4296 Targeting ERG Through Toll-Like Receptor 4 in Prostate Cancer

OBJECTIVES/GOALS: The objective of this research was to learn how the oncogenic transcription factor, ERG, is regulated in prostate cancer. If we could learn how ERG is regulated and which genes are important for its oncogenic phenotype in prostate cells, we could design new therapeutic strategies against ERG, which has proven to be difficult to target. METHODS/STUDY POPULATION: We conducted an shRNA screen in prostate cells to determine candidate genes and pathways that are important for ERG function. To validate the findings of the screen, we performed a variety of cell-based functional assays, including trans-well migration, wound healing, and clonogenic survival assays. To further investigate the mechanism between ERG and the genes revealed by the screen, we performed biochemical and molecular biology experiments such as Western blotting and qRT-PCR for protein and mRNA expression, co-immunoprecipitation assays to determine protein-protein interactions, and chromatin immunoprecipitation (ChIP-qPCR) to determine transcription factor binding to DNA sites. RESULTS/ANTICIPATED RESULTS: The screen revealed that genes involved in the toll-like receptor 4 (TLR4) pathway are important for ERG-mediated migration. We tested the effect of a TLR4 inhibitor on ERG function and observed decreased migration and clonogenic survival exclusively in ERG-positive cells. Expression of pMEK and pERG was reduced when TLR4 was inhibited, which suggests a mechanism in which TLR4 upregulates pMEK, leading to the phosphorylation and activation of ERG. This is supported by functional assays in which cells expressing a phosphomimetic ERG are resistant to the TLR4 inhibitor. We demonstrated that ERG drives the transcription of TLR4 and its endogenous ligands HSPA8 and BGN. Therefore, ERG can sensitize the cell to TLR4 activation by increasing the number of receptors as well as providing the ligands needed for stimulation. DISCUSSION/SIGNIFICANCE OF IMPACT: This research provides a new therapeutic pathway for treating ERG-positive patients through TLR4 inhibition. This can be beneficial because many patients become resistant to the standard therapy, leaving very few treatment options. TLR4-based therapies could provide an alternative for patients who have developed resistance.

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Toll-like receptor 4 signaling activates ERG function in prostate cancer and provides a therapeutic target

NAR Cancer ◽

10.1093/narcan/zcaa046 ◽

2021 ◽

Vol 3 (1) ◽

Author(s):

Benjamin M Greulich ◽

Joshua P Plotnik ◽

Travis J Jerde ◽

Peter C Hollenhorst

Keyword(s):

Prostate Cancer ◽

Transcription Factor ◽

Transcriptional Activation ◽

Gene Activation ◽

Clonogenic Survival ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Critical Function ◽

Tlr4 Signaling ◽

Tlr4 Gene

Abstract The TMPRSS2–ERG gene fusion and subsequent overexpression of the ERG transcription factor occurs in ∼50% of prostate tumors, making it the most common abnormality of the prostate cancer genome. While ERG has been shown to drive tumor progression and cancer-related phenotypes, as a transcription factor it is difficult to target therapeutically. Using a genetic screen, we identified the toll-like receptor 4 (TLR4) signaling pathway as important for ERG function in prostate cells. Our data confirm previous reports that ERG can transcriptionally activate TLR4 gene expression; however, using a constitutively active ERG mutant, we demonstrate that the critical function of TLR4 signaling is upstream, promoting ERG phosphorylation at serine 96 and ERG transcriptional activation. The TLR4 inhibitor, TAK-242, attenuated ERG-mediated migration, clonogenic survival, target gene activation and tumor growth. Together these data indicate a mechanistic basis for inhibition of TLR4 signaling as a treatment for ERG-positive prostate cancer.

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Novel Roles in Human MD-2 of Phenylalanines 121 and 126 and Tyrosine 131 in Activation of Toll-like Receptor 4 by Endotoxin

Journal of Biological Chemistry ◽

10.1074/jbc.m705994200 ◽

2007 ◽

Vol 283 (3) ◽

pp. 1257-1266 ◽

Cited By ~ 38

Author(s):

Athmane Teghanemt ◽

Fabio Re ◽

Polonca Prohinar ◽

Richard Widstrom ◽

Theresa L. Gioannini ◽

...

