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1.
Sci Rep ; 12(1): 19660, 2022 11 16.
Article in English | MEDLINE | ID: mdl-36385153

ABSTRACT

Multiple myeloma (MM) remains an incurable malignancy of plasma cells despite constantly evolving therapeutic approaches including various types of immunotherapy. Increased arginase activity has been associated with potent suppression of T-cell immune responses in different types of cancer. Here, we investigated the role of arginase 1 (ARG1) in Vκ*MYC model of MM in mice. ARG1 expression in myeloid cells correlated with tumor progression and was accompanied by a systemic drop in ʟ-arginine levels. In MM-bearing mice antigen-induced proliferation of adoptively transferred T-cells was strongly suppressed and T-cell proliferation was restored by pharmacological arginase inhibition. Progression of Vκ*MYC tumors was significantly delayed in mice with myeloid-specific ARG1 deletion. Arginase inhibition effectively inhibited tumor progression although it failed to augment anti-myeloma effects of bortezomib. However, arginase inhibitor completely prevented development of bortezomib-induced cardiotoxicity in mice. Altogether, these findings indicate that arginase inhibitors could be further tested as a complementary strategy in multiple myeloma to mitigate adverse cardiac events without compromising antitumor efficacy of proteasome inhibitors.


Subject(s)
Multiple Myeloma , Mice , Animals , Bortezomib/pharmacology , Bortezomib/therapeutic use , Multiple Myeloma/drug therapy , Arginase/metabolism , Cardiotoxicity , Proteasome Inhibitors/pharmacology
2.
Cancers (Basel) ; 15(1)2022 Dec 24.
Article in English | MEDLINE | ID: mdl-36612114

ABSTRACT

Chimeric antigen receptor (CAR)-modified T cell therapy has been rapidly developing in recent years, ultimately revolutionizing immunotherapeutic strategies and providing significant anti-tumor potency, mainly in treating hematological neoplasms. However, graft-versus-host disease (GVHD) and other adverse effects, such as cytokine release syndromes (CRS) and neurotoxicity associated with CAR-T cell infusion, have raised some concerns about the broad application of this therapy. Natural killer (NK) cells have been identified as promising alternative platforms for CAR-based therapies because of their unique features, such as a lack of human leukocyte antigen (HLA)-matching restriction, superior safety, and better anti-tumor activity when compared with CAR-T cells. The lack of CRS, neurotoxicity, or GVHD, in the case of CAR-NK therapy, in addition to the possibility of using allogeneic NK cells as a CAR platform for "off-the-shelf" therapy, opens new windows for strategic opportunities. This review underlines recent design achievements in CAR constructs and summarizes preclinical studies' results regarding CAR-NK therapies' safety and anti-tumor potency. Additionally, new approaches in CAR-NK technology are briefly described, and currently registered clinical trials are listed.

3.
Cancer Res ; 81(23): 6029-6043, 2021 12 01.
Article in English | MEDLINE | ID: mdl-34625423

ABSTRACT

The family of PIM serine/threonine kinases includes three highly conserved oncogenes, PIM1, PIM2, and PIM3, which regulate multiple prosurvival pathways and cooperate with other oncogenes such as MYC. Recent genomic CRISPR-Cas9 screens further highlighted oncogenic functions of PIMs in diffuse large B-cell lymphoma (DLBCL) cells, justifying the development of small-molecule PIM inhibitors and therapeutic targeting of PIM kinases in lymphomas. However, detailed consequences of PIM inhibition in DLBCL remain undefined. Using chemical and genetic PIM blockade, we comprehensively characterized PIM kinase-associated prosurvival functions in DLBCL and the mechanisms of PIM inhibition-induced toxicity. Treatment of DLBCL cells with SEL24/MEN1703, a pan-PIM inhibitor in clinical development, decreased BAD phosphorylation and cap-dependent protein translation, reduced MCL1 expression, and induced apoptosis. PIM kinases were tightly coexpressed with MYC in diagnostic DLBCL biopsies, and PIM inhibition in cell lines and patient-derived primary lymphoma cells decreased MYC levels as well as expression of multiple MYC-dependent genes, including PLK1. Chemical and genetic PIM inhibition upregulated surface CD20 levels in an MYC-dependent fashion. Consistently, MEN1703 and other clinically available pan-PIM inhibitors synergized with the anti-CD20 monoclonal antibody rituximab in vitro, increasing complement-dependent cytotoxicity and antibody-mediated phagocytosis. Combined treatment with PIM inhibitor and rituximab suppressed tumor growth in lymphoma xenografts more efficiently than either drug alone. Taken together, these results show that targeting PIM in DLBCL exhibits pleiotropic effects that combine direct cytotoxicity with potentiated susceptibility to anti-CD20 antibodies, justifying further clinical development of such combinatorial strategies. SIGNIFICANCE: These findings demonstrate that inhibition of PIM induces DLBCL cell death via MYC-dependent and -independent mechanisms and enhances the therapeutic response to anti-CD20 antibodies by increasing CD20 expression.


