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1.
Nucleic Acids Res ; 52(9): 5016-5032, 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38471819

RESUMO

Viruses are master remodelers of the host cell environment in support of infection and virus production. For example, viruses typically regulate cell gene expression through modulating canonical cell promoter activity. Here, we show that Epstein Barr virus (EBV) replication causes 'de novo' transcription initiation at 29674 new transcription start sites throughout the cell genome. De novo transcription initiation is facilitated in part by the unique properties of the viral pre-initiation complex (vPIC) that binds a TATT[T/A]AA, TATA box-like sequence and activates transcription with minimal support by additional transcription factors. Other de novo promoters are driven by the viral transcription factors, Zta and Rta and are influenced by directional proximity to existing canonical cell promoters, a configuration that fosters transcription through existing promoters and transcriptional interference. These studies reveal a new way that viruses interact with the host transcriptome to inhibit host gene expression and they shed light on primal features driving eukaryotic promoter function.


Assuntos
Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Iniciação da Transcrição Genética , Replicação Viral , Humanos , Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno , Regiões Promotoras Genéticas , TATA Box , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Transcrição Gênica , Proteínas Virais/metabolismo , Proteínas Virais/genética , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/virologia
2.
Clin Immunol ; 260: 109915, 2024 03.
Artigo em Inglês | MEDLINE | ID: mdl-38286172

RESUMO

The increased risk for acquiring secondary illnesses in people living with HIV (PLWH) has been associated with immune dysfunction. We have previously found that circulating monocytes from PLWH display a trained phenotype. Here, we evaluated the metabolic profile of these cells and found increased mitochondrial respiration and glycolysis of monocyte-derived macrophages (MDMs) from PLWH. We additionally found that cART shifted the energy metabolism of MDMs from controls toward increased utilization of mitochondrial respiration. Importantly, both downregulation of IKAROS expression and inhibition of the mTOR pathway reversed the metabolic profile of MDMs from PLWH and cART-treated control-MDMs. Altogether, this study reveals a very specific metabolic adaptation of MDMs from PLWH, which involves an IKAROS/mTOR-dependent increase of mitochondrial respiration and glycolysis. We propose that this metabolic adaptation decreases the ability of these cells to respond to environmental cues by "locking" PLWH monocytes in a pro-inflammatory and activated phenotype.


Assuntos
Infecções por HIV , Humanos , Macrófagos , Monócitos , Fenótipo , Serina-Treonina Quinases TOR/metabolismo
3.
Front Immunol ; 14: 1244159, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37901240

RESUMO

Introduction: Triple-negative breast cancer (TNBC) comprises a heterogeneous group of clinically aggressive tumors with high risk of recurrence and metastasis. Current pharmacological treatment options remain largely limited to chemotherapy. Despite promising results, the efficacy of immunotherapy and chemo-immunotherapy in TNBC remains limited. There is strong evidence supporting the involvement of Notch signaling in TNBC progression. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Notch inhibitors, including g-secretase inhibitors (GSIs), are quite effective in preclinical models of TNBC. However, the success of GSIs in clinical trials has been limited by their intestinal toxicity and potential for adverse immunological effects, since Notch plays key roles in T-cell activation, including CD8 T-cells in tumors. Our overarching goal is to replace GSIs with agents that lack their systemic toxicity and ideally, do not affect tumor immunity. We identified sulindac sulfide (SS), the active metabolite of FDA-approved NSAID sulindac, as a potential candidate to replace GSIs. Methods: We investigated the pharmacological and immunotherapeutic properties of SS in TNBC models in vitro, ex-vivo and in vivo. Results: We confirmed that SS, a known γ-secretase modulator (GSM), inhibits Notch1 cleavage in TNBC cells. SS significantly inhibited mammosphere growth in all human and murine TNBC models tested. In a transplantable mouse TNBC tumor model (C0321), SS had remarkable single-agent anti-tumor activity and eliminated Notch1 protein expression in tumors. Importantly, SS did not inhibit Notch cleavage in T- cells, and the anti-tumor effects of SS were significantly enhanced when combined with a-PD1 immunotherapy in our TNBC organoids and in vivo. Discussion: Our data support further investigation of SS for the treatment of TNBC, in conjunction with chemo- or -chemo-immunotherapy. Repurposing an FDA-approved, safe agent for the treatment of TNBC may be a cost-effective, rapidly deployable therapeutic option for a patient population in need of more effective therapies.