Keyword(s):

Protein Interactions ◽

Cell Activation ◽

Toll Like Receptor ◽

Wild Type ◽

Protein Protein Interactions ◽

Lipopolysaccharide Binding Protein ◽

Toll Like Receptor 4 ◽

Dependent Cell ◽

Cellular Responsiveness ◽

Tlr4 Activation

Potent mammalian cell activation by Gram-negative bacterial endotoxin requires sequential protein-endotoxin and protein-protein interactions involving lipopolysaccharide-binding protein, CD14, MD-2, and Toll-like receptor 4 (TLR4). TLR4 activation requires simultaneous binding of MD-2 to endotoxin (E) and the ectodomain of TLR4. We now describe mutants of recombinant human MD-2 that bind TLR4 and react with E·CD14 but do not support cellular responsiveness to endotoxin. The mutants F121A/K122A MD-2 and Y131A/K132A MD-2 react with E·CD14 only when co-expressed with TLR4. Single mutants K122A and K132A each react with E·CD14 ± TLR4 and promote TLR4-dependent cell activation by endotoxin suggesting that Phe121 and Tyr131 are needed for TLR4-independent transfer of endotoxin from CD14 to MD-2 and also needed for TLR4 activation by bound E·MD-2. The mutant F126A MD-2 reacts as well as wild-type MD-2 with E·CD14 ± TLR4. E·MD-2F126A binds TLR4 with high affinity (Kd ∼ 200 pm) but does not activate TLR4 and instead acts as a potent TLR4 antagonist, inhibiting activation of HEK/TLR4 cells by wild-type E·MD-2. These findings reveal roles of Phe121 and Tyr131 in TLR4-independent interactions of human MD-2 with E·CD14 and, together with Phe126, in activation of TLR4 by bound E·MD-2. These findings strongly suggest that the structural properties of E·MD-2, not E alone, determine agonist or antagonist effects on TLR4.

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Lycopene Inhibits Toll-Like Receptor 4-Mediated Expression of Inflammatory Cytokines in House Dust Mite-Stimulated Respiratory Epithelial Cells

Molecules ◽

10.3390/molecules26113127 ◽

2021 ◽

Vol 26 (11) ◽

pp. 3127

Author(s):

Jiyeon Choi ◽

Joo Weon Lim ◽

Hyeyoung Kim

Keyword(s):

Epithelial Cells ◽

A549 Cells ◽

Cytokine Expression ◽

Ros Production ◽

Toll Like Receptor ◽

Respiratory Epithelial Cells ◽

Toll Like Receptor 4 ◽

Mitochondrial Ros ◽

Tlr4 Activation ◽

Respiratory Epithelial

House dust mites (HDM) are critical factors in airway inflammation. They activate respiratory epithelial cells to produce reactive oxygen species (ROS) and activate Toll-like receptor 4 (TLR4). ROS induce the expression of inflammatory cytokines in respiratory epithelial cells. Lycopene is a potent antioxidant nutrient with anti-inflammatory activity. The present study aimed to investigate whether HDM induce intracellular and mitochondrial ROS production, TLR4 activation, and pro-inflammatory cytokine expression (IL-6 and IL-8) in respiratory epithelial A549 cells. Additionally, we examined whether lycopene inhibits HDM-induced alterations in A549 cells. The treatment of A549 cells with HDM activated TLR4, induced the expression of IL-6 and IL-8, and increased intracellular and mitochondrial ROS levels. TAK242, a TLR4 inhibitor, suppressed both HDM-induced ROS production and cytokine expression. Furthermore, lycopene inhibited the HDM-induced TLR4 activation and cytokine expression, along with reducing the intracellular and mitochondrial ROS levels in HDM-treated cells. These results collectively indicated that the HDM induced TLR4 activation and increased intracellular and mitochondrial ROS levels, thus resulting in the induction of cytokine expression in respiratory epithelial cells. The antioxidant lycopene could inhibit HDM-induced cytokine expression, possibly by suppressing TLR4 activation and reducing the intracellular and mitochondrial ROS levels in respiratory epithelial cells.

