Correction: Characterization of HBV integration patterns and timing in liver cancer and HBV-infected livers.

[This corrects the article DOI: 10.18632/oncotarget.25308.].

Loss-of-function mutations in Zn-finger DNA-binding domain of HNF4A cause aberrant transcriptional regulation in liver cancer.

Hepatocyte nuclear factors (HNF) are transcription factors that crucially regulate cell-specific gene expression in many tissues, including the liver. Of these factors, HNF4A acts both as a master regulator of liver organogenesis and a tumor suppressor in the liver. In our whole genome sequencing analysis, we found seven somatic mutations (three Zn-finger mutations, three deletion mutants, and one intron mutation) of HNF4A in liver cancers. Interestingly, three out of seven mutations were clustered in its Zn-finger DNA-binding domain; G79 and F83 are positioned in the DNA recognition helix and the sidechain of M125 is sticking into the core of domain. These mutations are likely to affect DNA interaction from a structural point of view. We then generated these mutants and performed in-vitro promoter assays as well as DNA binding assays. These three mutations reduced HNF4 transcriptional activity at promoter sites of HNF4A-target genes. Expectedly, this decrease in transcriptional activity was associated with a change in DNA binding. RNA-Seq analysis observed a strong correlation between HNF4A expression and expression of its target genes, ApoB and HNF1A, in liver cancers. Since knockdown of HNF4A caused a reduction in ApoB and HNF1A expression, possibly loss of HNF4 reduces the expression of these genes and subsequently tumor growth is triggered. Therefore, we propose that HNF4A mutations G79C, F83C, and M125I are functional mutations found in liver cancers and that loss of HNF4A function, through its mutation, leads to a reduction in HNF1A and ApoB gene expression with a concomitant increased risk of liver tumorigenesis.

Characterization of HBV integration patterns and timing in liver cancer and HBV-infected livers.

Integration of Hepatitis B virus (HBV) into the human genome can cause genetic instability, leading to selective advantages for HBV-induced liver cancer. Despite the large number of studies for HBV integration into liver cancer, little is known about the mechanism of initial HBV integration events owing to the limitations of materials and detection methods. We conducted an HBV sequence capture, followed by ultra-deep sequencing, to screen for HBV integrations in 111 liver samples from human-hepatocyte chimeric mice with HBV infection and human clinical samples containing 42 paired samples from non-tumorous and tumorous liver tissues. The HBV infection model using chimeric mice verified the efficiency of our HBV-capture analysis and demonstrated that HBV integration could occur 23 to 49 days after HBV infection via microhomology-mediated end joining and predominantly in mitochondrial DNA. Overall HBV integration sites in clinical samples were significantly enriched in regions annotated as exhibiting open chromatin, a high level of gene expression, and early replication timing in liver cells. These data indicate that HBV integration in liver tissue was biased according to chromatin accessibility, with additional selection pressures in the gene promoters of tumor samples. Moreover, an integrative analysis using paired non-tumorous and tumorous samples and HBV-related transcriptional change revealed the involvement of TERT and MLL4 in clonal selection. We also found frequent and non-tumorous liver-specific HBV integrations in FN1 and HBV-FN1 fusion transcript. Extensive survey of HBV integrations facilitates and improves the understanding of the timing and biology of HBV integration during infection and HBV-related hepatocarcinogenesis.

Genomic landscape of colitis-associated cancer indicates the impact of chronic inflammation and its stratification by mutations in the Wnt signaling.

