Alcohol-associated Hepatocellular Carcinoma Research Articles

Alcohol-associated liver cancer.

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.

Establishment and characterization of a highly metastatic hepatocellular carcinoma cell line

ABSTRACT The prevalence of alcohol-related hepatocellular carcinoma (HCC) has been increasing during the last decade. Cancer research requires cell lines suitable for both in vitro and in vivo assays. However, there is a lack of cell lines with a high in vivo metastatic capacity for this HCC subtype. Herein, a new HCC cell line was established, named HCC-ZJ, using cells from a patient diagnosed with alcohol-related HCC. The karyotype of HCC-ZJ was 46, XY, del (p11.2). Whole-exome sequencing identified several genetic variations in HCC-Z that occur frequently in alcohol-associated HCC, such as mutations in TERT, CTNNB1, ARID1A, CDKN2A, SMARCA2, and HGF. Cell counting kit-8 assays, colony formation assays, and Transwell assays were performed to evaluate the proliferation, migration, and sensitivity to sorafenib and lenvatinib of HCC-Z in vitro. HCC-ZJ showed a robust proliferation rate, a weak foci-forming ability, a strong migration capacity, and a moderate invasion tendency in vitro. Finally, the tumorigenicity and metastatic capacity of HCC-Z were evaluated using a subcutaneous xenograft model, an orthotopic xenograft model, and a tail-veil injection model. HCCZJ exhibited strong tumorigenicity in the subcutaneous xenograft and orthotopic tumor models. Moreover, HCC-ZJ spontaneously formed pulmonary metastases in the orthotopic tumor model. In summary, a new HCC cell line derived from a patient with alcohol-related HCC was established, which showed a high metastatic capacity and could be applied for in vitro and in vivo experiments during pre-clinical research. Highlights • An alcohol-related HCC cell line, HCC-ZJ, was established • HCC-ZJ was applicable for in vitro functional experiment and gene editing • HCC-ZJ was applicable for in vivo tumor growth and spontaneous metastasis models

High producer variant of lipoprotein lipase may protect from hepatocellular carcinoma in alcohol-associated cirrhosis

Progression of alcohol-associated liver disease (ALD) is driven by genetic predisposition. The rs13702 variant in the lipoprotein lipase (LPL) gene is linked to non-alcoholic fatty liver disease. We aimed at clarifying its role in ALD. Patients with alcohol-associated cirrhosis, with (n= 385) and without hepatocellular carcinoma (HCC) (n= 656), with HCC attributable to viral hepatitis C (n= 280), controls with alcohol abuse without liver damage (n= 366), and healthy controls (n= 277) were genotyped regarding the LPL rs13702 polymorphism. Furthermore, the UK Biobank cohort was analysed. LPL expression was investigated in human liver specimens and in liver cell lines. Frequency of the LPL rs13702 CC genotype was lower in ALD with HCC in comparison to ALD without HCC both in the initial (3.9% vs. 9.3%) and the validation cohort (4.7% vs. 9.5%; p <0.05 each) and compared with patients with viral HCC (11.4%), alcohol misuse without cirrhosis (8.7%), or healthy controls (9.0%). This protective effect (odds ratio [OR]= 0.5) was confirmed in multivariate analysis including age (OR= 1.1/year), male sex (OR= 3.0), diabetes (OR= 1.8), and carriage of the PNPLA3 I148M risk variant (OR= 2.0). In the UK Biobank cohort, the LPL rs13702C allele was replicated as a risk factor for HCC. Liver expression of LPL mRNA was dependent on LPL rs13702 genotype and significantly higher in patients with ALD cirrhosis compared with controls and alcohol-associated HCC. Although hepatocyte cell lines showed negligible LPL protein expression, hepatic stellate cells and liver sinusoidal endothelial cells expressed LPL. LPL is upregulated in the liver of patients with alcohol-associated cirrhosis. The LPL rs13702 high producer variant confers protection against HCC in ALD, which might help to stratify people for HCC risk. Hepatocellular carcinoma is a severe complication of liver cirrhosis influenced by genetic predisposition. We found that a genetic variant in the gene encoding lipoprotein lipase reduces the risk for hepatocellular carcinoma in alcohol-associated cirrhosis. This genetic variation may directly affect the liver, because, unlike in healthy adult liver, lipoprotein lipase is produced from liver cells in alcohol-associated cirrhosis.

