RESUMO
The progressive decline of CD8 T cell effector function-also known as terminal exhaustion-is a major contributor to immune evasion in cancer. Yet, the molecular mechanisms that drive CD8 T cell dysfunction remain poorly understood. Here, we report that the Kelch-like ECH-associated protein 1 (KEAP1)-Nuclear factor erythroid 2-related factor 2 (NRF2) signaling axis, which mediates cellular adaptations to oxidative stress, directly regulates CD8 T cell exhaustion. Transcriptional profiling of dysfunctional CD8 T cells from chronic infection and cancer reveals enrichment of NRF2 activity in terminally exhausted (Texterm) CD8 T cells. Increasing NRF2 activity in CD8 T cells (via conditional deletion of KEAP1) promotes increased glutathione production and antioxidant defense yet accelerates the development of terminally exhausted (PD-1+TIM-3+) CD8 T cells in response to chronic infection or tumor challenge. Mechanistically, we identify PTGIR, a receptor for the circulating eicosanoid prostacyclin, as an NRF2-regulated protein that promotes CD8 T cell dysfunction. Silencing PTGIR expression restores the anti-tumor function of KEAP1-deficient T cells. Moreover, lowering PTGIR expression in CD8 T cells both reduces terminal exhaustion and enhances T cell effector responses (i.e. IFN-γ and granzyme production) to chronic infection and cancer. Together, these results establish the KEAP1-NRF2 axis as a metabolic sensor linking oxidative stress to CD8 T cell dysfunction and identify the prostacyclin receptor PTGIR as an NRF2-regulated immune checkpoint that regulates CD8 T cell fate decisions between effector and exhausted states.
RESUMO
Infusion of 13C-labeled metabolites provides a gold standard for understanding the metabolic processes used by T cells during immune responses in vivo. Through infusion of 13C-labeled metabolites (glucose, glutamine, and acetate) in Listeria monocytogenes-infected mice, we demonstrate that CD8 T effector (Teff) cells use metabolites for specific pathways during specific phases of activation. Highly proliferative early Teff cells in vivo shunt glucose primarily toward nucleotide synthesis and leverage glutamine anaplerosis in the tricarboxylic acid (TCA) cycle to support adenosine triphosphate and de novo pyrimidine synthesis. In addition, early Teff cells rely on glutamic-oxaloacetic transaminase 1 (Got1)-which regulates de novo aspartate synthesis-for effector cell expansion in vivo. CD8 Teff cells change fuel preference over the course of infection, switching from glutamine- to acetate-dependent TCA cycle metabolism late in infection. This study provides insights into the dynamics of Teff metabolism, illuminating distinct pathways of fuel consumption associated with CD8 Teff cell function in vivo.
Assuntos
Acetatos , Linfócitos T CD8-Positivos , Isótopos de Carbono , Glutamina , Glutamina/metabolismo , Animais , Linfócitos T CD8-Positivos/metabolismo , Acetatos/metabolismo , Camundongos , Listeriose/metabolismo , Listeriose/imunologia , Listeriose/microbiologia , Listeria monocytogenes , Ciclo do Ácido Cítrico , Glucose/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Melanomas reprogram their metabolism to rapidly adapt to therapy-induced stress conditions, allowing them to persist and ultimately develop resistance. We report that a subpopulation of melanoma cells tolerate MAPK pathway inhibitors (MAPKis) through a concerted metabolic reprogramming mediated by peroxisomes and UDP-glucose ceramide glycosyltransferase (UGCG). Compromising peroxisome biogenesis, by repressing PEX3 expression, potentiated the proapoptotic effects of MAPKis via an induction of ceramides, an effect limited by UGCG-mediated ceramide metabolism. Cotargeting PEX3 and UGCG selectively eliminated a subset of metabolically active, drug-tolerant CD36+ melanoma persister cells, thereby sensitizing melanoma to MAPKis and delaying resistance. Increased levels of peroxisomal genes and UGCG were found in patient-derived MAPKi-relapsed melanomas, and simultaneously inhibiting PEX3 and UGCG restored MAPKi sensitivity in multiple models of therapy resistance. Finally, combination therapy consisting of a newly identified inhibitor of the PEX3-PEX19 interaction, a UGCG inhibitor, and MAPKis demonstrated potent antitumor activity in preclinical melanoma models, thus representing a promising approach for melanoma treatment.
