Advances and challenges in molecular understanding, early detection, and targeted treatment of liver cancer

Abstract

In this review, we explore the application of next-generation sequencing in liver cancer research, highlighting its potential in modern oncology. Liver cancer, particularly hepatocellular carcinoma, is driven by a complex interplay of genetic, epigenetic, and environmental factors. Key genetic alterations, such as mutations in TERT, TP53, and CTNNB1, alongside epigenetic modifications such as DNA methylation and histone remodeling, disrupt regulatory pathways and promote tumorigenesis. Environmental factors, including viral infections, alcohol consumption, and metabolic disorders such as nonalcoholic fatty liver disease, enhance hepatocarcinogenesis. The tumor microenvironment plays a pivotal role in liver cancer progression and therapy resistance, with immune cell infiltration, fibrosis, and angiogenesis supporting cancer cell survival. Advances in immune checkpoint inhibitors and chimeric antigen receptor T-cell therapies have shown potential, but the unique immunosuppressive milieu in liver cancer presents challenges. Dysregulation in pathways such as Wnt/β-catenin underscores the need for targeted therapeutic strategies. Next-generation sequencing is accelerating the identification of genetic and epigenetic alterations, enabling more precise diagnosis and personalized treatment plans. A deeper understanding of these molecular mechanisms is essential for advancing early detection and developing effective therapies against liver cancer.

Key Words: Liver cancer; Molecular mechanisms; Next-generation sequencing; Early detection; Wnt/β-catenin signaling

Core Tip: This review delves into the complexities of hepatocellular carcinoma (HCC); a highly aggressive form of liver cancer with a low 5-year survival rate and limited treatment options. It examines various factors contributing to HCC, highlights the need for improved early detection using biomarkers and advanced techniques like next-generation sequencing, and emphasizes the importance of understanding the molecular mechanisms involved, particularly the Wnt/β-catenin signaling pathway, to provide evidence for the development of more effective and personalized therapies.

INTRODUCTION

Liver cancer, primarily hepatocellular carcinoma (HCC), is a severe global health issue. According to the World Health Organization, it ranks sixth in prevalence and fourth in cancer-related mortality worldwide. Characterized by its rapid progression and poor prognosis, the 5-year survival rate for liver cancer patients is a dismal 3%, severely impacting their quality of life. Current treatment options, including surgical interventions, interventional therapies, local ablation, and targeted immunotherapies, have limited effectiveness, with survival rates remaining low for many patients even after treatment[1-3].

The origins of liver cancer are frequently tied to underlying conditions, which vary significantly across different regions. Major contributing factors include hepatitis B virus (HBV) infection, poor dietary habits, sedentary lifestyles, and exposure to fungal toxins[4]. Despite advancements in understanding these risk factors, the molecular mechanisms driving liver cancer remain poorly defined[5-8]. This lack of clarity hampers the development of effective treatments and underscores the need for more comprehensive research into the pathogenesis of HCC[9]. Efforts have focused on elucidating the intricate molecular events leading to liver cancer by exploring various dimensions of tumor biology, including genetic mutations[10,11], the microbiome [12,13], transcriptome alterations[14,15], epigenetic modifications[16,17], signaling pathways[18], and the tumor microenvironment. However, the complexity of liver cancer biology, marked by genetic heterogeneity and dynamic interactions between tumor cells and their surrounding environment, presents significant challenges. Given the aggressive nature of liver cancer, early detection and prediction are crucial in improving patient outcomes.

