Wilms’ Tumor Gene 1 (WT1) Silencing Inhibits Proliferation of Malignant Peripheral Nerve Sheath Tumor sNF96.2 Cell Line

Wilms’ tumor gene 1 (WT1) plays complex roles in tumorigenesis, acting as tumor suppressor gene or an oncogene depending on the cellular context. WT1 expression has been variably reported in both benign and malignant peripheral nerve sheath tumors (MPNSTs) by means of immunohistochemistry. The aim of the present study was to characterize its potential pathogenetic role in these relatively uncommon malignant tumors. Firstly, immunohistochemical analyses in MPNST sNF96.2 cell line showed strong WT1 staining in nuclear and perinuclear areas of neoplastic cells. Thus, we investigated the effects of silencing WT1 by RNA interference. Through Western Blot analysis and proliferation assay we found that WT1 knockdown leads to the reduction of cell growth in a time- and dose-dependent manner. siWT1 inhibited proliferation of sNF96.2 cell lines likely by influencing cell cycle progression through a decrease in the protein levels of cyclin D1 and inhibition of Akt phosphorylation compared to the control cells. These results indicate that WT1 knockdown attenuates the biological behavior of MPNST cells by decreasing Akt activity, demonstrating that WT1 is involved in the development and progression of MPNSTs. Thus, WT1 is suggested to serve as a potential therapeutic target for MPNSTs.     

The Role of Diacylglycerol Kinase ζ and Phosphatidic Acid in the Mechanical Activation of Mammalian Target of Rapamycin (mTOR) Signaling and Skeletal Muscle Hypertrophy

The activation of mTOR signaling is essential for mechanically induced changes in skeletal muscle mass, and previous studies have suggested that mechanical stimuli activate mTOR (mammalian target of rapamycin) signaling through a phospholipase D (PLD)-dependent increase in the concentration of phosphatidic acid (PA). Consistent with this conclusion, we obtained evidence which further suggests that mechanical stimuli utilize PA as a direct upstream activator of mTOR signaling. Unexpectedly though, we found that the activation of PLD is not necessary for the mechanically induced increases in PA or mTOR signaling. Motivated by this observation, we performed experiments that were aimed at identifying the enzyme(s) that promotes the increase in PA. These experiments revealed that mechanical stimulation increases the concentration of diacylglycerol (DAG) and the activity of DAG kinases (DGKs) in membranous structures. Furthermore, using knock-out mice, we determined that the ζ isoform of DGK (DGKζ) is necessary for the mechanically induced increase in PA. We also determined that DGKζ significantly contributes to the mechanical activation of mTOR signaling, and this is likely driven by an enhanced binding of PA to mTOR. Last, we found that the overexpression of DGKζ is sufficient to induce muscle fiber hypertrophy through an mTOR-dependent mechanism, and this event requires DGKζ kinase activity (i.e. the synthesis of PA). Combined, these results indicate that DGKζ, but not PLD, plays an important role in mechanically induced increases in PA and mTOR signaling. Furthermore, this study suggests that DGKζ could be a fundamental component of the mechanism(s) through which mechanical stimuli regulate skeletal muscle mass.     

Akt-dependent Activation of mTORC1 Complex Involves Phosphorylation of mTOR (Mammalian Target of Rapamycin) by IκB Kinase α (IKKα)

The serine/threonine protein kinase Akt promotes cell survival, growth, and proliferation through phosphorylation of different downstream substrates. A key effector of Akt is the mammalian target of rapamycin (mTOR). Akt is known to stimulate mTORC1 activity through phosphorylation of tuberous sclerosis complex 2 (TSC2) and PRAS40, both negative regulators of mTOR activity. We previously reported that IκB kinase α (IKKα), a component of the kinase complex that leads to NF-κB activation, plays an important role in promoting mTORC1 activity downstream of activated Akt. Here, we demonstrate IKKα-dependent regulation of mTORC1 using multiple PTEN null cancer cell lines and an animal model with deletion of IKKα. Importantly, IKKα is shown to phosphorylate mTOR at serine 1415 in a manner dependent on Akt to promote mTORC1 activity. These results demonstrate that IKKα is an effector of Akt in promoting mTORC1 activity.     

