1 2842 138 FREQUENT CONCOMITANT EPIGENETIC SILENCING OF SOX1 AND SECRETED FRIZZLED-RELATED PROTEINS (SFRPS) IN HUMAN HEPATOCELLULAR CARCINOMA. BACKGROUND AND AIM: EXCEPT FOR GENETIC MUTATIONS, EPIGENETIC CHANGES ARE ALSO INVOLVED IN THE DEVELOPMENT OF HUMAN CANCERS. RECENTLY, WE HAVE IDENTIFIED SOX1, SRY (SEX DETERMINING REGION Y)-BOX 1, IS HYPERMETHYLATED IN CERVICAL CANCER AND OVARIAN CANCER. THEREFORE, WE INVESTIGATED WHETHER PROMOTER HYPERMETHYLATION OF SOX1 IS COMMON IN HEPATOCELLULAR CARCINOMA (HCC). METHODS: WE USED METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION (MS-PCR) AND BISULFITE SEQUENCING TO ANALYZE THE METHYALTION LEVEL OF THE SOX1 PROMOTER IN SEVEN HCC CELL LINES, 54 CLINICAL HCCS, 42 CIRRHOTIC LIVERS, 21 LIVERS WITH CHRONIC HEPATITIS, AND 15 CONTROL LIVERS. THEN, WE EMPLOYED QUANTITATIVE MS-PCR (QMSP) TO VALIDATE IN AN INDEPENDENT SET OF SAMPLES (60 PAIRED HCCS AND 30 CONTROL LIVERS). FINALLY, WE USED LUCIFERASE REPORTER AND COLONY FORMATION ASSAY TO CHECK THE EFFECT OF SOX1 IN HCC. RESULTS: PROMOTER METHYLATION OF SOX1 WAS SIGNIFICANTLY FREQUENT IN HCC CELL LINES AND CLINICAL HCCS, CIRRHOTIC LIVERS, BUT NOT IN CONTROL LIVERS (P < 0.0001). THERE IS A SIGNIFICANT CORRELATION BETWEEN DOWNREGULATION OF SOX1 EXPRESSION AND PROMOTER METHYLATION. QMSP RESULTS CONFIRMED THAT PROMOTER HYPERMETHYLATION OF SOX1 IS SIGNIFICANTLY MORE FREQUENT IN HCCS THAN CONTROL LIVERS (P < 0.0001). THE FREQUENCY OF SOX1 METHYLATION IN PATIENTS WITH SECRETED FRIZZLED-RELATED PROTEINS (SFRPS) METHYLATION IS SIGNIFICANTLY HIGHER THAN IN PATIENTS WITHOUT SFRPS METHYLATION (P < 0.0001). FURTHERMORE, ECTOPIC EXPRESSION OF SOX1 COULD SUPPRESS T-CELL FACTOR-DEPENDENT TRANSCRIPTIONAL ACTIVITY AND COLONY FORMATION NUMBER IN HCCS. CONCLUSIONS: CONCOMITANT EPIGENETIC SILENCING OF SOX1 AND SFRPS THROUGH PROMOTER HYPERMETHYLATION IS FREQUENT IN HCCS, AND THIS MIGHT CONTRIBUTE TO ABNORMAL ACTIVATION OF CANONICAL WNT SIGNAL PATHWAY. 2013 2 4903 34 P16 PROMOTER HYPERMETHYLATION IN HUMAN HEPATOCELLULAR CARCINOMA WITH OR WITHOUT HEPATITIS VIRUS INFECTION. BACKGROUND: EPIGENETIC ALTERATION THROUGH METHYLATION IS ONE OF THE MOST IMPORTANT STEPS IN CARCINOGENESIS. HOWEVER, THE RELATION BETWEEN HEPATITIS VIRUS INFECTION AND EPIGENETIC ALTERATIONS IS POORLY UNDERSTOOD. METHODS: SIXTEEN PATIENTS WITHOUT HEPATITIS B VIRUS (HBV) AND HEPATITIS C VIRUS (HCV) AND 35 PATIENTS WITH HBV OR HCV WHO UNDERWENT LIVER RESECTION FOR HEPATOCELLULAR CARCINOMA (HCC) WERE STUDIED. MUTATION OF P53 WAS DETECTED BY DIRECT SEQUENCING. METHYLATION STATUS OF P16 WAS EVALUATED IN TUMOR AND NONCANCEROUS LIVER TISSUES BY METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION. RESULTS: IN HCC WITHOUT HBV AND HCV, P53 MUTATIONS WERE DETECTED IN 5 (31%) OF 16 HCCS. METHYLATION OF P16 PROMOTER WAS DETECTED IN 2 (25%) OF 8 MODERATELY DIFFERENTIATED HCCS, 6 (75%) OF 8 POORLY DIFFERENTIATED HCCS, AND NONE OF 16 NONCANCEROUS TISSUE SPECIMENS. IN HCC WITH HBV OR HCV, P53 MUTATIONS WERE DETECTED IN 8 (23%) OF 35 HCCS. METHYLATION OF P16 PROMOTER WAS DETECTED IN 2 (100%) OF 2 WELL-DIFFERENTIATED HCCS, 13 (76%) OF 17 MODERATELY DIFFERENTIATED HCCS, 12 (75%) OF 16 POORLY DIFFERENTIATED HCCS, AND 9 (26%) OF 35 NONCANCEROUS LIVER TISSUE SPECIMENS. CONCLUSIONS: OUR RESULTS SUGGEST THAT HEPATITIS VIRUSES MIGHT INDUCE METHYLATION OF P16 PROMOTER IN LIVER WITH CHRONIC INFLAMMATION, BEFORE APPEARANCE OF HCC. 2004 3 6770 46 [ABERRANT METHYLATION OF MULTIPLE GENES AND ITS CLINICAL IMPLICATION IN HEPATOCELLULAR CARCINOMA]. OBJECTIVE: TO INVESTIGATE THE METHYLATION FREQUENCIES OF MULTIPLE TUMOR SUPPRESSOR GENES (TSGS) IN HEPATOCELLULAR CARCINOMA (HCC) AND THE CLINICAL IMPLICATION OF ABERRANT DNA METHYLATION IN MOLECULAR CARCINOGENESIS OF HCC. METHODS: SIXTY SAMPLES OF HCC AND THE PAIRED ADJACENT LIVER TISSUE, 16 SAMPLES FROM POST-HEPATITIS CIRRHOTIC LIVERS, 5 FROM LIVERS WITH CHRONIC HEPATITIS AND 5 FROM NORMAL LIVERS WERE COLLECTED. EIGHT TSGS FREQUENTLY SILENCED BY HYPERMETHYLATION OF THEIR PROMOTERS IN VARIOUS TYPES OF DIGESTIVE TUMORS WERE SELECTED, INCLUDING APC, RASSF1A, P16, GSTP1, MGMT, DAPK, SOCS-1 AND RIZ1. THE STATUS OF PROMOTER METHYLATION IN THESE 8 GENES WAS INVESTIGATED USING METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION. THE CLINICOPATHOLOGICAL DATA OF HCC WERE ALSO ANALYZED IN ORDER TO EVALUATE THE CLINICAL IMPLICATION OF ABERRANT METHYLATION IN HCC. RESULTS: METHYLATION OF THE 8 TSGS WAS QUITE FREQUENT IN HCC, WITH A METHYLATION RATE OF 95.0% IN RASSF1A, 90.0% IN APC, 73.3% IN GSTP1, 65.0% IN P16, 61.6% IN RIZ1 AND 60.0% IN MGMT. METHYLATION OF THE 6 GENES WAS MORE FREQUENT IN HCC THAN THAT IN ADJACENT TISSUES (P < 0.05). THE METHYLATION RATE OF MGMT, GSTP1 AND RIZ1 IN THE ADJACENT TISSUES WAS 41.6%, 40.0% AND 25.0%, RESPECTIVELY, SIGNIFICANTLY HIGHER THAN THAT IN CIRRHOTIC LIVER (P < 0.05). P16 METHYLATION WAS MORE FREQUENTLY OBSERVED IN HCC IN ELDERLY PATIENTS. THE FREQUENCY OF MGMT METHYLATION WAS TENDED TO BE HIGHER IN GIANT HCC THAN THAT IN THE OTHER TYPES OF HCC. PATIENTS WITH MGMT METHYLATION IN THE TUMOR WERE FOUND TO HAVE A SHORTER DISEASE FREE SURVIVAL. CONCLUSION: DIFFERENT FREQUENCY OF METHYLATION IN HEPATOCELLULAR CARCINOMAS, ADJACENT LIVER TISSUES AND CIRRHOTIC LIVERS IMPLIES THAT EPIGENETIC ALTERATION IN THE HEPATOCELLULAR CARCINOGENESIS MAY BE A GRADUALLY PROGRESSIVE PROCESS. METHYLATION STATUS OF MGMT, GSTP1 AND RIZ1 MAY BE PROMISING IN RISK ASSESSMENT OF HEPATOCELLULAR CARCINOMA AND IN EARLY DIAGNOSIS. FURTHERMORE, MGMT METHYLATION MIGHT BE ALSO USED AS A POTENTIAL PROGNOSTIC BIOMARKER FOR HEPATOCELLULAR CARCINOMA PATIENTS. 