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. 2011 Feb 8:11:59.
doi: 10.1186/1471-2407-11-59.

SFRP1 reduction results in an increased sensitivity to TGF-β signaling

Kelly J Gauger  1 , Kerry L Chenausky, Molly E Murray, Sallie S Schneider
Affiliations

SFRP1 reduction results in an increased sensitivity to TGF-β signaling

Kelly J Gauger et al. BMC Cancer. .

Abstract

Background: Transforming growth factor (TGF)-β plays a dual role during mammary gland development and tumorigenesis and has been shown to stimulate epithelial-mesenchymal transition (EMT) as well as cellular migration. The Wnt/β-catenin pathway is also implicated in EMT and inappropriate activation of the Wnt/β-catenin signaling pathway leads to the development of several human cancers, including breast cancer. Secreted frizzled-related protein 1 (SFRP1) antagonizes this pathway and loss of SFRP1 expression is frequently observed in breast tumors and breast cancer cell lines. We previously showed that when SFRP1 is knocked down in immortalized non-malignant mammary epithelial cells, the cells (TERT-siSFRP1) acquire characteristics associated with breast tumor initiating cells. The phenotypic and genotypic changes that occur in response to SFRP1 loss are consistent with EMT, including a substantial increase in the expression of ZEB2. Considering that ZEB2 has been shown to interact with mediators of TGF-β signaling, we sought to determine whether TGF-β signaling is altered in TERT-siSFRP1 cells.

Methods: Luciferase reporter assays and real-time PCR analysis were employed to measure TGF-β transcriptional targets. Western blot analysis was used to evaluate TGF-β-mediated ERK1/2 phosphorylation. Migration chamber assays were utilized to quantify cellular migration. TERT-siSFRP1 cells were transfected with Stealth RNAi™ siRNA in order to knock-down the expression of ZEB2.

Results: TERT-siSFRP1 cells exhibit a significant increase in both TGF-β-mediated luciferase activity as well as TGF-β transcriptional targets, including Integrin β3 and PAI-1. Phosphorylation of ERK1/2 is increased in TERT-siSFRP1 cells in response to enhanced TGF-β signaling. Furthermore, when the TGF-β pathway is blocked with a TGF-βR antagonist (LY364947), cellular migration is significantly hindered. Finally, we found that when ZEB2 is knocked-down, there is a significant reduction in the expression of exogeneous and endogenous TGF-β transcriptional targets and cellular migration is impeded.

Conclusions: We demonstrate that down-regulation of SFRP1 renders mammary epithelial cells more sensitive to TGF-β signaling which can be partially ameliorated by blocking the expression of ZEB2.

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Figures

Figure 1
Figure 1
Loss of SFRP1 enhances TGF-β signaling activity. TERT-pSUPER and TERT-siSFRP1 cells were transfected with the CAGA-Luc and CMV-Renilla reporter vectors and relative luciferase activity was measured after an overnight incubation in the presence or absence of the 2.5 ng/ml TGF-β1. Bars represent mean ± SEM of relative luciferase activity (firefly luciferase activity/renilla luciferase activity) normalized to the relative luciferase activity in TERT-pSUPER cells treated with vehicle. ***p < 0.001 [significantly different from corresponding TERT-pSUPER cell line using students's t-test]
Figure 2
Figure 2
TGF-β responsive gene expression is augmented in response to SFRP1 loss. After an overnight treatment with 2.5 ng/ml TGF-β1 (A), or an overnight treatment with 10 μM LY364947 (B), total RNA was isolated from three separate harvests for real-time PCR analysis of β3 Integrin and PAI-1. All real-time PCR results are from two separate experiments performed in triplicate and results were normalized to amplification of β-actin mRNA. Bars represent mean ± SEM and are expressed as fold change with respect to TERT-pSUPER cells. *p < 0.05, **p < 0.01, ***p < 0.001 (significantly different from indicated data set using student's t-test)
Figure 3
Figure 3
Loss of SFRP1 increases TGF-β mediated ERK1/2 acivation and migration. TERT-pSUPER and TERT-siSFRP1 cells were serum starved overnight and treated with 5 μM U0126 (A) or 2.5 ng/ml TGF-β1 and/or 10 μM LY364947 (B). Cell lysates were analyzed for phospho-ERK1/2, ERK1/2, and Actin protein expression by western blot (A-B). TERT-pSUPER and TERT-siSFRP1 cells were plated in BD BioCoat™ control inserts and the cells capable of migrating through the 8 μm pore towards a chemoattractant were stained with 10% crystal violet and counted. Images were captured at 10X magnification (C). Experiments were repeated the number of cells within a representative 10X field from each experiment were counted and bars represent mean ± SEM cell number (D). *p < 0.05, (significantly different from vehicle treated cells using a student's t-test).
Figure 4
Figure 4
Knockdown of ZEB2 partially blocks TGF-β signaling in TERT-siSFRP1 cells. Stealth RNAi™ siRNA negative controls (62.5 nM) were transfected into TERT-pSUPER and TERT-siSFRP1 cells [TERT-pSUPER (control) and TERT-siSFRP1 (control)] and ZEB2 Stealth RNAi™ siRNA (62.5 nM) was transfected into TERT-siSFRP1 cells [TERT-siSFRP1 (siZEB2)]. Forty-eight hours after transfection, total RNA was isolated from each cell line for real-time PCR analysis. The levels of ZEB2 mRNA (A) β3 Integrin mRNA and PAI-1 mRNA (C) were normalized to amplification of β-actin mRNA, which was performed in parallel wells for each cell line. Bars represent mean ± SEM and are expressed as relative expression of TERT-pSUPER (control) cells. *p < 0.05, ***p < 0.001 (significantly different from TERT-pSUPER (control) cell line using Bonferroni's t-test following 1-way ANOVA). (B) TERT-siSFRP1 cells were transfected with either a Stealth RNAi™ siRNA negative control (62.5 nM) or ZEB2 Stealth RNAi™ siRNA (62.5 nM) and CAGA-Luc plus CMV-Renilla reporter vectors. The relative luciferase activity was measured after an overnight incubation in the presence or absence of the 2.5 ng/ml TGF-β1. Bars represent mean ± SEM of relative luciferase activity (firefly luciferase activity/renilla luciferase activity) normalized to the relative luciferase activity in TERT-siSFRP1 (control) cells treated with vehicle. ***p < 0.001 [significantly different from corresponding TERT-siSFRP1 (control) cell line using students's t-test]
Figure 5
Figure 5
Knockdown of ZEB2 diminishes the migratory phenotype of TERT-siSFRP1 cells. Stealth RNAi™ siRNA negative controls (62.5 nM) were transfected into TERT-pSUPER and TERT-siSFRP1 cells and ZEB2 Stealth RNAi™ siRNA (62.5 nM) was transfected into TERT-siSFRP1 cells. After 24 hours, cells were lifted from culture dishes and plated in BD BioCoat™ control inserts and the cells capable of migrating through the 8 μm pore towards a chemoattractant were stained with 10% crystal violet and counted. Images were captured at 10X magnification (A). Experiments were repeated the number of cells within a representative 10X field from each experiment were counted and bars represent mean ± SEM cell number (B). *p < 0.05 (significantly different from TERT-pSUPER (control) cell line using Bonferroni's t-test following 1-way ANOVA).
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