Исследование генома рака печени | ПРЕЦИЗИОННАЯ ОНКОЛОГИЯ

Исследование генома рака печени

Exploration of liver cancer genomes

Tatsuhiro Shibata & Hiroyuki Aburatani // Nature Reviews Gastroenterology & Hepatology  11, 340–349 (2014)

Введение

Рак печени — третья основная причина рак-ассоциированной смерти во всем мире. (1) Гепатоцеллюлярная карцинома (HCC) является наиболее распространенной формой рака печени с далее следующей внутрипеченочной холангиокарциномой (IHCC). (1) Хроническое повреждение печени, например, вызываемое хроническим гепатитом, циррозом печени и жировой болезнью печени, связано с раком печени. Заражение вирусом гепатита (например HBV, HCV и др.), экспозиция афлатоксина В, потребление алкоголя и другие метаболические заболевания (такие как ожирение, сахарный диабет и гемохроматоз) является известными факторами риска развития рака печени. (2, 3, 4), Дополнительно, паразиты, подобные печеночной двуустке, ассоциированы с IHCC в Юго-восточных азиатских странах. (5, 6)

Инцидент рака печени повышен в восточноазиатских и африканских странах. (1, 2, 3, 5) HBV инфекция более преобладает в африканских и азиатских странах (кроме Японии) относительно других географических регионов. (3) Однако, число инфицированных HCV быстро нарастает в Японии и странах Запада, особенно в США, где вирусный гепатит ассоциирован с злоупотреблением наркотиками. (2, 3)

Соматические альтерации генома рака печени

Геном рака печени содержит множественные типы соматических альтераций, включая мутации (такие как замены единичных нуклеотидов и небольшие инсерции и делеции), изменения числа копий гена(утрата, усиление и амплификация копий), и интрахромосомные и межхромосомные перестройки (большие делеции, инверсии, тандемные дупликации и транслокации).

Анализ числа копий всего генома


Introduction

Liver cancer is the third leading cause of cancer-related death worldwide.1 Hepatocellular carcinoma (HCC) is the most common form of liver cancer, followed by intrahepatic cholangiocarcinoma (IHCC).(1) Chronic liver damage, such as that caused by chronic hepatitis, liver cirrhosis and fatty liver disease, is closely associated with the occurrence of liver cancers. Hepatitis virus infection (for example HBV, HCV and others), aflatoxin B exposure, alcohol intake, and other metabolic diseases (such as obesity, diabetes mellitus and haemochromatosis) are well-known risk factors for liver cancer.(2, 3, 4) In addition, parasites such as liver fluke are associated with IHCC in Southeast Asian countries.(5, 6)

The incidence of liver cancer is high in East Asian and African countries.(1, 2, 3, 5) HBV infection is more prevalent in Africa and Asian countries (except Japan) than other regions of the world.3 However, the number of patients infected with HCV has been rapidly increasing in Japan and Western countries, especially in the USA where viral hepatitis infection is partly mediated through drug abuse.2, 3 In this Review, we mainly focus on HCC, as HCC and IHCC showed distinctive genomic alterations and fairly little is known about the IHCC genome alterations at present.

Somatic alterations in the liver cancer genome

The liver cancer genome contains multiple types of somatic alterations, including mutations (such as single nucleotide substitutions, and small insertions and deletions), changes of gene copy numbers (copy number loss, gain and amplification), and intra-chromosomal and inter-chromosomal rearrangements (large deletion, inversion, tandem duplication and translocation).

Genome-wide copy number analysis

Copy number changes in human cancers have been analysed mainly by array-based comparative genome hybridization methods. Bacterial artificial chromosome (BAC) clone DNA or oligonucleotide probe arrays (microarray-based comparative genomic hybridization) have been used in a number of studies to search for copy number changes in liver cancer.(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) Table 1 summarizes recurrent copy number alterations in HCC. In addition to well-known oncogenes, such as MYC and CCND1, and tumour suppressor genes, such as TP53 and RB, liver cancers harbour multiple chromosomal amplifications and deletions.

