Alterations of the OXPHOS system in neuroblastic tumors

NBs, like most other solid tumors, show high glucose uptake on PET examination (37,38). The Warburgian tendency to glycolysis brings about an elevated need for glucose, and excessive lactate production (6). Accordingly, NBs show a high rate of lactic acid production and a low rate of oxygen consumption, reflecting the switch from mitochondrial OXPHOS to glycolysis (39).

There is strong evidence that the Warburg effect in NBs is not caused by a low mitochondrial mass per cell. Electron microscopy showed numerous round and oval or elongated mitochondria in NB cells (40). Consistent with this observation, we recently reported equal levels of citrate synthase activity and porin expression in human NB samples and normal kidney tissue, indicating a similar mitochondrial mass (41). Furthermore, MitoTracker Green analysis indicated similar amounts of mitochondria in NBs, cerebral cortex and retinal ganglion cells (42). Strikingly, a significant reduction in mtDNA copy number and an overall reduction of OXPHOS enzyme activities were observed in human NB tissue; in comparison with cortical kidney tissue, the activity of succinate dehydrogenase (SDH; or complex II) was most affected (41). Interestingly, a similar phenotype was observed in adult renal cell carcinoma which is characterized by different genetic alterations (7).

Ganglioneuromas (GNs) and ganglioneuroblastomas (GNBs), like NBs, are classified as pNTs that originate from immature sympathetic neuroblasts. Intermixed GNBs are histologically and clinically situated between NBs and the benign GNs, while nodular GNBs are composed of nodules of NB cells in the background of intermixed GNBs. According to the Shimada classification, GNs are Schwannian stroma-dominant tumors, whereas NBs are considered as Schwannian stroma-poor neoplasias (43). Surprisingly, GNs show a severe loss of mitochondria, as indicated by loss of porin and significant reduction of citrate synthase; therefore, their loss of all OXPHOS complexes compared to normal adrenal gland and kidney cortex tissue is likely secondary to mitochondrial loss (44).

The nexus between common genetic alterations in NB and energy metabolism

Deletions of chromosome 1p36 occur in approximately 20-40% of primary NBs. As noted above, LOH of 1p is also highly correlated with MYCN amplification (32,33). Succinate dehydrogenase subunit B (SDHB) maps to chromosome 1p36, a region of frequent LOH in NB (31). There may be a correlation between 1p36 LOH and the low activity of SDH reported by our laboratory (41). Sequencing of all four subunits of SDH and two assembly factors showed no pathological mutations in human NB samples (41). These findings are in agreement with those of two other studies, which also failed to detect pathogenic mutations in SDHB and SDHB in NB. Furthermore, epigenetic studies also could not demonstrate evidence for silencing of SDHB expression (45,46). However, rare cases of NB with SDHB and SDHD mutations have been reported (46-48). NB cells transfected with different SDHB-mutated constructs showed down-regulation of mitochondrial and cytoplasmic metabolism as well as higher growth rates (49).

Deletion of the long arm of chromosome 11 is observed in high-risk NBs with an unfavorable prognosis (33). The SDHD gene is located on 11q23. A significant reduction in SDHD mRNA expression in NBs compared to both normal fetal neuroblasts and various normal adult tissues was demonstrated (46).

Gain of chromosome 17 is also a frequent chromosomal abnormality detected in advanced-stage NBs. Two patterns of gain are seen for this chromosome: either the whole chromosome 17 or only 17q is multiplied (34). NBs with a 17q gain overexpress BIRC5/Survivin, an anti-apoptotic protein (50). It was reported that the survivin protein is involved in reprogramming metabolism in NB cells by inducing fragmentation of mitochondria and reducing mitochondrial respiration. Survivin shifts the energy production from OXPHOS to aerobic glycolysis through inhibition of complex I and induction of mitochondrial fission via recruitment of dynamin 1-like protein/dynamin-related protein 1 (DNM1L/Drp1). NB cells with elevated survivin levels showed faster glucose metabolism and higher lactate production. Furthermore, inhibition of complex I by survivin prevented reactive oxygen species (ROS) accumulation and Forkhead box O3 (FOXO3) activity (50). Feichtinger et al. reported low complex I activity in NBs, although no data regarding gain of chromosome 17 were available (41).

