RESUMEN
Formation of membrane pores/channels regulates various cellular processes, such as necroptosis or stem cell niche signaling. However, the roles of membrane lipids in the formation of pores and their biological functions are largely unknown. Here, using the cellular stress model evoked by the sphingolipid analog drug FTY720, we show that formation of ceramide-enriched membrane pores, referred to here as ceramidosomes, is initiated by a receptor-interacting Ser/Thr kinase 1 (RIPK1)-ceramide complex transported to the plasma membrane by nonmuscle myosin IIA-dependent trafficking in human lung cancer cells. Molecular modeling/simulation coupled with site-directed mutagenesis revealed that Asp147 or Asn169 of RIPK1 are key for ceramide binding and that Arg258 or Leu293 residues are involved in the myosin IIA interaction, leading to ceramidosome formation and necroptosis. Moreover, generation of ceramidosomes independently of any external drug/stress stimuli was also detected in the plasma membrane of germ line stem cells in ovaries during the early stages of oogenesis in Drosophila melanogaster Inhibition of ceramidosome formation via myosin IIA silencing limited germ line stem cell signaling and abrogated oogenesis. In conclusion, our findings indicate that the RIPK1-ceramide complex forms large membrane pores we named ceramidosomes. They further suggest that, in addition to their roles in stress-mediated necroptosis, these ceramide-enriched pores also regulate membrane integrity and signaling and might also play a role in D. melanogaster ovary development.
Asunto(s)
Membrana Celular/metabolismo , Ceramidas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Motoras Moleculares/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Necrosis/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Células A549 , Animales , Línea Celular , Membrana Celular/patología , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Humanos , Neoplasias Pulmonares/patología , Simulación del Acoplamiento Molecular , Necrosis/patología , Oogénesis , Ovario/crecimiento & desarrolloRESUMEN
Signaling pathways regulated by mutant Fms-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD), which mediate resistance to acute myeloid leukemia (AML) cell death, are poorly understood. Here, we reveal that pro-cell death lipid ceramide generation is suppressed by FLT3-ITD signaling. Molecular or pharmacologic inhibition of FLT3-ITD reactivated ceramide synthesis, selectively inducing mitophagy and AML cell death. Mechanistically, FLT3-ITD targeting induced ceramide accumulation on the outer mitochondrial membrane, which then directly bound autophagy-inducing light chain 3 (LC3), involving its I35 and F52 residues, to recruit autophagosomes for execution of lethal mitophagy. Short hairpin RNA (shRNA)-mediated knockdown of LC3 prevented AML cell death in response to FLT3-ITD inhibition by crenolanib, which was restored by wild-type (WT)-LC3, but not mutants of LC3 with altered ceramide binding (I35A-LC3 or F52A-LC3). Mitochondrial ceramide accumulation and lethal mitophagy induction in response to FLT3-ITD targeting was mediated by dynamin-related protein 1 (Drp1) activation via inhibition of protein kinase A-regulated S637 phosphorylation, resulting in mitochondrial fission. Inhibition of Drp1 prevented ceramide-dependent lethal mitophagy, and reconstitution of WT-Drp1 or phospho-null S637A-Drp1 but not its inactive phospho-mimic mutant (S637D-Drp1), restored mitochondrial fission and mitophagy in response to crenolanib in FLT3-ITD+ AML cells expressing stable shRNA against endogenous Drp1. Moreover, activating FLT3-ITD signaling in crenolanib-resistant AML cells suppressed ceramide-dependent mitophagy and prevented cell death. FLT3-ITD+ AML drug resistance is attenuated by LCL-461, a mitochondria-targeted ceramide analog drug, in vivo, which also induced lethal mitophagy in human AML blasts with clinically relevant FLT3 mutations. Thus, these data reveal a novel mechanism which regulates AML cell death by ceramide-dependent mitophagy in response to FLT3-ITD targeting.
