DNA hydroxymethylation is one of the major epigenetic mechanisms mediating the development of several human cancers. This study aimed to identify key hydroxymethylated genes and transcription factors (TFs) associated with alpha-fetoprotein (AFP)-negative hepatocellular carcinoma (HCC) using whole-genome DNA hydroxymethylation profiling. A total of 615 differentially hydroxymethylated regions (DHMRs) were identified from AFP-negative HCC tissues compared to adjacent normal tissues. DHMR-associated genes were significantly enriched in gene ontology functions associated with actin binding, cell leading edge, and blood vessel morphogenesis and pathways such as MAPK signaling pathway, neuroactive ligand-receptor interaction, and axon guidance. Moreover, protein–protein interaction (PPI) network analysis showed that PH domain and leucine-rich repeat protein phosphatase 1 (PHLPP1) and SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily A, member 2 (SMARCA2) had higher degrees and were hub nodes. Furthermore, TF prediction analysis showed that TFs, such as nuclear factor I C (NFIC) and GATA binding protein 3 (GATA3), regulated many DHMR-associated genes. Our findings reveal that key hydroxymethylated genes such as PHLPP1 and SMARCA2, as well as TFs such as NFIC and GATA, may be involved in the development of AFP-negative HCC. These molecules may be potential biomarkers for AFP-negative HCC.
Get full access to this article
View all access options for this article.
References
1.
AghoramR., CaiP., and DickinsonJ.A. (2012). Alpha-foetoprotein and/or liver ultrasonography for screening of hepatocellular carcinoma in patients with chronic hepatitis B. Cochrane Database Syst, Rev, CD002799; DOI: 10.1002/14651858.CD002799.pub2.
2.
AshburnerM., BallC.A., BlakeJ.A., BotsteinD., ButlerH., CherryJ.M., et al. (2000). Gene ontology: tool for the unification of biology. Nat Genet, 25, 25–29.
3.
BaigJ.A., AlamJ.M., MahmoodS.R., BaigM., ShaheenR., SultanaI., et al. (2009). Hepatocellular carcinoma (HCC) and diagnostic significance of α-fetoprotein (AFP). J Ayub Med Coll Abbottabad, 21, 72–75.
4.
BruixJ., and ShermanM. (2011). Management of hepatocellular carcinoma: an update. American association for the study of liver diseases (AASLD) practice guideline. Hepatology, 53, 1020–1035.
DongL., JinL., TsengH.Y., WangC.Y., WilmottJ.S., YosufiB., et al. (2014). Oncogenic suppression of PHLPP1 in human melanoma. Oncogene 33,4756.
7.
GanW., HuangJ.L., ZhangM.X., FuY.P., YiY., JingC.Y., et al. (2018). New nomogram predicts the recurrence of hepatocellular carcinoma in patients with negative preoperative serum AFP subjected to curative resection. J Surg Oncol, 117, 1540–1547.
8.
GronostajskiR.M. (2000). Roles of the NFI/CTF gene family in transcription and development. Gene, 249, 31–45.
9.
GuZ., GuL., EilsR., SchlesnerM., and BrorsB. (2014). Circlize implements and enhances circular visualization in R. Bioinformatics 30,2811.
10.
GuerreromartínezJ.A., and ReyesJ.C. (2018). High expression of SMARCA4 or SMARCA2 is frequently associated with an opposite prognosis in cancer. Sci Rep 8,2043.
11.
HaffnerM.C., ChauxA., MeekerA.K., EsopiD.M., GerberJ., PellakuruL.G., et al. (2011). Global 5-hydroxymethylcytosine content is significantly reduced in tissue stem/progenitor cell compartments and in human cancers. Oncotarget, 2, 627–637.
12.
HoffmanG.R., RahalR., BuxtonF., XiangK., McallisterG., FriasE., et al. (2014). Functional epigenetics approach identifies BRM/SMARCA2 as a critical synthetic lethal target in BRG1-deficient cancers. Proc Natl Acad Sci U S A 111,3128.
13.
