ABSTRACT
Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. Metastasis is the major cause of TNBC mortality. Angiogenesis facilitates TNBC metastases. Many TNBCs also form vascular channels lined by tumor cells rather than endothelial cells, known as 'vasculogenic mimicry' (VM). VM has been linked to metastatic TNBC behavior and resistance to anti-angiogenic agents. Epidermal growth factor receptor (EGFR) is frequently expressed on TNBC, but anti-EGFR antibodies have limited efficacy. We synthesized an anti-EGFR antibody-endostatin fusion protein, αEGFR IgG1-huEndo-P125A (αEGFR-E-P125A), designed to deliver a mutant endostatin, huEndo-P125A (E-P125A), to EGFR expressing tumors, and tested its effects on angiogenesis, TNBC VM, and motility in vitro, and on the growth and metastasis of two independent human TNBC xenograft models in vivo. αEGFR-E-P125A completely inhibited the ability of human umbilical vein endothelial cells to form capillary-like structures (CLS) and of TNBC cells to engage in VM and form tubes in vitro. αEGFR-E-P125A treatment reduced endothelial and TNBC motility in vitro more effectively than E-P125A or cetuximab, delivered alone or in combination. Treatment of TNBC with αEGFR-E-P125A was associated with a reduction in cytoplasmic and nuclear ß-catenin and reduced phosphorylation of vimentin. αEGFR-E-P125A treatment of TNBC xenografts in vivo inhibited angiogenesis and VM, reduced primary tumor growth and lung metastasis of orthotopically implanted MDA-MB-468 TNBC cells, and markedly decreased lung metastases following intravenous injection of MDA-MB-231-4175 lung-tropic TNBC cells. Combined inhibition of angiogenesis, VM, and TNBC motility mediated by αEGFR-E-P125A is a promising strategy for the prevention of TNBC metastases.
Subject(s)
Angiogenesis Inhibitors/therapeutic use , Endostatins/metabolism , ErbB Receptors/antagonists & inhibitors , Immunoglobulin G/metabolism , Recombinant Fusion Proteins/therapeutic use , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/pathology , Angiogenesis Inhibitors/pharmacology , Animals , Antibody-Dependent Cell Cytotoxicity/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , ErbB Receptors/metabolism , Female , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Matrix Metalloproteinases/metabolism , Mice , Neoplasm Metastasis , Neovascularization, Pathologic/drug therapy , Phosphorylation/drug effects , Phosphoserine/metabolism , Recombinant Fusion Proteins/pharmacology , Vimentin/metabolism , Wnt Signaling Pathway/drug effectsABSTRACT
Chondrosarcoma is the second most common primary malignant bone tumor and is resistant to chemotherapy and radiation. Inadequate treatment response and poor prognosis requires novel therapeutic approaches. Prolinerich polypeptide1 (PRP1), synthesized by brain neurosecretory cells, has demonstrated antitumor properties in JJ012cells; however, its underlying molecular mechanism remains unclear. The present study aimed to investigate the epigenetic regulation by which PRP1 inhibits chondrosarcoma cancer stem cell (CSC) proliferation and to elucidate additional CSC biomarkers in human chondrosarcoma other than ALDH1A1. Human chondrosarcoma JJ012cells were treated with PRP1 prior to performing an Aldefluor™ assay and fluorescenceactivated cell sorting in order to determine aldehyde dehydrogenase (ALDH) expression levels and isolate ALDHhigh and ALDHlow cell populations. ALDH is an established marker of CSCs in several neoplasms, including chondrosarcoma. The cells were collected and lysed for gel electrophoresis, followed by western blot analysis. The Aldefluor™ assay was used to assess the expression levels of wellestablished CSC biomarkers, including CD133, CD4, CD10, CD144, CD177, CD221, CD271, leucinerich repeatcontaining G proteincoupled receptor 5, SOX2 and B lymphoma MoMLV insertion region 1 homolog (BMI1), within the ALDHhigh population of JJ012 cells. The results confirmed that ALDHA1 was the biomarker for chondrosarcoma CSCs. PRP1 was demonstrated to inhibit the ALDHhigh population colony and sarcosphere formation; 5 µg/ml PRP1 was indicated to be the optimum concentration in eliminating colonies formed by JJ012 cells (92%, P<0.001) and by the ALDHhigh CSCpopulation (80.5%, P<0.001) in the clonogenic doseresponse assay. Spheroid growth unequivocally decreased with an increase in PRP1 dose. In order to determine the molecular mechanism by which PRP1 decreased the CSC population, the regulation of the mammalian Switch/sucrose nonfermenting (SWI/SNF) complex, also referred to as BRG1associated factor (BAF) complex, which either activates or represses transcription, thus acting as an oncogene or tumor suppressor in human cells, was analyzed. PRP1 was demonstrated to decrease the expression levels of BRG, BAF170 and BRM; therefore, in JJ012 cells, these key players of the SWI/SNF (BAF) complex served an oncogenic role. The results of the present study demonstrated that PRP1 targets chromatinremodeling complexes; therefore, future efforts will be directed towards determining the interconnection between CSC maintenance, selfrenewal capacity and BAF complexes.
