Abstract
To date, the consequences of succinate dehydrogenase (SDH) impairment on overall mitochondrial functions are still obscure. In this study, we evaluated SDH activity and expression and mitochondrial homeostasis in 57 tissue samples of pheochromocytoma (PHEO)/paraganglioma (PGL) obtained from patients genotyped for PHEO/PGL susceptibility genes. The resulted SDH activity and content always decreased in SDH-mutated tumors, in one out of two MAX-mutated patients and in four patients resulted wild type (wt) at genetic screening. All these four wt patients were further screened for large deletions in SDH genes, TMEM127 and MAX and resulted wt but two had somatic SDHD mutations. The RT-PCR in the MAX-mutated sample suggests that the decrease in SDH depends on complex instability and not on a reduced SDHB expression. SDH mutations neither alter citrate synthase (CS) activity nor the content of voltage-dependent anion channel (VDAC) while the expression of the mitochondrial complex IV (cytochrome c oxidase (COX)) was found extremely variable in all (mutated and wt) samples suggesting an impairment of mitochondrial cristae in these tumors. In conclusion, tumors from patients with germ line SDH mutations invariably show decreased enzymatic activity and content, but an SDH impairment may also depend on SDH somatic mutations or, seemingly, on MAX mutations. The impaired SDH activity in the two wt tissues suggests mutations in other still unknown susceptibility genes. Finally, the extreme variability in COX expression levels is yet to be explained and this strongly suggests to evaluate other mitochondrial features to better understand the mitochondrial role in the pathogenesis of these tumors.
Introduction
Pheochromocytoma (PHEO)/paraganglioma (PGL) are neural crest-derived tumors. According to their differentiation, they can be classified into two types: PGLs that are sympathetic in origin, catecholamine secreting, and mostly located in the abdomen (mainly the adrenals) and in the thorax (sPGL) and PGLs that are parasympathetic in origin, nonsecreting or poorly secreting, and mostly located in the head and neck region (HN-PGL). In the last years, it has been demonstrated that at least 30% of all the PGLs are indeed due to a germ line mutation in one of the susceptibility genes (Neumann et al. 2002).
In addition to the tumor suppressor gene VHL (Latif et al. 1993, Walther et al. 1999), the proto-oncogene RET (Neumann et al. 1993, Eng et al. 1994, Eng 1996), the tumor suppressor gene NF1 (White & O'Connell 1991), the newly discovered TMEM127 (Qin et al. 2010), and the lately found MAX (Comino-Méndez et al. 2011), this group of genes also includes the genes encoding the four subunits of the succinate dehydrogenase (SDH), enzyme involved in the tricarboxylic acid cycle (Baysal et al. 2000, Niemann & Müller 2000, Astuti et al. 2001, Burnichon et al. 2010) and in the mitochondrial electron transport chain, and the recently identified gene SDHAF2 (SDH5) that is responsible for the flavination of the SDHA subunit (Hao et al. 2009).
Mitochondrial SDH is formed by four subunits: SDHA, a flavoprotein, and SDHB, an iron–sulfur protein, are the two catalytic subunits that are anchored to the inner mitochondrial membrane by two smaller structural subunits (SDHC and SDHD), containing cytochrome b and ubiquinone binding sites. These subunits are entirely encoded by nuclear genes located on chromosomes 5p15 for SDHA (with a pseudogene on 3q23), 1p36.13 for SDHB, 1q21.23 for SDHC, and 11q23 for SDHD.
It has also been described in another gene, named SDHAF1, which encodes a protein responsible for the SDH assembling (Ghezzi et al. 2009), but the correlation between this gene and the onset of PGL is yet to be established.
Germ line mutations in SDHB, SDHC, SDHD, and SDHAF2 genes are responsible for the occurrence of syndromes named PGL4, PGL3, PGL1, and PGL2 respectively. PGL1 presents mostly benign HN-PGL possibly associated with sPGL (Neumann et al. 2004, Simi et al. 2005). PGL2 is rare, only found in two families, and exclusively presents HN-PGL (Hao et al. 2009). PGL3 is mainly characterized by single HN-PGL (Schiavi et al. 2005) but the occurrence of abdominal sPGL has also been reported (Mannelli et al. 2007). PGL4 presents sPGL, which, at variance with tumors developed in PGL1 and PGL3, is malignant in more than 30% of cases, leading to metastatic disease (Linnoila et al. 1990, Amar et al. 2005, Pham et al. 2006, Timmers et al. 2007).
The two most common PGL syndromes, namely PGL1 and PGL4, also differ for penetrance (higher in PGL1) and mode of genetic transmission (maternal genomic imprinting in PGL1).
Heterozygous mutations in SDHA have been shown to cause the occurrence of abdominal sPGL (Burnichon et al. 2010).
