ABSTRACT
ZEB1 transcription factor is important in both development and disease, including many TGFß-induced responses, and the epithelial-to-mesenchymal transition (EMT) by which many tumors undergo metastasis. ZEB1 is differentially phosphorylated in different cell types; however the role of phosphorylation in ZEB1 activity is unknown. Luciferase reporter studies and electrophoresis mobility shift assays (EMSA) show that a decrease in phosphorylation of ZEB1 increases both DNA-binding and transcriptional repression of ZEB1 target genes. Functional analysis of ZEB1 phosphorylation site mutants near the second zinc finger domain (termed ZD2) show that increased phosphorylation (due to either PMA plus ionomycin, or IGF-1) can inhibit transcriptional repression by either a ZEB1-ZD2 domain clone, or full-length ZEB1. This approach identifies phosphosites that have a substantial effect regulating the transcriptional and DNA-binding activity of ZEB1. Immunoprecipitation with anti-ZEB1 antibodies followed by western analysis with a phospho-Threonine-Proline-specific antibody indicates that the ERK consensus site at Thr-867 is phosphorylated in ZEB1. In addition to disrupting in vitro DNA-binding measured by EMSA, IGF-1-induced MEK/ERK phosphorylation is sufficient to disrupt nuclear localization of GFP-ZEB1 fusion clones. These data suggest that phosphorylation of ZEB1 integrates TGFß signaling with other signaling pathways such as IGF-1. J. Cell. Physiol. 231: 2205-2217, 2016. © 2016 Wiley Periodicals, Inc.
Subject(s)
Zinc Finger E-box-Binding Homeobox 1/metabolism , Animals , CHO Cells , Cricetulus , Epithelial-Mesenchymal Transition , Insulin-Like Growth Factor I/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Phosphorylation , Signal Transduction/physiology , Zinc Finger E-box-Binding Homeobox 1/genetics , Zinc FingersABSTRACT
During progression of breast cancer, CCN6 protein exerts tumor inhibitory functions. CCN6 is a secreted protein that modulates the insulin-like growth factor-1 (IGF-1) signaling pathway. Knockdown of CCN6 in benign mammary epithelial cells triggers an epithelial to mesenchymal transition (EMT), with upregulation of the transcription factor ZEB1/δEF1. How CCN6 regulates ZEB1 expression is unknown. We hypothesized that CCN6 might regulate ZEB1, EMT and breast cancer invasion by modulating IGF-1 signaling. Exogenously added human recombinant CCN6 protein was sufficient to downregulate ZEB1 mRNA and protein levels in CCN6-deficient (CCN6 KD) HME cells and MDA-MB-231 breast cancer cells. Recombinant CCN6 protein decreased invasion of CCN6 KD cells compared with controls. We discovered that knockdown of CCN6 induced IGF-1 secretion in HME cells cultivated in serum-free medium to higher concentrations than found in MDA-MB-231 cells. Treatment with recombinant CCN6 protein was sufficient to decrease IGF-1 protein and mRNA to control levels, rescuing the effect of CCN6 knockdown. Specific inhibition of IGF-1 receptors using the pharmacological inhibitor NVP-AE541 or short hairpin shRNAs revealed that ZEB1 upregulation due to knockdown of CCN6 requires activation of IGF-1 receptor signaling. Recombinant CCN6 blunted IGF-1-induced ZEB1 upregulation in MDA-MB-231 cells. Our data define a pathway in which CCN6 attenuates IGF-1 signaling to decrease ZEB1 expression and invasion in breast cancer. These results suggest that CCN6 could be a target to prevent or halt breast cancer invasion.
