Cancer Research Annual Meeting 2010 Protein Translation and Cancer
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Cancer Research 68, 4058, June 1, 2008. doi: 10.1158/0008-5472.CAN-07-5580
© 2008 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Cells Silenced for SDHB Expression Display Characteristic Features of the Tumor Phenotype

Ana M. Cervera1,2, Nadezda Apostolova2, Francisco Luna Crespo1,2, Manuel Mata3 and Kenneth J. McCreath1,2

1 Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain and 2 Unidad Mixta de Investigación Centro Nacional de Investigaciones Cardiovasculares-Universitat de Valencia; 3 Unidad Central de Investigación, Facultad de Medicina, Universitat de Valencia, Valencia, Spain

Requests for reprints: Kenneth J. McCreath, Department of Regenerative Cardiology, Centro Nacional de Investigaciones Cardiovasculares, Melchor Fernandez Almagro 3, Madrid 28029, Spain. Phone: 34-914531200; Fax: 34-914531265; E-mail: kmccreath{at}cnic.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 Conclusion
 Disclosure of Potential...
 References
 
Recently, enzymes of the tricarboxylic acid (TCA) cycle have emerged as novel tumor suppressors. In particular, mutations in the nuclear-encoded subunits of succinate dehydrogenase (SDHB, SDHC, and SDHD) cause paragangliomas and pheochromocytomas. Although the mechanism(s) by which disruption of mitochondrial metabolism leads to neoplasia is largely unknown, increasing evidence points to an activation of pseudohypoxia. In this study, we have shown that silencing of SDHB using DNA-based small interfering RNA resulted in major impairments in cellular proliferation, respiration, and a corresponding shift to glycolysis. The levels of reactive oxygen species, however, were unchanged. As expected, hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) and HIF-2{alpha} were up-regulated in chronically silenced cells, suggesting that a pseudohypoxic state was attained. In addition, the c-Jun amino-terminal kinase and p38 kinase stress signaling proteins were hyperphosphorylated in SDHB-silenced cells. Microarray analysis showed that >400 genes were influenced (6-fold or more up-regulation or down-regulation) by silencing of SDHB, confirming the importance of the TCA cycle in cellular metabolism. Examples of dysregulated genes included those involved in proliferation, adhesion, and the hypoxia pathway. Of interest, SDHB-silenced cells had a greater capacity to adhere to extracellular matrix components, including fibronectin and laminin, than control cells, thus suggesting a possible mechanism of tumor initiation. Although transient silencing of the HIF-1{alpha} transcription factor in SDHB-silenced cells had little effect on the expression of a subset of up-regulated genes, it partially reversed the adhesion phenotype to fibronectin, pointing to a potentially important role for HIF-1 in this process. [Cancer Res 2008;68(11):4058–67]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Conclusion
 Disclosure of Potential...
 References
 
The tricarboxylic acid (TCA) cycle, discovered over 70 years ago, occupies a central position in the pathways of carbohydrate catabolism, occurring between glycolysis and oxidative phosphorylation. The TCA cycle or Krebs cycle has recently gained attention in the field of tumor biology with the demonstration that two of its components, succinate dehydrogenase (SDH) and fumarate hydratase (FH), are authentic tumor suppressors (for a recent review, see ref. 1). Interest was first sparked by the identification of one of the four SDH subunits, SDHD, as the gene responsible for hereditary paraganglioma, a disorder characterized by slow growing, mostly benign, but highly vascularized, tumors in the carotid body sites of the head and neck region (2). SDHD was later found to also play a role in the pathogenesis of pheochromocytoma tumors, which are derived from chromaffin cells of the adrenal medulla (3). Other paraganglioma susceptibility genes were later found to be identical to SDH subunits SDHC (4) and SDHB (5). The FH gene, which is mutated in individuals with hereditary leiomyomatosis and renal cell cancer was identified recently as the fourth TCA cycle tumor suppressor gene (6).

Genetic analysis of a hereditary paraganglioma, which was traced to an inactivating mutation of SDHD, revealed an association between mutations in SDH and activation of the hypoxia-response pathway (7). Immunohistochemical analysis showed strong staining of both hypoxia-inducible factor-1{alpha} (HIF-1{alpha}), HIF-2{alpha}, and also the angiogenic factor, vascular endothelial growth factor. These observations were later confirmed in a malignant sporadic pheochromocytoma caused by a germline missense mutation in the SDHB gene (8). These results and others (9) suggest that activation of the hypoxia-response pathway is a common theme underlying SDH (and also FH) loss of function (10).

Regions of hypoxia exist in most solid tumors (11, 12), and the detection of HIF proteins in paragangliomas is therefore not surprising. Oxygen deprivation stabilizes HIF-1{alpha} due to a failure of a key hydroxylation event at two conserved proline residues in the HIF oxygen-dependent degradation domain. Hydroxylation of HIF is carried out by a family of prolyl-4-hydroxylase (PHD) enzymes, of which three have been described in detail (13, 14). This hydroxylation event leads to recognition of HIF by a component of E3 ubiquitin-ligase, von Hippel Lindau protein (pVHL), which designates HIF for degradation via the 26S proteosome (15). Interestingly, PHD enzymes belong to a family of dioxygenases that metabolize oxygen and use 2-oxoglutarate (a TCA cycle metabolite) as a cosubstrate (16). During the hydroxylation reaction, 2-oxoglutarate undergoes oxidative decarboxylation to generate succinate, and a scenario develops, in which excess amounts of succinate can act as product inhibitors of the initial prolyl-hydroxylation reaction (15). This, in turn, would signal the presence of pseudohypoxia, leading to HIF stabilization and downstream adaptive responses. Recently, Gottlieb and colleagues showed in vitro that when the SDH enzyme is transiently inactivated [by RNA interference (RNAi) silencing of SDHD], cells accumulate succinate and HIF is stabilized due to a reduction of PHD activity (17). Increased succinate metabolite concentrations in SDH tumors (10) support this observation and provide a plausible explanation as to why HIF is detected in these tumors. Nevertheless, activation of HIF per se may not be the overriding mechanism of tumorigenesis, and recent evidence, at least in the case of genetic pathways in pheochromocytoma, would suggest that other levels of complexity may be operating (18). Moreover, succinate accumulation, although an attractive proposal for HIF activation, is not the only hypothesis put forward; reactive oxygen species (ROS) formation (resulting from mitochondrial injury) is another mechanism of HIF activation (19) and is observed in some SDH inactivation scenarios (20, 21).

