SDH defective cancers: molecular mechanisms and treatment strategies

Abstract

Succinate dehydrogenase (SDH), considered as the linkage between tricarboxylic acid cycle (TCA cycle) and electron transport chain, plays a vital role in adenosine triphosphate (ATP) production and cell physiology. SDH deficiency is a notable characteristic in many cancers. Recent studies have pinpointed the dysregulation of SDH can directly result its decreased catalytic activity and the accumulation of oncometabolite succinate, promoting tumor progression in different perspectives. This article expounds the various types of SDH deficiency in tumors and the corresponding pathological features. In addition, we discuss the mechanisms through which defective SDH fosters carcinogenesis, pioneering a categorization of these mechanisms as being either succinate-dependent or independent. Since SDH-deficient and cumulative succinate are regarded as the typical features of some cancers, like gastrointestinal stromal tumors, pheochromocytomas and paragangliomas, we summarize the presented medical management of SDH-deficient tumor patients in clinical and preclinical, identifying the potential strategies for future cancer therapeutics.

Graphical abstract

Introduction

Tumor initiation and progression are related to extensive altered cellular energy metabolism, which provides cancer cells with adequate energy and indispensable metabolic intermediates to support uncontrolled proliferation (Kroemer and Pouyssegur 2008; Hanahan 2022). The tricarboxylic acid (TCA) cycle couples with the oxidative phosphorylation (OXPHOS), that are two primary metabolic pathways of adenosine triphosphate (ATP) production in eukaryotes (Martinez-Reyes and Chandel 2020). Although Otto Warburg indicated that cancer cells acquired ATP bypass the TCA cycle and tend to utilize aerobic glycolysis, emerging evidence demonstrates that certain cancer cells, especially those with deregulated expression of oncogene and tumor suppressor, rely heavily on the metabolism of TCA cycle (Anderson et al. 2018).

The SDH complex, also known as mitochondrial complex II (CII), is the only enzyme that participates both the TCA cycle and the electron transport chain (ETC), thus plays a substantial role in energy generation and respiratory adaptation (Bezawork-Geleta et al. 2017; Vercellino and Sazanov 2022). SDH is a hetero-tetrameric nuclear encoded protein complex, consists of two catalytic subunits (SDHA, SDHB) and two anchoring subunits (SDHC, SDHD). The maturation and assembly of the matrix subunits is assisted by four assembly factors (SDHAF1 - 4). SDHA is covalently bound by flavin-adenine dinucleotide (FAD) and generates flavin adenine dinucleotide (FADH2) (Cao et al. 2023). Meanwhile, the catalytic bind pocket for succinate in SDHA help to oxidize succinate into fumarate in the TCA cycle. SDHB contains three Fe-S clusters that accept the electrons from FADH2 and channel them to ubiquinone (Q). By binding ubiquinone, the membrane embedded SDHC and SDHD produce two protons to help its reduction to ubiquinol (QH2) and funneled the electrons to mitochondrial complex III to produce ATP sequentially. In this procedure, SDHAF2 regulates the flavin acylation of SDHA, whereas SDHAF4 protects the FAD-SDHA dimer from oxidative stress, driving SDHA maturation and the formation of the SDHA-SDHB bond. The maturation of SDHB involves the insertion of Fe–S clusters, primarily induced by SDHAF1 and SDHAF3. Once SDHA and SDHB are matured, SDHC and SDHD dimerize in the inner mitochondrial membrane. While the assembly factors are released from subunits, SDHA-SDHB associates with SDHC-SDHD to construct an integral SDH complex (Rasheed and Tarjan 2018; Nolfi-Donegan et al. 2020).

The study of SDH deficiency diseases has expanded in the past decades, including inherited primary mitochondrial disorders and tumors. Leigh syndrome (a progressive neurodegenerative disorder), cardiomyopathy and infantile leukodystrophies are the typical diseases attribute to SDH dysfunction, which mainly initiates in childhood and influence ontogeny. Tumorigenesis associated with SDH deficiency is linked to familial paragangliomas (PGLs), pheochromocytomas (PHEOs), gastrointestinal stromal tumors (GISTs), pituitary adenomas (PAs), renal cell carcinomas (RCCs), and less commonly thyroid tumors and neuroblastomas (Gill 2018; Tsai and Lee 2019; Blay et al. 2021; Pitsava et al. 2021). Meanwhile, studies have demonstrated that both the subunits of SDH and its metabolite succinate can operate as the biomarker among several tumors, indicating the diagnostic value of SDH complex in clinical (Dalla Pozza et al. 2020). In this review, we present an overview of the linkage between SDH activity and cancer malignancy. Firstly, we discuss the pathological implications of abnormal SDH subunits and the diverse manifestations of SDH deficiency in various tumors. Subsequently, we put insight into the specific molecular mechanisms underlying SDH deficiency leading to carcinogenesis and the ideal medical management for cancer therapy in different perspectives.

Types of SDH defects in cancer

SDH works as an integral, any pathogenic germline variants in its four subunits (SDHA/B/C/D) or assembly factors disrupts the normal complex, thereby inducing its dysfunction and disease progression. SDHx mutations are heterozygous mutations inherited in an autosomal dominant fashion and tumorigenesis is initiated following bi-allelic loss as Knudson’s “two-hit” hypothesis (MacFarlane et al. 2020). Tumorigenesis linked to SDH deficiency usually arises when a germline mutation occurs in one allele, followed by a random mutation in the remaining wild-type allele over the individual's lifetime, resulting in the biallelic (germline plus somatic) mutation of SDH, which eventually evolves into a tumor by clonal expansion (Fullerton et al. 2020). In addition to genetic mutations, epigenetic modulation of SDHx genes and dysregulated enzymatic activity caused by post-translational modifications (PTMs) had been proven to associate with SDH deficiency and contribute to pathological states of tumor (Table 1).

Table 1.