Subject(s)
Breast Neoplasms/metabolism , Homeodomain Proteins/metabolism , Insulin-Like Growth Factor Binding Proteins/metabolism , Receptor, IGF Type 1/metabolism , Transcription Factors/metabolism , Breast Neoplasms/pathology , CCN Intercellular Signaling Proteins , Cell Dedifferentiation/drug effects , Cell Line, Tumor , Down-Regulation , Epithelial-Mesenchymal Transition/drug effects , Female , Fluorescent Antibody Technique , Gene Knockdown Techniques , Homeodomain Proteins/biosynthesis , Homeodomain Proteins/genetics , Humans , Insulin-Like Growth Factor Binding Proteins/deficiency , Insulin-Like Growth Factor Binding Proteins/genetics , Insulin-Like Growth Factor Binding Proteins/pharmacology , Insulin-Like Growth Factor I/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptor, IGF Type 1/antagonists & inhibitors , Recombinant Proteins/pharmacology , Signal Transduction/drug effects , Transcription Factors/biosynthesis , Transcription Factors/genetics , Up-Regulation , Zinc Finger E-box-Binding Homeobox 1ABSTRACT
Due to the key role of DNA in cell life and pathological processes, the design of specific chemical nucleases, DNA probes and alkylating agents is an important research area for the development of new therapeutic agents and tools in Biochemistry. Hence, the interaction of small molecules with DNA has attracted in particular a great deal of attention. The aim of this study was to investigate the ability of [Cr(phen)(2)(dppz)](3+) to associate with DNA and to characterize it as photocleavage reagent for Photodynamic Therapy (PDT). Chromium(III) complex [Cr(phen)(2)(dppz)](3+), (dppz = dipyridophenazine, phen = 1,10-phenanthroline), where dppz is a planar bidentate ligand with an extended pi system, has been found to bind strongly to double strand oligonucleotides (ds-oligo) and plasmid DNA with intrinsic DNA binding constants, K(b,) of (3.9+/-0.3)x10(5) M(-1) and (1.1+/-0.1)x10(5) M(-1), respectively. The binding properties to DNA were investigated by UV-visible (UV-Vis) absorption spectroscopy and electrophoretic studies. UV-Vis absorption data provide clearly that the chromium(III) complex interacts with DNA intercalatively. Competitive binding experiments show that the enhancement in the emission intensity of ethidium bromide (EthBr) in the presence of DNA was quenched by [Cr(phen)(2)(dppz)](3+), indicating that the Cr(III) complex displaces EthBr from its binding site in plasmid DNA. Moreover, [Cr(phen)(2)(dppz)](3+), non-covalently bound to DNA, promotes the photocleavage of plasmid DNA under 457 nm irradiation. We also found that the irradiated Cr(III)-plasmid DNA association is able to impair the transforming capacity of bacteria. These results provide evidence confirming the responsible and essential role of the excited state of [Cr(phen)(2)(dppz)](3+) for damaging the DNA structure. The combination of DNA, [Cr(phen)(2)(dppz)](3+) and light, is necessary to induce damage. In addition, assays of the photosensitization of transformed bacterial suspensions suggest that Escherichia coli may be photoinactivated by irradiation in the presence of [Cr(phen)(2)(dppz)](3+). In sum, our results allow us to postulate the [Cr(phen)(2)(dppz)](3+) complex as a very attractive candidate for DNA photocleavage with potential applications in Photodynamic Therapy (PDT).
Subject(s)
Chromium/chemistry , DNA/chemistry , Intercalating Agents/chemistry , Photochemotherapy , Binding, Competitive , DNA/metabolism , Ligands , Organometallic Compounds/chemistry , Phenanthrolines/chemistry , Phenazines , PhotolysisABSTRACT
The CCN family of matricellular proteins is essential for cell communication and mediation of epithelial stromal cross-talks with roles in development and cancer. In particular, loss of CCN6 messenger RNA expression has been recognized in highly aggressive breast cancers, especially in inflammatory breast cancer and breast cancers with axillary lymph node metastasis. Recent findings can better explain the relevance of CCN6's reduced expression on human invasive breast carcinomas. CCN6 has been shown to play a role in the process of epithelial to mesenchymal transition (EMT), which converts epithelial cells into migratory mesenchymal-like cells with invasive abilities. Although the mechanism by which CCN6 promotes EMT and invasion has not been fully elucidated, current data suggest that it involves the recruitment of the transcriptional regulators Snai1 and ZEB1 to the E-cadherin promoter.
