Role of ten‐eleven translocation proteins and 5‐hydroxymethylcytosine in hepatocellular carcinoma

Abstract In mammals, methylation of the 5th position of cytosine (5mC) seems to be a major epigenetic modification of DNA. This process can be reversed (resulting in cytosine) with high efficiency by dioxygenases of the ten‐eleven translocation (TET) family, which perform oxidation of 5mC to 5‐hydroxymethylcytosine (5hmC), 5‐formylcytosine and 5‐carboxylcytosine. It has been demonstrated that these 5mC oxidation derivatives are in a dynamic state and have pivotal regulatory functions. Here, we comprehensively summarized the recent research progress in the understanding of the physiological functions of the TET proteins and their mechanisms of regulation of DNA methylation and transcription. Among the three TET genes, TET1 and TET2 expression levels have frequently been shown to be low in hepatocellular carcinoma (HCC) tissues and received most attention. The modulation of TET1 also correlates with microRNAs in a post‐transcriptional regulatory process. Additionally, recent studies revealed that global genomic 5hmC levels are down‐regulated in HCC tissues and cell lines. Combined with the reported results, identification of 5hmC signatures in HCC tissues and in circulating cell‐free DNA will certainly contribute to early detection and should help to design therapeutic strategies against HCC. 5hmC might also be a novel prognostic biomarker of HCC. Thus, a detailed understanding of the molecular mechanisms resulting in the premalignant and aggressive transformation of TET proteins and cells with 5hmC disruption might help to develop novel epigenetic therapies for HCC.


| INTRODUC TI ON
Hepatocellular carcinoma (HCC) ranks as the sixth most common cancer and the third leading cause of cancer-associated deaths globally, with estimated 70 000 deaths annually. 1,2 The most significant risk factor of HCC is chronic fibrotic liver disease, mainly caused by hepatitis virus (B and C) infection and alcohol abuse. 2 HCC is highly refractory to chemotherapy. Even after complete HCC tumour resection or ablation, approximately 70% of patients experience tumour recurrence within 5 years, which will continue to progress into incurable advanced-stage disease. 3,4 Despite intensive research, the prognosis of HCC patients remains dismal, with an overall 5-year survival rate <15%. 5 There is an urgent need for novel biomarkers to develop preventive strategies and therapeutic interventions based on an improved understanding of molecular hepatocarcinogenesis.
It has been shown that both genetic and epigenetic alterations are crucial for the initiation of HCC, thus making epigenetics a promising and attractive field for identifying the subset of patients at a high risk of recurrence and with dismal survival outcomes. [6][7][8][9] In mammals, methylation of the fifth position of cytosine (5mC) in DNA has been one of the most broadly investigated epigenetic modifications and has critical functions in development and diseases. 10,11 60%-80% of CpG sites in the mammalian genome are modified by 5mC. 10 Some of the well-known functions of 5mC include regulation of genomic imprinting, X-chromosome inactivation, inhibition transposable elements and participation in transcription. 12 In the past decades, DNA methylation has been thought to be highly chemically and genetically stable. 13 Nonetheless, mammalian 5mC can be reversed to its unmodified form in multiple ways.
Increasing evidence indicates that these 5mC oxidation products are not short-term transient intermediates in an enzymatic pathway participating in DNA methylation. They can be steadily incorporated into the genomic DNA with high stability and can perform unique and significant regulatory functions in the mammalian genome. [18][19][20][21] Disruption of epigenetic patterns, including DNA methylation landscapes, is a hallmark of cancer. In this review, we summarize current advancements in the understanding of the machinery and functions of TET-mediated DNA demethylation. Additionally, recent studies indicate that genomic 5hmC is globally down-regulated in HCC tissues and cell lines. In combination with the existing scientific data, evaluation of 5hmC signatures in HCC tissues and in circulating cell-free DNA should certainly contribute to early detection and help to devise therapeutic strategies against HCC. Furthermore, 5hmC might be a novel prognostic biomarker for HCC patients. Thus, precise modulation of DNA methylation landscapes, which is partly regulated by TET proteins, is crucial for HCC initiation and progression and offers a fundamental protection against malignant cellular transformation.

