MCF-7, MDA-MB-231 and T-47D breast cancer cell lines, and the MCF-10 healthy breast cell line were procured from the American Type Culture Collection (Manassas, VA, USA). MCF-7 and MDA-MB-231 cells were grown in Minimum Essential Media (MEM), T-47D cells were grown in RPMI-1640 media and MCF-10 cells were grown in Mammary Epithelial Cell Growth Medium (Lonza, Basel, Switzerland). MEME and RPMI-1640 were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Media were supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and cells were cultured in the presence of 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in an atmosphere containing 5% CO₂. AMPK activators AICAR (Sigma-Aldrich, Merck Millipore, Darmstadt, Germany) and A23187 (Calbiochem, EMD Millipore, Billerica, MA, USA) were used to treat MCF-7 cells. AICAR was used at 0, 1 and 2 mM and A23187 at 0, 2 and 4 mM for 24 h followed by protein isolation and western blotting. Another AMPK activator, metformin (Sigma-Aldrich, Merck Millipore) was used to treat MCF-7 cells at 0, 20 and 40 mM concentrations for 24 h followed by subsequent analyses. An AMPK inhibitor, Compound C (Sigma-Aldrich, Merck Millipore) was used to pre-treat MCF-7 cells at 5 mM for 2 h followed by subsequent treatments and analyses.

For expression of green fluorescent protein (GFP)/DVL3 protein, a GFP-tagged-DVL3 expressing construct was generated by amplifying DVL3 using primer 1 (5′-GTGCTGGAATTCCCGAGGCC-3′) and primer 2 (5′-GCTCACATTGGATCCACAAAG-3′), with pEGFP-C1 plasmid (Addgene, Cambridge, MA, USA) serving as the negative control. Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used for MCF-7 and MDA-MB-231 cell transfection according to the manufacturer's protocol. Transfected cells were selected with antibiotic G418 (Sigma-Aldrich, Merck Millipore) for 2 weeks. Western blotting was performed to verify the cells that stably expressed GFP-DVL3.

Total RNA was extracted from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Reverse transcription reagent kit (Invitrogen; Thermo Fisher Scientific, Inc.) was used to synthesize cDNA according to the manufacturer's protocol. The expression of DVL genes was assessed by RT-qPCR using the ABI PRISM™ 7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.) with Taqman Gene Expression Assays, as per manufacturer's protocol. The TaqMan gene expression assays contained prevalidated primers and TaqMan probes for the individual genes specifically custom synthesized by Applied Biosystems. For amplification of DVL genes, two sets of primers and Taqman probes were used. The probes were labeled at the 5′ end with the reporter molecule 6-carboxy-fluorescein and labeled at the 3′ end with the quencher molecule 6-carboxy-tetramethylrhodamine. Primer sequences were: DVL2: 5′-TGAGCAACGATGACGCTGTG-3′ (forward) and 5′-GCAGGGTCAATTGGCTGGA-3′ (reverse); DVL3: 5′-ACAATGCCAAGCTACCATGCTTC-3′ (forward) and 3′-AGCTCCGATGGGTTATCAGCAC-5′ (reverse); and DVL-1: 5′-CCTTCCATCCAAATGTTGC-3′ (forward) and 5′-GTGACTGACCATAGACTCTGTGC-3′ (reverse). The RT-PCR amplification conditions were set at: 95°C for 5 min, 95°C for 1 min, 58°C for 30 sec, and 72°C for 1 min, for 40 cycles. Relative quantification of gene expression was performed as described by Applied Biosystems using 18S rRNA as an internal control. The comparative CT (cycle threshold) method was used for relative quantification of DVL genes' mRNA (18). PCR was performed in accordance with the manufacturer's protocol, in triplicate, on an ABI 7500 real-time PCR system (Applied Biosystems).

