**2. Epigenetic and SDH-deficiency: a connection with metastatic potential**

The metastatic cascade involves a succession of cell phenotypic alterations that spans from the acquisition of local invasive activity, the intravasation of cancer cells into blood and lymphatic vessels, their subsequent extravasation in the parenchyma of distant tissues and finally their growth forming macroscopic tumors. How a primary PPGL-tumor cell becomes metastatic and what are the molecular events involved in this process remain to be known. With the emergence of genomic profiling technologies, single gene/protein or multi-gene "signature"-based assays have been introduced to measure specific molecular pathway deregulations in cancer which could be used as clinically useful biomarkers. In PPGLs' patients, it is well established that the presence of inactivating germline mutations in the

**27**

PPGLs [12, 13].

the trigger for metastasis development.

epigenetic deregulations.

**SDH-deficient PPGLs**

*Metastatic Paragangliomas and Pheochromocytomas: An Epigenetic View*

*SDHB* gene is the most important molecular predictor of malignancy. More than 40% of patients with metastatic PPGLs (especially extra-adrenal tumors) carry germline *SDHB* mutations [6, 7]. Although mutations in other PPGL-predisposing genes, such as *FH*, *SDHC*, *SDHD*, *SDHA,* and *TMEM127* have been found in some patients with metastatic PPGLs, these mutations account for only <5% of cases. The mitochondrial 2-oxoglutarate/malate carrier *SLC25A11* gene has been proposed as a novel gene that can confer a predisposition to metastatic PPGLs but the number of patients harboring *SLC25A11*-germline mutations was rather limited to definitely assigned it a role in metastasis development [8]. Thus, *SDHB* gene germline mutation remains as the most reliable risk factor for metastasis. Nonetheless, metastases are developed in only 30% of the *SDHB*-mutation carriers and it is not known what are the mechanisms that either tip the balance towards the metastatic process or prevent it in these patients. Recent studies have pointed to several cancer-related genetic deregulations in metastatic PPGLs, especially prevalent in *SDHB*-related tumors. These include activation of telomerase and over-expression of genes involved in epithelial to mesenchymal transition [9–11]. However, these molecular alterations have been found in limited number of metastatic PPGLs and it is not known what their role is as triggers of the metastatic process. Aside *SDHB*-related metastatic PPGLs, the specific genetic traits involved in the development of the remaining 60% of metastatic PPGLs are not known. Somatic mutations in *ATRX* and *SETD2* genes, and fusions of *MAML3* gene have been identified in metastatic

One of the most relevant hints on the molecular mechanisms involved in metastasis came from the The Cancer Genome Atlas (TCGA) Program. These studies revealed that metastatic *SDHx*-mutated PPGLs do not accumulate more gene mutations at the somatic level than no-metastatic PPGLs [13]. It is now becoming increasingly evident that epigenetic changes play a key role in providing properties to the primary cancer cell that have a major contribution to the metastatic process. Relevant studies revealed that PPGLs, developed in patients with mutations in *SDHx* genes, harbor a DNA hypermethylation phenotype which is not present in PPGLs developed in patients with other genetic backgrounds [14]. Although these variations are commonly found in benign and metastatic *SDHB*mutated PPGLs, qualitative and/or quantitative deviations could cooperate to set

Epigenetics is defined as heritable changes in gene expression that do not involve a change in DNA sequence. Epigenetic changes occur in many types of cancer cells and include DNA methylation, histone modification, and small RNAs. Aberrant hypermethylation can lead to silencing of tumor-suppressor genes, histone modifications control the accessibility of the chromatin and transcriptional activities inside a cell, and microRNAs (miRNAs) can negatively control their target gene expression post-transcriptionally. Herein, we provide a perspective on the recent advances and challenges in our understanding of how epigenetic deregulations may underlie the progression of SDH-deficient PPGLs towards a metastatic disease and highlight promising therapeutic avenues that may be used to counteract those

**3. Succinate: an oncometabolite driving epigenetic deregulation in** 

The SDH complex links the tricarboxylic acid cycle (TCA) and the mitochondria respiratory chain by the coupling of succinate oxidation to fumarate to the reduction of ubiquinone to ubiquinol at the mitochondrial complex II (**Figure 1**). The

*DOI: http://dx.doi.org/10.5772/intechopen.96126*

*Metastatic Paragangliomas and Pheochromocytomas: An Epigenetic View DOI: http://dx.doi.org/10.5772/intechopen.96126*

