RESEARCH ARTICLE

Loss of BAP1 expression is associated withgenetic mutation and can predict outcomes ingallbladder cancer
Takashi Hirosawa ID1, Masaharu Ishida ID1*, Kentaro Ishii1, Keigo Kanehara1,
Katsuyoshi Kudo1, Shinobu Ohnuma1, Takashi Kamei1, Fuyuhiko Motoi1,
Takeshi Naitoh ID1, Florin M. Selaru2, Michiaki Unno1
1 Department of Surgery, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan,
2 Department of Medicine, Division of Gastroenterology and Hepatology, Johns Hopkins University,
Baltimore, Maryland, United States of America* mishida@surg.med.tohoku.ac.jp

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Abstract
Background

OPEN ACCESS
Citation: Hirosawa T, Ishida M, Ishii K, Kanehara K,
Kudo K, Ohnuma S, et al. (2018) Loss of BAP1
expression is associated with genetic mutation andcan predict outcomes in gallbladder cancer. PLoS
ONE 13(11): e0206643. https://doi.org/10.1371/
journal.pone.0206643

BRCA-1 associated protein (BAP1) is a de-ubiquitinating enzyme that regulates gene
expression. Recently, the BAP1 mutation and its involvement in cancer survival have beenreported in a range of tumor types, including uveal melanoma, mesothelioma, renal cancers,
and biliary tract cancers. However, the frequency of BAP1 mutation and down-regulation
varies among tumor types, and little is known about the function of BAP1 silencing in cancer
cells. Gallbladder carcinoma (GBC) is a type of biliary tract cancer with a poor prognosis.
Few mutational studies have investigated the role of BAP1 in GBC, and no functional study
in vitro-, or clinical studies about cancer survival have been done.

Editor: Aamir Ahmad, University of South Alabama
Mitchell Cancer Institute, UNITED STATES
Received: July 16, 2018
Accepted: October 16, 2018
Published: November 5, 2018
Copyright: © 2018 Hirosawa et al. This is an open
access article distributed under the terms of theCreative Commons Attribution License, which
permits unrestricted use, distribution, andreproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data arewithin the paper and its Supporting Information
files.
Funding: This work was supported in part by
KAKENHI Grant Numbers JP16K19912.
Competing interests: The authors have declaredthat no competing interests exist.

Methods
GBC cells were studied by following the small interfering RNA mediated silencing of BAP1
with regard to proliferation, migration, invasion, and drug sensitivity. We carried out genomic, epigenomic and immunohistochemical analyses to detect somatic BAP1 alterations in
47 GBC patients undergoing surgical resection.

Results
BAP1 depletion resulted in increased migration and invasion, but not proliferation, and also
resulted in decreased sensitivity to bortezomib, a proteasome inhibitor. Suppressed expression of BAP1 occurred in 22 GBC cases (46.8%) and showed a strong trend toward a worse
median survival time of 13.3 months (95% CI, 17.6–62.6) (p = 0.0034). Sanger sequencing
revealed a loss-of-function mutation of BAP1 in 11 out of these 22 GBC cases (50%) with
low BAP1 expression, whereas 2 out of 25 GBC cases (8%) were detected in cases withhigh BAP1 expression. Partial changes in methylation were observed in 6 out of 47 cases,
but methylation did not show a strong relationship to BAP1 expression or to the prognosis.

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BAP1 and gallbladder cancer

Conclusion
Our findings showed that genetic mutations are involved in BAP1 down-regulation, leading
to promotion of the invasive character of cancer cells and poor prognosis in GBC.

Introduction
BRCA-1 -associated protein (BAP1) is a de-ubiquitinating enzyme (DUB), a member of theubiquitin carboxyl-terminal hydrolase (UCH) subfamily, and is involved in cell cycle progression, gene transcription and DNA repair [1]. BAP1 was identified as a protein binding to the
BRCA1 RING finger domain and is encoded by the BAP1 gene at 3p21 [2]. No involvement of
BAP1 in breast cancer has been found [3], however, and BAP1 has not been studied in the context of cancer for some time.
Recently, various mutations of BAP1 have been found in several tumors, including uveal
melanoma (UM) [4], mesothelioma [5], renal cell carcinoma (RCC) [6, 7], and intrahepaticcholangiocarcinoma (ICC) [8, 9], but the frequency of BAP1 mutations varies widely among
different tumor types. Van de Nes JA et al. [4] detected somatic BAP1 mutations in 50.7% of
UM specimens (33 of 65), whereas Nasu M et al [5] detected mutations in 63.6% of mesotheliomas (14 of 22). Hakimi AA et al. [6] reported that BAP1 showed mutations in 11 out of 185
clear cell RCC cases (5.9%). Simbolo M et al. [9] revealed BAP1 mutations in 10 out of 70 ICC
cases (14.3%) and 1 out of 26 cases (3.8%) of gallbladder carcinoma (GBC), whereas Jiao.Y
et al. [8] revealed mutations in 8 out of 32 ICC cases (25%; discovery screen) and 1 out of 8
GBC cases (13%).
The germline BAP1 mutation is associated with an increased risk of UM [10, 11, 12, 13],
mesothelioma [11, 13, 14], cutaneous melanoma [11, 13], meningioma [12], RCC [13, 15] andMBAITs [11] (melanocytic BAP1-mutated atypical intradermal tumors) and is known as
BAP1 hereditary cancer predisposition syndrome [13].
Gallbladder carcinoma is a biliary tract cancer derived from the gallbladder mucosa, and isa malignant disease with a poor prognosis, as is ICC [16]. The incidence of GBC is high innorthern India, the Republic of Chile and Japan. The incidence in Japan is estimated at 7 per
100,000 [16]. Currently, the relationship between BAP1 mutations and the prognosis of GBC
is unknown, and no functional analysis of BAP1 in GBC cell lines has yet been reported.
DNA methylation is known as one of the epigenetic mechanisms that controls and maintains gene expression without changing the DNA base sequence [17]. Methylation of the
genome of BAP1 was analyzed in melanoma [18], malignant mesothelioma [5], and RCC [19],but almost no decrease in BAP1 expression due to methylation was found. There are also noreports that review the methylation of BAP1 in ICC or GBC.
In this study, we performed functional analysis in vitro and investigated the prognosis of
GBC according to the BAP1 expression in clinical specimens to elucidate the clinical significance of BAP1. The mechanism of the down-regulation of BAP1 was also examined in termsof the genomic mutation and DNA methylation.

