Methylation Mechanisms in Process of Cancer

*DNA Methylation Mechanism*

10.1038/ng.187

molcel.2011.08.032

gad.320069.118

[61] Mackay DJ et al. Hypomethylation of multiple imprinted loci in individuals with transient neonatal diabetes is associated with mutations in ZFP57. Nature Genetics. 2008;**40**:949-951. DOI:

[62] Quenneville S et al. In embryonic stem cells, ZFP57/KAP1 recognize a methylated hexanucleotide to affect chromatin and DNA methylation of imprinting control regions. Molecular Cell. 2011;**44**:361-372. DOI: 10.1016/j.

[63] Takahashi N et al. ZNF445 is a primary regulator of genomic imprinting. Genes & Development. 2019;**33**:49-54. DOI: 10.1101/

[64] Unoki M, Nishidate T, Nakamura Y. ICBP90, an E2F-1 target, recruits HDAC1 and binds to methyl-CpG through its SRA domain. Oncogene. 2004;**23**: 7601-7610. DOI: 10.1038/sj.onc.1208053

[65] Unoki M, Nakamura Y. Methylation at CpG islands in intron 1 of EGR2 confers enhancer-like activity. FEBS Letters. 2003;**554**:67-72. DOI: 10.1016/

S0014-5793(03)01092-5

**74**

**Chapter 5**

**Abstract**

in these malignances is reviewed.

**1. Introduction**

**77**

DNA Hydroxymethylation in the

Regulation of Gene Expression in

Chromatin in cancer undergoes chemical and structural changes that alter gene expression patterns. One of the chemical modifications that impacts gene regulation is 5-hydroxymethylcytosine (5hmC), also called DNA hydroxymethylation. 5hmC is a stable mark that is commonly associated with transcriptional activation. In cancer, the global loss of 5hmC is a hallmark. In addition, the deregulation of 5hmC in specific regions of the genome, such as enhancers, promoters, and body of the gene, alters the expression of genes in cancer. These alterations have been detected by the improvement in the mapping of 5hmC at genomic scale, which has allowed us to evaluate the sites where 5hmC alterations occur and the genes that are affected. In this chapter, the recent knowledge about the status of 5hmC in genome specific sites of human solid cancers, the relationship with enzymes ten-eleven translocation (TET) and **isocitrate dehydrogenase (**IDH) involved in the dynamic regulation of 5hmC levels, and the impact of the 5hmC aberrant changes on the genic expression

*Sofia L. Alcaraz-Estrada, Gabriela Leija-Montoya,*

*Claudia E. Millán-Testa, Mónica Sierra-Martínez, Magali Blanco-Morales and Jorge Sandoval-Basilio*

**Keywords:** DNA hydroxymethylation, 5hmC, cytosine modifications, DNA demethylation, epigenetics, chromatin, gene expression, cancer

In carcinogenesis, genetic alterations are necessary along with the deregulation

of the epigenetic phenomena. Epigenetics could be defined as the study of the mechanisms that control gene expression without modifying the DNA sequence [1]. In cancer, epigenetic changes can be used to identify the site of origin of the tumor, detect malignant tumors in the earliest stages, and also allow the identification of more aggressive tumors and predict the response to drug therapy [2, 3]. On the other hand, they can be used as therapeutic targets in epigenetic therapy [4]. DNA methylation is a widely studied epigenetic phenomenon, and it occurs predominantly (80%) in a CpG context, where cytosine (C) is methylated in carbon 5, generating 5-methylcytosine (5mC). 5mC constitutes approximately 1% of all

Human Solid Cancer

*Nicolás Serafín-Higuera, Silvia García,*

**Chapter 5**

## DNA Hydroxymethylation in the Regulation of Gene Expression in Human Solid Cancer

*Sofia L. Alcaraz-Estrada, Gabriela Leija-Montoya, Nicolás Serafín-Higuera, Silvia García, Claudia E. Millán-Testa, Mónica Sierra-Martínez, Magali Blanco-Morales and Jorge Sandoval-Basilio*

#### **Abstract**

Chromatin in cancer undergoes chemical and structural changes that alter gene expression patterns. One of the chemical modifications that impacts gene regulation is 5-hydroxymethylcytosine (5hmC), also called DNA hydroxymethylation. 5hmC is a stable mark that is commonly associated with transcriptional activation. In cancer, the global loss of 5hmC is a hallmark. In addition, the deregulation of 5hmC in specific regions of the genome, such as enhancers, promoters, and body of the gene, alters the expression of genes in cancer. These alterations have been detected by the improvement in the mapping of 5hmC at genomic scale, which has allowed us to evaluate the sites where 5hmC alterations occur and the genes that are affected. In this chapter, the recent knowledge about the status of 5hmC in genome specific sites of human solid cancers, the relationship with enzymes ten-eleven translocation (TET) and **isocitrate dehydrogenase (**IDH) involved in the dynamic regulation of 5hmC levels, and the impact of the 5hmC aberrant changes on the genic expression in these malignances is reviewed.

**Keywords:** DNA hydroxymethylation, 5hmC, cytosine modifications, DNA demethylation, epigenetics, chromatin, gene expression, cancer

#### **1. Introduction**

In carcinogenesis, genetic alterations are necessary along with the deregulation of the epigenetic phenomena. Epigenetics could be defined as the study of the mechanisms that control gene expression without modifying the DNA sequence [1]. In cancer, epigenetic changes can be used to identify the site of origin of the tumor, detect malignant tumors in the earliest stages, and also allow the identification of more aggressive tumors and predict the response to drug therapy [2, 3]. On the other hand, they can be used as therapeutic targets in epigenetic therapy [4].

DNA methylation is a widely studied epigenetic phenomenon, and it occurs predominantly (80%) in a CpG context, where cytosine (C) is methylated in carbon 5, generating 5-methylcytosine (5mC). 5mC constitutes approximately 1% of all

DNA bases [5] and is associated with the regulation of gene expression, recruitment of transcription factors, nucleosome positioning, splicing, and imprinting. Specifically, in cancer, the overall decrease of 5mC is a general brand and is enriched in specific areas such as the promoter and the first exon, which generates gene silencing and is associated with the development of cancer [6]. Although 5mC is a stable mark, 5mC was shown to oxidize to 5-hydroxymethylcytosine (5hmC). In mammals, the conversion of 5mC to 5hmC is catalyzed by members of the ten-eleven translocation family (TET1, TET2, and TET3), α-ketoglutarate (αKG), and Fe2+ dependent dioxygenases. TET enzymes also oxidize 5hmC to 5-formylcytosine (5fC) and 5-carboxycytosine (5cC) [7].

hand, the increase in chromatin-modifying enzymes, such as Ezh2 methyltransferase, has been published [21]. A significant decrease in members of the TET enzyme family has been reported, which correlates with low global levels of 5hmC [17–22]. Thus, altered patterns of 5hmC in melanomas have been observed, and in this sense, the analysis of 5hmC in melanomas showed a hypo-hydroxymethylation in the body of the Ras-related C3 botulinum toxin substrate 3 (RAC3) gene, the type 1 insulin-like growth factor receptor (IGF1R), and tissue inhibitor of metalloproteinases 2 (TIMP2) (**Table 1**). However, the effect at the expression level was not determined. In two studies published independently, they reported that in melanoma the expression levels of IGF1R and TIMP2 are high [23, 24], suggesting that hypo-5hmC in the body of the gene probably contributes to the high expression of the IGF1R and TIMP2 gene. On the other hand, it has been observed that the overexpression of TIMP2 in B16F10 melanoma cells reduces invasion and angiogenesis and inhibits

*DNA Hydroxymethylation in the Regulation of Gene Expression in Human Solid Cancer*

Gliomas are the most common brain tumors, being classified by grades (I–IV), based on differentiation status, malignant potential, response to treatment, and patient survival rate. Grades III and IV are referred as high-grade glioma and have the worst prognosis with a median survival for grade III of 2–3 years, while grade IV, named glioblastoma (GBM), has approximately 15 months [45]. In 2016, the

**position**

RAC3, IGF1R, TIMP2 Hypo-5hmC Gene body ND Melanoma [17] SOX2-OT, CHD2 Hyper-5hmC TSS1500 Activation Glioma [25]

PTEN, hMLH1, IRX1 Hypo-5hmC Promoter Repression Gastric [28]

TBX15 Hyper-5hmC Promoter Activation HCC [30] COMT, FMO3, LCAT Hypo-5hmC Promoter Repression HCC [30]

VHL, SETD2 Hypo-5hmC Gene body Repression ccRCC [14]

TESC, TGFBI, BMP7, NKD2 Hyper-5hmC Loci Activation Colon [32] GLO1 Hyper-5hmC Promoter Activation Endometrial [43] LZTS1 Hypo-5hmC Loci Repression Breast [44] *\*The effect of 5hmC was not determined in these genes. 5hmC, 5-hydroxymethylcytosine; TSS1500, 1500 bases upstream transcription start site; 50 UTR, 50 untranslated region; HCC, Hepatocellular carcinoma; HuRCSS, Kidney renal stem cells;*

gene body

promoter

**Effect Target cancer Refs.**

Activation Pancreas [29]

Activation HuRCSC [31]

UTR Activation Glioma [25]

Hyper-5hmC ND Activation Neuroblastoma [26, 27]

Hypo-5hmC Loci, promoter Repression Colon [32–42]

apoptosis [24].

**2.2 5hmC status in glioma cancer**

*DOI: http://dx.doi.org/10.5772/intechopen.92016*

**Name of gene 5hmC status 5hmC variable**

GATA6, MMP11, VAV2, LATS2 Hyper-5hmC Promoter and

CCNY, CDK16 Hyper-5hmC Loci and

, GHRHR\* ,

*ccRCC, Clear cell renal cell carcinoma; ND, not determined.*

*Genes with aberrant 5hmC in different solid human neoplasms.*

LSMEM1 Hyper-5hmC 5<sup>0</sup>

v-myc, FAM49A, DDX1, IL-2, IL-

CA2, FMN2, PDCD4, PKIB, SLC26A2, ALOX15\*

, TKTL1\*

15, PRC2

TFP12\*

**Table 1.**

**79**

Currently, it is reported that DNA hydroxymethylation is a stable epigenetic mark that the cells can inherit to its daughter's cells rather than just a transition state [8]. This mark represents the second most abundant C variant in the mammalian genome, always at levels below 5mC, but on the other hand, the 5hmC is 10–100 times higher than the 5fC and 5cC [9, 10].

In mammals, 5hmC occurs almost always (99.89%) in a CpG context [11] and, interestingly, is more enriched in distal regulatory elements of the promoter (46.4%), the body of the gene, and near the cis elements of transcription factors and is less abundant in the promoter region [9–11], suggesting that the hydroxymethylation of DNA has an extensive function in gene regulation. In addition, the enrichment of 5hmC in the different functional elements has been associated with gene transcriptional activation [9].

In humans, the presence of 5hmC would vary significantly between tissues: in the brain (0.67%), rectum (0.57%), liver (0.46%), colon (0.45%), and kidney (0.38%), the 5hmC levels are high, while in the lung (0.14%), they are relatively low and very low in the heart (0.05%), breast (0.05%), and placenta (0.06%) [12]. Compared to normal tissue, many solid neoplasms (e.g., breast, colon, prostate, and melanoma) are characterized by the overall loss of 5hmC. In some isolated cases of cancer, it was reported that 5hmC usually increases; however, the general trend is a global decrease of 5hmC in carcinogenesis [13]. Importantly, hypo-5hmC in cancer occurs in the body of the gene, enhancers, and near cis elements of transcription factors, altering gene expression [14, 15]. However, so far, the role of hydroxymethylation of DNA in cancer biology is not completely clear, and more studies are needed that provide deeper information on functions or potential applications as biomarkers. The purpose of this chapter is to provide current knowledge of the deregulation of 5hmC in genome specific sites, the relationship with enzymes ten-eleven translocation (TET) and **isocitrate dehydrogenase (**IDH) involved in the dynamic regulation of 5hmC levels, and its impact on gene expression in different human cancers.

#### **2. 5hmC status in solid cancer**

#### **2.1 5hmC status in melanoma**

Melanoma is a type of melanocyte neoplasm that is considered highly aggressive [16]. As one of the most aggressive human tumors, it can perform distal and lethal metastases despite the volume of the tumor being 1 mm<sup>3</sup> [17].

Melanoma is a complex disease influenced by genetic and epigenetic alterations. Importantly, epigenetic phenomena in this tumor include hypermethylation of the phosphatase and tensin homolog (PTEN) promoter and p16ink4, which is associated with the silencing of tumor suppressor genes [18, 19]. In addition, repressive chromatin marks that silence the TGF-pathway have been reported [20]. On the other

#### *DNA Hydroxymethylation in the Regulation of Gene Expression in Human Solid Cancer DOI: http://dx.doi.org/10.5772/intechopen.92016*

hand, the increase in chromatin-modifying enzymes, such as Ezh2 methyltransferase, has been published [21]. A significant decrease in members of the TET enzyme family has been reported, which correlates with low global levels of 5hmC [17–22]. Thus, altered patterns of 5hmC in melanomas have been observed, and in this sense, the analysis of 5hmC in melanomas showed a hypo-hydroxymethylation in the body of the Ras-related C3 botulinum toxin substrate 3 (RAC3) gene, the type 1 insulin-like growth factor receptor (IGF1R), and tissue inhibitor of metalloproteinases 2 (TIMP2) (**Table 1**). However, the effect at the expression level was not determined. In two studies published independently, they reported that in melanoma the expression levels of IGF1R and TIMP2 are high [23, 24], suggesting that hypo-5hmC in the body of the gene probably contributes to the high expression of the IGF1R and TIMP2 gene. On the other hand, it has been observed that the overexpression of TIMP2 in B16F10 melanoma cells reduces invasion and angiogenesis and inhibits apoptosis [24].

#### **2.2 5hmC status in glioma cancer**

DNA bases [5] and is associated with the regulation of gene expression, recruitment of transcription factors, nucleosome positioning, splicing, and imprinting. Specifically, in cancer, the overall decrease of 5mC is a general brand and is enriched in specific areas such as the promoter and the first exon, which generates gene silencing and is associated with the development of cancer [6]. Although 5mC is a stable mark, 5mC was shown to oxidize to 5-hydroxymethylcytosine (5hmC). In mammals, the conversion of 5mC to 5hmC is catalyzed by members of the ten-eleven translocation family (TET1, TET2, and TET3), α-ketoglutarate (αKG), and Fe2+ dependent dioxygenases. TET enzymes also oxidize 5hmC to 5-formylcytosine

Currently, it is reported that DNA hydroxymethylation is a stable epigenetic mark that the cells can inherit to its daughter's cells rather than just a transition state [8]. This mark represents the second most abundant C variant in the mammalian genome, always at levels below 5mC, but on the other hand, the 5hmC is 10–100

In mammals, 5hmC occurs almost always (99.89%) in a CpG context [11] and,

In humans, the presence of 5hmC would vary significantly between tissues: in the brain (0.67%), rectum (0.57%), liver (0.46%), colon (0.45%), and kidney (0.38%), the 5hmC levels are high, while in the lung (0.14%), they are relatively low and very low in the heart (0.05%), breast (0.05%), and placenta (0.06%) [12]. Compared to normal tissue, many solid neoplasms (e.g., breast, colon, prostate, and melanoma) are characterized by the overall loss of 5hmC. In some isolated cases of cancer, it was reported that 5hmC usually increases; however, the general trend is a global decrease of 5hmC in carcinogenesis [13]. Importantly, hypo-5hmC in cancer occurs in the body of the gene, enhancers, and near cis elements of transcription factors, altering gene expression [14, 15]. However, so far, the role of hydroxymethylation of DNA in cancer biology is not completely clear, and more studies are needed that provide deeper information on functions or potential applications as biomarkers. The purpose of this chapter is to provide current knowledge of the deregulation of 5hmC in genome specific sites, the relationship with enzymes ten-eleven translocation (TET) and **isocitrate dehydrogenase (**IDH) involved in the dynamic regulation of 5hmC levels, and its impact on gene

Melanoma is a type of melanocyte neoplasm that is considered highly aggressive [16]. As one of the most aggressive human tumors, it can perform distal and lethal

Melanoma is a complex disease influenced by genetic and epigenetic alterations. Importantly, epigenetic phenomena in this tumor include hypermethylation of the phosphatase and tensin homolog (PTEN) promoter and p16ink4, which is associated with the silencing of tumor suppressor genes [18, 19]. In addition, repressive chromatin marks that silence the TGF-pathway have been reported [20]. On the other

metastases despite the volume of the tumor being 1 mm<sup>3</sup> [17].

interestingly, is more enriched in distal regulatory elements of the promoter (46.4%), the body of the gene, and near the cis elements of transcription factors

and is less abundant in the promoter region [9–11], suggesting that the hydroxymethylation of DNA has an extensive function in gene regulation. In addition, the enrichment of 5hmC in the different functional elements has been

(5fC) and 5-carboxycytosine (5cC) [7].

*DNA Methylation Mechanism*

times higher than the 5fC and 5cC [9, 10].

expression in different human cancers.

**2. 5hmC status in solid cancer**

**2.1 5hmC status in melanoma**

**78**

associated with gene transcriptional activation [9].

