In 1927, Otto Warburg described what would be called the “Warburg effect,” in which tumour cells exhibited characteristic changes in metabolism, particularly the use of glycolysis rather than oxidative phosphorylation, despite the presence of adequate amounts of oxygen [1, 2]. Warburg believed that this process was the actual cause of neoplastic transformation [3].
Tumour development is now known to be driven by genetic damage. However, mutations in some metabolic enzymes, such as succinate dehydrogenase (SDH) and fumaratehydratase (FH), both parts of the tricarboxylic acid (TCA) cycle, can drive neoplastic transformation, and intermediate products of metabolism can also promote neoplastic progression [4].
Proliferating cancer cells have a high energy requirement to maintain homeostatic cellular processes. The shift in energy production to aerobic glycolysis, while allowing for more rapid production of adenosine triphosphate (ATP), yields far less energy than oxidative phosphorylation: there are two net molecules of ATP per glucose molecule in glycolysis versus 36 molecules of ATP via oxidative phosphorylation [5].
The reasons for the glycolytic energy dependence of proliferating tumour cells are still being debated. Initially, it was believed that the mitochondria in tumours were intrinsically defective. However, it was determined that tumour mitochondria are actually functional, retaining the capacity for oxidative phosphorylation and consuming oxygen at similar rates to normal tissues [6], although it should be appreciated that a degree of variability in mitochondrial activities exists across different neoplasms. Alternatively, high rates of glycolysis might be co-selected with factors that promote the expression of hypoxia-related genes (such as those required for angiogenesis) as an oxygen-independent energy source. Finally, increased intermediate products of glycolysis can easily be shunted into the biosynthetic pathways required for serine and nucleotide synthesis [7].
According to Folkman’s original theory [8], the onset of hypoxia in tumour triggers angiogenesis, which in turn is essential for supplying neoplastic cells with nutrients and oxygen and evacuating metabolic waste and carbon dioxide. The best understood hypoxia signaling mechanism is the stabilization and post-transcription activation of the hypoxia-inducible factor (HIF) proteins, which lead to the activation of many different pathways, including the vascular endothelial growth factor (VEGF) pathway. The VEGF pathway prompts and supports neoangiogenesis and glycolysis. Hypoxia-inducible pathway activation also has other effects, which include reducing the activity of mammalian target of rapamycin (mTOR), which in turn can reignite autophagy and promote survival under stress [9].
HIF is a heterodimer of an alpha subunit that is unstable in normoxia and a constitutively present and stable beta subunit. The hypoxia activation of HIF causes the heterodimer to bind to DNA at specific locations, called hypoxic response elements (HREs), eliciting the transcriptional up-regulation of genes required to respond appropriately to hypoxia [9].
In addition to triggering the VEGF pathway, the ubiquitously expressed HIF1 isoform promotes the transcription of glucose transporter 1 (GLUT1), which activates glucose transport inside the cell, lactate dehydrogenase-A (LDH-A), which is involved in the glycolytic pathway, erythropoietin (EPO), which enhances erythropoiesis, and nitric oxide synthase (NOS), which promotes angiogenesis and vasodilatation [9].
HIF1 also prevents the entry of pyruvate into the TCA cycle by inducing the expression of pyruvate dehydrogenase kinase 1 (PDK1), thus altering the expressed isoform of cytochrome c and inhibiting mitochondrial biogenesis. This process causes reduced levels of oxygen consumption and a shift away from oxidative phosphorylation. Interestingly, HIF1 can also be activated under normoxic conditions by a variety of oncogenic pathways, such as phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), and by mutations in von Hippel-Lindau tumour suppressor (VHL), SDH, and FH [10].
In the classic angiogenic pathway, VEGF binds to VEGF receptor 2 (VEGFR2) on endothelial cells, increasing the expression of the Notch ligand Delta-like 4 (DLL4) on the same cells. DLL4 then binds to its receptor Notch on the adjacent endothelium. Further expression of VEGFR2 and VEGFR1, as well as a smaller amount of VEGFR3, then follows, leading to triggering/amplification of the downstream phospholipase C familyγ (PLCγ)–protein kinase C (PKC)–Raf kinase–MAP kinase-ERK kinase (MEK)–mitogen-activated protein kinase (MAPK) pathway, concomitantly prompting cell proliferation and cell survival throughout the phosphoinositide 3-kinase (PI3 K)/protein kinase B (AKT) pathway [11].
The switch to glycolysis in neoplasia was, according to Warburg, irreversible [3], yet a more complex picture has emerged over the last decade. There have been observed instances in which oxidative phosphorylation predominates during neoplastic transformation [12]. This variation between OxPhos and glycolysisin cancer cells has been increasingly linked to specific disturbances in cell signaling pathways [13].
Additionally, tumours of the same genetic lineage can develop different metabolic adaptations depending on the host tissue from which they arise, suggesting that the stromal environment might play a crucial role in shaping the metabolic profile [14]. The different molecular mechanisms being postulated to explain this variability of the Warburg effect include the following: inhibition of pyruvate dehydrogenase (PDH) by PDK1, reduction of mitochondrial biogenesis and inhibition of oxidative phosphorylation, both are caused by P53 inactivation and mutations [15].
Warburg raised two important issues: first, how tumour cells are supplied with glucose; and second, how they are supplied with oxygen [1]. Folkman’s work addressed the latter question with the hypothesis that tumour growth is strictly angiogenesis-dependent [16]. The work undertaken to test this hypothesis led to the inclusion of “angiogenesis” as one of the hallmarks of cancer [8].
Although there is strong evidence that angiogenesis frequently occurs in cancer, we also now know that this event does not always occur. Indeed, some tumours, called “non-angiogenic tumours,” can grow without triggering new vessel formation by co-opting preexisting vessels [17, 18].
Non-angiogenic growth was first identified by histology in primary and metastatic lung carcinomas because neoplastic cells filled the alveolar spaces, co-opting the pre-existing capillary network and exhibiting a characteristic “chicken-wire” appearance [17]. A gene expression signature for non-angiogenic non-small cell lung cancer (NSCLC) was published in 2005 [19]. Surprisingly, rather than the classic angiogenesis-related genes, the differentially expressed genes were involved in mitochondrial metabolism, transcription, protein synthesis, and the cell cycle. Lack of differential mRNA expression between tumour phenotypes was noted for genes classically associated with hypoxia and angiogenesis. This result suggested that the response to hypoxia does not necessarily trigger neovascularisation, as would be observed in angiogenic tumours, but could actually be dependent on the genetic background of neoplastic cells, and in some instances, it could lead to metabolic reprogramming [19]. We therefore postulated that the degree to which a tumour will rely on angiogenic or non-angiogenic growth could be associated with a variety of events, including hypoxia, pseudo-hypoxia, and metabolic re-programming.
In the first part of the present study, we investigated whether there were truly no differences in the expression of hypoxia-and angiogenesis-related proteins between angiogenic and non-angiogenic tumours, as suggested by mRNA profiling. We also investigated the degree of these proteins expression and the expression of some mitochondrial biogenesis proteins via immunohistochemistry. Notably high cytoplasmic P53 expression in non-angiogenic tumours, compared to angiogenic tumours, was found after completing the first part of this study. We therefore performed a second investigation, in which we examined and sequenced the p53 gene in these tumours.