Cells were prepared as per experimental requirement, and harvested and pelleted for protein isolation. Cell pellets were lysed with 1X lysis buffer composed of 20 mM Tris pH 7.5, 1 mM EDTA, 150 mM NaCl, 2.5 mM sodium pyrophosphate, 1% Triton X-100, 1% sodium vanadate, 1 mM PMSF together with protease inhibitor cocktail (1:100; including phenylmethane sulfonyl fluoride). The cell lysates were centrifuged at 12, 000 × g for 20 min at 4°C, supernatant was collected and total protein content was estimated by using BCA protein assay kit (Sigma-Aldrich, Merck Millipore). For western blot analysis, 20 µg protein for each sample was separated by 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. These membranes were then subsequently blocked with 5% skimmed milk (fat-free) for 30 min and incubated with specific primary antibodies overnight at 4°C. The primary antibodies used were procured from Cell Signaling Technology, Inc. (Danvers, MA, USA), Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and EMD Millipore. Anti-phosphorylated (p) AMPKα (catalog no. cs 2531; 1:1,000), anti-AMPKα (catalog no. cs 2532; 1:1,000), anti-c-Myc (catalog no. cs 5605; 1:1,000), anti-cyclin D1 (catalog no. cs 2978; 1:1,000), anti-DVL1 (catalog no. sc-8025; 1:1,000), anti-DVL2 (catalog no. sc-8026; 1:1,000), anti-DVL3 (catalog no. sc-8027; 1:1,000), anti-β-catenin (catalog no. sc-7963; 1:1,000), and anti-GAPDH antibody (catalog no. EMD AB2302; 1:2,000) as a loading control. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary anti-mouse (catalog no. cs 7076) or anti-rabbit antibodies (catalog no. cs 7074), obtained from Cell Signaling Technology, Inc. in accordance with the manufacturer's protocol. An enhanced chemiluminescence solution (Invitrogen; Thermo Fisher Scientific, Inc.) was used for signal detection using photographic film followed by membrane scanning.

IHC was performed in healthy and cancerous breast tissue (n=5/each). Cancerous breast tissue samples and their corresponding healthy tissue samples were procured at the time of surgical resection, from patients presenting at the Department of Breast Surgery, The Third Hospital of Nanchang, Jiangxi, China. The study was approved by the ethics committee of The First Affiliated Hospital of Nanchang University, (Nanchang, China; Reference no. 2123432524AK) and written informed consent was obtained from patients or their family. Patients included in the study were Chinese adult women, mean age, 40.2±5.67. The patients had never received chemotherapy prior to surgical resection. For IHC analysis, surgically resected tissue parts were fixed in normal buffered formalin followed by serial dehydration in graded alcohol then embedded in paraffin. Tissues embedded in paraffin blocks were cut into 4 µM thick sections. Tissue sections were de-paraffinized and treated with primary antibodies for anti-DVL3 (1:200 dilution) and anti-β-catenin (1:200 dilution) for 24 h at 4°C, while Tris-buffered saline served as a negative control. Antibodies were procured from Novus Biologicals, LLC (Littleton, CO, USA) and BD Biosciences (Franklin Lakes, NJ, USA), respectively. Sections were visualized under a light microscope (Leica Microsystems GmbH, Wetzlar, Germany) at 40x magnification and manual scoring of the expression levels of proteins was calculated from immunopositive staining area (0–100%). The intensity of staining was recorded on the scale of: +1, weak; +2, moderate; +3, intense; and +4, very intense. The fold-change in the expression level of each protein was calculated by normalization to the expression level of the same protein in the healthy breast section. The IHC analysis from each tissue section was examined and scored independently by two investigators. The breast tumor array (BC08022) analysis for DVL3 and β-catenin was performed by US Biomax, Inc. (Rockville, MD, USA). The expression levels of DVL3 and β-catenin were represented as fold-change of each gene as compared to normal tissue. The cutoff point (6-folds) of both genes was determined by its expression levels and statistical significance.

The XTT cell proliferation kit (Invitrogen; Thermo Fisher Scientific, Inc.) was used to measure cell viability. The assay was performed according to the manufacturer's protocol. Each cell line was assayed in triplicate and three independent experiments were performed.

Cells were grown and seeded for 24 h. Media was removed after 24 h, followed by incubation with methanol for 30 min. Cells were then stained with crystal violet for 1 h at room temperature, and subsequently washed with PBS followed by cell counting under a light microscope (Leica Microsystems GmbH) at 40x magnification using Image J software version 1.4 (U.S. National Institutes of Health, Bethesda, USA). The experiment was performed in triplicate and three independent experiments were performed.

