David Tweardy - Academia.edu (original) (raw)

Papers by David Tweardy

eLife, 2013

While a first pregnancy before age 22 lowers breast cancer risk, a pregnancy after age 35 signifi... more While a first pregnancy before age 22 lowers breast cancer risk, a pregnancy after age 35 significantly increases life-long breast cancer risk. Pregnancy causes several changes to the normal breast that raise barriers to transformation, but how pregnancy can also increase cancer risk remains unclear. We show in mice that pregnancy has different effects on the few early lesions that have already developed in the otherwise normal breast—it causes apoptosis evasion and accelerated progression to cancer. The apoptosis evasion is due to the normally tightly controlled STAT5 signaling going astray—these precancerous cells activate STAT5 in response to pregnancy/lactation hormones and maintain STAT5 activation even during involution, thus preventing the apoptosis normally initiated by oncoprotein and involution. Short-term anti-STAT5 treatment of lactation-completed mice bearing early lesions eliminates the increased risk after a pregnancy. This chemoprevention strategy has important impli...

Journal of Biological Chemistry, 2004

Cancer Research, 2004

Stat3 is a signaling molecular and oncogene activated frequently in many human malignancies inclu... more Stat3 is a signaling molecular and oncogene activated frequently in many human malignancies including the majority of prostate, breast, and head and neck cancers; yet, no current chemotherapeutic approach has been implemented clinically that specifically targets Stat3. We recently developed G-rich oligodeoxynucleotides, which form intramolecular G-quartet structures (GQ-ODN), as a new class of Stat3 inhibitor. GQ-ODN targeted Stat3 protein directly inhibiting its ability to bind DNA. When delivered into cells using polyethyleneimine as vehicle, GQ-ODN blocked ligand-induced Stat3 activation and Stat3-mediated transcription of antiapoptotic genes. To establish the effectiveness of GQ-ODN as a potential new chemotherapeutic agent, we systemically administered GQ-ODN (T40214 or T40231) plus polyethyleneimine or polyethyleneimine alone (placebo) by tail-vein injection into nude mice with prostate and breast tumor xenografts. Whereas the mean volume of breast tumor xenografts in placebo-...

Blood, 2001

Signal transducer and activator of transcription 3 (STAT3) is an oncogene and a critical regulato... more Signal transducer and activator of transcription 3 (STAT3) is an oncogene and a critical regulator of multiple cell-fate decisions, including myeloid cell differentiation. Two isoforms of STAT3 have been identified: α (p92) and β (p83). These differ structurally in their C-terminal transactivation domains, resulting in distinct functional activities. The cis genetic elements that regulate the ratio of α to β messenger RNA (mRNA) are unknown. In this study, cloning, sequencing, and splicing analysis of the human and murine STAT3 genes revealed a highly conserved 5′ donor site for generation of both α and β mRNA and distinct branch-point sequences, polypyrimidine tracts, and 3′ acceptor sites (ASs) for each. The β 3′ AS was found to be located 50 nucleotides downstream of the α 3′ AS in exon 23. Two additional cryptic 3′ ASs (δ and ε) were also identified. Thus, we identified for the first time the cisregulatory sequences responsible for generation of STAT3α and STAT3β mRNA.

Frontiers in Bioscience, 2009

Introduction 3. Structure of SH2 domain 4. SH2 domains and phosphopeptide binding 5. Energetics o... more Introduction 3. Structure of SH2 domain 4. SH2 domains and phosphopeptide binding 5. Energetics of phosphopeptide binding 5.1. Phosphotyrosine 5.2. Residues C-terminal to the phosphotyrosine 6. Classification of SH2 Domains 7. STAT SH2 domains 7.1. STAT1 binding model 7.2. STAT3 binding model 7.3. STAT5 Binding model 8. Therapeutic targeting of STAT proteins at their SH2 domains 8.1. Selective targeting of STAT3 vs. STAT1

