RESUMEN
Polyploid giant cancer cells (PGCC) are frequently detected in tumors and are increasingly recognized for their roles in chromosomal instability and associated genome evolution that leads to cancer recurrence. We previously reported that therapy stress promotes polyploidy, and that acid ceramidase plays a role in depolyploidization. In this study, we used an RNA-seq approach to gain a better understanding of the underlying transcriptomic changes that occur as cancer cells progress through polyploidization and depolyploidization. Our results revealed gene signatures that are associated with disease-free and/or overall survival in several cancers and identified the cell cycle inhibitor CDKN1A/p21 as the major hub in PGCC and early progeny. Increased expression of p21 in PGCC was limited to the cytoplasm. We previously demonstrated that the sphingolipid enzyme acid ceramidase is dispensable for polyploidization upon therapy stress but plays a crucial role in depolyploidization. The current study demonstrates that treatment of cells with ceramide is not sufficient for p53-independent induction of p21 and that knockdown of acid ceramidase, which hydrolyzes ceramide, does not interfere with upregulation of p21. In contrast, blocking the expression of p21 with UC2288 prevented the induction of acid ceramidase and inhibited both the formation of PGCC from parental cells as well as the generation of progeny from PGCC. Taken together, our data suggest that p21 functions upstream of acid ceramidase and plays an important role in polyploidization and depolyploidization.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Células Gigantes , Neoplasias , Poliploidía , Humanos , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células Gigantes/metabolismo , Células Gigantes/patología , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , TranscriptomaRESUMEN
Ubiquitin (Ub) E1 initiates the Ub conjugation cascade by activating and transferring Ub to tens of different E2s. How Ub E1 cooperates with E2s that differ substantially in their predicted E1-interacting residues is unknown. Here, we report the structure of S. pombe Uba1 in complex with Ubc15, a Ub E2 with intrinsically low E1-E2 Ub thioester transfer activity. The structure reveals a distinct Ubc15 binding mode that substantially alters the network of interactions at the E1-E2 interface compared to the only other available Ub E1-E2 structure. Structure-function analysis reveals that the intrinsically low activity of Ubc15 largely results from the presence of an acidic residue at its N-terminal region. Notably, Ub E2 N termini are serine/threonine rich in many other Ub E2s, leading us to hypothesize that phosphorylation of these sites may serve as a novel negative regulatory mechanism of Ub E2 activity, which we demonstrate biochemically and in cell-based assays.
Asunto(s)
Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Enzimas Activadoras de Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina/metabolismo , Sitios de Unión , Línea Celular , Humanos , Modelos Moleculares , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Relación Estructura-Actividad , Transfección , Enzimas Activadoras de Ubiquitina/química , Enzimas Activadoras de Ubiquitina/genética , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Oncogenic multidrug resistance is commonly intrinsic to renal cancer based on the physiological expression of detoxification transporters, particularly ABCB1, thus hampering chemotherapy. ABCB1 activity is directly dependent on its lipid microenvironment, localizing to cholesterol- and sphingomyelin (SM)-rich domains. As ceramides are the sole source for SMs, we hypothesized that ceramide synthase (CerS)-derived ceramides regulate ABCB1 activity. Using data from RNA-Seq databases, we found that patient kidney tumors exhibited increased CerS2 mRNA, which was inversely correlated with CerS6 mRNA in ABCB1+ clear cell carcinomas. Endogenous elevated CerS2 and lower CerS5/6 mRNA and protein resulted in disproportionately higher CerS2 to CerS5/6 activities (approximately twofold) in chemoresistant ABCB1high (A498, Caki-1) compared with chemosensitive ABCB1low (ACHN, normal human proximal convoluted tubule cell) cells. In addition, lipidomics analyses by HPLC-MS/MS showed bias toward CerS2-associated C20:0/C20:1-ceramides compared with CerS5/6-associated C14:0/C16:0-ceramides (2:1). SMs were similarly altered. We demonstrated that chemoresistance to doxorubicin in ABCB1high cells was partially reversed by inhibitors of de novo ceramide synthesis (l-cycloserine) and CerS (fumonisin B1) in cell viability assays. Downregulation of CerS2/6, but not CerS5, attenuated ABCB1 mRNA, protein, plasma membrane localization, rhodamine 123+ efflux transport activity, and doxorubicin resistance. Similar findings were observed with catalytically inactive CerS6-H212A. Furthermore, CerS6-targeting siRNA shifted ceramide and SM composition to ultra long-chain species (C22-C26). Inhibitors of endoplasmic reticulum-associated degradation (eeyarestatin I) and the proteasome (MG132, bortezomib) prevented ABCB1 loss induced by CerS2/6 downregulation. We conclude that a critical balance in ceramide/SM species is prerequisite to ABCB1 expression and functionalization, which could be targeted to reverse multidrug resistance in renal cancers.