Browsing by Author "Lee, Yan-Hwa Wu"

Now showing 1 - 13 of 13

Critical roles of RNA helicase DDX3 and its interactions with eIF4E/PABP1 in stress granule assembly and stress response
(2012-01-01)
Shih, Jing-Wen
;
Wang, Wei-Ting
;
Tsai, Tsung-Yuan
;
Kuo, Chu-Yun
;
Li, Hao-Kang
Upon environmental insults, SGs (stress granules) aid cell survival by serving as sites of translational silencing. RNA helicase DDX3 was reported to associate with SGs. However, its role in SG physiology remains undefined. We have demonstrated previously that DDX3 acts as an eIF4E (eukaryotic initiation factor 4E)-inhibitory protein to suppress translation. In the present study, we indentified the SG marker PABP1 [poly(A)-binding protein 1] as another direct interaction partner of DDX3. We established various stimuli as novel stressors that direct DDX3 with eIF4E and PABP1 into SGs, but not to processing bodies. Interestingly, down-regulation of DDX3 interfered with SG assembly, led to nuclear accumulation of PABP1 and reduced cell viability following stress. Conversely, supplementation with a shRNA (short hairpin RNA)-resistant DDX3 restored SG formation, the translocation of PABP1 into SGs and cell survival. Notably, the SG-inducing capacity of DDX3 is independent of its ATPase and helicase activities, but mapped to the eIF4E-binding region. Moreover, the eIF4E-binding-defective mutant DDX3 was impaired in its SG-inducing ability and protective effect on cell survival under adverse conditions. All together, the present study has characterized DDX3 as a pivotal SG-nucleating factor and illustrates co-ordinative roles for DDX3, eIF4E and PABP1 in integrating environmental stress with translational regulation.
DDX3 localizes to the centrosome and prevents multipolar mitosis by epigenetically and translationally modulating p53 expression
(2017-08-25)
Chen, Wei-Ju
;
Wang, Wei-Ting
;
Tsai, Tsung-Yuan
;
Li, Hao-Kang
;
Lee, Yan-Hwa Wu
The DEAD-box RNA helicase DDX3 plays divergent roles in tumorigenesis, however, its function in mitosis is unclear. Immunofluorescence indicated that DDX3 localized to centrosome throughout the cell cycle and colocalized with centrosome-associated p53 during mitosis in HCT116 and U2OS cells. DDX3 depletion promoted chromosome misalignment, segregation defects and multipolar mitosis, eventually leading to G2/M delay and cell death. DDX3 prevented multipolar mitosis by inactivation and coalescence of supernumerary centrosomes. DDX3 silencing suppressed Ser(15) phosphorylation of p53 which is required for p53 centrosomal localization. Additionally, knockout of p53 dramatically diminished the association of DDX3 with centrosome, which was rescued by overexpression of the centrosomal targeting-defective p53 S15A mutant, indicating that centrosomal localization of DDX3 is p53 dependent but not through centrosomal location of p53. Furthermore, DDX3 knockdown suppressed p53 transcription through activation of DNA methyltransferases (DNMTs) along with hypermethylation of p53 promoter and promoting the binding of repressive histone marks to p53 promoter. Moreover, DDX3 modulated p53 mRNA translation. Taken together, our study suggests that DDX3 regulates epigenetic transcriptional and translational activation of p53 and colocalizes with p53 at centrosome during mitosis to ensure proper mitotic progression and genome stability, which supports the tumor-suppressive role of DDX3.
DDX3 Represses Stemness by Epigenetically Modulating Tumor-suppressive miRNAs in Hepatocellular Carcinoma
(2016-06-27)
Li, Hao-Kang
;
Mai, Ru-Tsun
;
Huang, Hsien-Da
;
Chou, Chih-Hung
;
Chang, Yi-An
Studies indicate that the presence of cancer stem cells (CSCs) is responsible for poor prognosis of hepatocellular carcinoma (HCC) patients. In this study, the functional role of DDX3 in regulation of hepatic CSCs was investigated. Our results demonstrated that reduced DDX3 expression was not only inversely associated with tumor grade, but also predicted poor prognosis of HCC patients. Knockdown of DDX3 in HCC cell line HepG2 induced stemness gene signature followed by occurrence of self-renewal, chemoreisistance, EMT, migration as well as CSC expansion, and most importantly, DDX3 knockdown promotes tumorigenesis. Moreover, we found positive correlations between DDX3 level and expressions of tumor-suppressive miR-200b, miR-200c, miR-122 and miR-145, but not miR-10b and miR-519a, implying their involvement in DDX3 knockdown-induced CSC phenotypes. In addition, DDX3 reduction promoted up-regulation of DNA methyltransferase 3A (DNMT3A), while neither DNMT3B nor DNMT1 expression was affected. Enriched DNMT3A binding along with hypermethylation on promoters of these tumor-suppressive miRNAs reflected their transcriptional repressions in DDX3-knockdown cells. Furthermore, individual restoration of these tumor-suppressive miRNAs represses DDX3 knockdown-induced CSC phenotypes. In conclusion, our study suggested that DDX3 prevents generation of CSCs through epigenetically regulating a subset of tumor-suppressive miRNAs expressions, which strengthens tumor suppressor role of DDX3 in HCC.