Materials and methods
Cell lines
Human gallbladder cancer (GBC) cell lines, G-415 [20] and OCUG-1 [21], were used in ourstudy. G-415 was obtained from the Cell Resource Center for Biomedical Research, Tohoku

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BAP1 and gallbladder cancer

University (Sendai, Japan). OCUG-1 was purchased from JCRB Cell Bank (Osaka, Japan). The
cell lines were obtained directly from these institutions and were passaged in our laboratory
for less than 6 months after receipt. The cell lines were cultured at 37˚C in a 5% CO2 humidified incubator with RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) plus 10% fetal
bovine serum (FBS) (Biowest, Nuaillé, France) and 1% penicillin-streptomycin (Gibco by Life
Technologies, Grand Island, NY, USA).

Reverse Transcription (RT) PCR
RNA was extracted using RNeasy Mini Kit (QIAGEN, Hilden, Germany) and analyzed by
Nanodrop (Thermo Scientific, Wilmington, DE, USA). RNA (0.5 μg) was transcribed into
cDNA (complementary DNA) using PrimeScript High Fidelity RT-PCR Kit (Takara Bio,
Kyoto, Japan) or PrimeScript RT reagent Kit (Takara Bio), according to the manufacturer’s
instructions. Complementary DNA was amplified using optimal PCR conditions and the
product was confirmed by electrophoresis on a 3% agarose gel. The sequences of the primer
pairs used in this study are shown in S1 Table.

Quantitative real-time PCR
Quantitative real-time PCR was performed using a Step One Plus Real-Time PCR system(Applied Biosystems, Foster City, CA, USA) with FAST SYBR Green Master Mix (Applied
Biosystems). The relative quantification of mRNA within the samples was performed using the
2−ΔΔCt method, and the results were normalized to the expression of GAPDH (glyceraldehyde
3-phosphate dehydrogenase) as an internal control in each sample. The sequences of theprimer pairs were the same as those used in the RT PCR.

Western blotting
Antibodies used for western blotting were as follows: as primary antibodies, mouse antihuman BAP1 monoclonal antibodies (sc-28383) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and rabbit monoclonal antibodies against human E-cadherin(#3195), vimentin (#5741) and GAPDH (#2118) were obtained from Cell Signaling Technology (Danvers, MA, USA); as secondary antibodies, anti-mouse IgG HRP-conjugated secondary antibody (#7076) and anti-rabbit IgG HRP-conjugated secondary antibody (#7074) werealso purchased from Cell Signaling Technology.
To isolate total protein, cells were lysed in radio-immunoprecipitation assay (RIPA) buffer(Thermo Fisher Scientific, Waltham, MA, USA), consisting of 25 mM Tris–HCl [pH 7.6], 150
mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% sodium dodecyl sulfate (SDS). Protein densitometry was performed using Pierce BCA Protein Assay Reagent A and Reagent B(Thermo Scientific, Rockford, IL, USA) and NanoDrop2000 (Thermo Scientific). The celllysate was diluted with 4x Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) and NuPAGE
sample reducing agent (Invitrogen, Carlsbad, CA, USA). Equal amounts of protein sample
were applied on polyacrylamide gels (Mini-PROTEAN TGX Precast Gels) (Bio-Rad), separated by electrophoresis and electroblotted onto polyvinylidene difluoride (PVDF) membranes(Trans-Blot Turbo Transfer Pack) (Bio-Rad). After blocking with SuperBlock blocking buffer
in tris buffered saline (TBS) (Thermo Scientific) and washing with TBS containing Tween 20(TBS-T), the membranes were incubated overnight with primary antibodies at 4˚C and thenincubated with IgG HRP-conjugated secondary antibodies for 60 minutes at room temperature. Signals were detected using the Clarity Western ECL Substrate (Bio-Rad). Protein bands
were visualized using an ImageQuant LAS 4000 mini system (GE Healthcare, Buckinghamshire, England, UK).

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BAP1 and gallbladder cancer

siRNA transfection
BAP1 was knocked-down using specific small interfering RNA (siRNA) oligonucleotides(ON-TARGET plus Human BAP1 siRNA; Dharmacon, Lafayette, CO, USA). ON-TARGET
plus Control Pool Non-Targeting Pool (Dharmacon) were used as the negative control. For
transfection, the cationic lipid-mediated transfection method, using Lipofectamine RNAiMAX
Reagent (Invitrogen), was adopted according to the manufacturer’s instructions. The
sequences of the siRNA used in this study are shown in S2 Table.