2008 4 1617 40 DNA METHYLTRANSFERASE EXPRESSION AND DNA METHYLATION IN HUMAN HEPATOCELLULAR CARCINOMA AND THEIR CLINICOPATHOLOGICAL CORRELATION. ABERRANT DNA METHYLATION ON CPG ISLANDS IS ONE OF THE MOST CONSISTENT EPIGENETIC CHANGES IN HUMAN CANCERS, AND THE METHYLATION PROCESS IS CATALYZED BY DNA METHYLTRANSFERASE (DNMT). WE EVALUATED I) THE MRNA LEVELS OF THREE DNMTS; DNMT1, DNMT3A AND DNMT3B, IN 25 HEPATOCELLULAR CARCINOMAS (HCCS), IN THEIR CORRESPONDING NON-CANCEROUS LIVER TISSUES AND IN 7 NORMAL LIVERS BY USING REAL-TIME REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION; II) NUCLEAR EXPRESSION OF DNMT1 AND DNMT3A PROTEINS IN THE HCCS BY IMMUNOHISTOCHEMISTRY, III) THE METHYLATION STATUS OF 5 GENES; P16, P15, E-CADHERIN, HIC-1 AND RASSF1A IN THE SAME TISSUES, AND IV) THE RELATIONSHIPS BETWEEN THE ABOVE RESULTS AND THE CLINICOPATHOLOGICAL CHARACTERISTICS, INCLUDING PROGNOSIS. THE DIFFERENCES IN MRNA EXPRESSION LEVELS FOR DNMT1, DNMT3A AND DNMT3B WERE STATISTICALLY SIGNIFICANT BETWEEN HCC AND NORMAL LIVERS (P<0.001), HCC AND CHRONIC HEPATITIS (P<0.001) AND HCC AND CIRRHOSIS (P<0.001). AN INCREASE IN MRNA EXPRESSION LEVELS OF >4-FOLD FOR DNMT3B IN HCCS WAS SIGNIFICANTLY ASSOCIATED WITH A POORER OVERALL SURVIVAL (P=0.027) AND SHORTER METASTASIS-FREE SURVIVAL (P=0.0299). A POORER RECURRENCE-FREE SURVIVAL WAS NOTED IN HCCS WITH A >4-FOLD INCREASE IN DNMT3A MRNA (P=0.0120). THE AVERAGE NUMBERS OF METHYLATED GENES WERE 0, 1.27, 1.38 AND 2.72 FOR NORMAL LIVERS, CHRONIC HEPATITIS, CIRRHOSIS AND HCCS, RESPECTIVELY, AND THIS PROGRESSIVE INCREASE FROM NORMAL LIVERS TO CHRONIC HEPATITIS/CIRRHOSIS THROUGH HCC MAY SUGGEST THAT TUMOR SUPPRESSOR GENE METHYLATION IS AN EARLY EVENT IN HEPATOCARCINOGENESIS. THESE RESULTS FIRST SUGGEST THAT HEPATOCARCINOGENESIS INVOLVES AN INCREASED EXPRESSION OF DNMT1, DNMT3A AND DNMT3B MRNA AND A PROGRESSIVE INCREASE IN THE NUMBER OF METHYLATED GENES FROM NORMAL LIVER, CHRONIC HEPATITIS/CIRRHOSIS TO HCC AND SECONDLY THAT AN INCREASE IN THE DNMT3A AND DNMT3B MRNA LEVELS IN HCCS RELATIVE TO THEIR NON-CANCEROUS TISSUES MAY BE A PREDICTOR OF POOR SURVIVAL. 2007 5 3264 42 HEPATITIS VIRUS INFECTION AFFECTS DNA METHYLATION IN MICE WITH HUMANIZED LIVERS. BACKGROUND & AIMS: CELLS OF TUMORS ASSOCIATED WITH CHRONIC INFLAMMATION FREQUENTLY HAVE ALTERED PATTERNS OF DNA METHYLATION, INCLUDING HEPATOCELLULAR CARCINOMAS. CHRONIC HEPATITIS HAS ALSO BEEN ASSOCIATED WITH ABERRANT DNA METHYLATION, BUT LITTLE IS KNOWN ABOUT THEIR RELATIONSHIP. METHODS: PYROSEQUENCING WAS USED TO DETERMINE THE METHYLATION STATUS OF CULTURED HUH7.5.1 HEPATOMA CELLS AFTER HEPATITIS C VIRUS (HCV) INFECTION. WE ALSO STUDIED MICE WITH SEVERE COMBINED IMMUNODEFICIENCY CARRYING THE UROKINASE-TYPE PLASMINOGEN ACTIVATOR TRANSGENE CONTROLLED BY AN ALBUMIN PROMOTER (UROKINASE-TYPE PLASMINOGEN ACTIVATOR/SEVERE COMBINED IMMUNODEFICIENT MICE), IN WHICH UP TO 85% OF HEPATOCYTES WERE REPLACED BY HUMAN HEPATOCYTES (CHIMERIC MICE). MICE WERE GIVEN INTRAVENOUS INJECTIONS OF HEPATITIS B VIRUS (HBV) OR HCV, LIVER TISSUES WERE COLLECTED, AND DNA METHYLATION PROFILES WERE DETERMINED AT DIFFERENT TIME POINTS AFTER INFECTION. WE ALSO COMPARED METHYLATION PATTERNS BETWEEN PAIRED SAMPLES OF HEPATOCELLULAR CARCINOMAS AND ADJACENT NONTUMOR LIVER TISSUES FROM PATIENTS. RESULTS: NO REPRODUCIBLE CHANGES IN DNA METHYLATION WERE OBSERVED AFTER INFECTION OF HUH7.5.1 CELLS WITH HCV. LIVERS FROM HBV- AND HCV-INFECTED MICE HAD GENOME-WIDE, TIME-DEPENDENT CHANGES IN DNA METHYLATION, COMPARED WITH UNINFECTED UROKINASE-TYPE PLASMINOGEN ACTIVATOR/SEVERE COMBINED IMMUNODEFICIENT MICE. THERE WERE CHANGES IN 160 +/- 63 GENES IN HBV-INFECTED AND 237 +/- 110 GENES IN HCV-INFECTED MICE. METHYLATION OF 149 COMMON GENES INCREASED IN HBV- AND HCV-INFECTED MICE; METHYLATION OF SOME OF THESE GENES ALSO INCREASED IN HEPATOCELLULAR CARCINOMA SAMPLES FROM PATIENTS COMPARED WITH NONTUMOR TISSUES. EXPRESSION OF IFNG, WHICH IS EXPRESSED BY NATURAL KILLER CELLS, INCREASED SIGNIFICANTLY IN CHIMERIC LIVERS, IN CONCORDANCE WITH INDUCTION OF DNA METHYLATION, AFTER INFECTION WITH HBV OR HCV. INDUCTION OF IFNG WAS REDUCED AFTER ADMINISTRATION OF AN INHIBITOR OF NATURAL KILLER CELL FUNCTION (ANTI-ASIALO GM1). CONCLUSIONS: IN CHIMERIC MICE WITH HUMANIZED LIVERS, INFECTION WITH HBV AND HCV APPEARS TO ACTIVATE A NATURAL KILL CELL-DEPENDENT INNATE IMMUNE RESPONSE. THIS CONTRIBUTES TO THE INDUCTION AND ACCUMULATION OF ABERRANT DNA METHYLATION IN HUMAN HEPATOCYTES. 