Subject(s)
Antimicrobial Cationic Peptides/pharmacology , Bone Neoplasms/metabolism , Chondrosarcoma/metabolism , Neoplastic Stem Cells/metabolism , Proto-Oncogene Proteins B-raf/metabolism , Aldehyde Dehydrogenase 1 Family/metabolism , Bone Neoplasms/drug therapy , Cell Line, Tumor , Cell Survival/drug effects , Chondrosarcoma/drug therapy , Chromatin/drug effects , Chromatin/genetics , Epigenesis, Genetic/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Neoplastic Stem Cells/drug effects , Retinal Dehydrogenase/metabolismABSTRACT
Chondrosarcomas are a heterogeneous group of malignant bone tumors that produce hyaline cartilaginous matrix. Mutations in isocitrate dehydrogenase enzymes (IDH1/2) were recently described in several cancers, including conventional and dedifferentiated chondrosarcomas. These mutations lead to the inability of IDH to convert isocitrate into α-ketoglutarate (α-KG). Instead, α-KG is reduced into D-2-hydroxyglutarate (D-2HG), an oncometabolite. IDH mutations and D-2HG are thought to contribute to tumorigenesis due to the role of D-2HG as a competitive inhibitor of α-KG-dependent dioxygenases. However, the function of IDH mutations in chondrosarcomas has not been clearly defined. In this study, we knocked out mutant IDH1 (IDH1mut) in two chondrosarcoma cell lines using the CRISPR/Cas9 system. We observed that D-2HG production, anchorage-independent growth, and cell migration were significantly suppressed in the IDH1mut knockout cells. Loss of IDH1mut also led to a marked attenuation of chondrosarcoma formation and D-2HG production in a xenograft model. In addition, RNA-Seq analysis of IDH1mut knockout cells revealed downregulation of several integrin genes, including those of integrin alpha 5 (ITGA5) and integrin beta 5 (ITGB5). We further demonstrated that deregulation of integrin-mediated processes contributed to the tumorigenicity of IDH1-mutant chondrosarcoma cells. Our findings showed that IDH1mut knockout abrogates chondrosarcoma genesis through modulation of integrins. This suggests that integrin molecules are appealing candidates for combinatorial regimens with IDH1mut inhibitors for chondrosarcomas that harbor this mutation.
ABSTRACT
Tumor necrosis factor receptor-associated factor 6 (TRAF6), an (K63) E3-ligase, plays a role in many biological processes and its activity is relevant in diffuse large B cell lymphoma (DLBCL) biology. Although molecules that trigger TRAF6 activation have been defined, those that stabilize TRAF6 and/or enhance TRAF6 function remain largely unclear. We found that TRAF6 amplifies pAKT signaling in DLBCL. Moreover, TRAF6 activation and stabilization of its ubiquitination profile are facilitated by smoothened (SMO), signal transducer of canonical Hedgehog signaling. Here, we report that SMO is needed to facilitate and maintain TRAF6-dependent elevated pAKT levels, and that the SMO/TRAF6 axis contributes to doxorubicin resistance in DLBCL. Mechanistically, we found that SMO, through its C-terminal tail, stabilizes and protects TRAF6 from degradation, an effect mediated by ubiquitin-specific protease-8. Moreover, this functional link between SMO and TRAF6 is reflected in DLBCL patients where high expression of both molecules correlates with poor prognosis. In summary, our study reveals a novel cell survival mechanism in which SMO stabilizes and protects TRAF6 from degradation. The axis SMO/TRAF6/AKT is highly relevant in the biology of DLBCL and is involved in doxorubicin resistance.