Table 1: Amplified and deleted genes in HCC

Gene name Locus Function
Recurrently amplified genes in HCC
MDM4 1q32.1 p53 pathway
BCL9 1q21.1 WNT pathway
ARNT 1q21.2 Xenobiotics metabolism
ABL2 1q25.2 Proliferation
MET 7q31.2 Proliferation
COPS5 8q13.1 Proteolysis
MTDH 8q22.1 Metastasis
COX6C 8q22.2 Mitochondria
MYC 8q24.21 Proliferation
CCND1 11q13.2 Proliferation
FGF19 11q13.2 WNT pathway
RPS6KB1 17q23.1 Proliferation
EEF1A2 20q13.33 Translation
Recurrently deleted genes in HCC
TNFRSF14 1p36.33 Immune response
CDKN2C 1p36.11 Cell cycle
ARID1A 1p36.11 Chromatin remodelling
TNFAIP3 6q26 NF-κB pathway
CSMD1 8p23.2 Immune response
DLC1 8p22 Small GTPase
SORBS3 8p21.3 Migration
WRN 8p21.3 DNA repair
SH2D4A 8p21.2 Proliferation
PROSC 8p11.2 Unknown
CDKN2A 9p21.3 Cell cycle
CDKN2B 9p21.3 Cell cycle
PTEN 10q23.31 Proliferation
SPRY2 13q31.1 Proliferation
BRCA2 13q13.1 DNA repair
RB1 13q14.3 Cell cycle
XPO4 13q11 Nuclear export
SMAD4 18q21.31 TGF-β signalling

 

The identification of target genes solely by copy number data has been challenging. Therefore, strategies based on integrative analysis of genetic alterations, gene expression profiling and oncogenic function of candidate genes might be an effective approach. Zender  et al.21 selected potential tumour suppressor genes using data from copy number analyses of human HCC, and functionally identified novel tumour suppressor genes, including XPO4, by in vivo short hairpin RNA screening in a mosaic mouse model. Sawey  et al.22 extracted genes located in chromosomal regions of recurrent focal amplification in human HCC and tested their oncogenic activity using a mouse hepatoblast model. These authors identified 18 tumour-promoting genes, including FGF19, which is located next to the CCND1 gene on 11q13.3. FGF19 and CCND1 cooperatively promote tumour formation through the CTNNB1 pathway.(22)

Katoh  et al.13 attempted to define a molecular classification of HCC on the basis of the copy number alteration profiles of 87 HCC tumours, including HBV-associated and HCV-associated cases. Two molecular subgroups were identified that are associated with virus status, the presence of intrahepatic metastasis and patient prognosis. The researchers also reported six distinctive combinations of copy number alterations in HCC.13 In another study, copy number changes in 63 HCCs of various aetiologies (viral and nonviral) were analysed and 8q24 copy number gains associated with MYC overexpression were identified that were unique to viral and alcohol-related HCCs.15 Amplification of MDM4 (1q32.1) and copy number gain of EEF1A2 (20q13.33) were shown to be frequent and aetiology-independent molecular events in HCC.15 A meta-analysis of four independent microarray comparative genomic hybridization datasets, including 169 samples, identified chromosomal gains in five broad (1q, 6p, 8q, 17q, and 10q) and two narrow (5p15.33 and 9q34.2–34.3) regions, and 88 significant losses frequently present in 4q, 6q, 8p, 9p, 13q, 14q, 16q and 17p.18 Wang  et al.19 reported the results of copy number analysis of 286 HCC tumours by single nucleotide polymorphism array, which identified 29 recurrently amplified and 22 recurrently deleted regions, as well as BCL9 and MTDH as novel amplified oncogenes in HCC.19

Whole-exome sequencing

Innovations in sequencing technologies have enabled researchers to explore the liver cancer genome in more depth. The capture or enrichment of DNA fragments containing the exonic region followed by massively parallel sequencing can determine somatic mutations in the whole exon domain (exome).23, 24 This approach enables the comprehensive detection of somatic alterations in the protein-coding region, and has led to the discovery of many novel genes implicated in liver cancer. Exomic sequencing of 10 HCV-positive HCCs and subsequent analysis of an additional tumour cohort of various aetiological backgrounds identified recurrent inactivating mutations of the ARID2 gene in 18.2% of HCV-associated HCCs.25 Guichard  et al.26 performed copy number analysis of 125 HCC cases and whole-exome sequencing of 24 of these cases and found new recurrent alterations in four genes (ARID1A, RPS6KA3, NFE2L2 and IRF2). Huang  et al.27 performed whole-exome sequencing of nine pairs of HCCs and their intrahepatic metastases. Although most substitutions (94.2%) were common in both primary and metastatic tumours, a fraction of mutations were only detected in primary (1.1%) or metastatic (4.7%) tumours. Among them, KDM6A, CUL9, FGD6, AKAP4 and RNF139 were found only in the metastatic tumours of three individuals.

