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Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets
Abstract
Unlabelled:
Hepatocellular carcinomas (HCCs) are a heterogeneous group of tumors that differ in risk factors and genetic alterations. We further investigated transcriptome-genotype-phenotype correlations in HCC. Global transcriptome analyses were performed on 57 HCCs and 3 hepatocellular adenomas and validated by quantitative RT-PCR using 63 additional HCCs. We determined loss of heterozygosity, gene mutations, promoter methylation of CDH1 and CDKN2A, and HBV DNA copy number for each tumor. Unsupervised transcriptome analysis identified 6 robust subgroups of HCC (G1-G6) associated with clinical and genetic characteristics. G1 tumors were associated with low copy number of HBV and overexpression of genes expressed in fetal liver and controlled by parental imprinting. G2 included HCCs infected with a high copy number of HBV and mutations in PIK3CA and TP53. In these first groups, we detected specific activation of the AKT pathway. G3 tumors were typified by mutation of TP53 and overexpression of genes controlling the cell cycle. G4 was a heterogeneous subgroup of tumors including TCF1-mutated hepatocellular adenomas and carcinomas. G5 and G6 were strongly related to beta-catenin mutations that lead to Wnt pathway activation; in particular, G6 tumors were characterized by satellite nodules, higher activation of the Wnt pathway, and E-cadherin underexpression.
Conclusion:
These results have furthered our understanding of the genetic diversity of human HCC and have provided specific identifiers for classifying tumors. In addition, our classification has potential therapeutic implications because 50% of the tumors were related to WNT or AKT pathway activation, which potentially could be targeted by specific inhibiting therapies.
... Thus, there is an urgent need to explore the molecular targets involved in HCC development. Genomic studies have reported that PI3K and Akt contribute to signaling upstream of the mTOR pathway and play a notable role in network signaling pathways in HCC [18,19]. The cell cycle, proliferation, apoptosis, and metabolism are all regulated by the PI3K/Akt/mTOR pathway [20,21]. ...
... The cell cycle, proliferation, apoptosis, and metabolism are all regulated by the PI3K/Akt/mTOR pathway [20,21]. Moreover, the PI3K/ Akt pathway is vital to tumor progression [18,22]. Intriguingly, efforts to target protein kinases for treating malignancies have increased dramatically [23]. ...
The medicinal plant, Azadirachta indica (Neem), has been found to interfere with the carcinogenesis process and act as a tumor suppressor. This study evaluated the anti-cancer properties of methanol extract from Azadirachta Indica against the human hepatocellular carcinoma cell line (HepG2) using the in vitro MTT assay. The results demonstrated that the methanol extract of Azadirachta Indica exhibited significant cytotoxicity against HepG2 cells, with an IC50 value of 81.17 µg/ml. Furthermore, an in silico approach was used to investigate the interaction between Azadirachta indica and Akt1, a protein kinase implicated in the pathogenesis of hepatocellular car-cinoma (HCC) via the PI3K/Akt/mTOR pathway. The study identified two promising Akt1 inhibitors, 2′,3′-Dehydrosalannol, and Quercetin, with an improved binding affinity (ΔGbind) and a safe pharmacological profile. These compounds also showed hepatoprotective actions. Molecular dynamics simulations, specifically the MM-GBSA model, were used to further scrutinize the inhibitory potential of the docked bio-constituents. The results suggest that the proposed in silico pipeline can be useful in identifying lead candidates for HCC through early-stage rational drug design. These candidates can then be verified in vitro and in vivo before being tested in clinical trials as a mainstream target against HCC.
... It exhibits chromosomal instability, global DNA hypomethylation, and further subdivision into two subclasses: a "Wnt-TGFb subclass" and a "progenitor subclass" [97]. The "non-proliferation class" of HCC is more heterogeneous and linked to HCV or alcohol-related etiology, including less aggressive, chromosomally stable tumors retaining hepatocyte-like features [98]. This class comprises at least two subclasses, with the well-defined 'G6' subclass harboring catenin beta-1 (CTNNB1) mutations, leading to greater Wnt/b-catenin pathway activation, telomerase reverse transcriptase (TERT) promoter mutations, and the hypermethylation of genes encoding for cyclin-dependent kinase inhibitor 2A (CDKN2A) and E-cadherin (CDH1) promoters [98]. ...
... The "non-proliferation class" of HCC is more heterogeneous and linked to HCV or alcohol-related etiology, including less aggressive, chromosomally stable tumors retaining hepatocyte-like features [98]. This class comprises at least two subclasses, with the well-defined 'G6' subclass harboring catenin beta-1 (CTNNB1) mutations, leading to greater Wnt/b-catenin pathway activation, telomerase reverse transcriptase (TERT) promoter mutations, and the hypermethylation of genes encoding for cyclin-dependent kinase inhibitor 2A (CDKN2A) and E-cadherin (CDH1) promoters [98]. CTNNB1-mutated HCCs in this subclass exhibit immunological cold characteristics and specific histological features [16]. ...
Hepatocellular carcinoma (HCC) remains a global health challenge with limited treatment options and a poor prognosis for advanced-stage patients. Recent advancements in cancer immunotherapy have generated significant interest in exploring novel approaches to combat HCC. One such approach involves the unique and versatile subset of T cells known as γδ T cells. γδ T cells represent a distinct subset of T lymphocytes that differ from conventional αβ T cells in terms of antigen recognition and effector functions. They play a crucial role in immunosurveillance against various malignancies, including HCC. Recent studies have demonstrated that γδ T cells can directly recognize and target HCC cells, making them an attractive candidate for immunotherapy. In this article, we aimed to explore the role exerted by γδ T cells in the context of HCC. We investigate strategies designed to maximize the therapeutic effectiveness of these cells and examine the challenges and opportunities inherent in applying these research findings to clinical practice. The potential to bring about a revolutionary shift in HCC immunotherapy by capitalizing on the unique attributes of γδ T cells offers considerable promise for enhancing patient outcomes, warranting further investigation.
... This proliferation class is associated with HBV infection and α-fetoprotein overexpression 4 [5]. Nonproliferation classes often have activation of Wnt signaling transducer β-catenin (CTNNB1) and more TERT promoter mutations. ...
... Thus, β-catenin accumulates in the cytoplasm and is translocated into the nucleus, where it activates the transcription of downstream genes [17]. It is the oncogenic pathway most triggered in HCC by CTNNB1 activating mutations (11-37%) and AXIN1 inactivating mutations (5-15%) or adenomatous polyposis coli gene (APC) (1-2%) [5]. CTNNB1 mutations, which code for β-catenin, are in-frame deletions or substitutions at a hotspot in the domain targeted by the APC/AXIN1/GSK3B inhibitory complex. ...
... Multiomic analyses have classified HCC based on genetic, metabolic, immune, and chromosomal profiles. Many groups have reported molecular classification of HCC based on genomic profiles [28][29][30][31] that can ultimately be grouped into two major molecular types [32]. One (proliferation class) is broadly characterized by signal enhancement related to cell proliferation and cell cycle progression and is generally associated with a more aggressive phenotype. ...
Hepatocellular carcinoma (HCC) is the most common primary malignancy in the liver and is the third cause of cancer-related death worldwide. Surveillance with abdominal ultrasound should be offered to individuals at high risk for developing HCC. Accurate diagnosis, staging, and liver function are crucial when determining the optimal therapeutic approach. The BCLC staging system is widely endorsed in Western countries. Managing this pathology requires a multidisciplinary, personalized approach, generally with a multimodal strategy. Surgery remains the only curative option, albeit local and systemic therapy may also increase survival when surgery is not suitable. In advanced disease, systemic treatment should be offered to patients with ECOG/PS 0-1 and Child–Pugh class A.
... Clinically, such tumors exhibit a less aggressive phenotype, including better histological differentiation, lower alpha-fetoprotein, and lack of enrichment in adverse prognostic features [31]. HCV and alcohol-related HCC are more common in this subtype [32]. ...
Simple Summary
Hepatocellular carcinoma is the second leading cause of cancer-related deaths and the seventh most common cancer worldwide. Although there have been rapid developments in the treatment of hepatocellular carcinoma over the past decade, the incidence and mortality rates of hepatocellular carcinoma remain challenging. Only about 30% of patients can be treated with curative methods, while over 50% of patients require systemic treatment to prolong survival, with a limited benefit. Molecular targeted therapy and immunotherapy have brought about a revolution in hepatocellular carcinoma systemic treatment. Nevertheless, the treatment of hepatocellular carcinoma is still a challenge due to significant drug resistance, tumor heterogeneity, lack of druggable mutation targets, and lack of effective biomarkers. To improve outcomes of hepatocellular carcinoma patients, we need to gain a deeper understanding of the hepatocellular carcinoma genome and explore more combination treatment regimens.
Abstract
Hepatocellular carcinoma (HCC) is the most common form of liver cancer, accounting for ~90% of liver neoplasms. It is the second leading cause of cancer-related deaths and the seventh most common cancer worldwide. Although there have been rapid developments in the treatment of HCC over the past decade, the incidence and mortality rates of HCC remain a challenge. With the widespread use of the hepatitis B vaccine and antiviral therapy, the etiology of HCC is shifting more toward metabolic-associated steatohepatitis (MASH). Early-stage HCC can be treated with potentially curative strategies such as surgical resection, liver transplantation, and radiofrequency ablation, improving long-term survival. However, most HCC patients, when diagnosed, are already in the intermediate or advanced stages. Molecular targeted therapy, followed by immune checkpoint inhibitor immunotherapy, has been a revolution in HCC systemic treatment. Systemic treatment of HCC especially for patients with compromised liver function is still a challenge due to a significant resistance to immune checkpoint blockade, tumor heterogeneity, lack of oncogenic addiction, and lack of effective predictive and therapeutic biomarkers.
... To examine whether our clustering results are consistent with known transcriptomic subtypes from the literature 39,40,65,66 , we downloaded pathways associated with the transcriptomic subtypes curated in MSigDB 67 and applied GSVA analysis to each sample to compute a pathway score for each sample. Samples were then assigned to the subtype with the highest pathway score. ...
Solid tumors are complex ecosystems with heterogeneous 3D structures, but the spatial intra-tumor heterogeneity (sITH) at the macroscopic (i.e., whole tumor) level is under-explored. Using a phylogeographic approach, we sequence genomes and transcriptomes from 235 spatially informed sectors across 13 hepatocellular carcinomas (HCC), generating one of the largest datasets for studying sITH. We find that tumor heterogeneity in HCC segregates into spatially variegated blocks with large genotypic and phenotypic differences. By dissecting the transcriptomic heterogeneity, we discover that 30% of patients had a “spatially competing distribution” (SCD), where different spatial blocks have distinct transcriptomic subtypes co-existing within a tumor, capturing the critical transition period in disease progression. Interestingly, the tumor regions with more advanced transcriptomic subtypes (e.g., higher cell cycle) often take clonal dominance with a wider geographic range, rejecting neutral evolution for SCD patients. Extending the statistical tests for detecting natural selection to many non-SCD patients reveal varying levels of selective signal across different tumors, implying that many evolutionary forces including natural selection and geographic isolation can influence the overall pattern of sITH. Taken together, tumor phylogeography unravels a dynamic landscape of sITH, pinpointing important evolutionary and clinical consequences of spatial heterogeneity in cancer.
... Among the 25% of subjects with advancedstage HCC who respond to ICI treatment, it has been documented that viral-related HCC showed an overall higher survival rate compared to non-viral-related HCC [23]. The chronic inflammation that accompanies the development of HCC, which is one of the most heterogenous cancers [24][25][26][27], increases the complexity of the TME [28][29][30], highlighting the need to characterize the TME associated with HCC to better understand the host immune response against cancer cells. Furthermore, most of the studies so far available on the TME have been based on transcriptomic profiles of immune cells in tumor tissues from HCC of different etiologies. ...
Simple Summary
Chronic hepatitis B virus (HBV) infection remains a major public health problem and the most common risk factor for the development of hepatocellular carcinoma (HCC). The prognosis of HCC is still ominous because diagnosis is usually made at advanced stages and therapeutic options are limited. Immunotherapy is increasingly used for treatment of solid tumors, including advanced HCC. However, most HCC patients do not respond to immunotherapy. The tumor microenvironment (TME) plays a crucial role in intratumor heterogeneity and evolution, treatment failure, and, ultimately, disease outcome. However, there is very limited information on the TME of HBV-HCC. Our study provides evidence that HBV-HCC is characterized by two distinct immune subtypes, immune-high and immune-low. We documented a high expression of CTLA-4 in the immune-high subtype. Our results may have implications in the context of new treatment combinations for HCC to identify patients who might benefit the most from immunotherapy.
