Epigenetic silencing of the WNT antagonist Dickkopf 3 disrupts normal Wnt/β-catenin signalling and apoptosis regulation in breast cancer cells

Cell lines and tumour samples

Breast tumour cell lines (BT549, MDA-MB-231, MDA-MB-435, MDA-MB-468, MCF-7, T47D, SK-BR-3, YCC-B1, YCC-B3 and ZR-75-1) were used 27. Human mammary epithelial cell lines HMEpC (Cat. no. CA-830-05a; Applied Biosystems, Foster City, CA, USA) and HMEC were used as controls. All carcinoma cell lines were maintained in RPMI 1640 (Gibco-BRL, Karlsruhe, Germany) supplemented with 10% foetal bovine serum (FBS; PAA Laboratories, Linz, Austria), 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. HMEpC and HMEC were cultured as previously described 28. RNA samples of human normal adult breast tissue were purchased commercially (Stratagene, La Jolla, CA, USA; Millipore Chemicon, Billerica, MA, USA and BioChain Institute, Hayward, CA, USA). DNA and RNA samples were obtained from various primary breast tumour tissues, breast tumour surgical margin tissues and normal breast tissues as described previously 29. Fresh cancer tissues and normal breast tissues were obtained from patients who underwent primary surgery at the Surgery Department of the First Affiliated Hospital of Chongqing Medical University. Clinical and pathological data of all the participants were obtained, and their demographies are summarized in Table 2. This research was approved by the Institutional Ethics Committees of the First Affiliated Hospital of Chongqing Medical University.

5-aza-2′-deoxycytidine (Aza) and trichostatin A (TSA) treatment

DNA demethylation treatment of breast cancer cell lines was performed as described previously 28. Cell lines were treated with 10 μmol/l 5-aza-2′-deoxycytidine (Sigma-Aldrich, Steinheim, Germany) for 3 days and further treated with 100 nmol/l trichostatin A (Sigma-Aldrich, Deisenheim, Germany) for additional 16 hrs.

Nucleic acid extraction

Genomic DNA and total RNA were isolated from cell lines and tissues using DNAzol and Trizol reagents (Invitrogen, Rockville, MD, USA), respectively, according to the manufacturer's recommendations 30. Tumour material was snap-frozen in liquid nitrogen immediately within 1/2 hr after surgery. Haematoxylin/eosin-stained sections were prepared for assessing the percentage of tumour cells where samples with only >70% tumour cells were selected. Normal breast tissues were similarly prepared. Spectrophotometry ND2000 was used to determine the concentration of DNA and RNA, and their integrity was assessed by gel electrophoresis.

Reverse transcriptase–polymerase chain reaction

First-strand cDNA was synthesized from 1 μg of total RNA using MuLV Reverse Transcriptase (Cat. no. N8080018; ABI, Foster City, CA, USA) to a final volume of 20 μl. For RT-PCR 30, samples were assayed in a 12.5 μl reaction mixture containing 2.5 μl of cDNA. Glyceraldehyde-3- phosphate dehydrogenase (GAPDH) was used as control.

Methylation-Specific PCR analysis of DKK3 promoter

Methylation-Specific PCR (MSP) was used to determine the DKK3 promoter methylation status, as described previously 31. Bisulfite modified DNA was amplified by two different primer pairs specific to the unmethylated (u) and methylated (m) promoter sequences respectively. The methylation-specific primers are m3: 5′-TTTCGGGTAT CGGCGTTGTC, m4: 5′-ACTAAACCGAATTACGCTACG; The unmethylation-specific primers are u3: 5′-GTTTTTTTGGGTATTGGTGTTGTT, u4: 5′-CAACTAAACCAAATTACACTACA. PCR amplification was performed for a total of 40 cycles with an annealing temperature of 60°C and 58°C respectively. Non-methylated and methylated human DNA were used as negative and positive controls respectively. MSP products were then analysed by a 2% agarose gel containing 100 bp DNA markers (MBI Fermentas, Vilnius, Lithuania).