Inflammatory bowel disease (IBD) increases the risk of colorectal cancer, known as colitis-associated cancer (CAC). It is still unclear what driver mutations are caused by chronic inflammation and lead to CAC development. To get insight into this issue, we investigated somatic alterations in CAC. We performed exome sequencing of 22 fresh CACs and targeted sequencing of 43 genes on 90 archive specimens from Japanese CAC patients, of which 58 were ulcerative colitis (UC) and 32 were Crohn's disease (CD). Consistently with the previous reports, TP53 was commonly mutated (66%) whereas APC, KRAS and SMAD4 were mutated less frequently (16%, 11% and 11%, respectively). Mucinous CD-CACs in the anus, an Asian-specific subtype of CD-CAC, had less somatic mutations in our target genes. We also found that RNF43, a negative regulator of the Wnt signaling, was somatically mutated in a significant fraction of CACs (10 of 90; 11%). Two lines of evidence indicated that somatic mutations of RNF43 were related to chronic inflammation. First, somatic mutations of RNF43 were significantly associated with longer duration of IBD. Second, clinico-pathological features suggested many of the APC-mutated CACs were actually sporadic colorectal cancer whereas RNF43-mutated CACs did not have this tendency. RNA-Seq analysis showed that RNF43-mutated CACs had elevated expression of c-Myc and its target genes, suggesting that RNF43 is a bona fide driver of CAC development. This study provides evidence that somatic mutation of RNF43 is the driver genetic alteration that links chronic inflammation and cancer development in about 10% of CACs.

Genomic characterization of biliary tract cancers identifies driver genes and predisposing mutations.

BACKGROUND & AIMS: Biliary tract cancers (BTCs) are clinically and pathologically heterogeneous and respond poorly to treatment. Genomic profiling can offer a clearer understanding of their carcinogenesis, classification and treatment strategy. We performed large-scale genome sequencing analyses on BTCs to investigate their somatic and germline driver events and characterize their genomic landscape. METHODS: We analyzed 412 BTC samples from Japanese and Italian populations, 107 by whole-exome sequencing (WES), 39 by whole-genome sequencing (WGS), and a further 266 samples by targeted sequencing. The subtypes were 136 intrahepatic cholangiocarcinomas (ICCs), 101 distal cholangiocarcinomas (DCCs), 109 peri-hilar type cholangiocarcinomas (PHCs), and 66 gallbladder or cystic duct cancers (GBCs/CDCs). We identified somatic alterations and searched for driver genes in BTCs, finding pathogenic germline variants of cancer-predisposing genes. We predicted cell-of-origin for BTCs by combining somatic mutation patterns and epigenetic features. RESULTS: We identified 32 significantly and commonly mutated genes including TP53, KRAS, SMAD4, NF1, ARID1A, PBRM1, and ATR, some of which negatively affected patient prognosis. A novel deletion of MUC17 at 7q22.1 affected patient prognosis. Cell-of-origin predictions using WGS and epigenetic features suggest hepatocyte-origin of hepatitis-related ICCs. Deleterious germline mutations of cancer-predisposing genes such as BRCA1, BRCA2, RAD51D, MLH1, or MSH2 were detected in 11% (16/146) of BTC patients. CONCLUSIONS: BTCs have distinct genetic features including somatic events and germline predisposition. These findings could be useful to establish treatment and diagnostic strategies for BTCs based on genetic information. LAY SUMMARY: We here analyzed genomic features of 412 BTC samples from Japanese and Italian populations. A total of 32 significantly and commonly mutated genes were identified, some of which negatively affected patient prognosis, including a novel deletion of MUC17 at 7q22.1. Cell-of-origin predictions using WGS and epigenetic features suggest hepatocyte-origin of hepatitis-related ICCs. Deleterious germline mutations of cancer-predisposing genes were detected in 11% of patients with BTC. BTCs have distinct genetic features including somatic events and germline predisposition.

Whole genome sequencing discriminates hepatocellular carcinoma with intrahepatic metastasis from multi-centric tumors.

BACKGROUND & AIMS: Patients with hepatocellular carcinoma (HCC) have a high-risk of multi-centric (MC) tumor occurrence due to a strong carcinogenic background in the liver. In addition, they have a high risk of intrahepatic metastasis (IM). Liver tumors withIM or MC are profoundly different in their development and clinical outcome. However, clinically or pathologically discriminating between IM and MC can be challenging. This study investigated whether IM or MC could be diagnosed at the molecular level. METHODS: We performed whole genome and RNA sequencing analyses of 49 tumors including two extra-hepatic metastases, and one nodule-in-nodule tumor from 23 HCC patients. RESULTS: Sequencing-based molecular diagnosis using somatic single nucleotide variation information showed higher sensitivity compared to previous techniques due to the inclusion of a larger number of mutation events. This proved useful in cases, which showed inconsistent clinical diagnoses. In addition, whole genome sequencing offered advantages in profiling of other genetic alterations, such as structural variations, copy number alterations, and variant allele frequencies, and helped to confirm the IM/MCdiagnosis. Divergent alterations between IM tumors with sorafenib treatment, long time-intervals, or tumor-in-tumor nodules indicated high intra-tumor heterogeneity, evolution, and clonal switching of liver cancers. CONCLUSIONS: It is important to analyze the differences between IM tumors, in addition to IM/MC diagnosis, before selecting a therapeutic strategy for multiple tumors in the liver. LAY SUMMARY: Whole genome sequencing of multiple liver tumors enabled the accuratediagnosis ofmulti-centric occurrence and intrahepatic metastasis using somatic single nucleotide variation information. In addition, genetic discrepancies between tumors help us to understand the physical changes during recurrence and cancer spread.

