ABSTRACT
Immune cells need to sustain a state of constant alertness over a lifetime. Yet, little is known about the regulatory processes that control the fluent and fragile balance that is called homeostasis. Here we demonstrate that JAK-STAT signaling, beyond its role in immune responses, is a major regulator of immune cell homeostasis. We investigated JAK-STAT-mediated transcription and chromatin accessibility across 12 mouse models, including knockouts of all STAT transcription factors and of the TYK2 kinase. Baseline JAK-STAT signaling was detected in CD8+ T cells and macrophages of unperturbed mice-but abrogated in the knockouts and in unstimulated immune cells deprived of their normal tissue context. We observed diverse gene-regulatory programs, including effects of STAT2 and IRF9 that were independent of STAT1. In summary, our large-scale dataset and integrative analysis of JAK-STAT mutant and wild-type mice uncovered a crucial role of JAK-STAT signaling in unstimulated immune cells, where it contributes to a poised epigenetic and transcriptional state and helps prepare these cells for rapid response to immune stimuli.
Subject(s)
Homeostasis , Janus Kinases , Macrophages , Mice, Knockout , STAT Transcription Factors , Signal Transduction , Animals , Mice , Macrophages/immunology , Macrophages/metabolism , Janus Kinases/metabolism , STAT Transcription Factors/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , STAT1 Transcription Factor/metabolism , STAT1 Transcription Factor/genetics , Mice, Inbred C57BL , Interferon-Stimulated Gene Factor 3, gamma Subunit/metabolism , Interferon-Stimulated Gene Factor 3, gamma Subunit/genetics , TYK2 Kinase/metabolism , TYK2 Kinase/genetics , Gene Expression RegulationABSTRACT
Tyrosine kinase 2 (TYK2) is a member of the Janus kinase/signal transducer and activator of transcription pathway, which is central in cytokine signaling. Previously, germline TYK2 mutations have been described in two patients developing de novo T-cell acute lymphoblastic leukemias (T-ALL) or precursor B-ALL. The mutations (P760L and G761V) are located within the regulatory pseudokinase domain and lead to constitutive activation of TYK2. We demonstrate the transformation capacity of TYK2 P760L in hematopoietic cell systems including primary bone marrow cells. In vivo engraftment of TYK2 P760L-expressing cell lines led to development of leukemia. A kinase inhibitor screen uncovered that oncogenic TYK2 acts synergistically with the PI3K/AKT/mTOR and CDK4/6 pathways. Accordingly, the TYK2-specific inhibitor deucravacitinib (BMS986165) reduces cell viability of TYK2 P760L-transformed cell models and ex vivo cultured TYK2 P760L-mutated patient- derived xenograft cells most efficiently when combined with mTOR or CDK4/6 inhibitors. Our study thereby pioneers novel treatment options for patients suffering from TYK2-driven acute leukemia.
Subject(s)
Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , TYK2 Kinase , Humans , Cell Line , Cyclin-Dependent Kinase 4 , Phosphatidylinositol 3-Kinases , TOR Serine-Threonine Kinases , TYK2 Kinase/genetics , TYK2 Kinase/metabolismABSTRACT
Tyrosine kinase 2 (TYK2) is a widely expressed receptor-associated kinase that is involved in signaling by a variety of cytokines with important immune regulatory activities. Absence of TYK2 in mice results in impaired NK cell maturation and antitumor activity, although underlying mechanisms are largely unknown. Using conditional ablation of TYK2 in NK cells we show that TYK2 is required for IFN-γ production by NK cells in response to IL-12 and for an efficient immune defense against Listeria monocytogenes Deletion of TYK2 in NK cells did not impact NK cell maturation and IFN-γ production upon NK cell activating receptor (actR) stimulation. Similarly, NK cell-mediated tumor surveillance was unimpaired upon deletion of TYK2 in NK cells only. In line with the previously reported maturation-associated Ifng promoter demethylation, the less mature phenotype of Tyk2-/- NK cells correlated with an increased CpG methylation at the Ifng locus. Treatment with the DNA hypomethylating agent 5-aza-2-deoxycytidine restored the ability of Tyk2-/- NK cells to produce IFN-γ upon actR but not upon IL-12 stimulation. NK cell maturation was dependent on the presence of TYK2 in dendritic cells and could be rescued in Tyk2-deficient mice by treatment with exogenous IL-15/IL-15Rα complexes. IL-15 treatment also rescued the in vitro cytotoxicity defect and the impaired actR-induced IFN-γ production of Tyk2-/- NK cells. Collectively, our findings provide the first evidence, to our knowledge, for a key role of TYK2 in the host environment in promoting NK cell maturation and antitumor activity.
