Liu, Chen

Background and Aims: Disruption of the epigenome is a hallmark of human disease, including liver cirrhosis and HCC. While genetic heterogeneity is an established effector of pathologic phenotypes, epigenetic heterogeneity is less well understood. Environmental exposures alter the liver-specific DNA methylation landscape and influence the onset of liver cancer. Given that currently available treatments are unable to target frequently mutated genes in HCC, there is an unmet need for novel therapeutics to prevent or reverse liver damage leading to hepatic tumorigenesis, which the epigenome may provide. Approach and Results: We performed genome-wide profiling of DNA methylation, copy number, and gene expression from multiple liver regions from 31 patients with liver disease to examine their crosstalk and define the individual and combinatorial contributions of these processes to liver disease progression. We identified epigenetic heterogeneity hotspots that are conserved across patients. Elevated epigenetic heterogeneity is associated with increased gene expression heterogeneity. Cirrhotic regions comprise 2 distinct cohorts—one exclusively epigenetic, and the other where epigenetic and copy number variations collaborate. Epigenetic heterogeneity hotspots are enriched for genes central to liver function (eg, HNF1A) and known tumor suppressors (eg, RASSF1A). These hotspots encompass genes including ACSL1, ACSL5, MAT1A, and ELFN1, which have phenotypic effects in functional screens, supporting their relevance to hepatocarcinogenesis. Moreover, epigenetic heterogeneity hotspots are linked to clinical measures of outcome. Conclusions: Substantial epigenetic heterogeneity arises early in liver disease development, targeting key pathways in the progression and initiation of both cirrhosis and HCC. Integration of epigenetic and transcriptional heterogeneity unveils putative epigenetic regulators of hepatocarcinogenesis.

There is a paucity of human in vitro models available to study the effects of alcohol in hepatocellular carcinoma (HCC). HCC cell lines are often deficient in either alcohol dehydrogenase (ADH) or cytochrome P450 2E1 (CYP2E1). VL‐17a is currently the most widely studied model available. VL‐17a is derived from a HepG2 cell line engineered to overexpress mouse ADH and human CYP2E1. LH86 is a novel HCC cell line previously developed in our laboratory. Here we compare the LH86 cell line with the current model to identify a novel tool to study the effects of alcohol in HCC. The aim of this study is to evaluate the LH86 cell line as a novel human in vitro model to study the effects of alcohol in HCC. The cell lines were cultured with alcohol and gene expression and epigenomic changes were identified. The LH86 cell line was observed to have unique functional properties indicating that it is a potential novel tool to study the effects of alcohol in HCC. Support or Funding Information R01 AA19976 TO KR AND CL

Abstract Background and Aims Chronic HCV infection is a leading etiologic driver of cirrhosis and ultimately HCC. Of the approximately 71 million individuals chronically infected with HCV, 10%‐20% are expected to develop severe liver complications in their lifetime. Epigenetic mechanisms including DNA methylation and histone modifications become profoundly disrupted in disease processes including liver disease. Approach and Results To understand how HCV infection influences the epigenome and whether these events remain as “scars” following cure of chronic HCV infection, we mapped genome‐wide DNA methylation, four key regulatory histone modifications (H3K4me3, H3K4me1, H3K27ac, and H3K27me3), and open chromatin in parental and HCV‐infected immortalized hepatocytes and the Huh7.5 HCC cell line, along with DNA methylation and gene‐expression analyses following elimination of HCV in these models through treatment with interferon‐α (IFN‐α) or a direct‐acting antiviral (DAA). Our data demonstrate that HCV infection profoundly affects the epigenome (particularly enhancers); HCV shares epigenetic targets with interferon‐α targets; and an overwhelming majority of epigenetic changes induced by HCV remain as “scars” on the epigenome following viral cure. Similar findings are observed in primary human patient samples cured of chronic HCV infection. Supplementation of IFN‐α/DAA antiviral regimens with DNA methyltransferase inhibitor 5‐aza‐2’‐deoxycytidine synergizes in reverting aberrant DNA methylation induced by HCV. Finally, both HCV‐infected and cured cells displayed a blunted immune response, demonstrating a functional effect of epigenetic scarring. Conclusions Integration of epigenetic and transcriptional data elucidate key gene deregulation events driven by HCV infection and how this may underpin the long‐term elevated risk for HCC in patients cured of HCV due to epigenome scarring.