*Pheochromocytoma, Paraganglioma and Neuroblastoma*

sues that can, eventually, become metastatic.

a significant clinical impact.

essential, and likely unique, role in the neuroendocrine tissues conforming human paraganglia such that its deregulation cause development of neoplasia in these tis-

One of the peculiarities of PPGLs is that they are generally slow growing, indolent tumors that are not life-threatening. However, 10–30% (according to different studies) of the PPGLs metastasize and once metastasis occurs, treatment options are rather limited and patients have poor prognosis, often with less than 50% surviving at 5 years [3]. Surgery can improve the prognosis but standard chemotherapeutic regimen with cyclophosphamide, vincristine, and dacarbazine, or radionuclide therapy with 131 Iodine-radiolabelled metaiodobenzylguanidine result in only partial responses. Thus, there is still a long road to reach therapeutic improvements. Further challenges for clinicians come from the fact that, in half of the cases, metastases are not present during the initial treatment of the patient but emerge over a period of undetermined time, which may even exceed 10 years after diagnosis of the primary tumor. For this reason, these patients receive longterm, post-treatment surveillance. However, the duration as well as the interval of the follow-up screening is poorly defined. Following these reasonings, the WHO 2017 Classification of Tumors of Endocrine Organs stated that PPGLs should be considered as tumors of undetermined biologic potential and should not be termed benign but should be classified as metastatic or not metastatic [4]. Given that all PPGLs are recognized as exhibiting malignant potential to some extent, the risk for malignant behavior must be determined to be able to pinpoint cases at risk of future metastases directly in the early post-operative period, a knowledge that would have

Despite overwhelming advances in understanding the molecular mechanisms of PPGL development made in the last decade, the factors governing the emergence of metastasis are still very poorly understood. Considerable efforts have been made in identifying histopathological features suggestive of metastatic behavior using pre-defined algorithms. The Pheochromocytoma of the Adrenal Gland Scaled Score (PASS) and the Grading System for Adrenal Pheochromocytoma and Paraganglioma (GAPP), rely on different histopathologic features or on a combination of histopathologic, immunohistochemical (Ki-67 index) and biochemical (catecholamine production) parameters, respectively, as tools to distinguish PPGLs with potential for aggressive behavior [5]. However, these algorithms lack accuracy and have a high degree of inter-observer variability thus complicating their clinical roll-out. Hence, the guiding of therapeutic decision-making by using predictive biomarkers in PPGL patients require in-depth knowledge of the biology of this

**2. Epigenetic and SDH-deficiency: a connection with metastatic** 

The metastatic cascade involves a succession of cell phenotypic alterations that spans from the acquisition of local invasive activity, the intravasation of cancer cells into blood and lymphatic vessels, their subsequent extravasation in the parenchyma of distant tissues and finally their growth forming macroscopic tumors. How a primary PPGL-tumor cell becomes metastatic and what are the molecular events involved in this process remain to be known. With the emergence of genomic profiling technologies, single gene/protein or multi-gene "signature"-based assays have been introduced to measure specific molecular pathway deregulations in cancer which could be used as clinically useful biomarkers. In PPGLs' patients, it is well established that the presence of inactivating germline mutations in the

**26**

neoplasia.

**potential**

*SDHB* gene is the most important molecular predictor of malignancy. More than 40% of patients with metastatic PPGLs (especially extra-adrenal tumors) carry germline *SDHB* mutations [6, 7]. Although mutations in other PPGL-predisposing genes, such as *FH*, *SDHC*, *SDHD*, *SDHA,* and *TMEM127* have been found in some patients with metastatic PPGLs, these mutations account for only <5% of cases. The mitochondrial 2-oxoglutarate/malate carrier *SLC25A11* gene has been proposed as a novel gene that can confer a predisposition to metastatic PPGLs but the number of patients harboring *SLC25A11*-germline mutations was rather limited to definitely assigned it a role in metastasis development [8]. Thus, *SDHB* gene germline mutation remains as the most reliable risk factor for metastasis. Nonetheless, metastases are developed in only 30% of the *SDHB*-mutation carriers and it is not known what are the mechanisms that either tip the balance towards the metastatic process or prevent it in these patients. Recent studies have pointed to several cancer-related genetic deregulations in metastatic PPGLs, especially prevalent in *SDHB*-related tumors. These include activation of telomerase and over-expression of genes involved in epithelial to mesenchymal transition [9–11]. However, these molecular alterations have been found in limited number of metastatic PPGLs and it is not known what their role is as triggers of the metastatic process. Aside *SDHB*-related metastatic PPGLs, the specific genetic traits involved in the development of the remaining 60% of metastatic PPGLs are not known. Somatic mutations in *ATRX* and *SETD2* genes, and fusions of *MAML3* gene have been identified in metastatic PPGLs [12, 13].

