Research Article
Functional and Clinical Evidence for NDRG2 as a Candidate
Suppressor of Liver Cancer Metastasis
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Dong Chul Lee, Yun Kyung Kang, Woo Ho Kim, Ye Jin Jang, Dong Joon Kim, In Young Park,
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Bo Hwa Sohn, Hyun Ahm Sohn, Hee Gu Lee, Jong Seok Lim, Jae Wha Kim, Eun Young Song,
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Dong Min Kim, Mi-Ni Lee, Goo Taeg Oh, Soo Jung Kim, Kyung Chan Park,
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Hyang Sook Yoo, Jong Young Choi, and Young Il Yeom
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Medical Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejon, Korea; 2Department of Pathology,
Inje University Seoul Paik Hospital; 3Department of Pathology, Seoul National University College of Medicine; 4Department of Biological
Sciences, Sookmyung Women’s University; 5Department of Molecular Life Sciences, Ewha Women’s University; and 6Department of
Internal Medicine, Kangnam St. Mary’s Hospital, Seoul, Korea
Abstract
Introduction
We searched for potential suppressors of tumor metastasis by
identifying the genes that are frequently down-regulated in
hepatocellular carcinomas (HCC) while being negatively
correlated with clinical parameters relevant to tumor metastasis, and we report here on the identification of N-myc
downstream regulated gene 2 (NDRG2) as a promising
candidate. NDRG2 expression was significantly reduced in
HCC compared with nontumor or normal liver tissues [87.5%
(35 of 40) and 62% (62 of 100) at RNA and protein levels,
respectively]. Reduction of NDRG2 expression was intimately
associated with promoter hypermethylation because its
promoter region was found to carry extensively methylated
CpG sites in HCC cell lines and primary tumors. Immunohistochemical analysis of NDRG2 protein in 100 HCC patient
tissues indicated that NDRG2 expression loss is significantly
correlated with aggressive tumor behaviors such as late
tumor-node-metastasis (TNM) stage (P = 0.012), differentiation grade (P = 0.024), portal vein thrombi (P = 0.011),
infiltrative growth pattern (P = 0.015), nodal/distant metastasis (P = 0.027), and recurrent tumor (P = 0.021), as well as
shorter patient survival rates. Ectopically expressed NDRG2
suppressed invasion and migration of a highly invasive cell
line, SK-Hep-1, and experimental tumor metastasis in vivo,
whereas small interfering RNA–mediated knockdown resulted
in increased invasion and migration of a weakly invasive cell
line, PLC/PRF/5. In addition, NDRG2 could antagonize transforming growth factor B1–mediated tumor cell invasion by
specifically down-regulating the expression of matrix metalloproteinase 2 and laminin 332 pathway components, with
concomitant suppression of Rho GTPase activity. These results
suggest that NDRG2 can inhibit extracellular matrix–based,
Rho-driven tumor cell invasion and migration and thereby
play important roles in suppressing tumor metastasis in HCC.
[Cancer Res 2008;68(11):4210–20]
Tumor metastasis and recurrence represent the most critical
factors affecting the prognosis of cancer patients. However, most
solid tumors suffer from the lack of therapies to control metastasis
formation and tumor recurrence. Metastasis is a complex,
multifaceted process involving tumor invasion, tumor cell dissemination, colonization of distant organs, and metastatic outgrowth
(1–4). These processes are mediated by the genes regulating diverse
molecular pathways such as cell invasiveness, motility, survival,
proliferation, angiogenesis, and others (2–4). Efforts to define
tumor metastasis in molecular terms have identified a number of
metastasis-promoting genes, such as transforming growth factor
(TGF)-h (5), vascular endothelial growth factor (6), and matrix
metalloproteinases (MMP; ref. 7), as well as metastasis suppressors,
such as KAI1 (8), NM23 (9), and KISS1 (10); yet, the understanding
of the molecular mechanism underlying metastasis is far from
complete.
Both the positive and negative regulators of tumor metastasis
have a clinical significance as the diagnostic marker and/or target
of therapeutic intervention. Positive regulators are usually upregulated in metastatic cancers, and thus preferred over metastasis
suppressors as the marker for predicting metastasis (4, 11). On the
other hand, both inhibiting the positive regulators of metastasis
and activating the metastasis suppressors are regarded as valid
means of preventing metastasis formation because disruption of
any step in the metastatic cascade could render metastatic cells
nonmetastatic (4, 12).
Several different approaches have been used to identify
metastasis suppressor genes (13). Typically, candidate metastasis
suppressors were initially discovered from differential gene
expression analyses [differential display (14), microarray (15), and
subtractive hybridization (9)], genetic analysis (microcell-mediated
chromosome transfer; ref. 16), clinical correlation analysis (17), or
combinations thereof (10). They were then characterized in vitro
and/or in vivo for their effects on invasiveness, motility, tumor
growth, and metastasis.
As an attempt to identify novel metastasis suppressor genes
having a clinical relevance with hepatocellular carcinoma (HCC), we
searched for the genes frequently down-regulated in HCC that are
also negatively associated with clinical parameters predictive of poor
prognosis, such as recurrence and tumor grade. We identified the
N-myc downstream regulated gene 2 (NDRG2) as one such candidate, and we provide in this report the biological evidence that it can
function as a negative regulator of tumor cell invasion and migration
and the clinical evidence that its down-regulation is associated with
aggressive tumor behaviors and poor prognosis of HCC.
Note: Supplementary data for this article are available at Cancer Research Online
(http://cancerres.aacrjournals.org/).
D.C. Lee and Y.K. Kang contributed equally to this work.
Requests for reprints: Young Il Yeom, Medical Genomics Research Center, Korea
Research Institute of Bioscience and Biotechnology, Daejon 305-333, Korea. Phone: 8242-860-4109; Fax: 82-42-879-8119; E-mail: [email protected].
