Overexpression of AGR2vH, an oncogenic AGR2 spliced transcript, potentiates tumorigenicity and proteomic alterations in cholangiocarcinoma cell

2021 ◽  
Author(s):  
Juthamas Yosudjai ◽  
Chaturong Inpad ◽  
Phattarin Pothipan ◽  
Saowaluk Saisomboon ◽  
Damrasamon Surangkul ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Pathway Activation ◽  
Tumorigenic Potential ◽  
Cholangiocarcinoma Cell ◽  
Overexpressing Cell ◽  
Xenograft Mice ◽  
Cell Proteome

ABSTRACT The upregulation of Anterior gradient 2 (AGR2) has been observed in cholangiocarcinoma (CCA) cells, nras-mutant zebrafish and specimens derived from CCA patients. Our previous study reported AGR2 splicing into AGR2vH to facilitate CCA cell aggressiveness, while this work aims to investigate the molecular mechanisms underlying AGR2vH. Firstly, AGR2vH upregulation was demonstrated in CCA tissues derived from patients. For in vitro studies, established AGR2vH-overexpressing KKU-213A cells were found to exhibit increased proliferation and clonogenicity. In vivo tumorigenicity assessed in a mouse model represented higher tumorigenic potential in AGR2vH-overexpressing cell xenograft mice. Next, LC-MS/MS was analyzed, and indicating that AGR2vH may be associated with CCA cell proliferation via Wnt/β-catenin signaling pathway activation, which was verified by β-catenin expression and nuclear translocation. The current results provide evidence that AGR2vH upregulation promotes tumorigenicity in CCA cells linked with an alteration of CCA cell proteome.

Overexpression of oncogenic spliced-AGR2vH potentiates cholangiocarcinoma cell tumorigenicity with proteomic alterations

2021 ◽  
Author(s):  
Juthamas Yosudjai ◽  
Chaturong Inpad ◽  
Damrasamon Surangkul ◽  
Suchada Phimsen ◽  
Saowaluk Saisomboon ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Liquid Chromatography Mass Spectrometry ◽  
Tumor Tissues ◽  
Tumorigenic Potential ◽  
Cell Metastasis ◽  
Cholangiocarcinoma Cell ◽  
Xenograft Mice ◽  
Cell Proteome

Abstract Upregulated expression of Anterior gradient 2 (AGR2) has been observed in cells, highly metastatic mouse models, nras-mutant zebrafish and cholangiocarcinoma (CCA) specimens derived from patients. Our previous study reported that AGR2 splicing into AGR2vH can promote CCA cell metastasis and survivability. This present study aimed to investigate the molecular mechanisms underlying AGR2vH tumorigenicity in vitro and in vivo. AGR2vH was determined in patient tissues and presented the upregulation in CCA tumor tissues compared with matched-normal adjacent tissues. During the in vitro studies, AGR2vH was ectopically overexpressed in KKU-213A cells. Established AGR2vH-overexpressing CCA cells were found to exhibit increased proliferative and clonogenic ability. For in vivo tumorigenicity, a higher tumorigenic potential was identified in AGR2vH-overexpressing cells xenograft mice. Moreover, liquid chromatography-mass spectrometry with protein bioinformatics was used to examine the proteomic alteration. The CCA cell proteome was altered, and it was indicated that AGR2vH may be associated with CCA cell proliferation via the activation of Wnt/β-catenin signaling pathway, which was verified via the comparative immunoblotting of β-catenin in cytoplasmic and nuclear fractionated proteins. These present results provided evidence that the upregulation of AGR2vH promotes the tumorigenicity of CCA cells, which was associated with an alteration of the CCA cell proteome.

