**3.2 Long-range hypermethylation of clustered protocadherin genes in metastatic** *SDHB***-mutated PPGLs**

*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

#### **Figure 4.**

*High level long-range hypermethylation of the clustered PCDH genes in metastatic SDHx-mutated PPGLs. Schematic representation of the genomic organization of the clustered PCDHA, PCDHB and PCDHG genes. For PCDHA and PCDHG genes, only the first exons (blue and orange rectangles, respectively) are represented. For PCDHB genes, rectangles represent the whole gene. Inverted gray triangles point to CpG hypermethylation sites detected in the SDHx-mutated PPGLs included in the TCGA database. Graphics represent DNA methylation levels of the indicated CpG islands (CpGI) according to their genotype. Data from patients without or with metastasis are represented in blue and red, respectively. SDHx-WT: PPGLs lacking mutations in any of the SDHx genes (include PPGLs with and without mutations in other PPGLsusceptibility genes); SDHB/D-Mut: Metastatic PPGLs from patients with germline mutations in* SDHB *or SDHD genes; SDHB*-*Mut: PPGLs from patients with germline mutations in SDHB genes; SDHB-WT: Metastatic PPGLs lacking mutations in SDHB genes. \*\** P *< 0.01; \*\*\** P *< 0.001; \*\*\*\** P *< 0.0001.*

present in various human malignant tumors, such as Wilms tumor, neuroblastoma, breast, prostate, colon cancer, gastric and biliary tract cancers, and astrocytoma suggesting that this process plays roles in regulating cancer development and/or progression [34–37]. By using one of the largest cohorts of epigenetically studied *SDHB*-mutated PPGLs, we have recently found that the epigenetic silencing of one of the clustered *PCDH* genes, *PCDHGC3*, is putatively involved in the metastatic behavior of these tumors [24]. Methylation of *PCDHGC3* promoter were found to be null in normal paraganglia, null or low in most *SDHB*-mutated PPGLs that do not metastasize, high in *SDHB*-mutated metastatic PPGLs, and much higher in the metastatic tissues derived from these tumors. Similar findings have been reported in colorectal cancer, showing that *PCDHGC3* is methylated and silenced during the adenoma-to-carcinoma transition [37]. These data suggest that this epigenetic trait is progressively amplified during the transformation of the tumor cells from benign state to the invasive and metastatic states, as suggested for other oncogenes and tumor suppressor genes [38].

We also found that, not only *PCDHGC3*, but the other clustered *PCDH* genes are highly methylated in metastatic SDHx-mutated PPGLs. Indeed, the *in-silico* analysis of DNA methylation data reported by TCGA confirmed the hypermethylation of the clustered *PCDH* genes (**Figure 4**) in *SDHx*-mutated PPGLs and allowed further analysis of this phenomena. As in our report, methylation of different CpG islands were detected in the three clustered *PCDHs*, being more highly enriched in the *PCDHG* cluster. **Figure 4** shows analysis of three different CpG regions in that cluster revealing that, similarly to our findings in *PCDHGC3* promoter region, methylation levels were higher in *SDHx*-mutated PPGLs than in PPGLs that did not harbor *SDHx* mutations. More importantly, among the *SDHB*-mutated PPGLs, those having a metastatic behavior had a significantly higher levels of methylation than tumors that had not developed metastasis at the last follow-up date. Analysis of the RNAseq data confirmed the epigenetic silencing of, not only *PCDHGC3* [24], but also *PCDHGC4* gene (**Figure 5**). The *PCDHGC4* mRNA levels were found significantly decreased in *SDHx*-mutated PPGLs as compared with tumors with other

**33**

*Metastatic Paragangliomas and Pheochromocytomas: An Epigenetic View*

genotypes. More importantly, downregulation was significantly more dramatic in metastatic than in benign *SDHx*-mutated tumors. Thus, the corrupted epigenetic changes in this chromosomal region seems to amplify the positive selection of the most metastatic cells and the evolutionary capacity of cancer to spread out of the tissue of origin. Interestingly, only the PCDHGC4 isoform, among the clustered PCDH proteins, have been shown to be strictly required for postnatal viability and

*SDHB genes; SDHB-WT: Metastatic PPGLs lacking mutations in SDHB genes. \*\*\*\** P *< 0.0001.*

