ABSTRACT
Amplification of the CCNE1 locus on chromosome 19q12 is prevalent in multiple tumour types, particularly in high-grade serous ovarian cancer, uterine tumours and gastro-oesophageal cancers, where high cyclin E levels are associated with genome instability, whole-genome doubling and resistance to cytotoxic and targeted therapies1-4. To uncover therapeutic targets for tumours with CCNE1 amplification, we undertook genome-scale CRISPR-Cas9-based synthetic lethality screens in cellular models of CCNE1 amplification. Here we report that increasing CCNE1 dosage engenders a vulnerability to the inhibition of the PKMYT1 kinase, a negative regulator of CDK1. To inhibit PKMYT1, we developed RP-6306, an orally bioavailable and selective inhibitor that shows single-agent activity and durable tumour regressions when combined with gemcitabine in models of CCNE1 amplification. RP-6306 treatment causes unscheduled activation of CDK1 selectively in CCNE1-overexpressing cells, promoting early mitosis in cells undergoing DNA synthesis. CCNE1 overexpression disrupts CDK1 homeostasis at least in part through an early activation of the MMB-FOXM1 mitotic transcriptional program. We conclude that PKMYT1 inhibition is a promising therapeutic strategy for CCNE1-amplified cancers.
Subject(s)
Cyclin E , Membrane Proteins , Ovarian Neoplasms , Protein Serine-Threonine Kinases , Protein-Tyrosine Kinases , CDC2 Protein Kinase , Cyclin E/genetics , Female , Gene Amplification , Gene Expression Regulation, Neoplastic , Humans , Membrane Proteins/genetics , Neoplasms/genetics , Ovarian Neoplasms/pathology , Protein Serine-Threonine Kinases/genetics , Protein-Tyrosine Kinases/genetics , Synthetic Lethal MutationsABSTRACT
GPR105, a G protein-coupled receptor for UDP-glucose, is highly expressed in several human tissues and participates in the innate immune response. Because inflammation has been implicated as a key initial trigger for type 2 diabetes, we hypothesized that GPR105 (official gene name: P2RY14) might play a role in the initiation of inflammation and insulin resistance in obesity. To this end, we investigated glucose metabolism in GPR105 knockout (KO) and wild-type (WT) mice fed a high-fat diet (HFD). We also examined whether GPR105 regulates macrophage recruitment to liver or adipose tissues by in vivo monocyte tracking and in vitro chemotaxis experiments, followed by transplantation of bone marrow from either KO or WT donors to WT recipients. Our data show that genetic deletion of GPR105 confers protection against HFD-induced insulin resistance, with reduced macrophage infiltration and inflammation in liver, and increased insulin-stimulated Akt phosphorylation in liver, muscle, and adipose tissue. By tracking monocytes from either KO or WT donors, we found that fewer KO monocytes were recruited to the liver of WT recipients. Furthermore, we observed that uridine 5-diphosphoglucose enhanced the in vitro migration of bone marrow-derived macrophages from WT but not KO mice, and that plasma uridine 5-diphosphoglucose levels were significantly higher in obese versus lean mice. Finally, we confirmed that insulin sensitivity improved in HFD mice with a myeloid cell-specific deletion of GPR105. These studies indicate that GPR105 ablation mitigates HFD-induced insulin resistance by inhibiting macrophage recruitment and tissue inflammation. Hence GPR105 provides a novel link between innate immunity and metabolism.
