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MYC-driven medulloblastoma (MB) is a highly aggressive cancer type with poor prognosis and limited treatment options. Through CRISPR-Cas9 screening across MB cell lines, we identified the Mediator-associated kinase CDK8 as the top dependence for MYC-driven MB. Loss of CDK8 markedly reduces MYC expression and impedes MB growth. Mechanistically, we demonstrate that CDK8 depletion suppresses ribosome biogenesis and mRNA translation. CDK8 regulates occupancy of phospho-Polymerase II at specific chromatin loci facilitating an epigenetic alteration that promotes transcriptional regulation of ribosome biogenesis. Additionally, CDK8-mediated phosphorylation of 4EBP1 plays a crucial role in initiating eIF4E-dependent translation. Targeting CDK8 effectively suppresses cancer stem and progenitor cells, characterized by increased ribosome biogenesis activity. We also report the synergistic inhibition of CDK8 and mTOR in vivo and in vitro . Overall, our findings establish a connection between transcription and translation regulation, suggesting a promising therapeutic approach targets multiple points in the protein synthesis network for MYC-driven MB.
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Sepsis is the leading cause of death in intensive care units worldwide. Current treatments of sepsis are largely supportive and clinical trials using specific pharmacotherapy for sepsis have failed to improve outcomes. Here, we used the lipopolysaccharide (LPS)-stimulated mouse RAW264.7 cell line and AlphaLisa assay for TNFa as a readout to perform a supervised drug repurposing screen for sepsis treatment with compounds targeting epigenetic enzymes, including kinases. We identified the SCH772984 compound, an extracellular signal-regulated kinase (ERK) 1/2 inhibitor, as an effective blocker of TNFa production in vitro. RNA-Seq of the SCH772984-treated RAW264.7 cells at 1, 4, and 24 h time points of LPS challenge followed by functional annotation of differentially expressed genes highlighted the suppression of cellular pathways related to the immune system. SCH772984 treatment improved survival in the LPS-induced lethal endotoxemia and cecal ligation and puncture (CLP) mouse models of sepsis, and reduced plasma levels of Ccl2/Mcp1. Functional analyses of RNA-seq datasets for kidney, lung, liver, and heart tissues from SCH772984-treated animals collected at 6 h and 12 h post-CLP revealed a significant downregulation of pathways related to the immune response and platelets activation but upregulation of the extracellular matrix organization and retinoic acid signaling pathways. Thus, this study defined transcriptome signatures of SCH772984 action in vitro and in vivo, an agent that has the potential to improve sepsis outcome.
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Antiinflamatorios/farmacología , Endotoxemia/tratamiento farmacológico , Indazoles/farmacología , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Piperazinas/farmacología , Piridinas/farmacología , Pirrolidinas/farmacología , Triazoles/farmacología , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Línea Celular , Quimiocina CCL2/sangre , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Reposicionamiento de Medicamentos , Endotoxemia/mortalidad , Regulación de la Expresión Génica/efectos de los fármacos , Lipopolisacáridos/toxicidad , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Activación Plaquetaria/efectos de los fármacos , Células RAW 264.7 , Transcriptoma/genéticaRESUMEN
Melanomas commonly undergo a phenotype switch, from a proliferative to an invasive state. Such tumor cell plasticity contributes to immunotherapy resistance; however, the mechanisms are not completely understood and thus are therapeutically unexploited. Using melanoma mouse models, we demonstrated that blocking the MNK1/2-eIF4E axis inhibited melanoma phenotype switching and sensitized melanoma to anti-PD-1 immunotherapy. We showed that phospho-eIF4E-deficient murine melanomas expressed high levels of melanocytic antigens, with similar results verified in patient melanomas. Mechanistically, we identified phospho-eIF4E-mediated translational control of NGFR, a critical effector of phenotype switching. Genetic ablation of phospho-eIF4E reprogrammed the immunosuppressive microenvironment, exemplified by lowered production of inflammatory factors, decreased PD-L1 expression on dendritic cells and myeloid-derived suppressor cells, and increased CD8+ T cell infiltrates. Finally, dual blockade of the MNK1/2-eIF4E axis and the PD-1/PD-L1 immune checkpoint demonstrated efficacy in multiple melanoma models regardless of their genomic classification. An increase in the presence of intratumoral stem-like TCF1+PD-1+CD8+ T cells, a characteristic essential for durable antitumor immunity, was detected in mice given a MNK1/2 inhibitor and anti-PD-1 therapy. Using MNK1/2 inhibitors to repress phospho-eIF4E thus offers a strategy to inhibit melanoma plasticity and improve response to anti-PD-1 immunotherapy.