Amplification of MYCN is found in half of all high-risk NBs with poor prognosis and resistance to therapy (51). Studies showed that MYCN is associated with regulation of energy metabolism through direct or indirect activation of genes involved in glycolysis, glutamine metabolism, fatty acids metabolism and mitochondrial function (52-54). Amplification of MYCN supports the Warburg effect in NBs by activating the transcription of several glycolytic genes. Knock-down of MYCN expression in MYCN-amplified NB cell lines caused down-regulation of glycolytic metabolism. Depletion of MYCN led to lower expression of the genes encoding glucose transporter 1 (GLUT1), HK2, PDK1, phosphoglycerate kinase 1 (PGK1), aldolase A (ALDA), and LDHA and leads to decreased proliferation of MYCN-amplified NBs (52).

Interestingly, mitochondrial inhibition by metaiodobenzylguanidine (MIBG) caused destabilization of MYCN and MYC in MYCN-amplified and non-MYCN-amplified NB cells and suppressed NB cell growth (55). MIBG also reduced the ATP to ADP ratio in NB cells (56).

Abundant glutamine is necessary for the TCA cycle in MYCN-amplified NB. Deprivation of glutamine led to depletion of TCA-cycle intermediates (54). During anaplerosis, mitochondrial glutamine metabolism can replenish oxaloacetate (OAA) for the TCA cycle (57). Provision of sufficient levels of glutamine for NB cells relies on activation of amino-acid transporter ASCT2, a glutamine transporter. High expression of MYCN, ATF4 and ASCT2 was observed in MYCN-amplified NB. The elevated level of ASTC2 expression observed in high-stage NB correlates with MYCN and ATF4 expression that promotes aggressive NB progression. Knockdown of MYCN led to a significant reduction in ASCT2 abundance (54).

The expression of the aryl hydrocarbon receptor (AHR), a receptor for dioxin-like compounds, correlates with a higher degree of differentiation of NBs. An inverse correlation between AHR and MYCN levels was reported (58). Members of the E2F family are transcription factors that regulate cell-cycle progression and influence MYCN expression in NBs (59). Ectopic expression of AHR correlates with down-regulation of MYCN and E2F1 expression in NB cells. Silencing of AHR enhanced the expression of both E2F1 and MYCN in NB cells. It is proposed that AHR is an upstream regulator of MYCN expression (58). It was further suggested that AHR and E2F1 are involved in mitochondrial function. Interaction between AHR and ATP5α1, a subunit of complex V, was reported, from which the authors hypothesized that AHR contributes to homeostasis of mitochondrial function and cellular energy metabolism (60). Knockdown of E2F1 in HeLa cells produced highly elevated mRNA levels of genes involved in mitochondrial biogenesis (61). Concurrent findings also support a role for E2F1 in regulating energy homeostasis and mitochondrial function: E2F1 causes a shift from oxidative to glycolytic metabolism under stress conditions (62).

The monocarboxylate transporter (MCT) family member MCT1 can transport both lactate and pyruvate. High activity of MCT1 was correlated with MYCN amplification in NBs. Inhibition of MCT1 causes acidosis and a significant reduction in viability of NB cells (63).

HIF-1α was shown to be expressed in both MYCN-amplified and non-amplified NBs. It seems HIF-1α in association with MYCN fortifies aerobic glycolysis in NBs. Knockdown of HIF1-α selectively decreased the expression of genes encoding GAPDH, HK2, PDK1, ALDA and LDHA in NBs. Concurrent knockdown of both MYCN and HIF-1α further reduced HK2 and LDHA expression (52).

Other regulators of metabolism in NB

Transient receptor potential cation channel, subfamily M, member 2 (TRPM2) is an ion channel that is frequently expressed in cancers. Full-length TRPM2 (TRPM2-L) protected NBs from moderate oxidative stress. The other form of TRPM2 is a dominant-negative short isoform (TRPM2-S). NBs which expressed TRPM2-S showed a significant reduction in tumor growth compared to TRPM2-L-expressing NBs. Significantly reduced expression of HIF-1/2α was detected in TRPM2-S-expressing NB cells. Interestingly, the target proteins regulated by HIF-1/2α in mitochondria (e.g., BNIP3, NDUFA4L2, COX4.1, and COX4.2) were significantly down-regulated in these cells (64).

It was demonstrated that HIF-2α also can play an indirect role in NB metabolism. Over-expressed HIF-2α but weak HIF-1α expression was detected in well-vascularized areas of NBs (65,66). Vascularization, which enables access to more oxygen and metabolites like glucose, glutamine, etc., is necessary for tumor cells. Moreover, blood vessels help tumor cells to get rid of metabolic waste products.