Although the precise link between Krebs cycle dysfunction and the propensity for tumorigenesis remains to be elucidated, we aimed to gain a better understanding of the wider consequences of chronic SDH inactivation by creating stable cell lines in which the SDHB gene is silenced by RNAi. Here, we provide the first demonstration that inactivation of SDH activity results in defective cellular proliferation and respiration, but that cellular redox status is unchanged. Additionally, microarray analysis of SDH-inactivated cells identified several candidate genes that may be involved in the tumor phenotype, notably those involved in extracellular matrix (ECM) processing and pseudohypoxic drive. Consistent with this analysis, we found that SDHB-silenced cells have a greater ability to adhere to ECM components, thus possibly contributing to the tumor phenotype.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 Conclusion
 Disclosure of Potential...
 References
 
An expanded methods section can be found in Supplementary Procedures.

Materials. Culture media, fetal bovine serum (FBS) and Lipofectamine 2000 were from Invitrogen Life Technologies. MitoTracker Red was from Molecular Probes (Invitrogen). All remaining materials, unless otherwise noted, were from Sigma Chemical Co.

Cell culture and transfection. The Hep3B and HeLa cell lines were maintained as described (22). The gastric adenocarcinoma cell line AGS was grown in Ham's F12K medium with L-glutamine and 10% FBS. The pVHL-deficient renal clear cell carcinoma cell line 786-0, overexpressing either wild-type VHL (VHL+) or mutant VHL (VHL–), was a gift from Dr. William Kaelin, Jr. (Dana-Farber Cancer Institute) and were maintained as described (23). Cell cultures were propagated at 37°C in a humidified 5% CO2/95% air atmosphere. All cultures were supplemented with penicillin and streptomycin. Transient transfections were carried out on 90% confluent cell cultures using Lipofectamine reagent according to the manufacturer's instructions. Unless stated otherwise, for transient small interfering RNA (siRNA) transfection studies, cells were transfected twice at 24-h intervals and processed 72 h after the second transfection. To establish stable silenced lines, cells were transfected with a 10:1 ratio of the siRNA cassette and empty pcDNA vector to allow antibiotic selection. Cells were maintained under selection (400 µg/mL geneticin) until colonies were visible (~14 d); individual clones were then isolated with cloning cylinders and amplified under selection conditions.

Biochemical analysis. Growth assessment was measured by cell counting. For measurement of extracellular lactate, conditioned medium from cultures was collected and assayed using the Lactic Acid Assay kit (Sigma) according to the manufacturer's instructions. SDH (complex II) activity was measured in permeabilized cells as described (17). For quantitative assessment of cellular ROS, we used the Amplex Red Hydrogen Peroxide/Peroxidase Assay (Molecular Probes). Oxygen consumption in whole cells was recorded as described previously (23).

SDHB silencing. A vector-based system was used for delivery of siRNA duplexes (24). Four independent complementary DNA oligonucleotides were synthesized and annealed by a melt and slow cool procedure. The target sequences were as follows: siSucA 5'-GATTAAGAATGAAGTTGACTC-3, siSucB 5'-GAGCAACTTCTATGCACAGTA-3 (which target two independent regions of the coding sequence), siSucC 5'-GCTCAGAGCTGAACATAATT-3, and siSucD 5'-GAGTTCCTTTAAAGATCTTGG-3' (targeting the 3' untranslated region of SDHB). As a control for silencing, we constructed an siRNA directed against green fluorescent protein (GFP; ref. 22).

RNA extraction and reverse transcription–PCR. Total cellular RNA was isolated using the RNeasy Mini kit from Qiagen. Total cellular RNA (1 µg) was reverse transcribed with 100 units of SuperScript II reverse transcriptase (Invitrogen) using an oligo-dT primer according to the manufacturer's instructions. For reverse transcription–PCR (RT-PCR) analysis, the following forward and reverse primers (respectively) were designed using the PrimerSelect kit from DNASTAR: for succinate receptor 1 (GPR91) (SUCNR1), 5'-TATGGTTTAACTCAGCAGAAT-3' and 5' GTATAGAGGTTGGCATGAAGCACA-3' (Genbank accession NM_033050); for dual specificity phosphatase 4 (DUSP4), 5'-GCCGAGCGCACCGACATCTG-3'and 5'-GCTGCGGCGCTGCTTAACGAACTC-3' (Genbank accession NM_001394); and for carbonic anhydrase IX (CAIX), 5'-GGGTGTCATCTGGACTGTGTT-3' and 5'-CTTCTGTGCTGCCTTCTCATC-3' (Genbank accession NM_001216). For SDHB, we used SDHB-F and SDHB-R, as described above. The human cyclophilin primer pair (5'-CGTCTCCTTTGAGCTGTTTG-3' and 5'-GGTGATCTTCTTGCTGGTCT-3') was kindly provided by Dr. Maria Dolores Barrachina. The HIF-1{alpha} primer pair was described (23). Other primer pairs are given in Supplementary Table S1. For semiquantitative analysis, the PCR conditions were established to yield linear reaction rates.