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP , Neoplasias Renales , Proteínas de la Membrana , Esfingolípidos , Esfingosina N-Aciltransferasa , Proteínas Supresoras de Tumor , Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Ceramidas/metabolismo , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Degradación Asociada con el Retículo Endoplásmico , Femenino , Humanos , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , ARN Mensajero/genética , Esfingolípidos/metabolismo , Esfingosina N-Aciltransferasa/genética , Esfingosina N-Aciltransferasa/metabolismo , Espectrometría de Masas en Tándem , Microambiente TumoralRESUMEN
Multiple myeloma (MM) remains an incurable disease and there is an unmet medical need for novel therapeutic drugs that do not share similar mechanisms of action with currently available agents. Sphingosine kinase 2 (SK2) is an innovative molecular target for anticancer therapy. We previously reported that treatment with SK2 inhibitor opaganib inhibited myeloma tumor growth in vitro and in vivo in a mouse xenograft model. In the current study, we performed a phase I study of opaganib in patients with relapsed/refractory multiple myeloma (RRMM). Thirteen patients with RRMM previously treated with immunomodulatory agents and proteasome inhibitors were enrolled and treated with single-agent opaganib at three oral dosing regimens (250 mg BID, 500 mg BID, or 750 mg BID, 28 days as a cycle). Safety and maximal tolerated dose (MTD) were determined. Pharmacokinetics, pharmacodynamics, and correlative studies were also performed. Opaganib was well tolerated up to a dose of 750 mg BID. The most common possibly related adverse event (AE) was decreased neutrophil counts. There were no serious AEs considered to be related to opaganib. MTD was determined as at least 750 mg BID. On an intent-to-treat basis, one patient (7.7%) in the 500 mg BID dose cohort showed a very good partial response, and one other patient (7.7%) achieved stable disease for 3 months. SK2 is an innovative molecular target for antimyeloma therapy. The first-in-class SK2 inhibitor opaganib is generally safe for administration to RRMM patients, and has potential therapeutic activity in these patients. Clinicaltrials.gov: NCT02757326.
Asunto(s)
Mieloma Múltiple , Humanos , Animales , Ratones , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , Fosfotransferasas (Aceptor de Grupo Alcohol)/uso terapéutico , Inhibidores de Proteasoma/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , DexametasonaRESUMEN
Adoptive transfer of tumor epitope-reactive T cells has emerged as a promising strategy to control tumor growth. However, chronically-stimulated T cells expanded for adoptive cell transfer are susceptible to cell death in an oxidative tumor microenvironment. Because oxidation of cell-surface thiols also alters protein functionality, we hypothesized that increasing the levels of thioredoxin (Trx), an antioxidant molecule facilitating reduction of proteins through cysteine thiol-disulfide exchange, in T cells will promote their sustained antitumor function. Using pre-melanosome protein (Pmel)-Trx1 transgenic mouse-derived splenic T cells, flow cytometry, and gene expression analysis, we observed here that higher Trx expression inversely correlated with reactive oxygen species and susceptibility to T-cell receptor restimulation or oxidation-mediated cell death. These Trx1-overexpressing T cells exhibited a cluster of differentiation 62Lhi (CD62Lhi) central memory-like phenotype with reduced glucose uptake (2-NBDGlo) and decreased effector function (interferon γlo). Furthermore, culturing tumor-reactive T cells in the presence of recombinant Trx increased the dependence of T cells on mitochondrial metabolism and improved tumor control. We conclude that strategies for increasing the antioxidant capacity of antitumor T cells modulate their immunometabolic phenotype leading to improved immunotherapeutic control of established tumors.