Subject(s)
Bacterial Infections/immunology , Immunity, Innate/immunology , Immunologic Surveillance/immunology , Killer Cells, Natural/immunology , Neoplasms/immunology , TYK2 Kinase/immunology , Animals , Lymphocyte Activation/immunology , Mice , Mice, KnockoutABSTRACT
Human melanocytic nevi (moles) are benign lesions harboring activated oncogenes, including BRAF. Although this oncogene initially acts mitogenically, eventually, oncogene-induced senescence (OIS) ensues. Nevi can infrequently progress to melanomas, but the mechanistic relationship with OIS is unclear. We show here that PTEN depletion abrogates BRAF(V600E)-induced senescence in human fibroblasts and melanocytes. Correspondingly, in established murine BRAF(V600E)-driven nevi, acute shRNA-mediated depletion of PTEN prompted tumor progression. Furthermore, genetic analysis of laser-guided microdissected human contiguous nevus-melanoma specimens recurrently revealed identical mutations in BRAF or NRAS in adjacent benign and malignant melanocytes. The PI3K pathway was often activated through either decreased PTEN or increased AKT3 expression in melanomas relative to their adjacent nevi. Pharmacologic PI3K inhibition in melanoma cells suppressed proliferation and induced the senescence-associated tumor suppressor p15(INK4B). This treatment also eliminated subpopulations resistant to targeted BRAF(V600E) inhibition. Our findings suggest that a significant proportion of melanomas arise from nevi. Furthermore, these results demonstrate that PI3K pathway activation serves as a rate-limiting event in this setting, acting at least in part by abrogating OIS. The reactivation of senescence features and elimination of cells refractory to BRAF(V600E) inhibition by PI3K inhibition warrants further investigation into the therapeutic potential of simultaneously targeting these pathways in melanoma.
Subject(s)
Cellular Senescence , Melanoma/pathology , Nevus/pathology , PTEN Phosphohydrolase/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins B-raf/metabolism , Skin Neoplasms/pathology , Amino Acid Substitution , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p15/metabolism , Enzyme Activation , Fibroblasts/metabolism , Fibroblasts/pathology , Glutamic Acid/genetics , Glutamic Acid/metabolism , Humans , Melanocytes/metabolism , Melanocytes/pathology , Melanoma/metabolism , Nevus/metabolism , PTEN Phosphohydrolase/genetics , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins c-akt/metabolism , Skin Neoplasms/metabolism , Valine/genetics , Valine/metabolismABSTRACT
In response to tenacious stress signals, such as the unscheduled activation of oncogenes, cells can mobilize tumour suppressor networks to avert the hazard of malignant transformation. A large body of evidence indicates that oncogene-induced senescence (OIS) acts as such a break, withdrawing cells from the proliferative pool almost irreversibly, thus crafting a vital pathophysiological mechanism that protects against cancer. Despite the widespread contribution of OIS to the cessation of tumorigenic expansion in animal models and humans, we have only just begun to define the underlying mechanism and identify key players. Although deregulation of metabolism is intimately linked to the proliferative capacity of cells, and senescent cells are thought to remain metabolically active, little has been investigated in detail about the role of cellular metabolism in OIS. Here we show, by metabolic profiling and functional perturbations, that the mitochondrial gatekeeper pyruvate dehydrogenase (PDH) is a crucial mediator of senescence induced by BRAF(V600E), an oncogene commonly mutated in melanoma and other cancers. BRAF(V600E)-induced senescence was accompanied by simultaneous suppression of the PDH-inhibitory enzyme pyruvate dehydrogenase kinase 1 (PDK1) and induction of the PDH-activating enzyme pyruvate dehydrogenase phosphatase 2 (PDP2). The resulting combined activation of PDH enhanced the use of pyruvate in the tricarboxylic acid cycle, causing increased respiration and redox stress. Abrogation of OIS, a rate-limiting step towards oncogenic transformation, coincided with reversion of these processes. Further supporting a crucial role of PDH in OIS, enforced normalization of either PDK1 or PDP2 expression levels inhibited PDH and abrogated OIS, thereby licensing BRAF(V600E)-driven melanoma development. Finally, depletion of PDK1 eradicated melanoma subpopulations resistant to targeted BRAF inhibition, and caused regression of established melanomas. These results reveal a mechanistic relationship between OIS and a key metabolic signalling axis, which may be exploited therapeutically.
