-
Cancer Biology and Signal Transduction
Identification of Transmembrane Protein 98 as a
NovelChemoresistance-Conferring Gene in HepatocellularCarcinoma
Kevin Tak-Pan Ng1,2,3, Chung Mau Lo1,3, Dong Yong Guo1, Xiang
Qi1,3, Chang Xian Li1, Wei Geng1,2,3,Xiao Bing Liu1,2,3, Chang Chun
Ling1, Yuen Yuen Ma1,3, Wai Ho Yeung1,2,3, Yan Shao1,3,Ronnie
Tung-Ping Poon1,2,3, Sheung Tat Fan1,2,3, and Kwan Man1,2,3
AbstractChemoresistance is one of the major obstacles in
systemic chemotherapy and targeted therapy for
patients with advanced hepatocellular carcinoma. To identify
novel chemoresistance-associated targets in
hepatocellular carcinoma, chemoresistant hepatocellular
carcinoma cell lines were established. By com-
paring the global gene expression profiles between
chemoresistant and chemosensitive cell lines, eight
novel chemoresistance-associated genes were identified to be
significantly associated with the commonly
augmented chemoresistance of hepatocellular carcinoma cells. One
upregulated candidate named trans-
membrane protein 98 (TMEM98) was found to be overexpressed in 80
of 118 (67.80%) of patients with
hepatocellular carcinoma. TMEM98 mRNA in tumor tissues was
significantly higher than nontumor
tissues of patients with hepatocellular carcinoma (P <
0.0001). Upregulation of TMEM98 was significantlycorrelated with
advanced tumor stage (P ¼ 0.048), high incidence of early tumor
recurrence (P ¼ 0.005),poor overall survival (P ¼ 0.029), and poor
disease-free survival (P ¼ 0.011) of patients with
hepatocellularcarcinoma after hepatectomy. Importantly,
upregulation of TMEM98 mRNA in patients with hepatocel-
lular carcinoma who received transarterial chemoembolization
(TACE) treatment was significantly higher
than in patients without TACE treatment (P ¼ 0.046). Moreover,
patients with poor response to TACEtreatment had higher degree of
TMEM98 upregulation than the responsive patients. In vitro and in
vivo
studies showed that suppression of TMEM98 in chemoresistant
hepatocellular carcinoma cells restored
their chemosensitivity, while forced overexpression of TMEM98
enhanced their chemoresistance. The
mechanism of TMEM98 in conferring chemoresistance of
hepatocellular carcinoma might be possibly
through activation of the AKT pathway and deactivation of p53.
In conclusion, we identified a panel of
novel common chemoresistance-associated genes and demonstrated
that TMEM98 is a chemoresistance-
conferring gene in hepatocellular carcinoma. Mol Cancer Ther;
13(5); 1285–97. �2014 AACR.
IntroductionLiver cancer is the sixth most common cancer and
the
thirdmost common cause of cancer-related deathsworld-wide
(1).Hepatocellular carcinoma represents 70% to 85%of the total
liver cancer (1). Up to 70% of patients withhepatocellular
carcinoma have been suffering from lim-ited treatment options
because of late diagnosis and/or
advanced stage of the disease when, however, surgicaltreatments
including liver transplantation and hepatecto-my as well as
regional therapy are not feasible. Currently,there is no proven
effective conventional systemic che-motherapy for patients with
advanced hepatocellularcarcinoma because of the inherent
chemoresistant natureof hepatocellular carcinoma and with
intolerable cytotox-icity, resulting in thedismalprognosis of
thesepatients (2–4).Hepatocellular carcinoma is a
heterogeneousdisease interms of etiology, molecular, and
carcinogenic mechan-isms as well as biologic behaviors, which can
collectivelycontribute to diverse mechanisms of
chemoresistanceamong patients with hepatocellular carcinoma (5).
Recentsuccess of clinical trial of single-agent sorafenib in
treatingadvanced patients with hepatocellular carcinoma hasshed
lights for future development of targeted therapyfor patients with
advanced hepatocellular carcinoma (6).However, most of the targeted
agents have demonstratedavery lowresponse rate, including sorafenib
(3, 7), leavingthe problem of chemoresistance to be solved.
Therefore,
Authors' Affiliations: 1Department of Surgery; 2Center for
CancerResearch; and 3State Key Laboratory for Liver Research, the
Universityof Hong Kong, Pokfulam, Hong Kong SAR, China
Note: Supplementary data for this article are available at
Molecular CancerTherapeutics Online
(http://mct.aacrjournals.org/).
Corresponding Author: Kwan Man, Department of Surgery, Centre
forCancer Research and State Key Laboratory for Liver Research,
TheUniversity of Hong Kong, Room L9-55, Li Ka Shing Faculty of
MedicineBuilding, 21 Sassoon Road, Pokfulam, Hong Kong, China.
Phone: 86-852-28199646; Fax: 86-852-28199634; E-mail:
[email protected]
doi: 10.1158/1535-7163.MCT-13-0806
�2014 American Association for Cancer Research.
MolecularCancer
Therapeutics
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identification of novel molecular targets must be veryimportant
for understanding the underlying mechanismsof chemoresistance of
hepatocellular carcinoma and even-tually fordevelopingmore
effective therapeutic regimens.
The development of chemoresistance in hepatocellularcarcinoma
could be either intrinsic and/or acquired.There have been
severalmolecular targets associatedwithchemoresistance of
hepatocellular carcinoma. Chemore-sistance of hepatocellular
carcinoma can be achieved byupregulation of the drug transporter
family known as theadenosine triphosphate-binding cassette (ABC)
transpor-ters such as ABCB1, ABCC1, ABCC2, and ABCC3 (8–11),leading
to increment of drugs efflux system to removedrugs out of cells.
High levels ofMRP2,MRP3,MRP4, andMRP5 genes have been found in an
acquired cisplatin-resistant hepatocellular carcinoma cell line
(12). More-over, recent findingshave suggested that liver cancer
stemcells contribute to chemoresistance of hepatocellular
car-cinoma. For instance, upregulation of Octamer 4 gene,
atranscriptional factor of pluripotent cells, can
significantlyaugment chemoresistance of hepatocellular
carcinomacells via the Oct4–AKT–ABCG2 pathway (13). CD133þ
hepatocellular carcinoma cancer stem cells can also con-tribute
to chemoresistance through activation of the Akt/PKB pathway (14).
