Aberrant methylation of DACT1 and DACT2 are associated with tumor progression and poor prognosis in esophageal squamous cell carcinoma
Guo et al. Journal of Biomedical Science
Aberrant methylation of DACT1 and DACT2 are associated with tumor progression and poor prognosis in esophageal squamous cell carcinoma
Yan-li Guo 0
Bao-En Shan 0
Wei Guo 0
Zhi-Ming Dong 0
Zhen Zhou 0
Su-Peng Shen 0
Xin Guo 0
Jia Liang 0
Gang Kuang 0
0 Laboratory of Pathology, Hebei Cancer Institute, The Fourth Hospital of Hebei Medical University , Jiankanglu 12, Shijiazhuang 050011, Hebei , China
Background: The DACT (Dishevelled-associated antagonist of β-catenin) family of scaffold proteins may play important roles in tumorigenesis. However, the epigenetic changes of DACT1, 2, 3 and their effect on esophageal squamous cell carcinoma (ESCC) have not been investigated so far. The aim of this study was to investigate the promoter methylation and expression of DACT family, in order to elucidate more information on the role of DACT with regard to the progression and prognosis of ESCC. Methods: MSP and BGS methods were respectively applied to examine the methylation status of DACT; RT-PCR, Western blot and immunohistochemistry methods were respectively used to determine the mRNA and protein expression of DACT; MTT, Colony-formation and Wound-healing assay were performed to assess the effect of DACT1 and DACT2 on proliferation and migration of esophageal cancer cells. Results: Frequent reduced expression of DACT1, DACT2 and DACT3 were found in esophageal cancer cell lines and the expression levels of DACT1 and DACT2 were reversed by 5-Aza-Dc. Decreased mRNA and protein expression of DACT1 and DACT2 were observed in ESCC tumor tissues and were associated with the methylation status of transcription start site (TSS) region. The hypermethylation of CpG islands (CGI) shore region in DACT1 was observed both in tumor and corresponding adjacent tissues but wasn't related to the transcriptional inhibition of DACT1. The methylation status of TSS region in DACT1 and DACT2 and the protein expression of DACT2 were independently associated with ESCC patients' prognosis. Conclusions: The TSS region hypermethylation may be one of the main mechanisms for reduced expression of DACT1 and DACT2 in ESCC. The simultaneous methylation of DACT1 and DACT2 may play important roles in progression of ESCC and may serve as prognostic methylation biomarkers for ESCC patients.
Esophageal squamous cell carcinoma; DACT gene; Methylation; Prognosis
Esophageal cancer is the eighth most common malignancy
in the world and the overall prognosis for esophageal
cancer patients is very poor [1–3]. According to the WHO
report in 2012, the world’s new cases of esophageal cancer
were 455 thousands, and 223 thousands of them occurred
in China, accounting for 49% . In china, esophageal
squamous cell carcinoma (ESCC) was the dominant
histological type of esophageal malignances. Based on two
national mortality surveys conducted in 1970s and 1990s,
ESCC has a striking geographic distribution in China, with
higher prevalence in Taihang mountain areas of North
China . There was also a strong tendency toward
familial aggregation of ESCC in these high-risk areas ,
suggesting that multiple genetic and epigenetic events
may contribute to the occurrence and progression of
ESCC. However, the precise molecular mechanisms of the
development and progression of ESCC still remained
unknown. Therefore, additional elucidation of the molecular
mechanisms involved in ESCC and the discovery of early
detect biomarkers are urgently needed for more effective
chemoprevention or treatment.
The DACT (Dishevelled-associated antagonist of
βcatenin) family of scaffold proteins, including DACT1,
DACT2 and DACT3, were isolated by a screen for proteins
interacting with Dishevelled protein central to Wnt
signaling [7–9]. DACT encode a series of vertebrate intracellular
proteins those can regulate intercellular signaling
pathways [10–12]. According to previous reports, DACT
proteins act as tumor suppressors in different tumor types
[13–15]. In addition, decreased expression and promoter
hypermethylation of DACT1 and DACT2 have been found
in some primary tumors and tumor cell lines, including
hepatocellular carcinoma , breast cancer , lung
cancer , colorectal cancer  and some other
cancers. However, Schussel et al.  showed quite a different
result which the promoter methylation of DACT1 and
DACT2 may not be a common event in oral squamous
cell carcinoma. Although the promoter of DACT3 also
has a large CpG Island, but the study in colorectal cancer
indicates that the histone modification, rather than the
aberrant, may be the main regulated mechanism for
inactivation of this gene . To our best knowledge, the
regulation mechanism of DACT1, DACT2 and DACT3
and their function in ESCC remains unknown. In the
present study, we attempted to detect the role and
methylation status of DACT1, DACT2 and DACT3 in ESCC,
and to elucidate the effect of their methylation status on
progression / prognosis of ESCC patients.