Whole-genome mutational landscape and characterization of noncoding and structural mutations in liver cancer.

Liver cancer, which is most often associated with virus infection, is prevalent worldwide, and its underlying etiology and genomic structure are heterogeneous. Here we provide a whole-genome landscape of somatic alterations in 300 liver cancers from Japanese individuals. Our comprehensive analysis identified point mutations, structural variations (STVs), and virus integrations, in noncoding and coding regions. We discovered mutational signatures related to liver carcinogenesis and recurrently mutated coding and noncoding regions, such as long intergenic noncoding RNA genes (NEAT1 and MALAT1), promoters, CTCF-binding sites, and regulatory regions. STV analysis found a significant association with replication timing and identified known (CDKN2A, CCND1, APC, and TERT) and new (ASH1L, NCOR1, and MACROD2) cancer-related genes that were recurrently affected by STVs, leading to altered expression. These results emphasize the value of whole-genome sequencing analysis in discovering cancer driver mutations and understanding comprehensive molecular profiles of liver cancer, especially with regard to STVs and noncoding mutations.

Whole-genome mutational landscape of liver cancers displaying biliary phenotype reveals hepatitis impact and molecular diversity.

Intrahepatic cholangiocarcinoma and combined hepatocellular cholangiocarcinoma show varying degrees of biliary epithelial differentiation, which can be defined as liver cancer displaying biliary phenotype (LCB). LCB is second in the incidence for liver cancers with and without chronic hepatitis background and more aggressive than hepatocellular carcinoma (HCC). To gain insight into its molecular alterations, we performed whole-genome sequencing analysis on 30 LCBs. Here we show, the genome-wide substitution patterns of LCBs developed in chronic hepatitis livers overlapped with those of 60 HCCs, whereas those of hepatitis-negative LCBs diverged. The subsequent validation study on 68 LCBs identified recurrent mutations in TERT promoter, chromatin regulators (BAP1, PBRM1 and ARID2), a synapse organization gene (PCLO), IDH genes and KRAS. The frequencies of KRAS and IDHs mutations, which are associated with poor disease-free survival, were significantly higher in hepatitis-negative LCBs. This study reveals the strong impact of chronic hepatitis on the mutational landscape in liver cancer and the genetic diversity among LCBs.

Circulating Tumor DNA Analysis for Liver Cancers and Its Usefulness as a Liquid Biopsy.

BACKGROUND & AIMS: Circulating tumor DNA (ctDNA) carrying tumor-specific sequence alterations has been found in the cell-free fraction of blood. Liver cancer tumor specimens are difficult to obtain, and noninvasive methods are required to assess cancer progression and characterize underlying genomic features. METHODS: We analyzed 46 patients with hepatocellular carcinoma who underwent hepatectomy or liver transplantation and for whom whole-genome sequencing data was available. We designed personalized assays targeting somatic rearrangements of each tumor to quantify serum ctDNA. Exome sequencing was performed using cell-free DNA paired primary tumor tissue DNA from a patient with recurrent liver cancer after transcatheter arterial chemoembolization (TACE). RESULTS: We successfully detected ctDNA from 100 µL of serum samples in 7 of the 46 patients before surgery, increasing with disease progression. The cumulative incidence of recurrence and extrahepatic metastasis in the ctDNA-positive group were statistically significantly worse than in the ctDNA-negative group (P = .0102 and .0386, respectively). Multivariate analysis identified ctDNA (OR 6.10; 95% CI, 1.11-33.33, P = .038) as an independent predictor of microscopic vascular invasion of the portal vein (VP). We identified 45 nonsynonymous somatic mutations in cell-free DNA after TACE and 71 nonsynonymous somatic mutations in primary tumor tissue by exome sequencing. We identified 25 common mutations in both samples, and 83% of mutations identified in the primary tumor could be detected in the cell-free DNA. CONCLUSIONS: The presence of ctDNA reflects tumor progression, and detection of ctDNA can predict VP and recurrence, especially extrahepatic metastasis within 2 years. Our study demonstrated the usefulness of ctDNA detection and sequencing analysis of cell-free DNA for personalized treatment of liver cancer.