Global epidemiology of alcohol-associated cirrhosis and HCC: trends, projections and risk factors.

Heavy alcohol consumption is a major cause of morbidity and mortality. Globally, alcohol per-capita consumption rose from 5.5 litres in 2005 to 6.4 litres in 2016 and is projected to increase further to 7.6 litres in 2030. In 2019, an estimated 25% of global cirrhosis deaths were associated with alcohol. The global estimated age-standardized death rate (ASDR) of alcohol-associated cirrhosis was 4.5 per 100,000 population, with the highest and lowest ASDR in Africa and the Western Pacific, respectively. The annual incidence of hepatocellular carcinoma (HCC) among patients with alcohol-associated cirrhosis ranged from 0.9% to 5.6%. Alcohol was associated with approximately one-fifth of global HCC-related deaths in 2019. Between 2012 and 2017, the global estimated ASDR for alcohol-associated cirrhosis declined, but the ASDR for alcohol-associated liver cancer increased. Measures are required to curb heavy alcohol consumption to reduce the burden of alcohol-associated cirrhosis and HCC. Degree of alcohol intake, sex, older age, obesity, type 2 diabetes mellitus, gut microbial dysbiosis and genetic variants are key factors in the development of alcohol-associated cirrhosis and HCC. In this Review, we discuss the global epidemiology, projections and risk factors for alcohol-associated cirrhosis and HCC.

Association between Hepatic Oxidative Stress Related Factors and Activation of Wnt/β-Catenin Signaling in NAFLD-Induced Hepatocellular Carcinoma

Simple SummaryActivation of the Wnt/β-catenin signaling pathway may reduce the efficacy of immune checkpoint inhibitors, which are first-line therapeutic agents for the treatment of hepatocellular carcinoma (HCC). Following gadoxetate-enhanced MRI, HCC lesions may exhibit equal or higher signal intensities in the hepatobiliary phase than normal tissue. Thus, MRI could be used to monitor the therapeutic effect of antitumor agents. In this study, we investigated the relationship between intrahepatic iron overload markers and oxidative stress and activation of the Wnt/β-catenin signaling pathway. We found that for nonalcoholic fatty liver disease-induced HCC, MRI yielded a sensitivity of 57.2% and a specificity of 100%. Serum ferritin > 77.5 ng/mL had a sensitivity of 85.7% and a specificity of 85.7%. We conclude that serum ferritin levels may further improve the accuracy with which activation of Wnt/β-catenin signaling can be predicted.We investigated the association between iron overload, oxidative stress (8-oxo-7,8-dihydroguanine: 8-oxo-dG scores), Wnt/β-catenin pathway activation (expression of glutamine synthetase: GS), and tumor hyperintensity in the Gd-EOB-DTPA-enhanced MRI hepatobiliary phase (relative enhancement ratio: RER). This was a retrospective analysis of 94 hepatocellular carcinoma (HCC) patients who underwent surgical resection. In HBV-, HCV-, and alcohol-associated HCC, serum ferritin levels in the high and low RER groups were equivalent. In contrast, ferritin levels were elevated in the ‘high RER’ group of patients with nonalcoholic fatty liver disease (NAFLD)-HCC. As predictors of GS positivity, high RER had a sensitivity of 57.2% and a specificity of 100%. High serum ferritin had a sensitivity of 85.7% and a specificity of 85.7%. All cases with serum ferritin ≥275.5 ng/mL and high RER were 8-oxo-dG- and iron staining-positive. Additionally, GS positivity was seen in all cases with “serum ferritin levels above the upper limits or iron staining-positive” and ‘8-oxo-dG high’ cases. Therefore, combining serum ferritin levels with RER may increase the accuracy with which activated Wnt/β-catenin signaling is predicted in NAFLD-HCC. We suggest that 8-oxo-dG accumulates following increased oxidative stress due to hepatic tissue iron deposition; this may activate Wnt/β-catenin signaling and trigger carcinogenesis.

