RESUMO
Fibrosis or accumulation of extracellular matrix is an evolutionarily conserved mechanism adopted by an organism as a response to chronic injury. Excessive fibrosis, however, leads to disruption of organ homeostasis and is a common feature of many chronic diseases. G protein-coupled receptors (GPCRs) are important cell signaling mediators and represent molecular targets for many Food and Drug Administration-approved drugs. To identify new targets for fibrosis, we used a synthetic GPCR system named designed receptors exclusively activated by designer drugs (DREADDs) to probe signaling pathways essential for fibrotic response. We found that upon expression in human lung fibroblasts, activation of Gq- and Gs-DREADDs abrogated the induction of TGFß-induced fibrosis marker genes. Genome-wide transcriptome analysis identified dysregulation of multiple GPCRs in lung fibroblasts treated with TGFß To investigate endogenous GPCR modulating TGFß signaling, we selected 13 GPCRs that signal through Gq or Gs and activated them by using specific agonists. We examined the impact of each agonist and how activation of endogenous GPCR affects TGFß signaling. Among the agonists examined, prostaglandin receptor agonists demonstrated the strongest inhibitory effect on fibrosis. Together, we have demonstrated that the DREADDs system is a valuable tool to identify beneficial GPCR signaling for fibrosis. This study in fibroblasts has served as a proof of concept and allowed us to further develop in vivo models for fibrosis GPCR discovery. SIGNIFICANCE STATEMENT: Fibrosis is the hallmark of many end-stage cardiometabolic diseases, and there is an unmet medical need to discover new antifibrotic therapies, reduce disease progression, and bring clinically meaningful efficacy to patients. Our work utilizes designed receptors exclusively activated by designer drug chemogenetic tools to identify beneficial GPCR signaling for fibrosis, providing new insights into GPCR drug discovery.
Assuntos
Drogas Desenhadas/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Fibrose , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Receptores de Prostaglandina/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
This study reports the establishment of a bone marrow mononuclear cell (BMMC) 3D culture model and the application of this model to define sensitivity and resistance biomarkers of acute myeloid leukaemia (AML) patient bone marrow samples in response to Cytarabine (Ara-C) treatment. By mimicking physiological bone marrow microenvironment, the growth conditions were optimized by using frozen BMMCs derived from healthy donors. Healthy BMMCs are capable of differentiating into major hematopoietic lineages and various types of stromal cells in this platform. Cryopreserved BMMC samples from 49 AML patients were characterized for ex vivo growth and sensitivity to Ara-C. RNA sequencing was performed for 3D and 2D cultures to determine differential gene expression patterns. Specific genetic mutations and/or gene expression signatures associated with the ability of the ex vivo expansion and response to Ara-C were elucidated by whole-exome and RNA sequencing. Data analysis identified unique gene expression signatures and novel genetic mutations associated with sensitivity to Ara-C treatment of proliferating AML specimens and can be used as predictive therapeutic biomarkers to determine the optimal treatment regimens. Furthermore, these data demonstrate the translational value of this ex vivo platform which should be widely applicable to evaluate other therapies in AML.
Assuntos
Citarabina/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Modelos Biológicos , Adulto , Idoso , Idoso de 80 Anos ou mais , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Citarabina/farmacologia , Feminino , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Masculino , Pessoa de Meia-Idade , Mutação/genética , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Resultado do TratamentoRESUMO
Metabolic dysregulation and mitochondrial dysfunction are important features of acute and chronic tissue injury across species, and human genetics and preclinical data suggest that the master metabolic regulator 5'-adenosine monophosphate-activated protein kinase (AMPK) may be an effective therapeutic target for chronic kidney disease (CKD). We have recently disclosed a pan-AMPK activator, MK-8722, that was shown to have beneficial effects in preclinical models. In this study we investigated the effects of MK-8722 in a progressive rat model of diabetic nephropathy to determine whether activation of AMPK would be of therapeutic benefit. We found that MK-8722 administration in a therapeutic paradigm is profoundly renoprotective, as demonstrated by a reduction in proteinuria (63% decrease in MK-8722 10 mg/kg per day compared with vehicle group) and a significant improvement in glomerular filtration rate (779 and 430 µl/min per gram kidney weight in MK-8722 10 mg/kg per day and vehicle group, respectively), as well as improvements in kidney fibrosis. We provide evidence that the therapeutic effects of MK-8722 may be mediated by modulation of renal mitochondrial quality control as well by attenuating fibrotic and lipotoxic mechanisms in kidney cells. MK-8722 (10 mg/kg per day compared with vehicle group) achieved modest blood pressure reduction (10 mmHg lower for mean blood pressure) and significant metabolic improvements (decreased plasma glucose, triglyceride, and body weight) that could contribute to renoprotection. These data further validate the concept that targeting metabolic dysregulation in CKD could be a potential therapeutic approach. SIGNIFICANCE STATEMENT: We demonstrate in the present study that the pharmacological activation of AMPK using a small-molecule agent provided renoprotection and improved systemic and cellular metabolism. We further indicate that modulation of renal mitochondrial quality control probably contributed to renoprotection and was distinct from the effects of enalapril. Our findings suggest that improving renal mitochondrial biogenesis and function and attenuating fibrosis and lipotoxicity by targeting key metabolic nodes could be a potential therapeutic approach in management of CKD that could complement the current standard of care.