Subject(s)
Inflammation/metabolism , Insulin Resistance/immunology , Obesity/metabolism , Receptors, Purinergic P2/metabolism , Animals , Chemotaxis, Leukocyte/immunology , Diet, High-Fat/adverse effects , Flow Cytometry , Immunoblotting , Inflammation/immunology , Macrophages/immunology , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Obesity/etiology , Obesity/immunology , Receptors, Purinergic P2/immunology , Receptors, Purinergic P2Y , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
ATR is a key kinase in the DNA-damage response (DDR) that is synthetic lethal with several other DDR proteins, making it an attractive target for the treatment of genetically selected solid tumors. Herein we describe the discovery of a novel ATR inhibitor guided by a pharmacophore model to position a key hydrogen bond. Optimization was driven by potency and selectivity over the related kinase mTOR, resulting in the identification of camonsertib (RP-3500) with high potency and excellent ADME properties. Preclinical evaluation focused on the impact of camonsertib on myelosuppression, and an exploration of intermittent dosing schedules to allow recovery of the erythroid compartment and mitigate anemia. Camonsertib is currently undergoing clinical evaluation both as a single agent and in combination with talazoparib, olaparib, niraparib, lunresertib, or gemcitabine (NCT04497116, NCT04972110, NCT04855656). A preliminary recommended phase 2 dose for monotherapy was identified as 160 mg QD given 3 days/week.
Subject(s)
Neoplasms , Humans , Ataxia Telangiectasia Mutated Proteins , Neoplasms/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , GemcitabineABSTRACT
Ataxia telangiectasia and Rad3-related (ATR) kinase protects genome integrity during DNA replication. RP-3500 is a novel, orally bioavailable clinical-stage ATR kinase inhibitor (NCT04497116). RP-3500 is highly potent with IC50 values of 1.0 and 0.33 nmol/L in biochemical and cell-based assays, respectively. RP-3500 is highly selective for ATR with 30-fold selectivity over mammalian target of rapamycin (mTOR) and more than 2,000-fold selectivity over ataxia telangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK), and phosphatidylinositol 3-kinase alpha (PI3Kα) kinases. In vivo, RP-3500 treatment results in potent single-agent efficacy and/or tumor regression in multiple xenograft models at minimum effective doses (MED) of 5 to 7 mg/kg once daily. Pharmacodynamic assessments validate target engagement, with dose-proportional tumor inhibition of phosphorylated checkpoint kinase 1 (pCHK1) (IC80 = 18.6 nmol/L) and induction of phosphorylated H2A.X variant histone (γH2AX), phosphorylated DNA-PK catalytic subunit (pDNA-PKcs), and phosphorylated KRAB-associated protein 1 (pKAP1). RP-3500 exposure at MED indicates that circulating free plasma levels above the in vivo tumor IC80 for 10 to 12 hours are sufficient for efficacy on a continuous schedule. However, short-duration intermittent (weekly 3 days on/4 days off) dosing schedules as monotherapy or given concomitantly with reduced doses of olaparib or niraparib, maximize tumor growth inhibition while minimizing the impact on red blood cell depletion, emphasizing the reversible nature of erythroid toxicity with RP-3500 and demonstrating superior efficacy compared with sequential treatment. These results provide a strong preclinical rationale to support ongoing clinical investigation of the novel ATR inhibitor, RP-3500, on an intermittent schedule as a monotherapy and in combination with PARP inhibitors as a potential means of maximizing clinical benefit.
Subject(s)
Ataxia Telangiectasia , Poly(ADP-ribose) Polymerase Inhibitors , Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors , DNA-Activated Protein Kinase/metabolism , Humans , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic useABSTRACT
PKMYT1 is a regulator of CDK1 phosphorylation and is a compelling therapeutic target for the treatment of certain types of DNA damage response cancers due to its established synthetic lethal relationship with CCNE1 amplification. To date, no selective inhibitors have been reported for this kinase that would allow for investigation of the pharmacological role of PKMYT1. To address this need compound 1 was identified as a weak PKMYT1 inhibitor. Introduction of a dimethylphenol increased potency on PKMYT1. These dimethylphenol analogs were found to exist as atropisomers that could be separated and profiled as single enantiomers. Structure-based drug design enabled optimization of cell-based potency. Parallel optimization of ADME properties led to the identification of potent and selective inhibitors of PKMYT1. RP-6306 inhibits CCNE1-amplified tumor cell growth in several preclinical xenograft models. The first-in-class clinical candidate RP-6306 is currently being evaluated in Phase 1 clinical trials for treatment of various solid tumors.