Subject(s)
Cellular Senescence/genetics , Mitochondria/enzymology , Oncogenes/genetics , Pyruvate Dehydrogenase Complex/metabolism , Animals , Cell Line , Citric Acid Cycle , Disease Models, Animal , Enzyme Activation , Glycolysis , Humans , Melanoma/drug therapy , Melanoma/enzymology , Melanoma/genetics , Melanoma/pathology , Mice , Mice, Inbred NOD , Mice, SCID , Mitochondria/metabolism , Molecular Targeted Therapy , Oxidative Phosphorylation , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/deficiency , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins B-raf/genetics , Pyruvate Dehydrogenase (Lipoamide)-Phosphatase/metabolism , Pyruvate Dehydrogenase Acetyl-Transferring Kinase , Signal TransductionABSTRACT
Signal transducer and activator of transcription (STAT) 1 is part of the Janus kinase (JAK)/STAT signalling cascade and is best known for its essential role in mediating responses to all types of interferons (IFN). STAT1 regulates a variety of cellular processes, such as antimicrobial activities, cell proliferation and cell death. It exerts important immune modulatory activities both in the innate and the adaptive arm of the immune system. Based on studies in mice and data from human patients, STAT1 is generally considered a tumour suppressor but there is growing evidence that it can also act as a tumour promoter. This review aims at contrasting the two faces of STAT1 in tumourigenesis and providing an overview on the current knowledge of the underlying mechanisms or pathways.
Subject(s)
Adaptive Immunity , Immunity, Innate , Neoplasms/immunology , STAT1 Transcription Factor/immunology , Signal Transduction/immunology , Tumor Suppressor Proteins/immunology , Animals , Humans , Neoplasms/genetics , Neoplasms/pathology , STAT1 Transcription Factor/genetics , Signal Transduction/genetics , Tumor Suppressor Proteins/geneticsABSTRACT
Innate lymphoid cells (ILC) play a significant role in the intestinal immune response and T-bet+ CD127+ group 1 cells (ILC1) have been linked to the pathogenesis of human inflammatory bowel disease (IBD). However, the functional importance of ILC1 in the context of an intact adaptive immune response has been controversial. In this report we demonstrate that induced depletion of T-bet using a Rosa26-Cre-ERT2 model resulted in the loss of intestinal ILC1, pointing to a post-developmental requirement of T-bet expression for these cells. In contrast, neither colonic lamina propria (cLP) ILC2 nor cLP ILC3 abundance were altered upon induced deletion of T-bet. Mechanistically, we report that STAT1 or STAT4 are not required for intestinal ILC1 development and maintenance. Mice with induced deletion of T-bet and subsequent loss of ILC1 were protected from the induction of severe colitis in vivo. Hence, this study provides support for the clinical development of an IBD treatment based on ILC1 depletion via targeting T-bet or its downstream transcriptional targets.
Subject(s)
Intestinal Mucosa/immunology , Lymphocytes/immunology , T-Box Domain Proteins/immunology , Animals , Citrobacter rodentium , Colitis/chemically induced , Colitis/immunology , Dextran Sulfate , Enterobacteriaceae Infections/immunology , Female , Immunity, Innate , Male , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , STAT1 Transcription Factor/genetics , STAT1 Transcription Factor/immunology , STAT4 Transcription Factor/genetics , STAT4 Transcription Factor/immunology , Tamoxifen/pharmacology , Trichinella spiralis , Trichinellosis/immunologyABSTRACT
Ablation of the Raf-1 protein causes fetal liver apoptosis, embryonic lethality, and selective hypersensitivity to Fas-induced cell death. Furthermore, Raf-1-deficient cells show defective migration as a result of the deregulation of the Rho effector kinase Rok-alpha. In this study, we show that the kinase-independent modulation of Rok-alpha signaling is also the basis of the antiapoptotic function of Raf-1. Fas activation stimulates the formation of Raf-1-Rok-alpha complexes, and Rok-alpha signaling is up-regulated in Raf-1-deficient cells. This leads to increased clustering and membrane expression of Fas, which is rescued both by kinase-dead Raf-1 and by interfering with Rok-alpha or its substrate ezrin. Increased Fas clustering and membrane expression are also evident in the livers of Raf-1-deficient embryos, and genetically reducing Fas expression counteracts fetal liver apoptosis, embryonic lethality, and the apoptotic defects of embryonic fibroblasts. Thus, Raf-1 has an essential function in regulating Fas expression and setting the threshold of Fas sensitivity during embryonic life.