Abstract
HBV is the most common risk factor for HCC development, accounting for almost 50% of cases worldwide. Despite significant advances in immunotherapy, there is limited information on the HBV-HCC tumor microenvironment (TME), which may influence the response to checkpoint inhibitors. Here, we characterize the TME in a unique series of liver specimens from HBV-HCC patients to identify who might benefit from immunotherapy. By combining an extensive immunohistochemistry analysis with the transcriptomic profile of paired liver samples (tumor vs. nontumorous tissue) from 12 well-characterized Caucasian patients with HBV-HCC, we identified two distinct tumor subtypes that we defined immune-high and immune-low. The immune-high subtype, seen in half of the patients, is characterized by a high number of infiltrating B and T cells in association with stromal activation and a transcriptomic profile featuring inhibition of antigen presentation and CTL activation. All the immune-high tumors expressed high levels of CTLA-4 and low levels of PD-1, while PD-L1 was present only in four of six cases. In contrast, the immune-low subtype shows significantly lower lymphocyte infiltration and stromal activation. By whole exome sequencing, we documented that four out of six individuals with the immune-low subtype had missense mutations in the CTNNB1 gene, while only one patient had mutations in this gene in the immune-high subtype. Outside the tumor, there were no differences between the two subtypes. This study identifies two distinctive immune subtypes in HBV-associated HCC, regardless of the microenvironment observed in the surrounding nontumorous tissue, providing new insights into pathogenesis. These findings may be instrumental in the identification of patients who might benefit from immunotherapy.
... Based on molecular characteristics, HCC, a highly heterogeneous cancer, can be divided into two groups, including proliferative and non-proliferative subtypes [9][10][11] . Recently, cytokeratin 19 (CK19) has become an important marker of proliferation subtypes [12][13][14] . ...
Background and Objective: In clinical practice, CK19 can be an important predictor for the prognosis of HCC. Because the high incidence and mortality rates of HCC, more effective and practical prognostic prediction models are needed to be developed urgently.
Methods: A total of 1168 HCC patients, who underwent radical surgery at the Guangxi Medical University Cancer Hospital, between January 2014 and July 2019, were recruited with clinicopathological data collected. Of the clinicopathological data, the optimal cutoff value of CK19-positive HCC was determined by calculating the area under the curve (AUC) using survival analysis and time-dependent receiver operating characteristic (timeROC) curve analysis. The predictors were screened using univariate and multivariate COX regression and least absolute shrinkage and selection operator (LASSO) regression to construct nomogram prediction models, and their predictive potentials were assessed using calibration curves and AUC values.
Results: The 0% positive rate of CK19 was considered the optimal cutoff value to predict the poor prognosis of CK19-positive HCC. The survival analysis of 335 CK19-positive HCC showed no significant statistical differences in the overall survival (OS) and disease-free survival (DFS) of CK19-positive HCC patients. A five-factor risk (CK19, CA125, Edmondson, BMI and tumor number) scoring model and an OS nomograph model were constructed and established, and the OS nomograph model showed a good predictive performance and was subsequently verified.
Conclusion: A 0% expression level of CK19 protein may be an optimal threshold for predicting the prognosis of CK19-positive HCC. Based on this CK19 marker a good nomogram model was constructed to prediction of HCC prognosis.
... Recent studies have reported that NUP155 expression is correlated with the prognosis of various cancers [18,19]. Additionally, NUP155 activates the cell cycle protein-dependent kinase inhibitor p21 in the p53 (tumor suppressor) pathway and has a key role in the transcriptional response to DNA damage [20,21]. Basit et al. demonstrated that the cGAS-STING-TBK1-IRF3 signaling-mediated regulation of p21 in the innate immune response affected chromosomal stability [22]. ...
NUP155 is reported to be correlated with tumor development. However, the role of NUP155 in tumor physiology and the tumor immune microenvironment (TIME) has not been previously examined. This study comprehensively investigated the expression, immunological function, and prognostic significance of NUP155 in different cancer types. Bioinformatics analysis revealed that NUP155 was upregulated in 26 types of cancer. Additionally, NUP155 upregulation was strongly correlated with advanced pathological or clinical stages and poor prognosis in several cancers. Furthermore, NUP155 was significantly and positively correlated with DNA methylation, tumor mutational burden, microsatellite instability, and stemness score in most cancers. Additionally, NUP155 was also found to be involved in TIME and closely associated with tumor infiltrating immune cells and immunoregulation-related genes. Functional enrichment analysis revealed a strong correlation between NUP155 and immunomodulatory pathways, especially antigen processing and presentation. The role of NUP155 in breast cancer has not been examined. This study, for the first time, demonstrated that NUP155 was upregulated in breast invasive carcinoma (BRCA) cells and revealed its oncogenic role in BRCA using molecular biology experiments. Thus, our study highlights the potential value of NUP155 as a biomarker in the assessment of prognostic prediction, tumor microenvironment and immunotherapeutic response in pan-cancer.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12885-024-12039-6.
... Although the authors did not evaluate the intratumoral steatosis of the TGFβ-Wnt subclass in their study, the results suggest that our SBC-HCC may be closely related to the TGFβ-Wnt subclass. Interestingly, among the non-proliferation classes associated with HCV infection and alcohol, Boyault's G4 subclass and Murai's Class II subclass are characterized by steatohepatic HCC, a well differentiated tumor and good prognosis, with an active immune response, and lower TP53 mutation rates [28][29][30] . This is similar to the steatotic HCC subtype we identified in the TCGA HCC cohort. ...
Abnormal lipid metabolism promotes hepatocellular carcinoma (HCC) progression, which engenders therapeutic difficulties owing to unclear mechanisms of the phenomenon. We precisely described a special steatotic HCC subtype with HBV-related cirrhosis and probed its drivers. Hematoxylin-eosin (HE) staining of 245 HCC samples revealed a special HCC subtype (41 cases) characterized by HBV-related cirrhosis and intratumoral steatosis without fatty liver background, defined as steatotic HCC with HBV-related cirrhosis (SBC-HCC). SBC-HCC exhibits a larger tumor volume and worse prognosis than non-SBC-HCC. Screening for driver genes promoting fatty acid (FA) biosynthesis in the Gao’s HBV-related cirrhosis HCC cases and GSE121248’ HBV-related HCC cases revealed that high expression of SOCS5 predicts increased FA synthesis and that SOCS5 is upregulated in SBC-HCC. Through proteomics, metabolomics, and both in vivo and in vitro experiments, we demonstrated that SOCS5 induces lipid accumulation to promote HCC metastasis. Mechanistically, through co-IP and GST-pulldown experiments, we found that the SOCS5-SH2 domain, especially the amino acids Y413 and D443, act as critical binding sites for the RBMX-RRM domain. SOCS5-RBMX costimulates the promoter of SREBP1, inducing de novo lipogenesis, while mutations in the SH2 domain, Y413, and D443 reverse this effect. These findings precisely identified SBC-HCC as a special steatotic HCC subtype and highlighted a new mechanism by which SOCS5 promotes SBC-HCC metastasis.
... One of the molecular characteristics of HCC is the transcriptomic imbalance that drives rapid cell cycle progression and uncontrolled cell proliferation (Lee et al., 2004;Boyault et al., 2007;Calderaro et al., 2019;Rebouissou and Nault, 2020). This imbalanced transcriptional program generally requires (1) a DNA binding, TF-driven, cell cycle-related transcriptional profile to globally increase pre-RNA synthesis and (2) RBPs to modulate the posttranscriptional profile to regulate cell cyclerelated RNA metabolism and protein production. ...
The cell cycle is a highly regulated process in which proteins involved in cell cycle progression exhibit periodic expression patterns, controlled by specific mechanisms such as transcription, translation, and degradation. However, the precise mechanisms underlying the oscillations of mRNA levels in cell cycle regulators are not fully understood. In this study, we observed that the stability of cyclin D1 (CCND1) mRNA fluctuates during the cell cycle, with increased stability during interphase and decreased stability during the M phase. Additionally, we identified a key RNA binding protein, positive coactivator 4 (PC4), which plays a crucial role in stabilizing CCND1 mRNA and regulating its periodic expression. Moreover, the binding affinity of PC4 to CCND1 mRNA is modulated by two cell cycle–specific posttranslational modifications: ubiquitination of K68 enhances binding and stabilizes the CCND1 transcript during interphase, while phosphorylation of S17 inhibits binding during the M phase, leading to degradation of CCND1 mRNA. Remarkably, PC4 promotes the transition from G1 to S phase in the cell cycle, and depletion of PC4 enhances the efficacy of CDK4/6 inhibitors in hepatocellular carcinoma, suggesting that PC4 could serve as a potential therapeutic target. These findings provide valuable insights into the intricate regulation of cell cycle dynamics.
... 5,6 To finely tune its gene repertoire, b-catenin is able to cooperate with a plethora of histone modifiers and chromatin remodelers, 7 numbers of them being mutated in HCC. 8 In HB, additional mutations can also affect chromatin modifiers or long non-coding RNAs (lncRNAs) produced from parentally imprinted clusters, such as H19 or MEG3. 9,10 The DLK1/DIO3 locus encodes the largest cluster of non-coding RNAs (ncRNAs), including 54 microRNAs (miRNAs), several small nucleolar RNAs (snoRNAs), and lncRNAs (e.g., MEG3, MEG8/RIAN, MEG9/MIRG) expressed from the maternal allele but also encoding paternally expressed RNAs such as DLK1, RTL1, and DIO3 ( Figure 1A). ...
The CTNNB1 gene, encoding β-catenin, is frequently mutated in hepatocellular carcinoma (HCC, ∼30%) and in hepatoblastoma (HB, >80%), in which DLK1/DIO3 locus induction is correlated with CTNNB1 mutations. Here, we aim to decipher how sustained β-catenin activation regulates DLK1/DIO3 locus expression and the role this locus plays in HB and HCC development in mouse models deleted for Apc (ApcΔhep) or Ctnnb1-exon 3 (β-cateninΔExon3) and in human CTNNB1-mutated hepatic cancer cells. We identified an enhancer site bound by TCF-4/β-catenin complexes in an open conformation upon sustained β-catenin activation (DLK1-WRE) and increasing DLK1/DIO3 locus transcription in β-catenin-mutated human HB and mouse models. DLK1-WRE editing by CRISPR/Cas9 approach impaired DLK1/DIO3 locus expression and slowed tumor growth in subcutaneous CTNNB1-mutated tumor cell grafts, ApcΔhep HB and β-cateninΔExon3 HCC. Tumor growth inhibition resulted either from increased FADD expression and subsequent caspase-3 cleavage in the first case, or from decreased expression of cell cycle actors regulated by FoxM1 in the others. Therefore, the DLK1/DIO3 locus is an essential determinant of FoxM1-dependent cell proliferation during β-catenin-driven liver tumorigenesis. Targeting the DLK1-WRE enhancer to silence the DLK1/DIO3 locus might thus represent an interesting therapeutic strategy to restrict tumor growth in primary liver cancers with CTNNB1 mutations.
... 15 Besides, liver tumors found in NASH patients have been classified as poorly proliferating tumors. 41 Accordingly, Ki67 immunolabeling led to very weak or no staining inside or outside liver tumors of mice included in our experimental protocol (data not shown). ...
The global pandemic of metabolic diseases has increased the incidence of hepatocellular carcinoma (HCC) in the context of non‐alcoholic steatohepatitis (NASH). The downregulation of the E3 ubiquitin ligase TRIM21 has been linked to poor prognosis in different cancers including HCC. In order to investigate the role of TRIM21 in liver cancer progression on NASH, Trim21+/+ and Trim21−/− male mice were injected with streptozotocin at the neonatal stage. The hypoinsulinemic mice were then fed with a high‐fat high‐cholesterol diet (HFHCD) for 4, 8 or 12 weeks. All mice developed NASH which systematically resulted in HCC progression. Interestingly, compared to the Trim21+/+ control mice, liver damage was worsened in Trim21−/− mice, with more HCC nodules found after 12 weeks on HFHCD. Immune population analysis in the spleen and liver revealed a higher proportion of CD4⁺PD‐1⁺ and CD8⁺PD‐1⁺ T cells in Trim21−/− mice. The liver and HCC tumors of Trim21−/− mice also exhibited an increase in the number of PD‐L1⁺ and CD68⁺ PD‐L1⁺ cells. Thus, TRIM21 limits the emergence of HCC nodules in mice with NASH by potentially restricting the expression of PD‐1 in lymphocytes and PD‐L1 in tumors.
... Transcriptomic profiling of HCC tumors reveals a subset of HCCs that are less differentiated expressing progenitor markers IGF2, EpCAM, and AFP (ref. [36][37][38]). Hepatocytes might dedifferentiate into progenitor-like cells before transforming into HCC (ref. ...
