2.1 Pathophysiology
When we consider the above pathogenic mechanisms, then it is natural to think of ways of testing them out experimentally. Biologists have spent much time working on ‘model organisms’ which can be studied in the laboratory. This approach can work because the basics of life (including SDH) are common between humans and the most primitive and simple organisms. Many of these model methods have been applied to this problem. So far most of the studies pointed out that there is no sufficient animal model to test SDH functions. More suitable in vivo models are needed to unravel underlying pathogenic mechanisms and to develop therapeutic strategies. Emerging models in different organisms have been developed that we will summarize below. Both invertebrate and vertebrate models have been used to investigate and characterize SDHx functions.
Table
2 summarizes the advantages and limitations of the various models used for PPGL research.
Table 2
Comparisons of different model systems
Yeast | 40% | 47% | Haploid SDHB KO is viable | Unicellular |
Nematode/worm | 52% | 52% | Metabolically, very similar to human; can be used for large scale drug screens | Has only a few neuroendocrine cells. Does not develop tumors |
Fruit fly | 60% | 53% | Metabolically, very similar to human; can be used for large scale drug screens | Germline SDHx-mutated do not develop tumors |
Zebrafish | 70% | 77% | Similar metabolic consequences to human and nematode | Germline SDHx-mutated do not develop tumors |
Mouse | 92% | 90% | Applicable xenograft models | Germline SDHx-mutated do not develop tumors |
Rat | 92% | 89% | Can develop PPGL tumors with germline sdhb mutations; RS0: best xenograft model to date | To date, poorly investigated |
Dog | 94% | 88% | Spontaneously develops PPGL tumors, with similar germline risk genes | Expensive to maintain dog colonies; but possible to exploit routine domestic dog veterinary cases |
We will consider the models in ascending order of laboratory complexity. First, unicellular systems (cultured cells, yeast), then invertebrates (the nematode
Caenorhabditis elegans, the fruit fly), then non-mammalian vertebrates (Zebrafish), finally, mammals (mouse, rat and dog).
2.1.1
Cultured cells. Cultured cells, immortalised in some way, have a major role in biological research, and the study of SDHx mutations in PPGL has been no exception.
Generation of cell lines derived from PPGL-type tumors has presented multiple challenges, partly because of the histological complexity of these tumors, but also because pheochromocytomas and paragangliomas occur relative rarely spontaneously in rodents [
51], although they can be induced chemically or by irradiation in mice and rats (reviewed most recently by Bayley and Devilee 2020 [
52]).
Table
3 lists the cell lines which have been used in PPGL research related to
SDHx genes. Section
1 of the table lists cell lines has germline
SDHx modifications and which can be considered authentically ‘Cluster 1 pheochromocytoma-like’. These have been the most difficult to make. The Italian team immortalised cells from an
SDHC tumor and an
SDHD, using hTERT and/or SV40 large tumor antigen [
53] and have exploited these cells extensively, increasing understanding of the pathological nature of PPGL tumors [
9,
54]. The French team made immortalized cells from a mouse bearing a ‘floxed’
Sdhb gene [
36] and these too have been investigated in depth, especially in the field of epigenetics, to demonstrate the ‘hypermethylator’ phenotype [
55]. They were also used to study metabolism [
56]. The exact nature of these cells has been questioned [
52], although the epigenetic observations are undoubtedly very cogent to PPGL.
Table 3
Model systems: cultured cells. The table is in 3 sections: (i) pheo lines with SDHx knocked out, (ii) pheo lines with or without SDHx silencing, and (iii) non-pheo lines that have been used
Pheo or pheo-like lines with germline SDHx knocked out or mutated |
PTJ64i PTJ86i | Human | Immortalized cell lines derived from SDHC- and SDHD-mutated human patients. Immortalised with retroviral-mediated transduction with hTERT and/or simian virus 40 large tumor antigen | On flow cytometry, showed ‘immature mesenchymal, hypoxic, vasculoneural markers’ similar to tumors | Derived from SDHC c.43C > T (p.Arg15*) and SDHD c.27delC (p.Val10Phefs*5) human tumors | - | Importance of NOTCH signaling [ 53]. Importance of miR-200a,b,c and miR-34. Effects of PPAR-alpha inhibition [ 54] | |
imCC | Mouse | Adrenal cells derived from mice bearing a ‘floxed’ SDHB exon 2 | Doubt as to whether these are true ‘chromaffin cells’. See [ 52] | Deletion of SDHB exon 2 | Excess succinate, depleted fumarate | Hypermethylation phenotype | |
RSO; RS1/2 | Rat | Sdhb +/- rats were irradiated and some developed pheochromocytomas. Tissue from tumors was injected into NSG mice to give xenografts. Cells from these were incubated in hypoxic media and two lines were prepared: RS0 (sdhb-/-) and RS1/2 (sdhb +/-) | Convincingly ‘chromaffin’ | sdhb-/- (RS0) sdhb +/- (RS1/2) | Sensitivity to oxygen; excess succinate and lactate; high dopamine | Successfully used in mouse xenografts | |
Phaeochromocytoma lines with or without SDHx silencing or KO |