Data were analyzed using one-way analysis of variance followed by Tukey's post hoc test, and the Chi-square test. Data are presented as the mean ± standard deviation. SPSS v16 was used for statistical analyses. P<0.05 was considered to indicate a statistically significant difference.

It has previously been demonstrated that DVLs are key in the upregulation of the expression of β-catenin and promotion of cell growth in various cancers, such as colorectal cancer (19), malignant pleural mesothelioma (20) and non-small-cell lung cancer (21). The present study first examined the expression patterns of DVL1, 2 and 3, in MCF-7, MDA-MB-231 and T-47D breast cancer cell lines, and the MCF-10 healthy breast cell line by western blot analysis. The results demonstrated that DVL3 was notably upregulated in breast cancer cell lines, compared with the healthy breast cell line (Fig. 1A). These data suggested that DVL3 is upregulated in breast cancer cells. The MCF-7 cells revealed greater levels of DVL3 compared with the other breast cancer cell lines. The mRNA expression levels of DVL genes in breast cancer cell lines and the healthy breast cell line were then determined by RT-qPCR. The gene expression analysis demonstrated similar results to those presented in Fig. 1A. The mRNA expression levels of DVL3 were considerably greater in breast cancer cell lines compared with healthy breast cells (Fig. 1B). Furthermore, the mRNA expression levels of DVL1 and DVL2 were reduced in each cell line, compared with DVL3. These findings primarily indicated that DVL3 was the major isoform among DVLs that was recurrently upregulated in breast cancer cells. The results also suggested that MCF-7 and MDA-MB-231 cell lines were more susceptible to alterations in DVL3 levels, compared with T-47D cells. MCF-7 and MDA-MB-231 cell lines were therefore selected for further studies.

The significance of DVL3 and β-catenin expression level alterations in breast cancer was investigated by examining the expression levels of these proteins using IHC and tissue array analysis in breast cancer and healthy breast tissues. The IHC analysis of breast cancer and healthy breast tissues revealed a greater level of staining of DVL3 and β-catenin in breast cancer tissues compared with healthy breast tissue (Fig. 2A). The relative protein expression levels of DVL3 and β-catenin were estimated from tissue sections. DVL3 expression exhibited a >2-fold (P=0.03) increase in breast cancer tissues compared with in healthy breast tissue (Fig. 2B). β-Catenin expression exhibited a >3-fold (P=0.022) increase in breast cancer tissues compared with in healthy breast tissue (Fig. 2B). DVL3 and β-catenin were revealed to be overexpressed in breast cancer tissue compared with healthy breast tissue. The tissue array analysis revealed an increase in the expression of DVL3 and β-catenin in breast tumor samples (Table I). The data obtained were categorized ≤ or >6-fold for a comparative analysis with healthy tissue. A greater level of β-catenin (>6-fold) was noted as statistically significant in breast cancer tissue samples. Statistical analysis revealed that expression levels of DVL3 (P=0.044) and β-catenin (P=0.038) were significantly increased (>6-fold) in breast cancer tissue at an advanced stage of the disease. In addition, the levels of DVL3 and β-catenin were significantly increased (>6-fold) in breast tissue with metastatic cancer (Table I).

DVL3 has previously been suggested to act as an important signal transduction molecule, which mediates the Wnt/β-catenin signaling pathway and controls cell growth (3,4). The association of DVL3 and β-catenin signaling in breast carcinogenesis was investigated, in order to examine the role of DVL3 in cell proliferation via Wnt/β-catenin signal pathway activation in breast cancer. MCF-7 and MDA-MB-231 breast cancer cell lines were transfected with a human GFP-tagged DVL3 expression plasmid for stable expression of DVL3. Western blotting revealed that β-catenin was significantly elevated in cells transfected with GFP-DVL3 stable clones (termed GC01 and GC02) (Fig. 3A). GC01 demonstrated a reduced expression of GFP-DVL3, compared with GC02; however, the expression analysis was statistically significant in the two cell lines (Fig. 3B). The XTT cell proliferation assay revealed that MCF-7 and MDA-MB-231 cells with ectopic expression of DVL3 demonstrated a more significant time-dependent rate of proliferation compared with the vector control (Fig. 3B). Furthermore, a clonogenic assay revealed a 1.6–2.5-fold increase in the number of colonies in MCF-7 and MDA-MB-231 cells stably expressing GFP-DVL3 compared with their respective vector controls (Fig. 3C). These results suggested that breast cancer cell proliferation is promoted by DVL3-mediated increases in Wnt/β-catenin signaling activity.