Overexpression of granulin epithelinprecursor andABCB5 in liver
cancer stem cells can lead tosignificant increment of
chemoresistance (15). Besides, astudy from acquired
doxorubicin-resistant hepatocellu-lar carcinoma cell lines has
identified a panel of differ-entially overexpressed genes and
subsequently charac-terized that upregulation of TOP2A gene is one
of thecontributors of acquired doxorubicin-resistance
inhepatocellular carcinoma (16). Yet, the molecular infor-mation
governing chemoresistance in hepatocellularcarcinoma so far is far
away from achieving effectivetherapeutic regimens.
To search for novel targets,we started
fromestablishingchemoresistant sublines from a human metastatic
hepa-tocellular carcinomacell line and further identifiedapanelof
common differential genes linking to the developmentof acquired
chemoresistance of hepatocellular carcinomacells. Moreover, we
investigated one of the upregulatedcandidates, transmembrane
protein 98 (TMEM98), tounveil its clinical significance and roles
in chemoresis-tance of hepatocellular carcinoma.
Materials and MethodsPatients
Onehundred and eighteen patientswith hepatocellularcarcinoma who
underwent liver resection betweenDecember 1999 and May 2009 were
recruited fromDepartment of Surgery, Queen Marry Hospital, the
Uni-versity of Hong Kong. Twelve normal liver tissues wererecruited
from living donors at the same hospital. The lastfollow-up date for
the patients with hepatocellular carci-noma was on October 2010.
Twenty-five clinical sampleswere obtained from patients with
hepatocellular carcino-ma who received TACE treatment and they were
subse-
quently subject for other treatments, including hepatec-tomy and
liver transplantation. The technical details ofTACE protocol have
been described in previous paper(17). The periods of TACE treatment
for these patientsmay vary, from 2 to 105 months. The
responsivenessafter TACE treatment were defined by our
pathologistbased on the pathologic report from the last CT
scanwhich were defined into 5 categories by our medicalspecialists:
1, no abnormality; 2, appearance of newtumor; 3, tumor same size;
4, decrease in tumor load;and 5, increase in tumor load. We defined
the category 1and 4 as "responsive group," while categories 2, 3,
and 5as "poorly responsive group." All clinical samples
wereobtained from patients with signed consent. The studywas
approved by the Ethics Committee of the Univer-sity of Hong
Kong.
Cell linesHuman hepatocellular carcinoma cell lines, MHCC97L
(metastatic), PLC (nonmetastatic), and Hep3B (nonmeta-static),
were cultured at Dulbecco’s modified Eagle medi-um (DMEM) medium
with 10% FBS (Invitrogen). Toestablish chemoresistant cells,
MHCC97L/CisR, andMHCC97L/DoxR, MHCC97L was chronically
incubatedwith increased concentrations of cisplatin
(PharmachemieBV) or doxorubicin (Sigma-Aldrich) for 12
months,starting from concentration of 100 ng/mL of cisplatinor 20
ng/mL of doxorubicin. On average, the concen-tration of cisplatin
or doxorubicin was increased forevery 2 to 3 weeks. Proliferation
rates of the chemore-sistant sublines were examined for each month.
BeforecDNA microarray analysis, the chemoresistant sublineswere
cultured in DMEM medium without drug for1 month. MHCC97L/CisR2 and
MHCC97L/DoxR2were sublines under exposure of cisplatin and
doxoru-bicin, respectively, for additional of 2 months.
Cloning and transfectionFull length of human TMEM98 cDNA was
cloned into
pcDNA3.1(þ) vector (Invitrogen). The primers for cloningincluded
forward primer: 50-GTACCAGGATCCAGCATGGAGACTGTGGTGATTGTT-30; reverse
primer: 50-CTCGAGTCTAGA CTAAATTGCAGACTGCTCCTGCA-30. Transfection of
plasmids to cells was performed byLipofectamine-2000 (Invitrogen).
Stable transfectants wereselected fromG418-containingmedium for
2weeks. siRNAof targeting human TMEM98 mRNA and negative
controlsiRNA were purchased from Invitrogen. siRNAs weretransfected
to cells by using Lipofectamine RNAiMAX(Invitrogen) according to
manufacturer’s instruction.
Proliferation assaysMTT (Invitrogen) assay and colony formation
assay
were performed as previously described (18). Cells
weretreatedwithdrugs for 72hours and2weeks forMTTassayand colony
formation assay, respectively. Each experi-ment consisted of 3
replications and at least 3 individualexperiments were carried
out.
Ng et al.
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cDNA microarray analysiscDNA microarray profiles of MHCC97L,
MHCC97L/
CisR, andMHCC97L/DoxRwere conducted by gene chipsystem Human
U133 Plus 2.0 (Affymetrix Inc.), byGenome Research Centre, The
University of Hong Kong(18). Microarray data (GEO accession number:
GSE54175)were analyzed by GeneSpring Version 10 (Agilent
Tech-nologies). A 3-fold difference was used to select
differen-tial genes.
Quantitative reverse transcription PCRTotal RNA from cells and
liver tissue samples were
purified by TRizol regent (Invitrogen). Method of quan-titative
reverse transcription PCR (qRT-PCR) analysiswasdescribed as in
previous study (19). The expression of 18Sribosomal RNA was used as
internal control. Primersused in this study were listed in
Supplementary Table S1.
Western blot analysisMethod of Western blot analysis was
described in
previous study (18). TMEM98 antibody was purchasedfrom
Sigma-Aldrich Corporation. Antibodies, includingAKT,
phospho-AKT(Ser473), BCL-XL, phospho-GSK-3b(Ser9), p21, p53,
phospho-p53(Ser6), phospho-p53(Ser9),phospho-p53(Ser15),
phospho-p53(Ser20), and phospho-p53(Ser392), were purchased from
Cell Signaling Tech-nology. b-Actin antibodywas purchased from
Santa CruzBiotechnology.
Apoptosis assayCells (3 � 105) were seeded onto 6-well plate for
24
hours. The cells were harvested and stained withAnnexin-V Fluor
Staining Kit (Roche) according to man-ufactory’s instruction and
analysis by flow cytometer.Early apoptosis was defined as Annexin
V–positive andpropidium iodide (PI)-negative cells. Late apoptosis
wasdefined as Annexin V–positive and PI-positive cells. Thenumber
of apoptotic cells included early and late apopto-sis. Each
experimentwas analyzed in triplicate and at least3 independent
experiments were performed.