DNA Damage, Liver Injury, and Tumorigenesis: Consequences of DDX3X Loss
(2019-02-01)
Chan, Chieh-Hsiang
;
Chen, Chun-Ming
;
Lee, Yan-Hwa Wu
;
You, Li-Ru
;
交大名義發表
The pleiotropic roles of DEAD-box helicase 3, X-linked (DDX3X), including its functions in transcriptional and translational regulation, chromosome segregation, DNA damage, and cell growth control, have highlighted the association between DDX3X and tumorigenesis. However, mRNA transcripts and protein levels of DDX3X in patient specimens have shown the controversial correlations of DDX3X with hepatocellular carcinoma (HCC) prevalence. In this study, generation of hepatocyte-specific Ddx3x-knockout mice revealed that loss of Ddx3x facilitates liver tumorigenesis. Loss of Ddx3x led to profound ductular reactions, cell apoptosis, and compensatory proliferation in female mutants at 6 weeks of age. The sustained phosphorylation of histone H2AX (gamma H2AX) and significant accumulation of DNA single-strand breaks and double-strand breaks in liver indicated that the replicative stress occurred in female mutants. Further chromatin immunoprecipitation analyses demonstrated that DDX3X bound to promoter regions and regulated the expression of DNA repair factors, DDB2 and XPA, to maintain genome stability. Loss of Ddx3x led to decreased levels of DNA repair factors, which contributed to an accumulation of unrepaired DNA damage, replication stress, and eventually, spontaneous liver tumors and DEN-induced HCCs in Alb-Cre/+Ddx3xflox/flox mice. Implications: These data identify an important role of DDX3X in the regulation of DNA damage repair to protect against replication stress in liver and HCC development and progression.
Functional Characterization of Glycine N-Methyltransferase and Its Interactive Protein DEPDC6/DEPTOR in Hepatocellular Carcinoma
(2012-02-01)
Yen, Chia-Hung
;
Lu, Yao-Cheng
;
Li, Chung-Hsien
;
Lee, Cheng-Ming
;
Chen, Chia-Yen
Glycine N-methyltransferase (GNMT) is a tumor suppressor for hepatocellular carcinoma (HCC). High rates of Gnmt knockout mice developed HCC. Epigenetic alteration and dysregulation of several pathways including wingless-type MMTV integration site (Wnt), mitogen-activated protein kinase (MAPK) and Janus kinase and signal transducer and activator of transcription (JAK-STAT) are associated with HCC development in Gnmt knockout mice. We hypothesized that GNMT may regulate signal transduction through interacting with other proteins directly. In this report, we identified a mammalian target of rapamycin (mTOR) inhibitor (DEP domain containing MTOR-interacting protein (DEPDC6/DEPTOR)) as a GNMT-binding protein by using yeast two-hybrid screening. Fluorescence resonance energy transfer assay demonstrated that the C-terminal half of GNMT interact with the PSD-95/DIg1/ZO-1 (PDZ) domain of DEPDC6/DEPTOR. Immunohistochemical staining showed that 27.5% (14/51) of HCC patients had higher expression levels of DEPDC6/DEPTOR in the tumorous tissues than in tumor-adjacent tissues, especially among HCC patients with hepatitis B viral infection (odds ratio 10.3, 95% confidence interval (CI) 1.05-11.3) or patients with poor prognosis (death hazard ratio 4.51, 95% CI 1.60-12.7). In terms of molecular mechanism, knockdown of DEPDC6/DEPTOR expression in HuH-7 cells caused S6K and 4E-BP activation, but suppressed Akt. Overexpression of DEPDC6/DEPTOR activated Akt and increased survival of HCC cells. Overexpression of GNMT caused activation of mTOR/raptor downstream signaling and delayed G2/M cell cycle progression, which altogether resulted in cellular senescence. Furthermore, GNMT reduced proliferation of HuH-7 cells and sensitized them to rapamycin treatment both in vitro and in vivo. In conclusion, GNMT regulates HOC growth in part through interacting with DEPDC6/DEPTOR and modulating mTOR/raptor signaling pathway. Both GNMT and DEPDC6/DEPTOR are potential targets for developing therapeutics for HCC. Online address: http://www.molmed.org doi: 10.2119/molmed.2011.00331
Functional Characterization of Glycine N-Methyltransferase and Its Interactive Protein DEPDC6/DEPTOR in Hepatocellular Carcinoma (vol 18, pg 286, 2012)
(2012-03-01)
Yen, Chia-Hung
;
Lu, Yao-Cheng
;