Gliomas are the most common brain tumors, being classified by grades (I–IV), based on differentiation status, malignant potential, response to treatment, and patient survival rate. Grades III and IV are referred as high-grade glioma and have the worst prognosis with a median survival for grade III of 2–3 years, while grade IV, named glioblastoma (GBM), has approximately 15 months [45]. In 2016, the


*\*The effect of 5hmC was not determined in these genes. 5hmC, 5-hydroxymethylcytosine; TSS1500, 1500 bases upstream transcription start site; 50 UTR, 50 untranslated region; HCC, Hepatocellular carcinoma; HuRCSS, Kidney renal stem cells; ccRCC, Clear cell renal cell carcinoma; ND, not determined.*

#### **Table 1.**

*Genes with aberrant 5hmC in different solid human neoplasms.*

World Health Organization (WHO) introduced a new classification where molecular markers were taken into account. In this classification glioma are divided into subtypes based on the isocitrate dehydrogenase 1 **(**IDH1) gene mutation status [46]. IDH can affect DNA hypermethylation at certain promoter regions, resulting in a glioma CpG island methylator phenotype [47]. In glioma, it has been described as an aberrant 5mC status in CpG island shores and is 5hmC-dependent, and it correlates with disease progression [15]. Moreover, in a previous report, the 5hmC patterns were analyzed in GBM samples. They observed a 3.5 reduction in the total 5hmC content and of what was present, localized primarily in super-enhancers and cis elements of transcription factors associated with proliferation. Also, they observed a significant enrichment of 5hmC sites in active transcribed genes in GBM. They reported a total of 2121 active transcribed genes of which 146 have the highest proportion of 5hmC.

Risk assessment based on several clinical and biological features, including age, stage, avian myelocytomastosis viral oncogene (v-myc) status, ploidy, and histology, classified the patients into three groups, low-risk (LR), intermediate-risk (IR), and high-risk (HR) disease [26]. Comparison of the 5hmC profile in LR vs. HR permitted the identification of 3320 genes with differential 5hmC levels between the groups. In the LR group, genes with 5hmC enrichment and increased expression were v-myc, family with sequence similarity 49 member A (FAM49A), and DEADbox helicase 1 (DDX1) (**Table 1**). Regarding the HR group, the genes with hyper-5hmC and high expression include genes involved in inflammation (IL-2 and IL-15)

*DNA Hydroxymethylation in the Regulation of Gene Expression in Human Solid Cancer*

Parathyroid cancer is a rare, indolent, and slowly progressive tumor, being the rarest cause of primary hyperparathyroidism. Surgery is the option of treatment; thus the early identification in the preoperative period is vital [55]. Clinical characteristics of parathyroid cancer can overlap with benign parathyroid disease [55, 56]. Barazeghi E. et al. showed reduced global levels of 5hmC in samples of parathyroid carcinoma compared with samples of normal tissues as well as benign parathyroid adenomas; thus, it was suggested that 5hmC level could be a marker to differentiate between benign and malign tumors [56]. Analysis of TET protein expression indicated variable expression of TET1 in parathyroid adenomas and carcinomas, and additionally reduced or absent expression of TET2 was observed in parathyroid carcinomas as compared with normal parathyroid tissue in concordance with the reduced levels of 5hmC reported in parathyroid carcinoma [56, 57]. Furthermore, increased levels of methylation in promoter CpG islands from TET2 were reported [57]. Analysis of genes regulated by hydroxymethylation on this cancer has not been

Thyroid cancer is the most common tumor of the endocrine organs, accounting for 90% of endocrine tumors. In general, thyroid cancer is originated of follicular cells being divided in papillary thyroid carcinoma and follicular thyroid carcinoma; a reduced percent of thyroid cancers is generated from parafollicular C cells being classified as medullary thyroid carcinomas [58]. The information about 5hmC status is extremely limited, but it has been observed that levels of 5hmC are reduced in papillary thyroid carcinomas as compared with control tissue [59]. However, information about expression of TET proteins and regulation by hydroxymethylation or

Cancer of the oral cavity is the most common tumor worldwide, the squamous cell carcinoma being the most common histopathology type [60]. A decreased expression of TET2 and a reduction of 5mhC levels in samples of oral squamous cell carcinomas have been reported compared with healthy oral tissues by immunohistochemistry [61]. Also, 5hmC levels decreased progressively from benign oral mucosal lesions to oral squamous cell carcinoma [62]. Expression analysis of

target genes regulated by 5hmC or mechanisms implicated have not been

and in the polycomb repressive complex 2 (PRC2) [27].

**2.4 5hmC status in parathyroid cancer**

*DOI: http://dx.doi.org/10.5772/intechopen.92016*

reported, yet.

reported yet.

**81**

**2.5 5hmC status in thyroid cancer**

**2.6 5hmC status in oral cancer**

target genes in this cancer or its subtypes is absent.

As an example, genes with hyper-5hmC in 1500 bases upstream of the transcription star site (TSS1500) that are transcriptionally active are SOX2 overlapping transcript (SOX-OT) and chromodomain-helicase-DNA-binding protein 2 (CHD2) (**Table 1**). Additionally, the gene that encodes the leucine-rich single-pass membrane protein 1 (LSMEM1, also called C7orf53) is transcriptionally active and also presented elevated levels of 5hmC in the 5<sup>0</sup> untranslated region (5<sup>0</sup> UTR) (**Table 1**) [25]**.**

#### **2.3 5hmC status in pediatric embryonal tumors**

The pediatric embryonal tumors are a rare type of childhood cancers that derive from neuroectodermal tissue and share related histopathological features despite distinct anatomical locations and diverse clinical outcomes [48]. These tumors can originate in many parts of the body, the ones that are derived from the sympathetic nervous system are called neuroblastoma, and the ones that are derived in the brain are called medulloblastoma [49].

#### *2.3.1 Medulloblastoma*

Medulloblastoma (MB) is the most common malignant brain tumor of childhood, the overall 5-year disease-free survival remains low (36%) for patients with dissemination, and prognosis remains poor for patients with recurrent MB [50]. In addition, majority of survivors exhibit long-term neurocognitive and neuroendocrine complication as a result of therapy [51, 52].

The analysis of the 5hmC levels in MB showed a reduction, in comparison to non-neoplastic cerebellum [53]. This finding agrees with other reports where the loss of 5hmC is a common event in other brain tumors as well as tumors of different origins [54]. With this discovery, the expression of TET1, TET2, TET3, IDH1, and IDH2 was analyzed, but the profile could not explain the reduction of 5hmC [53], although the difference in expression of this genes did correlate with the different MB molecular subgroups, suggesting a possible role for TET and IDH genes in the control of specifically developmental pathways activated in MB subgroups. This inability to associate the overall reduction of 5hmC levels and the expression of TET and IDH genes could be due to the method of selection of the 5hmC screening and the lack of analysis of the genomic distribution of 5hmC.

#### *2.3.2 Neuroblastoma*

Neuroblastoma is accountable for more than 7% of malignancies in patients younger than 15 years and is responsible for 15% of all pediatric oncology deaths. *DNA Hydroxymethylation in the Regulation of Gene Expression in Human Solid Cancer DOI: http://dx.doi.org/10.5772/intechopen.92016*

Risk assessment based on several clinical and biological features, including age, stage, avian myelocytomastosis viral oncogene (v-myc) status, ploidy, and histology, classified the patients into three groups, low-risk (LR), intermediate-risk (IR), and high-risk (HR) disease [26]. Comparison of the 5hmC profile in LR vs. HR permitted the identification of 3320 genes with differential 5hmC levels between the groups. In the LR group, genes with 5hmC enrichment and increased expression were v-myc, family with sequence similarity 49 member A (FAM49A), and DEADbox helicase 1 (DDX1) (**Table 1**). Regarding the HR group, the genes with hyper-5hmC and high expression include genes involved in inflammation (IL-2 and IL-15) and in the polycomb repressive complex 2 (PRC2) [27].

#### **2.4 5hmC status in parathyroid cancer**

World Health Organization (WHO) introduced a new classification where molecular markers were taken into account. In this classification glioma are divided into subtypes based on the isocitrate dehydrogenase 1 **(**IDH1) gene mutation status [46]. IDH can affect DNA hypermethylation at certain promoter regions, resulting in a glioma CpG island methylator phenotype [47]. In glioma, it has been described as an aberrant 5mC status in CpG island shores and is 5hmC-dependent, and it correlates with disease progression [15]. Moreover, in a previous report, the 5hmC patterns were analyzed in GBM samples. They observed a 3.5 reduction in the total 5hmC content and of what was present, localized primarily in super-enhancers and cis elements of transcription factors associated with proliferation. Also, they observed a significant enrichment of 5hmC sites in active transcribed genes in GBM. They reported a total of 2121 active transcribed genes of which 146 have the highest

As an example, genes with hyper-5hmC in 1500 bases upstream of the transcription star site (TSS1500) that are transcriptionally active are SOX2 overlapping

(CHD2) (**Table 1**). Additionally, the gene that encodes the leucine-rich single-pass membrane protein 1 (LSMEM1, also called C7orf53) is transcriptionally active and

The pediatric embryonal tumors are a rare type of childhood cancers that derive from neuroectodermal tissue and share related histopathological features despite distinct anatomical locations and diverse clinical outcomes [48]. These tumors can originate in many parts of the body, the ones that are derived from the sympathetic nervous system are called neuroblastoma, and the ones that are derived in the brain

Medulloblastoma (MB) is the most common malignant brain tumor of childhood, the overall 5-year disease-free survival remains low (36%) for patients with dissemination, and prognosis remains poor for patients with recurrent MB [50]. In addition, majority of survivors exhibit long-term neurocognitive and neuroendo-

The analysis of the 5hmC levels in MB showed a reduction, in comparison to non-neoplastic cerebellum [53]. This finding agrees with other reports where the loss of 5hmC is a common event in other brain tumors as well as tumors of different origins [54]. With this discovery, the expression of TET1, TET2, TET3, IDH1, and IDH2 was analyzed, but the profile could not explain the reduction of 5hmC [53], although the difference in expression of this genes did correlate with the different MB molecular subgroups, suggesting a possible role for TET and IDH genes in the control of specifically developmental pathways activated in MB subgroups. This inability to associate the overall reduction of 5hmC levels and the expression of TET and IDH genes could be due to the method of selection of the 5hmC screening and

Neuroblastoma is accountable for more than 7% of malignancies in patients younger than 15 years and is responsible for 15% of all pediatric oncology deaths.

UTR)

transcript (SOX-OT) and chromodomain-helicase-DNA-binding protein 2

also presented elevated levels of 5hmC in the 5<sup>0</sup> untranslated region (5<sup>0</sup>

**2.3 5hmC status in pediatric embryonal tumors**

crine complication as a result of therapy [51, 52].

the lack of analysis of the genomic distribution of 5hmC.

are called medulloblastoma [49].

*2.3.1 Medulloblastoma*

*2.3.2 Neuroblastoma*

**80**

proportion of 5hmC.

*DNA Methylation Mechanism*

(**Table 1**) [25]**.**

Parathyroid cancer is a rare, indolent, and slowly progressive tumor, being the rarest cause of primary hyperparathyroidism. Surgery is the option of treatment; thus the early identification in the preoperative period is vital [55]. Clinical characteristics of parathyroid cancer can overlap with benign parathyroid disease [55, 56]. Barazeghi E. et al. showed reduced global levels of 5hmC in samples of parathyroid carcinoma compared with samples of normal tissues as well as benign parathyroid adenomas; thus, it was suggested that 5hmC level could be a marker to differentiate between benign and malign tumors [56]. Analysis of TET protein expression indicated variable expression of TET1 in parathyroid adenomas and carcinomas, and additionally reduced or absent expression of TET2 was observed in parathyroid carcinomas as compared with normal parathyroid tissue in concordance with the reduced levels of 5hmC reported in parathyroid carcinoma [56, 57]. Furthermore, increased levels of methylation in promoter CpG islands from TET2 were reported [57]. Analysis of genes regulated by hydroxymethylation on this cancer has not been reported, yet.

#### **2.5 5hmC status in thyroid cancer**

Thyroid cancer is the most common tumor of the endocrine organs, accounting for 90% of endocrine tumors. In general, thyroid cancer is originated of follicular cells being divided in papillary thyroid carcinoma and follicular thyroid carcinoma; a reduced percent of thyroid cancers is generated from parafollicular C cells being classified as medullary thyroid carcinomas [58]. The information about 5hmC status is extremely limited, but it has been observed that levels of 5hmC are reduced in papillary thyroid carcinomas as compared with control tissue [59]. However, information about expression of TET proteins and regulation by hydroxymethylation or target genes in this cancer or its subtypes is absent.

#### **2.6 5hmC status in oral cancer**

Cancer of the oral cavity is the most common tumor worldwide, the squamous cell carcinoma being the most common histopathology type [60]. A decreased expression of TET2 and a reduction of 5mhC levels in samples of oral squamous cell carcinomas have been reported compared with healthy oral tissues by immunohistochemistry [61]. Also, 5hmC levels decreased progressively from benign oral mucosal lesions to oral squamous cell carcinoma [62]. Expression analysis of target genes regulated by 5hmC or mechanisms implicated have not been reported yet.

#### **2.7 5hmC status in gastric cancer**

Gastric cancer is the third cause of cancer death. Environment factors, infections, and genetic and epigenetic alterations are related with development of this cancer [63]. Decreased expression of TET1 mRNA and protein has been reported in gastric cancer, while the expression of TET2 and TET3 did not show differences in expression compared with control tissue. In addition, diminished expression of TET1 has been associated with decreased levels of 5hmC in the promoter of PTEN, human mutL homolog 1 (hMLH1), and iroquois homeobox 1 (IRX1), correlating with their reduced expression levels (**Table 1**) [28]. Thus, reduced expression of TET1 and decreased levels of 5hmC in gastric cancer could be related with the decreased expression of suppressor tumors genes. An in-depth analysis of genes regulated by hydroxymethylation on this cancer could allow new therapeutic strategies.

(TBX15) with enrichment in the 5hmC promoter region was associated with transcriptional activation, while those that presented a decrease of 5hmC were low [30].

*DNA Hydroxymethylation in the Regulation of Gene Expression in Human Solid Cancer*

RCC is the most common parenchymal neoplasm in adults. Among urogenital tumors, it is the second with the highest incidence, which represents 2 to 3% of cancer in humans and 80 to 90% of kidney neoplasms. The most common subtypes

Recently, it was reported that kidney cancer and ccRCC have global decreased levels of 5hmC with respect to normal tissue [14]. Paradoxically in kidney renal stem cells (HuRCSC), hyper-5hmC patterns were detected in specific regions such as the cyclin Y (CCNY) promoter and loci and cyclin-dependent kinase-16 (CDK16) (**Table 1**), which was associated with a transcriptional activation. Likewise, the increased levels of TET1 in HuRCSC were detected, which probably explains the

In another study in which ccRCC was analyzed, it was shown that low levels of

methyltransferase are associated with low expression [14] (**Table 1**). In ccRCC loss of SETD2 is associated with genomic instability, aberrant transcriptional program, RNA processing defects, and impacts on cell proliferation, differentiation, and cell death [71]. Additionally, it has been observed that the IDH1 enzyme was significantly downregulated in ccRCC compared to normal kidney cells. Thus, in ccRCC the reduction of IDH1 can be a mechanism for the loss of 5hmC through the

The colon cancer or colorectal cancer is the third most common cancer in the

Decreased levels of global hydroxymethylation has been reported in colon cancer [32]. In accordance, decreased expression of TET1 has been reported in samples of human colorectal cancer tissue [33]. Moreover, loss of nuclear expression of TET2 has been observed in colorectal cancer tissue [34]. Thus, aberrant patterns of hydroxymethylation in colorectal cancer has been observed. In this sense, analysis of 5hmC status in colon cancer tissues showed hypo-hydroxymethylation in the

• Carbonic anhydrase 2 (CA2), which belongs to a group of zinc-binding

prevents degradation of p21, promoting cell cycle arrest [32–36]

• Programmed cell death 4 (PDCD4), considered a tumor suppressor in colorectal cancer, which can inhibit proliferation and invasion, preventing

• cAMP-dependent protein kinase inhibitor beta (PKIB), which promotes

function of PKIB in colon cancer remains to be determined) [32–39]

activation of Akt, contributing to cell growth and proliferation (however, the

enzymes, which catalyzes the reversible hydration of CO2 to bicarbonate, that

• Formin 2 (FMN2), involved in cell polarity and cytoskeleton organization and

of RCC are clear cell carcinoma (ccRCC) and papillary carcinoma [31].

5hmC in the body of the von Hippel–Lindau gene (VHL) and SETD2

world with more incidence in developed countries [72, 73].

is important for maintenance of pH [32–35]

AP-1 transcription and inhibiting mTOR/Akt [37]

**2.10 5hmC status in renal cell carcinoma (RCC)**

*DOI: http://dx.doi.org/10.5772/intechopen.92016*

increase of 5hmC in HuRCSC [31].

downregulation of 2-keto glutarate [14].

**2.11 5hmC status in colon cancer**

promoter region of genes such as:

**83**

#### **2.8 5hmC status in pancreatic cancer**

Pancreatic cancer is a disease with high mortality rate, being the fourth cause of cancer-related deaths in the United States and most developed countries. Different types of pancreatic cancer can rise; however, the subtype termed pancreatic ductal adenocarcinoma (PDAC) is the most common, which accounts for about 85–90% of cases [64, 65]. The absence of early detection methods, delay in diagnosis, and unsuccessful treatments contribute to the high mortality of this cancer [65].

Low global levels of 5hmC have been reported in pancreatic cancer cell lines and in samples of human tumors compared with healthy pancreatic cells [29]. The reduced levels of 5hmC in samples of human pancreatic tumor tissues correlated with the decreased expression of TET1 [66]. A redistribution of 5hmCs was observed in pancreatic cancer, with enrichment in genomic specific regions as promoters and gene body, particularly of the transcriptional factor GATA6, matrix metallopeptidase 11 (MMP11), vav guanine nucleotide exchange factor 2 (VAV2), and large tumor suppressor kinase 2. The enrichment of 5hmC in this genes is in accordance with the increased expression in human samples of pancreatic cancer [29]. Additionally, it has been suggested that pancreatic cancer patients with high GATA6 survive longer so that GATA6 was proposed as a prognosis marker [67]. On the other hand, high levels of MMP11 were associated with poor prognosis of pancreatic cancer patients [68].

