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Tumor-associated macrophages (TAMs) are the major component of the tumor microenvironment (TME), where they sustain tumor progression and or-tumor immunity. Due to their plasticity, macrophages can exhibit anti- or pro-tumor functions through the expression of different gene sets leading to distinct macrophage phenotypes: M1-like or pro-inflammatory and M2-like or anti-inflammatory. NF-κB transcription factors are central regulators of TAMs in cancers, where they often drive macrophage polarization toward an M2-like phenotype. Therefore, the NF-κB pathway is an attractive therapeutic target for cancer immunotherapy in a wide range of human tumors. Hence, targeting NF-κB pathway in the myeloid compartment is a potential clinical strategy to overcome microenvironment-induced immunosuppression and increase anti-tumor immunity. In this review, we discuss the role of NF-κB as a key driver of macrophage functions in tumors as well as the principal strategies to overcome tumor immunosuppression by targeting the NF-κB pathway.
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FN-kappa B , Neoplasias , Humanos , FN-kappa B/genética , FN-kappa B/metabolismo , Macrófagos/metabolismo , Neoplasias/genética , Neoplasias/terapia , Neoplasias/metabolismo , Subunidad p50 de NF-kappa B , Fenotipo , Microambiente Tumoral/genéticaRESUMEN
In vivo apoptosis of human mesenchymal stromal cells (MSCs) plays a critical role in delivering immunomodulation. Yet, caspase activity not only mediates the dying process but also death-independent functions that may shape the immunogenicity of apoptotic cells. Therefore, a better characterization of the immunological profile of apoptotic MSCs (ApoMSCs) could shed light on their mechanistic action and therapeutic applications. We analyzed the transcriptomes of MSCs undergoing apoptosis and identified several immunomodulatory factors and chemokines dependent on caspase activation following Fas stimulation. The ApoMSC secretome inhibited human T cell proliferation and activation, and chemoattracted monocytes in vitro. Both immunomodulatory activities were dependent on the cyclooxygenase2 (COX2)/prostaglandin E2 (PGE2) axis. To assess the clinical relevance of ApoMSC signature, we used the peripheral blood mononuclear cells (PBMCs) from a cohort of fistulizing Crohn's disease (CD) patients who had undergone MSC treatment (ADMIRE-CD). Compared with healthy donors, MSCs exposed to patients' PBMCs underwent apoptosis and released PGE2 in a caspase-dependent manner. Both PGE2 and apoptosis were significantly associated with clinical responses to MSCs. Our findings identify a new mechanism whereby caspase activation delivers ApoMSC immunosuppression. Remarkably, such molecular signatures could implicate translational tools for predicting patients' clinical responses to MSC therapy in CD.
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Enfermedad de Crohn , Células Madre Mesenquimatosas , Humanos , Enfermedad de Crohn/genética , Enfermedad de Crohn/terapia , Dinoprostona/metabolismo , Leucocitos Mononucleares/metabolismo , Secretoma , Células Madre Mesenquimatosas/metabolismo , Inmunomodulación , Apoptosis , CaspasasRESUMEN
NF-κB transcription factors are major drivers of tumor initiation and progression. NF-κB signaling is constitutively activated by genetic alterations or environmental signals in many human cancers, where it contributes to almost all hallmarks of malignancy, including sustained proliferation, cell death resistance, tumor-promoting inflammation, metabolic reprogramming, tissue invasion, angiogenesis, and metastasis. As such, the NF-κB pathway is an attractive therapeutic target in a broad range of human cancers, as well as in numerous non-malignant diseases. Currently, however, there is no clinically useful NF-κB inhibitor to treat oncological patients, owing to the preclusive, on-target toxicities of systemic NF-κB blockade. In this review, we discuss the principal and most promising strategies being developed to circumvent the inherent limitations of conventional IκB kinase (IKK)/NF-κB-targeting drugs, focusing on new molecules that target upstream regulators or downstream effectors of oncogenic NF-κB signaling, as well as agents targeting individual NF-κB subunits.
