Integrative Array-Based Approach Identiﬁes MZB1 as a ... · DNA hypermethylation in hepatocellular carcinoma (HCC). Experimental Design: We conducted integrative array-based approach

Human Cancer Biology

Integrative Array-Based Approach Identifies MZB1 as aFrequently Methylated Putative Tumor Suppressor inHepatocellular Carcinoma

Satoshi Matsumura1,5, Issei Imoto1,7, Ken-ichi Kozaki1, Takeshi Matsui2, Tomoki Muramatsu1,3,Mayuko Furuta1,3, Shinji Tanaka5, Michiie Sakamoto6, Shigeki Arii5, and Johji Inazawa1,3,4

AbstractPurpose: The aim of this study was the identification of novel tumor suppressor genes (TSG) silenced by

DNA hypermethylation in hepatocellular carcinoma (HCC).

Experimental Design: We conducted integrative array-based approach for genome-wide screening of

methylation targets using a methylated DNA immunoprecipitation-CpG island microarray and expression

array in three universal hepatoma cell lines and normal liver tissue. Through detailed expression and

functional analyses using hepatoma cell lines and primary HCC samples, we isolated novel TSGs for HCC.

Results:A total of 642 geneswere identified asmethylated in three hepatoma cell lines but unmethylated

in normal liver tissue, whereas 204 genes on autosomes were identified as genes unexpressed but restored

after treatment with 5-aza-20-deoxycytidine in these cell lines and expressed in normal tissue. Through the

integrationof results of the two-array analyses and further validation analyses of expression andmethylation

status in 17 cell lines and 30 primary tumors of hepatoma, we identifiedMZB1,marginal zone B and B1 cell-

specific protein, encoding an endoplasmic reticulum protein, as a putative TSG frequently methylated within

its CpG island in hepatoma. Among 162 patients with primary HCC, silencing of MZB1 protein was

significantly and independently associated with a worse outcome. Restoration of MZB1 expression in

hepatoma cells reduced cell proliferation in vitro and in vivo through G1-arrest.

Conclusions: These results suggest thatmethylation-mediated silencing ofMZB1 expression leads to loss

of its tumor-suppressive activity, which may be a factor in the hepatocarcinogenesis, and is a useful

prognosticator in HCC. Clin Cancer Res; 18(13); 3541–51. �2012 AACR.

IntroductionHepatocellular carcinoma (HCC), one of the most com-

mon malignancies worldwide, is associated with hepatitisvirus infections, dietary aflatoxin, chronic alcohol/tobaccoconsumption, and cirrhosis. Genomic alterations, such asthe gain or loss of chromosomal regions and specific genemutations, have been frequently noted in hepatocarcino-

genesis (1). Furthermore, epigenetic abnormalities, such asDNA methylation and chromosome remodeling, may alsopromote tumorigenesis (2, 3). DNA hypermethylation ofpromoter CpG islands leads to the inactivation of tumorsuppressor genes (TSG) and critical cancer-related genes inhuman cancers including HCC (4, 5). DNA methylationchanges have been reported to be specific to the canceroustissuemaking it possible to distinguish HCC and surround-ing nontumorous tissues (6). Indeed, abnormalDNAmeth-ylation of several TSGs, such as RASSF1A, CDKN2A,CRABP1, GSTP1, CHRNA3, DOK1, SFRP1, GAAD45a, andCDKN2B, is reported to be associated with HCC, andhypermethylation of specific genes, such as CHFR and SYKis detected in advanced stages of HCC (6). However, thenumber of reportedmethylation-target genes is far fewer forHCC than for colon cancer or gastric cancer (7). Therefore,further identification of remaining targets for methylationmay clarify the specific molecular events involved in HCCprogression, enabling the prevention, diagnosis, and treat-ment of HCC to be approached as potential clinical appli-cations of DNA methylation signature at a molecular level.

To discover novel methylation-target sequences withhigh specificity and sensitivity in a genome-wide manner,large-scale screening methods, which have the potential to

Authors' Affiliations: 1Department of Molecular Cytogenetics, 2MedicalTop Track Program, Medical Research Institute and School of BiomedicalScience, 3Global Center of Excellence Program for International ResearchCenter for Molecular Science in Tooth and Bone Diseases, 4Department ofGenome Medicine, Hard Tissue Genome Research Center, and 5Depart-ment of Hepato-Biliary-Pancreatic Surgery, Graduate School of Medicine,TokyoMedical and Dental University; 6Department of Pathology, School ofMedicine, Keio University, Tokyo; and 7Department of Human Genetics,Institute of Health Biosciences, The University of Tokushima GraduateSchool, Kuramoto-cho, Tokushima, Japan

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Johji Inazawa, Department of Molecular Cytoge-netics,Medical Research Institute, TokyoMedical andDental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. Phone: 81-3-5803-5820; Fax: 81-3-5803-0244; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-11-1007

�2012 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 3541

on December 2, 2020. © 2012 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 9, 2012; DOI: 10.1158/1078-0432.CCR-11-1007

http://clincancerres.aacrjournals.org/

find novel methylation targets in a whole range of cancers,have been developed. A combination of 3 types of pretreat-ments, such as enzyme digestion, affinity enrichment, orsodium bisulfite, was followed by different analytical steps,such as gel-, array-, or next-generation sequencing-basedanalysis (8). Among them, methylated-DNA immunopre-cipitation (MeDIP; ref. 9) in combination with an oligo-nucleotide array or next-generation sequencing allows forrapid and efficient genome-wide assessment of DNAmeth-ylation, although these methodologies generally result in alist of several hundred candidate genes. Although an anal-ysis of possible promoters or dense CpG islands is used tonarrow down the number of candidate genes, the list is stilltoo long. Pharmacologic unmasking expressionmicroarrayapproaches are also used widely to identify methylationtargets (10), although they are also prone to give false-positive genes that are indirect methylation targets them-selves and not considered to be a reliable gauge of DNAmethylation at a given locus.

To identify genes that are downregulated because of DNAhypermethylation and to concentrate those genes mostfrequently involved in HCC, we applied the followingintegrative array-based approach to 3 hepatoma cell lines:(i) MeDIP in combination with CpG island-array (MeDIP-chip) analysis to identify genes methylated in a cancer-specificmanner, (ii) expressionmicroarray analysis to iden-tify genes downregulated in a cancer-specific mannerand reactivated upon treatment with 5-aza-20deoxycytidine(5-aza-dCyd), and (iii) a combination of data from bothapproaches. Through further examination of a subset ofobtained candidates, we identified marginal zone B and B1cell-specific protein (MZB1), also known as proapoptotic

caspase adaptor protein (PACAP), pERp1, or MGC29506(11),whose inactivation is relatedwith aworse prognosis inprimary tumors as a possible TSG for HCC.