Identification of 40S ribosomal protein S8 as a novel biomarker for alcohol‑associated hepatocellular carcinoma using weighted gene co‑expression network analysis.

Alcohol-associated hepatocellular carcinoma (HCC) is a subtype of HCC with poor prognosis. The present study aimed to identify key biomarkers for alcohol-associated HCC. The gene data profiles and corresponding clinical traits of patients with alcohol-associated HCC were downloaded from The Cancer Genome Atlas (TCGA) database. Firstly, good genes and good samples were identified, which were subsequently used to conduct weighted gene co-expression network analysis (WGCNA). Hub genes in the significant modules were selected following Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, and from constructing a protein-protein interaction (PPI) network. Real hub genes among hub genes were determined following progression, survival analysis and gene set enrichment analysis (GSEA), as well as reverse transcription-quantitative PCR and immunohistochemical staining of non-alcohol- and alcohol-associated HCC samples. In total, 64 good samples of alcohol-associated HCC with height score <160 were selected, from which 15,195 good genes were identified and used to conduct WGCNA; 8 gene co-expressed modules were identified using WGCNA, while 3 modules (including pink, magenta and turquoise) were significantly associated with Child-Pugh score, T-stage and body weight. Following GO and KEGG analysis and construction of the PPI network, a total of 30 hub genes were identified in the aforementioned 3 gene co-expressed modules, while 16 hub genes (including AURKB, BUB1, BUB1B, CCNB1, CCNB2, CDC20, CDCA8, CDK1, PLK1, RPS5, RPS7, RPS8, RPS14, RPS27, RPSA and TOP2A) were associated with the development of alcohol-associated HCC, and had a significant prognosis value. Among these genes, only RPS8 was highly expressed in alcohol-associated HCC, but not in non-alcohol-associated HCC, while RPS5 was not significantly associated in either alcohol- or non-alcohol-associated HCC. GSEA demonstrated that 10 pathways, including RNA polymerase and ribosome pathways were enriched in alcohol-associated HCC samples where RPS8 was highly expressed. Taken together, the results of the present study demonstrate that RPS8 may be a novel biomarker for the diagnosis of patients with alcohol-associated HCC.

Differential alternative splicing regulation among hepatocellular carcinoma with different risk factors

BackgroundHepatitis B virus (HBV), hepatitis C virus (HCV), and alcohol consumption are predominant causes of hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying how differently these causes are implicated in HCC development are not fully understood. Therefore, we investigated differential alternative splicing (AS) regulation among HCC patients with these risk factors.MethodsWe conducted a genome-wide survey of AS events associated with HCCs among HBV (n = 95), HCV (n = 47), or alcohol (n = 76) using RNA-sequencing data obtained from The Cancer Genome Atlas.ResultsIn three group comparisons of HBV vs. HCV, HBV vs. alcohol, and HCV vs. alcohol for RNA seq (ΔPSI> 0.05, FDR < 0.05), 133, 93, and 29 differential AS events (143 genes) were identified, respectively. Of 143 AS genes, eight and one gene were alternatively spliced specific to HBV and HCV, respectively. Through functional analysis over the canonical pathways and gene ontologies, we identified significantly enriched pathways in 143 AS genes including immune system, mRNA splicing-major pathway, and nonsense-mediated decay, which may be important to carcinogenesis in HCC risk factors. Among eight genes with HBV-specific splicing events, HLA-A, HLA-C, and IP6K2 exhibited more differential expression of AS events (ΔPSI> 0.1). Intron retention of HLA-A was observed more frequently in HBV-associated HCC than HCV- or alcohol-associated HCC, and intron retention of HLA-C showed vice versa. Exon 3 (based on ENST00000432678) of IP6K2 was less skipped in HBV-associated in HCC compared to HCV- or alcohol-associated HCC.ConclusionAS may play an important role in regulating transcription differences implicated in HBV-, HCV-, and alcohol-related HCC development.