Asunto(s)
Bencimidazoles/farmacología , Ceramidas , Resistencia a Antineoplásicos , Leucemia Mieloide Aguda , Mitofagia , Mutación , Piperidinas/farmacología , ARN Interferente Pequeño/farmacología , Transducción de Señal , Tirosina Quinasa 3 Similar a fms , Animales , Ceramidas/genética , Ceramidas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Dinaminas , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mitofagia/efectos de los fármacos , Mitofagia/genética , Fosforilación/efectos de los fármacos , Fosforilación/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
Folate, an important nutrient in the human diet, has been implicated in cancer, but its role in metastasis is not established. We have shown previously that the withdrawal of medium folate leads to the inhibition of migration and invasion of A549 lung carcinoma cells. Here we have demonstrated that medium folate regulates the function of Rho GTPases by enabling their carboxyl methylation and translocation to plasma membrane. Conversely, the lack of folate leads to the retention of these proteins in endoplasmic reticulum. Folate also promoted the switch from inactive (GDP-bound) to active (GTP-bound) GTPases, resulting in the activation of downstream kinases p21-activated kinase and LIM kinase and phosphorylation of the actin-depolymerizing factor cofilin. We have further demonstrated that in A549 cells two GTPases, RhoA and Rac1, but not Cdc42, are immediate sensors of folate status: the siRNA silencing of RhoA or Rac1 blocked effects of folate on cofilin phosphorylation and cellular migration and invasion. The finding that folate modulates metastatic potential of cancer cells was confirmed in an animal model of lung cancer using tail vein injection of A549 cells in SCID mice. A folate-rich diet enhanced lung colonization and distant metastasis to lymph nodes and decreased overall survival (35 versus 63 days for mice on a folate-restricted diet). High folate also promoted epithelial-mesenchymal transition in cancer cells and experimental mouse tumors. Our study provides experimental evidence for a mechanism of metastasis promotion by dietary folate and highlights the interaction between nutrients and metastasis-related signaling.
Asunto(s)
Adenocarcinoma/enzimología , Cofilina 1/metabolismo , Ácido Fólico/administración & dosificación , Neoplasias Pulmonares/enzimología , Proteína de Unión al GTP rac1/fisiología , Proteína de Unión al GTP rhoA/fisiología , Adenocarcinoma/secundario , Administración Oral , Animales , Línea Celular Tumoral , Membrana Celular/enzimología , Movimiento Celular/efectos de los fármacos , Supervivencia Celular , Suplementos Dietéticos , Retículo Endoplásmico/enzimología , Transición Epitelial-Mesenquimal , Ácido Fólico/farmacología , Humanos , Neoplasias Pulmonares/patología , Metástasis Linfática , Masculino , Metilación , Ratones SCID , Trasplante de Neoplasias , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Transducción de Señal , Proteína de Unión al GTP cdc42/metabolismo , Quinasas p21 Activadas/química , Proteína de Unión al GTP rac1/químicaRESUMEN
Programmed death ligand 1, PD-L1 (CD274), facilitates immune evasion and exerts pro-survival functions in cancer cells. Here, we report a mechanism whereby internalization of PD-L1 in response to alterations of bioactive lipid/ceramide metabolism by ceramide synthase 4 (CerS4) induces sonic hedgehog (Shh) and transforming growth factor ß receptor signaling to enhance tumor metastasis in triple-negative breast cancers (TNBCs), exhibiting immunotherapy resistance. Mechanistically, data showed that internalized PD-L1 interacts with an RNA-binding protein, caprin-1, to stabilize Shh/TGFBR1/Wnt mRNAs to induce ß-catenin signaling and TNBC growth/metastasis, consistent with increased infiltration of FoxP3+ regulatory T cells and resistance to immunotherapy. While mammary tumors developed in MMTV-PyMT/CerS4-/- were highly metastatic, targeting the Shh/PD-L1 axis using sonidegib and anti-PD-L1 antibody vastly decreased tumor growth and metastasis, consistent with the inhibition of PD-L1 internalization and Shh/Wnt signaling, restoring anti-tumor immune response. These data, validated in clinical samples and databases, provide a mechanism-based therapeutic strategy to improve immunotherapy responses in metastatic TNBCs.
Asunto(s)
Antígeno B7-H1 , Ceramidas , Inmunoterapia , Metástasis de la Neoplasia , Transducción de Señal , Antígeno B7-H1/metabolismo , Ceramidas/metabolismo , Humanos , Animales , Inmunoterapia/métodos , Ratones , Línea Celular Tumoral , Femenino , Proteínas Hedgehog/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/inmunologíaRESUMEN
Mechanisms by which Porphyromonas gingivalis (P. gingivalis) infection enhances oral tumor growth or resistance to cell death remain elusive. Here, we determined that P. gingivalis infection mediates therapeutic resistance via inhibiting lethal mitophagy in cancer cells and tumors. Mechanistically, P. gingivalis targets the LC3B-ceramide complex by associating with LC3B via bacterial major fimbriae (FimA) protein, preventing ceramide-dependent mitophagy in response to various therapeutic agents. Moreover, ceramide-mediated mitophagy is induced by Annexin A2 (ANXA2)-ceramide association involving the E142 residue of ANXA2. Inhibition of ANXA2-ceramide-LC3B complex formation by wild-type P. gingivalis prevented ceramide-dependent mitophagy. Moreover, a FimA-deletion mutant P. gingivalis variant had no inhibitory effects on ceramide-dependent mitophagy. Further, 16S rRNA sequencing of oral tumors indicated that P. gingivalis infection altered the microbiome of the tumor macroenvironment in response to ceramide analog treatment in mice. Thus, these data provide a mechanism describing the pro-survival roles of P. gingivalis in oral tumors.