I2008 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-07-5040
Cancer Res 2008; 68: (11). June 1, 2008
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NDRG2 Is a Novel Metastasis Suppressor of Liver Cancer
Materials and Methods
Reagents. TGF-h1 was obtained from R&D Systems, Inc. Synthetic small
interfering RNAs (siRNA) for NDRG2 (sense, 5¶-CCUACAUCCUGGCGAGAUATT-3¶ and antisense, 5¶-UAUCUCGCCAGGAUGUAGGTT-3¶) and green
fluorescent protein (GFP; sense, 5¶-GUUCAGCGUGUCCGGCGAGTT-3¶ and
antisense, 5¶-CUCGCCGGACACGCUGAACTT-3¶) were purchased from
Samchully Pharm Co., and EZ-Detect Rho activation kit was from Pierce,
Inc. Monoclonal antibody (mAb) to NDRG2 protein was prepared from a
hybridoma cell line established in-house by fusing the splenocytes of BALB/
c mice immunized with recombinant NDRG2 protein with the SP2/0-Ag14
murine myeloma cells, and described in U.S. patent 11/3938979.
Generation of stable transfectants overexpressing NDRG2. For
overexpression of NDRG2, the coding region (nucleotides 150–1,222) of
NDRG2 cDNA was inserted in-frame into pcDNA3.1/myc-his vector
(Invitrogen). To prepare stable transfectants, SK-Hep-1 or PLC/PRF/5 cells
were seeded in a six-well dish at 1 105 per well and transfected with
pcDNA3.1/myc-his vector containing NDRG2 cDNA or a control vector
using Lipofectamine Plus reagent (Invitrogen). After culturing in complete
medium containing G418 (Invitrogen) for f2 wk, individual clones were
isolated. For in vivo metastasis experiments, SK-Hep-1 cell lines stably
overexpressing NDRG2 were established using a commercial lentiviral
expression system following the supplier’s instruction (Invitrogen).
Reporter assay. Cells were seeded at a density of 5 104 per well in
12-well plates and transfected with 0.5 Ag DNA (0.4 Ag 3TP-Luc and 0.1 Ag
renilla-Luc) per well using Lipofectamine Plus reagent according to the
supplier’s protocol. The next day, cells were stimulated with TGF-h1 and,
24 h later, harvested and lysed in 100 AL of reporter lysis buffer (Promega).
Luciferase activity was measured using the luciferase assay system
(Promega) in a luminometer (Lumat LB9501, Berthold). Renilla luciferase
was used as the internal control for normalization of transfection efficiency.
Luciferase assays were done in duplicates, and each experiment was
repeated at least twice.
Tissue arrays and immunohistochemical analysis. For tissue array,
surgically resected samples from patients with pathologically defined HCC
were collected by Inje University Seoul Paik Hospital, Seoul, Korea. All
tissues were routinely fixed in 10% buffered formalin and embedded in
paraffin blocks. Contents of the experiments involving human tissue
specimen were reviewed and approved by the Internal Review Board. Core
tissue biopsies (2 mm in diameter) were taken from individual paraffinembedded HCC (donor blocks) and arranged in a new recipient paraffin
block (tissue array block) using a trephin apparatus (Superbiochips
Laboratories). The resulting tissue array blocks (TA 128 and TA129)
contained 100 HCCs and 20 nonneoplastic liver tissues. Four-micrometerthick sections of the tissue array blocks were subjected to immunohistochemical study. Following deparaffinization and antigen retrieval, the slides
were labeled with a mAb to NDRG2. Labeling was detected using the
avidin-biotin complex (ABC) method. 3,3¶-Diaminobenzidine was used as
a chromogen. Normal saline was used as a substitute for the primary
antibody for the negative control reaction. NDRG2 expression was scored as
positive if z10% of the cells showed moderate to strong staining, weak if
either cytoplasmic or membranous staining was noted in <10%, and
negative if neither cytoplasmic nor membranous staining was seen. For the
statistical analysis of the tissue array data, categorical variables were
compared using the m2 test, and analyzed with SPSS statistical software
version 11.0.1. Survival rates were compared by Kaplan-Meier test ( for
univariate analysis) and Cox proportional hazards regression ( for
multivariate analysis) using the same software. P < 0.05 was regarded as
significant.
Reverse transcription-PCR. Reverse transcription-PCR (RT-PCR)
experiments were carried out according to the standard protocol. The
sequences of PCR primers are described in Supplementary Table S1. The
optimized PCR conditions were as follows: 1 cycle at 95jC for 30 s;
30 (semiquantitative) or 45 (real-time) cycles at 95jC for 30 s, 60jC for 30 s,
and 72jC for 30 s; and a final extension at 72jC for 10 min. Specificity of the
real-time RT-PCR amplification was checked by a melting curve analysis
( from 50jC to 90jC) using SYBR premix Ex Tag (TaKaRa Bio) in Exicycler
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(Bioneer). Relative gene expression levels were calculated as 2 DCT after
normalization against h-actin, where C T was defined as the threshold cycle
number of PCR at which amplified product was first detected.
Western blotting. Cells were lysed in a lysis buffer [1% Triton X-100,
150 mmol/L NaCl, 100 mmol/L KCl, 20 mmol/L HEPES (pH 7.9), 10 mmol/L
EDTA, 1 mmol/L sodium orthovanadate, 10 Ag/mL aprotinin, 10 Ag/mL
leupeptin, and 1 Amol/L phenylmethylsulfonyl fluoride] on ice. Western blot
analyses were carried out according to the standard protocol using
nitrocellulose membranes (Bio-Rad). For immunoblotting, membranes were
incubated with the primary antibody (1:5,000) for 2 h, followed by
1-h incubation with a 1:5,000 dilution of horseradish peroxidase (HRP)–
linked secondary antibody. Finally, the immunoreactive proteins were
detected by enhanced chemiluminescence assay with HRP (Pierce).
Bisulfite sequencing and methylation-specific PCR. Genomic DNA
modification with sodium bisulfite was done as previously reported (18).