#### **2.9 5hmC status in hepatocellular cancer**

Globally, hepatocellular carcinoma (HCC) is the sixth most prevalent cancer and the third leading cause of cancer-related death, estimated to cause the death of 500,000–600,000 people per year [69]. The factors that contribute to mortality in HCC are the 5-year recurrence rates standing at 70% after tumor resection and 15–30% posttransplant [70].

Hepatic B virus (HBV) and hepatitis C (HCV) are the main cause of HCC. For HCC related to HBV, epigenetic alterations play vital roles in hepatocarcinogenesis through direct and indirect mechanisms initiated by HBV [69].

Low global levels of 5hmC have been reported in HCC [69]. A redistribution of 5hmCs was observed in HCC, with an enrichment in specific genomic region as promoters. In this same report, a decrease in the levels of hydroxymethylation was observed in the promoter of different genes, this catechol-O-methyltransferase (COMT), the flavin-containing monooxygenase 3 (FMO3), and lecithin-cholesterol acyltransferase **(**LCAT) [30] (**Table 1**). The gene T-Box transcription factor 15

(TBX15) with enrichment in the 5hmC promoter region was associated with transcriptional activation, while those that presented a decrease of 5hmC were low [30].

#### **2.10 5hmC status in renal cell carcinoma (RCC)**

**2.7 5hmC status in gastric cancer**

*DNA Methylation Mechanism*

**2.8 5hmC status in pancreatic cancer**

pancreatic cancer patients [68].

15–30% posttransplant [70].

**82**

**2.9 5hmC status in hepatocellular cancer**

strategies.

Gastric cancer is the third cause of cancer death. Environment factors, infections, and genetic and epigenetic alterations are related with development of this cancer [63]. Decreased expression of TET1 mRNA and protein has been reported in gastric cancer, while the expression of TET2 and TET3 did not show differences in expression compared with control tissue. In addition, diminished expression of TET1 has been associated with decreased levels of 5hmC in the promoter of PTEN, human mutL homolog 1 (hMLH1), and iroquois homeobox 1 (IRX1), correlating with their reduced expression levels (**Table 1**) [28]. Thus, reduced expression of TET1 and decreased levels of 5hmC in gastric cancer could be related with the decreased expression of suppressor tumors genes. An in-depth analysis of genes regulated by hydroxymethylation on this cancer could allow new therapeutic

Pancreatic cancer is a disease with high mortality rate, being the fourth cause of cancer-related deaths in the United States and most developed countries. Different types of pancreatic cancer can rise; however, the subtype termed pancreatic ductal adenocarcinoma (PDAC) is the most common, which accounts for about 85–90% of cases [64, 65]. The absence of early detection methods, delay in diagnosis, and unsuccessful treatments contribute to the high mortality of this cancer [65].

Low global levels of 5hmC have been reported in pancreatic cancer cell lines and

observed in pancreatic cancer, with enrichment in genomic specific regions as promoters and gene body, particularly of the transcriptional factor GATA6, matrix metallopeptidase 11 (MMP11), vav guanine nucleotide exchange factor 2 (VAV2), and large tumor suppressor kinase 2. The enrichment of 5hmC in this genes is in accordance with the increased expression in human samples of pancreatic cancer [29]. Additionally, it has been suggested that pancreatic cancer patients with high GATA6 survive longer so that GATA6 was proposed as a prognosis marker [67]. On the other hand, high levels of MMP11 were associated with poor prognosis of

Globally, hepatocellular carcinoma (HCC) is the sixth most prevalent cancer and

Hepatic B virus (HBV) and hepatitis C (HCV) are the main cause of HCC. For HCC related to HBV, epigenetic alterations play vital roles in hepatocarcinogenesis

Low global levels of 5hmC have been reported in HCC [69]. A redistribution of 5hmCs was observed in HCC, with an enrichment in specific genomic region as promoters. In this same report, a decrease in the levels of hydroxymethylation was observed in the promoter of different genes, this catechol-O-methyltransferase (COMT), the flavin-containing monooxygenase 3 (FMO3), and lecithin-cholesterol acyltransferase **(**LCAT) [30] (**Table 1**). The gene T-Box transcription factor 15

the third leading cause of cancer-related death, estimated to cause the death of 500,000–600,000 people per year [69]. The factors that contribute to mortality in HCC are the 5-year recurrence rates standing at 70% after tumor resection and

through direct and indirect mechanisms initiated by HBV [69].

in samples of human tumors compared with healthy pancreatic cells [29]. The reduced levels of 5hmC in samples of human pancreatic tumor tissues correlated with the decreased expression of TET1 [66]. A redistribution of 5hmCs was

RCC is the most common parenchymal neoplasm in adults. Among urogenital tumors, it is the second with the highest incidence, which represents 2 to 3% of cancer in humans and 80 to 90% of kidney neoplasms. The most common subtypes of RCC are clear cell carcinoma (ccRCC) and papillary carcinoma [31].

Recently, it was reported that kidney cancer and ccRCC have global decreased levels of 5hmC with respect to normal tissue [14]. Paradoxically in kidney renal stem cells (HuRCSC), hyper-5hmC patterns were detected in specific regions such as the cyclin Y (CCNY) promoter and loci and cyclin-dependent kinase-16 (CDK16) (**Table 1**), which was associated with a transcriptional activation. Likewise, the increased levels of TET1 in HuRCSC were detected, which probably explains the increase of 5hmC in HuRCSC [31].

In another study in which ccRCC was analyzed, it was shown that low levels of 5hmC in the body of the von Hippel–Lindau gene (VHL) and SETD2 methyltransferase are associated with low expression [14] (**Table 1**). In ccRCC loss of SETD2 is associated with genomic instability, aberrant transcriptional program, RNA processing defects, and impacts on cell proliferation, differentiation, and cell death [71]. Additionally, it has been observed that the IDH1 enzyme was significantly downregulated in ccRCC compared to normal kidney cells. Thus, in ccRCC the reduction of IDH1 can be a mechanism for the loss of 5hmC through the downregulation of 2-keto glutarate [14].

#### **2.11 5hmC status in colon cancer**

The colon cancer or colorectal cancer is the third most common cancer in the world with more incidence in developed countries [72, 73].

Decreased levels of global hydroxymethylation has been reported in colon cancer [32]. In accordance, decreased expression of TET1 has been reported in samples of human colorectal cancer tissue [33]. Moreover, loss of nuclear expression of TET2 has been observed in colorectal cancer tissue [34]. Thus, aberrant patterns of hydroxymethylation in colorectal cancer has been observed. In this sense, analysis of 5hmC status in colon cancer tissues showed hypo-hydroxymethylation in the promoter region of genes such as:


• Solute carrier family 26 member 2 (SLC26A2) (low expression of this protein promotes proliferation in vitro observed in colon cancer cell lines) [32–41]

breast tissue. These results coincide with the low expression of TET1. The results suggest that low levels of 5hmC in the locus of LZTS1 are probably due to the decrease in TET1 [44]. In human cancer, loss of LZTS1 expression has been associ-

*DNA Hydroxymethylation in the Regulation of Gene Expression in Human Solid Cancer*

In the tumors researched in this document, in most of them, an overall global reduction of 5hmC, with accumulation in certain genetic locations or genes, is reported. Some are also related to the overexpression of genes, taking into account that 5hmC is an epigenetic mark of transcription activation. However, the methods used to determine the 5hmC vary between the studies, and not all the methods can be considered reliable to distinguish between 5mC and 5hmC with confidence. Moreover, an area that has not been explored is the effect that 5hmC has on the expression in miRNAs and lncRNA. These observations can be taken to reassess the

Expression analyses of TET family members and correlation with 5hmC status have been performed in a wide variety of cancers. However, the biological effect of this aberrant changes in 5hmC levels has not been deeply determined. For example, targeted genes regulated by hydroxymethylation in many cancers have not been established, characterization of the hydroxymethylation patterns in regulator regions of target genes is not determined in some cases, and their expression levels are not corroborated with 5hmC status. Importantly, the effects resulting in the modification or the return to the original state of the hydroxymethylation patterns in cancer are extensively unknown. In addition, since hydroxymethylation can be regulated in different physiological process in health and disease, modifications in 5hmC status could generate undesirable side effects. Thus, more studies are necessary to have a comprehensive understanding of the biological effects and dynamic changes of the hydroxymethylation in cancer, which could allow new therapeutic

We acknowledge support from Universidad Hipócrates and Consejo Nacional de

ated with tumor progression, metastasis, and poor prognosis [82].

*DOI: http://dx.doi.org/10.5772/intechopen.92016*

role of DNA hydroxymethylation status in tumorigenesis.

**3. Conclusion**

strategies in the future.

**Acknowledgements**

**Conflict of interest**

C Cytosine

**85**

Ciencia y Tecnología CONACyT.

**Appendices and nomenclature**

bHlH Basic helix–loop–helix

CA2 Carbonic anhydrase 2 CCNY Cyclin Y promoter and loci ccRCC Clear cell carcinoma cell renal

The authors declare no conflict of interest.

ALOX15 Arachidonate 15-lipoxygenase BMP7 Bone morphogenetic protein 7


Related to 5hmC status and expression, in some cases, the decreased levels of expression of these genes were corroborated. Additionally, hyperhydroxymethylation and increased expression of different genes have been observed in samples of human primary colon cancer [32–42]. The genes with hyper-5hmC status and high expression include tescalcin (TESC), a calcium-binding protein involved in the promotion of tumorigenesis in colorectal cancer, which activates Akt-dependent NF-κB pathway, promoting proliferation and also contributing to invasion and metastasis in colon cancer [32–42, 74, 75]; transforming growth factor-beta-induced (TGFBI) that promotes cell proliferation, migration, metastasis, and inflammation [32–42, 74–76]; and bone morphogenetic protein 7 (BMP7) that has an augmented expression, but in this case there has been a controversy because in colorectal cancer it has also been reported to have low levels. Additionally, it has been reported that, in colorectal cancer, BMP7 has anticancer activity [32–42, 74–78] and naked cuticle homolog 2 (NKD2) [32].

#### **2.12 5hmC status in endometrial cancer**

Endometrial cancer is the most common tumor in the genital tract in developed countries [43]. A report showed a decreased level of 5hmC in samples of endometrial cancer by an ELISA-like reaction. In addition, decreased expression levels of TET1 and TET2 mRNA and increased levels of TET3 mRNA were observed [79]. However, in another report, increased levels of TET1 protein and increased levels of 5hmC in endometrial cancer and hyperplasia using immunohistochemistry were indicated [43]. This discrepancy could be associated to the different levels of regulation of TET1 expression. Interestingly, the authors determined that TET1 could promote the accumulation of 5hmC in the promoter of the glyoxalase I (GLO1) gene (**Table 1**), resulting in the increased expression of GLO1 in endometrial cancer [43]. GLO1 promotes proliferation and chemotherapeutic resistance and contributes to progestin resistance used in the treatment of endometrial cancer [43, 79, 80]. Moreover, it was reported that metformin treatment reduced the expression of TET1 and 5hmC levels, promoting the reduction of GLO1 expression and increasing the sensitivity to progestin in a model in vitro [43].

#### **2.13 5hmC status in breast cancer**

Breast cancer (BC) is the most frequent neoplasia in women worldwide; data from the WHO suggests that it comprises 16% of the total of cancer cases. In addition, every year 138 million new cases are detected, and approximately 458,000 deaths occur due to the pathology [81].

In breast cancer, low levels of 5hmC in the locus of the gene leucine zipper putative tumor suppressor (LZTS1) (**Table 1**) have been reported. In addition, the level of LZTS1 expression was low in breast cancer samples compared to normal

#### *DNA Hydroxymethylation in the Regulation of Gene Expression in Human Solid Cancer DOI: http://dx.doi.org/10.5772/intechopen.92016*

breast tissue. These results coincide with the low expression of TET1. The results suggest that low levels of 5hmC in the locus of LZTS1 are probably due to the decrease in TET1 [44]. In human cancer, loss of LZTS1 expression has been associated with tumor progression, metastasis, and poor prognosis [82].

#### **3. Conclusion**

• Solute carrier family 26 member 2 (SLC26A2) (low expression of this protein promotes proliferation in vitro observed in colon cancer cell lines) [32–41]

Related to 5hmC status and expression, in some cases, the decreased levels of

observed in samples of human primary colon cancer [32–42]. The genes with hyper-5hmC status and high expression include tescalcin (TESC), a calcium-binding protein involved in the promotion of tumorigenesis in colorectal cancer, which acti-

transforming growth factor-beta-induced (TGFBI) that promotes cell proliferation, migration, metastasis, and inflammation [32–42, 74–76]; and bone morphogenetic protein 7 (BMP7) that has an augmented expression, but in this case there has been a controversy because in colorectal cancer it has also been reported to have low levels. Additionally, it has been reported that, in colorectal cancer, BMP7 has anti-

Endometrial cancer is the most common tumor in the genital tract in developed countries [43]. A report showed a decreased level of 5hmC in samples of endometrial cancer by an ELISA-like reaction. In addition, decreased expression levels of TET1 and TET2 mRNA and increased levels of TET3 mRNA were observed [79]. However, in another report, increased levels of TET1 protein and increased levels of 5hmC in endometrial cancer and hyperplasia using immunohistochemistry were indicated [43]. This discrepancy could be associated to the different levels of regulation of TET1 expression. Interestingly, the authors determined that TET1 could promote the accumulation of 5hmC in the promoter of the glyoxalase I (GLO1) gene (**Table 1**), resulting in the increased expression of GLO1 in endometrial cancer [43]. GLO1 promotes proliferation and chemotherapeutic resistance and contributes to progestin resistance used in the treatment of endometrial cancer [43, 79, 80]. Moreover, it was reported that metformin treatment reduced the expression of TET1 and 5hmC levels, promoting the reduction of GLO1 expression and increasing

Breast cancer (BC) is the most frequent neoplasia in women worldwide; data from the WHO suggests that it comprises 16% of the total of cancer cases. In addition, every year 138 million new cases are detected, and approximately 458,000

In breast cancer, low levels of 5hmC in the locus of the gene leucine zipper putative tumor suppressor (LZTS1) (**Table 1**) have been reported. In addition, the level of LZTS1 expression was low in breast cancer samples compared to normal

• Arachidonate 15-lipoxygenase (ALOX15)

*DNA Methylation Mechanism*

**2.12 5hmC status in endometrial cancer**

the sensitivity to progestin in a model in vitro [43].

**2.13 5hmC status in breast cancer**

deaths occur due to the pathology [81].

**84**

• Growth hormone-releasing hormone (GHRHR)

• Inhibitor of the tissular factor pathway 2 (TFPI2)

• Transketolase-like protein 1 (TKTL1) (**Table 1**).

expression of these genes were corroborated. Additionally, hyper-

hydroxymethylation and increased expression of different genes have been

vates Akt-dependent NF-κB pathway, promoting proliferation and also contributing to invasion and metastasis in colon cancer [32–42, 74, 75];

cancer activity [32–42, 74–78] and naked cuticle homolog 2 (NKD2) [32].

In the tumors researched in this document, in most of them, an overall global reduction of 5hmC, with accumulation in certain genetic locations or genes, is reported. Some are also related to the overexpression of genes, taking into account that 5hmC is an epigenetic mark of transcription activation. However, the methods used to determine the 5hmC vary between the studies, and not all the methods can be considered reliable to distinguish between 5mC and 5hmC with confidence. Moreover, an area that has not been explored is the effect that 5hmC has on the expression in miRNAs and lncRNA. These observations can be taken to reassess the role of DNA hydroxymethylation status in tumorigenesis.

Expression analyses of TET family members and correlation with 5hmC status have been performed in a wide variety of cancers. However, the biological effect of this aberrant changes in 5hmC levels has not been deeply determined. For example, targeted genes regulated by hydroxymethylation in many cancers have not been established, characterization of the hydroxymethylation patterns in regulator regions of target genes is not determined in some cases, and their expression levels are not corroborated with 5hmC status. Importantly, the effects resulting in the modification or the return to the original state of the hydroxymethylation patterns in cancer are extensively unknown. In addition, since hydroxymethylation can be regulated in different physiological process in health and disease, modifications in 5hmC status could generate undesirable side effects. Thus, more studies are necessary to have a comprehensive understanding of the biological effects and dynamic changes of the hydroxymethylation in cancer, which could allow new therapeutic strategies in the future.