Subject(s)
Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-raf/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Signal Transduction , Animals , Apoptosis/genetics , Apoptosis/physiology , Cells, Cultured , Cytoskeletal Proteins/metabolism , Death Domain Receptor Signaling Adaptor Proteins , Dose-Response Relationship, Drug , Embryonic Development/physiology , Fibroblasts/metabolism , Flow Cytometry , Fluorescent Antibody Technique , Genes, Lethal , Intracellular Signaling Peptides and Proteins , Liver/embryology , Liver/metabolism , Membranes/metabolism , Mice , Mice, Mutant Strains/embryology , Mice, Mutant Strains/metabolism , Models, Biological , Proto-Oncogene Proteins c-raf/genetics , Sensitivity and Specificity , Tumor Necrosis Factor Receptor-Associated Peptides and Proteins/metabolism , rho-Associated KinasesABSTRACT
The cell dose in umbilical cord blood units is a major determinant for the outcome of hematopoietic cell transplantation. Prostaglandin analogs and dipeptidylpeptidase-4 (DPP4/CD26)-inhibitors enhance the ability of hematopoietic stem cells (HSCs) to reconstitute hematopoiesis. Here we explored the synergism between treprostinil, a stable prostaglandin agonist, and the DPP4/CD26-inhibitor vildagliptin. The combination of treprostinil and forskolin caused a modest but statistically significant increase in the surface levels of DPP4/CD26 on hematopoietic stem and progenitor cells (HSPCs) derived from murine bone and human cord blood. Their migration towards stromal cell-derived factor-1 (SDF-1/CXCL12) was enhanced, if they were pretreated with treprostinil and forskolin, and further augmented by vildagliptin. Administration of vildagliptin rescued 25% of lethally irradiated recipient mice injected with a limiting number of untreated HSPCs, but 90 to 100% of recipients injected with HSPCs preincubated with treprostinil and forskolin. The efficacy of vildagliptin surpassed that of treprostinil (60% rescue). Surprisingly, concomitant administration of vildagliptin and treprostinil resulted in poor survival of recipients indicating mutual antagonism, which was recapitulated when homing of and colony formation by HSPCs were assessed. These observations of regimen-dependent synergism and antagonism of treprostinil and vildagliptin are of translational relevance for the design of clinical trials. KEY MESSAGES: Pretreatment with treprostinil increases surface levels of DPP4/CD26 in HSPCs. Vildagliptin enhances in vitro migration of pretreated HSPCs. Vildagliptin enhances in vivo homing and engraftment of pretreated HSPCs. Unexpected mutual antagonism in vivo by concomitant administration of vildagliptin and treprostinil.
Subject(s)
Antihypertensive Agents/administration & dosage , Dipeptidyl-Peptidase IV Inhibitors/administration & dosage , Epoprostenol/analogs & derivatives , Hematopoietic Stem Cell Transplantation , Vildagliptin/administration & dosage , Animals , Cells, Cultured , Dipeptidyl Peptidase 4/metabolism , Drug Antagonism , Drug Synergism , Epoprostenol/administration & dosage , Fetal Blood/cytology , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/metabolism , Humans , MiceABSTRACT
Innate lymphoid cells (ILC) play a significant immunological role at mucosal surfaces such as the intestine. T-bet-expressing group 1 innate lymphoid cells (ILC1) are believed to play a substantial role in inflammatory bowel disease (IBD). However, a role of T-bet-negative ILC3 in driving colitis has also been suggested in mouse models questioning T-bet as a critical factor for IBD. We report here that T-bet deficient mice had a greater cellularity of NKp46-negative ILC3 correlating with enhanced expression of RORγt and IL-7R, but independent of signaling through STAT1 or STAT4. We observed enhanced neutrophilia in the colonic lamina propria (cLP) of these animals, however, we did not detect a greater risk of T-bet-deficient mice to develop spontaneous colitis. Furthermore, by utilizing an in vivo fate-mapping approach, we identified a population of T-bet-positive precursors in NKp46-negative ILC3s. These data suggest that T-bet controls ILC3 cellularity, but does do not drive a pathogenic role of ILC3 in mice with a conventional specific pathogen-free microbiota.