Assuntos
Sulindaco , Neoplasias de Mama Triplo Negativas , Humanos , Animais , Camundongos , Sulindaco/farmacologia , Sulindaco/uso terapêutico , Secretases da Proteína Precursora do Amiloide , Neoplasias de Mama Triplo Negativas/metabolismo , Anti-Inflamatórios não Esteroides/uso terapêutico , Modelos Animais de Doenças
4.
Cancers (Basel) ; 15(15)2023 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-37568775

RESUMO

BACKGROUND: The efficacy of CB-103 was evaluated in preclinical models of both ER+ and TNBC. Furthermore, the therapeutic efficacy of combining CB-103 with fulvestrant in ER+ BC and paclitaxel in TNBC was determined. METHODS: CB-103 was screened in combination with a panel of anti-neoplastic drugs. We evaluated the anti-tumor activity of CB-103 with fulvestrant in ESR1-mutant (Y537S), endocrine-resistant BC xenografts. In the same model, we examined anti-CSC activity in mammosphere formation assays for CB-103 alone or in combination with fulvestrant or palbociclib. We also evaluated the effect of CB-103 plus paclitaxel on primary tumors and CSC in a GSI-resistant TNBC model HCC1187. Comparisons between groups were performed with a two-sided unpaired Students' t-test. A one-way or two-way ANOVA followed by Tukey's post-analysis was performed to analyze the in vivo efficacy study results. THE RESULTS: CB-103 showed synergism with fulvestrant in ER+ cells and paclitaxel in TNBC cells. CB-103 combined with fulvestrant or paclitaxel potently inhibited mammosphere formation in both models. Combination of CB-103 and fulvestrant significantly reduced tumor volume in an ESR1-mutant, the endocrine-resistant BC model. In a GSI-resistant TNBC model, CB-103 plus paclitaxel significantly delayed tumor growth compared to paclitaxel alone. CONCLUSION: our data indicate that CB-103 is an attractive candidate for clinical investigation in endocrine-resistant, recurrent breast cancers with biomarker-confirmed Notch activity in combination with SERDs and/or CDKis and in TNBCs with biomarker-confirmed Notch activity in combination with taxane-containing chemotherapy regimens.

6.
Oncogene ; 41(47): 5076-5091, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36243802

RESUMO

Treatment of patients with triple-negative breast cancer (TNBC) has been challenging due to the absence of well-defined molecular targets and the highly invasive and proliferative nature of TNBC cells. Current treatments against TNBC have shown little promise due to high recurrence rate in patients. Consequently, there is a pressing need for novel and efficacious therapies against TNBC. Here, we report the discovery of a novel small molecule inhibitor (NSC33353) with potent anti-tumor activity against TNBC cells. The anti-proliferative effects of this small molecule inhibitor were determined using 2D and 3D cell proliferation assays. We found that NSC33353 significantly reduces the proliferation of TNBC cells in these assays. Using proteomics, next generation sequencing (NGS), and gene enrichment analysis, we investigated global regulatory pathways affected by this compound in TNBC cells. Proteomics data indicate a significant metabolic reprograming affecting both glycolytic enzymes and energy generation through oxidative phosphorylation. Subsequently, using metabolic (Seahorse) and enzymatic assays, we validated our proteomics and NGS analysis findings. Finally, we showed the inhibitory and anti-tumor effects of this small molecule in vitro and confirmed its inhibitory activity in vivo. Doxorubicin is one of the most effective agents in the treatment of TNBC and resistance to this drug has been a major problem. We show that the combination of NSC33353 and doxorubicin suppresses the growth of TNBC cells synergistically, suggesting that NSC33353 enhances TNBC sensitivity to doxorubicin. In summary, our data indicate that the small molecule inhibitor, NSC33353, exhibits anti-tumor activity in TNBC cells, and works in a synergistic fashion with a well-known chemotherapeutic agent.


Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/genética , Linhagem Celular Tumoral , Proliferação de Células , Apoptose , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
7.
EBioMedicine ; 77: 103910, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35248994

RESUMO

BACKGROUND: Low-density neutrophils (LDN) are increased in several inflammatory diseases and may also play a role in the low-grade chronic inflammation associated with obesity. Here we explored their role in obesity, determined their gene signatures, and assessed the effect of bariatric surgery. METHODS: We compared the number, function, and gene expression profiles of circulating LDN in morbidly obese patients (MOP, n=27; body mass index (BMI) > 40 Kg/m2) and normal-weight controls (NWC, n=20; BMI < 25 Kg/m2) in a case-control study. Additionally, in a prospective longitudinal study, we measured changes in the frequency of LDN after bariatric surgery (n=36) and tested for associations with metabolic and inflammatory parameters. FINDINGS: LDN and inflammatory markers were significantly increased in MOP compared to NWC. Transcriptome analysis showed increased neutrophil-related gene expression signatures associated with inflammation, neutrophil activation, and immunosuppressive function. However, LDN did not suppress T cells proliferation and produced low levels of reactive oxygen species (ROS). Circulating LDN in MOP significantly decreased after bariatric surgery in parallel with BMI, metabolic syndrome, and inflammatory markers. INTERPRETATION: Obesity increases LDN displaying an inflammatory gene signature. Our results suggest that LDN may represent a neutrophil subset associated with chronic inflammation, a feature of obesity that has been previously associated with the appearance and progression of co-morbidities. Furthermore, bariatric surgery, as an efficient therapy for severe obesity, reduces LDN in circulation and improves several components of the metabolic syndrome supporting its recognized anti-inflammatory and beneficial metabolic effects. FUNDING: This work was supported in part by grants from the National Institutes of Health (NIH; 5P30GM114732-02, P20CA233374 - A. Ochoa and L. Miele), Pennington Biomedical NORC (P30DK072476 - E. Ravussin & LSU-NO Stanley S. Scott Cancer Center and Louisiana Clinical and Translational Science Center (LACaTS; U54-GM104940 - J. Kirwan).


