the mitochondrial intermembrane space are released into the cytosol upon apoptotic insults. These include cytochrome c, Smac/Diablo, Omi, AIF and Endonuclease G. The release of cytochrome c from mitochondria induces the assembly of the apoptosome, a cytosolic complex comprising cytochrome c, Apaf-1 and caspase-9 that activate processes leading to cell death. In addition, changes in mitochondrial physiology have been demonstrated during many apoptotic processes but the relation of these changes to apoptosis as well as the nature of their biochemical effectors were unknown. Evidence that supports a direct role for energy metabolism in the regulation of apoptosis has accumulated recently with the discoveries that many pro- and anti-apoptotic proteins have an immediate effect on mitochondrion physiology. These include the observations that Bcl-xL and Bcl-2 improve oxidative phosphorylation and that Akt and Bad stimulate glucose metabolism. Nevertheless, none of these studies explained how changes in energy metabolism regulate cell death (Downward, Nature 2003; 424: 896).

Based on electron microscopy tomography, it was recently shown that structural changes in mitochondria are required for rapid and complete cytochrome c release during apoptosis (Scorrano et al, Dev Cell, 2002; 2: 55). We studied the relationship between energy metabolism and mitochondrial structure and have shown that following apoptosis induction, structural mitochondrial changes, namely matrix condensation and cristae unravelling, result from a decline in oxidizable substrates and consequently in mitochondrial membrane potential (Gottlieb et al., Cell Death Differ, 2003; 10: 709). We are now studying the roles of energy metabolism and ion flow across the mitochondrial inner membrane in the regulation of mitochondrial structure during apoptosis (Figure 1). A decrease in glycolysis is the primary cause for the decline in mitochondrial membrane potential and the adoption of a condensed configuration. Though glycolysis has been suggested to regulate apoptosis in several systems, the relationship between glycolysis and mitochondrial structure is the first direct biochemical link between glucose metabolism and apoptosis.

Fig. 1: Mitochondria change their density and morphology when energy metabolism is disrupted. Is that the link between mitochondrion physiology and apoptosis?

TCA cycle dysfunction and pseudo-hypoxia in cancer
As early as the 1930s, Otto Warburg described a direct link between defects in mitochondrial physiology and tumourigenesis. More recently (2000s) it was shown that several nuclear-encoded mitochondrial proteins are tumour suppressors, involved in cancers due to either hereditary or somatic mutations (Eng et al., Nature Reviews Cancer, 2003; 3: 193). These genes include succinate dehydrogenase (SDH) and fumarate hydratase, both members of the tricarboxylic acid (TCA) cycle that connects glucose metabolism in the cytosol to oxidative phosphorylation in the mitochondria. Inherited or somatic mutations in subunits B, C or D of the SDH genes are associated with the development of phaeochromocytoma, paraganglioma or renal cell carcinoma. Furthermore, mutations in SDH were shown to induce the hypoxia-response pathway in tumours. In particular, elevated levels of Hypoxia Inducible Factor-1α (HIF-1α) and the induction of hypoxia-inducible genes were observed in paraganglioma carrying an SDHD mutation. The HIF-α subunit is the oxygen-regulated component of the HIF transcription factor, a heterodimer comprised of HIF-α and HIF-β subunits. The physiological function of HIF is to promote adaptation of cells to low oxygen by inducing neovascularization and glycolysis. It was recently demonstrated that HIF activation resulting from VHL mutations promotes metastasis by inducing the expression of met and CXCR4. The VHL gene product (pVHL) is part of an E3 ubiquitin ligase complex that binds to the oxygen-dependent degradation (ODD) domain of HIF-α in an oxygen-dependent manner and targets it for degradation. Therefore, in tumours carrying VHL mutations that can no longer bind to and destabilize HIF-1α, HIF-1α is stabilized even under normoxic conditions.

The binding of pVHL to HIF-α is regulated by the hydroxylation of two specific prolyl residues in the ODD domain. Hydroxylation at the 4-position of Pro-402 and Pro-564 of HIF-1α enables formation of two hydrogen bonds to pVHL and increases the binding of pVHL to HIF-1α by several order of magnitude. This post-translational modification is catalyzed by the HIF-α-prolyl hydroxylases (PHD1-3). PHD activity is dependent on molecular oxygen and is considered to be an important oxygen sensing mechanism in animal cells. In addition to oxygen, the PHDs utilize α-ketoglutarate as a co-substrate and require ferrous iron (Fe²⁺) and ascorbate as cofactors. The PHD isozymes belong to the Fe²⁺ and α-ketoglutarate-dependent family of dioxygenases that split molecular oxygen to hydroxylate their substrates and, in parallel, oxidize and decarboxylate α-ketoglutarate to succinate. Significantly, both α-ketoglutarate and succinate are TCA cycle intermediates; in the TCA cycle, succinate is converted to fumarate by SDH. Succinate moves freely between the mitochondria and the cytosol via the dicarboxylic acid translocator in the mitochondrial inner membrane and the voltage-dependent anion channel (VDAC/porin) in the mitochondrial outer membrane.

Fig. 2: The role of succinate in mitochondrion-to-cytosol signalling. Succinate accumulates in mitochondria due to SDH inhibition and transports to the cytosol. Elevated succinate inhibits PHD and thus HIF-1α hydroxylation. Consequently, pVHL binding to HIF-1α decreases leading to HIF activation and the induction of genes that promote tumour aggressiveness.

Recently, we identified a novel pathway that links the down-regulation of SDH to the induction of HIF-1α (Selak et al., Cancer Cell, in press). We showed that accumulation of succinate, due to SDH inhibition, transmits an "oncogenic" signal from the mitochondria to the cytosol. Once in the cytosol, succinate inhibits PHD activity leading to HIF-1α induction (Figure 2). Thus, a mitochondrial metabolite can, under some circumstances, modulate nuclear events by inducing HIF target genes that facilitate angiogenesis, metastasis and metabolism, ultimately leading to tumour progression (Figure 2). This mitochondrion-to-cytosol pathway identifies succinate for the first time as an intracellular messenger. Moreover, demonstrating a mitochondrial metabolite as a functional regulator in the cytosol opens a new window in biochemical research and suggests new approaches for therapy.