Abstract
BackgroundTransforming 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.MethodsLuciferase 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.ResultsTERT-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.ConclusionsWe 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.
Highlights
Transforming growth factor (TGF)-b 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
We demonstrate that down-regulation of Secreted frizzledrelated protein 1 (SFRP1) renders mammary epithelial cells more sensitive to TGF-b signaling which can be partially ameliorated by blocking the expression of Zinc finger E-box-binding homeobox 2 (ZEB2)
SFRP1 loss stimulates the TGFb signaling pathway Our initial screen of genes affected by SFRP1 loss in HMECs identified several upregulated genes within the TGF-b pathway, which suggested that there may be an alteration in this signaling pathway in TERTsiSFRP1 cells (Additional File 1)
Summary
Transforming growth factor (TGF)-b 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. Transforming growth factor (TGF)-b is a multifunctional cytokine that regulates a variety of physiological processes and plays a dual role during mammary gland development and tumorigenesis. TGF-b signaling is manifested by way of TGF-b receptor activation and the canonical pathway involves the subsequent phosphorylation of receptor-associated Smad and Smad proteins which form complexes with Smad. TGF-b signaling is manifested by way of TGF-b receptor activation and the canonical pathway involves the subsequent phosphorylation of receptor-associated Smad and Smad proteins which form complexes with Smad4 These activated Smad complexes accumulate in the nucleus where, along with co-activators and cell-specific DNA-binding factors, they regulate gene expression. The neoplastic tumor cell environment transforms TGF-b into an oncogenic cytokine which actively contributes to the acquisition and development of metastatic phenotypes in part through its ability to stimulate EMT and cellular migration [4,5,6,7]
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