At present, very few information is available on the biochemical/functional modifications induced by gene mutations in these tumors. Indeed, to date, the correlation between SDH mutations and SDH activity has been studied only in a French family mutated for SDHD and in two case reports mutated for SDHB and SDHA respectively (Gimenez-Roqueplo et al. 2001, 2002, Burnichon et al. 2010) or in 22 cases of HN-PGL (Douwes Dekker et al. 2003).
In the last few years, we have diagnosed and treated many patients affected by sporadic as well as familial PHEO/PGL. Some of these patients were found to be SDHB, SDHC, or SDHD mutation carriers. The type of mutations ranged from nonsense to missense, frame-shift, splicing alteration. In addition, some patients presented a clinical picture suggesting a PGL syndromic disease (i.e. multiple or recurrent PGLs) but, unexpectedly, resulted wild type (wt) at genetic analysis. Here, we discuss the results obtained on mitochondrial function in 57 cases of sPGL/HN-PGL, in which SDH activity and assembling have been evaluated.
Materials and methods
Tissue specimens
According to a protocol approved by our Local Ethics Committee, we have collected many tissue specimens as well as blood samples for DNA and mRNA analyses and biochemical and morphological assays. All patients provided informed consent. In the present work, we evaluated 57 cases of sPGL/HN-PGL (Table 1). After surgery, tissues were immediately frozen in liquid nitrogen and stored at −80 °C.
Schematic representation of the 57 patients analyzed
| No. of cases | Genetic analysis | Mutations | |
|---|---|---|---|
| sPGL wt | 32 | 22 wt | |
| 10 (SDHBa and SDHD) polymorphisms | |||
| sPGL mut | 10 | 3 SDHD | p.Gln109X (2) |
| p.Pro81Leu | |||
| 1 SDHC | p.Trp42X | ||
| 1 SDHB | p.Thr193His Fs28X | ||
| 2 RET | p.Cys634Tyr | ||
| 1 VHL | p.Leu198Val | ||
| 2 MAX | p.Leu94Pro | ||
| p.Arg33X | |||
| HN-PGL wt | 4 | 4 wt | |
| HN-PGL somatic mut | 2 | 2 SDHD | p.Asp113MetfsX21 |
| p.Leu7CysfsX7 | |||
| HN-PGL mut | 9 | 7 SDHD | p.Pro81Leu (3) |
| p.Gln109X | |||
| p.Tyr114Cys | |||
| p.IVS2-1G>T | |||
| Large deletion | |||
| 2 SDHB | p.Cys191Tyr | ||
| p.Met1Ile | |||
| Total | 57 |
One patient is polymorphic for both SDHB and MAX.
As control, we measured SDH enzymatic activity in four different adrenal glands obtained at surgery from donors for kidney transplantation and in the normal part of one kidney surgically removed for a tumor of the upper pole.
Mutation analysis
DNA was extracted from the peripheral blood leukocytes of each patient with the NucleoSpin Blood L kit (Macherey-Nagel, Düren, Germany) and analyzed for germ line mutations of SDHA (all exons), SDHB (all exons), SDHC (all exons), SDHD (all exons), SDHAF1 (all exons), SDHAF2 (all exons), MAX (all exons), TMEM127 (all exons), VHL (all exons), and RET (exons 5, 8, 10, 11, 13, 14, 15, and 16). For each gene, coding regions and exon–intron boundaries were amplified by PCR as described previously (Astuti et al. 2001). PCR products, purified with a commercial kit (PCR purification kit, Qiagen), were subjected to 2% agarose gel electrophoresis with ethidium bromide staining and subsequently sequenced with a genetic analyzer (ABI PRISM 310; Applied Biosystems, Milan, Italy). In all the germ line-mutated patients, loss of heterozygosity (LOH) analysis was investigated. Moreover, all the wt patients showing a decreased SDH activity and expression were also screened for somatic mutation for SDHA, SDHB, SDHC, SDHD, SDHAF1, SDHAF2, VHL, TMEM127, and MAX and for large deletion for SDHB, SDHC, SDHD, SDHAF1, SDHAF2, and VHL.
Multiplex ligation-dependent probe amplification reactions
For genomic rearrangements analysis, we used commercial kits for multiplex ligation-dependent probe amplification (MLPA)-based assays (SALSA MLPA P016B VHL and SALSA MLPA P226 SDHD from MRC-Holland, Amsterdam, The Netherlands) following manufacturers' instructions.
Amplification products were diluted in HiDi formamide containing 500TAMRA internal size standards (MRC-Holland) and then separated by size with an ABI PRISM 310 Genetic Analyser (Applied Biosystems). Electropherograms were analyzed using Coffalyser MLPA DAT Software (MRC-Holland).