One of the most relevant hints on the molecular mechanisms involved in metastasis came from the The Cancer Genome Atlas (TCGA) Program. These studies revealed that metastatic *SDHx*-mutated PPGLs do not accumulate more gene mutations at the somatic level than no-metastatic PPGLs [13]. It is now becoming increasingly evident that epigenetic changes play a key role in providing properties to the primary cancer cell that have a major contribution to the metastatic process. Relevant studies revealed that PPGLs, developed in patients with mutations in *SDHx* genes, harbor a DNA hypermethylation phenotype which is not present in PPGLs developed in patients with other genetic backgrounds [14]. Although these variations are commonly found in benign and metastatic *SDHB*mutated PPGLs, qualitative and/or quantitative deviations could cooperate to set the trigger for metastasis development.

Epigenetics is defined as heritable changes in gene expression that do not involve a change in DNA sequence. Epigenetic changes occur in many types of cancer cells and include DNA methylation, histone modification, and small RNAs. Aberrant hypermethylation can lead to silencing of tumor-suppressor genes, histone modifications control the accessibility of the chromatin and transcriptional activities inside a cell, and microRNAs (miRNAs) can negatively control their target gene expression post-transcriptionally. Herein, we provide a perspective on the recent advances and challenges in our understanding of how epigenetic deregulations may underlie the progression of SDH-deficient PPGLs towards a metastatic disease and highlight promising therapeutic avenues that may be used to counteract those epigenetic deregulations.

### **3. Succinate: an oncometabolite driving epigenetic deregulation in SDH-deficient PPGLs**

The SDH complex links the tricarboxylic acid cycle (TCA) and the mitochondria respiratory chain by the coupling of succinate oxidation to fumarate to the reduction of ubiquinone to ubiquinol at the mitochondrial complex II (**Figure 1**). The

#### **Figure 1.**

*Schematic representation of the SDH-mediated connection between the Krebs cycle and the mitochondrial respiratory chain. The succinate dehydrogenase complex is part of both, the Krebs cycle at the mitochondria matrix and the mitochondria respiratory chain in the inner mitochondrial membrane. It is composed of four subunits (SDHA, SDHB, SDHC and SDHD) that couples the succinate oxidation to fumarate to the reduction of ubiquinone (coenzyme Q: CoQ ) to ubiquinol via FAD at the mitochondrial complex II. The mitochondria respiratory chain consists of four membrane-bound, multimeric protein complexes (complexes I, II, III, and IV) that catalyzes the oxidation of reducing equivalents, mainly nicotinamide adenine dinucleotide (NADH), using the terminal electron acceptor oxygen. This electron transfer is linked to the ATP synthase, which generates ATP.*

fumarate/succinate ratio and the redox state of the ubiquinone pool act as signal transducers known to modulate the regulatory programs that control cell fate. Loss of SDH activity leads to dramatic elevation of its natural substrate, succinate. The succinate generated in the mitochondrial matrix is exported to the cytosol where it can inhibit 2-oxoglutarate (2OG)-dependent dioxygenases such as ten-eleven translocation (TET) DNA cytosine-oxidizing enzymes and prolyl hydroxylases (PHD) [15].