MTS assay
MTS assay using CellTiter 96 Aqueous One Solution Reagent (Promega, Madison, WI, USA),
containing a tetrazolium compound [(3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent(phenazine ethosulfate; PES) was adopted as a colorimetric method for determining the number of viable cells in the cell proliferation or cytotoxicity assays. The absorbance at 490nm wasrecorded using a Multiskan FC Microplate Photometer (Thermo Scientific).
For the proliferation assay, BAP1 knocked-down GBC cells were seeded on 96-well plates
and measured with the MTS assay on days 0, 1, 3, 5, and 7. The data were presented as relative
increases of the average intensity compared to the control group.
For drug sensitivity, BAP1 knocked-down cells were incubated on 96-well plates for 3 days
after administration of the drugs, gemcitabine (GEM; Wako, Osaka, Japan), fluorouracil(5-FU; Wako), cisplatin (CDDP; Wako), sodium valproate (Wako), 5-azacytidine (Wako), andbortezomib (Wako). Cell viability was measured by MTS assay and the obtained data wereanalyzed by calculating the area under the curve (AUC) of each group using GraphPad Prism(ver.7.0, GraphPad Software, San Diego, CA, USA).

Scratch assay
Migration assays were performed using a scratch assay. A “scratch” was created with a
1–200 μL pipet tip on a monolayer of confluent cells cultured on 6-well cell plates. The imagesof the scratches were captured at the beginning and at 24 hours after incubation with RPMI1640 medium not containing fetal bovine serum (FBS) during the cell migration. Digital
images of the gap closure were obtained under a microscope (BZ-9000, KEYENCE, Tokyo,
Japan). The migration area was analyzed using ImageJ software [22], and the data were presented as the relative increase in average migration area compared to the control group.

Invasion assay
Invasion assays were performed using a Corning BioCoat Matrigel Invasion Chamber (Corning, NY, USA) according to the manufacturer’s instructions. The BAP1 knocked-down GBC
cells that invaded through the pores to the lower surface of the filters after 20 hours were fixedand stained using 1% crystal violet (Wako, Osaka, Japan). The cell invasion was defined as thepercentage of cell density at 20 hours compared to that seeded at 0 hours in five selected microscope fields. The data were presented as the relative increase of the average cell density to thecontrol group.

Human samples
Human samples were obtained from 10% formalin-fixed paraffin-embedded (FFPE) specimens, which were surgically resected from patients who received surgery under a diagnosis ofgallbladder cancer (n = 47) at the Department of Surgery in Tohoku University Hospital

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BAP1 and gallbladder cancer

between January 2005 and December 2016. Tissue specimens were encoded to protect patient
confidentiality and processed under protocols approved by the Ethics Committee of Tohoku
University Graduate School of Medicine.

Immunohistochemistry
The Histofine Kit (Nichirei Biosciences, Tokyo, Japan), which uses the streptavidin–biotin
amplification method, was adopted for immunohistochemical analysis. Mouse monoclonal
antibodies targeting human BAP1 (Santa Cruz, CA, USA) were used for immunohistochemistry. The antigen–antibody complex was visualized with 3, 30 -diaminobenzidine solution consisting of 3, 30 -diaminobenzidine, Tris–HCl buffer, and H2O2. The sections were thencounterstained with hematoxylin, dehydrated in a graded series of alcohol, permeated inxylene, mounted and observed under a microscope (BZ-9000).
Immunostained sections were analyzed using a HistoFAXS image cytometer (TissueGnostics, Vienna, Austria) and the specific density of diaminobenzidine in the gallbladder cancer
cells was compared to that in hepatocytes using HistoQuest software (TissueGnostics). The
density of diaminobenzidine in the gallbladder cancer cell samples not containing liver tissue
in the specimen (N = 14) was compared to the median value of that in hepatocytes of the other33 samples containing liver tissue in the specimen.

DNA sequencing analysis
Genomic DNA was extracted from manually-collected samples of the cancer tissue portion in
the FFPE tissue specimens using the QIamp DNA mini kit (QIAGEN) according to the FFPE
protocols. DNA yield and quality were determined using Nanodrop (Thermo Scientific). For
sequencing the 17 coding exons of BAP1, the primers listed in S4 Table were designed using
Primer-BLAST (National Center for Biotechnology Information, Bethesda, MD, USA). PCR
amplification was performed in a total volume of 25μl containing 100ng DNA, 10pmol of eachprimer and 0.625 units of PrimeSTAR HS Premix (Takara Bio). DNA amplification was performed in a PCR Thermal cycler Dice (Takara Bio). The PCR was started with 10 seconds at
98˚C followed by 40 cycles of denaturation for 10 seconds at 98˚C, annealing at 60˚C for 15
seconds and extension at 72˚C for 90 seconds followed by a final extension at 72˚C for 90 seconds and cooling down for 10 minutes at 4˚C. All PCR products were purified with QIAquick
PCR Purification Kit (QIAGEN).
Capillary Sanger sequencing was conducted using an Applied Biosystems 3730xl DNA analyzer (Applied Biosystems) in Macrogen Japan Corp. (Kyoto, Japan)

Methylation analysis
Methylation-specific PCR (MSP) was adopted for methylation analysis of the CpG island of
BAP1. The UCSC Genome Browser (http://genome.ucsc.edu/cgi-bin/hgGateway) was used forsearching the CpG island of BAP1, and MethPrimer (http://www.urogene.org/methprimer/
index1.html) was utilized for the methylated-specific primer (M primer) and unmethylatedspecific primer (UM primer). Sequences of the M primer and UM primer sets are shown in S3
Table.
Bisulfite modification of 2μg of genomic tumor DNA was performed using EpiTect Fast
Bisulfite Conversion Kits (QIAGEN) following the manufacturer’s instructions. Approximately 100ng of bisulfite-modified DNA were used as a template for PCR amplification with
the M primer and UM primer. The PCR product was confirmed by electrophoresis on a 3%
agarose gel. For the control of methylated and unmethylated DNA, EpiTect PCR Control
DNA Set (QIAGEN) was used.

