© 2006 Japanese Cancer Association
Cancer Sci|February 2007|vol. 98|no. 2|169–173
Blackwell Publishing Asia
Downregulation of RECK by promoter methylation
correlates with lymph node metastasis in non-small
cell lung cancer
Huang-Chou Chang,1 Chun-Yu Cho2 and Wen-Chun Hung2,3,4
1Department of Chest Surgery, Kaohsiung Veterans General Hospital, 386, Ta-Chung 1st Road, Kaohsiung 813; 2Institute of Biomedical Sciences, National Sun
Yat-Sen University, 70, Lien-Hai Road, Kaohsiung 804; 3National Sun Yat-Sen University-Kaohsiung Medical University Joint Research Center, 70, Lien-Hai Road,
Kaohsiung 804, Taiwan
(Received September 3, 2006/Revised October 7, 2006/Accepted October 16, 2006/Online publication November 27, 2006)
In the present study, we addressed the molecular mechanism of the
downregulation of reversion-inducing-cysteine-rich protein with
Kazal motifs (RECK), a critical tumor suppressor that can potently
inhibit angiogenesis and metastasis, in non-small cell lung cancer
and its clinical significance. The methylation status of the RECK gene
promoter was studied by methylation-specific polymerase chain
reaction. RECK mRNA and protein levels were investigated by
reverse transcription–polymerase chain reaction and western blot
analysis. Downregulation of RECK was observed in 60% of the 55
tumors analyzed. Using methylation-specific polymerase chain
reaction analysis methylation of the RECK promoter was detected
in 63.6% (35/55) of the tumor tissues. A strong correlation between
downregulation and promoter methylation was found in these
tumors (P = 0.000005). More importantly, downregulation of RECK
significantly correlated with lymph node metastasis (P = 0.038).
Mutation of codon 12 of the K-ras gene was detected in 25.5% (14/
55) of lung tumor tissues. Statistical analysis indicated that K-ras
mutation was linked with RECK promoter methylation (P = 0.047)
and downregulation (P = 0.023). Promoter methylation was also
detected in human lung cancer cell lines, and the DNA methyltransferase
inhibitor 5′ ′ ′ ′-azacytidine reversed the expression of RECK and reduced
the invasive ability of these cell lines. Collectively, our results
suggest that downregulation of the metastasis suppressor RECK is
caused by promoter methylation in non-small cell lung cancer and is
associated with K-ras mutation and lymph node metastasis. (Cancer
Sci 2007; 98: 169–173)
gene that induced flat reversion in v-K-ras-transformed NIH/3T3
cells.(1) This gene encodes a membrane-anchored glycoprotein that
can negatively regulate matrix metalloproteinase-2 (MMP-2)
and MMP-9 activities and inhibit tumor angiogenesis and
metastasis.(2,3) Whereas RECK mRNA is highly expressed in
most normal human tissues and untransformed cells, it is
downregulated or undetectable in many tumor cell lines or in
cells ectopically expressing active oncogenes.(2) Pathological
studies have demonstrated that RECK downregulation is found
in human cancers including pancreatic cancer, breast cancer,
non-small cell lung cancer and colon cancer.(4–7) In addition,
reduced RECK expression correlates with poor prognosis in
these cancers. However, the molecular mechanism that causes
gene silencing of RECK in cancer cells is still unknown.
Mutations in the K-ras proto-oncogene are found frequently
in non-small cell lung cancer.(8) Aberrant activation of this oncogene
has been implicated in many aspects of malignant phenotypes
including proliferation, transformation, invasion and metastasis.
Numerous studies have shown that oncogenic ras increases the
metastatic ability of transformed cells.(9–11) However, the underly-
ing mechanism is poorly characterized. Recently, we addressed
he reversion-inducing-cysteine-rich protein with Kazal motifs
(RECK) gene was isolated as a transformation suppressor
the effect of oncogenic ras on RECK expression using an in vitro
inducible system.(12) Our results showed that ras activation
stimulates the expression of DNA methyltransferase 3b (DNMT3b),
promotes DNMT3b binding to the RECK promoter and
represses RECK expression via methylation of the promoter. We
also found that human lung cancer cells harboring K-ras muta-
tions exhibit increased promoter methylation and reduced
expression of the RECK gene.(12) To verify the results observed
in our previous study, we examined the methylation status of the
RECK promoter in 55 lung tumor tissues. Moreover, we studied
whether RECK promoter methylation correlated with K-ras
mutation and other clinicopathological parameters.
