Methylation of DACT2 promotes breast cancer development by activating Wnt signaling

Breast cancer is the most common malignant tumor in women worldwide. To explore the role of DACT2 in breast cancer, 5 cell lines and 153 cases of primary cancer were studied. The expression of DACT2 was detected in BT474, MDA-MB-231 and BT549 cells, while no expression was found in MDA-MB-468 and HBL100 cells. Complete methylation of DACT2 was found in MDA-MB-468 and HBL100 cells, partial methylation was observed in BT474 and BT549 cells, and no methylation was detected in MDA-MB-231 cells. Restoration of DACT2 expression was induced by 5-Aza in MDA-MB-468 and HBL100 cells. DACT2 was methylated in 49.7% (76/153) of primary breast cancer samples. Methylation of DACT2 was significantly associated with tumor size (P < 0.05). Reduced DACT2 expression was significantly associated with promoter region methylation in primary breast cancer (P < 0.05). DACT2 suppressed breast cancer cell growth and induced G1/S phase arrest in breast cancer cells. DACT2 inhibited Wnt/β-catenin signaling in human breast cancer cells and suppressed breast cancer cell tumor growth in xenograft mice. In conclusion, our results demonstrate that DACT2 is frequently methylated in human breast cancer, methylation of DACT2 activates Wnt signaling, and DACT2 suppresses breast cancer cell growth both in vitro and in vivo.

found in MDA-MB-468 and HBL100 cells, partial methylation was observed in BT474and BT549 cells, and no methylation was detected in MDA-MB-231 cells (Fig. 1B). These results indicate that DACT2 was silenced by promoter region methylation.
To further validate that the expression of DACT2 was regulated by promoter region methylation, breast cancer cell lines were treated with 5-Aza. Re-expression of DACT2 was found in MDA-MB-468 and HBL100 cell lines after 5-Aza treatment. Increased expression of DACT2 was induced by 5-Aza in BT474 and BT549 cell lines. No expression changes in DACT2 were found in MDA-MB-231 cells before and after 5-Aza treatment (Fig. 1A). These results demonstrate that the expression of DACT2 is regulated by promoter region methylation. To validate the efficiency of the MSP primers, bisulfite sequencing was employed. Dense methylation was observed in the promoter region of DACT2 in MDA-MB-468 and HBL100 cells, and unmethylation was found in MDA-MB-231 cells and normal breast tissue samples. Partial methylation was found in BT549 cells (Fig. 1C).
DACT2 is frequently methylated in human breast cancer. Methylation of DACT2 was examined in 153 cases of human primary breast cancer and 5 cases of normal breast tissue samples ( Fig. 2A). DACT2 was methylated in 49.7% (76/153) of human primary breast cancer, and no methylation was found in normal breast tissue samples. Methylation of DACT2 was significantly associated with tumor size (P < 0.05, Table 1). No association was found between DACT2 methylation and age, tumor grade, tumor stage, lymph node metastasis and the expression of progesterone receptor (PR), estrogen receptor (ER) or Human epidermal growth factor receptor 2 (HER2) (all P > 0.05).
The expression of DACT2 was analyzed by immunohistochemistry in 33 cases of available matched breast cancer and adjacent tissue samples. DACT2 staining was found mainly in cytoplasm (Fig. 2B). The levels of DACT2 expression were significantly lower in cancer tissues compared to adjacent normal tissue samples ( Fig. 2C, P < 0.001). Low level expression of DACT2 was found in 24 cases of cancer samples, 16 of which were methylated. Reduced DACT2 expression was significantly associated with promoter region hypermethylation ( Fig. 2D, P < 0.05). These results further suggest that the expression of DACT2 is regulated by promoter region methylation in breast cancer.

Restoration of DACT2 expression suppresses cell growth in human breast cancer cells.
To evaluate the effects of DACT2 on breast cancer cell proliferation, the MTT assay was employed in MDA-MB-468 and HBL100 cells. The OD values were 0.94 ± 0.10 vs. 0.69 ± 0.01 (P < 0.001) in MDA-MB-468 cells and 0.54 ± 0.04 vs. 0.38 ± 0.02 (P < 0.001) in HBL100 cells before and after restoration of DACT2 expression. The effect of DACT2 on cell growth was further validated by knocking down DACT2 in MDA-MB-231 cells. The OD values were 0.66 ± 0.02 vs. 0.72 ± 0.01 (P < 0.01) before and after knockdown DACT2 in MDA-MB-231 cells (Fig. 3A).
The effects of DACT2 on cell cycle were further validated by evaluating the expression levels of cyclin D1 and cyclin E1. Under western blot detection, the expression levels of cyclin D1 and cyclin E1 were reduced after re-expression of DACT2 in MDA-MB-468 and HBL100 cells. The expression levels of cyclin D1 and cyclinE1 were increased after knock down of DACT2 in MDA-MB-231 cells (Fig. 5A). These results further demonstrate that DACT2 induced the G1/S checkpoint arrest in breast cancer cells.