Assuntos
Cirurgia Bariátrica , Obesidade Mórbida , Cirurgia Bariátrica/métodos , Estudos de Casos e Controles , Humanos , Estudos Longitudinais , Neutrófilos/metabolismo , Obesidade Mórbida/complicações , Obesidade Mórbida/metabolismo , Obesidade Mórbida/cirurgia , Estudos Prospectivos
8.
Front Immunol ; 12: 785905, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34917094

RESUMO

Persons living with HIV (PLWH) are at higher risk of developing secondary illnesses than their uninfected counterparts, suggestive of a dysfunctional immune system in these individuals. Upon exposure to pathogens, monocytes undergo epigenetic remodeling that results in either a trained or a tolerant phenotype, characterized by hyper-responsiveness or hypo-responsiveness to secondary stimuli, respectively. We utilized CD14+ monocytes from virally suppressed PLWH and healthy controls for in vitro analysis following polarization of these cells toward a pro-inflammatory monocyte-derived macrophage (MDM) phenotype. We found that in PLWH-derived MDMs, pro-inflammatory signals (TNFA, IL6, IL1B, miR-155-5p, and IDO1) dominate over negative feedback signals (NCOR2, GSN, MSC, BIN1, and miR-146a-5p), favoring an abnormally trained phenotype. The mechanism of this reduction in negative feedback involves the attenuated expression of IKZF1, a transcription factor required for de novo synthesis of RELA during LPS-induced inflammatory responses. Furthermore, restoring IKZF1 expression in PLWH-MDMs partially reinstated expression of negative regulators of inflammation and lowered the expression of pro-inflammatory cytokines. Overall, this mechanism may provide a link between dysfunctional immune responses and susceptibility to co-morbidities in PLWH with low or undetectable viral load.


Assuntos
Suscetibilidade a Doenças/imunologia , Infecções por HIV/imunologia , Fator de Transcrição Ikaros/metabolismo , Macrófagos/imunologia , Fator de Transcrição RelA/metabolismo , Fármacos Anti-HIV/administração & dosagem , Estudos de Casos e Controles , Citocinas/metabolismo , Retroalimentação Fisiológica , Feminino , Regulação da Expressão Gênica/imunologia , HIV/imunologia , HIV/isolamento & purificação , Infecções por HIV/sangue , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Voluntários Saudáveis , Humanos , Inflamação/sangue , Inflamação/imunologia , Lipopolissacarídeos/imunologia , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Fator de Transcrição RelA/genética , Carga Viral/efeitos dos fármacos , Carga Viral/imunologia
9.
Mol Cancer Ther ; 20(7): 1295-1304, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33879557

RESUMO

Immune-checkpoint inhibitor (ICI) therapy has been widely used to treat different human cancers, particularly advanced solid tumors. However, clinical studies have reported that ICI immunotherapy benefits only ∼15% of patients with colorectal cancer, specifically those with tumors characterized by microsatellite instability (MSI), a molecular marker of defective DNA mismatch repair (dMMR). For the majority of patients with colorectal cancer who carry proficient MMR (pMMR), ICIs have shown little clinical benefit. In this study, we examined the efficacy of sulindac to enhance the response of pMMR colorectal cancer to anti-PD-L1 immunotherapy. We utilized a CT26 syngeneic mouse tumor model to compare the inhibitory effects of PD-L1 antibody (Ab), sulindac, and their combination on pMMR colorectal cancer tumor growth. We found that mice treated with combination therapy showed a significant reduction in tumor volume, along with increased infiltration of CD8+ T lymphocytes in the tumor tissues. We also demonstrated that sulindac could downregulate PD-L1 by blocking NF-κB signaling, which in turn led to a decrease in exosomal PD-L1. Notably, PD-L1 Ab can be bound and consumed by exosomal PD-L1 in the blood circulation. Therefore, in combination therapy, sulindac downregulating PD-L1 leads to increased availability of PD-L1 Ab, which potentially improves the overall efficacy of anti-PD-L1 therapy. We also show that low-dose sulindac does not appear to have a systemic inhibitory effect on prostaglandin E2 (PGE2). In conclusion, our findings provide unique insights into the mechanism of action and efficacy for sulindac as an immunomodulatory agent in combination with anti-PD-L1 therapy for the treatment of pMMR colorectal cancer.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Antineoplásicos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Inibidores de Checkpoint Imunológico/farmacologia , Sulindaco/farmacologia , Animais , Antineoplásicos Imunológicos/uso terapêutico , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos
10.
J Immunother Cancer ; 9(1)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33495297