Materials and Methods
Tissues. Fifty-five paired normal and lung tumor tissues were
obtained from patients who underwent resection of tumors at the
Kaohsiung Veterans General Hospital. Detailed data about
patient- and tumor-related variables were collected by reviewing
the patients’ medical charts. Before acquisition of these tissues,
the investigational nature of this study was explained to the
patients, and informed consent was obtained. Portions of resected
tissues were quickly placed into RNAlater solution (Ambion,
Austin, TX, USA) and stored at −20°C until use. Tissues were
subjected to isolation of genomic DNA, total RNA and proteins
using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) accord-
ing to the manufacturer’s instructions.
Cell lines and 5′ ′ ′ ′-azacytidine treatment. Five cell lines were used
in the present study. MRC-5 cells are human embryonic lung
fibroblasts and were used as a normal control. H226 and H520
are squamous cell carcinoma cell lines and H358 and A549 are
adenocarcinoma cell lines. Cells were maintained routinely in
Dulbecco’s modified Eagle’s medium/F12 medium supplemented
with 10% fetal calf serum (FCS) and antibiotics. 5′-Azacytidine
(5 µM) was added into the culture medium and incubated for
48 h. Genomic DNA, total RNA and cellular proteins were ex-
tracted using TRIzol reagent and subjected to different analyses.
RNA extraction and reverse transcription–polymerase chain reaction.
Expression of RECK mRNA was investigated using the OneStep
reverse transcription–polymerase chain reaction (RT-PCR) kit
according to the manufacturer’s protocol (Qiagen, Valencia, CA,
USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
was used as an internal control to check the efficiency of cDNA
synthesis and PCR amplification. cDNA synthesis was carried
out at 50°C for 30 min with the following PCR conditions: 30
cycles of denaturation (94°C/1 min), annealing (60°C/1 min)
and extension (72°C/1 min), and one final cycle of extension
(72°C/10 min). The predicted sizes of the RECK and GAPDH
4To whom correspondence should be addressed. E-mail: email@example.com
© 2006 Japanese Cancer Association
PCR products were 477 and 512 bp, respectively. The sequences
of the primers were: RECK-forward, 5′-CCTCAGTGAGCACAG
TTCAGA-3′ RECK-reverse, 5′-GCAGCACACACACTGCTGTA-3′
GAPDH-reverse, 5′-TGTGGTCATGAGTCCTTCCA-3′. After
reaction, PCR products were separated on a 2% agarose
gel, stained with ethidium bromide and visualized under
Western blot analysis. Equal amounts of cellular proteins were
subjected to sodium dodecylsulfate–polyacrylamide gel electro-
phoresis as described previously.(13) Proteins were transferred
to nitrocellulose membranes and the blots were probed with
various primary antibodies. Enhanced chemiluminescence
reagents were used to detect the proteins on the membranes.
Anti-RECK antibody (Clone 32C10A) was purchased from
MBL (Nagoya, Japan) and anti-actin antibody (Clone C4) was
obtained from Chemicon (Temecula, CA, USA).
Methylation-specific PCR analysis. Genomic DNA was modified
with sodium bisulfite and analyzed according to the procedures
of the CpGenome DNA Modification Kit (Chemicon). We used
the following primers for the detection of human RECK promoter
methylation: M-sense, 5′-AATAAAGAGTTTTGGTACGGGGTAC-
3′ and M-antisense, 5′-AAAACCGCGAAATACTCGAA-3′ for the
methylated sequence of the human RECK promoter; U-sense,
5′-TAAAGAGTTTTGGTATGGGGTATGT-3′ and U-antisense,
5′-CTCCAAACCACAAAATACTCAAA-3′ for the unmethylated
sequence of the human RECK promoter. The predicted products
for methylated and unmethylated DNA were 195 and 199 bp,
respectively. Modified DNA was amplified in 50-µL reaction
mixtures containing 5 µL of 10× PCR buffer, 14 µL of 25 mM
MgCl2, 2.5 µL of 25 mM dNTP, 1 µL of each primer (300 ng/µL),
and 0.5 units of AmpliTaq Gold DNA polymerase (Roche,
Basel, Switzerland). PCR was carried out in a thermal cycler for
35 cycles (denaturation at 95°C for 1 min, annealing at 56°C for
2 min and extension at 72°C for 1 min), followed by a final 5-
min extension at 72°C. PCR products were separated on 2%
agarose gels, stained with ethidium bromide and visualized
under ultraviolet illumination.