Subject(s)
Neoplasms , Protein-Tyrosine Kinases , Cell Line, Tumor , Cell Proliferation , Humans , Membrane Proteins , Neoplasms/pathology , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Protein Serine-Threonine KinasesABSTRACT
DNA polymerase theta (Polθ) is an attractive synthetic lethal target for drug discovery, predicted to be efficacious against breast and ovarian cancers harboring BRCA-mutant alleles. Here, we describe our hit-to-lead efforts in search of a selective inhibitor of human Polθ (encoded by POLQ). A high-throughput screening campaign of 350,000 compounds identified an 11 micromolar hit, giving rise to the N2-substituted fused pyrazolo series, which was validated by biophysical methods. Structure-based drug design efforts along with optimization of cellular potency and ADME ultimately led to the identification of RP-6685: a potent, selective, and orally bioavailable Polθ inhibitor that showed in vivo efficacy in an HCT116 BRCA2-/- mouse tumor xenograft model.
Subject(s)
DNA-Directed DNA Polymerase , Ovarian Neoplasms , Animals , DNA Replication , DNA-Directed DNA Polymerase/metabolism , Drug Design , Drug Discovery , Female , Humans , MiceABSTRACT
Our series of competitive antagonists against the G-protein coupled receptor P2Y(14) were found to be highly shifted in the presence of serum (>99% protein bound). A binding assay using 2% human serum albumin (HSA) was developed to guide further SAR studies and led to the identification of the zwitterion 2, which is substantially less shifted (18-fold) than our previous lead compound 1 (323-fold). However, as the bioavailability of 2 was low, a library of ester pro-drugs was prepared (7a-7j) and assessed in vitro. The most interesting candidates were then profiled in vivo and led to the identification of the pro-drug 7j, which possesses a substantially improved pharmacokinetic profile.
Subject(s)
Prodrugs/chemistry , Purinergic P2 Receptor Antagonists/chemistry , Receptors, Purinergic P2/chemistry , Biological Availability , Humans , Microsomes, Liver/metabolism , Prodrugs/chemical synthesis , Prodrugs/pharmacokinetics , Protein Binding , Purinergic P2 Receptor Antagonists/chemical synthesis , Purinergic P2 Receptor Antagonists/pharmacokinetics , Receptors, Purinergic P2/metabolism , Structure-Activity RelationshipABSTRACT
A weak, UDP-competitive antagonist of the pyrimidinergic receptor P2RY(14) with a naphthoic acid core was identified through high-throughput screening. Optimization provided compounds with improved potency but poor pharmacokinetics. Acylglucuronidation was determined to be the major route of metabolism. Increasing the electron-withdrawing nature of the substituents markedly reduced glucuronidation and improved the pharmacokinetic profile. Additional optimization led to the identification of compound 38 which is an 8 nM UDP-competitive antagonist of P2Y(14) with a good pharmacokinetic profile.
Subject(s)
Carboxylic Acids/chemical synthesis , Naphthalenes/chemical synthesis , Purinergic P2 Receptor Antagonists/chemical synthesis , Receptors, Purinergic P2 , Uridine Diphosphate , Animals , Binding, Competitive , Carboxylic Acids/chemistry , Carboxylic Acids/pharmacokinetics , Carboxylic Acids/pharmacology , Mice , Molecular Structure , Naphthalenes/chemistry , Naphthalenes/pharmacokinetics , Naphthalenes/pharmacology , Pan troglodytes , Protein Binding/drug effects , Purinergic P2 Receptor Antagonists/chemistry , Purinergic P2 Receptor Antagonists/pharmacokinetics , Purinergic P2 Receptor Antagonists/pharmacology , Receptors, Purinergic P2Y , Structure-Activity RelationshipABSTRACT
A weak antagonist of the pyrimidinergic receptor P2Y(14) containing a dihydropyridopyrimidine core was identified through high-throughput screening. Subsequent optimization led to potent, non-UTP competitive antagonists and represent the first reported non-nucleotide antagonists of this receptor. Compound 18q was identified as a 10 nM P2Y(14) antagonist with good oral bioavailability and provided sufficient exposure in mice to be used as a tool for future in vivo studies.