Using whole-exome sequencing of 87 HCC cases, Cleary  et al.28 identified recurrent alterations in the NFE2L2–KEAP1 and KMT2A (also known as MLL) pathways, and other genes (C16orf62 and RAC2) with lower mutation frequencies. Eight fluke-associated cholangiocarcinomas (the predominant type of liver cancer in northern Thailand and neighbouring countries) were analysed, and showed that the number of coding mutations per tumour ranged from 19 to 34, with an average of 26 mutations per sample.29 In addition to TP53 and KRAS, recurrent inactivating mutations in the MLL3, ROBO2, RNF43 and PEG3 genes were identified, and activating mutations were found in the GNAS gene.

Whole-genome sequencing

Several research groups have sequenced the full liver cancer genome in further attempts to identify all somatic driver events related to hepatocarcinogenesis, including substitutions in noncoding regions, structural rearrangements, and viral genome integration. Totoki  et al.30 first performed whole-genome sequencing of one HCV-associated HCC case (tumour genome and corresponding normal genome) and identified >16,000 somatic mutations and 26 intra-chromosomal and inter-chromosomal rearrangements generating four fusion transcripts. Among them, one in-frame fusion transcript (BCORL1-ELF4), generated by a small inversion on the X chromosome, showed reduced transcriptional repression activity compared to wild-type BCORL1, which encodes a tumour suppressor gene.

Fujimoto  et al.31 reported the results of whole-genome sequencing of 27 HCCs and matched normal genomes, 25 of which were associated with HBV or HCV infection. The average number of somatic point mutations at the whole-genome level was 4.2 per Mb. One tumour that contained an exceptionally large number of somatic mutations (24,147 substitutions) showed a DNA mismatch-repair defect caused by a somatic nonsense mutation in the MLH1 gene. Furthermore, mutations in several chromatin regulators, including ARID1A, ARID1B, ARID2, KMT2A and MLL3, were detected in ~50% of the tumours.

Whole-genome sequencing of 88 HCC tumour and normal tissue pairs, including 81 HBV-positive and no HCV-positive cases showed an average somatic mutation rate of 3.69 per Mb and a mean protein-altering mutation rate of 1.8 per Mb, which are mid-range among different cancer types.32 In this study, the WNT/CTNNB1 and JAK/STAT pathways were shown to be major oncogenic drivers in HCC and activating JAK1 mutations were identified in 9.1% of total cases, suggesting that these pathways could be novel therapeutic targets in HCC.

HBV genome integrations in the host genome

HBV is a DNA virus whose genome is integrated into the host genome. The integration of the viral genome affects host gene expression near the integration site and its effect on the integrity of the host genome is associated with virus-mediated hapatocarcinogenesis.33 In the past, Southern blot analysis or inverse PCR was applied to identify viral genome integration sites. However, current genome sequencing technology can detect virus integration events more comprehensively and at higher resolution than previously.

Jiang  et al.(34) performed high-depth (>80× and 240× coverage of the genome, two or three times more than that used for conventional whole-genome sequencing) whole-genome and transcriptome sequencing of four pairs of HBV-positive HCCs and identified 225 HBV genome integration sites by taking advantage of paired reads mapping to both human and viral genomes. A variety of genomic aberrations near viral integration sites were found, including direct gene disruption, viral promoter-driven gene transcription, viral–human transcript fusion, and DNA copy number alterations. Frequent HBV integration in TERT and MLL4 loci has also been reported.(31) Sung  et al.35 conducted whole-genome sequencing, at >30× coverage on average, of 81 HBV-positive and seven HBV-negative HCC samples. Analysis of HBV integration sites identified 399 integration breakpoints (4.9 per case). Frequent HBV integration breakpoints were observed in the TERT, KMT2D (also known as MLL4), CCNE1 and FN1 genes.