Subject(s)
Gene Expression Regulation, Neoplastic/drug effects , Lymphoma, Large B-Cell, Diffuse/drug therapy , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Rituximab/pharmacology , Animals , Antigens, CD20 , Antineoplastic Agents, Immunological/pharmacology , Apoptosis , Cell Proliferation , Female , Humans , Lymphoma, Large B-Cell, Diffuse/genetics , Lymphoma, Large B-Cell, Diffuse/metabolism , Lymphoma, Large B-Cell, Diffuse/pathology , Mice , Mice, SCID , Phosphorylation , Proto-Oncogene Proteins c-myc/metabolism , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
4.
Front Immunol ; 12: 642166, 2021.
Article in English | MEDLINE | ID: mdl-34163468

ABSTRACT

The incidence and prevalence rate of chronic inflammatory disorders is on the rise in the pediatric population. Recent research indicates the crucial role of interactions between the altered intestinal microbiome and the immune system in the pathogenesis of several chronic inflammatory disorders in children, such as inflammatory bowel disease (IBD) and autoimmune diseases, such as type 1 diabetes mellitus (T1DM) and celiac disease (CeD). Here, we review recent knowledge concerning the pathogenic mechanisms underlying these disorders, and summarize the facts suggesting that the initiation and progression of IBD, T1DM, and CeD can be partially attributed to disturbances in the patterns of composition and abundance of the gut microbiota. The standard available therapies for chronic inflammatory disorders in children largely aim to treat symptoms. Although constant efforts are being made to maximize the quality of life for children in the long-term, sustained improvements are still difficult to achieve. Additional challenges are the changing physiology associated with growth and development of children, a population that is particularly susceptible to medication-related adverse effects. In this review, we explore new promising therapeutic approaches aimed at modulation of either gut microbiota or the activity of the immune system to induce a long-lasting remission of chronic inflammatory disorders. Recent preclinical studies and clinical trials have evaluated new approaches, for instance the adoptive transfer of immune cells, with genetically engineered regulatory T cells expressing antigen-specific chimeric antigen receptors. These approaches have revolutionized cancer treatments and have the potential for the protection of high-risk children from developing autoimmune diseases and effective management of inflammatory disorders. The review also focuses on the findings of studies that indicate that the responses to a variety of immunotherapies can be enhanced by strategic manipulation of gut microbiota, thus emphasizing on the importance of proper interaction between the gut microbiota and immune system for sustained health benefits and improvement of the quality of life of pediatric patients.


Subject(s)
Celiac Disease/immunology , Diabetes Mellitus, Type 1/immunology , Gastrointestinal Microbiome/immunology , Inflammatory Bowel Diseases/immunology , Animals , Celiac Disease/microbiology , Child , Diabetes Mellitus, Type 1/microbiology , Female , Humans , Inflammatory Bowel Diseases/microbiology , Male
5.
Cell Commun Signal ; 17(1): 171, 2019 12 26.
Article in English | MEDLINE | ID: mdl-31878945

ABSTRACT

BACKGROUND: Lymphotoxin ß receptor (LTßR) plays important roles in the development of the immune system and immune response. At the cellular level, ligand-bound LTßR activates the pro-inflammatory NF-κB pathway but the detailed mechanisms regulating its signaling remain unknown. Understanding them is of high importance since LTßR and its ligands are promising therapeutic targets. Here, we studied the consequences of perturbed cellular cholesterol content on LTßR-induced NF-κB signaling. METHODS: To modulate cholesterol availability and/or level in lung carcinoma A549 and H2228, and endothelial HUVEC cells different treatment regimens with filipin, methyl-ß-cyclodextrin and simvastatin were applied. LTßR localization was studied by confocal microscopy. The activity of LTßR-induced NF-κB pathway was assessed by measuring the levels of NF-κB pathway inhibitor IκBα and phosphorylation of RelA transcription factor by Western blotting. The NF-κB transcriptional response, production of chemokines and adhesion molecules were examined by qRT-PCR, ELISA, and Western blotting, respectively. Adherence of different types of primary immune cells to epithelial A549 cells and endothelial HUVECs was measured fluorometrically. Interactions of LTßR with its protein partners were investigated by immunoprecipitation. RESULTS: We showed that filipin-mediated sequestration of cholesterol or its depletion from the plasma membrane with methyl-ß-cyclodextrin impaired LTßR internalization and potentiated LTßR-dependent activation of the canonical branch of the NF-κB pathway. The latter was manifested by enhanced degradation of IκBα inhibitor, elevated RelA phosphorylation, substantial increase in the expression of NF-κB target genes encoding, among others, cytokines and adhesion molecules known to play important roles in immune response. It was followed by robust secretion of CXCL8 and upregulation of ICAM1, that favored the adhesion of immune cells (NK and T cells, neutrophils) to A549 cells and HUVECs. Mechanistically, we showed that cholesterol depletion stabilized interactions of ligand-stimulated LTßR with modified forms of TRAF2 and NEMO proteins. CONCLUSIONS: Our results showed that the reduction of the plasma membrane content of cholesterol or its sequestration strongly potentiated signaling outcome initiated by LTßR. Thus, drugs modulating cholesterol levels could potentially improve efficacy of LTßR-based therapies. Video abstract.


