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Cited by 3Epigenetics and DNA MethylationCancer-related molecular mechanisms researchGenomics and Chromatin Dynamics

Multi-omic analysis of hepatocellular carcinoma reveals aberrant cis-regulatory changes and dysregulated retrotransposons with prognostic potential

Open access
Cheng, Clooney C. Y., Cheung, Ming Fung, Lee, Ah Young, Wu, Qiong, Chow, Savio Ho-Chit, Ang, Julie Y. J., Riquelme Medina, Ignacio, Lo, Grace +6 more·2025·Communications Biology
DOI 10.1038/s42003-025-09154-wPMC PMC12717160
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Abstract

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer and has limited therapeutic options. Epigenetic dysregulation plays a crucial role in hepatocarcinogenesis; however, its impact on cis-regulatory elements (CREs) and retrotransposons remains underexplored. Here, we investigated the epigenetic changes underlying the aberrant CRE and retrotransposon activity in HCC. We show that focal DNA hypomethylation of these elements is associated with transcriptional reprogramming. Notably, we uncover a dual regulatory mechanism for GPC3, a key diagnostic biomarker and immunotherapeutic target in HCC. This requires the concomitant reactivation of a fetal liver super-enhancer (SE) and DNA hypomethylation of CpG islands. Furthermore, DNA methylation loss drives cryptic activation of retrotransposons, some of which exhibit prognostic potential. Intriguingly, we identify a HERVE-int-derived long non-coding RNA that shows higher expression in patients with more aggressive tumors, poorer disease outcomes, and is correlated with molecular signatures associated with improved response to immunotherapy. Collectively, our findings reveal widespread epigenomic dysregulation of CREs and retrotransposons in HCC, highlighting new potential therapeutic strategies and biomarkers.

AI snapshot

Written from the paper’s full text.

TL;DR

This multi-omic study integrates DNA methylomes, histone modification profiles, and transcriptomes from paired HBV-associated HCC and adjacent normal liver to map dysregulated cis-regulatory elements and retrotransposons. It identifies a dual SE/CGI hypomethylation mechanism for GPC3 and a HERVE-int-derived lncRNA associated with aggressive tumor features but immunotherapy-response signatures.

Key findings

  • HCC showed widespread DNA methylation loss, with ~78% of hypomethylated regions overlapping partially methylated domains; hypermethylated regions were enriched at promoters, UTRs, and exons.
  • CRE reprogramming included 3,637 activated and 2,924 inactivated typical enhancers, plus 234 activated and 86 inactivated super-enhancers; GPC3 overexpression required both fetal liver SE reactivation and DNA hypomethylation of promoter and intragenic CGIs, with CRISPRi of constituent enhancers reducing GPC3 by at least 40% and targeted CGI methylation reducing it by 45–55%.
  • Retrotransposon dysregulation included 3,167 RT-typical enhancers and 1,361 RT-promoters; 24 de-repressed retrotransposons overlapped activated RT-promoters, 20 were LTR elements, and 11 overlapped annotated lncRNA promoters.
  • HERVE-int-High patients (top 25%, n = 93) had poorer progression-free survival, higher aneuploidy score, higher AFP, higher TMB, higher CD274/PD-L1 and CTLA-4, and upregulation of 6 of 9 atezolizumab+bevacizumab response signature genes; THE1D intermediate/high expression was also associated with worse outcomes.

Methods

  • Seven paired HBV-associated HCC and tumor-adjacent normal tissues underwent WGBS, ChIP-seq for H3K27ac, H3K4me3, H3K9me3, and H3K27me3, and stranded total RNA-seq; missing ChIP-seq datasets were imputed with Avocado, and four public TAN methylomes plus TCGA-LIHC data were included.
  • Bioinformatics included methylKit DMR calling, methpipe PMD calling, DESeq2 differential analysis, ROSE super-enhancer calling, HOMER motif analysis, GREAT/Metascape GO, WGCNA, TCGA survival/Cox/TMB analyses, and Hi-C interaction analysis.
  • Functional validation used luciferase assays, shRNA knockdown, CRISPRi, CRISPR-SunTag-DNMT3A1 targeted methylation, CRISPR-SunTag-TET1CD targeted demethylation, and RT-qPCR in HCC cell lines.

Limitations

  • The paper states that no statistical methods were used to predetermine sample size, investigators were not blinded, experiments were not randomized, and luciferase assays were repeated twice.
  • The paper notes that due to sample availability some experiments were omitted and missing ChIP-seq data were imputed; it also states that further studies are needed to evaluate the safety and efficacy of combined DNA-demethylating/immunotherapy strategies and that more preclinical studies are needed for HERVE-int as an immunotherapy predictor.
  • Evident from the text: the discovery cohort is small (seven paired HBV-associated HCC/TAN samples), and several key clinical associations rely on TCGA-LIHC or other public datasets, which may limit generalizability.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s42003-025-09154-w

Cites

134 of 134 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

47 of 64 cited sources checked · 17 supported · 0 flagged · accuracy not yet measured

  • Findingown data

    25% of upregulated genes (n=120) in HCC are proliferation class signature genes.