RESUMEN
Repeat concussions (or repetitive mild traumatic brain injury [rmTBI]) are complex pathological processes consisting of a primary insult and long-term secondary complications and are also a prerequisite for chronic traumatic encephalopathy (CTE). Recent evidence implies a significant role of autophagy-mediated dysfunctional mitochondrial clearance, mitophagy, in the cascade of secondary deleterious events resulting from TBI. C18-ceramide, a bioactive sphingolipid produced in response to cell stress and damage, and its synthesizing enzyme (CerS1) are precursors to selective stress-mediated mitophagy. A transporter, p17, mediates the trafficking of CerS1, induces C18-ceramide synthesis in the mitochondrial membrane, and acts as an elimination signal in cell survival. Whether p17-mediated mitophagy occurs in the brain and plays a causal role in mitochondrial quality control in secondary disease development after rmTBI are unknown. Using a novel repetitive less-than-mild TBI (rlmTBI) injury paradigm, ablation of mitochondrial p17/C18-ceramide trafficking in p17 knockout (KO) mice results in a loss of C18-ceramide-induced mitophagy, which contributes to susceptibility and recovery from long-term secondary complications associated with rlmTBI. Using a ceramide analog with lipid-selenium conjugate drug, LCL768 restored mitophagy and reduced long-term secondary complications, improving cognitive deficits in rlmTBI-induced p17KO mice. We obtained a significant reduction of p17 expression and a considerable decrease of CerS1 and C18-ceramide levels in cortical mitochondria of CTE human brains compared with age-matched control brains. These data demonstrated that p17/C18-ceramide trafficking is an endogenous neuroprotective mitochondrial stress response following rlmTBI, thus suggesting a novel prospective strategy to interrupt the CTE consequences of concussive TBI.
RESUMEN
The spleen is an important mediator of both adaptive and innate immunity. As such, attempts to modulate the immune response provided by the spleen may be conducive to improved outcomes for numerous diseases throughout the body. Here, biomimicry is used to rationally design nanomaterials capable of splenic retention and immunomodulation for the treatment of disease in a distant organ, the postinfarct heart. Engineered senescent erythrocyte-derived nanotheranostic (eSENTs) are generated, demonstrating significant uptake by the immune cells of the spleen including T and B cells, as well as monocytes and macrophages. When loaded with suberoylanilide hydroxamic acid (SAHA), the nanoagents exhibit a potent therapeutic effect, reducing infarct size by 14% at 72 h postmyocardial infarction when given as a single intravenous dose 2 h after injury. These results are supportive of the hypothesis that RBC-derived biomimicry may provide new approaches for the targeted modulation of the pathological processes involved in myocardial infarction, thus further experiments to decisively confirm the mechanisms of action are currently underway. This novel concept may have far-reaching applicability for the treatment of a number of both acute and chronic conditions where the immune responses are either stimulated or suppressed by the splenic (auto)immune milieu.