2014 6 153 41 ABERRANT METHYLATION OF MULTIPLE TUMOR SUPPRESSOR GENES IN AGING LIVER, CHRONIC HEPATITIS, AND HEPATOCELLULAR CARCINOMA. ABERRANT DNA METHYLATION IS AN IMPORTANT EPIGENETIC ALTERATION IN HEPATOCELLULAR CARCINOMA (HCC). HOWEVER, THE MOLECULAR PROCESSES UNDERLYING THE METHYLATOR PHENOTYPE AND THE CONTRIBUTION OF HEPATITIS VIRUSES ARE POORLY UNDERSTOOD. THE CURRENT STUDY IS A COMPREHENSIVE METHYLATION ANALYSIS OF HUMAN LIVER TISSUE SPECIMENS. A TOTAL OF 176 LIVER TISSUES, INCLUDING 77 PAIRS OF HCCS AND MATCHING NONCANCEROUS LIVER AND 22 NORMAL LIVERS, WERE ANALYZED FOR METHYLATION. METHYLATION OF 19 EPIGENETIC MARKERS WAS QUANTIFIED, AND THE RESULTS WERE CORRELATED WITH DIFFERENT DISEASE STATES AND THE PRESENCE OR ABSENCE OF HEPATITIS B VIRUS (HBV) AND HEPATITIS C VIRUS (HCV) INFECTIONS. BASED ON METHYLATION PROFILES, THE 19 LOCI WERE CATEGORIZED INTO 3 GROUPS. NORMAL LIVER TISSUES SHOWED METHYLATION PRIMARILY IN GROUP 1 LOCI (HIC-1, CASP8, GSTP1, SOCS1, RASSF1A, P16, APC), WHICH WAS SIGNIFICANTLY HIGHER THAN GROUP 2 (CDH1, RUNX3, RIZ1, SFRP2, MINT31) AND GROUP 3 MARKERS (COX2, MINT1, CACNA1G, RASSF2, MINT2, REPRIMO, DCC) (P < 0.0001). NONCANCEROUS LIVERS DEMONSTRATED INCREASED METHYLATION IN BOTH GROUP 1 AND GROUP 2 LOCI. METHYLATION WAS SIGNIFICANTLY MORE ABUNDANT IN HCV-POSITIVE LIVERS COMPARED WITH NORMAL LIVER TISSUES. CONVERSELY, HCC SHOWED FREQUENT METHYLATION AT EACH LOCUS INVESTIGATED IN ALL 3 GROUPS. HOWEVER, THE GROUP 3 LOCI SHOWED MORE DENSE AND FREQUENT METHYLATION IN HCV-POSITIVE CANCERS COMPARED WITH BOTH HBV-POSITIVE CANCERS AND VIRUS-NEGATIVE CANCERS (P < 0.0001). CONCLUSION: METHYLATION IN HCC IS FREQUENT BUT OCCURS IN A GENE-SPECIFIC AND DISEASE-SPECIFIC MANNER. METHYLATION PROFILING ALLOWED US TO DETERMINE THAT ABERRANT METHYLATION IS COMMONLY PRESENT IN NORMAL AGING LIVERS, AND SEQUENTIALLY PROGRESSES WITH ADVANCING STAGES OF CHRONIC VIRAL INFECTION. FINALLY, OUR DATA PROVIDE EVIDENCE THAT HCV INFECTION MAY ACCELERATE THE METHYLATION PROCESS AND SUGGESTS A CONTINUUM OF INCREASING METHYLATION WITH PERSISTENT VIRAL INFECTION AND CARCINOGENESIS IN THE LIVER. 2008 7 3190 43 HCV-INDUCED EPIGENETIC CHANGES ASSOCIATED WITH LIVER CANCER RISK PERSIST AFTER SUSTAINED VIROLOGIC RESPONSE. BACKGROUND & AIMS: CHRONIC HEPATITIS C VIRUS (HCV) INFECTION IS AN IMPORTANT RISK FACTOR FOR HEPATOCELLULAR CARCINOMA (HCC). DESPITE EFFECTIVE ANTIVIRAL THERAPIES, THE RISK FOR HCC IS DECREASED BUT NOT ELIMINATED AFTER A SUSTAINED VIROLOGIC RESPONSE (SVR) TO DIRECT-ACTING ANTIVIRAL (DAA) AGENTS, AND THE RISK IS HIGHER IN PATIENTS WITH ADVANCED FIBROSIS. WE INVESTIGATED HCV-INDUCED EPIGENETIC ALTERATIONS THAT MIGHT AFFECT RISK FOR HCC AFTER DAA TREATMENT IN PATIENTS AND MICE WITH HUMANIZED LIVERS. METHODS: WE PERFORMED GENOME-WIDE CHIPMENTATION-BASED CHIP-SEQ AND RNA-SEQ ANALYSES OF LIVER TISSUES FROM 6 PATIENTS WITHOUT HCV INFECTION (CONTROLS), 18 PATIENTS WITH CHRONIC HCV INFECTION, 8 PATIENTS WITH CHRONIC HCV INFECTION CURED BY DAA TREATMENT, 13 PATIENTS WITH CHRONIC HCV INFECTION CURED BY INTERFERON THERAPY, 4 PATIENTS WITH CHRONIC HEPATITIS B VIRUS INFECTION, AND 7 PATIENTS WITH NONALCOHOLIC STEATOHEPATITIS IN EUROPE AND JAPAN. HCV-INDUCED EPIGENETIC MODIFICATIONS WERE MAPPED BY COMPARATIVE ANALYSES WITH MODIFICATIONS ASSOCIATED WITH OTHER LIVER DISEASE ETIOLOGIES. UPA/SCID MICE WERE ENGRAFTED WITH HUMAN HEPATOCYTES TO CREATE MICE WITH HUMANIZED LIVERS AND GIVEN INJECTIONS OF HCV-INFECTED SERUM SAMPLES FROM PATIENTS; MICE WERE GIVEN DAAS TO ERADICATE THE VIRUS. PATHWAYS ASSOCIATED WITH HCC RISK WERE IDENTIFIED BY INTEGRATIVE PATHWAY ANALYSES AND VALIDATED IN ANALYSES OF PAIRED HCC TISSUES FROM 8 PATIENTS WITH AN SVR TO DAA TREATMENT OF HCV INFECTION. RESULTS: WE FOUND CHRONIC HCV INFECTION TO INDUCE SPECIFIC GENOME-WIDE CHANGES IN H3K27AC, WHICH CORRELATED WITH CHANGES IN EXPRESSION OF MRNAS AND PROTEINS. THESE CHANGES PERSISTED AFTER AN SVR TO DAAS OR INTERFERON-BASED THERAPIES. INTEGRATIVE PATHWAY ANALYSES OF LIVER TISSUES FROM PATIENTS AND MICE WITH HUMANIZED LIVERS DEMONSTRATED THAT HCV-INDUCED EPIGENETIC ALTERATIONS WERE ASSOCIATED WITH LIVER CANCER RISK. COMPUTATIONAL ANALYSES ASSOCIATED INCREASED EXPRESSION OF SPHK1 WITH HCC RISK. WE VALIDATED THESE FINDINGS IN AN INDEPENDENT COHORT OF PATIENTS WITH HCV-RELATED CIRRHOSIS (N = 216), A SUBSET OF WHICH (N = 21) ACHIEVED VIRAL CLEARANCE. CONCLUSIONS: IN AN ANALYSIS OF LIVER TISSUES FROM PATIENTS WITH AND WITHOUT AN SVR TO DAA THERAPY, WE IDENTIFIED EPIGENETIC AND GENE EXPRESSION ALTERATIONS ASSOCIATED WITH RISK FOR HCC. THESE ALTERATIONS MIGHT BE TARGETED TO PREVENT LIVER CANCER IN PATIENTS TREATED FOR HCV INFECTION. 