Integrated analysis of whole genome and transcriptome sequencing reveals diverse transcriptomic aberrations driven by somatic genomic changes in liver cancers.

Recent studies applying high-throughput sequencing technologies have identified several recurrently mutated genes and pathways in multiple cancer genomes. However, transcriptional consequences from these genomic alterations in cancer genome remain unclear. In this study, we performed integrated and comparative analyses of whole genomes and transcriptomes of 22 hepatitis B virus (HBV)-related hepatocellular carcinomas (HCCs) and their matched controls. Comparison of whole genome sequence (WGS) and RNA-Seq revealed much evidence that various types of genomic mutations triggered diverse transcriptional changes. Not only splice-site mutations, but also silent mutations in coding regions, deep intronic mutations and structural changes caused splicing aberrations. HBV integrations generated diverse patterns of virus-human fusion transcripts depending on affected gene, such as TERT, CDK15, FN1 and MLL4. Structural variations could drive over-expression of genes such as WNT ligands, with/without creating gene fusions. Furthermore, by taking account of genomic mutations causing transcriptional aberrations, we could improve the sensitivity of deleterious mutation detection in known cancer driver genes (TP53, AXIN1, ARID2, RPS6KA3), and identified recurrent disruptions in putative cancer driver genes such as HNF4A, CPS1, TSC1 and THRAP3 in HCCs. These findings indicate genomic alterations in cancer genome have diverse transcriptomic effects, and integrated analysis of WGS and RNA-Seq can facilitate the interpretation of a large number of genomic alterations detected in cancer genome.

A practical method to detect SNVs and indels from whole genome and exome sequencing data.

The recent development of massively parallel sequencing technology has allowed the creation of comprehensive catalogs of genetic variation. However, due to the relatively high sequencing error rate for short read sequence data, sophisticated analysis methods are required to obtain high-quality variant calls. Here, we developed a probabilistic multinomial method for the detection of single nucleotide variants (SNVs) as well as short insertions and deletions (indels) in whole genome sequencing (WGS) and whole exome sequencing (WES) data for single sample calling. Evaluation with DNA genotyping arrays revealed a concordance rate of 99.98% for WGS calls and 99.99% for WES calls. Sanger sequencing of the discordant calls determined the false positive and false negative rates for the WGS (0.0068% and 0.17%) and WES (0.0036% and 0.0084%) datasets. Furthermore, short indels were identified with high accuracy (WGS: 94.7%, WES: 97.3%). We believe our method can contribute to the greater understanding of human diseases.

The tumor-suppressive miR-497-195 cluster targets multiple cell-cycle regulators in hepatocellular carcinoma.

MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression and commonly deregulated in carcinogenesis. To explore functionally crucial tumor-suppressive (TS)-miRNAs in hepatocellular carcinoma (HCC), we performed integrative function- and expression-based screenings of TS-miRNAs in six HCC cell lines. The screenings identified seven miRNAs, which showed growth-suppressive activities through the overexpression of each miRNA and were endogenously downregulated in HCC cell lines. Further expression analyses using a large panel of HCC cell lines and primary tumors demonstrated four miRNAs, miR-101, -195, -378 and -497, as candidate TS-miRNAs frequently silenced in HCCs. Among them, two clustered miRNAs miR-195 and miR-497 showed significant growth-suppressive activity with induction of G1 arrest. Comprehensive exploration of their targets using Argonute2-immunoprecipitation-deep-sequencing (Ago2-IP-seq) and genome-wide expression profiling after their overexpression followed by pathway analysis, revealed a significant enrichment of cell cycle regulators. Among the candidates, we successfully identified CCNE1, CDC25A, CCND3, CDK4, and BTRC as direct targets for miR-497 and miR-195. Moreover, target genes frequently upregulated in HCC in a tumor-specific manner, such as CDK6, CCNE1, CDC25A and CDK4, showed an inverse correlation in the expression of miR-195 and miR-497, and their targets. These results suggest the molecular pathway regulating cell cycle progression to be integrally altered by downregulation of miR-195 and miR-497 expression, leading to the aberrant cell proliferation in hepatocarcinogenesis.

