Phenotype of vigilin expressing breast cancer cells binding to the 69 nt 3'UTR element in CSF-1R mRNA.

Vigilin, a nucleocytoplasmic shuttling protein, post-transcriptionally suppresses proto-oncogene c-fms expression (encoding CSF-1R) in breast cancer by binding to a 69 nt cis-acting 3-UTR element in CSF-1R mRNA. CSF-1R is an important mediator of breast cancer development, metastasis, and survival. We confirm that vigilin decreases in vitro reporter luciferase activity as well as the translation rate of target mRNAs. We further explore the mechanism of suppression of CSF-1R. We show that the 69 nt binding element has profound effects on translation efficiency of CSF-1R mRNA, not seen in the presence of mutation of the element. Also, mutation of the 69 nt element in the CSF-1R mRNA 3'UTR both interferes with direct vigilin binding and obviates effect of vigilin overexpression on translational repression of CSF-1R. We show that stable vigilin binding requires the full length 69 nt CSF-1R element, including the 26 nt pyrimidine-rich core. Furthermore, titration of endogenous vigilin and other proteins which bind the 69 nt element, by exogenously introduced CSF-1R mRNA 3'UTR containing the pyrimidine-rich sequence, increases the adhesion, motility, and invasion of breast cancer cells. This phenotypic effect is not seen when the 69 nt element is deleted. Lastly, we are the first to show that human breast tissues exhibit strong vigilin expression in normal breast epithelium. Our pilot data suggest decreased vigilin protein expression, along with shift from the nucleus to the cytoplasmic location, in the transition to ductal carcinoma in situ.

Anti-CD137 Suppresses Tumor Growth by Blocking Reverse Signaling by CD137 Ligand.

CD137 (4-1BB) is a T-cell costimulatory molecule, and agonstic CD137 antibodies are currently being evaluated in the clinic as cancer immunotherapy. Recently, it was found that CD137-/- mice or mice injected with agonistic anti-CD137 antibodies exhibit heightened antitumor responses, contrary to expectations based on other knowledge of CD137 function. Here, we report findings related to reverse signaling by CD137 ligand (CD137L) in antigen-presenting dendritic cells (DC) in tumors that address these paradoxical results. Specifically, CD137L suppressed intratumoral differentiation of IL12-producing CD103+ DC and type 1 tumor-associated macrophages (TAM). Differentiation of these cell types is important because they are required to generate IFNγ-producing CD8+ cytotoxic T lymphocytes (Tc1). Notably, CD137L blockade increased levels of IL12 and IFNγ, which promoted intratumoral differentiation of IFNγ-producing Tc1, IL12-producing CD103+ DC, and type 1 TAM within tumors. Our results offer an explanation for the paradoxical effects of CD137 blockade, based on differential immunomodulatory effects of CD137 signaling and reverse signaling in T cells and DC, respectively. Further, they show how CD137L blockade can seed a forward-feedback loop for activation of CD103+ DC/type 1 TAM and Tc1 that can create a self-perpetuating cycle of highly effective immunosurveillance. Cancer Res; 77(21); 5989-6000. ©2017 AACR.

Expression of the cytoplasmic nucleolin for post-transcriptional regulation of macrophage colony-stimulating factor mRNA in ovarian and breast cancer cells.

The formation of the mRNP complex is a critical component of translational regulation and mRNA decay. Both the 5' and 3'UTRs of CSF-1 mRNA are involved in post-transcriptional regulation. In CSF-1 mRNA, a small hairpin loop structure is predicted to form at the extreme 5' end (2-21nt) of the 5'UTR. Nucleolin binds the hairpin loop structure in the 5'UTR of CSF-1 mRNA and enhances translation, while removal of this hairpin loop nucleolin binding element dramatically represses translation. Thus in CSF-1 mRNA, the hairpin loop nucleolin binding element is critical for translational regulation. In addition, nucleolin interacts with the 3'UTR of CSF-1 mRNA and facilitates the miRISC formation which results in poly (A) tail shortening. The overexpression of nucleolin increases the association of CSF-1 mRNA containing short poly (A)n≤26, with polyribosomes. Nucleolin both forms an mRNP complex with the eIF4G and CSF-1 mRNA, and is co-localized with the eIF4G in the cytoplasm further supporting nucleolin's role in translational regulation. The distinct foci formation of nucleolin in the cytoplasm of ovarian and breast cancer cells implicates the translational promoting role of nucleolin in these cancers.