Clinical characteristics analysis of 1180 patients with hepatocellular carcinoma secondary to hepatitis B, hepatitis C and alcoholic liver disease.

ObjectiveTo determine the clinical and liver stiffness characteristics of a cohort of Chinese patients with Hepatocellular carcinoma in different stages of Barcelona clinic liver cancer.MethodsDetails of 1180 patients with Hepatocellular carcinoma referred from October 2014 to November 2017 were collected retrospectively. Demographic data, etiology, clinical, and biochemical details were retrospectively analyzed. The changes of liver stiffness in different etiologies and different stages of Barcelona clinic liver cancer were especially analyzed.ResultsThe onset age was 60.33 ± 9.11 (range 24‐84) years, 9 cases were ≤40 years, 572 cases were 41‐60 years, males accounted for 83.92%, females accounted for 16.08%; 599 cases were ≥61 years, males accounted for 78.25%, females accounted for 21.75%. Compared with males, the proportion of females ≥61 is higher than that of men. Majority (n = 787; 66.69%) had HBV infection; second commonest cause was HCV infection (n = 217; 18.39%). More patients with HBV infection were 41‐60 years (69.06%) and were younger than HCV patients. There was no statistical difference in etiology, age, gender, and distribution of diabetes mellitus among different Barcelona clinic liver cancer stages (P > .05). The overall Hepatocellular carcinoma (HCC) was found to be positively correlated with alkaline phosphatase, γ‐glutamyltransferase, and alpha‐fetoprotein and liver stiffness measurement values from stage A to stage D (P < .05). ANOVA analysis showed that the overall liver stiffness measurement among the four BCLC stages was found to be statistically significant different in HBV‐infected and HCV‐infected HCC patients.ConclusionMajority (99.24%) were patients aged >40 years old. Male is a high incidence population. In etiological analysis, HBV dominates HCC occurrence, HBV‐, HCV‐, and alcohol‐associated HCC have distinct clinical and biochemical characteristics, necessitating different screening policies to optimize HCC surveillance and management.

ALDH2 deficiency promotes alcohol-associated liver cancer by activating oncogenic pathways via oxidized DNA-enriched extracellular vesicles

Excessive alcohol consumption is one of the major causes of hepatocellular carcinoma (HCC). Approximately 30-40% of the Asian population are deficient for aldehyde dehydrogenase 2 (ALDH2), a key enzyme that detoxifies the ethanol metabolite acetaldehyde. However, how ALDH2 deficiency affects alcohol-related HCC remains unclear. ALDH2 polymorphisms were studied in 646 patients with viral hepatitis B (HBV) infection, who did or did not drink alcohol. A new model of HCC induced by chronic carbon tetrachloride (CCl4) and alcohol administration was developed and studied in 3 lines of Aldh2-deficient mice: including Aldh2 global knockout (KO) mice, Aldh2*1/*2 knock-in mutant mice, and liver-specific Aldh2 KO mice. We demonstrated that ALDH2 deficiency was not associated with liver disease progression but was associated with an increased risk of HCC development in cirrhotic patients with HBV who consumed excessive alcohol. The mechanisms underlying HCC development associated with cirrhosis and alcohol consumption were studied in Aldh2-deficient mice. We found that all 3 lines of Aldh2-deficient mice were more susceptible to CCl4 plus alcohol-associated liver fibrosis and HCC development. Furthermore, our results from in vivo and in vitro mechanistic studies revealed that after CCl4 plus ethanol exposure, Aldh2-deficient hepatocytes produced a large amount of harmful oxidized mitochondrial DNA via extracellular vesicles, which were then transferred into neighboring HCC cells and together with acetaldehyde activated multiple oncogenic pathways (JNK, STAT3, BCL-2, and TAZ), thereby promoting HCC. ALDH2 deficiency is associated with an increased risk of alcohol-related HCC development from fibrosis in patients and in mice. Mechanistic studies reveal a novel mechanism that Aldh2-deficient hepatocytes promote alcohol-associated HCC by transferring harmful oxidized mitochondrial DNA-enriched extracellular vesicles into HCC and subsequently activating multiple oncogenic pathways in HCC. Alcoholics with an ALDH2 polymorphism have an increased risk of digestive tract cancer development, however, the link between ALDH2 deficiency and hepatocellular carcinoma (HCC) development has not been well established. In this study, we show that ALDH2 deficiency exacerbates alcohol-associated HCC development both in patients and mouse models. Mechanistic studies revealed that after chronic alcohol exposure, Aldh2-deficient hepatocytes produce a large amount of harmful oxidized mitochondrial DNA via extracellular vesicles, which can be delivered into neighboring HCC cells and subsequently activate multiple oncogenic pathways, promoting HCC.