The bisulfite-modified DNA was resuspended in 30 AL of sterile water, and
10 nanograms were amplified in 5% DMSO, 20 mmol/L Tris-HCl (pH 8.8),
2 mmol/L MgCl2, 10 mmol/L KCl, 1.25 mmol/L deoxynucleotide
triphosphates, 400 nmol/L of primer pair, and 5 units of Taq DNA
polymerase (Roche). Cycling conditions were as follows: preincubation at
94jC for 5 min, followed by 35 cycles of 94jC for 30 s, 54jC for 30 s, and
72jC for 30 s, and a final extension of 5 min at 72jC. PCR products were
purified and cloned into pGEM-T-Easy Vector System (Promega) for DNA
sequencing analysis. Primer sequences for the bisulfite sequencing and
methylation-specific PCR of the NDRG2 promoter region are described in
Supplementary Table S1. PCR reaction using the unmethylated primer set
was carried out at the following conditions: hot start at 95jC for 6 min,
followed by 35 cycles of 94jC for 30 s, 52jC for 30 s, and 72jC for 30 s, and
final extension at 72jC for 10 min. The PCR conditions for methylated
primer set were the same except for the annealing temperature (56jC).
Gelatin zymography. Gelatin zymography was carried out to determine
the activities of MMP2 and MMP9 expressed in mock- or NDRG2transfected PLC/PRF/5 cells after stimulating them with TGF-h1
(5 ng/mL) for 48 h. Briefly, spent culture media were loaded to 10% SDSpolyacrylamide gel containing 1 mg/mL gelatin (Sigma) under nonreducing
conditions. Following electrophoresis, the gels were washed twice with 2.5%
Triton X-100 (Sigma) to remove SDS and then incubated in a zymographic
buffer [0.5 mol/L Tris-HCl (pH 7.5), 50 mmol/L CaCl2 and 2 mol/L NaCl]
overnight at 37jC. Gels were stained with 0.05% Coomassie brilliant blue
R-250. Gelatinase activity was visualized as clear bands against the bluestained gelatin background.
Determination of Rho GTPase activity. Rho GTPase assay was done
according to the manufacturer’s protocol (Pierce). Briefly, cells were serum
starved for 24 h and then stimulated with TGF-h1 (5 ng/mL) for 15 min.
The cells were washed with ice-cold TBS [25 mmol/L Tris-HCl (pH 7.5) and
150 mmol/L NaCl] and lysed in lysis/binding/wash buffer [25 mmol/L TrisHCl (pH 7.5), 150 mmol/L NaCl, 5 mmol/L MgCl2, 1% NP40, 1 mmol/L DTT,
and 5% glycerol]. Cell lysates (500 Ag) were incubated with glutathione
S-transferase-rhotekin-Rho binding domain (400 Ag) at 4jC for 24 h. The
GTP-bound form of Rho protein was detected by immunoblotting with antiRho antibody.
Migration and invasion assays. Cell migration and invasion assays
were done essentially as previously described (19). For migration assay, the
under surface of Transwell filter inserts (Corning Costar) was precoated
with fibronectin at a concentration of 20 Ag/mL. The Transwells were then
assembled in a 24-well plate, with the lower chamber filled with 800 AL of
serum-free medium and the upper chamber inoculated with 200 AL of cells
(5 104/mL). For the invasion assay, cells (5 104/mL) were seeded onto
Transwell filters coated with Matrigel (upper side of the filter) and
fibronectin (underside of the filter). The Transwell was then left in a CO2
incubator for 24 h. Cells that invaded into the lower surface of the filters
were fixed with methanol and stained with H&E (Sigma-Aldrich). The
number of migrated or invaded cells per microscopic field was counted
at 50 magnification.
Metastasis analysis in a mouse model. In vivo metastasis experiments
were done using 4-wk-old female BALB/c nu/nu mice (Japan SLC). Mice
were anesthetized by inhalation with diethyl ether, and spleens exteriorized
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Figure 1. Pattern of NDRG2 expression in HCC. A, analysis of the expression pattern of NDRG2 in nontumor (NT ) and tumor (T ) tissues of HCC using cDNA
microarrays. The NDRG2 expression value reflects the log 2 value for the ratio of NDRG2 mRNA level in HCC relative to that in normal liver (NL ), which was used
as the common control. B, validation of the cDNA microarray data for NDRG2 by real-time RT-PCR. A total of 20 sets of paired tumor-nontumor samples of HCC
were used in the analysis (10 pairs each from recurrent and nonrecurrent HCCs). The relative expression value (T/NT) was calculated after normalizing the mRNA
level of NDRG2 against that of h-actin in each sample as described (50). Grade 1 to Grade 4, Edmondson’s histologic tumor grades. C, Western blot analysis of
NDRG2 protein expression in HCC tissues. Specificity of the anti-NDRG2 mAb was confirmed against transiently overexpressed, Myc-tagged NDRG2 with antibodies
against Myc epitope or recombinant NDRG2 protein (left ). Right, results of Western blot analysis of NDRG2 protein in paired tumor-nontumor tissues of HCC with
the anti-NDRG2 antibody. Two normal liver samples were included as the control. h-Actin was used as a loading control.
via a flank incision. SK-Hep-1 cells mock infected or infected with NDRG2lenti virus (1 106 in 100 AL) were injected into the lower polar side of
spleen through a 27-gauge needle, and splenectomies done 1 min later. All
animals were sacrificed at 4 wk, and the metastatic nodules in the liver
counted.
