Molecular pathogenesis of tumorigenesis caused by succinate dehydrogenase defect

Highlights

• Malfunction of succinate dehydrogenase (SDH), leads to tumorigenesis.

• Succinate is linked to metabolite and GPCR signalling, and inflammatory response.

• Moderate generation of ROS leads to tumorigenesis.

• Succinate levels and ROS are therefore the denominators of tumorigenesis.

Abstract

Succinate dehydrogenase (SDH), also named as complex II or succinate:quinone oxidoreductases (SQR) is a critical enzyme in bioenergetics and metabolism. This is because the enzyme is located at the intersection of oxidative phosphorylation and tricarboxylic acid cycle (TCA); the two major pathways involved in generating energy within cells. SDH is composed of 4 subunits and is assembled through a multi-step process with the aid of assembly factors. Not surprisingly malfunction of this enzyme has marked repercussions in metabolism leading to devastating tumors such as paraganglioma and pheochromocytoma. It is already known that mutations in the genes encoding subunits lead to tumorigenesis, but recent discoveries have indicated that mutations in the genes encoding the assembly factors also contribute to tumorigenesis. The mechanisms of pathogenesis of tumorigenesis have not been fully understood. However, a multitude of signaling pathways including succinate signaling was determined. We, here discuss how defective SDH may lead to tumor development at the molecular level and describe how yeast, as a model system, has contributed to understanding the molecular pathogenesis of tumorigenesis resulting from defective SDH.

Introduction

Succinate dehydrogenase (SDH) also named as complex II or succinate:quinone oxidoreductases (SQR) is positioned at the intersection of oxidative phosphorylation and tricarboxylic acid cycle (TCA) (Fig. 1), the essential pathways of bioenergetics in many organisms. SDH comprises 4 subunits (Fig. 1) that are assembled together through a multi-step process which is facilitated by accessory proteins. The details of structure, function and assembly of SDH have been recently reviewed by our group in a separate article and interested readers are referred to that article (Moosavi et al., 2019).

Mutations in the genes encoding 4 subunits lead to tumorigenesis. Additionally recently various types of tumors have been caused by defects in accessory (assembly) factors.

Tumorigenesis is not only the consequence of energy production deregulation and metabolism; an essence of the function of SDH but also the result of signaling events, as this enzyme plays diverse signaling roles, some of which recently discovered. This, therefore, makes understanding the tumorigenesis difficult. Further complexity is added to that because SDH, on the one hand, is a sensor of apoptosis and therefore its proper function is required for apoptosis and on the other hand, its defect causes apoptosis through various mechanisms. We here, put together the pathways which contribute to tumorigenesis as a consequence of SDH defect and present a clearer picture of the molecular details.

For simplicity, the mutations are divided into two groups; mutations in SDH subunits and in assembly factors.

SDH impairment is implicated in tumors such as hereditary paraganglioma, and pheochromocytoma (Astuti et al., 2001; Baysal et al., 2000; Niemann and Müller, 2000; Yankovskaya et al., 2003). Succinate dehydrogenase (SDH) and fumarate hydratase (FH; the enzyme which catalyzes the next step in the Krebs cycle ensuing SDH) mutations follow the hereditary pattern of tumor suppressor genes. The inflicted cells inherit a germline loss of function mutation in one allele while the other wild-type allele is lost through somatic deletion or chromosomal loss (termed loss of heterozygosity) leading to tumor development (Bayley and Devilee, 2010; Zhou et al., 2018).

Although initially it was thought that SDHA mutations were responsible for Leigh syndrome (LS), a neurodegenerative disease, recent data indicate SDHA mutations can also cause sporadic paragangliomas and pheochromocytomas (Burnichon et al., 2010; Dwight et al., 2013; Korpershoek et al., 2011) and even recently an SDHA mutant has been reported to give rise to both disorders (Renkema et al., 2015). Furthermore, mutations in the SDH assembly factor SDHAF1 (Ghezzi et al., 2009a), SDHB (Alston et al., 2012) and SDHD (Jackson et al., 2013) were shown to induce LS or LS-like symptoms.

