Introduction
Breast cancer is the leading cause of cancer death among females [
1], and results from accumulated genetic and epigenetic alterations of various cancer genes, including tumor-suppressor genes (TSGs) and oncogenes [
2]. Epigenetic alterations, especially CpG promoter methylation, play important roles in the initiation and progression of multiple cancers including breast cancer. Hypermethylation of CpG island (CGI) in the promoter regions of TSGs is an alternative mechanism for TSG silencing and could occur early in tumorigenesis, thus serving as a promising tumor marker for breast cancer diagnosis [
3]. Aberrant promoter methylation of some TSGs, such as
RASSF1A,
BRCA1,
TWIST,
Cyclin D2, and
p16, has been shown to be good biomarkers for the early detection or for a therapeutic target in breast cancer [
4,
5].
Wnt/β-catenin signaling plays an important role in multiple tumorigenesis, including breast cancer [
6]. Epigenetic silencing of negative regulators of WNT signaling is crucial for the aberrant activation of WNT/β-catenin signaling in tumor pathogenesis [
5,
7].
DACT1, a homologue of Dapper, located at chromosomal region 14q23.1, was first identified as a Dishevelled (Dvl)-associated antagonist of Wnt/β-catenin and JNK signaling pathways [
8,
9],
DACT1 is expressed during embryonic development in the adult brains of mice [
10,
11], but studies on its role in tumorigenesis are scanty.
DACT1 has been shown to be reduced in several tumors, such as gastrointestinal stromal tumors [
12] and non-small cell lung cancer (NSCLC) [
13], but overexpressed in some other tumors [
14,
15]. Dysregulated DACT1 was associated with poor prognosis in tumor patients [
13].
DACT1 was also reported to be a novel inhibitor of the WNT/β-catenin signaling in hepatocellular carcinoma (HCC) [
16]. However, its expression and biologic functions in breast cancer pathogenesis are unknown.
We identified DACT1 as a methylated target in the MB231 breast cancer cell line as compared with normal tissue in our pilot cancer epigenome study. Here, we further examined the expression and promoter methylation of DACT1 in multiple breast cell lines and primary tumors, and evaluated its potential as a tumor biomarker for breast cancer. We further demonstrated the biologic functions of DACT1 in breast cancer cells in vivo and in vitro in the context of the Wnt/β-catenin signaling pathway.
Materials and methods
Cell lines, tumor samples, and normal tissues
Several breast cancer cell lines (BT549, MDA-MB-231, MDA-MB-468, MCF-7, T47D, SK-BR-3, YYC-B1, YCC-B3, and ZR-75-1) were studied. All cell lines were maintained at 37°C in RPMI 1640 supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), 100 U/ml of penicillin, and streptomycin.
Normal human breast tissue RNA samples were purchased commercially (Stratagene, La Jolla, CA, USA; Millipore Chemicon, Billerica, MA, USA; BioChain Institute, Hayward, CA, USA). Primary breast tumor samples, paired surgical-margin tissues, and normal breast tissues were obtained from the First Affiliated Hospital of Chongqing Medical University, or elsewhere, as described previously [
17‐
19]. All samples were evaluated and subject to histologic diagnosis by pathologists. Clinical information, including age, tumor grade, tumor size, follow-up data after initial diagnosis, and treatment, was obtained for the majority of tumor cases. Grading of tumors was achieved by staining with hematoxylin and eosin (H&E). All patients provided written consent for the study. The study was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (Approval notice: 2010/2012(23)).
Semiquantitative RT-PCR analysis
Total RNA was isolated from cell lines by using TRI Reagent (Molecular Research Center, Cincinnati, OH, USA). Semiquantitative RT-PCR was performed as described previously [
20].
GAPDH was amplified as a control. Primer sequences are listed in Table
1. RT-PCR was performed with 32 cycles for
DACT1, and 23 cycles for
GAPDH, by using Go-Taq (Promega, Madison, WI, USA).
Table 1
List of primers used in this study
MSP | DACT1m3 | CGGGATAGTAGTAGTCGGC | 118 | 41 | 60 |
| DACT1m4 | CGCTAAAACTACGACCGCG | | | |
| DACT1u3 | GTTGGGATAGTAGTAGTTGGT | 123 | 41 | 58 |
| DACT1u4 | AAACACTAAAACTACAACCACA | | | |
RT-PCR | DACT1-F | AGGAGAAGTTCTTGGAGGAG | 179 | 32 | 55 |
| DACT1-R | TGAGCTAGGCCGACTGTCTG | | | |
| GAPDH-F | GGAGTCAACGGATTTGGT | 206 | 23 | 55 |
| GAPDH-R | GTGATGGGATTTCCATTGAT | | | |
Real-time PCR | DACT1-F | GACGAGCAGAGCAATTACACC | 158 | 40 | 60 |
| DACT1-R | ACCGTTTGAATGGGCAGA | | | |
| β-actin-F | CCTGTGGCATCCACGAAACT | 314 | 40 | 60 |
| β-actin-R | GAAGCATTTGCGGTGGACGAT | | | |
5-Aza-2'-deoxycytidine and trichostatin A treatment
Cell lines were treated with 10 mM 5-aza-2'-deoxycytidine (Aza; Sigma-Aldrich, St Louis, MO, USA) for 3 days or further treated with 100 nM trichostatin A (TSA; Cayman Chemical Co., Ann Arbor, MI, USA) for 14 hours.