Somatic change of retrotransposons in HCC

The human genome contains a variety of repetitive sequences, including tandem repeats (such as satellite DNA and microsatellite DNA) and retrotransposons, such as short interspersed nuclear elements (SINEs) and long interspersed nuclear elements (LINEs). In the human genome, Alu and LINE-1 are major forms of SINEs and LINEs, respectively. Given that current massive parallel sequencing technologies can produce only short reads (~200 bp), repetitive sequences, which constitute ~20% of the human genome, remain to be explored in genome sequencing.(36)

Retrotransposon capture sequencing applied to HCC samples revealed two LINE-1-mediated somatic changes associated with liver tumorigenesis.(37) One was a germline retrotransposon insertion in the MCC gene, a tumour suppressor gene that is known to be mutated in colorectal cancers. This retrotransposon insertion was found to downregulate MCC expression and activate the WNT/CTNNB1 pathway. The other event, a tumour-specific LINE-1 insertion, activates a potential oncogene, ST18, in liver tumours.

Mutation signatures and aetiological factors

There are six patterns of somatic substitution (C>A/G>T, C>G/G>C, C>T/G>A, T>A/A>T, T>C/A>G and T>G/A>C) in the cancer genome and they are affected by exogenous or endogenous mutagens, such as oxidative stress, exposure to chemicals or UV, and defects in the DNA repair machinery.38 Whole-genome sequencing in cancer can identify large numbers of neutral mutations and is more appropriate for the analysis of mutation signatures in an unbiased manner than is whole-exome sequencing.

The first whole-genome sequencing study of a Japanese HCV-positive HCC case showed a distinct mutation signature (dominance in C>T/G>A and T>C/A>G) in the liver cancer genome.(30) A similar substitution pattern was also reported in Asian HBV-positive HCC cases.(32, 33) Guichard  et al.26 reported the over-representation of C>A/G>T substitutions in HCC in a Western population with multiple aetiological backgrounds, although their data was obtained using whole-exome sequencing.(26) Using whole-genome sequencing, a study of 27 HCC cases of different aetiological backgrounds demonstrated a dominance of T>C/A>G transitions as well as C>A/G>T transversions and C>T/G>A transitions, particularly at CpG sites.(31) As C>T/G>A transitions are commonly found in other cancers, T>C/A>G transitions and C>A/G>T transversions could be characteristic mutational signatures of HCC genomes. Habitual alcohol drinking and the occurrence of synchronous or metachronous multiple liver nodules were significantly associated with the principal components of the somatic substitution patterns.31 Somatic substitutions in IHCC associated with liver fluke are predominantly C>T/G>A transitions, the majority of which are identified in the context of a CpG-to-TpG change as the result of 5-methylcytosine deamination.(29)

In addition to six different substitutions, information on the bases immediately 5′ and 3′ to each mutation has been used to identify context-dependent mutation patterns in a wide range of cancers. Among 22 mutational signatures identified by this cross-tumour analysis, HCC contained five distinct signatures, which was the highest number among the 30 tumour types and indicates that a complex mutagenesis process operates in this tumour (Figure 1).(39)

Exploration of liver cancer genomes1

Figure 1: Multiple aetiological factors and ethnic differences affect somatic mutation signatures in liver cancer.  Five characteristic mutation signatures identified in the liver cancer genome are shown.39

Epigenetic alterations in HCC

HCC is a heterogeneous disease in terms of aetiology and cell of origin.40 Various environmental agents and lifestyles known to be risk factors for HCC are suspected to promote its development by eliciting epigenetic changes, which have a key role in a wide range of human malignancies.41

DNA methylation in HCC

Altered DNA methylation is an early event in HCC development. Global hypomethylation mainly affects intergenic regions of the genome and has a critical role in increasing chromosomal instability.42 DNA methylation of gene promoters, which is important in transcriptional regulation and the cellular differentiation process,43 is a common mechanism of gene silencing in cancer cells. Furthermore, CpG island hypermethylation phenotypes have been reported in various types of cancers, such as colorectal,(44) uterine,(45) glioma,(46) and renal(47) cancers. However, the presence of such phenotypes is still controversial in HCC.(48, 49)