#### **Acknowledgements**

We acknowledge support from Universidad Hipócrates and Consejo Nacional de Ciencia y Tecnología CONACyT.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Appendices and nomenclature**



RNA Ribonucleic acid

TESC Tescalcin

TRDN Triadin

50

**87**

fate transporter)

TGFBI Transforming growth factor-beta-induced

TIMP2 Tissue inhibitor of metalloproteinase 2

v-myc Avian myelocytomastosis viral oncogene VAV2 Vav guanine nucleotide exchange factor 2

SOX2-OT SOX2 overlay transcript

*DOI: http://dx.doi.org/10.5772/intechopen.92016*

TET Ten-eleven translocation TET1 Ten-eleven translocation 1 TET2 Ten-eleven translocation 2 TET3 Ten-eleven translocation 3 TFPI2 Tissue factor pathway inhibitor 2 TGF Transforming growth factor

TKTL1 Transketolase-like protein1

VHL Von Hippel–Lindau gene

5hmC 5-Hydroxymethylcytosine

UTR 5<sup>0</sup> untranslated region

5fC 5-Formylcytosine 5cC 5-Carboxycytosine 5mC 5-Methylcytosine

αKG α-Ketoglutarate

SETD2 SET domain containing 2, histone lysine methyltransferase SLC26A2 The solute carrier family 26 member 2 (diastrophic dysplasia sul-

*DNA Hydroxymethylation in the Regulation of Gene Expression in Human Solid Cancer*

*DNA Hydroxymethylation in the Regulation of Gene Expression in Human Solid Cancer DOI: http://dx.doi.org/10.5772/intechopen.92016*


CHD2 Chromodomain-helicase-DNA-binding protein 2

FAM49A Family with sequence similarity 49 member A

G9a Euchromatic histone-lysine N-methyltransferase 2 (EHMT2)

CDK16 Cyclin-dependent kinase-16 COMT Catechol O-methyltransferase CpG Islands cytokine phophate guanine

Ezh2 Histone-lysine methyltransferase

FMO3 Flavin-containing monooxygenase 3

GHRHR Growth hormone-releasing hormone

HIF1α Hypoxia-inducible factor 1 alpha subunit HIF2α Hypoxia-inducible factor 2 alpha subunit

H3K36me3 Trimethylation of lysine 36 of histone H3

IGF1R Insulin-like growth factor 1 receptor

LCAT Lecithin-cholesterol acyltransferase LATS2 Suppressor kinase of large tumors 2

MRPL50 Mitochondrial ribosomal protein L50 MYCN Proto-oncogene, bHLH transcription factor

PCTAIRE1 Serine/threonine protein kinase (PCTK1) PDAC Pancreatic ductal adenocarcinoma

PKIB CAMP-dependent protein kinase inhibitor beta PPP1R3A Protein phosphatase 1 regulatory subunit 3A

RAC3 Ras-related C3 botulinum toxin substrate 3

LSMEM1 Leucine-rich single-pass membrane protein 1

DDX1 DEAD-box helicase 1 DNA Deoxyribonucleic acid

GATA6| GATA binding protein 6

hMLH1 Human mutL homolog 1

HuRCSC Kidney renal stem cell

IDH Isocitrate dehydrogenase IDH1 Isocitrate dehydrogenase 1 IDH2 Isocitrate dehydrogenase 2

lncRNA Long noncoding RNAs IR Intermediate-risk IRX1 Iroquois homeobox 1

hypo-5hmC Hypo-5 hydroxymethylcytosine

Fe2+ Iron 2+ FMN2 Formin 2

*DNA Methylation Mechanism*

GBM Glioblastoma

GLO1 Glyoxalase I

HR High-risk

LR Low-risk

MB Medulloblastoma mm3 Cubic millimeter mRNA Messenger RNA

NDRG3 Neuregulin 3

p16ink Protein 16 ink

**86**

RCC Renal cell carcinoma

MMP11 Matrix metalloproteinase-1

NKD2 Naked cuticle homolog 2 NF-κB Nuclear factor kappa B

PDCD4 Programmed cell death 4

PRC2 Polycomb repressive complex 2 PTEN Phosphatase and tensin homolog

### **Author details**

Sofia L. Alcaraz-Estrada1†, Gabriela Leija-Montoya2†, Nicolás Serafín-Higuera<sup>3</sup> , Silvia García<sup>4</sup> , Claudia E. Millán-Testa<sup>5</sup> , Mónica Sierra-Martínez<sup>6</sup> , Magali Blanco-Morales<sup>5</sup> and Jorge Sandoval-Basilio<sup>5</sup> \*

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*DNA Hydroxymethylation in the Regulation of Gene Expression in Human Solid Cancer*

1 Genomic Medicine Division, National Medical Center, 20 de Noviembre ISSSTE, Mexico City, Mexico

2 Medical School Campus Mexicali, Autonomous University of Baja California, Mexicali, Mexico

3 Cell Biology Laboratory, Health Sciences Unit, Dentistry Faculty, Autonomous University of Baja California, Mexicali, Mexico

4 Clinical Research Service, National Medical Center, 20 de Noviembre ISSSTE, Mexico City, Mexico

5 Molecular Biology Laboratory, Hipocrates University, Acapulco de Juarez, Guerrero, Mexico

6 Laboratory of Genetics and Molecular Diagnostics, Juarez Hospital of México, Mexico City, Mexico

\*Address all correspondence to: jorgesandoval@uhipocrates.edu.mx; sanbajl83@gmail.com

† Both authors have contributed equally to this work.

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*DNA Hydroxymethylation in the Regulation of Gene Expression in Human Solid Cancer DOI: http://dx.doi.org/10.5772/intechopen.92016*

#### **References**

**Author details**

*DNA Methylation Mechanism*

Mexico City, Mexico

Mexico City, Mexico

Guerrero, Mexico

Mexico City, Mexico

sanbajl83@gmail.com

**88**

Mexicali, Mexico

Silvia García<sup>4</sup>

Sofia L. Alcaraz-Estrada1†, Gabriela Leija-Montoya2†, Nicolás Serafín-Higuera<sup>3</sup>

1 Genomic Medicine Division, National Medical Center, 20 de Noviembre ISSSTE,

2 Medical School Campus Mexicali, Autonomous University of Baja California,

3 Cell Biology Laboratory, Health Sciences Unit, Dentistry Faculty, Autonomous

4 Clinical Research Service, National Medical Center, 20 de Noviembre ISSSTE,

6 Laboratory of Genetics and Molecular Diagnostics, Juarez Hospital of México,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

5 Molecular Biology Laboratory, Hipocrates University, Acapulco de Juarez,

\*Address all correspondence to: jorgesandoval@uhipocrates.edu.mx;

† Both authors have contributed equally to this work.

provided the original work is properly cited.

, Mónica Sierra-Martínez<sup>6</sup>

\*

,

, Claudia E. Millán-Testa<sup>5</sup>

Magali Blanco-Morales<sup>5</sup> and Jorge Sandoval-Basilio<sup>5</sup>

University of Baja California, Mexicali, Mexico

,

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hydroxymethylcytosine is linked to gene body hypermethylation in kidney cancer. Cell Research [Internet]. 2016; **26**(1):103-118. Available from: http:// www.nature.com/articles/cr2015150

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melanoma: Past and future. Epigenetics

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[Internet]. 2015;**10**(2):103-121.

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ve/pii/S0092867412010124

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epigenetic regulator of multiple invasion-suppressor microRNAs by targeting ten eleven translocation 1 in invasive human hepatocellular carcinoma tumors. Hepatology [Internet]. 2015;**62**(2):466-480. Available from: http://doi.wiley.com/ 10.1002/hep.27816

Available from: https://www.mdpi.com/ 2072-6694/12/1/129 [Cited 5 February

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[65] Qian L, Li Q, Baryeh K, Qiu W, Li K, Zhang J, et al. Biosensors for early diagnosis of pancreatic cancer: A review. Translational Research [Internet]. 2019;**213**:67-89. Available from: https://linkinghub.elsevier.com/ retrieve/pii/S1931524419301604

[66] Wu J, Li H, Shi M, Zhu Y, Ma Y, Zhong Y, et al. TET1-mediated DNA hydroxymethylation activates inhibitors of the Wnt/β-catenin signaling pathway to suppress EMT in pancreatic tumor cells. Journal of Experimental & Clinical Cancer Research [Internet]. 2019;**38**(1): 348. Available from: https://jeccr. biomedcentral.com/articles/10.1186/ s13046-019-1334-5 [Cited 23 January

[67] Martinelli P, Carrillo-de Santa Pau E, Cox T, Sainz B, Dusetti N, Greenhalf W, et al. GATA6 regulates EMT and tumour dissemination, and is a

marker of response to adjuvant

**39**(11):5963-5971

196-208

chemotherapy in pancreatic cancer. Gut [Internet]. 2017;**66**(9):1665-1676. Available from: http://gut.bmj.com/ lookup/doi/10.1136/gutjnl-2015-311256

[68] Lee J, Lee J, Kim JH. Identification of matrix metalloproteinase 11 as a prognostic biomarker in pancreatic cancer. Anticancer Research. 2019;

[69] Liu J, Jiang J, Mo J, Liu D, Cao D,

[70] Chuang K-H, Whitney-Miller CL, Chu C-Y, Zhou Z, Dokus MK, Schmit S,

et al. MicroRNA-494 is a master

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2020]

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Oncology Letters [Internet]. 2019;**18**(3): 2304-2309. Available from: http:// www.spandidos-publications.com/

[60] Montero PH, Patel SG. Cancer of the oral cavity. Surgical Oncology Clinics of North America [Internet]. 2015;**24**(3):491-508. Available from: https://linkinghub.elsevier.com/retrie

papillary thyroid carcinoma.

*DNA Methylation Mechanism*

10.3892/ol.2019.10531

ve/pii/S1055320715000319

[61] Jäwert F, Hasséus B, Kjeller G, Magnusson B, Sand L, Larsson L. Loss of 5-hydroxymethylcytosine and TET2 in

Anticancer Research [Internet]. 2013; **33**(10):4325-4328. Available from: http://www.ncbi.nlm.nih.gov/pubmed/

[62] Cuevas-Nunez MC, CBF G,Woo S-B,

hydroxymethylcytosine in oral epithelial

carcinoma. Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology [Internet]. 2018;**125**(1):59.e2-73.e2. Available from: https://linkinghub.elsevier. com/retrieve/pii/S2212440317308453

Ramsey MR, Chen XL, Xu S, et al. Biological significance of 5-

dysplasia and oral squamous cell

[63] Fattahi S, Kosari-Monfared M, Golpour M, Emami Z, Ghasemiyan M, Nouri M, et al. LncRNAs as potential diagnostic and prognostic biomarkers in gastric cancer: A novel approach to personalized medicine. Journal of Cellular Physiology [Internet]. 2020; **235**(4):3189-3206. Available from: https://onlinelibrary.wiley.com/doi/abs/

[64] Jelski W, Mroczko B. Biochemical diagnostics of pancreatic cancer -

10.1002/jcp.29260

**94**

oral squamous cell carcinoma.

24122999

2020]

[71] González-Rodríguez P, Engskog-Vlachos P, Zhang H, Murgoci A-N, Zerdes I, Joseph B. SETD2 mutation in renal clear cell carcinoma suppress autophagy via regulation of ATG12. Cell Death & Disease [Internet]. 2020;**11**(1): 69. Available from: http://www.ncbi. nlm.nih.gov/pubmed/31988284 [Cited 6 February 2020]

[72] Tiwari A, Saraf S, Jain A, Panda PK, Verma A, Jain SK. Basics to advances in nanotherapy of colorectal cancer. Drug Delivery and Translational Research [Internet]. 2019:1-20. Available from: http://link.springer.com/10.1007/ s13346-019-00680-9

[73] Jahanafrooz Z, Mosafer J, Akbari M, Hashemzaei M, Mokhtarzadeh A, Baradaran B. Colon cancer therapy by focusing on colon cancer stem cells and their tumor microenvironment. Journal of Cellular Physiology [Internet]. 2019. Available from: http://doi.wiley.com/ 10.1002/jcp.29337 [Cited 23 January 2020]

[74] Kang J, Kang YH, Oh BM, Uhm TG, Park SY, Kim TW, et al. Tescalcin expression contributes to invasive and metastatic activity in colorectal cancer. Tumour Biology: The Journal of the International Society for Oncodevelopmental Biology and Medicine [Internet]. 2016;**37**(10): 13843-13853. Available from: http:// www.ncbi.nlm.nih.gov/pubmed/ 27485112 [Cited 23 January 2020]

[75] Kang YH, Han SR, Kim J-T, Lee S-J, Il YY, Min J-K, et al. The EF-hand calcium-binding protein tescalcin is a potential oncotarget in colorectal

cancer. Oncotarget [Internet]. 2014; **5**(8):2149-2160. Available from: http:// www.ncbi.nlm.nih.gov/pubmed/ 24811141 [Cited 23 January 2020]

[76] Zhu J, Chen X, Liao Z, He C, Hu X. TGFBI protein high expression predicts poor prognosis in colorectal cancer patients. International Journal of Clinical and Experimental Pathology [Internet]. 2015;**8**(1):702-710. Available from: http://www.ncbi.nlm.nih.gov/pub med/25755764 [Cited 23 January 2020]

[77] Liu R-X, Ren W-Y, Ma Y, Liao Y-P, Wang H, Zhu J-H, et al. BMP7 mediates the anticancer effect of honokiol by upregulating p53 in HCT116 cells. International Journal of Oncology [Internet]. 2017;**51**(3):907-917. Available from: http://www.ncbi.nlm. nih.gov/pubmed/28731124 [Cited 23 January 2020]

[78] Pellatt AJ, Mullany LE, Herrick JS, Sakoda LC, Wolff RK, Samowitz WS, et al. The TGFβ-signaling pathway and colorectal cancer: Associations between dysregulated genes and miRNAs. Journal of Translational Medicine [Internet]. 2018;**16**(1):191. Available from: http://www.ncbi.nlm.nih.gov/pub med/29986714 [Cited 23 January 2020]

[79] Ciesielski P, Jóźwiak P, Wójcik-Krowiranda K, Forma E, Cwonda Ł, Szczepaniec S, et al. Differential expression of ten-eleven translocation genes in endometrial cancers. Tumor Biology [Internet]. 2017;**39**(3): 101042831769501. Available from: http://journals.sagepub.com/doi/ 10.1177/1010428317695017

[80] Zhang Z, Dong L, Sui L, Yang Y, Liu X, Yu Y, et al. Metformin reverses progestin resistance in endometrial cancer cells by downregulating GloI expression. International Journal of Gynecologic Cancer [Internet]. 2011;**21**(2):213-221. Available from: http://ijgc.bmj.com/ lookup/doi/10.1097/IGC.0b013e 318207dac7 [Cited 05 February 2020]

[81] Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians [Internet]. 2018;**68**(6): 394-424. Available from: http://doi. wiley.com/10.3322/caac.21492

**Chapter 6**

**Abstract**

Research Progress of DNA

*Zhu Gaohong and Xie Lijun*

targeted cancer treatment.

**1. Introduction**

**97**

Methylation in Thyroid Cancer

We have summarized increasing data from all kinds of experiment results of papers in recent years, which are associated with tumor suppressor genes, oncogenes, and thyroid-specific genes and attempt to elucidate the importance of epigenetic modifications and the mechanisms of aberrant DNA methylation in thyroid cancer in this review. The results showed that current articles have revealed the importance of epigenetic modifications and the different types of mechanisms in thyroid cancer. The mechanisms of DNA methylation related to thyroid cancer demonstrate that acquired epigenetic abnormalities together with genetic changes play an important role in alteration of gene expression patterns. Aberrant DNA methylation has been well known in the CpG regions. Among the genes identified, we have shown the status of DNA promoter methylation in papillary, follicular, medullary, and anaplastic thyroid cancer. It suggested that thyroid cancer subtypes present differential promoter methylation signatures, which will encourage potential thyroid cancer detection in its early stages, assessment of prognosis, and

**Keywords:** thyroid carcinoma, DNA methylation, epigenetic inheritance, tumor

Thyroid cancer is the most frequent endocrine neoplasia. The National Cancer Institute estimated that there would be 44,670 new cases of thyroid cancer (TC) with 1690 deaths in 2010, and with an overall estimate of 56,870 new cases by 2017, and its incidence has been increasing in recent decades. Compared with other adult cancers, TC tends to occur in younger people between the ages of 20 and 60. It is three times more common in women than men [1] and has the fastest rising incidence rates in women and the second fastest in men with an annual percentage change of approximately 5%, making TC the sixth most common cancer in women [2]. There are four main types of which papillary and follicular (PTC, FTC) types together account for >90% followed by medullary thyroid cancers (MTC) with 3–5% and anaplastic carcinomas (ATC) making up <3% [3]. Reasons for this trend have been attributed to improvement in imaging (ultrasound technology) that is allowing the identification of ever smaller thyroid nodules. However, with this gain in detection, determining which benign nodules (adenomas) will progress to cancer cannot be determined on the basis of histology alone, underscoring the need for

suppressor genes, oncogene genes, thyroid-specific genes

[82] Vecchione A, Baldassarre G, Ishii H, Nicoloso MS, Belletti B, Petrocca F, et al. Fez1/Lzts1 absence impairs Cdk1/ Cdc25C interaction during mitosis and predisposes mice to cancer development. Cancer Cell [Internet]. 2007;**11**(3):275-289. Available from: https://linkinghub.elsevier.com/ retrieve/pii/S153561080700030X

#### **Chapter 6**

[81] Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians [Internet]. 2018;**68**(6): 394-424. Available from: http://doi. wiley.com/10.3322/caac.21492

*DNA Methylation Mechanism*

[82] Vecchione A, Baldassarre G, Ishii H, Nicoloso MS, Belletti B, Petrocca F, et al. Fez1/Lzts1 absence impairs Cdk1/ Cdc25C interaction during mitosis and

development. Cancer Cell [Internet]. 2007;**11**(3):275-289. Available from: https://linkinghub.elsevier.com/ retrieve/pii/S153561080700030X

predisposes mice to cancer

**96**

## Research Progress of DNA Methylation in Thyroid Cancer

*Zhu Gaohong and Xie Lijun*

#### **Abstract**

We have summarized increasing data from all kinds of experiment results of papers in recent years, which are associated with tumor suppressor genes, oncogenes, and thyroid-specific genes and attempt to elucidate the importance of epigenetic modifications and the mechanisms of aberrant DNA methylation in thyroid cancer in this review. The results showed that current articles have revealed the importance of epigenetic modifications and the different types of mechanisms in thyroid cancer. The mechanisms of DNA methylation related to thyroid cancer demonstrate that acquired epigenetic abnormalities together with genetic changes play an important role in alteration of gene expression patterns. Aberrant DNA methylation has been well known in the CpG regions. Among the genes identified, we have shown the status of DNA promoter methylation in papillary, follicular, medullary, and anaplastic thyroid cancer. It suggested that thyroid cancer subtypes present differential promoter methylation signatures, which will encourage potential thyroid cancer detection in its early stages, assessment of prognosis, and targeted cancer treatment.