Assuntos
Nefropatias Diabéticas/metabolismo , Hipoglicemiantes/uso terapêutico , Imidazóis/uso terapêutico , Proteínas Quinases/metabolismo , Piridinas/uso terapêutico , Quinases Proteína-Quinases Ativadas por AMP , Idoso , Animais , Benzimidazóis , Glicemia/metabolismo , Pressão Sanguínea , Células Cultivadas , Nefropatias Diabéticas/tratamento farmacológico , Feminino , Taxa de Filtração Glomerular , Humanos , Hipoglicemiantes/farmacologia , Imidazóis/farmacologia , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Piridinas/farmacologia , Ratos , Ratos Zucker , Triglicerídeos/sangueRESUMO
GPR40 and GPR120 are fatty acid sensors that play important roles in glucose and energy homeostasis. GPR40 potentiates glucose-dependent insulin secretion and demonstrated in clinical studies robust glucose lowering in type 2 diabetes. GPR120 improves insulin sensitivity in rodents, albeit its mechanism of action is not fully understood. Here, we postulated that the antidiabetic efficacy of GPR40 could be enhanced by coactivating GPR120. A combination of GPR40 and GPR120 agonists in db/db mice, as well as a single molecule with dual agonist activities, achieved superior glycemic control compared with either monotherapy. Compared with a GPR40 selective agonist, the dual agonist improved insulin sensitivity in ob/ob mice measured by hyperinsulinemic-euglycemic clamp, preserved islet morphology, and increased expression of several key lipolytic genes in adipose tissue of Zucker diabetic fatty rats. Novel insights into the mechanism of action for GPR120 were obtained. Selective GPR120 activation suppressed lipolysis in primary white adipocytes, although this effect was attenuated in adipocytes from obese rats and obese rhesus, and sensitized the antilipolytic effect of insulin in rat and rhesus primary adipocytes. In conclusion, GPR120 agonism enhances insulin action in adipose tissue and yields a synergistic efficacy when combined with GPR40 agonism.
Assuntos
Tecido Adiposo/metabolismo , Diabetes Mellitus Experimental/metabolismo , Lipólise , Receptores Acoplados a Proteínas G/metabolismo , Tecido Adiposo/efeitos dos fármacos , Animais , Células CHO , Cricetinae , Cricetulus , Diabetes Mellitus Experimental/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Resistência à Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/fisiopatologia , Lipólise/efeitos dos fármacos , Masculino , Camundongos , Ratos , Receptores Acoplados a Proteínas G/agonistasRESUMO
Selective peroxisome proliferator-activated receptor γ (PPARγ) modulators (SPPARγMs) have been actively pursued as the next generation of insulin-sensitizing antidiabetic drugs, because the currently marketed PPARγ full agonists, pioglitazone and rosiglitazone, have been reported to produce serious adverse effects among patients with type 2 diabetes mellitus. We conducted extensive transcriptome profiling studies to characterize and to contrast the activities of 70 SPPARγMs and seven PPARγ full agonists. In both 3T3-L1 adipocytes and adipose tissue from db/db mice, the SPPARγMs generated attenuated and selective gene-regulatory responses, in comparison with full agonists. More importantly, SPPARγMs regulated the expression of antidiabetic efficacy-associated genes to a greater extent than that of adverse effect-associated genes, whereas PPARγ full agonists regulated both gene sets proportionally. Such SPPARγM selectivity demonstrates that PPARγ ligand regulation of gene expression can be fine-tuned, and not just turned on and off, to achieve precise control of complex cellular and physiological functions. It also provides a potential molecular basis for the superior therapeutic window previously observed with SPPARγMs versus full agonists. On the basis of our profiling results, we introduce two novel, gene expression-based scores, the γ activation index and the selectivity index, to aid in the detection and characterization of novel SPPARγMs. These studies provide new insights into the gene-regulatory activity of SPPARγMs as well as novel quantitative indices to facilitate the identification of PPARγ ligands with robust insulin-sensitizing activity and improved tolerance among patients with type 2 diabetes, compared with presently available PPARγ agonist drugs.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Hipoglicemiantes/farmacologia , PPAR gama/agonistas , PPAR gama/metabolismo , Transcriptoma/genética , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Células COS , Chlorocebus aethiops , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Perfilação da Expressão Gênica/métodos , Resistência à Insulina/genética , Ligantes , Masculino , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Transcriptoma/efeitos dos fármacosRESUMO