RESUMO

BACKGROUND: Poly(ADP-ribose) polymerase (PARP) inhibitors (eg, olaparib) are effective against BRCA-mutated cancers at/near maximum tolerated doses by trapping PARP-1 on damaged chromatin, benefitting only small patient proportions. The benefits of targeting non-DNA repair aspects of PARP with metronomic doses remain unexplored. METHODS: Colon epithelial cells or mouse or human bone marrow (BM)-derived-myeloid-derived suppressor cells (MDSCs) were stimulated to assess the effect of partial PARP-1 inhibition on inflammatory gene expression or immune suppression. Mice treated with azoxymethane/four dextran-sulfate-sodium cycles or APCMin/+ mice bred into PARP-1+/- or treated with olaparib were used to examine the role of PARP-1 in colitis-induced or spontaneous colon cancer, respectively. Syngeneic MC-38 cell-based (microsatellite instability, MSIhigh) or CT-26 cell-based (microsatellite stable, MSS) tumor models were used to assess the effects of PARP inhibition on host responses and synergy with anti-Programmed cell Death protein (PD)-1 immunotherapy. RESULTS: Partial PARP-1 inhibition, via gene heterozygosity or a moderate dose of olaparib, protected against colitis-mediated/APCMin -mediated intestinal tumorigenesis and APCMin -associated cachexia, while extensive inhibition, via gene knockout or a high dose of olaparib, was ineffective or aggravating. A sub-IC50-olaparib dose or PARP-1 heterozygosity was sufficient to block tumorigenesis in a syngeneic colon cancer model by modulating the suppressive function, but not intratumoral migration or differentiation, of MDSCs, with concomitant increases in intratumoral T cell function and cytotoxicity, as assessed by granzyme-B/interferon-γ levels. Adoptive transfer of WT-BM-MDSCs abolished the protective effects of PARP-1 heterozygosity. The mechanism of MDSC modulation involved a reduction in arginase-1/inducible nitric oxide synthase/cyclo-oxygenase-2, but independent of PARP-1 trapping on chromatin. Although a high-concentration olaparib or the high-trapping PARP inhibitor, talazoparib, activated stimulator of interferon gene (STING) in BRCA-proficient cells and induced DNA damage, sub-IC50 concentrations of either drug failed to induce activation of the dsDNA break sensor. STING expression appeared dispensable for MDSC suppressive function and was not strictly required for olaparib-mediated effects. Ironically, STING activation blocked human and mouse MDSC function with no additive effects with olaparib. A metronomic dose of olaparib was highly synergistic with anti-PD-1-based immunotherapy, leading to eradication of MSIhigh or reduction of MSS tumors in mice. CONCLUSIONS: These results support a paradigm-shifting concept that expands the utility of PARP inhibitor and encourage testing metronomic dosing of PARP inhibitor to enhance the efficacy of checkpoint inhibitor-based immunotherapies in cancer.


Assuntos
Colite/complicações , Neoplasias do Colo/tratamento farmacológico , Inibidores de Checkpoint Imunológico/administração & dosagem , Ftalazinas/administração & dosagem , Piperazinas/administração & dosagem , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Administração Metronômica , Animais , Azoximetano/efeitos adversos , Linhagem Celular Tumoral , Colite/induzido quimicamente , Neoplasias do Colo/etiologia , Sulfato de Dextrana/efeitos adversos , Sinergismo Farmacológico , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Camundongos , Células Supressoras Mieloides/metabolismo , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
11.
J Vis Exp ; (154)2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31885371

RESUMO

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. While antiretroviral therapy efficiently lowers systemic viral load and restores normal CD4+ T cell counts, it does not reconstitute a completely functional immune system. A dysfunctional immune system in HIV-infected individuals undergoing cART may be characterized by immune activation, early aging of immune cells, or persistent inflammation. These conditions, along with comorbid factors associated with HIV infection, add complexity to the disease, which cannot be easily reproduced in cellular and animal models. To investigate the molecular events underlying immune dysfunction in these patients, a system to culture and manipulate human primary monocytes in vitro is presented here. Specifically, the protocol allows for the culture and transfection of primary CD14+ monocytes obtained from HIV-infected individuals undergoing cART as well as from HIV-negative controls. The method involves isolation, culture, and transfection of monocytes and monocyte-derived macrophages. While commercially available kits and reagents are employed, the protocol provides important tips and optimized conditions for successful adherence and transfection of monocytes with miRNA mimics and inhibitors as well as with siRNAs.