Tissue homogenates and lysates
Unless specified, all reagents were purchased from Sigma–Aldrich. Homogenates were prepared as already described (Kirby et al. 2007) with minor modifications. Briefly, dissected tissues (50–150 mg) were finely chopped; resuspended in a solution containing 120 mM KCl, 20 mM HEPES, 2 mM MgCl2, 1 mM EGTA, and 5 mg/ml BSA; and homogenized in a hand-held glass/glass homogenizer. The homogenate was centrifuged at 800 g for 10 min at 4 °C and the enzyme assays were performed on the supernatant. For western blot analysis, chopped tissues were lysed in lysis buffer (containing 50 mM Tris–HCl, pH=7.5, 120 mM NaCl, 1 mM EGTA, 6 mM EDTA, 15 mM Na4P2O7, 20 mM NaF, 1% Triton X-100, and protease inhibitor cocktail) homogenized in a hand-held glass/glass homogenizer and incubated for 30 min on ice. Lysates were clarified by centrifugation at 10 000 g for 15 min at 4 °C. Supernatants were quantified for protein content (Coomassie Blue reagent was from Bio-Rad; Rapizzi et al. 2009). All procedures were carried out on ice or at 4 °C.
SDH and citrate synthase activity
Tissue homogenates (50 μg) were incubated in a phosphate buffer containing sodium azide, 2,6-dichlorophenolindophenol (DCPIP), sodium succinate, and phenazine methosulfate. Complex II-specific activity was evaluated by measuring the decrease in absorbance due to the oxidation of DCPIP at 600 nm (Kirby et al. 2007). Citrate synthase (CS) activity was measured by incubating the homogenates (25 μg) in a Tris buffer containing acetyl coenzyme A, 5,5′-dithiobis(2-nitrobenzoic acid), and oxaloacetate and measuring the increase in absorbance at 412 nm (Kirby et al. 2007). The enzymatic activities were evaluated by photometry using the Victor3 1420 Multilabel Counter (Packard Instruments, Perkin-Elmer, Waltham, MA, USA).
In all the experiments, homogenates and lysates extracted from different healthy tissues, such as liver, kidney, and adrenal gland, were used as controls. As no relevant differences were observed among these tissues, we decided to show in the figures only the results obtained from the adrenal gland (considered to be 100%).
Western blot
Samples of clarified lysates (30 μg proteins) were separated by SDS/PAGE and transferred onto PVDF (Immobilon, Millipore, MA, USA) as described previously (Rapizzi et al. 2009). Bound antibodies were detected using ECL reagents (Immobilon) and analyzed using a Bio-Rad ChemiDoc Imaging System for dedicated chemiluminescent image acquisition (Imaging and Analysis Software by Bio-Rad, Quantity-One). The polyclonal anti-SDHB was from Atlas Antibodies (Stockholm, Sweden) and the monoclonal anti-subunit IV of complex IV was from Invitrogen (Molecular Probes, Inc., Eugene, OR, USA). The polyclonal anti-VDAC1, the polyclonal anti-actin, and all the secondary antibodies, such as the anti-rabbit, the anti-mouse, and the anti-goat IgG conjugated to HRP, were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
RNA isolation and quantitative real-time RT-PCR
Total RNA was extracted from one normal adrenal gland and from the MAX-mutated (p.Leu94Pro) patient using RNeasy Mini Kit (Qiagen). Total RNA was reverse transcribed using TaqMan RT PCR kit (Applied Biosystems). Gene expression measurement was performed by TaqMan using the 7900HT Sequence Detection Systems (Applied Biosystems, Foster City, CA, USA) as detailed elsewhere (Lombardi et al. 2008). Primers/probes for SDHA (Hs00417200_m1), SDHB (Hs01042482_m1), SDHC (Hs01698067_s1), and SDHD (Hs01098144_g1; TaqMan Gene Expression Assays; Applied Biosystems, Warrington, UK) were used. The amount of target, normalized to the human endogenous reference GAPDH (Pre-Developed TaqMan Assay Reagents, Applied Biosystems) and relative to a calibrator (quantitative PCR human reference total RNA; Stratagene, Santa Clara, CA, USA), was given by 
Statistical analysis
Statistical analysis was performed using Student's t-test, and P<0.05 was considered significant. Data are reported as mean±s.d. of at least three experiments with similar results.
