Chronic infection with the hepatitis C virus (HCV) causes epigenetic changes in liver cells that alter gene expression patterns and continue to affect risk for hepatocellular carcinoma (HCC), regardless of patients’ response to direct-activating antiviral (DAA) agents, researchers report in the June issue of Gastroenterology. These alterations might be targeted to prevent liver cancer in patients receiving treatment for HCV infection.
Treatment with DAA agents can cure more than 90% of patients with chronic HCV infection. Although curing the infection decreases the overall HCC risk in HCV-infected patients, it does not eliminate virus-induced HCC risk — especially in patients with advanced fibrosis. The absolute risk of HCC remains high for patients with cirrhosis even if they had a sustained virologic response (SVR) to therapy.
It is not clear how HCC develops after an SVR to treatment. HCV-mediated liver disease and carcinogenesis involve chronic hepatic inflammation and fibrogenesis, with formation of neoplastic clones that arise and progress in the carcinogenic environment. A gene expression signature was identified in liver tissues from HCV-infected patients that was associated with HCC risk and mortality, so the virus appears to induce changes in transcription that promote hepatocarcinogenesis. Epigenetic changes have also been identified in HCC tissues, but little is known about their role in virus-associated hepatocarcinogenesis.
Epigenetic alterations affect the expression of genes, without changing the nucleotide sequence, via many mechanisms, including DNA methylation, nucleosome remodeling, and histone modification. Histone modifications include acetylation of lysine (K) residues (eg, K27 of histone H3). H3K27ac is a modification that is associated with open chromatin and increased gene expression.
Nourdine Hamdane et al performed genome-wide 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 (HBV) infection, and 7 patients with nonalcoholic steatohepatitis (NASH) in Europe and Japan.
The authors found that HCV, HBV, and NASH all induced modifications of histones (H3K27ac). However, HCV-induced H3K27ac persisted even after clearance of HCV infection by DAA or interferon treatment. There was a correlation between H3K27ac and changes in gene expression in livers from HCV-infected and DAA-cured patients. So, these epigenetic changes deregulate transcription, and this deregulation persists after cure. For example, HCV-induced H3K27ac altered expression of genes whose products regulate the tumor necrosis factor (TNF), inflammatory , epithelial–mesenchymal transition, phosphoinositide 3-kinase, AKT, and MTOR pathways. Approximately 900 of the genes with epigenetic modifications were directly linked with carcinogenesis.
The analysis showed that changes in H3K27ac observed in HCV-infected patients were partly reversed in cured patients with stage F2–3 fibrosis. This group shared 2259 of the 5318 (42.5%) modified genes in the HCV-infected group. In contrast, patients with advanced liver disease (F4) still HCV-induced changes in H3K27ac after their infection was cured with DAAs, in 96.6% of modified genes (5140 of 5318 genes).
Hamdane et al observed that 52% of H3K27ac-modified genes were specific to tumor tissues, 31% were specific to adjacent nontumorous tissues, and 17% were common to the paired tissues. The epigenetic alterations therefore persist from advanced fibrosis to HCC and might be involved in hepatocarcinogenesis before and after cure.
To determine which fraction of the observed changes depended on interactions between HCV and hepatocytes, the authors performed studies of mice with humanized livers (with engrafted human hepatocytes). These livers can be infected with HCV and develop inflammation and fibrosis, similar to patients with hepatitis. Hamdane et al observed significant changes in levels of H3K27ac in livers of the HCV-infected mice, which persisted after cure of the infection with DAAs. The H3K27ac modifications also increased expression of genes in the TNF pathway, as in human livers.
A comparative analysis of genes with persistent H3K27ac modifications in patients and mice showed a set of 65 commonly modified genes. Genes upregulated in mice and humans after after HCV infection and DAA cure, compared with noninfected samples, included KRAS, TNF, IL2, STAT5, and genes in the epithelial–mesenchymal transition, apoptosis, glycolysis, and inflammation pathways.
Increased expression of sphingosine kinase 1 (SPHK1), and the transcription factor SOX9, was confirmed at the protein level in patients with HCV before and after cure, as well as in tumor samples. Increased levels of SPHK1 were associated with development of HCC in a cohort of patients with cirrhosis and patients with HCV cured by DAA treatment.
The authors conclude that chronic HCV infection induces H3K27ac modifications and changes in transcription that are associated with HCC risk and persist after HCV cure. They state that there have been few studies of epigenetic changes following HCV infection, and that this is the first integrative genome-wide approach that combines analyses in patient liver tissues and mice with humanized livers.
In an editorial that accompanies the article, Volker Lohmann and Ralf Bartenschlager point out that the the epigenetic alterations were not dependent on the type of therapy (DAA or interferon). They write that the findings reveal the lasting impact of chronic HCV infection on the liver and provide a compelling reason to treat patients with HCV early, before the accumulation of these epigenetic changes.