**4. microRNA and lncRNA**

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

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

the genotype of the tumor [23, 63]. Higher levels of miR-483-5p have been also in metastatic tumors compared with benign tumors [23, 64]. Given the rarity of PPGLs, in general, and of metastatic PPGLs with *SDHB* mutations, in particular, the putative involvement of SDH-deficiency mediated miRNA deregulation in metastasis development is yet unknown.

miR-210 is one of the best characterized miRNAs downstream HIF1α activation and a candidate tumor-driver of metabolic reprogramming in cancer [65]. Some studies have proposed that up-regulation of miR-210 is a hallmark of the *VHL*/*SDHx*-mutated PPGLs [66] whereas others have ascribed it a role exclusively in *VHL*-mutated tumors [18]. One of the targets of miR-210 is the gene that codifies the iron–sulfur cluster assembly enzyme (*ISCU*) required for the assembly of maturation of Fe-S clusters, critical bioinorganic prosthetic groups essential for electron transport and multiple metabolic processes [67]. The miR-210-*ISCU* signaling pathway, a hallmark of the HIF activation in cancer, is activated in *SDHx* and *VHL*-mutated PPGLs [18]. However, the role of miR-210 in metastasis predisposition of *SDHB*-mutated PPGLs is not known. A recent report showed that the serum levels of miR-210 are decreased in metastatic PPGLs [68] although these data were grounded in a very limited number of samples and has not been confirmed in publicly available databases. For example, *in silico* analysis of the TCGA database confirms previous reports showing that miR-210 is highly over-expressed in *VHL*-mutated PPGLs and moderately up-regulated in *SDHB*-PPGLs although this was independent on whether the tumor had or not metastatic behavior (**Figure 6**). Similarly, *ISCU* mRNA levels more dramatically decreased in *VHL*-mutated than in *SDHx*-mutated PPGLs. Among *SDHB*-mutated PPGLs, the differences of *ISCU* levels were not significant enough to assign it a role as biomarker of metastasis development. miR-210 was not found over-expressed, neither was *ISCU* underexpressed, in tumors carrying somatic mutations of the gene encoding the HIF2α subunit (*EPAS1*) of the HIF transcription factor thus confirming previous reports showing that this miRNA is a substrate of HIF1α but not HIF2α [69], at least, in the context of paraganglionar tissues. Thus, the available data suggest that miR-210 should not be used as a biomarker of metastatic *SDHB*-mutated PPGLs.

#### **Figure 6.**

*miR-210-ISCU signaling is moderately activated in SDHx-mutated PPGLs irrespective of their benign or metastatic behavior. miR-210-3p and ISCU levels in PPGLs included in the TCGA database are represented according to their genotype. Data from patients without or with metastasis are represented in blue and red, respectively. SDHB/D: PPGLs from patients with germline mutations in SDHB or SDHD genes; VHL*: *PPGLs with mutations in VHL; EPAS1*: *PPGLs with mutations in EPAS1; others: PPGLs with or without mutations in other PPGL-susceptibility genes; SDHB: metastatic PPGLs from patients with germline mutations in SDHB gene; \*\*\*\** P *< 0.0001.*

**37**

*Metastatic Paragangliomas and Pheochromocytomas: An Epigenetic View*

potentially metastatic tumors in patients carrying *SDHB* mutation.

with SDH-deficiency under phase II clinical trial.

benefited by the use of these epigenetic drugs [24].

lncRNAs are usually defined as non-coding RNAs greater than 200nucleotides [70]. Although their functions are not well understood they seem to have key roles in gene regulation which depend on their localization and their specific interactions with DNA, RNA and proteins. Their tissue-specific and condition-specific expression patterns suggest that lncRNAs could be potential biomarkers. Recent reports described DGCR9, FENDRR, HIF1A-AS2, MIR210HG [71] and BC063866 [21] with significantly elevated expression in metastatic compared to benign PPGLs. Expression of BC063866 was found significantly elevated in *SDHx*-mutated metastatic PPGLs and, if validated in larger series, could be a novel biomarker to identify

**5. Epigenetic drugs as therapeutic strategies for patients with metastatic** 

Among epigenetic drugs, despite their limitations, DNA methyltransferase (DNMT) inhibitors are the most effective epigenetic therapy developed to date. Azacitidine and decitabine are cytidine analogues that incorporate themselves into replicating DNA and inhibit DNMTs. This implies that these inhibitors have broad cellular effects leading to global loss of DNA methylation. Hence their use as epigenetic drugs have to deal with strategies to minimize the off-target effects. The use of effective methods for drug delivery reduces side effects and attains a higher therapeutic index. There are various delivery systems like nanocarriers (nanogels, liposomes, dendrimers, and polymeric nanoparticles) that enhance drug stability, permeability and retention. Low doses have received regulatory approval for the treatment of myelodysplastic syndrome and acute myeloid leukemia who are not candidates for conventional induction chemotherapy. The use of the DNMT inhibitor, guadecitabine, is currently been evaluated in patients with PPGLs associated