PC12 | Rat | Derived from a rat pheochromocytoma and passaged in further rats | Classic neuronal cells. In NGF, will differentiate into neuron-type cells. Contain chromaffin granules and many characteristic catecholamine-related enzymes | Mutation in Max, gene coding for Myc partner protein Max [ 58] | Expresses many enzymes of catecholamine metabolism | Innumerable studies in general neuroscience | |
MPC 4/30 | Mouse | Pheochromocytoma cell line derived from irradiated Nf1 KO heterozygote mouse | Show chromosomal instability | Derived from mouse with heterozygous knockout of neurofibromatosis gene Nf1 | Not SDH-deficient, but sdhb was knocked down by one group [ 60] | sdhb knock-down used to investigate expression of iron-transport proteins [ 60] | |
MTT cells ‘mouse tumor cells’ | Mouse | More aggressive derivative of MPC | Pheo-like | Ultimately derived from mouse with heterozygous knockout of neurofibromatosis gene Nf1. No Sdhx variation | Catecholamine secreting | Later used in allograft [ 62] | |
hPheo1 | Human | Human pheo-derived cell line. Sporadic case, no known germline risk gene present. Immortalised with lentivirus-h TERT | Express chromogranin, COMT, TH | Deletion at 16p (does not affect SDHA,B,C,D) | Do not synthesise catecholmines | | |
N2a, sdhb silenced | Mouse | Neuroblastoma (pheo-like) N2a silenced in Sdhb (Non sdhb-silenced MPC and MTT also studied) | Neuron-like | SDHB KD | SDHB very much reduced on western blots | Effect of piperlongumine | |
PC12 with sdhb knockdown | Rat | Modified PC12 cell line | | Sdhb knockdown | Catecholamine oversecretion | Knockdown of SDHB led to increased catecholamine synthesis, increased ROS, and HIF stabilization | |
MTT, sdhb silenced | Mouse | Mouse pheo line | | Nf1 +/- | 60% reduction in SDH activity; increased succinate | Effect of fibroblast co-culture | |
hPheo1 SDHB KD | hpheo1 shRNA SDHB | SDHB knock-down of hPheo1 cells | ditto | SDHB -/- | Increased succinate, decreased SDH on western blots | Used to study polyamine pathway and its inhibition | |
Non-neuroendocrine cell lines that have been used, for gene knock-out, gene silencing or pharmacological inhibition of SDHx |
SDHC E69 | Mouse | Mouse fibroblasts. A copy of the C elegans point mutation in sdhc known as mev-1, which reduces but does not abolish, sdhc activity, and causes generation of ROS | A fibroblast and not an adrenal or neuroendocrine cell | sdhc point mutant p.V69 > E | Evidence of increased ROS | Sensitivity to oxygen; increased superoxide; increased apoptosis | |
HEK293 human embryonic fibroblasts | Human | Human embryonic kidney line. SDHD knockdown | Fibroblast | SDHD KD | SDH activity reduced 50% | To study effect of SDH silencing on HIF-alpha prolyl hydroxylase | |
Hep3B HeLa | Human | Human hepatoma and cervical cells, SDHB silenced | Non-pheo | SDHB KD | SDH much reduced on western blotting | Increased HIF-1alpha. Major microarray mRNA expression changes consistent with tumor measurements | |
HEK293 Hep3B | Human | Human embryonic kidney and hepatoma cell lines. SDH inhibited by 2-thenoyltrifluoroacetone (TTFA); Sdhd was silenced by siRNA | Non-pheo | SDHD KD | SDH much reduced on western blotting | Epigenetic hypermethylator changes | |
HEK293 | Human | Human embryonic kidney line. SDHB knockdown | Non-pheo | SDHB KD | Diminished SDH on western blots | Effects of hypoxia on alpha ketoglutarate dependent dioxygenases | |
iMEFs sdhb -/- (irradiated mouse embryonic fibroblasts) | Mouse | Immortalised mouse cells with SDHB KO. Prepared from a conditionally knocked-out Sdhc mouse | Non-pheo | SDHB -/- | Diminished SDH on western blots; increased succinate, decreased fumarate; decreased SDH activity | ditto | |
iMK Sdhb -/- (immortalized mouse kidney) | Mouse | ditto | ditto | ditto | ditto | ditto | |
HCT116 SDHB KO 7, 23 | Human | Colo-rectal cancer cells, SDHB removed by CRISPR | Non-pheo | SDHB -/- | Accumulated succinate, decreased fumarate | Very comprehensive metabolomics survey. Dependence on glutaminolysis. Susceptibility to BET inhibitors | |
More recently, the Tischler-Powers American team, in a major
tour de force of experimental technique, have prepared two rat-derived cell lines containing
sdhb deletions: ‘RSO’ is
sdhb-/-, while ‘RS1/2’ is
sdhb +/- [
57]. A series of steps starting with a floxed
sdhb rat and running through a mouse xenograft stage followed by novel cell cultures in hypoxic and low-serum conditions allowed the isolation of these cells, which seem set to provide an important resource.
In Section
3 of the table, there are listed pheo-like cell lines in which
SDHx mutations are not fundamental. The classic is the PC12, a line derived from a spontaneous rat tumor of (as we understand it) unknown precise genomic composition, although it is known to contain a mutation in
Max, encoding a partner protein for Myc [
58]. (
Max is a risk gene for human PPGL in Cluster 3[
25].) PC12 cells display dense chromaffin-like granules and synthesize catecholamine neurotransmitters dopamine and norepinephrine. This line has been used extensively in general neuronal research as well as in PPGL research, where
sdhb has been knocked down by RNA silencing, e.g. to study catecholamine metabolism [
66]. The heterozygous
Nf1 +/-mouse [
73] provided the ‘MPC’ (mouse pheochromocytoma) line, derived from a pheochromocytoma in the mouse [
61].