DACT2 suppresses cell migration and invasion in breast cancer cells.
To evaluate the effects of DACT2 on cell migration and invasion, the transwell assays were used. The number of migratory cells was 1102.33 ± 76.57 vs. 483.00 ± 3.61 for MDA-MB-468 cells and 262.00 ± 5.00 vs. 112.00 ± 16.00 for HBL100 cells before and after restoration of DACT2 expression. The cell number was reduced significantly after re-expression of DACT2 in MDA-MB-468 and HBL100 cells (both P < 0.001, Fig. 4A). The number of migratory cells was 216.00 ± 9.85 vs. 382.33 ± 42.12 before and after knockdown of DACT2 in MDA-MB-231 cells. The cell number was increased significantly after knockdown of DACT2 in MDA-MB-231 cells (P < 0.01, Fig. 4A Fig. 4B). These results suggest that DACT2 suppresses breast cancer cell migration and invasion.
DACT2 is a Wnt/β-catenin signaling pathway inhibitor in human breast cancer. DACT2 was found to be involved in Wnt/β-catenin signaling in different cancers in our previous reports [11][12][13][14][15] . The mechanism of DACT2 in human breast cancer remains unclear. The effects of DACT2 on Wnt/β-catenin signaling were analyzed by DACT2 overexpression and siRNA knockdown techniques in human breast cancer cells. The levels of non-phospho (active) β-Catenin were reduced and the levels of p-β-catenin were increased while the total level of β-catenin was not changed after re-expression of DACT2 in MDA-MB-468 and HBL100 cells. The levels of downstream target genes, myc and cyclinD1, were reduced after restoration of DACT2 expression (Fig. 5A). The levels of active-β-catenin, myc and cyclinD1 increased, the levels of p-β-catenin decreased, and the levels of total β-catenin did not change after knockdown of DACT2 in MDA-MB-231 cells (Fig. 5A). These results suggest that DACT2 inhibits the Wnt/β-catenin signaling pathway in human breast cancer cells.

DACT2 inhibits breast cancer cell growth in vivo.
To further validate the effects of DACT2 on breast cancer cell growth, DACT2 unexpressed and re-expressed MDA-MB-468 cell xenograft mouse models were employed (Fig. 5B). The tumor volumes were 160.7 ± 12.8 mm 3

Discussion
The connection of wnt signaling and human cancer was first reported by Kinzler et al. in 1991 16 .The DACT family of scaffold proteins was discovered by virtue of their binding to Dvl proteins, central to Wnt and Planar Cell Polarity (PCP) signaling 9 . In zebrafish, DACT1 has a greater impact on β-catenin-dependent signaling and DACT2 has a greater impact on the β-catenin-independent process called planar cell polarity/ convergent-extension signaling 17 . DACT3 has been reported to be a negative regulator of canonical Wnt signaling both in mice development and human colorectal cancer 18,19 . A recent study suggests that there are two Dact3 paralogs (dact3a and dact3b) in zebrafish. The dact3a and dact3b paralogs have not been well studied in development and human diseases. A novel member of DACT gene family, DACT4, was identified in zebrafish in a recent report 20,21 . The importance of these DACT members in development has been gradually recognized. DACT2 is the best studied DACT member in human diseases [11][12][13][14][15]22 .
In this study, we found that DACT2 is frequently methylated in human breast cancer, and the expression of DACT2 is regulated by promoter region methylation. Re-expression of DACT2 suppresses breast cancer cell growth in vitro and in vivo. We demonstrated that DACT2 suppresses human breast cancer growth by inhibiting the Wnt/β-catenin signaling pathway. DACT2 methylation is associated with tumor size. Recently, Xiang et al. found that DACT2 inhibits EMT by antagonizing wnt signaling. They also found that DACT2 suppresses breast cancer cell migration and invasion by inducing actin cytoskeleton regorganization 23 . In this study, we also found that DACT2 suppresses breast cancer cell migration and invasion. In conclusion, our data indicate that DACT2 is frequency methylated in human breast cancer and the expression of DACT2 is regulated by promoter region methylation. DACT2 suppresses breast cancer development by inhibiting canonical Wnt signaling. Therefore, DACT2 methylation is a potential breast cancer detection marker.

5-Aza-2'-deoxycytidine (5-Aza) treatment.
Breast cancer cell lines were split to low density (30% confluence) 12 hours before treatment. Cells were treated with 5-Aza-2′-deoxycytidine (Sigma, St. Louis, MO) at a concentration of 2 µM in the growth medium. The growth medium was exchanged every 24 hours for a total of 96 hours treatment. RNA Isolation and Semi-quantitative RT-PCR. Total RNA was extracted using Trizol Reagent (Life Technology, MD, USA). Agarose gel electrophoresis and spectrophotometric analysis were used to detect RNA quality and quantity. First strand cDNA was synthesized according to manufacturer's instructions (Invitrogen, Carlsbad, CA). A total of 5 µg RNA was used to synthesize first strand cDNA. The reaction mixture was diluted to 100 µl with water, then 2.5 µl of diluted cDNA was used for 25 µl PCR reaction. The sequences of PCR primers for DACT2 are as follows: 5′-GGC TGA GAC AAC AGG ACA TCG-3′ (F) and 5′-GAC CGT CGC TCA TCT CGT AAAA-3′ (R). RT-PCR was amplified for 35 cycles. GAPDH was amplified for 25 cycles as an internal control. The primer sequences of GAPDH are as follows: 5′-GAC CAC AGT CCA TGC CAT CAC-3′ (F), and 5′-GTC CAC CAC CCT GTT GCT GTA-3′ (R). The amplified PCR products were examined by 1.5% agarose gels.