(Invitrogen, Carlsbad, CA, USA) supplemented with 10%
heat-inactivated fetal bovine serum (FBS, Invitrogen,
Carlsbad, CA, USA), 100 U/mL penicillin, and 100 μg/
mL streptomycin. Treatment of these four cells with
DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine
(5-Aza-Dc, 5 μmol/L, Sigma, St Louis, MO, USA) and
histone deacetylase (HDAC) inhibitor trichostatin A
(TSA,0.3 μmol/L, Cayman Chemical Co, Ann Arbor, MI,
USA) was performed as previously reports [20, 21].
Control cells received no drug treatment. DNA, RNA and
protein were isolated from these cells.
Patients and specimens
One hundred fifty-nine paired primary tumor and
corresponding adjacent non-cancerous tissues of ESCC were
collected from the Fourth Affiliated Hospital, Hebei
Medical University between the years of 2005 and 2009.
All procedures performed in this study were in accordance
with the ethical standards of the institutional research
committee and with the 1964 Helsinki declaration and its
later amendments or comparable ethical standards. The
study was approved by the ethics committee of Hebei
Medical University Fourth Affiliated Hospital, and the
informed consent was obtained from all of the patients.
All study subjects were ethnically homogeneous Han
nationality and were from the same areas, which were the
high-risk areas of upper gastrointestinal cancers (UGIC)
in Hebei province. The incidence of UGIC in this area was
over one hundred per one hundred thousands [22, 23].
Complete clinicopathologic characteristics and UGIC
family history of these cases were available before
operation and during follow-up. Individuals with at least one
first-degree relative or at least two second-degree relatives
having esophageal/cardia/gastric cancer were defined as
having family history of UGIC. Distribution of
clinicopathological data in the study cohort was shown in Table 1.
For this study, all of the subjects were re-examined and
confirmed by professional pathologists for
histopathological diagnosis. All the esophageal cancer were
squamous cell carcinoma and the adjacent non-cancerous
tissues were normal tissues or hyperplasia tissues,
which confirmed by microscope examination. The
survival data were ascertained through the Tumor Registry
and Hospital chart review. Fourteen patients were lost
to follow up.
Stable gene transfections
Transfection was done using FuGENE HD transfection
reagent (Roche, Swiss) as recommended by the
manufacturer’s instructions. Full-length DACT1 and DACT2 cDNA
were cloned into pcDNA3.1 and pCMV6 vector. Then,
exponentially grown TE1 and TE13 cells were transfected
with DACT1 and DACT2 expression plasmid
(pcDNA3.1DACT1 and pCMV6-DACT2) or the relevant empty
positive n (%) p
positive n (%) p
The TE1 or TE13 cells, the stable transfected cells
(pcDNA3.1-DACT1/TE1,TE13 and pCMV6-DACT2/
TE1,TE13) and the cells treated with 5-Aza-Dc for
72 h were seeded in 24-well plates at the
concentration of 2 × 105 per well and allowed to form a
confluent monolayer for 24 h, and then scratched with a
pipette tip and washed to remove floating cells. Cells
were photographed at the same field every 2 h.
DACT1, DACT2, DACT3 mRNA expression via
semiquantitative reverse transcription-polymerase chain
reaction (RT-PCR) and quantitative real-time RT-PCR assays
Total RNA was isolated from cell lines and tissues by
Trizol reagent (Invitrogen, Carlsbad, CA) according to
the manufacturer’s instructions. Agarose gel
electrophoresis and spectrophotometric analysis (A260: 280 nm
ratio) were used to evaluate RNA quality and quantity.
RT-for-PCR kit (Clontech, Palo Alto, CA) with random
priming as recommended in the protocol provided. All
primers and reaction conditions are listed in Additional
file 1: Table S1. The glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) gene was used as an internal control.
For RT-PCR, the PCR products were separated in 2%
agarose gel in electrophoresis and visualized with
ethidium bromide staining, and quantified using an image
analysis system (Gel work-2ID). Real time PCR reactions
were performed by the Stepone Plus Thermal Cycler
(Applied Biosystems, Foster City, CA, USA) and SYBR
green PCR Master Mix (Life Technology, Foster City, CA,
USA). The expression levels of target genes were
normalized with GAPDH using the 2-△△CT method . The
reaction was repeated in triplicate with each of the
samples for quality control.
Western blot analysis
Western blot was performed according to previous
report . Whole cell lysates from cell lines were
prepared by lysing the cells in ice-cold RIPA buffer.
