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Wnt signaling blockage inhibits cell proliferation and migration, and induces apoptosis in triple-negative breast cancer cells.

Bilir B, Kucuk O, Moreno CS - J Transl Med (2013)

Bottom Line: In addition, treatment with iCRT-3 resulted in increased apoptosis in vitro.Knockdown of the Wnt pathway transcription factor, SOX4 in triple negative BT-549 cells resulted in decreased cell proliferation and migration, and combination treatment of iCRT-3 with SOX4 knockdown had a synergistic effect on inhibition of cell proliferation and induction of apoptosis.These data suggest that targeting SOX4 and/or the Wnt pathway could have therapeutic benefit for TNBC patients.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia, USA. cmoreno@emory.edu.

ABSTRACT

Background: Triple-negative breast cancer (TNBC) is an aggressive clinical subtype of breast cancer that is characterized by the lack of estrogen receptor (ER) and progesterone receptor (PR) expression as well as human epidermal growth factor receptor 2 (HER2) overexpression. The TNBC subtype constitutes approximately 10%-20% of all breast cancers, but has no effective molecular targeted therapies. Previous meta-analysis of gene expression profiles of 587 TNBC cases from 21 studies demonstrated high expression of Wnt signaling pathway-associated genes in basal-like 2 and mesenchymal subtypes of TNBC. In this study, we investigated the potential of Wnt pathway inhibitors in effective treatment of TNBC.

Methods: Activation of Wnt pathway was assessed in four TNBC cell lines (BT-549, MDA-MB-231, HCC-1143 and HCC-1937), and the ER+ cell line MCF-7 using confocal microscopy and Western blot analysis of pathway components. Effectiveness of five different Wnt pathway inhibitors (iCRT-3, iCRT-5, iCRT-14, IWP-4 and XAV-939) on cell proliferation and apoptosis were tested in vitro. The inhibitory effects of iCRT-3 on canonical Wnt signaling in TNBC was evaluated by quantitative real-time RT-PCR analysis of Axin2 and dual-luciferase reporter assays. The effects of shRNA knockdown of SOX4 in combination with iCRT-3 and/or genistein treatments on cell proliferation, migration and invasion on BT-549 cells were also evaluated.

Results: Immunofluorescence staining of β-catenin in TNBC cell lines showed both nuclear and cytoplasmic localization, indicating activation of Wnt pathway in TNBC cells. iCRT-3 was the most effective compound for inhibiting proliferation and antagonizing Wnt signaling in TNBC cells. In addition, treatment with iCRT-3 resulted in increased apoptosis in vitro. Knockdown of the Wnt pathway transcription factor, SOX4 in triple negative BT-549 cells resulted in decreased cell proliferation and migration, and combination treatment of iCRT-3 with SOX4 knockdown had a synergistic effect on inhibition of cell proliferation and induction of apoptosis.

Conclusions: These data suggest that targeting SOX4 and/or the Wnt pathway could have therapeutic benefit for TNBC patients.

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Related in: MedlinePlus

SOX4 knockdown inhibits proliferation, migration and invasion of BT-549 cells, and cooperates with iCRT-3 to inhibit proliferation. Cells that were transduced with scrambled shRNA or SOX4 shRNA lentiviral particles were treated with vehicle (DMSO) or iCRT-3 (25 μM), and cell index measurements were continuously taken for 48 hours in (A) proliferation assay, and 24 hours in (B) migration and (C) invasion assays using an xCELLigence instrument. Data represent mean ± SEM of three independent experiments (**p < 0.01).
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Figure 6: SOX4 knockdown inhibits proliferation, migration and invasion of BT-549 cells, and cooperates with iCRT-3 to inhibit proliferation. Cells that were transduced with scrambled shRNA or SOX4 shRNA lentiviral particles were treated with vehicle (DMSO) or iCRT-3 (25 μM), and cell index measurements were continuously taken for 48 hours in (A) proliferation assay, and 24 hours in (B) migration and (C) invasion assays using an xCELLigence instrument. Data represent mean ± SEM of three independent experiments (**p < 0.01).

Mentions: To characterize the mechanism underlying the synergism between SOX4 knockdown and iCRT-3 treatment, we next assessed the effect of SOX4 knockdown in combination with iCRT-3 treatment on cell proliferation, migration and invasion in BT-549 cells using the xCELLigence system. Cells that were transduced with scrambled or SOX4 shRNAs were treated with DMSO or 25 μM iCRT-3, and monitored continuously for 48 hours in a proliferation assay, and for 24 hours in migration and invasion assays. As shown in Figure 6, SOX4 knockdown inhibited cell proliferation and migration. Effects on invasion were not statistically significant, although the trend was towards lower invasion with SOX4 knockdown. Combination treatment of iCRT-3 with SOX4 knockdown induced further decrease in cell proliferation, but had no increased effect over SOX4 knockdown in cell migration or invasion.