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Linfocitos T CD8-positivos/inmunología , Factor 4E Eucariótico de Iniciación/inmunología , Inmunidad Celular , Sistema de Señalización de MAP Quinasas/inmunología , Melanoma Experimental/inmunología , Proteínas Serina-Treonina Quinasas/inmunología , Animales , Antígeno B7-H1/genética , Antígeno B7-H1/inmunología , Línea Celular Tumoral , Factor 4E Eucariótico de Iniciación/genética , Inmunoterapia , Sistema de Señalización de MAP Quinasas/genética , Melanoma Experimental/genética , Melanoma Experimental/terapia , Ratones , Ratones Transgénicos , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Proteínas Serina-Treonina Quinasas/genética , Receptor de Factor de Crecimiento Nervioso/genética , Receptor de Factor de Crecimiento Nervioso/inmunologíaRESUMEN
Enhancer of zeste homolog 2 (EZH2), a catalytic component of polycomb repressive complex 2 (PRC2), is commonly overexpressed or mutated in many cancer types, both of hematological and solid nature. Till now, plenty of EZH2 small molecule inhibitors have been developed and some of them have already been tested in clinical trials. Most of these inhibitors, however, are effective only in limited cases in the context of EZH2 gain-of-function mutated tumors such as lymphomas. Other cancer types with aberrant EZH2 expression and function require alternative approaches for successful treatment. One possibility is to exploit synthetic lethal strategy, which is based on the phenomenon that concurrent loss of two genes is detrimental but the deletion of either of them leaves cell viable. In the context of EZH2/PRC2, the most promising synthetic lethal target seems to be SWItch/Sucrose Non-Fermentable chromatin remodeling complex (SWI/SNF), which is known to counteract PRC2 functions. SWI/SNF is heavily involved in carcinogenesis and its subunits have been found mutated in approximately 20% of tumors of different kinds. In the current review, we summarize the existing knowledge of synthetic lethal relationships between EZH2/PRC2 and components of the SWI/SNF complex and discuss in detail the potential application of existing EZH2 inhibitors in cancer patients harboring mutations in SWI/SNF proteins. We also highlight recent discoveries of EZH2 involvement in tumor microenvironment regulation and consequences for future therapies. Although clinical studies are limited, the fundamental research might help to understand which patients are most likely to benefit from therapies using EZH2 inhibitors.
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Proteína Potenciadora del Homólogo Zeste 2 , Neoplasias , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Microambiente TumoralRESUMEN
The mitogen-activated protein kinase (MAPK)-interacting kinases 1 and 2 (MNKs 1/2) and their downstream target eIF4E, play a role in oncogenic transformation, progression and metastasis. These results provided rationale for development of first MNKs inhibitors, currently in clinical trials for cancer treatment. Inhibitors of the MNKs/eIF4E pathway are also proposed as treatment strategy for inflammatory conditions. Here we present results of optimization of indazole-pyridinone derived MNK1/2 inhibitors among which compounds 24 and 26, selective and metabolically stable derivatives. Both compounds decreased levels of eIF4E Ser206 phosphorylation (pSer209-eIF4E) in MOLM16 cell line. When administered in mice compounds 24 and 26 significantly improved survival rates of animals in the endotoxin lethal dose challenge model, with concomitant reduction of proinflammatory cytokine levels - TNFα and IL-6 in serum. Identified MNK1/2 inhibitors represent a novel class of immunomodulatory compounds with a potential for the treatment of inflammatory diseases including sepsis.