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The procurement and management of nutrients and ability to fight infections are fundamental requirements for survival. These defense responses are bioenergetically costly, requiring the immune system to balance protection against pathogens with the need to maintain metabolic homeostasis. NF-κB transcription factors are central regulators of immunity and inflammation. Over the last two decades, these factors have emerged as a pivotal node coordinating the immune and metabolic systems in physiology and the etiopathogenesis of major threats to human health, including cancer, autoimmunity, chronic inflammation, and others. In this review, we discuss recent advances in understanding how NF-κB-dependent metabolic programs control inflammation, metabolism, and immunity and how improved knowledge of them may lead to better diagnostics and therapeutics for widespread human diseases.
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FN-kappa B , Neoplasias , Autoinmunidad , Homeostasis , Humanos , InflamaciónRESUMEN
Cancer cells reprogram lipid metabolism to fuel cell division, adaptation to stress, and metastatic dissemination. NF-κB transcription factors control this mechanism in aggressive Consensus Molecular Subtype (CMS)4 of colorectal carcinoma (CRC) via triacylglycerol (TAG) lipase, carboxylesterase 1 (CES1), thereby linking obesity-associated inflammation with metabolic adaptation and cytoprotection from lipid-induced toxicity. Our findings identify a potential therapeutic route to treat patients with metastasis-prone CRC and provide an example for targeting core tumor subtype-based vulnerabilities in cancers beyond CRC.
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Cell fractionation is a method used to study different cellular events like protein translocation and sequestration by disrupting cells and fractionating their contents, thus allowing an enrichment of the protein of interest. Using different concentrations of sucrose or detergent buffer formulations in combination with centrifugations, the cell fractions are separated based on their density and size.
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Fraccionamiento Celular , FN-kappa B , Fracciones SubcelularesRESUMEN
Immunohistochemistry (IHC) is a technique aimed at detecting specific antigens on tissue sections by the use of targeting reagents labeled with reporter molecules. This technique allows a snapshot of the structure of tissue and determines the cellular and subcellular localization of a target antigen. This chapter describes how to identify and localize NF-κB proteins in human tissue using immunohistochemical staining on formalin-fixed paraffin-embedded and frozen tissue.
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FN-kappa B/metabolismo , Antígenos , Formaldehído , Humanos , Inmunohistoquímica , Adhesión en Parafina , Fosforilación , Fijación del TejidoRESUMEN
The reprogramming of cell metabolism is a hallmark of cancer. NF-κB transcription factors coordinate the host defense responses to stress, injury, and infection. They also play a central role in oncogenesis, at least in part by regulating cell metabolism and the adaptation to energy stress conditions in various types of cancer, such as colorectal carcinoma (CRC). Here, we describe the XF Cell Mito Stress Test methodology aimed at characterizing the metabolic and bioenergetic profile of CRC cells following the silencing of the essential NF-κB subunit, RelA. This methodology may also be applied to other cancers to reveal novel core vulnerabilities of malignant cells.
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Neoplasias Colorrectales , Neoplasias Colorrectales/metabolismo , Metabolismo Energético , Humanos , Mitocondrias/metabolismo , FN-kappa B/metabolismo , Respiración , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismoRESUMEN
Peptides are emerging as an increasingly dependable class of therapeutics in the treatment of cancer and metabolic and cardiovascular diseases, which are all areas of high interest to the pharmaceutical industry. The global market for peptide therapeutics was valued at about 25 billion USD in 2018 and is estimated to reach 57.2 billion USD by the end of 2027. Here, we describe a method for the screening and deconvolution of combinatorial peptide libraries to discover compounds that target discrete signaling components of the NF-κB pathway. Recently, we used this approach to specifically disrupt the interaction between the JNK-activating kinase, MKK7, and the NF-κB-regulated antiapoptotic factor, GADD45ß, in multiple myeloma (MM). We showed that the GADD45ß/MKK7 complex is a functionally critical survival module downstream of NF-κB in MM cells and as such provides an attractive therapeutic target to selectively inhibit NF-κB antiapoptotic signaling in cancer cells. By integrating the library screening and deconvolution methods described here with a rational chemical optimization strategy, we developed the first-in-class GADD45ß/MKK7 inhibitor, DTP3 (a D-tripeptide), which is now being trialed in MM and diffuse large B-cell lymphoma (DLBCL) patients. The same drug discovery approach may be generally applied to therapeutically target other key components of the NF-κB pathway in cancers beyond MM and DLBCL, as well as in non-malignant NF-κB-driven diseases.