Sdhb has been knocked down in this line [
60], to study the potential therapeutic effect of ascorbic acid, a key cofactor for α-KG-dependent dioxygenases [
32]. The ‘MTT’ (mouse tumor cells) line was derived from MPC and is more metastatically aggressive [
63]. This too has had its
sdhb silenced to study growth and invasiveness under
sdhb knockdown [
67]. Silencing led to a 60% reduction in SDHB expression, and it was observed that the
SDHB-silenced MTT cells took up lactate secreted by fibroblasts when they were co-cultured with them in a monolayer. Lactate produced by cancer-associated fibroblasts was also taken up by pheochromocytoma spheroids when a 3D-co-culture was performed and
SDHB was silenced. In addition, in the latter case the migratory potential of spheroids became increased, as a result of
SDHB silencing.
The ‘hPheo1’ (human pheo 1) was derived from a sporadic human pheochromocytoma of unknown genomic landscape, using lentivirus h-TERT immortalization [
64]. This too has been
sdhb silenced, to study the effect of polyamine compounds [
68].
In the third part of Table
3, Non-chromaffin cell lines that have been used for gene knockout, gene silencing or pharmacological inhibition of SDHx’, there are listed a series of non-chromaffin cells that have been employed in SDHx research. A number have been produced from conditional knockout
sdhx mice, including iMEF
sdhb-/- (immortalized mouse embryonic fibroblasts) and iMK
sdhb-/- [
71], and MEF and BMK (baby mouse kidney) by the Spanish team [
74,
75]. The Japanese team mimicked the
C elegans mev-1 oxygen-sensitive point mutation in mouse fibroblasts, introducing to these cells the pV69 > E mutation in
sdhc [
69]. One of the key advances in the understanding of PPGL pathophysiology (that an alpha-ketoglurate-dependent dioxygenase could be inhibited by excess succinate) was made in the HEK293 (human embryonic fibroblast) line, subjected to
SDHB knockdown, by Selak and others [
31].
Small-molecule SDH inhibition has been used in HeLa cells and H295R adrenocortical cells [
76]. The SDH inhibitors itaconate and atpenin, or siRNA treatment specific for
SDHB, did not negatively affect chromaffin cells, in contrast to HeLa or H295R cells. After SDH inhibition or silencing, chromaffin-type PC12 cells showed increased expression of glutaminase-1 (GLS-1). BPTES, a GLS-1 inhibitor was able significantly to inhibit proliferation of SDH-defective PC12 cells [
76]. This is potentially important and may suggest a mechanism by which SDH germline mutations confer susceptibility to neuroendocrine tumors: that chromaffin-type cells may be able to survive knock-out of both SDH alleles due to their ability to use glutamine as an anaplerotic substrate.
Kitazawa and others deleted
SDHB in HCT116 cells (of colorectal cancer origin) by CRISPR to make an effective drug-screening survey [
72].
2.1.2
Saccharomyces cerevisiae, or yeast, has contributed significantly to understanding the molecular pathogenesis of tumorigenesis resulting from defective SDH [
77]. Yeast studies have shed much light on one of the proposed pathophysiological mechanisms, inhibition of DNA demethylation [
37].
Table
4 shows yeast, and worm, fruit fly and zebra fish models.
Table 4
Model systems in SDHx PPGL: yeast S. cerevisiae, worm, C. elegans, fruit fly, D melanogaster, zebrafish Danio rerio
Yeast |
sdh2 KO | Saccharomyces cerevisiae (yeast) | KO mutation in Sdh-2 (yeast SDHB) | Succinate accumulation; increased ROS production but without evidence of mutagenic DNA damage. Due to excess succinate, showed inhibition of alpha-ketoglutarate-dependent dioxygenases Jlp1 (sulphur metabolism) and Jmj-C domain histone demethylases | |
Copy of human glomus tumor mutation | S. cerevisiae | Conversion of yeast SDH to equivalent of the C191Y human mutation | Increase in ROS production and mitochondrial DNA mutability. Showed that this mutation abolished SDH activity and was truly deleterious | |
Sdh-2 (yeast SDHB) | S. cerevisiae | KO mutation Sdh-2 | High throughput drug screening. The main upshot was the potential benefit of LDH inhibition | |
Copies of human mutations | S. cerevisiae | Putting human mutations into yeast SDHx to see if pathogenic | Was correlation in damaging effects between yeast and human equivalent mutations | |
Sdh6 (SDHAF1) disruption | S. cerevisiae | Disruption of the SDHAF1 homologue Sdh6 | SDH deficiency, succinate accumulation, and prevented OXPHOS-dependent growth | |
Sdh5 (SDHAF2) disruption | S. cerevisiae | Disruption of the SDHAF2 homologue Sdh5 | Succinate accumulation; failure in respiratory-dependent growth and reduction in oxygen consumption | |
Sdh8 (SDHAF4) disruption | S. cerevisiae | Disruption of the SDHAF4 homologue Sdh8 | Succinate accumulation, but also maintains approximately 40% of wild type SDH activity | |
Worm |
Complete sdhb KO | Caenorhabditis elegans | Complete sdhb-1 KO | Lethal at L2 larval stage. Sensitivity to hyperoxia. Succinate accumulation, damaged oxygen consumption | |
Cell-specific sdhb-1(RNAi) | C. elegans | Cell-specific sdhb-1(RNAi) | Also used DMOG to suppress SDH. Crosses with HIF mutants | |