PCDHGC4 *gene silencing in SDHB-mutated PPGLs that developed metastasis. PCDHGC4 mRNA levels in metastatic (red) and not-metastatic (blue) PPGLs included in the TCGA database are represented according to their genotype. SDHx-WT: PPGLs lacking mutations in any of the SDHx genes (include PPGLs with and without mutations in other PPGL-susceptibility genes); SDHB/D-Mut: PPGLs from patients with germline mutations in SDHB or SDHD genes; SDHB-Mut: Metastatic PPGLs from patients with germline mutations in* 

Consistent with previous findings in colon cancer cell lines [37], we found in that decreased *PCDHGC3* gene expression in two different cancer cell lines resulted in significant increases in cell proliferation, cell migration, and collective cell invasion. Silencing of *PCDHGC3* gene also resulted in increased tumor growth in studies of xenograft tumor models *in vivo*. Consistent with this, the current published data showed that PCDHs regulate pathways for cell proliferation and death. In tumor tissues derived from PPGLs, loss of *PCDH* expression is an indicator of poor prognosis, as revealed by our data and the *in silico* analysis of published data. Importantly, in *SDHB*-mutated metastatic PPGLs with high levels of *PCDHGC3* methylation, diagnosis of primary tumor and metastatic disease was synchronous in most cases, but some patients had a metastasis-free time ranging from 1 to 19 years. Thus, it is possible that epigenetic alterations of *PCDHGC3* during tumor initiation do not automatically lead to the manifestation of full metastatic potential. Rather, metastatic potential likely evolves through quantitative amplification, ultimately providing the cell with metastatic fitness. Thus, *PCDHGC3* acts as a tumor suppressor gene in PPGLs, could be an efficient biomarker of malignancy,

Targeting any of the protocadherin genes is challenging given that they are highly expressed in nervous system where exert relevant functions for the establishment and maintenance of specific neuronal connections. It is imperative, thus, to unravel the signaling pathways downstream *PCDHGC3* to identify potential therapeutic targets activated in the absence of *PCDHGC3* expression. Current published data have shown that PCDHs are tightly linked to several major signaling pathways, including the Wnt/β-catenin and receptor tyrosine kinase signaling

and could represent a novel target for personalized medicine.

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

survival of many neuronal subsets [39].

**Figure 5.**

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

#### **Figure 5.**

*Pheochromocytoma, Paraganglioma and Neuroblastoma*

present in various human malignant tumors, such as Wilms tumor, neuroblastoma, breast, prostate, colon cancer, gastric and biliary tract cancers, and astrocytoma suggesting that this process plays roles in regulating cancer development and/or progression [34–37]. By using one of the largest cohorts of epigenetically studied *SDHB*-mutated PPGLs, we have recently found that the epigenetic silencing of one of the clustered *PCDH* genes, *PCDHGC3*, is putatively involved in the metastatic behavior of these tumors [24]. Methylation of *PCDHGC3* promoter were found to be null in normal paraganglia, null or low in most *SDHB*-mutated PPGLs that do not metastasize, high in *SDHB*-mutated metastatic PPGLs, and much higher in the metastatic tissues derived from these tumors. Similar findings have been reported in colorectal cancer, showing that *PCDHGC3* is methylated and silenced during the adenoma-to-carcinoma transition [37]. These data suggest that this epigenetic trait is progressively amplified during the transformation of the tumor cells from benign state to the invasive and metastatic states, as suggested for other oncogenes and

*High level long-range hypermethylation of the clustered PCDH genes in metastatic SDHx-mutated PPGLs. Schematic representation of the genomic organization of the clustered PCDHA, PCDHB and PCDHG genes. For PCDHA and PCDHG genes, only the first exons (blue and orange rectangles, respectively) are represented. For PCDHB genes, rectangles represent the whole gene. Inverted gray triangles point to CpG hypermethylation sites detected in the SDHx-mutated PPGLs included in the TCGA database. Graphics represent DNA methylation levels of the indicated CpG islands (CpGI) according to their genotype. Data from patients without or with metastasis are represented in blue and red, respectively. SDHx-WT: PPGLs lacking mutations in any of the SDHx genes (include PPGLs with and without mutations in other PPGLsusceptibility genes); SDHB/D-Mut: Metastatic PPGLs from patients with germline mutations in* SDHB *or SDHD genes; SDHB*-*Mut: PPGLs from patients with germline mutations in SDHB genes; SDHB-WT: Metastatic PPGLs lacking mutations in SDHB genes. \*\** P *< 0.01; \*\*\** P *< 0.001; \*\*\*\** P *< 0.0001.*