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Statistical analysis
Quantitative data (mean ± SEM) were analyzed using Student’s t-test, Fisher’s exact test, oneway ANOVA, two-way ANOVA, and Mann–Whitney U test. Drug sensitivity was comparedwith the AUC of each group. Overall survival was calculated by Kaplan–Meier analysis andcompared by log-rank tests. The Cox proportional hazards model was used to examine prognostic factors. Differences were considered significant when p < 0.05.

Results
BAP1 expression in GBC cell lines
BAP1 expression in the GBC cell lines G-415 and OCUG-1 was evaluated by RT-PCR and western blotting. In RT-PCR, the band corresponding to BAP1 was recognized in G-415 but hardly
confirmed in OCUG-1 (S1 Fig). This indicated the expression of BAP1 mRNA in G-415 and thedown-regulation in OCUG-1. Western blotting showed the same result as RT-PCR. The band of
BAP1 was observed strongly in G-415 but hardly recognized in OCUG-1 (S2 Fig). From theabove results, BAP1 is considered to be expressed strongly in G-415 but scarcely at all in OCUG1. The BAP1 positive GBC cell line G-415 was used for further analysis using siRNA for BAP1.

Down-regulation of BAP1 in GBC cell line by siRNA
The GBC cell line G-415 was transfected with siRNA against BAP1, and western blotting using
anti-BAP1 antibody was performed (S3A Fig). The expression of BAP1 was measured as theaverage luminance of the target band by ImageJ and compared with the control (luminance:
113.8). Suppression of BAP1 expression was observed in siRNA1 (luminance: 51.06), siRNA2(luminance: 56.73), and siRNA3 (luminance: 67.87) (S3B Fig). siRNA1 and siRNA2, whichhave strong inhibitory effects on expression, were used for functional analysis of the proliferation, migration, and invasion.

Proliferation was not affected by BAP1 down-regulation
To investigate the change in cell proliferation by the down-regulation of BAP1, MTS assay was
performed on BAP1 knocked-down G-415 cells. The proliferative ability on day 1, day 3, day
5, and day 7 was 1, 2.00 ± 0.02, 6.22 ± 3.29, and 10.68 ± 8.50 fold, respectively, in the control
group; 1, 2.05 ± 0.15, 6.03 ± 2.76, 9.90 ± 6.50 fold, respectively, in the siRNA1 group; 1,
1.99 ± 0.02, 6.24 ± 3.96, and 11.8 ± 10.65 fold, respectively, in the siRNA 2 group (control vs
siRNA1; p = 0.99, control vs siRNA2; p = 0.99) (Fig 1). From the above results, it was considered that suppression of the BAP1 expression did not significantly affect the proliferation ability in the GBC cell line.

Migration was promoted by BAP1 down-regulation
To investigate the migratory ability of the BAP1 down-regulated cells, scratch assay was performed on BAP1 knocked-down G-415 cells (Fig 2A). Compared to the control group, thescratch area significantly decreased by 2.14 ± 0.25 fold (p < 0.05) in the siRNA1 group and2.27 ± 0.32 fold (p < 0.05) in the siRNA2 group (Fig 2B). The suppression of BAP1 expression
was thought to enhance cell migration.

Invasion was promoted by BAP1 down-regulation
To investigate the invasive ability, invasion assay using a Matrigel invasion chamber was performed on BAP1 knocked-down G-415 cells (Fig 3A). Compared to the control group, thePLOS ONE | https://doi.org/10.1371/journal.pone.0206643 November 5, 20186 / 19

BAP1 and gallbladder cancer

Fig 1. Proliferation assay in BAP1 knockdown GBC cell line. BAP1 expression of the GBC cell line G-415 was downregulated by siRNA and the proliferative ability was measured by MTS assay. No significant difference was observed between
each siRNA group and the control. The vertical axis shows the relative intensity compared to the intensity at day 1, and thehorizontal axis shows the number of cell culture days.
https://doi.org/10.1371/journal.pone.0206643.g001

number of cells on the lower surface of the chamber increased to 2.16 ± 0.36 fold (p < 0.05) inthe siRNA1, and 1.85 ± 0.36 fold in the siRNA2 (p = 0.08) (Fig 3B). Since proliferation was notaffected by BAP1 down-regulation, the difference in the number of cells was mainly due to thecells that had passed through the chamber, which means the invasive ability was enhanced bysuppressing the expression of BAP1.

EMT was not involved in the elevated migration and invasion of BAP1
down-regulation
We suspected that the main mechanism of the enhanced ability of migration and invasion in
BAP1 knocked-down GBC cells was epithelial mesenchymal transition (EMT). EMT was evaluated by examining the expression of E-cadherin as an epithelial marker and vimentin as amesenchymal marker in western blotting. Compared to the control group, no difference wasobserved in the expression of E-cadherin or vimentin in BAP1 knocked-down G-415 cells(S4 Fig).

Sensitivity to bortezomib was attenuated by BAP1 down-regulation
The MTS assay was performed on BAP1 knocked-down G-415 cells to investigate whether thesensitivity to drugs was affected by the suppression of BAP1. No significant change wasobserved between BAP1 knocked-down cells and the control under the anticancer drugs for
the GBCs: gemcitabine (control vs siRNA1; p = 0.76, control vs siRNA2; p = 0.12, Fig 4A), cisplatin (CDDP) (control vs siRNA1; p = 0.92, control vs siRNA2; p = 0.82, Fig 4B), and fluorouracil (5-FU) (control vs siRNA1; p = 0.68, control vs siRNA2; p = 0.80, Fig 4C). Sodium
valproate, a histone deacetylase inhibitor [23], (control vs siRNA1; p = 0.35, control vs
siRNA2; p = 0.56, Fig 4D) and 5-azacytidine, a DNA methyltransferase inhibitor [24], (control

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Fig 2. Migration assay in BAP1 knockdown GBC cell line. (a) A diagram immediately after scratching in the confluently cultured cells (day 1) and the narrowed
gap by migrating cells (day 2) are shown. (b) The relative increase of the migration area compared to the control group after 24 hours was significantly higher inthe BAP1 knocked-down cells.
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vs siRNA1; p = 0.26, control vs siRNA2; p = 0.71, Fig 4E) also had no significant effect on thesensitivity. A significant decrease in susceptibility to bortezomib, a proteosome inhibitor, wasobserved in BAP1 knocked-down cells compared with the control group (control vs siRNA1;
p < 0.001, control vs siRNA2; p < 0.001, Fig 4F).