A molecular mechanism of active DNA demethylation has been identified and shown to be involved in tumorigenesis,50 particularly in glioma and haematological malignancies. Hydroxymethylcytosine is present at a considerable level in normal adult liver tissues and is often decreased in tumour tissues;51 however, its role in liver carcinogenesis remains unknown. IDH1 and IDH2 mutations are frequent in IHCCs and have been detected in 34 of 326 cases (10%).(52) Tumours containing mutations in IDH1 or IDH2 had lower 5-hydroxymethylcytosine and higher 5-methylcytosine levels compared with those without mutations, and 50% of hypermethylated genes overlapped with DNA hypermethylation in IDH1-mutant glioblastomas.(51)

To investigate DNA methylation patterns comprehensively, aberrantly methylated genes are identified by methylated DNA immunoprecipitation (meDIP) followed by tiling array53 or next-generation sequencing. Deng  et al.54 applied the meDIP-chip method to identify 15 genes preferentially methylated in HCV-related HCCs. Alternatively, a genome-wide DNA methylation assay that was developed on Beadchip™ (Illumina Inc., San Diego, CA) technology55 can measure methylation levels quantitatively at single CpG sites, and yield largely comparable results to meDIP sequencing56 and whole-genome bisulphite sequencing. This assay has been applied to methylation profiling in various cancers and in the cancer genome atlas project.57 A few distinct epigenetic subtypes identified on the basis of the methylation pattern have been detected in HCCs and will be integrated with genetic alteration data.

Shen  et al.58 used a 27K Infinium™ array (Illumina) to analyse 62 HCC cases and identified 2,324 differentially methylated CpG sites, of which 684 hypermethylation markers could be utilized for plasma DNA diagnostics. They also analysed 66 HCC cases using a 450K array in which the top 500 significant CpG sites that were differentially methylated were able to distinguish HCC from adjacent tissues.59 Meanwhile, Tao  et al.60 analysed noncancerous tissues of HBV-associated HCC on a 27K array and identified hypermethylated genes. Accumulation of such methylations would form “an epigenetic field for cancerization”.61

An early study62 showed that extensive methylation is associated with CTNNB1 mutations, while HCC with a TP53 mutation is often characterized by chromosomal instability. Given that CTNNB1 and TP53 mutations are mutually exclusive in HCCs, such distinct methylation patterns could be associated with particular genetic alterations.

Promoter CpG islands of the CDKN2A and CDKN2B tumour suppressor genes are frequently hypermethylated, leading to inactivation of the RB pathway.63 Methylation of the CDKN2A gene promoter occurs in 73% of HCC tissues,64 56% of HBV-related HCC, and 84% of HCV-related HCC.65 RASSF1A is methylated in up to 85% of HCCs,66 GSTP1 in 50–90%,67, 68 and MGMT in 40%.69

Transcriptome analysis and beyond

RNA sequencing technology has enabled not only transcriptomic profiling, but also the identification of rearranged transcripts, such as translocations and inversions, and tumour-specific expression of noncoding RNAs, although the latter analysis requires deep coverage of sequencing reads. No recurrent fusion genes have been reported in HCC to date.

Classification based on gene expression, copy number and DNA methylation profiling data would help elucidate the correlation between mutation profiles and molecular subclasses.70, 71 Gene expression profiles in cancer are the result of genetic and epigenetic alterations. Therefore, an integrated genomic analysis is necessary to determine how these genetic and epigenetic alterations affect cancer phenotypes, because the combination of somatic mutations, promoter methylation, and chromosomal loss might lead to gene inactivation.72

Vetter  et al.73 reported on the association between the increase of splicing variants of the KLF6 gene and increased hepatocarcinogenesis. Splicing variants in HCCs have been reported in several genes,74 including LLGL1 (also known as HUGL1),75 TCF4,76 and p73.77 Transcriptome sequencing of HCC samples combined with genotyping validation identified a frequent adenosine-to-inosine RNA editing event in the AZIN1 gene in HCC.78, 79 This editing induces a serine-to-glycine amino acid change that confers gain-of-function activity and a stronger affinity of the edited protein to antizyme. Increased AZIN1 (antizyme inhibitor 1) protein stability could promote cell proliferation, presumably through the neutralization of ornithine decarboylase (ODC) and G1/S-specific cyclin-D1 (CCND1) degradation mediated by antizyme. Adenosine-to-inosine RNA editing will contribute to more transcriptome diversity and liver carcinogenesis.