Animal modelXenograft ectopic liver tumor model in nude mice
was
adopted (18). For drug treatment, single dose of
cisplatin(5mg/kg) or doxorubicin (5mg/kg)was administrated tonude
mice at day 5 after subcutaneous injection of cells (5� 105
cells/100 mL). Tumor volume was calculated as thefollowing
equation: tumor volume (cm3)¼ length�width� thickness. At least 6
mice were performed for eachexperimental group. Animal study was
specificallyapproved byAnimal (Control of Experiments)
OrdinanceChapter 340, the Department of Health, Hong Kong Spe-cial
Administrative Region [Ref.: (12–63) in DH/HA&P/8/2/3 Pt.
37].
Statistical analysisTMEM98 mRNA of clinical samples was
analyzed
by Prism Version 5.01 (Graphpad). The difference of
TMEM98 mRNA between tumor and nontumor tissuesof each patient
with hepatocellular carcinoma wasdetermined as: DDDCt(TMEM98) ¼
DDCt(tumor) �DDCt(nontumor). Statistical analysis of clinical
para-meters was carried out using SPSS 16 for Windows(SPSS Inc.).
Receiver operating characteristic (ROC)curve was generated to
analyze the sensitivity and 1-specificity of DDDCt(TMEM98) value to
predict overallsurvival of patient with hepatocellular carcinoma
afterhepatectomy. Youden index was used to determine
highderegulation (High group) and low deregulation (Lowgroup) of
patient with hepatocellular carcinoma. Theassociation of TMEM98
deregulation and clinicopatho-logic parameters was analyzed by a x2
test. The prog-nostic value of TMEM98 mRNA for predicting
overalland disease-free survivals of patient with
hepatocellularcarcinoma after hepatic resection was calculated
byKaplan–Meier analysis with the log-rank test. For dis-ease-free
survival analysis, patient with hepatocellularcarcinoma under the
category of hospital mortality wereexcluded. Cox proportional
hazard regression modelwas performed to test factors that were
significantlyassociated with the overall survival or
disease-freesurvival of the patient with hepatocellular carcinoma.P
value < 0.05 was considered to be statisticallysignificant.
ResultsEstablishment of chemoresistant hepatocellularcarcinoma
sublines
After continuous incubation of increasing concentra-tions of
cisplatin or doxorubicin to a human metastatichepatocellular
carcinoma cell line named MHCC97L for12 months, 2 chemoresistant
sublines, MHCC97L/CisRand MHCC97L/DoxR, were established. The
morpholo-gy of MHCC97L/CisR and MHCC97L/DoxR sublineswas similar to
MHCC97L. In a nondrug culture medium,these sublines grew slightly
faster than MHCC97L (Sup-plementary Fig. S1). The in vivo growth
rate of thesesublines without drug was similar to MHCC97L
(Supple-mentary Fig. S2).
The in vitro chemoresistance of MHCC97L/CisR andMHCC97L/DoxR
sublines was significantly increasedcompared with MHCC97L. MTT
assay showed that che-moresistance of MHCC97L/CisR to cisplatin
(IC50 ¼20 mg/mL) was nearly 10-folds higher than MHCC97L(IC50¼
2.2mg/mL).MHCC97L/DoxRexhibitedmore than25-fold increase of
resistance to doxorubicin comparedwith MHCC97L (IC50 of
doxorubicin: 40 mg/mL vs.1.5 mg/mL; Fig. 1A). The colony-forming
ability ofMHCC97/CisR and MHCC97L/DoxR retained
similarcolony-forming ability at 1,000 ng/mL of cisplatin(Fig. 1B)
and 200 ng/mL of doxorubicin (Fig. 1C), respec-tively, whereas
MHCC97L was dramatically suppressedin >500 ng/mL of cisplatin or
>50 ng/mL of doxorubicin(Fig. 1B and C). The IC50 of
MHCC97L/CisR2and MHCC97L/DoxR2 was approximately 1.3- and1.2-fold
of the MHCC97L/CisR and MHCC97L/DoxR,
The Role of TMEM98 in Chemoresistance of Hepatocellular
Carcinoma
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MTT AssayA
B
C
D E
MHCC97L
Cisplatin (ng/mL)
Cisplatin concentration (ng/mL)
0
0 50 100 200 300 500
0 50
Time (day)0 5 10 15 20 25 30
Time (day)0 5 10 15 20 25 30
100 200 300 500
500 800 1,000 2,000 3,000
Cisplatin (ng/mL)
Colony formation assay
Colony formation assay
Doxorubicin (ng/mL)
Doxorubicin concentration (ng/mL)
In vivo xenograftmodel under cisplatin treatment
In vivo xenograftmodel under doxorubicin treatment
Doxorubicin (ng/mL)
0 5,000 10,000 15,000 20,000 20,0000 40,000 60,000 80,00025,000
30,000
MHCC97L/CisR
MHCC97LMHCC97L/CisR
MHCC97LMHCC97L/CisR
MH
CC
97L
MH
CC
97L/
Cis
R
0 500 800 1,000 2,000 3,000
Num
ber
of c
olon
ies
250
200
150
100
50
0
Num
ber
of c
olon
ies
250
200
150
100
50
0
MH
CC
97L
MH
CC
97L/
Cis
R
MHCC97L
MHCC97L/DoxR
MHCC97LMHCC97L/DoxR
MHCC97LMHCC97L/Dox
MH
CC
97L
MH
CC
97L/
Dox
R
MH
CC
97L
MH
CC
97L/
Dox
RP
erce
ntag
e of
via
ble
cells
(%
)1009080706050403020100
Tum
or v
olum
e (m
m3 )
1009080706050403020100 Tu
mor
vol
ume
(mm
3 )
1009080706050403020100
Per
cent
age
of v
iabl
e ce
lls (
%)
1009080706050403020100
MTT Assay
Ng et al.
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respectively (data not shown). The in vivo chemoresistanceof
MHCC97/CisR and MHCC97L/DoxR was increasedcompared with MHCC97L
under cisplatin and doxorubi-cin treatment, respectively (Fig. 1D
and E).
Identification of common chemoresistance-associated genesThe
experimental procedures to identify common che-
moresistant candidates were illustrated in Supplementa-ry Fig.