PHD enzymes catalyze the prolyl-hydroxylation of the hypoxia-inducible factors HIF1α and HIF2α which transcriptionally regulates HIFα-responsive genes and conform the major hub involved in oxygen-sensing (**Figure 2**). These genes serve to adapt cells to oxygen deficiencies and their over-activation under pathologic conditions may also have pro-tumorigenic activity. HIFα proteins are degraded under physiological conditions by a mechanism requiring active PHD enzymes. PHDcatalyzed prolyl-hydroxylation of HIFα proteins is required by their recognition by VHL, subsequent ubiquitination and proteasomal degradation. Low oxygen levels and succinate repress PHD activities thus leading to the stabilization and functional activation of HIFα proteins. This oxygen-sensing pathway has long been considered a driver mechanism of metastasis in tumors with SDH-deficiencies [16]. However, although HIF1α protein and HIF1α-responsive genes are over-expressed in PPGLs carrying *SDHx* mutations, this signature is much weaker than that of PPGLs carrying *VHL*-loss-of-function mutations which rarely metastasize [17, 18]. Moreover, nuclear HIF2α does accumulate in all paragangliomas of the head and neck which very scarcely develop metastasis. These observations argue against nuclear HIFα

**29**

**Figure 2.**

*Metastatic Paragangliomas and Pheochromocytomas: An Epigenetic View*

proteins as the triggers of malignant transformation of *SDHx*-mutant PPGLs. Further research is required to demonstrate whether any, both or none of the HIFα

*Oxygen and oncometabolite dependent regulation of HIF*α*. Under physiological conditions, prolyl hidroxylases (PHD) hydroxylate two proline residues in HIF*α *subunits thus allowing their recognition by the von Hipple-Lindau protein (VHL). VHL is a component of a ubiquitination protein complex that ubiquitinate (Ub) prolyl-hydroxylated HIF*α *for degradation by the proteasome. PHDs activity rely on oxygen (O2) and oxoglutarate (2-OG). When oxygen concentration diminishes below physiological levels the activity of PHDs is inhibited leading to the dissociation of VHL from HIF*α *which results in HIF*α *stabilization that is transported to the nucleus, binds to HIF*β *and activates transcription of target genes by binding to hypoxia-responsive elements (HRE) in their promoter regions. Succinate, as well as fumarate, structurally mimics 2-OG and inhibits PHDs (product inhibition) when present at elevated concentrations, as observed in tumor cells* 

In addition to PHDs, succinate, which can accumulate to millimolar levels in *SDHx*-mutant PPGLs, is a potent inhibitor of TET enzymes and the Jumonji domain-containing histone demethylases [19]. TET enzymes hydroxylate DNAmethylcytosines into 5-hydroxymethylcytosine leading to DNA demethylation. Increased DNA methylation in or near promoter regions, and subsequent decreased gene expression, has been associated with oncogenesis in a number of tumor types including PPGLs carrying *SDHx*-mutations [14]. A role for TET enzymes in this

In addition to DNA epigenetic alterations, metastasis in PPGLs patients has also been shown to be associated with other epigenetic traits such as aberrant expression of long non-coding RNA (lncRNA) [21] and microRNAs (miRNAs) [22, 23] although these deregulations are not specific of SDH-deficient metastatic PPGLs.

Site-specific DNA hypermethylation in regions of DNA with a high density of cytosine-guanine (CpG) dinucleotides in promoters represent a common feature of the cancer-associated epigenetic landscape. These CpG hypermethylations are linked with repressive chromatin modifications and silencing of tumor suppressor

proteins are required for malignant transformation of PPGLs.

*carrying inactivating mutations-driven disfunction of SDH or fumarate hydratase.*

phenotype has been recently demonstrated [20].

**3.1 Succinate-induced DNA hypermethylation**

*DOI: http://dx.doi.org/10.5772/intechopen.96126*

*Metastatic Paragangliomas and Pheochromocytomas: An Epigenetic View DOI: http://dx.doi.org/10.5772/intechopen.96126*

#### **Figure 2.**

*Pheochromocytoma, Paraganglioma and Neuroblastoma*

fumarate/succinate ratio and the redox state of the ubiquinone pool act as signal transducers known to modulate the regulatory programs that control cell fate. Loss of SDH activity leads to dramatic elevation of its natural substrate, succinate. The succinate generated in the mitochondrial matrix is exported to the cytosol where it can inhibit 2-oxoglutarate (2OG)-dependent dioxygenases such as ten-eleven translocation (TET) DNA cytosine-oxidizing enzymes and prolyl hydroxylases