DNA bisulfite treatment and methylation-specific PCR
Genomic DNA was extracted from tumors, normal tissues, and cell pellets by using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Bisulfite modification of DNA and methylation-specific PCR (MSP) were performed as described previously [
21,
22]. Bisulfite-treated DNA was amplified by MSP with
DACT1 methylation-specific primer set for
DACT1 promoter (Table
1), by using AmpliTaq-Gold DNA Polymerase (Applied Biosystems, Foster City, CA, USA). Methylated and unmethylated MSP primer sets target the same CpG sites and do not amplify genomic DNA with no bisulfite treatment.
Quantitative reverse transcription polymerase chain reaction
Real-time PCR (rtPCR) was performed by using Maxima SYBR Green/ROX qPCR Master Mix (MBI Fermentas, St. Leon-Rot, Germany) (Table
1). Thermal-cycling reaction was performed in the 7500 Real-Time PCR System (Applied Biosystems). Melting-curve analysis and agarose gel electrophoresis of PCR products were further performed. Relative expression levels of
DACT1 in breast tissues were standardized to β-actin levels.
Tissue microarray and immunohistochemistry
To evaluate DACT1 expression in breast tissues, tissue microarray (TMA) was used as described previously, containing 30 pairs primary tumors and corresponding tumor-margin tissues [
18]. Immunohistochemistry was performed by using a two-step method. In brief, after deparaffinization, sections were hydrated and underwent sodium citrate antigen retrieval. Sections were then incubated with 3% hydrogen peroxide to block endogenous peroxidase activity. A rabbit polyclonal antibody against human DACT1 protein (Ab104.4; Abcam, Cambridge, UK) was used. Sections were incubated with primary antibody (1:200 dilution) overnight at 4°C, detected by using polyperoxidase-anti-rabbit IgG (Jackson Immunoresearch Laboratories, West Grove, PA, USA), and counterstained with hematoxylin. To eliminate nonspecific staining, a negative control was performed with PBS.
All immunohistochemical photographs were analyzed by using Image Pro Plus (IPP, version 6.0; Media Cybernetics, Silver Spring, MD, USA), as described previously [
18]. The mean optical density (OD), as a quantitative measure of stain intensity, was analyzed to determine average protein expression.
Immunofluorescence staining
Cells transfected with pcDNA3.1-DACT1 or pcDNA3.1 plasmid were grown on glass coverslips. Transfected cells were washed with PBS, fixed with 4% paraformaldehyde in PBS for 15 minutes, permeabilized with 0.5% Triton X-100 for 30 minutes, and blocked with 3% bovine serum albumin in PBS at 37°C for 60 minutes. Cells were incubated with primary antibodies diluted in TBST at 4°C for 12 hours, washed twice with PBS, and then incubated with DyLight-conjugated anti-rabbit or anti-mouse antibody (CoWin Biotech Co., Ltd. (CWBIO), Beijing, China) for an additional 50 minutes. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Roche, Palo Alto, CA, USA).
Cell-proliferation assay
MB231 cells were cultured in six-well plates at a density of 1 × 10
4 cells/well and allowed to grow overnight. Cultures were then transfected with pcDNA3.1-DACT1 or pcDNA3.1 plasmid by using Lipofectamine-2000 (Invitrogen). At 24, 48, and 72 hours, cell proliferation was measured by using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) [
23].
Colony-formation assay was performed by using a monolayer culture. Cells (MB231 and MCF7) were plated in six-well plates and transfected with pcDNA3.1-DACT1 or pcDNA3.1 (4 μg each) plasmid by using Lipofectamine 2000 (Invitrogen). At 48 hours after transfection, cells were collected, replated, and selected for 2 weeks in the presence of G418 (0.4 mg/ml). Surviving colonies (≥50 cells/colony) were counted after staining with gentian violet. All experiments were performed 3 times.
Wound-healing assay
DACT1 and vector-expressing cells (MB231 and MCF7) were selected by using G418, and then cultured in six-well plates until confluent. After scratching the monolayer, cells were photographed at 0, 12, 24, 36, 42, and 48 hours under a 10× objective (Leica DMI4000B, Milton Keynes, Bucks, UK).
Caspase-3 colorimetric assay
Cells were seeded in six-well plates and transfected with pcDNA3.1-DACT1 or pcDNA3.1 plasmid, and then collected and lysed for protein purification at 24 or 48 hours. Total protein extraction was performed for analyzing active caspase-3 by using Caspase-3 Colorimetric Assay Kits (KeyGen Biotech Co, Nanjing, China).