Early-stage liver cancer is often asymptomatic, making it challenging to diagnose until the disease has progressed to an advanced stage[19]. This emphasizes the need for effective screening programs and predictive models to identify at-risk individuals and detect cancer at a more treatable stage. Advances in molecular biology have opened new avenues for early detection and prediction of liver cancer. Biomarkers such as alpha-fetoprotein (AFP)[20-22], des-gamma-carboxy prothrombin (DCP)[23,24], and the glypican-3 (GPC3) protein[25,26] have shown promise in identifying early-stage HCC[27]. AFP, which was identified over 60 years ago, remains the most common serological marker of HCC[28]. The AFP gene, part of the albumin gene family, includes two distinct enhancer and silencer regions. In liver cancer, inhibition of the enhancer or deletion of the silencer can impair these regulatory elements, causing activation of the AFP promoter and leading to the overexpression of AFP, then the expression level and the protein level of AFP serve as a valuable biomarker for liver cancer[29]. DCP has been extensively studied over the past two decades, primarily in Asian countries, where it is recognized as a useful marker for liver tumors. Elevated DCP levels are commonly observed in HCC patients[30]. Normalization of DCP levels following effective cancer treatment strongly suggests that the tumor is the primary source of its production. GPC3 is a membrane proteoglycan that is typically absent in healthy adult liver but is overexpressed in HCC[31]. This overexpression is linked to a poor prognosis and contributes to HCC progression by interacting with Wnt signaling proteins and growth factors[32]. However, AFP, DCP, and GPC3 have notable limitations. AFP and DCP may lack specificity and sensitivity, often showing elevated levels in various liver conditions, while GPC3 may not be consistently expressed in all HCC cases and can be affected by the tumor microenvironment.

In addition to these classic biomarkers, non-classic markers like gamma-glutamyl transferase (GGT) and interleukin (IL)-8 have shown potential in assisting with tumor diagnosis and prognostication. Elevated GGT levels are associated with liver inflammation and oxidative stress, and recent studies have linked high GGT levels with poorer outcomes in HCC patients, suggesting its role as a marker of liver tumor progression and therapeutic resistance[33-35]. Similarly, IL-8, a pro-inflammatory cytokine, is elevated in the serum of HCC patients and has been implicated in promoting angiogenesis and immune evasion in the tumor microenvironment[36]. Elevated IL-8 levels are correlated with more aggressive HCC phenotypes and are associated with poor prognosis, making IL-8 a valuable indicator in assessing tumor progression and patient prognosis[36-38]. The addition of these non-classic markers to conventional biomarker panels may enhance diagnostic accuracy and provide more nuanced prognostic information, ultimately improving personalized treatment approaches for HCC.

Next-generation sequencing (NGS) has emerged as a transformative tool in this context, offering unprecedented insights into the molecular underpinnings of liver cancer[39-41]. NGS enables high-throughput and high-precision analysis of the genome, transcriptome, and epigenome, revealing critical mutations, gene expression patterns, and epigenetic modifications that drive tumorigenesis. circulating tumor DNA[42,43] and miRNAs[44,45] are emerging as potential noninvasive biomarkers for liver cancer screening. These biomarkers can be detected through blood tests, offering a feasible and less-invasive alternative to traditional diagnostic methods. By elucidating these molecular mechanisms, NGS provides valuable information for early detection, prognostic assessment, and the identification of novel therapeutic targets. The application of NGS in liver cancer research is rapidly advancing our understanding of the disease and holds promise for developing more effective and personalized treatment strategies.

METABOLIC AND PARANEOPLASTIC INDICATORS IN HCC

HCC is frequently associated with paraneoplastic syndromes, which manifest through metabolic abnormalities, including altered serum lipid and blood glucose levels[46-48]. These metabolic changes are byproducts of liver dysfunction and reflect the systemic impact of HCC. For instance, dyslipidemia is often seen in HCC due to the central role of the liver in lipid metabolism, while hyperglycemia may arise as a paraneoplastic syndrome or from insulin resistance exacerbated by chronic liver disease[49,50]. Elevated blood glucose levels, especially in patients with underlying diabetes, have been linked to poorer HCC prognosis and heightened risk of recurrence after treatment[51,52]. Paraneoplastic syndromes, such as erythrocytosis[53,54], hypercalcemia[55,56], and hypoglycemia[57-59], are significant clinical considerations as they often emerge alongside advanced HCC and complicate disease management.