We also found that, not only *PCDHGC3*, but the other clustered *PCDH* genes are highly methylated in metastatic SDHx-mutated PPGLs. Indeed, the *in-silico* analysis of DNA methylation data reported by TCGA confirmed the hypermethylation of the clustered *PCDH* genes (**Figure 4**) in *SDHx*-mutated PPGLs and allowed further analysis of this phenomena. As in our report, methylation of different CpG islands were detected in the three clustered *PCDHs*, being more highly enriched in the *PCDHG* cluster. **Figure 4** shows analysis of three different CpG regions in that cluster revealing that, similarly to our findings in *PCDHGC3* promoter region, methylation levels were higher in *SDHx*-mutated PPGLs than in PPGLs that did not harbor *SDHx* mutations. More importantly, among the *SDHB*-mutated PPGLs, those having a metastatic behavior had a significantly higher levels of methylation than tumors that had not developed metastasis at the last follow-up date. Analysis of the RNAseq data confirmed the epigenetic silencing of, not only *PCDHGC3* [24], but also *PCDHGC4* gene (**Figure 5**). The *PCDHGC4* mRNA levels were found significantly decreased in *SDHx*-mutated PPGLs as compared with tumors with other

**32**

tumor suppressor genes [38].

**Figure 4.**

PCDHGC4 *gene silencing in SDHB-mutated PPGLs that developed metastasis. PCDHGC4 mRNA levels in metastatic (red) and not-metastatic (blue) PPGLs included in the TCGA database are represented according to their genotype. SDHx-WT: PPGLs lacking mutations in any of the SDHx genes (include PPGLs with and without mutations in other PPGL-susceptibility genes); SDHB/D-Mut: PPGLs from patients with germline mutations in SDHB or SDHD genes; SDHB-Mut: Metastatic PPGLs from patients with germline mutations in SDHB genes; SDHB-WT: Metastatic PPGLs lacking mutations in SDHB genes. \*\*\*\** P *< 0.0001.*

genotypes. More importantly, downregulation was significantly more dramatic in metastatic than in benign *SDHx*-mutated tumors. Thus, the corrupted epigenetic changes in this chromosomal region seems to amplify the positive selection of the most metastatic cells and the evolutionary capacity of cancer to spread out of the tissue of origin. Interestingly, only the PCDHGC4 isoform, among the clustered PCDH proteins, have been shown to be strictly required for postnatal viability and survival of many neuronal subsets [39].

Consistent with previous findings in colon cancer cell lines [37], we found in that decreased *PCDHGC3* gene expression in two different cancer cell lines resulted in significant increases in cell proliferation, cell migration, and collective cell invasion. Silencing of *PCDHGC3* gene also resulted in increased tumor growth in studies of xenograft tumor models *in vivo*. Consistent with this, the current published data showed that PCDHs regulate pathways for cell proliferation and death. In tumor tissues derived from PPGLs, loss of *PCDH* expression is an indicator of poor prognosis, as revealed by our data and the *in silico* analysis of published data. Importantly, in *SDHB*-mutated metastatic PPGLs with high levels of *PCDHGC3* methylation, diagnosis of primary tumor and metastatic disease was synchronous in most cases, but some patients had a metastasis-free time ranging from 1 to 19 years. Thus, it is possible that epigenetic alterations of *PCDHGC3* during tumor initiation do not automatically lead to the manifestation of full metastatic potential. Rather, metastatic potential likely evolves through quantitative amplification, ultimately providing the cell with metastatic fitness. Thus, *PCDHGC3* acts as a tumor suppressor gene in PPGLs, could be an efficient biomarker of malignancy, and could represent a novel target for personalized medicine.