Alterations of the energy metabolism in WT

The role of fluoro-D-glucose (FDG)-PET in WTs has not been well established (82). However, there are some case reports demonstrating the WTs can be monitored by PET examination (83). In classical triphasic WT, the epithelial, blastemal and stromal elements differ in terms of mitochondrial energy metabolism. Normal mitochondrial mass was reported for the blastemal and epithelial areas. In contrast, the stroma regions showed severe loss of mitochondria compared to normal kidney tissue, resulting in loss of all OXPHOS complexes, as indicated by low porin expression and citrate synthase activity. The mtDNA copy number was also reduced in WT, which can partly explain the reduction in OXPHOS enzymes (84). These observations are supported by ultrastructural studies, which showed that mesenchymal cells contain few mitochondria, whereas primitive epithelial cells possess numerous mitochondria (85).

In agreement with these data, Hammer and colleagues detected a general decrease of mitochondrial metabolism in WTs. By mass spectrometric analyses of formalin-fixed paraffin-embedded tissue sections, they showed that proteins involved in OXPHOS and other mitochondrial functions (OXPHOS complex components and TCA cycle enzymes) were present at lower levels in WT samples compared with normal kidney tissue. In general, many other metabolic proteins were found to be decreased in WTs compared to normal tissue, for example aldehyde dehydrogenases and proteins related to propanoate and butanoate metabolism, long-chain fatty acid metabolism, and degradation of the branched-chain amino acids valine, leucine and isoleucine. However, because the tissue samples were obtained after chemotherapy treatment, it is unclear if these changes are also present in primary, untreated WT (86).

In WTs, HIF-1α has been found to be expressed at higher levels compared to normal kidney. Moreover, localization of HIF-1α in the tumor tissue is predominantly nuclear, whereas in normal kidney it is cytoplasmic (87,88). As previously mentioned, HIF-1α plays an important role in tumor development in different ways. For example, HIF-1α increases the expression and activity of many different glycolytic protein isoforms, including the transporters GLUT1 and GLUT3 that transport glucose across the plasma membrane, enhancing tumor glycolytic flux. HIF-1α seems also to be involved in the modulation of mitochondrial function and oxygen consumption by inactivating the pyruvate dehydrogenase (PDH) complex in some tumor types (89). Therefore, overexpression of HIF-1α and its nuclear localization, together with the reductions in mitochondrial biogenesis and/or OXPHOS activity found in WTs, suggest that these types of tumors are characterized by the metabolic switch to aerobic glycolysis.

Interestingly, there are no further studies available which have investigated the metabolism of WT in detail regarding the metabolic pathways known to be altered in a range of cancers (see “Regulation of cancer metabolism”). There is only indirect evidence from the genes altered in WT that there are alterations of cell metabolism.

The nexus between genetic alterations in WT and energy metabolism

About 5-6% of WTs present with TP53 mutations. Interestingly, TP53 mutations are mostly found in anaplastic WT, indicating an association with a more aggressive tumor phenotype (58,90,91). TP53 is a tumor suppressor gene involved in the development of many types of cancer. It plays a role in the regulation of many genes, including acting as a transcriptional repressor of GLUT1, and therefore it is important in glycolysis regulation. In anaplastic WT a stronger expression of GLUT1 compared to non-anaplastic WT was observed (92). Moreover, as previously mentioned, GLUT1 is stimulated by HIF-1α. Since p53 mediates the repression of HIF-1α, HIF-1α and p53 seem to cooperate in establishing the Warburg effect in anaplastic WTs and thereby could be responsible for the development of an aggressive phenotype. Interestingly, Zawacka-Pankau et al. showed that pharmacological re-activation of p53 in tumor cell lines and xenografts decreases the levels of HIF-1α, both in normoxic and hypoxic conditions, and they also found that p53 binds to the promoter of HIF-1α, inhibiting its expression. Moreover, they showed that the pharmacological re-activation of p53 is able to down-regulate metabolic genes, including the gene responsible for the transcription of GLUT1, confirming the interdependence of p53 and HIF-1α in the regulation of the Warburg effect in cancer cells (93). The morphology and physiology of WTs resemble those of the developing kidney (94). For example, the WNT pathway is required during embryonic development to maintain the pluripotency and plasticity of human embryonic stem cells (71). Activation of the WNT/β-catenin pathway is an important hallmark of stemness of cancer stem cells, and β-catenin mutations have been found in 15% of WTs (95,96). In WTs, mutations affecting CTNNB1, the gene encoding β-catenin, usually result in constitutive activation of the WNT/β-catenin signaling pathway (95). In one study, the authors reported that at least 50% of tumors with WT1 mutations also had a β-catenin mutation (97,98).