The innate immune agonist STING (STimulator of INterferon Genes) binds its natural ligand 2'3'-cGAMP (cyclic guanosine-adenosine monophosphate) and initiates type I IFN production. This promotes systemic antigen-specific CD8+ T-cell priming that eventually provides potent antitumor activity. To exploit this mechanism, we synthesized a novel STING agonist, MSA-1, that activates both mouse and human STING with higher in vitro potency than cGAMP. Following intratumoral administration of MSA-1 to a panel of syngeneic mouse tumors on immune-competent mice, cytokine upregulation and its exposure were detected in plasma, other tissues, injected tumors, and noninjected tumors. This was accompanied by effective antitumor activity. Mechanistic studies in immune-deficient mice suggested that antitumor activity of intratumorally dosed STING agonists is in part due to necrosis and/or innate immune responses such as TNF-α activity, but development of a robust adaptive antitumor immunity is necessary for complete tumor elimination. Combination with PD-1 blockade in anti-PD-1-resistant murine models showed that MSA-1 may synergize with checkpoint inhibitors but can also provide superior tumor control as a single agent. We show for the first time that potent cyclic dinucleotides can promote a rapid and stronger induction of the same genes eventually regulated by PD-1 blockade. This may have contributed to the relatively early tumor control observed with MSA-1. Taken together, these data strongly support the development of STING agonists as therapy for patients with aggressive tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 treatment by enhancing the anti-PD-1 immune profile.
Assuntos
Imunidade Inata/imunologia , Imunoterapia/métodos , Interferons/metabolismo , Neoplasias/imunologia , Animais , Linhagem Celular Tumoral , Feminino , Humanos , CamundongosRESUMO
Targeting the programmed death 1/programmed death ligand 1 (PD-1/PD-L1) pathway with immunotherapy has revolutionized the treatment of many cancers. Somatic tumor mutational burden (TMB) and T-cell-inflamed gene expression profile (GEP) are clinically validated pan-tumor genomic biomarkers that can predict responsiveness to anti-PD-1/PD-L1 monotherapy in many tumor types. We analyzed the association between these biomarkers and the efficacy of PD-1 inhibitor in 11 commonly used preclinical syngeneic tumor mouse models using murinized rat anti-mouse PD-1 DX400 antibody muDX400, a surrogate for pembrolizumab. Response to muDX400 treatment was broadly classified into three categories: highly responsive, partially responsive, and intrinsically resistant to therapy. Molecular and cellular profiling validated differences in immune cell infiltration and activation in the tumor microenvironment of muDX400-responsive tumors. Baseline and on-treatment genomic analysis showed an association between TMB, murine T-cell-inflamed gene expression profile (murine-GEP), and response to muDX400 treatment. We extended our analysis to investigate a canonical set of cancer and immune biology-related gene signatures, including signatures of angiogenesis, myeloid-derived suppressor cells, and stromal/epithelial-to-mesenchymal transition/TGFß biology previously shown to be inversely associated with the clinical efficacy of immune checkpoint blockade. Finally, we evaluated the association between murine-GEP and preclinical efficacy with standard-of-care chemotherapy or antiangiogenic agents that previously demonstrated promising clinical activity, in combination with muDX400. Our profiling studies begin to elucidate the underlying biological mechanisms of response and resistance to PD-1/PD-L1 blockade represented by these models, thereby providing insight into which models are most appropriate for the evaluation of orthogonal combination strategies.