Copy of human mutation in SDHB | C. elegans | Arg244His point mutant (corresponding to Arg230His in human SDHB) | Succinate accumulation, damaged oxygen consumption, increased pyruvate and lactate levels, increased LDH activity. Sterile adults | |
mev-1 | C. elegans | G71E in SDHC | mev-1 was discovered in a worm screen for oxygen sensitivity. G71E mutants display oxygen hypersensitivity, decreased lifespan and brood size phenotypes. G71E substitution did not affect succinate-to-fumarate conversion, but led to electron leakage | |
sdha-1 variations | C. elegans | Over- and under-expression of sdha-1 | Loss-of-function mutations in sdha-1 increases, while sdha-1 over-expression decreases phosphoenolpyruvate carboxykinase: an inverse correlation between mitochondrial function and the levels of anabolic processes | |
Fruit fly |
SDHB KO | Drosophila melanogaster (fruit fly) | Sdhb-/- | Defective complex II. Increased hydrogen- peroxide. Hypersensitive to oxygen, progeroid, early death, abnormal flight muscle, mitochondrial abnormalities | |
SDHA and SDHB KOs | D. melanogaster | Sdha -/- Sdhb -/- | Rapamycin treatment improved climbing ability. Rapamycin treatment (inhibits mTor) Extends life of mutants. Rapamycin increased SDH enzymatic activity but no decrease in ROS levels | |
Deletion of the SDHAF4 ortholog dSdhaf4 | D. melanogaster | Deletion of dSdhaf4 | Neurodegenerative phenotypes, early-adult lethality and sensitivity to oxidative stress. Significant succinate accumulation and almost 90% decrease in SDH activity | |
Deletion of the SDHAF3 ortholog dSdhaf3 | D. melanogaster | Deletion of the SDHAF3 ortholog dSdhaf3 | Hypersensitive to oxidative stress; muscular and neuronal dysfunction. Impaired SDH activity and reduced SDHB levels | |
Zebrafish |
Complete KO of SDHB | Danio rerio | Complete KO of sdhb | Decreased survival; abnormal development (swim bladders). Increased succinate level | |
A key advantage of yeast is that it can be haploid. Loss of the
SDHB (
Sdh2 in yeast) subunit
Sdh2(-) in a yeast model resulted in increased ROS production but without evidence of mutagenic DNA damage [
37]. In the mutant yeast strain, succinate accumulated significantly, which was shown to inhibit two alpha-KG-dependent enzymes: Jlp1, involved in sulfur metabolism and Jhd1, which is a JmjC-domain-containing histone demethylase (JHDMs) enzyme. Smith and colleagues also showed that mammalian JmjC-domain histone demethylases were also sensitive to succinate inhibition
in vitro and in cultured cells, proposing that any alpha-KG-dependent enzyme might be dysregulated by excess succinate leading to tumorigenesis and or/tumor progression. These findings were underpinned by
in vitro studies performed by Xiao et al. [
33] on human α-KG-dependent enzymes, showing that the structurally similar succinate and fumarate anions can also function as alpha-KG antagonists. As a consequence of inhibiting multiple a-KG-dependent dioxygenases, including the JMJD family KDMs and the TET family of 5mC hydroxylases, excess succinate and fumarate might cause genome-wide alterations in histone and DNA methylation patterns.
The effect of a pathogenic missense mutation (C191Y) in the
SDHB gene associated with a glomus tumor (i.e. paraganglioma) was examined in yeast. The homologous mutation resulted in abolished SDH activity with increased sensitivity to oxidative stress. In addition, the
sdh2C184Y mutant allele (equivalent to human
SDHBC191Y) was not able to rescue the defective OXPHOS phenotype of the
Δsdh2 null, or knockout, mutant. The authors concluded that the C191Y mutation leads to both an increase in ROS production and to mitochondrial DNA mutability [
78].
Panizza et al. analyzed a series of missense mutations in
SDHB,
SDHC and
SDHD homologous genes and demonstrated that yeast functions as a good model to validate the pathogenic significance of these mutations, in case they concern conserved amino acid residues in conserved domains [
80]. This illustrates how yeast may be used to examine ‘variants of unknown significance’ found in sequencing of the human genome.
In the basic biochemistry, pioneering studies in yeast contributed to the elucidation of the SDH assembly pathway. SDHAF1 was the first SDH assembly factor to be identified. Homozygous
SDHAF1 mutations were detected in patients suffering in infantile leukoencephalopathy, a neurodegenerative disorder similar to Leigh Syndrome [
95]. The patients showed elevated succinate levels detectable by
in vivo proton MR spectroscopy [
95]. Disruption of the SDHAF1 yeast homologue
Sdh6 caused SDH deficiency and prevented OXPHOS-dependent growth [
81]. Yeast
sdh5, the homologue of human
SDHAF2, was first discovered as an uncharacterized mitochondrial protein, which was highly conserved throughout eukaryotes [
14]. Deletion of
sdh5 in yeast caused failure in respiratory-dependent growth and reduction in oxygen consumption. Furthermore, Hao et al. identified Sdh1/SDHA as binding partners of Sdh5p, which is required for Sdh1p flavinylation. Subsequently, at least two families with familial paraganglioma have been found to have
SDHAF1 mutations, showing that mutations in SDH assembly factors can recapitulate the phenotype caused by the core subunit mutations [
14].