ACTG1 and TLR3 are biomarkers for alcohol-associated hepatocellular carcinoma.

Alcohol consumption is a risk factor for the development of hepatocellular carcinoma (HCC); however, the association between alcohol and HCC remains unknown. The present study aimed to identify key genes related to alcohol-associated HCC to improve the current understanding of the pathology of this disease. Alcohol-associated and non-alcohol-associated HCC samples in the GSE50579 dataset of the Gene Omnibus Database were analyzed to investigate altered gene expression. Integrated bioinformatics methods were employed to clarify the biological functions of the differentially expressed genes (DEGs), including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interactions (PPIs). The present study reported that candidate biomarker micro (mi)RNAs via TargetScan Human 7.1. DEGs and their associated miRNAs (according to bioinformatics analysis) were validated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Additionally, 284 EGs from the GSE50579 dataset were revealed. In GO term analysis, DEGs were closely associated with the ‘regulation of nucleic acid metabolism’. KEGG pathway analysis indicated that the DEGs were tightly engaged in the ‘VEGF and VEGF receptor signaling network’, ‘proteoglycan syndecan-mediated signaling events’, ‘erbB receptor signaling’ and ‘β1 integrin cell surface interactions’. According to the results of PPI and heat map analysis, the main hub genes were centrin 3 (CETN3), Toll-like receptor 3 (TLR3), receptor tyrosine-protein kinase (ERBB4), heat shock protein family member 8, actin γ1 (ACTG1) and α-smooth muscle actin. it was demonstrated that the ACTG1, TLR3, miR-6819-3p and miRΝΑ (miR)-6877-3P had undefined associations. Furthermore, RT-qPCR analysis revealed that miR-6819-3p and miR-6877-3P may enhance the expression levels of ACTG1 and inhibit the expression levels of TLR3 in alcohol-associated HCC tissues. TLR3 and ACTG1 were proposed as potential biomarkers of alcohol-associated HCC. Investigation into the regulatory functions of miR-6819-3p and miR-6877-3P may provide novel insights into the treatment of alcohol-associated HCC.

Methylation patterns of RASA3 associated with clinicopathological factors in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the sixth most common tumor worldwide. The relationship between the gene methylation accumulation and HCC has been widely studied. In our study, we used the Sequenom EpiTYPER assay to investigate the methylation levels of the RASA3 in 164 HCC samples and paired adjacent non-cancerous tissues, and the association between methylation level and clinicopathological features. The methylation level of the RASA3 in HCC samples was found significantly lower than that in the adjacent non-cancerous tissues (P<0.0001). Moreover, the hypomethylation of RASA3 in HCC samples was connected with the presence of tumornecrosis (P=0.029) and alcohol intake (P=0.002). Furthermore, it was found that the expression of RASA3 was significantly decreased in tumor tissues (P=0.0053), which was also correlated with the methylation levels of RASA3 gene. Thus, RASA3 hypomethylation is a common feature in HCC, and may be a potential mechanism for HCC development, and serves as a useful biomarker for the early detection, especially in alcohol-associated HCCs.

