scholarly journals TLR4-Mediated Recognition of Mouse Polyomavirus Promotes Cancer-Associated Fibroblast-Like Phenotype and Cell Invasiveness

Cancers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 2076
Author(s):  
Vaclav Janovec ◽  
Boris Ryabchenko ◽  
Aneta Škarková ◽  
Karolína Pokorná ◽  
Daniel Rösel ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
T Antigen ◽  
Cancer Associated Fibroblast ◽  
Inflammatory Mechanism ◽  
Stat3 Phosphorylation ◽  
Active Viral Replication ◽  
Culture Models ◽  
Capsid Protein Vp1 ◽  
Cell Invasiveness ◽  
Tlr4 Activation

The tumorigenic potential of mouse polyomavirus (MPyV) has been studied for decades in cell culture models and has been mainly attributed to nonstructural middle T antigen (MT), which acts as a scaffold signal adaptor, activates Src tyrosine kinases, and possesses transforming ability. We hypothesized that MPyV could also transform mouse cells independent of MT via a Toll-like receptor 4 (TLR4)-mediated inflammatory mechanism. To this end, we investigated the interaction of MPyV with TLR4 in mouse embryonic fibroblasts (MEFs) and 3T6 cells, resulting in secretion of interleukin 6 (IL-6), independent of active viral replication. TLR4 colocalized with MPyV capsid protein VP1 in MEFs. Neither TLR4 activation nor recombinant IL-6 inhibited MPyV replication in MEFs and 3T6 cells. MPyV induced STAT3 phosphorylation through both direct and MT-dependent and indirect and TLR4/IL-6-dependent mechanisms. We demonstrate that uninfected mouse fibroblasts exposed to the cytokine environment from MPyV-infected fibroblasts upregulated the expressions of MCP-1, CCL-5, and α-SMA. Moreover, the cytokine microenvironment increased the invasiveness of MEFs and CT26 carcinoma cells. Collectively, TLR4 recognition of MPyV induces a cytokine environment that promotes the cancer-associated fibroblast (CAF)-like phenotype in noninfected fibroblasts and increases cell invasiveness.

scholarly journals Intranuclear Crosstalk between Extracellular Regulated Kinase1/2 and Signal Transducer and Activator of Transcription 3 Regulates JEG-3 Choriocarcinoma Cell Invasion and Proliferation

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Diana M. Morales-Prieto ◽  
Stephanie Ospina-Prieto ◽  
Wittaya Chaiwangyen ◽  
Maja Weber ◽  
Sebastian Hölters ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Capacity ◽  
Mapk Signaling ◽  
Signaling Molecules ◽  
Signal Transducer ◽  
Stat3 Phosphorylation ◽  
Functional Implications ◽  
Choriocarcinoma Cells ◽  
Cell Invasiveness ◽  
Transcription 3

Invasiveness of trophoblast and choriocarcinoma cells is in part mediated via leukemia inhibitory factor- (LIF-) induced activation of signal transducer and activator of transcription 3 (STAT3). The regulation of STAT3 phosphorylation at its ser727 binding site, possible crosstalk with intracellular MAPK signaling, and their functional implications are the object of the present investigation. JEG-3 choriocarcinoma cells were cultured in presence/absence of LIF and the specific ERK1/2 inhibitor (U0126). Phosphorylation of signaling molecules (p-STAT3 (ser727 and tyr705) and p-ERK1/2 (thr 202/tyr 204)) was assessed per Western blot. Immunocytochemistry confirmed results, but also pinpointed the location of phosphorylated signaling molecules. STAT3 DNA-binding capacity was studied with a colorimetric ELISA-based assay. Cell viability and invasion capability were assessed by MTS and Matrigel assays. Our results demonstrate that LIF-induced phosphorylation of STAT3 (tyr705 and ser727) is significantly increased after blocking ERK1/2. STAT3 DNA-binding capacity and cell invasiveness are enhanced after LIF stimulation and ERK1/2 blockage. In contrast, proliferation is enhanced by LIF but reduced after ERK1/2 inhibition. The findings herein show that blocking ERK1/2 increases LIF-induced STAT3 phosphorylation and STAT3 DNA-binding capacity by an intranuclear crosstalk, which leads to enhanced invasiveness and reduced proliferation.