Yeast cells lacking
Sdh8 exhibit accumulation of succinate, but the magnitude of succinate accumulation is much less than that observed in
sdh5Δ mutants, and
sdh8Δ mutant yeast maintain approximately 40% of wild type SDH activity [
82]. These findings show that Sdh8 is involved in SDH biogenesis, but it is not absolutely required for SDH assembly.
Currently there are no publications associated with human mutations in SDHAF3 (sdh7) or SDHAF4 (sdh8) assembly factor genes.
Thus, yeast has been a very useful and instructive model for basic SDH biochemistry (suggesting new human genes for mutational study), for methylation effects, for metabolism, and for investigation of variants of unknown significance.
2.1.3
Caenorhabditis elegans (henceforth, ‘the worm’) is a nematode worm found in the soil. About 1 mm long, it has about 1000 somatic cells whose lineages are understood, just less than half of which form its very primitive nervous system. In as simple an organism as this, ‘neuroendocrine’ cells as such might not be expected, but in fact, the neuron known as ‘RID’ has many features of a neuroendocrine cell [
96].
The
C. elegans mutation originally known as
mev-1 was discovered in a screen for oxygen sensitivity [
86]. The name is derived from ‘methyl viologen’, the redox-active weedkiller also known as paraquat. It was later shown that the mutation lay in s
dh3 (
SDHC) [
87]. This homozygous point mutation (G71E in the worm) did not affect the metabolic function of the enzyme (namely to oxidize succinate to fumarate in the TCA cycle), but led to electron leakage, reflecting the role of the
sdh3/
SDHC subunit in electron transport [
88]. This manifests in oxygen hypersensitivity, decreased lifespan and brood size phenotypes, while a deletional allele of
mev-1 (
mev-(lf)) is lethal [
89]. These phenotypes have been replicated in a mouse model (see in ‘Rodent’ section below), supporting the idea that these processes are evolutionarily conserved [
97]. It is of interest that many agricultural pest control agents target SDHx as their mode of action [
98], and such agents are being considered for use as human anti-fungal treatments [
99].
Recently
mev-1 was used in a proof-of-concept study of miniSOG (‘singlet oxygen generator’)-mediated CALI (‘chromophore-assisted light inactivation’), which is a technique in which spatio-temporally controlled ROS generation is conducted. The use of miniSOG-mediated CALI is a suitable platform for instant inactivation of respiratory chain components [
100].
Huang and Lemire [
83] observed that different mutations in the
SDHB gene resulted in ‘superoxide generation and premature aging’. Important to note, that the
C.
elegans sdhb-1 null mutant’s development arrests midway in development at the L2 larval stage.
Very recently, a copy of the human SDHB Arg230His mutation that presented in a Scottish family has been created in
C. elegans (Arg244His in the worm) [
84]. The findings are at an early stage, but the data show that (i) the enzyme is definitely defective, in that there is an abnormal build-up of succinate; (ii) the worm develops abnormally and is sterile but lives to a later larval stage than the L2-arrested SDHB-deletion worm; and (iii) shows a different pattern of metabolic rearrangement (reminiscent of Warburg effect) compared to the complete SDHB-deletion worm.
Another worm group used convergent genetic and pharmacological approaches to study the effect of SDH deficits on the HIF system in the worm [
85]. One effect of HIF activation in the worm is a delay in egg-laying, or ‘egg retention’. To study this, they knocked out SDHB in a subset of neurons, including those thought to be responsible for egg-laying. They also used dimethyloxalylglycine (DMOG), a succinate analogue. Both manoeuvres retarded egg laying; but
not in HIF-1 knockout worms, showing that the delay in egg-laying is dependent on the HIF system. According to their model, the inhibition of EGL-9 by excess succinate prevents HIF-1 (human HIFα ortholog) hydroxylation; thereby promoting HIF-1 signalling, which eventually leads, among other processes, to the observed retention of eggs.
Lastly, in well-fed worms carrying loss-of-function mutations in
sdha-1, the expression of phosphoenolpyruvate carboxykinase (PEPCK;
pck-1 and
pck-2), which converts oxaloacetate to phosphoenolpyruvate and CO
2, and is part of the gluconeogenic pathway, is increased, while
sdha-1 over-expression has the opposite effect, suggesting an inverse correlation between mitochondrial function and the levels of anabolic (e.g. gluconeogenic, lipogenic) processes [
90]. The homozygous
sdha-1 loss-of-function nematodes are viable but show cell non-autonomous behavioral and developmental deficiencies: slower developmental rate from L2 to L3 larval stage; L4 males fail to remodel their anal depressor muscle and are therefore incapable of copulation; show slower movement; and show a slower rate of oxygen consumption. Their mitochondria are smaller and less networked [
90].
This illustrates an interesting metabolic re-routing in SDH deficiency, which is also seen in tumors in general [
101].
Thus,
C. elegans can reveal oxidative effects, metabolic effects and developmental abnormalities in SDHx mutants.
2.1.4
The fruit fly
Drosophila melanogaster is perhaps the longest-used model organism, having been studied (by Morgan and others) since about 1910. Although it has been used very extensively in other biological research, few studies have been done on succinate dehydrogenase. Walker and others [
91] were interested in molecular mechanisms that regulate the formation of reactive oxygen species and performed a screen for
Drosophila mutants that are hypersensitive to high oxygen levels (100%: room air is about 20%). They isolated and characterized an
SDHB mutant, which survived for only one day in 100% oxygen, while wild-type flies survived for about 7 days. Survival in room air was likewise curtailed. Measurements of hydrogen peroxide showed a 32% increase in the mutants. Mitochondrial structure was abnormal; tests of ageing showed premature ageing in the mutants.