Assuntos
Antígeno B7-H1 , Imunoterapia , Neoplasias , Receptor de Morte Celular Programada 1 , Animais , Antígeno B7-H1/antagonistas & inibidores , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Inibidores de Checkpoint Imunológico , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Microambiente TumoralRESUMO
Programmed cell death-1 (PD-1) blockade has a profound effect on the ability of the immune system to eliminate tumors, but many questions remain about the cell types involved and the underlying mechanisms of immune activation. To shed some light on this, the cellular and molecular events following inhibition of PD-1 signaling was investigated in the MC-38 colon carcinoma model using constitutive (PD-1 KO) and conditional (PD1cKO) mice and in wild-type mice treated with PD-1 antibody. The impact on both tumor growth and the development of tumor immunity was assessed. In the PD-1cKO mice, a complete deletion of Pdcd1 in tumor-infiltrating T cells (TILs) after tamoxifen treatment led to the inhibition of tumor growth of both small and large tumors. Extensive immune phenotypic analysis of the TILs by flow and mass cytometry identified 20-different T cell subsets of which specifically 5-CD8 positive ones expanded in all three models after PD-1 blockade. All five subsets expressed granzyme B and interferon gamma (IFNγ). Gene expression analysis of the tumor further supported the phenotypic analysis in both PD-1cKO- and PD-1 Ab-treated mice and showed an upregulation of pathways related to CD4 and CD8 T-cell activation, enhanced signaling through costimulatory molecules and IFNγ, and non-T-cell processes. Altogether, using PD-1cKO mice, we define the intrinsic nature of PD-1 suppression of CD8 T-cell responses in tumor immunity.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Experimentais/imunologia , Receptor de Morte Celular Programada 1/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Feminino , Inibidores de Checkpoint Imunológico/farmacologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Morte Celular Programada 1/deficiênciaRESUMO
Myeloid-derived suppressor cells (MDSC) are immature myeloid cells that accumulate in the tumor microenvironment (TME). MDSCs have been shown to dampen antitumor immune responses and promote tumor growth; however, the mechanisms of MDSC induction and their role in promoting immune suppression in cancer remain poorly understood. Here, we characterized the phenotype and function of monocytic MDSCs (M-MDSC) generated by coculture of human peripheral blood mononuclear cells with SK-MEL-5 cancer cells in vitro. We selected the SK-MEL-5 human melanoma cell line to generate M-MDSCs because these cells form subcutaneous tumors rich in myeloid cells in humanized mice. M-MDSCs generated via SK-MEL-5 coculture expressed low levels of human leukocyte antigen (HLA)-DR, high levels of CD33 and CD11b, and suppressed both CD8+ T-cell proliferation and IFNγ secretion. M-MDSCs also expressed higher levels of immunoglobulin-like transcript 3 (ILT3, also known as LILRB4) and immunoglobulin-like transcript 4 (ILT4, also known as LILRB2) on the cell surface compared with monocytes. Therefore, we investigated how ILT3 targeting could modulate M-MDSC cell function. Treatment with an anti-ILT3 antibody impaired the acquisition of the M-MDSC suppressor phenotype and reduced the capacity of M-MDSCs to cause T-cell suppression. Finally, in combination with anti-programmed cell death protein 1 (PD1), ILT3 blockade enhanced T-cell activation as assessed by IFNγ secretion. IMPLICATIONS: These results suggest that ILT3 expressed on M-MDSCs has a role in inducing immunosuppression in cancer and that antagonism of ILT3 may be useful to reverse the immunosuppressive function of M-MDSCs and enhance the efficacy of immune checkpoint inhibitors.
Assuntos
Melanoma/imunologia , Glicoproteínas de Membrana/imunologia , Monócitos/imunologia , Células Supressoras Mieloides/imunologia , Receptores Imunológicos/imunologia , Animais , Feminino , Xenoenxertos , Humanos , Melanoma/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Monócitos/metabolismo , Células Supressoras Mieloides/metabolismo , Receptores Imunológicos/metabolismoRESUMO
Prostaglandin E2 (PGE2), an arachidonic acid pathway metabolite produced by cyclooxygenase (COX)-1/2, has been shown to impair anti-tumor immunity through engagement with one or more E-type prostanoid receptors (EP1-4). Specific targeting of EP receptors, as opposed to COX-1/2 inhibition, has been proposed to achieve preferential antagonism of PGE2-mediated immune suppression. Here we describe the anti-tumor activity of MF-766, a potent and highly selective small-molecule inhibitor of the EP4 receptor. EP4 inhibition by MF-766 synergistically improved the efficacy of anti-programmed cell death protein 1 (PD-1) therapy in CT26 and EMT6 syngeneic tumor mouse models. Multiparameter flow cytometry analysis revealed that treatment with MF-766 promoted the infiltration of CD8+ T cells, natural killer (NK) cells and conventional dendritic cells (cDCs), induced M1-like macrophage reprogramming, and reduced granulocytic myeloid-derived suppressor cells (MDSC) in the tumor microenvironment (TME). In vitro experiments demonstrated that MF-766 restored PGE2-mediated inhibition of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production in THP-1 cells and human blood, and PGE2-mediated inhibition of interleukin (IL)-2-induced interferon (IFN)-γ production in human NK cells. MF-766 reversed the inhibition of IFN-γ in CD8+ T-cells by PGE2 and impaired suppression of CD8+ T-cells induced by myeloid-derived suppressor cells (MDSC)/PGE2. In translational studies using primary human tumors, MF-766 enhanced anti-CD3-stimulated IFN-γ, IL-2, and TNF-α production in primary histoculture and synergized with pembrolizumab in a PGE2 high TME. Our studies demonstrate that the combination of EP4 blockade with anti-PD-1 therapy enhances antitumor activity by differentially modulating myeloid cell, NK cell, cDC and T-cell infiltration profiles.