Subject(s)
Cholesterol/pharmacology , Lymphotoxin beta Receptor/antagonists & inhibitors , Lymphotoxin beta Receptor/metabolism , NF-kappa B/metabolism , Signal Transduction/drug effects , A549 Cells , Cells, Cultured , Humans , Jurkat Cells , Neutrophils/drug effects , Neutrophils/metabolism
6.
Leukemia ; 33(10): 2416-2428, 2019 10.
Article in English | MEDLINE | ID: mdl-30872780

ABSTRACT

The BCL-2 inhibitor venetoclax has only limited activity in DLBCL despite frequent BCL-2 overexpression. Since constitutive activation of the B cell receptor (BCR) pathway has been reported in both ABC and GCB DLBCL, we investigated whether targeting SYK or BTK will increase sensitivity of DLBCL cells to venetoclax. We report that pharmacological inhibition of SYK or BTK synergistically enhances venetoclax sensitivity in BCL-2-positive DLBCL cell lines with an activated BCR pathway in vitro and in a xenograft model in vivo, despite the only modest direct cytotoxic effect. We further show that these sensitizing effects are associated with inhibition of the downstream PI3K/AKT pathway and changes in the expression of MCL-1, BIM, and HRK. In addition, we show that BCR-dependent GCB DLBCL cells are characterized by deficiency of the phosphatase SHP1, a key negative regulator of the BCR pathway. Re-expression of SHP1 in GCB DBLCL cells reduces SYK, BLNK, and GSK3 phosphorylation and induces corresponding changes in MCL1, BIM, and HRK expression. Together, these findings suggest that SHP1 deficiency is responsible for the constitutive activation of the BCR pathway in GCB DLBCL and identify SHP1 and BCL-2 as potential predictive markers for response to treatment with a venetoclax/BCR inhibitor combination.


Subject(s)
Agammaglobulinaemia Tyrosine Kinase/metabolism , Antineoplastic Agents/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Lymphoma, Large B-Cell, Diffuse/drug therapy , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Sulfonamides/pharmacology , Syk Kinase/metabolism , Animals , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Humans , Lymphoma, Large B-Cell, Diffuse/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction/drug effects , Xenograft Model Antitumor Assays/methods
7.
Oncoimmunology ; 7(5): e1423183, 2018.
Article in English | MEDLINE | ID: mdl-29721381

ABSTRACT

Diminished overall survival rate of non-Hodgkin lymphoma (NHL) patients treated with a combination regimen of rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) has been recently linked to recurrent somatic mutations activating FOXO1. Despite of the clinical relevance of this finding, the molecular mechanism driving resistance to R-CHOP therapy remains largely unknown. Herein, we investigated the potential role of FOXO1 in the therapeutic efficacy of rituximab, the only targeted therapy included in the R-CHOP regimen. We found CD20 transcription is negatively regulated by FOXO1 in NHL cell lines and in human lymphoma specimens carrying activating mutations of FOXO1. Furthermore, both the expression of exogenous mutants of FOXO1 and the inhibition of AKT led to FOXO1 activation in lymphoma cells, increased binding to MS4A1 promoter and diminished CD20 expression levels. In contrast, a disruption of FOXO1 with CRISPR/Cas9 genome-editing (sgFOXO1) resulted in CD20 upregulation, improved the cytotoxicity induced by rituximab and the survival of mice with sgFOXO1 tumors. Accordingly, pharmacological inhibition of FOXO1 activity in primary samples upregulated surface CD20 levels. Importantly, FOXO1 was required for the downregulation of CD20 levels by the clinically tested inhibitors of BTK, SYK, PI3K and AKT. Taken together, these results indicate for the first time that the AKT-unresponsive mutants of FOXO1 are important determinant of cell response to rituximab-induced cytotoxicity, and suggest that the genetic status of FOXO1 together with its transcriptional activity need further attention while designing anti-CD20 antibodies based regimens for the therapy of pre-selected lymphomas.

8.
Blood ; 130(14): 1628-1638, 2017 10 05.
Article in English | MEDLINE | ID: mdl-28830887

ABSTRACT

Downregulation of CD20, a molecular target for monoclonal antibodies (mAbs), is a clinical problem leading to decreased efficacy of anti-CD20-based therapeutic regimens. The epigenetic modulation of CD20 coding gene (MS4A1) has been proposed as a mechanism for the reduced therapeutic efficacy of anti-CD20 antibodies and confirmed with nonselective histone deacetylase inhibitors (HDACis). Because the use of pan-HDACis is associated with substantial adverse effects, the identification of particular HDAC isoforms involved in CD20 regulation seems to be of paramount importance. In this study, we demonstrate for the first time the role of HDAC6 in the regulation of CD20 levels. We show that inhibition of HDAC6 activity significantly increases CD20 levels in established B-cell tumor cell lines and primary malignant cells. Using pharmacologic and genetic approaches, we confirm that HDAC6 inhibition augments in vitro efficacy of anti-CD20 mAbs and improves survival of mice treated with rituximab. Mechanistically, we demonstrate that HDAC6 influences synthesis of CD20 protein independently of the regulation of MS4A1 transcription. We further demonstrate that translation of CD20 mRNA is significantly enhanced after HDAC6 inhibition, as shown by the increase of CD20 mRNA within the polysomal fraction, indicating a new role of HDAC6 in the posttranscriptional mechanism of CD20 regulation. Collectively, our findings suggest HDAC6 inhibition is a rational therapeutic strategy to be implemented in combination therapies with anti-CD20 monoclonal antibodies and open up novel avenues for the clinical use of HDAC6 inhibitors.