The types of SDH deficiency in cancer and related tumor characteristics

Diseases	Alterations	Estimated Frequency of All SDH Deficient Cases	Location or Genome Site	Main Features or Main Effect	Reference
GIST	SDHA mutation	30%	Stomach	1. Epithelioid type 2. Young adults, children 3. Female predominance 4. Lymph node metastasis	(Blay et al. 2021; Kelly et al. 2021)
	SDHB/C/D mutation	20%
	SDHC promoter hypermethylation	50%
PPGL and PHEO	SDHB mutation	72%	intra-abdominal and extra-adrenal locations	1. Young adults 2. Predisposed to metastasis and recurrent	(Kantorovich et al. 2010; Favier et al. 2015; Jain et al. 2020; Nolting et al. 2022)
	SDHD mutation	9%
RCC	SDHB mutation	83%	Kidney	1. Median age ~ 40 years old 2. Slight male predominance 3. Cytoplasmic vacuoles or inclusions in histologic features	(Williamson et al. 2015; Wang and Rao 2018; Tsai and Lee 2019)
Thyroid cancers	SDHB/D mutation	6%	Thyroid	Unknown	(Ni et al. 2015)
	SDHC mutation	5%
Head and neck tumors	SDHC/D mutation	Unknown	Carotid body	High lifetime penetrance 2. Less aggressive behavior 3. Better survival compared to other subunit mutations	(Kantorovich et al. 2010; Favier et al. 2015; Jain et al. 2020)
Carney Triad	SDHC promoter hypermethylation	Rare	Mainly stomach and small intestine	1. Young Adults (median 18 years old) 2. Female predilection multifocality	(Pitsava et al. 2021)
Glioblastoma	SDHA phosphorylation	Unknown	Tyr215	Help to maintain cell survival	(Ogura et al. 2012)
Fibrosarcoma	SDHA acetylation	Unknown	Lys335	Deacetylation induces tumorigenesis and tumor growth	(Li et al. 2020a)
Clear cell renal cell carcinoma	SDHA succinylation	Unknown	Lys547	Desuccinylation induces Tumor metastasis and proliferation	(Ma et al. 2019)

SDHx mutation

The typical representative cancer with SDHA germline mutation is GIST. GIST is the most prevalent mesenchymal tumor in the gastrointestinal tract, with the majority of GISTs containing gain of function mutations in either KIT or PDGFRα (Kelly et al. 2021). The reported prevalence of SDH-deficient GIST varies from 10 to 15% among all GIST cases. SDH-deficient GIST mainly arise in the stomach and accompany of children, adolescent and young adults < 30 years of age, who characteristically have multinodular gastric masses and presents as female predilection (Blay et al. 2021). Additionally, SDH-deficient GISTs are associated with a high rate of distant metastasis (up to 82%), which tend to occur in liver. A strong genotype–phenotype correlation exists for SDHA germline variants in GIST, which occurs in about 30% of the SDH-deficient GIST cases. The remaining SDH-deficient GISTs can be divided into two parts, the one including the mutations of SDHB, SDHC or SDHD accounts for 20%, while the other approximately 50% is associated with CpG island hyper-methylation in the promoter region of the SDHC, also recognized as the SDHC epimutation (Gill 2018; Neppala et al. 2019).

PHEO and PPGL are the most common tumors arising in patients with germline SDHx mutations, nearly 40% of hereditary cases carried a pathogenic variant of SDHx (Nolting et al. 2022). SDHB mutations were reported most frequently (72%) in patients with primary PHEO and PPGL, secondly as SDHD (9%). PPGL patients carried a germline SDHB mutation have been demonstrated to present at a young age (< 20 years of age) and are predisposed to multiple and recurrent tumors with high metastatic spread (metastatic risk = 35–75%), often diagnosed as an intra-abdominal extra-adrenal cancer (Jain et al. 2020). In contrast, tumors with SDHD and SDHC germline variants that are almost always benign and commonly occur in the head and neck areas. Multiple patients with head and neck tumors or a hereditary PPGL are suggestive of SDHD-mutant (11q23), present a high lifetime penetrance (around 43% of patients manifest by 60 years old) and appear to be associated with less aggressive behavior and better survival compared to other subunit mutations (Kantorovich et al. 2010; Favier et al. 2015; Jain et al. 2020).

Besides PHEO and PPGL, SDHB mutations are also linked to malignant tumors as RCC and thyroid carcinoma. The SDH defective RCC almost due to SDH germline mutations, most commonly SDHB (83%), less commonly SDHC, and rarely SDHA or SDHD. Learn from a clinical statistic, SDHB-mutant RCC typically diagnose at mean age of 38 to 40 and with a slight male predominance (male to female ratio,1.75:1). Additionally, those SDHB-mutant RCC present special features morphologically, as they are composed of cuboidal cells with intracytoplasmic vacuoles or flocculent inclusions, and under a solid, nested or tubular growth pattern (Williamson et al. 2015; Wang and Rao 2018; Tsai and Lee 2019). Study about thyroid cancers suggested that approximately 6% cases have a germline mutation of SDHB or SDHD and a further 5% have somatic SDHC mutations (Ni et al. 2015).

In summary, mutations in all subunits of SDH are linked to cancer malignancy and are typically associated with highly aggressive tumors, a younger onset and a greater prevalence in females. Whether age-related factors or sex hormone are involved in the SDH-deficient neoplasia still need further study.

Epigenetic regulation of SDH

The core of epigenetics is the diverse repertoire of covalent modifications made to histone proteins and nucleic acids that cooperatively regulate chromatin structure and gene expression (Goldberg et al. 2007). Studies on epigenetic regulation mechanisms are limited to investigations into SDHC. SDHC-specific hypermethylation is considered as a molecular signature of Carney triad, defined by synchronous or metachronous GIST, PPGL and pulmonary chondroma. In a cohort of 37 Carney triad patients, comparative genomic hybridization studies showed that hypermethylation of the SDHC promoter and first exon contributed to deletions in 1q21–q23.3, which eventually induced the epigenetic inactivation of SDH (Pitsava et al. 2021). Additionally, Trombetti and colleagues have found that over-expression of GATA- 1 (a regulator of hematopoiesis) can mediate the high level of SDHC then promoted myeloid leukemia progression, mechanically due to the increased two isoforms produced by alternative splicing, named SDHC ∆3 and ∆5 ASV (lacking exon 3 or exon 5) (Trombetti et al. 2021).