Subject(s)
Purinergic P2 Receptor Antagonists/chemical synthesis , Pyrimidines/chemical synthesis , Receptors, Purinergic P2/chemistry , Administration, Oral , Animals , Biological Availability , Mice , Molecular Structure , Pan troglodytes , Purinergic P2 Receptor Antagonists/chemistry , Pyrimidines/administration & dosage , Pyrimidines/chemistry , Receptors, Purinergic P2Y , Structure-Activity RelationshipABSTRACT
Connexin43 (Cx43) is often deregulated in breast cancer tissue compared with normal adjacent tissue. Stable reexpression of Cx43 in cancer slows growth and renders the cells more sensitive to cytotoxic chemotherapeutics. Pseudogenes are often considered nonfunctional copies of DNA. The Cx43 pseudogene (PsiCx43) possesses all the features of an expressed gene and is exclusively transcribed in breast cancer cell lines and not in normal cells. PsiCx43 can be translated in vivo, and its protein exhibits growth-suppressive behavior similar to Cx43. We showed that PsiCx43 binds to the polyribosomes in breast cancer cells and that exogenous expression of PsiCx43 induces translational inhibition of Cx43. Furthermore, PsiCx43 is translated and binds more efficiently to the translational machinery than does Cx43 in an in vitro system. Following knockdown of PsiCx43 in breast cancer cells, we observed an increase in Cx43 RNA and protein. This results in increased cellular sensitivity to cytotoxic chemotherapy. Our results show that PsiCx43 acts as a posttranscriptional regulator of Cx43 in breast cancer cells, and that this represents an example of the regulation of genes by pseudogenes with potential therapeutic implications in cancer.
Subject(s)
Breast Neoplasms/genetics , Connexin 43/genetics , Pseudogenes , Antibiotics, Antineoplastic/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Blotting, Western , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Connexin 43/metabolism , Doxorubicin/pharmacology , Female , Humans , Paclitaxel/pharmacology , Polyribosomes , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
Gene regulation by transcriptional and post-translational mechanisms is implicated in the regulation of cellular homeostasis. Transcriptional deregulation has been largely documented in the etiology of diseases such as cancer, obesity and diabetes. During the past decade, the control of translation initiation by the PI3K/Akt/mTOR pathway in the development of these pathologies has been documented. Rapamycin, a specific inhibitor of mTOR, demonstrates considerable anti-proliferative activity against numerous cancer types. Recent studies also demonstrated that rapamycin may be beneficial in the treatment of obesity and diabetes. Rapamycin and its analogs seem destined for a promising future and will help in the development of novel therapeutic strategies.
Subject(s)
Protein Biosynthesis/genetics , Protein Kinases/physiology , Animals , Diabetes Mellitus/genetics , Humans , Neoplasms/genetics , Obesity/genetics , TOR Serine-Threonine KinasesABSTRACT
Over the years, studies have focused on the transcriptional regulation of oncogenesis. More recently, a growing emphasis has been placed on translational control. The Ras and Akt signal transduction pathways play a critical role in regulating mRNA translation and cellular transformation. The question arises: How might the Ras and Akt signaling pathways affect translation and mediate transformation? These pathways converge on a crucial effector of translation, the initiation factor eIF4E, which binds the 5'cap of mRNAs. This review focuses on the role of eIF4E in oncogenesis. eIF4E controls the translation of various malignancy-associated mRNAs which are involved in polyamine synthesis, cell cycle progression, activation of proto-oncogenes, angiogenesis, autocrine growth stimulation, cell survival, invasion and communication with the extracellular environment. eIF4E-mediated translational modulation of these mRNAs plays a pivotal role in both tumor formation and metastasis. Interestingly, eIF4E activity is implicated in mitosis, embryogenesis and in apoptosis. Finally, the finding that eIF4E is overexpressed in several human cancers makes it a prime target for anticancer therapies.