Microarray analysis. Triplicate independent sets of RNA samples were prepared from clones pU62 and D20, which were matched for growth conditions and passage number. Total RNA was prepared as above, and integrity was checked with a Bioanalyzer. Only RNA preparations with 28 s/18 s ratio near 2.0 were used. RNA (4 µg) was reverse-transcribed with the One-Cycle cDNA Synthesis kit. In total, 15 µg of cRNA were hybridized to the Human Genome U133 Plus 2.0 Chip (Affymetrix). Arrays were washed, stained, and scanned according to standard protocols supplied by the manufacturer. After scanning, files were normalized and compared using the invariant set method and modeled according to the PM/MM model (25). This comparison identified genes which were overexpressed and underexpressed, as well as those which were either present or absent in silenced cells. An ANOVA test was used to assess the statistical significance of changes. False-positive changes were excluded on the basis of false discovery rate calculations. Finally, significant changes were filtered using a fold change (absolute value) of >6. The results were then classified using the DNA chip analyzer software according to gene ontology terms. Where reported, expression values were described as log2 fold over control (FOC).

Western blotting. Preparation of total protein extracts and membrane transfer were carried out as described (23). Mitochondrial-enriched proteins were isolated with a Mitochondrial/Cytosolic Fractionation kit (Pierce Chemicals). Protein concentrations were determined with Pierce bicinchoninic acid protein assay (Pierce Chemicals). Primary antibodies used for immunoblot analysis were purchased as follows: GPR91 (SUCNR1), DUSP4, and HIF-2{alpha} from Abcam plc; HIF-1{alpha} and p67Phox (neutrophil cytosolic factor 2, NCF2) from BD Biosciences; Porin and SDHB from Molecular Probes (Invitrogen); apoptosis-inducing factor (AIF) from ProSci, Inc.; interleukin 8 (IL-8) from R&D Systems; phosphosporylated stress-activated protein kinase (SAPK)/c-Jun amino-terminal kinase (JNK), SAPK/JNK, phosphorylated p38, and p38 from Cell Signaling; tubulin, actin, FLAG, and S100A6 from Sigma. Protein bands were detected with species-specific peroxidase-conjugated secondary antibodies using the enhanced chemiluminescence method from Amersham.

Adhesion to ECM proteins. Assays were carried out using the following kits obtained from Cell Biolabs, Inc.: CytoSelect 48-well cell adhesion assay ECM array and CytoSelect 48-well cell adhesion assay containing fibronectin. Alternatively, gelatin (Sigma; 100 µg/mL), poly-L-lysine (Sigma; 1 mg/mL), or fibronectin (Sigma; 10 µg/mL) was coated onto 96-well plates. In all cases, cells (1 x 105) were seeded onto the wells and were incubated at 37°C for 60 min in a tissue culture incubator before washing and staining.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Conclusion
 Disclosure of Potential...
 References
 
Transient SDHB silencing does not affect ROS levels. To investigate the influence of SDHB function in pseudohypoxic metabolism, we targeted its expression in Hep3B cells. In a preliminary analysis, four distinct regions within the SDHB mRNA were chosen for silencing; two oligonucleotides targeted the SDHB coding region (siSucA/siSucB) and two oligonucleotides targeted the 3' noncoding region (siSucC/siSucD). All four silencing oligonucleotides provoked a significant reduction in the steady-state levels of SDHB protein but were without effect on tubulin protein levels (Fig. 1A ). Control transfections, either with the empty siRNA expression vector (pU6) or siGFP vector, produced no change in SDHB protein expression (Fig. 1A). Successful SDHB silencing was also shown in the AGS cell line, establishing the generality of this approach (Fig. 1A). To determine whether the reduction of SDHB protein resulted in altered ROS levels, we measured cellular peroxides in SDHB-silenced cells, using a quantitative assay (Fig. 1B). As a robust control for this assay, we included a silencing vector for AIF, a mitochondrial protein whose loss results in increased levels of mitochondrial ROS (22). The results showed that, whereas silencing of AIF increased cellular peroxide content, silencing of SDHB with two independent short hairpin RNA (shRNA) vectors did not lead to increased ROS levels despite a significant reduction in the amount of expressed SDHB protein. It is possible that ROS production was below the limit of detection in our assays; however, these results are consistent with similar knockdown strategies used for the mammalian SDHD subunit (17).


Figure 1
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  		Figure 1. Transient knockdown of SDHB does not dysregulate ROS production. A, Hep3B or AGS cells were transiently transfected with SDHB silencing vectors (siSucA–siSucD) or control vectors (pU62 or siGFP). At 72 h posttransfection, total protein extracts were subjected to Western blot analysis with anti-SDHB antibody. An antibody to tubulin served as a loading control. B, Hep3B cells were independently transfected as above with the silencing vectors siSucC, siSucD, siAIF, or the control siGFP construct. At 72 h posttransfection, half of the cells from each treatment flask were harvested for Western blot analysis with antibodies to SDHB, AIF, and tubulin (top). The remaining half of the cells were harvested for peroxide measurements (bottom). Peroxide measurements represent the means ± SE from three independent experiments; *, P < 0.05, compared with siGFP transfected control cells.