Materials and MethodsCell lines and primary tumor samples

A total of 17 hepatoma cell lines including 15 HCC lines(cHc4, Hep3B, Hep-Kano, Hep-TABATA, HLE, HLF, huH-1,HuH-7, JHH-1, JHH-4, JHH-5, JHH-7, Li-7, PLC/PRF/5, andSK-HEP-1) and 2 hepatoblastoma lines (HepG2 and HUH-6) were used (12). All 162 primary HCC samples wereobtained during surgery from patients with HCCs treatedat Tokyo Medical and Dental University (Tokyo, Japan)between 2000 and 2005. Relevant clinical and survival datawere available for all patients (Table 1). Themedian follow-up period for the surviving patients was 19months (rangingfrom 1 to 103 months). Samples from 17 of these patientswith HCC were immediately frozen in liquid nitrogen andstored at �80�C until required for DNA and RNA analyses.Normal liver tissues were obtained from surgical samples in2 patients (cases C20 and C40) with colon cancer metas-tasis. Written consent was obtained after approval by thelocal ethics committee.

MeDIP-chip analysisThe DNA methylation profiles were analyzed by MeDIP

using anti-5-methylcytidine antibody (Eurogentec) fol-lowed by the Human 244K CpG island microarray (Agi-lent),which contains 237,220probes, covering27,800CpGislands (97.5%ofUCSC annotatedCpG islands), accordingto themanufacturer’s instructions. Image analysis with dataextraction was done using Feature Extraction Software,version 9.0 (Agilent Technologies). The analytic procedureused for the results of theMeDIP-chip assaywasdescribed inthe Supplementary Experimental Procedure.1

Gene expression arrayGene expression was profiled using a 4 � 44K Human

Whole Genome Ver. 2.0 gene expression array (AgilentTechnologies) according to themanufacturer’s instructions.Total RNAs extracted from hepatoma cell lines and fromthese cells treated with 10 mmol/L 5-aza-dCyd for 5 dayswere used for conventional and pharmacologic unmaskinganalyses, respectively. Two normal livers (C20 and C40)were used as controls. All samples were analyzed twice.Image analysis with data extraction and the data analysiswere conducted using Feature Extraction Software, version9.0 and GeneSpring GX10 software (Agilent Technologies),respectively.1

Real-time reverse transcription PCRTo analyze the restored expression of genes, the cell lines

were cultured with 1 to 10 mmol/L 5-aza-dCyd for 5 days.Levels of mRNA expression were measured with ABI PRISM

Translational RelevanceAlthough hepatocellular carcinoma (HCC) is one of

the most common malignancies worldwide, the molec-ular mechanisms underlying hepatocarcinogenesisremain unclear. Epigenetic abnormalities, such as DNAmethylation, may promote tumorigenesis as well asgenomic alterations in hepatocarcinogenesis. To discov-er novel methylation-target sequences with high speci-ficity and sensitivity in a genome-wide manner, weconducted expression array analyses as well as methyl-ated-DNA immunoprecipitation in combination withan oligonucleotide array, which allows for rapid andefficient genome-wide assessment of DNA methylation,resulting in the identification of marginal zone B and B1cell-specific protein (MZB1) as a tumor suppressor genesilenced by DNA hypermethylation in hepatoma.Among 162 patients with primary HCC, silencing ofMZB1 protein was significantly and independently asso-ciated with a worse outcome. Moreover, restoration ofMZB1 expression in hepatoma cells reduced cell prolif-eration in vitro and in vivo throughG1 arrest.Our findingsprovide a novel insight into the prevention, diagnosis,and treatment of HCC.

1The entire microarray data set is available at http://www.ncbi.nlm.nih.gov/geo/info/linking.html under the data series accession no. GSE35313.

Matsumura et al.

Clin Cancer Res; 18(13) July 1, 2012 Clinical Cancer Research3542




7500 sequence detection System (Applied Biosystems)using TaqMan Gene Expression Assays (Hs01048042_m1for ANGPT2, Hs00174937_m1 for CCK, Hs00405322_m1for DERL3, Hs00417143_m1 for RADIL, Hs00191390_m1

forKCNK6, Hs00219458_m1 for L1TD1, Hs00414907_m1for MZB1, Hs00386153_m1 for FAR1, Hs00382235_m1for OCIAD2, Hs00257935_m1 for PBX4, andHs00610060_m1 for SFRP1; Applied Biosystems)

Table 1. Association between clinicopathologic characteristics and MZB1 expression

MZB1 immunoreactivity

n Negative (%) Positive (%) P a

Total 162 84 (51.9) 78 (48.1)GenderMale 122 66 (54.1) 56 (45.9) 0.4139Female 40 18 (45.0) 22 (55.0)

Age, yMean 65.7 65.7>65 98 49 (50.0) 49 (50.0) 0.5594<65 64 35 (54.7) 29 (45.3)

VirusHCV(þ) 79 39 (49.4) 40 (50.6) 0.2742HBV(þ) 38 25 (65.8) 13 (34.2)HCV(�), HBV(�) 45 20 (44.4) 25 (55.6)

AFP, ng/mLb 5,390 � 22,968 2,819 � 13,373 0.3982PIVKA-II, mAU/mLb 7,252 � 43,097 6,767 � 25,669 0.9325Aspartate aminotransferase, IU/Lb 48.6 � 25.3 54.7 � 34.2 0.1982Alanine aminotransferase, IU/Lb 42.8 � 32.6 53.7 � 34.8 0.0418Platelet (%)b 84.1 � 13.1 85.9 � 13.6 0.3862Total bilirubin, mg/dLb 0.86 � 0.44 0.86 � 0.40 0.9795Albumin, g/dLb 3.9 � 0.5 3.9 � 0.4 0.6972Child–Pugh scoreb 5.4 � 0.6 5.3 � 0.7 0.8199Tumor size, cmb 4.6 � 2.8 4.5 � 3.1 0.8411Tumor numberSingle 105 50 (47.6) 55 (52.4) 0.1940Multiple 57 34 (59.6) 23 (40.4)

Histopathologic gradingWell-differentiated 43 24 (55.8) 19 (44.2) 0.4560Moderately differentiated 90 43 (47.8) 47 (52.2)Poorly differentiated 29 17 (58.6) 12 (41.4)

Portal vein invasionAbsent 92 40 (43.5) 52 (56.5) 0.0222Present 70 44 (62.9) 26 (37.1)

Surgical marginAbsent 139 73 (52.5) 66 (47.5) 0.8478Present 23 11 (47.8) 12 (52.2)

Background liver parenchymaNormal liver 10 2 (20.0) 8 (80.0) 0.0696Chronic hepatitis 78 39 (50.0) 39 (50.0)Liver cirrhosis 74 43 (58.1) 31 (41.9)

Tumor stageI 13 3 (23.1) 10 (76.9) 0.0207II 57 25 (43.9) 32 (56.1)III 58 33 (56.9) 25 (43.1)IVA 34 23 (67.6) 11 (32.4)

NOTE: Statistically significant values are in boldface type.Abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus.aP values are from c2, Fisher exact, or Student t test and were statistically significant at <0.05.bMean � SD.