In vitro invasion assay. The in vitro invasion assay was carried
out using 24-well transwell units with polycarbonate filters
(pore size 8 µM) coated on the upper side with Matrigel (Becton
Dickinson Labware, Bedford, MA, USA). Cells were collected,
and 5 × 103 cells in 100 µL of medium containing 5′-azacytidine
(5 µM) or antibodies was placed in the upper part of the
transwell unit and allowed to invade for 24 h. The lower part of
the transwell unit was filled with medium containing 10% FCS.
After incubation, non-invaded cells on the upper part of the
membrane were removed with a cotton swab. Invaded cells on
the bottom surface of the membrane were fixed in formaldehyde,
stained with Giemsa solution and counted under a microscope.
Statistical analysis. The associations between RECK and
clinicopathological parameters were assessed using the χ2-test and
Fisher’s exact test. Statistical significance was defined as P < 0.05.
RECK expression is downregulated in lung cancers. Tissues of 55
patients were included in the present study and the patients’
characteristics are described in Table 1. We first examined the
expression of RECK in paired normal and lung tumor tissues by
RT-PCR and western blot analysis. The signal intensities on the
blots were calculated and compared. The RECK protein level of
most of the tumor tissues investigated in the present study was
very low. However, three cases showed a marginal reduction (7–
12%) of RECK protein in tumor tissues. Therefore, we decided
to use 20% reduction as a cut-off value in our study. More than
20% reduction of RECK protein levels in tumor tissues was
defined as downregulation. Our results indicated that RECK was
downregulated in 60% (33/55) of tumor tissues. Representative
data are shown in Fig. 1 and the distributions of the RECK/actin
ratio of normal and tumor groups is demonstrated in Fig. 1C.
We next tested the correlation between RECK expression and
clinicopathological parameters. Our data indicated that RECK
downregulation was not associated with sex, age, T-stage, stage
or histology (Table 1). On the contrary, RECK downregulation
was significantly associated with increased lymph node
metastasis (Table 1).
RECK downregulation is associated with promoter methylation.
We next addressed whether downregulation of RECK in tumor
tissues was caused by promoter methylation, an epigenetic
Table 1.Correlation between reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) and clinicopathological parameters
(n = 55)
No change Downregulation
Squamous cell carcinoma
Large cell carcinoma
Lymph node metastasis
*P < 0.05. a: Due to the small case number of large cell carcinoma, the statistical significance was tested between adenocarcinoma and squamous
cell carcinoma groups.
Chang et al.
Cancer Sci| February 2007| vol. 98| no. 2|
© 2006 Japanese Cancer Association
alteration that frequently leads to gene silencing. Methylation-
specific PCR (MSP) analysis showed that RECK promoter
methylation was found in 63.6% (35/55) of lung tumor
tissues. Figure 2 demonstrates the methylation status of the
RECK promoter of four paired normal and lung tumor tissues. We
tested the correlation between RECK expression and pro-
moter methylation and our data indicated that downregulation
of RECK was strongly correlated with promoter methylation
(P = 0.000005; Table 2).
K-ras mutation is correlated with RECK promoter methylation and
downregulation. Our previous study demonstrated that oncogenic
ras increases the expression of DNMT3b and induces promoter
methylation of the RECK gene.(12) However, these findings
have not been verified in primary tumor tissues. Therefore, we
investigated the correlation between K-ras mutation and RECK
promoter methylation in lung tumors. Our results showed that
K-ras mutation was detected in 25.5% (14/55) of the 55 tumors
tested in the present study and was significantly correlated with
RECK promoter methylation (P = 0.047; Table 3). In addition, K-ras
mutation also correlated with RECK downregulation (P = 0.023).