Hepatocarcinogenesis is a multi-step process. However, the regulators of hepatocellular carcinoma (HCC) initiation are understudied. Adult liver-specific gene expression was globally downregulated in HCC. We hypothesize that adult liver-specific genes, especially adult liver-enriched transcription factors may exert tumor-suppressive functions in HCC. In this study, we identify ZBTB7B, an adult liver-enriched transcription factor as a permissive regulator of HCC initiation. ZBTB7B is highly expressed in hepatocytes in adult livers, compared to fetal livers. To evaluate the functions of ZBTB7B in hepatocarcinogenesis, we performed hepatocyte-specific ZBTB7B knockout in hydrodynamic oncogene transfer-induced mouse liver cancer models. Hepatocyte-specific knockout of ZBTB7B promotes activated Akt and N-Ras-induced HCC development. Moreover, ZBTB7B deficiency sensitizes hepatocytes to a single oncogene Akt-induced oncogenic transformation and HCC initiation, which is otherwise incompetent in inducing HCC. ZBTB7B deficiency accelerates HCC initiation by down-regulating adult liver-specific gene expression and priming livers to a fetal-like state. The molecular mechanism underlying ZBTB7B functions in hepatocytes was investigated by integrated transcriptomic, phosphoproteomic, and chromatin immunoprecipitation-sequencing analyses. Integrative multi-omics analyses identify c-Jun as the core signaling node in ZBTB7B-deficient liver cancer initiation. c-Jun is a direct target of ZBTB7B essential to accelerated liver cancer initiation in ZBTB7B-deficient livers. Knockdown of c-Jun expression or dominant negative c-Jun expression delays HCC development in ZBTB7B-deficient livers. In addition, ZBTB7B competes with c-Jun for chromatin binding. Ectopic ZBTB7B expression attenuates the tumor-promoting functions of c-Jun. Expression of ZBTB7B signature, composed of 140 genes co-regulated by ZBTB7B and c-Jun, is significantly downregulated in early-stage HCCs compared to adjacent normal tissues, correlates to liver-specific gene expression, and is associated with good prognosis in human HCC. Thus, ZBTB7B functions as a permissive regulator of HCC initiation by directly regulating c-Jun expression and function.
... To compare our four subtypes with previously identified molecular subtypes of HCC, 368 HCC samples were classified according to molecular signatures from previous studies. Seven published HCC classifications were collected for comparison: cholangiocarcinomalike HCC (CLHCC) [19], Chiang classification [23], metastasis gene signature [22], activation and silencing of the Hippo pathway [18], Hoshida subtypes [21], Boyault classification [63], and 65-gene-based risk score (65RS), calculated via the method described in this study [24]. Signatures for calculating ssGSEA score to explore the correlation of distinctive characterization between subtypes were derived from previous studies (Supplementary Table S7). ...
Hepatocellular carcinoma (HCC) is a highly detrimental cancer type and has limited therapeutic options, posing significant threats to human health. The development of HCC has been associated with a disorder in bile acid (BA) metabolism. In this study, we employed an integrative approach, combining various datasets and omics analyses, to comprehensively characterize the tumor microenvironment in HCC based on genes related to BA metabolism. Our analysis resulted in the classification of HCC samples into four subtypes (C1, C2a, C2b, and C3). Notably, subtype C2a, characterized by the highest bile acid metabolism score (BAMS), exhibited the highest survival probability. This subtype also demonstrated increased immune cell infiltration, lower cell cycle scores, reduced AFP levels, and a lower risk of metastasis compared to subtypes C1 and C3. Subtype C1 displayed poorer survival probability and elevated cell cycle scores. Importantly, the identified subtypes based on BAMS showed potential relevance to the gene expression of drug targets in currently approved drugs and those under clinical research. Genes encoding VEGFR (FLT4 and KDR) and MET were elevated in C2, while genes such as TGFBR1, TGFB1, ADORA3, SRC, BRAF, RET, FLT3, KIT, PDGFRA, and PDGFRB were elevated in C1. Additionally, FGFR2 and FGFR3, along with immune target genes including PDCD1 and CTLA4, were higher in C3. This suggests that subtypes C1, C2, and C3 might represent distinct potential candidates for TGFB1 inhibitors, VEGFR inhibitors, and immune checkpoint blockade treatments, respectively. Significantly, both bulk and single-cell transcriptome analyses unveiled a negative correlation between BA metabolism and cell cycle-related pathways. In vitro experiments further confirmed that the treatment of HCC cell lines with BA receptor agonist ursodeoxycholic acid led to the downregulation of the expression of cell cycle-related genes. Our findings suggest a plausible involvement of BA metabolism in liver carcinogenesis, potentially mediated through the regulation of tumor cell cycles and the immune microenvironment. This preliminary understanding lays the groundwork for future investigations to validate and elucidate the specific mechanisms underlying this potential association. Furthermore, this study provides a novel foundation for future precise molecular typing and the design of systemic clinical trials for HCC therapy.
... The S1 subtype was characterized by aberrant activation of the WNT oncogenic signal, S2 was typi ed by elevated plasma alpha-fetoprotein (AFP) levels and concurrent AKT activation, and S3 was associated with increased expression of genes related to hepatic function and improved patients' prognosis (33). A further six molecular subtypes of HCC were identi ed by Sandrine Boyault et al., in which the G3 subtype exhibited TP53 mutations and overexpression of cell cycle regulation genes, indicating a poorer prognosis for these patients (31). It was observed that the signi cant correlation between our consensus subtype CS3 and SURVIVAL_DOWN, Hoshida_S1/S2, as well as Boyault_G3, con rming the depressing prognostic outcomes of CS3 and emphasizing the need for more attention on these patients' clinical treatments. ...
O-glycosylation exerts significant influence on cellular physiological processes and disease regulation by modulating the structure, function, and stability of proteins. However, there is still a lack of research focusing on O-glycosylation in relation to the prognosis of HCC patients. Here we explored expression and function of O-glycosylation gene in HCC from both bulk and single-cell perspectives. The multi-omics data associated with O-glycosylation, identified through the Weighted Gene Co-expression Network Analysis (WGCNA), combined with ten distinct clustering algorithms to define the molecular subgroups of HCC. CS1 was characterized by significant genomic variation, moderate immune cell infiltration and immune function enrichment. CS2 performed a better prognosis, and was featured by stable genomic structure, an immune-hot phenotype with rich immune cell infiltration and sensitive to immunotherapy. CS3 was characterized by a poor prognosis, outstanding genomic instability, an immune-cold phenotype, but can benefit more from treatment with drugs such as sorafenib, cisplatin, paclitaxel, and gemcitabine. Ultimately, we re-emphasized O-glycosylation genes in individual HCC patients, deploying 59 types of machine learning to construct and evaluate the prognostic signature. The microarray results indicated a pronounced upregulation of Oglycosylation hub genes involved in HCC stratification and modeling within HCC tumorous tissues. In conclusion, we have highlighted the significant impacts of O-glycosylation on HCC by redefining the subtypes of HCC as well as constructing the CMLS. This research has established an optimized decision-making platform that enables precise stratification of HCC patients, refines tumor treatment plans, and predicts patient survivability holding broad clinical implications.
... Macrotrabecular-massive hepatocellular carcinoma (MTM-HCC) is a histopathological subtype defined by the World Health Organization (WHO) that has received significant clinical attention due to its association with poor prognosis [1,2]. According to genomic profiling and next-generation sequencing, HCC with varied histologic features was divided into G1-G6 classification [3]. MTM-HCC belongs to the G3 subtype, which is known for its clinical and genetic features such as TP53 mutations, FGF19 amplifications, high alphafetoprotein (AFP) serum level, satellite nodules, and macroor microvascular invasion [4]. ...
To evaluate the efficacy of MRI-based radiomics and clinical models in predicting MTM-HCC. Additionally, to investigate the ability of the radiomics model designed for MTM-HCC identification in predicting disease-free survival (DFS) in patients with HCC.
A total of 336 patients who underwent oncological resection for HCC between June 2007 and March 2021 were included. 127 patients in Cohort1 were used for MTM-HCC identification, and 209 patients in Cohort2 for prognostic analyses. Radiomics analysis was performed using volumes of interest of HCC delineated on pre-operative MRI images. Radiomics and clinical models were developed using Random Forest algorithm in Cohort1 and a radiomics probability (RP) of MTM-HCC was obtained from the radiomics model. Based on the RP, patients in Cohort2 were divided into a RAD-MTM-HCC (RAD-M) group and a RAD-non-MTM-HCC (RAD-nM) group. Univariate and multivariate Cox regression analyses were employed to identify the independent predictors for DFS of patients in Cohort2. Kaplan–Meier curves were used to compare the DFS between different groups pf patients based on the predictors.
The radiomics model for identifying MTM-HCC showed AUCs of 0.916 (95% CI: 0.858–0.960) and 0.833 (95% CI: 0.675–0.935), and the clinical model showed AUCs of 0.760 (95% CI: 0.669–0.836) and 0.704 (95% CI: 0.532–0.843) in the respective training and validation sets. Furthermore, the radiomics biomarker RP, portal or hepatic vein tumor thrombus, irregular rim-like arterial phase hyperenhancement (IRE) and AFP were independent predictors of DFS in patients with HCC. The DFS of RAD-nM group was significantly higher than that of the RAD-M group (p < .001).
MR-based clinical and radiomic models have the potential to accurately diagnose MTM-HCC. Moreover, the radiomics signature designed to identify MTM-HCC also can be used to predict prognosis in patients with HCC, realizing the diagnostic and prognostic aims at the same time.
... Advances in omics technologies permit the large-scale analysis of the molecular characteristics for HCC and have defined several clinal patient subtypes. Transcriptomic subtyping has revealed HCC subclasses that differ in the expression of genes related to proliferation, stemness, metabolism, hepatocyte differentiation, and liver function (8)(9)(10)(11)(12)(13). Among these transcriptome-based subtyping, Sia et al. divided HCC into two subtypes according to distinct TME characteristics, where "immune active" subtype demonstrated favorable prognosis and "immune exhausted" subtype had a bad prognosis. ...
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide, ranking fourth in frequency. The relationship between metabolic reprogramming and immune infiltration has been identified as having a crucial impact on HCC progression. However, a deeper understanding of the interplay between the immune system and metabolism in the HCC microenvironment is required. In this study, we used a proteomic dataset to identify three immune subtypes (IM1-IM3) in HCC, each of which has distinctive clinical, immune, and metabolic characteristics. Among these subtypes, IM3 was found to have the poorest prognosis, with the highest levels of immune infiltration and T-cell exhaustion. Furthermore, IM3 showed elevated glycolysis and reduced bile acid metabolism, which was strongly correlated with CD8 T cell exhaustion and regulatory T cell accumulation. Our study presents the proteomic immune stratification of HCC, revealing the possible link between immune cells and reprogramming of HCC glycolysis and bile acid metabolism, which may be a viable therapeutic strategy to improve HCC immunotherapy.
... Still, combining publicly available whole-genome and transcriptome sequencing data, we demonstrated that MHCC97 cells Notably, all sequenced DIAMOND tumours show a consistent upregulation of Ctnnb1 and Lef1, while Tcf7l2 is not differentially regulated. This expression pattern of these genes is similar to human HCC subtype G6 36,37 and contrasts somewhat with tumours generated via targeted genomic deletions of Ctnnb1 exon 3 7 that give rise to two distinct tumour types with either all three genes upregulated or all three genes unchanged. This indicates that metabolic input may be an important modulator of the transcriptional response to aberrant Wnt-activation that should be considered in the clinical setting. ...
Several preclinical models have been recently developed for metabolic associated fatty liver disease (MAFLD) and associated hepatocellular carcinoma (HCC) but comprehensive analysis of the regulatory and transcriptional landscapes underlying disease in these models are still missing. We investigated the regulatory and transcriptional landscape in fatty livers and liver tumours from DIAMOND mice that faithfully mimic human HCC development in the context of MAFLD. RNA-sequencing and ChIP-sequencing revealed rewiring of the Wnt/β-catenin regulatory network in DIAMOND tumours, as manifested by chromatin remodelling and associated switching in the expression of the canonical TCF/LEF downstream effectors. We identified splicing as a major mechanism leading to constitutive oncogenic activation of β-catenin in a large subset of DIAMOND tumours, a mechanism that is independent on somatic mutations in the locus and that has not been previously shown. Similar splicing events were found in a fraction of human HCC and hepatoblastoma samples.
... In addition, we found that RMI2 expression was significantly correlated with satellite nodules in HCC patients. A study [36] that used transcriptome analysis to identify 6 subgroups of HCC associated with clinical and genetic characteristics reported that Group-6 tumors were characterized by satellite nodules and strongly related to β-catenin mutations that lead to Wnt pathway activation. This is consistent with our findings. ...
Background
The Wnt/β-catenin signaling pathway plays an important role in embryogenesis and tumorigenesis. In human cancer, abnormal activity of Wnt/β-catenin signaling pathway induces overexpressed of downstream genes, and initiate oncogene. There are several target genes known to be key players in tumorigenesis, such as c-myc, cyclin D1, MMPs or survivin. Therefore, identifying the target genes of Wnt/β-catenin signaling pathway is important to understanding Wnt/β-catenin-mediated carcinogenesis. In this study, we developed a combined bioinformatics and experimental approach to find potential target genes.
Methods
Luciferase reporter assay was used to analyze the promoter activity of RMI2. WST1 cell proliferation assays and transwell assays were performed to determine the proliferation and migration capacities of RMI2 overexpressing or knockdown stable hepatic cells. Finally, xenograft experiments were performed to measure the tumor formation capacity in vivo.