Subject(s)
Antigens, CD20/genetics , Antineoplastic Agents/pharmacology , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Lymphoma, Non-Hodgkin/drug therapy , Rituximab/pharmacology , Animals , Antigens, CD20/immunology , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , B-Lymphocytes/pathology , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic/drug effects , Histone Deacetylase 6 , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Lymphoma, Non-Hodgkin/genetics , Lymphoma, Non-Hodgkin/immunology , Lymphoma, Non-Hodgkin/pathology , Mice, Inbred BALB C , Mice, SCID , RNA, Messenger/genetics , Tumor Cells, Cultured , Up-Regulation/drug effects
9.
MAbs ; 6(5): 1300-13, 2014.
Article in English | MEDLINE | ID: mdl-25517315

ABSTRACT

Clinical trials with SRC family kinases (SFKs) inhibitors used alone or in a combination with anti-CD20 monoclonal antibodies (mAbs) are currently underway in the treatment of B-cell tumors. However, molecular interactions between these therapeutics have not been studied so far. A transcriptional profiling of tumor cells incubated with SFKs inhibitors revealed strong downregulation of MS4A1 gene encoding CD20 antigen. In a panel of primary and established B-cell tumors we observed that SFKs inhibitors strongly affect CD20 expression at the transcriptional level, leading to inhibition of anti-CD20 mAbs binding and increased resistance of tumor cells to complement-dependent cytotoxicity. Activation of the AKT signaling pathway significantly protected cells from dasatinib-triggered CD20 downregulation. Additionally, SFKs inhibitors suppressed antibody-dependent cell-mediated cytotoxicity by direct inhibition of natural killer cells. Abrogation of antitumor activity of rituximab was also observed in vivo in a mouse model. Noteworthy, the effects of SFKs inhibitors on NK cell function are largely reversible. The results of our studies indicate that development of optimal combinations of novel treatment modalities with anti-CD20 mAbs should be preceded by detailed preclinical evaluation of their effects on target cells.


Subject(s)
Antibodies, Monoclonal/immunology , Antigens, CD20/immunology , Neoplasms/immunology , Protein Kinase Inhibitors/immunology , src-Family Kinases/immunology , Animals , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal, Murine-Derived/immunology , Antibodies, Monoclonal, Murine-Derived/pharmacology , Antibody-Dependent Cell Cytotoxicity/drug effects , Antibody-Dependent Cell Cytotoxicity/immunology , Antigens, CD20/genetics , Antigens, CD20/metabolism , Blotting, Western , Cell Line, Tumor , Dasatinib , Disease Models, Animal , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/immunology , HEK293 Cells , Humans , K562 Cells , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Mice, Inbred C57BL , Neoplasms/drug therapy , Neoplasms/genetics , Oligonucleotide Array Sequence Analysis , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/immunology , Proto-Oncogene Proteins c-akt/metabolism , Pyrimidines/immunology , Pyrimidines/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Rituximab , Signal Transduction/drug effects , Signal Transduction/immunology , Thiazoles/immunology , Thiazoles/pharmacology , Transcriptome/drug effects , Transcriptome/immunology , src-Family Kinases/antagonists & inhibitors , src-Family Kinases/metabolism
10.
Cancer Res ; 74(5): 1371-8, 2014 Mar 01.
Article in English | MEDLINE | ID: mdl-24398474

ABSTRACT

How necrotic areas develop in tumors is incompletely understood but can impact progression. Recent findings suggest that the formation of vascular microthrombi contributes to tumor necrosis, prompting investigation of coagulation cascades. Here, we report that loss of tumor suppressor P14ARF can contribute to activating the clotting cascade in glioblastoma. P14ARF transcriptionally upregulated TFPI2, a Kunitz-type serine protease in the tissue factor pathway that inhibits the initiation of thrombosis reactions. P14ARF activation in tumor cells delayed their ability to activate plasma clotting. Mechanistically, P14ARF activated the TFPI2 promoter in a p53-independent manner that relied upon c-JUN, SP1, and JNK activity. Taken together, our results identify the critical signaling pathways activated by P14ARF to prevent vascular microthrombosis triggered by glioma cells. Stimulation of this pathway might be used as a therapeutic strategy to reduce aggressive phenotypes associated with necrotic tumors, including glioblastoma.


Subject(s)
Glioblastoma/genetics , Signal Transduction/genetics , Thromboplastin/genetics , Thrombosis/genetics , Tumor Suppressor Protein p14ARF/genetics , Genes, Tumor Suppressor , Humans , Promoter Regions, Genetic/genetics , Transcriptional Activation/genetics , Tumor Cells, Cultured , Up-Regulation/genetics
11.
Mol Oncol ; 7(1): 67-84, 2013 Feb.
Article in English | MEDLINE | ID: mdl-22989406