G-Quadruplex in the NRF2 mRNA 5' Untranslated Region Regulates De Novo NRF2 Protein Translation under Oxidative Stress.

Inhibition of protein synthesis serves as a general measure of cellular consequences of chemical stress. A few proteins are translated selectively and influence cell fate. How these proteins can bypass the general control of translation remains unknown. We found that low to mild doses of oxidants induce de novo translation of the NRF2 protein. Here we demonstrate the presence of a G-quadruplex structure in the 5' untranslated region (UTR) of NRF2 mRNA, as measured by circular dichroism, nuclear magnetic resonance, and dimethylsulfate footprinting analyses. Such a structure is important for 5'-UTR activity, since its removal by sequence mutation eliminated H2O2-induced activation of the NRF2 5' UTR. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics revealed elongation factor 1 alpha (EF1a) as a protein binding to the G-quadruplex sequence. Cells responded to H2O2 treatment by increasing the EF1a protein association with NRF2 mRNA, as measured by RNA-protein interaction assays. The EF1a interaction with small and large subunits of ribosomes did not appear to change due to H2O2 treatment, nor did posttranslational modifications, as measured by two-dimensional (2-D) Western blot analysis. Since NRF2 encodes a transcription factor essential for protection against tissue injury, our data have revealed a novel mechanism of cellular defense involving de novo NRF2 protein translation governed by the EF1a interaction with the G-quadruplex in the NRF2 5' UTR during oxidative stress.

Association of pain intensity, pain-related disability, and depression with hypothalamus-pituitary-adrenal axis function in female patients with chronic temporomandibular disorders.

Patients with temporomandibular disorders (TMD) commonly experience myofascial and joint pain, pain-related disability, and other pain conditions including depression. The present study was carried out to explore the function of the hypothalamus-pituitary-adrenal (HPA) axis in relation to variables of the Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) Axis II and comorbid depression in female patients with TMD. Cortisol and dehydroepiandrosterone (DHEA) levels were determined in saliva samples that had been collected at various periods after waking (0, 30, and 60min) and at nighttime (2100-2200h) from 52 female patients with chronic TMD pain and age- and gender-matched controls (n=54, 20-40 years old). There were no significant differences in the levels and diurnal patterns of cortisol and DHEA secretion between groups of patients with TMD and controls. In patients, the cortisol awakening response (CAR) or diurnal cortisol rhythm were not associated with any variables of the RDC/TMD Axis II or the Beck Depression Inventory (BDI)-II total scores. However, the ratio of overall cortisol secretion within the first hour after waking (CARauc) to overall DHEA secretion during the post-waking period (Daucawk), defined as CARauc/Daucawk, was significantly associated with pain-related RDC/TMD variables (pain intensity and pain-related disability) and BDI-II total scores. Pain intensity and pain-related disability scores were also significantly associated with BDI-II total scores. These results indicated that an increase in molar cortisol/DHEA ratio due to the dissociation between cortisol and DHEA secretion was associated with pain intensity, pain-related disability, and depression in female patients with TMD.

Depletion of Alloreactive T-Cells by Anti-CD137-Saporin Immunotoxin.