HouY., DengJ., ZhangL., XieX., GuoX., SunC., et al. (2015). Lower expression of PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1) association with poor prognosis of gastric cancer. Int J Clin Exp Med 8,20481.
14.
JonesP.A., and BaylinS.B. (2002). The fundamental role of epigenetic events in cancer. Nat Rev Genet, 3, 415–428.
15.
KanehisaM., and GotoS. (2000). KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res, 28, 27–30.
KriaucionisS., and HeintzN. (2009). The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science, 324, 929–930.
18.
LaiA.Y., and WadeP.A. (2011). Cancer biology and NuRD: a multifaceted chromatin remodelling complex. Nat Rev Cancer, 11, 588–596.
19.
LangmeadB., and SalzbergS.L. (2012). Fast gapped-read alignment with Bowtie 2. Nat Methods, 9, 357–359.
20.
LeeH.K., LeeD.S., and ParkJ.C. (2015). Nuclear factor I-C regulates E-cadherin via control of KLF4 in breast cancer. BMC Cancer, 15, 1–11.
21.
LiM., GaoF., XiaY., TangY., ZhaoW., JinC., et al. (2016). Filtrating colorectal cancer associated genes by integrated analyses of global DNA methylation and hydroxymethylation in cancer and normal tissue. Sci Rep, 6, 31826.
22.
LiaoW.T., LiT.T., WangZ.G., WangS.Y., HeM.R., YeY.P., et al. (2013). microRNA-224 promotes cell proliferation and tumor growth in human colorectal cancer by repressing PHLPP1 and PHLPP2. Clin Cancer Res, 19, 4662–4672.
23.
LienhardM., GrimmC., MorkelM., HerwigR., and ChavezL. (2014). MEDIPS: genome-wide differential coverage analysis of sequencing data derived from DNA enrichment experiments. Bioinformatics, 30, 284–286.
24.
LiverEAFTSOT. (2012) EASL-EORTC clinical practice guidelines: management of hepatocellular carcinoma. J Hepatol, 56, 908–943.
25.
LoC.M., NganH., TsoW.K., LiuC.L., LamC.M., PoonR.T.P., et al. (2002). Randomized controlled trial of transarterial lipiodol chemoembolization for unresectable hepatocellular carcinoma. Hepatology, 35, 1164–1171.
MaS., JiaoB., LiuX., YiH., KongD., GaoL., et al. (2010). Approach to radiation therapy in hepatocellular carcinoma. Cancer Treat Rev, 36, 157–163.
28.
MehraR., VaramballyS., DingL., ShenR., SabelM.S., GhoshD., et al. (2005). Identification of GATA3 as a breast cancer prognostic marker by global gene expression meta-analysis. Cancer Res 65,11259.
29.
MuffadalM., WangJ., MotiwalaT., FrankhouserD., YanP., JacobS.T., et al. (2015). Identification of 5-hydroxymethylated loci as potential novel biomarkers in liver cancer. Int J Mol Med 36,S21.
NilssonJ., HelouK., KovácsA., BendahlP.O., BjursellG., FernöM., et al. (2010). Nuclear Janus-activated kinase 2/nuclear factor 1-C2 suppresses tumorigenesis and epithelial-to-mesenchymal transition by repressing Forkhead box F1. Cancer Res, 70, 2020–2029.
32.
NitscheC., EdderkaouiM., MooreR.M., EiblG., KasaharaN., TregerJ., et al. (2012). The phosphatase PHLPP1 regulates Akt2, promotes pancreatic cancer cell death, and inhibits tumor formation. Gastroenterology, 142, 377.e5–387.e5.
33.
O'NeillA.K., NiederstM.J., and NewtonA.C. (2013). Suppression of Survival Signalling Pathways by the Phosphatase PHLPP. FEBS J, 280, 572–583.
34.
PagesH., AboyounP., GentlemanR., and DebroyS. (2014). Biostrings: string objects representing biological sequences, and matching algorithms. R package version 2.36.4. Available at http://bioconductor.org/packages/3.1/bioc/html/Biostrings.html (accessed October11, 2019).