Results
NDRG2 expression is significantly reduced in HCC. To
identify the potential suppressors of tumor metastasis in HCC,
we first analyzed the gene expression profiles of 57 tumor and 41
nontumor liver tissues of HCC patients using cDNA microarrays
(total 49K), and identified the genes that are frequently downregulated in HCC by significance analysis of microarray (unpaired
two-class; Supplementary Table S2; ref. 20). We then estimated the
potential clinical significance of these genes by correlating their
expression pattern with the clinical parameters relevant to tumor
metastasis (Supplementary Table S3). Based on the reasoning that
potential metastasis suppressors rank high in terms of the degree
and the clinicopathologic significance of down-regulation in
tumors, we chose NDRG2 as one of the best candidates fulfilling
Cancer Res 2008; 68: (11). June 1, 2008
these criteria (Fig. 1A; Supplementary Tables S2 and S3). The top
three most down-regulated genes, SLC22A3, LCAT, and LOC401022,
were not selected for further consideration because they were
much less significantly associated with the clinicopathology of
HCC than NDRG2 (Supplementary Table S3) and less likely to play
regulatory roles in tumor metastasis according to their known
functions or the functional structure of the predicted protein
product (data not shown). In contrast, NDRG2 exhibited an
extensive negative correlation with various clinical features of HCC
progression, such as metastasis (P = 0.01167 for extrahepatic),
recurrence (P = 0.0084), invasion (P = 0.00161–0.01887 for various
forms of invasion), extranodular growth (P = 0.00927), tumor size
(P = 0.00601), and Edmondson’s tumor grade (P = 0.00023;
Supplementary Table S3). Moreover, its amino acid sequence is
highly related to that of NDRG1 (57% homology), which is known to
have roles in stress responses, cell proliferation and differentiation,
and suppression of tumor metastasis (14, 21, 22). We carried out a
real-time RT-PCR analysis for 20 paired tumor-nontumor samples
from the HCC patients who had undergone partial hepatectomy,
and confirmed that NDRG2 expression was significantly reduced in
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NDRG2 Is a Novel Metastasis Suppressor of Liver Cancer
recurrent tumors (P = 0.00775) and advanced histologic grades
(P = 0.02142; Fig. 1B). We also found from a Western blot analysis
that the NDRG2 protein levels in recurrent and high-grade tumors
were mostly lower than those in nonrecurrent and low-grade
tumors (Fig. 1C). Therefore, we conclude that NDRG2 expression is
significantly down-regulated in HCC at both mRNA and protein
levels in a manner negatively associated with aggressive tumor
behaviors.
Evaluation of the clinical significance of NDRG2 by
immunohistochemical analysis of liver cancer tissues. We
extended our analysis on the clinical significance of NDRG2 by
immunohistochemically examining the protein expression in a
larger number of HCC tissues collected from a cohort distinct from
the one used for cDNA microarray analysis. In a preliminary
analysis with nonneoplastic liver tissues, NDRG2 protein was found
to be expressed in the cytoplasm and plasma membrane of
hepatocytes and bile duct epithelial cells with a moderate to strong
staining intensity (Fig. 2A, a). In tumor cells, NDRG2 showed
variable expression patterns, comparable to or much weaker
than its level in nonneoplastic liver tissues or not detectable at all
(Fig. 2A, b and c). Interestingly, in the boundary between HCC and
nonneoplastic liver tissues, NDRG2-negative HCC cells often
seemed to be infiltrating into adjacent nontumor tissues expressing
NDRG2 (Fig. 2A, d).
The clinicopathologic significance of NDRG2 down-regulation in
HCC was evaluated by correlating its expression pattern with
Figure 2. Immunohistochemical analysis
of NDRG2 protein expression in liver
tissues and survival analysis of HCC
patients in relation to NDRG2 expression
pattern. A, immunohistochemical analysis.
a, nonneoplastic liver tissue showing
strong positive stainings in cytoplasm and
membranes of hepatocytes. b and c,
HCCs with positive (b) and negative (c )
staining. d, image of NDRG2-negative
HCC cells (T ) infiltrating into adjacent
nonneoplastic liver tissue (NT ).
Immunoperoxidase staining was done with
the ABC method. Original magnification,
400 (a–c ); 200 (d). B, correlation
of the overall survival rate of HCC patients
with NDRG2 expression pattern. Curves
were estimated using the Kaplan-Meier
method (P = 0.017). Continuous line
NDRG2-positive group; dotted line
negative/weak group. C, multivariate
survival analysis for predictive factors of
overall survival using the Cox regression
model. The incorporated clinical variables
were selected from the factors showing
prognostic value by univariate analysis.
Variables such as tumor size, portal vein
thrombi, intrahepatic metastasis, and
lymph node/distant metastasis were
not included because they already
played a role in defining TNM stage
(American Joint Committee on Cancer,
http://www.cancerstaging.org/). *, liver
functional status: B/C (n = 13) versus A
(n = 87).
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Table 1. Clinicopathologic features of HCC in relation to
the NDRG2 expression pattern
Clinicopathologic parameter
NDRG2 expression*
Negative/weak
Age (y)
Young (V53)
Elderly (z54)
Sex
Male
Female
Tumor size
Small (V5)
Large (z5)
c
TNM stage
I-II
III-IV
Edmondson grade
Grade 2
Grade 3/4
Portal vein thrombi
Absent
Microscopic
Gross
Growth pattern
Expansile
Infiltrative
Intrahepatic metastasis
Absent
Present
Lymph node or distant metastasis
Absent
Present
Recurrent HCC
Absent
Present
Serum anti-HBV
Negative
Positive
Serum anti-HCV
Negative
Positive
P
Positive
32
30
15
23
0.240
51
11
35
3
0.171
30
32
21
17
0.507
23
39
24
14
0.012
13
49
16
22
0.024
20
20
22
20
13
5
0.011
40
22
33
5
0.015
35
26
27
11
0.174
46
16
35
3
0.027
13
41
18
20
0.021
18
44
10
28
0.770
54
6
35
2
0.427
*NDRG2 expression was determined by immunohistochemical staining of HCC tissues on a tissue microarray using a mAb to NDRG2.
cThe stage was determined by the TNM staging criteria of the 6th
edition of American Joint Committee on Cancer (http://www.
cancerstaging.org/).
clinical parameters (Table 1). Of the 100 HCCs examined, 38
exhibited a positive NDRG2 expression in tumor cells, whereas in
62 HCCs, the NDRG2 staining was not detectable at all (negative;
n = 24) or at a much lower level (weak; n = 38). For statistical
analysis, the HCC samples were categorized into two groups
(negative/weak or positive) according to the NDRG2 expression
level. We found a significant negative relationship between the
NDRG2 level and TNM stage (P = 0.012) or Edmondson’s histologic
grade (P = 0.024; Table 1). Down-regulation of NDRG2 expression
was also significantly associated with invasive tumor features such
as the presence of portal vein thrombi (P = 0.011), infiltrative tumor
Cancer Res 2008; 68: (11). June 1, 2008
growth pattern (P = 0.015), nodal/distant metastasis (P = 0.027),
and tumor recurrence (P = 0.021). However, NDRG2 expression was
not significantly associated with age, sex, tumor size, serum antiHBV, or serum anti-HCV.
