MOLECULAR ANALYSIS OF STAT IN BREAST CANCER CELL
Y Eugene Chin
Yale University 47 College Street, Ste 203 New Haven, Ct 065208047
Grant 1F32CA069741-01 from National Cancer Institute IRG: MEP
1F32CA069741-01 (1996): $35300
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Y Eugene Chin
ACETYLATION-DEPENDENT IFN SIGNAL TRANSDUCTION
Y Eugene Chin
Rhode Island Hospital, Providence, Ri 02903-4923
Grant 5R01GM087331-02 from National Institute Of General Medical Sciences
Abstract: Type I interferon (v affect cell differentiation, proliferation, and survival in nearly all kinds of cell types, and are efficacious in the treatment of malignancies such as chronic myeloid leukemia. In recent years, the usage of IFN¿ for solid cancer chemotherapy has received considerable attention. IFN¿-activated STAT2, STAT1, and IRF9 form the transcription factor IFN-stimulated gene factor-3 (ISGF3) complex, which binds to interferon stimulated response element (ISRE) sequence for transcriptional activation. IFN¿ activated STAT proteins also form homo- or hetero-dimer that bind to sis-inducible element (SIE) to regulate gene expression. IFN¿ receptor (IFN¿R) consist two cognate subunits IFN¿R1 and IFN¿R2. IFN¿ binds IFN¿R2 to instigate its association with IFN¿R1. IFN¿R1 and IFN¿R2 are cross-phosphorylated by their associated tyrosine kinases (Jak1 and Tyk2), followed by subsequent STAT2 and STAT1 recruitment and phosphorylational activation. The mechanism of how IFN¿ activates IRF9 is unknown. IRF9-dependent transcriptional activation can be enhanced by treating the cells with deacetylase inhibitors, suggesting IRF9 activation involves acetylation. We detected IFN¿R2 acetylation by recruiting tumor suppressor-like transcription cofactor CREB-binding protein (CBP) or its homologous p300. Acetylated IFN¿R2 can then recruit IRF9. IRF9 as well as STAT2 and STAT1 are all acetylated by CBP prior to forming the transcriptional active ISGF3 complex. IFN¿R also recruits deacetylases including SIRT and HDAC members. Although tyrosine phosphorylation has long been accepted to play a paramount role in cytokine receptor signal transduction, our findings based upon preliminary data and protein secondary structural analysis challenge this concept. The overarching hypothesis to be tested here is that CBP/p300-mediated acetylation cascade triggered by IFN¿ plays an indispensable role in signal transduction for anti-proliferation, proapotosis, and anti-metastasis gene regulation. Disrupting IFN¿R and deacetylase association with deacetylase inhibitors may improve the therapeutic effects of IFN¿ in cancer. We will apply site-directed mutagenesis, FRET technology, as well as the antibody array technology developed in our lab in order to (1) define the acetyltransferase activity associated with CBP/p300 on IFN¿R activation in intracellular signaling; (2) analyze IFN¿R, STAT1 and ISGF3 deacetylation by HDAC or SIRT in signal termination; and (3) determine whether acetylated STAT1 dimer or ISGF3 complex is critical for IFN¿- mediated anti-proliferation, proapoptosis, and antimetastasis gene regulation in cancer cells. These approaches will help elucidate the function of acetylation and deacetylation in the regulation of IFN¿R activation, STAT dimer, and ISGF3 complex formation for signal transduction and transcription, and allow us to maximize the therapeutic applications of IFN¿ for cancer
Keywords: ATP[{..}]protein-tyrosine O-phosphotransferase; Accounting; Acetylation; Acetyltransferase; Address; Affect; Alferon; Angiogenesis Antagonists; Angiogenesis Blockers; Angiogenesis Inhibitors; Angiogenetic Antagonists; Angiogenic Antagonists; Angiostatic Agents; Anti-Angiogenetic Agents; Anti-Angiogenic Agents; Anti-Angiogenic Drugs; Antiangiogenesis Agents; Antiangiogenic Agents; Antibodies; Antiviral Agents; Antiviral Drugs; Antivirals; Attention; Bears; Binding; Binding (Molecular Function); C-terminal; CBP protein; CBP protein, human (CREB binding protein); CREB Binding Protein; CREB binding protein, human; CREB-binding protein; CREBBP protein, human; Cancer Treatment; Cancers; Cell Communication and Signaling; Cell Differentiation; Cell Differentiation process; Cell Nucleus; Cell Signaling; Cell Surface Receptors; Cells; Chemotherapy Protocol; Chemotherapy Regimen; Chemotherapy, Cancer, General; Chemotherapy-Oncologic Procedure; Chronic Myeloid Leukemia; Combination Chemotherapy Regimen; Complex; Cytokine Receptors; Cytokine Signal Transduction; Cytokine