Asunto(s)
Linfocitos T/metabolismo , Tiorredoxinas/metabolismo , Animales , Antioxidantes/metabolismo , Línea Celular Tumoral , Transportador de Glucosa de Tipo 1/metabolismo , Selectina L/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias/metabolismo , Estrés Oxidativo , Fenotipo , Especies Reactivas de Oxígeno/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Linfocitos T/citología , Linfocitos T/inmunología , Tiorredoxinas/genética , Microambiente Tumoral , Antígeno gp100 del Melanoma/genética , Antígeno gp100 del Melanoma/metabolismoRESUMEN
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
The su(var)3-9, enhancer of zeste, trithorax (SET)/inhibitor 2 of protein phosphatase 2A (PP2A) oncoprotein binds and inhibits PP2A, composed of various isoforms of scaffolding, regulatory, and catalytic subunits. Targeting SET with a sphingolipid analog drug fingolimod (FTY720) or ceramide leads to the reactivation of tumor suppressor PP2A. However, molecular details of the SET-FTY720 or SET-ceramide, and mechanism of FTY720-dependent PP2A activation, remain unknown. Here, we report the first in solution examination of the SET-FTY720 or SET-ceramide complexes by NMR spectroscopy. FTY720-ceramide binding resulted in chemical shifts of residues residing at the N terminus of SET, preventing its dimerization or oligomerization. This then released SET from PP2ACα, resulting in PP2A activation, while monomeric SET remained associated with the B56γ. Our data also suggest that the PP2A holoenzyme, composed of PP2A-Aß, PP2A-B56γ, and PP2ACα subunits, is selectively activated in response to the formation of the SET-FTY720 complex in A549 cells. Various PP2A-associated downstream effector proteins in the presence or absence of FTY720 were then identified by stable isotope labeling with amino cells in cell culture, including tumor suppressor nonmuscle myosin IIA. Attenuation of FTY720-SET association by point mutations of residues that are involved in FTY720 binding or dephosphorylation of SET at Serine 171, enhanced SET oligomerization and the formation of the SET-PP2A inhibitory complex, leading to resistance to FTY720-dependent PP2A activation.-De Palma, R. M., Parnham, S. R., Li, Y., Oaks, J. J., Peterson, Y. K., Szulc, Z. M., Roth, B. M., Xing, Y., Ogretmen, B. The NMR-based characterization of the FTY720-SET complex reveals an alternative mechanism for the attenuation of the inhibitory SET-PP2A interaction.