Subject(s)
Lymphoma, Large B-Cell, Diffuse/genetics , Signal Transduction/genetics , Smoothened Receptor/genetics , TNF Receptor-Associated Factor 6/genetics , Antibiotics, Antineoplastic/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Doxorubicin/pharmacology , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , HEK293 Cells , Humans , Lymphoma, Large B-Cell, Diffuse/metabolism , Lymphoma, Large B-Cell, Diffuse/pathology , Protein Stability , Proteolysis , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , RNA Interference , Smoothened Receptor/metabolism , TNF Receptor-Associated Factor 6/metabolism , UbiquitinationABSTRACT
Signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) mutations in X-linked lymphoproliferative disease (XLP) lead to defective NKT cell development and impaired humoral immunity. Because of the redundancy of SLAM family receptors (SFRs) and the complexity of SAP actions, how SFRs and SAP mediate these processes remains elusive. Here, we examined NKT cell development and humoral immunity in mice completely deficient in SFR. We found that SFR deficiency severely impaired NKT cell development. In contrast to SAP deficiency, SFR deficiency caused no apparent defect in follicular helper T (TFH) cell differentiation. Intriguingly, the deletion of SFRs completely rescued the severe defect in TFH cell generation caused by SAP deficiency, whereas SFR deletion had a minimal effect on the defective NKT cell development in SAP-deficient mice. These findings suggest that SAP-dependent activating SFR signaling is essential for NKT cell selection; however, SFR signaling is inhibitory in SAP-deficient TFH cells. Thus, our current study revises our understanding of the mechanisms underlying T cell defects in patients with XLP.
Subject(s)
Natural Killer T-Cells/physiology , Signal Transduction/physiology , Signaling Lymphocytic Activation Molecule Family Member 1/physiology , Signaling Lymphocytic Activation Molecule Family/physiology , Animals , Antigens, Ly/physiology , CARD Signaling Adaptor Proteins/physiology , Immunity, Humoral , Kruppel-Like Transcription Factors/biosynthesis , Lymphoproliferative Disorders/genetics , Mice , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases/physiology , Promyelocytic Leukemia Zinc Finger Protein , Signaling Lymphocytic Activation Molecule Associated Protein/physiologyABSTRACT
Cell death and survival signaling pathways have opposed but fundamental functions for various cellular processes and maintain cell homeostasis through cross talk. Here we report a novel mechanism of interaction between these two pathways through the cleavage of RNF31 by caspases. RNF31, a component of the linear ubiquitin chain assembly complex (LUBAC), regulates cell survival by inducing linear ubiquitination of NF-κB signaling components. We found that RNF31 is cleaved under apoptosis conditions through various stimulations. The effector caspases caspase 3 and caspase 6 are responsible for this event, and aspartates 348, 387, and 390 were identified as target sites for this cleavage. Cleavage of RNF31 suppressed its ability to activate NF-κB signaling; thus, mutation of cleavage sites inhibited the induction of apoptosis by treatment with tumor necrosis factor alpha (TNF-α). Our findings elucidate a novel regulatory loop between cell death and the survival signal and may provide guidance for the development of therapeutic strategies for cancers through the sensitization of tumor cells to death-inducing drugs.
Subject(s)
Aspartic Acid/metabolism , Caspase 3/metabolism , Caspase 6/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Ubiquitin-Protein Ligase Complexes/metabolism , Ubiquitin-Protein Ligases/metabolism , A549 Cells , Apoptosis/drug effects , Aspartic Acid/genetics , Binding Sites/drug effects , Cell Line , Cell Survival/drug effects , Gene Expression Regulation , HCT116 Cells , HEK293 Cells , HT29 Cells , HeLa Cells , Humans , Mutation , NF-kappa B/metabolism , Proteolysis , Signal Transduction/drug effects , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , Ubiquitination/drug effectsABSTRACT
In this issue of Blood, Juilland and colleagues reveal the expression pattern and the role of different members of the activating transcription factor (ATF) family in survival of diffuse large B-cell lymphoma (DLBCL) cells.