Asunto(s)
Biomimética , Infarto del Miocardio , Humanos , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/patología , Corazón , Inmunidad Innata , InmunomodulaciónRESUMEN
The metabolic consequences of mitophagy alterations due to age-related stress in healthy aging brains versus neurodegeneration remain unknown. Here, we demonstrate that ceramide synthase 1 (CerS1) is transported to the outer mitochondrial membrane by the p17/PERMIT transporter that recognizes mislocalized mitochondrial ribosomes (mitoribosomes) via 39-FLRN-42 residues, inducing ceramide-mediated mitophagy. P17/PERMIT-CerS1-mediated mitophagy attenuated the argininosuccinate/fumarate/malate axis and induced d-glucose and fructose accumulation in neurons in culture and brain tissues (primarily in the cerebellum) of wild-type mice in vivo. These metabolic changes in response to sodium-selenite were nullified in the cerebellum of CerS1to/to (catalytically inactive for C18-ceramide production CerS1 mutant), PARKIN-/- or p17/PERMIT-/- mice that have dysfunctional mitophagy. Whereas sodium selenite induced mitophagy in the cerebellum and improved motor-neuron deficits in aged wild-type mice, exogenous fumarate or malate prevented mitophagy. Attenuating ceramide-mediated mitophagy enhanced damaged mitochondria accumulation and age-dependent sensorimotor abnormalities in p17/PERMIT-/- mice. Reinstituting mitophagy using a ceramide analog drug with selenium conjugate, LCL768, restored mitophagy and reduced malate/fumarate metabolism, improving sensorimotor deficits in old p17/PERMIT-/- mice. Thus, these data describe the metabolic consequences of alterations to p17/PERMIT/ceramide-mediated mitophagy associated with the loss of mitochondrial quality control in neurons and provide therapeutic options to overcome age-dependent sensorimotor deficits and related disorders like amyotrophic lateral sclerosis (ALS).
Asunto(s)
Malatos , Mitofagia , Ratones , Animales , Ceramidas/metabolismo , Neuronas Motoras/metabolismo , Fumaratos , Ubiquitina-Proteína LigasasRESUMEN
Crosstalk between metabolic and signaling events that induce tumor metastasis remains elusive. Here, we determine how oncogenic sphingosine 1-phosphate (S1P) metabolism induces intracellular C3 complement activation to enhance migration/metastasis. We demonstrate that increased S1P metabolism activates C3 complement processing through S1P receptor 1 (S1PR1). S1P/S1PR1-activated intracellular C3b-α'2 is associated with PPIL1 through glutamic acid 156 (E156) and aspartic acid 111 (D111) residues, resulting in NLRP3/inflammasome induction. Inactivation mutations of S1PR1 to prevent S1P signaling or mutations of C3b-α'2 to prevent its association with PPIL1 attenuate inflammasome activation and reduce lung colonization/metastasis in mice. Also, activation of the S1PR1/C3/PPIL1/NLRP3 axis is highly associated with human metastatic melanoma tissues and patient-derived xenografts. Moreover, targeting S1PR1/C3/PPIL1/NLRP3 signaling using molecular, genetic, and pharmacologic tools prevents lung colonization/metastasis of various murine cancer cell lines using WT and C3a-receptor1 knockout (C3aR1-/-) mice. These data provide strategies for treating high-grade/metastatic tumors by targeting the S1PR1/C3/inflammasome axis.
Asunto(s)
Inflamasomas , Melanoma , Humanos , Ratones , AnimalesRESUMEN
The E1 enzyme Uba6 initiates signal transduction by activating ubiquitin and the ubiquitin-like protein FAT10 in a two-step process involving sequential catalysis of adenylation and thioester bond formation. To gain mechanistic insights into these processes, we determined the crystal structure of a human Uba6/ubiquitin complex. Two distinct architectures of the complex are observed: one in which Uba6 adopts an open conformation with the active site configured for catalysis of adenylation, and a second drastically different closed conformation in which the adenylation active site is disassembled and reconfigured for catalysis of thioester bond formation. Surprisingly, an inositol hexakisphosphate (InsP6) molecule binds to a previously unidentified allosteric site on Uba6. Our structural, biochemical, and biophysical data indicate that InsP6 allosterically inhibits Uba6 activity by altering interconversion of the open and closed conformations of Uba6 while also enhancing its stability. In addition to revealing the molecular mechanisms of catalysis by Uba6 and allosteric regulation of its activities, our structures provide a framework for developing Uba6-specific inhibitors and raise the possibility of allosteric regulation of other E1s by naturally occurring cellular metabolites.
Asunto(s)
Enzimas Activadoras de Ubiquitina , Ubiquitina , Catálisis , Dominio Catalítico , Humanos , Ubiquitina/metabolismo , Enzimas Activadoras de Ubiquitina/metabolismo , Ubiquitinas/metabolismoRESUMEN
4'-Phosphopantetheinyl transferases (PPTs) catalyze the transfer of 4'-phosphopantetheine (4-PP) from coenzyme A to a conserved serine residue of their protein substrates. In humans, the number of pathways utilizing the 4-PP post-translational modification is limited and may only require a single broad specificity PPT for all phosphopantetheinylation reactions. Recently, we have shown that one of the enzymes of folate metabolism, 10-formyltetrahydrofolate dehydrogenase (FDH), requires a 4-PP prosthetic group for catalysis. This moiety acts as a swinging arm to couple the activities of the two catalytic domains of FDH and allows the conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. In the current study, we demonstrate that the broad specificity human PPT converts apo-FDH to holoenzyme and thus activates FDH catalysis. Silencing PPT by small interfering RNA in A549 cells prevents FDH modification, indicating the lack of alternative enzymes capable of accomplishing this transferase reaction. Interestingly, PPT-silenced cells demonstrate significantly reduced proliferation and undergo strong G(1) arrest, suggesting that the enzymatic function of PPT is essential and nonredundant. Our study identifies human PPT as the FDH-modifying enzyme and supports the hypothesis that mammals utilize a single enzyme for all phosphopantetheinylation reactions.