We next examined whether NDRG2 down-regulation is correlated with HCC patient survival and found that the ‘‘negative/weak’’
group had significantly less favorable overall survival rates than the
‘‘positive’’ group (P = 0.017; Fig. 2B). A multivariate analysis
including NDRG2 expression status, patient’s age, TNM stage,
histologic grade, growth pattern, and liver functional status (ChildPugh stage) also revealed that NDRG2 expression loss was a
statistically significant factor in predicting poor overall survival
(hazard ratio, 2.390; P = 0.038) but next only to TNM stage and
Child-Pugh stage (Fig. 2C; Supplementary Table S4).
These results confirm that NDRG2 down-regulation is associated
with advanced and aggressive tumor behaviors that are relevant to
tumor metastasis and survival in HCC.
Methylation pattern of the CpG sites of NDRG2 promoter
sequence in liver cancer cell lines and primary tumors. To
understand the molecular mechanism of the predominant
transcriptional repression of NDRG2 in HCC, we investigated the
DNA methylation status of a CpG-rich region encompassing the
proximal promoter and exon 1 of NDRG2 ( 421 to +100
nucleotides) through bisulfite sequencing and methylation-specific
PCR (Fig. 3A). Faithful cytosine-thymine conversion was confirmed
from the bisulfite sequencing data by the lack of cytosine residues
in non-CpG sites in HepG2 cells and normal liver tissue (Fig. 3B,
top). Several methylated CpG sites were detected within the
NDRG2 promoter in HepG2 cells but not in the normal liver tissue.
Hypermethylation of the NDRG2 promoter region was also
observed in SK-Hep-1, SNU-354, and SNU-368 cell lines (Fig. 3B,
bottom). The methylated CpG sites were highly concentrated in
CpG #1–16 in all the cell lines examined, allowing the design of a
methylation-specific PCR primer set for a simpler detection of
promoter hypermethylation. Further methylation-specific PCR
analysis using the primer set shown in Fig. 3A revealed that
Hep3B, PLC/PRF/5, and SNU-182 cell lines were also hypermethylated at the NDRG2 locus (Fig. 3C, top). RT-PCR analysis
showed that NDRG2 mRNA expression is much repressed in these
hypermethylated cell lines compared with normal liver (Fig. 3C,
bottom). These results indicate that the transcriptional repression
of NDRG2 in liver cancer cell lines is highly correlated with
promoter hypermethylation.
To determine whether the aberrant hypermethylation of the
NDRG2 promoter region also occurs in primary HCC tissues, we
investigated the DNA methylation status in nine paired tumornontumor tissues by methylation-specific PCR. The NDRG2
promoter sequences were frequently hypermethylated in tumor
tissues in contrast to the nontumor tissue of the same individual
(7 of 9 cases; Fig. 3D, top). Some methyl CpG sites were also
detected in nontumor tissues but the frequency was much lower
than in tumor tissues. We also found from a RT-PCR analysis that
the expression pattern of NDRG2 mRNA in HCC tissues was
inversely related to the extent of promoter methylation (Fig. 3D,
bottom). These findings indicate that promoter hypermethylation
of NDRG2 is tumor specific and represents one of the major
mechanisms for silencing NDRG2 expression in HCC.
NDRG2 can suppress tumor cell invasion and migration. The
expression pattern of NDRG2 in HCC and its association with
clinical parameters relevant to malignant tumor progression and
recurrence prompted us to explore its possible role in tumor
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NDRG2 Is a Novel Metastasis Suppressor of Liver Cancer
metastasis. We first examined the effect of NDRG2 on tumor cell
invasion in two different liver cancer cell lines, PLC/PRF/5 and SKHep-1, which were reported to be weakly and highly invasive,
respectively (23). As shown in Fig. 4A, forced expression of NDRG2
in these cell lines significantly suppressed their invasiveness
through Matrigel (P < 0.01). We also found that NDRG2 could
significantly suppress the migration of both cell lines (P < 0.01;
Fig. 4B). On the other hand, siRNA-mediated knockdown of
NDRG2 increased the motility and invasiveness of PLC/PRF/5 cells
to a significant level (Fig. 4C). The antimigratory activity of NDRG2
was confirmed by a wound healing assay in SK-Hep-1 cells that
stably express NDRG2 (data not shown).
We next examined the inhibitory effect of NDRG2 on the ability
of liver cancer cells to colonize the liver after entry into hepatic
portal system in vivo, using the splenic injection model (24). SKHep-1 cells injected into the spleen of nude mice metastasized to
the liver and formed a multitude of white colonies visible from
the surface (Fig. 4D). However, ectopic expression of NDRG2 in
these cells significantly reduced their colonization in the liver,
suggesting that NDRG2 can inhibit the metastasis formation of
SK-Hep-1 cells at a distant site in vivo. This result, together
with those of in vitro cell invasion and migration assays, indicates
that NDRG2 may be a novel suppressor of tumor metastasis
in HCC.