S3. To avoid overestimation of differential genesfrom cDNA
microarray analysis, 3-fold difference wasused as filter condition
rather than 2-fold difference.Comparing to cDNA microarray profile
of MHCC97L,537 and 678 differential genes were identified
inMHCC97L/CisR and MHCC97L/DoxR respectively, inwhich 235 genes
were commonly differentially expressedin both sublines. Among them,
164 genes were commonlyupregulated (Supplementary Table S2) and 71
geneswerecommonly downregulated (Supplementary Table S3).
Byapplying 10-fold difference as secondary selection crite-rion, 36
candidate genes were identified to be commonlydifferentially
expressed. After validation by qRT-PCRanalysis, 19 common
differential genes, 7 upregulatedand 12 downregulated, were
identified (SupplementaryTable S4).To examine whether the degree of
differential expres-
sion is correlated to the degree of chemoresistance
ofhepatocellular carcinoma cells, the expression of 19
dif-ferential genes were further verified in MHCC97L,MHCC97L/CisR,
MHCC97L/CisR2, MHCC97L/DoxR,and MHCC97L/DoxR2 cells. Among the
upregulatedgenes, the expression levels of APOB, RUNDC3B,SYK,
TMEM47, and TMEM98 genes were significantlyincreased along with the
increased degrees of chemore-sistance of cells, reaching the
highest level in bothMHCC97L/CisR2 and MHCC97L/DoxR2 comparedwith
MHCC97L/CisR andMHCC97L/DoxR, respective-ly (Supplementary Fig.
S4A). Among the downregulatedgenes, the expression levels of FGB,
ZNF284, and BTB11were significantly lowered along with increased
degreesof chemoresistance of cells, reaching the lowest level
inboth MHCC97L/CisR2 and MHCC97L/DoxR2 com-pared with MHCC97L/CisR
and MHCC97L/DoxR,respectively (Supplementary Fig. S4B). Totally, 8
che-moresistance-associated genes were identified to be
sig-nificantly associated with common chemoresistance
ofhepatocellular carcinoma cells.To identify hepatocellular
carcinoma–associated
candidate genes, mRNA expression levels of APOB,RUNDC3B, SYK,
TMEM47, and TMEM98 genes werepreliminarily investigated in 37 pairs
of tumor andadjacent nontumor liver tissues of patients with
hepa-tocellular carcinoma. One of them named TMEM98 was
found to be overexpressed in approximately 65% of thepatients
with hepatocellular carcinoma (SupplementaryTable S5). Owing to
chemoresistance- and hepatocellu-lar carcinoma–associated features
of TMEM98 gene, itwas selected for further characterization.
Deregulation of TMEM98 predicts poor prognosis ofpatients with
hepatocellular carcinoma
Among 118 patients with hepatocellular carcinoma, 80patients
(67.80%)were found to differentially overexpressTMEM98 mRNA
(DDDCt(TMEM98) � 1). The averagerelative DDCt(TMEM98) value among
tumor liver tissues,nontumor liver tissues, and healthy donor liver
tissueswere 7.00, 5.34, and 4.29, respectively (Fig. 2A).
Theexpression level of TMEM98mRNA in tumor liver tissuewas
significantly higher than in nontumor liver tissues(unpaired
2-tailed t test, P < 0.0001; paired 2-tailed t test,P <
0.0001) and healthy donor liver tissues (unpaired 2-tailed t test,
P¼ 0.0002). Agreedwith the result from qRT-PCR analysis, Western
blot analysis showed thatTMEM98 protein was overexpressed in tumor
tissue ofpatients with hepatocellular carcinoma compared
withnontumor tissues and healthy donor tissues (Fig. 2B).
Fifty-one (43.22%) and 67 (56.78%) patients with hepa-tocellular
carcinoma were defined as TMEM98 highderegulation group (High
group) and low deregulationgroup (Low group) respectively according
to Youdenindex analysis. High deregulation of TMEM98 mRNAwas
significantly correlated with the presence ofadvanced New AJCC
stage (P ¼ 0.001) and recurrenceof tumor within first year (P ¼
0.005; Table 1). Moreover,low deregulation of TMEM98 mRNA was
significantlycorrelated with early pathologic
tumor–node–metastasis(pTNM) stage (P ¼ 0.048).
Kaplan–Meier analysis illustrated that patients withhigh
deregulation of TMEM98 mRNA were significantlyassociated with poor
overall survival (log-rank ¼ 4.741,P ¼ 0.029) and poor disease-free
survival (log-rank ¼6.543, P ¼ 0.011; Fig. 2C). The mean periods of
overalland disease-free survival for the High group were 66.6and
38.8 months, whereas for the Low group were 85.8and 62.8
months.
Cox proportional hazard regression analysis was usedto find out
the independent predictors for predictingoverall survival and
disease-free survival of patients withhepatocellular carcinoma
after hepatectomy among select-ed 5 factors, including TMEM98 mRNA,
pTNM stage,venous infiltration, New AJCC stage, and serum
a-feto-protein (AFP) level, which were significantly associatedwith
the overall survival of patients with hepatocellularcarcinoma by
Kaplan–Meier analysis (SupplementaryTable S6). Univariable Cox
proportional hazard regressionanalysis showed that TMEM98 mRNA was
a significant
Figure 1. Establishment of chemoresistant hepatocellular
carcinoma sublines. A, MTT assay under cisplatin and doxorubicin
treatments. Colony formationassay under cisplatin treatment (B) and
doxorubicin treatment (C), D, xenograft ectopic nudemousemodel of
MHCC97L andMHCC97L/CisR under cisplatintreatment. E, xenograft
ectopic nude mouse model of MHCC97L and MHCC97L/DoxR under
doxorubicin treatment. ��, P < 0.01.