Asunto(s)
Proteína Transportadora de Acilo/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Secuencia de Bases , Biocatálisis , Ciclo Celular/genética , Muerte Celular/genética , Línea Celular , Clonación Molecular , Activación Enzimática , Silenciador del Gen , Humanos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Panteteína/análogos & derivados , Panteteína/metabolismo , ARN Interferente Pequeño/genética , Serina/metabolismo , Especificidad por Sustrato , Transferasas (Grupos de Otros Fosfatos Sustitutos)/biosíntesis , Transferasas (Grupos de Otros Fosfatos Sustitutos)/deficiencia , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genéticaRESUMEN
Cytosolic 10-formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1) is an abundant enzyme of folate metabolism. It converts 10-formyltetrahydrofolate to tetrahydrofolate and CO(2) in an NADP(+)-dependent reaction. We have identified a gene at chromosome locus 12q24.11 of the human genome, the product of which has 74% sequence similarity with cytosolic FDH. This protein has an extra N-terminal sequence of 22 amino acid residues, predicted to be a mitochondrial translocation signal. Transfection of COS-7 or A549 cell lines with a construct in which green fluorescent protein was introduced between the leader sequence and the rest of the putative mitochondrial FDH (mtFDH) has demonstrated mitochondrial localization of the fusion protein, suggesting that the identified gene encodes a mitochondrial enzyme. Purified pig liver mtFDH displayed dehydrogenase/hydrolase activities similar to cytosolic FDH. Real-time PCR performed on an array of human tissues has shown that although cytosolic FDH mRNA is highest in liver, kidney, and pancreas, mtFDH mRNA is most highly expressed in pancreas, heart, and brain. In contrast to the cytosolic enzyme, which is not detectable in cancer cells, the presence of mtFDH was demonstrated in several human cancer cell lines by conventional and real-time PCR and by Western blot. Analysis of genomes of different species indicates that the mitochondrial enzyme is a later evolutionary product when compared with the cytosolic enzyme. We propose that this novel mitochondrial enzyme is a likely source of CO(2) production from 10-formyltetrahydrofolate in mitochondria and plays an essential role in the distribution of one-carbon groups between the cytosolic and mitochondrial compartments of the cell.
Asunto(s)
Aldehído Deshidrogenasa/química , Aldehído Deshidrogenasa/genética , Mitocondrias/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Homología de Secuencia de Aminoácido , Aldehído Deshidrogenasa/metabolismo , Aldehído Deshidrogenasa Mitocondrial , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Clonación Molecular , Citosol/enzimología , Humanos , Ratones , Datos de Secuencia Molecular , Transporte de Proteínas , Ratas , Alineación de Secuencia , PorcinosRESUMEN
10-Formyltetrahydrofolate dehydrogenase (FDH) suppresses cancer cell proliferation through p53-dependent apoptosis but also induces strong cytotoxicity in p53-deficient prostate cells. In the present study, we have shown that FDH induces apoptosis in PC-3 prostate cells through simultaneous activation of the c-Jun-NH(2)-kinase (JNK) and extracellular signal-regulated kinase (ERK) pathways with JNK phosphorylating c-Jun and ERK1/2 phosphorylating Elk-1. The JNK1/2 inhibitor SP600125 or ERK1/2 inhibitor PD98059 prevented phosphorylation of c-Jun and Elk-1, correspondingly and partially protected PC-3 cells from FDH-induced cytotoxicity. Combination of the two inhibitors produced an additive effect. The contribution from the JNK cascade to FDH-induced apoptosis was significantly stronger than from the ERK pathway. siRNA knockdown of JNK1/2 or "turning off" the downstream target c-Jun by either siRNA or expression of the dominant-negative c-Jun mutant, TAM67, rescued PC-3 cells from FDH-induced apoptosis. The pull-down assays on immobilized c-Jun showed that c-Jun is directly phosphorylated by JNK2 in FDH-expressing cells. Interestingly, the FDH-induced apoptosis in p53-proficient A549 cells also proceeds through activation of JNK1/2, but the down-stream target for JNK2 is p53 instead of c-Jun. Furthermore, in A549 cells, FDH activates caspase 9, whereas in PC-3 cells, it activates caspase 8. Our studies indicate that the JNK pathways are common downstream mechanisms of FDH-induced cytotoxicity in different cell types, whereas the end point target in the cascade is cell type specific. JNK activation in response to FDH was inhibited by high supplementation of reduced folate leucovorin, further indicating a functional connection between folate metabolism and mitogen-activated protein kinase pathways.