Core liver cancer genes and pathways

Comprehensive analyses of the liver cancer genome have demonstrated that multiple cancer genes and molecular pathways are recurrently altered and have pivotal roles in hepatocarcinogenesis (Figure 2). Table 2 summarizes important mutated genes in liver cancer.

Exploration of liver cancer genomes2

Figure 2: Core oncogenic pathways in hepatocarcinogenesis.  Representative genes involved in each pathway are indicated.

Table 2: Candidate driver genes in hepatocellular carcinoma with recurrent genetic alterations

Gene Frequency (%) Total number of cases analysed Number of mutation-positive cases Genetic alteration Pathway
*Copy number change.
Abbreviation: LOH, loss of heterozygosity.
TP53 31 2,720 844 Mutation, LOH TP53
ARID1A 28.2 85 24 Mutation, LOH Chromatin modifying
CTNNB1 18.8 3,238 609 Mutation WNT
MTDH 14.7 286* 42 Amplification Cell adhesion
AXIN1 14.2 466 66 Mutation, LOH WNT
CDKN2A 11.7 686 80 Mutation, LOH Cell cycle
ARID2 10.9 202 22 Mutation, LOH Chromatin modifying
CHD1L 10.7 286* 31 Amplification Chromatin modifying
BCL9 8.7 286* 25 Amplification Chromatin modifying
NFE2L2 7.4 162 12 Mutation Oxidative stress
ATM 6.9 72 5 Mutation, LOH TP53
PIK3CA 6.3 631 40 Mutation Growth factor signalling
SMARCA4 6.2 129 8 Mutation, LOH Chromatin modifying
TSC2 5.2 77 4 Mutation, LOH Growth factor signalling
CCND1 4.7 286* 14 Amplification Cell cycle
APC 4.7 107 5 Mutation, LOH WNT
JAK2 4.7 85 4 Mutation Growth factor signalling
PTEN 4.4 451 20 Mutation, LOH Growth factor signalling
BRAF 4.4 360 16 Mutation Growth factor signalling
FGF19 4.3 286* 13 Amplification Growth factor signalling
RB1 4.3 94 4 Mutation, LOH Cell cycle
COL1A1 4.2 71 3 Mutation Cell adhesion
HNF1A 3.9 233 9 Mutation Chromatin modifying
KRAS 2.7 672 18 Mutation Growth factor signalling
NRAS 1.6 426 7 Mutation Growth factor signalling

 

TP53 pathway

TP53 is the top gene among recurrently mutated genes in HCC, and its mutation frequency varies between 18% and 35.2% (25.9% on average) of HCCs.80 Alterations of other genes located upstream and downstream on the TP53 pathway, such as recurrent mutations of the ATM (an upstream regulator of TP53 activation81) and CDKN1A (a target of TP5382) genes, have also been reported. Moreover, mutations of the IRF2 gene, which encodes a positive regulator of TP53 protein expression, are mutually exclusive to the TP53 mutation in a cohort of patients with HCC.(26)

Cell cycle regulation pathway

The G1/S cell cycle checkpoint and cell senescence are regulated by RB and CDKN2A. Inactivation of the RB and CDKN2A genes by homozygous deletion and promoter CpG hypermethylation or point mutations has been reported in HCC.83, 84 The tumour suppressing activity of RB in the liver was evaluated in a mouse model, and RB inactivation was found to be associated with both increased cell proliferation and chromosomal instability.85

TERT pathway

Activation of telomerase (encoded by the TERT gene), which is physiologically silenced in most normal cells, is required for infinite replication in cancer cells.86 Somatic mutations in the TERT gene promoter have been shown to promote TERT gene expression in melanoma.87, 88 Killela  et al.89 screened these mutations in >1,000 tumours of various organs and reported that 27% of HCC cases harboured these alterations. Nault  et al. reported TERT promoter mutations in 54% of human HCCs and 25% of cirrhotic preneoplastic nodules, suggesting that this alteration could be the earliest recurrent genetic event in hepatocarcinogenesis.90