Li, Chung-Hsien
;
Lee, Cheng-Ming
;
Chen, Chia-Yen
Human DExD/H RNA helicases: Emerging roles in stress survival regulation
(2014-09-25)
Shih, Jing-Wen
;
Lee, Yan-Hwa Wu
;
交大名義發表
;
生物科技學系
;
National Chiao Tung University
Environmental stresses threatening cell homeostasis trigger various cellular responses ranging from the activation of survival pathways to eliciting programmed cell death. Cellular stress response highly depends on the nature and level of the insult as well as the cell type. Notably, the interplay among all these responses will ultimately determine the fate of the stressed cell. Human DExD/H RNA helicases are ubiquitous molecular motors rearranging RNA secondary structure in an ATP-dependent fashion. These highly conserved enzymes participate in nearly all aspects of cellular process involving RNA metabolism. Although numerous functions of DExD/H RNA helicases are well documented, their importance in stress response is only just becoming evident. This review outlines our current knowledge on major mechanistic themes of human DExD/H RNA helicases in response to stressful stimuli, especially on emerging molecular models for the functional roles of these enzymes in the stress survival regulation. (C) 2014 The Authors. Published by Elsevier B.V.
Membrane protein-regulated networks across human cancers
(2019-07-16)
Lin, Chun-Yu
;
Lee, Chia-Hwa
;
Chuang, Yi-Hsuan
;
Lee, Jung-Yu
;
Chiu, Yi-Yuan
Alterations in membrane proteins (MPs) and their regulated pathways have been established as cancer hallmarks and extensively targeted in clinical applications. However, the analysis of MP-interacting proteins and downstream pathways across human malignancies remains challenging. Here, we present a systematically integrated method to generate a resource of cancer membrane protein-regulated networks (CaMPNets), containing 63,746 high-confidence protein-protein interactions (PPIs) for 1962 MPs, using expression profiles from 5922 tumors with overall survival outcomes across 15 human cancers. Comprehensive analysis of CaMPNets links MP partner communities and regulated pathways to provide MP-based gene sets for identifying prognostic biomarkers and druggable targets. For example, we identify CHRNA9 with 12 PPIs (e.g., ERBB2) can be a therapeutic target and find its anti-metastasis agent, bupropion, for treatment in nicotine-induced breast cancer. This resource is a study to systematically integrate MP interactions, genomics, and clinical outcomes for helping illuminate cancer-wide atlas and prognostic landscapes in tumor homo/heterogeneity.
RNA helicase DDX3 maintains lipid homeostasis through upregulation of the microsomal triglyceride transfer protein by interacting with HNF4 and SHP
(2017-01-27)
Tsai, Tsung-Yuan
;
Wang, Wei-Ting
;
Li, Hao-Kang
;
Chen, Wei-Ju
;
Tsai, Yu-Hong
Multifunctional RNA helicase DDX3 participates in HCV infection, one of the major causes of hepatic steatosis. Here, we investigated the role of DDX3 in hepatic lipid metabolism. We found that HCV infection severely reduced DDX3 expression. Analysis of intracellular triglyceride and secreted ApoB indicated that lipid accumulations were increased while ApoB secretion were decreased in DDX3 knockdown HuH7 and HepG2 cell lines. Down-regulation of DDX3 significantly decreased protein and transcript expression of microsomal triglyceride transfer protein (MTP), a key regulator of liver lipid homeostasis. Moreover, DDX3 interacted with hepatocyte nuclear factor 4 (HNF4) and small heterodimer partner (SHP), and synergistically up-regulated HNF4-mediated transactivation of MTP promoter via its ATPase activity. Further investigation revealed that DDX3 interacted with CBP/p300 and increased the promoter binding affinity of HNF4 by enhancing HNF4 acetylation. Additionally, DDX3 partially relieved the SHP-mediated suppression on MTP promoter by competing with SHP for HNF4 binding which disrupted the inactive HNF4/SHP heterodimer while promoted the formation of the active HNF4 homodimer. Collectively, these results imply that DDX3 regulates MTP gene expression and lipid homeostasis through interplay with HNF4 and SHP, which may also reveal a novel mechanism of HCV-induced steatosis.