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Fig 3. Invasion assay in BAP1 knockdown GBC cell line. (a) The GBC cell line G-415 was transfected with siRNA against BAP1 and was seededon a Matrigel invasion chamber and the infiltrated cells on the lower surface of the chamber were stained. (b) Compared to the control group, thenumber of infiltrated cells tended to increase in both siRNA1 and siRNA2, and the number of infiltrated cells was significantly higher in thesiRNA1.
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Low expression of BAP1 was associated with poor survival in GBC
Immunohistochemistry of BAP1 in 47 cases of GBC revealed 25 cases with high BAP1 expression and 22 cases with low BAP1 expression (Table 1). The average age was 68 (46–86) years in

Fig 4. Drug sensitivity test in BAP1 knockdown GBC cell line. Cell viability of BAP1 knocked-down GBC cell line G-415 under the agents of gemcitabine(GEM), cisplatin (CDDP), fluorouracil (5-FU), sodium valproate, 5-azacytidine, and bortezomib was measured by MTS assay. No change in sensitivity wasobserved in GEM, CDDP, 5-FU, sodium valproate or 5-azacytidine. Bortezomib showed a significant decrease in sensitivity in the siRNA1 and siRNA2 groupcompared to the control.
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BAP1 and gallbladder cancer

Table 1. Clinical characteristics of the 47 cases of GBC patients.
High expression

Low expression

25

22

68.0 (46–86)

69.1 (46–83)

0.71

12: 13

9: 13

0.77

Stage (0 : I : II : III : IV)

2 : 4 : 3 : 11 : 5

1 : 2 : 3 : 6 : 10

0.41

R0 surgery

72% (18/25)

50% (11/22)

0.14

Number of cases
Age (range)
Sex (M 21:F 26)

P valuehttps://doi.org/10.1371/journal.pone.0206643.t001

the high BAP1 expression group (BAP1-H), 69.1 (46–83) years in the low BAP1 expression
group (BAP1-L), and there was no significant difference (p = 0.71). There were 12 men and 13
women in the BAP1-H group, and 9 men and 13 women in the BAP1-L group, with no significant difference (p = 0.77). The stage classification of gallbladder cancer according to the UICC
system [25] was 2:4:3:11:5 (stage 0:I:II:III:IV) in the BAP1-H group and 1:2:3:6:10 (stage 0:I:II:
III:IV) in the BAP1-L group. The proportion of stage IV GBC was higher in the BAP1-L groupcompared to the BAP1-H group, but no significant difference was observed (p = 0.41). The R0(no residual tumor) resection rate was 72% in the BAP1-H group and 50% in the BAP1-L
group, with no significant difference (p = 0.14).
A Kaplan–Meier survival curve was generated from the data of 41 patients, excluding inhospital deaths and deaths from other diseases, and the prognosis was significantly poorer
for the BAP1-L group (p < 0.01, Fig 5) (50% survival time was 13.3 months in the BAP1-L
group, and it could not be calculated in the BAP1-H group because it was 50% or more). The
survival analysis for 28 cases of advanced stage III and IV showed a similar result of significantly poorer prognosis in the BAP1-L group (p < 0.01, S5 Fig). (The 50% survival time was
11.7 months for the BAP1-L group and 85.8 months for the BAP1-H group). Multivariate analysis was performed with the primary tumor site (T), the regional lymph node involvement(N), the presence or otherwise of distant metastatic spread (M), the extent of residual diseaseFig 5. Kaplan–Meier survival analysis of BAP1 expression. Kaplan–Meier survival curves for 41 GBC cases except for inhospital deaths and deaths from other diseases show a significantly poorer prognosis in the BAP1 low expression group.
https://doi.org/10.1371/journal.pone.0206643.g005

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BAP1 and gallbladder cancer(R), differentiation, infiltration, histological type, and BAP1 expression by the Cox proportional hazards model; N (p < 0.001), M (p < 0.01), R (p < 0.001), and BAP1 expression(P < 0.05) were detected as significant prognostic factors.