WNT pathway

Aberrant activation of WNT signalling is a driving molecular event in a wide range of tumours, including liver cancers.91 Somatically acquired missense mutations in exon 3 of the CTNNB1 gene are frequently reported in HCC (10.0−32.8% in genome-wide sequencing studies).92 In addition to CTNNB1, alterations of APC and AXIN1, which are tumour suppressor genes that negatively regulate catenin β-1 (CTNNB1) protein levels in a post-transcriptional manner, have been recurrently reported in HCC and hepatoblastoma.93, 94, 95 Frequent epigenetic inactivation of SFRPs and SOX1, both of which are negative regulators of WNT signalling, has also been detected.96, 97 Alterations in the CTNNB1, APC and AXIN1 genes occur in a mutually exclusive way and activate downstream signals, including transcriptional activation of the MYC and CCND1 genes, which are also amplified in HCC.98, 99, 100 CTNNB1 mutation is reported to be associated with HCV-related HCC.28

Chromatin modifying factors

DNA is tightly associated with proteins, mainly various types of histones, and compactly packed in the nucleus. This DNA-protein complex is called chromatin, and its structure (open or closed) or position is dynamically regulated by histone modifications or ATP-dependent mobilization, which affect gene expression and convey epigenetic information beyond DNA replication. The SWI/SNF (switch/sucrose non-fermentable) protein complex regulates chromatin structure by altering the position of the nucleosome, the basic unit of the DNA-histone complex, and participates in a wide range of biological phenomena, such as differentiation, growth, DNA repair, and reprogramming.101, 102 ARID1A, ARID1B and ARID2 encode core proteins of SWI/SNF complexes and are frequently altered in HCC.26, 31 Alterations of these ARID family members have been reported in other tumour types, including ovarian cancer, renal cell cancer and gastric cancer.103 In addition, the presence of frame-shift mutations, copy number loss and homozygous deletions observed in in vitro studies demonstrated that members of the ARID family function as tumour suppressor genes.

Alterations of other epigenetic regulators have also been reported in HCC. As an epigenetic writer (functioning in histone modification), mutations in the gene encoding histone-lysine N-methyltransferase 2A (KMT2A; also known as MLL)104, 105 and its family members (MLL3 and MLL4) are frequent.28 A group of genes encoding epigenetic readers (specifically recognizing histone modification) including BPTF106 and other histone binding proteins (RNF20 [also known as BRE1A] and BRDT) are also altered in certain HCCs.31 Alterations in these epigenetic regulators account for >50% of HCC cases.31

Growth factor signalling pathway

Copy number analyses of HCC identified focal gene amplification of the genes encoding the receptor tyrosine kinase MET, FGF19 (which is a ligand for FGFR4), and downstream signalling components (MYC and RPS6KB1). Furthermore, HCC genome sequencing studies have revealed recurrent somatic mutations in genes encoding other kinases (RPS6KA3 and JAK1). Epigenetic silencing of SOCS-1, a negative regulator of the JAK/STAT pathway, occurs frequently in HCC.107 Compared to other epithelial cancers, such as lung or colorectal cancer, activating mutations in the RAS (KRAS, NRAS and HRAS) and PIK3CA genes are rarely reported in HCC, but occur more frequently in IHCC.108, 109, 110 Activation of other growth factors including TGF-β,111 IGF112 and VEGF113 are also involved in hepatocarcinogenesis. These genomic alterations, especially JAK1/PIK3CA mutations,32 are potential therapeutic targets in liver cancer.

KEAP1–NFE2L2 pathway

The NFE2L2 gene encodes a sequence-specific transcriptional factor that upregulates genes associated with oxidative stress and other metabolic pathways.114 The level of the NFE2L2 protein is regulated by the ubiquitin-proteasome pathway, and KEAP1 functions as an E3 ubiquitin ligase. Activating missense mutations in the NFE2L2 gene,115 which disrupt direct NFE2L2–KEAP1 interaction, or inactivating mutations of the KEAP1 gene are recurrently reported in HCC.26, 28 These alterations result in the accumulation of the NFE2L2 protein and promote aberrant activation of downstream genes that confer resistance to oxidative stress and induce metabolic transformation in cancer cells.114, 116