scholarly journals A Transformation-Defective Polyomavirus Middle T Antigen with a Novel Defect in PI3 Kinase Signaling

2016 ◽  
Vol 91 (2) ◽  
Author(s):  
Deborah Denis ◽  
Cecile Rouleau ◽  
Brian S. Schaffhausen
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
T Antigen ◽  
Pi3 Kinase ◽  
Binding Motif ◽  
Wild Type ◽  
Pi3k Activity ◽  
Murine Polyomavirus ◽  
Middle T Antigen

ABSTRACT Middle T antigen (MT), the principal oncoprotein of murine polyomavirus, transforms by association with cellular proteins. Protein phosphatase 2A (PP2A), YAP, Src family tyrosine kinases, Shc, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-γ1 (PLCγ1) have all been implicated in MT transformation. Mutant dl1015, with deletion of residues 338 to 347 in the C-terminal region, has been an enigma, because the basis for its transformation defect has not been apparent. This work probes the dl1015 region of MT. Because the region is proline rich, the hypothesis that it targets Src homology domain 3 (SH3) domains was tested, but mutation of the putative SH3 binding motif did not affect transformation. During this work, two point mutants, W348R and E349K, were identified as transformation defective. Extensive analysis of the E349K mutant is described here. Similar to wild-type MT, the E349K mutant associates with PP2A, YAP, tyrosine kinases, Shc, PI3 kinase, and PLCγ1. The E349K mutant was examined to determine the mechanism for its transformation defect. Assays of cell localization and membrane targeting showed no obvious difference in localization. Src association was normal as assayed by in vitro kinase and MT phosphopeptide mapping. Shc activation was confirmed by its tyrosine phosphorylation. Association of type 1 PI3K with MT was demonstrated by coimmunoprecipitation, showing both PI3K subunits and in vitro activity. Nonetheless, expression of the mutants failed to lead to the activation of two known downstream targets of PI3K, Akt and Rac-1. Strikingly, despite normal association of the E349K mutant with PI3K, cells expressing the mutant failed to elevate phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in mutant-expressing cells. These results indicate a novel unsuspected aspect to PI3K control. IMPORTANCE The gene coding for middle T antigen (MT) is the murine polyomavirus oncogene most responsible for tumor formation. Its study has a history of uncovering novel aspects of mammalian cell regulation. The importance of PI3K activity and tyrosine phosphorylation are two examples of insights coming from MT. This study describes new mutants unable to transform like the wild type that point to novel regulation of PI3K signaling. Previous mutants were defective in PI3K because they failed to bind the enzyme and bring the activity to the membrane. These mutants recruit PI3K activity like the wild type, but fail to elevate the cellular level of PIP3, the product used to signal downstream of PI3K. As a result, they fail to activate either Akt or Rac1, explaining the transformation defect.

scholarly journals Nuclear-capture of endosomes drives depletion of nuclear G-actin to promote SRF/MRTF gene expression and cancer cell invasiveness

2020 ◽  
Author(s):  
Sergi Marco ◽  
Matthew Neilson ◽  
Madeleine Moore ◽  
Arantxa Pérez-García ◽  
Holly Hall ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Import ◽  
Tyrosine Kinases ◽  
Target Genes ◽  
Serum Response Factor ◽  
Actin Binding ◽  
Cell Scattering ◽  
Nuclear Localisation Sequence ◽  
Related Transcription Factor ◽  
Cell Invasiveness