DNA sequencing detected a number of genomic mutations in BAP1 in GBC
The results of the sequencing of 17 exons of BAP1 of the GBC cell lines and clinical specimens
are shown in Table 2. Neither G-415 nor OCUG-1 showed genetic mutations of BAP1. In theclinical specimens, 13 of the 47 patients had genetic nonsynonymous mutations of BAP1, consisting of 2 out of 25 cases in the BAP1-H group and 11 out of 22 cases in the BAP1-L group.
Representative examples of BAP1 mutation and BAP1 expression are shown in Fig 6 and S6
Fig. Mutations are described according to the Human Genome Variation Society guidelines(http://www.hgvs.org/mutnomen/).
The mutations found in the BAP1-H group were: ID2, c.705C>A (p.Pro235 = ), andc.2166C>A (p.Arg722 = ); ID3, c.651C>T (p.Ala217 = ); ID6, c.747G>A (p.Lys249 = ); ID10,
c.591G>A (p.Gly197 = ); ID16, c.652A>T (p.Thr218Ser), and c.1219G>T (p.Asp407Tyr);
ID38, c.1012C>T (p.Pro338Ser), c.1020C>T (p.Gly340Gly), and c.1024A>T (p.Ser342Cys);
ID45, c.249C>T (p.Ala83 = ). Seven mutations in five cases (ID2, ID3, ID6, ID10, and ID45)
in the BAP1-H group showed synonymous substitutions in which the encoded amino acid was
not altered.
On the other hand, the mutations in the BAP1-L group were: ID11, c.163G>T (p.Glu55Ter), and c.747G>A (p. Lys249 = ); ID12, c.128T>A (p.Val43Glu), c.197T>A (p.Val66Glu),and c.587G>A (p.Trp196Ter); ID14, c.616G>A (p.Ala206Thr), c.697_698delinsTA (p.Val233Ter), and c.1729G>T (p.Glu577Ter); ID17, c.232A>T (p.Asn78Tyr), c.243C>A (p.Phe81Leu),
c.985C>T (p.Pro329Ser), c.1021A>T (p.His341Leu) and c.1903C>A (p. Leu 635Met); ID18,
c.996C>T (p.Pro332 = ); ID19, c.1027C>A (p.Leu343Ile), and c.1048C>A (p.Pro350Thr);
ID24, c.733C>T (p.Leu245 = ) and c.1036G>T (p.Val346Phe); ID28, c.746_747delinsTA (p.
Lys249Ile); ID32, c.4_5insC c.9_10GG>TA (p.Asn2ThrfsTer3) and c.1831G>T (p.Glu611Ter);
ID35, c.131A>T (p.Tyr44Phe), and c.1002A>G (p.Leu334 = ); ID40, c.721T>A (p.Tyr241Asn),and c.746_747delinsTA (p.Lys249Ile); ID46, c.965A>T (p.Gln322Leu). Nonsynonymous substitutions and/or frame shifts were found in eleven cases, with one case showing a synonymous
substitution. The genetic mutations affecting the protein expression of BAP1 were significantly
higher in the BAP1-L group (11 cases, 50%) than in the BAP1-H group (2 cases, 8%) (p =0.003). Synonymous substitution mutations found in three cases of the BAP1-H group and twocases of the BAP1-L group are reported in polymorphic databases (dbSNP http://www.ncbi.
nlm.nih In .gov / projects / SNP /); these were thought to be single-nucleotide polymorphisms(SNPs).
In addition, in three cases of the BAP1-L group (ID 18, ID 27, ID 46), homozygous deletion
was suspected due to the lack of PCR products in the consecutive exons (Table 2).
The summary of mutations and homozygous deletion in the 47 cases of GBC patients areshown in Table 3.

Partial methylation of CpG island of BAP1 was detected in GBC
To investigate whether methylation is involved in the suppression of BAP1, DNA methylation
analysis of the CpG island was performed in GBC cell lines and 47 GBC specimens (Table 2).
In the clinical specimens of GBC, 41 of 47 cases did not show bands for the M product (S7A
Fig) and the other six showed weak bands for the M product (ID4, ID6, ID25, ID39, ID40,
ID43) (S7B Fig), suggesting that partial methylation had occurred. Among six cases, two cases(ID4, ID6) were in the BAP1-H group and four cases (ID25, ID39, ID40, ID43) were in the

PLOS ONE | https://doi.org/10.1371/journal.pone.0206643 November 5, 201812 / 19

BAP1 and gallbladder cancer

Table 2. Summary of clinical characteristics, BAP1 expression, mutation, homozygous deletion and methylation in the GBC patients.
ID

Age sex(year)

St. BAP1 Expression BAP1 Mutation Affected Exon

ID1

60

F

3

High

No

ID2

65

M

1

High

Yes

Description of Mutation

Homozygous Deletion Methylation(Suspected)
UM

Exon9

c.705C>A (p.Pro235 = )Exon17c.2166C>A (p.Arg722 = )Exon8c.651C>T (p.Ala217 = )

UM

ID3

73

F

3

High

Yes

ID4

71

M

3

High

No

PM

ID5

70

M

3

High

No

UM

ID6

72

M

4

High

Yes

ID7

76

F

3

Low

No

UM

ID8

72

F

3

High

No

UM

ID9

72

F

3

High

No

ID10

66

M

3

High

Yes

Exon8

c.591G>A (p.Gly197 = )

UM

ID11

66

F

4

Low

Yes

Exon4

c.163G>T (p.Glu55Ter)

UM

Exon9

c.747G>A (p. Lys249 = )Exon4

c.128T>A (p.Val43Glu)
c.197T>A(p.Val66Glu)

Exon8c.587G>A (p.Trp196Ter)

Exon8c.616G>A (p.Ala206Thr)

Exon9c.697_698delinsTA(p.Val233Ter)

Exon14c.1729G>T (p.Glu577Ter)

Exon8c.652A>T (p.Thr218Ser)

Exon12c.1219G>T (p.Asp407Tyr)

Exon4c.232A>T (p.Asn78Tyr)c.243C>A (p.Phe81Leu)

Exon11c.985C>T (p.Pro329Ser)
c.1021A>T (p.His341Leu)

Exon15c.1903C>A (p. Leu 635Met)

ID12

74

F

3

Low

Yes

ID13

66

M

4

Low

No

ID14

62

F

3

Low

Yes

ID15

70

M

4

High

No

ID16

66

F

4

High

Yes

ID17

80

M

4

Low

Yes

Exon9

UM

c.747G>A (p.Lys249 = )

PM

UM

UM

UM
UM

UM
UM
UM

ID18

72

M

4

Low

Yes

Exon11c.996C>T (p.Pro332 = )