The Role of TMEM98 in Chemoresistance of Hepatocellular
Carcinoma
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A B
C
E
G
F
D
15
10
5
0
1.0
0.8
0.6
0.4
0.2
0.0
HCC-T
HC
C #
1
HC
C #
2
HC
C #
3
HC
C #
4
HC
C #
5
HC
C #
6
HC
C #
7
HCC-NT
Liver tissues
0.00 25.00
Overall survival (month) Disease-free survival (month)
Patients with TACE treatment
Poorly responsive Responsive
n = 18
n = 3
50.00 75.00 100.00 125.00 0.00 25.00 50.00
P = 0.029* P = 0.011*
Low, n = 67
Low, n = 65
High, n = 51
High, n = 51
75.00 100.00 125.00
Cum
sur
viva
l
Healthy donor
Hea
lthy
dono
rs
TMEM98
10
5
0
–5
–10
5
4
3
2
1
0
1 2 T NT NT NT NTT T T NTT NTT NTT
HCC HCC HCC HCC
TACE
With TACE
876543210
0 500 0 50 100 200 500
Rel
ativ
e ra
tio (
fold
) 6
5
4
3
2
1
0Rel
ativ
e ra
tio (
fold
)
1,000
Cisplatin concentration (ng/mL) Doxorubicin concentration
(ng/mL)
2,000 3,000
Expression level of TMEM98 mRNA respondingto cisplatin
treatment
Expression level of TMEM98 mRNA respondingto doxorubicin
treatment
Without TACE
MHCC97L
PLC
Hep3B
MHCC97L
PLC
Hep3B
Non-TACE
NTT NT NT NTT T T
β-Actin
TMEM98
β-Actin
Rel
ativ
e ex
pres
sion
leve
lto
TM
EM
98 m
RN
A (
log 2
)
Rel
ativ
e T
/NT
rat
ioof
TM
EM
98 m
RN
A (
log 2
)
Rel
ativ
e T
/NT
rat
ioof
TM
EM
98 m
RN
A (
log 2
)
1.0
0.8
0.6
0.4
0.2
0.0
Cum
sur
viva
l
Figure 2. Clinical significance of TMEM98 in hepatocellular
carcinoma. A, TMEM98 mRNA in liver tissues of patients with
hepatocellular carcinoma andhealthy donors. B, expression of TMEM98
protein in 5 pairs of tumor and nontumor tissues of patients with
hepatocellular carcinoma. C, Kaplan–Meieranalysis of overall and
disease-free survival of patients with hepatocellular carcinoma. D,
Western blot analysis of TMEM98 protein betweennon-TACE and TACE
treatment groups. E, comparison of tumor to nontumor ratio (T/NT)
of TMEM98 mRNA between patients with and withoutTACE treatment. F,
comparison of tumor to nontumor ratio (T/NT) of TMEM98 mRNA between
patients with hepatocellular carcinoma with poorlyresponsive and
responsive effects in the TACE treatment group. G, TMEM98mRNA in
different hepatocellular carcinoma cells in responding to
cisplatinor doxorubicin treatment. �, P < 0.05; ��, P <
0.01.
Ng et al.
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predictor for both of overall survival (HR ¼ 1.818;
95%confidence interval (CI), 1.05–3.14; P¼ 0.032) and disease-free
survival (HR ¼ 1.816; 95% CI, 1.14–2.89; P ¼ 0.012)of patients with
hepatocellular carcinoma after hepatecto-my (Supplementary Table
S6). Multivariable Cox propor-tional hazard regression analysis
revealed that TMEM98was not an independent factor for overall or
disease-freesurvival of patients with hepatocellular carcinoma.To
determine whether the deregulation of TMEM98
is correlated to increased chemoresistance of patientswith
hepatocellular carcinoma, the expression levels ofTMEM98 mRNA and
protein patients with hepatocellularcarcinoma received TACE
treatment (TACE group) was
examined. The expression level of TMEM98 protein in theTACE
groupwas found to be higher than in the non-TACEgroup (Fig. 2D).
Eighty percent of patients with hepatocel-lular carcinoma were
found to overexpress TMEM98mRNAin tumor tissues afterTACE
treatment. The averagetumor to nontumor (T/NT) ratio of TMEM98 mRNA
inthe TACE group was significantly higher than patientswithout TACE
treatment (P ¼ 0.046; Fig. 2E). Moreover,in the TACE group,
patients with poor response to TACEtreatment showed a higher degree
of T/NT ratio ofTMEM98 mRNA [DDDCt(TMEM98) ¼ 2.92] comparedwith
patients responded to TACE treatment [DDDCt(TMEM98) ¼ 1.42; Fig.
2F].
Table 1. Correlation analysis of TMEM98 mRNA and
clinicopathologic features of patients withhepatocellular
carcinoma
TMEM98 mRNA (n)
Clinicopathologic features Number (n) Low High P
SexMale 94 54 40 0.772Female 24 13 11
Age� 55 years 64 33 31 0.213> 55 years 54 34 20
pTNM stagea
Early stage (I–II) 34 24 10 0.048c
Advanced stage (III–IV) 83 42 41New AJCCa
Stage I to II 78 53 25 0.001c
Stage III to IV 39 13 26Venous infiltrationAbsent 51 34 17
0.059Present 67 33 34
Cirrhosisa
No 42 20 22 0.169Yes 74 45 29
Encapulationa
Absent 23 14 9 0.668Present 22 12 10
Tumor sizea
< 5 cm 33 20 13 0.598� 5 cm 69 38 31
AFP levela
� 1000 ng/ml 91 55 36 0.141> 1000 ng/ml 27 12 15
Hepatitis B surface antigena
Negative 17 10 7 0.854Positive 101 57 44
Recurrence within first yearb
No 68 46 22 0.005c
Yes 46 19 27
aTotal number less than 118 because of missing data.bFour
patients were excluded because of their death within the first year
without recurrence.c, P < 0.05.