MZB1 Methylation in Hepatocellular Carcinoma

www.aacrjournals.org Clin Cancer Res; 18(13) July 1, 2012 3543




according to the manufacturer’s instructions. Gene expres-sion values are given as ratios between the genes of interestand an internal reference gene (Hs99999903_m1 forACTB;Applied Biosystems) that provides an internal normaliza-tion factor, and subsequently normalized with the value inthe controls (relative expression level). The assay was con-ducted twice for each sample.

Methylation analysisSodium bisulfite-treated genomic DNA was subjected

to PCR using primer sets to amplify regions of interest(Supplementary Table S1). For the combined bisulfiterestriction analysis (COBRA), PCR products were digestedwith BstU1, Taq1, or Hha1 and electrophoresed (13). Theintensity of methylated alleles as a percentage on theethidium bromide-stained gels was calculated, and amethylation density cutoff point of 20% was consideredsignificant as described elsewhere (14). For bisulfite geno-mic sequencing (BGS), PCR products were subcloned andsequenced.

ImmunohistochemistryIndirect immunohistochemistry was conducted with for-

malin-fixed, paraffin-embedded tissue sections using anautomated immunostainer (Benchmark XT; Ventana Med-ical Systems) with heat-induced epitope retrieval, anti-MZB1 (1:50; 11454-1-AP; Protein Tech), anti-PCNA(1:1,000; #2586; Cell Signaling Technology), or anti-Ki-67 antibodies (1:100; M7240; Dako). The slides werecounterstained with Mayer’s hematoxylin, and analyzedunder a light microscope by 2 pathologists blinded toclinical characteristics and outcomes. Twenty representativefields per slide were examined, and the percentage of thetotal cell population that expressedMZB1was evaluated foreach case at �200 magnification. Expression of MZB1protein was graded as either positive (�10% of tumor cellsshowing immunopositivity) or negative (�10% of tumorcells showing immunopositivity or no staining). Plasmacells and bile duct epithelial cells were used as positive andnegative controls, respectively.

Western blottingWestern blotting was conducted as described elsewhere

(12). Anti-FLAG-tag and anti-b-actin antibodies were pur-chased from Sigma-Aldrich, and the anti-cleaved caspase-3antibody (#9661) was purchased from Cell SignalingTechnology.

Fluorescent immunocytochemistryThe plasmid expressing C-terminally FLAG-tagged

MZB1 (pCMV-3Tag3A-MZB1) was obtained by cloningthe full coding sequence of MZB1 in-frame along withthe 3xFLAG-epitope into the pCMV-3Tag3A vector (Stra-tagene). Cells were fixed in 10% trichloroacetic acid,permeabilized with 0.2% Triton X-100, and treated withblocking solution (1% bovine serum albumin in PBS).After incubation with the primary antibodies (anti-MZB1,1:100 and/or anti-Calnexin, 1:100) for 1 hour, the bound

antibody was visualized using a Cy3-conjugated orfluorescein isothiocyanate (FITC)-conjugated secondaryantibody (1:1,000). After being mounted with DAPI(40,60-diamidino-2-phenylindole) to stain nuclei, thecells were observed under a fluorescence microscope(BZ-8100; Keyence).

In vitro and in vivo growth assayColony formation assays using cells transiently intro-

duced with pCMV-3Tag3A-MZB1 or the empty vector(pCMV-3Tag3A-mock) was conducted as described else-where (15). The expression ofMZB1 protein was confirmed48 hours after transfection by Western blotting and fluo-rescent immunocytochemistry.

Stable MZB1 transfectants and control counterpartswere obtained by introducing pCMV-3Tag3A-MZB1 orpCMV-3Tag3A-mock into cells with G418 selection, and2.5 � 104 cells were seeded in 24-well plates. The num-bers of viable cells were assessed 24 to 72 hours afterseeding by the water-soluble tetrazolium salt assay. Thecell cycle was evaluated 48 hours after seeding by afluorescence-activated cell sorting (FACS) as describedelsewhere (15).

The in vivo tumor-suppressive ability of MZB1 was inves-tigated by conducting tumor xenograft experiments. Six-week-old female severe combined immunodeficient(SCID) mice were injected subcutaneously in the lowerback with MZB1-expressing or control mock-transfectedcells (4 � 107). All procedures involving animals wereapproved by and conformed to the guidelines of our Insti-tutional Animal Care and Use Committee. Tumor forma-tion in SCID mice was monitored daily and the recipientmice were sacrificed for tumor weight evaluation and pro-tein expression analyses 5 weeks postinjection.

Statistical analysisThe c2 or Fisher’s exact test was used to test for differences

between groups. Kaplan–Meier method and log-rank testwere used for the survival analyses. Univariate and multi-variate survival analyses were conducted using the likeli-hood ratio test of the stratified Cox proportional-hazardsmodel. Differences between subgroups were tested with theStudent t test. For multiple group comparisons, ANOVAfollowed by Scheff�e post-hoc test was used. Differences wereassessedwith a2-sided test, and considered significant at theP < 0.05 level.

ResultsScreening in the MeDIP-chip analysis

For the screening of aberrantly methylated genes byMeDIP-chip analysis, we used a CpG island microarray, inwhich 11,229 genes harbor CpG islands upstream orwithin, with the algorithm shown in the SupplementaryExperimental Procedure and Supplementary Fig. S1.Among 11,229 genes, CpG islands of 2,476 genes wereunmethylated in normal liver tissue (case C20) but meth-ylated in at least 1 of 3 hepatoma cell lines (Hep3B,

Matsumura et al.