ID19

56

M

4

Low

Yes

Exon11c.1027C>A (p.Leu343Ile)c.1048C>A (p.Pro350Thr)

Exon13,14,15

UM

ID20

67

M

1

High

No

UM

ID21

75

M

4

Low

No

UM

ID22

54

F

4

High

No

UM

ID23

83

F

2

Low

No

ID24

76

F

3

Low

Yes

UM

UM
Exon9

c.733C>T(p.Leu245 = )Exon11c.1036G>T (p.Val346Phe)

UM

ID25

78

F

1

Low

No

PM

ID26

70

F

1

High

No

UM

ID27

66

F

3

Low

No

ID28

76

F

2

Low

Yes

ID29

70

F

4

Low

No

UM

ID30

77

F

3

High

No

UM

ID31

86

M

1

High

No

ID32

73

M

4

Low

Yes

ID33

78

M

2

High

Exon1,2,3,4,5
Exon9c.746_747delinsTA (p.Lys249Ile)

UM
UM

UM
Exon1

c.4_5insC c.9_10GG>TA (p.Asn2ThrfsTer3)

Exon14c.1831G>T (p.Glu611Ter)

No

UM
UM
(Continued )PLOS ONE | https://doi.org/10.1371/journal.pone.0206643 November 5, 201813 / 19

BAP1 and gallbladder cancer

Table 2. (Continued)
ID

Age sex(year)

St. BAP1 Expression BAP1 Mutation Affected Exon

Description of Mutation

Homozygous Deletion Methylation(Suspected)

ID34

53

F

3

High

No

ID35

47

M

2

Low

Yes

UM

ID36

74

F

0

High

No

ID37

70

F

3

High

No

ID38

49

M

0

High

Yes

ID39

70

M

4

Low

No

ID40

82

F

0

Low

Yes

ID41

68

M

2

High

No

UM

ID42

71

M

2

High

No

UM

ID43

46

F

3

Low

No

PM

ID44

61

F

1

Low

No

UM

ID45

82

F

3

High

Yes

Exon4

c.249C>T (p.Ala83 = )

ID46

66

M

4

Low

Yes

Exon11c.965A>T (p.Gln322Leu)

ID47

46

F

4

High

No

Exon4

c.131A>T (p.Tyr44Phe)

Exon11c.1002A>G (p.Leu334 = )

UM
UM
UM

Exon9

c.1012C>T(p.Pro338Ser)
c.1020C>T(p.Gly340Gly)c.1024A>T (p.Ser342Cys)

Exon9c.721T>A (p.Tyr241Asn)
c.746_747delinsTA (p.Lys249Ile)

UM

PM
PM

UM
Exon13,14,15,16,17

UM
UMSt., Stage; UM, Unmethylated; PM, Partially methylated. Synonymous substitution is highlighted in yellow, and non-synonymous substitution is in orange.
https://doi.org/10.1371/journal.pone.0206643.t002

BAP1-L group. There was no significant relationship between methylation in the GBC specimens and BAP1 expression (p = 0.40) or prognosis. (P = 0.71).
OCUG-1 showed strong bands, not only for the unmethylation-specific product (UM product), but also for the methylation-specific product (M product), while little M product was
detected in G-415 (S7C Fig). To investigate whether methylation is involved in the regulationof BAP1 expression in OCUG-1, BAP1 expression was measured by quantitative real-time
PCR with demethylation by 5-azacytidine (5-AZA). The expression level of BAP1 in OCUG-1
after demethylation was 1.83 ± 0.35 fold higher than that in the control (S8 Fig). The suppressed expression of BAP1 in OCUG-1 was therefore thought to be due to methylation.

Discussion
The functional role of BAP1 down-regulation in cancer cells remains controversial. In a studyof UM cell lines, the suppression of BAP1 by siRNA did not affect proliferation, migration orinvasion [26]. Meanwhile, the suppression of BAP1 by siRNA in bile duct cancer cell lines
enhanced the proliferative capacity [27]. In our study by knocking-down the expression of
BAP1 by siRNA, the cell proliferation ability did not change, while the migratory and invasive
abilities were enhanced, though we use one kind of GBC cell line. This result supports our clinical outcomes of more advanced stages of GBC and the poor prognosis for patients who showlow BAP1 expression.
In order to investigate the mechanism of the enhanced ability of migration and invasion by
BAP1 down-regulation, we analyzed EMT in BAP1 knocked-down cells, but failed to prove
the positive involvement of EMT in which epithelial cells lost cell polarity and cell adhesion
function with the surrounding cells and acquired mesenchymal properties [28]. As for themechanism of invasion and migration, Onken et al. have reported that the down-regulation of
BAP1 increases the transmigration of cancer cells, but does not affect intercalation in uveal

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BAP1 and gallbladder cancer

Fig 6. BAP1 expression and detected mutations in clinical GBC (ID 12). (a) Since the BAP1 staining concentration was low in the cancer part (Ca40.4 < Liver 70.3), ID12 was categorized to the BAP1 low expression group. (b) The nonsense mutation on exon 8 (c.587G>A (p.Trp96Ter)) and the missense
mutation on exon 4 (c.128T>A (p.Val43Glu), c.197T>A (p.Val66Glu)) were observed.
https://doi.org/10.1371/journal.pone.0206643.g006

melanoma UM cells [29]. Further in vitro studies are needed to elucidate the functional role of
BAP1 down-regulation in cancer cells.

