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DACT1, an antagonist to Wnt/β-catenin signaling, suppresses tumor cell growth and is frequently silenced in breast cancer.

Yin X, Xiang T, Li L, Su X, Shu X, Luo X, Huang J, Yuan Y, Peng W, Oberst M, Kelly K, Ren G, Tao Q - Breast Cancer Res. (2013)

Bottom Line: We found that DACT1 expression was silenced in eight (88.9%) of nine breast cancer cell lines, and its protein levels were obviously reduced in breast tumors compared with paired surgical-margin tissues.Functional assays showed that ectopic expression of DACT1 could inhibit breast tumor cell proliferation in vivo and in vitro through inducing apoptosis, and further suppress tumor cell migration through antagonizing the Wnt/β-catenin signaling pathway.Our study demonstrates that DACT1 could function as a tumor suppressor but was frequently downregulated in breast cancer.

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ABSTRACT

Introduction: Aberrant activation of Wnt/β-catenin signaling plays an important role in the pathogenesis of breast cancer. DACT1 (Dapper/Frodo) has been identified as involved in antagonizing Wnt/β-catenin signaling through interacting with Dishevelled (Dvl), a central mediator of Wnt signaling, whereas its role in breast tumorigenesis remains unclear.

Methods: We examined DACT1 expression in breast cancer cell lines and primary tumors with semiquantitative or quantitative RT-PCR and immunochemistry, and further evaluated the promoter methylation of DACT1 with methylation-specific PCR (MSP). We also explored the tumor-suppressive functions of DACT1 in vivo and in vitro, and its related mechanism in breast cancer.

Results: We identified DACT1 as a methylated target in our breast cancer epigenome study. Here, we further investigated DACT1 expression in multiple breast cell lines and primary tumors, and further studied its function and molecular mechanisms. We found that DACT1 expression was silenced in eight (88.9%) of nine breast cancer cell lines, and its protein levels were obviously reduced in breast tumors compared with paired surgical-margin tissues. Promoter CpG methylation of DACT1 was detected in five (55.6%) of nine breast cancer cell lines and 40 (29.9%) of 134 primary tumors, but not in surgical-margin tissues and normal breast tissues. Demethylation treatment of breast cancer cell lines restored DACT1 expression along with promoter demethylation, suggesting that an epigenetic mechanism mediates DACT1 silencing in breast cancer. Functional assays showed that ectopic expression of DACT1 could inhibit breast tumor cell proliferation in vivo and in vitro through inducing apoptosis, and further suppress tumor cell migration through antagonizing the Wnt/β-catenin signaling pathway.

Conclusions: Our study demonstrates that DACT1 could function as a tumor suppressor but was frequently downregulated in breast cancer.

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Tumor-suppressive functions of DACT1 in breast cancer cells. (A) Subcellular localization of DACT1 in MB231 cells was detected with immunofluorescence. (B) Representative colony-formation assay of DACT1 in MB231 and MCF7 (left panel). Quantitative analyses of colony numbers are shown as values of mean ± SD. *P < 0.05 (right panel, upper). DACT1 expression as measured with RT-PCR in vector- and DACT1-transfected MB231 and MCF7 cells (right panel, lower). (C) Cell Counting Kit-8 (CCK8) assay assessed the effect of DACT1 on cell proliferation in vector-, and DACT1- expressing MB231 cells. The values are shown as the mean ± SD. *P < 0.05; **P < 0.01. (D) The colorimetric assay for active caspase-3 was performed in vector-, and DACT1-expressing MB231 and MCF7 breast cancer cells at 24 and 48 hours.
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Figure 4: Tumor-suppressive functions of DACT1 in breast cancer cells. (A) Subcellular localization of DACT1 in MB231 cells was detected with immunofluorescence. (B) Representative colony-formation assay of DACT1 in MB231 and MCF7 (left panel). Quantitative analyses of colony numbers are shown as values of mean ± SD. *P < 0.05 (right panel, upper). DACT1 expression as measured with RT-PCR in vector- and DACT1-transfected MB231 and MCF7 cells (right panel, lower). (C) Cell Counting Kit-8 (CCK8) assay assessed the effect of DACT1 on cell proliferation in vector-, and DACT1- expressing MB231 cells. The values are shown as the mean ± SD. *P < 0.05; **P < 0.01. (D) The colorimetric assay for active caspase-3 was performed in vector-, and DACT1-expressing MB231 and MCF7 breast cancer cells at 24 and 48 hours.

Mentions: DACT1 repression by promoter methylation in breast cancer indicated that DACT1 is likely a tumor suppressor. Immunostaining showed that DACT1 is located mainly in the cytoplasm and membrane of DACT1-expressing MB231 cells (Figure 4A). Colony-formation assay and CCK-8 cell-proliferation assay were further performed to assess the effect of DACT1 on cell proliferation of breast cancer. About 40% to 60% reduction of colony numbers was observed in DACT1-transfected MB231 and MCF7 cells, compared with controls (*P < 0.05) (Figure 4B). Cell viability was significantly decreased at 24, 48, and 72 hours after transfection with DACT1 in MB231 cells (**P < 0.01; *P < 0.05) (Figure 4C).