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Transducción de Señal , Apoptosis , Humanos , Linfoma de Células B Grandes Difuso , Mieloma Múltiple/tratamiento farmacológico , FN-kappa B/metabolismo , Biblioteca de Péptidos , Péptidos , Mapeo de Interacción de ProteínasRESUMEN
The ability to adapt to low-nutrient microenvironments is essential for tumor cell survival and progression in solid cancers, such as colorectal carcinoma (CRC). Signaling by the NF-κB transcription factor pathway associates with advanced disease stages and shorter survival in patients with CRC. NF-κB has been shown to drive tumor-promoting inflammation, cancer cell survival, and intestinal epithelial cell (IEC) dedifferentiation in mouse models of CRC. However, whether NF-κB affects the metabolic adaptations that fuel aggressive disease in patients with CRC is unknown. Here, we identified carboxylesterase 1 (CES1) as an essential NF-κB-regulated lipase linking obesity-associated inflammation with fat metabolism and adaptation to energy stress in aggressive CRC. CES1 promoted CRC cell survival via cell-autonomous mechanisms that fuel fatty acid oxidation (FAO) and prevent the toxic build-up of triacylglycerols. We found that elevated CES1 expression correlated with worse outcomes in overweight patients with CRC. Accordingly, NF-κB drove CES1 expression in CRC consensus molecular subtype 4 (CMS4), which is associated with obesity, stemness, and inflammation. CES1 was also upregulated by gene amplifications of its transcriptional regulator HNF4A in CMS2 tumors, reinforcing its clinical relevance as a driver of CRC. This subtype-based distribution and unfavorable prognostic correlation distinguished CES1 from other intracellular triacylglycerol lipases and suggest CES1 could provide a route to treat aggressive CRC.
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Hidrolasas de Éster Carboxílico/metabolismo , Neoplasias Colorrectales/enzimología , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/metabolismo , Triglicéridos/metabolismo , Hidrolasas de Éster Carboxílico/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Femenino , Humanos , Masculino , Proteínas de Neoplasias/genética , Triglicéridos/genéticaRESUMEN
Cancer cells can survive chemotherapy-induced stress, but how they recover from it is not known. Using a temporal multiomics approach, we delineate the global mechanisms of proteotoxic stress resolution in multiple myeloma cells recovering from proteasome inhibition. Our observations define layered and protracted programs for stress resolution that encompass extensive changes across the transcriptome, proteome, and metabolome. Cellular recovery from proteasome inhibition involved protracted and dynamic changes of glucose and lipid metabolism and suppression of mitochondrial function. We demonstrate that recovering cells are more vulnerable to specific insults than acutely stressed cells and identify the general control nonderepressable 2 (GCN2)-driven cellular response to amino acid scarcity as a key recovery-associated vulnerability. Using a transcriptome analysis pipeline, we further show that GCN2 is also a stress-independent bona fide target in transcriptional signature-defined subsets of solid cancers that share molecular characteristics. Thus, identifying cellular trade-offs tied to the resolution of chemotherapy-induced stress in tumor cells may reveal new therapeutic targets and routes for cancer therapy optimization.
