RESUMO
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.
Assuntos
Avaliação Pré-Clínica de Medicamentos/normas , Proteínas de Fluorescência Verde/metabolismo , Domínios PDZ/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/química , Transcrição Gênica/efeitos dos fármacos , Motivos de Aminoácidos , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Dobutamina/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Etanolaminas/análise , Etanolaminas/farmacologia , Genes Reporter , Proteínas de Fluorescência Verde/genética , Células HEK293 , Compostos Heterocíclicos com 3 Anéis/análise , Compostos Heterocíclicos com 3 Anéis/farmacologia , Via de Sinalização Hippo , Humanos , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Piridinas/análise , Piridinas/farmacologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tioureia/análogos & derivados , Tioureia/análise , Tioureia/farmacologia , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , ortoaminobenzoatos/análise , ortoaminobenzoatos/farmacologiaRESUMO
The mammalian Hippo pathway comprises mammalian Ste20-like kinases (MST1/2) and large tumor suppressor kinases (LATS1/2). LATS1/2, which are activated by MST1/2, phosphorylate a transcriptional co-activator, yes-associated protein (YAP), and induce the recruitment of YAP by 14-3-3 to cytoplasm, so that the TEAD-dependent gene transcriptions are turned off. Although the core components of the Hippo pathway are well conserved in metazoans, it has been discussed that Caenorhabditis elegans lacks YAP ortholog, we found that F13E6.4 gene encodes a protein that shows sequence similarities to YAP in the N-terminal TEAD-binding domain and in the WW domain. We designated this gene as yap-1. YAP-1 is widely expressed in various cells such as epithelial cells, muscles, hypodermal cells, gonadal sheath cells, spermatheca, and hypodermal cells. YAP-1 is distributed in cytoplasm and nuclei. wts-1 (LATS ortholog) and ftt-2 (14-3-3 ortholog) knockdowns cause nuclear accumulation of YAP-1, supporting that the subcellular localization of YAP-1 is regulated in a similar way as that of YAP. Heat shock also causes the nuclear accumulation of YAP-1 but after heat shock, YAP-1 translocates to cytoplasm. Knockdowns of DAF-21 (HSP90 ortholog) and HSF-1block the nuclear export of YAP-1 during this recovery. YAP-1 overexpression is beneficial for thermotolerance, whereas YAP-1 hyperactivity induced by wts-1 and ftt-2 knockdowns is deleterious on thermal response and yap-1 deficiency promotes health aging. In short, YAP-1 partially shares basal characters with mammalian YAP and plays a role in thermal stress response and healthy aging.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Envelhecimento , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Fosfoproteínas/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Citoplasma/metabolismo , Humanos , Ligação Proteica/fisiologia , Transdução de Sinais/fisiologia , Temperatura , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAPRESUMO
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.