| S TRUC TURE AND FUN C TI ON OF TE T PROTEIN S IN REL ATI ON TO THE REG UL ATION OF DNA ME THYL ATION PAT TE R N S
Three mammalian TET proteins have been identified so far, which catalyse the sequential oxidation of 5mC to 5hmC, 5fC and 5caC (collectively called oxidized 5mC). [22][23][24] Furthermore, oxygen, Fe (II) and α-ketoglutarate (α-KG) are indispensable for the TET enzymes to perform the successive oxidation of 5mC and of its two intermediate oxidized derivatives, including 5hmC and 5fC. The final oxidized product is 5caC. 23,25 TET proteins are multi-domain enzymes with the molecular mass ranging from 180 to 230 kDa. The core catalytic domain at the C-terminus of all three TET proteins typically consists of a cysteine-rich domain, a conserved double-stranded β-helix (DSBH) domain. 26 Fe (II), α-KG and 5mC are brought together by the DSBH domain for subsequent catalytic oxidization, whereas the cysteine-rich domain surrounding the DSBH core makes the integrated catalytic core and TET DNA interaction stable. The TET DNA contact is not dependent on the methyl group, meaning that TET proteins accommodate various patterns of modified cytosine. 27 Structural studies indicate that the core catalytic domain preferentially binds cytosines in a CpG-dependent manner but does not interact with the surrounding DNA bases and shows little or no specificity for flanking DNA sequences. 27,28 Apart from their catalytic region, TET1 and TET3 share an N-terminal CXXC zinc finger domain that can bind DNA. 29,30 The catalytic region of the C-terminus alone can be targeted for relocation to the nucleus and implement the oxidization of 5mC. 22,29,31 TET proteins are obviously involved in the pathway of DNA demethylation. 26,32 Firstly, under the conditions where the maintenance methyltransferase DNMT1 is absent or prevented from accessing newly generated DNA, 5mC could be diluted during DNA replication, a process referred to as passive DNA demethylation.
Secondly, 5hmC and probably other oxidized 5mC forms interact F I G U R E 1 A schematic outline of the association between 5mC and 5hmC. 5mC generated from post-duplicative transfer of the methyl group to cytosine via the catalysis by DNMTs, which utilize S-adenosyl methionine (SAM) as a methyl donor. There are three known mammalian TET proteins at present, which catalyse the sequential oxidation of 5mC to 5hmC, 5fC and 5caC (collectively known as oxidized 5mC). Furthermore, oxygen, Fe(II) and α-KG are indispensable for the TET enzymes to perform the successive oxidation of 5mC and of its two intermediate oxidized derivatives, 5hmC and 5fC. The final oxidized product is 5caC with maintenance methylation by inhibiting DNA binding with the UHRF1-DNMT1 complex; thus, TET enzymes are highly likely to contribute to passive DNA demethylation. 33,34 Thirdly, TET proteins are implicated in active demethylation, an enzymatic process in which the bases of 5mC as well as their oxidized forms are replaced with unmethylated cytosines in a replication-independent pattern. Even though multiple machineries have been proposed, the signalling cascade involving TDG-mediated base excision and DNA BER is now considered the main driver of DNA demethylation. 18 Remarkably, dysfunction of TET proteins does not always result in up-regulation of 5mC due to aberrant DNA demethylation, indicating that oxidized intermediates of 5mC could serve as stable epigenetic biomarkers regardless of their functions in the DNA methylation process, probably by replacing methyl CpG-binding proteins, by targeting specific proteins that interact with oxidized forms of 5mC or by changing the epigenetic modification of histones. 35,36 A schematic outline of the association between 5mC and 5hmC has been shown in Figure 1.