Subject(s)
CARD Signaling Adaptor Proteins/metabolism , Gene Expression Regulation, Neoplastic , Guanylate Cyclase/metabolism , Lymphoma, Large B-Cell, Diffuse/metabolism , Myeloid Differentiation Factor 88/metabolism , Transcription Factor AP-1/metabolism , HumansABSTRACT
GLI1 oncogene has been implicated in the pathobiology of several neoplasms including diffuse large B-cell lymphoma (DLBCL). However, mechanisms underlying GLI1-increased activity in DLBCL are poorly characterized. Herein, we demonstrate that IKKß phosphorylates GLI1 in DLBCL. IKKß activation increased GLI1 protein levels and transcriptional activity, whereas IKKß silencing decreased GLI1 levels and transcriptional activity. Tumor necrosis factor-α (TNFα) mediated IKKß activation-impaired GLI1 binding with the E3 ubiquitin ligase-ITCH, leading to decreased K48-linked ubiquitination/degradation of GLI1. We found 8 IKKß-dependent phosphorylation sites that mediate GLI1 stability. Mutating or deleting these residues facilitated GLI1-ITCH interaction and decreased the protective effect of TNFα on GLI1 stability. IKKß-GLI1 crosstalk is significant because combined inhibition of both molecules resulted in synergistic suppression of DLBCL viability in vivo and in vitro. By linking IKKß-mediated nuclear factor-κB activity with GLI1, we identified a crosstalk between these 2 pathways that can inform the design of novel therapeutic strategies in DLBCL.
Subject(s)
I-kappa B Kinase/metabolism , Lymphoma, Large B-Cell, Diffuse/metabolism , Transcription Factors/metabolism , Cell Line, Tumor , Cell Survival , Humans , Lymphoma, Large B-Cell, Diffuse/genetics , NF-kappa B/metabolism , Phosphorylation , Protein Stability , Repressor Proteins/metabolism , Transcription Factors/chemistry , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Zinc Finger Protein GLI1ABSTRACT
Extensive reprogramming of cellular energy metabolism is a hallmark of cancer. Despite its importance, the molecular mechanism controlling this tumour metabolic shift remains not fully understood. Here we show that 14-3-3σ regulates cancer metabolic reprogramming and protects cells from tumorigenic transformation. 14-3-3σ opposes tumour-promoting metabolic programmes by enhancing c-Myc poly-ubiquitination and subsequent degradation. 14-3-3σ demonstrates the suppressive impact on cancer glycolysis, glutaminolysis, mitochondrial biogenesis and other major metabolic processes of tumours. Importantly, 14-3-3σ expression levels predict overall and recurrence-free survival rates, tumour glucose uptake and metabolic gene expression in breast cancer patients. Thus, these results highlight that 14-3-3σ is an important regulator of tumour metabolism, and loss of 14-3-3σ expression is critical for cancer metabolic reprogramming. We anticipate that pharmacologically elevating the function of 14-3-3σ in tumours could be a promising direction for targeted anticancer metabolism therapy development in future.
Subject(s)
14-3-3 Proteins/genetics , Biomarkers, Tumor/genetics , Breast Neoplasms/genetics , Energy Metabolism/genetics , Exoribonucleases/genetics , Gene Expression Regulation, Neoplastic , Proto-Oncogene Proteins c-myc/metabolism , 14-3-3 Proteins/metabolism , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/metabolism , Breast Neoplasms/metabolism , Cell Line, Tumor , Disease-Free Survival , Exoribonucleases/metabolism , Female , Gene Knockout Techniques , Glutamine/metabolism , Glycolysis/genetics , HCT116 Cells , Humans , Middle Aged , Organelle Biogenesis , Prognosis , Proteolysis , Ubiquitination/genetics , Young AdultABSTRACT
Lymphomas develop and progress in a specialized tissue microenvironment such as bone marrow as well as secondary lymphoid organs such as lymph node and spleen. The lymphoma microenvironment is characterized by a heterogeneous population of stromal cells, including fibroblastic reticular cells, nurse-like cells, mesenchymal stem cells, follicular dendritic cells, and inflammatory cells such as macrophages, T- and B-cells. These cell populations interact with the lymphoma cells to promote lymphoma growth, survival and drug resistance through multiple mechanisms. Angiogenesis is also recognized as an important factor associated with lymphoma progression. In recent years, we have learned that the interaction between the malignant and non-malignant cells is bidirectional and resembles, at least in part, the pattern seen between non-neoplastic lymphoid cells and the normal microenvironment of lymphoid organs. A summary of the current knowledge of lymphoma microenvironment focusing on the cellular components will be reviewed here.
