ABSTRACT
The transcriptional coactivator with PDZ-binding motif (TAZ) (WWTR1) induces epithelial-mesenchymal transition and enhances drug resistance in multiple cancers. TAZ has been shown to interact with transcription factors in the nucleus, but when phosphorylated, translocates to the cytoplasm and is degraded through proteasomes. Here, we identified a compound TAZ inhibitor 4 (TI-4) that shifted TAZ localization to the cytoplasm independently of its phosphorylation. We used affinity beads to ascertain a putative target of TI-4, chromosomal segregation 1 like (CSE1L), which is known to be involved in the recycling of importin α and as a biomarker of cancer malignancy. We found that TI-4 suppressed TAZ-mediated transcription in a CSE1L-dependent manner. CSE1L overexpression increased nuclear levels of TAZ, whereas CSE1L silencing delayed its nuclear import. We also found via the in vitro coimmunoprecipitation experiments that TI-4 strengthened the interaction between CSE1L and importin α5 and blocked the binding of importin α5 to TAZ. WWTR1 silencing attenuated CSE1L-promoted colony formation, motility, and invasiveness of human lung cancer and glioblastoma cells. Conversely, CSE1L silencing blocked TAZ-promoted colony formation, motility, and invasiveness in human lung cancer and glioblastoma cells. In human cancer tissues, the expression level of CSE1L was found to correlate with nuclear levels of TAZ. These findings support that CSE1L promotes the nuclear accumulation of TAZ and enhances malignancy in cancer cells.
Subject(s)
Cell Nucleus/metabolism , Cellular Apoptosis Susceptibility Protein/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Neoplasms/metabolism , Neoplasms/pathology , Trans-Activators/metabolism , Cell Line, Tumor , Cell Movement , Cell Proliferation , Gene Expression Regulation, Neoplastic , Gene Silencing , Green Fluorescent Proteins/metabolism , Humans , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Models, Biological , Neoplasm Invasiveness , Neoplasms/genetics , Phosphorylation , Photobleaching , Protein Binding , Protein Transport , Subcellular Fractions/metabolism , Transcriptional Coactivator with PDZ-Binding Motif Proteins , Tumor Stem Cell Assay , alpha Karyopherins/metabolismABSTRACT
Yes-associated protein 1 (YAP1) interacts with TEAD transcription factor in the nucleus and upregulates TEAD-target genes. YAP1 is phosphorylated by large tumor suppressor (LATS) kinases, the core kinases of the Hippo pathway, at 5 serine residues and is sequestered and degraded in the cytoplasm. In human cancers with the dysfunction of the Hippo pathway, YAP1 becomes hyperactive and confers malignant properties to cancer cells. We have observed that cold shock induces protein kinase C (PKC)-mediated phosphorylation of YAP1. PKC phosphorylates YAP1 at 3 serine residues among LATS-mediate phosphorylation sites. Importantly, PKC activation recruits YAP1 to the cytoplasm even in LATS-depleted cancer cells and reduces the cooperation with TEAD. PKC activation induces promyelocytic leukemia protein-mediated SUMOylation of YAP1. SUMOylated YAP1 remains in the nucleus, binds to p73, and promotes p73-target gene transcription. Bryostatin, a natural anti-neoplastic reagent that activates PKC, induces YAP1/p73-mediated apoptosis in cancer cells. Bryostatin reverses malignant transformation caused by the depletion of LATS kinases. Therefore, bryostatin and other reagents that activate PKC are expected to control cancers with the dysfunction of the Hippo pathway.
Subject(s)
Signal Transduction , Humans , Bryostatins , Phosphoproteins/metabolism , Protein Kinase C/metabolism , Serine , Signal Transduction/genetics , YAP-Signaling ProteinsABSTRACT
Transcriptional co-activator with PDZ-binding motif (TAZ) plays versatile roles in cell proliferation and differentiation. It is phosphorylated by large tumor suppressor kinases, the core kinases of the tumor-suppressive Hippo pathway. Phosphorylation induces the cytoplasmic accumulation of TAZ and its degradation. In human cancers, the deregulation of the Hippo pathway and gene amplification enhance TAZ activity. TAZ interacts with TEA domain family members (TEAD), and upregulates genes implicated in epithelial-mesenchymal transition. It also confers stemness to cancer cells. Thus, TAZ activation provides cancer cells with malignant properties and worsens the clinical prognosis. Therefore, TAZ attracts attention as a therapeutic target in cancer therapy. We applied 18 606 small chemical compounds to human osteosarcoma U2OS cells expressing GFP-fused TAZ (GFP-TAZ), monitored the subcellular localization of GFP-TAZ, and selected 33 compounds that shifted GFP-TAZ to the cytoplasm. Unexpectedly, only a limited number of compounds suppressed TAZ-mediated enhancement of TEAD-responsive reporter activity. Moreover, the compounds that weakened TEAD reporter activity did not necessarily decrease the unphosphorylated TAZ. In this study, we focused on three compounds that decreased both TEAD reporter activity and unphosphorylated TAZ, and treated several human cancer cells with these compounds. One compound did not show a remarkable effect, whereas the other two compounds compromised the cell viability in certain cancer cells. In conclusion, the GFP-TAZ-based assay can be used as the first screening for compounds that inhibit TAZ and show anticancer properties. To develop anticancer drugs, we need additional assays to select the compounds.