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Toll-Like Receptor 4 (TLR4)/Cyclooxygenase-2 (COX-2) Regulates Prostate Cancer Cell Proliferation, Migration, and Invasion by NF-κB Activation

Medical Science Monitor ◽

10.12659/msm.906857 ◽

2018 ◽

Vol 24 ◽

pp. 5588-5597 ◽

Cited By ~ 7

Author(s):

Wei Wang ◽

Jiye Wang

Keyword(s):

Prostate Cancer ◽

Cell Proliferation ◽

Cancer Cell ◽

Prostate Cancer Cell ◽

Cyclooxygenase 2 ◽

Migration And Invasion ◽

Cancer Cell Proliferation ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Cox 2

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The Role of Toll-Like Receptor 4 in Infectious and Noninfectious Inflammation

Mediators of Inflammation ◽

10.1155/2016/6978936 ◽

2016 ◽

Vol 2016 ◽

pp. 1-9 ◽

Cited By ~ 117

Author(s):

Monica Molteni ◽

Sabrina Gemma ◽

Carlo Rossetti

Keyword(s):

Tissue Injury ◽

Sterile Inflammation ◽

Research Progress ◽

Toll Like Receptor ◽

Proinflammatory Response ◽

Toll Like Receptor 4 ◽

The Family ◽

Tlr4 Activation ◽

Molecular Patterns

Toll-like receptor 4 (TLR4) belongs to the family of pattern recognition receptors (PRRs). They are highly conserved receptors that recognize conserved pathogen-associated molecular patterns (PAMPs), thus representing the first line of defense against infections. TLR4 has been long recognized as the sensing receptor for gram-negative lipopolysaccharide (LPS). In addition, it also binds endogenous molecules produced as a result of tissue injury. Hence, TLR4 represents a key receptor on which both infectious and noninfectious stimuli converge to induce a proinflammatory response. TLR4-mediated inflammation, triggered by exogenous or endogenous ligands, is also involved in several acute and chronic diseases, having a pivotal role as amplifier of the inflammatory response. This review focuses on the research progress about the role of TLR4 activation in infectious and noninfectious (e.g., sterile) inflammation and the effects of TLR4 signaling in some pathological conditions.

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Toll-like receptor 4 ligation confers chemoresistance to docetaxel on PC-3 human prostate cancer cells

Cell Biology and Toxicology ◽

10.1007/s10565-012-9221-2 ◽

2012 ◽

Vol 28 (4) ◽

pp. 269-277 ◽

Cited By ~ 19

Author(s):

Yuntao Zhang ◽

Yong Wang ◽

Jianlin Yuan ◽

Weijun Qin ◽

Fei Liu ◽

...

Keyword(s):

Prostate Cancer ◽

Cancer Cells ◽

Human Prostate ◽

Human Prostate Cancer ◽

Toll Like Receptor ◽

Prostate Cancer Cells ◽

Toll Like Receptor 4

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Synthetic molecules and functionalized nanoparticles targeting the LPS-TLR4 signaling: A new generation of immunotherapeutics

Pure and Applied Chemistry ◽

10.1351/pac-con-11-10-35 ◽

2011 ◽

Vol 84 (1) ◽

pp. 97-106 ◽

Cited By ~ 4

Author(s):

Francesco Peri ◽

Valentina Calabrese ◽

Matteo Piazza ◽

Roberto Cighetti

Keyword(s):

Myeloid Differentiation ◽

Vaccine Adjuvants ◽

Toll Like Receptor ◽

Differentiation Antigen ◽

Toll Like Receptor 4 ◽

Tlr4 Signaling ◽

Bacterial Endotoxins ◽

Pharmacological Interest ◽

Tlr4 Activation ◽

New Generation

Toll-like receptor 4 (TLR4), the receptor of bacterial endotoxins in mammalians, plays a pivotal role in the induction of innate immunity and inflammation. TLR4 activation by bacterial lipopolysaccharide (LPS) is achieved by the coordinate and sequential action of three other proteins, the lipopolysaccharide binding protein (LBP), the cluster differentiation antigen CD14, and the myeloid differentiation protein (MD-2) receptors, that bind LPS and present it in a monomeric form to TLR4 by forming the activated [TLR4·MD-2·LPS]2 complex. Small molecules and nanoparticles active in modulating the TLR4 signal by targeting directly the MD-2·TLR4 complex or by interfering in other points of the TLR4 signaling are presented in this paper. These compounds have great pharmacological interest as vaccine adjuvants, immunotherapeutics, anti-sepsis, and anti-inflammatory agents.