The protein concentration was determined using the
BCA Protein Assay Kit (Pierce, Rockford, IL, USA),
and then, equal amount of proteins were separated by
SDS-PAGE and electroblotted onto PVDF membranes
(Millipore, Billerica, MA, USA). The membranes were
blocked in 5% nonfat milk and incubated with Rabbit
anti-human polyclonal antibody for DACT1 (1 μg/ml,
ab72078, Abcam, UK) and DACT2(2 μg/ml, ab79042,
Abcam, UK), and mouse anti-human monoclonal
antibody for β-actin (1:5000 dilution, ab6276, Abcam,
UK) and visualization with enhanced
chemiluminescence (ECL) detection reagents (Beyotime, Haimen,
Family history of UGIC
vector (pcDNA3.1/TE1 and pCMV6/TE1) as control. The
resistant cell clones were selected with G418 expanded for
Cell proliferation assay
Cell proliferation was measured with MTT assay. The TE1
or TE13 cells, the stable transfected cells
(pcDNA3.1DACT1/TE1,TE13 and pCMV6-DACT2/TE1,TE13) and
the cells treated with 5-Aza-Dc for 72 h were seeded in
96well plates (3 × 103) in 200 μl of RPMI 1640 with 10% FBS
under standard culture conditions (six wells/group). MTT
(Sigma-Aldrich, Saint Louis, MO, USA) in PBS (5 mg/ml)
was added (20 μl /well) and plates were incubated at 37 °C
for 4 h, after which culture media was removed, DMSO
was added and the absorbance was measured at 570 nm.
This experiment was repeated three times.
Colony formation assay
For colony assay, The TE1 or TE13 cells, the stable
transfected cells (pcDNA3.1-DACT1/TE1,TE13 and
pCMV6-DACT2/TE1,TE13) and the cells treated with
5Aza-Dc for 72 h were seeded at density 500 cells/35 mm
dish (three dishes/group), and incubated in normal
growth medium two weeks later, cell colonies were
stained with hematoxylin and counted.
DACT1, DACT2 protein expression via
immunohistochemical staining (IHC)
The protein expression of DACT1, DACT2 and DACT3 in
tumor and corresponding adjacent tissues of ESCC was
detected using the immunostaining method of
avidinbiotin complex immunoperoxidase . After blocking of
endogenous peroxidase and non-specific reactions, the
slides were incubated with rabbit anti-human polyclonal
antibody for DACT1 (1:100 dilution, abcam, UK), DACT2
(1:100 dilution, abcam, UK) and DACT3 (1:50 dilution,
AmyJet Scientific), and then incubated with biotinylated
secondary antibody and ABC reagent. 3,
3′-Diaminobenzidine (Sigma, St Louis, MO) was used as the chromogen,
and counterstaining was done using hematoxylin. In
negative controls, the primary antibody was replaced with
nonimmune serum for the negative control. Slides with
positive staining of DACT1, DACT2 and DACT3 were
used as positive control. Protein expression of DACT1,
DACT2 and DACT3 were evaluated according to a scoring
method reported previously . Scoring accounted for
both intensity and the percentage of positive cells. All
slides were examined and scored by three independent
observers, who were blinded to the clinical data.
Methylation analysis of DACT via methylation-specific
polymerase chain reaction (MSP) and bisulfite genomic
sequencing (BGS) methods
Total DNA was isolated from cell lines using DNAzol
(Invitrogen, Carlsbad, CA, USA) and the genomic DNA
from tissues of ESCC was prepared using the Proteinase K
digestion method. Genomic DNA was treated with Epitect
Fast Bisulfite Conversion Kits (Qiagen, Germany)
according to the manufacturer’s instructions. Unmethylated
cytosines was converted to uracil by bisulfite treatment,
whereas methylated cytosine couldn’t be converted and
remained as cytosine. Based on this potential difference in
the DNA sequence between methylated and unmethylated
alleles after bisulfite treatment, we designed MSP primers
to analyze two regions of DACT1, and one region of
DACT2 and DACT3. One of the regions in DACT1 was
encompassing TSS (region 2, from −16 ~ 105 bp) and
the other was located in the CGI shore (region 1, from
−540 ~ −419 bp), which was the flank of the traditional
CpG islands and had relatively low CpG density. The
methylation status of DACT2 (from −30 ~ 126 bp) and
DACT3 (from −154 ~ 28 bp) were determined in the
region encompassing TSS (Fig. 1a, b, c). The MSP
products were analyzed on 2% agarose gels with ethidium
bromide and visualized under UV illumination.