 
Metabolic consequences for chronic SDHB silencing. To achieve prolonged (chronic) silencing of SDH, we separately transfected the shRNA plasmids siSucC and siSucD into Hep3B cells, together with a plasmid containing a neomycin selection marker. Shown in Fig. 2A , steady-state SDHB protein levels were severely reduced in multiple independent SDHB-silenced clones compared with the vector control cell line (pU62) and a stable AIF-silenced cell line (AIF-1-10), and hence, these cells were considered SDHB deficient. SDH activity in SDHB-silenced cells was reduced to 10% to 20% of control levels (Fig. 2A, middle), establishing that the loss of SDHB protein severely retards SDH activity in SDHB-silenced cells. In comparison, SDH activity caused by either SDHB or SDHD mutations in clinical tumor samples, is undetectable (7, 8). To validate SDHB knockdown in long-term culture, we reintroduced the wild-type SDHB gene into silenced cells as a separate trancription unit; thus, clone D11 was transfected with wild-type SDHB cDNA or a control vector and SDH activity was measured after 48 h. Reintroduction of SDHB significantly increased SDH activity in knockdown cells, but did not increase the activity in pU62 control cells (Fig. 2A, bottom). The percentage of SDH recovery (60%) was in line with Lipofectamine transfection efficiencies in Hep3B cells (data not shown).


Figure 2
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  		Figure 2. Metabolic consequences of SDH inactivation in stably SDHB-silenced cells. A, Hep3B cells were transfected with either siSucC or siSucD together with pcDNA3, and single colonies were expanded under neomycin selection. Top, Western blot analysis with anti-SDHB antibody of total cell extracts from different SDHB-silenced clones. The tubulin protein served as a loading standard. Controls included pU62 cells and the stable AIF-silenced cell line AIF-1-10. Middle, SDH activity in SDHB-silenced clones. Activities are presented as the percentage of the activity measured in clone pU62 and are the means ± SE of three independent experiments. Bottom, pU62 and D11 cells were transfected with either empty vector or a vector encoding wild-type SDHB. At 48 h posttransfection, cells were collected and assayed for SDH activity. Columns, means of three experiments; bars, SE. B, pU62, D11, and D20 cells were plated at 200,000 per well in six-well plates, and proliferation was measured over 5 d by total cell counting after trypsinization and collection. Note that the growth profiles of clones D11 and D20 are similar. Points, means from three experiments; bars, SE. Oxygen consumption rates of clones pU62, C21, and D20; *, P < 0.05, compared with pU62 cells (n = 4–5 experiments). Lactate extrusion in cell cultures grown for 3 d without medium change. Columns, means of three replicate samples; bars, SE. Representative of two independent experiments. Peroxide production measured by the Amplex Red reaction. AIF-1-10 cells were used as a positive control for increased ROS production; *, P < 0.05, compared with pU62 cells. Columns, means of three experiments; bars, SE. C, Western blot analysis of HIF-1{alpha} and HIF-2{alpha} expression in total cell extracts of SDHB-silenced clones D11 and D20 compared with pU62 cells. Anti-SDHB antibody was used to confirm repression in silenced but not control cells, and tubulin was used as a loading standard. Hep3B cells were treated with 500 µmol/L TTFA in complete medium for 6 h, and nuclear extracts were analyzed by Western analysis with antibodies to HIF-1{alpha} and HIF-2{alpha}. Asterisk denotes a nonspecific band used as a loading marker. D, Hep3B and AGS cells were treated in complete medium with CoCl2 as indicated. Western blots of total protein extracts were probed sequentially with antibodies to HIF-1{alpha} and SDHB. Antitubulin staining was used as a loading standard.

 
Reasoning that loss of a mitochondrial component would have metabolic consequences for cell growth and oxygen consumption, we next investigated these variables in the SDHB-silenced cell lines. Compared with control cells, the growth rates of clones D11 and D20, as measured by total cell counts over 5 days, were reduced by ~30% (Fig. 2B). This was consistent with results from other independent cell clones (data not shown). Cell cycle analysis of the SDHB-silenced cell lines showed that a greater proportion of the silenced cells were in the G0-G1 phase of the cell cycle compared with control cells, with a corresponding decrease in the percentage of cells at S-M and G2-M phase (Supplementary Fig. S1), suggestive of a cell cycle block at G1. The oxygen consumption rates of clones C21 and D20 were also reduced by 20% to 30% [2.7 ± 0.2 (n = 3) and 3.0 ± 0.2 (n = 3) nmol oxygen consumed/min/106 cells, respectively] compared with pU62 cells (3.8 ± 0.2, n = 3; Fig. 2B), indicating that cellular respiration was significantly perturbed. Furthermore, extruded lactate production was increased by >2-fold in the silenced clones during extended culture (Fig. 2B), suggesting that glycolysis was up-regulated to compensate for reduced oxidative phosphorylation (26). Chronic repression of SDHB did not lead to increased ROS levels compared with pU62 controls (Fig. 2B); however, the AIF-silenced cell line AIF-1-10 was found to contain increased peroxide levels, confirming our previous results (22).