Assuntos
Linfócitos T CD8-Positivos , Receptores de Prostaglandina E Subtipo EP4 , Animais , Ciclo-Oxigenase 2 , Dinoprostona , Macrófagos , CamundongosRESUMO
Inhibition of cannabinoid receptor 1 (CB1) has shown efficacy in reducing body weight and improving metabolic parameters, with the effects correlating with target engagement in the brain. The peripheral effects of inhibiting the CB1 receptor has been appreciated through studies in diet-induced obese and liver-specific CB1 knockout mice. In this article, we systematically investigated gene expression changes in peripheral tissues of diet-induced obese mice treated with the CB1 inverse agonist AM251 [1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl)pyrazole-3-carboxamide]. CB1 receptor inhibition led to down-regulation of genes within the de novo fatty acid and cholesterol synthetic pathways, including sterol regulatory element binding proteins 1 and 2 and their downstream targets in both liver and adipose tissue. In addition, genes involved in fatty acid beta-oxidation were up-regulated with AM251 treatment, probably through the activation of peroxisome proliferator-activated receptor alpha (PPARalpha). In adipose tissue, CB1 receptor inhibition led to the down-regulation of genes in the tumor necrosis factor alpha signal transduction pathway and possibly to the activation of PPARgamma, both of which would result in improved insulin sensitivity.
Assuntos
Agonistas de Receptores de Canabinoides , Resistência à Insulina/genética , Tecido Adiposo/metabolismo , Animais , Regulação para Baixo/efeitos dos fármacos , Homozigoto , Insulina/genética , Insulina/metabolismo , Insulina/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Obesos , PPAR alfa/agonistas , PPAR alfa/genética , PPAR alfa/metabolismo , PPAR gama/agonistas , PPAR gama/genética , PPAR gama/metabolismo , Piperidinas , Pirazóis/metabolismo , Pirazóis/farmacologia , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Receptores de Canabinoides/genética , Receptores de Canabinoides/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/agonistas , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Ativação TranscricionalRESUMO
PURPOSE: Adipocytes represent one of the most abundant constituents of the mammary gland. They are essential for mammary tumor growth and survival. Metabolically, one of the more important fat-derived factors ("adipokines") is adiponectin (APN). Serum concentrations of APN negatively correlate with body mass index and insulin resistance. To explore the association of APN with breast cancer and tumor angiogenesis, we took an in vivo approach aiming to study its role in the mouse mammary tumor virus (MMTV)-polyoma middle T antigen (PyMT) mammary tumor model. EXPERIMENTAL DESIGN: We compared the rates of tumor growth in MMTV-PyMT mice in wild-type and APN-null backgrounds. RESULTS: Histology and micro-positron emission tomography imaging show that the rate of tumor growth is significantly reduced in the absence of APN at early stages. PyMT/APN knockout mice exhibit a reduction in their angiogenic profile resulting in nutrient deprivation of the tumors and tumor-associated cell death. Surprisingly, in more advanced malignant stages of the disease, tumor growth develops more aggressively in mice lacking APN, giving rise to a larger tumor burden, an increase in the mobilization of circulating endothelial progenitor cells, and a gene expression fingerprint indicative of more aggressive tumor cells. CONCLUSIONS: These observations highlight a novel important contribution of APN in mammary tumor development and angiogenesis, indicating that APN has potent angio-mimetic properties in tumor vascularization. However, in tumors deprived of APN, this antiangiogenic stress results in an adaptive response that fuels tumor growth through mobilization of circulating endothelial progenitor cells and the development of mechanisms enabling massive cell proliferation despite a chronically hypoxic microenvironment.
Assuntos
Neoplasias Mamárias Experimentais/irrigação sanguínea , Neoplasias Mamárias Experimentais/genética , Neovascularização Patológica/genética , Adiponectina/sangue , Adiponectina/genética , Adiponectina/metabolismo , Animais , Antígenos Virais de Tumores/genética , Apoptose , Western Blotting , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Glucose/farmacocinética , Masculino , Neoplasias Mamárias Experimentais/patologia , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , PPAR gama/agonistas , PPAR gama/metabolismo , Polyomavirus/genética , Tomografia por Emissão de Pósitrons , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tiazolidinedionas/farmacologia , Fatores de Tempo , Carga Tumoral/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Fibrosis, or the accumulation of extracellular matrix, is a common feature of many chronic diseases. To interrogate core molecular pathways underlying fibrosis, we cross-examine human primary cells from various tissues treated with TGF-ß, as well as kidney and liver fibrosis models. Transcriptome analyses reveal that genes involved in fatty acid oxidation are significantly perturbed. Furthermore, mitochondrial dysfunction and acylcarnitine accumulation are found in fibrotic tissues. Substantial downregulation of the PGC1α gene is evident in both in vitro and in vivo fibrosis models, suggesting a common node of metabolic signature for tissue fibrosis. In order to identify suppressors of fibrosis, we carry out a compound library phenotypic screen and identify AMPK and PPAR as highly enriched targets. We further show that pharmacological treatment of MK-8722 (AMPK activator) and MK-4074 (ACC inhibitor) reduce fibrosis in vivo. Altogether, our work demonstrate that metabolic defect is integral to TGF-ß signaling and fibrosis.