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Factores Inmunológicos/síntesis química , Indazoles/química , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Piridonas/química , Choque Séptico/tratamiento farmacológico , Secuencia de Aminoácidos , Animales , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Endotoxinas/metabolismo , Factor 4E Eucariótico de Iniciación/metabolismo , Humanos , Factores Inmunológicos/farmacología , Ratones , Simulación del Acoplamiento Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Choque Séptico/inducido químicamente , Transducción de Señal , Relación Estructura-ActividadRESUMEN
Burkitt lymphoma (BL) is a rapidly growing tumor, characterized by high anabolic requirements. The MYC oncogene plays a central role in the pathogenesis of this malignancy, controlling genes involved in apoptosis, proliferation, and cellular metabolism. Serine biosynthesis pathway (SBP) couples glycolysis to folate and methionine cycles, supporting biosynthesis of certain amino acids, nucleotides, glutathione, and a methyl group donor, S-adenosylmethionine (SAM). We report that BLs overexpress SBP enzymes, phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase 1 (PSAT1). Both genes are controlled by the MYC-dependent ATF4 transcription factor. Genetic ablation of PHGDH/PSAT1 or chemical PHGDH inhibition with NCT-503 decreased BL cell lines proliferation and clonogenicity. NCT-503 reduced glutathione level, increased reactive oxygen species abundance, and induced apoptosis. Consistent with the role of SAM as a methyl donor, NCT-503 decreased DNA and histone methylation, and led to the re-expression of ID4, KLF4, CDKN2B and TXNIP tumor suppressors. High H3K27me3 level is known to repress the MYC negative regulator miR-494. NCT-503 decreased H3K27me3 abundance, increased the miR-494 level, and reduced the expression of MYC and MYC-dependent histone methyltransferase, EZH2. Surprisingly, chemical/genetic disruption of SBP did not delay BL and breast cancer xenografts growth, suggesting the existence of mechanisms compensating the PHGDH/PSAT1 absence in vivo.
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In oncology, the "Warburg effect" describes the elevated production of energy by glycolysis in cancer cells. The ubiquitous and hypoxia-induced 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) plays a noteworthy role in the regulation of glycolysis by producing fructose-2,6-biphosphate (F-2,6-BP), a potent activator of the glycolysis rate-limiting phosphofructokinase PFK-1. Series of amides and sulfonamides derivatives based on a N-aryl 6-aminoquinoxaline scaffold were synthesized and tested for their inhibition of PFKFB3 in vitro in a biochemical assay as well as in HCT116 cells. The carboxamide series displayed satisfactory kinetic solubility and metabolic stability, and within this class, potent lead compounds with low nanomolar activity have been identified with a suitable profile for further in vivo evaluation.
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Amidas/química , Fosfofructoquinasa-2/antagonistas & inhibidores , Quinoxalinas/química , Quinoxalinas/farmacología , Sulfonamidas/química , Células HCT116 , Humanos , Cinética , SolubilidadRESUMEN
Energy and biomass production in cancer cells are largely supported by aerobic glycolysis in what is called the Warburg effect. The process is regulated by key enzymes, among which phosphofructokinase PFK-2 plays a significant role by producing fructose-2,6-biphosphate; the most potent activator of the glycolysis rate-limiting step performed by phosphofructokinase PFK-1. Herein, the synthesis, biological evaluation and structure-activity relationship of novel inhibitors of 6-phosphofructo-2-kinase/fructose-2,6-biphosphataseâ 3 (PFKFB3), which is the ubiquitous and hypoxia-induced isoform of PFK-2, are reported. X-ray crystallography and docking were instrumental in the design and optimisation of a series of N-aryl 6-aminoquinoxalines. The most potent representative, N-(4-methanesulfonylpyridin-3-yl)-8-(3-methyl-1-benzothiophen-5-yl)quinoxalin-6-amine, displayed an IC50 of 14â nm for the target and an IC50 of 0.49â µm for fructose-2,6-biphosphate production in human colon carcinoma HCT116 cells. This work provides a new entry in the field of PFKFB3 inhibitors with potential for development in oncology.