Abstract Signals are relayed from receptor tyrosine kinases (RTKs) at the cell surface to effector systems in the cytoplasm and nucleus, and coordination of this process is important for the execution of migratory phenotypes, such as cell scattering and invasion. The endosomal system influences how RTK signalling is coded, but the ways in which it transmits these signals to the nucleus to influence gene expression are not yet clear. Here we show that an RTK, cMET promotes Rab17-dependent endocytosis of EphA2, another RTK, followed by centripetal transport of EphA2-positive endosomes. EphA2 then mediates physical capture of endosomes on the outer surface of the nucleus; a process involving interaction between the nuclear import machinery and a nuclear localisation sequence in EphA2’s cytodomain. Nuclear capture of EphA2 promotes RhoG-dependent phosphorylation of the actin-binding protein, cofilin to oppose nuclear import of G-actin. The resulting depletion of nuclear G-actin drives transcription of Myocardin-related transcription factor (MRTF)/serum-response factor (SRF)-target genes to implement cell scattering and the invasive behaviour of cancer cells.

scholarly journals Atiprimod Triggered Apoptotic Cell Death via Acting on PERK/eIF2a/ATF4/CHOP and STAT3/NF-KB axis in MDA-MB-231 and MDA-MB-468 Breast Cancer Cells

2021 ◽  
Author(s):  
Ajda Coker-Gurkan ◽  
Esin Can ◽  
Semanur Sahin ◽  
PINAR OBAKAN YERLIKAYA ◽  
Elif-Damla ARISAN
Keyword(s):  
Breast Cancer ◽  
Cell Death ◽  
Er Stress ◽  
Cancer Cells ◽  
Tyrosine Kinases ◽  
Breast Cancer Cells ◽  
Apoptotic Cell ◽  
Pivotal Role ◽  
Type I ◽  
Stat3 Phosphorylation

Abstract Purpose: The constitutive activation of STAT3 through receptor tyrosine kinases triggered breast cancer cell growth, and invasion-metastasis. Atiprimod impacts anti-proliferative, anti-carcinogenic effects in hepatocellular carcinoma, lymphoma, multiple myeloma via hindering the biological activity of STAT3. Dose-dependent atiprimod evokes first autophagy as a survival mechanism and then apoptosis due to prolonged ER stress in pituitary adenoma cells. The therapeutic efficiency and mechanistic action of atiprimod in breast cancer cells have not been investigated yet. Thus, we aimed to modulate the pivotal role of ER stress in atiprimod-triggered apoptosis in MDA-MB-231 and MDA-MB-468 breast cancer cells. Results: Dose- and time-dependent atiprimod treatment inhibits cell viability and colony formation in MDA-MB-468 and MDA-MB-231 breast cancer cells. A moderate dose of atiprimod (2 mM) inhibited STAT3 phosphorylation at Tyr705 residue and also suppressed the total expression level of p65. In addition, nuclear localization of STAT1, 3 and NF-kB was prevented by atiprimod exposure in MDA-MB-231 and MDA-MB-468 cells. Atiprimod evokes PERK, BiP, ATF-4, CHOP upregulation, and PERK (Thr980), eIF2a (Ser51) phosphorylation’s. However, atiprimod suppressed IRE1a-mediated Atg-3, 5, 7, 12 protein expressions and no alteration were observed on Beclin-1, p62 expression levels. PERK/eIF2a/ATF4/CHOP axis pivotal role in atiprimod-mediated G1/S arrest and apoptosis via Bak, Bax, Bim and PUMA upregulation in MDA-MB-468 cells. Moreover, atiprimod renders MDA-MB-231 more vulnerable to type I programmed cell death by plasmid-mediated increased STAT3 expression. Conclusion: Atiprimod induced prolonged ER stress-mediated apoptosis via both activating PERK/eIF2a/ATF4/CHOP axis and suppressing STAT3/NF-kB transcription factors nuclear migration in TBNC cells.

scholarly journals Stoichiometry of cellular and viral components in the polyomavirus middle-T antigen-tyrosine kinase complex.