As described for SDHD, inactivation of SDH activity by gene silencing can result in elevated expression of HIF (17). We therefore analyzed the steady-state levels of HIF proteins in two independent silenced SDHB cell lines. As shown in Fig. 2C, both D11 and D20 cells had increased steady-state levels of HIF-1{alpha}, and also HIF-2{alpha}, compared with control. Pharmacologic suppression of SDH activity in Hep3B cells with the complex II inhibitor 2-thenoyltrifluoroacetone (TTFA) also increased steady-state levels of HIF-1{alpha} and HIF-2{alpha} (Fig. 2C), indicating that elevated HIF expression is a general consequence of SDH inactivation. Interestingly, Dahia and colleagues (9) have recently suggested that HIF-1{alpha} itself plays a central role in the expression of the SDHB subunit. Wondering if this was a general phenomenon, we treated both Hep3B and AGS cells with cobalt chloride (CoCl2), a hypoxia mimetic, and examined SDHB protein expression. As expected, CoCl2 increased the steady-state levels of HIF-1{alpha} protein (Fig. 2C). At the same time, SDHB protein levels were reduced significantly in both independent cell lines, in agreement with previous observations (9). Notably, HIF-1 has recently been shown to regulate mitochondrial respiration during hypoxia by modulating expression of the cytochrome oxidase IV complex subunits cyclooxygenase 4-1 (COX4-1) and COX4-2 (27). Moreover, HIF-1 can limit pyruvate entry into the TCA cycle via up-regulation of PDK1, which decreases pyruvate dehydrogenase activity and so reduces the conversion of pyruvate to acetyl-CoA (28, 29). A simple explanation for the decrease in oxygen consumption in SDH-silenced cells would be a reduced amount of electron donors available for the electron transport chain, although it is evident that HIF-1 may have multiple roles in the process of mitochondrial respiration.

Expression of missense SDHB mutations does not affect ROS levels. In recent years, an increasing number of missense mutations of SDHB (and also other SDH subunits) have been identified during genetic analyses in pheochromocytomas (30). As it was formally conceivable that expression of mutant SDH subunits, rather than silencing of SDH activity in itself, might alter ROS levels (20, 21), we overexpressed three clinically validated missense mutant SDHB genes in a background of reduced SDH activity. To our knowledge, the mitochondrial localization sequence of SDHB has not yet been empirically determined. We therefore first assessed whether mutant SDHB proteins showed altered intracellular localization by tracking epitope-tagged versions of these proteins. Addition of the FLAG epitope resulted in slower migration of the SDHB band in lysates of transfected Hep3B cells, because the size had increased by ~1 kDa (Fig. 3A ). Confocal microscopy of transfected HeLa cells showed that the overexpressed proteins exhibited a punctate staining reminiscent of mitochondria (Fig. 3B, left). Superposition of MitoTracker Red costaining images revealed a considerable degree of overlap (Fig. 3B, middle and right). Mitochondrial localization of the SDHB proteins was further confirmed in fractionation studies, because FLAG-tagged proteins were exclusively detected in the mitochondrial fraction of Hep3B cells, as defined by the mitochondrial protein marker porin (Fig. 3C). Identical results were obtained in HeLa cells (not shown). These findings suggest that the missense mutated SDHB proteins can reach the mitochondrion and localize normally. Having confirmed this, we next determined the effect of missense mutated SDHB proteins on ROS levels when expressed in the SDHB-silenced D11 cell clone (Fig. 3D). Cellular peroxide levels showed no differences compared with cells transfected with empty vector, and so, consistent with previous results (17), we can find no evidence for ROS increases either when SDHB is silenced or when SDHB mutants are dominantly expressed.


Figure 3
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  		Figure 3. Mutant SDHB proteins reach the mitochondria but do not increase ROS production. A, Hep3B cells were transfected independently with control vector, wild-type SDHB (pWT), wild-type SDHB-FLAG (pWT-F), SDHB (R48Q)-FLAG (pR48Q-F), SDHB (P197R)-FLAG (pP197R-F), and SDHB (R242C)-FLAG (pR242C-F). At 48 h posttransfection, total cell extracts were collected and analyzed by Western blot with antibodies to SDHB and FLAG. Tubulin was used as a loading control. Note that addition of FLAG epitope results in a slower migrating band. B, confocal imaging of HeLa cells transiently transfected with pWT-F, pR48Q-F, pP197R-F, or pR242C-F. HeLa cells were stained 48 h posttransfection, as described in Materials and Methods with anti-FLAG monoclonal antibody and MitoTracker Red. Individual images were merged digitally. Overlapping red and green pixels appear as orange/yellow. Representative of three performed. C, Western blot analysis of Hep3B mitochondrial (M) and cytosolic (C) fractions. Cells were transfected with the indicated vectors as in A. At 48 h posttransfection, mitochondrial and cytosolic extracts were prepared and analyzed by Western analysis with anti-FLAG antibody. Mitochondrial enrichment was confirmed with an antibody to the mitochondrial-specific protein porin, and tubulin was used as a loading standard. D, SDHB-silenced D11 cells were transiently transfected with the indicated nontagged wild-type and mutant SDHB constructs. At 48 h posttransfection, cells were collected for Western blot analysis (top) and ROS measurement (bottom). Columns, mean of three replicate samples; bars, SE. Representative of two independent experiments.

 
Global expression analysis upon SDHB silencing. To gain insight into the differential effects of SDHB silencing, samples of RNA, derived from D20 cells and pU62 controls, were hybridized to Affymetrix HG-U133 Plus 2.0 microarray chips. After background subtraction and data normalization, using a cutoff value of 3-fold up-regulation or down-regulation, interrogation of the microarray detected >900 differentially expressed gene targets. To reduce the quantity of differentially expressed genes to a more manageable number, targets with a fold change of >6 at P < 0.05 were considered. It is acknowledged that this artificially high cutoff could miss important changes of lower magnitude, but this was chosen to examine gross changes in expression. This yielded a final sample set of ~400 targets, the majority of which (70%) were up-regulated in response to SDHB silencing (Supplementary Table S2). A full listing of the microarray results have been deposited in the National Center for Biotechnology Information GEO database, under the accession number GSE10289. Analysis of gene expression levels, calculated as present or absent according to the PM/MM difference model included in the dCHIP analysis software (25), showed that, interestingly, ~50% of dysregulated genes in this subset were uniquely expressed in either control pU62 cells or D20 cells. We have defined these genes as being either turned on (Supplementary Table S2A) or turned off (Supplementary Table S2B) in SDHB-silenced cells (31). The remaining 50% of genes were present but differentially regulated in both control and silenced cells (Supplementary Table S2C). Using gene ontology classification analysis, we were able to assign ~300 of these genes to biological functions (Fig. 4A ). Notably, genes involved in catalytic activity, cell proliferation, and cell adhesion were well represented in this analysis. In keeping with the trend of a general up-regulation of gene transcription, we found that the majority of the 300 target sample set were also up-regulated (Fig. 4B).