    “We found that 25% of the upregulated genes (n = 120) were defined as proliferation class signature genes (Supplementary Fig. 3d)53,54”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [53] Hoshida, Y. et al. Integrative transcriptome analysis reveals common molecular subclasses of human hepatocellular carcinoma. Cancer Res. 69, 7385–7392 (2009). source
    • cites [54] Chiang, D. Y. et al. Focal gains of VEGFA and molecular classification of hepatocellular carcinoma. Cancer Res. 68, 6779–6788 (2008). source
  • Findingown data

    Activated typical enhancers in HCC are associated with developmental GO terms and enriched for binding motifs of development-related TFs such as CEBPα/β, NK2-1, and ZIC3.

    “The activated typical enhancers were associated with developmental gene ontology (GO) terms and were enriched for binding motifs of development-related TFs such as CEBPα/β, NK2-1, and ZIC3 (Fig. 2a, b)55–57.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [55] Liu, Y. et al. TET2 is recruited by CREB to promote Cebpb, Cebpa, and Pparg transcription by facilitating hydroxymethylation during adipocyte differentiation. iScience 26, 108312 (2023). source
    • cites [56] Schmidt, L., Heyes, E. & Grebien, F. Gain-of-function effects of N-terminal CEBPA mutations in acute myeloid leukemia. Bioessays 42, e1900178 (2020). source

      The Introduction (page unavailable) identifies C/EBPα as a transcription factor necessary for the development of the liver and other tissues, supporting its description as development-related. The paper discusses C/EBPα binding motifs in leukemia, but does not report activated typical enhancers, developmental GO terms, or motif enrichment in HCC; it also does not substantiate the other named factors. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [57] Varma, S. et al. The transcription factors Grainyhead-like 2 and NK2-homeobox 1 form a regulatory loop that coordinates lung epithelial cell morphogenesis and differentiation. J. Biol. Chem. 287, 37282–37295 (2012). source
  • Findingcitation

    GPC3 is highly expressed in HCC relative to other cancer types.

    “Importantly, GPC3 is highly expressed in HCC relative to other cancer types (Fig. 3a)40,61.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [40] Yamauchi, N. et al. The glypican 3 oncofetal protein is a promising diagnostic marker for hepatocellular carcinoma. Mod. Pathol. 18, 1591–1598 (2005). source

      The Hepatocyte lineage section reports diffuse GPC3 staining in 47 of 56 HCC cases (84%), while Other cell lineages reports little or no staining in the colorectal metastases, cholangiocellular carcinomas, and carcinoid tumor examined. The comparison does not establish higher expression than other cancer types generally: the paper also reports diffuse GPC3 staining in hepatoblastoma. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [61] Hsu, H. C., Cheng, W. & Lai, P. L. Cloning and expression of a developmentally regulated transcript MXR7 in hepatocellular carcinoma: Biological significance and temporospatial distribution. Cancer Res. 57, 5179–5184 (1997).

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Findingown data

    SEs in both HCC and TAN are associated with GO terms related to metabolic genes (e.g. CYP3A4, SLC38A4, IGFBP1).

    “SEs in both HCC and TAN were associated with GO terms related to metabolic genes (e.g. CYP3A4, SLC38A4, and IGFBP1) (Fig. 2c)20,21,58.”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [20] Hnisz, D. et al. Super-enhancers in the control of cell identity and disease. Cell 155, 934–947 (2013). source

      In Results, ‘Super-enhancers in many cell types,’ the paper reports that Gene Ontology analysis links super-enhancer-associated genes to cell- and tissue-defining biological processes. It does not report this GO finding for both HCC and tumor-adjacent normal tissue (TAN), or identify metabolic terms or the three named genes in that comparison. The copy provides section labels but no page numbers. (LLM, conf=0.79)

      Experimental — accuracy not yet measured
    • cites [21] Whyte, W. A. et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 153, 307–319 (2013). source

      The Results section, “Super-enhancers are cell type-specific and mark key cell identity genes” (page unavailable), reports GO analysis across several cell types, but not HCC or TAN. Searches for HCC, TAN, the three named genes, and liver metabolism found no passage supporting the specific association claimed. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
    • cites [58] Loven, J. et al. Selective inhibition of tumor oncogenes by disruption of super-enhancers. Cell 153, 320–334 (2013). source

      The Results section on other cancers examines super-enhancers in glioblastoma and small-cell lung cancer, not HCC or tumor-adjacent normal tissue. Searches for HCC, TAN, the three named genes, and metabolic GO terms found no passage addressing the claimed association. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
  • Findingown data

    GPC3 overexpression in HCC is accompanied by significant 3D interactions among constituent enhancers and between constituent enhancers and the promoter.