Results
In this study, 42 tissue samples of sPGL and 15 samples of HN-PGL (Table 1) were evaluated. At genetic analysis, 32 sPGLs resulted wt (22 without any germ line mutations and ten with polymorphic variants) and ten resulted with germ line mutations (three SDHD, one SDHC, one SDHB, two RET, one VHL, and two MAX) while among the 15 HN-PGLs, four resulted wt, nine resulted with germ line mutations (seven SDHD and two SDHB), and two with SDHD somatic mutations. A significant decrease in SDH activity, ranging from 60 to 80%, was found in all the SDH-mutated sPGLs, and as shown in Fig. 1A, a decrease in SDH activity, ranging from 40 to 80%, was also found in two out of 32 wt sPGLs and one MAX-mutated sPGL. Tumors mutated for RET and VHL presented a SDH activity comparable with that of controls.
SDH activity and expression in sPGL. (A) The histogram represents the SDH activities expressed as the percentage of the activity measured in tissue homogenates. (B) Blot is representative of three independent experiments, and SDHB protein expression values were normalized on actin (blot not showed). Bars are the mean±s.d. of three independent experiments, *P<0.05 compared with control. Two out of 32 wt patient tumors (dark gray bars), one MAX-mutated out of five tumors mutated for other susceptibility genes (black and white bars), and all SDH-mutated tissues (white bars) show a significantly decreased SDH activity (A) and SDHB expression (B) compared with control (black bar). Bar (a) is the mean±s.d. of SDH activity and expression of the 30 wt sPGL showing no significant differences compared with control. Bars (b and c) represent the highest and lowest level of SDH activity (A) and expression (B) measured in wt sPGL.
Citation: Endocrine-Related Cancer 19, 3; 10.1530/ERC-11-0263
Similar results were obtained in HN-PGL tissues (Fig. 2A). In fact, all the SDH-mutated tumors showed a significant decrease in SDH activity, ranging from 50 to 90%. The two tissue samples showing a decreased SDH activity and classified as wt at genetic analysis were found to have SDHD somatic mutations. One of these mutations (p.Asp113MetfsX21) has been previously described by Cascon et al. (2002), while the second is a novel mutation (p.Leu7CysfsX7; Fig. 3).
SDH activity and expression in HN-PGL. (A) The histogram represents the SDH activities expressed as the percentage of the activity measured in tissue homogenates. (B) Blot is representative of three independent experiments, and SDHB protein expression values were normalized on actin (blot not showed). Bars are the mean±s.d. of three independent experiments, *P<0.05 compared with control. The two patients with the somatic mutations (light gray bars) and all SDH-mutated tissues (white bars) show a significantly decreased SDH activity (A) and SDHB expression (B) compared with control (black bar).
Citation: Endocrine-Related Cancer 19, 3; 10.1530/ERC-11-0263
Electrophoresis patterns of the two somatic SDHD mutations. The DNA mutations are indicated by the small arrow.
Citation: Endocrine-Related Cancer 19, 3; 10.1530/ERC-11-0263
The SDH expression in tumor samples, as evaluated by western blot analysis, closely correlated with the results of enzymatic assay, either in sPGL or in HN-PGL samples (Figs 1B and 2B respectively). LOH was found only in 70% of the patients with a decreased SDH activity and expression, while the remaining 30% showed a loss of SDH protein and function even in the absence of LOH.
To investigate the mechanism responsible for the decreased SDH activity and expression in one of the two MAX-mutated samples (p.Leu94Pro), we performed an RT-PCR study on the four SDH subunit transcript levels. While in the control normal adrenal tissue, mRNA expression of the four subunits resulted, as expected, similar, in the MAX-mutated tissue, the transcript levels of SDHB, SDHC, and SDHD, were found slightly increased compared with control (Fig. 4).
mRNA expression levels of SDH subunits. Real-time PCR analysis in a normal adrenal gland and in the MAX-mutated (p.Leu94Pro) tissues. Quantitative mRNA analysis was performed by simultaneous amplification of the target SDHA, SDHB, SDHC, and SDHD transcripts. Results are expressed as fold changes according to the 
Citation: Endocrine-Related Cancer 19, 3; 10.1530/ERC-11-0263
At variance with SDH activity and expression, the activity of CS, which is an enzyme present in the mitochondrial matrix and is involved in the TCA cycle (Fig. 5), and the expression of voltage-dependent anion channel (VDAC), a protein located on mitochondria outer membrane (Fig. 6), were found normal and comparable among all the tissues both in sPGL and in HN-PGL while the expression of cytochrome c oxidase (COX), which is an enzyme of the respiratory chain located in the inner mitochondrial membrane as mitochondrial complex IV, has been found extremely variable among the different tissue samples (Fig. 7).
Citrate synthase (CS) activity in sPGL (A) and HN-PGL (B). The histogram represents the CS activities expressed as the percentage of the activity measured in tissue homogenates. Bars are the mean±s.d. of three independent experiments (each of them conducted in duplicate samples). In both sPGL and HN-PGL, all wt (dark gray bars), RET-, VHL-, MAX- (black and white bars), and SDH-mutated (light gray or white bars) patient tumors show a CS activity comparable with control (black bar). In A, bar (a) is the mean±s.d. of CS activity of the 32 wt sPGL, bars (b and c) are the highest and lowest CS activity, respectively, measured in wt tumors.