Results
The results showed that RMI2 mRNA was upregulated after LiCl treatment and Wnt3a-conditioned medium in a culture of SK-hep-1 cell lines. A chromatin immunoprecipitation (ChIP) assay showed that the β-catenin/T cell-specific factor (TCF) complex binds to the putative TCF binding site of the RMI2 promoter. We then found a TCF binding site at − 333/− 326 of the RMI2 promoter, which is crucial for β-catenin responsiveness in liver cell lines. RMI2 was overexpressed in hepatoma tissue and cell lines, and it promoted the migration and invasion of HCC cells. Moreover, RMI2 upregulated the expression of epithelial-mesenchymal transition (EMT) markers and the Wnt3a/β-catenin-related genes, but silencing RMI2 had the opposite effects. Notably, the expression of RMI2 was positively correlated with the clinical data of HCC patients who had significantly shorter overall survival (OS) and disease-free survival (DFS) (Both: P < 0.05). In addition, a total of 373 HCC patients’ data from the Caner Genome Atlas project (TCGA) were used to validate our findings.
Conclusions
Taking all these findings together, we determined that RMI2 was a new target gene of the Wnt/β-catenin signaling pathway. We also found that RMI2 promotes EMT markers, HCC cell invasion, and metastasis, which indicated that RMI2 is a potential target for preventing or at least mitigating the progression of HCC.
Hepatocellular carcinoma (HCC) often develops from chronic liver inflammation. Inflammation within a tumor can either promote cancer progression or activate an immune response against it. This study aims to determine the clinical significance of enhanced inflammation in HCC.
Data from 655 HCC patients across four cohorts (TCGA, GSE6764, GSE76427, GSE89377) were examined. Inflammatory response was quantified using a scoring system derived from the gene set variation analysis of the “INFLAMMATORY_RESPONSE” gene set.
A stepwise increase in inflammatory response was noted from normal liver to cirrhosis, with consistently lower levels in HCC across both GSE6764 and GSE89377 cohorts (both p < 0.001). Similar trends were observed in interferon response, pathways such as IL6/JAK/STAT3 and complement signaling, coagulation cascade, and allograft rejection (all p < 0.02). HCCs with high inflammatory response were associated with increased immune cell infiltrations (p < 0.01) and cytolytic activity (p < 0.001). Interestingly, these HCCs had reduced mutation rates, no relationship with cell proliferation, and displayed both immune responses and pro-cancerous signals including epithelial-mesenchymal transition, KRAS, and hypoxia. Further, a high inflammatory score correlated with improved disease-free survival in TCGA (p = 0.034) and overall survival in GSE76427 (p = 0.008).
HCC with higher levels of inflammatory response demonstrated increased immune cell infiltration, enhanced immune-related and other pro-cancerous-related signaling, and showed a trend toward a better patient prognosis.
Abstract
Background
Standardized pathological evaluation based on immunohistochemical (IHC) analysis could improve HCC diagnoses worldwide. We evaluated differences in clinicopathological subgroups in HCCs from two academic institutions in Tokyo-Japan, and Jakarta-Indonesia.
Methods
Clinicopathological parameters and molecular expression patterns were evaluated in 35 HCCs from Indonesia and 41 HCCs from Japan. IHC analysis of biliary/stem-cell (B/S) markers (cytokeratin 19, sal-like protein 4, epithelial cell adhesion molecule) and Wnt/β-catenin (W/B) signaling-related molecules (β-catenin, glutamine synthetase) could determine the IHC-based subgroups. For immunosubtypes categorization, CD3/CD79α double immunohistochemistry was done to evaluate the infiltration of T and B cells. CD34 staining allowed identification of vessels that encapsulated tumor clusters (VETC).
Results
Indonesian HCC patients were mostly <60 years old (66%) with a hepatitis B virus (HBV) background (82%), in contrast to Japanese HCC patients (8% and 19%, respectively, both P<0.001). In comparison with Japanese, Indonesian cases more frequently had >5 cm tumor size (74% vs 23%, P=0.001), poor differentiation (40% vs 24%), portal vein invasion (80% vs 61%), and AFP levels >500 ng/ml (45% vs 13%, P=0.005). No significant differences were found in the proportions of B/S, W/B, and -/- subgroups from both countries. No Immune-high tumors were observed among Indonesian cases, and Immune-low tumors (66%) were more common than in Japanese cases (54%). VETC-positive tumors in Indonesia were significantly more common (29%), and most were in the HBV (90%) and -/- subgroups (90%), whereas Japanese VETC cases (10%, P=0.030) were nonviral (100%) and W/B subgroups (75%).
Conclusion
IHC-based analysis more precisely reflected the clinicopathological differences of HCCs in Japan and Indonesia. These findings provide new insights into standardization attempts and HCC heterogeneity among countries.
Hepatocellular carcinoma (HCC) is the dominant type of liver cancers and is one of the deadliest health threats globally. The conventional therapeutic options for HCC are hampered by low efficiency and intolerable side effects. Gene therapy, however, now offers hope for the treatment of many disorders previously considered incurable, and gene therapy is beginning to address many of the shortcomings of conventional therapies. Herein, we summarize the involvement of genes in the pathogenesis and prognosis of HCC, with a special focus on dysregulated signaling pathways, genes involved in immune evasion, and non-coding RNAs as novel two-edged players, which collectively offer potential targets for the gene therapy of HCC. Herein, the opportunities and challenges of HCC gene therapy are discussed. These include innovative therapies such as genome editing and cell therapies. Moreover, advanced gene delivery technologies that recruit nanomedicines for use in gene therapy for HCC are highlighted. Finally, suggestions are offered for improved clinical translation and future directions in this area of endeavor.
Immune checkpoint inhibitors have produced encouraging results in cancer patients. However, the majority of ß-catenin mutated tumors have been described as lacking immune infiltrates and resistant to immunotherapy. The mechanisms by which the oncogenic ß-catenin affects immune surveillance remain unclear. Herein, we highlighted the involvement of ß-catenin in the regulation of the exosomal pathway and in the immune/cancer cell communication in hepatocellular carcinoma (HCC). We showed that mutated ß-catenin represses SDC4 and RAB27A expression, two main actors of exosome biogenesis, in both liver cancer cell lines and HCC patient samples. Using nanoparticle tracking analysis and live-cell imaging, we further demonstrated that activated ß-catenin represses exosome release. Then, we demonstrated in 3D spheroid models that activation of β-catenin promotes a decrease of immune cell infiltration through a default in exosome secretion. Taken together, our results provide the first evidence that oncogenic ß-catenin plays a key role in exosome biogenesis. Our study gives new insight into the impact of ß-catenin mutations on tumor microenvironment remodeling, which could lead to the development of new strategies to enhance immunotherapy response.
Background/aim:
Advancements in genetic analysis technologies have led to establishment of molecular classifications systems for primary liver cancers. The correlation between pathological morphology and genetic mutations in hepatocellular carcinoma (HCC) is becoming increasingly evident. To construct appropriate experimental models, it is crucial to select cell lines based on their morphology and genetic mutations. In this study, we conducted comprehensive genetic analyses of primary liver cancer cell lines and examined their correlations with morphology.
Materials and methods:
Thirteen primary liver cancer cell lines established in our Department were investigated. Eleven cell lines were HCC cell lines, whereas 2 were combined hepatocellular-cholangiocarcinoma (CHC) cell line characteristics. Whole exome sequencing and fusion gene analyses were conducted using a next generation sequencing platform. We also examined correlations between cell mutations and morphological findings and conducted experiments to clarify the association between morphological findings and genetic alterations.
Results:
Mutations in TP53, HMCN1, PCLO, HYDIN, APOB, and EYS were found in 11, 5, 4, 4, 3, and 3 cell lines, respectively. CTNNB1 mutation was not identified in any cell line. The original tumor of four cell lines (KYN-1, KYN-2, KYN-3, and HAK-6) showed morphologically macrotrabecular massive patterns and these cell lines harbor TP53 mutations. Two cell lines (KYN-2 and KMCH-2) showed an extremely high tumor mutation burden. These two cell lines possess ultra-mutations associated with DNA repair and/or DNA polymerase.
Conclusion:
The study identified correlations between morphological findings and genetic mutations in several HCC cell lines. Cell lines with unique genetic mutations were found. This information will be a valuable tool for the selection of suitable experimental models in HCC research.
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. HCC incidence is on the rise, while treatment options remain limited. Thus, a better understanding of the molecular pathways involved in HCC development has become a priority to guide future therapies. While previous studies implicated the Activator Protein-1 (AP-1) (Fos/Jun) transcription factor family members c-Fos and c-Jun in HCC formation, the contribution of Fos-related antigens (Fra-) 1 and 2 is unknown. Here, we show that hepatocyte-restricted expression of a single chain c-Jun~Fra-2 protein, which functionally mimics the c-Jun/Fra-2 AP-1 dimer, results in spontaneous HCC formation in c-Jun~Fra-2 hep mice. Several hallmarks of human HCC, such as cell cycle dysregulation and the expression of HCC markers are observed in liver tumors arising in c-Jun~Fra-2 hep mice. Tumorigenesis occurs in the context of mild inflammation, low-grade fibrosis, and Pparγ-driven dyslipidemia. Subsequent analyses revealed increased expression of c-Myc, evidently under direct regulation by AP-1 through a conserved distal 3′ enhancer. Importantly, c-Jun~Fra-2-induced tumors revert upon switching off transgene expression, suggesting oncogene addiction to the c-Jun~Fra-2 transgene. Tumors escaping reversion maintained c-Myc and c-Myc target gene expression, likely due to increased c-Fos. Interfering with c-Myc in established tumors using the Bromodomain and Extra-Terminal motif inhibitor JQ-1 diminished liver tumor growth in c-Jun~Fra-2 mutant mice. Thus, our data establish c-Jun~Fra-2 hep mice as a model to study liver tumorigenesis and identify the c-Jun/Fra-2-Myc interaction as a potential target to improve HCC patient stratification and/or therapy.
Simple Summary
Intrahepatic cholangiocarcinoma (ICCA) is the second most common type of primary liver malignancy after hepatocellular carcinoma (HCC). ICCA is characterized by molecular heterogeneity and a diverse histopathological spectrum. Generally, the prognosis for patients diagnosed with ICCA is poor. Recent advances have improved our understanding of the molecular genetics and histological subtypes of ICCA. An accurate diagnosis of ICCA is important for patient management and prognosis. This review aims to provides an updated overview of the pathology of ICCA, with a particular focus on its molecular genetics, histological subtypes, and the diagnostic approaches necessary to distinguish it from other diseases.
Abstract
Intrahepatic cholangiocarcinoma (ICCA) is a malignant epithelial neoplasm characterized by biliary differentiation within the liver. ICCA is molecularly heterogeneous and exhibits a broad spectrum of histopathological features. It is a highly aggressive carcinoma with high mortality and poor survival rates. ICCAs are classified into two main subtypes: the small-duct type and large-duct types. These two tumor types have different cell origins and clinicopathological features. ICCAs are characterized by numerous molecular alterations, including mutations in KRAS, TP53, IDH1/2, ARID1A, BAP1, BRAF, SAMD4, and EGFR, and FGFR2 fusion. Two main molecular subtypes—inflammation and proliferation—have been proposed. Recent advances in high-throughput assays using next-generation sequencing have improved our understanding of ICCA pathogenesis and molecular genetics. The diagnosis of ICCA poses a significant challenge for pathologists because of its varied morphologies and phenotypes. Accurate diagnosis of ICCA is essential for effective patient management and prognostic determination. This article provides an updated overview of ICCA pathology, focusing particularly on molecular features, histological subtypes, and diagnostic approaches.
Heavy alcohol intake induces a wide spectrum of liver diseases ranging from steatosis, steatohepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Although alcohol consumption is a well-known risk factor for the development, morbidity, and mortality of HCC globally, alcohol-associated HCC (A-HCC) is poorly characterized compared to viral hepatitis-associated HCC. Most A-HCCs develop post alcohol-associated cirrhosis, but the direct carcinogenesis from ethanol and its metabolites to A-HCC remains obscure. The differences between A-HCC and HCCs caused by other etiologies have not been well investigated in terms of clinical prognosis, genetic or epigenetic landscape, molecular mechanisms, and heterogeneity. Moreover, there is a huge gap between basic research and clinical practice due to the lack of pre-clinical models of A-HCC. In the current review, we discuss the pathogenesis, heterogeneity, preclinical approaches, epigenetic and genetic profiles of A-HCC, and discuss the current insights into and the prospects for future research on A-HCC. The potential effect of alcohol on cholangiocarcinoma and liver metastasis are also discussed.