1990 ◽  
Vol 10 (10) ◽  
pp. 5569-5574 ◽  
Author(s):  
S H Cheng ◽  
P C Espino ◽  
J Marshall ◽  
R Harvey ◽  
A E Smith
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Cellular Proliferation ◽  
T Antigen ◽  
Phosphatidylinositol Kinase ◽  
Polyomavirus Middle T Antigen ◽  
Viral Components ◽  
Middle T Antigen

Our results indicate that only one type of tyrosine kinase is present within each middle-T antigen-tyrosine kinase complex, suggesting that middle-T antigen forms separate complexes with different tyrosine kinases. Furthermore, we determined that there is only one molecule of middle-T antigen within any one of these complexes. We interpret this to mean that in any given cell, polyomavirus transformation involves, at least in part, the simultaneous deregulation of a number of separate pathways controlling cellular proliferation. Finally, we also demonstrate that the separate middle-T:pp60c-src and middle-T:pp59c-fyn complexes are each able to interact with the same cellular p81/85-kDa phosphoprotein, a possible component of the phosphatidylinositol kinase.

Genistein Inhibits Cell Proliferation and Induces Mitochondrial and Nuclear Alterations That Indicate Mechanisms Involved in Apoptosis

2000 ◽  
Vol 6 (S2) ◽  
pp. 850-851
Author(s):  
Geoffrey Gobert ◽  
Ed Curren ◽  
Wade Welshons ◽  
Qing-yuan Sun ◽  
Heide Schatten
Keyword(s):  
Breast Cancer ◽  
Cell Proliferation ◽  
Tyrosine Kinases ◽  
High Concentration ◽  
Transmission Electron ◽  
Low Concentrations ◽  
Culture Models ◽  
High Concentrations ◽  
Underlying Mechanisms ◽  
Nuclear Alterations

A highly significant correlation between reduced incidence of breast cancer in Asian countries and consumption of soy suggests that specific components in soy may have anticarcinogen activity. The soy ingredients genistein and daidzein have been found to inhibit induced breast tumors in animal and cell culture models. These isoflavones are known to be both agonists and antagonists of estrogen activity but only genistein is also a potent inhibitor of tyrosine kinases which are the primary intracellular signalling mechanisms associated with the regulation of cell proliferation.Genistein promotes cell proliferation in breast cancer cells at low concentrations in its function as estrogen agonist but inhibits cell proliferation at high concentrations (30 μM). In order to investigate the underlying mechanisms by which high concentration of genistein inhibit cell proliferation we treated MCF-7 cells with increasing concentrations of genistein and analyzed cells by immunofluorescence and transmission electron microscopy (TEM).

scholarly journals MicroRNA-122 supports robust innate immunity in hepatocytes by suppressing STAT3 phosphorylation

2018 ◽  
Author(s):  
Hui Xu ◽  
Shi-Jun Xu ◽  
Shu-Juan Xie ◽  
Yin Zhang ◽  
Jian-Hua Yang ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Tyrosine Kinases ◽  
Large Scale ◽  
Type I Interferons ◽  
Poly I:C ◽  
Hcv Rna ◽  
Type I ◽  
Type Iii ◽  
Stat3 Phosphorylation ◽  
Potential Strategy

ABSTRACTThe intrinsic innate immunity of hepatocytes is essential for the control of hepatitis viruses and influences the outcome of antiviral therapy. MicroRNA-122 (miR-122) is the most abundant microRNA in hepatocytes and is a central player in liver biology and disease. However, little is known about the role of miR-122 in hepatocyte innate immunity. Herein, we show that restoring miR-122 levels in hepatoma cells markedly increased the activation of both type III and type I interferons (IFNs) in response to hepatitis C virus (HCV) RNA or poly(I:C). We determined that miR-122 promotes IFN production through down-regulating the tyrosine (Tyr705) phosphorylation of STAT3. We show that STAT3 represses IFN activation by inhibiting interferon regulatory factor 1 (IRF1), which is rate-limiting for maximal IFN expression, especially type III IFNs. Through large-scale screening, we identified that miR-122 targets MERTK, FGFR1 and IGF1R, three oncogenic receptor tyrosine kinases that directly promote STAT3 phosphorylation. These findings reveal a previously unknown role for miR-122 in hepatic immunity and indicate a new potential strategy for treating hepatic infections through targeting STAT3.