Assuntos
Fibrose/genética , Fibrose/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Adenilato Quinase/metabolismo , Animais , Benzimidazóis/farmacologia , Células Cultivadas , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Humanos , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Piridinas/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Transcriptoma/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Immunotherapy fails to cure most cancer patients. Preclinical studies indicate that radiotherapy synergizes with immunotherapy, promoting radiation-induced antitumor immunity. Most preclinical immunotherapy studies utilize transplant tumor models, which overestimate patient responses. Here, we show that transplant sarcomas are cured by PD-1 blockade and radiotherapy, but identical treatment fails in autochthonous sarcomas, which demonstrate immunoediting, decreased neoantigen expression, and tumor-specific immune tolerance. We characterize tumor-infiltrating immune cells from transplant and primary tumors, revealing striking differences in their immune landscapes. Although radiotherapy remodels myeloid cells in both models, only transplant tumors are enriched for activated CD8+ T cells. The immune microenvironment of primary murine sarcomas resembles most human sarcomas, while transplant sarcomas resemble the most inflamed human sarcomas. These results identify distinct microenvironments in murine sarcomas that coevolve with the immune system and suggest that patients with a sarcoma immune phenotype similar to transplant tumors may benefit most from PD-1 blockade and radiotherapy.
Assuntos
Sarcoma/terapia , Análise de Célula Única/métodos , Microambiente Tumoral/imunologia , Animais , Antineoplásicos Imunológicos/farmacologia , Transplante de Medula Óssea , Linfócitos T CD8-Positivos/imunologia , Proteínas de Ligação a DNA/genética , Resistencia a Medicamentos Antineoplásicos/imunologia , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoterapia , Camundongos Endogâmicos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Sarcoma/genética , Sarcoma/imunologia , Evasão Tumoral , Microambiente Tumoral/genética , Sequenciamento do ExomaRESUMO
Bromodomain and extraterminal domain (BET) proteins help direct the differentiation of helper T cell subsets, but their role in activated T cell function has not been described in detail. In this study, we investigate various consequences of epigenetic perturbation in human T lymphocytes using MK-8628, a potent and highly selective inhibitor of BET proteins. MK-8628 reduces the expression of canonical transcripts directing the proliferation, activation, and effector function of T lymphocytes. Treatment with MK-8628 abolishes the expression of key cyclins required for cell cycle progression and induces G1 cell cycle arrest in TCR-activated lymphocytes. This antiproliferative phenotype partially results from T lymphocyte apoptosis, which is exacerbated by MK-8628. In naive and memory T cell subsets, MK-8628 antagonizes T cell activation and suppresses polyfunctional cytokine production. Collectively, our results describe potent immunosuppressive effects of BET inhibition on human T cell biology. These results have important implications for immune modulatory targeting of BET proteins in the settings of T cell-driven autoimmune inflammation.