Myeloid cells are abundant and plastic immune cell subsets in the liver, to which pro-tumorigenic, inflammatory and immunosuppressive roles have been assigned in the course of tumorigenesis. Yet several aspects underlying their dynamic alterations in hepatocellular carcinoma (HCC) progression remain elusive, including the impact of distinct genetic mutations in shaping a cancer-permissive tumor microenvironment (TME). Here, in newly generated, clinically-relevant somatic female HCC mouse models, we identify cancer genetics’ specific and stage-dependent alterations of the liver TME associated with distinct histopathological and malignant HCC features. Mitogen-activated protein kinase (MAPK)-activated, NrasG12D-driven tumors exhibit a mixed phenotype of prominent inflammation and immunosuppression in a T cell-excluded TME. Mechanistically, we report a NrasG12D cancer cell-driven, MEK-ERK1/2-SP1-dependent GM-CSF secretion enabling the accumulation of immunosuppressive and proinflammatory monocyte-derived Ly6Clow cells. GM-CSF blockade curbs the accumulation of these cells, reduces inflammation, induces cancer cell death and prolongs animal survival. Furthermore, GM-CSF neutralization synergizes with a vascular endothelial growth factor (VEGF) inhibitor to restrain HCC outgrowth. These findings underscore the profound alterations of the myeloid TME consequential to MAPK pathway activation intensity and the potential of GM-CSF inhibition as a myeloid-centric therapy tailored to subsets of HCC patients.
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality globally. HCC is highly heterogenous with diverse etiologies leading to different driver mutations potentiating unique tumor immune microenvironments. Current therapeutic options, including immune checkpoint inhibitors and combinations, have achieved limited objective response rates for the majority of patients. Thus, a precision medicine approach is needed to tailor specific treatment options for molecular subsets of HCC patients. Lipid nanovesicle platforms, either liposome- (synthetic) or extracellular vesicle (natural)-derived present are improved drug delivery vehicles which may be modified to contain specific cargos for targeting specific tumor sites, with a natural affinity for liver with limited toxicity. This mini-review provides updates on the applications of novel lipid nanovesicle-based therapeutics for HCC precision medicine and the challenges associated with translating this therapeutic subclass from preclinical models to the clinic.
Large-scale transcriptomic data are crucial for understanding the molecular features of hepatocellular carcinoma (HCC). Integrated 15 transcriptomic datasets of HCC clinical samples, the first version of HCCDB (HCC database) was released in 2018. Through the meta-analysis of differentially expressed genes and prognosis-related genes across multiple datasets, it provides a systematic view of the altered biological processes and the inter-patient heterogeneities of HCC with high reproducibility and robustness. With four years having passed, the database now needs integration of recently published datasets. Furthermore, the latest single-cell and spatial transcriptomics have provided a great opportunity to decipher complex gene expression variations at the cellular level with spatial architecture. Here, we present HCCDB v2.0, an updated version that combines bulk, single-cell, and spatial transcriptomic data of HCC clinical samples. It dramatically expands the bulk sample size by adding 1656 new samples from 11 datasets to the existing 3917 samples, thereby enhancing the reliability of transcriptomic meta-analysis. A total of 182,832 cells and 69,352 spatial spots were added to the single-cell and spatial transcriptomics sections, respectively. A novel single-cell level and 2-dimension (sc-2D) metric was proposed as well to summarize cell type-specific and dysregulated gene expression patterns. Results are all graphically visualized in our online portal, allowing users to easily retrieve data through a user-friendly interface and navigate between different views. With extensive clinical phenotypes and transcriptomic data in the database, we show two applications for identifying prognosis-associated cells and tumor microenvironment. HCCDB v2.0 is available at http://lifeome.net/database/hccdb2.
Hepatocellular carcinoma (HCC) is a malignant tumor with heterogenous nature and high mortality worldwide. RNA processing has been implicated in tumorigenesis, progression, and prognosis to develop early diagnosis and targeted therapy for cancer, including HCC. This study aimed to identify and validate a novel RNA processing gene signature for predicting HCC prognosis. Transcriptome and clinical data of HCC samples were obtained from Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC), Gene Expression Omnibus databases, and RNA processing factors from the AmiGO database. The least absolute shrinkage and selection operator and multivariate Cox regression analysis were performed to examine prognostic gene signatures. Correlation analysis of prognostic models with clinical features and HallMark pathway-based Genomic Variation Analysis enrichment analysis were performed. We identified seven distinguished RNA processing factors to build the prognostic model for HCC patients. ROC curve analysis of the TCGA-HCC, ICGC-HCC, and GSE76427 cohorts determined the ability to predict prognosis through a sevengene-based model (AUC > 0.7). A nomogram to forecast the overall survival among HCC patients was established. In addition, 7 cell types (CD3+ cells, CD8+ T cells, macrophage/monocyte, monocyte, myeloid dendritic cells, neutrophils and cancer-associated fibroblast) significantly differed between the two risk groups. Furthermore, enrichment analysis showed significant enrichment in fatty acid metabolism, lipogenesis, pancreatic beta cells, and bile acid metabolism in the low-risk group, while in the high-risk group, DNA repair, protein secretion, and mitotic pathways were significantly enriched. Analyzing the Tumor Immune Dysfunction and Exclusion results demonstrated that immune checkpoint blockade therapy was poorly efficacious in the high-risk group. This study constructed and validated a novel prognostic signature related to RNA processing factors in HCC, improving therapeutic strategies for HCC. Building such prognostic signatures could pave the way for developing targeted therapy and precision medicine in HCC.
Hepatocellular carcinoma (HCC) is a serious neoplastic disease with increasing incidence and mortality, accounting for 90% of all liver cancers. Hepatitis viruses are the major causative agents in the development of HCC. Hepatitis A virus (HAV) primarily causes acute infections, which is associated with HCC to a certain extent, as shown by clinicopathological studies. Chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infections lead to persistent liver inflammation and cirrhosis, disrupt multiple pathways associated with cellular apoptosis and proliferation, and are the most common viral precursors of HCC. Mutations in the HBV X protein (HBx) gene are closely associated with the incidence of HCC, while the expression of HCV core proteins contributes to hepatocellular lipid accumulation, thereby promoting tumorigenesis. In the clinical setting, hepatitis D virus (HDV) frequently co-infects with HBV, increasing the risk of chronic hepatitis. Hepatitis E virus (HEV) usually causes acute infections. However, chronic infections of HEV have been increasing recently, particularly in immuno-compromised patients and organ transplant recipients, which may increase the risk of progression to cirrhosis and the occurrence of HCC. Early detection, effective intervention and vaccination against these viruses may significantly reduce the incidence of liver cancer, while mechanistic insights into the interplay between hepatitis viruses and HCC may facilitate the development of more effective intervention strategies. This article provides a comprehensive overview of hepatitis viruses and reviews recent advances in research on aberrant hepatic immune responses and the pathogenesis of HCC due to viral infection.
N6-methyladenosine (m6A) RNA modification is the most common and conserved epigenetic modification in mRNA and has been shown to play important roles in cancer biology. As the m6A reader YTHDF1 has been reported to promote progression of hepatocellular carcinoma (HCC), it represents a potential therapeutic target. In this study, we evaluated the clinical significance of YTHDF1 using human HCC samples and found that YTHDF1 was significantly upregulated in HCCs with high stemness scores and was positively associated with recurrence and poor prognosis. Analysis of HCC spheroids revealed that YTHDF1 was highly expressed in liver cancer stem cells (CSC). Stem cell–specific conditional Ythdf1 knockin (CKI) mice treated with diethylnitrosamine showed elevated tumor burden as compared with wild-type mice. YTHDF1 promoted CSCs renewal and resistance to the multiple tyrosine kinase inhibitors lenvatinib and sorafenib in patient-derived organoids and HCC cell lines, which could be abolished by catalytically inactive mutant YTHDF1. Multiomic analysis, including RNA immunoprecipitation sequencing, m6A methylated RNA immunoprecipitation sequencing, ribosome profiling, and RNA sequencing identified NOTCH1 as a direct downstream of YTHDF1. YTHDF1 bound to m6A modified NOTCH1 mRNA to enhance its stability and translation, which led to increased NOTCH1 target genes expression. NOTCH1 overexpression rescued HCC stemness in YTHDF1-deficient cells in vitro and in vivo. Lipid nanoparticles targeting YTHDF1 significantly enhanced the efficacy of lenvatinib and sorafenib in HCC in vivo. Taken together, YTHDF1 drives HCC stemness and drug resistance through an YTHDF1–m6A–NOTCH1 epitranscriptomic axis, and YTHDF1 is a potential therapeutic target for treating HCC.
Significance
Inhibition of YTHDF1 expression suppresses stemness of hepatocellular carcinoma cells and enhances sensitivity to targeted therapies, indicating that targeting YTHDF1 may be a promising therapeutic strategy for liver cancer.
Purpose of review
The purpose of this review is to both summarize the current knowledge of hepatocellular carcinoma molecular biology and to suggest a framework in which to prospectively translate this knowledge into patient care. This is timely as recent guidelines recommend increased use of these technologies to advance personalized liver cancer care.
Recent findings
The main themes covered here address germline and somatic genetic alterations recently discovered in hepatocellular carcinoma, largely owing to next generation sequencing technologies, and nascent efforts to translate these into contemporary practice.
Summary
Early efforts of translating molecular profiling to hepatocellular carcinoma care demonstrate a growing number of potentially actionable alterations. Still lacking are a consensus on what biomarkers and technologies to adopt, at what scale and cost, and how to integrate them most effectively into care.
Primary liver cancer (PLC) consists of two main histological subtypes; hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA). The role of transcription factors (TFs) in malignant hepatobiliary lineage commitment between HCC and iCCA remains underexplored. Here, we present genome-wide profiling of transcription regulatory elements of 16 PLC patients using single-cell assay for transposase accessible chromatin sequencing. Single-cell open chromatin profiles reflect the compositional diversity of liver cancer, identifying both malignant and microenvironment component cells. TF motif enrichment levels of 31 TFs strongly discriminate HCC from iCCA tumors. These TFs are members of the nuclear/retinoid receptor, POU, or ETS motif families. POU factors are associated with prognostic features in iCCA. Overall, nuclear receptors, ETS and POU TF motif families delineate transcription regulation between HCC and iCCA tumors, which may be relevant to development and selection of PLC subtype-specific therapeutics.
This review aims to provide a comprehensive understanding of the molecular mechanisms underlying autophagy and mitophagy in hepatocellular carcinoma (HCC). Autophagy is an essential cellular process in maintaining cell homeostasis. Still, its dysregulation is associated with the development of liver diseases, including HCC, which is one of leading causes of cancer-related death worldwide. We focus on elucidating the dual role of autophagy in HCC, both in tumor initiation and progression, and highlighting the complex nature involved in the disease. In addition, we present a detailed analysis of a small subset of autophagy- and mitophagy-related molecules, revealing their specific functions during tumorigenesis and the progression of HCC cells. By understanding these mechanisms, we aim to provide valuable insights into potential therapeutic strategies to manipulate autophagy effectively. The goal is to improve the therapeutic response of liver cancer cells and overcome drug resistance, providing new avenues for improved treatment options for HCC patients. Overall, this review serves as a valuable resource for researchers and clinicians interested in the complex role of autophagy in HCC and its potential as a target for innovative therapies aimed to combat this devastating disease.
Hepatocellular carcinoma (HCC) is a primary liver cancer characterized by hepatocellular differentiation. HCC is molecularly heterogeneous with a wide spectrum of histopathology. The prognosis of patients with HCC is generally poor, especially in those with advanced stages. HCC remains a diagnostic challenge for pathologists because of its morphological and phenotypic diversity. However, recent advances have enhanced our understanding of the molecular genetics and histological subtypes of HCC. Accurate diagnosis of HCC is important for patient management and prognosis. This review provides an update on HCC pathology, focusing on molecular genetics, histological subtypes, and diagnostic approaches.
The different cell types of the liver provide the framework for a variety of benign and malignant tumor entities, each of which shows a more or less defined spectrum of associated molecular alterations. Among the malignant neoplasm, carcinomas are prevailing, with malignant mesenchymal tumors being exceedingly rare. Hepatocellular carcinoma (HCC) is one of the most frequent tumors worldwide, and, like intrahepatic cholangiocarcinoma (iCCA), it shows a rising incidence. For both tumor types, specific risk factors and molecular subgroups have been identified. Hepatocellular adenoma is a paradigm entity in terms of morphomolecular subclassification, which has recently been adopted to facilitate the subtyping of some hepatocellular carcinomas. Molecular markers support the differential diagnosis of highly differentiated hepatocellular tumors, and the detection of genetic rearrangement may be helpful in subtyping mesenchymal liver tumors. The role of predictive (therapy-guiding) molecular markers is rising in malignant liver tumors and already has its position in iCCA; concerning HCC, this field is still emerging.
Immune checkpoint inhibitors have produced encouraging results in patients with hepatocellular carcinoma (HCC). However, the majority of β-catenin mutated HCC have been described as lacking immune infiltrates and resistant to immunotherapy. The mechanisms by which the oncogenic β-catenin affects immune surveillance remain unclear. Herein, we highlighted the involvement of β-catenin in the regulation of the exosomal pathway and in the immune/cancer cell communication. We showed that mutated β-catenin represses SDC4 and RAB27A expression in liver cancer cell lines and in HCC patient samples. Using nanoparticle tracking and live-cell imaging, we further demonstrated that activated β-catenin represses exosome release. Using 3D spheroid models, we demonstrated that activation of β-catenin promotes a decrease of immune cell infiltration through a default in exosome secretion. Thus, our results provide new insights into the impact of β-catenin mutations on the liver tumor microenvironment and may enable the development of new strategies to increase immunotherapy response.
