ABSTRACT
BACKGROUND: Gliomas are highly aggressive and lack of efficient targeted therapy. YAP, as a Hippo pathway downstream effector, plays a key role in promoting tumor development through the interaction with transcription factor TEAD on the NH3-terminal proline-rich domain. Therefore, targeting TEAD-interacting domain of YAP may provide a novel approach for the treatment of gliomas. MATERIALS AND METHODS: We generated a truncated YAP protein which includes the TEAD-binding domain (YAPBD), and supposed YAPBD can interact with endogenous TEAD but lost the function to activate YAP target gene expressions. The association of YAP expression with the malignant characters of glioma tissues were determined by immunohistochemistry. TEAD-binding capacity of YAPBD was determined by co-immunoprecipitation. The cell proliferation and migration were determined by MTT assay, xenograft assay, wound healing assay and transwell assay, respectively. YAP target genes were detected by Western blot. RESULTS: YAP was highly expressed in glioma tissues and associated with tumor malignancy. YAPBD could block the TEAD-YAP complex formation by competing with YAP binding to TEAD. YAPBD could inhibit glioma cell growth both in vitro and in vivo, through the induction of cell cycle arrest and apoptosis. The cell cycle-related gene cyclin D1 and c-myc, and anti-apoptotic gene Bcl-2, Bcl-xL and survivin were inhibited after YAPBD overexpression. Furthermore, YAPBD also decreased cell migration and invasion, and repressed epithelial-mesenchymal transition. CONCLUSION: YAPBD can block glioma cell survival and repress YAP-dependent gene expressions, indicating gene therapy which targets TEAD-YAP complex would be a potential and significant novel approach for human malignant gliomas.
Subject(s)
Cell Cycle Proteins/pharmacology , Cell Survival/drug effects , Central Nervous System Neoplasms/pathology , Glioma/pathology , Recombinant Proteins/pharmacology , Transcription Factors/pharmacology , Animals , Binding, Competitive , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Central Nervous System Neoplasms/diagnosis , Central Nervous System Neoplasms/genetics , Codon, Nonsense/genetics , Cohort Studies , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Neoplastic , Glioma/diagnosis , Glioma/genetics , Humans , Mice , Mice, Nude , Nuclear Proteins/metabolism , Protein Binding , Protein Interaction Domains and Motifs/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , TEA Domain Transcription Factors , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Transcriptional coactivator with PDZ-binding motif (TAZ), a Hippo pathway downstream effector, promotes tumor progression by serving as a transcriptional coactivator with TEAD. Here, we introduced a new construct which can express the TEAD-binding domain of TAZ protein (TAZBD), and determined its antitumor effect in malignant glioma both in vitro and in vivo. We first observed that TAZ was upregulated in glioma tissues and related to malignant clinicopathologic characteristic, indicating the crucial role of TAZ during glioma progression. In U87 and U251 cells, TAZBD expression increased the proportion of apoptotic cells, and suppressed the colony formation and tumorigenicity. Further, TAZBD also decreased cell metastasis through the repression of epithelial-mesenchymal transition. The mechanistic study showed that TAZBD suppression of glioma cells was predominantly through blocking the TAZ-TEAD complex formation by competing with endogenous TAZ. Thus, the gene therapy of malignant glioma through blocking TAZ-TEAD complex by TAZBD may provide a new way for the targeted therapy of glioma.
Subject(s)
Apoptosis , Brain Neoplasms/secondary , DNA-Binding Proteins/metabolism , Epithelial-Mesenchymal Transition , Glioma/pathology , Nuclear Proteins/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Animals , Biomarkers, Tumor , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Cell Movement , Cell Proliferation , DNA-Binding Proteins/genetics , Gene Expression Regulation, Neoplastic , Glioma/genetics , Glioma/metabolism , Humans , Mice , Mice, Nude , Neoplasm Invasiveness , Nuclear Proteins/genetics , Prognosis , TEA Domain Transcription Factors , Trans-Activators/genetics , Transcription Factors/genetics , Transcriptional Coactivator with PDZ-Binding Motif Proteins , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
The eukaryotic initiation factor (eIF)4Ebinding proteins (4EBPs) regulate capdependent protein translation and control the assembly of the eIF4F complex. In the present study, a phosphorylationdeficient truncated 4EBP2 (eIF4FD) was constructed into the eukaryotic expression vector pSecTag2, and the in vitro and in vivo effects on malignant glioma survival were determined through inhibiting eIF4F complex assembly. Cell cycle distribution analysis and TUNEL staining show that overexpression of eIF4FD suppressed cell proliferation and induced apoptosis in U251 cells. Western blotting showed that the cell cyclerelated genes cyclin D1 and Cmyc, and antiapoptotic genes Bcell lymphoma 2 (Bcl2), Bclextra large and survivin were reduced following the overexpression of eIF4FD. Furthermore, eIF4FD suppressed glioma vascularization via reductions in the expression of ßcatenin and vascular endothelial growth factor. In the orthotopic xenograft model, the stable expression of eIF4FD in U251 cells attenuated cell growth and increased the rate of apoptosis. Accordingly, pSecTag2PTDeIF4FD injection via the tail vein of mice also lead to cell growth inhibition and the induction of apoptosis. Therefore, the study showed that phosphorylationdeficient truncated 4EBP2 efficiently inhibited eIF4E and prevented the formation of the eIF4F complex, which further contributed to the inhibition of cell proliferation and vascularization, and the induction of apoptosis. Therefore, the 4EBP2based phosphorylationdeficient truncation designed in the present study may represent a novel approach for the targeted therapy of human malignant glioma though inhibition of the translation initiation complex.
Subject(s)
Apoptosis , Cell Proliferation , Eukaryotic Initiation Factor-4F/antagonists & inhibitors , Glioma/prevention & control , Animals , Biomarkers, Tumor , Cell Cycle , Eukaryotic Initiation Factor-4F/genetics , Eukaryotic Initiation Factor-4F/metabolism , Glioma/metabolism , Glioma/pathology , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Translation initiation factors (eIFs) are over-activated in many human cancers and may contribute to their progression. The small molecule 4EGI-1, a potent inhibitor of translation initiation through disrupting eIF4E/eIF4G interaction, has been shown to exert anti-cancer effects in human cancer cells. The goal of the present study was to evaluate the anti-cancer effects of 4EGI-1 in human glioma U251 cells. We found that 4EGI-1 impaired the assembly of the eIF4F complex, and inhibited proliferation of U251 cells via inducing apoptosis. 4EGI-1 treatment induced collapse of mitochondrial membrane potential (MMP) and production of intracellular reactive oxygen species (ROS), which were prevented by the ROS scavenger N-acetyl-cysteine (NAC). In addition, 4EGI-1 inhibited mitochondrial ATP synthesis via suppressing complex I activity, but had no effects on mitochondrial biogenesis. The results of fluorescence staining showed that 4EGI-1 indeed fragmented the mitochondrial network of U251 cells. We found a significant decrease in optic atrophy type 1 (Opa-1) and mitofusin 1 (Mfn-1) related to fusion proteins as well as an increase in fission protein dynamin-related protein 1 (Drp-1). Furthermore, the anti-cancer effects of 4GI-1 were partially nullified by knock down of Drp-1 using siRNA. These data indicate that the use of inhibitors that directly target the translation initiation complex eIF4F could represent a potential novel approach for human glioma therapy.