Hepatocellular carcinoma (HCC) is the second most common cause of cancer deaths worldwide. HCC is known to be a CpG Island methylator Phenotype (CIMP) cancer. Tumor development in CIMP cancers is characterized by epigenomic changes including changes in DNA methylation. We have previously shown that overlapping methylation abnormalities are conserved between primary tissue and cell culture models of HCC. Alcohol consumption is known to increase the risk of development of HCC and has also been shown to alter DNA methylation in a manner that contributes to liver disease. Here we study the effect of alcohol as a driver to tumor progression in HCC. We have developed in vitro models of HCC to identify the gene targets altered by ethanol metabolism during tumor progression over an extended period of time.

No effective systemic treatment is available for patients with unresectable, recurrent, or metastatic mucoepidermoid carcinoma (MEC), the most common salivary gland malignancy. MEC is frequently associated with a t(11;19)(q14-21;p12-13) translocation that creates a CRTC1-MAML2 fusion gene. The CRTC1-MAML2 fusion exhibited transforming activity in vitro; however, whether it serves as an oncogenic driver for MEC establishment and maintenance in vivo remains unknown. Here, we show that doxycycline-induced CRTC1-MAML2 knockdown blocked the growth of established MEC xenografts, validating CRTC1-MAML2 as a therapeutic target. We further generated a conditional transgenic mouse model and observed that Cre-induced CRTC1-MAML2 expression caused 100% penetrant formation of salivary gland tumors resembling histological and molecular characteristics of human MEC. Molecular analysis of MEC tumors revealed altered p16-CDK4/6-RB pathway activity as a potential cooperating event in promoting CRTC1-MAML2–induced tumorigenesis. Cotargeting of aberrant p16-CDK4/6-RB signaling and CRTC1-MAML2 fusion–activated AREG/EGFR signaling with the respective CDK4/6 inhibitor Palbociclib and EGFR inhibitor Erlotinib produced enhanced antitumor responses in vitro and in vivo. Collectively, this study provides direct evidence for CRTC1-MAML2 as a key driver for MEC development and maintenance and identifies a potentially novel combination therapy with FDA-approved EGFR and CDK4/6 inhibitors as a potential viable strategy for patients with MEC.

Hepatocellular Carcinoma (HCC) is a highly fatal liver cancer with no effective treatments. HCC tumors, formed after long‐term viral or alcohol related inflammation and cirrhosis, have a surprisingly low number of genetic mutations. Prior research has found that epigenetic patterns, such as DNA methylation at gene promoter sites, are common in HCC patients. Recent improvements in genetic sequencing and online genetic databases, have allowed us to identify a set of gene targets, including FMO3, for HCC detection or treatment. The hypothesis is that FMO3 is reduced by epigenetic dysregulation, leading to tumor formation. To find the connection between FMO3, epigenetic events and HCC tumor pathways, our lab created novel HCV‐stable and non‐infected HCC cell lines. We also used Huh7 and Huh7.5 cells to show the epigenetic changes in stressed cells and methylation inhibited cells. To mimic inflammation and stress leading to epigenetic changes, Huh7 cells were exposed to heat shock at 40°C for 40 min. Huh7.5 cells were treated with epigenetic inhibitor 5‐Aza‐2 deoxycytidine. RT‐qPCR and Western Blot show that FMO3 is significantly reduced in stressed cells and upregulated when treated with the methylation inhibitor. These results indicate that FMO3 may be used for early cancer risk detection and/or to improve treatment options by identifying the relationship between epigenetic regulation and tumorigenesis. Support or Funding Information NIH NIDDK 1R01DK110024‐01A1