Signaling; Cytoplasm; Cytoplasmic Domain; Cytoplasmic Tail; DNA Binding Domain; Deacetylase; Deacetylation; Dimerization; Disease; Disorder; Dissociation; Drug Resistance, Multiple; Drug Resistant, Multiple; EC 2.7; EP300; EP300 gene; EPH- and ELK-Related Tyrosine Kinase; EPH-and ELK-Related Kinase; EPHA8; Elements; EphA8 Protein; Ephrin Type-A Receptor 8; Ephrin Type-A Receptor 8 Precursor; Event; Exhibits; FRET; Family member; Fluorescence Resonance Energy Transfer; G-interferon; Gene Action Regulation; Gene Activation; Gene Expression; Gene Expression Regulation; Gene Regulation; Gene Regulation Process; Gene Transcription; Generalized Growth; Genes; Genetic Transcription; Ginterferon; Growth; HDAC; HDAC Proteins; HEK3; Hand; HeLa; Hela Cells; Heterochromatin; Histone Acetylase; Histone Deacetylase; Histone H3; Histones; Homo; IFN; IFN Alpha; IFN-stimulated gene factor 3 complex; IFNa; ISFG-3; ISGF-3; ISGF3 factor; ISGF3 protein; Inhibitors, Angiogenetic; Inhibitors, Angiogenic; Interferon Activation; Interferon Alfa-n3; Interferon Receptor; Interferon Type I; Interferon, Leukocyte; Interferon, Lymphoblast; Interferon, Lymphoblastoid; Interferon-alpha; Interferons; Intracellular Communication and Signaling; Kinases; L-Lysine; L-Serine; L-tyrosine, dihydrogen phosphate (ester); Leukemia, Granulocytic, Chronic; Link; Lysine; Malignant Cell; Malignant Neoplasm Therapy; Malignant Neoplasm Treatment; Malignant Neoplasms; Malignant Tumor; Mass Spectrum; Mass Spectrum Analysis; Mediating; Metastasis; Metastasize; Metastatic Neoplasm; Metastatic Tumor; Modeling; Molecular Interaction; Multi-Drug Resistance; Multidrug Resistance; Mutagenesis, Site-Directed; Myelocytic Leukemia, Chronic; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Chronic; N-terminal; NH2-terminal; Neoplasm Metastasis; Neovascularization Inhibitors; Nuclear; Nuclear Export; Nuclear Translocation; Nucleus; P113; PTK; Pathway interactions; Phospho-CREB Binding Protein; Phosphorylation; Phosphotransferases; Phosphotyrosine; Photometry/Spectrum Analysis, Mass; Play; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Property; Property, LOINC Axis 2; Protein Dimerization; Protein Motifs, DNA-Binding; Protein Phosphorylation; Protein Tyrosine Kinase; Protein Tyrosine Kinase EEK; Proteins; Quimioterapia; RNA Expression; Receptor Activation; Receptor Protein; Receptor Signaling; Recruitment Activity; Regulation; Resistance to Multi-drug; Resistance to Multidrug; Resistance to Multiple Drug; Resistant to Multiple Drug; Resistant to multi-drug; Resistant to multidrug; Response Elements; Role; Rubinstein-Taybi syndrome protein, human; SH2 Domains; STAT protein; STAT1; STAT1 gene; STAT113; STAT2; STAT2 gene; STAT91; Secondary Neoplasm; Secondary Tumor; Series; Serine; Signal Transducer and Activator of Transcription; Signal Transduction; Signal Transduction Systems; Signaling; Site; Site-Directed Mutagenesis; Site-Specific Mutagenesis; Solid; Spectrometry, Mass; Spectroscopy, Mass; Spectrum Analyses, Mass; Spectrum Analysis, Mass; Targeted DNA Modification; Targeted Modification; Technology; Testing; Therapeutic; Therapeutic Effect; Tissue Growth; Transcription; Transcription Activation; Transcription, Genetic; Transcriptional Activation; Transphosphorylases; Tumor Cell Migration; Tumor Suppressor Proteins; Tyrosine Kinase; Tyrosine Phosphorylation; Tyrosine-O-phosphate; Tyrosine-Protein Kinase Receptor EEK; Tyrosine-Specific Protein Kinase; Tyrosylprotein Kinase; Up-Regulation; Ursidae; Ursidae Family; Viral Genes; Virus; Viruses, General; Work; ing; antiangiogenic; anticancer therapy; base; biological signal transduction; cancer cell; cancer chemotherapy; cancer metastasis; cancer therapy; cell type; cofactor; dimer; disease/disorder; extracellular; gene product; histone acetyltransferase; human CREBBP protein; hydroxyaryl protein kinase; improved; inhibitor; inhibitor/antagonist; interferon therapy; interferon-stimulated gene factor 3; malignancy; member; multi-drug resistant; multidrug resistant; neoplasm/cancer; nuclear protein CBP; ontogeny; p300; pathway; phospho-CREB-binding protein; protein protein interaction; receptor; recruit; response; social role; src Homology Region 2 Domain; theories; transcription factor; transcription factor ISGF3; tumor suppressor; tyrosyl protein kinase