Recently, it was shown that WNT signaling works in a context and tissue specific manner. So far 19 WNT genes were identified. WNT signaling can be divided into a canonical and a non-canonical pathway, which is β-catenin independent. In a recent paper it was shown that WNT3A (non-canonical) can activate mitochondrial biogenesis and oxygen consumption in mouse myotubes. WNT5A suppressed this action (99). In contrast, WNT3A was shown to increase glycolytic capacity during osteoclast differentiation (100). In addition, WNT5A expression correlated with good or bad prognosis depending on the analyzed tumor type (101-104). Activation of the canonical WNT/β-catenin signaling was shown to increase aerobic glycolysis through suppression of mitochondrial respiration (105,106).

An important downstream target of the WNT/β-catenin pathway is MYC, a proto-oncogene that functions as transcription factor for many genes involved in cell cycle. The MYC and MYCN genes have similar 5' noncoding exons and extensive homology in the coding regions, and they are both overexpressed in many WTs (107,108). As MYC promotes glycolysis and energy production and is a key regulator of glutaminolysis in transformed cells (109), and MYCN in association with HIF-1α increases the transcription of glycolytic enzymes (52), these observations indicate that the WNT signaling pathway influences cancer cell metabolism.

Expression of IGF-2 is increased in 69% of WTs (110). IGF-2 is an imprinted gene normally expressed only from the paternal allele. In sporadic WTs, this gene often exhibits bi-allelic expression (111). An increase of IGF-1 or IGF-2 leads to activation of the insulin signaling pathway. A cascade of events leads to the activation of the PI3K-mitogen activated protein kinase (MAPK)-Akt signaling pathway that results in an increase in glucose uptake and metabolism. Furthermore, activation of Akt signaling via down-regulation of PGC-1α decreases mitochondrial biogenesis (6). WT1, located on chromosome 11p13, plays an important role in genitourinary development, encoding a zinc finger transcription factor and acting as a tumor suppressor. WT1 loss-of-function mutations occur in 10-20% of WT cases (95,97). WTX, located on chromosome Xq11.1, was also found to be involved in WT development. The prevalence of WTX mutations is approximately 18% in WTs (112). Both WT1 and WTX mutations cause dysregulation of the WNT/β-catenin pathway, and consequently abnormal cell proliferation (97,112).

Mutations in the WT1 gene can affect E-cadherin expression. It can induce epithelial differentiation during mesenchymal-epithelial transition and E-cadherin expression in the embryonic kidney (113). Loss or reduction of mtDNA results in hypermethylation of the E-cadherin gene promoter (114). When the tumor cell lines, LNrho0-8 (derived from the LNCaP prostate cancer cell line) and MCFrho0 (derived from the MCF-7 breast cancer cell line) were depleted of mtDNA by treatment with ethidium bromide, the cells lost their epithelial features and gained a mesenchymal phenotype. Furthermore, repression of E-cadherin and induction of vimentin expression were observed. Down-regulation of E-cadherin in LNrho0-8 and MCFrho0 cells was shown to be due to hypermethylation of the E-cadherin promoter region (115). We propose that the reduction of mtDNA copy number leading to a decline in OXPHOS activity in WT induces epigenetic changes in nuclear DNA, influencing EMT. Because only 10-15% of sporadic (116,117) WTs carry a WT1 mutation, but many have low mtDNA levels, low mtDNA and OXPHOS might be the main trigger for low E-cadherin expression.

It has been shown in human embryonic kidney cells and murine mesonephric cells, that inducing or repressing WT1 expression affects the expression of genes involved in the synthesis of cholesterol and fatty acids (118). The mevalonate pathway is involved in the production of cholesterol, vitamin D, steroid hormones and bile acids. Transcription of these genes needs activation by sterol regulatory element-binding proteins (SREBPs). Rae et al. hypothesize that WT1 interacts with SREBP to block transcription of mevalonate pathway genes (118). Therefore, it is reasonable to speculate that, in tumors where WT1 is mutated, the synthesis of cholesterol and other fatty acids is increased. This would offer an advantage to the tumor since these are building blocks for cellular membranes and thus important for cell proliferation.

Funding: This work was supported by the Vereinigung zur Förderung der pädiatrischen Forschung und Fortbildung Salzburg, Children Cancer Foundation, the Austrian Research Promotion Agency (822782/THERAPEP) and the Mitochondrial European Educational Training (MEET), ITN MARIE CURIE PEOPLE (317433).