Potential of tumor-suppressive miR-596 targeting LGALS3BP as a therapeutic agent in oral cancer.

The incidence and mortality statistics for oral squamous cell carcinoma (OSCC) were 10th and 12th, respectively, in human cancers diagnosed worldwide in 2008. In this study, to identify novel tumor-suppressive microRNAs (TS-miRNAs) and their direct targets in OSCC, we performed methylation-based screening for 43 miRNAs encoded by 46 miRNA genes located within 500 bp downstream of 40 CpG islands and genome-wide gene expression profiling in combination with a prediction database analysis, respectively, in 18 cell lines, resulting in the identification of a novel TS-miRNA miR-596 directly targeting LGALS3BP/Mac-2 BP/90K. DNA hypermethylation of CpG island located 5'-upstream of miR-596 gene was frequently observed in OSCC cell lines (100% of 18 cell lines) and primary OSCC cases (46.2 and 76.3% of 26 Japanese and 38 Thais primary cases, respectively) in a tumor-specific manner. The ectopic transfection of double-stranded RNA (dsRNA) mimicking miR-596 or specific small interfering RNA for LGALS3BP significantly induced growth inhibition and apoptosis in cell lines lacking miR-596 expression or overexpressing LGALS3BP, respectively, in a manner associated with a suppression of ERK1/2 phosphorylation. Moreover, we also mention the effect of dsRNA mimicking miR-596 on the growth of an OSCC cell line in vivo. Our findings define a central role for miR-596 in OSCC and suggest the potential of miR-596 as an anticancer agent for miRNA replacement therapy in OSCC.

Integrative array-based approach identifies MZB1 as a frequently methylated putative tumor suppressor in hepatocellular carcinoma.

PURPOSE: The aim of this study was the identification of novel tumor suppressor genes (TSG) silenced by DNA hypermethylation in hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: We conducted integrative array-based approach for genome-wide screening of methylation targets using a methylated DNA immunoprecipitation-CpG island microarray and expression array in three universal hepatoma cell lines and normal liver tissue. Through detailed expression and functional analyses using hepatoma cell lines and primary HCC samples, we isolated novel TSGs for HCC. RESULTS: A total of 642 genes were identified as methylated in three hepatoma cell lines but unmethylated in normal liver tissue, whereas 204 genes on autosomes were identified as genes unexpressed but restored after treatment with 5-aza-2'-deoxycytidine in these cell lines and expressed in normal tissue. Through the integration of results of the two-array analyses and further validation analyses of expression and methylation status in 17 cell lines and 30 primary tumors of hepatoma, we identified MZB1, marginal zone B and B1 cell-specific protein, encoding an endoplasmic reticulum protein, as a putative TSG frequently methylated within its CpG island in hepatoma. Among 162 patients with primary HCC, silencing of MZB1 protein was significantly and independently associated with a worse outcome. Restoration of MZB1 expression in hepatoma cells reduced cell proliferation in vitro and in vivo through G(1)-arrest. CONCLUSIONS: These results suggest that methylation-mediated silencing of MZB1 expression leads to loss of its tumor-suppressive activity, which may be a factor in the hepatocarcinogenesis, and is a useful prognosticator in HCC.

miR-152 is a tumor suppressor microRNA that is silenced by DNA hypermethylation in endometrial cancer.

