DNA methylation in Hepatoblastoma-a literature review

Hepatoblastoma (HB) is the most common malignant liver tumor in children, accounting for more than 80% of all pediatric malignant liver tumors and 1% of all pediatric tumors. The incidence rate is 1.5 ppm, and the annual growth rate is 2.7% [1, 2]. The histological types of HB are diverse but mainly include epithelial cell types: pure fetal type, embryonic type, mixed type, and small-cell undifferentiated type. The varieties of different histological types suggest that the tumor cells are in different stages of differentiation and that the clinical characteristics are different. HB may originate from primary hepatoblasts or from less differentiated liver stem cells or human fetal liver multipotent progenitor cells (hFLMPCs) [3]. A meta-analysis revealed similarities between HB, early mouse embryonic liver, and hepatocyte-derived stem/progenitor cells, suggesting that HB may originate from hepatic precursor cells [4]. HB cells can express CD44, CD90, CD133, and other cancer stem cell markers, suggesting that cancer stem cells also exist in HB [5].

The specific mechanism for the transformation of normal hepatocytes into HB is unknown. Cells may begin to transform in an early stage of liver differentiation, or the transformation may be caused by gene mutation after liver development completed. The Wnt/β-catenin signaling pathway plays a key role in the process of tumorigenesis. The catenin beta 1 gene (CTNNB1) encodes the β-catenin protein, and mutations in CTNNB1 occur widely in HB. Mutations in CTNNB1 are closely related to HB and are most likely the original aberrations leading to tumorigenesis. In normal hepatocytes, the β-catenin N-terminal serine and threonine residues encoded by CTNNB1 exon 3 are phosphorylated by the APC regulator of WNT signaling pathway/Axin/ glycogen synthase kinase 3 beta (APC/Axin/GSK3β) complex and then recognized by the ubiquitin-conjugating complex and ubiquitinated. The protein is subsequently degraded by the proteasome, which reduces the β-catenin protein concentration in the cytoplasm and inactivates the Wnt/β-catenin signaling pathway [6]. Mutation of the CTNNB1 gene causes the β-catenin protein to be unable to be degraded, abnormally aggregate in the cytoplasm, and ectopically transferred to the nucleus [7]. In the nucleus, β-catenin associates with the T cytokine/lymphocyte enhancement factor binding factor (TCF/LEF) family. Transcription factors bind and recruit coactivators (BCL9, CBP/300, Pygo, etc.) to activate the transcription of downstream target genes [8]. Approximately 60–80% of HBs have CTNNB1 mutations, including point mutations and deletions in exon 3 [9]. Other genes with mutations in HBs include APC (20.51%), AXIN1 (1.67%), AXIN2 (3.75%), and leucine-rich repeat-containing G protein-coupled receptor 6 (LGR6) (12.5%) [10].

In the 1980s, Feinberg and Vogelstein first linked the epigenetic inheritance of genes to cancer [11]. Epigenetics alters the spatial structure of genes by modifying genes involved in processes such as DNA methylation, histone deacetylation, and RNA methylation, regulating gene transcription and translation and affecting gene expression. The expression of proteins is different between different organs, and the protein expression within the same organ is different at different developmental stages. Epigenetics plays an important role in this process. In the past few decades, an increasing number of studies have shown that epigenetic changes are widely involved in the occurrence, invasion, and metastasis of cancer [12] and promote tumorigenesis and tumor development by inactivating tumor suppressor genes (TSGs) and activating oncogenes. HB gene mutations are simple, and whole-exon sequencing shows that there are only 2.9 mutations per HB tumor on average [13], which is much lower than 35–66 mutations per tumor in liver cancer [14‐16], indicating that HBs are not only caused by the accumulation of gene mutations; in addition, most HBs occur in children, suggesting that epigenetic mechanisms that control development play an important role in the occurrence and development of HB. This article reviews the role of DNA methylation in HB occurrence, diagnosis, and treatment.