    “GPC3 overexpression was accompanied by significant 3D interactions amongst constituent enhancers as well as between constituent enhancers and the promoter (Fig. 3b)66,67.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [66] Zhao, J. & Faryabi, R. B. Spatial promoter-enhancer hubs in cancer: Organization, regulation, and function. Trends Cancer 9, 1069–1084 (2023). source

      The Abstract (page unavailable) describes promoter–enhancer hubs and their association with elevated gene expression generally, but does not mention GPC3 or HCC. Searches for GPC3 and for hepatocellular carcinoma with enhancer–promoter interactions found no passage addressing the specific claim. (LLM, conf=0.72)

      Experimental — accuracy not yet measured
    • cites [67] Uyehara, C. M. & Apostolou, E. 3D enhancer-promoter interactions and multi-connected hubs: Organizational principles and functional roles. Cell Rep. 42, 112068 (2023). source

      The Introduction section on highly multi-connected 3D hubs discusses enhancer–promoter interactions generally and gives examples at other loci, including PVT1. Searches for GPC3, HCC, and hepatocellular carcinoma found no passage in our copy addressing GPC3 overexpression or the claimed interactions among its enhancers and promoter. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
  • May combine two claims. 31
  • Classified as probably not a substantial claim. 13
  • The quote only partly states this claim. 2

  • May combine two claims.

    Findingown data

    PMDs in HCC are enriched with repressive histone modifications and associated with transcriptional repression.

    “These domains were enriched with repressive histone modifications and associated with transcriptional repression (Supplementary Fig. 2c, d)44–46.”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [44] Salhab, A. et al. A comprehensive analysis of 195 DNA methylomes reveals shared and cell-specific features of partially methylated domains. Genome Biol. 19, 150 (2018). source
    • cites [45] Hon, G. C. et al. Global DNA hypomethylation coupled to repressive chromatin domain formation and gene silencing in breast cancer. Genome Res. 22, 246–258 (2012). source

      The sections “Allelic basis of repressive chromatin and DNA methylation” and “Formation of repressive chromatin domains at hypomethylated genomic regions” link PMDs or hypomethylated genes to repressive histone marks and reduced expression. But the study examines HCC1954 breast cancer cells, not hepatocellular carcinoma (HCC), so it does not establish the claim for HCC. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [46] Berman, B. P. et al. Regions of focal DNA hypermethylation and long-range hypomethylation in colorectal cancer coincide with nuclear lamina-associated domains. Nat. Genet 44, 40–46 (2011). source

      The Main section (Fig. 5d; page unavailable) reports enrichment of the repressive H3K9me3 mark within IMR-90 fibroblast PMDs. It also reports reduced expression of two genes within colorectal-tumor PMDs (Fig. 5a–b). These findings support parts of the general association, but the paper does not establish the stated histone and transcriptional findings for PMDs in HCC. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    The genes with promoter DNA hypomethylation include AFP, several HCC-related lncRNAs (LINC01419, LINC02163, LINC02835), and tumor-associated antigens such as the MAGE gene family.

    “These genes included AFP, a diagnostic marker for HCC, several HCC-related lncRNAs (LINC01419, LINC02163, and LINC02835), and tumor-associated antigens such as the MAGE gene family35–37.”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [35] Unfried, J. P. et al. Long noncoding RNA NIHCOLE promotes ligation efficiency of DNA double-strand breaks in hepatocellular carcinoma. Cancer Res. 81, 4910–4925 (2021). source

      The Results section identifies LINC02163 as NIHCOLE, an lncRNA highly expressed in HCC. It does not report promoter DNA hypomethylation for LINC02163 or the listed genes, and the passages do not establish the other named genes as members of the claimed group. No page number is available in our copy. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [36] Wu, Y. et al. An LTR retrotransposon-derived long noncoding RNA lncMER52A promotes hepatocellular carcinoma progression by binding p120-catenin. Cancer Res. 80, 976–987 (2020). source
    • cites [37] Deng, B. et al. An LTR retrotransposon-derived lncRNA interacts with RNF169 to promote homologous recombination. EMBO Rep. 20, e47650 (2019). source

      The section “PRLH1 is an ERV9 LTR lncRNA regulated by p53” (page unavailable) identifies LINC01419 as a lncRNA upregulated in HCC. The copy does not establish promoter DNA hypomethylation for it or identify AFP, LINC02163, LINC02835, or the MAGE family as genes with hypomethylated promoters. A discussion of possible CpG methylation at the PRLH1 promoter does not establish the claimed hypomethylation. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    Inactivated typical enhancers in HCC are associated with collagen fibril organization and chemotaxis GO terms and enriched for binding motifs of immune-related TFs, including STAT1.