Asunto(s)
Genes p53 , Variación Genética , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Adenocarcinoma/enzimología , Adenocarcinoma/genética , Apoptosis , Línea Celular Tumoral , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Masculino , Fosforilación , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/genética , Interferencia de ARN , ARN Neoplásico/genética , ARN Interferente Pequeño/genética , Especificidad por Sustrato , Transfección , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
How lipid metabolism is regulated at the outer mitochondrial membrane (OMM) for transducing stress signaling remains largely unknown. We show here that this process is controlled by trafficking of ceramide synthase 1 (CerS1) from the endoplasmic reticulum (ER) to the OMM by a previously uncharacterized p17, which is now renamed protein that mediates ER-mitochondria trafficking (PERMIT). Data revealed that p17/PERMIT associates with newly translated CerS1 on the ER surface to mediate its trafficking to the OMM. Cellular stress induces Drp1 nitrosylation/activation, releasing p17/PERMIT to retrieve CerS1 for its OMM trafficking, resulting in mitochondrial ceramide generation, mitophagy and cell death. In vivo, CRISPR-Cas9-dependent genetic ablation of p17/PERMIT prevents acute stress-mediated CerS1 trafficking to OMM, attenuating mitophagy in p17/PERMIT-/- mice, compared to controls, in various metabolically active tissues, including brain, muscle, and pancreas. Thus, these data have implications in diseases associated with accumulation of damaged mitochondria such as cancer and/or neurodegeneration.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/fisiología , Mitocondrias/patología , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/fisiología , Mitofagia , Esfingosina N-Aciltransferasa/fisiología , Estrés Fisiológico , Animales , Sistemas CRISPR-Cas , Ceramidas/metabolismo , Retículo Endoplásmico/patología , Humanos , Metabolismo de los Lípidos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Proteínas Mitocondriales/antagonistas & inhibidores , Transporte de ProteínasRESUMEN
The dysregulation of Fbxo4-cyclin D1 axis occurs at high frequency in esophageal squamous cell carcinoma (ESCC), where it promotes ESCC development and progression. However, defining a therapeutic vulnerability that results from this dysregulation has remained elusive. Here we demonstrate that Rb and mTORC1 contribute to Gln-addiction upon the dysregulation of the Fbxo4-cyclin D1 axis, which leads to the reprogramming of cellular metabolism. This reprogramming is characterized by reduced energy production and increased sensitivity of ESCC cells to combined treatment with CB-839 (glutaminase 1 inhibitor) plus metformin/phenformin. Of additional importance, this combined treatment has potent efficacy in ESCC cells with acquired resistance to CDK4/6 inhibitors in vitro and in xenograft tumors. Our findings reveal a molecular basis for cancer therapy through targeting glutaminolysis and mitochondrial respiration in ESCC with dysregulated Fbxo4-cyclin D1 axis as well as cancers resistant to CDK4/6 inhibitors.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias Esofágicas/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Regulación Neoplásica de la Expresión Génica , Glutamina/metabolismo , Hipoglucemiantes/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Animales , Bencenoacetamidas/farmacología , Línea Celular Tumoral , Ciclina D1/genética , Ciclina D1/metabolismo , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/metabolismo , Resistencia a Antineoplásicos/genética , Sinergismo Farmacológico , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/metabolismo , Carcinoma de Células Escamosas de Esófago/patología , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Glutaminasa/antagonistas & inhibidores , Glutaminasa/genética , Glutaminasa/metabolismo , Glutamina/antagonistas & inhibidores , Humanos , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Metformina/farmacología , Ratones , Terapia Molecular Dirigida , Fenformina/farmacología , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Transducción de Señal , Tiadiazoles/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mechanistic details for the roles of sphingolipids and their downstream targets in the regulation of tumor growth, response to chemo/radiotherapy, and metastasis have been investigated in recent studies using innovative molecular, genetic and pharmacologic tools in various cancer models. Induction of ceramide generation in response to cellular stress by chemotherapy, radiation, or exogenous ceramide analog drugs mediates cell death via apoptosis, necroptosis, or mitophagy. In this chapter, distinct functions and mechanisms of action of endogenous ceramides with different fatty acyl chain lengths in the regulation of cancer cell death versus survival will be discussed. In addition, importance of ceramide subcellular localization, trafficking, and lipid-protein binding between ceramide and various target proteins in cancer cells will be reviewed. Moreover, clinical trials from structure-function-based studies to restore antiproliferative ceramide signaling by activating ceramide synthesis will also be analyzed. Future studies are important to understand the mechanistic involvement of ceramide-mediated cell death in anticancer therapy, including immunotherapy.