*Schematic representation of the SDH-mediated connection between the Krebs cycle and the mitochondrial respiratory chain. The succinate dehydrogenase complex is part of both, the Krebs cycle at the mitochondria matrix and the mitochondria respiratory chain in the inner mitochondrial membrane. It is composed of four subunits (SDHA, SDHB, SDHC and SDHD) that couples the succinate oxidation to fumarate to the reduction of ubiquinone (coenzyme Q: CoQ ) to ubiquinol via FAD at the mitochondrial complex II. The mitochondria respiratory chain consists of four membrane-bound, multimeric protein complexes (complexes I, II, III, and IV) that catalyzes the oxidation of reducing equivalents, mainly nicotinamide adenine dinucleotide (NADH), using the terminal electron acceptor oxygen. This electron transfer is linked to the ATP synthase, which* 

PHD enzymes catalyze the prolyl-hydroxylation of the hypoxia-inducible factors

HIF1α and HIF2α which transcriptionally regulates HIFα-responsive genes and conform the major hub involved in oxygen-sensing (**Figure 2**). These genes serve to adapt cells to oxygen deficiencies and their over-activation under pathologic conditions may also have pro-tumorigenic activity. HIFα proteins are degraded under physiological conditions by a mechanism requiring active PHD enzymes. PHDcatalyzed prolyl-hydroxylation of HIFα proteins is required by their recognition by VHL, subsequent ubiquitination and proteasomal degradation. Low oxygen levels and succinate repress PHD activities thus leading to the stabilization and functional activation of HIFα proteins. This oxygen-sensing pathway has long been considered a driver mechanism of metastasis in tumors with SDH-deficiencies [16]. However, although HIF1α protein and HIF1α-responsive genes are over-expressed in PPGLs carrying *SDHx* mutations, this signature is much weaker than that of PPGLs carrying *VHL*-loss-of-function mutations which rarely metastasize [17, 18]. Moreover, nuclear HIF2α does accumulate in all paragangliomas of the head and neck which very scarcely develop metastasis. These observations argue against nuclear HIFα

**28**

(PHD) [15].

*generates ATP.*

**Figure 1.**

*Oxygen and oncometabolite dependent regulation of HIF*α*. Under physiological conditions, prolyl hidroxylases (PHD) hydroxylate two proline residues in HIF*α *subunits thus allowing their recognition by the von Hipple-Lindau protein (VHL). VHL is a component of a ubiquitination protein complex that ubiquitinate (Ub) prolyl-hydroxylated HIF*α *for degradation by the proteasome. PHDs activity rely on oxygen (O2) and oxoglutarate (2-OG). When oxygen concentration diminishes below physiological levels the activity of PHDs is inhibited leading to the dissociation of VHL from HIF*α *which results in HIF*α *stabilization that is transported to the nucleus, binds to HIF*β *and activates transcription of target genes by binding to hypoxia-responsive elements (HRE) in their promoter regions. Succinate, as well as fumarate, structurally mimics 2-OG and inhibits PHDs (product inhibition) when present at elevated concentrations, as observed in tumor cells carrying inactivating mutations-driven disfunction of SDH or fumarate hydratase.*

proteins as the triggers of malignant transformation of *SDHx*-mutant PPGLs. Further research is required to demonstrate whether any, both or none of the HIFα proteins are required for malignant transformation of PPGLs.

In addition to PHDs, succinate, which can accumulate to millimolar levels in *SDHx*-mutant PPGLs, is a potent inhibitor of TET enzymes and the Jumonji domain-containing histone demethylases [19]. TET enzymes hydroxylate DNAmethylcytosines into 5-hydroxymethylcytosine leading to DNA demethylation. Increased DNA methylation in or near promoter regions, and subsequent decreased gene expression, has been associated with oncogenesis in a number of tumor types including PPGLs carrying *SDHx*-mutations [14]. A role for TET enzymes in this phenotype has been recently demonstrated [20].

In addition to DNA epigenetic alterations, metastasis in PPGLs patients has also been shown to be associated with other epigenetic traits such as aberrant expression of long non-coding RNA (lncRNA) [21] and microRNAs (miRNAs) [22, 23] although these deregulations are not specific of SDH-deficient metastatic PPGLs.

#### **3.1 Succinate-induced DNA hypermethylation**

Site-specific DNA hypermethylation in regions of DNA with a high density of cytosine-guanine (CpG) dinucleotides in promoters represent a common feature of the cancer-associated epigenetic landscape. These CpG hypermethylations are linked with repressive chromatin modifications and silencing of tumor suppressor

#### *Pheochromocytoma, Paraganglioma and Neuroblastoma*

genes. We discuss here the current understanding of the epigenetic basis of metastasis in *SDHB*-related PPGLs uncovered by our recent studies.