Depletion of alloreactive T-lymphocytes from allogeneic bone marrow transplants may prevent graft-versus-host disease (GVHD) without impairing donor cell engraftment, immunity, and the graft-versus-leukemia (GVL) effect. Alloreactive T-cells may be identified by their expression, upon activation, of CD137, a costimulatory receptor and putative surrogate marker for antigen-specific effector T-cells. In this context, we tested the use of anti-CD137-saporin immunotoxin to selectively deplete mouse and human alloreactive T-cells. Anti-CD137 antibodies were internalized by cells within 4 h of binding to the cell surface CD137, and anti-CD137-saporin immunotoxin effectively killed polyclonally activated T-cells or antigen-stimulated T-cells. Transfer of donor T-cells after allodepletion with anti-CD137-saporin immunotoxin failed to induce any evident expression of GVHD; however, a significant GVL effect was observed. Targeting of CD137 with an immunotoxin was also effective in killing polyclonally activated or alloreactive human T-cells. Our results indicate that anti-CD137-saporin immunotoxin may be used to deplete alloreactive T-cells prior to bone marrow transplantation and thereby prevent GVHD and the relapse of leukemia.

NVP-BEZ235, a dual PI3K/mTOR inhibitor synergistically potentiates the antitumor effects of cisplatin in bladder cancer cells.

The PI3K/Akt/mTOR pathway is a prototypic survival pathway and constitutively activated in many malignant conditions. Moreover, activation of the PI3K/Akt/mTOR pathway confers resistance to various cancer therapies and is often associated with a poor prognosis. In this study, we explored the antitumor effect of NVP-BEZ235, a dual PI3K/mTOR inhibitor in cisplatin-resistant human bladder cancer cells and its synergistic interaction with cisplatin. A human bladder cancer cell line with cisplatin resistance was exposed to escalating doses of NVP-BEZ235 alone or in combination with cisplatin and antitumor effects was determined by the CCK-8 assay. Based on a dose-response study, synergistic interaction between NVP-BEZ235 and cisplatin was evaluated by combination index (CI), three-dimensional model and clonogenic assay. The combination of NVP-BEZ235 and cisplatin caused significant synergistic antitumor effect in cisplatin-resistant bladder cancer cells over a wide dose range and reduced the IC50 of NVP-BEZ235 and cisplatin by 5.6- and 3.6-fold, respectively. Three-dimensional synergy analysis resulted in a synergy volume of 388.25 µM/ml2% indicating a strong synergistic effect of combination therapy. The combination therapy caused cell cycle arrest and caspase-dependent apoptosis. Although NVP-BEZ235 suppressed PI3K/mTOR signaling without any paradoxical induction of Akt activity, it caused MEK/ERK pathway activation. The present study demonstrated that the PI3K/mTOR dual inhibitor NVP-BEZ235 can synergistically potentiate the antitumor effects of cisplatin in cisplatin-resistant bladder cancer cells though the suppression of cell cycle progression and the survival pathway as well as induction of caspase-dependent apoptosis.

4-1BB-based isolation and expansion of CD8+ T cells specific for self-tumor and non-self-tumor antigens for adoptive T-cell therapy.

Adoptive T-cell therapy is a promising approach to the immunotherapy of cancer, but for it to be a general cancer therapy a simple and standardized procedure for producing tumor-specific CD8 T cells is needed. On the basis of a unique property of 4-1BB (CD137), the selective expression on activated T cells, we have developed a simple and practical protocol to produce antigen-specific CD8 T cells from peripheral blood mononuclear cells. We have proved the feasibility of this procedure by isolating and expanding cytomegalovirus-specific CD8 T cells, and applied the procedure to produce Epstein-Barr virus (EBV)-specific CD8 T cells. By using this procedure, we could readily produce 10-10 antigen-specific CD8 T cells from 30 to 50 mL of blood in about 4 weeks. Moreover, our protocol allowed us to produce, from solid cancer patients, CD8 T cells that were specific for self/tumor antigens such as human telomerase reverse transcriptase (hTERT). It is interesting to note that, we were unable to amplify hTERT-specific CD8 T cells from healthy donors. Our protocol can be readily translated into cGMP-compliant production and is currently being used to produce EBV-specific CD8 T cells for phase I clinical trial. We believe that our method will provide a practical and effective option for adoptive T-cell therapy in the clinic.