Asunto(s)
Apoptosis/efectos de los fármacos , Ceramidas/farmacología , Neoplasias/patología , Transducción de Señal/efectos de los fármacos , Animales , Transporte Biológico , Humanos , Neoplasias/tratamiento farmacológicoRESUMEN
Human papillomavirus (HPV) infection is linked to improved survival in response to chemo-radiotherapy for patients with oropharynx head and neck squamous cell carcinoma (HNSCC). However, mechanisms involved in increased HNSCC cell death by HPV signaling in response to therapy are largely unknown. Here, using molecular, pharmacologic and genetic tools, we show that HPV early protein 7 (E7) enhances ceramide-mediated lethal mitophagy in response to chemotherapy-induced cellular stress in HPV-positive HNSCC cells by selectively targeting retinoblastoma protein (RB). Inhibition of RB by HPV-E7 relieves E2F5, which then associates with DRP1, providing a scaffolding platform for Drp1 activation and mitochondrial translocation, leading to mitochondrial fission and increased lethal mitophagy. Ectopic expression of a constitutively active mutant RB, which is not inhibited by HPV-E7, attenuated ceramide-dependent mitophagy and cell death in HPV(+) HNSCC cells. Moreover, mutation of E2F5 to prevent Drp1 activation inhibited mitophagy in HPV(+) cells. Activation of Drp1 with E2F5-mimetic peptide for inducing Drp1 mitochondrial localization enhanced ceramide-mediated mitophagy and led to tumor suppression in HPV-negative HNSCC-derived xenograft tumors in response to cisplatin in SCID mice.
Asunto(s)
Antineoplásicos/administración & dosificación , Carcinoma de Células Escamosas/tratamiento farmacológico , Ceramidas/metabolismo , Cisplatino/administración & dosificación , Proteínas de la Membrana/metabolismo , Mitofagia , Proteínas E7 de Papillomavirus/metabolismo , Esfingosina N-Aciltransferasa/metabolismo , Animales , Antineoplásicos/metabolismo , Carcinoma de Células Escamosas/patología , Muerte Celular , Línea Celular Tumoral , Cisplatino/metabolismo , Modelos Animales de Enfermedad , Xenoinjertos , Humanos , Ratones SCID , Trasplante de Neoplasias , Proteínas E7 de Papillomavirus/genética , Resultado del TratamientoRESUMEN
Signaling by the transforming growth factor-ß (TGF-ß) receptors I and II (TßRI/II) and the primary cilia-localized sonic hedgehog (Shh) pathway promote cell migration and, consequently, tumor metastasis. In contrast, the sphingolipid ceramide inhibits cell proliferation and tumor metastasis. We investigated whether ceramide metabolism inhibited TßRI/II trafficking to primary cilia to attenuate cross-talk between TßRI/II and the Shh pathway. We found that ceramide synthase 4 (CerS4)-generated ceramide stabilized the association between TßRI and the inhibitory factor Smad7, which limited the trafficking of TßRI/II to primary cilia. Expression of a mutant TßRI that signals but does not interact with Smad7 prevented the CerS4-mediated inhibition of migration in various cancer cells. Genetic deletion or knockdown of CerS4 prevented the formation of the Smad7-TßRI inhibitory complex and increased the association between TßRI and the transporter Arl6 through a previously unknown cilia-targeting signal (Ala31Thr32Ala33Leu34Gln35) in TßRI. Mutating the cilia-targeting signal abolished the trafficking of TßRI to the primary cilia. Localization of TßRI to primary cilia activated a key mediator of Shh signaling, Smoothened (Smo), which stimulated cellular migration and invasion. TßRI-Smo cross-talk at the cilia in CerS4-deficient 4T1 mammary cancer cells induced liver metastasis from orthotopic allografts in both wild-type and CerS4-deficient mice, which was prevented by overexpression of Smad7 or knockdown of intraflagellar transport protein 88 (IFT88). Overall, these data reveal a ceramide-dependent mechanism that suppresses cell migration and invasion by restricting TßRI/II-Shh signaling selectively at the plasma membrane of the primary cilium.