Post-translational modifications (PTMs) of SDH

The PTMs of SDH complex in tumor cells are mainly comprised of phosphorylation, acetylation and succinylation. Ogura and coworkers demonstrated that mitochondrial tyrosine kinase c-Src phosphorylated SDHA at Tyr215 in glioblastoma cells, which was necessary to maintain mitochondrial respiratory system, and the disturbance of SDHA phosphorylation would induce reactive oxygen species (ROS) generation (Ogura et al. 2012). Additionally, study suggested that the dephosphorylation process mediated by PTEN-like mitochondrial phosphatase- 1 (PTPMT1) is connected to an adaption of glucose level and is involved in the deregulation of SDH activity (Nath et al. 2015). Concerning acetylation, SDHA has been identified as an acetylated protein with 13 acetylated lysine residues. The study by Li et al. unveiled that high expression of Myc promoted the SDHA acetylation at Lys335 by activating the SKP2-mediated degradation of sirtuin3 (SIRT3) deacetylase. The activity of hyperacetylated SDH is suppressed by the cumulative succinate, eventually facilitates H3 K4 me3 activation and tumor-specific gene expression in Fibrosarcoma (Li et al. 2020a, b). Another study identified that applying chidamide, a novel histone deacetylase inhibitor, can inhibit multiple myeloma cells proliferation by targeting SDHA (Sun et al. 2019). Succinylation is another PTM occur in lysine, we discuss it in detail in next part. The results of Du and colleagues suggested sirtuin5 (a critical desuccinylase) interacted with SDHA and desuccinylated it at lysine 547, which weakened its combination with the SDHAF2 and resulted in its lower activity. The damage of SDH enzymatic activity would promote the oncogenesis of clear cell renal cell carcinoma (Ma et al. 2019). It seems that different PTMs can exert different even opposite effect to SDH activity and tumor progression, more efforts are needed to explore the key factor that leads to difference.

Other mechanisms modulating SDH activity

In addition to the previously mentioned regulations of SDH, some direct effectors have been proved to mediate SDH activity. TRAP- 1, a molecular chaperone of the Hsp90 family in mitochondria, inhibited SDH catalytic activities through binding SDHB, which resulted in succinate accumulation and pseudohypoxia (Sciacovelli et al. 2013). Furthermore, survivin, an inhibitor of apoptosis has been identified to increase the stability of SDHB and SDHC subunits, thereby leading to the promotion of cellular respiration in tumor cells and supporting their motility and metastasis (Seo et al. 2016).

SDH deficiency induces tumor malignancy in a succinate-dependent manner

In many malignant tumors, the obvious consequence of SDH deficiency is the accumulation of the oncometabolite succinate. Excessive succinate leaks into the cytoplasm and is secreted into extracellular space. ‘Pseudohypoxia’ and ‘DNA hypermethylation’ are two primary characteristics in cancer environment attributed to the cumulative cytosolic succinate. The cumulative succinate competitively inhibits prolyl hydroxylases (PHD), resulting in decreased hydroxylation of hypoxia inducible factor- 1 (HIF- 1α) but increases combination of HIF-β in the nucleus (Selak et al. 2005; Kluckova and Tennant 2018). Therefore, HIF- 1α escapes from proteasome destiny, and functions with HIF-β counterpart as a complex to activate hypoxia respond pathways and induces angiogenesis and cell proliferation. In addition, the cumulative succinate works as the competitively antagonists of α-KG-dependent dioxygenases, including JMJD family and the TET family of 5 mC hydroxylases, which results in a genome-wide hypermethylation of promoter regions (CpG islands) and transcriptional gene silencing (Letouze et al. 2013). Regarding extracellular succinate, increasing evidence indicates that it accelerates tumor angiogenesis and enhances inflammation by activating succinate receptor- 1 (SUCNR- 1, also known as GRP91) (Gilissen et al. 2016). Except the pseudo-hypoxic signaling and epigenetic modifications, succinate has been reported to influence tumor progression via activating oncogenic signaling pathways, regulating protein function by succinylation, a novel PTM, as well as reconstituting the tumor microenvironment.

Succinate activates oncogenic signaling pathways

Over the past decades, numerous data have suggested that various oncogenic signaling cascades were stimulated by succinate to facilitate tumor progression. Excessive succinate can enhance cancer metastasis and invasion by interacting SUCNR1 or driving epithelial-mesenchymal transition (EMT). A study reported that cancer-derived succinate can drive this procedure and tumor-associated macrophages polarization via activating SUCNR1 triggered phosphoinositide 3-kinase (PI3 K)-HIF- 1α signaling axis in an autocrine and paracrine manners (Wu et al. 2020). Additionally, as a consequence of SDHB deficiency in colorectal cancer, transforming growth factor-β (TGF-β) signaling pathway was activated by up-regulating a tight-junction transcriptional repression complex SNAIL1-SMAD3/SMAD4, which contribute to lymphatic and distant metastasis and invasion (Wang et al. 2016). Of note, several studies reported that mitochondrial fission promoted certain cancer metastasis and invasion, including breast, hepatocellular and thyroid cancer (Lu et al. 2018; Kuo et al. 2022). Meanwhile, a research reveal that succinate can mediate Drp- 1 fission proteins activity through SUCNR1 in human mesenchymal stromal cells (Ko et al. 2017). Although there is no direct evidence of this linkage in cancer cells, this intriguingly phenomenon deserve further study.

Additionally, succinate contributed to abnormal proliferation in many cancers. It has been reported that impaired SDHA/B activity was responsible for the proliferation of hepatocellular carcinoma (HCC) (Yuan et al. 2023). Precisely, succinate stabilized and activated YAP/TAZ through promoting the deneddylation of Cullin1 and destroying its function in proteasome degradation. Furthermore, Li et al. reported that SDHC deficiency can mechanistically drive HCC growth via activating ROS/NF-κB signaling (Li et al. 2019). Continuous stimulation with succinate is found to result in an increase in the growth of uterine endometrial cancer cells by regulation of SGK- 1/KCNQ1 (Gu et al. 2020). In line with solid tumor, SDH deficient lead to the enhancement in clone formation abilities of acute myeloid leukemia. Meanwhile, high succinate level triggered ubiquitin-conjugating enzyme UBC12 phosphorylation (p-UBC12)/cullin pathways, which blocked function in cullins neddylation and thus comprehensively stabilize tumor-promoting proteins as the defense for AML cells against anticancer agents (Chen et al. 2024). However, the cognition of succinate in promoting tumor proliferation remains controversial. Both animal model of colorectal cancer and lung cancer demonstrated that inject extracellular succinate did not influence cancer cell viability or proliferation (Wu et al. 2020, Jiang et al. 2023). Future research needs to consider the different concentration of succinate, the different tumor types with different stages could lead to the diverse results. In summary, excessive succinate under SDH deficient circumstances have been confirmed as an important factor in regulation tumor migration, invasion and proliferation through activating signaling pathways.