The etiology and development of human cancers that remain little understood might be enlightened by defining tumor suppressor microRNAs (TS-miRNA). In this study, we identified TS-miRNAs silenced by aberrant DNA hypermethylation in endometrial cancer. Functional screening of 327 synthetic miRNAs in an endometrial cancer cell proliferation assay identified 103 miRNAs that inhibited cell growth. We then determined the sequence, DNA methylation status, and expression levels of these miRNAs in endometrial cancer cell lines and primary tumors. These determinations led to the identification of miR-152 as a candidate TS-miRNA gene in endometrial cancer. Epigenetic silencing documented in miR-152 was consistent with its location at 17q21.32 in intron 1 of the COPZ2 gene, which is also silenced often in endometrial cancer by DNA hypermethylation, and also with evidence that miR-152 targets the DNA methyltransferase DNMT1. Notably, restoration of miR-152 expression in endometrial cancer cell lines was sufficient to inhibit tumor cell growth in vitro and in vivo. We identified E2F3, MET, and Rictor as novel candidate targets of miR-152, suggesting how its epigenetic silencing can drive endometrial carcinogenesis. Our findings define a central role for miR-152 in endometrial cancer, and they also suggest its use in new therapeutic strategies to treat this cancer.

The tumor suppressive microRNA miR-218 targets the mTOR component Rictor and inhibits AKT phosphorylation in oral cancer.

The incidence of oral squamous cell carcinoma (OSCC) is rising rapidly in developed countries, posing a growing challenge due to the poor management of this type of malignancy at present. In this study, we profiled tumor suppressive microRNAs (miRNAs) that are silenced by DNA hypermethylation in OSCC using a function-based screening approach. This approach employed a cell proliferation assay for 327 synthetic miRNAs in two OSCC cell lines. Among the 110 miRNAs identified in this set that exhibited inhibitory properties, we compared DNA methylation and expression status in a wider panel of OSCC cell lines and primary tumor tissues, resulting in the identification of miR-218 and miR-585 as functionally significant miRNA genes that are frequently silenced in OSCC by DNA hypermethylation. Ectopic expression of miR-218 and miR-585 in OSCC cells lacking endogenous expression reduced cell growth in part through caspase-mediated apoptosis. Notably, miR-218 reduced levels of the rapamycin-insensitive component of mTOR, Rictor, in a manner associated with a suppression of Akt S473 phosphorylation. Together our findings define miR-585 as a tumor suppressive function that is often epigenetically silenced in OSCC, and they identify Rictor as a novel target of miR-218, suggesting that activation of the mTOR-Akt signaling pathway induced by Rictor contributes centrally to oral carcinogenesis.

miR-124 and miR-203 are epigenetically silenced tumor-suppressive microRNAs in hepatocellular carcinoma.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that, in general, negatively regulate gene expression. They have been identified in various tumor types, showing that different sets of miRNAs are usually deregulated in different cancers. Some miRNA genes harboring CpG islands undergo methylation-mediated silencing, a characteristic of many tumor suppressor genes. To identify such miRNAs in hepatocellular carcinoma (HCC), we first examined the methylation status of 43 loci containing CpG islands around 39 mature miRNA genes in a panel of HCC cell lines and non-cancerous liver tissues as controls. Among 11 miRNA genes frequently methylated in HCC cell lines but not in non-cancerous liver tissues, three miRNA genes, i.e. miR-124, miR-203 and miR-375, were selected as silenced miRNAs through CpG-island methylation by comparing methylation and expression status and evaluating restored expression after treatment with 5-aza-2'-deoxycytidine. In primary tumors of HCC with paired non-tumorous liver tissues, only miR-124 and miR-203 showed frequent tumor-specific methylation, and their expression status was inversely correlated with methylation status. Ectopic expression of miR-124 or miR-203 in HCC cells lacking their expression inhibited cell growth, with direct downregulation of possible targets, cyclin-dependent kinase 6 (CDK6), vimentin (VIM), SET and MYND domain containing 3 (SMYD3) and IQ motif containing GTPase activating protein 1 (IQGAP1) or ATP-binding cassette, subfamily E, member 1 (ABCE1), respectively. Our results suggest that miR-124 and miR-203 are novel tumor-suppressive miRNAs for HCC epigenetically silenced and activating multiple targets during hepatocarcinogenesis.