Orthologs of two SDH assembly factors,
dSdhaf3 and
dSdhaf4 were extensively characterized in
Drosophila. Loss of
SDHAF3/dSdhaf3 in the fruit fly results in impaired SDH activity and reduced SDHB levels, in addition mutant flies are hypersensitive to oxidative stress and display muscular and neuronal dysfunction phenotypes [
93]
Deletion of the
SDHAF4 ortholog
dSdhaf4 caused significantly more succinate accumulation and almost 90% decrease in SDH activity, coupled with neurodegenerative phenotypes, early-adult lethality and sensitivity to oxidative stress [
82].
Thus, the fruit fly confirms similar observation on ROS seen in
C. elegans and yeast.
Recently
Danio rerio has also shown potential to become an effective model in which to investigate the mechanisms behind PPGL [
94]. Homozygous mutant animals with truncated
sdhb showed shorter lifespans, incompletely or non-inflated swim bladders, defects in energy metabolism and swimming behaviour. In homozygous
sdhb-mutant larvae, the number and structure of the mitochondria showed no difference from the wild-type, and they observed decreased mitochondrial complex 2 activity and succinate accumulation, as in
SDHB-associated PPGLs. This novel model offers an opportunity to investigate both HIF activation and alpha-keto-glutarate-dependent dioxygenases, and to screen possible therapeutic targets.
2.1.6
Rodents have been very important in SDH research.
Table
5 shows the mouse and rat models.
Table 5
Mouse and rat models of PPGL
Mouse models |
SDHD-ESR TD-SDHD | Mouse | Two floxed sdhd mouse models. SDHD-ESR has a tamoxifen-inducible CRE recombinase, while TH-SDHD has a catecholaminergic, tissue specific inducer (tyrosine hydroxylase) | sdhd -/- | No tumors, but changes in catecholaminergic cell maturation. The SDHD-ESR mouse was later used to prepare tissues and two cell lines, as described by Millan-Ucles et al. 2014 [ 74] | |
Combined sdhb pten | Mouse | Cross between floxed pten+/- and sdhb +/-mice, targeted by PSA promoter | sdhb-/- pten -/- | Tumors occurred but did have residual SDH activity, suggesting that only those with attenuated cre activity, with persisting sdhb activity, had actually survived | |
Xenograft in NOD-scid (NSG) | Mouse with human xenograft | Viable tissue from human patient tumors injected into subcutaneous tissues of immunodeficient mice | As defined by original patient | Similar to human pathology. Technically difficult and slow process | |
iMCC SDHB-KO allograft | Allografts to mouse from iMCC cells | iMCC cells derived from a ‘floxed’ (sdhb exon 2) mouse injected into nude mouse | sdhb-/- | Useful imaging data obtained by PET-CT | |
MTT-SDHBKD | Mouse | Allograft of MTT cells with SDHB knock-down into nude mice | sdhb-/- | Definite reduction in both message and SDHB protein SDH, increased succinate. Study of NAD+/PARP pathway | |
N2a Sdhb knockdown allograft | Mouse | N2a (neuroblastoma) cells with Sdhb knocked down allografted into mice | sdhb KD | Used to study effect of alkaloid piperlongumine on SDH-deficient cells | |
RS0 xenograft | Rat-to-mouse xenograft | RS0 Sdhb-/- rat tumor cells xenografted into nude mice | sdhb-/- | First real sdhx KO mouse model; true zellballen architecture | |
Rat model |
MENX | Non-sdhx pheo model in the rat | Naturally occurring mutant | | Carries germline mutation in Cdkn1b, coding for cell cycle regulator p27kip1. Pseudohypoxic phenotype, but not sdhx-mutated | |
It is first important to say that it has proved impossible to generate, by conventional means, a complete knock-out of
sdhx in a mouse [
109], even with organ targeting etc. Sitting as SDH does at the very centre of the metabolism of every living thing on the planet, this is perhaps not too surprising.
The first stab was taken by the Spanish. They produced two floxed
sdhd mice. The first, SDHD-ESR, had a tamoxifen-inducible promoter, while TH-SDHD was under the control of tyrosine hydroxylase promoter, so that the wild-type alleles could be knocked out (by CRE recombinase) when desired (by tamoxifen) and in the right place (where catecholamines are made) [
102]. Regrettably, tumors were not forthcoming, but the SDHD-ESR mouse was used to make tissue for further study and for cell lines [
74].
The French group also made two heterozygous mouse models for subsequent mating: a floxed
pten+/- and a floxed
sdhb +/- , to try to exploit the propensity for
pten-mutated mice to develop tumors. When these were crossed, tumors did occur, but regrettably, residual SDH activity was still present, suggesting that the tissue had only survived because the expected CRE-mediated elimination of
sdhb had been incomplete [
103].
Across the ocean, the Tischler lab was able to xenograft viable human tumor tissue into immunodeficient, NSG, mice [
104]. This worked, but it is a slow and tricky process, totally dependent on material from human surgery.
The French were able allograft iMCC SDHB-KO cells into NMRI-nu nude mice, to obtain tumors, which allowed a novel form of magnetic resonance imaging aimed at the detection of succinate itself [
105].