Succinate induces protein succinylation

Succinylation is a naturally occurring PTM event that modulate the structural conformation and function of proteins by transferring the succinyl group (-CO-CH2-CH2-CO2H) to the lysine residues (Zhang et al. 2011) (Fig. 1). The succinyl groups mainly originate from succinyl-CoA, which is an immediate upstream metabolite of succinate and is confirmed to be in an equilibrium level with succinate (Li et al. 2015). While some scholars believe those succinyl moiety of succinylation, occurring in the cytoplasm and nucleus, can be derived from succinate (Tretter et al. 2016). There are mainly two perspectives for explaining the regulation of succinylation in shifting protein structure and properties. Firstly, it is a negatively charged lysine acylation that can neutralize the charge status from the positive (+ 1) to a net negative (− 1). Additionally, succinylation shift a relatively large mass (~ 100 Da) to a lysine residue than other typical covalent modification groups like acetylation (~ 40 Da), thus may impose an important impact of the target proteins (Sreedhar et al. 2020; Shen et al. 2023). The process of succinylation can be either enzymatically mediated by succinyltransferase (KAT2 A, CPT1 A and OXCT1) (Wang et al. 2017, Li et al. 2020b, Ma et al. 2024) and desuccinylase (SIRT5 and HDAC1/2/3) (Du et al. 2011; Li et al. 2023), or it can occur non-enzymatically in a pH and succinyl-CoA dose-dependent manner (Dai et al. 2022). Lysine succinylation reflects metabolic reprogramming in cancers and is involved in various cellular activities, including mitochondrial metabolism and genome integrity (Liu et al. 2023). It is noteworthy that succinylation plays diverse roles at different sites of action in different tumors, and there has no determined conclusion on its function yet.

Fig. 1.

Succinate induces substrate succinylation to facilitate tumor progression. The mechanisms of succinylation can be divided into nonenzymatic modulation and enzymatic modulation. Succinylation drives cancer traits via enhancing the stability and/or activity of protein or by altering their structure

Several studies indicate that the occurrence and progression of tumor are associated with lysine succinylation. As the consequence of alterative structure, Tong et al. presented data describing the succinylation at kidney-type glutaminase (GLS) K311, which resulted in GLS conformational changes, enhanced oligomerization, and increased its activity to maintain redox homeostasis, finally facilitating the PDAC proliferation by avoiding apoptosis caused by oxidative stress (Tong et al. 2021). Additionally, Chen et al. revealed the succinylation at multiple lysine residues stimulated the formulation of active acyl-CoA oxidase 1 (ACOX1) homodimer, which leads to oxidative DNA damage response and poor survival in HCC patients (Chen et al. 2018). Apart from the protein structure, studies have shown that profound functional changes happened after lysine succinylation, that mainly related to enhanced protein stability and activity. A series of studies has unveiled the competition between succinylation and ubiquitination at specific residues. For instance, studies of glioblastoma presented that succinylation of glycolytic enzyme phosphoglycerate kinase 1 (PGK1) at the K191 and K192 sites suppressed its proteasomal degradation, further contributes to tumor growth and immune escape by enhanced aerobic glycolysis and increased lactate amount (Yang et al. 2024). In gastric cancer, it is discovered K47 succinylation of S100 A10 regulated by CPT1 A was stabilized through suppressing the ubiquitylation and subsequent proteasomal degradation, facilitating the invasion and metastasis of tumor cells (Wang et al. 2019). In another GC study, the lactate dehydrogenase A (LDHA) was found highly succinylated at K222, thereby reduced its binding with sequestosome 1 (SQSTM1) and decreased its lysosomal degradation. The overexpressed K222-succinylated LDHA was proved to associate with the proliferation, invasion and migration of GC, also the poor prognosis in GC patients (Li et al. 2020b). Meanwhile, studies of prostate cancer demonstrated the K118 succinylation of LDHA is related to an increase of LDH activity, cell migration and proliferation (Kwon et al. 2023). In addition to facilitate tumor development, succinylation was identified contributing to onco-therapeutic resistance by maintenance of genome integrity of transformed cells. Human flap endonuclease 1 (FEN1) succinylation at K200 is necessary for the DNA damage repair process in HeLa cells, while defects of FEN1 succinylation led to the cumulative DNA damage and hypersensitive to fork-stalling agents (Shi et al. 2020).

Apart from functioned as the cancer driving factor mentioned above, several studies support opposite opinions of succinylation. For instance, hyposuccinylation is confirmed in primary esophageal squamous cell carcinoma (ESCC) specimens, further discovery found that ESCC malignant behaviors are suppressed when succinylation levels are restored (Guo et al. 2021). Another controversial point is about drug resistance. Studies demonstrate that hypersuccinylation of chromatin components are common upon SDH loss, leading to DNA repair defect, exhibited increased levels of the DNA damage marker gamma H2 A.X. This inherent DNA repair deficiency makes hypersuccinylated tumor cells more susceptible to genotoxic drugs like gemcitabine and etoposide (Smestad et al. 2018).

As a PTM associated closely to the initiation and progression of many tumors, succinylation provide a new direction for the development of oncology treatments. Besides applying inhibitors of oncoprotein that over-activated or over-expressed by succinylation, such as the GLS inhibitor CB- 839, modulating the regulatory enzyme of succinylation has been studied as a promising approach to impede tumor progression (Yang et al. 2021). For instance, Tong et al. suggested that the succinyltransferase activity-defective KAT2 A can interfere the proliferation and invasion of pancreatic carcinoma cells significantly (Tong et al. 2020).

Although accumulating evidence from lab research clarified the mechanism of succinylation in tumorigenesis, most of them still lack direct and definitive clinical sample for support. As research on lysine succinylation in cancer progresses, its dual function, onco-therapeutic strategies and clinical significance need deep discovery.