Genomic DNA, which was treated by CpG
methyltransferase (Sss I) following the manufacturer’s directions (New
England BioLabs, Inc, Beverly, MA), was used as
positive control. Water blank was used as a negative
control. Every sample was repeated methylation analysis in
duplicate in order to ensure the quality control. For
BGS, primers were designed to recognize sodium
bisulfite converted DNA and encompassing the MSP regions
(DACT1, BGS region 1 from −573 ~ −382 bp, BGS
region 2 from −196 ~ 376 bp; DACT2, from −30 ~ 226 bp;
DACT3, from −349 ~ 148 bp) (Fig. 1a, b, c). The primer
sequences of DACT1, DACT2, DACT3 and PCR
conditions (product size and annealing temperatures) were
shown in Additional file 1: Table S1. The target fragment
of PCR products for BGS were purified and cloned into
pGEM-T vectors (Promega, San Luis Obispo, CA) and 10
clones of each specimen were sequenced by automated
fluorescence-based DNA sequencing.
Statistical analysis was performed using SPSS19.0
software package (SPSS Company, Chicago, IL). The
methylation frequency of DACT1, DACT2, DACT3 between
ESCC and corresponding adjacent tissues were
compared using Chi-square test. The mRNA expression of
DACT genes was expressed as the mean ± standard
deviation and the means were compared using the Student’s
t test. Kaplan–Meier survival curves were constructed
and the Log-rank tests were used as needed for the
univariate comparison of DACT expression and
methylation categories. Cox’s multivariate test was used to adjust
for potentially confounding variables and to evaluate the
role of DACT as an independent predictor for patient
prognosis. Two-sided tests were used to determine
significance and P values < 0.05 was regarded as
statistically significant for all statistic tests.
Frequent silencing of DACT1, DACT2 and DACT3 in
esophageal cancer cell lines
The mRNA expression of DACT1, DACT2 and DACT3
were examined in four esophageal cancer cell lines by
RT-RCR and qRT-RCR methods. The mRNA expression
of DACT1 was silenced in TE13, T.Tn cell lines and
reduced in TE1, Eca109 cell lines; the mRNA expression
of DACT2 was silenced in TE1, T.Tn, Eca109 cell lines
and reduced in TE13 cell line; while the mRNA
expression levels of DACT3 remained relatively weak in all four
cell lines (Fig. 1d, e, f, g).
Up-regulation of DACT1 and DACT2, but not DACT3, by
5-Aza-Dc treatment in esophageal cancer cell lines
In order to clarify the possible epigenetic mechanism of
down-regulation of DACT1, DACT2 and DACT3
expression (such as methylation and acetylation), we further
detected the mRNA expression of DACT1,2,3 in cell lines
after 5-Aza-Dc (DNA methyltransferase inhibitor) or TSA
(histone deacetylase inhibitor) treatment. As shown in
Fig. 1d, e, f, g, the mRNA expression of DACT1 and
Fig. 1 The mRNA expression and methylation analysis of DACT1, DACT2 and DACT3 in esophageal cancer cell lines. a, b, c Schematic structure of CpG
islands in DACT1, DACT2 and DACT3 genes. The MSP and BGS-regions were shown in the schematic. The transcription start point was as +1. d The
changes of DACT1, 2, 3 mRNA expression in treated or untreated cells with 5-Aza-Dc or TSA by RT-PCR method; e, f, g Relative mRNA expression of
DACT1, 2, 3 in treated or untreated cells with 5-Aza-Dc or TSA by Quantitative realtime RT-PCR method. h The methylation status of DACT1, 2, 3 in four
cell lines with(+) or without(−) 5-Aza-Dc treatment detected by MSP method. M, methylated gene; U, unmethylated gene. i The methylation
status of every CpG site in two regions of DACT1, one region of DACT2 and one region of DACT3 in four tumor cell lines by BGS method. The color of
circles for each CpG site represents the percentage of methylation. The “F” and “R” represent forward and reverse primers for MSP, respectively
DACT2 were all significantly increased in four esophageal
cancer cells after treatment with 5-Aza-Dc, but not with
TSA. However, DACT3 mRNA was not significantly
upregulated after treatment with 5-Aza-Dc or TSA in four
esophageal cancer cell lines.