Role of the ligand in intracellular receptor function: receptor affinity determines activation in vitro of the latent dioxin receptor to a DNA-binding form

1991 ◽  
Vol 11 (1) ◽  
pp. 401-411
Author(s):  
S Cuthill ◽  
A Wilhelmsson ◽  
L Poellinger
Keyword(s):  
Dna Binding ◽  
Cellular Response ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Rank Order ◽  
Receptor Ligands ◽  
Free System ◽  
Dioxin Receptor

To reconstitute the molecular mechanisms underlying the cellular response to soluble receptor ligands, we have exploited a cell-free system that exhibits signal- (dioxin-)induced activation of the latent cytosolic dioxin receptor to an active DNA-binding species. The DNA-binding properties of the in vitro-activated form were qualitatively indistinguishable from those of in vivo-activated nuclear receptor extracted from dioxin-treated cells. In vitro activation of the receptor by dioxin was dose dependent and was mimicked by other dioxin receptor ligands in a manner that followed the rank order of their relative affinities for the receptor in vitro and their relative potencies to induce target gene transcription in vivo. Thus, in addition to triggering the initial release of inhibition of DNA binding and presumably allowing nuclear translocation, the ligand appears to play a crucial role in the direct control of the level of functional activity of a given ligand-receptor complex.

scholarly journals The PPARα pathway in Vγ9Vδ2 T cell anergy

2014 ◽  
Vol 19 (4) ◽  
Author(s):  
Mary Poupot ◽  
Frédéric Boissard ◽  
Delphine Betous ◽  
Laure Bardouillet ◽  
Séverine Fruchon ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Molecular Mechanisms ◽  
T Cell Response ◽  
T Cell Anergy ◽  
Pathway Activation ◽  
In Vitro Response ◽  
Vγ9vδ2 T Cells

AbstractPhosphoantigens (PAgs) activate Vγ9Vδ2 T lymphocytes, inducing their potent and rapid response in vitro and in vivo. However, humans and nonhuman primates that receive repeated injections of PAgs progressively lose their Vγ9Vδ2 T cell response to them. To elucidate the molecular mechanisms of this in vivo desensitization, we analyzed the transcriptome of circulating Vγ9Vδ2 T cells from macaques injected with PAg. We showed that three PAg injections induced the activation of the PPARα pathway in Vγ9Vδ2 T cells. Thus, we analyzed the in vitro response of Vγ9Vδ2 T cells stimulated with a PPARα agonist. We demonstrated that in vitro PPARα pathway activation led to the inhibition of the BrHPP-induced activation and proliferation of human Vγ9Vδ2 T cells. Since the PPARα pathway is involved in the antigen-selective desensitization of human Vγ9Vδ2 T cells, the use of PPARα inhibitors could enhance cancer immunotherapy based on Vγ9Vδ2 T cells.

scholarly journals Real-Time Analysis of Endogenous Wnt Signalling in 3D Mesenchymal Stromal Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Fatima Saleh ◽  
Alice Carstairs ◽  
S. Leah Etheridge ◽  
Paul Genever
Keyword(s):  
Time Course ◽  
Molecular Mechanisms ◽  
Adipogenic Differentiation ◽  
Wnt Signalling ◽  
Egfp Expression ◽  
Culture Techniques ◽  
Real Time Analysis ◽  
Pathway Activation

Wnt signalling has been implicated in the regulation of stem cell self-renewal and differentiation; however, the majority of in vitro studies are carried out using monolayer 2D culture techniques. Here, we used mesenchymal stromal cell (MSC) EGFP reporter lines responsive to Wnt pathway activation in a 3D spheroid culture system to mimic better the in vivo environment. Endogenous Wnt signalling was then investigated under basal conditions and when MSCs were induced to undergo osteogenic and adipogenic differentiation. Interestingly, endogenous Wnt signalling was only active during 3D differentiation whereas 2D cultures showed no EGFP expression throughout an extended differentiation time-course. Furthermore, exogenous Wnt signalling in 3D adipogenic conditions inhibited differentiation compared to unstimulated controls. In addition, suppressing Wnt signalling by Dkk-1 restored and facilitated adipogenic differentiation in MSC spheroids. Our findings indicate that endogenous Wnt signalling is active and can be tracked in 3D MSC cultures where it may act as a molecular switch in adipogenesis. The identification of the signalling pathways that regulate MSCs in a 3D in vivo-like environment will advance our understanding of the molecular mechanisms that control MSC fate.