Flow Cytometry analysis of cell cycle status

To assess cell cycle status, MB231 cells and BT549 cells were seeded (1 × 10⁶ cells/well) in 6-well plates and transfected with 4 μg of pMH-DKK3 or pMH empty vector using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol. After 48 hrs, cells were digested using 0.1% trypsin and centrifuged at 1000 r.p.m. for 5 min. Pellets were washed with PBS and fixed in ice-cold 70% ethanol for 1 hr, and treated with 100 μl of 50 mg/l propidium iodide for 30 min. at 4°C in the dark. Annexin V-FITC/PI staining was used for apoptosis analysis. Data were analysed with the CELL Quest software (BD Biosciences, San Jose, CA, USA).

Colony formation assay

Colony formation assays were performed as described previously 28. Cells were plated in six-well plates and transfected with 4 μg pMH-DKK3 vectors or with an empty vector using Lipofectamine™2000 (Invitrogen, Carlsbad) according to the manufacturer's instructions. At 48 hrs after transfection, cells were collected, re-plated and selected for 2 weeks in the presence of 0.2 mg/ml G418 in BT549 cell and 1.2 mg/ml G418 in MB231 cell. Surviving colonies were counted after staining with Giemsa.

Wound healing assay

Stably transfected cells were cultured in a six-well plate until confluent. The cell layer was carefully wounded using sterile pipette tips and washed twice with PBS, and re-suspended in fresh complete medium. After incubating for 12 and 24 hrs, cells were imaged using Nikon microscope with a low magnification 10× phase contrast objective lens. The experiments were triplicated.

Western blot analysis

Western blot was performed on protein extracts prepared at 48 hrs after transfection of the DKK3 expression vector or empty vector. Equal amounts of protein (50 μg/lane of total extract) were separated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred onto a PVDF membrane (Bio-Rad, Hercules, CA, USA). After blocking the membrane in 5% skim milk powder dissolved in PBS for 1 hr at room temperature, the antibodies used were β-catenin, JNK, and phosphor-JNK (Abcam, Cambridge, UK), active-β-catenin (Millipore), cyclin D1, c-Myc, E-Cadherin, vimentin (Epitomics Inc., Burlingame, CA, USA), and DKK3 (R&D systems, Minneapolis, MN, USA). The samples were subsequently incubated with a 1:2,000 dilution of an HRP-conjugated goat-antimouse IgG (Promega, Madison, WI, USA) for 1 hr. Bands were visualized using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). GAPDH was used as a control.

Statistical analysis

Statistical analyses were performed using SPSS software version 13 (SPSS Inc., Chicago, IL, USA). Chi-square test, t-test, non-parameter Spearman test and Fisher's exact test were used to compare experimental methylation statuses and clinical data. Differences were considered statistically significant when P < 0.05.

Expression of DKK family genes in breast cancer cells

We firstly examined the expression of DKK family genes in 10 breast cancer cell lines using semiquantitative RT-PCR. Result showed that DKK1 was detected in all the cell lines; DKK2 mRNA was absent in 9/10 cell lines but weakly expressed in BT549; DKK3 was silenced/down-regulated in five cell lines (BT549, MB231, MB435, MB468 and YCC-B1); DKK4 was significantly down-regulated in all the cell lines (Fig. 1A), while all DKK family genes were readily expressed in normal mammary tissues and cells. These results indicated that DKK2, -3 and -4 are frequently down-regulated in breast cancer cells.

DKK3 down-regulation in breast cancer correlated with its promoter methylation

We next analysed the promoters of DKK genes to evaluate whether silencing of DKK genes was because of promoter methylation. We found that DKK1, -2 and -3 contained typical CpG islands spanning the proximal promoter and exon regions (Fig. 1B, Figure S1), whereas no CpG island was observed in DKK4 promoter (data not shown). We further investigated the methylation status of DKK2 and DKK3 promoters. MSP analysis revealed that both DKK2 (data not shown) and DKK3 methylation were frequently detected in breast cancer lines. Specifically, DKK3 was methylated in eight of 10 (80%) breast cell lines with little or no expression, but not in HMEC and HMEpC cells with expression (Fig. 1C). We, thus chose to further study DKK3 in detail.

To further clarify whether DKK3 silencing was related to promoter methylation, cell lines treated with Aza and TSA were used. Demethylation of CpG-dinucleotides was examined by MSP analysis. Results showed that DKK3 expression was restored in response to 5-aza-dC treatment, along with increased unmethylated promoter alleles (Fig. 1D), suggesting that promoter methylation is directly responsible for DKK3 down-regulation in breast cancer cells.