**Keywords:** thyroid carcinoma, DNA methylation, epigenetic inheritance, tumor suppressor genes, oncogene genes, thyroid-specific genes

#### **1. Introduction**

Thyroid cancer is the most frequent endocrine neoplasia. The National Cancer Institute estimated that there would be 44,670 new cases of thyroid cancer (TC) with 1690 deaths in 2010, and with an overall estimate of 56,870 new cases by 2017, and its incidence has been increasing in recent decades. Compared with other adult cancers, TC tends to occur in younger people between the ages of 20 and 60. It is three times more common in women than men [1] and has the fastest rising incidence rates in women and the second fastest in men with an annual percentage change of approximately 5%, making TC the sixth most common cancer in women [2]. There are four main types of which papillary and follicular (PTC, FTC) types together account for >90% followed by medullary thyroid cancers (MTC) with 3–5% and anaplastic carcinomas (ATC) making up <3% [3]. Reasons for this trend have been attributed to improvement in imaging (ultrasound technology) that is allowing the identification of ever smaller thyroid nodules. However, with this gain in detection, determining which benign nodules (adenomas) will progress to cancer cannot be determined on the basis of histology alone, underscoring the need for

genetic markers of early detection for TC. Recently, epigenetic alterations have been shown to play a role in the development and progression of thyroid cancer.

With the deepening of tumor research, it has been gradually found that epigenetics plays an important role in the occurrence and development of tumors. Mechanisms of epigenetics include, but are not limited to, DNA methylation (methylating of ciliary carbon at position 5), posttranslation modification of histone, chromatin remodeling (structural change), gene imprinting, RNA interference (noncoding RNA or gene silencing), etc. The epigenetic mechanisms of tumor cells have lost a fine regulation, and the breakdown of epigenetic patterns will lead to tumor phenotype expression. These mechanisms have been reviewed elsewhere, and here we will focus on DNA methylation in thyroid cancer.

DNA methylation is an important epigenetic change, which is persistent and hereditary. The methylation of promoter CpG can regulate gene expression and maintain chromosome integrity and DNA recombination. Based on 789 samples from the Gene Expression Omnibus (GEO) and the Cancer Genome Atlas (TCGA) databases, the five-CpG signature could provide a novel biomarker with useful applications in thyroid cancer (PTC, FTC, ATC, and MTC) diagnosis and the diagnostic score formula on the condition of DNA methylation data [4]. However, the methylation pattern is unstable and can be reversed by small molecules and endogenous enzymes, leading to dedifferentiation and tumor heterogeneity. Abnormal DNA methylation, including a decrease in the overall methylation level of the genome, was accompanied by hypermethylation in some gene promoter regions. The hypermethylation of tumor suppressor gene promoter can reduce its expression, while the hypomethylation of tumor suppressor gene promoter can increase its expression, leading to tumorigenesis [5–8]. The following will describe the state of aberrant DNA methylation in different thyroid cancers.

#### **2. DNA methylation in PTC**

RET/PTC rearrangement and mutations in Ras and BRAF genes often occur in papillary thyroid carcinoma (PTC) [9]. In addition, many methylation of cancer suppressor genes are associated with BRAF gene mutations, such as Ras-association domain family 1A (RASSF1A), solute carrier family 5 member 8 gene (SLC5A8), retinoic acid receptor β2 (RARβ2), tissue inhibitor of metalloproteinase3 (TIMP3), phosphatase and tensin homolog deleted on chromosome ten (PTEN), metallothionein 1G (MT1G), ataxia-telangiectasia mutated (ATM), E-cadherin (ECAD), death-associated protein kinase (DAPK), multiple tumor suppressor 1 (MTS1 or P16), and mut-L homolog 1 (MLH1). Mutations of TSHR gene are not common in thyroid cancer, but high methylation and low expression of TSHR gene often occur. Recent studies have found that thyroid-specific genes (thyroidstimulating hormone receptor and sodium/iodide symporter (TSHR and NIS), thyroid transcription factor-1 (TTF-1)) play an important role in occurrence and development of PTC. This part summarizes the related research on methylation genes in PTC in recent years (see **Table 1**).

#### **2.1 DNA methylation of cancer suppressor genes in PTC**

#### *2.1.1 Ras association domain family 1 (RASSF1A)*

RASSF1A is a member of Ras superfamily, which is located at chromosome 3 (exactly on 3p21.3). Hypermethylation of CpG islands in the RASSF1A promoter region contributes to epigenetic inactivation. It is a tumor suppressor gene widely **Sample sources**

**99**

*Tumor suppressor genes* Patient tissue, normal

Western blotting analyses, RT-QPCR,

RASSF1A

SLC5A8

RARβ2

TIMP3

 High

 High

 High

 High

Low

Low

Low

Low

Low

Low

Low

Low

 +

Wnt/βcatenin

Mediate the adhesion of cells

 +

 +

 +

PI3K/Akt

 Inhibit PI3K/Akt pathway

Metallothionein

Regulate cell cycle

 +

 +

 +

 +

MAPK

 Stabilize the

Sodium transporter

Negative regulation of cell cycle

Inhibitor of

metalloproteinase

 [23]

*Research Progress of DNA Methylation in Thyroid Cancer*

 [26] [28]

[29]

 [8]

microtubules

 [12, 13]

[17]

 [20]

*DOI: http://dx.doi.org/10.5772/intechopen.91048*

fluorescent

 analysis

people tissue

Patient tissue, normal

Methylation-specific

 PCR, RT-QPCR

 PTEN MT1G

ATM

 High

 High

 High

> people tissue

Patient tissue, normal

Database query and

medical chart, RT-PCR

retrospective

ECAD

 High

> people tissue

*Thyroid-specific*

Patient tissue, normal

Western blotting analyses, RT-QPCR

 NIS TSHR

TTF-1

 High

 High

 High

Low

Low

Low

 +

 +

 +

TSHR/

Sodium transporter

[36]

[37]

cAMP

Thyrotropin

Inhibit the

response to pioglitazone

pro-adipogenic

[40]

 receptor

people tissue

*Other potential genes*

Patient tissue, thyroid

Western blotting analyses, RT-QPCR,

RASSF10

14-3-3σ

RIZ1

DACT2

 High

–

 –

 High

–

 –

High

–

 –

 High

–

 –

Others

 Inhibit cell Cell cycle regulation Epigenetic mechanisms

Inhibiting Wnt signaling

proliferation

[42]

[45]

[44]

[45]

bisulfite sequencing,

 and

methylation-specific

 PCR

cancer cell line

**Table 1.** *Aberrant methylated*

 *genes in PTC.*

 *genes*

 **Main method**

**Related**

**Methylation**

**Related gene**

**BRAF**

**Cell**

**Function**

**References**

**expression**

**mutation**

**pathway**

**(high/low)**

**(+/**

**)**

**(high/low)**

**gene**


#### *Research Progress of DNA Methylation in Thyroid Cancer DOI: http://dx.doi.org/10.5772/intechopen.91048*

genetic markers of early detection for TC. Recently, epigenetic alterations have been shown to play a role in the development and progression of thyroid cancer. With the deepening of tumor research, it has been gradually found that epige-

netics plays an important role in the occurrence and development of tumors. Mechanisms of epigenetics include, but are not limited to, DNA methylation (methylating of ciliary carbon at position 5), posttranslation modification of histone, chromatin remodeling (structural change), gene imprinting, RNA interference (noncoding RNA or gene silencing), etc. The epigenetic mechanisms of tumor cells have lost a fine regulation, and the breakdown of epigenetic patterns will lead to tumor phenotype expression. These mechanisms have been reviewed elsewhere,

DNA methylation is an important epigenetic change, which is persistent and hereditary. The methylation of promoter CpG can regulate gene expression and maintain chromosome integrity and DNA recombination. Based on 789 samples from the Gene Expression Omnibus (GEO) and the Cancer Genome Atlas (TCGA) databases, the five-CpG signature could provide a novel biomarker with useful applications in thyroid cancer (PTC, FTC, ATC, and MTC) diagnosis and the diagnostic score formula on the condition of DNA methylation data [4]. However, the methylation pattern is unstable and can be reversed by small molecules and endogenous enzymes, leading to dedifferentiation and tumor heterogeneity. Abnormal DNA methylation, including a decrease in the overall methylation level of the genome, was accompanied by hypermethylation in some gene promoter regions. The hypermethylation of tumor suppressor gene promoter can reduce its expression, while the hypomethylation of tumor suppressor gene promoter can increase its expression, leading to tumorigenesis [5–8]. The following will describe

RET/PTC rearrangement and mutations in Ras and BRAF genes often occur in papillary thyroid carcinoma (PTC) [9]. In addition, many methylation of cancer suppressor genes are associated with BRAF gene mutations, such as Ras-association domain family 1A (RASSF1A), solute carrier family 5 member 8 gene (SLC5A8), retinoic acid receptor β2 (RARβ2), tissue inhibitor of metalloproteinase3 (TIMP3),

and here we will focus on DNA methylation in thyroid cancer.

the state of aberrant DNA methylation in different thyroid cancers.

phosphatase and tensin homolog deleted on chromosome ten (PTEN),

metallothionein 1G (MT1G), ataxia-telangiectasia mutated (ATM), E-cadherin (ECAD), death-associated protein kinase (DAPK), multiple tumor suppressor 1 (MTS1 or P16), and mut-L homolog 1 (MLH1). Mutations of TSHR gene are not common in thyroid cancer, but high methylation and low expression of TSHR gene often occur. Recent studies have found that thyroid-specific genes (thyroidstimulating hormone receptor and sodium/iodide symporter (TSHR and NIS), thyroid transcription factor-1 (TTF-1)) play an important role in occurrence and development of PTC. This part summarizes the related research on methylation

RASSF1A is a member of Ras superfamily, which is located at chromosome 3 (exactly on 3p21.3). Hypermethylation of CpG islands in the RASSF1A promoter region contributes to epigenetic inactivation. It is a tumor suppressor gene widely

**2. DNA methylation in PTC**

*DNA Methylation Mechanism*

genes in PTC in recent years (see **Table 1**).

*2.1.1 Ras association domain family 1 (RASSF1A)*

**98**

**2.1 DNA methylation of cancer suppressor genes in PTC**

**Table 1.** *Aberrant methylated*

 *genes in PTC.*

expressed in various normal organs but is often deleted in tumors. It is speculated that BRAFV600E gene mutation in PTC regulated the RASSF1A-MST1-FoxO3 signaling pathway, which led to RASSF1A hypomethylation and affected the malignant degree of thyroid cancer. It is found that the methylation rate of RASSF1A in thyroid cancer is 15–75% [10]. Studies indicated RASSF1A methylation differed in PTC compared with normal thyroid and was correlated with extracapsular invasion inversely. It suggested that RASSF1A has a potential role as a molecular marker for characterization of PTC histopathology [11–15]. It is shown that hypermethylation of RASSF1A promoter region is 20–32% in PTC. Hypermethylation of RASSF1A in PTC was related to the multifocal and extracapsular invasion of tumors [16].

*2.1.5 Phosphatase and tensin homolog deleted on chromosome 10 (PTEN)*

*Research Progress of DNA Methylation in Thyroid Cancer*

*DOI: http://dx.doi.org/10.5772/intechopen.91048*

*2.1.6 Metallothionein 1G (MT1G)*

*2.1.7 Ataxia-telangiectasia mutated*

*2.1.8 E-cadherin*

**101**

The PTEN gene is located on chromosome 10 (especially on the region of 10q23), which could encode a specific phosphatidylinositol triphosphate 3 dephosphorylation and inhibit the activation of PI3K/Akt signaling pathway. PTEN expression alteration is crucial to the pathogenesis of cancer and other diseases. Low level of PTEN caused by homozygous deletions, frameshift, nonsense mutations or hypermethylation, or PTEN protein destability occurs frequently in various human cancers [24, 25]. It was shown that PTEN gene expression was low in thyroid cancer. One study found the methylation status of PTEN in FA, FTC, and PTC. The results indicated that PTEN methylation level was gradually increased in PTC (45.7%), FA (83.3%), and FTC (85.7%). PTEN methylation was related to mutations genes in PI3K/Akt signaling pathway, such as PIK3CA and Ras genes, suggesting that PTEN methylation and PI3K/Akt signaling pathway played an important role in the process of occurrence and development in PTC [26].

MT1G, a member of the metallothionein family, is a highly conserved cysteinerich small molecule, which is mainly involved in metal-related transport. MT1G exists in normal cells; it can regulate and maintain intracellular metal ion balance, cell proliferation, and apoptosis. MT1G promoter methylation is associated with decreasing gene expression, but not complete abrogation. Studies have shown that MT1G gene has abnormal methylation in thyroid cancer, liver cancer, colon cancer, and prostate cancer. It is confirmed that restoring MT1G gene expression could inhibit tumors growth in vivo and in vitro, suggesting that MT1G gene has anticancer effect [27]. It is shown that MT1G gene was abnormally methylated in thyroid cancer (30.3% in malignant tumors and 18.8% in benign tumors). Its expression was significantly decreased, and that methylation of MT1G gene was associated with its low expression. Further studies suggested that restoring MT1G gene expression could inhibit the growth and infiltration of PTC and induced cell cycle inhibition and apoptosis. The mechanism may inhibit PI3K/AKT pathway. In addition, hypermethylation of MT1G was also associated with lymph node metastasis [28].

ATM belongs to the PI3/PI4 enzyme family. Although there is emerging evidence for a role of ATM in promoting tumorigenesis, ATM signaling provides a barrier to activated oncogenes and tumor progression, rather than promoting cancer early in tumorigenesis. ATM is ubiquitous in human and other higher animal tissues and cells, such as testicular tissue. And a study showed that ATM was hypermethylated in PTC (50%) and 0% in normal thyroid tissue [29].

ECAD is a family of transmembrane glycoproteins responsible for calciumdependent cell adhesion. It is the key structural components of adherens junctions. It is reported that ECAD may also act as a gene transcriptional regulator. Two main mechanisms including hypermethylation of the promoter and microRNA imbalance have been widely studied under the ECAD regulation in head and neck tumors. The methylation of ECAD promoter region was accounted for 39.3% in PTC, and ECAD expression decreased in the early stage of tumorigenesis. The experiment data showed that methylation level of ECAD in thyroid cancer increased to 56% (18/32)

#### *2.1.2 Solute carrier gene family 5A, member 8 (SLC5A8)*

SLC5A8 is a passive iodine transporter located in the parietal membrane of thyroid follicular cells. SLC5A8 is not regulated by thyrotropin in normal thyroid tissues but methylated in thyroid tumors. Hypermethylation often occurred in the first exon of CpG islands in SLC5A8, which results in gene silencing and restoring expression inhibiting cancer cell growth. It has been pointed out that SLC5A8 was an anti-oncogene of colon cancer [17]. SLC5A8 was also frequently hypermethylated in thyroid cancer. Its function is unclear nowadays, but its hypermethylation might play a key role in the occurrence of thyroid cancer [18]. Studies revealed that SLC5A8 gene was highly methylated in typical PTC (90%) and only 20% in other types of PTC. In addition, low expression of SLC5A8 was also associated with BRAF T1796A, suggesting that SLC5A8 methylation may be important in MAPK pathway [19].

#### *2.1.3 Retinoic acid receptor beta2*

RARβ2 is a type of nuclear receptor that is activated by both all-trans retinoic acid and 9-cis retinoic acid, which has been shown to function as a tumor suppressor gene in different types of human tumors. It has been found that RARβ2 expression was decreased or deleted in tumors. It meant that RARβ2 inactivation was related to tumorigenesis. In the treatment of metastasis and recurrence of thyroid cancer, retinoic acid therapy could restore the iodine uptake ability of metastasis and then improved the efficacy of 131-I radiotherapy. Researches indicated that the methylation rate of RARβ2 in thyroid cancer was 14%, higher than that in normal thyroid tissues (7%). RARβ2 gene methylation was associated with BRAF gene mutation in Wnt/beta catenin pathway [20]. Studies found there was RARβ2 gene hypermethylation in thyroid cancer cell lines. And after treatment with 5 azacytidine, RARβ2 expression was significantly increased, and the growth of tumors was inhibited, while the inhibition still existed after removing 5-azacytidine. RARβ2 gene methylation took part in tumorigenesis and development in PTC [21].

#### *2.1.4 Tissue inhibitor of metalloproteinases-3 (TIMP3)*

TIMP3 can bind to matrix metalloproteinases (MMPs), inhibiting the activity of MMPs effectively [20, 22]. Methylation of TIMP3 promoter has been demonstrated in many malignant tumors. It is often associated with growth, invasion, and lymph node metastasis of malignant tumors. It is pointed out that BRAF mutation caused low expression of TIMP3 in PTC, which could cause invasion and progression of tumors. It was found that 38% of TIMP3 are hypermethylated in PTC [23].