Wnt signaling blockage inhibits cell proliferation and migration, and induces apoptosis in triple-negative breast cancer cells.

Bilir B, Kucuk O, Moreno CS - J Transl Med (2013)

SOX4 knockdown inhibits proliferation, migration and invasion of BT-549 cells, and cooperates with iCRT-3 to inhibit proliferation. Cells that were transduced with scrambled shRNA or SOX4 shRNA lentiviral particles were treated with vehicle (DMSO) or iCRT-3 (25 μM), and cell index measurements were continuously taken for 48 hours in (A) proliferation assay, and 24 hours in (B) migration and (C) invasion assays using an xCELLigence instrument. Data represent mean ± SEM of three independent experiments (**p < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4228255&req=5

Figure 6: SOX4 knockdown inhibits proliferation, migration and invasion of BT-549 cells, and cooperates with iCRT-3 to inhibit proliferation. Cells that were transduced with scrambled shRNA or SOX4 shRNA lentiviral particles were treated with vehicle (DMSO) or iCRT-3 (25 μM), and cell index measurements were continuously taken for 48 hours in (A) proliferation assay, and 24 hours in (B) migration and (C) invasion assays using an xCELLigence instrument. Data represent mean ± SEM of three independent experiments (**p < 0.01).
Mentions: To characterize the mechanism underlying the synergism between SOX4 knockdown and iCRT-3 treatment, we next assessed the effect of SOX4 knockdown in combination with iCRT-3 treatment on cell proliferation, migration and invasion in BT-549 cells using the xCELLigence system. Cells that were transduced with scrambled or SOX4 shRNAs were treated with DMSO or 25 μM iCRT-3, and monitored continuously for 48 hours in a proliferation assay, and for 24 hours in migration and invasion assays. As shown in Figure 6, SOX4 knockdown inhibited cell proliferation and migration. Effects on invasion were not statistically significant, although the trend was towards lower invasion with SOX4 knockdown. Combination treatment of iCRT-3 with SOX4 knockdown induced further decrease in cell proliferation, but had no increased effect over SOX4 knockdown in cell migration or invasion.

Bottom Line: In addition, treatment with iCRT-3 resulted in increased apoptosis in vitro.Knockdown of the Wnt pathway transcription factor, SOX4 in triple negative BT-549 cells resulted in decreased cell proliferation and migration, and combination treatment of iCRT-3 with SOX4 knockdown had a synergistic effect on inhibition of cell proliferation and induction of apoptosis.These data suggest that targeting SOX4 and/or the Wnt pathway could have therapeutic benefit for TNBC patients.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia, USA. cmoreno@emory.edu.

ABSTRACT

Background: Triple-negative breast cancer (TNBC) is an aggressive clinical subtype of breast cancer that is characterized by the lack of estrogen receptor (ER) and progesterone receptor (PR) expression as well as human epidermal growth factor receptor 2 (HER2) overexpression. The TNBC subtype constitutes approximately 10%-20% of all breast cancers, but has no effective molecular targeted therapies. Previous meta-analysis of gene expression profiles of 587 TNBC cases from 21 studies demonstrated high expression of Wnt signaling pathway-associated genes in basal-like 2 and mesenchymal subtypes of TNBC. In this study, we investigated the potential of Wnt pathway inhibitors in effective treatment of TNBC.

Methods: Activation of Wnt pathway was assessed in four TNBC cell lines (BT-549, MDA-MB-231, HCC-1143 and HCC-1937), and the ER+ cell line MCF-7 using confocal microscopy and Western blot analysis of pathway components. Effectiveness of five different Wnt pathway inhibitors (iCRT-3, iCRT-5, iCRT-14, IWP-4 and XAV-939) on cell proliferation and apoptosis were tested in vitro. The inhibitory effects of iCRT-3 on canonical Wnt signaling in TNBC was evaluated by quantitative real-time RT-PCR analysis of Axin2 and dual-luciferase reporter assays. The effects of shRNA knockdown of SOX4 in combination with iCRT-3 and/or genistein treatments on cell proliferation, migration and invasion on BT-549 cells were also evaluated.

Results: Immunofluorescence staining of β-catenin in TNBC cell lines showed both nuclear and cytoplasmic localization, indicating activation of Wnt pathway in TNBC cells. iCRT-3 was the most effective compound for inhibiting proliferation and antagonizing Wnt signaling in TNBC cells. In addition, treatment with iCRT-3 resulted in increased apoptosis in vitro. Knockdown of the Wnt pathway transcription factor, SOX4 in triple negative BT-549 cells resulted in decreased cell proliferation and migration, and combination treatment of iCRT-3 with SOX4 knockdown had a synergistic effect on inhibition of cell proliferation and induction of apoptosis.

Conclusions: These data suggest that targeting SOX4 and/or the Wnt pathway could have therapeutic benefit for TNBC patients.

Show MeSH
Related in: MedlinePlus