Subject(s)
Lymphoma/etiology , Lymphoma/pathology , Tumor Microenvironment , Animals , Cell Survival , Endothelial Cells/metabolism , Humans , Lymphoma/metabolism , Lymphoma, B-Cell/etiology , Lymphoma, B-Cell/metabolism , Lymphoma, B-Cell/pathology , Mesenchymal Stem Cells/metabolism , Neovascularization, Pathologic , Signal Transduction , Stromal Cells/metabolism , Stromal Cells/pathology , Tumor Microenvironment/genetics , Tumor Microenvironment/immunologyABSTRACT
Diffuse large B-cell lymphoma (DLBCL) is an aggressive disease with a high proliferation rate. However, the molecular and genetic features that drive the aggressive clinical behavior of DLBCL are not fully defined. Here, we have demonstrated that activated Jun signaling is a frequent event in DLBCL that promotes dissemination of malignant cells. Downregulation of Jun dramatically reduces lymphoma cell adhesion to extracellular matrix proteins, subcutaneous tumor size in nude mice, and invasive behavior, including bone marrow infiltration and interaction with bone marrow stromal cells. Furthermore, using a combination of RNA interference and gene expression profiling, we identified Jun target genes that are associated with disseminated lymphoma. Among them, ITGAV, FoxC1, and CX3CR1 are significantly enriched in patients with 2 or more extranodal sites. Our results point to activated Jun signaling as a major driver of the aggressive phenotype of DLBCL.
Subject(s)
Gene Expression Regulation, Neoplastic , Lymphoma, Large B-Cell, Diffuse/genetics , Lymphoma, Large B-Cell, Diffuse/pathology , Proto-Oncogene Proteins c-jun/metabolism , Animals , Cell Adhesion , Cell Line, Tumor , Extracellular Matrix Proteins/metabolism , Gene Expression Profiling , Gene Knockdown Techniques , Humans , Lymphoma, Large B-Cell, Diffuse/metabolism , Mice, Nude , Proto-Oncogene Proteins c-jun/genetics , Signal Transduction , Transcription Factor AP-1/genetics , Transcription Factors/metabolismABSTRACT
Glutamine has been implicated as an immunomodulatory nutrient, but how glutamine uptake is mediated during T cell activation is poorly understood. We have shown that naive T cell activation is coupled with rapid glutamine uptake, which depended on the amino acid transporter ASCT2. ASCT2 deficiency impaired the induction of T helper 1 (Th1) and Th17 cells and attenuated inflammatory T cell responses in mouse models of immunity and autoimmunity. Mechanistically, ASCT2 was required for T cell receptor (TCR)-stimulated activation of the metabolic kinase mTORC1. We have further shown that TCR-stimulated glutamine uptake and mTORC1 activation also required a TCR signaling complex composed of the scaffold protein CARMA1, the adaptor molecule BCL10, and the paracaspase MALT1. This function was independent of IKK kinase, a major downstream target of the CARMA1 complex. These findings highlight a mechanism of T cell activation involving ASCT2-dependent integration of the TCR signal and a metabolic signaling pathway.