The aberrant promoter methylation of DACT1 and DACT2
induce down-regulation of their mRNA expression in
esophageal cancer cell lines
To investigate whether the silencing of DACT1, 2, 3
were associated with their hypermethylation, the
MethPrimer program  and the CpG island searcher 
were firstly used to analyze the CpG islands within the
sequence of DACT genes. As shown in Fig. 1, DACT1,
DACT2 and DACT3 were all shown to have the CpG
islands. MSP results were shown in Fig. 1h, complete
methylation was found in TE13 and T.TN cell lines and
incomplete methylation was observed in TE1 and
Eca109 cell lines in the two regions of DACT1. The
complete methylation of DACT2 was detected in TE1,
T.TN, Eca109 cell lines and incomplete methylation was
detected in TE13 cell lines. No methylated bands of
DACT3 were detected in all four cell lines. The BGS
assay was used to reveal dense methylation of every
CpG sites. As shown in Fig. 1i, the methylation
frequency of DACT1 CpG sites were higher in TE13 and
T.TN cell lines than those in TE1 and Eca109 cell lines,
especially the sites which were contained in the primers.
The methylation frequency of DACT2 CpG sites were
higher in TE1, T.TN and Eca109 cell lines than those in
TE13 cell lines, especially the sites which were contained
in the primers. No methylation was found of DACT3 in
these four cell lines. The results indicated that the MSP
assay results accurately represent DACT1, DACT2 and
DACT3 methylation status in these cell lines. After
treatment with 5-Aza-Dc, demethylation of DACT1 and
DACT2 were observed in these cells, together with the
results of DACT1 and DACT2 expression was restored
or increased after 5-Aza-Dc treatment, indicating that
DACT1 and DACT2 expression was regulated by the
methylation status of the genes.
results suggested that DACT1 and DACT2 may act as
tumor suppressor genes in esophageal cancer through
inhibition of cell proliferation and migration.
Inhibition of proliferation and migration in TE1 and TE13
Cell lines after restoration of DACT1/DACT2 or treatment
TE1 and TE13 cells, which demonstrated low or
negative expression of DACT1 and DACT2 genes, were
adopted to transfect with DACT1 or DACT2 plasmid
and investigate effect of DACT1 or DACT2 on the cell
proliferation using MTT and Colony-formation assays
respectively. As shown in Fig. 2, stable transfection of
DACT1/DACT2 or treatment with 5-Aza-Dc resulted in
significant inhibition of the proliferation index and
reduction of colony numbers in TE1 and TE13 cell lines.
The Wound-healing assay was further employed to
evaluate the effect of DACT1 and DACT2 on cell
migration. Cell migration ability was significantly decreased at
12, 24, 36 and 48 h after transfection with DACT1/
DACT2 or treatment with 5-Aza-Dc (Fig. 2d). These
Decreased mRNA and protein expression of DACT1,
DACT2, DACT3 in ESCC
DACT1, DACT2, DACT3 mRNA expression in ESCC
tissues were reduced significantly compared with those
in corresponding adjacent non-cancerous tissues (tDACT1 =
16.548, P < 0.001; tDACT2 = 7.236, P < 0.001; tDACT3 = 7.124,
P < 0.001) (Fig. 3a, b). IHC method was used to evaluate
the protein expression of DACT1, DACT2, and DACT3 in
ESCC tissues. The pattern of immunohistochemical
staining of them was cytoplasmic (Fig. 3c). The frequency of
DACT1, DACT2, DACT3 positive expression in tumor
tissues was significantly lower than that in corresponding
adjacent non-cancerous tissues (Fig. 3d; P < 0.001). When
stratified for clinicopathologic characteristics, DACT1 and
DACT2 mRNA and protein expression was associated with
clinical stage, Lymph node metastasis and UGIC family
Fig. 2 The effect on cell proliferation, colony formation and immigration abilities in TE1 and TE13 cell lines after treated with 5-Aza-dC or stable
transfected of DACT1 /DACT2. a The protein expression of DACT1 and DACT2 in TE1 or TE13 cells after stable transfected with DACT1/DACT2 plasmid
or treated with 5-Aza-dC using western-blot method. b The results of MTT assay in treated or untreated TE1/TE13 cells with 5-Aza-dC or the stable
transfected TE1/TE13 cells; **Compared with untreated cell line, P < 0.01; c The results of colony formation assays in treated or untreated TE1/TE13
cells with 5-Aza-dC or the stable transfected TE1/TE13 cells; **Compared with untreated cell line, P < 0.01; d The relative scratch width of treated or
untreated TE1/TE13 cells or the stable transfected TE1/TE13 cells detected by Wound healing analysis. The initial width of the scratch was as 1
Fig. 3 The expression and methylation analysis of DACT1, DACT2 and DACT3 in ESCC and corresponding adjacent non-cancerous tissues. a
Representative RT-PCR results of DACT1, 2, 3 in 5 matched pairs (case1-case5) of ESCC tissues (T) and non-cancerous tissues (N). GAPDH
was used as an endogenous control. b Relative mRNA expression of DACT1, 2, 3 in ESCC and adjacent tissues. c Representative immunohistochemical
staining of DACT1, 2, 3 in ESCC tumor tissues (SP × 200). a: the positive expression of DACT1; b: the positive expression of DACT2; c: the positive expression
of DACT3; d Positive expression frequency of DACT1, 2, 3 proteins in ESCC and corresponding adjacent tissues. e, f Relative mRNA expression of DACT1
and DACT2 in different subgroups of ESCC cases, which was expressed as the mean ± standard deviation. * P < 0.05, **P < 0.01. g Representative MSP
results of DACT1, DACT2 and DACT3 in 3 matched pairs (case1-case3) of tumor tissue (T) and non-cancerous tissues (N). M, Methylation; U, Unmethylation;
PC, positive control; NC, negative control. h The methylation rate of DACT1, 2, 3 in ESCC tissues and non-cancerous tissues. **P < 0.01. i The relative mRNA
expression of DACT1 and DACT2 in methylated or unmethylated groups. The mRNA expression of DACT1 and DACT2 were all associated with methylation
status of their TSS-regions in ESCC. **P < 0.01
history (P < 0.05), but not with age, gender, histological
grade (Table 1; Fig. 3e, f ).