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Neoplasias/tratamiento farmacológico , Estrés Fisiológico/efectos de los fármacos , Antineoplásicos/farmacología , Autofagia/fisiología , Línea Celular Tumoral , Humanos , Metaboloma/genética , Mitocondrias/metabolismo , Mieloma Múltiple/metabolismo , Neoplasias/metabolismo , Neoplasias/fisiopatología , Inhibidores de Proteasoma/farmacología , Proteolisis , Proteoma/genética , Análisis de Sistemas , Transcriptoma/genéticaRESUMEN
Escaping programmed cell death is a hallmark of cancer. NF-κB transcription factors are key regulator of cell survival and aberrant NF-κB signaling has been involved in the pathogenesis of most human malignancies. Although NF-κB is best known for its antiapoptotic role, other processes regulating the life/death balance, such as autophagy and necroptosis, seem to network with NF-κB. This review discusses how the reciprocal regulation of NF-κB, autophagy and programmed cell death affect cancer development and progression.
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FN-kappa B/metabolismo , Neoplasias/genética , Autofagia , Humanos , Transducción de SeñalRESUMEN
GADD45ß/MKK7 complex is a non-redundant, cancer cell-restricted survival module downstream of the NF-kB survival pathway, and it has a pathogenically critical role in multiple myeloma, an incurable malignancy of plasma cells. The first-in-class GADD45ß/MKK7 inhibitor DTP3 effectively kills MM cells expressing its molecular target, both in vitro and in vivo, by inducing MKK7/JNK-dependent apoptosis with no apparent toxicity to normal cells. DTP3 combines favorable drug-like properties, with on-target-specific pharmacology, resulting in a safe and cancer-selective therapeutic effect; however, its mode of action is only partially understood. In this work, we have investigated the molecular determinants underlying the MKK7 interaction with DTP3 by combining computational, NMR, and spectroscopic methods. Data gathered by fluorescence quenching and computational approaches consistently indicate that the N-terminal region of MKK7 is the optimal binding site explored by DTP3. These findings further the understanding of the selective mode of action of GADD45ß/MKK7 inhibitors and inform potential mechanisms of drug resistance. Notably, upon validation of the safety and efficacy of DTP3 in human trials, our results could also facilitate the development of novel DTP3-like therapeutics with improved bioavailability or the capacity to bypass drug resistance.
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NF-κB plays a pivotal role in oncogenesis. This transcription factor is best known for promoting cancer cell survival and tumour-driving inflammation. However, several lines of evidence support a crucial role for NF-κB in governing energy homeostasis and mediating cancer metabolic reprogramming. Mitochondria are central players in many metabolic processes altered in cancer. Beyond their bioenergetic activity, several facets of mitochondria biology, including mitochondrial dynamics and oxidative stress, promote and sustain malignant transformation. Recent reports revealed an intimate connection between NF-κB pathway and the oncogenic mitochondrial functions. NF-κB can impact mitochondrial respiration and mitochondrial dynamics, and, reciprocally, mitochondria can sense stress signals and convert them into cell biological responses leading to NF-κB activation. In this review we discuss their emerging reciprocal regulation and the significance of this interplay for anticancer therapy.
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Mitocondrias/metabolismo , FN-kappa B/metabolismo , Neoplasias/metabolismo , Animales , Antineoplásicos/farmacología , Reprogramación Celular/efectos de los fármacos , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/patologíaRESUMEN
Aberrant NF-κB activity drives oncogenesis and cell survival in multiple myeloma (MM) and many other cancers. However, despite an aggressive effort by the pharmaceutical industry over the past 30 years, no specific IκBα kinase (IKK)ß/NF-κB inhibitor has been clinically approved, due to the multiple dose-limiting toxicities of conventional NF-κB-targeting drugs. To overcome this barrier to therapeutic NF-κB inhibition, we developed the first-in-class growth arrest and DNA-damage-inducible (GADD45)ß/mitogen-activated protein kinase kinase (MKK)7 inhibitor, DTP3, which targets an essential, cancer-selective cell-survival module downstream of the NF-κB pathway. As a result, DTP3 specifically kills MM cells, ex vivo and in vivo, ablating MM xenografts in mice, with no apparent adverse effects, nor evident toxicity to healthy cells. Here, we report the results from the preclinical regulatory pharmacodynamic (PD), safety pharmacology, pharmacokinetic (PK), and toxicology programmes of DTP3, leading to the approval for clinical trials in oncology. These results demonstrate that DTP3 combines on-target-selective pharmacology, therapeutic anticancer efficacy, favourable drug-like properties, long plasma half-life and good bioavailability, with no target-organs of toxicity and no adverse effects preclusive of its clinical development in oncology, upon daily repeat-dose administration in both rodent and non-rodent species. Our study underscores the clinical potential of DTP3 as a conceptually novel candidate therapeutic selectively blocking NF-κB survival signalling in MM and potentially other NF-κB-driven cancers.