Western blot
Transfected cells were lysed in M-PER Mammalian Protein Extraction Reagent (Pierce, Thermo Scientific, Cramlington, UK) containing a protease inhibitor cocktail (Sigma Aldrich). A total of 50 μg of protein lysate for each sample was separated by using sodium dodecylsulfate/polyacrylamide gel electrophoresis (SDS-PAGE). The lysates were then transferred to PVDF membranes for antibody incubation. After blocking with 5% nonfat milk and 0.1% Tween 20 in TBS, the membranes were incubated with DACT1 antibody (Abcam, Cambridge, UK), or antibodies to active β-catenin (Millipore, Billerica, MA, USA), cyclin D1, c-Myc, cleaved caspase 3, and cleaved PARP (Epitomics Inc., Burlingame, CA, USA). The immunoblots were visualized by using an enhanced chemiluminescence detection system. GAPDH was used as a control.
In vivo tumor model
Stable DACT1-expressing MB231 cells or controls (1 × 106 cells in 0.2 ml PBS) were subcutaneously injected into the right dorsal flank of female nude mice (6 weeks old, six mice per group). The weight of nude mice was measured every 7 days for 4 weeks. The xenograft tumor weight was assessed at the terminal time. The protocols for in vivo animal experiment were approved by the Committee on Ethical Use of Animals of the First Affiliated Hospital of Chongqing Medical University.
Statistical analysis
Statistical analyses were performed with SPSS version 16 (SPSS Inc., Chicago, IL, USA). Student t test was used to analyze the difference of DACT1 expression between breast cancer tissues and surgical margin tissues. χ2 test and Fisher Exact test were used to assess the correlation between DACT1 methylation and clinicopathologic parameters. For all the tests, P < 0.05 was considered statistically significant.
Discussion
From this study, we report that DACT1 is widely expressed in normal breast tissues but frequently downregulated/silenced by promoter methylation in breast cancer. DACT1 is methylated in 29.9% of primary breast tumors, but not in surgical-margin tissues and normal breast tissues. DACT1 inhibits breast cancer cell proliferation by inducing apoptosis, and further suppresses tumor-cell migration through downregulating β-catenin activity, thus functioning as a tumor suppressor for breast cancer.
Epigenetic disruption of TSGs, including promoter methylation and histone modification, is a key mechanism regulating cancer gene expression [
25,
26].
DACT1 was frequently downregulated by promoter methylation in HCC, whereas another DACT family member,
DACT3, was repressed by bivalent histone modifications in colon cancer [
16,
27]. We report that
DACT1 was frequently methylated in breast cancer cell lines and primary tumors, which was correlated with its downregulation/silencing. No methylation was detected in some breast cell lines with silenced
DACT1, suggesting that histone modifications or other mechanisms may be alternative mechanisms for
DACT1 downregulation in some settings.
The Wnt/β-catenin signaling pathway plays an important role in tumor proliferation and migration [
6]. Dact1 (Dapper), originally identified as an interacting protein for Dishevelled (Dvl), has been involved in distinct Wnt-dependent developmental processes of
Xenopus, zebrafish, and mice [
8,
11,
28‐
30]. Dact1 antagonizes Wnt signaling by binding with Dvl and promoting its degradation. Notably, this inhibitory activity is conserved from
Xenopus to humans [
8,
9,
31,
32]. DACT1 has been identified disrupting the expression and localization of β-catenin, thus dysregulating Wnt/β-catenin signaling in NSCLC [
13]. We found that DACT1, located mainly in the cytoplasm and membrane, reduced the expression of active β-catenin and its downstream target gene
c-MYC in breast cancer cells, thus inhibiting cell proliferation of breast cancer by inducing apoptosis, as well as tumor cell migration.
DACT1 has been reported as a potential tumor marker associated with poor prognosis of NSCLC [
13]. We observed tumor-specific methylation of
DACT1 in breast cancer, indicating its potential as a tumor marker, although no obvious correlation between its methylation and clinicopathologic features was found, which must be further confirmed by a large sample-sized study. Future study of circulating methylated
DACT1 in serum or in combination with other methylated TSGs may be performed for the detection of breast cancer [
5,
33,
34].
Conclusions
In summary, DACT1, as a Wnt/β-catenin signaling antagonist, is frequently downregulated/silenced in breast cancer, acting as a functional tumor suppressor in breast tumorigenesis, and may serve as a potential tumor marker for breast cancer.
Acknowledgements
We thank Dr. Sun Y. Rha for some breast cancer cell lines. This study was supported by National Natural Science Foundation of China (31171243, 81072148, and 81172582), Natural Science Foundation of Chongqing (2010BB5101) and the Group Research Scheme of The Chinese University of Hong Kong (3110085).
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
XY, TX, WXS, XS, XL, JH, YY, and WP acquired data. XY, TX, and LL analyzed data and drafted the manuscript. MO and KK provided material. GR reviewed the manuscript. QT conceived of and supervised the study, analyzed data, and finalized the manuscript. All authors read and approved the manuscript for publication.