2019 8 3240 28 HEPATIC LIPID ACCUMULATION ALTERS GLOBAL HISTONE H3 LYSINE 9 AND 4 TRIMETHYLATION IN THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR ALPHA NETWORK. RECENT DATA SUGGEST THAT THE ETIOLOGY OF SEVERAL METABOLIC DISEASES IS CLOSELY ASSOCIATED WITH TRANSCRIPTOME ALTERATION BY ABERRANT HISTONE METHYLATION. WE PERFORMED DNA MICROARRAY AND CHIP-ON-CHIP ANALYSES TO EXAMINE TRANSCRIPTOME PROFILING AND TRIMETHYLATION ALTERATIONS TO IDENTIFY THE GENOMIC SIGNATURE OF NONALCOHOLIC FATTY LIVER DISEASE (NAFLD), THE MOST COMMON FORM OF CHRONIC LIVER DISEASE. TRANSCRIPTOME ANALYSIS SHOWED THAT STEATOTIC LIVERS IN HIGH-FAT DIET-FED APOLIPOPROTEIN E2 MICE SIGNIFICANTLY ALTERED THE EXPRESSION OF APPROXIMATELY 70% OF TOTAL GENES COMPARED WITH NORMAL DIET-FED CONTROL LIVERS, SUGGESTING THAT HEPATIC LIPID ACCUMULATION INDUCES DRAMATIC ALTERATIONS IN GENE EXPRESSION IN VIVO. ALSO, PATHWAY ANALYSIS SUGGESTED THAT GENES ENCODING CHROMATIN-REMODELING ENZYMES, SUCH AS JUMONJI C-DOMAIN-CONTAINING HISTONE DEMETHYLASES THAT REGULATE HISTONE H3K9 AND H3K4 TRIMETHYLATION (H3K9ME3, H3K4ME3), WERE SIGNIFICANTLY ALTERED IN STEATOTIC LIVERS. THUS, WE FURTHER INVESTIGATED THE GLOBAL H3K9ME3 AND H3K4ME3 STATUS IN LIPID-ACCUMULATED MOUSE PRIMARY HEPATOCYTES BY CHIP-ON-CHIP ANALYSIS. RESULTS SHOWED THAT HEPATIC LIPID ACCUMULATION INDUCED ABERRANT H3K9ME3 AND H3K4ME3 STATUS IN PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR ALPHA AND HEPATIC LIPID CATABOLISM NETWORK GENES, REDUCING THEIR MRNA EXPRESSION COMPARED WITH NON-TREATED CONTROL HEPATOCYTES. THIS STUDY PROVIDES THE FIRST EVIDENCE THAT EPIGENETIC REGULATION BY H3K9ME3 AND H3K4ME3 IN HEPATOCYTES MAY BE INVOLVED IN HEPATIC STEATOSIS AND THE PATHOGENESIS OF NAFLD. THUS, CONTROL OF H3K9ME3 AND H3K4ME3 REPRESENTS A POTENTIAL NOVEL NAFLD PREVENTION AND TREATMENT STRATEGY. 2012 9 3646 37 INCREASED PROTEIN EXPRESSION OF DNA METHYLTRANSFERASE (DNMT) 1 IS SIGNIFICANTLY CORRELATED WITH THE MALIGNANT POTENTIAL AND POOR PROGNOSIS OF HUMAN HEPATOCELLULAR CARCINOMAS. ALTERATION OF DNA METHYLATION IS ONE OF THE MOST CONSISTENT EPIGENETIC CHANGES IN HUMAN CANCERS. DNA METHYLTRANSFERASE (DNMT) 1 IS A MAJOR ENZYME INVOLVED IN ESTABLISHING GENOMIC METHYLATION PATTERNS. MOST OF THE STUDIES CONCERNING DNMT1 EXPRESSION IN HUMAN CANCERS HAVE BEEN PERFORMED ONLY AT THE MRNA LEVEL. TO DIRECTLY EXAMINE DNMT1 PROTEIN EXPRESSION LEVELS DURING HUMAN HEPATOCARCINOGENESIS, 16 HISTOLOGICALLY NORMAL LIVER TISSUES, 51 NONCANCEROUS LIVER TISSUES EXHIBITING CHRONIC HEPATITIS OR CIRRHOSIS, WHICH ARE CONSIDERED TO BE PRECANCEROUS CONDITIONS, AND 53 HEPATOCELLULAR CARCINOMAS (HCCS) WERE SUBJECTED TO IMMUNOHISTOCHEMIC EXAMINATION. IF MORE THAN 20% OF THE CELLS EXHIBITED NUCLEAR DNMT1 STAINING, THE TISSUE SAMPLE WAS CONSIDERED TO BE DNMT1-POSITIVE. DNMT1 IMMUNOREACTIVITY WAS OBSERVED IN 23 (43%) OF THE HCCS, BUT IN NONE (0%) OF THE HISTOLOGICALLY NORMAL LIVER OR NONCANCEROUS LIVER TISSUES EXHIBITING CHRONIC HEPATITIS OR CIRRHOSIS. THE INCIDENCE OF INCREASED DNMT1 PROTEIN EXPRESSION IN HCCS CORRELATED SIGNIFICANTLY WITH POOR TUMOR DIFFERENTIATION (P = 0.0006) AND PORTAL VEIN INVOLVEMENT (P = 0.0002). MOREOVER, THE RECURRENCE-FREE (P = 0.0001) AND OVERALL (P < 0.0001) SURVIVAL RATES OF PATIENTS WITH HCCS EXHIBITING INCREASED DNMT1 PROTEIN EXPRESSION WERE SIGNIFICANTLY LOWER THAN THOSE OF PATIENTS WITH HCCS THAT DID NOT EXHIBIT INCREASED EXPRESSION. INCREASED DNMT1 PROTEIN EXPRESSION MAY PLAY A CRITICAL ROLE IN THE MALIGNANT PROGRESSION OF HCCS AND BE A BIOLOGIC PREDICTOR OF BOTH HCC RECURRENCE AND A POOR PROGNOSIS IN HCC PATIENTS. 2003 10 5357 42 REACTIVE OXYGEN SPECIES INDUCE EPIGENETIC INSTABILITY THROUGH THE FORMATION OF 8-HYDROXYDEOXYGUANOSINE IN HUMAN HEPATOCARCINOGENESIS. CHRONIC HEPATITIS C (CHC) TRIGGERS OXIDATIVE STRESS AND CONTRIBUTES TO THE EMERGENCE OF HEPATOCELLULAR CARCINOMA (HCC). WE PREVIOUSLY REPORTED THAT TUMOR SUPPRESSOR GENE (TSG) METHYLATION IS A CRITICAL FACTOR DURING THE EARLY STAGES OF HEPATOCARCINOGENESIS. IN THIS STUDY, WE CLARIFY THE ASSOCIATION BETWEEN OXIDATIVE STRESS AND EPIGENETIC ALTERATIONS DURING HEPATOCARCINOGENESIS. WE EXAMINED DNA OXIDATION AND METHYLATION PROFILES IN 128 LIVER BIOPSY SAMPLES FROM CHC PATIENTS. THE DNA OXIDATION AND METHYLATED TSG NUMBERS WERE QUANTIFIED USING IMMUNOHISTOCHEMICAL ANALYSIS OF 8-HYDROXYDEOXYGUANOSINE (8-OHDG) AND QUANTITATIVE PCR FOR 11 TSGS, RESPECTIVELY. THE QUANTITATIVE CHROMATIN IMMUNOPRECIPITATION-PCR (CHIP-QPCR) ASSAY IN HEPG2 AND FETAL LIVER HC CELLS TREATED WITH H2O2 WAS USED TO QUANTIFY TRIMETHYL-H3K4, ACETYLATED-H4K16 (AN ACTIVE CHROMATIN MARKER), TRIMETHYL-H3K27 (A REPRESSIVE CHROMATIN MARKER) AND 8-OHDG. WE ANALYZED 30 PROMOTERS OF 25 DIFFERENT TSGS BY QPCR. THE HIGH LEVELS OF 8-OHDG WAS THE ONLY VARIABLE THAT WAS SIGNIFICANTLY ASSOCIATED WITH THE INCREASED NUMBER OF METHYLATED TSGS IN CHC (P < 0.0001). THE CHIP-QPCR REVEALED THAT AFTER H2O2 TREATMENT OF THE CELL LINES, THE 8-OHDG-BOUND PROMOTERS SHOWED A MODIFICATION FROM AN ACTIVE CHROMATIN (TRIMETHYL-H3K4 AND ACETYLATED-H4K16 DOMINANT) TO A REPRESSIVE CHROMATIN (TRIMETHYL-H3K27 DOMINANT) STATUS. WE CONCLUDE THAT OXIDATIVE STRESS ALTERS THE CHROMATIN STATUS, WHICH LEADS TO ABNORMAL METHYLATION OF TSGS, AND CONTRIBUTES TO HEPATOCARCINOGENESIS IN CHC PATIENTS. 