Abstract
Hepatocellular carcinoma (HCC) is a highly fatal disease, usually occurring as a result of an underlying chronic liver dysfunction. Despite the growing attention to preventive medicine and the advancement of imaging techniques, HCC is often diagnosed in an advanced stage and represents a leading cause of death among malignancies. Currently available treatment options show important limits, such as high recurrence of HCC, small number of properly matched donors for liver transplantation and low efficacy of systemic chemotherapies. For these reasons, an in-depth knowledge of the molecular basis of this carcinoma can play a pivotal role in obtaining an accurate diagnosis and in order to apply new therapeutic strategies. Recent studies offer an accurate picture of the main genes involved in the malignant transformation of hepatocytes, while the detection of circulating tumor DNA in HCC is leading to encouraging results. In addition, since some mendelian disorders are known to predispose to the onset of HCC, the early diagnosis of affected patients is mandatory in order to permit the best chance for successful treatment and surveillance. At the same time, this approach makes it possible to identify and assess relatives with an increased risk of developing HCC.
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Keywords
- Hepatocellular carcinoma
- Cirrhosis
- Somatic mutations
- Early diagnosis
- Therapeutic strategies
- Personalized medicine
- Circulating tumor DNA
- TERT gene
- TP53 gene
- CTNNB1 gene
1 Introduction
The process promoting the development of hepatocellular carcinoma (HCC) is similar to that underlying other types of cancer. It is a multistep mechanism involving a mixture of genetic and environmental factors. Several exogenous risk factors are implicated in the development and progression of HCC, notably hepatitis infections, alcohol abuse, metabolic syndrome, obesity, diabetes and many others. All these conditions favor a chronic inflammatory state leading to the deposition of fibrotic tissue and playing a crucial role in the onset of liver cirrhosis, which frequently precedes the development of the tumor. It is indeed well known that dysplastic hepatocytes grow inside the regenerative nodules progressively acquiring multiple genetic mutations leading to the neoplasm [1].
2 Genetic Landscape of Hepatocellular Carcinoma
The understanding of the genetic landscape of HCC has improved over the past few years as a result of massive advances in genomic technologies. Next-generation-sequencing techniques have allowed us to obtain an in-depth picture of the most frequently mutated genes in HCC. Pathogenic variants in several genes have been found in HCC samples. The genes most frequently mutated in this tumor, ordered according to the mutation rate, are: TERT, TP53, CTNNB1, AXIN1, LAMA2, ARID1A, ARID2, WWP1, RPS6KA3, ATM, CDKN2A, KMT2D, NFE2L2, ERRFI1, ZIC3, ALB, KMT2C, IRF2, BAZ2B, UBR3, and others [2]. A recent study showed that alterations in these genes can be detected also in plasma cell-free tumor DNA of patients with HCC [3].
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TERT
The TERT (telomerase reverse transcriptase; MIM *187270) gene pathogenic variants are found in more than 68% of HCC samples and they involve the promoter region in 44–59% of cases, representing the most frequently occurring point mutations in HCC [4] and the earliest alterations in hepatocarcinogenesis related to cirrhosis [5, 6]. TERT, the catalytic subunit of the telomerase complex, plays a fundamental role in maintaining the length of telomere caps. Pathogenic variants in its promotor, through recruitment of the transcription factor GABP [7], upregulate both telomerase promoter activity and TERT transcription. While the reduced length of telomere caps at the ends of chromosomes is responsible for DNA double-strand breaks, genomic instability and cell senescence, increased telomerase expression is involved in carcinogenesis [8].
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TP53
Mutations in the TP53 (tumor protein p53; MIM *191170) gene are detected in about 35–50% of HCC cases. The transcription factor p53, a tumor suppressor known to be involved in several malignancies, regulates cell cycle arrest, apoptosis, senescence, DNA repair and changes in metabolism, maintaining genomic integrity. TP53 alterations are responsible for the survival of aneuploid cells, and they can cause centrosome amplification and chromosome instability [9]. High chromosome instability has been reported in HCC patients diagnosed with TP53 pathogenic variants. The same patients also presented poor differentiation status of neoplasms [10], correlating with a poor prognosis [11]. A large number of TP53 missense mutations detected in HCC cases are localized in the DNA-binding domain of TP53, leading to a lower affinity in binding target genes [4].