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Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Fosfofructoquinasa-2/antagonistas & inhibidores , Quinoxalinas/química , Quinoxalinas/farmacología , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Células HCT116 , Humanos , Ácido Láctico/antagonistas & inhibidores , Ácido Láctico/biosíntesis , Modelos Moleculares , Estructura Molecular , Fosfofructoquinasa-2/metabolismo , Quinoxalinas/síntesis química , Relación Estructura-ActividadRESUMEN
MAPK interacting kinase (MNK), a downstream effector of mitogen-activated protein kinase (MAPK) pathways, activates eukaryotic translation initiation factor 4E (eIF4E) and plays a key role in the mRNA translation of mitogenic and antiapoptotic genes in acute myeloid leukemia (AML) cells. We examined the antileukemic properties of a novel MNK inhibitor, SEL201. Our studies provide evidence that SEL201 suppresses eIF4E phosphorylation on Ser209 in AML cell lines and in primary patient-derived AML cells. Such effects lead to growth inhibitory effects and leukemic cell apoptosis, as well as suppression of leukemic progenitor colony formation. Combination of SEL201 with 5'-azacytidine or rapamycin results in synergistic inhibition of AML cell growth. Collectively, these results suggest that SEL201 has significant antileukemic activity and further underscore the relevance of the MNK pathway in leukemogenesis.
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Melanoma can be stratified into unique subtypes based on distinct pathologies. The acral/mucosal melanoma subtype is characterized by aberrant and constitutive activation of the proto-oncogene receptor tyrosine kinase C-KIT, which drives tumorigenesis. Treatment of these melanoma patients with C-KIT inhibitors has proven challenging, prompting us to investigate the downstream effectors of the C-KIT receptor. We determined that C-KIT stimulates MAP kinase-interacting serine/threonine kinases 1 and 2 (MNK1/2), which phosphorylate eukaryotic translation initiation factor 4E (eIF4E) and render it oncogenic. Depletion of MNK1/2 in melanoma cells with oncogenic C-KIT inhibited cell migration and mRNA translation of the transcriptional repressor SNAI1 and the cell cycle gene CCNE1. This suggested that blocking MNK1/2 activity may inhibit tumor progression, at least in part, by blocking translation initiation of mRNAs encoding cell migration proteins. Moreover, we developed an MNK1/2 inhibitor (SEL201), and found that SEL201-treated KIT-mutant melanoma cells had lower oncogenicity and reduced metastatic ability. Clinically, tumors from melanoma patients harboring KIT mutations displayed a marked increase in MNK1 and phospho-eIF4E. Thus, our studies indicate that blocking MNK1/2 exerts potent antimelanoma effects and support blocking MNK1/2 as a potential strategy to treat patients positive for KIT mutations.