Succinate regulates immune system

Research over the past several decades has shown that the abnormal immune system presents a compromised defense against cancer in the host. Immune cells are capable to sense the excessive succinate in the tumor microenvironment (TME) and turning on specific immune functions in response (Xia et al. 2021). The reduced activity of SDH in lung cancer resulted succinate accumulation and secretion, further stimulate the formation of polarized M2-like tumor-associated macrophage (TAM) and facilitated the migration of TAM via SUCNR1, which suppressed the anti-tumor immune response (Wu et al. 2020). Alternatively, a study suggested that M2-like TAMs can downregulate SDHD expression and induce excessive succinate in breast cancer through STAT and TGF-β pathways. This process further promotes angiogenesis and immune evasion by stabilizing HIF- 1α and enhancing its transcriptional activity in cancer cells (Gomez et al. 2020). The above samples give us a hint that there might be more mutual regulation between immune cells and SDH. Additionally, research of SDH deficient renal cell carcinoma revealed a cold TME, presented as a low infiltration of T cell, leukocyte and myeloid-derived immune cells, as well as the prominently upregulation of PD-L1 and TGFB1 in comparison to other RCCs (Neves et al. 2022). In an analysis of colon adenocarcinoma, data showed that the extent of immune infiltration by T helper cells and Th2 cells were closely and positively related to the expression of SDH, while the specific molecular mechanism needs further research (Nan et al. 2023).

In general, researchers believed that under a SDH deficient circumstance, succinate can mediate the immunosuppression in cancer. In contrast, a recent study pointed that antigen presentation and T cell–mediated killing ability in melanoma tumor can be increased when SDH activity was lost. The work demonstrated that moderate reduction of SDH activity and cumulative succinate in tumors not only boosts T cell engagement by upregulating the expression of major histocompatibility complex I (MHC-I) but also facilitates the selective expansion of protective T cell clones. Therefore, applying the SDH-rewiring approach to covert “cold” tumor to “hot” can be a potential way to activate anti-tumor responses and improve immunotherapy (Mangalhara et al. 2023). This study suggested to take the SDH deficient degree into consideration and get a more precise conclusion. As a recent review pointed out that tumor-intrinsic and tumor-promoting inflammation, along with its messengers, can exhibit both immune-supporting and immune-suppressing properties, depending on the time and context. This suggests that the role of succinate in cancer immunity may be closely correlated to the specific tumor background and the stage of tumor progression, warranting further investigation (Denk and Greten 2022). Meanwhile, work as a signal molecule, succinate itself has been learned widely in different types of immune cells. For example, a report indicated that succinate could induce CD8 + T cell cytotoxicity in an autocrine signaling (Elia et al. 2022). Succinate also mediated the chemotaxis of human dendritic cells and enhanced its capacity of antigen-presenting (Rubic et al. 2008; Inamdar et al. 2023). Whether these regulations still exist in a cancer background can be an intriguing question and needs further study.

Succinate induces tumor angiogenesis

Current understanding on the mechanisms by which succinate induces angiogenesis are based on two ways. Firstly, excessive succinate impairs PHD enzyme activity and subsequently stabilize HIF- 1α expression. This induces upregulation of target genes containing hypoxia response elements (HRE), including angiogenic genes like vascular endothelial growth factor (VEGF) (Li et al. 2018). Mounting evidence has demonstrated that tumors with SDH deficiency like hereditary paraganglioma (SDHD mutation) and pheochromocytoma (SDHB mutation), presents increased expression of HIF- 1α and associated angiogenic genes. Secondly, succinate can be secreted into the extracellular environment, where it activates SUCNR1, triggering a variety of responses, one of which is angiogenesis (Atallah et al. 2022). Research of gastric cancer suggested that succinate promote VEGF expression depended on the activation of signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase (ERK)1/2 via SUCNR1, operating through a HIF- 1α independent mechanism (Mu et al. 2017). Additionally, succinate can drive polarization of tumor associated macrophage through SUCNR1, which promote cancer angiogenesis through the release of several pro-angiogenic factors, including VEGFA, from TAM (Hsu and Sabatini 2008).

SDH deficiency induces tumor malignancy in a succinate-independent manner

As the component of electron transport chain, SDH deficiency results directly in mitochondria dysfunction, which induces the generation of excessive ROS and altered metabolic program. In this section, we will discuss how SDH deficiency influences tumor progression in a ROS-related way and reprograms cell metabolism (Fig. 2, Table 2).

Fig. 2.

SDH deficiency regulates tumor progression through metabolic reprogramming and ROS-related manner. SDH-deficient cells exhibit significant alterations in iron and copper homeostasis, due to the increased expression of some iron and copper transporters. Since SDH deficiency breaks the TCA cycle, pyruvate is diverted into aspartate biosynthesis by pyruvate carboxylase to maintain cell growth in Sdhb-/- cells. While with Sdhd-/- background, cells appear to increase their synthesis of aspartate and citrate using reductive carboxylation

Table 2.

SDH deficiency related metabolic effects in cancer

Metabolic Effects	Downstream consequence	References
Altered TCA Cycle Activity	Altered ATP production, Consumption of extracellular pyruvate, Accumulation of succinate	(Cardaci et al. 2015; Tseng et al. 2018)
Loss of mitochondrial membrane potential	Altered ATP production and ROS generation	(Siebels and Drose 2013; Robb et al. 2018)
Altered Amino acid Metabolism	Decreased asparagine synthesis, Increased cystine, serine, glycine and asparagine level	(Ryan et al. 2021)
Altered iron and copper homestasis	Increased mitochondrial copper (I) and cytosolic iron (II) levels	(Goncalves et al. 2021)

ROS-Related manner

Along with mitochondrial complex I and complex III, complex II/SDH is one of the main suppliers of mitochondrial ROS. SDH significantly contributes to ROS generation in cells both directly and indirectly way (Mills et al. 2016, Esteban-Amo et al. 2024). With high concentration of succinate, the overproduction of ubiquinol (QH2) by SDH is responsible for the generation of ROS in complex I through reverse electron transport (RET) (Liu et al. 2002; Robb et al. 2018). Specifically, the electrons from QH2 are forced back to the FMN of the CI, leading to superoxide production due to the reaction between O2 and reduced FMN. At a low concentration of succinate, the electron transport chain is obstructed at the Q site of SDH or complex III, reactive oxygen species (ROS) can be generated directly at the FAD site of SDHA (Siebels and Drose 2013). Additionally, other studies indicate that mutations in SDHB, SDHC, or SDHD subunits affect superoxide generation at the ubiquinone binding site in SDHD through intermolecular interactions (Guzy et al. 2008).