ABSTRACT

Some endocytic proteins have recently been shown to play a role in tumorigenesis. In this study, we demonstrate that APPL2, an adapter protein with known endocytic functions, is upregulated in 40% cases of glioblastoma multiforme, the most common and aggressive cancer of the central nervous system. The silencing of APPL2 expression by small interfering RNAs (siRNAs) in glioma cells markedly reduces cell survival under conditions of low growth factor availability and enhances apoptosis (measured by executor caspase activity). Long-term depletion of APPL2 by short hairpin RNAs (shRNAs), under regular growth factor availability, suppresses the cell transformation abilities, assessed by inhibited colony formation in soft agar and by reduced xenograft tumor growth in vivo. At the molecular level, the negative effect of APPL2 knockdown on cell survival is not due to the alterations in AKT or GSK3ß activities which were reported to be modulated by APPL proteins. Instead, we attribute the reduced cell survival upon APPL2 depletion to the changes in gene expression, in particular to the upregulation of apoptosis-related genes, such as UNC5B (a proapoptotic dependence receptor) and HRK (harakiri, an activator of apoptosis, which antagonizes anti-apoptotic function of Bcl2). In support of this notion, the loss of glioma cell survival upon APPL2 knockdown can be rescued either by an excess of netrin-1, the prosurvival ligand of UNC5B or by simultaneous silencing of HRK. Consistently, APPL2 overexpression reduces expression of HRK and caspase activation in cells treated with apoptosis inducers, resulting in the enhancement of cell viability. This prosurvival activity of APPL2 is independent of its endosomal localization. Cumulatively, our data indicate that a high level of APPL2 protein might enhance glioblastoma growth by maintaining low expression level of genes responsible for cell death induction.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Glioma/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Apoptosis/genetics , Apoptosis/physiology , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Blotting, Western , Cell Survival/genetics , Cell Survival/physiology , Endocytosis , Flow Cytometry , Fluorescent Antibody Technique , Glioma/genetics , Glioma/therapy , HeLa Cells , Humans , Immunohistochemistry , Mice , Mice, Inbred NOD , Mice, SCID , Xenograft Model Antitumor Assays
12.
J Cell Sci ; 125(Pt 17): 4090-102, 2012 Sep 01.
Article in English | MEDLINE | ID: mdl-22685329

ABSTRACT

APPL1 is a multifunctional adaptor protein that binds membrane receptors, signaling proteins and nuclear factors, thereby acting in endosomal trafficking and in different signaling pathways. Here, we uncover a novel role of APPL1 as a positive regulator of transcriptional activity of NF-κB under basal but not TNFα-stimulated conditions. APPL1 was found to directly interact with TRAF2, an adaptor protein known to activate canonical NF-κB signaling. APPL1 synergized with TRAF2 to induce NF-κB activation, and both proteins were necessary for this process and function upstream of the IKK complex. Although TRAF2 was not detectable on APPL endosomes, endosomal recruitment of APPL1 was required for its function in the NF-κB pathway. Importantly, in the canonical pathway, APPL1 appeared to regulate the proper spatial distribution of the p65 subunit of NF-κB in the absence of cytokine stimulation, since its overexpression enhanced and its depletion reduced the nuclear accumulation of p65. By analyzing the patterns of gene transcription upon APPL1 overproduction or depletion we found altered expression of NF-κB target genes that encode cytokines. At the molecular level, overexpressed APPL1 markedly increased the level of NIK, the key component of the noncanonical NF-κB pathway, by reducing its association with the degradative complex containing TRAF2, TRAF3 and cIAP1. In turn, high levels of NIK triggered nuclear translocation of p65. Collectively, we propose that APPL1 regulates basal NF-κB activity by modulating the stability of NIK, which affects the activation of p65. This places APPL1 as a novel link between the canonical and noncanonical machineries of NF-κB activation.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , NF-kappa B/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Cell Nucleus/metabolism , Endosomes/metabolism , Enzyme Stability , Gene Expression Regulation , Gene Knockdown Techniques , HEK293 Cells , Human Umbilical Vein Endothelial Cells , Humans , Mice , Protein Binding , Protein Transport , Signal Transduction , TNF Receptor-Associated Factor 2/metabolism , Transcription Factor RelA/metabolism , NF-kappaB-Inducing Kinase
13.
J Clin Invest ; 122(4): 1283-95, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22378045

ABSTRACT

Malignant gliomas are the most common and the most lethal primary brain tumors in adults. Among malignant gliomas, 60%-80% show loss of P14ARF tumor suppressor activity due to somatic alterations of the INK4A/ARF genetic locus. The tumor suppressor activity of P14ARF is in part a result of its ability to prevent the degradation of P53 by binding to and sequestering HDM2. However, the subsequent finding of P14ARF loss in conjunction with TP53 gene loss in some tumors suggests the protein may have other P53-independent tumor suppressor functions. Here, we report what we believe to be a novel tumor suppressor function for P14ARF as an inhibitor of tumor-induced angiogenesis. We found that P14ARF mediates antiangiogenic effects by upregulating expression of tissue inhibitor of metalloproteinase-3 (TIMP3) in a P53-independent fashion. Mechanistically, this regulation occurred at the gene transcription level and was controlled by HDM2-SP1 interplay, where P14ARF relieved a dominant negative interaction of HDM2 with SP1. P14ARF-induced expression of TIMP3 inhibited endothelial cell migration and vessel formation in response to angiogenic stimuli produced by cancer cells. The discovery of this angiogenesis regulatory pathway may provide new insights into P53-independent P14ARF tumor-suppressive mechanisms that have implications for the development of novel therapies directed at tumors and other diseases characterized by vascular pathology.