PPAR Gamma Coactivator 1 Beta (PGC-1β) Reduces Mammalian Target of Rapamycin (mTOR) Expression via a SIRT1-Dependent Mechanism in Neurons

Mammalian target of rapamycin (mTOR) is a key regulator of metabolism, cell growth, and protein synthesis. Since decreased mTOR activity has been found to slow aging in many species, the aim of this study was to examine the activity of mTOR and its phosphorylated form in in vitro and in vivo models mimicking Alzheimer’s disease (AD), and investigate the potential pathway of PGC-1β in regulating mTOR expression. Primary neurons and N2a cells were treated with Aβ_25–35, while untreated cells served as controls. The expression of mTOR, p-mTOR (Ser2448), and PGC-1β was determined with Western blotting and RT-PCR assay, and the translocation of mTOR was detected using confocal microscopy. Aβ_25–35 treatment stimulated the translocation of mTOR from cytoplasm to nucleus, and resulted in elevated expression of mTOR and p-mTOR (Ser2448) and reduced PGC-1β expression. In addition, overexpression of PGC-1β was found to decrease mTOR expression. The results of this study demonstrate that Aβ increases the expression of mTOR and p-mTOR at the site of Ser2448, and the stimulation of Aβ is likely to depend on sirtuin 1, PPARγ, and PGC-1β pathway in regulating mTOR expression.     

Glucose-induced gradual phenotypic modulation of cultured human glomerular epithelial cells may be independent of Wilms’ tumor 1 (WT1)

Background

Melatonin, a hormone-like substance involved in the regulation of the circadian rhythm, has been demonstrated to protect cells against oxidative DNA damage and to inhibit tumorigenesis.

Results

In the current study, we investigated the effect of melatonin on DNA strand breaks using the alkaline DNA comet assay in breast cancer (MCF-7) and colon cancer (HCT-15) cell lines. Our results demonstrated that cells pretreated with melatonin had significantly shorter Olive tail moments compared to non-melatonin treated cells upon mutagen (methyl methanesulfonate, MMS) exposure, indicating an increased DNA repair capacity after melatonin treatment. We further examined the genome-wide gene expression in melatonin pretreated MCF-7 cells upon carcinogen exposure and detected altered expression of many genes involved in multiple DNA damage responsive pathways. Genes exhibiting altered expression were further analyzed for functional interrelatedness using network- and pathway-based bioinformatics analysis. The top functional network was defined as having relevance for “DNA Replication, Recombination, and Repair, Gene Expression, [and] Cancer”.

Conclusions

These findings suggest that melatonin may enhance DNA repair capacity by affecting several key genes involved in DNA damage responsive pathways.     

Mammalian Target of Rapamycin (mTOR) Tagging Promotes Dendritic Branch Variability through the Capture of Ca2+/Calmodulin-dependent Protein Kinase II α (CaMKIIα) mRNAs by the RNA-binding Protein HuD