Oncogene, 2003

Nuclear mitotic apparatus protein-retinoic acid receptor a (NuMA-RARa) is the fourth of five fusi... more Nuclear mitotic apparatus protein-retinoic acid receptor a (NuMA-RARa) is the fourth of five fusion proteins identified in acute promyelocytic leukemia (APL) patients. The molecular basis for its oncogenic activity has not been delineated. In gel-shift assays, NuMA-RARa bound to retinoic acid response elements (RAREs) both as a homodimer and as a heterodimer with RXRa. The binding profile of NuMA-RARa to a panel of RAREs was very similar to PML-RARa and PLZF-RARa. In transient transfection assays using HepG2 cells, NuMA-RARa inhibited wild-type RARa transcriptional activity, while it augmented STAT3 transcriptional activity. In GST-pull down experiments, NuMA-RARa formed a complex with the corepressor SMRT, was released from the NuMA-RARa/SMRT complexes by all-trans retinoic acid (ATRA) at 10 À7-10 À6 M and became associated with the coactivator TRAM-1 at 10 À8 M ATRA. Studies comparing NuMA-RARa with NuMA-RARa(DCC) demonstrated that the dimerization or a-helical coiled-coil domain of NuMA was required for homodimer formation, transcriptional repression of wild-type RARa, transcriptional activation of STAT3, and stability of the NuMA-RARa/SMRT complex. Confocal fluorescent microscopy of HeLa cells was performed following transient expression of cyan fluorescent protein (CFP)-tagged proteins and incubation of cells with or without ATRA. Within the nucleus, CFP-NuMA-RARa exhibited a speckled pattern identical to that observed in cells transfected with CFP-NuMA. Furthermore, CFP-NuMA-RARa colocalized with yellow fluorescent protein-tagged (YFP)-NuMA. In contrast, CFP-NuMA-RARa(DCC) exhibited a diffuse granular pattern within the nucleus, similar to RARa. These results indicate that the dimerization domain of NuMA-RARa is critical for each of the known oncogenic activities of NuMA fusion proteins as well as its sequestration to nuclear sites normally occupied by NuMA and is distinct from RARa.

Molecular and Cellular Biology, 2004

Acute promyelocytic leukemia (APL) cells contain one of five chimeric retinoic acid α-receptor (R... more Acute promyelocytic leukemia (APL) cells contain one of five chimeric retinoic acid α-receptor (RARα) genes (X-RARα) created by chromosomal translocations or deletion; each generates a fusion protein thought to transcriptionally repress RARα target genes and block myeloid differentiation by an incompletely understood mechanism. To gain spatiotemporal insight into these oncogenic processes, we employed fluorescence microscopy and fluorescence recovery after photobleaching (FRAP). Fluorescence microscopy demonstrated that the intracellular localization of each of the X-RARα proteins was distinct from that of RARα and established which portion(s) of each X-RARα protein—X, RAR, or both—contributed to its altered localization. Using FRAP, we demonstrated that the intranuclear mobility of each X-RARα was reduced compared to that of RARα. In addition, the mobility of each X-RARα was reduced further by ligand addition, in contrast to RARα, which showed no change in mobility when ligand was ...

American Journal of Physiology-Endocrinology and Metabolism, 2020

Cellular mechanisms causing insulin resistance (IR) in chronic kidney disease (CKD) are poorly un... more Cellular mechanisms causing insulin resistance (IR) in chronic kidney disease (CKD) are poorly understood. One potential mechanism is that CKD-induced inflammation activates the signal transducer and activator of transcription 3 (Stat3) in muscle. We uncovered increased p-Stat3 in muscles of mice with CKD or mice fed high-fat diet (HFD). Activated Stat3 stimulates the expression of Fbxo40, a muscle-specific E3 ubiquitin ligase that stimulates ubiquitin conjugation leading to degradation of insulin receptor substrate 1 (IRS1). Evidence that Stat3 activates Fbxo40 includes 1) potential Stat3 binding sites in Fbxo40 promoters; 2) Stat3 binding to the Fbxo40 promoter; and 3) constitutively active Stat3 stimulating both Fbxo40 expression and its promoter activity. We found that IL-6 activates Stat3 in myotubes, increasing Fbxo40 expression with reduced IRS1 and p-Akt. Knockdown Fbxo40 using siRNA from myotubes results in higher levels of IRS1 and p-Akt despite the presence of IL-6. We tr...

The Journal of Immunology

Kupffer cells were the principal source of IL-6 produced in the livers of mice following i.v. ino... more Kupffer cells were the principal source of IL-6 produced in the livers of mice following i.v. inoculation of Listeria monocytogenes. IL-6 mRNA expression and the production of IL-6 were reduced drastically within the nonparenchymal liver cell population derived from mice rendered Kupffer cell depleted by pretreatment with liposome-encapsulated dichloromethylene diphosphonate. A sharp increase in the appearance of activated STAT3 occurred in extracts of purified hepatocytes derived from normal mice infected i.v. with Listeria. Remarkably, the kinetics of this increase overlapped IL-6 mRNA expression by Kupffer cells; each peaked at approximately 30 min postinfection. No increase in STAT3 activation was observed in IL-6-deficient or Kupffer cell-depleted animals. The results of these experiments indicate that the synthesis of IL-6 and the activation of STAT3 within hepatocytes are critical functions of Kupffer cells occurring very early during the course of systemic listerial infections.