Trends in renal function after radical nephrectomy: a multicentre analysis.

OBJECTIVE: To evaluate serial changes in renal function by investigating various clinical factors after radical nephrectomy (RN). PATIENTS AND METHODS: The study population consisted of 2068 consecutive patients who were treated at multiple institutions by RN for renal cortical tumour without metastasis between 1999 and 2011. We measured the serial change in estimated glomerular filtration rate (eGFR) and clinical factors during a 60-month follow-up period. The changes in eGFR over time were analysed according to baseline eGFR (eGFR ≥60 and 15-59 mL/min/1.73m(2) ) using a linear mixed model. The independent prognostic value of various clinical factors on the increase in eGFR was ascertained by multivariate mixed regression model. RESULTS: Overall, there was a subsequent restoration of renal function over the 60 months. The slope for the relationship between the eGFR and the time since RN was 0.082 (95% confidence interval [CI] 0.039-0.104; P < 0.001) and 0.053 (95% CI 0.006-0.100; P = 0.038) in each baseline group, indicating that each month after RN was associated with an increase in eGFR of 0.082 and 0.053 mL/min/1.73m(2) , respectively. When we analysed renal function based on various factors, postoperative eGFR of patients with diabetes mellitus, old age (≥70 years) or a preoperative eGFR of <30 mL/min/1.73 m(2) , was decreased or maintained at a certain level without any improvement in renal function. Preoperative predictors of an increase in eGFR after RN were young age, no DM, no hypertension, a preoperative eGFR of ≥30 mL/min/1.73m(2) and time after surgery (≥36 months). CONCLUSIONS: Renal function recovered continuously during the 60-month follow-up period after RN. However, the trends in functional recovery change were different according to various clinical factors and such information should be discussed with patients when being counselled about their treatment for renal cell carcinoma (RCC).

4-1BB signaling breaks the tolerance of maternal CD8+ T cells that are reactive with alloantigens.

4-1BB (CD137, TNFRSF9), a member of the activation-induced tumor necrosis factor receptor family, is a powerful T-cell costimulatory molecule. It generally enhances CD8(+) T responses and even breaks the tolerance of CD8(+) T cells in an antigen-specific manner. In the present study we found that it was expressed in the placentas of pregnant mice and that its expression coincided with that of the immunesuppressive enzyme indoleamine 2,3-dioxygenase (IDO). Therefore, we investigated whether 4-1BB signaling is involved in fetal rejection using agonistic anti-4-1BB mAb and 4-1BB-deficient mice. Treatment with agonistic anti-4-1BB mAb markedly increased the rate of rejection of allogeneic but not syngeneic fetuses, and this was primarily dependent on CD8(+) T cells. Complement component 3 (C3) seemed to be the effector molecule because 4-1BB triggering resulting in accumulation of C3 in the placenta, and this accumulation was also reversed by anti-CD8 mAb treatment. These findings demonstrate that 4-1BB triggering breaks the tolerance of CD8(+) T cells to alloantigens in the placenta. Moreover, triggering 4-1BB protected the pregnant mice from Listeria monocytogenes (LM) infection, but led to rejection of semi-allogeneic fetuses. Therefore, given the cross-recognition of alloantigen by pathogen-reactive CD8(+) T cells, the true function of 4-1BB may be to reverse the hypo-responsiveness of pathogen-reactive CD8(+) T cells in the placenta in cases of infection, even if that risks losing the fetus.

Reverse signaling through the costimulatory ligand CD137L in epithelial cells is essential for natural killer cell-mediated acute tissue inflammation.