The fate of a memory, whether stored or forgotten, is determined by the ability of an active or tagged synapse to undergo changes in synaptic efficacy requiring protein synthesis of plasticity-related proteins. A synapse can be tagged, but without the “capture” of plasticity-related proteins, it will not undergo long lasting forms of plasticity (synaptic tagging and capture hypothesis). What the “tag” is and how plasticity-related proteins are captured at tagged synapses are unknown. Ca²⁺/calmodulin-dependent protein kinase II α (CaMKIIα) is critical in learning and memory and is synthesized locally in neuronal dendrites. The mechanistic (mammalian) target of rapamycin (mTOR) is a protein kinase that increases CaMKIIα protein expression; however, the mechanism and site of dendritic expression are unknown. Herein, we show that mTOR activity mediates the branch-specific expression of CaMKIIα, favoring one secondary, daughter branch over the other in a single neuron. mTOR inhibition decreased the dendritic levels of CaMKIIα protein and mRNA by shortening its poly(A) tail. Overexpression of the RNA-stabilizing protein HuD increased CaMKIIα protein levels and preserved its selective expression in one daughter branch over the other when mTOR was inhibited. Unexpectedly, deleting the third RNA recognition motif of HuD, the domain that binds the poly(A) tail, eliminated the branch-specific expression of CaMKIIα when mTOR was active. These results provide a model for one molecular mechanism that may underlie the synaptic tagging and capture hypothesis where mTOR is the tag, preventing deadenylation of CaMKIIα mRNA, whereas HuD captures and promotes its expression in a branch-specific manner.     

Muscle Wasting in Fasting Requires Activation of NF-κB and Inhibition of AKT/Mechanistic Target of Rapamycin (mTOR) by the Protein Acetylase,GCN5

NF-κB is best known for its pro-inflammatory and anti-apoptotic actions, but in skeletal muscle, NF-κB activation is important for atrophy upon denervation or cancer. Here, we show that also upon fasting, NF-κB becomes activated in muscle and is critical for the subsequent atrophy. Following food deprivation, the expression and acetylation of the p65 of NF-κB on lysine 310 increase markedly in muscles. NF-κB inhibition in mouse muscles by overexpression of the IκBα superrepressor (IκBα-SR) or of p65 mutated at Lys-310 prevented atrophy. Knockdown of GCN5 with shRNA or a dominant-negative GCN5 or overexpression of SIRT1 decreased p65K310 acetylation and muscle wasting upon starvation. In addition to reducing atrogene expression, surprisingly inhibiting NF-κB with IκBα-SR or by GCN5 knockdown in these muscles also enhanced AKT and mechanistic target of rapamycin (mTOR) activities, which also contributed to the reduction in atrophy. These new roles of NF-κB and GCN5 in regulating muscle proteolysis and AKT/mTOR signaling suggest novel approaches to combat muscle wasting.     

Tumor necrosis factor-alpha (TNF-α)-mediated in vitro human retinal pigment epithelial (RPE) cell migration mainly requires Akt/mTOR complex 1 (mTORC1), but not mTOR complex 2 (mTORC2) signaling

When rhegmatogenous retinal detachment occurs, tumor necrosis factor-alpha (TNF-α) among other cytokines leaks into the subretinal space, induces resident retinal pigment epithelial (RPE) cells to migrate, which is the initial step of proliferative vitreoretinopathy (PVR). In the current study, we aim to understand how this is regulated by focusing the cellular mechanisms involved. Here we identified an Akt/Tuberous sclerosis protein 2 (TSC2)/mTOR complex1 (mTORC1) signaling pathway after TNF-α treatment to mediate RPE cell migration. Suppression of mTORC1 activation, either by its inhibitor rapamycin, or by activation of its suppressor AMP activated protein kinase (AMPK), inhibited TNF-α-mediated RPE cell migration, while RNA interference (RNAi)-mediated knocking-down of SIN1 or Rictor, two key components of mTOR complex 2 (mTORC2), had no significant effect on TNF-α-induced RPE cell migration. Our data provide initial evidence that TNF-α-mediated in vitro RPE cell migration mainly requires Akt/mTORC1, but not mTORC2 signaling. The results of this study may lead to indentify novel signaling targets against PVR.     