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Antineoplásicos/farmacología , Dasatinib/farmacología , Melanoma/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Animales , Línea Celular Tumoral , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Melanoma/enzimología , Melanoma/secundario , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Mutación Missense , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-kit/genética , Transducción de Señal , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Inhibition of oncogenic transcriptional programs is a promising therapeutic strategy. A substituted tricyclic benzimidazole, SEL120-34A, is a novel inhibitor of Cyclin-dependent kinase 8 (CDK8), which regulates transcription by associating with the Mediator complex. X-ray crystallography has shown SEL120-34A to be a type I inhibitor forming halogen bonds with the protein's hinge region and hydrophobic complementarities within its front pocket. SEL120-34A inhibits phosphorylation of STAT1 S727 and STAT5 S726 in cancer cells in vitro. Consistently, regulation of STATs- and NUP98-HOXA9- dependent transcription has been observed as a dominant mechanism of action in vivo. Treatment with the compound resulted in a differential efficacy on AML cells with elevated STAT5 S726 levels and stem cell characteristics. In contrast, resistant cells were negative for activated STAT5 and revealed lineage commitment. In vivo efficacy in xenotransplanted AML models correlated with significant repression of STAT5 S726. Favorable pharmacokinetics, confirmed safety and in vivo efficacy provide a rationale for the further clinical development of SEL120-34A as a personalized therapeutic approach in AML.
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Antineoplásicos/farmacología , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Leucemia Mieloide Aguda/metabolismo , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT5/metabolismo , Animales , Antineoplásicos/química , Línea Celular Tumoral , Quinasa 8 Dependiente de Ciclina/química , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Modelos Moleculares , Conformación Molecular , Fosforilación/efectos de los fármacos , Unión Proteica , Inhibidores de Proteínas Quinasas/química , Factor de Transcripción STAT1/química , Factor de Transcripción STAT5/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
MAP kinase-interacting kinases (MNK1 and MNK2) are often activated downstream of ERK and p38 MAPK in the MAP kinase family. The role of MNKs in the development and progression of solid tumors and hematological malignancies has been widely discussed, particularly in the context of cap dependent translation, regulated by phosphorylation of eIF4E. MNK/eIF4E axis is involved in the expression of pro angiogenic, antiapoptotic, cell cycle, and motility proteins, such as MCL1, VEGF, MMP3, SNAIL, SMAD2, ß-catenin or cyclin D1, and is essential during Ras and c Myc-induced transformation. MNK1/2 emerged as eligible targets for drug discovery in oncology, based on the antitumor effects observed in genetic knockout and RNA interference experiments and at the same time lack of adverse effects in dual knockout animals. There is a high interest in the development of pharmacological inhibitors of MNK1/2 as not only tools for further basic research studies but also potential drugs in diseases characterized by deregulated translation. Unfortunately, the role of MNK1/2 in cancer still remains elusive due to the absence of potent and selective probes. Moreover, in many instances, hypotheses have been built reliant upon unspecific MNK1/2 inhibitors such as CGP57380 or cercosporamide. Lately, the first two clinical programs targeting MNKs in oncology have been revealed (eFT508 and BAY 1143269), although several other MNK programs are currently running at the preclinical stage. This review aims to provide an overview of recent progress in the development of MNK inhibitors.
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Antineoplásicos/farmacología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Terapia Molecular Dirigida , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/química , Transducción de SeñalRESUMEN
Cyclin-dependent kinase (CDK) inhibitors have been developed as potential anticancer therapeutics and several nonselective compounds are currently in advanced clinical trials. This review is focused on the key biological roles of CDK8 kinase, which provide a proof-of-principle for continued efforts toward effective cancer treatment, targeting activity of this CDK family member. Among currently identified kinase inhibitors, several displayed significant selectivity for CDK8 and notably the effectiveness in targeting cancer specific gene expression programs. Structural features of CDK8 and available ligands were discussed from a perspective of the rational drug design process. Current state of the art confirms that further development of CDK8 inhibitors will translate into targeted therapies in oncology. This article is part of a Special Issue entitled:Inhibitors of Protein Kinases.