Subject(s)
Cell Transformation, Neoplastic , Eukaryotic Initiation Factor-4E/physiology , Protein Biosynthesis , Animals , Apoptosis , Cell Cycle , Cell Division , Cyclin D1/genetics , Genes, myc , Humans , MAP Kinase Signaling System , Vascular Endothelial Growth Factor A/geneticsABSTRACT
Primary effusion lymphoma (PEL) is a lymphoproliferative disease of B-cell origin that is associated with HHV-8 infection. PEL cells harbor a non-B, non-T phenotype and lack significant surface immunoglobulin (Ig) expression, a characteristic that has not been fully explained. In the present study, we demonstrate that PEL cells constitutively express interferon regulatory factor (IRF)-4, a transcription factor that regulates the activity of the immunoglobulin light-chain enhancer elements lambdaB and kappaE3' through binding to a composite Ets-IRF site. IRF-4 activity requires its physical interaction with PU.1, an Ets family member involved in the activation of genes essential for B-cell development. However, in PEL-derived B-cell lines, PU.1 expression was completely abrogated; expression of the B cell specific transcription factor Oct-2, which is known to regulate PU.1 expression, was also abolished. Moreover, the B-cell-specific coactivator of octamer factors, BOB-1/OcaB, was expressed at very decreased levels in PEL cells. Ectopic expression of Oct-2 was able to fully restore PU.1 promoter activity in the PEL cell line BCBL-1, while PU.1 expression also reconstituted the activity of the lambdaB Ets-IRF site. In addition, protein levels of BSAP/Pax-5 and IRF-8/ICSBP were undetectable in PEL cells. The pattern of transcription factor ablation observed in PEL was found to be comparable to that observed in classical Hodgkin's disease-derived cell lines, which also lack B-cell-specific surface markers. These observations indicate that disruption of the B-cell-specific transcriptional program is likely to contribute to the incomplete B-cell phenotype characteristic of PEL cells.
Subject(s)
B-Lymphocytes/metabolism , Gene Expression Regulation, Neoplastic , Herpesviridae Infections/genetics , Herpesvirus 8, Human/isolation & purification , Lymphoma, B-Cell/genetics , Neoplasm Proteins/genetics , Transcription Factors/genetics , Transcription, Genetic , Tumor Virus Infections/genetics , Body Fluids , Burkitt Lymphoma/genetics , Burkitt Lymphoma/metabolism , Burkitt Lymphoma/pathology , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Enhancer Elements, Genetic , Herpesviridae Infections/metabolism , Herpesviridae Infections/pathology , Hodgkin Disease/genetics , Hodgkin Disease/metabolism , Hodgkin Disease/pathology , Humans , Immunoglobulin Light Chains/genetics , Interferon Regulatory Factors , Lymphoma, B-Cell/metabolism , Lymphoma, B-Cell/pathology , Lymphoma, B-Cell/virology , Neoplasm Proteins/biosynthesis , Octamer Transcription Factor-2 , PAX5 Transcription Factor , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins/genetics , RNA, Messenger/biosynthesis , RNA, Neoplasm/biosynthesis , Recombinant Fusion Proteins/physiology , Repressor Proteins/biosynthesis , Repressor Proteins/genetics , Trans-Activators/biosynthesis , Trans-Activators/genetics , Transcription Factors/biosynthesis , Transfection , Tumor Virus Infections/metabolism , Tumor Virus Infections/pathologyABSTRACT
The human T cell leukemia/lymphotropic virus-1 (HTLV-I) is the etiologic agent of adult T cell leukemia (ATL), an aggressive and fatal leukemia of CD4+ T lymphocytes. Interferon regulatory factor-4 (IRF-4) was shown previously to be constitutively expressed in T cells infected with HTLV-1. In this study, we investigated the role of IRF-4 gene regulation in the context of HTLV-1 infection using gene array technology and IRF-4 expressing T cells. Many potential IRF-4 regulated genes were identified, the vast majority of which were repressed by IRF-4 expression. Cyclin B1, a G2-M checkpoint protein identified as an IRF-4 repressed gene in the array, was further characterized in the context of HTLV-1 infection. All HTLV-1 infected cell lines and ATL patient lymphocytes demonstrated a dramatic decrease in cyclin B1 levels; subsequent analysis of the cyclin B1 promoter identified two sites important in IRF-4 binding and repression of cyclin B1 expression. Furthermore, IRF-4-mediated repression of cyclin B1 led to a significant decrease in CDC2 kinase activity in HTLV-1 infected T cells. IRF-4 expression in HTLV-1 infected T cells also downregulated other genes implicated in the mitotic checkpoint as well as genes involved in actin cytoskeletal rearrangement, DNA repair, apoptosis, metastasis and immune recognition. Several of the identified genes are dysregulated in ATL and may provide important mechanistic information concerning pathways critical to the emergence of ATL.