The Bethesda team used a similar allograft approach, this time using MTT cells with
shdb knockdown (MTT-
SDHBKD) in nude mice, to investigate the action of olaparib, which targets a DNA repair pathway, in an attempt to exploit this action to sensitise the cells to chemotherapeutic agents, with some success [
62]. Again in Bethesda, Bullova and others used N2a neuroblastoma cells (which are neuroendocrine) with
Sdhb knockdown to allograft nude mice, to study the effect of the alkaloid piperlongumine on high-ROS tumor cells, with promising results [
65].
Lastly, Powers and others have used their RS0 (
Sdhb-/-) rat cell line successfully to xenograft into nude mice [
57], making a very promising model. The RS0 xenograft line showed the ‘zellballen’ architecture, did not express SDHB and produced dopamine with low levels of norepinephrine. Metabolic profiling of RS0 xenografts demonstrates succinate and lactate accumulation and transcriptomic analysis shows high expression of HIF2alpha regulatory network components [
57].
In the rat, the main mouse line that shows spontaneous formation of PPGL tumors is ‘MENX’, which is not an
Sdhb-mutated line, but is known to carry a mutation in the cell-cycle regulator p27kip1. However, it does show a pseudohypoxic phenotype [
107,
108], a useful feature for students of SDH.
Curiously the dog is susceptible to a very similar spectrum of malignant diseases as the human, and PPGL is no exception. Keeping dog colonies is prohibitively expensive, and reproduction rates are long in laboratory timescales, but research efforts based on a ‘breed-based genomic approach’ have been begun [
110].
Table
6 lists studies on canine PPGL. Canine PPGL is not uncommon in veterinary practice [
111]. PCC occurs more often in middle-aged to older dogs. In addition, the incidence of PCC does not depend on breed or gender. Dogs develop metastases in 13–28% of cases, most often in lymph nodes, in the liver, lung, spleen and bones in contrast to humans where the chance of metastases are approximately 10% and concern lymph nodes, liver, lung and bones [
112].
Table 6
Studies in the dog. The table summarises publications on canine PPGL. These are based on clinical observation by our veterinary colleagues. There have been no attempts to manipulate the dog genome
Collection of 61 cases. No sequencing | Ultrasound is a useful tool in clinical investigation. Metastasis was common | |
Six PCs and 2PGLs. Sequenced SDHD and SDHB | One germline sdhd mutation was found; many somatic sdhd and sdhb mutations. | |
Measurement of urinary and plasma catechols metanephrines as diagnostic tool | As in humans, the measurement of urinary caecholamines can be instructive | |
Clinical review | Some metastasise, some do not | |
Molecular study of 50 PPGLs. Immunocytochemistry and SDHB + SDHD sequencing | Much loss of chromosome 5. Likely pathogenic mutations in SDHB and SDHD found, although no definite germline cases | |
Occurrence of both Cushings (ACTH) and pheo is same dog | Case report describing a very unusual combination, which can also occur in human medicine | |
Survey of dog tumors from the vet clinic, by immunohistochemistry | A proportion are deficient in SDHA and SDHB | |
Diagnosis of PPGLs in dogs can be made by diagnostic imaging (ultrasonography, computerized tomography (CT) and magnetic resonance imaging (MRI)), as well as by biochemical testing [
112]. As in human patients, both plasma and urine samples can be tested biochemically. To differentiate pheochromocytoma from other adrenal conditions, measuring urinary normetanephrine:catecholamine ratio seems to be the best option [
114]. Urinary and plasma catecholamines and metanephrines in dogs with pheochromocytoma, hypercortisolism, nonadrenal disease and in healthy dogs.
Holt and others identified
SDHB (pArg38Gln) and
SDHD (pLys122Arg) mutations in canine PPGL [
113]. Korpershoek and others examined 25 PPGL samples by Sanger sequencing and found one case of
SDHB and 3 cases of
SDHD mutations [
115]. Immunocytochemical and DNA rearrangements were similar to those in human tumors. Abed and others examined 35 PCC samples by immunohistochemistry and the majority of the sections showed SDHx abnormalities: 25 samples lacked SDHB immunoreactivity, whereas 4 samples did not express either SDHA or SDHB [
117].
2.2 Model systems: in drug action
2.2.1
Current treatment of PPGL tumors
Aside from surgery, the most frequently used treatments for metastatic PPGL have been chemotherapy and radiation therapy [
24]. The most commonly used combination chemotherapy includes cyclophosphamide, vincristine and dacarbazine, known as ‘CVD’ [
118]. CVD treatment is commonly used as a first-line therapy for metastatic PPGL, as patients show response to the treatment and have an overall improvement of other symptoms (blood pressure, blood glucose level). However, only about half of the treated patients respond to CVD treatment and after discontinuation of the therapy, a large number of patients show tumor progression [
119‐
121].
Another well-used treatment for metastatic PPGLs is the radionuclide therapy, using radioactively tagged compounds that bind to the tumors.
131I-MIBG is one;
177Lu-DOTA-SSA is another. Although treatment with
131I-MIBG can cause several side-effects including nausea, anorexia, thrombocytopenia, lymphopenia and leukopenia [
122,
123], it has been proven that patients who respond to the treatment live longer [
124]. As progression of PPGLs can happen rapidly, early treatment after diagnosis has been advised [
123,
125,
126].