Other epigenetic drugs include the inhibitors of histone-lysine methyltransferases [72]. Multiple PRC2 inhibitors are currently being evaluated in ongoing phase I/II clinical trials in a range of cancers [73]. Most hypermethylated genes in metastatic *SDHB*-mutated PPGLs are PRC2 targets thus suggesting that patients could be

The findings that overproduction of succinate suppresses HDR provide a mechanistic basis for the use novel effective strategies to exploit these defects for therapeutic gain. HDR repression in SDH-deficient tumors enhances cellular dependence on alternative, poly [ADP-ribose] polymerase (PARP) dependent DNA repair mechanisms, which appears to offer a compelling opportunity for targeted therapeutic intervention in oncometabolite-driven cancers. A large body of scientific evidence and clinical trials led to FDA approval of PARP inhibitor monotherapy for the treatment of various cancers harboring mutations in HDR machinery, including those with BRCA1/2 loss [74]. It should be explored whether the HDR defect conferred by succinate accumulation is strong enough to put into practice this therapeutic strategy in SDH-deficient driven cancers. One interesting possibility will be to add DNA-damaging therapies to PARP antagonists to maximize therapeutic efficacy. Notably, the PARP inhibitor olaparib in combination with temozolamide is

currently undergoing testing in phase II clinical study in metastatic PPGLs. Hypersuccinylation can also be a target of therapy in metastatic PPGLs. Succinyl-CoA accumulated in SDH-deficient tumors can be condensed with glycine by D-aminolevulinate synthase 1 to form 5-aminolevulinate and enter the heme biosynthesis pathway. Therefore, glycine supplementation may facilitate removal of succinyl-CoA and inhibit succinylation. Relief of hypersuccinylation by glycine

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

**PPGLs**

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

*Pheochromocytoma, Paraganglioma and Neuroblastoma*

opment is yet unknown.

the genotype of the tumor [23, 63]. Higher levels of miR-483-5p have been also in metastatic tumors compared with benign tumors [23, 64]. Given the rarity of PPGLs, in general, and of metastatic PPGLs with *SDHB* mutations, in particular, the putative involvement of SDH-deficiency mediated miRNA deregulation in metastasis devel-

miR-210 is one of the best characterized miRNAs downstream HIF1α activation and a candidate tumor-driver of metabolic reprogramming in cancer [65]. Some studies have proposed that up-regulation of miR-210 is a hallmark of the *VHL*/*SDHx*-mutated PPGLs [66] whereas others have ascribed it a role exclusively in *VHL*-mutated tumors [18]. One of the targets of miR-210 is the gene that codifies the iron–sulfur cluster assembly enzyme (*ISCU*) required for the assembly of maturation of Fe-S clusters, critical bioinorganic prosthetic groups essential for electron transport and multiple metabolic processes [67]. The miR-210-*ISCU* signaling pathway, a hallmark of the HIF activation in cancer, is activated in *SDHx* and *VHL*-mutated PPGLs [18]. However, the role of miR-210 in metastasis predisposition of *SDHB*-mutated PPGLs is not known. A recent report showed that the serum levels of miR-210 are decreased in metastatic PPGLs [68] although these data were grounded in a very limited number of samples and has not been confirmed in publicly available databases. For example, *in silico* analysis of the TCGA database confirms previous reports showing that miR-210 is highly over-expressed in *VHL*-mutated PPGLs and moderately up-regulated in *SDHB*-PPGLs although this was independent on whether the tumor had or not metastatic behavior (**Figure 6**). Similarly, *ISCU* mRNA levels more dramatically decreased in *VHL*-mutated than in *SDHx*-mutated PPGLs. Among *SDHB*-mutated PPGLs, the differences of *ISCU* levels were not significant enough to assign it a role as biomarker of metastasis development. miR-210 was not found over-expressed, neither was *ISCU* underexpressed, in tumors carrying somatic mutations of the gene encoding the HIF2α subunit (*EPAS1*) of the HIF transcription factor thus confirming previous reports showing that this miRNA is a substrate of HIF1α but not HIF2α [69], at least, in the context of paraganglionar tissues. Thus, the available data suggest that miR-210

should not be used as a biomarker of metastatic *SDHB*-mutated PPGLs.