STAT3 is an important signaling molecule that modulates a wide range of genes by relaying extrace... more STAT3 is an important signaling molecule that modulates a wide range of genes by relaying extracellular signals from the plasma membrane to the nucleus in response to peptide hormone binding. It is known to play a prominent role in the initiation and progression of cancer, as it is constitutively activated in 25–100 % of more than 25 different malignancies and has been implicated in nearly all the hallmarks of cancer. In addition, STAT3 contributes to development and maintenance of cancer stem cells, as well as to cancer immune evasion and resistance to chemotherapy and radiotherapy, making it an even more attractive target for cancer therapy. In this chapter, we give an overview of strategies involved in targeting STAT3 and discuss recent advances in the development of STAT3 modulating agents.

Biochemical Pharmacology, 2021

This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Organic & Biomolecular Chemistry, 2020

Recurrence and drug resistance are major challenges in the treatment of acute myeloid leukemia (A... more Recurrence and drug resistance are major challenges in the treatment of acute myeloid leukemia (AML) that spur efforts to identify new clinical targets and active agents.

Rheumatology, 2017

Objective. SSc is an autoimmune disease characterized by progressive fibrosis of the skin and int... more Objective. SSc is an autoimmune disease characterized by progressive fibrosis of the skin and internal organs. IL-6 and related cytokines that signal through STAT3 have been implicated in the pathogenesis of SSc and mouse models of fibrosis. The aim of this study was to investigate the efficacy of inhibiting STAT3 in the development of fibrosis in two mouse models of skin fibrosis. Methods. Biopsy samples of skin from SSc patients and healthy control subjects were used to determine the expression pattern of phosphotyrosyl (pY705)-STAT3. C188-9, a small molecule inhibitor of STAT3, was used to treat fibrosis in the bleomycin-induced fibrosis model and Tsk-1 mice. In vitro studies were performed to determine the extent to which STAT3 regulates the fibrotic phenotype of dermal fibroblasts. Results. Increased STAT3 and pY705-STAT3 was observed in SSc skin biopsies and in both mouse models of SSc. STAT3 inhibition with C188-9 resulted in attenuated skin fibrosis, myofibroblast accumulation, pro-fibrotic gene expression and collagen deposition in both mouse models of skin fibrosis. C188-9 decreased in vitro dermal fibroblast production of fibrotic genes induced by IL-6 trans-signalling and TGFb. Finally, TGF-b induced phosphotyrosylation of STAT3 in a SMAD3-dependent manner. Conclusion. STAT3 inhibition decreases dermal fibrosis in two models of SSc. STAT3 regulates dermal fibroblasts function in vitro and can be activated by TGF-b. These data suggest that STAT3 is a potential therapeutic target for dermal fibrosis in diseases such as SSc.

Clinical cancer research : an official journal of the American Association for Cancer Research, Jan 22, 2017

The incidence of hepatocellular carcinoma (HCC) is increasing in the United States and liver canc... more The incidence of hepatocellular carcinoma (HCC) is increasing in the United States and liver cancer is the second leading cause of cancer-related mortality worldwide. Non-alcoholic steatohepatitis (NASH) is becoming an important risk for HCC and most patients with HCC have underlying liver cirrhosis and compromised liver function, which limit treatment options. Thus, novel therapeutic strategies to prevent or treat HCC in the context of NASH and cirrhosis are urgently needed. <br /><br />Experimental Design: Constitutive activation of signal transducer and activator of transcription 3 (STAT3) is frequently detected in HCC tumors. STAT3 signaling plays a pivotal role in HCC survival, growth, angiogenesis and metastasis. We identified C188-9, a novel small-molecule STAT3 inhibitor using computer-aided rational drug design. In this study, we evaluated the therapeutic potential of C188-9 for HCC treatment and prevention. <br /><br />Results: C188-9 showed antitum...

Blood, Dec 31, 2016

AD-HIES is caused by dominant negative mutations in STAT3; however, the molecular basis for mutan... more AD-HIES is caused by dominant negative mutations in STAT3; however, the molecular basis for mutant STAT3 allele dysfunction is unclear and treatment remains supportive. We hypothesized that AD-HIES mutations decrease STAT3 protein stability and that mutant STAT3 activity can be improved by agents that increase chaperone protein activity. We used computer modeling to characterize the effect of STAT3 mutations on protein stability. We measured STAT3 protein half-life (t1/2) and determined levels of STAT3 phosphorylated on tyrosine (Y) 705 (pY-STAT3) and mRNA levels of STAT3 gene targets in EBV-transformed B (EBV) cells, human peripheral blood mononuclear cells (PBMC), and mouse splenocytes incubated without or with chaperone protein modulators-HSF1A, a small-molecule TRiC modulator, or geranylgeranylacetone (GGA), a drug that upregulates heat shock protein (HSP) 70 and HSP90. Computer modeling predicted that 81% of AD-HIES mutations are destabilizing. STAT3 protein t1/2 in EBV cells f...