Subject(s)
DNA-Binding Proteins/physiology , Gene Expression Regulation , Human T-lymphotropic virus 1/physiology , Leukemia-Lymphoma, Adult T-Cell/genetics , Transcription Factors/physiology , Base Sequence , DNA-Binding Proteins/genetics , Down-Regulation , Humans , Interferon Regulatory Factors , Jurkat Cells , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic , Transcription Factors/geneticsABSTRACT
Human T cell leukemia virus (HTLV) is the causative agent of adult T cell leukemia (ATL), an aggressive and fatal leukemia of CD4+ T lymphocytes in which interferon regulatory factor-4 (IRF-4) becomes constitutively expressed, concomitant with major alterations in host gene expression. When constitutively expressed in uninfected T lymphocytes, IRF-4 caused reduced expression of critical DNA repair genes, including Rad51, XRCC1, Ung1, RPA, and proliferative cell nuclear antigen (PCNA), a transcriptional phenotype with striking similarities to the profile observed in HTLV-infected T lymphocytes. Concomitant with the inhibition of gene expression and defects in the DNA repair pathways, increased sensitivity of T lymphocytes to various genotoxic stresses that challenged all major DNA repair pathways were detected. Together, these results support a role for IRF- 4 in the repression of DNA repair activity and an increase in the risk of mutations. IRF-4 may thus represent a previously unidentified endogenous transcriptional repressor of DNA repair mechanisms.
Subject(s)
DNA Repair/physiology , DNA-Binding Proteins/metabolism , Human T-lymphotropic virus 1/metabolism , T-Lymphocytes/virology , Transcription Factors/metabolism , DNA/metabolism , DNA/radiation effects , DNA Damage , DNA Repair/genetics , Down-Regulation , Humans , Interferon Regulatory Factors , Jurkat Cells , Oxidative Stress , T-Lymphocytes/metabolism , Ultraviolet RaysABSTRACT
We summarize recent studies on the activation and regulation of interferon (IFN) regulatory factor-4 (IRF-4) and its function in activated T cells, human T cell lymphoma virus (HTLV-I)-infected T cells, and HTLV-I-induced adult T cell leukemia (ATL). We have examined the specific mechanisms underlying the expression and regulation of the IRF-4 transcription factor in HTLV-I-infected cells and have shown that constitutive IRF-4 expression is exclusive to the transformed, leukemic ATL phenotype as opposed to the nonleukemic HTLV-I associated myelopathies/tropical spastic paraparesis (HAM/TSP) phenotype. In contrast, IRF-4 is only transiently induced in T lymphocytes activated by signals that mimic stimulation through the T cell receptor (TCR). In vivo and in vitro analyses have identified several regulatory regions within the human IRF-4 promoter that interact with the transcriptional regulators NF-kappaB, NF-AT, and Sp-1 to drive IRF-4 production in HTLV-I-infected, ATL-derived cells. cDNA array analysis of an IRF-4-expressing T cell line has also provided valuable insight into potential IRF-4 target genes. Further investigation of these novel IRF-4-regulated genes will permit a mechanistic understanding of IRF-4 function in HTLV-I-induced leukemogenesis.