*miR-210-ISCU signaling is moderately activated in SDHx-mutated PPGLs irrespective of their benign or metastatic behavior. miR-210-3p and ISCU levels in PPGLs included in the TCGA database are represented according to their genotype. Data from patients without or with metastasis are represented in blue and red, respectively. SDHB/D: PPGLs from patients with germline mutations in SDHB or SDHD genes; VHL*: *PPGLs with mutations in VHL; EPAS1*: *PPGLs with mutations in EPAS1; others: PPGLs with or without mutations in other PPGL-susceptibility genes; SDHB: metastatic PPGLs from patients with germline mutations in SDHB* 

**36**

**Figure 6.**

*gene; \*\*\*\** P *< 0.0001.*

lncRNAs are usually defined as non-coding RNAs greater than 200nucleotides [70]. Although their functions are not well understood they seem to have key roles in gene regulation which depend on their localization and their specific interactions with DNA, RNA and proteins. Their tissue-specific and condition-specific expression patterns suggest that lncRNAs could be potential biomarkers. Recent reports described DGCR9, FENDRR, HIF1A-AS2, MIR210HG [71] and BC063866 [21] with significantly elevated expression in metastatic compared to benign PPGLs. Expression of BC063866 was found significantly elevated in *SDHx*-mutated metastatic PPGLs and, if validated in larger series, could be a novel biomarker to identify potentially metastatic tumors in patients carrying *SDHB* mutation.

## **5. Epigenetic drugs as therapeutic strategies for patients with metastatic PPGLs**

Among epigenetic drugs, despite their limitations, DNA methyltransferase (DNMT) inhibitors are the most effective epigenetic therapy developed to date. Azacitidine and decitabine are cytidine analogues that incorporate themselves into replicating DNA and inhibit DNMTs. This implies that these inhibitors have broad cellular effects leading to global loss of DNA methylation. Hence their use as epigenetic drugs have to deal with strategies to minimize the off-target effects. The use of effective methods for drug delivery reduces side effects and attains a higher therapeutic index. There are various delivery systems like nanocarriers (nanogels, liposomes, dendrimers, and polymeric nanoparticles) that enhance drug stability, permeability and retention. Low doses have received regulatory approval for the treatment of myelodysplastic syndrome and acute myeloid leukemia who are not candidates for conventional induction chemotherapy. The use of the DNMT inhibitor, guadecitabine, is currently been evaluated in patients with PPGLs associated with SDH-deficiency under phase II clinical trial.

Other epigenetic drugs include the inhibitors of histone-lysine methyltransferases [72]. Multiple PRC2 inhibitors are currently being evaluated in ongoing phase I/II clinical trials in a range of cancers [73]. Most hypermethylated genes in metastatic *SDHB*-mutated PPGLs are PRC2 targets thus suggesting that patients could be benefited by the use of these epigenetic drugs [24].

The findings that overproduction of succinate suppresses HDR provide a mechanistic basis for the use novel effective strategies to exploit these defects for therapeutic gain. HDR repression in SDH-deficient tumors enhances cellular dependence on alternative, poly [ADP-ribose] polymerase (PARP) dependent DNA repair mechanisms, which appears to offer a compelling opportunity for targeted therapeutic intervention in oncometabolite-driven cancers. A large body of scientific evidence and clinical trials led to FDA approval of PARP inhibitor monotherapy for the treatment of various cancers harboring mutations in HDR machinery, including those with BRCA1/2 loss [74]. It should be explored whether the HDR defect conferred by succinate accumulation is strong enough to put into practice this therapeutic strategy in SDH-deficient driven cancers. One interesting possibility will be to add DNA-damaging therapies to PARP antagonists to maximize therapeutic efficacy. Notably, the PARP inhibitor olaparib in combination with temozolamide is currently undergoing testing in phase II clinical study in metastatic PPGLs.

Hypersuccinylation can also be a target of therapy in metastatic PPGLs. Succinyl-CoA accumulated in SDH-deficient tumors can be condensed with glycine by D-aminolevulinate synthase 1 to form 5-aminolevulinate and enter the heme biosynthesis pathway. Therefore, glycine supplementation may facilitate removal of succinyl-CoA and inhibit succinylation. Relief of hypersuccinylation by glycine

supplementation, has been shown to result in inhibited growth of hypersuccinylated tumors [59], thus shedding lights on alternative approaches for *SDHx*-mutated-PPGLs.