Alpha‐1 antitrypsin deficiency (AATD) liver disease is characterized by marked heterogeneity in presentation and progression, despite a common underlying gene mutation, strongly suggesting the involvement of other genetic and/or epigenetic modifiers. Variation in clinical phenotype has added to the challenge of detection, diagnosis, and testing of new therapies in patients with AATD. We examined the contribution of DNA methylation (5‐methylcytosine [5mC]) to AATD liver disease heterogeneity because 5mC responds to environmental and genetic cues and its deregulation is a major driver of liver disease. Using liver biopsies from adults with early‐stage AATD and the ZZ genotype, genome‐wide 5mC patterns were interrogated. We compared DNA methylation among patients with early AATD, and among patients with normal liver, cirrhosis, and hepatocellular carcinoma derived from multiple etiologic exposures, and linked patient clinical/demographic features. Global analysis revealed significant genomic hypomethylation in AATD liver–impacting genes related to liver cancer, cell cycle, and fibrosis, as well as key regulatory molecules influencing growth, migration, and immune function. Further analysis indicated that 5mC changes are localized, with hypermethylation occurring within a background of genome‐wide 5mC loss and with patients with AATD manifesting distinct epigenetic landscapes despite their mutational homogeneity. By integrating clinical data with 5mC landscapes, we observed that CpGs differentially methylated among patients with AATD disease are linked to hallmark clinical features of AATD (e.g., hepatocyte degeneration and polymer accumulation) and further reveal links to well‐known sex‐specific effects of liver disease progression. Conclusion: Our data reveal molecular epigenetic signatures within this mutationally homogeneous group that point to ways to stratify patients for liver disease risk.

Disruption of epigenetic mechanisms has been intimately linked to the etiology of human cancer. Understanding how these epigenetic mechanisms (including DNA methylation [5mC], hydroxymethylation [5hmC], and histone post‐translational modifications) work in concert to drive cancer initiation and progression remains unknown. Hepatocellular carcinoma (HCC) is increasing in frequency in Western countries but lacks efficacious treatments. The epigenome of HCC remains understudied. To better understand the epigenetic underpinnings of HCC, we performed a genome‐wide assessment of 5mC, 5hmC, four histone modifications linked to promoter/enhancer function (H3K4me1, H3K27ac, H3K4me3, and H3K27me3), and transcription across normal, cirrhotic, and HCC liver tissue. Implementation of bioinformatic strategies integrated these epigenetic marks with each other and with transcription to provide a comprehensive epigenetic profile of how and when the liver epigenome is perturbed during progression to HCC. Our data demonstrate significant deregulation of epigenetic regulators combined with disruptions in the epigenome hallmarked by profound loss of 5hmC, locus‐specific gains in 5mC and 5hmC, and markedly altered histone modification profiles, particularly remodeling of enhancers. Data integration demonstrates that these marks collaborate to influence transcription (e.g., hyper‐5hmC in HCC‐gained active enhancers is linked to elevated expression) of genes regulating HCC proliferation. Two such putative epigenetic driver loci identified through our integrative approach, COMT and FMO3 , increase apoptosis and decrease cell viability in liver‐derived cancer cell lines when ectopically re‐expressed. Conclusion : Altogether, integration of multiple epigenetic parameters is a powerful tool for identifying epigenetically regulated drivers of HCC and elucidating how epigenome deregulation contributes to liver disease and HCC.

Pancreatic cancer has one of the lowest survival rates of all cancers. The mechanism underlying chemo-resistance of pancreatic cancer is not well understood. Our previous article reported that small molecule YM155 induced apoptosis in pancreatic cancer cells via activation of death receptor 5. In this study, we aim to continuously address death receptor 5–mediated apoptosis in chemo-resistant pancreatic carcinoma. We found that in comparison to paired pancreatic cancer tissues and adjacent normal tissues, five of the six cancer tissues had downregulated death receptor 5 and upregulated Bcl-xL. Mono treatment with lexatumumab was not sufficient to induce apoptosis in pancreatic cancer cells, whereas focal adhesion kinase inhibitor PF573228 significantly sensitized lexatumumab-induced apoptosis. Western blotting analysis revealed that lexatumumab and PF573228 combination treatment increased death receptor 5 but decreased Bcl-xL expression. Interestingly, pre-treatment with Bcl-xL inhibitor ABT263 reversed the insensitivity of panc-1 cells to lexatumumab or PF573228-induced apoptosis. Specific small interfering RNA-mediated gene silencing of Bcl-xL effectively sensitized pancreatic cancer cells to lexatumumab or PF573228-induced apoptosis. Furthermore, lexatumumab and PF573228 combination was shown to exhibit significant xenograft pancreatic tumor growth inhibition in SCID mice. Our data provide fundamental evidence to support the notion that lexatumumab and PF573228 co-treatment could be a potentially effective regime for patients with pancreatic cancer.