Targeting any of the protocadherin genes is challenging given that they are highly expressed in nervous system where exert relevant functions for the establishment and maintenance of specific neuronal connections. It is imperative, thus, to unravel the signaling pathways downstream *PCDHGC3* to identify potential therapeutic targets activated in the absence of *PCDHGC3* expression. Current published data have shown that PCDHs are tightly linked to several major signaling pathways, including the Wnt/β-catenin and receptor tyrosine kinase signaling

pathways [40–44]. In renal cancer cell lines, we have found that *PCDHGC3* loss of expression associates with increased mTOR activity. Several reports have shown activation of the mTOR pathway in PPGLs [45]. In addition, inhibition of this pathway exerts potent antitumor activity in a rat model of pheochromocytoma [46]. The epigenetic silencing of *PCDHGC3* could, thus, serve as a biomarker for the selection of patients appropriate for therapeutic options targeting the mTOR pathway.

### **3.3 Succinate-induced histone methylation**

Gene expression can also be altered by changes in chromatin structure via chemical modification of amino acids on histone tails. Accumulation of high levels of succinate in SDH-deficient PPGLs inhibits JmjC domain-containing histone demethylases (KDMs) [19, 47, 48]. These KDMs remove the methyl group on lysine in histone tails, which can either activate or repress transcription depending on the specifically modified lysine residues. Generally, H3K4, H3K36 and H3K79 methylations are considered to mark active transcription, whereas H3K9, H3K27 and H4K20 methylations are thought to be associated with silenced chromatin states [49].

Succinate increases methylation of H3K27 and H3K79 [19]. Trimethylation of H3K27 is a hallmark of repressed transcription. It is tightly associated with inactive gene promoters and also the gene promoters that were found hypermethylated in *SDHB*-mutated metastatic PPGLs. Instead, H3K79 methylation is linked to active transcription and may influence transcription elongation and genomic stability [50] (**Figure 4**).

Succinate induces inhibition of the activities of KDM4A which remove methylation on histone 3 lysine 9 (H3K9) [51, 52]. H3K9 methylation is the mark of heterochromatin, which is the condensed, transcriptionally inactive state of chromatin. Importantly, Sulkowski et al. have recently shown that increased succinate levels, induced by SDH silencing, can also repress homology-dependent DNA repair (HDR) by directly inhibiting the H3K9 demethylase KDM4B, 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 HDR [51] (**Figure 4**). This finding underscores the notion that decreased DNA repair acts as a key oncogenic mechanism in SDH-deficient PPGLs, similarly to the underlying mechanisms of the familial breast and ovarian cancer predisposition syndromes linked to the *BRCA1* and *BRCA2* genes.

#### **3.4 Succinate-induced loss of insulators**

DNA hypermethylation outside of gene promoters may also have significant impacts on PPGL pathophysiology, especially when hypermethylation occurs at the CCCTC-binding factor (CTCF) insulators. Insulators are DNA regulatory elements that block the interaction between gene enhancers and gene promoters. They block the spreading of enhancers action and thus insulate, or shield, gene promoters from unwanted regulation [53, 54]. CTCF dimerization, when it is bound to different DNA sequences, mediates long-range chromatin looping allowing the insulation of promoters from enhancer sequences (**Figure 4**). Many proto-oncogenes are isolated in such domains and thus protected from promiscuous enhancer interactions. The CTCF insulator is methylation-sensitive and may be displaced by DNA methylation. DNA hypermethylation at CTCF insulators is traduced in promiscuous enhancer-promoter interactions with the subsequent induction of the affected genes [53, 55].

**35**

**4. microRNA and lncRNA**

*Metastatic Paragangliomas and Pheochromocytomas: An Epigenetic View*

genome topology and the enhancer/promoter functions.

and activities of proteins involved in diverse cellular processes [57].

with *SDHB*-mutations and to develop drug therapies and targeted agents.

(miRNAs) and long-non-coding RNAs (lncRNAs), in metastatic PPGLs.

