Project description:Background & Aims. Hepatocellular carcinoma (HCC), the most prevalent form of liver cancer, is growing in incidence but treatment options remain limited, particularly for late stage disease. As liver cirrhosis is the principal risk state for HCC development, markers to detect early HCC within this patient population are urgently needed. Perturbation of epigenetic marks, such as DNA methylation (5mC), is a hallmark of human cancers, including HCC. Identification of regions with consistently altered 5mC levels in circulating cell free DNA (cfDNA) during progression from cirrhosis to HCC could therefore serve as markers for development of minimally-invasive screens of early HCC diagnosis and surveillance. Methods. To discover DNA methylation derived biomarkers of HCC in the background of liver cirrhosis, we profiled genome-wide 5mC landscapes in patient cfDNA using the Infinium HumanMethylation450k BeadChip Array. We further linked these findings to primary tissue data available from TCGA and other public sources. Using biological and statistical frameworks, we selected CpGs that robustly differentiated cirrhosis from HCC in primary tissue and cfDNA followed by validation in an additional independent cohort. Results. We identified CpGs that segregate patients with cirrhosis, from patients with HCC within a cirrhotic liver background, through genome-wide analysis of cfDNA 5mC landscapes. Lasso regression analysis pinpointed a panel of probes in our discovery cohort that were validated in two independent datasets. A panel of five CpGs (cg04645914, cg06215569, cg23663760, cg13781744, and cg07610777) yielded AUROCs of 0.9525, 0.9714, and 0.9528 in cfDNA discovery and tissue validation cohorts 1 and 2, respectively. Conclusions. 5mC markers derived from cfDNA robustly identify HCC within a cirrhotic liver background indicating that further validation is warranted. Our finding that 5mC markers derived from primary tissue did not perform well in cfDNA, compared to those identified directly from cfDNA, reveals potential advantages of starting with cfDNA to discover high performing markers for liquid biopsy development.

Project description:Abstract Rationale: Interferon (IFN) plays a key role in immunotherapy by acting through interferon-stimulated genes. Interferon resistance limits its efficacy. Ubiquitination modification of key proteins is a popular research direction in hepatocellular carcinoma (HCC), and it is also one of the important mechanisms of interferon resistance. This study is dedicated to discovering the link between the pathogenesis of interferon resistance and ubiquitination modifications in HCC. Methods: We first combined the TCGA LIHC dataset and genes from the iUUCD 2.0 database to screen the most promising ubiquitination-related gene, UBE2O, in HCC using a bioinformatics approach. A combination of proteomic analysis, mass spectrometry, and survival analysis was used to screen for pathways and proteins negatively regulated by UBE2O. The effect of silencing UBE2O on interferon sensitivity in HCC cells was assessed using in vitro clone formation, migration, and wound healing assays and in vivo subcutaneous tumor xenograft models. Changes in the half-life of substrates following inhibition of UBE2O were assessed using a cycloheximide method. Two databases were used to predict ubiquitination sites on IFIT3. After rescue experiments to exclude the effect of substrate, proteomic analysis was reperformed to analyze the effect on the interferon pathway and phenotype. The effect on substrate and interferon sensitivity was observed using arsenic trioxide (ATO) to inhibit UBE2O. Results: Initially, UBE2O was deduced from 807 UUCRGs in the TCGA LIHC dataset by six consecutive screening steps. UBE2O was significantly overexpressed in HCC cell lines and tissues compared to the corresponding normal group, and low expression of UBE2O was associated with a better prognosis. UBE2O was knocked down for proteomic analysis, and the results suggested that UBE2O negatively regulates the interferon-alpha/beta signaling (p＜0.01) and the interferon pathway (p＜0.01). IFIT3 in the interferon pathway was identified as a possible ubiquitinated substrate of UBE2O by combining mass spectrometry, the human protein atlas (HPA), and TCGA-based survival analysis. There was a significant negative correlation between UBE2O and IFIT3, including survival analysis showing opposite trends and a negative correlation in protein expression. UBE2O binds to IFIT3, and interferon-α promotes this binding. The stability of IFIT3 protein increased with decreasing UBE2O (p＜0.05), and these effects disappeared with the mutational inactivation of UBE2O. K236 of IFIT3 is the active site of ubiquitination. By increasing IFIT3, the knockdown of UBE2O increases the sensitivity of HCC cells to interferon, leading to reduced proliferation and migration (all p＜0.05). These effects disappeared after the elimination of IFIT3. Additionally, ATO inhibited UBE2O and therefore increased interferon sensitivity in HCC cells. Conclusions: UBE2O exerts a protumor role and targets a new substrate, IFIT3, for ubiquitination and degradation, impeding the interferon pathway activation and interferon sensitization in HCC. The findings will shed light on the interferon-based targeted or combination therapeutic strategies for HCC in the future.

Project description:Background: C3H mice have been frequently used in cancer studies as animal models of spontaneous liver tumors and chemically induced hepatocellular carcinoma (HCC). Epigenetic modifications, including DNA methylation, are among pivotal control mechanisms of gene expression leading to carcinogenesis. Although information on somatic mutations in liver tumors of C3H mice is available, epigenetic aspects are yet to be clarified. Methods: We performed next generation sequencing-based analysis of DNA methylation and microarray analysis of gene expression to explore genes regulated by DNA methylation. Overlaying these data, we selected cancer-related genes whose expressions are inversely correlated with DNA methylation levels in the associated differentially methylated regions (DMRs) located around transcription start sites (TSSs) (promoter DMRs). We further assessed mutuality of the selected genes for expression and DNA methylation in human HCC using the Cancer Genome Atlas (TCGA) database. Results: We obtained data on genome-wide DNA methylation profiles in the normal and tumor livers of C3H mice. We identified promoter DMRs of genes which are reported to be related to cancer and whose expressions are inversely correlated with the DNA methylation, including Mst1r, Slpi and Extl1. The association between DNA methylation and gene expression was confirmed using a DNA methylation inhibitor 5- aza-2'-deoxycytidine (5-aza-dC) in Hepa1c1c7 cells and Hepa1-6 cells. Overexpression of Mst1r in Hepa1c1c7 cells illuminated a novel downstream pathway via IL-33 upregulation. Database search indicated that gene expressions of Mstr1 and Slpi are upregulated and the TSS upstream regions are hypomethylated also in human HCC. These results suggest that DMRs, including those of Mst1r and Slpi, are involved in liver tumorigenesis in C3H mice, and also possibly in human HCC. Conclusions: Our study clarified genome wide DNA methylation landscape of C3H mice. The data provide useful information for further epigenetic studies of mice models of HCC. The present study particularly proposed novel DNA methylation-regulated pathways for Mst1r and Slpi, which may be applied not only to mouse HCC but also to human HCC.

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