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CTNNB1
The CTNNB1 (catenin, beta-1; MIM *116806) gene encodes β-catenin, an adherens junction protein, acting as a signaling molecule in the wingless-type (Wnt) pathway. Mutations in this gene cause an aberrant activation of the Wnt β-catenin pathway occurring in 20–40% of HCC samples [12]. β-catenin and Wnt-signaling activation can determine genomic instability, which becomes more evident in association with increased DNA damage or mismatch repair defects, frequently appearing in HCC development. Transient activation of the Wnt/β-catenin pathway can also induce TERT mRNA expression and an elevated telomerase activity in different cell lines, supporting the hypothesis that these genes interact in the process of hepatocarcinogenesis [13]. It is interesting to note that mutations in CTNNB1 are reported to be mutually exclusive with TP53 pathogenic variants [14].
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AXIN1
AXIN1 (axis inhibitor 1; MIM *603816) is a gene mutated in 5–10% of HCC cases. Its contribution to tumor growth is related to the activation of the Wnt β-catenin pathway [14]. Genetic alterations in CTNNB1 and AXIN1 are mutually exclusive, probably due to their opposite roles. AXIN1 is in fact a negative regulator of this cascade.
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LAMA2
LAMA2 (laminin, alpha-2; MIM *156225) encodes laminin-α2, a crucial component of the muscle basement membrane. It is expressed in skeletal muscle myoblasts and myotubes, where it promotes cell survival, myoblast fusion and myotube formation [15] and it seems to play a role in tumor suppression [2]. Biallelic germinal pathogenic variants of this gene have been reported in multiple types of muscular dystrophy. Somatic mutations of LAMA2, more frequently associated with other cancers, are reported in 5–12% of HCC patients [16].
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ARID1A and ARID2
ARID1A (AT-rich interactive domain-containing protein 1, MIM *603024) and ARID2 (AT-rich interactive domain-containing protein 2, MIM *609539) are mutated in up to 20% of tumoral tissue samples in patients with HCC. ARID1A is part of the BRG1-associated factor (BAF) complex [17] that regulates the chromatin structure mobilizing nucleosomes by sliding, expelling or inserting histones, modulating the accessibility of DNA to other systems involved in DNA transcription, replication and repair [4]. It is considered a tumor suppressor that is involved in the mismatch repair mechanism. The reduced expression of this gene is associated with a poor prognosis and facilitates HCC metastasis development. Hepatocyte-specific ARID1A knockout mice present with steatohepatitis and HCC [18].
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WWP1
WWP1 (WW domain-containing protein 1; MIM *602307) is an E3 ubiquitin ligase that plays a pivotal role in HCC tumorigenesis due to its function in regulation of signaling involving Smad4 and EGFR. WWP1 aberrant expression in HCC is associated with a poor prognosis [19].
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RPS6KA3
RPS6KA3 (ribosomal protein S6 kinase A3; MIM *300075) encodes a member of the ribosomal S6 family of serine/threonine kinases. Constitutional mutations of this gene cause Coffin–Lowry syndrome (MIM #303600), a rare X-linked dominant disease characterized by mental retardation, facial dysmorphisms, tapering fingers, small fingernails, hypotonia and skeletal anomalies. Ribosomal S6 kinase tumoral alterations interfere with p53 pathways implicated in DNA repair and in maintaining genomic stability [20].
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ATM
The ataxia telangiectasia mutated gene (ATM serine/threonine kinase; MIM *607585) is involved in DNA damage checkpoint and repair, together with p21, and its mutation rate amounts to 7% of HCC cases.
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CDKN2A
Mutations in the CDKN2A (cyclin-dependent kinase inhibitor 2A; MIM *600160) gene are detected in 6–30% of HCC cases. CDKN2A encodes two distinct proteins involved in the p53 and RB1 pathways, respectively, and represented by p16(INK4A), a cyclin-dependent kinase inhibitor and tumor suppressor that downregulates cell cycle progression, and p14(ARF), which plays a role in MDM2 stabilization. The impaired function of p16 can produce genomic instability, especially in tumors where defects in DNA checkpoint control and in repair mechanisms are already present [21].
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KMT2D
The KMT2D (lysine-specific methyltransferase 2D; MIM *602113) gene product methylates the Lys-4 position of histone H3 and it is considered a tumor suppressor due to its involvement in gene expression regulation. The mutation rate in HCC is around 6%. KMT2D seems to be involved in transcript elongation associated with histone H3K4 methylation [22]. Its mutations lead to genomic instability in genomic regions where early replicating fragile sites are located. Constitutional heterozygous pathogenic variants in KMT2D have been shown to cause Kabuki syndrome 1 (MIM #147920), a chromatinopathy characterized by peculiar facial dysmorphism, mental retardation, postnatal growth retardation, congenital heart disease and other anomalies [23].