Subject(s)
Brain Neoplasms/blood supply , Gene Expression Regulation, Neoplastic , Glioblastoma/blood supply , Neoplasm Proteins/physiology , Neovascularization, Pathologic/physiopathology , Tissue Inhibitor of Metalloproteinase-3/biosynthesis , Tumor Suppressor Protein p14ARF/physiology , Animals , Brain Neoplasms/metabolism , Cell Line, Transformed , Chemotaxis/drug effects , Corneal Neovascularization/physiopathology , Culture Media, Conditioned/pharmacology , Endothelial Cells/pathology , Fibroblasts/drug effects , Genes, p53 , Glioblastoma/metabolism , Humans , Mice , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Neovascularization, Pathologic/genetics , Proto-Oncogene Proteins c-mdm2/physiology , RNA Interference , RNA, Small Interfering/pharmacology , Recombinant Fusion Proteins/physiology , Sp1 Transcription Factor/physiology , Tissue Inhibitor of Metalloproteinase-3/antagonists & inhibitors , Tissue Inhibitor of Metalloproteinase-3/genetics , Tissue Inhibitor of Metalloproteinase-3/physiology , Transcription, Genetic , Tumor Cells, Cultured/metabolism , Up-Regulation
14.
Biochem J ; 423(3): 389-400, 2009 Oct 12.
Article in English | MEDLINE | ID: mdl-19686092

ABSTRACT

Multifunctional adaptor protein APPL1 [adaptor protein containing PH (pleckstrin homology) domain, PTB (phosphotyrosine binding) domain and leucine zipper motif] belongs to a growing group of endocytic proteins which actively participate in various stages of signalling pathways. Owing to its interaction with the small GTPase Rab5, APPL1 localizes predominantly to a subpopulation of early endosomes but is also capable of nucleocytoplasmic shuttling. Among its various binding partners, APPL1 was reported to associate with the nuclear co-repressor complex NuRD (nucleosome remodelling and deacetylase), containing both nucleosome remodelling and HDAC (histone deacetylase) activities, but the biochemical basis or functional relevance of this interaction remained unknown. Here we characterized the binding between APPL1 and NuRD in more detail, identifying HDAC2 as the key NuRD subunit responsible for this association. APPL1 interacts with the NuRD complex containing enzymatically active HDAC2 but not HDAC1 as the only deacetylase. However, the cellular levels of HDAC1 can regulate the extent of APPL1-NuRD interactions, which in turn modulates the nucleocytoplasmic distribution of APPL1. Increased binding of APPL1 to NuRD upon silencing of HDAC1 promotes the nuclear localization of APPL1, whereas HDAC1 overexpression exerts an opposite effect. Moreover, we also uncovered a NuRD-independent interaction of APPL1 with HDAC1. APPL1 overexpression affects the composition of the HDAC1-containing NuRD complex and the expression of HDAC1 target p21WAF1/CIP1. Cumulatively, these data reveal a surprising complexity of APPL1 interactions with HDACs, with functional consequences for the modulation of gene expression. In a broader sense, these results contribute to an emerging theme of endocytic proteins playing alternative roles in the cell nucleus.


Subject(s)
Carrier Proteins/metabolism , Cell Nucleus/metabolism , Cyclin-Dependent Kinase Inhibitor p21/biosynthesis , Gene Expression Regulation/physiology , Histone Deacetylases/metabolism , Active Transport, Cell Nucleus/physiology , Adaptor Proteins, Signal Transducing , Carrier Proteins/genetics , Cell Line , Cell Nucleus/genetics , Cyclin-Dependent Kinase Inhibitor p21/genetics , Gene Silencing , Histone Deacetylase 1 , Histone Deacetylase 2 , Histone Deacetylases/genetics , Humans , Mi-2 Nucleosome Remodeling and Deacetylase Complex , Protein Binding/physiology , Repressor Proteins/genetics , Repressor Proteins/metabolism
15.
Mol Oncol ; 3(4): 321-38, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19577966

ABSTRACT

Accumulating evidence argues that many proteins governing membrane sorting during endocytosis participate also in nuclear signaling and transcriptional regulation, mostly by modulating the activity of various nuclear factors. Some adaptors and accessory proteins acting in clathrin-mediated internalization, as well as endosomal sorting proteins can undergo nuclear translocation and affect gene expression directly, while for others the effects may be more indirect. Although it is often unclear to what extent the endocytic and nuclear functions are interrelated, several of such proteins are implicated in the regulation of cell proliferation and tumorigenesis, arguing that their dual-function nature may be of physiological importance.


Subject(s)
Endocytosis/physiology , Gene Expression Regulation/physiology , Neoplasms/metabolism , Signal Transduction/physiology , Transcription, Genetic/genetics , Animals , Endocytosis/genetics , Humans , Protein Transport/genetics , Protein Transport/physiology , Signal Transduction/genetics
16.
Cancer Res ; 65(2): 605-12, 2005 Jan 15.
Article in English | MEDLINE | ID: mdl-15695405