NDRG2 can antagonize TGF-B1 in inducing tumor cell
invasion. TGF-h1 is known to play pivotal roles in promoting
tumor cell invasion and metastasis and is overexpressed in
advanced cancers including HCC (25–27). We therefore examined
Figure 3. Methylation pattern of the promoter region of NDRG2 in HCC. A, schematic map of 32 CpG sites (vertical lines ) in the promoter region of the NDRG2
gene. The CpG sites were numbered from 421 bases upstream of the transcription initiation site (+1). The positions of methylation-specific PCR primers encompassing
five CpG sites are indicated by arrows (forward arrows , CpG 4, CpG 5, and 6; reverse arrows , CpG 11 and CpG 12). B, bisulfite sequencing of the NDRG2 promoter
region in normal liver tissue and liver cancer cell lines. Top, raw sequencing graphs for the bisulfite-treated NDRG2 promoter region. The original CpG sites are
underlined. Bottom, summarized results for bisulfite sequencing analysis of the NDRG2 promoter in normal liver and various liver cancer cell lines. Each circle indicates
a CpG site: open circle, unmethylated CpG; solid circle, methylated CpG. Numbers correspond to the position of CpG sites shown in A. C, methylation-specific
PCR analysis of the NDRG2 promoter in liver cancer cell lines (top ) and the corresponding RT-PCR data for the NDRG2 mRNA expressed in each cell line (bottom ).
The position of methylation-specific PCR primer set is indicated in A. U, unmethylated; M, methylated. D, methylation-specific PCR analysis of the NDRG2 promoter in
nine pairs of tumor (T ) and nontumor (NT ) tissues of HCC (top two rows ) and the corresponding expression pattern of NDRG2 mRNA as determined by RT-PCR
(last row ). Methylation-specific PCR primers are described in A . Normal liver was included as the control.
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Figure 4. NDRG2 suppresses tumor cell invasion and migration. A, invasion analysis of PLC/PRF/5 and SK-Hep-1 cells stably expressing NDRG2. Top, expression
pattern of NDRG2 protein in the cells stably transfected with a Myc-tagged NDRG2 expression vector. h-Actin was used as a loading control. Bottom, results of
invasion assays. The invasion ability was estimated using Transwell filters coated with Matrigel (upper side) and fibronectin (underside). Left, image of PLC/PRF/5 cells
that invaded through the Transwell filter (H&E staining); middle and right, quantified data for the invaded cells. TGF-h1 (2 ng/mL)–treated PLC/PRF/5 cells were
included as the positive control. **, P < 0.01, versus mock (t test). B, migration analysis of PLC/PRF/5 and SK-Hep-1 cells stably expressing NDRG2 . The migration
ability was estimated using Transwell filters coated with fibronectin. Left, image of PLC/PRF/5 cells that migrated through the Transwell filter (H&E staining);
middle and right, quantified data for the migrated cells. TGF-h1 (2 ng/mL)–treated PLC/PRF/5 cells were included as the positive control. **, P < 0.01, versus mock
(t test). C, analysis of cell migration and invasion after siRNA-mediated knockdown of NDRG2 in PLC/PRF/5 cells. The NDRG2 mRNA level in PLC/PRF/5 cells
transfected with siRNAs was analyzed by RT-PCR and shown with h-actin as control (top ). The effects of NDRG2-targeting siRNA on cell migration and invasion
were quantitatively estimated in comparison with those of GFP-targeting control siRNA (bottom ). *, P < 0.05, versus mock (t test). D, NDRG2 can suppress tumor
cell metastasis in vivo . Liver metastatic nodules were produced in BALB/c nu/nu mice by intrasplenic injection of SK-Hep-1 cells mock-infected or infected with
NDRG2 -lenti virus followed by splenectomy. Photograph of the liver of a mouse carrying metastatic colonies is shown along with the image of a tissue section stained
with H&E (left). Arrows, tumor nodules. Right, total number of liver metastatic nodules; n = 4 and 7 for the mock and NDRG2 transfectant groups, respectively.
*, P < 0.05, versus mock (t test). Expression of NDRG2 protein in SK-Hep-1 transfectants was analyzed by Western blotting and shown with that of h-actin control.
the regulatory relationship between TGF-h1 and NDRG2. TGF-h1
suppressed NDRG2 mRNA expression in PLC/PRF/5 cells while
augmenting the expression of the invasion/motility-inducing gene
plasminogen activator inhibitor type 1 (PAI-1; Fig. 5A, left). This
Cancer Res 2008; 68: (11). June 1, 2008
was also observed in HepG2 and HaCaT cell lines (data not shown).
In turn, NDRG2 inhibited the TGF-h1–mediated induction of PAI-1
expression. In a parallel experiment, we examined the effect of
NDRG2 on the TGF-h1 modulation of 3TP promoter, which was
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NDRG2 Is a Novel Metastasis Suppressor of Liver Cancer
derived from the PAI-1 gene promoter and contained multiple
copies of TGF-h responsive elements (28), and found that its
activity was significantly repressed by NDRG2 in PLC/PRF/5 cells
(Fig. 5A, right). We then examined the effect of NDRG2 on TGF-h1–
induced tumor cell invasion in PLC/PRF/5 cells and found that
NDRG2 could inhibit the TGF-h1 action in promoting tumor cell
invasion (Fig. 5B).
To understand the molecular basis of the NDRG2 antagonism
of TGF-h1 action, we analyzed the effect of NDRG2 on the
expression of the genes involved in tumor cell invasion in TGFh1–stimulated PLC/PRF/5 cells. TGF-h1–mediated expression of
epithelial-mesenchymal transition regulators such as E-cadherin,
snail, and slug was not changed by NDRG2 (data not shown).