Subject(s)
Immunity, Innate , Intestinal Mucosa/immunology , Lymphocytes/immunology , T-Box Domain Proteins/immunology , Animals , Colitis/genetics , Colitis/immunology , Colitis/pathology , Inflammatory Bowel Diseases/genetics , Inflammatory Bowel Diseases/immunology , Inflammatory Bowel Diseases/pathology , Intestinal Mucosa/pathology , Lymphocytes/pathology , Mice , Mice, Knockout , Nuclear Receptor Subfamily 1, Group F, Member 3/genetics , Nuclear Receptor Subfamily 1, Group F, Member 3/immunology , Receptors, Interleukin-7/genetics , Receptors, Interleukin-7/immunology , STAT1 Transcription Factor/genetics , STAT1 Transcription Factor/immunology , STAT4 Transcription Factor/genetics , STAT4 Transcription Factor/immunology , T-Box Domain Proteins/geneticsABSTRACT
Natural killer (NK) cells are important components of the innate immune defense against infections and cancers. Signal transducer and activator of transcription 1 (STAT1) is a transcription factor that is essential for NK cell maturation and NK cell-dependent tumor surveillance. Two alternatively spliced isoforms of STAT1 exist: a full-length STAT1α and a C-terminally truncated STAT1ß isoform. Aberrant splicing is frequently observed in cancer cells and several anti-cancer drugs interfere with the cellular splicing machinery. To investigate whether NK cell-mediated tumor surveillance is affected by a switch in STAT1 splicing, we made use of knock-in mice expressing either only the STAT1α (Stat1α/α) or the STAT1ß (Stat1ß/ß ) isoform. NK cells from Stat1α/α mice matured normally and controlled transplanted tumor cells as efficiently as NK cells from wild-type mice. In contrast, NK cells from Stat1ß/ß mice showed impaired maturation and effector functions, albeit less severe than NK cells from mice that completely lack STAT1 (Stat1-/- ). Mechanistically, we show that NK cell maturation requires the presence of STAT1α in the niche rather than in NK cells themselves and that NK cell maturation depends on IFNγ signaling under homeostatic conditions. The impaired NK cell maturation in Stat1ß/ß mice was paralleled by decreased IL-15 receptor alpha (IL-15Rα) surface levels on dendritic cells, macrophages and monocytes. Treatment of Stat1ß/ß mice with exogenous IL-15/IL-15Rα complexes rescued NK cell maturation but not their effector functions. Collectively, our findings provide evidence that STAT1 isoforms are not functionally redundant in regulating NK cell activity and that the absence of STAT1α severely impairs, but does not abolish, NK cell-dependent tumor surveillance.
Subject(s)
Killer Cells, Natural/cytology , Lymphopoiesis/physiology , STAT1 Transcription Factor/immunology , Animals , Bone Marrow Transplantation , Cell Line, Tumor , Cytotoxicity, Immunologic , Immunologic Surveillance/drug effects , Immunologic Surveillance/immunology , Interferon-Stimulated Gene Factor 3/deficiency , Interferon-Stimulated Gene Factor 3/genetics , Interferon-Stimulated Gene Factor 3/immunology , Interleukin-15/pharmacology , Interleukin-15 Receptor alpha Subunit , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Lymphocyte Depletion , Lymphoid Tissue/cytology , Lymphoma/immunology , Lymphoma/pathology , Lymphopoiesis/drug effects , Mice , Mice, Inbred C57BL , Mice, Knockout , Organ Specificity , Protein Isoforms/genetics , Protein Isoforms/immunology , Receptors, Interferon/deficiency , STAT1 Transcription Factor/deficiency , STAT1 Transcription Factor/genetics , Specific Pathogen-Free Organisms , Spleen/cytology , Interferon gamma ReceptorABSTRACT
STAT1 has a key role in the regulation of innate and adaptive immunity by inducing transcriptional changes in response to cytokines, such as all types of interferons (IFN). STAT1 exist as two splice isoforms, which differ in regard to the C-terminal transactivation domain (TAD). STAT1ß lacks the C-terminal TAD and has been previously reported to be a weaker transcriptional activator than STAT1α, although this was strongly dependent on the target gene. The mechanism of this context-dependent effects remained unclear. By using macrophages from mice that only express STAT1ß, we investigated the role of the C-terminal TAD during the distinct steps of transcriptional activation of selected target genes in response to IFNγ. We show that the STAT1 C-terminal TAD is absolutely required for the recruitment of RNA polymerase II (Pol II) and for the establishment of active histone marks at the class II major histocompatibility complex transactivator (CIIta) promoter IV, whereas it is dispensable for histone acetylation at the guanylate binding protein 2 (Gbp2) promoter but required for an efficient recruitment of Pol II, which correlated with a strongly reduced, but not absent, transcriptional activity. IFNγ-induced expression of Irf7, which is mediated by STAT1 in complex with STAT2 and IRF9, did not rely on the presence of the C-terminal TAD of STAT1. Moreover, we show for the first time that the STAT1 C-terminal TAD is required for an efficient recruitment of components of the core Mediator complex to the IFN regulatory factor (Irf) 1 and Irf8 promoters, which both harbor an open chromatin state under basal conditions. Our study identified novel functions of the STAT1 C-terminal TAD in transcriptional activation and provides mechanistic explanations for the gene-specific transcriptional activity of STAT1ß.