Figure 4
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  		Figure 4. Microarray analysis from the D20 SDHB-silenced clone. A, pie chart showing the gene ontology classifications of the ~300 target genes showing a 6-fold or greater (P < 0.05) change in expression levels compared with the pU62 control clone. Others represent gene ontology groups with few representatives (<5%) in the analysis; for example, calcium ion transporter activity, cytokine binding, digestion, DNA binding, and chymotrypsin activity. B, percentages of targets which are either up-regulated (+) or down-regulated (–) in the gene ontology analysis. C, top 15 overexpressed (top) and underexpressed (bottom) gene products from the microarray analysis.

 
A list of the most up-regulated (on) and down-regulated (off) gene targets is shown in Fig. 4C. Analysis of the results indicated that the most highly expressed gene in the silenced cells versus controls was S100A6. S100A6 (S100 calcium binding protein A6) belongs to a family of small divalent-cation binding proteins of the EF-hand type, which have been implicated in the regulation of a wide variety of intracellular processes, such as cell proliferation, intracellular signaling, and energy metabolism. Interestingly, S100A6 is considered a diagnostic marker for pancreatic cancer (32) and is found to be both positively (32) and negatively (33) associated with proliferation and invasion. Notably, S100A6 was also up-regulated during a microarray analysis of rat pheochromocytoma cells compared with terminally differentiated rat adrenomedullary chromaffin cells (34). Regarding other up-regulated genes, it is noteworthy that collagen 1{alpha}1 (COL1A1), insulin-like growth factor binding protein 2 (IGFBP2), IGFBP7, and lysyl oxidase (LOX)–like 2 (LOXL2) have all been implicated in the tumor phenotype. LOXL2 belongs to a gene family (of which LOX is the prototypic founder) whose members have been associated with cell motility, cross-link formation in stromal collagens and elastin, and tumor development and progression (35). Supporting an implication in the tumor phenotype, LOXL2 and S100A6 (both classified as switched on in our analysis) are among the top 50 up-regulated genes produced upon liver-specific inactivation of the von Hippel Lindau gene (VHL; ref. 36). In contrast, LOX (log2 FOC –2.5; not shown) was down-regulated in our analysis. This is in fact not surprising, given that, although LOX expression has been found to be indispensable for hypoxia-induced metastasis (37), its expression is also down-regulated in many cancers (ref. 38 and references therein). COL1A1, which produces a major component of type I collagen, is also up-regulated by hypoxia (39, 40). IGFBP2 (Supplementary Table S2A) and IGFBP7 (log2 FOC 4.6; Supplementary Table S2C) form part of a family of binding proteins that modulate the bioavailability of insulin-like growth factors. A positive correlation between tumor grade and the level of IGFBP2 expression has been reported in a variety of tumors (reviewed in ref. 41), and IGFBP2 transcription has been shown to be regulated by HIF (42). Overexpression of IGFBP2 results in up-regulation of integrin {alpha}5 (ITGA5; ref. 43), which was also up-regulated in our microarray analysis (log2 FOC 2.5; not shown). ITGA5 is also a recorded hypoxia-responsive gene (40). In contrast, high expression of IGFBP7 has been found to suppress cancer cell growth and may inhibit anchorage-independent growth while increasing adherence to ECM proteins (44). Of particular interest was the observation that a former orphan G-protein–coupled receptor (GPR91) was also significantly up-regulated (classified as on) in SDHB-silenced D20 cells. Ling and colleagues have recently shown in mice that this gene encodes the natural receptor for succinate, and it has been renamed SUCNR1 (45). Accordingly, SUCNR1 overexpression stimulates the release of renin from the kidney, which can culminate in the production of angiotensin II, leading to hypertension (45). This raises the interesting possibility that succinate acts as a signaling molecule.

To evaluate the significance of the microarray observations, we next performed gene-specific RT-PCR of a sample set of differentially regulated genes. We chose 17 genes, covering a range of both up-regulated and down-regulated targets and assessed their expression levels both in control pU6 cells and in the two independently SDHB-silenced clones D20 and D11 (Fig. 5A ). The genes confirmed by RT-PCR analysis to be down-regulated were SDHB, LOX, Meprin {alpha}, and Lysozyme. Among the mRNAs confirmed by the RT-PCR analysis to be up-regulated in SDHB-silenced cells were IL-8, CXCR4, ITGA5, CAIX, JunB, and p21, all of which are considered prototypical hypoxia and/or HIF response genes. The cyclin-dependent kinase inhibitor p21 acts clasically to block the cell cycle at G1, and overexpressed p21 detected in Vhl–/– fibrosarcoma cells and murine embryonic fibroblasts is partly responsible for the slower proliferation of these cells compared with their wild-type counterparts (46). This is in good agreement with our cell cycle analysis of silenced lines (Supplementary Fig. S1). Other genes confirmed to be up-regulated upon SDHB silencing include DUSP4 (alternatively named MKP2) and NCF2, a cytosolic component of NADPH oxidase.