DNMT inhibitor 5′ ′ ′ ′-azacytidine restores RECK expression and reduces
the invasive ability of human lung cancer cell lines. To examine the
role of promoter methylation in the silencing of the RECK gene
in lung cancer cells, we first investigated the methylation status
of the RECK promoter and its expression. As demonstrated in
Fig. 3A, MRC-5 human lung fibroblasts expressed high levels
of RECK mRNA and protein. RECK was also highly expressed
in H226 human lung cancer cells. In contrast, the expression of
RECK in H520, H358 and A549 lung cancer cells was very low
or undetectable. MSP analysis indicated that significant RECK
promoter methylation was found in these three lung cancer cells
but not in MRC-5 and H226 cells (Fig. 3B). We tested the effect
of the DNMT inhibitor 5′-azacytidine on H520, H358, A549
and H226 cells. Our results demonstrated that this drug restored
the expression of RECK and reduced the invasive ability of the
H520, H358 and A549 cell lines (Fig. 3C,D). Conversely, 5′-
azacytidine did not obviously increase the RECK protein level
in H226 cells (Fig. 3C). Only 20% inhibition of cell invasion
was observed in this cell line after drug treatment (Fig. 3D).
RECK is an important MMP inhibitor that is involved in the
regulation of various physiological and pathological processes.
Mice lacking RECK die in utero with developmental defects in
blood vessels, the neural tube and mesenchymal tissues.(3)
RECK is also a target of myogenic regulatory factors and
participates in the control of myogenesis.(14) These results
motifs (RECK) in human lung tumor tissues (T) and their normal
counterparts (N). Resected tissues placed in the RNAlater solution were
subjected to isolation of mRNA and proteins using TRIzol reagent.
(A) Reverse transcriptase–polymerase chain reaction assays were carried
out to investigate RECK mRNA levels. Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was used as an internal control. (B) The
protein level of RECK in N or T tissues was investigated by western blot
analysis and actin was used as an internal control. (C) The distribution
of the RECK/actin ratio of N and T tissues.
Expression of reversion-inducing-cysteine-rich protein with Kazal
with Kazal motifs (RECK) promoter in human lung tumor tissues.
Genomic DNA was isolated from lung tumors or their normal counterparts
and subjected to sodium bisulfite modification. Methylation-specific
polymerase chain reaction was used to detect the methylation status of
the human RECK promoter. The predicted products for methylated (M)
and unmethylated (U) DNA were 195 and 199 bp, respectively.
Methylation of the reversion-inducing-cysteine-rich protein
(RECK) downregulation was associated with promoter methylation
Reversion-inducing-cysteine-rich protein with Kazal motifs
No change Downregulation
cysteine-rich protein with Kazal motifs (RECK) downregulation and
K-ras mutation was associated with reversion-inducing-
Wild type Mutant
© 2006 Japanese Cancer Association
suggest that RECK is a key player in embryonic development.
Under pathological conditions, RECK has been shown to be
downregulated in human cancers.(4–7) Two recent studies demon-
strated that the 5-year survival rate of lung cancer patients with
tumors with strong RECK expression was significantly higher
than that of patients with weakly expressing tumors.(6,15) Therefore,
RECK is an important prognostic factor for lung cancer patients.
However, the molecular mechanism by which RECK was
downregulated in tumors has never been characterized. In the
present study, we provide the first evidence that silencing of the
RECK gene in lung cancer cells is caused by promoter methy-
lation. We also found that RECK downregulation is closely
linked with increased lymph node metastasis. In addition, we
showed that 5′-azacytidine (also named Vidaza), a DNMT
inhibitor that has recently been approved by the US Food and
Drug Administration (FDA) for treatment of myelodysplastic
syndrome,(16) may restore RECK expression and inhibit invasion
of cultured lung cancer cells.(12) Our results strongly support the
notion that RECK is a metastasis suppressor and a better
prognostic factor for lung cancer.