Hepatocellular carcinoma (HCC) is the major primary malignant tumor in the human liver, but the molecular changes leading
to liver cell transformation remain largely unknown. The Wnt-β-catenin pathway is activated in colon cancers and some melanoma
cell lines, but has not yet been investigated in HCC. We have examined the status of the β-catenin gene in different transgenic
mouse lines of HCC obtained with the oncogenes c-myc or H-ras. Fifty percent of the hepatic tumors in these transgenic mice had activating somatic mutations within the β-catenin gene
similar to those found in colon cancers and melanomas. These alterations in the β-catenin gene (point mutations or deletions)
lead to a disregulation of the signaling function of β-catenin and thus to carcinogenesis. We then analyzed human HCCs and
found similar mutations in eight of 31 (26%) human liver tumors tested and in HepG2 and HuH6 hepatoma cells. The mutations
led to the accumulation of β-catenin in the nucleus. Thus alterations in the β-catenin gene frequently are selected for during
liver tumorigenesis and suggest that disregulation of the Wnt-β-catenin pathway is a major event in the development of HCC
in humans and mice.
The Wnt signalling pathway is essential for development and organogenesis1,
2,
3. Wnt signalling stabilizes -catenin, which accumulates
in the cytoplasm, binds to T-cell factor (TCF; also known as lymphocyte enhancer-binding
factor, LEF) and then upregulates downstream genes4,
5,
6. Mutations
in CTNNB1 (encoding -catenin) or APC (adenomatous polyposis
coli) have been reported in human neoplasms including colon cancers and hepatocellular
carcinomas7,
8,
9,
10,
11,
12,
13 (HCCs). Because HCCs tend
to show accumulation of -catenin more often than mutations in CTNNB1
, we looked for mutations in AXIN1, encoding a key factor for Wnt
signalling, in 6 HCC cell lines and 100 primary HCCs. Among the 4 cell lines
and 87 HCCs in which we did not detect CTNNB1 mutations, we identified
AXIN1 mutations in 3 cell lines and 6 mutations in 5 of the primary HCCs.
In cell lines containing mutations in either gene, we observed increased DNA
binding of TCF associated with -catenin in nuclei. Adenovirus mediated
gene transfer of wild-type AXIN1 induced apoptosis in hepatocellular
and colorectal cancer cells that had accumulated -catenin as a consequence
of either APC, CTNNB1 or AXIN1 mutation, suggesting that
axin may be an effective therapeutic molecule for suppressing growth of hepatocellular
and colorectal cancers.
There is little information regarding the molecular mechanisms of hepatocarcinogenesis. We studied the p53 gene at the DNA, RNA, and protein level in seven human hepatocellular carcinoma (HCC)-derived cell lines; six of seven showed p53 abnormalities. By Southern blotting, the p53 gene was found to be partially deleted in Hep 3B and rearranged in SK-HEP-1 cells. Transcripts of the p53 gene were undetectable in Hep 3B as well as in FOCUS cells that had no apparent deletion or rearrangement of the p53 gene. Immunoprecipitation after [35S]methionine labeling of HCC cells demonstrated that p53 protein was absent in Hep 3B and FOCUS and reduced in concentration in PLC/PRF/5 cells. p53 synthesized by Mahlavu cells showed a slower migration on SDS/polyacrylamide gels suggesting it was an abnormal protein. In Huh7 cells, p53 protein had a prolonged half-life leading to its accumulation in the nuclei; increased levels of p53 protein were also found by immunoblotting. The p53 gene and its expression appeared to be unaltered in the hepatoblastoma-derived Hep G2 cell line. We found that the loss of p53 expression did not occur as a late in vitro event in the FOCUS cell line because p53 protein was also nondetectable at an early passage. We conclude that the loss of p53 expression or the presence of abnormal forms of the protein are frequently associated with HCC cell lines. These observations suggest that alterations in p53 may be important events in the transformation of hepatocytes to the malignant phenotype.
The generation of invasiveness in transformed cells represents an essential step of tumor progression. We show here, first, that nontransformed Madin-Darby canine kidney (MDCK) epithelial cells acquire invasive properties when intercellular adhesion is specifically inhibited by the addition of antibodies against the cell adhesion molecule uvomorulin; the separated cells then invade collagen gels and embryonal heart tissue. Second, MDCK cells transformed with Harvey and Moloney sarcoma viruses are constitutively invasive, and they were found not to express uvomorulin at their cell surface. These data suggest that the loss of adhesive function of uvomorulin (which is identical to E-cadherin and homologous to L-CAM) is a critical step in the promotion of epithelial cells to a more malignant, i.e., invasive, phenotype. Similar modulation of intercellular adhesion might also occur during invasion of carcinoma cells in vivo.
We screened 75 primary hepatocellular carcinomas for somatic mutations in the entire coding region of the beta-catenin gene. We detected somatic mutations in 14 tumors; 12 were considered to cause amino acid substitutions and 2 were interstitial deletions of 51 or 195 nucleotides of genomic DNA, corresponding to exon 3. Among the 12 point mutations, 6 occurred at potential serine/threonine phosphorylation residues of codons 33, 41, or 45. The remaining six tumors contained a mutation at codon 32 (aspartic acid) or 34 (glycine), flanking to the serine residue at codon 33. By Western blot analysis, we confirmed accumulation of beta-catenin in five tumors for which frozen tissues were available; the five included tumors in which amino acid alterations had occurred at codons 32, 34, or 45, and one with a 17-amino acid deletion. Our results suggested that accumulation of beta-catenin due to amino acid substitutions at potential serine/threonine phosphorylation residues or at their neighboring codons or interstitial deletions involving exon 3 could contribute to hepatocellular carcinogenesis.
Aberrant methylation of CpG islands acquired in tumor cells in promoter regions is one method for loss of gene function. We determined the frequency of aberrant promoter methylation (referred to as methylation) of the genes retinoic acid receptor beta-2 (RARbeta), tissue inhibitor of metalloproteinase 3 (TIMP-3), p16INK4a, O6-methylguanine-DNA-methyltransferase (MGMT), death-associated protein kinase (DAPK), E-cadherin (ECAD), p14ARF, and glutathione S-transferase P1 (GSTP1) in 107 resected primary non-small cell lung cancers (NSCLCs) and in 104 corresponding nonmalignant lung tissues by methylation-specific PCR. Methylation in the tumor samples was detected in 40% for RARbeta, 26% for TIMP-3, 25% for p16INK4a, 21% for MGMT, 19% for DAPK, 18% for ECAD, 8% for p14ARF, and 7% for GSTP1, whereas it was not seen in the vast majority of the corresponding nonmalignant tissues. Moreover, p16INK4a methylation was correlated with loss of p16INK4a expression by immunohistochemistry. A total of 82% of the NSCLCs had methylation of at least one of these genes; 37% of the NSCLCs had one gene methylated, 22% of the NSCLCs had two genes methylated, 13% of the NSCLCs had three genes methylated, 8% of the NSCLCs had four genes methylated, and 2% of the NSCLCs had five genes methylated. Methylation of these genes was correlated with some clinicopathological characteristics of the patients. In comparing the methylation patterns of tumors and nonmalignant lung tissues from the same patients, there were many discordancies where the genes methylated in nonmalignant tissues were not methylated in the corresponding tumors. This suggests that the methylation was occurring as a preneoplastic change. We conclude that these findings confirm in a large sample that methylation is a frequent event in NSCLC, can also occur in smoking-damaged nonmalignant lung tissues, and may be the most common mechanism to inactivate cancer-related genes in NSCLC.
Hepatocellular carcinoma (HCC) is a leading cause of death worldwide. Using cDNA microarrays to characterize patterns of gene expression in HCC, we found consistent differences between the expression patterns in HCC compared with those seen in nontumor liver tissues. The expression patterns in HCC were also readily distinguished from those associated with tumors metastatic to liver. The global gene expression patterns intrinsic to each tumor were sufficiently distinctive that multiple tumor nodules from the same patient could usually be recognized and distinguished from all the others in the large sample set on the basis of their gene expression patterns alone. The distinctive gene expression patterns are characteristic of the tumors and not the patient; the expression programs seen in clonally independent tumor nodules in the same patient were no more similar than those in tumors from different patients. Moreover, clonally related tumor masses that showed distinct expression profiles were also distinguished by genotypic differences. Some features of the gene expression patterns were associated with specific phenotypic and genotypic characteristics of the tumors, including growth rate, vascular invasion, and p53 overexpression.
One of the main regulatory pathways reported to be altered in hepatocellular carcinoma (HCC) is that of cell cycle control involving RB1 gene-related cell inhibitors. We investigated p14(ARF), p15(INK4B), p16(INK4A), p18(INK4C), and RB1 genes in a series of HCCs and associated cirrhosis with the goal of ascertaining their pattern of inactivation by gene methylation. Thirty-three HCCs, adjacent nonneoplastic cirrhotic tissues, and 6 HCC cell lines were studied. Cirrhoses (25 of 33, 76%), HCCs (31 of 33, 94%), and 3 of 6 (50%) cell lines showed 1 or more methylated genes. Cirrhoses (17 of 33, 51%) had more frequently than HCCs (11 of 33, 33%, P =.01) only 1 methylated gene. With the exception of p18(INK4C) the genes under study showed promoter methylation with frequency ranging from 82% (p16(INK4A) in HCC) to 33% and 39% (p15(INK4B) and p16(INK4A) in cirrhoses). In cases with only 1 methylated gene, p15(INK4B) in cirrhosis (8 of 17, 47%) and p16(INK4A) in HCC (10 of 11, 91%) were the more frequently altered. An optimal correlation was found between p15 and p16 gene methylation and complete protein loss in HCC detected by immunocytochemistry, whereas a partial loss of the same proteins was a feature of methylated cirrhoses. Inactivation by DNA methylation of several genes of the RB1 pathway is common to cirrhosis and HCC. An early pattern of methylatory events (1 methylated gene) is a feature of cirrhosis rather than HCC, whereas an advanced one (> or = 3 methylated genes) is characteristic of malignancy. Early methylation changes seem to involve p15(INK4B) and p16(INK4A) in cirrhosis and p16(INK4A) in HCC. In conclusion, a stepwise progression of methylating events is a feature of the sequence cirrhosis-HCC and contributes to the process of hepatic carcinogenesis with potential clinical implications.
Hepatocarcinogenesis is a slow process during which genomic changes progressively alter the hepatocellular phenotype to produce cellular intermediates that evolve into hepatocellular carcinoma. During the long preneoplastic stage, in which the liver is often the site of chronic hepatitis, cirrhosis, or both, hepatocyte cycling is accelerated by upregulation of mitogenic pathways, in part through epigenetic mechanisms. This leads to the production of monoclonal populations of aberrant and dysplastic hepatocytes that have telomere erosion and telomerase re-expression, sometimes microsatellite instability, and occasionally structural aberrations in genes and chromosomes. Development of dysplastic hepatocytes in foci and nodules and emergence of hepatocellular carcinoma are associated with the accumulation of irreversible structural alterations in genes and chromosomes, but the genomic basis of the malignant phenotype is heterogeneous. The malignant hepatocyte phenotype may be produced by the disruption of a number of genes that function in different regulatory pathways, producing several molecular variants of hepatocellular carcinoma. New strategies should enable these variants to be characterized.
Liver adenomas are benign tumors at risk of malignant transformation. In a genome-wide search for loss of heterozygosity (LOH) associated with liver adenomas, we found a deletion in chromosome 12q in five of ten adenomas. In most cases, LOH at 12q was the only recurrent genetic alteration observed, suggesting the presence of a tumor-suppressor gene in that region. A minimal common region of deletion was defined in 12q24 that included the gene TCF1 (transcription factor 1), encoding hepatocyte nuclear factor 1 (HNF1; refs 1,2). Heterozygous germline mutations of TCF1 have been identified in individuals affected with maturity-onset diabetes of the young type 3 (MODY3; ref. 3). Bi-allelic inactivation of TCF1 was found in 10 of 16 screened adenomas, and heterozygous germline mutation were present in three affected individuals. Furthermore, 2 well-differentiated hepatocellular carcinomas (HCCs) occurring in normal liver contained somatic bi-allelic mutations of 30 screened HCCs. These results indicate that inactivation of TCF1, whether sporadic or associated with MODY3, is an important genetic event in the occurrence of human liver adenoma, and may be an early step in the development of some HCCs.