HB is a rare tumor, and there is sparse literature on DNA methylation. In the PubMed database, only 97 articles were returned from a search using the keywords “hepatoblastoma” and “methylation”, which is far lower than the 2513 articles returned using the keywords “hepatocellular carcinoma” and “methylation”. This shows that methylation has been far less studied in HB than in other tumors. There are a large number of differentially methylated regions (DMRs) and differentially methylated sites (DMSs) in HB and normal liver cells. Using the HM450K methylation chip to analyze HB tissue and adjacent normal liver tissue, 1359 DMSs were detected, involving 765 genes. Of these DMSs, 58% were hypomethylated, and most of the hypermethylated sites were located in the CpG islands in the promoter region of the genes. These 1359 CpG sites overlapped highly with CpG sites that were altered during liver development, suggesting that some of the HBs were caused by methylation changes in the early stages of liver development [17]. According to GO and KEGG analyses of the different genes, the three most affected physiological processes were cell adhesion, blood coagulation, and nervous system development, and the three most affected cellular functions were protein binding, nucleotide binding and ATP binding [18]. The lower overall methylation levels in HB cells than in normal hepatocytes have also been confirmed by other experiments [18]. These data show that methylation in HB is characterized by low levels of global DNA methylation and high levels of methylation of specific genes, which is consistent with the methylation characteristics of other tumors [19].

DNA methylation changes, such as hypomethylation of oncogenes and hypermethylation of TSGs, regulate gene expression, acting as an inactivator of TSGs and an activator of oncogenes [20]. The regulation of imprinted genes involved in methylation also plays a role in tumorigenesis in HB [21]. DNA methylation regulates gene expression and affects the expression of downstream genes through various pathways, playing a role in inhibiting cancer cell apoptosis, promoting cancer cell proliferation and cell cycle dysfunction, and promoting tumor metastasis (Table 1).

DNA methylation changes, as a general change in tumor cells, can cause tumor cells to show specific changes that make them different from ordinary cells. In the initial diagnosis of HB, imaging screening and biopsy-based pathological diagnoses are mainly used. These methods are more reliable and specific than other methods. DNA methylation can be used as an index for screening for tumor recurrence and metastasis during postoperative review and as an index for judging prognosis. According to the literature reports, the RASSF1A, UHRF1, MT1G, and NDRG2 promoter regions are hypermethylated. The degree of methylation is related to postoperative recurrence and distant metastasis and can be used as a marker for diagnosis and prognosis [31, 41, 54, 73, 85].

For the treatment of DNA methylation aberrations, because DNA methylation is achieved by the function of DNMTs, DNMTs are a therapeutic target for DNA demethylation and restoration of TSG functions. The most studied demethylating drugs at present are mainly DNMT inhibitors [86]. The mechanism of antitumor action of DNMT inhibitors is mainly to inhibit the activity of DNMTs so that the duplicated DNA is not methylated, and all alleles would be demethylated so that the expression of TSGs would be restored and they could play their role in inhibiting tumor growth, achieving antitumor therapy [87]. Currently, the most widely used DNMT inhibitors are decitabine (5-aza-2′-deoxycytidine) and azacytidine [88], but there is no clinical information on the treatment of HB with either drug.

HB is a tumor that proliferates because hepatocyte differentiation is blocked in early development. Mutations of the CTNNB1 gene can be detected in most tumors, and DNA methylation plays an important role in the occurrence and development of tumors. Hypermethylation reduces the expression of specific TSGs, while global hypomethylation increases the expression of other genes, promoting tumor development through various pathways. The dysfunction of a variety of DNA methylation regulatory enzymes plays a key role in the hypermethylation of TSGs and the hypomethylation of oncogenes, especially the UHRF1 protein.

According to the review in this article, DNA methylation can affect the formation and metastasis of hepatoblastoma through different cell signaling pathways, and the metastasis of hepatoblastoma is an important factor affecting the prognosis. Therefore, further research on the mechanism of DNA methylation in hepatoblastoma formation and metastasis has important basic and clinical significance.

At present, various tumor cells show global hypomethylation and hypermethylation of specific TSGs. It is speculated that this wide range of methylation changes is accomplished by DNA methylation regulatory enzymes. Therefore, further research on the regulatory mechanism of DNA methylation regulatory enzymes and their role in tumor development should be a future direction. Second, the mechanism by which DNA methylation regulatory enzymes enhance the methylation of TSGs and reduce the methylation of other genes and whether other proteins play a role in this process needs to be further studied. Third, the main factors influencing the long-term prognosis of HB are vascular invasion and distant metastasis [89]. The methylation level of multiple TSGs is closely related to vascular invasion and distant metastasis, which can be studied with large sample sizes.