Aberrant methylation of DACT1 and DACT2 in ESCC
The methylation analysis of DACT1, DACT2 and DACT3
were successfully performed uding MSP method in all
specimens (Fig. 3g). The methylation frequency of DACT1
was different in two regions (Fig. 3h). Of primary ESCC
and corresponding non-cancerous tissues,
hypermethylation was observed in 54.1% (86/159) and 43.4% (69/159)
at region1, 47.8% (76/159) and 16.4% (26/159) at region2,
respectively. The methylation frequency of DACT1 in the
region1 and region 2 were all higher than those in
corresponding adjacent tissues, however, only region 2 showed
the statistical difference (Pregion1 > 0.05; Pregion2 < 0.001;
Fig. 3h). The methylation frequency of DACT2 was 52.2%
(83/159) in tumor tissues, which was higher than that in
corresponding adjacent tissues (21.4%, 34/159, P < 0.001;
Fig. 3h). The methylation frequency of DACT3 was very
low both in ESCC and corresponding adjacent tissues (3.8
and 5.7%; P = 0.598) (Fig. 3h).
When stratified for clinicopathologic characteristics,
the methylation status of region1 in DACT1 was not
associated with any characteristic, and the TSS-region in
DACT1 (region2) and DACT2 were only associated with
Table 2 Methylation status of DACT1 and DACT2 in ESCC cases
Family history of UGIC
M methylation, U unmethylation
UGIC family history. But the simultaneous methylation
frequency of DACT1 (region2) and DACT2 were
associated with clinical stage, LN metastasis and UGIC family
history (Table 2).
Association between expression and methylation status
of DACT1, DACT2
As show in Fig. 3i and Table 3, the mRNA and protein
expression of DACT1, DACT2 in ESCC tissues with
hypermethylation of TSS-region were significantly reduced
compared to those in ESCC tissues without methylation
of this region (P < 0.05); However, the mRNA and protein
expression of DACT1 was not different between the ESCC
tissues with or without hypermethylation of CGI-shore
region (region1) (P > 0.05). The results indicated that the
reduced expression of DACT1 and DACT2 were
associated with methylation status of the TSS-region, and the
Table 3 Correlation between the methylation status of DACT1,
DACT2 and its protein expression in ESCC cases
M methylation, U unmethylation
methylation status of CGI-shore region was not associated
with the transcriptional inhibition of DACT1.
Survival analysis of DACT1 and DACT2 in ESCC patients
As show in Fig. 4a and b, DACT1 and DACT2 protein
expression were positively correlated with ESCC patients’
survival (PDACT1 < 0.001, PDACT2 = 0.001, Log-rank test).
The simultaneous positive expression of DACT1 and
DACT2 showed the best prognosis (P < 0.001, Log-rank
test, Fig. 4c). The methylation status of region1 in DACT1
was not correlated with ESCC patients’ survival (P = 0.105,
Log-rank test; Fig. 4d) and TSS-region methylation status of
DACT1 and DACT2 were inversely correlated with ESCC
patients’ survival (PDACT1 < 0.001, PDACT2 < 0.001, Log-rank
test; Fig. 4e, f ). Furthermore, the ESCC patients with
simultaneous methylation of DACT1 and DACT2 showed worst
prognosis (P < 0.001, Log-rank test; Fig. 4g). ESCC patients
in stage III / IV or with positive UGIC family history, and
with DACT1 or DACT2 methylation showed poor
prognosis (Fig. 4h and i, P < 0.001, Log-rank test). To determine
which variables were independent predictors of ESCC
patients’ survival, a multivariate analysis was performed using
COX proportional hazard regression model. The results
indicated that the methylation status of TSS-region in
DACT1 or DACT2, the protein expression of DACT2,
TNM stage and UGIC family history were
independently associated with ESCC patients’ survival (Table 4).