2013 11 1393 40 DIAGNOSTIC VALUE OF THE HYPOMETHYLATION OF THE WISP1 PROMOTER IN PATIENTS WITH HEPATOCELLULAR CARCINOMA ASSOCIATED WITH HEPATITIS B VIRUS. WNT1-INDUCIBLE SIGNALING PATHWAY PROTEIN 1 (WISP1) REGULATES CELL PROLIFERATION, DIFFERENTIATION, ADHESION, MIGRATION AND SURVIVAL. ABNORMAL WISP1 EXPRESSION IS ASSOCIATED WITH THE CARCINOGENESIS OF HEPATOCELLULAR CARCINOMA (HCC). ABERRANT DNA METHYLATION IS ONE OF THE MAJOR EPIGENETIC ALTERATIONS IN HCC. HOWEVER, THE METHYLATION STATUS OF THE WISP1 PROMOTER IS STILL UNCLEAR. WE THEREFORE AIMED TO DETERMINE THE METHYLATION STATUS OF THE WISP1 PROMOTER AND EVALUATE ITS CLINICAL VALUE IN HCC. THE STUDY ENROLLED 251 PARTICIPANTS, INCLUDING 123 PARTICIPANTS WITH HCC, 90 PARTICIPANTS WITH CHRONIC HEPATITIS B (CHB) AND 38 HEALTHY CONTROLS (HCS). WISP1 METHYLATION STATUS, MRNA LEVELS AND PLASMA SOLUBLE WISP1 WERE DETECTED BY METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION (MSP), QUANTITATIVE REAL-TIME PCR (RT-QPCR) AND ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA), RESPECTIVELY. WE FOUND THAT THE METHYLATION FREQUENCY OF WISP1 IN PATIENTS WITH HCC WAS SIGNIFICANTLY LOWER THAN THAT IN PATIENTS WITH CHB AND HCS, WHILE THE RELATIVE EXPRESSION LEVELS OF WISP1 MRNA WERE MARKEDLY HIGHER IN PATIENTS WITH HCC THAN IN PATIENTS WITH CHB AND HCS. FURTHERMORE, THE PLASMA SOLUBLE WISP1 IN PATIENTS WITH HCC WAS OBVIOUSLY LOWER THAN IN THAT IN PATIENTS WITH CHB AND HCS. ALPHA-FETOPROTEIN (AFP) IS A WIDELY RECOGNIZED BIOMARKER TO DIAGNOSE HCC WHICH LACKS ENOUGH SENSITIVITY AND SPECIFICITY. WISP1 PROMOTER METHYLATION STATUS COMBINED WITH AFP SIGNIFICANTLY IMPROVED THE DIAGNOSTIC ABILITY IN DISCRIMINATING HCC FROM CHB COMPARED WITH AFP OR WISP1 METHYLATION STATUS ALONE. IN CONCLUSION, HYPOMETHYLATION OF THE WISP1 GENE PROMOTER MAY SERVE AS A NONINVASIVE BIOMARKER FOR DETECTING HBV-ASSOCIATED HCC. 2020 12 1342 43 DETECTING ABNORMAL METHYLATION OF TUMOR SUPPRESSOR GENES GSTP1, P16, RIZ1, AND RASSF1A IN HEPATOCELLULAR CARCINOMA AND ITS CLINICAL SIGNIFICANCE. HEPATOCELLULAR CARCINOMA (HCC) HAS A HIGH RATE OF MORTALITY. FURTHER STUDIES INTO EPIGENETIC CHANGES IN HCC, PARTICULARLY THE ABNORMAL METHYLATION OF TUMOR SUPPRESSOR GENES (TSGS), ARE REQUIRED, SINCE THESE CHANGES MAY PROVIDE NOVEL BIOMARKERS FOR EARLY SCREENING AND DIAGNOSIS OF HCC. BY USING METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION (MSP), THE PRESENT STUDY DETECTED THE METHYLATION STATUS IN THE PROMOTER REGION OF 4 CANDIDATE TSGS, GSTP1, P16, RIZ1, AND RASSF1A, RESPECTIVELY, IN 35 PAIRED HCC AND TUMOR-ADJACENT LIVER TISSUES IN ADDITION TO 20 NORMAL LIVER TISSUES. THEIR EFFECT ON THE INITIATION AND PROGRESSION OF HCC WAS ALSO INVESTIGATED BY ANALYZING THE CLINICOPATHOLOGICAL DATA. THE RESULTS OF THE PRESENT STUDY REVEALED THAT THE METHYLATION LEVEL OF RIZ1 AND GSTP1 GENES IN HCC WAS SIGNIFICANTLY INCREASED COMPARED WITH THAT IN THE ADJACENT TISSUES (P<0.01) AND THE NORMAL LIVER TISSUES (P<0.01). THE METHYLATION FREQUENCY OF P16 AND RASSF1A GENES WAS NOT SIGNIFICANTLY INCREASED COMPARED WITH THAT OBSERVED IN THE ADJACENT TISSUES (P>0.05) BUT WAS SIGNIFICANTLY INCREASED COMPARED WITH THE NORMAL TISSUES (P<0.01). IN HCC TISSUES, THE METHYLATION FREQUENCY OF THE GSTP1 GENE IN TUMORS WITH CAPSULAR INVASION WAS SIGNIFICANTLY INCREASED COMPARED WITH THAT IN TUMORS WITHOUT CAPSULAR INVASION (P<0.05). THE METHYLATION FREQUENCY OF P16 GENE IN HEPATITIS B SURFACE ANTIGEN (HBSAG)-POSITIVE HCC PATIENTS WAS SIGNIFICANTLY INCREASED COMPARED WITH THAT IN HBSAG-NEGATIVE PATIENTS (P<0.05). THE METHYLATION STATUS OF RIZ1 AND RASSF1A GENES WAS NOT SIGNIFICANTLY CORRELATED WITH THE CLINICOPATHOLOGICAL DATA (P>0.05). PREVIOUS STUDIES HAVE DEMONSTRATED THAT THE METHYLATION STATUS OF RIZ1 AND GSTP1 GENES IS HCC-SPECIFIC, AND THUS MAY BE USED AS A BIOMARKER TO ASSIST THE CLINICAL DIAGNOSIS OF HCC. WHILE THE METHYLATION OF GSTP1 GENE PROMOTER MAY ASSOCIATE WITH THE INVASIVENESS OF HCC, CHRONIC HEPATITIS B VIRUS INFECTION MAY BE THE CAUSE OF METHYLATION-INDUCED P16 INACTIVATION. 2015 13 5338 45 QUANTITATIVE EVALUATION OF RASSF1A METHYLATION IN THE NON-LESIONAL, REGENERATIVE AND NEOPLASTIC LIVER. BACKGROUND: EPIGENETIC CHANGES DURING AGEING AND THEIR RELATIONSHIP WITH CANCER ARE UNDER THE FOCUS OF INTENSE RESEARCH. RASSF1A AND NORE1A ARE NOVEL GENES ACTING IN CONCERT IN THE PROAPOPTOTIC PATHWAY OF THE RAS SIGNALLING. WHILE NORE1A HAS NOT BEEN PREVIOUSLY INVESTIGATED IN THE HUMAN LIVER, RECENT REPORTS HAVE SUGGESTED THAT RASSF1A IS FREQUENTLY EPIGENETICALLY METHYLATED NOT ONLY IN HCC BUT ALSO IN THE CIRRHOTIC LIVER. METHODS: TO ADDRESS WHETHER EPIGENETIC CHANGES TAKE PLACE IN CONNECTION TO AGE AND/OR TO THE UNDERLYING DISEASE, WE INVESTIGATED RASSF1A AND NORE1A GENE PROMOTER METHYLATION BY CONVENTIONAL METHYLATION SPECIFIC PCR AND REAL-TIME MSP IN A SERIES OF HEPATITIC AND NON-HEPATITIC LIVERS HARBORING REGENERATIVE/HYPERPLASTIC (CIRRHOSIS/FOCAL NODULAR HYPERPLASIA), DYSPLASTIC (LARGE REGENERATIVE, LOW AND HIGH GRADE DYSPLASTIC NODULES) AND NEOPLASTIC (HEPATOCELLULAR ADENOMA AND