However, NDRG2 significantly suppressed the TGF-h1–mediated
induction of MMP2, an extracellular matrix (ECM)–degrading
enzyme that plays roles in tumor cell invasion and activation of
latent extracellular TGF-h (ref. 7; Fig. 5C, top). We also examined
the biochemical activity of MMPs by gelatin zymography and
found that the culture supernatant of NDRG2-overexpressing
cells exhibited much weaker gelatinase activity compared with
control (Fig. 5C, bottom). We then analyzed the consequences of
NDRG2 expression on the laminin 332 ( formerly known as
laminin 5) pathway, which is known to play critical roles in
tumor cell invasion, migration, and survival during metastasis
(29). NDRG2 suppressed the TGF-h1 induction of h3 (LAMB3)
and g2 (LAMC2) subunits of laminin 332 and a3 chain (ITGA3)
of a3h1 integrin (Fig. 5D, left). Because the activation of Rho
GTPase activity by the engagement of a3h1 integrin with laminin
332 is crucial for tumor cell invasion (29), we examined the
effect of NDRG2 on Rho GTPase activity and found that both the
basal and TGF-h1–induced levels of GTP-bound, active Rho
protein were decreased by NDRG2 (Fig. 5D, right). These results
indicate that NDRG2 can block the Rho-driven tumor cell
invasion process mediated by TGF-h1 by suppressing the
induction of the genes involved in ECM degradation and ECMbased cell motility.
Figure 5. NDRG2 suppresses TGF-h1–induced tumor cell invasion. A, effect of NDRG2 on TGF-h1–induced gene expression. Mock- or NDRG2 -overexpressing
PLC/PRF/5 cells were stimulated with TGF-h1 (5 ng/mL) for 24 h, and the expression pattern of PAI-1 was analyzed by RT-PCR (left ). Right, effect of NDRG2
on the TGF-h1 modulation of 3TP-Luc reporter. Firefly luciferase activity was measured from the extracts of PLC/PRF/5 cells treated with the indicated amount of
TGF-h1 for 24 h. Renilla luciferase reporter was used as the internal control for transfection efficiency. Points, mean of three independent experiments; bars, SE.
B, effect of NDRG2 on TGF-h1–induced invasion of PLC/PRF/5 cells. The invasion ability of nonstimulated or TGF-h1 (5 ng/mL; 24 h)–stimulated cells was
estimated using Transwell filters coated with Matrigel (upper side) and fibronectin (underside). *, P < 0.05, versus mock (t test). C, effect of NDRG2 on the TGF-h1
modulation of gelatinase activities. Mock-transfected or NDRG2 -overexpressing PLC/PRF/5 cells were stimulated with TGF-h1 (5 ng/mL) for 24 h, and the mRNA
expression of gelatinase A (MMP2) and gelatinase B (MMP9 ) genes analyzed by RT-PCR (top ). Bottom, gelatin zymograms measuring the biochemical activity of
gelatinases in the corresponding culture supernatant. D, effect of NDRG2 on the TGF-h1 modulation of the laminin 332 pathway. PLC/PRF/5 cells were treated
with TGF-h1 as described in C , and the expression pattern of laminin 332 pathway components was analyzed by RT-PCR (left ). Right, effect of NDRG2 on the activity
of Rho GTPase, a critical intracellular signaling component of the laminin 332 pathway. The Rho GTPase activity was measured as the level of GTP-bound Rho
protein (Rho-GTP ) in the immunoprecipitated extracts of mock-transfected or NDRG2 -overexpressing PLC/PRF/5 cells. TGF-h1 stimulation was done at 5 ng/mL for
15 min and immunoblotting was carried out with anti-Rho antibody. Levels of total Rho protein in total cell extracts are shown as a loading control.
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Discussion
In this study, we investigated the biological function and clinical
significance of NDRG2 in HCC progression. We found that NDRG2
was frequently down-regulated in HCC and provided evidence
suggesting that this observation can have significant implications
in the negative regulation of tumor metastasis.
Several pieces of evidence suggest that NDRG2 might be a novel
candidate suppressor of tumor metastasis in HCC. First, downregulation of NDRG2 expression is frequently observed in primary
tumors and tumor cell lines. Our data showed that NDRG2
expression was significantly reduced in the majority of HCCs
compared with adjacent nontumor tissues or normal liver.
Moreover, NDRG2 remained down-regulated in the secondary
tumors formed during the metastatic progression of HCC
(Supplementary Fig. S1). Loss of NDRG2 expression has also been
reported in meningioma and glioma tissues and glioblastoma cell
lines (30, 31). Second, the loss of NDRG2 expression is significantly
associated with malignant tumor features with increased metastatic potential and poor prognosis. Immunohistochemical analysis
of NDRG2 protein expression in HCC by tissue microarray
indicated that NDRG2 expression loss is closely correlated with
portal vein thrombi, infiltrative growth pattern, nodal/distant
metastasis, and recurrence. We also found that recurrent tumors
derived from intrahepatic invasion exhibited significantly lower
levels of NDRG2 mRNA expression than those of multicentric
origins (P = 0.032; Supplementary Fig. S2). Previous reports showed
that reduced expression of metastasis repressors such as NM23
and KAI1 could be correlated with poor prognosis of tumors
(32, 33), and we found in the present study that NDRG2 downregulation is one of the independent poor prognostic factors by
multivariate survival analysis. Third, our functional studies showed
that NDRG2 can inhibit tumor cell invasion and migration on
reexpression or ectopic expression in highly invasive tumor cell
lines in vitro and in vivo. It could also antagonize TGF-h1–
mediated tumor cell invasion and migration with concomitant
down-regulation of molecular markers involved in ECM degradation and ECM-based cell motility. Finally, NDRG2 has an amino
acid sequence highly related to that of its homologue NDRG1 (57%
homology; ref. 22), which has been identified as a candidate
suppressor of metastasis in colon and prostate cancers (14, 21).
Together, these data indicate that NDRG2 might have a clinical
significance as a marker associated with negative regulation of
tumor metastasis in HCC.
The loss of NDRG2 expression could have been caused by a
number of genetic or epigenetic events during hepatocarcinogenesis. Frequent losses of chromosomes 1p, 4q, 6q, 8p, 13q, 16q,
and 17p have been reported in HCC, but the loss of 14q, where the
NDRG2 gene is located, was rarely observed (34). Therefore, the
major cause of NDRG2 expression loss in HCC could be either
changes in trans-acting factor activity or methylation-induced
promoter inactivation. Down-regulation of NDRG2 by N-Myc, as its
name implies, has not been reported yet, nor has there been any
report on the up-regulation of N-Myc in human HCC. Therefore,
N-Myc–mediated regulation is not likely a common mechanism of
NDRG2 down-regulation. Recently, c-Myc–mediated NDRG2 repression has been shown in HeLa and HEK293 cell lines (35).