Targeted inactivation of murine Ddx3x: essential roles of Ddx3x in placentation and embryogenesis
(2016-07-15)
Chen, Chia-Yu
;
Chan, Chieh-Hsiang
;
Chen, Chun-Ming
;
Tsai, Yin-Shuan
;
Tsai, Tsung-Yuan
The X-linked DEAD-box RNA helicase DDX3 (DDX3X) is a multifunctional protein that has been implicated in gene regulation, cell cycle control, apoptosis, and tumorigenesis. However, the precise physiological function of Ddx3x during development remains unknown. Here, we show that loss of Ddx3x results in an early post-implantation lethality in male mice. The size of the epiblast marked by Oct3/4 is dramatically reduced in embryonic day 6.5 (E6.5) Ddx3x(-)/Y embryos. Preferential paternal X chromosome inactivation (XCI) in extraembryonic tissues of Ddx3x heterozygous (Ddx3x(-/+)) female mice with a maternally inherited null allele leads to placental abnormalities and embryonic lethality during development. In the embryonic tissues, Ddx3x exhibits developmental-and tissue-specific differences in escape from XCI. Targeted Ddx3x ablation in the epiblast leads to widespread apoptosis and abnormal growth, which causes embryonic lethality in the Sox2-cre/+; Ddx3x(flox)/Y mutant around E11.5. The observation of significant increases in gamma H2AX and p-p53(Ser15) indicates DNA damage, which suggests that loss of Ddx3x leads to higher levels of genome damage. Significant upregulation of p21(WAF1/Cip1) and p15(Ink4b) results in cell cycle arrest and apoptosis in Ddx3x-deficient cells. These results have uncovered that mouse Ddx3x is essential for both embryo and extraembryonic development.
Targeted inactivation of murine Ddx3x: essential roles of Ddx3x in placentation and embryogenesis
(2016-07-15)
Chen, Chia-Yu
;
Chan, Chieh-Hsiang
;
Chen, Chun-Ming
;
Tsai, Yin-Shuan
;
Tsai, Tsung-Yuan
The X-linked DEAD-box RNA helicase DDX3 (DDX3X) is a multifunctional protein that has been implicated in gene regulation, cell cycle control, apoptosis, and tumorigenesis. However, the precise physiological function of Ddx3x during development remains unknown. Here, we show that loss of Ddx3x results in an early post-implantation lethality in male mice. The size of the epiblast marked by Oct3/4 is dramatically reduced in embryonic day 6.5 (E6.5) Ddx3x(-)/Y embryos. Preferential paternal X chromosome inactivation (XCI) in extraembryonic tissues of Ddx3x heterozygous (Ddx3x(-/+)) female mice with a maternally inherited null allele leads to placental abnormalities and embryonic lethality during development. In the embryonic tissues, Ddx3x exhibits developmental-and tissue-specific differences in escape from XCI. Targeted Ddx3x ablation in the epiblast leads to widespread apoptosis and abnormal growth, which causes embryonic lethality in the Sox2-cre/+; Ddx3x(flox)/Y mutant around E11.5. The observation of significant increases in gamma H2AX and p-p53(Ser15) indicates DNA damage, which suggests that loss of Ddx3x leads to higher levels of genome damage. Significant upregulation of p21(WAF1/Cip1) and p15(Ink4b) results in cell cycle arrest and apoptosis in Ddx3x-deficient cells. These results have uncovered that mouse Ddx3x is essential for both embryo and extraembryonic development.