#### *2.1.5 Phosphatase and tensin homolog deleted on chromosome 10 (PTEN)*

The PTEN gene is located on chromosome 10 (especially on the region of 10q23), which could encode a specific phosphatidylinositol triphosphate 3 dephosphorylation and inhibit the activation of PI3K/Akt signaling pathway. PTEN expression alteration is crucial to the pathogenesis of cancer and other diseases. Low level of PTEN caused by homozygous deletions, frameshift, nonsense mutations or hypermethylation, or PTEN protein destability occurs frequently in various human cancers [24, 25]. It was shown that PTEN gene expression was low in thyroid cancer. One study found the methylation status of PTEN in FA, FTC, and PTC. The results indicated that PTEN methylation level was gradually increased in PTC (45.7%), FA (83.3%), and FTC (85.7%). PTEN methylation was related to mutations genes in PI3K/Akt signaling pathway, such as PIK3CA and Ras genes, suggesting that PTEN methylation and PI3K/Akt signaling pathway played an important role in the process of occurrence and development in PTC [26].

#### *2.1.6 Metallothionein 1G (MT1G)*

expressed in various normal organs but is often deleted in tumors. It is speculated that BRAFV600E gene mutation in PTC regulated the RASSF1A-MST1-FoxO3 signaling pathway, which led to RASSF1A hypomethylation and affected the malignant degree of thyroid cancer. It is found that the methylation rate of RASSF1A in thyroid cancer is 15–75% [10]. Studies indicated RASSF1A methylation differed in PTC compared with normal thyroid and was correlated with extracapsular invasion inversely. It suggested that RASSF1A has a potential role as a molecular marker for characterization of PTC histopathology [11–15]. It is shown that hypermethylation of RASSF1A promoter region is 20–32% in PTC. Hypermethylation of RASSF1A in PTC was related to the multifocal and extracapsular invasion of tumors [16].

SLC5A8 is a passive iodine transporter located in the parietal membrane of thyroid follicular cells. SLC5A8 is not regulated by thyrotropin in normal thyroid tissues but methylated in thyroid tumors. Hypermethylation often occurred in the first exon of CpG islands in SLC5A8, which results in gene silencing and restoring expression inhibiting cancer cell growth. It has been pointed out that SLC5A8 was an anti-oncogene of colon cancer [17]. SLC5A8 was also frequently hypermethylated in thyroid cancer. Its function is unclear nowadays, but its hypermethylation might play a key role in the occurrence of thyroid cancer [18]. Studies revealed that SLC5A8 gene was highly methylated in typical PTC (90%) and only 20% in other types of PTC. In addition, low expression of SLC5A8 was also associated with BRAF T1796A, suggesting that SLC5A8 methylation may be impor-

RARβ2 is a type of nuclear receptor that is activated by both all-trans retinoic acid and 9-cis retinoic acid, which has been shown to function as a tumor suppressor gene in different types of human tumors. It has been found that RARβ2 expression was decreased or deleted in tumors. It meant that RARβ2 inactivation was related to tumorigenesis. In the treatment of metastasis and recurrence of thyroid cancer, retinoic acid therapy could restore the iodine uptake ability of metastasis and then improved the efficacy of 131-I radiotherapy. Researches indicated that the methylation rate of RARβ2 in thyroid cancer was 14%, higher than that in normal thyroid tissues (7%). RARβ2 gene methylation was associated with BRAF gene mutation in Wnt/beta catenin pathway [20]. Studies found there was RARβ2 gene

hypermethylation in thyroid cancer cell lines. And after treatment with 5 azacytidine, RARβ2 expression was significantly increased, and the growth of tumors was inhibited, while the inhibition still existed after removing 5-azacytidine. RARβ2 gene methylation took part in tumorigenesis and development in PTC [21].

TIMP3 can bind to matrix metalloproteinases (MMPs), inhibiting the activity of MMPs effectively [20, 22]. Methylation of TIMP3 promoter has been demonstrated in many malignant tumors. It is often associated with growth, invasion, and lymph node metastasis of malignant tumors. It is pointed out that BRAF mutation caused low expression of TIMP3 in PTC, which could cause invasion and progression of tumors. It was found that 38% of TIMP3 are hypermethylated in PTC [23].

*2.1.4 Tissue inhibitor of metalloproteinases-3 (TIMP3)*

*2.1.2 Solute carrier gene family 5A, member 8 (SLC5A8)*

tant in MAPK pathway [19].

*DNA Methylation Mechanism*

*2.1.3 Retinoic acid receptor beta2*

**100**

MT1G, a member of the metallothionein family, is a highly conserved cysteinerich small molecule, which is mainly involved in metal-related transport. MT1G exists in normal cells; it can regulate and maintain intracellular metal ion balance, cell proliferation, and apoptosis. MT1G promoter methylation is associated with decreasing gene expression, but not complete abrogation. Studies have shown that MT1G gene has abnormal methylation in thyroid cancer, liver cancer, colon cancer, and prostate cancer. It is confirmed that restoring MT1G gene expression could inhibit tumors growth in vivo and in vitro, suggesting that MT1G gene has anticancer effect [27]. It is shown that MT1G gene was abnormally methylated in thyroid cancer (30.3% in malignant tumors and 18.8% in benign tumors). Its expression was significantly decreased, and that methylation of MT1G gene was associated with its low expression. Further studies suggested that restoring MT1G gene expression could inhibit the growth and infiltration of PTC and induced cell cycle inhibition and apoptosis. The mechanism may inhibit PI3K/AKT pathway. In addition, hypermethylation of MT1G was also associated with lymph node metastasis [28].

#### *2.1.7 Ataxia-telangiectasia mutated*

ATM belongs to the PI3/PI4 enzyme family. Although there is emerging evidence for a role of ATM in promoting tumorigenesis, ATM signaling provides a barrier to activated oncogenes and tumor progression, rather than promoting cancer early in tumorigenesis. ATM is ubiquitous in human and other higher animal tissues and cells, such as testicular tissue. And a study showed that ATM was hypermethylated in PTC (50%) and 0% in normal thyroid tissue [29].

#### *2.1.8 E-cadherin*

ECAD is a family of transmembrane glycoproteins responsible for calciumdependent cell adhesion. It is the key structural components of adherens junctions. It is reported that ECAD may also act as a gene transcriptional regulator. Two main mechanisms including hypermethylation of the promoter and microRNA imbalance have been widely studied under the ECAD regulation in head and neck tumors. The methylation of ECAD promoter region was accounted for 39.3% in PTC, and ECAD expression decreased in the early stage of tumorigenesis. The experiment data showed that methylation level of ECAD in thyroid cancer increased to 56% (18/32)

and 0% (0/27) in normal thyroid tissue. Further studies found that there was no significant correlation between ECAD methylation and T stage of lymph node metastasis in thyroid cancer. After 2.6 years of follow-up, the recurrence of thyroid cancer associated with ECAD methylation has no correlation [8].

include TSHR, NIS, thyroglobulin (Tg), and thyroid peroxidase (TPO), which participate in thyroid iodine uptake and maintenance of normal thyroid function. Under normal conditions, TSH stimulates TSHR on thyroid follicular epithelial cells and activates NIS to ingest iodine into cells. TSH is produced by the pituitary thyrotrophs and stimulates thyroid functions using TSHR. The iodine ingested synthesizes thyroid hormones catalyzed by TPO and is stored in Tg. It is found that abnormal expression of these molecules is related to iodine metabolism in PTC. Studies have shown that the expression of TSHR, NIS, Tg, and TPO with BRAF mutated in thyroid cancer is decreased [36]. In PTC, both TSHR and NIS are abnormally methylated, and their expression is decreased. Low expression of TSHR and NIS may be related to the occurrence and development of tumors. It also reduces the uptake of iodine capacity in tumor cells. Scholars found TSHR and NIS become an important cause for PTC in 131-I radiotherapy [37, 38]. In human and rabbit thyroid cancer cells, BRAFV600E mutant, a carcinogenic homolog of murine sarcomatous virulent bacterium, could cause activation of BRAF/MEK/ MAPK signaling pathway and expression silencing of thyroid-specific genes including TPO, Tg, TSHR, and NIS. At last, it resulted in the reduction of iodine

*Research Progress of DNA Methylation in Thyroid Cancer*

*DOI: http://dx.doi.org/10.5772/intechopen.91048*

TTF-1 is known as thyroid-specific enhancer-binding protein (T/EBP). It is a transcription factor with homologous domains in the thyroid, lung, and central nervous system. TTF-1 gene is located in region 14q13.3 on chromosome 14. It is comprised of three exons and two introns. Under physiological conditions, TTF-1 is stable positive in thyroid tissue. TTF-1 can regulate the expression of thyroidrelated genes such as TG, TPO, TSHR, and NIS. Thus it acts a pivotal part in regulating growth, development, and function of thyroid. It showed hNIS mRNA expression loss might be related to methylation of thyroid-specific transcription factor genes. Abnormal methylation caused loss of transcription factor expression with indirect loss of hNIS mRNA expression through the KAT-5 and KAT-10 responses to 5-azacytidine treatment with acquisition of parallel TTF-1 and hNIS mRNA expression. It was found that insufficient expression of TTF-1 and Pax-8 may result in the decrease of activity of thyroglobulin gene promoter in thyroid cancer cells. Some confirmed that TTF-1 gene was expressed lowly in thyroid cancer [39]. Other researchers studied the methylation status of TTF-1 in thyroid cancer and found that TTF-1 gene was highly methylated and lowly expressed in

At present, BRAF gene mutation has been found in melanoma, ovarian serous tumor, colorectal cancer, glioma, liver cancer, and leukemia. A large number of studies also showed that BRAF gene mutation in PTC is closely related to methylation of tumor suppressor. BRAF gene is an important transducer for Ras/Raf/MEK/ ERK/MAPK pathways. About 90% of T1799A point mutation in BRAFV600E happen in PTC, which can increase BRAF activity. The BRAF gene plays its biological role by activating MEK/ERK signaling pathway. The result showed that PTC with BRAF gene mutation had strong tissue invasiveness and was easy to infiltrate tissues around thyroid gland [41]. Methylation of TIMP3 gene, SLC5A8 gene, and DAPK gene, which are tumor suppressor genes, are related to BRAF gene mutation and PTC invasion. The overexpression of BRAFV600E gene could lead to silencing of some thyroid-specific genes (like NIS, TG, TPO) in the activation state of

uptake in PTC.

**103**

*2.2.2 Thyroid transcription factor-1*

thyroid cancer cell lines, but not in normal thyroid [40].

**2.3 DNA methylation of oncogene gene in PTC**

#### *2.1.9 Death-associated protein kinase*

DAPK is a calmodulin-regulated ATK, which has an important role in the process of apoptosis. DAPK mechanism is largely due to promoter hypermethylation, leading to gene silencing. DAPK is ubiquitous in normal tissues. When the promoter of DAPK is methylated, it will cause an abnormal gene expression. Abnormal expression of DAPK can hinder the normal process of apoptosis and bring about tumorigenesis. And its low expression or deletion is one of the important mechanisms of cell carcinogenesis. It has been found that methylation of CpG island in the promoter region in DAPK is an important reason for expression silencing. The loss of DAPK protein in sporadic colorectal cancer is caused by the promoter hypermethylation. It existed in very small tumors. Therefore, the loss of DAPK gene plays an important role in the early stage of tumor formation. It is reported that DAPK promoter methylation accounts for 51% in PTC and abnormal methylation and DAPK gene silencing existed in many kinds of cancer cells [30]. It is pointed out that the high methylation level of DAPK gene was associated with tumor size and multiple lesions [31].

#### *2.1.10 Multiple tumor suppressor 1 (MTS1 or P16)*

MTS1 is hereafter called p16. It is an anti-oncogene in many tumors. The 5<sup>0</sup> -CpG fragment in the promoter region of P16 gene is the most susceptible to methylation, inhibiting its expression products. Abnormal expression of P16 gene can overactivate cyclin-dependent protein kinase 4 and stimulate abnormal cell proliferation, leading to tumorigenesis. Aberrant methylation of promoter region in P16 gene is the main cause for P16 gene inactivation [32]. Some scholars reported that the P16 gene was hypermethylated (35.9%) in PTC [33].

#### *2.1.11 Mut-L homolog 1*

MLH1 is one of the DNA mismatch repair genes located on the 3p21 region in chromosome 3. A correlation between MLH1 promoter methylation, specifically the 'C' region stops in MLH1 protein formation, can prevent the normal activation of DNA repair gene. Low expression of MLH1 gene was associated with BRAFV600E mutation and RET/PTC rearrangement. Hypermethylation of MLH1 promoter was found in colon cancer as well. A study indicated that abnormal methylation of MLH1 was significantly correlated with lymph node metastasis of PTC, suggesting that MLH1 might be a molecular marker of lymph node metastasis in PTC [34]. Another study found that there were abnormal methylation and low expression of MLH1 in thyroid cancer and MLH1 expression is associated with BRAF, IDH1, and NRAS gene mutations [35].

#### **2.2 DNA methylation of thyroid-specific genes in PTC**

#### *2.2.1 Thyroid-stimulating hormone receptor and sodium/iodide symporter*

On the other hand, methylation of thyroid-specific genes is also one of the causes for occurrence and development of PTC. Thyroid-specific genes mainly

#### *Research Progress of DNA Methylation in Thyroid Cancer DOI: http://dx.doi.org/10.5772/intechopen.91048*

and 0% (0/27) in normal thyroid tissue. Further studies found that there was no significant correlation between ECAD methylation and T stage of lymph node metastasis in thyroid cancer. After 2.6 years of follow-up, the recurrence of thyroid

DAPK is a calmodulin-regulated ATK, which has an important role in the process of apoptosis. DAPK mechanism is largely due to promoter hypermethylation, leading to gene silencing. DAPK is ubiquitous in normal tissues. When the promoter of DAPK is methylated, it will cause an abnormal gene expression. Abnormal expression of DAPK can hinder the normal process of apoptosis and bring about tumorigenesis. And its low expression or deletion is one of the important mechanisms of cell carcinogenesis. It has been found that methylation of CpG island in the promoter region in DAPK is an important reason for expression silencing. The loss

hypermethylation. It existed in very small tumors. Therefore, the loss of DAPK gene plays an important role in the early stage of tumor formation. It is reported that DAPK promoter methylation accounts for 51% in PTC and abnormal methylation and DAPK gene silencing existed in many kinds of cancer cells [30]. It is pointed out that the high methylation level of DAPK gene was associated with tumor size and

MTS1 is hereafter called p16. It is an anti-oncogene in many tumors. The 5<sup>0</sup>

fragment in the promoter region of P16 gene is the most susceptible to methylation, inhibiting its expression products. Abnormal expression of P16 gene can overactivate cyclin-dependent protein kinase 4 and stimulate abnormal cell proliferation, leading to tumorigenesis. Aberrant methylation of promoter region in P16 gene is the main cause for P16 gene inactivation [32]. Some scholars reported that

MLH1 is one of the DNA mismatch repair genes located on the 3p21 region in chromosome 3. A correlation between MLH1 promoter methylation, specifically the 'C' region stops in MLH1 protein formation, can prevent the normal activation of DNA repair gene. Low expression of MLH1 gene was associated with BRAFV600E mutation and RET/PTC rearrangement. Hypermethylation of MLH1 promoter was found in colon cancer as well. A study indicated that abnormal methylation of MLH1 was significantly correlated with lymph node metastasis of PTC, suggesting that MLH1 might be a molecular marker of lymph node metastasis in PTC [34]. Another study found that there were abnormal methylation and low expression of MLH1 in thyroid cancer and MLH1 expression is associated with BRAF, IDH1, and


cancer associated with ECAD methylation has no correlation [8].

of DAPK protein in sporadic colorectal cancer is caused by the promoter

*2.1.9 Death-associated protein kinase*

*DNA Methylation Mechanism*

multiple lesions [31].

*2.1.11 Mut-L homolog 1*

NRAS gene mutations [35].

**102**

*2.1.10 Multiple tumor suppressor 1 (MTS1 or P16)*

the P16 gene was hypermethylated (35.9%) in PTC [33].

**2.2 DNA methylation of thyroid-specific genes in PTC**

*2.2.1 Thyroid-stimulating hormone receptor and sodium/iodide symporter*

On the other hand, methylation of thyroid-specific genes is also one of the causes for occurrence and development of PTC. Thyroid-specific genes mainly include TSHR, NIS, thyroglobulin (Tg), and thyroid peroxidase (TPO), which participate in thyroid iodine uptake and maintenance of normal thyroid function. Under normal conditions, TSH stimulates TSHR on thyroid follicular epithelial cells and activates NIS to ingest iodine into cells. TSH is produced by the pituitary thyrotrophs and stimulates thyroid functions using TSHR. The iodine ingested synthesizes thyroid hormones catalyzed by TPO and is stored in Tg. It is found that abnormal expression of these molecules is related to iodine metabolism in PTC. Studies have shown that the expression of TSHR, NIS, Tg, and TPO with BRAF mutated in thyroid cancer is decreased [36]. In PTC, both TSHR and NIS are abnormally methylated, and their expression is decreased. Low expression of TSHR and NIS may be related to the occurrence and development of tumors. It also reduces the uptake of iodine capacity in tumor cells. Scholars found TSHR and NIS become an important cause for PTC in 131-I radiotherapy [37, 38]. In human and rabbit thyroid cancer cells, BRAFV600E mutant, a carcinogenic homolog of murine sarcomatous virulent bacterium, could cause activation of BRAF/MEK/ MAPK signaling pathway and expression silencing of thyroid-specific genes including TPO, Tg, TSHR, and NIS. At last, it resulted in the reduction of iodine uptake in PTC.