Fatty Liver Disease and Hepatocellular Carcinoma: The Pathologist's View.

Chronic alcohol misuse and progressed nonalcoholic fatty liver disease (NAFLD) due to the metabolic syndrome and resulting to nonalcoholic steatohepatitis (NASH) are prime causes of hepatocellular carcinoma (HCC) in Western industrialized countries. The incidence of HCC in NASH-cirrhosis is lower than that of HCC occuring in HCV-related or alcoholic cirrhosis. Up to 20% of cases of alcohol-associated HCC may develop in pre-cirrhotic liver while HCC is also increasingly recognised in pre-cirrhotic NASH raising questions on appropriate surveillance measures for these patient populations. The recently described steatohepatitic subtype of HCC presents with higher frequency in NAFLD compared to alcoholic liver disease (ALD) patients. This review will mainly focus on histopathology and summarize current data on the epidemiology, pathogenesis, diagnosis and management of NAFLD- and ALD-related HCC.

DNA methylation and gene expression profiles show novel regulatory pathways in hepatocellular carcinoma.

BackgroundAlcohol is a well-known risk factor for hepatocellular carcinoma (HCC), but the mechanisms underlying the alcohol-related hepatocarcinogenesis are still poorly understood. Alcohol alters the provision of methyl groups within the hepatic one-carbon metabolism, possibly inducing aberrant DNA methylation. Whether specific pathways are epigenetically regulated in alcohol-associated HCC is, however, unknown. The aim of the present study was to investigate the genome-wide promoter DNA methylation and gene expression profiles in non-viral, alcohol-associated HCC. From eight HCC patients undergoing curative surgery, array-based DNA methylation and gene expression data of all annotated genes were analyzed by comparing HCC tissue and homologous cancer-free liver tissue.ResultsAfter merging the DNA methylation with gene expression data, we identified 159 hypermethylated-repressed, 30 hypomethylated-induced, 49 hypermethylated-induced, and 56 hypomethylated-repressed genes. Notably, promoter DNA methylation emerged as a novel regulatory mechanism for the transcriptional repression of genes controlling the retinol metabolism (ADH1A, ADH1B, ADH6, CYP3A43, CYP4A22, RDH16), iron homeostasis (HAMP), one-carbon metabolism (SHMT1), and genes with a putative, newly identified function as tumor suppressors (FAM107A, IGFALS, MT1G, MT1H, RNF180).ConclusionsA genome-wide DNA methylation approach merged with array-based gene expression profiles allowed identifying a number of novel, epigenetically regulated candidate tumor-suppressor genes in alcohol-associated hepatocarcinogenesis. Retinol metabolism genes and SHMT1 are also epigenetically regulated through promoter DNA methylation in alcohol-associated HCC.Due to the reversibility of epigenetic mechanisms by environmental/nutritional factors, these findings may open up to novel interventional strategies for hepatocarcinogenesis prevention in HCC related to alcohol, a modifiable dietary component.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0077-1) contains supplementary material, which is available to authorized users.