HepG2, and HuH-7). Because CpG islands of 642 of those2,476 genes were methylated in all 3 cell lines(Fig. 1A), we selected them as candidates consistentlyhypermethylated in hepatomas.

Screening in the expression array analysisIn expression array analysis done in a duplicate man-

ner, we used only reproducible probes (the coefficient ofvariation, CV <50%) in each set of experiments to eval-uate obtained values. We focused on genes satisfying 2criteria: (a) genes whose expression was observed innormal liver tissue but repressed in hepatoma cells, and(b) genes whose expression was restored after treatmentwith 5-aza-dCyd in hepatoma cells. Among genesexpressed in normal liver tissue (C20 and C40), theexpression of 1,730, 1,158, and 1,521 genes was silenced

but restored by 5-aza-dCyd in Hep3B, HepG2, and HuH-7cells, respectively. Among them, 204 genes on autosomescommonly satisfied criteria in all 3 lines (Fig. 1A), indi-cating 204 genes to be candidates consistently silencedthrough methylation in hepatocarcinogenesis.

Integrationandvalidationof results in twomicroarray-based analyses

On the basis of the MeDIP-chip and expression arrayanalyses, 11 genes were selected as overlapping genes in 2different genome-wide array-based screeningmethods, sug-gesting them to be pharmacologically unmasked, tumor-specific methylation targets in all 3 hepatoma cell lines(Fig. 1A and Supplementary Table S2). Because these genesseem tobe silenced in a tumor-specificmanner, they are alsocandidates for TSG in hepatocarcinogenesis.

A

MZB1

ANGPT2

L1TD1

RADIL

CCK

SFRP1

PBX4

FAR1

DERL3

KCNK6

OCIAD2

Hu

H-7

hu

H-1

HU

H-6

JH

H-1

JH

H-4

JH

H-5

JH

H-7

PL

C/P

RF

/5

C20

SK

-HE

P-1

Li-

7

He

pG

2

Hep

3B

HL

F

cH

c4

C40

He

p-T

AB

AT

A

HL

E

He

p-K

AN

O

Hep

G2 +

5-a

za-d

Cyd

Hu

H-7

+ 5

-aza-d

Cyd

Hep

3B

+ 5

-aza-d

Cyd

Relative ratio(/C20)

100-10- 100

1.5- 10 0.5- 1.5 0.1- 0.5

0.01- 0.1 -

0.01

Level

M M M M M M M M M MM M M M

M

M M M

M

M

M M

M

M

M M

M MM M M M M M M

M

M

M

MM M

M

M

MM M M

M

M

MM M M

M

M

MM M M

MM M

MM M

MM M

M

MM

M

M

M

M

MM

M

M

M

M

M

M

M

M

MM

M

M

M

MM

M

MM

M

M

M

M

M

M

M

MM M MM

M

M

M M

MM

MM

MM M M M M

M

MM MMMM

M M MMM

M

Hu

H-7

hu

H-1

HU

H-6

JH

H-1

JH

H-4

JH

H-5

JH

H-7

PL

C/P

RF

/5

C2

0

SK

-HE

P-1

Li-

7

He

pG

2

He

p3

B

HL

F

cH

c4

C4

0

Hep

-TA

BA

TA

HL

E

He

p-K

AN

O

ANGPT2

L1TD1

1

2

3

4

5

6

7

8

1

2

1

2

3

1

2

1

2

3

4

1

2

1

2

3

4

1

2

3

4

1

2

OCIAD2

SFRP1

CCK

KCNK6

MZB1

PBX4

FAR1

RADIL

DERL3

%methylatedallele

10099-2019- 1

0

Level

B

1,730, 1,158, and 1,521 genes restoredin Hep3B, HepG2, and HuH-7, respectively (>4-fold)

26,242 probes commonly expressed in 2 normal liver

24,373, 23,165, and 24,413 probes decreased in Hep3B, HepG2, and HuH-7, respectively(vs. normal liver, >2-fold)

Total 41,057 probes on expression array

MeDIP on CpG island array Expression array

7,945 genes unmethylated in normal liver

642 genes commonly methylated in all 3 lines,but unmethylated in normal liver

2,476 genes methylated in at least 1 cell line,but unmethylated in normal liver

11,229 CpG island containing genes on array

•1 normal liver (case C20)•3 hepatoma cell lines (Hep3B, HepG2, HuH-7)

Total 25,412 genes on CpG island array

Normal liver

Hepatoma cell lines

Normal liver

204 autosomal genescommonly downregurated in hepatoma and restored after 5-aza-dcyd treatment

5-aza-dCyd treatment

•2 normal liver (cases C20 and C40)•3 hepatoma cell lines (Hep3B, HepG2, HuH-7)

+ 5-aza-dCyd

Overlapping

Hepatoma cell lines

Overlapping

11 overlapped genes among 2 analyses

CpG island

Figure 1. Identification of 11 genes as possible methylation targets. A, left top, overview of the screening approach using MeDIP-chip analyses in 3 hepatomacell lines (Hep3B, HepG2, and HuH-7) and control normal liver tissue (case C20); right top, overview of the screening approach using duplicate expressionarray analyses in the same 3 hepatoma cell lines without and with 5-aza-dCyd treatment and 2 normal liver tissues (cases C20 and C40); bottom, bycombining results obtained from 2 different genome-wide array-based screening methods, 11 genes overlapped between 2 approaches as possible genes,which are consistently silenced by tumor-specific CpG island methylation. B, top, summary of DNA methylation status of CpG islands around11 selected candidates in 17 hepatoma cell lines and normal liver tissue determined by COBRA. Each box indicates restricted status by enzymes(% methylated allele). A methylation density cutoff point of 20% was considered significant (13). Arrowheads indicate regions in which more than 50%(>9 of 17) of cell lines showed tumor-specific hypermethylation comparedwith normal liver tissue; bottom, profiles of expression of 11 candidates determinedby real-time RT-PCR in 17 hepatoma cell lines. Three (Hep3B, HepG2, and HuH-7) of 17 lines were treated with 5-aza-dCyd. Ratio relative to normalliver tissue (C20) is shown by a 7-gradient pattern. "M's" indicates that genes showed hypermethylation (see C, top) in each cell line: nonunderlined andunderlined "M's" indicate silenced (<0.01 compared with C20) and retained gene expression, respectively.