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Antineoplásicos/farmacología , Sistemas de Liberación de Medicamentos , Mieloma Múltiple , FN-kappa B/antagonistas & inhibidores , Proteínas de Neoplasias/antagonistas & inhibidores , Línea Celular Tumoral , Humanos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , FN-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Prueba de Estudio ConceptualRESUMEN
GADD45ß is selectively and constitutively expressed in Multiple Myeloma cells, and this expression correlates with an unfavourable clinical outcome. GADD45ß physically interacts with the JNK kinase, MKK7, inhibiting its activity to enable the survival of cancer cells. DTP3 is a small peptide inhibitor of the GADD45ß/MKK7 complex and is able to restore MKK7/JNK activation, thereby promoting selective cell death of GADD45ß-overexpressing cancer cells. Enzymatic MS foot-printing and diazirine-based chemical cross-linking MS (CX-MS) strategies were applied to study the interactions between GADD45ß and MKK7 kinase domain (MKK7_KD) and between DTP3 and MKK7_KD. Our data show that the binding between GADD45ß and MKK7 largely occurs between GADD45ß loop 2 (region 103-117) and the kinase enzymatic pocket. We also show that DTP3 interferes with this GADD45ß/MKK7 interaction by contacting the MKK7 peptides, 113-136 and 259-274. Accordingly, an MKK7_KD Δ(101-136) variant lacking Trp135 did not produce a fluorescence quenching effect upon the binding of DTP3. The assessment of the interaction between GADD45ß and MKK7 and the elucidation of the recognition surfaces between DTP3 and MKK7 significantly advance the understanding of the mechanism underlying the inhibition of the GADD45ß/MKK7 interaction by DTP3 and pave the way to the design of small-molecule DTP3 analogues.
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Antígenos de Diferenciación/química , MAP Quinasa Quinasa 7 , Complejos Multiproteicos , Péptidos/química , Inhibidores de Proteínas Quinasas/química , Humanos , MAP Quinasa Quinasa 7/antagonistas & inhibidores , MAP Quinasa Quinasa 7/química , Espectrometría de Masas , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/químicaRESUMEN
T-cell exclusion from the tumor microenvironment (TME) is a major barrier to overcoming immune escape. Here, we identify a myeloid-intrinsic mechanism governed by the NF-κB effector molecule GADD45ß that restricts tumor-associated inflammation and T-cell trafficking into tumors. In various models of solid cancers refractory to immunotherapies, including hepatocellular carcinoma and ovarian adenocarcinoma, Gadd45b inhibition in myeloid cells restored activation of proinflammatory tumor-associated macrophages (TAM) and intratumoral immune infiltration, thereby diminishing oncogenesis. Our results provide a basis to interpret clinical evidence that elevated expression of GADD45B confers poor clinical outcomes in most human cancers. Furthermore, they suggest a therapeutic target in GADD45ß for reprogramming TAM to overcome immunosuppression and T-cell exclusion from the TME.Significance: These findings define a myeloid-based immune checkpoint that restricts T-cell trafficking into tumors, with potentially important therapeutic implications to generally improve the efficacy of cancer immunotherapy. Cancer Res; 78(5); 1275-92. ©2017 AACR.