    “inactivated typical enhancers were associated with collagen fibril organization and chemotaxis GO terms and were enriched for binding motifs of immune-related TFs, including STAT1 (Fig. 2a, b)12,13.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [12] Chiappinelli, K. B. et al. Inhibiting DNA Methylation Causes an Interferon Response in Cancer via dsRNA Including Endogenous Retroviruses. Cell 162, 974–986 (2015). source

      The Results section on DNMT inhibitors and type I interferon signaling reports STAT1 upregulation in ovarian cancer cell lines, but does not analyze inactivated enhancers in HCC. Searches for collagen fibril organization, chemotaxis, and enhancer-binding motifs found no passage addressing the claimed associations. (LLM, conf=0.75)

      Experimental — accuracy not yet measured
    • cites [13] Roulois, D. et al. DNA-demethylating agents target colorectal cancer cells by inducing viral mimicry by endogenous transcripts. Cell 162, 961–973 (2015). source

      The Results section discusses interferon-responsive signaling and STAT1 in colorectal cancer cell experiments, not inactivated enhancers in HCC. Searches for collagen fibril organization, chemotaxis, and enhancer-binding motifs found no passage addressing those claims in our copy; page numbers were not provided. (LLM, conf=0.75)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Knockdown of SETDB1 in HCC cells led to the transcriptional derepression of two HERVE-int elements through loss of H3K9me3.

    “a recent study demonstrated that knockdown of SETDB1, the H3K9 methyltransferase, in HCC cells led to the transcriptional derepression of two HERVE-int elements through loss of H3K9me3”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [63] Igarashi, Y. et al. Identification and clinical implications of endogenous retrovirus elements suppressed by SETDB1 in hepatocellular carcinoma. JHEP Rep. 7, 101307 (2025). source

      The Results section (page not provided) reports that SETDB1 knockdown increased expression of four HERV elements in HCC cells and reduced H3K9me3 at their regions. It does not identify two HERVE-int elements: the four tested elements include one HERVE-int element, on chromosome 5. (LLM, conf=0.90)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingcitation

    LTR2B, LTR8B, and LTR10A are among over-represented LTR subfamilies in activated RT-typical enhancers and regulate genes critical in placental development.

    “These included LTR2B, LTR8B, and LTR10A, which regulate genes critical in placental development (Supplementary Fig. 8g and Supplementary Data 7)78,79.”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [78] Gao, L. et al. Single-cell analysis reveals transcriptomic and epigenomic impacts on the maternal-fetal interface following SARS-CoV-2 infection. Nat. Cell Biol. 25, 1047–1060 (2023). source

      The Results section, “LTR8B elements function as enhancers in placental cells” (page unavailable), reports that a subset of LTR8B elements act as enhancers regulating placental genes. The passages do not establish that LTR2B or LTR10A regulate such genes, or that all three subfamilies are over-represented in activated RT-typical enhancers. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [79] Frost, J. M. et al. Regulation of human trophoblast gene expression by endogenous retroviruses. Nat. Struct. Mol. Biol. 30, 527–538 (2023). source

  • May combine two claims.

    Findingcitation

    LTR12C is among over-represented LTR subfamilies in activated RT-promoters and is pervasively activated in HBV-associated HCC.

    “These include LTR12C, which is pervasively activated in HBV-associated HCC (Supplementary Fig. 8h and Supplementary Data 7)34.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [34] Hashimoto, K. et al. CAGE profiling of ncRNAs in hepatocellular carcinoma reveals widespread activation of retroviral LTR promoters in virus-induced tumors. Genome Res. 25, 1812–1824 (2015). source

      The Results section on aberrant LTR activation reports that LTR12C was preferentially activated in HCC, with 101 elements significantly up-regulated. The stratification Results section links high LTR expression overall to viral etiology, with the highest expression mostly in HBV-positive tumors. It does not establish that LTR12C specifically is pervasively activated in HBV-associated HCC. Page numbers were not supplied in our copy. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    The role of epigenetic dysregulation in driving the aberrant activity of CREs and retrotransposons in HCC remains incompletely understood.

    “the role of epigenetic dysregulation in driving the aberrant activity of CREs and retrotransposons in HCC remains incompletely understood50–52,93.”
    Quote in the paperSubstantial claim3 of 4 sources checked
    • cites [50] Cancer Genome Atlas Research Network. Electronic address, w. b. e. & Cancer Genome Atlas Research, N. Comprehensive and Integrative Genomic Characterization of Hepatocellular Carcinoma. Cell 169, 1327-1341 e1323 (2017).