scholarly journals TIE2 Associates with Caveolae and Regulates Caveolin-1 To Promote Their Nuclear Translocation

2017 ◽  
Vol 37 (21) ◽  
Author(s):  
Mohammad B. Hossain ◽  
Rehnuma Shifat ◽  
Jingyi Li ◽  
Xuemei Luo ◽  
Kenneth R. Hess ◽  
...  
Keyword(s):  
Dna Repair ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Subcellular Fractionation ◽  
Tyrosine Kinase Receptor ◽  
Histone H4 ◽  
Small Interfering Rnas ◽  
Caveolin 1

ABSTRACT DNA repair pathways are aberrant in cancer, enabling tumor cells to survive standard therapies—chemotherapy and radiotherapy. Our group previously reported that, upon irradiation, the membrane-bound tyrosine kinase receptor TIE2 translocates into the nucleus and phosphorylates histone H4 at Tyr51, recruiting ABL1 to the DNA repair complexes that participate in the nonhomologous end-joining pathway. However, no specific molecular mechanisms of TIE2 endocytosis have been reported. Here, we show that irradiation or ligand-induced TIE2 trafficking is dependent on caveolin-1, the main component of caveolae. Subcellular fractionation and confocal microscopy demonstrated TIE2/caveolin-1 complexes in the nucleus, and using inhibitor or small interfering RNAs (siRNAs) against caveolin-1 or Tie2 inhibited their trafficking. TIE2 was found in caveolae and directly phosphorylated caveolin-1 at Tyr14 in vitro and in vivo. This modification regulated the generation of TIE2/caveolin-1 complexes and was essential for TIE2/caveolin-1 nuclear translocation. Our data further demonstrate that the combination of TIE2 and caveolin-1 inhibitors resulted in significant radiosensitization of malignant glioma cells, which will guide the development of combinatorial treatment with radiotherapy for patients with glioblastoma.

scholarly journals CSIG-15. INHIBITION OF THE bHLH TRANSCRIPTIONAL NETWORKS BY A MUTATED E47 PROTEIN LEADS TO A STRONG ANTI-GLIOMA ACTIVITY IN VITRO AND IN VIVO

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi47-vi47
Author(s):  
Marilin Koch ◽  
Stefan Czemmel ◽  
Felix Lennartz ◽  
Sarah Beyeler ◽  
Justyna Przystal ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Clonogenic Survival ◽  
Dominant Negative ◽  
Molecular Profile ◽  
Transcriptional Networks ◽  
Molecular Alterations ◽  
Helix Loop Helix

Abstract OBJECTIVE The transcription factor E47 heterodimerizes with helix-loop-helix (HLH) and basic helix-loop-helix transcription (bHLH) factors like ID-1 and Olig2 that are overexpressed in glioblastoma. A dominant-negative variant of the E47 (dnE47) lacking the nuclear translocation signal, leads to cytoplasmatic sequestration of HLH and bHLH transcription factors. Here, we investigated combinations of dnE47-mediated inhibition of the bHLH transcriptional network with temozolomide and irradiation and explored the underlying molecular mechanisms. METHODS Long-term and stem cell glioma lines were transduced with a Doxycycline-inducible dnE47 lentivirus. Functional characterizations included immunocytochemistry, immunoblots, cytotoxicity and clonogenicity assays in vitro and latency until the onset of symptoms in vivo. CAGE and RNASeq were conducted for analyzing the dnE47-induced molecular profile. RESULTS The induction of dnE47 led to cytoplasmatic sequestration of HLH/bHLH transcription, reduced proliferation, increased cytotoxicity and reduced clonogenic survival in vitro and a prolonged latency until the onset of neurological symptoms in vivo. CAGE and RNASeq data revealed alterations in several cancer-relevant pathways. CONCLUSIONS A dnE47-mediated inhibition of the bHLH transcription network induced actionable molecular alterations in glioma cells that could be exploited for the design of novel therapies.