Fig. 4 Kaplan–Meier univariate survival analysis of DACT1 / DACT2 expression and methylation status in ESCC. a, b, c Kaplan–Meier curves for
cumulative survival stratified by DACT1 and DACT2 expression: showing a direct correlation between DACT1 or DACT2 expression and ESCC patient’s
survival. d, e, f, g Kaplan–Meier curves for cumulative survival stratified by the methylation status of two regions of DACT1 and one region of DACT2:
There was not correlation between the region 1 methylation status of DACT1 and the ESCC patient’s survival. The hypermethylation of TSS-region in
DACT1 and DACT2 were associated with the poor patient survival of ESCC cases; Simultaneous methylation of DACT1 and DACT2 showed worst
prognosis. (H) Kaplan–Meier curves for cumulative survival stratified by methylation status and UGIC family history: ESCC patients with positive UGIC
family history and DACT1 or DACT2 methylation showed poor patient survival. (I) Kaplan–Meier curves for cumulative survival stratified by methylation
status and TNM stages: Stage III and IV ESCC patients with DACT1 or DACT2 methylation showed the poor patient survival
In the present study, we found the expression of DACT1,
DACT2 and DACT3 were frequently silenced or
decreased in esophageal cancer cell lines. Up-regulation of
DACT1 and DACT2 but not DACT3 after treatment
with 5-Aza-Dc, inferring that DNA methylation may be
the main regulatory mechanism for the inactivation of
DACT1 and DACT2 but not DACT3 in esophageal
cancer cell lines, which further confirmed by direct BGS
and MSP analysis of promoter methylation status of
DACT1, 2, 3. However, the mRNA expression of the
three genes was not significantly up-regulated after
treatment with TSA in esophageal cancer cell lines,
indicating that histone acetylation may not play crucial roles
Table 4 Multivariate analysis of survival in ESCC cases (Cox’s test)
Odds ratio (95% CI)
MSP1 methylation of DACT1 −0.240 0.215 0.265 0.787(0.516–1.200)
MSP2 methylation of DACT1
0.744 0.333 0.026 2.104(1.095–4.043)
methylation of DACT2
Family history of UGIC
1.257 0.288 0.000 3.515(1.998–6.183)
0.520 0.298 0.081 1.681(0.937–3.016)
−0.804 0.261 0.002 0.448(0.269–0.747)
1.533 0.222 0.000 4.632(3.000–7.152)
0.737 0.207 0.000 2.089(1.391–3.137)
in the inactivation of DACT1, DACT2 and DACT3. The
similar results were observed in other carcinomas such
as DACT1 in breast cancer  and DACT2 in
hepatocellular carcinoma , gastric cancer , lung cance
, colon cancer , and also in esophageal cancer
. But the result about DACT3 was different from the
previous studies. The histone modification may be the
main regulated mechanism of DACT3 in colorectal
cancer . The difference may partly due to the fact that
histone modification has the tumor cell specific and the
precise mechanism of this gene inactivation need to be
further studied. In addition, the inhibition of
proliferation and migration was further detected in TE1 and
TE13 cells after treatment with 5-Aza-Dc or stable
transfection of DACT1 or DACT2 plasmid, indicating
the tumor suppressor role of DACT1 and DACT2 in
esophageal cancer cell lines.
Aberrant methylation of CpG islands in gene promoter
has been well-established as a major mechanism for the
inactivation of tumor suppressor genes in tumorigenesis
[31–33]. The hypermethylation of DACT1 in the region
near TSS within CpG islands was detected in multiple
breast cancer cell lines and primary breast tumors, and
the methylation status in this region was a main
epigenetic mechanism of DACT1 silencing in breast cancer
. Meanwhile, recent studies points out that DNA
methylation can directly silence genes with non-CpG
islands, such as CGI shore [34, 35]. The aberrant
methylation of DACT1 in CGI shore region had been detected
in primary gastric cancer and the methylated CpG site
count in this region had the significant applicability for
prognosis evaluation of gastric cancer patients . Two
regions including TSS-region and CGI shore-region
were selected to analyze the methylation status of
DACT1 in present study. The TSS-region methylation
frequency of DACT1 in tumor tissues was significantly
higher than that in adjacent tissues and was associated
with the decreased mRNA and protein expression of
DACT1. The methylation frequency of CGI shore in
DACT1 was higher both in tumor and adjacent tissues.