Discrepant data about SDH-deficient cell have been published showing ROS increase and others not. One hypothesis is that such differences may actually reflect the heterogeneity of the phenotype linked to the distinct subtypes of SDH subunits, with a general consensus that SDHB-mutated cells do show increased oxidative stress (Bezawork-Geleta et al. 2017). It has been reported that SDHB silencing increased ROS production and nuclear HIF1α stabilization, further elevated the levels of the anti-apoptotic protein Bcl- 2 and exert reduced apoptosis in pheochromocytoma cells (Saito et al. 2016). In addition, SDHC related deficiency has been demonstrated to cause increased ROS levels in HCC cells and regulate tumor growth and metastasis through ROS/nuclear factor (NF)-κB signaling (Li et al. 2019). Consequently, numerous compounds that impact SDH activity and promote apoptosis mediated by abundant ROS have been examined for their potential as anticancer agents, including α-TOS, MitoVES, Troglitazone, 3 NP, LND, DT- 010 and so on (Zhang and Lang 2023).

Metabolic reprogramming

SDH deficiency directly results in the blockage of the TCA cycle and the loss of the mitochondrial membrane potential, induced an energetic switch from aerobic respiration to glycolytic metabolism in cells to acquire ATP (Tseng et al. 2018). SDH dysfunction also disturb other metabolic procedures. It has been reported that acute inhibition of SDH resulted in a marked reduction in asparagine synthesis due to the inability to activate reductive carboxylation. In contrast, chronic disruption of SDH may trigger adaptive compensatory metabolic alterations, leading to elevated intracellular levels of cystine, serine, glycine, and asparagine (Ryan et al. 2021). In addition, Cardaci and his collaborators demonstrated that the ablation of SDH activity urged cells to consume extracellular pyruvate to sustain Warburg-like bioenergetic features, since pyruvate was diverted into aspartate biosynthesis by pyruvate carboxylase to maintain cell growth (Cardaci et al. 2015). Except amino acid metabolism, Goncalves et al. made a significant discovery when they reported that SDH-deficient cells, especially SDHB knock down, presented increased mitochondrial copper (I) and cytosolic iron (II) levels (Goncalves et al. 2021). These cells exhibited distinct expression patterns of transporters involved in the uptake of iron and copper into the cells and mitochondria: elevated levels of DMT1 (Slc11a2) and reduced levels of SLC25 A37 would support the increased cytosolic accumulation of iron (II), while decreased CTR1 (Slc31a1) and increased Slc25a3 would help maintain high levels of mitochondrial copper (I). The prominent alterations in iron and copper homeostasis can cause important oxidative stress and promote metastasis. Another study also pointed a transferrin-dependent ferrous iron overload in SDHB-mutant PPGL, while using pharmacologic ascorbic acid notably reduced SDHB-low metastatic lesions and extended the survival (Liu et al. 2020). Depicting the global landscape of metabolic network in SDH deficient tumor cell may provide a novel therapeutic strategy by targeting the metabolic vulnerability (Fig. 2).

Diagnosis and medical management for SDH defective tumor patient

Accurate and rapid identification of SDH deficiency is crucial in clinical diagnosis and cancer therapy. Several biomarkers and correlational methods have been developed based on assessment of SDH protein expression, SDHx variants, and its metabolite succinate. Any inactivation of SDH components can impair the stabilization of SDH complex and releasing the SDHB subunit into the cytoplasm where it degrades rapidly. This loss of expression of SDHB in cancer tissue can be visualized reliably and cheaply in clinic by simple immunohistochemistry (IHC) (van Nederveen et al. 2009; Gill et al. 2010). In contrast, retained SDHD staining has been identified as a characteristic marker of pathogenic SDHD mutations and is considered a valuable complement to SDHB IHC in specific situations, such as assessing the pathogenicity of SDHD gene variants in PPGL (Menara et al. 2015). With the advancement of genetic testing, targeted next-generation sequencing (NGS) analysis and whole genome analysis support to reveal the genetic variations of SDH. Although it still requires formalin-fixed paraffin-embedded tissues for detection like IHC, it provides a more comprehensive prospective for understanding other alterations (Toledo and Dahia 2015). Moreover, an innocuous and precise method, magnetic resonance spectroscopy (1H-MRS), is employed in analyzing succinate in vivo in the field of PPGL, PC and GIST, since succinate shows a specific peak appears at 2.4 ppm in the spectrum generally (Lussey-Lepoutre et al. 2020). The high sensitivity (87%) and specificity (100%) of this technology was validated in a cohort of PGL patients with defective SDH. For in vitro succinate measurement, utilizing liquid chromatography with mass spectrometry (LC–MS/MS) is feasible. In some cases, the tissue-specific succinate to fumarate ratio (SFR) was particularly high and can be recognized as a powerful support to the results of IHC (Wallace et al. 2020). Therefore, succinate and SDH have been recognized as the biomarker of certain cancers.

Beyond diagnosis, the medical managements of SDH-defective cancers, mainly targeted therapy, chemotherapy and medicine combination strategy, are worth study in clinical and pre-clinical investigation. In this section, we primarily discuss several representative cancers associated with SDH deficiency (Table 3).

Table 3.

Clinical therapy studies for SDH defective cancer

Disease	Drug	Mechanism	Phase	NCT number	Reference
GIST	Regorafenib	Inhibit Tyrosine kinase activity	II	NCT02638766	(Ben-Ami et al. 2016)
	Pazopanib		II	NCT01524848	(Ganjoo et al. 2014)
	Linsitinib		II	NCT01560260	(von Mehren et al. 2020)
	Temozolomide	Induce DNA methylation and damage	II	NCT03556384	(Yebra et al. 2022)
	BGJ- 398	Inhibit FGF1 - 4 ligands	I	NCT02257541	(Flavahan et al. 2019)
PPGL	Sunitinib	Inhibit Tyrosine kinase activity	II	NCT00843037	(O'Kane et al. 2019)
	Cabozantinib	Inhibit Tyrosine kinase activity	II	NCT02302833	(Wang et al. 2022)
	Guadecitabine (SGI- 110)	Inhibit DNA methylation	II	NCT03165721	(Ligon et al. 2023)
	Temozolomide Temozolomide with olaparib	Induce DNA methylation and damage	II II	NCT05885386 NCT04394858	(Hadoux et al. 2014)
RCC	Vandetanib and metformin	Inhibit glucose uptake, glycolysis, and fatty acid synthesis	II	NCT02495103	(Carlo et al. 2019)
	Talazoparib and avelumab	Induce DNA damage and immune checkpoint blockade	II	NCT04068831	(Kotecha et al. 2024)