DKK3 is frequently methylated in primary breast carcinomas

To investigate DKK3 methylation in primary breast tumour tissues, MSP analysis was employed to test 96 primary breast tumour tissues, 43 surgical marginal tissues and 16 normal breast tissues. The methylation status of DKK3 in these three different kinds of tissues was shown in Table 1 and Figure 2. DKK3 was methylated in 78% (75/96) of breast tumours and 12.5% of (2/16) normal breast tissues, but not in surgical margins examined (P < 0.05) (Fig. 2). These results indicated that DKK3 is methylated in a virtually tumour-specific manner.

We further analysed the association of DKK3 methylation with clinicopathological features. We found that DKK3 methylation was statistically correlated with clinical stage, lymph node metastasis and oestrogen receptor (ER) status (P < 0.05), but not associated with age, tumour size, progesterone receptor (PR) and hormone receptor (HR) status of breast cancer patients (Table 2).

DKK3 inhibits cell proliferation through inducing apoptosis in breast tumour cells

Silencing of DKK3 by promoter methylation in breast cancer indicated that it may function as a TSG. To elucidate the effect of DKK3 on cell proliferation, colony formation assay was performed. Results showed ~40–80% reduction in colony formation in DKK3-transfected MB231 and BT549 cancer cells, compared to controls (Fig. 3; P < 0.01). To evaluate the mechanism of DKK3 in suppressing cell proliferation, flow cytometric analyses of apoptosis and cell cycle were performed. We found that MB231 cells with DKK3 expression underwent significant apoptosis compared to controls, with 37% of cells staining positive for Annexin V (Fig. 4A). DKK3 significantly increased BT549 and MB231 cells with G0-G1 phase by 10% (P < 0.05), compared to controls (Fig. 4B). These results indicate that the inhibition of cell proliferation by DKK3 is most likely mediated by G0/G1 cell cycle arrest and apoptosis.

DKK3 suppresses epithelial-mesenchymal transition and migration of breast cancer cells

To assess the effect of DKK3 on cell migration, we firstly examined cell morphology changes. DKK3-transfected cells regained cell–cell contacts and adherence to each other, with less aggressive behaviour, whereas the vector-transfected cells exhibited unique irregular shapes and distinguishing spread features same as the original cells (Fig. 5A), suggesting that DKK3 most likely reversed tumour cell epithelial-mesenchymal transition (EMT). Western blot analysis showed up-regulated epithelial marker E-cadherin and down-regulated mesenchymal marker vimentin in DKK3-expressing cells (Fig. 6B), indicating that DKK3 indeed negatively regulates EMT.

As EMT is implicated in regulating stem cell properties, we further investigated the expression of some stem cell-associated markers in DKK3-transfected breast cancer cells. DKK3 down-regulated stem markers such as NANOG, ABCG2 and OCT4 (Fig. 6C), thus may reverse the stem cell-like phenotype of tumour cells.

Wound healing assay was further performed to uncover the effect of DKK3 on cell migration. Results showed that DKK3-expressing cells spread along the wound edges remarkably slower than the vector-transfected cells at 28 hrs (Fig. 5B), indicating that DKK3 inhibits cell migration.

DKK3 regulates canonical and non-canonical Wnt signalling in breast cancer cells

As Wnt/β-catenin signalling plays an important role in tumour metastasis, we thus investigated whether DKK3 counteracts this pathway for its tumour suppressive function. The effects of DKK3 on subcellular localization of β-catenin were performed in breast cancer cells with abundant level of endogenous β-catenin. Immunofluorescence staining showed increased concentration of β-catenin in cytoplasm and on the membrane in DKK3-transfected cells, compared to controls in which β-catenin was predominantly localized in nuclei. We also found significantly inhibited expression of active β-catenin and its downstream target genes, c-Myc and cyclin D1 in DKK3-expressing MB231 cells (Fig. 6A and B). Our findings suggest that DKK3 interferes with β-catenin activity and its localization, thus crippling the Wnt/β-catenin signalling pathway in breast tumorigenesis.

As JNK pathway plays an important role in non-canonical Wnt signalling, we further assessed the effect of DKK3 on JNK signalling in breast cancer cells. We found that phosphorylated JNK was increased in DKK3-expressing MB231 cells, but no obvious change was observed in the total amount of JNK protein (Fig. 6B), suggesting that DKK3 also regulates non-canonical Wnt signalling in breast tumorigenesis.