Subject(s)
DNA-Binding Proteins/metabolism , Human T-lymphotropic virus 1/pathogenicity , Leukemia-Lymphoma, Adult T-Cell/metabolism , Transcription Factors/metabolism , Animals , Gene Expression Regulation, Leukemic , HSP90 Heat-Shock Proteins/metabolism , Humans , Interferon Regulatory Factors , Leukemia-Lymphoma, Adult T-Cell/genetics , Leukemia-Lymphoma, Adult T-Cell/virology , Promoter Regions, Genetic , Protein Processing, Post-Translational , Receptors, Antigen, T-Cell/metabolism , Signal Transduction , T-Lymphocytes/metabolism , T-Lymphocytes/virology , Tacrolimus Binding Proteins/metabolism , Transcriptional ActivationABSTRACT
OBJECTIVE: Significant new data suggest that metabolic disorders such as diabetes, obesity, and atherosclerosis all posses an important inflammatory component. Infiltrating macrophages contribute to both tissue-specific and systemic inflammation, which promotes insulin resistance. The complement cascade is involved in the inflammatory cascade initiated by the innate and adaptive immune response. A mouse genomic F2 cross biology was performed and identified several causal genes linked to type 2 diabetes, including the complement pathway. RESEARCH DESIGN AND METHODS: We therefore sought to investigate the effect of a C3a receptor (C3aR) deletion on insulin resistance, obesity, and macrophage function utilizing both the normal-diet (ND) and a diet-induced obesity mouse model. RESULTS: We demonstrate that high C3aR expression is found in white adipose tissue and increases upon high-fat diet (HFD) feeding. Both adipocytes and macrophages within the white adipose tissue express significant amounts of C3aR. C3aR(-/-) mice on HFD are transiently resistant to diet-induced obesity during an 8-week period. Metabolic profiling suggests that they are also protected from HFD-induced insulin resistance and liver steatosis. C3aR(-/-) mice had improved insulin sensitivity on both ND and HFD as seen by an insulin tolerance test and an oral glucose tolerance test. Adipose tissue analysis revealed a striking decrease in macrophage infiltration with a concomitant reduction in both tissue and plasma proinflammatory cytokine production. Furthermore, C3aR(-/-) macrophages polarized to the M1 phenotype showed a considerable decrease in proinflammatory mediators. CONCLUSIONS: Overall, our results suggest that the C3aR in macrophages, and potentially adipocytes, plays an important role in adipose tissue homeostasis and insulin resistance.
Subject(s)
Adipose Tissue/immunology , Adipose Tissue/metabolism , Insulin Resistance/immunology , Macrophages/immunology , Receptors, Complement/immunology , Receptors, Complement/metabolism , 3T3-L1 Cells , Animals , Cell Movement/immunology , Dietary Fats/pharmacology , Homeostasis/immunology , Hypoglycemic Agents/pharmacology , Inflammation/immunology , Inflammation/metabolism , Insulin/pharmacology , Macrophages/cytology , Male , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Obesity/immunology , Obesity/metabolism , Phenotype , Receptors, Complement/geneticsABSTRACT
eIF4E, the mRNA 5' cap-binding translation initiation factor, is overexpressed in numerous cancers and is implicated in mechanisms underlying oncogenesis and senescence. 4E-BPs (eIF4E-binding proteins) inhibit eIF4E activity, and thereby act as suppressors of eIF4E-dependent pathways. Here, we show that tumorigenesis is increased in p53 knockout mice that lack 4E-BP1 and 4E-BP2. However, primary fibroblasts lacking 4E-BPs, but expressing p53, undergo premature senescence and resist oncogene-driven transformation. Thus, the p53 status governs 4E-BP-dependent senescence and transformation. Intriguingly, the 4E-BPs engage in senescence via translational control of the p53-stabilizing protein, Gas2. Our data demonstrate a role for 4E-BPs in senescence and tumorigenesis and highlight a p53-mediated mechanism of senescence through a 4E-BP-dependent pathway.