Defect in SDH expression has also been observed in a subset of gastrointestinal stromal tumors (GIST). GIST that lack KIT or platelet-derived growth factor receptor alpha (PDGFRA) mutations have been classified as KIT/PDGFRA wild type GIST (WT GIST) (Corless et al., 2004). 20–40% of all KIT/PDGFRA WT GIST is succinate dehydrogenase complex (SDH)-deficient GIST. In this disease, SDH expression is impaired usually as a result of germline and/or somatic loss-of-function mutations in any of the four SDH subunits (A, B, C, or D) (Corless et al., 2004; Nannini et al., 2017). Other forms of malignancy associated with impaired SDH function include renal and thyroid tumors, neuroblastoma, and testicular seminoma, implying its involvement in a wide range of tumors (Bardella et al., 2011).

Besides tumor and neurodegeneration, SDH defects are also implicated in diabetes, ageing, optic atrophy, ischemia reperfusion (IR) injury and ischemic preconditioning (IPC) (Rustin et al., 2002; Wojtovich et al., 2013). The broad spectrum of diseases is likely a reflection of several roles that SDH plays in cellular processes (Rustin et al., 2002).

So far at least 4 SDH assembly factors have been identified in humans including SDHAF1, SDHAF3, SDHAF2, and SDHAF4.

A soluble mitochondrial matrix protein (Iverson et al., 2012) which was initially identified as a specific human SDH assembly factor, defective in two families with specific infantile leukoencephalopathy syndrome. The diminished function of SDHAF1 in humans and its homolog in yeast cause reduced SDH activity and assembly but engender no effect on other mitochondrial respiratory chain complexes implying its specificity to the SDH assembly (Ghezzi et al., 2009a). Later more cases of infantile leukoencephalopathy syndrome were reported with similar or novel mutations in SDHAF1 gene (particularly Gly57Arg substitution) with varying clinical symptoms. All patients showed accumulation of succinate detectable by in vivo proton MR spectroscopy of the brain (Ohlenbusch et al., 2012; Taylor et al., 2013).

Studies have indicated that although yeast Sdh6 and human SDHAF2 have similar functions, they may differ in certain aspects (Ghezzi et al., 2009b). Sdh6 (in yeast) presumably interacts with Sdh1/Sdh2 subcomplex and the interface for the interaction lies within Sdh2 (Van Vranken et al., 2015). SDHAF1 (in humans) contains an LYR motif; LX(L/A)YRXX(L/I)(R/K) which is likely the signature for proteins involved in Fe-S metabolism. Thus SDHAF1 harboring LYR motifs may play a role in insertion or retention of the Fe-S centers within SDH structure. Consequently, failure in SDHAF1 incorporation into the apo-enzyme structure may undermine the holo-enzyme structure or prevent its formation (Ghezzi et al., 2009a). In line with this, defective SDHAF1 significantly debilitates the biogenesis of SDHB as a result of sequential rapid degradation by the mitochondrial protease, LONP1 (Maio et al., 2016).

A separate study using yeast, fly and mammalian cell culture has shown that SDHAF1 in concert with SDHAF3 (another assembly factor; see below) promotes maturation of SDH2 during oxidative metabolism and particularly protects against ROS damage, however, the details have not been fully investigated (Na et al., 2014).