Molecular subtyping of human hepatocellular carcinoma (HCC) with potential mechanistic and therapeutic impact has not been achieved thus far. We have analyzed the mRNA expression patterns of 43 different human HCC samples and 3 HCC cell lines in comparison with normal adult liver using high-density cDNA microarrays. Two main groups of HCC, designated group A (65%) and group B (35%), were distinguished based on clustering of the most highly varying genes. Group A HCCs were characterized by induction of a number of interferon (IFN)-regulated genes, whereas group B was characterized mainly by down-regulation of several apoptosis-relevant and IFN-regulated genes. The number of apoptotic tumor cells and tumor-infiltrating lymphocytes was significantly higher in tumors of group A as compared with those of group B. Based on the expression pattern, group B was further subdivided into two subgroups, designated subgroup B1 (6 of 43 tumors, 14%) and subgroup B2 (9 of 43 tumors, 21%). A prominent characteristic of subgroup B1 was high overexpression of insulin-like growth factor (IGF)-II. All tested HCC cell lines expressed equally high concentrations of IGF-II transcripts and co-segregated with group B1 in clustering. IGF-II overexpression and induction of IFN-related genes were mutually exclusive, even when analysis was extended to other cancer expression profile studies. Moreover, IFN-gamma treatment substantially reduced IGF-II expression in HCC cells. In conclusion, cDNA microarray analyses provided subtyping of HCCs that is related to intratumor inflammation and tumor cell apoptosis. This profiling may be of mechanistic and therapeutic impact because IGF-II overexpression has been linked to reduced apoptosis and increased proliferation and may be accessible to therapeutic intervention.
A recent report revealed that phosphoinositide-3-kinase, catalytic, alpha (PIK3CA) gene is somatically mutated in several types of human cancer, suggesting the mutated PIK3CA gene as an oncogene in human cancers. However, because the previous report focused the mutational search primarily on colon cancers, the data on PIK3CA mutations in other types of human cancers have been largely unknown. Here, we performed mutational analysis of the PIK3CA gene by polymerase chain reaction-single-strand conformation polymorphism assay in 668 cases of common human cancers, including hepatocellular carcinomas, acute leukemias, gastric carcinomas, breast carcinomas, and non-small-cell lung cancers. We detected PIK3CA somatic mutations in 26 of 73 hepatocellular carcinomas (35.6%), 25 of 93 breast carcinomas (26.9%), 12 of 185 gastric carcinomas (6.5%), one of 88 acute leukemias (1.1%), and three of 229 non-small-cell lung cancers (1.3%). Some of the PIK3CA mutations were detected in the early lesions of breast cancer carcinoma, hepatocellular carcinoma, and gastric carcinomas, suggesting that PIK3CA mutation may occur independent of stage of the tumors. The high incidence and wide distribution of PIK3CA gene mutation in the common human cancers suggest that alterations of lipid kinase pathway by PIK3CA mutations contribute to the development of human cancers.
The Wnt/beta-catenin signaling pathway is activated in many human hepatocellular carcinomas (HCC). We tried to identify the genes involved in carcinogenesis and progression of HCC with beta-catenin mutations. We used PCR-based subtractive hybridization to compare gene expression between malignant and benign components of a human HCC occurring in pre-existing adenoma activated for beta-catenin. Two of the genes identified belong to the Regenerating gene (REG) family. They encode the Regenerating islet-derived 3 alpha (REG3A/HIP/PAP/REG-III) and 1 alpha (REG1A) proteins, both involved in liver and pancreatic regeneration and proliferation. Using siRNA directed against beta-catenin, we demonstrated that REG3A is a target of beta-catenin signaling in Huh7 hepatoma cells. The upregulation of REG3A and REG1A expression is significantly correlated to the beta-catenin status in 42 HCC and 28 hepatoblastomas characterized for their beta-catenin status. Thus, we report strong evidence that both genes are downstream targets of the Wnt pathway during liver tumorigenesis.
The variability in the prognosis of individuals with hepatocellular carcinoma (HCC) suggests that HCC may comprise several distinct biological phenotypes. These phenotypes may result from activation of different oncogenic pathways during tumorigenesis and/or from a different cell of origin. Here we address whether the transcriptional characteristics of HCC can provide insight into the cellular origin of the tumor. We integrated gene expression data from rat fetal hepatoblasts and adult hepatocytes with HCC from human and mouse models. Individuals with HCC who shared a gene expression pattern with fetal hepatoblasts had a poor prognosis. The gene expression program that distinguished this subtype from other types of HCC included markers of hepatic oval cells, suggesting that HCC of this subtype may arise from hepatic progenitor cells. Analyses of gene networks showed that activation of AP-1 transcription factors in this newly identified HCC subtype might have key roles in tumor development.
Numerous genetic alterations are accumulated during the process of hepatocarcinogenesis. These genetic alterations can be divided into two groups. The first set of genetic alterations is specific of hepatocellular tumor risk factors. It includes integration of hepatitis B virus (HBV) DNA, R249S TP53 (tumor protein p53) mutation in aflatoxin B1-exposed patients, KRAS mutations related to vinyl chloride exposure, hepatocyte nuclear factor 1alpha (HNF1alpha) mutations associated to hepatocellular adenomas and adenomatosis polyposis coli (APC) germline mutations predisposing to hepatoblastomas. The second set of genetic alterations are etiological nonspecific, it includes recurrent gains and losses of chromosomes, alteration of TP53 gene, activation of WNT/beta-catenin pathway through CTNNB1/beta-catenin and AXIN (axis inhibition protein) mutations, inactivation of retinoblastoma and IGF2R (insulin-like growth factor 2 receptor) pathways through inactivation of RB1 (retinoblastoma 1), P16 and IGF2R. Comprehensive analyses of these genetic alterations have defined two pathways of hepatocarcinogenesis according to the presence or the absence of chromosomal instability. Hepatitis B virus and poorly differentiated tumors are related to chromosome instable tumors associated with frequent TP53 mutations, whereas non-HBV and well-differentiated tumors are related to chromosomal stable samples that are frequently beta-catenin activated. These classifications have clinical relevance as genetic alterations may also be related to prognosis.
Perturbations to the Wnt signaling pathway have been implicated in a large proportion of human hepatocellular carcinomas (HCCs). Activating beta-catenin mutations and loss of function mutations in Axin1 are thought to be functionally equivalent. We examined the Wnt pathway in HCC by comparing the expression of beta-catenin target genes and the level of beta-catenin-dependent transcriptional activation, in 45 HCC tumors and four cell lines. Among these samples, beta-catenin and AXIN1 were mutated in 20 and seven cases, respectively. We found a significant correlation between activated beta-catenin mutations and overexpression of mRNA for the target genes glutamine synthetase (GS), G-protein-coupled receptor (GPR)49 and glutamate transporter (GLT)-1 (P=0.0001), but not for the genes ornithine aminotransferase, LECT2, c-myc and cyclin D1. We also showed that GS is a good immunohistochemical marker of beta-catenin activation in HCC. However, we observed no induction of GS, GPR49 or GLT-1 in the five inactivated Axin1 tumors. Beta-catenin-dependent transcriptional activation in two Axin1-mutated HCC cell lines was much weaker than in beta-catenin-mutated cell lines. Our results strongly suggest that in HCC, contrary to expectation, the loss of function of Axin1 is not equivalent to the gain of function of beta-catenin. Our results also suggest that the tumor suppressor function of Axin1 in HCC may be related to another, non-Wnt pathway.
Using complementary DNAs of human insulin-like growth factors as probes, expressions of the insulin-like growth factors I and II mRNA were examined in seven human hepatoma tissues and their adjacent nontumorous livers. The level of insulin-like growth factor I mRNA in hepatoma was lower than that in the nontumorous liver control. This phenomenon was probably caused by the low expression of human growth hormone receptor in hepatoma tissues. The levels of insulin-like growth factor II mRNA vary among hepatomas. Some show elevated expression; some have diminished expression compared to their nontumorous liver counterparts. In four of the seven hepatomas, expression of fetal forms of insulin-like growth factor II transcripts was observed and may represent dedifferentiation of insulin-like growth factor II expression during hepatocarcinogenesis.
It is now evident that development of hepatocellular carcinoma (HCC) in human is associated with a long series of cellular and tissue changes that precede the ultimate development of the cancer. During recent years, enormous progress in molecular research on HCC has been made, particularly in the area of integration of HBV DNA to host cell and oncogene association with carcinogenicity. A high ratio of HCCs from patients in endemic area has integrated HBV DNA in cellular DNA and in some cases, chromosomal translocations associated with HBV integration were observed, suggesting that the integration or the results thereof are connected with cancer development. Employing a DNA-mediated transfection assay using NIH3T3 mouse fibroblasts with high molecular weight DNA, we detected three cellular transforming genes (lca, N-ras, hst) in primary human HCC. However, little is known as to the linkage between the activation of these genes and liver carcinogenesis. In most human primary HCC tissues, at least two oncogenes, c-myc and N-ras are overexpressed, while in some cases other oncogenes c-fos or lca are overexpressed. It is likely that multiple c-oncogens are important in HCC, but specific transcripts for the malignancy of HCC were not detected. At present, we could not find any relationship between the expression of c-oncogenes and integration of HBV, serological markers or the degree of differentiation. Of the experimental animals most frequently used for studies of liver cancer, the rat is best understood and mimics closely many of the lesions in humans. It is of interest to consider that the identification and elucidation of the mechanisms underlying carcinogenic processes in the rat may offer testable hypotheses for steps in the human.
Meta-analysis of seroprevalence surveys shows that horizontal transmission of hepatitis B virus (ie, that occurring without apparent parenteral, sexual, or perinatal exposure) is common in areas endemic for the virus. It occurs especially in pre-adolescent children. In developed countries, where endemicity of hepatitis B virus is low, horizontal transmission (probably via saliva or open wounds) may occur in households with a persistent carrier, but it is less efficient a means of infection than is sexual or perinatal transmission. Horizontal transmission also seems possible in pre-school day-care centres in developed countries, despite the small numbers of carriers in such places.
PIP
Primary tumors of the liver infrequently develop in patients with a normal liver or in those who have not been exposed to one of several tumor-producing compounds. Hepatocellular adenoma was one of the rarest liver tumors prior to the use of oral contraceptives (OCs). Now the annual incidence in longterm users is estimated at 3-4/100,000. An adenoma that follows OC use is one that often regresses with discontinuation. Focal nodular hyperplasia is a nonencapsulated solitary lesion that has a fibrotic stellate center in which large thick-walled arteries are the source of the blood supply, and occurs most often in women during the menstrual age, and there is no evidence that OCs have increased their frequency. Adenomatous hyperplasia occurs occasionally in patients with submassive necrosis and also in those with cirrhosis. Liver cysts present most often in middle aged women and the ratio of females to males is 4:1. In the US, metastatic carcinoma of the liver is some 18-20 times more frequent and about 85% of these arise in a cirrhotic or precirrhotic liver. Malignant mesenchymal tumors have been associated with exposure to vinyl chloride of injection of Thorotrast. Signs and symptoms of liver disease occur in about 50% of patients with hepatic metastases with hepatomegaly being the most common physical sign. Metastatic carcinoma most often produces multiple umbilicated nodules that involve the liver uniformly. Portal hypertension may be associated with a hepatic neoplasm.
Materno-fetal transmission of hepatitis B was studied in 97 healthy carriers of hepatitis B surface antigen (HBsAg). Antepartum transmission occurred in at least 10 per cent. Intrapartum transmission may have occurred in about 40 per cent as a result of swallowing of the infective fluid by the baby during delivery, and materno-fetal transfusion during labour. Person to person transmission after delivery played a minor role. The presence of hepatitis B associated e antigen (HBeAg) in 48 per cent of maternal serum correlated strongly with the subsequent presence of antigen in the infants. There was a linear association between the incidence of antigens in cord blood and the duration of the first stage of labour, with a significant association when labour exceeded nine hours. Caesarean section is recommended if mothers have HbeAg; likewise amniocentesis and breast feeding should be discouraged if mothers have HbeAg.
The mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF2R) functions in the intracellular trafficking of lysosomal enzymes, the activation of the potent growth inhibitor, transforming growth factor beta 2, and the degradation of IGF2 (ref. 1), a mitogen often overproduced in tumours. We have recently shown that 70% of human hepatocellular tumours have loss of heterozygosity (LOH) at the M6P/IGF2R locus which maps to chromosome 6q26-q27 (ref. 8). Using a coarse screen, we have now identified point mutations in the remaining allele of 25% of human hepatocellular carcinomas (HCCs) with LOH. These mutations give rise to truncated receptor protein and significant amino acid substitutions, and provide evidence that the M6P/IGF2R gene functions as a tumour suppressor in human liver carcinogenesis.
Liver cancer (LC) ranks fifth in frequency in the world with an estimated number of 437,000 new cases in 1990. In developing countries, incidence rates are two- to three-fold higher than in developed countries. The geographic areas at highest risk are located in Eastern Asia, with age-adjusted incidence rates (AAIRs) ranking from 27.6 to 36.6 per 100,000 in men; Middle Africa, with AAIRs ranking from 20.8 to 38.1 per 100,000 in men; and some countries of Western Africa, with AAIRs ranking from 30 to 48 per 100,000 in men. The geographic areas at lowest LC risk are Northern Europe, Australia, New Zealand, and the Caucasian populations in North and Latin America, with AAIRs below 5.0 per 100,000 in men. Excess of LC incidence among men compared to women is universal, with sex ratios between 1.5 and 3.0. Significant variations in LC incidence among different ethnic groups living in the same geographical area and among migrants of the same ethnic groups living in different areas have been extensively described. The variability of LC incidence rates between countries and within countries, strongly suggests differences in exposure to risk factors. The role of chronic infection with the Hepatitis B and hepatitis C viruses (HBV and HCV) in the etiology of LC is well established. The attributable risk estimates for LC for each of these hepatotropic viruses vary among countries but the combined effects of persistent HBV or HCV infections account for well over 80% of LC cases worldwide. Other documented risk factors such as aflatoxin exposure in diets, cigarette smoking, alcohol consumption, and oral contraceptives may explain the residual variation between and within countries. Interactions between some risk factors have been postulated, and are subject of active research. New laboratory techniques and biological markers such as polymerase chain reaction detection of HBV DNA and HCV RNA, as well as specific mutations related to aflatoxin exposure may help to provide quantitative estimates of the risk related to each these factors.