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NFE2L2
NFE2L2 (nuclear factor erythroid 2-like 2; MIM *600492) represents a leucine zipper transcription factor that binds to the antioxidant response element (ARE) [24], preventing cancer development. Dysregulation of the NFE2L2 gene alters its antineoplastic activity. Somatic pathogenic variants are detected in about 5% of HCC. The combination of mutation in other genes, such as ATM or TP53, together with NFE2L2 alterations, has an additive effect in causing HCC genomic instability.
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ERRFI1
ERRFI1 (ERBB receptor feedback inhibitor 1; MIM *608069) gene mutations are found in around 5% of HCC samples. This gene encodes a cytoplasmic protein that binds and inhibits growth factor receptor kinases and their related signaling. The EGRF-mitogen-inducible gene 6 (MIG6) signal is involved in the inhibition of the EGFR and HGF pathways and its defective activity can induce genomic instability, facilitating the onset of HCC.
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ALB
ALB (albumin; MIM *103600) encodes the most common protein in human blood, which is produced in the liver and acts as the main regulator of colloid osmotic pressure and as a carrier for multiple molecules. Mutations in ALB have been reported in 5% of HCC samples. Experimental studies have proposed that ALB alterations can contribute to oxidative stress, while decreased serum albumin might have a role in HCC prognosis [25].
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KMT2C
KMT2C (lysine-specific methyltransferase 2C; MIM *606833) pathogenic variants had been reported in around 4% of HCC samples. This gene encodes a tumor suppressor member of the myeloid/lymphoid or mixed-lineage leukemia family and it mediates histone H3 methylation at lysine 4 [26], being part of the ASC-2/NCOA6 histone–methyltransferase complex (ASCOM). It acts as a transcriptional coactivator, playing a key role in epigenetics [27], especially in genomic instability [28].
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IRF2
Somatic mutations of the IRF2 (interferon regulatory factor 2; MIM *147576) gene had been reported in HCC with a mutation rate of about 4%. IRF2 is an antagonistic repressor of α and β interferon transcriptional activation (an IRF1-mediated process). It is known that IRF2 upregulation is involved in neoplasm development in mice [29], by promoting cell transformation and genomic instability.
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BAZ2B
Mutations in the BAZ2B (bromodomain adjacent to zinc finger domain, 2B; MIM *605683) gene have been found in around 3% of HCC samples. The function of the corresponding protein remains not well known, but it is thought to be part, as the majority of bromodomain-containing proteins, of the chromatin-dependent regulation of the transcription complex [30].
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UBR3
The UBR3 (ubiquitin protein ligase E3 component N-recognin 3; MIM *613831) gene function consist in regulating molecules involved in DNA repair and transcription. In animal models, this gene seems to upregulate the Hedgehog signaling pathway [31], the alterations of which have been extensively studied in cancer. It is known that haploinsufficiency of Patched-1, an antagonist of Hedgehog activation, induces genomic instability, promoting carcinogenesis. UBR3 alterations can be detected in around 2% of HCC patients.
3 Hepatocellular Carcinoma and Mendelian Disorders
HCC is not frequently encountered in hereditary cancer predisposition syndromes. At the same time, multiple mendelian diseases are known to confer an increased risk for developing HCC. The principal monogenic disorders associated with HCC are: alpha-1 antitrypsin deficiency (SERPINA1 gene), tyrosinemia (FAH gene), glycogen storage disease type I (HNF1A gene), acute intermittent and cutanea tarda porphyria (HMBS and UROD genes), hereditary hemochromatosis (HFE, HAMP, HJV, TFR2, and SLC40A1 genes) and Wilson’s disease (ATP7B gene) [32]. Similarly, settings of medium/low penetrance single-nucleotide polymorphisms (SNPs) can represent a significant risk factor for HCC. Various SNPs in different genes, identified through genome-wide association studies, have been shown to have a possible role in HCC development (DEPDC5, GRIK1, KIF1B, STAT4, MICA, DLC1, DDX18, PNPLA3, and TM6SF2 genes). In particular, the role of specific SNPs of PNPLA3 and TM6SF2 has been confirmed in studies conducted in a sample of individuals with alcoholic liver disease but also in patients with otherwise healthy liver [33].
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Valiante, M., Grammatico, P. (2023). Molecular and Genetic Mechanisms of Hepatocellular Carcinoma. In: Ettorre, G.M. (eds) Hepatocellular Carcinoma. Updates in Surgery. Springer, Cham. https://doi.org/10.1007/978-3-031-09371-5_2
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