Pathophysiology of liver cancer

Liver cancer is a complex disease with multiple etiological factors and molecular mechanisms contributing to its development and progression. The underlying mechanisms can be categorized into genetic, epigenetic, and environmental factors, all of which interact to drive hepatocarcinogenesis. Genetic changes, including point mutations, gene amplifications, alterations in promoter regions, and copy number variations often triggered by viral infections or exposure to hepatotoxic stress transform hepatocytes and contribute to tumor development. Key oncogenes and tumor suppressor genes are frequently altered in HCC[60]. Alcohol-related HCC exhibits significant enrichment in genetic alterations involving CTNNB1, TERT, CDKN2A, SMARCA2 and HGF. Mutations in the TERT promoter, activate telomerase expression, occur in 60% of cases, are among the most common genetic alterations in liver cancer[61,62]. Additionally, mutations in TP53, a crucial tumor suppressor, and CTNNB1, which encodes β-catenin, are frequently observed. These mutations disrupt cell cycle regulation, apoptosis, and Wnt/β-catenin signaling, promoting uncontrolled cell proliferation and survival. CTNNB1 and TP53 mutations are mutually exclusive and define two HCC subtypes. CTNNB1-mutant tumors are large, well-differentiated, and cholestatic, with microtrabecular and pseudoglandular patterns and few inflammatory infiltrates. TP53-mutant tumors are poorly differentiated, compact, multinucleated, pleomorphic, and often show vascular invasion[62]. Epigenetic alterations, including histone, DNA, and RNA modifications, also contribute to liver cancer development[63]. Aberrant methylation of tumor suppressor gene promoters leads to their silencing, while global hypomethylation can activate oncogenes. Histone modification, including methylation, phosphorylation, acetylation, ubiquitylation, and sumoylation, controls gene expression by altering chromatin structure. Acetylation and methylation play a role in chromatin remodeling and gene expression regulation[64]. Alterations in histone acetylation and methylation enzymes affect gene expression in non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), contributing to changes in hepatic metabolism, apoptosis, and progression to HCC. DNA epigenetics indicate modifications that affect gene expression without altering the DNA sequence. Aberrant hypermethylation of tumor suppressor genes in HCC, including CDH1, RASSF1A, GSTP, SOCS1, SFRP1, and PTEN, as well as CDK inhibitors such as p16^INK4A, p21 and p27^kip1, results in the loss of cell cycle checkpoints and promotes increased cell proliferation[65]. m6A methylation has been identified in HBV RNA during viral infections. Recent research shows that m6A factors can influence the viral life cycle and significantly impact virus–host interactions. m6A modification can regulate viral replication both directly, by adding m6A to viral genomic RNA or mRNA, and indirectly, by affecting the expression of genes or the stability of RNA involved in viral replication[66]. Chronic liver inflammation, often due to HBV or hepatitis C virus infection, is a significant risk factor for HCC[67,68]. Persistent inflammation induces a regenerative response in hepatocytes, increasing the risk of genetic and epigenetic alterations. Additionally, liver fibrosis and cirrhosis, resulting from chronic liver injury, create a pro-tumorigenic environment[69,70]. Activated hepatic stellate cells and the extracellular matrix components promote tumor cell proliferation, invasion, and angiogenesis[71]. Metabolic disorders, such as NAFLD and NASH, are increasingly recognized as significant contributors to liver cancer[72,73]. Insulin resistance, obesity, and lipid accumulation in hepatocytes lead to oxidative stress and chronic inflammation, promoting hepatocarcinogenesis[4]. Dietary factors, including aflatoxin exposure from contaminated food, also play a role in liver cancer development[74]. Aflatoxin B1, a potent carcinogen, induces mutations in the TP53 gene, further enhancing cancer risk[75]. The tumor microenvironment in the liver plays a crucial role in cancer progression[13]. Immune cells, fibroblasts, endothelial cells, and the extracellular matrix interact with tumor cells, influencing their behavior. Tumor-associated macrophages and myeloid-derived suppressor cells create an immunosuppressive environment, aiding tumor evasion from immune surveillance[76]. Liver cancer arises from a complex interplay of genetic, epigenetic, and environmental factors. A comprehensive understanding of these mechanisms is crucial for developing effective therapeutic strategies. Integrating these factors is essential for advancing early detection methods and optimizing targeted therapeutic approaches.