β2-AR Activation promotes cleavage and nuclear translocation of Her2 and metastatic potential of cancer cells

2020 ◽  
Author(s):  
Dan Liu ◽  
Xiyue Xu ◽  
Shuci Liu ◽  
Xuan Zhao ◽  
Anqun Tang ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Signaling Pathways ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Biological Response ◽  
Metastatic Potential ◽  
Adrenergic Signaling ◽  
Signaling Activation

Abstract Background The prolonged hypersecretion of catecholamine induced by chronic stress may correlate with various steps of malignant progression of cancer and β2-AR overexpressed in certain cancer cells may translate the signals from neuroendocrine system to malignant signals by interacting with oncoproteins such as Her2. Crosstalk of the cell signaling pathways mediated by β2-AR and Her2 may promote a stronger or more sustained biological response. However, the molecular mechanisms underlying cross-communication between β2-AR and Her2 mediated signaling pathways are not fully understood. Methods In this study, the effects of adrenergic signaling on Her2 cleavage were evaluated by various assays, such as western blot, immunofluorescence and immunohistochemistry. In order to reveal the mechanism about Her2 cleavage triggered by β2-AR activation, the molecular and pharmacological means were employed. By using in vitro and in vivo assay, the influences of the crosstalk between β2-AR and Her2 on the bio-behaviors of tumor cells were demonstrated. Results Our data demonstrate that catecholamine stimulation activates the expression and proteolytic activity of ADAM10 by modulating the expression of miR-199a-5p and SIRT1 and also confirm that catecholamine induction triggers the activities of γ-secretase, leading to shedding of Her2 ECD by ADAM10 and subsequent intramembranous cleavage of Her2 ICD by presenilin-dependent γ-secretase, nuclear translocation of Her2 ICD and enhanced transcription of tumor metastasis-associated gene COX-2 . Chronic stimulation of catecholamine strongly promotes the invasive activities of cancer cells in vitro and spontaneous tumor lung metastasis in mice. Furthermore, the nuclear localization of Her2 was significantly correlated with overexpression of β2-AR in human breast cancer tissues. Conclusion This study illustrates that adrenergic signaling activation triggers Her2 cleavage, resulting in enhanced invasive and metastasis activities of cancer cells. Our data also reveal that an unknown mechanism by which the regulated intramembrane proteolysis (RIP) initiated by β2-AR activation controls a novel Her2-mediated signaling transduction under physiological and pathological conditions.

scholarly journals Role of the ligand in intracellular receptor function: receptor affinity determines activation in vitro of the latent dioxin receptor to a DNA-binding form.

1991 ◽  
Vol 11 (1) ◽  
pp. 401-411 ◽  
Author(s):  
S Cuthill ◽  
A Wilhelmsson ◽  
L Poellinger
Keyword(s):  
Dna Binding ◽  
Cellular Response ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Rank Order ◽  
Receptor Ligands ◽  
Free System ◽  
Dioxin Receptor

To reconstitute the molecular mechanisms underlying the cellular response to soluble receptor ligands, we have exploited a cell-free system that exhibits signal- (dioxin-)induced activation of the latent cytosolic dioxin receptor to an active DNA-binding species. The DNA-binding properties of the in vitro-activated form were qualitatively indistinguishable from those of in vivo-activated nuclear receptor extracted from dioxin-treated cells. In vitro activation of the receptor by dioxin was dose dependent and was mimicked by other dioxin receptor ligands in a manner that followed the rank order of their relative affinities for the receptor in vitro and their relative potencies to induce target gene transcription in vivo. Thus, in addition to triggering the initial release of inhibition of DNA binding and presumably allowing nuclear translocation, the ligand appears to play a crucial role in the direct control of the level of functional activity of a given ligand-receptor complex.

