RESUMEN
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.
Asunto(s)
Evaluación Preclínica de Medicamentos/normas , Proteínas Fluorescentes Verdes/metabolismo , Dominios PDZ/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/análisis , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/química , Transcripción Genética/efectos de los fármacos , Secuencias de Aminoácidos , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Dobutamina/farmacología , Evaluación Preclínica de Medicamentos/métodos , Etanolaminas/análisis , Etanolaminas/farmacología , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Compuestos Heterocíclicos con 3 Anillos/análisis , Compuestos Heterocíclicos con 3 Anillos/farmacología , Vía de Señalización Hippo , Humanos , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Piridinas/análisis , Piridinas/farmacología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/efectos de los fármacos , Tiourea/análogos & derivados , Tiourea/análisis , Tiourea/farmacología , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , ortoaminobenzoatos/análisis , ortoaminobenzoatos/farmacologíaRESUMEN
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.
Asunto(s)
Adipogénesis/efectos de los fármacos , Fármacos Antiobesidad/farmacología , Etacridina/farmacología , Péptidos y Proteínas de Señalización Intracelular/agonistas , Células Madre Mesenquimatosas/efectos de los fármacos , Proteína Fosfatasa 1/metabolismo , Proteína Fosfatasa 2/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/agonistas , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Genes Reporteros/efectos de los fármacos , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Fosfoproteínas/agonistas , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Proteína Fosfatasa 1/química , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 2/química , Proteína Fosfatasa 2/genética , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Transactivadores , Factores de Transcripción , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Señalizadoras YAPRESUMEN
The transcriptional coactivator with a PDZ-binding motif (TAZ) cooperates with various transcriptional factors and plays various roles. Immortalized human mammalian epithelial MCF10A cells form spheres when TAZ is overexpressed and activated. We developed a cell-based assay using sphere formation by TAZ-expressing MCF10A cells as a readout to screen 18,458 chemical compounds for TAZ activators. Fifty compounds were obtained, and 47 were confirmed to activate the TAZ-dependent TEAD-responsive reporter activity in HEK293 cells. We used the derived subset of compounds as a TAZ activator candidate minilibrary and searched for compounds that promote myogenesis in mouse C2C12 myoblast cells. In this study, we focused on one compound, IBS008738. IBS008738 stabilizes TAZ, increases the unphosphorylated TAZ level, enhances the association of MyoD with the myogenin promoter, upregulates MyoD-dependent gene transcription, and competes with myostatin in C2C12 cells. TAZ knockdown verifies that the effect of IBS008738 depends on endogenous TAZ in C2C12 cells. IBS008738 facilitates muscle repair in cardiotoxin-induced muscle injury and prevents dexamethasone-induced muscle atrophy. Thus, this cell-based assay is useful to identify TAZ activators with a variety of cellular outputs. Our findings also support the idea that TAZ is a potential therapeutic target for muscle atrophy.