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Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Transcripción Genética/efectos de los fármacos , Quinasa 8 Dependiente de Ciclina/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Estructura Molecular , Neoplasias/genética , Niacinamida/análogos & derivados , Niacinamida/química , Niacinamida/uso terapéutico , Compuestos de Fenilurea/química , Compuestos de Fenilurea/uso terapéutico , Conformación Proteica , Inhibidores de Proteínas Quinasas/química , Transducción de Señal/efectos de los fármacos , SorafenibRESUMEN
Tumor hypoxia confers resistance to many modalities of anticancer therapy. The endoplasmic reticulum (ER) is highly sensitive to severe hypoxic stress and results in the activation of the unfolded protein response. ATF4 is the main transcriptional regulator of the cellular hypoxic response to the Unfolded Protein Response (UPR) and activates genes that promote restoration of normal ER function and survival under hypoxia. Elevated expression of ATF4 is associated with resistance to current chemotherapeutic drugs including DNA-interactive and damaging agents, nonsteroidal anti-inflammatory drugs and proteasome inhibitors. ATF4 decreases the antitumor activity of chemotherapy by mechanisms involving expression of genes involved in oxidative stress resistance, redox homeostasis and inhibitors of apoptosis. ATF4 plays also a crucial role in resistance to proteasomal inhibitor bortezomib (PS-341) by the induction of prosurvival pathways, such as autophagy, that can relieve the protein overload in bortezomib treated cells. Inhibition of ATF4 represents an attractive stand-alone therapy as well as an opportunity to enhance the efficacy of current chemotherapeutic agents without causing high tissue toxicity to normal tissues.
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Factor de Transcripción Activador 4/fisiología , Autofagia , Neoplasias/metabolismo , Factor de Transcripción Activador 4/genética , Hipoxia de la Célula , Resistencia a Antineoplásicos , Retículo Endoplásmico/efectos de los fármacos , Humanos , Respuesta de Proteína Desplegada , eIF-2 Quinasa/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Tumour hypoxia contributes to failure of cancer treatment through its ability to protect against therapy and adversely influence tumour biology. In particular, several studies suggest that hypoxia promotes metastasis. Hypoxia-induced cellular changes are mediated by oxygen-sensitive signaling pathways that activate downstream transcription factors. We have investigated the induction and transcriptional regulation of a novel metastasis-associated gene, LAMP3 during hypoxia. MATERIALS AND METHODS: Microarray, quantitative PCR, Western blot analysis and immunohistochemistry were used to investigate hypoxic regulation of LAMP3. The mechanism for LAMP3 induction was investigated using transient RNAi and stable shRNA targeting components of the hypoxic response. Endoplasmic reticulum stress inducing agents, including proteasome inhibitors were assessed for their ability to regulate LAMP3. RESULTS: LAMP3 is strongly induced by hypoxia at both the mRNA and protein levels in a large panel of human tumour cell lines. Induction of LAMP3 occurs as a consequence of the activation of the PERK/eIF2alpha/ATF4 arm of the unfolded protein response (UPR) and is independent of HIF-1alpha. LAMP3 is expressed heterogeneously within the microenvironment of tumours, overexpressed in breast cancer, and increases in tumours treated with avastin. CONCLUSIONS: These data identify LAMP3 as a novel hypoxia-inducible gene regulated by the UPR. LAMP3 is a new candidate biomarker of UPR activation by hypoxia in tumours and is a potential mediator of hypoxia-induced metastasis.