Asunto(s)
Movimiento Celular , Ceramidas/metabolismo , Cilios/metabolismo , Metástasis de la Neoplasia/patología , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Células A549 , Animales , Línea Celular Tumoral , Proliferación Celular , Ceramidas/genética , Cilios/genética , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ratones Noqueados , Receptores de Factores de Crecimiento Transformadores beta/genética , Proteína smad7/metabolismo , Esfingosina N-Aciltransferasa/genética , Esfingosina N-Aciltransferasa/metabolismoRESUMEN
10-Formyltetrahydrofolate dehydrogenase (FDH) converts 10-formyltetrahydrofolate to tetrahydrofolate (THF). Expression of the enzyme in FDH-deficient cancer cells induces cytotoxicity that can be reversed by supplementation with high concentrations of a reduced folate, 5-formyl-THF (leucovorin). In contrast, non-tumor cells are resistant to FDH. The present study was undertaken to investigate mechanisms that could protect cells against FDH suppressor effects. Using 10 microM leucovorin supplementation of FDH-sensitive A549 cells transfected for FDH expression, we selected clones that have acquired resistance against FDH. Resistant cells expressed high levels of FDH and were capable of growing after withdrawal of leucovorin. These cells, however, have increased doubling time due to prolonged S phase. They also have significantly increased levels of total folate pool and THF/5,10-methylene-THF pool while the level of 10-formyl-THF was two-fold lower than in parental FDH-sensitive cells. We have shown that the FDH-catalyzed reaction proceeds at about a three-fold slower rate at the ratio of 10-formyl-THF/THF corresponding to the resistant cells than at the ratio corresponding to parental sensitive cells, due to product inhibition (KI is 2.35 microM). FDH-resistant cells have strongly up-regulated dihydrofolate reductase (DHFR) that is proposed to be a mechanism for the alteration of folate pools and a key component of the acquired resistance. Elevation of DHFR in A549 cells by transient transfection decreased sensitivity to FDH toxicity and allowed selection of FDH-resistant clones. DHFR-induced repression of FDH catalysis could be an S phase-related metabolic adjustment that provides protection against FDH suppressor effects.
Asunto(s)
Leucovorina/farmacología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/antagonistas & inhibidores , Tetrahidrofolato Deshidrogenasa/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Secuencia de Bases , Catálisis , Línea Celular , Cartilla de ADN , Resistencia a Medicamentos , Ácido Fólico/metabolismo , HumanosRESUMEN
A folate enzyme, FDH (10-formyltetrahydrofolate dehydrogenase; EC 1.5.1.6), is not a typical tumour suppressor, but it has two basic characteristics of one, i.e. it is down-regulated in tumours and its expression is selectively cytotoxic to cancer cells. We have recently shown that ectopic expression of FDH in A549 lung cancer cells induces G1 arrest and apoptosis that was accompanied by elevation of p53 and its downstream target, p21. It was not known, however, whether FDH-induced apoptosis is p53-dependent or not. In the present study, we report that FDH-induced suppressor effects are strictly p53-dependent in A549 cells. Both knockdown of p53 using an RNAi (RNA interference) approach and disabling of p53 function by dominant-negative inhibition with R175H mutant p53 prevented FDH-induced cytotoxicity in these cells. Ablation of the FDH-suppressor effect is associated with an inability to activate apoptosis in the absence of functional p53. We have also shown that FDH elevation results in p53 phosphorylation at Ser-6 and Ser-20 in the p53 transactivation domain, and Ser-392 in the C-terminal domain, but only Ser-6 is strictly required to mediate FDH effects. Also, translocation of p53 to the nuclei and expression of the pro-apoptotic protein PUMA (Bcl2 binding component 3) was observed after induction of FDH expression. Elevation of FDH in p53 functional HCT116 cells induced strong growth inhibition, while growth of p53-deficient HCT116 cells was unaffected. This implies that activation of p53-dependent pathways is a general downstream mechanism in response to induction of FDH expression in p53 functional cancer cells.