GIST

Tyrosine kinase inhibitors (TKIs), like imatinib and sunitinib, are the preferred and the first-line treatment in GIST with KIT/PDGFRA mutations and SDH competent (Mei et al. 2018). Unfortunately, due to the absence of gain-of-function tyrosine kinase mutations, the SDH defective GIST has primary resistance to general KIT/PDGFRA TKIs, only response to few TKIs. Available data presented that regorafenib, a third-line treatment for metastatic GIST with developed-resistance to imatinib and sunitinib, showed good activity to SDH-deficient GIST in a phase II trial, with partial response (n = 2) as the best response (33.3%) (Ben-Ami et al. 2016). Moreover, one GIST patient with SDH deficiency showed long-term disease control after 17 cycles of pazopanib treatment (Ganjoo et al. 2014). An oral IGF- 1R TKI linsitinib has been detected in phase II study, the clinical benefit rate (CBR) and progression-free survival (PFS) of 15 SDH-deficient GIST at 9 months were 40% and 52%, respectively, suggesting a potential benefit of linsitinib in this population (von Mehren et al. 2020). Based on the limited efficacy of extant TKIs and the highly vascularized phenotype in SDH deficient GIST, developing the newer generation of TKIs with a prominent antiangiogenic mechanism of action seems a promising choice.

Since SDH-deficient tumors tend to present elevated DNA double-strand breaks (DSBs) and DNA-damage-response foci, coupled with functional defects in DNA-repair pathways attributed to excessive succinate, the application of poly (ADP)-ribose polymerase inhibitors (PARPi) treatment has emerged as a synthetic lethal therapeutic strategy (Sulkowski et al. 2018). Additionally, a study showed that TKI-resistant SDH-deficient GIST models were sensitive to alkylating agent temozolomide, which induced on-target DNA damage (Yebra et al. 2022). Therefore, A phase II study of temozolomide is currently under way. For lab research, William et al. demonstrated an altered chromosomal topology in SDH-deficient GIST, which induced the topological reorganization of the FGF and KIT loci, then upregulating the fibroblast growth factor (FGF) ligands, especially FGF3 and FGF4. Further, usage of BGJ- 398 (an FGF1 - 4 inhibitor in clinical trial) and sunitinib in PDX model presented a durable tumor-suppressive efficacy, which may provide a novel interfering strategy (Flavahan et al. 2019).

PPGL

Considering that most of the PPGLs attributed to genetic mutations, genetic testing is applied generally to guide personalized medicine. The analysis of transcriptomic study divided PPGLs into two clusters: cluster 1 with pseudohypoxic phenotype and cluster 2 with activated kinase-linked signaling pathways, including MAPK and mTOR (Favier et al. 2015). SDH-deficient PPGL belongs to cluster 1, which is suggested applying an antiangiogenic approach. Candidate drugs, mostly TKIs, have been applied to block the activation of VEGF signaling. In a phase II SNIPP trial, SDHx-mutant PPGL patients presented partial response or stable disease to sunitinib (O'Kane et al. 2019). Moreover, a retrospective clinical trial indicated a partial response to sunitinib in 21% of all patients, with 62.5% of SDHB variant cases appeared stable disease or a partial response (Ayala-Ramirez et al. 2012). Another promising TKI cabozantinib has also been investigated in a clinical phase II trial in metastatic PPGL, for which SDHB mutant patient showed good response without severe hypertension or cardiovascular events (Jimenez et al. 2020). Preclinical study also corroborates this result (Wang et al. 2022).

Besides targeting angiogenesis, triggering epigenetic alteration can be an option. A prospective clinical phase II study investigating guadecitabine (SGI- 110), a dinucleotide containing the DNA methyltransferase inhibitor decitabine, presented a manageable toxicity but null-objective response in SDH-deficient PPGLs (Ligon et al. 2023). Considering chemotherapy, cyclophosphamide–vincristine–dacarbazine (CVD) chemotherapy is recommended as the standard for metastatic PPGL. It is reported that after CVD therapy, usage of temozolomide monotherapy as a maintenance was desirable for SDHB mutant PPGLs (Hadoux et al. 2014; Nolting et al. 2022).

There are many preclinical studies deserving attention, which may inspire new ideas for the development of medical treatment. Pang et al. found that SDHB mutant PPGLs developed a dependency on CI, which support the DNA repair pathway and led to chemoresistance (Pang et al. 2018). Thus, the combination of temozolomide with a PARP inhibitor could represent an innovative therapeutic strategy for SDHB-mutant pheochromocytomas and paragangliomas. A prospective randomized clinical trial is currently recruiting participants to compare temozolomide alone versus temozolomide plus the PARP inhibitor olaparib in metastatic pheochromocytomas (NCT04394858). Another intriguing perspective is suppressing OXPHOS in PPGL. Metformin, an inhibitor of OXPHOS by CI inhibition, was demonstrated to decrease the viability of PPGL cell lines (Li et al. 2017; Thakur et al. 2019). As therapeutic strategies continue to be refined and new medical treatments are introduced, the prognosis for SDH-deficient pheochromocytomas and paragangliomas appears to be optimistic.

RCC

Although SDH-deficient RCC is a certain category in kidney caners, therapeutic schemes aimed at these groups are rare. Some researchers believed SDH-deficient RCCs highly depended on glycolysis pathways, which provided an idea for targeting the metabolism (Chan et al. 2011). However, a correlated clinical trial investigating the combination of vandetanib with metformin for SDH-deficient RCCs and hereditary leiomyomatosis and RCC (HLRCCs) was terminated (Carlo et al. 2019). Recently, a phase II trial of talazoparib and avelumab in metastatic kidney cancer, including SDH deficient cases, neither presented clinical beneficial (Kotecha et al. 2024).

Concluding remarks and future perspectives

As the intersection of the TCA cycle and the ETC, SDH present irreplaceable function in ATP production and cell metabolism. Over the past years, increasing studies have demonstrated that SDH deficiency disturb the cell homeostasis, contribute to abnormal alterations of various vital biological progress, which were correlated tightly to tumor progression. In this review, we have categorized the types of SDH deficiency, described how dysfunction of SDH complex mediate tumor progression and summarize the diagnosis and medical treatments for certain SDH defective cancers.