Assuntos
Melanoma , Peroxissomos , Humanos , Peroxissomos/metabolismo , Metabolismo dos Lipídeos , Melanoma/genética , Ceramidas/farmacologia , Ceramidas/metabolismoRESUMO
Environmental nutrient availability influences T cell metabolism, impacting T cell function and shaping immune outcomes. Here, we identified ketone bodies (KBs)-including ß-hydroxybutyrate (ßOHB) and acetoacetate (AcAc)-as essential fuels supporting CD8+ T cell metabolism and effector function. ßOHB directly increased CD8+ T effector (Teff) cell cytokine production and cytolytic activity, and KB oxidation (ketolysis) was required for Teff cell responses to bacterial infection and tumor challenge. CD8+ Teff cells preferentially used KBs over glucose to fuel the tricarboxylic acid (TCA) cycle in vitro and in vivo. KBs directly boosted the respiratory capacity and TCA cycle-dependent metabolic pathways that fuel CD8+ T cell function. Mechanistically, ßOHB was a major substrate for acetyl-CoA production in CD8+ T cells and regulated effector responses through effects on histone acetylation. Together, our results identify cell-intrinsic ketolysis as a metabolic and epigenetic driver of optimal CD8+ T cell effector responses.
Assuntos
Linfócitos T CD8-Positivos , Histonas , Ácido 3-Hidroxibutírico/metabolismo , Ácido 3-Hidroxibutírico/farmacologia , Acetilação , Histonas/metabolismo , Corpos Cetônicos , Animais , CamundongosRESUMO
Promoting the macroautophagy/autophagy-mediated degradation of specific proteins and organelles can potentially be utilized to induce apoptosis in cancer cells or sensitize tumor cells to therapy. To examine this concept, we enriched for autophagosomes from histone deacetylase inhibitor (HDACi)-sensitive U937 lymphoma cells and isogenic HDACi-resistant cells. Mass spectrometry on autophagosome-enriched fractions revealed that HDACi-resistant cells undergo elevated pexophagy, or autophagy of the peroxisome, an organelle that supports tumor growth. To disturb peroxisome homeostasis, we enhanced pexophagy in HDACi-resistant cells via genetic silencing of peroxisome exportomer complex components (PEX1, PEX6, or PEX26). This consequently sensitized resistant cells to HDACi-mediated apoptosis, which was rescued by inhibiting ATM/ataxia-telangiectasia mutated (ATM serine/threonine kinase), a mediator of pexophagy. We subsequently engineered melanoma cells to stably repress PEX26 using CRISPR interference (CRISPRi). Melanoma cells with repressed PEX26 expression showed evidence of both increased pexophagy and peroxisomal matrix protein import defects versus single guide scrambled (sgSCR) controls. In vivo studies showed that sgPEX26 melanoma xenografts recurred less compared to sgSCR xenografts, following the development of resistance to mitogen-activated protein kinase (MAPK)-targeted therapy. Finally, prognostic analysis of publicly available datasets showed that low expression levels of PEX26, PEX6 and MTOR, were significantly associated with prolonged patient survival in lymphoma, lung cancer and melanoma cohorts. Our work highlighted that drugs designed to disrupt peroxisome homeostasis may serve as unconventional therapies to combat therapy resistance in cancer.Abbreviations: ABCD3/PMP70: ATP binding cassette subfamily D member 3; ACOX1: acyl-CoA oxidase 1; AP: autophagosome; COX: cytochrome c oxidase; CQ: chloroquine; CRISPRi: clustered regularly interspaced short palindromic repeats interference; DLBCL: diffuse large B-cell lymphoma; GO: gene ontology; dCas9: Cas9 endonuclease dead, or dead Cas9; HDACi: histone deacetylase inhibitors; IHC: Immunohistochemistry; LAMP2: lysosomal associated membrane protein 2; LCFAs: long-chain fatty acids; LFQ-MS: label-free quantitation mass spectrometry; LPC: lysophoshatidylcholine; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PBD: peroxisome biogenesis disorders; PTS1: peroxisomal targeting signal 1; ROS: reactive oxygen species; sgRNA: single guide RNA; VLCFAs: very-long chain fatty acids; Vor: vorinostat; WO: wash-off.