Subject(s)
Nuclear Proteins/genetics , Protein Domains/immunology , RNA Polymerase II/metabolism , STAT1 Transcription Factor/metabolism , Trans-Activators/genetics , Transcriptional Activation/immunology , Animals , Cells, Cultured , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Histone Code , Interferon-Stimulated Gene Factor 3, gamma Subunit/genetics , Interferon-Stimulated Gene Factor 3, gamma Subunit/metabolism , Macrophages , Mice , Mice, Inbred C57BL , Mice, Knockout , Nuclear Proteins/metabolism , Primary Cell Culture , Promoter Regions, Genetic/genetics , Protein Isoforms/genetics , Protein Isoforms/immunology , Protein Isoforms/metabolism , STAT1 Transcription Factor/genetics , STAT1 Transcription Factor/immunology , STAT2 Transcription Factor/genetics , STAT2 Transcription Factor/metabolism , Trans-Activators/metabolismABSTRACT
Tyrosine kinase 2 (TYK2) is a Janus kinase (JAK) that is crucially involved in inflammation, carcinogenesis and defense against infection. The cytotoxic activity of natural killer (NK) cells in TYK2-deficient (Tyk2-/-) mice is severely reduced, although the underlying mechanisms are largely unknown. Using Tyk2-/- mice and mice expressing a kinase-inactive version of TYK2 (Tyk2K923E ), we show that NK cell function is partly independent of the enzymatic activity of TYK2. Tyk2-/- and Tyk2K923E NK cells develop normally in the bone marrow, but the maturation of splenic Tyk2-/- NK cells (and to a lesser extent of Tyk2K923E NK cells) is impaired. In contrast, the production of interferon γ (IFNγ) in response to interleukin 12 (IL-12) or to stimulation through NK cell-activating receptors strictly depends on the presence of enzymatically active TYK2. The cytotoxic activity of Tyk2K923E NK cells against a range of target cells in vitro is higher than that of Tyk2-/- NK cells. Consistently, Tyk2K923E mice control the growth of NK cell-targeted tumors significantly better than TYK2-deficient mice, showing the physiological relevance of the finding. Inhibitors of TYK2's kinase activity are being developed for the treatment of inflammatory diseases and cancers, but their effects on tumor immune surveillance have not been investigated. Our finding that TYK2 has kinase-independent functions in vivo suggests that such inhibitors will leave NK cell mediated tumor surveillance largely intact and that they will be suitable for use in cancer therapy.
ABSTRACT
The activation of oncogenes in primary cells blocks proliferation by inducing oncogene-induced senescence (OIS), a highly potent in vivo tumor-suppressing program. A prime example is mutant BRAF, which drives OIS in melanocytic nevi. Progression to melanoma occurs only in the context of additional alteration(s) like the suppression of PTEN, which abrogates OIS. Here, we performed a near-genomewide short hairpin (sh)RNA screen for novel OIS regulators and identified by next generation sequencing and functional validation seven genes. While all but one were upregulated in OIS, depletion of each of them abrogated BRAF(V) (600E) -induced arrest. With genome-wide DNA methylation analysis, we found one of these genes, RASEF, to be hypermethylated in primary cutaneous melanomas but not nevi. Bypass of OIS by depletion of RASEF was associated with suppression of several senescence biomarkers including senescence-associated (SA)-ß-galactosidase activity, interleukins, and tumor suppressor p15(INK) (4B) . Restoration of RASEF expression inhibited proliferation. These results illustrate the power of shRNA OIS bypass screens and identify a potential novel melanoma suppressor gene.