To identify epigenetic alterations relevant for metastasis, we recently performed a comprehensive analysis of DNA methylation in metastatic PPGLs with and without *SDHB* mutations. This analysis revealed that over 1000 genes harbored promoter hypermethylation in the metastatic tumors but not in the not metastatic ones thus suggesting that those gene alterations have a role in the pathogenesis of the metastatic disease linked to *SDHB* mutations [24]. About 15% of these alterations had been also identified in *sdhb−/−* mouse chromaffin cells and in 41% of *SDHx*-mutated PPGLs analyzed by Letouzé et al. [14]. Although these authors did not make distinctions whether the PPGLs were or not metastatic, they did find that hypermethylation was stronger in *SDHB*-PPGLs. Therefore, it is likely that gene

#### **Figure 3.**

*Outline of the epigenetic changes induced by abnormal succinate accumulation due to SDHx-mutations. Mutations of the SDHx genes in PPGLs cause blockage of SDH activity and subsequent abnormal succinate and succinyl-CoA accumulation. Increased levels of succinate induce inhibition of 2-oxoglutarate-dependent dioxygenases such as TET enzymes and the Jumonji domain-containing histone demethylases leading to activation or repression of gene transcription. TET enzymes hydroxylate DNA-methylcytosines into 5-hydroxymethylcytosine leading to DNA demethylation. Increased DNA methylation due to impaired TET enzyme functions in or near promoter regions induces decreased gene transcription (D) that, when affects tumor suppressor genes, such as PCDHGC3, may trigger different aspects of the metastatic programs. Gene expression can also be inhibited by succinate-induced inhibition of Jumonji domain-containing histone demethylases that remove the methyl group on lysine in histone tails. Histone methylation occurs by the transfer of methyl groups from the methyl donor S-adenosylmethionine (SAM) to amino acids of histone proteins. This protein modification can either increase or decrease transcription of genes, depending on which amino acids are methylated, and how many methyl groups are attached. Succinate inhibition of demethylation of trimethylated-H3K27 by the Polycomb complex (PRC2) induce gene silencing and chromatin condensation (D). (C) Succinate also represses homology-dependent DNA repair by inhibiting the H3K9 demethylase, leading to global elevation of trimethylated H3K9 chromatin marks at loci surrounding DNA breaks. This masks a local H3K9 trimethylation signal that is essential for the proper execution of homology-dependent DNA repair. (A, B) Apart from repression of gene expression, abnormal succinate accumulation may induce gene transcription. This occurs when DNA methylation affects CTCF insulators which prevents CTCF binding to CTCF binding sites, CTCF dimerization and the assembly of long-range chromatin looping. This provokes promiscuous enhancer-promoter interactions and the subsequent induction of the affected genes (B). (A) Increased succinyl-CoA levels induce succinylation of histones associated with enhanced in vitro transcription. The figure shows the enzymatic succinylation of histone via the KAT2A histone succinyltransferase which associates with* α*-ketoglutarate dehydrogenase (*α*-KGDH).*

**31**

*Metastatic Paragangliomas and Pheochromocytomas: An Epigenetic View*

initiation and others involved in metastasis development.