Subject(s)
Amino Acid Transport System ASC/immunology , Glutamine/metabolism , Multiprotein Complexes/metabolism , Receptors, Antigen, T-Cell/immunology , TOR Serine-Threonine Kinases/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Adoptive Transfer , Amino Acid Transport System ASC/genetics , Amino Acid Transport System ASC/metabolism , Animals , B-Cell CLL-Lymphoma 10 Protein , Biological Transport , CARD Signaling Adaptor Proteins/metabolism , CD28 Antigens/immunology , Caspases/metabolism , Cell Differentiation/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/pathology , Enzyme Activation/immunology , Humans , Inflammation/immunology , Interleukin-2/biosynthesis , Jurkat Cells , Leucine/metabolism , Lymphocyte Activation/immunology , Mechanistic Target of Rapamycin Complex 1 , Mice , Mice, Inbred C57BL , Mice, Knockout , Minor Histocompatibility Antigens , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein , Neoplasm Proteins/metabolism , Signal Transduction/immunology , Th1 Cells/immunologyABSTRACT
The proto-oncogene c-Maf is a transcription factor that plays a critical role in the differentiation of various T helper (T(H)) cell subsets. The amount of c-Maf increases after stimulation of the T cell receptor (TCR), which results in the production of multiple cytokines. We showed that two essential regulators of the transcription factor nuclear factor κB (NF-κB), the scaffold protein CARMA1 and the kinase IKKß [inhibitor of NF-κB (IκB) kinase ß], are also critical for the activation of c-Maf. Although CARMA1 deficiency did not affect the TCR-dependent increase in c-Maf abundance in T cells, CARMA1-dependent activation of the IKK complex was required for the nuclear translocation of c-Maf and its binding to the promoters of its target genes. Consistent with a role for c-Maf in the development of T follicular helper (T(FH)) cells, which provide help to B cells in the germinal centers of the spleen, CARMA1- or IKKß-deficient mice immunized with peptide antigen had defects in the generation of T(FH) cells, formation of germinal centers, and production of antigen-specific antibodies. Together, these data suggest a mechanism by which c-Maf is regulated during T cell activation and differentiation.
Subject(s)
CARD Signaling Adaptor Proteins/metabolism , Guanylate Cyclase/metabolism , I-kappa B Kinase/metabolism , Proto-Oncogene Proteins c-maf/metabolism , Signal Transduction , CD4-Positive T-Lymphocytes/enzymology , Humans , Interleukins/biosynthesis , Jurkat Cells , Ovalbumin/administration & dosage , Proto-Oncogene MasABSTRACT
Mantle cell lymphoma (MCL) remains incurable due to its inevitable pattern of relapse after treatment with current existing therapies. However, the promise of a cure for MCL lies in the burgeoning area of novel agents. In this study, we elucidated the therapeutic effect and mechanism of carfilzomib, a novel long-acting second-generation proteasome inhibitor, in MCL cells. We found that carfilzomib induced growth inhibition and apoptosis in both established MCL cell lines and freshly isolated primary MCL cells in a dose-dependent manner. In contrast, carfilzomib was less toxic to normal peripheral blood mononuclear cells from healthy individuals. The carfilzomib-induced apoptosis of MCL cells was mediated by the activation of JNK, Bcl-2, and mitochondria-related pathways. In addition, carfilzomib inhibited the growth and survival signaling pathways NF-κB and STAT3. Interestingly, we discovered that expression of immunoproteasome (i-proteasome) subunits is required for the anti-MCL activity of carfilzomib in MCL cells. In MCL-bearing SCID mice/primary MCL-bearing SCID-hu mice, intravenous administration of 5 mg/kg carfilzomib on days 1 and 2 for 5 weeks slowed/abrogated tumor growth and significantly prolonged survival. Our preclinical data show that carfilzomib is a promising, potentially less toxic treatment for MCL. Furthermore, an intact i-proteasome, especially LMP2, appears to be necessary for its anti-MCL activity, suggesting that i-proteasome could serve as a biomarker for identifying patients who will benefit from carfilzomib.