Asunto(s)
Clorhidrato de Fingolimod/farmacología , N-Metiltransferasa de Histona-Lisina/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Proteína Fosfatasa 2/metabolismo , Moduladores de los Receptores de fosfatos y esfingosina 1/farmacología , Células A549 , Dimerización , Humanos , Unión ProteicaRESUMEN
Telomerase activation protects cells from telomere damage by delaying senescence and inducing cell immortalization, whereas telomerase inhibition mediates rapid senescence or apoptosis. However, the cellular mechanisms that determine telomere damage-dependent senescence versus apoptosis induction are largely unknown. Here, we demonstrate that telomerase instability mediated by silencing of sphingosine kinase 2 (SPHK2) and sphingosine 1-phosphate (S1P), which binds and stabilizes telomerase, induces telomere damage-dependent caspase-3 activation and apoptosis, but not senescence, in p16-deficient lung cancer cells or tumors. These outcomes were prevented by knockdown of a tumor-suppressor protein, transcription factor 21 (TCF21), or by ectopic expression of WT human telomerase reverse transcriptase (hTERT) but not mutant hTERT with altered S1P binding. Interestingly, SphK2-deficient mice exhibited accelerated aging and telomerase instability that increased telomere damage and senescence via p16 activation especially in testes tissues, but not in apoptosis. Moreover, p16 silencing in SphK2-/- mouse embryonic fibroblasts activated caspase-3 and apoptosis without inducing senescence. Furthermore, ectopic WT p16 expression in p16-deficient A549 lung cancer cells prevented TCF21 and caspase-3 activation and resulted in senescence in response to SphK2/S1P inhibition and telomere damage. Mechanistically, a p16 mutant with impaired caspase-3 association did not prevent telomere damage-induced apoptosis, indicating that an association between p16 and caspase-3 proteins forces senescence induction by inhibiting caspase-3 activation and apoptosis. These results suggest that p16 plays a direct role in telomere damage-dependent senescence by limiting apoptosis via binding to caspase-3, revealing a direct link between telomere damage-dependent senescence and apoptosis with regards to aging and cancer.
Asunto(s)
Apoptosis , Caspasa 3/metabolismo , Senescencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Neoplasias Pulmonares/patología , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Telómero/patología , Animales , Caspasa 3/genética , Proliferación Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Femenino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Lisofosfolípidos/metabolismo , Ratones , Ratones Noqueados , Ratones SCID , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Telómero/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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
Our previous study suggested that ceramide synthase 6 (CerS6), an enzyme in sphingolipid biosynthesis, is regulated by p53: CerS6 was elevated in several cell lines in response to transient expression of p53 or in response to folate stress, which is known to activate p53. It was not clear, however, whether CerS6 gene is a direct transcriptional target of p53 or whether this was an indirect effect through additional regulatory factors. In the present study, we have shown that the CerS6 promoter is activated by p53 in luciferase assays, whereas transcriptionally inactive R175H p53 mutant failed to induce the luciferase expression from this promoter. In vitro immunoprecipitation assays and gel shift analyses have further demonstrated that purified p53 binds within the CerS6 promoter sequence spanning 91 bp upstream and 60 bp downstream of the transcription start site. The Promo 3.0.2 online tool for the prediction of transcription factor binding sites indicated the presence of numerous putative non-canonical p53 binding motifs in the CerS6 promoter. Luciferase assays and gel shift analysis have identified a single motif upstream of the transcription start as a key p53 response element. Treatment of cells with Nutlin-3 or low concentrations of actinomycin D resulted in a strong elevation of CerS6 mRNA and protein, thus demonstrating that CerS6 is a component of the non-genotoxic p53-dependent cellular stress response. This study has shown that by direct transcriptional activation of CerS6, p53 can regulate specific ceramide biosynthesis, which contributes to the pro-apoptotic cellular response.
Asunto(s)
Proteínas de la Membrana/metabolismo , Mutación Missense , Motivos de Nucleótidos , Elementos de Respuesta , Esfingosina N-Aciltransferasa/metabolismo , Estrés Fisiológico , Transcripción Genética , Proteína p53 Supresora de Tumor/metabolismo , Células A549 , Sustitución de Aminoácidos , Ceramidas/biosíntesis , Ceramidas/genética , Humanos , Imidazoles/farmacología , Proteínas de la Membrana/genética , Piperazinas/farmacología , Esfingosina N-Aciltransferasa/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
A family of six ceramide synthases with distinct but overlapping substrate specificities is responsible for generation of ceramides with acyl chains ranging from â¼14-26 carbons. Ceramide synthase 6 (CerS6) preferentially generates C14- and C16-ceramides, and we have previously shown that down-regulation of this enzyme decreases apoptotic susceptibility. In this study, we further evaluated how increased CerS6 expression impacts sphingolipid composition and metabolism. Overexpression of CerS6 in HT29 colon cancer cells resulted in increased apoptotic susceptibility and preferential generation of C16-ceramide, which occurred at the expense of very long chain, saturated ceramides. These changes were also reflected in sphingomyelin composition. HT-CerS6 cells had increased intracellular levels of sphingosine, which is generated by ceramidases upon hydrolysis of ceramide. qRT-PCR analysis revealed that only expression of acid ceramidase (ASAH1) was increased. The increase in acid ceramidase was confirmed by expression and activity analyses. Pharmacological inhibition of JNK (SP600125) or curcumin reduced transcriptional up-regulation of acid ceramidase. Using an acid ceramidase promoter driven luciferase reporter plasmid, we demonstrated that CerS1 has no effect on transcriptional activation of acid ceramidase and that CerS2 slightly but significantly decreased the luciferase signal. Similar to CerS6, overexpression of CerS3-5 resulted in an â¼2-fold increase in luciferase reporter gene activity. Exogenous ceramide failed to induce reporter activity, while a CerS inhibitor and a catalytically inactive mutant of CerS6 failed to reduce it. Taken together, these results suggest that increased expression of CerS6 can mediate transcriptional activation of acid ceramidase in a JNK-dependent manner that is independent of CerS6 activity.