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [51] Song, M. A. et al. Elucidating the landscape of aberrant DNA methylation in hepatocellular carcinoma. PLoS One 8, e55761 (2013). source

      The Introduction says HCC methylation studies had been limited in scope, including studies of repetitive elements such as LINE1, and calls for further analysis. This supports the broader point that epigenetic changes in HCC remain incompletely understood, but the paper does not establish a role for epigenetic dysregulation in driving aberrant CRE or retrotransposon activity. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
    • cites [52] Shen, J. et al. Genome-wide DNA methylation profiles in hepatocellular carcinoma. Hepatology 55, 1799–1808 (2012). source

      The Introduction links genetic and epigenetic aberrations to HCC, and the Results describe altered DNA methylation in HCC tissue. The paper does not address whether epigenetic dysregulation drives aberrant activity of cis-regulatory elements or retrotransposons, or whether that role remains incompletely understood (searched for CREs, enhancers, and retrotransposons). (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [93] Hlady, R. A. et al. Integrating the epigenome to identify drivers of hepatocellular carcinoma. Hepatology 69, 639–652 (2019). source

      The Abstract says the HCC epigenome remains understudied and reports epigenetic remodeling of enhancers, supporting the claim about incomplete understanding of regulatory-element activity. The passages do not address retrotransposon activity, so that part of the claim is not supported by this source. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    DAC treatment could induce expression of retrotransposons normally silenced by DNA methylation, which could augment tumor immunogenicity.

    “DAC treatment could induce expression of retrotransposons normally silenced by DNA methylation, which could also augment tumor immunogenicity10–14,99.”
    Quote in the paperNot a substantial claim6 of 6 sources checked
    • cites [10] Goyal, A. et al. DNMT and HDAC inhibition induces immunogenic neoantigens from human endogenous retroviral element-derived transcripts. Nat. Commun. 14, 6731 (2023). source
    • cites [11] Brocks, D. et al. DNMT and HDAC inhibitors induce cryptic transcription start sites encoded in long terminal repeats. Nat. Genet 49, 1052–1060 (2017). source
    • cites [12] Chiappinelli, K. B. et al. Inhibiting DNA Methylation Causes an Interferon Response in Cancer via dsRNA Including Endogenous Retroviruses. Cell 162, 974–986 (2015). source
    • cites [13] Roulois, D. et al. DNA-demethylating agents target colorectal cancer cells by inducing viral mimicry by endogenous transcripts. Cell 162, 961–973 (2015). source
    • cites [14] Kong, Y. et al. Transposable element expression in tumors is associated with immune infiltration and increased antigenicity. Nat. Commun. 10, 5228 (2019). source
    • cites [99] Jiang, Q. et al. HIF regulates multiple translated endogenous retroviruses: Implications for cancer immunotherapy. Cell 188, 1807–1827 e1834 (2025). source

      The Results (Pharmacological Induction of HIF-responsive ERVs; page unavailable) show that the DNMT1 inhibitor GSK3685032 induced ERV4700 RNA in renal cancer cells. The Introduction says DNA methylation usually represses ERV transcription, and the Discussion links ERV expression to immune activation. The paper does not establish that DAC specifically produces these effects; it tested a different inhibitor. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingcitation

    Liver-restricted TFs (C/EBPα/β, HNF1A, HNF4A), pioneering TFs (CEBPα/β, FOXA2, FOXA3), and Wnt-signaling TFs (TCF7) bind at individual GPC3 constituent enhancers.

    “we detected binding of liver-restricted TFs (C/EBPα/β, HNF1A, and HNF4A), pioneering TFs (CEBPα/β, FOXA2, and FOXA3), and Wnt-signaling TFs (TCF7) at individual constituent enhancers (Fig. 3b)20,21.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [20] Hnisz, D. et al. Super-enhancers in the control of cell identity and disease. Cell 155, 934–947 (2013). source

      The Results section ('Transcription factors in ESCs'; page unavailable) reports transcription-factor enrichment at ESC super-enhancers, including the Wnt-pathway factor Tcf3, but not binding at GPC3 enhancers. Searches for GPC3 and the named liver, pioneer, and Wnt-signaling factors found no passage addressing the specific binding claim. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
    • cites [21] Whyte, W. A. et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 153, 307–319 (2013). source

      The Introduction and Results discuss transcription-factor binding at enhancers generally, including constituent enhancers in embryonic stem and pro-B cells (page unavailable). Searches for GPC3 and the named liver, pioneering, and Wnt-signaling factors found no passage reporting their binding at GPC3 constituent enhancers. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    Increased expression of ERVs, including a full-length HERV4700 element, was significantly associated with improved response to PD-1/PD-L1 blockade in clear cell renal cell carcinoma.