It has previously been demonstrated that AMPK activators induce growth inhibitory effects against human cancers, including cervical and breast cancer (13,22,23). The AMPK activators, AICAR and A23187, were used in the present study to analyze the effects of AMPK activation on the expression levels of DVL3 and β-catenin in breast cancer cells. MCF-7 and MDA-MB231 cell lines treated with AICAR demonstrated a marked reduction in DVL3 expression levels (Fig. 4A). A23187, a calcium ionophore, activates AMPK via its upstream calmodulin dependent protein kinase domain by increasing cytosolic calcium levels (24). In the present study, A23187 reduced DVL3 expression in MCF-7 and MDA-MB-231 cells in a dose-dependent manner (Fig. 4B). Treatment of breast cancer cell lines with these two AMPK activators resulted in an inhibition of DVL3 expression levels, which may act to further modulate the signaling events in cancer cell growth. The present study then aimed to decipher the underlying molecular mechanism of breast cancer cell growth inhibition, and its association with DVL3 reduction and AMPK activators by using metformin (another AMPK activator). Metformin is a commonly used AMPK activator and potent anticancer drug (13). Treatment of MCF-7 and MDA-MB-231 breast cancer cells with metformin resulted in a depletion of DVL3 in the cell lines in a dose-dependent manner (Fig. 4C). Treatment with metformin reduced the levels of DVL3 and β-catenin in a dose-dependent manner in the two cell lines. Metformin activated the levels of pAMPKα in a dose-dependent manner, but did not result in alteration to total AMPK levels (Fig. 4C). In addition, metformin dose-dependently inhibited expression of the two downstream transcriptional products of β-catenin, c-Myc and cyclin D1 (Fig. 4C). These results suggested that AMPK activators enhanced the phosphorylation of AMPK, resulting in an inhibition of the DVL3 and β-catenin crosslink, and suppressed cell growth in breast cancer cells as indicated by reduced colony number.

The AMPK activators primarily activate AMPK activity; however, it has previously been demonstrated that AMPK activators may be involved in cellular metabolic processes, such as energy metabolism (25,26). The present study subsequently examined whether AMPK activator-mediated DVL3 reduction is dependent on AMPK signaling. Compound C, which is a potent AMPK inhibitor, was used to counteract the effects of metformin on AMPK activation. Treatment of MFC-7 cells with 5 µM compound C resulted in a small increase in DVL3 levels, with a small inhibition of pAMPKα levels (Fig. 5A). Treatment with 20 mM metformin in MCF-7 cells induced a reduction in DVL3 expression and an increase in pAMPKα levels (Fig. 5A). The metformin-induced decrease in DVL3 and increase in pAMPKα levels were reversed following the addition of compound C (Fig. 5A). These results suggested that metformin-induced DVL3 reduction may be dependent on AMPK signaling. It was previously demonstrated that DVL3 ectopic expression may increase breast cancer cell proliferation. The present study elucidated the molecular basis of DVL3 reduction and Wnt/β-catenin signaling by AMPK activators and suppression of cell proliferation. MCF-7 and MDA-MB-231 cells were treated with metformin, and an XTT assay was performed to analyze cell proliferation. Cell proliferation analysis demonstrated that metformin significantly suppressed cell proliferation in MCF-7 and MDA-MB-231 cells in a dose- and time-dependent manner (Fig. 5B). The clonogenic assay supported the results of the cell proliferation assay by demonstrating a reduction in the number of colonies by 41 and 68% in MCF-7 cells, and 22 and 56% in MDA-MB-231 cells at 10 and 20 mM concentrations of metformin, respectively (Fig. 5C). Notably, metformin did not modulate the levels of DVL3 mRNA in the two breast cancer cell lines (Fig. 5D). A very high concentration of metformin (80 mM) resulted in a small decrease in DVL3 mRNA in MCF-7 cells (P=0.042). This observation suggested that metformin may have no interaction with the DVL3 gene and it may act at the post-transcriptional level. These results indicated that breast cancer cell growth inhibition via AMPK is associated with a reduction in DVL3 protein expression levels.