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Biomarcadores de Tumor/genética , Hipoxia/genética , Proteínas de Membrana de los Lisosomas/metabolismo , Proteínas de Neoplasias/metabolismo , Respuesta de Proteína Desplegada/genética , Animales , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Hipoxia/metabolismo , Inmunohistoquímica , Proteínas de Membrana de los Lisosomas/genética , Ratones , Ratones Endogámicos BALB C , Metástasis de la Neoplasia/genética , Proteínas de Neoplasias/genética , Reacción en Cadena de la Polimerasa , Probabilidad , Análisis por Matrices de Proteínas , ARN Interferente Pequeño/análisis , Valores de Referencia , Transfección , Células Tumorales CultivadasRESUMEN
The ubiquitin-proteasome system plays a key regulatory role in cellular homeostasis. The inhibition of the 26S proteasome by Bortezomib leads to the accumulation of misfolded proteins, resulting in endoplasmic reticulum stress followed by a coordinated cellular response called unfolded protein response (UPR). Endoplasmic reticulum stress is also a potent inducer of macroautophagy. Bortezomib is a selective and potent inhibitor of the 26S proteasome and is approved for the treatment of multiple myeloma. Clinical trials with Bortezomib have shown promising results for some types of cancers, but not for some others, including those of the breast. In this study, we show that Bortezomib induces the UPR and autophagy in MCF7 breast cancer cells. Surprisingly, Bortezomib did not induce phosphorylation of PERK, a key initial step of the UPR. We show that induction of autophagy by Bortezomib is dependent on the proteasomal stabilisation of ATF4 and up-regulation of LC3B by ATF4. We show that ATF4 and LC3B play a critical role in activating autophagy and protecting cells from Bortezomib-induced cell death. Our experiments also reveal that HDAC6 knockdown results in decreased LC3B protein and reduced autophagy. Our work shows that the induction of autophagy through ATF4 may be an important resistance mechanism to Bortezomib treatment in breast cancer, and targeting autophagy may represent a novel approach to sensitize breast cancers to Bortezomib.
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Factor de Transcripción Activador 4/metabolismo , Antineoplásicos/uso terapéutico , Autofagia/efectos de los fármacos , Ácidos Borónicos/uso terapéutico , Pirazinas/uso terapéutico , Factor de Transcripción Activador 4/efectos de los fármacos , Factor de Transcripción Activador 4/genética , Bortezomib , Neoplasias de la Mama/tratamiento farmacológico , División Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Citometría de Flujo , Humanos , Inmunohistoquímica , Proteínas de Neoplasias/efectos de los fármacos , Proteínas de Neoplasias/metabolismo , Reacción en Cadena de la Polimerasa , ARN Neoplásico/efectos de los fármacos , ARN Neoplásico/genética , ARN Interferente Pequeño/efectos de los fármacos , ARN Interferente Pequeño/genéticaRESUMEN
The lack of oxygen delivery to tumor cells has profound consequences for tumor growth and correlates with poor prognosis. Some tumors contain regions of very severe hypoxia called anoxia, which constitutes a functionally different state to hypoxia. In response to anoxia, mammalian cells induce coordinated cytoprotective programs that are critical for tumor survival: the unfolded protein response and integrated stress response. Therefore, targeting additional components of anoxic pathways, besides the hypoxia-inducible response, may be effective for future anticancer therapies.
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Hipoxia/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Biosíntesis de Proteínas , Factor de Transcripción Activador 4/metabolismo , Retículo Endoplásmico/enzimología , Humanos , Hipoxia/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Pliegue de Proteína , Transcripción Genética , eIF-2 Quinasa/metabolismoRESUMEN
The ADAM (a disintegrin and a metalloprotease) family includes the best-characterized proteins involved in gamete interaction and membrane fusion in mammals. Previous studies have shown that the murine testase 2 (ADAM 25) gene is expressed specifically in testis. We found two different restriction patterns of subcloned fragments of the gene, indicating the presence of two different testase 2 transcripts. Further experiments and Celera database searches demonstrated that the two transcripts are the products of two testase 2 genes, which are located on mouse chromosome 8 in close distance of 24 kb. They show high sequence similarity to the published testase 2 gene (87.8 and 95.5%, respectively). The genomic structure of both testase 2 genes (alpha and beta) is different from other ADAM family genes like that for cyritestin and fertilin. While these genes are composed of about 20 exons, the testase 2alpha and 2beta genes contain only two exons. The first exon is very short approximately 85 bp while the second exon is approximately 2.4 kb long. Both testase 2 genes are specifically expressed in testis and they exhibit the same temporal and spatial expression pattern during male germ cell differentiation with the onset of expression in haploid stages.