Alcohol Consumption, Folate Intake, Hepatocellular Carcinoma, and Liver Disease Mortality

Excessive alcohol consumption is a well-established risk factor for liver disease and hepatocellular carcinoma (HCC). Previous studies have found that increased alcohol consumption can lead to lower absorption of folate. Conversely, higher folate intake has been inversely associated with liver damage and HCC. In the current study, we investigate the effect of alcohol consumption and folate intake on HCC incidence and liver disease mortality in the NIH-American Association of Retired Persons Diet and Health Study. The study population included 494,743 participants who reported at baseline their dietary intake for the previous year. Alcohol and folate were analyzed with hazards ratios (HR) and 95% confidence intervals (CI) using multivariate Cox proportional hazards regression models adjusted for age, sex, race, education, smoking, body mass index, and diabetes. HCC incidence (n = 435) was determined through 2006 via linkage with cancer registries, and liver disease mortality (n = 789) was determined through 2008 via linkage to the U.S. Social Security Administration Death Master File and the National Death Index Plus by the National Center for Health Statistics. Consumption of more than three drinks per day was positively associated with both HCC incidence (HR: 1.92; 95%CI: 1.42-2.60) and liver disease mortality (HR: 5.84; 95%CI: 4.81-7.10), whereas folate intake was associated with neither outcome. Folate, however, modified the relationship between alcohol and HCC incidence (Pinteraction = 0.03), but had no effect on the relationship between alcohol and liver disease mortality (Pinteraction = 0.54). These results suggest that higher folate intake may ameliorate the effect of alcohol consumption on the development of HCC. Folate intake may be beneficial in the prevention of alcohol-associated HCC.

Elk1 and AP-1 sites in the TBP promoter mediate alcohol-induced deregulation of Pol III-dependent genes

The major risk factors for hepatocellular carcinoma (HCC) are chronic liver diseases that include hepatitis B, hepatitis C, alcoholic liver disease and non-alcoholic steatohepatitis. However, the mechanisms of alcohol-associated HCC remain to be elucidated. The products of RNA Pol III (RNA polymerase III) dependent genes are elevated in both transformation cells and tumor cells. TBP (TATA-box binding protein) is a central transcription factor, which regulates Pol I, Pol II and Pol III gene activity. Our studies have demonstrated that alcohol increases TBP expression and Pol III gene transcription to promote liver tumor formation. We continue to investigate how ethanol mediates TBP expression. Here, we report that ethanol induces TBP promoter activity and the induction is ethanol dose dependent. Blocking the JNK1 pathway by a chemical inhibitor and siRNA reduces this ethanol-induced activity. Furthermore, mutating G>A at a −46bp Elk1 binding site of the TBP promoter or mutating AP-1 binding site at −37bp (A>G) and −38bp (C>T) reduces the TBP promoter activity. Mutation of both Elk1 and AP-1 binding sites dramatically represses this induction. Together, these studies demonstrate that, for the first time, alcohol increases Pol III gene transcription through a response element, which is composed of the overlapping Elk1 and AP-1 binding sites of the TBP promoter and affected by alcohol. It suggests that these binding sites may play a critical role in alcohol-induced deregulation of Pol III genes in liver tumor development.

Non‐alcoholic fatty liver disease progresses to hepatocellular carcinoma in the absence of apparent cirrhosis

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in developed countries, and accumulating evidence suggests it as the hepatic manifestation of the metabolic syndrome (MS). Although the published prevalence of hepatocellular carcinoma (HCC) is low in NAFLD/NASH patients, most of these data have been derived from areas endemic for viral hepatitis. We recruited 162 adults with HCC between February 2007 and March 2008, investigated the underlying etiologies and determined the prevalence of the MS and related features within each group. Patients with NAFLD/NASH-associated HCC exhibited a higher prevalence of metabolic features (Type 2 diabetes mellitus, hypertension, dyslipidemia, coronary artery disease) compared to non-NAFLD/NASH-HCC. Intriguingly, a significant number (41.7%; p < 0.005) of individuals with NAFLD/NASH-HCC had no evidence of cirrhosis. Patients with alcohol-induced liver disease also displayed many features (14/19, 73.7%) of the MS, although, in contrast to NAFLD/NASH-HCC, alcohol-associated HCC was highly associated with cirrhosis (95.0%; p = 0.064). NAFLD/NASH as the hepatic entity of the MS may itself pose a risk factor for HCC, even in the absence of cirrhosis. The MS may also promote development of HCC among those with alcoholic liver disease. Increased awareness of liver manifestations in the MS may instigate early interventions against developing HCC.