mutations in *SDHB* induce epigenetic programs that may be involved in tumors

Gene set enrichment analysis revealed that the hypermethylated promoters in metastatic *SDHB*-mutated PPGLs were associated with developmental genes that are preferential targets of the polycomb repressive complex 2, PRC2. PRC2 catalyzes the mono-methylation, di-methylation and tri-methylation of histone H3 at lysine 27 required for PRC2-mediated gene silencing and for maintaining cellular identity during differentiation and development [25]. Specifically, PRC2 occupies a special set of developmental genes in embryonic stem cells that must be repressed to maintain pluripotency and that are poised for activation during cell differentiation. In cancer, aberrant promoter hypermethylation, or PRC-mediated repression, can inhibit differentiation programs, such that cancer cells are arrested at a proliferative state [26] (see **Figure 3**). In agreement with these observations, increasingly, metabolites, such as succinate, are recognized as important modulators of the regulatory programs that control cell fate [27]. Thus, it is tempting to speculate that the succinate 'oncometabolite' plays an essential role in the epigenetic reprograming of chromaffin cells such that, when reaching high enough levels, induces the transit of mature differentiated cells towards a less differentiated state that allow them to proliferate and generate a tumor mass. This could provide an explanation for tumorigenesis in SDH-deficient tumors. However, it cannot explain why some SDH-deficient PPGLs acquire metastatic fitness, but others do not. The identification of an epigenetic signature specific for metastatic SDH-deficient PPGLs, but not present in SDH-PPGLs that do not develop metastasis, provides some clues. Our recent study revealed that, in addition to the epigenetic changes in developmentally regulated genes, high level hypermethylation of genes involved in homophilic cellto-cell adhesion was present in metastatic but not in non-metastatic PPGLs *SDHB*mutated PPGLs. Loss of cell–cell adhesion is a hallmark of metastatic cells required for the transformation of immobile cells into motile cells providing them the ability to invade local tissues leading to metastasis at distant organs. Among these hypermethylated genes, we identified *CNTN2*, *SDK1*, *TENM1*, *TENM4* encoding neuronal cell adhesion molecules involved in the establishment of connections in the nervous system. More strikingly, the cell–cell adhesion set of hypermethylated genes included a 1 Mb-long chromosomal region that hold clustered protocadherin genes [designated as *PCDHA*, *PCDHB* and *PCDHG* (collectively, *PCDHs*)] encompassing 50 different genes at the chromosomal locus 5q31.3 [24] (**Figure 4**). One of the *PCDH* genes, *PCDHGC3*, has been further analyzed and found to be of clinical

*DOI: http://dx.doi.org/10.5772/intechopen.96126*

relevance in metastatic *SDHB*-mutated PPGLs.

**metastatic** *SDHB***-mutated PPGLs**

**3.2 Long-range hypermethylation of clustered protocadherin genes in** 

*PCDH* genes, organized into three closely linked gene clusters (*PCDHA*, *PCDHB* and *PCDHG*), span nearly 1 million base pairs [28] (**Figure 4**). The *PCDHA* and *PCDHG* clusters are organized into variable and constant exons. The generation of full-length *PCDHA* and *PCDHG* messenger RNA requires RNA splicing of each variable exon to three constant exons. Each of the variable exon promoters are randomly activated in individual neurons to generate individual cell-specific patterns of *PCDH* gene expression. In contrast, *PCDHB* mRNA consists of only the variable exon. These genes are involved in the regulation of neural development and engage in homophilic/heterophilic trans-interactions as multimers acting as cell-surface molecular barcodes [29–33]. Their unique genomic organization makes them sensitive to long range epigenetic silencing (LRES). Several recent studies have revealed that epigenetic silencing of clustered *PCDHs* is

#### *Metastatic Paragangliomas and Pheochromocytomas: An Epigenetic View DOI: http://dx.doi.org/10.5772/intechopen.96126*

*Pheochromocytoma, Paraganglioma and Neuroblastoma*

tasis in *SDHB*-related PPGLs uncovered by our recent studies.

genes. We discuss here the current understanding of the epigenetic basis of metas-

a comprehensive analysis of DNA methylation in metastatic PPGLs with and without *SDHB* mutations. This analysis revealed that over 1000 genes harbored promoter hypermethylation in the metastatic tumors but not in the not metastatic ones thus suggesting that those gene alterations have a role in the pathogenesis of the metastatic disease linked to *SDHB* mutations [24]. About 15% of these alterations had been also identified in *sdhb−/−* mouse chromaffin cells and in 41% of *SDHx*-mutated PPGLs analyzed by Letouzé et al. [14]. Although these authors did not make distinctions whether the PPGLs were or not metastatic, they did find that hypermethylation was stronger in *SDHB*-PPGLs. Therefore, it is likely that gene

To identify epigenetic alterations relevant for metastasis, we recently performed