SDHAF3/Sdh7 and SDHAF1/Sdh6 are assembly factors required for maturation of SDHB/ Sdh2. When Sdh7 in yeast and Drosophila is deleted SDH activity and Sdh2 levels are reduced. Drosophila without Sdh7 is hypersensitive to oxidative stress and suffers from muscular and neuronal dysfunction. Yeast Sdh7 and Sdh6 act together to assist in Sdh2 maturation through interaction with Sdh1/Sdh2 intermediate (interface of the reaction resides within Sdh2), protecting against oxidative damage. These data from yeast and Drosophila implied that SDHAF3 mutations might have caused SDH-associated disease in cases where no mutations in the genes encoding SDH subunits were identified (Van Vranken et al., 2015). This prediction recently came true as an impaired (hypomorphic) variant of SDHAF3, c.157 T > C (p.Phe53Leu) was identified in pheochromocytoma and paraganglioma patients. In agreement with that, the sdhaf3 mutant could not restore SDH function in yeast cells lacking Sdh7. Furthermore, although WT SDHAF3 could interact directly with SdhB in vitro, the defective variant could not (Dwight et al., 2017).

Studies in human initially revealed that germline loss-of-function mutations in SDHAF2 gene segregate with hereditary paraganglioma; a neuroendocrine tumor hitherto associated with mutations in genes encoding SDH subunits (Hao et al., 2009). Further clinical studies were carried out in sporadic patients with paraganglioma and pheochromocytoma who had no mutations in SDHD, SDHC, or SDHB. They revealed that investigating SDHAF2 mutations in young patients suffering from isolated head and neck paraganglioma without mutations in SDHD, SDHC, or SDHB, and in individuals with familial antecedents who are negative for mutations in all other risk genes is definitely justifiable and may reveal mutations in SDHAF2 gene (Bayley et al., 2010). Subsequently, more cases of head and neck paraganglioma and their association with various types of SDHAF2 mutations/deletion were reported (Hoekstra et al., 2017; Kunst et al., 2011; Piccini et al., 2012; Zhu et al., 2015). In one study a universal genetic screening approach was applied for sequencing all susceptibility genes for hereditary pheochromocytoma/paraganglioma. And a novel SDHAF2 mutation in association with pheochromocytoma was identified which was previously found to be linked to mutations in SDH subunits and other genes. This study illustrated that the application of this approach was particularly useful to detect new SDHAF2 mutations which might have been otherwise overlooked by assessing only phenotype (Casey et al., 2014).

Sdh8 is a mitochondrial matrix protein with two functions; first, it binds directly to Sdh1-FAD and acts as a chaperone to protect against ROS generated by solvent-accessible FAD covalently bound to Sdh1. Second, it assists in Sdh1-Sdh2 dimer formation and thereby stabilizes the SDH complex structure.

So far no human disease reported being associated with defective SDHAF4. However, the pathology of sdhaf4Δ in Drosophila can possibly be linked to the corresponding human diseases, as defects in various SDH subunits of model organisms typically cause analogous diseases in humans. This is in line with the reports of SDH-defective diseases such as Leigh’s syndrome and Wild Type gastrointestinal stromal tumors (WT GIST) without any mutations in all known genes encoding SDH subunits (Van Vranken et al., 2014).

Mutating SDH8 in yeast/fly and siRNA-mediated knockdown of SDHAF4 in mammalian cells reduce the levels of SDH complex and SDH enzyme activity. Nevertheless, Sdh8/SDHAF4 is not absolutely essential for SDH activity. Because SDH activity is still preserved to some extent even though Sdh8/SDHAF4 is lacking in yeast/fly/mammalian cell models. Surprisingly though the loss of SDHAF4 induces more drastic outcomes in Drosophila than in yeast and mammalian cells. This is partly because sdhaf4Δ Drosophila maintains only 10% of SDH activity and almost no SDH complex while the sdh8Δ yeast strain keeps 40% of SDH activity and complex formation. Additionally deletion of SDHAF4 in Drosophila, unlike yeast, destabilizes Sdh1 dramatically and leads to both muscular and behavioral dysfunction. The reason for these differences is not fully understood but it might be that SDHAF4/Sdh8 in mammalian cells/yeast cells is functionally redundant or that the yeast growth media may provide a buffering capacity against oxidative stress (Van Vranken et al., 2014).