Role of tumor microenvironment in liver cancer progression and therapy resistance

Despite significant advances in liver cancer research, a deeper understanding of the molecular and cellular components of the tumor microenvironment is essential, as it plays a central role in the progression of HCC. The tumor microenvironment comprises a complex network of cancer cells, innate and adaptive immune cells, stromal cells, endothelial cells, and cancer-associated fibroblasts[77-79]. Within this environment, macrophages infiltrate[9,80], fibroblasts proliferate[81,82], and angiogenesis is promoted[83]; all of which contribute to tumor formation, survival, and metastasis. Efforts are underway to develop targeted treatments that inhibit angiogenesis, cellular adhesion, and immune cell infiltration in the tumor microenvironment. Although earlier research predominantly focused on adaptive immune cells, particularly T lymphocytes due to their cytotoxic potential against cancer cells, recent therapies have shifted toward targeting innate immune responses[78,84]. This shift has led to innovative approaches, such as immune checkpoint inhibitors (ICIs) and chimeric antigen receptor T cell therapies[85-87]. In treating advanced HCC, immunotherapeutics, such as monoclonal antibodies targeting cytotoxic T-lymphocyte-associated protein (CTLA)-4 and programmed cell death protein (PD)-1, have shown promise by inhibiting immune checkpoints, thereby enhancing immune responses against tumor cells[88-90]. Notablr’s unique immunosuppressive environment, which is tolerogenic to gut-derived microbial metabolites, presents challenges in achieving effective immune responses. Since 2017, ICIs such as pembrolizumab[91,92] and nivolumab[93,94], which target PD-1, have been approved as second-line therapies for advanced HCC, despite demonstrating only limited improvements in survival compared to sorafenib. Additionally, the Food and Drug Administration has approved a combination of nivolumab and ipilimumab (an anti-CTLA-4 antibody) for advanced HCC. However, these treatments yield responses in only 20%-30% of patients, highlighting an urgent need to identify biomarkers to predict therapy response and to develop combination treatments that could overcome resistance[95].

Wnt/β-catenin pathway of liver cancer

The Wnt/β-catenin signaling pathway, essential for embryonic development and cellular processes, is often dysregulated in liver cancers[18]. The pathway includes canonical and non-canonical routes, with the canonical pathway involving β-catenin, a key factor in tumorigenesis encoded by CTNNB1. Mutations in this pathway are common in liver tumors, and β-catenin also interacts with other signaling pathways. Wnt proteins act as ligands that bind to frizzled receptors, activating intracellular signaling pathways[96,97]. The canonical Wnt/β-catenin pathway, crucial for development and homeostasis, involves β-catenin regulating cell adhesion and transcription[98]. In the absence of Wnt signals, β-catenin is degraded by a destruction complex composed of AXIN, APC, glycogen synthase kinase 3, and casein kinase 1. Dysregulation of this pathway can contribute to liver diseases, including cancer[99,100].