ABSTRACT

Hypoxia-inducible factor 1 (HIF-1) is the central mediator of cellular responses to low oxygen and has recently become an important therapeutic target for solid tumor therapy. Inhibition of HIF-1 is expected to result in the attenuation of hypoxia-inducible genes, which are vital to many aspects of tumor biology, including adaptative responses for survival under anaerobic conditions. To identify small molecules inhibiting the HIF-1 pathway, we did a biological screen on a 10,000-membered natural product-like combinatorial library. The compounds of the library, which share a 2,2-dimethylbenzopyran structural motif, were tested for their ability to inhibit the hypoxic activation of an alkaline phosphatase reporter gene under the control of hypoxia-responsive elements in human glioma cells. This effort led to the discovery of 103D5R, a novel small-molecule inhibitor of HIF-1alpha. 103D5R markedly decreased HIF-1alpha protein levels induced by hypoxia or cobaltous ions in a dose- and time-dependent manner, whereas minimally affecting global cellular protein expression levels, including that of control proteins such as HIF-1beta, IkappaBalpha, and beta-actin. The inhibitory activity of 103D5R against HIF-1alpha was clearly shown under normoxia and hypoxia in cells derived from different cancer types, including glioma, prostate, and breast cancers. This inhibition prevented the activation of HIF-1 target genes under hypoxia such as vascular endothelial growth factor (VEGF) and glucose transporter-1 (Glut-1). Investigations into the molecular mechanism showed that 103D5R strongly reduced HIF-1alpha protein synthesis, whereas HIF-1alpha mRNA levels and HIF-1alpha degradation were not affected. 103D5R inhibited the phosphorylation of Akt, Erk1/2, and stress-activated protein kinase/c-jun-NH(2)-kinase, without changing the total levels of these proteins. Further studies on the mechanism of action of 103D5R will likely provide new insights into its validity/applicability for the pharmacologic targeting of HIF-1alpha for therapeutic purposes.


Subject(s)
Benzopyrans/pharmacology , DNA-Binding Proteins/antagonists & inhibitors , Nuclear Proteins/antagonists & inhibitors , Transcription Factors/antagonists & inhibitors , Biological Factors/pharmacology , Breast Neoplasms/drug therapy , Cell Line, Tumor , Combinatorial Chemistry Techniques , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dose-Response Relationship, Drug , Glioblastoma/drug therapy , Humans , Hypoxia-Inducible Factor 1 , Hypoxia-Inducible Factor 1, alpha Subunit , Male , Nuclear Proteins/biosynthesis , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Prostatic Neoplasms/drug therapy , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Transcription Factors/biosynthesis , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic/drug effects , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/biosynthesis , Vascular Endothelial Growth Factor A/genetics
17.
Cell Biol Int ; 28(11): 773-80, 2004.
Article in English | MEDLINE | ID: mdl-15563399

ABSTRACT

Ca2+-regulated nuclear factor of activated T cell (NFAT) family members are transcription factors crucial for the expression of various cytokine and other immunoregulatory genes. Moreover, NFAT transcription factors are involved in the regulation of development, maturation and selection of thymocytes. Typically, the NFAT complex is made up of NFATc (NFATc1-4) protein and activator protein-1 (AP-1) transcription factor. AP-1 is a dimer consisting of two Jun proteins (homodimers) or Jun and Fos proteins (heterodimers). We have previously reported that NFAT DNA-binding activity significantly decreases in the thymus during glucocorticoid-induced apoptosis. In this study, we demonstrate that the expression and phosphorylation status of the NFAT proteins do not change during glucocorticoid-induced apoptosis. This suggests that glucocorticoids do not disturb a signal transduction pathway leading to the activation of NFATc proteins in thymocytes. Although the levels of particular Jun and Fos proteins do not decrease after glucocorticoid administration, the formation or DNA-binding activity of some AP-1 dimers is specifically abolished. Thus, the observed inhibition of NFAT transcription factor activity during glucocorticoid-induced apoptosis is likely to be a consequence of this perturbation or the lack of a proper AP-1 component.


Subject(s)
Apoptosis/drug effects , DNA-Binding Proteins/metabolism , Dexamethasone/pharmacology , Nuclear Proteins/metabolism , T-Lymphocytes/metabolism , Thymus Gland/cytology , Transcription Factor AP-1/metabolism , Transcription Factors/metabolism , Animals , DNA/metabolism , DNA-Binding Proteins/genetics , Dimerization , Electrophoresis, Polyacrylamide Gel , Electrophoretic Mobility Shift Assay , Female , Mice , NFATC Transcription Factors , Nuclear Proteins/genetics , Phosphoproteins/genetics , Phosphoproteins/metabolism , Phosphorylation/drug effects , Proto-Oncogene Proteins c-fos/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Regulatory Sequences, Nucleic Acid/genetics , Signal Transduction , Transcription Factors/genetics
18.
Brain Pathol ; 13(4): 539-53, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14655759

ABSTRACT

Many malignant glioma cells express death receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), yet some of these cells are resistant to TRAIL. Here, we examined signaling events in TRAIL-induced apoptosis and searched for therapeutic agents that could overcome TRAIL resistance in glioma cells. TRAIL induced apoptosis through death receptor 5 (DR5) and was mediated by caspase-8-initiated extrinsic and intrinsic mitochondrial pathways in sensitive glioma cell lines. TRAIL also triggered apoptosis in resistant glioma cell lines through the same pathways, but only if the cells were pretreated with chemotherapeutic agents, cisplatin, camptothecin and etoposide. Previous studies suggested that this was due to an increase in DR5 expression in wild-type TP53 cells, but this mechanism did not account for cells with mutant TP53. Here, we show that a more general effect of these agents is to downregulate caspase-8 inhibitor c-FLIP(S) (the short form of cellular Fas-associated death domain-fike interleukin-1-converting enzyme-inhibitory protein) and up-regulate Bak, a pro-apoptotic Bcl-2 family member, independently of cell's TP53 status. Furthermore, we showed that TRAIL alone or in combination with chemotherapeutic agents, induced apoptosis in primary tumor cultures from patients with malignant gliomas, reinforcing the potential of TRAIL as an effective therapeutic agent for malignant gliomas.