Citation: Endocrine-Related Cancer 19, 3; 10.1530/ERC-11-0263
Study of voltage-dependent anion channel (VDAC) expression by western blot analysis in sPGL and HN-PGL. Blot is representative of three independent experiments, and the bar graphs are the mean±s.d. of VDAC protein expression values normalized on actin (blot not showed). In both sPGL and HN-PGL, all wt (dark gray bars), SDH-polymorphic (light gray bars), RET-, VHL-, MAX- (black and white bars), and SDH-mutated (light gray or white bars) tumors show VDAC expression levels comparable with control (black bar). In A, bar (a) is the mean±s.d. of VDAC expression of the 32 wt sPGL, bars (b and c) are the highest and lowest VDAC levels, respectively, measured in wt tumors.
Citation: Endocrine-Related Cancer 19, 3; 10.1530/ERC-11-0263
Study of cytochrome c oxidase (COX) expression by western blot analysis in sPGL and HN-PGL. Blot is representative of three independent experiments, and the bar graphs are the mean±s.d. of COX protein expression values normalized on actin (blot not showed). *P<0.05 vs control. In sPGL, eight out of 32 wt tumors (dark gray bars), the two MAX-mutated tissues (black and white bars), and one SDH-mutated patient (white bars) show a significantly decreased COX expression compared with control (black bar), while three wt tumoral tissues (dark gray bars) and four out of five SDH-mutated tissues (white bars) show a significantly increased COX expression. In the HN-PGL, one out of four wt tumors (dark gray bars), one SDH somatic-mutated tumor (light gray bars), and four SDH-mutated patients (white bars) show a significantly decreased COX expression, while one SDH somatic-mutated tumor (light gray bars) and one SDH-mutated tumoral tissue (white bars) show a significantly increased COX expression compared with control (black bar). In A, bar (a) is the mean±s.d. of COX expression of the 21 wt sPGLs not showing significant differences compared with control. Among these patients, bars (b and c) represent the wt patients with highest and lowest COX levels respectively.
Citation: Endocrine-Related Cancer 19, 3; 10.1530/ERC-11-0263
Discussion
To the best of our knowledge, here for the first time SDH activity and expression have been systematically studied in sPGLs and HN-PGLs. Indeed, until now, only two research groups measured SDH activity in SDH-mutated tumors. In particular, Gimenez-Roqueplo et al. (2001) reported that in a French family, a mutation in SDHD results in the lack of SDH activity, and they also found a loss of SDH activity in two patients with SDHB and SDHA mutations (Gimenez-Roqueplo et al. 2002, Burnichon et al. 2010). Douwes Dekker et al. (2003) also investigated SDH expression beside the SDH activity, but only in HN-PGLs.
As expected, we also observed that in SDH-mutated patients, SDH enzymatic activity is always decreased and that this loss of activity closely correlates with a decrease in protein expression.
This observation is in accordance with that previously reported by Dahia et al. (2005) and by Favier et al. (2009). Indeed, as already suggested for the not flavinated subunit A (Hao et al. 2009), in presence of mutations in one of the SDH subunits, the nonfunctional assembled SDH complex might be unstable and, as a consequence, more susceptible to degradation.
Interestingly, in addition to the SDH-mutated samples, a decrease in SDH activity and expression was also found in three sPGLs, two classified as wt and one as MAX-mutated, and in two HN-PGLs classified as wt at peripheral DNA genetic screening.
Concerning the MAX-mutated tissue, some hypotheses can be assumed to explain the decreased complex II activity and expression. Indeed, we cannot exclude that this finding might be due to a mutation in another still unknown gene involved in the correct SDH assembling/function. Protein MAX is a member of the basic helix–loop–helix leucine zipper family of transcription factors and, depending on its association with MYC or Mad, the formed complex behaves as a transcriptional activator or a transcriptional repressor respectively. Accordingly, MAX might regulate the expression of some genes implicated in SDH formation, but this hypothesis is not confirmed by our RT-PCR experiments. Indeed, in the MAX-mutated tissue, the SDHB, SDHC, and SDHD transcripts were slightly increased not only in comparison with the SDHA transcript but also in comparison with the SDH transcripts of the healthy adrenal gland, thus suggesting an SDH complex instability and disassembling more than a negative transcriptional regulation in the MAX-mutated tissue.
As the other MAX-mutated tissue showed a normal SDH activity, a study on a larger number of MAX-mutated tissues is necessary to understand the relationship, if any, between SDH activity and MAX mutations.