The canonical Wnt/β-catenin signaling pathway is crucial in HCC pathogenesis. β-catenin activation occurs in 20%-35% of HCC cases[101], often due to mutations in the CTNNB1[102], which encode β-catenin and block its degradation, leading to its accumulation and nuclear translocation[103]. These mutations result in tumors with distinct features, including large size and well-differentiated structures[104]. While β-catenin alone is insufficient to induce HCC in mouse models, its activation can cooperate with other oncogenic pathways. In the mouse model, combined activation of insulin receptor substrate-1/mitogen-activated protein kinase and Wnt/β-catenin pathways are crucial for liver cancer development. Mice with a single transgene did not develop tumors, but double transgenic mice exhibited increased hepatocellular dysplasia and progressed to HCC. All transgenic lines showed significantly elevated mRNA levels of IGF-1, Wnt1, and Wnt3[105]. In another mouse model with a mutant β-catenin allele, isolated Wnt-activating β-catenin mutations are insufficient for liver cancer development. However, when combined with HRAS mutations, HCC develops with 100% incidence. While HRAS mutations alone cause large cell dysplasia, β-catenin mutations promote clonal expansion and progression to HCC[106]. In a mouse model with combined expression of c-MET-V5 and mutant β-catenin-Myc, HCC developed with significant similarity to clinical cases[107]. Additionally, mutations in AXIN1, present in 3%-16% of HCC cases, are often associated with HBV infection and chromosomal instability. Beyond genetic mutations, epigenetic factors and non-coding RNAs also contribute to aberrant Wnt/β-catenin activation in HCC. For example, SFRP3 promoter hypermethylation in HCC cell lines and tissues correlates with reduced SFRP3 expression. Demethylation restores SFRP3 levels, suggesting that hypermethylation contributes to HCC by downregulating SFRP3 and enhancing Wnt signaling[108]. miR-300 and various long noncoding RNAs modulate Wnt/β-catenin signaling, influencing tumor progression[109]. The tumor microenvironment, including autophagy and exosomes, further impacts Wnt/β-catenin signaling, affecting cancer cell metabolism, proliferation, and metastasis.

NGS and liver cancer

NGS has become crucial for identifying genetic variations and biomarkers in liver cancer. This technology enables comprehensive profiling of cancer genomes and transcriptomes, rapidly pinpointing potential driver mutations and therapeutic targets. NGS applications, including targeted NGS[110], whole-exome sequencing (WES)[111], and whole-genome sequencing (WGS)[112], aid in diagnosing, treatment planning, and risk assessment for liver cancer. Biomarkers for liver disease identified with protein and NGS were as summarized in Table 1.

Table 1 Biomarkers for liver disease identified through protein and sequencing analysis.

Applied method	Biomarkers (Target proteins and genes)	Ref.
Detection of protein content	AFP, DCP, GPC3, GGT, IL-8	Zhao et al[20]; Hu et al[21]; Hanif et al[22]; Lai et al[23]; Bertino et al[24]; Zhou et al[25]; Shimizu et al[26]
Epigenetic modification	CDH1, RASSF1A, GSTP, SOCS1, SFRP1, PTEN, p16, p21, p27 and m6A methylation of HBV RNA	Raggi and Invernizzi[65]; Kostyusheva et al[66]
Targeted NGS	TP53, TERT and TSC2	Lin et al[113]
WGS	CDKN2A, CCND1, APC, TERT, ASH1L, NCOR1, and MACROD2, ARID1A, TTC28, LRP1B and BCL9	Fujimoto et al[118]; Zhou et al[119]
WES	PCK2, HUWE1, ARID1A, RPS6KA3, NFE2L2, IRF2, APOB, P53, Keap1, CDKN1A, XPO1 and HIST1H1E	Gnirke et al[114]; Liu et al[115]; Guichard et al[116]; Zhou et al[117]
RNA-seq	PTPRC and FOXP3	Zhang et al[121]

NGS: Next-generation sequencing; WGS: Whole-genome sequencing; WES: Whole-exome sequencing; RNA-seq: RNA sequencing; AFP: Alpha-fetoprotein; DCP: Des-gamma-carboxy prothrombin; GPC3: Glypican-3; GGT: Gamma-glutamyl transferase; IL: Interleukin.

Targeted NGS: Targeted NGS is designed to focus on specific genes associated with liver cancer. These panels provide a detailed analysis of known cancer-related mutations, making them a cost-effective and rapid option for clinical use. However, their limitation lies in their narrow scope, as they miss mutations outside the targeted genes, potentially leading to incomplete risk assessments and missed novel biomarkers.