Asunto(s)
Ceramidasa Ácida/metabolismo , Apoptosis/efectos de los fármacos , Ceramidas/farmacología , Neoplasias del Colon/patología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteínas de la Membrana/metabolismo , Esfingosina N-Aciltransferasa/metabolismo , Ceramidasa Ácida/genética , Antimetabolitos Antineoplásicos/farmacología , Western Blotting , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Citometría de Flujo , Fluorouracilo/farmacología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteínas de la Membrana/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esfingolípidos/metabolismo , Esfingosina N-Aciltransferasa/genética , Células Tumorales CultivadasRESUMEN
Sphingolipids are bioactive lipid effectors, which are involved in the regulation of various cellular signaling pathways. Sphingolipids play essential roles in controlling cell inflammation, proliferation, death, migration, senescence, metastasis and autophagy. Alterations in sphingolipid metabolism have been also implicated in many human cancers. Macroautophagy (referred to here as autophagy) is a form of nonselective sequestering of cytosolic materials by double membrane structures, autophagosomes, which can be either protective or lethal for cells. Ceramide, a central molecule of sphingolipid metabolism is involved in the regulation of autophagy at various levels, including the induction of lethal mitophagy, a selective autophagy process to target and eliminate damaged mitochondria. In this review, we focused on recent studies with regard to the regulation of autophagy, in particular lethal mitophagy, by ceramide, and aimed at providing discussion points for various context-dependent roles and mechanisms of action of ceramide in controlling mitophagy.
Asunto(s)
Autofagia , Ceramidas/metabolismo , Mitocondrias/metabolismo , Mitofagia , Neoplasias/metabolismo , Animales , Humanos , Mitocondrias/patología , Neoplasias/patologíaRESUMEN
Sphingolipid metabolism is being increasingly recognized as a key pathway in regulating cancer cell survival and proliferation. However, very little is known about its role in multiple myeloma (MM). We investigated the potential of targeting sphingosine kinase 2 (SK2) for the treatment of MM. We found that SK2 was overexpressed in MM cell lines and in primary human bone marrow (BM) CD1381 myeloma cells. Inhibition of SK2 by SK2- specific short hairpin RNA or ABC294640 (a SK2 specific inhibitor) effectively inhibited myeloma cell proliferation and induced caspase 3mediated apoptosis. ABC294640 inhibited primary human CD1381 myeloma cells with the same efficacy as with MM cell lines. ABC294640 effectively induced apoptosis of myeloma cells, even in the presence of BM stromal cells. Furthermore, we found that ABC294640 downregulated the expression of pS6 and directed c-Myc and myeloid cell leukemia 1 (Mcl-1) for proteasome degradation. In addition, ABC294640 increased Noxa gene transcription and protein expression. ABC294640, per se, did not affect the expression of B-cell lymphoma 2 (Bcl-2), but acted synergistically with ABT-737 (a Bcl-2 inhibitor) in inducing myeloma cell death. ABC294640 suppressed myeloma tumor growth in vivo in mouse myeloma xenograft models. Our data demonstrated that SK2 provides a novel therapeutic target for the treatment of MM.This trial was registered at www.clinicaltrials.gov as #NCT01410981.