The presence of SDHD somatic mutations explains the reduced SDH activity and content in the two HN-PGLs resulted wt at peripheral DNA genetic screening. Such somatic mutations as well as large deletions were not found in the two sPGLs resulted with wt at genetic screening. Therefore, a possible explanation for such a finding might be a mutation in one so far undiscovered susceptibility gene involved in SDH assembling and function.
It is well known that mitochondria are dynamic organelles that can change in number and morphology. Mitochondrial integrity is maintained through a continuous process by which mitochondria divide and fuse forming a dynamic interconnecting network, which undergoes remarkable structural changes both during apoptosis and under oxidative stress. To investigate whether our patients were also showing other alterations in other mitochondrial enzyme activities, we studied the activity of the CS. The CS is an enzyme present in the mitochondrial matrix and belongings to the TCA. Interestingly, CS activity was comparable among all the tissues, thus suggesting that enzyme function within the mitochondrial matrix was not impaired by the presence of SDH mutations.
As SDH is a complex present in the inner mitochondrial membrane and personal preliminary data obtained by electron microscopy on SDH-mutated sPGL and HN-PGL seem to suggest a structural alteration of mitochondrial cristae, we also investigated the expression levels of VDAC, a channel present on mitochondrial outer membrane and of COX, an enzyme of the respiratory chain present in the inner mitochondrial membranes. Interestingly, the expression of COX has been found extremely variable in SDH-mutated as well as in SDH wt tumoral tissues, while VDAC content was comparable among all the samples. These observations suggest that in these tumors, only the mitochondrial cristae are altered, while the matrix and the outer membrane do not seem to be consistently modified.
In conclusion, an impairment of SDH activity can not only be due to germ line mutations in SDH-related genes but also due to somatic SDH mutations in tumor tissue and possibly due to germ line mutations in other known (i.e. MAX) or unknown susceptibility genes. Moreover, SDH impairment seems to alter neither the outer mitochondrial membrane, as shown by the normal VDAC expression, nor the mitochondrial matrix, as shown by the normal CS activity. At variance, the wide difference in COX activity, either in SDH-mutated or wt tissues, suggests an alteration in mitochondrial cristae in PHEO/PGL, but additional studies are necessary to explain such high variability and to fully understand the mitochondrial role in the pathogenesis of these tumors.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work has been supported by Fondazione Cassa di Risparmio di Pistoia e Pescia (Prot. 2010.0278), Istituto Toscano Tumori (ITT, Prot. AOOGRT/325462/Q.80.110) to M Mannelli and E Rapizzi is recipient of ITT fellowship.
Acknowledgements
E Rapizzi, T Ercolino, L Canu, and M Mannelli are members of the ENS@T (European Network for the Study of Adrenal Tumors).
References
Amar L, Bertherat J, Baudin E, Ajzenberg C, Bressac-de Paillerets B, Chabre O, Chamontin B, Delemer B, Giraud S & Murat A et al. 2005 Genetic testing in pheochromocytoma or functional paraganglioma. Journal of Clinical Oncology 23 8812–8818. doi:10.1200/JCO.2005.03.1484.
Astuti D, Latif F, Dallol A, Dahia PL, Douglas F, George E, Sköldberg F, Husebye ES, Eng C & Maher ER 2001 Gene mutations in the succinate dehydrogenase subunit SDHB cause susceptibility to familial pheochromocytoma and to familial paraganglioma. American Journal of Human Genetics 69 49–54.Erratum in: Am J Hum Genet 2002, 70 565 doi:10.1086/321282.
Baysal BE, Ferrell RE, Willett-Brozick JE, Lawrence EC, Myssiorek D, Bosch A, van der Mey A, Taschner PE, Rubinstein WS & Myers EN et al. 2000 Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma. Science 287 848–851. doi:10.1126/science.287.5454.848.
Burnichon N, Brière JJ, Libé R, Vescovo L, Rivière J, Tissier F, Jouanno E, Jeunemaitre X, Bénit P & Tzagoloff A et al. 2010 SDHA is a tumor suppressor gene causing paraganglioma. Human Molecular Genetics 19 3011–3020. doi:10.1093/hmg/ddq206.
Cascon A, Ruiz-Llorente S, Cebrian A, Telleria D, Rivero JC, Diez JJ, Lopez-Ibarra PJ, Jaunsolo MA, Benitez J & Robledo M 2002 Identification of novel SDHD mutations in patients with phaeochromocytoma and/or paraganglioma. European Journal of Human Genetics 10 457–461. doi:10.1038/sj.ejhg.5200829.