Another important finding of the present study is that RECK
promoter methylation and downregulation in lung cancer is
associated with K-ras mutation. We and others have demon-
strated that the ras oncogene represses RECK expression.(17,18)
Our recent study extended these works and revealed that
oncogenic ras acts through DNMT3b-mediated promoter
methylation to repress RECK expression.(12) Our hypothesis is
that oncogenic ras upregulates the expression of DNMT3b and
increases the binding of this protein to the RECK gene promoter,
which results in promoter methylation and gene silencing. To
verify this hypothesis, two issues should be proved. First, whether
ras mutation is indeed linked with RECK gene silencing in
primary tumors. We investigated this correlation in lung tumor
tissues and verified that K-ras mutation and RECK downregula-
tion are strongly correlated. Similar findings were observed
in cultured lung cancer cell lines. H358 and A549 cells have
been shown to harbor K-ras mutation.(19) In the present study,
the expression of RECK was low and the RECK promoter was
highly methylated. In contrast, H226 cells that harbor wild-type
K-ras exhibited high RECK expression and low promoter
methylation. The genetic alterations of H520 have been little
reported. H520 cells harbor the wild-type K-ras gene.(19) How-
ever, a very recent study demonstrated RASSF8 (RAS association
RalGDS/AF-6 domain family 8), a candidate tumor suppressor
gene involved in the RAS signaling pathway, is downregulated
in H520 cells.(20) Therefore, our results suggest that hyperactiva-
tion of the ras signaling pathway suppresses RECK expression
in vivo. The second issue is whether DNMT3b is upregulated in
human lung tumor tissues and whether its overexpression is
correlated with ras mutation. Two previous studies demon-
strated that DNMT3b promoter polymorphisms are linked with
an increased risk of lung cancer.(21,22) Recently, two independent
studies have shown that DNMT3b is overexpressed in human
lung cancer.(23,24) However, the status of ras mutation in tumor
tissues was not addressed in these two studies. Therefore, the
correlation between ras mutation and DNMT3b overexpression
is still unclear and needs to be investigated in future works.
Previous studies on the oncogenic activity of ras focused
mainly on its effect on cell proliferation. However, the effect of
ras on tumor metastasis is less clear. Recently, several potential
5′-azacytidine. (A) Human lung fibroblast (1, MRC-5) and lung cancer cell lines (2, H226; 3, H520; 4, H358; 5, A549) were harvested and subjected
to isolation of genomic DNA, RNA and protein by TRIzol reagent. Reverse transcription–polymerase chain reaction (RT-PCR) and western blot
analysis were carried out to investigate RECK expression. (B) The methylation status of the RECK promoter was also addressed by methylation-
specific polymerase chain reaction. M, methylated; U, unmethylated. (C) H520 (3), H358 (4), A549 (5) and H226 (2) cells were treated with 5′-
azacytidine for 48 h and RECK expression was examined by RT-PCR and western blot analysis. (D) In vitro cell invasion assays were carried out
and the data are shown as mean ± SD. A, 5′-azacytidine; C, control (0.1% dimethylsulfoxide). GAPDH, glyceraldehyde-3-phosphate
Downregulation of reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) in human lung cancer cell lines and its restoration by
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Cancer Sci|February 2007| vol. 98|no. 2|
© 2006 Japanese Cancer Association
mediators have been identified for ras-induced metastasis, including
cytoskeletal proteins,(25) integrins,(26) cadherins,(27) angiogenic
factors(28,29) and MMP. A genome-wide screening of ras targets
demonstrated that ras suppresses the expression of thrombospondin
1 and tissue inhibitor of metalloproteinase 1, two important
antimetastatic genes, to enhance cell invasion.(30) Pathological
studies have also shown that inactivation of RASSF1A, a
tumor suppressor that exhibits antimetastatic activity, is closely
linked with ras mutation in cancer cells.(31,32) We now identify
RECK as a molecular target for the ras oncogene and show that
downregulation of RECK is associated with K-ras mutation
and lymph node metastasis in non-small cell lung tumor tissues
This study was supported by grants VGHNSU94-003 and VGHNSU
95-001 to Huang-Chou Chang and a grant from National Sun Yat-Sen
University–Kaohsiung Medical University Joint Research Center to
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