Figure 5
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  		Figure 5. Analysis of differentially expressed genes and signaling pathways in SDHB-silenced cells. A, semiquantitative RT-PCR analysis of selected genes dysregulated in SDHB-silenced cells (clones D11 and D20) compared with control pU62 cells. B, semiquantitative RT-PCR analysis of Hep3B cells incubated for 48 h in the presence or absence of 25 mmol/L DMS (top), RT-PCR analysis of candidate gene expression in 786-0 VHL– versus VHL+ cell lines (middle), and RT-PCR analysis of Hep3B cells 48 h posttransfection with empty vector (pBABE) or HIF-2{alpha} (bottom). In all cases, the results shown are representative independent experiments of at least four performed. C, protein analysis of SDHB-silenced cells (clones D11 and D20) and the control cell line pU62. Left, Western blots of total cellular extracts were probed sequentially with antibodies to IL-8, SUCNR1, S100A6, and NCF2. Anti-SDHB antibody confirmed repression in silenced cells, but not control cells, and tubulin was used as a loading standard. Right, Immunocytochemistry analysis of DUSP4 localization. Note the coincidence of DUSP4 and Hoechst staining in cell nuclei. These results are representative of three independent experiments. D, semiquantitative RT-PCR analysis of selected gene expression after transient silencing of HIF-1{alpha}. pU62 and D11 were transiently transfected with either siCONT or siHIF-1{alpha}, and cells were collected 72 h posttransfection. In all cases, the results shown are representative from three independent transfection experiments. E, stress-activated signaling pathways are activated in SDHB-silenced cells. Levels of constitutively active p38 MAPK and JNK in D11 and D20 SDHB-silenced clones compared with pU62 control cells. The levels of active MAPKs were determined by Western blot of total cell lysates with phosphorylated specific anti-MAPK antibodies. Corresponding total MAPK expressions, detected with pan-specific antibodies, were used as loading controls. Representative of three independent experiments (top). Bottom, serum-stimulated phosphorylation of p38 and JNK MAPKs. After overnight serum starvation, cells were exposed to 10% serum for the times indicated. The levels of active MAPKs were determined as above.

 
Given that succinate accumulation is a by-product of SDH deficiency, we next tested whether exposure of wild-type cells to exogenous succinate could replicate the microarray findings. Hep3B cells were incubated with culture medium containing 25 mmol/L dimethyl succinate (DMS) for 48 h (17), and total RNA was extracted for RT-PCR analysis. Exposure to DMS increased the expression of S100A6, p21, and SUCNR1 mRNA, but did not affect the expression of cyclophilin (Fig. 5B, top). Control-matched mRNA or tissue samples from pheochromocytomas were not available to corroborate our findings; so as an alternative, we examined target gene expression in VHL-deficient cells. VHL tumors and SDHB/SDHD mutations share a common gene expression signature (9), and VHL-deficient cells have a general respiration deficiency which leads to a reduction in the activity of isolated complexes II + III (47). A subset of genes was thus analyzed by RT-PCR of RNA from 786-0 cells expressing mutant VHL (VHL–) or wild-type VHL (VHL+). Loss of VHL up-regulated a subset of genes similar to those affected by SDHB silencing, namely NCF2, S100A6, SUCNR1, and CXCR4 (Fig. 5B, middle). In the context of pseudohypoxia, 786-0 cells have been shown to overexpress HIF-2{alpha}, but not HIF-1{alpha} (36). Extending our results, we overexpressed a HIF-2{alpha} transcription unit in Hep3B cells and observed that SUCNR1 and also CXCR4 mRNA were up-regulated compared with cells transfected with empty vector (Fig. 5B, bottom). Taken together with the mRNA expression data from SDHB-silenced cells, it seems likely that SUCNR1 could be an HIF-responsive gene. In support of this notion, it is interesting that succinate levels in ischemic rat liver are increased 14-fold (48).

To verify whether our RT-PCR results were consistent with expression at the protein level, we next performed Western blot analysis on a sample of up-regulated gene products from control pU62 cells and the SDHB-silenced clones D20 and D11 (Fig. 5C, top). In agreement with the microarray and RT-PCR data, we observed increased steady-state levels of IL-8, SUCNR1, S100A6, and NCF2 proteins. DUSP4 protein expression, monitored by immunocytochemistry, was also increased in SDHB-silenced cells and exhibited a nuclear localization (Fig. 5C, bottom).

To assess the possibility that HIF might be responsible for some of these observed differences in gene expression levels, we next silenced expression of HIF-1{alpha} in D11 cells. Using ON-TARGETplus SMARTpool siRNA directed to HIF-1{alpha}, we first verified that HIF-1{alpha} could be substantially silenced in cobalt-treated Hep3B cells (Supplementary Fig. S2). Next, we transfected siRNA reagents into control pU62 and D11 cells and collected cells for RT-PCR analysis at 72 hours posttransfection (Fig. 5D). As expected, HIF-1{alpha} mRNA was substantially reduced after silencing in both pU62 and D11 cells compared with control siRNA. Further analysis also showed a modest (but reproducible) reduction in mRNA expression from both SUCNR1 and S100A6 gene products. Other gene products tested were not changed after HIF-1{alpha} silencing, reflecting perhaps the involvement of HIF-2{alpha} or other activating factors.