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BAP1 and gallbladder cancer

Table 3. Summary of mutations and homozygous deletion in the 47 cases of GBC patients.
High expression(25 cases)

Low expression(22 cases)

P value

Synonymous substitution (variant)

5 (20%)

1 (4.5%)0.19

Nonsynonymous substitution

2 (8%)

11 (50%)0.003

03 (13.6%)

0.09

2 (8%)

13 (59%)0.0003Homozygous deletion (HD)Nonsynonymous substitution
and/or HD
https://doi.org/10.1371/journal.pone.0206643.t003

Although BAP1 down-regulation did not affect the drug sensitivity of antitumor agentsused to treat GBC, we showed that BAP1 down-regulation attenuated the sensitivity to bortezomib, which is clinically applied to multiple myeloma and an inhibitor of proteasome that
regulates the accumulation of the abnormal protein and lethal stress in cells [30]. The mechanism involved in the acquisition of tolerance to bortezomib has not been clarified, but the
involvement of mutations of proteasome and chaperones in the endoplasmic reticulum has
been reported in multiple myeloma [31]. Although bortezomib did not show a prognostic
improvement in a phase II clinical trials for advanced cholangiocarcinoma [32], there is areport of the antitumor effects of bortezomib [33]. By selecting patients according to BAP1
expression, bortezomib could be a candidate for chemotherapy to treat GBC.
The frequency of mutation affecting protein expression in GBC detected in our study (8%in the BAP1-H group, 50% in the BAP1-L group, 27.6% in total) is higher than in previous
reports (13% [8], 3.8% [9]). The detected mutations tended to be more common in the firsthalf of BAP1, which contains domains such as UCH domain and BRCA 1 associated RING
domain 1 (BARD 1) binding domain [1]. UCH is involved in the de-ubiquitin activity of BAP1[2], and BAP1 interacts with BARD1 to inhibit the E3 ligase activity of BRCA1-BARD1 complex. BAP1 and BRCA1-BARD1 complex coordinately regulate ubiquitination during the
DNA damage response and the cell cycle [34, 35]. Accordingly, it is speculated that mutations
involved particularly in these regions greatly affect the decrease in the functional expression of
BAP1.
FFPE samples show frequent sequence changes due to DNA damage resulting from formalin fixation and storage, most commonly manifesting as a cytosine to thymine transition
caused by the deamination of cytosine (artifactual C>T and G>A transitions) [36]. We did
not use this method to remove artificial transitions, but even if we exclude this type of mutation, the frequency of detected mutations of GBC in our study is still higher than that reported
in the past. Our study showed significant involvement of BAP1 in GBC.
In addition to the genetic mutations found by DNA sequencing, homozygous deletion was
suspected in three cases (ID 18, ID 27, ID 46) in which PCR products were not detected in consecutive exons. In BAP1 low expression mesothelioma, homozygous deletion was recognizedin 76% of samples with fluorescence in situ hybridization (FISH) [37], so there might be a considerable number of deletions that we could not detect by DNA sequencing.
As for methylation, it might suppress the expression of BAP1 in OCUG-1 but, in clinicalsamples, methylation was occasional and weak and did not correlate with the BAP1 expression
or prognosis of GBC. From the above, it seems that DNA methyltransferase inhibitors are lesslikely to be useful for BAP1, as reported in other cancers [5, 18, 19].
Since the point mutation in the exon region is mainly studied in our study of BAP1 expression, the mutation in the intron region and other epigenetic mechanism such as histone acetylation and post-translational modification were not taken into consideration. Although we
could not demonstrate that the cause of low BAP1 expression was comprehensively analyzed,

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BAP1 and gallbladder cancer

our study has revealed that genomic mutations of BAP1 are strongly related to the suppressionof BAP1 expression in GBC.

Conclusion
Our study suggests that the suppressed expression of BAP1 mainly due to genetic mutations
promotes the migratory and invasive ability of GBC cells and consequently correlates with apoor prognosis. BAP1 is a tumor suppressor gene in GBC that affects the prognosis and could
be a target for therapy.

Supporting information
S1 Table. Sequences of primer of BAP1 and GAPDH for the RT-PCR.
(PDF)
S2 Table. Sequences of siRNA.
(PDF)
S3 Table. Sequences of M primer and UM primer.
(PDF)
S4 Table. Primer sequences used for 17 exon sequencing of DNA.
(PDF)
S1 Fig. Expression of BAP1 in the GBC cell lines by RT-PCR.
(PDF)
S2 Fig. Expression of BAP1 in the GBC cell lines by Western blotting.
(PDF)
S3 Fig. BAP1 down-regulation by siRNA in the GBC cell line.
(PDF)
S4 Fig. Epithelial mesenchymal transition (EMT) markers in the BAP1 knockdown GBC
cell line.
(PDF)
S5 Fig. Kaplan–Meier survival analysis of BAP1 expression (Stage III, IV).
(PDF)
S6 Fig. BAP1 expression and detected mutations in clinical GBC (ID 14).
(PDF)
S7 Fig. DNA methylation analysis of CpG island of BAP1.
(PDF)
S8 Fig. BAP1 expression of BAP1-methylated GBC cell line after treatment with 5-azacytidine.
(PDF)
S9 Fig. Schema of domains and detected mutations of BAP1.
(PDF)

Acknowledgments
The authors would like to thank F. Fujishima for lending their expertise in immunohistochemical analysis.

PLOS ONE | https://doi.org/10.1371/journal.pone.0206643 November 5, 201817 / 19

BAP1 and gallbladder cancer

This work was supported in part by KAKENHI Grant Numbers JP16K19912.

Author Contributions
Investigation: Takashi Hirosawa, Kentaro Ishii, Keigo Kanehara.
Supervision: Masaharu Ishida, Katsuyoshi Kudo, Shinobu Ohnuma, Takashi Kamei, Fuyuhiko Motoi, Takeshi Naitoh, Florin M. Selaru, Michiaki Unno.

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