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Hepatocellular carcinoma (HCC) is the most rapidly increasing cause of cancer-related mortality in the United States. Because of the lack of viable treatment options for HCC, prevention in high risk patients has been proposed as an alternative strategy. The main risk factor for HCC is cirrhosis and several lines of evidence implicate epidermal growth factor (EGF) in the progression of cirrhosis and development of HCC. We therefore examined the effects of the EGF receptor (EGFR) inhibitor erlotinib on liver fibrogenesis and hepatocellular transformation in three different animal models of progressive cirrhosis – a rat model induced by repeated, low dose injections of diethylnitrosamine (DEN), a mouse model induced by carbon tetrachloride (CCl4) and a rat model induced by bile duct ligation (BDL). Erlotinib reduced EGFR phosphorylation in hepatic stellate cells (HSC), and reduced the total number of activated HSC. Erlotinib also decreased hepatocyte proliferation and liver injury. Consistent with all these findings, pharmacological inhibition of EGFR signaling effectively prevented the progression of cirrhosis and regressed fibrosis in some animals. Moreover, by alleviating the underlying liver disease, erlotinib blocked the development of HCC and its therapeutic efficacy could be monitored with a previously reported gene expression signature predictive of HCC risk in human cirrhosis patients. Conclusion These data suggest that EGFR inhibition with FDA-approved inhibitors presents a promising therapeutic approach for reduction of fibrogenesis and prevention of HCC in high risk cirrhosis patients who can be identified and monitored by gene expression signatures.

SUMMARY Cirrhosis is a milieu that develops hepatocellular carcinoma (HCC), the second most lethal cancer worldwide. HCC prediction and prevention in cirrhosis are key unmet medical needs. Here we have established an HCC risk gene signature applicable to all major HCC etiologies: hepatitis B/C, alcohol, and non-alcoholic steatohepatitis. A transcriptome meta-analysis of >500 human cirrhotics revealed global regulatory gene modules driving HCC risk and lysophosphatidic acid pathway as a central chemoprevention target. Pharmacological inhibition of the pathway in vivo reduced tumors and reversed the gene signature, which was verified in organotypic ex vivo culture of patient-derived fibrotic liver tissues. These results demonstrate the utility of clinical organ transcriptome to enable a strategy, reverse-engineering precision cancer prevention.

infection is an important risk factor for hepatocellular carcinoma (HCC). Despite effective antiviral therapies, the risk for HCC is decreased but not eliminated after a sustained virologic response (SVR) to direct-acting antiviral (DAA) agents, and the risk is higher in patients with advanced fibrosis. We investigated HCV-induced epigenetic alterations that might affect risk for HCC after DAA treatment in patients and mice Gastroenterology 2019;156:2313-2329 BASIC AND TRANSLATIONAL LIVER with humanized livers. METHODS: We performed genomewide ChIPmentation-based ChIP-Seq and RNA-seq analyses of liver tissues from 6 patients without HCV infection (controls), 18 patients with chronic HCV infection, 8 patients with chronic HCV infection cured by DAA treatment, 13 patients with chronic HCV infection cured by interferon therapy, 4 patients with chronic hepatitis B virus infection, and 7 patients with nonalcoholic steatohepatitis in Europe and Japan. HCV-induced epigenetic modifications were mapped by comparative analyses with modifications associated with other liver disease etiologies. uPA/SCID mice were engrafted with human hepatocytes to create mice with humanized livers and given injections of HCV-infected serum samples from patients; mice were given DAAs to eradicate the virus. Pathways associated with HCC risk were identified by integrative pathway analyses and validated in analyses of paired HCC tissues from 8 patients with an SVR to DAA treatment of HCV infection. RESULTS:We found chronic HCV infection to induce specific genome-wide changes in H3K27ac, which correlated with changes in expression of mRNAs and proteins. These changes persisted after an SVR to DAAs or interferon-based therapies. Integrative pathway analyses of liver tissues from patients and mice with humanized livers demonstrated that HCV-induced epigenetic alterations were associated with liver cancer risk. Computational analyses associated increased expression of SPHK1 with HCC risk. We validated these findings in an independent cohort of patients with HCV-related cirrhosis (n ¼ 216), a subset of which (n ¼ 21) achieved viral clearance. CONCLUSIONS: In an analysis of liver tissues from patients with and without an SVR to DAA therapy, we identified epigenetic and gene expression alterations associated with risk for HCC. These alterations might be targeted to prevent liver cancer in patients treated for HCV infection.