scholarly journals The Protective Roles and Molecular Mechanisms of Troxerutin (Vitamin P4) for the Treatment of Chronic Diseases: A Mechanistic Review

2020 ◽  
Vol 19 (1) ◽  
pp. 97-110
Author(s):  
Mohammad Zamanian ◽  
Gholamreza Bazmandegan ◽  
Antoni Sureda ◽  
Eduardo Sobarzo-Sanchez ◽  
Hasan Yousefi-Manesh ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Therapeutic Potential ◽  
Acetylcholinesterase Activity ◽  
Nuclear Factor Κb ◽  
In Vivo Studies ◽  
Related Factor ◽  
Nuclear Factor

: Troxerutin (TRX), a semi-synthetic bioflavonoid derived from rutin, has been reported to exert several pharmacological effects including antioxidant, anti-inflammatory, antihyperlipidemic, and nephroprotective. However, the related molecular details and its mechanisms remain poorly understood. In the present review, we presented evidences from the diversity in vitro and in vivo studies on the therapeutic potential of TRX against neurodegenerative, diabetes, cancer and cardiovascular diseases with the purpose to find molecular pathways related to the treatment efficacy. TRX has a beneficial role in many diseases through multiple mechanisms including, increasing antioxidant enzymes and reducing oxidative damage, decreasing in proapoptotic proteins (APAF-1, BAX, caspases-9 and-3) and increasing the antiapoptotic BCL-2, increasing the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and downregulating the nuclear factor κB (NFκ). TRX also reduces acetylcholinesterase activity and upregulates phosphoinositide 3- kinase/Akt signaling pathway in Alzheimer’s disease models. Natural products such as TRX may develop numerous and intracellular pathways at several steps in the treatment of many diseases. Molecular mechanisms of action are revealing novel, possible combinational beneficial approaches to treat multiple pathological conditions.

scholarly journals Activation of the clock gene TIMELESS by H3k27 acetylation promotes colorectal cancer tumorigenesis by binding to Myosin-9

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Meng Cao ◽  
Yi Wang ◽  
Yijing Xiao ◽  
Dandan Zheng ◽  
Chunchun Zhi ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Clock Gene ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Clock Genes ◽  
Tumor Development ◽  
Creb Binding Protein ◽  
Functional Studies

Abstract Background Colorectal cancer (CRC) is a common tumor characterized by its high mortality. However, the underlying molecular mechanisms that drive CRC tumorigenesis are unclear. Clock genes have important roles in tumor development. In the present study, the expression and functions of clock gene TIMELESS (encoding the Timeless protein) in CRC were investigated. Methods Immunohistochemistry, cell proliferation, migration, invasion, EMT and xenograft tumor experiments were used to prove the function of Timeless in the tumorigenesis of CRC. Immunoprecipitation, mass spectrometry, Immunofluorescence and Chromatin immunoprecipitation (ChIP) were utilized to clarify the mechanism of Timeless in regulating CRC tumorigenesis. Results We found that Timeless was upregulated in CRC tissues compared with corresponding normal tissues and its expression was closely associated with the TNM stages and overall survival of CRC patients. Functional studies demonstrated that Timeless promoted the proliferation, invasion, and EMT of CRC cells in vitro and in vivo. Mechanistic investigations showed that Timeless activated the β-catenin signal pathway by binding to Myosin-9, which binds to β-catenin to induce its nuclear translocation. The upregulation of Timeless was attributed to CREB-binding protein (CBP)/p300-mediated H3K27 acetylation of the promoter region of Timeless. Conclusion Timeless regulates the tumorigenesis of CRC by binding to and regulating myosin-9, suggesting Timeless might be a potential prognostic biomarker and therapeutic target for CRC.

Sign in / Sign up

Export Citation Format

Share Document