Assuntos
Separação Celular/métodos , Monócitos/citologia , Transfecção , Animais , Polaridade Celular , Sobrevivência Celular , Células Cultivadas , Regulação para Baixo , Humanos , Ativação de Macrófagos , Macrófagos/citologia , MicroRNAs/genética , MicroRNAs/metabolismo , Fenótipo , RNA Interferente Pequeno/metabolismo
12.
Front Oncol ; 8: 575, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30564555

RESUMO

Triple negative breast cancer (TNBC) patients have high risk of recurrence and metastasis, and current treatment options remain limited. Cancer stem-like cells (CSCs) have been linked to cancer initiation, progression and chemotherapy resistance. Notch signaling is a key pathway regulating TNBC CSC survival. Treatment of TNBC with PI3K or mTORC1/2 inhibitors results in drug-resistant, Notch-dependent CSC. However, downstream mechanisms and potentially druggable Notch effectors in TNBC CSCs are largely unknown. We studied the role of the AKT pathway and mitochondrial metabolism downstream of Notch signaling in TNBC CSC from cell lines representative of different TNBC molecular subtypes as well as a novel patient-derived model. We demonstrate that exposure of TNBC cells to recombinant Notch ligand Jagged1 leads to rapid AKT phosphorylation in a Notch1-dependent but RBP-Jκ independent fashion. This requires mTOR and IKKα. Jagged1 also stimulates mitochondrial respiration and fermentation in an AKT- and IKK-dependent fashion. Notch1 co-localizes with mitochondria in TNBC cells. Pharmacological inhibition of Notch cleavage by gamma secretase inhibitor PF-03084014 in combination with AKT inhibitor MK-2206 or IKK-targeted NF-κB inhibitor Bay11-7082 blocks secondary mammosphere formation from sorted CD90hi or CD44+CD24low (CSCs) cells. A TNBC patient-derived model gave comparable results. Besides mitochondrial oxidative metabolism, Jagged1 also triggers nuclear, NF-κB-dependent transcription of anti-apoptotic gene cIAP-2. This requires recruitment of Notch1, IKKα and NF-κB to the cIAP-2 promoter. Our observations support a model where Jagged1 triggers IKKα-dependent, mitochondrial and nuclear Notch1 signals that stimulate AKT phosphorylation, oxidative metabolism and transcription of survival genes in PTEN wild-type TNBC cells. These data suggest that combination treatments targeting the intersection of the Notch, AKT and NF-κB pathways have potential therapeutic applications against CSCs in TNBC cases with Notch1 and wild-type PTEN expression.

13.
Mol Cell Biol ; 38(10)2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29483302

RESUMO

Insulin receptor substrate 1 (IRS-1) is a common cytosolic adaptor molecule involved in signal transduction from insulin and insulin-like growth factor I (IGF-I) receptors. IRS-1 can also be found in the nucleus. We report here a new finding of unique IRS-1 nuclear structures, which we observed initially in glioblastoma biopsy specimens and glioblastoma xenografts. These nuclear structures can be reproduced in vitro by the ectopic expression of IRS-1 cDNA cloned in frame with the nuclear localization signal (NLS-IRS-1). In these structures, IRS-1 localizes at the periphery, while the center harbors a key autophagy protein, LC3. These new nuclear structures are highly dynamic, rapidly exchange IRS-1 molecules with the surrounding nucleoplasm, disassemble during mitosis, and require a growth stimulus for their reassembly and maintenance. In tumor cells engineered to express NLS-IRS-1, the IRS-1/LC3 nuclear structures repress autophagy induced by either amino acid starvation or rapamycin treatment. In this process, IRS-1 nuclear structures sequester LC3 inside the nucleus, possibly preventing its cytosolic translocation and the formation of new autophagosomes. This novel mechanism provides a quick and reversible way of inhibiting autophagy, which could counteract autophagy-induced cancer cell death under severe stress, including anticancer therapies.


Assuntos
Proteínas Substratos do Receptor de Insulina/metabolismo , Proteínas Substratos do Receptor de Insulina/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Autofagia/fisiologia , Núcleo Celular/fisiologia , Sobrevivência Celular/genética , Glioblastoma/metabolismo , Células HeLa , Humanos , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/ultraestrutura , Fator de Crescimento Insulin-Like I/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Neoplasias , Fosfoproteínas , Receptor IGF Tipo 1/fisiologia , Transdução de Sinais
14.
Oncoimmunology ; 6(10): e1344804, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29123954