The Role of TMEM98 in Chemoresistance of Hepatocellular
Carcinoma
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MTT assay of MHCC97L/CisR subline MTT assay of MHCC97L/DoxR
sublineA
B
C
D
E
F
Cisplatin concentration (ng/mL)
Cisplatin concentration (μg/mL)
Cisplatin concentration (ng/mL)
Cis
plat
intr
eatm
ent
Dox
orub
icin
trea
tmen
t
0 5,000 10,000 15,000 20,000 0 5,000 10,000 15,000 20,000
Time (day)0 5 10 15 20 25 30
Time (day)0 5 10 15 20 25 30
Cisplatin (μg/mL)
0 1 3 5
0 1 3 5
0 1 3 5
0 1 3 5
si-Nilsi-Negsi-TMEM98
Si-TMEM98
Si-Ctr Si-TMEM98Si-Ctr
Si-TMEM98Si-Ctr
si-TMEM98
si-Ctr
si-Nilsi-Negsi-TMEM98
Doxorubicin concentration (ng/mL)
Doxorubicin concentration (μg/mL)
Doxorubicin concentration (ng/mL)
In vivo xenograftmodel under cisplatin treatment
In vivo xenograftmodel under doxorubicin treatment
Doxorubicin (μg/mL)
0 5,000 10,000 15,000 20,000 25,000 30,000 0 5,000 10,000 15,000
20,000 25,000 30,000
MH
CC
97L-
3.1
MH
CC
97L-
TM
EM
98
MH
CC
97L-
3.1
MH
CC
97L-
TM
EM
98
MHCC97L-3.1MHCC97L-TMEM98
MHCC97L-3.1MHCC97L-TMEM98
16
14
12
10
8
6
4
2
0
30
25
20
15
10
5
0
Perc
enta
ge o
f via
ble
cells
(%)
Perc
enta
ge o
f via
ble
cells
(%)
Perc
enta
ge o
f apo
ptot
ic c
ells
(%)
Apoptosis assay on MHCC97L/CisR
MTT assay of MHCC97L-derivedtransfectants in cisplatin
pcDNA3.1TMEM98-1TMEM98-2
pcDNA3.1TMEM98-1TMEM98-2
MTT assay of MHCC97L-derivedtransfectants in doxorubicin
Apoptosis assay on MHCC97L/DoxR
Perc
enta
ge o
f apo
ptot
ic c
ells
(%)
1009080706050403020100
Perc
enta
ge o
f via
ble
cells
(%)
1009080706050403020100 Pe
rcen
tage
of v
iabl
e ce
lls (%
)
1009080706050403020100
1009080706050403020100
Tum
or v
olum
e (m
m3 )
Tum
or v
olum
e (m
m3 )
1009080706050403020100
1009080706050403020100
Ng et al.
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TMEM98 is a novel chemoresistance-conferringtarget in
hepatocellular carcinomaTreatment with low dosages of cisplatin or
doxorubicin
in short period could upregulate TMEM98mRNA in bothMHCC97L and
PLC cells in a dose-dependent manner,whileHep3Bgained upregulation
ofTMEM98 in cisplatintreatment but not in doxorubicin treatment
(Fig. 2G).Upregulation of TMEM98 mRNA after cisplatin or
doxo-rubicin treatment could be persisted after removal of
drug(Supplementary Fig. S5).TMEM98 siRNA could significantly
suppress the
expression of TMEM98mRNA to 96 hours after transfec-tion in
MHCC97L/CisR and MHCC97L/DoxR (Supple-mentary Fig. S6). Suppression
of TMEM98 expressionresulted in significant decrement of the
chemoresistanceofMHCC97L/CisR to cisplatin (si-Neg: IC50¼ 15
mg/mL;si-TMEM98: IC50 ¼ 5 mg/mL; Fig. 3A) and MHCC97L/DoxR to
doxorubicin (si-Neg: IC50 > 30 mg/mL; si-TMEM98: IC50 ¼ 11
mg/mL; Fig. 3A). Suppression ofTMEM98 expression in MHCC97L/CisR or
MHCC97L/DoxR cells by siRNA could significantly enhance
theirapoptosis under cisplatin (Fig. 3B) or doxorubicin(Fig. 3C)
treatment compared with control cells. More-over, overexpression of
TMEM98 in MHCC97L cellscould significantly increase their
chemoresistance to bothcisplatin (MHCC97L-pcDNA3.1: IC50 ¼ 4.0
mg/mL;MHCC97L-TMEM98-1: IC50 ¼ 5.5 mg/mL; Fig. 3D) anddoxorubicin
(MHCC97L-pcDNA3.1: IC50 ¼ 4.2 mg/mL;MHCC97L-TMEM98-1: IC50 ¼ 10
mg/mL; Fig. 3D). Over-expression of TMEM98 in PLC cell line also
increased itschemoresistance (Supplementary Fig. S7). Most
important-ly, overexpression of TMEM98 in MHCC97L cell line
sig-nificantly enhanced its in vivo resistance to both
cisplatin(Fig. 3E) and doxorubicin (Fig. 3F). However,
overexpres-sion of TMEM98 in MHCC97L cell line could not
increasesorafenib chemoresistance (Supplementary Fig. S8).
TMEM98 modulates chemoresistance ofhepatocellular carcinoma
through AKT and p53pathwaysTo understand the molecular mechanisms
of TMEM98
on chemoresistance of hepatocellular carcinoma, 2 impor-tant
signaling pathways, including the AKT and p53pathways, were
investigated. Activation of the AKT[i.e., phospho-AKT(Ser473)]
pathway was found inMHCC97L/CisR and MHCC97L/DoxR compared
withMHCC97L (Fig. 4A). Suppression of TMEM98 by siRNAresulted in
repression of activation of AKT (Fig. 4B).Moreover, suppression of
TMEM98 in MHCC97L/CisRand MHCC97L/DoxR could repress EGF- and
IGF2-induced activation of AKT and its downstream targets,
including BCL-XL and phospho-GSK-3b(Ser9) (Fig. 4C).Furthermore,
suppression of TMEM98 inMHCC97L/CisRand MHCC97L/DoxR could inhibit
the activation of theAKT pathway under drug environment in a
dose-depen-dent manner (Fig. 4D). In addition, forced
overexpressionof TMEM98 in MHCC97L led to elevation of AKT
activa-tion under cisplatin or doxorubicin treatment (Fig. 4E).
Deactivation of p53, illustrated by deactivations
ofphospho-p53(Ser15) and phospho-p53(Ser392), wasfound in
MHCC97L/CisR and MHCC97L/DoxR, whileother forms of activated p53,
including phospho-p53(Ser20), phospho-p53(Ser6), and
phospho-p53(Ser9),could not be detected (Fig. 5A). Suppression of
TMEM98in chemoresistant sublines restored the activation of
phos-pho-p53(Ser15) and phospho-p53(Ser392) (Fig. 5B). Thedegree of
activation of the p53 signaling pathway inMHCC97L/CisR and
MHCC97L/DoxR was elevatedunder drug environment after suppression
of TMEM98(Fig. 5C). Meanwhile, forced-overexpression of
TMEM98inMHCC97L could repress p53 activation under cisplatinor
doxorubicin treatment (Fig. 5D).