Hepatocellular carcinoma (HCC) is associated with a poor prognosis due to late diagnoses and a lack of effective treatment options. Epidermal growth factor receptor (EGFR)-targeted therapies have been effective in other cancers. However, erlotinib and cetuximab have shown only modest efficacy in clinical trials of HCC. We examined epithelial-to-mesenchymal transition (EMT) as a determinant of sensitivity of HCC to EGFR inhibitors. A panel of 12 human hepatoma cell lines were classified as epithelial or mesenchymal based on their expression of E-cadherin and vimentin. The resulting classification correlated with a previous microarray analysis of human hepatoma cell lines whereby the mesenchymal cell lines were shown to have increased expression of genes involved in metastasis and invasion. Sensitivity to erlotinib, gefitinib, and cetuximab was assessed and the epithelial cell lines were found to be significantly more susceptible to all three agents. Analysis of the EGFR pathway showed that EMT status was independent of EGFR expression or downstream extracellular signal-regulated kinase activation and only the epithelial cell lines expressed ErbB3. Interestingly, mesenchymal cells resistant to EGFR inhibitors had increased AKT and signal transducer and activator of transcription-3 activation through elevated expression of integrin-linked kinase (ILK). Mesenchymal cell lines were therefore experimentally transformed with kinaseinactive ILK (KI-ILK) with a resulting decrease in ILK activity and activation of AKT. KI-ILK transformants showed increased sensitivity to EGFR inhibitors both in vitro and in an in vivo xenograft model. These data suggest that EMT predicts HCC sensitivity to EGFR-targeted therapies and that ILK is a novel target to overcome HCC resistance to EGFR inhibition.

Summary The incidence of hepatocellular carcinoma (HCC) is rapidly increasing due to the prevalence of obesity and non-alcoholic fatty liver disease, but the molecular triggers that initiate disease development are not fully understood. We demonstrate that mice with targeted loss of function point mutations within the AMP-activated protein kinase (AMPK) phosphorylation sites acetyl-CoA carboxylase 1 (ACC1 Ser79Ala) and ACC2 (ACC2 Ser212Ala) have increased liver de novo lipogenesis (DNL) and liver lesions. The same mutation in ACC1 also increases DNL and proliferation human liver cancer cells. Consistent with these findings, a novel, liver specific ACC inhibitor (ND-654), that mimics the effects of ACC phosphorylation, inhibits hepatic DNL and the development of HCC, improving survival of tumor-bearing rats when used alone and in combination with the multi-kinase inhibitor sorafenib. These studies highlight the importance of DNL and dysregulation of AMPK-mediated ACC phosphorylation in accelerating HCC and the potential of ACC inhibitors for treatment.

The EGF gene polymorphism genotype is associated with risk for development of hepatocellular carcinoma in liver cirrhosis through modulation of EGF levels.

Summary Hepatitis C virus (HCV) is one of the major etiologic agents that causes hepatocellular carcinoma (HCC) by generating an inflammatory, fibrogenic, and carcinogenic tissue microenvironment in the liver. HCV-induced HCC is a rational target for cancer preventive intervention because of the clear-cut high-risk condition, cirrhosis, associated with high cancer incidence (1% to 7% per year). Studies have elucidated direct and indirect carcinogenic effects of HCV, which have in turn led to identification of candidate HCC chemoprevention targets. Selective molecular targeted agents may enable personalized strategies for HCC chemoprevention. In addition, multiple experimental and epidemiological studies suggest the potential value of generic drugs or dietary supplements targeting inflammation, oxidant stress, or metabolic derangements as possible HCC chemopreventive agents. While the successful use of highly effective direct-acting antiviral agents will make important inroads into reducing long term HCC risk, there will remain an important role for HCC chemoprevention even after viral cure, given the persistence of HCC risk in persons with advanced HCV fibrosis as shown in recent studies. The successful development of cancer preventive therapies will be more challenging compared to cancer therapeutics because of the requirement for larger and longer clinical trials and the need for a safer toxicity profile given its use as a preventive agent. Molecular biomarkers to selectively identify high-risk population could help mitigate these challenges. Genome-wide, unbiased molecular characterization, high-throughput drug/gene screening, experimental model-based functional analysis, and systems-level in silico modeling are expected to complement each other to facilitate discovery of new HCC chemoprevention targets and therapies.