RESUMO

Myeloid-derived suppressor cells (MDSC) promote tumor growth by blocking anti-tumor T cell responses. Recent reports show that MDSC increase fatty acid uptake and fatty acid oxidation (FAO) to support their immunosuppressive functions. Inhibition of FAO promoted a therapeutic T cell-mediated anti-tumor effect. Here, we sought to determine the mechanisms by which tumor-infiltrating MDSC increase the uptake of exogenous lipids and undergo metabolic and functional reprogramming to become highly immunosuppressive cells. The results showed that tumor-derived cytokines (G-CSF and GM-CSF) and the subsequent signaling through STAT3 and STAT5 induce the expression of lipid transport receptors with the resulting increase in the uptake of lipids present at high concentrations in the tumor microenvironment. The intracellular accumulation of lipids increases the oxidative metabolism and activates the immunosuppressive mechanisms. Inhibition of STAT3 or STAT5 signaling or genetic depletion of the fatty acid translocase CD36 inhibits the activation of oxidative metabolism and the induction of immunosuppressive function in tumor-infiltrating MDSC and results in a CD8+ T cell-dependent delay in tumor growth. Of note, human tumor-infiltrating and peripheral blood MDSC also upregulate the expression of lipid transport proteins, and lipids promote the generation of highly suppressive human MDSC in vitro. Our data therefore provide a mechanism by which tumor-derived factors and the high lipid content in the tumor microenvironment can cause the profound metabolic and functional changes found in MDSC and suggest novel approaches to prevent or reverse these processes. These results could further enhance the efficacy of cancer immunotherapy.

15.
Oncotarget ; 7(14): 17565-78, 2016 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-27007050

RESUMO

The success of adoptive T cell-based immunotherapy (ACT) in cancer is limited in part by the accumulation of myeloid-derived suppressor cells (MDSC), which block several T cell functions, including T cell proliferation and the expression of various cytotoxic mediators. Paradoxically, the inhibition of CD8+ T cell differentiation into cytotoxic populations increased their efficacy after ACT into tumor-bearing hosts. Therefore, we aimed to test the impact of conditioning CD8+ T cells with MDSC on their differentiation potential and ACT efficacy. Our results indicate that MDSC impaired the progression of CD8+ T cells into effector populations, without altering their activation status, production of IL-2, or signaling through the T cell receptor. In addition, culture of CD8+ T cells with MDSC resulted in an increased ACT anti-tumor efficacy, which correlated with a higher frequency of the transferred T cells and elevated IFNγ production. Interestingly, activated CD62L+ CD8+ T cells were responsible for the enhanced anti-tumor activity showed by MDSC-exposed T cells. Additional results showed a decreased protein synthesis rate and lower activity of the mammalian/mechanistic target of rapamycin (mTOR) in T cells conditioned with MDSC. Silencing of the negative mTOR regulator tuberous sclerosis complex-2 in T cells co-cultured with MDSC restored mTOR activity, but resulted in T cell apoptosis. These results indicate that conditioning of T cells with MDSC induces stress survival pathways mediated by a blunted mTOR signaling, which regulated T cell differentiation and ACT efficacy. Continuation of this research will enable the development of better strategies to increase ACT responses in cancer.


Assuntos
Imunoterapia Adotiva/métodos , Células Mieloides/imunologia , Linfócitos T/imunologia , Animais , Carcinoma Pulmonar de Lewis/imunologia , Carcinoma Pulmonar de Lewis/terapia , Comunicação Celular/imunologia , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Feminino , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Timoma/imunologia , Timoma/terapia , Neoplasias do Timo/imunologia , Neoplasias do Timo/terapia
16.
Clin Sci (Lond) ; 129(11): 951-62, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26205779

RESUMO

Our laboratory established a role for poly(ADP-ribose)polymerase (PARP) in asthma. To increase the clinical significance of our studies, it is imperative to demonstrate that PARP is actually activated in human asthma, to examine whether a PARP inhibitor approved for human testing such as olaparib blocks already-established chronic asthma traits in response to house dust mite (HDM), a true human allergen, in mice and to examine whether the drug modulates human cluster of differentiation type 4 (CD4(+)) T-cell function. To conduct the study, human lung specimens and peripheral blood mononuclear cells (PBMCs) and a HDM-based mouse asthma model were used. Our results show that PARP is activated in PBMCs and lung tissues of asthmatics. PARP inhibition by olaparib or gene knockout blocked established asthma-like traits in mice chronically exposed to HDM including airway eosinophilia and hyper-responsiveness. These effects were linked to a marked reduction in T helper 2 (Th2) cytokine production without a prominent effect on interferon (IFN)-γ or interleukin (IL)-10. PARP inhibition prevented HDM-induced increase in overall cellularity, weight and CD4(+) T-cell population in spleens of treated mice whereas it increased the T-regulatory cell population. In CD3/CD28-stimulated human CD4 (+)T-cells, olaparib treatment reduced Th2 cytokine production potentially by modulating GATA binding protein-3 (gata-3)/IL-4 expression while moderately affecting T-cell proliferation. PARP inhibition inconsistently increased IL-17 in HDM-exposed mice and CD3/CD28-stimulated CD4(+) T cells without a concomitant increase in factors that can be influenced by IL-17. In the present study, we provide evidence for the first time that PARP-1 is activated in human asthma and that its inhibition is effective in blocking established asthma in mice.