Strain-induced Differentiation of Fetal Type II Epithelial Cells Is Mediated via the Integrin α6β1-ADAM17/Tumor Necrosis Factor-α-converting Enzyme (TACE) Signaling Pathway

Mechanical forces are critical for normal fetal lung development. However, the mechanisms regulating this process are not well-characterized. We hypothesized that strain-induced release of HB-EGF and TGF-α is mediated via integrin-ADAM17/TACE interactions. Employing an in vitro system to simulate mechanical forces in fetal lung development, we showed that mechanical strain of fetal epithelial cells actives TACE, releases HB-EGF and TGF-α, and promotes differentiation. In contrast, in samples incubated with the TACE inhibitor IC-3 or in cells isolated from TACE knock-out mice, mechanical strain did not release ligands or promote cell differentiation, which were both rescued after transfection of ADAM17. Cell adhesion assay and co-immunoprecipitation experiments in wild-type and TACE knock-out cells using several TACE constructs demonstrated not only that integrins α6 and β1 bind to TACE via the disintegrin domain but also that mechanical strain enhances these interactions. Furthermore, force applied to these integrin receptors by magnetic beads activated TACE and shed HB-EGF and TGF-α. The contribution of integrins α₆ and β₁ to differentiation of fetal epithelial cells by strain was demonstrated by blocking their binding site with specific antibodies and by culturing the cells on membranes coated with anti-integrin α₆ and β₁ antibodies. In conclusion, mechanical strain releases HB-EGF and TGF-α and promotes fetal type II cell differentiation via α₆β₁ integrin-ADAM17/TACE signaling pathway. These investigations provide novel mechanistic information on how mechanical forces promote fetal lung development and specifically differentiation of epithelial cells. This information could be also relevant to other tissues exposed to mechanical forces.     

Transforming Growth Factor ��-activated Kinase 1 (TAK1) Kinase Adaptor, TAK1-binding Protein 2, Plays Dual Roles in TAK1 Signaling by Recruiting Both an Activator and an Inhibitor of TAK1 Kinase in Tumor Necrosis Factor Signaling Pathway

Transforming growth factor β-activated kinase 1 (TAK1) kinase is an indispensable signaling intermediate in tumor necrosis factor (TNF), interleukin 1, and Toll-like receptor signaling pathways. TAK1-binding protein 2 (TAB2) and its closely related protein, TAB3, are binding partners of TAK1 and have previously been identified as adaptors of TAK1 that recruit TAK1 to a TNF receptor signaling complex. TAB2 and TAB3 redundantly mediate activation of TAK1. In this study, we investigated the role of TAB2 by analyzing fibroblasts having targeted deletion of the tab2 gene. In TAB2-deficient fibroblasts, TAK1 was associated with TAB3 and was activated following TNF stimulation. However, TAB2-deficient fibroblasts displayed a significantly prolonged activation of TAK1 compared with wild type control cells. This suggests that TAB2 mediates deactivation of TAK1. We found that a TAK1-negative regulator, protein phosphatase 6 (PP6), was recruited to the TAK1 complex in wild type but not in TAB2-deficient fibroblasts. Furthermore, we demonstrated that both PP6 and TAB2 interacted with the polyubiquitin chains and this interaction mediated the assembly with TAK1. Our results indicate that TAB2 not only activates TAK1 but also plays an essential role in the deactivation of TAK1 by recruiting PP6 through a polyubiquitin chain-dependent mechanism.     

Prediction of an HLA-DR-binding peptide derived from Wilms’ tumour 1 protein and demonstration of in vitro immunogenicity of WT1(124–138)-pulsed dendritic cells generated according to an optimised protocol