CARCINOMA) GROWTHS. RESULTS: IN THE HEPATITIC LIVER (CHRONIC HEPATITIC/CIRRHOSIS, HEPATOCELLULAR NODULES AND HCC) WE FOUND WIDESPREAD RASSF1A GENE PROMOTER METHYLATION WITH A METHYLATION INDEX THAT INCREASED FROM REGENERATIVE CONDITIONS (CIRRHOSIS) TO HEPATOCELLULAR NODULES (P < 0.01) TO HCC (P < 0.001). IN THE NON-HEPATITIC LIVER A CONSISTENT PATTERN OF GENE METHYLATION WAS ALSO FOUND IN BOTH LESIONAL (FOCAL NODULAR HYPERPLASIA AND HEPATOCELLULAR ADENOMA) AND NON-LESIONAL TISSUE. SPECIFICALLY, HEPATOCELLULAR ADENOMAS (HA) SHOWED A METHYLATION INDEX SIGNIFICANTLY HIGHER THAN THAT DETECTED IN FOCAL NODULAR HYPERPLASIA (FNH) (P < 0.01) AND IN NON-LESIONAL TISSUE (P < 0.001). IN NON-LESIONAL LIVER ALSO THE METHYLATION INDEX GRADUALLY INCREASED BY AGEING (P = 0.002), SUGGESTING A PROGRESSIVE SPREADING OF METHYLATED CELLS OVER TIME. AS OPPOSED TO RASSF1A GENE PROMOTER METHYLATION, NORE1A GENE WAS NEVER FOUND EPIGENETICALLY ALTERATED IN BOTH HEPATITIC AND NON-HEPATITIC LIVER. CONCLUSION: WE HAVE SHOWN THAT IN NON-LESIONAL, REGENERATIVE AND NEOPLASTIC LIVER THE RASSF1A GENE IS INCREASINGLY METHYLATED, THAT THIS CONDITION TAKES PLACE AS AN AGE-RELATED PHENOMENON AND THAT THE EARLY SETTING AND SPREADING OVER TIME OF AN EPIGENETICALLY METHYLATED HEPATOCYTE SUBPOPULATION, MIGHT BE RELATED TO LIVER TUMORIGENESIS. 2006 14 817 40 CHARACTERISTIC PATTERNS OF ALTERED DNA METHYLATION PREDICT EMERGENCE OF HUMAN HEPATOCELLULAR CARCINOMA. WE AIMED TO IDENTIFY THE SPECIFIC SUBSET OF TUMOR SUPPRESSOR GENES (TSGS) THAT ARE METHYLATION-SILENCED DURING THE EARLIEST STEPS OF HEPATOCARCINOGENESIS, AND TO FURTHER EVALUATE WHETHER THESE GENES CAN SERVE AS PREDICTIVE BIOMARKERS OF HEPATOCELLULAR CARCINOMA (HCC) EMERGENCE. A TOTAL OF 482 LIVER TISSUES INCLUDING 177 PAIRS OF HCCS AND MATCHED NONTUMOR LIVERS AND 128 LIVER BIOPSIES FROM CHRONIC HEPATITIS C (CHC) PATIENTS WERE ANALYZED FOR QUANTITATIVE METHYLATION ANALYSIS IN 24 TSG PROMOTERS AND THREE MINT LOCI. THE TUMORS WERE CLASSIFIED AS EARLY, LESS-PROGRESSED, AND HIGHLY PROGRESSED HCCS USING HISTOLOGY AND RADIOLOGICAL APPROACHES. A SUBSET OF TSGS THAT HARBORED DISTINCTLY HIGH LEVELS OF METHYLATION IN EARLY HCCS WERE SELECTED. BASED ON THE METHYLATION PROFILES OF THESE GENES, KAPLAN-MEIER ANALYSES WERE PERFORMED TO DETERMINE TIME-TO-HCC OCCURRENCE IN CHC PATIENTS. SUBSEQUENTLY, MULTIVARIATE ANALYSIS WAS PERFORMED USING AGE, GENDER, FIBROSIS STAGE, AND NUMBER OF METHYLATED TSGS AS COVARIATES. AMONG TSGS ANALYZED, A SUBSET OF EIGHT TSGS (HIC1, GSTP1, SOCS1, RASSF1, CDKN2A, APC, RUNX3, AND PRDM2) DEMONSTRATED A DISTINCT CLUSTER BY HIERARCHICAL CLUSTERING AND RECEIVER OPERATING CHARACTERISTIC ANALYSES. THIS SUBSET OF TSGS SHOWED SIGNIFICANTLY HIGHER METHYLATION LEVELS IN THE EARLY HCCS (P < 0.0001). IN THE CHC PATIENTS, METHYLATION FREQUENCIES IN THESE TSGS WERE ASSOCIATED WITH SHORTER TIME-TO-HCC OCCURRENCE (P < 0.0001), AND NUMBER OF METHYLATED GENES WAS AN INDEPENDENT RISK FACTOR FOR HCC (HAZARD RATIO = 5.21, 95% CONFIDENCE INTERVAL = 2.25-11.76, P = 0.0002). CONCLUSION: EPIGENETIC INACTIVATION OF A SUBSET OF TSGS PLAYS A CRITICAL ROLE IN THE EARLIEST STEPS OF HEPATOCARCINOGENESIS. FURTHERMORE, EPIGENETIC INACTIVATION OF THESE GENES IN CHC PROVIDES A PROGNOSTIC VALUE FOR DETERMINING THE RISK FOR DEVELOPING HCC LATER IN LIFE. 2012 15 4905 42 P16INK4A HYPERMETHYLATION IS ASSOCIATED WITH HEPATITIS VIRUS INFECTION, AGE, AND GENDER IN HEPATOCELLULAR CARCINOMA. PURPOSE: THE TUMOR SUPPRESSOR GENE P16INK4A IS MAINLY INACTIVATED BY AN EPIGENETIC CHANGE INVOLVING PROMOTER HYPERMETHYLATION IN HEPATOCARCINOGENESIS. THE POSSIBLE CLINICAL IMPACT OF P16INK4A METHYLATION AND THE POTENTIAL RISK FACTORS FOR THIS EPIGENETIC ALTERATION HAVE NOT BEEN THOROUGHLY INVESTIGATED. EXPERIMENTAL DESIGN: WE STUDIED THE METHYLATION STATUS AND MRNA AND PROTEIN EXPRESSION OF P16INK4A IN 50 HEPATOCELLULAR CARCINOMAS AND CORRESPONDING NONNEOPLASTIC LIVER LESIONS USING METHYLATION-SPECIFIC PCR, REVERSE TRANSCRIPTION-PCR, AND IMMUNOHISTOCHEMICAL TECHNIQUES. RESULTS: P16INK4A HYPERMETHYLATION WAS OBSERVED IN 58% (29 OF 50) OF THE HEPATOCELLULAR CARCINOMAS AND 16% (6 OF 38) OF THE CORRESPONDING CHRONIC HEPATITIS AND CIRRHOSIS TISSUE SAMPLES. P16INK4A METHYLATION WAS SIGNIFICANTLY ASSOCIATED WITH MRNA AND PROTEIN EXPRESSION (P <0.001 AND P=0.003, RESPECTIVELY). ALL OF THE P16INK4A-METHYLATED TUMORS WERE POSITIVE FOR HEPATITIS B VIRUS OR HEPATITIS C VIRUS MARKERS, BUT NONE OF THE VIRUS-NEGATIVE TUMORS EXHIBITED P16INK4A METHYLATION (P=0.006). THE FREQUENCY OF P16INK4A HYPERMETHYLATION TENDED TO BE HIGHER IN HEPATITIS C VIRUS-RELATED TUMORS (23 OF 32, 72%) THAN IN HEPATITIS B VIRUS-RELATED TUMORS (6 OF 13, 46%; P=0.1). ABERRANT METHYLATION OF P16INK4A WAS ALSO RELATED SIGNIFICANTLY TO INCREASING AGE, FEMALE GENDER, AND NORMAL LEVELS OF SERUM PIVKA-II (P=0.02, 0.04, AND 0.04, RESPECTIVELY). NO STATISTICALLY SIGNIFICANT DIFFERENCE IN SURVIVAL WAS OBSERVED BETWEEN PATIENTS WITH P16INK4A HYPERMETHYLATION AND THOSE WITHOUT. CONCLUSIONS: OUR OBSERVATIONS SUGGEST THAT P16INK4A HYPERMETHYLATION MAY CONTRIBUTE TO HEPATOCARCINOGENESIS FROM AN EARLY STAGE AND THAT MULTIPLE RISK FACTORS, SUCH AS VIRAL INFECTIONS, AGE, AND GENDER, MAY BE ASSOCIATED WITH P16INK4A HYPERMETHYLATION IN HEPATOCARCINOGENESIS. 