Nonalcoholic Fatty Liver Disease progresses to HCC in the absence of apparent Cirrhosis

Background and Aims: NAFLD is the most common liver disease in developed countries, and accumulating evidence suggests it as the hepatic manifestation of the metabolic syndrome. Although the published prevalence of hepatocellular carcinoma (HCC) is low in NAFLD/NASH patients, most of these data have been derived from areas endemic for viral hepatitis. Thus we aimed to analyze a european collective of patients with a significant proportion of NAFLD associated HCC. Methods: 162 adults with HCC between 02/2007 and 03/2008 were recruited into the study. The underlying etiologies, the prevalence of the metabolic syndrome and related features within each group were determined. Results: In patients with NAFLD/NASH-associated HCC a higher prevalence of metabolic features (type 2 diabetes mellitus, hypertension, dyslipidemia, coronary artery disease) was found, compared to non-NAFLD/NASH-HCC. Intriguingly, for a significant number (41.7%; p&lt;0.005) of individuals with NAFLD/NASH-HCC no evidence of cirrhosis could be obtained. Patients with alcohol-induced liver disease also displayed many features (14/19, 73.7%) of the metabolic syndrome, although, in contrast to NAFLD/NASH-HCC, alcohol-associated HCC was highly associated with cirrhosis (95.0%; p=0.064). Conclusion: NAFLD/NASH as the hepatic entity of the metabolic syndrome may itself pose a risk factor for HCC, even in the absence of cirrhosis. The metabolic syndrome may also contribute to development of HCC among those with alcoholic liver disease. Increased awareness of liver manifestations in patients exhibiting metabolic syndrome may warrant early screening for HCC.

Expanding the natural history of nonalcoholic steatohepatitis: From cryptogenic cirrhosis to hepatocellular carcinoma

Background & Aims: Nonalcoholic steatohepatitis (NASH) may progress to cirrhosis; whether NASH plays also a role in the development of hepatocellular carcinoma (HCC) is unknown. Methods: Among 641 cirrhosis-associated HCCs, we retrospectively identified 44 patients with cryptogenic cirrhosis (CC). Of these, 23 were actively followed up and were compared in a case-control study with viral- and alcohol-associated HCC. Family and personal history of diabetes, hypertension, coronary heart disease, dyslipidemia, obesity, and biochemical data were compared between groups. Iron status and presence of mutations in the HFE gene of familiar hemochromatosis were also determined. Results: Family history was not different in relation to etiology. The prevalence of obesity and diabetes was significantly higher in patients with CC. Although liver function was similar, CC patients had higher glucose, cholesterol, and triglyceride plasma levels, increased parameters of insulin resistance, and lower aminotransferase levels. Iron status and prevalence of mutations in the HFE gene did not differ. Logistic regression analysis identified in sequence hypertriglyceridemia, diabetes, and normal aminotransferases as independent factors associated with HCC arising in CC. Conclusions: Features suggestive of NASH are more frequently observed in HCC arising in patients with CC than in age- and sex-matched HCC patients of well-defined viral or alcoholic etiology. HCC may represent a late complication of NASH-related cirrhosis.GASTROENTEROLOGY 2002;123:134-140

Interactions Between Alcohol and Hepatitis Viruses in the Liver

There is a high frequency of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections among individuals with alcoholic liver disease (ALD) and alcohol-associated hepatocellular carcinoma (HCC), suggesting that these viruses are implicated in the pathogenesis of these diseases. Alcohol may act synergistically by promoting the development and progression of liver disease. The interactions of alcohol with infected hepatocytes and with antiviral immunity may result in altered patterns of virus gene expression and replication, making diagnosis difficult in some cases of ALD and HCC. The potential association of ALD and alcoholic HCC with serologically negative virus variants raises major difficulties in the epidemiologic assessment of cause and effect, providing major challenges for the future.