    “increased expression of ERVs, including a full-length HERV4700 element, was significantly associated with improved response to PD-1/PD-L1 blockade in clear cell renal cell carcinoma32,33.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [32] Smith, C. C. et al. Endogenous retroviral signatures predict immunotherapy response in clear cell renal cell carcinoma. J. Clin. Invest 128, 4804–4820 (2018). source

      The paper title (unpaginated in our copy) says endogenous retroviral signatures predict immunotherapy response in clear cell renal cell carcinoma. The available passages do not establish that increased expression predicts improved response, identify a full-length HERV4700 element, or specify a significant association with PD-1/PD-L1 blockade. Searches for HERV4700 and treatment-response terms returned no Results passages. (LLM, conf=0.68)

      Experimental — accuracy not yet measured
    • cites [33] Panda, A. et al. Endogenous retrovirus expression is associated with response to immune checkpoint blockade in clear cell renal cell carcinoma. JCI Insight 3. https://doi.org/10.1172/jci.insight.121522 (2018). source

      The Introduction and Figure 3 caption (page not available in our copy) report that higher ERV3-2 expression was significantly associated with response to PD-1/PD-L1 blockade in clear cell renal cell carcinoma. The cited paper does not identify ERV3-2 as a full-length HERV4700 element or report a response association for HERV4700; a search for HERV4700 found no passage addressing it. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    Inactivated typical enhancers were broadly shared between most HCC samples, whereas substantial numbers of activated typical enhancers were restricted to specific patients.

    “while inactivated typical enhancers were broadly shared between most HCC samples, substantial numbers of the activated typical enhancers were restricted to specific patients.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    RNA-seq revealed upregulation of cell cycle regulators and downregulation of immune- and extracellular matrix-related genes in HCC.

    “RNA-seq revealed upregulation of cell cycle regulators and downregulation of immune- and extracellular matrix-related genes (Fig. 1c and Supplementary Fig. 3a,b,c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    234 SEs were activated and 86 SEs were inactivated in HCC.

    “We identified activated and inactivated SEs in HCC (n = 234 and n = 86, respectively) (Fig. 2d, Supplementary Fig. 4c, and Supplementary Data 6).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    GPC3 is broadly silenced in normal adult livers but becomes overexpressed in more than 70% of HCC.

    “While GPC3 is broadly silenced in normal adult livers, it becomes overexpressed in more than 70% of HCC, which we validated in both our patient samples and the TCGA-LIHC cohort (Supplementary Fig. 5a)61.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [61] Hsu, H. C., Cheng, W. & Lai, P. L. Cloning and expression of a developmentally regulated transcript MXR7 in hepatocellular carcinoma: Biological significance and temporospatial distribution. Cancer Res. 57, 5179–5184 (1997).

  • May combine two claims.

    Findingown data

    Chromatin accessibility of the GPC3 SE positively correlates with that of the GPC3 promoter and with GPC3 gene expression.

    “The chromatin accessibility of the SE positively correlated with that of the GPC3 promoter and its gene expression (Supplementary Fig. 5b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Activated SEs in HCC exhibit heterogeneity across HCC samples and are associated with metabolism, early development, and extracellular matrix formation GO terms.

    “The activated SEs also exhibited heterogeneity across HCC samples and were associated with metabolism, early development, and extracellular matrix formation GO terms (Fig. 2d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    There was no significant change in H3K27me3 at dysregulated typical enhancers, but loss of H3K9me3 was observed at activated typical enhancers.

    “While there was no significant change in H3K27me3, we observed loss of H3K9me3 at the activated typical enhancers.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    3637 typical enhancers gained activity and 2924 lost activity in HCC.

    “We defined 3637 and 2924 typical enhancers that gained and lost activity, respectively (Fig. 2a, Supplementary Fig. 4a, and Supplementary Data 6).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The GPC3 SE is differentially reactivated in HCC but not in other analyzed cancer types.

    “Importantly, this SE was differentially reactivated in HCC but not in other analyzed cancer types (Supplementary Fig. 5c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A HERVE-int-derived lncRNA shows higher expression in patients with more aggressive tumors and poorer disease outcomes.

    “we identify a HERVE-int-derived long non-coding RNA that shows higher expression in patients with more aggressive tumors, poorer disease outcomes”
    • Also stated in: § DNA hypomethylation leads to cryptic activation of retrotransposon-driven transcripts associated with poorer disease outcomes · plain
      “Patients with high HERVE-int expression (HERVE-int-High, top 25%, n = 93) were associated with poorer progression-free survival (Fig. 5c).”
    • Also stated in: § DNA hypomethylation leads to cryptic activation of retrotransposon-driven transcripts associated with poorer disease outcomes · plain
      “The expression of HERVE-int also increased across tumor grades (Fig. 5d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The study demonstrated widespread epigenetic changes at CREs and retrotransposons, coupled with transcriptional reprogramming in HCC.

    “We demonstrated the widespread epigenetic changes at CREs and retrotransposons, which are coupled with transcriptional reprogramming in HCC.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    HERVE-int exhibited a highly restricted expression pattern in HCC but not in other cancer types, and is more highly expressed in HCC compared to other cancer types.

    “HERVE-int exhibited a highly restricted expression pattern in HCC but not in other cancer types (Fig. 6f).”
    • Also stated in: § Discussion · plain
      “HERVE-int is more highly expressed in HCC compared to other cancer types”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    HERVE-int-High patients had higher AFP expression than HERVE-int-Low patients.