**30**

*associates with* α*-ketoglutarate dehydrogenase (*α*-KGDH).*

**Figure 3.**

*Outline of the epigenetic changes induced by abnormal succinate accumulation due to SDHx-mutations. Mutations of the SDHx genes in PPGLs cause blockage of SDH activity and subsequent abnormal succinate and succinyl-CoA accumulation. Increased levels of succinate induce inhibition of 2-oxoglutarate-dependent dioxygenases such as TET enzymes and the Jumonji domain-containing histone demethylases leading to activation or repression of gene transcription. TET enzymes hydroxylate DNA-methylcytosines into 5-hydroxymethylcytosine leading to DNA demethylation. Increased DNA methylation due to impaired TET enzyme functions in or near promoter regions induces decreased gene transcription (D) that, when affects tumor suppressor genes, such as PCDHGC3, may trigger different aspects of the metastatic programs. Gene expression can also be inhibited by succinate-induced inhibition of Jumonji domain-containing histone demethylases that remove the methyl group on lysine in histone tails. Histone methylation occurs by the transfer of methyl groups from the methyl donor S-adenosylmethionine (SAM) to amino acids of histone proteins. This protein modification can either increase or decrease transcription of genes, depending on which amino acids are methylated, and how many methyl groups are attached. Succinate inhibition of demethylation of trimethylated-H3K27 by the Polycomb complex (PRC2) induce gene silencing and chromatin condensation (D). (C) Succinate also represses homology-dependent DNA repair by inhibiting the H3K9 demethylase, leading to global elevation of trimethylated H3K9 chromatin marks at loci surrounding DNA breaks. This masks a local H3K9 trimethylation signal that is essential for the proper execution of homology-dependent DNA repair. (A, B) Apart from repression of gene expression, abnormal succinate accumulation may induce gene transcription. This occurs when DNA methylation affects CTCF insulators which prevents CTCF binding to CTCF binding sites, CTCF dimerization and the assembly of long-range chromatin looping. This provokes promiscuous enhancer-promoter interactions and the subsequent induction of the affected genes (B). (A) Increased succinyl-CoA levels induce succinylation of histones associated with enhanced in vitro transcription. The figure shows the enzymatic succinylation of histone via the KAT2A histone succinyltransferase which* 

mutations in *SDHB* induce epigenetic programs that may be involved in tumors initiation and others involved in metastasis development.

Gene set enrichment analysis revealed that the hypermethylated promoters in metastatic *SDHB*-mutated PPGLs were associated with developmental genes that are preferential targets of the polycomb repressive complex 2, PRC2. PRC2 catalyzes the mono-methylation, di-methylation and tri-methylation of histone H3 at lysine 27 required for PRC2-mediated gene silencing and for maintaining cellular identity during differentiation and development [25]. Specifically, PRC2 occupies a special set of developmental genes in embryonic stem cells that must be repressed to maintain pluripotency and that are poised for activation during cell differentiation. In cancer, aberrant promoter hypermethylation, or PRC-mediated repression, can inhibit differentiation programs, such that cancer cells are arrested at a proliferative state [26] (see **Figure 3**). In agreement with these observations, increasingly, metabolites, such as succinate, are recognized as important modulators of the regulatory programs that control cell fate [27]. Thus, it is tempting to speculate that the succinate 'oncometabolite' plays an essential role in the epigenetic reprograming of chromaffin cells such that, when reaching high enough levels, induces the transit of mature differentiated cells towards a less differentiated state that allow them to proliferate and generate a tumor mass. This could provide an explanation for tumorigenesis in SDH-deficient tumors. However, it cannot explain why some SDH-deficient PPGLs acquire metastatic fitness, but others do not. The identification of an epigenetic signature specific for metastatic SDH-deficient PPGLs, but not present in SDH-PPGLs that do not develop metastasis, provides some clues. Our recent study revealed that, in addition to the epigenetic changes in developmentally regulated genes, high level hypermethylation of genes involved in homophilic cellto-cell adhesion was present in metastatic but not in non-metastatic PPGLs *SDHB*mutated PPGLs. Loss of cell–cell adhesion is a hallmark of metastatic cells required for the transformation of immobile cells into motile cells providing them the ability to invade local tissues leading to metastasis at distant organs. Among these hypermethylated genes, we identified *CNTN2*, *SDK1*, *TENM1*, *TENM4* encoding neuronal cell adhesion molecules involved in the establishment of connections in the nervous system. More strikingly, the cell–cell adhesion set of hypermethylated genes included a 1 Mb-long chromosomal region that hold clustered protocadherin genes [designated as *PCDHA*, *PCDHB* and *PCDHG* (collectively, *PCDHs*)] encompassing 50 different genes at the chromosomal locus 5q31.3 [24] (**Figure 4**). One of the *PCDH* genes, *PCDHGC3*, has been further analyzed and found to be of clinical relevance in metastatic *SDHB*-mutated PPGLs.