We thendetermined the status ofmethylation and expres-sion for all 11 candidates through COBRA and real-timereverse transcription PCR (RT-PCR), respectively, in a panelof 17 hepatoma cell lines and 2 normal liver tissues (Fig.1B). Among them, ANGPT2was highly methylated in boththehepatoma cells andnormal liver tissue. Among the other10 genes, 8 genes (L1TD1, MZB1, PBX4, FAR1, RADIL,DERL3, SFRP1, and CCK) harbored at least 1 hypermethy-lated region in >50% of hepatoma cell lines compared withnormal liver tissue, whereas OCIAD2 and KCNK6 wereinfrequently methylated in the hepatoma lines. Amongthose 8 genes, only MZB1 and FAR1 were downregulated(<0.1 relative to normal liver) in all cell lines with theirhypermethylation, whereas the other 6 genes wereexpressed even in cell lines with their hypermethylation,suggesting MZB1 and FAR1 to be possible methylationtargets for gene silencing in hepatoma cells, although theexpression of all 11 genes was more or less restored by 1 to

10mmol/L of 5-aza-dCyd treatment in eachof the 3 cell lines(Supplementary Fig. S2).

MZB1 is frequently silenced through CpG islandmethylation in primary tumors

We next determined the methylation and expressionstatus of MZB1 and FAR1 in 17 paired tumorous andnontumorous tissues from primary HCCs (cases L81-L104; Fig. 2A and Supplementary Fig. S3). Tumor-specificMZB1 (region 8) and FAR1 (region 3) hypermethylationwas observed in 9 (52.9%) and 8 (47.1%) cases, respec-tively. In those cases with hypermethylation, tumor-specificdownregulation of MZB1 and FAR1 expression wasobserved in 9/9 (100%) and 5/8 (62.5%) cases, respective-ly, suggestingMZB1 to be themost probable candidate for agene silenced through tumor-specific methylation. In addi-tional 13 cases (Fig. 2A, cases L07-L79), tumor-specificMZB1 hypermethylation was observed in 6 (46.2%) cases

B

MZB1 methylation (+) expression (-)

79L48L

MZB1 methylation (-) expression (+)

L82 L104

A

Methylation 15/30 (50.0%)

Region 8

Silencing 22/30 (73.3%)

BstU1

Re

lati

ve

mR

NA

ex

pre

ss

ion

(/p

air

ed

N)

MZB 1

Tumor-specific hypermethylation (T > 20%, N < 20%)

>50% decreased expression in tumor tissue

N T

L81

N T

L82

N T

L83

N T

L84

N T

L85

N T

L86

N T

L87

N T

L88

N T

L89

N T

L92

N T

L97

N T

L98

N T

L99

N T

L100

N T

L101

N T

L102

N T

L104

- +- + - +- + - +- +- +- + - +- +- +- +- +- + - +- + - +- +- +- +- + - + - +- +- +- + - +- + - +- +- +- + - +- + DW

Primary HCCN T

L07

N T

L09

N T

L25

N T

L31

N T

L34

N T

L50

N T

L57

N T

L62

N T

L67

N T

L71

N T

L74

N T

L78

N T

L79

- +- + - + - + - +- +- +- + - +- +- +- +- +- + - +- + - +- +- +- +- +- +- +- +- +- +

0

1

2

MZB1 (5q31.2)

Tel. Cen.200 bp

CpG island

Bisulfite PCR 1 3 5 7

Exon1

CpG site

Hep3B

HepG2

HuH-7

C20

C40

L87N

L87T

(+)

(-)

(-)

(-)

(-)

(+)

(+)

Expression

(↓BstU1 site) Methylation (%)

87.5

33.1

24.1

93.5

66.6

26.7

30.4

33.7

M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M

M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M MM M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M MM M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M MM M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M MM M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M MM M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M

M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M

M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M y y M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M y M M M M y M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M y M M M M y M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M y M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M y M y M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M

4 6 82

100

73.5

34.8

95.8

87.9

67.4

52.7

31.8

Total Region 8

C

M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M

M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M

M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M

M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M

HepG2+5-aza-dCyd (+)

M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M

Hep3B+5-aza-dCyd (+)

HuH-7+5-aza-dCyd (+)

53.0 61.4

20.5 45.8

M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M \\ M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M

M M M M M M M M M M M M M M M M

Figure 2. Correlation of methylation and expression status of MZB1 in primary HCCs. A, representative results of the methylation status of region 8 within theMZB1 CpG island determined by COBRA (top) and the relative level of MZB1 mRNA expression determined by real-time RT-PCR (bottom) inprimary HCC tumors (T) and corresponding noncancerous liver tissues (N). Arrowheads, fragments specifically restricted at sites recognized as methylatedCpGs; arrow, undigested fragments indicating unmethylated CpGs. Amethylation density of cutoff point of 20%was considered significant (14). Closed andopen circles indicate samples with tumor-specific methylation (positive in tumor but negative in corresponding nontumorous tissue) and those with thereduced mRNA expression (>50% decreased expression) in tumor tissue compared with paired nontumorous tissue, respectively. B, representativeresults of immunohistochemical analysis ofMZB1 protein expression in primary HCCs indicate thatmethylation status ofMZB1 is inversely correlatedwith itsmRNA and protein expression. Bars, 20 mm. Magnifications are�200. C, top, a schematic map of the CpG-rich region around the CpG island and exon 1 ofMZB1. Vertical ticks, CpG sites on the expanded axis. Gray box, CpG island. Horizontal closed arrows, the regions examined in the COBRA and BGS.Downward arrows, restriction sites (BstU1) for the COBRA. Bottom, representative results of BGS of theMZB1 CpG island in 3 hepatoma cell lines withoutand with 5-aza-dCyd, 2 normal livers (C20 and C40), and a paired representative case (cases L87) examined in the COBRA. Open and filled squaresrepresent unmethylated andmethylatedCpG sites, respectively, and each row represents a single clone. Restriction sites are indicated by downward arrows.