The Anastomotic Angle of Hemodialysis Arteriovenous Fistula Is Associated With Flow Disturbance at the Venous Stenosis Location on Angiography
(2020-07-23)
Yang, Chih-Yu
;
Li, Ming-Chia
;
Lan, Chien-Wen
;
Lee, Wang-Jiun
;
Lee, Chen-Ju
The juxta-anastomotic stenosis of an arteriovenous fistula (AVF) is a significant clinical problem in hemodialysis patients with no effective treatment. Previous studies of AV anastomotic angles on hemodynamics and vascular wall injury were based on computational fluid dynamics (CFD) simulations using standardized AVF geometry, not the real-world patient images. The present study is the first CFD study to use angiographic images with patient-specific outcome information, i.e., the exact location of the AVF stenotic lesion. We conducted the CFD analysis utilizing patient-specific AVF geometric models to investigate hemodynamic parameters at different locations of an AVF, and the association between hemodynamic parameters and the anastomotic angle, particularly at the stenotic location. We analyzed 27 patients who used radio-cephalic AVF for hemodialysis and received an angiographic examination for juxta-anastomotic stenosis. The three-dimensional geometrical model of each patient's AVF was built using the angiographic images, in which the shape and the anastomotic angle of the AVF were depicted. CFD simulations of AVF hemodynamics were conducted to obtain blood flow parameters at different locations of an AVF. We found that at the location of the stenotic lesion, the AV angle was significantly correlated with access flow disturbance (r= 0.739;p< 0.001) and flow velocity (r= 0.563;p= 0.002). Furthermore, the receiver operating characteristic (ROC) curve analysis revealed that the AV angle determines the lesion's flow disturbance with a high area under the curve value of 0.878. The ROC analysis also identified a cut-off value of the AV angle as 46.5 degrees, above or below which the access flow disturbance was significantly different. By applying CFD analysis to real-world patient images, the present study provides evidence that an anastomotic angle wider than 46.5 degrees might lead to disturbed flow generation, demonstrating a reference angle to adopt during the anastomosis surgery.
Y-Box Binding Protein 1 Stabilizes Hepatitis C Virus NS5A via Phosphorylation-Mediated Interaction with NS5A To Regulate Viral Propagation
(2015-11-01)
Wang, Wei-Ting
;
Tsai, Tsung-Yuan
;
Chao, Chi-Hong
;
Lai, Bo-Ying
;
Lee, Yan-Hwa Wu
Replication of hepatitis C virus (HCV) is dependent on virus-encoded proteins and numerous cellular factors. DDX3 is a well-known host cofactor of HCV replication. In this study, we investigated the role of a DDX3-interacting protein, Y-box binding protein 1 (YB-1), in the HCV life cycle. Both YB-1 and DDX3 interacted with the viral nonstructural protein NS5A. During HCV infection, YB-1 partially colocalized with NS5A and the HCV replication intermediate double-stranded RNA (dsRNA) in HCV-infected Huh-7.5.1 cells. Despite sharing the same interacting partners, YB-1 participated in HCV RNA replication but was dispensable in steady-state HCV RNA replication, different from the action of DDX3. Moreover, knockdown of YB-1 in HCV-infected cells prevented infectious virus production and reduced the ratio of hyperphosphorylated (p58) to hypophosphorylated (p56) forms of NS5A, whereas DDX3 silencing did not affect the ratio of the p58 and p56 phosphoforms of NS5A. Interestingly, silencing of YB-1 severely reduced NS5A protein stability in NS5A-ectopically expressing, replicon-containing, and HCV-infected cells. Furthermore, mutations of serine 102 of YB-1 affected both YB-1-NS5A interaction and NS5A-stabilizing activity of YB-1, indicating that this Akt phosphorylation site of YB-1 plays an important role in stabilizing NS5A. Collectively, our results support a model in which the event of YB-1 phosphorylation-mediated interaction with NS5A results in stabilizing NS5A to sustain HCV RNA replication and infectious HCV production. Overall, our study may reveal a new aspect for the development of novel anti-HCV drugs. IMPORTANCE Chronic hepatitis C virus (HCV) infection induces liver cirrhosis and hepatocellular carcinoma. The viral nonstructural protein NS5A co-opting various cellular signaling pathways and cofactors to support viral genome replication and virion assembly is a new strategy for anti-HCV drug development. NS5A phosphorylation is believed to modulate switches between different stages of the HCV life cycle. In this study, we identified the cellular protein YB-1 as a novel NS5A-interacting protein. YB-1 is a multifunctional protein participating in oncogenesis and is an oncomarker of hepatocellular carcinoma (HCC). We found that YB-1 protects NS5A from degradation and likely regulates NS5A phosphorylation through its phosphorylation-dependent interaction with NS5A, which might be controlled by HCV-induced signaling pathways. Our observations suggest a model in which HCV modulates NS5A level and the ratio of the p58 and p56 phosphoforms for efficient viral propagation via regulation of cellular signaling inducing YB-1 phosphorylation. Our finding may provide new aspects for developing novel anti-HCV drugs.