Succinate is the main product of SDH deficiency, also regarded as acknowledged oncometabolite, takes a dominant part in SDH-defective cancer malignancy. Apart from its classic mediation to cancer by modulating pseudohypoxia and DNA hypermethylation, we summarize other noteworthy mechanisms in a succinate dependent or independent way. There is no doubt that excessive succinate facilitates cancer cell invasion and metastasis in different animal models. However, whether succinate can promote tumor proliferation still in dispute. Except considering the tumor stages and types in future study, optimizing the SDH-deficient/high succinate model seems a promising method. Since most studies choose to inject extracellular succinate into tumor bearing animal at intervals to mimic succinate accumulation (Wu et al. 2020, Jiang et al. 2023), which may fail to reflect a sustainable high succinate level in reality. Meanwhile, as a novel PTM identified in 2011, succinylation has gained lots of attention. With the continuous findings of catalytic enzymes and potential oncoprotein in succinylation procedure, there still lack direct and powerful evidence from clinic data to support lysine succinylation facilitate tumor malignancy. Implementing integrity genome profile of clinic tumor tissue with high succinylation and retrospective analysis may help to decipher this problem better.

The mechanisms by which different cancers respond to SDH dysfunction are distinct, and there remains a lack of clear explanations summarizing the regular patterns of these responses. It can be hypothesized that the stage of tumor progression plays a dominant role. At the beginning, excessive succinate leaks into the cytoplasm, which mainly induces pseudohypoxia and promotes angiogenesis. Following the secretion of succinate into the extracellular environment, additional mechanisms become possible, such as the activation of signaling cascades reliant on SUCNR1 and the disturbance of immune hemostasis, which can induce immune escape. Currently, magnetic resonance spectroscopy (1H-MRS) is regarded as a precise method for analyzing succinate levels in vivo in the field of PPGL, PC and GIST patients. Developing and utilizing the real-time metabolic profiling to examine changes in the metabolic landscape of SDH-deficient patients would be a valuable direction for future study.

Although immune therapy has involved in various cancer therapy guide, we found few immunotherapies were applied in SDH-deficient cancers, which may attribute to the unclear of current study. Several studies suggested a “cold” TME in SDH defective cancer, mainly presented as decreased T cell infiltration and increased M2-like macrophages. In contrast, once the SDH deficient and/or excessive succinate occurs in immune cells, the situation appears in adverse. The research conducted by Chen and his colleagues revealed that intracellular succinate could promote pro-inflammatory signature in SDHB-deficient T cells, but it suppressed their proliferation by improperly allocating carbon resource (Chen et al. 2022). Given the heterogeneous patterns and limited understanding of immune profiles in SDH-defective/succinate-cumulative cancer, it is essential to investigate the immune landscape utilizing multi-omics methodologies in future studies. Elucidating the cancer immune profiles will not only shed light on how succinate/SDH dysfunction disturb the immune phenotype but may also help identify the biomarkers that predict response to immunotherapy. Finally, exploring the mechanism underlying the differing roles of SDH in both tumor tissue and immune cells could provide valuable insights and will be pivotal in advancing this field.

Current medical treatments focused on SDH deficient patients are wide and broad, like modulating angiogenesis or DNA damage repair, which leaves a lot of blank spaces for developing targeted therapy. To achieve that, constructing the in vitro or in vivo models that represent the characteristics of SDH-deficient tumor patients, combined with the genome-wide CRISPR screening, could serve as an effective approach for target identification. In addition, given the complex and interconnected networks in SDH-deficient cancers, it is essential to identified targets with high specificity that occupy key positions within the regulatory network. For instance, cumulative succinate relies on its receptor SUCNR1 to activate various downstream oncogenic signaling pathways. Therefore, interfere SUCNR1 can be a promising way to block succinate function in upstream. In a model of intestinal ischemia/reperfusion(I/R), knockdown of Sucnr1 or blockage of SUCNR1 in vitro and in vivo reversed the effects of succinate in exacerbating I/R induced acute lung injury (Wang et al. 2023). Moreover, how to overcome the drug resistance and improve an on-target therapeutic scheme, like to recover SDH activity directly with low toxicity need to be considered in future research. Additionally, clinical trials investigating novel therapies should be consistently pursued. Taken as a whole, deepening our exploration of SDH-deficient cancers will provide new insights into cancer metabolism research and precision therapies.

Abbreviations

α-KG

α-Ketoglutarate

ACOX1

Active acyl-CoA oxidase 1

ATP

Adenosine triphosphate

Bcl- 2

B cell lymphoma 2

CI

Mitochondrial complex I

CII

Mitochondrial complex II

CIII

Mitochondrial complex III

CVD

Cyclophosphamide–vincristine–dacarbazine

DSBs

DNA double-strand breaks

ERK1/2

Extracellular regulated protein kinases

FADH2

Reduced flavin adenine dinucleotide

FGF

Fibroblast growth factor

GC

Gastric cancer

GISTs

Gastrointestinal stromal tumors

HCC

Hepatocellular carcinoma

HIF- 1α

Hypoxia inducible factor- 1

HPPS

Hereditary paraganglioma/pheochromocytoma syndrome

LDHA

Lactate dehydrogenase A

OXPHOS

Oxidative phosphorylation

PARPi

Poly (ADP)-ribose polymerase inhibitors

PGLs

Familial paragangliomas

PHD

Prolyl hydroxylases

PHEOs

Pheochromocytomas

PI3 K

Phosphoinositide 3-kinase

PPGL

Pheochromocytomas and paragangliomas

PTMs

Post-translational modifications

RCCs

Renal cell carcinomas

RET

Reverse electron transfer

ROS

Reactive oxygen species

SDH

Succinate dehydrogenase

SQSTM1

Sequestosome 1

TAM

Tumor-associated macrophage

TCA cycle

Tricarboxylic acid cycle

TKIs

Tyrosine kinase inhibitors

VEGF

Vascular endothelial growth factor

Author contributions

Jiaer Wang: Writing – original draft, review & editing, Visualization, Revision Tao Yuan: Funding acquisition. Bo Yang: Supervision, Funding acquisition. Qiaojun He: Supervision, Conceptualization. Hong Zhu:Supervision, Conceptualization. All authors reviewed the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (U21 A20420 to B. Y. and 82304517 to T. Y.) and Zhejiang Provincial Natural Science Foundation (LQ23H310009 to T. Y.).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

The author declares that all work described here has not been published before and that its publication has been approved by all co-authors.

Competing interests

The authors declare no competing interests.