2004 16 3825 40 INVESTIGATION OF CTNNB1 GENE MUTATIONS AND EXPRESSION IN HEPATOCELLULAR CARCINOMA AND CIRRHOSIS IN ASSOCIATION WITH HEPATITIS B VIRUS INFECTION. HEPATITIS B VIRUS (HBV), ALONG WITH HEPATITIS C VIRUS CHRONIC INFECTION, REPRESENTS A MAJOR RISK FACTOR FOR HEPATOCELLULAR CARCINOMA (HCC) DEVELOPMENT. HOWEVER, MOLECULAR MECHANISMS INVOLVED IN THE DEVELOPMENT OF HCC ARE NOT YET COMPLETELY UNDERSTOOD. RECENT STUDIES HAVE INDICATED THAT MUTATIONS IN CTNNB1 GENE ENCODING FOR BETA-CATENIN PROTEIN LEAD TO ABERRANT ACTIVATION OF THE WNT/ BETA-CATENIN PATHWAY. THE MUTATIONS IN TURN ACTIVATE SEVERAL DOWNSTREAM GENES, INCLUDING C-MYC, PROMOTING THE NEOPLASTIC PROCESS. THE PRESENT STUDY EVALUATED THE MUTATIONAL PROFILE OF THE CTNNB1 GENE AND EXPRESSION LEVELS OF CTNNB1 AND C-MYC GENES IN HBV-RELATED HCC, AS WELL AS IN CIRRHOTIC AND CONTROL TISSUES. MUTATIONAL ANALYSIS OF THE BETA-CATENIN GENE AND HBV GENOTYPING WERE CONDUCTED BY DIRECT SEQUENCING. EXPRESSION OF BETA-CATENIN AND C-MYC GENES WAS ASSESSED USING REAL-TIME PCR. AMONG THE HCC CASES, 18.1% SHOWED MISSENSE POINT MUTATION IN EXON 3 OF CTNNB1, MORE FREQUENTLY IN CODONS 32, 33, 38 AND 45. THE FREQUENCY OF MUTATION IN THE HOTSPOTS OF EXON 3 WAS SIGNIFICANTLY HIGHER IN NON-VIRAL HCCS (29.4%) RATHER THAN HBV-RELATED CASES (12.7%, P = 0.021). THE EXPRESSION OF BETA-CATENIN AND C-MYC GENES WAS FOUND UPREGULATED IN CIRRHOTIC TISSUES IN ASSOCIATION WITH HBV INFECTION. MUTATIONS AT BOTH PHOSPHORYLATION AND NEIGHBORING SITES WERE ASSOCIATED WITH INCREASED ACTIVITY OF THE WNT PATHWAY. THE RESULTS DEMONSTRATED THAT MUTATED BETA-CATENIN CAUSED ACTIVATION OF THE WNT PATHWAY, BUT THE RATE OF CTNNB1 GENE MUTATIONS WAS NOT RELATED TO HBV INFECTION. HBV FACTORS MAY DEREGULATE THE WNT PATHWAY BY CAUSING EPIGENETIC ALTERATIONS IN THE HBV-RELATED HCC. 2020 17 2792 28 FAT10 IS AN EPIGENETIC MARKER FOR LIVER PRENEOPLASIA IN A DRUG-PRIMED MOUSE MODEL OF TUMORIGENESIS. THERE IS CLINICAL EVIDENCE THAT CHRONIC LIVER DISEASES IN WHICH MDBS (MALLORY DENK BODIES) FORM PROGRESS TO HEPATOCELLULAR CARCINOMA. THE PRESENT STUDY PROVIDES EVIDENCE THAT LINKS MDB FORMATION INDUCED BY CHRONIC DRUG INJURY, WITH PRENEOPLASIA AND LATER TO THE FORMATION OF TUMORS, WHICH DEVELOP LONG AFTER DRUG WITHDRAWAL. EVIDENCE INDICATED THAT THIS LINK WAS DUE TO AN EPIGENETIC CELLULAR MEMORY INDUCED BY CHRONIC DRUG INGESTION. MICROARRAY ANALYSIS SHOWED THAT THE EXPRESSIONS OF MANY MARKERS OF PRENEOPLASIA (UBD, ALPHA FETOPROTEIN, KLF6 AND GLUTATHIONE-S-TRANSFERASE MU2) WERE INCREASED TOGETHER WHEN THE DRUG DDC WAS REFED. THESE CHANGES WERE SUPPRESSED BY S-ADENOSYLMETHIONINE FEEDING, INDICATING THAT THE DRUG WAS AFFECTING DNA AND HISTONES METHYLATION IN AN EPIGENETIC MANNER. THE LINK BETWEEN MDB FORMATION AND NEOPLASIA FORMATION WAS LIKELY DUE TO THE OVER EXPRESSION OF UBD (ALSO CALLED FAT10), WHICH IS UP REGULATED IN 90% OF HUMAN HEPATOCELLULAR CARCINOMAS. IMMUNOHISTOCHEMICAL STAINING OF DRUG-PRIMED MOUSE LIVERS SHOWED THAT FAT10 POSITIVE LIVER CELLS PERSISTED UP TO 4 MONTHS AFTER DRUG WITHDRAWAL AND THEY WERE STILL FOUND IN THE LIVERS OF MICE, 14 MONTHS AFTER DRUG WITHDRAWAL. THE REFEEDING OF DDC INCREASED THE PERCENT OF FAT10 HEPATOCYTES. 2008 18 4231 42 METHYLATION OF PROTOCADHERIN 10, A NOVEL TUMOR SUPPRESSOR, IS ASSOCIATED WITH POOR PROGNOSIS IN PATIENTS WITH GASTRIC CANCER. BACKGROUND & AIMS: BY USING METHYLATION-SENSITIVE REPRESENTATIONAL DIFFERENCE ANALYSIS, WE IDENTIFIED PROTOCADHERIN 10 (PCDH10), A GENE THAT ENCODES A PROTOCADHERIN AND IS SILENCED IN A TUMOR-SPECIFIC MANNER. WE ANALYZED ITS EPIGENETIC INACTIVATION, BIOLOGICAL EFFECTS, AND PROGNOSTIC SIGNIFICANCE IN GASTRIC CANCER. METHODS: METHYLATION STATUS WAS EVALUATED BY COMBINED BISULFITE RESTRICTION ANALYSIS AND BISULFITE SEQUENCING. THE EFFECTS OF PCDH10 RE-EXPRESSION WERE DETERMINED IN GROWTH, APOPTOSIS, PROLIFERATION, AND INVASION ASSAYS. PCDH10 TARGET GENES WERE IDENTIFIED BY COMPLEMENTARY DNA MICROARRAY ANALYSIS. RESULTS: PCDH10 WAS SILENCED OR DOWN-REGULATED IN 94% (16 OF 17) OF GASTRIC CANCER CELL LINES; EXPRESSION LEVELS WERE RESTORED BY EXPOSURE TO DEMETHYLATING AGENTS. RE-EXPRESSION OF PCDH10 IN MKN45 GASTRIC CANCER CELLS REDUCED COLONY FORMATION IN VITRO AND TUMOR GROWTH IN MICE; IT ALSO INHIBITED CELL PROLIFERATION (P < .01), INDUCED CELL APOPTOSIS (P < .001), AND REPRESSED CELL INVASION (P < .05), UP-REGULATING THE PRO-APOPTOSIS GENES FAS, CASPASE 8, JUN, AND CDKN1A; THE ANTIPROLIFERATION GENE FGFR; AND THE ANTI-INVASION GENE HTATIP2. PCDH10 METHYLATION WAS DETECTED IN 82% (85 OF 104) OF GASTRIC TUMORS COMPARED WITH 37% (38 OF 104) OF PAIRED NONTUMOR TISSUES (P < .0001). IN THE LATTER, PCDH10 METHYLATION WAS HIGHER IN PRECANCEROUS LESIONS (27 OF 45; 60%) THAN IN CHRONIC GASTRITIS SAMPLES (11 OF 59; 19%) (P < .0001). AFTER A MEDIAN FOLLOW-UP PERIOD OF 16.8 MONTHS, MULTIVARIATE ANALYSIS REVEALED THAT PATIENTS WITH PCDH10 METHYLATION IN ADJACENT NONTUMOR AREAS HAD A SIGNIFICANT DECREASE IN OVERALL SURVIVAL. KAPLAN-MEIER SURVIVAL CURVES SHOWED THAT PCDH10 METHYLATION WAS ASSOCIATED SIGNIFICANTLY WITH SHORTENED SURVIVAL IN STAGE I-III GASTRIC CANCER PATIENTS. CONCLUSIONS: PCDH10 IS A GASTRIC TUMOR SUPPRESSOR; ITS METHYLATION AT EARLY STAGES OF GASTRIC CARCINOGENESIS IS AN INDEPENDENT PROGNOSTIC FACTOR. 