Asunto(s)
Adamantano/análogos & derivados , Inhibidores Enzimáticos/farmacología , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/enzimología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/metabolismo , Piridinas/farmacología , Adamantano/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Mieloma Múltiple/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , ARN Interferente Pequeño/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Sphingolipid molecules act as bioactive lipid messengers and exert their actions on the regulation of various cellular signaling pathways. Sphingolipids play essential roles in numerous cellular functions, including controlling cell inflammation, proliferation, death, migration, senescence, tumor metastasis and/or autophagy. Dysregulated sphingolipid metabolism has been also implicated in many human cancers. Macroautophagy (referred to here as autophagy) "self-eating" is characterized by nonselective sequestering of cytosolic materials by an isolation membrane, which can be either protective or lethal for cells. Ceramide (Cer), a central molecule of sphingolipid metabolism, has been extensively implicated in the control of autophagy. The increasing evidence suggests that Cer is highly involved in mediating two opposing autophagic pathways, which regulate either cell survival or death, which is referred here as autophagy paradox. However, the underlying mechanism that regulates the autophagy paradox remains unclear. Therefore, this review focuses on recent studies with regard to the regulation of autophagy by Cer and elucidates the roles and mechanisms of action of Cer in controlling autophagy paradox. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.
Asunto(s)
Autofagia , Ceramidas/metabolismo , Transducción de Señal , Animales , HumanosRESUMEN
BACKGROUND & AIMS: gp96, or grp94, is an endoplasmic reticulum (ER)-localized heat shock protein 90 paralog that acts as a protein chaperone and plays an important role for example in ER homeostasis, ER stress, Wnt and integrin signaling, and calcium homeostasis, which are vital processes in oncogenesis. However, the cancer-intrinsic function of gp96 remains controversial. METHODS: We studied the roles of gp96 in liver biology in mice via an Albumin promoter-driven Cre recombinase-mediated disruption of gp96 gene, hsp90b1. The impact of gp96 status on hepatic carcinogenesis in response to diethyl-nitrosoamine (DENA) was probed. The roles of gp96 on human hepatocellular carcinoma cells (HCC) were also examined pharmacologically with a targeted gp96 inhibitor. RESULTS: We demonstrated that gp96 maintains liver development and hepatocyte function in vivo, and its loss genetically promotes adaptive accumulation of long chain ceramides, accompanied by steatotic regeneration of residual gp96+ hepatocytes. The need for compensatory expansion of gp96+ cells in the gp96- background predisposes mice to develop carcinogen-induced hepatic hyperplasia and cancer from gp96+ but not gp96- hepatocytes. We also found that genetic and pharmacological inhibition of gp96 in human HCCs perturbed multiple growth signals, and attenuated proliferation and expansion. CONCLUSIONS: gp96 is a pro-oncogenic chaperone and an attractive therapeutic target for HCC.