An ultra-deep targeted NGS was applied to analyze the 31 spatially distinct regions from 11 resected multifocal HCC samples. Truncal and branchy drivers were both economically and effectively identified. TP53 and TERT were the most commonly altered truncal drivers in multifocal HCC, and TSC2 was the most frequently mutated branchy driver[113].

WES covers: Whole protein-coding exons of the human genome (30-40 Mb, approximately 1%-2% of the human genome)[114] capture a broad range of genetic mutations relevant to liver cancer. WES was the most common platform for the Cancer Genome Atlas projects. The coverage of the target regions is usually more than 100 ×, which enables more accurate detection of detection of single nucleotide variants and short indels.

By whole-exome sequencing of a pleomorphic cell-type HCC tissue and its matched normal tissue, it is suggested that PCK2 and HUWE1 are associated with carcinoma cell proliferation in HCC[115].

WES identified 24 HCC tumors, revealing 135 homozygous deletions and 994 somatic mutations in genes with predicted functional consequences. Additionally, new recurrent alterations were discovered in four genes: ARID1A, RPS6KA3, NFE2L2, and IRF2[116]. Cell lines from HCC patients were analyzed using WES, showing an average of 16.55 (range, 15.38%-18.17%) heterogeneous mutations in each line. The main observed substitutions were C:G > T:A and T:A > C:G transitions, Seven reported driver gene mutations were observed in the three HCC cells, including APOB, P53, Keap1, CDKN1A, XPO1, ARID1A and HIST1H1E[117].

WGS: WGS provides a comprehensive view of the entire genome, including both coding and non-coding regions, structural variations, and epigenetic changes. This method identifies all types of genetic alterations, including novel and unexpected variants. The genetic changes were analyzed in 300 liver cancers, including point mutations, structural variations, and virus integrations. There was a correlation between structural variations and DNA replication timing, impacting known cancer-related genes such as CDKN2A, CCND1, APC, and TERT, as well as new genes such as ASH1L, NCOR1, and MACROD2, altering gene expression. The analysis identified recurrent structural variations targeting key driver genes such as TERT, CDKN2A, APC, and ARID1A, as well as newly identified genes such as TTC28, MACROD2, and LRP1B, leading to changes in gene expression in liver cancer genomes[118]. WGS of 40 pairs of primary and early-recurrent HBV-related HCC tumors from patients showed that the mutations and copy-number gains in BCL9 activated Wnt/β-catenin signaling and created an immune-excluded tumor microenvironment. BCL9 could be a new therapeutic target for recurrent HCC[119].

RNA sequencing: RNA sequencing (RNA-seq) has become a powerful tool in defining the transcriptomic changes related to HCC. A transcriptome-wide study of HCC in a group of United States Caucasian patients using RNA-seq technology revealed significant up-regulation of oxidative phosphorylation as the primary signal in HCC development, and signals related to DNA damage were also identified in HCC samples[120]. Single-cell RNA-seq (scRNA-seq) is a powerful tool for studying cellular components, cell-cell interactions, transition in cellular status, and clonal evolution of the tumor. A recent study conducted scRNA-seq on samples taken from eight patients with HCC. Researchers were able to identify three distinct HCC subtypes characterized by varying immune statuses. This analysis also shed light on the expression levels of chemokines and cytokines, as well as the metabolic features unique to each subtype, they also use PTPRC and FOXP3 to differentiate HCCs into the three subtypes[121]. According to a study on the spatial heterogeneity of the immune microenvironment in HCC, the researchers observed a gradual increase in the number of CD4 + effector memory T cells from the non-tumor region to the tumor core. Conversely, CD8 + effector memory T cells showed a decreasing trend in the same regions[122].

CONCLUSION

The integration of comprehensive molecular studies with emerging sequencing technologies is key to tackling the complexities of liver cancer. Continued research in this domain is essential for developing effective screening programs, early detection methodologies, and novel therapeutic approaches, ultimately aiming to improve survival rates and quality of life for liver cancer patients.