Assuntos
Antialérgicos/farmacologia , Antiasmáticos/farmacologia , Antígenos de Dermatophagoides , Asma/prevenção & controle , Pulmão/efeitos dos fármacos , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Asma/enzimologia , Asma/imunologia , Asma/fisiopatologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Ativação Enzimática , Humanos , Mediadores da Inflamação/metabolismo , Pulmão/enzimologia , Pulmão/imunologia , Pulmão/fisiopatologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/deficiência , Poli(ADP-Ribose) Polimerases/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/enzimologia , Linfócitos T Reguladores/imunologia , Células Th2/efeitos dos fármacos , Células Th2/enzimologia , Células Th2/imunologia
17.
Cancer Immunol Res ; 3(11): 1236-47, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26025381

RESUMO

Myeloid-derived suppressor cells (MDSC) promote tumor growth by inhibiting T-cell immunity and promoting malignant cell proliferation and migration. The therapeutic potential of blocking MDSC in tumors has been limited by their heterogeneity, plasticity, and resistance to various chemotherapy agents. Recent studies have highlighted the role of energy metabolic pathways in the differentiation and function of immune cells; however, the metabolic characteristics regulating MDSC remain unclear. We aimed to determine the energy metabolic pathway(s) used by MDSC, establish its impact on their immunosuppressive function, and test whether its inhibition blocks MDSC and enhances antitumor therapies. Using several murine tumor models, we found that tumor-infiltrating MDSC (T-MDSC) increased fatty acid uptake and activated fatty acid oxidation (FAO). This was accompanied by an increased mitochondrial mass, upregulation of key FAO enzymes, and increased oxygen consumption rate. Pharmacologic inhibition of FAO blocked immune inhibitory pathways and functions in T-MDSC and decreased their production of inhibitory cytokines. FAO inhibition alone significantly delayed tumor growth in a T-cell-dependent manner and enhanced the antitumor effect of adoptive T-cell therapy. Furthermore, FAO inhibition combined with low-dose chemotherapy completely inhibited T-MDSC immunosuppressive effects and induced a significant antitumor effect. Interestingly, a similar increase in fatty acid uptake and expression of FAO-related enzymes was found in human MDSC in peripheral blood and tumors. These results support the possibility of testing FAO inhibition as a novel approach to block MDSC and enhance various cancer therapies.


Assuntos
Neoplasias do Colo/terapia , Ácidos Graxos/metabolismo , Neoplasias Pulmonares/terapia , Linfócitos do Interstício Tumoral/imunologia , Células Mieloides/imunologia , Animais , Antineoplásicos/uso terapêutico , Antioxidantes/uso terapêutico , Neoplasias do Colo/imunologia , Neoplasias do Colo/metabolismo , Terapia Combinada , Avaliação Pré-Clínica de Medicamentos/métodos , Metabolismo Energético/imunologia , Compostos de Epóxi/uso terapêutico , Feminino , Humanos , Tolerância Imunológica/imunologia , Imunoterapia Adotiva/métodos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Camundongos Endogâmicos C57BL , Oxirredução/efeitos dos fármacos , Microambiente Tumoral/imunologia
18.
J Biol Chem ; 290(13): 8067-80, 2015 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-25645911

RESUMO

Glioblastoma is one of the most aggressive brain tumors. We have previously found up-regulation of growth differentiation factor 15 (GDF15) in glioblastoma cells treated with the anticancer agent fenofibrate. Sequence analysis of GDF15 revealed the presence of a microRNA, miR-3189, in the single intron. We then asked whether miR-3189 was expressed in clinical samples and whether it was functional in glioblastoma cells. We found that expression of miR-3189-3p was down-regulated in astrocytoma and glioblastoma clinical samples compared with control brain tissue. In vitro, the functionality of miR-3189-3p was tested by RNA-binding protein immunoprecipitation, and miR-3189-3p coimmunoprecipitated with Argonaute 2 together with two of its major predicted gene targets, the SF3B2 splicing factor and the guanine nucleotide exchange factor p63RhoGEF. Overexpression of miR-3189-3p resulted in a significant inhibition of cell proliferation and migration through direct targeting of SF3B2 and p63RhoGEF, respectively. Interestingly, miR-3189-3p levels were increased by treatment of glioblastoma cells with fenofibrate, a lipid-lowering drug with multiple anticancer activities. The attenuated expression of miR-3189-3p in clinical samples paralleled the elevated expression of SF3B2, which could contribute to the activation of SF3B2 growth-promoting pathways in these tumors. Finally, miR-3189-3p-mediated inhibition of tumor growth in vivo further supported the function of this microRNA as a tumor suppressor.


Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , MicroRNAs/genética , Animais , Sequência de Bases , Sítios de Ligação , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Glioblastoma/genética , Glioblastoma/patologia , Fator 15 de Diferenciação de Crescimento/genética , Fator 15 de Diferenciação de Crescimento/metabolismo , Humanos , Camundongos Nus , Transplante de Neoplasias , Interferência de RNA , Fatores de Processamento de RNA , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/genética , Fatores de Troca de Nucleotídeo Guanina Rho/biossíntese , Fatores de Troca de Nucleotídeo Guanina Rho/genética
19.
Mol Cell Biol ; 35(1): 182-98, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25332241

RESUMO

Fenofibrate (FF) is a common lipid-lowering drug and a potent agonist of the peroxisome proliferator-activated receptor alpha (PPARα). FF and several other agonists of PPARα have interesting anticancer properties, and our recent studies demonstrate that FF is very effective against tumor cells of neuroectodermal origin. In spite of these promising anticancer effects, the molecular mechanism(s) of FF-induced tumor cell toxicity remains to be elucidated. Here we report a novel PPARα-independent mechanism explaining FF's cytotoxicity in vitro and in an intracranial mouse model of glioblastoma. The mechanism involves accumulation of FF in the mitochondrial fraction, followed by immediate impairment of mitochondrial respiration at the level of complex I of the electron transport chain. This mitochondrial action sensitizes tested glioblastoma cells to the PPARα-dependent metabolic switch from glycolysis to fatty acid ß-oxidation. As a consequence, prolonged exposure to FF depletes intracellular ATP, activates the AMP-activated protein kinase-mammalian target of rapamycin-autophagy pathway, and results in extensive tumor cell death. Interestingly, autophagy activators attenuate and autophagy inhibitors enhance FF-induced glioblastoma cytotoxicity. Our results explain the molecular basis of FF-induced glioblastoma cytotoxicity and reveal a new supplemental therapeutic approach in which intracranial infusion of FF could selectively trigger metabolic catastrophe in glioblastoma cells.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Fenofibrato/farmacologia , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Trifosfato de Adenosina/metabolismo , Animais , Antineoplásicos/farmacologia , Astrócitos/citologia , Neoplasias Encefálicas/metabolismo , Morte Celular , Linhagem Celular Tumoral , Transporte de Elétrons , Feminino , Glioblastoma/metabolismo , Glicólise , Humanos , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Nus , Mitocôndrias/metabolismo , Transplante de Neoplasias , Oxigênio/metabolismo , Consumo de Oxigênio , PPAR alfa/metabolismo , Transdução de Sinais
20.
Cancer Res ; 75(2): 275-83, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25406192

RESUMO

Enzymatic depletion of the nonessential amino acid l-Arginine (l-Arg) in patients with cancer by the administration of a pegylated form of the catabolic enzyme arginase I (peg-Arg I) has shown some promise as a therapeutic approach. However, l-Arg deprivation also suppresses T-cell responses in tumors. In this study, we sought to reconcile these observations by conducting a detailed analysis of the effects of peg-Arg I on normal T cells. Strikingly, we found that peg-Arg I blocked proliferation and cell-cycle progression in normal activated T cells without triggering apoptosis or blunting T-cell activation. These effects were associated with an inhibition of aerobic glycolysis in activated T cells, but not with significant alterations in mitochondrial oxidative respiration, which thereby regulated survival of T cells exposed to peg-Arg I. Further mechanistic investigations showed that the addition of citrulline, a metabolic precursor for l-Arg, rescued the antiproliferative effects of peg-Arg I on T cells in vitro. Moreover, serum levels of citrulline increased after in vivo administration of peg-Arg I. In support of the hypothesis that peg-Arg I acted indirectly to block T-cell responses in vivo, peg-Arg I inhibited T-cell proliferation in mice by inducing accumulation of myeloid-derived suppressor cells (MDSC). MDSC induction by peg-Arg I occurred through the general control nonrepressed-2 eIF2α kinase. Moreover, we found that peg-Arg I enhanced the growth of tumors in mice in a manner that correlated with higher MDSC numbers. Taken together, our results highlight the risks of the l-Arg-depleting therapy for cancer treatment and suggest a need for cotargeting MDSC in such therapeutic settings.


Assuntos
Arginina/imunologia , Carcinoma Pulmonar de Lewis/imunologia , Células Mieloides/imunologia , Linfócitos T/imunologia , Animais , Arginina/administração & dosagem , Arginina/deficiência , Arginina/metabolismo , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/terapia , Metabolismo Energético/efeitos dos fármacos , Imunoterapia Adotiva , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Polietilenoglicóis/administração & dosagem , Linfócitos T/efeitos dos fármacos
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