NOTCH pathway

The role of the NOTCH cascade in solid tumours is controversial. Comparative functional genomics integrating transcriptome data from mice and human HCC samples indicate that NOTCH is activated in this cancer,117, 118 whereas other reports identified activation of NOTCH signalling as a suppressor feedback mechanism during HCC progression.119, 120 These contradictions suggest that biological activities of NOTCH signalling during hepatocarcinogenesis largely depend on the cellular contexts, as reported in other tumour types.121

Genomic changes during tumour progression

Midorikawa  et al.72 analysed copy number changes during multistep hepatocarcinogenesis and found that 1q21.3–44 gain and loss of heterozygosity on 1p36.21–36.32 and 17p13.1–13.3 were frequently observed in the early stage of HCC, whereas the combination of chromosomal gains on 5q11.1–35.3 and 8q11.1–24.3 and loss of heterozygosity on 4q11–34.3 and 8p11.21–23.3 are late molecular events in advanced HCC.

Roessler  et al.20 combined array comparative genomic hybridization and gene expression data in 76 HBV-positive HCCs and attempted to elucidate genomic signatures associated with tumour progression and the prognosis of patients. These authors found a substantial correlation between copy number aberration and gene expression. In particular, a cluster of six genes located on chromosome 8p were deleted in tumours from patients with a poor prognosis; these genes included PROSC, SH2D4A and SORBS3, which showed tumour suppressive activities, along with DLEC1 (also known as DLC1), a known tumour suppressor gene.

Classification and prognosis prediction

In clinical settings, prognosis assessment and decisions regarding treatment are made on the basis of various tumour staging systems. The Edmondson–Steiner grading system has been applied to assess tumour aggressiveness in HCC, but data supporting its independent prognostic impact are quite weak.122 Therefore, new approaches and methodologies are needed to develop independent prognostic and predictive tools that might finally assist the clinical decision-making process to further improve curative strategies in HCC.

Genomic profiling, such as gene expression profiling, has been applied to classify HCCs.123, 124 Copy number alterations have also been integrated for classification and therapeutic target identification.125 In prognosis prediction, the expression pattern from the adjacent non-tumour tissue, which reflects “carcinogenic field effect”,126 was previously reported to correlate with patient survival.127 A large collection of human HCC samples from patients undergoing curative resection was analysed by microarray profiling. A panel of five genes, including TAF9, RAMP3, HN1, KRT19 and RAN, showed the strongest prognostic relevance and was selected for further analysis.128 The five-genes score was further validated in an independent, large cohort and also increased its prognostic accuracy when combined with the expression pattern in non-tumour tissues as described above.127

Integrative genomic analysis with gene mutation profiles will enable us to elucidate the genetic and epigenetic mechanism of HCC for better classification and to construct a better scoring system for prognosis prediction and treatment selection.

Conclusions

As summarized in this Review, advances in sequencing technologies have enabled the examination of liver cancer genomes at high resolution. In addition to copy number changes and mutations, analyses have identified additional genome alterations, including structural alterations, HBV integration, and retrotransposon changes. Integrated analyses of trans-omics data (genome, transcriptome and methylome data) have identified multiple critical genes and pathways implicated in hepatocarcinogenesis.

These comprehensive genomic analyses have already identified many potential therapeutic targets in liver cancer, including growth factor signalling/kinases (MET, FGF9/FGFR, PIK3CA/AKT/mTOR and JAK/STAT), the NFE2L2-mediated oxidative pathway and chromatin modifying factors. Functional analysis of these targets and the identification of novel potential driver mutations, and the construction of in vitro and in vivo therapeutic models to evaluate new molecular-targeting compounds are necessary for effective translational research connecting basic molecular science to the clinic.

The aetiological factors associated with liver cancer (for example hepatitis infection, alcohol and obesity) are well known, and ethnic differences in the prevalence of this disease are prominent. However, the effect of these factors on the accumulation of somatic changes in the liver and the influence of ethnic variation in risk factors on the susceptibility to this tumour remain unknown. In this sense, the international collaboration of cancer genome sequencing projects, such as the International Cancer Genome Consortium (ICGC), will contribute to an improved understanding of this tumour.

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