Given that mitogen-activated protein kinases (MAPK) are key participants in signal transduction pathways in response to a variety of physiologic stresses, we examined whether stress-induced signaling was activated in SDHB-silenced cells. In contrast with pU62 control cells, both SDHB-silenced clones had increased nonstimulated levels of activated JNK and p38 kinase (p38), as detected with phosphorylated specific antibodies (Fig. 5E, top). Moreover, serum stimulation of quiescent SDHB-silenced D11 cells triggered a more robust increase in the levels of activated JNK and p38 (Fig. 5E, bottom). These findings indicate that in addition to activating pseudohypoxic drive, SDH inactivation also results in an activation of the MAPK phosphorelay system. In support of this finding, complex II inactivation by malonate has been shown to activate p38 signaling in neuroblastoma cells (49). In addition, PC12 cells, which are used as a model cell line for carotid body type I cells can respond to hypoxia by an activation of p38 (50).

SDHB-silenced cells have superior adherence to ECM. Finally, to examine the biological implications of overexpression of adhesion-related genes in SDHB-silenced cells detected in the microarray analysis, we monitored the ability of SDHB-silenced cells to attach to ECM with in vitro adhesion assays. As anticipated, SDHB-silenced cells had an increased ability to attach to a range of ECM components, but adhesion to the negative control bovine serum albumin (BSA) did not differ between SDHB-silenced cells and pU62 controls (Fig. 6A ). Similar results were obtained in two independent SDHB-silenced cell lines (Fig. 6B). Importantly, adhesion of cells to poly-L-lysine, a matrix that does not engage ECM, was similar regardless to SDHB silencing (Fig. 6B). To assess the potential contribution of HIF-1{alpha} to this phenotype, we repeated transient silencing of HIF-1{alpha} in both pU62 and D11 cells and measured adhesion to fibronectin. Interestingly, whereas HIF-1{alpha} silencing had no effect on control cell adhesion, we found a statistically significant decrease in adhesion of D11 cells to fibronectin upon HIF-1{alpha} silencing (Fig. 6C). Given that S100A6 (and SUCNR1) were down-regulated after HIF-1{alpha} silencing, we overexpressed an S100A6 transcription unit in Hep3B cells and found that adhesion was increased compared with the control (Fig. 6D). Taking these results together and considering the importance of cell-ECM interactions for tumor initiation, these observations suggest a possible HIF-1{alpha}–dependent mechanism, potentially involving S100A6, whereby SDH-deficient cells could have an advantage in the initial stages of the disease progression.


Figure 6
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  		Figure 6. Adhesion of SDHB-silenced cells to ECM components. Cells were grown to semiconfluence in culture medium and then plated (1 x 105 cells per well) on 48 or 96-well plates coated with ECM components. Plates were then incubated in a tissue culture incubator for 60 min at 37°C. After incubation, plates were washed and adherent cells stained according to protocols supplied by the manufacturer. Absorbance was read at 570 nm. A, comparison of adhesion of control cells (pU62) and SDHB-silenced cells (clone D20) to the following ECM components: fibronectin (FN), collagens I and IV (COL I + IV), laminin (LN), fibrinogen (FB). BSA was used as non-ECM negative control. B, comparison of adhesion of control cells (pU62) and two independent SDHB-silenced cell lines (D11 and D20) to fibronectin, gelatin (GT), and poly-L-lysine (PL). C, comparison of adhesion of control pU62 and D11 cells to fibronectin (10 µg/mL) after transient silencing (72 h) of HIF-1{alpha} (***, P = 0.0009). D, Hep3B cells were transfected with control vector or a vector containing an S100A6 transcription unit. At 72 h posttransfection, half of the cells from each treatment flask were harvested for Western blot analysis with antibodies to S100A6 and tubulin. The remaining half of the cells was used for adhesion measurement (***, P < 0.0001). Columns, mean of at least three replicates; bars, SE. Representative of at least two independent experiments.

 

   Conclusion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusion
Disclosure of Potential...
 References
 
By creating cell lines in which the SDHB gene was silenced, we provide the first analysis of the physiologic consequences for chronic loss of SDH activity. Although we could find no changes in the levels of ROS, silencing of SDHB generated defects both in cellular proliferation and in oxygen consumption. Both of these phenotypes are observed in human VHL-deficient cells lines (47) and probably arise, in part, from impaired mitochondrial activity. Of note, pheochromocytomas are classified as slow-growing tumors (51). Analysis of the global gene changes upon silencing of SDHB resulted in the discovery of multiple differentially expressed genes, some of which have known roles in the tumor phenotype and could be possible targets for therapeutic intervention. Very recently, a family of histone demethylase enzymes that operate via 2-oxoglutarate–dependent dioxygenation has been described (reviewed in ref. 52). Although relatively little is known yet about the physiologic roles of these enzymes, histone methylation has long been known to affect gene transcription. As these enzymes will most probably be affected by the 2-oxoglutarate/succinate ratio in cells, they could also be potentially dysregulated in SDH deficiencies. Finally, our novel discovery of SUCNR1 responsiveness in SDHB-silenced cells and appreciation of succinate as a signaling molecule, as well as the activation of the MAPK stress response, add new tiers of complexity to the links between the TCA cycle and cancer.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: Instituto de Salud Carlos III, Fondo de Investigacion Sanitaria grant PI0600299 (K.J. McCreath), FPU fellowship from Ministerio de Educacion, Cultura y Deporte (N. Apostolova), and "Ramon y Cajal" fellowship program (K.J. McCreath). Centro Nacional de Investigaciones Cardiovasculares is supported by the Spanish Ministry of Health and Consumer Affairs and Pro-CNIC Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Angel Ortega (University of Valencia) for help with microscopy and Simon Bartlett for helpful comments on the manuscript.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 9/21/07. Revised 1/29/08. Accepted 3/ 3/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusion
 Disclosure of Potential...
 References
 

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