| D ISCOVERY AND DOWN -REG UL ATI ON OF 5HM C
Recently, it was demonstrated that enzymatic oxidation of 5mC to 5hmC might serve as a common modification of DNA. Downstream erasure of 5hmC could actually be part of a sophisticated process of epigenetic gene regulation. 15 5hmC was first identified in the DNA of Teven bacteriophages and detected in the genomic DNA of mammalian organs such as the brain and liver during the 1970s, but its functional significance was not appreciated comprehensively. 37,38 In 2009, the catalytic function of the TET proteins was unambiguously proven: conversion of 5mC to 5hmC, which has been rediscovered in substantial amounts in the murine brain, embryonic stem cells, Purkinje cells and granule cells. 22,39 5hmC has been thought to perform an essential function in cellular differentiation and embryonic stem cells. 31 It has been reported that genomic 5hmC content is reduced overall in solid carcinoma tissues including HCC. 18,[40][41][42] Mutations and down-regulation of TET genes could result in lower amounts of 5hmC in cancer tissues as compared to normal controls. [43][44][45] Nonetheless, a full understanding of the exact molecular significance of 5hmC in HCC is still lacking.

| P OS T-TR ANSCRIP TIONAL REG UL ATI ON OF TE T1 BY MULTIPLE MICRORNA S
MiRNAs consist of a broad spectrum of small highly conserved noncoding RNAs that bind to the 3' untranslated regions (UTRs) of protein-coding mRNAs to inhibit protein expression. 46 Emerging evidence reveals that miRNAs can serve as tumour suppressors or oncogenic promoters, depending on the function of their target gene within a certain cell or tissue type. 47 Zhang et al have shown that TET1 was the direct target of miR-29a through the 3′-UTR of mRNA. 50 Further evidence has revealed that the miR-29 family modulates active DNA demethylation through direct targeting of TET1 in multiple solid cancers. 51,52 Lin et al have investigated the potential function of the negative feedback of miR-29-TET1 in HCC tumorigenesis and its potential mechanisms. 53 Furthermore, miR-29a overexpression up-regulates DNA methylation of the suppressor of cytokine signalling 1 (OSCS1) promoter; this process has been implicated in HCC metastasis in vitro and in vivo. In terms of mechanism, miR-29a down-regulates anti-metastatic OSCS1 by directly targeting the TET family, thus leading to repression of OSCS1 promoter demethylation. Finally, miR-29a up-regulation is associated with dismal clinical outcomes and with repression of TET-SOCS1-matrix metalloproteinase (MMP) 9 axis in HCC patients.
These findings mean that miR-29a is a crucial epigenetic regulator, facilitating HCC metastasis via the TET-SOCS1-MMP9 axis silencing. 54 Aside from the miR-29 family, there are other miRNAs participating in TET regulation in HCC. MiR-494 is up-regulated and involved in the silencing of several invasion-suppressor miRNAs by direct targeting of TET1, thus resulting in vascular invasion of a tumour. 55 Moreover, TET1 has been reported to be the direct target of miRNA-191, whose aberrant overexpression reduces the expression of TET1 in intrahepatic cholangiocarcinoma. The decreased TET1 expression helps the methylated CpG-rich segments at a promoter site of the p53 gene to stay methylated, resulting in down-regulation of p53, thereby disrupting the anti-cancer function of p53. Overall, the up-regulated miR-191 is related to intrahepatic cholangiocarcinoma progression via the miR-191/TET1/p53 pathway. 56 Furthermore, miR-520b inhibits cell proliferation by reducing the expression of TET1. 57 In this process, however, TET1 may act as an oncogene, and this notion contradicts results of a previous study. The paradoxical function has yet to be elucidated and might be partly attributed to a multitude of mechanisms of HCC aetiology and the complexity of clinical specimens. 57 Increasing evidence has shown that TET1 expression can be modified by microRNAs, but TET1 can also modulate the miRNA expression pattern via its demethylation activity. Not only can TET1 be modulated by microRNAs, but TET1 can also induce several invasion-suppressor microRNA genes through DNA demethylation in their proximal CpG locus. 49,55,58 Hence, it is difficult but necessary to investigate the relevant regulatory machineries that orchestrate transcriptional and/or post-transcriptional regulation during the initiation and progression of HCC. Multiple studies have revealed that the expression of TET1 proteins is significantly decreased in HCC tissues compared with normal noncancerous counterparts. 43,53,54,[59][60][61][62] Moreover, the mRNA levels of all three TET proteins are dramatically decreased in the adult liver when compared to foetal liver. 63  this observation is consistent with some reports indicating that TET1 silencing increases mammary and prostatic cancer invasion and metastasis. 65 Evidence has shown that miR-494-induced HCC cell invasion can be abrogated by increased expression of TET1 because it could trigger several invasion-suppressor miRNA genes by DNA methylation in their proximal CpG regions, implying that TET1 might function as a pivotal gene target in the regulation of the inhibitory effect of miR-494 on HCC cell invasiveness. 55 In intrahepatic cholangiocarcinoma, the decreased expression of TET1 can compromise the anti-cancer potential of p53. Mechanistically, TET1 directly targets the transcriptional start site of the p53 gene and promotes the expression of p53 by demethylating the CpG islands of the transcriptional start site within the p53 gene. 56