#### *2.2.2 Thyroid transcription factor-1*

TTF-1 is known as thyroid-specific enhancer-binding protein (T/EBP). It is a transcription factor with homologous domains in the thyroid, lung, and central nervous system. TTF-1 gene is located in region 14q13.3 on chromosome 14. It is comprised of three exons and two introns. Under physiological conditions, TTF-1 is stable positive in thyroid tissue. TTF-1 can regulate the expression of thyroidrelated genes such as TG, TPO, TSHR, and NIS. Thus it acts a pivotal part in regulating growth, development, and function of thyroid. It showed hNIS mRNA expression loss might be related to methylation of thyroid-specific transcription factor genes. Abnormal methylation caused loss of transcription factor expression with indirect loss of hNIS mRNA expression through the KAT-5 and KAT-10 responses to 5-azacytidine treatment with acquisition of parallel TTF-1 and hNIS mRNA expression. It was found that insufficient expression of TTF-1 and Pax-8 may result in the decrease of activity of thyroglobulin gene promoter in thyroid cancer cells. Some confirmed that TTF-1 gene was expressed lowly in thyroid cancer [39]. Other researchers studied the methylation status of TTF-1 in thyroid cancer and found that TTF-1 gene was highly methylated and lowly expressed in thyroid cancer cell lines, but not in normal thyroid [40].

#### **2.3 DNA methylation of oncogene gene in PTC**

At present, BRAF gene mutation has been found in melanoma, ovarian serous tumor, colorectal cancer, glioma, liver cancer, and leukemia. A large number of studies also showed that BRAF gene mutation in PTC is closely related to methylation of tumor suppressor. BRAF gene is an important transducer for Ras/Raf/MEK/ ERK/MAPK pathways. About 90% of T1799A point mutation in BRAFV600E happen in PTC, which can increase BRAF activity. The BRAF gene plays its biological role by activating MEK/ERK signaling pathway. The result showed that PTC with BRAF gene mutation had strong tissue invasiveness and was easy to infiltrate tissues around thyroid gland [41]. Methylation of TIMP3 gene, SLC5A8 gene, and DAPK gene, which are tumor suppressor genes, are related to BRAF gene mutation and PTC invasion. The overexpression of BRAFV600E gene could lead to silencing of some thyroid-specific genes (like NIS, TG, TPO) in the activation state of

BRAF/MEK/MAPK pathway. It could cause iodine uptake activity decrease and ineffectiveness of radioiodine therapy. Therefore, it could affect thyroid cancer progression.

**3.1 DNA methylation of tumor suppressor genes in FTC**

*Research Progress of DNA Methylation in Thyroid Cancer*

*DOI: http://dx.doi.org/10.5772/intechopen.91048*

PTEN negatively regulates AKT/PKB signaling pathway. It is involved in regulation of cell cycle, cell growth inhibition, and rapid division [47]. Aberrant DNA methylation in this gene is also mostly reported in FTC. It confirmed that PTEN promoter hypermethylation was detected in six of seven (85%) FTC and five of six (83.3%) follicular adenomas. The results showed a high frequency of PTEN promoter hypermethylation, especially in follicular tumors. It means that it has a possible role in thyroid cancer [25]. Studies found methylation status of PTEN in FA and FTC. And PTEN methylation level was gradually increased in FA (83.3%) and FTC (85.7%). Authors have shown that methylation of PTEN promoter plays an

Differential expression of RASSF1A gene is related to occurrence of thyroid cancer. Aberrant DNA methylation is an important mechanism of RASSF1A gene inactivation. Studies found 44% benign adenomas, 75% follicular thyroid cancers tumors, and 20% PTC tumors harbored promoter methylation in greater than or equal to 25% of RASSF1A alleles by real-time quantitative methylation-specific PCR [39–42]. Methylation frequency was higher in invasive thyroid cancer. It was found to be 70% of the RASSF1A methylation rate in FTC, 80% in MTC, and 78% in UTC compared with benign PTC [14]. Inactivation of RASSF1A in different stages of thyroid cancer was detected by tumor metastasis classification, and compared with FTC, only a small part of RASSF1A methylation in PTC is abnormal. These studies indicated follicular cell-derived thyroid tumorigenesis may be an early step [15, 49].

RAP1GAP gene encodes a type of GTPase-activating protein that downregulates Ras-related protein activity. Ras oncoproteins are very important for both development and maintenance of many tumor types. RAP1GAP is involved in the regulation of mitosis and carcinogenesis in thyroid cells. Researchers aimed to determine the global patterns of aberrant DNA methylation in thyroid cancer using DNA methylation arrays [50]. And the study identified 262 and 352 hypermethylated and 13 and 21 hypomethylated genes in PTC and FTC, respectively. In addition, 86 and 131 hypermethylated genes were identified. Among these genes, four potential oncogenes (INSL4, DPPA2, TCL1B, and NOTCH4) were frequently regulated by

Besides, a member of the serine protease inhibitor superfamily named Maspin is a unique tumor suppressor gene encoding SERPINB5 gene. Epigenetic changes of Maspin expression occurred in the 5<sup>0</sup> regulatory region of Maspin gene and involved cytosine methylation, histone deacetylation, and chromatin accessibility. The epigenetic deregulation frequently participates in tumorigenesis by inactivation of tumor suppressor genes. The association of promoter hypermethylation and gene silencing is an established oncogenic process in cancer. Promoter methylation of Maspin gene could lead to gene silencing in thyroid cancer, breast cancer, skin cancer, and colon cancer. Studies have indicated that overexpression of Maspin in

*3.1.1 PTEN*

important role in FTC [48].

*3.1.3 Rap1-GTPase-activating protein*

aberrant methylation in primary thyroid tumors [51].

*3.1.4 Mammary serine protease inhibitor (Maspin)*

*3.1.2 RASSF1A*

**105**

#### **2.4 Other potentially aberrant methylation genes in PTC**

In recent years, DNA methylation has become a new research hotspot. Many genes have been studied as potential abnormal methylation sites, including Ras association domain family 2 (RASSF2), Ras-association domain family 10 (RASSF10), disheveled-binding antagonist of beta-catenin 2 (DACT2), retinoblastoma protein-interacting zinc finger gene 1 (RIZ1), 14-3-3 protein family (14-3-3 sigma), and other signaling pathways such as JAK-STAT pathway, NF-kappa B pathway, HIF1 alpha pathway, and Notch pathway in thyroid cancer [42–45]. However, there are relatively few reports on these genes and pathways in thyroid cancer. Most of the mechanisms are not clear at present and need further study.

#### **3. DNA methylation in FTC**

DNA methylation analysis revealed 2130 and 19 differentially methylated CpGs in PTC and follicular thyroid carcinoma (FTC), respectively [46]. Aberrant DNA methylation of tumor suppressor genes is common in FTC and ATC. Certain specific tumor suppressor genes are mainly PTEN, RASSF1A, Rap1-GTPase activating protein, and thyroid-specific gene TRSH in FTC. This part summarizes the related research on methylation genes in FTC in recent years (see **Table 2**).


**Table 2.** *Aberrant methylated genes in FTC.*

#### **3.1 DNA methylation of tumor suppressor genes in FTC**

#### *3.1.1 PTEN*

BRAF/MEK/MAPK pathway. It could cause iodine uptake activity decrease and ineffectiveness of radioiodine therapy. Therefore, it could affect thyroid cancer

In recent years, DNA methylation has become a new research hotspot. Many genes have been studied as potential abnormal methylation sites, including Ras association domain family 2 (RASSF2), Ras-association domain family 10

(RASSF10), disheveled-binding antagonist of beta-catenin 2 (DACT2), retinoblastoma protein-interacting zinc finger gene 1 (RIZ1), 14-3-3 protein family (14-3-3 sigma), and other signaling pathways such as JAK-STAT pathway, NF-kappa B pathway, HIF1 alpha pathway, and Notch pathway in thyroid cancer [42–45]. However, there are relatively few reports on these genes and pathways in thyroid cancer. Most of the mechanisms are not clear at present and need further study.

DNA methylation analysis revealed 2130 and 19 differentially methylated CpGs in PTC and follicular thyroid carcinoma (FTC), respectively [46]. Aberrant DNA methylation of tumor suppressor genes is common in FTC and ATC. Certain specific tumor suppressor genes are mainly PTEN, RASSF1A, Rap1-GTPase activating protein, and thyroid-specific gene TRSH in FTC. This part summarizes the related

[50] RAP1GAP RAP1GAPase-activating protein ↑ + 38% of FTC

[58] NIS Sodium transporter ↑ + 53.8% of

[52, 53] Maspin A member of serine protease inhibitor ↑ + 100% of

[61] DMCpG — ↑ — 84% of FTC [61] RASAL1 — ↑ — 4.88% of FTC [57] COL4A2 — ↑ — 56% of FTC [64] RASSF10 — ↑ — 50% of FTC

[58] TSHR Thyrotropin receptor ↑ + 47% of FTC

cycle and preventing cells from growing and

**methylation**

**BRAFE Incidence%**

thyroid cancers

WDTC

↑ + 75% of FTC

↑ + 85.7% of FTC

research on methylation genes in FTC in recent years (see **Table 2**).

**References Gene Function DNA**

promotes their stabilization

[39–42] RASSFIA RASSF1A localizes to microtubules and

[25] PTEN PTEN is involved in the regulation of cell

dividing rapidly

**2.4 Other potentially aberrant methylation genes in PTC**

progression.

*DNA Methylation Mechanism*

**3. DNA methylation in FTC**

*Tumor suppressor genes*

*Thyroid-specific genes*

*Oncogene genes*

*Other genes*

**Table 2.**

**104**

*Aberrant methylated genes in FTC.*

PTEN negatively regulates AKT/PKB signaling pathway. It is involved in regulation of cell cycle, cell growth inhibition, and rapid division [47]. Aberrant DNA methylation in this gene is also mostly reported in FTC. It confirmed that PTEN promoter hypermethylation was detected in six of seven (85%) FTC and five of six (83.3%) follicular adenomas. The results showed a high frequency of PTEN promoter hypermethylation, especially in follicular tumors. It means that it has a possible role in thyroid cancer [25]. Studies found methylation status of PTEN in FA and FTC. And PTEN methylation level was gradually increased in FA (83.3%) and FTC (85.7%). Authors have shown that methylation of PTEN promoter plays an important role in FTC [48].

#### *3.1.2 RASSF1A*

Differential expression of RASSF1A gene is related to occurrence of thyroid cancer. Aberrant DNA methylation is an important mechanism of RASSF1A gene inactivation. Studies found 44% benign adenomas, 75% follicular thyroid cancers tumors, and 20% PTC tumors harbored promoter methylation in greater than or equal to 25% of RASSF1A alleles by real-time quantitative methylation-specific PCR [39–42]. Methylation frequency was higher in invasive thyroid cancer. It was found to be 70% of the RASSF1A methylation rate in FTC, 80% in MTC, and 78% in UTC compared with benign PTC [14]. Inactivation of RASSF1A in different stages of thyroid cancer was detected by tumor metastasis classification, and compared with FTC, only a small part of RASSF1A methylation in PTC is abnormal. These studies indicated follicular cell-derived thyroid tumorigenesis may be an early step [15, 49].

#### *3.1.3 Rap1-GTPase-activating protein*

RAP1GAP gene encodes a type of GTPase-activating protein that downregulates Ras-related protein activity. Ras oncoproteins are very important for both development and maintenance of many tumor types. RAP1GAP is involved in the regulation of mitosis and carcinogenesis in thyroid cells. Researchers aimed to determine the global patterns of aberrant DNA methylation in thyroid cancer using DNA methylation arrays [50]. And the study identified 262 and 352 hypermethylated and 13 and 21 hypomethylated genes in PTC and FTC, respectively. In addition, 86 and 131 hypermethylated genes were identified. Among these genes, four potential oncogenes (INSL4, DPPA2, TCL1B, and NOTCH4) were frequently regulated by aberrant methylation in primary thyroid tumors [51].

#### *3.1.4 Mammary serine protease inhibitor (Maspin)*

Besides, a member of the serine protease inhibitor superfamily named Maspin is a unique tumor suppressor gene encoding SERPINB5 gene. Epigenetic changes of Maspin expression occurred in the 5<sup>0</sup> regulatory region of Maspin gene and involved cytosine methylation, histone deacetylation, and chromatin accessibility. The epigenetic deregulation frequently participates in tumorigenesis by inactivation of tumor suppressor genes. The association of promoter hypermethylation and gene silencing is an established oncogenic process in cancer. Promoter methylation of Maspin gene could lead to gene silencing in thyroid cancer, breast cancer, skin cancer, and colon cancer. Studies have indicated that overexpression of Maspin in

gastric cancer, pancreatic cancer, and ovarian cancers resulted from CpG promoter of Maspin demethylation. A study detected DNA methylation status in Maspin promoter region, indicating that overexpression of the gene was the result of DNA hypomethylation [52]. It was closely related to the morphological dedifferentiation of thyroid cancer. Another study found 100% Maspin hypermethylation was closely associated with morphological dedifferentiation in thyroid cancers [52, 53].

thyroid tumors, including PTEN mutations, various subtypes of Ras mutations, PIK3CA mutations, and amplification [66, 67]. One study analyzed 24 genetic alterations in the major genes of MAPK and PI3K-AKT pathways in 48 ATC samples and found that the majority of (81%) samples that harbored genetic alterations could be likely activated in both pathways [68]. Accordingly, another DNA methylation pan-cancer study focused on promoters found that thyroid carcinoma

hypermethylation events. And ATC exhibits a high frequency of DNA methylation alterations (tenfold higher than PTC) [69, 70]. A recent pan-cancer analysis on whole exome sequencing revealed that the mutation frequency in PTC was one of the lowest (approximately 1 change/Mb across the entire exome) among solid tumors, while the mutation frequency in ATC was at the opposite extreme and was closer to that in melanoma and lung cancer, exceeding 100 changes/Mb [71, 72]. In addition, solute carrier family 26, member 4 (SLC26A4) gene, encodes a transmembrane protein named pendrin with up to 15 predicted membrane spanning domains and affects the flow of iodine into follicular lumen. The following were reported: 71% of ATC, 44% of benign tumors, 46% of FTC, and 71% of PTC, with abnormal SLC26A4 gene methylation in 64 cases of primary thyroid tumors

exhibited one of the lowest frequencies in both hypomethylation and

*Research Progress of DNA Methylation in Thyroid Cancer*

*DOI: http://dx.doi.org/10.5772/intechopen.91048*

and 6 cases of thyroid tumor cell lines [18, 73]. In addition, 81.5% of

tors) is frequently overexpressed in thyroid tumors [75, 76].

**Authors Gene Function DNA**

by reciprocal translocations

plays a role in a variety of developmental processes

[70] TCL1B An oncogene frequently activated

[70] NOTCH4 A Member of notch family, which

recent years (see **Table 3**).

*Oncogene genes*

*Thyroid-specific genes*

*Tumor suppressor genes*

*Aberrant methylated genes in ATC.*

**Table 3.**

**107**

hypermethylated genes and 89% of hypomethylated genes were also present in nondifferentiated primary tumors (MTCs and ATCs) and nondifferentiated thyroid carcinoma-derived cancer cell lines [74], while Ras protein activator like-1 gene (RASAL1) displayed MAPK- and PI3K-suppressing and thyroid tumor-suppressing activities, which can be impaired by the mutations. Hypermethylation and mutations of RASAL1 were found in 33.33% (n = 10 of 30) of ATCs and in 0 of 20 (0%) of benign thyroid tumors [62]. However, ATC showed more hypomethylation than hypermethylation events, indicating that hypomethylation is related to dedifferentiation [70]. The authors validated four genes (NOTCH4 and TCL1B in ATCs, INSL4 and DPPA2 in MTCs) that become aberrantly hypomethylated in nondifferentiated thyroid tumors. All of them have been proposed to have an oncogenic role in cancer. And NOTCH4 (a member of the Notch family of transmembrane recep-

This part summarizes the related research on methylation genes in ATC in

[18, 73] SLC26A4 Dysfunctional pendrin ↑ — 71% ATC

[25] PTEN PI3K–AKT pathway ↑ — 81% of ATC [62] RASAL1 MAPK- and PI3K-suppressing ↑ — 33.33% of ATC

**methylation**

**BRAFE Incidence%**

↑ + 64% of ATC

↑ + 45% of ATC

#### **3.2 DNA methylation of thyroid-specific genes in FTC**

In addition to tumor suppressor genes and oncogenes, hypermethylation could lead to NIS and TSHR gene silencing in FTC, too. NIS methylation is of great significance in treatment of thyroid cancer. Therefore, abnormal methylation of these genes may be the pathogenesis or progression factor in FTC [54, 55]. Aberrant promoter methylation was examined in 24 tumor suppressor genes using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay and methylation-specific PCR (MSP). In thyroid cancers, CASP8 (caspase-8), RASSF1, and NIS were methylated in 9/13, 10/13, and 7/13, respectively [56]. Some researches also found combination gene panels TPO and UCHL1 (ROC = 0.607, sensitivity 78%) discriminated FTC from FA and RASSF1 and TPO (ROC = 0.881, sensitivity 78%) discriminated FTC from normal. Methylation of TSHR distinguished PTC from FTC (ROC = 0.701, sensitivity 84%) and PTC from FA (ROC = 0.685, sensitivity 70%) [57]. And the six-gene panel of TIMP3, RARβ2, SERPINB5, RASSF1, TPO, and TSHR, which differentiates PTC from normal thyroid, had the best combination sensitivity (91%) and specificity (81%) of the panels addressing discrimination of cancer tissue by quantitative methylation-specific polymerase chain reaction (QMSP) in a retrospective cohort of 329 patients [58, 59].