Subject(s)
Apoptosis/physiology , Intracellular Signaling Peptides and Proteins , Membrane Glycoproteins/physiology , Signal Transduction/physiology , Tumor Necrosis Factor-alpha/physiology , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Apoptosis Inducing Factor , Apoptosis Regulatory Proteins , Blotting, Northern , Blotting, Western , Brain Neoplasms/pathology , CASP8 and FADD-Like Apoptosis Regulating Protein , Carrier Proteins/pharmacology , Caspases/metabolism , Caspases/pharmacology , Cytochromes c , Dose-Response Relationship, Drug , Drug Interactions , Enzyme Inhibitors/pharmacology , Flavoproteins/metabolism , Flow Cytometry , Glioma/pathology , Humans , Membrane Proteins/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , Mutation , Proteins/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Signal Transduction/drug effects , Subcellular Fractions/drug effects , Subcellular Fractions/metabolism , TNF-Related Apoptosis-Inducing Ligand , Temperature , Time Factors , Tumor Cells, Cultured , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , X-Linked Inhibitor of Apoptosis Protein , bcl-2 Homologous Antagonist-Killer Protein , rho GTP-Binding Proteins/metabolism
19.
Oncogene ; 22(48): 7617-27, 2003 Oct 23.
Article in English | MEDLINE | ID: mdl-14576824

ABSTRACT

Activation of Akt signalling pathway is frequently found in glioma cells and may contribute to their resistance to undergo apoptosis in response to conventional therapies. We found that cyclosporin A (CsA) induces apoptosis of C6 glioma cells, which is associated with transcriptional activation of fasL. In the present paper, we investigated an involvement of Akt signalling in the regulation of FasL expression in CsA-induced apoptosis. We demonstrated that the level of active Akt decreases significantly after CsA treatment, which results in the decrease of Forkhead phosphorylation and its translocation to the nucleus. It correlated with an increase of binding to the Forkhead-responsive element FHRE from the FasL promoter, as demonstrated by gel-shift assays. Although treatment with LY294002, a specific inhibitor of PI3 K, decreased the phosphorylation of Akt and increased Fkhr translocation to the nucleus, these events were not sufficient to induce FasL expression and apoptosis of C6 glioma cells. Interference with Akt/Forkhead signalling by membrane-targeted Akt or removal of the FKHR-binding sites from the FasL promoter significantly abolished its activation. These results indicate that downregulation of Akt signalling and activation of Forkhead is a prerequisite for the induction of FasL promoter. It may be clinically important for pharmacological intervention in gliomas.


Subject(s)
Apoptosis , Glioma/metabolism , Glioma/pathology , Membrane Glycoproteins/genetics , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/antagonists & inhibitors , Signal Transduction , Transcription Factors/metabolism , Up-Regulation , Animals , Apoptosis/drug effects , Cell Line, Tumor , Cyclosporine/pharmacology , Fas Ligand Protein , Forkhead Transcription Factors , Glioma/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Signal Transduction/drug effects , Transcription, Genetic , Up-Regulation/drug effects
20.
J Biol Chem ; 277(16): 14102-8, 2002 Apr 19.
Article in English | MEDLINE | ID: mdl-11827957

ABSTRACT

The tumor suppressor p53 can induce growth arrest and cell death via apoptosis in response to a number of cellular stresses. We have shown previously that the immunosuppressant cyclosporin A (CsA) induces programmed cell death with typical features of apoptosis in rat glioma cells. We report that CsA treatment results in increased level of the p53 tumor suppressor, its nuclear accumulation, and transcriptional activation of p53-dependent genes. The increase of p53 correlates with the elevation of p21(Waf1) and Bax protein expression. The increased level of Bax protein was accompanied with changes in its subcellular localization and association with mitochondria. Importantly, we demonstrate that glioma cells stably transfected with a mutant p53 (p53Val135) fail to increase p21 and Bax protein levels and are less sensitive to CsA-induced apoptosis. Furthermore, primary fibroblasts from p53-/- knockout mice are significantly more resistant to CsA-induced apoptosis compared with their corresponding counterparts containing functional p53. Together, our results suggest that the apoptotic program activated by CsA can be mediated by activation of p53 tumor suppressor and potentiation of its ability to initiate apoptosis.


Subject(s)
Apoptosis , Cyclosporine/pharmacology , Proto-Oncogene Proteins c-bcl-2 , Tumor Suppressor Protein p53/metabolism , Animals , Blotting, Northern , Blotting, Western , Cell Division , Cell Line , Cell Nucleus/metabolism , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/biosynthesis , Fibroblasts/metabolism , Genes, Dominant , Glioma/metabolism , Immunoblotting , Kinetics , Mice , Mice, Knockout , Microscopy, Fluorescence , Mitochondria/metabolism , Mitogen-Activated Protein Kinases/metabolism , Plasmids/metabolism , Proto-Oncogene Proteins/biosynthesis , RNA/metabolism , Rats , Time Factors , Transcription, Genetic , Transfection , Tumor Cells, Cultured , Up-Regulation , bcl-2-Associated X Protein , p38 Mitogen-Activated Protein Kinases
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