DiscussionSystemic chemotherapy has been adopted to treat
advanced patients with hepatocellular carcinoma formore than 30
years, but the survival outcome of thesepatients remained
unsatisfactory (3, 4). The emergence oftargeted therapy, encouraged
by improved survival ben-efits from sorafenib, will become a major
strategy fortreatment of advanced patients with hepatocellular
car-cinoma. However, inadequate knowledge on the molec-ular
mechanisms of chemoresistance of hepatocellularcarcinoma from
intrinsic and acquired pathways hindersthe development of effective
targeted therapy on eradi-cating cancer cells (7, 20). Therefore,
identificationofnoveltargets becomes an important task not only to
understandmolecular mechanism of chemoresistance of hepatocellu-lar
carcinoma but to eventually develop new effectivetherapeutic
strategies for advanced patients with hepato-cellular carcinoma
(21).
We applied a step-by-step approach to identify
novelchemoresistance-associated genes. The in vitro and in
vivogrowth rates of the chemoresistant sublines without
drugtreatment were similar to parental MHCC97L cell line,indicating
that the selection process did not alter theproliferation and
tumorigenesis of the sublines. The invitro and in vivo
chemoresistance of the chemoresistantsublines significantly higher
than MHCC97L indicatedthat the acquired molecular changes of these
sublinesunder drug selection, therefore, may be mainly prone
todevelop their chemoresistance. There were 235 commonly
Figure 3. Functional role of TMEM98 in chemoresistance of
hepatocellular carcinoma. A, MTT assay of MHCC97L/CisR and
MHCC97L/DoxR sublines incisplatin or doxorubicin treatment after
suppression of TMEM98 expression. B, apoptosis assay ofMHCC97L/CisR
in cisplatin treatment after suppression ofTMEM98 expression. C,
apoptosis assay of MHCC97L/DoxR in doxorubicin treatment after
suppression of TMEM98 expression. D, MTT assay of
TMEM98-overexpressing MHCC97L cells in cisplatin or doxorubicin
treatment. Cisplatin treatment (E), or doxorubicin treatment (F) on
in vivo ectopic model ofMHCC97L-3.1 and MHCC97L-TMEM98. �, P <
0.05; ��, P < 0.01.
The Role of TMEM98 in Chemoresistance of Hepatocellular
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differential genes, resting more than 300 and 400 geneswere
distinctly differential in MHCC97L/CisR andMHCC97L/DoxR,
respectively. These data indicated thatthe acquired chemoresistance
of hepatocellular carcinomais contributed by acquired genetic or
epigenetic changes ofa variety of genes, which were either or both
drug-specificand nonspecific. Hepatocellular carcinoma can resist
todifferent chemotherapeutic and targeting agents (2,
20).Identification of common differential genes is indispens-able
for understanding the molecular mechanism of che-moresistance of
hepatocellular carcinoma on differentdrugs.
The first question we would like to ask was whetherchange in
expression of these genes was correlated to thechanges of
chemoresistance in hepatocellular carcinoma.Among 19 highly
differential genes, 5 genes were foundto have significantly
increasing upregulation and 3 geneswere found to have significantly
increasing downregula-tion in both MHCC97L/CisR2 and
MHCC97L/DoxR2comparing to MHCC97L/CisR and
MHCC97L/DoxR,respectively, indicating that the increasing
differentialexpressions of these genes are significantly correlated
tothe acquired chemoresistance in hepatocellular carcino-ma. So
far, the roles of these genes in chemoresistance of
Figure 4. The roles of TMEM98 inthe AKT pathway. A, Western
blotanalysis of TMEM98, AKT, andphospho-AKT(Ser473) protein
inMHCC97L and chemoresistantsublines. B, Western blot analysisof
TMEM98, AKT, and phospho-AKT(Ser473) upon suppression ofTMEM98 in
chemoresistantsublines. C, suppression ofTMEM98 inhibits EGF- and
IGF2-mediated AKT activation inMHCC97L/CisR and MHCC97L/DoxR
sublines. D, suppression ofTMEM98 in chemoresistantsublines
suppressedAKT signalingunder cisplatin or doxorubicintreatment. E,
overexpression ofTMEM98 in MHCC97L cell lineenhanced AKT activation
undercisplatin or doxorubicin treatment.�, si-Ctr; þ,
si-TMEM98.
Ng et al.
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hepatocellular carcinoma is not yet clear, suggestingthat they
may be novel chemoresistance-associatedcandidates.Genes involved in
tumorigenesis have been also found
to contribute to chemoresistance of hepatocellular carci-noma
(13, 15, 22, 23). Identification of
hepatocellularcarcinoma–associated targets is thus critical not
only forunderstanding the molecular mechanisms of both intrin-sic
and acquired chemoresistance but also for developinghepatocellular
carcinoma–associated targeted therapy.From preliminary examination
of clinical samples, oneof the differential genes, TMEM98, was
found to be over-
expressed in most of the hepatocellular carcinoma tissue.Human
TMEM98 protein, composed of 226 amino acidresidues, is a potential
single-pass transmembrane pro-tein located in endoplasmic
reticulum. TMEM98 has beenfound to be overexpressed in
adenocarcinoma subtype ofadenosquamous carcinoma whose patients
have relativepoor prognosis than other subtypes (24).TMEM98mRNAhas
been included into adenocarcinoma-like expressionsignature, which
is associated with an epithelial mesen-chymal transition and
activated b-catenin pathway (24).So far the function of TMEM98 is
unclear. In our study,TMEM98 mRNA was found to be overexpressed in
early
Figure 5. The roles of TMEM98 inthe p53 pathway. A, Western
blotanalysis of p53 and differentphosphorylated p53 proteins
inMHCC97L and chemoresistantsublines. B, Western blot analysisof
p53 and differentphosphorylated p53 proteins inchemoresistant
sublines aftersuppression of TMEM98. C,suppression of TMEM98
enhancedp53 activation in chemoresistantsublines under drug
treatments. D,overexpression of TMEM98 inMHCC97Lcell line
suppressedp53activation under drug treatments.�, si-Ctr; þ,
si-TMEM98.
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The Role of TMEM98 in Chemoresistance of Hepatocellular
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70% of tumor tissues of patients with hepatocellularcarcinoma,
indicating that this gene is hepatocellularcarcinoma–associated and
its deregulation in hepatocel-lular carcinoma may also possibly
contribute to intrinsicchemoresistance of hepatocellular carcinoma.