Subject(s)
Cellular Senescence , Melanoma/metabolism , Proto-Oncogene Proteins B-raf/metabolism , RNA Interference , Tumor Suppressor Proteins/metabolism , ras Guanine Nucleotide Exchange Factors/metabolism , Amino Acid Substitution , Cell Cycle Checkpoints/genetics , Cell Line, Tumor , Humans , Melanoma/genetics , Melanoma/pathology , Mutation, Missense , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , Proto-Oncogene Proteins B-raf/genetics , Tumor Suppressor Proteins/genetics , ras Guanine Nucleotide Exchange Factors/geneticsABSTRACT
Oncogene-induced senescence (OIS) is a growth arrest triggered by the enforced expression of cancer-promoting genes and acts as a barrier against malignant transformation in vivo. In this study, by a combination of in vitro and in vivo approaches, we investigate the role of OIS in tumours originating from the thyroid epithelium. We found that expression of different thyroid tumour-associated oncogenes in primary human thyrocytes triggers senescence, as demonstrated by the presence of OIS hallmarks: changes in cell morphology, accumulation of SA-ß-Gal and senescence-associated heterochromatic foci, and upregulation of transcription of the cyclin-dependent kinase inhibitors p16(INK4a) and p21(CIP1). Furthermore, immunohistochemical analysis of a panel of thyroid tumours characterised by different aggressiveness showed that the expression of OIS markers such as p16(INK4a), p21(CIP1) and IGFBP7 is upregulated at early stages, and lost during thyroid tumour progression. Taken together, our results suggest a role of OIS in thyroid carcinogenesis.
Subject(s)
Aging , Proto-Oncogenes , Thyroid Neoplasms/metabolism , Adult , Aged , Carcinoma , Carcinoma, Papillary , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p16/biosynthesis , Cyclin-Dependent Kinase Inhibitor p21/biosynthesis , Female , HEK293 Cells , Humans , Insulin-Like Growth Factor Binding Proteins/biosynthesis , Male , Middle Aged , Proto-Oncogenes/genetics , Thyroid Cancer, Papillary , Thyroid Carcinoma, Anaplastic , Thyroid Gland/cytology , Transduction, GeneticABSTRACT
Ras activation is common to many human cancers and promotes cell proliferation and survival by initiating multiple signaling cascades. Accordingly, Ras-transformed cells are generally considered too resourceful to become addicted to a single effector. In contrast to this tenet, we now demonstrate an absolute, cell autonomous requirement for Raf-1 in the development and maintenance of Ras-induced skin epidermis tumors. Mechanistically, Raf-1 functions as an endogenous inhibitor dimming the activity of the Rho-dependent kinase Rok-alpha in the context of a Ras-induced Raf-1:Rok-alpha complex. Raf-1-induced Rok-alpha inhibition allows the phosphorylation of STAT3 and Myc expression and promotes dedifferentiation in Ras-induced tumors. These data link the Raf-1:Rok-alpha complex to STAT3/Myc activation and delineate a pathway crucial for cell fate decision in Ras-induced tumorigenesis.
Subject(s)
Carcinoma, Squamous Cell/enzymology , Proto-Oncogene Proteins c-raf/physiology , Skin Neoplasms/enzymology , Animals , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Cell Differentiation , Cell Proliferation , Gene Deletion , Mice , Mice, Transgenic , Phosphorylation , Proto-Oncogene Proteins c-myc/metabolism , Proto-Oncogene Proteins c-raf/genetics , Proto-Oncogene Proteins c-raf/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Son of Sevenless Proteins/genetics , rho-Associated Kinases/metabolismABSTRACT
The activity of Raf-1 and Rok-alpha kinases is regulated by intramolecular binding of the regulatory region to the kinase domain. Autoinhibition is relieved upon binding to the small guanosine triphosphatases Ras and Rho. Downstream of Ras, Raf-1 promotes migration and tumorigenesis by antagonizing Rok-alpha, but the underlying mechanism is unknown. In this study, we show that Rok-alpha inhibition by Raf-1 relies on an intermolecular interaction between the Rok-alpha kinase domain and the cysteine-rich Raf-1 regulatory domain (Raf-1reg), which is similar to Rok-alpha's own autoinhibitory region. Thus, Raf-1 mediates Rok-alpha inhibition in trans, which is a new concept in kinase regulation. This mechanism is physiologically relevant because Raf-1reg is sufficient to rescue all Rok-alpha-dependent defects of Raf-1-deficient cells. Downstream of Ras and Rho, the Raf-1-Rok-alpha interaction represents a novel paradigm of pathway cross talk that contributes to tumorigenesis and cell motility.