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Carcinogénesis , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Hígado/metabolismo , Glicoproteínas de Membrana/metabolismo , Alquilantes/farmacología , Animales , Carcinogénesis/efectos de los fármacos , Carcinogénesis/metabolismo , Línea Celular Tumoral , Dietilnitrosamina/farmacología , Retículo Endoplásmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Ratones , Modelos Animales , Chaperonas Moleculares/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
FTY720 (Fingolimod, Gilenya) is a sphingosine analog used as an immunosuppressant in multiple sclerosis patients. FTY720 is also a potent protein phosphatase 2A (PP2A)-activating drug (PAD). PP2A is a tumor suppressor found inactivated in different types of cancer. We show here that PP2A is inactive in polycythemia vera (PV) and other myeloproliferative neoplasms characterized by the expression of the transforming Jak2(V617F) oncogene. PP2A inactivation occurs in a Jak2(V617F) dose/kinase-dependent manner through the PI-3Kγ-PKC-induced phosphorylation of the PP2A inhibitor SET. Genetic or PAD-mediated PP2A reactivation induces Jak2(V617F) inactivation/downregulation and impairs clonogenic potential of Jak2(V617F) cell lines and PV but not normal CD34(+) progenitors. Likewise, FTY720 decreases leukemic allelic burden, reduces splenomegaly, and significantly increases survival of Jak2(V617F) leukemic mice without adverse effects. Mechanistically, we show that in Jak2(V617F) cells, FTY720 antileukemic activity requires neither FTY720 phosphorylation (FTY720-P) nor SET dimerization or ceramide induction but depends on interaction with SET K209. Moreover, we show that Jak2(V617F) also utilizes an alternative sphingosine kinase-1-mediated pathway to inhibit PP2A and that FTY720-P, acting as a sphingosine-1-phosphate-receptor-1 agonist, elicits signals leading to the Jak2-PI-3Kγ-PKC-SET-mediated PP2A inhibition. Thus, PADs (eg, FTY720) represent suitable therapeutic alternatives for Jak2(V617F) MPNs.
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Janus Quinasa 2/metabolismo , Leucemia/tratamiento farmacológico , Glicoles de Propileno/farmacología , Proteína Fosfatasa 2/metabolismo , Esfingosina/análogos & derivados , Animales , Línea Celular Transformada , Línea Celular Tumoral , Células Cultivadas , Fosfatidilinositol 3-Quinasa Clase Ib , Proteínas de Unión al ADN , Activación Enzimática/efectos de los fármacos , Clorhidrato de Fingolimod , Chaperonas de Histonas , Humanos , Immunoblotting , Inmunosupresores/farmacología , Janus Quinasa 2/genética , Estimación de Kaplan-Meier , Leucemia/genética , Leucemia/patología , Ratones , Ratones SCID , Mutación , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Proteína Fosfatasa 2/genética , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Esfingosina/farmacología , Resultado del TratamientoRESUMEN
We have investigated the role of ceramide in the cellular adaptation to folate stress induced by Aldh1l1, the enzyme involved in the regulation of folate metabolism. Our previous studies demonstrated that Aldh1l1, similar to folate deficiency, evokes metabolic stress and causes apoptosis in cancer cells. Here we report that the expression of Aldh1l1 in A549 or HCT116 cells results in the elevation of C16-ceramide and a transient up-regulation of ceramide synthase 6 (CerS6) mRNA and protein. Pretreatment with ceramide synthesis inhibitors myriocin and fumonisin B1 or siRNA silencing of CerS6 prevented C16-ceramide accumulation and rescued cells supporting the role of CerS6/C16-ceramide as effectors of Aldh1l1-induced apoptosis. The CerS6 activation by Aldh1l1 and increased ceramide generation were p53-dependent; this effect was ablated in p53-null cells. Furthermore, the expression of wild type p53 but not transcriptionally inactive R175H p53 mutant strongly elevated CerS6. Also, this dominant negative mutant prevented accumulation of CerS6 in response to Aldh1l1, indicating that CerS6 is a transcriptional target of p53. In support of this mechanism, bioinformatics analysis revealed the p53 binding site 3 kb downstream of the CerS6 transcription start. Interestingly, ceramide elevation in response to Aldh1l1 was inhibited by silencing of PUMA, a proapoptotic downstream effector of p53 whereas the transient expression of CerS6 elevated PUMA in a p53-dependent manner indicating reciprocal relationships between ceramide and p53/PUMA pathways. Importantly, folate withdrawal also induced CerS6/C16-ceramide elevation accompanied by p53 accumulation. Overall, these novel findings link folate and de novo ceramide pathways in cellular stress response.