Subject(s)
Antineoplastic Agents/therapeutic use , Lymphoma, Mantle-Cell/drug therapy , Molecular Targeted Therapy/methods , Oligopeptides/therapeutic use , Proteasome Endopeptidase Complex/metabolism , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Resistance, Neoplasm , Humans , Lymphoma, Mantle-Cell/pathology , Male , Mice , Mice, SCID , Mitochondria/metabolism , Oligopeptides/pharmacology , Proteasome Endopeptidase Complex/genetics , Signal Transduction/drug effects , Signal Transduction/geneticsABSTRACT
Reversible ubiquitin modification of cell signaling molecules has emerged as a critical mechanism by which cells respond to extracellular stimuli. Although ubiquitination of TGF-ß-activated kinase 1 (TAK1) is critical for NF-κB activation in T cells, the regulation of its deubiquitination is unclear. We show that USP18, which was previously reported to be important in regulating type I interferon signaling in innate immunity, regulates T cell activation and T helper 17 (Th17) cell differentiation by deubiquitinating the TAK1-TAB1 complex. USP18-deficient T cells are defective in Th17 differentiation and Usp18(-/-) mice are resistant to experimental autoimmune encephalomyelitis (EAE). In response to T cell receptor engagement, USP18-deficient T cells exhibit hyperactivation of NF-κB and NFAT and produce increased levels of IL-2 compared with the wild-type controls. Importantly, USP18 is associated with and deubiquitinates the TAK1-TAB1 complex, thereby restricting expression of IL-2. Our findings thus demonstrate a previously uncharacterized negative regulation of TAK1 activity during Th17 differentiation, suggesting that USP18 may be targeted to treat autoimmune diseases.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Endopeptidases/metabolism , MAP Kinase Kinase Kinases/metabolism , NF-kappa B/metabolism , NFATC Transcription Factors/metabolism , Th17 Cells/cytology , Th17 Cells/metabolism , Animals , Autoimmune Diseases/immunology , Autoimmune Diseases/metabolism , Catalysis , Cell Differentiation/genetics , Cell Differentiation/immunology , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/metabolism , Endopeptidases/genetics , Gene Expression , Gene Knockout Techniques , Interleukin-2/antagonists & inhibitors , Interleukin-2/metabolism , Mice , Mice, Knockout , Protein Binding , Receptors, Antigen, T-Cell/metabolism , Signal Transduction , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Th17 Cells/immunology , Ubiquitin Thiolesterase , UbiquitinationABSTRACT
Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults. Aberrant activation of Hedgehog (Hh) and nuclear factor (NF)-κB pathways is ubiquitously observed and known to mediate tumor growth, survival, and chemoresistance in DLBCL. Here, we find that activation of Hh signaling is positively correlated with NF-κB pathway in DLBCL tumors, and that smoothened (SMO), the signal transducer subunit of Hh pathway, contributes to NF-κB activation through recruiting G protein subunits Gαi and Gα12 to activate PKCß/CARMA1/TRAF6/NEMO signaling axis followed by assembling of the CARMA1/BCL10/MALT1/TRAF6 complex to SMO. Moreover, functional inhibition of SMO enhances the cytotoxic effects of NF-κB inhibitor. Altogether, our study reveals a noncanonical Hh signaling pathway in which SMO activates trimeric G proteins and CARMA1-associated signaling complex, leading to NF-κB activation. This signaling cascade contributes to the survival of DLBCL and may serve as a potential target for combination therapies in DLBCL.
Subject(s)
CARD Signaling Adaptor Proteins/metabolism , Guanylate Cyclase/metabolism , Hedgehog Proteins/metabolism , Heterotrimeric GTP-Binding Proteins/metabolism , Lymphoma, Large B-Cell, Diffuse/pathology , NF-kappa B/metabolism , Receptors, G-Protein-Coupled/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Adult , Apoptosis , B-Cell CLL-Lymphoma 10 Protein , Blotting, Western , CARD Signaling Adaptor Proteins/genetics , Caspases/genetics , Caspases/metabolism , Cell Proliferation , Cytokines/metabolism , Electrophoretic Mobility Shift Assay , Enzyme-Linked Immunosorbent Assay , Guanylate Cyclase/genetics , Hedgehog Proteins/genetics , Heterotrimeric GTP-Binding Proteins/genetics , Humans , I-kappa B Kinase/genetics , I-kappa B Kinase/metabolism , Immunoenzyme Techniques , Immunoprecipitation , Luciferases/metabolism , Lymphoma, Large B-Cell, Diffuse/genetics , Lymphoma, Large B-Cell, Diffuse/metabolism , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein , NF-kappa B/genetics , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Protein Kinase C/genetics , Protein Kinase C/metabolism , Protein Kinase C beta , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Real-Time Polymerase Chain Reaction , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Smoothened Receptor , TNF Receptor-Associated Factor 6/genetics , TNF Receptor-Associated Factor 6/metabolism , Tissue Array Analysis , Tumor Cells, CulturedABSTRACT
Activation of NF-κB transcription factor is crucial for survival, proliferation, and differentiation of T cells. In this issue of Immunity, Paul et al. (2012) demonstrate that autophagy is a pathway by which TCR-activated NF-κB is turned over.
