According to the International Neuroblastoma (NB) Pathology Classification, NBs are defined as embryonal tumors of the sympathetic nervous system, derive from the neural crest and arise in the adrenal medulla, paravertebral sympathetic ganglia, and sympathetic paraganglia [1
]. Paraganglioma and pheochromocytoma are histologically related to NB as they are all neural crest derived. NBs mainly consist of immature neuroblasts, whereas pheochromocytomas and paragangliomas contain mature chromaffin cells.
Pheochromocytomas and paragangliomas frequently exhibit mutations in the succinate dehydrogenase (SDH) subunits SDHB, SDHC, SDHD indicating that these SDH subunits act as tumor suppressors in neuroendocrine tissues [2
]. The SDH complex is composed of four subunits and contains a flavin molecule (FAD), non-heme iron centers and a b-type cytochrome as prosthetic groups. The complex is anchored by a large SDHC and a small SDHD subunit that together comprise the membrane-spanning heme protein cyt b
]. In addition, two assembly factors of SDH SDHAF1 and SDH5 have been reported recently. SDH5 is a gene required for flavination of SDH [4
]. Pathogenic mutations in SDH5 have been identified in paragangliomas [5
Mutations in the PHOX2B and anaplastic lymphoma kinase (ALK) genes are linked to a predisposition for neuroblastoma [6
]. Mutations in PHOX2B have been found in a minority of familial neuroblastoma cases. So far no clear link to the energy metabolism has been demonstrated for these two genes.
Mutations in the VHL
gene cause von Hippel-Lindau disease, a dominantly inherited familial cancer syndrome predisposing to a variety of malignant and benign neoplasms, including clear cell renal carcinoma and pheochromocytoma [11
]. Loss of VHL protein in tumors results in an accumulation of hypoxia-inducible factor-1α (HIF-1α) during normoxia. Such accumulation of HIF-1α in turn induces expression of various genes containing hypoxia-responsive elements, thereby decreasing the expression of components of aerobic energy metabolism [14
]. Accordingly, not only is SDHB protein suppressed in tumors with mutations in SDHB and SDHD, but also in a subset of tumors with VHL mutations which act through HIF1α [18
]. In addition, alterations of proteins involved in Fe-S cluster biogenesis, including Nitrogen fixation-1 homolog (nfs-1), LYR motif containing 4 (LYRM4; isd11), and frataxin, can lead to a reduction of SDH activity [20
]. For example, disrupted expression of frataxin in murine hepatocytes causes decreased oxidative phosphorylation (OXPHOS) paralleled by reduced activity of iron-sulfur cluster-containing proteins [20
]. During the process of OXPHOS ATP is formed as electrons are transferred from NADH or FADH2 to molecular oxygen (O2) by a series of electron carriers. The energy released by catabolic biochemical processes, such as glycolysis, citric acid cycle, and fatty acid oxidation is stored as the reduced coenzymes NADH or FADH2. There is a step by step transfer of electrons from NADH or FADH2 to specific protein complexes, which are part of the electron transport chain. Electrons are transferred from these reduced equivalents, through the electron transport chain (ETC), to O2
. The ETC consists of four multisubunit complexes namely complex I - IV. SDH is both part of the ETC as well as the citric acid cycle. The liberated energy is used to create a proton gradient over the inner mitochondrial membrane. In the final reaction of OXPHOS, the reflux of protons is used by the complex V (ATP synthase) to produce ATP.
The aerobic use of glucose as an energy source through glycolysis is a feature common to most solid tumors, in turn leading to a lesser dependence on OXPHOS, which is called the Warburg effect [25
]. The downregulation of OXPHOS in tumor cells seems to be achieved by different mechanisms. First, profound hypoxia can be the cause of compensatory upregulation of glycolysis in most tumors. Secondly, it is becoming more and more evident that the loss of tumor suppressor genes such as VHL
or activation of oncogenes results in downregulation of OXPHOS [27
]. Finally, direct inactivation of components of OXPHOS has been detected in a minority of tumors. Besides the association of SDH and tumor development, loss of NADH: ubiquinone oxidoreductase (complex I) of the respiratory chain has also been shown in oxiphilic tumors [29
The aim of the present study was to determine if there are specific alterations of aerobic energy metabolism in NBs, especially of SDH, or if there is an overall downregulation of OXPHOS complexes.
Although it is generally accepted that solid cancers exhibit in most cases a shift from oxidative phosphorylation to glycolysis the type of alteration has not been investigated in many types of cancers, and to our knowledge not in neuroblastic tumors.