The Wilms' tumour 1 (WT1) protein is over-expressed in several types of cancer including leukaemias and might therefore constitute a novel target for immunotherapy. Recently, human leucocyte antigen (HLA) class I-binding WT1 peptides have been identified and shown to stimulate CD8(+) T cells in vitro. For maximal CD8 cell efficacy, CD4(+) helper T cells responding to major histocompatibility complex (MHC) class II-binding epitopes are required. Here, we report that scanning the WT1 protein sequence using an evidence-based predictive computer algorithm (SYFPEITHI) yielded a peptide WT1(124-138) predicted to bind the HLA-DRB1*0401 molecule with high affinity. Moreover, synthetic WT1(124-138)-peptide-pulsed dendritic cells (DC), generated according to a protocol optimised in the present study, sensitised T cells in vitro to proliferate and secrete interferon-gamma (IFN-gamma) when rechallenged with specific peptide-pulsed DC, but not with peripheral blood mononuclear cells (PBMC). These results suggest that the WT1 protein may yield epitopes immunogenic to CD4 as well as CD8 T cells, and therefore constitute a novel potential target for specific immunotherapy.     

HIV-1 Tat Promotes Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) vIL-6-Induced Angiogenesis and Tumorigenesis by Regulating PI3K/PTEN/AKT/GSK-3β Signaling Pathway

Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) is etiologically associated with KS, the most common AIDS-related malignancy. KS is characterized by vast angiogenesis and hyperproliferative spindle cells. We have previously reported that HIV-1 Tat can trigger KSHV reactivation and accelerate Kaposin A-induced tumorigenesis. Here, we explored Tat promotion of KSHV vIL-6-induced angiogenesis and tumorigenesis. Tat promotes vIL-6-induced cell proliferation, cellular transformation, vascular tube formation and VEGF production in culture. Tat enhances vIL-6-induced angiogenesis and tumorigenesis of fibroblasts and human endothelial cells in a chicken chorioallantoic membrane (CAM) model. In an allograft model, Tat promotes vIL-6-induced tumorigenesis and expression of CD31, CD34, SMA, VEGF, b-FGF, and cyclin D1. Mechanistic studies indicated Tat activates PI3K and AKT, and inactivates PTEN and GSK-3β in vIL-6 expressing cells. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a specific inhibitor of PI3K, effectively impaired Tat’s promotion of vIL-6-induced tumorigenesis. Together, these results provide the first evidence that Tat might contribute to KS pathogenesis by synergizing with vIL-6, and identify PI3K/AKT pathway as a potential therapeutic target in AIDS-related KS patients.     

Binding of Galectin-3, a β-Galactoside-binding Lectin,to MUC1 Protein Enhances Phosphorylation of Extracellular Signal-regulated Kinase 1/2 (ERK1/2) and Akt,Promoting Tumor Cell Malignancy

Both mucin 1 (MUC1) and galectin-3 are known to be overexpressed in various malignant tumors and associated with a poor prognosis. It has been extensively reported that MUC1 is involved in potentiation of growth factor-dependent signal transduction. Because some carbohydrate moieties carried on MUC1 change to preferable ones for binding of galectin-3 in cancer cells, we speculated that MUC1-mediated signaling may occur through direct binding of galectin-3. Immunochemical studies showed that the distribution of galectin-3 coincided with that of MUC1 in various human tumor tissues but not in human nonmalignant tissues, and the level of galectin-3 retained on the surface of various cancer cells paralleled that of MUC1. Treatment of MUC1-expressing cells with galectin-3 induced phosphorylation of ERK1/2 and Akt following enhanced phosphorylation of MUC1 C-terminal domain, consistently promoting tumor cell malignancy. It is also noted that this enhanced phosphorylation occurred independently of EGF receptor-mediated signaling in both EGF receptor- and MUC1-expressing cells, and multivalency of galectin-3 was important for initiation of MUC1-mediated signaling. Expectedly, both silencing of endogenous galectin-3 and treatment with galectin-3 antagonists down-regulated cell proliferation of MUC1-expressing cells. These results suggest that the binding of galectin-3 to MUC1 plays a key role in MUC1-mediated signaling. Thus, constitutive activation of MUC1-mediated signaling in an autocrine/paracrine manner caused by ligation of galectin-3 promotes uncontrolled tumor cell malignancy. This signaling may be another MUC1-mediated pathway and function in parallel with a growth factor-dependent MUC1-mediated signaling pathway.