Assuntos
Acetanilidas/farmacologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Compostos Heterocíclicos com 3 Anéis/farmacologia , Imunossupressores/farmacologia , Proteínas/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Voluntários Saudáveis , Humanos , Ativação Linfocitária/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Physical activity promotes metabolic and cardiovascular health benefits that derive in part from the transcriptional responses to exercise that occur within skeletal muscle and other organs. There is interest in discovering a pharmacologic exercise mimetic that could imbue wellness and alleviate disease burden. However, the molecular physiology by which exercise signals the transcriptional response is highly complex, making it challenging to identify a single target for pharmacological mimicry. The current studies evaluated the transcriptome responses in skeletal muscle, heart, liver, and white and brown adipose to novel small molecule activators of AMPK (pan-activators for all AMPK isoforms) compared to that of exercise. A striking level of congruence between exercise and pharmacological AMPK activation was observed across the induced transcriptome of these five tissues. However, differences in acute metabolic response between exercise and pharmacologic AMPK activation were observed, notably for acute glycogen balances and related to the energy expenditure induced by exercise but not pharmacologic AMPK activation. Nevertheless, intervention with repeated daily administration of short-acting activation of AMPK was found to mitigate hyperglycemia and hyperinsulinemia in four rodent models of metabolic disease and without the cardiac glycogen accretion noted with sustained pharmacologic AMPK activation. These findings affirm that activation of AMPK is a key node governing exercise mediated transcription and is an attractive target as an exercise mimetic.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Tecido Adiposo/metabolismo , Fígado/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Animais , Metabolismo Energético , Ativação Enzimática/efeitos dos fármacos , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Homeostase , Camundongos Endogâmicos C57BL , Oxirredução , Condicionamento Físico AnimalRESUMO
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by pathologic T cell-B cell interactions and autoantibody production. Defining the T cell populations that drive B cell responses in SLE may enable design of therapies that specifically target pathologic cell subsets. Here, we evaluated the phenotypes of CD4+ T cells in the circulation of 52 SLE patients drawn from multiple cohorts and identified a highly expanded PD-1hiCXCR5-CD4+ T cell population. Cytometric, transcriptomic, and functional assays demonstrated that PD-1hiCXCR5-CD4+ T cells from SLE patients are T peripheral helper (Tph) cells, a CXCR5- T cell population that stimulates B cell responses via IL-21. The frequency of Tph cells, but not T follicular helper (Tfh) cells, correlated with both clinical disease activity and the frequency of CD11c+ B cells in SLE patients. PD-1hiCD4+ T cells were found within lupus nephritis kidneys and correlated with B cell numbers in the kidney. Both IL-21 neutralization and CRISPR-mediated deletion of MAF abrogated the ability of Tph cells to induce memory B cell differentiation into plasmablasts in vitro. These findings identify Tph cells as a highly expanded T cell population in SLE and suggest a key role for Tph cells in stimulating pathologic B cell responses.
Assuntos
Linfócitos B/imunologia , Interleucinas/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Proteínas Proto-Oncogênicas c-maf/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Adulto , Idoso , Antígeno CD11c/metabolismo , Sistemas CRISPR-Cas/genética , Estudos de Casos e Controles , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/genética , Comunicação Celular/imunologia , Técnicas de Cultura de Células , Separação Celular , Células Cultivadas , Técnicas de Cocultura , Feminino , Citometria de Fluxo , Técnicas de Inativação de Genes , Humanos , Interleucinas/antagonistas & inibidores , Lúpus Eritematoso Sistêmico/sangue , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Masculino , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/metabolismo , Proteínas Proto-Oncogênicas c-maf/genética , RNA-Seq , Receptores CXCR5/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismoRESUMO
Adipose tissue is a metabolically responsive endocrine organ that secretes a myriad of adipokines. Antidiabetic drugs such as peroxisome proliferator-activated receptor (PPAR) gamma agonists target adipose tissue gene expression and correct hyperglycemia via whole-body insulin sensitization. The mechanism by which altered gene expression in adipose tissue affects liver and muscle insulin sensitivity (and thus glucose homeostasis) is not fully understood. One possible mechanism involves the alteration in adipokine secretion, in particular the up-regulation of secreted factors that increase whole-body insulin sensitivity. Here, we report the use of transcriptional profiling to identify genes encoding for secreted proteins the expression of which is regulated by PPARgamma agonists. Of the 379 genes robustly regulated by two structurally distinct PPARgamma agonists in the epididymal white adipose tissue (EWAT) of db/db mice, 33 encoded for known secreted proteins, one of which was FGF21. Although FGF21 was recently reported to be up-regulated in cultured adipocytes by PPARgamma agonists and in liver by PPARalpha agonists and induction of ketotic states, we demonstrate that the protein is transcriptionally up-regulated in adipose tissue in vivo by PPARgamma agonist treatment and under a variety of physiological conditions, including fasting and high fat diet feeding. In addition, we found that circulating levels of FGF21 protein were increased upon treatment with PPARgamma agonists and under ketogenic states. These results suggest a role for FGF21 in mediating the antidiabetic activities of PPARgamma agonists.