Assuntos
Autofagia , Melanoma , ATPases Associadas a Diversas Atividades Celulares/genética , Autofagia/genética , Resistência a Medicamentos , Ácidos Graxos/metabolismo , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/metabolismo , Proteínas de Membrana/metabolismo , Peroxissomos/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
The BRAFV600E mutation occurs in more than 50% of cutaneous melanomas, and results in the constitutive activation of the mitogen-activated protein kinases (MAPK) pathway. MAP kinase-interacting serine/threonine-protein kinase 1 and 2 (MNK1/2) are downstream effectors of the activated MAPK pathway, and important molecular targets in invasive and metastatic cancer. Despite the well-known role of MNK1 in regulating mRNA translation, little is known concerning the impact of its aberrant activation on gene transcription. Here, we show that changes in the activity, or abundance, of MNK1 result in changes in the expression of pro-oncogenic and pro-invasive genes. Among the MNK1-upregulated genes, we identify Angiopoietin-like 4 (ANGPTL4), which in turn promotes an invasive phenotype via its ability to induce the expression of matrix metalloproteinases (MMPs). Using a pharmacologic inhibitor of MNK1/2, SEL201, we demonstrate that BRAFV600E-mutated cutaneous melanoma cells are reliant on MNK1/2 for invasion and lung metastasis.
Assuntos
Proteína 4 Semelhante a Angiopoietina/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Pulmonares/genética , Melanoma/tratamento farmacológico , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Xenoenxertos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Melanoma/genética , Melanoma/patologia , Camundongos , Mutação/genética , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Transcriptoma/genética , Melanoma Maligno CutâneoRESUMO
Cancer is irrevocably linked to aberrant metabolic processes. While once considered a vestigial organelle, we now know that peroxisomes play a central role in the metabolism of reactive oxygen species, bile acids, ether phospholipids (e.g. plasmalogens), very-long chain, and branched-chain fatty acids. Immune system evasion is a hallmark of cancer, and peroxisomes have an emerging role in the regulation of cellular immune responses. Investigations of individual peroxisome proteins and metabolites support their pro-tumorigenic functions. However, a significant knowledge gap remains regarding how individual functions of proteins and metabolites of the peroxisome orchestrate its potential role as a pro-tumorigenic organelle. This review highlights new advances in our understanding of biogenesis, enzymatic functions, and autophagic degradation of peroxisomes (pexophagy), and provides evidence linking these activities to tumorigenesis. Finally, we propose avenues that may be exploited to target peroxisome-related processes as a mode of combatting cancer.
Assuntos
Neoplasias/metabolismo , Peroxissomos/metabolismo , Antineoplásicos/farmacologia , Autofagia , Carcinogênese , Humanos , Neoplasias/imunologia , Biogênese de Organelas , Peroxissomos/efeitos dos fármacos , Peroxissomos/enzimologia , Peroxissomos/imunologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Peroxisomes are a critical rheostat of reactive oxygen species (ROS), yet their role in drug sensitivity and resistance remains unexplored. Gene expression analysis of clinical lymphoma samples suggests that peroxisomes are involved in mediating drug resistance to the histone deacetylase inhibitor (HDACi) Vorinostat (Vor), which promotes ROS-mediated apoptosis. Vor augments peroxisome numbers in cultured lymphoma cells, concomitant with increased levels of peroxisomal proteins PEX3, PEX11B, and PMP70. Genetic inhibition of peroxisomes, using PEX3 knockdown, reveals that peroxisomes protect lymphoma cells against Vor-mediated cell death. Conversely, Vor-resistant cells were tolerant to elevated ROS levels and possess upregulated levels of (1) catalase, a peroxisomal antioxidant, and (2) plasmalogens, ether glycerophospholipids that represent peroxisome function and serve as antioxidants. Catalase knockdown induces apoptosis in Vor-resistant cells and potentiates ROS-mediated apoptosis in Vor-sensitive cells. These findings highlight the role of peroxisomes in resistance to therapeutic intervention in cancer, and provide a novel modality to circumvent drug resistance.