#### **3.3 Other abnormal methylation genes in FTC**

Nowadays, there are a few studies on methylation of FTC genes. However, many genes still have been studied as potential abnormal methylation sites in FTC. For example, one of the ZIC families called ZIC1 (C2H2-type zinc finger proteins) is frequently hypermethylated in FTCs [60]. Moreover, 3564 differentially methylated CpGs (DMCpG) were detected in FTC and 84% hypermethylated with respect to normal controls. It is suggested that perturbed DNA methylation, in particular hypermethylation, is a component of the molecular mechanisms leading to FTC formation and that DNA methylation profiling might help in differentiating FTCs from their benign counterpart [61]. Also, others identified the presence of RASAL1 mutations, with a prevalence of 4.88% (n = 2 of 41) in FTC and 16.67% (n = 5 of 30) in ATC [62]. Studies found a more detailed analysis showing that 53.9% of the hypermethylated and 81.5% of the hypomethylated CpG sites identified in differentiated primary tumors (PTCs and FTCs) were also present in differentiated thyroid carcinoma-derived cancer cell [61]. Aside from that, COL4A2 was hypermethylated in 56% of the FTC samples by array measurement in the discovery series [57, 63]. Another study indicated that RASSF10 was frequently hypermethylated in thyroid cancer. It showed 50% of methylation frequency of RASSF10 in FTC and the highest (100%) in MTCs [64].

#### **4. DNA methylation in ATC**

Promoter methylation of PTEN is also common in anaplastic thyroid cancer (ATC) [65]. PTEN methylation is related to gene changes of PI3K Akt pathway in

#### *Research Progress of DNA Methylation in Thyroid Cancer DOI: http://dx.doi.org/10.5772/intechopen.91048*

gastric cancer, pancreatic cancer, and ovarian cancers resulted from CpG promoter of Maspin demethylation. A study detected DNA methylation status in Maspin promoter region, indicating that overexpression of the gene was the result of DNA hypomethylation [52]. It was closely related to the morphological dedifferentiation of thyroid cancer. Another study found 100% Maspin hypermethylation was closely

In addition to tumor suppressor genes and oncogenes, hypermethylation could

Nowadays, there are a few studies on methylation of FTC genes. However, many genes still have been studied as potential abnormal methylation sites in FTC. For example, one of the ZIC families called ZIC1 (C2H2-type zinc finger proteins) is frequently hypermethylated in FTCs [60]. Moreover, 3564 differentially methylated CpGs (DMCpG) were detected in FTC and 84% hypermethylated with respect to normal controls. It is suggested that perturbed DNA methylation, in particular hypermethylation, is a component of the molecular mechanisms leading to FTC formation and that DNA methylation profiling might help in differentiating FTCs from their benign counterpart [61]. Also, others identified the presence of RASAL1 mutations, with a prevalence of 4.88% (n = 2 of 41) in FTC and 16.67% (n = 5 of 30) in ATC [62]. Studies found a more detailed analysis showing that 53.9% of the hypermethylated and 81.5% of the hypomethylated CpG sites identified in differentiated primary tumors (PTCs and FTCs) were also present in differentiated thyroid carcinoma-derived cancer cell [61]. Aside from that, COL4A2 was

hypermethylated in 56% of the FTC samples by array measurement in the discovery

hypermethylated in thyroid cancer. It showed 50% of methylation frequency of

Promoter methylation of PTEN is also common in anaplastic thyroid cancer (ATC) [65]. PTEN methylation is related to gene changes of PI3K Akt pathway in

series [57, 63]. Another study indicated that RASSF10 was frequently

RASSF10 in FTC and the highest (100%) in MTCs [64].

**4. DNA methylation in ATC**

**106**

associated with morphological dedifferentiation in thyroid cancers [52, 53].

lead to NIS and TSHR gene silencing in FTC, too. NIS methylation is of great significance in treatment of thyroid cancer. Therefore, abnormal methylation of these genes may be the pathogenesis or progression factor in FTC [54, 55]. Aberrant

promoter methylation was examined in 24 tumor suppressor genes using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay and methylation-specific PCR (MSP). In thyroid cancers, CASP8 (caspase-8), RASSF1, and NIS were methylated in 9/13, 10/13, and 7/13, respectively [56]. Some researches also found combination gene panels TPO and UCHL1 (ROC = 0.607, sensitivity 78%) discriminated FTC from FA and RASSF1 and TPO (ROC = 0.881, sensitivity 78%) discriminated FTC from normal. Methylation of TSHR distinguished PTC from FTC (ROC = 0.701, sensitivity 84%) and PTC from FA (ROC = 0.685, sensitivity 70%) [57]. And the six-gene panel of TIMP3, RARβ2, SERPINB5, RASSF1, TPO, and TSHR, which differentiates PTC from normal thyroid, had the best combination sensitivity (91%) and specificity (81%) of the panels addressing discrimination of cancer tissue by quantitative methylation-specific polymerase chain reaction (QMSP) in a retrospective cohort of 329 patients [58, 59].

**3.2 DNA methylation of thyroid-specific genes in FTC**

*DNA Methylation Mechanism*

**3.3 Other abnormal methylation genes in FTC**

thyroid tumors, including PTEN mutations, various subtypes of Ras mutations, PIK3CA mutations, and amplification [66, 67]. One study analyzed 24 genetic alterations in the major genes of MAPK and PI3K-AKT pathways in 48 ATC samples and found that the majority of (81%) samples that harbored genetic alterations could be likely activated in both pathways [68]. Accordingly, another DNA methylation pan-cancer study focused on promoters found that thyroid carcinoma exhibited one of the lowest frequencies in both hypomethylation and hypermethylation events. And ATC exhibits a high frequency of DNA methylation alterations (tenfold higher than PTC) [69, 70]. A recent pan-cancer analysis on whole exome sequencing revealed that the mutation frequency in PTC was one of the lowest (approximately 1 change/Mb across the entire exome) among solid tumors, while the mutation frequency in ATC was at the opposite extreme and was closer to that in melanoma and lung cancer, exceeding 100 changes/Mb [71, 72].

In addition, solute carrier family 26, member 4 (SLC26A4) gene, encodes a transmembrane protein named pendrin with up to 15 predicted membrane spanning domains and affects the flow of iodine into follicular lumen. The following were reported: 71% of ATC, 44% of benign tumors, 46% of FTC, and 71% of PTC, with abnormal SLC26A4 gene methylation in 64 cases of primary thyroid tumors and 6 cases of thyroid tumor cell lines [18, 73]. In addition, 81.5% of hypermethylated genes and 89% of hypomethylated genes were also present in nondifferentiated primary tumors (MTCs and ATCs) and nondifferentiated thyroid carcinoma-derived cancer cell lines [74], while Ras protein activator like-1 gene (RASAL1) displayed MAPK- and PI3K-suppressing and thyroid tumor-suppressing activities, which can be impaired by the mutations. Hypermethylation and mutations of RASAL1 were found in 33.33% (n = 10 of 30) of ATCs and in 0 of 20 (0%) of benign thyroid tumors [62]. However, ATC showed more hypomethylation than hypermethylation events, indicating that hypomethylation is related to dedifferentiation [70]. The authors validated four genes (NOTCH4 and TCL1B in ATCs, INSL4 and DPPA2 in MTCs) that become aberrantly hypomethylated in nondifferentiated thyroid tumors. All of them have been proposed to have an oncogenic role in cancer. And NOTCH4 (a member of the Notch family of transmembrane receptors) is frequently overexpressed in thyroid tumors [75, 76].


This part summarizes the related research on methylation genes in ATC in recent years (see **Table 3**).

#### **Table 3.**

*Aberrant methylated genes in ATC.*

#### **5. DNA methylation in MTC**

Medullary thyroid cancer is a neuroendocrine tumor originating from parafollicular C cells, and it is highly resistant to chemo- and radiotherapy [77]. Spry1 is a candidate tumor-suppressor gene in MTC. The hyper-activation of PI3K/ Akt/mTOR cascade has a relevant role in the pathogenesis and progression of MTC. In fact, most of pro-oncogenic effects of RET and Ras mutations are modulated by the activation of PI3K/Akt/mTOR pathway [78, 79]. For patients with metastatic disease, standard treatment modalities include local treatments (radiofrequency ablation, radiation therapy, embolization) and systemic treatment chemotherapy and more recently tyrosine kinase inhibitors (TKIs) targeting RET protein. As experience has been limited to case reports or case series, response rates (RR) ranged from 0 to 25% for periods of up to a few months. In nondifferentiated thyroid tumors, INSL4 and DPPA2 become aberrantly hypomethylated, both of which have been proposed to have an oncogenic role in MTC.

unknown. The aberrant overexpression of INSL4 in breast tumors, together with the aberrant promoter hypomethylation reported in this study, suggests that promoter demethylation might be a frequent mechanism of activation of INSL4 onco-

DPPA2 is expressed early in the embryo's development [87] but also in some tumor types [88]. Although the underlying molecular mechanism has not been reported yet, the authors' data indicated that promoter hypomethylation might play an important role. The frequent promoter hypomethylation observed in nondifferentiated tumors might be relevant for treatment with demethylating drugs [51, 88].

More and more researches have realized that the occurrence of tumors is not only entirely determined by genes but also epigenetics. The changes of epigenetics in thyroid cancer are mainly manifested in the aberrant methylation of tumor suppressor genes and thyroid-related genes. Numerous studies on DNA methylation in thyroid cancer have improved our understanding of thyroid carcinogenesis. Some of the recent findings, including the huge catalog of DNA methylation alterations, the association of DNA hypomethylation with cancer progression and dedifferentiation, the existence of different methylomes related to different clinical and molecular phenotypes, and the influence of immune-infiltrating cells in tumor DNA methylation patterns, are most likely to lead the direction of future research in the field of DNA methylation in thyroid cancer. A large number of studies confirmed the importance of DNA methylation as a source of novel biomarkers for early diagnosis, therapeutic perspective, and prognosis evaluation in thyroid cancer. In addition, the design of specific target demethylation drugs, which reactivate the function of tumor suppressor genes, is expected to become a new scheme for cancer treatment. Therefore, further functional experiments in vitro and in vivo are necessary for better understanding of the meaning and potential mechanism of DNA methylation changes in thyroid cancer as well as the evaluation of candidate bio-

**5.3 Developmental pluripotency-associated 2 (DPPA2)**

*Research Progress of DNA Methylation in Thyroid Cancer*

*DOI: http://dx.doi.org/10.5772/intechopen.91048*

markers through case-control studies and prospective trials.

\*Address all correspondence to: 1026909611@qq.com

provided the original work is properly cited.

Department of Nuclear Medicine, First Affiliated Hospital of Kunming Medical

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

gene activation in cancer.

**6. Conclusion**

**Author details**

**109**

Zhu Gaohong\* and Xie Lijun

University, Kunming, China

#### **5.1 Sprouty1 (Spry1)**

Sprouty (Spry) family of genes is composed of four members in mammals (Spry1–4). The Spry1 promoter is frequently methylated in MTC, and that Spry1 expression is consequently decreased. These findings identify Spry1 as a candidate tumor-suppressor gene in MTC. In mammals, the situation is more complicated as Spry proteins have been shown to be activators or inhibitors of receptor tyrosine kinase signaling depending on the cellular context or the receptor tyrosine kinase analyzed [80, 81]. Spry family members have been proposed to function as tumorsuppressor genes in a growing list of cancerous malignancies, including prostate and hepatocellular carcinoma, B-cell lymphoma, or neuroblastoma [82]. Finally, the authors found that SPRY1 promoter is frequently methylated and its expression decreased in human MTC. The mRNA levels of Spry1 are detected in murine C cells of thyroid with real-time RT-PCR. In situ hybridization showed expression of Spry1 mRNA in the fourth pharyngeal pouch, where thyroid C-cells originate and betagalactosidase staining of thyroids from 1-month-old Spry1LacZ/þ mice. One study used the hypomethylating agent 5<sup>0</sup> -aza-deoxycytidine (5<sup>0</sup> -Aza-dC) to confirm a causal relationship between promoter methylation and Spry1 expression [83]. As expected, TT cells treated with 5<sup>0</sup> -aza-dC showed an increase of approximately sixfold in the levels of Spry1 mRNA when compared to vehicle-treated cells. 50 -Aza-deoxycytidine (AZA), a demethylating agent, is in combination with the mTOR inhibitor everolimus in MTC cells (MZ-CRC-1 and TT). An innovative bioinformatic pipeline identified four potential molecular pathways implicated in the synergy between AZA and everolimus: PI3K-Akt signaling, the neurotrophin pathway, ECM-receptor interaction, and focal adhesion. Among these, the neurotrophin signaling pathway was most directly involved in apoptosis, through NGFR and Bax gene overexpression. Increased expression of genes involved in the NGFR-MAPK10-TP53-Bax/Bcl2 pathway during incubation with AZA plus everolimus was validated by western blotting in MZ-CRC-1 cells [84].

#### **5.2 Insulin-like 4 (INSL4)**

INSL4 (pro-EPIL) belongs to the insulin and insulin-like growth factor family and is expressed strongly during the first trimester of pregnancy by the differentiated syncytiotrophoblast [85]. It has been shown to be overexpressed in breast tumors with an aggressive phenotype [86], but the underlying mechanisms are still unknown. The aberrant overexpression of INSL4 in breast tumors, together with the aberrant promoter hypomethylation reported in this study, suggests that promoter demethylation might be a frequent mechanism of activation of INSL4 oncogene activation in cancer.

#### **5.3 Developmental pluripotency-associated 2 (DPPA2)**

DPPA2 is expressed early in the embryo's development [87] but also in some tumor types [88]. Although the underlying molecular mechanism has not been reported yet, the authors' data indicated that promoter hypomethylation might play an important role. The frequent promoter hypomethylation observed in nondifferentiated tumors might be relevant for treatment with demethylating drugs [51, 88].

### **6. Conclusion**

**5. DNA methylation in MTC**

*DNA Methylation Mechanism*

**5.1 Sprouty1 (Spry1)**

used the hypomethylating agent 5<sup>0</sup>

expected, TT cells treated with 5<sup>0</sup>

**5.2 Insulin-like 4 (INSL4)**

50

**108**

Medullary thyroid cancer is a neuroendocrine tumor originating from parafollicular C cells, and it is highly resistant to chemo- and radiotherapy [77]. Spry1 is a candidate tumor-suppressor gene in MTC. The hyper-activation of PI3K/ Akt/mTOR cascade has a relevant role in the pathogenesis and progression of MTC. In fact, most of pro-oncogenic effects of RET and Ras mutations are modulated by the activation of PI3K/Akt/mTOR pathway [78, 79]. For patients with metastatic disease, standard treatment modalities include local treatments (radiofrequency ablation, radiation therapy, embolization) and systemic treatment chemotherapy and more recently tyrosine kinase inhibitors (TKIs) targeting RET protein. As experience has been limited to case reports or case series, response rates (RR) ranged from 0 to 25% for periods of up to a few months. In nondifferentiated thyroid tumors, INSL4 and DPPA2 become aberrantly hypomethylated, both of

Sprouty (Spry) family of genes is composed of four members in mammals (Spry1–4). The Spry1 promoter is frequently methylated in MTC, and that Spry1 expression is consequently decreased. These findings identify Spry1 as a candidate tumor-suppressor gene in MTC. In mammals, the situation is more complicated as Spry proteins have been shown to be activators or inhibitors of receptor tyrosine kinase signaling depending on the cellular context or the receptor tyrosine kinase analyzed [80, 81]. Spry family members have been proposed to function as tumorsuppressor genes in a growing list of cancerous malignancies, including prostate and hepatocellular carcinoma, B-cell lymphoma, or neuroblastoma [82]. Finally, the authors found that SPRY1 promoter is frequently methylated and its expression decreased in human MTC. The mRNA levels of Spry1 are detected in murine C cells of thyroid with real-time RT-PCR. In situ hybridization showed expression of Spry1 mRNA in the fourth pharyngeal pouch, where thyroid C-cells originate and betagalactosidase staining of thyroids from 1-month-old Spry1LacZ/þ mice. One study



causal relationship between promoter methylation and Spry1 expression [83]. As


sixfold in the levels of Spry1 mRNA when compared to vehicle-treated cells.

NGFR-MAPK10-TP53-Bax/Bcl2 pathway during incubation with AZA plus everolimus was validated by western blotting in MZ-CRC-1 cells [84].

INSL4 (pro-EPIL) belongs to the insulin and insulin-like growth factor family and is expressed strongly during the first trimester of pregnancy by the differentiated syncytiotrophoblast [85]. It has been shown to be overexpressed in breast tumors with an aggressive phenotype [86], but the underlying mechanisms are still


which have been proposed to have an oncogenic role in MTC.

More and more researches have realized that the occurrence of tumors is not only entirely determined by genes but also epigenetics. The changes of epigenetics in thyroid cancer are mainly manifested in the aberrant methylation of tumor suppressor genes and thyroid-related genes. Numerous studies on DNA methylation in thyroid cancer have improved our understanding of thyroid carcinogenesis. Some of the recent findings, including the huge catalog of DNA methylation alterations, the association of DNA hypomethylation with cancer progression and dedifferentiation, the existence of different methylomes related to different clinical and molecular phenotypes, and the influence of immune-infiltrating cells in tumor DNA methylation patterns, are most likely to lead the direction of future research in the field of DNA methylation in thyroid cancer. A large number of studies confirmed the importance of DNA methylation as a source of novel biomarkers for early diagnosis, therapeutic perspective, and prognosis evaluation in thyroid cancer. In addition, the design of specific target demethylation drugs, which reactivate the function of tumor suppressor genes, is expected to become a new scheme for cancer treatment. Therefore, further functional experiments in vitro and in vivo are necessary for better understanding of the meaning and potential mechanism of DNA methylation changes in thyroid cancer as well as the evaluation of candidate biomarkers through case-control studies and prospective trials.

### **Author details**

Zhu Gaohong\* and Xie Lijun Department of Nuclear Medicine, First Affiliated Hospital of Kunming Medical University, Kunming, China

\*Address all correspondence to: 1026909611@qq.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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**117**

Section 4

Experimental Methods in

Methylation Researchs

Section 4