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Safflower polysaccharides activate the transcription factor NF-κB via Toll-like receptor 4 and induce cytokine production by macrophages

International Immunopharmacology ◽

10.1016/s1567-5769(02)00076-0 ◽

2002 ◽

Vol 2 (8) ◽

pp. 1155-1162 ◽

Cited By ~ 92

Author(s):

Izuru Ando ◽

Yoshinori Tsukumo ◽

Tetsuya Wakabayashi ◽

Sachiko Akashi ◽

Kensuke Miyake ◽

...

Keyword(s):

Transcription Factor ◽

Cytokine Production ◽

Toll Like Receptor ◽

Toll Like Receptor 4

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Respiratory Syncytial Virus Fusion Protein-Induced Toll-Like Receptor 4 (TLR4) Signaling Is Inhibited by the TLR4 Antagonists Rhodobacter sphaeroides Lipopolysaccharide and Eritoran (E5564) and Requires Direct Interaction with MD-2

mBio ◽

10.1128/mbio.00218-12 ◽

2012 ◽

Vol 3 (4) ◽

Cited By ~ 64

Author(s):

Prasad Rallabhandi ◽

Rachel L. Phillips ◽

Marina S. Boukhvalova ◽

Lioubov M. Pletneva ◽

Kari Ann Shirey ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Rhodobacter Sphaeroides ◽

Dependent Manner ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Tlr4 Signaling ◽

F Protein ◽

Terminal Domain ◽

Syncytial Virus ◽

Tlr4 Activation

ABSTRACTRespiratory syncytial virus (RSV) is a leading cause of infant mortality worldwide. Toll-like receptor 4 (TLR4), a signaling receptor for structurally diverse microbe-associated molecular patterns, is activated by the RSV fusion (F) protein and by bacterial lipopolysaccharide (LPS) in a CD14-dependent manner. TLR4 signaling by LPS also requires the presence of an additional protein, MD-2. Thus, it is possible that F protein-mediated TLR4 activation relies on MD-2 as well, although this hypothesis has not been formally tested. LPS-free RSV F protein was found to activate NF-κB in HEK293T transfectants that express wild-type (WT) TLR4 and CD14, but only when MD-2 was coexpressed. These findings were confirmed by measuring F-protein-induced interleukin 1β (IL-1β) mRNA in WT versus MD-2−/−macrophages, where MD-2−/−macrophages failed to show IL-1β expression upon F-protein treatment, in contrast to the WT. BothRhodobacter sphaeroidesLPS and synthetic E5564 (eritoran), LPS antagonists that inhibit TLR4 signaling by binding a hydrophobic pocket in MD-2, significantly reduced RSV F-protein-mediated TLR4 activity in HEK293T-TLR4–CD14–MD-2 transfectants in a dose-dependent manner, while TLR4-independent NF-κB activation by tumor necrosis factor alpha (TNF-α) was unaffected.In vitrocoimmunoprecipitation studies confirmed a physical interaction between native RSV F protein and MD-2. Further, we demonstrated that the N-terminal domain of the F1 segment of RSV F protein interacts with MD-2. These data provide new insights into the importance of MD-2 in RSV F-protein-mediated TLR4 activation. Thus, targeting the interaction between MD-2 and RSV F protein may potentially lead to novel therapeutic approaches to help control RSV-induced inflammation and pathology.IMPORTANCEThis study shows for the first time that the fusion (F) protein of respiratory syncytial virus (RSV), a major cause of bronchiolitis and death, particularly in infants and young children, physically interacts with the Toll-like receptor 4 (TLR4) coreceptor, MD-2, through its N-terminal domain. We show that F protein-induced TLR4 activation can be blocked by lipid A analog antagonists. This observation provides a strong experimental rationale for testing such antagonists in animal models of RSV infection for potential use in people.

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