35.
PascualS., HerreraI., and IrurzunJ. (2016). New advances in hepatocellular carcinoma. World J Hepatol, 8, 421–438.
36.
PaskaA.V., and HudlerP. (2014). Aberrant methylation patterns in cancer: a clinical view. Biochem Med, 25, 161–176.
37.
RobinsonM., MccarthyD., and SmythG.K. (2010). edgeR: differential expression analysis of digital gene expression data. J Hosp Palliat Nurs, 4, 206–207.
38.
ShannonP., MarkielA., OzierO., BaligaN.S., WangJ.T., RamageD., et al. (2003). Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res, 13, 2498–2504.
39.
ShannonP., RichardsM. (2012). MotifDb: an annotated collection of protein-DNA binding sequence motifs. Available at https://bioconductor.org/packages/release/bioc/html/MotifDb.html (accessed December28, 2017).
40.
ShenK., XiZ., XieJ., WangH., XieC., LeeC.S., et al. (2016). Guttiferone K suppresses cell motility and metastasis of hepatocellular carcinoma by restoring aberrantly reduced profilin 1. Oncotarget, 7, 56650–56663.
41.
SilvaJ.P., GormanR.A., BergerN.G., TsaiS., ChristiansK.K., ClarkeC.N., et al. (2017). The prognostic utility of baseline alpha-fetoprotein for hepatocellular carcinoma patients. J Surg Oncol, 116, 831–840.
42.
SzklarczykD., FranceschiniA., WyderS., ForslundK., HellerD., HuertacepasJ., et al. (2015). STRING v10: protein–protein interaction networks, integrated over the tree of life. Nucleic Acids Res 43,D447.
43.
TzartzevaK., ObiJ., RichN.E., ParikhN.D., MarreroJ.A., YoppA., et al. (2018). Surveillance imaging and alpha fetoprotein for early detection of hepatocellular carcinoma in patients with cirrhosis: a meta-analysis. Gastroenterology, 154, 1706.e1–1718.e1.
44.
VaquerizasJ.M., KummerfeldS.K., TeichmannS.A., and LuscombeN.M. (2009). A census of human transcription factors: function, expression and evolution. Nat Rev Genet 10,252.
45.
YeC., TaoR., CaoQ., ZhuD., WangY., WangJ., et al. (2016). Whole-genome DNA methylation and hydroxymethylation profiling for HBV-related hepatocellular carcinoma. Int J Oncol 49,589.
46.
YoonN.K., MareshE.L., ShenD., ElshimaliY., AppleS., HorvathS., et al. (2010). Higher levels of GATA3 predict better survival in women with breast cancer. Human Pathol, 41, 1794–1801.
47.
YuG., WangL.G., HanY., and HeQ.Y. (2012). clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS, 16, 284–287.
48.
YuG., WangL.G., and HeQ.Y. (2015). ChIPseeker: an R/Bioconductor package for ChIP peak annotation, comparison and visualization. Bioinformatics, 31, 2382–2383.
49.
YuY., DaiM., LuA., YuE., and MerlinoG. (2018). PHLPP1 mediates melanoma metastasis suppression through repressing AKT2 activation. Oncogene, 37, 2225–2236.
50.
ZaazaaA.M., LokmanM.S., ShalbyA.B., AhmedH.H., and EltoumyS.A. (2018). Ellagic acid holds promise against hepatocellular carcinomain an experimental model: mechanisms of action. Asian Pac J Cancer Prev 19,387.
51.
ZhangY., LiuT., MeyerC.A., JérômeE., JohnsonD.S., BernsteinB.E., et al. (2008). Model-based analysis of ChIP-Seq (MACS). Genome Biol 9,137.
52.
ZhangZ., ZhangY., WangY., XuL., and XuW. (2016). Alpha-fetoprotein-L3 and Golgi protein 73 may serve as candidate biomarkers for diagnosing alpha-fetoprotein-negative hepatocellular carcinoma. Onco Targets Ther, 9, 123–129.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