Renal ischemia-reperfusion injury (IRI) after kidney transplantation is a major cause of delayed graft function. Even though IRI is recognized as a highly coordinated and specific process, the pathways and mechanisms through which the innate response is activated are poorly understood. In this study, we used a mouse model of acute kidney IRI to examine whether the interactions of costimulatory receptor CD137 and its ligand (CD137L) are involved in the early phase of acute kidney inflammation caused by IRI. We report here that the specific expressions of CD137 on natural killer cells and of CD137L on tubular epithelial cells (TECs) are required for acute kidney IRI. Reverse signaling through CD137L in TECs results in their production of the chemokine (C-X-C motif) receptor 2 ligands CXCL1 and CXCL2 and the subsequent induction of neutrophil recruitment, resulting in a cascade of proinflammatory events during kidney IRI. Our findings identify an innate pathogenic pathway for renal IRI involving the natural killer cell-TEC-neutrophil axis, whereby CD137-CD137L interactions provide the causal contribution of epithelial cell dysregulation to renal IRI. The CD137L reverse signaling pathway in epithelial cells therefore may represent a good target for blocking the initial stage of inflammatory diseases, including renal IRI.

HDAC activity is required for efficient core promoter function at the mouse mammary tumor virus promoter.

Histone deacetylases (HDACs) have been shown to be required for basal or inducible transcription at a variety of genes by poorly understood mechanisms. We demonstrated previously that HDAC inhibition rapidly repressed transcription from the mouse mammary tumor virus (MMTV) promoter by a mechanism that does not require the binding of upstream transcription factors. In the current study, we find that HDACs work through the core promoter sequences of MMTV as well as those of several cellular genes to facilitate transcriptional initiation through deacetylation of nonhistone proteins.

4-1BB functions as a survival factor in dendritic cells.

4-1BB (CD137) is expressed on dendritic cells (DCs) and its biological function has remained largely unresolved. By comparing 4-1BB-intact (4-1BB(+/+)) and 4-1BB-deficient (4-1BB(-/-)) DCs, we found that 4-1BB was strongly induced on DCs during the maturation and that DC maturation was normal in the absence of 4-1BB. However, DC survival rate was low in the absence of 4-1BB, which was due to the decreased Bcl-2 and Bcl-x(L) in 4-1BB(-/-) DCs compared with 4-1BB(+/+) DCs after DC maturation. Consistent with these results, 4-1BB(-/-) DCs showed an increased turnover rate in steady state and more severely decreased in spleen by injecting LPS compared with 4-1BB(+/+) DCs. When OVA-pulsed DCs were adoptively transferred to recipient mice along with OVA-specific CD4(+) T cells, 4-1BB(-/-) DCs did not properly migrate to the T cell zone in lymph nodes and poorly induced proliferation of CD4(+) T cells, although both DCs comparably expressed functional CCR7. Eventually, 4-1BB(-/-) DCs generated a reduced number of OVA-specific memory CD4(+) T cells compared with 4-1BB(+/+) DCs. To further assess the role of 4-1BB on DC longevity in vivo, 4-1BB(+/+) and 4-1BB(-/-) C57BL/6 were administrated with Propionibacterium acnes that develop liver granuloma by recruiting DCs. Number and size of granuloma were reduced in the absence of 4-1BB, but the inflammatory cytokine level was comparable between the mice, which implied that the granuloma might be reduced due to the decreased longevity of DCs. These results demonstrate that 4-1BB on DCs controls the duration, DC-T interaction, and, therefore, immunogenicity.

Mad1 is a transcriptional repressor of Bcl-6.