2009 19 2759 37 EXPRESSION OF IL-17 AND ITS GENE PROMOTER METHYLATION STATUS ARE ASSOCIATED WITH THE PROGRESSION OF CHRONIC HEPATITIS B VIRUS INFECTION. TO EXPLORE INTERLEUKIN-17 (IL-17) AND ITS EPIGENETIC REGULATION DURING THE PROGRESSION OF CHRONIC HEPATITIS B VIRUS (HBV) INFECTION.A TOTAL OF 162 PATIENTS WITH CHRONIC HBV INFECTION, INCLUDING 75 WITH CHRONIC HEPATITIS B (CHB), 54 WITH HEPATITIS B-ASSOCIATED LIVER CIRRHOSIS AND 33 WITH HEPATITIS B-ASSOCIATED HEPATOCELLULAR CARCINOMA (HBV-HCC), WERE ENROLLED IN THIS STUDY. THIRTY HEALTHY ADULTS OF THE SAME ETHNICITY WERE ENROLLED IN THE CONTROL GROUP. WHOLE VENOUS BLOOD WAS OBTAINED FROM THE PATIENTS AND NORMAL CONTROLS (N = 30). CLINICAL AND LABORATORY PARAMETERS WERE ASSESSED, AND WE PERFORMED ENZYME-LINKED IMMUNOSORBENT ASSAY AND QUANTITATIVE REAL-TIME PCR TO MEASURE THE SERUM LEVELS AND RELATIVE MRNA EXPRESSION OF IL-17, RESPECTIVELY. IL-17 PROMOTER METHYLATION IN PERIPHERAL BLOOD MONONUCLEAR CELLS WAS ASSESSED BY METHYLATION-SPECIFIC PCR. WE ANALYZED THE SERUM AND MRNA LEVELS OF IL-17 AND IL-17 PROMOTER METHYLATION IN THE 4 GROUPS AS WELL AS THE EFFECT OF METHYLATION ON SERUM IL-17 LEVELS. CORRELATIONS BETWEEN THE IL-17 PROMOTER METHYLATION STATUS AND CLINICAL PARAMETERS WERE ANALYZED BY SPEARMAN CORRELATION ANALYSIS.COMPARED TO THE NORMAL CONTROL GROUP, THE PATIENT GROUPS EXHIBITED SIGNIFICANTLY HIGHER SERUM AND RELATIVE MRNA LEVELS OF IL-17. THE METHYLATION DISTRIBUTION AMONG THE PATIENTS WAS SIGNIFICANTLY LOWER THAN THAT AMONG THE NORMAL CONTROLS (P < .05), WITH THE HBV-HCC GROUP SHOWING THE LOWEST IL-17 GENE METHYLATION FREQUENCY. THE AVERAGE IL-17 PROMOTER CG METHYLATION LEVEL WAS NEGATIVELY CORRELATED WITH IL-17 MRNA EXPRESSION (R = -0.39, P = .03), AND NEGATIVE CORRELATIONS BETWEEN IL-17 PROMOTER METHYLATION AND PROTHROMBIN TIME ACTIVITY (R = -0.585, P = .035), ALANINE AMINOTRANSFERASE (R = -0.522, P < .01), ASPARTATE AMINOTRANSFERASE (R = -0.315, P < .05), AND THE MODEL FOR END-STAGE LIVER DISEASE SCORE (R = -0.461, P < .05) WERE OBSERVED. IL-17 SERUM LEVELS IN THE METHYLATED-PROMOTER GROUPS WERE SIGNIFICANTLY LOWER THAN THOSE IN THE UNMETHYLATED-PROMOTER GROUPS.IL-17 EXPRESSION AND PROMOTER METHYLATION WERE ASSOCIATED WITH CHRONIC HBV INFECTION PROGRESSION, ESPECIALLY IN THE HBV-HCC GROUP. THE IL-17 PROMOTER STATUS MAY HELP CLINICIANS INITIATE THE CORRECT TREATMENT STRATEGY AT THE CHB STAGE. 2019 20 5717 38 SIRTUIN-6-DEPENDENT GENETIC AND EPIGENETIC ALTERATIONS ARE ASSOCIATED WITH POOR CLINICAL OUTCOME IN HEPATOCELLULAR CARCINOMA PATIENTS. SIRTUIN 6 (SIRT6) IS A MEMBER OF THE SIRTUIN FAMILY OF NAD+-DEPENDENT DEACETYLASES. GENETIC DELETION OF SIRT6 IN MICE RESULTS IN A SEVERE DEGENERATIVE PHENOTYPE WITH IMPAIRED LIVER FUNCTION AND PREMATURE DEATH. THE ROLE OF SIRT6 IN DEVELOPMENT AND PROGRESSION OF HEPATOCELLULAR CARCINOMA IS CURRENTLY UNKNOWN. WE FIRST INVESTIGATED SIRT6 EXPRESSION IN 153 PRIMARY HUMAN LIVER CANCERS AND IN NORMAL AND CIRRHOTIC LIVERS USING MICROARRAY ANALYSIS. SIRT6 WAS SIGNIFICANTLY DOWN-REGULATED IN BOTH CIRRHOTIC LIVERS AND CANCER. A SIRT6 KNOCKOUT (KO) GENE EXPRESSION SIGNATURE WAS GENERATED FROM PRIMARY HEPATOCTYES ISOLATED FROM 3-WEEK-OLD SIRT6-DEFICIENT ANIMALS. SIRT6-DEFICIENT HEPATOCYTES SHOWED UP-REGULATION OF ESTABLISHED HEPATOCELLULAR CARCINOMA (HCC) BIOMARKERS ALPHA-FETOPROTEIN (AFP), INSULIN-LIKE GROWTH FACTOR 2 (IGF2), H19, AND GLYPICAN-3. FURTHERMORE, DECREASED SIRT6 EXPRESSION WAS OBSERVED IN HEPATOMA CELL LINES THAT ARE KNOWN TO BE APOPTOSIS-INSENSITIVE. RE-EXPRESSION OF SIRT6 IN HEPG2 CELLS INCREASED APOPTOSIS SENSITIVITY TO CD95-STIMULATION OR CHEMOTHERAPY TREATMENT. LOSS OF SIRT6 WAS CHARACTERIZED BY ONCOGENIC CHANGES, SUCH AS GLOBAL HYPOMETHYLATION, AS WELL AS METABOLIC CHANGES, SUCH AS HYPOGLYCEMIA AND INCREASED FAT DEPOSITION. THE HEPATOCYTE-SPECIFIC SIRT6-KO SIGNATURE HAD A PROGNOSTIC IMPACT AND WAS ENRICHED IN PATIENTS WITH POORLY DIFFERENTIATED TUMORS WITH HIGH AFP LEVELS AS WELL AS RECURRENT DISEASE. FINALLY, WE DEMONSTRATED THAT THE SIRT6-KO SIGNATURE POSSESSED A PREDICTIVE VALUE FOR TUMORS OTHER THAN HCC (E.G., BREAST AND LUNG CANCER). CONCLUSION: LOSS OF SIRT6 INDUCES EPIGENETIC CHANGES THAT MAY BE RELEVANT TO CHRONIC LIVER DISEASE AND HCC DEVELOPMENT. DOWN-REGULATION OF SIRT6 AND GENES DYSREGULATED BY LOSS OF SIRT6 POSSESS ONCOGENIC EFFECTS IN HEPATOCARCINOGENESIS. OUR DATA DEMONSTRATE THAT DEFICIENCY IN ONE EPIGENETIC REGULATOR PREDISPOSES A TUMORIGENIC PHENOTYPE THAT ULTIMATELY HAS RELEVANCE FOR OUTCOME OF HCC AND OTHER CANCER PATIENTS. 2013