Matsumura et al.





and all of those cases also showed tumor-specific down-regulation of this gene. Among 12 cases available forimmunohistochemical staining of MZB1 protein, MZB1was expressed in nontumorous tissues and the methylationstatus and protein expression status of MZB1 matched in 8tumorous tissues (Fig. 2B).To clarify the methylation status of the CpG island of

MZB1, we conducted BGS in cell lines and tumorous andnontumorous tissues of HCC as well as normal liver tissue.CpG sites within the CpG island tended to be differentiallymethylated among the cell lines and primary samples:MZB1-nonexpressing cell lines except HuH-7 and primarytumors showed a highlymethylated patternwithin the CpGisland, whereas MZB1-expressing normal liver tissue andnontumorous tissue of HCC case showed partially meth-ylated (Fig. 2C). Although total %methylation of CpG siteswithin the CpG island of HuH-7 cells is higher than butclose to those in MZB1-expressiong samples, dense meth-ylation without unmethylated allele was observed in spe-cific regions within CpG island in HuH-7 cells. In addition,we confirmed that treatment with 5-aza-dCyd partiallyrestored methylation within the CpG island of MZB1 inHepG2, Hep3B, and HuH-7 cells, whose expression ofMZB1 was restored after the same treatment (Fig. 1B),suggesting that methylation within the CpG islandupstream toMZB1 occurs in cell lines and primary tumorsof hepatoma and at least partly contributes to the silenc-ing of its expression of the mRNA and protein level.Notably, 5-aza-dCyd-treated hepatoma cells and C20showed similar methylation level of the MZB1 CpGisland (Fig. 2C), whereas 5-aza-dCyd-treated hepatomacells showed much higher MZB1 expression level com-

pared with C20 (Fig. 1B), suggesting that 5-aza-dCydtreatment indirectly activates transcription of MZB1through demethylation of transcription factors/cofactorsfor MZB1, which might downregulated in normal hepa-tocytes or other mechanisms.

Immunohistochemical staining of MZB1 in primaryHCCs

To determine clinicopathologic significance of theMZB1downregulation in primary HCCs, we conducted animmunohistochemical analysis of theMZB1 in162primarycases (Fig. 3 and Table 1). In the tumorous regions, 78(48.1%) showed immunoreactivity to MZB1 (positivein Table 1), whereas 84 (51.9%) did not (negativein Table 1). In the nontumorous regions, on the otherhand, 135 (83.3%) showed immunoreactivity to MZB1,whereas 27 (16.7%) did not. Negative MZB1 immunore-activity was more frequent in cases with portal invasion(P ¼ 0.0222) and in higher tumor stages (P ¼ 0.0207).However, the MZB1 protein expression in each tumor wasnot associated with other characteristics.

In Kaplan–Meier survival curves, univariate analyses ofoverall and nonrecurrent survival with log-rank testsshowed a significant association between negative MZB1immunoreactivity and a poor survival rate of patients(P ¼ 0.0031 and 0.0044, respectively; Fig. 3B). In theCox proportional hazard regression model (Table 2),univariate analyses showed that negative MZB1 immu-noreactivity, a-fetoprotein (AFP), tumor size, tumornumber, portal vein invasion, background liver paren-chyma, and tumor stage were significantly associated withoverall survival. Multivariate analysis using a stepwise

A

B

Overa

ll s

urv

ival

pro

bab

ilit

y

0

0.2

0.4

0.6

0.8

1.0

0 10 20 30 40 50 60 70 80

MZB1-negative (n = 84)

MZB1-positive (n = 78)

Log-rank test, P = 0.0031

Time after operation (mo)

Recu

rren

ce-f

ree s

urv

iva

l p

rob

ab

ilit

y

0

0.2

0.4

0.6

0.8

.1.0

0 10 20 30 40 50 60 70

MZB1-negative (n = 84)

MZB1-positive (n = 78)

Log-rank test, P = 0.0044

Time after operation (mo)

0807302#1707903#2083297#15

Nontumor

Tumor

MZB1 (-) MZB1 (+)Normal liver

Figure 3. A, representative results of immunohistochemical staining of MZB1 protein in normal liver tissue, nontumorous liver tissue, and tumorous tissuesin HCCs. Both normal and nontumorous hepatocytes showed MZB1 immunopositivity, whereas tumor cells showed either negative or positiveimmunoreactivity. Bars, 25 mm. Magnifications are �200. B, Kaplan–Meier curves for overall survival (left) and recurrence-free survival (right) rates of 162patients with primary HCCs. Negative MZB1 immunoreactivity of tumor cells was significantly associated with worse overall and recurrence-free survivals(P ¼ 0.0031 and 0.0044, respectively; log-rank test).






Cox regression procedure revealed that MZB1 immuno-reactivity, tumor size, and tumor stage were independent-ly selected as predictive factors for overall survival in bothforward and backward procedures (P ¼ 0.0234, 0.0319,and 0.0301, respectively).

MZB1 reexpression suppresses proliferation andtumor formation of cancer cells in vitro and in vivo

To investigate the biologic significance of MZB1 inhepatocarcinogenesis, MZB1 expression was transientlyor stably restored in hepatoma cells lacking MZB1 expres-sion. We then measured the proliferation and tumorformation of those cells in comparison with the controlcounterparts transduced with an empty vector in vitro andin vivo.

In colony-formation assays using transiently trans-fected cells, the occupancy of the stained area of coloniesproduced by MZB1-transfected HLE cells (Fig. 4A), whichshow MZB1 hypermethylation pattern (SupplementaryFig. S4), and other hepatoma cells (data not shown)decreased compared with those of control counterparts.In an in vitro proliferation assay using stably transfected

cells, cells expressing MZB1 protein, which was predom-inantly colocalized with an endoplasmic reticulum (ER)marker in the cytoplasm, grew slightly but significantlyslower than the control cells (Fig. 4B). In a FACS analysisto examine the mode of action of MZB1 in the cell cycle,an accumulation of cells in G0–G1 phase and a decrease inS and G2–M phase cells but no increase in sub-G1 phasecells was observed among MZB1-transfected cells com-pared with mock-transfected counterparts (Fig. 4C), sug-gesting that MZB1 contributes to the arrest of hepatomacells at the G1-S checkpoint without inducing apoptosis.Indeed, a similar expression pattern of cleaved caspase 3,one of markers of apoptosis, was observed between stableMZB1 transfectants and control counterparts even aftertreatment with CDDP for induction of apoptosis (Fig.4C). Subcutaneous tumor growth experiments using sta-ble transfectants showed that restored expression ofMZB1 in hepatoma cells correlated with reduced tumorvolume and weight in vivo probably because of a decreasein cell proliferation shown by lower PCNA and Ki-67positivities in MZB1-transfected cells (Fig. 4D). Inresected tumors, no induction of apoptosis detected by