| THE D IS RUP TI ON OF TE T2 OR TE T3 IN H CC
Compared with TET1 disruption, somatic alterations and functions of TET2 and TET3 are comparatively rare in patients with HCC.
Nevertheless, the expression and activity of proteins TET2 and TET3 proteins in the progression of HCC were recently investigated. Liu and colleagues have demonstrated that a decrease in 5hmC amounts is related to the progression of HCC via down-regulation of the TET1 protein. 18 In contrast, Gao and colleagues 62 Table 1. Understanding the function of oxidization patterns of 5mC, and particularly 5hmC, in the control over liver progenitor cell differentiation will uncover molecular mechanisms of regenerative activity in liver tissue and novel therapeutic targets in HCC.

| AC TIVE DNA DEME THYL ATION IN HEPATI C PLURIP OTEN C Y AND D IFFERENTIATI ON
Given that 5mC, 5hmC and 5caC amounts are highly dynamic during early embryonic development, little is known about their pre-

| H CC IS A SSO CIATED WITH DECRE A S ED 5HM C LE VEL S
It has been demonstrated that the global 5hmC content is reduced overall in cancer specimens. 18,40,43,85 The

| A SSO CIATI ON OF 5HM C WITH CLINI C AL AND B I O CHEMI C AL CHAR AC TERIS TI C S OF H CC PATIENTS
Evidence has shown that reduced 5hmC amounts in genomic DNA may correlate with HCC development. Therefore, the association of 5hmC content with clinical characteristics in HCC has also been extensively investigated. Chen et al have demonstrated that the 5hmC content is dramatically associated with HCC TNM stage, and a reduced 5hmC amount correlates with tumour progression. 54 Likewise, Chen et al have discovered that the 5hmC staining score is inversely associated with the TNM stage. Particularly, 5hmC levels can be utilized to discriminate HCC patients at TNM stage I from stage II-III. 42 These results are in agreement with the findings that a global reduction in 5hmC contents in HCC genomic DNA takes place in the very early stage (BCLC stage 0) and correlates with HBV infection. 61 In another study, Liu et al have evaluated the clinical relevance of 5hmC expression in two separated cohorts containing 318 patients and 328 patients who underwent surgical resection. 86 In the training group, the 5hmC expression was associated with sex and the AFP levels. In the validation group, 5hmC expression was associated with sex, age, the AFP level, tumour number and TNM stage. Furthermore, Shen et al have found that 5hmC is related to tumour size. According to the subgroup analyses depending on tumour size, HCC subjects with a tumour size ≤5.0 cm have higher 5hmC content and lower levels of fasting plasma aspartate aminotransferase, the ratio of alanine aminotransferase to aspartate aminotransferase, c-glutamyl transferase and AFP in comparison with the patients with tumour size ≥5 cm. 87 These findings suggest that 5hmC is associated with the damage liver function. Therefore, application of 5hmC in combination with routine testing of liver functional parameters may serve as a novel biomarker for early detection of HCC.