    “HERVE-int-High, but not THE1D-Intermediate or -High patients, had higher aneuploidy score and AFP expression. (Fig. 6c, d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    THE1D-Intermediate or -High patients did not have higher aneuploidy score or AFP expression.

    “HERVE-int-High, but not THE1D-Intermediate or -High patients, had higher aneuploidy score and AFP expression. (Fig. 6c, d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    LINEs are over-represented in dysregulated RT-typical enhancers, while LTRs are enriched in both dysregulated RT-typical enhancers and RT-promoters.

    “whereas Long Interspersed Nuclear Elements (LINEs) were over-represented in dysregulated RT-typical enhancers, LTRs were enriched in both dysregulated RT-typical enhancers and RT-promoters.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    24 de-repressed retrotransposons overlap with activated RT-promoters, of which 20 are LTR elements.

    “identifying 24 candidates, of which, 20 were LTR elements (Fig. 5a).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    HERVE-int-High patients had higher aneuploidy score than HERVE-int-Low patients.

    “HERVE-int-High, but not THE1D-Intermediate or -High patients, had higher aneuploidy score and AFP expression. (Fig. 6c, d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both HERVE-int-High and THE1D-High patients were enriched in the iClust3 subclass.

    “We found that both HERVE-int-High and THE1D-High patients were enriched in the iClust3 subclass (Supplementary Fig. 10i).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [50] Cancer Genome Atlas Research Network. Electronic address, w. b. e. & Cancer Genome Atlas Research, N. Comprehensive and Integrative Genomic Characterization of Hepatocellular Carcinoma. Cell 169, 1327-1341 e1323 (2017).

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Findingown data

    The HERVE-int element functions as both an enhancer and a promoter.

    “Luciferase assays validated that the HERVE-int element functioned as both an enhancer and a promoter”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    3,167 dysregulated retrotransposon-derived typical enhancers (RT-typical enhancers) and 1,361 RT-promoters were identified in HCC.

    “We identified 3,167 and 1,361 dysregulated retrotransposon-derived typical enhancers (RT-typical enhancers) promoters (RT-promoters), respectively (Supplementary Fig. 8a, b and Supplementary Data 7).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    WGCNA of the TCGA-LIHC cohort generated 84 modules.

    “Briefly, the network was constructed using the blockwiseModules function using the parameters -power = 14, mergeCutHeight = 0.1 and deepSplit = 4, generating 84 modules.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    HERVE-int is a potential prognostic biomarker and predictor of patient response to immunotherapy for HCC.

    “Collectively, our findings highlight HERVE-int as a potential prognostic biomarker and predictor of patient response to immunotherapy for HCC.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Both the aberrant reactivation of a fetal liver SE and DNA methylation levels of the two GPC3 CGIs are required for GPC3 overexpression in HCC.

    “Both events are required for GPC3 overexpression in HCC.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    HERVE-int-High patients may be more likely to benefit from ICIs, VEGF inhibitors, and/or their combined therapeutic regimens.

    “these data suggest that HERVE-int-High patients may be more likely to benefit from ICIs, VEGF inhibitors, and/or their combined therapeutic regimens”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Due to sample availability, some experiments were omitted.

    “Due to sample availability, some experiments were omitted.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data

    For ARID3A and FOXA3, knockdown efficiencies varied substantially between replicates; therefore, C/EBPα, MYBL2, and SOX13 were focused on because they showed significant and reproducible knockdown efficiencies.

    “For ARID3A and FOXA3, knockdown efficiencies varied substantially between replicates; therefore, we focused on C/EBPα, MYBL2, and SOX13, which showed significant and reproducible knockdown efficiencies (Supplementary Fig. 6d).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    No statistical methods were used to predetermine sample size.

    “No statistical methods were used to predetermine sample size.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Patients with high expression of a HERVE-int element exhibited molecular signatures of more favorable responses to immunotherapy, including higher TMB and expression of key immune checkpoint molecules and VEGFA/B.

    “we discovered that patients with high expression of a HERVE-int element exhibited molecular signatures of more favorable responses to immunotherapy, including higher TMB and expression of key immune checkpoint molecules and VEGFA/B.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Luciferase assays were repeated twice with similar results.

    “Luciferase assays were repeated twice with similar results obtained.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    All ethical regulations relevant to human research participants were followed, informed consent was obtained from all patients, and the study protocol was approved by the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee.

    “All ethical regulations relevant to human research participants were followed. Informed consent was obtained from all patients and the study protocol was approved by the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The investigators were not blinded to allocation during experiments and outcome assessment.

    “The investigators were not blinded to allocation during experiments and outcome assessment.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationreasoning

    Integrating specific retrotransposons, such as HERVE-int, into existing predictive biomarker panels might improve identification of patients most likely to benefit from combined therapy.