Comino-Méndez I, Gracia-Aznárez FJ, Schiavi F, Landa I, Leandro-García LJ, Letón R, Honrado E, Ramos-Medina R, Caronia D & Pita G et al. 2011 Exome sequencing identifies MAX mutations as a cause of hereditary pheochromocytoma. Nature Genetics 43 663–667. doi:10.1038/ng.861.
Dahia PL, Ross KN, Wright ME, Hayashida CY, Santagata S, Barontini M, Kung AL, Sanso G, Powers JF & Tischler AS et al. 2005 A HIF1alpha regulatory loop links hypoxia and mitochondrial signals in pheochromocytomas. PLoS Genetics 1 72–80. doi:10.1371/journal.pgen.0010008.
Douwes Dekker PB, Hogendoorn PC, Kuipers-Dijkshoorn N, Prins FA, van Duinen SG, Taschner PE, van der Mey AG & Cornelisse CJ 2003 SDHD mutations in head and neck paragangliomas result in destabilization of complex II in the mitochondrial respiratory chain with loss of enzymatic activity and abnormal mitochondrial morphology. Journal of Pathology 201 480–486. doi:10.1002/path.1461.
Eng C 1996 Seminars in medicine of the Beth Israel Hospital, Boston. The RET proto-oncogene in multiple endocrine neoplasia type 2 and Hirschsprung's disease. New England Journal of Medicine 335 943–951. doi:10.1056/NEJM199609263351307.
Eng C, Smith DP, Mulligan LM, Nagal MA, Healey CS, Ponder MA, Gardner E, Scheumann GFW, Jackson CE & Tunnacliffe A et al. 1994 Point mutation within the tyrosine kinase domain of the RET proto-oncogene in multiple endocrine neoplasia type 2B and related sporadic tumours. Human Molecular Genetics 3 237–241.Erratum in: Hum Mol Genet 1994, 3 686 doi:10.1093/hmg/3.2.237.
Favier J, Brière JJ, Burnichon N, Rivière J, Vescovo L, Benit P, Giscos-Douriez I, De Reyniès A, Bertherat J & Badoual C et al. 2009 The Warburg effect is genetically determined in inherited pheochromocytomas. PLoS ONE 4 e7094 doi:10.1371/journal.pone.0007094.
Ghezzi D, Goffrini P, Uziel G, Horvath R, Klopstock T, Lochmüller H, D'Adamo P, Gasparini P, Strom TM & Prokisch H et al. 2009 SDHAF1, encoding a LYR complex-II specific assembly factor, is mutated in SDH-defective infantile leukoencephalopathy. Nature Genetics 41 654–656. doi:10.1038/ng.378.
Gimenez-Roqueplo AP, Favier J, Rustin P, Mourad JJ, Plouin PF, Corvol P, Rötig A & Jeunemaitre X 2001 The R22X mutation of the SDHD gene in hereditary paraganglioma abolishes the enzymatic activity of complex II in the mitochondrial respiratory chain and activates the hypoxia pathway. American Journal of Human Genetics 69 1186–1197. doi:10.1086/324413.
Gimenez-Roqueplo AP, Favier J, Rustin P, Rieubland C, Kerlan V, Plouin PF, Rötig A & Jeunemaitre X 2002 Functional consequences of a SDHB gene mutation in an apparently sporadic pheochromocytoma. Journal of Clinical Endocrinology and Metabolism 87 4771–4774. doi:10.1210/jc.2002-020525.
Hao HX, Khalimonchuk O, Schraders M, Dephoure N, Bayley JP, Kunst H, Devilee P, Cremers CW, Schiffman JD & Bentz BG et al. 2009 SDH5, a gene required for flavination of succinate dehydrogenase, is mutated in paraganglioma. Science 325 1139–1142. doi:10.1126/science.1175689.
Kirby DM, Thorburn DR, Turnbull DM & Taylor RW 2007 Biochemical assay of the respiratory chain complex activity. Methods in Cell Biology 80 93–119. doi:10.1016/S0091-679X(06)80004-X.
Latif F, Tory K, Gnarra J, Yao M, Duh F-M, Orcutt ML, Stackhouse T, Kuzmin I, Modi W & Geil L et al. 1993 Identification of the von Hippel–Lindau disease tumor suppressor gene. Science 260 1317–1320. doi:10.1126/science.8493574.
Linnoila RI, Keiser HR, Steinberg SM & Lack EE 1990 Histopathology of benign versus malignant sympathoadrenal paragangliomas: clinicopathologic study of 120 cases including unusual histologic features. Human Pathology 21 1168–1180. doi:10.1016/0046-8177(90)90155-X.
Livak KJ & Schmittgen TD 2001 Analysis of relative gene expression data using real time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25 402–408. doi:10.1006/meth.2001.1262.