Table 2. Cox proportional hazard regression analysis for overall survival

UnivariateMultivariatea

Factor HR (95% CI) Pa Pb

GenderMale vs. female 0.990 (0.509–1.925) 0.9764 X

Age, y>65 vs. <65 0.957 (0.527–1.738) 0.8859 X

AFP>200 vs. <200 ng/mL 2.202 (1.214–3.995) 0.0094 X

Tumor size>3 vs. <3 cm 3.548 (1.498–8.401) 0.0040 0.0319

Tumor numberMultiple vs. single 2.531 (1.406–4.554) 0.0019 X

Histopathologic gradingPoor-moderate vs. well 2.312 (1.074–4.975) 0.0321 X

Portal vein invasionPresent vs. absent 2.309 (1.136–3.716) 0.0173 X

Surgical marginPresent vs. absent 1.948 (0.963–3.940) 0.0637 X

Background liver parenchymaLC vs. CH þ NL 1.825 (1.009–3.300) 0.0468 X

StageIII þ IVA vs. I þ II 3.466 (1.710–7.024) 0.0006 0.0301

MZB1 expressionc

Negative vs. positive 2.532 (1.338–4.791) 0.0043 0.0234

NOTE: Statistically significant values are in boldface type.Abbreviations: CH, chronic hepatitis; LC, liver cirrhosis; NL, normal liver.aForward and backward stepwise analyses were used for multivariate analysis.bP values are from 2-sided tests and were statistically significant at <0.05.cMZB1 expression was evaluated by immunohistochemical analysis as described in Materials and Methods.

Matsumura et al.





cleaved caspase 3 expression in MZB1-transfetced cellswas observed compared with mock-transfected counter-parts in vivo (Fig. 4D).

DiscussionEpigenetic silencing of TSGs plays an important role

in the carcinogenesis (16), including hepatomagenesis(5–7). Although many studies have reported aberranthypermethylation of genes in HCC, e.g. CDH1, RASSF1A,

GSTP, SOCS1, SFRP1, and PTEN identified as TSGssilenced by hypermethylation, most of these studieswere limited to the analysis of a single or a few genes(5–7, 17). Because the number of methylation-targetTSGs identified to date is far fewer for HCC than forother cancers possibly because of fewer attempts to con-duct genome-wide analysis (18), there remain manygenes hypermethylated in HCC. With advancements inmicroarray technology, the number of genes found to behypermethylated in HCC in a cancer-specific manner is

A

mock#2 MZB1#2

pCMV-3Tag3A

0

1

2

3

mock MZB1

#1 #2 #1 #2

pCMV-3Tag3A

a,ba,b

Tu

mo

r w

eig

ht

(g)

X20

X120

Calnexin(ER marker) FLAG-MZB1 Merged

pCMV-3Tag3A-MZB1 (HepG2)B

DC

Rela

tive g

row

th r

ati

o (

/ W

T 2

4 h

)

24 48 72 96 120

MZB1#1

MZB1#2

mock#2

mock#1

Stable pCMV-3Tag3A

a,b

a,b

a,b

a

Time after plating (h)

0

1

2

3

4

5 HepG2pCMV-3Tag3A

MZB1mock

FLAG

24

17

ACTB

pCMV-3Tag3A

MZ

B1

mo

ck

KDHLE

Occu

pan

cy o

fsta

ined

are

a (

%)

0

10

20

30

40

50

mock MZB1

pCMV-3Tag3A

∗∗

cCASP3

FLAG

MZB1

ACTB

NT

CD

DP

NT

CD

DP

mock#2

pCMV-3Tag3A

MZB1#2

FLAG

MZB1

ACTB

cCASP3

mo

ck#2

pCMV-3Tag3AM

ZB

1#2

mock#2

pCMV-3Tag3A

sub-G1: 0.68G0–G1: 54.44

S: 21.69G2–M: 23.44

sub-G1: 0.69G0–G1: 60.74

S: 16.45G2–M: 21.97

040

80

120

160

200

Co

un

ts

0 200 400 600 800 1000FL2-A

050

100

150

200

250

Co

un

ts

0 200 400 600 800 1000FL2-A

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Figure 4. Tumor-suppressive effects of restoration of MZB1 expression in hepatoma cells in vitro and in vivo. A, HLE cells lacking MZB1 expression weretransiently transfected with pCMV-3Tag3A-MZB1 or pCMV-3Tag3A-mock and selected with G418 for 2 weeks. MZB1 expression was confirmed byimmunoblotting using 10 mg of protein extract and anti-MZB1 antibody (left). The drug-resistant colonies formed by the MZB1-transfected cells were morenumerous than those formed by control counterparts (top right). Occupancy of the stained colony area was calculated (bottom right). Columns,means of 3 separate experiments, each conducted in triplicate; bars, SD (histogram). �, P < 0.05 versus mock control (Student t test). Similar results wereobtained in the Hep3B and SK-HEP-1 cell lines (data not shown). B, stable HepG2 transfectants were established by transfection of pCMV-3Tag3A-MZB1 orpCMV-3Tag3A-mockwith G418 selection. Immunofluorescence cytochemistry showed almost all pCMV-3Tag3A-MZB1–transfected cells to express FLAG-tagged MZB1 predominantly with an endoplasmic reticulum (ER) marker (calnexin, left). After plating into 24-well plates (2.5 � 104 cells per well), theproliferative activity of stablyMZB1-transfected cells was found to be less than that of the control counterparts. Results of relative growth ratio are shownwiththemean�SD for 3 separate experiments, each done in triplicate (right). Differenceswere analyzed by 1-way ANOVAwith subsequent Scheff�e tests: a and b,P < 0.05 versus mock #1 and #2, respectively. C, top left, representative results of the population in each phase of the cell cycle in stable transfectantsdescribed in (B) assessed by FACS. Bottom left, columns, means of 3 separate clones, each conducted in triplicate; bars, SD (histogram). �, P < 0.05versus mock control (Student t test). Right, representative results of immunoblotting of MZB1 protein and cleaved caspase-3 (cCASP3), one of apoptoticmarkers, without (no treatment, NT) or with cisplatinum (CDDP, 20 mmol/L) treatment for 48 hours. D, representative results of tumors formed inSCIDmice following injection of stablyMZB1-transfected cells.MZB1-transfectedHepG2 cells ormock-transfected cells (4� 107) were injected into the rightand left dorsal flanks. SCIDmicewere sacrificed for tumor weight evaluation 5weeks postinjection (top left). Results of tumor weight are shownwith themean� SD for 3 separate experiments, each done in triplicate (top right). Differences were analyzed as described in (B). Bottom left, representativeresults of immunohistochemical analysis of MZB1 as well as PCNA and Ki-67, markers for cell proliferation, in resected tumors. A positive MZB1immunoreactivity was detected inMZB1-transfetced cells but not inmock-transfected counterparts, and both PCNA and Ki-67 immunoreactivities were lessfrequently observed in MZB1-transfetced cells compared with mock-transfected counterparts. Bars, 25 mm. Magnifications are �200. Bottom right,representative results of immunoblotting of MZB1 protein and cCASP3 in resected tumors.






expected to increase (8, 19). Although the functionalconsequence of promoter hypermethylation is transcrip-tional silencing of the associated gene, this assumptionoften goes untested, as few have concurrently investigatedboth methylation and expression (6, 7). On the basis ofthese hypotheses and background, we conductedgenome-wide screening of methylation-target TSGsusing a combination of 2 microarray-based approaches:MeDIP-chip analysis in hepatoma cell lines and expres-sion array analysis for genes pharmacologically un-masked in the same lines. As a result of this approach,several genes were newly identified as candidate methyl-ation targets, and among them MZB1 was showed to bethe most possible TSG, which is silenced through meth-ylation and contributes to the hepatocarcinogenesis.