Relevance: NARRATIVE Type I interferons (IFN,) have been widely used for treatment of cancer and virus-infected diseases. Our recent findings suggest that IFN-alpha can have a previous totally unknown signaling event, i.e., lysine acetylation cascade, leading to anti-proliferation and anti-viral gene activation. In this proposal, we want to address in detail, the precise mechanism of how acetylation cascade works in turning on genes involved in anti-proliferation and anti-metastasis in response to IFN treatment in cancer cells
Project start date: 2009-09-30
Project end date: 2011-08-31
Budget start date: 1-SEP-2010
Budget end date: 31-AUG-2011
PFA/PA: PA-07-070
5R01GM087331-02 (2010): $298100
1R01GM087331-01 (2009): $291476
STAT In TNF Alpha Induced Apoptosis
Y Eugene Chin
Pathology And Lab Medicinebrown University
164 Angell Street
providence, Ri 02912
Grant 1R01CA082549-01A2 from National Cancer Institute IRG: ET
Abstract: Tumor necrosis factor-a (TNFa), a pleiotropic cytokine derived from activated macrophages and other cells, plays pivotal roles in some inflammatory, cardiovascular, and systemic diseases. TNFa is a potential anticancer agent. However, TNFa has dual but opposing effects on cell growth/survival it inhibits growth and induces apoptosis in some cancer cells but stimulates growth and maintains survival in others. By binding to its cell surface receptors (TNFR1/TNFR2), TNJFa elicits several signaling events including activation of the Caspase cascade and NF-KB. Caspase activation or over-expression of a component(s) of this pathway often leads to apoptosis. In contrast, NF-KB activation is critical for promoting cell survival and suppressing apoptosis. Thus, TNFa´s output on cell is decided by the balance between NF-KB and Caspase activation. To trigger apoptosis, TNFa needs not only to activate Caspases but also to minimize or even shut down NF-KB activation. Little is known about how TNFa suppresses NF-xB activation while triggering Caspase activation. The role of protein tyrosine phosphorylation in signal transduction has been confirmed for many cytokines but remains unclear for TNFa. The JAK (Janus kinase)-STAT (signal transducer and activator of transcription) pathway is a tyrosine phosphorylation-signaling route used by interferon and other cytokines. Several lines of evidence suggest that JAK/STAT may play an important role in TNFa´s signaling events. First, TNF receptors recruit JAK/Stati especially in TNFa-sensitive cells. Second, both Jaki- and Stati-deficient cells become TNFa resistant. Third, TNFR1 signaling adapter TRADD transfection-induced apoptosis is sensitized by co-transfection with Stati. These findings lead to the following working hypothesis phosphoStati is a signal adapter associated with TNFR1/TRADD complex. It favors TNFa-induced cell death by stabilizing the death signaling complex formation and inhibiting NF-kB activation and its subsequent gene regulation. In the proposed study, the specific aims are designed to exploit this new information as follows (1) TNFR-mediated JAK/Stat 1 activation and the role of Stati as a signal adapter in death signal transduction will be examined. (2) The interaction between Stati and TRADD in stabilizing death-signaling complex (TNFR1-TRADD-FADD) formation will be analyzed. (3) The inhibitory role of Stati on NF-KB-mediated gene regulation will be further explored. These complimentary approaches will elucidate the fundamental role of Stati in apoptosis induction by TNa and might eventually provide a boost to cancer therapies
Keywords: apoptosis, cell growth regulation, cytokine receptor, transcription factor, tumor necrosis factor alpha JAK kinase, binding site, gene induction /repression, gene mutation, nuclear factor kappa beta, phosphoprotein, proline, protein protein interaction, serine, tyrosine cell line
Project start date: 2001-03-01
Project end date: 2005-02-28
1R01CA082549-01A2 (2001): $243993
5R01CA082549-05 (2004): $242550
MOLECULAR ANALYSIS OF STAT IN BREAST CANCER CELL
Y Eugene Chin
Yale University 47 College Street, Ste 203 New Haven, Ct 065208047
Grant 5F32CA069741-02 from National Cancer Institute IRG: MEP