Bcl-6, a major regulator of B lymphocyte function that contributes to neoplastic transformation of B cells, is expressed in activated germinal center (GC) B cells and down-regulated during terminal differentiation to plasma cells. Regulation of Bcl-6 expression is incompletely characterized. Terminal B cell differentiation is associated with down-regulation of Bcl-6, activation of Blimp-1, modulation of Myc, and specifically with the up-regulation of the Mad1 and Mad4 transcription factors, which play a critical role in cell differentiation and cell cycle regulation. Because the Mad E-box consensus binding site is present in the upstream promoter of Bcl-6, we investigated whether Bcl-6 may be under control of the Mad1 transcription factor. Anti-sense Mad1 oligonucleotides abrogated the down-regulation of Bcl-6 expression that occurred during in vitro differentiation of mouse splenic B cells induced by dextran-conjugated anti-IgD Ab and IL-5. Transduction of the WEHI 231 B cell line with retroviruses expressing Mad1 down-regulated Bcl-6 expression. Expression of the 5' upstream promoter region of Bcl-6 was down-regulated by co-expression of Mad1. Last, chromatin immunoprecipitation assays with anti-Mad1 Ab demonstrated in vivo interaction of Mad1 with the Bcl-6 promoter region. The findings suggest that Mad1 is a transcriptional repressor of Bcl-6.

4-1BB (CD137) is required for rapid clearance of Listeria monocytogenes infection.

4-1BB (CD137), a member of the tumor necrosis factor receptor superfamily, is a T-cell-costimulatory receptor that is expressed on activated T cells, dendritic cells, and NK cells. Little has been reported about its role in early host defense against bacterial infection. In this study, we report that 4-1BB-deficient (4-1BB(-/-)) mice are much more susceptible to Listeria monocytogenes (intracellular bacteria) infections than wild-type mice. Upon L. monocytogenes infection, 4-1BB(-/-) mice showed a lower survival rate, a higher bacterial burden in organs, and larger hepatic microabscesses than 4-1BB(+/+) mice. 4-1BB(-/-) mice also had impairment in clearance of bacteria from the bloodstream. Neutrophils from 4-1BB(+/+) mice constitutively expressed 4-1BB, which could be activated to induce intracellular Ca(2+) influx by ligation with anti-4-1BB antibody. On the other hand, neutrophils from 4-1BB(-/-) mice were defective in reactive oxygen species generation, phagocytic activities, and intracellular Ca(2+) mobilization. In addition, mice pretreated with anti-4-1BB monoclonal antibody were much more resistant to L. monocytogenes infection than control antibody-treated mice. Our results support the notion that 4-1BB may play a major role in host defense against intracellular pathogens through neutrophil activation.

Multichannel magnetic stimulation system design considering mutual couplings among the stimulation coils.

We introduce some simulation and experiment results of the multichannel magnetic stimulator development that has been carried out as an initial attempt to realize a multichannel functional magnetic stimulator. For efficient functional magnetic stimulations, precise spatial localization of stimulation sites without any movements of the stimulation coils is very important. We have found that the mutual coupling effect among the adjacent stimulation coils in the coil array has to be considered in the determination of the charge voltages in some coil array configurations. Experimental results obtained with a 4-channel magnetic stimulator are presented.

Activation of terminal B cell differentiation by inhibition of histone deacetylation.

A role for histone acetylation, which can alter the accessibility of DNA to transcriptional regulatory proteins and contribute to gene expression, in regulating terminal B cell differentiation was investigated in the mature B lymphoma L10A and mouse splenic B cells. Incubation of the L10A cells with the histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and butyrate increased expression of Blimp-1, J chain, and mad genes, decreased expression of c-myc and BSAP/Pax-5 genes, increased the expression of surface CD43 and Syndecan-1, and decreased surface IgM. Incubation of splenic B cells with TSA and dextran conjugated anti-IgD Ab increased Blimp-1 gene and Syndecan-1 surface expression. The alteration in gene expression and cell surface markers was consistent with induction of the onset of terminal B cell differentiation. Co-incubation of L10A cells with TSA and cycloheximide (CHX) abrogated the up-regulation of Blimp-1 expression, indicating that TSA-activated Blimp-1 expression required synthesis of a transcriptional activator. In contrast, mad expression was increased in L10A cells cultured with TSA and cycloheximide or cycloheximide alone, suggesting mad expression may occur independent of Blimp-1 expression and is regulated by a labile, HDAC associated transcriptional repressor. The results demonstrate that histone acetylation regulates transcription of genes controlling terminal B cell differentiation.