Among 11 genes we identified through genome-globalscreening of methylation-mediated silenced genes in hep-atoma cells, 10 were newly identified candidates and onlySFRP1 was known as possible methylation-target TSG inHCC (20, 21). The remarkable reduction in the number ofcandidate genes and lack of various known HCC-relatedmethylation targets within candidates may be because of asmall number of commonly methylated and/or silencedgenes among the 3 cell lines used in this study. Indeed,several frequently methylated genes, such as SLIT2, PTGS2(COX2), and HHIP, for which methylation data are avail-able in all 3 hepatoma cell lines, showed different methyl-ation patterns among the cell lines (22–24). These varia-tionsmay come from the different backgrounds of the 3 celllines, such as hepatitis B virus infection in Hep3B and nohepatitis virus infection in the other 2 lines, because hyper-methylated genes in HCC tumors are known to exhibitremarkably distinct patterns depending on associated riskfactors (25, 26). Therefore, it is suggested that the candidategenes identified and validated in the present study maycontribute to functional pathways shared among differentsubtypes of HCC regardless of associated risk factors, andthis might be the reason why MZB1 protein expressionstatus was not statistically associated with the status ofhepatitis virus infection and background liver parenchymain our analysis.

One of striking findings of our immunohistochemicalanalysis ofMZB1using a panel of primary tumor samples ofHCC was that immunoreactivity to the MZB1 protein ineach sample was significantly associated with a worse clin-ical outcome even after stratification with other clinico-pathologic characteristics. This result indicates that MZB1might be useful as an independent prognosticator inpatients with HCC, although MZB1 immunoreactivity ineach case was significantly associated with portal invasionand in tumor stages. Because MZB1 seems to be down-regulated in a cancer-specific manner in HCC and itsexpression is observed in most normal human tissues(11), it will be interesting whether MZB1 works as a TSGin specific tissues including liver or in various tissues.

MZB1 was first identified as a caspase-2–binding mol-ecule through a yeast 2–hybrid system using a humanB lymphocyte cDNA library, which was conducted to

determine the mechanism of activation of caspase-2 inapoptosis of B cells triggered by ligation of the antigenreceptor (11). Although MZB1 was shown to bind cas-pase-2 and -9 in vitro and in vivo and be triggered upon thetransient transfection of human kidney cells and Rat-1fibroblasts and stable transfection of human B cell lines(11), the precise mechanisms by which it exerts proapop-totic activity remain unclear because of the absence ofstructural hallmarks besides a CXXC thioredoxin motifand no homology with other molecules in apoptoticpathways. Recently, it was shown that MZB1 occurs inthe luminal ER and affects multiple cellular processes,such as (a) the oxidative folding and assembly andsecretion of immunoglobulin in plasma cells (27, 28)and (b) the regulation of Ca2þ homeostasis and ER Ca2þ

stores, integrin-mediated adhesion, and antibody secre-tion in marginal zone B cells of the spleen and innate-likeB cells (B1 cells, ref. 29). Because those processes areassociated with the functional differentiation of B cells, itis possible that the MZB1-induced assembly of severaltarget proteins including integrin may contribute to theantiproliferative effect of MZB1 on hepatoma cells with-out induction of apoptosis observed in this study. Theexpression of this gene is observed not only in the B-celllineage including plasma cells, marginal zone B cells, orB1 cells (27–29), but also in most normal human tissuesexcept the placenta constitutively even in the absence ofan apoptotic stimulus (11), suggesting MZB1 to affectvarious biologic processes in different tissues possiblythrough interaction with various proteins and/or thetargeting of various molecules. Indeed, MZB1 proteinwas reported to be downregulated in intestinal-type gas-tric cancer, although the clinicopathologic and biologicsignificance was not analyzed (30), suggesting MZB1 toact as a TSG at least in some tissues including stomachand liver tissue. Further examination will be required toclarify the mechanisms of the antiproliferative effect ofMZB1.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: S. Matsumura, I. Imoto, J. InazawaDevelopment of methodology: S. Matsumura, I. Imoto, T. Matsui,J. InazawaAcquisition of data: S. Matsumura, M. Furuta, S. TanakaAnalysis and interpretation of data: S. Matsumura, I. Imoto, K. Kozaki,J. InazawaWriting, review, and/or revision of the manuscript: S. Matsumura,I. Imoto, J. InazawaAdministrative, technical, or material support: I. Imoto, T. Muramatsu,M. Sakamoto, J. InazawaStudy supervision: I. Imoto, T. Matsui, S. Arii, J. Inazawa

AcknowledgmentsThe authors thank Ayako Takahashi, Rumi Mori, and Ayumi Shioya for

technical assistance.

Grant SupportThis study was supported in part by Grant-in-Aid for Scientific Research

(A) and (C), and Scientific Research on Priority Areas and Innovative Areas,

Matsumura et al.





and a Global Center of Excellence (GCOE) Program for InternationalResearch Center for Molecular Science in Tooth and Bone Diseases fromtheMinistry of Education, Culture, Sports, Science, and Technology, Japan; aHealth andLabour SciencesResearchGrant by theMinistry ofHealth, Labourand Welfare, Japan; a grant from the New Energy and Industrial TechnologyDevelopment Organization (NEDO).

The costs of publication of this article were defrayed in part by the pay-ment of page charges. This article must therefore be hereby marked advertise-ment in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 28, 2011; revised April 17, 2012; accepted April 27, 2012;published OnlineFirst May 9, 2012.

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2012;18:3541-3551. Published OnlineFirst May 9, 2012.Clin Cancer Res Satoshi Matsumura, Issei Imoto, Ken-ichi Kozaki, et al. CarcinomaMethylated Putative Tumor Suppressor in Hepatocellular Integrative Array-Based Approach Identifies MZB1 as a Frequently

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