Assuntos
Tecido Adiposo/metabolismo , Fatores de Crescimento de Fibroblastos/biossíntese , PPAR gama/fisiologia , Regulação para Cima/fisiologia , Células 3T3-L1 , Tecido Adiposo/fisiologia , Sequência de Aminoácidos , Animais , Fatores de Crescimento de Fibroblastos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Dados de Sequência Molecular , PPAR gama/genética , Coelhos , Regulação para Cima/genéticaRESUMO
The use of the thiazolidinedione insulin sensitizers rosiglitazone and pioglitazone for the treatment of type 2 diabetes mellitus in recent years has proven to be effective in helping patients resume normal glycemic control. However, their use is often associated with undesirable side effects including peripheral edema, congestive heart failure and weight gain. Here, we report the identification and characterization of a novel selective PPARgamma modulator, SPPARgammaM5 ((2S)-2-(2-chloro-5-{[3-(4-chlorophenoxy)-2-methyl-6-(trifluoromethoxy)-1H-indol-1-yl]methyl} phenoxy)propionic acid), which has notable insulin sensitizing properties and a superior tolerability profile to that of rosiglitazone. SPPARgammaM5 is a potent ligand of human PPARgamma with high selectivity versus PPARalpha or PPARdelta in receptor competitive binding assays. In cell-based transcriptional activation assays, SPPARgammaM5 was a potent partial agonist of human PPARgamma in comparison to the PPARgamma full agonist rosiglitazone. Compared to rosiglitazone or the PPARgamma full agonist COOH (2-(2-(4-phenoxy-2-propylphenoxy)ethyl)indole-5-acetic acid), SPPARgammaM5 induced an attenuated PPARgamma-regulated gene expression profile in fully differentiated 3T3-L1 adipocytes and white adipose tissue of chronically treated db/db mice. SPPARgammaM5 treatment also reduced the insulin resistance index by homeostasis model assessment (HOMA), suggesting an improvement in insulin resistance in these db/db mice. Treatment of obese Zucker rats with either rosiglitazone or SPPARgammaM5 resulted in an improvement in selected parameters that serve as surrogate indicators of insulin resistance and hyperlipidemia. However, unlike rosiglitazone, SPPARgammaM5 did not cause significant fluid retention or cardiac hypertrophy in these rats. Thus, compounds such as SPPARgammaM5 may offer beneficial effects on glycemic control with significantly attenuated adverse effects.
Assuntos
Acetatos/farmacologia , Doenças Cardiovasculares/induzido quimicamente , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/farmacologia , Indóis/farmacologia , Resistência à Insulina , PPAR gama/efeitos dos fármacos , Propionatos/farmacologia , Tiazolidinedionas/farmacologia , Células 3T3-L1 , Acetatos/efeitos adversos , Acetatos/metabolismo , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/metabolismo , Animais , Células COS , Cardiomegalia/induzido quimicamente , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatologia , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/fisiopatologia , Chlorocebus aethiops , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Agonismo Parcial de Drogas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Hemodiluição , Humanos , Hipoglicemiantes/efeitos adversos , Hipoglicemiantes/metabolismo , Indóis/efeitos adversos , Indóis/metabolismo , Resistência à Insulina/genética , Masculino , Camundongos , Camundongos Endogâmicos , PPAR alfa/efeitos dos fármacos , PPAR alfa/metabolismo , PPAR delta/efeitos dos fármacos , PPAR delta/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Propionatos/efeitos adversos , Propionatos/metabolismo , Ligação Proteica , Ratos , Ratos Zucker , Rosiglitazona , Tiazolidinedionas/efeitos adversos , Tiazolidinedionas/metabolismo , Ativação Transcricional/efeitos dos fármacos , Transfecção , Equilíbrio Hidroeletrolítico/efeitos dos fármacosRESUMO
Similar efficacy of the cathepsin K inhibitor odanacatib (ODN) and the bisphosphonate alendronate (ALN) in reducing bone turnover markers and increasing bone mineral density in spine and hip were previously demonstrated in ovariectomized (OVX)-monkeys treated for 20 months in prevention mode. Here, we profiled RNA from tibial metaphysis and diaphysis of the same study using Affymetrix microarrays, and selected 204 probe sets (p < 0.001, three-group ANOVA) that were differentially regulated by ODN or ALN versus vehicle. Both drugs produced strikingly different effects on known bone-related genes and pathways at the transcriptional level. Although ALN either reduced or had neutral effects on bone resorption-related genes, ODN significantly increased the expression of osteoclast genes (eg, APC5, TNFRSF11A, CTSK, ITGB3, and CALCR), consistent with previous findings on the effects of this agent in enhancing the number of nonresorbing osteoclasts. Conversely, ALN reduced the expression of known bone formation-related genes (eg, TGFBR1, SPP1, RUNX2, and PTH1R), whereas ODN either increased or had neutral effects on their expression. These differential effects of ODN versus ALN on bone resorption and formation were highly correlative to the changes in bone turnover markers, cathepsin K (Catk) target engagement marker serum C-terminal cross-linked telopeptide (1-CTP) and osteoclast marker tartrate resistant acid phosphatase isoform 5b (TRAP5b) in the same monkeys. Overall, the molecular profiling results are consistent with the known pharmacological actions of these agents on bone remodeling and clearly differentiate the molecular mechanisms of ODN from the bisphosphonates.