Lombardi A, Cantini G, Piscitelli E, Gelmini S, Francalanci M, Mello T, Ceni E, Varano G, Forti G & Rotondi M et al. 2008 A new mechanism involving ERK contributes to rosiglitazone inhibition of tumornecrosis factor-alpha and interferon-gamma inflammatory effects in human. Arteriosclerosis, Thrombosis, and Vascular Biology 28 718–724. doi:10.1161/ATVBAHA.107.160713.
Mannelli M, Ercolino T, Giachè V, Simi L, Cirami C & Parenti G 2007 Genetic screening for pheochromocytoma: should SDHC gene analysis be included? Journal of Medical Genetics 44 586–587. doi:10.1136/jmg.2007.051045.
Neumann HP, Berger DP, Sigmund G, Blum U, Schmidt D, Parmer RJ, Volk B & Kirste G 1993 Pheochromocytomas, multiple endocrine neoplasia type 2, and von Hippel–Lindau disease. New England Journal of Medicine 329 1531–1538.Erratum in: N Engl J Med 1994, 331 1535 doi:10.1056/NEJM199311183292103.
Neumann HP, Bausch B, McWhinney SR, Bender BU, Gimm O, Franke G, Schipper J, Klisch J, Altehoefer C & Zerres K et al. 2002 Germ line mutations in non syndromic pheochromocytoma. New England Journal of Medicine 346 1459–1466. doi:10.1056/NEJMoa020152.
Neumann HP, Pawlu C, Peczkowska M, Bausch B, McWhinney SR, Muresan M, Buchta M, Franke G, Klisch J & Bley TA et al. 2004 Distinct clinical features of paraganglioma syndromes associated with SDHB and SDHD gene mutations. Journal of the American Medical Association 292 943–951. doi:10.1001/jama.292.8.943.
Niemann S & Müller U 2000 Mutations in SDHC cause autosomal dominant paraganglioma, type 3. Nature Genetics 26 268–270. doi:10.1038/81551.
Pham TH, Moir C, Thompson GB, Zarroug AE, Hamner CE, Farley D, van Heerden J, Lteif AN & Young WF Jr 2006 Pheochromocytoma and paraganglioma in children: a review of medical and surgical management at a tertiary care center. Pediatrics 118 1109–1117. doi:10.1542/peds.2005-2299.
Qin Y, Yao L, King EE, Buddavarapu K, Lenci RE, Chocron ES, Lechleiter JD, Sass M, Aronin N & Schiavi F et al. 2010 Germline mutations in TMEM127 confer susceptibility to pheochromocytoma. Nature Genetics 42 229–233. doi:10.1038/ng.533.
Rapizzi E, Taddei ML, Fiaschi T, Donati C, Bruni P & Chiarugi P 2009 Sphingosine 1-phosphate increases glucose uptake through trans-activation of insulin receptor. Cellular and Molecular Life Sciences 66 3207–3218. doi:10.1007/s00018-009-0106-3.
Schiavi F, Boedeker CC, Bausch B, Peçzkowska M, Gomez CF, Strassburg T, Pawlu C, Buchta M, Salzmann M & Hoffmann MM et al. 2005 Predictors and prevalence of paraganglioma syndrome associated with mutations of the SDHC gene. Journal of the American Medical Association 294 2057–2063. doi:10.1001/jama.294.16.2057.
Simi L, Sestini R, Ferruzzi P, Gaglianò MS, Gensini F, Mascalchi M, Guerrini L, Pratesi C, Pinzani P & Nesi G et al. 2005 Phenotype variability of neural crest derived tumours in six Italian families segregating the same founder SDHD mutation Q109X. Journal of Medical Genetics 42 e52 doi:10.1136/jmg.2004.030353.
Timmers HJ, Kozupa A, Eisenhofer G, Raygada M, Adams KT, Solis D, Lenders JW & Pacak K 2007 Clinical presentations, biochemical phenotypes, and genotype–phenotype correlations in patients with SDHB-associated pheochromocytomas and paragangliomas. Journal of Clinical Endocrinology and Metabolism 92 326–332. doi:10.1210/jc.2006-2315.
Walther MM, Reiter R, Keiser HR, Choyke PL, Venzon D, Hurley K, Gnarra JR, Reynolds JC, Glenn GM & Zbar B et al. 1999 Clinical and genetic characterization of pheochromocytoma in von Hippel–Lindau families: comparison with sporadic pheochromocytoma gives insight into natural history of pheochromocytoma. Journal of Urology 162 659–664. doi:10.1097/00005392-199909010-00004.
White R & O'Connell P 1991 Identification and characterization of the gene for neurofibromatosis type 1. Current Opinion in Genetics & Development 1 15–19. doi:10.1016/0959-437X(91)80034-J.