Oncotarget, Jan 25, 2016

While STAT3 has been validated as a target for treatment of many cancers, including head and neck... more While STAT3 has been validated as a target for treatment of many cancers, including head and neck squamous cell carcinoma (HNSCC), a STAT3 inhibitor is yet to enter the clinic. We used the scaffold of C188, a small-molecule STAT3 inhibitor previously identified by us, in a hit-to-lead program to identify C188-9. C188-9 binds to STAT3 with high affinity and represents a substantial improvement over C188 in its ability to inhibit STAT3 binding to its pY-peptide ligand, to inhibit cytokine-stimulated pSTAT3, to reduce constitutive pSTAT3 activity in multiple HNSCC cell lines, and to inhibit anchorage dependent and independent growth of these cells. In addition, treatment of nude mice bearing xenografts of UM-SCC-17B, a radioresistant HNSCC line, with C188-9, but not C188, prevented tumor xenograft growth. C188-9 treatment modulated many STAT3-regulated genes involved in oncogenesis and radioresistance, as well as radioresistance genes regulated by STAT1, due to its potent activity agai...

The Journal of allergy and clinical immunology, Jul 29, 2016

During IgE-mediated immediate hypersensitivity reactions, vascular endothelial cells permeabilize... more During IgE-mediated immediate hypersensitivity reactions, vascular endothelial cells permeabilize in response to mast cell mediators. We have demonstrated previously that patients and mice with signal transducer and activator of transcription 3 (STAT3) mutations (autosomal dominant hyper-IgE syndrome [AD-HIES]) are partially protected from anaphylaxis. We sought to study the mechanism by which STAT3 contributes to anaphylaxis and determine whether small-molecule inhibition of STAT3 can prevent anaphylaxis. Using unaffected and STAT3-inhibited or genetic loss-of-function samples, we performed histamine skin prick tests, investigated the contribution of STAT3 to animal models of anaphylaxis, and measured endothelial cell permeability, gene and protein expression, and histamine receptor-mediated signaling. Although mouse mast cell degranulation was minimally affected by STAT3 blockade, mast cell mediator-induced anaphylaxis was blunted in Stat3 mutant mice with AD-HIES and in wild-type...

Journal of Biological Chemistry, 2015

AML1-ETO is the most common fusion oncoprotein causing acute myeloid leukemia (AML), a disease wi... more AML1-ETO is the most common fusion oncoprotein causing acute myeloid leukemia (AML), a disease with a 5-year survival rate of only 24%. AML1-ETO functions as a rogue transcription factor, altering the expression of genes critical for myeloid cell development and differentiation. Currently, there are no specific therapies for AML1-ETO-positive AML. While known for decades to be the translational product of a chimeric gene created by the stable chromosome translocation t(8;21)(q22;q22), it is not known how AML1-ETO achieves its native and functional conformation or whether this process can be targeted for therapeutic benefit. Here, we show that the biosynthesis and folding of the AML1-ETO protein is facilitated by interaction with the essential eukaryotic chaperonin TRiC (or CCT). We demonstrate that a folding intermediate of AML1-ETO binds to TRiC directly, mainly through its ␤-strand rich, DNA-binding domain (AML-(1-175)), with the assistance of HSP70. Our results suggest that TRiC contributes to AML1-ETO proteostasis through specific interactions between the oncoprotein's DNA-binding domain, which may be targeted for therapeutic benefit.

International Journal of Molecular Sciences, 2015

The folding of newly synthesized proteins and the maintenance of pre-existing proteins are essent... more The folding of newly synthesized proteins and the maintenance of pre-existing proteins are essential in sustaining a living cell. A network of molecular chaperones tightly guides the folding, intracellular localization, and proteolytic turnover of proteins. Many of the key regulators of cell growth and differentiation have been identified as clients of molecular chaperones, which implies that chaperones are potential mediators of oncogenesis. In this review, we briefly provide an overview of the role of chaperones, including HSP70 and HSP90, in cancer. We further summarize and highlight the emerging the role of chaperonin TRiC (T-complex protein-1 ring complex, also known as CCT) in the development and progression of cancer mediated through its critical interactions with oncogenic clients that modulate growth deregulation, apoptosis, and genome instability in cancer cells. Elucidation of how TRiC modulates the folding and function of oncogenic clients will provide strategies for developing novel cancer therapies.