However, we did not find a significant negative correlation
between the mRNA levels of c-Myc and NDRG2 in HCC tissues
(Supplementary Fig. S3). In the present study, we showed that the
CpG sites in the NDRG2 promoter were mostly hypermethylated in
Cancer Res 2008; 68: (11). June 1, 2008
liver cancer cell lines and primary tumors, whereas those in
normal liver tissues remained unmethylated. Loss of NDRG2
expression due to promoter hypermethylation has also been
reported in meningioma (30) although the position of CpG
methylation sites was somewhat different from what we observed
in HCC. We also found that the loss of NDRG2 expression in
primary HCC is carried on to the secondary tumors formed during
metastatic progression, likely due to inheritance of the promoter
hypermethylation pattern at NDRG2 locus (Supplementary Fig. S1).
Therefore, we conclude that methylation-induced promoter
inactivation might be the major cause of the frequent loss of
NDRG2 expression in HCC. However, for the other cases, NDRG2
expression loss could be due to changes in trans-acting factor
activities.
Our data provided the first functional evidence that NDRG2
can inhibit the invasion and migration of hepatoma cells. HCC is
known as an invasive cancer, with liver itself being the most
frequent target of metastasis (36–39). However, in HCC, tumor
cells grow embedded in a microenvironment with a high content
of ECM proteins, such as laminin, collagen, vitronectin and
fibronectin, as a consequence of the development of cirrhosis
(40). Therefore, crossing the ECM barriers by tumor cells is
regarded to be particularly important for metastatic progression
of HCC. Among the many different categories of molecules
regulating tumor metastasis, we focused on the effect of NDRG2
on the expression of the genes responsible for tumor invasion
such as epithelial-mesenchymal transition regulators, ECM
proteins and their cell-surface receptors, and ECM-degrading
enzymes. We found that NDRG2 can antagonize the function of
TGF-h1 in promoting tumor invasion and migration by
abrogating the induction of gelatinase activity and laminin 332
pathway. Gelatinases (MMP2 and MMP9) are important players
of tumor invasion that degrade ECM components and activate
the latent TGF-h present in extracellular space (7, 41). The
laminin 332 pathway is also known to play critical roles in tumor
cell invasion, migration, and survival during metastasis (42, 43).
The proteolytic processing of laminin 332 g2 chain via MMP2
exposes a cryptic promigratory site on laminin 332, providing a
trigger for cell migration and invasion (44, 45). Laminin 332 is
expressed do novo in HCC, absent in the peritumoral tissues (23),
and differentially expressed in metastatic and nonmetastatic
HCCs (46, 47). Major extracellular components of the laminin 332
pathway include laminin 332, integrins a3h1 and a6h4, collagen
XVII, and CD151 (29), and HCC cells expressing a3h1 integrin can
use laminin 332 as a preferential route to invade surrounding
tissues whereas those not expressing a3h1 integrin do not
migrate and invade (23, 36). In addition, recent reports suggested
that the a3 chain of integrin, but not a6, causes tumor cell
invasion and MMP activation in HCC, and that the TGF-h1
induction of a3 induces migratory and invasive phenotypes in
noninvasive HCC cell lines (48, 49). Our data showed that NDRG2
could suppress the TGF-h1–induced expression of a3h1 integrin
and h3/g2 subunits of laminin 332 in HCC cells. Moreover, in the
absence of TGF-h1 treatment, the basal levels of a3 integrin and
g2 subunit of laminin 332 were also reduced by NDRG2 in PLC/
PRF/5 cells (data not shown). As a result, NDRG2 blocked the
activation of Rho GTPase activity, which accompanies the
engagement of a3h1 integrin with laminin 332. However, NDRG2
did not alter the expression pattern of a6h4 integrin. These data
strongly suggest that the specific down-regulation of laminin 332
pathway components (a3h1 integrin and laminin 332) and MMP2
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NDRG2 Is a Novel Metastasis Suppressor of Liver Cancer
by NDRG2 collectively inhibits the ECM-based, Rho-driven tumor
cell invasion and migration, eventually playing crucial roles in
suppressing tumor metastasis in HCC. Our results also suggest
that NDRG2 may play important roles in antagonizing the
invasion-promoting activity of TGF-h1 during metastasis. We
think that these results might apply to a number of additional
cancer types other than HCC because NDRG2 is frequently downregulated in many other cancer types as well, according to public
gene expression databases such as CGAP7 and SOURCE.8 In fact,
we have observed that NDRG2 can also suppress the metastasis
formation of melanoma (B16F10) and colon cancer (SW480) cells
in mouse models of tumor metastasis (data not shown).
In summary, we reported the frequent loss of NDRG2 expression
in HCC caused by hypermethylation of promoter CpG sites, the
7
http://cgap.nci.nih.gov/Genes/RunUniGeneQuery?PAGE=1&ORG=Hs&SYM=
&PATH=&TERM=NDRG2
8
http://smd.stanford.edu/cgi-bin/source/expressionSearch?option=cluster&
criteria=Hs.525205&dataset=9&organism=Hs
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Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
Received 8/24/2007; revised 1/30/2008; accepted 3/27/2008.
Grant support: Grant-in-Aid for the 21C Frontier Human Genome Functional
Analysis Project from the Ministry of Science and Technology of Korea and by Korea
Research Institute of Bioscience and Biotechnology Research Initiative Program.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
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Functional and Clinical Evidence for NDRG2 as a Candidate
Suppressor of Liver Cancer Metastasis
Dong Chul Lee, Yun Kyung Kang, Woo Ho Kim, et al.
Cancer Res 2008;68:4210-4220.
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