| THE P OTENTIAL OF 5HM C A S A D IAG NOS TI C B I OMARK ER OF H CC
Chronic liver inflammation has always preceded the majority of Furthermore, chronic liver inflammation leads to hypermethylation of specific CpG islands, which may influence both non-hepatocytes and hepatocytes in the liver. In particular, methylation of specific CpG islands is higher during the progression from chronic hepatitis to cirrhosis and to HCC, leading to the inhibition of some tumour suppressor genes. These alterations indicate that 5hmC may serve as a potential diagnostic biomarker of a higher regenerative activity and of a late precancerous stage in the chronically inflamed liver.
5hmC is a major mammalian DNA epigenetic mark that has been associated with global gene regulation and tumorigenesis. Via a sensitive chemical labelling-dependent low-input shotgun sequenc-

| CLINI C AL IMPLI C ATI ON S OF DECRE A S ED 5HM C LE VEL S IN H CC PROG NOS IS AND THER APY
Multiple studies have revealed that a decrease in 5hmC levels in HCC tissues correlates with tumour stage and poor prognosis. 42,54,59,61,86,87 Subjects with lower 5hmC content in HCC specimens experience shorter overall survival and disease-free survival.
Furthermore, the reduced level of 5hmC in HCC and non-tumour tissues correlates with early tumour recurrence after surgical resection.
Available data on 5hmC and IDH2 expression alterations in HCC revealed that low 5hmC and IDH2 expression correlates with aggressive properties of HCC. Reduced 5hmC or IDH2 amounts individually and combined 5hmC and IDH2 expression correlates with shorter overall survival and higher cumulative recurrence rates. Multiple analyses suggest that 5hmC or IDH2 and 5hmC + IDH2 are independent predictive factors for overall survival and disease-free survival, thereby confirming that 5hmC may be a biological biomarker with high prognostic and predictive value. 86 In addition, ALDOB and multiple miRNAs, some of which are up-regulated in HCC, have been demonstrated to directly target TET proteins. [53][54][55]57,64 Decreased expression of TET proteins and lower 5hmC levels are general hallmarks of multiple cancer types, including HCC. 18 These pieces of evidence imply that miRNA repression or substitution might offer us a novel therapeutic approach to inhibit HCC invasion and distant metastasis. In particular, miR-29a promotes a metastatic phenotype and its up-regulation correlates with dismal clinical outcomes among HCC patients. Additionally, miR-29a could be a useful indicator for evaluation of metastatic patterns of HCC.
This notion indicates that miR-29a repression by specific miR-29a inhibitors in highly metastatic cells decreases the metastatic potential as well as increase the expression of TET proteins and SOCS1; this phenomenon represents a novel scheme for a therapeutic intervention that inhibits HCC metastatic phenotypes. Chuang  A schematic photograph of applications of 5hmC in circulating cell-free DNA in the course of HCC management is present in  61 Additionally, studies suggest that a decrease in 5hmC content correlates with the poor prognosis of HCC.
The genome-wide epigenetics-based technology continues to hold tremendous promise for both deepening our understanding of HCC progression at the molecular biological level and for the development of an effective, minimally invasive diagnostic strategy with direct clinical application. 61,92,102 Not only are these epigenetic profiles strongly disturbed during the early stages of HCC progression, but increasing evidence also indicates that these are unique to specific cancer types. These data are suggestive of a potential application to the stratification of patients into optimal downstream therapeutic regimes. Linking these stratification schemes with joint testing of relevant clinical characteristics will be greatly useful. In contrast to genetic mutations, which were encoded in the DNA sequence, epigenetic alterations are potentially reversible. Therapeutic interventions against this early abnormality in patients with hematopoietic cancers by means of demethylating agents (decitabine and azacytidine) have not yielded conclusive results. [103][104][105][106] Although some studies suggest that TET2 mutations are associated with a better clinical response, such a treatment does not provide a survival benefit. Nevertheless, disruption of TET2 enhances the therapeutic efficacy of CD19-targeted T cells, leading to deep molecular remission in a 78-year-old patient with chronic lymphocytic leukaemia. 107 Thus, the development of refined drugs that reverse normal epigenetic signatures is greatly attractive in the field of cancer research, because such drugs have substantial clinical applications as effective therapies in combination treatments. Connecting genome-wide epigenetics-based agents with recent developments in CRISPR-Cas9 technologies and expansion and persistence of CAR T cells will strongly contribute to future research into cancer progression and to identification of innovative cancer therapies.

CO N FLI C T S O F I NTE R E S T
No potential conflicts of interest existed.