These results suggested that hypermethylation of
TSSregion was more cancer-specific and was associated with
the transcriptional inhibition of DACT1. The TSS-region
may be regarded as the critical CpG region of DACT1.
The CGI shore-region was more likely to be methylated
but may be not necessary for the tumor formation. The
TSS-region was selected to analyze the methylation
status of DACT2 and there was a significant
concordance between the hypermethylation of this gene and its
reduced expression in the present study. By using the
same MSP primers as we used in this study, Yu et al.
 found DACT2 was frequently methylated in human
gastric cancer and aberrant methylation may be a main
mechanism of DACT2 inactiviton. The similar results
were also reported in hepatocellular carcinoma ,
lung cancer , colon cancer  and also in
esophageal cancer . When stratified for clinicopathologic
characteristics, the methylation status of DACT1 or
DACT2 was only associated with UGIC family history,
but the simultaneous methylation of DACT1 and
DACT2 were associated with clinical stage, LN
metastasis and UGIC family history, suggesting that multiple
gene methylation may be an ideal cancer biomarker in
progression of ESCC and may have definite value on
estimating prognosis of ESCC patients.
The relationship between DACT1/DACT2 protein
expression, methylation status and the 5-year overall
survival rate was further analyzed to evaluate their value in
ESCC patients’ prognosis. In the present study, we
showed that protein expression and methylation status
of DACT1 and DACT2 were significantly and directly
correlated with ESCC patients’ survival, suggesting that
the inactivation of DACT1 and DACT2 via
hypermethylation may confer a growth advantage in ESCC.
Therefore, the hypermethylation and inactivation of DACT1
and DACT2 may be considered to be the poor
prognostic factors to ESCC patients. But the methylation status
of CGI shore-region in DACT1 was not associated with
ESCC patients’ survival, mainly due to the reason that
this region was not the critical CpG region of DACT1.
This result was different from a previous study  and
the further studies with a larger number of patients may
be needed to verify the results. Furthermore, the survival
rate of ESCC was closely associated with the stage in
which the malignancy is diagnosed . The stage III/IV
ESCC patients with DACT1 or DACT2 methylation
showed the worse survival, which further indicated the
methylation status of DACT1 and DACT2 could be
considered as the useful markers for evaluating the
prognosis of ESCC. UGIC family history was one of the
important factors on the occurrence of ESCC in the high
incidence regions of North China [6, 23, 38]. In the
present study, the ESCC patients both with positive
UGIC family history and DACT1/DACT2 methylation
had been showed the worst survival, indicating the
poorer prognosis of DACT1/DACT2 methylation in the
high risk populations. In multivariate analysis, the
methylation status of TSS-region in DACT1 and DACT2,
negative expression of DACT2, tumor stage, and positive
UGIC family history provided independent predictive
information on ESCC patients’ poor survival.
In conclusion, the present study suggests that the TSS
region hypermethylation may be one of the main
mechanisms for reduced expression of DACT1 and DACT2 in
ESCC. The CGI shore region in DACT1 is more likely to
be methylated but is not cancer-specific and isn’t related
to the transcriptional inhibition of DACT1. The
simultaneous methylation of DACT1 and DACT2 may play
important roles in progression of ESCC, and may serve
as prognostic biomarkers for ESCC patients.
Additional file 1: Table S1. Primer sequences, annealing temperature
and product size of DACT gene Family. (DOC 56 kb)
This work was supported by the National Natural Science Foundation of
China (No. 81472335, to W.G; No. 81572441, to Z.M.D); Natural Science
Foundation of Hebei Province of China (No.H2013206315, to Y.L.G; No.
H2015206196, to W.G; No. H2015206420, to Z.M.D); The focal point Project of
Hebei medical research (No.20130543, to Y.L.G).
Availability of data and materials
Data and materials related to this work are available upon request.
BS and WG designed the study and applied for Research Ethics Board
approval. YG performed the experiments, analyzed the data and prepared
the manuscript. ZD provided some theoretical and experimental guidance
for the design and performing the experiments. ZZ, SS and GK recruited the
patients and collected the data. XG and JL prepared draft figures and tables.
All authors read and approved the final manuscript.
Consent for publication
All authors approve the manuscript for publication.
Ethics approval and consent to participate
All procedures performed in this study were in accordance with the ethical
standards of the institutional and/or national research committee and with
the 1964 Helsinki declaration and its later amendments or comparable
ethical standards. The study was approved by the ethics committee of Hebei
Medical University Fourth Affiliated Hospital, and the informed consent was
obtained from all of the patients.
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