Assuntos
Apoptose/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Linfoma/patologia , Peroxissomos/metabolismo , Catalase/metabolismo , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Inativação Gênica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/metabolismo , Ácidos Hidroxâmicos/farmacologia , Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Modelos Biológicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , VorinostatRESUMO
Post-translational mechanisms regulating cell-matrix adhesion turnover during cell locomotion are not fully elucidated. In this study, we uncovered an essential role of Y118 site-specific tyrosine phosphorylation of paxillin, an adapter protein of focal adhesion complexes, in paxillin recruitment to autophagosomes to trigger turnover of peripheral focal adhesions in human breast cancer cells. We demonstrate that the Rab-7 GTPase is a key upstream regulator of late endosomal sorting of tyrosine118-phosphorylated paxillin, which is subsequently recruited to autophagosomes via the cargo receptor c-Cbl. Essentially, this recruitment involves a direct and selective interaction between Y118-phospho-paxillin, c-Cbl, and LC3 and is independent from c-Cbl E3 ubiquitin ligase activity. Interference with the Rab7-paxillin-autophagy regulatory network using genetic and pharmacological approaches greatly impacted focal adhesion stability, cell locomotion and progression to metastasis using a panel of human breast cancer cells. Together, these results provide novel insights into the requirement of phospho-site specific post-translational mechanism of paxillin for autophagy targeting to regulate cell-matrix adhesion turnover and cell locomotion in breast cancer cells.
Assuntos
Autofagossomos/metabolismo , Neoplasias da Mama/metabolismo , Endossomos/metabolismo , Matriz Extracelular/metabolismo , Paxilina/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Autofagia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Progressão da Doença , Técnicas de Silenciamento de Genes , Humanos , Proteínas Associadas aos Microtúbulos , Metástase Neoplásica , Fosforilação , Ligação Proteica , Proteólise , Transdução de Sinais , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
In eukaryotic cells, the rate-limiting component for cap-dependent mRNA translation is the translation initiation factor eIF4E. eIF4E is overexpressed in a variety of human malignancies, but whether it has a role in melanoma remains obscure. We hypothesized that eIF4E promotes melanoma cell proliferation and facilitates the development of acquired resistance to the BRAF inhibitor vemurafenib. We show that eIF4E is overexpressed in a panel of melanoma cell lines, compared with immortalized melanocytes. Knockdown of eIF4E significantly repressed the proliferation of a subset of melanoma cell lines. Moreover, in BRAF(V600E) melanoma cell lines, vemurafenib inhibits 4E-BP1 phosphorylation, thus promoting its binding to eIF4E. Cap-binding and polysome profiling analysis confirmed that vemurafenib stabilizes the eIF4E-4E-BP1 association and blocks mRNA translation, respectively. Conversely, in cells with acquired resistance to vemurafenib, there is an increased dependence on eIF4E for survival; 4E-BP1 is highly phosphorylated and thus eIF4E-4E-BP1 associations are impeded. Moreover, increasing eIF4E activity by silencing 4E-BP1/2 renders vemurafenib-responsive cells more resistant to BRAF inhibition. In conclusion, these data suggest that therapeutically targeting eIF4E may be a viable means of inhibiting melanoma cell proliferation and overcoming vemurafenib resistance.
Assuntos
Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos/fisiologia , Indóis/farmacologia , Melanoma/patologia , Neoplasias Cutâneas/patologia , Sulfonamidas/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos/genética , Técnicas de Silenciamento de Genes , Inativação Gênica , Humanos , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas B-raf , RNA Interferente Pequeno/farmacologia , VemurafenibRESUMO
Darinaparsin (Dar; ZIO-101; S-dimethylarsino-glutathione) is a promising novel organic arsenical currently undergoing clinical studies in various malignancies. Dar consists of dimethylarsenic conjugated to glutathione (GSH). Dar induces more intracellular arsenic accumulation and more cell death than the FDA-approved arsenic trioxide (ATO) in vitro, but exhibits less systemic toxicity. Here, we propose a mechanism for Dar import that might explain these characteristics. Structural analysis of Dar suggests a putative breakdown product: dimethylarsino-cysteine (DMAC). We show that DMAC is very similar to Dar in terms of intracellular accumulation of arsenic, cell cycle arrest, and cell death. We found that inhibition of γ-glutamyl-transpeptidase (γ-GT) protects human acute promyelocytic leukemia cells (NB4) from Dar, but not from DMAC, suggesting a role for γ-GT in the processing of Dar. Overall, our data support a model where Dar, a GSH S-conjugate, is processed at the cell surface by γ-GT, leading to formation of DMAC, which is imported via xCT, xAG, or potentially other cystine/cysteine importing systems. Further, we propose that Dar induces its own import via increased xCT expression. These mechanisms may explain the enhanced toxicity of Dar toward cancer cells compared with ATO.