RNA-based mechanisms of epigenetic regulation are less well understood than mechanisms involved on DNA methylation and histones but have also profound roles in gene regulation, development and tumorigenesis. Several recent studies have analyzed the pattern of expression of non-coding RNAs, including microRNAs

Mature miRNAs (~22nucleotides long) base-pair with target mRNAs to inhibit translation or direct mRNA degradation. Several studies have shown over-expression of miR-183 in metastatic compared with non-metastatic PPGLs, irrespective of

**3.5 Succinate-induced protein succinylation**

Recent studies of SDH-deficient gastrointestinal stromal tumors (GISTs) have uncovered the frequent hypermethylation of CTCF insulators where DNA methylation replaces CTCF binding [55, 56]. This ubiquitous insulator losses leads SDH-deficient cells to acquire promiscuous enhancer-promoter interactions and an altered genome topology promoting expression of genes such as *FGF4* or *KIT* involved in the oncogenic programs activated in GIST. This discovery raises the interesting possibility that SDH-deficiency in PPGLs may drive oncogenic programs, in the absence of DNA mutations, by epigenetic modifications that alter

SDH inactivation induces accumulation of the immediate upstream metabolite, succinyl-CoA. Succinyl-CoA is the substrate used for the succinylation of proteins, in which succinyl group is transferred to a lysine residue of a protein. It is a recently identified common and widespread posttranslational modification that directly couples TCA cycle metabolism, via succinyl-CoA, to alterations in the structures

Lysine succinylation can occur by a non-enzymatic chemical reaction. This suggests that the abundance of succinyl-CoA would be one of the main governing factors of protein succinylation. A recent study have demonstrated that knockdown of *SDHB* leads to global lysine hyper-succinylation in multiple cellular compartments, especially mitochondria, coupled with increased succinyl-CoA levels [58]. Succinate-induced hypersuccinylation results in apoptosis resistance suggesting a relevant role in tumorigenesis and metastasis development. Succinylation can also occur at the nuclei. In this regard, Wang et al. have demonstrated that the lysine acetyltransferase 2A (KAT2A) may also act as a histone succinyltransferase by forming a complex with α-ketoglutarate dehydrogenase (α-KGDH) that catalyzes the conversion of α-ketoglutarate (α-KG) to succinyl-CoA in the promoter regions of genes [59] (**Figure 4**). Indeed, more than one-third of nucleosomes, including histone and non-histone chromatin components, have been shown to be lysine succinylated in the absence of functional SDH activity suggesting that SDH loss has significant effects on chromatin structure and function and subsequent gene expression [60]. These succinyl marks in chromatin coincide with H3K4me3 chromatin marks, but not with H3K27me3-chromatin marks, suggesting that succinylation of chromatin at active gene promoters is functionally meaningful. Histone succinylation induces widespread gene expression changes that promote tumor growth [61, 62]. However, how histone and nonhistone protein succinylation affects tumorigenesis remains largely unexplored and deserve in-depth characterization to unravel their putative involvement in metastasis development in patients

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

Recent studies of SDH-deficient gastrointestinal stromal tumors (GISTs) have uncovered the frequent hypermethylation of CTCF insulators where DNA methylation replaces CTCF binding [55, 56]. This ubiquitous insulator losses leads SDH-deficient cells to acquire promiscuous enhancer-promoter interactions and an altered genome topology promoting expression of genes such as *FGF4* or *KIT* involved in the oncogenic programs activated in GIST. This discovery raises the interesting possibility that SDH-deficiency in PPGLs may drive oncogenic programs, in the absence of DNA mutations, by epigenetic modifications that alter genome topology and the enhancer/promoter functions.

## **3.5 Succinate-induced protein succinylation**

*Pheochromocytoma, Paraganglioma and Neuroblastoma*

**3.3 Succinate-induced histone methylation**

tion syndromes linked to the *BRCA1* and *BRCA2* genes.

subsequent induction of the affected genes [53, 55].

**3.4 Succinate-induced loss of insulators**

pathway.

states [49].

[50] (**Figure 4**).

pathways [40–44]. In renal cancer cell lines, we have found that *PCDHGC3* loss of expression associates with increased mTOR activity. Several reports have shown activation of the mTOR pathway in PPGLs [45]. In addition, inhibition of this pathway exerts potent antitumor activity in a rat model of pheochromocytoma [46]. The epigenetic silencing of *PCDHGC3* could, thus, serve as a biomarker for the selection of patients appropriate for therapeutic options targeting the mTOR