    “Integrating specific retrotransposons, such as HERVE-int, into the existing predictive biomarker panels might improve identification of patients most likely to benefit from combined therapy.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The experiments were not randomized.

    “The experiments were not randomized.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    To mitigate amplification bias, four WGBS libraries were prepared per sample.

    “To mitigate amplification bias, four WGBS libraries were prepared per sample.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Cell lines were routinely tested and found to be free of mycoplasma contamination.

    “Cell lines were routinely tested and found to be free of mycoplasma contamination by the MycoStripTM Test Kit (InvivoGen).”
    Quote in the paperNot a substantial claim
45 of 45

  • The paper relies on this without support.

    The seven HBV-associated HCC and matched tumor-adjacent normal tissue samples are representative of HCC tumors and normal liver tissues generally.

    “We performed whole genome bisulfite sequencing (WGBS), chromatin immunoprecipitation followed by sequencing (ChIP-seq), and stranded total RNA-sequencing on seven pairs of HBV-associated HCC and matched tumor-adjacent normal (TAN) tissues (Fig. 1a and Supplementary Data 1).”

  • The paper relies on this without support.

    The four publicly available DNA methylomes of TAN tissues are comparable to the study's TAN samples.

    “To bolster our analyses, we included four publicly available DNA methylomes of TAN tissues42.”

  • The paper relies on this without support.

    The TCGA-LIHC cohort is representative of the study's HCC population for validating molecular and clinical findings.

    “we stratified patients in the TCGA-LIHC cohort based on the expression of each of the 24 candidates.”

  • The paper relies on this without support.

    Molecular signatures associated with improved response to immunotherapy indicate actual improved response to immunotherapy.

    “is correlated with molecular signatures associated with improved response to immunotherapy.”

  • The paper relies on this without support.

    GPC3 has two CpG islands: one proximal to the promoter and one within its first intron.

    “GPC3 has two CpG islands (CGIs): one proximal to the promoter (promoter CGI) and the other within its first intron (intragenic CGI).”
5 of 5

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Conflicting counts of activated typical enhancers

    Minor

    The Results report 3,637 activated typical enhancers, whereas the Methods report 3,367 using the stated differential-analysis criteria.

  • TF shortlist conflicts with the stated overlap

    Minor

    The text says 137 of the 271 enhancer-enriched TFs were positively correlated with GPC3. If the 137 are already members of the 271, overlapping those lists cannot yield only five TFs.

2 of 2

Cites

Flagged first, then by citations

Research works printed but not linked: 1

  1. Bastian, M., Heymann, S. & Jacomy, M. in Proceedings of the international AAAI conference on web and social media. 361-362.

Cited by

Still being checked.

Authors
Cheng, C. C. Y., Cheung, M. F., Lee, A. Y., Wu, Q., Chow, S. H.-C., Ang, J. Y. J., Riquelme Medina, I., Lo, G., Wu, H., Yang, W., Lai, P. B. S., Yip, K. Y., Cheng, A. S., & Leung, D.
Affiliations
  • Hong Kong University of Science and Technology (HK)
  • Chinese University of Hong Kong (HK)
  • Sanford Burnham Prebys Medical Discovery Institute (US)
Corresponding author
  • Yip, Kevin Y.
  • Cheng, Alfred S.
  • Leung, Danny
ORCID
Venue
Communications Biology
Publisher
Springer Science and Business Media LLC
Published
2025-12-13
Volume
8
Issue
1
Pages
1792-1792
ISSN
2399-3642 (ISSN-L)
DOI
10.1038/s42003-025-09154-w
PMC
PMC12717160
OpenAlex
W4417296500
Semantic Scholar
3ed2d6556ed99cbdaa0f49af5ea87b73a7cb6583
Cited by
3
References
134
Citations by year
2026: 3
FWCI
0.89
Type
journal article
Language
en
Open access
Licence
Locations
  • Communications Biology (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • Rare & Special e-Zone (The Hong Kong University of Science and Technology) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Croucher Foundation (CIA16SC02)
  • Research Grants Council, University Grants Committee (16103721)
  • The Hong Kong Epigenome Project funded by The Lo Ka Chung Charitable Foundation
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer-related molecular mechanisms research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
Keywords
hepatocellular carcinoma, cis-regulatory elements, retrotransposons, DNA hypomethylation, super-enhancers, CpG islands, glypican-3, long non-coding RNA, immunotherapy response, prognostic factors, epigenetic dysregulation, transcriptional reprogramming
MeSH terms
Multiomics, Carcinoma, Hepatocellular, Humans, Liver Neoplasms, Prognosis, Regulatory Sequences, Nucleic Acid, Biomarkers, Tumor, Gene Expression Regulation, Neoplastic, Retroelements, CpG Islands, DNA Methylation, Epigenesis, Genetic, Cell Line, Tumor, Glypicans, RNA, Long Noncoding

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-27.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-27.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

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