Subject(s)
Cell Transformation, Neoplastic/genetics , Eukaryotic Initiation Factor-4E/genetics , Tumor Suppressor Protein p53/genetics , Animals , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Cellular Senescence/genetics , Eukaryotic Initiation Factor-4E/metabolism , Mice , Mice, Knockout , Tumor Suppressor Protein p53/metabolismABSTRACT
BACKGROUND: Translation deregulation is an important mechanism that causes aberrant cell growth, proliferation and survival. eIF4E, the mRNA 5' cap-binding protein, plays a major role in translational control. To understand how eIF4E affects cell proliferation and survival, we studied mRNA targets that are translationally responsive to eIF4E. METHODOLOGY/PRINCIPAL FINDINGS: Microarray analysis of polysomal mRNA from an eIF4E-inducible NIH 3T3 cell line was performed. Inducible expression of eIF4E resulted in increased translation of defined sets of mRNAs. Many of the mRNAs are novel targets, including those that encode large- and small-subunit ribosomal proteins and cell growth-related factors. In addition, there was augmented translation of mRNAs encoding anti-apoptotic proteins, which conferred resistance to endoplasmic reticulum-mediated apoptosis. CONCLUSIONS/SIGNIFICANCE: Our results shed new light on the mechanisms by which eIF4E prevents apoptosis and transforms cells. Downregulation of eIF4E and its downstream targets is a potential therapeutic option for the development of novel anti-cancer drugs.
Subject(s)
Epigenesis, Genetic , Eukaryotic Initiation Factor-4E/physiology , RNA, Messenger/biosynthesis , 5' Untranslated Regions/genetics , Animals , Apoptosis , Apoptosis Regulatory Proteins/biosynthesis , Apoptosis Regulatory Proteins/genetics , Cell Division/genetics , Eukaryotic Initiation Factor-4E/genetics , Intercellular Signaling Peptides and Proteins/biosynthesis , Intercellular Signaling Peptides and Proteins/genetics , Mice , NIH 3T3 Cells/cytology , Oligonucleotide Array Sequence Analysis , Polyribosomes/metabolism , Protein Biosynthesis , RNA, Messenger/genetics , Recombinant Fusion Proteins/physiology , Regulatory Sequences, Ribonucleic Acid/genetics , Ribosomal Proteins/biosynthesis , Ribosomal Proteins/geneticsABSTRACT
IL-15Ralpha mRNA and protein levels are increased in human T cell lymphotropic virus type-I (HTLV-I)-associated adult T cell leukemia. Previously, we demonstrated that IL-15Ralpha expression was activated by HTLV-I Tax, in part, through the action of NF-kappaB. However, there appeared to be additional motifs within the IL-15Ralpha promoter that were responsive to HTLV-I Tax. In this study, we demonstrated that IL-15Ralpha mRNA expression was activated in human monocytes by IFN treatment, suggesting a role for IFN regulatory factors (IRFs) in IL-15Ralpha transcription. In addition, an IRF element within the Tax-responsive element of the IL-15Ralpha promoter was necessary for maximal Tax-induced activation of this promoter. Furthermore, we demonstrated that IRF-4, a transcription factor known to be elevated in HTLV-I-infected cells, activated the IL-15Ralpha promoter. Inhibition of IRF-4 action lead to reduced Tax-induced activation of the IL-15Ralpha promoter, while inhibition of both IRF-4 and NF-kappaB severely inhibited the Tax-induced activation of this promoter. These findings suggest a role for both NF-kappaB and IRF-4 in the transcriptional regulation of IL-15Ralpha by HTLV-I Tax. It is possible that the HTLV-I Tax-mediated induction of IL-15Ralpha and IL-15 may lead to an autocrine cytokine-mediated stimulatory loop leading to the proliferation of HTLV-I infected cells. This loop of proliferation may facilitate viral propagation and play a role in HTLV-I-mediated disease progression.