Gene expression can also be altered by changes in chromatin structure via chemical modification of amino acids on histone tails. Accumulation of high levels of succinate in SDH-deficient PPGLs inhibits JmjC domain-containing histone demethylases (KDMs) [19, 47, 48]. These KDMs remove the methyl group on lysine in histone tails, which can either activate or repress transcription depending on the specifically modified lysine residues. Generally, H3K4, H3K36 and H3K79 methylations are considered to mark active transcription, whereas H3K9, H3K27 and H4K20 methylations are thought to be associated with silenced chromatin

Succinate increases methylation of H3K27 and H3K79 [19]. Trimethylation of H3K27 is a hallmark of repressed transcription. It is tightly associated with inactive gene promoters and also the gene promoters that were found hypermethylated in *SDHB*-mutated metastatic PPGLs. Instead, H3K79 methylation is linked to active transcription and may influence transcription elongation and genomic stability

Succinate induces inhibition of the activities of KDM4A which remove methylation on histone 3 lysine 9 (H3K9) [51, 52]. H3K9 methylation is the mark of heterochromatin, which is the condensed, transcriptionally inactive state of chromatin. Importantly, Sulkowski et al. have recently shown that increased succinate levels, induced by SDH silencing, can also repress homology-dependent DNA repair (HDR) by directly inhibiting the H3K9 demethylase KDM4B, 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 HDR [51] (**Figure 4**). This finding underscores the notion that decreased DNA repair acts as a key oncogenic mechanism in SDH-deficient PPGLs, similarly to the underlying mechanisms of the familial breast and ovarian cancer predisposi-

DNA hypermethylation outside of gene promoters may also have significant impacts on PPGL pathophysiology, especially when hypermethylation occurs at the CCCTC-binding factor (CTCF) insulators. Insulators are DNA regulatory elements that block the interaction between gene enhancers and gene promoters. They block the spreading of enhancers action and thus insulate, or shield, gene promoters from unwanted regulation [53, 54]. CTCF dimerization, when it is bound to different DNA sequences, mediates long-range chromatin looping allowing the insulation of promoters from enhancer sequences (**Figure 4**). Many proto-oncogenes are isolated in such domains and thus protected from promiscuous enhancer interactions. The CTCF insulator is methylation-sensitive and may be displaced by DNA methylation. DNA hypermethylation at CTCF insulators is traduced in promiscuous enhancer-promoter interactions with the

**34**

SDH inactivation induces accumulation of the immediate upstream metabolite, succinyl-CoA. Succinyl-CoA is the substrate used for the succinylation of proteins, in which succinyl group is transferred to a lysine residue of a protein. It is a recently identified common and widespread posttranslational modification that directly couples TCA cycle metabolism, via succinyl-CoA, to alterations in the structures and activities of proteins involved in diverse cellular processes [57].

Lysine succinylation can occur by a non-enzymatic chemical reaction. This suggests that the abundance of succinyl-CoA would be one of the main governing factors of protein succinylation. A recent study have demonstrated that knockdown of *SDHB* leads to global lysine hyper-succinylation in multiple cellular compartments, especially mitochondria, coupled with increased succinyl-CoA levels [58]. Succinate-induced hypersuccinylation results in apoptosis resistance suggesting a relevant role in tumorigenesis and metastasis development. Succinylation can also occur at the nuclei. In this regard, Wang et al. have demonstrated that the lysine acetyltransferase 2A (KAT2A) may also act as a histone succinyltransferase by forming a complex with α-ketoglutarate dehydrogenase (α-KGDH) that catalyzes the conversion of α-ketoglutarate (α-KG) to succinyl-CoA in the promoter regions of genes [59] (**Figure 4**). Indeed, more than one-third of nucleosomes, including histone and non-histone chromatin components, have been shown to be lysine succinylated in the absence of functional SDH activity suggesting that SDH loss has significant effects on chromatin structure and function and subsequent gene expression [60]. These succinyl marks in chromatin coincide with H3K4me3 chromatin marks, but not with H3K27me3-chromatin marks, suggesting that succinylation of chromatin at active gene promoters is functionally meaningful. Histone succinylation induces widespread gene expression changes that promote tumor growth [61, 62]. However, how histone and nonhistone protein succinylation affects tumorigenesis remains largely unexplored and deserve in-depth characterization to unravel their putative involvement in metastasis development in patients with *SDHB*-mutations and to develop drug therapies and targeted agents.