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Apoptosis , Ceramidas/biosíntesis , Ácido Fólico/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteínas de la Membrana/biosíntesis , Esfingosina N-Aciltransferasa/biosíntesis , Estrés Fisiológico , Proteína p53 Supresora de Tumor/metabolismo , Aldehído Deshidrogenasa/genética , Aldehído Deshidrogenasa/metabolismo , Sustitución de Aminoácidos , Proteínas Reguladoras de la Apoptosis/biosíntesis , Proteínas Reguladoras de la Apoptosis/genética , Línea Celular Tumoral , Ceramidas/genética , Humanos , Proteínas de la Membrana/genética , Mutación Missense , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Elementos de Respuesta/genética , Esfingosina N-Aciltransferasa/genética , Proteína p53 Supresora de Tumor/genética , Regulación hacia Arriba/genéticaRESUMEN
Mechanisms by which autophagy promotes cell survival or death are unclear. We provide evidence that C(18)-pyridinium ceramide treatment or endogenous C(18)-ceramide generation by ceramide synthase 1 (CerS1) expression mediates autophagic cell death, independent of apoptosis in human cancer cells. C(18)-ceramide-induced lethal autophagy was regulated via microtubule-associated protein 1 light chain 3 ß-lipidation, forming LC3B-II, and selective targeting of mitochondria by LC3B-II-containing autophagolysosomes (mitophagy) through direct interaction between ceramide and LC3B-II upon Drp1-dependent mitochondrial fission, leading to inhibition of mitochondrial function and oxygen consumption. Accordingly, expression of mutant LC3B with impaired ceramide binding, as predicted by molecular modeling, prevented CerS1-mediated mitochondrial targeting, recovering oxygen consumption. Moreover, knockdown of CerS1 abrogated sodium selenite-induced mitophagy, and stable LC3B knockdown protected against CerS1- and C(18)-ceramide-dependent mitophagy and blocked tumor suppression in vivo. Thus, these data suggest a new receptor function of ceramide for anchoring LC3B-II autophagolysosomes to mitochondrial membranes, defining a key mechanism for the induction of lethal mitophagy.
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Autofagia , Ceramidas/farmacología , Mitofagia/efectos de los fármacos , Fagosomas/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Humanos , Lípidos/química , Microscopía ConfocalRESUMEN
Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer resistant to current therapies, including oxaliplatin (Oxa). Growing evidence supports the ability of cancers to harness sphingolipid metabolism for survival. Sphingosine-1-phosphate (S1P) is an anti-apoptotic, pro-survival mediator that can influence cellular functions such as endoplasmic reticulum (ER) stress. We hypothesize that PDAC drives dysregulated sphingolipid metabolism and that S1P inhibition can enhance ER stress to improve therapeutic response to Oxa in PDAC. Methods: RNA sequencing data of sphingolipid mediators from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression Project (GTEx) datasets were analyzed. Murine and human PDAC cell lines were treated with small interfering RNA (siRNA) against sphingosine kinase-2 (SPHK2) or ABC294640 (ABC) and incubated with combinations of vehicle control or Oxa. In an orthotopic syngeneic KPC PDAC model, tumors were treated with either vehicle control, Oxa, ABC, or combination therapy. Results: RNA sequencing analysis revealed multiple significantly differentially expressed sphingolipid mediators (P < 0.05). In vitro, both siRNA knockdown of SPHK2 and ABC sensitized cells to Oxa therapy (P < 0.05), and induced eukaryotic initiation factor 2α (eIF2α) and protein kinase RNA-like endoplasmic reticulum kinase (PERK) phosphorylation, hallmarks of ER stress. In vitro therapy also increased extracellular high mobility group box 1 (HMGB1) release (P < 0.05), necessary for immunogenic cell death (ICD). In vivo combination therapy increased apoptotic markers as well as the intensity of HMGB1 staining compared to control (P < 0.05). Conclusions: Our evidence suggests that sphingolipid metabolism is dysregulated in PDAC. Furthermore, S1P inhibition can sensitize PDAC to Oxa therapy through increasing ER stress and can potentiate ICD induction. This highlights a potential therapeutic target for chemosensitizing PDAC as well as an adjunct for future chemoimmunotherapy strategies.
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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.