DACT1, an antagonist to Wnt/β-catenin signaling, suppresses tumor cell growth and is frequently silenced in breast cancer.

Yin X, Xiang T, Li L, Su X, Shu X, Luo X, Huang J, Yuan Y, Peng W, Oberst M, Kelly K, Ren G, Tao Q - Breast Cancer Res. (2013)

Tumor-suppressive functions of DACT1 in breast cancer cells. (A) Subcellular localization of DACT1 in MB231 cells was detected with immunofluorescence. (B) Representative colony-formation assay of DACT1 in MB231 and MCF7 (left panel). Quantitative analyses of colony numbers are shown as values of mean ± SD. *P < 0.05 (right panel, upper). DACT1 expression as measured with RT-PCR in vector- and DACT1-transfected MB231 and MCF7 cells (right panel, lower). (C) Cell Counting Kit-8 (CCK8) assay assessed the effect of DACT1 on cell proliferation in vector-, and DACT1- expressing MB231 cells. The values are shown as the mean ± SD. *P < 0.05; **P < 0.01. (D) The colorimetric assay for active caspase-3 was performed in vector-, and DACT1-expressing MB231 and MCF7 breast cancer cells at 24 and 48 hours.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Tumor-suppressive functions of DACT1 in breast cancer cells. (A) Subcellular localization of DACT1 in MB231 cells was detected with immunofluorescence. (B) Representative colony-formation assay of DACT1 in MB231 and MCF7 (left panel). Quantitative analyses of colony numbers are shown as values of mean ± SD. *P < 0.05 (right panel, upper). DACT1 expression as measured with RT-PCR in vector- and DACT1-transfected MB231 and MCF7 cells (right panel, lower). (C) Cell Counting Kit-8 (CCK8) assay assessed the effect of DACT1 on cell proliferation in vector-, and DACT1- expressing MB231 cells. The values are shown as the mean ± SD. *P < 0.05; **P < 0.01. (D) The colorimetric assay for active caspase-3 was performed in vector-, and DACT1-expressing MB231 and MCF7 breast cancer cells at 24 and 48 hours.
Mentions: DACT1 repression by promoter methylation in breast cancer indicated that DACT1 is likely a tumor suppressor. Immunostaining showed that DACT1 is located mainly in the cytoplasm and membrane of DACT1-expressing MB231 cells (Figure 4A). Colony-formation assay and CCK-8 cell-proliferation assay were further performed to assess the effect of DACT1 on cell proliferation of breast cancer. About 40% to 60% reduction of colony numbers was observed in DACT1-transfected MB231 and MCF7 cells, compared with controls (*P < 0.05) (Figure 4B). Cell viability was significantly decreased at 24, 48, and 72 hours after transfection with DACT1 in MB231 cells (**P < 0.01; *P < 0.05) (Figure 4C).

Bottom Line: We found that DACT1 expression was silenced in eight (88.9%) of nine breast cancer cell lines, and its protein levels were obviously reduced in breast tumors compared with paired surgical-margin tissues.Functional assays showed that ectopic expression of DACT1 could inhibit breast tumor cell proliferation in vivo and in vitro through inducing apoptosis, and further suppress tumor cell migration through antagonizing the Wnt/β-catenin signaling pathway.Our study demonstrates that DACT1 could function as a tumor suppressor but was frequently downregulated in breast cancer.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: Aberrant activation of Wnt/β-catenin signaling plays an important role in the pathogenesis of breast cancer. DACT1 (Dapper/Frodo) has been identified as involved in antagonizing Wnt/β-catenin signaling through interacting with Dishevelled (Dvl), a central mediator of Wnt signaling, whereas its role in breast tumorigenesis remains unclear.

Methods: We examined DACT1 expression in breast cancer cell lines and primary tumors with semiquantitative or quantitative RT-PCR and immunochemistry, and further evaluated the promoter methylation of DACT1 with methylation-specific PCR (MSP). We also explored the tumor-suppressive functions of DACT1 in vivo and in vitro, and its related mechanism in breast cancer.

Results: We identified DACT1 as a methylated target in our breast cancer epigenome study. Here, we further investigated DACT1 expression in multiple breast cell lines and primary tumors, and further studied its function and molecular mechanisms. We found that DACT1 expression was silenced in eight (88.9%) of nine breast cancer cell lines, and its protein levels were obviously reduced in breast tumors compared with paired surgical-margin tissues. Promoter CpG methylation of DACT1 was detected in five (55.6%) of nine breast cancer cell lines and 40 (29.9%) of 134 primary tumors, but not in surgical-margin tissues and normal breast tissues. Demethylation treatment of breast cancer cell lines restored DACT1 expression along with promoter demethylation, suggesting that an epigenetic mechanism mediates DACT1 silencing in breast cancer. Functional assays showed that ectopic expression of DACT1 could inhibit breast tumor cell proliferation in vivo and in vitro through inducing apoptosis, and further suppress tumor cell migration through antagonizing the Wnt/β-catenin signaling pathway.

Conclusions: Our study demonstrates that DACT1 could function as a tumor suppressor but was frequently downregulated in breast cancer.

Show MeSH
Related in: MedlinePlus