To evaluate how characterization of genetic alterations can help in the elucidation of liver carcinogenesis pathways, 137 tumors were analyzed.
High-density allelotype, p53, Axin1, and beta-catenin gene mutations were determined. Alterations were analyzed according to clinical parameters.
Tumors could be divided into 2 groups according to chromosome stability status. In the first group, demonstrating a chromosome stability, beta-catenin mutation associated with chromosome 8p losses were frequently found as the single genetic alterations. beta-catenin mutations were associated with large tumor size and with negative hepatitis B virus status. In the second group, demonstrating a chromosome instability, the most frequent allelic losses were on chromosome 1p, 4q, 6q, 9p, 13q, 16p, 16q, and 17p; Axin1 and p53 were frequently mutated. All of these alterations, except losses on 6q and 9p, were associated with hepatitis B virus infection. P53 mutations, 17p, 13q losses, and a high value of the fractional allelic loss index were associated with poor differentiated tumors, independently of risk factors. Finally, in the whole series, chromosome 9p and 6q losses were associated with poor prognosis.
Two main pathways defined by genetic alterations show different risk factors and clinical characteristics. Furthermore, loss of chromosome 9p or 6q is an independent prognostic indicator.
p16, cyclin D1 and retinoblastoma protein (pRB) regulate G1 to S transition and are commonly targeted in various cancers. However, few studies have simultaneously examined all components of the p16/cyclin D1/pRB pathway (RB pathway) in hepatocellular carcinoma (HCC). To clarify the role of the disruption of the RB pathway in HCC, we analyzed p16, pRB and cyclin D1 in 47 HCCs. Inactivation of p16 was detected in 30 of 47 HCCs (64%) by Western blot analysis and significantly correlated with hypermethylation of the promoter of this gene. pRB expression was found to be absent in 13 of 47 HCCs (28%) by immunohistochemistry. We found that 38 of 47 HCCs (81%) contained at least one inactivation in either pRB or p16. Furthermore, there was a significant inverse correlation between p16 and pRB inactivation (p = 0.041). Overexpression of cyclin D1 was detected in 5 of 47 HCCs (11%) by immunohistochemistry. The cases with cyclin D1 overexpression exhibited an advanced clinicopathological appearance and also contained inactivation of pRB and/or p16. These findings suggest that inactivation of pRB and/or p16 is a major event in human hepatocarcinogenesis, while cyclin D1 overexpression may confer additional growth advantages to the tumor in addition to pRB and/or p16 inactivation in HCC.
Inappropriate activation of the Wnt/beta-catenin signaling has been implicated in the development of hepatocellular carcinoma (HCC), but exactly how beta-catenin works remains to be elucidated. To identify, in vivo, the target genes of beta-catenin in the liver, we have used the suppression subtractive hybridization technique and transgenic mice expressing an activated beta-catenin in the liver that developed hepatomegaly. We identified three genes involved in glutamine metabolism, encoding glutamine synthetase (GS), ornithine aminotransferase (OAT) and the glutamate transporter GLT-1. By Northern blot and immunohistochemical analysis we demonstrated that these three genes were specifically induced by activation of the beta-catenin pathway in the liver. In different mouse models bearing an activated beta-catenin signaling in the liver known to be associated with hepatocellular proliferation we observed a marked up-regulation of these three genes. The cellular distribution of GS and GLT-1 parallels beta-catenin activity. By contrast no up-regulation of these three genes was observed in the liver in which hepatocyte proliferation was induced by a signal-independent of beta-catenin. In addition, the GS promoter was activated in the liver of GS(+/LacZ) mice by adenovirus vector-mediated beta-catenin overexpression. Strikingly, the overexpression of the GS gene in human HCC samples was strongly correlated with beta-catenin activation. Together, our results indicate that GS is a target of the Wnt/beta-catenin pathway in the liver. Because a linkage of the glutamine pathway to hepatocarcinogenesis has already been demonstrated, we propose that regulation of these three genes of glutamine metabolism by beta-catenin is a contributing factor to liver carcinogenesis.
To identify the genes responsible for carcinogenesis and progression of hepatocellular carcinoma (HCC), we screened differentially expressed genes in several human HCC cell lines. Among these genes, Gpr49 was up-regulated in PLC/PRF/5 and HepG2. Gpr49 is a member of the glycoprotein hormone receptor subfamily, which includes the thyroid-stimulating hormone receptor (TSHR). However, Gpr49 remains to be an orphan G-protein-coupled receptor. By real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis, overexpression (>3-fold increase compared with the corresponding noncancerous liver tissue) of Gpr49 mRNA was observed in 18 of 38 (47%) HCCs compared with corresponding noncancerous livers. Clinicopathologically, overexpression of Gpr49 was frequently observed in HCC with mutation in beta-catenin exon 3 (14 of 16 cases, 87.5%). Moreover, introduction of mutant beta-catenin into mouse hepatocytes in culture caused up-regulation of the Gpr49 mouse homologue. Therefore, Gpr49 is likely to be a target gene activated by Wnt-signaling in HCC. In conclusion, although much is still unknown, Gpr49 may be critically involved in the development of HCCs with beta-catenin mutations and has the potential to be a new therapeutic target in the treatment of HCC.
We analyzed global gene expression patterns of 91 human hepatocellular carcinomas (HCCs) to define the molecular characteristics of the tumors and to test the prognostic value of the expression profiles. Unsupervised classification methods revealed two distinctive subclasses of HCC that are highly associated with patient survival. This association was validated via 5 independent supervised learning methods. We also identified the genes most strongly associated with survival by using the Cox proportional hazards survival analysis. This approach identified a limited number of genes that accurately predicted the length of survival and provides new molecular insight into the pathogenesis of HCC. Tumors from the low survival subclass have strong cell proliferation and antiapoptosis gene expression signatures. In addition, the low survival subclass displayed higher expression of genes involved in ubiquitination and histone modification, suggesting an etiological involvement of these processes in accelerating the progression of HCC. In conclusion, the biological differences identified in the HCC subclasses should provide an attractive source for the development of therapeutic targets (e.g., HIF1a) for selective treatment of HCC patients. Supplementary material for this article can be found on the HEPATOLOGY Web site (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html)
Chronic infections with hepatitis B virus (HBV) and hepatitis C virus (HCV) are the most important risk factors for the development of hepatocellular carcinoma (HCC) in humans. HBV is the primary cause of HCC in high-risk areas including China and Africa, whereas in developed countries such as the United States, HCV plays a more prominent role and is at least partially responsible for the increase in HCC incidence in this country. Humans are exposed to hepatocarcinogenic aflatoxins through ingestion of moldy foods, a consequence of poor storage of susceptible grains. Highly exposed populations are primarily in sub-Sahara Africa and Asia, where dietary aflatoxins significantly enhance the carcinogenic effects of viral hepatitis. Heavy, long-term alcohol use is a risk factor for HCC, whereas moderate use (1-3 drinks/day) is not. Constituents of cigarette smoke are hepatic carcinogens in animals, and there is mounting evidence that the liver is an organ susceptible to tobacco carcinogenicity. Diabetic patients are at risk for HCC probably as a result of the hepatic injury, fibrosis, and eventual cirrhosis resulting from fatty liver disease. Given the current epidemic of obesity and diabetes in the United States, this risk factor will be increasingly important. Increased risk for HCC is evident in young noncirrhotic users of oral contraceptives in the United States and Europe. In summary, risk factors for HCC are identifiable in most patients and primarily are associated with chronic hepatic injury.
Chronic infection with the hepatitis B virus (HBV) is a major risk factor for development of hepatocellular carcinoma (HCC). The pathogenesis of cancer in HBV infection has been extensively analyzed, and multiple factors appear to play a role. A major factor is chronic inflammation and the effects of cytokines in the development of fibrosis and liver cell proliferation. Also important is the role of integration of HBV DNA into host cellular DNA, which, in some situations, acts to disrupt or promote expression of cellular genes that are important in cell growth and differentiation. In addition, expression of HBV proteins may have a direct effect on cellular functions, and some of these gene products can favor malignant transformation. Several HBV genes have been found in infected tissues more frequently than others, including truncated pre-S2/S, hepatitis B X gene, and a novel spliced transcript of HBV, referred to as the hepatitis B spliced protein. The proteins expressed from these integrated genes have been shown to have intracellular activities that may account for their association with HCC, including effects on cellular growth and apoptosis. Finally, some patients with HCC have no detectable hepatitis B surface antigen in serum but do have low levels of HBV DNA in serum and integrated molecules of HBV DNA in tissue. Occult HBV infection may account for a proportion of cases of HCC that occur in patients without serologic markers for hepatitis B and C and may be a cofactor in HCC in patients with chronic hepatitis C who have coexistent occult HBV infection.
Estimates from the year 2000 indicate that liver cancer remains the fifth most common malignancy in men and the eighth in women worldwide. The number of new cases is estimated to be 564,000 per year, including 398,000 in men and 166,000 in women. In high-risk countries, liver cancer can arise before the age of 20 years, whereas, in countries at low risk, liver cancer is rare before the age of 50 years. Rates of liver cancer in men are typically 2 to 4 times higher than in women. The incidence of primary liver cancer is increasing in several developed countries, including the United States, and the increase will likely continue for some decades. The trend is a result of a cohort effect related to infection with hepatitis B and C viruses, the incidence of which peaked in the 1950s to 1980s. In selected areas of some developing countries, the incidence of primary liver cancer has decreased, possibly as a result of the introduction of hepatitis B virus vaccine. The geographic variability in incidence of primary liver cancer is largely explained by the distribution and the natural history of the hepatitis B and C viruses. The attributable risk estimates for the combined effects of these infections account for well over 80% of liver cancer cases worldwide. Primary liver cancer is the first human cancer largely amenable to prevention using hepatitis B virus vaccines and screening of blood and blood products for hepatitis B and C viruses.
To determine the methylation profile of multiple tumor-related genes during multistep hepatocarcinogenesis, we investigated the methylation status of CpG islands of 9 genes, using methylation-specific polymerase chain reaction for 60 paired hepatocellular carcinoma (HCC) and non-HCC liver tissue samples, 22 dysplastic nodule (DN), 30 liver cirrhosis (LC), 34 chronic hepatitis (CH) and 20 normal liver samples. The methylation status of 9 genes was correlated to the clinicopathological findings of HCC patients. All HCC samples showed methylation of at least one gene, whereas it was shown in 72.7% of DN and 40% of LC, but was not shown in CH and normal liver samples (P < 0.001). The number of genes methylated showed a stepwise increase with the progression of stages (0 for normal liver and CH, 0.5 for LC, 1.5 for DN, and 3.7 for HCC (P < 0.001)). The genes frequently methylated in HCC were APC (81.7%), GSTP1 (76.7%), RASSF1A (66.7%), p16 (48.3%), COX-2 (35%), and E-cadherin (33.3%). COX-2, p16, RASSF1A, and TIMP-3 were not methylated in LC and CH from patients without concurrent HCC. Chronic liver diseases with concurrent HCC showed higher methylation frequencies of the tested genes, and a higher number of methylated genes than those without concurrent HCC. HCC patients with methylation of E-cadherin or GSTP1 showed poorer survival than those without (P = 0.034 and 0.043, respectively). In conclusion, our results indicated that CpG island methylation of tumor-related genes is an early and frequent event, and accumulates step-by-step during a multistep hepatocarcinogenesis. CpG island methylation of E-cadherin or GSTP1 might serve as a potential biomarker for prognostication of HCC patients.
Immunostaining was performed using monoclo-nal anti--catenin as previously described
- Pierce
Pierce). Immunostaining was performed using monoclo-nal anti--catenin as previously described. 17
Somatic mutations of the beta-catenin gene are frequent in mouse and human hepatocellular carcinomas
- La A Coste
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M6P/IGF2R gene is mutated in human hepatocellular carcinomas with loss of heterozygosity
- De Souza
Genetic alterations associated with hepatocellular carcinomas define distinct pathways of hepatocarcinogenesis
- Laurent-Puig
Primary liver cancer: worldwide incidence and trends
- Bosch
AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1
- Satoh
Transmission of hepatitis B antigens from symptom free carrier mothers to the fetus and the infant
- Wong
Somatic mutations of the beta-catenin gene are frequent in mouse and human hepatocellular carcinomas
- de La Coste
Gene expression patterns in human liver cancers
- Chen