Studies on
SDHB-deficient models showed that
SDHB deletion or knockdown results in an increase of reactive oxygen species. A novel option in PPGL treatment is to further elevate ROS levels in tumor cells, which leads to their apoptosis through DNA damage. Such an approach is to use ascorbic acid to increase the oxidative burden of tumor cells.
SDHBKD allograft-bearing mice were treated with pharmacologic ascorbic acid and showed suppressed tumor growth and longer overall survival [
60].
Another way to exert increased oxidative stress on tumor cells is by blocking NRF2, which plays a role in glutathione synthesis. Glutathione is a substrate of glutathione peroxidase, a key enzyme in eliminating ROS. In a recent study Brusatol, an NRF2 inhibitor, resulted in longer survival and suppressed metastatic lesions of PPGL allograft mice. Thus, Brusatol is another promising therapeutic agent in treating PPGLs [
127].
As currently used therapies are mainly palliative and not all patients respond, there is a need for new possible treatments concerning PPGL patients. Anti-angiogenic treatment options are under development, such as sunitinib, carbozantinib, axitinib, levantinib and pazopanib [
126]. Ongoing PPGL-related clinical trials can be checked on the US Government Clinical Trials website (clinicaltrials.gov) [
128,
129].
In parallel with recent chemotherapic and radiotherapic treatments, novel targeted treatment strategies are also under development. Many of these studies are in a preclinical phase, performed on murine and human cell lines and spheroids. The novel agents include BYL719 (a PI3K alpha inhibitor), sunitinib (a receptor tyrosine kinase inhibitor) and everolimus (an mTORC1 inhibitor) [
130]. Some of these drugs have been tried in combination. The most promising of these combinations seems to be that of BYL719 and everolimus: the combined effects of these drugs include shrinkage or collapse of tumor cell spheroids, in addition to GSK3 inhibition, cyclinD1/D3 downregulation in PCC cell lines [
130]. The HIF1 inhibitor, belzutifan, has recently been shown to be useful in VHL disease [
131] and Pacak-Zhuang disease [
132] and we understand that clinical trials of this drug are in progress in PPGL.
Pseudohypoxia alters immune system functions; for example, it leads to inactivation of cytotoxic T-cell lymphocytes and increased expression of the immune checkpoint protein programmed death-ligand 1 (PD-L1) and its receptor [
133]. PD-L1/PD-1, as important immune checkpoint proteins have been often targeted by novel immune therapies in the last decade. This pathway, among others, functions in the recognition of cancer cell by the immune system. A phase 2 clinical trial of the PD-1 inhibitor pembrolizumab for patients with metastatic pheochromocytoma and paraganglioma is currently ongoing (NCT02721732) [
134].
2.2.2
Treatment strategies developed in animal models
Novel treatment strategies are still in need because of the recurrence of PPGLs following treatment. Simple and more complex model systems are frequently used as first options to test new therapeutic drugs in parallel to tumor cell lines and/or mice. We only show some examples of how different model systems have been applied to develop novel treatments or to test the effect of drug candidates.
Yeast SDH mutant cells were also used to conduct high-throughput drug screens by Bancos et al. [
79]. These authors screened more than 200,000 compounds to find drugs that are differentially toxic to mutant yeast and identified several inhibitors of alcohol dehydrogenase, which is the yeast equivalent of human lactate dehydrogenase: both regenerate NAD
+ when TCA cycle function is deranged. Therefore, the authors treated SDH-deficient human HEK293 cells with a lactate dehydrogenase inhibitor (LDHI) and found that they were sensitive to LDHI. Thus, lactate dehydrogenase might be a novel therapeutic target.
Another possible treatment option is the mTOR inhibitor rapamycin, also known as sirolimus (everomilus is a cousin), which was tested in
Drosophila sdhA and
sdhB mutants. Following rapamycin treatment, the mutant flies lived longer than the non-treated ones. Treated sdhB mutants also showed improved climbing ability and had increased SDH enzymatic activity. However rapamycin treatment did not decrease ROS levels in mutant flies [
92].
Vascularisation is an important component of tumor growth and a target for therapy; the NOTCH antagonist delta-like 1 homologue (DLK1) was previously identified as a tumor pericyte-associated antigen in various carcinomas [
135], also in renal cell carcinomas [
136]. Inhibition of DLK-1 (e.g. vaccination against DLK-1) led to tumor vascular normalization [
135].
Verginelli et al. found that Imatinib (Gleevec) that targets endothelial-mural signalling, blocked paraganglioma xenograft formation [
8,
9]. In addition, these authors showed that inhibition of DLK1 signaling at the level of PDGFR kinase by imatinib prevented the formation of paraganglioma tumors in nude mice [
9], providing a mechanistic rationale for investigating this treatment.
Themozolomide (TMZ), a chemotherapeutic agent combined with the PARP (Poly (ADP-ribose)-polymerase) inhibitor Olaparib (Ola) was tested on a mouse allograft model. In this model athymic nude mice were injected intravenously by
SDHB-silenced MTT cells. The mechanism behind depends on the fact that the absence of complex II (SDH) in the respiratory chain results in overactivation of complex I, also known as NADH:ubiquinone oxidoreductase, leading to excess NAD
+ production. PARP, which is known to repair DNA breaks after genotoxic stress, uses the elevated NAD
+ levels as a cofactor [
137,
138]. When the NAD
+/PARP DNA repair pathway was inhibited by the TMZ/Ola combination, mice survived longer and had less metastatic lesions [
62]. Recently, TMZ and Ola treatment option has been translated into an NCI Clinical Trial [
139].