Stoichiometry of cellular and viral components in the polyomavirus middle-T antigen-tyrosine kinase complex

1990 ◽  
Vol 10 (10) ◽  
pp. 5569-5574
Author(s):  
S H Cheng ◽  
P C Espino ◽  
J Marshall ◽  
R Harvey ◽  
A E Smith
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Cellular Proliferation ◽  
T Antigen ◽  
Phosphatidylinositol Kinase ◽  
Polyomavirus Middle T Antigen ◽  
Viral Components ◽  
Middle T Antigen

Our results indicate that only one type of tyrosine kinase is present within each middle-T antigen-tyrosine kinase complex, suggesting that middle-T antigen forms separate complexes with different tyrosine kinases. Furthermore, we determined that there is only one molecule of middle-T antigen within any one of these complexes. We interpret this to mean that in any given cell, polyomavirus transformation involves, at least in part, the simultaneous deregulation of a number of separate pathways controlling cellular proliferation. Finally, we also demonstrate that the separate middle-T:pp60c-src and middle-T:pp59c-fyn complexes are each able to interact with the same cellular p81/85-kDa phosphoprotein, a possible component of the phosphatidylinositol kinase.

Expression of Toll-like receptor 4 is associated with enteroviral replication in human myocarditis

2003 ◽  
Vol 104 (6) ◽  
pp. 577-584 ◽  
Author(s):  
Mamoru SATOH ◽  
Motoyuki NAKAMURA ◽  
Tomonari AKATSU ◽  
Junji IWASAKA ◽  
Yudai SHIMODA ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Systolic Function ◽  
Immunohistochemical Analysis ◽  
Minus Strand ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Tlr4 Mrna ◽  
Cellular Source ◽  
Active Viral Replication ◽  
Capsid Protein Vp1

Previous studies have demonstrated that inflammatory cytokine expression associated with enteroviral (EV) infection may play an important role in human myocarditis. However, the mechanism of the host immune response against viral pathogens has not been fully understood. The aim of the present study was to determine whether Toll-like receptor 4 (TLR4) and EV RNA are present in human myocarditis. Endomyocardial biopsy samples were obtained from 44 patients with myocarditis and five controls. Levels of plus- and minus-strand EV RNAs and TLR4 mRNA were measured by real-time reverse transcriptase–PCR. Immunohistochemical analysis was performed to identify the cellular source of TLR4 and the EV capsid protein VP1. EV RNA was present in 21 patients with myocarditis and these patients were defined as having either active viral replication (n=15) or latent viral persistence (n=6). Neither strand of EV RNA was detected in controls. TLR4 mRNA expression levels were higher in myocarditis patients than in controls (TLR4/glyceraldehyde-3-phosphate dehydrogenase ratio 1.48±0.17 compared with 0.08±0.06, P<0.001). A positive correlation was found between EV RNA and TLR4 levels (plus-strand vs TLR4: r=0.66, P<0.001; minus-strand vs TLR4: r=0.48, P<0.001). TLR4 immunostaining was observed in infiltrating cells and myocytes in patients with myocarditis. The EV capsid protein VP1 was also found in myocytes. The myocarditis group with EV replication and high levels of TLR4 showed significantly lower systolic function. The present study has shown that increased expression of TLR4 is associated with EV replication and that these RNA levels are related to cardiac dysfunction in human myocarditis.

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