Subject(s)
Drug Evaluation, Preclinical/standards , Green Fluorescent Proteins/metabolism , PDZ Domains/drug effects , Small Molecule Libraries/analysis , Small Molecule Libraries/pharmacology , Transcription Factors/antagonists & inhibitors , Transcription Factors/chemistry , Transcription, Genetic/drug effects , Amino Acid Motifs , Cell Line, Tumor , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cell Survival/drug effects , Cytoplasm/drug effects , Cytoplasm/metabolism , Dobutamine/pharmacology , Drug Evaluation, Preclinical/methods , Ethanolamines/analysis , Ethanolamines/pharmacology , Genes, Reporter , Green Fluorescent Proteins/genetics , HEK293 Cells , Heterocyclic Compounds, 3-Ring/analysis , Heterocyclic Compounds, 3-Ring/pharmacology , Hippo Signaling Pathway , Humans , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation/drug effects , Protein Binding/drug effects , Protein Serine-Threonine Kinases/metabolism , Pyridines/analysis , Pyridines/pharmacology , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction/drug effects , Thiourea/analogs & derivatives , Thiourea/analysis , Thiourea/pharmacology , Time Factors , Transcription Factors/genetics , Transcription Factors/metabolism , ortho-Aminobenzoates/analysis , ortho-Aminobenzoates/pharmacologyABSTRACT
Yes-associated protein 1 (YAP1) and transcriptional co-activator with PDZ-binding motif (TAZ) co-operate with numerous transcription factors to regulate gene transcriptions. YAP1 and TAZ are negatively regulated by the tumor suppressive Hippo pathway. In human cancers, the Hippo pathway is frequently deregulated and YAP1 and TAZ escape the inhibition by the Hippo pathway. The upregulation of YAP1 and TAZ induces epithelial-mesenchymal transition and increases drug resistance in cancer cells. TAZ is implicated in cancer stemness. In consequence cancers with hyperactive YAP1 and TAZ are associated with poor clinical prognosis. Inhibitors of YAP1 and TAZ are reasoned to be beneficial in cancer therapy. On the other hand, since YAP1 and TAZ play important roles in the regulation of various tissue stem cells and in tissue repair, activators of YAP1 and TAZ are useful in the regenerative medicine. We discuss the potential application of inhibitors and activators of YAP1 and TAZ in human diseases and review the progress of drug screenings to search for them.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Neoplasms/metabolism , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Drug Resistance, Neoplasm , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Neoplastic , Hippo Signaling Pathway , Humans , Molecular Targeted Therapy , Neoplasms/drug therapy , Neoplastic Stem Cells/drug effects , Prognosis , Regenerative Medicine , Signal Transduction , Trans-Activators , Transcription Factors , Transcriptional Coactivator with PDZ-Binding Motif Proteins , YAP-Signaling ProteinsABSTRACT
Membrane-associated guanylate kinase with an inverted arrangement of protein-protein interaction domains (MAGI)2 (also called synaptic scaffolding molecule (S-SCAM), atrophin-1-interacting protein 1, activin receptor-interacting protein 1) is a scaffold protein that binds a wide variety of receptors, cell adhesion molecules and signalling molecules. It also interacts with other scaffold proteins and adaptors, and forms a protein network that supports cell junctions. As it is highly expressed in brain, the study on its roles in synaptic organization initially preceded. However, mounting evidence indicates that MAGI2/S-SCAM functions as a tumour suppressor and plays essential roles to maintain the integrity of cell structures in non-neuronal tissues. We review the articles regarding to MAGI2/S-SCAM outside brain and discuss future perspectives for the research of MAGI family proteins.
Subject(s)
Carrier Proteins/metabolism , Adaptor Proteins, Signal Transducing , Animals , Brain Neoplasms/physiopathology , Carrier Proteins/chemistry , Guanylate Kinases , Humans , Models, Biological , Signal Transduction , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/metabolismABSTRACT
Transcriptional co-activator with PSD-95/Dlg-A/ZO-1 (PDZ)-binding motif (TAZ) regulates in cell proliferation and differentiation. In mesenchymal stem cells it promotes osteogenesis and myogenesis, and suppresses adipogenesis. TAZ activators are expected to prevent osteoporosis, obesity and muscle atrophy. TAZ activation induces epithelial-mesenchymal transition, confers stemness to cancer cells and leads to poor clinical prognosis in cancer patients. In this point of view, TAZ inhibitors should contribute to cancer therapy. Thus, TAZ attracts attention as a two-faced drug target. We screened for TAZ modulators by using human lung cancer A549 cells expressing the fluorescent reporter. Through this assay, we obtained TAZ activator candidates. We unexpectedly found that ethacridine, a widely used antiseptic and abortifacient, enhances the interaction of TAZ and protein phosphatases and increases unphosphorylated and nuclear TAZ. Ethacridine inhibits adipogenesis in mesenchymal C3H10T1/2 cells through the activation of TAZ. This finding suggests that ethacridine is a bona fide TAZ activator and supports that our assay is useful to discover TAZ activators.