Subject(s)
Proto-Oncogene Proteins c-raf/physiology , rho-Associated Kinases/metabolism , Animals , Cell Movement , Cells, Cultured , Enzyme Activation , Feedback, Physiological , Mice , Protein Structure, Tertiary , Proto-Oncogene Proteins c-raf/chemistry , Proto-Oncogene Proteins c-raf/metabolism , rho-Associated Kinases/antagonists & inhibitorsABSTRACT
RASSF1A is a tumor suppressor gene that is epigenetically silenced in a wide variety of sporadic human malignancies. Expression of alternative RASSF1 isoforms cannot substitute for RASSF1A-promoted cell-cycle arrest and apoptosis. Apoptosis can be driven by either activating Bax or by activation of MST kinases. The Raf1 proto-oncogene binds to MST2, preventing its activation and proapoptotic signaling. Here we show that key steps in RASSF1A-induced apoptosis are the disruption of the inhibitory Raf1-MST2 complex by RASSF1A and the concomitant enhancement of MST2 interaction with its substrate, LATS1. Subsequently, RASSF1A-activated LATS1 phosphorylates and releases the transcriptional regulator YAP1, allowing YAP1 to translocate to the nucleus and associate with p73, resulting in transcription of the proapoptotic target gene puma. Our results describe an MST2-dependent effector pathway for RASSF1A proapoptotic signaling and indicate that silencing of RASSF1A in tumors removes a proapoptotic signal emanating from p73.
Subject(s)
Apoptosis , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Transcription, Genetic , Tumor Suppressor Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Cell Line, Tumor , Cell Nucleus/metabolism , Humans , Models, Biological , Phosphoproteins/metabolism , Phosphorylation , Protein Binding , Protein Transport , Proto-Oncogene Mas , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-raf/metabolism , Serine-Threonine Kinase 3 , Signal Transduction , Transcription Factors , Tumor Protein p73 , Tumor Suppressor Protein p53/metabolism , YAP-Signaling ProteinsABSTRACT
Normal erythropoiesis critically depends on the balance between the renewal of precursor cells and their differentiation. If the renewal phase is shortened, the decrease in the precursor pool results in anemia; conversely, impaired differentiation increases the number of proliferating progenitors and the potential risk of leukemic transformation. Using gene ablation, we have discovered 2 self-sustaining signal transduction loops that antagonize each other and regulate erythroid progenitor proliferation and differentiation, respectively. We identify Raf-1 as the main activator of the MEK/ERK cascade and as the key molecule in maintaining progenitor proliferation. Differentiation, in contrast, is mediated by Fas via the activation of both the ASK1/JNK/p38 module and the caspase cascade. The point of convergence between the 2 cascades is activated ERK, which positively feeds back on the proliferation pathway by maintaining the expression of Raf-1, while inhibiting the expression of Fas and therefore differentiation. In turn, Fas, once expressed, antagonizes proliferation by exerting a negative feedback on ERK activation and Raf-1 expression. Simultaneously, Fas-mediated caspase activation precipitates differentiation. These results identify Raf-1 and Fas as the key molecules whose expression finely tunes erythropoiesis and the extent of ERK activation as the switch that tips the balance between them.
Subject(s)
Erythroblasts/drug effects , Proto-Oncogene Proteins c-raf/genetics , Proto-Oncogene Proteins c-raf/pharmacology , fas Receptor/genetics , fas Receptor/pharmacology , Animals , Caspases/metabolism , Cell Differentiation/drug effects , Erythroblasts/cytology , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Extracellular Signal-Regulated MAP Kinases/metabolism , Flavonoids/pharmacology , Gene Expression Profiling , Mice , Mice, Inbred C57BL , Mice, Inbred MRL lpr , Mice, Knockout , Reverse Transcriptase Polymerase Chain Reaction/methods , Structure-Activity RelationshipABSTRACT
The kinases of the Raf family have been intensively studied as activators of the mitogen-activated protein kinase kinase/extra-cellular signal-regulated kinase (ERK) module in regulated and deregulated proliferation. Genetic evidence that Raf is required for ERK activation in vivo has been obtained in lower organisms, which express only one Raf kinase, but was hitherto lacking in mammals, which express more than one Raf kinase. Ablation of the two best studied Raf kinases, B-Raf and Raf-1, is lethal at midgestation in mice, hampering the detailed study of the essential functions of these proteins. Here, we have combined conventional and conditional gene ablation to show that B-Raf is essential for ERK activation and for vascular development in the placenta. B-Raf-deficient placentae show complete absence of phosphorylated ERK and strongly reduced HIF-1alpha and VEGF levels, whereas all these parameters are normal in Raf-1-deficient placentae. In addition, neither ERK phosphorylation nor development are affected in B-raf-deficient embryos that are born alive obtained by epiblast-restricted gene inactivation. The data demonstrate that B-Raf plays a nonredundant role in ERK activation during extraembyronic mammalian development in vivo.