ABSTRACT
The scaffold protein CARMA1 is required for the TCR-induced lymphocyte activation. In this study, we show that CARMA1 also plays an essential role in T cell differentiation. We have found that the adoptive transfer of bone marrow cells expressing constitutively active CARMA1 results in lung inflammation, eosinophilia, and elevated levels of IL-4, IL-5, and IL-10 in recipient mice. In contrast, CARMA1-deficient T cells are defective in TCR-induced expression of Th2 cytokines, suggesting that CARMA1 preferentially directs Th2 differentiation. The impaired cytokine production is due to reduced expression of JunB and GATA3 transcription factors. CARMA1 deficiency affects JunB stability resulting in its enhanced ubiquitination and degradation. In contrast, TCR-dependent induction of GATA3 is suppressed at the transcriptional level. We also found that supplementation with IL-4 partially restored GATA3 expression in CARMA1-deficient CD4(+) splenocytes and subsequently production of GATA3-dependent cytokines IL-5 and IL-13. Therefore, our work provides the mechanism by which CARMA1 regulates Th2 cell differentiation.
Subject(s)
CARD Signaling Adaptor Proteins/physiology , GATA3 Transcription Factor/biosynthesis , Interleukins/metabolism , Proto-Oncogene Proteins c-jun/biosynthesis , Th2 Cells/immunology , Animals , CARD Signaling Adaptor Proteins/deficiency , CARD Signaling Adaptor Proteins/genetics , GATA3 Transcription Factor/genetics , Gene Expression Regulation , Genetic Vectors/genetics , Humans , Interleukin-4/pharmacology , Interleukins/biosynthesis , Interleukins/genetics , Lymphocyte Activation , Mice , Mice, Knockout , Ovalbumin/toxicity , Proto-Oncogene Proteins c-jun/genetics , Pulmonary Eosinophilia/genetics , Radiation Chimera , Recombinant Fusion Proteins/physiology , Sequence Deletion , Specific Pathogen-Free Organisms , Th2 Cells/drug effects , Th2 Cells/metabolism , TransfectionABSTRACT
The NF-κB family of transcription factors plays a crucial role in cell activation, survival and proliferation. Its aberrant activity results in cancer, immunodeficiency or autoimmune disorders. Over the past two decades, tremendous progress has been made in our understanding of the signals that regulate NF-κB activation, especially how scaffold proteins link different receptors to the NF-κB-activating complex, the IκB kinase complex. The growing number of these scaffolds underscores the complexity of the signaling networks in different cell types. In this review, we discuss the role of scaffold molecules in signaling cascades induced by stimulation of antigen receptors, G-protein-coupled receptors and C-type Lectin receptors, resulting in NF-κB activation. Especially, we focus on the family of Caspase recruitment domain (CARD)-containing proteins known as CARMA and their function in activation of NF-κB, as well as the link of these scaffolds to the development of various neoplastic diseases through regulation of NF-κB.
Subject(s)
CARD Signaling Adaptor Proteins/metabolism , Guanylate Cyclase/metabolism , Membrane Proteins/metabolism , NF-kappa B/metabolism , Adaptor Proteins, Signal Transducing/metabolism , B-Cell CLL-Lymphoma 10 Protein , CARD Signaling Adaptor Proteins/chemistry , Caspases/metabolism , Guanylate Cyclase/chemistry , Humans , Membrane Proteins/chemistry , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein , NF-kappa B/physiology , Neoplasm Proteins/metabolism , Receptors, Antigen, T-Cell/metabolism , Signal TransductionABSTRACT
Activation of transcription factor nuclear factor-kappaB (NF-kappaB) and Jun N-terminal kinase (JNK) play the pivotal roles in regulation of lymphocyte activation and proliferation. Deregulation of these signaling pathways leads to inappropriate immune response and contributes to the development of leukemia/lymphoma. The scaffold protein CARMA1 [caspase-recruitment domain (CARD) membrane-associated guanylate kinase (MAGUK) protein 1] has a central role in regulation of NF-kappaB and the JNK2/c-Jun complex in both B and T lymphocytes. During last several years, tremendous work has been done to reveal the mechanism by which CARMA1 and its signaling partners, B cell CLL-lymphoma 10 and mucosa-associated lymphoid tissue 1, are activated and mediate NF-kappaB and JNK activation. In this review, we summarize our findings in revealing the roles of CARMA1 in the NF-kappaB and JNK signaling pathways in the context of recent advances in this field.