Further-more, high deregulation of TMEM98 in
hepatocellularcarcinoma was found to be significantly associated
withadvanced New AJCC stage. Low deregulation ofTMEM98 in
hepatocellular carcinoma was significantlyassociatedwith early
pTNMstage andnearly significantlyassociated with absence of venous
infiltration. Thesedemonstrated a positive correlation between
TMEM98deregulation and progressive phenotype of hepatocellu-lar
carcinoma. Moreover, high deregulation of TMEM98in hepatocellular
carcinoma was found to be significantlycorrelated with higher
incidence of early tumor recur-rence and poor overall and
disease-free survivals ofpatients with hepatocellular carcinoma
after hepatecto-my, suggesting that TMEM98 mRNA may be a
potentialprognostic marker for patients with hepatocellular
carci-noma. The diagnostic value of TMEM98 protein inpatients with
hepatocellular carcinoma is also valuablefor further
characterization.
TACE has been widely used to improve the survivalof patients
with unresectable hepatocellular carcinoma(25). Although a recent
study demonstrated improvedsurvival rates and low mortality rate
using TACE withlipiodol in 8,510 patients with unresectable
hepatocellu-lar carcinoma (26), the survival rate of advanced
hepato-cellular carcinoma from TACE treatment thus far hasbeen
unsatisfactory (25, 27) suggesting a pressing needto identify
molecular mechanism linking to acquiredchemoresistance during TACE
treatment. Our resultshowed that a significantly increasing
upregulation ofTMEM98 mRNA in tumor was found in patients
withhepatocellular carcinoma received TACE treatment com-pared with
patients with hepatocellular carcinomawithout TACE treatment,
indicating that hepatocellularcarcinoma tumor cells acquired higher
changes ofTMEM98 expression than nontumor cells after
chemo-therapy. In addition, the degree of deregulation ofTMEM98 in
patients who were poorly responsive toTACE treatment was higher
than that in TACE-respon-sive patients, implying that acquired
overexpression ofTMEM98 in hepatocellular carcinoma may be
linkingto the acquired chemoresistance of patients with
hepato-cellular carcinoma after chemotherapy. The in vitrostudy
indicated that TMEM98 is responsive to chemo-therapeutic
agents.Altogether, the abovedata suggestedapossible association of
acquired TMEM98 expressionin development of chemoresistance in
hepatocellular car-cinoma. However, the sample size of patients
receivedTACE treatment in this study was insufficient to reachsolid
conclusion because of the lack of patients withhepatocellular
carcinoma who were subjected for TACEas the first treatment option
before surgical treatment.
In our functional study, suppression of TMEM98expression could
trim down chemoresistance of chemore-
sistant sublines, while forced overexpression of TMEM98could
increase chemoresistance of different hepatocellu-lar carcinoma
cells. These data indicated that TMEM98may plays important roles in
the development and main-tenance of chemoresistance in
hepatocellular carcinomaand targeting suppression of TMEM98 in
hepatocellularcarcinoma may be a potential strategy to overcome
che-moresistance of hepatocellular carcinoma.
Several lines of evidence illustrated that activation ofAKT and
deactivation of p53 play important roles onchemoresistance of
cancers and hepatocellular carcinomaleading to important targets
for treating advancedpatientswith hepatocellular carcinoma (28–32).
In our study, thechemoresistant sublines exhibitedhigher level of
activatedAKT and lower levels of activated forms of p53
thanparental MHCC97L, indicating that AKT and p53 may beinvolved in
the development of chemoresistance.Ourdatademonstrated that TMEM98
could modulate chemoresis-tance of hepatocellular carcinoma cells
throughmodifyingthe status of the AKT and p53 pathways.
In conclusion, our clinical and experimental evidencessuggested
that TMEM98 is not only a prognostic markerfor patients with
hepatocellular carcinoma but also anovel molecular target
associated with intrinsic andacquired chemoresistance of
hepatocellular carcinoma.Furthermore, TMEM98 may confer
chemoresistance ofhepatocellular carcinoma by activation of the AKT
sig-naling pathway and deactivation of p53. These findingsshould
provide important information for developingeffective strategy in
the future to overcome chemoresis-tance of hepatocellular
carcinoma.
Disclosure of Potential Conflicts of InterestNo potential
conflicts of interest were disclosed.
Authors' ContributionsConception and design: K.T.-P. Ng, C. Mau
Lo, K. ManDevelopment of methodology: K.T.-P. Ng, K. ManAcquisition
of data (provided animals, acquired and managed patients,provided
facilities, etc.):K.T.-P.Ng,C.MauLo,D.Y.Guo,C.X. Li, X.B. Liu,Y.Y.
Ma, R.T.-P. PoonAnalysis and interpretation of data (e.g.,
statistical analysis, biostatis-tics, computational analysis):
K.T.-P. Ng, C.X. Li, X.B. Liu, Y.Y. Ma,K. ManWriting, review,
and/or revision of the manuscript: K.T.-P. Ng, C. MauLo, S.T. Fan,
K. ManAdministrative, technical, or material support (i.e.,
reporting or orga-nizing data, constructing databases): K.T.-P. Ng,
X. Qi, W. Geng,C.C. Ling, W.H. Yeung, Y. Shao, K. ManStudy
supervision: C. Mau Lo, R.T.-P. Poon, S.T. Fan, K. Man
Grant SupportThis study was supported by the Collaborative
Research Funds
(HKU5/CRF/08 & HKU3/CRF/11R by C.M. Lo and K. Man) of
theResearch Grant Council of Hong Kong, and the Small Project
Funding(201109176183 by K.T.-P. Ng and K. Man) and the Seed Funding
Pro-gramme for BasicResearch (201306159004 byK.T.-P.NgandK.Man) of
theUniversity of Hong Kong.
The costs of publication of this article were defrayed in part
by thepayment of page charges. This article must therefore be
hereby markedadvertisement in accordance with 18 U.S.C. Section
1734 solely to indicatethis fact.
Received September 25, 2013; revised February 6, 2014; accepted
Feb-ruary 17, 2014; published OnlineFirst March 7, 2014.
Mol Cancer Ther; 13(5) May 2014 Molecular Cancer
Therapeutics1296
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www.aacrjournals.org Mol Cancer Ther; 13(5) May 2014 1297
The Role of TMEM98 in Chemoresistance of Hepatocellular
Carcinoma
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Chemoresistance-Conferring Gene in Hepatocellular
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