Abstract: The aberrant action of growth factors and their receptors is one of major determinants in the uncontrolled proliferation and evolution of normal breast epithelium to cancer cells. Many clinical, cellular, and molecular biology studies have indicated that abnormal expression of epidermal growth factor (EGF), transforming growth factor alpha (TGFalpha), and their shared receptor (EGFR or the related receptors erbB2/neu and erbB3) is frequently associated with malignant transformation of breast cells. Dr. Fu and others have recently demonstrated that EGF can induce rapid tyrosine phosphorylation and nuclear translocation of transcription factor p9l (p9l, or STAT1alpha). Through its SH2 domain, p9l directly interacts with EGFR and is activated in a ligand-dependent manner. Activated p9l translocates to the nucleus and stimulates specific transcription of the oncogene c-fos in vitro and in vivo. These studies suggest that EGF can use a direct signaling pathway to control nuclear transcriptional events (Fu and Zhang, 1993). Recently, studies from other laboratories have demonstrated that EGF, PDGF, CSF-l, IL-3, IL-5, IL-6, and IL-1O all use the similar direct pathway in controlling nuclear transcriptional events (reviewed by Darnell et al., 1994). A fundamental question in breast cancer research is whether and how this direct signaling pathway is involved in abnormal cell growth and transformation of breast cells. My recent results have shown that p9l seems to be very important in signaling oncogene expression in breast cancer cells, especially those cells which over-express EGFR. A deeper understanding of the role of these signaling events may eventually suggest a basis for the treatment of breast cancer. This fellowship will allow me to further investigate the role of this direct signaling pathway in breast cancer cells, especially, in response to an autocrine- and/or paracrine- acting growth factor such as EGF.
Keywords: biological signal transduction, breast neoplasm, neoplastic cell, transcription factor, epidermal growth factor, gene expression, growth factor receptor, transforming growth factor, tissue /cell culture
5F32CA069741-02 (1997): $35300
STAT3 Acetylation And Deacetylation In Metastasis
Y Eugene Chin
Rhode Island Hospital (providence, Ri) Providence, Ri 029034923
Grant 5R01CA102128-03 from National Cancer Institute IRG: TPM
Abstract: Signal transducer and activator of transcription (STAT) activation and inactivation are controlled by protein tyrosine kinase (i.e., JAK) and protein tyrosine phosphatase (i.e., SHP-2) activities, respectively. C-terminal region phosphorylated STAT dimerize and translocate into nuclei where it binds to DNA for transcriptional activation. To achieve maximum transcriptional activity, STAT needs to interact with other nuclear factors. Evidence from our laboratory shows that both p300/CBP and HDAC family members are capable of forming complexes with STAT3 but exert opposite effects on STAT3-dependent transcription while p300/CBP enhances STATS s activity in transcription, overexpression of the HDAC family member HDAC3 in 293T cells was highly effective in blocking STAT3-dependent transcription. Thus, p300/CBP and HDAC activities may play key roles in regulating STAT3 activity. Recently, STAT3 has been found to play an important role in regulating cell migration and tumor metastasis although the mechanism has yet to be determined. In this regard, STAT3 constitutive phosphorylation has been widely detected in both metastatic and non-metastatic cells. Thus, an additional post-translational modification event seems to be required for STAT3 to regulate gene transcription relevant to acquisition of the metastatic phenotype. The overarching hypothesis to be tested in this proposal is that STAT activity is under the control of acetylation and deacetylation mediated by HAT and HDAC, respectively. It is hypothesized further that constitutive STAT3 acetylation may play a central role in development of metastasis. To fully test these hypotheses, experiments will use our antibody array technology, specific gene-deficiency and/or down regulation, and mass-spectroscopy in order to (1) explore HAT/HDAC as components of STAT3 signaling complex; (2) test STAT3 acetylation in STAT3 dimerization and resisting to inactivation/degradation; and (3) test the hypothesis that STAT3 acetylation plays a role in metastasis-related gene regulation and constitutive STAT3 acetylation is responsible for metastatic phenotype in vivo. These complimentary approaches will elucidate the role of uncontrolled STAT3 acetylation/deacetylation in causing cancer cell invasion and metasasis.
Keywords: acetylation, genetic regulation, metastasis, neoplasm /cancer invasiveness, neoplasm /cancer genetics, clinical research, human subject, laboratory mouse, mass spectrometry
Project start date: 2005-06-01
Project end date: 2010-04-30
5R01CA102128-03 (2007): $280899
5R01CA102128-02 (2006): $289289
1R01CA102128-01A2 (2005): $295329